A Translational Medicine Appraisal of Specialized Andrology Testing in Unexplained Male Infertility

Int Urol Nephrol (2014) 46:1037–1052 DOI 10.1007/s11255-014-0715-0

UROLOGY – REVIEW

A translational medicine appraisal of specialized andrology testing in unexplained male infertility

Sandro C. Esteves • Rakesh K. Sharma • Jaime Gosa´lvez • Ashok Agarwal

Received: 13 March 2014 / Accepted: 5 April 2014 / Published online: 27 April 2014 Ó Springer Science+Business Media Dordrecht 2014

Abstract The diagnostic and prognostic validity of sperm Keywords Andrology Male infertility Diagnostic function biomarkers is particularly relevant for males with techniques and procedures Translational medical unexplained infertility in which routine semen analysis research Review fails to detect subcellular sperm dysfunctions. In this general review, we examine the role and significance of specialized andrology laboratory tests from past to present Introduction and provide a glance toward the future. We concluded that the assessment of sperm DNA damage and oxidative stress Male factor is responsible for about 50 % of infertility provide a relatively independent measure of fertility that cases; 20 % as sole reason and 30–40 % as contributory yields diagnostic and prognostic information complemen- [1]. When at least two semen analyses are normal and there tary to, but distinct and more significant than, standard is no identifiable cause after a thorough workup including sperm parameters. Since none of the available methods for history, physical examination and endocrine laboratory such testing have been fully translated, further research is testing, the patients are categorized as having unexplained necessary to evaluate their cost-effectiveness when applied male infertility (UMI) [2]. Female factor infertility should in large scale to daily medical practice. Application of have been ruled out with at least a detailed history and translational medicine concepts would also be useful to clinical examination, and no demonstrable tubal patency, accelerate the clinical application of recent discoveries in uterine anatomy and ovulatory function abnormalities [3]. the fields of genomics, proteomics and metabolomics. The concept of UMI itself has only recently emerged and should be differentiated from idiopathic male infertility (IMI). Both conditions share similar traits but abnormal semen analyses are characteristic of IMI. The reported prevalence of UMI varies widely from 6 to & S. C. Esteves ( ) 27 % [2], thus indicating that the percentage of men suffering Androfert, Andrology and Human Reproduction Clinic, Av. Dr. Heitor Penteado, 1464, Campinas, Saõ Paulo 13075-460, Brazil from this condition depends on the extent to which they have e-mail: [email protected] been investigated. Ideally, the patient needs to seek evaluation by a specialist for being diagnosed with unexplained R. K. Sharma A. Agarwal infertility but in the Western world only about half of infertile Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH, USA men will present to a specialist for an evaluation [4, 5]. e-mail: [email protected] Economic, social, geographical and religious are important A. Agarwal determinants of whether a man will seek specialist’s evalu- e-mail: [email protected] ation and thus could affect the reported prevalence of UMI. Potential causes that have been associated with the J. Gosa´lvez ‘‘diagnosis’’ of UMI, and which are frequently unexplored, Genetics Unit, Department of Biology, Universidad Autońoma de Madrid, Madrid, Spain include immune, genetic and other defects at the sperm e-mail: [email protected] subcellular level related to oxidative stress (OS) and 123 1038 Int Urol Nephrol (2014) 46:1037–1052 chromatin integrity [6]. Specialized andrology testing is test systems, and quality assurance during all testing pro- aimed at shedding light on these problems though factors cesses [13]. such as tests’ complexity, difficulties in interpretation, Despite being helpful, routine semen analysis is limited uncertainties about their clinical validity and lack of treat- in its ability to predict the male fertility potential unless ment options limit their routine application [7, 8]. In fact, parameters are at extreme low levels [14]. Among infertile assisted reproductive techniques (ART) are often the treat- men, semen analysis results are normal in up to 40 % of the ment many couples prefer to solve their unexplained infer- cases [12, 15, 16]. In addition, it rarely provides a diagnosis tility problem provided they are overall successful and its usefulness to recommend a treatment modality has irrespective of the underlying infertility cause [9]. As a result, been challenged by the recently released World Health the andrology clinical laboratory is not very much regarded Organization (WHO) reference standards [14, 17–19]. Of beyond its role in performing a routine semen analysis. note, the reference limits of sperm morphology (strict cri- Notwithstanding, novel information from both basic and teria; Tygerberg method) were lowered to 4 % in contrast clinical andrology research has emerged, and the factors to 14 % as in the previous standards [18, 19]. Infertility that contribute to sperm dysfunction are now better specialists have relied on strict morphology cutoff points to understood [8]. The challenge is how to translate these new recommend treatment modalities since 1988, when its evidence and technology advances to the clinical scenario. relevance to in vitro fertilization (IVF) and intrauterine In an era of increasing cost consciousness, it is paramount insemination (IUI) success rates was demonstrated by for clinicians and healthcare providers alike to be able to Kruger and colleagues [20]. Specifically, values below 4 % investigate couples with unexplained infertility in a cost- have been used to recommend intracytoplasmic sperm effective and evidence-based manner. This approach may injection (ICSI) rather than conventional IVF or IUI due to not only provide a better understanding of the underlying the markedly lower pregnancy outcomes of these two infertility cause but is also likely to lead to better, more treatment methods when using semen with low proportion cost-effective and less invasive treatments. In this review, of normal sperm [21, 22]. Interestingly, the distribution of we discuss the role and clinical significance of specialized semen analysis results of fertile men in centiles, as shown andrology tests for men with unexplained infertility taking by the new WHO standards, demonstrated that even though into consideration a translational medicine (TM) approach. 5 % of the studied men had morphology values below the We hope that the thoughts expressed here may clarify some 4 % cutoff point, they still were able to initiate an unas- misconceptions on the matter concerned and be of broader sisted pregnancy within 12 months of unprotected inter- use for doctors treating infertility patients. course, thus contradicting the current practice [17, 18]. It should be noted, however, that reference values, as proposed by the WHO, merely represent the distribution of Why we need to go beyond the routine semen analysis semen parameters of a limited group of recent fathers. As the WHO standards have been adopted by most andrology Semen analysis, often ordered by the gynecologist during laboratories over the years, more men will be placed into the female infertility workup, is the most widely used the category of UMI due to the lowered reference limits. biomarker to predict the male fertility potential [10]. It As noted by Esteves et al. [17], it is still unclear whether provides information on the functional status of the semi- the newly released WHO reference standards will result in niferous tubules, epididymis and accessory sex glands, and a more cost-effective evaluation of the infertile couple or in its results are often taken as a surrogate measure of a man’s a delay in the definitive diagnosis and management of the ability to father a pregnancy. Routine semen analysis infertile couple. Collectively, these findings indicate that include as follows: (a) physical characteristics of semen, physicians treating infertile couples should exercise cau- including liquefaction, viscosity, pH, color and odor; tion when interpreting the results of routine semen analysis. (b) specimen volume; (c) sperm concentration; (d) sperm Semen analysis alone is usually insufficient for diagnosis, motility and progression; (e) sperm morphology; (f) leu- as it does not account for sperm dysfunction, such as kocyte quantification; and (g) fructose detection in cases immature chromatin, OS and DNA damage. where no spermatozoa are found and ejaculate volume is low [11]. Intraindividual variation in semen parameters is common, and therefore, at least two semen specimens Specialized andrology tests: what are they? should be evaluated [12]. Since it is a complex test, semen analysis should ideally be carried out in andrology labo- The past ratories. Minimum standards for laboratories performing semen analyses include the presence of experienced tech- For many years, particularly before the advent of ICSI, nicians, internal and external quality control, validation of tests that assessed antisperm antibodies (ASA), sperm 123 Int Urol Nephrol (2014) 46:1037–1052 1039 hyperactivation and acrosome reaction (AR), sperm bind- their ability to truly assess the multiphasic nature of HA ing and penetration to the human zona pellucida (ZP) were [32]. Hence, its role in fertility prediction remains pri- used both to investigate males with unexplained infertility marily investigational. The AR was also used as a bio- and to predict the fertilizing potential of sperm in con- marker of male infertility. The sperm acrosome contains ventional IVF. ASA, the hallmark of autoimmune male the enzymes required for oocyte penetration that cannot be infertility, were one of the first biomarkers used in clinical released prematurely or too late [33]. Fluorescent lectins practice [8]. ASA develop after accidental or iatrogenic are often used to visualize the sperm acrosome; such stains breach of the blood–testis barrier and are implicated in bind to either the outer membrane or the acrosome con- sperm dysfunction by several mechanisms, including tents, thus discriminating sperm populations with intact or altered sperm penetration into the cervical mucus, ZP reacted acrosomes [34]. AR defects have been associated binding, AR and sperm–oocyte fusion [23, 24]. As these with unexplained infertility as affected sperm fail to pen- alterations were implicated in infertility, it would then be etrate the ZP and have low rates of fertilization with con- useful to detect ASA and treat the couples accordingly. In ventional IVF [35]. Given that these patients achieve an early study, lower spontaneous conception rates (15.3 normal fertilization and pregnancy rates with ICSI and as vs. 66.7 %, p \ 0.05) were observed in couples with treatments for AR defects do not currently exist, the clin- autoimmunity to sperm compared with those without ical use of AR testing has been abandoned [36]. clinically relevant levels of ASA [25]. Recent data have Binding of the spermatozoa to the ZP is species specific otherwise indicated that ASA were not associated with and triggers the AR [37]. Defective binding can be eval- spontaneous, IVF or ICSI pregnancy rates [23, 26, 27]. uated by both the hemizona assay and the sperm–zona ASA should interfere with sperm function in order to binding ratio test [38]. The hemizona assay involves human impair fertility by altering sperm–mucus penetration, oocytes from which a single ZP is isolated and split in half. sperm–zona pellucida binding and AR [2]. Hence, the One half is incubated with fertile donor sperm (positive value of testing solely for ASA is of limited value. Even if control) and the other half is incubated with patient sperm. the detrimental effect of ASA to sperm function were In the sperm–zona binding ratio test, a complete zona is confirmed, there would be not much else we could offer incubated with equal numbers of motile spermatozoa from rather than ART. Attempts of treating ASA with medica- control and test populations, each labeled with a different tion were abandoned due to poor efficacy and serious fluorescent dye. Irrespective of the method, the number of adverse effects [28]. In contrast, microinjection of the spermatozoa bounded to the zona is counted, and the ASA-compromised spermatozoa into the oocyte cytoplasm results are expressed as a ratio of the number of test sperm not only bypasses sperm–oocyte membrane interaction but to that of control. Abnormal zona binding was correlated to ICSI outcomes are also unaffected by the levels of ASA poor fertilization rates using conventional IVF, and the bound to sperm [23]. Collectively, this information casts tests were primarily used in such cases [39]. While the doubt on the clinical relevance of ordering ASA testing in hemizona assay solely assesses sperm–zona binding, the UMI. optimized sperm penetration assay (SPA; zona-free ham- Other biomarkers that evaluate specific aspects of sperm ster oocyte penetration assay) collectively assesses the function were also explored. Sperm kinematics involves spermatozoon’s ability to undergo capacitation, AR, fusion the use of computerized motion analysis (CASA) in con- and penetration through the oolema, and decondensation junction with a kinematics module to distinguish different within the oocyte’s cytoplasm [40]. In SPA, hamster subpopulations of motile spermatozoa. The system recon- oocytes from which the ZP is removed are incubated with structs sperm trajectories in successive video frames, and sperm specimens. The zona-free eggs are examined using the movement parameters are derived. Then, the analyzer phase contrast microscope. The test is scored by calculating segregates the subpopulation of spermatozoa that meets the percentage of ova that are penetrated or the average operator-defined kinematics criteria. Hyperactivation (HA), number of sperm penetrations per ovum. The SPA has a characterized by high velocity, large amplitude, and fla- high positive predictive value to determine the presence of gellar waves, creates a characteristic sperm motion pattern normal fertilization in conventional IVF and the achieve- that is quantified by CASA [29]. The percentage of hy- ment of pregnancy in males with unexplained infertility peractivated sperm was shown to be markedly lower in [41, 42]. In fact, SPA had been clinically used for many patients with unexplained infertility in comparison with years to predict the fertilizing potential of sperm during normal men [30]. Early studies also indicated that HA IVF [43]. As patients with defective hemizona binding and positively correlated with IVF fertilization rates [31]. altered SPA results have no treatment option other than Despite the aforementioned evidence, it was argued that ICSI, in which the steps of capacitation, AR, sperm zona the determination of HA using ‘‘snapshot’’ of sperm tra- and membrane binding and ova penetration are bypassed, jectories, as routinely performed by CASA, is limited in the tests are now rarely used [8]. 123 1040 Int Urol Nephrol (2014) 46:1037–1052

The present Several assays have been developed to assess sperm DNA damage. These assays are based on different princi- Our increased knowledge of the molecular mechanisms ples and therefore differ in their ability to detect DNA regulating sperm function allowed the development of new damage [51, 65]. In general, these methods can be grouped diagnostic tests. Specifically, the importance of OS levels into (1) assays that measure DNA damage by incorporating and nuclear DNA integrity in determining the functional DNA probes or modified nucleotides at the site of damage; competence of human spermatozoa has been emphasized (2) assays that measure the susceptibility of DNA to [44–46]. The aforementioned parameters cannot be detec- denaturation, that is, their principle is to first promote DNA ted by the routine semen analysis but seems to better cor- denaturation and then measure DNA damage by detecting relate with the male fertility status than the latter [47, 48]. the formation of single-strand DNA from native double- As a result, tests to measure DNA integrity and seminal OS strand DNA; and (3) assays that indirectly measure the have become clinically available [49, 50]. In this section, level of chromatin compaction. In Table 1, we summarize we discuss the relevance of sperm DNA integrity and the most commonly used assays principle, methodology, seminal OS to male fertility, the available tests and how clinical utility and drawbacks. they are performed, and the reasons limiting their full translation into the clinical practice. Reactive oxygen species

Sperm chromatin integrity Reactive oxygen species (ROS) are by-products of oxygen metabolism and energy production that act as regulators of Sperm chromatin is characterized by the absence of both vital physiological intracellular processes. In the male single and double DNA strand breaks and nucleotide reproductive tract, the primary sources of ROS are leuko- modifications or base loss. It thus gives rise to a nonlinear cytes and spermatozoa. Small quantities of ROS have disposition of the nucleotides along each single DNA physiologic role in sperm capacitation, AR, hyperactiva- strand and chromosome. Loss of DNA integrity is termed tion and the fusion with the oocyte [66]. In sperm, ROS are ‘‘DNA damage,’’ and it may occur at any level during generated by both the NADPH oxidase and NADH- spermatogenesis, spermiogenesis, epididymis transit, or dependent oxido-reductase systems at the plasma mem- when spermatozoa are used in vitro prior to fertilization brane and mitochondrial levels, respectively [67]. When [51]. Sperm DNA damage is a broad term that accounts for ROS levels disproportionately increase, mainly by the many defects in the DNA structure including (1) single or presence of superoxide (O2–), hydroxyl radicals (OH) or double DNA strand breaks, (2) base deletion or modifica- nitric derivatives (NO), compared with the neutralizing tion, (3) interstrand or intrastrand DNA cross-linkage and capacity of intracellular and extracellular antioxidants, or (4) protamine mispackage via defective DNA–protein when a reduction in the antioxidant capacity occurs, OS crosslinking [52]. Sperm DNA fragmentation refers to the usually follows. breaks occurring at the DNA strands, and they are termed Reactive oxygen species primarily target lipids, proteins single- (ss) or double-strand breaks (ds). Single-strand and DNA through a variety of mechanisms [66]. Owed to breaks give rise to free 50–30 ends affecting only one DNA an available valence electron, oxidative species interact strand, while its template remains intact. In contrast, blunt with the cell membrane and cause lipid peroxidation of 50–30 ends affecting both DNA strands characterize double- which the final product is malondialdehyde, a measurable strand DNA breaks. Post-meiotically initiated abortive compound used to determine the extent of ongoing oxi- apoptosis, unresolved strand breaks during spermiogenesis, dative modification of membrane-polyunsaturated fatty OS, and abortive apoptosis triggered after detection of acids (PFAs). The peroxidation of PFAs has detrimental DNA missrepair or initiation of oxidative damage have all effects on the structure of the sperm head and midpiece been implicated as potential sources of this damage [2, 47]. membranes, causing changes in the sperm membrane flu- Sperm with damaged DNA are released in the semen, idity that ultimately lead to suboptimal sperm motility and and despite the likely result of infertility, these defective fertilization [66]. Oxidative species also target proteins. As cells may still retain the ability to fertilize. Sperm DNA a result, carbonyl (CO) groups are produced on protein side damage has been associated with several infertility phe- chains of oxidized Pro, Arg, Lys and Thr residues and can notypes including unexplained infertility, idiopathic infer- be used as biomarkers of OS [68]. In addition, ROS can tility, repeated IUI and IVF failure, and recurrent inflict serious damage to DNA, including point mutations, miscarriage [53–62]. It has also been suggested that the polymorphisms, deletions, chromosomal rearrangements, offspring generated from such defective sperm have an frame shifts and single-stranded or double-stranded breaks increased risk of imprinting defects and cancer [63, 64]. [69]. The OS-induced sperm damage has been suggested to

123 n rlNprl(04 46:1037–1052 (2014) Nephrol Urol Int Table 1 Characteristics of the methods commonly used for assessing sperm DNA damage Assay Principle How results are Normal limits Interpretation of Clinical Utility in UMI Drawbacks expressed abnormal results in UMI

1. In situ nick Direct incorporation of Percentage of sperm \19 % for TUNEL when DNA damage is a Identify patients in whom Both methods are labor translation modified nucleotides at with DNA damage, used to discriminate significant DNA damage is an intensive and require (ISNT) assay the site of damage (single represented by those fertile from unselected contributor to the important infertility fluorescence microscopy 2. Terminal or double-strand DNA with the probes infertile men with 70 % infertility problem of factor; Monitor the effect or flow cytometry; No deoxy breaks) incorporated to DNA accuracy [101] the couple of medical interventions; international nucleotide breaks Tool for selecting the best standardization and transferase- management strategy and external quality control; mediated ART method Relatively low sensitivity dUTP nick end as the accessibility of the labeling probes to the breaks in a (TUNEL) tightly packed sperm assay genome is limited because the methods do not include a lysis step; The value of the assays for both diagnosis and natural pregnancy prediction in UMI has not been established 3. Sperm Differential susceptibility Percentage of sperm \30 % or \40 % for SCSA Couples in whom the Identify patients in which SCSA require expensive chromatin of DNA to denaturation with fragmented DNA when used to discriminate husband has a high DNA damage is an equipment; Interobserver dispersion test according to the level of (SCD, SCSA, AO) fertile from unselected percentage of important infertility subjectivity to categorize (SCD) DNA damage (SCD, Degree of DNA infertile couples with a spermatozoa with factor; Monitor the effect the halos is a limitation of 4. Sperm SCSA, AO) and fragmentation in a positive predictive value DNA damage have of medical interventions; SCD; Comet is labor chromatin controlled protein single spermatozoon of 73 % and negative reduced natural Tool for selecting the best intensive and requires structure assay depletion (SCD) as assessed by the predictive value of 68 % fertility or a management strategy and expensive software for (SCSA) Selective partial DNA percentage of DNA in [99, 103, 107] prolonged time to ART method imaging analysis; variable pregnancy Comet protocols and pH 5. Comet assay; denaturation after neutral the tail of the comet, or alkaline lysis tail length and conditions affect assay 6. Acridine treatment, with formation intensity of staining performance; Comet has orange (AO) of single-strand (ss) DNA (Comet) high interobserver test from native double-strand subjectivity; No (ds) or single-strand (ss), international in sperm with a damaged standardization and chromatin (Comet) external quality control; The value of the assays for both diagnosis and natural pregnancy prediction in UMI has not

123 been established 1041 1042 Int Urol Nephrol (2014) 46:1037–1052

be a signiﬁcant contributing factor in 30–80 % of all cases of male infertility [70]. The methods used for ROS detection are broadly divi- ded into two major categories based on their ability to directly or indirectly measure oxidative radicals [71]. Indirect measurements involve the assessment of lipid peroxidation products (malondialdehyde), protein oxida-

chromatin packaging rather than DNA fragmentation; Intra- and interobserver variability due to intermediate colors of staining and fading; CMA3 is not amarker speciﬁc for defective protamination; Thresholds not standardized tion products (carbonyl groups) and oxidized DNA (8- hydroxy-20-deoxyguanosine [8-OHdG]), whereas direct oxidative-stress measurements include total or speciﬁc ROS level in semen and total antioxidant capacity (TAC) [72–74]. The assays principle, methodology, clinical utility and drawbacks are summarized in Table 2.

The future

Clinical Utility in UMI Drawbacks Novel genomic, proteomic and metabolomics techniques are under investigation and may add to the existing tests to more precisely diagnose men with unexplained infertility. While genomic techniques are ideally carried out in the genetic laboratory, proteomics and metabolomics methods could be incorporated into the clinical andrology laboratory [8]. abnormal results in UMI The genomic study of single-gene polymorphisms could illicit whether the genetic background confers protection against ROS. In this field, two different gene families have been investigated, glutathione S-transferase (GST) and 8-oxo-guanine DNA glycosylase-1 (OGG1). Its cell detoxifying properties characterizes GST gene family, localized in chromosome 14. The primary detoxification mechanism involves GST enzyme activity, which promotes Not defined Not defined Not defined Methods indirectly measure Normal limits Interpretation of conjugation of reactive compounds with glutathione [75]. Two variants of the GTS gene family, GSTT1 and GSTM1, have been described. While the null genotype for each gene is associated with a higher risk of cancer, carriers of this mutation have an increased vulnerability to cytogenetic damage and oxidative DNA damage [75–77]. Recent studies suggest that the presence of GSTM1 null genotype

with loose chromatin packing is a risk factor for male infertility, as increased levels of OS expressed Percentage of sperm in both sperm and seminal plasma have been described in men with unexplained infertility harboring such mutations [78, 79]. 8-Oxo-guanine DNA glycosylase-1 gene, localized in chromosome 3, encode for the OGG1 enzyme, which has a key role in repairing oxidative DNA damage [80]. This repair pathway involves base excision, which is essential for recognition of Oxo-G:C base pairs, and ca- talyzation of both Oxo-G residues exclusion and DNA probes to nuclear chromatin Direct incorporation of backbone cleavage [81]. OGG1 gene polymorphisms have been described in humans, including Ser326Ser and Cys326Cys originating from C?G exon-transversion, continued which result in amino acid substitution from serine to cysteine in codon 326. It has been shown that cigarette staining (AB) blue (TB); A3 (CMA3) 7. Aniline blue 8. Toluidine 9. Chromomicyn Table 1 Assay Principle How results are smokers harboring the variant Cys326Cys have higher 123 n rlNprl(04 46:1037–1052 (2014) Nephrol Urol Int Table 2 Characteristics of the methods commonly used for assessing oxidative stress Assay Principle Specimen How results are Normal limits Interpretation Clinical Utility in UMI Drawbacks expressed of abnormal results in UMI

1. Thiobarbituric acid Malondialdehyde (MDA), a Semen nmoL MDA/ 0.0287 ± 0.0162 nmol/ Increased lipid Select subgroups of TBARS, ROS and TAC reactive substances byproduct of lipid and 10 9 107 sperm, 108 sperm and peroxidation patients in which requires expensive (TBARS) peroxidation, condenses seminal nmol MDA 0.65 ± 0.17 nmol/ of sperm oxidative stress is an equipment; No international with two equivalents plasma mL-1 seminal mL-1 seminal plasma membranes important standardization and external thiobarbituric acid and give plasma, or nmoL [72] contributory quality control; TAC not a fluorescent red derivative MDA/total infertility factor; specific for type of deficient that can be assayed seminal plasma Monitor the effect of seminal antioxidant; The spectrophotometrically. medical value of the assays for both Absorbance at 532 nm is interventions diagnosis and natural recorded pregnancy prediction in UMI has not been established 2. ROS by Intra- and extracellular ROS Semen 9106 counted B0.0185 9 106 cpm/ Increased ROS Chemiluminescence levels (mainly H2O2, O2-, photons per 20 9 106 sperm [73] levels and OH-) react with probes minute (cpm) per and emit photons that are 20 9 106 sperm/ measured using a mL luminometer. The final chemiluminescent signal is the integrated sum of the partial signals generated by every spermatozoon 3. Seminal total Capacity of the antioxidants Seminal Molar Trolox [2,000 micromoles of Excessive antioxidant capacity in a given sample to prevent plasma equivalents Trolox [74] ROS or (TAC) by enhanced ABTS oxidation is decreased chemiluminescence proportional to their seminal concentration. Suppression plasma of absorbance at 750 nm is antioxidants measured and compared with that of standard Trolox, a water-soluble tocopherol analog 123 1043 1044 Int Urol Nephrol (2014) 46:1037–1052

levels of sperm 8-OHdG, a marker of OS, than the most common homozygote carriers Ser326Ser [82]. Genomics applied to andrology would be important not only from a diagnosis viewpoint but also for treatment management. If the genome condition is contributory to the ﬁnal OS effect, this aspect will need to be taken into account when treating patients with antioxidant cocktails, since the desired effects could be dependent not only on the superoxide anion only; Thresholds not standardized

Qualitative analysis of intensity of external aggressions, but also from the indi- vidual genome condition. Proteomic analysis, which involves the use of one- or two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) coupled with mass spectrometry, could be used not only to conﬁrm the presence of a given protein but also to measure its quantity in different

Clinical Utility in UMI Drawbacks isoforms. At present, most proteomic investigation focuses on discovering various proteins present in the semen. A database listing thousands of proteins in the semen has been recently reviewed [83]. The most abundant proteins

levels of free superoxide anion identified so far in the seminal plasma include fibronectin, Increased of abnormal results in UMI lactoferrin, laminin, albumin and semenogelin, whose molecular functions are associated with catalytic enzymatic activity, protein metabolism, RNA processing/transcrip- tion, cell transport/structure and signal transduction [84]. Efforts are now focused on the identification of what proteins could be used as biomarkers of male fertility and infertility. In this sense, heat shock protein 2 and SPACA1 precipitate Light pink or white Normal limits Interpretation (sperm acrosome membrane-associated protein) are examples of proteins with different expression in sperm of fertile and infertile males [85, 86]. Heat shock protein 2 and SPACA1 are found in the sperm acrosome and seem to be involved in the mediation of sperm–oocyte fusion. Recent reports have also highlighted other proteins in precipitates is compared to a reference card

expressed sperm and seminal plasma that are differentially expressed in men with increased OS [87, 88]. One example is pro- lactin-induced protein that was found to be more abun- dantly present in men with increased OS levels [88]. The Sperm Color intensity of signiﬁcance of these proteins as biomarkers of male fertility is yet to be determined. Metabolomics, on the other hand, is the study of small, low molecular weight molecular metabolites that are the -azino-di-(3-ethylbenzthiazoline sulfonate) 0 products of cell metabolism [89, 90]. It has been conceived

2,2 with an expectation that body-ﬂuid analysis can be optimized to create a low-cost, informative and medically ABTS relevant means of measuring metabolic changes, even

soluble nitro-substituted aromatic tetrazolium compound that reacts with cellular superoxide ions to form a colored intermediate and visible formazan derivative. Depending on the concentration of superoxide anion and the cellular content of various oxidoreductases, a precipitate with a colorvary that from light pinkpurple to or dark almost blackappear will when standard clinical chemistry markers are within nor- NBT is a yellow water- mal limits [91]. These biomarkers are quantiﬁed by various forms of analytical, biochemical and spectral analysis to establish the quantitative lists or signatures of the metabolites for healthy control population and test subjects with continued speciﬁc illnesses [92]. Measurement of metabolic markers reactive oxygen species, of OS can be included in this category. Sperm and seminal tetrazolium reduction test (NBT) 4. Nitroblue ROS Table 2 Assay Principle Specimen How results are plasma metabolomics overlaps with the methods used for 123 Int Urol Nephrol (2014) 46:1037–1052 1045

ROS assessments. Other methods used in metabolomics and unanswered questions that explain the reluctance of include gas chromatography, high-performance liquid clinicians against their use into the routine workup of males chromatography (HPLC) and capillary electrophoresis for with unexplained infertility. First, none of the tests enable separation of metabolites, whereas mass spectrometry, us to depict the exact etiology and pathogenesis associated Fourier-transform infrared and Raman or near-infrared with infertility. For example, even though sperm DNA spectroscopy are used for identification and quantification damage is mostly oxidatively induced, it may also occur as of the metabolites [90]. Metabolomics testing in the field of a result of apoptosis during spermiogenesis, alterations in andrology is just beginning but its potential to contribute to chromatin remodeling during spermiogenesis, as well as the identification of biomarkers in men with unexplained exposure to environmental toxicants and gonadotoxins infertility is well recognized. such as chemotherapy and radiotherapy [97]. Second, the methods cannot discriminate between physiological and pathological damage as not all DNA damage is detrimental Why it has been difficult to translate specialized [47]. In mammalian species comprising 3,000–4,000 andrology testing from the bench to bedside megabases, about 30 % of the genome has DNA related to genes, while the remaining 70 % are pseudogenes, spacer Translational medicine aims to close the gap between sequences and highly or moderately repetitive DNA ‘‘what we know and what we practice’’ [93–95]. This sequences. Within the 30 % of gene-related sequences, means the transfer of diagnostic and therapeutic advances only about 3 % are DNA sequences directly involved in proven effective in large well-conducted trials to daily amino acid coding sequences while the remaining comprise medical practice [95, 96]. An integral part of TM is regulatory sequences, introns and non-translated sequen- translational research, which encompasses a complexity of ces, as well as active repetitive DNA sequences such as scientific, financial, ethical, regulatory, legislative and ribosomal, RNAs and tRNAs. DNA damage in non-coding practical obstacles that need to be addressed at several regions are not expected to be as detrimental as damage levels to make the process efficient [93, 94]. In the field of occurring in a coding region of the genome, although andrology, TM could be defined as the integrated and cost- epigenetic defects may negatively impact the reproductive effective application of clinical and laboratory biomarkers process due to disrupted gene control [64, 69]. Third, there to improve the understanding and management of male is no consensus with regard to the best method to assess infertility. According to the road map to TM, once a bio- sperm DNA damage and OS, the threshold values to dis- marker is identified and shown to be relevant, it should criminate fertile and infertile men, the ideal way to inter- then move from the bench to the bedside [8, 94]. As such, pret and manage abnormal results and lastly the correlation TM has the potential to deliver practical benefits to patients among tests in reporting abnormal results [47, 48, 51, 99, as it allows the understanding of how interventions are 100]. Proposed threshold values vary among tests and also being implemented in ‘‘real-world’’ settings and their by different authors using the same methods (Table 3)[55, effectiveness; it involves several well-coordinated steps 61, 98, 101–114]. Lastly, there is lack of information on and would require government approval, which is both how the results of OS and sperm DNA tests correlate with expensive and time consuming [93, 95]. past sperm function tests (SFT) in men with unexplained The specialized andrology tests that have been used over infertility. the years worked their way into the clinical field from basic Notwithstanding, there is growing evidence of an asso- research. Despite being translated at different extents, none ciation between both increased sperm DNA damage and of them have fully fulfilled the criteria of TM. Given the OS and reduced fertility, both in vivo and in vitro, and with complexity to perform most of the tests, the difficulties in pregnancy loss after IVF and ICSI [62]. Unlike ASA and interpreting their results, the lack of treatment options for fertilization defects, sperm DNA damage and OS are the detected defects, and in view of the overall success of potential targets for therapeutic interventions including the ART, there is little incentive for infertility clinics to invest use of antioxidants and lifestyle modifications [115]. in these methods. Hence, there is an urgent need to refine these methods and The clinical validity of assessing sperm-mediated validate their cutoff points in different subsets of patients, immunity and fertilization defects has not been confirmed, as well as in different interventions. and nowadays the methods are rarely used. These tests Concerning the translation of novel biomarkers, the should remain primarily on the bench until new develop- challenges are manifold. In spite of the wide number of ments were made that would allow correlation with current potential biomarkers been discovered, their clinical validity and forthcoming tests’ results. is still uncertain. The use of semen for proteomic and As far as the currently used tests are concerned, sperm metabolomics analysis is complex because it contains DNA damage and OS assessments, there are limitations sperm as well as seminal plasma that represent a 123 1046 Int Urol Nephrol (2014) 46:1037–1052

Table 3 Cutoff points of sperm DNA assays to predict in vivo and in vitro fertility Authors, year [ref.] Method Cutoff point (%) Prediction

Evenson et al. 1999 [99] SCSA 30 Discriminate infertile from fertile males Giwercman et al. 2010 [108] SCSA 20 Discriminate infertile from fertile males Spano et al. 2000 [54] SCSA 40 Discriminate infertile from fertile males Sharma et al. 2010 [101] TUNEL 19 Discriminate infertile from fertile males Duran et al. 2002 [113] TUNEL 12 Pregnancy in IUI Bungum et al. 2004 [103] SCSA 27 Pregnancy in IUI, IVF and ICSI Bungum et al. 2007 [109] SCSA 30 Pregnancy in IUI, IVF and ICSI Seli et al. 2004 [106] TUNEL 20 Pregnancy in IVF and ICSI Huang et al. 2005 [105] TUNEL 10 Pregnancy in IVF and ICSI Boe-Hansen et al. 2006 [107] SCSA 27 Pregnancy in IVF and ICSI Borini et al. 2006 [98] TUNEL 10 Pregnancy in IVF and ICSI Benchaib et al. 2007 [104] TUNEL 15 Pregnancy in IVF and ICSI Greco et al. 2005 [114] TUNEL 15 Pregnancy in ICSI Micinski et al. 2009 [110] SCSA 15 Pregnancy in ICSI Simon et al. 2011 [111] Alkaline Comet 25 Pregnancy in IVF Nun˜ez-Calonge et al. 2012 [61] SCD 17 Pregnancy in ICSI with egg donation and selected sperm Gosa´lvez et al. 2013 [112] SCD 24.5; 17.5 Pregnancy in ICSI with egg donation and neat or selected sperm SCSA sperm chromatin structure assay, TUNEL terminal deoxynucleotidyl transferase-mediated digoxigenin-dUTP nick-end labeling technique, SCD sperm chromatin dispersion test combination of prostate, seminal vesicles and bulbourethral laboratory site as the time frame from ejaculation to ana- gland components. Moreover, ‘‘omics’’ analysis of semen lysis can also influence the results [119]. In addition, it is is difficult due to the physiological sperm modifications important to verify whether leukocytospermia is present after ejaculation, which can be further complicated by because leukocytes can be activated by infection and intra- and interindividual variation and other factors such inflammation, in which case they are capable of producing as age [8]. high levels of ROS. Though leukocytospermia is defined by a concentration of white blood cells above 1 million per milliliter of semen, leukocytes below the aforesaid When to recommend testing and how to handle threshold can also be harmful [120]. abnormal results At present, there is fair evidence to recommend that sperm chromatin and OS levels are assessed as part of the Availability of assays to measure DNA damage and OS initial clinical investigation of all males with unexplained varies widely among clinical andrology laboratories and infertility. Determination of OS levels can select subgroups infertility clinics, thus making it difficult to estimate to of patients in which OS is an important infertility factor. what extent they have been implemented. It seems sound to These patients may benefit of antioxidant supplementation speculate, however, that sperm DNA assays are in advan- and life-style modifications including cessation of smoking tage as an easy-to-use land low-cost commercial kit has and weight loss [121, 122]. The OS assays can also help to been available since 2005 [102]. Assays to measure OS are monitor the effect of these interventions. Notwithstanding, more technically demanding, as they require a spectro- clinical trials are necessary to identify in which extension photometer that is an expensive instrument. A ready-to-use these interventions would decrease OS, as well as other colorimetric kit has been developed to detect superoxide important aspects such as composition, dose and duration anion in sperm based on the nitroblue tetrazolium reduction of antioxidant therapy [11]. test, but the test is not yet commercially available [116]. Determination of sperm DNA damage can also provide Practical recommendations for DNA damage and OS useful information for an individualized management. In assays include the ones commonly used in routine semen one study, the probability of fertilization in vivo was close analysis. Special attention should be given to avoid longer to zero when the proportion of spermatozoa showing DNA abstinence periods before semen collection since they were damage exceeded 30 % as detected by SCSA [103]. Sim- shown to increase OS and DNA damage [117, 118]. ilarly, semen specimens with [12 % spermatozoa with Patients should be instructed to collect samples at the DNA damage by the TUNEL assay resulted in no

123 Int Urol Nephrol (2014) 46:1037–1052 1047 pregnancies by IUI [113]. This information is particularly increased pregnancy rate during ART cycles (pooled OR 4.18; relevant for young couples with short duration of infertility 95 % CI 2.65–6.59; p \ 0.00001). Interestingly, sperm DNA who are usually advised to take an expectant approach to damage was decreased by 13.8 % (95 % CI -17.5 to their infertility problem [123]. In the face of abnormal -10.1 %) after oral antioxidant therapy in one of the included results, an active management would be advisable. Treat- studies [124]. In another study, a significantly higher preg- ment of the underlying condition, if possible, and IVF and nancy rate was obtained when sperm injections were carried ICSI have been proposed as the best options. Successful out with testicular rather than ejaculated spermatozoa pregnancies have been obtained using sperm fractions with obtained from men with increased sperm DNA damage (44.4 a high proportion of DNA damage. Of note, significantly vs. 6 %; p \ 0.05) [114]. The aforementioned findings seem higher clinical pregnancy rates (52.9 vs. 22.2 %) and to be related to the threefold lower DNA damage in testicular delivery rates (47.1 vs. 22.2 %) were obtained after ICSI as (13.3 %) compared with ejaculated spermatozoa (39.7 %) in compared to IVF when semen samples with high levels of menwithhighspermDNAdamage[125]. Lastly, recent sperm DNA damage were used [103]. However, even if evidence indicates that the proportion of ejaculated sperm ICSI were applied successfully an increased incidence of with DNA damage can be reduced in approximately 90 % of pregnancy loss would be expected. In a recent meta-ana- men by frequent ejaculation and shorter abstinence periods lysis involving 2,969 couples, the risk of pregnancy loss before semen collection for ART, with positive results on was increased by 2.16-fold when semen specimens with an pregnancy rates [117, 118, 126]. In summary, these findings abnormally high proportion of DNA damage was used indicate that assays detecting DNA damage could be used as a (95 % confidence interval: 1.54–3.03; p \ 0.00001) [52]. tool for both counseling and management purposes. Collectively, these observations indicate that men with We therefore advocate that the current sperm functional high proportions of sperm with DNA damage have lower tests (SFT), ROS and/or sperm DNA damage, are ordered at reproductive potential regardless of the method used for the initial workup of males with unexplained infertility. conception. Abnormal test results would identify couples with a higher Likewise increased OS, medical interventions aimed to risk of remaining childless if an expectant management has alleviate sperm DNA damage can be considered. As DNA been taken. In addition, testing would be useful to monitor damage is the result of both intrinsic and extrinsic distur- interventions and to define which couples are suitable for bances usually associated with OS, it has been proposed that IUI. For those patients already enrolled in ART programs, sperm DNA damage could be alleviated by antioxidant sup- test results would be useful not only to recommend the best plementation [11, 115, 121]. In a recent Cochrane study treatment modality, IVF or ICSI, but also the optimal involving 15 studies and 964 participants, the use of oral abstinence period before semen collection and the source of antioxidants by the male partner was associated with an sperm, ejaculated or testicular in the cases of ICSI (Fig. 1).

Fig. 1 Algorithm for management of males with unexplained oxidative-stress underlying conditions (e.g. subclinical genital tract infertility using current SFT, ROS and/or sperm DNA damage. infections, clinical varicocele). Monitoring of tests’ results should be Testing should be ordered at the initial workup. Abnormal test results undertaken at 2–3 month intervals after interventions. ART are would identify couples at higher risk of remaining childless if an recommended for patients with abnormal results, particularly after expectant management is taken. Interventions aimed to decrease OS interventions. Testicular spermatozoa should be considered for sperm and sperm DNA damage include antioxidant therapy, life-style injections in such cases modiﬁcations (cessation of smoking, weight loss) and treatment of

123 1048 Int Urol Nephrol (2014) 46:1037–1052

Even though the determination of sperm OS levels and more possibilities for targeted therapies, but they still have DNA damage allows clinicians to make more informed a long way to reach the bedside. As part of TM, the decisions in their daily practice and take action based on application of the novel ‘omics’ testing in UMI should start quantitative results, values above a given cutoff point do not with specific objectives and selection criteria that may be preclude the achievement of full-term unassisted pregnancy suggested by the results of previous and current functional [127]. The consequences of sperm DNA damage upon tests. Then, studies using general and organelle specific reproductive outcome depend not only on the severity and omics could be designed in exploration of subcellular site of damage, but also on age-related oocyte capacity to defects that could explain the alterations seen with previous repair sperm DNA strand breaks [128, 129]. Hence, caution and current SFT. should be exercised when interpreting abnormal results since Our proposed working definition of TM applied to the none of the methods have been fully translated yet. clinical andrology laboratory implies that a precisely defined, clinically relevant question drives the process. A key question in the era of assisted reproduction is whether Conclusions any underlying seminal problem in men with unexplained infertility would be cost-effective of investigating and Conventional semen analysis alone is insufficient in the treating rather than applying ART to overcome infertility. laboratory evaluation of men with unexplained infertility. Until we respond to it with certainty, the unrestrictive As routinely performed, it does not account for sperm acceptability and widespread application of specialized dysfunctions including DNA damage and OS. These con- SFT will be halted. ditions are usually contributory for unexplained male infertility. Specialized andrology testing is aimed at shedding light on these subcellular problems though factors Review criteria such as tests’ complexity, difficulties in interpretation, uncertainties about their clinical validity and lack of An extensive search of studies examining the relationship treatment options limit their routine application. Tests that between specialized andrology tests and unexplained male assess ASA, sperm hyperactivation and AR, sperm binding infertility was performed using search engines such as and penetration to the human ZP are rarely used nowadays. ScienceDirect, OVID, Google Scholar, PubMed and Difficulties related to setting-up the assays in the clinical MEDLINE. The overall strategy for study identification andrology laboratory and validation of test results have and data extraction was basedonthefollowingkeywords: limited their application. Moreover, there is no effective ‘‘semen analysis,’’ ‘‘unexplained infertility,’’ ‘‘male infer- treatment for such sperm function defects, and since the tility,’’ ‘‘sperm DNA damage,’’ ‘‘sperm DNA fragmenta- critical steps for fertilization are bypassed by ICSI, these tion,’’ ‘‘oxidative stress,’’ ‘‘autoimmune infertility,’’ issues are no longer a major concern. ‘‘antisperm antibodies,’’ ‘‘fertilization defects,’’ ‘‘acrosome Consensus is growing about the clinical utility of sperm reaction,’’ ‘‘hyperactivation,’’ ‘‘hemizona assay,’’ ‘‘sperm DNA damage and seminal OS assays in males with unex- penetration assay,’’ ‘‘sperm genomics,’’ ‘‘sperm proteo- plained infertility. The application of such assays would be mics,’’ ‘‘sperm metabolomics’’ and ‘‘pregnancy rate.’’ advantageous provided an opportunity for an individualized Articles published in English only were considered. Data management is warranted in the face of abnormal results. that were solely published in conference or meeting pro- From the available evidence, it seems sound to consider these ceedings, websites or books were not included. Book- tests for counseling young couples with short duration of chapter and websites citations providing conceptual content unexplained infertility about their reproductive potential. The were used. assays also can be used to identify and monitor males with increased OS who are candidates for antioxidant therapy. Conflict of interest The authors declare that they have no conflict Equally important for couples considering to embark in ART of interest. would be to know the sperm DNA integrity status. Test results might help to define the best treatment strategy including the References optimal ART method and how the sperm should be collected and handled. Despite all that, clinicians should be aware that 1. 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