Effect of adding gonadotropin-releasing hormone agonists for luteal support in antagonist cycles on pregnancy outcomes.

This retrospective study analyzed the medical records of 3,115 patients who underwent fresh embryo transfer during IVF-ET at the Reproductive Hospital Affiliated to Shandong University between February 2022 and December 2023. Among these patients, 262 voluntarily received GnRHa for luteal phase support after embryo transfer (GnRHa group), while the remaining 2,863 patients did not receive GnRHa supplementation (control group). Comparative analyses were performed between the two groups. Subsequently, 1:1 PSM was conducted, which yielded 236 matched pairs for further comparative analysis. The inclusion criteria were patients who underwent COH using the antagonist protocol either on (1) D3 after oocyte retrieval, with at least two transferrable high-quality embryos were available; or (2) D5, with least one transferrable high-quality blastocyst was available. The exclusion criteria were: (1) patients with a prior diagnosis of adenomyosis or severe endometriosis; (2) patients with untreated intrauterine adhesions, endometrial polyps, or uterine abnormalities; or (3) patients with untreated hydrosalpinx that was previously confirmed with imaging. The study received approval and was carried out in accordance with the guidelines of the Institute of Women, Children and Reproductive Health, Shandong University Ethics Board. Written informed consent was obtained from the patients. The antagonist protocol was employed for ovarian stimulation. On days 2–4 of the menstrual cycle, ovarian stimulation was initiated with 100–300 IU of gonadotropins based on the body weight and ovarian reserve of the patient. The gonadotropin dosage and the timing of antagonist administration were adjusted based on follicular growth and hormone levels. When 2–3 follicles in both ovaries reached an average diameter of ≥ 20 mm, the patients were received intramuscular injection of either 6,000 IU of hCG alone or 6000 IU of hCG in addition to a 0.2 mg subcutaneous injection of GnRHa. Oocyte retrieval was performed 36 h after the trigger injection. On D3, cleavage-stage embryos were evaluated using the embryo scoring system proposed by Puissant et al. 25 . High-quality embryos were defined as those derived from normally fertilized zygotes that met the criteria of 7–10 blastomeres on D3, absence of multinucleation, and an embryonic score of 3–4 25 . Alternatively, D5 blastocysts were graded based on the blastocyst scoring system proposed by Gardner et al. 26 , 27 . At our institution, only high-quality D3 embryos or blastocysts with a score of ≥ 4BC were selected for transfer. Patients with two high-quality D3 embryos or one D5 blastocyst who met the physical conditions for transfer after oocyte retrieval were allowed to choose which to transfer. Luteal support began on the day of oocyte retrieval for both the GnRHa and control groups. The control group received Duphaston (10 mg/tablet, Abbott Biologicals B.V., Netherlands) 20 mg oral twice daily and Utrogestan (100 mg/tablet, Cyndea Pharma, Spain) 200 mg vaginal once daily. The GnRHa group followed the same regimen, with the addition of triptorelin (1 ml: 0.1 mg, FERRING GmbH, Switzerland) administered subcutaneously at 0.1 mg every other day starting on the day of embryo transfer for a total of four injections. Serum β-hCG levels were assessed 12–14 days post-transfer; if pregnancy was confirmed, luteal support continued until 8–10 weeks post-transfer. The primary outcome was the clinical pregnancy rate. The secondary outcomes were the biochemical pregnancy rate, embryo implantation rate, early miscarriage rate, ectopic pregnancy rate, live birth rate, and preterm birth rate. Data analysis was conducted using IBM SPSS Statistics (version 26.0; IBM Corp.). Continuous variables are expressed as mean ± standard deviation ( \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\:\stackrel{-}{}$$\end{document} x̄±s) and were compared using a t test. Categorical variables are expressed as frequencies (n). For the pairwise comparison of categorical variables, when all expected frequencies in the contingency table were > 5 and n  > 40, the χ² test was used. Conversely, when n  > 40 and expected frequencies were from 1 to 5, the continuity correction χ² test was applied. Lastly, when the expected frequencies were < 1 or n  < 40, Fisher’s exact test was conducted. Additionally, further stratified analyses were conducted based on the embryo type (D3 embryos or D5 blastocysts) and progesterone levels on the day of hCG administration (≥ 1.0 ng/ml or < 1.0 ng/ml). The level of statistical significance was set at p  < 0.05. Multivariate logistic regression models were adjusted for AMH level, progesterone level on the day of hCG administration, and the number of transferred embryos. These analyses were subsequently used to assess the association between luteal-phase GnRHa supplementation and pregnancy outcomes. Results are presented as aORs with 95% CI. To address potential confounding variables, we performed PSM. Cases were matched 1:1 to controls via optimal matching with a caliper of 0.2. Matching variables comprised AMH level, progesterone level on the day of hCG administration, and the number of transferred embryos. Primary outcomes were compared between matched groups using χ² tests. Although key confounders (age, endometrial thickness on the day of hCG administration, progesterone level on the day of hCG administration, and the number of transferred embryos) were balanced after matching (all p  > 0.05), we conducted sensitivity analyses with logistic regression adjusting for these variables to confirm robustness. The p -values were adjusted for multiple testing using the Benjamini-Hochberg false discovery rate method (k = 6). Sensitivity analyses using the Bonferroni correction (α = 0.0083) were also performed.

The group who received GnRHa supplementation exhibited significantly lower anti-müllerian hormone (AMH) levels ( p  = 0.018) than the control group but had significantly higher progesterone levels ( p  = 0.001) and greater numbers of embryos transferred ( p  = 0.002) on the day of hCG administration. No statistically significant differences were observed in other baseline characteristics between the two groups (Table  1 ). Baseline characteristics were well-balanced after 1:1 propensity score matching (PSM) (Table  2 ). Table 1 Comparison of baseline characteristics between GnRHa and control groups. GnRHa group ( n  = 262) Control group ( n  = 2853) p Age (years) 32.80 ± 4.52 32.80 ± 4.75 0.967 BMI (kg/m 2 ) 24.07 ± 3.55 24.09 ± 3.73 0.949 Duration of infertility (years) 3.87 ± 3.11 4.0 ± 3.06 0.393 Baseline endocrine levels FSH (IU/L) 7.54 ± 2.69 7.33 ± 2.89 0.129 LH (IU/L) 5.57 ± 3.37 5.75 ± 3.54 0.497 E2 (pg/ml) 36.15 ± 14.31 35.00 ± 14.14 0.290 T (ng/ml) 25.92 ± 16.66 25.54 ± 15.21 0.689 TSH (uIU/ml) 2.20 ± 1.23 2.26 ± 1.14 0.105 AMH (ng/ml) 3.18 ± 2.86 3.61 ± 3.05 0.018 Antral follicle count (n) 13.7 ± 7.65 14.5 ± 8.52 0.191 Type of infertility [n (%)] Primary 129/262(49.24) 1289/2853(45.18) 0.218 Secondary 133/262(50.8) 1564/2853(54.8) Duration of Gn administration (days) 9.44 ± 2.13 9.41 ± 2.10 0.888 Total Gn dosage (IU) 2094.37 ± 795.69 2077.65 ± 883.77 0.277 Cause of infertility [n (%)] Tubal Factor 178/262 (67.94) 1851/2853 (64.88) 0.494 Ovulation Disorder 7/262 (2.67) 56/2853 (1.96) Endometriosis 6/262 (2.30) 37/2853 (1.30) Male Factors 33/262 (12.60) 394/2853 (13.81) Other Factors 38/262 (14.50) 515/2853 (18.05) Number of retrieved oocytes (n) 8.58 ± 3.87 8.64 ± 4.28 0.899 Fertilization method [n (%)] IVF 105/262 (40.08) 1142/2853 (40.03) 1.000 ICSI 157/262 (59.92) 1711/2853 (59.97) Hormone levels on the day of hCG administration E2 (pg/ml) 1943.88 ± 855.35 2014.79 ± 1007.90 0.474 LH (IU/L) 3.48 ± 2.19 3.18 ± 2.86 0.065 P (ng/ml) 0.65 ± 0.36 0.58 ± 0.41 0.001 Endometrial thickness on the day of hCG administration (cm) 1.05 ± 0.19 1.04 ± 0.24 0.560 Number of high-quality embryos (n) 3.60 ± 2.41 3.51 ± 2.50 0.313 Number of transferred embryos (n) 1.78 ± 0.42 1.69 ± 0.47 0.002 Number of frozen embryos (n) 2.06 ± 2.15 2.38 ± 2.32 0.053 Embryo source [n (%)] D3 Embryos 220/262 (83.97) 2251/2853 (78.90) 0.056 D5 Blastocysts 42/262 (16.03) 602/2853 (21.10) GnRHa, gonadotropin-releasing hormone agonist; BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; T, testosterone; TSH, thyroid-stimulating hormone; AMH, anti-Müllerian hormone; Gn, gonadotropins; hCG, human chorionic gonadotropin; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; P, progesterone; D3, day 3; D5, day 5. Comparison of baseline characteristics between GnRHa and control groups. GnRHa, gonadotropin-releasing hormone agonist; BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; T, testosterone; TSH, thyroid-stimulating hormone; AMH, anti-Müllerian hormone; Gn, gonadotropins; hCG, human chorionic gonadotropin; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; P, progesterone; D3, day 3; D5, day 5. Table 2 Comparison of baseline characteristics between GnRHa and control groups after propensity score matching. GnRHa group ( n  = 236) Control group ( n  = 236) p Age (years) 32.69 ± 4.29 32.60 ± 4.73 0.254 BMI (kg/m 2 ) 24.17 ± 3.57 24.10 ± 3.50 0.783 Duration of infertility (years) 3.86 ± 3.01 3.99 ± 3.11 0.386 Baseline endocrine levels FSH (IU/L) 7.36 ± 2.35 7.43 ± 2.60 0.830 LH (IU/L) 5.57 ± 3.35 5.65 ± 3.45 0.771 E2 (pg/ml) 36.15 ± 14.31 34.60 ± 15.39 0.238 T (ng/ml) 25.94 ± 16.71 25.26 ± 15.02 0.153 TSH (uIU/ml) 2.20 ± 1.23 2.07 ± 1.03 0.456 AMH (ng/ml) 3.18 ± 2.86 3.19 ± 1.23 0.891 Antral follicle count (n) 14.39 ± 7.40 13.78 ± 8.71 0.585 Type of infertility [n (%)] Primary 119/236 (50.42) 98/236 (41.53) 0.167 Secondary 117/236 (49.58) 138/236 (58.47) Duration of Gn administration (days) 9.46 ± 2.18 9.57 ± 1.87 0.497 Total Gn dosage (IU) 2074.52 ± 799.71 2178.31 ± 868.60 0.088 Cause of infertility [n (%)] Tubal Factor 165/236 (69.91) 167/236 (70.76) 0.061 Ovulation Disorder 7/236 (2.97) 0 Endometriosis 6/236 (2.54) 3/236 (1.27) Male Factors 28/236 (11.86) 30/236 (12.71) Other Factors 30/236 (12.71) 36/236 (15.25) Number of retrieved oocytes (n) 8.78 ± 3.87 8.78 ± 4.15 0.434 Fertilization method [n (%)] IVF 92/236 (38.98) 97/236 (41.10) 0.639 ICSI 144/236 (61.02) 139/236 (58.90) Hormone levels on the day of hCG administration E2 (pg/ml) 1964.96 ± 850.01 2071.25 ± 1040.10 0.115 LH (IU/L) 3.48 ± 2.23 3.67 ± 2.39 0.648 P (ng/ml) 0.65 ± 0.36 0.60 ± 0.34 0.467 Endometrial thickness on day of hCG administration (cm) 1.05 ± 0.18 1.05 ± 0.17 0.115 Number of high-quality embryos (n) 3.74 ± 2.47 3.50 ± 2.23 0.142 Number of transferred embryos (n) 1.78 ± 0.42 1.78 ± 0.41 1.000 Number of frozen embryos (n) 2.15 ± 2.21 2.47 ± 2.27 0.112 Embryo source [n (%)] D3 Embryos 199/236 (84.32) 200/236 (84.75) 0.899 D5 Blastocysts 37/236 (15.68) 36/236 (15.25) GnRHa, gonadotropin-releasing hormone agonist; BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; T, testosterone; TSH, thyroid-stimulating hormone; AMH, anti-Müllerian hormone; Gn, gonadotropins; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; P, progesterone; D3, day 3; D5, day 5. Comparison of baseline characteristics between GnRHa and control groups after propensity score matching. GnRHa, gonadotropin-releasing hormone agonist; BMI, body mass index; FSH, follicle-stimulating hormone; LH, luteinizing hormone; E2, estradiol; T, testosterone; TSH, thyroid-stimulating hormone; AMH, anti-Müllerian hormone; Gn, gonadotropins; IVF, in vitro fertilization; ICSI, intracytoplasmic sperm injection; P, progesterone; D3, day 3; D5, day 5. After adjusting for AMH level, progesterone level on the day of hCG administration, and the number of transferred embryos, the GnRHa group demonstrated significantly higher clinical pregnancy rates than the control group (59.54% vs. 53.80%, respectively; adjusted odds ratio [aOR] 1.34, 95% confidence interval [CI] 1.01–1.77, p  = 0.042). No significant differences were observed between groups in early miscarriage, ectopic pregnancy, live birth, or preterm delivery rates (Table  3 ). Following PSM, the GnRHa group maintained significantly higher clinical pregnancy rates compared to the control group ( p  = 0.026) (Table  4 ). Sensitivity analyses that adjusted for clinically relevant predictors yielded consistent results (aOR = 1.65, 95%CI 1.12–2.41, p  = 0.001). Table 3 Comparison of pregnancy outcomes between GnRHa and control groups [n (%)] GnRHa group Control group p OR (95%CI) Clinical pregnancy rate 156/262 (59.54) 1535/2853 (53.80) 0.042 1.34 (1.01–1.77) Early miscarriage rate 16/156 (10.26) 189/1535 (12.31) 0.901 0.97 (0.56–1.67) Ectopic pregnancy rate 7/156 (4.49) 39/1535 (2.54) 0.281 1.63 (0.67–3.95) Live birth rate 120/262 (45.80) 1306/2853 (45.78) 0.965 0.99 (0.76–1.31) Preterm birth rate 23/120 (19.17) 313/1306 (23.97) 0.173 0.72 (0.45–1.16) GnRHa, gonadotropin-releasing hormone agonist; OR, odds ratio; CI, confidence interval. Comparison of pregnancy outcomes between GnRHa and control groups [n (%)] GnRHa, gonadotropin-releasing hormone agonist; OR, odds ratio; CI, confidence interval. Table 4 Comparison of the pregnancy outcomes between the GnRHa and control groups [n (%)] after propensity score matching. GnRHa group Control group p Clinical pregnancy rate 145/236 (61.44) 121/236 (51.27) 0.026 Early miscarriage rate 16/145 (11.03) 7/121 (5.79) 0.129 Ectopic pregnancy rate 6/145 (4.14) 5/121 (4.13) 0.998 Live birth rate 115/236 (48.73) 103/236 (43.64) 0.268 Preterm birth rate 23/115 (20.00) 22/103 (21.34) 0.804 GnRHa, gonadotropin-releasing hormone agonist. Comparison of the pregnancy outcomes between the GnRHa and control groups [n (%)] after propensity score matching. GnRHa, gonadotropin-releasing hormone agonist. Following PSM, the patients were divided into two groups based on their progesterone levels on the day of hCG administration (progesterone level < 1.0 ng/ml group, n  = 419; progesterone level ≥ 1.0 ng/ml group, n  = 53). In the progesterone level < 1.0 ng/ml group, patients who received GnRHa showed significantly higher clinical pregnancy rates ( p  = 0.020). However, the rate of preterm births was significantly lower in the progesterone level ≥ 1.0 ng/ml group ( p  = 0.015) (Table  5 ). Table 5 Comparison of pregnancy outcomes based on progesterone levels the day of human chorionic gonadotropin administration [n (%)]. Progesterone level < 1.0 ng/ml Progesterone level ≥ 1.0 ng/ml GnRHa group ( n  = 205) Control group ( n  = 214) p GnRHa group ( n  = 31) Control group ( n  = 22) p Clinical pregnancy rate 133/205 (64.88) 115/214 (53.73) 0.020 12/31 (38.71) 6/22 (27.27) 0.386 Embryo implantation rate 160/362 (44.20) 144/379 (37.99) 0.086 14/58 (24.14) 8/41 (19.51) 0.586 Early miscarriage rate 16/133 (12.03) 6/115 (5.22) 0.060 0/12 (0) 1/6 (16.67) 0.333 Live birth rate 105/205 (51.22) 99/214 (46.26) 0.310 10/31 (32.23) 4/22 (18.18) 0.407 Preterm birth rate 21/105 (20.00) 18/99 (18.18) 0.741 2/10 (20.00) 4/4 (100.00) 0.015 GnRHa, gonadotropin-releasing hormone agonist. Comparison of pregnancy outcomes based on progesterone levels the day of human chorionic gonadotropin administration [n (%)]. GnRHa, gonadotropin-releasing hormone agonist. The participants were also categorized into a day 3 (D3) embryo group and a day 5 (D5) blastocyst group. In the D5 group, patients who were administered GnRHa demonstrated significantly higher clinical pregnancy rates, embryo implantation rates, and live birth rates ( p  = 0.003, 0.024, and 0.036, respectively). Conversely, in the D3 group, no statistically significant differences were observed in the clinical pregnancy rates, embryo implantation rates, early miscarriage rates, ectopic pregnancy rates, live birth rates, or preterm birth rates between the GnRHa and non-GnRHa groups (Table  6 ). Following false discovery rate (FDR) adjustment in patients undergoing D5 single blastocyst transfer, the GnRHa group maintained significantly higher rates of clinical pregnancy (q = 0.018), embryo implantation (q = 0.048), and live birth (q = 0.048) compared to the control group, although neither group demonstrated significant differences in early miscarriage or ectopic pregnancy rates (both q = 1.000) (Table  7 ). Table 6 Comparison of pregnancy outcomes based on embryo type [n (%)]. D3 Embryos D5 Blastocysts GnRHa group ( n  = 199) Control group ( n  = 200) p GnRHa group ( n  = 37) Control group ( n  = 36) p Clinical pregnancy rate 120/199 (60.30) 109/200 (54.50) 0.241 25/37 (67.57) 12/36 (33.33) 0.003 Embryo implantation rate 153/383 (39.95) 141/384 (36.72) 0.358 21/37 (56.76) 11/36 (30.56) 0.024 Early miscarriage rate 10/120 (8.33) 5/109 (4.59) 0.252 6*/25 (24.00) 2/12 (16.67) 1.000 Ectopic pregnancy rate 5/120 (4.17) 4/109 (3.67) 1.000 1/25 (4.00) 1/12 (8.33) 1.000 Live birth rate 97/199 (48.74) 94/200 (47.00) 0.727 18/37 (48.65) 9/36 (25.00) 0.036 Preterm birth rate 22/97 (22.68) 21/94 (22.34) 0.955 1/18 (5.56) 1/9 (11.11) 1.000 GnRHa, gonadotropin-releasing hormone agonist; D5, day 5. * one resulted from a traffic accident, and another due to fetal encephalocele. Comparison of pregnancy outcomes based on embryo type [n (%)]. GnRHa, gonadotropin-releasing hormone agonist; D5, day 5. * one resulted from a traffic accident, and another due to fetal encephalocele. Table 7 Treatment outcomes in day 5 single blastocyst transfer (adjusted for false discovery rate) [n (%)] D5 Blastocysts GnRHa Group ( n  = 37) Control Group ( n  = 36) RR (95%CI) Raw p FDR-corrected q Clinical pregnancy rate 25/37 (67.57) 12/36 (33.33) 2.03 (1.20–3.43) 0.003 0.018 Embryo implantation rate 21/37 (56.76) 11/36 (30.56) 1.86 (1.06–3.25) 0.024 0.048 Early miscarriage rate 6/25 (24.00) 2/12 (16.67) 1.44 (0.34–6.10) 1.000 1.000 Ectopic pregnancy rate 1/25 (4.00) 1/12 (8.33) 0.48 (0.03–7.18) 1.000 1.000 Live birth rate 18/37 (48.65) 9/36 (25.00) 1.95 (1.02–3.70) 0.036 0.048 Preterm birth rate 1/18 (5.56) 1/9 (11.11) 0.50 (0.03–7.45) 1.000 1.000 GnRHa, gonadotropin-releasing hormone agonist; D5, day 5; FDR, false discovery rate. Treatment outcomes in day 5 single blastocyst transfer (adjusted for false discovery rate) [n (%)] GnRHa, gonadotropin-releasing hormone agonist; D5, day 5; FDR, false discovery rate.

The results of this study revealed that using GnRHa as luteal support in the antagonist cycle during IVF-ET significantly increased clinical pregnancy rates, particularly in patients undergoing D5 blastocyst transfer. Supplementation with GnRHa also notably improved rates of clinical pregnancy, embryo implantation, and live birth. Multiple studies have suggested that the clinical pregnancy rates following fresh embryo transfer are often lower using the antagonist protocol than when using the traditional long agonist protocol. These observations might be due to the use of GnRHant, which could compromise endometrial receptivity. Therefore, in this study, further investigations were conducted. In our study, we determined that, during antagonist cycles, fresh embryo transfers using blastocysts yielded higher clinical pregnancy and live birth rates than those that used cleavage-stage embryos. This difference was likely because of a better synchronization between the timing of blastocyst transfer and the endometrial window of receptivity. As a result, the addition of GnRHa as a luteal support might have further improved endometrial receptivity, thereby enhancing pregnancy outcomes in blastocyst transfers. Natural conception relies on progesterone secretion by the corpus luteum. However, In IVF-ET, ovarian stimulation involves the use of gonadotropin-releasing hormone (GnRH) analogs (GnRHa or GnRHant), which often results in luteal phase insufficiency. Additionally, in controlled ovarian hyperstimulation (COH) cycles, multiple corpora lutea produce supraphysiological levels of estrogen and progesterone, which suppress the hypothalamic-pituitary axis and inhibit LH secretion. This hormonal disruption can lead to inadequate progesterone levels, thereby lowering clinical pregnancy and embryo implantation rates while increasing the risk of early miscarriage. Therefore, luteal support is an essential component of IVF-ET protocols. The antagonist protocol is one type of COH approach that offers several advantages. These include a shorter duration of drug action, reduced risk of unintended early pregnancy, prevention of ovarian cyst formation, and a lower incidence of ovarian hyperstimulation syndrome (OHSS) 13 . However, this protocol is more likely to result in luteal phase insufficiency than the traditional long agonist protocol 4 . These patterns have prompted clinical interest in whether enhanced luteal support can further improve fresh embryo transfer rates in patients ndergoing the antagonist protocol. Although existing evidence has verified the efficacy of exogenous progesterone supplementation for luteal support, the use of hCG considerably increases the risk of OHSS 14 . Meanwhile, the role of estradiol supplementation in pregnancy outcomes remains controversial. Consequently, there is a growing focus on identifying novel approaches to enhance luteal support and optimize clinical outcomes. In 1993, Wilshire discovered that the application of GnRHa during early pregnancy did not negatively affect pregnancy outcomes 15 . This finding prompted studies regarding whether GnRHa could be utilized as a type of luteal support to enhance pregnancy outcomes. For example, Zafardoust et al. demonstrated that subcutaneous injection of 0.1 mg triptorelin on day 6 after oocyte retrieval markedly improved embryo implantation and pregnancy rates in patients undergoing antagonist protocols for intracytoplasmic sperm injection (ICSI) 16 . Similarly, in 2005, Pirard et al. 17 found that GnRHa could serve as an alternative to progesterone for luteal support. In a 2015 prospective randomized controlled trial 6 , Pirard et al. further demonstrated that the daily intranasal administration of GnRHa as the only luteal support in non-suppression cycles achieved favorable clinical outcomes. More recently, Bar et al. conducted a retrospective analysis involving 1,479 women and 2,529 antagonist cycles 18 . Their results revealed that 1,436 cycles using daily intranasal GnRHa as the only luteal support yielded significantly higher live birth rates than the remaining 1,093 cycles that had received conventional progesterone support. The difference was particularly pronounced in women over 35 years of age. Kung et al. 19 also showed that a single dose of GnRHa on day 6 after ICSI substantially improved embryo implantation, ongoing pregnancy, and live birth rates, and that even greater benefits were observed in patients with elevated follicle-stimulating hormone levels or reduced follicle counts. The mechanisms by which the use of GnRHa as a luteal support improves pregnancy outcomes remain uncertain. However, some researchers have proposed that GnRHa enhances endometrial receptivity and luteal function. Specifically, Maggi et al. 20 suggested that GnRH receptors were widely expressed across reproductive tissues (e.g., ovaries, uterus), and that they performed distinct biological roles at different sites. For example, GnRHa binds to GnRH receptors on the endometrium to promote the motility of stromal cells 21 . Additionally, GnRHa can improve endometrial receptivity by modulating the DNA methylation levels of Hoxa10. The GnRH/GnRH receptor system is also expressed in cytotrophoblasts and syncytiotrophoblasts, in addition to playing a role in regulating the synthesis and secretion of placental hCG 22 . Moreover, GnRHa may enhance luteal function by acting on GnRH receptors located on granulosa lutein cells in the ovaries. Walters et al. 22 demonstrated that GnRHa enhances luteal function by targeting granulosa lutein cells through: (1) high-affinity receptor binding (Kd = 2.3 nM), (2) PLCβ-mediated calcium signaling (3.1-fold increase), (3) upregulation of progesterone synthesis enzymes (+ 68% 3β-HSD activity), and (4) anti-apoptotic effects (-41% caspase-3). These mechanisms explain the efficiency of GnRHa in luteal support. However, our study did not provide direct evidence supporting these mechanisms. Notably, several studies have reported that there were no significant benefits to the use of GnRHa as luteal support in patients undergoing antagonist protocols for fresh embryo transfer. For example, in 2017, Benmachiche et al. 23 conducted a randomized controlled trial with 328 patients (165 in the GnRHa supplementation group vs. 163 controls). Their exclusion criteria consisted of: age ≥ 40 years, baseline follicle-stimulating hormone ≥ 12 mIU/mL, high OHSS risk, and poor ovarian responders (as defined by Bologna criteria). They found no statistically significant differences between the GnRHa supplementation and control groups for implantation rates (27% vs. 23%, respectively; p = 0.35) or clinical pregnancy rates (37% vs. 31%, respectively; p = 0.25). However, we did not stratify participants by ovarian function, age, or hormonal status, in our study; this might have accounted for the different results between our results and those of Benmachiche et al. A 2021 retrospective study by Eftekhar et al. 12 analyzed 168 antagonist protocol cycles (84 cycles each in the intervention and control groups, excluding OHSS-prone patients). They also found that GnRHa supplementation during the luteal phase did not significantly improve implantation or clinical pregnancy rates. The different results in our study might have occurred because we did not stratify patients by predisposition to OHSS. In this study, we did not conduct longitudinal follow-up of offspring health or systematically collect drug-related adverse event data, which precluded definitive safety conclusions regarding GnRHa supplementation. However, pregnancy outcomes suggested that GnRHa as luteal support did not significantly increase risks of early miscarriage, ectopic pregnancy, or preterm birth. These observations aligned with the 2017 cohort study by Zhou et al. 24 , which reported comparable efficacy between GnRHa and standard progesterone in pregnancy and delivery with neonatal outcomes. Despite transient side effects (e.g., hot flashes, headaches), no long-term developmental impacts were observed at 1-year follow-up, which supported the safety profile of GnRHa. This study had some limitations. First, as a retrospective analysis with a relatively small sample size, there might have been bias introduced. Although PSM was performed, the findings were based on post hoc analyses, and numerous confounding factors could not be further measured or compared due to sample size constraints. Future studies should consider expanding the sample size or conducting multi-center prospective randomized controlled trials to supplement our findings. In conclusion, for patients undergoing ovarian stimulation with the antagonist protocol, initiating alternate-day GnRHa supplementation on D3 post-egg retrieval enhanced luteal support and improved clinical pregnancy rates during fresh embryo transfer. This approach was particularly beneficial for patients with progesterone levels < 1.0 ng/ml on the day of hCG administration and for those undergoing fresh blastocyst transfer.

In vitro fertilization and embryo transfer (IVF-ET; in vitro fertilization, IVF) is a cornerstone technique for treating infertility. Despite significant advancements over the past half-century, IVF continues to be a challenging procedure. For example, embryo implantation rates remain low 1 and live birth rates per transfer cycle hover around 30% 2 . Improving these pregnancy remains a critical issue in reproductive medicine. Progress has been made in ovarian stimulation and embryo culture techniques; therefore, the next focus for enhancing pregnancy rates has become luteal support. Proper luteal phase support is essential for achieving pregnancy. Insufficient progesterone secretion or premature luteolysis disrupts the secretory transformation of the endometrium, which causes asynchrony between endometrial and embryonic development. In addition, endometrial receptivity is reduced, which increases the risk of miscarriage or infertility. In IVF-ET cycles, factors such as the use of gonadotropin-releasing hormone antagonists (GnRHant), high doses of exogenous human chorionic gonadotropin (hCG), and supraphysiological levels of estrogen in the early luteal phase can all contribute to luteal phase deficiency 3 . The antagonist protocol has gained widespread use in recent years, although it is more likely to induce luteal phase deficiency than the traditional long agonist protocol 4 . However, one study has indicated that strengthening luteal support during antagonist cycles can help achieve pregnancy and live birth rates comparable to those of the long agonist protocol 5 . Various forms of progesterone, such as gels, capsules, and injections, are common luteal support agents. Recent studies have suggested that the use of gonadotropin-releasing hormone agonists (GnRHa) as luteal support may improve pregnancy outcomes 6 – 8 . One of the proposed mechanisms is to stimulate pituitary luteinizing hormone secretion to maintain luteal function or to directly act on the ovaries, endometrium, or embryos 9 – 11 . However, one study reported that using GnRHa as luteal support did not improve implantation or clinical pregnancy rates 12 . The present study aimed to clarify whether GnRHa supplementation in luteal support enhanced pregnancy rates.