Genetic Studies In Women With Oocyte Maturation Defects

A mature oocyte is a major determinant of successful fertilization and embryonic developmental capacity.Oocyte maturation defects affect fertilization and early embryonic development.Our study is to investigate the causative genes of oocyte maturation defects by whole-exome sequencing and Sanger sequencing.In the first chapter,a homozygous mutation in TBPL2 was identified as a contributory genetic factor in families with oocyte maturation defects;in the second chapter,we further investigated the role of ZP genes in patients with oocyte degeneration based on our previous studies;in the third chapter,we summarized the characteristics of variants in TUBB8 gene in patients with oocyte maturation defects in Shandong province.Chapter IA homozygous variant in TBPL2 was identified in women with oocyte maturation defects and infertilityObjective:A mature oocyte is a key factor for a successful pregnancy.Oocyte maturation defects could lead to fertilization failure and embryonic development arrest.With the application of assisted reproductive technology in the treatment of infertility,we identified some rare patients with repeated failed cycles of defects in oocyte maturation and/or early embryonic development.The causative genes and mechanisms of oocyte maturation defects are still unclear.Recent studies have suggested that mutations in genes related to oocyte maturation and early embryonic development are the causes of the disease.However,the known pathogenic mutations could account for only about 30%of individuals with oocyte maturation defects,and the cause of a large proportion of individuals with oocyte maturation defects is still unknown.Thus,the aim of this study is to identify novel causative genes in infertile families with oocyte maturation defects.Methods:Whole-exome sequencing was performed on the members of the two unrelated pedigrees characterized by oocyte maturation defects.Variants were filtered according to type,frequency,pathogenicity prediction,gene expression and function.and Sanger sequencing was performed for data validation.Conservation analysis was performed by Clustal Omega and pathogenicity of variants was predicted by bioinformatics software.Variants were classified according to the standards and guidelines recommended by the American College of Medical Genetics and Genomics(ACMG).To verify whether the disease allele was independent in the two families,short tandem repeat(STR)analysis was performed in the probands of these two families.The effect of the splicing mutation on mRNA integrity was assessed by minigene assay;single oocyte cDNA sequencing was used to further verify the effect of mutation on splicing.Single-oocyte RNA sequencing was performed to analyze the effect of the mutation on oocyte transcriptome.Results:A homozygous splicing variant c.788+3A>G,p.R233X in TATA-box binding protein like 2(TBPL2)was identified in two unrelated families characterized by oocyte maturation defects.The variant was located only in the C-terminal core domain and the position of this variant is conserved among different species.Bioinformatics predicted that the variant might affect splicing.According to the standards and guidelines recommended by ACMG,the variant was classified as pathogenic.STR analysis indicated that the disease allele origins of these two families were independent.The variant disrupted the integrity of TBPL2 mRNA,caused exon 4 skipping and resulted in a truncated protein of 232 amino acids that lacks most of the core domain.TBPL2 is a vertebrate oocyte-specific general transcription factor and it could bind to promoters of active genes in oocyte and regulate these genes expression.Transcriptome sequencing of affected oocytes showed that the number of downregulated genes was significantly higher than that of upregulated genes,and the downregulated genes were significantly enriched in transcription-related pathways.Vital genes for oocyte maturation,fertilization and early embryonic development including BMP15,GDF9,ZP1,ZP4,TLE6,and KHDC3L were widely and markedly downregulated,suggesting a mutation in the transcriptional factor,TBPL2,led to global gene alterations in oocytes.Conclusion:Our findings highlight the critical role of TBPL2 in female reproduction.The variant c.788+3A>G impaired TBPL2 mRNA integrity,caused a premature termination codon,and decreased the expression of many oocyte-specific genes that are important for oocyte maturation,fertilization and early embryonic development,which may ultimately induce defects in oocyte maturation and early embryonic development.Chapter II The critical role of ZP genes in female infertility characterized by empty follicle syndromeObjective:Our previous study identified that a ZP3 mutation destroyed the binding of ZP3 and ZP2 protein,hindered the formation of zona pellucida,resulting in oocyte maturation defects characterized by oocyte degeneration.Patients with oocyte maturation defects caused by this mutation showed that only degenerated or collapsed oocytes were obtained after the induction of ovulation despite apparently normal size and number of follicles monitored by ultrasound and normal estrogen levels,which is known as genuine empty follicle syndrome syndrome(EFS).However,human zona pellucida glycoproteins contain ZPI-4,except for ZP3 mutations,whether other zona pellucida(ZP)gene mutations caused oocyte degeneration needs further study.Methods:We recruited the peripheral blood of the largest sample size to date-35 individuals diagnosed with genuine EFS.They underwent assisted reproduction treatment for one to six cycles at different hospitals,but only degenerated or collapsed oocytes were retrieved.Genomic DNA was extracted from peripheral blood of patients,and the whole exome sequencing and Sanger sequencing were performed.Variants were filtered according to stringent criteria including type,frequency,pathogenicity prediction,gene expression and function,and Sanger sequencing was performed on the patients and other family members to validate the selected variants.Clustal Omega software was used for sequence conservation analysis and PyMOL software was used to predict the functional effect.Variants were evaluated and classified according to the standards and guidelines recommended by the American College of Medical Genetics and Genomics(ACMG).Wild-type and mutant plasmids were constructed,and transfected into CHO-K1 cells.The effects of variants on protein expression and localization were investigated by Western Blot and immunofluorescenceResults:By screening and validating the sequencing results,we found that 18 individuals with genuine EFS carried ZP mutations,including 20 mutations in ZP1,2 mutations in ZP2 and 1 previously reported mutation in ZP3.ZP variants may account for more than half(51.43%;18 of 35)of the genuine EFS cohort.The ZP1 variants were inherited in an autosomal recessive pattern;the ZP2 and ZP3 variants followed an autosomal dominant inheritance pattern.Conservation analysis showed that all variants were highly conserved among different species.Variants were classified as likely pathogenic or pathogenic according to ACMG standards.By PyMOL software prediction,variants p.C80G and p.W83R in ZP1 and p.R642Q in ZP2 might alter amino acid interaction and affect protein structure.Functional studies in CHO-K1 cells suggested that most ZP1 variants led to increased intracytoplasmic protein and some variants influenced the intracellular transportation of other ZP proteins.Variant p.R642Q of ZP2 caused the secretion of ZP2 protein with an increased molecular weight,suggesting altered protein modification.Variant p.I619N of ZP2 resulted in increased ZP2 protein in cell lysate and decreased ZP2 protein in culture medium.Taken together,these results showed that different ZP variants might block the intracellular transportation and/or secretion of ZP proteins and disrupt the zona pellucida.Conclusion:We identified novel variants of ZP genes in more than half the cohort with genuine EFS and oocyte degeneration.Variants of ZP genes caused protein intracellular sequestration and failure to assemble the ZP filaments,resulting in EFS and female infertility.Our findings not only reveal the critical roles of ZP genes but also pave the way for the efficient molecular diagnosis of females with genuine EFS and oocyte degeneration.Chapter III Mutation analysis of tubulin beta 8 class VIII in infertile females with oocyte or embryonic defectsObjective:Oocyte maturation is a prerequisite for successful fertilization and subsequent embryonic development.It involves nuclear and cytoplasmic maturation.Nuclear maturation is the meiotic process characterized by germinal vesicle breakdown and the extrusion of the first polar body,while cytoplasmic maturation accompanying the nuclear maturation includes a series of molecular events required for meiosis,fertilization,and early embryo development.Abnormal meiosis can lead to oocyte maturation arrest at different stages.Tubulin beta 8 class VIII(TUBB8)is a special ?-tubulin isotype,which is specifically expressed in oocytes and early embryos.TUBB8 is the first reported Medelian pathogenic gene responsible for human oocyte maturation arrest and mutations in TUBB8 could lead to various phenotypes including oocyte maturation arrest and early embryonic arrest.To further investigate the prevalence and phenotypic spectra of TUBB8 variants,we performed Sanger sequencing of TUBB8 on individuals with oocyte maturation defects and/or early embryonic arrest in Shandong Province.Methods:We recruited 115 infertile women with repeated failed cycles with more than half of the oocytes exhibiting maturation defect and/or embryonic defect and 200 healthy controls from Shandong Province for Sanger sequencing of TUBB8 gene by extracting peripheral blood genomic DNA.The pathogenicity of variants was predicted by SIFT and Polyphen-2 software and assessed according to the standards and guidelines recommended by the American College of Medical Genetics and Genomics(ACMG).Conservation analysis and protein structure was performed with Clustal Omega and PyMOL,respectively.The morphologies of the oocytes and embryos were evaluated by light microscopy.The effect of mutations on spindle morphology was detected by immunofluorescence and visualized under a confocal microscope.Results:Overall,we identified 31 variants in the TUBB8 gene from 36 individuals,accounting for 31.3%(36/115)of the cohort with oocyte and/or embryonic defects.All of the variants included heterozygous/homozygous missense variants and a heterozygous frameshift insertion variant.Among which,15 variants were novel;two variants were known while having very low frequencies;and 14 variants were previously reported.All variants were absent in the matched controls and the positions at which the variants were located were shown to be conserved among different primate species.Almost all of the variants were predicted to be deleterious and variants were classified as likely pathogenic or pathogenic by ACMG standards.Novel variants might affect microtubule stability and spindle assembly by PyMOL prediction.The results of immunofluorescence showed that mutations p.G98R,p.A393D and p.R241H resulted in undetectable spindle and disordered DNA.Additionally,these variants had diverse phenotypic effects,including not only oocyte maturation arrest,fertilization failure,and early embryonic arrest,but also multi-pronuclei formation,which is a new phenotype associated with TUBB8 variants.Conclusion:Overall,this study reveals a large number of variants of the TUBB8 gene in infertile females with oocyte or embryonic defects from Shandong Province with a proportion of 31.3%.Our results not only broaden the mutational and phenotypic spectra of TUBB8 variants,but also further confirm the critical role of TUBB8 in oocyte maturation,fertilization,and early embryonic development.For individuals with oocyte or embryonic defects,mutation screening of TUBB8 is recommended,which should improve the efficiency of genetic diagnosis and treatment.