Clinical And Genetic Study Of Patients With Fertilization Or Embryo Development Failure

Chapter ?.Clinical study of patients with in vitro fertilization failureSection ?.Analysis of causes for patients with no available embryo to transfer after IVF/ICSI treatmentObjective:The types of patients with no available embryo to transfer after in vitro fertilization(IVF)/intracytoplasmic sperm injection(ICSI)treatment included abnormal fertilization(i.e.fertilization failure and polyspermy),oocytes activation failure,oocytes cleavage arrest,and poor quality of embryos.The pathogenesis for abnormal fertilization and/or poor quality of embryos may include abnormrsaal oocyte quality and quantity and sperm abnormalities.The purpose of this part is to identify the causes and related factors of patients with no available embryo to transfer after IVF/ICSI treatmentMethods:In this study,241 patients with no available embryo to transfer after IVF/ICSI treatment were recruited as study subjects,and 1048 patients with successful pregnancy and live birth after the first IVF and/or ICSI cycle were enrolled as the control group.The basal clinical characteristics,the data and outcomes of ovarian stimulation for subjects were collected and analyzed by statistical methods And binary logistic regression analysis was used to identify the relevant factors that may affect the poor outcome of IVF/ICSI as no available embryo to transfer.Results:We found that 33.6%,20.3%,13.7%,15.8%and 16.6%of the patients with no available embryo to after in vitro fertilization treatment were caused by poor embryo quality,cleavage arrest,complete fertilization failure,partial fertilization failure and non-2PN fertilization,respectively.The basal characteristics,clinical characteristics,ovarian response parameters and laboratory data between two groups were statistically significant differences.Case group was younger(31.21+3.96 vs 32.1±4.28,P<0.05).And the proportions of patients with no ovulation/ovulation dysfunction,endometriosis,recurrent spontaneous abortion(RSA)/assisted reproductive technology(ART)failure history,and unexplained infertility were higher in case group(9.54%vs 2.77%,P<0.05;4.98%vs 3.15%,P<0.05;12.97%vs 5.91.P<0.05;? 7.88%vs 1.81%,P<0.05),the male factor infertility patients were in low proportion(10.37%vs 30.06%,P<0.05).AFC of case group is less than control group[12(9-18)vs 14(11-18),P=0.004].Compared with the control group,the case group had higher proportion of GnRH-Ant protocol(28.22%vs 19.66%,P<0.05),and fewer oocytes with ?>14mm on the HCG day[8(5-12)vs 10(7-12),P<0.05].Males from case group were younger 31.94±4.63 vs 32.95±5.23,P=0.033),sperm parameters were better(P<0.05).In case group,no high-quality embryos were obtained,the number of retrieved oocytes[8(4-13)vs 10(7-13),P<0.05],the number of normal pronuclei zygotes[4(2-7)vs 7(5-10),P<0.05]and the number of cleavage zygotes[2(1-5)vs 6(4-9),P<0.05]were significantly lower than those in control group.After adjusted by confounding factors in binary logistic regression,the analysis shown that RSA/ART failure history(OR=3.007,95%confidence interval[CI]:1.406-3.606,P=0.007),unexplained infertility(OR=4.594.95%CI:1.566-13.478,P=0.005),using GnRH-Ant protocol(OR=1.953,95%CI:1.038-3.675,P=0.083)and higher proportion of non-2PN zygotes(OR=1.300,95%CI:1.114-1.516,P=0.001)had a significant positive effect on no available embryo to transfer.There was a negative correlation between the number of cleavage zygotes(OR=0.716,95%CI:0.599-0.857,P<0.001),the number of high-quality embryos(OR=0.200,95%CI:0.154-0.26,P<0.001)and no available embryo to transfer.Conclusion:This study analyzed the causes and related factors of patients with no available embryo to transfer after IVF/ICSI treatment and found that the proportion of patients with poor embryo quality was the highest.Patients with RSA/ART failure history,unexplained infertility,using GnRH-Ant protocol,higher proportion of non-2PN zygotes,and few cleavage zygotes and high-quality embryos indicated that the risk of no available embryo to transfer increasedSection ?.Analysis of causes for patients with in vitro fertilization failure and the pregnancy outcomes after rescue ICSIObjective:In vitro fertilization failure means that the oocytes retrieved in IVF treatment cannot fuse with sperm to form a zygote.The main reasons can be summarized as follows:sperm failed to bind to zona pellucida or inject into oocyte,zygote arrest caused by oocyte structure abnormalities or abnormal activation of oocytes.The mechanism of ICSI is to inject a sperm into oocyte bypass the biological barrier,so ICSI is preferred as a remedy for fertilization failure.The aim of this study was to identify the causes of patients with in vitro fertilization failure and explore the effects of rescue ICSI for the improvement of pregnancy outcomesMethods:We recruited 844 females with fertilization failure,and 13442 matched controls.All subjects underwent their first IVF treatment.The clinical characteristics and pregnancy outcomes between cases and controls were compared.Statistical analysis was used for data comparison.Results:The proportions of primary infertility and unexplained infertility in fertilization failure group were higher than control group(69.11%vs 49.00%,P<0.05;6.39%vs 2.63%,P<0.05;37.04%vs 27.37%,P<0.05.The percentage of forward motile sperm in the case group was lower(37.42±15.80 vs 42.92±14.44.P<0.05).The female ovarian response parameters and fertilization outcomes were significantly difference between two groups(P<0.05).Compared with the control group,the case group had higher proportion of GnRH-a short protocol(17.28%vs 21.15%,P<0.05),thicker endometrium(1.13 ± 0.16 vs 1.10±0.20,P<0.05)and fewer retrieved oocytes 11(8-15)vs 12(8-16),P<0.05],2PN zygotes[0(0-1)vs 7(4-11),P<0.05]and high-quality embryo[3(2-4)vs 4(2-6),P<0.05].After rescue ICSI,there were no significant differences in clinical pregnancy rate,first trimester abortion rate and live birth rate between two groups(53.15%vs 53.43%,P=0.931;14.07%vs 14.95%,P=0.778;48.26%vs 45.38%,P=0.452).But day 3 high-quality embryo rate and implantation rate were lower in case group(42.11%vs 55.29%,P<0.05;33.73%vs 38.73%,P<0.05).Conclusion:This study suggested that primary infertility,unexplained infertility,low proportion of forward motile sperm,and few retrieved oocytes may lead to the failure of in vitro fertilization.Rescue ICSI can effectively improve the pregnancy outcomes of patients with IVF fertilization failure.Chapter ?.Mutational analysis of IZUMO1R in women with fertilization failure and polyspermy after in vitro fertilizationObjective:The etiology of fertilization failure and polyspermy during ART remains elusive.The aim of this study was to determine whether mutations in the IZUMO1 receptor(IZUMO1R)gene,which is essential for mammalian fertilization,contribute to the pathogenesis of fertilization failure or polyspermy in humans.Methods:We recruited 215 female subjects with fertilization failure/poor fertilization,330 females with polyspermy,and 300 matched controls.All subjects underwent IVF treatment.Peripheral blood DNA of cases was extracted and screened for mutations in IZUMO1R gene.Results:Four rare single nucleotide polymorphisms(SNPs)of the IZUMO1R were identified among specimens from patients with fertilization failure and polyspermy but were absent in the 300 control subjects.These included a missense SNP(rs76779571 in exon 4),which was found in two fertilization failure patients,and a nonsynonymous SNP(rs61742524 in exon 1)and two synonymous SNPs(rs76781645 in exon 1 and rs377369966 in intron 2),which were found among three polyspermy cases.Conclusion:The variations in IZUMO1R might play a role in the pathogenesis of fertilization failure and polyspermy,and the putative functions and effects of these rare variants require further studiesChapter ?.The Effects and Mechanisms of WEE2 gene in the pathogenesis of fertilization failureSection I.Mutational analysis of WEE2 in women with recurrent in vitro fertilization failureObjective:The aim of this study was to determine whether mutations in WEE2 gene,which is essential for oocyte meiosis,contribute to the pathogenesis of human recurrent in vitro fertilization failureMethods:Twenty-five female subjects with recurrent in vitro fertilization failure were enrolled.Peripheral blood DNA of cases was extracted and screened for mutations in WEE2 gene.We analyzed sequencing results to identify whether there are site mutations,deletions,insertions,repeats nucleotide or frameshift mutations in subjects with recurrent in vitro fertilization failureResults:In the cohort of 25 cases with recurrent in vitro fertilization failure,compound-heterozygous/homozygous variants in WEE2 gene were detected in two subjects.Case F3 with compound-heterozygous missense variant:[c.416 C>T(p.Thr139Ile)and c.585G>C(p.Lys195Asn)];Case F17 homozygous missense variant:c.821G>A(p.Arg274His).The sites of variants were conserved among different species.These variants were predicted to be deleterious by disease databaseConclusion:The compound-heterozygous/homozygous mutations in WEE2 might play a role in the pathogenesis of fertilization failure.And this study expands on the mutation spectrum of WEE2,and provides theoretical basis for genetic counseling of female infertility.Section ?.Model study of Wee2 knockout mouseObjective:WEE2,also known as Wee1B,is an important oocyte-specific kinase and belongs to the WEE kinase protein family,and acts as a key regulator of oocyte meiosis.More and more evidences show that missense mutations in WEE2 may lead to in vitro fertilization failure.The sequence of Wee2 is highly conserved between human and mouse.This study aimed to construct Wee2 knockout mouse to investigate the phenotype of fertility and identify how Wee2 gene defects contribute to the pathogenesis of fertilization failure.Methods;The Wee2 knockout mouse model was constructed by CRISPR/CAS9 technique.The genotypes were identified after the mating test.Wee2 mRNA expression efficiency in Wee2-/-female mouse was detected by real-time quantitative polymerase chain reaction(PCR).The fertility of Wee2-/-and Wee2+/-female mouse was observed.Hematoxylin-Eosin(HE)staining was performed in the Wee2-/-and Wee2+/-mouse ovary sections to observe the morphology differences of ovary and follicles.Results:Wee2-/-and Wee2+/-mouse were bred after Wee2 knockout by CRISPR/CAS9.Real-time qPCR results showed that Wee2 was almost knockout in ovary of mouse.But Wee2-/-female mouse shows a normal reproductive phenotype and normal morphology of ovary and follicles compared with Wee2+/-female mouseConclusion:Wee2 knockout in mouse did not affect the fertility and morphology of ovary and follicles.