Effect of different trigger strategies on pregnancy outcomes in letrozole-stimulated and modified natural frozen-thawed embryo transfer cycles: a retrospective cohort study

Abstract

Background

This study evaluated the effects of different ovulation trigger strategies on pregnancy outcomes in letrozole-stimulated and modified natural frozen–thawed embryo transfer (L-FET and mNC-FET) cycles.

Methods

A total of 2053 eligible cycles were retrospectively analyzed, including 773 L-FET and 1280 mNC-FET cycles. Participants were grouped by ovulation-triggering method: (1) dual trigger—0.1 mg gonadotropin-releasing hormone agonist (GnRHa) plus 2,000 IU of human chorionic gonadotropin (hCG); (2) hCG trigger—4,000–10,000 IU hCG alone, and (3) GnRHa trigger—0.1 mg GnRHa alone. In L-FET cycles, 105, 430, and 238 women received dual, hCG, and GnRHa triggers, respectively. In mNC-FET cycles, 914 women received hCG and 366 received GnRHa. Binary logistic regression identified independent predictors of pregnancy outcomes, and subgroup analyses were performed for L-FET cycles according to hCG trigger dose and serum luteinizing hormone (LH) levels on trigger day.

Results

In mNC-FET cycles, clinical pregnancy rates (CPR), pregnancy loss rates (PLR), and live birth rates (LBR) were comparable between the hCG and GnRHa trigger groups in both unadjusted and adjusted analyses. In L-FET cycles, after adjusting for confounders, both GnRHa (adjusted odds ratio [aOR] = 0.484, 95% confidence interval [CI], 0.244–0.962, P = 0.038) and hCG (aOR = 0.395, 95% CI 0.196–0.798, P = 0.010) triggers were associated with significantly lower miscarriage rates than the dual trigger group. CPR and LBR did not differ significantly among groups. Subgroup analysis revealed no significant differences in pregnancy outcomes across hCG trigger dose groups in L-FET cycles (P > 0.05). When stratified by LH level (< 20 IU/L vs. ≥20 IU/L), pregnancy outcomes were similar except for a higher PLR with the dual trigger in the LH surge (≥ 20 IU/L) subgroup (30.0% vs. 11.9%, P < 0.05).

Conclusions

In mNC-FET, hCG and GnRHa triggering yielded similar pregnancy outcomes. In L-FET, dual triggering increased miscarriage risk, particularly in the LH surge group (≥ 20 IU/L). Routine dual trigger use in L-FET is therefore not recommended. Luteal support should be individualized, and a 4000 IU hCG trigger may represent a cost-effective alternative for patients undergoing L-FET.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12884-026-09041-0.

Background

Over the past few decades, advances in embryo vitrification, culture media, and cryopreservation techniques have markedly increased the number of frozen-thawed embryo transfer (FET) cycles [1], making FET an indispensable component of assisted reproduction [2]. Compared with fresh embryo transfer, FET more closely mimics the hormonal environment of natural pregnancy and avoids the adverse effects of controlled ovarian hyperstimulation on embryo-endometrium synchronization [3, 4].

However, the optimal endometrial preparation protocol for FET remains a subject of debate. A recent Cochrane review found no single protocol to be superior in terms of reproductive outcomes [5]. Currently, three main endometrial preparation protocols are used: the natural cycle (NC, including true NC and modified NC), artificial cycle (AC), and stimulated cycle (STC).

For women with regular menstrual cycles and normal ovulation, NC-FET is generally the preferred, simplest, and safest method of endometrial preparation. Conversely, AC-FET may achieve better clinical outcomes in patients with irregular menstruation, ovulatory dysfunction, or a thin endometrium during the natural cycle. AC protocols also allow more flexible scheduling and greater patient convenience [6]. Nevertheless, the medications used in AC-FET can be expensive, and prolonged use of exogenous estrogen and progesterone may increase the risk of thrombosis, dizziness, nausea, and liver dysfunction [7]. For patients with endometrial dysplasia during natural or hormone replacement cycles, or those with irregular ovulation or anovulation, STC-FET often yields better pregnancy outcomes [8, 9].

Letrozole (LE), a potent and reversible aromatase inhibitor, lacks anti-estrogenic effects, does not adversely affect cervical mucus or endometrial morphology [10], and may enhance luteal function [11]. It is considered an ideal agent for ovulation induction and endometrial preparation [12]. In a large retrospective cohort study involving 110,722 single FET cycles, letrozole-stimulated FET (L-FET) was associated with higher clinical pregnancy rates (CPR), live birth rates (LBR), and lower pregnancy loss rates (PLR) than both NC and AC protocols [13]. Similarly, a systematic review and meta-analysis that included eight retrospective studies and one randomized controlled trial (RCT) reported a significantly higher CPR in the L-FET group compared with AC-FET [14]. More recently, an RCT in women with polycystic ovary syndrome found comparable CPR between L-FET and AC-FET. However, LE appeared more beneficial in patients with normal body weight or androgen levels [15].

Pregnancy outcomes in STC-FET depend on accurate detection of ovulation and precise estimation of the optimal window of endometrial receptivity. Human chorionic gonadotropin (hCG) is commonly administered to mimic the endogenous luteinizing hormone (LH) surge, inducing final oocyte maturation and ovulation approximately 36–38 h after injection [16]. Gonadotropin-releasing hormone agonists (GnRHa) have also been shown to effectively trigger ovulation [17], although this approach may lead to luteolysis; this can be mitigated by appropriate luteal phase support (LPS) [18]. To address this limitation, combining GnRHa with a reduced hCG dose—known as a dual trigger—has been introduced during superovulation to optimize in vitro fertilization (IVF) cycle outcomes. Previous studies have demonstrated that dual triggers can increase the number of oocytes retrieved, improve embryo quality, enhance CPR, and LBR while reducing the risk of ovarian hyperstimulation syndrome [19, 20].

Chen et al. reported that in LE-human menopausal gonadotropin (hMG) intrauterine insemination (IUI) cycles, CPR were similar across hCG, GnRHa, and dual-trigger groups. However, miscarriage rates differed, being lower with GnRHa and higher with dual-trigger, with no significant differences in neonatal outcomes. These findings suggest that trigger strategy may influence early pregnancy loss, but has a limited impact on neonatal outcomes [21]. However, evidence regarding the effect of different trigger methods on pregnancy outcomes during the endometrial preparation phase of FET in ovulation induction cycles remains limited.

A 2015 retrospective study involving 785 women compared pregnancy outcomes between GnRHa (n = 96) and hCG (n = 689) triggers in L-FET cycles. With standardized LPS (daily 40 mg progesterone injection), the GnRHa group showed a trend toward a higher miscarriage rate than the hCG group, although the difference was not statistically significant. In contrast, CPR and LBR were higher in the GnRHa group [22]. At that time, however, it was not yet recognized that GnRHa triggering could cause corpus luteum deficiency and luteal phase defects.

In modified NC-FET (mNC-FET), ovulation is triggered using either hCG or GnRHa [23], which simplifies timing and reduces the challenge of detecting spontaneous LH surges inherent to true NC-FET [24]. Nevertheless, the effect of these two triggering methods on pregnancy outcomes in mNC-FET remains controversial. Some studies suggest that hCG triggering offers no advantage over natural ovulation, aside from timing convenience, and may even impair endometrial receptivity, leading to lower pregnancy rates [25, 26]. In contrast, in NC-FET using GnRHa to induce ovulation followed by repeated daily GnRHa supplementation has been associated with satisfactory luteal phase steroid levels and higher ongoing pregnancy rates and LBR [23].

Given this context, the present study aimed to evaluate how different triggering strategies influence pregnancy outcomes in women undergoing L-FET and mNC-FET cycles, to identify optimal endometrial preparation protocols for FET.

Materials and methods

Population and study design

This retrospective study included women who underwent L-FET or mNC-FET at the Department of Reproduction and Genetics of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine, between January 1, 2017, and January 1, 2023. In total, 773 L-FET and 1,280 mNC-FET cycles were analyzed. The study was approved by the Reproductive Ethics Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine (approval no. AF/SC-08/02.0) and conducted in accordance with relevant institutional and national guidelines and regulations. All participants provided informed consent before inclusion, and follow-up was conducted at 12 months. Clinical data were retrieved from the hospital’s electronic medical record system.

Inclusion criteria were as follows: women aged 20–45 years undergoing endometrial preparation for FET using either a LE-stimulated cycle or a modified natural cycle, with at least one cryopreserved day-3 embryo available for transfer. Exclusion criteria were: (1) severe tubal hydrosalpinx or endometriosis; (2) untreated systemic or endocrine disorders, such as diabetes mellitus or Cushing’s syndrome; (3) congenital uterine malformations or untreated endometrial pathology; (4) karyotypic abnormalities in either partner; (5) pre-implantation genetic diagnosis/screening cycles; and (6) cycle cancellation due to unsuccessful embryo thawing or survival (Fig. 1).

Fig. 1.

Workflow of study enrollment

Ovarian stimulation and laboratory protocols

Each participant underwent IVF or intracytoplasmic sperm injection (ICSI) as clinically indicated. Controlled ovarian hyperstimulation (COH) was performed in accordance with our institutional clinical protocols, which included the use of GnRH antagonists, GnRHa, and mild stimulation regimens for patients with reduced ovarian reserve. The selection of COH protocols and gonadotropin dosage was individualized based on patient characteristics, including age, body mass index (BMI), basal follicle-stimulating hormone (FSH) levels, and antral follicle count.

When ultrasound confirmed the presence of ≥ 2 dominant follicles measuring ≥ 18 mm or ≥ 3 dominant follicles measuring ≥ 17 mm, along with progesterone and estradiol (E₂) levels, final oocyte maturation was triggered using hCG (Lizhu, Zhuhai, China) and/or triptorelin acetate injection (DECAPEPTYL^®, Ferring Pharmaceuticals). Oocyte retrieval was performed 34–36 h after triggering. Fertilization of the retrieved oocytes was achieved via IVF or ICSI, depending on semen characteristics. Subsequently, either a fresh embryo transfer or a freeze-all strategy was implemented in accordance with clinical guidelines.

High-quality embryos that were not used for fresh transfer were cryopreserved using a closed vitrification system. Cleavage-stage (day 3) embryos were graded according to the Cummins criteria [27], with Grade I and II embryos classified as high quality and selected for vitrification. Suboptimal day 3 embryos (grades III–IV) were cultured to the blastocyst stage. Blastocyst quality was assessed according to Gardner’s scoring system [28], and embryos graded ≥3BB were considered high-quality blastocysts. The decision to transfer one or two embryos was made by the attending clinician before the procedure. Vitrified embryos were thawed on the morning of the transfer day, following previously described protocols [29].

Endometrial preparation prior to embryo transfer

In L-FET cycles, patients received daily doses of 2.5–5 mg LE (Jiangsu Hengrui Medicine Co., China) from days 3 to 5 of the spontaneous or progesterone-withdrawal menstrual cycle, for five consecutive days. Transvaginal sonography and serum hormone measurements were initiated on day 10 and repeated as needed. If the dominant follicle diameter was < 14 mm on day 10, daily doses of 75–150 IU hMG (Anhui Fengyuan Pharmaceutical Co., China) were added to stimulate follicular growth, with dosage adjusted according to BMI and ovarian response. When a dominant follicle reached ≥ 14 mm on day 10, no additional medication was administered until ovulation triggering. Ovulation was triggered once the dominant follicle attained ≥ 17 mm, the endometrial thickness was ≥ 7 mm, and serum E₂ levels exceeded 150 pg/mL.

In mNC-FET cycles, transvaginal ultrasound and serum hormone monitoring started on day 10 of the menstrual cycle and continued daily or every other day, depending on follicular size and serum hormone levels. Ovulation triggering was determined by the detection of an LH surge (LH ≥ 20 IU/L and at least twice the mean of the previous 2 days). Upon LH surge detection, final oocyte maturation was induced with 4000–10,000 IU hCG, 0.1 mg GnRHa, or 0.1 mg GnRHa plus 2000 IU hCG, at the discretion of the attending physician. Cleavage-stage embryos were transferred 4 days after triggering, and blastocysts 6 days after.

Luteal support commenced the day after triggering daily intramuscular progesterone (40 mg; Zhejiang Xianju Pharmaceutical Co., China) and oral dydrogesterone (30 mg/day, 10 mg tablets; Abbott Co., USA) or progesterone vaginal sustained-release gel (90 mg, Merck Serono, Germany). In cycles without an LH surge (LH < 20 IU/L), triggering was performed at 21:00 with hCG, GnRHa, or both, and embryo transfer was scheduled 5 days later for cleavage-stage embryos or 7 days later for blastocysts. Progesterone supplementation commenced 2 days after the triggering event. Cycles were canceled if the endometrial thickness remained < 7 mm after 14 days of stimulation, regardless of the dominant follicle size. For confirmed pregnancies, progesterone supplementation was continued until 10 weeks of gestation.

Outcome measures

The primary outcome of this study was the LBR, while secondary outcomes included positive pregnancy rate (PPR), CPR, PLR, and ectopic pregnancy rate (EPR). A positive pregnancy was defined as a serum beta (β-hCG) level ≥ 25 IU/L at 14 days after embryo transfer. If the β-hCG result was positive, clinical pregnancy was confirmed by ultrasound detection of at least one gestational sac within the uterine cavity 4–6 weeks after FET. A live birth was defined as the delivery of a viable neonate at or beyond 28 weeks of gestation. Pregnancy loss referred to the loss of a clinical pregnancy within the first 20 weeks. Ectopic pregnancy was diagnosed by ultrasonography, surgical visualization, or histopathology and was defined as the implantation of a fertilized ovum outside the uterine cavity.

Statistical analysis

All statistical analyses were performed using SPSS version 21.0 (SPSS Corp., Armonk, NY, USA). Continuous variables following a normal distribution were expressed as mean ± standard deviation, whereas non-normally distributed data were presented as median (interquartile range, 25%–75%). Comparisons between two groups were performed using the independent samples t-test or the Mann-Whitney U test, as appropriate. For comparisons among three or more groups, one-way analysis of variance with post-hoc analysis or the Kruskal-Wallis rank sum test was employed. Categorical variables were compared using the chi-square (χ2) test or Fisher’s exact test, and results were expressed as frequencies and percentages.

Binary logistic regression analysis was utilized to assess the impact of potential confounding factors on pregnancy outcomes in FET cycles. Covariates included: female age, duration and type of infertility (primary or secondary), BMI, history of abortion, basal serum FSH levels, method of laboratory insemination (IVF/ICSI), whether fresh embryo transfer was performed, embryo quality (good vs. suboptimal), and endometrial thickness prior to FET. A P-value < 0.05 was deemed indicative of statistical significance.

To evaluate the effect of hCG dosage on pregnancy outcomes in L-FET cycles, the hCG trigger group was further divided into four subgroups: A (4000 IU), B (6000 IU), C (8000 IU), and D (10000 IU). Additionally, subgroup analysis based on LH levels on the trigger day (LH < 20 IU/L vs. LH ≥ 20 IU/L) was performed to explore how different triggering strategies, administered before or after the LH surge, affected pregnancy outcomes in L-FET cycles.

Results

Baseline characteristics of patients with different triggering strategies in L-FET and mNC-FET cycles

A total of 773 L-FET cycles were included in this study. Among these, 105 cycles (13.6%) received a dual trigger consisting of 0.1 mg GnRHa and 2000 IU hCG, 430 cycles (55.6%) were triggered with hCG alone, and 238 cycles (30.8%) were triggered with 0.1 mg GnRHa alone. In addition, 1,280 mNC-FET cycles were analyzed, comprising 914 cycles (71.4%) triggered with hCG and 366 cycles (28.6%) triggered with GnRHa.

The baseline characteristics of the study population are summarized in Table 1. No significant differences were observed among the groups with different triggering strategies in either L-FET and mNC-FET cycles in terms of age, BMI, infertility type or duration, gravidity, parity, history of abortion, baseline hormone levels (FSH, LH, E₂, progesterone), method of laboratory insemination, number of oocytes retrieved, total number of embryos, number and type of transferred embryos, and endometrial thickness before FET (all P > 0.05).

Table 1.

Baseline characteristics of patients with different triggering strategies in L-FET and mNC-FET cycles

Variables	L-FET				mNC-FET
	dual trigger (n = 105)	hCG trigger (n = 430)	GnRHa trigger (n = 238)	P value	hCG trigger (n = 914)	GnRHa trigger (n = 366)	P value
Age of woman (years)	33.39 ± 5.01	33.21 ± 5.25	32.72 ± 4.53	0.251	32.68 ± 4.67	32.79 ± 4.63	0.088
Infertility duration(years)	3.38 ± 2.30	3.69 ± 2.73	3.41 ± 2.36	0.229	3.51 ± 2.17	3.47 ± 2.37	0.361
BMI (kg/m2)	23.55 ± 2.59	23.82 ± 3.31	23.40 ± 3.17	0.179	23.73 ± 3.51	23.67 ± 3.55	0.144
Gravidity (n)	1 (0, 2)	1 (0, 2)	1 (0, 2)	0.113	1 (0, 2)	1 (0, 2)	0.936
Parity (n)	0 (0, 1)	0 (0, 1)	0 (0, 1)	0.786	0 (0, 1)	0 (0, 1)	0.459
Abortion (n)	0 (0, 1)	0 (0, 1)	0 (0, 1)	0.489	0 (0, 1)	0 (0, 1)	0.275
Type of infertility				0.174			0.527
Primary infertility	32.4% (34/105)	38.4% (165/430)	42.9% (102/238)		35.0% (320/914)	36.9% (135/366)
Secondary infertility	67.6% (71/105)	61.6% (265/430)	57.1% (136/238)		65.0% (594/914)	63.1% (231/366)
Baseline hormonal profile
FSH (mIU/mL)	7.17 ± 2.06	7.18 ± 1.91	7.22 ± 1.72	0.922	7.19 ± 2.29	7.13 ± 1.82	0.722
LH (mIU/mL)	5.53 ± 3.08	5.75 ± 3.15	6.09 ± 2.97	0.126	5.04 ± 3.17	5.91 ± 3.14	0.094
E2 (pg/ml)	42.68 ± 11.86	45.76 ± 16.45	46.39 ± 17.77	0.141	50.91 ± 16.82	45.53 ± 16.36	0.083
P (ng/ml)	0.65 ± 0.39	0.60 ± 0.41	0.64 ± 0.42	0.304	0.85 ± 0.35	0.63 ± 0.43	0.077
Laboratory insemination				0.337			0.755
IVF	78.1% (82/105)	77.4% (333/430)	72.7% (173/238)		73.7% (674/914)	74.6% (273/366)
ICSI	21.9% (23/105)	22.6% (97/430)	27.3% (65/238)		26.3% (240/914)	25.4% (93/366)
Oocytes retrieved	16.20 ± 7.87	16.54 ± 8.97	17.39 ± 9.28	0.275	16.88 ± 8.67	16.42 ± 8.43	0.689
Total number of embryos	5.62 ± 2.69	5.67 ± 2.92	6.08 ± 3.03	0.092	5.38 ± 2.84	5.22 ± 2.56	0.448
Protocol in fresh cycle				0.482			0.122
Fresh embryo transfer	11.4% (12/105)	12.8% (55/430)	9.7% (23/238)		13.6% (124/914)	16.9% (62/366)
Freeze-all strategy	88.6% (93/105)	87.2% (375/430)	90.3% (215/238)		86.4% (790/914)	83.1% (304/366)
Stimulated protocol for FET				0.288			N/A
Letrozole	22.9% (24/105)	25.1% (108/430)	29.8% (71/238)		N/A	N/A
Letrozole + HMG	77.1% (81/105)	74.9% (322/430)	70.2% (167/238)		N/A	N/A
Number of embryos transferred				0.086			0.294
Single	32.4% (34/105)	28.8% (124/430)	22.3% (53/238)		44.7% (409/914)	41.5% (152/366)
Double	67.6% (71/105)	71.2% (306/430)	77.7% (185/238)		55.3% (505/914)	58.5% (214/366)
Type of transferred embryos				0.158			0.098
Cleavage embryo	72.4% (76/105)	73.7% (317/430)	79.8% (190/238)		64.6% (590/914)	69.4% (254/366)
Blastocyst	27.6% (29/105)	26.3% (113/430)	20.2% (48/238)		35.4% (324/914)	30.6% (112/366)
Good-quality embryos				0.428			0.571
Yes	51.4% (54/105)	54.4% (234/430)	58.4% (139/238)		47.2% (431/914)	48.9% (179/366)
No	48.6% (51/105)	45.6% (196/430)	41.6% (99/238)		52.8% (483/914)	51.1% (187/366)
Endometrium thickness prior to FET (mm)	10.09 ± 1.71	10.30 ± 1.57	10.28 ± 1.57	0.459	10.10 ± 1.65	10.19 ± 1.23	0.681

Data are presented as mean ± SD for continuous variables and percentage (number/total) for dichotomous variables. N/A: not applicable

BMI=body mass index; E₂=estradiol; FSH=follicle stimulating hormone; FET=Frozen-thawed embryo transfer; HMG=human menopausal gonadotropin; ICSI=intracytoplasmic sperm injection; IVF = in vitro fertilization; LH=luteinizing hormone; P=progesterone; L-FET=letrozole-stimulated frozen-thawed embryo transfer; mNC-FET=modified natural cycle frozen-thawed embryo transfer

Pregnancy outcomes among different triggering strategies in L-FET and mNC-FET cycles

In L-FET cycles, the PPR, CPR, and EPR were slightly higher in the dual-trigger group than in the GnRHa and hCG trigger groups. However, these differences were not statistically significant (P = 0.764, 0.892, and 0.705, respectively). The PLR in the dual-trigger group (25.5%) was significantly higher than that in the GnRHa (13.6%) and hCG (11.5%) trigger groups (P = 0.029). Conversely, the LBR was slightly higher in the hCG trigger group (40.2%) than in the GnRHa (38.2%) and dual trigger groups (34.3%), although the difference was not statistically significant (P = 0.521) (Fig. 2 and Additional file 1).

Fig. 2.

Pregnancy outcomes among the three triggering methods in L-FET cycles

Note: Data are presented as percentages (numbers/total). *Compared to the dual-trigger group, P < 0.05

In mNC-FET cycles, the CPR, LBR, and PLR were higher in the hCG trigger group compared with the GnRHa trigger group, whereas the PPR and EPR were lower than in the GnRHa trigger group. Nevertheless, none of these differences reached statistical significance (all P > 0.05) (Fig. 3 and Additional file 2).

Fig. 3.

Pregnancy outcomes of the two triggering methods in mNC-FET cycles

Note: Data are presented as a percentage (number/total)

Variables independently associated with CPR, PLR, and LBR in L-FET and mNC-FET cycles

Binary logistic regression analysis was performed to identify factors independently associated with CPR, PLR, and LBR in both L-FET and mNC-FET cycles. In L-FET cycles (Table 2), compared with the dual-trigger group, the PLR was significantly lower in both the GnRHa trigger group (adjusted odds ratio [aOR] = 0.484, 95% confidence interval [CI] 0.244–0.962, P = 0.038) and the hCG trigger group (aOR = 0.395, 95% CI 0.196–0.798, P = 0.010). Additionally, maternal age was positively associated with PLR (aOR = 1.063, 95% CI = 1.005–1.124, P = 0.034).

Table 2.

Binary logistics regression analysis of CPR, PLR, and LBR in L-FET cycles

Variables	Clinical pregnancy rate		Pregnancy loss rate		Live birth rate
	Adjusted OR(95% CI)	P value	Adjusted OR(95% CI)	P value	Adjusted OR(95% CI)	P value
Age of woman (years)	0.927 (0.898–0.958)	< 0.001	1.063 (1.005–1.124)	0.034	0.914 (0.883–0.946)	< 0.001
Infertility duration(years)	0.985 (0.928–1.045)	0.612	1.059 (0.940–1.151)	0.273	0.923 (0.858–1.085)	0.542
Type of infertilitya(primary/secondary infertility)	1.027 (0.744–1.418)	0.870	0.737 (0.409–1.330)	0.311	1.289 (0.925–1.797)	0.134
BMI (kg/m2)	0.986 (0.965–1.049)	0.768	1.036 (0.992–1.081)	0.383	0.964 (0.887–1.047)	0.113
Abortion (n)	0.903 (0.765–1.066)	0.229	1.029 (0.783–1.352)	0.840	0.908 (0.760–1.086)	0.291
Basal serum FSH level(mIU/mL)	0.983 (0.909–1.062)	0.662	1.052 (0.823–1.201)	0.507	0.980 (0.903–1.063)	0.626
Laboratory inseminationa(IVF/ICSI)	0.880 (0.628–1.233)	0.459	0.980 (0.529–1.815)	0.948	0.841 (0.593–1.194)	0.333
Fresh embryo transfera(no/yes)	1.328 (0.855–2.063)	0.207	1.711 (0.864–3.386)	0.123	1.113 (0.708–1.752)	0.642
Good-quality embryosa(no/yes)	3.787 (2.866–5.006)	< 0.001	0.943 (0.858–1.027)	0.167	2.365 (1.535–3.643)	< 0.001
Endometrium thickness prior to FET (mm)	1.045 (0.957–1.141)	0.325	0.975 (0.937–1.179)	0.255	1.019 (0.931–1.116)	0.679
Trigger ovulation methodsa
dual trigger	Ref		Ref		Ref
hCG trigger	0.843 (0.531–1.338)	0.469	0.395 (0.196–0.798)	0.010	1.173 (0.727–1.895)	0.513
GnRHa trigger	0.710 (0.447–1.127)	0.147	0.484 (0.244–0.962)	0.038	0.964 (0.596–1.558)	0.880

^a represents type of infertility with primary as reference, laboratory fertilization mode with IVF as reference, fresh embryo transfer before FET as reference, FET without good-quality embryos as reference, trigger ovulation methods with dual trigger as reference

BMI=body mass index; FSH=follicle stimulating hormone; FET=Frozen-thawed embryo transfer; OR=Odds ratio; CI=Confidence intervals; ICSI=intracytoplasmic sperm injection; IVF = in vitro fertilization

After adjusting for potential confounding factors, no significant association was observed between different ovulation-triggering strategies and CPR or LBR. The transfer of high-quality embryos emerged as a strong positive predictor for both CPR (aOR = 3.787, 95% CI 2.866–5.006, P < 0.001) and LBR (aOR = 2.365, 95% CI 1.535–3.643, P < 0.001), whereas increasing maternal age was identified as a negative predictor for both CPR (aOR = 0.927, 95% CI 0.898–0.958, P < 0.001) and LBR (aOR = 0.914, 95% CI 0.883–0.946, P < 0.001).

In mNC-FET cycles (Table 3), even after adjusting for potential confounding factors, no significant differences were observed between the hCG and GnRHa trigger groups in terms of CPR, PLR, and LBR (P = 0.526, 0.388, and 0.408, respectively). Maternal age was associated with a higher PLR (aOR = 1.064, 95% CI 1.007–1.120, P = 0.025) and lower CPR (aOR = 0.970, 95% CI 0.944–0.997, P = 0.029) and LBR (aOR = 0.952, 95% CI 0.925–0.980, P = 0.001). The transfer of good-quality embryos was a positive predictor of both CPR (aOR = 1.296, 95% CI 1.033–1.628, P = 0.025) and LBR (aOR = 1.380, 95% CI 1.087–1.752, P = 0.008). Conversely, elevated basal FSH levels were identified as negative predictors for both CPR (aOR = 0.893, 95% CI 0.845–0.942, P < 0.001) and LBR (aOR = 0.884, 95% CI 0.834–0.938, P < 0.001). Furthermore, a longer duration of infertility was an important negative factor for CPR (aOR = 0.948, 95% CI 0.900–0.998, P = 0.042).

Table 3.

Binary logistics regression analysis of CPR, PLR, and LBR in mNC-FET cycles

Variables	Clinical pregnancy rate		Pregnancy loss rate		Live birth rate
	Adjusted OR (95% CI)	P value	Adjusted OR (95% CI)	P value	Adjusted OR (95% CI)	P value
Age of woman (years)	0.970 (0.944–0.997)	0.029	1.064 (1.007–1.120)	0.025	0.952 (0.925–0.980)	0.001
Infertility duration(years)	0.948 (0.900-0.998)	0.042	0.914 (0.822–1.017)	0.099	0.961 (0.910–1.015)	0.158
Type of infertilitya(primary/secondary infertility)	0.985 (0.760–1.278)	0.912	1.118 (0.658–1.899)	0.681	0.921 (0.702–1.209)	0.554
BMI (kg/m2)	1.024 (0.992–1.058)	0.144	1.020 (0.959–1.086)	0.528	1.022 (0.988–1.057)	0.212
Abortion (n)	0.932 (0.767–1.131)	0.474	0.631 (0.375–1.062)	0.083	1.053 (0.862–1.287)	0.611
Basal serum FSH level(mIU/mL)	0.893 (0.845–0.942)	< 0.001	0.993 (0.900-1.095)	0.882	0.884 (0.834–0.938)	< 0.001
Laboratory inseminationa(IVF/ICSI)	0.970 (0.944–0.997)	0.356	0.889 (0.765–1.013)	0.476	0.967 (0.752–1.181)	0.237
Fresh embryo transfera(no/yes)	1.165 (0.878–1.452)	0.467	1.163 (1.105–1.221)	0.165	1.050 (0.965–1.134)	0.473
Good-quality embryosa(no/yes)	1.296 (1.033–1.628)	0.025	0.882 (0.563–1.382)	0.585	1.380 (1.087–1.752)	0.008
Endometrium thickness prior to FET (mm)	1.042 (0.916–1.167)	0.139	0.868 (0.802–0.934)	0.231	1.134 (0.987–1.281)	0.089
Trigger ovulation methodsa
hCG trigger	Ref		Ref		Ref
GnRHa trigger	0.919 (0.709–1.192)	0.526	1.199 (0.769–1.629)	0.388	0.891(0.678–1.171)	0.408

^a represents type of infertility with primary as reference, laboratory fertilization mode with IVF as reference, fresh embryo transfer before FET as reference, FET without good-quality embryos as reference, trigger ovulation methods with hCG trigger as reference

BMI=body mass index; FSH=follicle stimulating hormone; FET=Frozen-thawed embryo transfer; OR=Odds ratio; CI=Confidence intervals; ICSI=intracytoplasmic sperm injection; IVF = in vitro fertilization

Subgroup analysis based on hCG trigger dose in L-FET cycles

The subgroup analysis of the hCG trigger group in L-FET cycles included a total of 430 cycles, divided into subgroup A (n = 160), subgroup B (n = 82), subgroup C (n = 81), and subgroup D (n = 107) (Table 4). There were no significant differences in the baseline characteristics of patients among the four subgroups (all P > 0.05). Similarly, the PPR, CPR, EPR, PLR, and LBR did not differ significantly among the four hCG dose subgroups (all P > 0.05). The results were presented in Fig. 4 and Additional file 3.

Table 4.

Baseline characteristics of patients with hCG trigger in L-FET cycles

Variables	Subgroup A	Subgroup B	Subgroup C	Subgroup D	F/ χ2 value	P value
Number of patients	160	82	81	107
Age of woman (years)	33.77 ± 4.98	32.99 ± 5.96	33.41 ± 5.20	32.38 ± 5.08	1.580	0.194
Infertility duration(years)	3.92 ± 3.3	3.73 ± 2.53	3.72 ± 3.01	3.28 ± 2.09	1.187	0.314
BMI (kg/m2)	23.84 ± 3.08	23.96 ± 3.71	23.41 ± 3.49	24.68 ± 3.75	2.034	0.110
Gravidity (n)	1 (0, 2)	1 (0, 2)	1 (0, 2)	1 (0, 2)	2.985	0.394
Parity (n)	0 (0, 1)	0 (0, 1)	0 (0, 1)	0 (0, 1)	1.127	0.771
Abortion (n)	0 (0, 0)	0 (0, 1)	0 (0, 1)	0 (0, 1)	2.160	0.092
Type of infertility					0.764	0.858
Primary infertility	36.3% (58/160)	39.0% (32/82)	42.0% (34/81)	38.3% (41/107)
Secondary infertility	63.8% (102/160)	61.0% (50/82)	58.0% (47/81)	61.7% (66/107)
Baseline hormonal profile
FSH (mIU/mL)	7.25 ± 1.89	7.31 ± 2.32	7.15 ± 1.62	6.99 ± 1.79	0.568	0.637
LH (mIU/mL)	5.66 ± 3.62	5.70 ± 2.97	5.49 ± 2.58	6.12 ± 2.94	0.725	0.538
E2 (pg/ml)	44.89 ± 16.08	46.06 ± 17.35	44.85 ± 17.43	47.52 ± 15.59	0.645	0.587
P (ng/ml)	0.56 ± 0.37	0.58 ± 0.41	0.59 ± 0.36	0.67 ± 0.50	1.507	0.212
Laboratory insemination					1.654	0.647
IVF	79.4% (127/160)	73.2% (60/82)	75.3% (61/81)	79.4% (85/107)
ICSI	20.6% (33/160)	26.8% (22/82)	24.7% (20/81)	20.6% (22/107)
Oocytes retrieved	15.98 ± 8.44	15.17 ± 9.69	17.00 ± 8.08	17.81 ± 9.05
Total number of embryos	5.40 ± 3.00	5.37 ± 2.86	6.12 ± 2.57	6.10 ± 2.60	2.361	0.071
Protocol in fresh cycle					2.864	0.413
Fresh embryo transfer	15.0% (24/160)	12.2% (10/82)	14.8% (12/81)	8.4% (9/107)
Freeze-all strategy	85.0% (136/160)	87.8% (72/82)	85.2% (69/81)	91.6% (98/107)
Number of embryos transferred					3.785	0.286
Single	33.8% (54/160)	29.3% (24/82)	25.9% (21/81)	23.4% (25/107)
Double	66.2% (106/160)	70.7% (58/82)	74.1% (60/81)	76.6% (82/107)
Type of transferred embryos					4.193	0.241
Cleavage embryo	68.75% (110/160)	73.2% (60/82)	76.5% (62/81)	79.4% (85/107)
Blastocyst	31.25% (50/160)	26.8% (22/82)	23.5% (19/81)	20.6% (22/107)
Good-quality embryos					1.544	0.672
Yes	58.1% (93/160)	52.4% (43/82)	50.6% (41/81)	53.3% (57/107)
No	41.9% (67/160)	47.6% (39/82)	49.4% (40/81)	46.7% (50/107)
Endometrium thickness prior to FET (mm)	10.47 ± 1.50	10.42 ± 1.70	10.14 ± 1.29	10.07 ± 1.72	1.917	0.126

Data are presented as mean ± SD for continuous variables and percentage (number/total) for dichotomous variables

BMI=body mass index; E₂=estradiol; FSH=follicle stimulating hormone; FET=Frozen-thawed embryo transfer; ICSI=intracytoplasmic sperm injection; IVF=in vitro fertilization; LH=luteinizing hormone; P=progesterone

Fig. 4.

Pregnancy outcomes among the four hCG subgroups in L-FET cycles

The results of the binary logistic regression analysis for the four hCG dose subgroups are presented in Table 5. No significant associations were found between hCG dose and CPR, PLR, or LBR (all P > 0.05). Consistent with earlier findings, the transfer of high-quality embryos remained a positive predictor of both CPR (aOR = 1.782, 95% CI 0.978–2.990, P = 0.042) and LBR (aOR = 4.749, 95% CI 3.007–7.499, P < 0.001). In contrast, increasing maternal age was identified as a negative predictor for both CPR (aOR = 0.910, 95% CI 0.865–0.956, P < 0.001) and LBR (aOR = 0.894, 95% CI 0.848–0.942, P < 0.001).

Table 5.

Binary logistics regression analysis of CPR, PLR, and LBR in the four hCG subgroups

Variables	Clinical pregnancy rate		Pregnancy loss rate		Live birth rate
	Adjusted OR(95% CI)	P value	Adjusted OR(95% CI)	P value	Adjusted OR(95% CI)	P value
Age of woman (years)	0.910 (0.865–0.956)	< 0.001	1.070 (0.978–1.170)	0.034	0.894 (0.848–0.942)	< 0.001
Infertility duration(years)	0.992 (0.901–1.091)	0.865	1.004 (0.911–1.107)	0.933	0.977 (0.815–1.170)	0.798
Type of infertilitya(primary/secondary infertility)	1.067 (0.628–1.812)	0.810	1.188 (0.419–3.367)	0.745	1.391 (0.820–2.359)	0.221
BMI (kg/m2)	0.970 (0.842–1.118)	0.676	1.075 (0.816–1.416)	0.606	0.938 (0.813–1.082)	0.381
Abortion (n)	0.881 (0.675–1.150)	0.353	1.053 (0.846–1.311)	0.556	0.883 (0.669–1.163)	0.375
Basal serum FSH level(mIU/mL)	0.978 (0.816–1.173)	0.535	1.050 (0.843–1.308)	0.662	0.967 (0.854–1.095)	0.699
Laboratory inseminationa(IVF/ICSI)	0.759 (0.443-1.300)	0.315	1.544 (0.615–3.877)	0.355	0.631 (0.364–1.094)	0.101
Fresh embryo transfera(no/yes)	0.975 (0.509–1.868)	0.939	0.930 (0.255–3.390)	0.913	1.160 (0.601–2.237)	0.658
Good-quality embryosa(no/yes)	1.782 (0.978–2.990)	0.042	0.928 (0.897–0.958)	0.048	4.749 (3.007–7.499)	< 0.001
Endometrium thickness prior to FET (mm)	1.026 (0.965–1.090)	0.408	0.971 (0.858–1.098)	0.637	1.050 (0.987–1.116)	0.119
Subgroupa
Subgroup A	Ref		Ref		Ref
Subgroup B	0.886 (0.483–1.626)	0.696	1.375 (0.553–2.331)	0.072	0.508 (0.272–0.950)	0.054
Subgroup C	1.301 (0.702–1.766)	0.403	1.137 (0.982–1.213)	0.117	0.798 (0.431–1.477)	0.473
Subgroup D	0.739 (0.416–1.310)	0.300	1.057 (0.575–2.466)	0.345	0.739 (0.417–1.308)	0.299

^a represents type of infertility with primary as reference, laboratory fertilization mode with IVF as reference, fresh embryo transfer before FET as reference, FET without good-quality embryos as reference, subgroup with subgroup A as reference

BMI=body mass index; FSH=follicle stimulating hormone; FET=Frozen-thawed embryo transfer; OR=Odds ratio; CI=Confidence intervals; ICSI=intracytoplasmic sperm injection; IVF=in vitro fertilization

Subgroup analysis based on trigger-day LH Levels in L-FET cycles

As shown in Table 6, subgroup analysis based on trigger-day LH levels in L-FET cycles demonstrated that clinical outcomes were generally comparable across the three trigger protocols in both the LH < 20 IU/L and LH ≥ 20 IU/L groups. No significant differences were observed in the PPR, CPR, LBR, or EPR (all P > 0.05). However, the dual trigger protocol was associated with a higher PLR in both LH subgroups, reaching statistical significance in the LH (≥ 20 IU/L) cohort compared with the hCG trigger group (30.0% vs. 11.9%, P < 0.05).

Table 6.

Subgroup analysis results based on trigger-day LH levels in L-FET cycles

	LH<20IU/L (N = 501)			LH≥20IU/L (N = 272)
	dual trigger	hCG trigger	GnRHa trigger	dual trigger	hCG trigger	GnRHa trigger
Number of patients	66	267	168	39	163	70
Positive pregnancy rate	59.1% (39/66)	53.2% (142/267)	52.4% (88/168)	53.8% (21/39)	54.6% (89/163)	51.4% (38/70)
Clinical pregnancy rate	53.0% (35/66)	49.8% (133/267)	51.2% (86/168)	51.3% (20/39)	51.5% (84/163)	45.7% (32/70)
Ectopic pregnancy rate	8.6% (3/35)	5.3% (7/133)	4.7% (4/86)	5.0% (1/20)	6.0% (5/84)	3.1%% (1/32)
Pregnancy loss rate	22.9% (8/35)	11.3% (15/133)	14.0% (12/86)	30.0% (6/20)	11.9% (10/84)*	12.5% (4/32)
Live birth rate	34.8% (23/66)	39.7% (106/267)	39.3% (66/168)	33.3% (13/39)	41.1% (67/163)	35.7% (25/70)
Singletons	27.3% (18/66)	30.3% (81/267)	28.6% (48/168)	25.6% (10/39)	32.5% (53/163)	30.0% (21/70)
Twins	7.6% (5/66)	9.4% (25/267)	10.7% (18/168)	7.7% (3/39)	8.6% (14/163)	5.7% (4/70)

Data are presented as percentage (number/total). * Compared with dual trigger group, P < 0.05

LH=luteinizing hormone

Discussion

To the best of our knowledge, this is the largest study to date to provide a detailed analysis of pregnancy outcomes associated with different triggering strategies in L-FET and mNC-FET cycles. In this study, triggering in mNC-FET cycles included hCG or GnRHa, whereas in L-FET cycles, a dual trigger protocol was also applied in addition to these two methods. Our findings showed that in both L-FET and mNC-FET cycles, the hCG trigger group exhibited higher CPR and LBR and a lower PLR than the GnRHa trigger group, although none of these differences reached statistical significance.

Similarly, Le et al. reported that in hMG-stimulated IUI cycles supported by luteal phase supplementation, the PPR (14.8% vs. 22.2%) and CPR (13.3% vs. 23.2%) were lower in the GnRHa trigger group than in the hCG trigger group, though the differences were not statistically significant [30]. Previous studies have shown that, following hCG triggering, concentrations of progesterone, inhibin, and amphiregulin in the follicular fluid were significantly higher than those observed after GnRHa triggering [31–33]. Additionally, differences in gene expression profiles of granulosa and cumulus cells, as well as early luteal endometrial gene expression, have been observed when comparing GnRHa and hCG triggers, with LH suggested as the upstream regulatory factor underlying these differences [34, 35]. These differences likely reflect the fact that a standard hCG trigger induces supraphysiological LH-like activity, thereby enhancing ovarian steroidogenesis more strongly than GnRHa triggering. In contrast, the stimulatory effect of a GnRHa trigger on steroidogenesis is lower than that of the natural mid-cycle gonadotropin surge, although full oocyte maturation can still be achieved [36].

In a large retrospective study, Chen et al. analyzed 6,075 LE-hMG IUI cycles and found comparable CPR among the hCG, GnRHa, and dual-trigger groups (15.8%, 16.0%, and 15.9%, respectively; P = 0.964). Notably, none of the three groups in that study received LPS [21]. In contrast, Huang et al. reported that in L-FET cycles with standardized LPS, both CPR (53.12% vs. 47.0%) and LBR (43.75% vs. 38.89%) were higher in the GnRHa trigger group than in the hCG trigger group, although the differences were not statistically significant (both P > 0.05) [22].

Most previous studies on the dual trigger have focused primarily on its ability to increase the number of retrieved and mature oocytes and to improve pregnancy outcomes in GnRH antagonist protocols for IVF [20, 37]; however, its effects in stimulated FET cycles have not been investigated. In the present analysis of 773 L-FET cycles, the results suggested that the dual trigger group achieved higher PPR and CPR than the hCG and GnRHa groups, although these differences did not reach statistical significance. Several mechanisms have been proposed to explain the beneficial effects of the dual trigger. These include the induction of both LH and FSH surges that mimic the physiological conditions of natural ovulation, as well as the direct action of GnRHa on its receptors [20]. Specifically, GnRHa stimulates the pituitary gland to release LH and FSH. The LH surge acts synergistically with hCG to activate LH receptors. At the same time, FSH promotes nuclear maturation and cumulus expansion by maintaining open gap junctions between the oocyte and cumulus cells and by enhancing LH receptor expression in luteinizing granulosa cells [19]. In addition to these pituitary-mediated effects on gonadotropin release, GnRHa can directly regulate ovulation by acting on ovarian granulosa cells [38]. Furthermore, GnRH promotes adhesion between endometrial epithelial cells and the embryo [39]. By activating endocrine–paracrine mechanisms, it may enhance endometrial receptivity and facilitate embryo–endometrium communication [40]. Interestingly, crosstalk between the endocrine and immune systems, possibly mediated by GnRH and its receptor, may also play an essential role in human embryonic implantation [41]. A previous study reported that adding a low dose of GnRHa on the day of hCG administration during ovulation therapy could enhance CPR by increasing serum progesterone levels, improving endometrial blood perfusion, and increasing endometrial thickness [42].

Additionally, the present study found that the dual trigger group exhibited a significantly higher PLR (P < 0.05) and a lower LBR (P > 0.05) compared with the hCG and GnRHa trigger groups. This pattern was particularly pronounced in the LH surge (≥ 20 IU/L) subgroup, where the PLR was significantly higher in the dual trigger group than in the hCG trigger group (P < 0.05). Similarly, a retrospective study conducted in 2021 involving 466 patients undergoing either STC or NC-FET reported that the combined use of GnRHa and low-dose hCG for ovulation induction significantly increased the risk of early PLR [43]. Chen et al. also demonstrated that, in LE-hMG IUI cycles, the PLR was higher in the dual-trigger group than in the hCG and GnRHa trigger groups (P = 0.045) [21].

Previous research has highlighted that LPS is a critical determinant of reproductive outcomes in GnRHa-triggered cycles, rather than the GnRHa trigger protocol itself [44]. Luteal phase defects may result in impaired endometrial responsiveness to progesterone [40]. In natural menstrual cycles, the spontaneous LH surge is characterized by a short ascending phase (≈ 14 h), a peak plateau (≈ 14 h), and a prolonged descending phase (≈ 20 h) [45]. By contrast, hCG has a significantly longer half-life (32–33 h) than LH, maintaining luteal function for 7–10 days. The LH surge induced by GnRHa, however, is transient, with a short ascending limb (> 4 h) and a long descending limb (> 20 h) [46], and results in a lower total release of circulating LH and FSH after the trigger. Moreover, a potential direct luteolytic effect of GnRHa has been proposed, supported by in vitro studies showing that GnRHa can initiate apoptotic cascades in granulosa cells [47, 48]. Therefore, a reinforced or optimized LPS regimen is crucial for achieving favorable pregnancy outcomes in GnRHa-triggered cycles.

Another study reported that the flare-up effect of GnRHa induces a physiological increase in both FSH and LH levels, which may positively influence cumulus cell growth and distribution [49]. Moreover, continuous hCG stimulation can lead to supraphysiological luteal steroid levels, suppressing the release of endogenous gonadotropins essential for corpus luteum support and ultimately impairing endometrial receptivity [50]. Physiologically, it has been proposed that a refractory period follows the initial GnRHa trigger, during which granulosa cells become unresponsive to subsequent LH-like exogenous stimulation. This hypothesis was supported by a pilot study in which a low dose of hCG (1500 IU) was administered either 12–35 h after the GnRHa trigger, followed by standard LPS [51]. In the control group, ovulation was triggered with hCG (10,000 IU). Compared with the 12-h group, the 35-h group demonstrated higher mid-luteal progesterone levels and improved pregnancy rates. Notably, no significant difference in pregnancy outcomes was observed between the GnRHa + 35-h hCG group and the hCG only group. These findings may help explain the higher miscarriage rate observed in the dual-trigger group compared with the hCG and GnRHa groups, despite the use of a small dose of hCG and progesterone supplementation intended to counteract luteolysis. Furthermore, the relatively small sample size of the dual-trigger group may have introduced bias into the statistical analysis, potentially contributing to the observed increase in PLR.

In the present study, the hCG trigger group exhibited the lowest PLR among the L-FET cycles. To further investigate this observation, subgroup analyses were conducted to evaluate the impact of different hCG trigger doses on pregnancy outcomes. Interestingly, no significant differences were found among the various hCG dose groups. Evidence from animal studies has shown that ovulation requires approximately 85% of the LH-like activity generated by the natural mid-cycle gonadotropin surge. In contrast, oocyte maturation can occur when follicles are exposed to only about one-third of this activity [52]. Therefore, the use of an hCG trigger (e.g., 10,000 IU, considered the gold standard) produces supraphysiological LH-like activity and provides a signal for final oocyte maturation that far exceeds physiological requirements [53]. This conclusion is consistent with several previous studies [54, 55]. For instance, a retrospective cohort study in high responders demonstrated that a reduced hCG dose of 3300 IU yielded comparable numbers of mature and fertilized oocytes, as well as similar pregnancy rates to those achieved with a 5000 IU dose [54]. Similarly, another retrospective study comparing hCG doses of 3300, 4000, 5000, and 10,000 IU found no significant differences in oocyte maturation, CPR, or LBR across the groups [55].

During the natural menstrual cycle, progesterone peaks around the time of implantation, approximately 8 days after ovulation, triggered by the LH surge. A mid-luteal progesterone concentration of ≥ 25 nmol/L is considered evidence of ovulation and a functional corpus luteum [56]. During early pregnancy, trophoblast-derived hCG maintains corpus luteum function until the luteo-placental shift occurs around 7 weeks of gestation [57, 58]. In assisted reproductive technology (ART), hCG is widely used as a surrogate for endogenous LH to induce final oocyte maturation and ovulation. A randomized controlled trial demonstrated that higher hCG trigger doses significantly increased endogenous progesterone levels during the mid-to-late luteal phase in women undergoing GnRH antagonist IVF cycles [59]. Endogenous progesterone plays multiple essential roles during the luteal phase, including supporting early implantation. It also regulates the implantation window through the secretory transformation of the endometrium, enhances endometrial vascularity, modulates the immune response, and reduces uterine contractions before implantation [56, 60]. Nevertheless, that trial found no corresponding improvement in the CPR [59].

In ART, luteal phase abnormalities are often attributed to supraphysiological steroid concentrations produced by multiple corpora lutea following exogenous gonadotropin stimulation. These elevated steroid levels suppress the pituitary release of LH and FSH through negative feedback on the hypothalamic-pituitary axis, which can lead to corpus luteum regression if not supported [60, 61]. In L-FET, small doses of ovulation-inducing agents do not produce such supraphysiological steroid concentrations and therefore do not cause severe luteal defects. LE enhances luteal function by stimulating corpus luteum activity and progesterone secretion [11]. In this context, an hCG dose of 4000 IU may be sufficient to trigger oocyte maturation and provide adequate luteal support for pregnancy. Consistent with this, increasing the hCG trigger dose did not improve pregnancy outcomes in our study. Given the inherent limitations of retrospective analyses, prospective studies are warranted to further clarify the relationship between hCG trigger dose and reproductive outcomes in L-FET cycles.

With respect to its strengths, this retrospective study, which compares different trigger methods in L-FET and mNC-FET cycles, represents the largest sample size reported to date, with a 100% follow-up rate at the time of reporting. Additionally, subgroup analyses were conducted for L-FET cycles based on hCG trigger dose and serum LH levels on the trigger day. These findings indicated that an appropriate hCG dose can optimize luteal function and endometrial receptivity, thereby providing clinicians with practical guidance for selecting trigger strategies and dosages. Such optimization may help reduce the risk of early pregnancy loss while balancing efficacy and safety. Furthermore, this study highlights the need to individualize trigger strategies in L-FET cycles and provides a foundation for future multicenter prospective studies.

Nevertheless, certain limitations should be acknowledged. First, the retrospective design meant that patients were assigned to different regimens according to clinical practice, which may have introduced selection bias. Second, although binary logistic regression was applied to minimize confounding, unmeasured factors such as psychological state, physical activity, use of nutritional supplements, and dietary habits could not be fully controlled. Third, this study was conducted at a single reproductive center; therefore, validation of these findings through a multicenter clinical trial would enhance their generalizability. Fourth, analysis of cycle cancellation rates was not performed. Finally, a more comprehensive assessment would require follow-up of obstetric and neonatal outcomes to strengthen the clinical relevance of the results.

Conclusion

This study demonstrated that neither hCG nor GnRHa triggers significantly affected pregnancy outcomes in women undergoing mNC-FET. Therefore, clinicians may select a triggering method based on individual patient characteristics and clinical judgement. In women undergoing L-FET, a dual trigger comprising 0.1 mg GnRHa combined with 2000 IU hCG was associated with an increase in PLR, particularly in the presence of an LH surge. Consequently, the routine use of a dual trigger in L-FET cycles is not recommended. If luteal support is applied, it should be administered cautiously and tailored to individual needs.

Furthermore, the hCG trigger group showed a significantly lower PLR, and variations in hCG trigger dose did not significantly influence pregnancy outcomes. No significant differences were observed in PPR, CPR, or LBR among the different trigger strategies. Taken together, these findings suggest that a 4000 IU hCG trigger may represent a cost-effective and clinically feasible option for patients undergoing L-FET cycles. Nevertheless, large-scale, multicenter, prospective studies are warranted to validate and expand upon these findings.

Abbreviations

aOR

adjusted odds ratio

ART

assisted reproductive technology

CI

confidence interval

CPR

clinical pregnancy rate

EPR

ectopic pregnancy rate

FET

frozen–thawed embryo transfer

FSH

follicle-stimulating hormone

GnRHa

gonadotropin-releasing hormone agonist

hCG

human chorionic gonadotropin

IUI

intrauterine insemination

LBR

live birth rate

LE

letrozole

LH

luteinizing hormone

L-FET

letrozole-stimulated frozen–thawed embryo transfer

LPS

luteal phase support

mNC-FET

modified natural cycle frozen–thawed embryo transfer

OR

odds ratio

PLR

pregnancy loss rate

PPR

positive pregnancy rate

Authors’ contributions

LG wrote the manuscript. MY contributed to data collection. HW guided the design and reviewed the manuscript. YL assisted in data analysis. SX and FL guided the design and implementation of the study. All authors read and approved the final manuscript.

Funding

This study was funded by the National Natural Science Foundation of China (82474560, 81974577, 82174429) and the Natural Science Foundation Project of Shandong Province (ZR2023MH112). This work was supported by the National Natural Science Foundation of China (General Program, Grant No. 82474560), which also covers the article processing charges.

Data availability

The datasets analyzed in this study will be available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

This study received approval from the Institutional Review Board and the Institutional Ethics Committee of the Reproductive Medical Center at the affiliated Hospital of Shandong University of Traditional Chinese Medicine (ref approval no. AF/SC-08/02.0). All procedures were conducted in accordance with relevant guidelines and regulations. Furthermore, informed consent was obtained from all participants prior to the study.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.