The Clinical Value Of Monitoring Serum Hormone Levels In Frozen-thawed Embryo Transfer Cycles

BACKGROUNDSince1983, the first frozen-thawed embryo transfer achieved clinical pregnancy in the world, FET technology has been widely used in the field of human assisted reproductive technology (ART). How to improve the success rate of FET is one of the hot researches in ART. The pregnancy outcome of FET are associated with embryo quality、synchronization of the embryo and endometrium、and endometrial receptivity. The cycle of frozen simulates normal physiological states, which has a good synchronization of embryo and endometrium. So, embryo quality and endometrial receptivity are particularly important in FET cycle. The levels of serum estradiol and progesterone indirectly reflect the quality of oocytes and embryos in the control of ovarian hyperstimulation. Endometrium is the target organ of estrogen and progesterone, only the strictly regulation of estrogen and progesterone, the embryo can be successfully implanted. And also, endometrium is the target organ of luteinizing hormone (LH), through the feedback systems of the estrogen and progesterone, the level of LH can be changed, LH may become a new biological marker of endometrial receptivity. Serum hormones are the key factors of affecting endometrial receptivity. Therefore, the serum hormones play a crucial role in ART, which are the important indicators for the clinician to determine program adjustments and the predictive factors of IVF-ET pregnancy outcome.At present, there are many researches on the relations of sreum hormones and the outcome of IVF-ET. However, whether the serum hormones levels affect the outcome of FET are less, and the conclusions are not consistent. So, this study will explore the relationship of serum hormones levels and FET pregnancy outcome, in order to find predictive indicators for FET pregnancy outcome, provide the basis for the improvement of monitoring indicators, and provide a reference to determine the timing of frozen-thawed embryo transfer.PART-I The predictive value of serum hormones levels on hCG administration day to the outcome of frozen-thawed embryo transfer cyclesOBJECTIVETo investigate the influence of serum hormones levels on hCG administration day on the FET pregnancy outcome, in order to provide the basis for the improvement of monitoring clinical indicators and provide a reference to determine the timing of embryo transfer.METHOD1.Patients:Retrospectively analyzed the clinical data of466in the first FET cycles from July2009to December2011in the Center of Reproductive Medicine Nanfang Hospital, incluing299natural cycles,167hormone replacement therapy cycles without pituitary down-regulation.1.1The inclusion criteria:(to meet all the following conditions)(1)age<40years old;(2)use a standard mid-luteal phase long protocol in ovarian stimulation cycle;(3)patients with failure or cancel transplantation in ovarian stimulation cycle who were transferred frozen embryos after2to3months;(4)transfer2or3frozen-thawed embryos;(5)not taking hormone drugs before frozen-thawed embryos transfer at least two months;(6)basal FSH<10mIU/mL in both ovarian stimulation cycles and frozen-thawed cycles;1.2The exclusion criteria:(to meet one of the following)(1)affect endometrial environmental factors, eg:endometriosis, adenomyosis, intrauterine adhesions, uterine effusion, endometrial polyps,endometrial tuberculosis or endometrial hyperplasia;(2)use coasting program in ovarian stimulation cycle;(3)use assisted hatching in frozen-thawed embryo transfer cycle;(4)transfer blastocyst or two-steps in frozen-thawed embryo transfer cycle;2.Method of determination the FET pregnancy outcome indicators:Receiver operating characteristic curve (ROC).3.Grouping:According to serum E2level on hCG administration day, we classified them to four groups:groupA1,<2000.00pg/ml; groupA2,2000.01～3000.00pg/ml; group A3,3000.01～4000.OOpg/ml; group A4,>4000.01pg/ml. According to the methods of endometrial preparation for frozen-thawed embryos transfer, the patients were conducted into natural cycle (group B1) and hormone replacement therapy cycle without pituitary down-regulation (group B2). If the E2level on hCG administration day exceed3000.00pg/ml, according to the reason for FET, we grouped them into two groups:group C1, failure of fresh cycles, group C2, whole embryos freezing.4.The criteria of administrating hCG:When at least2follicles reached18mm in diameter, hCG5,000-10,000IU was administered intramuscularly at night. 5.Measurement of hormones:Basal FSH、LH、E2were measured in the early follicular phase. E2、LH and P were measured on hCG administration day. Hormones were determined by automatic chemical immune luminescence, intra coefficients of variation were less than5%. Serum E2levels exceed4300pg/ml, diluted determination.6.The definition of high ovarian response:Patients who had serum E2levels on hCG administration day≥3000.00pg/mL, and/or≥15retrieved oocytes.7.Methods of endometrial preparation:Natural cycle or hormone replacement therapy cycle without pituitary down-regulation.8.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, two or three good quality embryos were transplanted, surplus embryos were be frozen. Embryos cryopreservation and recovery, luteal supporting and pregnancy diagnosis were best seen by reference to the conventional methods of our center.9.Statistical analysis:We used SPSS13.0software package for statistical analysis. Results were showed by mean±standard deviation. ROC curve was used to analysis the relationship between serum hormones levels on hCG administration day and FET pregnancy outcome. When data 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 choiced; χ2test was applied to rate comparison. When data 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. RESULTSl.The serum E2levels on hCG administration day can predict the FET pregnancy outcome (AUC=0.57, P=0.011), but the accuracy was low; The serum LH and P levels can’t predict the FET pregnancy outcome (AUC=0.50, AUC=0.48, respectively, P>0.05).2.As the E2levels on hCG administration day increasing, the P levels, the number of oocytes, mature oocytes, the high quality embryos and frozen embryos were more and more, the rate of canceling fresh embryos transfer and transferring high quality frozen embryos were higher, but the application of Gn was significantly reduced (P<0.05); Compared to group A1and group A2, there were a higher clinical pregnancy rate and implantation rate in group A3and group A4, the differences were significant (P<0.05).3.The E2levels on hCG administration day was not associated with basal FSH、 age and the initial of Gn (P>0.05); was negatively correlated with the total of Gn (r=-0.232, P<0.001), but the correlation was weak; was positively correlated with the number of oocytes retrieved (r=0.609, P<0.001), was positively correlated with mature oocytes (r=0.596, P<0.001), was positively correlated with high qulity embryos (r=0.319, P<0.001) and was positively correlated with frozen embryos (r=0.558, P<0.001), the correlations between the two variables were very close.4.Compared the natural cycles and HRT cycles, the difference in endometrium thickness was significantly thicker in natural cycles (P=0.003), but they can achieve similar implantation rate and clinical pregnancy rate (P>0.05). In the natural cycles, there was a significant difference in clinical pregnancy rate among the four groups (P=0.042), and the E2level on hCG administration day was controlled of3000-4000pg/ml, the pregnancy rate was highest. In HRT cycles, as the E2levels on hCG administration day increasing, the implantation rate and clinical pregnancy rate were higher, but the difference in clinical pregnancy rate was not statistically significant (P=0.207); The difference in implantation rate was statistically significant (P=0.003).5.Compared the high ovarian responser who underwent freezing transplantation after whole embryo freezing or failure of fresh cycles, in the whole embryo freezing group, basal FSH and the application of Gn were lower, the E2and P levels on hCG administration day were higher, the number of oocytes, mature oocytes, the high quality embryos and frozen embryos were more, the number of embryos transferred was significantly reduced (P=0.000), the rate of transferring high quality frozen embryos were higher (P=0.000), and the implantation rate was significantly higher (P=0.004); But they had a similar clinical pregnancy rate (P=0.775).6.Compared the methods of endometrial preparation for frozen-thawed embryos transferred in high ovarian responser, In natural cycles, the difference in endometrium thickness was significantly thickner (P=0.006), but there were no significant differences in implantation rate and clinical pregnancy rate between the two groups (P>0.05).CONCLUSION1.The serum E2levels on hCG administration day are associated with ovarian response and embryo quality, which can predict the FET pregnancy outcome, the serum E2levels exceed3000pg/ml can obtain a better implantation rate and clinical pregnancy rate in FET cycles.2.The key factor that affects frozen embryo transfer pregrancy outcome is embryo quality (transfer the rate of high quality embryo), the natural cycle and HRT cycle can achieve similar implantation rate and clinical pregnancy rate. According to the specific conditions of the patients, we should take individualized treatment plan. PART II The influence of serum E2and LH level on the pregnancy outcome in HRT-FET cycles without pituitary down-regulationOBJECTIVETo investigate the influence of serum E2and LH level on timing of embryo transfer and pregnancy outcome in hormone replacement therapy-frozen thawed embryo transfer cycles without pituitary down-regulation.METHOD1.Patients:Retrospective analyzed170HRT-FET cycles without pituitary down-regulation from March2010to December2011in our center.1.1The inclusion criteria:(to meet all the following conditions)(1)age<40years old;(2)use a standard mid-luteal phase long protocol in ovarian stimulation cycle;(3)basal FSH<10mIU/mL in ovarian stimulation cycle;(4)have at least3months interval of FET cycle and ovarian stimulation cycle;(5)have embryos cryopreservation and transfer2or3frozen-thawed embryos;(6)not taking hormone drugs before frozen-thawed embryo transfer at least two months;(7)serum FSH, LH and E2were in the early follicular phase, transvaginal ultrasound uterine adnexa were normal.1.2The exclusion criteria:(to meet one of the following)(1)affect endometrial environmental factors, eg:endometriosis, adenomyosis, intrauterine adhesions, uterine effusion, endometrial polyps,endometrial tuberculosis or endometrial hyperplasia;(2)oocyte donation or acception;(3)use coasting program in ovarian stimulation cycle;(4)use assisted hatching in frozen embryo transfer cycle; (5)transfer blastocyst or two-steps in frozen-thawed embryo transfer cycle;(6)have dominant follicle growth on progesterone initiation day;(7)the endometrium thickness has not to7mm when taking estradiol valerate over21days;(8) the anovulation patients who caused by organic disease.2.Grouping:According to the percentile of E2level on progesterone initiation day, the patients were grouped into three groups:A1, percentile <25th (<221.70pg/ml), A2, percentile25th～75th (221.70～394.05pg/ml), A3, percentile>75th (>394.05pg/ml). According to the percentile of LH level on progesterone initiation day, the patients were grouped into three groups:B1, percentile<25th (<10.38IU/L), B2, percentile25th-75th (10.38～20.33IU/L), B3, percentile>75th (>20.33IU/L). According to previous menstrual cycle, the patients were divided into ovulation group and anovulation group. The clinical characteristics and pregnancy outcomes in ovarian stimulation cycle and frozen-thawed embryo transfer cycle were compared among groups.3.Measurement of hormones:Basal FSH、LH、E2were measured after serum sample was collected in the early follicular phase. E2、LH and P were measured on progesterone initiation day. Hormones were determined by automatic chemical immune luminescence, intra coefficients of variation were less than5%.4.Laboratory processing, embryo cryopreservation and recovery, luteal supporting and pregnancy diagnosis:The same to part I.5.The criteria of administrating progesterone:Patients who took estradiol valerate medicine≥12days, and endometrial thickness≥7mm on progesterone initiation day.6.Statistical analysis:We used SPSS13.0software package for statistical analysis. Results were showed by mean±standard deviation. When data 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 choiced;χ2test was applied to rate comparison. All P values<0.05were considered to be statistically significant.RESULTS1.There were no significant differences in clinical characteristics in ovarian stimulation cycles and frozen-thawed embryo transfer cycles among the A1, A2, A3groups (P>0.05). As the percentile of E2increasing, clinical pregnancy rate and implantation rate were reduced, but not significantly (P>0.05).2.But for the total of oral estradiol, there were no significant differences in clinical characteristics and pregnancy outcomes in ovarian stimulation cycles and frozen-thawed embryo transfer cycles among the B1, B2, B3groups (P>0.05).3.Compared with the ovulation group, the patients’age were younger, basal FSH were lower, the number of embryos transferred was smaller in anovulation group. But there were no significant differences in the serum E2and LH levels、 endometrium thickness on progesterone initiation day、the rate of LH surge in two groups, and they can obtain similar implantation rate and pregnancy rate.4.If LH>10.00IU/L, compared with the ovulation group, basal FSH and the rate of uterine surgery were lower, the number of embryos transferred was smaller, the rate of whole embryo freezing was higher, the number of oocytes, mature oocytes, the high quality embryos, frozen embryos and endometrium thickness on progesterone initiation day were significantly higher in anovulation group (P<0.05), but there were no significant differences in implantation rate and clinical pregnancy rate between the two groups (P>0.05).CONCLUSION1.The range of serum E2and L H levels allowing endometrium to convert the window of receptivity may be large in HRT-FET cycles without pituitary down-regulation; The serum E2and LH levels on progesterone initiation day may not influence pregnancy outcomes.2.The patients with ovulation or anovulation can be a LH surge, and the proportion is similar in HRT-FET cycles without pituitary down-regulation.3.Whether there is LH peak have no effect on pregnancy outcome in HRT-FET cycles without pituitary down-regulation.4.The HRT-FET cycles without pituitary down-regulation can be used in patients with ovulation or anovulation.