Toward clinical exomes in diagnostics and management of male infertility.

Infertility, affecting ∼10% of men, is predominantly caused by primary spermatogenic failure (SPGF). We screened likely pathogenic and pathogenic (LP/P) variants in 638 candidate genes for male infertility in 521 individuals presenting idiopathic SPGF and 323 normozoospermic men in the ESTAND cohort. Molecular diagnosis was reached for 64 men with SPGF (12%), with findings in 39 genes (6%). The yield did not differ significantly between the subgroups with azoospermia (20/185, 11%), oligozoospermia (18/181, 10%), and primary cryptorchidism with SPGF (26/155, 17%). Notably, 19 of 64 LP/P variants (30%) identified in 28 subjects represented recurrent findings in this study and/or with other male infertility cohorts. NR5A1 was the most frequently affected gene, with seven LP/P variants in six SPGF-affected men and two normozoospermic men. The link to SPGF was validated for recently proposed candidate genes ACTRT1, ASZ1, GLUD2, GREB1L, LEO1, RBM5, ROS1, and TGIF2LY. Heterozygous truncating variants in BNC1, reported in female infertility, emerged as plausible causes of severe oligozoospermia. Data suggested that several infertile men may present congenital conditions with less pronounced or pleiotropic phenotypes affecting the development and function of the reproductive system. Genes regulating the hypothalamic-pituitary-gonadal axis were affected in >30% of subjects with LP/P variants. Six individuals had more than one LP/P variant, including five with two findings from the gene panel. A 4-fold increased prevalence of cancer was observed in men with genetic infertility compared to the general male population (8% vs. 2%; p = 4.4 × 10-3). Expanding genetic testing in andrology will contribute to the multidisciplinary management of SPGF.

Meiotic Chromosome Structure, the Synaptonemal Complex, and Infertility.

In meiosis, homologous chromosome synapsis is mediated by a supramolecular protein structure, the synaptonemal complex (SC), that assembles between homologous chromosome axes. The mammalian SC comprises at least eight largely coiled-coil proteins that interact and self-assemble to generate a long, zipper-like structure that holds homologous chromosomes in close proximity and promotes the formation of genetic crossovers and accurate meiotic chromosome segregation. In recent years, numerous mutations in human SC genes have been associated with different types of male and female infertility. Here, we integrate structural information on the human SC with mouse and human genetics to describe the molecular mechanisms by which SC mutations can result in human infertility. We outline certain themes in which different SC proteins are susceptible to different types of disease mutation and how genetic variants with seemingly minor effects on SC proteins may act as dominant-negative mutations in which the heterozygous state is pathogenic.

Whole-exome sequencing improves the diagnosis and care of men with non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is a severe and frequent cause of male infertility, often treated by testicular sperm extraction followed by intracytoplasmic sperm injection. The aim of this study is to improve the genetic diagnosis of NOA, by identifying new genes involved in human NOA and to better assess the chances of successful sperm extraction according to the individual's genotype. Exome sequencing was performed on 96 NOA-affected individuals negative for routine genetic tests. Bioinformatics analysis was limited to a panel of 151 genes selected as known causal or candidate genes for NOA. Only highly deleterious homozygous or hemizygous variants were retained as candidates. A likely causal defect was identified in 16 genes in a total of 22 individuals (23%). Six genes had not been described in man (DDX25, HENMT1, MCMDC2, MSH5, REC8, TDRKH) and 10 were previously reported (C14orf39, DMC1, FANCM, GCNA, HFM1, MCM8, MEIOB, PDHA2, TDRD9, TERB1). Seven individuals had defects in genes from piwi or DNA repair pathways, three in genes involved in post-meiotic maturation, and 12 in meiotic processes. Interestingly, all individuals with defects in meiotic genes had an unsuccessful sperm retrieval, indicating that genetic diagnosis prior to TESE could help identify individuals with low or null chances of successful sperm retrieval and thus avoid unsuccessful surgeries.

The piRNA-pathway factor FKBP6 is essential for spermatogenesis but dispensable for control of meiotic LINE-1 expression in humans.

Infertility affects around 7% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with SPGF and detected rare bi-allelic loss-of-function variants in FKBP6 in five additional persons. All six individuals had no or extremely few sperm in the ejaculate, which were not suitable for medically assisted reproduction. Evaluation of testicular tissue revealed an arrest at the stage of round spermatids. Lack of FKBP6 expression in the testis was confirmed by RT-qPCR and immunofluorescence staining. In mice, Fkbp6 is essential for spermatogenesis and has been described as being involved in piRNA biogenesis and formation of the synaptonemal complex (SC). We did not detect FKBP6 as part of the SC in normal human spermatocytes, but small RNA sequencing revealed that loss of FKBP6 severely impacted piRNA levels, supporting a role for FKBP6 in piRNA biogenesis in humans. In contrast to findings in piRNA-pathway mouse models, we did not detect an increase in LINE-1 expression in men with pathogenic FKBP6 variants. Based on our findings, FKBP6 reaches a "strong" level of evidence for being associated with male infertility according to the ClinGen criteria, making it directly applicable for clinical diagnostics. This will improve patient care by providing a causal diagnosis and will help to predict chances for successful surgical sperm retrieval.

Large-scale analyses of the X chromosome in 2,354 infertile men discover recurrently affected genes associated with spermatogenic failure.

Although the evolutionary history of the X chromosome indicates its specialization in male fitness, its role in spermatogenesis has largely been unexplored. Currently only three X chromosome genes are considered of moderate-definitive diagnostic value. We aimed to provide a comprehensive analysis of all X chromosome-linked protein-coding genes in 2,354 azoospermic/cryptozoospermic men from four independent cohorts. Genomic data were analyzed and compared with data in normozoospermic control individuals and gnomAD. While updating the clinical significance of known genes, we propose 21 recurrently mutated genes strongly associated with and 34 moderately associated with azoospermia/cryptozoospermia not previously linked to male infertility (novel). The most frequently affected prioritized gene, RBBP7, was found mutated in ten men across all cohorts, and our functional studies in Drosophila support its role in germ stem cell maintenance. Collectively, our study represents a significant step towards the definition of the missing genetic etiology in idiopathic severe spermatogenic failure and significantly reduces the knowledge gap of X-linked genetic causes of azoospermia/cryptozoospermia contributing to the development of future diagnostic gene panels.

STK33 Phosphorylates Fibrous Sheath Protein AKAP3/4 to Regulate Sperm Flagella Assembly in Spermiogenesis.

Spermatogenesis defects are important for male infertility; however, the etiology and pathogenesis are still unknown. Herein, we identified two loss-of-function mutations of STK33 in seven individuals with non-obstructive azoospermia. Further functional studies of these frameshift and nonsense mutations revealed that Stk33-/KI male mice were sterile, and Stk33-/KI sperm were abnormal with defects in the mitochondrial sheath, fibrous sheath, outer dense fiber, and axoneme. Stk33KI/KI male mice were subfertile and had oligoasthenozoospermia. Differential phosphoproteomic analysis and in vitro kinase assay identified novel phosphorylation substrates of STK33, fibrous sheath components A-kinase anchoring protein 3 and A-kinase anchoring protein 4, whose expression levels decreased in testis after deletion of Stk33. STK33 regulated the phosphorylation of A-kinase anchoring protein 3/4, affected the assembly of fibrous sheath in the sperm, and played an essential role in spermiogenesis and male infertility.

Germ Cell-Specific Proteins AKAP4 and ASPX Facilitate Identification of Rare Spermatozoa in Non-Obstructive Azoospermia.

Non-obstructive azoospermia (NOA), the most severe form of male infertility, could be treated with intracytoplasmic sperm injection, providing spermatozoa were retrieved with the microdissection testicular sperm extraction (mTESE). We hypothesized that testis-specific and germ cell-specific proteins would facilitate flow cytometry-assisted identification of rare spermatozoa in semen cell pellets of NOA patients, thus enabling non-invasive diagnostics prior to mTESE. Data mining, targeted proteomics, and immunofluorescent microscopy identified and verified a panel of highly testis-specific proteins expressed at the continuum of germ cell differentiation. Late germ cell-specific proteins AKAP4_HUMAN and ASPX_HUMAN (ACRV1 gene) revealed exclusive localization in spermatozoa tails and acrosomes, respectively. A multiplex imaging flow cytometry assay facilitated fast and unambiguous identification of rare but morphologically intact AKAP4+/ASPX+/Hoechst+ spermatozoa within debris-laden semen pellets of NOA patients. While the previously suggested markers for spermatozoa retrieval suffered from low diagnostic specificity, the multistep gating strategy and visualization of AKAP4+/ASPX+/Hoechst+ cells with elongated tails and acrosome-capped nuclei facilitated fast and unambiguous identification of the mature intact spermatozoa. AKAP4+/ASPX+/Hoechst+ assay may emerge as a noninvasive test to predict retrieval of morphologically intact spermatozoa by mTESE, thus improving diagnostics and treatment of severe forms of male infertility.

Whole transcriptome analysis to identify non-coding RNA regulators and hub genes in sperm of non-obstructive azoospermia by microarray, single-cell RNA sequencing, weighted gene co-expression network analysis, and mRNA-miRNA-lncRNA interaction analysis.

BACKGROUND: The issue of male fertility is becoming increasingly common due to genetic differences inherited over generations. Gene expression and evaluation of non-coding RNA (ncRNA), crucial for sperm development, are significant factors. This gene expression can affect sperm motility and, consequently, fertility. Understanding the intricate protein interactions that play essential roles in sperm differentiation and development is vital. This knowledge could lead to more effective treatments and interventions for male infertility. MATERIALS AND METHODS: Our research aim to identify new and key genes and ncRNA involved in non-obstructive azoospermia (NOA), improving genetic diagnosis and offering more accurate estimates for successful sperm extraction based on an individual's genotype. RESULTS: We analyzed the transcript of three NOA patients who tested negative for genetic sperm issues, employing comprehensive genome-wide analysis of approximately 50,000 transcript sequences using microarray technology. This compared gene expression profiles between NOA sperm and normal sperm. We found significant gene expression differences: 150 genes were up-regulated, and 78 genes were down-regulated, along with 24 ncRNAs up-regulated and 13 ncRNAs down-regulated compared to normal conditions. By cross-referencing our results with a single-cell genomics database, we identified overexpressed biological process terms in differentially expressed genes, such as "protein localization to endosomes" and "xenobiotic transport." Overrepresented molecular function terms in up-regulated genes included "voltage-gated calcium channel activity," "growth hormone-releasing hormone receptor activity," and "sialic acid transmembrane transporter activity." Analysis revealed nine hub genes associated with NOA sperm: RPL34, CYB5B, GOL6A6, LSM1, ARL4A, DHX57, STARD9, HSP90B1, and VPS36. CONCLUSIONS: These genes and their interacting proteins may play a role in the pathophysiology of germ cell abnormalities and infertility.

Homozygous mutations in C14orf39/SIX6OS1 cause non-obstructive azoospermia and premature ovarian insufficiency in humans.

Human infertility is a multifactorial disease that affects 8%-12% of reproductive-aged couples worldwide. However, the genetic causes of human infertility are still poorly understood. Synaptonemal complex (SC) is a conserved tripartite structure that holds homologous chromosomes together and plays an indispensable role in the meiotic progression. Here, we identified three homozygous mutations in the SC coding gene C14orf39/SIX6OS1 in infertile individuals from different ethnic populations by whole-exome sequencing (WES). These mutations include a frameshift mutation (c.204_205del [p.His68Glnfs∗2]) from a consanguineous Pakistani family with two males suffering from non-obstructive azoospermia (NOA) and one female diagnosed with premature ovarian insufficiency (POI) as well as a nonsense mutation (c.958G>T [p.Glu320∗]) and a splicing mutation (c.1180-3C>G) in two unrelated Chinese men (individual P3907 and individual P6032, respectively) with meiotic arrest. Mutations in C14orf39 resulted in truncated proteins that retained SYCE1 binding but exhibited impaired polycomplex formation between C14ORF39 and SYCE1. Further cytological analyses of meiosis in germ cells revealed that the affected familial males with the C14orf39 frameshift mutation displayed complete asynapsis between homologous chromosomes, while the affected Chinese men carrying the nonsense or splicing mutation showed incomplete synapsis. The phenotypes of NOA and POI in affected individuals were well recapitulated by Six6os1 mutant mice carrying an analogous mutation. Collectively, our findings in humans and mice highlight the conserved role of C14ORF39/SIX6OS1 in SC assembly and indicate that the homozygous mutations in C14orf39/SIX6OS1 described here are responsible for infertility of these affected individuals, thus expanding our understanding of the genetic basis of human infertility.

Comparative single-cell analysis of biopsies clarifies pathogenic mechanisms in Klinefelter syndrome.

Klinefelter syndrome (KS), also known as 47, XXY, is characterized by a distinct set of physiological abnormalities, commonly including infertility. The molecular basis for Klinefelter-related infertility is still unclear, largely because of the cellular complexity of the testis and the intricate endocrine and paracrine signaling that regulates spermatogenesis. Here, we demonstrate an analysis framework for dissecting human testis pathology that uses comparative analysis of single-cell RNA-sequencing data from the biopsies of 12 human donors. By comparing donors from a range of ages and forms of infertility, we generate gene expression signatures that characterize normal testicular function and distinguish clinically distinct forms of male infertility. Unexpectedly, we identified a subpopulation of Sertoli cells within multiple individuals with KS that lack transcription from the XIST locus, and the consequence of this is increased X-linked gene expression compared to all other KS cell populations. By systematic assessment of known cell signaling pathways, we identify 72 pathways potentially active in testis, dozens of which appear upregulated in KS. Altogether our data support a model of pathogenic changes in interstitial cells cascading from loss of X inactivation in pubertal Sertoli cells and nominate dosage-sensitive factors secreted by Sertoli cells that may contribute to the process. Our findings demonstrate the value of comparative patient analysis in mapping genetic mechanisms of disease and identify an epigenetic phenomenon in KS Sertoli cells that may prove important for understanding causes of infertility and sex chromosome evolution.

A homozygous missense variant in YTHDC2 induces azoospermia in two siblings.

Male infertility is a complex multifactorial reproductive disorder with highly heterogeneous phenotypic presentations. Azoospermia is a medically non-manageable cause of male infertility affecting ∼1% of men. Precise etiology of azoospermia is not known in approximately three-fourth of the cases. To explore the genetic basis of azoospermia, we performed whole exome sequencing in two non-obstructive azoospermia affected siblings from a consanguineous Pakistani family. Bioinformatic filtering and segregation analysis of whole exome sequencing data resulted in the identification of a rare homozygous missense variant (c.962G>C, p. Arg321Thr) in YTHDC2, segregating with disease in the family. Structural analysis of the missense variant identified in our study and two previously reported functionally characterized missense changes (p. Glu332Gln and p. His327Arg) in mice showed that all these three variants may affect Mg2+ binding ability and helicase activity of YTHDC2. Collectively, our genetic analyses and experimental observations revealed that missense variant of YTHDC2 can induce azoospermia in humans. These findings indicate the important role of YTHDC2 deficiency for azoospermia and will provide important guidance for genetic counseling of male infertility.

Profiling of circulating extracellular vesicle microRNAs reveals diagnostic potential and pathways in non-obstructive and obstructive azoospermia.

The accurate diagnosis of non-obstructive azoospermia (NOA) and obstructive azoospermia (OA) is crucial for selecting appropriate clinical treatments. This study aimed to investigate the pivotal role of miRNAs in circulating plasma extracellular vesicles (EVs) in distinguishing between NOA and OA, as well as uncovering the signaling pathways involved in azoospermia pathogenesis. In this study, differential expression of EV miR-513c-5p and miR-202-5p was observed between NOA and OA patients, while the selenocompound metabolism pathway could be affected in azoospermia through Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The predictive power of these microRNAs was evaluated using ROC-AUC analysis, demonstrating promising sensitivity, specificity, and area under the curve values. A binomial regression equation incorporating circulating plasma levels of EVs miR-202-5p and miR-513c-5p along with follicle-stimulating hormone was calculated to provide a clinically applicable method for diagnosing NOA and OA. This study presents a potentially non-invasive testing approach for distinguishing between NOA and OA, offering a possibly valuable tool for clinical practice.

Dysregulation of MTFR2, ATP5IF1 and BAK1 in Sertoli cells relates to idiopathic non-obstructive azoospermia via inhibiting mitochondrial fission and inducing mitochondrial dysfunction†.

Non-obstructive azoospermia affects more than 10% of infertile men with over 70% patients are idiopathic with uncharacterized molecular mechanisms, which is referred as idiopathic non-obstructive azoospermia. In this study, we checked the morphology of Sertoli cell mitochondria in testis biopsies from patients with idiopathic non-obstructive azoospermia and patients with obstructive azoospermia who have normal spermiogenesis. The expression of 104 genes controlling mitochondria fission and fusion were analyzed in three gene expression datasets including a total of 60 patients with non-obstructive azoospermia. The levels of 7 candidate genes were detected in testis biopsies from 38 patients with idiopathic non-obstructive azoospermia and 24 patients with obstructive azoospermia who have normal spermatogenesis by RT-qPCR. Cell viability, apoptosis, mitochondria membrane potential, adenosine triphosphate production, oxygen consumption, and mitochondria morphology were examined in primary human Sertoli cells. Mouse spermatogonial stem cells were used to detect the cell supporting capacity of Sertoli cells. We observed that patients with idiopathic non-obstructive azoospermia had elongated mitochondria. MTFR2 and ATP5IF1 were downregulated, whereas BAK1 was upregulated in idiopathic non-obstructive azoospermia testis and Sertoli cells. Sertoli cells from patients with idiopathic non-obstructive azoospermia had reduced viability, mitochondria membrane potential, adenosine triphosphate production, oxygen consumption rate, glycolysis and increased apoptosis. Knockdown MTFR2 in Sertoli cells increased the mitochondria size. Knockdown ATP5IF1 did not change mitochondrial morphology but increased adenosine triphosphate hydrolysis. Overexpression of BAK1 reduced membrane potential and upregulated cell apoptosis. The dysregulation of all these three genes contributed to the dysfunction of Sertoli cells, which provides a clue for idiopathic non-obstructive azoospermia treatment.

Deleterious variants in X-linked RHOXF1 cause male infertility with oligo- and azoospermia.

Oligozoospermia and azoospermia are two common phenotypes of male infertility characterized by massive sperm defects owing to failure of spermatogenesis. The deleterious impact of candidate variants with male infertility is to be explored. In our study, we identified three hemizygous missense variants (c.388G>A: p.V130M, c.272C>T: p.A91V, and c.467C>T: p.A156V) and one hemizygous nonsense variant (c.478C>T: p.R160X) in the Rhox homeobox family member 1 gene (RHOXF1) in four unrelated cases from a cohort of 1201 infertile Chinese men with oligo- and azoospermia using whole-exome sequencing and Sanger sequencing. RHOXF1 was absent in the testicular biopsy of one patient (c.388G>A: p.V130M) whose histological analysis showed a phenotype of Sertoli cell-only syndrome. In vitro experiments indicated that RHOXF1 mutations significantly reduced the content of RHOXF1 protein in HEK293T cells. Specifically, the p.V130M, p.A156V, and p.R160X mutants of RHOXF1 also led to increased RHOXF1 accumulation in cytoplasmic particles. Luciferase assays revealed that p.V130M and p.R160X mutants may disrupt downstream spermatogenesis by perturbing the regulation of doublesex and mab-3 related transcription factor 1 (DMRT1) promoter activity. Furthermore, ICSI treatment could be beneficial in the context of oligozoospermia caused by RHOXF1 mutations. In conclusion, our findings collectively identified mutated RHOXF1 to be a disease-causing X-linked gene in human oligo- and azoospermia.

Timing of Testicular Biopsy in Relation to Oocyte Retrieval and the Outcomes of Intracytoplasmic Sperm Injection.

PURPOSE: We evaluate microscopic (micro) testicular sperm extraction (TESE) timing relative to oocyte retrieval on intracytoplasmic sperm injection outcome. MATERIALS AND METHODS: Couples with nonobstructive azoospermia who underwent intracytoplasmic sperm injection with freshly retrieved spermatozoa were analyzed based on whether micro-TESE was performed at least 1 day prior to oocyte retrieval (TESE-day-before group) or on the day of oocyte retrieval (TESE-day-of group). Embryology and clinical outcomes were compared. RESULTS: The percentage of patients who underwent a successful testicular sperm retrieval was significantly lower in the TESE-day-before cohort (62%) than in the TESE-day-of cohort (69%; odds ratio [OR] 1.4, 95% CI [1.1, 1.7], P < .001). The fertilization rate was also found to be significantly lower in the TESE-day-before group (45%) than in the TESE-day-of group (53%; OR 1.4, 95% CI [1.2, 1.7], P = .01). Although the association between the cleavage rate and TESE timing was not statistically significant, the implantation rate was found to be significantly higher in the day-before cohort (28%) than in the day-of cohort (22%; OR 0.7, 95% CI [0.6, 0.9], P = .01). Nevertheless, it was found that the clinical pregnancy and delivery rates were not statistically significantly associated with the TESE timing. CONCLUSIONS: Although sperm retrieval and fertilization rates were lower in the TESE-day-before cohort, the 2 cohorts showed comparable embryologic and clinical outcomes. Micro-TESE can be performed before oocyte harvesting to provide physicians ample time to decide between cancelling oocyte retrieval or retrieving oocytes for cryopreservation.

Whole genome sequencing identifies a homozygous splicing variant in TDRKH segregating with non-obstructive azoospermia in an Iranian family.

Non-obstructive azoospermia (NOA) resulting from primary spermatogenic failure represents one of the most severe forms of male infertility, largely because therapeutic options are very limited. Beyond their diagnostic value, genetic tests for NOA also hold prognostic potential. Specifically, genetic diagnosis enables the establishment of genotype-testicular phenotype correlations, which, in some cases, provide a negative predictive value for testicular sperm extraction (TESE), thereby preventing unnecessary surgical procedures. In this study, we employed whole-genome sequencing (WGS) to investigate two generations of an Iranian family with NOA and identified a homozygous splicing variant in TDRKH (NM_001083965.2: c.562-2A>T). TDRKH encodes a conserved mitochondrial membrane-anchored factor essential for piRNA biogenesis in germ cells. In Tdrkh knockout mice, de-repression of retrotransposons in germ cells leads to spermatogenic arrest and male infertility. Previously, our team reported TDRKH involvement in human NOA cases through the investigation of a North African cohort. This current study marks the second report of TDRKH's role in NOA and human male infertility, underscoring the significance of the piRNA pathway in spermatogenesis. Furthermore, across both studies, we demonstrated that men carrying TDRKH variants, similar to knockout mice, exhibit complete spermatogenic arrest, correlating with failed testicular sperm retrieval.

Novel MEIOB pathogenic variants including a homozygous non-canonical splicing variant, cause meiotic arrest and human non-obstructive azoospermia.

Non-obstructive azoospermia (NOA) is the most severe form of human male infertility, and the genetic causes of NOA with meiotic arrest remain largely unclear. In this study, we identified novel compound heterozygous MEIOB variants (c.814C > T: p.R272X and c.976G > A: p.A326T) and a previously undescribed homozygous non-canonical splicing variant of MEIOB (c.528 + 3A > C) in two NOA-affected individuals from two irrelevant Chinese families. MEIOB missense variant (p.A326T) significantly reduced protein abundance and nonsense variant (p.R272X) produced a truncated protein. Both of two variants impaired the MEIOB-SPATA22 interaction. The MEIOB non-canonical splicing variant resulted in whole Exon 6 skipping by minigene assay, which was predicted to produce a frameshift truncated protein (p.S111Rfs*32). Histological and immunostaining analysis indicated that both patients exhibited a similar phenotype as we previously reported in Meiob mutant mice, that is, absence of spermatids in seminiferous tubules and meiotic arrest. Our study identified three novel pathogenic variants of MEIOB in NOA patients, extending the mutation spectrum of the MEIOB and highlighting the contribution of meiotic recombination related genes in human fertility.

Semen analysis and reproductive hormones in boys with classical Hodgkin lymphoma treated according to the EuroNet-PHL-C2 protocol.

STUDY QUESTION: What is the impact of the EuroNet-PHL-C2 treatment for boys with classical Hodgkin lymphoma (cHL) on semen parameters? SUMMARY ANSWER: More than half of the patients (52%, n = 16/31) had oligozoospermia or azoospermia at 2 years from cHL diagnosis; particularly boys treated for advanced-stage cHL had low sperm counts and motility. WHAT IS KNOWN ALREADY: Chemotherapy and radiotherapy to the inguinal region or testes can impair spermatogenesis and result in reduced fertility. The EuroNet-PHL-C2 trial aims to minimize radiotherapy in standard childhood cHL treatment, by intensifying chemotherapy. The present study aims to assess the (gonadotoxic) impact of this treatment protocol on semen parameters and reproductive hormones in boys aged ≤18 years. STUDY DESIGN, SIZE, DURATION: This international, prospective, multi-centre cohort study was an add-on study to the randomized phase-3 EuroNet-PHL-C2 trial, where the efficacy of standard cHL treatment with OEPA-COPDAC-28 (OEPA: vincristine, etoposide, prednisone, and doxorubicin; COPDAC-28: cyclophosphamide, vincristine, prednisone, and dacarbazine) was compared to intensified OEPA-DECOPDAC-21 chemotherapy (DECOPDAC-21: COPDAC with additional doxorubicin and etoposide and 25% more cyclophosphamide). Patients were recruited between January 2017 and September 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: Eligibility criteria included male patients, diagnosed with classical HL before or at the age of 18 years, and treated according to the EuroNet-PHL-C2 protocol in any of the 18 participating sites in the Netherlands, Germany, Belgium, Czech Republic, and Austria. Sperm parameters (sperm concentration, progressive motility, sperm volume, and calculated total motile sperm count) were assessed at diagnosis and 2 years after diagnosis in (post)pubertal boys. Laboratory measurements (serum follicle-stimulating hormone (FSH) and inhibin B) were performed in samples drawn at diagnosis, during treatment (2-3 times), and at 2 years post-diagnosis, and (age-adjusted) analyses were conducted separately for pre-pubertal and (post)pubertal boys. Outcomes were compared between the treatment levels (TL1, TL2, and TL3) and consolidation treatment schemes (COPDAC-28 and DECOPDAC-21). MAIN RESULTS AND THE ROLE OF CHANCE: In total, 101 boys were included in the present analysis: 73 were (post)pubertal (median age 15.4 years, (IQR 14.4; 16.6), 10 TL1, 29 TL2, 34 TL3, 62% of TL2/3 patients received COPDAC-28) and 28 boys were pre-pubertal (median age 9.6 years (IQR 6.6; 11.4), 4 TL1, 7 TL2, 17 TL3, 38% of TL2/3 patients received COPDAC-28). The study included six boys who had received pelvic radiotherapy; none were irradiated in the inguinal or testicular area. At diagnosis, 48 (post)pubertal boys delivered semen for cryopreservation; 19 (40%) semen samples were oligospermic and 4 (8%) were azoospermic. Low sperm concentration (<15 mil/ml) appeared to be related to the HL disease itself, with a higher prevalence in boys who presented with B symptoms (76% vs 26%, aOR 2.3 (95% CI 1.0; 3.8), P = 0.001) compared to those without such symptoms. At 2 -years post-diagnosis, 31 boys provided semen samples for analysis, of whom 12 (39%) boys had oligozoospermia and 4 (13%) had azoospermia, while 22 boys (71%) had low total motile sperm counts (TMSC) (<20 mil). Specifically, the eight boys in the TL3 group treated with DECOPDAC-21 consolidation had low sperm counts and low progressive motility after 2 years (i.e. median sperm count 1.4 mil/ml (IQR <0.1; 5.3), n = 7 (88%), low sperm concentration, low median progressive motility 16.5% (IQR 0.0; 51.2), respectively). Age-adjusted serum FSH levels were significantly raised and inhibin B levels (and inhibin B:FSH ratios) were decreased during chemotherapy in (post)pubertal boys, with subsequent normalization in 80% (for FSH) and 60% (for inhibin B) of boys after 2 years. Only 4 out of the 14 (post)pubertal boys (29%) with low sperm concentrations after 2 years had elevated FSH (>7.6 IU/l), while 7 (50%) had low inhibin B levels (<100 ng/l). In pre-pubertal boys, reproductive hormones were low overall and remained relatively stable during chemotherapy. LIMITATIONS, REASONS FOR CAUTION: The present analyses included sperm and laboratory measurements up to 2 years post-diagnosis. Long-term reproductive outcomes and potential recovery of spermatogenesis remain unknown, while recovery was reported up to 5- or even 10-year post-chemotherapy in previous studies.Boys who were pre-pubertal at diagnosis were still too young and/or physically not able to deliver semen after 2 years and we could not assess a potential difference in gonadotoxicity according to pubertal state at the time of treatment. Overall, the statistical power of the analyses on sperm concentration and quality after 2 years was limited. WIDER IMPLICATIONS OF THE FINDINGS: Results of the semen analyses conducted among the 31 boys who had provided a semen sample at 2 years post-treatment were generally poor. However, additional long-term and adequately powered data are crucial to assess the potential recovery and clinical impact on fertility. The participating boys will be invited to deliver a semen sample after 5 years. Until these data become available, benefits of intensified chemotherapy in cHL treatment to reduce radiotherapy and lower risk for development of secondary tumours should be carefully weighed against potentially increased risk of other late effects, such as diminished fertility due to the increased chemotherapy burden. Boys with newly diagnosed cHL should be encouraged to deliver sperm for cryopreservation whenever possible. However, patients and clinicians should also realize that the overall state of disease and inflammatory milieu of cHL can negatively affect sperm quality and thereby reduce chance of successful fertility preservation. Furthermore, the measurement of FSH and inhibin B appears to be of low value in predicting low sperm quality at two years from cHL treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was funded by the Dutch charity foundation KiKa (project 257) that funds research on all forms of childhood cancer. C.M.-K., D.K., W.H.W., D.H., MC, A.U., and A.B. were involved in the development of the EuroNet-PHL-C2 regimen. The other authors declare no potential conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Systematic molecular analyses for 115 karyotypically normal men with isolated non-obstructive azoospermia.

STUDY QUESTION: Do copy-number variations (CNVs) in the azoospermia factor (AZF) regions and monogenic mutations play a major role in the development of isolated (non-syndromic) non-obstructive azoospermia (NOA) in Japanese men with a normal 46, XY karyotype? SUMMARY ANSWER: Deleterious CNVs in the AZF regions and damaging sequence variants in eight genes likely constitute at least 8% and approximately 8% of the genetic causes, respectively, while variants in other genes play only a minor role. WHAT IS KNOWN ALREADY: Sex chromosomal abnormalities, AZF-linked microdeletions, and monogenic mutations have been implicated in isolated NOA. More than 160 genes have been reported as causative/susceptibility/candidate genes for NOA. STUDY DESIGN, SIZE, DURATION: Systematic molecular analyses were conducted for 115 patients with isolated NOA and a normal 46, XY karyotype, who visited our hospital between 2017 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: We studied 115 unrelated Japanese patients. AZF-linked CNVs were examined using sequence-tagged PCR and multiplex ligation-dependent probe amplification, and nucleotide variants were screened using whole exome sequencing (WES). An optimized sequence kernel association test (SKAT-O), a gene-based association study using WES data, was performed to identify novel disease-associated genes in the genome. The results were compared to those of previous studies and our in-house control data. MAIN RESULTS AND THE ROLE OF CHANCE: Thirteen types of AZF-linked CNVs, including the hitherto unreported gr/gr triplication and partial AZFb deletion, were identified in 63 (54.8%) cases. When the gr/gr deletion, a common polymorphism in Japan, was excluded from data analyses, the total frequency of CNVs was 23/75 (30.7%). This frequency is higher than that of the reference data in Japan and China (11.1% and 14.7%, respectively). Known NOA-causative AZF-linked CNVs were found in nine (7.8%) cases. Rare damaging variants in known causative genes (DMRT1, PLK4, SYCP2, TEX11, and USP26) and hemizygous/multiple-heterozygous damaging variants in known spermatogenesis-associated genes (TAF7L, DNAH2, and DNAH17) were identified in nine cases (7.8% in total). Some patients carried rare damaging variants in multiple genes. SKAT-O detected no genes whose rare damaging variants were significantly accumulated in the patient group. LIMITATIONS, REASONS FOR CAUTION: The number of participants was relatively small, and the clinical information of each patient was fragmentary. Moreover, the pathogenicity of identified variants was assessed only by in silico analyses. WIDER IMPLICATIONS OF THE FINDINGS: This study showed that various AZF-linked CNVs are present in more than half of Japanese NOA patients. These results broadened the structural variations of AZF-linked CNVs, which should be considered for the molecular diagnosis of spermatogenic failure. Furthermore, the results of this study highlight the etiological heterogeneity and possible oligogenicity of isolated NOA. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by Grants from the Japan Society for the Promotion of Science (21K19283 and 21H0246), the Japan Agency for Medical Research and Development (22ek0109464h0003), the National Center for Child Health and Development, the Canon Foundation, the Japan Endocrine Society, and the Takeda Science Foundation. The results of this study were based on samples and patient data obtained from the International Center for Reproductive Medicine, Dokkyo Medical University Saitama Medical Center, Koshigaya, Japan. The authors have no conflicts of interest to disclose. TRIAL REGISTRATION NUMBER: N/A.