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

ABSTRACT This study aimed to evaluate the predictive value of relative change in E2 levels during controlled ovarian stimulation (COS) on embryo development and pregnancy outcomes in

assisted reproductive technology (ART). We retrospectively analyzed 9,376 patients who underwent their first fresh ART cycle from January 1, 2020, to December 31, 2022. Patients were

classified into four groups based on relative change in E2 levels: low response group, moderate response group, moderate-high response group, and high response group. The primary outcomes

were blastocyst formation rate, clinical pregnancy rate, and live birth rate, while secondary outcomes included miscarriage rate and ectopic pregnancy rate. Most cycles (96.5%) demonstrated

an increase in E2 levels during COS. The blastocyst formation rate significantly increased across the groups (low response group: 0.13, moderate response group: 0.21, moderate-high response

group: 0.28, high response group: 0.34; _P_ < 0.001). Multivariable logistic regression showed significantly higher blastocyst formation rates in the moderate response group (adjusted OR 

= 2.012, 95% CI: 1.687–2.399), moderate-high response group (adjusted OR = 4.613, 95% CI: 3.853–5.523), and high response group (adjusted OR = 11.295, 95% CI: 9.192–13.880) compared to the

low response group. Both 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

(clinical pregnancy rate: 54.5% and 61.5% vs. 35.5%, adjusted RR = 1.21 [95% CI: 1.03–1.42] and 1.27 [95% CI: 1.08–1.51]; live birth rate: 44.9% and 52.0% vs. 25.7%, adjusted RR = 1.27 [95%

CI: 1.06–1.52] and 1.35 [95% CI: 1.11–1.64]). However, no significant differences were observed in either clinical pregnancy rate or live birth rate between the moderate response group and

low response group (clinical pregnancy rate: adjusted RR = 1.07 [95% CI: 0.91–1.25]; live birth rate: adjusted RR = 1.11 [95% CI: 0.92–1.33]). No significant differences in miscarriage rate

or ectopic pregnancy rate were observed across the groups. Higher E2 responses were associated with improved embryo development and better pregnancy outcomes. SIMILAR CONTENT BEING VIEWED BY

OTHERS THE ROLE OF PEAK SERUM ESTRADIOL LEVEL IN THE PREVENTION OF MULTIPLE PREGNANCIES IN GONADOTROPIN STIMULATED INTRAUTERINE INSEMINATION CYCLES Article Open access 15 November 2022

ELEVATED ESTRADIOL LEVELS IN FROZEN EMBRYO TRANSFER HAVE DIFFERENT EFFECTS ON PREGNANCY OUTCOMES DEPENDING ON THE STAGE OF TRANSFERRED EMBRYOS Article Open access 04 April 2022 EFFECTS OF

SERUM ESTROGEN LEVELS BEFORE FROZEN-THAWED BLASTOCYST TRANSFER ON PREGNANCY OUTCOMES IN HORMONE REPLACEMENT CYCLES Article Open access 21 January 2023 INTRODUCTION Successful ART depends on

the development of at least one embryo that can progress to the blastocyst stage, successfully implant in the uterus, and continue developing until a live birth. However, most human oocytes

in ART cycles fail to develop into blastocysts suitable for transfer. To improve the likelihood of obtaining viable embryos, COS with exogenous gonadotropins is commonly used to promote the

synchronized development of multiple follicles, yielding a higher number of mature oocytes in one cycle. Serum estradiol (E2) levels are an important component of assessing COS response, but

the predictive value of changes in E2 levels for ART outcomes remains controversial. Several studies have shown that higher E2 levels during COS may be associated with better in vitro

fertilization/intracytoplasmic sperm injection (IVF/ICSI) success rates1,2, while excessively high E2 levels during COS may create suboptimal conditions in the preimplantation uterine

environment, leading to abnormal placentation3and increasing the risk of adverse neonatal outcomes such as small–for–gestational–age (SGA) infants4and low birth weight (LBW)5. Other

researchers argue that E2 concentrations on the day of hCG administration alone are poor predictors of IVF/ICSI outcomes6,7and may not independently increased the risk of LBW and SGA8. It

remains unclear whether E2 levels during COS can independently predict IVF/ICSI outcomes or if a critical threshold exists that significantly affects live birth rates. Previous studies have

indicated that E2 levels are significantly correlated with the number of preovulatory follicles, retrieved oocytes, and resulting embryos9,10, and have suggested that these levels can be

used to predict follicle and embryo development during assisted reproduction. However, relatively few studies adopt arbitrary E2 thresholds for stratified analysis to predict embryo

development and pregnancy outcomes1,8,9,11,12, but these studies often yield inconsistent conclusions, limiting the reliability of E2 levels as a prognostic marker. While E2 levels in ART

has been widely studied, few investigations have examined how changes in E2 levels predict pregnancy outcomes13. This study explores the predictive value of relative E2 changes during COS on

embryo development and ART outcomes. By investigating these relative changes, we hope to provide a more accurate assessment of E2 dynamics to optimize the prognostic value of E2 in IVF/ICSI

treatment. MATERIALS AND METHODS STUDY DESIGN AND PARTICIPANTS In this retrospective cohort study, we identified all women aged 21–52 years who underwent fresh assisted reproductive cycles

at the Reproductive Center of Liuzhou Hospital, Guangzhou Women and Children’s Medical Center, between January 1, 2020 and December 31, 2022. This study only included patients undergoing

their own egg stimulation cycles, and no donor egg cycles were included. The analysis was restricted to patients who entered their first cycle with complete clinical data available (_n_ =

9,376), representing 81.1% of the total cycles during this period. We excluded cycles with inaccurate medical records or missing data, as well as patients with polycystic ovary syndrome

(PCOS) or endometriosis due to their potential to introduce heterogeneity in ovarian response and pregnancy outcomes, which could confound the interpretation of our findings (Fig. 1). This

study included all fresh assisted reproductive cycles for the analysis of embryological outcomes, without specific differentiation of PGT cycles. However, for the analysis of pregnancy

outcomes, only fresh transfer cycles were included, and PGT cycles, which result in cryopreservation following biopsy, were excluded. Patients’ clinical characteristics such as age and

duration of infertility were self-reported. Infertility diagnosis, medication protocols, and fertilization plans were recorded by physicians. Height and weight were measured and recorded

on-site prior to the appointment, from which BMI was calculated (weight kilograms divided by the square of height in meters). Cycle-specific information, including oocyte and embryo

development, days of embryo transfer, and the number of embryos transferred, was documented by the reproductive laboratory. Only cycles with at least one embryo transfer at the cleavage or

blastocyst stage were included in the clinical pregnancy analysis. Serum hormone values were obtained from the databases of the Clinical Laboratory and Genetics Laboratory. CONTROLLED

OVARIAN STIMULATION PROTOCOLS The GnRH agonist protocol was employed for patients with good ovarian reserve (AFC ≥ 5) and normal baseline hormone levels, which were defined as FSH < 10

mIU/ml, AMH between 1.0 and 1.4 ng/ml, and AFC between 6 and 15. Downregulation began one week after ovulation using subcutaneous GnRH-a injections, such as triptorelin (0.1 mg/day or

long-acting 3.75 mg) or leuprorelin (3.75 mg). Downregulation success was defined by specific hormone levels (E2 < 50 pg/ml, LH < 5 mIU/ml, FSH < 5 mIU/ml) and uterine ultrasound

criteria, including an endometrial thickness < 5 mm and ovarian follicles of < 8 mm. Once downregulation was achieved, gonadotropin stimulation was initiated with doses ranging from

100 to 300 IU/day, adjusted based on age, BMI, and AFC. Follicular growth was monitored via transvaginal ultrasound and serum hormone levels every 2–3 days. Ovulation was triggered when

dominant follicles reached ≥ 18 mm using HCG (5,000–10,000 IU), GnRH agonists, or a dual trigger (HCG + GnRH agonist), followed by egg retrieval 34–36 h later. Luteal support began on the

day of egg retrieval, with progesterone administered vaginally, orally, or intramuscularly, and continued for 10 weeks if pregnancy was confirmed. The GnRH antagonist protocol is suitable

for patients with varying ovarian responses, including those with good, moderate, or poor ovarian reserve. Baseline assessments are performed on cycle days 2–3, including hormone levels

(FSH, LH, E2, AMH) and transvaginal ultrasound to assess AFC and the presence of any ovarian cysts or abnormalities. For patients with good ovarian reserve, the classification criteria are

FSH < 10 mIU/ml, AMH ≥ 1.0 ng/ml, and AFC ≥ 5. For patients with lower ovarian reserve or poor responders, the gonadotropin dose is adjusted accordingly based on baseline assessments, and

for poor responders, a higher starting dose of gonadotropins (≥ 300 IU/day) may be used to stimulate follicular development effectively. Gonadotropin stimulation commenced with initial

doses of 150–450 IU/day, adjusted according to patient-specific factors. A GnRH antagonist (e.g., cetrorelix 0.25 mg/day or ganirelix 0.25 mg/day) was added when the lead follicle reached 14

mm to prevent premature LH surges. Ovulation was triggered with HCG, GnRH agonists, or a dual trigger, followed by egg retrieval 34–36 h later. Luteal support was initiated on the day of

egg retrieval and continued as described above. The progestin-primed ovarian stimulation (PPOS) protocol was primarily used for patients with diminished ovarian reserve, repeated

implantation failure, or specific uterine conditions requiring embryo freezing14. For these patients, the classification criteria include AMH ≤ 1.0 ng/ml or a history of repeated

implantation failure or specific uterine conditions. Medroxyprogesterone acetate (MPA, 10 mg/day) was administered together with gonadotropins (150–300 IU/day), and follicular development

was monitored via ultrasound and blood tests. Ovulation was triggered when the dominant follicle reached ≥ 18 mm, and all embryos were cryopreserved for subsequent transfer cycles. The

minimal stimulation protocol was reserved for patients with diminished ovarian reserve or previous poor response to stimulation. The classification criteria include patients with AMH < 

1.0 ng/ml or a history of poor ovarian response to stimulation. This protocol is also indicated for patients with a high risk of ovarian hyperstimulation syndrome (OHSS), as it uses lower

doses of gonadotropins. Oral clomiphene citrate (50–100 mg/day) or letrozole (2.5–5 mg/day) was started on cycle days 2–3, often in combination with low-dose gonadotropins (HMG 75 IU/day).

Follicular growth was monitored every 2–3 days using ultrasound, and medications were adjusted accordingly. Ovulation was triggered when dominant follicles reached ≥ 18 mm, followed by egg

retrieval 34–36 h later. Luteal support was provided as described in the other protocols. ENDOCRINE HORMONE MEASUREMENTS Hormone levels (FSH, LH, E2, P4) were measured at multiple time

points: baseline levels during the early follicular phase (cycle day 2–3 for antagonist protocols) or after achieving downregulation (E2 < 50 pg/ml for GnRH agonist protocols), on the day

of HCG administration, and 12–14 days post-transfer to confirm pregnancy. These measurements were conducted using a chemiluminescent immunoassay (CLIA) on an Abbott i2000SR analyzer (Abbott

Park, Illinois, USA) in a certified clinical laboratory with intra- and inter-assay coefficients of variation maintained below 10%, ensuring reliable and accurate results. EXPOSURES The

exposure factor is defined as the ratio of trigger day E2 to baseline E2, calculated using the formula (trigger day E2-baseline E2)/baseline E2. Based on this ratio, patients were divided

into four groups: Low E2 response group (< 13.52), moderate response group (13.52–34.24), moderate-high response group (34.24–70.05), and high response group (> 70.05). As this is the

first study to evaluate relative changes in E2 levels during controlled ovarian stimulation, no established thresholds exist in the literature. Therefore, we adopted a quartile-based

approach to ensure objective and statistically robust groupings. Patients in the lowest quartile (< 13.52) were designated as the control group for reference. ENDOMETRIAL PREPARATION

PROTOCOL In all fresh transfer cycles, the endometrial preparation protocol was standardized. Endometrial thickness was monitored using transvaginal ultrasound, starting from the mid-to-late

follicular phase of controlled ovarian stimulation. The criterion for transfer eligibility was an endometrial thickness ≥ 7 mm on the day of ovulation trigger (HCG administration). Luteal

support was initiated on the day of oocyte retrieval and included vaginal progesterone (e.g., 200 mg twice daily) or intramuscular progesterone (e.g., 40 mg daily), which was continued until

10 weeks of gestation in patients with confirmed pregnancy. OUTCOMES The primary outcomes were blastocyst formation rate, clinical pregnancy rate, and live birth rate. The blastocyst

formation rate was defined as the ratio of successfully cultured blastocysts to either the number of follicle punctures or 2PN oocytes. The clinical pregnancy is defined as the presence of

an intrauterine gestational sac observed via ultrasound, or records of live birth or miscarriage. The live birth is defined as the birth of one or more live infants. Secondary outcomes

included oocyte retrieval rate, fertilization rate, miscarriage rate, and ectopic pregnancy rate. The number of follicle punctures refers to the total number of follicles attempted to be

aspirated for oocyte retrieval. The number of oocytes retrieved refers to the total number of oocytes obtained after each puncture. The number of mature oocytes refers to the number of

mature oocytes (MII stage). The number of 2PN oocytes refers to the number of normally fertilized oocytes with two pronuclei. The miscarriage rate is defined as the loss of an intrauterine

pregnancy before 20 weeks of gestation. The ectopic pregnancy rate refers to the proportion of pregnancies occurring outside the uterine cavity. STATISTICAL METHODS Given that the data did

not conform to a normal distribution, baseline and cycle characteristics were presented as medians with interquartile ranges (IQR). Continuous variables, including hormone levels, were

compared between groups using the Mann-Whitney U test. To control for the impact of covariates with statistically significant differences between groups, analysis of covariance (ANCOVA) was

employed. Chi-square tests were used to analyzed blastocyst formation and pregnancy outcomes between groups, and relative risk (RR) with 95% confidence intervals (Cl) was calculated using

log-binomial regression models. Both crude and adjusted analyses were performed to account for potential covariates that could influence pregnancy outcomes. Statistical significance was set

at 0.05, and all tests were two-tailed. All statistical analyses were conducted using SPSS 26.0 and the R statistical language in the RStudio. ETHICS APPROVAL This study was approved by the

Ethics Committee of Guangzhou Women and Children’s Medical Center Liuzhou Hospital (Approval No. 2024 − 211), and the committee waived the requirement for individual informed consent, as the

data were de-identified and this was a retrospective analysis in compliance with relevant ethical guidelines and regulations. RESULTS We analyzed a total of 9,376 fresh, first-time assisted

reproductive cycles. During COS, the majority of assisted reproductive cycles showed an increase in E2 levels compared to baseline (_n_ = 9,047, 96.5%). However, a minority of cycles

experienced a decrease in E2 levels following COS (_n_ = 329, 3.5%). In this study, patients were divided into four groups based on the ratio of (E2 level on the day of trigger - baseline E2

level)/baseline E2 level). The groups were classified as follows: low response (ratio < 13.25), moderate response (13.52 ≤ ratio < 34.24), moderately-high response (34.24 ≤ ratio <

 70.05), and high response (ratio ≥ 70.05), with 2,344 cases in each group. EMBRYOLOGICAL OUTCOMES Among these four response groups, there was a decreasing trend in patient age: 39.0 years

in the low response group, 37.0 years in the moderate response group, 35.0 years in the moderate-high response group, and 33.0 years in the high response group. The differences in age

between groups were statistically significant (_p_ < 0.05). Although the differences were small, the BMI in the low response group was statistically different from that in the

moderate-high response group and high response group (_p_ < 0.05). Additionally, baseline FSH levels and LH levels on the day of trigger were significantly higher in the low response

group compared to other groups, while P4 levels on the day of trigger were significantly lower than those in the higher response groups. Significant differences were also observed between

groups in terms of infertility diagnosis and ovarian stimulation protocols; pelvic factor infertility was more prevalent in patients with high E2 response, while diminished ovarian reserve

(DOR) was more common in patients with low E2 response (Table 1). Figure 2; Table 2 present the outcomes of 85,463 oocytes retrieved from 93,363 follicle punctures. There was a significant

weak positive correlation between the degree of E2 response and the ratio of oocytes, M2 oocytes, and 2PN oocytes retrieved per follicle puncture, and a moderate positive correlation with

the blastocyst formation rate (Spearman’s rho = 0.516, _p_ < 0.01). Pairwise comparisons among the four E2 response groups showed significant differences in blastocyst formation rate

between each group (_p_ < 0.001). After adjusting for covariates based on significant differences in baseline characteristics between groups, these significant differences in blastocyst

formation rate persisted (_p_ < 0.001). When simplifying the E2 response into two groups, the “low E2 response group” included patients with a ratio ≤ 34.24, and the “high E2 response

group” included those with a ratio > 34.24. Chi-square tests revealed a significant difference in the blastocyst formation rate per puncture between the two groups (0.2645 vs. 0.7875; _p_

 < 0.001). Univariable logistic regression demonstrated that the blastocyst formation rate was significantly higher in the moderate response group (OR = 3.65, 95% CI: 3.17–4.21, _p_ <

0.001), moderate-high response group (OR = 13.14, 95% CI: 11.38–15.19, _p_ < 0.001), and high response group (OR = 44.75, 95% CI: 37.72–53.08, _p_ < 0.001) compared to the low response

group (Supplementary Table 1). As illustrated in Fig. 2, this relationship is nearly linear, indicating that as E2 response increases, the blastocyst formation rate rises progressively.

Subsequently, variables found to be significant in the univariable logistic regression were included in a multivariable logistic regression model to control for potential confounders. The

results showed that the blastocyst formation rate remained significantly higher in the moderate response group (adjusted OR = 1.60, 95% CI: 1.04–2.47, _p_ = 0.033), moderate-high response

group (adjusted OR = 3.68, 95% CI: 2.38–5.68, _p_ < 0.001), and high response group (adjusted OR = 8.99, 95% CI: 5.75–14.08, _p_ < 0.001) compared to the low response group, suggesting

an independent association between E2 response levels and blastocyst formation rate (Supplementary Table 1). Supplementary Tables 2 and 3 display the embryological outcomes of oocytes

sourced from patients younger than 35 years or older, respectively. Supplementary Fig. 2 illustrates that the trend in blastocyst formation rate per follicle puncture is similar across these

two age groups. Supplementary Tables 4 and 5 show the embryological outcomes of oocytes sourced from patients with BMI below 25 and those with a BMI of 25 or higher, respectively.

Supplementary Fig. 3 indicates that trend in blastocyst formation rate follicle puncture is also similar across these two BMI groups. Regarding of age or BMI, the blastocyst formation rate

steadily increased with higher E2 response. PREGNANCY OUTCOMES The four E2 response groups were comparable in terms of age, hormone levels, and infertility diagnosis, with no differences in

infertility duration or fertilization method. A total of 4,456 fresh transfer cycles were confirmed, including (17.5%) in the low response group, 1,222 (27.4%) in the moderate response

group, 1,415 (31.8%) in the moderate-high response group, and 1,041 (23.4%) in the high response group (Table 3). When simplified into two E2 response groups (low and high response), a

chi-square test demonstrated a significant difference in clinical pregnancy rates between the two groups (0.401 vs. 0.575; _p_ < 0.001). As shown in Table 4, the clinical pregnancy rate

was significantly higher in the moderate-high response group and high response group compared to the low response group (54.5% and 61.5% vs. 35.5%; absolute differences of 19% and 26%,

respectively; adjusted relative risks [aRR] of 1.21 [95% CI: 1.03–1.42] and 1.27 [95% CI: 1.08–1.51], respectively). These differences remained significant after adjusting for age, baseline

FSH, LH and P4 levels on the day of hCG, infertility diagnosis, ovarian stimulation protocols, and day of transfer. Given the significant differences in embryo transfer type (Day 3 vs. Day

5/6) among E2 response groups, we conducted an interaction analysis to evaluate its potential modifying effect on clinical pregnancy outcomes. The results showed no significant interactions

(e.g., moderate-high response group: Day5, Estimate = −0.2261, _p_ = 0.278; high response group: Day5, Estimate = −0.2209, _p_ = 0.294), indicating that the associations between E2 response

and clinical pregnancy outcomes are consistent across embryo transfer types. There was no significant difference in clinical pregnancy rate between the moderate response group and the low

response group after controlling for confounders (aRR: 1.07 [95% CI: 0.91–1.25]). The live birth rate in the moderate-high response group and high response group was also significantly

higher than in the low response group (44.9% and 52.0% vs. 25.7%; absolute differences of 19.2% and 26.3%; aRRs of 1.27 [95% CI: 1.06–1.52] and 1.35 [95% CI: 1.11–1.64], respectively).

Although the live birth rate in the moderate response group was higher than in the low response group (34.5% vs. 25.7%; absolute difference of 8.8% [95% CI: −3.9–21.5%]; aRR of 1.11 [95% CI:

0.92–1.33]), the difference was not statistically significant. There were no significant differences in the miscarriage rate between the low response group and the moderate response group,

moderate-high response group, and high response group (10.8%, 12.1%, 12.7% vs. 12.7%; absolute differences of 1.9%, 0.6%, and 0%; aRRs of 0.85 [95% CI: 0.65–1.33], 1.04 [95% CI: 0.78–1.39],

and 1.21 [95% CI: 0.88–1.67], respectively). Similarly, there were no significant differences in ectopic pregnancy rate between the low response group and the other response groups.

DISCUSSION 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 understood15,16. 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 invasion24,25,26. It also influences immune cells like uterine natural killer (uNK) cells and decidual

macrophages, contributing to endometrial remodeling and creating a favorable implantation environment27,28,29. It also influences immune cells like uNK cells and decidual macrophages,

contributing to endometrial remodeling and creating a favorable implantation environment30,31. 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 pregnancies32,33. 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. CONCLUSION In this retrospective cohort study, the relative change in E2 response during COS was significantly

associated with blastocyst formation rate, clinical pregnancy rate, and live birth rate. Higher E2 responses were predictive of improved pregnancy outcomes. However, the interpretation of

these results is limited by the retrospective design and potential biases due to selection and residual confounding. Future prospective studies are needed to further validate the predictive

value of E2 response and to identify the optimal range of E2 levels for maximizing pregnancy success. DATA AVAILABILITY The data that support the findings of this study are not publicly

available due to privacy concerns regarding patient confidentiality but may be made available from the corresponding author upon reasonable request. REFERENCES * Khalaf, Y., Taylor, A. &

Braude, P. Low serum estradiol concentrations after five days of controlled ovarian hyperstimulation for in vitro fertilization are associated with poor outcome. _Fertil. Steril._ 74(1),

63–66 (2000). Article  CAS  PubMed  Google Scholar  * Siddhartha, N. et al. Correlation of serum estradiol level on the day of ovulation trigger with the reproductive outcome of

intracytoplasmic sperm injection. _J. Hum. Reprod. Sci._ 9(1), 23–27 (2016). Article  CAS  PubMed  PubMed Central  Google Scholar  * Farhi, J. et al. High serum oestradiol concentrations in

IVF cycles increase the risk of pregnancy complications related to abnormal placentation. _Reprod. Biomed. Online_. 21(3), 331–337 (2010). Article  CAS  PubMed  Google Scholar  * Imudia, A.

N. et al. Peak serum estradiol level during controlled ovarian hyperstimulation is associated with increased risk of small for gestational age and preeclampsia in Singleton pregnancies after

in vitro fertilization. _Fertil. Steril._ 97(6), 1374–1379 (2012). Article  CAS  PubMed  Google Scholar  * Liu, S. et al. High oestradiol concentration after ovarian stimulation is

associated with lower maternal serum beta-HCG concentration and neonatal birth weight. _Reprod. Biomed. Online_. 35(2), 189–196 (2017). Article  PubMed  Google Scholar  * Kyrou, D. et al.

Does the estradiol level on the day of human chorionic gonadotrophin administration have an impact on pregnancy rates in patients treated with rec-FSH/GnRH antagonist? _Hum. Reprod._ 24(11),

2902–2909 (2009). Article  CAS  PubMed  Google Scholar  * Palmerola, K. L., Rudick, B. J. & Lobo, R. A. Low estradiol responses in oocyte donors undergoing gonadotropin stimulation do

not influence clinical outcomes. _J. Assist. Reprod. Genet._ 35(9), 1675–1682 (2018). Article  PubMed  PubMed Central  Google Scholar  * Huang, J. et al. Association between peak serum

estradiol level during controlled ovarian stimulation and neonatal birthweight in freeze-all cycles: a retrospective study of 8501 Singleton live births. _Hum. Reprod._ 35(2), 424–433

(2020). Article  CAS  PubMed  Google Scholar  * Bose, R. & Mahadevan, M. M. Embryo associated immunosuppressor factor(s) secreted by preembryo and serum estradiol levels are predictive

of pregnancy outcome: effect of gonadotropin releasing hormone agonist (GnRHa) treatment of patients undergoing in vitro fertilization and embryo transfer (IVF-ET). _Immunol. Invest._ 21(1),

25–38 (1992). Article  CAS  PubMed  Google Scholar  * Rehman, R., Jawaid, S., Gul, H. & Khan, R. Impact of peak estradiol levels on reproductive outcome of intracytoplasmic sperm

injection. _Pak J. Med. Sci._ 30(5), 986–991 (2014). PubMed  PubMed Central  Google Scholar  * Kolibianakis, E. M. et al. Exposure to high levels of luteinizing hormone and estradiol in the

early follicular phase of gonadotropin-releasing hormone antagonist cycles is associated with a reduced chance of pregnancy. _Fertil. Steril._ 79(4), 873–880 (2003). Article  PubMed  Google

Scholar  * Kumbak, B., Oral, E., Karlikaya, G., Lacin, S. & Kahraman, S. Serum oestradiol and beta-HCG measurements after day 3 or 5 embryo transfers in interpreting pregnancy outcome.

_Reprod. Biomed. Online_. 13(4), 459–464 (2006). Article  CAS  PubMed  Google Scholar  * Kondapalli, L. A., Molinaro, T. A., Sammel, M. D. & Dokras, A. A decrease in serum estradiol

levels after human chorionic gonadotrophin administration predicts significantly lower clinical pregnancy and live birth rates in in vitro fertilization cycles. _Hum. Reprod._ 27(9),

2690–2697 (2012). Article  CAS  PubMed  PubMed Central  Google Scholar  * Ata, B. & Kalafat, E. Progestin-primed ovarian stimulation: for whom, when and how? _Reprod. Biomed. Online_.

48(2), 103639 (2024). Article  CAS  PubMed  Google Scholar  * Orzołek, I., Sobieraj, J., Domagała-Kulawik, J. & Estrogens Cancer and Immununity. _Cancers (Basel). _14(9), 2265 (2022). *

Toriola, A. T. et al. Determinants of maternal sex steroids during the first half of pregnancy. _Obstet. Gynecol._ 118(5), 1029–1036 (2011). Article  CAS  PubMed  PubMed Central  Google

Scholar  * Parisi, F. et al. The pathophysiological role of estrogens in the initial stages of pregnancy: molecular mechanisms and clinical implications for pregnancy outcome from the

periconceptional period to end of the first trimester. _Hum. Reprod. Update_. 29(6), 699–720 (2023). Article  CAS  PubMed  PubMed Central  Google Scholar  * Zavy, M. T. et al. In high

responding patients undergoing an initial IVF cycle, elevated estradiol on the day of hCG has no effect on live birth rate. _Reprod. Biol. Endocrinol._ 12, 119 (2014). Article  PubMed 

PubMed Central  Google Scholar  * Joo, B. S. et al. Serum estradiol levels during controlled ovarian hyperstimulation influence the pregnancy outcome of in vitro fertilization in a

concentration-dependent manner. _Fertil. Steril._ 93(2), 442–446 (2010). Article  CAS  PubMed  Google Scholar  * Wang, M. et al. Effect of elevated estradiol levels on the hCG administration

day on IVF pregnancy and birth outcomes in the long GnRH-agonist protocol: analysis of 3393 cycles. _Arch. Gynecol. Obstet._ 295(2), 407–414 (2017). Article  ADS  MathSciNet  CAS  PubMed 

Google Scholar  * Morales, H. S. G. et al. Serum estradiol level on the day of trigger as a predictor of number of metaphase II oocytes from IVF antagonist cycles and subsequent impact on

pregnancy rates. _JBRA Assist. Reprod._ 25(3), 447–452 (2021). PubMed  PubMed Central  Google Scholar  * Beker, A. R., Colenbrander, B. & Bevers, M. M. Effect of 17beta-estradiol on the

in vitro maturation of bovine oocytes. _Theriogenology_ 58(9), 1663–1673 (2002). Article  CAS  PubMed  Google Scholar  * Kim, J. S. et al. Supplementation with estradiol-17β improves Porcine

oocyte maturation and subsequent embryo development. _Fertil. Steril._ 95(8), 2582–2584 (2011). Article  CAS  PubMed  Google Scholar  * Critchley, H. O. D., Maybin, J. A., Armstrong, G. M.

& Williams, A. R. W. Physiology of the endometrium and regulation of menstruation. _Physiol. Rev._ 100(3), 1149–1179 (2020). Article  PubMed  Google Scholar  * Simón, C. et al.

Cytokines-adhesion molecules-invasive proteinases. The missing paracrine/autocrine link in embryonic implantation? _Mol. Hum. Reprod._ 2(6), 405–424 (1996). Article  PubMed  Google Scholar 

* Brayman, M., Thathiah, A. & Carson, D. D. MUC1: a multifunctional cell surface component of reproductive tissue epithelia. _Reprod. Biol. Endocrinol._ 2, 4 (2004). Article  PubMed 

PubMed Central  Google Scholar  * Lee, S. et al. Fluctuation of peripheral blood T, B, and NK cells during a menstrual cycle of normal healthy women. _J. Immunol._ 185(1), 756–762 (2010).

Article  CAS  PubMed  Google Scholar  * Wang, F., Qualls, A. E., Marques-Fernandez, L. & Colucci, F. Biology and pathology of the uterine microenvironment and its natural killer cells.

_Cell. Mol. Immunol._ 18(9), 2101–2113 (2021). Article  CAS  PubMed  PubMed Central  Google Scholar  * Tian, X., Eikmans, M. & Hoorn, M. V. The role of macrophages in oocyte donation

pregnancy: A systematic review. _Int. J. Mol. Sci._ 21(3), 939 (2020). * Ajayi, A. F. & Akhigbe, R. E. Staging of the estrous cycle and induction of estrus in experimental rodents: an

update. _Fertil. Res. Pract._ 6, 5 (2020). Article  PubMed  PubMed Central  Google Scholar  * Huhtinen, K. et al. Intra-tissue steroid profiling indicates differential progesterone and

testosterone metabolism in the endometrium and endometriosis lesions. _J. Clin. Endocrinol. Metab._ 99(11), E2188–E2197 (2014). Article  CAS  PubMed  Google Scholar  * Fritz, R., Jindal, S.,

Feil, H. & Buyuk, E. Elevated serum estradiol levels in artificial autologous frozen embryo transfer cycles negatively impact ongoing pregnancy and live birth rates. _J. Assist. Reprod.

Genet._ 34(12), 1633–1638 (2017). Article  PubMed  PubMed Central  Google Scholar  * Wu, Z. et al. Effect of HCG-day serum progesterone and oestradiol concentrations on pregnancy outcomes

in GnRH agonist cycles. _Reprod. Biomed. Online_. 24(5), 511–520 (2012). Article  CAS  PubMed  Google Scholar  * Chen, Y. C., Lai, Y. J., Su, Y. T., Tsai, N. C. & Lan, K. C. Higher

gestational weight gain and lower serum estradiol levels are associated with increased risk of preeclampsia after in vitro fertilization. _Pregnancy Hypertens._ 22, 126–131 (2020). Article 

PubMed  Google Scholar  * Mackens, S. et al. Impact of serum estradiol levels prior to progesterone administration in artificially prepared frozen embryo transfer cycles. _Front. Endocrinol.

(Lausanne)_. 11, 255 (2020). Article  PubMed  Google Scholar  Download references FUNDING This research was supported by the Guangxi Science and Technology Plan Project (GuiKe AD22035223),

Project of Liuzhou Science and Technology Bureau (Liu Ketong [2023] No. 40), the Self-funded by the Health Commission of Guangxi Zhuang autonomous Region (Z-B20221568), Optimization and

Application of Preimplantation Genetic Diagnosis Platform for Thalassemia in Guangxi Region (2023YRZ0103), the Open Project of NHC Key Laboratory of Thalassemia Medicine (GJWJWDP202206), and

Medical Youth Reserve Talent Training Program of the Health Commission of Guangxi Zhuang Autonomous Region (Gui Wei Ren Fa [2025] No. 5). The funders had no role in study design, data

collection and analysis, decision to publish, or preparation of the manuscript. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Department of Reproductive Medicine, Guangzhou Women and

Children’s Medical center Liuzhou Hospital, Liuzhou, Guangxi, China Wenjie Huang, Liuyan Wei, Juan Tang, Liuying Nong, Ni Tang, Jingjing Li & Li Fan * Liuzhou maternity and Child

Healthcare Hospital, Liuzhou, Guangxi, China Wenjie Huang, Qiuyue Wen, Zuxing Qin, Lixiang Xu, Jingjing Li & Li Fan * Guangxi Clinical Research Center for Obstetrics and Gynecology,

Liuzhou, Guangxi, China Jingjing Li & Li Fan * Liuzhou Key Laboratory of Gynecologic Tumor, Liuzhou, Guangxi, China Jingjing Li & Li Fan Authors * Wenjie Huang View author

publications You can also search for this author inPubMed Google Scholar * Liuyan Wei View author publications You can also search for this author inPubMed Google Scholar * Juan Tang View

author publications You can also search for this author inPubMed Google Scholar * Liuying Nong View author publications You can also search for this author inPubMed Google Scholar * Ni Tang

View author publications You can also search for this author inPubMed Google Scholar * Qiuyue Wen View author publications You can also search for this author inPubMed Google Scholar *

Zuxing Qin View author publications You can also search for this author inPubMed Google Scholar * Lixiang Xu View author publications You can also search for this author inPubMed Google

Scholar * Jingjing Li View author publications You can also search for this author inPubMed Google Scholar * Li Fan View author publications You can also search for this author inPubMed 

Google Scholar CONTRIBUTIONS Conceptualization, Wenjie Huang, Jingjing Li and Li Fan; Formal analysis, Wenjie Huang, Liuyan Wei, Juan Tang, Liuying Nong and Ni Tang; Funding acquisition,

Jingjing Li and Li Fan; Investigation, Qiuyue Wen and Lixiang Xu; Methodology, Liuyan Wei, Liuying Nong and Ni Tang; Supervision, Jingjing Li and Li Fan; Validation, Qiuyue Wen, Zuxing Qin

and Lixiang Xu; Writing – original draft, Wenjie Huang; Writing – review & editing, Jingjing Li and Li Fan. CORRESPONDING AUTHORS Correspondence to Wenjie Huang, Jingjing Li or Li Fan.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION PUBLISHER’S NOTE Springer Nature remains neutral with regard to jurisdictional

claims in published maps and institutional affiliations. ELECTRONIC SUPPLEMENTARY MATERIAL Below is the link to the electronic supplementary material. SUPPLEMENTARY MATERIAL 1 RIGHTS AND

PERMISSIONS OPEN ACCESS This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing,

distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and

indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third

party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the

article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright

holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/. Reprints and permissions ABOUT THIS ARTICLE CITE THIS ARTICLE Huang, W., Wei, L., Tang, J.

_et al._ Impact of relative estradiol changes during ovarian stimulation on blastocyst formation and live birth in assisted reproductive technology. _Sci Rep_ 15, 15617 (2025).

https://doi.org/10.1038/s41598-025-00200-5 Download citation * Received: 21 October 2024 * Accepted: 25 April 2025 * Published: 05 May 2025 * DOI: https://doi.org/10.1038/s41598-025-00200-5

SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article. Copy to

clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * IVF/ICSI outcomes * Estrogen * Controlled ovarian stimulation * Ovarian response * Pregnancy

outcomes