A Study On Clinical Characteristics And Pathogenetic-related Genes Of Infertile Patients With Narrow Perivitelline Space

Background:With the development of in vitro fertilization/intracytoplasmic sperm injection（IVF/ICSI）and sequencing technology,some studies found that a part of abnormal development of human oocytes or embryos belonged to Mendelian genetic diseases.However,although 22 genes have been reported as causative genes,the etiological factors remain unclear in the most of infertile patients with abnormal development of oocytes or embryos.Oocytes with narrow perivitelline space（NPVS）belongs to a kind of oocyte matruation arrest,which is characterized as oocytes with NPVS and intended zona pellucida.Those patients with NPVS always undergo multiple IVF/ICSI failures and endure huge economic burden and mental stress because they can hardly obtain available embryos.However,for pathogeny of NPVS oocytes is elusive,there is no effective clinical therapy at present.Thus,analyzing clinical characteristics of infertile patients with NPVS,and applying whole exome sequencing（WES）to investigate possible disease genes play important roles in early diagnosing,treating,and providing genetic counseling for those infertile patients with NPVS.Objectives:1.To investigate the clinical characteristics of infertile patients with NPVS,by retrospectively analyzing laboratory and clinical materials of them.2.To investigate clinical therapy for infertile patients with NPVS.3.To provide a new direction for future diagnosis and treatment by finding possible disease genes for oocytes with NPVS.Methods:1.Incidence was confirmed by retrospectively analyzing clinical data of infertile patients with NPVS in Department of Reproductive Medicine,Xiangya Hospital,Central South University.321 cycles with normal perivitelline space which accepted transvaginal ovum pick-up with NPVS group as the same day was as control group.At first,laboratory and clinical data of 37 cycles with NPVS was compared with control group to confirm general clinical characteristics of NPVS infertility.Then,according to different fertilization methods,the aforementioned NPVS group and control group were analyzed separately,to confirm the characteristics of NPVS infertility at different fertilization methods.Independent-samples T test,one-way ANOVA,Chi-square or Fisher’s exact test was used to conduct statistical analysis.2.The efficacy of ICSI combined with laser-assisted hatching technology was confirmed by retrospectively analyzing clinical materials of two infertile females with NPVS in Steptermber,2020 and November,2020.3.Peripheral blood from 11 infertile patients with NPVS was collected and WES bioinformatics was applied to find possible disease genes.Sanger sequencing was applied in two families who carried a possible disease gene to confirm the genetic pattern.1000 Genome Project database（1000G）and Exome Aggregation Consortium（Ex AC）were used to confirm the mutation frequency.Meta SVM,SIFT,Polyphen-2,Mutation Taster and PROVEAN software were used to predict the deleteriousness.Exon-Intron Graphic Maker,Pfam,Clustal Omega and SWISS MODEL tools were used to analyze the gene structure,two-dimensional,three-dimensional structure of proteins and conservation of the mutation amino acid.In addition,Real-time quantitative reverse transcription polymerase chain reaction（RT-q PCR）,oocyte sequencing data from the public database and immunofluorescence（IF）were applied to confirm the gene expression.4.Wild-type and mutant CPEB2 plasmids were constructed and transiently transfected into He La cells.Then IF was used to confirm the location change in cells after the possible disease gene mutated.RT-q PCR and Western Blot were used to confirm the expression change in cells.Cycloheximide（CHX）experiment was used to confirm the change in protein stability.Results:1.The incidence of which all oocytes obtained in one cycle presented as narrow perivitelline space in IVF/ICSI patients in our center from December,1^st,2013 to November,30^th,2020 was 0.12%.Compared with controls,NPVS group had significantly increased infertile years（9.00±6.33 vs 4.60±3.57,P=0.000）,primary infertility rate（100%vs48.3%,P=0.000）and no available embryo cycle rate（35.1%vs 5.9%,P=0.022）,as well as reduced average oocyte retrieval number（6.68±5.48vs 9.30±6.03,P=0.012）,MII rate（0.46±0.35 vs 0.80±0.20,P=0.000）,fertilization rate（0.55±0.37 vs 0.72±0.23,P=0.014）and 2PN rate（0.49±0.34 vs 0.67±0.24,P=0.007）.2.According to different fertilization methods,NPVS and control group were analyzed seperately.In IVF cycles,NPVS group included 21cycles,and control group had 259 cycles.Compared to control group,NPVS group had reduced average oocyte retrieval number（5.86±5.21 vs9.71±6.16,P=0.006）,MII rate（36.6%vs 78.2%,P=0.000）,fertilization rate（15.4%vs 66.5%,P=0.000）,2PN rate（9.8%vs 61.0%,P=0.000）and2PN cleavage rate（75.0%vs 97.7%,P=0.000）.In the aforementioned IVF cycles,NPVS group had 11 cycles and control group had 22 cycles which accepted early rescue ICSI.NPVS group had significantly increased fertilization rate（82.1%vs 62.2%,P=0.019）and 2PN rate（82.1%vs 59.0%,P=0.007）,but significantly reduced MII rate（41.5%vs74.6%,P=0.000）.In ICSI cycles,NPVS group included 16 cycles and control group included 62 cycles.Compared to control group,NPVS group had significantly reduced MII rate（53.2%vs 80.0%,P=0.000）,available embryo rate（40.0%vs 62.6%,P=0.004）,good-quality embryo rate（33.3%vs 50.2%,P=0.037）,but there were no significant difference in fertilization rate（75.8%vs 73.3%,P=0.673）,2PN rate（68.2%vs70.6%,P=0.687）,2PN cleavage rate（100%vs 96.3%,P=0.389）.3.Two infertile patients with NPVS accepted ICSI combined lase-assisted hatching technology.No.01 patient obtained one 5BC blastocyst and did not establish pregnancy after transferring.No.02 patient obtained two 8c/II embryos and two 5BC blastocysts.The patient gave birth to a baby after two 8c/II embryos were transferred.4.CPEB2 was a possible causative gene by analyzing WES in 11infertile patients with NPVS and searching literature.Two CPEB2mutations（p.Q678delins QV and p.C158Y）were found in two families.Sanger sequencing confirmed an autosomal dominant pattern.The mutation frequency of p.C158Y was 0.00009301 in Ex AC and SIFT predicted that it was deleterious.Clustal Omega predicted that the two mutation sites had high amino acid sequencing conservation and the functions of protein were affected after mutation.What is more,CPEB2was expressed in human GV and MII oocytes and was located in cytoplasm.5.IF experiment in He La cells showed that p.C158Y would cause CPEB2 mutants shifted into nucleus from cytoplasm,causing that morphology of overexpressed cells shrank.Although p.Q678delins QV did not affect the location,but the overexpressed cells also shrank.RT-q PCR conformed that human CPEB2 wild type and mutants have no different expression change in m RNA level,but WB showed that compared to WT,the two mutant protein expression level decreased significantly.Furthermore,CHX experiment indicated that protein stability of wide-type and mutated CPEB2 was different:p.Q678delins QV>p.C158Y>wild type.Conclusions:1.Infertility of which all oocytes obtained in one IVF/ICSI cycle presented as NPVS was rare,and the incidence in IVF/ICSI patients in our center was 0.12%.It was commonly seen in patients diagnosed as primary infertility and had a long infertility year.The fertilization rate by IVF in patients with NPVS was low,but ICSI could increase the fertilization rate in NPVS oocytes.2.ICSI combined lase-assisted hatching technology might be an effective treatment,increasing the chance of obtaining available embryos and pregnancy for infertile patients with NPVS.3.CPEB2 might be one of disease genes and had an autosomal dominant pattern,which expressed in human oocyte cytoplam.4.In vitro function experiments confirmed that p.C158Y and p.Q678delins QV in CPEB2 could affect its protein location,expression level and stability in cells.