ABSTRACT
Infertility, affecting â¼10% 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.
Subject(s)
Infertility, Male , Humans , Male , Infertility, Male/genetics , Adult , Exome Sequencing , Steroidogenic Factor 1/genetics , Azoospermia/genetics , Oligospermia/genetics , Mutation , Spermatogenesis/genetics , Cohort StudiesABSTRACT
Nonobstructive azoospermia is the leading cause of male infertility. Abnormal levels of transmembrane protein 225 (TMEM225), a testis-specific protein, have been found in patients with nonobstructive azoospermia, suggesting that TMEM225 plays an essential role in male fertility. Here, we generated a Tmem225 KO mouse model to explore the function and mechanism of TMEM225 in male reproduction. Male Tmem225 KO mice were infertile. Surprisingly, Tmem225 deletion did not affect spermatogenesis, but TMEM225-null sperm exhibited abnormalities during epididymal maturation, resulting in reduced sperm motility and an abnormal hairpin-loop configuration. Furthermore, proteomics analyses of cauda sperm revealed that signaling pathways related to mitochondrial function, the glycolytic pathway, and sperm flagellar morphology were abnormal in Tmem225 KO sperm, and spermatozoa lacking TMEM225 exhibited high reactive oxygen species levels, reduced motility, and flagellar folding, leading to typical asthenospermia. These findings suggest that testicular TMEM225 may control the sperm maturation process by regulating the expression of proteins related to mitochondrial function, glycolysis, and sperm flagellar morphology in epididymal spermatozoa.
Subject(s)
Azoospermia , Humans , Male , Mice , Animals , Azoospermia/metabolism , Sperm Maturation , Sperm Motility , Semen , Spermatozoa/metabolism , Testis/metabolism , Spermatogenesis , Fertility , Mice, KnockoutABSTRACT
The maintenance of genome integrity in the germline is crucial for mammalian development. Long interspersed element type 1 (LINE-1, L1) is a mobile genetic element that makes up about 17% of the human genome and poses a threat to genome integrity. N6-methyl-adenosine (m6A) plays an essential role in regulating various biological processes. However, the function of m6A modification in L1 retrotransposons and human germline development remains largely unknown. Here we knocked out the m6A methyltransferase METTL3 or the m6A reader YTHDF2 in human embryonic stem cells (hESCs) and discovered that METTL3 and YTHDF2 are crucial for inducing human spermatogonial stem cells (hSSCs) from hESCs in vitro. The removal of METTL3 or YTHDF2 resulted in increased L1 retrotransposition and reduced the efficiency of SSC differentiation in vitro. Further analysis showed that YTHDF2 recognizes the METTL3-catalyzed m6A modification of L1 retrotransposons and degrades L1 mRNA through autophagy, thereby blocking L1 retrotransposition. Moreover, the study confirmed that m6A modification in human fetal germ cells promotes the degradation of L1 retrotransposon RNA, preventing the insertion of new L1 retrotransposons into the genome. Interestingly, L1 retrotransposon RNA was highly expressed while METTL3 was significantly downregulated in the seminal plasma of azoospermic patients with meiotic arrest compared to males with normal fertility. Additionally, we identified some potentially pathogenic variants in m6A-related genes in azoospermic men with meiotic arrest. In summary, our study suggests that m6A modification serves as a guardian of genome stability during human germline development and provides novel insights into the function and regulatory mechanisms of m6A modification in restricting L1 retrotransposition.
Subject(s)
Azoospermia , Retroelements , Male , Animals , Humans , Retroelements/genetics , RNA , Azoospermia/genetics , Cell Differentiation/genetics , Methyltransferases/genetics , Methyltransferases/metabolism , RNA, Messenger/genetics , Mammals/metabolismABSTRACT
Spermatogonial stem cells (SSCs) are capable of transmitting genetic information to the next generations and they are the initial cells for spermatogenesis. Nevertheless, it remains largely unknown about key genes and signaling pathways that regulate fate determinations of human SSCs and male infertility. In this study, we explored the expression, function, and mechanism of USP11 in controlling the proliferation and apoptosis of human SSCs as well as the association between its abnormality and azoospermia. We found that USP11 was predominantly expressed in human SSCs as shown by database analysis and immunohistochemistry. USP11 silencing led to decreases in proliferation and DNA synthesis and an enhancement in apoptosis of human SSCs. RNA-sequencing identified HOXC5 as a target of USP11 in human SSCs. Double immunofluorescence, Co-immunoprecipitation (Co-IP), and molecular docking demonstrated an interaction between USP11 and HOXC5 in human SSCs. HOXC5 knockdown suppressed the growth of human SSCs and increased apoptosis via the classical WNT/ß-catenin pathway. In contrast, HOXC5 overexpression reversed the effect of proliferation and apoptosis induced by USP11 silencing. Significantly, lower levels of USP11 expression were observed in the testicular tissues of patients with spermatogenic disorders. Collectively, these results implicate that USP11 regulates the fate decisions of human SSCs through the HOXC5/WNT/ß-catenin pathway. This study thus provides novel insights into understanding molecular mechanisms underlying human spermatogenesis and the etiology of azoospermia and it offers new targets for gene therapy of male infertility.
Subject(s)
Apoptosis , Cell Proliferation , Spermatogenesis , Thiolester Hydrolases , Wnt Signaling Pathway , Humans , Male , Adult Germline Stem Cells/metabolism , Apoptosis/genetics , Azoospermia/metabolism , Azoospermia/genetics , Azoospermia/pathology , beta Catenin/metabolism , beta Catenin/genetics , Cell Proliferation/genetics , Homeodomain Proteins/metabolism , Homeodomain Proteins/genetics , Spermatogenesis/genetics , Spermatogonia/metabolism , Spermatogonia/cytology , Testis/metabolism , Testis/cytology , Thiolester Hydrolases/genetics , Thiolester Hydrolases/metabolism , Wnt Signaling Pathway/geneticsABSTRACT
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.
Subject(s)
Azoospermia , Gene Expression Profiling , Gene Regulatory Networks , MicroRNAs , RNA, Long Noncoding , RNA, Messenger , Single-Cell Analysis , Spermatozoa , Humans , Male , Azoospermia/genetics , Azoospermia/metabolism , Spermatozoa/metabolism , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , MicroRNAs/genetics , MicroRNAs/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Analysis, RNA , Transcriptome , Oligonucleotide Array Sequence AnalysisABSTRACT
INTRODUCTION: Parthenogenetic chimera is an extremely rare condition in human. Very few patients with parthenogenetic chimerism with XX/XY cells have been identified. CASE PRESENTATION: We report the clinical findings and molecular analysis of chimerism with a 46,XX/46,XY karyotype in a patient presenting idiopathic oligoasthenoteratozoospermia (OAT). To clarify the mechanism of chimera formation, short tandem repeat analysis using 21 loci was carried out. Quantitation of alleles in D6S1043, D12S391, fibrinogen alpha chain, and amelogenin revealed double paternal and one maternal genetic contribution to the patient, which is consistent with a parthenogenetic chimerism. The likely mechanism of chimerism formation was also discussed, followed by a literature review. CONCLUSION: This is the first documented case of parthenogenetic chimerism in an adult male with XX/XY cells presenting OAT. Improved cell sampling and more sensitive and specific detection methods are necessary to identify more patients with XX/XY chimerism for systematic studies on this condition in the future.
Subject(s)
Chimerism , Humans , Male , Adult , Oligospermia/genetics , Parthenogenesis/genetics , Microsatellite Repeats/genetics , Chromosomes, Human, Y/genetics , Chromosomes, Human, X/genetics , Azoospermia/genetics , KaryotypingABSTRACT
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.
Subject(s)
Azoospermia , Infertility, Male , Oligospermia , Humans , Male , Azoospermia/genetics , Azoospermia/pathology , Genes, X-Linked , HEK293 Cells , Infertility, Male/genetics , Oligospermia/genetics , SemenABSTRACT
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.
Subject(s)
Azoospermia , Sperm Injections, Intracytoplasmic , Pregnancy , Female , Humans , Male , Oocyte Retrieval , Testis/pathology , Semen , Azoospermia/therapy , Azoospermia/pathology , Spermatozoa/pathology , Sperm Retrieval , Biopsy , Retrospective StudiesABSTRACT
PURPOSE: We sought to examine sperm retrieval and testicular histology in males of different ages with Klinefelter syndrome. MATERIALS AND METHODS: We identified all males with Klinefelter syndrome who underwent microdissection testicular sperm extraction at our institution from 1995 to 2020. Patients were divided into adolescent (<20 years) and adult (≥20 years) cohorts. Histology and sperm retrieval were compared using chi-square statistics. Multivariable logistic regression models were used to examine factors associated with successful sperm retrieval. RESULTS: We identified 217 males with Klinefelter syndrome, of whom 59 were adolescents and 158 were adults. Adults were stratified into 10-year groupings (20-29 years, n = 62; 30-39 years, n = 88; ≥40 years, n = 8). Approximately 17% of adolescents had testis histology containing germ cells compared with 15% of the 20 to 29-year cohort, 14% of the 30 to 39-year cohort, and 0% over 40 years. In comparison to adolescents (53%), the sperm retrieval rate was significantly higher in the 20 to 29-year cohort (71%, P = .04) and lower in the ≥40-year cohort (13%, P = .03). In multivariable analysis, the presence of hypospermatogenesis on testis biopsy (OR 5.8, P = .03) was associated with higher odds of successful sperm retrieval. CONCLUSIONS: Younger males more frequently had germ cell-containing testis histology, however this finding was not associated with a higher odds of sperm retrieval. Reproductive urologists should counsel azoospermic males with Klinefelter syndrome that sperm retrieval during adolescence for fertility preservation is not required and can be performed in young adulthood.
Subject(s)
Azoospermia , Klinefelter Syndrome , Adult , Adolescent , Humans , Male , Young Adult , Testis/pathology , Klinefelter Syndrome/complications , Klinefelter Syndrome/pathology , Sperm Retrieval , Semen , Azoospermia/pathology , Spermatozoa , Retrospective StudiesABSTRACT
Nonobstructive azoospermia (NOA), the most severe manifestation of male infertility, lacks a comprehensive understanding of its genetic etiology. Here, a bi-allelic loss-of-function variant in REC114 (c.568C > T: p.Gln190*) were identified through whole exome sequencing (WES) in a Chinese NOA patient. Testicular histopathological analysis and meiotic chromosomal spread analysis were conducted to assess the stage of spermatogenesis arrested. Co-immunoprecipitation (Co-IP) and Western blot (WB) were used to investigate the influence of variant in vitro. In addition, our results revealed that the variant resulted in truncated REC114 protein and impaired interaction with MEI4, which was essential for meiotic DNA double-strand break (DSB) formation. As far as we know, this study presents the first report that identifies REC114 as the causative gene for male infertility. Furthermore, our study demonstrated indispensability of the REC114-MEI4 complex in maintaining DSB homoeostasis, and highlighted that the disruption of the complex due to the REC114 variant may underline the mechanism of NOA.
Subject(s)
Azoospermia , Infertility, Male , Humans , Male , Azoospermia/genetics , Azoospermia/pathology , Loss of Heterozygosity , Infertility, Male/genetics , Infertility, Male/pathology , Testis/pathology , Meiosis/genetics , Cell Cycle Proteins/geneticsABSTRACT
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.
Subject(s)
Azoospermia , Infertility, Male , Humans , Male , Mice , Animals , Azoospermia/genetics , Azoospermia/pathology , Infertility, Male/genetics , Mutation/genetics , Proteins/genetics , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Meiosis/genetics , DNA-Binding Proteins/geneticsABSTRACT
Oligoasthenoteratozoospermia (OAT) is a common type of male infertility; however, its genetic causes remain largely unknown. Some of the genetic determinants of OAT are gene defects affecting spermatogenesis. BCORL1 (BCL6 corepressor like 1) is a transcriptional corepressor that exhibits the OAT phenotype in a knockout mouse model. A hemizygous missense variant of BCORL1 (c.2615T > G:p.Val872Gly) was reported in an infertile male patient with non-obstructive azoospermia (NOA). Nevertheless, the correlation between BCORL1 variants and OAT in humans remains unknown. In this study, we used whole-exome sequencing to identify a novel hemizygous nonsense variant of BCORL1 (c.1564G > T:p.Glu522*) in a male patient with OAT from a Han Chinese family. Functional analysis showed that the variant produced a truncated protein with altered cellular localization and a dysfunctional interaction with SKP1 (S-phase kinase-associated protein 1). Further population screening identified four BCORL1 missense variants in subjects with both OAT (1 of 325, 0.31%) and NOA (4 of 355, 1.13%), but no pathogenic BCORL1 variants among 362 fertile subjects. In conclusion, our findings indicate that BCORL1 is a potential candidate gene in the pathogenesis of OAT and NOA, expanded its disease spectrum and suggested that BCORL1 may play a role in spermatogenesis by interacting with SKP1.
Subject(s)
Exome Sequencing , Infertility, Male , Repressor Proteins , Male , Humans , Repressor Proteins/genetics , Infertility, Male/genetics , Infertility, Male/pathology , Oligospermia/genetics , Oligospermia/pathology , Adult , Pedigree , Azoospermia/genetics , Azoospermia/pathology , Loss of Function Mutation/genetics , Genetic Predisposition to Disease , Protein-Arginine N-Methyltransferases/genetics , Mutation, Missense/genetics , Spermatogenesis/geneticsABSTRACT
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.
Subject(s)
Azoospermia , Cell Cycle Proteins , DNA Copy Number Variations , Humans , Azoospermia/genetics , Male , Exome Sequencing , Adult , Mutation , Japan , KaryotypingABSTRACT
STUDY QUESTION: Do the genetic determinants of idiopathic severe spermatogenic failure (SPGF) differ between generations? SUMMARY ANSWER: Our data support that the genetic component of idiopathic SPGF is impacted by dynamic changes in environmental exposures over decades. WHAT IS KNOWN ALREADY: The idiopathic form of SPGF has a multifactorial etiology wherein an interaction between genetic, epigenetic, and environmental factors leads to the disease onset and progression. At the genetic level, genome-wide association studies (GWASs) allow the analysis of millions of genetic variants across the genome in a hypothesis-free manner, as a valuable tool for identifying susceptibility risk loci. However, little is known about the specific role of non-genetic factors and their influence on the genetic determinants in this type of conditions. STUDY DESIGN, SIZE, DURATION: Case-control genetic association analyses were performed including a total of 912 SPGF cases and 1360 unaffected controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: All participants had European ancestry (Iberian and German). SPGF cases were diagnosed during the last decade either with idiopathic non-obstructive azoospermia (n = 547) or with idiopathic non-obstructive oligozoospermia (n = 365). Case-control genetic association analyses were performed by logistic regression models considering the generation as a covariate and by in silico functional characterization of the susceptibility genomic regions. MAIN RESULTS AND THE ROLE OF CHANCE: This analysis revealed 13 novel genetic association signals with SPGF, with eight of them being independent. The observed associations were mostly explained by the interaction between each lead variant and the age-group. Additionally, we established links between these loci and diverse non-genetic factors, such as toxic or dietary habits, respiratory disorders, and autoimmune diseases, which might potentially influence the genetic architecture of idiopathic SPGF. LARGE SCALE DATA: GWAS data are available from the authors upon reasonable request. LIMITATIONS, REASONS FOR CAUTION: Additional independent studies involving large cohorts in ethnically diverse populations are warranted to confirm our findings. WIDER IMPLICATIONS OF THE FINDINGS: Overall, this study proposes an innovative strategy to achieve a more precise understanding of conditions such as SPGF by considering the interactions between a variable exposome through different generations and genetic predisposition to complex diseases. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the "Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020)" (ref. PY20_00212, P20_00583), the Spanish Ministry of Economy and Competitiveness through the Spanish National Plan for Scientific and Technical Research and Innovation (ref. PID2020-120157RB-I00 funded by MCIN/ AEI/10.13039/501100011033), and the 'Proyectos I+D+i del Programa Operativo FEDER 2020' (ref. B-CTS-584-UGR20). ToxOmics-Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, is also partially supported by the Portuguese Foundation for Science and Technology (Projects: UIDB/00009/2020; UIDP/00009/2020). The authors declare no competing interests. TRIAL REGISTRATION NUMBER: N/A.
Subject(s)
Azoospermia , Oligospermia , Male , Humans , Genome-Wide Association Study , Genetic Predisposition to Disease , Azoospermia/genetics , Oligospermia/genetics , Environmental ExposureABSTRACT
Fertility restoration using autologous testicular tissue transplantation is relevant for infertile men surviving from childhood cancer and, possibly, in men with absent or incomplete spermatogenesis resulting in the lack of spermatozoa in the ejaculate (non-obstructive azoospermia, NOA). Currently, testicular tissue from pre-pubertal boys extracted before treatment with gonadotoxic cancer therapy can be cryopreserved with good survival of spermatogonial stem cells. However, strategies for fertility restoration, after successful cancer treatment, are still experimental and no clinical methods have yet been developed. Similarly, no clinically available treatments can help men with NOA to become biological fathers after failed attempts of testicular surgical sperm retrieval. We present a case of a 31-year-old man with NOA who had three pieces of testis tissue (each â¼2 × 4 × 2 mm3) extracted and cryopreserved in relation to performing microdissection testicular sperm extraction (mTESE). Approximately 2 years after mTESE, the thawed tissue pieces were engrafted in surgically created pockets bilaterally under the scrotal skin. Follow-up was performed after 2, 4, and 6 months with assessment of reproductive hormones and ultrasound of the scrotum. After 6 months, all engrafted tissue was extracted and microscopically analyzed for the presence of spermatozoa. Furthermore, parts of the extracted tissue were analyzed histologically and by immunohistochemical analysis. Active blood flow in the engrafted tissue was demonstrated by doppler ultrasound after 6 months. No spermatozoa were found in the extracted tissue. Histological and immunohistochemical analysis demonstrated graft survival with intact clear tubules and normal cell organization. Sertoli cells and spermatocytes with normal morphology were located near the basement membrane. MAGE-A and VASA positive spermatogonia/spermatocytes were detected together with SOX9 positive Sertoli cells. Spermatocytes and/or Sertoli cells positive for γH2AX was also detected. In summary, following autologous grafting of frozen-thawed testis tissue under the scrotal skin in a man with NOA, we demonstrated graft survival after 6 months. No mature spermatozoa were detected; however, this is likely due to the pre-existing spermatogenic failure.
Subject(s)
Azoospermia , Testis , Adult , Humans , Male , Child , Testis/pathology , Semen , Spermatozoa/pathology , Spermatogonia , Sertoli Cells , Azoospermia/surgery , Azoospermia/pathology , Sperm RetrievalABSTRACT
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.
Subject(s)
Azoospermia , Oligospermia , Testicular Neoplasms , Adolescent , Young Adult , Humans , Male , Child , Azoospermia/epidemiology , Azoospermia/genetics , Azoospermia/diagnosis , Oligospermia/epidemiology , Oligospermia/genetics , Retrospective Studies , Pedigree , Risk Factors , Testicular Neoplasms/epidemiology , Testicular Neoplasms/geneticsABSTRACT
STUDY QUESTION: Are there subgroups among patients with cryptozoospermia pointing to distinct etiologies? SUMMARY ANSWER: We reveal two distinct subgroups of cryptozoospermic (Crypto) patients based on testicular tissue composition, testicular volume, and FSH levels. WHAT IS KNOWN ALREADY: Cryptozoospermic patients present with a sperm concentration below 0.1 million/ml. While the etiology of the severely impaired spermatogenesis remains largely unknown, alterations of the spermatogonial compartment have been reported including a reduction of the reserve stem cells in these patients. STUDY DESIGN, SIZE, DURATION: To assess whether there are distinct subgroups among cryptozoospermic patients, we applied the statistical method of cluster analysis. For this, we retrospectively selected 132 cryptozoospermic patients from a clinical database who underwent a testicular biopsy in the frame of fertility treatment at a university hospital. As controls (Control), we selected 160 patients with obstructive azoospermia and full spermatogenesis. All 292 patients underwent routine evaluation for endocrine, semen, and histological parameters (i.e. the percentage of tubules with elongated spermatids). Moreover, outcome of medically assisted reproduction (MAR) was assessed for cryptozoospermic (n = 73) and Control patients (n = 87), respectively. For in-depth immunohistochemical and histomorphometrical analyses, representative tissue samples from cryptozoospermic (n = 27) and Control patients (n = 12) were selected based on cluster analysis results and histological parameters. PARTICIPANTS/MATERIALS, SETTING, METHODS: This study included two parts: firstly using clinical parameters of the entire cohort of 292 patients, we performed principal component analysis (PCA) followed by hierarchical clustering on principal components (i.e. considering hormonal values, ejaculate parameters, and histological information). Secondly, for histological analyses seminiferous tubules were categorized according to the most advanced germ cell type present in sections stained with Periodic acid Schif. On the selected cohort of 39 patients (12 Control, 27 cryptozoospermic), we performed immunohistochemistry for spermatogonial markers melanoma-associated antigen 4 (MAGEA4) and piwi like RNA-mediated gene silencing 4 (PIWIL4) followed by quantitative analyses. Moreover, the morphologically defined Adark spermatogonia, which are considered to be the reserve stem cells, were quantified. MAIN RESULTS AND THE ROLE OF CHANCE: The PCA and hierarchical clustering revealed three different clusters, one of them containing all Control samples. The main factors driving the sorting of patients to the clusters were the percentage of tubules with elongated spermatids (Cluster 1, all Control patients and two cryptozoospermic patients), the percentage of tubules with spermatocytes (Cluster 2, cryptozoospermic patients), and tubules showing a Sertoli cells only phenotype (Cluster 3, cryptozoospermic patients). Importantly, the percentage of tubules containing elongated spermatids was comparable between Clusters 2 and 3. Additional differences were higher FSH levels (P < 0.001) and lower testicular volumes (P < 0.001) in Cluster 3 compared to Cluster 2. In the spermatogonial compartment of both cryptozoospermic Clusters, we found lower numbers of MAGEA4+ and Adark spermatogonia but higher proportions of PIWIL4+ spermatogonia, which were significantly correlated with a lower percentage of tubules containing elongated spermatids. In line with this common alteration, the outcome of MAR was comparable between Controls as well as both cryptozoospermic Clusters. LIMITATIONS, REASONS FOR CAUTION: While we have uncovered the existence of subgroups within the cohort of cryptozoospermic patients, comprehensive genetic analyses remain to be performed to unravel potentially distinct etiologies. WIDER IMPLICATIONS OF THE FINDINGS: The novel insight that cryptozoospermic patients can be divided into two subgroups will facilitate the strategic search for underlying genetic etiologies. Moreover, the shared alterations of the spermatogonial stem cell compartment between the two cryptozoospermic subgroups could represent a general response mechanism to the reduced output of sperm, which may be associated with a progressive phenotype. This study therefore offers novel approaches towards the understanding of the etiology underlying the reduced sperm formation in cryptozoospermic patients. STUDY FUNDING/COMPETING INTEREST(S): German research foundation CRU 326 (grants to: SDP, NN). Moreover, we thank the Faculty of Medicine of the University of Münster for the financial support of Lena Charlotte Schülke through the MedK-program. We acknowledge support from the Open Access Publication Fund of the University of Münster. The authors have no potential conflicts of interest. TRIAL REGISTRATION NUMBER: N/A.
Subject(s)
Follicle Stimulating Hormone , Spermatogenesis , Testis , Humans , Male , Adult , Retrospective Studies , Testis/pathology , Follicle Stimulating Hormone/blood , Azoospermia/pathology , Sperm Count , Spermatozoa/pathology , Cluster Analysis , Oligospermia/pathology , Infertility, Male/pathology , Infertility, Male/etiologyABSTRACT
In brief: Since available therapeutic approaches for chemotherapy-induced non-obstructive azoospermia (NOA) patients are not enough efficient, an urgent need for treatment alternatives is felt. This study shows that adipose tissue-derived mesenchymal stem cells-derived exosome (AD-Exo) treatment is more effective in ameliorating busulfan-induced NOA rat models compared to platelet-rich plasma (PRP). Abstract: Patients with non-obstructive azoospermia (NOA) are unable to have their children. Therefore, there is an urgent need for additional treatment alternatives for these patients. Recently, novel treatments based on the exosomes derived from mesenchymal stem cells (MSCs) as the agents responsible for exerting the paracrine effects and consequently biological functions of MSCs are proposed. Besides, platelet-rich plasma (PRP) as a significant blood byproduct has been therapeutically applied in several male infertility studies. In this study, we compared the effects of PRP and exosome treatment on spermatogenesis restoration in NOA rat models. Exosomes and PRP were isolated from the adipose tissue-derived MSCs (AD-MSCs) collected from conditioned medium and peripheral blood of human volunteers, respectively. Non-obstructive azoospermia (NOA) induction was done through two doses of busulfan at a 21-day interval. Thirty-five days after NOA induction, intratesticular injection of AD-MSCs-derived exosome (AD-Exo), PRP, and PBS was performed. The control group did not receive any treatment. Two months later, the rats were euthanized for further analysis. Our results revealed that both AD-Exo and PRP treatments improved the size and weight of testis, modulated the expression level of Dazl, Ddx4, Stra8, Pwil1, and Ccna1, and ameliorated the serum level of LDH, SOD, and GR enzymes in NOA rats. Moreover, the AD-Exo group showed improved testosterone, GPx, MAD, and CAT serum levels, sperm motility, and protein levels of DAZL and DDX4. This investigation verified the more efficient effects of AD-Exo treatment in comparison to PRP in ameliorating busulfan-induced NOA rat models.
Subject(s)
Azoospermia , Busulfan , Disease Models, Animal , Exosomes , Mesenchymal Stem Cells , Platelet-Rich Plasma , Spermatogenesis , Male , Animals , Exosomes/metabolism , Exosomes/transplantation , Azoospermia/therapy , Azoospermia/pathology , Azoospermia/chemically induced , Spermatogenesis/drug effects , Mesenchymal Stem Cells/metabolism , Rats , Busulfan/pharmacology , Platelet-Rich Plasma/metabolism , Humans , Testis/metabolism , Testis/pathology , Rats, Sprague-DawleyABSTRACT
RESEARCH QUESTION: Can artificial intelligence (AI) improve the efficiency and efficacy of sperm searches in azoospermic samples? DESIGN: This two-phase proof-of-concept study began with a training phase using eight azoospermic patients (>10,000 sperm images) to provide a variety of surgically collected samples for sperm morphology and debris variation to train a convolutional neural network to identify spermatozoa. Second, side-by-side testing was undertaken on two cohorts of non-obstructive azoospermia patient samples: an embryologist versus the AI identifying all the spermatozoa in the still images (cohort 1, nâ¯=â¯4), and a side-by-side test with a simulated clinical deployment of the AI model with an intracytoplasmic sperm injection microscope and the embryologist performing a search with and without the aid of the AI (cohort 2, nâ¯=â¯4). RESULTS: In cohort 1, the AI model showed an improvement in the time taken to identify all the spermatozoa per field of view (0.02 ± 0.30 â¯×⯠10-5s versus 36.10 ± 1.18s, P < 0.0001) and improved recall (91.95 ± 0.81% versus 86.52 ± 1.34%, P < 0.001) compared with an embryologist. From a total of 2660 spermatozoa to find in all the samples combined, 1937 were found by an embryologist and 1997 were found by the AI in less than 1000th of the time. In cohort 2, the AI-aided embryologist took significantly less time per droplet (98.90 ± 3.19 s versus 168.7 ± 7.84 s, P < 0.0001) and found 1396 spermatozoa, while 1274 were found without AI, although no significant difference was observed. CONCLUSIONS: AI-powered image analysis has the potential for seamless integration into laboratory workflows, to reduce the time to identify and isolate spermatozoa from surgical sperm samples from hours to minutes, thus increasing success rates from these treatments.
Subject(s)
Artificial Intelligence , Azoospermia , Sperm Injections, Intracytoplasmic , Spermatozoa , Humans , Male , Azoospermia/diagnosis , Azoospermia/therapy , Sperm Injections, Intracytoplasmic/methods , Neural Networks, Computer , Proof of Concept Study , Sperm Retrieval , AdultABSTRACT
BACKGROUND: Both non-obstructive azoospermia (NOA) and primary ovarian insufficiency (POI) are pathological conditions characterized by premature and frequently complete gametogenesis failure. Considering that the conserved meiosis I steps are the same between oogenesis and spermatogenesis, inherited defects in meiosis I may result in common causes for both POI and NOA. The present research is a retrospective investigation on an Iranian family with four siblings of both genders who were affected by primary gonadal failure. METHODS: Proband, an individual with NOA, was subjected to clinical examination, hormonal assessment, and genetic consultation. After reviewing the medical history of other infertile members of the family, patients with NOA went through genetic investigations including karyotyping and assessment of Y chromosome microdeletions, followed by Whole exome sequencing (WES) on the proband. After analyzing WES data, the candidate variant was validated using Sanger sequencing and traced in the family. RESULTS: WES analysis of the proband uncovered a novel homozygote nonsense variant, namely c.118C>T in MSH4. This variant resulted in the occurrence of a premature stop codon in residue 40 of MSH4. Notably, the variant was absent in all public exome databases and in the exome data of 400 fertile Iranian individuals. Additionally, the variant was found to co-segregate with infertility in the family. It was also observed that all affected members had homozygous mutations, while their parents were heterozygous and the fertile sister had no mutant allele, corresponding to autosomal recessive inheritance. In addition, we conducted a review of variants reported so far in MSH4, as well as available clinical features related to these variants. The results show that the testicular sperm retrieval and ovarian stimulation cycles have not been successful yet. CONCLUSION: Overall, the results of this study indicate that the identification of pathogenic variants in this gene will be beneficial in selecting proper therapeutic strategies. Also, the findings of this study demonstrate that clinicians should obtain the history of other family members of the opposite sex when diagnosing for POI and/or NOA.