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Research Article *Corresponding author Susana Fernandes, Department of Genetics, Faculty of Medicine of University of Porto, Al. Prof. Hernâni Emerging Genetic Alterations Monteiro, 4200 - 319 Porto, Portugal; Tel: 351 22 551 36 47; Email: Submitted: 12 October 2016 Linked to Male Infertility: Accepted: 10 February 2017 Published: 14 February 2017 X- Copy Number Copyright © 2017 Fernandes et al. Variation and Spermatogenesis OPEN ACCESS Keywords Regulatory ’ Expression • Male infertility • Spermatogenesis Catarina Costa1, Maria João Pinho1,2, Alberto Barros1,2,3, and • DNA copy number variations • expression Susana Fernandes1,2* 1Department of Genetics, Faculty of Medicine of University of Porto, Portugal 2i3S – Instituto de Investigação e Inovação em Saúde, University of Porto, Portugal 3Centre for Reproductive Genetics A Barros, Portugal

Abstract The etiopathogenesis of primary testicular failure remains undefined in 50% of cases. Most of these idiopathic cases probably result from genetic mutations/anomalies. Novel causes, like Copy Number Variation and gene expression profile, are being explored thanks to recent advances in the field of genetics. Our aim was to study Copy Number Variation (CNV) 67, a patient-specific CNV related to spermatogenic anomaly and evaluate the expression of regulatory genes AKAP4, responsible for sperm fibrous sheet assembly, and STAG3, essential for sister chromatid cohesion during meiosis. One hundred infertile men were tested for CNV67 with quantitative PCR (qPCR). Quantitative real-time PCR was performed to evaluate gene expression patterns of the two mentioned genes in testicular biopsies from 22 idiopathic infertile patients. CNV67 deletion was found in 2% of patients, with the same semen phenotype described in previous studies. Expression levels of AKAP4 and STAG3 were down regulated in infertile patients when compared to control group (p<0.05). Resulting data reinforce the role of CNV67 in male infertility etiology. Its frequency is significantly higher in oligo/azoospermic men and evidence indicates consistency of phenotype. Down regulation of AKAP4 and STAG3 cellular transcript levels was observed in the testicular biopsies, suggesting that the gene expression is altered, contributing to unsuccessful sperm production. As one continues to better understand about the genetics of male infertility, there will be undoubtedly a shift towards better diagnosis and treatment for those patients presenting idiopathic infertility.

INTRODUCTION environmental factors. Spermatogenesis is a highly complex process controlled by several regulatory genes which assure Background the correct maturation steps, from spermatogonia to sperm [3]. An estimated 15% of couples are infertile, not achieving a clinical pregnancy after 1 year of unprotected intercourse, genes involved must either be only expressed or be functionally These men are otherwise usually healthy, suggesting that any with a great impact on the individual, couple and society [1,2]. required for spermatogenesis [3]. Male reproductive dysfunction is the sole or contributory cause During the last years, novel tests and diagnostic tools have been of infertility in half of the couples [3,4], with abnormalities employed to identify rare genetic mutations and polymorphism of sperm number (azoospermia, oligozoospermia), motility (asthenozoospermia) and morphology (terathozoospermia) declining cost and increased power of whole-genome sequencing being frequently diagnosed [5,6]. Male infertility can be clinically studies,with putative including direct evaluation or indirect of theeffects increasingly on spermatogenesis. important inter The divided in three main categories: acquired, congenital and genic regions of the genome, is leading to nascent paths of research and likely indicate that, in the future, such studies will be used still represents 50% of the cases of primary spermatogenic failure on daily-basis [13]. Likewise, genetic testing of Copy Number idiopathic, when no cause is identified [6,7]. The idiopathic group in humans [2,8] and presently, due to the lack of pathophysiological Variation and spermatogenesis’s regulatory genes expression may reveal the etiology of idiopathic patients and, consequently, underlying causes are thought to be genetic [6,10,11], mainly increase the likelihood of successful paternity and reduce understanding, no specific treatment is offered [9]. Most of the due to spermatogenesis defects [3,12], correlated, or not, with potential risks to the progeny [13,14]. Copy Number Variation

Cite this article: Costa C, Pinho MJ, Barros A, Fernandes S (2017) Emerging Genetic Alterations Linked to Male Infertility: X-Chromosome Copy Number Variation and Spermatogenesis Regulatory Genes’ Expression. Ann Reprod Med Treat 2(1): 1008. Fernandes et al. (2017) Email:

Central Bringing Excellence in Open Access complexes, a large ring-shaped proteinaceous structure which a DNA tethers sister chromatids, providing cohesion to the structure segment,(CNV) has 1 raisedkb or longer, a considerable that is present interest in amonga variable scientific number and of [22,26]. Its deletion has been related to a Premature Ovary medical communities. CNV is conventionally defined as Failure (POF). Interestingly, STAG3 comprehensive CNV map of the , in 2006, several a severe defect in synapses and premature loss of centromeric diseasescopies in have the genome,been linked between to CNVs, individuals mainly due [15]. to Since disruption the first of cohesion during the early stages of -deficientprophase maleI, which mice causes display an functional elements (either genes or regulatory elements). In arrest during the zygotene-like stage, leading to infertility [22, fact, it is well established that Y chromosome CNVs in the AZF region are linked to spermatogenic impairment and are routinely in27]. a group The aim of infertileof this study men andis to consolidate explore these the emerging pathophysiology genetic alterations by quantifying the copy number variation of CNV67 (increasedanalysed for number genetic of DNA male copies infertility compared diagnosis to reference [9,16]. genome) These of spermatogenesis regulatory genes AKAP4 and STAG3 will be andunbalanced losses (reduction quantitative or deletion variants compared can be to classified reference into genome) gains evaluatedwhich links in CNV67 infertile to men male testicular infertility. biopsies In addition, and correlated the expression with the (in)fertility status.

[15]. Recently, high-resolution X-chromosome specific array- MATERIALS AND METHODS genescomparative are particularly genomic tempting hybridization because (aCGH) men are identified hemizygous CNVs for which could be related with male infertility [17]. the X-genes. Since compensation by a normal allele is impossible, AKAP4 and STAG3. Each analysis design it is more likely that a mutation may affect the fertility of an willThis be explained study includes separately. two distinct analyses – CNV67 screening and expression profile of the most promising candidates, resembling AZF deletions of the CNV67 Screening Analysis individual [6,10]. From the reported CNVs, CNV67 was one of Patient samples: Peripheral blood samples were collected in infertile patients at a frequency of 1.1% (p<0.01), ranging from 100 Portuguese idiopathic infertile men, with different Y chromosome [16,17]. CNV67 deletion was exclusively found patient’s phenotypes from azoospermia due to Sertoli-Cell-Only grades of spermatogenic impairment - 44 azoospermic (AZO), Syndrome (SCOS) to oligozoospermia. It is localized in Xq28 and is likely to be maternally inherited [16]. It has been suggested were47 severe selected oligozoospermic on the basis of (SOZ), a comprehensive 4 oligozoospermic andrological (OZ) examinationand 5 normozoospermic including medical (N) men history, (Table semen 1). Infertile analysis, patients scrotal that CNV67 deletion linked to spermatogenic failure may be ultrasound, and hormonal and genetic analysis. Patients with related to highly duplicated genes of X-Cancer Testis Antigen abnormal karyotype or Y chromosome microdeletion were (CTA) family, the most represented X-linked testis specific family. excluded. Normal controls were fertile normozoospermic In fact, X-CTA genes comprise 10% of all X-linked genes and are (MAGEA9B), volunteers. expressedexpression specifically level in spermatocytes in testis [18]. and In particular, in some tumour CNV67 cell deletion lines. mayIt may remove also affect the chromosome melanoma antigen X open familyreading A, frame 9B (CXorf40A), Genomic DNA (gDNA) Extraction: situated at < 1Mb from the deletion and regulation elements of mL) was collected through vein puncture from all participants. Peripheral blood (3–5 (HSFX1/2) High molecular weight DNA was isolated using a salting out method. Heat Shock Transcription Factor Family, X-Linked 1/2 Quantitative PCR (qPCR): [16,17]. on each sample was determined by Quantitative PCR (qPCR). Gene expression profile can be used as a basis for identification ® probes were designedT he by number the manufacturer of copies of (Applied CNV67 of candidate genes that contribute to male infertility [19-21]. To date, genetic studies in mice have identified more than 200 genes TaqMan Biosystems, Foster City, USA) and were chosen to target specific that are specifically or. AKAP4 preferentially and STAG3 involved are strong in the candidate complex regulation of fertility and some are specifically expressed in the Pregions. (labeled Hs03323870_cn with VIC) and used was as selected the reference for the gene. target Reactions CNV67 (labeled with FAM) and Hs03323870 was selected for RNase (germAKAP4 line), an [6,10,11,19] X-linked member of the AKAP gene family, encodes genes for male infertility [7,22]. The A-kinase anchor 4 were performed in triplicate in a final volume of 20 µL according® a cyto skeletal structure surrounding the region of the principal to the manufacturer’s instructions. Briefly,® the components of the most abundant protein of theAKAP4 spermatozoon’s anchors cAMP-dependent fibrous sheet, Copy Number Assay, the reaction mix ® were: Copy 4Number µL genomic Reference DNA, Assay 10 µL (RNase 2X TaqMan P) and Genotyping Master Mix, 1 µL 20X TaqMan essentialpiece of sperm for sperm flagellum capacitation, [23]. playing a central role in the 1 µL 20X TaqMan regulationprotein kinase of normal A (PKA) sperm to themotility sperm [11,24,25]. fibrous sheet,In fact, which studies is 4 µL nuclease-free water. qPCR was carried out on a Step One have shown that in AKAP4 Plus™ Real-Time PCR System (Applied Biosystems). The thermal was not reduced, they were immotile, resulting in male infertility -deficient mice, though sperm count Table 1: Clinical description of the study population. AKAP4 Patient’s semen phenotype (n=100) [6,11]. Furthermore,AKAP4 is another likely required study verified for the no structural detection and of Azoospermic 44 immunolabeling in man with 0% sperm mobility [19]. Severe oligozoospermic (<5x106 Oligozoospermic (5-15x106 4 Therefore,( STAG3 Normal /mL) 475 functional integrity of the fibrous sheath [25]. Stromalin 3 /mL) ) is a component of all meiosis-specific cohesion Ann Reprod Med Treat 2(1): 1008 (2017) 2/6 Fernandes et al. (2017) Email:

Central Bringing Excellence in Open Access cycling conditions were as follows: Initial enzyme activation for Table 2: Clinical description of the study population. ° C, 40 cycles were performed, each one consisting Patients ° ° ID Diagnosis TESE Clinical history C and 60 seconds at 60 C. (n=29) 10 minutes at 95 Cases w1 AZS SCOS Normal ofData 15 seconds Analysis at 95 (n=22) Software v2.0 was used AZS MA Normal to determine the copy number statusTM of each target region, Hypospadias, Orchitis (28yo, w113w77 AZS MA andApplied calculations Biosystems were performed Copy Caller according to the maximum- likelihood algorithm of the software. Raw copy value (RCV) AZS MA Left testicular torsion 29yo) represents a non-integer number of copy calculated, whereas w131 AZS HP Normal w119 w140 AZS MA Normal AZS MA Normal locatedpredicted on copy X-chromosome, number (PCN) normal is defined females as will an displayinteger PCNnumber of 2 AZS SCOS Stroke w147 andof copy normal determined males PCN by theequal algorithm to 1. In (0,the 1, case 2, or of 3+). male As alteration, CNV67 is w152b AZS SCOS Parotiditis w149 w154 AZS SCOS of 0 is regarded as CN loss. w160 AZS HP Normal Inguinal Hernia (6yo, 7yo) Copy Number (CN) gain is defined as PCN higher than 1, and PCN w162 AZS SCOS Normal AKAP4 and STAG3 Expression w165 AZS HP Normal Patient samples: w166 AZS HP Epilepsy idiopathic infertile men with AZS (used as cases). Seven men with AZS MA Normal Testicular samples were collected from 22 AZS SCOS Normal w175 w183 AZS HP Leftscrotalhydrocele secondary infertility were used as controls. Testicular biopsies w176 w186 AZS SCOS Normal were obtained to confirm the clinical diagnosis (diagnostic AZS MA Normal biopsy) or for sperm retrieval (Testicular Sperm Extraction: Patients with abnormal karyotype or Y chromosome micro Left varicocele, Orchitis TESE) to intracytoplasmic sperm injection (treatment biopsy). AZS SCOS deletion were excluded. Clinical information of each sample is w187 (24yo) w220w195 AZS SCOS Normal AZS HP Hepatitis C, ex-alcoholic shownEach in sample Table (2). was divided into three aliquots: one was reserved Control ANE- for histological analysis, the second (100-200mg) was processed w58w227 HP Paraplegy for sperm extraction and the third (10mg) was immediately (n=7) ® ® ANE- transferred to a 1.5mL tube with mRNA later solution (Ambion , JACUL HP Diabetes Mellitus

w90 ANE- studies. w103 JACUL HP Foster City, USA) and stored at -80ºC for further gene expression RNA isolation and reverse transcription (RT) reaction: ANE- Psycologicanejaculation w106 JACUL SCOS Paraplegy After thawing the frozen pellets, cells were lysed on ice with ANE- w116 JACUL HP Paraplegy 1000μL of TriPure Isolation Reagent (Roche Diagnostics, OLIGO- w128 JACUL HP Normal associatedIndianapolis, protocol. USA) and At passed the end, several RNA pellettimes through was resuspended a syringe and needle. The total mRNA was then extracted according to the OLIGO- w164 ASTHE HP Normal - in 50μL of diethylpyro carbonate (DEPC)-treated RNase-free ASTHE water (Promega, Wisconsin, USA) and incubated for 1h on ice. ANEJACUL: Anejaculation; AZS: Azoospermia;Shaded samples HP- Hypospermatogen had no expression RNA was then quantified in a Biotech Photometer UV 1101 was reverse transcribed to complementary DNA (cDNA) using foresis; at MA: least Maturation one of the arrest;studied OLIGO-ASTHE: genes. Oligo-asthenozoospermia; qScript(WPA, Cambridge, UK). 1µg of mRNA in a total volume, Gaithersburg, of 10μL SCOS: Sertoli-Cell-Only Syndrome; TM TM according cDNAto the SuperMixmanufacturer’s (Quanta, instructions. Biosciences USA), with random hexamers as the priming method and qRT-PCR was performed in a volume of 10μL, using 2μL of cDNA, Gene Expression Analysis by Quantitative Real-Time 2.5µL of Nuclease® Free-water, 5µL of 2xKAPA probe Master Mix PCR (Qrt-PCR): ® Gene Expression Assays were used Gene Expression Assay for each gene, using a Fast (Kappa Biosystems, Boston, Massachusetts, USA) and 0,5µL of for both targeted experimental genes (AKAP4 20X TaqMan ° and STAG3 TaqMan ® probes were labeled initial enzyme activation for 2 minutes at 50 C and 20 seconds - Hs00275849_m1 Protocol°C, 40 according cycles were to manufacturer performed, instructions. each one consisting Briefly, after of 3 ° ° RNA 18S Ribosomal - Hs00429370_m1). (18S) was All used TaqMan as the housekeeping gene C and 20 seconds at 60 C. Standard curves were at 95 with FAM dye® Gene and Expression were purchased Assay was from also Applied utilized. Biosystems. seconds at 95 and TaqMan genesperformed were withrun together five points, with in a duplicates.negative control. Each PCR for relative quantification was run in triplicate (technical replicates) and all RNA expression levels were analysed by qRT-PCR on a StepOnePlus™ Real- Time PCR System (Applied Biosystems). Ann Reprod Med Treat 2(1): 1008 (2017) 3/6 Fernandes et al. (2017) Email:

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up andData down was analyzed regulation using for geneREST expression 2009 (Relative studies Expression ( Software Tool), which is) a standalone software tool that estimates http:// www.qiagen.com/rest The purpose of this software is to determine whether there are significant differences between samples and controls, while thetaking probability in account of issues the alternate of reaction hypothesis efficiency thatand reference the difference gene betweennormalization. the sample The obtained and control hypothesis groups istest due P(H1) only represents to chance.

Real time PCR-negativity was defined by the absence of amplified Figure 1 Y3803. Whiskers refer to maximum and minimum of copies calculated. As expressionsproduct after values, 40 cycles we proposed and because that the REST value softwareof the last usescycle Ctof Copy Number Variation 67 deletion of infertile man Y3790 and values and reaction efficiency for calculations instead of relative Number of Copies (PCN) of 2 and normal males PCN equal to 1. In the case of CNV67 is located on X-chromosome, normal females will display Predicted usedamplification for the statistical (Ct = 40 cycles)analysis should (StatView correspond for Windows) to the withvalue the of absence of relative expression. Wilcoxon Signed Rank Test was male alteration, Copy Number (CN) gain is defined as PCN higher than 1, and PCN of 0 is regarded as CN loss. YF154 – Fertile man control. significanceRESULTS levelAND set DISCUSSION at p < 0.05. * * CNV67 Screening Analysis studied from infertile men with different sperm phenotypes and In order to screen CNV67 deletion, 100 samples were concentration, as previously described, by RT-PCR (Table 1). Two SCOSindividuals after biopsy – Y3790 and and Y3803 Y3803 presented – were founda severe to oligozoospermiahave deletion (0 (2copies) x106 of CNV67 (2%). Y3790 was azoospermic, diagnosed with

sperm/mL) (Figure 1). Our data supports the sperm phenotypes related to CNV67 deletion (azoospermia in a clinical population,context of SCOS even or though oligozoospermia). our sample wasMoreover, quite thesmall findings when indicate a significantly higher frequency in our Portuguese deletion is directly responsible for the altered sperm phenotype (eithercompared affecting to previous gene expression studies [16, or 17]. regulatory Whether elements) the observed or is

AKAP4related to and increased STAG3 genomic Expression instability remains uncertain [17]. Figure 2 Expresion levels of AKAP4 and STAG3 in testicular samples. cDNA expression was normalized using a housekeeping gene (18S). - Quantification of testicular mRNA levels of genes AKAP4expression and was carried out by qRT-PCR in individuals showing spermati WhiskersBoxes represents represent the the interquartile minimum range, and maximum or the middle observations. 50% of STAG3 observations. The dotted line represents the median gene expression. respectively.genic failure. Clinical Two spermatogenesis and pathologic information related genes on the - cases and – were analyzed in 20 and 22 testicular biopsies samples, Significance differences between groups are represented as: * p<0.05. The data was analysed by Mann-Whitney-Wilcoxon test. controls are presented in Table (2). controls. Results indicated that the AKAP4 and STAG3 were down referenceAnalysis gene of normalization, the qRT-PCR 18S results housekeeping was completed gene was by usingused. REST 2009 software. The findings are summarized in Table 3. For regulated with statistical significance (p<0.05) in the case group using the following formula: compared to the control group (Table 3, Figure 2). Sample expression ratios were calculated with REST software AKAP4 and w220 patient did not express STAG3Interestingly, 6 cases (w147, w149, w154, w176, w186, ÷ Geometric mean (concentration of reference gene 1, ofw195) gene didexpression not express in spermatogenic failure patients could not be Relative Expression = Concentration of Gene of interest concentration of reference gene 2,…) exclusively(see Tableattributed 2). It tohas a beendecreased demonstrated number thatof germ the reductioncells, but With the use of this software, the up or down regulation the contribution of the reduced cellular expression should be also for each gene expression was estimated comparing cases with taken in account [19]. Ann Reprod Med Treat 2(1): 1008 (2017) 4/6 Fernandes et al. (2017) Email:

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Table 3 : AKAP4and STAG3 expression results. Gene Type RxnEffic. Expression Std. Error 95% C.I. P(H1) Result 18S REF 1.000

AKAP4 0.9558 0.011 0.000 - 0.645 0.007 DOWN STAG3 TRG 0.9346 0.050 0.001 0.0-114.4260.0-62.299 0.038 DOWN TRG 0.9829 - 2.076 alternative hypothesis that difference between sample and control groups is due only to chance REF: Reference; TRG:Target. RxnEffic: Reaction efficiency; Std. Error: Standard Error; 95% C.I. – 95% confidence interval; P(H1): Probability of AKAP4 and its regulation [28], and is exclusively expressed in germ cells, encodes a protein involved in fibrous sheet assembly emergingmale infertility genetic that alteration are currently with implications classified asin idiopathicmale infertility. [13]. AKAP4 expression exhibit a statistically The present study also explores gene expression profile as an during the post-meiotic phase of spermatogenesis [25,29]. The genes AKAP4 and STAG3 on infertile men testicular biopsies. Interestingly,findings of this nostudy expression on was detected in several SCOS OurTherefore data reports we assessed altered the expression expression of profile germ-line of regulatory patients.significant However, difference the with 2 oligo-asthenozoospermic down regulation in the However, case group. the genes, providing an initial glimpse into the complex regulatory 2 oligo-asthenozospermic patients used as controls expressed network controlling germ line development. Further analyses AKAP4, contradicting the data published in a previous study [22]. in larger series are required to better understand the biological patients used as controls expressed AKAP4, contradicting the data implications of these differences. Although the importance of published in a previous study. Similarly, down regulation of STAG3 diagnosing genetic factors is fully recognized, the diagnostic STAG3 encodes a predominant workup of infertility in men is still limited to a few genetic tests wasspermatocytes, found in the participating case group. Thein the telomere attachment to the diagnosis and help to prevent miscarriage and iatrogenic nuclearSTAG protein periphery, component telomere ofmaintenance, cohesin complexes chromosome in primarypairing, transmission[17]. Genetic of testing genetic allowsdefects clarifyingto the offspring an obscure through infertility Assisted chromosome synapses and maintenance of sister chromatid that efforts should be made in order to identify potential genetic causesReproduction of infertility Techniques and, in this (ART) way, [3,4].aid couples Therefore, to make we informed believe ofcohesion candidate [27]. genes This that protein contribute is exclusively to spermatogenic expressed impairment. in meiosis. decisions, optimize genetic testing and provide therapeutic OneGene must expression emphasize profiles that can an be inherent used as problem a basis for in identificationinvestigating targets [2,31]. testicular expression changes is the cellular complexity of the organ [21]. Here we analyzed the transcriptional changes in a complete organ, with distinct germ cell types. One advantage is workupIn conclusion, and the role our of AKAP4 findings and merit STAG3 further in male investigation infertility. in that we revealed complex transcriptional changes related to the order to elucidate the potential of CNV67 in routine fertility Ethics Approval and Consent to Participate the inherent disadvantage, compared with isolated cell fractions, aswhole we testis cannot during directly germ identify cell differentiation. the locus of The expression same point change. has Furthermore, whether the observed differential expression study.The Informed local Ethical consent Committees was obtained of the from Faculty the patients of Medicine before of beingUniversity included of Porto/ in this Centro study during Hospitalar their S. reproductive João approved medical the treatment. inprofiles delineating represent the patterns the cause of orgene consequence expression ofinvolved spermatogenic in male impairment remains to be elucidated. This data should be useful germline, which may contribute to understanding male infertility. ACKNOWLEDGEMENTS CONCLUSION We would like to thank the patients involved for their Classic male infertility tests, like karyotyping, Y chromosome micro deletions and FISH analysis at somatic and germ cell levels, onparticipation, statistical analysis. Nuno Barros and Joaquina Silva for providing patients samples and their clinical data, and Sofia Dória for help of genome disorders on male infertility. A wide range of molecular REFERENCES methodsare no longer are required sufficient for to better investigate understanding the potential of male contribution infertility causes and, therefore, increase the potential offer for a better 1. Zegers-Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, et al. International Committee for Monitoring treatment for infertile patients [30].

Assisted Reproductive Technology (ICMART) and the World Health of potential clinical relevance in the etiology of male infertility Organization (WHO) revised glossary of ART terminology, 2009. Novel genetic alterations have been identified which may be in the medium term, like Copy Number Variation (CNV). Of all 2. FertilityEsteves SC.and ASterility. clinical 2009; appraisal 92: 1520-1524. of the genetic basis in unexplained

3. male infertility. J Hum Reprod Sci. 2013; 6: 176-182. frequency.CNVs related It is to likely male that infertility, rare single X-CNV67 nucleotide was onepolymorphisms of the most (SNPs)interesting and CNVs,ones [16], although with arare consistent on an individual phenotype basis, and collectively significant Elliott DJ, Cooke HJ. The molecular genetics of male infertility. 4. Bioessays.McLachlan 1997;RI, Krausz 19: 801-809. C. Clinical evaluation of the infertile male: new they can contribute to explain a significant number of cases of Ann Reprod Med Treat 2(1): 1008 (2017) 5/6 Fernandes et al. (2017) Email:

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5. options, new challenges. Asian J Androl. 2012; 14: 3-5. 20. 596-606. Cooper T, Castilla JA. Who Laboratory Manual For The Examination Chalmel F, Lardenois A, Evrard B, Mathieu R, Feig C, Demougin P, et 6. And Processing Of Human Semen. Journal of Andrology. 2009; 30: 9. al. Global human tissue profiling and protein network analysis reveals distinct levels of transcriptional germline-specificity and identifies Stouffs K, Tournaye H, Liebaers I, Lissens W. Male infertility and the 21. targetFeig C, genesKirchhoff for maleC, Ivell infertility. R, Naether Hum O, Schulze Reprod. W, 2012; Spiess 27: AN, 3233-3248. et al. A new involvement of the X chromosome. Hum Reprod Update. 2009; 15: 623-637. disturbed human spermatogenesis. Molecular human reproduction. paradigm for profiling testicular gene expression during normal and 7. Krausz C. Male infertility: Pathogenesis and clinical diagnosis. Best Practice & Research Clinical Endocrinology & Metabolism. 2011; 25: 22. 2007; 13: 33-43. 8. 271-285.Lee JY, Dada R, Sabanegh E, Carpi A, Agarwal A. Role of genetics in Llano E, Gomez-H L, Garcia-Tunon I, Sanchez-Martin M, Caburet S, Luis Barbero J, et al. STAG3 is a strong candidate gene for male infertility. azoospermia. Urology. 2011; 77: 598-601. 23. Hum Mol Genet. 2014; 23: 3421-3431. 9. McLachlan RI, O’Bryan MK. Clinical Review: State of the art for genetic Localization of AKAP4 and tubulin in sperm with reduced Moretti E, Scapigliati G, Pascarelli NA, Baccetti B, Collodel G. 10. Zhengtesting K,of infertile Yang F, men. Wang J Clin PJ. RegulationEndocrinol ofMetab. male 2010; fertility 95: by1013-1024. X-linked 24. motility. Asian J Androl. 2007; 9: 641-649. 11. genes.Stouffs J K,Androl. Lissens 2010; W. X31: chromosomal 79-85. mutations and spermatogenic hAKAP82.Turner RM, Genomic Johnson organization, LR, Haig-Ladewig protein L, kinase Gerton A-RII GL, Moss binding, SB. and An X-linked gene encodes a major human sperm fibrous sheath protein, 12. failure.Krausz Biochim C, Giachini Biophys C. Genetic Acta. 2012; risk factors1822: 1864-1872. in male infertility. Arch 25. distribution ... J Biol Chem. 1998; 273: 32135-32141. protein 4 has a conserved role in mammalian spermatogenesis. 13. Androl. 2007; 53: 125-33. Hu Y, Yu H, Pask AJ, O’Brien DA, Shaw G, Renfree MB. A-kinase anchoring

350.Hotaling J, Carrell DT. Clinical genetic testing for male factor infertility: 26. Reproduction. 2009; 137: 645-653. current applications and future directions. Andrology. 2014; 2: 339- 14. CentromereHopkins J, Hwang Chromatid G, Jacob Cohesion, J, Sapp N,and Bedigian Required R, Okafor DNAK, et al.Repair Meiosis- and Specific Cohesin Component, Stag3 Is Essential for Maintaining Wosnitzer MS. Genetic evaluation of male infertility. Transl Androl 15. 10. Urol. 2014; 3: 17-26. Synapsis between Homologous . Plos Genetics. 2014; Ayobami Ward JH, Matthew Mckay, Steve Murray and Philip W. Fanciulli M, Petretto E, Aitman TJ. Gene copy number variation and 16. Lo Giacco D, Chianese C, Ars E, Ruiz-Castañé E, Forti G, Krausz C, et al. common human disease. Clin Genet. 2010; 77: 201-213. 27. Recurrent X chromosome-linked deletions: discovery of new genetic Jordan. Genetic interactions between the meiosis-specific cohesion 28. components, STAG3, REC8 and RAD21L. 3G. 2016; 6:1713-1724. Krausz C, Giachini C, Lo Giacco D, Daguin F, Chianese C, Ars E, et al. Mutation analysis in patients with total sperm immotility. Journal of factors in male infertility. J Med Genet. 2014; 51: 340-344. Pereira R, Oliveira J, Ferraz L, Barros A, Santos R, Sousa M, et al. 17. High resolution X chromosome-specific array-CGH detects new CNVs Assisted Reproduction and Genetics. 2015; 32: 893-902. 18. in infertile males. PLoS One. 2012; 7: 44887. 29. Miki K, Willis WD, Brown PR, Goulding EH, Fulcher KD, Eddy EM. biology of cancer testis antigens: Putative function, regulation and Fratta E, Coral S, Covre A, Parisi G, Colizzi F, Danielli R, et al. The Targeted disruption of the Akap4 gene causes defects in sperm 30. flagellum and motility. Dev Biol. 2002; 248: 331-342. et al. Emerging molecular methods for male infertility investigation. therapeutic potential. Molecular Oncology. 2011; 5: 164-182. Benkhalifa M, Montjean D, Belloc S, Dalleac A, Ducasse M, Boyer P, expression signature of early stages of the germ line supports the pre- 19. Bonache S, Algaba F, Franco E, Bassas L, Larriba S. Altered gene 31. Expert review of molecular diagnostics. 2014; 14: 37-45. meiotic origin of human spermatogenic failure. Andrology. 2014; 2: Dunn TM, McGovern MM, Bar-Chama N, Kardon NB. Genetic evaluation of male factor infertility. Am J Hum Genet. 1999; 65: 214-A.

Cite this article Costa C, Pinho MJ, Barros A, Fernandes S (2017) Emerging Genetic Alterations Linked to Male Infertility: X-Chromosome Copy Number Variation and Spermato- genesis Regulatory Genes’ Expression. Ann Reprod Med Treat 2(1): 1008.

Ann Reprod Med Treat 2(1): 1008 (2017) 6/6