We demonstrated that the serum E2 level on the progesterone initiation day was significantly higher in the OP/LB group than in the non-OP/LB group during artificial FET cycles with cleavage-stage embryo transfer. In addition, elevated E2 levels in the 91st-100th percentile had detrimental effects on embryo implantation, clinical pregnancy and OP/LB compared to E2 levels in the 1st-10th percentile. However, such effects of E2 levels on embryo implantation, clinical pregnancy and OP/LB were not observed in frozen-thawed blastocyst-stage embryo transfer cycles. This is the first study to find that serum E2 levels on progesterone initiation day have different effects on pregnancy outcomes between cleavage-stage embryo transfers and blastocyst-stage embryo transfers during artificial FET cycles.
Although estrogen levels in normal natural cycles reach 300–400 pg/ml before ovulation, a study on donor cycles revealed that the E2 requirement for embryo implantation is low (< 100 pg/ml) . Our study supports this conclusion, showing appropriately 40% of conceptions occurring in patients with E2 levels < 100 pg/ml either in cleavage-stage embryo transfers (95/233) or in blastocyst-stage embryo transfer cycles (65/165). On the other hand, successful conception in fresh embryo transfer cycles implies that embryo implantation can occur in an environment with the supraphysiological levels of E2 caused by ovarian stimulation. This can be explained by the fact that serum E2 levels were not associated with pregnancy outcomes in blastocyst transfer cycles in the present study. Similarly, in a study by Özdemir et al., E2 levels did not have significant effects on the pregnancy and miscarriage rate in autologous day 5 embryo transfer cycles using HRT . Our data showed that pregnancies could occur in a wide range of serum E2 levels from low levels of 10 pg/ml to a supraphysiological level of > 1000 pg/mL.
Interestingly, regarding cleavage-stage embryo transfer, we found that elevated E2 levels in the 91st-100th percentile on progesterone initiation day had detrimental effects on CPR and OP/LB rates in HRT cycles compared to the low E2 levels in the 1st-10th percentile. Fritz et al. had a similar finding that OP/LB decreased six-fold in FET cycles with the highest 10% E2 concentrations compared to those cycles with the lowest 10% E2 concentrations . The detrimental effects of an elevated E2 level on pregnancy have been reported in fresh in vitro fertilization cycles [4, 5, 15]. Experiments with a mouse model revealed that high levels of estrogen rapidly close the receptivity window for embryo implantation by altering endometrial gene expression, which causes the uterus to become unreceptive for implantation [16, 17]. As a consequence of this shortened receptivity window caused by a high E2 level, the receptivity window may be missed for a late-growing blastocyst developed from a day-3 embryo, resulting in a low chance of implantation; however, a blastocyst transfer can result in immediate implantation within the receptivity window.
In contrast to our findings regarding high E2 levels in cleavage-stage FET cycles, some authors have not found an adverse effect of high E2 levels on pregnancy outcomes. Niu et al. reported that E2 concentrations did not appear to be associated with the pregnancy rate in autologous day-3 FET cycles . They grouped E2 concentrations on progesterone initiation day into the 0–25th, 25th–75th, and 75th–100th percentile groups, and the average E2 concentration in the highest group (299 ± 48.9 pg/ml) was not in the range of the highest 10th percentile of our study (508.4–951.0 pg/ml). When we regrouped E2 levels according to their study, no differences in pregnancy or implantation between groups were observed. Classifying E2 levels into five groups, Remohi et al. did not find elevated E2 levels on the oocyte donation day to be associated with the implantation or pregnancy rate . This may be due to the high quality of embryos derived from donor oocytes that can be implanted within a narrow receptive window in conditions of elevated E2 levels, which was similar to our findings for blastocyst-stage embryo transfer cycles.
In contrast to our finding of a significant difference in E2 levels between the OP/LB group and the non-OP/LB group for cleavage-stage embryo transfer cycles, this difference in E2 levels was not found by several authors [8, 11, 18]. This discrepancy may be have two main causes. First, these authors used transdermal and intramuscular E2 for E2 supplementation, whereas we used oral estrogen. Oral estrogen is metabolized and transformed into estrone in the intestine and liver, which has a lower binding affinity for estrogen receptors than E2 . Second, the study by Fritz et al. pooled day-3 and day-5 embryos for analysis , whereas we analyzed data for cleavage-stage embryo transfer and blastocyst-stage embryo transfer separately.
Multivariable logistic regression was conducted to analyze E2 levels on progesterone initiation day in cleavage-stage embryo transfer cycles. We found that the E2 level was an independent variable affecting OP/LB after adjustments for female age. However, ROC curve analysis revealed that the E2 level on progesterone initiation day had no predictive power for pregnancy outcomes in either cleavage-stage or blastocyst-stage embryo transfer cycles. Although we acknowledge that the retrospective nature is a limitation in this study, recruitment of women under 40 years old with two good embryos being transferred into a normal endometrium eliminated the bias caused by other confounding variables. Another limitation involving the small sizes of the highest and lowest E2 levels indicates the need for larger sample sizes in prospective studies in the future.