Screening Zona Pellucida Genes In Women With Oocyte Anomalies

BackgroundA mature oocyte is a prerequisite for successful fertilization,the growth and development of embryos,and infertility treatment.During the course of oocyte maturation,ZP(zona pellucida)genes,which code for the ZP glycoproteins,are expressed exclusively in growing and fully grown oocytes.In humans,the ZP consists of ZP1-ZP4.In mice,the ZP matrix is composed of ZP1,ZP2,and ZP3.It is first observed from primary follicles and increases in thickness concomitantly with oocyte diameter increase.During the growth of mouse oocytes,the absolute rate of protein synthesis increases about 40-fold,and the number of copies of ZP messenger RNAs(mRNAs)increases dramatically.The momentous functions of the ZP matrix during oocyte growth and fertilization have been further demonstrated by knockout mice.Abnormal organization in the ZP may result in abnormal oocyte-cumulus cell interactions,thereby influencing oogenesis and oocyte maturation.To our knowledge,sequencing of the ZP1-4 genes has rarely been reported in sporadic patients with repeated cycles of oocyte maturation arrest or morphologic defects,especially in Han Chinese women.Our assumption is that the ZP is connected to oocyte maturation,and we examined variations in the ZP genes that may be implicated in oocyte maturation arrest or oocyte morphologic defects.ObjectiveTo detect ZP gene(ZP1-ZP4)mutations in patients with oocyte anomalies.Materials and MethodsA total of 92 infertile patients with repeated cycles of oocyte maturation arrest(group Ⅰ,n=49)or oocyte morphologic defect(group Ⅱ,n=43)as well as 373 healthy controls.Human oocyte maturation arrest occurred in different phases of the cell-division cycle including germinal vesicle arrest,metaphase Ⅰ arrest,or the mix.The oocyte morphologic parameters mainly comprised the polar body,cytoplasm,PVS,and ZP.All patients were ≤ 40 years of age,and their blood samples were collected from the Center for Reproductive Medicine,Shandong University.Genomic DNA extracted from peripheral blood and coding regions of ZP genes amplified by polymerase chain reaction and sequenced by a DNA analyzer.ResultsIn group Ⅰ with oocyte maturation arrest,no novel variants were found.In groupⅡ with oocyte morphologic defects,four novel variants,two in the ZP1 gene[c.247T>C(p.W83R)and c.1413G>A(p.W471X)]and two in the ZP2 gene[c.1599G>T(p.R533S)and c.1696T>C(p.C566R)]were detected in 4 of 43 patients(approximately 9%)but were absent from the controls.Protein alignments showed that the four variants were highly conserved among different species,and all four variants were predicted to be deleterious by gene software predictions.ConclusionZP gene variants may account for patients with oocyte morphologic abnormalities but not for those with oocyte maturation arrest.Further functional studies should be performed to prove the potentially deleterious effects of these variants.