ARRDC5 deficiency impairs spermatogenesis by affecting SUN5 and NDC1.

Sperm with normal morphology and motility are essential for successful fertilization, and the strong attachment of the sperm head-tail coupling apparatus to the nuclear envelope during spermatogenesis is required to ensure the integrity of sperm for capacitation and fertilization. Here, we report that Arrdc5 is associated with spermatogenesis. The Arrdc5 knockout mouse model showed male infertility characterized by a high bent-head rate and reduced motility in sperm, which led to capacitation defects and subsequent fertilization failure. Through mass spectrometry, we found that ARRDC5 affects spermatogenesis by affecting NDC1 and SUN5. We further found that ARRDC5 might affect the vesicle-trafficking protein SEC22A-mediated transport and localization of NDC1, SUN5 and other head-tail coupling apparatus-related proteins that are responsible for initiating the attachment of the sperm head and tail. We finally performed intracytoplasmic sperm injection as a way to explore therapeutic strategies. Our findings demonstrate the essential role and the underlying molecular mechanism of ARRDC5 in anchoring the sperm head to the tail during spermatogenesis.

PATL2 regulates mRNA homeostasis in oocytes by interacting with EIF4E and CPEB1.

The accumulation and storage of maternal mRNA is crucial for oocyte maturation and embryonic development. PATL2 is an oocyte-specific RNA-binding protein, and previous studies have confirmed that PATL2 mutation in humans and knockout mice cause oocyte maturation arrest or embryonic development arrest, respectively. However, the physiological function of PATL2 in the process of oocyte maturation and embryonic development is largely unknown. Here, we report that PATL2 is highly expressed in growing oocytes and couples with EIF4E and CPEB1 to regulate maternal mRNA expression in immature oocytes. The germinal vesicle oocytes from Patl2-/- mice exhibit decreasing maternal mRNA expression and reduced levels of protein synthesis. We further confirmed that PATL2 phosphorylation occurs in the oocyte maturation process and identified the S279 phosphorylation site using phosphoproteomics. We found that the S279D mutation decreased the protein level of PATL2 and led to subfertility in Palt2S279D knock-in mice. Our work reveals the previously unrecognized role of PATL2 in regulating the maternal transcriptome and shows that phosphorylation of PATL2 leads to the regulation of PATL2 protein levels via ubiquitin-mediated proteasomal degradation in oocytes.

Bi-allelic variants in ASTL cause abnormal fertilization or oocyte maturation defects.

Fertilization is a fundamental process of development, and the blocking mechanisms act at the zona pellucida (ZP) and plasma membrane of the egg to prevent any additional sperm from binding, permeating and fusing after fertilization. In clinical practice, some couples undergoing recurrent IVF failures that mature oocytes had abnormal fertilization for unknown reason. Ovastacin encoded by ASTL cleave the ZP protein ZP2 and play a key role in preventing polyspermy. Here, we identified bi-allelic variants in ASTL that are mainly characterized by fertilization problems in humans. All four independent affected individuals had bi-allelic frameshift variants or predicted damaging missense variants, which follow a Mendelian recessive inheritance pattern. The frameshift variants significantly decreased the quantity of ASTL protein in vitro. And all missense variants affected the enzymatic activity that cleaves ZP2 in mouse egg in vitro. Three knock-in female mice (corresponding to three missense variants in patients) all show subfertility due to low embryo developmental potential. This work presents strong evidence that pathogenic variants in ASTL cause female infertility and provides a new genetic marker for the diagnosis of fertilization problems.

CCDC28A deficiency causes sperm head defects, reduced sperm motility and male infertility in mice.

Mature spermatozoa with normal morphology and motility are essential for male reproduction. The epididymis has an important role in the proper maturation and function of spermatozoa for fertilization. However, factors related to the processes involved in spermatozoa modifications are still unclear. Here we demonstrated that CCDC28A, a member of the CCDC family proteins, is highly expressed in testes and the CCDC28A deletion leads to male infertility. We found CCDC28A deletion had a mild effect on spermatogenesis. And epididymal sperm collected from Ccdc28a-/- mice showed bent sperm heads, acrosomal defects, reduced motility and decreased in vitro fertilization competence whereas their axoneme, outer dense fibers, and fibrous sheath were all normal. Furthermore, we found that CCDC28A interacted with sperm acrosome membrane-associated protein 1 (SPACA1) and glycogen synthase kinase 3a (GSK3A), and deficiencies in both proteins in mice led to bent heads and abnormal acrosomes, respectively. Altogether, our results reveal the essential role of CCDC28A in regulating sperm morphology and motility and suggesting a potential marker for male infertility.

Bi-allelic missense variants in MEI4 cause preimplantation embryonic arrest and female infertility.

Preimplantation embryonic arrest is an important pathogenesis of female infertility, but little is known about the genetic factors behind this phenotype. MEI4 is an essential protein for DNA double-strand break formation during meiosis, and Mei4 knock-out female mice are viable but sterile, indicating that MEI4 plays a crucial role in reproduction. To date, MEI4 has not been found to be associated with any human reproductive diseases. Here, we identified six compound heterozygous and homozygous MEI4 variants-namely, c.293C > T, p.(Ser98Leu), c.401C > G, p.(Pro134Arg), c.391C > G, p.(Pro131Ala), c.914A > T, p.(Tyr305Phe), c.908C > G, p.(Ala303Gly), and c.899A > T, p.(Gln300Leu)-in four independent families that were responsible for female infertility mainly characterized by preimplantation embryonic arrest. In vitro, we found that these variants reduced the interaction between MEI4 and DNA. In vivo, we generated a knock-in mouse model and demonstrated that female mice were infertile and were characterized by developmental defects during oogenesis. Our findings reveal the important roles of MEI4 in human reproduction and provide a new diagnostic marker for genetic counseling of clinical infertility patients.

Identification novel mutations and phenotypic spectrum expanding in PATL2 in infertile women with IVF/ICSI failure.

AIM: Abnormalities in oocyte maturation, fertilization, and early embryonic development are major causes of primary infertility in women who are undergoing IVF/ICSI attempts. Although many genetic factors responsible for these abnormal phenotypes have been identified, there are more additional pathogenic genes and variants yet to be discovered. Previous studies confirmed that bi-allelic PATL2 deficiency is an important factor for female infertility. In this study, 935 infertile patients with IVF/ICSI failure were selected for whole-exome sequencing, and 18 probands carrying PATL2 variants with a recessive inheritance pattern were identified. METHODS: We estimated that the prevalence contributed by PATL2 was 1.93% (18/935) in our study cohort. RESULTS: 15 novel variants were found in those families, including c.1093C > T, c.1609dupA, c.1204C > T, c.643dupG, c.877-2A > G, c.1228C > G, c.925G > A, c.958G > A, c.4A > G, c.1258T > C, c.1337G > A, c.1264dupA, c.88G > T, c.1065-2A > G, and c.1271T > C. The amino acids altered by the corresponding variants were highly conserved in mammals, and in silico analysis and 3D molecular modeling suggested that the PATL2 mutants impaired the physiologic function of the resulting proteins. Diverse clinical phenotypes, including oocyte maturation defect, fertilization failure, and early embryonic arrest might result from different variants of PATL2. CONCLUSIONS: These results expand the spectrum of PATL2 variants and provide an important reference for genetic counseling for female infertility, and they increase our understanding of the mechanisms of oocyte maturation arrest caused by PATL2 deficiency.

ADGB variants cause asthenozoospermia and male infertility.

Asthenozoospermia is one of the main factors leading to male infertility, but the genetic mechanisms have not been fully elucidated. Variants in the androglobin (ADGB) gene were identified in an infertile male characterized by asthenozoospermia. The variants disrupted the binding of ADGB to calmodulin. Adgb-/- male mice were infertile due to reduced sperm concentration (< 1 × 106 /mL) and motility. Spermatogenesis was also abnormal, with malformation of both elongating and elongated spermatids, and there was an approximately twofold increase in apoptotic cells in the cauda epididymis. These exacerbated the decline in sperm motility. It is surprising that ICSI with testicular spermatids allows fertilization and eventually develops into blastocyst. Through mass spectrometry, we identified 42 candidate proteins that are involved in sperm assembly, flagella formation, and sperm motility interacting with ADGB. In particular, CFAP69 and SPEF2 were confirmed to bind to ADGB. Collectively, our study suggests the potential important role of ADGB in human fertility, revealing its relevance to spermatogenesis and infertility. This expands our knowledge of the genetic causes of asthenozoospermia and provides a theoretical basis for using ADGB as an underlying genetic marker for infertile males.

Homozygous variants in CDC23 cause female infertility characterized by oocyte maturation defects.

Oocyte maturation defects are major phenotypes resulting in female infertility. Although many genetic factors have been found to be responsible for these phenotypes, the underlying pathogenic genes and variants remain to be identified. The anaphase promoting complex or cyclosome (APC/C) is known to be essential in the metaphase-to-anaphase transition. In this study, we identified two homozygous missense variants (c.986A > G, p.Y329C and c.988C > T, p.R330C) in CDC23 that are responsible for female infertility characterized by oocyte maturation defects in three infertile individuals. CDC23 (cell division cycle 23) is one of the core subunits of the APC/C. In vitro experiments showed that the variant c.986A > G (p.Y329C) led to a decrease in CDC23 protein level and the variant c.988C > T (p.R330C) changed the localization of CDC23 in HeLa cells and mouse oocytes. In vivo studies showed that Cdc23Y329C/Y329C mice successfully mimicked the patients' phenotype by causing low expression of CDC23 and APC4 and the accumulation of securin and cyclin B1 in oocytes. AZ3146 treatment was able to rescue the phenotype. Taken together, our findings reveal the important roles of CDC23 in human oocyte maturation and provide a new genetic marker for female infertility.

Homozygous Mutations in BTG4 Cause Zygotic Cleavage Failure and Female Infertility.

Zygotic cleavage failure (ZCF) is a unique early embryonic phenotype resulting in female infertility and recurrent failure of in vitro fertilization (IVF) and/or intracytoplasmic sperm injection (ICSI). With this phenotype, morphologically normal oocytes can be retrieved and successfully fertilized, but they fail to undergo cleavage. Until now, whether this phenotype has a Mendelian inheritance pattern and which underlying genetic factors play a role in its development remained to be elucidated. B cell translocation gene 4 (BTG4) is a key adaptor of the CCR4-NOT deadenylase complex, which is involved in maternal mRNA decay in mice, but no human diseases caused by mutations in BTG4 have previously been reported. Here, we identified four homozygous mutations in BTG4 (GenBank: NM_017589.4) that are responsible for the phenotype of ZCF, and we found they followed a recessive inheritance pattern. Three of them-c.73C>T (p.Gln25Ter), c.1A>G (p.?), and c.475_478del (p.Ile159LeufsTer15)-resulted in complete loss of full-length BTG4 protein. For c.166G>A (p.Ala56Thr), although the protein level and distribution of mutant BTG4 was not altered in zygotes from affected individuals or in HeLa cells, the interaction between BTG4 and CNOT7 was abolished. In vivo studies further demonstrated that the process of maternal mRNA decay was disrupted in the zygotes of the affected individuals, which provides a mechanistic explanation for the phenotype of ZCF. Thus, we provide evidence that ZCF is a Mendelian phenotype resulting from mutations in BTG4. These findings contribute to our understanding of the role of BTG4 in human early embryonic development and provide a genetic marker for female infertility.

Bi-allelic Missense Pathogenic Variants in TRIP13 Cause Female Infertility Characterized by Oocyte Maturation Arrest.

Normal oocyte meiosis is a prerequisite for successful human reproduction, and abnormalities in the process will result in infertility. In 2016, we identified mutations in TUBB8 as responsible for human oocyte meiotic arrest. However, the underlying genetic factors for most affected individuals remain unknown. TRIP13, encoding an AAA-ATPase, is a key component of the spindle assembly checkpoint, and recurrent homozygous nonsense variants and a splicing variant in TRIP13 are reported to cause Wilms tumors in children. In this study, we identified homozygous and compound heterozygous missense pathogenic variants in TRIP13 responsible for female infertility mainly characterized by oocyte meiotic arrest in five individuals from four independent families. Individuals from three families suffered from oocyte maturation arrest, whereas the individual from the fourth family had abnormal zygote cleavage. All displayed only the infertility phenotype without Wilms tumors or any other abnormalities. In vitro and in vivo studies showed that the identified variants reduced the protein abundance of TRIP13 and caused its downstream molecule, HORMAD2, to accumulate in HeLa cells and in proband-derived lymphoblastoid cells. The chromosome mis-segregation assay showed that variants did not have any effects on mitosis. Injecting TRIP13 cRNA into oocytes from one affected individual was able to rescue the phenotype, which has implications for future therapeutic treatments. This study reports pathogenic variants in TRIP13 responsible for oocyte meiotic arrest, and it highlights the pivotal but different roles of TRIP13 in meiosis and mitosis. These findings also indicate that different dosage effects of mutant TRIP13 might result in two distinct human diseases.

A novel homozygous variant in ZFP36L2 cause female infertility due to oocyte maturation defect.

Normal oocyte maturation is an important requirement for the success of human reproduction, and defects in this process will lead to female infertility and repeated IVF/ICSI failures. In order to identify genetic factors that are responsible for oocyte maturation defect, we used whole exome sequencing in the affected individual with oocyte maturation defect from a consanguineous family and identified a homozygous variant c.853_861del (p.285_287del) in ZFP36L2. ZFP36L2 is a RNA-binding protein, which regulates maternal mRNA decay and oocyte maturation. In vitro studies showed that the variant caused decreased protein levels of ZFP36L2 in oocytes due to mRNA instability and might lead to the loss of its function to degrade maternal mRNAs. Previous study showed that the pathogenic variants in ZFP36L2 were associated with early embryonic arrest. In contrast, we identified a novel ZFP36L2 variant in the affected individual with oocyte maturation defect, which further broadened the mutational and phenotypic spectrum of ZFP36L2, suggesting that ZFP36L2 might be a genetic diagnostic marker for the affected individuals with oocyte maturation defect.

Genetic screening in patients with ovarian dysfunction.

Ovarian dysfunction, including premature ovarian insufficiency and decreased ovarian reserve, affects the ovarian reserve and is one of the leading causes of female infertility. More and more cases of ovarian dysfunction are associated with genetic factors. Here, we identified eight potential variants in five genes (MSH4, HFM1, SYCE1, FSHR, and C14orf39) from six independent families by exome sequencing. The splice-site variants in SYCE1 and MSH4 affected canonical splicing isoforms, leading to missing protein domains or premature termination. Our findings expand the mutational spectrum of ovarian dysfunction and provide potential biomarkers for future genetic counseling and for more personalized treatments. Exome sequencing was shown to be a useful tool to better dissect the genetic basis for ovarian dysfunction and yielded a genetic diagnosis in about 5.0% (6/124) of cases in a cohort of 124 patients with ovarian dysfunction.

IQUB deficiency causes male infertility by affecting the activity of p-ERK1/2/RSPH3.

STUDY QUESTION: Can new genetic factors responsible for male infertility be identified, especially for those characterized by asthenospermia despite normal sperm morphology? SUMMARY ANSWER: We identified the novel pathogenetic gene IQ motif and ubiquitin-like domain-containing (IQUB) as responsible for male infertility characterized by asthenospermia, involving sperm radial spoke defects. WHAT IS KNOWN ALREADY: To date, only a few genes have been found to be responsible for asthenospermia with normal sperm morphology. Iqub, encoding the IQUB protein, is highly and specifically expressed in murine testes and interacts with the proteins radial spoke head 3 (RSPH3), CEP295 N-terminal like (CEP295NL or DDC8), glutathione S-transferase mu 1 (GSTM1) and outer dense fiber of sperm tails 1 (ODF1) in the yeast two-hybrid system. STUDY DESIGN, SIZE, DURATION: The IQUB variant was identified by whole-exome sequencing in a cohort of 126 male infertility patients with typical asthenospermia recruited between 2015 and 2020. Knockout (KO) and knockin (KI) mouse models, scanning and transmission electron microscopy (TEM), and other functional assays were performed, between 2019 and 2021. PARTICIPANTS/MATERIALS, SETTING, METHODS: The IQUB variant was identified by whole-exome sequencing and confirmed by Sanger sequencing. Iqub KO and KI mice were constructed to mimic the phenotype of the affected individual. After recapitulating the phenotype of human male infertility, scanning and TEM were performed to check the ultrastructure of the sperm. Western blot and co-immunoprecipitation were performed to clarify the pathological mechanism of the IQUB variant. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a homozygous nonsense IQUB variant (NM_001282855.2:c.942T> G(p.Tyr314*)) from an infertile male. Iqub KO and KI mice mimicked the infertility phenotype and confirmed IQUB to be the pathogenetic gene. Scanning and TEM showed that sperm of both the mouse models and the affected individual had radial spoke defects. The functional assay suggested that IQUB may recruit calmodulin in lower Ca2+ environments to facilitate the normal assembly of radial spokes by inhibiting the activity of RSPH3/p-ERK1/2 (a nontypical AKAP (A-Kinase Anchoring Protein) forming by RSPH3 and phosphorylation of extracellular signal-regulated kinase 1 and 2 (p-ERK1/2)). LIMITATIONS, REASONS FOR CAUTION: Additional cases are needed to confirm the genetic contribution of IQUB variants to male infertility. In addition, because no IQUB antibody is available for immunofluorescence and the polyclonal antibody we generated was only effective in western blotting, immunostaining for IQUB was not performed in this study. Therefore, this study lacks direct in vivo proof to confirm the effect of the variant on IQUB protein level. WIDER IMPLICATIONS OF THE FINDINGS: Our results suggest a causal relation between IQUB variants and male infertility owing to asthenospermia, and partly clarify the pathological mechanism of IQUB variants. This expands our knowledge of the genes involved in human sperm asthenospermia and potentially provides a new genetic marker for male infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2021YFC2700100), the National Natural Science Foundation of China (32130029, 82171643, 81971450, 82001538, and 81971382) and the Guangdong Science and Technology Department Guangdong-Hong Kong-Macao Joint Innovation Project (2020A0505140003). There are no competing interests to declare. TRIAL REGISTRATION NUMBER: N/A.

Heterozygous loss-of-function variants in LHX8 cause female infertility characterized by oocyte maturation arrest.

PURPOSE: The genetic causes of oocyte maturation arrest leading to female infertility are largely unknown, and no population-based genetic analysis has been applied in cohorts of patients with infertility. We aimed to identify novel pathogenic genes causing oocyte maturation arrest by using a gene-based burden test. METHODS: Through comparison of exome sequencing data from 716 females with infertility characterized by oocyte maturation arrest and 3539 controls, we performed a gene-based burden test and identified a novel pathogenic gene LHX8. Splicing event was evaluated using a minigene assay, expression of LHX8 protein was assessed in HeLa cells, and nuclear subcellular localization was determined in both HeLa cells and mouse oocytes. RESULTS: A total of 5 heterozygous loss-of-function LHX8 variants were identified from 6 independent families (c.389+1G>T, c.412C>T [p.Arg138∗], c.282C>A [p.Cys94∗]; c.257dup [p.Tyr86∗]; and c.180del, [p.Ser61Profs∗30]). All the identified variants in LHX8 produced truncated LHX8 protein and resulted in loss of LHX8 nuclear localization in both HeLa cells and mouse oocytes. CONCLUSION: By combining genetic evidence and functional evaluations, we identified a novel pathogenic gene LHX8 and established the causative relationship between LHX8 haploinsufficiency and female infertility characterized by oocyte maturation arrest.

Novel biallelic mutations in PADI6 in patients with early embryonic arrest.

Peptidyl arginine deiminase, type VI (PADI6) is a member of the subcortical maternal complex (SCMC), which plays vital roles in mammalian embryogenesis. Most mutations in SCMC members have been reported to cause human embryonic arrest, and a total of 15 mutations in PADI6 have been shown to be responsible for early embryonic arrest according to previous studies. However, the genetic factors behind this phenotype remain to be understood in further detail. Here, we identified 13 novel mutations and 4 previously reported mutations of PADI6 in 14 patients who were diagnosed with abnormal embryonic development caused by early arrest, embryonic fragmentation, and recurrent implantation failure. Most of the mutations were predicted by in silico analysis to be deleterious or damaging to the function of PADI6. In addition, the total and East Asian population frequencies of the mutations were low or absent in the gnomAD database. Our study expands the mutational spectrum in PADI6 and will provide precise targets for genetic counseling in the future.

Bi-allelic mutations in MOS cause female infertility characterized by preimplantation embryonic arrest.

STUDY QUESTION: Are mutations in MOS (MOS proto-oncogene, serine/threonine kinase) involved in early embryonic arrest in infertile women? SUMMARY ANSWER: We identified mutations in MOS that may cause human female infertility characterized by preimplantation embryonic arrest (PREMBA), and the effects of the mutations in human embryonic kidney 293T (HEK293T cells) and mouse oocytes provided evidence for a causal relation between MOS and female infertility. WHAT IS KNOWN ALREADY: MOS, an activator of mitogen-activated protein kinase, mediates germinal vesicle breakdown and metaphase II arrest. Female MOS knockout mice are viable but sterile. Thus, MOS seems to be an important part of the mammalian cell cycle mechanism that regulates female meiosis. STUDY DESIGN, SIZE, DURATION: Whole-exome sequencing, bioinformatics filtering analysis and genetic analysis were performed to identify two different biallelic mutations in MOS in two independent families. The infertile patients presenting with early embryonic arrest were recruited from October 2018 to June 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: The female patients diagnosed with primary infertility were recruited from the reproduction centres of local hospitals. Genomic DNA from the affected individuals, their family members and healthy controls was extracted from peripheral blood. We performed whole-exome sequencing in patients diagnosed with PREMBA. Functional effects of the mutations were investigated in HEK293T cells by western blotting and in mouse oocytes by microinjection and immunofluorescence. MAIN RESULTS AND THE ROLE OF CHANCE: We identified the homozygous missense mutation c.285C>A (p.(Asn95Lys)) and the compound heterozygous mutations c.467delG (p.(Gly156Alafs*18)) and c.956G>A (p.(Arg319His)) in MOS in two independent patients. The mutations c.285C>A (p.(Asn95Lys)) and c.467delG (p.(Gly156Alafs*18)) reduced the protein level of MOS, and all mutations reduced the ability of MOS to phosphorylate its downstream target, extracellular signal-regulated kinase1/2. In addition, the identified mutations reduced the capacity of exogenous human MOS to rescue the metaphase II exit phenotype, and the F-actin cytoskeleton of mouse oocytes was affected by the patient-derived mutations. LIMITATIONS, REASONS FOR CAUTION: Owing to the lack of in vivo data from patient oocytes, the exact molecular mechanism affected by MOS mutations and leading to PREMBA is still unknown and should be further investigated using knock-out or knock-in mice. WIDER IMPLICATIONS OF THE FINDINGS: We identified recessive mutations in MOS in two independent patients with the PREMBA phenotype. Our findings reveal the important role of MOS during human oocyte meiosis and embryonic development and suggest that mutations in MOS may be precise diagnostic markers for clinical genetic counselling. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Natural Science Foundation of China (81725006, 81822019, 81771581, 81971450, 81971382,82001538 and 82071642), the project supported by the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), the Project of the Shanghai Municipal Science and Technology Commission (19JC1411001), the Natural Science Foundation of Shanghai (19ZR1444500 and 21ZR1404800), the Shuguang Program of the Shanghai Education Development Foundation and the Shanghai Municipal Education Commission (18SG03), the Foundation of the Shanghai Health and Family Planning Commission (20154Y0162), the Capacity Building Planning Program for Shanghai Women and Children's Health Service and the collaborative innovation centre project construction for Shanghai Women and Children's Health. The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Bi-allelic variants in KCNU1 cause impaired acrosome reactions and male infertility.

STUDY QUESTION: Are there new genetic factors responsible for male infertility with normal sperm quantity and morphology? SUMMARY ANSWER: We identified the bi-allelic variants in KCNU1 and confirmed it a novel pathogenetic gene for male infertility mainly due to impaired sperm acrosome reactions (ARs). WHAT IS KNOWN ALREADY: Until now, the underlying genetic determinants for male affected individuals exhibiting normal sperm quantity and morphology have been largely unknown. Potassium/calcium-activated channel subfamily U member 1 (KCNU1) is a sperm-specific potassium channel. The Kcnu1 null mutation in male mice causes infertility due to the impaired progressive motility and AR. STUDY DESIGN, SIZE, DURATION: We recruited a cohort of 126 male infertility individuals with typical asthenospermia or fertilization failure and focused on two infertile males from two consanguineous families from 2015 to 2020; whole-exome sequencing and homozygosity mapping were performed. We identified a homozygous missense variant (c.2144A>G, p.His715Arg) and a homozygous donor splice-site variant (c.1295 + 3A>C, p.Val405Glyfs*8) in KCNU1. Then, we generated a knock-in (KI) mouse model in September 2020 and have now carried out functional studies and possible treatment strategies. PARTICIPANTS/MATERIALS, SETTING, METHODS: The affected individuals with infertility were recruited from the Shanghai Ninth Hospital affiliated to Shanghai Jiao Tong University. Genomic DNA from the affected individual was extracted from peripheral blood. Whole-exome sequencing, homozygosity mapping and in silico analyses were used to screen and identify KCNU1 variants, and the variants were confirmed by Sanger sequencing. We used C57BL/6N mouse to construct KI mouse model to mimic the reproductive phenotype in vivo. We performed functional experiments by western blotting, AR assay and immunofluorescent Staining. Finally, we performed IVF and ICSI to explore the treatment strategies. MAIN RESULTS AND THE ROLE OF CHANCE: We identified a homozygous missense variant (c.2144A>G, p.His715Arg) and a homozygous donor splice-site variant (c.1295 + 3A>C, p.Val405Glyfs*8) in KCNU1 in two infertile males. We demonstrated that the splice-site variant affected normal alternative splicing of KCNU1, thus leading to the loss of function of KCNU1. Meanwhile, the missense pathogenic variant reduced the KCNU1 protein levels in sperm of both the affected individual and the KI mouse model, resulting in impaired ARs and male infertility. Intracytoplasmic sperm injection was able to rescue the deficiencies. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The exact molecular mechanism of KCNU1 and pathways need to be further explore in the future. WIDER IMPLICATIONS OF THE FINDINGS: This is the first report that establishes a causal relationship between KCNU1 deficiency and male infertility, confirming the critical role of KCNU1 in human reproduction. Our findings expand our knowledge of the genes that play critical roles in the human sperm AR and provide a new genetic marker for infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the SHIPM-pi fund no. JY201801 from the Shanghai Institute of Precision Medicine, Ninth People's Hospital Shanghai Jiao Tong University School of Medicine, the National Natural Science Foundation of China (81725006, 81771649, 81822019, 81771581, 81971450, 81971382, 82001538 and 82071642). The authors declare no conflict of interest. TRIAL REGISTRATION NUMBER: N/A.

Homozygous Mutations in WEE2 Cause Fertilization Failure and Female Infertility.

Fertilization is a fundamental process of development and is a prerequisite for successful human reproduction. In mice, although several receptor proteins have been shown to play important roles in the process of fertilization, only three genes have been shown to cause fertilization failure and infertility when deleted in vivo. In clinical practice, some infertility case subjects suffer from recurrent failure of in vitro fertilization and intracytoplasmic sperm injection attempts due to fertilization failure, but the genetic basis of fertilization failure in humans remains largely unknown. Wee2 is a key oocyte-specific kinase involved in the control of meiotic arrest in mice, but WEE2 has not been associated with any diseases in humans. In this study, we identified homozygous mutations in WEE2 that are responsible for fertilization failure in humans. All four independent affected individuals had homozygous loss-of-function missense mutations or homozygous frameshift protein-truncating mutations, and the phenotype of fertilization failure was shown to follow a Mendelian recessive inheritance pattern. All four mutations significantly decreased the amount of WEE2 protein in vitro and in affected individuals' oocytes in vivo, and they all led to abnormal serine phosphorylation of WEE2 and reduced tyrosine 15 phosphorylation of Cdc2 in vitro. In addition, injection of WEE2 cRNA into affected individuals' oocytes rescued the fertilization failure phenotype and led to the formation of blastocysts in vitro. This work presents a novel gene responsible for human fertilization failure and has implications for future therapeutic treatments for infertility cases.

Novel biallelic mutations in MEI1: expanding the phenotypic spectrum to human embryonic arrest and recurrent implantation failure.

STUDY QUESTION: Are any novel mutations and corresponding new phenotypes, other than recurrent hydatidiform moles, seen in patients with MEI1 mutations? SUMMARY ANSWER: We identified several novel mutations in MEI1 causing new phenotypes of early embryonic arrest and recurrent implantation failure. WHAT IS KNOWN ALREADY: It has been reported that biallelic mutations in MEI1, encoding meiotic double-stranded break formation protein 1, cause azoospermia in men and recurrent hydatidiform moles in women. STUDY DESIGN, SIZE, DURATION: We first focused on a pedigree in which two sisters were diagnosed with recurrent hydatidiform moles in December 2018. After genetic analysis, two novel mutations in MEI1 were identified. We then expanded the mutational screening to patients with the phenotype of embryonic arrest, recurrent implantation failure, and recurrent pregnancy loss, and found another three novel MEI1 mutations in seven new patients from six families recruited from December 2018 to May 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: Nine primary infertility patients were recruited from the reproduction centers in local hospitals. Genomic DNA from the affected individuals, their family members, and healthy controls was extracted from peripheral blood. The MEI1 mutations were screened using whole-exome sequencing and were confirmed by the Sanger sequencing. In silico analysis of mutations was performed with Sorting Intolerant From Tolerant (SIFT) and Protein Variation Effect Analyzer (PROVEAN). The influence of the MEI1 mutations was determined by western blotting and minigene analysis in vitro. MAIN RESULTS AND THE ROLE OF CHANCE: In this study, we identified five novel mutations in MEI1 in nine patients from seven independent families. Apart from recurrent hydatidiform moles, biallelic mutations in MEI1 were also associated with early embryonic arrest and recurrent implantation failure. In addition, we demonstrated that protein-truncating and missense mutations reduced the protein level of MEI1, while the splicing mutations caused abnormal alternative splicing of MEI1. LIMITATIONS, REASONS FOR CAUTION: Owing to the lack of in vivo data from the oocytes of the patients, the exact molecular mechanism(s) involved in the phenotypes remains unknown and should be further investigated using knock-out or knock-in mice. WIDER IMPLICATIONS OF THE FINDINGS: Our results not only reveal the important role of MEI1 in human oocyte meiosis and early embryonic development, but also extend the phenotypic and mutational spectrum of MEI1 and provide new diagnostic markers for genetic counseling of clinical patients. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2018YFC1003800, 2017YFC1001500, and 2016YFC1000600), the National Natural Science Foundation of China (81725006, 81822019, 81771581, 81971450, and 81971382), the project supported by the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), the Project of the Shanghai Municipal Science and Technology Commission (19JC1411001), the Natural Science Foundation of Shanghai (19ZR1444500), the Shuguang Program of the Shanghai Education Development Foundation and the Shanghai Municipal Education Commission (18SG03), the Shanghai Health and Family Planning Commission Foundation (20154Y0162), the Strategic Collaborative Research Program of the Ferring Institute of Reproductive Medicine, Ferring Pharmaceuticals and the Chinese Academy of Sciences (FIRMC200507) and the Chongqing Key Laboratory of Human Embryo Engineering (2020KFKT008). No competing interests are declared. TRIAL REGISTRATION NUMBER: N/A.

FBXO43 variants in patients with female infertility characterized by early embryonic arrest.

STUDY QUESTION: Can any new genetic factors responsible for early embryonic arrest in infertile patients be identified, together with the mechanism of pathogenic variants? SUMMARY ANSWER: We identified three homozygous variants in the F-box protein 43 gene (FBXO43) in infertile patients and studies on the effects of the variants in HEK293T cells and mouse oocytes provided evidence for a causal relation between FBXO43 and female infertility. WHAT IS KNOWN ALREADY: FBXO43, an inhibitor of the anaphase-promoting complex/cyclosome, mediates Metaphase II arrest as a component of the cytostatic factor in oocytes. Both male and female Fbxo43 knockout mice are viable but sterile. FBXO43, therefore, appears to be an essential component of the mammalian cell-cycle machinery that regulates both male and female meiosis. Until now, only one article has reported a homozygous FBXO43 variant associated with teratozoospermia, but the causal relationship was not established with functional evidence. STUDY DESIGN, SIZE, DURATION: Whole-exome sequencing (WES) and homozygosity mapping were performed in 24 probands from consanguineous families who suffered from early embryonic arrest, and two different homozygous variants in FBXO43 were identified in two independent families. WES data from a further 950 infertile women with early embryonic arrest were screened for homozygous and compound heterozygous variants in FBXO43, and a third individual with an additional homozygous variant in FBXO43 was identified. The infertile patients presenting with early embryonic arrest were recruited from August 2016 to May 2020. PARTICIPANTS/MATERIALS, SETTING, METHODS: The women diagnosed with primary infertility were recruited from the reproduction centers of local hospitals. Genomic DNA samples from the affected individuals, their family members, and healthy controls were extracted from peripheral blood. The FBXO43 variants were identified using WES, homozygosity mapping, in silico analysis, and variant screening. All of the variants were confirmed by Sanger sequencing, and the effects of the variants were investigated in human embryonic kidney (HEK) 293T cells by western blotting and in mouse oocytes by complementary RNA injection. MAIN RESULTS AND THE ROLE OF CHANCE: We identified three homozygous variants in FBXO43 (NM_001029860.4)-namely, c.1490_1497dup (p.(Glu500Serfs*2)), c.1747C>T (p.(Gln583*)), and c.154delG (p.(Asp52Thrfs*30))-in three independent families. All of the homozygous variants reduced the protein level of FBXO43 and reduced the level of its downstream target Cyclin B1 in HEK293T cells. In addition, the variants reduced the ability of exogenous human FBXO43 to rescue the parthenogenetic activation phenotype in Fbxo43 knockdown mouse oocytes. LIMITATIONS, REASONS FOR CAUTION: Owing to the lack of in vivo data from the oocytes of patients, the exact molecular mechanism remains unknown and should be further investigated using knock out or knock in mice. WIDER IMPLICATIONS OF THE FINDINGS: Our study has identified three pathogenic variants in FBXO43 that are involved in human early embryonic arrest. These findings contribute to our understanding of the role of FBXO43 in human early embryonic development and provide a new genetic marker for female infertility. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by the National Key Research and Development Program of China (2018YFC1003800, 2017YFC1001500, and 2016YFC1000600), the National Natural Science Foundation of China (81725006, 81822019, 81771581, 81971450, 81971382, and 82001552), the project supported by the Shanghai Municipal Science and Technology Major Project (2017SHZDZX01), the Project of the Shanghai Municipal Science and Technology Commission (19JC1411001), the Natural Science Foundation of Shanghai (19ZR1444500), the Shuguang Program of the Shanghai Education Development Foundation and the Shanghai Municipal Education Commission (18SG03), the Foundation of the Shanghai Health and Family Planning Commission (20154Y0162), the Capacity Building Planning Program for Shanghai Women and Children's Health Service, and the collaborative innovation center project construction for Shanghai Women and Children's Health. None of the authors have any competing interests. TRIAL REGISTRATION NUMBER: N/A.