Clinical outcomes following GnRH antagonist protocol change to PPOS protocol versus GnRH antagonist protocol in patients with pre-elevated progesterone: a propensity score matching study.

This retrospective cohort study included patients who underwent superovulation treatment initiated with the antagonist protocol in the Department of Reproductive Genetics of the First Affiliated Hospital of Kunming Medical University from January 2020 to December 2024. Inclusion criteria: The first in vitro fertilization (IVF)/Intracytoplasmic sperm injection (ICSI) cycle, the population with elevated progesterone ≥ 0.9 ng/ml during the process of ovulation induction, fresh embryo transfer was canceled due to elevated progesterone, and antagonist protocol cycle at the initiation ( n = 2797). The exclusion criteria are as follows: Chromosomal abnormalities, recurrent miscarriage, endometriosis disease, frozen egg cycles, patients undergoing Rescue intracytoplasmic sperm injection (RICSI) for IVF non-fertilization, patients undergoing Preimplantation genetic testing (PGT) for assisted reproduction, and patients with elevated progesterone ≥ 0.9 ng/ml on the Human chorionic gonadotropin (HCG) injection day ( n = 1154). Additionally, patients with missing measurement of hormone levels on the HCG day and those with failed follow-up were excluded ( n = 978). Ultimately, 231 patients whose P increased ≥ 0.9 ng/ml when initiated with the GnRH antagonist protocol were switched to the PPOS protocol, while 345 patients maintained the original antagonist protocol after P increased ≥ 0.9 ng/ml. Since there were differences in demographic characteristics between the two groups of patients, we used propensity score matching to control confounding bias [ 17 ]. Its principle is to calculate the conditional probability of being assigned to the experimental group based on the values of covariates and thereby identify a control group with similar characteristics [ 18 ]. The tolerance value we adopted was 0.001. Through propensity matching, we obtained 188 patients with consistent demographic characteristics for each group. This study was approved by the Ethics Committee of the First Affiliated Hospital of Kunming Medical University, and informed consent was waived. A flowchart of the study is shown in Fig. 1 . Fig. 1 Flowchart of patient inclusion and exclusion. PPOS, progestin-primed ovarian stimulation; n, number of participants; RICSI, Rescue intracytoplasmic sperm injection; PGT, Preimplantation Genetic Testing Flowchart of patient inclusion and exclusion. PPOS, progestin-primed ovarian stimulation; n, number of participants; RICSI, Rescue intracytoplasmic sperm injection; PGT, Preimplantation Genetic Testing The Hormone measurem was performed using the automated Elecsys immunoassay system from Roche Diagnostics (Mannheim, Germany). The intra- and inter-assay coefficients of variation for Follicle-stimulating hormone (FSH), Luteinizing hormone (LH), Estradiol (E2), Testosterone (T) and P were all less than 2% and 3%, respectively. In the GnRH antagonist protocol, from the 1 st to the 4th day of the menstrual cycle, regardless of the natural or artificial cycle, all patients received daily injections of gonadotropins. The gonadotropins utilized were recombinant follicle-stimulating hormone (rFSH) (Gonal-F, Merck Serono, Italy, or Puregon, Organon, Oss, the Netherlands), highly purified urinary follicle-stimulating hormone (Urofollitropin for Injection, Zhuhai Lizhu Pharmaceutical Co., Ltd.) or human menopausal gonadotropins (HMG; Zhuhai Lizhu Pharmaceutical Co., Ltd.). The initial dose of gonadotropins, ranging from 75 to 300 units per day, was determined based on age, antral follicles (AFCs), Anti-Müllerian hormone (AMH) levels, body mass index (BMI), and the clinician’s judgment. Follicular development was monitored via transvaginal ultrasound and serum sex hormone levels. Within the first 4 days of initiating ovarian stimulation, the dose of gonadotropins was adjusted according to the ovarian response. When the average diameter of the dominant follicle reaches 13 mm (flexible protocol) [ 15 ], gonadotropin-releasing hormone (GnRH) antagonists (Cetrotide, Merck Serono, Italy, or Ganirelix Acetate Injection, Organon, the Netherlands) was added until the triggering day (Fig. 2 ). Meanwhile, fresh embryo transfer was cancelled, and all embryos underwent complete freezing. Fig. 2 Schematic diagram of the Antagonist to PPOS Protocol and Antagonist groups Schematic diagram of the Antagonist to PPOS Protocol and Antagonist groups This group of patients were initially designated to the antagonist protocol. The determination of the Gonadotropin (Gn) starting dose was consisted with the antagonist protocol. Follicular development was monitored by serum sex hormone levels and transvaginal ultrasound. Within the first 4 days of initiating ovarian stimulation, the dose of gonadotropin was adjusted according to the ovarian response. If the progesterone level rose to more than 0.9 ng/ml at any point during or after the addition of the antagonist, the antagonist was stopped, and 10 mg medroxyprogesterone acetate (MPA, Shanghai Xinyi Pharmaceutical Co.) or dydrogesterone (Abbott BiologicalsB.V., Netherlands.) was added twice or three times one day, and the GN drugs were uniformly changed to HMG. Considering that the effective FSH activity of HMG is lower than that of Recombinant Follicle-Stimulating Hormone (r-FSH), after the adjustment to HMG, the corresponding GN dose was maintained at the original level or increased by 75–150 units based on the development of follicular, the antagonist protocol was then converted to the PPOS protocol. To prevent patients from experiencing anxiety and distress due to premature menstruation resulting from early discontinuation of luteal support following oocyte retrieval, and to maintain a physiologically regular menstrual pattern, progesterone was maintained until 10 days after oocyte retrieval and subsequently discontinued. Meanwhile, fresh embryo transfer was cancelled and all embryos were cryopreserved(Fig.  2 ). When there were two or more follicles measuring at least 18 mm in diameter, or minimum of three follicles with diameters of 17 mm or greater, ovulation was considered to be triggered. For inducing ovulation, either human chorionic gonadotropin (hCG, Zhuhai Lizhu Pharmaceutical Co., Ltd.) at doses of 8000 or 10,000 IU, or recombinant chorionic gonadotropin (Ovidrel from Merck Serono) at a dose of 250 µg is administered. In cases where serum estradiol (E2) levels were equal to or exceed 5000 pg/mL, a GnRH agonist triptorelin (Gonapeptyl by Ferring) at a dosage of 0.2 mg may be given alone or combined with urinary hCG at a dose of 2000 IU to mitigate the risk for Ovarian hyperstimulation syndrome (OHSS). Oocyte retrieval guided by transvaginal ultrasound guidance was carried out within 34–36 h following ovulation trigger. In the natural cycle and mild stimulation cycle, hCG was used to induce the final ovulation, and patients took oral dydrogesterone (Abbott Biologicals B.V.) at a dose of 20 mg twice daily and progesterone vaginal capsules (Angitan, Besins Manufacturing Belgium.) at a dose of 0.2 g twice daily to support the luteal phase after the ovulation day [ 19 ]. The timing for frozen-thawed embryo transfer was three to five days after ovulation. For patients with thin endometrium, hormone replacement therapy or GnRH-a down-regulation followed by hormone replacement therapy was recommended. For cycles without ovulation, oral fermartin progesterone capsules (4 mg; Solvay Pharmaceutical Co., Ltd.) were taken twice daily at 2 mg and soft progesterone vaginal capsules (0.2 g) twice daily after the endometrial conversion, respectively, 3 and 5 days after exogenous progesterone exposure, frozen-thawed embryos were transferred. When pregnancy was achieved, exogenous progesterone supplementation was continued until 10 weeks of pregnancy. For the statistical analysis of the matched data, the primary outcome is the clinical pregnancy rate (CPR), and the secondary outcomes is the number of high-quality embryos. In addition, we also analyzed indicators such as High-quality embryo formation rate, and Blastocyst formation rate et, al. The specific calculation formulas for the corresponding outcome indicators are as follows: High-quality embryo formation rate = The number of high-quality embryos/The number of retrieved oocytes. Blastocyst formation rate = The number of blastocysts/The number of blastocysts cultivated. High-quality blastocysts fomation rate = The number of high-quality blastocysts/The number of blastocysts cultivated. CPR of the first frozen embryo transfer cycle = The number of patients achieved clinical pregnancy/The number of patients undergoing first FET. Early miscarriage rate of the first frozen embryo transfer cycle = The number of patients with early miscarriage/The number of patients achieved clinical pregnancy. CPR of all transferred cycle = The number of cycles achieved clinical pregnancy/Total number of current transferred cycles. According to the Shapiro-Wilk test, the current continuous data fail to pass the normality test, variables are represented by the median and interquartile range. Additionally, categorical variables are presented as counts and percentages. The Mann-Whitney U test is employed for the measurement data, and the chi-square test or Fisher’s exact test is utilized for the comparison between proportions. Due to the differences between the two groups in the baseline data, we further adopt the propensity score matching (PSM) method for grouping. Generalized linear models (GLM) were used to correct the data that could affect the results and further explain the impact of the protocal on the ART outcome, for the analysis of continuous outcomes, the identity link function is used, and for the analysis of binomial distribution measurements, the logit link function is adopted [ 20 ]. When analyzing the number of retrieved oocytes, the model was adjusted for female age, duration of infertility, type of infertility, infertility diagnosis, BMI, AMH, and protocol. When analyzing embryo outcomes, the model was further adjusted for fertilization method (in vitro fertilization or intracytoplasmic sperm injection). When analyzing blastocyst culture results, the model was further adjusted for the number of blastocysts cultured. When analyzing the clinical outcomes of the first frozen embryo transfer cycle (clinical pregnancy rate, early pregnancy loss rate), the model was additionally adjusted for endometrial preparation protocol, endometrial thickness, whether blastocysts were transferred, and the number of embryos transferred. When analyzing cumulative frozen embryo transfer cycles for clinical pregnancy, the model was further adjusted for the number of frozen embryo transfer cycles. Further, logistic regression was established to confirm the impact of the intervention on the primary outcomes.

Because the basal luteinizing hormone was different between the two groups of patients, this study employed propensity score matching to match the patients in a 1:1 ratio. The factors used for matching included age, BMI, AMH, basal FSH, basal LH, type of infertility, and fertilization method (IVF/ICSI). A total of 188 pairs (a total of 376 samples) were successfully matched after PSM matching (tolerance is 0.001), and there were no significant differences in the baseline conditions between the two groups of patients after matching (Table  1 ). Table 1 Characteristics of the included participants Characteristics Before PSM After PSM Antagonist ( N  = 345) Antagonist-to-PPOS ( N  = 231) p Antagonist ( N  = 188) Antagonist-to-PPOS ( N  = 188) p General condition Female age, years, n (%) 0.94 0.80  20–29 104 (30.14%) 68 (29.44%) 46 (24.47%) 50 (26.60%)  30–34 143 (41.45%) 97 (41.99%) 78 (41.49%) 79 (42.02%)  35–37 60 (17.39%) 36 (15.58%) 40 (21.28%) 33 (17.55%)  38–40 23 (6.67%) 18 (7.79%) 11 (5.85%) 15 (7.98%)  >40 15 (4.35%) 12 (5.19%) 13 (6.91%) 11 (5.85%) BMI, kg/m 2 , median (IQR) 21.60(19.80, 23.83) 22.06(19.92, 24.46) 0.25 22.22(20.20, 24.61) 22.03(19.94, 24.03) 0.47 AMH, ng/mL, median (IQR) 3.34(2.27, 5.31) 3.70(2.11, 5.89) 0.62 3.79(2.37, 5.31) 4.02(2.25, 5.83) 0.49 Basal reproductive hormones FSH, IU/mL 6.04 (4.77, 7.82) 6.44(5.27, 8.42) 0.08 5.96 (4.89, 7.62) 6.34 (5.14, 8.82) 0.06 LH, IU/mL 4.99 (3.42, 8.20) 5.89 (4.05, 8.45) 0.01* 4.97 (3.53, 7.81) 5.75 (3.82, 8.17) 0.12 E2, pg/mL 45.28 (34.96, 58.49) 43.90 (33.86, 57.73) 0.76 P, ng/mL 0.33 (0.21, 0.78) 0.34 (0.20, 0.69) 0.84 0.32 (0.18, 0.71) 0.35 (0.20, 0.67) 0.54 Testosterone, ng/dL 0.27 (0.19, 0.36) 0.28 (0.19, 0.38) 0.38 0.27 (0.19, 0.37) 0.27 (0.19, 0.35) 0.76 Type of infertility, n (%) 0.14 0.18  Primary 168 (48.70%) 127 (54.98%) 80 (42.55%) 94 (50.00%)  Secondary 177 (51.30%) 104 (45.02%) 108 (57.45%) 94 (50.00%) Infertility diagnosis, n (%) 0.06 0.06  Female 238 (68.99%) 147 (63.64%) 119 (63.30%) 141 (75.00%)  Male 24 (6.96%) 26 (11.26%) 18 (9.57%) 15 (7.98%)  Combined 68 (19.71%) 54 (23.38%) 50 (26.60%) 31 (16.49%)  Unexplained 15 (4.35%) 4 (1.73%) 1 (0.53%) 1 (0.53%) Fertilization, n (%) 0.39 0.29  IVF 281 (81.45%) 181 (78.35%) 134 (71.28%) 144 (76.60%)  ICSI 64 (18.55%) 50 (21.65%) 54 (28.72%) 44 (23.40%) Ovulation induction condition GN dosage (IU), n (%) 0.41 0.31   3000 97 (28.12%) 83 (35.93%) 45 (23.94%) 57 (30.32%) GN duration, median (IQR) 10 (9, 11) 11 (9, 12) 0.02* 10 (9, 11) 11 (9, 12) 0.04*  FSH on hCG trigger day (IU/mL), median (IQR) 15.17 (11.75, 19.85) 15.69 (10.23, 20.61) 0.52 14.99 (11.93, 19.68) 16.49 (10.94, 21.90) 0.13  E2 on hCG trigger day (pg/mL), median (IQR) 3297 (2405.50, 4528) 3747 (2335.50, 5350) 0.04* 3108 (2292.75, 4510.25) 3571 (2122, 5071) 0.053 P on hCG trigger day (ng/mL), median (IQR) 1.35 (1.02, 1.72) 2.02 (1.35, 4.31) <0.001* 1.34 (0.98, 1.60) 2.02 (1.35, 4.31) <0.001*  LH on hCG trigger day (IU/mL), median (IQR) 2.02 (1.21, 3.20) 3.84 (2.55, 5.74) <0.001* 1.96 (1.15, 3.10) 3.64 (2.44, 5.49) <0.001* Characteristics of the included participants On the trigger day, there was no significant difference in the levels of follicle-stimulating hormone (FSH) (14.99 IU/mL vs. 16.49 IU/mL), estradiol (E2) (3108 pg/mL vs. 3571 pg/mL) between the antagonist protocol group and the antagonist-to-PPOS protocol group after PSM matching. Nevertheless, the level of LH in the antagonist-to-PPOS protocol group on the HCG day was higher than that in the antagonist group after PSM matching (3.64 IU/mL vs. 1.96 IU/mL, P < 0.001), with a similar tendency before PSM matching, which might be attributed to the addition of antagonists in the antagonist group to suppress LH. LH can affect oocyte maturation and quality, and high serum LH levels are associated with higher implantation rates in the GnRH antagonist cycles [ 21 ]. The proportion of patients divided by different GN dosage levels was similar between the two groups. The GN duration (11 days VS 10 days)in the antagonist-to-PPOS protocol group exceed those in the antagonist protocol (PSM matching, P = 0.04; before PSM matching, P = 0.02). Among the patients included in our study, neither group experienced ovarian hyper-stimulation syndrome (Table 1 ). Before adjusted by Generalized linear models, the number of retrieved oocytes (13 vs. 13, P  = 0.61) and the number of 2PN fertilized eggs (8.5 for antagonist-to-PPOS group vs. 7 for antagonist group, P  = 0.37) were comparable between antagonist-to-PPOS group and antagonist group. Comparison of the embryo culture conditions between the antagonist-to-PPOS and control groups suggests that, The number of available embryos (9 for antagonist-to-PPOS group versus 7 for tagonist group, P  = 0.07) was comparable in these two groups, but the number of high-quality embryos (4 vs. 3, P  = 0.02), high-quality embryo formation rate (31.01% vs. 25%, P  = 0.02), the number of blastocysts cultivated (8 vs. 6, P  = 0.007), the number of blastocysts formationed (5 vs. 4, P  = 0.002), blastocyst formation rate (62.50% vs. 50.00%, P  = 0.004), the number of high-quality blastocysts (4 vs. 2, P  < 0.001) and high-quality blastocysts fomation rate (33.33% vs. 22.22%, P  = 0.002) were higher in the antagonist-to-PPOS group compared to antagonist group (Supplementary Table 1 ). After adjusted for age, duration of infertility, type of infertility, infertility diagnosis, BMI, AMH, and protocol by Generalized linear model, the number of retrieved oocytes seemed to be higher in the antagonist group (13.59 [13.06–14.13] vs. 12.80 [12.29–13.13], P  = 0.03). However, similar to before GLM correction, despite adjusting for age, duration of infertility, type of infertility, infertility diagnosis, BMI, AMH, protocol, method of fertilization (IVF/ICSI), and number of blastocysts cultivated, the number of blastocysts (4.52 [4.18–4.89] vs3.73 [3.39–4.10], P  = 0.002), the number of high-quality blastocysts per person (3.28 [2.98–3.63] vs. 2.45 [2.16–2.79], P  < 0.001), and the high-quality blastocyst rate (32.15 [28.36–35.94]% vs. 26.25 [22.44–30.05]%, P  = 0.03) were higher in the antagonist-to-PPOS group than in the antagonist group (Table  2 ). Table 2 ART outcomes between patients stimulated with GnRH antagonist and those changed to PPOS after adjusted by GLM Laboratory outcomes GnRH antagonist protocol ( n  = 188) Antagonist changed to the PPOS protocol ( n  = 188) P value The number of oocytes retrieved, mean [CI] a 13.59 [13.06–14.13] 12.80 [12.29–13.13] 0.03* The number of 2PN fertilized eggs, mean [CI] b 8.31 [7.90–8.74] 8.01[7.60–8.44] 0.31 The number of available embryos, mean [CI] b 7.96 [7.56–8.38] 7.79 [7.39–8.22] 0.57 The number of high-quality embryos, mean [CI] b 3.50 [3.24–3.78] 3.72 [3.45–4.01] 0.25 High-quality embryo rate (%) b 26.60 [23.64–29.55] 30.04 [27.10–32.98.10.98] 0.11 The number of blastocysts cultivated, mean [CI] b 7.33[6.91–7.77] 6.77[6.37–7.20] 0.07 The number of blastocysts, mean [CI] c 3.73 [3.39–4.10] 4.52 [4.18–4.89] 0.002* Blastocyst formation rate (%) c 50.08 [45.26–54.90] 57.11 [52.31–61.91] 0.05 The number of high-quality blastocysts per person, mean[CI] c 2.45 [2.16–2.79] 3.28 [2.98–3.63] <0.001* High-quality blastocysts rate (%) c 26.25 [22.44–30.05] 32.15 [28.36–35.94] 0.03* Pregnancy outcome GnRH antagonist protocol ( n  = 157) Antagonist changed to the PPOS protocol ( n  = 119) CPR of the first frozen embryo transfer cycle (%) d 58.00 [49.00–66.00] 45.00 [35.00–55.00] 0.05 Early miscarriage rate of the first FET cycle (%) d 6.00 [2.00–14.00] 13.00 [5.00–27.00] 0.16 CPR of all completed FET cycles (%) e 55.00 [48.00–62.00] 50.00 [42.00–59.00] 0.37 e: Data are presented as adjusted values [CI]. Generalized linear regression models were used for data adjustment a: model adjusted for age, duration of infertility, type of infertility, infertility diagnosis, BMI, AMH, and protocol; b: model adjusted for age, duration of infertility, type of infertility, infertility diagnosis, BMI, AMH, protocol, and method of fertilization (IVF/ICSI); c: model adjusted for age, duration of infertility, type of infertility, infertility diagnosis, BMI, AMH, protocol, method of fertilization (IVF/ICSI), and number of blastocysts cultivated; d: model adjusted for age, infertility type, BMI, AMH, protocol, endometrial preparation protocol, endometrial thickness, transfer of blastocysts, and number of embryos transferred; e: model adjusted for age, infertility type, BMI, AMH, protocol, endometrial preparation protocol, endometrial thickness, transfer of blastocysts, number of embryos transferred, and number of frozen embryo transfer cycles ART outcomes between patients stimulated with GnRH antagonist and those changed to PPOS after adjusted by GLM GnRH antagonist protocol ( n  = 157) Antagonist changed to the PPOS protocol ( n  = 119) e: Data are presented as adjusted values [CI]. Generalized linear regression models were used for data adjustment a: model adjusted for age, duration of infertility, type of infertility, infertility diagnosis, BMI, AMH, and protocol; b: model adjusted for age, duration of infertility, type of infertility, infertility diagnosis, BMI, AMH, protocol, and method of fertilization (IVF/ICSI); c: model adjusted for age, duration of infertility, type of infertility, infertility diagnosis, BMI, AMH, protocol, method of fertilization (IVF/ICSI), and number of blastocysts cultivated; d: model adjusted for age, infertility type, BMI, AMH, protocol, endometrial preparation protocol, endometrial thickness, transfer of blastocysts, and number of embryos transferred; e: model adjusted for age, infertility type, BMI, AMH, protocol, endometrial preparation protocol, endometrial thickness, transfer of blastocysts, number of embryos transferred, and number of frozen embryo transfer cycles Among the two groups of patients, 156 patients in the antagonist group chose to freeze one tube of high-quality day 3 (D3) embryos (1–2 embryos) and the remaining embryos for blastocyst culture. In the antagonist-to-PPOS group, 157 patients made the same choice. As of December 13, 2024, a total of 366 frozen embryo transfer cycles were completed by 276 patients with PSM matches. Among them, 119 patients underwent 155 frozen embryo transfer cycles using the antagonist-to-PPOS protocol, and 157 patients underwent 211 frozen embryo transfer cycles using the antagonist protocol. There were no significant differences in the average age of patients in the transplantation cycle, type of infertility, duration of infertility, type of transplanted embryos, and thickness of the transplanted endometrium. However, the proportion of non-down-regulation protocols in the first FET cycle of the antagonist protocol group was significantly higher than that of the antagonist-to-PPOS group (70.06% vs. 56.30%, P  = 0.02, Supplementary Table 2 ), and the proportion of two embryos transferred was significantly higher than that of the antagonist-to-PPOS group (64.33% vs. 47.90%, P  = 0.01, Supplementary Table 2 ). And for the completed FET cycles, the BMI of the antagonist protocol group was higher than the antagonist-to-PPOS protocol (22.40 kg/m 2 VS 21.48 kg/m 2 , P  = 0.01, Supplementary Table 2 ), and the endometrium of the antagonist-to-PPOS protocol was thicker than the antagonist protocol (1.00 cm VS 0.90 cm, P  = 0.04, Supplementary Table 2 ). The proportion of two embryos transferred in the antagonist protocol group was higher than that in the antagonist-to-PPOS group (66.82% VS 53.55%, P  = 0.01, Supplementary Table 2 ). Before adjusted by GLM, there was no significant difference in the clinical pregnancy rate between the two groups in the first frozen embryo transplantation cycle (56.05% for the antagonist protocol and 47.90% for the antagonist-to-PPOS protocol, P  = 0.18) and the completed FET cycles (54.03% for the antagonist protocol and 50.97% for the antagonist-to-PPOS protocol, P  = 0.60, Supplementary Table 1 ), but it is worth noting that the early miscarriage rate appeared to be slightly higher in the antagonist-to-PPOS protocol group than in the antagonist protocol group, although there was no statistical difference (7.95% for the antagonist protocol and 15.38% for the antagonist-to-PPOS protocol, P  = 0.15, Supplementary Table 1 ). After adjusted for age, infertility type, BMI, AMH, protocol, endometrial preparation protocol, endometrial thickness, transfer of blastocysts, and number of embryos transferred by GLM, the CPR of the first frozen embryo transfer cycle appeared to be slightly higher in the antagonist protocol group than in the antagonist-to-PPOS protocol group, but there was no statistical difference (58.00 [49.00–66.00]% VS 45.00 [35.00–55.00]%, P  = 0.05, Table  2 ). The early miscarriage rate of the first FET cycle (6.00 [2.00–14.00]% VS 13.00 [5.00–27.00]%, P  = 0.16, Table  2 ) were comparble between the GnRH antagonist protocol group and the antagonist-to-PPOS protocol group, and after adjusted for age, infertility type, BMI, AMH, protocol, endometrial preparation protocol, endometrial thickness, transfer of blastocysts, number of embryos transferred, and number of frozen embryo transfer cycles, the CPR of all completed FET cycles are comparable between the GnRH antagonist protocol and the antagonist-to-PPOS protocol group (55.00 [48.00–62.00]% VS 50.00 [42.00–59.00]%, P  = 0.37, Table  2 ). For the first frozen embryo transfer cycle and completed FET cycles, a logistic regression was conducted based on whether the patient achieved clinical pregnancy. The following factors were included: patient age, BMI, AMH, infertility type (primary/secondary infertility), application of down-regulation protocol, endometrial thickness, Cleavage/blastocyst transferred, the number of transferred embryos, and the ovarian stimulation cycle protocol (GnRH antagonist protocol/antagonist-to-PPOS protocal). The patients’ age affected the clinical pregnancy rate of the first FET cycle (β coefficient= −0.10, P  = 0.01) and the completed FET cycles (β coefficient= −0.10, P  = 0.003, Table  3 ), so was the cleavage transferred (first frozen embryo transfer cycle: OR = 0.33, P  < 0.001 and the completed FET cycles: OR = 0.32, P  < 0.001,Table  3 ), while other factors had no statistically significant impact on clinical pregnancy. This suggests that antagonist-to-PPOS protocol does not affect the clinical pregnancy rate of the first FET cycle (OR = 1.56, P  = 0.11) and completed FET cycles (OR = 1.24, P  = 0.37, Table  3 ). Table 3 Logistic regression analysis for the clinical pregnancy rate Variables B Standard error Wald P value Odds ratio OR 95% confidence interval The first frozen embryo transfer cycle  Age (years) −0.10 0.04 7.74 0.01* 0.91 0.85–0.97 Protocol (antagonist protocol) 0.45 0.28 2.62 0.11 1.56 0.92–2.69 Infertility type (Secondary infertility) 0.07 0.29 0.05 0.82 1.07 0.61–1.89  BMI (kg/m²) 0.01 0.04 0.02 0.89 1.01 0.93–1.09  AMH(ng/mL) 0.06 0.06 0.97 0.33 1.06 0.95–1.19 Non-GnRH-a endometrial preparation protocal −0.22 0.28 0.60 0.44 0.80 0.46–1.40  Cleavage transferred −1.11 0.33 11.16 < 0.001* 0.33 0.17–0.63  Number of transplanted embryos 0.19 0.31 0.39 0.53 1.22 0.66–2.23  Thickness of endometrium(cm) 0.73 0.77 0.90 0.34 2.08 0.49–9.42 Completed FET cycles  Age (years) −0.09 0.03 9.34 0.002* 0.91 0.86–0.97 Protocol (antagonist protocol) 0.21 0.24 0.78 0.38 1.23 0.78–1.95 Infertility type (Secondary infertility) −0.03 0.25 0.01 0.91 0.96 0.60–1.58  BMI (kg/m²) 0.03 0.05 0.57 0.45 1.03 0.96–1.10  AMH (ng/mL) 0.03 0.05 0.38 0.54 1.03 0.94–1.13 Non-GnRH-a endometrial preparation protocal −0.13 0.24 0.32 0.57 0.88 0.55–1.39  Cleavage transferred −1.16 0.28 17.46 < 0.001* 0.31 0.18–0.54  Number of transplanted embryos 0.32 0.27 1.47 0.23 1.38 0.82–2.33  Thickness of endometrium(cm) 0.09 0.14 0.47 0.50 1.10 0.84–1.43  Number of FET cycles 0.19 0.24 0.62 0.43 1.21 0.76–1.93 B: β coefficient *Statistically significant correlations ( p  < 0 0.05) are highlighted by Age and Cleavage transferred Logistic regression analysis for the clinical pregnancy rate B: β coefficient *Statistically significant correlations ( p  < 0 0.05) are highlighted by Age and Cleavage transferred

Progesterone level can cumulatively increase even prior to the trigger injection due to the rapid rate of steroid hormone production by multiple developing follicles in a stimulated ovary, and this phenomenon is particularly prevalent when estradiol levels are significantly elevated [ 1 ]. As a result, there may be an inconsistency between the embryo and the receptive window of the endometrium, leading a lower live birth rates in Assisted Reproductive Technology [ 2 ]. When progesterone (P) levels rise above a certain threshold, frozen embryo transfer was considered a better option than fresh embryo transfer [ 3 ]. The Gonadotropin-releasing hormone (GnRH) antagonist protocol is currently one of the most widely used superovulation protocols and is applicable to individuals with poor ovarian reserve, normal ovarian reserve, and high response [ 4 – 6 ]. Researchers have reported premature luteinization [ 7 – 9 ] in the GnRH antagonist protocol, which indicates the premature elevation of progesterone. The incidence of premature luteinization ranges from 2% to 35% in the GnRH antagonist protocol [ 9 , 10 ]. Although sometimes the elevated progesterone may decrease due to the addition of antagonists, but because the endometrium is prematurely exposed to high levels of progesterone, the endometrial receptivity is damaged, leading to a reduction in the success rate of fresh embryo transfer [ 9 , 11 ]. Therefore, for these patients, total embryo freezing and frozen embryo transfer are frequently considered [ 3 ]. In progesterone-primed ovarian stimulation (PPOS) protocal, progesterone is added in the early follicular phase to simulate the physiological situation where there is almost no spontaneous ovulation in the luteal phase, thereby blocking the surge of luteinizing hormone (LH) through progesterone [ 12 ]. Current studies indicate that the ovulation induction effect and clinical pregnancy outcome of the PPOS protocol are not inferior to those of the antagonist protocol and the GnRH-a long protocol, furthermore, since expensive antagonists and down-regulation preparations are not necessary, the PPOS protocol is more economically efficient in terms of cost-effectiveness [ 13 , 14 ]. However, the PPOS protocol is not applicable for fresh embryo transfer as the endometrium is prematurely exposed to elevated levels of progesterone, this protocol mandates that all embryos be cryopreserved. According to the literature reports, the serum P level threshold that negatively impacts live birth rates in fresh elective Single Embryo Transfer (eSET) cycles using the GnRH antagonist protocol is 0.8 ng/ml ~ 1.5 ng/ml [ 15 , 16 ], and in our center, the threshold for P elevation that requires the cancellation of fresh embryo transfer is ≥ 0.9 ng/mL in antagonist protocols. We endeavoured to adapt the antagonist protocol to the PPOS protocol in the population where there was a premature progesterone increase ≥ 0.9 ng/mL in the antagonist protocol, and explore the influence of this alteration in the ovulation induction and clinical outcomes. We compared it with the population that still persisted in using the antagonist protocol after a premature progesterone increase of ≥ 0.9 ng/mL to examine whether antagonist-to-PPOS protocol holds more advantages in the population with a premature progesterone increase.

Data from over 14,000 ART cycles were reported separately by two reaserches, showing a negative correlation between premature P increases and the chance of getting pregnant [ 22 , 23 ], the threshold of how much the progesterone level would affect embryo implantation ranged from P >0.9 ng/mL to 3.0 ng/mL [ 16 , 23 , 24 ]. In GnRH antagonist cycles, the incidence of elevated progesterone levels ranges from 2% to 35% [ 9 , 10 , 25 ]. During superovulation, elevated levels of FSH may induce multifollicular development, as follicular diameter increases, intrafollicular progesterone concentrations rise, and the conversion of progesterone to androgens in theca cells is delayed, leading to increased progesterone accumulation [ 26 – 28 ]. Infertile women with late-onset congenital adrenal hyperplasia and patients with diminished ovarian reserve who are supplemented with dehydroepiandrosterone may also experience premature elevation of progesterone during superovulation [ 29 , 30 ]. However, premature progesterone elevation during ovarian stimulation is not fully understood [ 31 ], in some antagonist protocol cycles, progesterone rise is unexpected. The PPOS protocol introduces progesterone early to suppress ovulation and reduce treatment costs, and achieves superovulation outcomes comparable to the antagonist protocol [ 13 ], but whether switching to PPOS benefits patients undergoing the antagonist protocol with elevated progesterone remains unclear. In the group of patients with premature progesterone elevation in the antagonist protocol, we selected a part of patients and added medroxyprogesterone acetate or dydrogesterone to switch to the PPOS protocol. Our research indicated that the number of retrieved oocytes was comparable between the group of antagonist-to-PPOS group and antagonist group before GLM. Although the antagonist protocol group demonstrated a certain advantage in the number of retrieved oocytes after adjusting for age, duration of infertility, type of infertility, infertility diagnosis, BMI, AMH, and stimulation protocol, patients undergoing the antagonist-to-PPOS protocol yielded a higher number of blastocysts, a greater number of high-quality blastocysts per patient, and an elevated high-quality blastocyst rate compared to those in the antagonist protocol group. These findings suggest that the antagonist-to-PPOS protocol is not inferior to the standard antagonist protocol with respect to blastocyst development outcomes, despite a marginally lower oocyte yield after adjustment using the Generalized Linear Model. When further comparing the clinical pregnancy rates of the two protocols, the switch from the antagonist protocol to the PPOS protocol had no adverse impact on the clinical pregnancy rate in the first frozen embryo transfer cycle and completed FET cycles, this is similar to the results of a recent RCT study on infertile women with normal ovarian reserve, which suggests that the pregnancy outcomes, including implantation rates, clinical pregnancy rates, and abortion rates, are similar between the PPOS and antagonist protocol groups [ 32 ]. It is notable that in a study focusing on the elderly population, it was discovered that the blastocyst formation rate and euploidy in the PPOS group were lower than those in the antagonist protocol, however, in the young subgroup, there was no significant difference in the blastocyst formation rate and euploidy compared with the antagonist [ 33 ]. M del Mar Vidal et al. also conducted a study on the euploidy rate of oocytes in the PPOS protocol, they adopted a self-controlled study of repeated cycles, and the study results suggested that the PPOS protocol could obtain more oocytes at a lower cost without damaging the embryo ploidy rate [ 34 ]. In our study, the median ages of the two groups were 33 and 32 years old respectively. But it is worth noting that, certain studies asserted that the PPOS protocol adversely affected granulosa cell function [ 35 , 36 ] and follicular growth [ 37 ], and contrary to our research, in contrast to the antagonist protocol, the PPOS protocol might even result in a lower cumulative live birth rate in certain patient cohorts [ 38 ]. Meanwhile, a recent meta-analysis included 2156 patients with PCOS and compared the pregnancy outcomes between the PPOS and the antagonist protocols, which is similar to our study results, the study revealed that the PPOS protocol and the antagonist protocol were comparable in terms of ovum quality and clinical outcomes [ 5 ]. It should be mentioned that, our study was unable to calculate the cumulative pregnancy rate due to the fact that most patients did not complete all embryo transfers and the majority of pregnant patients are at the early/middle stages of gestation and have not delivered yet. Therefore, whether the PPOS protocol affect live birth rate requires further research. This study possesses certain limitations that need to be recognized. The incorporation of a restricted number of samples from a sole center might induce bias. The limited sample size is capable of handling only a few confounding factors, and if significant confounding factors are not encompassed in the analysis, it could result in bias. Additionally, if data from other centers are utilized for analysis, the outcomes might vary. And simultaneously, propensity matching might lead to the loss of some sample size. Finally, our study did not include cumulative live birth rates because the number of patients who have completed follow-up is limited. Whether the antagonist protocol changed to the PPOS protocol is suitable for this part of the population requires further study. Obtaining representative random samples and maintaining an adequate sample size could be conducive to attaining impartial results in future studies. The treatment protocol has been elaborated in detail, enabling other centers to replicate the outcomes of this study.

In patients who have an elevated progesterone level in the antagonist protocol, switching to the PPOS protocol can increase the number of high-quality embryos and blastocysts, and does not affect the success rate of the first cycle frozen embryo transfer, and the research findings still need to be confirmed by results from a larger sample of studies. The dataset presented in this paper is not readily accessible since, due to ethical constraints, such data are not made publicly available. Requests for accessing the dataset should be directed to [email protected].

Supplementary Material 1. Supplementary Material 1.