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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.

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.

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 Human 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 coupled with EIF4E and CPEB1 to regulate maternal mRNA expression in immature oocytes. The GV oocytes from Patl2−/- mice exhibit decreasing maternal mRNA expression and reduced levels of protein synthesis. We further confirmed PATL2 phosphorylation in the oocyte maturation process and identified the S279 phosphorylation site using phosphoproteomics. We found 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.

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.

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.

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.

Meiotic spindle assembly ensures proper chromosome segregation in oocytes. However, the mechanisms behind spindle assembly in human oocytes remain largely unknown. We used three-dimensional high-resolution imaging of more than 2000 human oocytes to identify a structure that we named the human oocyte microtubule organizing center (huoMTOC). The proteins TACC3, CCP110, CKAP5, and DISC1 were found to be essential components of the huoMTOC. The huoMTOC arises beneath the oocyte cortex and migrates adjacent to the nuclear envelope before nuclear envelope breakdown (NEBD). After NEBD, the huoMTOC fragments and relocates on the kinetochores to initiate microtubule nucleation and spindle assembly. Disrupting the huoMTOC led to spindle assembly defects and oocyte maturation arrest. These results reveal a physiological mechanism of huoMTOC-regulated spindle assembly in human oocytes.

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.

PURPOSE: To identify the genetic factors responsible for asthenozoospermia, which is a major cause of male infertility characterized by immotile and malformed spermatozoa. METHODS: Whole-exome sequencing was performed in two brothers from a family with asthenozoospermia to identify pathogenic variants. The functional effect of the identified variant was investigated in HEK293T cells using a minigene assay. RESULTS: We identified a novel homozygous splicing variant c.6311-2A>G in DNAH8 from two affected brothers belonging to the same consanguineous family. The splicing variant altered a consensus splice acceptor site of DNAH8 intron 44, which led to the deletion of exon 45 and resulted in a frameshift and a predicted truncated protein (p.G2104Efs*19). Although most spermatozoa from the patients presented with reduced sperm motility, intracytoplasmic sperm injection was able to overcome the inability of the spermatozoa to reach the ovum and thus produce a healthy child for the proband. CONCLUSIONS: This finding expands the mutational spectrum of DNAH8, making it a potential genetic diagnostic marker for those suffering from primary male infertility. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10815-021-02116-1.

PURPOSE: To screen novel mutations in LHCGR responsible for empty follicle syndrome and explore the pathological mechanism of mutations. METHODS: Four affected individuals diagnosed with infertility-associated anovulation or oligo-ovulation from three independent families were recruited. Sanger sequencing was used to identify the LHCGR mutations in affected individuals. Western blot was performed to evaluate the effects of mutations on LHCGR protein levels. Immunofluorescence was done to explore the effects of mutations on LHCGR subcellular localization. The ATP levels were measured to infer the functional effects of the mutations on LHCGR. RESULTS: In the present study, three novel biallelic mutations in LHCGR were identified in four affected individuals from three independent families with empty follicle syndrome or oligo-ovulation. All biallelic mutations were inherited from the proband of their parents. The western blot showed that the identified mutations decreased LHCGR protein level and altered the glycosylation pattern. The immunofluorescence showed an ectopic subcellular localization of LHCGR in cultured HeLa cells. Besides, the mutations in LHCGR also reduced the cellular ATP consumption. CONCLUSION: These findings confirm previous studies and expand the mutational spectrum of LHCGR, which will provide genetic diagnostic marker for patients with empty follicle syndrome. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01931-2) contains supplementary material, which is available to authorized users.

PURPOSE: We aimed to identify novel variants in TUBB8 and corresponding new abnormal phenotypes in oocytes/fertilization/ embryonic development responsible for female infertility. METHODS: Sanger sequencing of TUBB8 was performed in infertile women with abnormalities in oocyte maturation or embryonic development. The effects of the variants were evaluated in patients’ oocytes by morphological observations and immunofluorescence. RESULTS: We identified 34 novel variants of TUBB8 in 51 patients who were diagnosed with abnormalities in oocyte maturation or early embryonic development. We found a novel phenotype in which large polar bodies were present in three independent patients possibly associated with a recurrent variant. Moreover, we identified a novel type of TUBB8 variant consisting of an in-frame deletion-insertion, which has not been previously reported. CONCLUSIONS: Our present study identified 34 novel variants in TUBB8 in 51 patients. These patients show oocyte maturation arrest, oocytes with large polar body, fertilization failure, early embryonic arrest or embryonic implantation failure. These results expand the kinds of variants and phenotypic spectrum of TUBB8 variants with regard to female infertility. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-020-01830-6) contains supplementary material, which is available to authorized users.

PURPOSE: “Zygotic cleavage failure” is an embryonic phenotype that causes female infertility and failure of in vitro fertilization and/or intracytoplasmic sperm injection. We aimed to identify pathogenic variants in a female infertility patient from a consanguineous family with the “zygotic cleavage failure” phenotype. METHODS: Whole-exome sequencing was performed in the affected patient; Sanger sequencing was used to confirm the identified variant. The functional effect of the identified variant was further investigated in HeLa cells. RESULTS: We identified a novel homozygous missense mutation in BTG4 (c.285G > C, p.W95C) in the affected individual. Co-immunoprecipitation in HeLa cells showed the complete loss of the interaction between the p.W95C BTG4 variant protein and CNOT7. CONCLUSION: This study confirms our previous research and expands the mutational spectrum of BTG4. Our findings complement the diagnostic genetic basis for “zygotic cleavage failure” patients and suggest that BTG4 may be a good target for future therapeutic strategies.

BackgroundAbnormal pronuclear formation during fertilisation and subsequent early embryonic arrest results in female infertility. In recent years, with the prevalence of assisted reproductive technology, a few genes have been identified that are involved in female infertility caused by abnormalities in oocyte development, fertilisation and embryonic development. However, the genetic factors responsible for multiple pronuclei formation during fertilisation and early embryonic arrest remain largely unknown.ObjectiveWe aim to identify genetic factors responsible for multiple pronuclei formation during fertilisation or early embryonic arrest.MethodsWhole-exome sequencing was performed in a cohort of 580 patients with abnormal fertilisation and early embryonic arrest. Effects of mutations were investigated in HEK293T cells by western blotting and immunoprecipitation, as well as minigene assay.ResultsWe identified a novel homozygous missense mutation (c.397T>G, p.C133G) and a novel homozygous donor splice-site mutation (c.546+5G>A) in the meiotic gene REC114. REC114 is involved in the formation of double strand breaks (DSBs), which initiate homologous chromosome recombination. We demonstrated that the splice-site mutation affected the normal alternative splicing of REC114, while the missense mutation reduced the protein level of REC114 in vitro and resulted in the loss of its function to protect its partner protein MEI4 from degradation.ConclusionsOur study has identified mutations in REC114 responsible for human multiple pronuclei formation and early embryonic arrest, and these findings expand our knowledge of genetic factors that are responsible for normal human female meiosis and fertility.

Background Millions of people around the world are plagued by hearing loss. More than 50% of congenital or pre-lingual deafness is associated with genetic factors and has highly genetic heterogeneity. To date, although hundreds of genes have been found to be implicated in non-syndromic deafness, there are still lots of genes or loci that we need to verify. Methods In this study, we performed target sequencing and Sanger sequencing in a Kazakh consanguineous family with autosomal recessive non-syndromic hearing loss. Following that, functional and structural studies predicted the pathogenic effect of novel mutations by use of the online tools. Results We identified a novel homozygous mutation p.R3191C in MYO15A gene causing deafness in this family. The mutation p.R3191C co-segregated with the disease phenotype in this family and was not present in any public databases. Automatic tools predict that the novel mutation makes a great impact on the function and structure of MYO15A protein. Conclusions This is a novel mutation of MYO15A causing deafness and also the first report of MYO15A mutations causing deafness in the Kazakh families. This finding expanded the spectrum of MYO15A mutations, making it more precise for future genetic diagnosis in patients with deafness.

BackgroundSuccessful human reproduction requires normal spermatogenesis, oogenesis, fertilisation and early embryonic development, and abnormalities in any of these processes will result in infertility. Early embryonic arrest is commonly observed in infertile patients with recurrent failure of assisted reproductive technology (ART). However, the genetic basis for early embryonic arrest is largely unknown.ObjectiveWe aim to identify genetic causes of infertile patients characterised by early embryonic arrest.MethodsWe pursued exome sequencing in a proband with embryonic arrest from the consanguineous family. We further screened candidate genes in a cohort of 496 individuals diagnosed with early embryonic arrest by Sanger sequencing. Effects of mutations were investigated in HeLa cells, oocytes and embryos.ResultsWe identified five independent individuals carrying biallelic mutations in NLRP2. We also found three individuals from two families carrying biallelic mutations in NLRP5. These mutations in NLRP2 and NLRP5 caused decreased protein expression in vitro and in oocytes and embryos.ConclusionsNLRP2 and NLRP5 are novel mutant genes responsible for human early embryonic arrest. This finding provides additional potential diagnostic markers for patients with recurrent failure of ART and helps us to better understand the genetic basis of female infertility characterised by early embryonic arrest.

The human zona pellucida (ZP) is an extracellular glycoprotein matrix composed of ZP1, ZP2, ZP3, and ZP4 surrounding the oocyte, and it plays an important role in sperm–egg interactions during fertilization. Structural and functional changes in the ZP can influence the process of fertilization and lead to female infertility. Previous studies have identified mutations in ZP1, ZP2, and ZP3 that lead to female infertility caused by oocyte degeneration, empty follicle syndrome, or in vitro fertilization failure. Here we describe seven patients from six independent families who had several abnormal oocytes or suffered from empty follicle syndrome, similar to the previously reported phenotypes. By whole-exome sequencing and Sanger sequencing, we identified several novel mutations in these patients. These included three homozygous mutations in ZP1 (c.1708G > A, p.Val570Met; c.1228C > T, p.Arg410Trp; c.507del, p.His170Ilefs*52), two mutations in a compound heterozygous state in ZP1 (c.1430 + 1G > T, p.Cys478X and c.1775-8T > C, p.Asp592Glyfs*29), a homozygous mutation in ZP2 (c.1115G > C, p.Cys372Ser), and a heterozygous mutation in ZP3 (c.763C > G, p.Arg255Gly). In addition, studies in CHO cells showed that the mutations in ZP1, ZP2, and ZP3 might affect the corresponding protein expression, secretion, and interaction, thus providing a mechanistic explanation for the phenotypes. Our study expands the spectrum of ZP gene mutations and phenotypes, and provides a further understanding of the pathogenic mechanism of ZP gene mutations in vitro.

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

PANX1, one of the members of the pannexin family, is a highly glycosylated channel-forming protein. Recently, we identified heterozygous variants in PANX1 that follow an autosomal dominant inheritance pattern and cause female infertility characterized by oocyte death. In this study, we screened for novel PANX1 variants in patients with the phenotype of oocyte death and discovered a new type of inheritance pattern accompanying PANX1 variants. We identified two novel homozygous missense variants in PANX1 [NM_015368.4 c.712T>C (p.(Ser238Pro) and c.899G>A (p.(Arg300Gln))] associated with the oocyte death phenotype in two families. Both of the homozygous variants altered the PANX1 glycosylation pattern in cultured cells, led to aberrant PANX1 channel activation, and resulted in mouse oocyte death after fertilization in vitro. It is worth noting that the destructive effect of the two homozygous variants on PANX1 function was weaker than that caused by the recently reported heterozygous variants. Our findings enrich the variational spectrum of PANX1 and expand the inheritance pattern of PANX1 variants to an autosomal recessive mode. This highlights the critical role of PANX1 in human oocyte development and helps us to better understand the genetic basis of female infertility due to oocyte death.

PURPOSE: We aimed to identify pathogenic variants in two infertile sisters in a family with a thin zona pellucida (ZP) phenotype. METHODS: Whole-exome sequencing was performed in the two affected sisters, and Sanger sequencing was used to confirm the identified variants. The effects of the identified variant were further investigated in mouse oocytes and Chinese hamster ovary (CHO) cells. RESULTS: We identified a novel homozygous frameshift variant in ZP2 (c.1235_1236del, p.Q412Rfs*17) in the two affected individuals. Immunoblotting demonstrated that the variant produced a truncated ZP2 protein that was expressed at low levels in CHO cells. Immunofluorescence in mouse oocytes confirmed the decreased protein level of mutant ZP2, although the subcellular localization was not affected. In addition, immunoprecipitation showed that the pathogenic variant reduced the interaction between ZP2 and ZP3. CONCLUSION: This study identified a novel pathogenic variant in ZP2 that produces a truncated ZP2 protein. The variant might disrupt the assembly of ZP2-ZP3 dimers, thus resulting in a thin ZP and female infertility.

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.

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.

Successful human reproduction initiates from normal gamete formation, fertilization and early embryonic development. Abnormalities in any of these steps will lead to infertility. Many infertile patients undergo several failures of IVF and intracytoplasmic sperm injection (ICSI) cycles, and embryonic developmental arrest is a common phenotype in cases of recurrent failure of IVF/ICSI attempts. However, the genetic basis for this phenotype is poorly understood. The subcortical maternal complex (SCMC) genes play important roles during embryonic development, and using whole-exome sequencing novel biallelic mutations in the SCMC genes TLE6, PADI6 and KHDC3L were identified in four patients with embryonic developmental arrest. A mutation in TLE6 was found in a patient with cleaved embryos that arrested on day 3 and failed to form blastocysts. Two patients with embryos that arrested at the cleavage stage had mutations in PADI6, and a mutation in KHDC3L was found in a patient with embryos arrested at the morula stage. No mutations were identified in these genes in an additional 80 patients. These findings provide further evidence for the important roles of TLE6, PADI6 and KHDC3L in embryonic development. This work lays the foundation for the genetic diagnosis of patients with recurrent IVF/ICSI failure.

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.

Oocyte maturation arrest results in female infertility, but the genetic determinants of human oocyte maturation arrest remain largely unknown. Previously, we identified TUBB8 mutations responsible for human oocyte maturation arrest, indicating the important role of genetic factors in the disorder. However, TUBB8 mutations account for only around 30% of individuals with oocyte maturation arrest; thus, the disorder is likely to involve other genetic factors that are as yet unknown. Here, we initially identified a homozygous nonsense mutation of PATL2 (c.784C>T [p.Arg262∗]) in a consanguineous family with a phenotype characterized by human oocyte germinal vesicle (GV) arrest. Subsequent mutation screening of PATL2 in a cohort of 179 individuals identified four additional independent individuals with compound-heterozygous PATL2 mutations with slight phenotypic variability. A genetic burden test further confirmed the genetic contribution of PATL2 to human oocyte maturation arrest. By western blot in HeLa cells, identification of splicing events in affected individuals’ granulosa cells, and immunostaining in affected individuals’ oocytes, we provide evidence that mutations in PATL2 lead to decreased amounts of protein. These findings suggest an important role for PATL2 mutations in oocyte maturation arrest and expand our understanding of the genetic basis of female infertility.

PURPOSE: The present study was intended to identify genetic causes of infertile patients with recurrent failure of in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) attempts. METHODS: Infertile patients with recurrent IVF/ICSI failure from Shanghai Ji Ai Genetics & IVF Institute and the Ninth Hospital affiliated with Shanghai Jiao Tong University were recruited. Genomic DNA samples were extracted from their peripheral blood. Whole-exome sequencing and Sanger validation were performed to identify candidate variants. RESULTS: We identified novel transducin-like enhancer of split 6 (TLE6) gene mutations in three patients with recurrent IVF/ICSI failure. One patient carried a homozygous missense mutation (c.1226G>A; p.Arg409Gln) with subsequent fertilization failure, while the other two patients carried either a homozygous missense mutation (c.1621G>A; p.Glu541Lys) or a compound heterozygous missense mutation (c.388G>A/c.1507G>A; p.Asp130Asn/p.Val503Ile) and had viable but low-quality embryos. CONCLUSIONS: Our study expands the mutational and phenotypic spectrum of TLE6 and suggests the important role of TLE6 during embryonic development. Our findings have implications for the genetic diagnosis of female infertility with recurrent IVF/ICSI failure. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s10815-019-01653-0) contains supplementary material, which is available to authorized users.

Fertilization involves a series of molecular events immediately following egg–sperm fusion; Ca2+ oscillations are the earliest signaling event, and they initiate the downstream reactions including pronucleus formation. Successful human reproduction requires normal fertilization. In clinical IVF or ICSI attempts, some infertile couples suffer from recurrent fertilization failure. However, the genetic reasons for fertilization failure are largely unknown. Here, we recruited several couples diagnosed with fertilization failure even though their gametes are morphologically normal. Through whole-exome sequencing and Sanger sequencing, we identified biallelic mutations in gene-encoding phospholipase C zeta 1 (PLCZ1) in four independent males in couples diagnosed with fertilization failure. Western blotting showed that missense mutations decreased the level of PLCZ1 and that nonsense or frameshift mutations resulted in undetectable or truncated proteins. Expression of these mutations in mice significantly reduced the levels of oocyte activation. Artificial oocyte activation in patient oocytes could rescue the phenotype of fertilization failure and help establish pregnancy and lead to live birth. Our findings expand the spectrum of PLCZ1 mutations that are responsible for human fertilization failure and provide a potentially feasible therapeutic treatment for these patients.

Abnormal pronuclear formation during fertilisation and subsequent early embryonic arrest results in female infertility. In recent years, with the prevalence of assisted reproductive technology, a few genes have been identified that are involved in female infertility caused by abnormalities in oocyte development, fertilisation and embryonic development. However, the genetic factors responsible for multiple pronuclei formation during fertilisation and early embryonic arrest remain largely unknown.We aim to identify genetic factors responsible for multiple pronuclei formation during fertilisation or early embryonic arrest.Whole-exome sequencing was performed in a cohort of 580 patients with abnormal fertilisation and early embryonic arrest. Effects of mutations were investigated in HEK293T cells by western blotting and immunoprecipitation, as well as minigene assay.We identified a novel homozygous missense mutation (c.397TG, p.C133G) and a novel homozygous donor splice-site mutation (c.546+5GA) in the meiotic geneOur study has identified mutations in

Context The LH/chorionic gonadotropin receptor (LHCGR) is mainly expressed in gonads and plays important roles in estradiol production, ovulation, and luteal formation. Women with pathogenic LHCGR variants suffer from infertility, and successful fertility treatments for such women have never been reported. Objective The purpose of this study was to determine whether women with pathogenic LHCGR variants can achieve successful pregnancies through in vitro fertilization. Design Three women with LH resistance and infertility and their parents underwent exome sequencing. The biochemical characteristics and functional effects of LHCGR mutation were assessed in transfected human embryonic kidney -293T cells and primary granulosa cells. Results All affected women harbored pathogenic LHCGR variants. The LHCGR variants lacked cell surface localization and signal transduction abilities in vitro and in vivo. After dual triggering and prolonging the interval between triggering and oocyte pick-up, all three patients achieved oocytes and high-quality embryos. After frozen embryo transfer, one woman successfully birthed twins, and one woman successfully birthed a live boy. Apart from difficulties in oocyte retrieval, no obvious abnormalities in fertilization or during embryo development and pregnancy were identified in these patients. Conclusions This study is, to our knowledge, the first to report successful assisted reproductive treatment of women with pathogenic LHCGR variants using their own oocytes. Our results supported that defects in LHCGR disrupted ovulation but had no effect on fertilization and embryo development.

Intellectual disability (ID) describes a wide range of serious human diseases caused by defects in central nervous system development and function. Some mutant genes have been found to be associated with these diseases, but not all cases can be explained, thus suggesting that other disease-causing genes have not yet been discovered. Sialic acid is involved in a number of key biological processes, including embryo formation, nerve cell growth, and cancer cell metastasis, and very recently it has been suggested that N-acetylneuraminic acid synthase-mediated synthesis of sialic acid is required for brain and skeletal development. CMP-sialic acid synthetase (CMAS) is one of four enzymes involved in NeuNAc metabolism, as it catalyzes the formation of CMP-NeuNAc. Before the present study, no links between mutations in CMAS and incidences of human ID had been reported. In the current study, we recruited a recessive nonsyndromic ID pedigree with consanguineous marriage in which all patients have typical clinical manifestations of ID. We identified the NM_018686.3:c.563G > A (p.Arg188His) substitution in CMAS as being responsible for the disease in this family. Conservation analysis, structural prediction, and enzyme activity experiments demonstrated that (p.Arg188His) influences protein dimerization and alters CMAS enzyme activity. Our results offer a new orientation for future research and clinical diagnosis.

Successful human reproduction requires normal spermatogenesis, oogenesis, fertilisation and early embryonic development, and abnormalities in any of these processes will result in infertility. Early embryonic arrest is commonly observed in infertile patients with recurrent failure of assisted reproductive technology (ART). However, the genetic basis for early embryonic arrest is largely unknown.We aim to identify genetic causes of infertile patients characterised by early embryonic arrest.We pursued exome sequencing in a proband with embryonic arrest from the consanguineous family. We further screened candidate genes in a cohort of 496 individuals diagnosed with early embryonic arrest by Sanger sequencing. Effects of mutations were investigated in HeLa cells, oocytes and embryos.We identified five independent individuals carrying biallelic mutations in

Early embryonic arrest is one of the major causes of female infertility. However, because of difficulties in phenotypic evaluation, genetic determinants of human early embryonic arrest are largely unknown. With the development of assisted reproductive technology, the phenotype of early human embryonic arrest can now be carefully evaluated. Here, we describe a consanguineous family with a recessive inheritance pattern of female infertility characterized by recurrent early embryonic arrest in cycles of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). We have identified a homozygous PADI6 nonsense mutation (c.1141C>T [p.Gln381 ∗ ]) that is responsible for the phenotype. Mutational analysis of PADI6 in a cohort of 36 individuals whose embryos displayed developmental arrest identified two affected individuals with compound-heterozygous mutations (c.2009_2010del [p.Glu670Glyfs ∗ 48] and c.633T>A [p.His211Gln]; c.1618G>A [p.Gly540Arg] and c.970C>T [p.Gln324 ∗ ]). Immunostaining indicated a lack of PADI6 in affected individuals' oocytes. In addition, the amount of phosphorylated RNA polymerase II and expression levels of seven genes involved in zygotic genome activation were reduced in the affected individuals' embryos. This phenotype is consistent with Padi6 knockout mice. These findings deepen our understanding of the genetic basis of human early embryonic arrest, which has been a largely ignored Mendelian phenotype. Our findings lay the foundation for uncovering other genetic causes of infertility resulting from early embryonic arrest.

Successful fertilization is fundamental for sexual reproduction. After undergoing a series of molecular and morphological changes, the haploid sperm fuses with the haploid oocyte to create a diploid zygote. Defects in this process might lead to human fertilization failure. We have previously found homozygous mutations in WEE2 that are responsible for human fertilization failure, but the genetic basis of human fertilization failure requires further investigation. In the present study, we screened for WEE2 mutations in a new cohort of patients with fertilization failure. Through Sanger sequencing of WEE2 exons, we identified seven novel mutations and two reported mutations in WEE2 from six affected individuals. Morphologically normal PB1 oocytes can be retrieved from all patients. However, most of the oocytes cannot be fertilized successfully. These findings confirmed our previous research and expanded the mutational spectrum of WEE2, making it a potential genetic diagnostic marker for those suffering from human fertilization failure.

Connexins and pannexins are two protein families that play an important role in cellular communication. Pannexin 1 (PANX1), one of the members of pannexin family, is a channel protein. It is glycosylated and forms three species, GLY0, GLY1, and GLY2. Here, we describe four independent families in which mutations in PANX1 cause familial or sporadic female infertility via a phenotype that we term "oocyte death." The mutations, which are associated with oocyte death, alter the PANX1 glycosylation pattern, influence the subcellular localization of PANX1 in cultured cells, and result in aberrant PANX1 channel activity, ATP release in oocytes, and mutant PANX1 GLY1. Overexpression of a patient-derived mutation in mice causes infertility, recapitulating the human oocyte death phenotype. Our findings demonstrate the critical role of PANX1 in human oocyte development, provide a genetic explanation for a subtype of infertility, and suggest a potential target for therapeutic intervention for this disease.

Human oocyte maturation is a precondition for fertilization and ensuing embryonic development. Previously, we identified TUBB8 variants as a genetic determinant of human oocyte maturation arrest and showed that these variants cause variable and mixed phenotypes in oocyte maturation and early embryo development. We also estimated that rare inherited or de novo variants in the TUBB8 gene accounted for 30% of individuals in a small cohort of patients affected by oocyte maturation arrest. In the present study, we recruited a further 87 patients from unrelated families diagnosed with oocyte maturation or early embryonic arrest and identified 30 patients carrying TUBB8 variants. The corresponding phenotypes not only include oocyte maturation arrest, failure of fertilization, and early embryonic arrest, but also extend to the new phenotype of failure of embryo implantation. These observations provide the most detailed mutational and phenotypic spectrum of TUBB8, further extend the spectrum of variants and dysfunctional oocyte and embryo phenotypes caused by TUBB8 variants, and confirm previous findings for a critical role of TUBB8 during oocyte maturation and early embryonic development. Thus, TUBB8 mutation screening might not only be a genetic diagnostic marker for patients with oocyte maturation arrest, but might also have clinical implications for evaluating the competence of patients’ functional oocytes with first polar body (PB1).

Arthrospira platensis is an ideal organism for a proteome study under conditions of environmental stress. Herein, we report the differential expression of proteins in A. platensis strain YZ responding to 48 h of continuous dark or light conditions by two-dimensional gel electrophoresis and peptide mass fingerprinting techniques. There were 102 proteins retrieved with significant differential expression between light and dark incubation, among which 96 protein spots were found with high homology to Arthrospira genus (67 proteins in A. platensis strain Paraca and 29 in A. maxima strain CS-328). The other six proteins belonged to other microorganisms. Using a clusters of orthologous groups database, we classified the 102 differentially expressed positive proteins into 19 types based on their function and localized them in their respective KEGG metabolic pathways. The proteins were involved in 47 metabolic pathways, such as photosynthesis, glycolysis/gluconeogenesis pathway, oxidative phosphorylation, and ami...