Preliminary Study On TBP2 Gene And Prognosis In Patients With Premature Ovarian Failure

Part Ⅰ The study of TBP2 gene in patients with premature ovarian failureObjective:It has previously been reported that TBP2 gene expressed exclusively in ovary and female TBP2 null mice were sterile.We aimed to sequence the 7 exons of TBP2 gene so that to assess the association between the TBP2 gene and primary ovarian insufficiency(POI)in a Chinese population.Methods:A cohort of 60 Chinese POF patients matched with 60 fertile controls(with normal menstrual cycle and sex hormone levels,matched for age,ethnic background and sex with the patients)was recruited by the First Affiliated Hospital of Nanjing Medical University.Their peripheral blood samples were collected and there were no interventions.Genomic DNA was extracted from peripheral leukocytes with Axprep DNA Blood Kit.The entire coding region and splice sites of the TBP2 gene were amplified by polymerase chain reaction(PCR)using primers designed by UCSC ExonPrimer online software.All the PCR products were directly sequenced according the Sanger sequencing method.The sequences of TBP2 gene in both POF and control groups were synthetically analyzed by BioEdit Sequence Alignment Editor,Mutation Surveyor DNA Variant Analysis Software and Hardy-Weinberg equilibrium.Web-based programs ClustalX and PolyPhen 2 were used to predict the potential functional or structural impacts of the missense variants on the TBP2 gene.Chi-square was used to assess the genotype distribution and allele frequency between cases and controls.Results:A known single nucleotide polymorphism(c.92G>C)was identified in both POI and control groups.There was no difference in the genotype distribution or allelic frequencies between the control group and the POI group(P>0.05).No plausible pathogenic mutations were identified.Conclusion:Our study indicates that the SNP(c.92G>C)of TBP2 gene is not responsible for idiopathic POI in the Chinese Han population.Part Ⅱ The preliminary exploration of assisted reproductive protocols in women of premature ovarian failure with infertilityObjective:Premature ovarian failure is a complex endocrine disorder.Women with POF are difficult to acquire pregnancy spontaneously.This study aims to analyze the pregnancy outcomes in the 50 POF women with infertility by using natural cycle protocol or micro-stimulation cycle protocol of in vitro fertilization-embryo transplantation(IVF-ET)and to evaluate the curative effects of the two assisted reproductive protocols,so that the effects of different assisted reproductive protocols on clinical patients can be discussed.Study Design:172 infertile patients with POF which included 74 patients by natural cycle protocol and 98 patients by micro-stimulation cycle protocol,they were collected during the recent four years in Reproductive Center of the First Affiliated Hospital of Nanjing Medical University and 50 of them were successfully pregnant after using IVF-ET.The 50 patients all met the POF diagnostic criteria and were divided into two groups according to different therapeutic protocols of IVF,17 patients by natural cycle protocol were in group A and 33 cases with micro-stimulation cycle protocol were in group B.All the subjects,with normal chromosome karyotype,were absent of pelvic factors or other systemic diseases and they were accompanied with fertile husbands.The age,age of menarche,body mass index(BMI),years of infertility,serum hormone(FSH),luteinizing hormone(LH),estrogen(E2),testosterone(T),prolactin(PRL),antral follicle count(AFC)and thyroid function of all the subjects were recorded.Their pregnancy outcomes were analyzed after 3 to 4 years of following-up so that to analyze the curative effects of the two assisted reproductive protocols.Results:The level of FSH exceeded 40IU/L(met the POF diagnostic criteria)in both two groups before the treatment of assisted reproductive technology.In general condition,there was no significant difference of age,age of menarche,BMI,years of infertility,times of pregnancy and numbers of basic follicle between two groups(P>0.05).Moreover,by comparing the sex hormone levels of FSH,LH,E2 in A and B groups[A group:FSH(10.39±5.83)IU/L,LH(3.75±2.07)IU/L and E2(44.53±63.00)pg/ml,B group:FSH(12.18±6.06)IU/L,LH(4.19±1.80)IU/L and E2(43.40±61.35)pg/ml],there was no significant difference on the third day of withdraw bleeding after more than three months’ combination estrogen and progesterone hormone replacement therapy(HRT).Besides,there was one case of natural cycle protocol and seven cases of micro-stimulation cycle protocol with abnormal thyroid function.And each of the two groups had one case of hyperprolactinemia.In the pregnancy outcomes,14 cases with natural cycle protocol had clinical pregnancy and all of them were alive,so the rate of clinical pregnancy in natural cycle group was 18.9%(14/74).While 29 cases with micro-stimulation cycle protocol had clinical pregnancy and 25 of them were alive so the rate of clinical pregnancy in micro-stimulation cycle group was 29.6%(29/98).We found that there was no significant difference of the live birth rate between the two groups(P>0.05).In addition,there was no miscarriage or ectopic pregnancy in group A while there were 4 cases of miscarriage and 1 case of ectopic pregnancy in group B.Conclusion:Women with POF extremely rarely achieve pregnancy spontaneously and most of them get pregnancy by oocyte donation and IVF.Our study showed that natural cycle protocol and micro-stimulation cycle protocol of IVF-ET might be effective in the fertility treatment of patients with POF/POI.