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Objective: To assess the association between ethnicity and fertility outcomes for men in a statewide cohort., Methods: We linked data from the Utah Population Database and Subfertility Health Assisted Reproduction and Environment database, to comprise a cohort of sub-fertile men who underwent semen analysis between 1998 and 2017 in Utah. A multivariable Cox proportional hazard model was constructed to understand the impact of ethnicity on fertility outcomes in our cohort., Results: A total of 11,363 men were included. 1039 (9.1%) were Hispanic. 39.7% of men in the lowest socioeconomic status group were Hispanic (P <.001). When controlling for demographic and clinical factors, the number of live births was reduced for Hispanic men (hazard ratios [HR] = 0.62 [0.57-0.67], P <.001). Though fertility treatment had a positive effect (HR 1.242 [1.085-1.421], P <.001), in competing risks models, Hispanic men were less likely to use fertility treatment (HR = 0.633 [0.526-0.762], P <.001)., Conclusion: Hispanic ethnicity is significantly associated with a lower likelihood of successful fertility outcomes in Utah. Hispanic men had nearly a 40% reduced likelihood of live births when controlling for sociodemographic factors. Our results indicate that, depending on age, Hispanic men have up to approximately 14 fewer live births per 100 men per year, pointing to a significant disparity in fertility outcomes in the state of Utah. Given 15.1% of Utah's population identifies as Hispanic and 18.7% of the United States population identifies as Hispanic on the 2020 Census, a better understanding of the association of ethnicity and fertility outcomes is imperative., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Physical activity is a first-line treatment for polycystic ovary syndrome (PCOS). Resistance or aerobic exercise improves metabolic complications, reproductive outcomes, and quality of life in PCOS. DNA methylation reprogramming during exercise may be the major modifier behind these changes. We sought to evaluate genome-wide DNA methylation changes after supervised resistance and aerobic exercise in women with PCOS. Exercises were performed in 56 women with PCOS (resistance, n  = 30; aerobic, n  = 26), for 16 weeks (wks), three times per week, in 50-minute to one-hour sessions. Anthropometric indices and hormonal and metabolic parameters were measured before and after training. Genome-wide leukocyte DNA methylation was analysed by Infinium Human MethylationEPIC 850K BeadChip microarrays (Illumina). Both resistance and aerobic exercise improved anthropometric indices, metabolic dysfunction, and hyperandrogenism in PCOS after the training programme, but no differences were observed between the two exercises. Resistance and aerobic exercise increased genome-wide DNA methylation, although resistance changed every category in the CpG island context (islands, shores, shelve, and open sea), whereas aerobic exercise altered CpG shores and the open sea. Using a stringent FDR (>40), 6 significantly differentially methylated regions (DMRs) were observed in the resistance exercise cohort and 14 DRMs in the aerobic cohort, all of which were hypermethylated. The increase in genome-wide DNA methylation may be related to the metabolic and hormonal changes observed in PCOS after resistance and aerobic exercise. Since the mammalian genome is hypermethylated globally to prevent genomic instability and ageing, resistance and aerobic exercise may promote health and longevity through environmentally induced epigenetic changes.

Dynein complexes are large, multi-unit assemblies involved in many biological processes via their critical roles in protein transport and axoneme motility. Using next-generation sequencing of infertile men presenting with low or no sperm in their ejaculates, we identified damaging variants in the dynein-related gene AXDND1. We thus hypothesised that AXDND1 is a critical regulator of male fertility. To test this hypothesis, we produced a knockout mouse model. Axdnd1 -/- males were sterile at all ages but presented with an evolving testis phenotype wherein they could undergo one round of histologically replete spermatogenesis followed by a rapid depletion of the seminiferous epithelium. Marker experiments identified a role for AXDND1 in maintaining the balance between differentiation-committed and self-renewing spermatogonial populations, resulting in disproportionate production of differentiating cells in the absence of AXDND1 and increased sperm production during initial spermatogenic waves. Moreover, long-term spermatogonial maintenance in the Axdnd1 knockout was compromised, ultimately leading to catastrophic germ cell loss, destruction of blood-testis barrier integrity and immune cell infiltration. In addition, sperm produced during the first wave of spermatogenesis were immotile due to abnormal axoneme structure, including the presence of ectopic vesicles and abnormalities in outer dense fibres and microtubule doublet structures. Sperm output was additionally compromised by a severe spermiation defect and abnormal sperm individualisation. Collectively these data identify AXDND1 as an atypical dynein complex-related protein with a role in protein/vesicle transport of relevance to spermatogonial function and sperm tail formation in mice and humans. This study underscores the importance of studying the consequences of gene loss-of-function on both the establishment and maintenance of male fertility., (© 2024. The Author(s).)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

RASopathies are syndromes caused by congenital defects in the Ras/mitogen-activated protein kinase (MAPK) pathway genes, with a population prevalence of 1 in 1,000. Patients are typically identified in childhood based on diverse characteristic features, including cryptorchidism (CR) in >50% of affected men. As CR predisposes to spermatogenic failure (SPGF; total sperm count per ejaculate 0-39 million), we hypothesized that men seeking infertility management include cases with undiagnosed RASopathies. Likely pathogenic or pathogenic (LP/P) variants in 22 RASopathy-linked genes were screened in 521 idiopathic SPGF patients (including 155 CR cases) and 323 normozoospermic controls using exome sequencing. All 844 men were recruited to the ESTonian ANDrology (ESTAND) cohort and underwent identical andrological phenotyping. RASopathy-specific variant interpretation guidelines were used for pathogenicity assessment. LP/P variants were identified in PTPN11 (two), SOS1 (three), SOS2 (one), LZTR1 (one), SPRED1 (one), NF1 (one), and MAP2K1 (one). The findings affected six of 155 cases with CR and SPGF, three of 366 men with SPGF only, and one (of 323) normozoospermic subfertile man. The subgroup "CR and SPGF" had over 13-fold enrichment of findings compared to controls (3.9% vs. 0.3%; Fisher's exact test, p = 5.5 × 10 -3 ). All ESTAND subjects with LP/P variants in the Ras/MAPK pathway genes presented congenital genitourinary anomalies, skeletal and joint conditions, and other RASopathy-linked health concerns. Rare forms of malignancies (schwannomatosis and pancreatic and testicular cancer) were reported on four occasions. The Genetics of Male Infertility Initiative (GEMINI) cohort (1,416 SPGF cases and 317 fertile men) was used to validate the outcome. LP/P variants in PTPN11 (three), LZTR1 (three), and MRAS (one) were identified in six SPGF cases (including 4/31 GEMINI cases with CR) and one normozoospermic man. Undiagnosed RASopathies were detected in total for 17 ESTAND and GEMINI subjects, 15 SPGF patients (10 with CR), and two fertile men. Affected RASopathy genes showed high expression in spermatogenic and testicular somatic cells. In conclusion, congenital defects in the Ras/MAPK pathway genes represent a new congenital etiology of syndromic male infertility. Undiagnosed RASopathies were especially enriched among patients with a history of cryptorchidism. Given the relationship between RASopathies and other conditions, infertile men found to have this molecular diagnosis should be evaluated for known RASopathy-linked health concerns, including specific rare malignancies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Juchnewitsch, Pomm, Dutta, Tamp, Valkna, Lillepea, Mahyari, Tjagur, Belova, Kübarsepp, Castillo-Madeen, Riera-Escamilla, Põlluaas, Nagirnaja, Poolamets, Vihljajev, Sütt, Versbraegen, Papadimitriou, McLachlan, Jarvi, Schlegel, Tennisberg, Korrovits, Vigh-Conrad, O’Bryan, Aston, Lenaerts, Conrad, Kasak, Punab and Laan.)

Background: Single-cell RNA-seq (scRNA-Seq) has been widely adopted to study gene expression of the human testis. Several datasets of scRNA-Seq from human testis have been generated from different groups processed with different informatics pipelines. An integrated atlas of scRNA-Seq expression constructed from multiple donors, developmental ages, and fertility states would be widely useful for the testis research community., Objective: To describe the generation and use of the human infertility single-cell testis atlas (HISTA), an interactive web tool for understanding human spermatogenesis through scRNA-Seq analysis., Methods: We obtained scRNA-Seq datasets derived from 12 donors, including healthy adult controls, juveniles, and several infertility cases, and reprocessed these data using methods to remove batch effects. Using Shiny, an open-source environment for data visualization, we created numerous interactive tools for exploring the data, some of which support simple statistical hypothesis testing. We used the resulting HISTA browser and its underlying data to demonstrate HISTA's value for testis researchers., Results: A primary application of HISTA is to search by a single gene or a set of genes; thus, we present various analyses that quantify and visualize gene expression across the testis cells and pathology. HISTA also contains machine-learning-derived gene modules ("components") that capture the entire transcriptional landscape of the testis tissue. We show how the use of these components can simplify the highly complex data in HISTA and assist with the interpretation of genes with unknown functions. Finally, we demonstrate the diverse ways HISTA can be used for new data analysis, including hypothesis testing., Discussion and Conclusions: HISTA is a research environment that can help scientists organize and understand the high-dimensional transcriptional landscape of the human testis. HISTA has already contributed to published testis research and can be updated as needed with input from the research community or downloaded and modified for individual needs., (© 2024 American Society of Andrology and European Academy of Andrology.)

Study Question: Can we simultaneously assess risk for multiple cancers to identify familial multicancer patterns in families of azoospermic and severely oligozoospermic men?, Summary Answer: Distinct familial cancer patterns were observed in the azoospermia and severe oligozoospermia cohorts, suggesting heterogeneity in familial cancer risk by both type of subfertility and within subfertility type., What Is Known Already: Subfertile men and their relatives show increased risk for certain cancers including testicular, thyroid, and pediatric., Study Design, Size, Duration: A retrospective cohort of subfertile men (N = 786) was identified and matched to fertile population controls (N = 5674). Family members out to third-degree relatives were identified for both subfertile men and fertile population controls (N = 337 754). The study period was 1966-2017. Individuals were censored at death or loss to follow-up, loss to follow-up occurred if they left Utah during the study period., Participants/materials, Setting, Methods: Azoospermic (0 × 106/mL) and severely oligozoospermic (<1.5 × 106/mL) men were identified in the Subfertility Health and Assisted Reproduction and the Environment cohort (SHARE). Subfertile men were age- and sex-matched 5:1 to fertile population controls and family members out to third-degree relatives were identified using the Utah Population Database (UPDB). Cancer diagnoses were identified through the Utah Cancer Registry. Families containing ≥10 members with ≥1 year of follow-up 1966-2017 were included (azoospermic: N = 426 families, 21 361 individuals; oligozoospermic: N = 360 families, 18 818 individuals). Unsupervised clustering based on standardized incidence ratios for 34 cancer phenotypes in the families was used to identify familial multicancer patterns; azoospermia and severe oligospermia families were assessed separately., Main Results and the Role of Chance: Compared to control families, significant increases in cancer risks were observed in the azoospermia cohort for five cancer types: bone and joint cancers hazard ratio (HR) = 2.56 (95% CI = 1.48-4.42), soft tissue cancers HR = 1.56 (95% CI = 1.01-2.39), uterine cancers HR = 1.27 (95% CI = 1.03-1.56), Hodgkin lymphomas HR = 1.60 (95% CI = 1.07-2.39), and thyroid cancer HR = 1.54 (95% CI = 1.21-1.97). Among severe oligozoospermia families, increased risk was seen for three cancer types: colon cancer HR = 1.16 (95% CI = 1.01-1.32), bone and joint cancers HR = 2.43 (95% CI = 1.30-4.54), and testis cancer HR = 2.34 (95% CI = 1.60-3.42) along with a significant decrease in esophageal cancer risk HR = 0.39 (95% CI = 0.16-0.97). Thirteen clusters of familial multicancer patterns were identified in families of azoospermic men, 66% of families in the azoospermia cohort showed population-level cancer risks, however, the remaining 12 clusters showed elevated risk for 2-7 cancer types. Several of the clusters with elevated cancer risks also showed increased odds of cancer diagnoses at young ages with six clusters showing increased odds of adolescent and young adult (AYA) diagnosis [odds ratio (OR) = 1.96-2.88] and two clusters showing increased odds of pediatric cancer diagnosis (OR = 3.64-12.63). Within the severe oligozoospermia cohort, 12 distinct familial multicancer clusters were identified. All 12 clusters showed elevated risk for 1-3 cancer types. An increase in odds of cancer diagnoses at young ages was also seen in five of the severe oligozoospermia familial multicancer clusters, three clusters showed increased odds of AYA diagnosis (OR = 2.19-2.78) with an additional two clusters showing increased odds of a pediatric diagnosis (OR = 3.84-9.32)., Limitations, Reasons for Caution: Although this study has many strengths, including population data for family structure, cancer diagnoses and subfertility, there are limitations. First, semen measures are not available for the sample of fertile men. Second, there is no information on medical comorbidities or lifestyle risk factors such as smoking status, BMI, or environmental exposures. Third, all of the subfertile men included in this study were seen at a fertility clinic for evaluation. These men were therefore a subset of the overall population experiencing fertility problems and likely represent those with the socioeconomic means for evaluation by a physician., Wider Implications of the Findings: This analysis leveraged unique population-level data resources, SHARE and the UPDB, to describe novel multicancer clusters among the families of azoospermic and severely oligozoospermic men. Distinct overall multicancer risk and familial multicancer patterns were observed in the azoospermia and severe oligozoospermia cohorts, suggesting heterogeneity in cancer risk by type of subfertility and within subfertility type. Describing families with similar cancer risk patterns provides a new avenue to increase homogeneity for focused gene discovery and environmental risk factor studies. Such discoveries will lead to more accurate risk predictions and improved counseling for patients and their families., Study Funding/competing Interest(s): This work was funded by GEMS: Genomic approach to connecting Elevated germline Mutation rates with male infertility and Somatic health (Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD): R01 HD106112). The authors have no conflicts of interest relevant to this work., Trial Registration Number: N/A., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

In the male mouse germ line, PIWI-interacting RNAs (piRNAs), bound by the PIWI protein MIWI2 (PIWIL4), guide DNA methylation of young active transposons through SPOCD1. However, the underlying mechanisms of SPOCD1-mediated piRNA-directed transposon methylation and whether this pathway functions to protect the human germ line remain unknown. We identified loss-of-function variants in human SPOCD1 that cause defective transposon silencing and male infertility. Through the analysis of these pathogenic alleles, we discovered that the uncharacterized protein C19ORF84 interacts with SPOCD1. DNMT3C, the DNA methyltransferase responsible for transposon methylation, associates with SPOCD1 and C19ORF84 in fetal gonocytes. Furthermore, C19ORF84 is essential for piRNA-directed DNA methylation and male mouse fertility. Finally, C19ORF84 mediates the in vivo association of SPOCD1 with the de novo methylation machinery. In summary, we have discovered a conserved role for the human piRNA pathway in transposon silencing and C19ORF84, an uncharacterized protein essential for orchestrating piRNA-directed DNA methylation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

An increasing number of genes are being described in the context of non-syndromic male infertility. Linking the underlying genetic causes of non-syndromic male infertility with clinical data from patients is important to establish new genotype-phenotype correlations. This process can be facilitated by using universal nomenclature, but no standardized vocabulary is available in the field of non-syndromic male infertility. The International Male Infertility Genomics Consortium aimed at filling this gap, providing a standardized vocabulary containing nomenclature based on the Human Phenotype Ontology (HPO). The "HPO tree" was substantially revised compared with the previous version and is based on the clinical work-up of infertile men, including physical examination and hormonal assessment. Some causes of male infertility can already be suspected based on the patient's clinical history, whereas in other instances, a testicular biopsy is needed for diagnosis. We assembled 49 HPO terms that are linked in a logical hierarchy and showed examples of morphological features of spermatozoa and testicular histology of infertile men with identified genetic diagnoses to describe the phenotypes. This work will help to record patients' phenotypes systematically and facilitate communication between geneticists and andrologists. Collaboration across institutions will improve the identification of patients with the same phenotypes, which will promote the discovery of novel genetic causes for non-syndromic male infertility., (© 2023. Springer Nature Limited.)

Objective: To determine whether severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection affects male reproductive health, considering the many potential factors that contribute to declines in male fertility on a semiglobal scale., Design: In total, 64 human semen samples-32 treatment and 32 control-were laboratory processed and bioinformatically analyzed to assess differences in DNA methylation patterns. Implementing multiple bioinformatic tools, the analyses conducted will elicit between-group differences with respect to epigenetic age, epigenetic instability, semiglobal, and regional methylation, in addition to methylation patterns as a function of time since infection., Setting: University hospital., Patients: The study cohort of 64 individuals was drawn from a larger population of 94 volunteer participants recruited at the Human Reproduction Center at the Clinical Hospital of the Ribeirao Preto Medical School-University of São Paulo between June 2021 and January 2022 as well as in accordance with the ethical guidelines established by the Declaration of Helsinki., Intervention: Exposure to SARS-CoV-2., Main Outcome Measure(s): Effects on male reproductive health were reported as differences in DNA methylation measured using an array. Mean β values at key regulatory loci for human spermatocytes were analyzed and compared between groups. Further analysis of β values using epigenetic age, instability, semiglobal, and regional methylation tools provided an analysis with substantial breadth and depth., Results: In all analyses, there were no differences between groups. Considering these results, it can be inferred that infection with SARS-CoV-2 does not alter the epigenome of human spermatocytes in significant and/or persistent ways. Tangentially, these data also suggest that human male reproductive health is minimally altered by the virus, or that it is altered in a way that is independent of epigenetic programming., Conclusion: Infection with SARS-CoV-2 has been reportedly associated with alterations in male fertility. This study asserts that such alterations do not have an epigenetic basis but are likely a result of concomitant symptomatology, i.e., fever and inflammation. Across the multiple bioinformatic analyses conducted, the results of this test did not detect any differences in DNA methylation patterns between coronavirus disease 2019 and noncoronavirus disease semen donor groups., Competing Interests: Declaration of interests I.S. has nothing to disclose. M.R.S. reports funding from Instituto Nacional de Ciência e Tecnologia – Hormônio e Saúde da Mulher (CNPq), number 176841/2022-9 Clinical data and human semen samples to the research for the submitted work. C.F. has nothing to disclose. R.M.D.R. reports funding from Instituto Nacional de Ciência e Tecnologia – Hormônio e Saúde da Mulher (CNPq), number 176841/2022-9 Clinical data and human semen samples to the research for the submitted work. K.I.A. reports patents for Methods of identifying male fertility status and embryo quality and Systems and methods for determining the impact of age-related changes in sperm epigenome on offspring phenotype outside the submitted work. R.P.S. has nothing to disclose. T.G.J. has nothing to disclose., (Copyright © 2023 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Objective: To investigate the power of DNA methylation variability in sperm cells in assessing male fertility potential., Design: Retrospective cohort., Setting: Fertility care centers., Patients: Male patients seeking infertility treatment and fertile male sperm donors., Intervention: None., Main Outcome Measures: Sperm DNA methylation data from 43 fertile sperm donors were analyzed and compared with the data from 1344 men seeking fertility assessment or treatment. Methylation at gene promoters with the least variable methylation in fertile patients was used to create 3 categories of promoter dysregulation in the infertility treatment cohort: poor, average, and excellent sperm quality., Results: After controlling for female factors, there were significant differences in intrauterine insemination pregnancy and live birth outcomes between the poor and excellent groups across a cumulative average of 2-3 cycles: 19.4% vs. 51.7% (P=.008) and 19.4% vs. 44.8% (P=.03), respectively. Live birth outcomes from in vitro fertilization, primarily with intracytoplasmic sperm injection, were not found to be significantly different among any of the 3 groups., Conclusion: Methylation variability in a panel of 1233 gene promoters could augment the predictive ability of semen analysis and be a reliable biomarker for assessing intrauterine insemination outcomes. In vitro fertilization with intracytoplasmic sperm injection appears to overcome high levels of epigenetic instability in sperm., Competing Interests: Declaration of interests R.H.M. is an employee of Inherent Biosciences with ability to exercise stock options in company; listed as inventor on patent application for this work. E.A.D. has nothing to disclose. K.R.B. is an employee of Inherent Biosciences; inventor on patents associated with publication; Inherent Biosciences Stock option holder at Inherent Biosciences. C.R.N. is involved with Inherent Biosciences in one or a combination of the following capacities: employee, intern, advisor, equity-holder. He also reports funding from Inherent Biosciences for the submitted work. C.A.P. reports that during the creation of this manuscript, he was a paid intern/employee of Inherent Biosciences. B.R.E. is involved with Inherent Biosciences in one or a combination of the following capacities: employee, intern, advisor, equity-holder. K.I.A. is involved with Inherent Biosciences in one or a combination of the following capacities: employee, intern, advisor, equity-holder. He also reports patents for Methods of identifying male fertility status and embryo quality and Systems and methods for determining impact of age related changes in sperm epigenome on offspring phenotype; shareholder in Inherent Biosciences outside the submitted work. J.M.H. is involved with Inherent Biosciences in one or a combination of the following capacities: employee, intern, advisor, equity-holder. He also reports funding from Inherent Biosciences for the submitted work; patent for IP from andrology lab licensed to Inherent bio; stock options from Inherent Biosciences and Paterna Biosciences outside the submitted work. T.G.J. is involved with Inherent Biosciences in one or a combination of the following capacities: employee, intern, advisor, equity-holder. He also reports patents for Methods of identifying male fertility status and embryo quality and Systems and methods for determining impact of age related changes in sperm epigenome on offspring phenotype; shareholder in Inherent Biosciences outside the submitted work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Objective: To understand how chronic exposure to industrial air pollution is associated with male fertility through semen parameters., Design: Retrospective cohort study., Patients: Men in the Subfertility, Health, and Assisted Reproduction cohort who underwent a semen analysis in the two largest healthcare systems in Utah from 2005-2017 with ≥1 measured semen parameter (N = 21,563)., Intervention(s): Residential histories for each man were constructed using locations from administrative records linked through the Utah Population Database. Industrial facilities with air emissions of nine endocrine-disrupting compound chemical classes were identified from the Environmental Protection Agency Risk-Screening Environmental Indicators microdata. Chemical levels were linked with residential histories for the 5 years before each semen analysis., Main Outcome Measures: Semen analyses were classified as azoospermic or oligozoospermic (< 15 M/mL) using World Health Organization cutoffs for concentration. Bulk semen parameters such as concentration, total count, ejaculate volume, total motility, total motile count, and total progressive motile count were also measured. Multivariable regression models with robust standard errors were used to associate exposure quartiles for each of the nine chemical classes with each semen parameter, adjusting for age, race, and ethnicity, as well as neighborhood socioeconomic disadvantage., Results: After adjustment for demographic covariates, several chemical classes were associated with azoospermia and decreased total motility and volume. For exposure in the 4th relative to 1st quartile, significant associations were observed for acrylonitrile (β total motility = -0.87 pp), aromatic hydrocarbons (odds ratio [OR] azoospermia = 1.53; β volume = -0.14 mL), dioxins (OR azoospermia = 1.31; β volume = -0.09 mL; β total motility = -2.65 pp), heavy metals (β total motility = -2.78pp), organic solvents (OR azoospermia = 1.75; β volume = -0.10 mL), organochlorines (OR azoospermia = 2.09; β volume = -0.12 mL), phthalates (OR azoospermia = 1.44; β volume = -0.09 mL; β total motility = -1.21 pp), and silver particles (OR azoospermia = 1.64; β volume = -0.11 mL). All semen parameters significantly decreased with increasing socioeconomic disadvantage. Men who lived in the most disadvantaged areas had concentration, volume, and total motility of 6.70 M/mL, 0.13 mL, and 1.79 pp lower, respectively. Count, motile count, and total progressive motile count all decreased by 30-34 M., Conclusion(s): Several significant associations between chronic low-level environmental exposure to endocrine-disrupting compound air pollution from industrial sources and semen parameters were observed. The strongest associations were seen for increased odds of azoospermia and declines in total motility and volume. More research is needed to further explore additional social and exposure factors as well as expand on the risk posed to male reproductive health by the studied chemicals., (Copyright © 2023 American Society for Reproductive Medicine. All rights reserved.)

Background: As the average age of fatherhood increases worldwide, so too does the need for understanding effects of aging in male germline cells. Molecular change, including epigenomic alterations, may impact offspring. Age-associated change to DNA cytosine methylation in the cytosine-guanine (CpG) context is a hallmark of aging tissues, including sperm. Prior studies have led to accurate models that predict a man's age based on specific methylation features in the DNA of sperm, but the relationship between aging and global DNA methylation in sperm remains opaque. Further clarification requires a more complete survey of the methylome with assessment of variability within and between individuals., Results: We collected sperm methylome data in a longitudinal study of ten healthy fertile men. We used whole-genome bisulfite sequencing of samples collected 10 to 18 years apart from each donor. We found that, overall, variability between donors far exceeds age-associated variation. After controlling for donor identity, we see significant age-dependent genome-wide change to the methylome. Notably, trends of change with age depend on genomic location or annotation, with contrasting signatures that correlate with gene density and proximity to centromeres and promoter regions., Conclusions: We uncovered epigenetic signatures that reflect a stable process which begins in early adulthood, progressing steadily through most of the male lifespan, and warrants consideration in any future study of the aging sperm epigenome., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Complex diseases have multifactorial etiologies making actionable diagnostic biomarkers difficult to identify. Diagnostic research must expand beyond single or a handful of genetic or epigenetic targets for complex disease and explore a broader system of biological pathways. With the objective to develop a diagnostic tool designed to analyze a comprehensive network of epigenetic profiles in complex diseases, we used publicly available DNA methylation data from over 2,400 samples representing 20 cell types and various diseases. This tool, rather than detecting differentially methylated regions at specific genes, measures the intra-individual methylation variability within gene promoters to identify global shifts away from healthy regulatory states. To assess this new approach, we explored three distinct questions: 1) Are profiles of epigenetic variability tissue-specific? 2) Do diseased tissues exhibit altered epigenetic variability compared to normal tissue? 3) Can epigenetic variability be detected in complex disease? Unsupervised clustering established that global epigenetic variability in promoter regions is tissue-specific and promoter regions that are the most epigenetically stable in a specific tissue are associated with genes known to be essential for its function. Furthermore, analysis of epigenetic variability in these most stable regions distinguishes between diseased and normal tissue in multiple complex diseases. Finally, we demonstrate the clinical utility of this new tool in the assessment of a multifactorial condition, male infertility. We show that epigenetic variability in purified sperm is correlated with live birth outcomes in couples undergoing intrauterine insemination (IUI), a common fertility procedure. Men with the least epigenetically variable promoters were almost twice as likely to father a child than men with the greatest number of epigenetically variable promoters. Interestingly, no such difference was identified in men undergoing in vitro fertilization (IVF), another common fertility procedure, suggesting this as a treatment to overcome higher levels of epigenetic variability when trying to conceive., Competing Interests: RM, CP, KB, AO, RB, LL, JH, KA, and TJ are involved with Inherent Biosciences in one or a combination of the following capacities: employee, intern, advisor, equity-holder. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer XC declared a shared affiliation with the author LL to the handling editor at the time of review., (Copyright © 2023 Miller, Pollard, Brogaard, Olson, Barney, Lipshultz, Johnstone, Ibrahim, Hotaling, Schisterman, Mumford, Aston and Jenkins.)

Non-obstructive azoospermia, the absence of sperm in the ejaculate due to disturbed spermatogenesis, represents the most severe form of male infertility. De novo microdeletions of the Y-chromosomal AZFa region are one of few well-established genetic causes for NOA and are routinely analysed in the diagnostic workup of affected men. So far, it is unclear which of the three genes located in the AZFa chromosomal region is indispensible for germ cell maturation. Here we present four different likely pathogenic loss-of-function variants in the AZFa gene DDX3Y identified by analysing exome sequencing data of more than 1,600 infertile men. Three of the patients underwent testicular sperm extraction and revealed the typical AZFa testicular Sertoli cell-only phenotype. One of the variants was proven to be de novo. Consequently, DDX3Y represents the AZFa key spermatogenic factor and screening for variants in DDX3Y should be included in the diagnostic workflow., (© 2023. The Author(s).)

Non-obstructive azoospermia (NOA) is the most severe form of male infertility and typically incurable. Defining the genetic basis of NOA has proven challenging, and the most advanced classification of NOA subforms is not based on genetics, but simple description of testis histology. In this study, we exome-sequenced over 1000 clinically diagnosed NOA cases and identified a plausible recessive Mendelian cause in 20%. We find further support for 21 genes in a 2-stage burden test with 2072 cases and 11,587 fertile controls. The disrupted genes are primarily on the autosomes, enriched for undescribed human "knockouts", and, for the most part, have yet to be linked to a Mendelian trait. Integration with single-cell RNA sequencing data shows that azoospermia genes can be grouped into molecular subforms with synchronized expression patterns, and analogs of these subforms exist in mice. This analysis framework identifies groups of genes with known roles in spermatogenesis but also reveals unrecognized subforms, such as a set of genes expressed across mitotic divisions of differentiating spermatogonia. Our findings highlight NOA as an understudied Mendelian disorder and provide a conceptual structure for organizing the complex genetics of male infertility, which may provide a rational basis for disease classification., (© 2022. The Author(s).)

We determine whether a suspected seasonal variability in semen quality affect subsequent live birth rates. This is a retrospective, cohort analysis of men who provided semen analyses as part of fertility workup through a large andrology lab between 1996 and 2013 and corresponding birth rates using the Utah Population Database (UPDB). Semen parameters were analysed including total motile count (TMC), total sperm count, sperm concentration and progressive motility. Corresponding live births reflect those born in the state of Utah and were derived from birth certificate data available in the UPDB. Descriptive statistics were reported along with linear regression analysis with mixed effected models to test for an interaction between seasonal variation in semen quality and birth rates, accounting for age at the time of the semen analysis and abstinence time. A total of 11,929 patients and 14,765 semen samples were included. Only 3597 men (39% of men) had one or more values outside the World Health Organization reference range for their semen parameters. Linear regression demonstrated a consistent U-shaped relationship between TMC, total sperm count, and sperm concentration and season, with spring and winter yielding the highest values with a decline in the summer and fall. 7319 of these males had recorded live births for a total of 13,502 live births during the study period after a median follow-up of 7.2 years (IQR: 3.9-11.0). We did not find a significant interaction between specific semen parameters for a specific season and subsequent live births. Semen quality was the highest in the spring and winter, however there was no interaction between seasonal variability in semen quality and subsequent births. This is one of the largest studies describing seasonal variation in semen quality in humans., (© 2022 Wiley-VCH GMbH.)

Male infertility is a common condition affecting at least 7% of men worldwide and is often genetic in origin. Using whole exome sequencing, we recently discovered three hemizygous, likely damaging variants in DDB1- and CUL4-associated factor 12-like protein 1 (DCAF12L1) in men with azoospermia. DCAF12L1 is located on the X-chromosome and as identified by single cell sequencing studies, its expression is enriched in human testes and specifically in Sertoli cells and spermatogonia. However, very little is known about the role of DCAF12L1 in spermatogenesis, thus we generated a knockout mouse model to further explore the role of DCAF12L1 in male fertility. Knockout mice were generated using CRISPR/Cas9 technology to remove the entire coding region of Dcaf12l1 and were assessed for fertility over a broad range of ages (2-8 months of age). Despite outstanding genetic evidence in men, loss of DCAF12L1 had no discernible impact on male fertility in mice, as highlighted by breeding trials, histological assessment of the testis and epididymis, daily sperm production and evaluation of sperm motility using computer assisted methods. This disparity is likely due to the parallel evolution, and subsequent divergence, of DCAF12 family members in mice and men or the presence of compounding environmental factors in men., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Thousands of genes are expressed during spermatogenesis and male infertility has a strong genetic component. Within this study, we focus on the role of Zfr2 in male fertility, a gene previously implicated in human male fertility. To date, very little is known about the role of ZFR2 in either humans or mice. To this end, the requirement for ZFR2 in male fertility was assessed using a knockout mouse model., Results: Zfr2 was found to be expressed in the testes of both humans and mice. Deletion of Zfr2 was achieved via removal of exon 2 using CRISPR-Cas9 methods. The absence of Zfr2 did not result in a reduction in any fertility parameters assessed. Knockout males were capable of fostering litter sizes equal to wild type males, and there were no effects of Zfr2 knockout on sperm number or motility. We note Zfr2 knockout females were also fertile., Conclusions: The absence of Zfr2 alone is not sufficient to cause a reduction in male fertility in mice., (Copyright © 2022. Published by Elsevier Inc.)

Long-acting testosterone replacement therapy (TRT) suppresses spermatogenesis. A short-acting TRT, Natesto, maintains spermatogenesis in some men. This study evaluated hormonal and semen parameters converting men from long-acting TRT to Natesto. Baseline hormones, again on long-acting TRT and 1 month after converting to Natesto, as well as semen parameters 3 months after converting to Natesto were assessed. Twenty-seven men were directly converted from long-acting forms of TRT to Natesto. Mean duration on long-acting TRT was 24.3 ± 19 months. Testosterone levels were similar on long-acting forms of TRT and Natesto, however; E2 levels were significantly lower on Natesto. Ten men had semen analyses demonstrating azoospermia while on long-acting TRT, the remainder were presumed to be azoospermic or severely oligospermic which has been well established as an effect of long-acting TRT. All 27 men had resumption of spermatogenesis with a mean sperm concentration of 50.7 million/ml after converting to Natesto, considered within the fertile range. One couple achieved a pregnancy 4 months after converting to Natesto. Hypogonadal men on long-acting TRT interested in resumption of spermatogenesis may convert directly to Natesto for an opportunity to do so while remaining on a form of TRT and achieving lower E2 levels., (© 2022 The Authors. Andrologia published by Wiley-VCH GmbH.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022. Published by Elsevier Inc.)

Study Question: What is the load, distribution and added clinical value of secondary findings (SFs) identified in exome sequencing (ES) of patients with non-obstructive azoospermia (NOA)?, Summary Answer: One in 28 NOA cases carried an identifiable, medically actionable SF., What Is Known Already: In addition to molecular diagnostics, ES allows assessment of clinically actionable disease-related gene variants that are not connected to the patient's primary diagnosis, but the knowledge of which may allow the prevention, delay or amelioration of late-onset monogenic conditions. Data on SFs in specific clinical patient groups, including reproductive failure, are currently limited., Study Design, Size, Duration: The study group was a retrospective cohort of patients with NOA recruited in 10 clinics across six countries and formed in the framework of the international GEMINI (The GEnetics of Male INfertility Initiative) study., Participants/materials, Setting, Methods: ES data of 836 patients with NOA were exploited to analyze SFs in 85 genes recommended by the American College of Medical Genetics and Genomics (ACMG), Geisinger's MyCode, and Clinical Genome Resource. The identified 6374 exonic variants were annotated with ANNOVAR and filtered for allele frequency, retaining 1381 rare or novel missense and loss-of-function variants. After automatic assessment of pathogenicity with ClinVar and InterVar, 87 variants were manually curated. The final list of confident disease-causing SFs was communicated to the corresponding GEMINI centers. When patient consent had been given, available family health history and non-andrological medical data were retrospectively assessed., Main Results and the Role of Chance: We found a 3.6% total frequency of SFs, 3.3% from the 59 ACMG SF v2.0 genes. One in 70 patients carried SFs in genes linked to familial cancer syndromes, whereas 1 in 60 cases was predisposed to congenital heart disease or other cardiovascular conditions. Retrospective assessment confirmed clinico-molecular diagnoses in several cases. Notably, 37% (11/30) of patients with SFs carried variants in genes linked to male infertility in mice, suggesting that some SFs may have a co-contributing role in spermatogenic impairment. Further studies are needed to determine whether these observations represent chance findings or the profile of SFs in NOA patients is indeed different from the general population., Limitations, Reasons for Caution: One limitation of our cohort was the low proportion of non-Caucasian ethnicities (9%). Additionally, as comprehensive clinical data were not available retrospectively for all men with SFs, we were not able to confirm a clinico-molecular diagnosis and assess the penetrance of the specific variants., Wider Implications of the Findings: For the first time, this study analyzed medically actionable SFs in men with spermatogenic failure. With the evolving process to incorporate ES into routine andrology practice for molecular diagnostic purposes, additional assessment of SFs can inform about future significant health concerns for infertility patients. Timely detection of SFs and respective genetic counseling will broaden options for disease prevention and early treatment, as well as inform choices and opportunities regarding family planning. A notable fraction of SFs was detected in genes implicated in maintaining genome integrity, essential in both mitosis and meiosis. Thus, potential genetic pleiotropy may exist between certain adult-onset monogenic diseases and NOA., Study Funding/competing Interest(s): This work was supported by the Estonian Research Council grants IUT34-12 and PRG1021 (M.L. and M.P.); National Institutes of Health of the United States of America grant R01HD078641 (D.F.C., K.I.A. and P.N.S.); National Institutes of Health of the United States of America grant P50HD096723 (D.F.C. and P.N.S.); National Health and Medical Research Council of Australia grant APP1120356 (M.K.O'B., D.F.C. and K.I.A.); Fundação para a Ciência e a Tecnologia (FCT)/Ministério da Ciência, Tecnologia e Inovação grant POCI-01-0145-FEDER-007274 (A.M.L., F.C. and J.G.) and FCT: IF/01262/2014 (A.M.L.). J.G. was partially funded by FCT/Ministério da Ciência, Tecnologia e Ensino Superior (MCTES), through the Centre for Toxicogenomics and Human Health-ToxOmics (grants UID/BIM/00009/2016 and UIDB/00009/2020). M.L.E. is a consultant for, and holds stock in, Roman, Sandstone, Dadi, Hannah, Underdog and has received funding from NIH/NICHD. Co-authors L.K., K.L., L.N., K.I.A., P.N.S., J.G., F.C., D.M.-M., K.A., K.A.J., M.K.O'B., A.M.L., D.F.C., M.P. and M.L. declare no conflict of interest., Trial Registration Number: N/A., (© The Author(s) 2022. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: To investigate the impact of chemotherapy on the uterus., Design: Cross-sectional pilot study., Setting: Single university fertility clinic., Patients: Twelve patients with a history of alkylating agent chemotherapy exposure after Hodgkin lymphoma (cancer) vs. 12 normally menstruating women (controls)., Interventions: The inclusion criteria were age of 18-45 years and consent for endometrial biopsy. The exclusion criteria were the absence of the uterus, completed pelvic radiation, uterine or cervical cancer, and metastatic cancer. Each participant underwent endometrial biopsy and pelvic ultrasound. All study visits were conducted in the late proliferative phase of the menstrual cycle., Main Outcome Measures: Uterine volume, blood flow, endometrial thickness, histology, deoxyribonucleic acid methylation pattern, and relative ribonucleic acid (RNA) expression level during the same phase of the menstrual cycle., Results: In the study group, visits were conducted at a median of 31.5 (13.5-42.5) months after chemotherapy. The median uterine volume among cancer survivors was 36 (11.3-67) cm 3 , and that of the general population controls was 39 (13-54) cm 3 . On histologic examination, there were no cytologic or architectural atypia. The RNA-sequencing analysis revealed poor clustering of both control and treatment samples. However, we identified 3 differentially expressed genes on RNA-sequencing, but there was no concordance found among the differentially expressed genes and deoxyribonucleic acid methylation changes suggesting most likely false-positive results., Conclusions: Approximately 2.5 years after chemotherapy, a time at which several survivors of Hodgkin lymphoma may resume family-building, endometrial thickness and endometrial histology were not significantly affected by a history of alkylating agent chemotherapy exposure., (© 2022 The Authors.)

Aging men display reduced reproductive health; however, testis aging is poorly understood at the molecular and genomic levels. Here, we utilized single-cell RNA-seq to profile over 44,000 cells from both young and older men and examined age-related changes in germline development and in the testicular somatic cells. Age-related changes in spermatogonial stem cells appeared modest, whereas age-related dysregulation of spermatogenesis and somatic cells ranged from moderate to severe. Altered pathways included signaling and inflammation in multiple cell types, metabolic signaling in Sertoli cells, hedgehog signaling and testosterone production in Leydig cells, cell death and growth in testicular peritubular cells, and possible developmental regression in both Leydig and peritubular cells. Remarkably, the extent of dysregulation correlated with body mass index in older but not in younger men. Collectively, we reveal candidate molecular mechanisms underlying the complex testicular changes conferred by aging and their possible exacerbation by concurrent chronic conditions such as obesity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Testicular-derived inhibin B (α/β B dimers) acts in an endocrine manner to suppress pituitary production of follicle-stimulating hormone (FSH), by blocking the actions of activins (β A/B/β A/B dimers). Previously, we identified a homozygous genetic variant (c.1079T>C:p.Met360Thr) arising from uniparental disomy of chromosome 2 in the INHBB gene (β B-subunit of inhibin B and activin B) in a man suffering from infertility (azoospermia). In this study, we aimed to test the causality of the p.Met360Thr variant in INHBB and testis function. Here, we used CRISPR/Cas9 technology to generate InhbbM364T/M364T mice, where mouse INHBB p.Met364 corresponds with human p.Met360. Surprisingly, we found that the testes of male InhbbM364T/M364T mutant mice were significantly larger compared with those of aged-matched wildtype littermates at 12 and 24 weeks of age. This was attributed to a significant increase in Sertoli cell and round spermatid number and, consequently, seminiferous tubule area in InhbbM364T/M364T males compared to wildtype males. Despite this testis phenotype, male InhbbM364T/M364T mutant mice retained normal fertility. Serum hormone analyses, however, indicated that the InhbbM364T variant resulted in reduced circulating levels of activin B but did not affect FSH production. We also examined the effect of this p.Met360Thr and an additional INHBB variant (c.314C>T: p.Thr105Met) found in another infertile man on inhibin B and activin B in vitro biosynthesis. We found that both INHBB variants resulted in a significant disruption to activin B in vitro biosynthesis. Together, this analysis supports that INHBB variants that limit activin B production have consequences for testis composition in males., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (© 2022. The Author(s).)

Background: Human male infertility has a notable genetic component, including well-established diagnoses such as Klinefelter syndrome, Y-chromosome microdeletions and monogenic causes. Approximately 4% of all infertile men are now diagnosed with a genetic cause, but a majority (60-70%) remain without a clear diagnosis and are classified as unexplained. This is likely in large part due to a delay in the field adopting next-generation sequencing (NGS) technologies, and the absence of clear statements from field leaders as to what constitutes a validated cause of human male infertility (the current paper aims to address this). Fortunately, there has been a significant increase in the number of male infertility NGS studies. These have revealed a considerable number of novel gene-disease relationships (GDRs), which each require stringent assessment to validate the strength of genotype-phenotype associations. To definitively assess which of these GDRs are clinically relevant, the International Male Infertility Genomics Consortium (IMIGC) has identified the need for a systematic review and a comprehensive overview of known male infertility genes and an assessment of the evidence for reported GDRs., Objective and Rationale: In 2019, the first standardised clinical validity assessment of monogenic causes of male infertility was published. Here, we provide a comprehensive update of the subsequent 1.5 years, employing the joint expertise of the IMIGC to systematically evaluate all available evidence (as of 1 July 2020) for monogenic causes of isolated or syndromic male infertility, endocrine disorders or reproductive system abnormalities affecting the male sex organs. In addition, we systematically assessed the evidence for all previously reported possible monogenic causes of male infertility, using a framework designed for a more appropriate clinical interpretation of disease genes., Search Methods: We performed a literature search according to the PRISMA guidelines up until 1 July 2020 for publications in English, using search terms related to 'male infertility' in combination with the word 'genetics' in PubMed. Next, the quality and the extent of all evidence supporting selected genes were assessed using an established and standardised scoring method. We assessed the experimental quality, patient phenotype assessment and functional evidence based on gene expression, mutant in-vitro cell and in-vivo animal model phenotypes. A final score was used to determine the clinical validity of each GDR, across the following five categories: no evidence, limited, moderate, strong or definitive. Variants were also reclassified according to the American College of Medical Genetics and Genomics-Association for Molecular Pathology (ACMG-AMP) guidelines and were recorded in spreadsheets for each GDR, which are available at imigc.org., Outcomes: The primary outcome of this review was an overview of all known GDRs for monogenic causes of human male infertility and their clinical validity. We identified a total of 120 genes that were moderately, strongly or definitively linked to 104 infertility phenotypes., Wider Implications: Our systematic review curates all currently available evidence to reveal the strength of GDRs in male infertility. The existing guidelines for genetic testing in male infertility cases are based on studies published 25 years ago, and an update is far overdue. The identification of 104 high-probability 'human male infertility genes' is a 33% increase from the number identified in 2019. The insights generated in the current review will provide the impetus for an update of existing guidelines, will inform novel evidence-based genetic testing strategies used in clinics, and will identify gaps in our knowledge of male infertility genetics. We discuss the relevant international guidelines regarding research related to gene discovery and provide specific recommendations to the field of male infertility. Based on our findings, the IMIGC consortium recommend several updates to the genetic testing standards currently employed in the field of human male infertility, most important being the adoption of exome sequencing, or at least sequencing of the genes validated in this study, and expanding the patient groups for which genetic testing is recommended., (© The Author(s) 2021. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

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., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are short (21 to 35 nucleotides in length) and noncoding and are found almost exclusively in germ cells, where they regulate aberrant expression of transposable elements and postmeiotic gene expression. Critical to the processing of piRNAs is the protein poly(A)-specific RNase-like domain containing 1 (PNLDC1), which trims their 3' ends and, when disrupted in mice, causes azoospermia and male infertility., Methods: We performed exome sequencing on DNA samples from 924 men who had received a diagnosis of nonobstructive azoospermia. Testicular-biopsy samples were analyzed by means of histologic and immunohistochemical tests, in situ hybridization, reverse-transcriptase-quantitative-polymerase-chain-reaction assay, and small-RNA sequencing., Results: Four unrelated men of Middle Eastern descent who had nonobstructive azoospermia were found to carry mutations in PNLDC1 : the first patient had a biallelic stop-gain mutation, p.R452Ter (rs200629089; minor allele frequency, 0.00004); the second, a novel biallelic missense variant, p.P84S; the third, two compound heterozygous mutations consisting of p.M259T (rs141903829; minor allele frequency, 0.0007) and p.L35PfsTer3 (rs754159168; minor allele frequency, 0.00004); and the fourth, a novel biallelic canonical splice acceptor site variant, c.607-2A→T. Testicular histologic findings consistently showed error-prone meiosis and spermatogenic arrest with round spermatids of type Sa as the most advanced population of germ cells. Gene and protein expression of PNLDC1, as well as the piRNA-processing proteins PIWIL1, PIWIL4, MYBL1, and TDRKH, were greatly diminished in cells of the testes. Furthermore, the length distribution of piRNAs and the number of pachytene piRNAs was significantly altered in men carrying PNLDC1 mutations., Conclusions: Our results suggest a direct mechanistic effect of faulty piRNA processing on meiosis and spermatogenesis in men, ultimately leading to male infertility. (Funded by Innovation Fund Denmark and others.)., (Copyright © 2021 Massachusetts Medical Society.)

The clinical effect of sperm DNA damage in assisted reproduction has been a controversial topic during recent decades, leading to a variety of clinical practice recommendations. While the latest European Society of Human Reproduction and Embryology (ESHRE) position report concluded that DNA damage negatively affects assisted reproduction outcomes, the Practice Committee of the American Society for Reproductive Medicine (ASRM) does not recommend the routine testing of DNA damage for in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Herein, our aim was to perform a systematic review and meta-analysis of studies investigating whether sperm DNA damage affects clinical outcomes in IVF and ICSI, in order to contribute objectively to a consistent clinical recommendation. A comprehensive systematic search was conducted according to PRISMA guidelines from the earliest available online indexing year until March 2020, using the MEDLINE-PubMed and EMBASE databases. We included studies analysing IVF and/or ICSI treatments performed in infertile couples in which sperm DNA damage was well defined and assessed. Studies also had to include information about pregnancy, implantation or live birth rates as primary outcomes. The NHLBI-NIH quality assessment tool was used to assess the quality of each study. Meta-analyses were conducted using the Mantel-Haenszel method with random-effects models to evaluate the Risk Ratio (RR) between high-DNA-damage and control groups, taking into account the 95% confidence intervals. Heterogeneity among studies was evaluated using the I 2 statistic. We also conducted sensitivity analyses and post-hoc subgroup analyses according to different DNA fragmentation assessment techniques. We identified 78 articles that met our inclusion and quality criteria and were included in the qualitative analysis, representing a total of 25639 IVF/ICSI cycles. Of these, 32 articles had sufficient data to be included in the meta-analysis, comprising 12380 IVF/ICSI cycles. Meta-analysis revealed that, considering IVF and ICSI results together, implantation rate (RR = 0.74; 95% CI = 0.61-0.91; I 2  = 69) and pregnancy rate (RR = 0.83; 0.73-0.94; I 2  = 58) are negatively influenced by sperm DNA damage, although after adjustment for publication bias the relationship for pregnancy rate was no longer significant. The results showed a non-significant but detrimental tendency (RR = 0.78; 0.58-1.06; I 2  = 72) on live birth rate. Meta-analysis also showed that IVF outcomes are negatively influenced by sperm DNA damage, with a statistically significant impact on implantation (RR = 0.68; 0.52-0.89; I 2  = 50) and pregnancy rates (RR = 0.72; 0.55-0.95; I 2  = 72), although the latter was no longer significant after correction for publication bias. While it did not quite meet our threshold for significance, a negative trend was also observed for live birth rate (RR = 0.48; 0.22-1.02; I 2  = 79). In the case of ICSI, non-significant trends were observed for implantation (RR = 0.79; 0.60-1.04; I 2  = 72) or pregnancy rates (RR = 0.89; 0.78-1.02; I 2  = 44), and live birth rate (RR = 0.92; 0.67-1.27; I 2  = 70). The current review provides the largest evidence to date supporting a negative association between sperm DNA damage and conventional IVF treatments, significantly reducing implantation and pregnancy rates. The routine use of sperm DNA testing is therefore justified, since it may help improve the outcomes of IVF treatments and/or allow a given couple to be advised on the most suitable treatment. Further well-designed controlled studies on a larger number of patients are required to allow us to reach more precise conclusions, especially in the case of ICSI treatments., (© 2021 Cambridge Philosophical Society.)

Background: Male infertility is a prevalent clinical presentation for which there is likely a strong genetic component due to the thousands of genes required for spermatogenesis. Within this study we investigated the role of the gene Scrn1 in male fertility. Scrn1 is preferentially expressed in XY gonads during the period of sex determination and in adult Sertoli cells based on single cell RNA sequencing. We investigated the expression of Scrn1 in juvenile and adult tissues and generated a knockout mouse model to test its role in male fertility., Results: Scrn1 was expressed at all ages examined in the post-natal testis; however, its expression peaked at postnatal days 7-14 and SCRN1 protein was clearly localized to Sertoli cells. Scrn1 deletion was achieved via removal of exon 3, and its loss had no effect on male fertility or sex determination. Knockout mice were capable of siring litters of equal size to wild type counterparts and generated equal numbers of sperm with comparable motility and morphology characteristics., Conclusions: Scrn1 was found to be dispensable for male fertility, but this study identifies SCRN1 as a novel marker of the Sertoli cell cytoplasm., (© 2021 American Association of Anatomists.)

Immotile and rare sperm isolation from a complex cell background is an essential process for infertility treatment. The traditional sperm collection process from a biopsy sample requires long, tedious searches, yet still results in low sperm retrieval. In this work, a high recovery, high throughput sperm separation process is proposed for the clinical biopsy sperm retrieval process. It is found that sperm have different focusing positions compared with non-sperm cells in the inertial flow, which is explained by a sperm alignment phenomenon. Separation in the spiral channel device results in a 95.6% sperm recovery in which 87.4% of non-sperm cells get removed. Rare sperm isolation from a clinical biopsy sample is performed with the current approach. The chance of finding sperm is shown to increase 8.2 fold in the treated samples. The achieved results highly support this method being used for the development of a rapid biopsy sperm sorting process. In addition, the mechanism was proposed and can be applied for the high-efficiency separation of non-spherical particles in general.

Male infertility is a common and complex disease, manifesting as a wide range of phenotypes, ranging from apparently normal semen parameters with an inexplicable inability to conceive, to the complete absence of sperm production. The diversity of male infertility phenotypes, coupled with the extreme complexity of spermatogenesis has significantly confounded the identification of the underlying genetic causes for these conditions, though incremental progress has been made, particularly in the past decade. In this review, we discuss the progress that has been made to date, tools and resources that have proven effective in accelerating discovery of novel genetic markers for male infertility, and areas in which we see the greatest potential for advancing the field in the coming years. These include the development and use of robust phenotyping tools, the continued development of in vitro and animal models for variant validation, increased utilization and refinement of whole genome approaches for discovery, and further expansion of consortia that assemble groups of clinicians and basic researchers with the unified goal of disentangling the complex genetic architecture of male infertility. As these resources mature, and funding agencies increasingly recognize the importance of these efforts for improving human health, the discovery of novel genetic markers for male infertility will certainly continue to accelerate., Competing Interests: Conflicts of Interest: The authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tau.2020.03.43). The series “Genetic Causes and Management of Male Infertility” was commissioned by the editorial office without any funding or sponsorship. The authors have no other conflicts of interest to declare., (2021 Translational Andrology and Urology. All rights reserved.)

The present updated systematic review and meta-analysis aims to summarize the evidence from published studies with low risk for any important bias (based on methodological quality assessment) investigating the potential associations of adiposity with sperm quality and reproductive hormones. We conducted a systematic search of the literature published in MEDLINE-PubMed and EMBASE through June 2019. Based on the criteria in our review, 169 eligible publications were used for data abstraction. Finally, 60 articles were included in the qualitative analysis and 28 in the quantitative analysis. Our systematic review results indicated that overweight and/or obesity were associated with low semen quality parameters (i.e., semen volume, sperm count and concentration, sperm vitality and normal morphology) and some specific reproductive hormones (e.g., inhibin B, total testosterone and sex hormone-binding globulin). Overweight and/or obesity were also positively associated with high estradiol concentrations. Meta-analysis indicated that overweight and/or obesity categories were associated with lower sperm quality (i.e., semen volume, sperm count and concentration, sperm vitality, total motility and normal morphology), and underweight category was likewise associated with low sperm normal morphology. In conclusion, our results suggest that maintaining a healthy body weight is important for increasing sperm quality parameters and potentially male fertility., (© 2020 World Obesity Federation.)

Background: Many environmental and lifestyle factors have been implicated in the decline of sperm quality, with diet being one of the most plausible factors identified in recent years. Moreover, several studies have reported a close association between the alteration of specific sperm DNA methylation signatures and semen quality., Objectives: To evaluate the effect of tree nut consumption on sperm DNA methylation patterns in healthy individuals reporting eating a Western-style diet., Material and Methods: This is a post hoc analysis conducted in a subset of participants (healthy, non-smoking, and young) from the FERTINUTS 14-wk randomized-controlled, parallel trial, recruited between December 2015 and February 2017. The participants included in the current study (n = 72) were randomly selected in a proportion 2:1 from the original FERTINUTS trial between the 98 participants that completed the entire dietary intervention (nut group, n = 48; control group, n = 24). Sperm DNA methylation patterns were examined at baseline and after 14 weeks in 48 individuals consuming 60 g/d of mixed nuts (nut group) and in 24 individuals following the usual Western-style diet avoiding consumption of nuts (control group)., Results: Over the course of the trial, no significant changes in global methylation were observed between groups. However, in the nut group, we identified 36 genomic regions that were significantly differentially methylated between the baseline and the end of the trial and 97.2% of the regions displayed hypermethylation. We identified no such change in the control group over the same period of time. We also utilized the recently developed germ line age calculator to determine if nut consumption resulted in alterations to the epigenetic age of cells and no significant differences were found., Discussion and Conclusion: Adding nuts to a regular Western-style diet subtly impacts sperm DNA methylation in specific regions, demonstrating that there are some sperm epigenome regions that could respond to diet., (© 2020 American Society of Andrology and European Academy of Andrology.)