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.

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.

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.

Testicular function in males with infantile nephropathic cystinosis.

STUDY QUESTION: Do males with the rare lysosomal storage disease infantile nephropathic cystinosis (INC) have a chance of biological fatherhood? SUMMARY ANSWER: Cryostorage of semen could be an option for approximately 20% of young males with INC, with surgical sperm retrieval from the centre of the testes providing additional opportunities for fatherhood. WHAT IS KNOWN ALREADY: Biallelic mutations in the cystinosin (CTNS) gene in INC cause dysfunction in cystine transport across lysosomal membranes and cystine accumulation throughout the body. Spontaneous paternity in cystinosis has not been described, despite the availability of cysteamine treatment. Azoospermia has been diagnosed in small case series of males with INC. ART using ICSI requires few spermatozoa, either from semen or extracted surgically from the testes of azoospermic men. However, there is limited evidence to suggest this could be successful in INC. STUDY DESIGN, SIZE, DURATION: In this prospective cohort study performed between 2018 and 2019, we performed a cross-sectional investigation of 18 male patients with INC to delineate endocrine and spermatogenic testicular function. PARTICIPANTS/MATERIALS, SETTING, METHODS: Serum hormone levels, semen samples (according to World Health Organization 2010 standards), and testicular ultrasound images were analysed in 18 male patients aged 15.4-40.5 years. Surgical sperm extraction was performed in two, and their testicular biopsies were investigated by light and electron microscopy. Past adherence to cysteamine treatment was assessed from medical record information, using a composite scoring system. MAIN RESULTS AND THE ROLE OF CHANCE: Adherence to cysteamine treatment was high in most patients. Testicular volumes and testosterone levels were in the normal ranges, with the exception of two and three older patients, respectively. Serum LH levels were above the normal range in all subjects aged ≥20 years. FSH levels were elevated in all but four males: three with spermatozoa in semen and one adolescent. Inhibin B levels were shown to be lower in older men. Testicular ultrasound revealed signs of obstruction in 67% of patients. Reduced fructose and zinc seminal markers were found in 33%, including two patients with azoospermia who underwent successful surgical sperm retrieval. Histology identified fully preserved spermatogenesis in the centre of their testes, but also tubular atrophy and lysosomal overload in Sertoli and Leydig cells of the testicular periphery. LIMITATIONS, REASONS FOR CAUTION: Limitations of this study are the small number of assessed patients and the heterogeneity of their dysfunction in cystine transport across lysosomal membranes. WIDER IMPLICATIONS OF THE FINDINGS: This study suggests that testicular degeneration in cystinosis results from the lysosomal overload of Sertoli and Leydig cells of the testicular periphery, and that this can possibly be delayed, but not prevented, by good adherence to cysteamine treatment. Endocrine testicular function in INC may remain compensated until the fourth decade of life; however, azoospermia may occur during adolescence. Cryostorage of semen could be an option for approximately 20% of young males with INC, with surgical sperm retrieval providing additional opportunities for biological fatherhood. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the Cystinosis Foundation Germany. The authors have no competing interests to declare. TRIAL REGISTRATION NUMBER: n/a.

Serum concentrations of dihydrotestosterone are associated with symptoms of hypogonadism in biochemically eugonadal men.

PURPOSE: Symptoms of hypogonadism are often reported by subjects with normal serum testosterone (T) levels. We aimed to assess the association between clinical symptoms in andrological outpatients and sex steroids levels. METHODS: This is a retrospective cross-sectional cohort study in an Academic clinic and research unit. International Index of Erectile Function (IIEF, EF domain) and Aging Males Symptoms scale (AMS) questionnaires were completed by 635 and 574 men, respectively (mean age: 47.3 ± 13.9 and 47.4 ± 13.8 years, p = 0.829), free of interfering medications with complaints possibly related to hypogonadism. RESULTS: Serum total/free T as well as dihydro-T (DHT) was associated with IIEF-EF and AMS scores in the overall population using univariate analyses. Multivariate approaches revealed DHT concentrations in subjects with normal T levels (n = 416, Total T > 12 nmol/L) to be significant predictors of AMS scores. A 0.1 nmol/l serum DHT increase within the eugonadal range was associated with a 4.67% decrease in odds of having worse symptoms (p = 0.011). In men with biochemical hypogonadism (Total T < 12 nmol/L), total and free T rather than DHT were associated with AMS results. This association was not found for IIEF-EF scores. Indirect effects of age and BMI were seen for relations with hormone concentrations but not questionnaire scores. CONCLUSION: DHT can be associated with symptoms of hypogonadism in biochemically eugonadal men. Serum DHT measurement might be helpful once the diagnosis of hypogonadism has been ruled out but should not be routinely included in the primary diagnostic process.

The European Academy of Andrology (EAA) ultrasound study on healthy, fertile men: Scrotal ultrasound reference ranges and associations with clinical, seminal, and biochemical characteristics.

BACKGROUND: Scrotal color Doppler ultrasound (CDUS) still suffers from lack of standardization. Hence, the European Academy of Andrology (EAA) has promoted a multicenter study to assess the CDUS characteristics of healthy fertile men (HFM) to obtain normative parameters. OBJECTIVES: To report and discuss the scrotal organs CDUS reference ranges and characteristics in HFM and their associations with clinical, seminal, and biochemical parameters. METHODS: A cohort of 248 HFM (35.3 ± 5.9years) was studied, evaluating, on the same day, clinical, biochemical, seminal, and scrotal CDUS following Standard Operating Procedures. RESULTS: The CDUS reference range and characteristics of the scrotal organs of HFM are reported here. CDUS showed a higher accuracy than physical examination in detecting scrotal abnormalities. Prader orchidometer (PO)- and US-measured testicular volume (TV) were closely related. The US-assessed TV with the ellipsoid formula showed the best correlation with the PO-TV. The mean TV of HFM was ~ 17 ml. The lowest reference limit for right and left testis was 12 and 11 ml, thresholds defining testicular hypotrophy. The highest reference limit for epididymal head, tail, and vas deferens was 12, 6, and 4.5 mm, respectively. Mean TV was associated positively with sperm concentration and total count and negatively with gonadotropins levels and pulse pressure. Subjects with testicular inhomogeneity or calcifications showed lower sperm vitality and concentration, respectively, than the rest of the sample. Sperm normal morphology and progressive motility were positively associated with epididymal head size/vascularization and vas deferens size, respectively. Increased epididymis and vas deferens sizes were associated with MAR test positivity. Decreased epididymal tail homogeneity/vascularization were positively associated with waistline, which was negatively associated with intratesticular vascularization. CDUS varicocele was detected in 37.2% of men and was not associated with seminal or hormonal parameters. Scrotal CDUS parameters were not associated with time to pregnancy, number of children, history of miscarriage. CONCLUSIONS: The present findings will help in better understanding male infertility pathophysiology, improving its management.

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.

[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.

Fertility preservation in boys: recent developments and new insights †.

BACKGROUND: Infertility is an important side effect of treatments used for cancer and other non-malignant conditions in males. This may be due to the loss of spermatogonial stem cells (SSCs) and/or altered functionality of testicular somatic cells (e.g. Sertoli cells, Leydig cells). Whereas sperm cryopreservation is the first-line procedure to preserve fertility in post-pubertal males, this option does not exist for prepubertal boys. For patients unable to produce sperm and at high risk of losing their fertility, testicular tissue freezing is now proposed as an alternative experimental option to safeguard their fertility. OBJECTIVE AND RATIONALE: With this review, we aim to provide an update on clinical practices and experimental methods, as well as to describe patient management inclusion strategies used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss. SEARCH METHODS: Based on the expertise of the participating centres and a literature search of the progress in clinical practices, patient management strategies and experimental methods used to preserve and restore the fertility of prepubertal boys at high risk of fertility loss were identified. In addition, a survey was conducted amongst European and North American centres/networks that have published papers on their testicular tissue banking activity. OUTCOMES: Since the first publication on murine SSC transplantation in 1994, remarkable progress has been made towards clinical application: cryopreservation protocols for testicular tissue have been developed in animal models and are now offered to patients in clinics as a still experimental procedure. Transplantation methods have been adapted for human testis, and the efficiency and safety of the technique are being evaluated in mouse and primate models. However, important practical, medical and ethical issues must be resolved before fertility restoration can be applied in the clinic.Since the previous survey conducted in 2012, the implementation of testicular tissue cryopreservation as a means to preserve the fertility of prepubertal boys has increased. Data have been collected from 24 co-ordinating centres worldwide, which are actively offering testis tissue cryobanking to safeguard the future fertility of boys. More than 1033 young patients (age range 3 months to 18 years) have already undergone testicular tissue retrieval and storage for fertility preservation. LIMITATIONS REASONS FOR CAUTION: The review does not include the data of all reproductive centres worldwide. Other centres might be offering testicular tissue cryopreservation. Therefore, the numbers might be not representative for the entire field in reproductive medicine and biology worldwide. The key ethical issue regarding fertility preservation in prepubertal boys remains the experimental nature of the intervention. WIDER IMPLICATIONS: The revised procedures can be implemented by the multi-disciplinary teams offering and/or developing treatment strategies to preserve the fertility of prepubertal boys who have a high risk of fertility loss. STUDY FUNDING/COMPETING INTERESTS: The work was funded by ESHRE. None of the authors has a conflict of interest.

Immunohistochemical characterization of the anti-Müllerian hormone receptor type 2 (AMHR-2) in human testes.

PURPOSE: In males, AMH is secreted by immature Sertoli cells; following exposure to endogenous androgens, Sertoli cells undergo a process of maturation which ultimately inhibits AMH expression to undetectable levels in the serum. However, expression of AMH receptor (AMHR-2) has never been studied in human testes, and high intratubular concentrations of AMH have been reported in recent literature. We therefore assessed expression of AMHR-2 in several testicular tissue samples by immunohistochemistry (IHC). METHODS: The IHC method was first validated on tissue samples from healthy human testis (n = 2) and from marmoset ovary (n = 1). The same method was then used for assessment on testicular histopathology specimens from patients with mixed atrophy (MA, n = 2), spermatogenetic arrest (SA, n = 2), Sertoli cell-only syndrome (SCO, n = 1), Klinefelter syndrome (KS, n = 1), and nonseminomatous germ cell tumors (NSGCT, n = 1). Tissue samples from two subjects at different pubertal stages (AndroProtect (AP), aged 5 and 14 years) with hematological malignancies were also retrieved. RESULTS: In adult men, AMHR-2 was expressed on peritubular mesenchymal cells, with patterns closely mirroring α-smooth muscle actin expression. Similar patterns were preserved in almost all conditions; however, in nonseminomatous germ cell tumors the tissue architecture was lost, including AMHR-2 expression. More positive and diffuse staining was observed in tissue samples from prepubertal testes. CONCLUSIONS: In specimens from both healthy and affected testes, AMHR-2 expression appears weaker in adult than in prepubertal tissue sections. The persistence of AMHR-2 expression seemingly hints at a possible effect of intratesticular AMH on the tubular walls.

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.

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.

Ageing in men with normal spermatogenesis alters spermatogonial dynamics and nuclear morphology in Sertoli cells.

BACKGROUND: Ageing in men is believed to be associated with fertility decline and elevated risk of congenital disorders for the offspring. The previous studies also reported reduced germ and Sertoli cell numbers in older men. However, it is not clear whether ageing in men with normal spermatogenesis affects the testis and germ cell population dynamics in a way sufficient for transmitting adverse age effects to the offspring. OBJECTIVES: We examined men with normal spermatogenesis at different ages concerning effects on persisting testicular cell types, that is the germ line and Sertoli cells, as these cell populations are prone to be exposed to age effects. MATERIAL AND METHODS: Ageing was assessed in testicular biopsies of 32 patients assigned to three age groups: (i) 28.8 ± 2.7 years; (ii) 48.1 ± 1 years; and (iii) 70.9 ± 6.2 years, n = 8 each, with normal spermatogenesis according to the Bergmann-Kliesch score, and in a group of meiotic arrest patients (29.9 ± 3.8 years, n = 8) to decipher potential links between different germ cell types. Besides morphometry of seminiferous tubules and Sertoli cell nuclei, we investigated spermatogenic output/efficiency, and dynamics of spermatogonial populations via immunohistochemistry for MAGE A4, PCNA, CREM and quantified A-pale/A-dark spermatogonia. RESULTS: We found a constant spermatogenic output (CREM-positive round spermatids) in all age groups studied. In men beyond their mid-40s (group 2), we detected increased nuclear and nucleolar size in Sertoli cells, indirectly indicating an elevated protein turnover. From the 7th decade (group 3) of life onwards, testes showed increased proliferation of undifferentiated spermatogonia, decreased spermatogenic efficiency and elevated numbers of proliferating A-dark spermatogonia. DISCUSSION AND CONCLUSION: Maintaining normal sperm output seems to be an intrinsic determinant of spermatogenesis. Ageing appears to affect this output and might provoke compensatory proliferation increase in A spermatogonia which, in turn, might hamper germ cell integrity.

Immunolocalization of DMRTB1 in human testis with normal and impaired spermatogenesis.

BACKGROUND: The transcription factor DMRTB1 plays a pivotal role in coordinating the transition between mitosis and meiosis in murine germ cells. No reliable data are available for human testis. OBJECTIVES: The present study aims to examine the testicular expression pattern of DMRTB1 in men showing normal and impaired spermatogenesis. MATERIALS AND METHODS: Immunohistochemistry was performed using 54 human testicular biopsy specimens and a commercial rabbit polyclonal anti-DMRTB1 primary antibody. RT-PCR complemented immunohistochemistry. To further characterize immunopositive cells and possible co-localization, the proliferation marker Ki-67, the tumor marker PLAP, and an anti-DMRT1 antibody were used. RESULTS: In men with normal spermatogenesis, a strong immunoreactivity was detectable in a subset of spermatogonia (38.34 ± 2.14%). Some spermatocytes showed a weak immunostaining. Adjacent Sertoli cells were immunonegative. Compared with a hematoxylin and eosin overview staining, these immunopositive cells were almost exclusively identified as Apale and B spermatogonia and primary spermatocytes in (pre-)leptotene, zygotene, and pachytene stages. In patients with spermatogenic arrest at spermatogonial level, an altered staining pattern was found. No immunoreactivity was detected in Sertoli cells in Sertoli cell-only syndrome. In germ cell neoplasia in situ (GCNIS) tubules, except for a few (0.4 ± 0.03%), pre-invasive tumor cells were immunonegative. Seminoma cells showed no immunostaining. DISCUSSION: According to previous findings in mice, it seems reasonable that DMRTB1 is expressed in these normal germ cell populations. Moreover, altered staining pattern in spermatogenic arrest at spermatogonial stage suggests a correlation with mitosis and transformation into B spermatogonia. The absence of DMRTB1 in GCNIS cells and tumor cells might be associated with uncontrolled neoplastic cell proliferation and progression into invasive germ cell tumors. Further research is required to elucidate, for example, the role of DMRTB1 in the malignant transformation of human germ cells. CONCLUSION: Our data indicate a relevant role for DMRTB1 regarding the entry of spermatogonia into meiosis in men.

AMH and INSL3 in testicular and extragonadal pathophysiology: what do we know?

BACKGROUND: It is commonly accepted that testicular function is prevalently regulated by the hypothalamic-pituitary-gonadal axis: The pulsatile secretion of GnRH by the hypothalamus induces pituitary expression of the two gonadotropins FSH and LH, which then stimulate Sertoli and Leydig cells, respectively, therefore regulating steroidogenesis and spermatogenesis. However, a growing body of evidence has recently suggested that other hormones act on the reproductive tract since the early phases of fetal development. Anti-Müllerian hormone and INSL3 are still largely used only for research purposes despite being increasingly recognized as markers of Sertoli and Leydig cells function, respectively. OBJECTIVES: Provide an up-to-date review of the role of anti-Müllerian hormone and INSL3 in human pathophysiology according to current evidence. MATERIALS AND METHODS: A thorough literature review was performed on PubMed, OVID MEDLINE/EMBASE and Google Scholar for papers concerning anti-Müllerian hormone and INSL3 in human males. RESULTS: INSL3 is not acutely regulated by the hypothalamic-pituitary axis but is constitutively secreted by Leydig cells, therefore representing a valid marker for their number and status. Anti-Müllerian hormone expression, on the other hand, is downregulated by androgens, therefore occurring mostly at the early stages of testicular differentiation and before the onset of puberty. Several conditions affecting testicular development, such as male hypogonadotropic hypogonadism, and their treatment have been associated to specific pattern of INSL3 and anti-Müllerian hormone expression, proving a role for both hormones in the diagnostic and therapeutic management. Recent reports suggest a role for both anti-Müllerian hormone and INSL3 in extra gonadal physiology, such as cardiovascular and bone health. CONCLUSION: Anti-Müllerian hormone and INSL3 are markers of Sertoli and Leydig cells maturation, respectively, usually involved in the pathogenesis of disorders of sexual differentiation. However, their role in testicular pathology has only been hinted at in the last decades. Recent evidence supports an involvement of both anti-Müllerian hormone and INSL3 in extragonadal pathophysiology as well.

Seminal plasma (SP) induces a rapid transforming growth factor beta 1 (TGFß1)-independent up-regulation of epithelial-mesenchymal transdifferentiation (EMT) and myofibroblastic metaplasia-markers in endometriotic (EM) and endometrial cells.

PURPOSE: To study if short-term exposure (2 h and 6 h) of endometrial/endometriotic tissues and cells to 10% seminal plasma (SP) can induce EMT/metaplasia. METHODS: Basic research experimental study was carried out in a University hospital-based fertility center. Semen samples, peritoneal fluid (PF) from endometriosis patients, endometrial biopsy from premenopausal women, immortalized endometriotic epithelial cell line (12Z), and immortalized endometrial stromal cell line (St-T1b) were studied. Rapid stain identification test (RSID), TGFß1 immunofluorescence of washed sperms, TGFß1-ELISA of SP and PF, in vitro study (2 h and 6 h incubation) and real-time PCR of endometrial tissue and cell lines to analyze gene expression of EMT/metaplasia markers and mediators were done. RESULTS: SP is still detectable in washed semen. TGFß1 was expressed on the plasma membrane of the sperms and was significantly more concentrated in SP (88.17 ng/ml) than PF. 10% SP induced an up-regulation of alpha smooth muscle actin expression in endometrial tissue (p = 0.008) and in 12Z cells (p = 0.05), mostly TGFß1-independent. TWIST expression was persistently significantly down-regulated while Snail1 and 2 were up-regulated, though insignificant. CONCLUSION: Our results provide novel evidence to support that even in semen washed twice, SP is still detectable. The changes in EMT/metaplasia markers and mediators give a new insight into a possible effect of SP on the pathogenesis of endometriosis.

Activin A target genes are differentially expressed between normal and neoplastic adult human testes: clues to gonocyte fate choice.

BACKGROUND: Human testicular germ cell tumours (TGCT) arise from germ cell neoplasia in situ (GCNIS) cells that originate from foetal germ cell precursors. Activin A is central to normal foetal testis development, and its dysregulation may contribute to TGCT aetiology. OBJECTIVE: (i) To test whether the expression profiles of activin A targets in normal and neoplastic human testes indicates functional links with TGCT progression. (ii) To investigate whether activin A levels influence MMP activity in a neoplastic germ cell line. MATERIALS AND METHODS: (1) Bouin's fixed, paraffin-embedded human testes were utilized for PCR-based transcript analysis and immunohistochemistry. Samples (n = 5 per group) contained the following: (i) normal spermatogenesis, (ii) GCNIS or (iii) seminoma. CXCL12, CCL17, MMP2 and MMP9 were investigated. (2) The human seminoma-derived TCam-2 cell line was exposed to activin A (24 h), and target transcripts were measured by qRT-PCR (n = 4). ELISA (n = 4) and gelatin zymography (n = 3) showed changes in protein level and enzyme activity, respectively. RESULTS: (i) Cytoplasmic CXCL12 was detected in Sertoli and other somatic cells, including those surrounding seminoma cells. Anti-CCL17 labelled only the cytoplasm of Sertoli cells surrounding GCNIS, while anti-MMP2 and anti-MMP9 labelled germline and epithelial-like cells in normal and neoplastic testes. (ii) Exposing TCam-2 cells to activin A (50 ng/mL) elevated MMP2 and MMP9 transcripts (fourfold and 30-fold), while only MMP2 protein levels were significantly higher after activin A (5 ng/mL and 50 ng/mL) exposure. Importantly, gelatin zymography revealed activin A increased production of activated MMP2. DISCUSSION: Detection of CCL17 only in GCNIS tumours may reflect a change in Sertoli cell phenotype to a less mature state. Stimulation of MMP2 activity by activin A in TCam-2 cells suggests activin influences TGCT by modulating the tumour niche. CONCLUSION: This knowledge provides a basis for understanding how physiological changes that influence activin/TGF-ß superfamily signalling may alter germ cell fate.

A history of undescended testes in young men with Klinefelter syndrome does not reduce the chances for successful microsurgical testicular sperm extraction.

Klinefelter syndrome (KS) and undescended testes (UDT) are known etiologies for non-obstructive azoospermia (NOA), and coexistence of both etiologies is not uncommon. Patients with both KS and a history of UDT are therefore considered to have extremely reduced chances for paternity. We aimed to analyze the impact of previous surgically corrected unilateral or bilateral UDT on sperm retrieval rates (SRRs) by microsurgical testicular sperm extraction (mTESE) in azoospermic men with KS. Age, testicular volumes, and hypothalamo-pituitary-gonadal axis function were investigated in relation to SRRs in 29 non-mosaic KS patients (47,XXY) with a history of UDT (group 1) who underwent mTESE between 2008 and 2016 in our center and compared to the data of age- and serum testosterone-matched non-mosaic KS controls with eutopic testes at birth (group 2), and to those of 51 men with NOA and a normal male karyotype (46,XY), but previous UDT (group 3). SRRs in KS patients with surgically corrected UDT during childhood were comparable to SRRs of KS patients with eutopic testes at birth: 31% (35% in unilateral and 22% in bilateral UDT) vs. 38% (p = 0.581). SRRs and Leydig cell function in group 1 were negatively correlated with age. Significantly higher SRRs (66%) were found in euploid azoospermic men with surgically corrected UDT (p < 0.001). A history of UDT does not preclude chances for future fatherhood in young azoospermic males with KS. In one of three men with previous unilateral UDT and in one of 4-5 in those with previous bilateral UDT, spermatozoa can be harvested by mTESE during late adolescence or young adulthood for immediate or future use in assisted reproduction.