Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation
2021; Springer Nature; Volume: 13; Issue: 12 Linguagem: Inglês
10.15252/emmm.202114887
ISSN1757-4684
AutoresYinli Zhang, Wei Zheng, Peipei Ren, Huiling Hu, Xiaomei Tong, Shuoping Zhang, Xiang Li, Haichao Wang, Jun‐Chao Jiang, Jiamin Jin, Weijie Yang, Lan-Rui Cao, Yuanlin He, Yerong Ma, Yingyi Zhang, Yifan Gu, Liang Hu, Keli Luo, Fei Gong, Guangxiu Lu, Ge Lin, Heng‐Yu Fan, Songying Zhang,
Tópico(s)Gynecological conditions and treatments
ResumoArticle15 November 2021Open Access Source DataTransparent process Biallelic mutations in MOS cause female infertility characterized by human early embryonic arrest and fragmentation Yin-Li Zhang Yin-Li Zhang orcid.org/0000-0001-8353-9615 Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China These authors contributed equally to this work Search for more papers by this author Wei Zheng Wei Zheng orcid.org/0000-0002-1397-4176 Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China These authors contributed equally to this work Search for more papers by this author Peipei Ren Peipei Ren Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Huiling Hu Huiling Hu Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Xiaomei Tong Xiaomei Tong Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Shuo-Ping Zhang Shuo-Ping Zhang Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Xiang Li Xiang Li Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Haichao Wang Haichao Wang Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Jun-Chao Jiang Jun-Chao Jiang Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Jiamin Jin Jiamin Jin Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Weijie Yang Weijie Yang Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Lanrui Cao Lanrui Cao Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Yuanlin He Yuanlin He Department of Epidemiology, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, China Search for more papers by this author Yerong Ma Yerong Ma Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Yingyi Zhang Yingyi Zhang Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Yifan Gu Yifan Gu Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Liang Hu Liang Hu Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Keli Luo Keli Luo Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Fei Gong Fei Gong Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Guang-Xiu Lu Guang-Xiu Lu Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Ge Lin Corresponding Author Ge Lin [email protected] orcid.org/0000-0002-3877-2546 Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Heng-Yu Fan Corresponding Author Heng-Yu Fan [email protected] orcid.org/0000-0003-4544-4724 Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Songying Zhang Corresponding Author Songying Zhang [email protected] orcid.org/0000-0001-8044-6237 Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Yin-Li Zhang Yin-Li Zhang orcid.org/0000-0001-8353-9615 Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China These authors contributed equally to this work Search for more papers by this author Wei Zheng Wei Zheng orcid.org/0000-0002-1397-4176 Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China These authors contributed equally to this work Search for more papers by this author Peipei Ren Peipei Ren Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Huiling Hu Huiling Hu Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Xiaomei Tong Xiaomei Tong Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Shuo-Ping Zhang Shuo-Ping Zhang Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Xiang Li Xiang Li Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Haichao Wang Haichao Wang Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Jun-Chao Jiang Jun-Chao Jiang Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Jiamin Jin Jiamin Jin Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Weijie Yang Weijie Yang Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Lanrui Cao Lanrui Cao Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Yuanlin He Yuanlin He Department of Epidemiology, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, China Search for more papers by this author Yerong Ma Yerong Ma Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Yingyi Zhang Yingyi Zhang Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Yifan Gu Yifan Gu Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Liang Hu Liang Hu Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Keli Luo Keli Luo Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Fei Gong Fei Gong Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Guang-Xiu Lu Guang-Xiu Lu Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Ge Lin Corresponding Author Ge Lin [email protected] orcid.org/0000-0002-3877-2546 Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China Search for more papers by this author Heng-Yu Fan Corresponding Author Heng-Yu Fan [email protected] orcid.org/0000-0003-4544-4724 Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Life Sciences Institute, Zhejiang University, Hangzhou, China Search for more papers by this author Songying Zhang Corresponding Author Songying Zhang [email protected] orcid.org/0000-0001-8044-6237 Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China Search for more papers by this author Author Information Yin-Li Zhang1,2, Wei Zheng3,4, Peipei Ren1,2, Huiling Hu3,4, Xiaomei Tong1,2, Shuo-Ping Zhang3,4, Xiang Li1,2, Haichao Wang1,2, Jun-Chao Jiang5, Jiamin Jin1,2, Weijie Yang1,2, Lanrui Cao5, Yuanlin He6, Yerong Ma1,2, Yingyi Zhang1,2, Yifan Gu3,4, Liang Hu3,4, Keli Luo3,4, Fei Gong3,4, Guang-Xiu Lu3,4, Ge Lin *,3,4, Heng-Yu Fan *,2,5 and Songying Zhang *,1,2 1Assisted Reproduction Unit, Department of Obstetrics and Gynecology, Sir Run Run Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China 2Key Laboratory of Reproductive Dysfunction Management of Zhejiang Province, Hangzhou, China 3Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of CITIC-Xiangya, Changsha, China 4Laboratory of Reproductive and Stem Cell Engineering, NHC Key Laboratory of Human Stem Cell and Reproductive Engineering, Central South University, Changsha, China 5Life Sciences Institute, Zhejiang University, Hangzhou, China 6Department of Epidemiology, Center for Global Health, School of Public Health, Nanjing Medical University, Nanjing, China *Corresponding author. Tel: +86 13187057355; E-mail: [email protected] *Corresponding author. Tel: +86 13600540360; E-mail: [email protected] *Corresponding author. Tel: +86 13805727588; E-mail: [email protected] EMBO Mol Med (2021)13:e14887https://doi.org/10.15252/emmm.202114887 See also: L Wang & Q Sang (December 2021) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Early embryonic arrest and fragmentation (EEAF) is a common phenomenon leading to female infertility, but the genetic determinants remain largely unknown. The Moloney sarcoma oncogene (MOS) encodes a serine/threonine kinase that activates the ERK signaling cascade during oocyte maturation in vertebrates. Here, we identified four rare variants of MOS in three infertile female individuals with EEAF that followed a recessive inheritance pattern. These MOS variants encoded proteins that resulted in decreased phosphorylated ERK1/2 level in cells and oocytes, and displayed attenuated rescuing effects on cortical F-actin assembly. Using oocyte-specific Erk1/2 knockout mice, we verified that MOS-ERK signal pathway inactivation in oocytes caused EEAF as human. The RNA sequencing data revealed that maternal mRNA clearance was disrupted in human mature oocytes either with MOS homozygous variant or with U0126 treatment, especially genes relative to mitochondrial function. Mitochondrial dysfunction was observed in oocytes with ERK1/2 deficiency or inactivation. In conclusion, this study not only uncovers biallelic MOS variants causes EEAF but also demonstrates that MOS-ERK signaling pathway drives human oocyte cytoplasmic maturation to prevent EEAF. SYNOPSIS Biallelic variants in MOS gene cause recurrent early embryonic arrest and fragmentation (EEAF) and female infertility. MOS variants impair activation of MOS-ERK signal cascade, prevent substantial maternal mRNAs decay and hamper embryonic development both in human and mice. Four rare variants in MOS gene were identified in three Chinese infertile females displaying EEAF followed recessive inheritance pattern. Mutant MOS proteins failed to activate ERK1/2 cascade, and Erk1/2-deficient mice also exhibited EEAF, confirming the human diagnostic. MOS-ERK1/2 signal cascade is required for maternal mRNA clearance during human oocyte maturation, especially of transcripts relative to mitochondrial function. Inactivation of human or mouse MOS-ERK1/2 signal cascade caused mitochondrial dysfunction in mature oocytes. The paper explained Problem Early embryonic arrest and fragmentation (EEAF) is a compound phenotype of cleavage arrest and embryo fragmentation, leading to recurrent failure of assisted reproductive technology treatment and female infertility. In recent years, several pathogenic variants in maternal genes could account for 10–15% of the etiology of embryonic arrest. However, the underlying genetic determinants and mechanism of EEAF are largely unknown. Results We identified three patients bearing biallelic mutations in Moloney sarcoma oncogene (MOS) gene displaying EEAF. They carry homozygous missense variant c.285C>A (p. Asn95Lys), homozygous nonsense variant c.960C>A (p. Cys320Ter), and compound heterozygous missense variants c.416T>C (p. Met139Thr) and c.737G>A (p. Arg246His), respectively. Compared with wild-type MOS, these MOS variants impaired ERK1/2 activation, resulting in oocyte meiotic resumption and cytoskeleton assembly defects. RNA-sequencing results revealed that substantial maternal mRNAs' clearance during oocyte maturation was retarded either in mature oocytes carrying MOSAsn95Lys variant or in U0126-treated oocytes, in which the most affected transcripts were enriched in mitochondrial biological process. Furthermore, inactivation of MOS-ERK signal cascade caused mitochondrial dysfunction of mature oocytes. These results indicate MOS-ERK1/2 signal pathway is vital for embryo development through driving oocyte cytoplasmic maturation in human and mouse. Clinical impact This study firstly provides direct evidence that biallelic MOS mutation is associated with EEAF phenotype and female infertility, which may serve a target gene for early diagnosis and therapeutic strategy development. Introduction Early embryo fragmentation is defined as anucleate cell fragments derived from the blastomere, and it is a common feature of in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles. Embryos with more than 25% fragmentation are closely associated with deleterious outcomes, including decreased blastocyst formation and implantation rate, and increased malformation rate after pregnancy (Ebner et al, 2001). Several hypotheses for pathogenesis of embryo fragmentation concerns on apoptosis, telomere length, cytoskeleton abnormality, increasing maternal age, and DNA fragment in sperm, with no definitive conclusion having been drawn (Fujimoto et al, 2011). Recurrent early embryonic arrest with fragmentation (EEAF) is a severe type of embryo developmental failure that results in no embryo transfer, which performed as a compound phenotype of cleavage arrest and embryo fragmentation. Despite increasing studies support that EEAF may be of maternal origin defects, however, the causative gene and the underlying mechanism remain largely unknown. In recent years, variants in single maternal-effect gene, including TUBB8 (MIM: 616768), PADI6 (MIM: 610363), TLE6 (MIM: 612399), KHDC3L (MIM: 611687), NLRP2 (MIM: 609364), and NLRP5 (MIM: 609658) (Alazami et al, 2015; Feng et al, 2016; Xu et al, 2016; Mu et al, 2019; Zhang et al, 2019b; Zheng et al, 2020a), have been found to be responsible for human recurrent embryonic arrest in IVF/ICSI attempts. Additionally, we previously indicated variants in human BTG4 (MIM: 605673), causing large-scale maternal-effect gene decay defect, resulted in zygotic cleavage failure (Zheng et al, 2020b). However, none of these genes can explain the EEAF mechanistically. The Moloney sarcoma oncogene (Mos) gene encodes a serine/threonine protein kinase, which was first identified as a cytostatic factor to maintain oocyte metaphase II (MII) arrest via activation of the ERK pathway by phosphorylating MEK (Sagata et al, 1989; Verlhac et al, 2000). MOS-ERK signal pathway-mediated MII arrest relied on EMI2-mediated APCCDC20 inhibition to prevent cyclin B1 from degradation (Shoji et al, 2006; Suzuki et al, 2010; Sako et al, 2014). The MOS mRNA is highly expressed in oocyte, and MOS protein is actively translated during oocyte maturation and rapidly degraded after fertilization, in several vertebrates, including human (Sagata et al, 1988; Watanabe et al, 1991; Sha et al, 2020b). Despite that MOS protein is nearly untranslated in oocyte at GV stage, precocious activation MOS-ERK signal cascade by microinjection of Mos mRNAs in mouse immature oocytes could promote cyclin B1 translation and maturation promoting factor (MPF) activation, leading to oocyte meiotic maturation resumption (Choi et al, 1996b; Cao et al, 2020). Deficiency of Mos or Erk1/2 in mice results in oocyte MII arrest failure, spindle abnormality, large polar body, and early embryo developmental arrest (Colledge et al, 1994; Hashimoto et al, 1994; Araki et al, 1996; Choi et al, 1996a; Zhang et al, 2015). Until now, MOS (OMIM ID: 190060; NM_005372.1) has not been found to be associated with any human disease definitely. In this study, we identified four pathogenic variants in MOS that are responsible for human recurrent EEAF. Both affected individuals had biallelic variants that followed a Mendelian recessive inheritance pattern. We verified the pathogenic effects of MOS variants on protein expression and ERK1/2 activation. Inactivation of MOS-ERK signal pathway in oocyte caused abnormal F-actin assembly disorder, maternal-effect genes decay defect, and mitochondrial dysfunction, resulting in EEAF. Our findings established the causal relationship between MOS and the phenotype of EEAF in human, which may provide targets for therapeutic strategies in future. Results Clinical characteristics of the affected individuals We recruited three independent female probands with infertility of unknown causes. All affected individuals belong to Chinese Han population and are from three families without potential fertility problems in China. They had normal menstrual cycles and sex hormone levels (Appendix Table S1). Proband II-1 from a consanguineous family (family 1) had undergone one IVF and two ICSI failed attempts. All 15 oocytes retrieved in the three attempts were mature. However, half of the retrieved oocytes displayed multiple polar bodies or enriched granules in the cytoplasm, with a low normal fertilization (2PN) rate (20%, 25%, and 50%, respectively), and nine zygotes were cleaved at the two- to seven-cell stage on day 3 and arrested during subsequent blastocyst culture. Three of nine arrested embryos were accompanied by severe fragmentation at the cleavage stage (Table 1 and Fig 1D). Proband II-1 in family 2 had undergone four separate ICSI attempts, and three previous failed attempts in other hospitals were diagnosed as embryonic arrest (arrested at the two- to six-cell stage, with embryo fragmentation). In her fourth ICSI attempt, we used time-lapse to observe embryonic development. Overall, 11 of the 14 oocytes retrieved were mature, and the couple chose to freeze four MII oocytes in this attempt. Then, three 2PN, two 1PN, and two 3PN zygotes were formed; all zygotes consistently arrested at the two- to five-cell stage, and six of seven arrested embryos with severe fragmentation were produced during the first cleavage (Table 1 and Fig 1D). Proband II-1 from a consanguineous family (family 3) had undergone one IVF with ICSI rescue in other hospital and one ICSI attempt in our hospital. In her two attempts, partial immature oocytes were observed, including 2 metaphase I (MI) oocytes of 12 retrieved oocytes and 2 MI oocytes of 5 retrieved oocytes. In her first attempt, four zygotes were derived yet only one developed to five-cell stage accompanied by severe fragmentation and other three were arrested at one-cell stage. In her second attempt, all three 2PN zygotes arrested at two- and three-cell stage (Table 1 and Fig 1D). Table 1. Oocyte and embryo characteristics of IVF and ICSI attempts for the affected individuals. Family NO. Age (years) Duration of infertility (years) IVF/ICSI Attempt Retrieved oocytes Immature oocytes MII oocytes Oocyte with abnormal morphology Fertilized outcomes (2PN + 1PN + MPN + 0PN) Cleaved/Fragmented embryo Embryo outcomes 1 40 12 IVF 5 0 5 multi-polar body 1 + 0+1 + 3 3/0 2*3-cell, 1*2-cell, arrested ICSI 4 0 4 multi-polar body, dark cytoplasm 1 + 0+0 + 3 3/2 1*6-cell, 1*4-cell, 1*2-cell, arrested ICSI 6 0 6 multi-polar body, dark cytoplasm 3 + 0+0 + 3 3/1 1*7-cell, 1*4-cell, 1*5-cell, arrested 2 31 4 ICSI 9 0 9 N/A NA 3/N/A All arrested at 2- to 6-cella a The attempt at other hospitals. ICSI 13 0 13 N/A NA 2/2 All arrested at 2- to 6-cella a The attempt at other hospitals. ICSI+AOA 16 0 16 4 degradations 4 + 5+1 + 2 6/N/A All arrested at 2- to 6-cella a The attempt at other hospitals. ICSI 14 3 11 (4 frozen) N/A 3 + 2+2 + 0 7/6 3*5-cell, 4*2-cell, arrested 3 34 7 IVF+Rescue ICSI 12 2 10 N/A 4 1/1 1*5-cell, arrested a ICSI 5 2 3 N/A 3 + 0+0 + 0 3/0 2*3-cell, 1*2-cell, arrested Arrested, arrested during subsequent blastocyst culture; MII, Metaphase II; MPN, multi-pronucleus; N/A, not available; PN, pronucleus. a The attempt at other hospitals. Figure 1. Identification of MOS variants in female patients with early embryonic arrest and fragmentation Three pedigrees and four MOS variants were identified, including homozygous missense variant Asn95Lys in patient 1 (family 1), compound heterozygous missense variants Met139Thr and Arg246His in patient 2 (family 2), and homozygous nonsense variant Cys320Ter in patient 3 (family 3). The four MOS variants were inherited and identified via Sanger sequencing. WT indicates wild-type. Distribution of MOS variants in genome and the corresponding amino acid sequences. Multiple sequence alignment of four segments of MOS and the orthologs, with mutated residue marked in yellow. MOS variants encoding amino acid disrupted the ion pairs formed by wild-type MOS protein. The morphology of oocytes and embryos from female patients with MOS variants. The day of oocyte retrieval was defined as day 0. Polar bodies were indicated by black arrows. Scale bar = 20 µm. Source data are available online for this figure. Source Data for Figure 1 [emmm202114887-sup-0005-SDataFig1.zip] Download figure Download PowerPoint Identification of MOS pathogenic variants Whole-exome sequencing (WES) was used to identify pathogenic variants. After filtering using the criteria described in the materials and methods section, only biallelic variants of MOS (OMIM ID: 190060; NM_005372.1) were found in all affected individuals yet absent in our 100 controls undergoing natural conception, suggesting the po
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