The Open Reproductive Science Journal, 2011, 4, 27-41 27 Open Access The Role of Contemporary Andrology in Unraveling the Mystery of Unexplained Male Infertility Alaa Hamada1, Sandro C. Esteves2 and Ashok Agarwal*,1 1Center for Reproductive Medicine, Cleveland Clinic, Cleveland, USA 2ANDROFERT – Andrology and Human Reproduction Clinic, Campinas, Brazil Abstract: Unexplained male infertility is a condition in which infertile men have normal semen analyses on multiple occasions with no obvious physical or endocrine abnormality when female partner infertility has been ruled out. In addition to erectile problems and coital factors, immune causes and dysfunctional sperm may contribute to such condition. Contemporary andrology may help analyze the unexplained male fertility problem on the basis of cellular and sub-cellular mechanisms and select the proper management. This review highlights the concept of unexplained male infertility and discusses the potential causes and its proper management in the era of modern andrology and assisted reproductive techniques. Keywords: Andrology, idiopathic, UMI, sexual dysfunction, cancer, immunogenic, antisperm antibodies. INTRODUCTION as unexplained reduction in semen quality in terms of sperm count, motility, morphology in males who are normal on Infertility is defined as the inability of the couples to physical examination and endocrine testing [5]. This achieve pregnancy after 12 months of unprotected inter- subcategory accounts for 31% of infertile men [5]. Table 1 course. This condition affects 13-15% of the couples world- clearly shows the prevalence of the two conditions [7]. wide [1]. However, the prevalence of such problem differs between countries being higher in the underdeveloped ones Table 1. Distribution of Diagnostic Categories in a Male [2]. This difference may be attributed to disparity in the Infertility Clinic resources allocated for diagnosis and treatment. Male factor infertility is responsible for more than 50% of the problem of Category Frequency the infertility [2]. Male factor is the sole reason in 20% and contributory role in 30-40% [2]. Immunological - While impairment in the male reproductive potential can Idiopathic 32.6% be attributed to various congenital and acquired etiologies Varicocele 26.6% such as cryptorchidism, Klinefelter syndrome, vasal atresia, varicocele, genital infection, endocrine disturbance etc. [3], Obstruction 15.3% no identifiable cause is found in 37-58% of cases of male Normal female factor (unexplained male infertility) 10.7% infertility [4-6]. This category is called male infertility of Cryptorchidism 2.7% unknown origin that is specifically defined as the impairment in the male reproductive function with spontaneous occur- Ejaculatory failure 2.0% rence or as a result of obscure or unknown cause. Endocrinologic 1.5% Generally, male infertility of unknown origin is divided Drug/radiation 1.4% into two groups: idiopathic and unexplained varieties. This classification is different from infertility of unknown origin Genetic 1.2% in women as the idiopathic and unexplained point out the Testicular failure 1.1% same condition. Unexplained male infertility UMI is a sub- Sexual dysfunction 0.7% category of infertility of unknown origin and accounts for 6- 27% of infertile men [4]. The term UMI is reserved for Pyospermia 0.5% infertile men with normal semen analyses on two or more Cancer 0.4% occasions with no demonstrable physical or endocrine abnormalities and female factor infertility has been ruled out Systemic disease 0.3% [7]. On the other hand, idiopathic male infertility is defined Infection 0.2% Torsion 0.1% Ultrastructural 0.1% *Address correspondence to this author at the Center for Reproductive Medicine, Cleveland Clinic, Desk A19.1, 9500 Euclid Avenue, Cleveland, Total 100.0% Ohio44195, USA; Tel: 2163752679; E-mail: [email protected] 1874-2556/11 2011 Bentham Open 28 The Open Reproductive Science Journal, 2011, Volume 3 Hamada et al. The initial assessment of infertile men includes detailed recognition. This protection is achieved by the integrity of history and physical examination with two proper semen the blood-testis barrier. Moreover, the integrity of the analyses and endocrine testing. This initial evaluation is able epithelial barriers augmented by local cellular immunosupp- to identify the cause of male infertility in roughly half of the ressive barriers account for the isolation of the sperm in the patients. However, many others will need to go through the other regions of the male genital tract [10]. Interestingly, novel and sophisticated andrological tests to discover hidden there are three postulated theories that explain the origin of problems. This article will discuss the potential etiologies loss of immune tolerance to the sperm and mounting of and the possible contemporary andrological management immune reactions against sperm as foreign cells. The first outline of the unexplained male infertility problem. theory hypothesizes that newly developing haploid sperm have different chromosomal make up from somatic cells [11]. The second theory claims that sperm are not present CONCEALED ETIOLOGIES OF UNEXPLAINED during the embryonic life when immune tolerance occurs to MALE INFERTILITY (UMI) self antigens [12]. While the third theory, called the In general, normal semen analyses do not guarantee immunosuppressive theory, postulates that T-suppressor fecundity outcomes. The predictive value of normal semen lymphocytes, which are responsible for inhibition of the testing in anticipation of natural pregnancy is only 60% [8]. immune response against sperm, may be down regulated by Table 2 shows the frequency of semen analysis abnormalities continuous leakage of spermatozoal antigens from the in 8,758 infertile patient’s attending a fertility clinic [7]. As a genital tract [13]. The two arms of the immune response, matter of fact, it is a seemingly critical situation for both the humoral and cellular, may be implicated in the etiology of clinician and the couples with unexplained infertility to find UMI. everything is normal on initial lab testing and the couples are Humoral Immune Infertility childless for a long duration. Antisperm antibodies (ASA) are the whole mark of Table 2. Distribution of Abnormalities of Semen Parameters in humoral immune infertility. These antibodies are directed to 8758 Patients [8] various sperm antigens and implicated in sperm dysfunction. About 10% of infertile men have ASA versus 2% of normal Abnormality in semen parameters Frequency fertile men [14]. Nevertheless, Moghissi et al. noticed that the presence of ASA was significantly higher (42.5%) Azoospermia 4% among patients with unexplained and persistent infertility Predominance of a single abnormal parameter 29% [9]. Motility 18% The disruption of blood-testis barrier or epithelial barriers, an immunosuppression defect or genital tract insults Volume 2% are the primary mechanisms responsible for leakage of Morphology 7% sperm antigens and formation of ASA. However, it is still unknown whether ASA are locally formed within the genital Density 2% tract or merely transuded from the serum. Specifically Defects in two or more parameters 37% speaking, ASA are found in three locations in the serum, All parameters normal seminal plasma, and sperm-bound. Among these, sperm 30% bound are the most clinically relevant. Moreover, the (Unexplained male infertility) immunoglobulin (Ig) classes of ASA, namely, IgG which is both locally derived and transuded from serum, and IgA For men with unexplained infertility and normal semen which is thought to be purely locally produced are the most analyses the following possibilities should be considered: i) frequently implicated in the pathogenesis of humoral presence of a female factor, ii) inappropriate coital habits, iii) immune infertility [15]. To add further confusion, 7% to erectile dysfunction, iv) the presence of antisperm antibodies 17% of infertile women can also produce antisperm (autoimmune infertility) and iv) sperm dysfunction [7]. To antibodies in their cervical fluids [16,17]. exclude the first three conditions, thorough history taking as ASA interfere with various sperm functions such as well as a complete gynecological evaluation are needed induction of apoptosis and acrosome reaction prematurity. whereas the modern andrology could aid in managing the ASA may also hinder fertilization event by inhibition of last two conditions. cervical mucus penetration, zona pellucida binding or sperm- oocyte fusion. Furthermore, ASA may change some macro- Immunologic Infertility molecular and sub-cellular function by altering chaperon Immunogenic or autoimmune infertility is defined as an function, protein folding and disulphide bonds [18]. The end improper bodily immune response, whether humoral or result is that pregnancy rates may be reduced by ASA [19]. cellular, against sperm antigens that causes sperm functional Currently, there is sparse evidence, if ever, can be drawn dysfunction and renders the male infertile. Generally, it from traditional semen analysis for the presence of ASA. accounts for 4.5% of the total male fertility problem [5]. Correspondingly, elevated titers of ASA can be found even However, this percentage may rise to 40% among men with in the face of normal semen parameters [20]. Sperm UMI [9]. agglutination is the only highly suggestive phenomenon that Testis is immunologically privileged site to protect the can be seen in semen analysis in cases of elevated ASA titers newly developing haploid spermatozoa from immune [19]. However, this phenomenon is time dependent and Mystery of Unexplained Male Infertility The Open Reproductive Science Journal, 2011, Volume 3 29 rarely involves a large proportion of motile spermatozoa testis in the face of absence of ASA [29,30]. Moreover, cell soon after liquefaction, even when all ejaculated spermato- mediated immunoreactivity has been detected in 50% of zoa are antibody coated [19]. In the same manner, the patients with unilateral and 80% with bilateral surgically immobilizing and apoptogenic impacts of ASA on the sperm repaired cryptorchidism [31]. Meanwhile, sperm exposed to require complement activation which is prevented by the cytokines such as tumor necrosis factor (TNF) and interferon potent anticomplementary substances in the semen [21,22]. gamma show impairment in motility and inability to Notwithstanding, adequate amount of complement is present penetrate hamster eggs [32,33]. Despite these evidences, the in cervical fluid which can be activated through antibody full blown spectrum of cellular immune infertility is difficult antigen reaction and exert toxic effect on sperm. to demonstrate in the lab and the role of this type of immunogenic infertility in UMI patients is still speculative. The diagnosis of immunological infertility requires two Lastly, more sophisticated investigations are needed to detect conditions to be satisfied [23]: the impact of cellular immunity in men with unexplained a) Fifty percent or more of the motile spermatozoa infertility. (progressive and non-progressive) have attached beads. It should be noted, however, that particle Sperm Dysfunction binding restricted to the tail tip is not associated with impaired fertility and can be present in fertile men. In the light of the fact that conventional semen para- meters such as sperm count, motility, vitality and morpho- b) Sperm-bound antibodies interfere with sperm func- logy are inadequate indicators of sperm function and fertili- tion; this is usually demonstrated by using functional zation potentials [34], contemporary sperm function tests tests such as the sperm–mucus penetration test, zona may be of help in delineating these issues. Specifically binding assays and the acrosome reaction. speaking, sperm function tests may provide more clinically Currently, the most popular tests to identify sperm-bound useful prognostic and/or diagnostic information. Such tests ASA are both the direct immunobead test (IBT) and the may be used to distinguish between fertile and infertile men direct mixed agglutination reaction (MAR) [24]. In direct and to aid in revealing the cause of male subfertility and IBT, beads coated with covalently-bound rabbit anti-human suggesting therapeutics. Sperm function tests available in the immunoglobulins against IgG or IgA are mixed directly with andrology armamentarium include assays that investigate washed spermatozoa. The binding of beads with anti-human sperm DNA integrity, seminal reactive oxygen species, acro- IgG or IgA to motile spermatozoa indicates the presence of some reaction, hyperactivated motility and zona pellucida IgG or IgA antibodies on sperm surface [23]. IBT is more binding and penetration. time consuming but it identifies the proportion of antibody- Covert Genetic Causes of UMI bound sperm in a given sample, the antibody class and the location of antibodies on sperm surface. On the other hand, On completion of the meiotic process, germ cells produce direct MAR test is an inexpensive, quick and sensitive 4 haploid spermatids. Meanwhile, Shuffling of some genes screening test where sheep erythrocytes are used instead of occurs between homologous chromosomes during the same immunobeads to detect and localize antibody-bound sperm process giving rise to genetic diversity. Non-disjunction [25,26]. Frequently, antibody-coated sperm may appear as a events during gametogenesis results in either extra or mis- poor postcoital test [27]. Complement, which is normally sing chromosome leading to aberrations in the numerical found in higher amounts in the cervical mucus than in the chromosomal complement called “aneuploidy”. On the other seminal plasma, is needed to immobilize spermatozoa. The hand, structural chromosomal complement defects such as antibody complement reaction may take at least 6 hours to deletions, translocations and inversions also occur in sperm manifest. Physicians performing a postcoital test (PCT) or eggs due to chromosomal insults. Both structural and within 2h after intercourse or using in vitro mucus penetra- numerical chromosomal complement defects can give rise to tion assays may miss the immobilizing antibodies [27]. dysfunctional sperm. Furthermore, the 4 haploid spermatids Consequently, these patients may appear as having an undergo the process of spermiogenesis to form mature and absence of male factor infertility. It is therefore advisable to motile sperm. Interestingly, during spermiogenesis, the hap- perform a PCT after at least 6 h following intercourse [27]. loid sperm chromatin undergoes significant changes in ASA can cause infertility without obvious problems with which most histones are replaced first by transition proteins, cervical mucus penetration. Such antibodies may interfere then by positively charged protamines [35]. By this remodel- with the acrosome reaction and inhibit sperm penetration ing process the sperm DNA condenses so tightly that it is into the zona-pellucida and fusion with the oocyte [28]. resistant to mechanical stresses such as sonication and even to boiling, which destroy the DNA in somatic cells [36]. The Not only the exact site and mechanism for ASA produc- condensation of sperm DNA protects it during its transit tion in UMI patients are still unknown but also the impli- through the male and female reproductive tracts. Cytogenetic cated sperm antigens are not identified yet. Improvement in analysis and molecular biology genetic testing may identify recent techniques in sperm surface proteomics may help subfertile males misdiagnosed as having unexplained and pinpoint these antigens, improve our understanding of idiopathic infertility. Abnormalities causing male infertility immune infertility and may suggest specific therapy later on. include: Cellular Immune Infertility  Alterations in chromosomal complement Evidence for cellular immune infertility emerges from a  Gene mutation and polymorphisms variety of human and animal studies on torsion that were  DNA integrity defects able to detect inflammatory cell infiltrate in the contralateral 30 The Open Reproductive Science Journal, 2011, Volume 3 Hamada et al. Alterations in Chromosomal Complement analysis is rarely performed in the evaluation of male infertility [52]. In animal studies involving the mice, up to The possibility of having sperm chromosomal aneuploidy 300 null mutations and 50 conditional targeted deletions is inversely related to sperm concentration and total prog- have produced models of male infertility. Not only the DNA ressive motility [37,38]. Increased sperm aneuploidy rates sequence has an effect on male fertility but there is also a may impact male fertility and pregnancy viability. The exact role for epigenetic events and modifiers of gene expression. causes of aneuploidy are mostly unknown, but smoking, alcohol, chemotherapy and ageing may play a role. The It is likely that specific genes play a role in patients with overall frequency of chromosomally abnormal sperm in the unexplained male infertility since they control meiosis general population is estimated to be 7%. Moreover, the events, spermiogenesis, remodeling, motility, capacitation mean frequency of disomic sperm (presence of two copies of and fertilization. It is now possible to monitor the expression a chromosome) for autosomes and sex chromosomes are of thousands of genes simultaneously with DNA microarray 0.13% and 0.37%, respectively, while the rate for diploid analysis. In a comparative analytic study on spermatozoa sperm (two copies of each chromosome) is 0.2% [39,40]. For from normospermic infertile men and fertile healthy controls normospermic infertile males, the corresponding figures are using microarray technology, hundreds of gene sequences 0.11%, 0.44% and 0.3-1%, respectively [39,40]. Whereas, (targets) were differentially expressed between these two the actual figures of sperm disomy and diploidy for those groups; there are few genes that are overexpressed whereas men with poor semen quality are even higher. all others are underexpressed in infertile men [53]. Interestingly, the inter-chromosomal variation in the rates Novel genetic studies are needed to decipher specific of disomies has been observed with sex chromosomes and genes that may be implicated in UMI patients. Due to the chromosomes 21 and 22; the higher rate of abnormalities fact that environmental factors may also influence genetic related to such chromosomes may be due to their lower rate expression in various ways and may even alter the posttrans- of meiotic recombination which renders them more prone to lational modifications of their products, the imperative non-disjunction [41]. dynamic interactions between human genes and environ- mental factors must be taken in consideration in analyzing Equally important, both morphologically normal and gene related fertility problems. abnormal spermatozoa can be disomic or diploid [42], or contain damaged DNA [43]; as such, selecting normally DNA Integrity Defects looking spermatozoa for ART does not guarantee the Sperm DNA integrity is increasingly being distinguished absence of chromosomal abnormalities. However, abnormal as an important marker of fertilizing efficiency, and it is spermatozoa that retain excess cytoplasm (ERC) exhibit a associated with better diagnostic and prognostic values than greater extent of aneuploidy and diploidy than those without standard sperm parameters [54]. Saleh et al. reported that an ERC from the same ejaculate whether selected by density increase of spermatozoa with abnormal chromatin structure gradient centrifugation [44], swim-up [45] or binding to or DNA damage (expressed as DNA fragmentation index, hyaluronic acid [46]. Structural chromosomal aberrations DFI) is negatively correlated with intracytoplasmic sperm such as inversions, deletions, balanced or unbalanced injection (ICSI) and in vitro fertilization (IVF) outcomes translocations and Y-chromosome microdeletions are often [55]. associated with abnormal semen parameters and higher rates of abortion and, in some cases, with higher risk for the birth Significantly, sperm with DNA damage are more often of a severely handicapped child [47]. seen in subfertile/infertile men than in fertile ones [56-58]. Successful fertilization of the human ova with spermatozoa Surprisingly, in Y-chromosome microdeletions related with damaged DNA may lead to paternal transmission of infertility, the AZFc region is prone to many smaller sub- defective genetic material with adverse consequences to deletions that are thought to be caused by intrachromosomal embryo development [59,60]. Approximately 8% of infertile recombination [48]. These partial deletions produce a wide men have abnormal DNA integrity despite normal semen array of phenotypes, ranging from normospermia to azoo- parameters [61]. The etiologies of such defects are due to spermia, due to factors that include the interaction of the variety of extrinsic and intrinsic factors. Heat, smoking, environment and the genetic background [49]. alcohol, radiation and other gonadotoxins are examples of Most chromosomal abnormalities may be detected by extrinsic factors. The intrinsic factors such as protamine using one of the following methods: (i) Sperm karyotyping deficiency, mutations that affect DNA packaging, reactive for detection of numerical chromosomal abnormalities; (ii) oxygen species and ageing are the main known factors [61]. Fluorescence in situ hybridization analysis [50], which can DNA damage is often assessed by the determination of be used to assess numerical and structural chromosomal chromatin compaction or DNA fragmentation. The former changes by using specific probes; and (iii) Quantitative examines the accessibility of dyes (acridine blue, aniline Polymerase Chain Reaction (Q-PCR), which is a promising blue and chromomycin A3) to nucleoproteins or chromatin technique to detect and quantify damage to nuclear and after challenging spermatozoa with physical insults; as such, mitochondrial DNA [51]. it reflects how susceptible the DNA is, or has been, to Specific Gene Defect (Mutations and Polymorphisms) noxious agents [62]. Toluidine and aniline blue stains binds to lightly packed chromatin and to lysine residues of acridine The sequencing of nucleotide bases in human genome that are not fully replaced by protamines, respectively. has widely opened the door for recognition of human fertility Chromomycin A3 (CMA3) binding is specific for related genes. Further research has embarked to analyze protamine-deficient areas because of its affinity to guanine- these genes in the near future. However, DNA sequence cytosine (G-C)-rich areas of DNA [63]. On the other hand, Mystery of Unexplained Male Infertility The Open Reproductive Science Journal, 2011, Volume 3 31 DNA fragmentation is measured by detecting single or nescence and flow cytometry, and indirect methods such as double strand DNA breaks. TUNEL (Transferase-mediated the colorimetric one. Chemiluminescence uses the probes dTUP nick-end labeling), Comet assay, acridine orange test lucigenin or luminol to detect ROS [78,79]. Luminol and SCSA (Sperm Chromatin Structure Assay) are methods (C H N O ) is a versatile chemical that exhibits chemilumi- 8 7 3 2 clinically available to detect DNA fragmentation. Although nescence with photons emission when mixed with an they differ in costs and methods, most of the mentioned tests appropriate oxidizing agent. It can penetrate inside the cell are clinically significant and correlates with sperm function and react with intracellular reactive oxygen species in and fertility [64]. addition to extracellular ones [79]. Photons produced are converted to an electrical signal and measured by lumino- Oxidative Stress meter, with ROS generation being measured as counted The Imbalance between the production of reactive photons per minute (cpm) [80]. The normal range is oxygen species (ROS) and the natural antioxidant defense <0.2×106 cpm per 20 million spermatozoa [81]. Intracellular system is termed as oxidative stress (OS). OS has a ROS can be measured by flow cytometry using different pathological effect on sperm function by causing damage to fluorescent probes such as 2’, 7’-dichlorofluorescin-dia- sperm DNA in the nucleus and mitochondria, and inducing cetate, hydroethidine that react with ROS to emit a red lipid peroxidation in the sperm plasma membrane as well as fluorescence [82]. The colorimetric technique is also widely denaturing cellular protein [65]. Mammalian spermatozoa used for indirectly quantifying ROS. It is based on the are redox cells that are able to produce reactive oxygen principle of spectrophotometry and measures lipid peroxide species and to export them to the extracellular medium [66- end products, mainly malondialdehyde, lipid hydroperoxides 70]. ROS are specifically defined as a group of metabolites and isoprostanes [83]. formed by reduction of oxygen including free radicals Various methods for total antioxidant capacity measure- (molecules with unpaired electron) such as superoxide anion ments are available such as enhanced chemiluminescence (O –•), the hydroxyl radical (OH•) as well as powerful oxi- 2 assays, spectrophotometric methods, fluorometric methods dants such as hydrogen peroxide (H O ). The origin of ROS 2 2 such as ORAC assay and electrochemical methods such as in semen is mainly from immature spermatozoa and seminal coulometry, voltammetry or electron spin resonance assay. leukocytes [65]. Some of these methods are commercially available ROS in low levels have a physiological role. They are particularly, spectrophotometric methods, fluorometric required by sperm to attain their functional maturity and are methods [84]. essential for capacitation, hyperactivation and acrosome Impairments in Fertilization Process reaction. ROS also exert their effect on sperm-oocyte inter- action. Low levels of lipid peroxidation cause modifications The ability of the sperm to fertilize the ova is related to of plasma membranes facilitating sperm adhesion to the its potentials of undergoing capacitation, which includes the oocyte [71,72]. However, the true physiological ROS levels acquisition of hyperactivated motility, and the acrosomal are still undetermined. reaction to penetrate the zona pellucida and its fusogenic ability with oolema. The competent sperm can successfully In fact, sperm are extremely susceptible to the effect OS achieve all these physiological processes culminating in because of their high content of polyunsaturated fatty acids fruitful fertilization outcome. It has been assumed that nor- (PUFA) in plasma membranes as well as their limited mospermic infertile men may have defective (incompetent) antioxidant defense [73]. sperm that are unable to fertilize. This assumption is ROS are usually associated with poor semen quality and strengthened by the observation of low success rates of IVF male infertility. It has been shown that 40%–88% of and intrauterine insemination (IUI) in certain cases of nonselected infertile patients have high levels of seminal unexplained infertility. Various studies examined the ROS [74]. However, recent reports found that normospermic defective fertilizing performance of the sperm from different infertile men have higher ROS levels and reduced total angles encompassing all the stages of sperm-egg antioxidant capacity (TAC) levels than the normospermic interactions. fertile counterpart [75]. The exact prevalence of OS in Zona Pellucida Binding Defects normospermic infertile men is unknown. Nevertheless, a controlled study on limited number of patients detected that Human ZP (hZP) is composed of four major 11% of normospermic men have OS [75]. glycoproteins (hZP1, hZP2, hZP3 and hZP4) [85]. Of these, ZP3 of human oocyte is believed to be the primary receptor Interestingly, ROS may seriously impair male reproduct- for capacitated acrosome intact sperm binding [85,86]. ive potential without causing gross deteriorations in the semen parameters. This impairment may be explained by Typically, sperm binding to ZP is through complimentary, deleterious effect of ROS on genetic material and on sperm species-specific, receptors or binding sites on the surface of capacitation and acrosome reaction [76]. In fact, ROS are the gametes [87-89]. A number of candidate sperm proteins have been found to be able to interact with either solubilized considered independent markers of male factor infertility or intact ZP. However, it is not clear whether or not they are [77]. the primary receptors for binding to the ZP [86,90-92]. In The most often used methods for detecting oxidative fact, sperm binding to ZP3 induces signal transduction stress include methods to measure ROS and methods to pathways within the spermatozoon, involving multiple measure total antioxidant capacity (TAC). In an andrology proteins, particularly protein-kinases A and C, that lead to setting, ROS measurement techniques are divided into two the acrosome reaction [93]. Moreover, acrosome-reacted major categories, i.e., direct methods such as chemilumi- spermatozoa are believed to bind to ZP2 that facilitates the 32 The Open Reproductive Science Journal, 2011, Volume 3 Hamada et al. penetration to the zona matrix and progression into the piece of the sperm flagellum is the prime mechanism for perivitelline space [94]. hyperactivation [106-108]. This entry is induced by intra- cellular alkalinization due to extrusion of H+ through voltage Defective ZP bound sperm are present in approximately gated proton pumps which are also located in the principal 15% and 25% of subfertile men with a normal semen piece of the flagellum [106]. Increased intracellular pH and analysis and with abnormal ones, respectively [95-97]. Such intracellular Ca+ regulate not only the HA process but also individuals have a reduced chance of achieving successful the acrosome reaction and the ability of the sperm to fertilize fertilization when undergoing IVF [97]. Mackenna et al. the egg [106]. Molecular studies on CatSper ion channel reported that two out of 18 men with unexplained infertility reveal that it is a novel protein complex that is composed of showed lack of sperm binding to ZP despite having sperm six subunits. Of these, four are α subunits (CatSper1-4) with morphology and hyperactivation status similar to fertile calcium selective pore and two are transmembrane proteins subjects [98]. The presence of defective sperm-zona with large extracellular domains, called CatSperβ and pellucida binding (DSZPB) in infertile men with normal CatSperγ, of unknown functions [109,110]. semen may be due to defective signal transduction pathways upstream of protein kinase A and C. However, most DSZPB The extent of hyperactivated motility in a population is infertile men with normal semen and those with severe positively correlated with the extent of zona binding, the teratozoospermia are likely to have downstream disorders, acrosome reaction, zona-free oocyte penetration and fertili- structural defects or absence of sperm receptors for binding zing capacity in vitro [111]. However, only a small propor- the ZP. tion of the sperm population may be hyperactivated at each time [105]. Additionally, only the capacitated sperm are able Two tests of sperm binding to the human zona have been to migrate towards the ovum site, under thermotaxis and described: i) the hemizona assay and ii) the sperm-zona chemotaxis stimuli [112,113]. binding ratio test. In the former, a single zona is bisected and each zona half is incubated with control and patient sperm There are specific kinetic concepts that characterize the suspensions [99]. In the latter, a complete zona is incubated motility dynamics of HA. These kinetic concepts are with equal numbers of motile spermatozoa from control and measured in vitro by using computerized motion analysis in test populations, each labeled with a different fluorescent conjunction to kinematics module to distinguish different dye [100]. In each case the number of spermatozoa from subpopulations of motile spermatozoa. HA sperm have high each population bound per whole or half zona is counted and curvilinear velocity (VCL), low linearity (LIN) and large the number of test sperm expressed as a ratio of that of the amplitude of the lateral head displacement. The clinical control. significance of such data is reflected by their correlation with IVF outcomes and spontaneous pregnancy rates [114]. Capacitation Defects Mirihasimu et al. showed that there is a significant Capacitation is a complex combination of two concomi- decrease in the percentage of hyperactivated sperm, sperm tant processes; mainly, the hyperactivation (HA) in which motility, progressive motility, and curvilinear velocity the sperm acquire a new motility pattern known as (VCL) from infertile men in comparison to sperm from hyperactivated motility and the acrosomal reaction (AR) fertile donors after overnight incubation with capacitating [101]. Defects in capacitation may explain subfertility in conditions while LIN was increased in the former [115]. some normospermic infertile men. Computerized assessment of follicular fluid (FF)-induced Hyperactivation (HA) is considered the first step of the hyperactivation (HA) has been proved to be significantly complex capacitation process. It involves a typical swim- lower in patients with unexplained infertility in comparison ming pattern of movement shown by most sperm retrieved with normal fertile men [99]. The absence of HA after the from the oviductal ampulla at the time of fertilization [102]. addition of FF was observed in 39% of patients with Hyperactivated sperm exhibit high amplitude and asym- unexplained infertility [98], it is likely that spermatozoa metrical flagellar bending movement. Hyperactivation is from such patients have reduced ability to penetrate through characterized by switching of sperm movement from prog- the oocyte vestments and ZP as a result of this abnormal HA ressive motility to more vigorous (non-progressive) flagellar response to FF. Matthew et al. discovered that male patients motion. The role of hyperactivation is to enhance the ability with mutated CatSper1gene are infertile with poor HA of sperm to detach from the oviduct wall, to move around in response despite their normal sperm count, morphology and its labyrinthine lumen, to penetrate into the cumulus even their initial motility [116]. Furthermore, an animal oophorus and, finally, to efficiently drill zona pellucida and study on mice concluded that mutation in each of CatSper reach the oolema [103]. It is speculated that specific signals ion channel protein can lead to infertility despite of normal and physiologic stimuli appear within the oviduct shortly semen parameters, normal testicular histology, size and before ovulation and act to induce hyperactivation. These weight [117]. Interestingly, there are two known CatSper2 include hormones (e.g. progesterone), ions and secretions in gene related mutations in humans that cause male infertility, the oviduct luminal fluid [104]. Moreover, when the oocyte termed CatSper-related nonsyndromic male infertility and enters the oviduct, it usually brings along cumulus oophorus deafness-infertility syndrome [118]. However, both syn- and follicular fluid that have been shown to influence dromes are associated with gross semen abnormalities. hyperactivation sperm motility as well [105]. Further investigation is needed to disclose the genetic and molecular nature of fertilization in patients with defective Recently, it has been suggested that increased intra- HA response and unexplained infertility. Moreover, minor cellular calcium entry through voltage gated calcium chan- mutations in human CatSper genes are yet to be deciphered nels (CatSper1-4; Cation channel of Sperm) in the principal in males with unexplained infertility. Mystery of Unexplained Male Infertility The Open Reproductive Science Journal, 2011, Volume 3 33 Acrosome Reaction Defects Defective ZPIAR can be caused by different mechanisms in men with or without severe sperm morphological defects. The acrosome reaction (AR) is defined as the process of In men with normal sperm morphology, defective ZPIAR is fusion of sperm plasma membrane with outer acrosomal most likely to be due to subtle biochemical or molecular membrane leading to release of exocytotic proteolytic defects in ZP receptors, signal transduction pathways, enzymes (acrosine and hyaluronidase) in response to sperm– inefficient cholesterol or zinc removal from the plasma zona pellucida binding. Human sperm initiate primary membrane during capacitation, actin polymerization or binding to the ZP with intact acrosome [119]. ZP3 is acrosomal enzyme activation [89,133-138]. Furthermore, it considered the natural stimulus for the AR which leads to the is possible that other prostatic secretions (e.g., citric acid) proteolytic dissolution of the zona pellucid [120]. might affect zinc levels or even sperm function [127]. Zinc There are two types of defective acrosome reaction levels determination in the seminal plasma is unlikely to be which have clinical significance. The first is the acrosome clinically useful for the prediction of defective ZPIAR; reaction prematurity which is defined as high level of currently, only the spermatozoa–ZP interaction tests using spontaneous acrosome reaction (>20% of spermatozoa human oocyte have been shown to be accurate [127]. exhibiting spontaneous AR) [121]. The second is the AR To assess the inducibility of AR, artificial stimuli used in insufficiency which is defined as poor responsiveness to AR vitro to challenge the acrosome reaction such as calcium stimulants (when <15% of spermatozoa responded to AR ionophore A23187 and progesterone. Under normal stimulants) [121]. Both conditions are associated with poor conditions, >15% AR in response to ionophore treatment is fertilization capacity on conventional IVF treatment. AR expected [139]. Visualization of acrosome reacted sperm can prematurity is usually associated with gross semen be achieved using different techniques as follows: abnormalities such as poor sperm motility and abnormal morphology and may be easily diagnosed [122-124]. 1) Birefringence characteristics of acrosomally reacted spermatozoa under polarized light microscopy [140] Some patients with unexplained infertility that have normal sperm–ZP binding have defective ZP-induced AR 2) Fluorescence microscopy after staining with a (AR insufficiency), which will result in reduced sperm–ZP fluoresceinated lectins [141,142]. penetration and failure of fertilization [125]. Patients with 3) Flow cytometry after addition of fluoresceinated this condition usually have a long duration of unexplained antiCD-46 monoclonal antibody [143]. infertility, normal semen analysis, and normal sperm–ZP binding, but show failure of ZP penetration by sperm and Defective Fusogenic Ability of the Acrosome Reacted have zero or low rates of fertilization with standard IVF Sperm with the Oolema [125]. The diagnostic feature is that very low proportions of The fusogenic potential of the capacitated sperm is repre- sperm undergo AR after binding to the ZP [126]. However, sented by ability of the equatorial region of the acrosome- those patients achieve high fertilization and pregnancy rates reacted human sperm to fuse with the vitelline membrane of with ICSI [126]. Although the frequency of defective ZP the oocyte. The fusogenic ability is usually tested by using induced AR (ZPIAR) was high in subfertile men with the sperm penetration assay (SPA) also known as the zona- idiopathic oligozoospermia (65%) and severe teratozoosper- free hamster oocyte penetration test. Although this test does mia (62%, strict normal sperm morphology ≤ 5%), defective not assess sperm-ZP interaction, it measures the spermato- ZPIAR was found in only 25% of normozoospermic zoon’s ability to undergo capacitation, acrosome reaction, subfertile men [96]. fusion and penetration through the oolema, and deconden- Although the exact mechanisms of AR insufficiency are sation within the cytoplasm of an oocyte. Basically, the zona unknown, defective ZPIAR is more likely to be related to pellucida is removed from a hamster oocyte, which is then major structural defects of the sperm head, such as small or incubated with human spermatozoa. In the original test abnormal acrosome, or associated abnormalities in the scoring is achieved by calculating the percentage of ova overlying plasma membrane in severe teratozoospermic which are penetrated; normal sperm are able to penetrate 10- subfertile men. In normozoospermic men, it has been shown 30% of hamster ova [47]. Recent refinement of this test is that the seminal zinc concentration was significantly higher performed by incubating sperm in a more potent capacitating in men with defective ZPIAR [127]. Zinc is considered one media which allow the majority of ova to be penetrated; of the decapacitating factors normally present in semen to scores are obtained by calculating the number of sperm that counteract AR prematurity [128]. Decapacitation factors penetrate each ovum [47]. Aitken et al. reported that 34.1% such as zinc, cholestryl sulphate and other proteins are of patients with unexplained infertility had <10% oocyte usually adsorbed on and stabilize the plasma membranes penetration against 0% in a control group of fertile men during sperm journey in the male and female reproductive [144]. Various studies have evaluated the ability of the SPA tract until reaching the oviduct [128]. The addition of these to predict success or failure of IVF. Some investigators have decapacitating factors to the culture medium inhibits sperm shown no correlation with an abnormal test [145], whereas capacitation and hyperactivated motility, as well as others have claimed 100% predictability [146]. Taking an spermatozoa–ZP binding and penetration in vitro [129-132]. average from different studies, a normal SPA may have 70% Therefore, high seminal zinc concentration may have an predictability of fertilization in vitro [27]. Nonetheless, adverse effect on the ZPIAR. It is also possible that zinc semen samples which fail to fertilize hamster ova usually are binding to the sperm plasma membrane affects calcium unable to fertilize human ova [47]. Although the SPA is influx through ion competition during capacitation. considered a research tool, it may be of clinical value for 34 The Open Reproductive Science Journal, 2011, Volume 3 Hamada et al. men with unexplained infertility with poor fertilization rate assay, capacitation, hyperactivation motility, inducibility of on IVF. acrosome reaction and the ability of sperm to fuse with the vitelline membrane (zona-free hamster egg penetration test) may be used. Fig. (1) depicts the management plan for A RATIONAL WORK UP PLAN FOR THE unexplained male infertility. MANAGEMENT OF MALES WITH UNEXPLAINED INFERTILITY TREATMENT STRATEGIES The initial test for couples with unexplained male infertility is the post-coital test (PCT). PCT is a technically The treatment of males with unexplained infertility does challenging test that must be appropriately timed and not follow the typical rules for standard clinical practice performed. Cervical mucus is normally hostile to sperm, decision making. Such a convention requires a specific except near the time of ovulation. The absence of sperm on a scientific plan to identify and correct a known defect and the postcoital test in the presence of normal semen parameters calculation of risks versus benefits. Due to the facts that no suggests incorrect coital technique or failure to ejaculate into recognizable reason for infertility is identified and few vagina while the presence of normal sperm numbers but randomized clinical trials are available, no uniform protocol reduced motility or a shaking motion on a postcoital test is could be followed. Counseling is an important part of suggestive of the presence of antisperm antibodies [147]. management particularly with regard to orientation about the The finding of a normal postcoital test raises the possibility physiology of ovulation and the need to time intercourse of a functional sperm defect. Assessment of sperm function with the periovualtory period. A detailed medical history can be divided into two steps. The first step should be to may help to disclose any hidden problems such as sexual check the competence of the sperm before fertilization event dysfunction and inadequate coitus habits. by measuring the levels of ROS as well as DNA and chromatin integrity defects. The second step should include Watchful Waiting the assessment of the fertilization potential of sperm Watchful waiting is advised for young couples with short especially for those patients with history of prior failure of duration of infertility. Pregnancy may occur spontaneously conventional IVF. These tests include: sperm-ZP binding without any interventions in cases of unexplained infertility Fig. (1). Work up plan for unexplained male infertility. ROS (reactive oxygen species), ICSI (intracytoplasmic sperm injection), ART (assisted reproductive techniques). Mystery of Unexplained Male Infertility The Open Reproductive Science Journal, 2011, Volume 3 35 [148]. Hull et al. found a cumulative pregnancy rate (PR) centrifugation. However, the authors observed that sperm ranging from 50-80% over a 3-year period as a function of washing was ineffective to remove ASA in approximately female age and 30–80% PR as a function of infertility 30% of the cases, and advise that the potential benefit of this duration [149]. Cumulative pregnancy rates of 60% may be strategy has to be tested individually [158]. Microinjection achieved within 2 years [148]. However, infertility periods of the compromised spermatozoa into the oocyte cytoplasm longer than 3 years are associated with very low PR of 1-3% (ICSI) bypasses sperm-oocyte membrane interaction, and particularly if the female partner is aged 35 years or older ICSI has been shown to increase fertilization when compared [148]. For couples whose time to conceive is longer than to conventional IVF in cases of male immunologic three years, the cumulative PR decreases by 2% for each infertility. Nagy et al. analyzed the outcome of ICSI in men year of age after 25.7 years [150]. Due to the costs of with a proportion of antisperm antibody-bound spermatozoa infertility treatments and given high proportion of couples of 80% or higher [159]. They concluded that fertilization, with unexplained infertility who spontaneously conceive cleavage and pregnancy rates after ICSI were not influenced within a 2-year period, it is advisable to defer treatment of by the percentage of ASA-bound spermatozoa, by the couples in this time period unless the female partner is aged dominant type of antibodies present, or by the location of 35 years or older. ASA on the spermatozoa. However, embryo quality was lower in the ASA-positive group [160]. In another study, Interventional Management similar results were observed but a higher rate of first trimester pregnancy loss was observed in the ASA positive Interventions, which include medication and/or surgery group. Clarke et al. and Check et al. studied 39 patients with or assisted conception, are justified in cases of unexplained a strong positivity on IBT (>80%) and 93 patients with infertility of long duration and/or advanced maternal and various degrees of autoantibodies, respectively [161,162]. paternal age. They found that fertilization and pregnancy rates were Immunological Infertility comparable between different levels of ASA on sperm. Esteves et al. analyzed a large cohort of 351 patients and Treatment of immune infertility includes methods to either confirmed that fertilization, cleavage and pregnancy rates decrease ASA production or to remove sperm-bound ASA. after ICSI were not influenced by the ASA levels on sperm ASA titers may be decreased by using condoms and [163]. These authors neither observed the negative impact of systemic steroid. Condoms are of theoretical benefit because ASA on embryo quality and cleavage rate nor an increase in they may help to lessen the chances for frequent exposure of pregnancy loss, as reported by others. They also compared sperm to female reproductive tract and hence decrease the ICSI outcomes between patients with ASA positivity and a sensitization and formation of ASA in cervical mucus [151]. group of patients in which ICSI was indicated for other Immunosuppressive therapy had been tried in early years reasons. Fertilization, embryo development, pregnancy but it is seldom used nowadays mainly because of the high success and miscarriage rates after ICSI in men exhibiting incidence of side-effects [152]. Moreover, efficacy of varying levels of autoimmunity against spermatozoa were steroids remains unclear as most studies lack appropriate within the same range as our population of ICSI patients placebo controls or have used different regimens and drugs. with severely abnormal seminal parameters. The authors Despite these shortcomings, two prospective and randomized conclude by suggesting that ASA may become inactive placebo-controlled studies were conducted and showed within the ooplasm after microinjection, or that a segregation conflicting results. In the study of Hendry et al, 40 mg of process may take place during the first cleavage divisions, prednisolone was given for a 6-month period from cycle similar to the inactivation and segregation processes that also days 1–10 of the female partner, and then tapered rapidly for occur with the acrosome and sperm tail after microinjection. the next 2 days [152]. The PR of treated and untreated Excessive Oxidative Stress groups was 31% and 9%, respectively. In another study, the authors reported similar PR after administration of Men with unexplained infertility may have higher methylprednisolone for three cycles despite a significant oxidative stress than controls [75,164]. Lines of therapy decrease in sperm-associated immunoglobulin IgG (but not include lifestyle habits modification, use of antioxidants and IgA) in the steroid treatment group [153]. It has been shown ART. Patients are advised to quit smoking, eat antioxidant- that steroids may be only effective in removing sperm-bound rich food and avoid pollutant environmental conditions. An ASA in presence of low antibodies titer [154]. Treatment antioxidant therapy has attracted attention in the recent years. with high dose steroids for long time is associated with side- Antioxidants are compounds and reactants which dispose, effects that include mood changes, fluid retention, dyspepsia, scavenge and suppress the formation of ROS, or oppose their gastrointestinal bleeding, aseptic necrosis of the hip joint, actions. Various antioxidants such as carnitine, vitamin C, and significant decrease of bone mineral density in up to vitamin E, coenzyme Q10, selenium, glutathione, N-acetyl 60% of the patients [152,155-156]. cysteine, carotenoids and trace metals are available. A recent Cochrane review on the use of antioxidants for male Alternatively, methods to remove ASA already bound to subfertility suggests that antioxidant supplementation may sperm include sperm washing and IgA protease treatment. improve the outcomes of live birth and pregnancy rate for Effectiveness of these techniques in recovering antibodies- subfertile couples undergoing ART cycles, but further head free spermatozoa are conflicting; most reports show limited to head comparisons are necessary to identify the superiority success due to the difficulty of eluting the sperm cell surface of one antioxidant over another [165]. Additionally, by any washing method [157]. Schneider et al. demonstrated therapeutic dosing, duration and the toxic levels of reactive that the population of antibodies-free spermatozoa was oxygen species (ROS) are still to be determined. increased by 29% after discontinuous colloidal gradient 36 The Open Reproductive Science Journal, 2011, Volume 3 Hamada et al. DNA Damage these conditions remains low, current data is limited and study populations are heterogenic [177-180]. It is therefore The management of unexplained male subfertility due to recommended that well-defined groups of couples under- DNA damage often requires ART. The probability of fertili- going ICSI with ejaculated sperm, ICSI with epididymal zation in vivo and by intrauterine insemination (IUI) seems sperm and ICSI with testicular sperm, and a control group of to be low when the proportion of sperm cells with DNA naturally conceived children are closely followed up. damage exceeds 30% and 12%, as detected respectively by SCSA or TUNEL [166,167]. Sperm DNA damage is nega- Fertilization Defects tively correlated with embryo quality and blastocyst forma- ART is indicated for fertilization defects involving sperm tion in in vitro fertilization (IVF) cycles and with fertiliza- capacitation, sperm-ZP interaction or sperm-oocyte fusion. tion rates both in IVF and ICSI cycles [168]. However, Couples should be advised that significantly higher rate of successful pregnancies in IVF/ICSI cycles can be obtained successful pregnancy achieved with IVF-ICSI compared to using semen samples with a high proportion of DNA dam- conventional IVF and IUI in such cases [95,126,181]. age. Bungum et al. demonstrated that significantly higher clinical pregnancy rates (52.9 % vs. 22.2 %) and delivery Donor Insemination rates (47.1 % vs. 22.2 %) were obtained after ICSI as com- Donor insemination is an alternative when all the above pared to IVF when semen samples with high levels of sperm treatment options fail. DNA damage were used, as previously suggested [169]. For infertile men with sperm DNA damage with history of multiple failures on IVF/ICSI, it is recommended to use EXPERT COMMENTARY testicular, rather than ejaculated sperm, harvested by either Remarkable developments have been achieved in the TESE or TESA [170]. Pregnancy rates in first cycles of field of andrology in the recent years which significantly TESA–ICSI are relatively high in these couples [170] and it improved our understanding of sperm physiology. Novel has been suggested that if pregnancy does not take place in diagnostic tools envision real time sperm function and may the first TESA–ICSI cycle, the cause of infertility in these aid in revealing sperm hidden alterations possibly related to couples may lie elsewhere. Interestingly, it has been hypo- infertility. Men facing unexplained infertility are charac- thesized that the degree of sperm DNA damage increases terized by being childless despite presence of normal semen with time after Sertoli cell release of testicular sperm. Other parameters and normal female partner evaluation. Obviously, techniques with variable success have been used to select classical detailed history taking and physical examination is sperm with lower DNA fragmentation index such the use of always necessary to disclose erectile dysfunction problem or Annexin-V columns [171,172], and hyaluronic acid binding irregular coital timing with regard to the periovualtory [46]. period. However, when all these measures fail, there is still Additionally, other sperm selecting techniques based on necessary to go through more sophisticated and expensive high resolution imaging of sperm include the selection of tests monitor sperm function in more detail. ART may solve spermatozoa devoid of surface vacuoles by high-magni- the problem of unexplained male infertility and bypass all fication ICSI [173] and the recently introduced confocal light the natural barriers that a dysfunctional sperm must face to absorption scattering spectroscopy [174] technology achieve fertilization. However, as interventional therapy, (CLASS). This novel technique allows for the noninvasive ART is not without complications and further studies are visualization of sperm subcellular structures such as intact needed to refine its role and decrease its impacts on the chromatin to be microinjected by ICSI. offspring. The activation of embryonic genome expression occurs at Normal semen parameters do not guarantee fecundity. the four- to eight-cell stage in human embryos. Therefore, This concept is important for all clinicians involved in the the paternal genome may not be effective until that stage and management of the subfertile male population. Nowadays, it is speculated that an elevated level of sperm DNA strand one of the main goals of scientists working in the andrology breaks seems to be of importance in the later stages of field is to find a diagnostic tool which correlates efficiently embryonic development. Aitken and Krausz [175] proposed with sperm fertilizing potential. The conceptualization of the that sperm DNA damage is promutagenic and can give rise in vivo process of human fertilization and sperm egg to mutations after fertilization, as the oocyte attempts to interaction in vitro is the key to disclose sperm functional repair DNA damage prior to the initiation of the first clea- alterations with tremendous impact on diagnosis and vage. Mutations occurring at this point will be fixed in the treatment of male infertility. germline and may be responsible for the induction of infertility, childhood cancer in the offspring and higher risk FIVE-YEAR REVIEW of imprinting diseases [176]. So far, however, follow-up studies of children born after ICSI compared with children The understanding of sperm physiology and fertilization born after conventional IVF have not been conclusive is far from complete. However, molecular and genetic regarding the risks of congenital malformations, imprinting studies are on the pace to give a detailed and thorough diseases and health problems in general. In vitro fertilization, perception of the entire process of human fertilization. in general, is associated with multiple gestations and Consequently, this perception may suggest, in the future, increased risk of congenital abnormalities (including specific molecular therapy or even genetic target needed to hypospadias) [177]. ICSI in particular, carries an increased be precisely modified to improve male reproductive risk of endocrine abnormalities, as well as epigenetic potential. imprinting effects [177]. Although the absolute risk of any of