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

Can any new genetic factors responsible for early embryonic arrest in infertile patients be identified, together with the mechanism of pathogenic variants? 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. 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. 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. 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. 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. 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. 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. 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. N/A.

Wang W ，Wang W ，Xu Y ，Shi J ，Fu J ，Chen B ，Mu J ，Zhang Z ，Zhao L ，Lin J ，Du J ，Li Q ，He L ，Jin L ，Sun X ，Wang L ，Sang Q ... -  《-》

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

Are mutations in MOS (MOS proto-oncogene, serine/threonine kinase) involved in early embryonic arrest in infertile women? 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. 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. 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. 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. 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. 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. 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. 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. N/A.

Zeng Y ，Shi J ，Xu S ，Shi R ，Wu T ，Li H ，Xue X ，Zhu Y ，Chen B ，Sang Q ，Wang L ... -  《-》

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

Are any novel mutations and corresponding new phenotypes, other than recurrent hydatidiform moles, seen in patients with MEI1 mutations? We identified several novel mutations in MEI1 causing new phenotypes of early embryonic arrest and recurrent implantation failure. 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. 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. 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. 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. 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. 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. 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. N/A.

Dong J ，Zhang H ，Mao X ，Zhu J ，Li D ，Fu J ，Hu J ，Wu L ，Chen B ，Sun Y ，Mu J ，Zhang Z ，Sun X ，Zhao L ，Wang W ，Wang W ，Zhou Z ，Zeng Y ，Du J ，Li Q ，He L ，Jin L ，Kuang Y ，Wang L ，Sang Q ... -  《-》

A heterozygous SPRY4 variant identified in female infertility characterized by reduced oocyte potential and early embryonic arrest.

Can novel genetic factors contributing to early embryonic arrest in infertile patients be identified, along with the underlying mechanisms of the pathogenic variant? We identified a heterozygous variant in the SPRY4 (sprouty RTK signaling antagonist 4) in infertile patients and conducted in vitro and in vivo studies to investigate the effects of the variant/deletion, highlighting its critical role in female reproductive health. SPRY4 acts as a negative regulator of receptor tyrosine kinases (RTKs) and functions as a tumor suppressor. Its abnormal expression can lead to recurrent miscarriage by affecting trophoblast function. In mice, Spry4 knockout (KO) leads to craniofacial anomalies and growth defects. A human study links the SPRY4 variant to a male patient with isolated hypogonadotropic hypogonadism (IHH), hypothetically impacting gonadotropin-releasing hormone (GnRH) neurons, and causing reproductive dysfunctions. SPRY4 is thus potentially integral in regulating endocrine homeostasis and reproductive function. To date, no study has reported SPRY4 variants associated with female fertility, and a causal relationship has not been established with functional evidence. Whole-exome sequencing (WES) was performed in 392 infertile women who suffered from primary infertility of unknown reason, and the heterozygous SPRY4 variant were identified in one independent family. The infertile patients presenting were recruited from July 2017 to November 2023. Women diagnosed with primary infertility were recruited from the Reproduction Center of Zhongshan Hospital, Fudan University. Genomic DNA was extracted from peripheral blood for WES analysis. The SPRY4 variant were identified through WES, in silico analysis, and variant screening. All variants were confirmed by Sanger sequencing. The effects of the variants were investigated in human embryonic kidney (HEK) 293T (HEK293T) cells via western blotting, and in mouse oocytes and embryos through complementary RNA (cRNA) injection, RNA sequencing, fluorescence, absorbance, and RT-qPCR assays. Gene function was further examined in Spry4 KO mice via histology, western blotting, ELISA, and RT-qPCR assays. We identified a missense heterozygous pathogenic variant in SPRY4 (GRCh38, GenBank: NM_030964.5, c.157C>T p.(Arg53Trp), rs200531302) that reduces SPRY4 protein levels in HEK293T cells and disrupts the redox system and mitochondrial function in mouse oocyte, and perturbs developmental potential in mouse embryos. These phenotypes could be partially reversed by the exogenous addition of Nrf1 cRNA. Additionally, Spry4-/- mice exhibit ovarian oxidative stress and decreased ovarian function. Due to the limited WES data and population, we identified only one family with a SPRY4 mutation. The deeper mechanism and therapeutic strategy should be further investigated through mutant mice and recovery experiment. Our study has identified a pathogenic variant in SPRY4 associated with early embryonic arrest in humans. These findings enhance our understanding of the role of SPRY4 in early embryonic development and present a new genetic marker for female infertility. This work was supported by the National Natural Science Foundation of China (82071643 and 82171655) and Natural Science Foundation of Shanghai (22ZR1456200). None of the authors have any competing interests. N/A.

Xia L ，Huang J ，Che Q ，Zhang J ，Zhang Z ，Shen Y ，Wang D ，Zhong Y ，Liu S ，Du J ... -  《-》

Biallelic variants in MOS cause large polar body in oocyte and human female infertility.

What is the genetic basis of female infertility involving abnormal oocyte morphology with the production of a large first polar body (PB1)? The homozygous missense variant (c.791C>G) and compound missense variants (c.596A>T and c.875C>T) in MOS proto-oncogene, serine/threonine kinase (MOS) (Online Mendelian Inheritance in Man (OMIM) reference: 190060; NM_005372.1) are responsible for abnormal oocyte morphology with the production of a large PB1 to cause infertility in women. MOS, an oocyte-specific gene, encodes a serine/threonine-protein kinase that directly phosphorylates mitogen-activated protein kinase (MAPK) kinase (MEK) to activate MAPK (also called extracellular-signal-regulated kinase (ERK)) signal cascade in the oocyte. Female mice lacking Mos remained viable, but infertile because of oocyte symmetric division, spontaneous parthenogenetic activation and early embryonic arrest. Recently, two independent studies demonstrated that female infertility with early embryonic arrest and fragmentation can be caused by biallelic mutations in MOS. However, so far, MOS variants have not been associated with the phenotype of large PB1 extrusion in human oocytes to contribute to female infertility. Two independent infertile families characterized by the presence of large PB1 in oocytes were recruited between December 2020 and February 2022. Genomic DNA was extracted from the peripheral blood samples of the subjects for whole-exome sequencing. Pedigree analysis was validated by Sanger sequencing. Then, the pathogenic effects of the MOS variants on MOS protein properties and ERK1/2 activation were determined in HEK293 cells and mouse oocytes. We identified three rare missense variants in MOS, including a homozygous missense variant (c.791C>G) from Patient 1 in Family 1 and two compound missense variants (c.596A>T and c.875C>T) from twin sisters in Family 2. The MOS variants followed a recessive inheritance pattern in infertile patients. All three patients displayed a high percentage of large PB1 extrusion in the oocytes. The three MOS variants could not activate MEK1/2 and ERK1/2 in oocytes and HEK293 cells. In addition, when compared with wild-type MOS, the MOS variants decreased the MOS protein level and attenuated the binding capacity with MEK1. Microinjection of wild-type human MOS complementary RNAs (cRNAs) reversed the symmetric division of oocytes after siMos treatment. In contrast, the three MOS variants demonstrated no rescuing ability. N/A. Owing to the scarcity of human oocyte samples and the associated ethical restrictions, we could not perform the rescue attempt for the study patients. Our findings expand the genetic and phenotypic spectrum of MOS variants in causing female infertility. Our study findings facilitate the early genetic diagnosis of abnormal oocyte morphology characterized as large PB1 that eventually causes infertility in women. This study was supported by the National Natural Science Foundation of China (82071640 and 82001633), Natural Science Foundation of Zhejiang Province (LD22C060001), the Key Projects Jointly Constructed by the Ministry and the Province of Zhejiang Medical and Health Science and Technology Project (WKJ-ZJ-2005), China Postdoctoral Science Foundation (2020M682575 and 2021T140198), the Changsha Municipal Natural Science Foundation (kq2007022) and Hunan Provincial Grant for Innovative Province Construction (2019SK4012). None of the authors declare any competing interests. N/A.

Zhang YL ，Zheng W ，Ren P ，Jin J ，Hu Z ，Liu Q ，Fan HY ，Gong F ，Lu GX ，Lin G ，Zhang S ，Tong X ... -  《-》