Journal für Reproduktionsmedizin und Endokrinologie – Journal of and Endocrinology –

Andrologie • Embryologie & Biologie • Endokrinologie • Ethik & Recht • Genetik Gynäkologie • Kontrazeption • Psychosomatik • Reproduktionsmedizin • Urologie

Andrology 2020 12th International/11th European/ 32nd German Congress of 5–9 December 2020 DIGITAL Abstracts J. Reproduktionsmed. Endokrinol 2020; 17 (Supplementum 1), 5-85

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Andrology 2020 12th International/11th European/ 32nd German Congress of Andrology 5–9 December 2020 DIGITAL Abstracts* DGA-Abstracts Vorträge geladener Redner

Saturday, 5 December 2020 check-list has to be answered to fulfill the cri- Some andrological diseases can be treated teria for payment. There are still some major causally such as hypogonadotropic hypo­ 10:00–10:45 h deficits in the present regulations, e.g. how gonadism, hyperprolactinemia, infections or to deal with the prepubertal boys with only ejaculatory disorders. Kryokonservierung von Sper­ experimental treatment options available: in It seems to be logical, to treat male immuno- mien – Relevantes und Neues prepubertal boys, immature testicular tissue SEINE TESTOSTERON- logical infertility with glucocorticosteroids; samples offer the chance for cryopreservation however, doubtless proof of evidence is still Cryopreservation of germ cells of spermatogonial stem cells. The later use missing. in German law and guidelines – of these cells may include methods such as THERAPIE SOLLTE the right to preserve ? grafting or in-vitro maturation of spermato- In some cases the cause of male infertility is known (non-obstructive azoospermia due to S. Kliesch zoa. Therefore, several networking European AZF gene deletions) without causal therapy Center of Reproductive Medicine and Andrology Centers offer this kind of fertility preserva- GENAU SO SEIN WIE ER: available. (CeRA), Department of Clinical and Surgical Androlo- tion to boys by now. In Germany the network Androprotect cares for the boy, while the gy, WHO collaboration center for research in male A significant percentage of male infertility girls get help by the network FertiProtekt. reproduction, Training Center of the European Aca- is still considered idiopathic and a variety demy of Andrology (EAA), University Clinic Münster Although further discussion of unresolved of empiric therapies has been used in these INDIVIDUELL (UKM), Germany questions is needed, the present regulatory cases, even if active principle and effective- guideline offers an important progress and ness are not proven. Fertility preservation in males and females advantage for patients suffering from an ill- facing a potentially gonadotoxic treatment ness which by itself or by the necessary treat- Antiestrogens such as tamoxifen an clomifen is highly relevant with a high impact on pa- ment will threat their reproductive capacity. have an exceptional position in andrological tients´ quality of life. In after therapy. Although treatment of infertile males spermarche and adults in their reproductive is an off lable use, their pharmacological ef- age, fertility preservation should be routinely 16:00–16:30 h fects are known (inhibition of hypothalamic performed by cryopreservation of ejaculated Therapie der Infertilität – End­ and pituitary estrogen receptors and stimula- or testicular (after surgical sperm ex- punkt Schwangerschaft tion of testosterone production and spermato- traction, TESE, in case of azoospermia or genesis by increase of LH and FSH). anejaculation). In Germany, the costs of these Medikamentöse Therapieoptionen Apart from drugs or factors related to life- procedures have to be paid by the patients und „life style“ Veränderungen – style such as alcohol and tobacco smoke, var- themselves. However, in May 2019 a new Wie kann man den Patienten be­ ious environmental and occupational agents, law was set up and cryopreservation will be raten? both chemical and physical, may impair male covered by the health insurances from now reproductive functions. Reproductive toxici­ on. The detailed regulations for refunding of F.-M. Köhn ty may evolve at the hypothalamic-pituitary, 1 fertility preservation measures are nearly set Andrologicum Munich, Germany testicular, or post-testicular level; endpoints • Individuell dosierbar up by now, but still under discussion. In its Andrological therapy should improve male comprise deterioration of essentials, all patients with diseases and treat- 1 fertility and – if necessary – ability for sexual and sperm function as well as endocrine dis- ments leading to the loss of gonads or to a • Geringe Gelmenge intercourse. orders and sexual dysfunction. With regard to potential failure of gonadal function will have the complex regulation of the male reproduc- access to the refunding of fertility preserva- The first intention is achieved by 1 tive system, the available information con- tion by their health insurances. This is not natural way; however, optimization of semen • Einfache Anwendung cerning single exogenous factors and their restricted to oncological diseases only. How- quality may also be worthwhile in order to mechanisms of action in humans is limited. ever, prior to cryopreservation a consultation perform less invasive assisted reproductive This is also due to the fact, that extrapolation by the specialized doctor treating the disease technologies (intrauterine in- Referenzen: stead of in vitro fertilization). of results obtained from experimental animal ® and an expert in the reproductive field (for 1. Aktuelle Fachinformation Testogel Dosiergel 16,2 mg/g Gel, Stand: Januar 2020. or in-vitro studies remains difficult. females a gynecologist trained in reproduc- Basis of the andrological workup is the cor- tive medicine and for males a doctor trained rect diagnosis and the estimation of the suc- Considering these aspects history-taking is an DR. KADE / BESINS Pharma GmbH, Berlin. Testogel® Dosiergel 16,2 mg/g Gel Wirkstoff : Testosteron. Verschreibungspfl ichtig. Zus.: 1 g Gel enthält 16,2 mg Testosteron. Eine Betätigung der Dosierpumpe in andrology) is mandatory and a kind of cess of therpeutical strategies. important basis of andrological diagnosis. liefert 1,25 g Gel, das 20,25 mg Testosteron enthält. Sonst. Bestandt.: Carbomer 980, Isopropylmyristat (Ph. Eur.), Ethanol 96 %, Natriumhydroxid, gereinigtes Wasser. Anw.: Testosteronersatztherapie bei männlichem Hypogonadismus, wenn der Testosteronmangel klinisch und labormedizinisch bestätigt wurde. Gegenanz.: Prostata- od. Brustkarzinom bzw. Verdacht auf diese Erkrankungen; Überempfi nd- lichkeit gg. den Wirkstoff od. einen der sonst. Bestandteile. Nebenw.: Emotionale Symptome (Stimmungsschwank., aff ektive Störung, Wut, Aggression, Ungeduld, Schlafl osigkeit, abnorme Träume, gestei- gerte Libido), Hautreaktionen (Akne, Alopezie, trockene Haut, Hautläsionen, Kontaktdermatitis, veränderte Haarfarbe, Hautausschlag, Überempfi ndlichkeit an der Applikationsstelle, Juckreiz an der Appli- kationsstelle), erhöhte PSA-, Hämatokrit-, Hämoglobin-Werte, maligne Hypertonie, Hitzewallungen, Venenentzünd., Durchfall, Blähungen, Schmerzen im Mund, Gynäkomastie, Brustwarzenbeschwerden, Hodenschmerzen, häufi gere Erektionen, eindrückbares Ödem. Weitere Nebenwirkungen aus Spontanbe- *Begutachtet und zusammengestellt vom wissenschaftlichen Komitee. Ein Verzeichnis der präsentierenden Autoren finden Sie auf Seite 86 f, alphabetisch richten mit Testogel bzw. von anderen testosteronhalt. Produkten gemeldete Nebenwirkungen: Polyzythämie, Anämie, Depression, Angst, Kopfschmerzen, ­geordnet nach Erstautoren. Mit Genehmigung von Wiley Doppelpublikation der Abstracts in „Andrology“ und „JRE“ Schwindel, Parästhesien, Vasodilatation (Hitzewallungen), tiefe Venenthrombose, Dyspnoe, Übelkeit, Schwitzen, Hypertrichose, Schmerzen des Bewe- gungsapparates, Probleme beim Wasserlassen, Hodenerkrankungen, Prostatavergrößerung, Oligospermie, benigne Prostatahyperplasie, Asthenie, Ödeme, Unwohlsein, Gewichtszunahme. Warnhinw.: Enthält Ethanol. Packungsbeilage beachten. Weit. Hinw. s. Fach- u. Gebrauchsinfo. Stand: 01/2020 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 5

TED 061 Rocker_210x297mm_3mm.indd 1 25.11.20 10:58 Andrology 2020 – Abstracts

16:30–17:00 h Sunday, 6 December 2020 tion (micro-TESE-ICSI) treatment that used Therapie der Infertilität – End­ fresh or cryopreserved sperm in patients with 10:06–10:15 h nonobstructive azoospermia (NOA). A total punkt Schwangerschaft of 338 NOA patients with 344 consecutive Der Patient mit Azoospermie – Surgical sperm collection – gold cycles received treatment in the reproductive praxisrelevante Empfehlungen medicine center of Peking University Third standard microTESE – is conven­ zur Diagnostik und Therapie Hospital in Beijing, China, from January tional testicular biopsy out? 2014 to December 2017. Fresh and I. Hoffmann Was macht eine gute Hodenhisto­ fresh sperm were used in 222 patients with Center of Reproductive Medicine and Andrology logie aus und warum? 234 cycles (Group A). Fresh oocytes and cry- opreserved sperm were used in 116 patients (CeRA), Department of Clinical and Surgical Androlo- D. Fietz with 110 cycles (Group B). We compared pa- gy, University Clinic Münster (UKM), Germany Institute for Veterinary Anatomy, Histology and Em- tient characteristics, embryonic development, bryology, Justus-Liebig-University Giessen, Germany Surgical extraction of spermatozoa from tes- and pregnancy outcomes between Groups A ticular tissue can be carried out using the fol- Following EAU guidelines, testis biopsies are and B. There was no statistical difference in lowing surgical procedures: indicated in case of azoospermia (obstructive the patient characteristics, and no differences – Microscopic Testicular Sperm Extraction or non-obstructive), Klinefelter syndrome, were observed with fertilization or qual-

DGA-Abstracts (mTESE ) in suspicion of testicular tumours ity embryo rates between Groups A and B. – Conventional Testicular Sperm Extraction (TGCTs) and adult cryptorchism. Multiple The rates of clinical pregnancy and live birth (cTESE ) testicular biopsies from each testis enhance were both higher for Group A than those for the chances to detect a) elongated Group B (both P < 0.05). In conclusion, fresh – Microscopic epididymal sperm aspiration for assisted reproduction (in combination testicular sperm appears to produce better (MESA) with testicular sperm extraction, TESE) and/ ICSI outcomes than cryopreserved testicular The principle of testicular sperm extraction is or b) TGCTs. Testicular histology is essential sperm in patients with NOA. based on the fact that the testicular spermato- for the evaluation of biopsies – but only if genesis a certain threshold beyond, in order done correctly. for spermatozoa can be found in the ejaculate Sampling and fixation of testis tissue is of [Silver 1999]. crucial importance: Squeezing artefacts need 14:00–14:30 h While testicular and epididymal spermatozoa to be avoided and a fixation with formalin (as can be extracted with a high degree of prob- performed commonly in pathologies) is not Clinical use of FSH in male inferti­ ability in obstructive azoospermia, surgical recommended. The latter leads to shrinking lity testicular sperm extraction remains a chal- artefacts and a decreased histological quality H. M. Behre lenge in non-obstructive azoospermia. In the in regard to structural preservation and nu- Center for Reproductive Medicine and Andrology, microscopic surgical technique, the semi­ clear morphology. Therefore, use of Bouin’s University Hospital Halle (Saale), Martin Luther Uni- niferous tubules are visualized and assessed or Stieve’s solution is recommended. As versity Halle-Wittenberg, Germany using a surgical microscope at a magnifica- standard staining, hematoxylin and eosin or tion of up to 20 times. A relationship between Periodic Acid Schiff staining are sufficient For several decades, the established clinical the diameter of a tubule and the probability as both enable qualitative and quantitative use of FSH in male infertility is the treat- of finding sperm is known [Schlegel 1999]. evaluation of germ cell development (nor- ment of male patients with hypogonadotropic Through the targeted collection, the mTESE mal or reduced spermatogenesis, maturation hypogonadism. Successful therapy will be increases the probability of identifying the arrests, germ cell aplasia and TGCTs) and stimulation of spermatogenesis in the testis spermatogenesis islands obtained in non- somatic cells (Sertoli and Leydig cells, im- and appearance of viable spermatozoa in the obstructive azoospermia and of extracting mune cells). Additionally, further analyses as ejaculate - and finally the induction of a clini- sperm for ICSI therapy. Many studies have e.g. immunohistochemistry as indicated for cal pregnancy in the female partner and the shown a significantly higher sperm extraction TGCT diagnosis is possible with Bouin-fixed birth of a healthy child. rate of the mTESE compared to the cTESE material. The last step in histological evalu- Several clinical studies with various FSH [Schlegel 1999, Bernie et al., 2015 etc.]. This ation is the use of an objective score count preparations in combination with hCG have also applies to certain groups of patients, analysis to predict the chances for a positive demonstrated the high treatment efficacy such as patients with a AZFc deletion [Black TESE outcome for the clinic. regarding these clinical endpoints. Shortcom- et al., 2015], patients with Klinefelter‘s syn- Only if all these aspects are taken under care- ings of this treatment are the long duration of drome [Sabbaghian et al., 2011], patients af- ful consideration, testicular histology is sig- therapy – that might last longer than 2 years ter already unsuccessfully performed cTESE nificant and with this an essential part of tes- in some patients – and the inconvenience of [Kalsi et al., 2014]. ticular biopsy and andrological examination. FSH injections every second or third day. A recent meta-analysis has shown that sperm Relevant improvements of FSH therapy in extraction rate at the cTESE and mTESE are male patients can be expected with modern equal [Corona et al., 2019]. Interpreting the 13:30–14:00 h FSH treatment options already available for study results the heterogeneity of patient pop- Pre Congress Course, clinical assisted reproduction treatment in the female ulations and study designs should be consid- patient. ered, particularly the significantly different Arguments for using fresh or In most countries, FSH treatment of patients risk profile between the groups of patients, cryopreserved sperm when with normogonadotropic idiopathic infertil- inclusion of randomized and non-randomized ­offering TESE in NOA patients ity and oligozoospermia is still considered trials and studies without information on his- experimental. Recent meta-analyses have H. Lin, H. Zhang, J. Mao, H. Jiang tological findings. shown that FSH can significantly increase Department of Urology, Peking University Third Hos- pregnancy rates in the female partners of pital, Peking, China these patients, but the effect-size is relatively Using fresh or cryopreserved sperm when of- low. Therefore, predictive factors for treat- fering TESE in NOA patients is controversial. ment success have to be identified, including We performed a study to evaluate the clinical FSH pharmacogenetics, to select the right outcomes of microdissection testicular sperm normogonadotropic patients with idiopathic extraction-intracytoplasmic sperm injec- infertility for FSH therapy.

6 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

15:30–16:00 h Systematic diagnosis of the infer- tile male S. Kliesch Center of Reproductive Medicine and Andrology (CeRA), Department of Clinical and Surgical Androlo- gy, WHO collaboration center for research in male reproduction, Training Center of the European Aca- demy of Andrology (EAA), University Clinic Münster (UKM), Germany The diagnostic work-up in the infertile male aims to identify underlying reasons for in- fertility and may lead either to a causative treatment or at least to a better understanding of the disease. Both will facilitate adequate councelling of the couple and influence the fi- DGA-Abstracts nal treatment decision in patients with couple infertility. The diagnostic workflow follows a systematic approach to elucidate previous Figure 1. D. Ježek factors influencing fertility and the present © D. Ježek status. Medical history, preferably taken in the presence of the female partner, clinical ex- amination with focus on gonadal phenotype form of male infertility that affects 8–20% of the testis parenchyma. Apart from routine and function as well as laboratory testing of of infertile patients. It has two forms, i.e. ob- hemalaun-eosin staining, immunohistochem- (including gonadotropins and an- structive and non-obstructive form (OA, ob- istry (IHC) is mandatory in order to check for drogens) and semen analysis according to the structive azoospermia, NOA, non-obstructive GCNIS. Most commonly used IHC mark- World Health Organization (WHO) standards azoospermia). In the case of OA, the vast ma- ers include placental alkaline phosphatase form the diagnostic basis. The final results jority of testicular parenchyma is preserved. (PLAP) and octamer-binding transcription may trigger further investigations such as A significant number of seminiferous tubules factor 4 (OCT-4). Using our technique of tis- genetic testing or sperm function tests. Apart have full spermatogenesis. In contrast to OA, sue handling, detailed histological analysis from sperm count, motility and morphology, NOA is much more severe type of azoo- and freezing-thawing, we were able to isolate additional tests have been developed in recent spermia, with extensive changes in the struc- spermatozoa in more than 68% of cases with years to improve the understanding of sperm ture of the male sex gland and impaired sper- azoospermia (Fig. 1). functional capability. DNA fragmentation matogenesis. However, in 60–70% of patients References: analysis has gained evidence and tests on with NOA, there are foci of spermatogenesis - Kern SQ, Speir RW, Akgul M, Cary C. Rare benign and sperm motility by computer-assisted analysis with a maintained production of mature sper- malignant testicular lesions: histopathology and manage- have been introduced in the diagnostic work matids and/or spermatozoa (“mixed atrophy ment. Curr Opin Urol 2020; 30: 235–44. up recently. Finally, the differential diagnosis of seminiferous tubules”). These cells can be - Corona G, Minhas S, Giwercman A, Bettocchi C, et al. of hypothalamic–pituitary, testicular mal- used for ICSI after the application of micro- Sperm recovery and ICSI outcomes in men with non-ob- function or congenital reproductive failure surgical procedure - open biopsy of the testis structive azoospermia: a systematic review and meta- will finally determine the possible treatment with cryopreservation. This procedure is both analysis. Hum Reprod Update 2019; 25: 733–57. options prior to the use of assisted repro- a diagnostic and a therapeutic procedure dur- - Lopes LS, Esteves SC. Testicular sperm for intracyto- plasmic sperm injection in non-azoospermic men: a para- ductive techniques (ART). After systematic ing which several pieces of the tissue (usually digm shift. Panminerva Med 2019; 61: 178–86. diagnostic work-up, 30% of patients will be from both ) are taken for histological identified with distinct diagnosis which are analysis and cryopreservation. Due to the worthwhile to be treated prior to ART. En- cryo­preservation, a mini-bank of testicular bi- 16:30–17:00 h docrine, surgical, or empirical therapeutic opsies for a given patient is formed. Testicu- options may be performed as stand alone lar sperm extraction (TESE) and ICSI pro- Genetic causes of spermatogenic treatment or in combination with ART after cedure may be repeated several times, using defect – Novel diagnostics in the interdisciplinary consultation of both part- frozen biopsy material. Thus, the patient with clinical workup azoospermia, as a rule, is subjected to a single ners, male and female, by the andrologist and F. Tüttelmann surgical procedure. A detailed histological a specialized gynecologist. This effort may Institute of Reproductive Genetics, University of analysis involves determining the degree of finally result in an optimized patient-centered Münster, Germany treatment approach for couple infertility. preservation of spermatogenesis and routine detection of possible germ cell neoplasia Impaired spermatogenesis is the most com- in situ (GCNIS) using immunohistochemi- mon cause for male infertility. Quantitative 16:00–16:30 h cal methods. In our setting, after excision, defects – namely oligo-/azoospermia – can each testicular biopsy is processed immedi- go hand in hand with qualitative defects of The importance of histological ately in the operation theatre. The excised sperm motility and morphology, while these analysis in diagnosing male infer- tissue is immersed in the transport medium can also occur in cases with normal sperm tility that preserves from further degrada- numbers. In both quantitative and qualita- tion. The biopsies are brought to the sterile tive spermatogenic failure, genetic causes D. Ježek cabinet inside the operation room. Within the increase with severity of the phenotype. For Dept. Histology and Embryology, University of cabinet, each biopsy is divided into two parts: example, azoospermia and especially the sub- ­Zagreb, School of Medicine, Dept. of Transfusion one part is plunged into the freezing medium type of meiotic arrest is clearly a genetic dis- Medicine and Transplantation Biology, University whereas the other part is fixed for the histo- order. Fittingly, a steadily increasing number Hospital Zagreb, Croatia logical analysis. Thus, “paired” or “matched” of associated genes has been described. Like- Histological analysis in andrology/male biopsies are formed. After fixation and par- wise, the genetic cause can be identified in infertility is mostly applicable for cases of affin embedding, an extensive cutting of the men with severely and specifically impaired azoospermia, where there is a need to analyse paraffin block is necessary to provide a de- motility and/or morphology. The former is testicular biopsies. Azoospermia is a difficult tailed insight into the histological structure genetically overlapping with primary ciliary

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 7 Andrology 2020 – Abstracts

dyskinesia (PCD), while the latter is recog- a time of day dependent manner, and that ‘Plus Paternal Project’ is currently develop- nised as the phenotypic entity of MMAF – treatment of obstructive sleep apnea by con- ing a roadmap for action. Multiple Morphologically Abnormalities of tinuous positive airway pressure probably HM is now well-recognized as a trusted re- the Sperm Flagellum. increases overnight testosterone concentra- source in all sectors: its innovative, respon- For many years, chromosome and AZF dele- tions. The impact of circadian misalignment sive and flexible approach is a model of inde- tion analyses were the only frequently applied on testosterone has been inadequately stud- pendent evidence-based men’s health advice genetic tests. However, these provide only ied, but does not appear to influence mean and training targeted to the needs of consum- ~20% of diagnoses in azoospermic men and testosterone levels. ers and health professionals. ~4% in all men in infertile couples. In fact, male infertility genetics has been lagging be- hind almost all other fields. Fortunately, the Symposium 1 – Genetics in increased application of large-scale genetic Monday, 7 December 2020 Male Reproduction analyses triggered by the development of next-generation sequencing (NGS) has now Plenary Lecture 1 09:30–09:50 h also arrived to Andrology. As such, exome sequencing has lately led to the continuous 08:30–09:15 h The identification and validation of genetic causes of male in­ discovery of genes associated with impaired The Healthy Male – evolution DGA-Abstracts spermatogenesis. Now, these findings to- fertility gether with NGS technology (exome or gene of Australia’s men’s health pro­ gramme M. O’Bryan panel sequencing) need to be translated into Brendan Houston and the International Male In­ the diagnostic setting. Since 2017, we have R. McLachlan fertility Genomics Consortium, The School of Bio- pioneered prospective exome sequencing Medical Director, Healthy Male [Andrology Austra- Sciences, The University of Melbourne, Australia in azoo-/cryptozoospermic men as well as lia], Director of Andrology Services, Hudson Institute in men with specific motility/morphology of Medical Research, Dept of Endocrinology, Mo- As explored earlier in the congress, the aeti- defects. This has significantly increased the nash Health, Adjunct Professor of Andrology; Dept. ology of human male infertility is frequently diagnostic yield, which is not only beneficial of Obstetrics & Gynaecology, Monash University. thought to be genetic. Unfortunately, proving in itself for affected men but also increasingly Consultant Andrologist, Monash IVF Group, Australia this is the case is often extremely difficult. therapeutically relevant, e.g., to assess suc- This difficulty arises for several reasons, in- cess for testicular sperm extraction (TESE) Healthy Male [formerly Andrology Aus- cluding that infertility, by definition, will lim- prior to the biopsy. tralia] was created in 2000 to improve com- it family size, thus making traditional linkage munity and professional awareness and edu- studies extremely difficult. Further, the num- cation in male reproductive health [MRH]. ber of genes expressed during spermatogen- 17:30–18:00 h Supported by the Federal Government, this esis (>19,000 in humans, including >2,000 national ‘virtual centre’ brings together ex- that are testis-enriched) means that offspring Sleep and Andrological Health perts to implement a collaborative strategy. infertility is likely a common consequence P. Y. Liu Initially focussing on MRH issues [androgen of mutagenesis within the germ line. At a Lundquist Institute at Harbor-ULCA Medical Center, use/abuse, infertility, ED, testis cancer, pros- practical level this means that the frequency David Geffen School of Medicine University of Cali- tate disease] HM recognised the linkages to of male infertility within a population will be fornia Los Angeles, CA, United States chronic disease [cardiovascular disease, dia- relatively high, but individual gene-specific betes, mental health] and expanded its focus. causes of male infertility will be low. Thus, It has long been recognized that the environ- The overall approach has been to first un- while identifying a potential genetic cause ment impacts reproduction and reproductive derstand consumer needs, then assess the of infertility is now technically feasible, health in all mammalian species through evidence, educate and upskill health profes- finding the second and third infertile patient seasonal, infradian, diurnal, and ultradian sionals and finally ‘close the loop’ through containing mutations in the same gene will processes. Sleep is one tightly regulated community education of what and how help be very laborious. To meet this challenge, the diurnal process which, when disordered, is can be accessed. Although not resourced as a International Male Infertility Genomics Con- recognized to cause hypersomnolence and research organisation the longitudinal ‘Men sortium is working collectively to assemble neuropsychological deficits, alter inflamma- in Australia telephone survey’ [MATeS, DNA samples from large numbers of clini- tory pathways, and unltimately lead to car- Lancet 2006] identified knowledge gaps for cally well described infertile men so as to diometabolic ill health. Disordered sleep may future attention. allow cross-referencing of sequencing data be insufficient as occurs with the modern across countries. In a parallel test of causality, Core material were generated by expert advi- 24/7 lifestyle, misaligned to the environment and as a step towards defining the mechanism sory groups and collaborative programs de- due to shift work or jet lag, or disrupted from of gene action, the role of individual genes in veloped with primary health practitioners and obstructive sleep apnea. Epidemiological and male infertility is being tested using a range disadvantaged groups, such as indigenous interventional data are accumulating to show of model systems ranging from cell lines, to people. Strong demand from grassroots or- that insufficient, misaligned and disrupted flies, to zebrafish, to mice. Each model sys- ganisations for evidence-based information sleep in men may lead to hypogonadism, tem has its advantages and disadvantages. has occurred for local and national use. Re- erectile dysfunction, and infertility; although Within this presentation we will explore the branding to ‘Healthy Male’ in 2018, a new more research is needed to determine the ex- analytical pipeline used to prioritize male in- website and social media engagement ex- act mechanisms by which the hypothalamo- fertility candidate genes identified via whole panded its profile. pituitary testicular axis in young and older exome/genome sequencing, the production of men is impacted. This symposium talk will Most effort has been given to the primary models and their phenotyping as a means to highlight the clinical research methodolo- care workforce, but specialist issues are be- validate the genetic causes of male infertility. gies utilized to interrogate these processes, ing addressed e.g. IVF gynaecologists in in order to contextualize and summarize the male fertility care, the Endocrine Society re available epidemiological and interventional androgen guidelines, and HM trainee fellow- 09:50–10:10 h studies performed. Intricate chronobiological ships. A close relationship has been forged Translational aspects of novel study designs and mathematical deconvolu- with the Federal Health Department with HM gene mutations affecting human tion of time series to determine as a trusted resource for policy development, hormone secretion, pulsatility and circadian notably overseeing National Men’s Health male fertility influences will be outlined where relevant. In Strategy 2020–2030. Recognizing the need M. Laan summary, available studies show that sleep to better support young men from preconcep- Institute of Biomedicine and Translational Medicine, restriction decreases 24 hour testosterone in tion though their first year of fatherhood, the University of Tartu, Estonia

8 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

Infertility affects ~7% of men, caused by are maintained, an intrinsic ageing process showed statistically significant differences congenital or acquired spermatogenic im- continues which is specific to germ cells, between the three groups and also discrimi- pairment due to either testicular damage or interfering with DNA integrity and different nated low testosterone values in the cohort defective per se. Today, up to from somatic ageing. Moreover, healthy age- of patients analyzed. The power of the candi- >50% of cases remain unexplained. Current ing enables full spermatogenesis during life- date proteins for discriminating IR, CVR and andrological workup includes limited genetic time and this seems to be an intrinsic deter- MetS were in general similar or better than tests with clear value in patient management minant. However, to maintain normal sperm testosterone. – standard karyotyping, analysis of AZF dele- output compensatory mechanisms such as an Conclusion New testosterone dependent tions and CFTR gene mutations. Insufficient increased proliferation of type-A spermato- proteins were identified which could form knowledge of mechanisms underlying infer- gonia, the reserve testicular stem cells, are the basis for more accurately measuring the tility limits primary management and patient- taking place. Overall, healthy ageing effects biological androgen acitivty. centred clinical care. Active research has male germ cell integrity at different levels been ongoing to identify novel genetic factors and could be responsible for the observed re- 14:09–14:27 h in male factor infertility, uncovering far more duced fecundity and poorer health prognosis diverse genetic heterogeneity than initially for the offspring of elderly fathers. Fertility awarness – Time to focus thought. Even for the extreme phenotypes, Grants: Supported by the German Research on the male partner such as azoospermia, only a small number of Foundation and internal CeRA grants. M. Bodin

genes have been identified with recurrent mu- DGA-Abstracts tations explaining the lack of . Leader- Centre for Sexology and Sexuality Studies, Malmö ship in translating the findings of monogenic Symposium 4 − Microbiome University, Sweden causes for the immediate patient benefit con- and Male Health Measures to improve reproductive health cerns rare conditions, congenital hypogon- through health promotion usually focus on adotropic hypogonadism and disorders of sex 13:51–14:09 h women and their behaviors and responsi- development with the diagnostic yield ~50%. Identification of new protein bilities. Although men’s fertility health is For oligozoospermia, representing­ the major- ­markers of biological androgen equally important, men have become the sec- ity of infertility cases, current known single ond sex in reproduction. Consequently, many gene defects mostly do not apply. Extensive activity in humans men have poor knowledge about, and feel research is urgently needed to capture the A. Giwercman1, I. Pla1, K. B. Sahlin1, K. Pawłowski2, distanced from, their reproductive functions ‘genetic landscape’ of reduced sperm counts, C. Fehniger1, Y. Lundberg Giwercman1, and bodies. By interviewing young and adult e.g. possibly explained by e.g. combined I. Leijonhufvud 1, R. Appelqvist1, G. Marko-Varga1, men, I found that most men were unaccus- effect of several low-penetrant variants or A. Sanchez1, J. Malm1 tomed to talk about their fertility, especially uncovered structural variants that destabilize 1Lund University, Sweden, and 2Warsaw University with friends. In cases of reproductive deci- chromosomal stability in spermatogenesis. of Life Sciences SGGW, Poland sion-making, only women’s health and age Data indicates that targeted analysis of single were of concern. Many men did, however, or few genes would not serve as an efficient Introduction The testosterone concentra- appreciate talking about their reproductive approach to majorly improve the diagnostics tion in plasma does not reflect the biological health when given the opportunity, which im- in male infertility. Instead, a favored long- androgenic activity (BAA). Hypogonadism is plies that men’s fertility awareness can easily term goal could be setting up a clinical qual- a predictor of infertility but also several other be raised merely by including them in the ity pipeline for genome-wide approaches, diseases, e.g. metabolic syndrome, diabetes, discussion. I also found that there is great un- e.g. exome sequencing analysis. Improved cardio-vascular disease, and osteoporosis, as certainty among men about what truly affects molecular diagnostics would not only impact well as premature mortality. Understanding fertility, pointing to a need for more studies patients’ clinical management and reproduc- the biology of androgen action may therefore on the relationship between lifestyle factors tive decision making, but also assessment of contribute to clarifying pathogenetic mecha- and male fertility, but also public information accompanying health-related issues and bet- nisms linking androgen deficiency to these that separates myths from facts. My conclu- ter estimates for potential congenital health diseases. sion is that fertility education is something risks of the offspring. Patients and Methods 30 healthy men that could start already in school, but we also (19–32 years) were castrated with an GnRH- need to find a format for education/counsel- 10:10–10:30 h antagonist and three weeks later given testos- ling that appeals to young and adult men. terone. Blood samples were collected at base- Impact of healthy ageing on male line, and three and five weeks after baseline, germ cells and used for proteomic studies. Symposium 5 − Male Ageing J. Gromoll Testosterone dependent proteins were ana- Centre of Reproductive Medicine and Andrology, lyzed in serum samples from 75 males (37.8 15:05–15:25 h Münster, Germany ± 5.5 years) recruited among men from in- What happens to the DNA of fertile couples. Comorbidities in this cohort Male germ cell ageing is an important, but were insulin resistance (IR), cardiovascular sperm as men age? grossly understudied question. Life-long risk, diabetes (DM) and low bone density S. Laurentino sperm production led to the assumption that (LBD). Center of Reproductive Medicine and Andrology male fertility remains unchanged through- (CeRA), Department of Clinical and Surgical Andro­ out life. However, there are indications that Samples were analyzed using mass spectrom- logy, University Clinic Münster (UKM), Germany advanced age has profound consequences to etry and results using various statistical and male fertility and offspring health, such as bioinformatic methods. Although males are capable of producing increased time to pregnancy and miscarriage Results In total, the levels of expression of sperm throughout their entire adult lives, rates, and higher incidence of certain disor- forty-six of 676 proteins varied statistically male fertility decreases with increasing age. ders in the offspring. The fact that some com- significantly with testosterone concentra- In addition, advanced paternal age is as- mon age-associated somatic diseases affect tion. Levels of thirty-seven proteins were sociated with increased miscarriage rates male fertility hinders the assessment of the positively associated, whereas, the remain- and the offspring of older men has a higher true effects of ageing on male reproduction. ing nine markers were negatively associated. incidence of so-called Therefore, we aim to assess effects of “pure” Eighty percent of the proteins (37 out of 46) disorders and poor perinatal health. The ageing on germ cells by studying healthy significantly distinguished the low testoster- reasons behind these adverse outcomes may men at different ages. We noticed that while one group from the normal ones. Three of the lie within the sperm DNA. While sperm it- normal classical male reproductive param- proteins were selected as potential candidate self is approximately 74 days old, the male eters, e.g. hormones and sperm parameters, markers for biological androgen activity and is the result of spermatogonial stem

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 9 Andrology 2020 – Abstracts

cells that remain in the gonad from early em- Life expectancy is increasing worldwide, as compounds with androgenic or androgenic- bryonic development and support continuous are birth rates, especially in Sub-Saharan Af- progestogenic properties, dimethandrolone spermatogenesis for decades from rica and South Asia, resulting in ever increas- undecanoate (DMAU) and 11β-methyl- onwards. These cells can be negatively af- ing world population growth. Since 1950 the 19-nortestosterone17β-dodecylcarbonate fected by advancing age both directly (e.g. world population has quadrupled and the cur- (11-βMNTDC), show promise in early hu- due to repeated cell divisions) and indirectly rent 7.7 billion inhabitants are predicted to man trials. These steroids, derivatives of (e.g. through the action of an altered somatic reach 11 billion by 2100. Overpopulation and 19-nortestosterone, are both well-tolerated environment). Ageing ultimately results in a increasing standards of living for all threaten when administered orally to men, without plethora of male germline-specific molecular the existence of mankind by contributing to liver toxicity, and with pharmacokinetics and effects which include a high de novo mutation climate change and damaging the environ- pharmacodynamics suggesting effectiveness rate, increased genomic instability and DNA ment. Would new methods of male contra- with once daily dosing. These characteristics damage, elongating telomeres, and profound ception be a game changer in population are desirable for an effective “male pill”, a changes in sperm DNA methylation. These growth? Before the “pill” and other modern method many men prefer. In parallel studies, molecular changes occur even in healthy men female methods, men were fully in charge of DMAU administered intramuscularly shows with normal sperm and hormonal parameters contraception by using condoms, withdrawal similar promise, with a goal towards develop- and they may contribute to the poorer repro- and vasectomy and even today 25% of con- ing a reversible male contraceptive adminis- ductive and offspring health outcomes expe- traceptive use consists of these male meth- tered every 3-6 months. Early clinical studies

DGA-Abstracts rienced by older men. ods. From these facts it can be concluded that of both DMAU and 11-βMNTDC suggest men would be ready to use new male contra- side effects are mild and may include acne, Symposium 6 – Sperm Produc­ ceptives if they would be reversible, free of modest weight gain and changes in serum tion and Function serious side effects and affordable. Several cholesterol. Longer term studies are needed opinion polls conducted on men participat- to evaluate the safety and efficacy of these CatSper & Co: How ion channels ing in clinical trials for male contraception prototype products, including evaluation of modulate sperm function have shown high approval rates. A study potential impacts on mood, libido and sexual performed in 9 countries extrapolated that function, with a goal towards expanding the L. Ded1,3, J. Y. Hwang1, K. Miki4, H. F. Shi1, 44 million men in these countries would use method mix and uptake of male contracep- J.-Ju Chung1,2 the tested male hormonal contraceptive. New tives. 1Department of Cellular & Molecular Physiology; male methods would also help to reduce the 2Department of Obstetrics, Gynecology, and Repro- unplanned and undesired conceptions, esti- ductive Sciences, Yale School of Medicine, New mated to amount to about half of the 1 million 18:15–18:30 h ­Haven, CT, United States; 3Laboratory of Reproductive­ daily conceptions worldwide. However, these Progress in Efficacy and Safety Biology, Institute of Biotechnology, Czech Academy of methods are not yet available and intensive Sciences, BIOCEV, Vestec, Czech Republic;­ 4Boston research by the pharma industry would be Study with NES-T gel Children’s Hospital, Boston, MA, United States necessary. Existential economic and social C. Wang Out of millions of ejaculated sperm, only a threats to livelihood and family life caused by Clinical and Translational Science Institute, few reach the fertilization site in mammals. the current pandemic may increase pressure The Lundquist Institute; Division of Endocrinology, Flagellar Ca2+ signaling nanodomains, or- for new male methods. The green activists ­Harbor-UCLA Medical Center, CA, United States have to recognize the link between overpopu- ganized by multi-subunit CatSper calcium On behalf of the Investigators of “Clinical channel complexes, are pivotal for sperm lation and climatic effects. Governmental financing might be necessary. Education and Evaluation of Daily Application of Nestoro- migration in the female tract, implicating ® family planning programs should not only ne (NES) and Testosterone (T) Combination CatSper-dependent mechanisms in sperm Gel for Male Contraception (CCN017) selection. Here, using biochemical and phar- address women, but include males, especially macological studies, we demonstrate that adolescents. Finally, male contraception The Contraceptive Development Program, CatSper1 is an O-linked glycosylated protein, needs politicians and celebrities as protago- Eunice Kennedy Shriver National Institute undergoing capacitation-induced process- nists. Altogether, this might bring about the of Child Health and Human Developing, ing dependent on Ca2+ and phosphorylation desired game change. National Institutes of Health, United States cascades. CatSper1 processing correlates together with the Population Council initiat­ with protein tyrosine phosphorylation (pY) ed planning of the study on the efficacy of ® development in sperm cells capacitated in 18:00–18:15 h daily application of Nestorone (NES) and vitro and in vivo. Using 3D in situ molecular Testosterone (T) gel in 2016 through the imaging and ANN-based automatic detection The Male Hormonal Contraceptive Contraceptive Clinical Trial Network Male of sperm distributed along the cleared female Pipeline Centers. The is the first user controlled male hormonal contraceptive study where preven- tract, we demonstrate that all spermatozoa S. T. Page tion of pregnancy in the female is the primary past the UTJ possess intact CatSper1 signals. University of Washington School of Medicine, endpoint of the study. All other hormonal Together, we reveal that fertilizing mouse ­WA, United States spermatozoa in situ are characterized by in- male contraceptive efficacy studies utilized tact CatSper channel, lack of pY, and reacted Nearly 40% of global are un- injections or implants. This late phase 2 hor- acrosomes. These findings provide molecular planned, a reflection of significant unmet monal male contraceptive study is based on insight into sperm selection for successful contraceptive needs. Men play a significant a phase 1 study that determined the optimal fertilization in the female reproductive tract. role in effective family planning, account- dose of NES and an early phase 2 study that ing for nearly one-quarter of all contracep- demonstrated the combination of NES+T ap- Symposium 8 – Male contra­ tive use worldwide; thus, the development plied daily on the skin will suppresses sperm of novel male contraceptive methods that output to levels that are compatible with ception are efficacious, reliable, safe and revers- preventing pregnancy in the female partner. ible could make a significant impact on the NES/T gel was manufactured containing 17:45–18:00 h health of both men and women. Exogenous NES 8 mg and T62 mg applied daily to the Population growth – Will male androgens form the basis of male hormonal skin and recruitment started in the US cent- contraception create a game contraceptives (MHC), but combinations ers (Los Angeles and Seattle) at the end of of testosterone plus progestins are more ef- 2018 and in Santiago (Chile), Edinburgh, change? fective. While a novel contraceptive gel is Manchester (UK), Stockholm (Sweden) and E. Nieschlag currently under Phase 2 efficacy evaluation, Nairobi (Kenya) in the spring of 2019. In Center for Reproductive Medicine and Andrology, efforts are ongoing towards expanding hor- the summer of 2020, four additional centers University of Münster, Germany monal contraceptive options for men. Novel (Portland, David, Norfolk, and Philadelphia)

10 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts in the US were recruited to participate in the of female vs. male puberty to metabolic cues. esis in vitro. However, important practical, center. Currently the study has enrolled 169 In this presentation, I will recapitulate what medical and ethical issues must be resolved couples with 94 that are in efficacy phase is known about the basic neuroendocrine before fertility restoration can be applied in where the couple relies on NE/T gel as their mechanisms for pubertal control in males, the clinic. sole method of contraception. There are very with particular emphasis of current gaps of Conclusions Considering the progress that few adverse events. The study incorporates a knowledge and open questions regarding the has been made and the barriers that still have detailed assessment of both male and female brain control of male puberty. Recognition of to be overcome, the first patients eligible for partners’ behavior and attitude to male con- such limitations should illuminate new areas autologous transplantation of testicular tis- traception and the study. Our goal is to recruit of research that may help to better manage sue or cell suspensions are likely to be those 420 couples, the enrollment is anticipated to pubertal disorders, also in boys. treated for solid non-metastatic tumours or complete by 2021 and end in 2023.The plan non-malignant diseases, as for these patients is to plan for a phase 3 study 2022–23 to meet there is no known risk of re-introducing ma- the regulatory agencies guidelines for an ac- Plenary Lecture 5 lignant cells into the testis. However, until ceptable contraception if NES/T gel is safe, successful clinical trials demonstrating safety well tolerated, efficacious and acceptable to 13:00–13:45 h and efficacy of fertility restoration have been the couple. Fertility Preservation – translation conducted, testicular tissue cryopreservation for fertility preservation should remain ex- into the clinic perimental. DGA-Abstracts Tuesday, 8 December 2020 E. Goossens Biology of the Testis lab, Vrije Universiteit Brussel, Symposium 11 – Imaging and Plenary Lecture 4 Belgium Surgery in Andrology 09:30–10:15 h Introduction Infertility is an important side effect of treatments used for cancer and other 14:20–14:40 h Neuroendocrine regulation of non-malignant conditions in males. This may male puberty: Certainties, as­ be due to the loss of spermatogonial stem Diagnostic value of Dual-Energy sumptions and open questions cells (SSCs) and/or altered functionality of CT angiography in the diagnosis testicular somatic cells (e.g. Sertoli cells, of arteriogenic erectile dysfunc­ M. M. Tena-Sempere Leydig cells). Whereas sperm cryopreserva- tion Instituto Maimónides de Investigación Biomédica de tion is the first-line procedure to preserve fer- Cordoba (IMIBIC); Department of Cell Biology, Phy- tility in post-pubertal males, this option does M. Wang1, X. Zhang2 siology and Immunology, University of Cordoba; Hos- not exist for pre-pubertal boys. For patients 1Clinical Medical College, Anhui Medical University, pital Universitario Reina Sofia; and CIBER Fisiopato- unable to produce sperm and at high risk of Hefei, China; 2Department of Urology, The First Affi- logía de la Obesidad y Nutrición, Instituto de Salud losing their fertility, testicular tissue freezing liated Hospital of Anhui Medical University, Hefei, Carlos III, Cordoba, Spain; and FiDiPro Program, De- is now proposed as an alternative experimen- China partment of Physiology, University of Turku, tal option to safeguard their fertility. Kiinamyllynkatu­ 10, FIN-20520 Turku, Finland The purpose of this study was to investigate This presentation will provide an update on the diagnostic value of Dual-Energy CT Puberty, as key maturational event in the clinical practices and experimental methods, angiography(DE-CTA) in patients with arte- lifetime of any individual, is under the pre- as well as on patient management inclusion rial erectile dysfunction. cise control of complex neuroendocrine strategies used to preserve and restore the Methods A total of 49 patients with sus- regulatory networks, which impinge onto the fertility of pre-pubertal boys at high risk of pected arteriogenic ED were enrolled.Colour upper levels of the so-called hypothalamic- fertility loss. duplex Doppler ultrasonography (CDDU) pituitary-gonadal (HPG) axis. These path- Methods Based on the expertise of Europe- was taken to evaluate arterial insufficiency ways transmit the regulatory effects of a large an and North American centres offering fer- of penile.After induction of an with number of endogenous and environmental tility preservation programs and a literature prostaglandin E,DE-CTA performed with the signals, including metabolic and nutritional search of the progress in clinical practices, latest generation 256-slice CT scanner was cues, that allow to timely and precisely patient management strategies and experi- used to obtain angiograms of the pelvis and modulate the full activation of the secretory mental methods used to preserve and restore penis. The arterial system supplying the pe- activity of GnRH (gonadotropin-releasing the fertility of pre-pubertal boys at high risk nis (internal pudendal artery, common penile hormone) neurons in the basal forebrain, as of fertility loss were identified. artery, penile dorsal artery, and cavernous major driving force for pubertal progression. artery) was evaluated with Multiple maxi- While notable chronological and phenotypic Results Since the first publication on mu- rine SSC transplantation in 1994, remarkable mum intensity projection (MIP) and volume differences exist between male and female rendering (VR). puberty, there are also important common- progress has been made towards clinical ap- alities, including the central role of GnRH plication: cryopreservation protocols for tes- Result Of the 49 patients,47 (95.9%) had neurons, and some of their major upstream ticular tissue have been developed in animal pelvic images of sufficient quality for evalu- regulators, in the neuroendocrine control of models, and are now offered to patients in ation. A total of 376 segments were finally puberty. The latter include kisspeptins, the clinics, however, still as an experimental pro- obtained and 163 segments were considered products of Kiss1 neurons, which have been cedure. The implementation of testicular tis- as abnormal. The distribution was 29 (17.8%) recently recognized as major gatekeepers and sue cryopreservation as a means to preserve in internal pudendal artery segment, 31 indispensable regulators of puberty onset the fertility of pre-pubertal boys is increasing. (19.0%)in common penile artery segment, 42 in both sexes, by virtue of their capacity to In 2019, more than 1033 young patients (up (25.8%) in penile dorsal artery segment and activate GnRH neuro-secretion. Admittedly, to the age of 18 years) had already undergone 61 (37.4%) in cavernous artery segment. The however, most of our current understanding testicular tissue retrieval and storage for fer- diagnostic validity of DE-CTA with CDDU on the ultimate mechanisms whereby puberty tility preservation. as gold standard was sensitivity = 85.4% and is centrally controlled stems from preclini- At the same time, important scientific pro- specificity = 83.3%. cal studies conducted mainly in females; key gress has been made concerning fertility Conclusion DE-CTA can detect macro­ aspects of the neurohormonal basis of male restoration methods. The technique of SSC angiopathy and delineate small vessels clear- puberty being mostly inferred from female transplantation has been translated to hu- ly, and thus can provide additional anatomic data. This is especially relevant when ad- man cadaver testis, proof-of-concept has evaluation of artery compared with CDDU. dressing the control of puberty by nutritional been obtained for testicular tissue grafting DE-CTA has the potential to become a reli- cues, where direct inference of is hampered in non-human primates, and important steps able method in the diagnosis of arteriogenic by the intrinsic differences in the sensitivity have been taken in establishing spermatogen- erectile dysfunction.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 11 Andrology 2020 – Abstracts

Symposium 12 − WHO semen erectile dysfunction (ED), and is also charac- operating nurse by type, as well as by stages analysis manual terized by a high level of their psychosexual of surgical intervention. satisfaction, sexual and social rehabilitation. In addition, the main tasks of an operating 14:20–14:40 h Knowledge of modern models of penile nurse at the intraoperative stage include: prosthesis, as well as individual choice of the When will sperm DNA testing be­ – strict adherence to the rules of asepsics and optimal device, is crucial for both the patient antiseptics throughout the entire operation come mandatory? and surgeon. – timely warning of the surgeon about any E. Baldi, S. Marchiani, L. Tamburrino, M. Muratori In addition, the urologist should have suf- violation of aseptics Depts. of Experimental and Clinical Medicine and of ficient information about possible complica- – restriction of movement in the operating Biomedical and Clinical Sciences “Mario Serio”, Uni- tions and difficulties that are inherent in pros- room of persons who are not involved in versity of Florence, Italy thetic surgery, have a complete understanding the operation (students, residents) of the patient‘s preparation, as well as about Routine evaluation of semen has been demon- – the presence of double gloves for the sur- strated to be insufficient to assess accurately the features of management in the postopera- tive period. geon and his assistant, as well as monitor- male fertility status. Sperm DNA fragmenta- ing their change at certain stages of the tion (sDF) is one of the most frequent among Preoperative preparation of the patient should operation include: defects found in DNA of spermatozoa of sub- – frequent irrigation of the operating wound DGA-Abstracts infertile men. Several meta-analyses dem- – somatic pathology examination; with an antiseptic solution (NaCl 0.9% + onstrated a clear association between sDF – a urine culture with definition of sensitivity rifampicin/gentamicin) throughout the levels and outcomes of natural and assisted course of surgery. reproduction and frequency of miscarriage. to antibiotics; sDF is moderately associated with semen – implementation of antibiotic prophylaxis; It is worth noting that careful treatment of quality, thus prospecting as a good parameter skin areas close to the operating field is man- – wash and shave the operating field on the datory, as well as treatment of the operating to be added to routine semen analysis­ in the day of the operation. diagnosis of male infertility. sDF may origi- field itself with an antiseptic solution. This nate in the testis, due to defective spermato- Special attention should be paid to patients takes into account the exposure time of the genesis and testicular apoptosis, and because with diabetes, since the sugar level above the skin antiseptic, which should be at least 10– of elevated oxidative stress, the latter likely maximum permissible may cause rejection of 15 minutes. The use of an alcoholic solution damaging sperm after spermiation. In addi- the implant and become a direct contraindi- of chlorhexidine reduces the risk of wound tion, there is evidence that sperm manipula- cation to surgery. Another serious factor that infection by 40% [Darouiche R. et al., 2010]. tion during assisted reproduction techniques, also needs to be considered is varicose veins After installing the urethral catheter, surgical may increase the damage to DNA. Despite of the lower extremities, which is associated gloves must be replaced. Just as before im- the progress in research on sDF, its introduc- with a high risk of developing thromboem- plantation of the components of the fallopros- tion in clinical setting remains highly de- bolism. thesis, it is mandatory to replace the surgical bated, mostly because of lack of standardized­ When preparing a patient for genital surgery gloves of each member of the operating team. methods and of effective therapies to reduce it is most preferable to remove the scrotal hair It is important to note that before implanta- sDF levels. Presently, sDF can be measured with a safety razor on the day of the opera­ tion, the components of the penile prosthesis with several assays, among which, the most tion. So, urologists from the University of are abundantly irrigated with an antiseptic so- popular are TUNEL (terminal transferase- Toronto studied the frequency of skin damage lution (rifampicin 10 mg/ml and gentamicin mediated dUTP end labelling), SCSA (sperm and the degree of scrotal hair removal when 1 mg/ml) with an exposure of at least three chromatin structure analysis), Comet or shaving this area of the body before surgery minutes. And after dilation of the cavernous SCGE (single-cell gel electrophoresis) and on the male genitals, concluded that any bodies, an antiseptic solution (gentamicin/ SCD (sperm chromatin dispersion). These as- violations of the integrity of the skin before rifampicin) is also introduced into them in says show different cut-off values, and, with intervention on the external genitals, as well order to sanitize the cavernous channels the exception of SCSA, are not or only poorly as the presence of hair can increase the likeli- Postoperative management should include standardized. In addition, they detect differ- hood of infectious and inflammatory compli- antibiotic prophylaxis, removal of the ure- ent types of nuclear damage making difficult cations [Grober ED, Domes T, Fanipour M, thral catheter on the first postoperative day, the comparison among the several studies Copp JE. Preoperative Hair Removal on the as well as daily dressings with cleanliness of present in the literature. In such a situation, Male Genitalia: Clippers vs. Razors. J Sex the postoperative wound, exclusion of post- the only possibility for laboratories evaluat- Med 2013; 10: 589-9]. operative hematomas with immediate evacu- ing sDF is to set their own cut-off values with Performing surgery for a penile prosthesis ation if present, and, finally, sexual rest for the established method by comparing fertile requires both all members of the operating one month (in patients with somatic burden, and sub-fertile men. Clearly, efforts should be team and the operating nurse to have suf- this period can be extended to one and a half done to establish the gold standard technique ficient experience in penile implantation months). to evaluate sDF, to find effective therapies to surgery, as well as knowledge of a variety of It is also worth noting the importance of an decrease DNA damage in vivo and to estab- special operational and technical techniques. lish correct procedure to limit the damage individual approach to each case, since to during in vitro manipulation. It is worth considering that to perform a date there are no specific clinical recommen- penile prosthesis requires a special surgical dations on the timing of antibiotic prophy- instruments: laxis and the drugs used. ESAU Symposium 2 – Surgery – ring Scott‘s retractor; in Sexual Medicine – diver and ensembler hooks; Symposium 13 – Fertility Pre­ 15:30–15:38 h – bougienage of the distal and proximal parts servation of the cavernous channels is performed by Preparing the patient for phallo­ Brooks bougie; 15:50–16:10 h endoprosthesis – in the presence of cavernous fibrosis, Ro- Who are the patients? A need for O. Ivanovic Apolikhin sello cavernotomes are used; fertility preservation beyond on­ N.A. Lopatkin Research Institute of Urology and in- – the length of corpus cavernosum is meas- cological diseases terventional Radiology, Moscow, Russian Federation ured using a Furlow ruler. K. Jahnukainen Penile prosthesis is a highly effective and safe All surgical instruments necessary for per- Children´s Hospital, Helsinki University Hospital and method of final-line treatment of patients with forming prosthetic surgery are laid out by the University of Helsinki, Finland

12 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

Introduction Allogeneic hematopoietic I have been working on in vitro spermatogen- Besides sex chromosome defects, an increas- stem cell transplantation (HSCT) is an es- esis since 2007, mainly using mice. In 2011, ing number of recessive mutations in autoso- tablished therapeutic procedure for severe our group succeeded in generating functional mal genes have been reported in NOA. Some hematological disorders including leukemia, mouse sperm from spermatogonial stem cells of these autosomal genes play a crucial role lymphoma, and benign hematological dis- using conventional organ culture method. in meiosis, therefore LoF mutations cause eases like aplastic anemia, sickle cell disease, The key to this success was commercially TESE negative NOA. From a diagnostic thalassemia and bone marrow failure syn- available serum replacements, KSR or Albu- point of view, TEX11 is already classified as dromes. Improvements in treatment modali- MAX, supplemented to the culture medium. a definitive disease-causing gene, whereas ties and supportive care have led to a growing At the time, I thought that the method could several autosomal genes have reached strong population of long-term survivors and knowl- be applied to animals other than mice, even or moderate clinical evidence opening a new edge on late effects is increasingly needed. to human. However, it did not work in other era towards the development of pre-TESE Infertility is of major concern following pedi- animals and not sufficiently effective even gene panel. atric HSCT with high impact on quality of in rats. To make matters worse, the serum life. Total body irradiation and high doses of replacements, which is effective for in vitro alkylating chemotherapy are acknowledged spermatogenesis in mice, contained various 17:36–17:48 h risk factors for gonadal dysfunction after unknown substances, and we were unable Sperm recovery and ICSI outco­ HSCT. Host genetic background may also in- to improve the medium because we had no mes in men with NOA

fluence the acute and long term toxicity after idea what components were actually effec- DGA-Abstracts HSCT and could at least partially explain the tive for in vitro spermatogenesis. Therefore, S. Kliesch inter-individual variability in outcome. we embarked on a project to identify these Center of Reproductive Medicine and Andrology Patients and Methods Cross-sectional important factors, substances, and chemicals (CeRA), Department of Clinical and Surgical Androlo- follow-up study enrolled 2 national cohorts and formulated a chemically defined me- gy, WHO collaboration center for research in male of adult males (≥ 18 years) treated with my- dium (CDM). The CDM would enable us to reproduction, Training Center of the European Aca- eloablative allogeneic pediatric HSCT before improve the culture medium and optimize it demy of Andrology (EAA), University Clinic Münster age 17 between 1980–2010 in Denmark and for each species. In this presentation, I would (UKM), Germany like to present advances in the composition of Finland. Gonadal function at long-term fol- Patients with non-obstructive azoospermia low-up was studied. media for in vitro spermatogenesis that could be an important step towards human in vitro (NOA) are a challenge for diagnosis and Spermatogonial numbers were evaluated spermatogenesis. treatment. The differential diagnose of NOA in cryopreserved immature testicular tissue comprises primary testicular failures, endo- from patients with non-malignant diseases crine failures and genetic disorders. While in the frame of the fertility preservation pro- EAA/ESAU Symposium 3 − hypothalamic-pituitary deficits result in tes- grams Androprotect and Nordfertil. Management of NOA: Current ticular failure of spermatogenesis and will be Results Late spermatogenic recovery is Practice and Thoughts for The successfully treated by gonadotropins, AZF a or AZF b deletions of the Y-chromosome possible 10–30 years following myeloabla- Future tive HSCT but depends on cumulative doses render successful sperm retrieval (nearly) of alkylating agents and irradiation. Pre-pu- 17:12–17:24 h impossible. Careful diagnostic assessment bertal status at HSCT increases the risk for has to rule out access to successful causative later testosterone substitution. Patients with Pre-TESE prognostic genetic treatment options in azoospermic patients sickle cell disease have a decreased spermat- tests: where we are now? and to differentiate those who should get ogonial numbers in cryopreserved testicular primary access to (microsurgical) techniques C. Krausz tissue prior to HSCT. of testicular sperm retrieval (TESE). Obstruc- Department of Experimental and Clinical Biomedical tive azoospermia (OA) has to be carefully The risk of male gonadal dys- Conclusion Sciences “Mario Serio”, University of Florence, Italy elucidated, as reconstructive surgery could be function after pediatric HSCT is high and a valuable option and 1 out of 6 azoospermic depends primarily on the cumulative tes- Over the last 20 years genetic screening man may be identified with OA instead of ticular irradiation dose and pubertal stage at became part of the routine diagnostic work- NOA. However, differentiation may be dif- transplant. Our findings pinpoint the need of up of non obstructive azoospermia (NOA) ficult with normal clinical phenotype (normal fertility preservation before HSCT as well patients. Numerical chromosomal altera- testis volume, normal endocrine profile, nor- as prolonged follow-up of pediatric HSCT tions, such as 47, XXY and structural al- mal seminal volume). Only genetic analysis patients into adulthood. Severe hematologi- terations, such as the Y chromosome-linked of the newly described genes (as TEX 11, cal disorders can have direct effects on sper- AZF deletions show a cause-effect relation- STAG 3 or TEX 14) may hint at meiotic arrest matogonial quantity and options for fertility ship with NOA. With the advent of Next of spermatogenesis and finally make TESE preservation. Generation Sequencing (NGS) monogenic causes started to be discovered, allowing to unnecessary [Tüttelmann et al., Andrology define the genetic etiology of an increasing 2019, Hum Reprod 2019; van der Bijl et al. 16:10–16:30 h proportion of idiopathic NOA. Apart from Hum Reprod 2019]. Even more individual- ised prognostic aspects may gain rele­vance, In vitro approaches to obtain their diagnostic value, some genetic factors have also a prognostic value in terms of tes- as polymorphisms of the FSH gene may spermatozoa: Experimental ticular sperm retrieval. For instance, carriers influence the TESE outcome [Busch et al. ­approaches with a clinical per­ of a complete AZFa or AZFb deletion have JCEM 2019]. Moreover, hypogonadism is spective poor TESE prognosis with virtually zero observed in 1/3 of NOA patients and poten- chance of testicular sperm retrieval. For an tial pretreatment options to increase endogen T. Ogawa appropriate prediction of TESE outcome in a testosterone levels to at least 8 nmol/l may Yokohama City University, Japan suspected complete AZFb deletion, it is fun- influence TESE outcome [Ramasamy et al. If human in vitro spermatogenesis is possible, damental to test for a specific distal marker 2009, Rohayem et al. 2015]. it would be a valuable technique for studying (sY1192), since the presence of this STS is In NOA, sperm recovery is recommended to human spermatogenesis in detail. In addition, compatible with positive sperm retrieval. An be performed by TESE either as a multifocal in vitro spermatogenesis would serve for pro- X chromosome-linked gene, TEX11, is sus- conventional or a microsurgical (microscopic ducing sperm from cryopreserved immature ceptible to intragenic deletions which lead to assisted) approach. Especially the most se- testis. Thus, it actually serves for preserving spermatocytic arrest. While TEX11 deletions vere patient groups with significantly reduced fertility of boys with malignant diseases such have a negative pre-TESE prognostic value, testicular volume, high FSH levels and severe as leukemia. However, human in vitro sper- the clinical interpretation of the non-loss of testicular damage may benefit most from the matogenesis is still unsuccessful, even today. function (LoF) mutations is more complex. microTESE approach as it facilitates to iden-

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 13 Andrology 2020 – Abstracts

tify focal spots of spermatogenesis in semi- the seminiferous tubuli of the contralateral­ symptoms onset [4]. Why some viruses such niferous tubules by magnification. However, testis to the neoplastic testis. At several as ZIKV escape from immune surveillance a very recent systematic review and meta- months after autotransplantation, semen sam- and persists in human testis? In response to analysis of the ESAU group revealed the ples should be evaluated for the presence of viral stimuli, the production by human tes- shortcomings of published data Altogether 56 spermatozoa. Autotransplantation of testicu- tis/germ cells of IFN-I, a key cytokine for studies on conventional, 43 studies on micro- lar frozen/ thawed germ cells post-chemo- antiviral defense, is very low and transient, TESE and another 18 studies with combined therapy may represent a means of colonizing which may prevent germ cells apoptosis and surgical approaches in heterogenous patient the human testis with its own cells, having sterility induced by IFN-I [5]. However, in cohorts failed to clarify the hypothesized ad- as an overall target the appearance of sper- the absence of a robust antiviral response, vantage of microTESE, as the studies were matozoa in the ejaculate. On the other hand, germ cells may represent an ideal shelter for not comparable, had uncontrolled and mostly the autotransplanted SSCs may undergo cel- viruses, since they are naturally segregated retrospective study designs. Nevertheless, lular divisions within the recipient patient’s from immune cells and antibodies. In this the overall sperm retrieval rate in NOA pa- testis. Thus the number of the non-exposed talk, I will summarize the state of the art on tients is 47% in 117 studies with more than to chemotherapy- SSCs may increase in vivo. the mechanisms of viral persistence in the 21.000 patients analysed. The TESE-ICSI Even if the final semen samples are negative testis and on SARS-CoV-2 interactions with procedures finally resulted in a cumulative for spermatozoa, the recipient testis may be the MGT. pregnancy rate of 29% per cycle (reported in positive for spermatozoa that have been de- References:

DGA-Abstracts 42/117 studies), with 1096 biochemical preg- rived by the autotransplanted SSC cellular nancies and 569 reported life births (cumula- subpopulation. The latter spermatozoa may 1. Le Tortorec et al, Physiological Reviews 2020. tive life birth rate of 24%) with no significant be used for ICSI techniques. 2. Entezami et al, EClinical Medicine 2020. differences between conventional and micro- 3. Matusali et al, Journal of Clinical Investigation 2018. surgical TESE [Corona et al. HRU 2019]. 4. Mahé et al, Lancet Infectious Diseases 2020. Symposium 14 – Sexually 5. Satie et al, Journal of Biological Chemistry 2011. 17:48–18:00 h transmitted diseases 17:40–18:00 h How close are we to spermato­ 17:20–17:40 h Viruses and testis cancer gonia stem cell autotransplanta­ Zika, Covid and beyond – Do we tion for the treatment of sub­ need to be concerned? A. Garolla populations of non-obstructed Department of Medicine, Unit of Andrology and N. Dejucq-Rainsford ­Reproductive Medicine, University of Padova, Italy azoospermic men? Inserm, École des hautes études en santé publique N. Sofikitis (EHESP), Institut de recherche en santé, environne- Testicular cancer represents the more fre- Department of Urology, Ioannina University School ment et travail (Irset), Université de Rennes, France quent solid tumor affecting males aged of Medicine, Greece 15–35 years. In the last decades, its incidence While several viruses such as HIV or HBV showed a progressive increased probably due Previous research efforts have indicated that are well-known vectors of sexually transmit- to genetic and environmental factors. Despite syngeneic or xenogeneic spermatogonia stem ted chronic diseases, lately, emerging viruses exposure to some viruses such as HIV, HCV, cell (SSCs) transplantation efforts into the triggering acute infections were unexpectedly EBV, and HPV is frequently related to can- seminiferous tubuli of immunodeficient ani- associated with long- term shedding in se- cer development, there are no studies aimed mals may result in the development of donor men and sexual transmission despite systemic to evaluate the possible implication of viral spermatogenesis within the recipient testis. It clearance. The sexual transmission of the infections in the pathogenesis of testicular has been suggested that the recovered donor deadly Ebola virus by survivors over 1 year af- cancer. In this study, we analyzed sperm pa- spermatozoa from the recipient testis can be ter recovery led to the resurgence of epidemic rameters and prevalence of HPV on sperm used for assisted reproductive techniques for foci, while that of the teratogenic mosquito- in 155 testicular cancer patients at diagnosis the treatment of non-obstructive azoospermia transmitted Zika virus led to its dissemination (T−1), after orchiectomy (T0) and after 12 [NOA; Update, 2003: in 14 countries outside the mosquito vector months from surgery or from the end of ad- 1 9, 291]. However ethical barriers, genetic area . In addition to diseases dissemina- juvant treatments (T12). All patients showed barriers, and virus-related transfer risks may tion, viral infections of the male genital tract a significantly higher prevalence of semen in- impede the utilization of donor human sper- (MGT) can cause male infertility, hormonal fection than controls (9.5% and 2.4% respec- matozoa generated in a host syngeneic or disturbances and viral transmission to the em- tively,) and altered sperm parameters both at xenogeneic testis for subsequent ICSI tech- bryo, affecting fetal development and preg- T−1 and T0. Considering sperm parameters, 1 niques for the treatment of NOA. nancy outcome . Worryingly, reports of semi- at T−1 we observed a reduction of progres- On the other hand, employing similar micro­ nal shedding and sexual transmission of other sive motility, and after orchiectomy patients surgical procedures, frozen/thawed human emerging viruses, including lethal ones, are showed a reduction of sperm concentration testicular germ cells or SSCs can be autotrans- accumulating [1]. The tropism for the MGT and count and a further worsening of motility. planted back to the patient’s testis without of SARS-CoV-2, the virus responsible­ for the Thereafter, patients were assigned to three using a recipient animal testis as a surrogate Covid19 pandemic, is currently unknown. A groups on the basis of medical option after organ. Candidates may be men with NOA single study reported SARS-CoV-2 in semen surgery: S = surveillance, R = radiotherapy, and unilateral testicular cancer who are about from infected men in the acute or recovery and C = chemotherapy +/− radiotherapy. At to undergo unilateral radical orchiectomy. At stage, whereas others failed to detect seminal T12, untreated patients had an improvement the time of such surgery, germ cells can be excretion [2]. The interplay between SARS- of sperm parameters while R group and even isolated from the contralateral healthy testis. CoV-2 and male hormones is an important more C group had a strong decrease of sperm In vitro culture techniques may increase the question. Indeed, testosterone is suspected to number (p < 0.01 both vs. T0 and S group). subpopulation of recovered SSCs [Hum Re- participate to the increased disease severity in Moreover, patients who received radio and/or prod Update, 2005: 11, 229]. In addition, it is men over women by boosting virus entry into chemotherapy had a very high prevalence of important that biopsies from the contralateral the cells. Deciphering the mechanisms for HPV semen infection (S = 7.7%, R = 30.8%, testis show an absence of neoplasia; fractions persistence in the MGT of emerging viruses and C = 61.5%). In conclusion, patients with of isolated/cultured testicular germ cells or and their interactions with MGT functions is testicular cancer had frequently altered sperm SSCs from the healthy testis will be then therefore of prime importance. parameters and higher prevalence of HPV se- frozen. Several months after completion of We recently demonstrated that ZIKV infects men infection that were worsened after radio chemotherapy in men with advanced testicu- the human testis ex vivo [3] and that persis- and chemotherapy. Because HPV infection lar cancer, frozen/thawed germ cells or SSCs tently infected human testicular germ cells is a risk factor for cancer development and can be transplanted back to the rete testis or are released in semen up to 160 days post- it may further reduce fertility, we suggest

14 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts screening for HPV in testicular cancer pa- in the primitive uSG cell cluster defined by (e.g. spermatogonial stem cell (SSC) renewal, tients at diagnosis and particularly after ad- scRNAseq were candidates to specifically SSC niche formation, and blood-testis-barrier juvant treatments. label SSCs. Immunofluorescence and im- formation). munohistochemical analysis revealed several Therefore, the focus of testicular cell culture- of these genes encode proteins selectively based research has been the development of Wednesday, 9 December 2020 expressed in uSG. We chose one of these – novel three-dimensional culture conditions. PLPPR3 – to purify uSG by FACS. Xenograft By transferring knowledge obtained from Symposium 15 − Emerging germ-cell transplantation analysis demon- novel cell-culture methodologies established Technologies strated that these cell-surface PLPPR3+ hu- recently in other fields of medical research, man testicular cells are 38-fold enriched for as for example organoids, new strategies 08:00–08:20 h SSCs, demonstrating that PLPPR3 is a highly have been designed to provide new tools for selective human SSC marker. Comparative more defined research approaches regarding Shedding light on signaling in RNAseq analysis of these PLPPR3+ highly gametogenesis and its failures [4]. Organoids sperm flagella enriched SSCs with differentiating (d) SPG are small cell aggregates similar to organs (KIT+ cells) revealed the full complement of D. Wachten found in vivo, but generated from single genes that shift expression during the uSG- cell suspension containing stem cells. These Institute of Innate Immunity, Medical Faculty, Uni- to-dSG developmental transition. Among versity of Bonn, Germany organ-like structures are also defined by their

these dynamically regulated genes were functionality, which makes them a valuable DGA-Abstracts Introduction Mammalian oocytes are en- genes encoding key components in the AKT, research model and most likely a useful clini- veloped by the zona pellucida (ZP), an extra- GDNF, JAK-STAT, and TGFβ signaling path- cal tool in future. cellular matrix of glycoproteins. To fertilize ways. Manipulating these signaling pathways the , sperm have to penetrate the ZP. To in cultured human SPG revealed that GDNF References: this end, interaction with ZP proteins evokes and BMP8B broadly support human SPG 1. Krausz. Best Pract Res Clin Endocrinol Metab 2011; certain sperm behavioral responses. How- culture, while Activin A selectively supports 25: 271–85. ever, the mechanisms underlying ZP action in more advanced human SPG. One condition 2. Agarwal, et al. Reprod Biol Endocrinol 2015; 26: 37. sperm are only ill-defined. – AKT pathway inhibition – had the unique 3. Sato, et al. Nature 2011; 471: 504–7. ability to selectively support the culture of 4. Alves-Lopes, Stukenborg. Hum Reprod Update 2018; Results Here, we delineate the sequence 24: 176–91. of ZP-signaling events in mouse sperm. We primitive human uSPG with the characteris- show that ZP proteins evoke a rapid intracel- tics of SSCs. This raises the possibility that lular pHi increase that rests predominantly supplementation with an AKT inhibitor could Symposium 16 − Failure of on Na /H exchange by NHA1 and requires be used to culture human SSCs in vitro for Testis Development cAMP synthesis by the soluble adenylyl cy- therapeutic applications. clase sAC as well as a hyperpolarized mem- 08:00–08:20 h brane potential set by the spem-specific K 08:40–09:00 h Trends in human semen quality channel Slo3. The alkaline-activated CatSper Testicular organoids − Valid ex­ channel translates the ZP-induced pHiin- after nearly a half century of crease into a Ca2 response. pectation for clinical andrology? ­literature Conclusion Our findings reveal the mo- J.-B. Stukenborg B. Jegou lecular components underlying ZP action on NORDFERTIL Research Lab Stockholm; Childhood Inserm researcher emeritus, director of research at mouse sperm, opening up new avenues for un- Cancer Research Unit; Department of Women’s and EHESP, University of Rennes, France derstanding the basic principles of sperm func- Children’s Health; Karolinska Institutet, and Karolins- tion and, thereby, mammalian fertilization. ka University Hospital, Stockholm, Sweden The theme of this presentation is the results of a critical appraisal and analysis that we Infertility is a multifactorial and complex have performed based on a stringent focus on 08:20–08:40 h problem. It affects approximately one out of the study designs applied in the literature on AKT Signaling Inhibition Favors seven couples in Europe [1] and at least 7.5 % trends in semen quality. This was based on of men between 15 and 60 years of age [2]. Culture of Human Undifferentiat­ a Medline search of the relevant article from The lack of detailed knowledge regarding the 1974 until April 2020, which was carried ed Spermatogonia biological processes important for human ga- out using a set of cross-keywords followed M. F. Wilkinson metogenesis, and therefore the lack of proper by selecting the sole publications relating Department of reproductive medicine, UC San Diego treatment options for these patients, highlights to spatial and temporal variations in semen School of Medicine, CA, United States the need of novel treatment ­methodologies, characteristics. including ex vivo approaches, to differentiate Spermatogonial stem cells (SSCs) are self- This critical analysis of the literature was mature gametes from immature germ cells or based on a careful distinction within the cor- renewing undifferentiated spermatogonia even pluripotent stem cells. (uSG). SSCs are essential for the continu- pus of available studies between those cor- ous production of sperm and have potential Strategies employed to study male fertility, responding to (i) multicenter investigations therapeutic value for treating male infertility, and thereby establish treatment strategies for categorized as studies based on mean values which afflicts > 100 million men world-wide. sub- and infertile men, will therefore require or estimates; (ii) studies based on retrospec- There are several bottlenecks in developing both, existing biobank material as well as tives data from individuals in a given area, as SSC therapy, including identifying human registry data to identify factors related to the well as (iii) studies covering the paradigm of SSC-specific markers and developing meth- disease, and ex vivo approaches to differen- geographical differences on semen quality. ods for human SSCs long-term culture. Here, tiate mature gametes from immature germ We conclude that plausible decreasing trends we report the identification of a selective hu- cells or even pluripotent stem cells. have occurred in some demarcated urban man SSC marker that allowed us to define The successful production of murine sperm areas. Furthermore, geographical contrasts the transcriptome of highly enriched SSCs, in vitro using testicular explant culture con- have been strongly establish. However, stud- which, in turn, led us to identify signaling ditions, reported for the first time in 2013 ies based on aggregating multiple data from pathways that influence the fate of uSG and [3], can be considered as a real breakthrough countries and / or continents cannot estab- thereby develop a method for their short-term study. However, this culture condition lacks lished a global conclusion that Human semen culture. To screen for SSC marker genes, we requirements enabling controlled monitor- quality has been deteriorating in Western employed single-cell RNA sequencing (scR- ing of endo- and paracrine pathways needed countries. To move way forward on this sen- NAseq) to analyze adult human testicular to create a robust model to study specific sitive topic, prospective studies that minimize cells. Genes exhibiting enriched expression aspects crucial to the spermatogenic process the impact of the key well-described factors

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 15 Andrology 2020 – Abstracts

of bias should be combined with the assess- Conclusion FEPOS is the world’s largest – Androgen replacement therapy in patients ment of men’s exposomes to improve our un- population-based male-offspring cohort spe- with biochemical and clinical signs of derstanding of the relations between Human cifically designed to investigate prenatal de- ­hypogonadism, after completion of the fer- semen quality and the environment. terminants of male fertility. Extensive data on tility treatment. maternal exposures combined with biological fertility markers in their sons allow for an- 08:40–09:00 h swers to long sought questions. Health behavior and environmen­ References: Keglberg Hærvig K, Bonde JP, Ramlau-Hansen CH, et al. Symposium 18 − Young Andro­ tal exposure during pregnancy Fetal programming of semen quality (FEPOS) cohort – A logists and reproductive function in the DNBC male-offspring cohort. Clin Epidemiol 2020; 12: adult son (FEPOS Cohort) 757–70. 10:30–10:50 h Sa. S. Tøttenborg1, K. Keglberg Hærvig1,2, K. Sørig Comparative investigations of the 2,3 4 5 Hougaard , C. Høst Ramlau-Hansen , G. Toft , sperm metabolome in fertility pa­ C. Lindh6, J. P. Ellekilde Bonde1, 2 Symposium 17 − Guidelines tients and healthy subjects 1Department of Occupational and Environmental EAA ­Medicine, Bispebjerg & Frederiksberg Hospital, Den- K. M. Engel1,2, J. Blaurock1, U. Rolle-Kampczyk3, DGA-Abstracts mark; 2Department of Public Health, The Faculty of 10:30–10:50 h M. von Bergen3, S.Grunewald1 Health Sciences, University of Copenhagen, Den- 1EAA Training Center of Andrology, Department of mark; 3National Research Centre for the Working En- Management of oligo-astheno-te­ Dermatology, Venerology and Allergology, Leipzig vironment, Denmark; 4Department of Public Health, ratozoo-spermia University, University Hospital Leipzig, Germany; Research Unit for Epidemiology, Aarhus University, S. Francavilla 2­Institute for Medical Physics and Biophysics, Leipzig Denmark; 5Department of Clinical Epidemiology, Department of Life, Health and Environmental Scien- University, Germany; 3Department of Molecular Sys- ­Aarhus University Hospital, Denmark; 6Division of ces, University of L’ Aquila, Italy tems Biology, Helmholtz Centre for Environmental Occupational and Environmental Medicine, Depart- Research, Germany ment of Laboratory Medicine, Lund University, Oligo-astheno-teratozoospermia (OAT) is Because of a high number of idiopathic in- Sweden­ frequently reported in men from infertile couples. Its etiology remains, in the majority fertilities among couples observed in fertility Introduction The hypothesis that disrup- of cases, unknown with a variety of factors clinics, the common spermiogram parameters tion of the fetal programming of the testicles to contribute to its pathogenesis. The aim of do not seem to be sufficient for the indication may result in reduced sperm count and infer- this presentation is to present the European of male fertility. Thus, the search for biomark- tility in adulthood has since the 1980s shifted Academy of Andrology (EAA) guideline to ers to predict semen quality with methods al- focus from exposures in adulthood to those discuss available management options. lowing deeper investigations of the ejaculate, in prenatal life. With few exeptions, however, such as “omics” methods is highly relevant. PubMed was searched for papers in English it has been impossible to put the hypothesis Analyzing the molecular composition of a for articles with search terms: male infertility to a critical test in epidemiological studies as cell by metabolomics gives more detailed and oligo-astheno-teratozoospermia. For evi- appropriate prospective data on time-specific information than a superficial investigation dence-based recommendations, the GRADE exposures combined with clinical follow- by common spermiogram analysis and could system was applied. up of sons when they reach adulthood have provide reasons for an otherwise undetected been lacking. A new male-offspring cohort For men with OAT, the EAA recommends: fertility disorder. – the Fetal Programming of Semen Qual- – A general physical examination to assess In a pilot study the concentrations of 180 ity (FEPOS)­ cohort [1] – created within the signs of hypogonadism. metabolites – among them amino acids, auspices of ReproUnion serves an unprec- – A scrotal physical examination to assess i) biogenic amines, acyl carnitines, lipids and edented chance for clarification. the testes and epididymes for volume and hexoses – of sperm and seminal plasma from Materials and Methods Young men ≈19 consistency, ii) deferent ducts for total or 10 smoking and 10 non-smoking subjects years of age born to women in the Danish partial absence and iii) occurrence of vari- have been elucidated by a targeted LC-MS/ National Birth Cohort for who two inter- cocele. MS-based metabolomics approach to ex- views and a blood sample obtained during – Performing two (2) semen analyses, ac- clude a possible confounder, namely smok- pregnancy were available, were enrolled cording to World Health Organization ing. Furthermore, conventional spermiogram 2017–2019. Participants answered a compre- (WHO) guidelines to define an OAT. analyses and flow cytometric analyses of cas- hensive questionnaire, underwent a physical pase-3 activity (apoptosis), CD46 (acrosome examination, and provided biospecimens. – An endocrine evaluation. reaction) and TUNEL (DNA fragmentation) Blood from mothers and sons were analyzed – A scrotal ultrasound (US) as part of routine have been performed. The results show that for biomarkers of per-and polyfluorinated investigation. the conventional spermiogram is not altered substances (PFAS), several phthalate metabo- – Karyotype analysis and assessment of in smokers compared to non-smokers. How- lites, triclosan, acetaminophen, and cotinine. Yq microdeletions in infertile men with a ever, the deeper investigation reveals an acti- Reproductive biomarkers included semen sperm concentration ≤ 5 × 106/mL. vated caspase cascade as well as an activated volume, concentration, total count, morphol- – Cystic fibrosis transmembrane conduct- nitrogen oxide synthase in sperm of smoking ogy, motility, DNA fragmentation index, and ance regulator (CFTR) gene evaluation in subjects. Both are signs for oxidative stress reproductive hormones. case of suspicion for incomplete congenital and are also detectable in the sperm metabo- Results Of 21,623 eligible sons 5,697 were obstruction of the genital tract. lome. These molecular foot prints together with an inconspicuous spermiogram show invited of whom 1,058 participated (19%). – Against quitting physical activity in order All xenobiotic chemicals except triclosan and that the standard spermiogram might lead to to improve the chance of achieving preg- rash conclusions regarding the putative fertil- acetaminophen were detected in quantifiable nancy. concentrations > 90% of women. The con- ity of an ejaculate while the metabolome is trast was large, exemplified by PFOA with – Against androgen replacement therapy in more differentiated and could provide causes 10th to 90th percentiles 2.5–7.2 ng/ml (median order to improve the chance of achieving for an otherwise undetected fertility disorder. 4,6 ng/ml). Other first-trimester exposures of pregnancy. In a subsequent study sperm and seminal interest were also prevalent e.g. 23% reported – Assisted Reproduction Techniques (ART) plasma of idiopathic and oligoastheno- tobacco smoking (19% light and 4% heavy in order to improve the chance of achieving zoospermic patients will be investigated in smokers) and 42% to be burdened by life or pregnancy, in case other treatment options comparison to healthy subjects by the above emotional stress. are not available or not efficient. named methods.

16 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

11:10–11:30 h Realization of reproductive potential is im- – creating a preventive environment in the portant not only for the reproduction of the field of reproductive health with responsi- Metabolic cooperation between population, but also, for guarantees of na- bility of the doctor and the patient as “sub- testicular cells is essential for tional and social stability and security of the jects of law” spermatogenesis state itself. – support for natural reproductive behavior M. G. Alves At reproductive age, women have marked in collaboration with traditional religious Department of Microscopy, Laboratory of Cell Bio­ regressive reproductive behavior (a large denominations logy, Institute of Biomedical Sciences Abel Salazar number of abortions), which depends on the – active involvement of employers as “sub- (ICBAS), University of Porto, Portugal; Unit for Multi- behavior of men, since the potentiating force jects of law” in the protection of family disciplinary Research in Biomedicine (UMIB), Uni- in the birth of children both in marriage and reproductive health versity of Porto, Portugal outside of it is the man. In the case of an out- – recognition of men’s health as an integral of-wedlock pregnancy, 90% of decisions to Male fertility issues have been overlooked factor in protecting the reproductive health keep the child depend on the man. The de- of the population of the CIS countries. for many years. Although statistics from the cision to start a family also depends on the World Health Organization (WHO) clearly man. show that sperm quality is decreasing world- 12:20–12:40 h wide to dangerous levels that can promise According to experts, behavioral risk factors Male Reproductive Health Initia­ natural conception, the molecular mecha- have a huge impact on reproductive potential. DGA-Abstracts nisms that control spermatogenesis remain Smoking increases the risk of infertility by tive – MRHI in global perspective 340%, and alcohol increases the risk of pre- as a technically challenging topic. Spermato- C. J. De Jonge genesis is a complex and highly regulated maturity by 300%. Therefore, further actions to preserve reproductive health should be Director, Andrology Program, University of Minneso- process orchestrated by testicular cells. In ta Medical Center; Adjunct Professor, Department of th largely aimed at combating risk factors that the 19 century, Enrico Sertoli described for Urology, University of Minnesota, MN, United States the first time irregularly shaped somatic cells reduce the likelihood of conception and safe in the testis and suggested that they did not birth of a child, especially the second and the Introduction A spotlight is shining more produce spermatozoa but support their pro- third. brightly on male reproductive health because duction. Indeed, those cells form the blood In addition, in the CIS countries, there is a of concerns about globally declining sperm testis barrier. This way, the Sertoli cells (SCs) high mortality rate among the working-age counts and, more recently, emerging data separate the interstitial fluid from the intratu- population, especially at the age of 40–65 showing an association between somatic and bular fluid. Later it was shown that in addi- years, and the majority of these deaths are reproductive health. While there is argument tion to controlling the passage of substances male. Therefore, the position of health au- regarding the certainty of sperm count de- and metabolites for the compartment where thorities should be particularly active in rela- cline the opposite is true for the link between germ cells develop, SCs also establish a strict tion to men aged 40–65 years, most of whom reproductive and somatic health. Adding to metabolic cooperation with developing germ are fathers and breadwinners of the family. concern is emerging data that the reproduc- cells. The distinct energetic needs during the tive health of the father can have an inter- different stages of spermatogenesis are con- Since men aged 40–65 years are experienced generational impact on the wellness of future trolled by the SCs and they also produce the and valuable employees at any enterprise, offspring. Perhaps more basic is the seem- lactate, used as main substrate energy source there is no doubt that large employers are ing diminished status of the semen analysis by developing germ cells. This topic has been interested in maintaining the social activity for assessing male fertility as evidenced by overlooked even though there is an increase of their employees. Therefore, we consider global­ly increased use of ICSI for non-male on the prevalence of metabolic diseases. it appropriate to involve them in preventive factor cases. Given the breadth and complex- Most studies aim to correlate the incidence programs. ity of all the aforementioned, which includes of metabolic diseases with sperm quality and Since the main potentiating role in realization basic, clinical and social science research there is not yet a consensus. Recently, several of the nation’s reproductive potential and in evidence, discussion amongst opinion lead- authors showed the impact of hormonal dys- maintaining reproductive activity is played by ers initiated about what can and needs to be function and metabolite shifting caused by men, it is men who should be the main focus done globally to address these issues and to those diseases in the testis, particularly on the of efforts. One of the aims should be creation ultimately benefit patients and their families. metabolic cooperation established between of preventive environment and development Materials and Methods Topical materials testicular cells. Thus, it is crucial to under- of principles of medical examinations in the in the scientific literature database and popu- stand how those mechanisms are sensitive system of male reproductive health care – not lar media were scoped. to energy homeostasis regulating hormones only the use of expensive high-tech medical A review of the evidence dem- and dietary habits. Overall, the study of SCs Results technologies. onstrated that male reproductive health is is an emerging field for researchers inter- globally in crisis and worsening in the virtual ested in the understanding of the molecular The proposed prevention paradigm should absence of effective healthcare policies, re- mechanisms responsible for male (in)fertility. also be family oriented. search funding, societal awareness and edu- Ultimately, those studies may highlight new Protection of the population’s reproductive cation. Evidence of a significant gender gap therapeutic targets for the control of male health should take place only through the in healthcare policies and initiatives demon- fertility. formation of natural reproductive behavior. strates the urgent and critical need for global Here, preventive measures, prevention of re- gender-inclusive healthcare strategies. productive losses and preservation of repro- Conclusions The Male Reproductive ductive potential are the key factors. This is Health Initiative (MRHI) was established Symposium 19 − Public Aware­ particularly important when the system of re- with a mission of advancing the science and ness productive health protection is implemented practice of male reproductive medicine. The in the CIS countries in the mass. MRHI aims to accomplish its mission by 12:00–12:20 h The demographic situation in the country promoting and contributing to high quality Preventive approach to male re­ is such that the window of opportunity for research in both basic and clinical science, productive potential in Russian decisive action is 3–5 years. For the full re- forging collaborative relationships with pro- alization and preservation of reproductive fessional societies to contribute to a mission Federation potential, as well as active social longevity, of education, and through advocacy of MRHI O. Ivanovich Apolikhin a separate concept of “Male reproductive to all stakeholders including patient support N. Lopatkin Research Institute of Urology & Interven- health” is necessary, which should be based groups, funding agencies, non-for-profit or- tional Radiology, Moscow, Russian Federation on the following principles: ganizations, industry, and the public.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 17 Andrology 2020 – Abstracts

Post Congress Course Basic lishment of organoids from gonadal somatic customized human sperm methylC-capture Science cells. For rodent models standardized­ proto- panel. With the panel, MTHFR 677TT men cols already exist to derive various stages of as compared to MTHFR 677CC men, showed 14:15–14:45 h primordial germ cells and precursors of male both hyper- and hypomethylation in their and female germ cells. It is relevant to expose sperm. Sperm hypomethylation in MTHFR Viral delivery systems for making the cells in vitro to sequentially changing 677TT men was increased by high dose fo- rapid somatic cell transgenics in cocktails of specific growth factors. These lic acid supplements. For both the MTHFR the testis conditions mimick the in vivo induction of genotype and folic acid supplement effects, primordial germ cells in specialized­ regions > 80% of the alterations in DNA methyla- A. Darbey1, P. Brown1, K. Kilcoyne1, D. Rebourcet2, of the early embryo. In parallel, various cell tion occurred in the dynamic CpGs targeted M.l Curley1, L. B. Smith1,2 culture systems have been developed for a uniquely by our panel. 1MRC Centre for Reproductive Health, University number of organs to derive highly organized Conclusions Dynamic sites in the human of Edinburgh, Edinburgh. United Kingdom; 2Faculty multicellular structures resembling many sperm DNA methylome appear to be par- of Science, University of Newcastle, Callaghan, aspects of normal physiological function. ticularly susceptible to alterations in folate Australia­ Gonadal organoids can also be created in metabolism and high dose folic acid sup- Introduction The production of transgenic many experimental setting like three dimen- plements. Approaches such as the use of the mice has permitted the functional investiga- sional culture systems, hanging drops, mi- customized human sperm methylC-capture

DGA-Abstracts tion of thousands of genes. However, these crofluidic chips and rotating flasks or wells. panel described here will likely improve our methods are costly in time, money and ani- Primary cells from fetal gonads show the ability to explore the effects of additional mals required to obtain a consistent pedigree. desired ability to undergo spontaneous organ environmental exposures on the sperm DNA In addition, developmental impacts often pre- devlopment. Bringing together these techno- methylome. (Supported by CIHR). clude analysis at later ages. logical advances germ cells established from pluripotent precursors developed into mature Materials and Methods Development of gametes. As yet mouse and sperm de- 16:45–17:15 h tools capable of gene manipulation at any giv- rived in such systems were used to generate en time of life obviate developmental issues Single cell RNA seq to study sper­ mouse offspring. The importance of the so- matogonial (dys-) function and permit rapid production of genetic models matic tissues to guide germ cells into the cor- from wild-type stock. We have undertaken an rect pathway is one of the important lesson N. Neuhaus empirical evaluation of different viral vector learned. In this talk the field will be reviewed Centre of Reproductive Medicine and Andrology, delivery systems to identify the pros and cons and the state of the art is described. Future Münster, Germany of each system for manipulation of somatic directions and the transfer to human applica- Spermatogonial stem cells (SSCs) form the cells and testis function in vivo. tions will be discussed. Results Methodologies to target Sertoli basis of human spermatogenesis. These are cells for robust, long term delivered transgene the least differentiated germ cells in the adult expression are now well established, and we 16:15–16:45 h human testis and are a subpopulation of the have utilised this efficacy to demonstrate diploid spermatogonia. SSCs are defined Epigenetic changes and their based on their functional properties, specifi- lineage tracing, knockout, knock-down and ­roles in male germ cells rescue of gene function in vivo. Leydig cells cally their ability to self-renew and to give rise have also been targeted with different viral J. Trasler, D. Chan1 to differentiating germ cells, which ultimately vectors. However, until now, no one study has Research Institute of the McGill University Health form sperm. Due to the complexity of hu- demonstrated robust and long-term transgene Centre and Departments of Pediatrics, Human Gene- man testicular tissues and the lack of marker expression. Here, we also characterise and tics and Pharmacology & Therapeutics, McGill Uni- genes to isolate specific spermatogonial sub- compare three commonly used and readily versity, Canada populations, their molecular properties have available viral vectors and define their effica- largely remained unknown. The advent of Introduction Sperm-DNA methylation pat- cy for delivery of transgenes to Leydig cells. high-throughput single cell RNA sequencing terns are unique and play a key role in the Together these tool kits provide options for strategies (scRNA-Seq) has facilitated the health of the next generation. Both folate rapid generation of somatic cell transgenics analysis of testicular cell suspensions, provid- in the diet and enzymes such as 5,10-meth- in the testis. ing hitherto unequalled insights with regard ylenetetrahydrofolate reductase (MTHFR) to the cell-type-specific expression profiles, Conclusion he wide availability of viral impact the delivery of methyl groups for including those of the spermatogonial-­ vectors now available and ease of delivery to DNA methylation. Our objectives were to as- subpopulations. Moreover, by comparison the intra-tubular and interstitial compartment sess which areas of the human sperm DNA with scRNA-Seq datasets obtained from tes- of the testis, means that rapid generation of ­methylome are most susceptible to altered ticular cells suspensions from infertile men, transgenic mice now takes just days from ini- folate and MTHFR levels. crucial insights have been gained regarding tial design to final analysis. The approach cir- Methods Whole genome bisulfite sequenc- the transcriptional changes of testicular cell cumvents developmental phenotypes whilst ing (WGBS) was performed on a sperm pool types associated with infertility. In this talk, simultaneously reducing cost, time, and num- from 30 men to identify regions of variable current scRNA-Seq datasets obtained from bers of animals required. These technologies sperm DNA methylation. The WGBS results human testicular tissues will be reviewed with will change the pace of genetic research in were used to create a customized human a particular focus on the novel insights regard- testis function and have potential as bespoke sperm methylC-capture panel to probe sperm ing the spermatogonial compartment in situa- therapeutics for the treatment of male repro- from individual MTHFR 677CC or MTHFR tions of spermatogonial (dys)function. ductive disorders. 677TT (50% decrease MTHFR activity) men and test the impact of high dose folic acid 18:15–18:45 h 15:15–15:45 h supplements (5 mg/day for 6 months) on the In vitro organogenesis of the testis sperm DNA methylome. Oxidative stress and the structure Results With WGBS we discovered 1 mil- and function of human spermato­ S. Schlatt lion sperm CpGs characterized as having Centre of Reproductive Medicine and Andrology, zoa intermediate methylation levels (20–80%); Münster, Germany J. R. Drevet these sites were significantly more variable in GReD Institute, INSERM U1103/CNRS UMR6293/ In recent years many breakthroughs in cel- sperm than regions of < 20% or > 80% meth- Université Clermont Auvergne, France lular research have opened fascinating sce- ylation and were termed dynamic sites. These narios for the ex vivo generation of germ cells dynamic sites, along with 2 million com- The integrity of the paternal and maternal ge- from pluripotent stem cells as well as estab- monly targeted CpGs, were used to create the netic material and their respective epigenetic

18 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts information is the guarantee of reproductive levels. Diagnostic and therapeutic aspects tory M2 macrophage phenotype. Epididymal success, the health of the offspring and the will also be briefly discussed. macrophages (EM) are less well studied, but sustainability of the species. The oxidative also comprise the largest cohort of resident metabolism that sustains the life of all aero- leukocytes in this organ. They can be found in bic cells is at the crossroads of responses to the epididymal interstitium, but also intraepi- the various environmental stimuli to which 18:45–19:15 h thelially. In the epididymis, which consists organisms are exposed, whatever their nature, of a single duct, the opposing ends face dif- whether physical, chemical or biological. The The immune system of the testis ferent immunological challenges. Tolerance gametes are no exception to this situation and, and epididymis: mastering para­ against the neoantigens on spermatozoa is the due to its characteristics, the spermatozoa is doxical tasks predominant task at the proximal end, whilst particularly exposed to oxidative alterations. an active immune defense is required at the A. Meinhardt To date, the links between oxidative damage distal end to combat ascending pathogens. Institute of Anatomy and Cell Biology, Justus-Liebig to the spermatozoa and functional impacts The different local microenvironments lead University of Giessen, Germany (motility, gamete interaction, capacitation) to a varied immunopathological response in have been clearly established. However, it is Testicular macrophages (TM) are heteroge- acute bacterial epididymo-orchitis with per- necessary to consider another level of oxi- neous and represent the largest immune cell manent damage occurring in the epididymal dative alteration of the male gamete which population in the male gonad. TM are located cauda, little to no obvious pathology in the

concerns the paternal nucleus. In this talk, in the testicular interstitial space and play an caput and regeneration of tissue damage in DGA-Abstracts combining fundamental research carried out essential role in maintaining normal organ the testis. This talk will describe similarities on mouse models and clinical investigations, functions, i.e. steroidogenesis and spermato- in the immunopathology in mice and men a presentation of oxidative damage on the genesis. TM contribute to the establishment and the organ specific identity of TM and EM sperm nucleus will be made with its possible and maintenance of the testicular immune as well as crucial factors necessary for their consequences at the genetic and epigenetic privilege by displaying an immunoregula- maintenance.

Freie Vorträge

O 01 previously reported genes –TEX14, DMRT1, ity is not anymore limited to karyotype or TEX11, SYCE1, MEIOB, MEI1 and STAG3 – Y chromosome microdeletion. Indeed, an Genetic dissection of spermato­ allowing to upgrade the clinical significance increasing list of human genes involved in genic arrest through exome ana­ of these genes for diagnostic purposes. Our infertility is now available. In order to trans- lysis: clinical implications for the meiotic studies provide novel insight into the late this research field to clinical application, management of azoospermic men functional consequences of the variants, sup- we set up a new diagnostic activity offering porting their pathogenic role. the analysis of a panel of 51 genes involved A. Riera-Escamilla1, D. Moreno-Mendoza1, Diagnosing complete MA in different form of non-syndromic human K. Holleman2, F. Cioppi3, F. Algaba1, M. Pybus1, Conclusions based on a genetic test is clinically relevant infertility. The panel encloses 34 genes for C. Friedrich4, M. J. Wyrwoll4, E. Casamonti3, because affected patients should not undergo male infertility, 15 genes for female infertil- S. Pietroforte3, L. Nagirnaja5, A. M. Lopes6, invasive testis surgery (TESE). Our findings ity and 2 genes causing both male and female S. Kliesch7, A. Pilatz8, D. T. Carrell9, D. F. Conrad5,10, contribute substantially to the development infertility. We present here the results of our E. Ars1, E. Ruiz-Castañé1, K. I. Aston9, of a pre-TESE prognostic gene panel. Wider new diagnostic activity. W. M. Baarends2, F. Tüttelmann4, C. Krausz1,3 implications include the understanding of 79 males and 15 1Fundació Puigvert, Barcelona, Spain; 2Erasmus MC Patients and Methods potential genetic links between NOA and females with non-syndromic infertility were University Medical Centre, Rotterdam, Netherlands; cancer predisposition, and between NOA and recruited. Five patients with known single 3University of Florence, Florence, Italy; 4University premature ovarian failure. gene mutations were used as positive con- of Münster, Münster, Germany; 5Oregon Health & trols. Sequencing libraries were prepared Science University, Portland, OR, United States; using the Agilent SureSelectQXT Target 6Universidade do Porto, Porto, Portugal; 7University Enrichment system. Multiplex sequencing Hospital Münster, Münster, Germany; 8Justus-Liebig- O 02 has been performed on Illumina NextSeq 550 University, Giessen,­ Germany; 9University of Utah with 2x75bp reads for total 51 genes in a se- School of Medicine, Salt Lake City, UT, United Diagnostic interests of a custom ries of 30 samples. Variant analysis has been States; 10Washington University School of Medicine, designed panel for the analysis of achieved using our in house bioinformatics St. Louis, MO, United States 51 genes involved in non-syndro­ pipeline (STARK). mic human infertility Introduction Azoospermia affects 1% of Result With a mean coverage of 457X and men and it can be the consequence of sper- O. Okutman1,2, J. Tarabeux3, J. Muller3,4,5, S. Viville1,2 99,8% of target bases successfully sequenced matogenic maturation arrest (MA). Although 1Hôpitaux Universitaires de Strasbourg, Laboratoire with a depth coverage over 30X, we prove the etiology of MA is likely to be of genetic de Diagnostic Génétique UF de génétique de l’infer- the robustness and the quality of our panel. origin, only 13 genes have been reported as tilité, Strasbourg, France; 2IPPTS, Université de In total, we identified causative mutations in recurrent potential causes of MA. Strasbourg, Strasbourg, France; 3Hôpitaux Universi- 8 patients (8.5%), five (6.3%) for the male co- Patients and Methods Exome sequencing taires de Strasbourg, Laboratoires de Diagnostic hort and three (20%) for the female patients. in 147 selected MA patients (discovery co- ­Génétique, UF de génétique moléculaire, Strasbourg, Such a yield is higher of the one reported so hort and two validation cohorts). France; 4Université de Strasbourg Faculté de médeci- far about sequencing custom panel of genes ne de Strasbourg, Laboratoire de Génétique Médica- Results We found strong evidence for related to infertility. le, INSERM, UMRS_1112, Institut de Génétique Médi- 5 novel genes likely responsible for MA Conclusion Our cohort proved the ability of cale d’Alsace (IGMA), Strasbourg, France; 5Hôpitaux (ADAD2, TERB1, SHOC1, MSH4, and our panel to detect various types of variants Universitaires de Strasbourg, Unité Fonctionnelle de ­RAD21L1), for which mouse knockout (KO) including substitution, indel and CNV. The Bioinformatique Médicale appliquée au diagnostic models are concordant with the human phe- genetic of infertility allow precise diagnosis, (UF7363), Strasbourg, France notype. Four of them were validated in the which allow to offer a genetic council to the two independent MA cohorts. In addition, Introduction With the advance of genome patient and his family and to personalize his 9 patients carried pathogenic variants in 7 wide analysis, the genetic of male infertil- treatment.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 19 Andrology 2020 – Abstracts

O 03 1Università di Roma “Sapienza”, Dipartimento di cally relevant threshold. However, the predic- Medicina Sperimentale, Roma, Italy; 2Università Fe- tive value of the mixed antiglobulin reaction Pituitary-gonadal axis in male derico II di Napoli, Dipartimento di Medicina Clinica (MAR) test positivity degree above such a ­patients affected by type 1 diabe­ e Chirurgia, Sezione di Endocrinologia, Napoli, Italy cut-off on the occurrence of natural pregnan- tes mellitus: comparison between cies remains undetermined. Furthermore, the Patients with Adrenal Insuffi- Context effectiveness of intrauterine insemination continous subcutaneous insulin ciency (AI) show an impaired quality of life, (IUI) in cases of strong sperm autoimmunisa- infusion vs multiple daily injecti­ metabolic status, immune function and bone tion is not yet established. ons: preliminary data metabolism. Data on sexual function in these patients are scarce and often contradictory. Patients and Methods his was a retrospec- C. Bruno, E. Vergani, G. E. Rizzo, A. Rizzi, tive cohort study on 108 men with MAR test to evaluate sexual dysfunction (SD) in L. Tartaglione, G. Piunno, A. Pontecorvi, Aim + ≥50%. A post-coital test (PCT) was per- female and male patients with primary and D. Pitocco, A. Mancini formed in all couples. Data on subsequent secondary AI and to investigate the effects Università Cattolica del Sacro Cuore, UOC Endocri- pregnancies were obtained by contacting men of once-daily glucocorticoid replacement on nologia e Diabetologia, Rome, Italy by telephone. sexual function. Introduction It is well known that in type Results A total of 84 men (77.8%) were 89 AI pa- 1 diabetes mellitus (T1DM), continuous sub- Participants and Intervention enrolled: 44 men with 100%+ MAR test and tients and 25 controls were enrolled in the cutaneous insulin injection (CSII) improves 40 with 50%-99%+ MAR test. Couples with

DGA-Abstracts DREAM trial. AI patients taking conven- metabolic control and reduces the occurrence 100%+ MAR test showed a lower natural tional multiple times a day glucocorticoid of hypoglycemia when compared to multiple live birth rate per couple (LBR) than that therapy were randomly assigned to continue daily insulin injections (MDIs). HbA , body observed with lower positivity degrees (4.5% 1c their therapy or to switch to an equivalent mass index (BMI) and inflammatory para­ vs 30%; p = 0.00001). A significant differ- dose of once-daily, modified-release hydro- meters represent the most important meta- ence between the two groups was observed cortisone. 63 patients (29 males) completed bolic outcomes; however, few data have been only for the PCT outcome, which was poor the questionnaires for sexual function evalua- reported on pituitary-gonadal axis, despite in 100%+ MAR test group. Better PCT out- tion at baseline and at 24 weeks. the impact of sexual hormones on metabolic comes were positively associated with the status. Therefore, our aim was to study hypo- Results SD was increased in female and occurrence of natural live births, for which thalamus-pituitary-gonadal axis response in male AI patients compared to the general the sole independent negative predictor was male patients under CSII or MDII, evaluating population. In the females, sexual health the sperm autoimmunisation degree. IUI was levels of total (T) and free Testosterone (fT), positively correlated with duration of disease performed as the first-line treatment in 38 luteinizing hormone (LH), sexual hormones (p = 0. 005) and estrogen levels (p = 0.007). out of 44 couples with a 100%+ MAR test, binding protein (SHBG) in a cohort of T1DM Questionnaire’s items for “arousal” and yielding 14 live births (36.8%). In couples male patients, comparing this two different “desire” negatively correlated with age. In with lower MAR test positivity, the LBR after therapeutical modalities. pre-menopausal women, there was no cor- IUI (26.9%) was similar to the natural LBR relation with androgens. In post-menopausal Patients and Methods We enrolled 40 (30%). patients there was a positive correlation of T1DM male patients, aged 19–55 ys, 27 Conclusions The routine use of MAR test sexual function with testosterone (p = 0.008). treated with MDIs and 13 with CSII. The in basic fertility workup is justified as it in- In males, erectile dysfunction correlated two groups were matched for age, BMI and fluences decision making. A 100%+ MAR with quality of life (p = 0.020), while there years from diagnosis; we evaluated T, fT, test can represent the sole cause of a cou- was no correlation with age, androgens and LH, SHBG, HbA1c, fasting glucose in all pa- ple’s infertility, which could be successfully metabolic profile. At 24 weeks there was no tients. HbA were measured by IFCC-NGSP treated with IUI. Nevertheless, a lower degree 1c detectable difference in sexual function be- standardized method, hormonal parameters of positivity may only represent a contribut- tween study groups. were determined by ECLIA method. fT was ing factor to a couple’s infertility, and so the calculated by Vermeulen formula. Conclusions Patients with AI show an in- decision to treat or wait also depends on the creased prevalence of sexual dysfunction. Results Although similar levels of HbA evaluation of conventional prognostic factors 1c Sexual health correlated with estrogen levels (mean ± 7.8 ± 0.1% in MDII, 7.5 ± 0.1% in including the PCT outcome. and duration of disease in female patients CSII), a statistically significant difference and with quality of life in males. The lack of was found in LH values between the two correlation with androgens in males and pre- groups, with higher levels in CSII vs MDIs O 06 menopausal women and the independency (mean ± SEM 4.68 ± 0.62 vs 2.55 ± 0.36 mU/ from commonly associated factors such as Follow-up after testicular tissue ml, respectively; p < 0.05) and a trend toward age and metabolic status suggests that sexual high T (9.47 ± 0.91 vs 7.23 ± 0.67 ng/ml) biopsy for fertility preservation in dysfunction in AI patients is characterized by SHBG (79.27 ±1 4.07 nmol/l vs 54.49 ± 4.04 Klinefelter boys and adolescents different mechanisms and patterns. nmol/l) and fT (0.12 ± 0.01 vs 0.11±0.01 ng/ A. Braye1, I. Gies2, E. Goossens1 ml) in the same group. 1Vrije Universiteit Brussel, Reproduction, Genetics Conclusions This preliminary data seem O 05 and Regenerative Medicine, Brussels, Belgium to indicate a better pituitary-gonadal axis 2Universitair Ziekenhuis Brussel, Pediatrics, Brussels, response in male T1DM patients under CSII Relationship between natural and Belgium compared to the ones under MDIs, with intrauterine insemination-assisted Introduction Klinefelter syndrome (KS- higher LH, T, fT levels in the first group, de- live births and the degree of 47,XXY) is the most common sex-chro- spite similar glucose control. Further studies sperm autoimmunisation mosomal aberration found in men affecting to confirm this observation and to evaluate its 1-2 in 1000 males. Although early testis impact on real clinical practice are needed. A. Barbonetti, C. Castellini, G. Cordeschi, development appears normal in KS boys, S. D’Andrea, M. Totaro, S. Francavilla, spermatogonial stem cell (SSC) depletion F. Francavilla occurs before puberty leading to infertility. University of L’Aquila, Andrology Unit, Department of O 04 Cryopreservation of SSCs before puberty is Life, Health and Environmental Sciences, L’Aquila, the only fertility preservation option for KS Sexual function in adrenal insuf­ Italy ficiency: data from the DREAM boys and adolescents without ongoing sper- Introduction The WHO recommends a matogenesis. Therefore, KS patients need ­trial cohort screening for antisperm antibodies as an to undergo testicular tissue biopsy (TTB) at V. Hasenmajer1, E. Sbardella1, C. Pozza1, C. Simeoli2, integral part of semen analysis, with 50% young age. The surgical complications after V. Sada1, C. Lecis1, R. Pivonello2, A. Isidori1, A. Lenzi1 antibody-coated motile sperm as the clini- TTB are rare (1%); however, the long-term

20 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts effects are unknown. This retrospective study were collected and compared to identify the the malondialdehyde (MDA) level and sperm aimed to investigate the long-term impact strengths and weaknesses of the current AI nDNA fragmentation were observed. Also, of TTB on the pubertal development of KS system and identify areas for improvement. the levels of testicular mRNA transcript patients. QC microbead testing validates the micro- and protein levels of steroidogenesis-related Patients and Methods KS patients fol- scope and protocol, with no observational genes [androgen receptor (AR), luteinizing lowed between 2009–2020 at the Universi- error considering the interval of confidence. hormone receptor (LHR), steroidogenic acute tair Ziekenhuis Brussel and offered fertility Correlating the CASA vs. manual concentra- regulatory protein (StAR), cytochrome P450 preservation were included. Exclusion crite- tion results yields a Pearson/Spearman score enzyme (CYP11A1), 3β-hydroxysteroid de- ria were mosaic cases, cryptorchidism and of 0.96/0.96 with a mean relative error near hydrogenase (HSD) and 17β-HSD] in the testosterone replacement therapy prior to the WHO LRL of 13%. Deeper inspection of testes decreased significantly. Treatment with TTB. Retrospectively collected data on tes- the AI shows false positives classifications bee bread significantly decreased leptin level, ticular volume, reproductive hormones, bone leading to over-estimation of concentration increased adiponectin level, upregulated ster- age and density were compared between KS and motility. In conclusion, mojo”s robot- oidogenesis-related genes and protein levels, patients who underwent TTB (biopsy group) ics platform and protocols show promise attenuated spermatogenesis impairment, and those who did not (control group). for clinical use. The AI software is proven and decreased sperm DNA fragmentation in capable of measuring sperm concentration HFD-induced obese rats. Results Of 72 KS patients included, 23 with high confidence. During the 2 month had testicular tissue biopsied (mostly be- Discussion and Conclusion Bee bread im-

study, AI performance increased from a base proved spermatogenesis and steroidogenesis DGA-Abstracts tween 12–16 years), while 49 refused fertil- Spearman of 0.52. Next, the AI will be fur- ity preservation. After TTB, no statistically decline by upregulating steroidogenic genes. ther clinically trained, reducing false positive Therefore, bee bread may be regarded as a significant difference in testicular volume incidence of the CNN. Motility classification was found between the biopsied and con- prospective remedy for subfertility in obese tuning will be performed to improve progres- individuals. tralateral non-biopsied testes. No statisti- sive and immotile grading. cally significant differences were found after TTB between the biopsy and control group for the reproductive hormones as well as for O 08 O 09 bone age and density. Testicular growth ar- rest/regression and hypergonadotropic hypo­ Obesity-induced steroidogenesis Sperm small non-coding RNA gonadism became clear after puberty onset in and spermatogenesis decline: the profile obtained by NGS and se­ both groups. protective role of Heterotrigona men quality among healthy Conclusion No differences were found be- itama bee bread young adults Updates on T-trials tween the biopsy and control group for the O. Sergeyev1,2, S. Victoria1, V. Naumov3, V. Bezuglov4, J. Suleiman1,2, V. Nna3, Z. Othman1,4, Z. Zakaria1, different parameters evaluated. These results M. Logacheva1,5, L. Smigulina1,2, Y. Dikov1,2, T. Deni- A. Abu Bakar1, M. Mohamed1,5 are reassuring and suggest that TTB has no sova1,2, A. Suvorov1,6, J. R. Pilsner6, R. Hauser7 1Universiti Sains Malaysia, Department of Physio­ additional impact on the pubertal develop- 1A.N. Belozersky Research Institute of Physico-Che- logy, School of Medical Sciences, Kubang Kerian, ment of KS patients on the long term. mical Biology Moscow State University, Moscow, Kelantan, Malaysia; 2Akanu Ibiam Federal Polytech- Russian Federation; 2Chapaevsk Medical Associa- nic, Unwana, Science Laboratory Technology, Afikpo, tion, Chapaevsk, Russian Federation; 3Kulakov Natio- Nigeria; 3University of Calabar, Department of Phy- O 07 nal Medical Research Center of Obstetrics, Gyneco- siology, Faculty of Basic Medical Sciences, College logy & Perinatology, Ministry of Health of the Clinical evaluation of male factor of Medical Sciences, P.M.B. 1115 Calabar, Cross River Russian Federation, Moscow, Russian Federation; infertility with artificial intelli­ State, Nigeria; 4Universiti Sultan, Zainal Abidin, Unit 4Moscow State University, Moscow, Russian Federa- gence integrated Computer of Physiology, Faculty of Medicine, Kuala Terengga- tion; 5Center for Data-Intensive Biomedicine & Bio- nu, Terengganu, Malaysia; 5Universiti Sains ma­ Assist­ed Sperm Analysis (CASA) technology, Skolkovo Institute of Science & Techno- laysia, Unit of Integrative Medicine, School of Medi- tools logy, Moscow, Russian Federation; 6University of cal Sciences, Kubang Kerian, Kelantan, Malaysia L. Björndahl1, J. Flanagan1, D. Thomas2, R. Maillot2, Massachusetts, Department of Environmental M. Monteiro2, Z. Simon2, M. Taha2 Background Decline in steroidogenesis and Health Sciences, School of Public Health & Health 7 1ANOVA Karolinska University Hospital, Stockholm, spermatogenesis have been observed in obese Sciences, Amherst, MA, United States; Harvard T.H. Sweden; 2mojo (Nanovare SAS), Lyon, France men. Chan School of Public Health, Department of Objectives The present study investigated Environ­mental Health, Boston, MA, United States The task remains to build an automated se- the protective effect of bee bread on obesity- men analysis system that upholds the confi- Introduction For the identification of small induced decline in steroidogenesis and sper- non-coding RNA (sncRNA) as prognostic dence intervals required by andrology inves- matogenesis. tigation for male factor infertility. This study markers of fertility subjects are usually re- presents and analyzes the validation of a new Methods Thirty-two adult male Sprague- cruited through infertility clinics. We ex- automated system using robotics and AI that Dawley rats weighing between 200–300 g amine the association of semen quality with conform to the WHO guidelines. An automo- were divided into four groups (n = 8/group), sncRNA profile in young healthy adults. tive robotic microscope named Mojo was de- namely; normal control (NC), high-fat diet Patients and Methods We selected 49 veloped for scanning wet semen preparations. (HFD), HFD plus bee bread or orlistat ad- subjects from the prospective cohort Rus- This platform uses an AI software based on ministered 6 weeks after induction of obesity sian Children’s Study (n = 516) enrolled at a unique convolution neural network (CNN) (HFD+BB and (HFD+O groups, respective- 8–9 years of age. Puberty was evaluated in that measures sperm concentration and motil- ly). Bee bread (0.5 g/kg) or Orlistat (10mg/ annual follow-ups and semen samples were ity. Mojo sperm counting chambers were used kg) was suspended in distilled water and collected at 18–20 years. Fresh semen was for validation. To rule out sampling error, given by oral gavage daily for 6 weeks after evaluated for volume, sperm concentration, videos of Hamilton Thorne accubeads+ were induction of obesity. and motility according to ESHRE manual. captured using the mojo counting chambers Results The HFD group showed significant Sperm RNA was extracted from 2 layers and microscope. The concentration of mi- decreases in follicle stimulating hormone after centrifugation with 50% and 90% den- crobeads was manually assessed from videos (FSH), luteinising hormone (LH), testoster- sity gradient. Libraries of sncRNA were con- and compared to their known measures. In one and adiponectin levels, and an increase structed using NEBNext Kit, n = 34 (NEB) this study, we analyzed semen samples from in leptin level compared to NC group. Fur- and NEXTFLEX® Kit, n=15 (Perkin-Elmer) 44 patients and compared standard manual thermore, sperm count, viability, motility and sequenced on NextSeq 500 (Illumina). assessment to mojo AI CASA assessment and normal morphology and epididymal an- Reads were mapped to hg38 in a sequential for concentration and motility. The results tioxidants decreased as well as increases in order: ribRNA > miRNA > piRNA > tRNA.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 21 Andrology 2020 – Abstracts

DESeq2 R package was used for the analysis was not associated with sperm concentration, measured within distinct epididymal regions of differentially expressed sncRNA. motility or morphology in the native ejacu- in untreated mice (by RT-qPCR) and in UP- Result Medians (IQR) for age, semen vol- late, a lower PIWI-LIKE 2 mRNA expression EC-infected mice (by whole transcriptome ume, sperm concentration, total motile sperm was significantly associated with a fertiliza- RNA-Seq analysis at day 10 post infection) and total progressive motile sperm (TPMS) tion rate ≥ 50% (p = 0.02, Mann-Whitney- exhibited striking region-specific differences. were: 18.3 (18.2–19.1) years, 3.2 (2.5–5) ml, U-Test). Furthermore, a lower PIWI-LIKE 1 Overall, these data suggest that the cauda 38.5 (22–66.5) mill/ml, 89.4 (51.0–160.2) mRNA expression was significantly associ- is more susceptible to an inflammatory re- mill, and 78.8 (46.4–135.7) mill. Mean num- ated with a fertilization rate ≥ 50% (p = 0.05, sponse compared to the caput, indicating that ber of sequenced reads was 7.97 mill, with Mann-Whitney-U-Test). the different immunological milieus might 63% alignment to sncRNA. 2332 sncRNA Conclusion The level of spermatozoal play a critical role in the magnitude of the were identified with≥ 10 mean counts: 378 PIWI-LIKE 1 and 2 mRNA expression ex- region-specific immune responses. miRNA, 1580 piRNA and 374 tRNA. In hibited a significant association to fertiliza- models adjusted for library kit and sperm tion rate in ART cycles. A detailed charac- layer we identified 431 sncRNA significantly terisation of the mechanisms and regulatory differentially expressed by any of 4 semen pathways of PIWI-LIKE 1 and 2 during sper- O 12 parameters: 70 miRNA, 278 piRNA and 83 matogenesis and of the predictive potential Pathogenic T cell function induced tRNA, FDR< 0.05. piR-7725 and piR-32962 for successful fertilization and implantation chronic epididymo-orchitis in the DGA-Abstracts overlapped with all semen parameters. 35 is highly needed. sncRNA were significantly associated with T cell transferred Rag1-/- Mice TPMS, FDR < 0.05. Q. Zeng1, J. Gong1, W. Yeung1, D. Yu2, Y. G. Duan1 1The University of Hong Kong - ShenZhen Hospital, Conclusion To our knowledge, this is the O 11 first study to examine the association of sn- Shenzhen Key Laboratory of Fertility Regulation, cRNA profile and semen quality in a cohort Discerning the role of different im­ Center of Assisted Reproduction and Embryology, of healthy young men. We suggest some can- mune cell populations in the pa­ Shenzhen, China 2University of Queensland Diamantina Institute, didate markers for evaluating semen quality thogenesis of epididymitis in young adults. Woolloongabba, Australia C. Pleuger1,2, S. Bhushan1,2, D. Bohnert1, M. Hoppe1, Funding RSF #18-15-00202; for parent Chronic epididymo-orchitis is thought to be M. Fijak1,2, S. Günther3, R. Middendorff1,2, K. Love- RCS – NIEHS #R01 ES014370. an inflammatory autoimmune disease with land1,4,5, M. Hedger4,5, A. Meinhardt1,2,4 the testis and epididymis lesions, including 1Justus-Liebig-University Giessen, Institute of Ana- germ cells loss, and dilated seminiferous tu- tomy and Cell Biology, Giessen, Germany; 2Justus- O 10 bules, aggressive and dysregulated structure Liebig-University, Hessian Centre of Reproductive remodeling, and impaired spermatogenesis. Spermatozoal PIWI-LIKE 2 mRNA Medicine, Giessen, Germany; 3Max Planck Institute Although sperm antigen-specific T cells have expression as predictor of fertili­ for Heart and Lung Research, ECCPS Bioinformatics been described in the pathogenesis of auto- zation success in ART and Deep Sequencing Platform, Bad Nauheim, Ger- immune animal models, the independent many; 4Hudson Institute of Medical Research, Centre T. Greither, M. Giebler, D. Handke, P. Kaltwaßer, role of T cells in the orchitis and epididymi- for Reproductive Health, Clayton, Australia; 5Monash G. Seliger, H. M. Behre tis pathogenesis remains unclearly deline- University, Department of Molecular and Translatio- Martin Luther University Halle-Wittenberg, Center ated. Here, we report that T cells transfer can nal Sciences, Clayton, Australia for Reproductive Medicine and Andrology, Halle mediate and exacerbate murine testicular and (Saale), Germany Previous data have shown that distinct sub- epididymis inflammation and pathogenesis sets of mononuclear phagocytes reside within in immunodeficiency mice. Naïve CD4+ or Introduction PIWI-LIKE 1–4 play a pivotal role in stem cell maintenance and transposon the different epididymal regions and could CD8+T cells transfer reduced the sperm repression in the human germline. Therefore, provide a rationale for obvious paradoxical motility and viability, widened seminiferous the dysregulation of these genes negatively immunological tasks of the epididymis, i.e. tubules, and impaired spermatogenesis with influence genetic stability of the respective maintenance of peripheral immune tolerance severe spontaneous testis and epididymitis germ cell and subsequent development and within the caput and the preservation of an histopathology that comparable in Rag1-/- maturation. Recently, we demonstrated that immune responsive environment within the mice with no T cells transfer. T cells trans- a lower PIWI-LIKE 2 mRNA expression in cauda. Using a murine model of acute bac- ferred testis and epididymitis mice had a ejaculated spermatozoa is more frequent in terial epididymitis (C57BL/6J WT strain, lower survival rate of sperm, the less capac- men with oligozoospermia [Giebler et al. bilateral injection of uropathogenic E. coli ity to fertilize, inflammatory cells infiltrated, Asian J Androl 2018; 20: 260–4]. In this (UPEC) into the ligated vas deferens), we and severe intestine histopathology with in- study, we aimed to analyse PIWI-LIKE 1-4 analyzed the immune responses within the creased permeability. Besides, in the T cells mRNA expression in ejaculated spermatozoa different epididymal regions in association transferred orchitis and epididymitis model, to predict ART outcome. with the ascension of UPEC (traced by agar CD25+Foxp3-CFP Treg cells with naïve plating and immunofluorescence) at days 1, T cells co-transfer ameliorated testis and Material and Methods From 160 ART cy- 3, 5, 7 and 10 post infection. While the caput epididymis pathology and improve the sperm cles, portions of the swim-up spermatozoa remained mostly unaffected, the cauda under- quantity and quality. We further evaluated the used for fertilization were collected and total went dramatic morphological alterations (im- role of gut microbiota in the immunopatho- RNA was isolated via TRIzol method. After mune cell infiltration, epithelial detachment, genesis of T cells dependent orchitis and cDNA synthesis, PIWI-LIKE 1-4 mRNA fibrosis, obstruction) as shown by Masson- epididymitis. The results demonstrated that expression was measured by qPCR using Goldner/ Azan staining, immunofluorescence gut microbiota diversity decreased in naïve T TaqMan probes with GAPDH as reference and morphometric evaluation (n=4 per ex- cell transfer mice, with antibiotics treatment gene. Relative mRNA expression was corre- perimental group). Flow cytometry analyses alleviated T cell transfer-induced testis and lated to ejaculate values and fertilization rate. revealed a massive infiltration of neutrophils epididymis. Using this transfer model, we Results PIWI-LIKE 1, 2 and 4 mRNA (Ly6g+) starting at day 3, followed by mono- validated that Stat3 deficiency attenuates that expression was significantly correlated with cytes (Ly6chi, F4/80lo, CD64lo, MHCint) T cell-induced testis and epididymis tissue percentage of motile spermatozoa (rS = 0.78; and monocyte-derived macrophages (Ly6chi, damage and protects the sperm loss and func- p < 0.001). PIWI-LIKE 4 mRNA expression F4/80hi, CD64hi, MHC-IIhi) at day 5 in the tion decrease. Our study reveals that T cells was inversely correlated to PIWI-LIKE 1 cauda, but not in the caput (n = 4 per group). are pathogenic in orchitis and epididymitis, and 2 mRNA expression (rS = –0.25 and rS In support, levels of mRNA transcripts en- and the transfer model serves as a valuable = –0.28, p < 0.001). While PIWI-LIKE 1-4 coding diverse immunomodulatory factors tool to evaluate pathways modulating T cells mRNA expression in motile spermatozoa (e.g. lipocalins, cystatin 11, lactotransferrin) in chronic epididymo-orchitis.

22 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

O 13 O 14 secondary hypogonadism, PH/SH) regarding bone density. Effects of LPS-induced epididymi­ Testosterone levels are independ­ Methods Registry data of 2–5 years com- tis on Wfdc gene expression in ently associated to sub-clinical prising 189 patients including 67 men with mice ­atherosclerosis in men with chro­ PH (mean age 32.0 ± 10.1 years), 49 with A. Dorth de Andrade1, N. Aparecida Partelli Mariani1, nic spinal cord injury SH (mean age 30.4 ± 9.4 years) and 73 with A. Andrew dos Santos Silva1, T. Rocha Fanti S. D’Andrea1, M. Totaro1, A. Parisi1, C. Castellini1, FH (mean age 44.8 ± 11.6 years) all receiv- Raimundo­ 1, H. Kushima1, M. A. Spadella2, G. Felzani2, S. Francavilla1, F. Francavilla1, ing uniform treatment using intramuscular T M. C. Werneck de Avellar3, E. José Ramo da Silva1 A. Barbonetti1 undecanoate (1000 mg). All patients received 1Instituto de Biociências de Botucatu, Universidade 1University of L’Aquila, Andrology Unit, Department annual assessments of bone density using Estadual Paulista “Júlio de Mesquita Filho”, Biophy- of Life, Health and Environment Sciences, L’Aquila, dual-energy X-ray absorptiometry. sics and Pharmacology, Botucatu, Brazil; 2Faculdade Italy; 2San Raffaele Institute, Spinal Unit, Sulmona, Results Serum T concentrations increased de Medicina de Marília, Discipline of Human Em- Italy from 6.8 ± 2.7 nmol/L to 18.1 ± 2.9 nmol/L in bryology, Marília, Brazil; 3Universidade Federal de men with PH/SH and from 7.9 ± 2.5 nmol/L São Paulo, Escola Paulista de Medicina, Pharmaco- Introduction Men suffering from spinal to 17.3 ± 3.2 nmol/L in men with FH. logy, São Paulo, Brazil cord injury (SCI) are at increased risk for cardiovascular (CV) diseases as documented There was an initial difference in bone den- sity between patient groups (T-Score lumbar The Whey-acidic protein four-disulfide core by a higher value of ultrasonographic carotid DGA-Abstracts domain (Wfdc) locus on mouse chromosome intima media thickness (cIMT), a surrogate spine, PH: –1.8 ± 0.3, SH: –2.3 ± 0.4, FH: 2 spans 16 Wfdc genes in its centromeric and marker of sub-clinical atherosclerosis. Fur- –1.2 ± 0.3, all p < 0.001 vs each other) and telomeric subloci. These genes play putative thermore, when compared to able-bodied (T-Score hip, PH: –1.4 ± 0.3, SH: –1.7 ± 0.4, roles in antimicrobial, immune and reproduc- general population, SCI men also exhibit FH: –1.1 ± 0.3, all p < 0.001 vs each other). tive homeostasis. Although Wfdc genes are a very higher prevalence of androgen defi- A significant increase in bone density was highly expressed in the epididymis their con- ciency, which can contribute to CV risk. We observed for all patient groups (lumbar spine tributions to epididymal health and disease aimed to verify the possible relationship be- and hip, both p < 0.001), albeit the effect remain obscure. Here, we tested the hypothe- tween testosterone levels and cIMT in men was least pronounced in FH and strongest sis that Wfdc genes are effectors downstream with chronic SCI. in SH (lumbar spine: p = 0.008 and hip: p = the inflammatory responses to lipopolysac- 0.01, post-hoc tests). Stepwise multiple Cox Patients and Methods cIMT of 60 men charide (LPS) in the epididymis. We induced regression models could attribute these dif- with chronic (> 1 years) SCI, aged 56.0 epididymitis in mice via interstitial or intra- ferences to the different baseline characteris- (25–75th: 46.0–67.2) years, was evaluated vasal LPS (50 µg) injection, targeting the ini- tics between groups including age and delta with neck ultrasonography (US). All patients tial segment (IS) or cauda epididymidis (CE), T levels. There was no difference between underwent a complete neurological exam, as respectively. Mice were euthanized 6, 24 and groups for the overall increase in hematocrit. well as biochemical and hormonal assess- 72 h after LPS injection and tissues were pro- Changes in PSA levels were more likely to ment. Comorbidity was scored by Charlson cessed for RT-qPCR assays. In the IS, LPS occur in FH (hazard ratio 1.4 [1.2–1.6], p = comorbidity index (CCI). downregulated one centromeric (Wfdc15b) 0.004). and five telomeric (Wfdc6a, Eppin, Wfdc8, Results The cIMT median value was of Conclusions This study provides major Wfdc11 and Wfdc16) Wfdc transcripts at 1.3 (1.0–1.8) mm. At the univariate linear new findings regarding effects T therapy in 6 h. At later time-points (24 and 72 h) LPS regression analyses, cIMT showed a signifi- different groups of hypogonadal men. Pa- upregulat­ed all centromeric (Slpi, Wfdc5, cant positive association with age (beta=0.09, tients with classical forms of hypogonadism Wfdc12, Wfdc15a and Wfdc15b) and five p = 0.008), calculated LDL cholesterol level have a more pronounced increase in bone telo­meric (Wfdc2, Wfdc3, Wfdc6b, Wfdc10 (beta = 0.09, p = 0.03), and HOMA-index of density than patients with FH. and Wfdc13) Wfdc transcripts. Pretreatment insulin resistance (beta = 0.03, p = 0.04) and with the NFKB inhibitor PDTC (100 mg/ a negative association with total testosterone kg, i.p.) prevented LPS-induced upregula- (TT) levels (beta = –0.04, p = 0.001). At the O 16 tion of telomeric but not centromeric Wfdc multivariable linear regression model, includ- transcripts at 24 h. In the CE, LPS upregu- ing the significant putative predictors identi- Low testosterone levels predict lated one centromeric (Wfdc5) and one telo- fied by univariate analyses, only TT levels clinical adverse outcomes in meric (Wfdc2) Wfdc transcripts at 24 h, while showed an independent association with SARS-CoV-2 pneumonia patients cIMT (beta= –0.03, p = 0.009). downregulated one centromeric (Wfdc15b) G. Rastrelli1, V. Di Stasi1, F. Inglese2, M. Beccaria2, and three telomeric (Wfdc6a, Wfdc11 and Conclusion In patients with chronic SCI, M. Garuti2, D. Di Costanzo2, F. Spreafico2, Wfdc16) Wfdc transcripts at 72 h. Pretreat- lower TT levels represent significant and G. F. Greco2, G. Cervi2, A. Pecoriello2, A. Magini1, ment with PDTC effectively prevented LPS- independent predictors of subclinical athero- T. Todisco1, S. Cipriani1, E. Maseroli1, G. Corona1, induced upregulation of Wfdc5 and Wfdc2 sclerosis, despite the very high prevalence of A. Salonia3, A. Lenzi4, M. Maggi1, G. De Donno2, transcripts in the CE. traditional CV risk factors in this population. L. Vignozzi1 Our findings indicate that members of the 1University of Florence, Experimental and Clinical Wfdc locus are differentially regulated by Biomedical Sciences, Florence, Italy; 2ASTT Carlo LPS/TLR4/NFKB signaling pathway in the O 15 Poma, Intensive Care Respiratory Unit and Pneumo- proximal and distal epididymis, suggesting Differential effects of testosterone logia, Mantua, Italy; 3IRCCS Ospedale San Raffaele, that WFDC family members could play roles treatment on bone density in men Urological Research Institute, Division of Experimen- 4 in epididymal responses to acute inflamma- with classical vs functional hypo­ tal Oncology, Milan, Italy; Sapienza University of tory stimuli. Rome, Department of Experimental Medicine, Rome, gonadism Support: FAPESP (2015/08227-0, Italy M. Zitzmann1, A. Traish2, S. Kliesch1 2017/20102-3) and CAPES. Ethics approval: Background The pandemic of new severe 1University Clinics Münster, Center for Reproductive 1029-CEUA. acute respiratory syndrome (SARS) due to Medicine / Clinical and Surgical Andrology, Münster, coronavirus (CoV) 2 (SARS-CoV-2) has Germany; 2Boston University School of Medicine, stressed the importance of effective diag- Urology, Boston, MA, United States nostic and prognostic biomarkers of clinical Introduction and Objective There are lim- worsening and mortality. Epidemiological ited data on Testosterone (T) therapy in men data showing a differential impact of SARS- with functional hypogonadism (FH) com- CoV-2 infection on women and men has sug- pared to those with classical forms (primary/ gested a potential role for testosterone (T) in

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 23 Andrology 2020 – Abstracts

determining gender-disparity in the SARS- ing single cell solution is screened for the functionally analyzed in our PGCLC differ- CoV-2 clinical outcomes. occurrence of spermatozoa. Depending on entiation system. Objectives To estimate the association the result, one or more TESE samples are For proof of concept, we used DND1 (DND between T level and SARS-CoV-2 clinical thawed and processed for TESE-ICSI and microRNA-mediated repression inhibitor 1), outcomes (defined as conditions requiring the spermatozoa yield is counted again. The a regulator of germ cell maintenance and dif- transfer to higher or lower intensity of care absolute number is recorded if less than 100 ferentiation in vertebrates. While DND1 is or death) in a cohort of patients admitted in spermatozoa are found, otherwise the result is extensively studied in zebrafish and mouse, the Respiratory Intensive Care Unit (RICU) displayed as being 100 as a maximum value. little is known about its role in human PGC These data were analysed for equality. Methods A consecutive series of 31 male differentiation. patients affected by SARS-CoV-2 pneumo- Results Sperm retrieval results of 872 Mutations were introduced using CRISPR/ nia and recovered in the Respiratory Inten- mTESE samples thawed on the day of ICSI Cas9 and three homozygous knockout sive Care Unit (RICU) of the “Carlo Poma” were compared to the respective TS. Compar- (KO) lines were analyzed for differences in Hospital in Mantua were analyzed. Several ing test and TESE-ICSI preparations 73.6% gene expression and differentiation capac- biochemical risk factors (i.e., blood count were identical or had a minor deviation of ± ity. DND1 KO lines exhibited pluripotency and leucocyte formula, C- Reactive Protein 5 spermatozoa, while in 12.9% of the sam- gene expression, and kept their ability to (CRP), procalcitonin (PCT), Lactic Dehydro- ples less and 13.4% more spermatozoa were differentiate towards all three germ layers in genase (LDH), Ferritin, D-Dimer, Fibrino- found at point of ICSI. Moreover, if no sperm vitro. During PGCLC induction, we derived DGA-Abstracts gen, Interleukin 6 (IL-6)) as well as total were found initially this was confirmed in reduced numbers of PGCLCs with varying testosterone (TT), calculated free T (cFT), 93.1% (268/288) of the samples. TS with 1–4 reduction depending on the hiPSCs line. KO Sex Hormone Binding Globulin (SHBG), and spermatozoa had a 27.2% chance to result in PGCLCs are currently analyzed for differ- Luteinizing Hormone (LH) were determined. a complete absence of spermatozoa. ences in gene and protein expression using Results Lower TT and cFT were found Conclusion A small TS analysed on the RNAseq and mass spectrometry to elucidate in the transferred to ICU/deceased in RICU day of TESE reliably reflects the probability pathways affected by DND1 KO during group vs. groups of patients transferred to IM of finding comparable numbers of sperm at ­hiPSCs differentiation. or maintained in the RICU in stable condi- time of ICSI. However, if only 1-4 sperm are tion. Both TT and cFT showed a negative in the TS, there is a relevant risk that no sper- significant correlation with biochemical risk matozoa can be retrieved for TESE-ICSI and O 19 factors (i.e. the neutrophil count, LDH and the couple should be carefully advised before start of treatment. Increased sperm DNA damage PCT) but a positive association with the leads to poor embryo quality and lymphocyte count. Likewise, TT was also negatively associated with CRP and ferritin subfertility of male rats treated levels. A steep increase of both ICU transfer O 18 with methylphenidate hydrochlo­ or mortality risk was observed in men with The role of Dead end 1 in early ride in adolescence TT < 5 nmol/L or cFT < 100 pmol/L. PGC development: combining A. Gomes, S. Dia, C. Bellin, T. Rosa, M. Araújo, Conclusion Our study demonstrates for the ­human iPSC differentiation with T. Mendes, V. Vendramini first time that lower baseline levels of TT and genome editing Federal University of São Paulo, Morfologia e cFT levels predict poor prognosis and mortal- ­Genética, São Paulo, Brazil ity in SARS-CoV-2 infected men admitted to E. M. Mall1,2, N. Rotte3, J. Emich3, M. Stehling2, RICU. H. Drexler2, E. Raz4, F. Tüttelmann3, S. Schlatt1, Methylphenidate hydrochloride (Ritalin®) is H. Schöler2 a psychostimulant that has been widely used 1University Hospital Münster, Centre of Reproductive in the treatment of Attention Deficit Hyper- O 17 Medicine and Andrology, Münster, Germany;2Max activity Disorder (ADHD). Although the Planck Institute for Molecular Biomedicine, Münster, treatments are able either to alleviate or extin- Does the sperm retrieval rate Germany;3Institute of Reproductive Genetics, Müns- guish the symptoms, the disorder is chronic (SRR) result of a small test ter, Germany;4Institute of Cell Biology, Münster, Ger- and has a 60% chance of accompanying the ­sample of each single mTESE many individual throughout life. Currently, little (microsurgical testicular sperm is known about the possible side effects of Primordial germ cells (PGCs) are the earli- methylphenidate (MPH) on male fertility and extraction) sample reliably predict est progenitors of the mammalian germline the impact of post-treatment paternal inherit- the SRR on the day of ICSI? and thus the requisite cells for transmitting ance. The present study aimed to investigate genetic and epigenetic information from one S. Kappes, F. Macke, S. Kliesch, V. Nordhoff whether MPH poses a risk to the reproductive generation to the next. Several differentia- Centre of Reproductive Medicine and Andrology, capacity and/or quality of embryos conceived tion protocols have been established to derive Münster, Germany after treatment during the adolescence of rats. early PGC-like cells (PGCLCs) from human Twenty four Wistar rats at 38 days of age Introduction The combination of mTESE induced pluripotent stem cells (hiPSCs). were treated with distilled water, by gavage, and intracytoplasmic sperm injection (ICSI) These cells resemble migrating PGCs in in daily doses using an adequate volume for has become the standard treatment of patients terms of epigenetic and transcriptomic pro- the animal weight (control group: n = 12); with azoospermia. For optimal management, files and represent the best available model to or 5 mg/kg body weight of MPH, in a single we developed a stepwise approach for analy- mimic early human developmental processes daily dose, via gavage (treated group: n = 12). sis of tissue samples. At day of surgery, a in vitro. Both groups were treated for 30 days. From small piece of each of the eight TESE sam- This study aimed to combine PGCLC dif- the 100 days of age the animals were present- ples per testis (test sample, TS) is analysed ferentiation with gene editing to elucidate ed to cohabitation with untreated females. for sperm occurrence. The outcome of the TS the role of genes associated with male Pregnant females were divided into 2 groups, may help to assess the chances for ICSI and to infertility during early germ cell develop- according to the stage of embryonic develop- decide how many samples have to be thawed ment. Within our clinical research unit, both ment: group E3 (2.5 dpc) and E20 (20 dpc). at time of ICSI. Here we evaluate, if the re- clinical and genomic data of infertile patients The effects of the paternal inheritance on the trieved sperm numbers of the TS correspond are available. Currently, more than 1000 embryonic development in the pre- and post- to the number of sperm in the cryopreserved exome datasets are available, mainly of in- implantation phases were analyzed. After the specimen later on the day of TESE-ICSI. fertile azoospermic men. Several interesting statistical analysis, the results showed that the Methods Of each TESE sample, about novel candidate genes have been prioritized group of males treated with MPH during ado- 1/10 is removed and digested immediately through multiple levels of in silico evidence. lescence presented alterations in most of the with collagenase for pre-analysis. The result- Prospectively, these candidate genes will be parameters of paternal inheritance analyzed,

24 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts namely: number of embryos containing 2-3 O 21 Results The majority of SCD patients symmetric blastomeres at 2.5 dpc, level of (n = 15) had S/T values below the reference embryo fragmentation, number of live pups Hydroxyurea is not the major values of healthy boys and therefore, a Z- at 20 dpc, number of implantations at 20 dpc cause for the depletion of germ score below –2. One patient scored above and rate of implantation at 20 dpc. Thus, it cells in immature testicular tis­ the reference values (Z-score above 2), and is concluded that the subchronic use of MPH sues of patients with sickle cell the remaining seven patients had spermato- during male pre-pubertal phase may cause gonial counts within the normal range. No reduction of reproductive capacity and de- disease correlation was observed between HU dose crease in embryo quality, probably due to the K. Benninghoven-Frey1, H. A. M. Ba Omar2, (p = 0,936), exposure time (p = 0,816), and production of irreparable genomic damage A. Jarisch3, S. Kliesch1, C. Krallmann1, age at treatment initiation (p = 0,051). 4,5 6 1 that took place during spermatogenesis. A. K. Lahtinen , C. Langenskiöld , V. Nordhoff , Conclusion The results suggest that HU J. Portela7, S. Schlatt1, M. Sundin8,9, therapy is not the major cause of the reduction J. B. Stukenborg2, K. Jahnukainen2,10, N. Neuhaus1 of spermatogonia in SCD patients. Therefore, O 20 1 University of Münster, Centre of Reproductive Medi- other factors intrinsic to the disease, such as Both comorbidity burden and low cine and Andrology, Institute of Reproductive and the disease severity or a prenatal influence of testosterone can explain symp­ Regenerative Biology, Münster, Germany; 2Karolins- SCD on the testis need to be considered. toms and signs of testosterone ka Institute and University Hospital, NORDFERTIL

Research Lab Stockholm, Department of Women’s DGA-Abstracts deficiency in men consulting for and Children´s Health, Stockholm, Sweden; 3Univer- O 22 sexual dysfunction sity Hospital Frankfurt, Johann Wolfgang Goethe University, Division of Stem Cell Transplantation and Safety of spermatogonial stem G. Rastrelli, G. Corona, M. Maggi cell transplantation in mice: University of Florence, Experimental and Clinical Bio- Immunology, Department of Children and Adolescent 4 medical Sciences, Florence, Italy Medicine, Frankfurt am Main, Germany; University health at different life stages of Helsinki, Faculty of Medicine, Applied Tumor across two generations in a Introduction Low testosterone (T) is fre- ­Genomics Research Program, Helsinki, Finland ­blinded longitudinal study quent in men with chronical illnesses. The 5University of Helsinki, Faculty of Medicine, Depart- clinical features of T deficiency (TD) overlap ment of Medical and Clinical Genetics / Medicum, J. B. Serrano1, R. van Eekelen1, C. M. de Winter-­ with those of chronic diseases. The aim of Helsinki, Finland; 6The Queen Silvia Children’s Korver1, S. K. M. van Daalen1, N. C. Tabeling1, this study is to evaluate the relative contribu- ­Hospital, Department of Paediatric Oncology, M. J. J. Gijbels2,3, C. L. Mulder1, A. M. M. van Pelt1 tion of chronic disease score (CDS) and low Gothenburg,­ Sweden; 7University of Amsterdam, 1Amsterdam UMC, Center for Reproductive Medi­ T to the presence of TD symptoms. Amsterdam UMC, Center of Reproductive Medicine, cine, Amsterdam, Netherlands; 2Amsterdam UMC, Methods A consecutive series of 3862 Research Institute Reproduction and Development, Department of Medical Biochemistry, Amsterdam, 3 men (aged 52.1 ± 13.1 years) consulting Amsterdam, Netherlands; 8Karolinska Institute, Divi- Netherlands; Maastricht University, Department of for sexual dysfunction was studied. Several sion of Paediatrics, Department of Clinical Science, Pathology, Maastricht, Netherlands clinical and biochemical parameters were Intervention and Technology, Stockholm, Sweden; Introduction Spermatogonial stem cell 9 collected, including the structured interview Karolinska University Hospital, Astrid Lindgren Chil- (SSC) in vitro propagation followed by ANDROTEST, for the assessment of TD dren’s Hospital, Section of Haematology, Immunolo- auto­transplantation (SSCT) is proposed as a 10 symptoms. Penile color Doppler ultrasound gy and HCT, Stockholm, Sweden; University of Hel- restorative fertility treatment for childhood (PCDU) was also performed. Based on the sinki and Helsinki University Hospital, New cancer survivors. Limited data is available medications taken, the CDS was calculated. Children’s Hospital, Paediatric Research Centre, on the impact of SSCT on the health of the For a subset of 1687 men, information on Helsinki,­ Finland offspring. mortality was collected (follow-up of 4.3 ± Introduction Cryopreservation of imma- Materials and Methods Neonatal mice 2.6 years). ture testicular tissues is explored worldwide SSCs (DBA/2J) were cultured and trans- Results Higher CDS was associated with for prepubertal boys as a fertility preservation planted into sterile males (W/W-v). Control lower free and total T (TT) as well as with strategy. Sickle cell disease (SCD) patients (DBA/2J) and transplanted (W/W-v) males higher ANDROTEST score. When introduc- constitute a growing cohort in cryopreserva- were placed in breeding with control females ing CDS and TT in multivariable models ad- tion programs and are usually treated with (DBA/2J) and bred for 2 generations (n=153 justed for age, severe erectile dysfunction and hydroxyurea (HU). This drug is suspected to control, n=126 F1 SSCT, n=117 F2 SSCT) to impaired morning were associated damage germ cells, including spermatogonia, assess health in a full-blinded study. At birth, with both CDS (odds ratio [OR] = 1.25 [1.13; which form the basis for spermatogenesis fol- the pups were checked for congenital abnor- 1.37] and 1.38 [1.29; 1.48], respectively) and lowing puberty. The aim of this study was to malities. During the first 28 days, weight, low TT (OR = 1.11 [1.00; 1.23] and 1.13 evaluate whether the number of spermatogo- length, ear-opening, eye-opening, fur growth [1.06; 1.21], respectively). Similar results nia in SCD patients was impaired and wheth- and incisor eruption were checked, along were obtained for PCDU parameters. Hypo- er this correlated with HU treatment. with behavioral reflex testing of negative geo- active sexual desire was associated with low Patients and Methods We included 23 taxis, grasp and righting reflexes. Fertility of TT (OR = 1.21 [1.13; 1.30]), whereas it was SCD patients (age range: 2–15 years) which the F1 animals was assessed by their ability inversely related with CDS (OR = 0.91[0.84; are part of the cryopreservation programs in to generate F2 and histological confirmation. 0.97]). When considering mortality for major Germany, Androprotect (n = 17), and in the The welfare and survival of the F1 animals cardiovascular events, TT < 8 nmol/L, but not Nordic countries, Nordfertil (n = 6). Two were followed up to 18 months. CDS, was a significant predictor (hazard ratio independent testicular tissue sections were Results At birth and during childhood, no = 5.57 [1.51; 20.63]). stained with the germ cell-specific marker significant differences were found between Conclusions Chronic illnesses are associ- MAGE A4 to identify spermatogonia in control and SSCT offspring, for the majority ated with an overt TD. Both chronic diseases round tubules. An age-independent Z-score of physical and behavioral tests. Congeni- and low T can be involved in determining was calculated by comparing the numbers of tal abnormalities were rare events (n = 8), symptoms present in subjects complaining spermatogonia per round tubule (S/T) of SCD with no statistically significant differences for sexual dysfunction. This should be con- patients to published values of healthy boys between groups in either generation (OR: sidered in the diagnostic workup for TD. (n = 309, age range: 0–18 years). Moreover, 3.57 (95%CI: [0.13; 293.33]) for F1 and clinical parameters were collected, including 4.19 (95%CI: [0.60–82.95]) for F2). During HU dose, exposure time and age at treatment the first 28 days, SSCT F2 weight was lower initiation. Considering all the clinical para­ than control (95%CI: [–0.42; –0.09]), while meters and spermatogonial numbers, a Spear- the eruption of their upper incisor occurred man correlation was performed. 1 day earlier (95% CI [–2.161; –0.167]). All

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 25 Andrology 2020 – Abstracts

animals were fertile and life expectancy was evaluate the in vitro effects of most com- Devices were fabricated from PDMS (poly- similar between groups (p = 0.22). mon bacteria of the male genital tract (MGT) dimethylsiloxane) using soft-lithography and Conclusion The general health and devel- (Proteus m., Pseudomonas a., E. coli (25922 a 3D-printed mold, and bonded to thin glass opment of SSCT derived pups during birth, – 35218), Klebsiella p. (700608 – 13833), slides to facilitate optical visualization of the childhood and adulthood were similar to con- E. cloacae, E. aerogens) on sperm functions. samples during culture. To preserve/activate trol pups, in both generations. Thus, our pre- Methods Semen samples were divided in the cellular and clonal arrangements of the clinical study takes an important step towards equal aliquots and incubated with bacterial tubular sub-compartments, intact marmoset clinical translation of SSCT and improving strains (at a concentration ten times greater seminiferous tubules from prepubertal mar- quality of life in prepubertal male cancer sur- than spermatozoa) for 1 and 3 hours at 37° C, moset monkeys, and adult human seminifer- ous tubules (sourced from gender dysphoria vivors. 5%CO2. Sperm motility, viability, oxidative stress (by MitoSOX™), caspase 3 and 7 ac- patients) were loaded in a confined culture tivity (by FLICA™) and DNA fragmentation chamber to promote a higher concentration O 23 (by Tunel) were evaluated. of autocrine secretions around the tubules. Culture was maintained for 9–12 days at Uropathogenic Escherichia coli in­ Results All bacteria, except Pseudomonas 35 ˚C under perfusion of fresh medium from duced infiltration of immune cells a., reduced significantly sperm progressive side channels, in a shear-free manner and in and total motility (n = 16). Sperm viability stimulatory (supplemented with FSH, LH) disturbs testicular function was affected only by Proteus m. and E. coli

DGA-Abstracts and non-stimulatory culture conditions. On- M. Wang, C. Pleuger, M. Fijak, A. Meinhardt, 35218 which determined also an increase chip live imaging, off-chip live-dead cell as- S. Bhushan of sperm oxidative stress, apoptosis and says and histological analysis demonstrated Institute of Anatomy and Cell Biology, Department of DNA fragmentation. To explain the possible viability of tissues, preserved structural in- Reproduction Biology, Giessen, Germany mechanism responsible for decrease of motil- tegrity in prepubertal marmosets and irregu- ity, spermatozoa were incubated with E. coli The testis is an immune-compromised or- lar maintenance of cultured human tubules, knockout for genes of Sperm Immobilizing gan, however, infection and inflammation mostly due to the variation in initial matura- Factor (SIF) and with the corresponding wild could lead to diminished fertility. Immuno- tion status of the patient testicular samples. type (MG1655 strand). After incubation with logical and infectious infertility are major Testosterone measurements by ELISA assay E. Coli wt, a reduction in sperm motility, but contributors (13–15%) to male infertility. demonstrated higher testosterone readouts in not in sperm viability, was observed, whereas Macrophages are important effector cells of stimulated culture samples, compared to non- incubation with KO did not affect both sperm the innate immune system and play a criti- supplemented culture samples. Our testes-on- parameters. The sequence analysis of align- cal role in host defense. They also contribute chip model can be further used to understand ment for SIF coding genes (respect to E. coli to the maintenance of tissue homeostasis and spermatogenesis and endocrine regulation in MG1655) showed the presence of homolo- promote inflammation resolution and tissue primates under stimulatory conditions. gous genes in all bacterial isolates (range of repair. In a mouse model of acute bacterial identity: 76–99%) except Pseudomonas a. testicular inflammation, we demonstrated a massive infiltration of immune cells such as Conclusions Our results demonstrated that neutrophils (Ly6G+), monocytes (Ly6Chi bacteria of MGT have a detrimental effect on O 26 MHC IIhi), and inflammatory macrophages sperm motility, likely due to the presence of An invertebrate in vivo platform (F4/80lo CD11bhi) into the interstitial space SIF. Loss of viability, increase of oxidative for the high-throughput analysis stress, apoptosis and DNA damage are likely of the testes after 1, 3, 5, 7, and 10 days of in- of evolutionarily-conserved sper­ fection with uropathogenic Escherichia Coli mediated by other bacterial factors requiring (UPEC). The infiltration of immune cells re- further investigation. Overall, our data sug- matogenesis genes sulted in the disturbance of spermatogenesis gest that bacterial semen infection may nega- J. Almeida1,2, R. Bratting Correia2,3, C. Shekhar with a concomitant significant reduction in the tively influence fertility status. Misra­ 2,4, J. D. Becker2,4, P. Navarro-Costa1,2 weight of the infected testes in comparison to 1Faculty of Medicine, University of Lisbon, ISAMB - control from 5 days of infection. Importantly, Institute of Environmental Health, Lisbon, Portugal; in Ccr2 deficient mice, which lack peripheral O 25 2Instituto Gulbenkian de Ciência, Oeiras, Portugal; blood monocytes due to a defective egress of Developing testes-on-chip model 3CAPES Foundation, Brasília, Brazil; 4Universidade Ly6Chi monocytes from the bone marrow, to study in vitro primate sperma­ Nova de Lisboa, Instituto de Tecnologia Química e the number of blood monocytes/inflamma- Biológica, Oeiras, Portugal tory macrophages were reduced and disease togenesis and endocrine dyna­ progression was attenuated in the testis after mics Introduction Recent advances in genome sequencing techniques have led to an in- 7 days of infection with UPEC. These find- 1 2 1 S. Sharma , B. Venzac , R. Sandhowe-Klaverkamp , creasingly higher number of genes being ings suggest that blood monocytes/inflamma- 2 1 S. Le Gac , S. Schlatt tentatively linked to spermatogenic func- tory macrophages impair testicular function 1 Centrum für Reproduktionsmedizin und Andrologie, tion. Nevertheless, generating the functional under inflammatory conditions which may Münster, Germany; 2Applied Microfluidics for lead to reduced fertility. data to confirm these associations remains BioEngineering Research, MESA+ Institute for Nano- costly and time-consuming. In this study we technology and TechMed Centre, University of tested the feasibility of using a simple inver- ­Twente, Enschede, Netherlands O 24 tebrate organism (the fruit fly Drosophila Testes evolved as a bi-functional and multi- ­melanogaster) as a fast, cost-effective screen- Effect of bacteria, present in the compartmental organ, functionally regu- ing tool to identify new spermatogenesis male genital tract, on human lated by the HPG axis-via gonadotropins. genes. sperm function: an in vitro study Conventional in vitro culture approaches Methods We identified 908 evolutionar- like gas-liquid interphase systems, failed to ily-conserved genes whose expression is S. Marchiani, G. Mattiuz, A. Antonelli, recapitulate the complex in vivo structural dynamically regulated during human male L. Tamburrino, S. Nicolò, I. Baccani, M. Muratori, physiology, endocrine regulation and stem- meiosis. We silenced each one of them in G. M. Rossolini, M. G. Torcia, E. Baldi cell niche-driven microenvironment of the fruit flies (in vivo RNAi), specifically as male University of Florence, Experimental and clinical primate testes. To address this research gap, germ cells prepare to enter meiosis (using the ­medicine, Florence, Italy we aimed to control the tissue micro-environ- bam-GAL4 driver). The reproductive fitness Introduction Microbial communities, pre­ ment to simulate in vivo-like physiological of these gene-silenced males was assessed sent in male genital tract, could impact male conditions and functional organization of the by four independent fertility tests, with those fertility status. However, such issue is little primate testes by using an organ-on-a-chip having average fertility rates below 75% be- investigated. The aim of the study was to approach to develop testes-on-chip models. ing selected for subsequent testicular pheno-

26 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts type characterization via differential interfer- the influence of activin A on the production ­Münster, Münster, Germany;3Centre of Reproductive ence contrast microscopy. Phenotypes were of fibronectin and MMPs. BMDMs cultured Medicine and Andrology, University Hospital Müns- ascribed to four main classes (pre-meiotic, in the presence of 50ng/ml activin A or with ter, Münster, Germany meiotic, post-meiotic or gametic defects) conditioned medium from cultured Sertoli The sperm-specific phospho- and results were integrated with all publicly- cells (SCCM) stimulated with TNF were ana- Introduction lipase C (PLC ) plays an important role available information on the human and lyzed by qRT-PCR and zymography. Prelimi- ζ ζ during fertilization. Upon sperm-oocyte fu- mouse orthologs of these genes. nary results show TNF-stimulated SCCM in- sion, PLC is released from the sperm head duced fibronectin mRNA in BMDMs (n = 3). ζ Results Out of the 908 genes analysed, we into the oocyte. PLC -induced repetitive Moreover, activin A increased MMP-2 and ζ identified 244 that are required for male fertil- increases of the intracellular Ca2+ concentra- MMP-14 mRNA levels with a concomitant ity, 177 of which have never been previously tion orchestrate the activation and develop- decrease of MMP-9 and TIMP1 in BMDMs associated with spermatogenetic functions in ment of the oocyte. Patients with reduced or (n = 8). Activin A also upregulated the enzy- any of the three species. Most of the recorded lacking PLC activity suffer from sub- or matic activity of MMP-2, and downregulated ζ phenotypes were at the post-meiotic stage infertility, also involving fertilization failure MMP-9 activity (n = 5). Follistatin, a potent (113/244), suggesting a link between meiotic upon ICSI. However, PLC activity is cur- activin A antagonist, inhibited these effects. ζ expression and later functions in spermio­ rently not routinely assessed in sperm from Taken together, these data indicate that CCR2 genesis. Of note, we found 32 cases where patients undergoing assisted reproduction. the fruit fly phenotype recapitulates the previ- and activin A may interact to regulate fibrosis Patients and Methods We developed a ously reported infertility of the corresponding during testicular inflammation. DGA-Abstracts fluorescence-based assay to quantify the PLC mammalian ortholog. Of these, 20 represent activity in human sperm samples. Further- new invertebrate models of known causes of more, we analysed the prevalence of altera- human and mouse infertility. O 28 Reconstitution of prospermatogo­ tions in the PLCZ1 gene in 27 selected pa- Conclusion Our data support Drosophila tients with a low fertilization rate in ICSI via melanogaster as a simple, high-throughput nial specification in vitro from hu­ PCR and Sanger Sequencing. ancillary tool to accelerate research in genet- man induced pluripotent stem Using the PLC assay, we quanti- ics of spermatogenesis. Results cells fied PLC activity in sperm samples from Y. S. Hwang1,2, S. Suzuki3, Y. Seita2, J. Ito2,4, healthy donors under different experimental O 27 K. Sato2,4, B. Hermann2,4,5, K. Sasaki1 conditions, and we compared PLC activity Loss of CCR2 inhibits the develop­ 1University of Pennsylvania, Department of Patho­ between sperm from healthy donors and pa- tients lacking PLC ζ activity. This revealed ment of testicular fibrosis – a pos­ logy and Laboratory Medicine, Philadelphia, PA, United­ States; 2University of Pennsylvania, Phila­ that the assay detects the global PLC activity sible role for activin A delphia, PA, United States; 3University of Texas in sperm, which involves several PLC iso- W. Peng1, A. C. Kauerhof1,2, E. Wahle1, C. Pleuger1, San ­Antonio, Biology, San Antonio, United States; forms including PLC ζ. Moreover, the genetic S. Bhushan1, K. Loveland3,2, M. Wygrecka4, A. Mein- 4University of Tokyo, Tokyo, Japan; 5University of screening identified two patients with novel hardt1,3, M. Hedger3,2, M. Fijak1 ­Texas San Antonio, San Antonio, TX, United States PLCZ1 mutations that are currently analysed 1 in more detail. Justus-Liebig-University of Giessen, Institute of Establishment of spermatogonia throughout We describe a fluorescence- Anatomy and Cell Biology, Giessen, Germany; the fetal and postnatal period is essential for Conclusion 2 based assay to measure PLC activity in sperm Monash University, Department of Molecular & production of spermatozoa and male fertility. 3 as well as of recombinant PLC protein. This Translational Sciences, Clayton, Australia; Hudson Here, we established a protocol for in vitro assay may help to better understand the func- Institute of Medical Research, Centre for Reproduc­ reconstitution of human prospermatogo- 4 tional impact of mutations in the PLCZ1 tive Health, Clayton, Australia; Universities of Gies- nial specification whereby human primordial gene. sen and Marburg Lung Center, Department of Bio- germ cell (PGC)-like cells (hPGCLCs) dif- chemistry, Giessen, Germany ferentiated from human induced pluripotent Experimental autoimmune orchitis (EAO) is stem cells were further induced into M- O 30 a mouse model of chronic testicular inflam- prospermatogonia-like cells (MLCs) and T1 Transcriptomic differences be­ mation that features fibrosis, an important prospermatogonia-like cells (T1LCs) using characteristic of testicular disturbances in long-term cultured xenogeneic reconstituted tween fibrotic and non-fibrotic men. Pro-inflammatory and pro-fibrotic me- testes. Single cell RNA-sequencing was used testicular tissue diators, such as TNF, CCR2 (MCP-1) and ac- to delineate the lineage trajectory leading M. Willems1, C. Olsen2, D. Croes2, B. Caljon2, tivin A, are increased during EAO followed to T1LCs, which closely resemble human J. De Schepper1,3, H. Tournaye1,4, E. Goossens1, by the infiltration of leukocytes into the tes- T1-prospermatogonia in vivo and exhibited D. Van Saen1 ticular interstitium. Sertoli cells produce ac- gene expression related to spermatogenesis 1Vrije Universiteit Brussel, Biology of the testis tivin A upon stimulation with inflammatory and diminished proliferation, a hallmark of (BITE), Jette, Belgium; 2UZ Brussel, BrightCore, factors, including TNF. Recruited fibrocytes ­quiescent T1 prospermatogonia. Notably, this Brussel,­ Belgium; 3UZ Brussel, Pediatrics depart- expressing hematopoietic markers and extra- system enabled us to visualize the dynamic ment, Brussel, Belgium;4UZ Brussel, Center for re- cellular matrix proteins as well as dysregu- and stage-specific regulation of transposable productive medicine, Brussel, Belgium lated expression of matrix metalloproteases elements during human prospermatogonial (MMPs) contribute to fibrogenesis in many specification. Together, our findings pave the Introduction Klinefelter syndrome (KS; organs. In this study, the interaction of the way for understanding and reconstructing hu- 47,XXY) affects 1–2 in 1000 newborn CCR2 axis and activin A on the development man male germline development in vitro. males. Most (95%) KS men suffer from of testicular fibrosis was investigated. EAO azoospermia due to a loss of spermatogonial was induced by active immunization with stem cells. Aditionally, testicular fibrosis is testicular antigens in WT and CCR2-/- mice O 29 detected from puberty onwards. However, (n=5-8/group). Organ damage due to EAO Development of an assay to mechanisms responsible for fibrosis and germ cell loss remain unknown. The aim of and collagen deposition detected by Sirius ­measure PLC ζ activity in human red staining were reduced in CCR2-/- testes. this study is to identify factors which may be Flow cytometry indicated that the induction sperm involved in the fibrotic remodeling of KS tes- by EAO of CD45+ fibrocytes expressing col- J. Rips1, C. Friedrich2, V. Nordhoff3, S. Kliesch3, tes by analyzing the transcriptome of fibrotic lagen I was also reduced in the CCR2-/- testes F. Tüttelmann2, T. Strünker1, C. Brenker1 testicular tissue. compared with WT testes. Bone marrow-de- 1Centre of Reproductive Medicine and Andrology, Patients and Methods RNA sequencing rived macrophages (BMDMs) were used as University of Münster, Münster, Germany; 2Institute was performed to compare the genetic pro- a surrogate of testicular macrophages to test of Reproductive Genetics, University Hospital file of testicular biopsies from patients with

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 27 Andrology 2020 – Abstracts

(KS and testis atrophy patients) and without fewer germ cells at birth, a higher proportion resulted to be differentially expressed be- (Sertoli cell only patients and fertile controls) of cells were GFRA1+ and Ki67+, indicating tween the patient groups. In conclusion, we testicular fibrosis. Five testicular samples that their development was advanced in fetal found that impaired spermatogenesis is as- were included in each group. Differentially stages by high activin A. By P3, these param- sociated with DNA methylation changes in expressed genes (DEGs) were considered eters did not differ, suggesting overexpres- testicular germ cells at functionally relevant significant when p2. To gain insight in the po- sion of activin A doesn’t interfere with the regions of the genome, which points to an tential functions of the DETs, gene-ontology capacity of surviving germ cells to develop important role of DNA methylation in nor- and KEGG analyses were performed. further. Altered levels of transcripts associ- mal spermatogenesis. We hypothesize that Results A total of 167 upregulated and ated with both SSCs (Eomes, Etv5, Bcl6b) the described DNA methylation changes may 567 downregulated DEGs were identified and differentiated spermatogonia (Stra8, Kit) reflect or contribute to premature abortion of between the two groups. Gene ontology in P0 Inha KO testes were consistent with spermatogenesis and therefore not appear in analysis revealed that the top downregulated the hypothesis of accelerated germ cell de- the mature, motile sperm. biological functions mostly included func- velopment prior to birth, and also indicated tions related to spermatogenesis and fertili- altered retinoic acid signalling activity. Our zation. In the top 10 upregulated biological observation that systemic activin A elevation O 33 modulates the pace of germ cell development functions, we could find DEGs involved in Prediabetes induces transgenera­ the extracellular structure organization, in- in mice prior to birth suggests that pregnancy

DGA-Abstracts cluding decorin, lumican and vascular cell conditions with elevated activin A may affect tional effects in testicular metabo­ adhesion molecule 1 (VCAM1). In addition, male reproductive health by influencing the lome and sperm quality: a silent the upregulation of delta like non-canonical male germline. comorbidity? notch ligand 1 (DLK1) was found. L. Crisóstomo1, R. A. Videira1,2, I. Jarak1,2, Conclusion We compared DETs between O 32 K. Starcˇevic´3, T. Mašek4, L. P. Rato2, J. F. Raposo5,6, fibrotic and non-fibrotic testis tissue to find 7 8 1 Whole-genome methylation ana­ R. L. Batterham , P. F. Oliveira , M. G. Alves genetic components related to the initiation 1Institute for Biomedical Sciences Abel Salazar, Uni- of testicular fibrosis. Several genes were lysis of testicular germ cells from versity of Porto, Department of Microscopy and Unit found upregulated, including DLK1, which cryptozoospermic men points to for Multidisciplinary Research in Biomedicine, Porto, was also found in previous KS-related gene recurrent and functionally rele­ Portugal;2Faculty of Health Sciences, University of expression studies. Furthermore, VCAM1, vant DNA methylation changes Beira Interior, Covilhã, Portugal;3Faculty of Veterinary which plays a role in the inflammation path- 1 2 3 3 Medicine, University of Zagreb, Department of Che- way, as well as other genes involved in the S. Di Persio , E. Leitão , M. Wöste , T. Tekath , 4 4 3 5 mistry and Biochemistry, Zagreb, Croatia; Faculty of extracellular matrix composition were found J. F. Cremers , M. Dugas , X. Li , G. Meyer zu Veterinary Medicine, University of Zagreb, Depart- differentially expressed between the two Hörste­ 5, S. Kliesch4, S. Laurentino1, B. Horsthemke2, 1 ment of Animal Nutrition and Dietetics, Zagreb, groups. N. Neuhaus 5 1 Croatia; NOVA Medical School – New University Lis- Centre of Reproductive Medicine and Andrology, 6 2 bon, Lisbon, Portugal; APDP – Diabetes Portugal, Münster, Germany; Institute of Human Genetics, 7 3 Lisbon, Portugal; University College London, Centre Essen,­ Germany; Institute of Medical Informatics, for Obesity Research, Rayne Institute; Centre for O 31 4 Münster, Germany; Centre of Reproductive Medicine Weight Management and Metabolic Surgery & Na- Activin A modulates the pace of and Andrology, Department of Clinical and Surgical 5 tional Institute of Health Research, London, United germ cell development at the on­ Andrology, Münster, Germany; Institute of Trans­ Kingdom;8University of Aveiro, QOPNA & LAQV, De- set of spermatogenesis lational Neurology, Department of Neurology, Müns- partment of Chemistry, Aveiro, Portugal ter, Germany P. Whiley1, M. Luu1, R. Hobbs1, K. Loveland1,2 Adoption of fat-rich diets, often starting at an 1Hudson Institute of Medical Research, Centre for In the last 15 years several studies have de- early age, is a main cause for “fat epidemic” Reproductive Health (CRH), Clayton, Australia; scribed a prevalence of DNA methylation and related comorbidities. Several studies 2Monash University, Department of Molecular and changes in sperm of infertile men. However, have reported a decrease in sperm quality and Translational Sciences, School of Clinical Sciences, more recently using whole genome bisulfite correlate it with the onset of metabolic dis- Clayton, Australia sequencing we were able to refute this theory eases. Recently, evidence has emerged about by demonstrating that somatic DNA con- the intergenerational (sons) and transgen- Male infertility and testicular cancer are tamination and genetic variation confound erational (grandsons) effects of ancestral thought to result from disruptions to testis methylation studies in sperm of severely oli- paternal fat-rich diet and metabolic status in development in utero. Physiological pertur- gozoospermic men. As we cannot exclude the sperm parameters. Hereby, we postulate that bations during human pregnancy can feature existence of testicular germ cells (TGCs) car- a prediabetic state induced by a fat-rich diet is high activin A levels, and this study investi- rying aberrant DNA methylation in such pa- related to sperm defects and testicular meta- gates how elevated activin affects the initial tients, we compared the TGC methylomes of bolic remodeling, for several generations. stages of sperm formation. In fetal mouse tes- four men with cryptozoospermia (CZ) and of tes, activin A affects Sertoli cell function and four men with obstructive azoospermia, who Our methodology is schematically described local steroid production. After birth, germ served as controls (CTR). By analyzing the in Figure 2. cells resume proliferation and transform into methylation levels at the imprinted regions, The consumption of fat-rich diets in F0 in- differentiating spermatogonia which initiate we confirmed that the samples were free of duces a prediabetic state that is reversed by the first round of spermatogenesis, or into the somatic cell DNA. Although there was no dif- dietary intervention. However, both mice fed spermatogonial stem cells (SSCs) vital to on- ference in the global DNA methylation level, with fat-rich diet (FR) and mice subjected to going spermatogenesis in adults. We studied we detected 271 differentially methylated re- diet intervention (DI) presented more sperm a mouse model with elevated activin A bioac- gions (DMRs) between the two groups, which defects and lower sperm viability and motili- tivity (Inha KO; lacks the inhibin a subunit, a are associated with 132 genes. To evaluate­ ty than mice fed with standard chow (CTRL). potent activin inhibitor), to discern whether the relevance of these DMR associated genes Even transient consumption of a fat-rich diet this selectively affected establishment of SSC during germ cell development, we analyzed affected testicular metabolism, particularly or spermatogonia initiating the first spermato­ single cell RNA sequencing datasets of hu- aminoacids metabolism, the energy-obtain- genic wave. Immunofluorescence was used man testicular germ cells from CZ and CTR ing pathways, and the lipid precursors. The to enumerate germ cells (SALL4+), nascent samples (n = 3 each) as well as two published sons (F1) of FR and DI mice have not dis- undifferentiated spermatogonia (GFRA1+), RNA-seq datasets [Guo et al., 2018; Her- played sperm defects, but testicular metabol- and proliferation (KI67+) in testis sections mann et al., 2018]. By this, we found that 65 ic profile remained abnormal. Interestingly, from Inha WT and KO mice at P0 and P3. of these genes are expressed at various stages grandsons (F2) of F0 mice fed a fat-rich diet Although Inha KO testes have significantly of spermatogenesis. Furthermore, 12 of them (both transiently and permanently), presented

28 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

O 35 Tumour infiltrating T lymphocytes in human testis cancer – identifi- cation and functional analysis R. Islam1, J. Heyer1,2, S. Indumathy1,3, K. Hartmann1, C. Pleuger4, M. Fijak4, S. Kliesch5, F. Dittmar2, F. Wagenlehner2, S. Herold6,7, M. Heiner6,7, M. Hedger3, B. Loveland8, K. Loveland3,9, H. C. Schuppe2, D. Fietz1 1Justus-Liebig-University, Institute for Veterinary Anatomy, Histology and Embryology, Giessen, Germany;­ 2Justus-Liebig-University, Dept. of Urology, Pediatric Urology and Andrology, Giessen, Germany; 3Centre for Reproductive Health, Hudson Institute for Medical Research, Clayton, Victoria, Australia; 4

Justus-Liebig-University, Institute of Anatomy and DGA-Abstracts Cell Biology, Giessen, Germany; 5University of Müns- ter, Centre of Reproductive Medicine and Andrology, Münster, Germany; 6Universities of Giessen and Marburg Lung Center (UGMLC), Department of Inter- nal Medicine II for Pulmonary and Critical Care ­Medicine and Infectious Diseases, Giessen, Ger­ many; 7German Center for Lung Research (DZL), Figure 2. L. Crisóstomo, et al. © L. Crisóstomo Giessen, Germany; 8Burnet Institute, Melbourne, Australia; 9Monash University, School of Clinical Sciences, Clayton, Victoria, Australia lower sperm counts and a higher proportion cline in Google queries for sex work (search T cells are critical to tumour development and of sperm defects than the CTRL counterparts. query: “Escort”) and dating apps (search associated immune surveillance. Different Those results were found to be correlated query: “Tinder”), and whether reversal of types of T cells, specifically, CD4+ regula- with abnormal testicular metabolic signatures such trends occurred at the end of lockdown. tory (Treg) and follicular helper T cells (Tfh) of ancestors. Materials and Methods Analysis was con- are strongly associated with poor prognosis Our findings show that a prediabetic state ducted on data from Google Trends, a tool of different cancers, but their involvement in promoted by unhealthy food intake induces to explore the relative popularity of selected human testicular germ cell tumours (TGCT) irreversible metabolic signatures in testis, search queries in selected regions over a spe- is unknown. This study aim is to identify which are related to sperm defects. Testicular cific duration. Non-parametric regression via and characterise Treg and Tfh involvement metabolome remodeling is inheritable via pa- Mann-Kendall trend test and pairwise com- in TGCT development and progression. For ternal lineage for up to grandsons, perpetuat- parisons via Wilcoxon rank sum test with this, human testis samples with seminoma ing sperm defects. Prediabetes may cause a Benjamini-Hoch correction were used to (SE; n = 10), or germ cell neoplasia in situ silent transgenerational imprinting that com- measure differences in trends before, during (GCNIS) with/without lymphocytic infil- promises male fertility potential. and after lockdown. trates (LY) (n = 10, each) were compared to samples revealing normal spermatogenesis Results Data between January 1 and Sep- (NSP; n = 10), or hypospermatogenesis with tember 4, 2020 were retrieved. All search focal infiltrates (HYP+LY; n = 9). Bouin- O 34 items showed significant trends (Pornhub: fixed paraffin-embedded specimens were p < 0.0001; Escort: p < 0.0001; Tinder: Online searches for pornography, processed for immunohistochemistry (IHC)/ p = 0.0187). Queries for Pornhub were more immunofluorescence (IF), and cryo-preserved dating apps and sex work during prevalent during lockdown than either before samples were used for RNA extraction and and after lockdown measures for or after (p < 0.0001 and p = 0.0003, respec- RT-qPCR. Furthermore, flow cytometry was COVID-19 in Italy tively), and more prevalent after than before performed on fresh TGCT samples (n = 4, in lockdown (p < 0.0001). Queries for Escort a range of 3 x 103–4.9 x 104 cells). A panel of A. Sansone1, A. Cignarelli2, G. Ciocca3, E. Colonnello1, significantly declined during lockdown antibodies against CD3, CD4, CD8, CD20cy, E. Limoncin1, D. Mollaioli1, E. A. Jannini1 (p < 0.0001) and steadily increased after CD68, CD25, FOXP3, CXCR5 and BCL6 1University of Rome Tor Vergata, Chair of Endocrino- measures were withdrawn (p < 0.0001), de- was used to detect and sort specific immune logy and Medical Sexology (ENDOSEX), Department spite being still lower than before (p < 0.012). cells. Both IHC and flow cytometry showed of Systems Medicine, Rome, Italy; 2University of Bari Queries for Tinder decreased during lock- a high abundance of T cells relative to other Aldo Moro, Department of Emergency and Organ down (p < 0.0001) and increased soon after immune cell types in SE. Flow cytometry Transplantation Section of Internal Medicine, Endo- (p < 0.0001), with no significant change be- data revealed that, besides the predominant crinology, Andrology and Metabolic Diseases, Bari, tween before and after lockdown (p = 0.15). CD4+ and CD8+ T cell subsets (36–60% and Italy;3”Sapienza” University of Rome, Department of Conclusions Interest in regards to pornog- 21–44% of CD45+ cells, resp.), Treg and Tfh Dynamic and Clinical Psychology, Rome, Italy raphy increased in Italy during lockdown, cells were detected in all tumour samples (up Introduction Lockdown and isolation and, while declining, remained high once to 0.5% of CD45+ cells), suggesting that Treg measures have been necessary to contain lockdown measures were withdrawn. Inter- and Tfh cells might play a role in TGCT bio­ spread of the SARS-CoV-2 coronavirus dis- est concerning dating apps and sex work logy. RT-qPCR confirmed that Treg and Tfh ease (COVID-19). In Italy, the most stringent declined abruptly during lockdown, and associated cytokines/chemokines are highly lockdown measures were kept from March increased once again following the end of expressed in SE. This study has demonstrated 9 to May 18, 2020. In the meantime, an restrictions. the complexity and indicated the possible im- increase in global traffic on several porno­ portance of rarer T cell subtypes in these tu- graphy websites was reported. We aimed to mours. Future experiments will use sorted T investigate whether this trend (search query: cell populations to extend our understanding “Pornhub”) was also associated with a de- of their functional roles in TGCT.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 29 Andrology 2020 – Abstracts

Grants Supported by DFG IRTG GRK genetic etio­logy is still undiscovered. Dis- sequences. Autosomal dominant and reces- 1871/2. ruption of the DNA Repair genes results in sive model have been applied to the obtained the dysfunction of the overall repair system. variants (n = 657 SNVs, 214 indels). Bioin- Germline mutations in the DNA Mismatch formatic analyses allowed the prioritization O 36 Repair (MMR) gene family lead to Lynch of the most promising variants. Whole-exome sequencing in testi­ syndrome (LS), which is a cancer (cc) prone A heterozygous frameshift deletion cular germ cell tumor: identifica­ disease. The most frequent malignancies (c.2906_2907delAT) in the MSH6 gene was associated with LS are colorectal and en- identified in patient 18-1040, whose mother tion of novel genetic factors relat­ dometrial cc, while less commonly affected ed to Lynch syndrome had endometrial cc and his grandfather had organs are hepatobiliary tract, urinary tract. pancreas cc. This variant has already been V. Rosta1, A. Riera-Escamilla2, D. Moreno-Mendoza2, Recently, TGCT has also been linked to LS. reported in association with LS. In patient E. Casamonti1, M. Gilardi1, S. Pietroforte1, Our objective was to test the diagnostic A2301, a novel heterozygous non-frameshift M. Vannucci1, C. Krausz1,2 performance of Whole Exome Sequencing deletion (c.3232_3237delTGTACT) of MLH3 1University of Florence, Andrology Department, (WES) in identifying monogenic causes of was found. In his family endometrial, Florence,­ Italy; 2Fundació Puigvert, Universitat Autò- TGCT in patients with family history of mul- and pancreas cc has been detected. noma de Barcelona, Instituto de Investigaciones Bio- tiple ccs. Our findings provide novel evidence for a ge- médicas Sant Pau (IIB-Sant Pau), Andrology Depart- DNA was extracted from peripheral blood netic link between TGCT and LS, indicating DGA-Abstracts ment, Barcelona, Spain lymphocytes of two unrelated patients (18- that TGCT may be part of the LS associated Testicular Germ Cell Tumor (TGCT) is a 1040, A2301) affected by TGCT and both urological malignancies. We therefore, advise multifactorial and polygenic disease. Al- of them with two or more family members an onco-andrological screening of the male though there is strong evidence for a sig- suffering from different types of cc. WES family members of LS families and genetic nificant hereditary component of TGCT, the was carried out on the variants in the coding testing for MMR status in TGCT patients.

Poster

P 1.1 Results In STRING, a cluster of E3 ent cohorts and one consanguineous family ubiquitin ligases was discovered, includ- [Wyrwoll, et al. Am J Hum Genet 2020; 107: Loss-of-function mutations in ing TRIM71. In this gene specifically, we 342–51]. The common phenotype between TRIM71 as novel cause of early identified a heterozygous duplication in an all men was non-obstructive azoospermia germ cell depletion in men and SCO patient, leading to a frameshift and mainly associated with meiotic arrest, but mice a premature stop codon. Several missense sporadically spermatids and rarely very few variants in TRIM71 were found in other pa- spermatozoa in the semen were observed. J. Emich1, L. Torres-Fernández2, M. Wöste3, tients. TRIM71 is highly expressed in human Combined with a testis-specific mRNA S. Mitschka2, Y. Port2, C. Friedrich1, S. Kliesch4, and mouse testes and, within the adult testis, expression pattern and a knockout mouse H. Schorle5, W. Kolanus2, F. Tüttelmann1 expression is restricted to spermatogonia in model that had been previously described to 1University of Münster, Institute of Reproductive Ge- both species. Trim71 gonad-specific Nanos3- be infertile, M1AP displays a highly promis- netics, Münster, Germany; 2University of Bonn, Life conditional knockout (KO) mice of both ing candidate gene relevant for spermato- and Medical Sciences Institute, Molecular Immuno- sexes are infertile. Homozygous KO males genesis in mice and men. However, hardly logy and Cell Biology, Bonn, Germany; 3University of present with reduced testicular volumes and anything is known about its cellular role and Münster, Institute of Medical Informatics, Münster, an SCO-like phenotype in histological stain- no protein motifs or domains were identified Germany; 4University Hospital Münster, Centre of ings, while heterozygous males also exhibit a yet, making it impossible to reliably predict Reproductive Medicine and Andrology, Department significant reduction in testis size. The SCO- molecular functions. Addressing this gap, of Clinical and Surgical Andrology, Münster, Ger­ like phenotype is already apparent in new- we first aim to investigate M1AP’s subcel- many; 5University of Bonn Medical School, Institute born mice, and since female KO mice also lular localisation in relation to cellular com- of Pathology, Department of Developmental Patho­ present with drastically reduced ovary size, partments such as the plasma membrane or logy, Bonn, Germany the presence of TRIM71 may be essential in mitochondria. Heterologous transfection of early germ cell development. DYK-tagged M1AP cDNA for protein over- Introduction Infertility affects ~15% of Conclusion These results point to a poten- expression enables detection of M1AP even couples worldwide. Sertoli cell-only (SCO) in the absence of a reliable commercial an- phenotype is a subtype of azoospermia with- tial role of TRIM71 in causing early germ cell depletion in mice and men. tibody. COS-7 cells present a suitable model out any germ cells and, if universally present, for this attempt due to their large cell body. no chance to father children. Despite its sever- Grants This work was supported by the Assumed impaired protein function related to ity and suspected genetic origin, the majority DFG Clinical Research Unit 326. aberrant localisation of mutated M1AP will of causes remain unknown. Candidate gene be identified by generating patient-specific approaches have been largely unsuccessful, variants via site-directed mutagenesis. With substantiating the need for more systematic this approach, we were already able to de- approaches, e.g. whole exome sequencing P 1.2 scribe protein truncation for the frameshift (WES). variant c.676dup (p.Trp226LeufsTer4), Patients and Methods We applied bio- Characterisation of the infertility- lacking 57% of its full-length as shown by informatic tools to WES data of 226 well- associated gene M1AP Western Blot. In perspective, this system will phenotyped SCO patients from the Male Re- be transferred to other cells simulating the N. Rotte, M. J. Wyrwoll, C. Friedrich, F. Tüttelmann productive Genomics (MERGE) study. Gene in vivo situation more closely, like TCam-2 or Institute of Reproductive Genetics, Münster, filtering was conducted using the in-house NTERA-2 cells. By in-depth characterisation Germany­ software Sciobase and a newly developed of the M1AP protein, we will contribute to data analysis tool termed Haystack, yield- We recently identified bi-allelic variants in understand its role and general mechanisms ing 706 candidate genes carrying loss-of- the gene M1AP encoding meiosis 1 arresting of meiotic processes, indisputably needed for function variants. Protein-protein interactions protein by whole exome sequencing in 14 the assessment of identified variants associ- were assessed employing STRING. individuals originating from four independ- ated with male infertility.

30 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

P 1.3 nation and sex differentiation of male genita- So, knocking out Tet1 has potential impacts lia. Several genes on the X chromosome and on Suz12 and Eed.Eed mutant mice are lethal The genetic portrait of male infer­ other autosomes have been shown to be anti- at gastrulation and Tet1 might be a potential tility: an in silico ethnic evaluation testes and have a detrimental effect on the de- regulator which leads to infertility.Analy- of haplotype distribution among velopment process of the normal male genital sis of putative TET1-binding sites and their populations system. The addition of X chromosomes to methylation status will be performed for se- the 46, XY karyotype results in seminifer- lected genes (e.g. HAND1, ESX1L) to deter- D. Santi, G. Spaggiari, L. Pagliai, M. Simoni, ous tubules dysgenesis, hypogonadism, and mine if there is a link between male infertility L. Casarini malformed genitalia. We report an infant and the interaction of TET1 and PRC2. University of Modena and Reggio Emilia, Unit of male with 49, XXXXY syndrome presenting ­Endocrinology, Department of Biomedical, Metabolic with ambiguous genitalia and multiple extra- and Neural Sciences, Modena, Italy gonadal anomalies. P 1.6 Background Male infertility is a complex Disorders in meiosis, spermato­ and multifactorial disease with an increasing P 1.5 genic cells and Sertoli cells in rats incidence worldwide. Among the possible etiopathogenetic factors, a genetic cause is Ten-eleven-translocation enzyme with experimentally induced me­ recognized in about the 15% of cases and 1 (TET1) and polycomb repressive tabolic syndrome 2 2 3 34% of them remain idiopathic, representing complex 2 (PRC2): Roles in sper­ O. Kolomiets , A. Kashintsova , Y. Korolev , DGA-Abstracts a diagnostic challenge, in which the genetic matogenesis and male fertility V. Spangenberg1, E. Bragina2 contribution is likely underestimated. The aim 1N.I. Vavilov Institute of General Genetics RAS, S. A. Shiplu1,2, H. C. Schuppe1, P. Western3, D. Fietz4, of the study was to analyse the world-wide dis- ­Moswoc, Russian Federation; 2A. N. Belozersky Re- K. Steger1,5, U. Schagdarsurengin1,2 tribution of genotypes associated with male in- search Institute of Physico-Chemical Biology MSU, 1Justus-Liebig-University Giessen, Clinic of Urology, fertility, evaluating their evolutionary conser- department of electron microscopy, Moscow, Rus­ Pediatric Urology and Andrology, Giessen, Germany; vation and selection in different populations. sian Federation; 3National Medical Research Center 2Justus-Liebig-University Giessen, Epigenetics of Methods A literature search (PubMed data­ for Rehabilitation and Balneology, Moscow, Russian Urogenital System, Giessen, Germany; 3Monash Uni- base) was performed to identify all single Federation versity, Germ Cell Development and Epigenetics, nucleotide polymorphisms (SNPs) associated Melbourne, Australia; 4Justus-Liebig-University Introduction Metabolic syndrome (MS) is with male infertility. SNPs with linkage dis- Giessen, Institute for Veterinary Anatomy, Histology characterized by the formation of a complex equilibrium (LD) value higher than 0.8 were and Embryology, Giessen, Germany; 5Justus-Liebig- of disorders among which the most charac- used as markers of a specific haplotype, re­ University Giessen, Molecular Andrology, Giessen, teristic is a combination of abdominal obe- presentative of the entire genomic region, and Germany sity, hyperglycemia. MS can cause impaired submitted to genetic analysis. Selected SNP spermatogenesis and lower sperm counts. data of individuals from 26 populations were TET1 and PRC2 regulate DNA and histone However, the details of the effect of MS on downloaded from the 1000 Genomes online methylation (H3K27me3), respectively. In spermatogenic cells remain unclear. The aim database. According to the genetic homology mice, TET1 and PRC2 dysregulations are as- of this work was to study some spermato­ calculated using SNPs associated with male sociated with male infertility. TET1 expres- genesis characteristics in rats with an experi- infertility, Bayesian clustering of individuals sion level was significantly downregulated mental MS. was performed. The genetic distance (FST) in infertile patients. We hypothesize, TET1 of each population from the Kenyan “LWK” interacts with PRC2 and promotes DNA de- Materials and Methods Eight male rats was calculated. LWK is assumed to be repre- methylation at H3K27me3-enriched genes. were divided into two groups: group I rats re- sentative of the ancestral genotype populat- We aim to clarify, where TET1 and PRC2 ceived a standart diet, group II received a diet ing African regions from which ancient hu- co-express during spermatogenesis, and co- high in glucose and fat for 60 days. Methods man migrations began. FST and geographic localize in the mature sperm genome and of immunocytochemistry and electron mi- distance were then interconnected with linear how this interaction is changed in the sperm croscopy were used. regression analyses. of subfertile men and Tet1-KO mice. Results The study of meiosis was carried Results Two hundred thirty-three SNPs re- Immunohistochemical analyses (IHC) have out in spreading nuclei of I-order spermato- ferring to 2504 sample individuals from 26 been performed for TET1, H3K27me3, core cytes (Sp) with antibodies to SCP3 – the different populations were included in the PRC2 components (EED, EZH2, SUZ12) protein of the synaptonamal complex (Sc). final database. The genetic analysis revealed on testis sections from men with obstructive Significant disturbances of the nuclear archi- that the FST calculated for 124 SNPs signifi- azoospermia and mice (wild-type wt and tectonics were found in pachyten Sp of rat cantly increased together with the geographic Tet1-KO). Western blot (WB) analyses of with MS as well as ring structure and Sc distance, suggesting to have undergone a core PRC2 components and H3K27me3 have fragmentation. In 50% of Sp defects of the natural selection during human evolution. also been done on human sperm. sexual body structure ware observed, which Discussion The study demonstrated that is manifestation of pachitene arrest resulting In humans, IHC results showed that TET1, in apoptosis [Turner et al., 2005]. SNPs associated with male infertility have H3K27me3, and EZH2 co-express in the ethnic-based distribution worldwide and spermatogonia B, leptotene The staining of the crushed cells of the natural selection impacted more than 100 and round spermatids. Compared to wt, in ­seminiferous tubules with antibodies to SNPs likely contributing to modulate male Tet1 homozygous KO mice, Tet1 showed LC3B protein, a marker of autophagosomes, reproductive functioning. no expression in germ cells. H3K27me3 ex- as well as ultrastructure research revealed pression had shifted from stage V–VI sper- many autophagosomes­ in Sertoli cells, Sp P 1.4 matogonia B to stage VII–VIII pre-leptotene and spermatids of group II rats. Electron spermatocytes, and from stage IX–XI elon- ­microscopic examination of the Sertoli cells 49, XXXXY syndrome: An infant gating to stage X–XI elongating of group II rats, in contrast to group I, re- presenting with ambiguous geni­ spermatids. EZH2 had gained expression in vealed lipid drops and numerous phagolys- talia stage VII–VIII pre-leptotene spermatocytes, osomes. in stage VI–X pachytene spermatocytes Conclusion MS can be the cause of a vio- P. Patel1, R. Nerli2, S. Ghagane2 whereas SUZ12 had shown new expression lation of meiosis at the pachytene stage. It 1Jawaharlal Nehru Medical College, Belagavi, Uro­ pattern in the late elongating spermatids and has been hypothesized that the activation of logy, Belagavi, India; 2KLES Dr. Prabhakar Kore Hos- elongated spermatids.EED lost expression autophagy is an adaptive mechanism that pital and research centre, Urology, Belagavi, India completely. However, WB analyses have removes of spermatogenic cells remnants The presence of normal genes on the Y chro- shown that PRC2 components were not pre- selected as a result of MS. Meiosis disorders mosome is essential for normal sex determi- served in the sperm except H3K27me3. and the autophagy activation may lead to a

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 31 Andrology 2020 – Abstracts

decrease in sperm count found by some re- Introduction A key enzyme of the folate ing CFTR genotypes: F508del/3849+10kbC searchers in experimental MS. metabolism is methylenetetrahydrofolate > T (n = 3), CFTRdele2,3/3849+10kbC > T reductase (MTHFR). MTHFR is required (n = 1), 3849+10kbC > T/Q493R (n = 1). for uninterrupted supply of S-adenosyl me- Pathozoospermia was found in 4 patients P 1.7 thionine for methylation reactions, replenish- (asthenoteratozoospermia, AT, n = 2; as- TREGEL – a shiny tool to identify ment of glutathione and nucleotide synthesis. thenozoospermia, A, n = 1; oligoasten- type, composition and location of Decreased activity of MTHFR enzyme can oteratozoospermia, OAT, n = 1), the patient be due to diminished expression of MTHFR. with 3849+10kbC > T/Q493R genotype gene regulatory elements Aberrant DNA methylation with oxidative had normozoospermia. Semen volume was S. Berres1, S. Laurentino1, M. Wöste2, M. Dugas2, DNA damage can decline semen quality. 2.5 ± 2.3 (0.3–6.0) ml, sperm concentra- J. Gromoll1 Yoga, a mind body intervention is gaining tion –149.2 ± 78.0 (64–243) mln/ml, total 1Centre of Reproductive Medicine and Clinical An- wide acceptance globally due to its benefits sperm count –432.5 ± 485.6 (19.2–1224) drology, Münster, Germany; 2Institute of Medical on chronic lifestyle disorders including male mln. Oligospermia was in 2 patients with ­Informatics, Münster, Germany infertility. Yoga practice lessens free radical F508del/3849+10kbC > T genotype (OAT, levels and up-regulate antioxidant defence V = 0.3 ml; AT, V = 1 ml). The fructose was Hormones can act through signal transduc- capacity of body and also improves DNA in- low in the sample with lowest volume. In- tion pathways or through modulation of tegrity. Yoga upregulates anti-inflammatory creased viscosity was found in one sample; gene expression, which can be accompa-

DGA-Abstracts & DNA repair genes and downregulates pH was in normal range (7.0–7.8). Leuko- nied by DNA methylation changes. Sertoli pro-inflammatory genes. Present study was spermia (2.0–2.2 mln/ml) was detected in 3 cells, which play a major role in supporting aimed to examine the impact of 3 weeks of samples. QKA of IGCs revealed increased % spermatogenesis, are the target of the folli- yoga based lifestyle intervention (YBLI) on (3–8.5%) of spermatocytes I at meiotic pro- cle-stimulating hormone (FSH). We have MTHFR gene expression in infertile men. phase, decreased % of spermatocytes II and analysed transcriptional changes associated increased % of degenerated IGCs. High per- with FSH stimulation of the SK11 Sertoli Materials and Methods 30 infertile men were enrolled for the prospective clinical centage of abnormal heads, 97-100% (n = 3), cell line. RNA-seq analysis revealed a total activated acrosome (n = 3), “immature” chro- of 185 genes at least 2-fold up- or downregu- trial. Supervised daily sessions were con- ducted for 2 hours per day by a certified yoga matin (n = 2), also, intra-gamete viral infec- lated (p ≤ 0.01) upon stimulation with FSH. tion (n = 2) and bacteria (n = 1) were found by We performed an analysis­ of the regulatory instructor. Blood and semen samples were analyzed pre and post intervention for the TEM. Sperm DNA fragmentation was high elements in the genome involved in these (32%) in 1 of 2 AT samples. 3849+10kbC transcriptional changes. This type of analysis expression of MTHFR gene and estimation of seminal reactive oxygen species (ROS). > T mutation is compatible with the absence is tedious and often difficult. Therefore, we of CBAVD in CF patients. The patients have developed the novel tool TREGEL (Tran- cDNA was synthesized by Agilent kit by re- verse transcribing total RNA extracted from various spermatology with no no specific scription Regulatory Elements) to search sperm abnormalities. Oligospermia and low for regulatory elements in a set of genes. It blood samples by phenol chloroform method. Expression of MTHFR in subject’s blood fructose indicate impaired seminal vesicles does so by using public available databases in some CF patients with no CBAVD. Signs (UCSC, Ensembl, JASPAR) and internal R samples were determined by quantitative real time polymerase chain reaction. of impaired prophase I of meiosis and sperm routines which calculate overlaps between ultrastructure defects were found. annotations and input genes. This tool al- Results Expression MTHFR gene was up- lowed us to identify genes containing CpG regulated with 5.517974 axis fold change sites and transcription factor binding sites after 21 days of YBLI in comparison to day- P 1.10 close to their promoters. The gene list was 0. Seminal ROS decreased significantly (p- further restricted by supplying TREGEL value < 0.05) post intervention. HDACi drugs hamper experimen­ with a list of Sertoli cell-specific transcrip- Conclusions This is the first report of up- tal mouse teratoma development tion factors. Using TREGEL, we obtained regulation of MTHFR gene following prac- J. Krasic1,2,3, L. Škara1,2,3, A. Katušic´ Bojanac1,3, F. Bu- information about type, composition and tice of yoga. This simple and easy to adopt lic Jakus1,3, D. Ježek3,4, M. Ulamec2,3,5, N. Sincˇic´1,2,3 location of regulatory elements for the re- lifestyle intervention can be integrated as an 1School of Medicine University of Zagreb, Zagreb, sulting 88 candidate genes. TREGEL also adjunct to the standard therapy in a resource Croatia; 2EpiMark, Group for Research on Epigenetic offers the option of uploading a user-curated limited set up considering the burden of infer- Biomarkers, Zagreb, Croatia; 3Centre of Excellence list of regulatory­ elements. This feature has tility and cost of treatment. for Reproductive and Regenerative Medicine, School the potential to improve current understand- of Medicine, University of Zagreb, Zagreb, Croatia; ing of how each gene is regulated. Results 4School of Medicine University of Zagreb, Depart- are visualized through an UCSC genome P 1.9 ment of Histology and Embryology, Zagreb, Croatia; browser interface and can be conveniently 5University Hospital Center Sestre milosrdnice, obtained as image and table files. With the CFTR genotypes and spermato­ ­Ljudevit Jurak Clinical Department of Pathology and help of this tool, we are now able to conduct logy in Cystic Fibrosis patients Cytology, Zagreb, Croatia a follow up analyses and validation to bet- without CBAVD ter understand Sertoli cell regulation. In the Testicular Germ Cell Tumours (TGCT) are future, we intend to share this tool, which we V. Chernykh, M. Shtaut, A. Sedova, E. Bragina, the most frequent malignancies in young believe is useful to other researchers studying S. Repina, E. Marnat, T. Sorokina, L. Kurilo males, driven by epimutations. Teratoma hormone-dependent transcriptional changes. Research Centre for Medical Genetics, Laboratory are TGCT composed of all three germ layer of Genetics of Reproduction Disorders, Moscow, tissues. Mouse teratoma model, due to its Russian Federation heterogeneity and pluripotent core, is a very P 1.8 We evaluate the semen parameters, sperm reliable in vitro model for investigating the Upregulation of MTHFR gene ex­ ultrastructure and chromatin in 5 pancreatic- effect of cancer drugs, including histone dea- pression by 3 weeks of yoga sufficient cystic fibrosis (PS-CF) patients cetylase inhibitors, on the epigenetic regula- tion of tumorigenicity. ­based lifestyle intervention in without bilateral obstruction of the vas def- erens (CBAVD), aged from 16 to 29 (22.6 After isolation, mouse cylinders were ­infertile men ± 5.0) years. Semen analysis, fructose test, treated for two hours with Trichostatin-A D. Borthakur1, M. Kumar1, H. Batra1, R. Kumar2, quantitative karyological analysis (QKA) of (TSA) and Valproate (VPA), respectively. R. Dada1 immature germ cells (IGCs) from the ejacu- Developing teratomas were cultured in vitro 1All India Institute of Medical Sciences, Anatomy, late sediment (the patent № 2328736, Russia, and measured for seven days. Proliferation New Delhi, India; 2All India Institute of Medical 2007) and transmission electronic microscopy (Ki-67) and apoptosis (Caspase-3) were Sciences, Urology, New Delhi, India (TEM) were done. The patients have follow- analysed by immunohistochemistry. Gene

32 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts expression of stemness/differentiation genes haircuts and enjoyed active sport and boxing. cancer. This finding revealed a first case of was analysed by qPCR. At present the patient is undergoing process- male infertility with inherited FANCM vari- TSA and VPA induced strong growth retarda- es to affirm a gender identity different from ant from healthy father coupled with latter tion despite unchanged proliferative or apop- previous. Chromosome analysis of peripheral cancer development. totic activity. Treatments exhibited opposite blood lymphocytes showed 46,XY karyo- Grants This study is a part of project “In- effects on gene expression compared to con- type. Despite the normal male karyotype the tegrated test for detection of genetic variants trol. TSA upregulates while VPA downregu- presence of undefined 46,XY DSD form is causing male infertility” funded by European lates stemness/differentiation genes. evidence against female-to-male transsexual- Regional Development Fund and co-support- ism. Due to the high genetic heterogeneity ed by the Center of Excellence for Repro- VPA downregulation of investigated genes and non-specific clinical features of many could be responsible for reducing teratoma ductive and Regenerative Medicine, Croatia genital abnormalities, the overlapping of phe- (grant KK.01.1.1.01.0008, “Reproductive growth, initiating senescence. TSA upregu- notypes, reaching a diagnosis is often com- lation of these genes points to a loss of ex- and Regenerative Medicine – Exploring New plicated, especially in 46,XY DSD patients. Platforms and Potentials”). pression regulation, with consequent growth Early differential diagnosis using complex retardation possibly being due to general ter- cytogenetic and molecular-genetic evaluation atoma dysregulation. This research confirms is the best strategy for management of vari- P 1.13 the effect of VPA and TSA strongly hamper- ous DSD forms. ing cancer development. These treatments Reproductive decline across gene­ DGA-Abstracts producing the same phenotypical effect of rations due to paternal MTHFR growth inhibition despite different molecu- P 1.12 lar effects and no change in proliferative deficiency and linked to demethy­ and ­mitotic activity illustrate the complex Whole exome sequencing of lation of young retrotransposons relationship between histone acetylation and ­Sertoli cell only syndrome patient G. Karahan1,2, D. Chan2, K. Shirane3, S. Janssen3, regulation of biological processes. provides novel insights on genetic M. Lorincz3, J. Trasler1,2,4,5 Grants This study was supported by the background on male infertility 1McGill University, Human Genetics, Montreal, Scientific Center of Excellence for Re- Canada;­ 2McGill University Health Centre, Research T. Maric´1,2, L. Trgovec-Greif3, M. Logara4, L. Zunic4, productive and Regenerative Medicine, Institute, Montreal, Canada; 3University of British R. Beluzic3, O. Vugrek3, M. Barbalic4,5, A. Katušic´ Republic of Croatia, and by the European Columbia, Department of Medical Genetics, Van­ ­Bojanac1,2 Union through the European Regional De- couver, Canada; 4McGill University, Department of 1School of Medicine University of Zagreb, Depart- velopment Fund, under grant agreement No. Pharma­cology and Therapeutics, Montreal, Canada; ment of Medical Biology, Zagreb, Croatia; 2School of KK.01.1.1.01.0008, project “Reproductive 5McGill University, Department of Pediatrics, Medicine University of Zagreb, Scientific Centre of and Regenerative Medicine – Exploring New Montreal,­ Canada Excellence of Reproductive and Regenerative Medi- Platforms and Potentials”. cine, Zagreb, Croatia; 3Rud¯er Boškovic´ Institute, Introduction Methylenetetrahydrofolate re­ ­Laboratory for Advanced Genomics, Zagreb, Croatia; ductase (MTHFR) is a crucial enzyme in one- 4Genom Ltd, Zagreb, Croatia; 5School of Medicine carbon metabolism with an important role in P 1.11 University of Split, Department of Medical Biology, the production of S-adenosyl methionine and A case 46,XY DSD in female Split, Croatia methyl groups for cellular processes includ- ­patient with pseudotrans­ ing DNA methylation. As mouse and human Infertility affects around 15% of couples and studies have shown that MTHFR deficiency sexualism male factor is responsible in up to 50% of cas- can impact male fertility and sperm DNA es. Genetic factor explains around 15–30% of S. Khayat1, L. Kurilo1, T. Sorokina1, N. Kibrik2, methylation, there is the potential for the cases, where some patients have well-defined V. Chernykh1,3 intergenerational passage of epimutations. abnormalities such as Klinefelter syndrome 1Research Centre for Medical Genetics, Laboratory Here, our aim was to determine whether the (XXY), deletions in AZF region or rear- of Genetics of Reproduction Disorders, Moscow, effect of MTHFR deficiency on the testis rangements in other infertility related genes Russian Federation; 2Psychiatry Moscow Research or sperm DNA methylation was similar or such as CFTR, SRY, AR, NR5A1. However, Institute, Moscow, Russian Federation; 3Pirogov Rus- exacerbat­ed from one generation to the next. vast majority of cases remains unexplained. sian National Research Medical University, Moscow, The aim of this study was to examine idio­ Materials and Methods First generation Russian Federation -/- pathic Sertoli cell only syndrome (SCOS) in (F1) C57BL/6 Mthfr male mice were bred +/- Genital abnormalities and disorders of sex infertile patient and healthy father by whole with Mthfr females to produce F2 genera- -/- development (DSD) represent rare congenital ­exome sequencing (WES) to determine tion Mthfr sons. Reproductive parameters diseases in which anatomical, chromosomal whether genetic mutations or other abnormal- were evaluated and sperm were collected or gonadal sex is atypical. We report on ities could provide explanation of the SCOS and used for genome-wide DNA methylation 46,XY DSD patient, presented herself as fe- diagnosis. WES was performed on DNA analysis. male-to-male transsexual, who was referred isolated from peripheral blood samples using Results While F1 Mthfr-/- fathers had minor for cytogenetic examination. Chromosomal NovaSeq6000 platform. Variants were anno- effects on testis weights and sperm counts analysis was not performed early. The patient tated and filtered based on family structure, with a small increase in abnormal tubules was 40-years old female with below average functional impact and phenotype matches us- (20%) in the testis, F2 Mthfr-/- sons showed stature and height, normal intelligence. There ing human and model organism data. Results a further deterioration in reproductive para­ were no psychiatric diseases mentioned in showed that son was compound heterozy- meters with more of a decrease in testis the anamnesis and family history. Neonatal gote with missense variants (p.Gln317Arg; weights and sperm counts and an increase medical sex assessment was failed, because rs375644492/p.Leu57Phe; rs142007602) in in abnormal tubules. F1 sperm DNA methy­ of genitalia ambiguous. The patient had in- both alleles while in father one allelic vari- lation was dramatically affected, with nearly tersex phenotype with severe hypospadias, ant (p.Leu57Phe; rs142007602) was found. 30,000 CpGs affected, most (99.2%) show- urogenital sinus and no palpable gonads. At Subsequently to infertility examination, our ing a loss of methylation. Compared to their the age of 3–12 years the patient underwent patient developed brain tumor – astrocytoma. fathers, > 80% of F2 sperm DNA methyla- several surgical corrections of genitalia ab- Recent findings suggest FANCM to play a tion defects overlapped with regions affected normalities. After that she was medically as- role in telomere length regulation showing in F1 sperm suggesting that there are regions signed as a girl and raised as female. From that loss of FANCM leads to telomeric re­ consistently susceptible to MTHFR deficien- early childhood she felt herself in the “wrong plication stress. Association of FANCM vari- cy. These regions were in genomic loci that body” and played with stereotypically male ants with astrocytoma has not been reported are normally methylated late during prenatal toys. Since teenage years, she expressed her- to date, however, mutations in FANCM had germ cell development and highly enriched in self in an opposite gender manner, got short been recently associated with SCOS and young retrotransposons.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 33 Andrology 2020 – Abstracts

Conclusions The worsening of reproduc- of unique reads for a sample to be included nied by a decrease in the concentration and tive parameters in MTHFR-deficient sons in that study was set at > 250,000. This was actively motile sperm. versus their fathers suggests that epigenetic observed in 11 (46%) samples prepared by So, that’s why we definitely can make the fol- defects can accumulate across generations. NEXTFlex and 20 (83%) by NEBNext. Ac- lowing conclusions: Loss of methylation at retrotransposons could cording to BA analysis of 8 pairs that met contribute to this effect, findings reminiscent sequencing quality, the average concordance 1. Sonoelastography become widely used in of epigenetic inheritance (funded by CIHR). correlation coefficient was 0.52 for miRNA, andrology, reproductology. 0.58 for piRNA and 0.65 for tRNA, p < 0.05. 2. Application of such a method of instru- mental diagnostic allows to reveal reproduc- P 1.14 Conclusion Using NEBNext kit we able to generate libraries with higher number of tive potential damage in men on the stage of Small RNA library preparation for reads uniquely mapped to sncRNAs. The preliminary ultrasound screening. human sperm of young adults highest reproducibility between kits was found for tRNA and lowest for miRNA. V. Shtratnikova1, V. Naumov2, V. Bezuglov3, P 2.2 M. Logacheva1,4, L. Smigulina1,5, Y. Dikov1,5, Funding: RSF #18-15-00202; for parent X-ray endovascular treatments of 1,5 1,6 6 RCS – NIEHS #R01 ES014370. T. Denisova , A. Suvorov , J. R. Pilsner , vascular erectile dysfunction R. Hauser7, S. A. Krawetz8, O. Sergeyev1,5 1A.N. Belozersky Research Institute of Physico-­ O. B. Zhukov1,2, V. A. Ukolov1,2 DGA-Abstracts Chemical Biology Moscow State University, P 2.1 1RUDN University, Moscow, Russian Federation; ­Moscow, Russian Federation; 2Kulakov National 2Association of Vascular Urologists and Reproducto- ­Medical Research Center of Obstetrics, Gynecology Ultrasonic sonoelastography of logists, Moscow, Russian Federation & Perinatology, Ministry of Health of the Russian scrotum in the diagnosis of male 3 Objective Penile prosthesis is the method ­Federation, Moscow, Russian Federation; Moscow fertility of choice in the surgical treatment of vascular State University, Moscow, Russian Federation; 1,2 4 O. B. Zhukov erectile dysfunction. However, in early stages Center for Data-Intensive Biomedicine & Biotechno- 1RUDN University, Moscow, Russian Federation; 2As- of disease it seems appropriate to apply a new logy, Skolkovo Institute of Science & Technology, 5 sociation of Vascular Urologists and Reproductolo- endovascular treatment. Moscow, Russian Federation; Chapaevsk Medical gists, Moscow, Russian Federation Association, Chapaevsk, Russian Federation; 6Uni- Method We have operated 14 patients with versity of Massachusetts, Department of Environ- Sonoelastographic method is widely used in vascular ED at the age of 25–48 (mean age mental Health Sciences, School of Public Health & urology for the past 9 years. With it assessed 36.4 ± 7.4). 9 of them had veno-occlusive Health Sciences, Amherst, MA, United States; the extent of fibrotic processes and dif­ erectile disruption, 3 of them had arterial in- 7Harvard T.H. Chan School of Public Health, Depart- ferentiate neoplastic diseases of the urinary sufficiency of cavernous body and 2 of them ment of Environmental­ Health, Boston, MA, United organs (bladder, kidney), prostate, penile and were with arteriovenous ED. States; 8Wayne State Uni­versity School of Medicine, scrotum. The diagnosis is based on Doppler ultrasound Department of Obstetrics­ and Gynecology, Center for The aim of our study was to determine the study of the corpora cavernous of the penis Molecular Medicine and Genetics, Detroit, MI, Uni- possibility of using this method for detecting with penile farmtests, Multispiral computed ted States the scrotum in assessing reproductive func- pharmacocavernosography and multispiral tion of men. computed tomography of the arteries of the Introduction The isolation of small non- pelvis . The study is performed on the unit This research was conducted in men aged from coding sperm RNA (sncRNA) and library “Toshiba Aquilion 64”. preparation for NGS challenging from se- 20 to 44 years old. They were divided into men with a small quantity of RNA present in three groups. The first group of men consisted Results We have made vesels stanting in sperm and the need to use DNAse. This was of 25 patients with secretory infertility. The isolated proximal atherosclerotic lesion iliac addressed by comparing two sncRNA library second group included 25 male with infertility vessels. Unilateral puncture access was used. preparation kits. caused by varicocele. The control group was Baloon dilatation was used when defeat of pudendal arteries was length. We used ba- Patients and Methods 24 sperm samples presented by 12 men in the age range from 22 to 31 years old with the implementation of the loon catheter Sim2 № Fr 5 with the pressure from the Russian Children’s Study biobank 10 atm in balloon and time for exposition was were prospectively collected at 18–20 years reproductive function of 1 or more children. The study was performed by ultrasonic scan- 4-5min . The operation was performed on the of age. After density gradient centrifugation periphery of the proximal area of the vessel. sperm RNA was extracted and treated by Qia- ner expert class Acuson S2000 manufacture of the company Siemens, was used micro- In vienocclusive abnormality of erection in gen DNAse and by TurboDNAse. 24 pairs of the form of abnormal venous drainage of the NEBNext and NEXTFlex sncRNA libraries wave sensor 18L6 HD. Sex hormones were studied on immunochemiluminescence ana- corpora cavernosa of the penis proximal di- were constructed and sequenced on NextSeq rection we were taking venous embolization. 500. Unique sncRNA reads were profiled, in- lyzer Access 2 production Beckman Coulter cluded miRNA, piRNA and tRNA, and the 8 (USA). Obtaining and study of ejaculate was Conclusions These techniques of opera- pairs that had > 240 different sncRNAs with carried out according to Recommendations of tions are effective ways of sexual rehabilita- th ≥ 10 counts were analyzed. Variance stabi- the WHO (5 edition). tion of patients with erectile dysfunction. lizing transformation (VST) was applied to For every group were identified the follow- the sncRNA counts for Bland-Altman (BA) ing indicators: speed of spread of shear wave, P 2.3 analysis. mapping the area of “soft” tissue, the concen- Results Additional treatment by Turbo tration of spermatozoids, mobility (rapid for- The effect of salubrinal on ER DNase decreased the yield of RNA by 42%. ward movement, slow forward movement), stress pathway in heat-stressed The median input sperm count and miRNA volume of ejaculate and testosterone. spermatogonial cells in vitro yielded was 28.4 (15.9–44.7) mill and 0.65 In the end we obtained the following results. S. Karadeniz Saygili1, M. K. Özbilgin2 (0.31–13.1) ng/ul, respectively. Although The application of research shear transverse 1Kütahya University of Health Sciences, Histology level of input miRNA measured by Qubit in wave Vs men with secretory infertility and and Embryology, Kütahya, Turkey; 2Manisa Celal both the NEBNext and NEXTFlex libraries varicocele is accompanied by the increase of Bayar University, Histology and Embryology, Manisa, was the same, 7.2 (2.5–105) ng, the concen- its value to 1.5–1.6 m/s and is characterized­ Turkey tration of DNA in library was significantly by 35–45% from similar parameters of re- higher using NEBNext, 11.8 (4.9–21.3) nM, productive healthy men. In half of the cases Aim It was aimed to investigate the effect in comparison with NEXTFlex, 0.2 (0–2.1) studies such an increase in performance and of SAL on endoplasmic reticulum (ER) stress nM. Low library quantity was critical for suc- decrease in the area of mapping the so-called on experimental in vitro heat stress model cessful sequencing. The minimum number “soft” areas of the epididymis is accompa- (HSM) of spermatogenic cells.

34 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

Materials and Methods In the study AP was simulated in the whole-cell model that astaxanthin has a protective effect on tes- Mouse (GC1) and sper- by applying an external stimulus current ticular torsion-detorsion (ischemia-reperfu- matocyte (GC2) cell lines were used. IC50 (10–30 pA), as a brief square pulse of 10 ms sion) injury in rats. dose of SAL were calculated via MTT assay. duration. The AP exhibits depolarization, Each cell lines were separated into 4 different repolarization, and hyperpolarization phases groups which are; control (GC1-C, GC2-C), as found in experiments. The simulated ionic P 2.6 SAL treated (GC1-SAL, GC2-SAL), HSM currents and AP show good agreement with The Novoglan-01 clinical trial of a experimental model (GC1-HSM, GC2-HSM) the experimental recordings. Therefore, this and SAL treated HSM model (GC1-HSM- electrophysiological model can be a powerful conservative treatment for adult SAL, GC2-HSMSAL). Control cells were platform to investigate the various electrical phimosis incubated under standard culture conditions. properties of VDSM cells in both normal and D. Gillatt, E. Chung, H. Mazure, A. James HSM model group cells were incubated un- pathological conditions. Macquarie University, Macquarie University Hospi- der 43oC for 60 minutes. In the SAL group, tal, Macquarie University, Australia cells were incubated with 20 µM SAL con- taining culture medium for 24 hours. After P 2.5 Adult phimosis can affect up to 10% of the the experiments, all groups were stained male population over 18. Common con- with immünoflorescence probes of p-PERK, Protective effect of astaxanthin on servative treatments with steroid creams are GRP78, p-eIF2 , HSP70 antibodies. More­ testicular ischemia-reperfusion in­ frequently unsuccessful, requiring repeat

α DGA-Abstracts over, mRNA levels of GRP78, PERK, eIF2α jury in rats interventions. Circumcision in adults is were evaluated with qRT-PCR. much more delicate than in children, with M. Baškovic´1, A. Katušic´ Bojanac2, N. Sincˇic´2, potential scarring and other undesirable side Results It was observed that HSM model 2 2 3 M. Himelreich , D. Krsnik , D. Ježek effects. Foreskin tissue expanders have been has cytotoxicity. In the experimental model 1 Children’s Hospital Zagreb, Department of pediatric advocated as a conservative treatment for of HSM cells, all the markers showed elevat- urology, Zagreb, Croatia; 2University of Zagreb, ed levels of immünoreactivity, that they were adult phimosis and an alternative to steroid School of Medicine, Department of Medical Biology, creams or circumcision for several years but agreed upon to have ER stress. With the treat- Zagreb, Croatia; 3University of Zagreb, School of ment of SAL, decreased levels of ER stress their relative safety and efficacy has never ­Medicine, Department of Histology and Embriology, been proven. Conservative treatments such detected. GRP78, PERK and eIF2α mRNA Zagreb, Croatia ć levels were upregulated in the HSM model as foreskin tissue expanders are particularly group. Even though there was a downregula- Introduction Testicular torsion is one of the attractive in the current pandemic when elec- tion following SAL treatment, the difference conditions of the acute scrotum that requires tive surgeries such as circumcision are often was not statistically significant. immediate surgical intervention. If not recog- unavailable or avoided. The Novoglan prod- nized at time, it can result of ischemic injuries uct is a foreskin tissue expander commercial- Conclusions In the view of such findings, ised for over 15 years with a very high safety it was thought that heat stress triggered ER and testicular loss. Astaxanthin (C40H52O4) is a pigment from the xanthophyll family, record. We describe the Novoglan-01 clinical stress on spermatogenic cells, and SAL has trial, started in 2019 at Macquarie Univer- effect on these markers. Studies of ER-relat- oxygenated derivatives of carotenoids whose synthesis in plants originates from lycopene. sity Hospital, Sydney, Australia. In line with ed stress mechanisms on spermatogenic cells Good Clinical Guidelines, the Novoglan-01 will be important for the therapeutical strate- The antioxidant activity of astaxanthin is 10 times higher than zeaxanthin, lutein, cantax- clinical trial aims to assess the safety, toler- gies with the forthcoming advanced molecu- ability and efficacy of the Novoglan product lar analyses. anthin, β-carotene and 100 times higher than α-tocopherol. Since to date there is no drug in treating adult phimosis. A second study given to patients with torsion-detorsion tes- site at Princess Alexandra Hospital, Brisbane, ticular injury, we have investigated the effect Australia has recently been set up. The study P 2.4 of this powerful antioxidant. is expected to conclude and report its findings in 2021. Electrophysiological modeling of Materials and Methods Thirty-two male Vas deferens smooth muscle cells: Fischer prepubertal rats were divided into 4 Role of ion channels in generating groups of 8 individuals. Group 1 underwent P 2.7 sham surgery to determine basal values for electrical activities histological evaluation. In group 2 (torsion- Machine learning applications in C. Mahapatra detorsion group), right testis was twisted at the domain of male infertility: University of California San Francisco, School of 720° for 90 min. After 90min of reperfusion, ­development of a prediction mo­ ­Medicine, San Francisco, CA, United States the testis was removed. Astaxanthin was del for Klinefelter Syndrome in administered intraperitoneally at the time The coordinated activation of several ion of detorsion (group 3) and 45 minutes after azoospermic men channels generates a wide range of electrical detorsion (group 4) in the treatment groups. H. Krenz1, A. Sansone2,3, C. Krallmann3, M. Zitz- activities in the Vas deferens smooth muscle Using software ImageJ®, histological mor- mann3, M. Dugas1, S. Kliesch3, F. Tüttelmann4, (VSDM) cell. Any mutation or dysfunction phometric values were measured. J. Gromoll3 to VSDM ion channels causes premature 1University of Münster, Institute of Medical Infor­ , which is a male sexual disorder. Results There is a statistically signifi- matics, Münster, Germany; 2University of Rome Tor To elucidate the quantitative contribution of cant difference in all observed parameters; 2 Vergata, Chair of Endocrinology and Medical Sexo­ individual ionic current to the action potential MSTD (193,489 ± 21,127) (χ = 55,733, DF logy (ENDOSEX), Department of Systems Medicine, (AP) generation, a biophysically constrained = 3, p < 0.0001), MSLD (89,057 ± 15,187) 2 Rome, Italy; 3University Hospital Münster, Centre of single guinea-pig VDSM cell model is pro- (χ = 65,687, DF = 3, p < 0.0001), epithelial 2 Reproductive Medicine and Andrology, Münster, posed. The model includes voltage-gated Na+ height (47,877 ± 12,800) (χ = 66,321, DF Germany; 4University of Münster, Institute of Repro- channel, Ca2+ channel, K+ channel, Ca2+ acti- = 3, p < 0.0001), tubular area (30920,292 ± 2 ductive Genetics, Münster, Germany vated K+ channel and nonselective cation cur- 4901,310) (χ = 59,290, DF = 3, p < 0.0001), rent (NSC). The contributions of each mem- luminal area (6361,205 ± 2040,415) Introduction With successful digitalization 2 brane current were investigated by sensitivity (χ = 67,882, DF = 3, p < 0.0001), Johnsen in hospitals over the past years, large sets of 2 analysis and modification of the current pa- score (6,563 ± 1,101) (χ = 71,018, DF = 3, patient records labelled with diagnoses are rameters. The ion channel conductances are p < 0.0001). Post-hoc analysis found that a now becoming available, facilitating the ap- set to maintain the resting membrane poten- statistically significant difference existed be- plication of machine learning (ML) based tial (RMP) at –50 mV as it is documented tween all groups (p < 0.0001). models to predict conditions of patients. ML that the resting membrane potential (RMP) in Conclusion By measuring all histological based models could present an ideal tool to VDSM cell varies between –45 to –70 mV. morphometric parameters it can be concluded improve diagnostic precision and therefore

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 35 Andrology 2020 – Abstracts

patient treatment. With the in-house devel- with density gradient (DG) method and Swim age was 36,2 ± 9,6 years (18–56 years). The oped database Androbase, the Centre of Up (SU) method using split human samples. study performed on a multislice computed Reproductive Medicine and Andrology (Uni- Methods This study included 35 men tomography “Toshiba Aquilion”. We used a versity Hospital Münster) has thousands of having normal semen parameters with a protocol Pelvis HCT Native; 120 KV; 60 mA; records on infertile men available that contain minimum of 80 million total sperm number. Rot. Time 0,5. After intracavernosal injection information on e.g. hormone levels, semen Spermatozoa isolated by SU, DG and MS 10–20 micrograms of “Caverject” penile analysis, and ultrasound. In order to evaluate methods were compared for sperm yield, Doppler was performed. When the maximum the potential of ML for identifying the Kline- vitality, motility and longevity as well as pharmacological response obtained, intra­ felter syndrome (KS) in azoospermic men, functional characteristics like mitochondrial cavernosally injected 5 ml of contrast “Vezi- multiple models from different algorithms integrity, protein tyrosine phosphorylation pak 320”, diluted with 15 ml of saline. have been trained and benchmarked. and acrosome reaction.The ability of selec- Results Abnormal venous leakeage was Patients (or Materials) and Methods Com- tion techniques in reducing spontaneous and confirmed in 72 (81.8%) patients, patho- putations were executed in R based on the radiation-induced sperm DNA lesions was logical shunts between the spongious and package MLR3. For this task, a cohort of assessed by TUNEL assay. cavernous bodies in 26 (29.5%), abnormal 1476 azoospermic patients (1099 controls Results MS-selected spermatozoa had structure of the corpora cavernosa in 13 with karyotype 46,XY and 377 KS with higher viability (p < 0.001), longevity in terms (14.8%) patients. We identified several types karyotype 47,XXY) was extracted from An- of total motility at the end of 6 and 18h post- of pathological venous leakage from corpora DGA-Abstracts drobase. A subset of patients (105 controls extraction (p < 0.001), and mitochondrial in- cavernosa during study: distal – via deep and 32 KS) was used for testing the model tegrity (p < 0.001) compared to those selected dorsal vein in 28 (31%), proximal – via deep performances and additionally evaluated by by DG. Furthermore, spontaneous DNA le- penile veins in 49 (55.7%), and mixed type 16 andrologists/urologists. sions were significantly reduced in MS and in 11 (12.6%). Additionally, revealed signs Results Comparison of the models regard- SU fractions compared to NE (p < 0.001). of sclerosis of the corpora cavernosa in 19 ing sensitivity, specificity, classification error Similarly, radiation-induced sperm DNA le- patients (21.5%), clarified the status of the and area under the curve (AUC) indicated sions were significantly lower in MS and SU- cavernous channels, urethra, and the integrity that support vector machines, random forests, fractions (p < 0.001) compared to DG. of the rigid and 3-component implants in 2 linear classifiers and logistic regression all (2.28%). The sensitivity of this method was Conclusion MS-based device offers a 98%, specificity –96%. perform particularly well on this task. For centrifugation-free, efficient, sperm selec- the independent test data set, a sensitivity of tion method, making it suitable for partially Conclusion This study demonstrated a 1 and a specificity of > 88% was achieved. equipped intra-uterine insemination (IUI) high diagnostic value of dynamic pharmaco­ The andrologists/urologists achieved simi- laboratories. cavernosography performed with computed lar results regarding specificity (~88%) but tomography in detecting pathological venous detected fewer KS patients than ML-based leakage from the cavernous pool and its ad- models (on average: 81%). P 2.9 vantage comparing ultrasound techniques. Conclusion These results indicate that ML Multispiral computed pharmaco- models might be able to increase the diagnos- cavernosography in the diagnosis tic rate of KS among azoospermic patients P 2.10 of venoocclusive erectile dysfunc- if respective supporting algorithms are rou- Satisfaction with the semirigid tinely included into database systems. tion penile prosthesis among couples O. Apolikhin, S. Krasnyak N.A. Lopatkin Research Institute of Urology and in- from a Semiurban Indian popula- P 2.8 terventional Radiology, Moscow, Russian Federation tion Evaluation of functional and ge- Introduction Causes of surgical treatment P. Gupta netic integrity of spermatozoa failure for venoocclusive erectile dysfunction JN Medical College, KLE Academy of Higher Educa- ­extracted by migration-sedimen- (ED) are not only the formation of new col- tion and Research (Deemed to be University), De- partment of Urology, Belagavi, India tation (MS) method laterals, but also the functioning of residual veins. The purpose of this study was visu- 1 1 1 2 Introduction Insertion of penile prosthesis H. Y. Meitei , S. Uppangala , G. Kalthur , S. Schlatt , alization of the veins, which are mainly in- for treatment of irreversible erectile dysfunc- S. K. Adiga1 volved in the blood leakage from the corpora 1 tion (ED) is a common and well-established Kasturba Medical College, Manipal, Manipal Aca- cavernosa. treatment in the western countries. Even in demy of Higher Education, Manipal, Department of 2 Methods and Materials Eighty-eight men the times of the newly available oral medi- Clinical Embryology, Manipal, India; University of with ultrasound signs of venous insufficiency cations, penile prostheses continue to have Munster, Centre of Reproductive Medicine and An- of the penis were examined. The average such a nonoptional place in the management drology, Munster, Germany Introduction Centrifugation-based sperm selection techniques used in assisted fertili- zation primarily involve selection of motile and viable sperm from centrifugal pellets of spermatozoa. The swim up (SU) method is routinely used either singly or in combination with density gradient (DG) method to select the most active and motile spermatozoa but these methods don”t replicate all complex natural barriers which sperm encounter in vivo. Migration-Sedimentation (MS) method isolates spermatozoa based on motility with sedimentation of gravity, thus, the deleterious effects of centrifugation are believed to be avoided. Hence, the main aim of this study is to understand the functional and genetic in- tegrity of spermatozoa extracted by migration -sedimentation (MS) method when compared Figure 3. P. Gupta

36 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts of severe ED. In this study, we have asses- Introduction The redistribution of glyco- der to identify early markers of efficacy/safety sed the satisfaction among semiurban Indian conjugates stands out as a major feature of for the therapy of ED with PDE5i, the basal couples following insertion of the semirigid sperm capacitation. However, terminal fucose clinical characterization of patients, integrat- penile prosthesis. changes during human in vitro capacitation ed with metabolomics analysis of serum and Materials and Methods Between Janu- remain poorly described. This study aimed to urine and genomic data, were here correlated ary 2000 and December 2015, 78 men with locate and quantify terminal fucose residues with the PDE5i efficacy and the occurrence ED underwent semirigid penile prosthesis on ejaculated and in vitro capacitated human ADR upon administration. implantation (PPI) at our hospital. The satis- sperm using fluorescence microscopy and Thirty-six males with new diagnosis of ED faction of patients and partners was evaluated field emission scanning electron microscopy were consecutively recruited and charac- using the ED Inventory of Treatment Satis- (FE-SEM). terized at basal for anthropometrics, blood faction (EDITS) questionnaire and EDITS Materials and Methods Sperm samples pressure, blood glucose, lipid profile, serum partner survey. were obtained from three normozoospermic levels of thyroid/sex hormones and erectile Results The mean age of the patients was donors and spermatozoa were capacitated function evaluated by IIEF-15 question- 44.84 ± 7.30 years. The mean duration of by swim-up during one hour. Two physio­ naire. Targeted Next Generation Sequencing time with ED in the preimplantation period logical sperm conditions were used, unca- (NGS) was applied to genes involved PDE5i was 38.69 ± 12.44 months. The satisfaction pacitated (UC) and one-hour capacitated pharmacodynamics and pharmacokinetics. of patients and partners as assessed by EDITS (C1). For fluorescence microscopy, surface Fasting metabolic profile of serum and urine questionnaire and EDITS partner survey was fucose residues were detected by Auleuria were assessed by NMR-based metabolomics DGA-Abstracts 80.66 ± 4.49 and 75.66 ± 6.57, respectively, ­aurantia agglutinin (AAA) lectin conjugated analysis. Patients were prescribed on-demand at 12 months after surgery and 71.73 ± 8.10 to fluorescein-5-isothiocyanate and a mini- therapy with Sildenafil oro-dispersible film and 65.6 ± 6.49, respectively, at 24 months mum of 300 cells were analyzed. For FE- and followed-up after 3 months from recruit- after surgery. SEM, AAA-binding sites were quantified by ment. Basal data were correlated with IIEF- indirect immunogold-labeling and a total of 15 score at follow-up and with the occurrence Conclusions This study showed a high de- 100 cells were included. Data were analyzed of ADR recorded by a dedicated question- gree of satisfaction among patients as well as statistically. naire. their partners with semirigid PPI. More than 80% of the men reported being very satis- Results Fluorescence microscopy identi- Twenty-eight patients were finally included fied with their penile implantation surgery fied a high subpopulation of cells (~45.0%) in the analysis. Serum LDL-cholesterol levels (Fig. 3). with terminal fucose residues distributed in were increased in those reporting ADR (143.3 a dotted way around de head in UC cells. ± 13.2 mg/dL ADR vs 133.1 ± 12.4 mg/dL However, after capacitation we observed No ADR; P = 0.046). NGS data showed that P 2.11 a significant increase in the percentage of specific variants of PDE11A and CYP2D7 spermatozoa with fluorescence in both acro- genes were more represented in drug re- Two piece inflatable penile somal and postacrosomal region (~70.0%; sponders (both relative risk=2.7 [0.9-5.1]; ­prosthesis surgery: operative p < 0.05). Otherwise, we quantified that the p = 0.04). NMR-based metabolomics showed steps number of colloidal gold particles decreases the highest association between serum LDL- significantly (p < 0.05) from ~65.0 in UC to cholesterol metabolites and the occurrence of P. Gupta ~40.0 in C1 by FE-SEM. ADR (Hazard ratio = 17.5; p = 0.019). JN Medical College, KLE Academy of Higher Educa- tion and Research (Deemed to be University), De- Conclusion Surface fucose residues under- The association between lipid profile and the partment of Urology, Belagavi, India go redistribution and decrease after in vitro ADR pattern suggests major cues in the tai- sperm capacitation. Overall, fluorescence loring of ED therapy with PDE5i. Introduction The placement of an inflat- microscopy combined with FE-SEM allows able penile prosthesis has been considered accurately identify the location pattern and the most reliable and effective treatment for the quantification of surface fucose residues P 3.1 erectile dysfunction in men whose medical on human spermatozoa. Handling temperature and time treatment has failed. interval prior to testicular-tissue Materials and Methods We report the use of this two-piece prosthesis in a 50 year old P 2.13 organotypic culture in prepubertal patient with erectile dysfunction and describe Evaluation of the patients’ geno­ mice have a differential impact on the surgical steps of the same. mic and metabolomic profile to cellular homeostasis Results The 2-piece system eliminates a improve the adherence to drug R. K. Pandya1, S. R. Salian1, S. L. Koulmane separate reservoir making the surgery easy 2 3 1 therapy: a study on PDE5 inhibi­ Laxminarayana­ , H. Krishnamurthy , A. Cheredath , for the treating surgeon/urologist . P. Tholeti1, S. Uppangala1, G. Kalthur1, S. Schlatt4, tors. Conclusion In an appropriately selected S. K. Adiga1 patient, the 2-piece penile prosthesis is a M. S. Rocca1, A. Vignoli2, L. Tenori3, M. Ghezzi1, 1Manipal Academy of Higher Education, Clinical Em- reliable, user-friendly prosthesis with high M. De Rocco Ponce4, G. Vatsellas5, D. Thanos5, bryology, Udupi, India; 2Manipal Academy of Higher patient and partner satisfaction rates. R. Padrini1, C. Foresta1, L. De Toni1 Education, Department of Pathology, Udupi, India; 1University of Padova, Department of Medicine, 3National Centre for Biological Sciences, TIFR, Cen- Padova,­ Italy, 2Consorzio interuniversitario Risonanze tral Imaging & Flow Cytometry Facility, Bengaluru, P 2.12 Magnetiche di Metallo Proteine, Sesto Fiorentino, India; 4Albert-Schweitzer-Campus 1, Gebäude Italy, 3Department of Chemistry “Ugo Schiff”, Uni- D11, Centre of Reproductive Medicine and Andro­ Fluorescence microscopy and versity of Florence, Sesto Fiorentino, Italy, 4Fundacio logy, Münster, Germany ­FE-SEM as complementary tools Puigvert, Andrology Department, Barcelona, Spain, Prepubertal boys undergoing chemotherapy to detect terminal fucose residues 5Biomedical Research Foundation Academy of are at high risk of gonadotoxicity. Cryopreser- Athens (BRFAA), Athens, Greece on human sperm glycocalyx vation of immature testicular tissue (ITT), yet L. Robles Gómez1, P. Sáez Espinosa1, Type V-phosphodiesterase-inhibitors (PDE5i) experimental, is the only recommended op- E. López Viloria1, N. Huerta Retamal1, A. Manuel2, are the first choice drugs used for the treat- tion for fertility preservation, prior to cancer A. Romero1, J. Aizpurua3, M. J. Gómez Torres1 ment of erectile dysfunction (ED), being ef- treatment. In optimizing the fertility resto- 1Universidad de Alicante, Biotecnología, Alicante, fective in 60–70% of patients. However, prox- ration procedures, care should be taken in Spain, 2Universidad de Murcia, Biología Celular e imately 50% patients per year discontinue the handling and manipulation of ITT in ex vivo Histología, Murcia, Spain, 3IVF SPAIN, Alicante, treatment PDEi reporting poor drug efficacy conditions, as a suitable holding condition is Spain or major adverse drug reactions (ADR). In or- of primary importance in the maintenance of

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 37 Andrology 2020 – Abstracts

cellular homeostasis, prior to cryopreserva- tion. To explore this, ITT from 6 day old mice were held and manipulated at ultra-profound- hypothermic, profound-hypothermic, and mild-warm-ischemic temperatures for vary- ing time periods, prior to 14-days organo- typic culture. Cell viability and functionality along with the expression of synaptonemal complex and chromatin condensing proteins were assessed. Holding of ITT at ultra- profound-hypothermic temperature up to 24 h did not change the cell viability, levels of testosterone, and in vitro proliferation ability, whereas ITT held at profound-hypothermic temperature for 24 h significantly reduced the number of viable cells (p < 0.01). However, the mild-warm-ischemic temperature was

DGA-Abstracts found detrimental for holding of ITT even up to 6 h. Furthermore, the holding of ITT at ultra-profound-hypothermic had no signifi- cant negative effect on 14-day organotypic culture, whereas ITTs from a profound hypo- thermic group showed reduced post-meiotic transcripts (p < 0.01). Therefore, holding of ITT at ultra-profound-hypothermic-tempera- ture was found to be most suitable; whereas profound-hypothermic-temperature may compromise post-meiotic germ cell yield, post in vitro culture. This data, albeit in the mouse model, will have immense value in hu- man prepubertal fertility restoration research.

P 3.2 The influence of different ways of education on the home injection of assisted reproduction patients H. Pan, L. Li, G. Liu Sixth Affiliated Hospital of Sun YatSen University, Reproductive Centre, Guangzhou, China Objective To compare the effects of differ- ent education methods on home injections of assisted reproduction patients to ensure the effectiveness and safety of home injections. Figure 4. H. Pan, et al. Methods 350 patients were randomly se- lected using the following methods: plain same time, nurses were able to observe the sultation guidance, psychological guidance, text, graphics and text, video, and nurse op- patient’s experience psychology during the online consultation guidance and nursing eration demonstration. The comparison of the health promotion process, and effectively services, and these managements were com- accuracy of the first injection after the educa- conduct psychological counseling to ensure pared with the follow-up managements be- tion was conducted between different educa- the medicine Effectiveness and safety during fore the epidemic. tion methods. And on the first day after the injection (Fig. 4). Results During COVID-19 epidemic, PGT home injection, the patients’ satisfaction with pregnancy patients who failed to arrive at different ways of education was followed up. the hospital on time increased. However, the Results After using different education P 3.3 number of people who visited online through methods, the nurses assessed the patient’s Follow-up management of inquiries and active contacts increased sig- injection preparation process, drug exhaust nificantly, with a follow-up rate of 100%. In method, dose adjustment, skin disinfection, ­pregnant women undergoing pre­ addition, through the way of online contact, injection site, injection method, drug storage, implantation genetic testing un- we can provide consultation, protection and needle handling accuracy, and patient satisfac- der the new coronavirus epidemic psychological guidance for PGT pregnant tion. The injection accuracy rate and patient women to ensure their safe pregnancy. H. Pan, L. Li, Y. Li satisfaction rate of patients with text education Sixth Affiliated Hospital of Sun YatSen University, Conclusion Medical staff can follow-up the were the lowest, while the injection accuracy Reproductive Centre, Guangzhou, China pregnant women with PGT through online rate and patient satisfaction of patients with consultation, and provide them with scien- video education combined with nurse opera- Objective To explore the appropriate fol- tific consultation guidance and psychological tion demonstration were the highest. low-up management of pregnant women with guidance during pregnancy, which not only Conclusion The use of video combined preimplantation genetic testing (PGT) during reduces the psychological burden of preg- with nurses’ demonstrations during the first COVID-19 epidemic. nant women with PGT during the epidemic, home injection of assisted reproductive Methods The follow-up managements of but also improves their compliance, so as to patients is more beneficial for patients to pregnant women with PGT was carried out ensure the maternal and infant health during master the correct injection method. At the during the epidemic period, including con- pregnancy.

38 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

P 3.4 quality when assessed by classical semen cultured on mouse embryonic fibroblasts, us- analysis indicated an IVF we decided to do ing a protocol adapted from Kanatsu-Shino- Understanding the role of selec­ a combined IVF/ICSI to avoid an expected hara et al. BoR 2003. Roughly 2×105 GSCs tive inhibition of Arachidonate failure. were plated in a 6-well plate, with medium 15-lipoxygenase (ALOX15) during Patients and Methods We used the stand- replaced every 3 days. At day 6, half plate human semen cryopreservation ard long protocol with 14 days GnRH agonist was exposed to cisplatin (1µg/ml) or drug- prior to a daily dose of 125IU recombinant free medium. At day 8, GSCs were harvested S. Uppangala, K. Rayalla, H. Y. Meitei, S. K. Adiga and sorted by flow cytometer, with DRAQ5™ Kasturba Medical College, MANIPAL, Manipal FSH for controlled ovarian hyperstimula- tion. Follicular development was monitored dye used to identify dead cells. Four repli- ­Academy of Higher Education, Clinical Embryology, cates were analysed using a paired t-test. Manipal, India by vaginal ultrasonography and was induced by 6500 IU recombinant hCG Results GSC colonies displayed the charac- Introduction One of the deleterious effect when at least one follicle reached a diameter teristic shape of proliferating cell chains and of semen cryopreservation is oxidative stress ≥20mm. Ovum pick-up was performed 36h a slow proliferating rate. Cell viability analy- which can compromise the sperm functional after hCG. sis found more than 95% cells were alive in and fertilizing ability. The oxidative stress Results We retrieved 20 oocyte-cumulus- both control and cisplatin group. Cisplatin in sperm can trigger lipid peroxidation complexes (COC) of which five were allocat- treatment significantly reduced the number of thereby producing cytotoxic aldehydes such ed to classical in-vitro fertilisation (IVF) and harvested cells, with an average drop of 56% 4 4 as malondialdehyde (MDA) and 4-hydro­ inseminated with >32,000 motile spermato- (control 13×10 cells vs cisplatin 7×10 cells; DGA-Abstracts xynonenal (4HNE). The metabolism of zoa. The remaining 15 oocytes were injected p < 0.05). 4HNE is through a class of lipoxygenase en- each with a single sperm (ICSI). Fertilisation Conclusion Dead cell count is unreliable as zymes mainly the human arachidonate 15-li- was checked 17h after IVF or ICSI. All 5 cell viability assay due to the early detach- poxygenase (ALOX15) which are potentially IVF oocytes showed no sign of fertilisation, ment of non-vital cells. Exposure to cisplatin, capable of disrupting sperm function. There- although the sperm were able to dissolve at a concentration within the range found in fore, the aim of this study was to understand the COCs and bound to the Zona pellucida. plasma of cancer patients, halved the number the effect of selective inhibition of ALOX15 The ICSI oocytes gave rise to 9 fertilised of mouse GSCs harvested within 48h. This enzyme by its inhibitor 6,11dihydro[1] oocytes (PN stages). Due to a mild ovarian cisplatin exposure condition (dose/time) will benzothiopyrano[4,3-b]indole (PD146176) hyperstimula­tion syndrome and because of be used as median lethal dose in an ongoing during human semen cryopreservation. the Corona pandemic all PN stages were toxicological study using mouse germline Methods This study included 20 men hav- frozen. In the meantime, 4 PN stages where stem cells. ing normal semen characteristics. The se- thawed and the transfer of two 6-cell stages men samples were pretreated with 0.25µM yielded in a biochemical pregnancy, which PD146176 for 20 min before rapid freezing. unfortunately, resulted in an early loss. P 3.7 Post thawing, spermatozoa were assessed for Conclusion Loss of CatSper channel func- Ex vivo testis explant culture of motility, morphology, mitochondrial poten- tion leads to infertility. CatSper-deficient tial, DNA damage and acrosome reaction. sperm fail to penetrate the Zona pellucida human intrapubertal testicular Results Ejaculates cryopreserved with and, thus to fertilise the egg both in vivo and tissue PD14616 showed a moderate but non-sig- in vitro. This indicates that in these normo- N. L. Aden1, A. Soave2, U. Kordes3, M. Bleeke3,4, nificant improvement in sperm motility and zoospermic patients ICSI is required to over- A. Salzbrunn1, K. von Kopylow1 mitochondrial potential whereas slight re- come CatSper-related infertility. 1University Medical Center Hamburg-Eppendorf, duction in morphological abnormalities and ­Department of Andrology, Hamburg, Germany; 2Uni- DNA damage was observed in comparison to P 3.6 versity Medical Center Hamburg-Eppendorf, Depart- the control. ment of Urology, Hamburg, Germany; 3University Conclusion The selective inhibition of Developing an in vitro model for Medical Center Hamburg-Eppendorf, Department of ALOX15 during human sperm cryopreser- toxicological study of chemo­ Pediatric Hematology and Oncology, Hamburg, Ger- 4 vation did not demonstrate any benefits in therapy on isolated mouse germ­ many; University Medical Center Hamburg-Eppen- improving the sperm functional and genetic dorf, Division of Pediatric Stem Cell Transplantation integrity indicating it is not useful in protect- line stem cells and Immunology, Hamburg, Germany ing sperm from freeze-thaw induced damage. F. Lopes1, A. Codino2, D. O’Carroll2, R. T. Mitchell1 Fertility preservation for boys 1University of Edinburgh, Queen’s Medical Research Introduction subjected to gonadotoxic therapy, e.g. for Institute, Edinburgh, United Kingdom; 2University of cancer treatment, is increasingly in demand P 3.5 Edinburgh, Centre for Regenerative Medicine, by patients and relatives, as long-term over- ­Edinburgh, United Kingdom Loss of CatSper-channel function all survival probability for pediatric cancer leads to male infertility and IVF Background The overall incidence of patients has risen to > 80% (German Child- failure: a case report mortality for childhood cancers is steadily hood Cancer Registry, 2018). Consequently, decreasing thanks to improved diagnosis and most of the patients reach the reproductive T. Pock1, C. Krallmann1, T. Sperlbaum2, treatments. Cisplatin is a chemotherapeutic age (nearly 50.000 in the GCCR long-term F. Tüttelmann3, H. M. Behre2, S. Kliesch1, drug used in paediatric oncology. Accumu- survivor cohort as of 2016). However, cryo- V. Nordhoff1 lating evidence shows that cisplatin impairs preservation of sperm, applicable for later 1Centre of Reproductive Medicine and Andrology, fertility in cancer survivors, by targeting assisted reproduction techniques (ART) is no Münster, Germany; 2Kinderwunschzentrum, Münster, the germ stem cells (GSCs) in the testis of option for prepubertal boys since no sperma- Germany; 3Institute of Reproductive Genetics, young patients. The absence of clinical op- tids are produced before puberty. Hence, the Münster,­ Germany tions to preserve children fertility means that only possibility to restore fertility of young Introduction The sperm-specific Ca2+ chan- strategies to protect the immature testis from cancer survivors is cryopreservation of tes- nel CatSper controls the intracellular Ca2+ chemotherapy-induced damage are urgently ticular tissue containing spermatogonial stem concentration and, thereby the swimming required. The work here aims to develop an cells (SSCs). behaviour of sperm. Genetic aberrations of in vitro model of GSCs to understand cis­ Material and Methods In a first attempt to genes encoding CatSper subunits are asso- platin cytotoxicity and identify key molecular test the potential of juvenile human testicular ciated with male infertility. We identified a pathways to be exploited for fertility preser- tissue, we cultivated a rice grain sized testicu- CATSPER2-deficident infertile patient that vation therapy. lar biopsy of a 13-year-old boy for 78 days. underwent with his female partner assisted Methods GSCs derived from pnd7 F1 The tissue was fixed in modified Davidson’s reproduction at our centre. Although semen (DBA/2 crossed C57BL/6) mouse testes were fluid and tissue cells were visualized using

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 39 Andrology 2020 – Abstracts

immunostaining with cell-specific markers. ability to re-organise into cord-like aggre- they allow to preserve the fluidity and integ- The initial cellular status of the testis tissue gates containing SOX9+ Sertoli cells. As we rity of the membranes, which has repercus- prior to culture was determined on toluidine currently observe patient-to-patient variation sions in a better mitochondrial status and blue-stained semi-thin sections of glutaral- in these cultures, we will first concentrate on with it, the sperm motility. dehyde-osmium-fixed and Epon-embedded increasing reproducibility. Then, hPGCLCs Acknowledgements Supported by FON- tissue. will be included to analyse male differentia- DECYT REGULAR N° 1180912, ANID, Results Both, germ cells, including SSCs, tion in this homologous organoid. Chilean Government. and somatic cells could be detected after the With this system, we have a model for early culture period. In one case, single PRM1+ fate decisions of human germ cells and a structures resembling spermatids were fo- platform to analyse genes associated with in- cally detected. These cells are most likely fertility. We will use hiPSCs with functional remnants of individual spermatozoa already knockout of respective candidate genes and P 3.10 present in the boy’s testicular tissue, as these analyse their interaction with the somatic transformation into were also identified in the testicular tissue surrounding and male differentiation in com- spermatogonial stem cells (SSC): before organ culture. parison to isogenic control hiPSCs. The key to understand infertility Conclusion These findings demonstrate the and malignancy of cryptorchidism potential to develop methods for in vitro sper- DGA-Abstracts R. Li, J. Hutson matogenesis and the propagation of human P 3.9 SSCs within cultured human juvenile testis Murdoch Children’s Research Institute, Surgical Re- biopsies. If these techniques can be success- The optimization of freezing me­ search, Parkville, Australia fully established, this would pave the way for dia with saccharides and/or anti­ Undescended testis (UDT) is a major health young male cancer survivors to fulfill their oxidants to improve the quality of problem, affecting over 2% of new-born wish for biological paternity in later life. cryopreserved boar spermatozoa boys with increased infertility (30–60%)

1 1 1 1 and testicular cancer (5–10 fold > normal R. Sánchez , F. Pezo , F. Zambrano , P. Uribe , males) later in life. We have studied animal J. Risopatron1, R. A. Burgos2 1 2 models in conjunction with human biopsies P 3.8 Universidad de La Frontera, Temuco, Chile; Univer- of UDT in order to understand the process of Novel organoid approaches to sidad Austral de Chile, Institute of Pharmacology gonocyte transformation into SSC to eluci- and Morphophysiology, Valdivia, Chile ­induce male differentiation of date how to prevent infertility and testicular ­human PGCLCs Introduction Boar semen cryopreservation cancer in cryptorchidism. We used testes remains a suboptimal reproductive techno­ from Oct4-promoter-driving GFP transgenic 1,2 3 1 E. M. Mall , N. Rotte , R. Sandhowe-Klaverkamp , logy, optimization of freezing media with mice, androgen receptor knockout (ARKO) 1 1 J. Wistuba , S. Schlatt saccharides and/or antioxidants (AOX) are mice, hypogonadal (hpg) mice, Bax knock- 1 University Hospital Münster, Centre of Reproductive part of the strategies used to improve the out (BaxKO) mice and human biopsies for 2 Medicine and Andrology, Münster, Germany; Max quality of cryopreserved sample. This study gene expression, immunohistochemistry and Planck Institute for Molecular Biomedicine, Münster, evaluated the combined effect of replace- confocal imaging analysis. Serum and testes 3 Germany; Institute of Reproductive Genetics, Müns- ment of lactose in the traditional medium by were collected for hormone analysis. We have ter, Germany 0.25 M of sucrose (S) or trehalose (T), and found that mouse transformed into The fetal gonad is characterised by its bi- the media supplementation with 2.0 mM of SSC between postnatal days 2-6 during min- potential nature, capable of developing butylhydroxytoluene (B) or 1 µM of mela- ipuberty when testosterone, FSH receptor into both, ovary or testis. The developing tonin (M). and Oct4 peaked. There was no difference for number of gonocytes transformed into SSC/ microenvironment precisely coordinates Material and Method The media used were the differentiation of primordial germ cells composed of medium A (saccharide/AOX/ tubule between ARKO mice and wild type (PGC) towards male gonocytes. However, egg yolk) and B (saccharide/AOX/egg yolk/ (WT) littermates. Germ cells/tubule were sig- fate decision towards the male lineage is not glycerol/Equex). One ejaculate from eight nificantly less in hpg mice comparing to WT. fully understood, since examination of these different boars were split and cryopreserved There were persisting gonocytes in BaxKO processes is limited for ethical and technical in the traditional freezing medium (Control) testicular tubules which were not present in reasons. and four experimental freezing mediums: WT. UDT biopsies showed empty tubules without germ cell significantly increased and We established a novel and standardized SB; SM; TB and TM. The sperm function test were evaluated by flow cytometry. Total number of germ cells decreased with increas- xeno-organoid approach combining somatic ing age of orchidopexy. There were persisting testicular cells from pre-pubertal rats and and progressive sperm motilities at 0, 30 and 60 min post-thaw were analyzed. gonocytes in testicular tubules of congenital iPSC-derived human PGC-like cells (hPG- UDT after gonocyte transformation. In con- CLC). These cells resemble early, migratory Results The groups TB and TM maintain clusion, we found that gonocytes transform PGCs and can recapitulate initial steps of sex the best viability, and only TM significantly into SSC at 2–6 days of age in mouse. Like differentiation. Our system enabled observa- decrease the membrane lipid disorder. The human minipuberty does exist in mouse and tion and manipulation of testicular cord-like SM, TB and TM groups presented a better coincides with gonocyte transformation into re-assembly and by adapting medium and mitochondrial membrane potential and sig- SSC. Gonocyte transformation in mouse is culture conditions, we facilitated hPGCLC nificantly lower production of 02- and perox- independent from androgen but gonodotro- integration into the somatic surrounding for ynitrite than the control, only TM decreased phin deficiency caused germ cell death. Dis- up to two weeks. Human PGCLCs main- the total ROS production, while SM reduced ruption of apoptosis regulator, Bax, caused tained their PGC-like morphology and ex- the lipid peroxidation. To total and progres- persisting gonocytes. Orchidopexy at older pressed the PGC markers OCT4, SOX17, and sive motility, the groups SM and TM were age showed significant germ cell depletion. AP2y. To assess their differentiation, we are significantly higher than the control at T0, These results suggest that gonocyte trans- analysing male specific gene expression and however, in all groups there was a negative formation into SSC is the key to understand changes in methylation. effect of the incubation time (T0 vs T60). infertility and malignancy of cryptorchidism. To transfer the organoid towards a homolo- Conclusion The replacement of lactose by gous setting, we applied previously published sucrose or trehalose and the supplementation protocols for the derivation of adult human of the medium with melatonin, diminish the testicular cells from testes obtained by gen- presence of nitrosative/oxidative stress mark- der-confirming surgery of male-to-female ers, since they counteract the harmful effects transgender patients. They maintain their of free radicals, in particular peroxynitrite,

40 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

P 4.1 P 4.2 Patient A 32-year-old man was referred for diagnostic workup due to infertility. Due to Novel model of sex steroid de­ Alphoma – a pituitary tumor intensifying visual disturbances and head- ficiency in mice to study the phy­ ­causing infertility aches, the patient went to an ophthalmolo- siological effects of delayed and K. Jankowska, P. Dudek, W. Zgliczyn´ski gist. A brain tumor was suspected based on suppressed puberty Centre of Postgraduate Medical Education, Depart- a fundus examination. Pituitary tumor was confirmed in an MRI scan. The patient was 1 1 1 1 ment of Endocrinology, Warsaw, Poland N. Kim , R. Khalil , K. David , L. Antonio , referred for further diagnostics to our Depart- D. Schollaert1, L. Deboel1, E. Van Herck1, Introduction Pituitary tumors can cause in- ment of Endocrinology. N. Wardenier2, M. Cools2, B. Decallonne1, fertility. Prolactin levels are not always very A contrast-enhanced MRI confirmed a 3 cm F. Claessens3, V. Dubois1, D. Vanderschueren1 high. Macroadenomas (tumors ≥ 10 mm) ad- pituitary tumor with pressure on the intersec- 1KU Leuven, Clinical and Experimental Endocrinology, ditionally give neurological symptoms like tion of the optic nerves. The tumor complete- Leuven, Belgium; 2Ghent University, Department of visual disturbances and headaches. ly destroyed the pituitary gland. Internal Medicine and Pediatrics, Ghent, Belgium; 3KU Leuven, Department of Cellular and Molecular Hyperprolactinemia in men most often leads Result A successful neurosurgical proce- Medicine, Leuven, Belgium to erectile dysfunction and decreased libido. dure was performed involving the excision Over time, gynecomastia may also develop. of a major portion of the tumor, thus saving Introduction Sex steroids are critical for This is caused by hypogonadism and a de- the patient’s sight and life. A histopathologi- skeletal development and maturation during crease in the testosterone to estrogen ratio, cal evaluation and clinical examinations have DGA-Abstracts puberty as well as skeletal maintenance dur- which causes mammary gland hyperplasia. allowed a new type of pituitary tumour – an ing adult life. However, the exact time during Testosterone substitution or treatment with alphoma, to be recognized. puberty when sex steroids have the highest dopamine agonists protects against the ef- Conclusion Alpha-subunit tests should also impact as well as the ability of bone to re- fects of hypogonadism. Erectile function and receive consideration in treating patients with cover from transient sex steroid deficiency libido return but there is no improvement a pituitary tumour. Perhaps the alpha-subunit is unclear. The latter is highly relevant in the in spermatogenesis. An operation and exo­ concentration in patients with infertility and clinical context of delayed puberty, since the genous gonadotropins can be used us an ef- hyperprolactinaemia should be measured impact of a delayed pubertal onset on adult fective therapy in such patients. (Fig. 5). bone health remains elusive. Surgical cas- tration is a common technique to study sex steroid effects in rodents, but it is irrevers- ible, invasive, and associated with metabolic and behavioral alterations. Hence, alternative approaches are needed to study timing and reversibility of sex steroid actions. Materials and Methods We used a low dose (LD) or a high dose (HD) of gonadotropin- releasing hormone antagonist to either tem- porarily or persistently suppress sex steroid­ action in male mice, respectively. Growth, body composition and bone parameters were determined. Results The LD group, a model for de- layed puberty, did not show changes in linear growth or body composition, but displayed reduced trabecular bone volume during pu- berty, which fully caught up at adult age. In contrast, the HD group, representing complete pubertal suppression, showed a phenotype reminiscent of that observed in surgically castrated rodents. Indeed, HD ani- mals exhibited severely impaired cortical and trabecular bone acquisition, decreased body weight and lean mass, and increased fat mass. In addition, the HD group was characterized by an increased linear growth, which is remi- niscent of the clinical observation in patients with hypogonadotropic hypogonadism. Conclusions We validated a new rodent model of chemical castration, which can be used as an alternative to surgical castration. Moreover, the transient nature of the inter- vention enables to study the effects of de- layed puberty and reversibility of sex steroid deficiency. Our work suggests that, at least in mice, a delayed pubertal timing is associated with bone loss during puberty, but this delete- rious effect does not persist at adult age.

Figure 5. K. Jankowska, et al.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 41 Andrology 2020 – Abstracts

Materials and Methods A systematic search was performed in PubMed, EMBASE, Cochrane Library, Web of science, Science Direct and CINAHL. Standardized mean differences (SMDs) and 95% confidence intervals (CIs) in total testosterone (TT) lev- els between men with 25-hydroxyvitamin D (25[OH] D) < 20 and ≥ 20 ng/mL were com- bined using random effects models. Funnel plot and trim-and-fill analysis were used to assess publication bias. Heterogeneity source was explored by a sub-group analysis accord- ing to health-related characteristics of the study populations. Results Eighteen included studies collec- tively gave information on 9892 men with vitamin D deficiency and 10675 controls.

DGA-Abstracts The pooled SMD revealed a slight, albeit just significant, positive association between 25(OH)D and TT (pooled SMD: -0.23, 95% CI: –0.45 to –0.01; p = 0.04) with a large be- tween-study heterogeneity (I-squared = 98%, p-value for heterogeneity < 0.00001). At the sub-group analysis, a significant positive as- sociation, along with noticeable decrease in heterogeneity, could only be demonstrated Figure 6. J. Rohayem, et al. © J. Rohayem in studies of patients with frailty states (pooled SMD: -0.19; 95% CI: –0.27, –0.10, P 4.3 and the testosterone (T) metabolites DHT and p < 0.0001; I-squared = 51%, p-value for 17β-estradiol (E2) were similar to controls. heterogeneity = 0.06). A sensitivity analysis The role of gonadotropins in testi- revealed a high stability of the result and the On gonadotropin replacement, 17-OHP, cular and adrenal androgen bio- trim-and-fill adjustment for publication bias 17-OH-pregnenolone, DHEAS and andros- did not affect the pooled estimate. synthesis pathway – insights from tenedione, as well as DHT, androstenediol, males with congenital hypogona- and all 11-oxygenated C19 androgens were Conclusions The results of the present normal. Progesterone (Δ4 pathway) was meta-­analysis support the notion that hypo­ dotropic hypogonadism on hCG/ vitaminosis D and androgen deficiency rFSH and on testosterone replace- slightly decreased, androstanediol (backdoor DHT pathway) and E2 (T metabolite) were should be regarded as markers of a poor ment increased. health status, sharing common underlying 1 1 2 aetiologies and risk factors. J. Rohayem , M. Zitzmann , S. Laurentino , Conclusions In males with CHH, serum S. Kliesch1, E. Nieschlag1, P. M. Holterhus3, A. Kulle3 steroid hormone profiles resemble those of 1Centre for Reproductive Medicine and Andrology, healthy men, if hCG/rFSH is used for sub- P 4.5 Clinical and Operative Andrology, Münster, Germany; stitution. Gonadotropins contribute to steroid 2 Centre for Reproductive Medicine and Andrology, hormone production along the classic Δ4 Gender dysphoria and hormonal Reproductive and Regenerative Biology, Münster, pathway and co-activate an alternative path- treatment: Ten years’ experience Germany; 3Children´s Hospital, University of Kiel, way of testosterone biosynthesis via andros- of a specialized center in Greece ­Pediatric Endocrinology and Diabetes, Kiel, Germany tenediol. Backdoor DHT biosynthesis, Δ5 I. Kakoulidis, I. Ilias, C. Milionis, A. Michou, Introduction By this case-control study we 17-OH-pregnenolone, DHEA(S) and andros- tenedione synthesis and 11-oxygenated C19 S. Stergiotis, S. Togias, A. Pappa, E. Venaki, aimed to delineate the role of gonadotropins E. Koukkou in male androgen biosynthesis pathways androgen production are activated indepen- dently of gonadotropins (Fig. 6). Elena Venizelou General and Maternity Hospital, De- Patients and Methods 25 males with con- partment of Endocrinology, Diabetes and Metabo- genital hypogonadotropic hypogonadism lism, Athens, Greece (CHH) underwent hCG/rFSH and testoster- P 4.4 one treatment sequentially. Serum steroid In response to the growing trend in the lit- hormone profiles (testosterone precursors and Relationship of vitamin D status erature on developing a management frame- metabolites) on both replacement regimens with testosterone levels: a sys- work for hormonal therapy (HT) in Gender dysphoria (GD), we present our department’s were analyzed, using liquid chromatography- tematic review and meta-analysis tandem mass spectrometry (LC-MS/MS) latest relevant experience. and compared to those of healthy controls, A. Barbonetti, S. D’Andrea, A. Martorella, F. Coccia, We conducted a retrospective case file study matched by age, BMI and serum testosterone. C. Castellini, M. Totaro, A. Parisi, F. Francavilla, on patients with GD, who attended our out- S. Francavilla On testosterone replacement, serum patient clinic from 2009–2019. In total 56 pa- Results University of L’Aquila, Andrology Unit, Department of concentrations of the classic 4 pathway hor- tients were included. 26 were trans-females/ Δ Life, Health and Environmental Sciences, L’Aquila, mones progesterone and 17-hydroxy-proges- TF (mean age ± SD 27.4 ± 10.0 years, HT Italy terone (17-OHP), and the marker steroid of an duration 3.2 ± 2.1 years) and 30 were trans- alternative pathway of testosterone synthesis Introduction In spite of the biological plau- males/TM (age 26.1 ± 5.6 years, HT duration (androstenediol) were decreased, compared sibility of a direct link between low vitamin 3.8 ± 2.6 years). to controls. Androstanediol, a marker of the D and androgen deficiency, the association HT in TFs was an antiandrogen/estrogen backdoor pathway of dihydrotestosterone remains inconclusive in epidemiological combination. GnRH agonist was also given (DHT) synthesis, was increased. 17-OH-preg- studies. Therefore, this systematic review and in 10 patients. No significant disturbance in nenolone, androstenedione and DHEAS (Δ5 meta-analysis of case-control studies aims to the lipid, haematological or hepatic profile pathway), three 11-oxygenated C19 androgens assess whether and in what populations such was noted. Total testosterone levels dur- (11-keto-A4, 11-keto-T and 11-keto-DHT) an association can be demonstrated. ing HT were 0.28 ± 0.2 ng/ml (pre-HT 6.0

42 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

± 5.2 ng/ml). TSH levels during treatment and adult KS (p < 0.001, = 0.01 and < 0.001, Conclusions Following LHRH stimula- were almost stable (from 2.3 ± 1.3 μIU/ml to respectively), whereas fT3 levels are reduced tion, T-allele carriers for the FSHB -211 G>T 2.8 ± 1.3 μIU/ml), while 25(OH)VitD levels in pubertal children and adults (p <0.001 and variant reached higher concentrations of LH were 17.37 ± 9.2 ng/ml. PRL levels tended = 0.01) and TSH values are lower in adult KS and lower concentrations of FSH than GG to increase at 32.4 ± 24.5 ng/ml, 6 months (p = 0.04). An increased prevalence of Ab- carriers, suggesting pituitary dysregulation. after HT (p = NS). In one case treatment Tg/TPO positivity is present in all age groups. Especially for the latter, this is in accordance with cabergoline was initiated. Osteoporosis A lower TV, alongside reduced echogenicity with reduced FSHB promoter activity leading treatment was initiated in one patient. Body and increased parenchymal inhomogeneity is to reduced FSH secretion and with different weight change was –0.27 ± 5.6 kg. present in KS, similar to CLT subjects. Tes- dynamics of LH release from the pituitary. HT in TMs was initially transdermal and then tosterone regulates the balance between thy- injectable forms of testosterone. In 4 patients, roid hormones, as evidenced by group differ- GnRH agonist was given for induction and ences according to gonadal status, confirmed maintenance of amenorrhea. Total testoster- by linear regression and a positive correlation P 4.8 between cfTe levels and the fT3/fT4 ratio. one levels, during HT, showed a tendency to Serum and seminal leptin levels, stabilize at 4.9 ± 2.3 ng/ml (pre-HT 0.9 ± 1.4 Conclusions KS is characterised by a com- serum testosterone levels and ng/ml). Mean Hemoglobin levels increased at bined form of hypothyrodism, already pre- 15.2 ± 1.0 g/dl (pre-HT 12.9 ± 1.1 g/dl). In sent in pre-pubertal children and potentially sperm parameters in obese men one case erythrocytosis occurred and treat- contributing to the overall clinical picture. V. Rilcheva1, K. Ananieva-Todorova2, F. Mehmedova1 DGA-Abstracts ment was stopped. Lipid and hepatic profile 1MC CIRM, Endocrinology, Pleven, Bulgaria; 2Medical were constant, while a slight decrease (p = University Pleven, University Clinic of Endocrinology NS) in TSH levels was observed during HT P 4.7 and Metabolic disease, Pleven, Bulgaria (from 3.22 ± 1.7 μIU/ml to 2.29 ± 1.1 μIU/ ml). 25(OH)VitD mean levels were 16.85 ± Pituitary response to LHRH stimu­ Introduction Leptin is a peptide hormone, 8.8 ng/ml. Body weight change was +3.6 ± lation tests in different FSHB secreted by white adipose tissue. The pres- 5.5 kg. -211G>T genotypes ence of leptin receptor on human spermatozoa and soluble leptin receptor in seminal plasma Five TFs underwent gender reassignment sur- 1 2 3 A. Sansone , M. Schubert , F. Tüttelmann , C. Krall- are well known. This receptor was signifi- gery (orchiectomy+colpoplasty) and 14 TMs 2 2 2 2 mann , M. Zitzmann , S. Kliesch , J. Gromoll cantly associated with the intactness of sperm respectively (7 mastectomy+hysterectomy, 5 1University of Rome Tor Vergata, Chair of Endocrino- mastectomy, 1 hysterectomy, and 1 mastecto plasma membranes. The aim of this study is logy and Medical Sexology (ENDOSEX), Department to investigate the relationship between serum my+hysterectomy+phalloplasty). 2 of Systems Medicine, Rome, Italy; University Hospi- and seminal plasma leptin levels, serum tes- tal Münster, Centre of Reproductive Medicine and 3 tosteron levels and sperm parameters in obese P 4.6 Andrology, Münster, Germany; University of Müns- and normal weigh ter, Institute of Reproductive Genetics, Münster, A combined form of hypothyroi­ Germany­ Patients and Methods The study includes dism is present in Klinefelter syn­ 60 obese men and 30 normal weight men, be- Introduction FSHB -211G>T variant drome from pre-pubertal children tween 20 and 50 years. Semen samples were (rs10835638) is known to affect pituitary analysed for sperm volume, concentration, to adult subjects: a longitudinal FSH output by impairing the transcriptional motility and morphology as well as for sperm retrospective study activity of FSHB. DNA fragmentation index. Serum levels of F. Carlomagno, P. Mazzotta, F. Sesti, M. Tenuta1, Materials and Methods This was a cross sex hormones were determined by immuno- M. Minnetti, C. Lecis, A. Aureli, M. Spaziani, sectional, retrospective study on 67 consecu- chemiluminiscence technique. Serum and C. Pozza, D. Gianfrilli, A. Radicioni, A. Lenzi, tive males undergoing LHRH stimulation test seminal plasma leptin levels as well as estra- A. M. Isidori for diagnostic purposes in cases of secondary diol and testosterone in seminal plasma levels Sapienza University of Rome, Department of Ex­ hypogonadism, performed by administering were tested by ELISA. perimental Medicine, Rome, Italy an intravenous bolus of 100 µg of the LHRH- Results The serum leptin level showed a analogue gonadorelin acetate, with blood sam- statistically significant difference between Introduction Hypothyroidism has been ples drawn immediately prior to injection (T0), the obese men group versus the normal proposed to contribute to the clinical picture and after 30 (T1) and 45 minutes (T2). Clinical weight men group (p < 0.05). In seminal of Klinefelter syndrome (KS). However, the and genetic data were retrieved from an elec- plasma, in regard of leptin, such a difference available studies are limited and fragmentary. tronic database. Linear longitudinal mixed- was not found. Seruml leptin levels in obese The present study aimed at describing thyroid effect models were used to assess the FSHB men correlate negatively with sperm motility. function and structure in KS, from pre-puber- genotype’s effects on FSH and LH levels over This study does not found a significant dif- ty throughout adulthood. time via additive and recessive models. ference in serum testosterone levels between Patients and Methods 254 KS individuals Results An overall marked increase in se- obese and normal weight men. were included, divided according to pubertal rum FSH and LH following administration status. Adults were further classified into The significant differences in the average of 100 µg of the LHRH-analogue was found sperm concentration and teratozoospermia eugonadal, hypogonadal and those receiv- (p < 0.0001 for linear trend, both models). ing testosterone replacement therapy. 300 index were observed by comparisson be- Peak levels of LH were significantly higher tween obese and non obese men. age-matched healthy and 61 adult chronic in TT than in GT and GG carriers (p = 0.012); lymphocytic thyroiditis (CLT) individuals no significant between-groups difference was Conclusion This pilot study showed that served as control groups. Serum TSH, fT3, found concerning stimulated FSH levels. In obesity adversely affects sperm parameters fT4, Ab-Tg/TPO, SHBG and testosterone both the additive and recessive models, the such as decreased sperm motility and in- concentrations were measured. The fT3/fT4 main effect of T allele(s) did not reach sta- creased teratozoospermal index. Plasma lep- ratio was derived and free testosterone (cfTe) tistical significance concerning FSH levels tin levels are significantly lower in patients was calculated. Thyroid ultrasound was (p = 0.8564 and p = 0.7520, respectively), yet with normal sperm parameters and showed in performed, and thyroid volume (TV) was interaction effects over time demonstrated versely proportional correlation with sperm measured, echogenicity was quantitatively an attenuated response in T-allele carriers motility. assessed while echotexture parameters were compared to the GG carriers (p = 0,0219 and The pandemic spread of obesity necessitates estimated through grey level co-occurrence p = 0,0276). Main and interaction effects further studies to clarify the additional links matrix (GLCM) analyses. for LH were significant in both the additive and mechanisms that are manifested at an Results Compared to healthy controls, fT4 (p = 0.017 and p = 0.0013) and recessive early stage and are related to violations of levels are reduced in pre-pubertal, pubertal model (p = 0.0019 and p = 0.0016). reproductive function in obese men.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 43 Andrology 2020 – Abstracts

P 4.9 tics in clinical routine as well as in research. published 10X genomics data, analysis of The current hierarchy of terms concerning single cell gene expression of human testicu- Action of progesterone on 2-ara­ infertile patients warrants improvements as lar WWC proteins revealed low WWC1 and chidonoylglycerol (2-AG) levels several phenotypes are not annotated and WWC3 levels. In contrast, WWC2 mRNA was and enzymes metabolizing 2-AG others are not linked in the correct hierarchy. highly expressed in various cellular subpopu- in human sperm Thus, we designed a revised HPO framework lations and its expression pattern overlapped with zygotene and pachytene 1 2 1 1 for male infertility that is easy to understand T. Depmeier , T. Strünker , M. Lehr , J. Fabian , clusters. WWC1 and WWC2 mRNA levels of 1 and straightforward in its application but does T. Philipps testis tissue were compared between patients 1Institute of Pharmaceutical and Medicinal Chemis- contain most relevant information to study the impact of an identified mutation on sper- with complete spermatogenesis (CS, n = 5) try, University of Münster, Münster, Germany; and those with Sertoli cell only (SCO, n = 2 matogenesis. To this end, an overview of dia­ Centre of Reproductive Medicine and Andrology, 5) or Klinefelter syndrome (KS, n = 3), both University of Münster, Münster, Germany gnoses for male infertility was made. From these, a branching structure was conceived: lacking spermatogenesis. WWC1 was uni- Introduction The sperm-specific CatSper the proposed “HPO tree”. formly expressed at low levels, while WWC2 2+ was highly expressed in testes with CS but controls the intracellular Ca concentration Currently, we focus on a framework for testis and, thereby, the function of sperm. Hu- significantly reduced in SCO or KS tissues. histopathology that is based on the presence Exome screening of patients with severe sper- man CatSper is activated in a non-genomic of germ cells and Sertoli cells, comprising

DGA-Abstracts matogenic failure (n = 735) revealed 20 rare ­fashion by oviductal steroids, e.g., progester- five main categories. In addition to Sertoli one. It has been proposed that progesterone heterozygous missense and 3 rare heterozy- cell-only phenotype, we add the classifica- gous splice variants (minor allele frequency stimulates the activity of membranous serine tion of tubular shadows, when also Sertoli hydrolase α/β hydrolase domain-containing < 0.01) in the WWC2 gene. Our data indicate cells are absent. When germ cells are present, specific expression patterns of WWC genes in protein 2 (ABHD2), which in turn degrades their stage of arrest, number and distribution 2-arachidonoylglycerol (2-AG) in the sperm testis. Wwc2 expression increases during mu- is informative about the effect of the causal rine postnatal development. Human WWC2 is membrane, relieving CatSper from inhibition gene with the causal mutation. Hence, we by 2-AG. expressed heterogeneously in spermatogenic propose to distinguish Germ Cell Arrest cell types. Testis tissues of patients lacking Material and Methods We investigated (GCA), in which no mature spermatozoa are spermatogenesis showed decreased WWC2 the progesterone-control of 2-AG turnover observed, from hypospermatogenesis (HS). expression and several heterozygous WWC2 in human sperm using the fluorescent 2-AG To establish a lower threshold for complete variants were identified in exomes of patients mimetic 1,3-dihydroxypropan-2-yl 4-pyren- spermatogenesis, we determined the number with unexplained infertility. Thus, WWC2 1-ylbutanoate (DPPB) and the serine-hydro- of tubules that contain elongating spermatids could be a potential regulator for germ cell lase inhibitor methyl arachidonyl fluorophos- (ES) in a collection of men with obstructive differentiation and male fertility. phonate (MAFP). Moreover, we analyzed azoospermia (N = 117). By these means, we the action of progesterone on 2-AG levels in introduce an evidence-based cut-off for tu- human sperm using liquid-chromatography bules containing ES to distinguish HS from combined with mass spectrometry (LC/MS) complete spermatogenesis. P 5.3 Results We show that human sperm me- We conclude that once this framework is Swine spermatozoa trigger tabolize DPPB. The turnover of DPPB is sup- incorporated in the HPO, the standardized ­neutrophil extracellular traps pressed by MAFP as well as by 2-AG, con- vocabulary will facilitate communication in (nets) leading to adverse effects firming that DPPB is metabolized by­serine clinical routine as well as in research between hydrolases and that DPPB competes with collaborating institutions and inform gene on sperm function 2-AG for the same active sites, respectively. discovery. R. Sánchez1, F. Zambrano1, C. Namuncura1, F. Pezo1, Progesterone does, however, not enhance P. Uribe1, M. Schulz1, R. A. Burgos2, A. Taubert3, DPPB turnover. Moreover, we show that C. Hermosilla3 2-AG levels in human sperm are similar be- P 5.2 1Universidad de La Frontera, Temuco, Chile; 2Univer- fore and after stimulation with progesterone. sidad Austral de Chile, Institute of Pharmacology Do WWC proteins have an impact 3 Conclusion We conclude that the enzymes and Morphophysiology, Valdivia, Chile; JL University metabolizing 2-AG in human sperm are, in on germ cell differentiation and of Giessen, Institute of Parasitology, Giessen, fact, insensitive to progesterone. In line with male fertility? Germany­ this finding, progesterone does not decrease V. Höffken1, N. Neuhaus2, F. Tüttelmann3, Introduction In pigs, the number of PMN 2-AG levels in human sperm. These result H. Pavenstädt1, J. Kremerskothen1 in uterus lumen increases within few hours contest the proposed mechanism of steroid- 1University Hospital Münster, Molecular Nephrology, after natural or artificial insemination result- 2+ activation of CatSper Ca channels. Internal Medicine D, Münster, Germany; 2University ing in early PMN-derived innate immune re- Hospital Münster, Centre of Reproductive Medicine actions. Sperm-NETs formation was recently and Andrology, Münster, Germany; 3University Hos- reported to occur in various mammalian spe- P 5.1 pital Münster, Institute of Reproductive Genetics, cies. The objective was to evaluate the direct Improving the HPO terms for non- Münster, Germany interactions of boar spermatozoa with swine PMN, the release of sperm-mediated NETs, syndromic male infertility Over 70% of male infertility cases remain and to assess NET-derived effects on sperm unexplained. At least 4% of diagnosed cases M. J. Wyrwoll1,2, G. W. van der Heijden3, L. Ramos3, functionality. underlie genetic reasons, hence research on S. Kliesch2, F. Tüttelmann1 genes affecting male fertility is needed. WWC Material and Method PMN/spermatozoa 1University of Münster, Institute of Human Genetics, proteins are known to regulate cell prolifera- were co-cultured in an incubator at 37° C. Münster, Germany; 2University Hospital Münster, tion, differentiation, and organ growth, thus The kinetic studies were evaluated for 1, 3 Centre of Reproductive Medicine and Andrology, being interesting candidates in context of and 5 h of exposure and with a cell ratio of Münster, Germany; 3Radboud University Medical 5 male fertility. Our project aims to elucidate 1:3 (PMN [2.5 × 10 ] and spermatozoa [7.5 × Center, Division of Reproductive Medicine, Depart- 5 the role of WWC proteins in spermatogenesis 10 ]). Sperm-triggered NETs were visualized ment of Obstetrics and Gynecology, Nijmegen, and male germ cell differentiation. qPCR by SEM- and immunofluorescence analyses. Netherlands­ analysis of murine testis revealed low Wwc1 Sperm-mediated NETosis was confirmed by The Human Phenotype Ontology (HPO) is but high Wwc2 mRNA expression. The Wwc2 presence of extruded DNA with global his- a formal dictionary of human disease phe- level increases during murine postnatal de- tones and NE. notypes and widely applied in genetics. It velopment, similar to expression patterns Results Sperm-mediated NETosis was allows phenotype driven differential diagnos- of germ cell markers (e.g. Ddx4). Based on confirmed by presence of extruded DNA

44 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts with global histones and NE. Largest sizes of Results We show that, with no exception, fied as non-smoking and men with levels sperm-mediated aggNETs were detected af- the steroids and prostaglandins identified in classified as smoking (DFI 13% vs 12 %, ter 5 h thereby resulting in effective massive FF and SF, respectively, serve as agonists that p = 0.48). sperm entrapment. The number of aggNETs activate CatSper via their respective binding Conclusion In the exposure situations of increased from 3 h onwards. Kinetic studies sites; yet, with different potency and effi- these men, smoking and PAH exposure did of swine sperm-mediated NETosis showed cacy. Moreover, we show that many of these not seem associated with DFI and potential to be a time-dependent cellular process. In steroids and prostaglandins are present in germ cell mutations, to the extent that DFI is addition, number of NETs-entrapped sper- FF or SF at concentrations that readily acti- an appropriate marker for the latter. matozoa increased at 3 h of exposure whilst vate ­CatSper. This suggests that they act as few free spermatozoa were detected after 3 chemical cues orchestrating the fertilization h. Anchored NETs also increased from 3 h process. Finally, we found that prostaglan- onwards. The cytotoxicity of NETs was con- din-, but not steroid-activation of CatSper is P 5.6 firmed by diminution of the total motility and suppressed by Zinc ions present in SF. This the progressive motility. Spermatozoa mem- might suppress premature prostaglandin- Indication of novel alternate brane integrity and function loss exposed to induced hyperactivation of sperm right after ­pathway of testosterone action in NETs was confirmed from 3 h. ejaculation. spermatogenic germ cells of male Conclusion NETs-derived induced damag- Conclusion Our results reveal the pro- sprague dawley rats ing effects on swine spermatozoa in mem- miscuous activation of CatSper by SF- and DGA-Abstracts brane integrity, -motility and functionality. FF-derived prostaglandins and steroids, re- A. Talapatra, N. Mills We hypothesize that swine sperm-triggered spectively, highlighting the physiological sig- Texas Womans’s University, Biology, Denton, TX, aggNETs might play a critical role in reduced nificance of these ligands. Furthermore, we United States fertility potential in swine reproductive tech- propose that the interplay of prostaglandins Introduction Leydig cell toxicant ethane nique. and Zinc in the ejaculate serves as a dilution dimethane sulfonate (EDS) triggers germ cell Acknowledgements Supported by FON- sensor that is crucial for the timing of motility apoptosis depleting testosterone (T) and in- DECYT REGULAR Project N° 1180912, responses in the female genital tract. terfering spermatogenesis. Testosterone acts ANID, Chilean Government and Post-Doc- via both canonical and non-canonical path- toral Grant, VRIP, Universidad de La Fron- ways primarily inducing nuclear androgen re- tera, Chile ceptors (nARs) canonically. We investigated P 5.5 existence of a functional non canonical path- way novel for testosterone in testicular germ Exposure to polycyclic aromatic cells. 10 days post-EDS treated male rats P 5.4 hydrocarbons and nicotine and were compared with testosterone-replaced sperm DNA strand breaks (EDS+extrinsic-T) and testosterone-supple- Pharmacology of the steroid- mented rats. Testosterone and progesterone J. Axelsson and prostaglandin-binding site have similar receptor binding site hence pres- Lund University, Translational Medcine, Malmö, ence of germ cell specific membrane proges- controlling the activity of CatSper Sweden channels in human sperm terone receptors (mPRs) were investigated. Introduction Smoking has been suggested mPRs in other tissues change ion influx via J. Jeschke1, C. Biagioni1, F. Börgel2, A. Schüring2,3, to cause mutations in sperm cells, and po- Progesterone Receptor Membrane Compo- A. Kulle4, P. M. Holterhus4, B. Wünsch2, V. Nordhoff1, tentially to be due to exposure to polycyclic nent 1 (PGRMC1). We further investigated T. Strünker1, C. Brenker1 aromatic hydrocarbons (PAH). Smoking whether testosterone binds mPRs in presence 1University Hospital Münster, University of Münster, has also been reported to be associated with of low level progesterone, mimicking normal Centre of Reproductive Medicine and Andrology, sperm DNA fragmentation, whereas DNA testicular environment. Münster, Germany; 2University of Münster, Depart- fragmentation more generally has been as- RT-qPCR, western ment of Pharmaceutical and Medicinal Chemistry, Materials and Methods sociated with the mutagenic potential of a blot, binding assay and IHC. Münster, Germany; 3University of Münster, UKM Kin- chemical. Accordingly, the aim of this study derwunschzentrum, Münster, Germany; 4Christian- was to study whether metabolites of PAHs Results EDS treatment caused significant Albrechts-University, Division of Pediatric Endocrino- and of nicotine, as biomarkers of exposure, reduction of 3βHSD2 and Insl3 transcrip- logy and Diabetes, Department of Pediatrics, Kiel, were associated with sperm DNA fragmenta- tion in Leydig cells along with testosterone Germany tion as a potential marker of mutations. depletion in serum and testes. The resultant testosterone loss increased germ cell apop- 2+ Introduction In human sperm, the Ca Patients and Methods From two cohorts of tosis significantly reducing testicular weight channel CatSper translates changes in the young men recruited from the general Swed- compromising blood-testes-barrier (BTB) in- chemical microenvironment into changes in ish population, the PAH metabolites 1-hy- tegrity. nARs were present in nuclei of Sertoli swimming behavior. As sperm pass through droxypyrene and 2-hydroxyphenanthrene,­ as & myoid cells and stripped cytoplasm from the male and female reproductive tract, they well as cotinine, were measured in urine from spermatids but absent in germ cells. Tran- are exposed to multiple chemical cues, such 381 men. Sperm DNA fragmentation index scription of five mPRs α( , β, γ, δ and ε) oc- as those present in seminal (SF) and follicular (DFI) was analysed by the Sperm Chromatin curred in male rat testes among which α and fluid (FF), which contain a complex mixture Structure Assay. Associations between me- β showed successful protein expression. Our of prostaglandins and steroids, respectively. tabolites as continuous variables, as well as novel results show successful identification It is well-established that CatSper is activated in quartiles, and DFI were studied by general of mPRs on isolated germ cells via IHC and by steroids and prostaglandins via two dis- linear models, adjusted for abstinence time. competitive binding assay shows successful tinct, extracellular binding sites. However, A similar analysis was done for cotinine cate- binding of testosterone with mPRs even in the pharmacology of the steroid and prosta- gorised as “non-smoker” (31 men), “environ- presence of low level progesterone. glandin binding site in human sperm is ill- mentally exposed” (141 men) and “smoker” defined. In particular, it is largely unknown (209 men). Conclusion Novel results imply presence of how the different steroids and prostaglandins an alternate pathway of testosterone action in Results No association was found between testes opening novel avenues to further inves- present in SF and FF affect the activity of any of the three biomarkers as continuous CatSper. tigate for alternate therapeutic targets in the variables and DFI (p = 0.35 to 0.99), and no future for solving male reproductive issues. Methods We studied the action of FF, SF difference found in DFI between the lowest and a set of different steroids and prostaglan- and highest quartile of the levels of the three dins identified in these fluids on CatSper in different biomarkers (p = 0.11 to 0.61), nor human sperm using Ca2+ fluorimetry. between men with levels of cotinine classi-

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 45 Andrology 2020 – Abstracts

P 5.7 strong rationale for the novel approaches in using a BD FACSMelody, we found higher conservation and fertilization techniques. levels of sperm DNA fragmentation (sDF) Identifying novel activin A targets The aim of the present study was to evaluate in the former, as assessed with Sperm Chro- in mouse gonocytes the physiological response of sperm mito- matin Dispersion test (mean, SD: 36.2,13.0 S. Moody1,2, P. Whiley1,2, P. Western1,2, K. Loveland1,2 chondria after sexual activity. vs 11.5,5.8; n = 4). This result is consistent 1Hudson Institute of Medical Research, Centre for The tissue samples from 29 weeks old male with the well-known link between sDF and Reproductive Health, Clayton, Australia; 2Monash Swiss Webster mice were collected on the OS and further validated the reliability of University, Department of Molecular and Trans­ next day after mating or from animals with- the new technique. We evaluated LOS in 69 lational Sciences, Clayton, Australia out sexual activity. The mitochondrial func- subfertile men (SM, consecutively collected tionality of the spermatozoa was evaluated among male partners of infertile couples) and Disruptions to fetal male germ cell (gono- using high-resolution fluorespirometry. Flux 10 donors (DM, recruited after excluding any cyte) development underpin infertility and control factors (FCF, specific contribution of condition, due to life style or medical his- reproductive disorders including testicular each substrate to mitochondrial oxygen con- tory, known to increase OS). We did not find germ cell tumours. We showed the TGF β sumption) were calculated. any difference between SM and DM (me- superfamily member activin A (encoded by dian [IQR]: LOS = 11.2 [8.1–18.9] and 12.6 Inhba) directly affects germline differen- After sexual activity, the spermatozoa [10.3–13.8] in SM and DM, respectively), tiation transcripts in a human gonocyte-like ­ROUTINE respiration was significantly however 28 out 69 SM (40%) showed a LOS cell. To reveal how testicular somatic cells higher. Moreover, mitochondrial complex th

DGA-Abstracts value higher than the 75 percentile of DM. support normal gonocyte development, this I-linked respiration at LEAK and oxida- We noticed that LOS was particularly high in study used mouse models to test the hypo­ tive phosphorylation (OXPHOS)-dependent SM with presence of bacteria in semen (4 out thesis that activin A acts directly on gono- states were significantly higher after sexual of 69). This result was confirmed by recruit- cytes to promote differentiation. The effects activity. However, there was no difference ing further subjects with bacteria in semen, of chronic activin A absence were assessed in ­OXPHOS coupling efficiency between where median LOS was 32.1 [12.8–37.4] (n = by RNA-Seq of gonocytes isolated by FACS groups, indicating on the proton leak after 18), much higher than the values found both from embryonic day (E) 13.5 and E15.5 tes- sexual activity, that was followed by increased in SM and in DM (p < 0.0001). In conclusion, tes from Inhba wildtype (WT) and knockout H2O2 (reactive oxygen species) production this study presents a new flow cytometric (KO) mice expressing the germline-specific rate in the sexually active group. In compari- technique to detect OS in live spermatozoa Oct4-Gfp transgene (n = 3–4). Also, whole son to mice without sexual activity, the FCF of native semen samples resulting sharply testis transcripts from E13.5 and E15.5 mice of glycerol 3-phosphate was decreased, while linked to sDF. This technique could be used lacking activin A (InhbaKO) and with elevat­ FCF of succinate, complex II substrate, was to identify SM with high levels of semen OS. ed activin A (InhaKO) were compared with increased in spermatozoa mitochondria of WT littermates (n = 3). To discern acute sexually active mice, indicating on energetic and direct effects of activin A and its inhibi- pathway switch after sexual activity. Besides, P 5.10 spermatozoa count was decreased in mice tion, isolated E13.5 gonocytes were cultured Optical recording of rapid non-­ 24h in media with either 5 ng/mL activin after mating. A, 10 µM SB431542 (activin/Nodal/TGFβ Our data show that spermatozoa mitochon- genomic progesterone signaling inhibitor) or vehicle for transcript analysis drial functionality is altered after sexual events in human sperm (n = 5–6). RNA-Seq revealed 27 and 92 dif- activity. The altered mitochondrial bio­ M. Kierzek1, E. Miller2,3,4, H. Wills4, T. Strünker1, ferentially expressed genes in KO vs WT energetics could be associated with increase C. Brenker1 gonocytes at E13.5 and E15.5, respectively. in immature spermatozoa fraction in sperm 1Centre of Reproductive Medicine and Andrology, Interestingly, Musashi-1 was significantly samples from sexually active mice. There- University of Münster, Münster, Germany; 2Universi- lower in both KO gonocytes at E15.5 and in fore, for the cryopreservation of the semen, ty of California, Berkeley, Department of Chemistry, SB431542-treated E13.5 gonocytes. Slc38a5 samples should not be collected on the next Berkeley, CA, United States; 3University of California, was lower in E15.5 KO gonocytes, InhbaKO day after the mating. Berkeley, Department of Molecular & Cell Biology, testes at E13.5 and E15.5, and increased in Berkeley, CA, United States; 4University of Califor- both InhaKO testes and activin A-treated nia, Berkeley, Neuroscience­ Institute, Berkeley, CA, E13.5 gonocytes. Further, in gonocyte cul- P 5.9 United States tures, activin A increased differentiation markers (Piwil4, Mov10l1, Nanos2), while Detection of oxidative stress in Introduction To navigate the oviduct SB431542 decreased these and increased an native semen samples: a new and fertilize the egg, human sperm rely early marker, Kit. This multifaceted approach flow cytometric technique on chemical cues provided by the female. revealed novel activin A targets in the male Chemo­sensory signaling in sperm involves L. Riley, O. Campolattano, R. Masti, E. Baldi, germline and demonstrated direct effects of changes in the membrane potential (Vm) and M. Muratori 2+ 2+ activin A that promote a differentiated pheno- intracellular Ca concentration [Ca ]i. The type. This platform can be used to investigate University of Florence, Experimental and Clinical Bio- sperm-specific Ca2+ channel CatSper controls medical Sciences, Florence, Italy 2+ + how disruptions to activin A signalling may [Ca ] i, while Slo3, the principle K channel 2+ underpin male reproductive pathologies. Oxidative stress (OS) has been largely asso- in sperm, sets Vm in a Ca -dependent man- ciated to male reproductive dysfunction and ner. Both CatSper and Slo3 are controlled by detection of ROS (Reactive Oxygen Species) the female steroid progesterone. We proposed 2+ P 5.8 in semen is important for the assessment of that the complex [Ca ]i responses triggered male infertility. Here, the percentage of live by progesterone in human sperm rest on a The effect of sexual activity on spermatozoa with oxidative stress (LOS) in CatSper-Slo3 interplay: progesterone-in- 2+ mice spermatozoa mitochondrial native semen samples was detected using a duced Ca influx via CatSper activates Slo3 new flow cytometric technique, coupling and the ensuing hyperpolarization deactivates functionality 2+ the staining of sperm mitochondrial ROS CatSper, thereby curtailing Ca influx. B. Švalbe1, M. Makrecka-Kuka1, E. Vavers1,2, with MitoSOX™ to that of dead cells using Materials and Methods To simultaneously M. Dambrova1,2, L. Zvejniece1 the LIVE/DEAD Fixable Dead Cell Stain monitor V and [Ca2+]i signals in sperm, we 1Latvian Institute of Organic Synthesis, Ri¯ga, Latvia; m kit (LD). As expected, the technique is able developed an optical multiplexing technique 2Riga Stradins University, Riga, Latvia to detect the increase of OS after challeng- called Frequency- and Spectrally-Tuned Recent studies on sperm physiology suggest ing spermatozoa with hydrogen peroxide Multiplexing (FASTM). Moreover, we estab- the mitochondrial function as a biomarker (1 mM), menadione (50 µM) and mercapto- lished a method to convert Vm-indicator fluo- of sperm health and fertility. Targeting mi- succinic acid (2 mM). In addition, after sort- rescence recorded using FASTM into absolute tochondrial bioenergetics would provide a ing live spermatozoa with and without OS changes in Vm (in millivolts). This enabled us

46 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

to study progesterone-induced Vm signals in a P 6.1 in WC (95.5 ± 9.5 vs 95 ± 8.5; p = 0.146) quantitative fashion. and BMI (26.48 ± 3.72 vs 24.67 ± 4.15; p = Novel Indices for the evaluation Results and Conclusion We show that 0.363) was not statistically significant among progesterone evokes a rapid, transient depo- of obesity in erectile dysfunction two groups. larization followed by an extended hyperpo- patients Conclusion VAI and LAP have a stronger larization. Signals recorded from CatSper-de- S. Jose, V. Vishal, R. AT, F. Cardoza correlation with ED severity than single an- ficient sperm confirm that the Vm responses Government Medical College Kozhikode, Department thropometric tools. Considering the simplic- are initiated by progesterone-activation of of Urology, Calicut, India ity and reliability, these novel indices should CatSper. Moreover, we found that buffer- be included in the evaluation of obese ED ing of [Ca2+]; by intracellular Ca2+ chelators Introduction The association between patients. Metabolic Syndrome (MetS) and erectile perturbs the Vm response, indicating that it is shaped by intracellular Ca2+ dynamics. Taken dysfunction (ED)is bidirectional. Inflamma- together, these results support the model that tory mediators from the visceral fat induce P 6.25 non-genomic progesterone signaling in hu- oxidative damages in the penile microvascu- man sperm involves an interplay of [Ca2+]; lature resulting in ED. Waist circumference Stereological properties of semi- (WC) is not a reliable indicator of visceral fat and Vm. niferous tubules in infertile men as it includes subcutaneous fat also. Greater with chromosomal and genetic

prevalence of MetS in Asian men compared DGA-Abstracts to African-American men with the same WC abnormalities P 5.11 is due to the relatively higher levels of viscer- D. Ježek1, M. Mokos1, A. Planinic´1, M. Himelreich al fat in them. Visceral Adiposity Index (VAI) Peric´2, A. Katušic´ Bojanac2, N. Sincˇic´2, F. Bulic Jakus2 Emerging roles of melanoma and Lipid Accumulation Product (VAP) are 1University of Zagreb, School of Medicine, Histology ­antigens in male germline stress- novel indices that include functional para­ and Embryology, Zagreb, Croatia; 2University of resistance meters along with anthropometric parameters ­Zagreb, School of Medicine, Medical Biology, J. Klein1, A. Lee1, R. P. Potts1, M. J. Oatley2, J. M. and gives a better assessment of visceral adi- Zagreb,­ Croatia 2 2 3 pose dysfunction. The purpose of the study is Oatley , T. Lord , K. Fon Tacer Chromosomal and genetic abnormalities are 1 to investigate the potential link between these St. Jude’s Children Research Hospital, Memphis, responsible for 15%–30% of male-factor 2 novel indices and ED severity. TN, United States; Washington State University, infertility. This study aimed at determining Pullman, WA, United States; 3Texas Tech University, Material and Methods In this observation- stereological properties of seminiferous tu- School of Veterinary Medicine, Amarillo, TX, United al cross-sectional study, ED patients were di- bules in infertile men with Klinefelter syn- States vided into mild (score > 11) and severe (score drome (KS, 9 patients), Y chromosome mi- ≤ 11) based on International Index of Erectile Genetically modified animals with loss-of- crodeletions (MYC, 14 patients) and CFRT Function-5 scores. WC, Body Mass Index gene mutation (CFTR, 3 patients), and to function of genes that encode melanoma (BMI) and lipid profile were obtained and antigens (Mages) provided important insights compare them with obstructive azoospermia VAI, LAP were calculated using formulas, of non-genetic origin (control group, 18 pa- into the enigmatic role of this protein family VAI = WC/[39.68+ (1.88 × BMI] × trigly­ in the male germline. MAGEs are proteins tients). The total volume, surface and length cerides/1.03 × 1.31/high density lipoprotein of seminiferous tubules were determined at that are restrictively expressed in testis but and LAP = [WC–65 ] × triglycerides. often aberrantly activated in cancer. Although the microscope magnification of 100×, using they have been intensively investigated for Result Of 116 men included in the study, the multipurpose Weibel test system with 42 60 had mild ED and 56 had severe ED. The test points. The surface area (At) of the test cancer therapy, the more fundamental physio­ 2 logical role in male germline remained over- mean age was 51.83 ± 6.2 for mild ED group system was 1,245 mm , individual test line shadowed. The expression of MAGEs at dis- and 52.16 ± 5.8 for severe ED group. Mean length (d) 0,185 mm, and the total length of crete stages of male germ cell differentiation VAI was statistically significantly higher in test lines was (Lt) 3,885 mm. All investigated suggested their functions throughout sper- severe ED group compared to mild ED group stereological parameters were significantly matogenesis. Intriguingly, knock out mouse (8.452 ± 2.33 vs 4.753 ± 1.52; p < 0.001). smaller in KS and MYC, respectively, com- models of different Mage genes suggested Mean LAP was also significantly higher in pared with CFTR and control group. There that they are dispensable for mouse fertil- severe ED group (89.427 ± 31.48 vs 52.21 were no differences in the results of stereo- ity. Intriguing, when males were exposed to ± 29.96; p < 0.001). Interesting, difference logical analysis when comparing histologi- diverse stressors, including DNA damaging busulfan, testis over-heating, or calorie re- striction, Mages provided stress protection to germ cells and reproductive advantage to animals. Given the recent evolutionary expansion of the MAGE gene family in pla- cental mammals, the phenotypes of Mage-a and Mage-b4 KOs imply that distinct MAGE genes evolved as they provided protection to a specific male germ cell population against a particular type of stress, including Mage- a against metabolic stress in differentiating spermatogonia and Mage-b4 against heat in undifferentiated spermatogonia. In addition to germ cells stress protection, Mages also regulate stemness of spermatogonial stem cells and enable faster regeneration of sper- matogenesis after stress-induced damage. Together, Mage KO mice exposed the im- portance of the stress-protective pathways unique to mammalian male germ cells, un- derstanding of which may catalyze novel strategies for male fertility preservation and cancer therapy. Figure 7. D. Ježek, et al.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 47 Andrology 2020 – Abstracts

cal subgroups (Sertoli cell-only syndrome; age-matched able-bodied men. Prospective 1Humanitas Research Hospital, Endocrinology & mixed atrophy) inside KS and MYC groups, studies are warranted to clarify whether this ­Medical Andrology Unit, Rozzamno (MI), Italy; respectively. Qualitative histological analysis condition is associated with a lower risk of 2F. Renzetti Hospital, Diabetology & Metabolic revealed significant impairment of spermato- age-related prostate proliferative diseases ­Disease Unit, Lanciano (CH), Italy; 3University of genesis in KS and MYC, whereas testicular despite many factors, peculiar to this popula- ­Naples, Federico II, Clinical Medicine and Surgery parenchyma was fully maintained in CFTR tion, with a possible role in promoting pros- Department, Naples, Italy; 4Sapienza, University of and control group. Spermatogenesis was tate growth. Rome, Section of Medical Pathophysiology, Center most seriously impaired in KS. In conclusion, of Rare Disease, Department of Experimental Medi- morphometric properties of seminiferous tu- cine, Roma, Italy; 5University of Padova, Department bules are linked to certain inherited factors P 6.4 of Medicine, Andrology and Reproductive Medicine (Fig. 7) Association of erectile dysfunc­ Unit, Padova, Italy; 6University of Florence, Sexual Grants This study was supported by the tion and type II diabetes mellitus Medicine and Andrology Unit, Department of Experi- Scientific Center of Excellence for Re- mental and Clinical Biomedical Sciences, Florence, at a tertiary care centre of South 7 productive and Regenerative Medicine, Italy; Second University of Naples, Department of Republic of Croatia, and by the European India Cardiothoracic and Respiratory Sciences, Endocrine 8 Union through the European Regional De- P. Patel1, R. Nerli2, S. Ghagane2, S. Nutalapati2 Unit, Naples, Italy; University of Catania, Depart- velopment Fund, under grant agreement No. 1Jawaharlal Nehru Medical College, Belagavi, Uro­ ment of Clinical and Experimental Medicine, Catani, 9 2 DGA-Abstracts KK.01.1.1.01.0008, project “Reproductive logy, Belagavi, India; KLES Dr. Prabhakar Kore Hos- Italy; Univerisity of Brescia, Department of Clinical 10 and Regenerative Medicine – Exploring New pital and research centre, Urology, Belagavi, India and Experimental Medicine, Brescia, Italy; Univer- Platforms and Potentials”. sity of L’Aquila, Department of Life Health and En- Introduction Erectile Dysfunction (ED) is vironmental Sciences, L’Aquila, Italy; 11Azienda ASL more common in diabetic men and, unfor- Bologna Maggiore – Bellaria Hospital, Endocrinology P 6.3 tunately, occurs at an earlier age in diabetic Uniti, Medical Department, Bologna, Italy patients when compared with the general Prostate volume in men with population. The study aims to evaluate the in- Introduction Low testosterone (T) in Kline- chronic spinal cord injury: a sys­ dependent predictors of ED in adult men with felter’s syndrome (KS) can contribute to typi- tematic review and meta-analysis type 2 diabetes mellitus (DM) at a tertiary cal features of the syndrome such as reduced of case-control studies care center of South India. bone mineral density, obesity, metabolic dis- turbances and increased cardiovascular risk. Materials and Methods A total of 720 men M. Totaro1, S. D’Andrea1, C. Castellini1, A. Parisi1, The aim of the present study is to review and G. Felzani2, S. Francavilla1, F. Francavilla1, aged 30–70 years who had been diagnosed with type 2 DM were enrolled for the study meta-analyze all available information re- A. Barbonetti1 garding possible differences in metabolic and 1University of L’Aquila, Andrology Unit, Department from January 2017 to January 2020 from the outpatient diabetes clinic of the Hospital. All bone homeostasis profile between T treated of Life, Health and Environment Science, L’Aquila, (TRT) or untreated KS and age-matched con- ItalyM 2Spinal Unit, San Raffaele, Sulmona, Italy patients completed the abridged version of the International Index of Erectile Function trols. Introduction Denervation and androgen (IIEF-5) questionnaire. Material and Methods We conducted a deficiency, peculiar to individuals with Results The mean age of the patients was random effect meta-analysis considering chronic spinal cord injury (SCI), could hin- (58.4 ± 7.8 years). 68.6% of subjects had all the available data from observational der to some extent both prostate growth ­varying degrees of erectile dysfunction, or randomized­ controlled studies compar- and activity. Although most of the available of which 54.6% had moderate to ­severe ing TRT-treated and untreated KS and age- scanty case-control studies revealed a smaller ED. 55.8% had poor glycemic control matched controls. Data were derived from an prostate volume in men with SCI than in age- extensive MEDLINE, Embase, and Cochrane (HbA1c ≥ 7%). Subjects with ED had a longer matched able-bodied controls, some authors duration of DM than those without ED (mean search. found no statistically significant differences. DM duration was 8.1 ± 4.9 years versus 4.4 Results Out of 799 retrieved articles, 21 In order to assess the relationship of SCI with ± 3.5 years; p < 0.001). Longer duration of observational and 22 interventional studies prostate volume comprehensively, we carried DM, poor glycemic control, hypertension, were included in the study. Retrieved trials out a meta-analysis of the available case–con- peripheral arterial disease, testosterone de- included 1144 KS subjects and 1284 healthy trol studies. ficiency were all independent predictors ED controls. Not-treated KS patients showed Methods A thorough search of MEDLINE, (p < 0.05). worse metabolic profiles (including higher Scopus and Web of Science databases was Conclusions A high incidence of erectile fasting glycemia and HOMA index as well as carried out to identify studies comparing dysfunction was observed in type 2 DM pa- reduced HDL-cholesterol and higher LDL- prostate size in men with and without SCI. tients attending the diabetic clinic, and over cholesterol) and body composition (higher Quality of the studies was assessed using the half of the people affected were of moderate- body mass index and waist circumference) Newcastle-Ottawa Scale. In presence of sig- to-severe in intensity. Poor glycemic control, and reduced bone mineral density (BMD) nificant heterogeneity, data were combined testosterone deficiency, peripheral arterial when compared to age-matched controls. using random effects models. Funnel plots disease were the modifiable risk factors for TRT in hypogonadal KS subjects was able to and trim-and-fill analysis were used to assess ED in diabetic subjects. At the same time, a improve body composition and BMD at spi- publication bias. longer duration of type 2 DM was noticed as nal levels but it was ineffective in ameliorat- Results Four studies met the strict inclu- a glaring non-modifiable risk factor, accord- ing lipid and glycemic profile. Accordingly, sion criteria and provided information on 278 ing to our study TRT-treated KS subjects still present worse men with chronic SCI and 1385 able-bodied metabolic parameters when compared to age- controls. The overall difference between the matched controls. P 6.5 two groups was statistically significant (com- Conclusion TRT outcomes observed in bined standardized mean difference: –1.31, Testosterone treatment in male KS regarding BMD, body composition and 95%CI: –2.59 to –0.03, p = 0.04) even in the patients with Klinefelter syndro­ glyco-metabolic control, are similar to those presence of high heterogeneity (P < 0.00001, observed in male with hypogonadism not re- 2 me: a systematic review and me­ I = 98%). The trim and fill analysis did not lated to KS. Moreover, body composition and identify possible missing studies in funnel ta-analysis BMD are better in treated than untreated hy- plot distribution, thus suggesting the absence W. Vena1, A. Pizzocaro1, F. Pelliccione2, R. Pivonello3, pogonadal KS. Larger and longer randomized of obvious publication bias. A. Radicioni4, R. Selice5, G. Rastrelli6, D. Pasquali7, placebo-controlled trials are advisable to bet- Conclusion In men with chronic SCI pros- A. E. Calogero8, A. Ferlin9, S. Francavilla10, ter confirm the present data, mainly derived tate volume is on average smaller than in A. Garolla5, G. Corona11 from observational studies.

48 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

P 6.6 2 diabetics (T2DM) and its relationship with than 150 cm. Further on physical examina- metabolic and clinical characteristics. tion, there were sparse facial, axillary and Safety of injected testosterone Materials and Methods A random cross- pubic hairs, bilateral gynaecomastia, strech undecanoate in the elderly male sectional study of men aged 45–60 years penile length of 4 cm and bilateral testicular J. Abildgaar1, A. K. Bang, L. Aksglaede, with a previously established diagnosis of volume of 3 mL. P. Christiansen1, A. Juul1, N. Jørgensen1 type 2 diabetes mellitus. The diagnosis of Result MRI of pituitary fossa demonstrated Rigshospitalet, Department of Growth and Repro- testosterone deficiency (hypogonadism) was empty sella with no pituitary mass lesion. duction, Copenhagen, Denmark established according to the recommenda- Laboratory investigations at the beginning tions of the EUA. Erectile dysfunction (ED) showed low FSH (0,05 mIU/mL), low LH Introduction Quarterly intramuscular depot injections with testosterone (T) undecanoate was calculated by scale IIEF –5. A total of (0 mIU/mL) and low testoterone (30,1 ng/ ® 30 patients were examined (mean age – 52.9 dL). After 11 month of testosterone gel ad- (TU; Nebido , Bayer) is considered more 2 convenient by most patients, and prevents ± 0.62 years, BMI –30.7 ± 1.2 kg/m , waist ministration, testosterone level rose into 497 daily fluctuations in serum T. However, use circumference (WC) –102.7 ± 2.9 cm, dura- ng/dL but there were no improvement in FSH of TU in elderly patients is limited due to lack tion of T2DM –7, 1 ± 1.23 years, HbA1c –7.4 and LH levels. After treatment discontinua- of safety and feasibility studies. ± 0.31%). According to the protocol, clinical tion, testosterone level dropped into 25 ng/ and laboratory data were studied, includ- dL. Here, we describe the results from our single- ing the presence of chronic non-infectious center longitudinal study. Conclusion Regarding his hypogonadal diseases, the severity of age-related andro- status, he was advised to continue hormonal DGA-Abstracts Methods Patients who initiated TU treat- gen deficiency according to the AMS scale. treatment and we are still doing challenge ment between 2005–2010 were included in The content of total testosterone, luteinizing test to estimate his probability of having the study. Elderly patients were defined as hormone, sex steroid binding globulin, and offspring, including any chance of gonado- born before 1955 and young patients between glycated hemoglobin in the blood was de- trophin therapy to initiate spermatogenesis in 1965–1990. termined by standard methods. Free testos­ this patient. T dose was adjusted through yearly visits in terone levels were determined by calculation. the out-patient clinic. Standard treatment was Results The prevalence of hypogonadism 1000 mg TU/12 weeks. Treatment adjust- was 46.7%, while the total testosterone level P 6.9 ments were performed based on 1) free T of less than 12 nmol/l was observed in 63.3% NADIR levels, 2) symptoms of T deficiency. of the surveyed, and the level of less than Male osteoporosis, a still over­ Supraphysiological hematocrit led to dose 8 nmol/l in 30% of the surveyed persons. looked issue: an identikit of adjustments independent of serum T levels. Moreover, impairment of libido was noted by ­patients seeking bone health eva­ Follow up was until April 1st, 2020 or discon- 70% of the surveyed, and impaired erectile luation at a tertiary academic tinuation of treatment. function –63.3%. The level of total testoster- ­medical centre Results Median age at time of TU initiation one in the blood significantly positively cor- 1,2 1,2 2 was 61 years (n = 63) in the elderly- and 30 related with the level of SHBG in the blood S. De Vincentis , A. Russo , E. Taliani , 2 1,2 2 1,2 years (n = 63) in the young patients. Median (r = +0.368). A. Ansaloni , D. Domenici , B. Madeo , V. Rochira 1 duration of treatment was 13 years for both University of Modena and Reggio Emilia, Unit of groups. ­Endocrinology, Department of Biomedical, Metabolic P 6.8 and Neural Sciences, Modena, Italy; 2Azienda Risk of polycythemia did not differ between ­Ospedaliero-Universitaria di Modena, Unit of Endo- elderly (n=22 [35 %]) and young patients Male infertility with empty sella crinology, Department of Medical Specialties, (n = 17 [27 %], (RR = 1.32, 95-%-CI: 0.70– syndrome: a case report Modena,­ Italy 2.48). Adjustments due to polycythemia was S. S. Suyono1, R. S. Marthasari2, N. Abdullah3 performed 43 times in the elderly vs. 45 times Male osteoporosis is under- 1Universitas Kristen Duta Wacana, Faculty of Medi- Background in the young (p = 0.74), due to increased free managed. The charateristics of men referring cine, Yogyakarta, Indonesia; 2Faculty of Medicine, T: 51 times in the elderly vs 34 in the young to health system for bone evaluation remains Universitas Katolik Widya Mandala, Biology, (p = 0.07) and due to a combination of the partially unknown. Surabaya,­ Indonesia; 3Faculty of Medicine, Uni­ two, 117 times in the elderly vs. 66 times in versitas Hasanuddin, Obstetrics and Gynecology, Aim To characterize from real-life data the young (p < 0.01). Makassar, Indonesia male patients seeking the first bone health Time between injections were prolonged in evaluation at a tertiary academic medical elderly patients (median: 1000 mg/15 weeks Introduction Empty sella syndrome, also center, referral for both andrological and vs 1000 mg/13 weeks after 10 years of TU, known as arachnoidocele, is a rare disorder bone diseases, over a 13-year observation p < 0.01) and accumulated dose of TU at time characterized by herniation of the arachnoid period. space within the sella which is often associat­ of polycythemia was lower in elderly – com- Methods Restrospective, cross-sectional pared to young patients (p < 0.05). ed with variable degrees of flatening of the pituitary gland. This condition is more com- study, including men (> 18 years) referring to Conclusion TU injections in the elderly pa- mon in women and associated with obesity. our Center from 2007 to 2020 for bone health tient does not increase risk of polycythemia, evaluation. Osteoporosis and osteopenia were however, accumulated dose of TU at time of Patient A 48-year-old man presented to defined considering DXA outcomes, accord- polycythemia is lower in the elderly male. the andrology clinic with 4 years of primary ing to WHO criteria. Thus, frequent treatment adjustments are infertility. He also complained erectile dys- Results A total of 455 men (mean age 62.5 needed at all ages. function and low desire. He revealed any his- tory of puberty at the age of 13 but he seldom + 15.1years) were included: 42 aged 18–40 showed interest in sex since puberty. He was years, 57 aged 40–50, 79 aged 50–60, 109 P 6.7 suffered from sinusitis for a long time. He de- aged 60–70, 122 aged 70–80, and 46 aged > 80. Overall, 125 patients (27.4%) were Testosterone deficiency and erec­ nied any sign and symptoms of cranial mass such as headache and visual disturbance. already followed at our Centre due to endo- tile dysfunction in middle-aged crinological/andrological diseases that are type 2 diabetics He has been diagnosed with empty sella syn- known to increase fracture risk (94 men) or drome (PES) since 2018 and had been treated not (31 men); general practioners and other V. Odin, O. Alekseenko, V. Didenko, V. Yudin with testosterone gel and got any improvement specialists asked for bone evaluation for 226 Military Medical Academy, Faculty Therapy, in his sexual heath. But the treatment had been (49.6%) and 101 (22.1%) men. DXA has St. Petersburg, Russian Federation discontinued due to Covid-19 pandemic. been performed for 354 patients. Prevalence Introduction To study the prevalence of He is extremely obese, 182 cm tall and 182kg of osteoporosis, osteopenia, and low bone testosterone deficiency in middle-aged type weight and his waist circumference is more mineral density for age were 25.9%, 26.4%

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 49 Andrology 2020 – Abstracts

and 13.2%, respectively. At least one fra- tion in AGHD males was 60%, with a higher Conclusion Family history is simple and gility fracture has already occurred in 213 erectile dysfunction (ED) prevalence in inexpensive information that should be part patients (46.8%), with higher prevalence in Group 2 (70%). A significant correlation was of the CV risk assessment in all men with ED non-endocrinological than endocrinological documented between QLS-H (R2 = 0.533, because it helps in the identification of those patients (56% vs 24%, p < 0.001). Consider- p < 0.005) and QoL-AGHDA (R2 = 0.221, who need lifestyle and risk factors modifi- ing fractured patients, 49 of them (23%) have p = 0.001) results and erectile function do- cations and whose risk would otherwise be never been treated with any anti-osteoporotic main, moreover serum IGF-1 resulted direct- overlooked. therapy, including calcium and vitamin D ly correlated to all IIEF-15 domain scores, supplementation. with the most significant relation with EF 2 Conclusions Male osteoporosis presents (R = 0.123, p = 0.019). P 6.12 with a high rate of fragility fractures (about Conclusions This study demonstrates a 50%) among patients referring to a tertiary high prevalence of sexual dysfunction in Insight on the intracrinology of academic medical center for bone health AGHD patients and that r-hGH treatment : androgen production evaluation. Most of fractured patients have seems to be associated to better sexual out- within human vagina not previously been evaluated by a clinician comes. Consequently, evaluation of sexual experienced in bone diseases or properly function should be integrated in the global V. Di Stasi1, I. Cellai1, P. Comeglio1, E. Maseroli1, treated, suggesting that consideration for male assessment of AGHD patients. T. Todisco1, C. Corno1, S. Filippi2, S. Cipriani1, 1 1 1 1 DGA-Abstracts osteoporosis should be reinforced in primary F. Sorbi , M. Fambrini , F. Petraglia , I. Scavello , healthcare setting in order to prevent fractures. G. Rastrelli1, G. Acciai1, F. Villanelli1, G. Danza1, P 6.11 E. Sarchielli3, G. Guarnieri3, A. Morelli3, M. Maggi1, L. Vignozzi1 Family history for cardio-meta­ 1 P 6.10 University of Florence, Italy, Experimental and Clini- bolic diseases: a predictor of cal Biomedical Sciences, Florence, Italy; 2University High prevalence of sexual dys­ ­major adverse cardiovascular of Florence, Italy, Neurosciences, Psychology, Drug function among patients with events in men with erectile dys­ Research and Child Health (NEUROFARBA), Florence, 3 Growth Hormone Deficiency function Italy; University of Florence, Italy, Experimental and Clinical Medicine, Florence, Italy (GHD): results from the Manage­ G. Rastrelli, D. Yannas, B. Mucci, G. Corona, ment of Adult GHD (MAGHD) M. Maggi Introduction In this study, we investigated Study. University of Florence, Experimental and Clinical Bio- the steroidogenic gene expression in hu- medical Sciences, Florence, Italy man vagina and verified the ability of hu- M. L. Monzani1,2, S. Pederzoli1,2, L. Volpi1, man vagina smooth muscle cells (hvSMCs) E. Magnani3, C. Diazzi2, V. Rochira1,2 Introduction Family history (FH) of car- to synthesize androgens from the upstream 1Università di Modena e Reggio Emilia, Dipartimento diovascular (CV) disease is a known CV risk precursor dehydroepiandrosterone (DHEA). di Scienze Biomediche, Metaboliche e Neuroscienze, factor. However, it is rarely considered for As readout for androgen receptor (AR) ac- Modena, Italy; 2Azienda Ospedaliero Universitaria di CV risk stratification. Furthermore, FH for tivation, we evaluated the expression of the Modena, Unità di Endocrinologia, Dipartimento di metabolic diseases is generally overlooked. androgen-dependent gene STAMP2. Specialità Mediche, Modena, Italy; 3AUSL-IRCCS di To evaluate, in a population of men with erec- hvSMCs were Reggio Emilia, Unità di Endocrinologia, Dipartimento tile dysfunction (ED), whether FH for cardio- Materials and Methods isolated from vagina tissues of women un- di Specialità Mediche, Reggio Emilia, Italy metabolic diseases could provide insights in metabolic and sexual features and predict the dergoing surgery for benign gynecological Introduction Adult growth hormone defi- occurrence of forthcoming major adverse CV diseases. In these cells, by real time RT-PCR, ciency (AGHD) affects quality of life (QoL). events (MACE). we evaluated mRNA expression of several Sexuality contributes considerably to well- steroidogenic enzymes and sex steroid recep- being and QoL, but studies on sexual func- Methods A consecutive series of 4,693 in- tors. Androgen production was quantified by tion in AGHD patients are lacking. dividuals (aged 51.3 ± 13.3 years) attending liquid chromatography tandem-mass spec- an Andrology outpatient clinic for ED was trometry (LC-MS/MS). Aim To investigate the prevalence of sexual studied. A subset of these (n = 1595) was dysfunction in AGHD patients referring to a evaluated retrospectively for MACE occur- Results In vaginal tissues, AR resulted sig- single endocrinological center and grouped rence. Several metabolic and sexual function- nificantly less expressed than estrogen recep- according to r-hGH therapy and to assess the related parameters­ were studied. For the tor α (ERα), while, in hvSMCs, it was higher correlation between sexual function and QoL retrospective study, information on incident than progestin and both estrogen receptors. scores, r-hGH treatment, clinical and hormo- MACE was collected over a mean follow-up In hvSMCs and in vaginal tissue, when com- nal parameters. of 4.2 ± 2.5 years. pared to ovary, pro-androgenic steroidogenic enzymes (HSD3 1/ 2, HSD17 3/ 5), along Patients and Methods A prospective, real- Results A greater number of cardio-meta- β β β β life, clinical trial involving AGHD patients bolic FH factors was associated with a worse with 5α-reductase isoforms and sulfo-trans- was performed in a tertiary, endocrinologi- metabolic profile, including higher waist ferase resulted more abundant. In addition, cal center. The 83 enrolled patients were di- circumference, triglycerides, glucose, glyco- enzymes involved in androgens inactivation vided in 2 groups according to AGHD treat- sylated hemoglobin, and diastolic blood pres- were less expressed than in the ovary. The ment: on long-term r-hGH therapy (Group 1, sure, as well as lower high-density lipopro- LC-MS/MS analysis revealed that, in hvS- n = 32) and not treated with r-hGH (Group tein (HDL) cholesterol. An increased number MCs, DHEA supplementation increased an- 2, n = 51). Clinical data and medical history of FH factors was associated with a worse drostenedione levels after short term in spent were collected. IGF-1, IGFBP-3, sex steroids erectile function, impaired penile dynamic medium, while increased testosterone (T) and and pituitary hormones were assayed. QoL peak systolic velocity and lower testosterone dihydrotestosterone (DHT) secretion were was assessed by Quality of Life Satisfaction levels. In the retrospective study, a positive detected after longer incubation. Finally, an- in Hypopituitarism (QLS-H) and QoL As- cardio-metabolic FH was associated with drogenic signaling activation was evaluated sessment of GHD in Adults (QoL-AGHDA) a significantly higher incidence of MACE, through STAMP2 mRNA expression after questionnaires. Index for Erectile Function-15 even after adjusting for age and comorbidi- DHEA and T stimulation. (IIEF-15) and Female Sexual Function Index ties. Interestingly, when dividing the sample Conclusion This study confirmed that hu- (FSFI) were employed to evaluate sexual into high- and low-risk categories accord- man vagina is an androgen-target organ with function in males and females, respectively. ing to several CV risk factors (age, previous the ability to synthesize androgens, thus pro- Results 83 AGHD patients (31 females, MACE, HDL-cholesterol and comorbidities), viding support for the use of androgens for 52 males, mean age 56.3 ± 14.7 years) were FH was confirmed as a predictor of incident local symptoms of genito-urinary syndrome evaluated. The prevalence of sexual dysfunc- MACE only among the low-risk individuals. of menopause.

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P 6.13 P 6.14 P 7.1 Self-reported premature ejacula­ Hyperhomocysteinemia and velo­ Testosterone, level of the lesion tion and related distress – occur­ cimetric parameters in patients and age are independently asso­ rence and clinical correlates: Re­ with erectile dysfunction under­ ciated with prostate volume sults from the European Male going penile echo-color doppler in men with chronic spinal cord Ageing Study G. Salvio, A. Ciarloni, M. Cutini, N. delli Muti, injury G. Corona1, G. Rastrelli1, G. Bartfai2, F. Casanueva3, F. Finocchi, L. Giovannini, G. Balercia A. Parisi1, M. Totaro1, S. D‘Andrea1, C. Castellini1, 1 A. Giwercman4, L. Antonio5, J. Slowikowska-­ Polytechnic University of Marche, Endocrinology G. Felzani2, S. Francavilla1, F. Francavilla1, Hilczer5,6, J. Tournoy5, M. Punab7, I. Huhtaniemi8, ­clinic, Ancona, Italy A. Barbonetti1 5 9 9 1 1 D. Vanderschueren , T. O’Neill , F. Wu , M. Maggi Introduction Erectile dysfunction (ED) University of L‘Aquila, Andrology Unit, Department 1 University of Florence, Experimental and Clinical can be influenced by the presence of athero- of Life, Health and Environment Sciences, L‘Aquila, 2 2 Biomedical Sciences, Florence, Italy; Albert Szent-­ sclerosis, a pathology of the vessels that also Italy; San Raffaele, Spinal Unit, Sulmona, Italy Gyorgy Medical University, Szeged, Hungary; affects the cavernous arteries and in which Introduction Although men with spinal 3Santiago de Compostela University, Santiago de hyperhomocysteinemia plays a central role. cord injury (SCI) exhibit a prostate volume Compostela, Spain; 4Malmö University Hospital, significantly smaller compared to age- Malmo, Sweden; 5Katholieke Universiteit Leuven, Objective We aimed to evaluate the associ- matched able-bodied men, the independent DGA-Abstracts Leuven, Belgium; 6Medical University of Lodz, Lodz, ation between homocysteine and velocimet- association of lower prostate volume with Poland; 7United Laboratories of Tartu University ric parameters detected by basal penile ECD its putative determinants has never been ­Clinics, Tartu, Estonia; 8Imperial College London, (peak systolic speed, PSV; acceleration time, analyzed in this population. This study was London, United Kingdom; 9University of Manchester, AT) in patients with ED. designed to identify variables independently Manchester, Germany Methods We conducted a cross-sectional study on ED patients who were studied from associated with prostate volume in men with Introduction There are few data which a clinical (medical history, BMI, blood pres- chronic SCI. have looked at the occurrence and clinical sure, smoking, antihypertensive therapy), Materials and Methods In this cross-sec- correlates of self-reported premature ejacula- biochemical (glycemia, HbA1c, lipid panel, tional study, prostate volume of 138 men with tion (PE) and its related distress in the general homocysteine, LH, total testosterone, SHBG chronic (> 1 years) SCI, aged 54.5 (25–75th: population. To determine the prevalence and and PSA) and instrumental (basal penile 36.0–66.0) years, was evaluated with trans- clinical correlates of self-reported PE and ECD) point of view. rectal ultrasonography. All patients under- PE- Related Distress in middle age and older Results 126 subjects (mean age 52.1 ± went a complete neurological exam, as well European men. 12.6 years; mean BMI 25.6 ± 4.0 kg/m2) as biochemical and hormonal assessment, Methods Subjects were recruited from were ­enrolled. We detected mean PSV values including measurement of total testosterone population samples of men aged 40–79 years of 13.1 ± 2.9 cm/s and mean AT of 2.28 ± (TT) levels. Free testosterone levels were cal- across 8 European centres. Self reported PE 0.70 m/s2. These parameters showed a strong culated (cFT) by the Vermeulen formula. and its related distress were derived from correlation among them (r = 0.690; p < 0.001), Results The median prostate volume was the EMAS sexual function questionnaire with frankly pathological values of PSV and 23.4 mL. At the univariate analysis, a larger (EMAS-SFQ). Becks depression Inven- AT in 5% and 59% of the subjects examined, prostate volume was significantly associ- tory (BDI) was used for the quantification respectively. Mean homocysteine levels were ated with higher TT (p < 0.001) and cFT of depressive symptoms, the Short Form 36 14.9 ± 9.5 µmol/l, with pathological values (p = 0.001), SCI level below T12 (p = 0.007), health survey (SF-36) for the assessment of present in 26% of the subjects examined. more advanced age (p = 0.04) and lower the quality of life, the International Prostate We found an inverse correlation between body mass index (p = 0.04). However, at the Symptom Score (IPSS) for the evaluation of homocysteine and PSV levels (r = –0.213; multiple regression analyses, an independent lower urinary tract symptoms. p = 0.03) and between homocysteine and AT positive association only persisted between the prostate volume with either TT or cFT 2,888 community dwelling men (r = –0.199, p = 0.05). Furthermore, we found Results levels, and, to a lesser extent, with age and a aged 40–79 years old (mean 58.9 ± 10.8 an inverse correlation between velocimetric level of spinal lesion below T12. A prostate years) were included in the analysis. Among parameters and age, BMI, blood glucose, volume below the median value was observed the subjects included, 889 (30.8%) self- HbA1c, LDL cholesterol and triglycerides and in 91.4% of patients with both biochemical reported PE. Among them, 211 (7.3%) a direct correlation between homocysteine androgen deficiency (TT < 264 ng/dL) and claimed to be distressed (5.9% and 1.4% and HDL cholesterol levels. Age showed a SCI level ≥ T12, but only in 16.5% of patients reported mild or moderate-severe distress, direct correlation with homocysteine levels with both normal androgen levels and SCI respectively). Increasing levels of PE-related (r = 0.287; p = 0.003), while no significant level below T12 (p < 0.001). distress were associated with a progressive correlations emerged between homocysteine worse sexual functioning and with couple and metabolic parameters. Conclusions Low testosterone levels and, impairment, along with a higher prevalence Conclusions Hyperhomocysteinemia is a to a lesser extent, a younger age, and a spinal of depressive symptoms (all p < 0.05). Fur- frequent condition in ED patients and its lesion level ≥T12 represent the only variables thermore, a worse quality of life and higher prevalence increases with age. The data from exhibiting an independent association with a IPSS score were associated with PE-related our study confirm the effect of hyperhomo- smaller prostate volume in men with SCI. distress (all p < 0.05). The aforementioned cysteinemia in the genesis of ED of arterial results were confirmed even when patients origin regardless of other metabolic factors. using drugs possibly interfering with ejacula- P 7.2 tion were excluded from the analysis. Specific function of the neonatal Conclusions Self-reported PE is relatively epididymis is reflected in its con­ common in European men aged more than tractile structures 40 years; however, its related distress is D. Weiser, C. Rager, S. Tasch, R. Middendorff relatively modest. Nonetheless, men with Justus-Liebig-University, Institute of Anatomy and any degree of self-reported PE show increas- Cell Biology, Giessen, Germany ing levels of depression, worse quality of life and worse couple satisfaction. The reported In adult epididymis throughout the whole limited PE-related distress may explain the duct histological structures of the smooth relatively low number of medical consulta- muscle layer (SML) reflect the specific func- tions for PE. tions of the organ. SML differs between the

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proximal transporting parts and the distal that ANP, CNP and also VNP significantly matogenesis. Future studies, using the liquid sperm-storing part. However, in the neo- reduced contraction frequency. Long-term ef- chromatography-tandem mass spectrometry, natal epididymis only one function exists: fects of CNP and VNP were analyzed in pri- could clarify the real steroidogenic potential waste disposal, i.e. the transport of exfoliated mary SMCs. We found that the cell number of both adrenal and testicular tissue in such epididymal epithelial cells out of the ductal was reduced after CNP or VNP treatment for patients, representing an in vivo model of lumen [Weiser et al. 2020]. 3 days. Thus, NPs affecting muscle tone and impaired lipid substrate for steroidogenesis. In this study we analysed the contractile proliferation could be developed to promis- ing drugs for BPH treatment as an alternative structures of the neonatal epididymal duct P 7.5 in regard to its function by immunohisto­ to tamsulosin with its side effects on ejacula- chemistry, life imaging and compared the tion. Generation of three-dimensional data with adult rats. In all regions of the neo- extracellular matrix (ECM-) de­ natal epididymal duct, SML showed a uni- P 7.4 rived scaffolds to reorganize form thickness and consistent spontaneous ­human testicular cells in vitro contractions. In the adult rats, SMLs signifi- Does reduced lipid substrate cantly increased from caput to cauda different mean impaired testicular func­ V. Höffken1,2, R. Sandhowe-Klaverkamp2, S. Schlatt2, to spontaneous contractions, which decreased tion? Androgen and sperm J. Wistuba2 from caput to cauda. 1University Hospital Münster, Molecular Nephrology, ­production in men with heterozy­ 2 DGA-Abstracts These contractile peculiarities of neona- Internal Medicine D, Münster, Germany University gous familial hypobetalipopro­ Hospital Münster, Centre of Reproductive Medicine tal epididymis reflect the underestimated teinemia epididymal function of waste disposal during and Andrology, Münster, Germany 1 2,3 2,3 development to prevent obstruction-induced G. Spaggiari , F. Nascimbeni , S. Lugari , Fertility preservation is important for patients 1 2,3 1,4 1,4 infertility. A. Granata , F. Carubbi , M. Simoni , D. Santi threatened by infertility like prepubertal boys 1 Azienda Ospedaliero-Universitaria of Modena, Unit suffering from malignant diseases unable to of Endocrinology, Department of Medical Special- cryopreserve sperm. Thus, immature testicu- P 7.3 2 ties, Modena, Italy; Azienda Ospedaliero-Universi- lar tissues likely containing germ cells are Natriuretic peptides as a future taria of Modena, Operating Unit of Internal and preserved, but systems for in vitro differen- 3 ­alternative for tamsulosin in BPH ­Metabolic Medicine, Modena, Italy; University of tiation are missing so far. Our study aimed Modena and Reggio Emilia, Operating Unit of Inter- at generating three-dimensional ECM-based treatment 4 nal and Metabolic Medicine, Modena, Italy; Uni- scaffolds derived from adult human testis of 1 2 1 M. A. Kuchta , F. Wagenlehner , R. Middendorff versity of Modena and Reggio Emilia, Unit of Endo- patients undergoing male to female gender 1 Justus-Liebig University, Institute for Anatomy and crinology, Department of Biomedical, Metabolic and confirming surgery as those natural matrices 2 Cell Biology, Giessen, Germany; Justus-Liebig Uni- Neural Sciences, Modena, Italy should provide factors supporting cell reor- versity, Department of Urology, Pediatric Urology and Heterozygous familial hypobetalipoprotein­ ganization and differentiation. Andrology, Giessen, Germany emia (FHBL) is a rare genetic condition To decellularize adult testicular tissues, two While the exact mechanisms for the de- mainly caused by truncating mutations in protocols were tested regarding ECM struc- velopment of benign prostatic hyperplasia apolipoprotein B (apoB) gene. The truncated tural maintenance and cell removal efficien- (BPH) remain unknown, it is suggested that apoB proteins determine extremely low plas- cy. Isolated human testicular cells were used hormonal regulation plays a critical role. In matic levels of apoB-containing lipoprotein for recolonization of generated scaffolds and BPH smooth muscle cells (SMCs) show an particles, which represent a fundamental sub- collagen sponges. Immunohistochemistry increase of muscle tone and cell proliferation. strate for steroidogenesis. The hypothesis that showed a protocol containing triton treatment Cyclic GMP (cGMP) signaling pathways a reduction of plasma lipoproteins could in- to be superior in ECM preservation, while regulate a variety of physiological functions, terfere with steroid hormone production was testicular cell removal was more effective by such as SMC relaxation or cell proliferation. investigated by few studies dating back to the another using 1%SDS. Flow cytometry ana­ Activators of this pathway are the natriuretic 80s considering only adrenal steroidogenesis lysis confirmed numbers of remaining cells peptides (NPs) ANP, BNP and CNP. High and enrolling maximum 4 patients. However, after treatments to be lower using 1%SDS concentrations of CNP were found in the the testicular steroidogenesis has never been (370 cells) than triton (22000 cells). Isolated prostate and seminal plasma. However, the investigated in FHBL. Additionally, male testicular cells successfully recolonized and function of CNP in prostatic SMCs is not mouse models of heterozygous FHBL ex- organized in artificial collagen sponges but clear. Since existing drugs such as tamsulosin hibited impaired sperm count, motility and were not able to do so in ECM scaffolds. Ad- lead to side effects like abnormal ejaculation survival time. With this in mind, we enrolled ditionally, hydrogel of homogenized ECM and even lead to discontinuation of therapy, 5 male patients (median age 41.6 [23.5–63.5] was used as coating for conventional testicu- research is focusing on new therapies. For years) with genetic-confirmed apoB-related lar cell culture to assess molecular cytocom- this, the hormonal influence of CNP on the heterozygous FHBL to assess the testicular patibility. The hydrogel promoted cellular cGMP signaling pathway and its signifi- function. At the clinical evaluation, they pre- survival, structure and reaggregation more cance for the development of BPH needs to sented normal testicular volume (right 16.9 efficient than common matrix coatings. Cell be investigated. We performed RT-qPCRs to [7.3–26.2] ml, left 16.6 [2.7–24.6] ml), while aggregates contained several germ cell types investigate the influence of NPs on the ex- in 2 cases (40%) testicular ultrasound inho- and somatic cells. Thus, supportive factors pression of cGMP pathway components in mogeneity was recorded. Androgen produc- for cellular reassembly were still present in cultured primary SMCs from human prostate. tion resulted within reference ranges for all the matrix derived hydrogel after processing. cGMP-ELISAs were used to show whether patients (median testosterone 4.5 [3.7–6.8] Our data show that human testicular ECM NPs activate this cGMP pathway. The ef- ng/ml), accompanied by normal levels of contains regulatory factors potentially pro- fects of natriuretic peptides and the synthetic luteinizing hormone 2.6 [1.2–6.0] mIU/ml. moting germ cell differentiation in vitro. A natriuretic peptide Vasonatrin (VNP) on On the contrary, 2 men resulted azoospermic, combination of collagen sponges and testicu- spontaneous contractility of prostate glands whereas seminal parameters were within the lar ECM components could be beneficial for were investigated by Live-Imaging. We reference range in other cases (sperm number development of in vitro system development found that CNP, more than ANP, activates 206.4 [68–360] millions, progressive motil- for male germ cell differentiation. cGMP production in primary prostatic SMC ity 50 [41–70]%). Thus, the testicular steroi- cultures in a dose-dependent manner. Inter- dogenic functionality seems not impaired in estingly, the expression of GC-B was higher heterozygous FHBL men. However, a high than that of GC-A in cell cultures, which may incidence of azoospermia (40%) was de- explain the higher production of cGMP after tected, suggesting a potential association be- CNP treatment. Furthermore, we showed tween heterozygous FHBL and altered sper-

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P 7.6 Introduction Testicular sperm are imma- University, Department of Urology, Lviv, Ukraine; ture cells unable to fertilize an oocyte. After 6Danylo Halytskyy Lviv National Medical University, Repair of penile fracture: how leaving the testis, sperm transit along the Department of Pediatric Surgery, Lviv, Ukraine long can it be delayed? epididymis to acquire motility and fertilizing Introduction This clinical retrospective 1 2 2 abilities. It has been proposed that the transfer P. Patel , R. Nerli , S. Ghagane study aimed to verify some current concepts 1 of information (proteins and RNAs) between Jawaharlal Nehru Medical College, Belagavi, Urol­ about possible molecular pathways responsi- 2 epididymis and sperm is mediated by extra- ogy, Belagavi, India; KLES Dr. Prabhakar Kore Hospi- ble for the onset of male infertility associated cellular vesicles, named epididymosomes, tal and research centre, Urology, Belagavi, India with scrotal hyperthermia. released by epididymal principal cells (PCs). Introduction “Penile fracture” is defined as However, direct protein transfer to sperm Patients and Methods To perform the the traumatic rupture of the tunica albuginea­ through epididymosomes is still poorly char- study, 226 men aged 22–40 were enrolled. of an erect penis. Patients typically describe acterized. The following research subgroups were immediate detumescence, severe pain, and distinguished: fertile men serving as con- Materials and Methods An in silico analy- swelling as a result of the injury. The ma- trol (n = 21), professional drivers (n = 52), sis using human and mouse proteomics and jority of patients can be diagnosed from infertile men with cryptorchidism in child- transcriptomics data was carried out to infer the history and physical examination alone. hood (n = 50), infertile men with varicocele proteins with a putative epididymis-origin Radiographic imaging studies, including ul- (n = 71), infertile men not exposed to pro- that are potentially transferred to sperm by trasonography, magnetic resonance imaging, longed genital heat stress (n = 32). Seminal epididymosomes. High-resolution confocal DGA-Abstracts and in some cases retrograde urethrography biochemical (neutral alpha-glucosidase microscopy of epididymis and testis tissue can aid in the diagnosis of unusual cases. – NAG, fructose, citric acid) and oxidative biopsies of human and mouse, and CFSE- Prompt surgical exploration and corporal re- stress (total antioxidant capacity – TAC, cata- fluorescently labeled epididymosomes were pair is the most efficacious therapy. lase activity, superoxide dismutase activity, used for experimental validation. Materials and Methods We retrospec- malondialdehyde level) parameters were de- Results Derived from the in silico analysis, tively reviewed the hospital inpatient and termined using commercially available kits. we identified 25 sperm proteins with a puta- outpatient records of all patients with penile In addition, the sperm chromatin structure as- tive epididymis-specific origin conserved in fractures, who underwent repair 8 days after say (SCSA), TUNEL assay and aniline blue both human and mouse species. The epididy- injury. This study was carried out following staining were used to assess sperm chromatin mal origin of 4 of these putative epididymis- permission obtained from the Institutional/ integrity. specific sperm proteins was validated by con- University ethical committee. The presenting The NAG activity was significantly focal microscopy. Specific protein expression Results symptoms, details of clinical examination, lower in cryptorchid men while drivers and patterns were observed in the different re- imaging records, and treatment plans were infertile men with varicocele exhibited signif- gions of the epididymis and, interestingly, recorded and analyzed. icantly lower TAC but higher catalase activity 1 of the 4 studied proteins was exclusively as well as sperm DNA fragmentation com- Results During a 10 year period, five pa- expressed in epididymal clear cells (CCs), pared to the control group. The NAG activity tients undergoing repair of penile fracture 8 while the others were expressed in both PCs was the main critical parameter correlated days after injury were identified. The mean and CCs. Finally, we showed that fluorescent- with basic sperm parameters in men exposed age was 28.6 and the mean period taken to ly labeled epididymosomes interacting with to active scrotal hyperthermia. Moreover, the present to us was 16.89.7 days. The duration sperm in vitro contain the 4 targeted sperm greatest number of correlations of antioxida- of time to repair was 189.7 days. The patients proteins with an epididymal origin. were evaluated at 12 weeks, 6 months, and 12 tive parameters with sperm DNA integrity Conclusion Our findings indicate that months after repair. There were no obvious were observed in varicocele patients. epididymosomes are capable of providing complications noted. All 5 patients experi- The biochemical status of spermatozoa with a set of epididymis-spe- Conclusion enced spontaneous erections within 12 weeks. semi­nal plasma may be responsible for low cific proteins that could be essential for the sperm quality in men exposed to both exter- Conclusions Surgical treatment/repair post-testicular maturation of sperm cells, and nal or internal genital heat stress. Dysregula- should always be offered to patients with pe- support the novel role of CCs in the transfer tion of seminal antioxidant components can nile fracture, irrespective of delayed presen- of proteins to sperm and in epididymosome be principally associated with the epididymal tation. Long term complications are rare and production. the patients in our series reported no erectile and prostate functions. Oxidative stress in- Grants Supported by PI16/00346 to RO; dysfunction at a minimum follow-up of 12 duced by local thermogenic factor can be one FPU15/02306, ASBMB, Lalor Foundation months. of the prominent mechanisms responsible for to FB. sperm DNA damage. Grants The study was financed by the P 7.7 National Science Centre, Poland (grant No P 7.8 Epididymosomes participate in 2015/19/B/NZ5/02241). Altered secretory function of male the transfer of a subset of epidi­ accessory organs and oxidative dymis-specific proteins to sperm stress may be involved in male in­ P 7.9 1,2 1 2 F. Barrachina , M. A. Battistone , J. Castillo , fertility evoked by scrotal heat C. Mallofré3, M. Jodar2,4, S. Breton1,5, R. Oliva2,4 TLR4/NFKB signaling pathway 1Massachusetts General Hospital and Harvard Medi- stress ­regulates epididymal embryonic cal School, Program in Membrane Biology, Nephro­ M. Fraczek1, L. Wojnar2, M. Kamieniczna1, development 3 3 3 4 logy Division, Department of Medicine, Boston, MA, M. Piasecka , K. Gill , M. Kups , V. Chopyak , 1 1 1 2 4 5 6 L. G. Ferreira , F. A. Nishino , S. G. Fernandes , United States; Institut d’Investigacions Biomèdiques A. Havrylyuk , J. Nakonechnyy , A. Nakonechnyy , 1 2 1 1 C. M. Ribeiro , B. T. Hinton , M. C. W. Avellar August Pi i Sunyer (IDIBAPS), Fundació Clínic per a la M. Kurpisz 1 1 Universidade Federal de São Paulo, Escola Paulista Recerca Biomèdica, Universitat de Barcelona, Institute of Human Genetics, Polish Academy of 2 2 de Medicina, São Paulo, SP, Brazil; University of ­Department of Biomedical Sciences, Faculty of Me- Sciences, Poznan, Poland; Poznan University of Me- ­Virginia, School of Medicine, Charlottesville, VA, dicine and Health Sciences, Barcelona, Spain; dical Sciences, Clinic of Urology and Oncological United States 3Universitat de Barcelona, Hospital Clínic, Depart- Urology, Poznan, Poland; 3Pomeranian Medical Uni- ment of Pathology, Barcelona, Spain; 4Hospital versity in Szczecin, Department of Histology and The Wolffian duct (WD) is an embryonic ­Clinic, ­Biochemistry and Molecular Genetics Service, ­Developmental Biology, Szczecin, Poland; 4Danylo tissue that undergoes androgen-induced Barcelona, Spain; 5Université Laval, Department of Halytskyy Lviv National Medical University, Depart- coiling and elongation to become the adult ­Obstetrics, Gynecology, and Reproduction, Faculty of ment of Clinical Immunology and Allergology, Lviv, epididymis. We showed a β-defensin (in- Medicine, Québec, Canada­ Ukraine; 5Danylo Halytskyy Lviv National Medical nate immunity component) as an androgen

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 53 Andrology 2020 – Abstracts

target in the developing WD. Indeed, im- Ang1 has an anti-inflammatory role, while on perineal area 5 minutes twice daily. A mune-/inflammatory mediators are required Ang2 has a pro-inflammatory effect.The aim Wilcoxon signed- rank test was performed to in developmental processes. β-defensins of this study was to evaluate Ang pathway as compare the effects of treatment at V0 and V1. and extracellular matrix (ECM) components an effective serum marker in BPH patients Results No differences between V0 and V1 regulate cellular responses by acting as Toll- who performed PEMF therapy. We provide were found in Ang1 and Ang2 levels, even like receptor 4 (TLR4) endogenous ligands. preliminary data of this study. if a trend in reduction in Ang1 was found: TLR4 is a player in the maintenance of adult Material and Methods This is a prospec- Ang1V0 1.04 (0.3; 2.1) vs Ang1V1 1.65 (0.58; epididymis, however its expression and tive interventional trial on 27 naïve patients 3.56), p = 0.054. However, data show a sig- functionality during WD morphogenesis is with LUTS. At baseline (V ) and after 28 nificative reduction in Ang2/Ang1 ratio from unknown. Here we studied TLR4 expression 0 days (V ) of PEMF therapy, all patients had V0 to V1: Ang2/Ang1V0 2.1 (0.7;5.0) vs and the regulation of TLR4-target genes dur- 1 blood tests, Ang-1 and Ang-2 serum levels, Ang2/Ang1V1 1.2 (0.7-2.9), p = 0.036. ing rat WD morphogenesis between embry- transrectal ultrasound and questionnaires Conclusion A deregulation of the balance onic days (e) 17.5 and 20.5. The functionality (IPSS). The PEMF device (Magcell® Micro- between Ang2 and Ang1 may be associated of TLR4/NFKB signaling and the morpho- circ, Physiomed Elektromedizin) was applied with inflammation. We demonstrated that, af- logical consequences of its activation were assessed using WD organotypic cultures challenged with LPS from E. coli (TLR4 ago-

DGA-Abstracts nist; 25 ng/mL) and PDTC (NFKB inhibitor; 100 μM). TLR4 was constitutively expressed in the WDs whereas Tlr4 mRNA levels in- creased between e17.5 (uncoiled WD) and e20.5 (coiled WD). A switch from a mesen- chymal to a predominant epithelial TLR4 im- munoreactivity also occurred in this period. Compared to controls, TLR4-target genes (inflammatory mediators) were differentially regulated and the IL1B immunodistribution changed in WDs challenged with LPS and/or PDTC, confirming the functionality of TLR4/ NFKB signaling in this tissue. TLR4 activa- tion by LPS also resulted in WDs with small- er length between bends and regions of ductal dilatation. These morphological disturbances were associated with laminin reassembly, implicating ECM as a participant in the regu- lation of WD morphogenesis by TLR4. Our results place TLR4/NFKB signaling as player during epididymal development. Activation of this signaling pathway during maternal infectious and inflammatory conditions may impact reproductive outcomes of male off- spring. Funding FAPESP/CNPq/CAPES. Ethics approval: CEUA-Unifesp #1776201213.

P 7.10 Angiopoietin pathway in benign prostatic hyperplasia after pulsed electromagnetic field therapy: evaluation of a new serum inflam- matory marker M. G. Tarsitano, M. Tenuta, F. Sciarra, P. Mazzotta, M. Minnetti, F. Sesti, V. Olivieri, R. Pofi, M. A. Venneri, D. Gianfrilli, A. Isidori Sapienza - Università di Roma, Rome, Italy Introduction Chronic low-grade inflamma- tion and endothelial activation seems to play an important role in Benign Prostatic Hyper- plasia (BPH) etiology, due to the consequent hypoxia. We recently demonstrated that pulsed electromagnetic field therapy (PEMF) is effective to reduce prostate volume (PV) and lower urinary tract symptoms (LUTS) after 28 days of therapy, because of its anti- inflammatory and pro-angiogenic effect. In this study, no changes were found in clas- sical inflammation markers. Angiopoietin (Ang) system regulates angiogenesis and is involved in the pathogenesis of inflammation: Figure 8. M. G. Tarsitano, et al.

54 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts ter 28 days of PEMF treatment, Ang2/Ang1 reduces significatively, as PV and IPSS. De- Figure 9. S. Chandra, et al. spite small sample size, these preliminary findings appear solid to identify Ang2/Ang1 as an effective serum marker of PEMF effi- cacy in BPH treatment (Fig 8: Angiopoietin pathway. Mod. from [van Meurs et al. Crit Care 2009; 13: 207]; Magcell® Microcirc, Physiomed Elektromedizin AG, Scnaittach, Germany set on frequency of 4–12 Hz and on an intensity of 1000 Gauss. A: (1) effective area; (2) start button; (3) status LED. B. Cor- rect position with the marked active surface placed on the perineal region; Characteristic of study population. Comparison between patients (n = 27) before (V0) and after 28 days of therapy (V1). Value are expressed in

median (IQR). Wicoxon test p-value reported DGA-Abstracts (* = p < 0.05).

P 7.11 P 7.12 decreases with the severity of hypospadias and hence is a marker for in-utero androgen Selection for female high fertility Anogenital distance: a marker action. Since the TDS disorders originate feedbacks on male reproductive of severity of hypospadias and from androgen disruption during the MPW, performance ­in-utero androgen production therefore an assessment of AGD is important in establishing testicular dysgenesis in new- 1 1 1 1 1 2 M. Michaelis , A. Sobczak , C. L. M. Ludwig , S. Chandra , R. Nerli , S. Ghagane borns as these culminate in low fertility and 2 1 1 M. Langhammer , J. M. Weitzel Jawaharlal Nehru Medical College, KLE University, testicular tumors in adulthood (Fig. 9). 1Leibniz Institute for Farm Animal Biology (FBN), Belagavi, Karnataka, India, Urology, Belagavi, India; ­Institute of Reproductive Biology, Dummerstorf, 2KLES Dr Prabhakar Kore Hospital and MRC, Urinary Germany;­ 2Leibniz Institute for Farm Animal Biology Biomarkers, Belagavi, India P 8.1 (FBN), Institute of Genetics and Biometry, Dummers­ torf, Germany Introduction Hypospadias is a congenital Male oncofertility: Awareness, anomaly in which the urethra opens onto knowledge and barriers among The Dummerstorf high fertility mouse lines the ventral aspect of the penis rather than oncologists in india FL1 and FL2 are selected for increased litter at the tip. Anogenital distance (AGD) is an size for more than 190 generations. Com- anthropometric measurement of genital de- P. Tholeti1, V. Bhat2, K. Udupa3, S. Uppangala1, pared to the unselected control mouse line, velopment. Hypospadias, cryptorchidism, G. Kalthur1, N. Spears4, T. Woodruff5, S. K. Adiga1 which derived from the same genetic back- testicular germ cell tumor and low sperm 1Manipal Academy of Higher Education, Clinical ground, both fertility lines almost doubled counts constitute the Testicular Dysgenesis ­Embryology, Manipal, India; 2Manipal Academy of the number of offspring per litter. Selection Syndrome (TDS) and have a common origin Higher Education, Pediatric Hematology & Oncology, for a high litter size did not only increase the in early fetal life caused by an abnormality Manipal, India; 3Manipal Academy of Higher Educa­ ovulation rate of FL1 and FL2, it is also an in the development of fetal testes. Genital tion, Medical Oncology, Manipal, India; 4University integral of other traits of female reproduction development is programmed during a critical of Edinburgh, Edinburgh, United Kingdom; 5North­ like , uterine capacity and time period, “Male Programming Window” western University, Chicago, IL, United States embryo survival. However, little is known (MPW). Disruption of androgen action dur- Despite numerous develop- about the effects of selection for exclusively ing this MPW results in hypospadias and re- Introduction ments in Oncofertility, there are barriers female high fertility on the male side. duced AGD. In this study, we intended to ex- amine the association of AGD with proximal which hinder the successful establishment Many genes essential for reproduction per- of a fertility preservation (FP) program. formance are shared by both sexes in their and distal hypospadias from all the newborn males. One of the barriers was found to be lack of reproductive organ. Hence, we assume that knowledge in both healthcare providers and long-term selection (45 years) for female Materials and Methods We examined all patient population worldwide, including In- high fecundity also affected male reproduc- male newborns in our hospital from January dia [Salama et al, JCO, Global Oncol 2020; tive performance. We found various altera- 2015 to December 2019. The anogenital dis- 6: 379]. Hence, there is a need to assess the tions the concentration of sex steroids, sperm tance was recorded in millimeters (mm) with awareness and identify the barriers among motility parameters and gene expression a sliding digital caliper, used for measuring healthcare providers to implement effective pattern in the testes. Our results indicate the distance between the base of the scrotum male FP program in the country. that FL1 and FL2 independently generated to the center of the anus; defined as AGD. Methods A nationwide survey was directed and justified their phenotype using different Results During the above mentioned pe- to oncologists attending various national con- molecular strategies. In addition, FL1 and riod, there were 28,426 (14,615 males and ferences or academic meetings between May FL2 display some elevated male- as well as 13,811 females) full-term live births in our and November 2019 with questions related to female-specific characteristics. hospital. The mean AGD was 21.06 ± 5.57 male oncofertility services. Informed consent Therefore, these genetic heterogeneous mm in newborns without hypospadias, 9.92 was taken at the beginning of the survey. The mouse models are likely to provide novel ± 1.90 mm in newborns with proximal hy- data was analysed by using descriptive sta- signatures for increased reproductive per- pospadias and 17.03 ± 1.95 mm with distal tistics. hypospadias. The shorter AGD in newborn formance and thus provide new insights into Results The response rate to the survey molecular and cellular complexity of highly males with proximal hypospadias in com- was 23% (49 of 214) from oncologists across fertile phenotypes. parison to distal hypospadias indicates AGD vari­ous subspecialties. Pertaining to knowl- to be a marker of the severity of in-utero an- edge of male FP options, 52% of them re- drogen production. ported adequate knowledge of sperm banking Conclusion Hypospadias is associated with but only 25% of them had awareness regard- reduced anogenital distance. AGD further ing immature testicular tissue (ITT) freezing.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 55 Andrology 2020 – Abstracts

When asked about the time required for each Although significantly higher MAGEC2 pro- T-cells, in tumor-central areas. Quantitative of the procedures, 71% reported that it took tein expression was detected in SE than in NS, RT-PCR of selected cytokines/chemokines < 1week for sperm banking while 36% re- according to our results MAGEC2 expression confirmed that TGCT is associated with a ported 2-4weeks for TT freezing and 14-28% was less sensitive for SE than previously re- pro-inflammatory milieu. The clinical data- were not aware of the time taken for either of ported. So, to consider MAGEC2 as a TGCT base of the study will allow tests for possible the procedures. The major barrier in oncofer- diagnostic biomarker further investigation of correlations between immunopathological tility services was cited as, “financial burden” expression and regulation at the DNA level patterns of TGCT and parameters of tumor (82%) on the patient. is required. stage and progression. By this comprehen- Conclusion The study demonstrated that Grants This study was supported by the sive approach, we aim to decipher the role majority of the oncologists agree on the im- Scientific Center of Excellence for Re- of “immune editing” during TGCT develop- portance of FP, however urged a need for productive and Regenerative Medicine, ment and progression/metastatic behaviour. oncofertility awareness programs. The survey Republic of Croatia and by the European Results will help to identify novel prognostic also highlights the major barriers for effective Union through the European Regional De- factors and/or immune-therapeutic concepts utilization of oncofertility services in India. velopment Fund, under grant agreement No. for human TGCT. One of the limitations of the study is lack of KK.01.1.1.01.0008, project “Reproductive Grant Supported by DFG GRK 1871/2. details pertaining to the types of cancers treat- and Regenerative Medicine – Exploring New ed by oncologists and the frequency of treating Platforms and Potentials

DGA-Abstracts adolescent and young adult cancer patients. P 8.4 Evidence of a dramatic alteration P 8.3 of the H4 acetylation pattern in P 8.2 Immunopathology of testicular testicular cancer patients prior to MAGEC2 as a biomarker for testi­ germ cell tumors – a clinical ap­ any treatment cular germ cell tumors 1 1 1,2 proach A. de la Iglesia , F. Barrachina , M. Jodar , D. Raos1,2, J. Krasic1,2, S. Masic3, M. Coric4, A. Soler-Ventura1, C. Mallofré3, L. Rodríguez-­ J. Heyer1,2, R. Islam2, F. Dittmar1, A. Pilatz1, B. Kruslin3,1,2, A. Katušic´ Bojanac1,2, F. Bulic Jakus1,2, Carunchio3, A. Goudarzi4, J. M. Corral5, K. Hartmann2, S. Kliesch3, F. Wagenlehner1, D. Ježek2,5, M. Ulamec3,2, N. Sincˇic´1,2 J. L. Ballescà6, J. Castillo1, R. Oliva1,2 M. Hedger4, B. Loveland5, K. Loveland4,6, D. Fietz2, 1University of Zagreb School of Medicine, Depart- 1Institut d’Investigacions Biomèdiques August Pi i H. C. Schuppe1 ment of Medical Biology, Zagreb, Croatia; 2Centre Sunyer (IDIBAPS), Molecular Biology of Reproduction 1Justus-Liebig-University, Giessen, Dept. of Urology, of Excellence for Reproductive and Regenerative and Development Research Group, Fundació Clínic Pediatric Urology and Andrology, Giessen, Germany; Medicine, University of Zagreb School of Medicine, per a la Recerca Biomèdica, Department of Biom­ 2Justus-Liebig-University, Giessen, Dept. of Veteri­ Zagreb, Croatia; 3University Hospital Center Sestre edical Sciences, Faculty of Medicine and Health nary Anatomy, Histology and Embryology, Giessen, milosrdnice, Ljudevit Jurak Clinical Department of Sciences, Universitat de Barcelona, Barcelona, Germany; 3University of Münster, Centre of Repro- Pathology and Cytology, Zagreb, Croatia; 4University Spain; 2Hospital Clínic, Biochemistry and Molecular ductive Medicine and Andrology, Münster, Germany; Hospital Centre Zagreb, Department of Pathology Genetics Service, Barcelona, Spain; 3Hospital Clínic, 4Hudson Institute for Medical Research, Centre for and Cytology, Zagreb, Croatia; 5University of Zagreb Department of Pathology, Barcelona, Spain; 4Shahid Reproductive Health, Clayton, Australia; 5Burnet Ins- School of Medicine, Department of Histology and Beheshti University of Medical Sciences, Depart- titute, Melbourne, Australia; 6Monash University, Embryology, Zagreb, Croatia ment of Clinical Biochemistry, School of Medicine, School of Clinical Sciences, Clayton, Australia Tehran, Iran; 5Hospital Clínic, Department of Urology, Testicular germ cell tumors (TGCTs) are the Barcelona, Spain; 6Hospital Clínic, Clinic Institute of most common malignancies among young Despite the presence of complex immuno- suppressive mechanisms contributing to im- Gynaecology, Obstetrics and Neonatology, Barcelo- men with increasing incidence leading to a na, Spain need for biomarkers for early detection and mune privilege in the testis, recruitment of immune cells with pro-inflammatory proper- diagnosis. TGCTs are divided into seminomas Introduction Histone H4 acetylation (SE) and nonseminomas (NS). MAGEC2 ex- ties is observed in testicular infections or tes- (H4ac) is a well-established epigenetic regu- pression is restricted to testis. However, mod- ticular germ cell tumors (TGCT). Although lator of spermatogenesis in many species, erate to high protein expression was seen in seminoma (SE) and pre-invasive germ cell particularly important during spermiogenesis malignant neoplasms and was already report- neoplasia in situ (GCNIS) are associated with for the histone-to-protamine transition. In this ed as a sensitive marker for SE. Still, investiga- immune cell infiltrates, infiltrate function and work, we aimed at gaining more understand- tions of DNA methylation pattern has not been how they affect TGCT progression and prog- ing about this epigenetic mark through the reported yet. We performed a comprehensive nosis, are unknown. As part of an ongoing study of its potential alterations in patients in silico bioinformatics analysis at the DNA prospective study, testicular tissue was ob- with testicular cancer. tained from 59 patients undergoing orchidec- methylation level and compared it with data of Patients and Methods We performed im- protein expression in SE and NS tissue. tomy for TGCT (age 18–69 years, mean 25.7; SE n = 33; non-SE TGCT n = 7; mixed tu- munohistochemistry in human testicular In 108 TGCT samples, MAGEC2 protein mors n=12; Leydig cell tumors n = 3; other ­biopsies (n=24) in order to compare the H4ac expression was analyzed by immunohisto- pathologies n = 4). A comparative analysis pattern between patients diagnosed with 5 chemistry in healthy testicular seminiferous of SE patients (n = 16) included Bouin-fixed different types of testicular cancer displaying tubule tissue, GCNIS, TGCTs, SE, and all and cryopreserved tissue from tumor-central, spermatogenic activity and control men with NS components. The immunoreactivity score -adjacent and -distant sites. Histopathology normal spermatogenesis. (IRS) was calculated with a cutoff value of showed considerable heterogeneity, display- Results We detected a drastic disruption of four for clinical diagnostic positivity. For ing SE (n = 10), GCNIS (n = 7), preserved the H4ac pattern in all subgroups of testicular the analysis of DNA methylation in TGCT, spermatogenesis/germ cell aplasia (HYP/ cancer patients assessed prior to any treat- UALCAN was used. SCO) (n = 13), or combinations thereof ment as compared to controls. In particular, MAGEC2 showed linear, decreasing trend (SE+GCNIS n = 4, SE+GCNIS+HYP/SCO a global increase of the H4ac levels occurs in of protein expression from healthy testicular n = 2, SE+HYP/SCO n = 1, GCNIS+HYP/ the seminiferous tubules adjacent to the tu- tissue to GCNIS and to TGCTs, meaning it SCO n = 10). Using immunohistochemistry, mour during the first stages of the spermato- becomes suppressed with malignant progres- cells positive for CD68, CD11c, CD3, CD4, genesis up to later-stages round spermatids, sion. MAGEC2 expression was significantly CD8, and CD20, and markers for Treg and Tfh as well as in the somatic Sertoli cells. This higher in SE than in NS. DNA methylation cells (CD25, FOXP3, CXCR5, BCL6) were pattern is markedly different to that found in pattern was in line with MAGEC2 protein mainly identified in GCNIS and SE. Semi- controls, in which a gradual increase of H4ac expression, being significantly higher in NS quantitative scoring revealed the highest with a maximum at elongating spermatids than in SE. numbers of immune cells, especially CD3+ was detected consistent with previous reports.

56 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

Conclusion Our novel results suggest that, Republic of Croatia, and by the European P 8.7 despite presenting spermatogenic activ- Union through the European Regional De- ity, there is a global deregulation of H4ac velopment Fund, under grant agreement No. FXYD5/dysadherin is a new mar­ in testicular cancer patients, which would KK.01.1.1.01.0008, project “Reproductive ker of prostate cancer progression potentially result in molecular alterations of and Regenerative Medicine – Exploring New and aggressiveness associated to the male gamete. The consequences of this Platforms and Potentials”.) epithelial-to-mesenchymal transi­ epigenetic alteration might be relevant in tion testicular cancer patients expressing desire of fatherhood, since testicular cancer diagnosis P 8.6 M. V. Mencucci1, R. Bello2, M. Rosso1, C. Moiola1, is common during reproductive age. Future The Persistent Müllerian Duct M. J. Besso1, M. H. Vazquez-Levin1 1 studies would reveal the actual risk and con- Syndrome (PMDS) in an adult – Instituto de Biologia y Medicina Experimental sequences towards the progeny underlying (IBYME; CONICET-FIBYME), Laboratorio de Estudios fertility preservation of epigenetically altered case report de Interacción Celular en Reproducción y Cáncer, male germ cells, which is in many cases the J. K. Wolski1,2, T. Demkow2, R. Sosnowski2, Ciudad Autónoma de Buenos Aires, Argentina; 2Uni- only reproductive option available for testicu- T. Kalinowski2, B. Adamowicz2, K. Sikora3 versidad de Tres de Febrero, Saenz Peña, Argentina 1 lar cancer patients. Fertility Clinic Novum, Andrology/Urology Unit, nd 2 Prostate Cancer (PCa) is the 2 most frequent Grants Supported by PI16/00346 to RO, Warsaw, Poland; M. Sklodowska-Curie Memorial cancer diagnosed in men. Among mechanisms Cancer Centre and Institute of Oncology, Department CD17/00109 to JC, FI17/00224 to AI DGA-Abstracts 3 involved, Epithelial to Mesenchymal Transi- of Uro-oncology, Warsaw, Poland; M. Sklodowska- tion (EMT) stands up, and Epithelial Cadherin Curie Memorial Cancer Centre and Institute of Onco- (CDH1) expression/function loss is an EMT P 8.5 logy, Department of Pathology, Warsaw, Poland hallmark. Decreased CDH1 was related to miRNA in prostate cancer – (pre) Introduction Paramesonephric (Müllerian) FXYD5/Dysadherin (FXYD5/Dys) expres- analytical challenges Ducts (MD) form uterus, fallopian tubes, sion, a poor prognosis and metastasis marker upper 4/5 of the vagina. In the male fetus in other tumors. Expression of EMT markers I. Abramovic1, M. Ulamec2, N. Sincˇic´1 MD should disappear until the 9 week of and FXYD5/Dys in PCa and their association 1School of Medicine University of Zagreb, Depart- pregnancy due to anti-müller hormone (MIS, with tumor aggressiveness were evaluated ment of Medical Biology, Zagreb, Croatia; 2University müllerian inhibiting substance), produced by using bioinformatics, cell models and tumor Clinical Hospital Center Sestre milosrdnice, Depart- Sertoli cells of the fetal male gonads. The samples. Gene-disease association analysis ment of Pathology and Cytology, Zagreb, Croatia presence of MD is accompanied by the un- (DisGeNET) identified CDH1 among PCa 50 Prostate cancer (PCa) is the most commonly descending testicles, inguinal hernia (hernia top-genes, and mutation analysis (COSMIC) diagnosed neoplasia among men. MicroR- uteri inguinalis) with adequate developed pe- showed 2.54% CDH1 mutations (57/2243). NAs (miRNAs) in liquid biopsies and tissue nis, the presence of male gonads in the ovary EMT markers and FYXD5/Dys expression have emerged as potential biomarkers that position; sometimes accompanying external analysis done in human PCa cell lines (meta- could improve PCa diagnosis, prognosis, and genital pseudohermaphrodism female form. static potential null/low: LNCaP/C4-2/22Rv1; management. With growing body of research, Diagnosis is usually stated in the prepuber- medium/high: C4-2B/DU-145/PC3) revealed conflicting data was reported, and questions tal period during the correction of hernias or lower CDH1 mRNA levels and higher ZEB1, are being raised regarding diverse (pre)ana- cryptorchismus. Due to the risk of neoplasm SLUG, N-cadherin and Vimentin in PCa with lytical factors influencing miRNA analysis, of the dysgenetic testes and the MD should medium/high metastatic potential (P<0.05). hindering its translation into clinical practice. be removed. PC3 cells showed higher (P < 0.05) expression Materials and Methods 45 yo patient with of Delta34-Ecadvar, an EMT-related novel To analyze and address current problems in CDH1 splice variant (2/57 mutations around miRNA clinical research on PCa, a PubMed- azoospermia, bilateral undescended testis, after bilateral inguinal hernia surgery; hypo­ splicing site). PC3 cells had higher (P < 0.05) based literature search was conducted. Di- expression of FXYD5/Dys mRNA and a 50 verse experimental designs and (pre)analyti- gonadism; glomerulonephritis; IgA nephro­ pathy; chronic renal insufficiency. US: tumor kDa protein localized to the cell membrane/cy- cal factors influencing miRNA analysis were toplasm, and immunodetected in PC3 derived studied and compared across studies. of the right testis, mass in the left inguinal canal, no abnormalities in pelvis. Tumor were tumors in nude mice. PC3 cells treatment with We observed that studies widely differ in markers normal. Cryopreservation of testicu- a FXYD5/Dys siRNA resulted in reversed ex- design parameters such as control groups, lar tissue was planned. pression of EMT markers and higher cell-cell serum and plasma comparison, sample stor- adhesiveness. A data set (GSE7930) analysis age, quality control steps and data normali- Results No evident tumor inside right tes- of low/high metastatic potential PC3 cell- zation. There is an immense lack of data on tis, part of gonadal tissue was cryopreserved. derived tumors showed higher EMT mark- methodo­logy framing parameters used in In the left groin clinically oviduct with ac- ers and FXYD5/Dys expression in the more published studies. companying nodule (gonad in the oviductal aggressive ones. A USCXena TCGA PCa position), uterus and vagina were found. Pa- We propose following steps for improvement data set analysis showed association between thologist report: testis with the hypoplasia of higher FXYD5/Dys expression and lower of future research: 1) control groups should the parenchyma (without spermatogenesis); be age-matched, preferably benign prostate free disease survival, higher Gleason, Stage Leydig cells hyperplasia; testicular ducts and and Grade Group (P<0.05) and an EMT pro- hyperplasia patients; 2) sample collection vessels hypoplasia; the seminal cord tissue and handling should be explained in detail: file. A multiple regression analysis identified with a hamartomatic system; hypoplastic FXYD5/Dys as predictor of high Grade and sample timing, centrifugation, hemolysis vagina and uterus, in the light covered with monitoring, storage conditions; 3) plasma Gleason score. These studies characterized atrophic mucous membrane. A fragment of FXYD5/Dys expression in PCa, and its rela- and serum samples should be stored at -80° hypoplastic tissues of the uterine shaft with C up to 5 years before analysis, while FFPE tion with EMT markers, disease progression fallopian tube and a fragment of tissues with and aggressiveness. blocks up to 10 years and data adjusted ac- a hamartomatic system with a change of type: cording to block’s age; 5) both endogenous adenomatoid tumor. Diagnosis: The Persis- and exogenous miRNAs should be used for tent Müllerian Duct Syndrome. P 8.8 quality control; 6) miRNAs dysregulated in Short anogenital distance is as­ PCa should be avoided for data normaliza- Conclusions Every man with bilateral un- tion, and MIQE guidelines adhered when descended testis should have diagnostics for sociated with testicular germ cell publishing RT-qPCR results. PMDS due to the risk of neoplasia. tumour development Grants This study was supported by the D. Moreno-Mendoza1, E. Casamonti2, A. Riera-­ Scientific Center of Excellence for Re- Escamilla3, S. Pietroforte3, G. Corona4, productive and Regenerative Medicine, E. Ruiz Castañe1, C. Krausz1,2

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 57 Andrology 2020 – Abstracts

1Fundació Puigvert, Instituto de Investigaciones Bio- Introduction and Objectives Leydig cell Pharmacology and Therapeutics, Montreal, Canada; médicas Sant Pau (IIB-Sant Pau), Departament de tumor (LCT) is a relatively rare testicular tu- 4McGill University, Department of Surgery, Montreal, Medicina, Universitat Autònoma de Barcelona, An- mor, consisting the most common non-germ Canada; 5McGill University, Department of Urology, drology, Barcelona, Spain; 2University of Florence, cell testicular tumor, accounting for 1-3% of Montreal, Canada;6McGill University, Department Department of Biomedical, Experimental and Clinical all testicular malignancies. Most LCTs are Obstetrics and Gynecology, Montreal, Canada; Sciences „Mario Serio“, Florence, Italy; 3Fundacio benign, but 10% of them are malignant. Typi- 7McGill University, Department of Human Genetics, ­Puigvert. Instituto de Investigaciones Biomédicas cally, they present as a testicular mass, often Montreal, Canada Sant Pau (IIB-Sant Pau), Molecular Biology Laborato- associated with azoospermia. We present Although combination chemo­ ry, Barcelona, Spain; 4Azienda Usl Bologna Maggio- our single center experience on micro-TESE Background therapy has contributed to increased survival re-Bellaria Hospital, Endocrinology Unit, Medical De- along with Testis Sparing Surgery (TSS). from Hodgkin“s disease (HD) and testicular partment, Bologna, Italy Materials and Methods Three azoospermic cancer (TC), questions about sperm qual- Introduction Testicular germ cell tumour patients underwent surgical exploration for ity after treatments have arisen. Studies have (TGCT) is a multifactorial disease in which testicular tumors and micro-TESE with TSS shown nuclear chromatin damage post-treat- various genetic and environmental factors play intent in our center during the last 9 months. ment, however, the sperm epigenome has re- a role. TGCT is part of the testicular dysgenesis One patient presented with a unilateral­ lesion ceived little attention. Our objectives were to syndrome (TDS) which includes also cryptor- and the other two bilateral lesions. All the determine the impact of HD and TC, as well chidism, hypospadias, oligo/azoospermia and patients had negative tumor markers, such as their treatments, on sperm DNA methyla- DGA-Abstracts short anogenital distance (AGD). The relation- as β-hCG, αFP, and LDH, elevated FSH and tion. ship between AGD and different components LH levels and borderline testosterone levels. Abdomen and thorax CT were normal. In all Methods Sperm were collected from com- of TDS has been assessed in a number of stud- munity controls (CC) and men with HD or ies while information about short AGD and three cases an inguinal approach was used along with temporary cord occlusion and TC (before and after chemotherapy treat- TGCT are not available in the literature. The ments; n = 6–7). Sperm DNA methylation role of AR (CAG)n in the development of TDS cold ischemia of the testicles. Under micro- scopic magnification and intraoperative ultra- was assessed using genome-wide and locus- disorders and in relationship with AGD are specific approaches. scarce and inconclusive. We aimed to evaluate sound assistance all masses were enucleated. AGD in relationship with TGCT and to assess Frozen section (FS) analysis of the lesions Results Before treatment, using 450K ar- the AR (CAG)n polymorphism in relationship and random biopsies were obtained, in order rays, a subset of probes distinguished sperm with AGD and TGCT. to exclude other pathologies of the testis, in- from TC, HD and CC subjects. On com- cluding ITNGCU. Benign findings allowed paring altered sperm methylation between Material and Methods 156 TGCT patients for TSS. In all three cases bilateral micro- HD and TC patients versus CC men, twice and 110 tumour-free normozoospermic TESE was performed, allowing spermatozoa as many sites were affected in TC than HD controls of Spanish origin. All subjects un- extraction, confirmed by the embryologist in men; both groups demonstrated mostly hy- derwent full andrological workup (including the operating room. pomethylation. In TC patients, the promoter semen and hormone analysis) and genetic Results The enucleated lesions measured region of GDF2 contained a large region of analysis (Androgen Receptor (CAG)n). The differential methylation. To assess altera- main outcome measures were the anopenile 0.7–1.2 cm in the largest diameter. All of the lesions were sex cord-stromal tumors, benign tions in DNA methylation over time/post- distance (AGDap), the anoscrotal distance treatment, serial samples from individual (AGDas) and AR(CAG)n. Leydig cell type with low Ki 67 index and TSS was accomplished in these cases. Com- patients were compared. With 450K arrays, Results We observed significantly shorter plete concordance was observed between following chemotherapy, patients showed in- AGD in the group of TGCT patients in respect the results of FS and permanent sections. In creased alterations in DNA methylation, up to controls (p < 0.001) independently from all three cases spermatozoa were found and to ­several years post-treatment, when com- sperm count and testis histology. Threshold cryopreserved. Post-surgically none of the pared to CC. Similarly, using a high resolu- values were calculated for AGD with the best patients reported any complication, and their tion human sperm-specific assay, at includes sensitivity and specificity. Subjects with AG- testosterone levels remain unaffected. One assessment of environmentally-sensitive sites Dap and AGDas below threshold showed a live birth has been reported after a successful (dynamic sites), demonstrated altered sperm significantly increased risk for TGCT (OR = ICSI. DNA methylation in patients post-treatment 4.97, 95-%-CI = 2.01–12.33, p = 0.001 and and suggested preferential susceptibility of OR = 4.11, 95-%-CI = 1.89–8.92, p ≤ 0.001, Conclusions LCT consists 1–3% of all dynamic CpGs. respectively). No significant correlation was testicular tumors, with 90% of them being benign. Most of the cases appear unilateral, Conclusions Distinct sperm DNA methyla- observed between AR(CAG)n polymor- tion signatures were present pre-treatment in phism and AGDs. The median values of the but 3-9% of them are bilateral. Since azoo- spermia can be associated with testicular ma- men with HD and TC and may help explain AR(CAG)n were similar between cases and increases in birth defects reported in clinical controls. lignancy in up to 15% of the cases it is crucial for the urologist not to overlook such a possi- studies. Epigenetic defects in sperm of some Conclusions Ours is the first study focus- bility, even if the tumor found is benign such cancer survivors are present up to two years ing on AGD in TGCT patients. We identified as LCT. In well selected cases, organ spar- post-treatment. Abnormalities in the sperm short AGDs as a significant risk factor for ing surgery can be performed simultaneously epigenome both pre- and post-chemotherapy this disease. After further validation of our with micro-TESE, without compromising the may have detrimental effects on future health. preliminary data, AGD measurement could oncological outcome. Grants Funded by CIHR become part of TGCT screening in order to better define those individuals who would benefit from long-term active follow-up. P 8.10 P 9.1 First night impotence dilemma in P 8.9 Abnormalities in the sperm DNA methylome both pre- and post- man: a Malaysian Study Leydig cell tumor presenting as treatment in men with Hodgkin’s M. I. Mohd Tambi azoospermia: Testis sparing sur­ disease and testicular cancer Damai Service Hospital, Men‘s Wellness Clinic, gery and micro TESE. A single ­Kuala Lumpur, Malaysia D. Chan1, K. Klein2, C. O’Flaherty1,3,4, P. Chan1,5, center experience B. Robaire6,3, J. Trasler1,7,3 When the husband failed to do penetrative T. Charalampos1, C. Konstantinidis2, K. Makarounis1 1Research Institute of the McGill University Health sex on the wedding night, the condition is 1General Hospital of Patras "O Aghios Andreas", Centre, Montreal, Canada; 2Lady Davis Institute known as first night impotence. If failure to Patras,­ Greece; 2National Rehabilitation Center, for Medical Research, Jewish General Hospital, perform penetrative sex persists, then the Neuro-Urology, Athens, Greece Montreal, Canada; 3McGill University, Department of marriage is unconsummated. Considering the

58 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts husband’s role in intimacy is vital, failure to which sexually active: 985). By propen- taining a better ICI test response. In Cohort do it might trigger marital crisis and eventu- sity score matching (PSM), we investigated 2, patients interviewed by female physician ally marital breakdown. The Men’s Wellness whether prevalence of sexual dysfunction, as more frequently reported to have a conflictual Clinic handles male sexual and reproductive measured by IIEF score ≤ 21, was different couple relationship and a reduced frequency issues. Based on a one-year (2018) records, among COVID-19 patients once matched for of climax in their partners. However, they a total of 152 couples and husbands attended age and prevalence of anxiety and depression reported less difficulties in achieving and the Clinic.­ All the male partners registered as (assessed by General Anxiety Disorder-7 maintaining erection, higher frequency of having difficulty in performing penetrative [GAD-7] and Patient Health Questionnaire-9 auto­eroticism, lower occurrence of ED dur- sex. Each male went through history taking, [PHQ-9] validated questionnaires), using a ing and lower impairment in filling of (IIEF-5) questionnaire and scoring 1:3 ratio. morning erections. the penis hardness score scale. Majority of Results Among the 985 sexually ac- Conclusions Physician’s gender affects the the males were virgins and they attempted tive men, 25 reported having contracted results obtained during the ED diagnostic ­penetrative sex with their spouses with book ­COVID-19. 100 men were therefore included workup. Men interviewed by a female phy- or internet sources about sex. Their usual in analysis following PSM (25 COVID-19 sician describe a less severe ED probably as experiences include rapid ejaculation before patients, 75 controls). No significant differ- attempt to defend their own virility. On the penetration, rapid flaccidity before penetra- ence in age, BMI, GAD-7 and PHQ-9 scores other hand, the presence of a male physi- tion or loss of rigidity when the penis buckled were found following matching (p = 0.39, cian during ICI test is associated to a worse on pressing a wall-like barrier at the entrance p = 0.54, p = 0.41 and p = 0.21, respectively). response suggesting a possible unconscious DGA-Abstracts of the vagina. Further attempt at penetrative An overall 14% prevalence of erectile dys- competition. sex resulted in failure. Most of the male at- function (IIEF score ≤ 21) was found for the tendees had tried folklore and traditional study population: COVID-19 patients had medicines. Some even resorted to seeking higher prevalence (7/25, 28%) than controls P 9.4 help from local Samman believing they were (7/75, 9.3%; p = 0.0208). charmed by evil spirits. Some have even at- Shaeer‘s punch technique for tempted taking herbal capsules laced with Conclusions Prevalence of sexual dys- function is common in COVID-19 patients. trans-corporal excavation of PDE-5 inhibitors but failed in their sexual at- ­Peyronie’s plaques upon penile tempts. Based on the examinations, male at- As IIEF investigates erectile function, our tendees who were healthy and who got Grade findings corroborate the role of endothelial prosthesis implantation dysfunction due to COVID-19 in the patho­ 3–4 Penis Hardness score, with wives known O. Shaeer genesis of vasculogenic ED, even after to experience vaginal spasms, the wives were Kasr El Aini Faculty of Medicine, Cairo University, matching for presence of psychological is- referred to the Gynaecologists. After self- Andrology, Giza, Egypt vaginal dilatation and „reverse“ Kegel exer- sues. Cases with severe erectile cises, their sexual dilemma was overcome. Introduction dysfunction and severe Peyronie’s deformity Male attendees who were suffering from P 9.3 may require penile prosthesis implantation „performance anxiety“ were counselled and along with plaque surgery. The latter requires were give PDE-5 inhibitors. Male attendees The physicians gender influences considerable additional operative time and who were found to have weak pelvic floor the results of the diagnostic adds to the possibility of infection. Shaeer’s were taught Kegel’s exercises to firm up the work­up for erectile dysfunction Punch Technique is a minimally invasive muscles and the penis during erection. For 1 1 2 procedure for plaque de-bulking from within all these men first night impotence became a G. Rastrelli , S. Cipriani , A. Craparo , 2 2 2 the corpora cavernosa, with low or no risk of thing of the past. S. De Vincentis , A. Granata , G. Spaggiari , M. Simoni2, M. Maggi1, D. Santi2 urethral or nerve injury. 1University of Florence, Experimental and Clinical Patients and Methods The procedure was P 9.2 Biomedical Sciences, Florence, Italy; 2University of performed for 26 patients. Following corpo- Prevalence of self-reported sexual Modena and Reggio Emilia, Department of Biomedi- rotomy and dilatation, Peyronie’s plaques cal, Metabolic and Neural Sciences, Modena, Italy were punched-out using the punch forceps. dysfunctions among male The punch forceps is a reusable metal instru- Despite the well-known influ- ­COVID-19 patients in Italy Background ment widely used to cut-out vertebral bony ence of psychological and situational factors 1 1 2 1 protrusions in neurosurgery. After Peyronie’s A. Sansone , D. Mollaioli , G. Ciocca , E. Colonnello , on erectile dysfunction (ED), the influence 1 1 plaques were cleared out, penile prosthesis E. Limoncin , E. A. Jannini of the physician’s gender on the andrological 1 implantation proceeded as usual. University of Rome Tor Vergata, Chair of Endocrino- work-up has never been investigated so far. logy and Medical Sexology (ENDOSEX), Department To investigate physician’s gender influence Results Prior to excavation, deviation was of Systems Medicine, Rome, Italy; 2“Sapienza“ Uni- on the erectile dysfunction (ED) diagnostic evaluated in artificial erection and ranged versity of Rome, Department of Dynamic and Clinical workup. from 40–80 degrees, average 68 ± 2. Direc- Psychology, Rome, Italy tion of curvature was dorsal in 11 patients, Cross-sectional study with retro­ Methods dorsolateral in 7 and ventral/ventrolateral A growing body of evidence spective data collection. We evaluate a Introduction in 8. is supporting the notion that the novel coro- consecutive series of ED patients: 95 at the navirus disease (COVID-19) due to SARS- University of Modena and Reggio Emilia Following the Punch technique, all patients CoV-2 might be an endothelial disease. Given (UNIMORE) and 1808 at the University of were clear of Peyronie’s plaques as per pal- the well-known role of endothelial dysregula- Florence (UNIFI). In the UNIMORE cohort pation. Upon evaluation of deformity with tion in the pathogenesis of male sexual dys- (Cohort 1), intracavernousal injection (ICI) the implant in the erect state, all patients had functions, we aimed to investigate whether test was performed in case of suspected vas- a straight penis. No adjuvant measures were patients with a self-reported history of cular pathogenic component. In the UNIFI required. Average additional operative time COVID-19 had worse erectile function than cohort (Cohort 2), patients were evaluated by for Punch technique ranged from 5 to 10 healthy controls, as measured by psychomet- Structured Interview on Erectile Dysfunction minutes. ric tools, once psychological symptoms have (SIEDY) and ANDROTEST. Both cohorts Twenty-five implants survived adequately, been ruled out. were divided in 2 groups according to the with no residual or recurrent curvature. No Materials and Methods The Sex@Covid gender of the physician who performed the urethral complications or extrusions were study was a case-control study was performed ICI test or the structured interview. noted. Penile sensitivity was preserved as per through a web-based survey between April 7 Results In Cohort 1, patients who had the subjective reports. and May 4th, 2020 in Italy. 6821 question- ICI test performed by a female physician Conclusions Shaeer’s Punch Technique al- naires were collected (males: 2644, among had a significantly higher probability of ob- lows safe and expedited excavation of Peyro-

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 59 Andrology 2020 – Abstracts

nie’s plaques upon penile prosthesis implan- P 9.7 P 9.8 tation, obviating the need for mobilization of the neurovascular bundle or the spongiosum, Circumferential acquired macro­ The role of family history of and obviating the need for plaque excision penis: definition and proposal of a ­cardiovascular disease in andro­ and grafting. geometrically-based reduction logical patients in the late onto­ corporoplasty genesis P 9.6 E. Pescatori, B. Drei, S. Rabito V. Odin, E. Kochanova, V. Didenko Erectile dysfunction as an early Hesperia Hospital, Andrology, Modena, Italy Kirov Military Medical Academy, Saint Petersburg, Russian Federation marker of cardiovascular disease Introduction In the medical literature there are scattered but recurrent reports of penises Introduction Cardiovascular diseases (CVD) S. Krasnyak, O. Apolikhin, E. Efremov too wide to allow effective penetration. Our are the most common diseases in late ontogen- N. Lopatkin Scientific Research Institute of Urology purpose is both: to define a new medical en- esis and one of the most frequently associated and Interventional Radiology – Branch of the Natio- tity, the “circumferential acquired macrope- comorbidities condition in andrological pa- nal Medical Research Radiological Centre of the nis”: development over time of a penile girth tients. A special role in the occurrence of CVD ­Ministry of Health of the Russian Federation, De- enhancement that mechanically hampers is given to family anamnesis. Several evidences partment of ­Andrology and Human Reproduction, penetration, and to propose a mathematical established a link between erectile dysfunction Moscow, ­Russian Federation DGA-Abstracts model to perform a geometrically-based re- (ED) and CVD. It is interesting to investigate Background Cardiovascular diseases (CVD) duction corporoplasty. the association of ED and family history of have a very similar profile of risk factors with Patients and Methods A recent case of CVD. such a common reproductive disorder as acquired penile circumferential widening Patients and Methods The study included erectile dysfunction (ED). Therefore, we per- prompted us to a Medline PubMed and a 46 patients 44 to 86 years old (mean age ± SD formed several studies to define link between Google search literature review of similar 61 ± 10.1) who attended an andrology out- erectile dysfunction and social significand cases. patient clinic complaining of ED. Men were disease (CVD, diabetes, metabolic syndrome Results We identified seven published cases classified as have a positive family history etc.) of acquired penile circumferential widening; of CVD – group 1 (n = 23) (60 ± 8.3 years) Methods We present data from different in five ones intercourse was hindered by the or patients without family history of CVD – studies performed in Research Institute of shaft deformation. In the majority of cases group 2 (n = 23) (60 ± 10.6 years).The clinical Urology from 2010. Totally more than 3000 priapistic episodes were deemed the causal and laboratory surveys including International patients were included. Patients filled the factor of the morphologic anomaly; in other Prostate Symptom Score (IPSS), Aging Male IPSS and IIEF-5 questionnaires. Anamnestic cases, including ours, a clear etiology could Symptom (AMS) scale and International In- data, the results of anthropometry (height, not be identified. Surgically treated cases dex of Erectile Function (IIEF-5) score were weight, body mass index (BMI), waist cir- underwent bilateral elliptical albuginea exci- investigated. Family history of CVD was cumference), and laboratory research meth- sions. Workup in our case included: dynamic defined as having a first-degree relative with ods: plasma cholesterol and glucose levels infusion cavernosometry and cavernosogra- a cardiovascular event such as coronary heart were recorded. Selected categories of pa- phy (figure) that ruled out vascular erectile disease or ischemic stroke before the age of 60 tients underwent a comprehensive cardiologi- problems, and dynamic Magnetic Resonance years for female and 55 years for men. cal evaluation. Sometimes we used methods Imaging that depicted an aneurismatic dilata- Results 1.) The comparison of the andro- of predictive analytics (CHAID algorithm, tion of both corpora cavernosa. We developed logical parameters show that, aging male neural network) in addition to conventional a mathematical model to precisely define the symptoms were more pronounced in the statistics. amount of tunica albuginea to be resected. sample of patients with positive family his- Results Prevalence of ED was 51.04%. In We performed accordingly a geometrically tory of CVD (p < 0.05), where the mean of this case, a mild ED is observed in 38% of sound reduction corporoplasty. A thinned AMS score was 44.6 ± 11.8, and 37.7 ± 7.3 men, moderate and severe ED – in 10.5% and albuginea was present in the affected area; in in second group. As well there was a statisti- 2.5%, respectively. After applying the algo- order to prevent possible recurrences we ap- cal difference in IPSS (p < 0.05) 10.4 ± 7.9 rithm of CHAID decision tree, we found that plied a bovine pericardium patch above both and 5.6 ± 4.1, respectively. But there were no the most significant risk factor for ED occur- corpora from one paraurethral margin to the significant differences between total testos- rence is the severity of LUTS (an importance other. Follow-up is ongoing; at two months terone levels and the IIEF-5 score. factor of 0.64), then with a significant lag fol- patient resumed complete intercourse activ- 2.) While comparative anthropometric meas- low waist circumference (0.08), cholesterol ity, with full patient/partner satisfaction at 11 ures didn”t show any significant differences: level (0.05), hypertension (0.05). Accuracy of months. BMI in the first group was 26.5 ± 2.9 kg/m2, the ED prediction using a trained neural net- Conclusions Circumferential acquired ma­ and 27.9 ± 2.8 kg/m2 in the other group, waist work was 81.9%. Predictors of arterial hyper- cro­penis is a rare but defined medical entity circumference was 102.2 ± 11.40 cm and 98 tension development were pulse frequency, that prevents affected men from sexual pene­ ± 7.7 cm, respectively. severity of LUTS age and body weight. The tration. We propose a mathematical model to Conclusion Family history of CVD a good use of predictive analytics methods allowed perform a sound geometric surgical correc- predictor of the severity of clinical manifesta- generating an algorithm for predicting the de- tion. Follow-up is encouraging (Fig. 10). tions in andrological patients. velopment of cardiovascular diseases with an accuracy of 80.3%. Conclusion Using erectile dysfunction as P 9.9 an early marker of cardiovascular diseases, Long term penile perception urologist can not only improve the quality of scores and sexual health out­ life of a patient with erectile dysfunction, but also save his life comes in patients treated for ­ex­strophy bladder P. Gupta JN Medical College, KLE Academy of Higher Educa- tion and Research (Deemed to be University), De- partment of Urology, Belagavi, India Introduction and Objectives Children born Figure 10. E. Pescatori, et al. with bladder exstrophy (CBE) have achieved

60 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts a near normal life-expectancy over the past lateral and craniocaudal position of the fully 9, 12 and 24 months revealed a statistically four decades. These patients undergo a num- erect phallus. Clinic based assessment ICI significant mean reduction in degrees of cur- ber of reconstructive procedures to achieve (10ug) with angulation measurement and an vature (3 mo: –11,7, p < 0.0001; 6 mo: –14,1, closure of the exstrophy, gain continence erectile function questionnaire (IIEF-15). p < 0.0001; 9 mo: –16,9, p < 0.0001; 12 mo: and treat genital anomalies. All these proce- A paired-samples t-test was calculated to –20,5, p < 0.0001; 24 mo: –23,1, p < 0.0001), dures have a profound impact on the physi- compare the mean degree of curvature using PDQ (–8,9, p < 0.0001; –10,7, p < 0.0001; cal appearance, psychological and sexual photos to the mean degree of curvature using –12,1, p < 0.0001; –13,3, p < 0.0001; –14,9, well-being of the patients. As these patients alprostadil injection. p < 0.0001) and IIEF (5,47, p = 0.002; 4,12, grow, sexual function and fertility become an Results The mean degree of curvature p = 0.011; 5,35, p = 0.007; 6.38, p = 0.004) increasingly important aspect of their day-to- using AHP was 46.7° (sd = 12.6), and the from baseline. Common side effects included day lives. We prospectively assessed patients mean degree of curvature using ICI was 54° minor pain upon injection (59%) and minor with CBE having undergone treatment at our (sd = 16,7). A significant increase in angula- local hematoma. No allergic reaction, rupture centre previously (at-least 5 years prior) and tion from AHP to ICI was found (p < 0.001). of tunica albuginea or other severe AE were were 18 years or more in relation to their A subgroup analysis was conducted on pa- observed. sexual function and fertility status. tients with a curvature using photos of less Conclusions The treatment with CCH Materials and Methods Male patients 18 than 50° also showed significant increase injections for PD has a lasting positive im- years of age and having undergone surgical in angulation from photo to alprostadil pact on degree of curvature, patient’s erectile treatment at our centre for CBE (at-least 5 (p < .001). A multiple linear regression was function and quality of sexual life scores over DGA-Abstracts years previously) were prospectively as- calculated predicting the difference in the more than 24 months. Pain is a common ad- sessed for sexual function and fertility status. curvature based on patients’ age and IIEF. verse event of CCH injections and should be The patients were asked to answer validated The regression equation was not significant prevented by penile block prior CCH injec- questionnaires, including the Sexual Health (p > 0.05). tion. CCH therapy can be considered a last- Inventory for Men (SHIM), Penile Percep- Conclusions Homebased autophotography ing, effective and safe for treatment of PD. tion Score (PPS), and Male Sexual Health of the erect phallus underestimates the degree Questionnaire for assessing ejaculatory dys- and extent of curvature. Neither IIEF nor age function. are a significant predictor of the difference in P 10.1 Results and Observations During the pe- AHP and ICI induced erection. Clinic based Adenosine is a pro-inflammatory riod Jan. 2019 – Dec. 2019 a total of 28 male alprostadil injection should be performed for molecule in human testicular peri­ patients previously operated for exstrophy of assessment prior planning treatment strategy, tubular cells bladder attended the Urological services. Pe- especially prior surgical correction. nile perception scores ranged from very satis- A. Missel1, L. Walenta1, M. Trottmann2, U. Pickl2, fied to very dissatisfied. Nineteen (67.85%) F. M. Köhn3, A. Mayerhofer1 patients were sexually active and only three P 9.11 1LMU München, Biomedical Center - Anatomy III, 2 patients gave history of antegrade ejaculation. Collagenase Clostridium histo­ Cell Biology, Planegg-Martinsried, Germany; Uro- logie und Andrologie am Promenadenplatz, Munich, Conclusions Patients operated for bladder lyticum for the treatment of Germany; 3Andrologicum München, Munich, exstrophy present with sexual and relation- ­Peyronie’s disease: a prospective ship concerns similar to the population at Germany­ 24 month follow up study large. Though not unique, these concerns Human testicular peritubular cells (HTPCs), need to be addressed on a case to case basis. D. Schlager1, O. Cakir2, F. Chierigo3, F. Geiger1, together with extracellular matrix, form a D. Ralph2, U. Wetterauer1, C. Leiber1 compartment surrounding the seminifer- 1University Medical Center Freiburg, Urology and ous tubules of the human testis. HTPCs are P 9.10 ­Andrology, Freiburg, Germany; 2University College involved in sperm transport, cell-cell com- Autophotography underestimates London Hospitals & St Peter’s Andrology, Andrology, munication and have immunological roles. 3 clinical extent of Peyronie’s dis­ London, United Kingdom; Department of Urology, In many cases of impaired spermatogenesis, San Martino University Hospital – IST National the architecture of the wall and the phenotype ease for penile angulation assess­ ­Cancer Research Institute, Genoa, Italy of HTPCs change, implying a role of these ment cells in male (in)fertility. Extracellular ATP Objectives Collagenase Clostridium his- D. Schlager1, O. Cakir2, F. Chierigo3, F. Geiger1, tolyticum (CCH) injections are the only acts as a danger molecule and is involved in D. Ralph2, C. Leiber1 licensed medical treatment for Peyronie’s promoting a pro-inflammatory environment. 1University Medical Center Freiburg, Urology and disease (PD). Only few studies are available A previous study in HTPCs indicated such ­Andrology, Freiburg, Germany; 2University College assessing long term follow up in the current actions after treatment with ATP [Walenta London Hospitals & St Peter’s Andrology, Andrology, literature. Our study evaluated complications et al., Sci. Report, 2018]. ATP can, however, London, United Kingdom; 3Department of Urology, and functional outcomes during a 24 month be degraded by the two ectonucleotidases San Martino University Hospital – IST National Can- follow up after therapy. ENTPD1 and NT5E, which are expressed cer Research Institute, Genoa, Italy by HTPCs in situ and in vitro. Their func- Patients and Methods Baseline, treatment tionality was confirmed in vitro (Malachite Objectives Since Kelami introduced at and follow-up (FU) data of 34 consecutive Green assay) and inhibition of ENTPD1 (by home autophotography (AHP) for evalua- patients treated with CCH using the London POM-1) also reduced the increase of pro- tion of penile curvature in 1983, standardized protocol (6 intralesional injections of CCH inflammatory cytokine levels evoked by ATP photography of the patients” phallus in full 0.9 mg at 4-weekly intervals in addition to treatment. Hence, metabolites of ATP (ADP, erection is considered a tool for objective home modelling) were assessed. Interna- AMP and adenosine) are likely involved. We penile deformity assessment. With several tional Index of Erectile Function (IIEF), PD focused on adenosine (A), because it showed conservative and surgical treatment options questionnaires (PDQ), and autophoto-docu- strong pro-inflammatory actions and elevated available today, evaluation of precise degree mentation were performed at baseline and at levels of several cytokines in HTPCs. It ex- of angulation is essential for deciding further 3, 6, 9, 12 and 24 months to evaluate degree erts its actions via four ADORA receptors treatment strategy. Our study evaluated AHP of curvature and functional outcomes. (A1, A2A, A2B and A3). A2B is strongly to intravacernoasl alprostadil injections (ICI) Results Median age was 58 ys (53; 65,2). expressed at mRNA level in HTPCs and we prior treatment decision. Median penile length was 13 cm (12,5; 14) found expression of A2B in corresponding Methods We performed a prospective study and median degree of curvature was 55° cells of human testicular sections, notably of 55 consecutive patients with Peyronie’s­ (38,7°;70°). PDQ score before treatment was in fibrotically thickened peritubular walls. disease prior receiving treatment. All patients 31 (23; 42,3), median IIEF prior treatment Activation of the A2B receptor in HTPCs, provided standardized AHP from in front, was 46,5 (34; 56). Follow up data at 3, 6, using the specific A2B agonist BAY60-6583,

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 61 Andrology 2020 – Abstracts

resulted in significantly elevated transcript macrophages under different maturation and likely controlling and/or affecting lipid me- and protein levels of IL6 and CXCL8, a result activation states will elucidate the role that tabolism and steroidogenesis in LCT. also obtained with A. However, while A el- activin A plays in regulating the functional Grants Supported by a grant 2016/23/B/ evated levels of CCL7, BAY60-6583 did not. phenotype of tissue-resident macrophages NZ4/01788 (OPUS12 from the National This indicates that in addition to A2B, other in the testis and other organs where activin ­Science Centre, Poland ADORA receptors are also involved. These is produced. results pinpoint A as a pro-inflammatory fac- tor in HTPCs and indicate that interference­ P 10.4 with the generation of ATP-metabolites can reduce inflammatory effects in HTPCs. P 10.3 Indolamine deoxygenase-1 (Ido-1) expression and the effects of fol­ Grants Supported by DFG MA1080/23-2. Do G protein-coupled estrogen ­receptor (GPER) and peroxisome listatin treatment in murine auto­ proliferator-activated receptors immune epididymo-orchitis P 10.2 (PPARs) regulate lipid metabolism R. Wijayarathna1,2, H. Khande1, N. Nicolas1,2,3, 1,2 4 1,2 and steroidogenesis in Leydig cell S. Biniwale , P. Gregorevic , K. Loveland , Regulation of macrophage re­ A. Meinhardt1,2,3, M. Fijak3, M. Hedger1,2 sponses in bone marrow-derived tumors? 1Hudson Institute of Medical Research, Centre for DGA-Abstracts and cell line macrophages by B. Bilin´ska1, E. Górowska-Wójtowicz1, A. Miłon´1, Reproductive Health, Clayton, Australia; 2Monash ­activin A: implications for testis P. Pawlicki1, M. Kotula-Balak2, A. Hejmej1, University, Southern Clinical School, Melbourne, 3 3 immunology J. K. Wolski Australia; Justus-Liebig University, Institute of Ana- 1Institute of Zoology and Biomedical Research, tomy and Cell Biology, Giessen, Germany; 4University J. Bender1,2,3, S. Bhushan3, A. Mansell4,1, ­Faculty of Biology, Jagiellonian University, Depart- of Melbourne, School of Biomedical Sciences, Mel- K. Loveland1,2, A. Meinhardt3, M. Hedger1,2 ment of Endocrinology, Kraków, Poland; 2University bourne, Australia 1Monash University, Department of Molecular and Centre of Veterinary Medicine, University of Agricul- Epididymo-orchitis can cause chronic pain Translational Sciences, Melbourne, Australia; ture in Krakow, Krakow, Poland; 3nOvum Fertility 2 and infertility. The immunology underlying Hudson Institute of Medical Research, Centre for ­Clinic Warszawa, Poland Reproductive Health, Melbourne, Australia; 3Justus- this condition is poorly studied. An important Liebig University Giessen, Institute of Anatomy and Introduction In recent years, a marked in- factor may be Ido-1, a tolerogenic enzyme Cell Biology, Giessen, Germany; 4Hudson Institute of crease in the incidence of Leydig cell tumor highly expressed in the caput epididymis. Medical Research, Centre for Innate Immunity and (LCT; leydigioma) has been reported, how- Ido-1 is regulated by activin A, a pro-inflam- Infectious Diseases, Melbourne, Australia ever the molecular and biochemical charac­ matory cytokine. Its binding protein, follista- teristics of LCT is scarce [Gheorghisan- tin (FST), inhibits Activin A. We examined Regulation of the anti-inflammatory pheno- Galateanu, BMC Res Notes 2014; 7: 656]. Ido-1 expression and the effects of exogenous type of the testicular macrophages is not well Adequate hormonal balance within the testis FST treatment in a murine model of ex- understood. The immunoregulatory cytokine, plays a pivotal role for blocking hormone-­ perimental autoimmune epididymo-orchitis activin A, which is highly expressed by sev- secreting LCT development. The study aims (EAEO). eral testicular cell types, including the Ser- to determine a potential link between GPER Adult C57/Bl6 mice were immunised with toli cells, could potentially contribute to this and PPARs (α, β, γ) in LCTs, and involve- mouse testicular homogenate in complete regula­tion. However, activin has been report- ment of this link in lipid homeostasis. and incomplete Freund’s adjuvant, along with ed to exert both pro- and anti-inflammatory Material and Methods Residual tissues Bordetella pertussis toxin. Controls received effects in different studies. The possibility from testicular biopsy were collected from adjuvant only, or were untreated. Prior to im- that the effects of activin are dependent on the azoospermic patients (31–45 year-old; munisation, some mice were injected with origin and activation state of the macrophage n = 24). Scanning electron microscopy and a non-replicative recombinant adeno-asso- was investigated by comparing primary routine hematoxylin-eosin (H-E) staining ciated viral vector carrying a gene cassette CSF1-matured bone marrow-derived macro­ were carried out to analyze general structure for FST, which raised peripheral FST levels phages (BMDMs) with two macrophage cells and morphology of LCTs. Protein levels of 5-fold, or an empty vector as control. Tissues lines: immortalised bone marrow-derived GPER, PPARα, PPARβ, PPARγ and LHR were analysed 30 and 50 days after the first macrophages (iMACs) and RAW264.7 (lutropin receptor), PKA (protein kinase A), immunisation. macrophages.­ In BMDM cultures treated PLIN1 (perilipin 1), HSL (hormone-sensitive The cauda epididymis is highly susceptible with bacterial lipopolysaccharide (LPS), ac- lipase), StAR (steroidogenic acute regula- tivin A enhanced expression of pro-inflam- to inflammatory damage. Ido-1, which is tory protein), TSPO (translocator protein), expressed at relatively low levels in the nor- matory markers, such as Tnf, but decreased HMGCS and HMGCR (HMG-CoA synthase their expression in the cell lines. By contrast, mal testis and cauda epididymis, was not in- and reductase) were detected by western blot- creased in orchitis, but increased in the cauda activin inhibited the expression of the anti- ting. inflammatory cytokine, Il10, in response to in proportion to the severity of inflammation. LPS, but increased expression of the anti- Results Leydigioma tumors were compact In the cauda, immune cell markers such as inflammatory macrophage marker Arg1 in with tumor cells closely apposed and tightly CD45, F4/80 and CD80 were increased with all three cell types. Furthermore, preliminary adhering to one another. H-E staining re- increasing severity of epididymitis. Exo­ examination of immunometabolic changes of vealed a mixture of four cell types. Most cells genous FST, which has been shown to lower activated BMDMs and iMACs by Seahorse were polygonal with abundant cytoplasm, the severity of orchitis in this model, reduced oxygen consumption analysis indicates that indistinct cell borders, and prominent nuclei. the expression of Ido1 and immune cell genes activin may regulate the metabolic switch In LCTs, increased expression of GPER and such as Cx3cr1 and CD80 in the cauda during from glycolysis (inflammatory) to oxidative decreased expression of PPARs (α, β, γ) were epididymitis. phosphorylation (anti-inflammatory) in im- detected compared to controls. Moreover, The data indicate that Ido-1 expression is mortalised cells. In summary, these studies expression levels of LHR, PKA, PLIN1, selectively increased in the cauda epididymis indicate that the effect of activin is down- StAR, HMGCS, HMGCR were altered while during EAEO. This may be due to differences stream of inflammatory signalling induced by no changes in HSL and TSPO levels were in the immunological environment and func- LPS, exerts complex effects on different gene detected when compared to Leydig cells in tional role of Ido-1 within the testis and dif- responses, and that the effects of activin on control samples. ferent epididymal regions. Exogenous FST inflammatory activity are dependent on the Conclusion Changes in the expression of reduces inflammation and the subsequent maturation and activation status of the mac- steroidogenic- and lipid balance-regulating increase in Ido-1 expression in the cauda rophage under investigation. Further studies proteins indicate functional interplay be- epididymis, indicating its therapeutic poten- of the differences between the responses of tween GPER and PPAR-mediated signaling tial for treatment of this disease.

62 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

P 10.5 of Münster, Centre of Reproductive Medicine and Mol Cell Biol 2016; 17 722–35]. Our recent Andrology, Department of Clinical and Surgical An- findings show that androgens control the Testicular dysgenesis syndrome drology, Münster, Germany; 4University Hospital of expression of Notch pathway components has a stem cell basis Münster, Department of Neurology, Institute of in vivo [Kamińska, et al. Reprod Biol En- 5 A. Kaushik, D. Bhartiya Translational Neurology, Münster, Germany; Max docrinol 2020; 18: 30], however molecular National Institute for Research In Reproductive Planck Institute for Molecular Biomedicine, Bio­ mechanisms of this regulation are not known. Health, Stem Cell Biology, Mumbai, India analytical Mass Spectrometry Unit, Münster, The study was aimed to explore the role of Germany­ nuclear (AR) and membrane (ZIP9) androgen Incidence of infertility and testicular cancers receptors in the effect of testosterone (T) on In the last years, single cell RNA sequencing has increased in young men in recent times DLL1, DLL4 and JAG1 expression in Sertoli (scRNA-seq) of human testicular cells has with decreased sperm count. The underlying cells. etiology leading to these defects remains to allowed to characterize different spermato- Material and Methods TM4 (AR+, ZIP9+) be understood. Testicular dysgenesis syn- gonial subpopulations, to identify molecular and 15P-1 (AR-, ZIP9+) Sertoli cell lines drome (TDS) is suggested to have a genetic footprints of each meiotic step, to describe were treated with T or transfected with AR basis and possibly arises due to adverse en- transcriptional changes during spermatid siRNA or ZIP9 siRNA to knockdown andro- vironmental influences. Being immortal, development, and to classify the spectrum of gen receptors. Real-time RT-PCR, western altered biology of testicular stem cells due somatic cells in the human testis. Despite the blot and immunofluorescence were used for to perinatal disturbances may result in adult important breakthroughs, this approach had analyses of mRNA and protein expression of DGA-Abstracts onset of diseases including cancer. not been applied to study the testicular tissue of patients with impaired spermatogenesis. In DLL1, DLL4 and JAG1. Testes harbors two populations of stem cells the current study, we performed scRNA-seq Results DLL1 mRNA and protein expres- including pluripotent very small embryonic- of about 30,000 cells from cryptozoosper- sion was reduced by T only in TM4 cells like stem cells (VSELs) and spermatogonial mic and obstructive azoospermic men (n = 3 (p < 0.001), whereas AR knockdown abolish­ stem cells (SSCs). VSELs can be isolated each) undergoing therapeutic testicular sperm ed this effect, which indicates that AR is from single cell suspension obtained after extraction. Using 52 published markers we involved in the regulation of DLL1 by andro- enzymatic digestion by first spinning at 200- identified 9 different germ cell stages and 6 gens. Testosterone decreased JAG1 expres- 300g which allows the majority of cells to testicular somatic cell subpopulations. Com- sion in both cell lines (p < 0.05; p < 0.001), pellet down. However, the stem cells remain paring the proportion of the different cell but only ZIP9 knockdown abrogated T im- buoyant at this time and can be enriched by types in the two groups, we found no change pact on JAG1. This points to a role of ZIP9 further centrifuging the supernatant at 1000g. in the number of spermatogonia and early in the control of JAG1 expression. Testos- VSELs are 2-6 µm in size, express pluripo- spermatocytes. However, the cryptozoosper- terone increased DLL4 (p < 0.05; p < 0.01; tent markers (Oct-4A, Sox2, Nanog, Stella, mic samples displayed reduced numbers of p < 0.001) in TM4 and 15P-1, but this effect Fragilis) and can be enumerated by flow cy- the most advanced germ cells (from pachy- was independent on AR or ZIP9. tometry as viable LIN-CD45-SCA1+ cells. tene spermatocytes onwards) and increased Co-expression of OCT-4 with ER and ER Conclusion In Sertoli cells androgens act- α β numbers of somatic cells. Furthermore, dif- makes them directly vulnerable to endocrine ing via various signaling pathways regulate ferential gene expression and gene ontology disrupting chemicals. the expression of DLL and JAG ligands. analyses allowed the identification of two Our results demonstrated that activation of Treatment effects of diethylstilbestrol (DES, additional somatic cell types in the crypto- the AR is involved in the control of DLL1 2 μg/pup/day on days 1–5) were studied in zoospermic group, specifically the perivas- expression, while ZIP9 signaling influences adult mice on D100. Spermatogenesis was cular and immune cells. To unravel the mo- JAG1 expression. The mechanism of DLL4 disrupted associated with altered ploidy sta- lecular regulators of the different germ and regulation remains to be elucidated in further tus, reduced sperm and infertility. This was somatic cells and the potential alterations in studies. associated with 7 folds increase in VSELs the cryptozoospermic group, we performed numbers and marked reduction of c-KIT gene regulatory network analysis and identi- Grants Supported by a grant 2017/25/B/ positive spermatogonial cells by flow cyto­ fied 403 transcription factors regulating up to NZ4/01037 (OPUS13, National Science Cen- metry and confirmed by qRT-PCR. NP95 2,450 genes each. Thirteen of these regula- tre, Poland) (chromatin remodeling agent) expression tory networks were differentially activated was disrupted suggesting altered epigenetic between the two datasets, with 11 specifi- P 10.8 status of stem cells by endocrine disruption cally altered in the germ cells and two in the possibly results in their excessive self-renew- somatic cells. Overall the use of this new Initiation of testicular differentia­ al and blocked differentiation. Nine of ten technology allowed us to highlight the cel- tion in prepubertal marmoset mice treated with DES had signs of testicular lular and molecular alterations in this patient (Callithrix jacchus) testicular tis­ tumor with markedly increased expression cohort despite their heterogeneous testicular sue using an in vitro organ culture of embryonic markers (OCT-4, SSEA-1). tissue composition. To conclude, stem cell basis for TDS is de­ system lineated for first time and VSELs initiate S. Sharma, R. Sandhowe-Klaverkamp, J. Wistuba, cancers. P 10.7 S. Schlatt Delta-like and Jagged proteins are Centrum für Reproduktionsmedizin und Andrologie, re­gulated by androgens in mouse Münster, Germany P 10.6 Sertoli-cells in vitro The current study aims at achieving in vitro maintenance and differentiation of primate A journey into the testicular tis­ A. Hejmej, A. Kamin´ska, S. Marek, germ cells, using testicular tissue from prepu- sue of cryptozoospermic patients M. Brzoskwinia, B. Bilin´ska bertal marmoset (Callithrix jacchus) by em- using single cell RNA sequencing Institute of Zoology and Biomedical Research, ploying a gas-liquid interphase culture sys- ­Faculty of Biology, Jagiellonian University, Depart- analysis tem. Ethical approval for the use of marmosets ment of Endocrinology, Kraków, Poland S. Di Persio1, T. Tekath2, L. M. Siebert-Kuss1, was obtained according to German federal J. F. Cremers3, J. Wistuba1, X. Li4, G. Meyer zu Introduction Notch pathway mediates con- law. Testis from three-prepubertal marmosets Hörste­ 4, H. Drexler4,5, M. Dugas2, S. Kliesch3, tact-dependent intercellular signaling, which (co-placental triplet) were used. Monkeys S. Schlatt1, S. Laurentino1, N. Neuhaus1 plays important role in spermatogenesis received a deadly overdose of pentobarbital, 1University Hospital of Münster, Centre of Repro­ [Parekh, et al. FASEB J 2019; 33: 8423–35]. their body weights were recorded and blood ductive Medicine and Andrology, Münster, Germany; Delta-like (DLL) and Jagged (JAG) proteins was collected for serum testosterone analysis. 2University Hospital of Münster, Institute of Medical are transmembrane ligands activating Notch Left-testis from each monkey was de-capsu- Informatics, Münster, Germany; 3University Hospital pathway in neighboring cells [Bray. Nat Rev lated and the parenchyme dissected. Tissue

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 63 Andrology 2020 – Abstracts

fragments (approximate size 1–3 mm3) were gy, SA, CA, USA). Mast cell tryptase-positive nantly in EC component, while EC compo- cultured on polycarbonate trans-well mem- cells were found in human, wild boar and stal- nent in mouse TCa was mostly unstained. brane inserts (8 µm pore size) at 35C and 5 % lion testis and human, rat, dog and wild boar Proliferative activity of human and mouse CO2 for upto 12-days in two-different condi- epididymis. All species, except fox and cat TCa correspond and exhibit the same tissue tions: “basic-culture” and “complex-culture” had positively stained stereocilia of the ductal localization while their apoptotic activity sig- (supplemented with FSH and hCG). Cultured pseudostratified columnar epithelium in the nificantly differs in the EC component. This fragments were harvested at day-12 and fixed epididymis. Chymase-positive cells were discrepancy could be explained by different in Bouin”s solution for histology. Both, pre- found only in human testis and epididymis. microenvironment and requires more detailed culture control and cultured samples were an- Additional animal species should be further investigation of the models behavior. alysed using Periodic Acid-Schiff (PAS) and analysed, including several rat strains, to de- immunohistochemical staining for evaluating termine the optimal experimental model for Grants This study was supported by the testicular maturation. Results from complex- mast cell investigation. Scientific Center of Excellence for Re- culture fragments demonstrated improved productive and Regenerative Medicine, Grants This study was supported by the Republic of Croatia, and by the European maintenance of structural integrity, testicu- Scientific Center of Excellence for Re- lar organization, and epithelial arrangement Union through the European Regional De- productive and Regenerative Medicine, velopment Fund, under grant agreement No. compared to samples cultured under basic Republic of Croatia, and by the European conditions. Immunohistochemical characteri- KK.01.1.1.01.0008, project “Reproductive Union through the European Regional De- and Regenerative Medicine – Exploring New

DGA-Abstracts zation demonstrated more advanced testicu- velopment Fund, under grant agreement No. Platforms and Potentials”. lar maturation status in fragments cultured in KK.01.1.1.01.0008, project “Reproductive complex media compared to basic media and and Regenerative Medicine – Exploring New controls, i.e., improved epithelial arrange- Platforms and Potentials”. ment of Sertoli cells (SOX9+), encapsulation P 10.11 of cultured fragments with peritubular-myoid Formation of the testicular im­ cells (αSMA+), localization of spermatogo- P 10.10 munological barrier through im- nial stem cells (MAGEA4+) and presence of mune modulation by somatic pachytene spermatocytes (Boule+) indicating Comparative analysis of apopto- initiation of meiotic transition in vitro. This sis and proliferation in terato­ cells model represents a novel ex vivo approach to carcinoma and the experimental H. Kabbesh1–2, K. Loveland3, P. Stanton3, G. Scheiner- explore the functional activation of primate teratocarcinoma in vivo model Bobis1, L. Konrad2 testis during crucial developmental phases. 1Justus-Liebig-University Giessen, Deaprtment of 1,2 1,2 1,2 L. Škara , J. Krasic , A. Katušic´ Bojanac , F. Bulic ­veterinary Physiology and Biochemistry, Giessen, 1,2 2,3 1,2 2,4,5 Jakus , D. Ježek , N. Sincˇic´ , M. Ulamec Germany; 2Justus-Liebig-University Giessen, Center 1 P 10.9 School of Medicine, University of Zagreb, Depart­ of Gynecology and Obstetrics, Giessen, Germany; 2 ment of Medical Biology, zagreb, Croatia; Centre of 3 Presence and distribution of Monash University, Hudson Institute of Medical Excellence for Reproductive and Regenerative Medi­ ­Research, Melbourne, Australia; mast-cells in the testis and epidi- cine, Zagreb, Croatia; 3University of Zagreb School of dymis of ten species Medicine, Department of Histology, Zagreb, Croatia; The blood-testis-barrier (BTB), which is 4University of Zagreb, School of Medicine, Depart­ based upon Sertoli cells (SCs), divides the M. Himelreich Peric´1, A. Dudas1, M. Hohsteter2, ment of Pathology, Zagreb, Croatia; 5University Clini­ seminiferous epithelium into basal and ad- I. Mihikovic Buhin2, M. Kos3, A. Katušic´ Bojanac1, cal Hospital Center Sestre milosrdnice, Ljudevit luminal compartments. The main role of the D. Ježek4 ­Jurak Clinical Department of Pathology and Cyto­ BTB is to form an immunological barrier in 1University of Zagreb, School of Medicine, Medical logy, Zagreb, Croatia order to preserve the meiotic and post-meiot- Biology, Zagreb, Croatia; 2University of Zagreb, ic stages of the germ cells from the immune ­Faculty of Veterinary Medicine, Department of Teratocarcinoma (TCa) is a type of testicular system. The BTB is composed of a number ­Veterinary Pathology, Zagreb, Croatia; 3Clinical Hos­ germ cell tumor composed of teratoma and of tight junction proteins, mainly the clau- pital Centre “Sisters of Mercy”, “Ljudevit Jurak” embryonal carcinoma (EC). Experimental dins family. Disorder of the BTB’s integrity ­Clinical Department of Pathology, Zagreb, Croatia; mouse TCa are obtained by transplanting caused by any internal or external factors 4University of Zagreb, School of Medicine, Histology gastrulating mouse embryos underneath a might result in infertility in males. Even if the and Embryology, Zagreb, Croatia kidney capsule of a syngeneic animal. While SCs are absent, only few immune cells enter the model histologically corresponds to hu- Mast cells play a vital role in the intermediate the seminiferous tubules when peritubular man TCa, their molecular signature remains cells (PTC) are present. This suggests that type of hypersensitivity reaction, fibroblast obscure. The aim of present study is to com- activation and collagen synthesis in most hu- other cell types or factors contribute to the pare proliferative and apoptotic activity on testicular immunological barrier (TIB). man tissues and organs. They inhabit the in- the protein level in human TCa and mouse terstitial tissue of human testes from the fetal experimental TCa in order to asses growth Our study is aimed at elucidating the role of period onward and are also known to play a similarity. different cell combinations (mainly SCs and significant role in human testicular pathol- PTC) on the BTB integrity and to elucidate ogy, surpassing the blood-testis barrier. Sev- In total, 20 experimental TCa obtained 4 the contribution of each cell type to the TIB. eral studies have reported the absence of mast weeks after transplantation and 28 human Furthermore, we aimed to treat rat primary cells in the mouse testis and the results on rat TCa were used. Three surrounding human SCs with particular cytokines to address their testes are strain-dependant. Ex vivo studies of non-tumor testicular tissue samples and five effects on the BTB integrity and on the TJ mast cells in the testis are non-existent, so it is mouse testes were used as respective con- proteins. Our experiments showed that SCs important to find an animal model. Our goal trols. We have semi-quantitatively assessed are the main constituent of the BTB. Co-cul- is to determine the presence and distribution proliferation and apoptosis by immunohis- turing of both SCs and PTC on Matrigel only of mast cells in the testes and find an animal tochemistry (IHC) and Western blot (WB) had a negligible effect on the BTB integrity. species that could be used as an optimal ex- using antibodies against proliferation marker Treatment of 93RS2 rat SCs with bone mor- perimental model for mast cell investigation. PCNA and apoptotic marker caspase-3. phogenetic protein2 (BMP2) demonstrated We included human, mouse (C3H), rat (Fis- WB analysis found no statistically significant a negative effect on the BTB integrity. This cher), cat, serval, dog, stallion, fox, wild boar difference in PCNA and caspase-3 expression effect was reversed by pretreatment with a and bull archive FFPE samples. Testes and between TCa and their controls. In contrary BMPR1 inhibitor. Our results also showed epididymides of ten species were immunohis- to PCNA, IHC analysis for overall caspase-3 that the gene expression of the junctional tochemically stained using antibodies against found statistically significant difference be- adhesion molecule-3 (JAM-3), which is a mast cell tryptase and chymase (sc-59587, sc- tween human and mouse TCa. Human TCa well-known tight junction protein, was up- 59586, respectively, Santa Cruz Biotechnolo- exhibited higher caspase-3 staining, predomi- regulated after treatment of primary SCs with

64 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts testosterone via the classical or non-classical P 11.1 P 11.2 androgen pathway. Further research is needed to understand the physiological implications ITRAQ-based quantitative dif­ Genetic architecture of severe of the results obtained. ferential protein expression spermatogenic failure and male ­analysis revealed essential role infertility – time to advance stan­ of glycolytic pathway in flagellum dard of care P 10.12 energy balance in asthenozoo­ N. Köckerling1, C. Friedrich1, M. J. Wyrwoll1, Peritubular myoid cells in men spermic males J. Emich1, N. Rotte1, R. Wabschke1, M. Vockel2, 3 2 2 2 with normal and impaired sper­ 1,2 3 3 M. Wöste , C. Ruckert , J. Seggewiss , S. Ledig , B. Basent Baruah , R. Rana , S. Kola , 2 2 4 4 matogenesis: CLEC3B as novel 2 3 A. C. Tewes , Y. Stratis , J. Wistuba , C. Krallmann , P. Chaturvedi , A. Srinivasana 4 2 1 candidate for fibrotic remodeling 1 S. Kliesch , A. Röpke , F. Tüttelmann Mergen Biologics, Reproductive Biology, Guwahati, 1 2 Institute of Reproductive Genetics, University of 1 1 2 India; All India Institute of Medical Sciences, Repro- L. M. Siebert-Kuss , S. Di Persio , T. Tekath , 2 3 Münster, Münster, Germany; Institute of Human 3 1 4 ductive Biology, New Delhi, India; All India Institute J. F. Cremers , J. Wistuba , X. Li , G. Meyer zu ­Genetics, University of Münster, Münster, Germany; 4 2 3 1 of Medical Sciences, Biophysics, New Delhi, India Hörste­ , M. Dugas , S. Kliesch , S. Schlatt , 3Institute of Medical Informatics, University Hospital 1 1 S. Laurentino , N. Neuhaus Background The present study was carried Münster, Münster, Germany; 4Centre of Reproductive 1 University Hospital of Münster, Institute of Repro- to understand quantitative expression analy- Medicine and Andrology, University Hospital ductive and Regenerative Biology, Centre of Repro- sis of proteins in the tail fraction of the both ­Münster, Münster, Germany DGA-Abstracts ductive Medicine and Andrology, Münster, Germany; motile and non-motile asthenozoospermic 2University Hospital of Münster, Insitut for Medical human spermatozoa samples. We demon- Introduction Half of all infertility cases, Informatics, Münster, Germany; 3University Hospital strated protein profiling to differentiate head affecting 10–15% of couples, arise from of Münster, Department of Clinical and Surgical and tail sections of human spermatozoa. We male factors like crypto- and azoospermia ­Andrology, Centre of Reproductive Medicine and have also attempted to establish the relation- (very few or no sperm in the semen). Well- ­Andrology, Münster, Germany; 4University Hospital ship between the sperm motility and glyco- established genetic causes include Klinefelter of Münster, Department of Neurology, Institute of lytic pathway enzymes. syndrome (KS) and AZF microdeletions for Translational Neurology, Münster, Germany quantitative spermatogenic failure (SPGF) Material and Methods Semen samples of and CFTR mutations for obstructive azoo- The wall of the human seminiferous tubules normal and asthenozoospermic males were spermia (OA). However, although gene mu- is composed of layers of peritubular myoid used for the study. Electron microscopy tations are expected to explain most remain- cells (PMCs) and extracellular matrix pro- evaluation was performed to understand ul- ing cases, no respective analyses have yet teins. A disturbance of these components is trastructural abnormalities in non-motile informed clinical practice. associated with male infertility, however a spermatozoa such as mid piece, and principal Since 2017, we pro- full characterization of the seminiferous tu- piece. Sperm head and tail were separated Patients and Methods spectively included crypto- and azoospermic bule wall in infertile men is not available yet. by sucrose density gradient. The tail pro- men attending the Centre of Reproductive This study aimed at extending the analysis teins were trypsin digested and subjected to Medicine and Andrology (CeRA, Mün- of the PMC compartment in infertile men. ­iTRAQ labelling followed by identification ster) into our Male Reproductive Genomics We performed single cell RNA sequencing by LC/MS. Proteomics result was validated (MERGE) study. After excluding known (scRNA-seq) of testicular cell suspensions with Immunohistochemistry using FITC con- conditions leading to SPGF (e.g., previous from patients with obstructive azoospermia jugated antibodies. chemotherapy)­ and ruling out chromosomal (normal, n = 3) and cryptozoospermia (cryp- Results Transmission electron microscopic aberrations and AZF deletions, we offered ex- to, n = 3). The results were validated using examination of non-motile and fertile semen ome sequencing, analysed 48 genes with suf- immunofluorescence stainings of markers for revealed major changes in the morphology ficient clinical validity, and strictly assessed PMCs and fibrotic PMCs in an expanded pa- of spermatozoa. Variable forms of structural variants according to clinical guidelines. tient cohort (n = 6 per group). ScRNAseq re- abnormalities of spermatozoa were observed Results Chromosomal aberrations (mostly vealed that in normal patients only 6.4% and in non-motile sperms. Total 84 tail specific 1.3% of the cells are represented by PMC or KS) and AZF deletions were identified in proteins were identified in the normal motile 15.9% and 2.8% of cases. In 512 men without fibrotic PMC, respectively. In the cryptozoo- spermatozoa and 26 tail specific proteins in spermic group, we found an increased pro- such conditions, 11 patients (2.2%) with OA the non-motile (asthenozoospermic) indi- carried causal mutations in CFTR/ADGRG2. portion of PMCs and fibrotic PMCs account- vidual. ing for 26.3% and 15.1% of all captured cells, In 16 patients (3.1%) with SPGF, mutations respectively. Differential gene expression The differential tail proteins identified be- in nine genes were identified as causal. Seg- analysis between the fibrotic PMCs of the longed to cytoskeletal, flagellar and cell regation analyses helped in assessing the two patient cohorts resulted in identification movement proteins and energy and metabo- pathogenicity of some variants, highlighted of 188 differentially expressed genes, includ- lism proteins. Immunofluorescence study of by a de novo mutation in DMRT1. ing CLEC3B. Co-­localization analysis con- semen samples revealed significantly lower Conclusions The sequencing performed firmed the expression of CLEC3B protein in levels of Glyceraldehyde-3-Phosphate Dehy- here offers a diagnostic yield comparable to decorin-positive (fibrotic PMC marker) cells drogenase (GAPDH), Enolase and Pyruvate that of current genetic tests and some find- in both normal and crypto patients. However, kinase (PK) in tail and mid-piece region of ings are immediately relevant for infertility immunofluorescence analysis highlighted a non-motile spermatozoa to that of fertile counselling/treatment, as they may indicate heterogeneous distribution of CLEC3B in the males. the success of treatments like testicular sperm tubular wall of cryptozoospermic patients. In Conclusion Tail structural abnormalities extraction. Overall, panel/exome sequencing conclusion, scRNA-seq revealed an increased and reduced expression of glycolytic pathway now provides strong enough evidence to be proportion of PMCs and fibrotic PMCs in enzymes in the immotile sample could cause included in diagnostic workups of men with cryptozoospermic patients. We identified energy imbalance that potentially weakens crypto- and azoospermia. Including more CLEC3B as a novel marker for fibrotic PMCs sperm movement. These proteins may serve genes and functional assessments of vari- and demonstrated a distinct expression pat- as diagnostic biomarkers of infertility and ants of uncertain significance will quickly tern for this protein in the cryptozoospermic targets for male contraception. increase relevant diagnostic findings. group compared to the normal situation. Per- spectively, linking the expression of CLEC3B to the spermatogenic state of the tubules and the proliferative activity of spermatogonia might reveal altered mechanisms associated with idiopathic infertility.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 65 Andrology 2020 – Abstracts

P 11.3 Introduction The ubiquitous presence and (14.8%) secondary infertility (group #2). environmental persistence of bisphenol A Group #3 was made of 115 fertile men. Clinical and seminal parameters (BPA) along with its reputation of being an Groups #1 and #2 showed higher frequency associated with testis micro­ endocrine disruptor, have generated concerns of cryptorchidism and gonadotrophins and lithiasis and its severity in males about the possible links with a spectrum of lower testis volume than group #3. Group #1 from infertile couples human health disorders, including infertility. showed lower testis volume and higher FSH The increasing use of BPA analogs, such as than group #2. Group #3 showed better se- S. D’Andrea, C. Castellini, M. Totaro, G. Cordeschi, bisphenol S (BPS) and bisphenol F (BPF), is men parameters than groups #1 and #2, while A. Parisi, F. Francavilla, S. Francavilla, A. Barbonetti attracting interest to these new compounds, group #1 showed lower sperm concentra- University of L’Aquila, Andrology Unit, Department which, however, could share chemical and tion and progressive motility than group #2. of Life, Health and Environment Sciences, L’Aquila, biological properties similar to BPA. In vitro No difference in duration-of-infertility was Italy studies demonstrated that BPA induces sperm found between groups #1 and #2. However, Introduction Testis microlithiasis (TM), apoptotic/oxidative damages; however, ef- group #2-with-miscarriages showed higher representing an incidental finding in the scro- fects of the human sperm exposure to BPS duration-of-infertility than group #1. In tal ultrasonography (US), is associated with and BPF have not yet been investigated. group #1, duration-of-infertility was positive- male infertility and a higher risk for testicular Materials and Methods Motile sperm ly associated with male age ≥ 42 years. Re- cancer as compared to that in infertile males suspensions were exposed for 4 h to scalar garding psychological traits, groups #1 and

DGA-Abstracts without TM. This study aimed to evaluate concentrations of BPS or BPF (10–400 µM) #2 did not differ and showed higher MHQ whether TM and its severity were associated or 400 µM BPA, used as positive control. total and free-anxiety score than group #3. to a different degree of testicular dysfunction Mitochondrial membrane potential (MMP) However, group #2-with-miscarriages had in men from infertile couples. and mitochondrial generation of reactive higher phobic-anxiety than group #1. In grou p#1, azoo&oligoasthenoterato-spermic men Patients and Methods This study was an oxygen species (ROS) were assessed at flow showed higher depression than the rest of the observational, retrospective, case-control in- cytometry using JC-1 dye and MitoSOX red, sample, duration-of-infertility ≥ 3 years was vestigation involving males who underwent respectively. Sperm motility was analysed by positively associated with phobic-anxiety, se- diagnostic work-up for couple infertility at CASA and vitality was assessed by eosin test. men volume was negatively associated with an Andrology clinic, between January 2004 Results MMP and ROS generation were somatization. and December 2018. One hundred patients, not significantly affected by the exposure to out of the 2112 scored men, were found to scalar concentrations of BPS or BPF. Con- Conclusions Infertile men showed worse have TM during the US evaluation. TM was sistent with the lack of mitochondrial effects, clinical and psychopathological character- defined as limited TM (LTM) or classical no significant differences were observed for istics than fertile men. Group #1 showed TM (CTM), when the maximum number of sperm motility and vitality in sperm treated worse clinical characteristics than group #2, hyperecogenic spots per sonogram was < 5 with scalar concentrations of BPS or BPF. but group #2-with-miscarriages had higher or ≥ 5, respectively. CTM, LTM, and control As expected, when compared to untreated duration-of-infertility and phobic-anxiety groups were compared for clinical variables, samples, 400 µM BPA produced a significant than group #1. In group #1, severe seminal serum levels of follicle-stimulating hormone decrease in % of sperm with high MMP (7.7 features, duration-of-infertility ≥ 3 years, age (FSH), luteinizing hormone (LH), and total ± 7.2% vs 75.9 ± 10.2%, p < 0.0001), which ≥ 42 years were associated with psychopatho­ testosterone, as well for semen parameters was accompanied by an increased % of sperm logical traits. and scrotal US features. with mitochondrial ROS generation (85.7 ± Results Males with CTM showed a lower 7.6% vs 28.1 ± 6.5%, p < 0.0001), complete mean testis volume (p = 0.03) and a lower sperm immobilization and loss of viability P 11.6 sperm concentration (p = 0.03) as compared (viable sperm: 15.2 ± 12.5% vs 87.7 ± 4.5%, LIN28A and LIN28B expression in to the other two groups. A higher FSH level p < 0.0001). testicular fibrosis – a case study was observed in the CTM group as compared Conclusion When compared to BPA, the 1 1 1 to that in the LTM group (p = 0.02) and in D. Krsnik , A. Katušic´ Bojanac , M. Himelreich Peric´ , analogues BPS and BPF, at environmentally 1 2 controls (p = 0.009). The multiple logistic re- F. Bulic Jakus , D. Ježek relevant concentrations, seem to be safer for 1 gression analysis showed that only a smaller University of Zagreb, School of Medicine, Medical sperm biology as they exert a neutral effect 2 volume exhibited an independent sig- on sperm motility, viability and mitochon- Biology, Zagreb, Croatia; University of Zagreb, nificant association with a higher odd of de- drial function. School of Medicine, Histology and Embryology, tecting CTM (OR = 0.84, 95-%-CI: 0.75–0.94; Zagreb,­ Croatia p = 0.02). No significant differences were The mammalian genome encodes two Lin28 observed between groups in the prevalence P 11.5 paralogs, Lin28A and Lin28B. LIN28A has of risk factors for testicular cancer, or in the Clinical and psychological charac­ been shown to be expressed in SSC of adult prevalence of conditions associated with TM. teristics of men with primary and human testis, but also in malignant germ Conclusion Males from infertile couples secondary couple infertility cells of testicular cancer. LIN28B has been who exhibit a reduced testicular volume implicated in the timing of onset of human should undergo scrotal US to exclude CTM. F. Lotti, F. Frizza, A. Pisetta, M. Maggi puberty, while its expression was found in Significant evidence provided here is that the University of Florence, Experimental and Clinical Leydig cells of animal models. Furthermore, presence of a LTM has no clinical relevance ­Biomedical Sciences, Florence, Italy the disturbance of their expression was found in a mouse model of hypogonadism. How- in males from infertile couples. Introduction No previous study compared clinical and psychological characteristics of ever, data about comparative distribution of men with primary and secondary infertility LIN28A and LIN28B in human infertile tes- tis are limited. Here we examined their pro- P 11.4 and fertile men and evaluated associations between psychological and clinical para­ tein expression profiles in testis samples of a Are bisphenols analogues safer meters in these groups. patient with tubular fibrosis, with increased than bisphenol A for human FSH levels (33 U/L) or with maturation ar- Methods We evaluated clinical and psy- rest. FFPE samples of testicular biopsies of ­spermatozoa? An in vitro study chological (Middlesex Hospital Question- azoospermic men were obtained from KBC C. Castellini, S. D’Andrea, M. Totaro, A. Parisi, naire, MHQ) characteristics of 580 males of Zagreb Andrology clinic. The immunohis- G. Di Emidio, C. Tatone, S. Francavilla, F. Francavilla, infertile couples (38.0 ± 5.8years) and 115 tochemical labelling was performed in trip- A. Barbonetti fertile men (36.6 ± 5.3 years). licate, with positive and negative controls. University of L’Aquila, Department of Life, Health Results Among infertile men, 494 (85.2%) In patient with tubular fibrosis, LIN28A and Environmental Sciences, L’Aquila, Italy had primary infertility (group #1) and 86 was surprisingly found to be strongly co-

66 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts expressed with LIN28B in interstitial tissue, of RdgC, a calcium-dependent phosphatase P 11.9 with cytoplasmic signal for both markers in present in the retina of Drosophila, our study existing Leydig cell clusters (nodules). In is the first to investigate the contribution of Results of testicular biopsy testis of a patient with maturation arrest and this newly identified sperm calmodulin bind- evalua­tion and reproductive hor­ no nodules, strong LIN28A/B staining was ing protein in the context of reproductive bio­ mones analysis in infertile men found in tubules, with cytoplasmic expression logy and sperm function. Overall, this project with Sertoli-cell only syndrome of LIN28A mainly in spermatogonial popula- will help to better understand the molecular tion, while LIN28B was presented with nu- mechanisms underlying sperm capacitation, D. Adamczewska, J. Slowikowska-Hilczer, clear staining in most maturation stages. The a process controlling sperm fertilizing abil- K. Marchlewska, R. Walczak-Jedrzejowska interstitial cells were negative for LIN28A, ity. Ultimately, our findings might open new Medical University of Lodz, Department of Andrology­ but exibited weak LIN28B expression. We avenues on the development of non-hormonal and Reproductive Endocrinology, Lodz, Poland conclude that LIN28A with LIN28B show an male contraceptives targeting sperm en- Introduction The aim of the study was to overlap in human testis with tubular fibrosis, zymes. evaluate testes with Sertoli cell only syn- but subcellular distribution of LIN28 proteins drome (SCOS) focusing on morphometric may be dependent on histological diagnosis P 11.8 signs of testicular dysgenesis and markers of of infertile testis. Further studies are ongoing Leydig cell (LC) function in relation to hor- to elucidate this assertion. Characterization of human sperm monal status of studied men.

Grants This study was supported by the intracellular metabolites to un­ Materials and Methods Forty nine tes- DGA-Abstracts Scientific Center of Excellence for Repro- derstand their association with ticular biopsies of patients with SCOS and ductive and Regenerative Medicine, Croatia, functional ability and genetic in­ 15 controls with normal spermatogenesis and through the European Regional De- tegrity (NOR) were assessed for the seminiferous velopment Fund, under grant agreement tubule diameter (STD), thickness of tubular KK.01.1.1.01.0008. “Reproductive and Re- A. Cheredath1, S. Uppangala1, G. Kalthur1, membrane (TM), areal fraction of intertubu- generative Medicine – Exploring New Plat- H. S. Atreya2, S. K. Adiga1 lar space (AFIS) and LC number (LC-score). forms and Potentials”. 1Kasturba Medical College, Manipal Academy of The results of histological examination were Higher Education, Clinical Embryology, Manipal, correlated with serum levels of FSH, LH, tes- India;­ 2Indian Institute of Sciencce, NMR Research tosterone (T) and T/LH ratio. Centre, Bengaluru, India P 11.7 Results In SCOS testicular volume (Me- The protein phosphatase with EF- Introduction Male factor alone contributes­ dian (M): 16.0 vs 29.5; p < 0.001) and STD (M: 141.7 vs 190.2; p < 0.001) were lower, hand domain 1, PPEF1, is a Ca2+- to approximately 50% of the infertility cases. Although semen analysis is a primary dia­ while TM (M: 9.8 vs 6.4; p < 0.001) and dependent calmodulin binding gnostic test in understanding the male re- AFIS (M: 47.6 vs 27.6; p < 0.001) were protein present in mature sperma­ productive potential, it does not address the significantly higher in comparison to NOR. tozoa fertilizing capacity and genetic integrity of LC-score was higher in SCOS than in NOR spermatozoa. Since cellular metabolism is in- group (M: 2.2 vs 1.1; p < 0.001). Abnormal C. Lavoie-Ouellet, A. Saindon, M. Clark, J. Ruiz, fluenced by genetic factors and pathophysio­ AFIS and STD were present in 43% of SCOS C. Belleannée, P. Lecler1 logy involved with it, characterization of the biopsies and among them in 81% the in- CHU de Québec – Université Laval, Québec, Canada endogenous metabolites from spermatozoa creased LC-score was found. In SCOS group Sperm capacitation is a post-ejaculatory may help us in better understanding the hid- the subjects had significantly higher levels maturational process that takes place in the den cause of male infertility. of both gonadotropins (FSH, M: 19.9 vs 3.4; p < 0.001; LH, M: 7.1 vs 4.2; p < 0.001). female genital tract under the influence of Patients (or Materials) and Methods In calcium and its mediator, calmodulin. Al- this study, using 1H Nuclear magnetic reso- Total serum testosterone level did not differ though capacitation is associated with pro- nance (NMR) spectroscopy intracellular between studied groups, however, T/LH ratio tein phosphorylation/dephosphorylation, the metabolites of sperm cells from 14 normo- was significantly decreased in SCOS group identity and contribution of sperm calmodu- zoospermic men and their association with (M: 2.3 vs 3.8; p < 0.001). Negative correla- lin binding proteins remain to be established. functional and genetic parameters such as tion between LC-score and STD was observed Having identified theprotein phosphatase motility, viability and DNA damage and were in SCOS group ( r = –0.48; p < 0.001). AFIS with EF-hand domain 1 (PPEF1) as a studied. correlated positively with serum FSH level in sperm calmodulin­ binding protein, we hy- both groups (NOR, r = 0.53; p < 0.05; SCOS, Twenty-one metabolites from pothesize that this Ser/Thr phosphatase could Results r = 0.41; p < 0.05), while with LH, and nega- spermatozoa were identified, out of which be involved in the capacitation process and tively with T/LH ratio, only in SCOS (LH, the concentration of leucine, isoleucine, the control of sperm hyperactivation. The r = 0.37; p < 0.05; T/LH, r = –0.36; p < 0.05). valine, glucose, lactate, putrescine and etha- main objectives of our study were to 1) iden- nolamine were negatively associated with Conclusions Presented data confirm that tify and localize sperm PPEF1 isoforms by the spermatozoa motility. On the other hand, substantial number of subjects with SCOS using biochemical and molecular approaches arginine, betaine and glycerol were positively presents signs of testicular dysgenesis and and, to 2) determine PPEF1 contribution to correlated with sperm motility. Further ge- impaired function of LC. Increased serum sperm capacitation by in vitro functional as- netic integrity and viability were positively levels of LH and FSH may reflect LC dys- says. Different PPEF1 transcript variants correlated with all metabolites tested except function, and additionally significant histo- encoding 4 isoforms were identified by PCR putrescine which was negatively correlated logical changes in testicular structure. amplification of bull testis RNA. Western- with the viability. blot performed on sperm subcellular frac- tions indicated that PPEF1 was exclusively Conclusion Thus, spermatozoa metabolites P 11.10 associated with sperm membranes. Indirect can aid in understanding the sperm functional immunofluorescence staining revealed that and genetic integrity. Further, metabolomics Mapping men’s reproductive PPEF1 is located in the neck, the flagellum analysis of sperm may serve as a screening ­decision-making pathways and the acrosome of non-capacitated sper- diagnostic tool in elucidating the pathology A. White matozoa. Although a phosphatase activity associated with male infertility. University of Oxford, Nuffield Department of Primary was detected in immunoprecipitated PPEF1, Health Care Sciences, Oxford, United Kingdom further studies are required to determine whether PPEF1 localization and phosphatase Introduction While men are half of the activity are modulated during capacitation. reproductive equation, research examining Acknowledging that PPEF1 is an ortholog men’s reproductive decision-making has

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 67 Andrology 2020 – Abstracts

been limited. This research uses reproductive life histories to construct a schematic map of men’s decision-making pathways about preg- nancy and contraceptive (non)use. Methods Individual telephone interviews (n = 48) were conducted with cisgender, hetero­sexual men, ages 25–67, living in seven U.S. southern states from May–De- cember 2019. Men were recruited using tar- geted Facebook­ advertisements. Interviews explored men’s reproductive histories, in- cluding contraceptive use and contribution to pregnancies (average time 70 minutes). In- terviews were audio-recorded, professionally transcribed, and analyzed using a constructiv- ist approach to grounded theory. Results Participants’ narratives were used DGA-Abstracts to develop a map of questions and outcomes Figure 11. related to pregnancy and contraceptive use. M. Salehi Novin, et al. The schematic reveals complex reproductive pathways where men have few options to manage their fertility and need to negotiate contraceptive (non)use and potential parent- groups and subgroups. T2 was increased in is not enough for choosing the best sperm es- hood with their partners. Feedback loops em- group 2 compared to group 1 (p = 0,00012) pecially in male factor patients. In Annexin- phasize that men are repeatedly confronted while no statistically significant difference in V magnetic activated cell sorting (MACS) with these questions and negotiations until ADC and T1 values was observed between technique, apoptotic sperm are separated they or their female partner(s) are infertile; groups. Within group 1, ADC was increased from non-apoptotic one by negative selec- it is not a one-time process but a continuous in subgroup B (n = 4) compared to subgroup tion. So, this method can help selecting good journey lasting over men’s reproductive lives. A (n = 5) and the increase was statisti- quality sperm for ICSI. Conclusions Findings expand our under- cally significant (p = 0,019). No statistically Method Semen samples from 30 male standing of men’s reproductive life course signifi­cant difference in ADC values was factor infertile couples (DFI > 30%) were and demonstrate how men negotiate aspects observed between subgroups 1A and 2A, 1B selected and divided into two group in each of this journey with their partners. The map and 2B or 2A and 2B. In conclusion, T2 is patient. control was washed with DGC and provides a starting point for classifying significantly increased in frozen and thawed experimental one was selected by MACS- the continuum of pregnancy intentions and tissue compared to fresh tissue which should DGC. Retrieved eggs in each patient, were contraceptive use for men, who are often ex- be considered when interpreting data and divided in 2. Control and experimental group cluded from such work. Future research may could reflect the effect of freezing and thaw- were injected by DGC and MACS respec- draw on these pathways as areas of inquiry, ing on the consistency and increased water tively. Semen parameters and DFI (SCD test) thereby expanding the small body of research diffusion into the tissue. ADC could be a use- were analyzed before and after processing. on men’s reproductive lives and concerns that ful parameter in evaluating testicular tissue After ICSI, rate of fertilization and embryo need to be understood. and predicting sperm retrieval but should be development were evaluated. Comparison evaluated in a study with larger sample sizes. between results of control and experimental Grants This study was supported by the groups was assessed by SPSS analysis. Scientific Center of Excellence for Re- P 11.11 Results Results showed that, sperm motil- productive and Regenerative Medicine, ity and morphology after MACS method Ex vivo 7 T magnetic resonance Republic of Croatia, and by the European (45%, 1.7%) was significantly higher than imaging of testicular tissue: Union through the European Regional De- DGC method (40%, 1.1%) and before wash- ­preliminary results velopment Fund, under grant agreement No. ing (35%, 0.9%). Percent of DFI in MACS 1,2 3 1,2 KK.01.1.1.01.0008, project “Reproductive A. Planinic´ , S. Škokic´ , D. Ježek group (36%) was significantly decreased 1 and Regenerative Medicine – Exploring New Department of Histology and Embryology, School of compared to DGC (45%) and primitive group Platforms and Potentials”. Medicine University of Zagreb, Zagreb, Croatia; (55%). The number of oocytes were injected 2Scientific Center of Excellence for Reproductive and in DGC group was 93 and in MACS group Regenerative Medicine, Zagreb, Croatia; 3Croatian was 111. Fertilization rate in both groups was Institute for Brain Research, Zagreb, Croatia P 11.12 almost the same (72.07% in MACS vs 73.11 Men with azoospermia who wish to have in DGC). Rate of day 3 embryo with good Annexin-V MACS sperm selection grade in MACS group (72. 5%) was signifi- children undergo TESE which has a success method could be effective on rate of only about 60%. Our study aimed to cantly higher than DGC (51.47%, p < 0.05). assess ex-vivo MRI of testicular tissue, in- sperm parameter and embryo The pregnancy rate, from MACS embryos cluding diffusion-weighted imaging (DWI) quality in male factor patients was 35.4%. as a potential tool in developing a prediction with high DNA fragment Conclusion Results indicated, sperm se- model for sperm retrieval as well as assess the lection by MACS-DGC method can improve M. Salehi Novin1, Z. Zandie2, M. Bakhtiari1, differences between fresh tissue and frozen- sperm motility, morphology and reduce R. Aflatoonian3 thawed tissue. The study included 18 samples sperm DNA fragmentation. No significant 1Iran University of medical sciences, Anatomy, of testicular tissue obtained via TESE, 9 that difference was observed in fertilization rate, Tehran,­ Iran; 2Iran university of medical science, underwent 7 T MRI as fresh tissue (group 1) but the percent of high-quality embryo was ­Shahid Akbar-abadi clinical Research development and 9 that were frozen in liquid nitrogen, significantly higher by this method. All unit, Tehran, Iran; 3Royan institute for reproductive thawed and then imaged (group 2). Groups pregnant had very high DNA fragmenta- biomedicine, Endocrinology and female infertility, were divided into two subgroups, samples tion (> 45%), according to the mechanism Tehran, Iran with sperm (A) and without sperm (B). T1, of MACS method, it can be suggested as T2 and ADC values were measured in all Introduction Sperm selection based on a good choice for patients with high DFI samples and values were compared between morphology and motility in ART techniques, (Fig. 11).

68 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

P 11.13 P 11.14 P 12.1 Sperm Izumo1 immuno­ First Genome Wide Association In vitro effects of aqueous extract fluorescence comparison of Study (GWAS) in men with unex­ of unfermented rooibos on human ­unexplained­ infertile men and plained infertility – Identification of sperm function men without fertilization prob­ a genetic region determining fol­ N. Takalani1, G. Adefolaju2, R. Henkel3,4, C. Opuwari2 lems licle-stimulating hormone action 1University of Limpopo, Pathology and Medical Sciences, Polokwane, South Africa; 2University of S. E. Özkoçer1, C. M. Seymen1, A. Öcal2, M. Schubert1, L. Pérez Lanuza2, M. Wöste3, ˙ 3 1 Limpopo, Pre- Clinical Sciences, Polokwane, South I. Kaplanog˘lu , Ç. Elmas M. Dugas3, Y. Rassam1, S. Heilmann-Heimbach4, Africa; 3University of the Western Cape, Bellville, 1Gazi University Faculty of Medicine, Histology and F. Tüttelmann5, S. Kliesch1, J. Gromoll2 South Africa; 4American Centre for Reproductive Embryology, Ankara, Turkey; 2Etlik Zübeyde Hanım 1Centre of Reproductive Medicine and Andrology, Medicine, OH, United States Hospital, Urology, Ankara, Turkey; 3Etlik Zübeyde University Hospital Münster, Department of Clinical Hanım Hospital, In Vitro Fertilization, Ankara, Turkey and Surgical Andrology, Münster, Germany; 2Institute Introduction Aspalathus linearis (Rooibos) Unexplained infertility means that the cou- of Reproductive and Regenerative Biology, Centre of is a popular plant owing to its antioxidant ples unable to achieve pregnancy with no Reproductive Medicine and Andrology, Münster, properties. Animal studies have demonstrated Germany; 3Institute of Medical Informatics, Uni­ the beneficial effects of the extract on sperm reason after two years of unprotected inter- 4 course. Semen analysis is first diagnostic test versity of Münster, Münster, Germany; Institute of functions. This study aimed to investigate DGA-Abstracts Human Genetics, University of Bonn, Medical the effects of unfermented rooibos extract on for evaluation of the male fertility and normal 5 results are seen in the unexplained infertil- School, University Clinics, Bonn, Germany; Institute sperm function in vitro. ity. However most molecular impacts are not of Reproductive Genetics, University of Münster, Material and Methods Semen samples clear. For fertilization, Izumo1 redistribution Münster, Germany were collected after 3–5 day’s sexual absti- is necessary after acrosome reaction. Animal Introduction 30–40% of infertile men nence by masturbation from healthy donors experiments shows that the defects related to remain unexplained. Follicle stimulating (n = 25) at the University of the Western Izumo1 cause the fertility problems. From hormone (FSH) plays a key role in initiation Cape, and infertile patients (n = 25) attend- June 2017 to March 2018, experiments and maintenance of spermatogenesis; action ing Tygerberg and Vincent Pallotti Hospitals, are conducted fresh semen specimen of 48 might be hampered in some of these patients. South Africa for fertility problems. Samples males. 18 control whose partners get sponta- One well-studied single nucleotide poly- were liquefied, washed (300 x g; 10 min) neously pregnant within a year and 30 males morphism (SNP) (rs10835638) is associated with human tubular fluid in 1 % bovine serum of unexplained infertility. Semen analysis with FSHB mRNA transcription and directly albumin (HTF-BSA) and incubated with un- and chromatin condensation assessment were affects FSH serum levels, testicular volume fermented rooibos aqueous extract (0, 0.15, done with fresh sperm samples. After density and spermatogenesis. The aim was to identify 1.5, 150 µg/ml) for 1 h at 37 °C, where HTF- gradient centrifugation, acrosome reaction further SNPs associated with FSH levels in BSA served as control. was assessed with progesterone. Antibodies unexplained infertile men. Results Sperm motility, vitality and DNA against to Izumo1 and CD46, and DAPI used Retrospectively, fragmentation remained unchanged in both for triple immunofluorescence of sperm. Patients and Methods around 1900 characterized men with unex- groups (p > 0.05). Intracellular reactive Age, semen analysis and chromatin con- plained infertility were identified from our oxygen species (ROS) production (15 µg/ densation parameters haven”t differ statisti- clinical database Androbase®. In the dis- ml; p < 0.01) and acrosome reaction (1.5 µg/ cally significant among the groups. Sperms covery cohort of 742 men a genome wide ml; p < 0.0001) increased significantly, while were evaluated in 4 groups: triple positive, association study (GWAS) was performed the percentage of intact mitochondrial mem- one positive (DAPI+), Izumo1 negative (Illumina PsychArray_v1.3®) and analyzed brane potential (MMP; 150 µg/ml; p < 0.01) (Izumo1-, CD46+, DAPI+), DAPI negative (Illumina®GenomeStudio, PLINK and R). decreased in the donor group, but were unaf- (Izumo1+, CD46+).Triple positive sperms As validation, the SNPs rs1103005 and fected in the patient group (p > 0.05). were higher in the control group than the rs10835638 were genotyped in an independ- Conclusion Lower concentrations of aque- experiment group but statistically insignifi- ent cohort of 1127 patients by TaqMan SNP- ous extract of unfermented rooibos induced cant (p = 0.075). Izumo1 negative and DAPI PCR and association to clinical parameters ROS production and acrosome reaction in the negative sperms have observed in some of the was evaluated. donor group. However, higher concentrations samples. Some sperms with the anomalies of the extract appear to be harmful to MMP, like double nuclei also have Izumo1 immu- Results Imputation analysis revealed 9 SNPs with genome-wide significance which might impair sperm functions and fer- nopositivity. There is statistically significant tility thereof. negative correlation between the chromatin (p < 3,994e-07) at the FSHB locus on Chr. condensation and the triple positive sperms 11p.14.1. All SNPs, including rs10835638, were highly linked to one another (linkeage within the groups (p < 0.05). Even Izumo1 P 12.2 immunopositivity haven’t differed among the disequilibrium). A validation study on 1127 groups, sperms have reacted different to the patients for the newly identified rs1103005 Impact of metabolically healthy progesterone. The physiologic reactions of and for rs10835638 revealed a significant as- obesity in patients with andro­ sociation with FSH (p < 1.93e–06; p < 4.04e- the sperms are regulated with the molecular logical problems level. Understanding the acrosome reaction 7), FSH/LH ratio (p < 4.84e-13; p < 4.94 1 1 1 1 in molecular level will be helpful for the dia­ e–12) and bi-testicular volume (p < 7.36e-03 F. Lotti , G. Rastrelli , E. Maseroli , S. Cipriani , 1 1 1 1 gnosis of the male infertility as well as the and p < .29e-03). F. Guaraldi , C. Krausz , Y. Reisman , A. Sforza , 1 1 treatment options. Discussion Our study revealed that not one M. Maggi , G. Corona single SNP, but rather the FSHB gene region 1University of Florence, Experimental and Clinical is the main genetic determinant affecting Biomedical Sciences, Florence, Italy FSH action. This study is the first delineat- Introduction While the pathogenic role of ing this region as of crucial importance in the metabolically complicated obesity (MCO) in regulation of FSH serum levels in infertile erectile dysfunction (ED), major adverse car- men with unexplained infertility. We suggest diovascular events (MACE) and male infertil- to include one of these SNPs into routine dia­ ity has been widely studied, that of metaboli- gnostics in infertility workup, to identify a cally healthy obesity (MHO) has been poorly subgroup of so far unexplained infertile men, investigated. We herein evaluated the role of who putatively benefit from FSH treatment. MHO in ED pathogenesis, MACE prediction Grants Supported by the DFG CRU326. and male reproductive health.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 69 Andrology 2020 – Abstracts

Methods We studied a consecutive series tigated if smoking cessation before the fetal and Novosibirsk. All men were somatically of 4945 men (50.5 ± 13.5 years) with sexual masculinization window, starting in gesta- healthy. The collection and analysis of se- dysfunction (SD) (cohort 1) and 231 (37.9 tional week seven, can mitigate the adverse men was performed according to who rec- ± 9.1 years) males of infertile couples (co- effects on semen volume, sperm concentra- ommendations. it is found that in the group hort 2). A subset of SD patients (n = 1687) tion, total sperm count, motility, morphology of somatically healthy men living in the city was longitudinally investigated to evaluate and testicular size. of Irkutsk spermiogram characterized by the MACE. All patients underwent clinical, bio- Methods In 980 18-year old sons of women presence of higher sperm count (million per chemical, erectile function (EF) and flaccid from the Danish National Birth Cohort, we ml), 34.4% more than in men of Novosibirsk penile color-Doppler US (PCDU) assess- estimated the differences in semen charac- and 23.6% more than men Ulan-Ude. Thus, ment. Infertile men underwent also scrotal teristics and testicular volume among early in the group of men of Irkutsk revealed a and transrectal US, semen analysis (including quitters (prior to week seven), continuous significant increase in concentrations of IL-8), prostatitis-like symptoms (PLS) as- smokers and non-smokers. Maternal smok- α-tocopherol by 44% compared with men of sessment. MHO was defined as BMI > 30 kg/ ing was reported around gestational week 16. Ulan-Ude and by 38% compared with men 2 m with HDL > 40 mg/dl and absence of dia- Negative binomial regression analyses were of Novosibirsk. In the study of semen of men betes or hypertension. The rest of the obesity adjusted for parents’ ages at birth, maternal of Ulan-Ude is installed active high content sample was defined MCO. MHO or MCO pre-pregnancy BMI, maternal alcohol in- of spermatozoa in relation to this indica- were compared with the rest of the sample take, family socioeconomic status, partner’s tor in men of Novosibirsk, Irkutsk and 16%

DGA-Abstracts (normal weight (NW) men). smoking, and son’s abstinence time, place of and 11% respectively. For men Ulan-Ude is Results In cohort 1, 816 men (16.5%) were ejaculation (home or clinic) and recent fever. also characterized by a higher level of total obese, 181 (3.7%) MHO, 635 (12.8%) MCO. Analysis of motility was further adjusted for antioxidant activity, 34% higher than that in In cohort 2, 68 men (28.4%) were obese, interval from ejaculation to analysis. men of Novosibirsk and 13%, than men of Irkutsk. The studies of the quality of ejacu- 19(8.2%) MHO, 49 (21.2%) MCO. After Results Of 230 (24%) women who smoked adjusting for confounders, in both samples, late and lipid peroxidation processes in men during first trimester, 62 (27%) quit before of various Siberian cities show that the place either MHO or MCO showed lower testoster- week seven. Sons of early quitters had a 19% one and worse PCDU parameters, compared of residence and ecological and geographic lower sperm concentration (95-%-CI: –36%, position are the main causes that determine to NW. However, only MCO showed a worse 2%) and 23% lower total sperm count (95- EF compared to NW. In the longitudinal fertility problems. Regional peculiarities and %-CI: –41%, 0%) than sons of non-smoking ecological and geographical environment can study, both MHO and MCO were indepen- mothers. Similarly, sons of continuous smok- dently associated with a higher incidence of be important factors affecting the functioning ers had 18% lower sperm concentration of the reproductive system and determine the MACE, compared to NW (both p < 0.05). In (95-%-CI: –31%, –3%) and 37% lower total cohort 2, MHO and MCO showed a higher heterogeneity of male infertility under condi- sperm count (95-%-CI: –39%, –12%). Ma- tions of anthropogenic press. prostate volume, and MCO also higher US ternal smoking was not associated with other and biochemical (sIL-8) features of prostatic semen characteristics or testicular size. inflammation compared to NW, but no differ- ences in PLS or seminal parameters. Conclusion Smoking cessation before ges- P 12.5 tational week seven did not appear to mitigate Conclusions MHO is associated with sub- the negative impact of smoking on offspring Inhibition of autophagy impairs clinical ED, increased cardiovascular (CV) semen quality. While results are based on sperm quality and promotes cell risk and prostate enlargement. MHO men relatively small numbers, they suggest that death in human spermatozoa ex­ should be considered at high CV risk, as adverse effects are not only caused by acute posed to oxidative stress MCO, and followed-up for EF and prostate disruption of the development of fetal repro- 1,2 2 2,3 2,3 abnormalities. ductive organs during week seven to fifteen, P. Uribe , J. Meriño , M. Schulz , F. Zambrano , J. V. Villegas2,4, I. Conejeros5, A. Taubert5, but that other mechanisms, such as delayed 5 1,3 effects of smoking compounds, epigenetic C. Hermosilla , R. Sánchez 1University of La Frontera, Department of Internal P 12.3 changes or pre-conceptional effects, may be 2 at play. Medicine, Temuco, Chile; University of La Frontera, Does maternal smoking cessation Center of Excellence in Translational Medicine – before the fetal masculinization Scientific and Technological Bioresource Nucleus 3 window mitigate adverse effects P 12.4 (CEMT – BIOREN), Temuco, Chile; Universidad de La Frontera, Department of Preclinical Sciences, on offspring semen quality? Sperm quality and features of the Temuco,­ Chile; 4Universidad de La Frontera, Center K. Keglberg Hærvig1, K. Ugelvig Petersen1, antioxidant protection system in of Reproductive Biotechnology – Scientific and Tech- 1 2 3 J. P. Bonde , A. Giwercman , G. Toft , B. Bjerre men living in different regions of nological Bioresource Nucleus (CEBIOR – BIOREN), Høyer1, C. Lindh4, C. Høst Ramlau-Hansen5, Temuco, Chile; 5Justus-Liebig-University Giessen, A. M. Nybo Andersen6, K. Sørig Hougaard7, Siberia Institute of Parasitology, Giessen, Germany S. Søgaard Tøttenborg1 N. Kurashova, B. Dashiev, L. Kolesnikova Autophagy is a regulated pathway of lysoso- 1Bispebjerg & Frederiksberg Hospital, Occupation- Federal State Public Scientific Institution “Scientific mal degradation which helps eukaryotic cells and Environmental Medicine, Copenhagen NV, Centre for Family Health and Human Reproduction to maintain or restore homeostasis, being a Denmark;­ 2Molecular Reproductive Medicine, Problems”, Irkutsk, Russian Federation stress response mechanism. Oxidative stress ­Department of Translational Medicine, Malmø, Currently, one of the risk factors for disor- (OS) is a main cause of impaired sperm func- Sweden;­ 3Aarhus University Hospital, Department of ders of the is the tion linked to male infertility and previously Clinical Epidemiology, Aarhus, Denmark; 4Division of formation of reactive oxygen species (ROS) we demonstrated that human sperm subjected Occupational and Environmental Medicine, Depart- in cells and tissues. The formation of ROS to OS activate an autophagic response; how- ment of Laboratory Medicine, Lund, Sweden; is balanced by the action of various anti- ever, the impact of blocking autophagy in hu- 5Research Unit for Epidemiology, Aarhus University, oxidants. The complexity and intensity of man sperm under OS conditions is unknown. Department of Public Health, Aarhus, Denmark; the process of spermatogenesis sensitive to The objective here was to evaluate the impact 6The Faculty of Health Sciences, University of violations of the balance of vitamins, coen- of autophagy inhibition in human sperm ­Copenhagen, Department of Public Health, Copen­ zymes, trace ­elements, necessitates the deter- exposed to OS. For this, spermatozoa from hagen, Denmark; 7National Research Centre for the mination of the content of these substances normozoospermic donors were used. Chlo- Working Environment, Copenhagen, Denmark in the ejaculate. The study of antioxidant roquine was used as the autophagy blocker, Introduction The negative impact of mater- system, lipid peroxidation and the quality of since it has been reported as an autophagy in- nal smoking during pregnancy on offspring semen in men of reproductive age living in hibitor in different mammalian cells, includ- semen quality is well established. We inves- Ulan-Ude, Republic of Buryatia, in Irkutsk ing human sperm. Ionomycin and hydrogen

70 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

peroxide (H2O2) were used separately to in- and a significant increase of Sperm motility control groups (p = 0.453), however DFI was duce OS. Sperm cells were co-incubated with in no- nicotine group. Testosterone show sig- inversely correlated with total sperm count ionomycin plus chloroquine and separately nificant increase in e-cig groups, more evi- (ρ = –0.384, p = 0.025). No association was with H2O2 plus chloroquine. An untreated dent in the nicotine group. Catalase enzyme found between DFI, HDS and either MLH1 control and a control incubated only with the activity is SS decrease 31.10%, P<0.0001 in or MSH2 promoter methylation. OS inducer were included. Viability, mito- nicotine group. Conclusion These data suggest a poten- chondrial membrane potential (ΔΨm), phos- Conclusion The present study showed that tial association between MLH1 promoter phatidylserine (PtdSer) externalization and E-cig exposure is associated with decreased methyla­tion and varicocele-related infertility, caspase activation were evaluated by flow sperm concentration, increase abnormal but larger cohort studies are needed to verify cytometry. Metabolic parameters including forms, increase testosterone level more evi- the results. glycolysis, measured as extracellular acidifi- dent in nicotine group. E-cig exposure affects cation rate (ECAR) and oxidative phospho- ROS neutralization by decrease CAT enzyme rylation based on the oxygen consumption activity and induce histological testicular de- rate (OCR) were analyzed with a Seahorse generation of spermatogonia. The decrease P 12.8 XFp extracellular flux analyzer. The results in catalase activity and sperm concentration Paternal preconception metho­ showed that OS decreases viability and ΔΨm, can be attributed to nicotine rather than the while increases PtdSer externalization and trexate exposure and perinatal E-­liquid. On the other hand, as regards in- characteristics of the children – caspase activation. The OS-mediated impair- creased abnormalities, E-liquid exerts this DGA-Abstracts ment of sperm parameters is accentuated effect, but the effect is enhanced further by a nationwide register study in when autophagy is blocked. Also, autophagy nicotine. Sweden 2006–2014 inhibition directly impairs ECAR and OCR P. Zarén1, A. Giwercman1,2 and negatively affect metabolic response un- 1 der OS conditions. P 12.7 Lund University, Department of Translational ­Medicine, Malmö, Sweden; 2Skane University In conclusion, blocking of autophagy results Determination of promoter ­Hospital, Reproductive Medicine Centre, Malmö, in increased oxidative damage, which nega- ­methylation of MLH1 and MSH2 Sweden tively impacts the functionality and lifespan of sperm cells, suggesting a crucial role of genes and their effect on sperm Introduction Methotrexate (MTX) is a autophagy as a stress response mechanism by DNA fragmentation and sperm folate antagonist used at low doses in the male gametes. chromatin condensation in men treatment of inflammatory diseases. Due to a lack of evidence and the known teratogenic Grants Supported by FONDECYT with varicocele effect in women, men have been recommend- 11170758 1 1 2 2 N. Hekim , S. Gunes , E. Lahrmann , R. Da Costa , ed to cease MTX treatment 3 months prior 2 2 J. Gromoll , S. Laurentino to conception. In recent years, a number of 1 Ondokuz Mayis University, Medical Biology, studies investigating paternal preconception 2 P 12.6 Samsun,­ Turkey; Centre of Reproductive Medicine MTX exposure have failed to demonstrate and Andrology, Münster, Germany Impact of Electronic Cigarette any significantly increased risk for adverse pregnancy outcomes. However, the evidence Smoke Exposure on Testicular and Introduction MLH1 and MSH2 are DNA mismatch repair (MMR) enzymes which is still scarce and current national guidelines Spermatozoa Performances in Al­ have essential roles in DNA repair and meio- differ. The aim of this study was to further bino Rats sis. Studies have shown that polymorphisms investigate the effect of male preconception MTX exposure on offspring health. O. Shaeer1, W. EL-Nattat2, M. Farag1, S. Elshimy3, and mutations of MMR genes might have ef- K. Shaeer3 fect on male fertility. Varicocele is the most Patients and Methods A nationwide regis- 1Kasr El Aini Faculty of Medicine, Cairo University, frequent curable cause of male infertility ter study was carried out, analyzing singleton Andrology, Giza, Egypt; 2National Research Center, and may lead to sperm DNA fragmentation live births recorded in the Swedish Medical Cairo, Germany; 3Kasr El Aini Faculty of Medicine, (SDF). The aim of the study is to analyze the Birth Register (MBR) 2006–2014. By link- Cairo University, Giza, Egypt promoter methylation of MLH1 and MSH2 ing MBR to the Swedish Prescribed Drug genes, sperm DNA fragmentation index Register, 224 offspring to fathers with pre- Introduction This study was done to inves- (DFI) and high DNA stainability (HDS) and conception MTX exposure were identified, tigate the toxic impact of Electronic Ciga- to investigate probable associations among along with 810 178 offspring to unexposed rettes smoke exposure on the testicular func- methylation alterations, SDF and sperm chro- fathers. Preconception MTX exposure was tions, semen parameters and the antioxidant matin condensation in men with varicocele. defined as fulfilling both 1) at least one MTX status in Albino rats. Patients and Methods Semen samples withdrawal from pharmacies 0–3 months Materials and Methods A total of 24 adult from 27 infertile men with varicocele and prior to conception and 2) at least one more fertile Male Wistar albino rats were included 10 normozoospermic men as a control group withdrawal 0–12 months prior to conception. in the study, randomly distributed into three were included in this study. Pyrosequencing The effects of paternal MTX exposure on the groups as follows: control group (n = 8), E- was used to analyze methylation of MLH1 risk of major congenital anomalies, preterm cig exposed group without nicotine (n = 8) and MSH2 promoters quantitatively. DFI birth and small-for-gestational age were ana- and E-cig 6 mg/ml of nicotine exposed group and HDS were assessed by sperm chromatin lyzed using logistic regression, adjusted for (n = 8). Rats in group 2 and 3 were exposed structure assay (SCSA). medical and socioeconomic factors. to 2 cycles/hour. Each cycle included 15 min Results The means of total sperm counts Results Given paternal preconception exposure and another 15 min for recovery, 6 MTX exposure, the odds ratio (95% CI) for hours/day for 8 weeks. in infertile men with varicocele and control group were 42.14 million vs 88.31 mil- congenital anomalies was 0.99 (0.41–2.41), At the end of the study, the testes were iso- lion, respectively. MLH1 promoter meth- for preterm birth 0.98 (0.54–1.81) and for lated for sperm examination and histological ylation was significantly higher in infertile small-for-gestational age 1.04 (0.43–2.53). analysis, as well as blood sampling and bio- group than in controls (means 3.4886% vs Conclusion No increased risk for adverse chemical analysis for testosterone level and 2.7750%, p = 0.017). Methylation of MLH1 pregnancy outcomes was observed after pa- antioxidant activity. promoter was positively correlated with ternal preconception MTX exposure. This Results Result showed that e-cig exposure methylation of MSH2 promoter (ρ = 0.398, is the largest study to date investigating the led to alteration of semen parameters with p = 0.024). HDS was higher in infertile men safety of paternal MTX use prior to concep- a significant drop of sperm concentration in with varicocele than normozoospermic men tion. Along with a growing body of evidence, nicotine group, a significant increase of mor- (means 5.3355% vs 2.4559%, p = 0.018). this study suggests MTX can safely be con- phologically abnormal in both e-cig groups DFI did not differ between varicocele and tinued among men trying to conceive.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 71 Andrology 2020 – Abstracts

P 12.9 identified selected pesticides and biocides spermatogenesis (temperature, humidity, that induced Ca2+-signals in human sperm. pressure, sunshine duration, and related vari- Epididymis-on-a-chip approach to We hypothesized that multiple pesticides and ables) upon human semen quality. A large elucidate the impact of environ­ biocides may interact with the human sperm- cohort of men (n = 11657) was subjected mental plastic pollution on male specific CatSper Ca2+-channel. to a thorough semen evaluation (semen vol- fertility Materials and Methods We screened 53 ume, sperm concentration, motility, vitality, 2+ morphology, round cells, HOS test, nuclear E. Stoimenou1, K. Pondman1, B. Venzac1, T. Burgers1, pesticides and biocides for effects on Ca - 2+ maturity, and sperm kinematics: VSL, VCL, 2 1,2 1 signaling in human sperm cells using a Ca - S. Sharma , S. Schlatt , S. Le Gac VAP, LIN, STR, WOB, BCF, ALH, and 1 fluorimetric assay. Using the specific CatSper University of Twente, Enschede, Netherlands; MAD) following WHO 2010 guidelines and 2CeRA, Münster, Germany inhibitor RU1968 we examined whether an induced Ca2+-influx involved CatSper. Dose internal/external quality assurance standards The blood-testis and blood-epididymis bar- response relations were assessed for the at LAR andrology laboratory (Argentina). As rier (BTB and BEB), are amongst the strong- Ca2+-influx inducing pesticides and biocides. a result, age ≥ 40 years, obesity (body mass est barriers in the body, protecting the post- These chemicals were examined for effects index [BMI] ≥ 30), and spermatogenesis dur- mitotic germ cells from the immune system. on on sperm penetration into viscous media ing summer were individually found delete- Studying these barriers is crucial not only in and acrosomal exocytosis. rious to semen quality, as reflected by lower sperm routine and kinematic parameters in the field of reproductive medicine, but also to Results We found that 28 of the 53 pes-

DGA-Abstracts samples from men ≥ 40 years, BMI ≥ 30 or identify chemicals and pollutants such as en- ticides and biocides induced a mean Ca2+- docrine disruptors possibly disturbing these 2+ obtained in summer, when compared to those influx > mean Ca -influx of the negative of men < 40 years, normal BMI, or obtained barriers, as exposure to certain environmental controls ±3SD (10 µM, n = 7). The most cues is considered to contribute to the signifi- in winter. Moreover, several confounding efficacious chemicals Milbemectin A4 and factors (obesity, cigarette-smoking, alcohol- cantly increased prevalence of male infertility A3, as well as Chlorpyrifos induced mean [1]. 2+ consumption, and spermatogenesis in low Ca -influxes at 10 µM reaching 87%, 85% humidity/long sunshine duration periods) Due to the absence of valid test systems the 2+ and 76% respectively of the maximal Ca - contributed to worsen the already decreased adverse effects of environmental chemicals influx induced by progesterone. The major- sperm quality in men ≥ 40 years. Interesting- and pharmaceuticals on adult human male ity of the chemicals were found to induce ly, a predictor selection algorithm followed reproductive function could not be systemati- 2+ an Ca -influx by activating CatSper (n = 4). by a multiple linear regression analysis of cally investigated. Therefore, establishing a Dose response relations were assessed for the meteorological variables identified humid- robust and physiologically relevant in vitro 28 pesticides and biocides with mean EC50 ity and sunshine duration as the most fre- screening platform using human male go- values in the µM-range (n > 6). Several of the quent semen quality predictors. Altogether, nadal tissue is essential. Here we present an pesticides and biocides inhibited subsequent findings from this wide study highlight the epididymis-on-a-chip microfluidic system, CatSper-responses induced by progesterone ­relevance of risk factors studies for predict- for studying the BEB barrier, using samples and prostaglandin E1 (n = 3). Several pesti- ing reproductive health alterations associated obtained from gender dysphoria patients cides and biocides affected sperm penetration to diseases, lifestyle, and environment and for undergoing sex reassignment surgery. The into viscous media and acrosomal exocytosis professional counseling. epididymal tissue is clamped between pillars (n = 3). in a custom-designed 3D printed cartridge, Conclusions 28 of 53 examined pesticides which is inserted in a housing chip. In our and biocides induced Ca2+-influxes in hu- P 12.12 system we aim to study the possible impact man sperms, the majority by activation of of, e.g., environmental pollutants and chemi- CatSper. Some of the pesticides and biocides Are ZnO-NPs harmless to sperma­ cals on the integrity of the BEB. were found to interfere with sperm functions togenesis? Focusing cytoskeleton Presently, the system and culture conditions in vitro. Clinical studies are needed to investi- and nucleoskeleton dynamics in are being optimized for prolonged ex vivo gate if exposure pesticides and biocides have spermatogonia cells culture of epididymal tissues [2]. This model effects on human fertility. 1,2 1,3 4,5 will be thereafter employed as a screening A. R. Pinho , F. Martins , M. E. V. Costa , 4,5 1,3 platform to study the impact of environmen- A. M. R. Senos , O. A. B. da Cruz e Silva , 1,4 1,3 tal chemicals, e.g. degraded by-products of M. D. L. Pereira , S. Rebelo P 12.11 1 environmental plastics, microplastics and Aveiro University, Department of Medical Sciences, 2 endocrine disruptors on the BEB integrity. Associations between semen Aveiro, Portugal; CICECO-Aveiro Institute of Mate- 3 Later, this platform can be adapted for similar quality and age, meteorological rials, University of Aveiro, Aveiro, Germany; Neuro- exploratory studies on the BTB using semi- variables, clinical conditions and science and Signaling Laboratory, Institute of Bio- niferous tubules. medicine—iBiMED, University of Aveiro, Aveiro, lifestyles Portugal; 4CICECO-Aveiro Institute of Materials, Uni- References: G. L. Verón1, A. D. Tissera2, G. M. Estofan3, R. Bello4, versity of Aveiro, Aveiro, Portugal; 5Aveiro University, Sharpe RM, Franks S. Nat Cell Biol. & Nat. Medicine F. Beltramone3, R. I. Molina2, M. H. Vazquez-Levin1 Department of Materials and Ceramic Engineering, 2000. 1Instituto de Biología y Medicina Experimental (IBY- Aveiro, Portugal Venzac B. et al. µTAS 2018, Kaohsiung, Taiwan, 2018. ME), Ciudad Autónoma de Buenos Aires, Argentina; New era of nanomedicine has brought excel- 2Laboratorio de Andrología y Reproducción (LAR), lent alternatives that benefit diagnosis and Córdoba, Argentina; 3Centro Integral de Ginecología, P 12.10 medical treatment, due to the exceptional set Obstetricia y Reproducción (CIGOR), Córdoba, Argen- of characteristics of nanoparticles. Zinc Ox- Widely used pesticides and bio­ tina; 4Universidad de Tres de Febrero (UNTREF), ide Nanoparticles (ZnO-NPs) are used in a 2+ ­Ciudad Autónoma de Buenos Aires, Argentina cides interfere with Ca -signaling variety of biomedical applications. However, through effects on the CatSper Semen analysis is an indispensable tool for its small size and its capacity to increase ROS channel in human sperm cells assessing male fertility potential since it pro- levels leading to apoptosis are very useful vides relevant information on male genital in cancer therapy and microbial treatment, A. Rehfeld, A. M. Andersson, N. E. Skakkebæk tract functionality. The evaluation of associa- raising also concerns on its biosafety. ZnO- Rigshospitalet, Growth and Reproduction, Copenha- tions between risk factors and semen quality NPs, in addition to being cytotoxic to the gen, Denmark is fundamental for clinical management. This male reproductive system, can also interfere Introduction Pesticides and biocides are blind cross-sectional study assessed the im- with the cytoskeleton and nuclear structure widely used in modern agriculture and hu- pact of age, clinical (obesity) and lifestyle in different cell types. Since the dynamics mans are broadly exposed to these chemicals. (cigarette smoking, alcohol consumption) of the cytoskeleton and nucleoskeleton is Our previous study [Schiffer et al. 2014] as well as meteorological variables during extremely important for the progression of

72 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts meiosis in spermatogenesis, it is urgent to once or twice a day to mimic the treatments LH, testosterone, Estradiol, TSH, prolactin, clarify the consequences of ZnO-NPs in the applied in the experimental groups (control). DHEAS. cyto­skeleton and nucleoskeleton dynam- Once completed the treatment period, all Conclusions It seems that we can use the ics using cells in pre-meiotic stage, namely animals were exposed to cohabitation with value of inhibin B and FSH to assess the spermatogonia cells. Therefore, our proposal females (total n = 60) in estrus stage. Vaginal intensity of spermatogenesis. The decreased was to evaluate in vitro the cytotoxicity of smears were collected in the following morn- concentration of inhibin B correlates with the ­ZnO-NPs using spermatogonia cells focusing ing and sperm-positive females were submit- number of sperm (the lower the concentra- on cytoskeleton and nucleoskeleton altera- ted to laparotomy at gestational day 20. Three tion of inhibin B the lower the efficiency of tion. Pre-meiotic cell model of mouse sper- days after mating, males were anesthetized spermatogenesis) and with FSH (the higher ® matogonia stage GC-1 spg cells were used to by intraperitoneal injection with Dopalen FSH, the lower the sperm count). High lev- ® evaluate the effect of ZnO-NPs on key pro- / Anasedan solution and underwent lapa- els of FSH and reduced levels of inhibin B teins of the cytoskeleton (tubulin and actin) rotomy to collect the testes, epididymides, clearly indicate impairment of spermato- and of the nucleoskeleton (SUN1, nesprin-1, ventral prostate and seminal vesicles. Sperm genic function in addition to the testes. lamin A/C, and LAP1). Our results clearly in- samples were frozen for sperm DNA integ- dicate that higher concentrations of ZnO-NPs rity analysis. Male and female rats were The concentration of testosterone is not good have a cytotoxic effect on GC-1 cells, leading submitted to euthanasia following the current predictor of spermatogenesis. (Inhibin B to a decrease of viability with an increase of guidelines established by the local council and testosterone are produced from different types of cells in the testis).

cell death, a consequence of changes in cy- for animal experimentation control (CEUA, DGA-Abstracts toskeleton and nucleoskeleton dynamics. As protocol number 3829240419). The results a conclusion, ZnO-NPs, in a time and dose we have so far suggest that animals aged with P 13.2 dependent manner, impact the dynamics D-galactose showed detrimental effects to and structure of the GC-1 cell line in mouse male fertility, evidenced by a significant raise Differential tissue-specific­damage sperm cells, and its nuclear movement, posi- in embryonic resorption and pre-implantation caused by bacterial epididymo-­ tioning and stability, which can compromise losses, while animals co-treated with açai orchitis in the mouse spermatogenesis and, thus male fertility. berry generated a significant increase of live B. Klein1,2, S. Bhushan2, S. Günther3, R. Middendorff2, Grants Project CICECO-Aveiro Insti- fetuses, in addition to a lower number of pre- K. Loveland4,5, M. Hedger4,5, A. Meinhardt2,4 tute of Materials, UIDB/50011/2020 & and post-implantation losses. 1WWU Münster, ZTE, Münster, Germany; 2Justus- UIDP/50011/2020, national funds through Our results suggest a strong positive nutra- Liebig University of Giessen, Institute of Anatomy FCT/MCTES. ceutical effect of açai berry on sperm DNA and Cell Biology, Giessen, Germany; 3Max Planck quality, for improving paternal contribution ­Institute for Heart and Lung Research, ECCPS Bio­ to embryo development and pregnancy out- P 12.13 informatics and Deep Sequencing Platform, Bad come in a model known for causing a high Nauheim, Germany; 4Hudson Institute of Medical Lyophilized acai berry (Euterpe oxidative stress status. Research, Centre for Reproductive Health, Clayton, oleracea Martius) prevents sene­ Australia; 5School of Clinical Sciences, Monash Uni- scence-related decay in male re­ P 13.1 versity, Department of Molecular and Translational Sciences, Clayton, Australia productive outcome Inhibin B and FSH – The best male Ascending bacterial urinary tract infections Y. Ceragioli da Silva, T. C. Simões Ferreira, fertility report can cause epididymo-orchitis. In the cauda M. Mumcu, V. Vendramini K. Jankowska, M. Rabijewski, W. Zgliczyn´ski epididymidis, this frequently leads to per- Federal University of Sao Paulo, Laboratory of Repro- Centre of Postgraduate Medical Education, Depart- sistent tissue damage. Less coherent data is ductive and Developmental Biology, Department of ment of Endocrinology, Warsaw, Poland available concerning the functional conse- Morphology and Genetics, Sao Paulo, Brazil quences of epididymo-orchitis on testis and The use of functional foods with high an- Introduction Sperm cells are produced in caput epididymidis. In an in vivo study, the thocyanin index, such as açai berry (Euterpe­ the testes. Inhibin B is produced by the Ser- functional and spatial differences in respon- oleracea Mart.), has been an ally in the toli cells in tubules seminiferous in the testes siveness of murine epididymis and testis to treatment of many diseases related to Meta- of males. These cells are stimulated by FSH. infection with uropathogenic Escherichia bolic Syndrome and aging. The experimental Can Inhibin B be a good marker of spermato- coli (UPEC) were addressed. Whole tran- model of induction of senescence, using D- genesis? Should we use other marker (FSH?, scriptome analysis (WTA) was performed galactose-treated rodents, displays indica- testosterone? testis volume?) on testis, and on caput, corpus and cauda tive characteristics of cognitive, motor and Patients We examined 100 patients with epididymidis of adult C57BL/6 J wildtype reproductive impairment similarly to those infertility. mice. Following UPEC-induced epididymo- observed in natural aging mice. Thus, this Methods Semen analysis was performed orchitis in these mice, epididymal and tes- project seeks to determine the level of sper- according to World Health Organization ticular tissue damage was evaluated histo- matic alterations and the implications for pa- guidelines (WHO 2010). Hormone analyses logically and semi-quantitatively at 10 days ternal contribution to embryonic and fetal de- include: FSH (follicle stimulating hormone), and 31 days post-inoculation. Expression of velopment conceived by the animals induced LH (luteinizing hormone), testosterone, es- inflammatory markers and candidate antimi- to senescence; we also propose to verify the tradiol, prolactin, TSH, and inhibin B. We crobial genes were analysed by RT-qPCR. possibility of avoiding such damages with analysed the dependencies between semen WTA revealed distinct differences in gene the concomitant consumption of açai berry. parameters, testicular volume and hormons, signatures between caput and cauda epididy- For this, 18 male Wistar rats were used and especially inhibin B. midis, particularly amongst immunity- distributed into 3 groups (n = 6 each): one related genes. Cellular and molecular signs Results The sperm count was significantly group of animals (n = 12) was submitted to and positively correlated with Inhibin B (r = of testicular inflammation and disruption of accelerated aging undergoing treatment with 0.75, p < 0.001). The Inhibin B was negative- spermatogenesis were evident at day 10, but D-galactose for a period of 4 weeks, in daily ly correlated with FSH (r = 0.66, p < 0.001). recovery was observed by day 31. In contrast doses of 200 mg / kg of body weight, at mid- to the cauda, the caput epididymidis did not day, via gavage; half of these animals (n = 6) The lower was the concentration of inhibin reveal any signs of gross morphological dam- received, at the same day, supplementation B, the lower was the number of sperm in the age or presence of pro-inflammatory pro- with 200 mg / kg of lyophilized açai berry semen. There was also a relationship between cesses, despite confirmed infection. Known diluted in water, 5 times a week at 5 P.M., seminogram and FSH – the higher was the UPEC-associated antimicrobial peptides since the first week of aging induction; a FSH, the lower was the number of sperm. (AMPs), like Lcn2, Camp and Lypd8, were group of animals (n = 6) received, also via There was no relationship between the inherently highly expressed and upregu- gavage, 0.6 ml of distilled water for 4 weeks, number of sperm and the concentration of lated in the caput following infection, poten-

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 73 Andrology 2020 – Abstracts

tially allowing an early luminal protection Introduction The role of prolactin in male homozygous deletion of CATSPER2. The from UPEC. At both time points, the caput reproductive function is less known compared patients’ sperm lacked functional CatSper epididymidis exhibited no obvious infection/ to its role in female reproduction, however the channels, but according to standard semen inflammation-derived tissue damage. Studies measurement of prolactin in the investigation analysis, the patients were normozoospermic. addressing earlier time-points will examine of male infertility is considered necessary. Thus, a loss of CatSper function escapes cur- whether or not a pro-inflammatory response Our aim was to assess the prevalence of hy- rent methods used to assess male fertility. The in the caput epididymidis is essential for ef- perprolactinemia – as a possible cause of male CATSPER2-deficient patients failed to con- fective protection from UPEC. infertility – in the laboratory work-up required ceive naturally, upon IUI, and/or upon IVF; in patients attending a specialized andrology conception was achieved only through ICSI. clinic for infertility or erectile dysfunction. P 13.3 We show that this is due to the failure of Cat- Materials and Methods We studied 656 Sper-deficient sperm to hyperactivate, where- Medice, cura te ipsum: case report men, with mean age (SD): 34 (11) years, who fore the sperm fail to ­penetrate the egg coat. of a first-person experience with presented at the andrology clinic either due to Remarkably, four of the seven patients also male infertility dyspermia (n = 249) or due to erectile dysfunc- suffer from a deletion of the STRC gene and, tion (n = 396); plasma prolactin was measured thus, from the deafness-infertility syndrome. A. Sansone in 292 men. Assessment of normal/elevated Conclusion We conclude that human sperm University of Rome Tor Vergata, Chair of Endocrino- values (> 30 ng/mL) of prolactin (as the de- dysfunction may involve loss of CatSper logy and Medical Sexology (ENDOSEX), Department DGA-Abstracts pendent variable), and the patients’ group function, that CatSper-deficient sperm fail to of Systems Medicine, Rome, Italy (individuals with dyspermia/erectile dysfunc- penetrate the egg coat, and that the homozy- Case report The male partner accounts for tion), plasma testosterone & gonadotrophins, gous deletion of CATSPER2 represents the almost 50% of cases of couple infertility, as as well as age (as covariates/factors), was done most common genetic cause underlying loss either the only or a contributing factor. Genet- with the chi square test & logistic regression. of CatSper function. Our results also demon- ic causes are considered responsible for 15% Results In men with dyspermia 121 had strate that a deletion of CATSPER2 is often, of cases, and are most frequently associated prolactin within normal limits and 6 had but not always, associated with a deletion of with severe oligozoospermia or azoospermia. increased levels, while in men with erectile STRC, causing the deafness-infertility syn- In the present case report, the Author – an an- dysfunction 149 had prolactin within normal drome. drologist mostly working on male infertility limits and 16 had increased levels (p = 0.17). and sexual dysfunctions – describes his own No effect of age, testosterone or gonadotro- experience with the diagnosis of male infer- pins on prolactin was noted (p = 0.47). P 13.6 tility, and the relevant psychological conse- Conclusion The measurement of prolactin quences, as well as the consequent changes in The role of activin A and chemo­ is often considered to be necessary in the kines in the development of in­ patient management learnt from this experi- investigation of male factor infertility, how- ence. The Author/patient had a three years his- ever, it is not often found to be high due to flammatory responses during tory of failing to achieve a clinical pregnancy pathological causes (prolactinoma, second- chronic testicular inflammation in with a healthy partner two years younger than ary hypogonadism). mice him, despite normal semen analysis (sperm count: 98 × 106/ml, 3.2 ml, total 313.6 × 106; H. Hasan1, W. Peng1, S. Bhushan1, E. Wahle1, total motility 60%; normal sperm morphol- C. Pleuger1, K. Loveland2,3, A. Meinhardt1,2, P 13.5 2,3 1 ogy: 13%) and normal hormone levels (FSH M. Hedger , M. Fijak 2.71 mIU/mL, LH 5.48 mIU/mL, inhibin B The (patho)physiology of CatSper 1Justus-Liebig-Universität Giessen, Institute of Ana- 188 pg/mL, total testosterone 27.65 nmol/L). Ca2+-channels in human sperm tomy and Cell Biology, Giessen, Germany; 2Hudson Institute of Medical Research, Centre of Reproduc­ The Author/patient and his partner sought 1 1 1 2 S. Young , C. Schiffer , C. Brenker , F. Tüttelmann , 3 fertility treatment: during the course of pre- tive Health, Clayton, Germany; Monash University, A. Röpke2, A. Wagner1, M. Kierzek1, C. Krallmann3, FIVET evaluation, both underwent karyotype Department of Molecular and Translational S. Kliesch3, T. Strünker1 analysis, and a paracentric inversion of chro- ­Sciences, Clayton, Australia 1Centre of Reproductive Medicine and Andrology, mosome 15 [46,XY,inv(15)(q15q24)] was University of Münster, Münster, Germany; 2Institute Experimental autoimmune orchitis (EAO) found, explaining the cause of male infertil- of Human Genetics, University Hospital Münster, serves as a model of autoimmune-based ity. While pregnancy is largely feasible via Münster, Germany; 3Centre of Reproductive Me­ chronic testicular inflammation and shares preimplantation genetic diagnosis, receiving dicine and Andrology, University Hospital Münster, many features in common with male sub- the diagnosis had severe psychological con- Münster, Germany fertility and infertility. EAO is characterized sequences for the Author/patient, with the by severe immune cell infiltration into the onset of moderate anxiety (General Anxiety Introduction Male infertility is often due interstitium, testicular fibrosis and an in- Disorder-7 scores increased from 4 to 13) and to low sperm counts, impaired motility, and/ crease in the production of pro-inflammatory depression (Patient Health Questionnaire-9 or abnormal morphology. However, for about cytokines and chemokines, including TNF, increased from 1 to 7) which lasted for several 30% of infertile men, semen parameters are IL-6, activin A and CCL2. An increase in months. Diagnosis also changed the Author’s normal, and infertility is rather due to an un- CD4+CD8+ double positive T cells also has approach to infertile patients, with an in- explained sperm dysfunction. The underlying been observed in the EAO testis. However, creased tendency to investigate genetic causes mechanisms are enigmatic. We surmised that the influence of CCL2 and activin A on the 2+ of couple infertility, and to suggest assisted human sperm dysfunction involves the Ca - testicular immune response remains unclear. reproduction techniques earlier than before in channel CatSper that translates changes in the cases of unexplained infertility. chemical microenvironment into changes in Preliminary results using azan and Giemsa swimming behavior. staining established that CCR2-/- mice, defective in recruitment of monocytes dur- P 13.4 Patients and Methods We developed a ing inflammation, are protected from EAO rapid test to assess CatSper activity in sperm Prolactin in patients of a specializ­ damage. Since activin A is increased in from patients undergoing a fertility workup. orchitis, its effect on bone marrow derived ed andrology clinic We characterized seven patients and their macrophages (BMDM) as an in vitro sur- E. Venaki, I. Ilias, A. Michou, C. Milionis, sperm identified as CatSper-deficient using rogate for testicular macrophages was in- I. Kakoulidis, S. Togias, S. Stergiotis, A. Pappa, genetic tests, audiometry, semen analysis, vestigated. Results from five independent 2+ S. Nikopoulou, E. Koukkou electrophysiology, Ca -fluorimetry, and mo- experiments show that activin A (50 ng/ml) Elena Venizelou General and Maternity Hospital, tility analysis. decreased mRNA expression of CCL7 and ­Department of Endocrinology, Diabetes and Meta­ Results We show that all seven patients CCL8 ten-fold and increased CXCL16 and bolism, Athens, Greece feature a similar genetic aberration, i.e. a CXCL14 3-fold and 60-fold, respectively.

74 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

Our preliminary analysis by flow cytometry Conclusion Despite several limits (retro- P 13.9 revealed that the treatment of murine single- spective design, no external validation), this cell suspensions of splenocytes with activin multivariable prediction model presented a Impact of genetic risk scores on A for 4 days led to a proportional increase in good discrimination and open the way to a association between childless­ CD3+ T cell numbers. Within the population screening test for CUAVD. ness and the risk of cardiovascu­ of T cells, we observed a decrease in CD4+ T lar mortality cells and an increase in CD8+, CD8+CD25+ and CD4+CD25+ T cells. Further analysis by P 13.8 A. Elenkov, O. Mellander, P. M. Nilsson, flow cytometry showed that activin A added Are tests for sperm antibodies A. Giwercman Lund University, Malmo, Sweden for 2 days to phorbol-12-myristat-13-acetate useful in clinical routine to iden­ (PMA) and ionomycin-activated splenocytes tify men with immunologic cause In a population-based cohort of 22,000 men increased the expression of IL-17A in CD8+ for infertility? gathered in the 1970s, we previously showed T cells, TNF, IFN-γ and IL-17A in CD4+ T that childlessness can be regarded as a pre- cells and IL-17A, TNF, IFN-γ and IL-10 in P. Houska, L. Björndahl dictor of cardiovascular disease (CVD) as- CD4+CD8+ double positive T cells. Karolinska University Hospital and Karolinska sociated mortality independently of other Our initial observations identify a role of ­Institutet, ANOVA, Stockholm, Sweden well-known risk factors. Before the era of as- activin A and CCL2 in altering the immune Background­ The testing for ASAB (anti- sisted reproduction sub-fertile men remained cell phenotype, where activin A shows poten- sperm antibodies) has become part of routine childless, why the latter condition can be DGA-Abstracts tial to regulate the chemokine network, and workup of an infertile patient since an article considered as proxy of poor male fertility. CCL2 plays a crucial role in exacerbating in 1952 described a test for “antisperm ag­ We wished to explore if the higher risk for testicular inflammation. glutinins”. However, in the last years there CVD mortality among childless, and presum- has been a decline in published research ar- ably infertile men is due to a more common inheritance of genetic risk for CVD or, if not, P 13.7 ticles indicating diminishing interest in this topic. Moreover, the production of some to assess if the two factors work synergisti- Development of a multivariable commercial kits ceased, resulting in issues cally as predictors of CVD mortality. prediction model for congenital with inhouse preparation of reagents, valida- We used data from a population-based cohort unilateral absence of the Vas tion and external quality control. of 2130 middle-aged men from Southern Sweden, specifically designed to study the ­Deferens (CUAVD) Objective and Rationale A literature re- view of original research articles and review epidemiology of CVD. Among them 311 S. Hamdi, C. Brusq, R. Mieusset articles published in the last 20 years that use (14%) were childless at the age of 45 years. Universitary Hospital of Toulouse / Toulouse Univer- tests for ASAB as a method to identify men Two sets of genetic risk scores (GRS) based sity, Research Group in Human Fertility, Toulouse, with immunologic cause for infertility. on either 27 or 50 single nucleotide polymor- France phisms (SNPs) – GRS27 vs. GRS50 – were Search Methods The MEDLINE da- tested. In previous studies each of the in- Introduction CUAVD is a rare condition tabase was searched from inception until cluded SNPs has been shown to be associated that is associated with a high risk of CFTR August 2020 with following search terms: with CVD at genome-wide significance level. mutations and/or unilateral renal agenesis. “antisperm”, “antibody”, “mar” and “asa”, The GRS of each individual in the study was No seminal profile is specific of this condi- combined with “sperm”. A deselection of the calculated based on the previously reported tion and the gold standard for its diagnosis is articles was performed by both investigators risk estimate of each SNP.Six different study transrectal ultrasonography (TRUS). The aim separately, the differences were discussed to categories according to fatherhood status of this study was to develop a multivariable prevent any bias. Deselection criteria were: (+/- ) and 3 groups of GRS – high, intermedi- prediction model for CUAVD in a cohort of published before the year 2000, fulltext not ate or low genetic risk – were defined. male partners of infertile couples. available in English, other than human sper- GRS distribution did not differ between fa- Patients and Methods We conducted a ret- matozoa, publications not answering the ob- jective of this study. thers and childless men (p = 0.29 for GRS 27 rospective review of medical records of 147 and 0.49, for GRS 50) However, high GRS patients that had TRUS in a single tertiary- Outcomes Available data does not support, was a stronger predictor for CVD mortality centre. Amongst them, 69 had CUAVD and that ASAB testing is an essential part of the among childless men (HR: 3.12 [95-%-CI: 78 were controls. Thirty predictors were routine workup for infertility in an individual 1.39–7.04 for GRS50] and HR: 3.73 [95-%- explored through uni- and bivariate analysis. patient: CI: 1.75–7.99 for GRS27]), than it was case Selected predictors were used in a complete 1. Until present day, no causative treatment among fathers (HR:1.92 [95-%-CI: 1.1–3.36 multivariable logistic regression model. for ASAB has been found. for GRS50] and HR: 1.54 [95-%-CI: 0.87– Results Sperm concentration, total sperm 2. Presence of ASAB does not appear to in- 2.75 for GRS 27]). number, sperm progressive motility and vi- fluence the success of ICSI treatment of Thus, combining GRS with fertility status tality were not significantly different between the infertile couple. may improve identification of high-risk CUAVD patients and controls. CUAVD 3. Screening tests rely on the presence of group for CVD mortality and, thereby, help patients presented a lower semen volume defining individuals who might benefit from than controls (1.5 vs 2.4 mL respectively, motile spermatozoa, meaning that cyto- toxic antibodies that cause death of the early preventive measures aiming to improve p < 0.000). However, no difference of histo- the lifespan and quality of life. ries of fertility was recorded (23 vs 32% of spermatozoa cannot be detected with these previous successful pregnancies respectively, methods. p = 0.23). Wider implications Screening tests for P 13.10 After variables selection, we obtained a final ASAB should no longer be used in the rou- Does the FSHB c.-211G > T poly­ tine workup of an infertile patient. multivariate model based on the three fol- morphism impact the spermato­ lowing predictors: history of cryptorchid- genic potential in infertile male ism, total seminal fructose below 13 mmol/ ejaculate, total seminal alpha-1,4 glucosi- patients? dase (mUI/ejaculate). The ROC curve of the S. Kaldewey1, M. Schubert1, L. Pérez Lanuza2, model presented an AUC value (95-%-CI) of H. Krenz3, M. Dugas3, S. Berres2, S. Kliesch1, 0.866 (0.808–0.924). This was in favour of J. Wistuba2, J. Gromoll2 a good selection between cases and controls. 1Centre of Reproductive Medicine and Andrology, The model was then internally validated by Department of Clinical Andrology, Münster, bootstrapping. Germany;­ 2Centre of Reproductive Medicine and

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 75 Andrology 2020 – Abstracts

­Andrology, Münster, Germany; 3Institute of Medical men samples and the putative impact of sub- by Strict Criteria. DNA fragmentation index Informatics - Informatics for Personalized Medicine, sequent antibiotic therapy is unknown. (FI) was assessed by the Sperm Chromatin Münster, Germany Aim To study the association of seminal Dispersion tests and nuclear immaturity (IN) by Aniline blue. The RH (FSH, LH, albumin, Follicle stimulating hormone plasma infections and antibiotic therapy with Introduction Total, free and bioavailable Testosterone, (FSH) is a key driver of spermatogenesis ORP in semen samples of infertile men. sex hormone-binding globulin (SHBG) and in the male. Via the FSH receptor it binds Methods We prospectively measured the Estradiol) were determined by ECLIA. All at Sertoli cells (SC), which play a decisive ORP in semen samples of 25 idiopathic in- patients signed a written consent. Data were nursing role for maturing germ cells. A fertile men who visited our Center of Repro- showed as median and interquartile range single nucleotide polymorphism (SNP) (rs ductive Medicine and Andrology for control and analyzed by Wilcoxon signed rank test. 10835638 FSHB c.-211 G > T) reduces the visits after having been diagnosed as idio­ A p < 0.05 was considered significant. promoter activity, thereby leading to reduced pathic infertile. In addition, to standard se- FSH values and lowered sperm counts. The men analysis according to WHO guidelines, Results A decreased in post-surgery values histo-pathological mechanisms behind this we performed seminal bacterial analysis and was observed in: BMI 45.4 (8.1) vs 29.0 (5.4) 2 have not been studied so far. This project additional sperm function tests, e.g. DNA kg/m , p = 0.003; semen volume 3.5 (0.5) aimed to determine the impact of the FSHB fragmentation index (DFI), CASA motility vs 3.0 (1.5) mL, p = 0.04; total sperm count 6 c.-211 G > T SNP on human spermatogenic assessment and used the MiOXSYS® System 218 (186) vs 74 (170) × 10 sperm, p = 0.02; efficiency. Testicular tissue of azoospermic (Aytu Bioscience, Engelwood, CO, USA) curvilinear velocity 58.7 (6.8) vs 55.0 (16,40) DGA-Abstracts patients homozygous for the T- allele was for ORP analysis. Results of ORP were then um/s, p = 0.02 and amplitude of lateral head analysed and results compared to patients compared with the results of all semen pa- displacement 3.1 (0.3) vs 3.0 (0.8) um, homozygous for the G- allele with respect to rameters, specifically in relation to a coexist- p = 0.01. No significant differences on sperm their spermatogenic potential. ence of seminal plasma infections. progressive motility, viability, normal forms, deformity index, FI and IN were found. RH 62 patients ho- Patients and Methods Results Preliminary data exhibited a sig- showed an improvement in FSH 2.7 (1.3) vs mozygous for T or G, from our inhouse da- nificant association of ORP with acute infec- 3.3 (1.6) (mUI/ml), p = 0.02 and SHBG 27.4 tabase were selected, with testicular biopsies tions in seminal plasma in 2 of 25 patients (20.0) vs 69.4 (58.7) (nmol/l) p = 0.007. Vari- available. Patients were subgrouped into ob- 6 (mean ORP: 16.35 vs. 2.83 mv/10 sperm, ation in BMI correlates positively with semen structive azoospermia (OA) vs. non-obstruc- p < 0.001) and a relevant increase in ORP volume (r: 0.6, p = 0.05) and with SHBG tive (NOA) and adjusted according to their after antibiotical treatment in another 3 pa- (r: 0.6, p = 0.01). genotype: NOA: n = 26 with GG, n = 26 with tients, while no association was seen for TT; OA (serving as controls): n = 5 with GG, standard semen paramaters and DNA frag- Conclusion Semen quality may not be n = 5 with TT). Immunohistochemistry was mentation in this study population. improved after bariatric surgery despite suc- performed applying the following antibodies: cessful weight loss and the improvement Conclusion We want to study the associa- of reproductive hormones. Further studies SOX9 for SC, MAGE A4 for spermatogonia tion of ORP with seminal tract infections in a and CREM for round spermatids (RS). Quan- are required to discard possible nutritional larger cohort and follow up the ORP after an- deficiencies and/or very near post surgery tification of distinct germ cell types were tibiotic therapy. ORP might prove as a useful carried out using the Vectra® microscope by evaluation as causing the effects observed on marker indicating recovery of sperm function spermatogenesis (Fig. 12). Perkin Elmer, allowing subsequent calcula- after treatment of seminal infections. tion of the Sertoli cell Workload (SCWL), defined as the number of germ cells served for by one SC, was calculated. P 13.12 P 14.1 Results There were no significant differ- Effect of bariatric surgery on re­ Sperm retrieval and cryopreserva­ ences in SC-, spermatogonia- and RS- num- productive hormones and semen tion in testicular torsion patients: bers between patients carrying the wildtype Benefits and limitations FSHB c.-211 G > T (GG) and those with two quality 1 2 T-alleles (TT) within the NOA cohort. Nei- M. C. Craia1,2, C. Massoni3, M. Vicentin4, A. Rashed , K. Mekati 1 th ther was the SCWL significantly different M. Grimoldi4, A. Caille1, R. Tioni4, A. Marcolini4, October 6 University, Faculty of Medicine, Uro­logy/ 2 between the genotypes. A. Diego4, M. J. Svetaz3, M. Posadas2, M. J. Munuce1 Andrology, Giza, Egypt; Tanta Uni. Faculty of Medi­ 1 cine, Urology/Andrology, Tanta, Egypt Conclusion The polymorphism FSHB c. Reproductive Medicine Laboratory, School of Bio- -211 G > T does not limit the spermatogenic chemical and Pharmaceutical Sciences, National Purpose To assess the value of sperm re- potential by lowering Sertoli cell numbers or University of Rosario, Clinical Biochemistry, Rosario, trieval and cryopreservation in postpubertal 2 the Sertoli cell workload. Argentina; School of Medical Sciences, National testicular torsion (TT) patients. University of Rosario., Biology, Rosario, Argentina; Funded by DFG CRU 326. Patients and Methods This was a pro- Grants 3Centenario Hospital., Endocrinology Laboratory, spective study conducted on postpubertal Rosario,­ Argentina 4British Sanatorium, Bariatric and males with TT. Surgical exploration was ur- Metabolic Surgery Unit, Rosario, Argentina P 13.11 gently performed and detorsion with fixation Association of seminal plasma in­ Introduction Excess of adipose tissue or excision was done according to the intra­ fections with oxidative stress would produce greater conversion of testos- operative assessment of the viability of tes- terone into estradiol leading to secondary ticular tissue. Also, the contralateral testis was Y. Rassam, M. Zitzmann, S. Kliesch hiperestrogenic hypogonadism hipogonado­ fixed to prevent future torsion. Conventional Centrum of Reproduction and Andrology, Department tropic with possible effects in the produc- or microsurgical sperm retrieval was tried in of Clinical and Surgical Andrology, Münster, tion of reproductive hormones (RH) and all cases with the intention of cryopreserva- Germany­ spermato­genesis. Our aim was to evaluate tion. Patients came back to clinic 1 week after Introduction Oxidative stress in seminal the RH and seminal quality (SQ) of young surgery and monthly thereafter for 3 months plasma can negatively affect sperm function, obese patients that have undergone bariatric with new hormonal assay and sperm analysis. hence it can reduce male fertility. Infections surgery. Sperm retrieval rate, changes in hormonal and are able to increase local concentrations of Material and Methods This prospective sperm parameters were reported to assess the reactive oxygen species (ROS). Recent stud- matched study was performed in 9 young potential benefit of sperm retrieval in these ies describe the assessment of the oxidation- (40). Patients were evaluated before and 6 to patients at the time of scrotal exploration. reduction potential (ORP) as feasible method 10 months after bariatric surgery. The SQ was Results Out of the 99 patients initially as- to measure simultaneously both oxidants and analyzed according to WHO (2010) using a sessed in this study, a total of 62 were eligible antioxidants. However, the association of computerized motion analysis system. Nor- to be enrolled. The mean age was 19.5 ys. A seminal plasma infections with ORP in se- mal forms and deformity index was assessed total of 20 testicular units could be salvaged

76 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

observed in cytotoxic NOA (1/8; 12.5%) and in genetic NOA (6/22; 27.3%). Only AMH serum levels was predictive of a negative TESE results in genetic NOA (figure 1-A). Using the Receiver Operating Charac- teristic (ROC), we calculated a cut-off for se- rum AMH (below 2.6 pmol/l) which allowed us to predict the negative SR with 100 % sen- sitivity (Fig. 13). Conclusions: Our results suggest that se- rum AMH could help us to predict the nega- tive results of sperm retrievals at TESE in genetic NOA, and on the other hand, plasma AMH is a good marker of Sertoli cell altera- tion’s function and it decrease is related to the origin of NOA and specially in genetic NOA which was associated with negative results of sperm retrievals at TESE. DGA-Abstracts

P 14.3 Figure 12. M. C. Craia, et al. Centriolar defects, centrin 1 alte- (32.3%). Successful sperm retrieval was Background The aim of the present study rations and FISH studies in hu- achieved in 93.5% of patients. On follow up, is to evaluate the contribution of serum Anti- man spermatozoa of a man of a 33.8% of patients developed azoospermia Müllerian hormone (AMH) in the manage- couple that produces aneuploid and a similar proportion showed OAT. ment of infertile men with azoospermia as embryos in natural and artificial well as the prediction of the outcome of sur- Conclusions Pubertal TT patients may de- fertilization velop azoospermia after surgical intervention gical sperm extraction (TESE). D. Noto1, G. Collodel1, G. Belmonte1, M. Riccio1, and should benefit from sperm retrieval and Clinical data and Materials and Methods R. Guazzo2, E. Moretti1 cryopreservation. hormonal profiles (FSH, LH, inhibin B, 1University of Siena, Molecular and Developmental AMH, and total testosterone (T) plasma lev- Medicine, Siena, Italy; 2University of Siena, Medical els ) from 134 men with azoospermia submit- P 14.2 Biotechnology, Siena, Italy ted to TESE, between 2017 and 2019 in Lille Does Anti-Müllerian hormone University Hospital, were retrospectively The large maternal contribution to embryo have a place in the management analysed. Patients were classified according aneuploidies is known and overshadows the of azoospermia? A single-center to the aetiology of azoospermia and the result clinical relevance of the result- study of 134 patients of sperm retrieval (SR) at TESE. ing in a fair consideration of paternal influ- ence to this phenomenon. H. Benderradji1,2, J. Prasivoravong2, F. Marcelli2, Results AMH plasma levels were signifi- A. L. Barbotin3, C. Marchetti4, S. Catteau-Jonard5, catively lower in unexplained, cryptorchid- We studied sperm from a man of a cou- C. Guittard6, A. Villers2, P. Pigny7, C. Leroy2 ism and genetic nonobstructive azoospermia ple that obtained aneuploid embryo with 1Lille Neuroscience & Cognition research center, (NOA) compared to obstructive azoospermia natural and assisted fertilization. After con- Inserm­ U 1172, Lille, France; 2Lille University Hospital, (OA) (medians = 28.3; 21.6; 7.2 and p = 0.04; ventional sperm evaluation, repeated three Department of Andrology and Urology, Lille, France; p = 0.002; p < 0.0001, respectively). Lowest times, immunofluorescence analysis (IF) for 3Lille University Hospital, Reproductive Biology-­ values were observed in cases of genetic NOA localization of tubulin and centrin 1 and ultra- Spermiology—CECOS Institute,, Lille, France; (p = 0.005 compared to unexplained NOA). A structural study by transmission electron mi- 4Reproductive Biology center “DU BOIS”, Lille, significant positive correlation was observed croscopy (TEM) were carried out. The sperm France; 5Lille University Hospital, Department of Re- between AMH, T and inhibin B in all sub- chromosomal aneuploidies were assessed productive Medicine, Lille, France; 6Reproductive groups of NOA. The best sperm retrieval rate by fluorescence in situ hybridization (FISH) Biology Center “DU BOIS”, Lille, France; 7Lille Uni- (SRR) was obtained in OA (100%). Among analysis for chromosomes 18 and 9. Sperm versity Hospital, CNRS, Inserm, UMR9020 – NOA subgroups, cryptorchidism had the best of a proven fertility man were used as control. UMR-S 1277, Lille, France SRR (13/22; 59.1%). The lowest SRR were Sperm concentration ranged from 20 to 35.5 × 106 sperm/ml, progressive motility from 18% to 25%, normal morphology from 3% to 4%, viability was 70%–75%. In a dis- crete percentage of cells, an irregular head- tail attachment was observed together with the presence of isolated and coiled tails. Centrin 1 was rare in sperm of the patient, whereas in sperm from control it was visible as two distinct spots in the head-tail junc- tion. The labeling with anti-tubulin antibody indicated that the 80% of patient’s sperm showed two spots in the connecting piece area, whereas control sperm displayed a sin- gle spot, indicating the proximal centriole. By TEM analysis we demonstrated that the second spot detected in patient’s sperm was the centiolar adjunct, that appeared very long, distinctive tract of human sperm immaturity. Figure 13. H. Benderradj, et al. FISH analysis revealed high frequencies of

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 77 Andrology 2020 – Abstracts

sperm aneuploidies: 18 disomy 1.5%; 9 dis- omy: 2.1%, diploidy: 1%. In conclusion, the sperm of this naturally fertile patient showed alteration in the con- necting piece, immature centriolar adjunct and scarce presence of centrin 1, concomi- tant with high frequency of aneuploidies, for this reason a paternal contribute to sperm ­aneuploidies cannot be ruled out.

P 14.4 The functionality of the sperm cell in the presence of antisperm anti- bodies A. F. Silva1, M. I. Cristo1, A. P. Sousa1,2, T. Almeida- DGA-Abstracts Santos1,2,3, S. Schlatt4, J. Ramalho-Santos1,5, S. Amaral1 1CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; 2Reproduc- tive Medicine Unit, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal; 3Faculty of Medicine, University of Coimbra, Coimbra, Portugal; 4Centre for Reproductive Medicine and Andrology (CeRA), Uni- versity of Münster, Münster, Germany; 5Department of Life Sciences, Faculty of Sciences and Technology, University of Coimbra, Coimbra, Portugal Introduction The presence of antisperm antibodies (ASA) has been described as a significant contributor to male infertility. Yet, the literature in this topic is scarce and con- troversial, being the clarification of the exact impact of ASA on sperm and male reproduc- tive function imperative. Our aim is thus to Figure 14. M. Deknuydt, et al. characterize the sperm function in ASA pa- tients, using a comprehensive and integrated Nevertheless, the exact impact of ASA in the technique (ART) between January 2015 approach. sperm function warrants further investiga- and October 2018. Semen was collected by tions and we now focusing on different ap- PVS if a neurological lesion impaired the Materials and Methods Human semen proaches to obtain new insights on the impact glans sensitivity. If PVS failed, it was com- samples were divided in 4 groups based on of these antibodies on male fertility. bined with midodrine. Midodrine only was the presence of antisperm antibodies (IgG used in all other causes of ED. A testicular and IgA; MAR test): (1) ASA > 50%, patho- sperm extraction (TESE) was performed as a logical group; (2) ASA 10%–50%; (3) ASA P 14.5 last resort. Ejaculation occurred in 58 of 70 < 10%; (4) without ASA. Several sperm patients (83%) after midodrine and/or PVS functional parameters were assessed micro- Non-invasive management of ­ejaculatory dysfunction in with a median of 2 attempts (Interquartile scopically: motility, morphology, viability Range [IQR] 1–2). The most represented (eosinY), chromatin status (Diff-Quik), acro- ­patients referred for sperm cryo- cause was spinal cord injury (SCI) with an somal integrity (PSA-FITC) and capacitation preservation: a retrospective ejaculatory rate (ER) of 87%. The ER and status (phosphorylated tyrosines); and by study the cryopreservation rate were significantly flow cytometry: mitochondrial membrane M. Deknuydt1, F. Marcelli2, B. Ducrocq3, increased when the level of lesion was above potential (JC-1) and superoxide production T10 segment. The ER was of 92% in neuro- (MitoSOX-red). A. L. Barbotin3, G. Robin2,4, M. Leroy4, C. Proust1, H. Benderradji2, J. M. Rigot2, X. Biardeau2, pathic diseases. It reached 100% in case of Results The data collected so far has J. Prasivoravong2 bladder neck incompetence or in psychogenic showed a 3% prevalence of ASA pathologi- 1Lens Hospital, Reproductive medicine, Lens, France; AN, explaining the high level of ejaculated cal patients in our center. Even with a reduced 2Lille Hospital, Urology and andrology, Lille, France; sperm cryopreserved. After a pelvic surgery number of samples in the pathological group, 3Lille Hospital, Institut of Reproductive Biology-Sper- (PS), the ER rate was of 78%. The use of a it was clear that besides some conventional miology-CECOS, Lille, France; 4Jeanne de Flandre psychotropic treatment was associated with para­meters such as viability and motility, Hospital, Reproductive medicine, Lille, France 67% of ejaculation despite midodrine. Poorer also sperm capacitation was impaired in the results of ER (20%) were obtained from pa- presence of ASA. Additionally, the sperm Background The non-invasive management tients with MN who could not cryopreserve mitochondrial function of these patients of ejaculatory dysfunction (ED) that impact sperm. Specimens from 45 of 58 patients who seem to have some dysfunctionalities. In fact, fertility is poorly documented in literature. A ejaculated (78%) were frozen. TESE allowed our preliminary data has showed a tendency sperm retrieval procedure using penile vibra- semen cryopreservation in 7/12 (58%) more towards a higher mitochondrial superoxide tory stimulation (PVS) and/or midodrine was patients. 52 patients have frozen sperm with production in ASA patients and more data is studied in patients with ED causing infertility a median of 8 samples (IQR 0–13). Ten preg- being collected to allow for more definitive whatever the cause. nancies occurred. The pregnancy rate (PR) conclusions. Results Data about sperm retrieval were per cycle and per embryo transfer were of Conclusions Overall, we have obtained rel- retrospectively collected for 70 patients 28% and 23% respectively. evant and new information regarding the ef- with ED referred to Andrology Department Conclusion The non-invasive management fects of ASA on sperm function, identifying of Lille University Hospital for fertility of ED using PVS and/or midodrine was as- new compromised aspects in these patients. preservation or before assisted reproductive sociated with an ER of 83% (Fig. 14).

78 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

P 14.6 4Monash University Parkville, Monash Institute of male infertility cases are caused by genetic Pharmaceutical Sciences, Drug Discovery Biology,, abnormalities such as chromosomal defects Effect of vas ligation on TESE Melbourne, Australia or gene mutations, leading to decreased germ ­results in non obstructive azoo­ cell production or function. Somehow, only The adult rat epididymis can be segmented spermic rats a few clinically relevant gene mutations/ into 19 segments with S19 being the most polymorphisms or novel genes have been O. Shaeer, M. Ghaly, E. Ibrahim, distal part where the sperm is stored. Dur- identified so far. This study aims to identify M. Abdelmotaleb, K. Shaeer ing ejaculation the sperm is driven forward novel genes related to sperm morphology and Kasr El Aini Faculty of Medicine, Cairo University, through the vas deferens by strong contrac- motility by next generation sequencing. Giza, Egypt tions of the cauda epididymis. Patients with benign prostatic hyperplasia (BPH) medicat- Testicular biopsies from azoospermic men Introduction To this date, there is neither ed with alpha-1 receptor adrenergic blockers were classified as normal spermatogenesis a method to predict the results of sperm often suffer from ejaculatory disorders. (NSP, n=3), spermatid arrest (SDA, n = 4) retrieval in patients with non-obstructive and Sertoli cell only syndrome (SCO, n = 3) We investigated the contractile response to azoospermia, nor methods to enhance its re- by histology. RNA was extracted from ad- oxytocin (OT) (500 nM) and norepinephrine sults, with the exception of cases with hypo- jacent cryopreserved biopsies followed by (NA) (10 µM) in multiple segments of the gonadotropic hypogonadism and large grade RNA-Seq (Illumina NextSeq 500 sequencer). varicoceles. This work evaluates a proposed rat epididymis by using live-imaging with special focus on the last two segments (S18 Differentially expressed genes (DEGs, fil-

method for enhancing sperm accumulation DGA-Abstracts (not sperm production), by ligation of the vas and S19). The response of S19 to three dif- ter for significance: base mean≥ 5, –0.585 deferens, in an effort to enhance TESE results ferent doses of OT (1 nM, 10 nM, 100 nM) ≤ log2fc ≥ 0.585, FDR ≤ 0.05) were NSP in NOA rats by sperm accumulation. was tested. The effects of two OT-antagonists Vs SCO–10,253, NSP Vs SDA–1,873, SDA (atosiban and cligosiban), an arginine vaso- Vs SCO–4,017 and filtered based on Gene Materials and Methods Sixty mature male pressin antagonist (SR 49059), an alpha-1 Ontology and KEGG pathway terms related rats with equally sized testes were included in blocker (tamsulosin) and a PDE5 inhibitor to “spermatogenesis”, “calcium pathway”, this study. 50 were in the study group and 10 (tadalafil) on S19 were tested separately. “cAMP pathway”, “flagella proteins” and were in the control group. Bilateral testicular highly down-regulated DEGs between NSP Both OT and NA increased contractions biopsies were performed for the 60 rats to Vs SDA. From which, 11 genes potentially throughout the rat epididymis. In S19 both confirm equal spermatogenesis in all testes. involved in sperm morphology and motility OT (dose-dependently) and NA were able Non-obstructive azoopermia was induced have been identified: CFAP47, PDE4A, ZP1, to induce a very strong and complex series using DNG (40 mg/kg body weight [bw]) + SLC9C1, SLC8B1, SPATA31E1, ORAI1, of contractions which could not be found in TU (25 mg/kg bw) every 30 days for three CACNB2, TNC, TEKT3, and TMEM37. any other segment investigated. Atosiban or months. Monthly FNA’s confirmed NOA Genes were validated using RT-PCR and ini- cligosiban were able to completely block from all rats. All rats became azoospermic tial immunohistochemistry (IHC) for ORAI1 the strong reaction of S19 to OT (500 nM) after three months. Rats of the study group and SPATA31E1 were performed. Further ex- while SR 49059 was only able to block it par- were divided into 2 subgruops. In the first periments for IHC and quantitative RT-PCR tially. Tamsulosin could completely block the group, ligation of the right vas was performed using different pathologies will narrow down strong reaction of S19 to NA (10 µM) while (24 rats). In the second group, ligation of the the list of potential genes involved in defec- tadalafil could not. OT was then still able to left vas was performed (24 rats). Two rats tive male germ cell differentiation and to induce that strong reaction in the adrenergi- died afterwards, one from each group. On reveal its biological significance. Additional cally blocked S19. the other hand control group rats were not li- experiments utilizing high-resolution quanti- gated. After a period of 90 days (2 spermato- In S19 of the rat epididymis OT had a simi- tative mass spectrometry (Orbitrap Eclipse) genic cycles), TESE was performed for the lar strong effect to NA. OT was still able to will be performed using ejaculates showing 56 rats in the study group (46 rats) and rats contract tissue important for the ejaculatory either normal or disturbed motility/morpho­ in the control group (10 rats). The hormonal process which had been unresponsive to NA. logy. injection proceeded till the final TESE and This could indicate that OT-based medica- Grants Supported by DFG GRK 1871/1 scarification of all rats in all groups. tion might be a great treatment option for Results On day 90 TESE was performed ejaculatory disorders, especially in cases of for all rats. Sperm retrieval was compared pretreatment with alpha-1 blockers such as P 14.9 between different groups. Number of sperms tamsulosin which is common in patients with and round spermatid in biopsies were meas- BPH. S19 might be a great model to test new Genetics and spermatology in ured. Theses numbers were significantly medication for the treatment of ejaculatory CBAVD patients disorders and BPH. higher in the ligated side when compared to V. Chernykh, E. Marnat, A. Sedova, M. Shtaut, the other side or to the control group. E. Bragina, T. Sorokina, T. Adyan, A. Polyakov, Conclusion Ligation of the vas deferens in P 14.8 L. Kurilo rats with NOA could enhance the results of Research Centre for Medical Genetics, Laboratory Sperm morphology and motility sperm retrieval. of Genetics of Reproduction Disorders, Moscow, in mice and men: Investigations Russian Federation of defective male germ cell diffe­ P 14.7 Materials and Methods A group of 73 un- rentiation related Russian men of reproductive age with Oxytocin as a new treatment S. Kothalawala1, S. Günther2, T. Timm3, G. Lochnit3, CBAVD syndrome. The average age was 31.1 ­option for ejaculatory disorders – H. C. Schuppe4, A. Pilatz4, F. Wagenlehner4, D. Fietz1 ± 7.1 years. The examined men did not have S19 of the rat epididymis as a 1Justus-Liebig University of Giessen, Institute for any karyotype abnormalities or clinically sig- ­testing model ­Veterinary Anatomy, Histology and Embryology, nificant microdeletions of the long arm of the Giessen, Germany; 2Max Planck Institute, Bad Y chromosome. B. Stadler1,2, M. R. Whittaker3, C. J. Nowell4, Nauheim,­ Germany; 3Justus-Liebig University of B. Exintaris2, R. Middendorff1 Results The sample of men was divided Giessen, Protein Analytics, Biochemistry Institute, 1Justus-Liebig-University, Institute of Anatomy and into 2 groups: with mutations in the CFTR Giessen, Germany; 4Justus-Liebig University of Cell Biology, Giessen, Germany; 2Monash University gene and without disorders in this gene. A ­Giessen, Clinic of Urology, Pediatric Urology and Parkville, Faculty of Pharmacy and Pharmaceutical comparative analysis of hematological pa- ­Andrology, Giessen, Germany Sciences, Drug Discovery Biology, Melbourne, Aus- rameters between these two groups was per- tralia; 3Monash University Parkville, Monash Institu- Male infertility can be caused by disturbanc- formed. te of Pharmaceutical Sciences, Drug Delivery, Dyna- es of sperm count, morphology and motility The volume of ejaculate samples studied mics and Disposition, Melbourne, Australia; amongst other reasons. About 30–40% of in groups I and II varied from 0.2 to 2.0 ml

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 79 Andrology 2020 – Abstracts

and 0.3 to 1.5 ml, with an average of 0.73 ± 0.42 ml and 0.70 ± 0.31 ml, respectively. Oligospermia was observed in group I in 56 (94.9%) patients, in group II – in 12 (92.3%) patients. The viscosity of the ejaculate samples sig- nificantly varied from normal (1–20 mm) to elevated values (up to 100 mm), and averaged 12.3 mm for the group of patients with CFTR gene mutations and 25.6 mm for the group of patients without CFTR gene mutations, respectively. The acidity of the ejaculate varied from acidic to alkaline (pH from 5.5–7.7). In the first group, only one male (who had the mutCFTR/5T genotype) had a normal pH value. In the second group of patients, the DGA-Abstracts pH value was lower than normal and did not exceed 6.9. The concentration of white blood cells in the ejaculate was within the reference values Figure 15. W. Vena, et al. (not exceeding 1 million / ml), exceeding this indicator (leukospermia) was observed in 5 taria Careggi, Andrology, Women’s Endocrinology Conclusions This analysis shows that patients (6.76%). and Gender Inconguence Unit, Department of Experi- cTESE/mTESE in subjects with NOA results Conclusion In the studied sample of men, mental and Clinical Biomedical Sciences, Florence, in SRRs of up to 50%, with no differences 12 spermatological signs of VAS deferens ob- Italy; F. Renzetti Hospital, Diabetes and Metabo- when cTESE was compared to mTESE. Re- struction and aplasia/hypoplasia of seminal lism Unit, Department of Internal Medicine,, Lancia- trieved sperms resulted in a LR of up to 28% 13 vesicles were found, which are characteristic no (CH), Italy; SLK-Kliniken Heilbronn, University of ICSI cycle. Although no difference between of patients with CBAVD syndrome and more Heidelberg, Department of Urology, Heilbronn, techniques was found, to conclusively clari- 14 than 90% of men with CF: azoo-/crypto- Germany;­ Justus-Liebig-University of Giessen, fy if one technique is superior to the other, zoospermia, oligospermia, and a reduced ­Department of Urology, Pediatric Urology and Andro- there is a need for a sufficiently powered and 15 pH. There were no significant differences in logy, Giessen, Germany; Ioannina University School well-designed randomized controlled trial to spermogram parameters between groups of of Medicine, Department of Urology, Ioannina, compare mTESE to cTESE in men with NOA patients with mutations and/or the 5T allele Greece­ (Fig. 15). of the CFTR gene and without them. Introduction Factors affecting sperm re- trieval rate (SRR) or pregnancy rates (PR) P 14.11 P 14.10 after testicular sperm extraction (TESE) in Factors associated with abnormal patients with non-obstructive azoospermia Sperm recovery and ICSI out­ (NOA) have not been systematically evalu- epididymo-testicular unit in a comes in men with non-obstruc­ ated. In addition, although micro-TESE ­cohort of French boys operated tive azoospermia: a systematic (mTESE) has been advocated as the gold for unilateral cryptorchidism standard for sperm retrieval in men with ­review and meta-analysis S. Hamdi1, V. Eng1, C. Brusq1, R. Mieusset1, O. Abbo2 NOA, its superiority over conventional TESE 1 2 3 4 1Universitary Hospital of Toulouse/Toulouse Univer- W. Vena , G. Corona , S. Minhas , A. Giwercman , (cTESE) remains conflicting. C. Bettocchi5, M. Dinkelman-Smit6, G. Dohle6, sity, Research Group in Human Fertility, Toulouse, F. Fusco7, A. Kadioglu8, S. Kliesch9, Z. Kopa10, Materials and Methods An extensive Med- France; 2Universitary Hospital of Toulouse, Pediatric C. Krausz11, F. Pelliccione12, A. Pizzocaro1, line, Embase and Cochrane search was per- Surgery, Toulouse, France 13 5 11 14 formed. All trials reporting SRR derived from J. Rassweiler , P. Verze , L. Vignozzi , W. Weidner , Introduction Abnormalities of the epidi­ 11 15 cTESE or mTESE in patients with NOA and M. Maggi , N. Sofikitis dymo-testicular unit (ETU) are frequently 1 their specific determinants were included. Humanitas Research Hospital, Endocrinology & reported in cryptorchidism. The goal of this ­Medical Andrology Unit, Rozzamno (MI), Italy; Results 117 studies met the inclusion cri- 2 study was to identify the main factors associ- Azienda ASL Bologna Maggiore – Bellaria Hospital, teria for this study, enrolling 21,404 patients ated with these abnormalities in order to gain Endocrinology Uniti, Medical Department, Bologna, with a mean age (± SD) of 35.0 ± 2.7 years. more insight into their pathogenesis. Italy; 3Imperial College NHS Healthcare, Department cTESE and mTESE were used in 56 and 43 of Urology, London, United Kingdom; 4Lund Uni­ studies, respectively whereas 10 studies used Patients and Methods A retrospective re- versity, Molecular Reproductive Medicine, Depart- a mixed approach and 8 compared cTESE view of medical files of 396 boys undergo- ment of Translational Medicine, Faculty of Medicine, with mTESE. Overall, a SRR per procedure ing orchiopexy for unilateral nonsyndromic Malmo, Sweden; 5University of Bari, Department of of 47 % was found. No differences were ob- cryptorchidism was conducted. ETU was Urology, Andrology and Kidney Transplantation Unit, served when mTESE was compared to cTESE classified as normal or abnormal (for both Department of Emergency and Organ Transplanta- 46% for cTESE versus 46% for mTESE. complete and incomplete dissociation). Ad- tion, Bari, Italy; 6University Medical Center Rotter- Meta-regression demonstrated that SRR per justed odds ratio (aOR) with 95% confident dam, Department of Urology, Erasmus MC, Rotter- cycle was independent of age and hormonal intervals (95%CI) were calculated using mul- dam, Netherlands; 7University of Naples Federico II, parameters at enrolment. However, the SRR tivariate logistic regression. Department of Neurosciences, Human Reproduction increased as a function of testis volume (TV). Results Abnormal ETU was reported in and Odontostomatology, Naples, Italy; 8University of In particular, by applying ROC curve analy- 121 boys (30.6%). They were younger than Istanbul, Department of Urology, Istanbul Faculty of sis, a mean TV higher than 12.5 ml predicted those with normal ETU (2.7 vs 4.8 year, Medicine,, Istanbul, Turkey; 9Münster University SRR > 60% with an accuracy of 86.2%. A p < 0.001). Age was associated with a lower Hospital (UKM), Department of Clinical and Surgical total of 1096 biochemical pregnancies were probability of abnormal ETU (aOR 0.81; 95- Andrology, Centre of Reproductive Medicine and reported (cumulative PR = 29% per ICSI cy- %-CI 0.73–0.90). Conversely, small testis ­Andrology (CeRA), Münster, Germany; 10Semmelweis cle. No influence of male and female age, and short spermatic cord length were associ- University, Andrology Centre, Department of Urology, mean TV or hormonal parameters on both PR ated with a high probability of abnormal ETU Budapest, Hungary; 11Azienda Ospedaliera Universi- and LBR per ICSI cycle was observed. (aOR 5.40, 95-%-CI 3.19–9.25 and aOR

80 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

4.69, 95-%-CI 1.63–15.48, respectively). benefit of ATB treatment on the sperm qual- Thus, the study of sperm morphology is a Open peritoneo-vaginal duct was associated ity. However, we suppose that proportion of fundamental element in the seminal analy- with a lower probability of abnormal ETU apoptotic sperm, as well as leucocyte count sis, but its real meaning has been biased by (aOR 2.62, 95-%-CI 1.44–4.93). However, and antisperm-antibodies might decrease – the techniques used for its evaluation. These testis position (external inguinal ring, ingui- that will be studied in the next phase of our classical techniques involve dehydration nal canal or abdominal) was not associated study. and subsequent staining, which involves the with a probability of abnormal ETU. production of artifacts. The objective of the present work was to compare two methods Conclusion Our study pointed out some P 15.1 factors associated with abnormal ETU in for equid semen morphology evaluation. The ® unilateral cryptorchidism and shed light on Sperm motility subpopulation first one is the new technique Trumorph involved mechanisms. Further research is structure in stallions: breed dif­ analyzing the spermatozoa suspended in their needed to confirm these results. ferences seminal fluid and the second is eiosine-ne- grosin staining technique. For that purpose, S. Gacem1, A. Velverde2, J. Catalán1, J. Miró1, a total of 49 ejaculates from 20 stallions and P 14.12 C. Soler1,3 29 donkeys were used. Semen samples were 1Universitat Autonoma of Barcelona, Equine repro- obtained with artificial vagina. After semen Asymptomatic urogenital infec­ duction, Sabadell, Spain; 2Costa Rica Institute of collection and dilution, an aliquot was placed tions and their impact on male Technology, School of Agronomy, San Carlos Cam-

on the slide and covered with cover-slide that DGA-Abstracts and female fertility pus, Alajuela, Costa Rica; 3Departamento de Biología was placed in the Trumorph® device. The Celular, Biología Funcional y Antropología Física, observation was made with a 40× negative J. Novák1,2, T. Fürst3, A. Langerová4, V. Vik2,5, Universitat de Valencia, Valencia, Spain phase contrast objective. Another aliquot was Z. Krátká6 stained using eiosine -negrosine stain, and an- 1General University Hospital and First Faculty of The introduction of computer-assisted semen alysed using 100× bright field objective. The ­Medicine, Charles University, Prague, Dpt. of Uro­ analysis (CASA-Mot) systems allowed for general morphology was very similar in both logy, Praha, Czech Republic; 2Gennet, Urology and an unprecedented degree of sophistication in species showing no significant differences in Andrology, Praha, Czech Republic; 3Faculty of the study of sperm kinematics patterns. The the total amount of abnormalities compar- ­Science, Palacky University, Dpt. of Mathematical aim of this study was to analyze kinematic ing both techniques. Nevertheless, the use Analysis and Application of Mathematics, Olomouc, characteristics of two horse breeds and to cal- of Trumorph® technique rendered a perfect Czech Republic; 4Gennet, IVF, Praha, Czech Republic; culate the subpopulation structure based on differentiation of head (both acrosome and 5Institute of Clinical and Experimental Medicine, these parameters. A total of 42 fresh semen post-acrosome areas), midpiece and principal Transplant Surgery Dpt., Praha, Czech Republic; ejaculates (26 of Spanish breed and 16 Ara- piece of the spermatozoa allowing to a better 6Gennet, Laboratory of Immunology, Praha, Czech bian breed) were collected by artificial vagina determination of morpho-abnormalities. So, Republic and subsequently analyzed for kinematic pa- we can conclude that Trumorph® could be a rameters using the ISAS®v1.2 CASA-Mot good and simple alternative for morphology This research focuses on asymptomatic uro- system and a Spermtrack® 10 µm depth re- analysis that use alive sperms avoiding arti- genital infections (Ureaplasma urealyticum, usable counting chamber. Eight kinematic fact production. Mycoplasma hominis, Mycoplasma genitali- parameters were evaluated automatically at um, Chlamydia trachomatis) as a potential 250 frames per second. All kinematic param- cause of infertility. These infections might eters showed significant differences among P 15.3 have a negative impact on fertility by activat- breeds. Spanish horse had higher VAP, VCL ing pro-inflammatory immune responses in and BCF compare to Arabian horse. Spanish Topological distribution of func­ both males and females. It is assumed that horse sperm was faster but Arab horse was tional-biochemical features of antibiotic treatment (ATB) suppresses the im- more linear. Principal component (CP) analy- mune reaction, and thus increases the chance spermatozoa by Raman Micro­ sis was done as the first step of subpopulation spectroscopy of conception. On the other hand, ATB might analysis, rendering three CP named veloc- have a negative impact on the sperm quality. ity, linearity and oscillation. Subpopulation V. Zieger1, R. Da Costa1, S. Amaral1,2,3, S. Schlatt1 Aim of the study To assess how urogenital analysis showed three, being the dominant 1University of Münster, Centre for Reproductive infections and its treatment with ATB affect subpopulation for stallion that composed by ­Medicine and Andrology, Münster, Germany; 2Uni- the quality of sperm. Five hundred males and fast, straight and lineal motility with a high versity of Coimbra, Center for Neuroscience and Cell their female partners from infertile couples beat spermatozoa (41.7%). Slight differences Biology (CNC), Biology of Reproduction and Stem were examined. Pathogens were diagnosed in in the distribution of these three subpopula- Cell Group, Coimbra, Portugal; 3University of urine samples using cultivation and PCR. In tions were observed between Arabian and ­Coimbra, Institute for Interdisciplinary Research, 243 couples (48.6%) at least one of the four Spanish horses. In conclusion, higher frame Coimbra,­ Portugal infections was detected. All infected couples rate of video capturing permitted to have new Introduction Understanding the complex were treated with Doxycycline 100 mg bid interpretation of the sperm kinematic sub- biochemistry of spermatozoa is critical to for 10 days. population structure in stallion. explore their pathophysiology. Since this The couples were divided into 4 groups ac- complexity is ignored by conventional sperm cording to the presence/absence of any infec- P 15.2 analysis, alternative approaches have to be tion in the male/female partner. Interestingly, considered and implemented. In this regard, no significant difference in the quality of Equine sperm morphology analy­ Raman Microspectroscopy represents an in- sperm was found among the groups. In 235 sis: alive vs dead stained sperms novative tool for the assessment of biochemi- men (133 without infection and 102 after techniques cal features otherwise undetectable. Previ- ATB treatment), a follow-up sperm analysis ously, using this technique, different sperm G. Sabrina1, J. Catalán1, I. Yanez-Ortiz1, C. Soler1,2, was performed 29–149 days later (median 78 regions head, midpiece and tail, were eas- J. Miró1 days). Lower sperm concentration was found ily disentangled based on their biochemical 1Universitat Autonoma of Barcelona, Equine repro- in the group after ATB treatment (at the bor- components and several organelle-related duction, Sabadell, Spain; 2Departamento de Biología der of significance, p = 0.08). No other sig- bands were identified. These novel readouts Celular, Biología Funcional y Antropología Física, nificant difference was found. are of value for clinical settings especially if Universitat de Valencia, Valencia, Spain Conclusion At least one asymptomatic uro- their specificity could be confirmed within genital infection is present in almost half of The evaluation of the male fertility potential the sperm structures. Thus, this study was infertile couples. In women, it was previously is based on the analysis of the basic spermatic carried-out to disclose the topological distri- found that ATB improves the chance of con- characteristics (concentration, motility and butions of Raman organelle-specific features ception. In this study, we have not found any morphology), as well as the seminal fluid. of sperm.

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 81 Andrology 2020 – Abstracts

Materials and Methods Sperm suspen- Conclusion Embedded into an easy-to- P 15.6 sions from normozoospermic donors were operate automatic setup configuration, we smeared onto Suprasil slides. Raman multi- envisage the common use of “Rapid-Mixing- Effect of incubation and analysis spectral mappings with signals of 14 spectral CASA” to elucidate how human and other temperature on kinematic and traits were recorded in all spermatozoal com- mammalian sperm translate changes in their morphometric human semen va­ partments. local chemical microenvironment into chang- lues Results The average spectrum of the sperm es in their swimming behavior. A. García Molina1, N. Navarro2, D. Bompart1, head is characterized by bands ranging from C. Cerveró3, S. Sadeghi1, C. Soler1,3 600 to 1150 cm-1, mainly associated with nu- P 15.5 1Proyectes i Serveis R+D, Biology, Valencia, Spain; cleic acids. Five bands at 1094, 1180, 1373, 2Instituto de Infertilidad Valenciano (IVI), Andrology, 1483, and 1509 cm-1 showed to be specific to Assessment of DNA fragmenta­ Valencia, Spain;3University of Valencia, Department the post-acrosomal area of the nucleus. In the tion in live spermatozoa: a novel of Functional Biology and Physical Anthropology, midpiece, the spectra are dominated by bands and rapid application of flow Valencia,­ Spain from 1150 to 1700 cm-1, which are related ­cytometry for sperm analysis to mitochondrial components. One band as- Introduction Human semen analysis sociated with cytochrome-C at 1546 cm-1 R. Da Costa, K. Redmann, S. Schlatt should begin a simple inspection soon after showed high specificity for this area. The Centre of Reproductive Medicine and Andrology, liquefaction, preferably at 30 minutes, but

DGA-Abstracts signals regarding the sperm tail, due to the Münster, Germany no longer than 60 minutes after ejaculation. structural aspects, tend to be too weak to be Introduction Sperm DNA integrity has be- During liquefaction, the sample can conserve deeply evaluated. come one of the most discussed and prom- either in room temperature (RT 23 °C) or at Conclusions Raman bands associated with ising biomarkers for the assessment of male 37 °C. The incubation temperatures and its different functional elements were consist- fertility. However, an easy-to-apply method impact on sperm motility are crucial but nev- ently distributed along specific sperm regions capable of evaluating DNA fragmentation er considered. This study aims to examine the and represent potential markers of functional- in the live fraction of spermatozoa, which is effect of different incubation temperatures ity related to DNA integrity and mitochondri- the one used and wanted for assisted repro- on sperm parameters in both qualitative and al activity. Raman analysis has the potential to ductive technologies (ART), has remained quantitative analysis. Likewise kinematics be considered as a new strategy for biochemi- elusive, preventing this parameter from being and morphometrics which refers to the quan- cal sperm assessment in infertile patients. fully applied in clinical settings. Herein we titative analysis of form. propose a novel, rapid and easy to perform Material and Methods Seminal samples co-staining for the analysis of DNA fragmen- from thirteen donors were incubated semen P 15.4 tation in membrane-intact spermatozoa. samples of were obtained by masturbation Development of a “Rapid-Mixing- Materials and Methods Normozoospermic after 2–4 ejaculatory abstinence days and in- cubated for 30 min at 23 °C (RT) and at 37 °C CASA” technique to quantify semen samples from donors (n = 10), were used to validate a co-staining consisting of and prepared following WHO 2010 criteria. ­transient ligand-induced motility Morphometric and kinematic parameters Acridine Orange (AO) and LIVE/DEAD ® responses in sperm populations Stain (LD), against established methods for evaluated using an ISAS v1 CASA-Morph S. Young, C. Brenker, T. Strünker, C. Schiffer the evaluation of cell viability, propidium io- and CASA-Mot systems, respectively. University Hospital Münster, Centre of Reproductive dide stain (PI), and DNA fragmentation, the Results The obtained data show that there Medicine and Andrology, Molecular Reproductive Sperm Chromatin Structure Assay (SCSA), were no significant differences (P > 0.05) on Physiology, Münster, Germany to rule out cross-interference. Furthermore, the subjective sperm quality parameters. On the accuracy of the method was tested by the the other hand, the sperm head morphomet- For more than 30 years, com- Introduction evaluation of samples prepared with different ric parameters were significantly higher after puter-aided sperm analysis (CASA) has been amounts of membrane and DNA damage. room temperature incubation; additionally, used to quantify kinematics of sperm in both with lower ellipticity (P < 0.05). Furthermore, research and clinical environments. Replac- Results No significant differences were observed between the co-staining and the kinematic parameters were evaluated at two ing the subjective human eye with objective different temperatures (23 °C and 37 °C) dur- computation, CASA has provided unique established staining procedures (membrane integrity, P = 0.755; DNA fragmentation ing the incubation time and analysis process. insights into sperm physiology and the basics Mainly, the four temperature combinations of reproduction. However, in vivo, the motil- P = 0.976). Moreover, high R square values were obtained from the analysis of samples showed that the results of kinematic param- ity of sperm is modulated by chemical cues eters followed this order according to the provided by the female to assist sperm in nav- of known membrane (R2 = 0.9959) and DNA damage (R2 = 0.9843). Combining simulta- incubation and analysis temperatures 23°- igating across the oviduct and fertilizing the 23 °C < 23°–37 °C < 37°–37 °C < 37°– 23°C. egg. In the past decade, a picture has emerged neous assaying for sperm membrane integrity that these chemosensory motility responses and nuclear DNA fragmentation allowed the Conclusion Our data demonstrate the op- are both rapid and transient. Due to their low analysis of four relevant sperm categories timal correlation in kinematics parameters time resolution, classical CASA approaches and assess the proportion of membrane-intact between the incubation and analysis tem- are ill-suited to study the ligand-control spermatozoa with compromised DNA integ- perature is 37 °C and 23 °C, respectively. The of sperm motility. We developed a kinetic, rity. temperature control during both incubation “Rapid-Mixing CASA” technique to study, in Conclusions This novel co-staining showed and analysis is needed for accurate semen a quantitative fashion, ligand-evoked motility no cross-interference between its elements, analysis. responses in human sperm. providing sensitive, accurate, and reliable Materials and Methods We combined the results. This new protocol has the potential to P 15.7 well-established CASA approach with an ad- provide clinically relevant information about vanced microfluidics setup. After contacting the DNA fragmentation in membrane-intact Atlas of human sperm morpho­ sperm with a chemical stimulus by rapid mix- spermatozoa, improving the diagnostic of anomalies using Trumorph® male infertility and enabling a better under- ing, a virtually instantaneous onset of micro- A. García Molina1, N. Navarro2, E. Nácher3, standing of sperm dysfunction. scopic data acquisition for motility analysis D. Bompart1, S. Sadeghi1, C. Soler1,3 is enabled. 1Proyectes i Serveis R+D, Biology, Valencia, Spain; Results We used this “Rapid-Mixing 2Instituto de Infertilidad Valenciano (IVI), Andrology, CASA” setup to characterize motility re- Valencia, Spain; 3University of Valencia, Department sponses evoked by chemical stimuli in human of Functional Biology and Physical Anthropology, sperm. Valencia,­ Spain

82 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

Introduction The morphology of the sper- The aim of the present work was to define the Results We showed that esterification of the matozoa is defined along the process of sperm optimal FR, considering the curvilinear ve- carboxylic acid dramatically reduces the po- formation in the testicle (spermatogenesis and locity (VCL) as the most sensible parameter. tency of PGE1 to activate CatSper. As a proof spermiogenesis), epididymal transit matura- Material and Methods Semen samples of of concept, two photolabile caged-PGE1 tion and mixing with accessory glands secre- 12 adult volunteers were obtained by mastur- derivatives were synthesized and character- tions during ejaculation. The semen analysis bation after 2–4 ejaculatory abstinence days. ized. Next, other photolabile groups will be is used to assess the fertile potential of the For the study, a reusable counting chamber attached to PGE1 to optimize the solubility, male, based on the concentration, motility, Spermtrack® of 10 µm depth was used in quantum yield, uncaging kinetics and resist- and morphology of the spermatozoa. Most of combination with an ISAS®v1 CASA-Mot ance to hydrolysis. them, including that recommended by WHO system. Image sequences were recorded at Conclusion Our results show the feasibil- 2010, implies different fixation and staining 500 frames per second using a digital cam- ity of the approach to synthesize an effec- processes that involve the production of mor- tive caged PGE1 to investigate prostaglandin ® era able to capture at 500 fps. These original phology artifacts. The Trumorph is based ­videos were fragmented at different FRs (25, activation of CatSper and ensuing motility on the examination of in vivo preparations 50, 100, 150, 200, and 250) to define the as- responses. of a fresh semen drop of 3 µl between slide ymptotic value of the exponential curve that and a conventional coverslip, after increasing defines the behavior of VCL relative to the the temperature to 45 ºC in just 10 seconds FR. P 15.10 and subjected to a pressure of 5 kP. To ob- Novel perspective in human DGA-Abstracts tain a homogeneous distribution of the drop Results The a value of the exponential is achieved in the whole area of the cover, curve was 150,23 fps, corresponding to a ­spermatozoa RNA content assess­ with a height of approximately 6μm, which VCL of 130.58 m/s, long from the value of ment and morphology evaluation “forces” the cells to arrange themselves ac- 98.89 m/s corresponding to 50 fps (the high- with in vivo acridine orange cording to their maximum projection. est FR used by most of the present CASA- Mot systems). ­staining Material and Methods Samples from 30 1,2 2,3 4 2,5 Conclusion As a conclusion, almost 150 T. Maric´ , A. Fucˇic´ , V. Vicˇic´ Bocˇkor , D. Ježek voluntary donors were obtained by mastur- 1 fps must be used for capture and analysis of School of Medicine, Department of Medical Biology, bation after 2–4 ejaculatory abstinence days. Zagreb, Croatia; 2Scientific Centre of Exellence for After liquefaction, the Trumorph® device was human sperm kinematic studies to have con- fidence on the results. Reproductive and Regenerative Medicine, University used to prepare the sample. The observations of Zagreb School of Medicine, Zagreb, Croatia; were made with a 40x objective PHN and 3Institute for Medical Research and Occupational images were captured and recorded using an Health, Zagreb, Croatia; 4Faculty of Science, Uni­ ® P 15.9 ISAS v1 CASA system. versity of Zagreb, Division of Molecular Biology, Results The most representative images Synthesis of caged prostaglandin Zagreb,­ Croatia; 5University of Zagreb School of of each type of anomaly were classified ac- E1 derivatives to study the ligand- ­Medicine, Department of Histology and Embryology, cording to head, midpiece or neck, and tail control of CatSper Ca2+-channels Zagreb, Croatia defects. Since each sperm has these three 1,2 2 1 components, in each image can be occur T. Schierling , T. Strünker , B. Wünsch Introduction Transgenerational effect car- 1 combination of various morphoanomalies. University of Münster, Institute for Pharmaceutical ried by spermatozoa that fertilizes egg can 2 For this reason, the images are accompanied and Medicinal Chemistry, Münster, Germany; Uni- be attributed not only to DNA sequence by the corresponding explanatory tables, versity of Münster, Centre of Reproductive Medicine transmitted to offspring but also to epigenetic where the morphoanomalies to be indicated. and Andrology, Münster, Germany characteristics of the sperm. Even though In the other hand, other non-spermatic cell spermatozoa are considered transcriptionally Introduction The sperm-specific CatSper silenced, various types of RNA are actively types were evaluated. 2+ channel controls the intracellular Ca - coordinated in different compartments inside Conclusion Elaboration of the atlas of concentration and, thereby, the motility of the cell. Aim of our study was to evaluate morpho-abnormalities sperm using the new human sperm. Human CatSper is activated morphology, sperm DNA damage and local- ® technique Trumorph following, classifica- by rise in intracellular pH, depolarization of ize RNA in the sperm cell with In vivo acri- tion WHO 2010. the membrane, and by a range of small mol- dine orange (AO) staining that detects sperm ecules including steroids and prostaglandins. DNA damage and DNA/RNA content. P 15.8 To elucidate the mechanism underlying pros- taglandin activation of CatSper and to study Methods Ten men suffering from oligo­ Optimal capture frame rate for the ensuing motility responses, we planned asthenoteratozoospermia (OAT) syndrome ­kinematic human sperm analysis to synthesize a caged PGE1 derivative. Using and 10 normozoospermic fertile men were analyzed using in vivo AO staining. After using CASA-Mot systems caged-PGE1 as a tool, we want to quantify the kinetics of CatSper activation and the liquefaction, 10 µl of sperm at concentration A. García Molina1, D. Bompart1, N. Navarro2, swimming path of sperm while navigating 5 million/ml was dropped on slide pretreated R. Esteve3, S. Sadeghi1, C. Soler1,3 in a light-sculptured prostaglandin gradient. with AO stain. Sample was gently covered 1Proyectes i Serveis R+D, Biology, Valencia, Spain; PGE1 harbors several O-containing func- with cover slip and within 2 hours analyzed. 2Instituto de Infertilidad Valenciano (IVI), Andrology, tional groups, i.e. a car­boxylic acid, an allyl Microscopic analysis was performed by fluo- 3 Valencia, Spain; University of Valencia, Department alcohol, and a β-hydroxy ketone for chemical rescent and confocal fluorescent microscopy. of Functional Biology and Physical Anthropology, modification. Results Fluorescent microscopy imaging Valencia,­ Spain Methods Both alcohols are chemically showed classic sperm morphology and red, Introduction Conventional semen analysis unstable and, thus, difficult to functional- orange or green sperm staining with red in- suffers for lack of precision and repetitive- ize. Therefore, we identified and employed dicating damaged and green intact genome. ness, reducing its impact in predicting male a general procedure for esterification of the Confocal imaging showed clear contrast fertility. The introduction of computer as- carboxylic acid using aliphatic and benzylic between red and green staining on the level sisted semen analysis (CASA-Mot) systems halides and base. The residual activity of the of single sperm. With red coloring, we could precisely localize RNA in residual cytoplasm for the evaluation of sperm motility and kin- caged-PGE1 derivatives was characterized ematics has overpassed these problems. Nev- by kinetic (Ca2+) fluorimetry, using sperm in damaged and intact sperm. Furthermore, ertheless, former technological limitations loaded with the fluorescent Ca2+-indicator detailed morphological characteristics of the have restricted the generalization of the use Fluo-4 and a fluorescence plate reader. The spermatozoa could be determined. of CASA-Mot both for research and clini- photocleavability of photolabile groups was Conclusion In vivo AO staining of sperma- cal purposes. One of these limitations is the investigated by exposing the caged-PGE1 to tozoa and imaging with confocal microscopy frame rate (FR) at which images are captured. UV irradiation. provided precise insight into level of DNA

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 83 Andrology 2020 – Abstracts

damage, localization of RNA in the sperm served, which leaded us to hypothesize that P 15.13 and detailed morphology within single sperm. those circRNAs could co-exist with their cir- RAC1 controls progressive move­ This study was supported by the cRNA turnover machinery. Grants ment and competitiveness of Scientific Center of Excellence for Re- Grants Supported by the Ministry of Econ- productive and Regenerative Medicine, omy and Competitiveness PI16/00346 to RO. mammalian spermatozoa Republic of Croatia, and by the European A. Amaral1, B. G. Herrmann1,2 Union through the European Regional De- P 15.12 1Max Planck Institute for Molecular Genetics, velopment Fund, under grant agreement No. 2 2+ ­Developmental Genetics, Berlin, Germany; Institute KK.01.1.1.01.0008, project “Reproductive The Ca -channel CatSper is not for Medical Genetics, Charité – University Medicine and Regenerative Medicine – Exploring New activated by cAMP/PKA signaling Berlin, Berlin, Germany Platforms and Potentials”. but directly affected by chemicals Motility is essential for mammalian sperm to used to probe the action of cAMP reach their goal. Although the main mecha- P 15.11 and PKA nisms generating sperm motility are known, T. Wang1, S. Young2, H. Krenz3, F. Tüttelmann4, the complete molecular pathways controlling Unique features of human sperm A. Röpke5, C. Krallmann2, S. Kliesch2, X. H. Zeng1, progressive movement towards the eggs re- circular RNAs (circRNAs) T. Strünker2, C. Brenker2 main elusive.

DGA-Abstracts M. Jodar1,2, A. Soler-Ventura2, A. Odriozola2, 1Nanchang University, Institute of Life Science and Mice with the t-haplotype were used. Phar- J. Castillo2, D. Delgado-Dueñas2, J. M. Corral3, School of Life Science, Nanchang, China; 2University macologically tools to inhibit RAC1 were J. L. Ballescà2, R. Oliva1,2 of Münster, Centre of Reproductive Medicine and employed. Comprehensive sperm kinematics 1Hospital Clinic, Biochemistry and Molecular Gene- Andrology, Münster, Germany; 3University of analyses were performed by computer as- tics Service, Barcelona, Spain; 2Institut d’Investiga­ ­Münster, Institute of Medical Informatics, Münster, sisted sperm motility analysis (CASA). Sin- cions Biomèdiques August Pi i Sunyer (IDIBAPS), Germany; 4University of Münster, Institute of Repro- gle sperm with distinct progressiveness were Fundació Clínic per a la Recerca Biomèdica, Depart- ductive Genetics, Münster, Germany; 5University of genotyped by PCR. ment of Biomedical Sciences, Faculty of Medicine Münster, Institute of Human Genetics, Münster, Our data shows that RAC1 plays an important and Health Sciences, Universitat de Barcelona, Mo- Germany­ role in controlling progressive movement. lecular Biology of Reproduction and Development Introduction The sperm-specific CatSper Upon Rac inhibition of wild type sperm, pro- 3 Research Group, Barcelona, Spain; Hospital Clinic, channel controls the influx of Ca2+ into the fla- gressive movement is impaired. Moreover, Urology, Barcelona, Spain gellum and, thereby, the swimming behavior sperm from mice homozygous for the geneti- cally variant t-haplotype (t/t), which are ster- CircRNAs are a new type of of sperm. A hallmark of human CatSper is its Introduction ile, show strongly enhanced RAC1 activity in non-coding RNAs which are more stable than polymodal activation by membrane voltage, comparison to wild type (+/+) controls, and the linear RNAs. We aimed to characterize intracellular pH, and oviductal hormones. quickly become immotile in vitro. Sperm from human sperm circRNAs profile. Whether CatSper is also activated by signal- ing pathways involving an increase of cAMP heterozygous (t/+) males, on the other hand, Patients and Methods KNIFE algorithm and ensuing activation of protein kinase A display intermediate RAC1 activity, impaired was applied to predict circRNAs from human (PKA) is, however, a matter of controversy. progressiveness and transmission ratio distor- sperm RNA-seq datasets. Additionally, we tion (TRD) in favor of t-sperm. We show that have tested the resistance of these predicted Methods To shed light on this question, t/+-derived sperm consist of two subpopula- circRNAs to the RNAse R treatment in sperm we used kinetic ion-sensitive fluorimetry, tions, highly progressive and less progressive. and testis samples. patch-clamp recordings, and optochemistry The majority of highly progressive sperm to study transmembrane Ca2+ flux and mem- Results More than 700 circRNAs derived carry the t-haplotype, while most less progres- brane currents in human sperm from healthy from 545 different genes were predicted in all sive sperm contain the wild type (+) chromo- donors and from patients that lack functional sperm samples assessed. Three distinct types some. Dosage-controlled RAC1 inhibition in CatSper channels. of sperm circRNAs were defined according t/+ sperm rescues progressive movement in to their pattern of resistance to the RNase R Results We find that human CatSper is nei- (+)-sperm in vitro, directly demonstrating that treatment: 1) Resistance to the RNase R treat- ther activated by intracellular cAMP directly impairment of progressive movement in the ment only for divergent primers amplification nor indirectly by the cAMP/PKA-signal- latter is caused by enhanced RAC1 activity. indicating circRNAs and their linear cognates ing pathway. Instead, we show that non- The combined data show that RAC1 plays a co-exist; 2) Resistance to the RNase R treat- physiological concentrations of cAMP and pivotal role in controlling progressive motil- ment for both convergent and divergent prim- membrane-permeable cAMP analogs used to ity in sperm, and that enhanced or reduced ers amplification, indicating that the RNAs mimic the action of intracellular cAMP acti- RAC1 activity interferes with progressive were mainly circRNAs; 3) High sensitivity vate human CatSper from the outside via a movement. Differential RAC1 activity within to the RNase R treatment for both convergent hitherto unknown extracellular binding site. a sperm population impairs the competitive- and divergent primers amplification indicat- Finally, we demonstrate that the effects of ness of sperm expressing suboptimal RAC1 ing the decircularitzacion of the circRNA. common PKA inhibitors on human CatSper activity and thus their fertilization success, as Additionally, we demonstrated that this rest predominantly, if not exclusively, on off- demonstrated by t/+-derived sperm. decircularitzation is not a stochastic event. target drug actions on CatSper itself rather than on inhibition of PKA. Conclusion As a differential feature be- tween sperm and somatic cells, in which lin- Conclusion We conclude that the concept P 15.14 ear and circular forms derived from the same of an intracellular cAMP/PKA-activation Tumor necrosis factor-alpha recep­ gene co-exist, we have observed that many of CatSper is primarily based on unspecific tor-1 (TNFR1) is present in human effects of chemical probes used to interfere RNAs from the sperm are exclusively pre- spermatozoa but is not functional sent in their circular form. In addition, since, with cAMP signaling. Altogether, our find- the linear cognates of these sperm circRNAs ings solve several controversial issues and in triggering apoptosis pathways were detected in the human testis, it is sug- reveal a novel ligand-binding site controlling C. Castellini1, S. D’Andrea1, M. Totaro1, A. Parisi1, gested that while linear forms are gradually the activity of CatSper, which has important P. Palumbo2, F. R. Augello2, F. Lombardi2, B. Cinque2, 2+ degraded during spermatogenesis, the circu- bearings on future studies of cAMP and Ca S. Francavilla1, F. Francavilla1, A. Barbonetti1 lar forms are selectively retained in the ma- signaling in sperm. 1Andrology Unit, Department of Life, Health and ture sperm. However, some sperm circRNAs ­Environmental Sciences, University of L’Aquila, showed sensitivity to the RNAse R treatment. L’Aquila, Italy; 2University of L’Aquila, Department of Interestingly, a non-random decircularization Life, Health and Environmental sciences, L’Aquila, of those non-intact sperm cirRNAs was ob- Italy

84 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

Although TNF-alpha is detectable within ciated death domain (TRADD) and Fas-as- with Caveolin-1 on large areas of the princi- the male genital tract, especially under pro- sociated protein with death domain (FADD), pal piece. When the components of TNFR1 inflammatory conditions, its effects on sperm was investigated by Western blot analysis signaling cascade were explored by Western functions are controversial, and no direct where Jurkat cells were used as the positive blot analysis, similar to Jurkat cells, sperma- evidence has been produced to date for the control. TNFR1 was immunolocalized in hu- tozoa displayed TRADD; nevertheless, band presence of its receptor, TNFR1, in human man spermatozoa by confocal microscope. density in the FADD molecular size was gen- spermatozoa. Sperm motility and MMP were not signifi- erally faint and poorly detectable, suggesting Mitochondrial membrane potential (MMP) cantly affected by the exposure to increasing a potential defective signaling downstream of and activation of caspase-8 (receptor caspase) concentrations of TNF-alpha, which did not TNFR1/TRADD interaction. and caspase-3 (executioner caspase) were induce caspase-8 or caspase-3 activation. Although human spermatozoa display the assessed by flow cytometry in motile sperm The Western blot analysis of sperm extracts TNFR1, it appears to be not functional in suspensions exposed for 2 h to scalar concen- from different ejaculates demonstrated, in all triggering apoptosis pathways in these cells, trations of TNF-alpha (0.5–100 µM). Sperm samples, the presence of a single immunore- likely due to a defective FADD signaling. motility was analysed by CASA. The sperm active band of the expected molecular size of The claimed detrimental effects on male fer- expression of TNFR1 as well as adaptor pro- TNFR1 (~55 kDa). At the confocal micro- tility of TNF-alpha should be reconsidered in teins of its signalling pathway, TNFR1-asso- scope, TNFR1 appeared to be colocalized the light of the present findings. DGA-Abstracts

J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) 85 Andrology 2020 – Abstracts

Index of authors (only primary authors)

A Fon Tacer K...... 47 L Abildgaard J...... 49 Fraczek M...... 53 Laan M...... 8 Abramovic I...... 57 Francavilla S...... 16 Laurentino S...... 9 Adamczewska D...... 67 Lavoie-Ouellet C...... 67 Aden N. L...... 39 G Lin H...... 6 Almeida J...... 26 Gacem S...... 81 Li R...... 40 Alves M. G...... 17 Garolla A...... 14 Liu P. Y...... 8 Amaral A...... 84 Gillatt D...... 35 Lopes F...... 39 Apolikhin O...... 36 Gomes A...... 24 Lotti F...... 66, 69 Axelsson J...... 45 Goossens E...... 11 Greither T...... 22 M B Gromoll J...... 9 Mahapatra C...... 35 DGA-Abstracts Baldi E...... 12 Gupta P...... 36, 37, 60 Mall E. M...... 24, 40 Barbonetti A...... 20, 42 Malm J...... 9 Barrachina F...... 53 H Mancini A...... 20 Basent Baruah B...... 65 Hamdi S...... 75, 80 Marchiani S...... 26 Bašković M...... 35 Hasan H...... 74 Marić T...... 83 Behre H. M...... 6 Hasenmajer V...... 20 McLachlan R...... 8 Bender J...... 62 Hejmej A...... 63 Meinhardt A...... 19 Benderradji H...... 77 Hekim N...... 71 Meitei H. Y...... 36 Benninghoven-Frey K...... 25 Heyer J...... 56 Michaelis M...... 55 Berres S...... 32 Himelreich Perić M...... 64 Missel A...... 61 Bhushan S...... 26 Höffken V...... 44 Mohd Tambi M. I...... 58 Bilińska B...... 62 Hoffmann I...... 6 Monzani M. L...... 50 Björndahl L...... 21 Houska P...... 75 Moody S...... 46 Bodin M...... 9 Moreno-Mendoza D...... 57 Bompart D...... 82, 83 I Munuce M. J...... 76 Borthakur D...... 32 Muratori M...... 46 Islam R...... 29 Braye A...... 20 Ivanovic Apolikhin O...... 12 Ivanovich Apolikhin O...... 17 N C Neuhaus N...... 18, 28 Carlomagno F...... 43 J Nieschlag E...... 10 Castellini C...... 66, 84 Noto D...... 77 Jahnukainen K...... 12 Chan D...... 58 Novák J...... 81 Jankowska K...... 41, 73 Chandra S...... 55 Jegou B...... 15 Charalampos T...... 58 Jeschke J...... 45 O Cheredath A...... 67 Ježek D...... 7, 47 O’Bryan M...... 8 Chernykh V...... 32, 79 Jodar M...... 84 Odin V...... 49 Chung J.-Ju...... 10 Jose S...... 47 Ogawa T...... 13 Crisóstomo L...... 28 Opuwari C...... 69 Özkoçer S. E...... 69 D K Kabbesh H...... 64 Da Costa R...... 82 P Kaldewey S...... 75 D’Andrea S...... 23, 66 Kappes S...... 24 Page S. T...... 10 Darbey A...... 18 Karadeniz Saygili S...... 34 Pandya R. K...... 37 De Jonge C. J...... 17 Karahan G...... 33 Pan H...... 38 Dejucq-Rainsford N...... 14 Katušić Bojanac A...... 33 Parisi A...... 51 Deknuydt M...... 78 Kaushik A...... 63 Patel P...... 31, 48, 53 de la Iglesia A...... 56 Keglberg Hærvig K...... 70 Peng W...... 27 Depmeier T...... 44 Khayat S...... 33 Pescatori E...... 60 De Rocco Ponce M...... 37 Kierzek M...... 46 Pezo F...... 40 De Vincentis S...... 49 Kim N...... 41 Pinho A. R...... 72 Di Persio S...... 63 Klein B...... 73 Planinić A...... 68 Di Stasi V...... 50 Kliesch S...... 5, 7, 13 Pleuger C...... 22 Dorth de Andrade A...... 23 Kochanova E...... 60 Pock T...... 39 Drevet J. R...... 18 Köckerling N...... 65 Duan Y. G...... 22 Köhn F.-M...... 5 R Kolomiets O...... 31 E Raos D...... 56 Kothalawala S...... 79 Rashed A...... 76 Elenkov A...... 75 Krasic J...... 32 Rassam Y...... 76 Emich J...... 30 Krasnyak S...... 60 Rastrelli G...... 23, 25, 50, 51, 59 Engel K. M...... 16 Krausz C...... 13 Rehfeld A...... 72 Krenz H...... 35 Riera-Escamilla A...... 19 F Krsnik D...... 66 Rilcheva V...... 43 Ferreira L. G...... 53 Kuchta M. A...... 52 Rips J...... 27 Fietz D...... 6 Kurashova N...... 70 Robles Gómez L...... 37

86 J Reproduktionsmed Endokrinol 2020; 17 (Supplementum 1) Andrology 2020 – Abstracts

Rohayem J...... 42 Stoimenou E...... 72 Verón G. L...... 72 Rosta V...... 30 Stukenborg J.-B...... 15 Viville S...... 19 Rotte N...... 30 Suleiman J...... 21 Suyono S. S...... 49 W S Švalbe B...... 46 Wachten D...... 15 Sabrina G...... 81 Wang C...... 10 Salehi Novin M...... 68 T Wang T...... 84 Salvio G...... 51 Weiser D...... 51 Talapatra A...... 45 Whiley P...... 28 Sansone A...... 29, 43, 59, 74 Tarsitano M. G...... 54 Santi D...... 31 White A...... 67 Tena-Sempere M. M...... 11 Wijayarathna R...... 62 Sasaki K...... 27 Tholeti P...... 55 Schierling T...... 83 Wilkinson M. F...... 15 Totaro M...... 48 Willems M...... 27 Schiffer C...... 82 Tøttenborg Sa. S...... 16 Schlager D...... 61 Wistuba J...... 52 Trasler J...... 18 Wolski J. K...... 57 Schlatt S...... 18 Tüttelmann F...... 7 Schubert M...... 69 Wyrwoll M. J...... 44 Sergeyev O...... 21, 34

Serrano J. B...... 25 U Y DGA-Abstracts Shaeer O...... 59, 71, 79 Uppangala S...... 39 Young S...... 74 Sharma S...... 26, 63 Uribe P...... 70 Shiplu S. A...... 31 Z Siebert-Kuss L. M...... 65 Zambrano F...... 44 Silva A. F...... 78 V Zarén P...... 71 Škara L...... 64 Vazquez-Levin M. H...... 57 Zhang X...... 11 Sofikitis N...... 14 Venaki E...... 42, 74 Zhukov O. B...... 34 Spaggiari G...... 52 Vena W...... 48, 80 Zieger V...... 81 Stadler B...... 79 Vendramini V...... 24, 73 Zitzmann M...... 23

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