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Despite continuous expansion of the RNA binding protein (RBP) world, there is a lack of systematic understanding of RBPs in the mammalian testis, which harbors one of the most complex tissue transcriptomes. We adapted RNA interactome capture to mouse male germ cells, building an RBP atlas characterized by multiple layers of dynamics along spermatogenesis. Trapping of RNA-cross-linked peptides showed that the glutamic acid-arginine (ER) patch, a residue-coevolved polyampholytic element present in coiled coils, enhances RNA binding of its host RBPs. Deletion of this element in NONO (non-POU domain-containing octamer-binding protein) led to a defective mitosis-to-meiosis transition due to compromised NONO-RNA interactions. Whole-exome sequencing of over 1000 infertile men revealed a prominent role of RBPs in the human genetic architecture of male infertility and identified risk ER patch variants.

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., (© 2024 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Purpose: This study aimed to identify the genetic causes of male infertility and primary ciliary dyskinesia (PCD)/PCD-like phenotypes in three unrelated Han Chinese families., Methods: We conducted whole-exome sequencing of three patients with male infertility and PCD/PCD-like phenotypes from three unrelated Chinese families. Ultrastructural and immunostaining analyses of patient spermatozoa and respiratory cilia and in vitro analyses were performed to analyze the effects of SPEF2 variants. Intracytoplasmic sperm injection (ICSI) was administered to three affected patients., Results: We identified four novel SPEF2 variants, including one novel homozygous splicing site variant [NC_000005.10(NM_024867.4): c.4447 + 1G > A] of the SPEF2 gene in family 1, novel compound heterozygous nonsense variants [NC_000005.10(NM_024867.4): c.1339C > T (p.R447*) and NC_000005.10(NM_024867.4): c.1645G > T (p.E549*)] in family 2, and one novel homozygous missense variant [NC_000005.10(NM_024867.4): c.2524G > A (p.D842N)] in family 3. All the patients presented with male infertility and PCD/likely PCD. All variants were present at very low levels in public databases, predicted to be deleterious in silico prediction tools, and were further confirmed deleterious by in vitro analyses. Ultrastructural analyses of the spermatozoa of the patients revealed the absence of the central pair complex in the sperm flagella. Immunostaining of the spermatozoa and respiratory cilia of the patients validated the pathogenicity of the SPEF2 variants. All patients carrying SPEF2 variants underwent one ICSI cycle and delivered healthy infants., Conclusion: Our study reported four novel pathogenic variants of SPEF2 in three male patients with infertility and PCD/PCD-like phenotypes, which not only extend the spectrum of SPEF2 mutations but also provide information for genetic counseling and treatment of such conditions., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To identify the genetic causes of multiple morphological abnormalities in sperm flagella (MMAF) and male infertility in patients from two unrelated Han Chinese families., Methods: Whole-exome sequencing was conducted using blood samples from the two individuals with MMAF and male infertility. Hematoxylin and eosin staining and scanning electron microscopy were performed to evaluate sperm morphology. Ultrastructural and immunostaining analyses of the spermatozoa were performed. The HEK293T cells were used to confirm the pathogenicity of the variants., Results: We identified two novel homozygous missense ARMC2 variants: c.314C > T: p.P105L and c.2227A > G: p.N743D. Both variants are absent or rare in the human population genome data and are predicted to be deleterious. In vitro experiments indicated that both ARMC2 variants caused a slightly increased protein expression. ARMC2-mutant spermatozoa showed multiple morphological abnormalities (bent, short, coiled, absent, and irregular) in the flagella. In addition, the spermatozoa of the patients revealed a frequent absence of the central pair complex and disrupted axonemal ultrastructure., Conclusion: We identified two novel ARMC2 variants that caused male infertility and MMAF in Han Chinese patients. These findings expand the mutational spectrum of ARMC2 and provide insights into the complex causes and pathogenesis of MMAF., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Individuals with 46,XX/XY chimerism can display a wide range of characteristics, varying from hermaphroditism to complete male or female, and can display sex chromosome chimerism in multiple tissues, including the gonads. The gonadal tissues of females contain both granulosa and germ cells. However, the specific sex chromosome composition of the granulosa and germ cells in 46,XX/XY chimeric female is currently unknown. Here, we reported a 30-year-old woman with secondary infertility who displayed a 46,XX/46,XY chimerism in the peripheral blood. FISH testing revealed varying degrees of XX/XY chimerism in multiple tissues of the female patient. Subsequently, the patient underwent preimplantation genetic testing (PGT) treatment, and 26 oocytes were retrieved. From the twenty-four biopsied mature oocytes, a total of 23 first polar bodies (PBs) and 10 second PBs were obtained. These PBs and two immature metaphase I (MI) oocytes only displayed X chromosome signals with no presence of the Y, suggesting that all oocytes in this chimeric female were of XX germ cell origin. On the other hand, granulosa cells obtained from individual follicles exhibited varied proportions of XX/XY cell types, and six follicles possessed 100% XX or XY granulosa cells. A total of 24 oocytes were successfully fertilized, and 12 developed into blastocysts, where 5 being XY and 5 were XX. Two blastocysts were transferred with one originating from an oocyte aspirated from a follicle containing 100% XY granulosa cells. This resulted in a twin pregnancy. Subsequent prenatal diagnosis confirmed normal male and female karyotypes. Ultimately, healthy boy-girl twins were delivered at full term. In summary, this 46,XX/XY chimerism with XX germ cells presented complete female, suggesting that germ cells may exert a significant influence on the sexual determination of an individual, which provide valuable insights into the intricate processes associated with sexual development and reproduction., (© 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.)

Purpose: The preimplantation genetic testing for aneuploidy (PGT-A) platform is not currently available for small copy-number variants (CNVs), especially those < 1 Mb. Through strategies used in PGT for monogenic disease (PGT-M), this study intended to perform PGT for families with small pathogenic CNVs., Methods: Couples who carried small pathogenic CNVs and underwent PGT at the Reproductive and Genetic Hospital of CITIC-Xiangya (Hunan, China) between November 2019 and April 2023 were included in this study. Haplotype analysis was performed through two platforms (targeted sequencing and whole-genome arrays) to identify the unaffected embryos, which were subjected to transplantation. Prenatal diagnosis using amniotic fluid was performed during 18-20 weeks of pregnancy., Results: PGT was successfully performed for 20 small CNVs (15 microdeletions and 5 microduplications) in 20 families. These CNVs distributed on chromosomes 1, 2, 6, 7, 13, 15, 16, and X with sizes ranging from 57 to 2120 kb. Three haplotyping-based PGT-M strategies were applied. A total of 89 embryos were identified in 25 PGT cycles for the 20 families. The diagnostic yield was 98.9% (88/89). Nineteen transfers were performed for 17 women, resulting in a 78.9% (15/19) clinical pregnancy rate after each transplantation. Of the nine women who had healthy babies, eight accepted prenatal diagnosis and the results showed no related pathogenic CNVs., Conclusion: Our results show that the extended haplotyping-based PGT-M strategy application for small pathogenic CNVs compensated for the insufficient resolution of PGT-A. These three PGT-M strategies could be applied to couples with small pathogenic CNVs., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 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 this study, we report on mosaic variegated aneuploidy (MVA) syndrome with tetraploidy and predisposition to infertility in a family. Sequencing analysis identified that the CEP192 biallelic variants (c.1912C>T, p.His638Tyr and c.5750A>G, p.Asn1917Ser) segregated with microcephaly, short stature, limb-extremity dysplasia, and reduced testicular size, while CEP192 monoallelic variants segregated with infertility and/or reduced testicular size in the family. In 1,264 unrelated patients, variant screening for CEP192 identified a same variant (c.5750A>G, p.Asn1917Ser) and other variants significantly associated with infertility. Two lines of Cep192 mice model that are equivalent to human variants were generated. Embryos with Cep192 biallelic variants arrested at E7 because of cell apoptosis mediated by MVA/tetraploidy cell acumination. Mice with heterozygous variants replicated the predisposition to male infertility. Mouse primary embryonic fibroblasts with Cep192 biallelic variants cultured in vitro showed abnormal morphology, mitotic arresting, and disruption of spindle formation. In patient epithelial cells with biallelic variants cultured in vitro, the number of cells arrested during the prophase increased because of the failure of spindle formation. Accordingly, we present mutant CEP192, which is a link for the MVA syndrome with tetraploidy and the predisposition to male infertility., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Study Question: Are there other pathogenic genes for asthenoteratozoospermia (AT)?, Summary Answer: DNAH3 is a novel candidate gene for AT in humans and mice., What Is Known Already: AT is a major cause of male infertility. Several genes underlying AT have been reported; however, the genetic aetiology remains unknown in a majority of affected men., Study Design Size Duration: A total of 432 patients with AT were recruited in this study. DNAH3 mutations were identified by whole-exome sequencing (WES). Dnah3 knockout mice were generated using the genome editing tool. The morphology and motility of sperm from Dnah3 knockout mice were investigated. The entire study was conducted over 3 years., Participants/materials Setting Methods: WES was performed on 432 infertile patients with AT. In addition, two lines of Dnah3 knockout mice were generated. Haematoxylin and eosin (H&E) staining, transmission electron microscopy (TEM), immunostaining, and computer-aided sperm analysis (CASA) were performed to investigate the morphology and motility of the spermatozoa. ICSI was used to overcome the infertility of one patient and of the Dnah3 knockout mice., Main Results and the Role of Chance: DNAH3 biallelic variants were identified in three patients from three unrelated families. H&E staining revealed various morphological abnormalities in the flagella of sperm from the patients, and TEM and immunostaining further showed the loss of the central pair of microtubules, a dislocated mitochondrial sheath and fibrous sheath, as well as a partial absence of the inner dynein arms. In addition, the two Dnah3 knockout mouse lines demonstrated AT. One patient and the Dnah3 knockout mice showed good treatment outcomes after ICSI., Large Scale Data: N/A., Limitations Reasons for Caution: This is a preliminary report suggesting that defects in DNAH3 can lead to asthenoteratozoospermia in humans and mice. The pathogenic mechanism needs to be further examined in a future study., Wider Implications of the Findings: Our findings show that DNAH3 is a novel candidate gene for AT in humans and mice and provide crucial insights into the biological underpinnings of this disorder. The findings may also be beneficial for counselling affected individuals., Study Funding/competing Interests: This work was supported by grants from National Natural Science Foundation of China (82201773, 82101961, 82171608, 32322017, 82071697, and 81971447), National Key Research and Development Program of China (2022YFC2702604), Scientific Research Foundation of the Health Committee of Hunan Province (B202301039323, B202301039518), Hunan Provincial Natural Science Foundation (2023JJ30716), the Medical Innovation Project of Fujian Province (2020-CXB-051), the Science and Technology Project of Fujian Province (2023D017), China Postdoctoral Science Foundation (2022M711119), and Guilin technology project for people's benefit (20180106-4-7). The authors declare no competing interests., Competing Interests: All authors declare no conflict of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of European Society of Human Reproduction and Embryology.)

Objective: To evaluate the clinical outcomes in the carriers of insertional translocation (IT)., Design: Retrospective case series., Setting: University-affiliated reproductive medical center., Patients: Twenty-three couples with ITs., Intervention: No direct interventions were involved; however, this study included patients who underwent preimplantation genetic testing for structural chromosomal rearrangements (PGT-SR)., Main Outcome Measure: Outcome of preimplantation genetic testing for structural chromosomal rearrangements and percentage of blastocysts available for transfer., Results: Among 23 IT carriers, 15 were simple interchromosome ITs (type A), 3 were intrachromosome IT carriers (type B), and 5 were interchromosome IT carriers combined with other translocations (type C). A total of 190 blastocysts from 30 cycles were biopsied, 187 embryos were tested successfully, and only 57 blastocysts (30.5%) from 21 patients were available for transfer (normal or balanced). The unbalanced rearrangement rate of type C was 79.2% (42/53), and the proportion of type A was 50.0% (57/114), which was significantly higher than that of type B (5%, 1/20). In type A, the probability of embryos harboring unbalanced rearrangement in female carriers was 56.0% (51/91), which was higher than that in male carriers (26.1%, 6/23). Furthermore, the haploid autosomal length value of the inserted fragment was correlated linearly with the incidence of abnormal embryos. In type A gametes, most gametes produced by 2:2 separation without crossover, and no 3:1 separation gamete was observed., Conclusions: The chance of identifying normal or balanced and mosaic blastocysts per mature oocytes in patients with ITs are 16.6% (67/404). Greater IT complexity results in fewer transferable embryos. For simple interchromosome ITs, female carriers and those with higher haploid autosomal length values have a higher risk of producing embryos with unbalanced rearrangement., Competing Interests: Z.Z. has nothing to disclose. K.L. has nothing to disclose. Se.Z. has nothing to disclose. D.C. has nothing to disclose. L.H. has nothing to disclose. Y.-Q.T. has nothing to disclose. Sh.Z. has nothing to disclose. F.G. has nothing to disclose. P.X. has nothing to disclose. G.L. has nothing to disclose., (© 2023 The Authors.)

Male infertility is a major reproductive disorder, which is clinically characterized by highly heterogeneous phenotypes of abnormal sperm count or quality. To date, five male patients with biallelic loss-of-function (LOF) variants of PARN-like ribonuclease domain-containing exonuclease 1 ( PNLDC1 ) have been reported to experience infertility with nonobstructive azoospermia. The aim of this study was to identify the genetic cause of male infertility with oligo-astheno-teratozoospermia (OAT) in a patient from a Chinese Han family. Whole-exome and Sanger sequencing analyses identified a homozygous LOF variant (NM&#95;173516.2, c.142C>T, p.Gln48Ter) in PNLDC1 . Hematoxylin and eosin staining revealed that the spermatozoa of the patient with OAT had an irregular head phenotype, including microcephaly, head tapering, and globozoospermia. Consistently, peanut agglutinin staining of the spermatozoa revealed a complete or partial loss of the acrosome. Furthermore, the disomy rate of chromosomes in the patient's spermatozoa was significantly increased compared with that of a fertile control sample. We reported an LOF variant of the PNLDC1 gene responsible for OAT., (Copyright © 2023 Copyright: © The Author(s)(2023).)

Background: Spermatogenic impairments can lead to male infertility by different pathological conditions, such as multiple morphological abnormalities of the sperm flagella (MMAF) and non-obstructive azoospermia (NOA). Genetic factors are involved in impaired spermatogenesis., Methods and Results: Here, we performed genetic analyses through whole-exome sequencing in a cohort of 334 Han Chinese probands with severe MMAF or NOA. Biallelic variants of CFAP54 were identified in three unrelated men, including one homozygous frameshift variant (c.3317del, p.Phe1106Serfs*19) and two compound heterozygous variants (c.878G>A, p.Arg293His; c.955C>T, p.Arg319Cys and c.4885C>T, p.Arg1629Cys; c.937G>A, p.Gly313Arg). All of the identified variants were absent or extremely rare in the public human genome databases and predicted to be damaging by bioinformatic tools. The men harbouring CFAP54 mutations exhibited abnormal sperm morphology, reduced sperm concentration and motility in ejaculated semen. Significant axoneme disorganisation and other ultrastructure abnormities were also detected inside the sperm cells from men harbouring CFAP54 mutations. Furthermore, immunofluorescence assays showed remarkably reduced staining of four flagellar assembly-associated proteins (IFT20, IFT52, IFT122 and SPEF2) in the spermatozoa of CFAP54- deficient men. Notably, favourable clinical pregnancy outcomes were achieved with sperm from men carrying CFAP54 mutations after intracytoplasmic sperm injection treatment., Conclusion: Our genetic analyses and experimental observations revealed that biallelic deleterious mutations of CFAP54 can induce severe MMAF and NOA in humans., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Study Question: Can whole-exome sequencing (WES) reveal new genetic factors responsible for male infertility characterized by oligozoospermia?, Summary Answer: We identified biallelic missense variants in the Potassium Channel Tetramerization Domain Containing 19 gene (KCTD19) and confirmed it to be a novel pathogenic gene for male infertility., What Is Known Already: KCTD19 is a key transcriptional regulator that plays an indispensable role in male fertility by regulating meiotic progression. Kctd19 gene-disrupted male mice exhibit infertility due to meiotic arrest., Study Design, Size, Duration: We recruited a cohort of 536 individuals with idiopathic oligozoospermia from 2014 to 2022 and focused on five infertile males from three unrelated families. Semen analysis data and ICSI outcomes were collected. WES and homozygosity mapping were performed to identify potential pathogenic variants. The pathogenicity of the identified variants was investigated in silico and in vitro., Participants/materials, Setting, Methods: Male patients diagnosed with primary infertility were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. Genomic DNA extracted from affected individuals was used for WES and Sanger sequencing. Sperm phenotype, sperm nuclear maturity, chromosome aneuploidy, and sperm ultrastructure were assessed using hematoxylin and eosin staining and toluidine blue staining, FISH and transmission electron microscopy. The functional effects of the identified variants in HEK293T cells were investigated via western blotting and immunofluorescence., Main Results and the Role of Chance: We identified three homozygous missense variants (NM&#95;001100915, c.G628A:p.E210K, c.C893T:p.P298L, and c.G2309A:p.G770D) in KCTD19 in five infertile males from three unrelated families. Abnormal morphology of the sperm heads with immature nuclei and/or nuclear aneuploidy were frequently observed in individuals with biallelic KCTD19 variants, and ICSI was unable to rescue these deficiencies. These variants reduced the abundance of KCTD19 due to increased ubiquitination and impaired its nuclear colocalization with its functional partner, zinc finger protein 541 (ZFP541), in HEK293T cells., Limitations, Reasons for Caution: The exact pathogenic mechanism remains unclear, and warrants further studies using knock-in mice that mimic the missense mutations found in individuals with biallelic KCTD19 variants., Wider Implications of the Findings: Our study is the first to report a likely causal relationship between KCTD19 deficiency and male infertility, confirming the critical role of KCTD19 in human reproduction. Additionally, this study provided evidence for the poor ICSI clinical outcomes in individuals with biallelic KCTD19 variants, which may guide clinical treatment strategies., Study Funding/competing Interest(s): This work was supported by the National Key Research and Developmental Program of China (2022YFC2702604 to Y.-Q.T.), the National Natural Science Foundation of China (81971447 and 82171608 to Y.-Q.T., 82101961 to C.T.), a key grant from the Prevention and Treatment of Birth Defects from Hunan Province (2019SK1012 to Y.-Q.T.), a Hunan Provincial Grant for Innovative Province Construction (2019SK4012), and the China Postdoctoral Science Foundation (2022M721124 to W.W.). The authors declare no conflicts of interest., Trial Registration Number: N/A., (© The Author(s) 2023. 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.)

PIWI-clade proteins harness piRNAs of 24-33 nt in length. Of great puzzles are how PIWI-clade proteins incorporate piRNAs of different sizes and whether the size matters to PIWI/piRNA function. Here we report that a PIWI-Ins module unique in PIWI-clade proteins helps define the length of piRNAs. Deletion of PIWI-Ins in Miwi shifts MIWI to load with shorter piRNAs and causes spermiogenic failure in mice, demonstrating the functional importance of this regulatory module. Mechanistically, we show that longer piRNAs provide additional complementarity to target mRNAs, thereby enhancing the assembly of the MIWI/eIF3f/HuR super-complex for translational activation. Importantly, we identify a c.1108C>T (p.R370W) mutation of HIWI (human PIWIL1) in infertile men and demonstrate in Miwi knock-in mice that this genetic mutation impairs male fertility by altering the property of PIWI-Ins in selecting longer piRNAs. These findings reveal a critical role of PIWI-Ins-ensured longer piRNAs in fine-tuning MIWI/piRNA targeting capacity, proven essential for spermatid development and male fertility., (© 2023. Science China Press.)

Introduction: Tracing the genetic causes for male infertility due to asthenoteratozoospermia has revealed at least 40 causative genes, which provides valuable reference for the genetic testing of asthenoteratozoospermia in clinical practice. To identify deleterious variants in the human tetratricopeptide repeat domain 12 (TTC12) gene in a large cohort of infertile Chinese males with asthenoteratozoospermia. Methods: A total of 314 unrelated asthenoteratozoospermia-affected men were recruited for whole exome sequencing. The effects of the identified variants were evaluated by in silico analysis, and confirmed by in vitro experiments. Intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of assisted reproduction technique therapy. Results and Discussion: Novel homozygous TTC12 variants (c.1467&#95;1467delG (p.Asp490Thrfs*14), c.1139&#95;1139delA (p.His380Profs*4), and c.1117G>A (p.Gly373Arg)) were identified in three (0.96%) of the 314 cases. Three mutants were indicated to be damaging using in silico prediction tools, and were further confirmed by in vitro functional analysis. Hematoxylin and eosin staining and ultrastructural observation of the spermatozoa revealed multiple morphological abnormalities of flagella, with the absence of outer and inner dynein arms. Notably, significant mitochondrial sheath malformations were also observed in the sperm flagella. Immunostaining assays indicated that TTC12 is present throughout the flagella, and was strongly concentrated in the mid-piece in control spermatozoa. However, spermatozoa from TTC12 -mutated individuals exhibited almost no staining intensity of TTC12 and outer and inner dynein arms components. The three men accepted ICSI treatment using their ejaculated spermatozoa, and two female partners successfully delivered healthy babies. Our findings provide direct genetic evidence that homozygous variants in TTC12 cause male infertility with asthenoteratozoospermia by causing dynein arm complex defects and mitochondrial sheath malformations in the flagellar. We also demonstrated that TTC12 deficiency-mediated infertility could be overcome by ICSI technology., 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 © 2023 Meng, Liu, Tan, Xu, He, Hu, Tu, Li, Du, Zhang, Lu, Fan, Lin, Nie, Zhang and Tan.)

Male infertility affects approximately 7% of childbearing couples and is a major health issue. Although nearly 50% idiopathic infertile men are assumed to have a genetic basis, the underlying causes remain largely unknown in most infertility cases. Here, we report two rare homozygous variants in two previously uncharacterized genes, C9orf131 and C10orf120, identified in two unrelated men with asthenozoospermia. Both genes were predominantly expressed in the testes. Furthermore, C9orf131 and C10orf120 knockout mice were successfully generated using the CRISPR-Cas9 technology. However, both C9orf131 -/- and C10orf120 -/- adult male mice were fertile, with testis-to-body weight ratios comparable to those of wild-type mice. No overt differences were found between wild-type, C9orf131 -/- , and C10orf120 -/- mice regarding testicular/epididymal tissue morphology, sperm count, sperm motility, or sperm morphology. Moreover, TUNEL assays indicated that the number of apoptotic germ cells in testes was not significantly different between the three groups. In summary, these findings suggest that C9orf131 and C10orf120 are redundant genes in male infertility., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Primate-specific genes (PSGs) tend to be expressed in the brain and testis. This phenomenon is consistent with brain evolution in primates but is seemingly contradictory to the similarity of spermatogenesis among mammals. Here, using whole-exome sequencing, we identified deleterious variants of X-linked SSX1 in six unrelated men with asthenoteratozoospermia. SSX1 is a PSG expressed predominantly in the testis, and the SSX family evolutionarily expanded independently in rodents and primates. As the mouse model could not be used for studying SSX1, we used a non-human primate model and tree shrews, which are phylogenetically similar to primates, to knock down (KD) Ssx1 expression in the testes. Consistent with the phenotype observed in humans, both Ssx1-KD models exhibited a reduced sperm motility and abnormal sperm morphology. Further, RNA sequencing indicated that Ssx1 deficiency influenced multiple biological processes during spermatogenesis. Collectively, our experimental observations in humans and cynomolgus monkey and tree shrew models highlight the crucial role of SSX1 in spermatogenesis. Notably, three of the five couples who underwent intra-cytoplasmic sperm injection treatment achieved a successful pregnancy. This study provides important guidance for genetic counseling and clinical diagnosis and, significantly, describes the approaches for elucidating the functions of testis-enriched PSGs in spermatogenesis., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: The genetic causes for most male infertility due to severe oligoasthenoteratozoospermia (OAT) remain unclear., Objective: To identify the genetic cause of male infertility characterised by OAT., Methods: Variant screening was performed by whole-exome sequencing from 325 infertile patients with OAT and 392 fertile individuals. In silico and in vitro analyses were performed to evaluate the impacts of candidate disease-causing variants. A knockout mouse model was generated to confirm the candidate disease-causing gene, and intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of clinical treatment., Results: We identified biallelic CFAP61 variants (NM&#95;015585.4: c.1654C>T (p.R552C) and c.2911G>A (p.D971N), c.144-2A>G and c.1666G>A (p.G556R)) in two (0.62%) of the 325 OAT-affected men. In silico bioinformatics analysis predicted that all four variants were deleterious, and in vitro functional analysis confirmed the deleterious effects of the mutants. Notably, H&E staining and electron microscopy analyses of the spermatozoa revealed multiple morphological abnormalities of sperm flagella, the absence of central pair microtubules and mitochondrial sheath malformation in sperm flagella from man with CFAP61 variants. Further immunofluorescence assays revealed markedly reduced CFAP61 staining in the sperm flagella. In addition, Cfap61 -deficient mice showed the OAT phenotype, suggesting that loss of function of CFAP61 was the cause of OAT. Two individuals accepted ICSI therapy using their own ejaculated sperm, and one of them succeeded in fathering a healthy baby., Conclusions: Our findings indicate that CFAP61 is essential for spermatogenesis and that biallelic CFAP61 variants lead to male infertility in humans and mice with OAT., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Congenital bilateral absence of the vas deferens (CBAVD) is observed in 1%-2% of males presenting with infertility and is clearly associated with cystic fibrosis transmembrane conductance regulator (CFTR) mutations. CFTR is one of the most well-known genes related to male fertility. The frequency of CFTR mutations or impaired CFTR expression is increased in men with nonobstructive azoospermia (NOA). CFTR mutations are highly polymorphic and have established ethnic specificity. Compared with F508Del in Caucasians, the p.G970D mutation is reported to be the most frequent CFTR mutation in Chinese patients with cystic fibrosis. However, whether p.G970D participates in male infertility remains unknown. Herein, a loss-of-function CFTR p.G970D missense mutation was identified in a patient with CBAVD and NOA. Subsequent retrospective analysis of 122 Chinese patients with CBAVD showed that the mutation is a common pathogenic mutation (4.1%, 5/122), excluding polymorphic sites. Furthermore, we generated model cell lines derived from mouse testes harboring the homozygous Cftr p.G965D mutation equivalent to the CFTR variant in patients. The Cftr p.G965D mutation may be lethal in spermatogonial stem cells and spermatogonia and affect the proliferation of spermatocytes and Sertoli cells. In spermatocyte GC-2(spd)ts (GC2) Cftr p.G965D cells, RNA splicing variants were detected and CFTR expression decreased, which may contribute to the phenotypes associated with impaired spermatogenesis. Thus, this study indicated that the CFTR p.G970D missense mutation might be a pathogenic mutation for CBAVD in Chinese males and associated with impaired spermatogenesis by affecting the proliferation of germ cells., Competing Interests: None

Purpose: The aim of this study is to identify the genetic cause of primary ciliary dyskinesia (PCD) and male infertility in two unrelated Han Chinese families., Methods: We performed whole-exome sequencing in two unrelated male Han Chinese patients suffering from infertility and PCD to identify the pathogenic variants. Ultrastructural and immunostaining analyses of patient's spermatozoa were performed to characterize the effect of the variants. The pathogenicity of the variants was validated using patient's spermatozoa by western blotting and immunostaining analysis. Intracytoplasmic sperm injection (ICSI) was conducted in the affected families., Results: Three variants in leucine-rich repeat containing 6 (LRRC6) [patient 1(compound heterozygote): NM&#95;012472: c.538C > T, (p.R180*) and c.64dupT, (p.S22Ffs*19); patient 2 (homozygote): c.863C > A, (p.P288H)] were identified in two unrelated patients with PCD and male infertility. These variants were predicated deleterious and were absent or rare in human population genome data. LRRC6-mutant spermatozoa showed a highly aberrant morphology and ultrastructure with lacked inner and outer dynein arms. The LRRC6 protein was present along the normal sperm flagella, and was significantly decreased in the mutated spermatozoa. Interestingly, both patients were able to conceive through ICSI and birthed a healthy baby., Conclusion: Our results extend the LRRC6 variant spectrum and provide reproductive guidance to families suffering from PCD-linked infertility caused by LRRC6 variants., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

The establishment and maintenance of pregnancy are involved in maternal-fetal immune tolerance whose imbalance can lead to recurrent spontaneous abortion (RSA). RSA is defined as two or more clinically recognized pregnancy losses within 20-24 weeks of gestation with the same partner, including embryonic and fetal losses. However, approximately half of RSA cases are idiopathic, which may be related to immune aberrations. T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is an inhibitory checkpoint protein that plays a critical role in immune tolerance. Several studies have reported that Tim-3 expression in immune cells is important for maintaining maternal-fetal immune tolerance and that the abnormal expression of Tim-3 may be associated with RSA. To further understand the etiology and pathogenesis of RSA and inspire novel strategies for its diagnosis and treatment, we reviewed the research progress on the Tim-3-induced regulation of natural killer cells, T cells, macrophages, dendritic cells, and myeloid-derived suppressor cells in maternal-fetal immune tolerance and RSA., Competing Interests: Declaration of Competing Interest The authors declare that there is in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Background: Meiosis is an essential stage in the life cycle of sexually reproducing species, underlying formation of haploid gametes and serving as the basis of genetic diversity. A central mechanism of meiosis is recombination between homologous chromosomes, during which programmed DNA double-strand breaks (DSBs) are sequentially repaired to form the crossovers essential for faithful chromosomal segregation. Aberrant meiotic recombination often leads to gametogenic failure or produces aneuploid gametes resulting in subfertility or infertility, miscarriage or birth defects., Objective and Rationale: The goal of this review was to characterize the molecular mechanisms of meiotic recombination and related human infertility disorders, particularly male infertility caused by non-obstructive azoospermia (NOA)., Search Methods: Our search included PubMed database articles, focusing mainly on English-language publications dated between January 2016 and February 2022. The search term 'meiosis' was combined with the following keywords: meiotic initiation, chromosome pairing, homologous recombination, chromosome axis, DSB, DSB repair, crossover, meiotic sex chromosome inactivation, meiotic checkpoints, meiotic arrest, NOA, premature ovarian insufficiency (POI) or premature ovarian failure, treatment and cancer. In addition, references within these articles were used to identify additional studies., Outcomes: The preliminary search generated ∼3500 records. The majority of articles were identified as meeting abstracts or duplicates, contained non-English text or provided insufficient data and were therefore eliminated. A total of 271 articles associated with meiotic recombination were included in the final analysis. This review provides an overview of molecules and mechanisms involved in meiotic recombination processes, specifically meiosis-specific chromosome structures, DSB formation, homology search, formation of recombination intermediates and crossover formation. The cumulative results suggest that meiosis is regulated sequentially by a series of meiotic recombination genes and proteins. Importantly, mutations in these genes often affect meiotic progression, activating meiotic checkpoints, causing germ cell arrest and leading to subfertility or infertility. At least 26 meiotic recombination-related genes have been reported to be mutated in NOA in men, and 10 of these genes are mutated in POI in women. This suggests that variants of meiotic recombination-related genes can cause human subfertility or infertility, especially NOA., Wider Implications: Understanding the processes of homologous chromosome pairing, recombination and timely resolution of homologous chromosomes may provide guidance for the analysis of potential monogenetic causes of human subfertility or infertility and the development of personalized treatments. In clinical practice, we can develop a meiotic recombination-related gene panel to screen for gene mutations in individuals with subfertility or infertility. Testicular sperm extraction should not be recommended when an NOA-affected individual carries definite disease-causing mutations of a meiotic gene, so as to avoid the unnecessary invasive diagnosis. Risk of ovarian dysfunction should be evaluated if a woman carries meiotic recombination-related gene mutations. It may be possible to improve or restore fertility through manipulation of meiotic recombination-related genes in the future., (© 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.)

Purpose: This study aimed to investigate the spectrum and characteristics of segmental aneuploidies (SAs) of <10 megabase (Mb) length in human preimplantation blastocysts., Methods: Preimplantation genetic testing for aneuploidy was performed in 15,411 blastocysts from 5171 patients using a validated 1 Mb resolution platform. The characteristics and spectrum of SAs, including the incidence, sizes, type, inheritance pattern, clinical significance, and embryo distribution, were studied., Results: In total, 6.4% of the 15,411 blastocysts carried SAs of >10 Mb, 4.9% of embryos had SAs ranging between 1 to 10 Mb, and 84.3% of 1 to 10 Mb SAs were <5 Mb in size. Inheritance pattern analysis indicated that approximately 63.8% of 1 to 10 Mb SAs were inherited and were predominantly 1 to 3 Mb in size. Furthermore, 18.4% of inherited SAs and 51.9% de novo 1 to 10 Mb SAs were pathogenic or likely pathogenic (P/LP). Different from whole-chromosome aneuploidies, reanalysis indicated that 50% of the de novo 1 to 10 Mb SAs and 70% of the >10 Mb SAs arose from mitotic errors., Conclusion: Based on the established platform, 1 to 10 Mb SAs are common in blastocysts and include a subset of P/LP SAs. Inheritance pattern analysis and clinical interpretation based on the American College of Medical Genetics and Genomics/Association for Molecular Pathology guidelines contributed to determine the P/LP SAs., Competing Interests: Conflict of Interest The authors declare no conflict of interest., (Copyright © 2022 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Background: Numerous variants of uncertain significance (VUSs) have been identified by whole exome sequencing in clinical practice. However, VUSs are not currently considered medically actionable., Objective: To assess the splicing patterns of 49 VUSs in 48 families identified clinically to improve genetic counselling and family planning., Methods: Forty-nine participants with 49 VUSs were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. Bioinformatic analysis was performed to preliminarily predict the splicing effects of these VUSs. RT-PCR and minigene analysis were used to assess the splicing patterns of the VUSs. According to the results obtained, couples opted for different methods of reproductive interventions to conceive a child, including prenatal diagnosis and preimplantation genetic testing (PGT)., Results: Eleven variants were found to alter pre-mRNA splicing and one variant caused nonsense-mediated mRNA decay, which resulted in the reclassification of these VUSs as likely pathogenic. One couple chose to undergo in vitro fertilisation with PGT treatment; a healthy embryo was transferred and the pregnancy is ongoing. Three couples opted for natural pregnancy with prenatal diagnosis. One couple terminated the pregnancy because the fetus was affected by short-rib thoracic dysplasia and harboured the related variant. The infants of the other two couples were born and were healthy at their last recorded follow-up., Conclusion: RNA splicing analysis is an important method to assess the impact of sequence variants on splicing in clinical practice and can contribute to the reclassification of a significant proportion of VUSs. RNA splicing analysis should be considered for genetic disease diagnostics., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: Previous studies suggested that non-invasive preimplantation genetic testing (niPGT) for intracytoplasmic sperm injection (ICSI) blastocysts can be used to identify chromosomal ploidy and chromosomal abnormalities. Here, we report the feasibility and performance of niPGT for conventional in vitro fertilization (IVF) blastocysts., Methods: This was a prospective observational study. In the preclinical stage, whole genome amplification and NGS were performed using the sperm spent culture medium (SCM). Then, trophectoderm (TE) biopsies and corresponding SCM derived from 27 conventional IVF monopronuclear embryos were collected. In the clinical stage, samples from 25 conventional IVF cycles and 37 ICSI cycles from April 2020-August 2021 were collected for performance evaluation., Results: Preclinically, we confirmed failed sperm DNA amplification under the current amplification system. Subsequent niPGT from the 27 monopronuclear blastocysts showed 69.2% concordance with PGT results of corresponding TE biopsies. In the clinical stage, no paternal contamination was observed in any of the 161 SCM samples from conventional IVF. While maternal contamination was observed in 29.8% (48/161) SCM samples, only 2.5% (4/161) samples had a contamination ratio ≥ 50%. Compared with that of TE biopsy, the performances of NiPGT from 161 conventional IVF embryos and 122 ICSI embryos were not significantly different (P > 0.05), with ploidy concordance rates of 75% and 74.6% for IVF and ICSI methods, respectively. Finally, evaluation of the euploid probability of embryos with different types of niPGT results showed prediction probabilities of 82.8%, 77.8%, 62.5%, 50.0%, 40.9% and 18.4% for euploidy, sex-chromosome mosaics only, low-level mosaics, multiple abnormal chromosomes, high-level mosaics and aneuploidy, respectively., Conclusions: Our research results preliminarily confirm that the niPGT approach using SCM from conventional IVF has comparable performance with ICSI and might broadening the application scope of niPGT., (© 2022. The Author(s).)

Background: Infertility is a global health concern. MEIOB has been found to be associated with premature ovarian insufficiency (POI) and non-obstructive azoospermia (NOA), but its variants have not been reported in Chinese patients. The aim of this study was to identify the genetic aetiology of POI or NOA in three Han Chinese families. Methods: Whole-exome sequencing (WES) was used to identify candidate pathogenic variants in three consanguineous Chinese infertile families with POI or NOA. Sanger sequencing was performed to validate these variants in the proband of family I and her affected family members. In vitro functional analyses were performed to confirm the effects of these variants. Results: Two novel homozygous frameshift variants (c.258&#95;259del and c.1072&#95;1073del) and one novel homozygous nonsense variant (c.814C > T) in the MEIOB gene were identified in three consanguineous Han Chinese families. In vitro functional analyses revealed that these variants produced truncated proteins and affected their function. Conclusion: We identified three novel MEIOB loss-of-function variants in local Chinese patients for the first time and confirmed their pathogenicity using in vitro functional analyses. These results extend the mutation spectrum of the MEIOB gene and have important significance for genetic counselling in these families., 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 © 2022 Wang, Liu, Tan, Meng, Meng, Nie, Du, Lu, Lin, He and Tan.)

Purpose: To evaluate the cytogenetic risk of assisted reproductive technology (ART) by comparing the incidence of de novo chromosomal abnormalities between fetuses conceived via in vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) and natural conception., Materials and Methods: Prenatal invasive diagnostic testing (amniocentesis and cytogenetic analysis) was performed on 1496 fetuses conceived via IVF/ICSI (IVF/ICSI group) and 1396 fetuses from natural conception (NC group). The incidence of de novo chromosomal abnormalities (including aneuploidy and chromosomal structure abnormalities) was used to evaluate the cytogenetic risk of ART. For statistical analysis, χ 2 -test was used for binary dependent variable. The significance level was P < 0.05 and confidence interval was 95%., Result(s): The IVF/ICSI group displayed a modest increase in the overall de novo chromosomal abnormality rate compared with that in the NC group but with no statistical significance (6.75% vs. 6.16%; χ 2  = 0.42, P > 0.05). The incidence of abnormal karyotypes was also not significantly different between the IVF/ICSI and NC groups in different maternal ages, including ≥ 35 years group (7.55% vs. 9.60%, χ 2  = 1.40, P > 0.05) and < 35 years group (6.20% vs. 4.54%, χ 2  = 2.51, P > 0.05). Moreover, there was no difference in the proportion of aneuploid and structural abnormalities in detected karyotypes between the IVF/ICSI and NC groups. Logistic regression analysis showed no significant association between the method of pregnancy and de novo chromosomal abnormalities (odds ratio (OR) 1.03; 95% CI 0.71-1.50; P = 0.86) after adjusting for other confounding factors., Conclusion(s): Fetuses conceived via IVF/ICSI had a slight but not statistically significant increase in de novo abnormal karyotypes compared to those in naturally conceived fetuses. Our findings indicate no significant association between de novo fetal chromosomal abnormalities and the pregnancy method in high-risk pregnancies in the second trimester. For these pregnancies with a high risk but with a normal karyotype, further genetic testing is required for diagnosis., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Spermiogenesis is a complex and tightly regulated process, consisting of acrosomal biogenesis, condensation of chromatin, flagellar assembly, and disposal of extra cytoplasm. Previous studies have reported that sperm flagellar 2 (SPEF2) deficiency causes severe asthenoteratozoospermia owing to spermiogenesis failure, but the underlying molecular mechanism in humans remains unclear. Here, we performed proteomic analysis on spermatozoa from three SPEF2 mutant patients to study the functional role of SPEF2 during sperm tail development. A total of 1262 differentially expressed proteins were detected, including 486 upregulated and 776 downregulated. The constructed heat map of the differentially expressed proteins showed similar trends. Among these, the expression of proteins related to flagellar assembly, including SPEF2, sperm associated antigen 6 (SPAG6), dynein light chain tctex-type 1 (DYNLT1), radial spoke head component 1 (RSPH1), translocase of outer mitochondrial membrane 20 (TOM20), EF-hand domain containing 1 (EFHC1), meiosis-specific nuclear structural 1 (MNS1) and intraflagellar transport 20 (IFT20), was verified by western blot. Functional clustering analysis indicated that these differentially expressed proteins were specifically enriched for terms such as spermatid development and flagellar assembly. Furthermore, we showed that SPEF2 interacts with radial spoke head component 9 (RSPH9) and IFT20 in vitro, which are well-studied components of radial spokes or intra-flagellar transport and are essential for flagellar assembly. These results provide a rich resource for further investigation into the molecular mechanism underlying the role that SPEF2 plays in sperm tail development and could provide a theoretical basis for gene therapy in SPEF2 mutant patients in the future., Competing Interests: None

Reciprocal translocations are the most common structural chromosome rearrangements and may be associated with reproductive problems. Therefore, the objective of this study was to analyze factors that can influence meiotic segregation patterns in blastocysts for reciprocal translocation carriers. Segregation patterns of quadrivalents in 10,846 blastocysts from 2,871 preimplantation genetic testing cycles of reciprocal translocation carriers were analyzed. The percentage of normal/balanced blastocysts was 34.3%, and 2:2 segregation was observed in 90.0% of the blastocysts. Increased TAR1 (ratio of translocated segment 1 over the chromosome arm) emerged as an independent protective factor associated with an increase in alternate segregation (p = 0.004). Female sex and involvement of an acrocentric chromosome (Acr-ch) were independent risk factors that reduced alternate segregation proportions (p < 0.001). Notably, a higher TAR1 reduced the proportion of adjacent-1 segregation (p < 0.001); a longer translocated segment and female sex increased the risk of adjacent-2 segregation (p = 0.009 and p < 0.001, respectively). Female sex and involvement of an Acr-ch enhanced the ratio of 3:1 segregation (p < 0.001 and p = 0.012, respectively). In conclusion, autosomal reciprocal translocation carriers have reduced proportions of alternate segregation in blastocysts upon the involvement of an Acr-ch, female sex, and lower TAR1. These results may facilitate more appropriate genetic counseling for couples with autosomal reciprocal translocation regarding their chances of producing normal/balanced blastocysts., 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 © 2022 Xie, Hu, Peng, Tan, Luo, Gong, Lu and Lin.)

Meiosis is pivotal to gametogenesis and fertility. Meiotic recombination is a mandatory process that ensures faithful chromosome segregation and generates genetic diversity in gametes. Non-obstructive azoospermia (NOA) caused by meiotic arrest is a common cause of male infertility and has many genetic origins, including chromosome abnormalities, Y chromosome microdeletion and monogenic mutations. However, the genetic causes of the majority of NOA cases remain to be elucidated. Here, we report our findings of three Shortage in chiasmata 1 (SHOC1) bi-allelic variants in three NOA patients, of which two are homozygous for the same loss-of-function variant (c.231&#95;232del: p.L78Sfs*9), and one is heterozygous for two different missense variants (c.1978G>A: p.A660T; c.4274G>A: p.R1425H). Testicular biopsy of one patient revealed impairment of spermatocyte maturation. Both germ-cell-specific and general Shoc1-knockout mice exhibited similar male infertility phenotypes. Subsequent analysis revealed comprehensive defects in homologous pairing and synapsis along with abnormal expression of DMC1, RAD51 and RPA2 in Shoc1-defective spermatocyte spreads. These findings imply that SHOC1 may have a presynaptic function during meiotic recombination apart from its previously identified role in crossover formation. Overall, our results provide strong evidence for the clinical relevance of SHOC1 mutations in patients with NOA and contribute to a deeper mechanistic understanding of the role of SHOC1 during meiotic recombination., (© 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.)

Background: Amelogenesis imperfecta (AI) is known to be a monogenic genetic disease caused by a variety of genes demonstrating a wide spectrum of penetrance. FAM83H is reported to be involved in AI: however, whether FAM83H causes AI with incomplete penetrance is unclear., Methods: Whole-exome sequencing was performed on two patients with AI, and putative disease-related variants were validated by Sanger sequencing. Bioinformatic and in vitro functional analyses were performed to functionally characterize the identified disease-causing variants., Results: We identified a novel heterozygous nonsense variant of FAM83H (NM&#95;198488: c.1975G > T, p.Glu659Ter); in vitro functional analysis showed that this mutant produced mislocalized proteins and was deleterious. Surprisingly, the clinical manifestations of each of the six individuals carrying this variant were different, with one carrier appearing to be completely asymptomatic for AI., Conclusion: Our findings expand the variant spectrum for FAM83H and the phenotypic spectrum for FAM83H-associated AI and suggest that FAM83H-mediated AI exhibits incomplete penetrance., (© 2022 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Asthenoteratozoospermia, defined as reduced sperm motility and abnormal sperm morphology, is a disorder with considerable genetic heterogeneity. Although previous studies have identified several asthenoteratozoospermia-associated genes, the etiology remains unknown for the majority of affected men. Here, we performed whole-exome sequencing on 497 unrelated men with asthenoteratozoospermia and identified DNHD1 bi-allelic variants from eight families (1.6%). All detected variants were predicted to be deleterious via multiple bioinformatics tools. Hematoxylin and eosin (H&E) staining revealed that individuals with bi-allelic DNHD1 variants presented striking abnormalities of the flagella; transmission electron microscopy (TEM) further showed flagellar axoneme defects, including central pair microtubule (CP) deficiency and mitochondrial sheath (MS) malformations. In sperm from fertile men, DNHD1 was localized to the entire flagella of the normal sperm; however, it was nearly absent in the flagella of men with bi-allelic DNHD1 variants. Moreover, abundance of the CP markers SPAG6 and SPEF2 was significantly reduced in spermatozoa from men harboring bi-allelic DNHD1 variants. In addition, Dnhd1 knockout male mice (Dnhd1 ‒/‒ ) exhibited asthenoteratozoospermia and infertility, a finding consistent with the sperm phenotypes present in human subjects with DNHD1 variants. The female partners of four out of seven men who underwent intracytoplasmic sperm injection therapy subsequently became pregnant. In conclusion, our study showed that bi-allelic DNHD1 variants cause asthenoteratozoospermia, a finding that provides crucial insights into the biological underpinnings of this disorder and should assist with counseling of affected individuals., 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.)

Asthenoteratospermia is a common cause of male infertility. Recent studies have revealed that CFAP65 mutations lead to severe asthenoteratospermia due to acrosome hypoplasia and flagellum malformations. However, the molecular mechanism underlying CFAP65-associated sperm malformation is largely unclear. Here, we initially examined the role of CFAP65 during spermiogenesis using Cfap65 knockout (Cfap65-/-) mice. The results showed that Cfap65-/- male mice exhibited severe asthenoteratospermia characterized by morphologically defective sperm heads and flagella. In Cfap65-/- mouse testes, hyper-constricted sperm heads were apparent in step 9 spermatids accompanied by abnormal manchette development, and acrosome biogenesis was abnormal in the maturation phase. Moreover, subsequent flagellar elongation was also severely affected and characterized by disrupted assembly of the mitochondrial sheath (MS) in Cfap65-/- male mice. Furthermore, the proteomic analysis revealed that the proteostatic system during acrosome formation, manchette organization and MS assembly was disrupted when CFAP65 was lost. Importantly, endogenous immunoprecipitation and immunostaining experiments revealed that CFAP65 may form a cytoplasmic protein network comprising MNS1, RSPH1, TPPP2, ZPBP1 and SPACA1. Overall, these findings provide insights into the complex molecular mechanisms of spermiogenesis by uncovering the essential roles of CFAP65 during sperm head shaping, acrosome biogenesis and MS assembly., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

In humans, TDRD7 (tudor domain containing 7) mutations lead to a syndrome combining congenital cataracts (CCs) and non-obstructive azoospermia (NOA), characterized by abnormal lens development and spermiogenesis. However, the molecular mechanism underlying TDRD7's functions in eye and testicular development are still largely unknown. Here, we show that the depletion of this gene in mice and humans resulted in the accumulation of autophagosomes and the disruption of macroautophagic/autophagic flux. The disrupted autophagic flux in tdrd7- deficient mouse embryonic fibroblasts (MEFs) was caused by a failure of autophagosome fusion with lysosomes. Furthermore, transcriptome analysis and biochemical assays showed that TDRD7 might directly bind to Tbc1d20 mRNAs and downregulate its expression, which is a key regulator of autophagosome maturation, resulting in the disruption of autophagosome maturation. In addition, we provide evidence to show that TDRD7-mediated autophagosome maturation maintains lens transparency by facilitating the removal of damaged proteins and organelles from lens fiber cells and the biogenesis of acrosome. Altogether, our results showed that TDRD7 plays an essential role in the maturation of autophagosomes and that tdrd7 deletion results in eye defects and testicular abnormalities in mice, implicating disrupted autophagy might be the mechanism that contributes to lens development and spermiogenesis defects in human. Abbreviations : CB: chromatoid bodies; CC: congenital cataract; CTSD: cathepsin D; DMSO: dimethyl sulfoxide; LAMP1: lysosomal-associated membrane protein 1; LECs: lens epithelial cells; MAP1LC3/LC3/Atg8: microtubule-associated protein 1 light chain 3; MEFs: mouse embryonic fibroblasts; NOA: non-obstructive azoospermia; OFZ: organelle-free zone; RG: RNA granules; SQSTM1/p62: sequestosome 1; TBC1D20: TBC1 domain family member 20; TDRD7: tudor domain containing 7; TEM: transmission electron microscopy; WT: wild type.

Multiple morphological abnormalities of the sperm flagella (MMAF)-induced asthenoteratozoospermia is a common cause of male infertility. Previous studies have identified several MMAF-associated genes, highlighting the condition's genetic heterogeneity. To further define the genetic causes underlying MMAF, we performed whole-exome sequencing in a cohort of 643 Chinese MMAF-affected men. Bi-allelic DNAH10 variants were identified in five individuals with MMAF from four unrelated families. These variants were either rare or absent in public population genome databases and were predicted to be deleterious by multiple bioinformatics tools. Morphological and ultrastructural analyses of the spermatozoa obtained from men harboring bi-allelic DNAH10 variants revealed striking flagellar defects with the absence of inner dynein arms (IDAs). DNAH10 encodes an axonemal IDA heavy chain component that is predominantly expressed in the testes. Immunostaining analysis indicated that DNAH10 localized to the entire sperm flagellum of control spermatozoa. In contrast, spermatozoa from the men harboring bi-allelic DNAH10 variants exhibited an absence or markedly reduced staining intensity of DNAH10 and other IDA components, including DNAH2 and DNAH6. Furthermore, the phenotypes were recapitulated in mouse models lacking Dnah10 or expressing a disease-associated variant, confirming the involvement of DNAH10 in human MMAF. Altogether, our findings in humans and mice demonstrate that DNAH10 is essential for sperm flagellar assembly and that deleterious bi-allelic DNAH10 variants can cause male infertility with MMAF. These findings will provide guidance for genetic counseling and insights into the diagnosis of MMAF-associated asthenoteratozoospermia., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background and Aims: Propionic acidemia (PA) is an autosomal recessive metabolic disorder caused by a deficiency of propionyl-CoA carboxylase and mutations in the PCCA and PCCB genes. In this study, we investigated the clinical characteristics of individuals with PA and conducted genetic analyses to provide new genetic evidence for the diagnosis of PA., Materials and Methods: We conducted whole-exome sequencing and Sanger sequencing in four individuals with PA from three unrelated Chinese families. We also performed a structural analysis of the PCCB protein variants. Couples from the three families included in our study underwent in vitro fertilization with preimplantation genetic testing., Results: We found five variants of PCCB. These biallelic variants were inherited from heterozygous parental carriers and were located in the functional domain, absent in human population genome datasets, and predicted to be deleterious. These findings indicate that the variants might be responsible for the clinical features observed in these particular patients with PA. Through successful embryo transfer and implantation, one of the couples fortunately gave birth to a healthy child., Conclusion: Overall, our study can expand the mutation spectrum of PCCB and provide useful information for the prenatal diagnosis of PA and genetic counseling for affected individuals., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Research Question: What is the genetic cause of multiple congenital disabilities in a girl with a maternal balanced X-autosome translocation [t(X-A)]? Is preimplantation genetic testing (PGT), to distinguish non-carrier from euploid/balanced embryos and prioritize transfer, an effective and applicable strategy for couples with t(X-A)?, Design: Karyotype analysis, whole-exome sequencing and X inactivation analysis were performed for a girl with congenital cardiac anomalies, language impairment and mild neurodevelopmental delay. PGT based on next-generation sequencing after microdissecting junction region (MicroSeq) to distinguish non-carrier and carrier embryos was used in three couples with a female t(X-A) carrier (cases 1-3)., Results: The girl carried a maternal balanced translocation 46,X,t(X;1)(q28;p31.1). Whole-exome sequencing revealed no monogenic mutation related to her phenotype, but she carried a rare skewed inactivation of the translocated X chromosome that spread to the adjacent interstitial 1p segment, contrary to her mother. All translocation breakpoints in cases 1-3 were successfully identified and each couple underwent one PGT cycle. Thirty oocytes were retrieved, and 13 blastocysts were eligible for biopsy, of which six embryos had a balanced translocation and only four were non-carriers. Three cryopreserved embryo transfers with non-carrier status embryos resulted in the birth of two healthy children (one girl and one boy), who were subsequently confirmed to have normal karyotypes., Conclusions: This study reported a girl with multiple congenital disabilities associated with a maternal balanced t(X-A) and verified that the distinction between non-carrier and carrier embryos is an effective and applicable strategy to avoid transferring genetic and reproductive risks to the offspring of t(X-A) carriers., (Copyright © 2021 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved.)

Background: Mosaicism poses challenges for genetic counseling and preimplantation genetic testing for monogenic disorders (PGT-M). NGS-based PGT-M has been extensively used to prevent the transmission of monogenic defects, but it has not been evaluated in the application of PGT-M resulting from mosaicism., Methods: Four women suspected of mosaicism were confirmed by ultra-deep sequencing. Blastocyst trophectoderm cells and polar bodies were collected for whole genome amplification, followed by pathogenic variants detection and haplotype analysis based on NGS. The embryos free of the monogenic disorders were transplantable., Results: Ultra-deep sequencing confirmed that the four women harbored somatic mosaic variants, with the proportion of variant cells at 1.12%, 9.0%, 27.60%, and 91.03%, respectively. A total of 25 blastocysts were biopsied and detected during four PGT cycles and 5 polar bodies were involved in one cycle additionally. For each couple, a wild-type embryo was successfully transplanted and confirmed by prenatal diagnosis, resulting in the birth of four healthy infants., Conclusions: Mosaic variants could be effectively evaluated via ultra-deep sequencing, and could be prevented the transmission by PGT. Our work suggested that an NGS-based PGT approach, involving pathogenic variants detection combined with haplotype analysis, is crucial for accurate PGT-M with mosaicism., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Genetics-associated asthenoteratozoospermia is often seen in patients with multiple morphological abnormalities of the sperm flagella (MMAF). Although 24 causative genes have been identified, these explain only approximately half of patients with MMAF. Since sperm flagella and motile cilia (especially respiratory cilia) have similar axonemal structures, many patients with MMAF also exhibit respiratory symptoms, such as recurrent airway infection, chronic sinusitis, and bronchiectasis, which are frequently associated with primary ciliary dyskinesia (PCD), another recessive disorder. Here, exome sequencing was conducted to evaluate the genetic cause in 53 patients with MMAF and classic PCD/PCD-like symptoms. Two homozygous missense variants and a compound-heterozygous variant in the BRWD1 gene were identified in three unrelated individuals. BRWD1 staining was detected in the whole flagella and respiratory cilia of normal controls but was absent in BRWD1-mutated individuals. Transmission electron microscopy and immunostaining demonstrated that BRWD1 deficiency in human affected respiratory cilia and sperm flagella differently, as the absence of outer and inner dynein arms in sperm flagellum and respiratory cilia, while with a decreased number and outer doublet microtubule defects of respiratory cilia. To our knowledge, this is the first report of a BRWD1-variant-related disease in humans, manifesting as an autosomal recessive form of MMAF and PCD/PCD-like symptoms. Our data provide a basis for further exploring the molecular mechanism of BRWD1 gene during spermatogenesis and ciliogenesis.

Spermatogenesis is a highly sophisticated process that comprises of mitosis, meiosis, and spermiogenesis. RNF216 (ring finger protein 216), an E3 ubiquitin ligase, has been reported to be essential for spermatogenesis and male fertility in mice. However, the stages affected by Rnf216 deficiency and its underlying molecular pathological mechanisms are still unknown. In this study, we generated Rnf216-deficient mice (Rnf216 -/- ) using CRISPR-Cas9 technology. Knockout of Rnf216 led to infertility in male but not female mice. Rnf216 knockout affected the prophase of meiosis I, as no genotypic difference was observed until 12 dpp (days postpartum). Rnf216 -/- spermatocytes were incompletely arrested at the zygotene stage and underwent apoptosis at approximately the pachytene stage. The proportion of zygotene spermatocytes was significantly increased, whereas the proportion of pachytene spermatocytes was significantly decreased in Rnf216 -/- testes. Nevertheless, there was no significantly genotypic difference in the number of diplotene spermatocytes. We further revealed that the PKA catalytic subunit β (PRKACB) was significantly increased, which subsequently resulted in elevated PKA activity in testes from adult as well as 9 dpp Rnf216 -/- mice. RNF216 interacts with PRKACB and promotes its degradation through the ubiquitin-lysosome pathway. Collectively, our results revealed an important role for RNF216 in regulation of meiosis and PKA stability in the testes., (© 2021 Federation of American Societies for Experimental Biology.)

Purpose: The purpose of this study is to summarize the clinical outcomes of apparently balanced chromosome rearrangement (ABCR) carriers in preimplantation genetic testing (PGT) cycles by next-generation sequencing following microdissecting junction region (MicroSeq) to distinguish non-carrier embryos from balanced carriers., Methods: A retrospective study of 762 ABCR carrier couples who requested PGT for structural rearrangements combined with MicroSeq at the Reproductive and Genetic Hospital of CITIC-Xiangya was conducted between October 2014 and October 2019., Results: Trophectoderm biopsy was performed in 4122 blastocysts derived from 917 PGT-SR cycles and 3781 blastocysts were detected. Among the 3781 blastocysts diagnosed, 1433 (37.9%, 1433/3781) were balanced, of which 739 blastocysts were carriers (51.57%, 739/1433) and 694 blastocysts were normal (48.43%, 694/1433). Approximately 26.39% of cycles had both carrier and normal embryo transfer, and the average number of biopsied blastocysts was 6.7. In the cumulative 223 biopsied cycles with normal embryo transfer, all couples chose to transfer the normal embryos. In the 225 cycles with only carrier embryos, the couples chose to transfer the carrier embryos in 169/225 (75.11%) cycles. A total of 732 frozen embryo transfer cycles were performed, resulting in 502 clinical pregnancies. Cumulatively, 326 babies were born; all of these babies were healthy and free of any developmental issues., Conclusion: Our study provides the first evaluation of the clinical outcomes of a large sample with ABCR carrier couples undergoing the MicroSeq-PGT technique and reveals its powerful ability to distinguish between carrier and non-carrier balanced embryos.

Background: The genetic causes of the majority of cases of female infertility caused by premature ovarian insufficiency (POI) are unknown., Objective: To identify the genetic causes of POI in 110 patients., Methods: Whole-exome sequencing was performed on 110 patients with POI, and putative disease-causative variants were validated by Sanger sequencing. Bioinformatic and in vitro functional analyses were performed for functional characterisation of the identified candidate disease-causative variants., Results: We identified two homozygous variants (NM&#95;001040274: c.150&#95;151del (p.Ser52Profs*7), c.999A>G (p.Ile333Met)) in SYCP2L in two patients, which had co-segregated with POI in these families. Bioinformatic analysis predicted that the two variants are deleterious, and in vitro functional analysis showed that mutant SYCP2L proteins exhibited mislocalisation and loss of function., Conclusions: SYCP2L is a novel gene found to be responsible for human POI. Our findings provide a potential molecular marker for POI and improve the understanding of the genetic basis of female infertility., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Oligoasthenoteratozoospermia (OAT) refers to the combination of various sperm abnormalities, including a decreased sperm count, reduced motility, and abnormal sperm morphology. Only a few genetic causes have been shown to be associated with OAT. Herein, we identified a novel homozygous frameshift mutation in meiosis-specific nuclear structural 1 (MNS1; NM&#95;018365: c.603&#95;604insG: p.Lys202Glufs*6) by whole-exome sequencing in an OAT proband from a consanguineous Chinese family. Subsequent variant screening identified four additional heterozygous MNS1 variants in 6/219 infertile individuals with oligoasthenospermia, but no MNS1 variants were observed among 223 fertile controls. Immunostaining analysis showed MNS1 to be normally located in the whole-sperm flagella, but was absent in the proband's sperm. Expression analysis by Western blot also confirmed that MNS1 was absent in the proband's sperm. Abnormal flagellum morphology and ultrastructural disturbances in outer doublet microtubules were observed in the proband's sperm. A total of three intracytoplasmic sperm injection cycles were carried out for the proband's wife, but they all failed to lead to a successful pregnancy. Overall, this is the first study to report a loss-of-function mutation in MNS1 causing OAT in a Han Chinese patient., Competing Interests: None

Asthenoteratozoospermia characterized by multiple morphological abnormalities of the flagella (MMAF) has been identified as a sub-type of male infertility. Recent progress has identified several MMAF-associated genes with an autosomal recessive inheritance in human affected individuals, but the etiology in approximately 40% of affected individuals remains unknown. Here, we conducted whole-exome sequencing (WES) and identified hemizygous missense variants in the X-linked CFAP47 in three unrelated Chinese individuals with MMAF. These three CFAP47 variants were absent in human control population genome databases and were predicted to be deleterious by multiple bioinformatic tools. CFAP47 encodes a cilia- and flagella-associated protein that is highly expressed in testis. Immunoblotting and immunofluorescence assays revealed obviously reduced levels of CFAP47 in spermatozoa from all three men harboring deleterious missense variants of CFAP47. Furthermore, WES data from an additional cohort of severe asthenoteratozoospermic men originating from Australia permitted the identification of a hemizygous Xp21.1 deletion removing the entire CFAP47 gene. All men harboring hemizygous CFAP47 variants displayed typical MMAF phenotypes. We also generated a Cfap47-mutated mouse model, the adult males of which were sterile and presented with reduced sperm motility and abnormal flagellar morphology and movement. However, fertility could be rescued by the use of intra-cytoplasmic sperm injections (ICSIs). Altogether, our experimental observations in humans and mice demonstrate that hemizygous mutations in CFAP47 can induce X-linked MMAF and asthenoteratozoospermia, for which good ICSI prognosis is suggested. These findings will provide important guidance for genetic counseling and assisted reproduction treatments., (Copyright © 2021 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)