Infertility Role of Genetics in Joo Yeon Lee, Rima Dada, Edmund Sabanegh, Angelo Carpi, and Ashok Agarwal OBJECTIVE To review established genetic causes of azoospermia, the most severe form of male , and help clinicians, scientists, and infertile couples considering assisted reproductive technologies (ART) to understand the complexity of the disorder and to maximize the chances of having a healthy infant through proper counseling and treatment. METHOD An initial literature search was performed on PubMed using the key words “azoospermia” “oligospermia,” and “genetics.” The results were limited to the studies on humans and written in English, which were written within last 10 years. Although preliminary query results showed more than 900 articles, further queries using key words, such as “,” “monogenics,” “aneuploidy,” “mitochondrial DNA,” and “epigenetics,” along with “azoospermia,” narrowed the results to 30 papers, which were included in the present study. RESULTS Genetic defects causing azoospermia were categorized into two large categories: chromosomal and nonchromosomal. Chromosomal defects were further categorized into (1) structural abnor- malities, such as Y chromosome micro/macrodeletions, chromosomal inversions, and transloca- tions; and (2) numerical abnormalities, also known as aneuploidy. Nonchromosomal defects included sperm mitochondrial genome defects and epigenetic alterations of genome. CONCLUSIONS As a result of advancements in ART, understanding the potential implications of genetic disorders for infertile couples is critical. Analysis of a potential genetic role in azoospermia holds promise to expand our knowledge to evaluate and to guide treatments. UROLOGY 77: 598–601, 2011. © 2011 Elsevier Inc.

nfertility is defined as “a failure to conceive after 12 lia are the key components of evaluation.2 In 70% of months of unprotected sexual intercourse.”1 Male examined men, this preliminary evaluation will identify Ifactor is responsible for 30% to 50% of cases of the cause.2 The remaining 30% of men require additional infertility, and as many as 20% of infertile men are evaluation, such as genetic testing to elucidate the un- diagnosed as azoospermic.2 Azoospermia affects 1% of the derlying cause. male population. Some conditions directly cause azoosper- mia, whereas others are the result of complex gene environ- Classification of Azoospermia 3 ment interactions. Azoospermia is classified as (1) obstructive azoospermia Although advancements in reproductive medicine, (OA) caused by obstruction of the ejaculatory pathway such as intracytoplasmic sperm injection (ICSI) and mi- and (2) nonobstructive azoospermia (NOA) caused by crosurgical testicular sperm extraction (micro-TESE) al- failure of . Depending on the etiology, it low men with minimal spermatogenesis to successfully may also be pretesticular, testicular, or post-testicular. reproduce, genetic defects may be passed down from Various checkpoints in spermatogenesis are controlled 3 sperm. The present study summarizes contemporary by a multitude of genes and signaling pathways that knowledge of the role of genetic disorders in azoospermic regulate meiosis, mitosis, and sperm transport. OA is males. associated with a congenital bilateral absence of the vas deferens (CBAVD), inflammation, and obstruction, EVALUATION OF AZOOSPERMIA which cause physical barriers for normal spermatozoal transit.4 NOA may be associated with either intrinsic Diagnosis of Azoospermia testicular defects (also known as primary testicular failure) A thorough sexual and medical history, hormonal mea- or hypothalamic-pituitary-adrenal axis abnormalities surements, and physical examination of external genita- (also known as secondary testicular failure). The two dif- ferent etiologies are easily distinguished based on hor-

Center for Reproductive Medicine, Cleveland Clinic, Cleveland, OH; All India Insti- mone levels. tute of Medical Sciences, New Delhi, India; and Department of Reproduction and Aging, University of Pisa, Pisa, Italy Reprint requests: Ashok Agarwal, Ph.D., Professor and Director, Center for Repro- GENETIC CAUSES OF AZOOSPERMIA ductive Medicine, Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail: [email protected] Approximately 29% of azoospermic men have underlying 5 Submitted: July 26, 2010, accepted (with revisions): October 3, 2010 genetic abnormalities. Common genetic abnormalities 598 © 2011 Elsevier Inc. 0090-4295/11/$36.00 All Rights Reserved doi:10.1016/j.urology.2010.10.001 include chromosomal or gene defects (nuclear or nal and external male genitalia they lack the AZF region mitochondrial), and epigenetic alterations. and are azoospermic.13 The mitotic loss of 1 Y chromosome during early embryonic development produces 45,X/46,XY mosaic Chromosomal Disorders karyotype.3 Although 80% of 45,X/46,XY individuals Approximately 4% of males undergoing ICSI have chro- have a normal SRY gene, approximately 60% of these mosomal abnormalities—80% involve sex chromo- individuals present with mixed with a somes.6 Robertsonian translocations and Klinefelter syn- streak gonad and a testis, a uterus, and a vagina.9 This drome (KFS) are the most prevalent chromosomal karyotype is usually seen in cases harboring AZF dele- disorders affecting 10% to 20% of azoospermic men,6 tions, where, because of chromosome instability, the Y which will be discussed later in this manuscript. chromosome is lost, resulting in mosaicism. Researchers Chromosomal disorders may involve sex chromosomes have hypothesized that cytogenetic mosaicism or muta- or autosomes. Numerical sex chromosomal aneuploidies tions in SRY downstream testis–determining genes, are more commonly observed in azoospermic men. such as Wilms’ tumor suppressor, DAX1, and testatin Y Chromosome Microdeletion. Y chromosome microde- (20p11.2) may play a role in the gonadal failure in these letions are found in 5% to 15% of infertile men with men.15 azoospermia and are the most common cause of azoospermia.6,7 X Chromosome–Linked Abnormalities. The role of The region critical for germ cell development and genes on the X chromosome in male infertility remains differentiation on Y chromosome long arm (Yq) is the mostly unknown, partly because researchers have relied 7 on rodent studies, which may not accurately reflect hu- azoospermic factor region (AZF). The AZF region en- 16 compasses multiple gene families, AZFa, AZFb, and man expression patterns. Thus, the clinical relevance is AZFc.8 AZFc is the most commonly found (60%) dele- limited to less than 10 genes, such as the androgen receptor gene (AR), which is present in both human and tion compared with AZFa (5%), AZFb (16%), or com- 16 bined (14%).9 This is in part because the length of AZFc mice. Although studies on these genes remain incon- is four times longer than AZFa.9 clusive, X chromosome’s hemizygotic nature in males Men with deletions spanning more than one AZF loci are suggests that de novo X chromosome mutations have a 16 usually azoospermic, as are men who harbor an AZFa and definite impact. AZFb deletion.10 AZFc deletions are usually associated with TEX11 (Xq13.1) and TAF7L (Xq22.1) play critical a variable phenotype caused by the presence of autosomal roles in the promotion of synapsis and regulation of homologue and multiple copies. Luddi et al. (2009) reported crossover. The disruption of these genes causes segrega- that a deletion of the USP9Y gene on AZFc, caused both tion defects and azoospermia. azoospermia and severe oligospermia.11 The most widely studied X chromosome–linked gene is 16 Studies have shown that men with an AZFc deletion AR Xq11.2-12. Because the AR mediates the actions of experience a steady decline in sperm count and, over a androgens, mutation of the AR gene manifests various period, develop azoospermia.12 Men with an AZF dele- androgen insensitivity phenotypes ranging from 46,XY 15 tion are at an increased risk of losing the Y chromosome infertile female to 46, XY azoospermic males. In the because of Y chromosome instability.12 transactivation domain (exon 1) of the AR gene, 2 polymorphic triplet repeats—(CAG)n and (GGC)n,— Aneuploidy. KFS is the most frequent cause of sex are present.16 Although some studies have found an chromosome aneuploidy and leads to NOA. Approxi- inverse correlation between CAG repeat length and male 13 mately 14% of azoospermic males have KFS. Approxi- infertility,17 others have not,18 suggesting that further mately 95% of men with KFS have 47,XXY chromosomal investigations should examine both significantly longer 9 complement. KFS men have reduced testis size, elevated and/or shorter CAG repeats and their functionality. follicle-stimulating hormone (FSH) and luteinizing hor- Kallmann syndrome (KS) is a common infertility dis- mone (LH) levels, as well as low testosterone (T) levels. order that occurs in approximately 1 in 30,000 live births. As much as 69% of men with KFS can have successful About 11% of KS men have X chromosome–linked gene 7 surgical sperm retrieval via micro-Tese. Although re- mutations, whereas others show autosomal inheritance trieved spermatozoa are usually haploid, there have been pattern.9 KS is characterized by decreased secretion of reports of KFS in offspring, a risk that can be greatly gonadotrophin-releasing hormone during embryonic de- minimized with embryo screening using preimplantation velopment caused by a deletion in the KAL-1 gene on X genetic diagnosis (PGD) and fluorescence in situ hybrid- chromosome (Xp22.3). Low levels of sex steroid hor- 14 ization (FISH). mones, especially androgen, hinder normal spermatogen- The 46,XX male syndrome is a rare aneuploidy that esis, leading to azoospermia and underdeveloped male 3 occurs in 1 in 20,000 live births. Approximately 90% of genitalia in KS.19 these males have sex determining region on the Y chro- mosome (SRY) translocated to the X chromosome or an Autosomal Abnormality. Robertsonian translocations autosome.3 Although 46,XX males exhibit normal inter- (RTs) are the most frequent structural chromosomal ab-

UROLOGY 77 (3), 2011 599 normality. They occur 1 in 1000 live births.19 Reciprocal sperm contains mtDNA deletions, and many of them and RTs are nine times more common in infertile men have two to seven deletions, which increase with age and than in healthy males.20 Recent studies have found hot oxidative stress. Although multiple mtDNA deletions in spots for translocation in 265 infertile men at loci 1p31-33, azoospermic males have been detected, recent studies 3p21.1-9,7q31, Xq28, and 6p21,6p22.1 Some of these are have found that the incidence of mtDNA deletions are testis-specific genes and others may represent novel loci, not significant enough to be differentiated, but that the which may play a role in the regulation of testicular size of the deletions were larger in azoospermic males function. than in fertile males.29 Chromosomal inversions in autosomes 1, 3, 4, 6, 9, 10, and 21 are more common in infertile men.21 Inversions on chromosome 9 associated with azoospermia are eight MANAGEMENT AND PROGNOSIS times more common in infertile men.22 OA Cystic fibrosis (CF) is an autosomal-recessive disorder caused by mutations in the cystic fibrosis transmembrane Men with CBAVD are successfully treated with TESE. regulator (CFTR) gene (7q31.20). About 90% of men For CBAVD men, it is assumed they harbor a CFTR with CF have OA caused by CBAVD.9 The CFTR gene mutation. The partner should also be screened for 51 encodes for a CFTR protein that plays an important role common CFTR mutations if mutations are found; the in sodium/chloride balance in epithelial secretion regu- siblings of the CFTR male have a 50% chance of being a lated by cAMP.22 CF manifests with varying phenotype carrier. from vasal agenesis to full-spectrum CF symptoms.23 The delta F508 mutation is the most common mutation (50– 80%) on the CFTR gene.24 NOA Men with KS have a complete lack of testosterone and do not undergo puberty. Treatment in such men requires Nonchromosomal Disorders: Epigenetic Alterations replacement therapy with LH analogues until serum T Abnormal DNA methylation and histone modification levels are normal. FSH analogue is then started. After 12 affect normal embryogenesis via transcriptional control to 24 months of treatment, these men go through puberty and can ultimately cause azoospermia. Aberrant histone and have sperm in their ejaculate. modifications in male infertility has not been thoroughly In men with KFS, T treatment helps to develop sec- explored; the involvement of DNA methyltrasferases and ondary sexual characteristics but does not initiate sper- methyl-binding domain proteins play critical roles in the matogenesis. In mosaic KFS, sperm may be retrieved with 25 methylation of germ cells. Moreover, because of its micro-Tese but FISH should be considered to prevent high activity in the adult testis, the methylation status of aneuploidy. the promoter region of the methyltetrahydrofolate reduc- Men with large AZF deletions and deletions involving tase (MTHFR) gene has been investigated and abnormal AZFa and AZFb loci present with azoospermia and have hypermethylation in the CpG island within the promoter poor prognosis on ART. However, men harboring an region was detected in males with NOA, whereas the AZFc deletion may have adequate sperm found by TESE 26 same region was unmethylated in the controls. Thus, and can undergo ART after comprehensive counseling the hypermethylated promoter region may down-regulate about their risk of similar or even larger deletions in their MTHFR enzymatic activities, resulting in azoospermia. It male offspring. has also been reported that retrieval of germ cells from Men harboring an AZFc microdeletion should undergo testes may result in the collection of epigenetically im- 12 25 sperm cryopreservation. Men with large AZF deletions mature germ cells. are also at risk of losing the Y chromosome during fertil- The expression profiles of micro-RNAs, noncoding ization. In such cases, offspring will exhibit a 45,XO RNAs have been studied to investigate the correlation karyotype or will be mosaic and may have sexual ambi- between miRNA aberrant expression and their involve- guity. ment in spermatogenesis. Because 5= and 3= untranslated Men harboring mtDNA nucleotide variations usually regions of mRNA are regulated by miRNAs, they may have high levels of reactive oxygen species (ROS), which play an important role in post-transcriptional control of 27 leads to mt dysfunction. High ROS levels damage sperm gene expression. The role sperm miRNAs play in the nuclear and mtDNA.30 Thus, early diagnosis and prompt control of gene expression is very probable, given their antioxidant treatment in such men may prevent irrevers- presence in the early embryonic stages. ible DNA damage and help maintain adenosine tri phos- phate levels to sustain spermatogenesis.30 Nonchromosomal Disorders: mt Genome Genetics is one of the most important yet under- Sperm mtDNA provides energy for spermatogenesis and emphasized cause of azoospermia. Improved understand- sperm motility. mtDNA is prone to mutations because of ing of the genetics of infertility holds promise to define its close proximity to the electron transport chain and its the etiology of azoospermia and counsel cases that were lack of histones and introns.28 Approximately 85% of previously diagnosed with idiopathic azoospermia.

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