Male infertility

Approximately 15% of couples are infertile . Male factors contribute to infertility in over 50% of cases . Identifiable genetic abnormalities contribute to 15%–20% of the most severe forms of male infertility (azoospermia), while the majority 30%–60% are idiopathic and under investigation with a strong suspicion of genetic underpinnings . Among known genetic causes of male infertility, chromosomal abnormalities, y-microdeletions, x-linked and autosomal gene mutations have been previously described. In this chapter, we discuss known genetic etiologies of male infertility and comment on novel genetic-based biomarkers for male fertility.

Chromosomal analysis

The incidence of chromosomal abnormalities is inversely proportional to sperm production. Less than 1% of men with normal sperm concentration are identified to have chromosomal abnormalities, while 5% of men with severe oligospermia defined as a sperm concentration less than 5 million sperm per milliliter, will have chromosomal abnormalities, and 10%–15% of men with azoospermia will have chromosomal abnormalities . Therefore, chromosomal analysis is not routinely performed, except for men with severe oligospermia or azoospermia, recurrent pregnancy loss or unexplained repeat failures of assisted reproductive techniques . In such cases, a chromosomal analysis, i.e., karyotype, is obtained to evaluate men with insufficient spermatogenesis.

Chromosomal Abnormalities: Several forms of chromosomal abnormalities exist. Aneuploidies are defined by an abnormal number of chromosomes either more or less than the euploid state, i.e., 46,XY or 46,XX. This occurs because of nondysjunction of homologous chromosomes during meiosis I or chromatid pairs during mitosis or meiosis II; it may also be due to chromosomal lagging during anaphase resulting in loss . Most common aneuploidies involve the gonosomes (X and Y chromosomes), followed by autosomal disomies of chromosomes 13, 18, and 21 . Examples include Klinefelter syndrome (KS) (47, XXY) and mixed gonadal dysgenesis (MGD) (45,X/46,XY) with the latter being a mosaic of two different chromosomal numbers. Deletions of genetic material from the Y chromosome may result in a microdeletion within the Y chromosome that cannot be detected on karyotype (Y-microdeletion) and are the cause of up to 10% of men with nonobstructive azoospermia . These are further discussed in sections below.

Translocations may be balanced, i.e., reciprocal translocations or unbalanced, i.e., Robertsonian translocations (RTs). Reciprocal translocations involve an exchange of genetic material between 2 or more chromosomes. They are the most common chromosomal structural anomalies in humans and are 10 times more common among infertile men . RTs are the result of one portion of a chromosome translocating to another chromosome. RTs occur with an incidence of 0.9% of men with severe male factor infertility and occur because of the rearrangement of chromosomes with loss of genetic material resulting in a complement of 45 chromosomes. These occur among acrocentric chromosomes such as chromosomes 13, 14, 15, 21, and 22. Here, the long arms fuse, resulting in loss of genetic material among the chromosomal short arms. The most common translocations include t(13q;14q) and t(14q;21q) and carry an incidence of 0.9% or less . Here, “q” designates the long arm of the chromosome, and “t” designates a translocation of the chromosomes within the brackets. These men tend to be phenotypically normal; however, they may demonstrate impaired spermatogenesis with increased rates of sperm aneuploidy among those sperm produced .

Autosomal inversions can be considered intrachromosomal reciprocal translocations and involve structural derangements without loss of genetic material. Inversions relevant to male infertility include that of chromosome 9. This inversion accounts for up to 3%–5% of male infertility and results in a variable phenotype ranging from normospermia, oligospermia, azoospermia, and asthenospermia .

Chromosomal analysis

The incidence of chromosomal abnormalities is inversely proportional to sperm production. Less than 1% of men with normal sperm concentration are identified to have chromosomal abnormalities, while 5% of men with severe oligospermia defined as a sperm concentration less than 5 million sperm per milliliter, will have chromosomal abnormalities, and 10%–15% of men with azoospermia will have chromosomal abnormalities . Therefore, chromosomal analysis is not routinely performed, except for men with severe oligospermia or azoospermia, recurrent pregnancy loss or unexplained repeat failures of assisted reproductive techniques . In such cases, a chromosomal analysis, i.e., karyotype, is obtained to evaluate men with insufficient spermatogenesis.

Chromosomal Abnormalities: Several forms of chromosomal abnormalities exist. Aneuploidies are defined by an abnormal number of chromosomes either more or less than the euploid state, i.e., 46,XY or 46,XX. This occurs because of nondysjunction of homologous chromosomes during meiosis I or chromatid pairs during mitosis or meiosis II; it may also be due to chromosomal lagging during anaphase resulting in loss . Most common aneuploidies involve the gonosomes (X and Y chromosomes), followed by autosomal disomies of chromosomes 13, 18, and 21 . Examples include Klinefelter syndrome (KS) (47, XXY) and mixed gonadal dysgenesis (MGD) (45,X/46,XY) with the latter being a mosaic of two different chromosomal numbers. Deletions of genetic material from the Y chromosome may result in a microdeletion within the Y chromosome that cannot be detected on karyotype (Y-microdeletion) and are the cause of up to 10% of men with nonobstructive azoospermia . These are further discussed in sections below.

Translocations may be balanced, i.e., reciprocal translocations or unbalanced, i.e., Robertsonian translocations (RTs). Reciprocal translocations involve an exchange of genetic material between 2 or more chromosomes. They are the most common chromosomal structural anomalies in humans and are 10 times more common among infertile men . RTs are the result of one portion of a chromosome translocating to another chromosome. RTs occur with an incidence of 0.9% of men with severe male factor infertility and occur because of the rearrangement of chromosomes with loss of genetic material resulting in a complement of 45 chromosomes. These occur among acrocentric chromosomes such as chromosomes 13, 14, 15, 21, and 22. Here, the long arms fuse, resulting in loss of genetic material among the chromosomal short arms. The most common translocations include t(13q;14q) and t(14q;21q) and carry an incidence of 0.9% or less . Here, “q” designates the long arm of the chromosome, and “t” designates a translocation of the chromosomes within the brackets. These men tend to be phenotypically normal; however, they may demonstrate impaired spermatogenesis with increased rates of sperm aneuploidy among those sperm produced .

Autosomal inversions can be considered intrachromosomal reciprocal translocations and involve structural derangements without loss of genetic material. Inversions relevant to male infertility include that of chromosome 9. This inversion accounts for up to 3%–5% of male infertility and results in a variable phenotype ranging from normospermia, oligospermia, azoospermia, and asthenospermia .

XXY Klinefelter syndrome

KS was first described in 1942 in a series of 9 patients with a phenotype characterized with gynecomastia, testicular hypotrophy, azoospermia, and an elevated follicle stimulating hormone (FSH) . Nearly 2 decades later in 1959, men with KS were identified to have sex chromosome aneuploidy with an additional X chromosome. Approximately 80%–90% of KS men have a 47, XXY karyotype. Karyotypes among the remaining 10%–20% include: 48,XXXY; 48,XXYY; 47,iXq,Y or mosaicisms of 2 different genetic lines such as 47,XXY/46,XY . Here, iXq refers to an isochromosome where the X chromosome is structurally abnormal with the chromosomal arms being mirrors of the “q” long arm of the X chromosome rather than a normal long (q) and short (p) arm. The mechanism for the additional X chromosome is because of nondisjunction where the sex chromosomes fail to separate. This event may occur during oogenesis in meiosis I (50%) or meiosis II (10%) or during meiosis I (40%) in spermatogenesis . Infrequently, (3%) nondysjunction occurs during early embryogenesis in the fertilized egg .

KS is the most common X chromosome abnormality and the most common genetic cause of male infertility. It carries an incidence of 0.1%–0.2% of newborn male births and has a prevalence of 1%–2% of infertile males , 0.6% of men with severe oligospermia, and 10%–12% of azoospermic males . Variations in X inactivation and KS results in reduced androgen production, increased estrogen to testosterone ratios, and variable sensitivity of the androgen receptor (AR) that may be associated with different CAG repeats . Phenotypically, individuals with KS demonstrate substantial variability in the severity of classic features and worsen as patients’ age. The phenotypic severity is thought to be due to the severity of polysomy . Varying degrees of genes escaping from these extra X chromosome(s) may influence the heterogeneity of phenotype. Among humans with more than one X chromosome (i.e., female 46,XX or KS 47,XXY), one of the X chromosomes is typically inactivated. However, approximately 15% of genes escape inactivation and may affect the resulting phenotype especially for men with supernumary X chromosomes . Classically, KS men are of tall stature, have gynecomastia, gynoid hips, propensity toward obesity, sparse body hair, hypotrophic and firm testicles (<4 cc’s) . Less commonly, KS patients may have under virilized features such as micropenis, undescended testicles, bifid scrotum, and hypospadias. They are typically identified to have hypergonadotropic hypogonadism (primary hypogonadism), with 65%–85% of KS men with total testosterone levels <12 nmol/L . Thus, they express signs and symptoms of hypogonadism such as osteoporosis, reduced libido, erectile dysfunction, and infertility. Psychosocial, language, speech, and intelligence may also be affected, and deficits have been shown to correlate to increasing supernumary of the X chromosome . It is important to remember that for many KS patients, low testosterone levels do not result in symptoms. For these men, the minimally low testosterone levels in KS patients are the highest that these patients have experienced and are associated with normal libido and sexual function.

Infertility in KS is because of impaired spermatogenesis and typically presents with azoospermia or with occasionally severe oligospermia. Testicles of men with KS typically demonstrate progressive hyalinization, fibrosis, and degeneration of germ cells and sertoli cells most often resulting in sertoli cell only (SCO) syndrome. This is thought to worsen immediately after puberty . The degeneration of spermatogenesis appears to occur immediately after puberty and seems to remain stable thereafter in life. Fertility options are generally dependent upon in vitro fertilization intracytoplasmic sperm injection (IVF-ICSI). In rare cases where sperm are present in the ejaculate, they may be used. Most commonly, men with KS are azoospermic and require microscopic testicular sperm extraction (mTESE). Controversy exists regarding the timing of sperm retrieval from KS patients. Attempts of fertility preservation and sperm retrieval have been made in pre-pubertal, post-pubertal, and adult KS patients. It is the personal view of the authors that fertility preservation should be considered post-puberty for KS patients . Otherwise, sperm retrieval may be performed in adults seeking fertility. This is successful in up to 70% of cases at specialized centers of excellence thought to be due to niches of retained spermatogenesis. Although strong evidence exists that 47,XXY can produce sperm, it is also possible in rare patients that spermatogenesis may be due to focal mosaicism of normal karyotype, with germ cells that are able to progress through meiosis, mitosis and spermiation . The sperm retrieved are considered safe for IVF , but do carry a higher diploid sperm incidence of 6.3% .

47,XYY is a much more rare sex chromosome anomaly following KS with an incidence of 0.1% . Most patients are not diagnosed until later in life with a median age of 17.1 years . This is in part due to a typically normal phenotype. Studies have described behavioral problems, cognitive impairments such as learning, speech and language deficits, aggressive behavior, tall stature, clinodactyly, and hypertelorism. Increased rates of asthma, austism, tremors, and seizures have also been noted .

Fertility among these individuals is variable with semen analyses ranging from azoospermia to normal sperm counts . However, more commonly, these individuals present with severe oligospermia or azoospermia . Testicular biopsy findings among azoospermic men, most commonly demonstrate SCO or maturation arrest . FSH levels are typically elevated in response to impaired spermatogenesis, and testosterone levels are generally normal or may be elevated . Sperm produced from 47,XYY individuals typically have a normal karyotype ; however, several reports have identified an increased incidence of 47,XYY aneuploidy sperm, and somewhat variable reports have been published suggesting sperm mosaicism, hyperdiploidy, and aneuploidy ranging from 0.57% to 77.8% . The mechanism of aneuploidy resulting in 47,XYY is because of nondisjunction at meiosis II during spermatogenesis, resulting in the additional Y chromosome in 84% of instances or postzygotic mitotic error in 16% of instances . Some authors suggest that in cases where these individuals produce normal ploidy sperm, the extra Y chromosome is lost prior to meiosis . However, among many of those germ cells with genetic abnormalities, arrest of germ cell maturation may occur, thus failing to produce spermatozoa. These individuals will demonstrate a spectrum of histology, ranging from SCO to early or late maturation arrest.

Fertility treatment relies primarily on harvesting sperm from the ejaculate if present or from the testicle through mTESE with intracytoplasmic sperm injection (ICSI)-assisted reproduction. Genetic screening of embryos prior to transfer may be considered by the couple during this process.

Y-Chromosome microdeletions

Y-Chromosome microdeletions have been extensively studied because the recognition that Yq has factors important for spermatogenesis . In 1996, the azoospermia factor (AZF) region of the euchromatin long arm of the Y chromosome was genetically mapped by Vogt and colleagues. Deletions in this region were identified among 13 of 370 men presenting with severe oligospermia or azoospermia . Y chromosome microdeletions are clinically important because they are associated with severe male infertility, and likelihood of treatment success can be determined by the location of the deletion. The AZF loci harbor 14 protein coding genes critical for spermatogenesis . These genes are organized into 3 distinct locations: “a,” “b,” and “c.” Each of these regions may be deleted independently or in combination and are implicated as the cause of defective spermatogenesis for 5% of men presenting with severe oligospermia and 10% of men with NOA (non-obstructive azoospermia) . The 6 classic forms of AZF deletions and their corresponding phenotype, in order of decreasing severity include: AZFabc (SCO), AZFa (SCO), AZFbc (SCO/maturation arrest), AZFb (maturation arrest), AZFc (severe oligospermia to azoospermia), and partial AZFc (normal spermatogenesis to azoospermia) . Figure 1 demonstrates the genes lost with each AZF region deletion.

Distribution of Y-Chromosome microdeletions. Reprinted with permission from Elsevier: Seminars in Cell and Developmental Biology 2016. Neto FTL, Bach PV, Najari BB, Li PS, Goldstein M.

The AZFa region spans 1100 kb, and harbors 2 protein coding genes: USP9Y and DBY . The DBY gene encodes RNA helicase and has been demonstrated to play a major role in spermatogenesis . These deletions are because of intrachromosomal recombination between flanking repeating genetic sequences or pallindromes . Complete deletions of AZFa are rare, accounting for only 3% of Y-microdeletions. They carry the poorest prognosis with azoospermia in all men. Histology typically demonstrates SCO, with no previous reports identifying sperm with mTESE. However, partial AZFa deletions have been reported; among these cases, USP9Y is deleted in isolation. The clinical phenotypes were azoospermia and severe oligospermia, with histology demonstrating hypospermatogenesis .

The AZFb region contains 7 protein encoding genes experimentally shown to be implicated in spermatogenesis. These include EIF1AY, RPS4Y2, and SMCY located in the X-degenerate euchromatin, and HSFY, XKRY, PRY, and RBMY located in the ampliconic regions . Deletions of the AZFb region are large (4.96–6.92 Mbs) , and thought to be due to homologous recombination (HR) and nonhomologous recombination (NHR) among other yet to be described mechanisms . AZFb deletions account for 15% of Y microdeletions. Invariably, complete deletions result in azoospermia and SCO or early maturation arrest histology .

The AZFc region spans 4.5 Mb of euchromatin and contains five protein encoding genes shown to be implicated in spermatogenesis: BPY2, CDY, DAZ, CSPG4LY, and GOLGAZLY . The DAZ family of 4 genes has been most prolifically studied. This family encodes RNA-binding proteins expressed exclusively in germ cells and exists in palindromic sequences DAZ1/2 and DAZ3/4 . Deletions among this region are thought to occur through HR or NHR. Most frequently deletions occur through HR and involve ampliconic regions b2/b4, b1/b3, b2/b3, and gr/gr resulting in the deletion of several genes including DAZ genes. NHR accounts for deletions of ampliconic regions P3a, P3b, and P3c . AZFc deletions are the most common Y-microdeletions because it is composed of amplicons, which are particularly susceptible to deletions by the above methods . AZFc deletions account for 60% of all clinically relevant Y-microdeletions . Up to 70% of men with AZFc deletions have sperm in the ejaculate, typically less than 1 million sperm per milliliter for these cryptospermic men with AZFc deletions . In men with azoospermia and AZFc deletions, mTESE can be used to harvest sperm from the testicle in 50%–60% . Thus, fertility potential is present in some patients with AZFc deletions; however, Y-microdeletions are passed to male offspring. Reports indicate that offspring with Y-microdeletions typically have impaired spermatogenesis because their only source of Y chromosome-associated genes is from the partially deleted Y chromosome .

Furthermore, combinations of deletions in various AZF regions may occur. Combined AZFb + AZFc deletions are the most common because the two regions overlap and share 1.5 Mb . These deletions do not extend to the same proximal extent as isolated AZFb deletions. Specifically, CDY1 gene is composed of 2 copies; 1 copy exists in the AZFc region while the other in the area of AZFb overlap . This combination of deletions accounts for approximately 13% of Y-microdeletions. Patient phenotypes demonstrate SCO or maturation arrest, and sperm retrieval attempts are uniformly unsuccessful.

In summary, Y-microdeletions occur frequently and are indicated among patients with azoospermia and severe oligospermia. Men with complete AZFa and b deletions do not produce sperm in our experience, and successful surgical sperm retrieval has not been described. Furthermore, AZFc deletions are associated with severe oligospermia or azoospermia at presentation. These couples will likely require IVF-ICSI. Genetic counseling should be provided before assisted reproduction as the Y-microdeletion will be transmitted to the male offspring, with variable but adverse effects on spermatogenesis.

XY,X Mixed gonadal dysgenesis

MGD was first described in 1963 by Sohval . Men with this diagnosis rarely present initially with infertility. These individuals may present as children with ambiguous or abnormal genitalia or as adults with infertility, gonadal failure, or short stature . Phenotype is variable and has been shown to generally follow gonadal development and location. Gonads may develop into testicles, or undifferentiated streaks, and may be located in the respective scrotum, intra-abdominally, or along the path of descent in the inguinal canals. Individuals with bilateral scrotal testicles typically present as a male with short stature and gonadal failure; those with one scrotal testicle and an intra-abdominal streak tend to present with sexual ambiguity as an under virilized infant; individuals who present with a streak and intra-abdominal testicle tend to present as in infant with sexual ambiguity resembling female clitoromegaly, and those with bilateral intra-abdominal streaks tend to present as a phenotypic female infant . MGD is also associated with cardiac and renal malformations, gonadal blastomas, and germ cell tumors.

Genetically, those with MGD typically have a karyotype consisting of a mosaicism of 45,X/46,XY; however, variants and ring mosaicisms have also been described such as 45,X/46,X(r)Y . Here, “r” designates a ring configuration. These abnormalities are because of chromosomal misaggregation secondary to anaphase lag or chromosomal rearrangement during early embryonic mitosis .

Most MGD patients are infertile. Most have azoospermia, with 1 report of successful mTESE sperm extraction . There are limited reports of oligozoospermia among individuals with MGD . Testicular biopsies describe disorganized cytoarchitecture, hyalinization, and atrophy of the seminiferous tubules . Most have testicular failure with approximately 45% requiring testosterone replacement when they are adult .

X-Linked and autosomal gene mutations

Studies have identified greater than 3000 differentially expressed genes associated with defective spermatogenesis. However, less than 0.01% of these genes have been further explored, and patients affected by deletions clinically characterized . Gene mutations may occur among somatic cells or germ cells. Mutations among somatic cells may be systemically manifested as seen with Kallmann syndrome (KLS) or located exclusively in the testicle among Leydig cells or more likely sertoli cells that function to support germ cells and facilitate spermatogenesis. Mutations affecting germ cells are also possible as they are the cells that undergo spermatogenesis. In this section, we review a selection of well-described and clinically relevant x-linked and autosomal gene mutations.

Kallmann syndrome

KLS is characterized clinically by hypogonadotropic hypogonadism (HH) and anosmia . KLS has an incidence of 0.2% and a male gender predilection of 5:1. Beyond HH and midline defects such as cleft palate, anosmia, or hyposmia, other clinical features associate with KLS include: infertility, tall stature, cryptorchidism, unilateral renal agenesis, and neurogenic deafness .

KLS may be the result of several genetic defects; however, x-linked genetic abnormalities were first described in the gene KAL1, and autosomal-linked abnormalities in the FGFR1 gene have also been described as causes of HH. The former accounts for 14% of familial cases and 11% of sporadic cases, while the latter accounts for 10% of cases . The KAL-1 gene encodes the protein anosmin-1, which is a cell adhesion protein of the extracellular matrix. During embryogenesis, anosmin-1 is required for the organized migration of both olfactory axons and GnRH neurons from the olfactory placode through the cribriform plate and into the preoptic area of the hypothalamus. As such, defects result in anosmia and GnRH deficiency. GnRH deficiency results in subsequent lack of pulsatile luteinizing hormone (LH) that is required for normal gonadal function . FGFR1 gene encodes the FGFR1 receptor implicated in olfactory and GnRH neuron development . Aberrant FGFR1 functioning has been associated with midline defects, cleft palate, dental agenesis, and malformations of extremities observed in some cases of KLS .

Secondary testicular failure due to HH occurs because of a lack of stimulation of the testicle to induce testosterone production and subsequent spermatogenesis. As such, management goals for males are to promote normal puberty in younger individuals and normal hormonal functioning and fertility potential among adults. Several regimens exist. Testosterone replacement therapy may be used as maintenance therapy to induce puberty. When fertility is desired, GnRH or gonadotropins are required to induce spermatogenesis. Common regimens include: human chorionic gonadotropins (hCG) 1000–2500 international units (IU) injected intramuscular (IM) or subcutaneously (SC) twice per week with or without the addition of human menopausal gonadotropins (hMG) 75–150 IU injected 3 times per week; pulsatile GnRH 25–600 ng/KG SC, intravenous (IV) or by pump, with pulses spaced every 120 min. Recombinant FSH (rFSH) may be used in place of hMG but is significantly more costly . The use of testosterone above to induce puberty is associated with profound testicular hypotrophy that may limit subsequent potential for quantitatively productive spermatogenesis. The use of hCG in adolescents with KLS or idiopathic HH can both optimize testicular growth for future spermatogenesis and produce testosterone for puberty.

Cystic fibrosis transmembrane conductance regulator

Cystic fibrosis (CF) has a prevalence of 0.03% in the general population and is the most common lethal genetic diagnosis among Caucasians . Disease occurs as the result of autosomal recessive severe mutations (classes 1, 2, and 3) in both copies of the CF transmembrane conductance regulator (CFTR) gene. This gene encodes a salt homeostasis anion channel in epithelial cells; loss of function of this channel manifests with thickened secretions in the lungs and pancreas . There are greater than 1950 CFTR mutations, and fortunately only a subset results clinically in CF . Among men who carry a mild form (classes 4 and 5) of the CFTR gene mutation, they may be phenotypically normal or present with congenital bilateral absence of the vas deferens (CBAVD). These men will commonly have the caput of the epididymis with failure of development of the remaining epididymis and vas deferens. Other clinical manifestations include: nonpatent vas deferens or blind ending vas deferens, hypoplasia or absence of the seminal vesicles, epididymal obstruction or ejaculatory duct obstruction .

CBAVD carries a prevalence of 1% among infertile patients and 25% of men with primary obstructive azoospermia . Among men presenting with CBAVD, 78% carry at least 1 identifiable CFTR mutation, and 46% carry 2 mutations (compound heterozygotes) . The alleles most frequently mutated and implicated in CBAVD of Northern European men is F508del (17%), R117H (3%), and 5T (25%), with the former also commonly mutated in CF, homozygous F508del/F508del . The mechanism by which CFTR mutations cause CBAVD has been proposed to be the result of abnormal Wolfian duct formation . Patients presenting with CBAVD should receive CFTR testing primarily for the partner and the patient to have an informed discussion regarding risk of CF to the offspring. Men with vasal agenesis should have an abdominal US to evaluate for renal agenesis. These men are amenable to surgical sperm retrieval from the epididymal remnant through percutaneous epididymal sperm aspiration (PESA) or the approach that we prefer to obtain optimal sperm quality microsurgical epididymal sperm aspiration (MESA). The sperm is amenable for use in IVF-ICSI, with appropriate genetic counseling and consideration of preimplantation genetic testing.