Impact of relative estradiol changes during ovarian stimulation on blastocyst formation and live birth in assisted reproductive technology

This study analyzed first ART cycles, classifying patients into four response groups based on the ratio of E2 levels on the day of hCG administration to baseline E2 during COS. E2 response levels correlated significantly with patient characteristics (age, BMI, baseline hormones) and key outcomes (oocyte retrieval, blastocyst formation rate, pregnancy rates). As E2 response levels increased, patient age decreased, ovarian function parameters (such as FSH and LH) improved, and oocyte retrieval and blastocyst formation rate significantly increased. Additionally, clinical pregnancy rate and live birth rate were significantly higher in the moderate-high response group and high response group compared to the low response group. These results suggest that E2 response level is an important predictive indicator for embryological outcomes and pregnancy success in ART cycles.

Estrogens regulate reproductive functions, with E2 being dominant during reproductive years. While estrogen’s role in late pregnancy is well-studied, its function in early pregnancy is less understood^15,16. In ART, exogenous hormones are used to modulate female sex hormone levels, mimicking the natural hormonal fluctuations of the menstrual cycle to optimize ART outcomes. However, the lack of high-quality studies has limited the establishment of standardized protocols¹⁷. Existing research on the role of E2 in ART has shown mixed results. Zavy et al.¹⁸ divided patients into three groups based on E2 levels (< 2000 pg/ml, 2000–4000 pg/ml, > 4000 pg/ml) and found no significant differences in live birth rate or miscarriage rates between groups. Conversely, Joo et al.¹⁹ stratified patients into five E2 level groups (< 1000 pg/ml, 1000–2000 pg/ml, 2000–3000 pg/ml, 3000–4000 pg/ml, > 4000 pg/ml) and observed significant differences in implantation rates, pregnancy rates, and live birth rate across these groups. The variation in E2 cutoff values used in different studies may explain the inconsistent findings. Differences in sample sizes may also contribute to these discrepancies. Larger studies, such as Kondapalli et al.¹³ with 1712 patients and Wang et al.²⁰ with 3393 patients, have reported significant increases in live birth rate in higher E2 level groups, providing greater statistical power and more reliable results. In contrast, smaller studies by Zavy et al.¹⁸ and Morales et al.²¹, with 478 and 181 patients respectively, may have lacked the power to detect subtle associations between E2 levels and pregnancy outcomes.

In contrast to these studies, our research utilized the ratio of (trigger day E2 – baseline E2)/baseline E2) to categorize patients. This approach offers better control of individual baseline E2 differences and dynamically captures relative change in E2 levels, providing a more consistent evaluation metric. Previous studies have shown that E2 levels on the day of HCG administration can effectively predict oocyte retrieval, maturation, and subsequent fertilization²¹. Consistent with these findings, our study demonstrated a positive correlation between E2 response levels during COS and oocyte retrieval, maturation, and fertilization rates. After adjusting for confounding factors, the moderate-high response group and high response group still showed significantly better oocyte retrieval, maturation, and fertilization outcomes compared to the low response group. Some studies have suggested that E2 may negatively affect blastocyst formation, as seen in bovine oocyte in vitro maturation (IVM) studies where 1 µg/ml of E2 reduced nuclear maturation and blastocyst formation rate²², others have reported that E2 promotes blastocyst formation in pigs²³. Such differences may be attributed to variations in species, culture conditions, and E2 concentrations. Our data indicate a moderate positive correlation between E2 response levels during COS and blastocyst formation rate, with significant differences observed between the low response group (0.13), moderate response group (0.212), moderate-high response group (0.279), and high response group (0.34). Even after adjusting for confounding factors, these differences continued to be significant. When the response groups were simplified into “low” and “high” response categories, the high response group had a significantly higher blastocyst formation rate than the low response group, underscoring the importance of E2 response in blastocyst development. Furthermore, both univariate and multivariate logistic regression analyses supported this conclusion, indicating a strong and independent association between E2 response and blastocyst formation. Blastocyst formation analysis (Supplemental Fig. 1) also showed similar trends between oocytes retrieved from patients aged ≥ 35 years and those aged < 35 years. Similarly, comparisons between BMI < 25 kg/m² and BMI ≥ 25 kg/m² (Supplemental Fig. 2) revealed similar trends, indicating that neither advanced age nor obesity significantly impacted the relationship between E2 response and blastocyst formation.

Estrogen plays a crucial role in endometrial receptivity by promoting epithelial proliferation, tissue regeneration, and enhancing implantation factors such as leukemia inhibitory factor (LIF) and MUC-1, which support embryo attachment and decidual invasion^24,25,26. It also influences immune cells like uterine natural killer (uNK) cells and decidual macrophages, contributing to endometrial remodeling and creating a favorable implantation environment^27,28,29. It also influences immune cells like uNK cells and decidual macrophages, contributing to endometrial remodeling and creating a favorable implantation environment^30,31. Therefore, investigating E2 levels may provide valuable insights into understanding endometrial receptivity and its role in ART.

Notably, maintaining E2 levels within an optimal range during ART is crucial for achieving favorable reproductive outcomes. Studies suggest that both high and low E2 levels may negatively impact pregnancy outcomes, with higher E2 linked to reduced live birth rate and increased ectopic pregnancies^32,33. High E2 levels have also been linked to adverse neonatal outcomes, such as low birth weight⁵, placenta-related complications³, and preeclampsia⁴, suggesting that elevated E2 levels may negatively impact pregnancy progression. Conversely, low E2 levels can also affect pregnancy outcomes. Chen et al.³⁴ reported that E2 levels < 1200 pg/ml were associated with an increased risk of preeclampsia, and lower E2 levels might reduce endometrial receptivity, affecting embryo implantation and pregnancy maintenance³⁵.

In our study, based on the ratio of trigger day E2 to baseline E2 during COS, we found that as the E2 response ratio increased, clinical pregnancy rate and live birth rate significantly improved. The moderate-high response group and high response group had significantly higher clinical pregnancy rate and live birth rate compared to the low response group, and these differences remained significant after adjusting for potential confounders. However, for the moderate response group, although clinical pregnancy rate and live birth rate were higher than in the low response group, the differences were not statistically significant after adjusting for confounders, indicating that lower E2 response levels may not be sufficient to impact pregnancy outcomes. Furthermore, although miscarriage rate in the moderate-high response group and high response group showed statistical differences before adjustment, the absolute differences with the low response group were small, and after adjusting for confounders, these differences were not significant. Similarly, no significant differences in ectopic pregnancy rate were observed between the E2 response groups. Given the similar miscarriage rate and ectopic pregnancy rate across E2 response groups, differences in live birth rate may be attributed to differences in implantation rates rather than pregnancy loss. These data suggest that E2 response levels during COS may influence embryo implantation success. Although no differences in ectopic pregnancy rate were observed between the low and high response groups, the wide confidence intervals reflect the variability of such rare outcomes in this large cohort.

The findings of this study have important implications for clinical practice. To our knowledge, this is the largest study to explore the impact of E2 response during COS on embryo development and pregnancy outcomes. Based on a large dataset of 9376 fresh, first IVF/ICSI cycles, we provide a comprehensive analysis of E2 response as a predictor of embryo development and pregnancy outcomes in ART. Using the ratio of trigger day E2 to baseline E2 offers a dynamic and individualized assessment of E2 changes during COS, improving predictive accuracy over traditional absolute E2 measurements. This method, compared to traditional analyses based on absolute E2 values, allows for better control of individual variability and provides clinicians with a more reliable predictive tool for assessing patients’ responses to COS and forecasting embryo development and pregnancy outcomes. Additionally, the multivariate analysis in our study controlled for several key confounding factors, such as age, baseline FSH, LH, and P4 levels on the day of hCG, infertility diagnosis, and stimulation protocols, enhancing the reliability of our findings. We observed a significant association between E2 response and blastocyst formation rate, as well as live birth rate, and these associations remained significant even after adjusting for important covariates. This suggests that the relative change in E2 response could serve as an independent predictor of clinical outcomes, offering important clinical utility.

Despite the strengths of our study, there are some limitations to consider. First, as a retrospective analysis, the study design cannot establish causality but demonstrates only an association between E2 response and pregnancy outcomes. Future prospective studies are needed to confirm our findings and explore the causal mechanisms behind them. Second, although we assessed the E2 response using a ratio to reflect the relative changes during COS, the thresholds for categorizing E2 response groups were based on the quartile method from the cohort’s data distribution. While statistically robust, these thresholds lack established clinical cutoffs and require further validation in future studies. Additionally, the clinical applicability of this ratio still requires validation, especially across different populations and stimulation protocols, as variations in baseline E2 levels and individual physiological responses could impact its generalizability. Third, although we adjusted for ovarian stimulation protocols in our multivariable analysis, the inherent heterogeneity in these protocols may still influence outcomes. Different protocols create distinct hormonal environments that can affect both embryological outcomes and endometrial receptivity, especially in fresh embryo transfers, where endometrial conditions are more sensitive to supraphysiological hormone levels. Additionally, as this study focused exclusively on fresh IVF/ICSI cycles, the applicability of our findings to frozen-thawed embryo transfer (FET) cycles remain uncertain, which may limit the generalizability of our results. Fourth, patients with PCOS and endometriosis were excluded to reduce heterogeneity, but this limits the applicability of our findings to these subgroups, warranting further investigation. Finally, although we adjusted for several confounding factors, there may still be residual confounders, such as patient lifestyle, metabolic factors, or subtle differences in stimulation protocols, that could influence pregnancy outcomes but were not fully captured in our dataset.