Elevated estradiol levels on hCG trigger day adversely effects on the clinical pregnancy rates of blastocyst embryo transfer but not cleavage-stage embryo transfer in fresh cycles: a retrospective cohort study

Patient selection

This single-center, retrospective cohort study was performed at the Reproductive Medicine Center of the Third Affiliated Hospital of Sun-Yat Sen University. This study protocol was approved by the Institutional Ethics Committee of the Third Affiliated hospital of Sun Yat-Sen university. All participants had signed the informed consent (Institutional Review Board approval: II 2023-051-01). Patients who underwent fresh ET cycles from October 2015 to September 2021 were included. Figure 1 summarizes the flow chart of this study. A total of 9,841 fresh cycles were initially included. Cases were excluded if they met the following criteria: (i) cycles were canceled due to no oocyte retrieval or no ET for the physical condition; (ii) no top-level embryos were produced (based on embryo stage on the day of transfer, embryos were scored by their morphologic manifestation under the light microscope) (Alpha Scientists in Reproductive Medicine & ESHRE Special Interest Group of Embryology, 2011; Gardner, Lane & Schoolcraft, 2000) ; (iii) core data was missing (e.g., pregnancy record or E₂ level on the hCG trigger day); (iv) endometrial or uterine factors impacted pregnancy, including endometrial polyps, fibroid uterus, and uterine effusion; (v) patients experienced early-onset severe ovarian hyperstimulation syndrome (OHSS) during a fresh ET cycle (the severity grading of OHSS was based on clinical symptoms and laboratory indicators (Nelson, 2017). (vi) patients lost to follow-up. After screening, a total of 3,009 cycles were available for this study. According to the embryo stage at transfer day, all fresh ET cycles were then classified into the cleavage group (transfer on day 3) and the blastocyst group (transfer on day 5).

Controlled stimulation and embryo transfer procedures

Patients received one of two COS protocols: the gonadotropin releasing hormone agonist (GnRH-a) long protocol or the gonadotropin releasing hormone antagonist (GnRH-ant) protocol. The appropriate protocol was selected by the clinician for each patient on the basis of individual characteristics.

The GnRH-a long protocol included long-acting GnRH-a or short-acting GnRH-a, which were different in injection dosage during mid-luteal phases. Patients received one dose of 1.0 mg triptorelin (Gonapeptyl; Ferring, France) in the long-acting GnRH-a protocol. As for short-acting GnRH-a protocol, patients alternatively received a daily injection of 0.1 mg of triptorelin. Pituitary-ovarian suppression was established based on serum LH <5 mIU/ml and E₂ <50 pg/ml. Next, a daily gonadotropin dose of 100-300 IU was used for ovarian stimulation and the amount was adjusted according to the ovarian response until the day of hCG administration.

For the GnRH-ant protocol, ovarian stimulation commenced with daily 75-300 IU gonadotropin from days 2 to 3 of menstruation. The clinician assessed and adjusted the dosage of gonadotropin according to ultrasound readings and serum E₂ levels. A daily dose of 0.25 mg cetrorelix (Cetrotide; Serono, Geneva, Switzerland) was given when the dominant follicle reached 14 mm and lasted until the day of hCG administration.

When at least two follicles ≥ 18 mm diameter were identified, a dose of 4,000 to 10,000 IU hCG (Lizhu, Guangdong, China) was used to trigger the follicle maturation. Oocyte retrieval was scheduled at 34–36 h after the injection of hCG under the guidance of a vaginal ultrasound. After retrieval, oocytes were inseminated using the standard fertilization method, in-vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), depending on the sperm’s quality. The male partner in this study had to be present for semen analysis on at least two separate visits. Normally, semen samples were derived from ejaculated sperm. If the male partner had non-obstructive azoospermia, semen was acquired using testicular or percutaneous epididymal sperm aspiration methods. ICSI was performed if any of the following indications existed: severe oligospermia (sperm density <5 × 10⁶ ml in two out of three semen analysis); severe weak spermatozoa (progressive motility ratio <10% in two out of three semen analysis); severe teratospermia (according to the sperm morphological criteria, the ratio of normal morphological sperm was <1% in two out of three semen analysis) (Esteves, 2022; Gatimel et al., 2017).

The embryos were incubated at 37 °C in a humid gas phase with a 4% oxygen, 6% carbon dioxide, and 90% nitrogen mixture. Normal fertilization is defined as the appearance of two pronuclei and two polar bodies within 16–18 h of insemination (the fertilization rate of embryo in these cycles ranged from 55.6% to 90%). Embryos scoring standard referred to the Society for Assisted Reproductive Technology (SART) scoring system (Racowsky et al., 2011). Cleavage-stage embryo was graded according to the number of blastomeres, the percentage of fragments, and the size of the blastomeres. A good quality cleavage-stage embryo was described as having a blastomere with seven to nine cells of an A or B grade. The blastocyst scoring included the stage of blastocyst expansion, the density and number of cells in the inner cell mass, the regularity as well as the cohesion of the trophectoderm. A blastocyst embryo above 3BB on day 5 was defined as being of good quality.

Fresh ET was planned based on a receptive endometrium state (acceptable morphology; endometrial thickness ≥ 8 mm). Cleavage-stage embryos were transferred on day 3 and blastocysts were transferred on day 5 after ovum pick-up. The luteal phase was supported by daily vaginal or intramuscular progesterone after fresh ET. If the pregnancy was successful, this administration continued until ten weeks of gestation.

Data collection and outcome measurement

The baseline demographic and clinical characteristics of the study subjects were acquired from our database. Basal sexual hormone levels involved follicle stimulating hormone (FSH, mIU/ml), luteinizing hormone (LH, mIU/ml), and estradiol (E₂, pg/ml), detected on the first to third day of menstruation. COS indicators comprised the ovarian stimulation protocol, sexual hormone levels on the hCG trigger day, and the number of retrieved oocytes. Endometrial thickness (mm) on the hCG trigger day was measured by ultrasonography (Simeonov et al., 2020). The number of inviable embryos and embryos transferred was also noted.

CPR per fresh ET cycle was the primary measurement of the study. Clinical pregnancy was confirmed by ultrasound to detect gestational sacs. All pregnant cases were followed up to acquire the delivery data. The secondary outcomes included miscarriage rate (spontaneous loss of intrauterine pregnancy before 28 gestational weeks), ectopic pregnancy rate (gestational sac outside of uterine confirmed by ultrasound), live birth rate (the delivery of one or more viable infant), singleton live birth rate, preterm birth rate (delivery before 37 weeks), fetal weight, and the rates of LBW (defined as neonatal weight <2,500 g), and SGA (defines as an infant with a birthweight <10th percentile for its gestational age).

Statistical analysis

Patients of both embryo stage groups were categorized into four sets according to the serum E₂ on the hCG day: E₂ ≤ 2,000 pg/ml (set 1), E₂ = 2,001–3,000 pg/ml (set 2), E₂ = 3,001–4,000 pg/ml (set 3), E₂ > 4,000 pg/ml (set 4). The patients of set 1 (E₂ < 2,000 pg/ml) were taken as reference. Continuous variables were presented as means ± standard deviation (normally distributed variables) or median with quartile (non-normally distributed variables), and differences among different sets were compared by one-way analysis of variance or Kruskal–Wallis test, respectively. Categorical variables were presented as a number with percentage, and difference were compared by chi-square test.

Binary logistics analysis was established to assess the association between serum E₂ on the hCG trigger day and CPR. Firstly, univariate analysis was applied to screened the potential risk factors for CPR. Variables were treated as confounders in the next multivariate regression if they were significantly associated with CPR in univariate analysis, or they changed the matched odds ratios (ORs) by more than 10 percent. The results of the previous study and clinical experience were also considered (Brodin et al., 2009; Kahyaoglu et al., 2015; Sermondade et al., 2019; Wang et al., 2022a; Wang et al., 2022b). The following were selected as confounding factors: age, BMI, infertility duration, fertilization method, sterility classification, previous cycle attempts, basal FSH/LH/E₂ levels, FSH/LH/progesterone (P) levels on hCG trigger day, endometrial thickness on hCG trigger day, number of retrieved oocytes, the number of inviable embryos, and the number of embryos transferred. We assessed the non-linear relationship between serum E₂ and CPR based on the spline smoothing plots. The threshold effect of serum E₂ on CPR were evaluated using a two-piecewise linear regression and the inflection point was defined using a recurrence method. Crude and adjusted odds ratio (OR) with 95% confidence intervals (CIs) were calculated to present the results. P < 0.05 was considered to be statistically significant. Statistical analyses were performed using EmpowerStats software (X&Y Solution, Inc., Boston, MA, USA; https://www.empowerstats.net/en/).

Baseline characteristics

A total of 2,217 and 792 fresh cycles were included in cleavage group and blastocyst group, respectively. Table 1 shows a comparison of the clinical characteristics among the different sets of the cleavage group. The patients with E₂ ≤ 2,000 pg/ml on the trigger day were older on average (34.5 ± 5.6 years), had a higher BMI (21.64, Q1-Q3 = 20.03–23.73, kg/m2), had made more previous ET attempts (≥ once, 27.73%) as well as a higher proportion in secondary infertility (61.75%), compared to patients in other sets. As for ovarian stimulation, patients with lower E₂ levels showed a great chance of using the GnRH-ant protocol for the pituitary down regulation. In addition, patients with elevated E₂ levels on hCG trigger day exhibited a higher basal LH level, higher LH and P levels on hCG trigger day, a thicker endometrium, more retrieved oocytes and more transferred embryos. Table 2 lists the characteristics among different sets in the blastocyst group. The differences were statistically significant in age, BMI, basal LH level, LH and P levels on hCG trigger day, the number of retrieved oocytes, and the trends were the same as those in the cleavage group.

Descriptive analysis of clinical outcomes

The pregnancy and perinatal outcomes of both groups according to different E₂ levels are presented in Table 3. We observed the increasing trends in CPR (P < 0.001), live birth rate (P = 0.002), and singleton live birth rate (P = 0.035) from set 1 to set 4 of patients in the cleavage group. For the blastocyst group, CPR expressed a decreasing trend (P = 0.016), and patients in set 4 had a significantly lower CPR compared to those in set 1 (38.1% vs. 56.2%, P = 0.007). Although the rates of preterm birth and LBW showed an increasing trend in both embryo stage groups, the differences among E₂ level sets of the two groups were not statistically significant.

Binary logistics analysis of clinical pregnancy rate

The univariate analyses in both groups are listed in Table 4. In the cleavage group, endometrial thickness on trigger day, retrieved oocytes, inviable embryos, and transferred embryos were each positively correlated with CPR. While older age, more previous cycle attempts, longer infertility duration, higher basal FSH, and FSH on hCG trigger day were significantly associated with decreased CPR (all P < 0.05). As for the blastocyst group, the number of embryos transferred was positively associated with CPR, and covariates in terms of age, FSH and P on hCG trigger day were negatively correlated with CPR (all P < 0.05). E₂ levels on hCG trigger day showed different effects for the outcomes in both embryo stage groups. Compared to set 1 (E₂ ≤ 2,000 pg/ml), higher E₂ levels in the cleavage group appeared to have a positive relationship with CPR, with the crude ORs of 1.54 (95% CI [1.27−1.29], P < 0.001), 1.64 (95% CI [1.28−2.10], P < 0.001) and 1.23 (95% CI [0.83−1.84], P = 0.303) from set 2 to set 4. As opposed to the cleavage group, higher E₂ in the blastocyst group was a negative factor for CPR, with crude ORs of 0.75 (95% CI [0.52−1.18], P = 0.124), 0.97 (95% CI [0.66−1.43], P = 0.869), 0.52 (95% CI [0.31−0.08], P = 0.004) from set 2 to set 4.

Table 5 lists the multivariate regression analysis results of both groups after adjusting for the confounding covariates. The constructed model I adjusted for demographic factors (including age and BMI), and model II adjusted for all covariates. In the cleavage group, the adjusted ORs of model I and model II for CPR with patients in set 4 (E₂ >4,000 pg/ml) were 0.98 and 1.59, respectively, compared to those in the reference set 1 (E₂ ≤ 2,000 pg/ml). The statistical difference was not significant (P = 0.919 in model I, P = 0.294 in model II). The E₂ level on hCG trigger day in set 4 of the blastocyte group was an independent risk factor for CPR, with an adjusted OR of 0.50 (95% CI [0.28−0.90], P = 0.021) in model I and 0.43 (95% CI [0.24−0.97], P = 0.039) in model II.

Non-linear relationship between serum E₂ and clinical pregnancy rates

After adjusting for all demographic and clinical variables, we found a nonlinear relationship between the E₂ levels on trigger day and the CPR for the two groups using spline smoothing plots (Fig. 2). The threshold effects of serum E₂ on CPR were analyzed using the two-piecewise linear regression analysis (Table 6). Inflection points for the cleavage and blastocyst groups were calculated as 31.1 pg/dl and 39.7 pg/dl (1 pg/dl =100 pg/ml), respectively. In the cleavage group, the CPR was positively associated with serum E₂ when the serum E₂ was under 31.1 pg/dl (adjusted OR = 1.04, 95% CI [1.01−1.07], P = 0.022); however, when the E₂ levels were beyond 31.1 pg/dl, the correlation between E₂ and CPR was not statistical significantly (adjusted OR = 0.98, 95% CI [0.96−1.01], P = 0.297). In the blastocyst group, the CPR did not change significantly when E₂ levels were lower than 39.7 pg/dl (adjusted OR = 0.99, 95% CI [0.97−1.02], P = 0.846). However, when the E₂ levels were higher than 39.7 pg/dl, the CPR decreased by 17% with every 1 pg/dl increase in E₂ (adjusted OR = 0.83, 95% CI [0.72–0.96], P = 0.012).