Novel WEE2 Gene Variants Identified In Patients With Fertilization Failure And Female Infertility

Background:Fertilization is defined as the formation of a diploid zygote after interaction and fusion of capacitated,free-swimming spermatozoa and the ovulated oocyte.The process begins with species-specific binding of sperm to eggs,highlights with Ca2+ oscillations induced egg activation to complete the second meiotic division marked by the extrusion of the second polar body,ends with the formation and fusion of female/male pronuclei.Successful fertilization is assessed by the presence of the second polar body and formation of female/male pronuclei,which is therefore a prerequisite for successful embryo development and human reproduction.In IVF clinics,after ART?IVF and/or ICSI?treatment,mature oocytes fuse with sperm to form zygote and begin early embryonic development.However,there are some subjects who not forming pronuclei after fertilization,indicating repeat cycles of fertilization failure.As for these subjects without available zygotes to transfer,even AOA treatment still cannot rescue the phenotype of fertilization failure.And now,the genetic factors attributing to human fertilization failure are still not confirmed.WEE2?WEE1 homolog 2,also known as WEEIB?gene,which appears to be of maternal origin,encodes the WEE2 protein that belongs to WEE family and is conserved among different species.In 2011,Jeong Su Oh et al indicated that down-regulation of Wee2 gene in mouse oocytes by morpholino oligonucleotide?MO?led to failure of pronuclei formation after the treatment of parthenogenetically activated with strontium or in-vitro fertilization,mimicking the human fertilization failure phenotype.In this study,we sequenced the whole exons of WEE2 in fertilization failure patients in order to find variants that may contribute to infertile individuals with such phenotype.Objective:To explore whether WEE2 gene variants contribute to human fertilization failure.Materials and Methods:The study included 90 Han Chinese women who underwent IVF and/or ICSI with recurrent cycles?at least 2 cycles?of pronucleus formation failure.Besides,the enrolled subjects should meet the following criteria:all subjects were<40 years of age with normal chromosome constitution.In each stimulation cycle,at least 4 oocytes were retrieved while majority of them resulted in non-pronucleus zygotes with the presence of 2PB after fertilization.Cases with confirmed male infertility were excluded.And sperm collected from male partners were usually tested to be fertile with research only donated oocytes at the 2nd cycle of assisted reproductive technology?ART?when unfertilized oocytes presented at the 1st cycle.A total of 200 Han Chinese women underwent IVF/ICSI treatment,who had normal pronuclei formation and had already given birth to at least one healthy child,were recruited as the controls.All the controls in this study should satisfied with the following foxur rules:? have already given birth to at least one child and not underwent abortion before;?came to our center seeking for the help of ART because of fallopian tube obstruction;?)underwent IVF/ICSI treatment and not experience abnormal fertilization;?had given birth to another child with the help of ART.Genomic DNA was extracted from peripheral blood samples,the whole exons of WEE2 were amplified by PCR and performed Sanger sequencing.After collecting the final sequencing results,we analyzes whether there are abnormal base-pairs?mutations,insertions,deletions,repeats nucleotide?in individuals with fertilization failure.In order to identify inheritance pattern,we collect blood sample from subjects with WEE2 gene variants.Results:In the cohort of 90 cases with fertilization failure,homozygous/compound-heterozygous variants in WEE2 gene were detected in seven individuals in a recessive inheritance pattern.Case 1?from a consanguineous family?with homozygous frameshift variant:c.293₂94insCTGAGACACCAGCCCAACC?p.Pro98Pro fs×2?;case 2 with homozygous missense variant:c.1576 T>G?p.Tyr526Asp?;case 6 with homozygous frameshift variant:c.1008₁009insTA?p.His337Tyrfs×24?,the other three cases with compound-heterozygous variants,case3:c.991C>A?p.His331Asn?and c.1304₁307delCCAA?p.Thr435Metfs×31?;case 4:c.341₃42 del AA?p.Lys114Asn fs×20?and c.864G>C?p.Gln288His?;case 5:c.1A>G?p.0??and c.1261G>A?p.Gly421Arg?;case 7:c.585G>C?p.Lys195Asn?and c.625G>T?p.Glu209X?.Except c.1576 T>G?from case 2?and c.864G>C?from case 4?,which have been previously reported as rare single nucleotide polymorphisms?SNPs?,other variants were novel and were predicted to be deleterious by disease database.According to ACMG standard and pedigree analysis,the results indicate that homozygous/compound-heterozygous variants in WEE2 gene may contribute to fertilization failure,inherited in an autosomal recessive mode.Conclusion:Homozygous/compound-heterozygous variants in WEE2 gene may be related to recurrent fertilization failure and female infertility.And the specific molecular mechanism need to be testified by the future functional research.