Genes and Male Infertility: What Can Go Wrong?

Normal sperm production (spermatogenesis), maturation (spermiogenesis), delivery to and passage through the female genital tract (ejaculation and sperm transport), and function (capacitation, egg penetration, and decondensation of the head) are essential to achieve fertilization and early embryonic development. These are long and complex processes, which not only require an appropriate hormonal environment, but also well-balanced autocrine, paracrine, and juxtacrine signaling events between the various components of the male reproductive system. Accordingly, when the molecules involved in these signaling cascades fail, male infertility occurs (Figure 1). Unfortunately, there is a paucity of information available concerning the molecular events regulating these complex processes, hence an inability to properly diagnose the cause of infertility for many patients. In fact, nearly one-quarter of all male infertility patients are diagnosed as "idiopathic," meaning we do not understand the etiology. Today, it is thought that much of what we currently diagnose as idiopathic may have a genetic basis. This review focuses on our current understanding of the molecular control of male fertility and the few genetic tests available for its diagnosis. . Genetic etiologies of human male infertility. Developmental disorders causing human male infertility result from a failure of gonadal development or testis determination, endocrinopathies, well-known genetic syndromes, and numerical and structural chromosomal abnormalities (translocations, deletions and inversions). CBAVD, congenital bilateral absence of the vas deferens; PMDS, persistent Müllerian duct syndrome. (Reprint from Matzuk MM, Lamb DJ. Genetic dissection of mammalian fertility pathways Nature Cell Biology 2002:4(S1)33-S40). Chromosomal abnormalities are common in infertile men, with an incidence of 5.8% as compared to an incidence of 0.5% in the fertile population (Johnson, 1998). Chromosomal abnormalities can occur on several genetic levels: 4.2% of abnormalities occur on the sex chromosomes, whereas 1.5% occur on the autosomes. The gain or loss of an entire single chromosome results in aneuploidy, whereas a polyploid state occurs when the entire chromosomal content is multiplied (polyploid cells are most commonly associated with malignancies). Structural abnormalities include the rearrangement or translocation of fragments of chromosomes, as in Robertsonian translocations, or deletions of single genes or portions of a chromosome. The incidence of abnormalities in chromosomal number, such as Klinefelter syndrome (XXY male), is greater in infertile men. This is a defect that is present in the majority of the cells of the individual. Defects in chromosome number are also present with an increased incidence in the sperm of infertile men as compared to fertile men (Finkelstein et al, 1998). These infertile men demonstrate normal somatic karyotype by blood exam, indicating the presence of a mosaicism between somatic and nonsomatic tissues or the presence of a mitotic or meiotic defect during spermatogenesis. Multicolor fluorescence in situ hybridization (FISH) on morphologically normal sperm from infertile patients demonstrates an increased rate of nondisjunction and aneuploidy (Huang et al, 1999), suggesting that normal sperm morphology cannot be used to identify sperm with a normal chromosomal complement. Additionally, pregnancies after intracytoplasmic sperm injection (ICSI) with sperm from these patients result in an increased rate of autosomal trisomies and sex chromosomal aneuploidies in the offspring (Martin, 1996). Klinefelter syndrome is the most common numerical sex chromosomal abnormality, occurring at a rate of 1/500 live male births (Rao and Rao, 1977), while accounting for 14% of all cases of azoospermia. The etiology is related to paternal or maternal sex chromosomal nondisjunction during meiosis, resulting in 90% of patients with the 47XXY karyotype and about 10% with the mosaic 46XY/47XXY. Phenotypically, patients present with increased height, decreased intelligence, obesity, diabetes mellitus, small firm testes, decreased Leydig cell function, variable sperm production, and an increased risk of leukemia, extragonadal germ cell tumors, and breast cancer (Therman, 1993). Although rare pregnancies have been achieved through natural conception, most patients will require the assistance of reproductive techniques to conceive, with the potential of passing the defect to their offspring (Harari et al, 1995); however, all offspring to date have had a normal chromosomal complement (Poulakis et al, 2001). Other sex chromosome defects occur less commonly. An XYY male occurs in 1/1000 live births and presents with increased height, decreased intelligence, antisocial behavior, increased risk of leukemia, and Sertoli-cell-only syndrome (SCOS) or maturation arrest, although many of these men are fertile (Griffin and Wilson, 1992). Mixed gonadal dysgenesis patients may present phenotypically as male, female, or with ambiguous genitalia. The most common karyotype observed in these patients is the 45X/46XY mosaic and may result from the loss of a Y chromosome early in embryonic development. However, 33% of patients have a normal karyotype, suggesting that factors other than sex chromosomal aneuploidy may be involved. The testes lack germ cells and, when intra-abdominal, have an increased risk of malignancy (Wegner et al, 1994). Structural chromosomal abnormalities occur in azoospermia and severely oligospermic patients more frequently than in fertile men (Jaffe and Oates, 1996). A Robertsonian translocation occurs 8.5-fold more often in infertile patients and consists of the exchange of entire chromosomal arms between 2 chromosomes (Antonelli et al, 2000). A reciprocal translocation occurs with the same frequency as a Robertsonian translocation and is defined as the mutual exchange of fragments between 2 chromosomes (Antonelli et al, 2000). These patients may present with sperm densities ranging from normal to azoospermia. The XX male syndrome occurs in 1 of 20 000 live births. Theories of how this happens range from the translocation of the SRY on the Y chromosome to the homologous region on the × chromosome to the mutation or deletion of an × gene that inhibits an autosomal testis determining gene (Page et al, 1987; Andersson et al, 1988). As described below in greater detail, molecular analysis demonstrates that most of these men have a translocation of a small piece of the Y chromosome containing the SRY or "sex determining gene" to the × chromosome. These patients are azoospermic, with 10% of them presenting with ambiguous genitalia or hypospadias (Schweikert et al, 1982). Because abnormal karyotypes are present in approximately 7–13% of patients with idiopathic infertility, testing for chromosomal abnormalities using routine karyotyping with high-resolution banding cytogenetics is recommended for men suspected of having more severe defects such as sperm densities of less than 5 million per milliliter. FISH may be used to detect aneuploidy in the sperm of patients contemplating assisted reproduction, given that higher rate of sex chromosome aneuploidy has been found in the sperm of patients with severe spermatogenic defects. Finally, prenatal genetic diagnosis is recommended for those with known genetic abnormalities (Van Assche et al, 1996; Pauer et al, 1997; Huang et al, 1999). The Y chromosome is the smallest chromosome, with 60 million base pairs, and is divided into a long arm (Yq) and a short arm (Yp) (Morton, 1991). Although most of the chromosome does not undergo meiotic recombination, the pseudoautosomal regions located at both ends of the Y chromosome pair with the corresponding regions on the × chromosome during meiosis (Figure 2). The genetic information on the Y chromosome is important for male sex determination and for normal spermatogenesis. . Schematic representation of the Y chromosome. Yp = short arm; Yq = long arm. (Reprinted from Maduro MR, Lamb DJ. Understanding the new genetics of male Infertility. Journal of Urology 2002;168: 2199, Lippincott Williams & Williams The Y chromosome is crucial for testis formation in early development, with the testis-determining factor (SRY) located on the proximal portion of Yp (Koopman et al, 1991). A deficient or absent SRY results in a 46XY female (Jager et al, 1990). Alternatively, a different sex reversed locus (SRVX) on the × chromosome may explain why SRY is impaired in only a small portion of 46XY females (Ogata and Matsuo, 1994). In addition to SRY, other autosomal and X-linked genes, such as those for androgen synthesis and metabolism, are also required for normal male sexual differentiation. Although not critical for survival, the Y chromosome does contain genes that are not involved in male reproduction, such as those for stature, crown tooth size, and depression. The azoospermic factor region (AZF) located at Yq11 was hypothesized by Tiepolo and Zuffardi (1976) to contain genes that control spermatogenesis. Three distinct intervals comprise this region and are associated with variable degrees of spermatogenic impairment: AZFa, AZFb, and AZFc; an AZFd is debated to reside between AZFb and AZFc (Kent-First et al, 1999; Oates, 2003). The proximal portion of the AZFa region contains pseudogene sequences homologous to genes at Xp22. Other genes in the region include DFFRY (human homologue of the Drosophila development fat facets gene), dead box on the Y (DBY), ubiquitously transcribed tetretricopeptide repeat gene on the Y (UTY), and AZFaT1 (Sargent et al, 1999). DDFRY, recently renamed USP9Y, is widely expressed in various tissues, whereas the murine homologue is testis specific. A homologous copy of USP9Y also exists on the × chromosome. Deletions or point mutation of USP9Y results in hypospermatogenesis; with the thought that the gene may regulate protein turnover (Lahn and Page, 1997). Y chromosome analysis of infertile men shows that DBY is deleted more frequently than USP9Y, resulting in hypospermatogenesis or SCOS. Deletion of both USP9Y and DBY results in SCOS (Foresta et al, 2000). Located mainly in the AZFb region, RNA binding motif protein on the Y (RBMY) consists of 30 genes and pseudogenes and may be divided into 6 subclasses (RBMY1-RBMY6) (Chai et al, 1997). As its name suggests, RBM may function in RNA metabolism. Deletion of the murine homologue of RBM results in male sterility, suggesting a similar role for the gene when it is absent in a small percentage of infertile men (Delbridge et al, 1997). Chromodomain protein on the Y (CDY) is another multicopy gene that is expressed during spermatogenesis and is thought to be involved in chromatin modifications during meiosis (Lahn and Page, 1999). DAZ (deleted in azoospermia) was the first candidate spermatogenesis gene identified in the AZFc region (Reijo et al, 1995). DAZ represents a multigene family with considerable variation in number and sequence of repeats. The DAZ protein is expressed mainly in postmeiotic germ cells, suggesting a role in posttranscriptional control during the later stages of differentiation (Habermann et al, 1998). Deletion of the Drosophila equivalent, boule, results in azoospermia, suggesting a critical role in spermatogenesis (Eberhart et al, 1996). The murine homologue of DAZ, dazla, exists only on chromosome 17, not on the Y, with the thought that the DAZ gene migrated to the Y after the divergence of the New World monkeys (Gromoll et al, 1999). The human autosomal homologue of daz, DAZLA, is mapped to chromosome 3p24 and is expressed specifically in the testis and at lower levels in the ovary. The murine dazla is expressed in both male and female germ cells and when deleted results in absence of gamete production. Other genes in the AZFc region include testis transcript Y2 (TTY2) and basic protein Y2 (BPY2); although their exact function remains unknown, they share characteristics with DAZ in that they are Y-specific genes in multiple copies and exhibit testis-specific expression (Lahn and Page, 1997). Large deletions of the Y chromosome can be detected by high-resolution cytogenetics as reported by Tiepolo and Zufferdi (1976) 25 years ago. Used more commonly today, polymerase chain reaction (PCR) is a technique that amplifies a portion of DNA as defined by sequence tagged sites (STSs). The STSs flank a portion of DNA that is amplified by PCR and then identified by molecular size on an agarose gel. The number of STSs used by a clinical laboratory may vary but should at least span interval 6 and AZFa, b, c, d regions believed to be involved in spermatogenesis. Small microdeletions on the Y chromosome may not be detected by PCR; thus, a normal analysis indicates only that no large deletions of the Y chromosome are present. Human sexual maturation and spermatogenesis is intricately controlled by the hypothalamic—pituitary—gonadal (HPG) axis; hence, disorders affecting this delicate balance can severely impair male sexual development and fertility. These rare disorders are usually the result of mutations, small deletions, or polymorphic expansions within the regulatory genes involved in the biosynthesis of hormones, growth factors, the androgen receptors, and their associated signal transduction pathways. Therefore, a good understanding of the genetic defects leading to the disturbance of the HPG axis is essential in the evaluation and treatment of male infertility. Gonadotropin-releasing hormone (GnRH) from the hypothalamus regulates the production of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) by the pituitary gonadotropes. FSH and LH are dimeric molecules that share a common α subunit with human chorionic gonadotropin and thyroid-stimulating hormone, but each has specific β-subunits. Hypogonadotropic hypogonadism (HH) is characterized by a decreased output of GnRH and low circulating levels of FSH and LH, resulting in the deficient androgen secretion and spermatogenesis in the testis (Seminara et al, 2000). Kallmann syndrome is the most common X-linked HH disorder in male infertility. A mutation in KAL-1 gene, which encodes a neural cell adhesion molecule, is the basis for this disease and occurs in approximately 1 in 10 000–60 000 live births. Patients with Kallmann syndrome usually seek medical attention in their late teens for delayed puberty with small, nonfunctioning testes and a short penis. Clinical features include anosmia, congenital deafness, asymmetry of the cranium and face, cleft palate, cerebellar dysfunction, cryptorchidism, and renal abnormalities. On the other hand, infertility may be the sole phenotypic abnormality that leads to the diagnosis. Hormone replacement is the treatment of choice, and spermatogenesis and subsequent fertility has been reported with coordinated gonadotropin stimulation (Sigman, 1992). The Dax1 gene found on the × chromosome encodes an orphan nuclear hormone receptor that plays a critical role in the development of the hypothalamus, pituitary, adrenal, and gonads (Burris et al, 1996). It appears to function in the maintenance of the integrity of the testicular epithelium and spermatogenesis. Mutations in Dax1 can also lead to HH in association with congenital adrenal hyperplasia, a devastating condition that can lead to early infant death due to electrolyte imbalance. Mutation in PC1, or convertase-1 gene, has also been linked with HH in conjunction with obesity and diabetes. This gene is believed to play a role in GnRH secretion and release of the precursor molecule in the hypothalamus (Jackson et al, 1997). Prader-Willi syndrome (PWS) patients, whose predominant features are obesity, mild to moderate mental retardation, and infantile hypotonia, can also present with HH. The culprit for this disease is thought to be mutations or deletions of a specific locus within paternal chromosome 15 (Smeets et al, 1992). This syndrome also follows a phenomenon called genomic imprinting, where differential expression of the 2 alleles at an autosomal locus is based on their parent of origin; usually 1 allele is expressed and the other silenced. Thus, while mutation of paternal 15q11q13 is required for PWS to occur, maternal loss of 15q11q13 results in Angelman syndrome (Jiang et al, 1998). Mutations leading to direct abnormal synthesis and function of the gonadotropins FSH and LH can also result in male infertility. Though variable phenotypes ranging from complete virilization failure to less severe forms of hypogonadism have been observed, lack of optimal Leydig cell stimulation and absence of spermatogenesis are characteristic features of LH mutation (Park et al, 1976; Weiss et al, 1992). Rarely, deficiencies in FSH are reported in subfertile males. Although FSH β-subunit mutations have been suggested, the exact genetic causes of these abnormalities are yet to be identified (Matthews et al, 1993). The integrity of the LH and FSH receptors also plays a critical role in maintaining the HPG axis function. Constitutive activation of the LH receptor leads to male precocious pseudopuberty, whereas pseudohermaphroditism and Leydig cell agenesis are the clinical phenotypes of LH resistance (Wu et al, 2000). Though it is rare in humans, LH receptor deficiency has been reported in a "knock out" animal model that renders the genetically modified mice infertile with the anatomical features of hypogonadism (Lei et al, 2001). On the other hand, less is known about the FSH receptors; only 1 mutation has been reported in a hypophysectomized man who is fertile (Gromoll et al, 1996). Disruption in any genes involved in androgen biosynthesis, metabolism, and action can negatively affect male sexual development and spermatogenesis. Testosterone is the product of a complex biosynthetic pathway that starts with cholesterol biodegradation. Members of the steroid receptor superfamily and their transcriptional coactivators are pivotal in the regulation of reproductive functions. In the male, conversion of testosterone to dihydrotestosterone by 5α-reductase is crucial for external genitalia and prostate growth. Therefore, deficiencies in 5α-reductase are associated with male infertility as a result of underdeveloped external genitalia and the inability to effectively deliver sperm (Griffin and Wilson, 1992). Androgen insensitivity syndromes are X-linked disorders that present with a range of internal and external virilization defects. These diseases include Reifenstein syndrome, testicular feminization (male pseudohermaphroditism), Lub syndrome, and Rosewater syndrome (Quigley et al, 1995; Jaffe and Oates, 1996). Ambiguous genitalia, micropenis, and hypospadias are common features. Most androgen receptor (AR) abnormalities causing clinical syndromes result from point mutations or small deletions in the AR gene. Identification of the same mutation in different individuals does not necessarily result in the same clinical phenotype, suggesting that other modulating genes may be involved (McPhaul et al, 1992). Kennedy disease (X-linked spinal and bulbar to a family of diseases characterized by the abnormal in et al, 1991). It is a rare disease by a mutation in 1 of the AR gene. In addition to their dysfunction, patients with Kennedy disease present with androgen testicular and male infertility. The range in from mild and to severe and death by these diseases an called This is characterized by the of the genetic disease in associated with the number of repeats. The of the disease occurs in the This a in genetic of who to assisted reproductive Although the receptor is known to be required for male fertility in a model et al, less is known in A mutation in the receptor gene 2 that is similar to that in the model has been described in a male et al, 1994). However, this fertility is analysis of LH, and and levels are commonly available to reproductive and the evaluation in patients suspected of HPG axis are also available to specific and growth factors in but these are for an infertility Although other genetic testing for defects in the hormonal control of male are not of these disorders may in the treatment of male infertility in the of gene associated with abnormal sex determination and development have long been as the cause for and sperm which may lead to male infertility. for of these diseases the genetic and molecular are for the genes involved have been an and diagnosis and of the are of diseases for which sex genes have been to be or that are to be bilateral absence of the vas is the most abnormality of the and and 2000). This condition is of the phenotypic of an autosomal disorder by more than different mutations and 2 in the gene, which and the production of the protein et al, male patients often present with and leading to male infertility. However, most patients with do not have the severe and defects in the et al, 2000). spermatogenesis is usually not sperm from the testis or can be used to however, there is a of the gene mutation to the genetic for gene mutations in both the and the female are important to the risk of by the potential Because of the large gene size, only about 30 mutations are in the clinical Thus, although an abnormal result is to a a normal result does not a absence of mutations et al, 1999). Alternatively, a can also be to diagnose This which the of in the however, often lead to results when they into results are also such as adrenal diabetes or renal failure are present. 2 results on different are required to diagnose Finally, several mutations in the gene do not lead to which may to results and 1998). This both the and the of the present clinical laboratory tests in this genomic In all patients with are thought to have a genital of et al, 2000). Patients presenting with absence of the vas are also at risk and should undergo although the absence of a on the same a different etiology et al, 2001). agenesis on the same a duct agenesis is of the phenotype, a polymorphic region of in of the gene should also be as it be for a less of that results in a lower of protein et al, 2000). Müllerian duct syndrome is a of male pseudohermaphroditism that results from a or deficiency of the male hormone or its receptor during prenatal development et al, 1994). The lack of Müllerian duct in an phenotypically normal man often results in and fertility (Jaffe and Oates, 1996). and are linked to male infertility, which can also be associated with 1994). An abnormal of the lower during development is for the spermatogenesis is not patients may through sperm from the testis or by assisted reproductive a condition commonly known as is believed to be due to a currently genetic which causes a characteristic spinal This is frequently associated with or failure or with a on fertility. deficiencies have also been reported in patients, but their direct association with remains to be Jaffe and Oates, 1996). syndrome men and is most by a mutation in a gene involved in the of the A tract and are characteristic features. infertility is through and spermatogenic et al, 1994). expansions of an repeat within a gene a protein are associated with the most common cause of This which causes and is also characterized by with a on spermatogenesis et al, 1993). The features of syndrome include short stature, and and In men, fertility is of and FSH which negatively affect spermatogenesis et al, 1994). Although several deletions have been to be associated with the no candidate gene is available and are that result from deficiencies in defects or gonadal failure due to after multiple may impair testicular function and fertility in these patients and phenotypes include deafness, and infertility. these features are due to a defect in the or of cells resulting from gene defects in the gene and syndromes are 2 good of diseases with this et al, et al, 1993). In these patients, fertility is impaired through sperm However, spermatogenesis is not or in fertilization can be used to achieve a et al, with for the syndrome is most by a function which causes structural abnormalities of the resulting in sperm due to In patients, who also from and may present their fertility. This however, be reversed through et al, Jaffe and Oates, 1996). in embryonic cell which specific genomic in animal have in the of of genes to be related to infertility. involved in sex determination and development have not been an most the genes involved in mutations in and have all been to be related to deficient testicular in mice et al, 1995; and 1999; et al, 1999; et al, et al, of these genes are also related to in humans, but in a small percentage of patients. these an of first described in animal that will an understanding of the molecular basis of male infertility. The of assisted reproductive has many infertile to these also natural to fertilization by the current of genetic analysis of men with idiopathic male infertility, it is that many of these men have a genetic cause of their infertility. Thus, while the current on the of are our of the genetic causes of male infertility should