Association Between Progesterone Level on Trigger Day to Basal Progesterone Ratio and in vitro Fertilization-Embryo Transfer Outcomes in Antagonist Protocols

Introduction

The gonadotropin-releasing hormone antagonist (GnRH-ant) protocol is a commonly used protocol for ovulation induction in in vitro fertilization-embryo transfer (IVF-ET), offering several advantages such as shorter duration of ovulation induction, reduced total Gn usage, and a lower risk of ovarian hyperstimulation syndrome (OHSS).¹ However, GnRH-ant cycles have been associated with a higher likelihood of elevated progesterone levels on trigger day (P_trigger) compared to agonist protocols.² At present, the most widely used threshold to indicate elevated P_trigger is 1.5 ng/mL.^3–5 Some researchers demonstrated that a P_trigger level of 1.5 ng/mL or higher negatively affects endometrial receptivity, thereby diminishing the success rate of fresh embryo transfers, and they recommend replacing fresh embryo transfers with a full embryo freeze in such cases.^6,7

The P_trigger level is influenced by factors such as the basal progesterone (bP) levels, defined as progesterone levels measured on Days 2 or 3 of the menstrual cycle, multiple follicular development, and the total dose of Gn administered.^8,9 bP, which is primarily produced by the adrenal glands, varies between individuals. Adrenal gland-derived progesterone remains relatively stable throughout the follicular phase.¹⁰ Therefore, changes in P_trigger levels are inherently linked to bP levels. However, whether the extent of P_trigger elevation and IVF-ET outcomes are correlated remains unclear. If such a correlation exists, different participant profiles may have varying P_trigger thresholds.

The P_trigger/bP ratio reflects the extent of change in P_trigger levels relative to bP. Based on this, our aim in this study was to explore the relationship between the P_trigger/bP ratio and IVF-ET outcomes, with the goal of providing a reference for informing clinical decision-making.

Materials and Methods

Clinical Data

The clinical data of women who underwent IVF-ET assisted reproduction at the Reproductive Medicine Department of our hospital between January 2019 and December 2023 were retrospectively analyzed.

The inclusion criteria were as follows: (1) Women aged below 35 years with normal ovarian reserve function who were undergoing their first IVF-ET for assisted reproduction. (2) Women receiving the antagonist protocol for controlled ovarian hyperstimulation (COH). (3) Women with a P_trigger level < 1.5 ng/mL. (4) Women who received at least one high-quality embryo during the fresh embryo transfer.

The following were the exclusion criteria: (1) Women with ovarian diseases, a history of ovarian surgery, polycystic ovary syndrome, or diminished ovarian reserve. (2) Women with uterine abnormalities or malformations. (3) Women with a history of endocrine-related diseases, such as adrenal cortical hyperplasia, hypothyroidism, or hyperthyroidism. (4) Women with chromosomal abnormalities or a history of recurrent miscarriage.

All study participants signed the informed consent form. This study was approved by the Ethics Committee of the institution (No. 20190184).

Study Methods

Detection of Antral Follicle Count (AFC) and Sex Hormone Levels

On day 2 or 3 of the menstrual cycle, B-mode ultrasound was utilized to measure the size of antral follicles and antral follicle count (AFC) in the participants. On the same day, venous blood samples were collected to measure the levels of basal estradiol (bE₂), bP, basal follicle stimulating hormone (bFSH), and basal luteinizing hormone (bLH). All hormone measurements were performed using the chemiluminescence assay method.

Ovulation Induction Regimen and Embryo Transfer

A daily subcutaneous injection of recombinant follicle-stimulating hormone (Serono, Merck, Switzerland) at a dose of 125–300 U/day was initiated from day 3 of the menstrual cycle to stimulate ovulation. The dosage was adjusted as needed according to follicular development and serum levels of estradiol (E₂), luteinizing hormone (LH), and progesterone. The blood progesterone was measured on the ovulation trigger day. The hormone levels on the ovulation trigger day were measured using fasting blood samples collected at 8:00 AM on the trigger day. Starting on day 5 or 6 of ovulation induction, subcutaneous injection of an antagonist, cetrorelix acetate (Baxter Oncology GmbH, Germany), at 0.25 mg/day was added.

Trigger criteria: Ovulation was triggered when at least one follicle reached a diameter of ≥ 18 mm or at least three follicles measured ≥ 16 mm in diameter. On the trigger day, participants received a subcutaneous injection of 250 ug of recombinant human chorionic gonadotropin (hCG) (Serono, Merck, Switzerland). Oocyte retrieval was performed 36 to 37 hours post-trigger.

Three days after oocyte retrieval, the decision to proceed with fresh embryo transfer, freezing, or extended culture to the blastocyst stage was made based on the participants’ endometrial condition, embryo quality, and other clinical factors. Luteal phase support was provided to participants who underwent fresh embryo transfer.

Judgement of Pregnancy Outcome

A β-human chorionic gonadotropin (hCG) level > 10 U/L, measured 14 days post-embryo transfer, was defined as hCG-positive. For individuals with positive hCG results, a transvaginal ultrasound was performed between 28⁠ and 35 days post-embryo transfer to confirm a clinical pregnancy, indicated by the presence of a gestational sac within the uterine cavity.

Observation Indicators

Indicators compared between the groups in the study included general data such as age of the participants, bFSH, and anti-Müllerian hormone (AMH); indicators of ovulation induction status such as the estradiol (E₂) level on the trigger day, the total dose of Gn administered, and the number of oocytes retrieved; and the outcome of fresh embryo transfer. Embryo implantation rate = number of gestational sacs / total number of embryos transferred × 100%. Clinical pregnancy rate = number of clinical pregnancy cycles / number of transfer cycles × 100%.

Experimental Setting

Based on the P_trigger/bP ratio, the cycles were categorized into four groups: group A (P_trigger/bP < 1, n = 284), group B (P_trigger/bP ≥ 1 and < 2, n = 363), group C (P_trigger/bP ≥ 2 and < 3, n = 165), and group D (P_trigger/bP ≥ 3, n = 118). Participants were divided into a pregnant group and a non-pregnant group based on whether they achieved clinical pregnancy after embryo transfer. Each patient underwent a single transfer, and the number of embryos transferred ranged from one to two.

Statistical Analysis

Statistical analyses were performed using SPSS version 23.0 software. The normality of distribution for measurement data was assessed using the Kolmogorov–Smirnov test. Normally distributed measurement data were presented as the mean ± standard deviation and analyzed using independent-samples t-tests or one-way analysis of variance (ANOVA). Non-normally distributed measurement data were expressed as the median and interquartile range (M [P25, P75]) and analyzed using the Mann–Whitney U-test. Categorical data were reported as percentages (%) and compared using the chi-squared test or Fisher’s exact test.

Logistic regression analysis was employed to explore associations between various factors and clinical pregnancy following fresh embryo transfer. The predictive power of each influencing factor was evaluated using receiver operating characteristic (ROC) curve analysis, with an area under the curve (AUC) value > 0.5 indicating that the factor had diagnostic utility. A statistically significant difference was defined as a P value < 0.05 (P < 0.05).

Results

Comparison of Clinical Characteristics Between Pregnant and Non-Pregnant Women

A total of 930 assisted reproduction cycles were included in this study. Baseline characteristics: age: 30.49 ± 3.41 years (range: 20–34 years); duration of infertility: 4.55 ± 3.69 years (range: 1–16 years); bP level: 0.64 ± 0.47 ng/mL (range: 0.10–2.84 ng/mL); and P_trigger level: 0.76 ± 0.36 ng/mL (0.10–1.49 ng/mL). Compared to the non-pregnant group, women in the pregnant group were significantly younger, had a shorter duration of infertility, received a lower total Gn dose, had a higher AFC, and more oocytes were retrieved (P < 0.05). The two groups did not show any statistically significant differences in AMH, body mass index (BMI), or basal hormone levels (P > 0.05) (Table 1).

Table 1 Comparison of Clinical Characteristics of Women in the Pregnant and Non-Pregnant Groups

Multivariate Logistic Regression Analysis of Factors Linked to Clinical Pregnancy in IVF-ET Cycles

A multivariate logistic regression model was constructed using factors with statistically significant differences identified in the univariate analysis. These factors included age, duration of infertility, AFC, total Gn dose, and number of oocytes retrieved. The analysis showed that age was a significant factor associated with clinical pregnancy outcomes (P < 0.05) (Table 2).

Table 2 Logistic Regression Analysis of Factors Linked to Clinical Pregnancy

Comparison of Clinical Characteristics Between Groups with Varying P_trigger/bP Ratios

After categorizing participants based on their P_trigger/bP ratio, it was observed that bP levels followed a descending order across the groups: group A > group B > group C > group D. Conversely, P_trigger levels followed an ascending order: group A < group B < group C < group D. Groups A and B had significantly younger women, and their dose of total Gn was lower than those in groups C and D (P < 0.05). Additionally, E₂ levels on trigger day in groups A and B were significantly lower than those in groups C and group D (P < 0.05). The number of oocytes retrieved in groups A, B, and C was significantly lower than that in group D. The embryo implantation rate and clinical pregnancy rate among women in group A were significantly higher than those in group D (P < 0.05). No significant differences were found between Groups A, B, C, and D with respect to AMH, duration of infertility, or BMI (P > 0.05) (Table 3).

Table 3 Comparison of Clinical Characteristics Among Groups with Varying Ptrigger/bP Ratios

Comparison of Clinical Characteristics After Age Matching Between Groups A and D

Given the aforementioned significant differences between groups A and D with respect to pregnancy outcomes, as well as the observed difference in age, a 1:1 propensity score matching was performed between groups A and D to control for age-related confounding factors. Propensity score matching was conducted using R 4.1.1 software with a caliper value of 0.1 to analyze the relationship between P_trigger/bP and assisted reproduction outcomes.

The results showed that, prior to matching, women in group A were younger, received a lower total Gn dose, had lower trigger day E₂ levels, lower P_trigger levels, and fewer oocytes retrieved, compared to group D. Additionally, women in group A had higher bP levels, embryo implantation rates, and clinical pregnancy rates (P < 0.05). After matching, compared to group D, women in group A still had lower trigger day E₂ and P_trigger levels but higher bP levels (P < 0.05). The embryo implantation and clinical pregnancy rates were higher in group A than in group D, but the differences were not significant (P > 0.05).

Analysis of the Predictive Power of P_trigger/bP Ratio for Clinical Pregnancy After Fresh Embryo Transfer Using ROC Curve Analysis

An ROC curve was plotted to evaluate the diagnostic accuracy of the P_trigger/bP ratio for predicting clinical pregnancy after fresh embryo transfer. The analysis revealed that the AUC was 0.538, indicating limited predictive ability. The sensitivity and specificity were 0.850 and 0.270, respectively, with a 95% CI of 0.501⁠–0.575, (P = 0.044) (Figure 1).

Figure 1 ROC curve analysis of the predictive power of Ptrigger/bP ratio for clinical pregnancy after fresh embryo transfers.

Discussion

Most studies suggest that elevated P_trigger levels in antagonist protocols exert a negative impact on the success rate of fresh embryo transfer in IVF-ET.¹¹ The underlying mechanism proposed is that elevated P_trigger levels cause premature progression of the endometrium into the secretory phase, leading to a developmental asynchrony between the embryo and the endometrium. This disrupts the implantation process, thereby reducing clinical pregnancy rates and potentially lowering live birth rates.¹²

Various thresholds for elevated P_trigger levels have been reported in the literature, including 0.8 ng/mL, 1.4 ng/mL, and 1.5 ng/mL, among others,^11,13,14 with 1.5 ng/mL being the most frequently used. The discrepancies in threshold values reported across studies may relate to differences in basal progesterone levels, individual variations in progesterone secretion, heterogeneity in baseline characteristics of study populations, and differences in ovarian response to stimulation among women undergoing ovulation induction. Since bP levels vary among individuals, P_trigger levels change in accordance with bP. However, the relationship between the extent of P_trigger elevation and the outcome of IVF assisted reproduction remains poorly understood.

In this study, women with P_trigger values uniformly below 1.5 ng/mL were included to evaluate the success rate of fresh embryo transfer. The P_trigger/bP ratio was used to reflect changes in P_trigger relative to bP. Participants were divided into four groups based on the P_trigger/bP ratios of <1, ≥1 to ≤2, ≥2 to ≤3, and ≥3, respectively. Differences in assisted reproduction outcomes among different groups were analyzed. Our analysis revealed that with increases in the P_trigger/bP ratio, bP levels progressively decreased (group A > group B > group C > group D), while P_trigger levels simultaneously rose (group A < group B < group C < group D). Women with P_trigger/bP < 2 (groups A and B) were significantly younger, required less total Gn, and had lower trigger day E₂ levels compared to those with P_trigger/bP ≥ 2 (groups C and D). Furthermore, women with P_trigger/bP < 3 (groups A, B, and C) had significantly fewer oocytes retrieved compared to those with P_trigger/bP ≥ 3 (group D).

During controlled ovarian hyperstimulation (COH), large doses of exogenous gonadotropins promote the simultaneous development of multiple follicles. Granulosa cells within these follicles synthesize significantly higher amounts of progesterone compared to natural cycles,^15,16 leading to elevated progesterone levels, including P_trigger, in the late follicular phase.¹⁷ In this study, we found that, with variations in the P_trigger/bP ratio, significant inter-group differences in P_trigger levels were observed. Notably, the groups with a P_trigger/bP ratio of ≥ 2, especially those reaching a ratio of 3, showed a consistent trend of significantly higher levels of total Gn doses, E² levels on trigger day, and more retrieved oocytes than other groups.

We also found that an increase in the P_trigger/bP ratio was associated with a gradual decline in both the implantation rates (34.93%, 32.46%, 28.78%, and 23.19% in each group, respectively) and the clinical pregnancy rates (49.30%, 44.08%, 43.64%, and 33.90% in each group, respectively). Group D, where the P_trigger/bP ratio was ≥ 3, had significantly lower embryo implantation and clinical pregnancy rates than Group A, where the P_trigger/bP ratio was < 1. This result indicates a potential link between lower P_trigger/bP ratios and better outcomes in fresh embryo transfer (IVF-ET). Women with a P_trigger/bP ratio < 3, especially those with a P_trigger/bP ratio < 1, achieved favorable outcomes, while those with a P_trigger/bP ratio ≥ 3 tended to have poorer post-transfer outcomes. However, our analysis also highlighted the presence of various confounding factors, such as age and total Gn dosage, among other factors across the groups. Therefore, to isolate the effect of the Ptrigger/bP ratio on embryo transfer outcomes, the influence of these other variables needs to be considered and controlled.

It is well established that the outcome of fresh embryo transfers is related to several factors. In this study, participants were categorized into pregnant and non-pregnant groups to investigate factors related to clinical pregnancy. The results revealed that, compared to the non-pregnant group, women in the pregnant group were significantly younger, had a shorter duration of infertility, required lower total Gn, and had significantly higher AFC and more retrieved oocytes. Subsequent logistic regression analysis confirmed that age was a significant predictor of clinical pregnancy among women undergoing IVF. This finding is consistent with published reports.^18–20

Propensity score matching was utilized to control for the confounding effect of age between groups A and D. After matching, the results showed that women in group A (P_trigger/bP < 1) exhibited lower trigger day E₂ levels, lower P_trigger, and higher bP levels compared to those in group D (P_trigger/bP ≥ 3). Women in group A demonstrated higher embryo implantation and clinical pregnancy rates compared to those in group D (P_trigger/bP ≥ 3), but these differences were not statistically different (Table 4). Furthermore, ROC curve analysis showed that the AUC for the predictive power of P_trigger/bP ratio for clinical pregnancy after fresh embryo transfer was 0.538, indicating limited diagnostic utility. These findings suggest that a P_trigger/bP ratio < 1 may be associated with improved assisted reproduction outcomes, but the ratio’s overall ability to predict clinical pregnancy success in fresh embryo transfer cycles, particularly under an antagonist protocol, appears limited.

Table 4 Comparison of Clinical Characteristics After Age Matching Between Groups A and D

Conclusion

In conclusion, in this study among women with normal ovarian reserve undergoing ovulation induction with an antagonist protocol, those with a P_trigger/bP ratio < 1 tended to have better assisted reproductive outcomes. However, the predictive accuracy of the P_trigger/bP ratio for clinical pregnancy following fresh embryo transfer was found to be relatively low, highlighting the need for further investigation. This study has several limitations. 1) As a retrospective analysis, the results may be subject to inherent bias. 2) Study participants were not stratified based on bP levels, and this may not fully reflect the impact of P_trigger/bP on fresh embryo transfer outcomes. Future research should involve larger sample sizes and continued observation to further clarify the relationship between the P_trigger/bP ratio and IVF-ET outcomes in antagonist protocols.