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 ... -  《-》

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 ... -  《-》

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 ... -  《-》

Novel mutations and structural deletions in TUBB8: expanding mutational and phenotypic spectrum of patients with arrest in oocyte maturation, fertilization or early embryonic development.

Are there any new type of mutations and novel phenotypes in patients with arrest in oocyte maturation, fertilization or early embryonic development having tubulin beta eight class VIII (TUBB8) mutations? We identified new types of mutations in TUBB8 associated with maturation, fertilization and developmental arrest. We previously found heterozygous mutations and a homozygous frameshift/internal seven amino acid deletion in TUBB8 that are responsible for oocyte maturation arrest. We recruited 10 new primary infertility patients from 9 families from December 2015 to May 2016, most of which exhibited failures in oocyte maturation. Ten primary infertility patients were recruited from the reproduction centers in local hospitals. Genomic DNA samples from the affected individuals, their family members and healthy controls were extracted from peripheral blood. TUBB8 in the DNA samples were sequenced by Sanger sequencing. TUBB8 sequence was then aligned by CodonCode software to identify rare variants. ExAC database was used to search frequency of corresponding mutations. In silico analysis of mutations was used by Polyphen and PROVEAN. Phenotypes of oocytes and embryos were evaluated by light microscopy, polarization microscopy or immunolabeling. Besides several novel heterozygous missense mutations, we also identified other new types of genetic variants, including homozygous mutations and a de novo compound heterozygous mutation. We also found a patient with a homozygous deletion of the whole TUBB8 gene, which is the first reported case of a large structural variation in this gene. In addition, we found different mutations in TUBB8 that could result in variability in oocyte/embryo phenotypes, including oocyte maturation arrest, first polar body (PB1) oocytes that cannot be fertilized, and PB1 oocytes that can be fertilized but arrest at an early embryonic stage. The exact molecular mechanism has not been analyzed and should be further investigated in the future. In addition, immunostaining of more oocytes with mutations and checking spindle status of oocytes with mutations non-invasively by polarization microscopy needs to be done in order to determine exact stage of PB1 oocytes and the functional differences of these mutations. The results not only emphasize the important role of TUBB8 in oocyte maturation, fertilization and early embryonic development but they also provide a basis for determining the genetic variations in TUBB8 as a potential additional criterion for evaluating the quality of patients' functional PB1 oocytes. National Key R&D Program of China (2016YFC1000600); Basic Research Program of China (2015CB943300); National Natural Science Foundation of China (81270747 and 81571501). No competing interests declared.

Chen B ，Li B ，Li D ，Yan Z ，Mao X ，Xu Y ，Mu J ，Li Q ，Jin L ，He L ，Kuang Y ，Sang Q ，Wang L ... -  《-》

Biallelic variants in KCTD19 associated with male factor infertility and oligoasthenoteratozoospermia.

Can whole-exome sequencing (WES) reveal new genetic factors responsible for male infertility characterized by oligozoospermia? We identified biallelic missense variants in the Potassium Channel Tetramerization Domain Containing 19 gene (KCTD19) and confirmed it to be a novel pathogenic gene for male infertility. KCTD19 is a key transcriptional regulator that plays an indispensable role in male fertility by regulating meiotic progression. Kctd19 gene-disrupted male mice exhibit infertility due to meiotic arrest. We recruited a cohort of 536 individuals with idiopathic oligozoospermia from 2014 to 2022 and focused on five infertile males from three unrelated families. Semen analysis data and ICSI outcomes were collected. WES and homozygosity mapping were performed to identify potential pathogenic variants. The pathogenicity of the identified variants was investigated in silico and in vitro. Male patients diagnosed with primary infertility were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. Genomic DNA extracted from affected individuals was used for WES and Sanger sequencing. Sperm phenotype, sperm nuclear maturity, chromosome aneuploidy, and sperm ultrastructure were assessed using hematoxylin and eosin staining and toluidine blue staining, FISH and transmission electron microscopy. The functional effects of the identified variants in HEK293T cells were investigated via western blotting and immunofluorescence. We identified three homozygous missense variants (NM_001100915, c.G628A:p.E210K, c.C893T:p.P298L, and c.G2309A:p.G770D) in KCTD19 in five infertile males from three unrelated families. Abnormal morphology of the sperm heads with immature nuclei and/or nuclear aneuploidy were frequently observed in individuals with biallelic KCTD19 variants, and ICSI was unable to rescue these deficiencies. These variants reduced the abundance of KCTD19 due to increased ubiquitination and impaired its nuclear colocalization with its functional partner, zinc finger protein 541 (ZFP541), in HEK293T cells. The exact pathogenic mechanism remains unclear, and warrants further studies using knock-in mice that mimic the missense mutations found in individuals with biallelic KCTD19 variants. Our study is the first to report a likely causal relationship between KCTD19 deficiency and male infertility, confirming the critical role of KCTD19 in human reproduction. Additionally, this study provided evidence for the poor ICSI clinical outcomes in individuals with biallelic KCTD19 variants, which may guide clinical treatment strategies. This work was supported by the National Key Research and Developmental Program of China (2022YFC2702604 to Y.-Q.T.), the National Natural Science Foundation of China (81971447 and 82171608 to Y.-Q.T., 82101961 to C.T.), a key grant from the Prevention and Treatment of Birth Defects from Hunan Province (2019SK1012 to Y.-Q.T.), a Hunan Provincial Grant for Innovative Province Construction (2019SK4012), and the China Postdoctoral Science Foundation (2022M721124 to W.W.). The authors declare no conflicts of interest. N/A.

Wang W ，Su L ，Meng L ，He J ，Tan C ，Yi D ，Cheng D ，Zhang H ，Lu G ，Du J ，Lin G ，Zhang Q ，Tu C ，Tan YQ ... -  《-》