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BACKGROUND: Early embryonic arrest and fragmentation (EEAF) is a common cause of female infertility, but the genetic causes remain to be largely unknown. CIP2A encodes the cellular inhibitor of PP2A, playing a crucial role in mitosis and mouse oocyte meiosis. METHODS: Exome sequencing and Sanger sequencing were performed to identify candidate causative genes in patients with EEAF. The pathogenicity of the CIP2A variant was assessed and confirmed in cultured cell lines and human oocytes through Western blotting, semi-quantitative RT-PCR, TUNEL staining, and fluorescence localization analysis. FINDINGS: We identified CIP2A (c.1510C > T, p.L504F) as a novel disease-causing gene in human EEAF from a consanguineous family. L504 is highly conserved throughout evolution. The CIP2A variant (c.1510C > T, p.L504F) reduced the expression level of the mutant CIP2A protein, leading to the abnormal aggregation of mutant CIP2A protein and cell apoptosis. Abnormal aggregation of CIP2A protein and chromosomal dispersion occurred in the patient's oocytes and early embryos. We further replicated the patient phenotype by knockdown CIP2A in human oocytes. Additionally, CIP2A deficiency resulted in decreased levels of phosphorylated ERK1/2. INTERPRETATION: We first found that the CIP2A loss-of-function variant associate with female infertility characterized by EEAF. Our findings suggest the uniqueness and importance of CIP2A gene in human oocyte and early embryo development. FUNDING: This work was supported by National Key Research and Development Program of China (2023YFC2706302), the National Natural Science Foundation of China (81000079, 81170165, and 81870959), the HUST Academic Frontier Youth Team (2016QYTD02), and the Key Research of Huazhong University of Science and Technology, Tongji Hospital (2022A20).

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BACKGROUND: Infertility and pregnancy loss are longstanding problems. Successful fertilization and high-quality embryos are prerequisites for an ongoing pregnancy. Studies have proven that every stage in the human reproductive process is regulated by multiple genes and any problem, at any step, may lead to fertilization failure (FF) or early embryonic arrest (EEA). Doctors can diagnose the pathogenic factors involved in FF and EEA by using genetic methods. With the progress in the development of new genetic technologies, such as single-cell RNA analysis and whole-exome sequencing, a new approach has opened up for us to directly study human germ cells and reproductive development. These findings will help us to identify the unique mechanism(s) that leads to FF and EEA in order to find potential treatments. OBJECTIVE AND RATIONALE: The goal of this review is to compile current genetic knowledge related to FF and EEA, clarifying the mechanisms involved and providing clues for clinical diagnosis and treatment. SEARCH METHODS: PubMed was used to search for relevant research articles and reviews, primarily focusing on English-language publications from January 1978 to June 2023. The search terms included fertilization failure, early embryonic arrest, genetic, epigenetic, whole-exome sequencing, DNA methylation, chromosome, non-coding RNA, and other related keywords. Additional studies were identified by searching reference lists. This review primarily focuses on research conducted in humans. However, it also incorporates relevant data from animal models when applicable. The results were presented descriptively, and individual study quality was not assessed. OUTCOMES: A total of 233 relevant articles were included in the final review, from 3925 records identified initially. The review provides an overview of genetic factors and mechanisms involved in the human reproductive process. The genetic mutations and other genetic mechanisms of FF and EEA were systematically reviewed, for example, globozoospermia, oocyte activation failure, maternal effect gene mutations, zygotic genome activation abnormalities, chromosome abnormalities, and epigenetic abnormalities. Additionally, the review summarizes progress in treatments for different gene defects, offering new insights for clinical diagnosis and treatment. WIDER IMPLICATIONS: The information provided in this review will facilitate the development of more accurate molecular screening tools for diagnosing infertility using genetic markers and networks in human reproductive development. The findings will also help guide clinical practice by identifying appropriate interventions based on specific gene mutations. For example, when an individual has obvious gene mutations related to FF, ICSI is recommended instead of IVF. However, in the case of genetic defects such as phospholipase C zeta1 (PLCZ1), actin-like7A (ACTL7A), actin-like 9 (ACTL9), and IQ motif-containing N (IQCN), ICSI may also fail to fertilize. We can consider artificial oocyte activation technology with ICSI to improve fertilization rate and reduce monetary and time costs. In the future, fertility is expected to be improved or restored by interfering with or supplementing the relevant genes.

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Oocyte maturation is vital to attain full competence required for fertilization and embryogenesis. NLRP14 is preferentially expressed in mammalian oocytes and early embryos. Yet, the role and molecular mechanism of NLRP14 in oocyte maturation and early embryogenesis are poorly understood, and whether NLRP14 deficiency accounts for human infertility is unknown. Here, we found that maternal loss of Nlrp14 resulted in sterility with oocyte maturation defects and early embryonic arrest (EEA). Nlrp14 ablation compromised oocyte competence due to impaired cytoplasmic and nuclear maturation. Importantly, we revealed that NLRP14 maintained cytoplasmic UHRF1 abundance by protecting it from proteasome-dependent degradation and anchoring it from nuclear translocation in the oocyte. Furthermore, we identified compound heterozygous NLRP14 variants in women affected by infertility with EEA, which interrupted the NLRP14-UHRF1 interaction and decreased UHRF1 levels. Our data demonstrate NLRP14 as a cytoplasm-specific regulator of UHRF1 during oocyte maturation, providing insights into genetic diagnosis for female infertility.

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Oocyte maturation arrest, fertilization failure, and early embryonic arrest are important causes of female infertility, whereas the genetic events that contribute to these processes are largely unknown. Loss-of-function of PABPC1L in mice has been suggested to cause female infertility involved in the absence of mature oocytes or embryos in vivo or in vitro. However, the role of PABPC1L in human female reproduction remains largely elusive. In this study, we identified a homozygous missense mutation (c.536G>A, p.R179Q) and a compound heterozygous mutation (c.793C>T, p.R265W; c.1201C>T, p.Q401*) in PABPC1L in two unrelated infertile females characterized by recurrent oocyte maturation abnormalities and early embryonic arrest. These variants resulted in nonfunctional PABPC1L protein and were associated with impaired chromatin configuration and transcriptional silencing in GV oocytes. Moreover, the binding capacity of mutant PABPC1L to mRNAs related to oocyte maturation and early embryonic development was decreased significantly. Our findings revealed novel PABPC1L mutations causing oocyte maturation abnormalities and early embryonic arrest, confirming the essential role of PABPC1L in human female fertility.

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Background: Early embryonic arrest (EEA) leads to repeated cessation of fresh cycles among infertile women undergoing in vitro fertilization (IVF). Whether the levels of some essential trace elements [copper (Cu), zinc (Zn), selenium (Se) and cobalt (Co)] in the bodies of women are related to the risk of EEA warrants study. Objective: Our study aimed to investigate the associations of peripheral blood levels of Cu, Zn, Se, and Co and their mixtures with the risk of EEA. Methods: A total of 74 EEA cases (123 IVF cycles) and 157 controls (180 IVF cycles) from the reproductive center of the First Affiliated Hospital of Anhui Medical University in Hefei, China, between June 2017 and March 2020 were included in our study. Demographic and clinical data were collected from electronic medical records. Cu, Zn, Se, and Co levels were measured in blood samples collected on the day of oocyte retrieval when infertile women entered clinical treatment for the first time using an inductively coupled plasma mass spectrometer (ICP-MS). Generalized estimating equation (GEE) models were used to evaluate the associations of four essential trace element concentrations individually with the risk of EEA, and Bayesian kernel machine regression (BKMR) was used to explore the associations between four essential trace element mixtures and the risk of EEA. Results: Se concentrations of infertile women were significantly lower in the case group compared with the control group. Co levels were significantly higher in the case group compared with the control group. The differences in Cu and Zn concentrations between the two groups were not significant. Based on single-metal models, Co was positively associated with the risk of EEA before and after adjustment for all confounders (odd ratio (OR) = 1.72, 95% confidence interval (CI): 1.18-2.52; OR = 2.27, 95% CI: 1.37-3.77, respectively), and Se was negatively associated with the risk of EEA before adjustment for all confounders (OR = 0.18, 95% CI: 0.07-0.51). BKMR analyses showed that Se was significantly and negatively associated with the risk of EEA when all the other three metals (Cu, Zn, and Co) were fixed at the 25th, 50th, or 75th percentiles, whereas Zn displayed a significant and positive association with the risk of EEA when all the other three metals (Cu, Se and Co) were fixed at the 25th, 50th, or 75th percentiles. Co did not show any effect on the risk of EEA when all the other metals (Cu, Zn, and Se) were fixed at the 25th, 50th, or 75th percentiles. In addition, an increasing trend of the joint effect of four essential trace elements on the risk of EEA was found, although it was not statistically significant. Conclusion: The levels of essential trace elements (Cu, Zn, Se, and Co) might correlate with the risk of EEA to some extent. The present study might provide a real-world perspective on the relationship between essential trace elements and the risk of EEA when considering them as a single element or as mixtures.

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The subcortical maternal complex (SCMC), composed of several maternal-effect genes, is vital for the development of oocytes and early embryos. Variants of SCMC-encoding genes (NLRP2, NLRP5, TLE6, PADI6, and KHDC3L, but not OOEP and ZBED3) are associated with human oocyte maturation dysfunction, fertilization failure, and early embryonic arrest. In this study, we enrolled 118 Chinese patients who experienced recurrent preimplantation embryonic arrest during assisted reproductive technology treatments and performed whole-exome sequencing. We discovered compound heterozygous missense variants (c.110G>C and c.109C>G) in the OOEP gene in one patient who experienced recurrent preimplantation embryonic arrest. Arrested embryos from this affected patient were analyzed by single-cell RNA sequencing, which showed a downregulated transcriptome. In addition, six novel NLRP5 variants (c.971T>A, c.3341T>C, c.1575_1576delAG, c.1830_1831delGT, c.1202C>T, and c.2378T>G) were identified in four patients with arrested and severely fragmented embryos. These suspicious mutations were examined by in vitro studies in HEK293T cells. Western blot analysis and immunofluorescence experiments showed that OOEP and partial NLRP5 mutations caused decreased protein levels. Our findings first demonstrated that biallelic variants in OOEP gene could also cause human early embryonic arrest, similar to other SCMC components. We expanded the genetic mutation spectrum of SCMC genes related to early embryogenesis in humans, especially early embryonic arrest.

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Early embryonic arrest denotes premature termination of development in preimplantation embryos, which is one of the major phenotypes of recurrent assisted reproduction failure. Padi6 is proven to be a member of the subcortical maternal complex (SCMC) in mice, which is essential in oocyte maturation and embryogenesis. We and other groups previously found that biallelic mutations in PADI6 caused female infertility manifesting as early embryonic arrest. In this study, we identified two novel homozygous variants (p.Cys163Arg, and p. Trp475*) of PADI6 in two infertile patients from a cohort of 75 females with the phenotype of early embryonic arrest. An in vitro expression study indicated severe decrease of PADI6, which might destruct the stability of SCMC. Our study expands the mutational spectrum of PADI6 and further supports the causality between PADI6 mutations and female infertility.

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Early embryonic arrest (EEA) leads to cancelation of fresh cycles among infertile women undergoing in vitro fertilization (IVF), bringing a great challenge for IVF. Whether exposure to thallium (Tl) is associated with an increased risk of EEA, especially its interaction with polymorphisms of mitochondria DNA (mtDNA) gene, is worthy of study. A case-control design was performed, including 74 EEA cases with 123 IVF cycles and 157 age and BMI-matched controls with 180 IVF cycles. Levels of Tl and other toxic metals (lead (Pb), (mercury) Hg, and (arsenci) As) were assessed by measuring them in blood samples collected on the day of oocyte retrieval; PCR amplification and sequencing were performed to screen the polymorphic sites of mtDNA gene in D-loop region. Bayesian kernel machine regression (BKMR) was used to confirm that Tl played a leading role in the situation of combined exposure; generalized estimating equation (GEE) models were used to evaluate the associations of Tl concentrations, polymorphisms of mtDNA gene, and their interactions with the risk of EEA. The impact of Tl exposure or polymorphisms of mtDNA gene on the oogenesis and embryonic development was also evaluated. BKMR analysis revealed that PIP (posterior inclusion probability) value of T1 was 0.9096, indicating that it played a leading role in the situation of combined exposure. Compared to the first quartile of Tl, the adjusted ORs (95% CIs) of EEA risk were 0.66 (0.26, 1.70), 1.18 (0.52, 2.64), and 4.53 (2.11, 9.69) for the second, third, and fourth quartile, respectively (p trend < 0.001). Compared to the wild type of mtDNA 16,519 gene (T 16,519 T), the adjusted OR (95% CI) of EEA risk for the variant type (T 16,519 C) was 3.11 (1.70, 5.72), and the variant types of the other sites with a minor allele frequency > 10% were not significantly related with the risk of EEA after FDR (False Discovery Rate) correction. With respect to interaction, compared to women at low Tl exposure level & wild type of mtDNA 16,519 gene group, the adjusted OR (95% CI) of EEA risk for women at high Tl exposure level & variant type of mtDNA 16,519 gene group was 9.28 (3.33, 25.81). Additionally, Tl exposure and polymorphisms of mtDNA 16,519 gene are inversely associated with the outcomes of oogenesis and embryonic development significantly. Our study indicated that high Tl exposure level was associated with the increased risk of EEA and Tl played a leading role in the situation of combined exposure; the strength of association was much higher when Tl exposure interacted with polymorphism of 16,519 mtDNA gene. These relationships might originate from the impact of Tl exposure or polymorphism of 16,519 mtDNA gene on the oogenesis and early embryonic development in vitro. Infertile women should keep high vigilant against Tl exposure especially those with variant type of mtDNA 16,519 gene.

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BACKGROUND: Early embryonic arrest is a great challenge for in vitro fertilization. Whether exposure to toxic metals is associated with an increased risk of early embryonic arrest warrants investigation. OBJECTIVES: Here, we conducted a case-control study in infertile women to estimate the associations between blood barium (Ba), arsenic (As), mercury (Hg), and lead (Pb) exposure levels and the risk of early embryonic arrest. METHODS: Ba, As, Hg, and Pb exposure levels in fasting blood collected from 74 infertile women (123 cycles) with early embryonic arrest and 157 infertile women (180 cycles) without early embryonic arrest were measured by ICP-MS. Bayesian kernel machine regression (BKMR) was used to assess the association of exposure level of toxic metals mixture with the risk of early embryonic arrest as well as to evaluate which metal playing a leading role in the association, and then generalized estimating equations (GEEs) were used to evaluate the relationship between the selected harmful metal and the risk of early embryonic arrest. Finally, the potential causes of early embryonic arrest originating from the harmful metal exposure were explored. RESULTS: Blood Ba levels were significantly higher in the case group than that in the control group (p = 0.009) rather than As, Pb and Hg. Results from BKMR showed that exposure to toxic metals mixture increased the risk of early embryonic arrest, with Ba playing a leading role (PIP = 0.9612). GEE analysis showed that high Ba exposure level was related with the increased risk of early embryonic arrest (p < 0.05) and it impacted on the oogenesis significantly. CONCLUSIONS: Our study found that exposure to toxic metals mixture was associated with the increased risk of early embryonic arrest, and Ba contributed most to the increased risk. Higher Ba exposure in whole blood corresponds to a higher risk of early embryonic arrest and impacted on the oogenesis significantly.

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PURPOSE: This study aims to identify the genetic causes of 12 women with primary infertility characterized by primarily oocyte maturation abnormality and consequent early embryonic arrest. METHODS: Genomic DNA was isolated from peripheral blood samples. Whole-exome sequencing was performed on the probands, and the identified variants were confirmed by Sanger sequencing. The pathogenicity of the identified variants on the protein was accessed in silico. And we used qRT-PCR to detect the possible effects of the novel mutation on the mRNA level of NLRP5. RESULTS: A novel homozygous frameshift variant (p.V429Efs*30) in NLRP5 and compound heterozygous variants with a novel frameshift variant (p.A297Efs*20) and a recurrent variant (c. 223-14_223-2delCCCTCCTGTTCCA) in PATL2 were identified in two unrelated affected individuals. qRT-PCR showed an obvious decrease of the mutant NLRP5 mRNA. In addition, the truncated proteins of NLRP5 and PATL2 were predicted to be non-functional due to the deletion of the most or the whole region of the critical functional domain(s) respectively. CONCLUSIONS: This study identified novel mutations in NLRP5 and PATL2, further expanding the mutational and phenotypic spectrum of both genes. This is the first report of the NLRP5 mutations that associates with oocyte maturation abnormality in humans.

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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.

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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.

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The cell division cycle 20 (CDC20) protein is a co-activator of anaphase-promoting complex/cyclosome (APC/C), required for mitotic exit and also meiotic exit, containing seven WD40 repeats in the C-terminus responsible for protein-protein interactions. Recently, a previous study has shown that biallelic mutations in CDC20 are causative for female infertility with abnormalities in oocyte maturation and embryonic development. This study is to further identify new mutations of CDC20 and the prevalence of variants in our cohort. A cohort of 50 primary infertile females with oocyte maturation abnormality and early embryonic arrest were recruited. Genomic DNA was isolated from peripheral blood samples. Mutation screening of all the coding regions of CDC20 was performed by Sanger sequencing. The pathogenicity of the identified variants on the CDC20 protein was accessed in silico. Two CDC20 variants, a nonsense mutation p.R262* and a missense mutation p.A211T, identified in one female of 50 unrelated affected individuals, accounting for a relative small proportion of this cohort (2%). In silico analysis revealed that the p.R262* would cause no production of protein or a truncated protein lacking five WD40 repeats in the C-terminus; and that p.A211T may interfere with the formation of a deep hydrophobic pocket and thus disturb the binding of CDC20 protein to the substrates of APC/C. This study identified two novel mutations in CDC20, further expanding the mutation spectrum of this gene. Our findings further confirm that biallelic mutations in CDC20 occur in a proportion of infertile females with oocyte maturation abnormality and early embryonic arrest.

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BACKGROUND: 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. OBJECTIVE: We aim to identify genetic factors responsible for multiple pronuclei formation during fertilisation or early embryonic arrest. METHODS: 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. RESULTS: We 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. CONCLUSIONS: Our 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.

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Early Embryonic Arrest (EEA) is one of the major causes of female infertility. Genetic factors including specific genes and miRNAs may play pivotal roles on EEA. However, it is not well defined what genes and micro RNAs participate the pathophysiological alterations of EEA. In this work, we compared the Transcriptome -Seq and microRNA profiles from three pairs of villi (three EEA patients and three normal pregnancy, NP). We first confirmed the array data by qPCR with ten randomly selected differentially expressed genes and ten differentially expressed miRNAs in villi from 20 EEA and 20 NP controls. We next applied Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway analysis and found that these differentially expressed genes enriched in the PI3K-Akt signaling pathway, Jak-STAT signaling pathway, MAPK signaling pathway, Complement and coagulation cascades, Hypertrophic cardiomyopathy (HCM), Dilated cardiomyopathy (DCM). Interestingly, hsa-miR-6515-5p and its target genes NLRP3, UGP2 may regulate the Immune system and carbohydrate metabolism. Hsa-miRNA 518 and its target gene EGR1 may regulate cell proliferation, angiogenesis, and cell apoptosis to impact early embryonic development. Moreover, novel-m0045-5p and its target gene RMDN3 may regulate microtubule formation on the development of EEA. Our research provides novel biomarkers for EEA and establishes a foundation for further study of the mechanism of EEA.

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