Premature timing of progesterone luteal phase support initiation did not negatively impact live birth rates in modified natural frozen thawed embryo transfer cycles.

Jing-Yan Song: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data. Zhen-Gao Sun: Conceived and designed the experiments. Wen-Jing Jiang: Performed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

The demographic and baseline IVF characteristics of 756 mNC-FET patients were investigated ( Table 1 ). Luteal phase support was administered to 182 patients 24 h after hCG triggering, and to 574 patients 48 h afterwards. There were no significant differences between the two groups regarding demographics, cycle features, ovarian stimulation data, embryo outcomes, or cycle outcomes (P > 0.05). Table 1 Demographic and baseline IVF characteristics of the study groups. Table 1 Premature LPS (n = 182) Conventional LPS (n = 574) P-value Demographic characteristics Female age at oocyte retrieval (years), mean ± SD 32.3 ± 3.9 32.6 ± 4.1 0.365 <35 yrs. 121 (66.5) 364 (63.4) 0.452 ≥35 yrs. 61 (33.5) 210 (36.4) BMI (kg/m 2 ), mean ± SD 22.6 ± 2.9 22.9 ± 2.9 0.296 Cycle characteristics Cause of infertility 0.289 Tubal factor 94 (51.6) 263 (45.8) Male factor 33 (18.1) 110 (19.2) Mixed factor 30 (16.5) 77 (13.4) Ovulation disorder/PCOS 8 (4.4) 54 (9.4) Endometriosis 9 (4.9) 41 (7.1) DOR or AMA 4 (2.2) 14 (2.4) Unexplained 4 (2.2) 15 (2.6) Duration of infertility (years), median (IQR) 3 (1, 4) 3 (2, 4) 0.512 Nulliparous 73 (40.1) 248 (43.2) 0.462 Gravidity, median (IQR) 1 (0, 2) 1 (0, 2) 0.990 Parity, median (IQR) 0 (0, 1) 0 (0, 1) 0.760 Basic FSH (mIU/ml), mean ± SD 7.4 ± 4.9 7.5 ± 3.1 0.863 Basic LH (mIU/ml), mean ± SD 5.0 ± 2.7 5.0 ± 2.6 0.981 Basic E 2 (pg/ml), mean ± SD 40.6 ± 17.8 40.8 ± 17.7 0.903 Ovarian stimulation data Type of GnRH analog 0.152 GnRH-a 91 (50.0) 291 (50.7) GnRH-ant 80 (44.0) 223 (38.9) None (mini-stimulation) 11 (6.0) 60 (10.5) No of days of COS, mean ± SD 10.9 ± 2.3 10.7 ± 2.4 0.185 Total Gn dose administered (IU), median (IQR) 2475 (2025, 3066) 2325 (1800, 3000) 0.115 LH on trigger day (mIU/ml), median (IQR) 1.5 (1.0, 2.6) 1.7 (1.0, 2.8) 0.624 E 2 on trigger day (pg/ml), mean ± SD 3604 ± 1332 3575 ± 1450 0.811 P 4 on trigger day (ng/ml), median (IQR) 1.1 (0.8, 1.9) 1.1 (0.8, 1.5) 0.074 Trigger drugs 0.319 hCG 161 (88.5) 491 (85.5) GnRH-a and hCG 21 (11.5) 83 (14.5) Embryologic outcomes No of oocytes retrieved, mean ± SD 14.1 ± 7.7 14.0 ± 7.8 0.877 Fertilization methods 0.710 IVF 141 (77.5) 437 (76.1) ICSI 41 (22.5) 137 (23.9) 2 PN zygotes, mean ± SD 8.9 ± 5.8 8.4 ± 5.2 0.253 Embryos available for transfer, mean ± SD 4.7 ± 2.4 4.5 ± 2.5 0.489 Blastocysts available for transfer, median (IQR) 2 (1, 3.5) 3 (2, 5) 0.227 No of high-quality embryos, median (IQR) 1 (0, 2) 1 (0, 2) 0.124 Cycle outcomes 0.436 Fresh ETs 44 (24.2) 123 (21.4) Freeze-all cycles 138 (75.8) 451 (78.6) Abbreviation: LPS, luteal phase support; DOR, diminished ovarian reserve; AMA, advanced maternal age; IQR, interquartile range; BMI, body mass index; AMH, anti-müllerian hormone; FSH, follicle stimulating hormone; LH, luteinizing hormone; E 2 , estradiol; P 4 , progesterone; GnRH-a, gonadotropin releasing hormone agonist; GnRH-ant, GnRH antagonist; IVF, in-vitro fertilization; ICSI, intracytoplasmic sperm injection; 2 PN, double pronuclear fertilization; Gn, gonadotropin; IU, international units. Data are presented as numbers (%) unless otherwise noted. Demographic and baseline IVF characteristics of the study groups. Abbreviation: LPS, luteal phase support; DOR, diminished ovarian reserve; AMA, advanced maternal age; IQR, interquartile range; BMI, body mass index; AMH, anti-müllerian hormone; FSH, follicle stimulating hormone; LH, luteinizing hormone; E 2 , estradiol; P 4 , progesterone; GnRH-a, gonadotropin releasing hormone agonist; GnRH-ant, GnRH antagonist; IVF, in-vitro fertilization; ICSI, intracytoplasmic sperm injection; 2 PN, double pronuclear fertilization; Gn, gonadotropin; IU, international units. Data are presented as numbers (%) unless otherwise noted. In terms of baseline mNC-FET characteristics ( Table 2 ), there was a greater number of assisted hatching procedures in the premature LPS group than in the conventional LPS group (53.8% vs. 42.3%, P = 0.007). Nevertheless, there were no significant differences in the number of previous ET cycles, LPS protocol, serum E 2 , LH, P 4 levels on LH surge day, serum E 2 , P 4 levels on ET day, number of transferred embryos, embryo developmental stage, good-quality embryo transferring, physicians performing ET procedures, and endometrial thickness between the two groups (P > 0.05). Table 2 Baseline mNC-FET characteristics of the study groups. Table 2 Premature LPS (n = 182) Conventional LPS (n = 574) P-value Number of previous ET cycles 0.199 1 83 (45.6) 310 (54.0) 2 59 (32.4) 167 (29.1) 3 25 (13.7) 56 (9.8) ≥4 15 (8.3) 41 (7.1) Luteal support protocol 0.512 Injected progesterone 62 (34.1) 213 (37.1) Vaginal progesterone 39 (21.4) 135 (23.5) Dydrogesterone 23 (12.6) 76 (13.2) Mixed 58 (31.9) 150 (26.1) LH level on LH surge day (IU/L), median (IQR) 16.3 (8.6, 29.2) 16.3 (10.5, 32.1) 0.134 E 2 level on LH surge day (pg/ml), median (IQR) 177 (107, 255) 160 (97, 250) 0.135 P 4 level on LH surge day (ng/ml), median (IQR) 1.2 (0.8, 1.6) 1.3 (1.0, 1.6) 0.158 E 2 level on ET day (pg/ml), median (IQR) 124 (100, 169) 134 (98, 193) 0.213 P 4 level on ET day (ng/ml), median (IQR) 17.4 (13.3, 23.5) 17.7 (13.0, 23.1) 0.995 Assisted hatching 98 (53.8) 243 (42.3) 0.007 Number of transferred embryos 0.922 Single 45 (24.7) 144 (25.1) Double 137 (75.3) 430 (74.9) Embryo developmental stage 0.929 D3 30 (16.5) 93 (16.2) D5/D6 152 (83.5) 481 (83.8) Good-quality embryo transferring 84 (46.2) 256 (44.6) 0.713 Physicians performing ET procedures 0.585 Physician A 42 (23.1) 161 (28.1) Physician B 43 (23.6) 132 (23.0) Physician C 45 (24.7) 136 (23.7) Physician D 52 (28.6) 145 (25.3) Endometrial thickness (mm), mean ± SD 10.5 ± 1.7 10.3 ± 1.6 0.186 Abbreviation: mNC, modified natural cycle; LPS, luteal phase support; FET, frozen embryo transfer; IQR, interquartile range; LH, luteinizing hormone; E 2 , estradiol; P 4 , progesterone; SET, single embryo transfer; DET, double embryo transfer. Data are presented as numbers (%) unless otherwise noted. Statistically significant values are indicated in bold. Baseline mNC-FET characteristics of the study groups. Abbreviation: mNC, modified natural cycle; LPS, luteal phase support; FET, frozen embryo transfer; IQR, interquartile range; LH, luteinizing hormone; E 2 , estradiol; P 4 , progesterone; SET, single embryo transfer; DET, double embryo transfer. Data are presented as numbers (%) unless otherwise noted. Statistically significant values are indicated in bold. The primary outcome of LBR in the premature LPS group was non-inferior to the conventional LPS group (30.8% vs. 31.2%, OR 0.99, 95% CI 0.77–1.27, P = 0.916), as shown in Table 3 . Furthermore, no significant differences were seen in positive pregnancy rate (39.6% vs.38.2%, OR 1.06, 95% CI 0.75–1.49, P = 0.734), clinical pregnancy rate (36.8% vs.37.6%, OR 0.97, 95% CI 0.68–1.36, P = 0.843), clinical pregnancy loss (18.1% vs.16.0%, OR 1.13, 95% CI 0.63–2.01, P = 0.681), ectopic pregnancy rate (4.2% vs.2.3%, OR 1.83, 95% CI 0.45–7.45, P = 0.665), multiple pregnancy rate (including twins) (15.3% vs.15.5%, OR 0.98, 95% CI 0.53–1.84, P = 0.960), preterm delivery (13.4% vs.12.5%, OR 1.08, 95% CI 0.53–2.17, P = 0.841), small for gestational age (5.4% vs.6.1%, OR 0.87, 95% CI 0.25–3.02, P > 0.999), large-for-gestational-age infants (10.7% vs. 8.9%, OR 1.20, 95% CI 0.49–2.92, P = 0.691), gestational diabetes (4.5% vs. 6.4%, OR 0.65, 95% CI 0.19–2.16, P = 0.663), and gestational hypertension (7.5% vs. 8.8%, OR 0.85, 95% CI 0.33–2.19, P = 0.732) between premature LPS and conventional LPS groups. In terms of embryo implantation rate and singleton birth weight, the independent t -test findings revealed no significant difference between the two groups (P > 0.05). Table 3 Pregnancy and birth outcomes between the two study groups. Table 3 Premature LPS (n = 182) Conventional LPS (n = 574) Odds ratio (95% CI) P-value Positive pregnancy rate 72 (39.6) 219 (38.2) 1.06 (0.75 to 1.49) 0.734 Clinical pregnancy rate 67 (36.8) 216 (37.6) 0.97 (0.68 to 1.36) 0.843 Embryo implantation rate (%), mean ± SD 25.3 ± 36.3 25.0 ± 35.8 – 0.928 Pregnancy loss per positive pregnancy 13 (18.1) 35 (16.0) 1.13 (0.63 to 2.01) 0.681 Ectopic pregnancy per positive pregnancy 3 (4.2) 5 (2.3) 1.83 (0.45 to 7.45) 0.665 Live birth rate 56 (30.8) 179 (31.2) 0.99 (0.77 to 1.27) 0.916 Multiple pregnancy rate per positive pregnancy (including twins) 11 (15.3) 34 (15.5) 0.98 (0.53 to 1.84) 0.960 Preterm delivery among clinical pregnancies 9 (13.4) 27 (12.5) 1.08 (0.53 to 2.17) 0.841 Singleton birthweight (g), mean ± SD 3268.1 ± 484.1 3291.8 ± 478.8 – 0.720 Small for gestational age * 3 (5.4) 11 (6.1) 0.87 (0.25 to 3.02) >0.999 Large for gestational age * 6 (10.7) 16 (8.9) 1.20 (0.49 to 2.92) 0.691 Gestational diabetes among clinical pregnancies 3 (4.5) 15 (6.4) 0.65 (0.19 to 2.16) 0.663 Gestational hypertension among clinical pregnancies 5 (7.5) 19 (8.8) 0.85 (0.33 to 2.19) 0.732 Abbreviation: LPS, luteal phase support; CI, confidence interval. *Small and large for gestational age defined as less than or more than two standard deviation units from expected birth weight. Small and large for gestational age were calculated from growth curves for Scandinavian children adjusted for sex and gestational age. Data are presented as numbers (%). Pregnancy and birth outcomes between the two study groups. Abbreviation: LPS, luteal phase support; CI, confidence interval. *Small and large for gestational age defined as less than or more than two standard deviation units from expected birth weight. Small and large for gestational age were calculated from growth curves for Scandinavian children adjusted for sex and gestational age. Data are presented as numbers (%). According to Table 4 , binary logistic regression analysis (allowing for confounder correction) revealed no significant connection between timing of LPS initiation after hCG triggering in mNC-FET cycles and LBR in the adjusted model (adjusted OR 0.98, 95% CI 0.67–1.43, P = 0.913). Female age at oocyte retrieval (≥35 yrs. vs. < 35 yrs, adjusted OR 0.62, 95% CI 0.44–0.89, P = 0.009), BMI (≥25 kg/m 2 vs. < 25 kg/m 2 , adjusted OR 0.63, 95% CI 0.42–0.94, P = 0.024), embryo transfer stage (D5/D6 vs. D3, adjusted OR 5.32, 95% CI 2.05–13.81, P < 0.001), physicians of embryo transfer (physician D vs. physician A, adjusted OR 0.43, 95% CI 0.27–0.69, P < 0.001), and number of embryos transferred (Double vs. Single, adjusted OR 5.12, 95% CI 2.13–12.33, P < 0.001) were all independent factors in LBR. Table 4 Crude and adjusted OR for the timing of LPS initiation in mNC-FETs and other key potential confounders for LBRs. Table 4 Variable Crude OR (95% CI) * Adjusted OR (95% CI) Adjusted P-value Timing of LPS initiation in mNC-FETs Premature LPS group Reference Reference Conventional LPS group 0.98 (0.68 to 1.41) 0.98 (0.67 to 1.43) 0.913 Female age at oocyte retrieval <35 yrs. Reference Reference ≥35 yrs. 0.60 (0.43 to 0.84) 0.62 (0.44 to 0.89) 0.009 Body mass index (BMI) 10 mIU/ml 0.90 (0.54 to 1.50) 1.06 (0.61 to 1.86) 0.829 LH on menstrual cycle days 2 – 3 ≤7 mIU/ml Reference Reference >7 mIU/ml 0.63 (0.40 to 1.00) 0.65 (0.40 to 1.06) 0.082 Cycle outcomes Freeze-all cycles Reference Reference Fresh ETs 1.12 (0.77 to 1.61) 1.34 (0.90 to 2.01) 0.148 Number of previous ET cycles <3 Reference Reference ≥3 0.75 (0.50 to 1.14) 0.71 (0.45 to 1.11) 0.129 LH level on LH surge day <15 mIU/ml Reference Reference 15–24.9 mIU/ml 0.78 (0.51 to 1.21) 0.79 (0.50 to 1.25) 0.316 25–39.9 mIU/ml 0.96 (0.62 to 1.48) 1.01 (0.64 to 1.60) 0.955 ≥40 mIU/ml 1.27 (0.84 to 1.94) 1.31 (0.85 to 2.04) 0.225 P 4 level on LH surge day <1.0 ng/ml Reference Reference ≥1.0 ng/ml 0.99 (0.71 to 1.38) 0.95 (0.67 to 1.35) 0.774 Luteal phase support Non-mixed Reference Reference Mixed 1.18 (0.84 to 1.66) 1.21 (0.84 to 1.73) 0.303 P 4 level on ET day <10 ng/ml Reference Reference ≥10 ng/ml 1.27 (0.74 to 2.17) 1.15 (0.65 to 2.02) 0.633 Assisted hatching Yes Reference Reference No 1.11 (0.81 to 1.51) 1.15 (0.82 to 1.61) 0.425 Embryo transfer stage D3 Reference Reference D5/D6 1.04 (0.68 to 1.57) 5.32 (2.05 to 13.81) < 0.001 Number of embryos transferred Single Reference Reference Double 1.61 (1.11 to 1.35) 5.12 (2.13 to 12.33) < 0.001 Good-quality embryo transferring Yes Reference Reference No 0.74 (0.54 to 1.00) 0.74 (0.53 to 1.05) 0.094 Physicians of embryo transfer Physician A Reference Reference Physician B 0.82 (0.54 to 1.24) 0.77 (0.50 to 1.19) 0.241 Physician C 0.91 (0.60 to 1.39) 0.88 (0.57 to 1.37) 0.568 Physician D 0.42 (0.29 to 0.73) 0.43 (0.27 to 0.69) < 0.001 Endometrial thickness prior to FET <8 mm Reference Reference ≥8 mm 1.14 (0.69 to 1.65) 1.13 (0.67 to 1.93) 0.640 Abbreviation: LPS, luteal phase support; OR, odds ratio; CI, confidence interval; mNC, modified natural cycle; FET, frozen embryo transfer; LBR, live birth rate; FSH, follicle stimulating hormone; LH, luteinizing hormone. * No adjustments for other covariates. Statistically significant values are indicated in bold. Crude and adjusted OR for the timing of LPS initiation in mNC-FETs and other key potential confounders for LBRs. Abbreviation: LPS, luteal phase support; OR, odds ratio; CI, confidence interval; mNC, modified natural cycle; FET, frozen embryo transfer; LBR, live birth rate; FSH, follicle stimulating hormone; LH, luteinizing hormone. * No adjustments for other covariates. Statistically significant values are indicated in bold. No significant statistical differences were found between the premature and conventional LPS groups of subsequent sensitivity analyses conducted for LBR according to the serum LH and P 4 levels on LH surge day (P > 0.05 for all, see Table 5 ). Table 5 Sensitivity analysis according to the serum LH and P 4 levels on LH surge day. Table 5 Premature LPS Conventional LPS Odds ratio (95% CI) P-value Total No. Events (%) Total No. Events (%) Total 182 56 (30.8) 574 179 (31.2) 0.99 (0.77 to 1.27) 0.916 LH level on LH surge day <15 mIU/ml 88 27 (30.7) 264 83 (31.4) 0.97 (0.57 to 1.63) 0.894 15–24.9 mIU/ml 30 10 (33.3) 111 27 (24.3) 1.56 (0.65 to 3.73) 0.320 25–39.9 mIU/ml 35 8 (22.9) 97 32 (33.0) 0.60 (0.25 to 1.47) 0.264 ≥40 mIU/ml 29 11 (37.9) 102 37 (36.3) 1.07 (0.46 to 2.52) 0.870 P 4 level on LH surge day <1.0 ng/ml 131 44 (33.6) 392 119 (30.4) 1.16 (0.76 to 1.77) 0.489 ≥1.0 ng/ml 51 12 (23.5) 182 60 (33.0) 0.63 (0.31 to 1.28) 0.197 Abbreviation: LPS, luteal phase support; P 4 , progesterone; LH, luteinizing hormone; CI, confidence interval. Sensitivity analysis according to the serum LH and P 4 levels on LH surge day. Abbreviation: LPS, luteal phase support; P 4 , progesterone; LH, luteinizing hormone; CI, confidence interval.

Between January 2019 and August 2021, 756 mNC-FET cycles performed at the investigator's reproductive center were included in this retrospective cohort study (see Fig. 1 ). The study was authorized by the local institutional review board (Reproductive Ethics Committee of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine, SDTCM/E2208-01, dated August 15, 2022) and was carried out in a public tertiary care hospital. Fig. 1 Flowchart of the study cohort of premature LPS versus conventional LPS in modified natural frozen thawed embryo transfer cycles. LPS, luteal phase support; hCG, human chorionic gonadotropin. Fig. 1 Flowchart of the study cohort of premature LPS versus conventional LPS in modified natural frozen thawed embryo transfer cycles. LPS, luteal phase support; hCG, human chorionic gonadotropin. During this study, ovulatory women undergoing autologous mNC-FET cycles with an age younger than 42 years old were included. The following patients were not included in this investigation: (1) body mass index (BMI) ≥ 35 kg/m 2 ; (2) congenital or acquired uterine abnormalities; (3) recurrent pregnancy loss and recurrent implantation failure; (4) preimplantation aneuploidy genetic testing (PGT-A); (5) in vitro maturation (IVM); (6) cryopreserved oocytes and/or donor oocytes; (7) unfollowed or incompletely documented data. Frozen-thawed embryo transfer with true natural and hormone-replacement cycles were also disregarded. Included patients were divided into two groups of premature LPS group (progesterone initiation 24 h after hCG trigger, n = 182) and conventional LPS group (progesterone initiation 48 h after hCG trigger, n = 574). Premature LPS patients began receiving exogenous progesterone for luteal phase support (LPS) on the first day after hCG triggering, whereas conventional LPS patients begin 48 h after triggering. Patients underwent ovarian stimulation with gonadotropin-releasing hormone agonist (GnRH-a), GnRH antagonist (GnRH-ant), or mini-stimulation protocol as previously described, and oocyte retrieval via ultrasound-guided transvaginal ovarian puncture is carried out 36–37 h after hCG (Ovitrelle®, 250 μg, Merck) or GnRH-a combined with hCG (double-trigger) [ 22 , 23 ]. Following egg retrieval, fertilization was carried out using intracytoplasmic sperm injection (ICSI) or in vitro fertilization (IVF) depending on the quality of the male sperm. Under ultrasound guidance, a fresh embryo transfer was carried out, and any remaining good-quality embryos or blastocysts were vitrified. Alternatively, if clinically indicated, a freeze-all strategy was employed. Good-quality D3 embryos had ≥6 blastomeres and ≤20% fragmentation. Good-quality D5 blastocysts should have embryo scores ≥ 3BB. A detailed description of the vitrification and thawing processes can be found elsewhere [ 24 ]. The American Society for Reproductive Medicine committees’ opinions on guidance on the number of embryos to transfer were followed [ 25 ]. Transvaginal ultrasound and serum hormone measurements (luteinizing hormone (LH), estradiol (E 2 ) and P 4 ) were performed on days 8 through 10 of the menstrual cycle, depending on the duration of the menstrual cycle, to track endometrial thickness and follicle size until ovulation triggering conditions were reached. A single intramuscular injection of 8000 IU hCG (Lizhu Pharmaceutical Trading Co., China) was used to trigger ovulation when the dominant follicle diameter was more than 17 mm, the endometrial thickness was more than 7 mm, P 4 was less than 1.5 ng/ml, and E 2 was more than 150 pg/ml. At approximately 9:00 a.m., the hCG injection was administered. Patients who had unanticipated spontaneous ovulation while being monitored, as well as those who had no prominent follicles by day 25 of the menstrual cycle, were eliminated. In the conventional LPS group, P 4 LPS started 48 h after hCG triggering. Exogenous P 4 was started 24 h after hCG triggering in the premature LPS group. When LPS would be initiated was determined by the attending physician's clinical judgment, which was primarily determined by the specific day the physician was scheduled to perform the embryo transfer procedure, as well as avoiding weekends so that laboratory workload could be reduced. Progesterone (P 4 ) was given intramuscularly (20 mg, Zhejiang Xianju Pharmaceutical Co., Ltd.), vaginally (90 mg, 8% Crinone, Moxerano), orally (10 mg, dydrogesterone, Abbott Laboratories biologicals), or in combination. The manner of administration is determined by the clinical preferences of both physicians and patients. D3 embryos or blastocysts were resuscitated for transfer on Day 4 or Day 6 after P 4 administration. A prominent physician in the department performed the embryo transfer operation. After FET, daily P 4 medication treatment was continued until the pregnancy test came out negative. Luteal phase support was continued if clinical pregnancy was confirmed until the placenta entirely replaced luteal function at 8–10 weeks of gestation. The primary outcome was the live birth rate (LBR), which was defined as the delivery of a viable newborn at 28 weeks or more gestation. The key secondary outcomes included the positive pregnancy rate, clinical pregnancy rate, embryo implantation rate, pregnancy loss rate, ectopic pregnancy rate, and multiple pregnancy rate (including twins). Gestational diabetes, gestational hypertension, preterm delivery, singleton birth weight, and small or large for gestational age are among the maternal and fetal outcomes monitored for more than 24 weeks of gestation. The Shapiro-Wilk test was used to determine the data's normality. If the quantitative variables were regularly distributed, independent t-tests and means and standard deviations (SDs) within the groups of interest were utilized for comparisons. Where data did not have a normal distribution, the Mann-Whitney U test was used to compare median and interquartile range (IQR) among groups of interest. Pearson Chi-square or Fisher exact tests were used to compare qualitative variables, which included numerator and denominator values and were provided as a number or percentage of instances. We used multivariable logistic regression analysis to identify factors that could be linked with the LBR, with the LBR as the dependent variable and timing of LPS initiation after the hCG trigger in the mNC-FET as the key independent variable. Female age at oocyte retrieval, BMI, FSH and LH on menstrual cycle days 2–3, cycle outcomes, number of precious ET cycles, LH and P 4 level on LH surge day, luteal phase support, P 4 level on ET day, assisted hatching, embryo transfer stage, number of embryos transferred, good-quality embryo transferring, physicians of embryo transfer (A, B, C, D), and endometrial thickness were among the potential predictors taken into consideration in the analysis. With a 95% confidence interval (CI), the odds ratio (OR) was used to represent the risk of LBR. In order to confirm the reliability of the findings, we also carried out additional sensitivity analyses on LBR in both groups, taking into account the potential influence of LH (<15, 15–24.9, 25–39.9, ≥40 mIU/ml) and P 4 (<1.0, ≥1.0 ng/ml) levels on the LH surge day [ 26 ]. The SPSS software, version 26.0, was used for all data analyses (SPSS Inc., Chicago, USA). All statistical tests had a two-tailed alpha of 0.05, and a P value < 0.05 was regarded as statistically significant for the two groups.

In summary, our study demonstrated that adding exogenous P4 for LPS 24 h after hCG triggering does not adversely affect mNC-FET cycle outcomes provided enough time is allowed for endometrium exposure to exogenous P4. This provides clinicians with a more flexible choice regarding embryo transfer date scheduling. However, more prospective clinical trials need to be designed to further validate our findings.

We conducted this study to determine if the LBRs in mNC-FET are negatively impacted by the premature timing of LPS initiated by P4 24 h after hCG triggering. As a result, the data imply that the premature timing of P4 LPS initiation has no effect on the LBR in infertile patients using modified natural cycle to prepare endometrium for FET. In mNC-FE, premature LPS initiation had no negative impacts on the cycle outcomes. Thus, we draw the conclusion that patients undergoing mNC-FET can have flexible scheduling for embryo transfer procedure as long as sufficient time was allowed for endometrial exposure to exogenous progesterone. Recent studies suggested that mNC-FET is preferable to HRT-FET for patients with regular ovulation in terms of pregnancy outcomes [ 27 ]. In contrast to HRT-FET, mNC-FET has a lower miscarriage rate, according to several previous studies [ 28 , 29 , 30 , 31 ]. Additionally, mNC-FET is associated with a lower risk of pregnancy complications such as prenatal hypertension and intrahepatic cholestasis, as well as a lower risk of large-for-gestational-age babies in singletons and more favorable postnatal outcomes when compared to HRT-FET [ 32 , 33 ]. Nevertheless, mNC-FET has fewer scheduling options for embryo transfer dates than HRT-FET, which limits its clinical use. Therefore, we performed this retrospective analysis to explore the timing of progesterone LPS for mNC-FET in order to provide doctors and IVF labs greater scheduling flexibility in order to optimize patient benefit. In this study, all patients chose to revive D3 embryos or blastocysts for transfer on day 4 or 6 after P4 administration. Synchronous development of the embryo and receptive endometrium is essential for successful implantation [ 15 ]. Previous studies have shown that embryo implantation rates are significantly reduced when asynchrony of the embryo and endometrium is greater than ±1.5 days [ 34 ]. In 2021, Weiss et al. conducted a proof-of-concept trial in which 56 patients treated with NC-FET underwent D2 cleavage-stage embryo transfer two days following P4 injection, regardless of ovulation or LH surge, and achieved a promising pregnancy outcome [ 35 ]. This shows that superior pregnancy outcomes can be obtained even in the absence of ovulation, provided that synchronization between the embryo and endometrium is maintained. Moreover, a recent meta-analysis suggested that day 3 embryos and blastocysts should be transferred after a full 3 and 5 days of P4 treatment in order to achieve higher implantation rates and improve embryo and endometrial synchrony [ 15 ]. There may be an explanation for the previous finding of Kyrou et al. that adding LPS 24 h after the hCG trigger did not increase ongoing pregnancy rates (22% vs. 21%, p = 0.8) among FET patients compared with those without LPS treatment [ 21 ]. This may be due to the fact that the aforementioned study fixed the time of embryo transfer as 5 days after the hCG trigger, while the LPS group initiated LPS 24 h after the hCG trigger, which could result in asynchrony between the endometrium and the embryo. At the moment, the duration of LPS in clinical practice is ambiguous, and further research is needed to investigate this issue. Some clinics elected to discontinue LPS on the day of a positive pregnancy test, whereas 52% preferred to maintain LPS until 12 weeks' gestation [ 36 ]. Despite a paucity of relevant evidence, it appears that there is a preference for continuing P4 supplementation until 8–10 weeks of gestation [ 37 , 38 ]. In our center, if a clinical pregnancy is verified, LPS will be continued until the placenta completely replaces luteal function at 8–10 weeks of gestation. A debate exists regarding whether LH levels on the day of hCG triggering affect clinical outcomes, and there is no consensus as to the appropriate level of LH for hCG triggering during mNC-FET cycles [ 39 ]. In a retrospective study by Reichman et al. it was discovered that when hCG was administered within one day of the LH surge (LH > 17 IU/L, E2 level dropped), patients undergoing mNC-FET cycles achieved a superior ongoing pregnancy rate [ 40 ]. Another study involved 233 patients who underwent mNC-FET using D4 embryos. In all patients, hCG was triggered once the dominant follicle was greater than 17 mm in diameter and the endometrium was acceptable. Five days after hCG triggering, FET was performed. In their study, the researchers found that spontaneous LH surge (LH > 10 IU/L) did not impact pregnancy outcomes [ 41 ]. Subsequently, A retrospective study conducted by Kahraman and his colleagues in 2020 found that hCG can be administered between the start of the LH rise (15 IU/L) and the LH peak (40 IU/L) without negatively impacting the clinical outcome of mNC-FET [ 42 ]. However, in a study of modified natural cycle for frozen-thawed euploid blastocyst transfer, an increase in LH ≥ 13 mIU/ml before the administration of hCG was found to have a negative impact on clinical pregnancy rates, and the researchers advised that in the presence of LH level ≥13 mIU/ml, it is recommended to avoid hCG administration and to plan the embryo transfer only after spontaneous follicular ruptures have occurred [ 43 ]. A possible explanation for this is the extremely small sample size of the LH ≥ 13 group (n = 22). Consequently, we performed an additional sensitivity analysis to assess the potential impact of LH and P4 levels on LBR on hCG trigger day and to confirm the reliability of the results, and found no significant statistical difference between groups. For mNC-FET, there is now debate over when to add P4 following hCG triggering, and no research has explored its influence on LBR. Our study fills this need and is novel. In spite of this, there are some limitations to this study. Firstly, due to the fact that it is a retrospective analysis conducted by a single center, this study is biased. Furthermore, we do not anticipate monitoring patients for follicular rupture or ovulation after hCG triggering. Further research is required to corroborate our findings in the future. Despite these limitations, our study findings are still useful for doctors using customizable mNC-FET methods.

In the realm of assisted reproduction, embryo freezing and frozen-thawed embryo transfer (FET) have been extensively employed since the first embryo freezing, resuscitation, and transfer to achieve pregnancy in 1983. Frozen-thawed embryo transfer can successfully minimize the risk of ovarian hyperstimulation syndrome (OHSS), reduce the number of ovarian stimulation cycles, lower the danger of ectopic pregnancy, and maximize the cumulative pregnancy rate. Thus, the proportion of FETs in embryo transfer cycles has been increasing rapidly over the past few years [ 1 ]. Currently, natural cycle (NC-FET), hormone replacement treatment cycle (HRT-FET), and stimulation cycles are the most frequently employed endometrial preparation regimens in clinical practice. However, there is no consensus on the optimal endometrial preparation protocol [ 2 ]. Notably, NC-FET is indicated for patients who experience regular ovulation [ 3 , 4 ]. Although NC-FET does not require exogenous hormones and makes the process physiologically similar to the patients' natural states, it has the disadvantages of repetitive monitoring and high cycle cancellation rate [ 5 ]. Hence, in clinical practice, human chorionic gonadotropin (hCG) is routinely used to simulate endogenous LH surge to induce ovulation, which is also known as mNC-FET. Compared to NC-FET, mNC-FET minimized the need for ultrasound and serum hormone monitoring, shortened the monitoring period, made it easier for clinicians to schedule the timing of embryo transfer, and decreased cycle cancellation rates [ 6 , 7 ]. Furthermore, it improved patient acceptability and economic cost-effectiveness. Although NC-FET or mNC-FET patients have regular monthly cycles and normal serum progestogen (P4) levels, some still experience luteal phase defects (LPD), which are aberrant endometrial responses to acceptable amounts of P4 exposure or inadequate P4 generation during the luteal phase, which lead to unsuccessful embryo implantation and early abortion [ 8 ]. According to recent studies, luteal phase support (LPS) enhances the success of mNC-FET pregnancies [ [9] , [10] , [11] ]. In terms of the optimal timing for administering P 4 in mNC-FET, there is disagreement, and local clinic practices differ significantly throughout the globe. In clinical practice, most investigators believe that ovulation occurs 36–48 h after hCG injection, so current studies often start using P 4 for LPS 48 h after hCG triggering [ 9 , 10 , 12 , 13 ], and some investigators administer it 36 h after hCG triggering [ 14 , 15 ]. However, it has been shown that hCG triggering leads to an increase in progesterone in the early luteal phase [ 16 , 17 , 18 ], which advances the implantation window and leads to asynchrony between the embryo and endometrium, which may lead to implantation failure [ 19 , 20 ]. Therefore, a limited number of researchers have decided that P 4 administration should take place 24 h after hCG triggering in order to prevent the issue of decreased endometrial receptivity to embryos brought on by early elevations of P 4 following hCG triggering [ 21 ]. Additionally, no comparative studies have been done on the most appropriate time to provide P 4 LPS during mNC-FET cycles. Therefore, the purpose of this study was to retrospectively assess the relationship between the timing of the administration of P4 LPS and reproductive outcomes in mNC-FET and to explore the optimal timing of P4 initiation for providing clinicians and patients with more options.

The authors declare no conflict of interest.

Data will be made available on request