Current and future genetic screening for male infertility

Urol Clin N Am 29 (2002) 767–792

Current and future genetic screening for male infertility Paul J. Turek, MDa,*, Renee A. Reijo Pera, PhDb a

Departments of Urology, Obstetrics, Gynecology and Reproductive Sciences, University of California San Francisco, 2330 Post Street, San Francisco, California 94115, USA b Center for Reproductive Sciences, Departments of Obstetrics, Gynecology and Reproductive Sciences, Urology and Physiology, Programs in Human Genetics and Cancer Genetics, University of California San Francisco, Box 0546 HSW-1480, San Francisco, California 94143, USA

Ninety-nine percent of people don’t have an inkling about how fast this revolution is coming. Steve Fodor, President, Affymetrix. Male-factor infertility is a factor in over half of couples that cannot conceive [1]. As many as half of these men have an unknown or idiopathic cause of subfertility. Recently, scientific advances have made it possible to classify many disorders as genetic that were previously considered idiopathic. Added to this growing knowledge of genetic risk is the ability to offer paternity to many of these men using intracytoplasmic sperm injection (ICSI), a technique that by its nature may bypass natural selection barriers. Consequently, it is more important than ever to keep abreast of advances in genetic reproductive medicine. This article reviews both current and future screening used to diagnose genetic infertility in men. After a brief review of genetics, it outlines genetic conditions that are associated with male infertility, the role of genetic counseling, and the currently understood risks of ICSI to offspring. Finally, it discusses current and future technologies applicable to the diagnosis of genetic infertility.

* Corresponding author. Department of Urology, University of California San Francisco, 2330 Post Street, 6th Floor, San Francisco, California 94115-1695. E-mail address: [email protected] (P.J. Turek).

Basic genetic concepts The appreciation of genetic factors in disease has existed for centuries. There are Talmudic references to exemptions from circumcision in males who were born into families of bleeders. Gregor Mendel in 1865 offered several germinal ideas about heredity from his work with garden peas [2]. He proposed that ‘‘hereditary factors’’ and ‘‘units of inheritance’’ (now genes) assort and segregate independently and lead to mutations and evolution. These ideas have since become established laws of heredity still accepted to this day. DNA consists of base pairs of amino acids. Genes are segments or sequences of base pairs that can to form a protein after the processes of transcription and translation. There are at least 40,000 genes in the human genome as garnered from the initial draft of the Human Genome Project [3,4]. Approximately 3000 genes, roughly 10% of all genes, have known associations with disease. Mutations are stable alterations in the DNA that can be passed to offspring. Mutations are common and necessary for evolution, but hereditary variation that is clinically noticeable is called disease. Recognized genetic defects are generally divided into three categories [5]. The smallest and simplest are point mutations in single genes that follow the rules of mendelian genetics and are usually acquired and transmitted in one of four basic patterns: autosomal dominant or recessive or Xlinked dominant or recessive. An example of a disorder that results from a mutation in a single gene is cystic fibrosis. The second category of genetic

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defects is that of chromosomal disorders. These are changes in whole segments of chromosomes and are classified as either structural or numerical in type. Structural problems occur when chromosomes break and reunite. As a consequence, there can be a loss (deletion), a gain (duplication), or an exchange (balanced defect) of genetic material (Fig. 1). Numerical chromosomal defects result from the failure of paired or sister chromatids to separate correctly during cell division (Table 1). As a result, extra or missing chromosomes are present ranging from one (aneuploidy) to an entire set of chromosomes (triploidy). The third category is polygenic or multifactorial genetic defects. These defects are the most common and include most disorders of human biology. They occur as a result of abnormalities in many genes from various chromosomes or in abnormalities of single genes that show variable defects in response to environmental effects or genetic modifiers. This category of gen-

etic defects tends to put the individual at some risk for disease; the environment then influences whether or not the disease is expressed. A fourth, rare category of mutations, termed mitochondrial disorders, consists of mutations in nonchromosomal DNA within these subcellular organelles. Human nucleated cells contain 46 chromosomes (23 pairs) consisting of one pair of each of 22 autosomes and one pair of sex chromosomes, the normal, diploid state found in all somatic cells. Germ cells (sperm and oocytes) contain only 23 chromosomes and are therefore termed haploid. When two gametes join during fertilization, each haploid gamete contributes to the making of a normal, diploid embryo. Because females have two X sex chromosomes, the haploid oocyte by necessity has a single X chromosome. Sperm have either an X or Y chromosome to contribute to the embryo. When somatic cells replicate, the process is termed mitosis; no reduction in chromosome num-

Fig. 1. Examples of chromosomal structural abnormalities in a single chromosome. (A, B, C) and between chromosomes (D). The chromosomal material that is altered or exchanged is shaded in each figure.

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Table 1 Examples of numerical chromosomal abnormalities Category

Example

Normal

46, XY

Aneuploidy (extra/missing chromosome) Monosomy Trisomy Mosaic aneuploidy (within individual)

47, XXY 45, X 47, XY, þ18 mos 46, XY/47, XXY

Polyploidy (extra sets of 23 chromosomes) Triploidy Tetraploidy

69 chromosomes 92 chromosomes

ber occurs. When the sex cells are formed from their precursors, however, the replication process involves an extra cell division that reduces the number of chromosomes from 46 to 23 in the gamete (Fig. 2). Subsequently, a typical mitotic step occurs so that the single, initially diploid cell produces four haploid cells. This sex cell replication pathway is termed meiosis. The maturation process that newly minted haploid spermatids undergo to become sperm is termed spermiogenesis; it does not involve nuclear replication. Genetic conditions associated with male infertility To examine the various genetic disorders that have been associated with infertility, it is best to divide the reproductive tract into three components, pretesticular, testicular, and posttesticular, and to examine specific genetic conditions associ-

Fig. 2. The events of spermatogenesis. Meiosis involves a reduction-division in the first of its two stages, so that normal diploid chromosomal complement is halved in sperm (n ¼ haploid number of chromosomes).

ated with each category [5]. The clinical or molecular tools available to make the diagnosis for each condition are listed in the tables associated with each section. The order of discussion of each condition is from most to least prevalent, and prevalence values given with each disorder reflect the incidence in the general population. In general, the single best educational resource on genetic conditions can be found on-line at www.ncbi.nlm.nih.gov. At this website, searching OMIM (Online Mendelian Inheritance in Man) [172] accesses a comprehensive database of human genes and a catalogue of genetic disorders, including current references. Current information about molecular testing available for genetic disorders is found at www.genetests.org [171]. Genetic conditions with pretesticular effects Pretesticular effects on infertility are generally hormonal in nature, because spermatogenesis is controlled by the hypothalamic-pituitary-gonadal axis. Most conditions that affect this hormonal axis result from single gene deletions (Table 2). b-thalassemia (1:20–30) Patients with b-thalassemia are largely of Mediterranean or African descent and have mutations in the beta-globin gene that lead to reduced amounts of b-globin (not to defective beta-globin as with sickle cell disorders) and thus to an imbalance in alpha- and beta-globin composition of hemoglobin [6]. Mutations ranging from point to entire gene deletions on chromosome 11p15 have been associated with this disorder [7]. Clinical features range from mild anemia (trait) to hemolytic anemia and iron overload (major thalassemia). Infertility is believed to result from the deposition of iron in the pituitary gland and testes, as in sickle cell anemia. Infertility treatment for both conditions is similar.

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Table 2 Genetic diagnosis of pretesticular causes of infertility Diagnosis Condition

Genetics

Clinical

b-thalaessemia

11p15

Anemia, iron overload

Sickle cell anemia

11p15.5

Cogenital adrenal hyperplasia Kallman’s syndrome

6p21.3

Anemia, red blood cell sickling Variable

Kalig-1 Xp22.3

Prader-Willi syndrome

15q11q13

Bardet-Biedl syndrome

6 loci: 11q13, 15q22 16q21, 3p13 2q31, 20p12 Xq11-q12

Androgen receptor mutations Kennedy disease

Xq11–12

Ataxia and hypogonadism

None

Juvenile hemachromatosis

1q

FSH/LH hormone or receptor defects

LH 19q13 FSH 11p13

Delayed puberty anosmia, testis atrophy Obesity delayed puberty, small hands/feet Retinitis pigmentosa, polydactyly, delayed puberty Variable hypospadias female phenotype Muscule atrophy testis atrophy Speech, gait disturbances Iron overload, pain, arrhythmia Variable phenotype undervirilized

Metabolic

Molecular

Sickle cell test

Hemoglobin electrophoresis Mutation analysis 11p15 Mutation analysis 11p15.5 Mutation analysis 6p21.3 FISH analysis

Urine metabolites Low FSH, LH, testosterone Low FSH, LH, testosterone Low FSH, LH, testosterone

Genital skin fibroblast biopsy

Low FSH, LH, testosterone Low FSH, LH, testosterone Variable low testosterone

15q11q13 Mutation analysis FISH Mutation analysis (research)

Mutation analysis

CAG repeat analysis Not available Not available Sequencing B subunit for FSH (research)

Abbreviations: FISH, fluorescence in situ hybridization; LH, luteinizing hormone; FSH, follicle stimulating hormone; CAG, cytosine adenosine guanine nucleotide repeat.

Sickle cell anemia (1:600) This autosomal recessive disorder is associated with the inheritance of two beta-S globin genes and is found in African-Americans. The relevant mutation is a single DNA base pair change on chromosome 11 [6,7]. Sickling of red blood cells occurs at low oxygen tension and, as a result, blood viscosity increases, causing stasis of flow in small vessels. Men with sickle cell anemia have decreased testosterone levels and either increased or decreased levels of luteinizing hormone (LH) and follicle-stimulating hormne (FSH). Although the presence of high or low levels of gonadotropins would seem to suggest quite different disease processes, it is theorized that pituitary and testicular microinfarcts from sickle disease account for both presentations of secondary hypogonadism. Infertility may be treatable if a pituitary defect is diag-

nosed, but assisted reproductive technology is required for cases of primary testicular failure. Gene therapy is actively being pursued for this disorder, based on improving RNA repair [8] or creating an antisickling beta-A globin variant molecule introduced through lentiviral vectors into bone marrow [9]. Congenital adrenal hyperplasia (1:5000) Several syndromes result from hereditary defects in the adrenal enzymes essential to glucocorticoid and androgen formation. Five enzymes are involved in the conversion of cholesterol to testosterone, and all can be affected by mutations. Mutations tend to be transmitted in autosomal recessive or X-linked patterns. In the absence of overt adrenal insufficiency (salt-wasting), congenital adrenal hyperplasia (CAH) is usually not

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recognized in newborn males. The most common form of CAH (90%) involves a deficiency of 21 hydroxylase located on 6p21.3. Cortisol and aldosterone production is low, but testosterone production is normal. Excess testosterone, however, is made because of excessive adrenal drive (elevated corticotropin), and thus precocious virilization is possible, resulting in growth and maturation of external genitalia, development of secondary sex characteristics, hoarsening of the voice, frequent erections, and excessive muscular development. Precocious virilization can occur in boys aged 3 to 5 years and can be the first sign of the disorder in non–salt-wasting CAH [10]. The much less common, salt-losing forms of CAH that have earlier effects on the cholesterol pathway (3b-hydroxysteroid dehydrogenase [maps to 1p13.1] and 20, 22-desmolase [maps to 8p11.2] deficiencies) usually do not result in virilization, and in fact, depending on the totality of the enzymatic block, can result in failure of normal internal and external virilization in infants [11]. There are also two enzymes within the testis that govern testosterone synthesis (17,20 desmolase and 17b-hydroxysteroid dehydrogenase [maps to 9q22]) that can be affected in CAH but do not result in salt-wasting symptoms. Mutations in these enzymes generally result in findings similar to 5a-reductase deficiency and include ambiguous or female external genitalia and cryptorchid testis in the male [12]. Despite drastic alterations in normal testosterone production, spermatogenesis in CAH can result from any of the five possible enzyme defects [13,14]. Spermatogenesis varies with the severity of the disorder and with how well the metabolic disorder is managed. Spermatogenesis is usually, but not always, associated with virilization, which follows one of two patterns. Excessive adrenal androgens can inhibit FSH and LH production and result in testes that remain infantile despite masculinization. Men with this disorder have erections and ejaculation but are azoospermic. Alternatively, adrenal androgens can prematurely activate maturation of the hypothalamic-pituitary-gonadal axis and initiate a true precocious puberty, including spermatogenesis. In summary, CAH is a major metabolic disorder in which infertility occurs but is not the initial presenting symptom. Kallman syndrome (1:30,000) Idiopathic hypogonadotropic hypogonadism (IHH) or Kallman syndrome is characterized by hypogonadism. Most patients experience a delay

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in puberty, although those with less severe defects may present with infertility only. Other findings include anosmia, cleft palate, and small testes. When anosmia is not present, the condition is termed IHH. Testicular biopsies display a wide range of findings from germ cell aplasia to focal areas of complete spermatogenesis [15]. The condition is inherited as a familial disorder in one third of cases. Both X-linked and autosomal inheritance patterns have been described [16,17]. In the Xlinked recessive form, deletions occur in Kalig1(kallman-interval 1), a gene responsible for the migration of gonadotropin-releasing hormone (GnRH) neurons to the preoptic area of the hypothalamus during development [18]. As a consequence, there is failure of testicular stimulation by the anterior pituitary and hypothalamus and thus testis failure. Mutations in other genes have also been associated with the development of IHH including Dax1 on the X chromosome (associated with congenital adrenal hyperplasia) [19], the GnRH receptor [16], and PC1 (associated with diabetes and obesity) [20]. Infertility can be treated with gonadotropin (LH and FSH) replacement over 12 to 18 months that induces sperm in the ejaculate in 80% of affected men [21]. The clinical diagnosis of Kallman syndrome is confirmed by blood tests revealing a low total testosterone level associated with low LH and low FSH levels in combination with normal prolactin levels. Prader-Willi syndrome (1:20,000) Genetic obesity, retardation, small hands and feet, early hypotonia, and hypogonadism characterize Prader-Willi syndrome. As with Kallman syndrome, a deficiency of GnRH is the postulated reason for hypogonadism. The single gene deletion associated with this condition has been mapped to a locus on paternal chromosome 15q11q13 [22]. Loss of maternal chromosome 15 will not result in the disease. Spermatogenesis can be induced with exogenous FSH and LH. A clue to this diagnosis is obtained from results of pituitary-gonadal hormone testing that resemble results with other forms of IHH. Bardet-Biedl syndrome (rare) Another rare form of hypogonadotropic hypogonadism, Bardet-Biedl syndrome also results from GnRH deficiency. It is characterized by retinitis pigmentosa, polydactyly, renal cystic dysplasia, and hypogonadism and is found in Arab and Bedouin populations with an incidence of

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1:13,500. Similar to Prader-Willi syndrome and unlike Kallman syndrome, it is also associated with genetic obesity. This condition has been linked to at least six distinct genetic loci, suggesting that it is a genetically heterogeneous disorder. In one study of 29 families, approximately one third of patients showed linkage to 11q13, another one third linked to 15q22.2-q23, and 25% linked to 16q21. The remaining 10% of patients showed no linkage pattern [23]. Subsequently, linkages to 3p13, 2q31, and 20p12 have been identified [24]. It is currently thought that disease requires mutations in one of six loci along with an additional mutation in a second locus. The hypogonadism is probably hypothalamic in nature and can be treated with FSH and LH. Androgen receptor gene mutations (1:60,000) In androgen receptor gene mutations, the androgen receptor (AR) is absent or functionally altered so that testosterone or its more bioactive derivative, dihydrotestosterone, cannot activate target genes. Thus, androgens have little to no effect on tissues; both internal and external genitalia are affected. Depending on the severity of the AR defect, serum testosterone levels can be low, normal, or quite high. The androgen balance in each individual depends on the functional integrity of the androgen receptors within the pituitary and hypothalamus. The clinical picture of infertility is also variable (nomenclature includes Reifenstein’s, and Rosewater syndromes) and ranges from apparent females with testes (complete androgen insensitivity) to normal but infertile males (Table 3) [25]. Over 300 distinct (mainly point) mutations have been found in the androgen receptor, a large steroid receptor gene on the X chromosome (Xq11-q12) [17]. Given the widely variable phenotype and numerous mutations noted in the AR gene, it has been proposed that as many as 40% of men with low or no sperm counts may have subtle androgen receptor abnormalities as an

underlying cause [26]. It is not clear whether the infertility in the milder forms of this disorder is truly treatable despite a case report of success with exogenous testosterone supplementation [27]. Kennedy disease (rare) Kennedy disease, also termed spinal and bulbar muscular atrophy, is a debilitating, neurodegenerative condition that begins by age 30 years and consists of muscle cramping and atrophy as well as testicular atrophy. The genetic basis for the disease is not exactly a mutation but a variation in the length in a specific region (transcriptional activation domain) of the AR gene that results in decreased androgen-binding ability. The AR gene on Xq has eight exons, and it is clear that a critical region of cytosine adenosine guanine (CAG)nucleotide repeats, usually 15 to 30 in number, exists in exon 1. Elongation of the number of CAG repeats in this region to more than 50 generally results in clinically apparent disease [28]. The clinical infertility resembles that caused by mild androgen insensitivity and may include gynecomastia in addition to testis atrophy. Interestingly, some affected patients may have only mild oligospermia and thus may be able to conceive naturally or with ICSI. Men fertile early on may actually experience reduction of sperm counts with advancing age and disease progression. Importantly, affected couples should be counseled about the phenomenon of genetic anticipation that occurs with this disorder, in which offspring may in fact inherent an even larger number of CAG-nucleotide repeats than occur in the parent. Clinically, this anticipation translates into a more severe disease phenotype that is manifest earlier in life. Specialized laboratories offer exon 1 CAG- nucleotide repeat analysis of the AR gene. Ataxia and hypogonadism (rare) Ataxia and hypogonadism is a rare condition that largely results from consanguineous unions and is probably transmitted in an autosomal reces-

Table 3 Categories of androgen receptor deficiency Category

Fertility potential

Receptor negative (No DHT binding) Low receptor (decreased DHT binding)

46 XY; Sertoli-cell–only testes Similar to receptor-negative patients Spermatogenesis present Infertile males

Abnormal receptor (abnormal binding) Receptor positive (normal DHT binding) Abbreviation: DHT, dihydrotestosterone.

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sive manner [29]. Cerebellar involvement is implicated because of speech and gait abnormalities, which start to occur at about 20 years of age. Affected patients can look eunuchoid and have atrophic testes and low libido. On evaluation, pituitary gonadotropin (LH and FSH) and testosterone levels are generally low. Hypothalamicpituitary dysfunction is thought to be the cause of infertility, possibly because of atrophy or hypoplasia of brain matter [30]. Gonadotropin replacement may be of value in helping affected individuals to conceive. Juvenile hemachromatosis (rare) Unlike the relatively common hereditary hemachromatosis affecting white men, the type II or juvenile form affects both sexes and has an earlier onset. Clinical symptoms of iron overload usually begin before age 30 years, and patients usually present with abdominal pain, hypogonadotropic hypogonadism, arrhythmias, and heart failure. Unlike the classic hereditary form that maps to 6p21.3, the juvenile disorder probably maps to a different locus (1q) [31]. On evaluation, pituitary gonadotropin (LH and FSH) and testosterone levels are low, as in sickle cell anemia, and are probably caused by iron deposits in the pituitary [32]. Luteinizing hormone and follicle-stimulating hormone and receptor mutations (rare) The pituitary hormones LH and FSH are each composed of two subunits: a shared alpha subunit and different beta subunits (LH beta-subunit gene localizes to 19q13.2, and FSH beta-subunit gene maps to 11pter-p11.2) [33]. Beta-subunit mutations in LH have been described and can lead to a wide variety of phenotypic features, including infertility with absent spermatogenesis to complete virilization failure [34,35]. Mutations in FSH have not been formally identified but are suggested by findings in infertile women [36]. Abnormalities of the FSH and LH receptors might also be causes of male infertility. Luteinizing hormone resistance caused by receptor abnormalities has been described and is associated with male pseudohermaphroditism and Leydig cell agenesis [37]. In contrast, FSH resistance caused by receptor defects has been reported but has been associated only with mild IHH-type symptoms [38]. An estrogen-receptor mutation has also been described in an infertile male with estrogen resistance (elevated LH, FSH, and estradiol levels but normal testosterone levels), but its relationship to infertility is speculative [39].

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Genetic conditions with testicular effects Conditions that directly affect the testicle tend to result from structural or numerical chromosomal abnormalities (Table 4). Testicular genetic effects are, currently, largely untreatable. Assisted reproductive technology, however, can provide biologic children for affected men, virtually ensuring that the genetic disease will be transmitted. Klinefelter syndrome (1:500) Klinefelter syndrome is the most common genetic reason for azoospermia, accounting for 14% of cases. It is associated with a triad of findings: small, firm testes, azoospermia, and gynecomastia, although gynecomastia is not necessarily a typical feature. Klinefelter syndrome is an abnormality of chromosomal number in which 90% of men carry an extra X chromosome (47 XXY) and 10% are mosaic with a combination of XXY/XY chromosomes. It is thought that approximately half of XXY cases are paternally derived, and recent evidence suggests that its occurrence may correlate with advancing paternal age [40]. This syndrome may also be associated with increased height, decreased intelligence, varicosities, obesity, diabetes, leukemia, increased likelihood of extragonadal germ cell tumors, and breast cancer (20-fold higher than in normal males). Testis biopsies show sclerosis and hyalinization. Hormones usually demonstrate a low testosterone level and frankly elevated LH and FSH levels. Natural paternity with this syndrome is possible but almost exclusively with the mosaic or milder form of the disease. Biologic paternity in cases of pure XXY males is now possible with ICSI [41]. Despite a uniformly abnormal somatic genotype, 75% to 100% of mature sperm from 47, XXY patients harbor a normal haploid sex chromosome complement (X or Y) instead of XY or YY complements [42–44]. The lack of significant gonosomal aneuploidy in the presence of somatic aneuploidy suggests that abnormal germ cell lines may arrest at a meiotic checkpoint within the testis or that somatic-germ line mosaicism is more common than previously thought. XYY syndrome (1:1000) XYY syndrome is another abnormality of chromosomal number that can result in infertility. Typically, men with 47,XYY are tall, may show decreased intelligence, could have a higher risk of leukemia, and may exhibit aggressive, often

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Table 4 Genetic diagnosis of testicular causes of infertility Diagnosis Condition

Genetics

Clinical

Metabolic

Molecular genetic

Klinefelter syndrome XYY syndrome

47, XXY and variants 47, XYY and variants

Gynecomastia, testis atrophy Tall, decreased intelligence

Karyotype

Chromosome translocations/ inversion Y microdeletions Noonan syndrome

Variable

Normal, infertile

Yq11, AZFa,b,c 12q24.2

Normal, infertile short, webbed neck, low ears

Mytonic dystrophy

19q13.2

SRY gene defects

Yp11.3

Cataracts, muscle weakness, infertile Sex reversal

High FSH, LH low testosterone High FSH, normal LH, Testosterone High, FSH, normal LH, Testosterone None High FSH, normal LH, Testosterone None

XX male syndrome

46, XX, SRY positive 46, XY 46, XY/45, X 1p35.1

Mixed gonadal dysgenesis Immotile cilia syndrome Dysplasia of fibrous sheath

None identified

Normal, testis atrophy Streak gonad, cryptorchidism Sinusitis, bronchiectasis, immotile sperm Immotile sperm

None None None None None

Karyotype

Karyotype

Yq mutation analysis Mutation analysis (Research) CTG repeat analysis Mutation analysis, FISH Karyotype, FISH Karyotype FISH analysis for SRY Sperm electron microscopy Sperm electron microscopy

Abbreviations: FISH, fluorescence in situ hybridization; FSH, follicle stimulating hormone; LH, luteinizing hormone; CTG, cytosine thymine guanine.

antisocial behavior [45,46]. Hormone evaluation reveals an elevated FSH level and normal testosterone and LH levels. Semen analyses show either severe oligospermia or azoospermia. In azoospermic men, testis biopsies demonstrate maturation arrest or Sertoli-cell–only syndrome. Studies that have focused on the chromosomal complements in mature sperm from XYY men show findings similar to those in Klinefelter’s patients: very few sperm (\1%) harbor sex chromosomal disomy (YY, XX, XY) [47–49]. This finding supports the hypothesis that the extra Y chromosome is eliminated at meiotic checkpoints during spermatogenesis. Chromosomal translocations or inversions (1:600–1:1000) When segments of chromosomes are exchanged, a translocation results (see Fig. 1D). When a chromosome breaks in two places and the material between the breakpoints reverses orientation, an inversion results (see Fig. 1C). Such exchanges may either interrupt important genes at the breakpoint or interfere with nor-

mal chromosome pairing during meiosis because of imbalances in chromosomal mass. Many translocations have been associated with male infertility. In particular, reciprocal and Robertsonian translocations (in which material from chromosomes 13, 14, 15, 21, and 22 are involved in as many as 15 different translocations) are at least eightfold more common in infertile men than in newborn males [50]. Importantly, carriers of Robertsonian translocations are at risk for pregnancies that may miscarry and for offspring with mental retardation and birth defects. In addition, autosomal inversions, particularly those involving chromosome 9, are also eightfold more likely to occur in infertile than in fertile men [50]. These types of chromosomal derangements tend to be balanced, so that no genetic material is lost or gained, and result in phenotypically normal males, but they are associated with oligospermia or azoospermia. Y chromosome microdeletions (1:4000 men) Twenty-five years ago, Tiepolo and Zuffardi [51] showed that the Y chromosome (Yq11) contains an azoospermia factor (AZF). With the construction

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of Y chromosome deletion maps, Reijo et al [52] were among the first to show molecular microdeletions in the Y chromosome in azoospermic men, most obviously in a region designated DAZ (deleted in azoospermia). Subsequently, Vogt et al [53] described deletions associated with male infertility that occur in three distinct regions of the Y chromosome, which they termed azoospermia factor regions a, b, and c (AZFa, b, c). Most recently, the entire nucleotide sequence of the AZFc region was determined, revealing this area to be a remarkable complex of six repeated copies of seven separate genes (BPY2, DAZ, CDY1, CSPG4LY, GOLGA2LY, TTTY3, and TTTY4) resulting from tandem duplication and inversion during evolution [54]. Further confirming the significance of the AZF regions, an actual DNA point mutation (four base pairs) in a single Y-linked gene (DFFRY, AZFa region) has been observed to be associated with male infertility [55]. It is now clear that similar Yq microdeletions occur in 6% to 8% of severely oligospermic men as well as in 3% to 15% of azoospermic men [56]. Taken together, such deletions are the most common molecularly defined cause of male infertility in humans [57]. It is currently advised that infertile men with ejaculated sperm concentrations below 5 million/ mL undergo genetic counseling and be offered testing for Y chromosome microdeletions [57]. It has been shown conclusively that Y chromosome microdeletions, although not associated with risks to general health, may be passed to the male offspring from affected fathers through ICSI [58]. One recently noted complicating feature of Y chromosome microdeletion testing is that some men may actually be genetic mosaics and harbor DAZ deletions only in germ line (gamete) tissue and not in somatic cells [59]. Thus, the current technique of testing peripheral blood for Y chromosome deletions may not accurately reflect the Y chromosome status of sperm. Noonan syndrome (1:2000) Noonan syndrome presents phenotypically as a male Turner syndrome that is inherited in an autosomal domination pattern. The karyotype in these men is normal (46,XY), however. A suspected genetic locus has been reported at 12q24.2-q24.31 [60,61], which probably involves a gene that encodes for a protein-tyrosine phosphatase and plays a role in the cellular response to extracellular signaling [62]. A second type of Noonan syndrome (type 2) that seems to be transmitted in an autosomal recessive pattern has

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also been described [63]. Typically, these men have dysmorphic features such as a webbed neck, short stature, low-set ears, and wide-set eyes. Abnormalities may also include bleeding diathesis and cardiac anomalies. At birth, 77% will have cryptorchidism that limits fertility in adulthood [64]. If the testes are fully descended, fertility is likely. Myotonic dystrophy (1:8000) Myotonic dystrophy is the most common cause of adult-onset muscular dystrophy and usually presents with cataracts, muscle weakness and wasting, hyogonadism, ECG changes, diabetes (5% of cases), and cholelithiasis (25%). Symptoms usually become evident in middle life, but signs may be evident in the second decade. This autosomal dominant disorder involves the expansion of an unstable trinucleotide repeat cytosine thymine guanine (CTG) region of a serine-threonine protein kinase gene on 19q13.2 [65]. Because reduced gene function correlates to the degree of repeat expansion, the severity of the condition varies with the number of repeats: normal individuals have between 5 and 30 copies, mildly affected persons have between 50 and 80 copies, and severely affected patients can have 2000 or more copies. Like Kennedy disease, this disorder is characterized by anticipation, in which amplification of the disease is observed in parent-to-child transmission, especially from mother to offspring. A congenital form of myotonic dystrophy also exists and presents with neonatal hypotonia, mental retardation (60%), and facial abnormalities. The congenital form is almost uniformly transmitted maternally [66]. Most men (65%) affected with myotonic dystrophy have testis atrophy on physical examination [67]. In addition, it is common to see elevated FSH and LH levels with normal testosterone levels and testis biopsies that show significant seminiferous tubule damage in 75% of cases. In one study, despite these findings of primary testicular failure, 66% of married men with myotonic dystrophy conceived naturally [67]. SRY gene defects (rare) Located on the short arm of the Y chromosome (Yp11.3) is the sex-determining region Y (SRY) gene, important for determining ‘‘maleness’’ [68]. Both 46XX SRY–positive males and 46 XY SRY–negative females have been described in the literature and constitute a rare form of phenotypic sex reversal [69,70]. The SRY gene encodes a

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protein that harbors a high mobility group box (HMG) sequence, a highly conserved DNA-binding motif that helps kink DNA [71]. This DNAbending effect may alter the ability of target genes to be transcribed. SRY mutations are not always detected in 46,XY phenotypic females, suggesting that genes other than SRY and in locations other than on the Y chromosome are important for complete male gonadal differentiation [72]. Indeed, a sexreversing locus has been mapped to the X chromosome (Xp21.2-p22.1) that may alter SRY activity during development by suppressing downstream genes that induce testis differentiation [72]. The locus has been termed SRVX, and its copy number may account for some Xlinked cases of the 46,XY female phenotype [73]. Potentially, SRY presence may normally suppress SRVX activity and therefore allow normal testis differentiation. If the sex-reversing SRVX gene copy number is too large because of duplication, SRY may not be able to suppress SRVX gene activity fully, and testis differentiation fails, leading to dysgenesis. In the male, this condition is generally detected by cytogenetic analysis showing a shortened or absent Y chromosome. Further analysis with fluorescence in situ hybridization (FISH) can determine the presence or absence of the SRY gene within the existing chromosomal complement. With mutations to, or translocation of, this gene, spermatogenesis genes on Yq are usually affected and result in infertility. XX male syndrome (1:20,000) In a male, the XX structural and numerical chromosomal condition is associated with gynecomastia at puberty or with azoospermia later on [74]. Typically, there are normal male external and internal genitalia. Hormone evaluation reveals elevated FSH and LH levels and low or normal testosterone levels. Testis biopsy demonstrates an absence of spermatogenesis with hyalinization, fibrosis, and clumps of Leydig cells. The most obvious explanation for the disease is that the SRY or testis-determining region is translocated from the Y to the X chromosome so that testis differentiation is present. This mechanism has been suggested because of the high degree of homology (98.7%) that exists between the short arm of the X and Y-chromosomes [75]. The azoospermia gene (AZF) regions on the Y chromosome, however, are not similarly translocated, resulting in azoospermia.

Mixed gonadal dysgenesis (rare) In males and females, mixed gonadal dysgenesis is a heterogeneous condition characterized by a unilateral testis on one side and a streak gonad on the opposite side. Genotypically, patients are usually 46,XY or 45,X/46,XY, both of which are associated with impaired gonadal development. Interestingly, mutations in the SRY gene have not been detected in most patients (80% have normal SRY), suggesting that the gonadal dysgenesis may be caused by cytogenetic mosaicism or by mutations in testis organizing genes that are downstream to SRY. One of these genes may be the newly cloned human testatin gene (20p11.2), a putative cathepsin inhibitor that is expressed early in testis development, just after SRY expression [76,77]. Affected patients may present with ambiguous external genitalia, but almost all will have intraabdominal testes. Scrotal testes may be associated with inguinal hernias and almost uniformly reveal seminiferous tubules with Sertoli cell only and with normal Leydig cells [78]. The dysgenetic gonad is predisposed to malignant degeneration (25% incidence) to gonadoblastoma or dysgerminoma, typically before puberty. Immotile cilia syndromes (1:30,000) The hallmark of immotile cilia syndromes, a group of heterogeneous diseases, is impaired or absent ciliary or flagellar motility. Clinical manifestations include chronic cough and sinus infection, nasal polyposis, bronchiectasis, and infertility. Kartagener syndrome, a particular form of this disease (1:60,000) involves the clinical triad of situs inversus with dextrocardia, bronchiectasis, and chronic sinusitis. Immotile cilia syndromes are characterized by abnormal cell axonemes, both in cilia and sperm tails. The normal axonemal arrangement is nine outer microtubular doublets with inner and outer dynein arms surrounding two central microtubules. In this disease, various defects in both the microtubule and dynein arm assembly have been reported [79]. It is thought that cellular motility is controlled by the action of over 200 distinct genes. Although no specific genes have been linked to this disease, the inheritance pattern in family pedigrees suggests that it is likely to be autosomal recessive [5,79]. Recently, investigators have cloned and mapped (to 1p35.1) a human homologue of a flagellate algae gene that encodes for inner dynein arms, a finding that may be an early clue to the genetics of sperm motility [80].

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Although infertility is the rule with immotile cilia syndromes, ejaculated sperm can be motile, and sperm concentrations can be normal or high. With ICSI, clinical pregnancies and live births have been reported using affected sperm [81,82]. Because the gene defect is usually recessive, normal offspring are likely; still, genetic counseling is critical. A novel association between immotile cilia syndromes and autosomal dominant polycystic kidney (ADPK) disease has recently been observed in Japanese patients: fully 25% of men with immotile cilia syndromes in a single center (n ¼ 16 patients) also had ADPK [83]. Clinical suspicion of this diagnosis is confirmed by sperm electron microscopy revealing abnormalities of the flagellar dynein arms or microtubules [84]. In humans, no specific gene mutations have been associated with these disorders as yet. Dysplasia of the fibrous sheath (rare) Dysplasia of the fibrous sheath (DFS) is a primary disorder of severe sperm asthenospermia quite distinct from immotile cilia syndromes described earlier. Classically, sperm are completely (mean 99%) immotile and exhibit short, thick, and irregular tails [84]. When examined by electron microscopy, affected sperm reveal marked hypertrophy and hyperplasia of random fibrous sheath constituents that form thick rings around the microtubules. As in immotile cilia syndromes, missing axonemal central pairs and dynein arms are infrequently apparent [85]. These abnormal-

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ities have been observed to be stable over time within individuals. A familial incidence and ethnic associations have been reported [86,87], suggesting an underlying genetic cause. Currently, however, no associated genetic mutations have been discovered, and thus testing is limited to electron microscopy findings. Genetic conditions with posttesticular effects The posttesticular portion of the reproductive tract includes the epididymis, vas deferens, seminal vesicles, and associated ejaculatory apparatus. Genetic conditions affecting these organs are mainly of polygenic or multifactorial nature (Table 5). This discussion excludes some conditions that present at birth or early childhood with ambiguous genitalia. Mutations in the cystic fibrosis transmembrane regulator gene (1:20–1:2500) Cystic fibrosis (CF) is the most common autosomal recessive disease in whites with an incidence of 1:2500 births and a carrier frequency of 1:20 [5]. The cystic fibrosis gene, called the cystic fibrosis transmembrane regulator gene (CFTR; 7q31.2), was cloned in 1989 and encodes the cyclic adenosine monophosphate-regulated chloride channel found in many secretory epithelia [88]. More than 800 mutations have been identified in the CFTR gene [89]. Clinical features of cystic fibrosis include chronic pulmonary obstruction and infection,

Table 5 Genetic diagnosis of posttesticular causes of infertility Diagnosis Condition

Genetics

Clinical

Metabolic

Molecular genetic

CFTR mutations

CFTR 7q31.2 5T IVS8 PKD1 16p13.3 PKD2 4q21

Absent vas, sinusitis

Sweat-chloride test

Kidney, liver, pancreas, intracranial cysts, hypertension Neurologic Obstructive azoospermia Bladder extrophy, epispadias Lax abdominal muscles, cryptorchidism Severe hypospadias, vaginal pouch Hernia, with uterus, fallopian tube

Renal insufficiency

Mutation analysis, gene sequencing Mutation analysis, DHPLC analysis

ADPK

Myelodysplasia Young syndrome Exstrophy/epispadsia complex Prune belly syndrome

None identified None identified None identified

5a-reductase deficiency

2p23

Persistent mu¨llerian ducts

19p13.3, 12q13

None identified

None None None

None None None

None

None

Elevated urinary steroids (research) None

None None

Abbreviations: ADPK, Autosomal dominant polycystic kidney disease; DHPLC, Denaturing high-performance liquid chromatography.

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exocrine pancreatic insufficiency, neonatal meconium ileus, and male infertility. Over 95% of men have abnormalities in wolffian duct–derived structures manifesting most commonly as congenital bilateral absence of the vas deferens (CBAVD) [17,28]. Anatomically, the body and tail of the epididymis, vas, seminal vesicles, and ejaculatory ducts are affected or absent, but the testis efferent ducts and caput epididymis are always present. One percent to 2% of otherwise infertile men also have CBAVD, which is considered a genital form of cystic fibrosis [90,91]. These patients exhibit the same spectrum of wolffian duct defects as those with cystic fibrosis but generally lack the severe pulmonary, pancreatic, and intestinal problems. Spermatogenesis is normal in approximately 90% of men with CBAVD [92]. It is thought that CBAVD is based on allelic patterns (homozygous and compound heterozygous) similar to those observed in typical cystic fibrosis but involve less severe mutations [91]. Predictably then, CFTR mutations are found frequently in men with CBAVD (Fig. 3), with a high frequency (11%– 36%) of compound heterozygotes in which a different mutation exists in each of the two CFTR gene copies [93,94]. Congenital bilateral absence of the vas deferens without other clinical symptoms is considered an atypical form of cystic fibrosis, and it is recommended that affected patients undergo sweat-chloride testing and evaluation for subtle lung disease, which may include chronic cough, sinusitis, and nasal polyps [95,96].

Fig. 3. Prevalence of mutations and renal agenesis in a population of men (n ¼ 168) with congenital bilateral absence of the vas deferens (CBAVD). (From McCallum TJ, Milunsky JM, Munarriz R, et al. Unilateral renal agenesis associated with congenital bilateral absence of the vas deferens: phenotypic findings and genetic considerations. Hum Reprod 200l;16:282–8; with permission.)

In addition to formal CFTR gene mutations, another genetic abnormality that occurs in CBAVD patients consists of variations in the polythymidine tract of the splicing region of intron 8 (IVS8), a noncoding sequence of DNA within the CFTR gene [91,97]. Three alleles (5T, 7T, and 9T) have been detected in this region, and the efficiency of the splice acceptor site function is greatest with the 9T allele. Decreases in the length of this thymidine tract are found in 12% to 27% of CBAVD patients and in 5% of the general population worldwide [91,98]. A reduction to 5T decreases the efficiency of splicing of exon 9 and eventually leads to a 10% to 50% reduction in CFTR mRNA and presumably to a decrease in mature, functional CFTR protein [5]. Presumably this reduction in mRNA leads to CBAVD without other obvious features of cystic fibrosis. Although the relationship between 5T variants, classic CFTR mutations, and phenotype is quite complex, it seems that a coexisting 5T variant can turn an otherwise phenotypically mild CFTR mutation into a severe one [91,96]. Congenital unilateral absence of the vas deferens (CUAVD) is yet another male infertility phenotype associated with CFTR mutations [99]. Men with this condition are clinically similar to CBAVD patients, except that they have a palpable vas deferens on one side. Despite the unilateral vas deferens, many CUAVD patients present with azoospermia, suggesting the presence of additional, occult wolffian duct abnormalities on the side of the vas deferens [99]. Like CBAVD, CUAVD may be genetically heterogeneous: a large molecular study found that no men with CUAVD and sperm in the ejaculate (n ¼ 12) harbored common CFTR mutations, whereas most men with CUAVD and azoospermia (90%, n ¼ 9) did in fact harbor mutations [99]. Generalized wolffian duct anomalies unrelated to CF gene mutations can also result in CBAVD. Patients with these anomalies, constituting roughly 10% of CBAVD patients (Fig. 3), have associated ipsilateral renal hypoplasia or agenesis and generally do not demonstrate CFTR mutations on molecular testing [100]. CFTR mutations have also been found in nearly 50% of men with idiopathic obstructive azoospermia and normally palpable vas deferens [101]. Because of this finding, it has been suggested that all patients with CBAVD, CUAVD, or idiopathic obstruction be screened for CFTR gene mutations. An analysis of the 5T, 7T, or 9T variants should also be included in this testing. In addition, renal ultraso-

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nography should be performed on all men with CBAVD and CUAVD to evaluate for renal hypoplasia and agenesis, findings that have implications for the likelihood of associated CFTR mutations. Finally, female partners of affected men should also be tested for CFTR mutations to inform the couple fully about all phenotypic possibilities in offspring. Autosomal dominant polycystic kidney disease (1:400–1:1000) Autosomal dominant polycystic kidney disease is characterized by numerous large cysts of the kidneys, liver, pancreas, and spleen and a 10% to 40% chance of developing aneurysms in the brain. Because the syndrome is often asymptomatic until adulthood, the first clinical presentation may be infertility. Infertility is caused by cysts in the epididymis, seminal vesicle, or ejaculatory ducts that can compress the ductal system and cause obstruction [83]. Three separate genetic loci have been associated with ADPK. PKD1 accounts for 85% of disease and is found on chromosome 16p13.3. PKD2 has been mapped to chromosome 4q21, and PKD3 is currently unmapped [5,102]. Because ADPK patients can produce offspring with ICSI using sperm from the epididymis or testis, genetic counseling is important for these patients even though routine molecular testing is not readily available. Myelodysplasia (spinal dysraphism) (1:1000) Myelodysplasia is a poorly understood, possibly multifactorial disorder that affects neurologic development of the spine. No genetic locus has been associated with this disorder, although a genetic mechanism is suggested by the affected sibling rate of 2% to 5%. Infertility in patients with this disease is usually associated with faulty emission or ejaculation, with impairments related to the level of spinal cord involvement. Lesions above T10-L1 will probably cause complete ejaculatory failure; lesions below this cord level may lead to normal emission but poor ejaculation. There is some evidence that spermatogenesis may also be impaired in affected patients [103]. Young syndrome (1:1000) The clinical triad of obstructive azoospermia, chronic sinusitis, and bronchiectasis is termed Young syndrome [104]. Epididymal obstruction, thought to be caused by inspissated secretions, usually occurs at the caput-corpus junction and

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is amendable to surgical correction. Epididymal obstruction may occur well after puberty, allowing paternity in some individuals before the onset of obstruction [105]. Because of the clinical presentation, and because many otherwise healthy men with idiopathic obstructive azoospermia harbor mutations related to cystic fibrosis [101], an association of this condition with CFTR gene mutations also seems likely. Indeed, in a small study of seven affected white men, two carried the delF508 mutation [106], but in two other larger studies totaling 26 men, only 1 exhibited CFTR mutations [107,108]. Other investigators believe that subtle abnormalities of axonemal structure, including absent central pair microtubules, radial spokes, and inner dynein arms, may account for the findings in patients with this condition [109]. Bladder exstrophy/epispadias complex (1:20,000) Bladder exstrophy/epispadias complex results from abnormal development of the cloacal membrane and is associated with a broad range of findings. Generally, there is abnormal closure and fusion of the lower abdominal wall, pubic symphysis, bladder, and penis, presenting as bladder exstrophy, penile epispadias, or both. Inheritance is probably multifactorial, with a 1% incidence among siblings, but no genetic mutations have been identified [110]. Bladder and bone closure and urethral reconstruction are performed at birth but are associated with significant abnormalities of erection and ejaculation in adulthood. Testis, epididymal, vasal, seminal vesicle, and ejaculatory duct anatomy is normal, and natural fertility is possible in the absence of bladder neck reconstruction. Despite excellent erectile function and antegrade ejaculation, however, fertility is hampered in many patients after surgery [111,112]. Prune belly syndrome (1:35,000) Occurring almost exclusively in men, prune belly syndrome (PBS) is characterized by the triad of lax abdominal wall musculature, urinary tract dilation, and abdominal cryptorchidism. The reason for these findings is unclear but may involve urethral occlusion during early gestation. No genetic mutation has been associated with PBS, although an autosomal recessive transmission is suspected in some cases [113]. Fertility with this disorder is compromised for several reasons, including ejaculatory dysfunction caused by bladder neck incompetence and megalourethra, obstruction caused by absent or atretic vas deferens, and cryptorchidism. Cryptorchidism

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is assumed to be a mechanical problem related to bladder distension causing obstruction in the inguinal canals during development. Spermatogenic activity on biopsy has been reported to range from absent to normal in prepubertal boys [114,115], but natural fertility in adulthood (even after orchidopexy) has not been demonstrated. Biologic paternity with PBS has recently been achieved with in vitro fertilization (IVF)–ICSI using testis sperm from PBS patients [116]. 5a-Reductase deficiency (rare) Males affected with 5a-reductase deficiency, an autosomal recessive disorder, have normal internal genital ductal structures and testes but have incompletely virilized external genitalia. A form of male pseudohermaphroditism, this condition results from the lack of conversion of testosterone to dihydrotestosterone (DHT) because of a deficiency in one of two described 5a-reductase enzymes. Mutations in isoenzyme 2, located on 2p23 [117] lead to low or undetectable DHT levels, despite normal testosterone levels. Physiologically, DHT is important for the normal development of the prostate and external genitalia (scrotum and penis), whereas fetal testosterone is critical for the development of normal wolffian duct structures (vas deferens, seminal vesicle) (Fig. 4). Therefore, affected individuals exhibit normal testes and internal wolffian duct structures but have perineoscrotal hypospadias and often a vaginal pouch. There is absence of female internal genitalia. Generally, the testes are found in the labio-scrotal folds or the inguinal canal, the seminal vesicles are rudimentary, and the prostate may be absent. There may be some degree of phenotypic sex reversal at puberty, with muscular development, voice change, phallic enlargement, and semen production but no gynecomastia, because of dramatic increases in testosterone production. Infertility results from the structural abnormalities of the external genitalia. Inguinal or labial testes resemble cryptorchid testes histologically.

Fig. 4. Androgens and tissues involved in the development of the male internal and external genitalia.

Semen production is highly viscous and of low volume. Spermatogenesis has been described in descended testes [118]; however, natural fertility has not been reported in these patients. Paternity is possible with the use of assisted reproductive technology [119]. Persistent mu¨llerian duct syndrome (rare) Persistent mu¨llerian duct syndrome involves altered male sexual differentiation with an abnormal persistence of mu¨llerian duct structures. It is a familial X-linked or autosomal dominant condition in which there is absence or impaired action of anti-mu¨llerian hormone (AMH, mu¨llerian inhibiting substance, testis-secreted H-Y antigen, maps to 19p13.3 [120]) during development. This disorder can be caused by failure of Sertoli cells to elaborate AMH, by failure of end-organ response to AMH (AMH receptor, mapped to 12q13), or by defects in the timing of AMH release [121,122]. Affected males have normal male external and internal genitalia but also have a fully developed uterus and fallopian tubes as a result of incomplete involution of mu¨llerian derivatives. The condition presents clinically as a hernia that contains a uterus and fallopian tubes (hernia uteri inguinalis) or cryptorchidism. Cryptorchidism may be secondary to the tethering of the testes to the mu¨llerian structure and inhibition of normal descent. During hernia repair, all efforts should be made to preserve the integrity of the vas deferens and epididymis. The fertility and cancer risks of cryptorchid patients with this syndrome are similar to those of other cryptorchid individuals. Patients with scrotal testes can be fertile. Role of genetic counseling in infertility Initially, all infertile men should be seen by an urologist for a thorough medical history, physical examination, semen analysis, and appropriate medical testing. In addition, genetic counseling is critical to the care of couples at-risk for genetic infertility. The current approaches to counseling patients at risk for genetic infertility include prescriptive counseling, in which clinicians first offer the appropriate clinical or laboratory tests to at-risk individuals, followed by referral to a genetic counselor if the testing results show an abnormality, and nonprescriptive or classic counseling, in which patients at-risk for genetic conditions meet with a genetic counselor before laboratory testing [123]. The risks and benefits of laboratory testing are discussed with the patient, and the couple then

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decides whether or not to proceed with formal testing (Fig. 5). With this approach, the counselor will not instruct the patient what to do [82]. After testing, patients are counseled regarding the results, whether positive or negative. The nonprescriptive approach has added value in clinical disciplines such as infertility in which the complete spectrum of genetic conditions that cause the problem is incompletely understood. In other words, it is important to counsel patients that although formal testing may reveal no definable genetic abnormality, they may still be at risk for other, currently undefined, types of genetic infertility. Formal genetic counseling can also procure information about family history or pedigree that can lead to further diagnoses or testing [124]. The lack of a family history of infertility or other medical problems does not eliminate or reduce the risk of genetic infertility. In fact, a family history review will often be unremarkable.

Safety of intracytoplasmic sperm injection Over the last decade, the number of sperm needed to achieve a pregnancy has fallen from hundreds of thousands for IVF, to one viable sperm with ICSI. This reduction has led to the development of aggressive surgical techniques to procure sperm from azoospermic men. As a consequence, many kinds of male-factor infertility previously deemed untreatable are, for the first time

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in history, associated with biologic paternity. Although a revolutionary and enabling technique in this regard, ICSI raises several questions, because its safety record has not been determined by fundamental research in clinically relevant models. Does intracytoplasmic sperm injection bypass natural selection barriers? Because sperm is chosen by a technician (and not necessarily by God or Darwin), it is assumed that many natural selection barriers present during natural fertilization are bypassed with ICSI. Indeed the transmission of paternal Y chromosome microdeletions in male offspring has already been demonstrated with this technique [59]. The ultimate significance of this finding is currently unclear but is likely to be clinically important. Can intracytoplasmic sperm injection introduce foreign material into oocytes? During normal fertilization, the sperm acrosome and perinuclear structures are removed at the point of entry into the oocyte cytoplasm. This ‘‘molecular undressing’’ of the sperm by the egg is certainly altered by ICSI, in which unprocessed sperm are mechanically introduced directly into the oocyte cytoplasm. As demonstrated in the monkey ICSI model, there is abnormal sperm nuclear remodeling in ICSI compared with natural fertilization [125]. Indeed, these alterations in

Fig. 5. Algorithm for nonprescriptive genetic counseling and testing for infertility followed by the Program in the Genetics of Infertility (PROGENI) at the University of California San Francisco.

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sperm-egg interaction may disturb early meiotic checkpoints in embryo development [125,126] and conceivably lead to a delay in male genome replication and to chromosomal abnormalities [127]. Another observation from the monkey ICSI model raises concern for the safety of ICSI in humans. Recently, investigators demonstrated that foreign DNA bound to sperm membranes can be transmitted into monkey oocytes with ICSI but seem to remain outside of the oocyte with IVF [128]. Although normal ICSI births were reported in this study, this finding raises concern that ICSI might theoretically allow the unintended transmission of infectious particles into oocytes. Indeed, sperm-mediated DNA transfer is currently under investigation as an alternative route to create transgenic animals [129]. Does intracytoplasmic sperm injection induce or perpetuate birth defects? It is an accepted fact that children born after IVF are as healthy as those conceived normally. The additional risk that ICSI brings to bear on congenital birth defect rates has also been assessed in several centers. Most studies comparing perinatal and morphologic features of children born by IVF-ICSI with those from born to age-matched parents after IVF or those conceived naturally reveal major malformation rates with ICSI that range from 0.95% to 4.6% and suggest no statistical differences in findings [130–133]. One study, however, has disputed these results on the basis of how the birth defects were classified [134]. After birth defects were reclassified in a more standardized format, these authors suggested that the rates of major and minor birth defect in Belgian ICSI children were actually twice as high (7.4%) as originally proposed (3.3%) [130]. This difference was based mainly on the reclassification of 14 observed anomalies as major malformations that were originally considered minor. Another large cohort study from Sweden (N ¼ 1139 infants)

noted that the prevalence of hypospadias is statistically higher in ICSI offspring than in naturally conceived children [135]. In any such comparison, it must be realized that a bias may exist simply because of the detailed scrutiny that ICSI offspring undergo relative to their naturally conceived counterparts. Is intracytoplasmic sperm injection associated with a higher miscarriage rate? Pregnancy losses have been reported in 17% to 22% of ICSI cycles, a number apparently no higher than in IVF cycles [132–139]. This rate is higher than that observed with natural conception and is thought to result from the overall higher incidence of prematurity and multiple gestations associated with IVF [134,140,141] and not to be related to a high rate of prenatal diagnostic testing [136] or to the use of retrieved sperm [13,135,142] in these pregnancies. Does intracytoplasmic sperm injection induce or perpetuate chromosomal abnormalities in children? Intracytoplasmic sperm injection is associated with a statistically higher chance of producing offspring with chromosomal abnormalities than natural conception or IVF. The overall incidence of abnormal karyotypes observed in ICSI offspring is outlined in Table 6. Detailed analysis of the largest (N ¼ 1082) and best-studied cohort of ICSI children [143] reveals a twofold increase in autosomal anomalies (0.83%), and a fourfold increase in sex chromosomal abnormalities (0.83%) compared with natural conceptions (0.2%–0.6% and 0.20%, respectively). Among the chromosomal abnormalities detected, the incidences of structural autosomal and sex chromosomal anomalies were statistically significantly higher in ICSI children. Some problems that may occur with sex chromosome abnormalities include an increased risk of miscarriage of affected embryos, heart problems for affected infants, specific behavior or learning difficulties for affected children and adults, and

Table 6 Literature reports of chromosomal anomalies in ICSI conceptions Study (Reference #)

Number of ICSI babies

Abnormal karyotypes (%)

Aboulghar et al, 2001 [144] La Sala et al, 2000 [170] Bonduelle et al, 1999 [130] Loft et al, 1999 [132] Van Opstal et al, 1997 [145]

430 289 1082 206 71

3.5% 2.4% 2.6% 3.4% 12.8%

Abbreviation: ICSI, intracytoplasmic sperm injection.

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infertility as an affected adult. The higher prevalence of chromosomal anomalies in ICSI children seems not to be correlated with maternal age or consanguinity [144] but is more likely caused by either sperm-derived (paternal) factors [145] or by the ICSI procedure itself [127]. There is recent interest in the role of maternal chromosomal aberrations as a contributing cause, because 5.5% to 9.8% of infertile women harbor karyotype abnormalities on pre-ICSI screening (N ¼ 1611 women tested) [146,147]. Does intracytoplasmic sperm injection affect childhood development? The issue of whether ICSI children have a higher likelihood of developmental delay is far more controversial than the chromosomal findings. One study suggests that 17% of ICSI children (n ¼ 89) experience developmental delay at 1 year of age compared with natural (n ¼ 80) and IVF (n ¼ 84) conceptions [148]. This difference was noted in mental development but not in the psychomotor index of the Bayley Scales of Infant Development. Weaknesses of the study are that the ICSI offspring did not undergo karyotype analysis and that paternal educational levels were lowest in the ICSI cohort. These findings are disputed by the results from two other centers that report no difference in mental development at 1 to 2 years of age in a total of 324 children conceived with ICSI when compared with those conceived naturally [149,150]. The controversy emphasizes the necessity of ongoing follow up of ICSI children to determine the true risk-benefit profile of this technology. Current technology for genetic screening Genetic testing is currently performed with both cytogenetic and molecular genetic technologies. Karyotyping Karyotyping, or high-resolution banding cytogenetics, is a well-established technique of characterizing chromosomal number and structure. Individual cells are arrested in metaphase and stained to reveal typical banding patterns. The chromosomal stains from a single cell are photographed; images are captured; and, with the aid of computer analysis, the chromosomes are arranged in pairs and carefully studied to deduce abnormalities of chromosome number or structure. This procedure is most commonly used on

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peripheral blood cells for the evaluation of male infertility. Polymerase chain reaction assay Polymerase chain reaction (PCR) assay has been revolutionary in allowing the direct investigation of DNA for genetic defects. It is unlike older techniques for DNA analysis, such as Southern blotting, for which thousands of cells were required to obtain sufficient material (5 lg DNA) for evaluation. With PCR, single-cell analysis (5 pg DNA) is now possible because of the ability to amplify individual sequences. Currently, this technique is so powerful that even single copies of genes within a single cell can be amplified a billionfold very quickly. Indeed, it has become the technique of choice for detection or diagnosis of single-gene defects. Because it is based on the exponential duplication of certain DNA sequences, proper selection of primers and controls is crucial for meaningful results. Variations of PCR assay include nested PCR, in which only a small, selected portion of the original PCR product is amplified on second and subsequent rounds, and multiplex PCR, in which several different probes are used to identify several deletions at several different loci simultaneously. Multiplex PCR is commonly used for the diagnosis of Y chromosome microdeletions in male infertility [57]. The addition of fluorescent dyes to primers (fluorescent PCR) increases the reliability of signal detection over radioisotope-gel methods, and can improve technique sensitivity by 1000-fold. In quantitative PCR, the amount of PCR product is measured and compared with wild type or controls, a technique that can allow the calculation of the relative number of molecules present. Finally, in primer extension preamplification (PEP), the entire genome is amplified with PCR to create a more abundant amount of initial template (50–100 genomic copies instead of one) on which to base a detailed subsequent study of specific genes. Fluorescence in situ hybridization In FISH, enzymatic or fluorescent-labeled DNA probes are hybridized to DNA within chromosomes. As with karyotyping, individual cells on a slide are arrested in metaphase and then subjected to DNA denaturation. DNA probes are then hybridized in situ to denatured cellular DNA within metaphase chromosomes. This technique can be used for single-gene identification and even for chromosome painting, in which

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multiple probes are assessed in multiple genomic locations. Fluorescence in situ hybridization is commonly used to detect chromosomal rearrangements such as SRY translocations. Comparative genomic hybridization As an extension of FISH, comparative genomic hybridization (CGH) involves the cohybridization of total genomic DNA from cells of interest (test DNA) labeled with one color and control cell DNA labeled in a different color to denatured metaphase chromosomes within a normal cell. The relative binding of the test and control probes for any DNA sequence in the normal cell will determine the fluorescent color. For example, if a certain sequence is duplicated in the green test DNA relative to red control DNA, relatively more green-labeled DNA will bind to the normal cell on analysis. This technique may become important for the detection of chromosomal imbalances within single cells, for example in the setting of preimplantation genetic diagnosis. DNA sequencing With the recent development of automated DNA sequencers, complete DNA base-pair analysis can now be performed expediently by either chemical or enzymatic methods. This powerful technique is valuable for finding single base-pair mutations in DNA samples containing a mixture of both normal and mutated molecules. In addition, it is a technique that can identify complete and partial gene mutations that are much larger than single point mutations. Future concepts and technology for genetic screening The study of male genetic infertility is still in its infancy. To date, only a few genes have been linked to this condition, although several hundred to a thousand are probably involved. In addition, paradigms of genetic infertility are likely to undergo considerable change with time. Newer paradigms are outlined here, because they may form the basis for investigations of male genetic infertility in the future. The autosomes and Infertility Current clinical testing in genetic infertility has concentrated on examination of the Y chromosome for genes involved with spermatogenic fail-

ure. Abundant evidence, however, suggests that the autosomes are as important as the sex chromosomes in male genetic infertility. Indeed, karyotype-detected autosomal abnormalities are more common than sex chromosomal anomalies in men with oligospermia, occurring in 1.5% of patients [50]. Whether the infertility in these cases is caused by the interruption of critical genes at translocation breakpoints or is a simple result of the inability of chromosomes to pair correctly during meiosis remains to be determined. In addition, most syndromes and conditions outlined in this article (see Tables 2, 4, and 5) have been mapped to autosomal loci. Even stronger evidence for the role of autosomal genes in male infertility now stems from the discovery of specific genes associated with infertility in autosomal locations. These include a version of the DAZ gene, the DAZ-like (DAZL) gene on chromosome 3p, and a human homologue of the fly sterility gene BOULE, designated hBOULE or BOL, located on chromosome 2q [151,152]. Thus, as the study of genetic infertility progresses, the role of autosomal mutations in this condition will increase in significance. As more autosomal genes are discovered, refinements in genomic mutation screening technologies will be needed to allow mutation screening of multiple genes in multiple locations. A powerful new technique that may find application to this problem is the DNA microarray, a technique that allows simultaneous examination of the activity of thousands of genes in parallel [153]. Created with the help of sophisticated automated instrumentation used in computer chip technology, DNA microarrays are simply PCR-blot techniques applied on a massive scale. Gene sequences of interest are attached to glass slides at high density (thousands of sequences/cm). This array is then incubated with patient samples labeled with a color probe. If the patient’s particular gene sequence is normal, then binding to the identical gene sequence on the array occurs and is detected by a color change (Fig. 6). If the patient’s gene has a mismatched sequence, indicating a mutation, hybridization efficiency is reduced or absent for that gene sequence. Subtle changes in hybridization efficiency can be detected with powerful software packages currently available for use with arrays (see www.microarrays.org) [173]. This incredibly powerful technology can be tailored to the individual biologic system of interest and used to perform simultaneous genomic screening for thousands of mutations or to examine patterns of gene expression (using RNA instead of DNA).

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line mutation patterns improves. Indeed, FISH with chromosomal painting and PCR technologies have been applied to single-sperm genetic analysis in efforts to characterize germ line mosaicism more accurately [154,156,161]. Future technology will need to continue to address the genetic mutations within testis tissue or mature spermatozoa, to better to inform couples of genetic risk to offspring before embarking on expensive assisted reproductive technologies. Genetic pathways and infertility

Fig. 6. An example of cDNA microarray interpretation. Patient gene sequence is tagged with a label of one color and hybridized to the array that is spotted with the normal DNA sequence and labeled in a different color. If hybridization occurs, the array spot (A) will be a mixture of the two colors. If there is no binding, for example because of a mutation, the spot color (B) will be the color of the spotted DNA. Within the lower portion of the figure is part of an array showing several hundred ‘‘spots,’’ each representing an individual gene sequence.

Somatic germ line mosaicism The finding of Y chromosome deletions in the sons of infertile men in the absence of demonstrated Y mutations in their fathers suggests that genetic mosaicism exists within somatic cells and possibly within germ line cells in genetic infertility [59]. Similarly, significant degrees of sperm aneuploidy can exist in otherwise karyotypically normal men, both infertile and fertile [154–156], with rates among infertile men double those observed in fertile controls (5.5% versus 2.6%, respectively) [157]. Complicating this picture of mosaicism is the suggestion that the prevalence of sperm chromosomal anomalies may actually increase with paternal age [158,159]. In conditions other than infertility, in fact, it has been suggested that germ line mutation patterns are quite different from somatic patterns and, in addition, are highly variable [160]. Such findings certainly add complexity to the idea of genetic testing, because the true tissue of relevance in infertility is germ line, not somatic (currently blood leukocytes). The prediction of intergenerational behavior of genes is not possible without a complete understanding of the variability within the germ line (and not somatic) mutational pathways. For example, infertility mutations that are currently considered de novo may be demonstrated to be inherited in the future as the knowledge of germ

Male infertility may not only be related to simple deletions in genes important for spermatogenesis but in fact may be caused by defects in entire genetic pathways involved in cell health. On the basis of observation of infertility caused by maturation arrest among three of eight brothers in a single generation, Chaganti and German [162] reported in the 1970s that infertility might be caused by mutations in genes that regulate meiotic progression. Another report observed a reduced ability to repair induced DNA damage in lymphocytes of an azoospermic man with maturation or meiotic arrest on testis biopsy [163]. These observations suggest the possibility that human meiotic arrest may be linked to mutations in genes required for the repair of DNA. Recent work at the University of California San Francisco has shown that, in fact, azoospermic, infertile men with a particular form of maturation arrest pattern shown on histologic evaluation can demonstrate evidence of faulty DNA repair [164]. Using microsatellite DNA analysis, the authors examined the integrity of the DNA repair process in testis tissue and found significantly higher rates of variability in dinucleotide (‘‘microsatellite CA’’) repeat length in infertile testis tissue than in fertile tissue and also detected base substitutions within microsatellites in infertile testis tissue. These defects were found only in infertile men and only in germ line DNA (not in somatic cells). This finding is similar to observations in other organisms, in which mutations in the genes required for DNA repair have been shown to cause infertility characterized by arrest at critical meiotic checkpoints. Genes involved at these checkpoints include those responsible for the tight regulation of genetic recombination and for governing the repair of DNA breaks essential for chromosomal exchange and crossover during normal meiosis. The consequences of defective DNA repair in germ cells to the health of children conceived by

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ICSI are unknown. The answer is complex because DNA repair defects may be mosaic within an individual, existing solely in the germ line and not in somatic cells. It may be that potential DNA repair defects will not be translated into the birth of viable offspring. Alternatively, if children are conceived with ICSI with sperm carrying potential DNA defects and survive to term, they may not only be at risk for infertility but also may be at increased risk of tumor formation later in life. Certainly, such research suggests the need for sperm-based assays in the future that can examine the integrity of recombination and DNA mismatch repair. In addition, assays to check the integrity of other biologic pathways that may be involved in male infertility, including apoptosis and cell cycle regulation, may also prove valuable to the field. Sperm DNA integrity In addition to defects in specific genes or gene pathways, male infertility may also be caused by a more nonspecific phenomenon: breakage or damage to the DNA ladder structure within sperm. Two prime sources of such DNA damage are smoking and environmental toxins, both of which act to degrade or separate the doublestranded structure of DNA through reactive oxygen species and free radical generation [165]. Reduced DNA integrity, in turn, may be associated with poor fertilization outcome [166]. Several assay systems have been applied to assess DNA integrity and damage that may result from such exposures. The comet assay involves single-cell electrophoresis and can resolve DNA break frequencies up to several thousand per cell. Much as in conventional electrophoresis, alkalineunwound DNA fragments are distributed over the gel according to size. Typically, in sperm with DNA damage, there is a ‘‘comet body’’ on the gel that reflects intact DNA, and a length of ‘‘comet tail’’ trailing from body on the gel, representing the smaller fragments of DNA [167]. This assay demonstrates only endpoint damage and does not indicate cause. Another assay that seeks to assess DNA damage, the sperm chromatin structure assay (SCSA), involves the exposure of sperm to acid followed by acridine-orange staining to detect single-stranded or denatured DNA [168]. After staining, a shift in fluorescence from green (native DNA) to red (denatured DNA) on flow cytometric assessment indicates DNA damage and may in fact predict

subfertility despite its lack of correlation with routine semen parameters [168]. An assay of DNA damage induced by apoptosis has been developed for the examination of sperm and is termed the TUNEL (terminal deoxynucleotidyltransferase deoxyuridine triphosphate [dUTP]-biotin nick end-labeling) assay [169]. It can be used to study apoptosis-induced DNA damage in sperm and may in fact correlate inversely with sperm motility and morphology and in vitro fertilization rates [166]. Further refinements in such assay systems will aid the understanding of DNA damage and human male infertility in the future. Summary Although much of male infertility is currently unexplained, it is likely that underlying defects in critical genes or entire gene pathways are responsible. Because powerful technologies exist to bypass severe male-factor infertility, improving the diagnosis of genetic infertility is important for the infertile couple, not only to explain the problem but also to inform them of conditions potentially transmissible to offspring. Acknowledgments The authors thank Lauri Black, MS, CGC, for her valuable contributions to this work. References [1] Sigman M, Lipshultz LI, Howards SS. Evaluation of the subfertile male. In: Lipshultz LI, Howards SS, editors. Infertility in the male. 3rd edition. New York: Mosby; 1997. p. 173–93. [2] Correns C. Untersuchungen uber die xenien bei Zeamays. Benichte der Deutche Botanische Gesellschatt 1899;17:410–8. [3] Landers ES, Linton LM, Birren B, et al. Initial sequencing and analysis of the human genome. Nature 2001;409:860–921. [4] Venter JC, Adams MD, Myers EW, et al. The sequence of the human genome. Science 2001;29: 1304–51. [5] Mak V, Jarvi KA. The genetics of male infertility. J Urol 1996;156:1245–56. [6] Weksler BB, Moore A. Anemia. In: Andreoli TE, Carpenter CCJ, Plum F, et al, editors. Cecil’s essentials of medicine. 2nd edition. Philadelphia: WB Saunders; 1990. p. 343–62. [7] Morton CC, Kirsch IR, Taub R, et al. Localization of the beta-globin gene by chromosomal in situ hybridization. Am J Hum Genet 1984;36: 576–85.

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