Clinical Effects Of Embryo Development Speed And Post-thawing Culture Duration In FET Cycle

BackgroundEmbryo cryopreservation is a biological technology for the long-term preservation of embryo by freezing early embryos with special protective agents and cooling measures, making the metabolism of embryos blocked utterly or slowed enough in liquid nitrogen (-196℃) and recovered adequately after anabiosis. It’s a goal for all reproductive centers to grant transplantation priority to the embryo with better development potential, to improve the cultivation rate and to reduce the multiple-pregnancy rate, no matter in fresh cycle or in freeze-thaw cycle. Surplus embryos caused by stimulate ovulation treatment also gain worldwide attention. In1983, the embryo freezing-thawing technology was first applied to human embryos by Trounson and Mohr successfully, achieving clinical pregnancy. The technology is now widely used all around the world as innovation is taking place. About40%to60%of patients undergoing ovarian stimulation in IVF-ET have excess good embryos after fresh embryo transfer,while the advantages of embryo cryopreservation technology are:1.reasonably limit the number of embryos transferred thereby reduce multiple pregnancy occur.2.provide a opportunity to retransplantation for implantation failure and abortion patients.3. improve the success rate of IVF,reduce the pain and economic pressure produced by the repeated COH;4.Improve cumulative pregnancy rate of the patients who have OHSS risk,cannot transfer fresh embryo.There are a large number of factors affecting pregnancy of patients undergoing FET,such as embryo quality, number of transplanting embryo, receptivity of endometrium,embryonic development synchronization,survival of embryo, in-vitro culture time, etc. This study aims to find out the link between embryo developing speed or different culture time after thawing and pregnancy outcomes, and provides clinic references to choose embryo with better implantation and development potential that could increase pregnancy rate.Part I The impact of embryo development speed and post-thawing culture duration in Cleavage stage FET cycleObjectiveTo investigate the impact of Cleavage stage embryonic development rate and freezing-thawing with different vitro incubation time to the outcome of FETMethod1.Patients:Retrospctively analyzed the clinical date of661in the FET cycles from January to December2012in the Center of Reproductive Medicine Nanfang Hospital.The inclusion criteria:①women undergoing IVF/ICSI treatment;②use a standard mid-luteal phase long protocol or GnRH antagonist in ovarian stimulation cycle;③patients with failure or cancel transplantation in ovarian stimulation cycle who were transferred frozen embryos after2to3months;④transfer1to3frozen-thawed embryos. IVF indication include:tubal factor,ovulation failure,unexplained infertility; ICSI indication include:man with serious oligoasthenozoospermia, obstructive azoospermia, retrograde ejaculation, previous IVF fertilization failure;the sperm of ICSI comes from ejaculation,PESA, TESA.The exclusion criteria:use coasting program in ovarian stimulation cycle; transfer blastocyst who come form frozen-thawed embryo and two-steps in frozen-thawed embryo transfer cycle; abnormal fertilization;④affect endometrial environmental factors,eg:endometriosis, adenomyosis,submucosal fibroids, intrauterine adhesions, uterine effusion, endometrial polyps, endometrial hyperplasia and endometrial tuberculosis;⑤use assisted hatching in frozen-thawed embryo transfer cycle.2.Method of determination the FET pregnancy outcome related indicators: Bivariate correlation analysis3.Grouping:According to the number of blastomere of D3frozen embryos,we classified them to3groups,group A:6-8blastomere cell;group B:<6blastomere cell;group C:>8blastomere cell(except:the group with blastomere cell of embryo is different when transfer several embryos);the patients were conducted into group D and group E according to the incubation time in vitro of the embryos after thawing,group D：culture2～4h in vitro after thaw,D3transplantation,goup F:culture18-24h in vitro after thaw, D4transplantation. According to the recovery situation of embryonic blastomeres with culture2-4h in vitro after thaw,we divided group D into two group:group D1(all blastomeres of transplanted embryo were all survived),group D2(all blastomeres of transplanted embryo were part survived and part lost).According to the situation of embryo which recovery mitosis with culture18-24h in vitro after thaw, we divided group E into two group：group E1(all embryo recovery mitosis),groupE2(a part of embryo recovery mitosis).4.The reason of transplant in FET:failure or cancel transplantation in ovarian stimulation cycle.5.Methods of endometrial preparation:natural cycle, ovarian stimulation cycles(HMG),hormone replacement therapy cycle without pituitary down-regulation,natural Cycle with HCG.6.Laboratory processing,embryo cryopreservation and recovery,luteal supporting and pregnancy diagnosis:Oocytes were fertilizated through IVF/ICSI.According to the patient’s age,previous transplantation,and the presence of OHSS symptoms after72h Ovulation, one to there the best quality embryos were transplanted,surplus embryos were be frozen by Cryopreserved standard of our center.Embryos cryopreservation and recovery,luteal supporting and pregnancy diagnosis were diagnosis were best seen by reference to the conventional methods of our center.7.Statistical analysis:We use SPSS13.0software package for statistical analysis.Results were showed by mean±standard deviation or rate(%). When date meet the conditions of homogeneity of variance,independent sample t-test or one-way ANOVA were be used; Otherwise,the nonparametric Kruskal-Wallis H test was be choosed;χ2test was applied to rate comparison, Multivariate correlation analysis using binary logistic regression. When date meet the normal bivariate distribution,Pearson correlation. Coefficient was adopted for the bivariate correlation analysis; Otherwise, Spearman correlation coefficient was be used. All P values <0.05were considered to be statistically significant(directly record with "P" in the table), while, P values≥0.05when result has no significant(record with NS in the table).Result1. In this study, we analysed a total of661FET cycles. there are1634and1615embryos was thawed and recoveried, respectively.The recovery rate was98.8%. In all these cycles,1511embryos was transplanted, and423of them enbed. The embryo implantation rate was27.99%.281of the661cycles got clinical pregnancy, clinical pregnancy rate was42.5%.There is no obvious correlation in FET pregnancy outcomes with assisted reproduction scheme, fertilization method, the transplanting reason and endometrial preparation plan in FET5cycles(P>0.05); It has positive correlation with in vitro incubation time after embryos thawed (r=0.08, p=0.08), though not much relevance; besides,it was positively correlated with embryonic development speed in fresh cycles.(r=0.113, P=0.113)2. There is no statistical differences in patients, age、duration of infertility、 ovulation induction project constituent ratio and fertilization way constituent ratio in fresh cycle、endometrial preparation plan constituent ratio in FET cycle、the thickness of endometrium and the embryo number of each cycle in group A、B and C while there are statistical differences in embryo recovery rate (P=0.007)、clinical pregnancy rate (P=0.003)and embryo implantation rate (P=0.007) in those groups. The pregnancy outcome was compared of between cycles in which the blastomere was100%survival or not after thawing, we found that there are statistical differences in embryo implantation rate、clinical pregnancy rate and the natural abortion rate (P>0.05)3. After revising the influence of confounding factor such as ovulation induction project constituent ratio and fertilization way constituent ratio of fresh cycle, and endometrial preparation plan constituent ratio、the thickness of endometrium and the embryo number of each cycle in FET cycle, a Logistic regression anaysis was performed, and we found that there is no Statistical differences in embryo implantation rate(95%CI0.745;1.152, P=0.493)、clinical pregnancy rate (95%CI0.908;1.408, P=0.272)、the natural abortion rate (95%CI0.538;1.378, P=0.533), multiple pregnancy rate (95%CI0.861;1.727, P=0.265) and ongoing pregnancy rate (95%CI0.451;1.118, P=0.139) in group D and E.4. Statistical differences in embryo implantation rate (29.60%VS.27.60%, P>0.05),the natural abortion rate(12.50%VS.17.40%, P>0.05)and multiple pregnancy rate (39.70%VS.39.10%, P>0.05) between group D1and D2.5.There is no Statistical differences(P>0.05)in the natural abortion rate between group E1and E2,while there is Statistical differences in embryo implantation rate (30.40%VS.15.70%, P=0.000)、clinical pregnancy rate (45.50%VS.25.00%, P=0.001) and multiple pregnancy rate (35.30%VS.9.50%, P=0.020)Conclusion1. The key to get pregnancy after frozen embryos thawing graft is the speed of embryonic development before being frozen, and an appropriate embryonic development speed can obtain good implantation rate and pregnancy rate in FET cycle, and that has no obvious correlation with the lost of survival embryo cleavage ball. 2. The embryo incubation time in vitro after thawing dose not affect assisted reproduction outcome in FET cycle; In vitro culture of embryonic maldevelopment, doesn’t mean the same dysplasia in the body.3.Once the incubation time in vitro was extended, fully recovered embryos mitotic group can get preferable embryo implantation rate and clinical pregnancy rate; but it will increase the rise of multiple pregnancy which will result in the increasing rise of obstetrical complication in latter half of gestation.Part Ⅱ Clinical effect of blastocyst formation time and the number of transplantation in FET cycleObjectiveTo investigate fresh the cycle the blastocyst speed and freeze-thaw cycle number of transplant FET cycle pregnancy outcome, and thus provide a reference for clinical work.Methods1. Study:a retrospective study from January2012to December Nanfang Hospital, Center for Reproductive line patients with FET progesterone treatment of167transfer cycles.1.1inclusion criteria:①age<40years patients;②at our center the first line the blastocyst-FET, blastocysts1-2; the③fresh cycle of graft failure or cancel the transplant, an interval of at least two months re-FET;1.2Exclusion criteria:①fresh cycle line coasting program;②FET cycle blastocysts in fresh cycle training process development speed is inconsistent;③insemination abnormal;④presence of the patients with endometrial environmental factors;⑤frozen before being hatchedor has been completely hatched blastocyst.2. grouping criteria:In accordance with the development rate of blastocysts before freezing fresh cycle divided into three groups:group A frozen fifth day of the formation of the blastocyst; sixth day of blastocyst formation of the B group is frozen; C group is frozen seventh dayformed blastocysts; according to the same period per transplant blastocyst speed different from the number of transplant group A is divided into group A1(transplant a single blastocyst) and A2(transplant two blastocysts); divided into group B group B1(transplant a single blastocyst) and B2(transplant two blastocysts); divided into group C group C1(Portable single blastocyst) and C2(transplant two blastocysts).3.Controlled ovarian hyperstimulation, egg retrieval, semen collection, optimization of treatment and in vitro fertilization mode selection the FET cycle endometrial preparation programs:Same as the first part of the paper.4.blastocysts frozen and recovery after transplantation luteal support, clinical pregnancy diagnosis OUTCOME MEASURES:with the first part of the paper.5.Statistical methods:SPSS13.0software package for statistical analysis of the database, the results mean plus or minus standard deviation (±s) or rate (%). Measurement data to meet the homogeneity of variance test, using independent sample t-test or one-way ANOVA, does not meet the homogeneity of variance test, using the non-parametric Kruskal-Wallis H test; compared by chi-square test; bivariate correlation analysis bivariate normal distribution, the Pearson correlation coefficient; non-normal distribution of two-variable data using the Spearman correlation coefficient analysis, multivariate correlation analysis using binary logistic regression. P<0.05was statistically significant difference in the table directly to the P value of said P>0.05for no significant difference in the table for the NS.Result1, in this study, A total of163transplant period, average age (30.76+4.50), duration of infertility (4.65+3.12) years, were thawed embryos292, live286after recovery, the study of the thawing recovery rate is97.95%, embryo transfer A total of279, growing embryo number to69, embryo to grow at A rate of24.73%, transplantation163cycles,56cycles in pregnancy, the pregnancy rate was34.36%, the blastocyst transplant cycle, frozen embryos grow rate of A and B two groups of patients with statistical difference (P=0.042), and> group B, group A and clinical pregnancy rate and spontaneous abortion rate is no statistical difference (P>0.05).2, A1, A2two groups of patients’ age, duration of infertility, row of super fresh cycle promoting form, pick up eggs than after insemination forming ratio and mode of lining to FET cycle, endometrial thickness, blastula refrigerant recovery rate, embryo grow rate, clinical pregnancy rate and spontaneous abortion rate comparison between two groups had no statistical difference (P>0.05).3, B1, B2, age and duration of infertility patients with similar between the two groups has statistical significance (P=0.000, P=0.000), and are based on B1group> B2, the rest of the row of super fresh cycle promoting form, pick up eggs than after insemination forming ratio and mode of lining to FET cycle, endometrial thickness, blastula refrigerant recovery rate, embryo grow rate, clinical pregnancy rate and spontaneous abortion rate comparison had no statistical difference between two groups (P>0.05). Through correction for B1, B2group patient’s age and duration of infertility and other confounding factors on the pregnancy outcome evaluation of the impact of related indicators, line of logistic regression analysis found that two groups of comparison in the clinical pregnancy rate (95%CI0.152;0.966, P=0.966) statistically differences, and spontaneous abortion rate (95%CIO.159;15.205, P=15.205), no statistical difference.4, A1, B1two groups of patients’ age, duration of infertility, row of super fresh cycle promoting form, pick up eggs than after insemination forming ratio and mode of lining to FET cycle, endometrial thickness, blastula refrigerant recovery rate, embryo grow rate, clinical pregnancy rate and spontaneous abortion rate comparison had no statistical difference between two groups (P>0.05). However, embryos grow rate and clinical pregnancy rate between the two groups although there was no significant difference, but is bigger than the A1, B1.5, No statistical difference between A2, B2, two groups of patients age, duration of infertility, pick up eggs, after the insemination row of composition ratio, super fresh cycle promoting constituent ratio, endometrial thickness is no statistical difference between groups (P>0.05), and FET cycles endometrial preparation methods statistically difference between the two groups (P=0.042); Two groups of blastocyst refrigerant recovery rate, clinical pregnancy rate and spontaneous abortion rate comparison had no statistical difference between two groups (P>0.05), while embryo rate of two groups was statistically difference (P=0.042), and A2> B2group. A2, B2via calibration FET lining for two groups of patients scheme than the confounding factors of blastocyst-FET pregnancy outcome evaluation of the impact of related indicators, lines of logistic regression analysis to compare two groups of clinical pregnancy (95%CIO.192;0.976, P=0.976) between groups was statistically difference, the natural abortion rate (95%CI0.090;1.802, P=1.802).Conclusion1. D5, D6frozen-recovery after formation of the blastocyst, embryo survival rate had no significant difference, different development stage of phase3-4quality blastocyst recovery after vitrification cryopreservation does not affect its survival.2. D5blastocysts from D6blastula by vitrification freezing thawing after transplantation, can obtain higher embryo grow rate and clinical pregnancy rate and lower the risk of spontaneous abortion, so we should give priority to choose in the clinical practice work D5blastula, followed by the D6blastocysts.3.D5blastocyst frozen transplantation can consider to use single embryo transfer, in order to reduce the risk of multiple pregnancy and abortion, but also help to reduce the long-term complications of pregnant women and fetus; For D6blastula, should choose double blastocyst transplant in order to obtain more ideal treatment effect.