Clinical Characteristics Analysis And Pathogenic Gene Identification Of Oocyte Maturation Defect Syndrome

Assisted reproductive technology（ART）has been widely used in the treatment of various infertility,but there are still some infertility causes that cannot be clarified and solved,such as oocyte maturation defect（OMD）also known as oocyte maturation block which is a rare form of primary infertility.The clinical features of OMD are primary infertility,repeated generation of immature oocytes,and can not be cultured to maturity in vitro,always fail to fertilize by intracytoplasmic sperm injection.There is no accurate data of the global incidence of the OMD.The cause of OMD is not yet clear.The mechanisms leading to the disease have been identified,such as lack or incomplete luteinizing hormone（LH）peak,disturbance of signal transduction mechanism in surrounding cumulus cells,changes in intrinsic factors of oocytes,and gene mutations affecting meiosis.Recently,the common methods for exploring the pathogenic genes and identification of the disease caused by single gene inheritance are genome-wide association analysis or whole exon sequencing（WES）which refers to the use of sequence capture technology to genome-wide.WES is a genomic analysis method for high-throughput sequencing after DNA capture and enrichment in the exome region.The genetic variants that alter protein sequences can be identified by WES.The high efficiency and economic utility make this technology widely used in the study of genetic pathogenic genes.In this study,we explored the pathogenesis and genetic pathogenic factors of OMD from two aspects: clinical features and pathogenic gene identification.Part I Clinical characteristics analysis of oocyte maturation defect syndromeObjectiveBy retrospective analysis,we compare the clinical indicators of patients who suffered oocyte maturation defect（OMD）with normal patients（excluding ovarian dysfunction and other diseases affecting oocyte quality）,in this way we initially understand the clinical characteristics of OMD and it can help us understanding how OMD exists.Finally,this result provides more reference for consultation and treatment for patients with repeated oocyte maturation failure or low maturation rate.Subjects and MethodsAccording to the OMD diagnostic criteria,we collected and analyzed the clinical information of OMD patients who accepted clinical treatment in the reproductive center of the First Affiliated Hospital of Zhengzhou University from January 1,2010 to December 31,2018.The normal patients were randomly selected as the control group.SPSS statistical software was used to test the difference between groups in the measurement data group.The data conforming to the normality were tested by two independent samples t test,while the data not conforming to the normal distribution were tested by wilcoxon rank sum test.The risk factors leading to the disease were explored by Logistic regression analysis.ResultIn this study,29 OMD patients and 74 normal patients were finally included.Statistical results showed that a total of 78 treatment cycles in the OMD group were summarized,and it was found that oocyte development arrest（normal morphology） accounted for about 47% of the total OMD cycles,oocyte immature which accompanied by abnormal zona pellucida accounted for about 23% of the total cycle.According to the summary of 29 OMD patients’ oocyte maturation types per cycle,the proportion of patients with MI block was relatively high,accounting for 31%,and the proportion of mixed block was 24%.In addition,the difference of clinical data between the two groups was statistically significant in age,amh level and estrogen level.The Logistic regression model analysis showed that the risk of oocyte maturation disorder increased with the decrease of E2（OR=1.056,P= 0.027,95% CI: 1.006-1.108）.ConclusionsMI arrest accounts for a large proportion of OMD patients with assisted reproduction in our center.In addition,the incidence of zona pellucida abnormality/deletion and oocyte deletion is relatively high.The decrease of E2 level is the risk factor of OMD.Part II Pathogenic gene identification of oocyte maturation defect syndromeObjectiveIn this study,the pathogenic gene/mutation of OMD was defined by whole exon sequencing,so as to improve the pathogenic gene spectrum of OMD and provide more evidences for the etiological study of oocyte maturation defect.Subjects and MethodsIn this study,clinical information and blood samples of patients with OMD were collected from the data center and sample center of the reproductive center of the first affiliated hospital of Zhengzhou university.At the same time,more comprehensive medical history inquiry and family investigation were conducted.By collecting patients’ peripheral blood,WES was used to detect all exon sequences of patients,and database comparison and software analysis were used to find candidate disease-causing genes/mutations.The risk value of the mutation was calculated by polyphen-2,Mutation Taster and Provean prediction software,and the hazard and degree of the mutation were predicted.The distribution and genetic pattern of the gene in the family were confirmed by Sanger sequencing.ResultsAccording to the WES results,we detected an unreported missense mutation（NM₁77987.2）on exon 4 of TUBB8 gene in one OMD patient（c.730G>A.p.Gly244Ser）,which was predicted to be harmful and pathogenic.This missense mutation leading to MI stage arrest in this patient.Sanger sequencing confirmed that only the patient’s father carried the mutation in the patient’s family,but no such mutation was detected in other normal family members.ConclusionsThe missense mutation c.730G>A.p.Gly244 Ser in the exon 4 of TUBB8 gene lead to the occurrence of MI arrest of OMD.