The impact of changes in LH levels in flexible antagonist protocols on the clinical outcomes of fresh IVF/ICSI cycles in populations of POSEIDON groups 1 and 2: a retrospective cohort study

Research subjects and grouping

Retrospective analysis was performed using clinical data of POSEIDON groups 1 and 2 treated with the flexible IVF/ICSI regimen of GnRH-A at the Reproductive Medicine Center of Fujian Maternal and Child Health Hospital from January 2017 to December 2022. The inclusion criteria were as follows: (1) POSEIDON group 1 population [age < 35 years, normal ovarian reserve function (AFC ≥ 5 and AMH ≥ 1.2 ng/mL)] with unexpected ovarian low response or suboptimal response (number of retrieved eggs ≤ 9) or POSEIDON group 2 population [age ≥ 35 years and normal ovarian reserve function (AFC ≥ 5 and AMH ≥ 1.2 ng/mL)] with unexpected ovarian low response or suboptimal response (number of retrieved eggs ≤ 9); (2) flexible COS using antagonists. Exclusion criteria: (1) polycystic ovary syndrome; (2) multiple uterine fibroids (or single fibroid ≥ 5 cm); (3) history of intrauterine adhesions; (4) ovarian tumors; (5) uterine adenomyosis; (6) endometriosis uterine malformation; and (7) chromosomal abnormalities in either spouse causing recurrent implantation failure (RIF) or recurrent miscarriage (RSA).

According to the value of LH change [(LH level on the trigger day − baseline LH)/baseline LH] × 100%, the study subjects were divided into five groups: groups A, B, C, D and E with the value of LH change ≥100%, ≥ 50% to <100%, ≥0% to <50%, ≥−50% to 0%, and ≥−100% to −50%, respectively.

This study was performed in accordance with the Declaration of Helsinki and was approved by the Medical Research Ethics Committee of Fujian Maternity and Child Health Hospital (Ethics approval number: 2024KY057). All patients are exempt from informed consent.

Flexible GnRH-A regimens

Starting from the days 2–5 of menstruation, Gn (Recombinant Human Follitropin Alfa Solution for Injection; Merck, Germany; Procon, Merck, USA or urofollitropin for injection (Zhuhai Lizhu Pharmaceutical Company, Zhuhai, Guangdong, China)) was administered with an initial dose of 100–600 IU after considering the patient’s age, body mass index (BMI), AMH, basal follicle stimulating hormone (FSH), and antral follicle count (AFC) levels. After Gn activation, serum FSH, LH, estradiol (E2), and progesterone (Prog) levels were checked every 3–5 days. Antagonist was added when the diameter of the dominant follicle reached 14 mm or approximately 15 mm or when the diameter of the dominant follicle was >12 mm and E2 > 300 ng/L (1 ng/L = 3.672 pmol/L). The dosage was adjusted till trigger day based on the follicle size and hormone levels. When the diameter of three dominant follicles was ≥17 mm, that of two dominant follicles was ≥18 mm, or that of one dominant follicle was ≥20 mm, trigger was induced by administering recombinant human chorionic gonadotropin (hCG) (Aize, Merck, Switzerland) 250 μg or hCG (Zhuhai Lizhu Group Lizhu Pharmaceutical Factory, Guangdong, China) 6,000–10,000 IU. Egg retrieval was performed after 36 h.

Laboratory indicators

Pronuclei were assessed 16–18 h post-insemination (IVF/ICSI), with 2PN indicating normal fertilization. Embryo culture was performed in a benchtop incubator (COOK Medical, Bloomington, IN, USA) under tightly controlled conditions. Cleavage-stage embryos were graded according to the Istanbul consensus, with Grade I and II embryos classified as high quality (Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology, 2011). Blastocyst evaluation was performed using the Gardner scoring system, where blastocysts scoring ≥3BB (including AA, AB, BA, and BB) were considered high quality (Gardner et al., 2000).

The laboratory indicators included number of retrieved eggs, rate of egg retrieval, number of fertilization (2PN), fertilization rate, number of cleavage, cleavage rate, number of excellent embryos, rate of excellent embryos, number of blastocysts, and rate of blastocyst formation. The relevant indicators were assessed via referring to the consensus of experts on quality control of clinical key indicators of assisted reproductive technology, which included

egg retrieval rate = (the number of eggs retrieved/number of follicles penetrated) × 100%

fertilization rate (2PN fertilization rate) = (number of double pronuclear fertilizations/number of retrieved eggs) × 100%

cleavage rate = (number of cleavage/number of double pronuclear fertilization) × 100%

excellent embryo rate = (number of excellent embryos/number of cleavage) × 100%

blastocyst formation rate = (number of blastocysts/number of blastocyst cultures) × 100%.

Clinical indicators

Clinical indicators included fresh cycle clinical pregnancy rate and live birth rate, which were calculated as follows:

clinical pregnancy rate (per fresh transplant cycle) = (number of clinical pregnancy cycles/number of fresh transplant cycles) × 100%

live birth rate (per fresh transfer cycle) = (number of live births/number of fresh embryo transfer cycles) × 100%.

Statistical analysis

SPSS23.0 was used for statistical analysis. Quantitative data that conform to normal distribution were expressed as mean ± standard deviation. Further, homogeneity of variance testing was performed. Non-normally distributed data were represented by median and interquartile intervals. The Kruskal-Wallis H-test (KW test) was used for intergroup comparisons, and Bonferroni method was used for pairwise comparisons between groups. Counting data were calculated using the Chi-square test. P < 0.05 was considered significant. ${\chi }^2$ test was used for counting data, which was expressed in terms of composition ratio.

Comparison of general information of patients in each group

In POSEIDON groups 1, no significant differences were observed in terms of the age, types of infertility, years of infertility, initial Gn dose, total Gn, and Prog level and endometrial thickness on the trigger day among the groups (all P values ≥ 0.05). The differences were significant among groups in terms of BMI, time to antagonist addition, Gn days, baseline LH, AMH, LH on the trigger day (all P values < 0.05; Table 1a).

In POSEIDON groups 2, no significant differences were observed in terms of the age, BMI, types of infertility, years of infertility, total Gn, Gn days, AMH, and Prog level and endometrial thickness on the trigger day among the groups (all P values ≥ 0.05). The differences were significant among groups in terms of time to antagonist addition, initial Gn dose, baseline LH, LH on the trigger day (all P values < 0.05; Table 1b).

Comparison of laboratory indicators and clinical outcomes among various groups

In POSEIDON groups 1, no significant difference (P ≥ 0.05) was observed among the groups in terms of egg retrieval rate, fertilization rate, cleavage rate, number of excellent embryos, excellent embryo rate, blastocyst number, and blastocyst formation rate. However, significant difference (P value < 0.05) was observed in terms of number of retrieved eggs, mature egg number, fertilization number (2PN number), and number of cleavage (P < 0.05; Table 2a). Further grouping comparison indicated that in terms of number of double pronuclear fertilizations (2PN), only group E patients were significantly higher than group A patients (P = 0.037; Fig. 1).

In POSEIDON groups 2, no significant difference (P ≥ 0.05) was observed among the groups in terms of egg retrieval rate, fertilization rate, cleavage rate, excellent embryo rate, and blastocyst formation rate. However, significant difference (P value < 0.05) was observed in terms of number of retrieved eggs, mature egg number, fertilization number (2PN number), number of cleavage, number of excellent embryos, and blastocyst number (P < 0.05; Table 2b). In terms of the number of eggs retrieved, group A exhibited significantly lower number than groups D and E (P = 0.010 and 0.0000, respectively; Fig. 2). In terms of the number of mature eggs, group A exhibited significantly lower number than groups D and G (P = 0.008 and 0.0000, respectively; Fig. 2), group B exhibited significantly lower number than group E (P = 0.033; Fig. 2) and group C exhibited significantly lower number than group E (P = 0.021; Fig. 2). In terms of fertilization number (2PN number), group A exhibited significantly lower number than groups D and E (P = 0.042 and 0.002, respectively; Fig. 2). In terms of cleavage count, group E exhibited significantly higher value than groups A and C (P = 0.005 and 0.037, respectively; Fig. 2). In terms of excellent embryos, group B exhibited significantly lower number than groups E (P = 0.038; Fig. 2).

No significant difference was observed in terms of clinical pregnancy rate and live birth rate among the groups (all P ≥ 0.05; Tables 3a and 3b).

Limitations

This study has several limitations. LH is secreted in a pulsatile manner, and a single measurement may not accurately reflect its dynamic fluctuations. Although we assessed the trend of LH changes using the formula [(LH level on the trigger day − baseline LH)/baseline LH] × 100%, the analysis still relies on discrete time-point measurements.

Additionally, most laboratory parameters were continuous variables, and some subgroups categorized by LH changes had relatively small sample sizes. The data distribution across groups did not fully meet the assumptions of normality, necessitating non-parametric statistical tests. While we mitigated potential confounding effects through methods such as age stratification, some bias in the conclusions may persist. Future studies should aim to expand the sample size and employ more robust methodologies to further investigate the optimal control of key nodes in the flexible antagonist protocol.