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Spermatogenesis is an ongoing developmental process in adult testes that requires the coordinated expression of many genes. The genetic causes of spermatogenic failure in men remain largely unknown, though abnormalities in the sex chromosomes constitute a significant portion of them. In this review, we focus on 3 disorders that involve the sex chromosomes and are often screened in infertility clinics. These are Klinefelter syndrome, Y chromosome microdeletion, and XX male syndrome. We describe their prevalence, the associated phenotypes, and the molecular mechanisms underlying the disorders and discuss the difficulties in identifying the causal genes contributing to the spermatogenic defects. Currently, there are no effective therapies for the spermatogenic failure in the patients, and conception through assisted reproductive technology bears the risk of passing genetic abnormalities to the next generation.

A significant proportion of male infertility is accompanied by an abnormal semen analysis, azoospermia or severe oligozoospermia, which is generally assumed to be the result of spermatogenic failure. The genetic contribution in the process of spermatogenesis, particularly the role of the Y chromosome in determination of semen quality, is still obscure. In order to explore the relationship between Y chromosome haplogroup and spermatogenic failure, we collected 285 idiopathic infertile males with azoo-/oligozoospermia and 515 fertile men, adopted 12 binary markers and recruited the subjects (cases and controls) in the same region to test whether there is a possible susceptibility of certain Y haplogroups to spermatogenic failure in the Han Chinese population. The results indicated that the prevalences of hg K in the control and the case population were 0.78% (4/515) and 2.80% (8/285), respectively. The difference between the frequencies of the hg K in the infertile males and the normal control population was significant [odds ratio (OR) = 3.69; 95% confidence interval (CI) = 1.10-12.36] (P = 0.028). However, in the other haplogroups no significant differences were found. In conclusion, Y haplogroup-K might bear a risk factor of male infertility, and the individuals in the haplogroup need to be further examined.

The vast majority of cases of spermatogenic failure, defined as no sperm in semen or low semen quality, are idiopathic, although several genetic factors have been identified. In this thesis, we focused on the effect of Y-chromosome variation including TSPY copy number and AZFc deletions on semen quality and studied the effectiveness and safety of in vitro spermatogonial stem cell propagation for (SSC) transplantation as a potential treatment for spermatogenic failure by increasing the number of SSCs. We found that the variation in TSPY copy number is not associated with total motile sperm count and therefore has no functional consequences for semen quality. AZFc deletions are known to be common genetic causes of spermatogenic failure. We showed that in our previously established culture system, the AZFc-deleted spermatogonia behave similar as spermatogonia from non-deleted men. To further characterize the human SSCs in our culture, we investigated which marker is efficient in enriching for SSCs. We found that ITGA6 (using magnetic assisted cell sorting or MACS) is an efficient method for enrichment of SSCs. We then investigated the genetic and epigenetic stability of human SSCs after long-term culture and showed that genetic stability of SSCs is preserved during culture. However, the methylation status of some of the imprinted genes is changed, which requires further investigation. We concluded that in vitro propagation of human SSCs followed by transplantation could be a good potential treatment option in patients with a low number of functional SSCs due to genetically caused spermatogenic failure.

BACKGROUND: DAZ gene family is crucial for human spermatogenesis that requires the precise co-ordination of cell cycle events. CDC25A is recognized as the downstream substrate of DAZ gene family and is thought to function on the M-phase regulation of cell cycles. We investigated the expression profiles of CDC25A in the testes of infertile men and evaluated the relationship between CDC25A levels and testicular phenotype, clinical hormonal parameters and sperm retrieval results. METHODS: The protein and mRNA transcript levels of CDC25A in the testes of 40 azoospermic men were determined by immunohistochemistry and quantitative real-time-PCR. CDC25A in human spermatozoa was investigated by western blotting and immunofluorescence staining. RESULTS: The CDC25A protein was expressed mainly in spermatocyte, spermatid and spermatozoa. CDC25A transcript levels were significantly decreased (P = 0.0009) in patients with spermatogenic failure, especially in men with meiotic arrest and Sertoli cell-only syndrome. Significantly higher CDC25A transcript levels were detected in patients with successful sperm retrieval than in patients with failed sperm retrieval (P = 0.005). CONCLUSIONS: Decreased CDC25A is associated with spermatogenic failure and failed sperm retrieval in infertile men. Further studies are necessary to explore the functional roles of CDC25A in human spermatozoa.

BACKGROUND: Because of the common use of ICSI and the potential genetic aetiology of spermatogenic failure, concern has been raised about transmitting genetic disorders to ICSI offspring. However, to date, in only approximately 15% of all cases of spermatogenic failure, an underlying genetic cause can be identified. We have previously established an association between spermatogenic failure and chromosomal region 11p15. In this study, we set out to explore whether NALP14, a gene recently mapped to 11p15, has a function in spermatogenesis and whether mutations in NALP14 can cause spermatogenic failure. METHODS: We applied two different multiple tissue northern (MTN) blots to determine tissue specificity of NALP14 and performed immunohistochemistry on human testis with anti-NALP14 antiserum. To determine imprinting status of NALP14, we tested the expression pattern of two single-nucleotide polymorphisms (SNPs) in human testis. Finally, we performed a mutation screen of the NALP14 gene in 157 men with azoospermia or severe oligozoospermia by direct sequencing; 158 normospermic men served as controls. RESULTS: NALP14 was, as are the three other genes in 11p15, exclusively expressed in testis. Within the testis, the NALP14 protein was mainly expressed in A dark spermatogonia, mid and late spermatocytes and spermatids. The mutation screen revealed five mutations that occurred only in the patient group. One of these unique mutations introduced an early stop codon in the NALP14 sequence, predicted to result in a severely truncated protein. CONCLUSION: Our data suggest that NALP14 has a function in spermatogenesis and that mutations in this gene might cause spermatogenic failure

Piwi-interacting RNAs (piRNAs) are a broad group of noncoding small RNAs that have important biological functions in germline cells and can maintain genome integrity via silencing of retrotransposons. In this study, we aimed to explore the associations between genetic variants of important genes involved in piRNA biogenesis and male infertility with spermatogenic impairment. To this end, five single-nucleotide polymorphisms (SNPs) in the ASZ1, PIWIL1, TDRD1, and TDRD9 genes were genotyped by TaqMan allelic discrimination assays in 342 cases of nonobstructive azoospermia (NOA) and 493 controls. The SNP rs77559927 in TDRD1 was associated with a reduced risk of spermatogenic impairment. The genotypes TC and TC + CC showed odds ratios and 95 % confidence intervals of 0.73 (0.55–0.98, P = 0.034) and 0.73 (0.56–0.97, P = 0.030), respectively, in patients with NOA compared with those in the controls. Thus, our results provided the first epidemiological evidence supporting the involvement of TDRD1 genetic polymorphisms in piRNA processing genes in determining the risk of spermatogenic impairment in a Han Chinese population.

FONDECYT FONDECYT

Objective To determine the expression of the inhibitor of apoptosis survivin in men with and without spermatogenic failure. Design Prospective case study. Setting Two university-based infertility clinics. Patient(s) Forty-nine infertile men presenting with azoospermia. Intervention(s) Testicular biopsies for histopathological assessment and analyses of survivin expression levels by real-time reverse transcription–polymerase chain reaction. Survivin levels were normalized to expression of the housekeeping porphobilinogen deaminase gene. Main Outcome Measure(s) Correlation of the histological findings with normalized survivin expression levels. Results(s) Testicular survivin mRNA expression was highest in normal spermatogenesis (n = 14). Decreased expression was observed in patients with spermatogenesis disorders. The expression level correlated with the degree of spermatogenic failure. While it was reduced in postmeiotic maturation arrest (n = 11), a lack of expression was seen in most specimens (10 of 12) with premeiotic maturation arrest and in all of those with Sertoli cell–only syndrome (n = 12). Conclusion(s) These data indicate that survivin is expressed in human germ cells and might be involved in apoptosis control during spermatogenesis. Decreased survivin expression in spermatogenic disorders may contribute to the accelerated germ cell apoptosis observed in male idiopathic infertility.

Male infertility is most commonly caused by spermatogenic defects or insufficiencies, the majority of which are as yet cureless. Recently, we succeeded in cultivating mouse testicular tissues for producing fertile sperm from spermatogonial stem cells. Here, we show that one of the most severe types of spermatogenic defect mutant can be treated by the culture method without any genetic manipulations. The Sl/Sl d mouse is used as a model of such male infertility. The testis of the Sl/Sl d mouse has only primitive spermatogonia as germ cells, lacking any sign of spermatogenesis owing to mutations of the c-kit ligand (KITL) gene that cause the loss of membrane-bound-type KITL from the surface of Sertoli cells. To compensate for the deficit, we cultured testis tissues of Sl/Sl d mice with a medium containing recombinant KITL and found that it induced the differentiation of spermatogonia up to the end of meiosis. We further discovered that colony stimulating factor-1 (CSF-1) enhances the effect of KITL and promotes spermatogenesis up to the production of sperm. Microinsemination of haploid cells resulted in delivery of healthy offspring. This study demonstrated that spermatogenic impairments can be treated in vitro with the supplementation of certain factors or substances that are insufficient in the original testes.

Men with spermatogenic failure so profound that they are considered as having nonobstructive azoospermia occasionally have spermatozoa in the ejaculate. We compared intracytoplasmic sperm injection outcomes following the injection of ejaculated or surgically retrieved spermatozoa from these men.A study was performed of intracytoplasmic sperm injection cycles with no spermatozoa on initial semen analysis and 100 or fewer following centrifugation (cryptozoospermia). Only 16 couples that underwent intracytoplasmic sperm injection cycles with ejaculated spermatozoa and cycles with testicular spermatozoa were included.Initial analysis was done to compare outcomes between the 2 semen origins. There was no difference in the rate of normal or abnormal fertilization between the 2 groups. The rate of clinical pregnancies seemed to favor testicular spermatozoa (47.4% vs 20.8%), although results were not significant. When a comparison was performed between the first testicular cycle and the ejaculated cycle closest in time to the cycle with testicular spermatozoa, a higher rate of normal fertilization with testicular spermatozoa was observed (60.9% vs 48.5%, p0.05). Also, in this comparison a clear trend toward a higher percent of clinical pregnancies and deliveries in the testicular group was observed (50.0% vs 14.3%).Transit through the male genital tract did not enhance the ability of ejaculated spermatozoa to achieve fertilization with intracytoplasmic sperm injection compared to that of testicular spermatozoa in men with severely impaired production. In ejaculated samples a lower number of spermatozoa available resulted in an impaired chance of achieving pregnancy. Using testicular spermatozoa may be a reasonable alternative for couples in whom multiple attempts at intracytoplasmic sperm injection have failed using ejaculated sperm from men with cryptozoospermia.

Objective To determine the expression profiles of the DAZ gene family in men with and without spermatogenic failure. Design Prospective case study. Setting University-based reproductive clinics and genetics laboratory. Patient(s) Thirty-four infertile men presenting with azoospermia. Intervention(s) The mRNA transcript concentrations of the DAZ family genes (BOULE, DAZL, DAZ) and the housekeeping GAPDH gene in the testes of azoospermic men were examined by quantitative competitive–reverse transcription–polymerase chain reaction (QC-RT-PCR). The steady-state concentrations of mRNA encoding for each gene in each testicular sample were normalized by the amounts of GAPDH. Main outcome measure(s) Transcript ratios (gene/GAPDH) of BOULE, DAZL, and DAZ. Result(s) The transcript ratios for BOULE, DAZL, and DAZ were significantly decreased in tissues with spermatogenic failure (hypospermatogenesis, maturation arrest, and Sertoli cell–only). However, the ratios of BOULE/DAZL and DAZ/DAZL did not reveal any significant difference in all tissues. Three patients with DAZ deletion possess lower transcripts of BOULE and DAZL. Conclusion(s) All members of the DAZ gene family play important roles in human spermatogenesis. Decreased concentrations of DAZ family members in men with spermatogenic failure may be due to the secondary effect of germ cell loss, and transcriptional control of BOULE, DAZL, and DAZ are not altered in the various degrees of spermatogenic failure. Although the sample size is limited, no compensatory increase of DAZL or BOULE transcription was found in men with DAZ deletion.

The aim of this study was to compare anti-Mullerian hormone (AMH) plasma levels in patients with azoospermia according to the physiopathology. In a prospective clinical study from April 2008 to March 2009 in University Hospital, we measured AMH levels in 49 consecutive patients with azoospermia. AMH plasma levels were correlated with FSH, inhibin B, bioavailable testosterone plasma levels and testicular volume and compared between nonobstructive azoospermia (NOA) and obstructive azoospermia (OA) and within four physiopathological subgroups of NOA: genetic, cryptorchidism, cytotoxic and unexplained. AMH, FSH, inhibin B, bioavailable testosterone plasma levels and testicular volumes were all related to each other. AMH plasma levels were lower in NOA relatively to OA. Lowest values were observed in cases of genetic NOA and on the other hand, the values observed in case of cytotoxic NOA were as high as the values observed in OA. FSH, inhibin B, bioavailable testosterone and testicular volume were not different between genetic and cytotoxic NOA. These results suggest that the decrease in AMH plasma levels is related to the origin of NOA, with low values in genetic NOA and values similar to OA in cytotoxic NOA. Further studies will be useful to understand the fine regulation of AMH production.

Homozygosity for the Ter mutation in the RNA-binding protein Dead end 1 (Dnd1(Ter/Ter)) sensitizes germ cells to degeneration in all mouse strains. In 129/SvJ mice, approximately 10% of Dnd1(Ter/+) heterozygotes develop spermatogenic failure, and 95% of unilateral cases occur in the left testis. The first differences between right and left testes were detected at Postnatal Day 15 when many more spermatogonial stem cells (SSCs) were undergoing apoptosis in the left testis compared to the right. As we detected no significant left/right differences in the molecular pathway associated with body axis asymmetry or in the expression of signals known to promote proliferation, differentiation, and survival of germ cells, we investigated whether physiological differences might account for asymmetry of the degeneration phenotype. We show that left/right differences in vascular architecture are associated with a decrease in hemoglobin saturation and increased levels of HIF-1alpha in the left testis compared to the right. In Dnd1 heterozygotes, lower oxygen availability was associated with metabolic differences, including lower levels of ATP and NADH in the left testis. These experiments suggest a dependence on oxygen availability and metabolic substrates for SSC survival and suggest that Dnd1(Ter/+) SSCs may act as efficient sensors to detect subtle environmental changes that alter SSC fate.

Infertility is a global healthcare problem, and despite long years of assisted reproductive activities, a significant number of cases remain idiopathic. Our currently restricted understanding of basic mechanisms driving human gametogenesis severely limits the improvement of clinical care for infertile patients. Using exome sequencing, we identified a nonsense mutation leading to a premature stop in the TEX15 locus (c.2130T>G, p.Y710*) in a consanguineous Turkish family comprising eight siblings in which three brothers were identified as infertile. TEX15 displays testis-specific expression, maps to chromosome 8, contains four exons and encodes a 2789-amino acid protein with uncertain function. The mutation, which should lead to early translational termination at the first exon of TEX15, co-segregated with the infertility phenotype, and our data strongly suggest that it is the cause of spermatogenic defects in the family. All three affected brothers presented a phenotype reminiscent of the one observed in KO mice. Indeed, previously reported results demonstrated that disruption of the orthologous gene in mice caused a drastic reduction in testis size and meiotic arrest in the first wave of spermatogenesis in males while female KO mice were fertile. The data from our study of one Turkish family suggested that the identified mutation correlates with a decrease in sperm count over time. A diagnostic test identifying the mutation in man could provide an indication of spermatogenic failure and prompt patients to undertake sperm cryopreservation at an early age.

The aim of this study was to assess the cellular miRNA expression behaviour in testes with spermatogenic failure (SpF). We performed a high-throughput screen of 623 mature miRNAs by a quantitative RT-qPCR-based approach in histologically well-defined testicular samples with spermatogenic disruption at different germ-cell stages, which revealed altered patterns of miRNA expression. We focussed on the differentially expressed miRNAs whose expression correlated with the number of testicular mature germ-cells and described the combined expression values of a panel of three miRNAs (miR-449a, miR-34c-5p and miR-122) as a predictive test for the presence of mature germ-cells in testicular biopsy. Additionally, we determined decreased cellular miRNA content in developing germ-cells of SpF testis; this was more noticeable the earlier the stage of germ-cell differentiation was affected by maturation failure. Furthermore, we showed that the miRNA expression profile in mature sperm from mild SpF patients was widely altered. Our results suggest that the cellular miRNA content of developed germ-cells depends heavily on the efficacy of the spermatogenic process. What is more, spermatozoa that have fulfilled the differentiation process still retain the dysregulated miRNA pattern observed in the developing SpF germ-cells. This altered miRNA molecular signature may have functional implications for the male gamete.

Insulin-like peptide 6 (INSL6) is a newly identified insulin/relaxin family peptide hormone that is predominantly expressed by the male germ cells in testes. A recent murine study demonstrated that INSL6-knockout results in spermatogenic failure. In the present study, human INSL6 gene was screened for mutations that may contribute to human spermatogenic failure. Of 249 patients and 249 healthy control subjects, a heterozygous R171H missense mutation was found in one patient. The R171H mutation probably disturbed the in vivo processing of the INSL6 prohormone because it was located at the absolutely conserved tetrabasic cleavage site between the C-peptide and the A-chain, therefore the R171H missense mutation might be responsible for human spermatogenic failure.

High incidence of mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene is associated with congenital bilateral absence of the vas deferens (CBAVD) and is considered as the genital form of cystic fibrosis (CF). The CFTR gene may also be involved in the etiology of male infertility in cases other than CBAVD. The present study was conducted to identify the spectrum and frequency of CFTR gene mutations in infertile Indian males with non-CBAVD obstructive azoospermia (n=60) and spermatogenic failure (n=150). Conspicuously higher frequency of heterozygote F508del mutation was detected in infertile males with non-CBAVD obstructive azoospermia (11.6%) and spermatogenic failure (7.3%). Homozygous IVS(8)-5T allele frequency was also significantly higher in both groups in comparison to those in normal healthy individuals. Two mutations in exon 25 viz., R1358I and K1351R were identified as novel mutations in patients with non-CBAVD obstructive azoospermia. Mutation R1358I was predicted as probably damaging CFTR mutation. This is the first report from the Indian population, emphasizing increased frequency of CFTR gene mutations in male infertility other than CBAVD. Thus, it is suggested that screening of CFTR gene mutations may be required in infertile Indian males with other forms of infertility apart from CBAVD and willing for assisted reproduction technology.

The double sex and mab-3-related transcription factor 1 (DMRT1) gene has long been linked to sex-determining pathways across vertebrates and is known to play an essential role in gonadal development and maintenance of spermatogenesis in mice. In humans, the genomic region harboring the DMRT gene cluster has been implicated in disorders of sex development and recently DMRT1 deletions were shown to be associated with non-obstructive azoospermia (NOA). In this work, we have employed different methods to screen a cohort of Portuguese NOA patients for DMRT1 exonic insertions and deletions [by multiplex ligation probe assay (MLPA); n = 68] and point mutations (by Sanger sequencing; n = 155). We have found three novel patient-specific non-coding variants in heterozygosity that were absent from 357 geographically matched controls. One of these is a complex variant with a putative regulatory role (c.-223_-219CGAAAT), located in the promoter region within a conserved sequence involved in Dmrt1 repression. Moreover, while DMRT1 domains are highly conserved across vertebrates and show reduced levels of diversity in human populations, two rare synonymous substitutions (rs376518776 and rs34946058) and two rare non-coding variants that potentially affect DMRT1 expression and splicing (rs144122237 and rs200423545) were overrepresented in patients when compared with 376 Portuguese controls (301 fertile and 75 normozoospermic). Overall our previous and present results suggest a role of changes in DMRT1 dosage in NOA potentially also through a process of gene misregulation, even though DMRT1 deleterious variants seem to be rare.

The complex process of spermatogenesis requires the expression and precise coordination of a multitude of genes. Abnormal function of such genes is frequently associated with male infertility. Among these candidates is the human BOULE gene that is a possible fundamental mediator of meiotic transition. In this study, we describe for the first time the existence of three BOULE transcript variants (B1, B2 and B3). We investigated their tissue specificity and mRNA transcript levels in 23 testis biopsies from infertile men. B1, B2 and B3 differed solely in their N-terminal sequences, which are encoded by three alternatively spliced exons 1. In humans, all three isoforms are exclusively expressed in the testes in a relative proportion of 80:220:1 for B1, B2 and B3, respectively. RT-PCR quantification revealed significantly reduced mRNA expression of all three variants in testicular biopsies with meiotic arrest (MA) compared with those with qualitatively complete spermatogenesis. Alteration of the B1/B2 and B1/B3 transcript ratios was correlated with reduced meiotic capacity of spermatocytes to produce round spermatids as assessed by flow cytometry. Furthermore, BOULE mRNA reduction in biopsies with MA paralleled the absence of BOULE protein as analysed by immunohistochemistry. In conclusion, the relative proportions of B1, B2 and B3 may serve as predictive markers for meiotic efficiency and thus the probability of finding haploid cells in the human testis. Among the three isoforms, B2 might have the major role for meiotic completion.

BACKGROUND: Reciprocal translocations are often associated with infertility in male carriers. However, some carriers present normal semen profiles and are identified because of repetitive pregnancy failures. METHODS: Here, we report two different cases of reciprocal translocations. The first patient carried a t(10;14) and was normozoospermic. The second patient carried a t(13;20) and was azoospermic. Synaptonemal complexes from both carriers were analysed using immunocytogenetic techniques and multi-centromere fluorescent in situ hybridization (cenM-FISH). RESULTS: Associations between the quadrivalent and the sex body or other autosomes were seen only in the t(13;20) carrier. Heterosynapsis was observed only in the t(10;14) carrier. Synaptic pairing abnormalities were seen in 71% of the spreads in the t(13;20) carrier and 30% of the spreads in the t(10;14) carrier. Recombination frequency was decreased in the t(13;20) carrier, but not in the t(10;14) carrier. CONCLUSIONS: By comparing these two different translocation carriers with different fertility outcomes, we discuss the possible mechanisms by which translocations might cause the spermatogenesis process to fail.

SLC26A8 is an anion transporter that is solely expressed in the testes. It interacts with MgcRacGAP that shows strong structural similarity with the Drosophila protein RotundRacGAP, which is established to have an essential role for male fertility in the fruit fly. To explore whether the SLC26A8 gene has a role in human male infertility, we performed mutational analysis in the coding region of the SLC26A8 gene in 83 male infertility patients and two groups of controls using single-strand conformational polymorphism and direct sequencing methods. We found six novel coding sequence variations, of which five lead to amino acid substitutions. All variants were found with similar frequencies in both patients and controls, thus suggesting that none of them may be causally associated with infertility. We conclude that the SLC26A8 mutations are not a common cause of male infertility.

Objectives. As many as 10% of infertile men have azoospermia caused by spermatogenic failure or ductal obstruction. The histologic diagnoses associated with spermatogenic failure—Sertoli cell-only syndrome, maturation arrest, and hypospermatogenesis—do not necessarily represent global changes in the affected testis, as occasional seminiferous tubules may still produce mature germ cells. Intracytoplasmic sperm injection (ICSI) allows individual sperm that have been isolated from testicular tissue to fertilize oocytes. This study assessed whether mature germ cells (either round spermatids or spermatozoa) were present in the ejaculates of patients with spermatogenic failure. Methods. All semen analyses performed at our tertiary care institution from January 1993 through December 1995 were reviewed to identify azoospermic men with spermatogenic failure. During this period, our laboratory employed Nuclear-Fast Red and picroindigocarmine staining (NF-PICS) of cytospin slides to identify rare spermatozoa and spermatids in otherwise azoospermic ejaculates. Results. Of 3005 analyses reviewed, 20 azoospermic men whose infertility was solely attributable to spermatogenic failure were identified. The histologic diagnoses were germinal cell aplasia (n = 7), complete maturation arrest (n = 6), incomplete maturation arrest (n = 3), and hypospermatogenesis (n = 4). Using the NF-PICS technique, mature germ cells were identified in the ejaculates of 15 men (75%), and 9 men (45%) had fully formed spermatozoa present. Conclusions. In the clinical management of azoospermic infertile men, the NF-PICS technique may be used to identify men who have some degree of testicular spermatogenesis. This might obviate the need for the purely diagnostic testis biopsy that is performed before therapeutic biopsy for testicular sperm extraction in conjunction with ICSI.