Andrological findings in infertile men with two (biallelic) CFTR mutations: results of a multicentre study in Germany and Austria comprising 71 patients.

STUDY QUESTION: When should cystic fibrosis transmembrane conductance regulator (CFTR) mutation analysis be recommended in infertile men based on andrological findings? SUMMARY ANSWER: CFTR mutation analysis is recommended in all men with unexplained azoospermia in the presence of normal gonadotropin levels. WHAT IS KNOWN ALREADY: While 80-97% of men with congenital bilateral absence of the vas deferens (CBAVD) are thought to carry CFTR mutations, there is uncertainty about the spectrum of clinical and andrological abnormalities in infertile men with bilallelic CFTR mutations. This information is relevant for evidence-based recommendations to couples requesting assisted reproduction. STUDY DESIGN, SIZE, DURATION: We studied the andrological findings of patients with two CFTR mutations who were examined in one of the cooperating fertility centres in Germany and Austria. In the period of January till July 2019, the completed and anonymized data sheets of 78 adult male patients were returned to and analysed by the project leader at the Institute of Human Genetics in Innsbruck, Austria. PARTICIPANTS/MATERIALS, SETTING, METHODS: Minimum study entry criteria were the presence of two (biallelic) CFTR mutations and results of at least one semen analysis. Andrological assessments were undertaken by standardized data sheets and compared with normal reference values. Seventy-one patients were eligible for the study (n = 30, 42% from Germany, n = 26, 37% from Austria, n = 15, 21% other nations). MAIN RESULTS AND THE ROLE OF CHANCE: Gonadotropin levels (FSH, LH) were normal, 22% of patients had reduced testosterone values. Mean right testis volume was 23.38 ml (SD 8.77), mean left testis volume was 22.59 ml (SD 8.68) and thereby statistically increased compared to normal (P < 0.01). although the means remained in the reference range of 12-25 ml. Semen analysis revealed azoospermia in 70 of 71 (99%) patients and severe oligozoospermia <0.1 × 106/ml in one patient. Four semen parameters, i.e. ejaculate volume, pH, α-glucosidase and fructose values, were significantly reduced (P < 0.01). Only 18% of patients had a palpatory and sonographically diagnosed CBAVD, while in 31% the diagnosis of CBAVD was uncertain, in 12% patients, the vas deferens was present but hypoplastic, and in 39% the vas deferens was normally present bilaterally. Seminal vesicles were not detectable in 37% and only unilaterally present in 37% of patients. Apart from total testes volume, clinical findings were similar in patients with two confirmed pathogenic CFTR mutations (Group I) compared with patients who carried one pathogenic mutation and one CFTR variant of unknown significance (Group II). LIMITATIONS, REASONS FOR CAUTION: We could not formally confirm the in trans position of genetic variants in most patients as no family members were available for segregation studies. Nonetheless, considering that most mutations in our study have been previously described without other rare variants in cis, and in view of the compatible andrological phenotype, it is reasonable to assume that the biallelic genotypes are correct. WIDER IMPLICATIONS OF THE FINDINGS: Our study reveals that CFTR mutation analysis has a broader indication than just the absence of the vas deferens. We recommend to completely sequence the CFTR gene if there is a suspicion of obstructive azoospermia, and to extend this analysis to all patients with unexplained azoospermia in the presence of normal gonadotropin levels. STUDY FUNDING/COMPETING INTEREST(S): German Research Foundation Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG CRU326, grants to F.T.). There are no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Bi-allelic variants in DNA mismatch repair proteins MutS Homolog MSH4 and MSH5 cause infertility in both sexes.

STUDY QUESTION: Do bi-allelic variants in the genes encoding the MSH4/MSH5 heterodimer cause male infertility? SUMMARY ANSWER: We detected biallelic, (likely) pathogenic variants in MSH5 (4 men) and MSH4 (3 men) in six azoospermic men, demonstrating that genetic variants in these genes are a relevant cause of male infertility. WHAT IS KNOWN ALREADY: MSH4 and MSH5 form a heterodimer, which is required for prophase of meiosis I. One variant in MSH5 and two variants in MSH4 have been described as causal for premature ovarian insufficiency (POI) in a total of five women, resulting in infertility. Recently, pathogenic variants in MSH4 have been reported in infertile men. So far, no pathogenic variants in MSH5 had been described in males. STUDY DESIGN, SIZE, DURATION: We utilized exome data from 1305 men included in the Male Reproductive Genomics (MERGE) study, including 90 males with meiotic arrest (MeiA). Independently, exome sequencing was performed in a man with MeiA from a large consanguineous family. PARTICIPANTS/MATERIALS, SETTING, METHODS: Assuming an autosomal-recessive mode of inheritance, we screened the exome data for rare, biallelic coding variants in MSH4 and MSH5. If possible, segregation analysis in the patients' families was performed. The functional consequences of identified loss-of-function (LoF) variants in MSH5 were studied using heterologous expression of the MSH5 protein in HEK293T cells. The point of arrest during meiosis was determined by γH2AX staining. MAIN RESULTS AND THE ROLE OF CHANCE: We report for the first time (likely) pathogenic, homozygous variants in MSH5 causing infertility in 2 out of 90 men with MeiA and overall in 4 out of 902 azoospermic men. Additionally, we detected biallelic variants in MSH4 in two men with MeiA and in the sister of one proband with POI. γH2AX staining revealed an arrest in early prophase of meiosis I in individuals with pathogenic MSH4 or MSH5 variants. Heterologous in vitro expression of the detected LoF variants in MSH5 showed that the variant p.(Ala620GlnTer9) resulted in MSH5 protein truncation and the variant p.(Ser26GlnfsTer42) resulted in a complete loss of MSH5. LARGE SCALE DATA: All variants have been submitted to ClinVar (SCV001468891-SCV001468896 and SCV001591030) and can also be accessed in the Male Fertility Gene Atlas (MFGA). LIMITATIONS, REASONS FOR CAUTION: By selecting for variants in MSH4 and MSH5, we were able to determine the cause of infertility in six men and one woman, leaving most of the examined individuals without a causal diagnosis. WIDER IMPLICATIONS OF THE FINDINGS: Our findings have diagnostic value by increasing the number of genes associated with non-obstructive azoospermia with high clinical validity. The analysis of such genes has prognostic consequences for assessing whether men with azoospermia would benefit from a testicular biopsy. We also provide further evidence that MeiA in men and POI in women share the same genetic causes. STUDY FUNDING/COMPETING INTEREST(S): This study was carried out within the frame of the German Research Foundation sponsored Clinical Research Unit 'Male Germ Cells: from Genes to Function' (DFG, CRU326), and supported by institutional funding of the Research Institute Amsterdam Reproduction and Development and funds from the LucaBella Foundation. The authors declare no conflict of interest.

Mutations in the stromal antigen 3 (STAG3) gene cause male infertility due to meiotic arrest.

STUDY QUESTION: Are sequence variants in the stromal antigen 3 (STAG3) gene a cause for non-obstructive azoospermia (NOA) in infertile human males? SUMMARY ANSWER: Sequence variants affecting protein function of STAG3 cause male infertility due to meiotic arrest. WHAT IS KNOWN ALREADY: In both women and men, STAG3 encodes for a meiosis-specific protein that is crucial for the functionality of meiotic cohesin complexes. Sequence variants in STAG3 have been reported to cause meiotic arrest in male and female mice and premature ovarian failure in human females, but not in infertile human males so far. STUDY DESIGN, SIZE, DURATION: The full coding region of STAG3 was sequenced directly in a cohort of 28 men with NOA due to meiotic arrest. In addition, a larger group of 275 infertile men that underwent whole-exome sequencing (WES) was screened for potential STAG3 sequence variants. Furthermore, meiotic spreads, immunohistochemistry, WES and population sampling probability (PSAP) have been conducted in the index case. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included 28 infertile but otherwise healthy human males who underwent Sanger sequencing of the full coding region of STAG3. Additionally, WES data of 275 infertile human males with different infertility phenotypes have been screened for relevant STAG3 variants. All participants underwent karyotype analysis and azoospermia factor (AZF) screening in advance. In the index patient, segregation analysis, WES data, PSAP, lab parameters, testis histology and nuclear spreads have been added to suplort the findings. MAIN RESULTS AND THE ROLE OF CHANCE: Two compound-heterozygous variants in STAG3 (c.[1262T>G];[1312C>T], p.[(Leu421Arg)];[(Arg438Ter)]) have been found to cause male infertility due to complete bilateral meiotic arrest in an otherwise healthy human male. Compound heterozygosity was confirmed by Sanger sequencing of the parents and the patient's brother. Other variants which may affect spermatogenesis have been ruled out through analysis of the patient's WES data and application of the PSAP pipeline. As expected from Stag3 knockout-mice meiotic spreads, germ cells did not develop further than zygotene and showed drastic chromosome aberrations. No rare variants in STAG3 were found in the 275 infertile males with other phenotypes. Our results indicate that STAG3 variants that negatively affect its protein function are a rare cause of NOA (<1% of cases). LIMITATIONS, REASONS FOR CAUTION: We identified only one patient with compound-heterozygous variants in STAG3 causing NOA due to meiotic arrest. Future studies should evaluate STAG3 variants in larger cohorts to support this finding. WIDER IMPLICATIONS OF THE FINDINGS: Identification of STAG3 sequence variants in infertile human males should improve genetic counselling as well as diagnostics and treatment. Especially before testicular sperm extraction (TESE) for ICSI, STAG3 variants should be ruled out to prevent unnecessary interventions with frustrating outcomes for both patients and clinicians. STUDY FUNDING/COMPETING INTEREST(S): This work was carried out within the frame of the German Research Foundation (DFG) Clinical Research Unit 'Male Germ Cells: from Genes to Function' (CRU326). Work in the laboratory of R.J. is supported by a grant of the European Union H2020 program GermAge. The authors declare no conflicts of interest. TRIAL REGISTRATION NUMBER: Not applicable.

ESHRE Task Force on Ethics and Law 21: genetic screening of gamete donors: ethical issues.

This Task Force document explores the ethical issues involved in the debate about the scope of genetic screening of gamete donors. Calls for expanded donor screening arise against the background of both occasional findings of serious but rare genetic conditions in donors or donor offspring that were not detected through present screening procedures and the advent of new genomic technologies promising affordable testing of donors for a wide range of conditions. Ethical principles require that all stakeholders' interests are taken into account, including those of candidate donors. The message of the profession should be that avoiding all risks is impossible and that testing should remain proportional.

Y-chromosome microdeletions are not associated with SHOX haploinsufficiency.

STUDY QUESTION: Are Y-chromosome microdeletions associated with SHOX haploinsufficiency, thus representing a risk of skeletal anomalies for the carriers and their male descendents? SUMMARY ANSWER: The present study shows that SHOX haploinsufficiency is unlikely to be associated with Y-chromosome microdeletions. WHAT IS KNOWN ALREADY: Y-chromosome microdeletions are not commonly known as a major molecular genetic cause of any pathological condition except spermatogenic failure. However, it has been recently proposed that they are associated not only with infertility but also with anomalies in the pseudoautosomal regions (PAR), among which SHOX haploinsufficiency stands out with a frequency of 5.4% in microdeletion carriers bearing a normal karyotype. This finding implies that sons fathered by men with Y-chromosome defects will not only exhibit fertility problems, but might also suffer from SHOX-related conditions. STUDY DESIGN: Five European laboratories (Florence, Münster, Barcelona, Padova and Ancona), routinely performing Y-chromosome microdeletion screening, were enrolled in a multicenter study. PARTICIPANTS/MATERIALS, SETTING, METHODS: PAR-linked and SHOX copy number variations (CNVs) were analyzed in 224 patients carrying Y-chromosome microdeletions and 112 controls with an intact Y chromosome, using customized X-chromosome-specific array-CGH platforms and/or qPCR assays for SHOX and SRY genes. MAIN RESULTS AND THE ROLE OF CHANCE: Our data show that 220 out of 224 (98.2%) microdeletion carriers had a normal SHOX copy number, as did all the controls. No SHOX deletions were found in any of the examined subjects (patients as well as controls), thus excluding an association with SHOX haploinsufficiency. SHOX duplications were detected in 1.78% of patients (n = 4), of whom two had an abnormal and two a normal karyotype. This might suggest that Y-chromosome microdeletions have a higher incidence for SHOX duplications, irrespective of the patient's karyotype. However, the only clinical condition observed in our four SHOX-duplicated patients was infertility. LIMITATIONS, REASONS FOR CAUTION: The number of controls analyzed is rather low to assess whether the SHOX duplications found in the two men with Y-chromosome microdeletions and a normal karyotype represent a neutral polymorphism or are actually associated with the presence of the microdeletion. WIDER IMPLICATIONS OF THE FINDINGS: Men suffering from infertility due to the presence of Y-chromosome microdeletions can resort to artificial reproductive technology (ART) to father their biological children. However, infertile couples must be aware of the risks implied and this makes genetic counseling a crucial step in the patient's management. This study does not confirm previous alarming data that showed an association between Y-chromosome microdeletions and SHOX haploinsufficiency. Our results imply that deletion carriers have no augmented risk of SHOX-related pathologies (short stature and skeletal anomalies) and indicate that there is no need for radical changes in genetic counseling of Yq microdeletion carriers attempting ART, since the only risk established so far for their male offspring remains impaired spermatogenesis. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Italian Ministry of University (grant PRIN 2010-2012 to C.K.), Tuscan Regional Health Research Program ('Progetto Salute 2009') to G.F., the Spanish Ministry of Health (grant FIS-11/02254) and the European Union 'Reprotrain' Marie Curie Network (project number: 289880 to C.K.). The authors declare that no conflicting interests exist.

Pathogenic gene variants in CCDC39, CCDC40, RSPH1, RSPH9, HYDIN, and SPEF2 cause defects of sperm flagella composition and male infertility.

Primary Ciliary Dyskinesia (PCD) is a rare genetic disorder affecting the function of motile cilia in several organ systems. In PCD, male infertility is caused by defective sperm flagella composition or deficient motile cilia function in the efferent ducts of the male reproductive system. Different PCD-associated genes encoding axonemal components involved in the regulation of ciliary and flagellar beating are also reported to cause infertility due to multiple morphological abnormalities of the sperm flagella (MMAF). Here, we performed genetic testing by next generation sequencing techniques, PCD diagnostics including immunofluorescence-, transmission electron-, and high-speed video microscopy on sperm flagella and andrological work up including semen analyses. We identified ten infertile male individuals with pathogenic variants in CCDC39 (one) and CCDC40 (two) encoding ruler proteins, RSPH1 (two) and RSPH9 (one) encoding radial spoke head proteins, and HYDIN (two) and SPEF2 (two) encoding CP-associated proteins, respectively. We demonstrate for the first time that pathogenic variants in RSPH1 and RSPH9 cause male infertility due to sperm cell dysmotility and abnormal flagellar RSPH1 and RSPH9 composition. We also provide novel evidence for MMAF in HYDIN- and RSPH1-mutant individuals. We show absence or severe reduction of CCDC39 and SPEF2 in sperm flagella of CCDC39- and CCDC40-mutant individuals and HYDIN- and SPEF2-mutant individuals, respectively. Thereby, we reveal interactions between CCDC39 and CCDC40 as well as HYDIN and SPEF2 in sperm flagella. Our findings demonstrate that immunofluorescence microscopy in sperm cells is a valuable tool to identify flagellar defects related to the axonemal ruler, radial spoke head and the central pair apparatus, thus aiding the diagnosis of male infertility. This is of particular importance to classify the pathogenicity of genetic defects, especially in cases of missense variants of unknown significance, or to interpret HYDIN variants that are confounded by the presence of the almost identical pseudogene HYDIN2.

The future of testis research is turning 6! Six years of international network for young researchers in male fertility.

The 'International Network for Young Researchers in Male Fertility' has now turned 6 years old and offers a platform that stimulates scientific exchange as well as the development of international cooperation for young researchers. We report on our scope and the exciting achievements, amongst others, the continually increasing number of participants and the growing success of our annual meetings.

Analysis of copy number variation in men with non-obstructive azoospermia.

BACKGROUND: Recent findings demonstrate that single nucleotide variants can cause non-obstructive azoospermia (NOA). In contrast, copy number variants (CNVs) were only analysed in few studies in infertile men. Some have reported a higher prevalence of CNVs in infertile versus fertile men. OBJECTIVES: This study aimed to elucidate if CNVs are associated with NOA. MATERIALS AND METHODS: We performed array-based comparative genomic hybridisation (aCGH) in 37 men with meiotic arrest, 194 men with Sertoli cell-only phenotype, and 21 control men. We filtered our data for deletions affecting genes and prioritised the affected genes according to the literature search. Prevalence of CNVs was compared between all groups. Exome data of 2,030 men were screened to detect further genetic variants in prioritised genes. Modelling was performed for the protein encoded by the novel candidate gene TEKT5 and we stained for TEKT5 in human testicular tissue. RESULTS: We determined the cause of infertility in two individuals with homozygous deletions of SYCE1 and in one individual with a heterozygous deletion of SYCE1 combined with a likely pathogenic missense variant on the second allele. We detected heterozygous deletions affecting MLH3, EIF2B2, SLX4, CLPP and TEKT5, in one subject each. CNVs were not detected more frequently in infertile men compared with controls. DISCUSSION: While SYCE1 and MLH3 encode known meiosis-specific proteins, much less is known about the proteins encoded by the other identified candidate genes, warranting further analyses. We were able to identify the cause of infertility in one out of the 231 infertile men by aCGH and in two men by using exome sequencing data. CONCLUSION: As aCGH and exome sequencing are both expensive methods, combining both in a clinical routine is not an effective strategy. Instead, using CNV calling from exome data has recently become more precise, potentially making aCGH dispensable.

A de novo paradigm for male infertility.

De novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10-5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10-4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

Clinical experience with azoospermia: aetiology and chances for spermatozoa detection upon biopsy.

The clinical workup of the infertile male with azoospermia aims at determining the aetiology and estimating the chances of finding spermatozoa by testicular sperm extraction (TESE). To establish prognostic criteria, 1583 consecutive patients with azoospermia consulting the Centre of Reproductive Medicine and Andrology, Münster, a tertiary referral centre, between 1976 and 2009 comprising 9.8% of all patients providing a semen sample were included in this retrospective analysis. The frequencies of diagnoses were as follows: 21% genetic causes (14% Klinefelter syndrome, 1% other chromosomal aberrations, 2% Y-chromosomal microdeletions, 1% hypogonadotropic hypogonadism, 3% congenital bilateral absence of the vas deferens), 31% current or former maldescended testes, varicocele, urogenital infections, 15% malignancies, 11% obstructions, 7% endocrine or other chronic diseases and 12% idiopathic azoospermia. Receiver-operating characteristic curves for chances of finding spermatozoa by testicular biopsy were calculated for testicular volume, serum follicle-stimulating hormone (FSH) and the seminal markers α-glucosidase, fructose and zinc where these data were available (N=283). Histograms of the seminal markers comparing data from men with obstructive azoospermia and normozoospermia visualize their discriminating power. Evidence-based threshold values for high chances of positive testicular biopsy serving as surrogate marker for TESE were derived from the subgroup of men with obstructive azoospermia for testicular volume (≥21mL), FSH (≤10U/L) and seminal α-glucosidase (≤18mU/ejaculate). Fructose and zinc could not predict the chances of finding spermatozoa upon biopsy. Based on these three parameters, positive biopsy and presumably TESE success can be quickly and reliably estimated in everyday practice with the colour-coded figures constructed from these data. As a seminal α-glucosidase reference limit of 18mU/ejaculate can also be used to diagnose congenital bilateral absence of the vas deferens, α-glucosidase (rather than seminal fructose) should be determined as part of the clinical routine when counselling patients before testicular biopsy.

Novel genetic aspects of Klinefelter's syndrome.

Klinefelter's syndrome (KS) is the most common chromosome aneuploidy in males, characterized by at least one supernumerary X chromosome. Although extensively studied, the pathophysiology, i.e. the link between the extra X and the phenotype, largely remains unexplained. The scope of this review is to summarize the progress made in recent years on the role of the supernumerary X chromosome with respect to its putative influence on the phenotype. In principal, the parental origin of the X chromosome, gene-dosage effects in conjunction with (possibly skewed) X chromosome inactivation, and--especially concerning spermatogenesis--meiotic failure may play pivotal roles. One of the X chromosomes is inactivated to achieve dosage-compensation in females and probably likewise in KS. Genes from the pseudoautosomal regions and an additional 15% of other genes, however, escape X inactivation and are candidates for putatively constituting the KS phenotype. Examples are the SHOX genes, identified as likely causing the tall stature regularly seen in KS. Lessons learned from comparisons with normal males and especially females as well as other sex chromosomal aneuploidies are presented. In addition, genetic topics concerning fertility and counseling are discussed.

Idiopathic male infertility is strongly associated with aberrant methylation of MEST and IGF2/H19 ICR1.

Aberrant imprinting in spermatozoa in a subset of infertile men has been postulated to be a risk factor for congenital diseases in children conceived via assisted reproduction techniques (ART). Studies in clinically well characterized large cohorts, however, have been missing. Using bisulfite sequencing, we determined the degree of methylation of the IGF2/H19 imprinting control region 1 (ICR1) and MEST differentially methylated regions in swim-up purified spermatozoa from 148 idiopathic infertile men and 33 normozoospermic controls. All control individuals had a high degree of IGF2/H19 ICR1 and a low degree of MEST methylation. Low sperm counts were clearly associated with IGF2/H19 ICR1 hypomethylation and, even stronger, with MEST hypermethylation. MEST hypermethylation, but not IGF2/H19 ICR1 hypomethylation was found in idiopathic infertile men with progressive sperm motility below 40% and bad sperm morphology below 5% normal spermatozoa. Ageing could be ruled out as a cause for the observed methylation defects. Sequence analysis of the CTCFL gene in peripheral blood DNA from 20 men with severe methylation defects revealed several polymorphisms, but no bona fide mutation. We conclude that idiopathic male infertility is strongly associated with imprinting defects at IGF2/H19 ICR1 and MEST, with aberrant MEST methylation being a strong indicator for sperm quality. The male germ cell thus represents a potential source for aberrant epigenetic features in children conceived via ART.

Aquaporins in the human testis and spermatozoa - identification, involvement in sperm volume regulation and clinical relevance.

Despite the high water-permeability of human spermatozoa, little is known about the identity and the role of aquaporins (AQP) in them or germ cells. Using ejaculates from donors, sperm AQPs were identified by western blotting followed by the analysis of mRNA with RT-PCR. Protein expression in the testis and spermatozoa was localized by immunocytochemistry. Inhibitors were used to investigate the involvement of aquaporins in water transport when ejaculated spermatozoa were swollen in medium mimicking uterine hypo-osmolality by quinine that blocks volume regulation. Sperm AQP7 and AQP8 in 39 infertile patients and 11 healthy donors were quantified by flow cytometry. AQP1 was absent from spermatozoa. Sperm and testicular AQP7-9 had nucleotide sequences identical to those of somatic cells but AQP8 mRNA also showed shorter variants. AQP7 was expressed abundantly by round and elongated spermatids and ejaculated spermatozoa, AQP8 by all germ cells and spermatozoa, and AQP9 rarely by spermatocytes or Sertoli cells. Protein bands showed specificity by western blotting for AQP7 and AQP8 but not AQP9. The absence of sperm AQP9 was further suggested by the ineffectiveness of its inhibitor phloretin in blocking quinine-induced swelling, but HgCl(2,) which inhibits AQP8, was effective. Sperm AQP7 expression was correlated with progressive motility and was lower in patients than in donors. Sperm AQP8 expression was inversely correlated with the extent of sperm coiling, which is a swelling phenomenon, but showed no difference between patients and donors. In conclusion, AQP7 and AQP8 were identified in human spermatozoa and could play a role in glycerol metabolism and water transport respectively.

A common haplotype of protamine 1 and 2 genes is associated with higher sperm counts.

Sperm chromatin compaction in the sperm head is achieved when histones are replaced by protamines during spermatogenesis. Haploinsufficiency of the protamine 1 (PRM1) or PRM2 gene causes infertility in mice. However, the published data remain inconclusive about a role of PRM1/2 variants in male infertility and their association with semen parameters. By full sequence analysis, we assessed the frequency of sequence variations in PRM1 and PRM2 in three groups of Caucasian patients with idiopathic teratozoospermia and normal (n = 88) or reduced sperm concentration (n = 83) and in men with a high percentage of normal sperm morphology and normal concentrations (n = 77). Two rare (c.54G>A and c.102G>T) and one common SNP (c.230A>C) were identified in PRM1. In PRM2, some rare heterozygous mutations and the two common intronic SNPs 298G>C and 373C>A were detected. None of the PRM1/2 variants was associated with teratozoospermia or individually with other semen parameters. However, significant linkage disequilibrium was detected between the common SNPs of PRM1 and PRM2 which formed haplotypes. Analysis of the pooled group (n = 248) revealed that homozygous carriers of the common haplotype ACC had a twofold higher sperm concentration and count than men lacking this haplotype, with sperm counts of heterozygotes for ACC being midway between the homozygotes. This markedly decreased sperm output might either be caused by spermatozoa lacking the ACC haplotype not being viable, or subject to negative selection. In addition, a significant deviation from Hardy-Weinberg-Equilibrium of these SNPs might indicate natural selection in favour of the ACC allele which leads to higher sperm output and therefore better fertility. In conclusion, for the first time we describe an association of a common haplotype formed by PRM1 and PRM2 with sperm output in a large group of men.

[Andrological diagnostics prior to treatment by assisted reproduction]. / Andrologische Diagnostik vor einer reproduktionsmedizinischen Behandlung.

Infertility is defined as the inability of a couple to succeed in achieving a spontaneous pregnancy after 1 year. Male and female factors contribute to infertility with approximately 40% each. In the remaining cases factors that affect fertility can be found in both partners. The andrological work-up should be started simultaneously with the gynecological diagnostic procedure in order to identify and treat andrological factors related to infertility. Since the majority of intracytoplasmic sperm injection procedures are performed due to andrological infertility, andrological diagnostics can prevent a delay in assisted reproductive technology. The andrological work-up can be necessary before 12 months of unsuccessful conception if the female partner is older than 35 years or andrological factors are present that could impair male fertility.

Sequence analysis of 37 candidate genes for male infertility: challenges in variant assessment and validating genes.

BACKGROUND: The routine genetic analysis for diagnosing male infertility has not changed over the last twenty years, and currently available tests can only determine the etiology of 4% of unselected infertile patients. Thus, to create new diagnostic assays, we must better understand the molecular and genetic mechanisms of male infertility. Although next-generation sequencing allows for simultaneous analysis of hundreds of genes and the discovery of novel candidates related to male infertility, so far only a few gene candidates have enough sound evidence to support the gene-disease relationship. OBJECTIVE: Since complementary studies are required to validate genes, we aimed to analyze the presence of potentially pathogenic rare variants in a set of candidate genes related to azoospermia in a hitherto understudied South American population. SUBJECTS AND METHODS: We performed whole exome sequencing in a group of 16 patients with non-obstructive azoospermia from Ribeirão Preto, Brazil. Based on a recent systematic review of monogenic causes of male infertility, we selected a set of 37 genes related to azoospermia, Sertoli-Cell-Only histology, and spermatogenic arrest to analyze. The identified variants were confirmed by Sanger sequencing, and their functional consequence was predicted by in silico programs. RESULTS: We identified potential pathogenic variants in seven genes in six patients. Two variants, c.671A>G (p.(Asn224Ser)) in DMRT1 and c.91C>T (p.(Arg31Cys)) in REC8, have already been described in association with azoospermia. We also found new variants in genes that already have moderate evidence of being linked to spermatogenic failure (TEX15, KLHL10), in genes with limited evidence (DNMT3B, TEX14) and in one novel promising candidate gene that has no evidence so far (SYCE1L). DISCUSSION: Although this study included a small number of patients, the process of rationally selecting genes allowed us to detect rare potentially pathogenic variants, providing supporting evidence for validating candidate genes associated with azoospermia.

Coiled sperm from infertile patients: characteristics, associated factors and biological implication.

BACKGROUND: There is no systematic study on coiled sperm in semen, although they are commonly observed. This work characterizes coiled sperm in infertile men to understand the clinical implications and investigate the possible cause by osmotic swelling. METHODS: Coiled sperm in semen from 439 infertile patients were quantified and their ultrastructure examined by electron microscopy. Hypo-osmotic swelling (HOS) and demembranation tests were performed to elucidate the nature of the coiling. RESULTS: Semen from patients contained overall 3% of sperm with head-in-coil (HIC) and 8% other coiled forms, with 12% of patients having 20% or more such sperm. The percentage of coiled sperm (but not HIC) was correlated with age (R = 0.26, P = 0.003) and the epididymal secretory marker neutral alpha-glucosidase (R = 0.16, P < 0.001), and associated with heavy smoking and varicocele. Electron microscopy revealed coiling of tail filaments within the plasma membrane, resembling HOS. Some seminal coiled sperm and most sperm freshly coiled upon HOS could be opened by demembranation, while those that could not be opened were probably fixed in position by oxidation, which occurred more frequently in patients than semen donors. CONCLUSIONS: Sperm coiling in semen is common and independent of sperm quantity or hormonal status. Whereas HIC may have a genetic background, other coiled forms may be associated with a hostile endogenous milieu in the epididymis that causes swelling.

[Genetics of male infertility]. / Genetik der männlichen Infertilität.

Genetic causes of male infertility increase in frequency with decreasing sperm concentration (oligo-/azoospermia). The decision about genetic tests should be made after a complete andrological work-up. Common causes comprise chromosomal aberrations (including Klinefelter syndrome), microdeletions of the AZF loci of the Y chromosome, mutations in the gene responsible for cystic fibrosis (CFTR) causing CBAVD and in genes involved in hypogonadotropic hypogonadism (including Kallmann syndrome). Every genetic investigation should be accompanied by comprehensive genetic counselling to help with the interpretation of results and support the patient/the couple concerning consequences for their family planning and treatment options.