Abstract
Background
Progestin-primed ovarian stimulation (PPOS) has emerged as an alternative to GnRH-antagonist protocols in IVF, yet its impact on embryo chromosomal competence remains controversial. Evidence is particularly limited regarding whether PPOS influences blastocyst euploidy rates and key clinical outcomes in PGT-A cycles, and whether these effects vary according to maternal age and ovarian reserve status.
Methods
This retrospective cohort study included 1,843 PGT-A cycles (666 PPOS, 1,177 GnRH antagonist) performed at a single tertiary IVF center between January 2016 and January 2024. Controlled ovarian stimulation was performed with either oral medroxyprogesterone acetate or daily cetrorelix, followed by vitrification of all biopsied blastocysts and subsequent frozen embryo transfer. Primary outcomes were blastocyst euploidy and clinical pregnancy rates. Secondary outcomes included embryological parameters, miscarriage, live birth, and cumulative pregnancy rates. Subgroup analyses were performed according to maternal age and ovarian reserve.
Results
The mean age was 37.64±4.48 years in the PPOS group and 37.80±3.49 years in the GnRH-antagonist group; age and other baseline characteristics were comparable between groups. PPOS cycles had a shorter stimulation duration (median 8 vs. 9 days, p = 0.001), with comparable gonadotropin requirements and embryological outcomes. Euploidy (32.6% vs. 32.1%, p = 0.653), mosaicism, and aneuploidy rates did not differ significantly. Clinical pregnancy, live birth, and cumulative pregnancy rates were equivalent.
Conclusion
PPOS and GnRH antagonist protocols yield comparable embryological and clinical outcomes in PGT-A cycles, including in advanced maternal age and diminished ovarian reserve subgroups. With its shorter stimulation duration and cost-effectiveness, PPOS is a valid alternative when fresh transfer is not planned.
Keywords: Progestin-primed ovarian stimulation, Preimplantation genetic testing, Aneuploidy, Pregnancy outcomes, Euploid blastocyst, Progesterone
Background
With the increasing use of vitrification technology, recent years have witnessed notable changes in clinical practice within in vitro fertilization (IVF) procedures. A consistent upward trend in freeze–thaw cycles has been observed. It is now well established that frozen embryo transfer (FET) yields pregnancy outcomes comparable to, and in some respects superior to, those of fresh embryo transfer [1–3]. These developments have contributed to a growing preference among clinicians for employing freeze–thaw cycles in scenarios where fresh embryo transfer is not feasible, such as preimplantation genetic testing (PGT) cycles, fertility preservation, and in high responder patients at risk of developing ovarian hyperstimulation syndrome.
In this context, the progestin-primed ovarian stimulation (PPOS) strategy, introduced in 2015 as a more cost-effective ovulation induction protocol, has gained increasing attention [4]. In the PPOS protocol, progesterone is administered to prevent the luteinizing hormone (LH) surge; however, as a result, fresh transfer cannot be performed. Several studies have reported no significant differences in outcomes between PPOS and GnRH antagonist regimens [5–7]. In PGT-A cycles, where transfer is delayed until genetic results are available, the principal drawback of PPOS—its adverse effect on endometrial receptivity—is clinically irrelevant, allowing for its more liberal use.
Nevertheless, debate persists regarding whether elevated follicular-phase progesterone concentrations might adversely affect embryo quality. High progesterone levels have been proposed to influence granulosa cell function and follicular development [8, 9]. Some reports suggest that PPOS may be associated with lower cumulative pregnancy rates compared with antagonist protocols [10], potentially due to negative effects on embryo quality and euploidy [11, 12]. Conversely, other studies have found no significant impact of PPOS on blastocyst euploidy rates [13, 14].
The present study aims to compare the clinical outcomes of PPOS and GnRH antagonist protocols and to investigate whether the choice of regimen affects blastocyst euploidy rates in PGT-A cycles. Given the limited number and scope of existing studies, current evidence remains insufficient to resolve this debate. By contributing additional data, our study seeks to enhance the available literature and provide further evidence to guide clinical decision-making.
Methods
Study design and participants
Following approval from the institutional ethics committee (Approval no: 2025-10/382), this retrospective cohort study included PGT-A cycles performed between January 2016 and January 2024 at our IVF unit, in which either a GnRH antagonist protocol or a progestin-primed ovarian stimulation (PPOS) protocol was used.
Data extracted from the hospital information system included demographic variables (age, body mass index [BMI], smoking status) and infertility-related characteristics (etiology, anti-Müllerian hormone [AMH] level, follicle-stimulating hormone [FSH] level, antral follicle count), as well as stimulation parameters (total gonadotropin dose, stimulation duration, estradiol level on trigger day). Embryological parameters were also recorded, including the number of mature (MII) oocytes retrieved, the number of two-pronuclear (2PN) embryos, total blastocyst count, and blastocyst quality. Blastocysts reaching day 5 or day 6 underwent trophectoderm biopsy for PGT-A. Chromosomal analysis was performed using next-generation sequencing (NGS). Indications for PGT-A included advanced maternal age (≥ 37 years), recurrent pregnancy loss (≥ 2 consecutive miscarriages), and recurrent implantation failure (≥ 4 embryo transfers or ≥ 2 blastocyst transfers without achieving pregnancy) [15].
Exclusion criteria
Exclusion criteria were chronic systemic disease, history of alcohol or substance abuse, chromosomal abnormalities or monogenic disease carrier status in either partner, untreated moderate-to-severe intrauterine adhesions, endometrial polyps, hydrosalpinx, ultrasound-confirmed endometriomas, or a known diagnosis of endometriosis. Cases involving the transfer of more than one embryo were also excluded from the study.
Embryo assessment and transfer
PGT-A results were categorized as euploid, mosaic, or aneuploid. Blastocyst quality was graded on day 5 according to the Gardner classification; embryos graded ≥3BB were considered high quality [16]. Only cycles involving the transfer of a single, high-quality day 5 or day 6 embryo were included. Cycles with double embryo transfer or inadequate-quality embryos were excluded.
Clinical, demographic, embryological, and pregnancy outcomes were compared between the PPOS and GnRH antagonist groups. Subgroup analyses were performed according to maternal age and ovarian reserve.
Outcomes and definitions
Biochemical pregnancy
Positive serum β-hCG above the laboratory threshold (≥ 10 IU/L) without ultrasound evidence of an intrauterine gestational sac.
Clinical pregnancy rate (per transfer)
Presence of ≥1 intrauterine gestational sac with fetal heartbeat at 6–7 weeks divided by the number of embryo transfer (ET) procedures performed in the group.
Miscarriage rate (per clinical pregnancy)
Loss of a clinically confirmed intrauterine pregnancy before 20 weeks divided by the number of clinical pregnancies.
Live birth rate (per transfer)
Number of ET procedures resulting in ≥ 1 live-born infant at ≥ 24 weeks’ gestation (or per national definition) divided by total ET procedures. Multiple births from a single ET are counted as one event.
Cumulative live birth rate per initiated PGT-A cycle
The proportion of ovarian stimulation cycles that resulted in at least one live-born infant after accounting for all subsequent frozen embryo transfers derived from the index stimulation cycle. All initiated PGT-A cycles were retained in the denominator regardless of embryo availability, transfer cancellation, or treatment discontinuation. This definition was selected to reflect real-world clinical effectiveness and to minimize transfer-based selection bias.
Ovarian stimulation, endometrial preparation, and embryo transfer
Controlled ovarian stimulation was initiated on day 2 or 3 of the menstrual cycle with recombinant FSH (rFSH; Gonal-F®, Merck Serono Inc.) and/or human menopausal gonadotropin (HMG; Meriofert®, IBSA, Switzerland), with doses adjusted according to age, BMI, antral follicle count, and AMH level.
In the PPOS group, oral medroxyprogesterone acetate (10 mg/day; Tarlusal®, Deva A.Ş., Turkey) was started concurrently with gonadotropins to suppress LH surge. In the GnRH antagonist group, daily cetrorelix acetate (0.25 mg; Cetrotide®, Merck Serono Inc., Switzerland) was initiated between stimulation days 4–6, based on follicular development assessed by transvaginal ultrasound. In our flexible antagonist protocol, initiation typically coincided with the first observation of a leading follicle ≥ 14 mm on ultrasound and was continued daily until the ovulation trigger. Gonadotropin doses were adjusted as needed.
Final oocyte maturation was triggered when at least one follicle reached ≥ 18 mm or ≥ 3 follicles reached ≥ 17 mm, using either subcutaneous triptorelin 0.2 mg (0.1 mg; Gonapeptyl®, Ferring Pharmaceuticals, Germany), subcutaneous hCG (7,500 U; Ovitrelle®, Merck Serono Inc., Switzerland), or a dual trigger (0.2 mg triptorelin plus 7,500 U hCG). Oocyte retrieval was performed 34–36 h post-trigger under transvaginal ultrasound guidance. Figure 1 illustrate stimulation protocols and medication timelines.
Fig. 1.
Schematic overview of ovarian stimulation protocols and medication timelines. The diagram illustrates the timing and sequence of medications used in the progestin-primed ovarian stimulation (PPOS) and GnRH antagonist protocols. Controlled ovarian stimulation was initiated on cycle day 2–3 with recombinant FSH with or without HMG. In the PPOS protocol, oral medroxyprogesterone acetate was administered from stimulation start, whereas in the antagonist protocol, cetrorelix was initiated flexibly according to follicular development. Final oocyte maturation was triggered using GnRH agonist, hCG, or dual trigger, followed by oocyte retrieval 34–36 h later
Frozen–thawed embryo transfer (FET) was performed in patients with at least one euploid embryo. Endometrial preparation was most commonly achieved using hormone replacement therapy (HRT) supported by a GnRH agonist; however, agonist-free HRT or natural cycles were also employed when clinically appropriate. In our clinical practice, an endometrial thickness of ≥ 7 mm and ≤ 14 mm with a trilaminar or homogeneous pattern was generally considered acceptable for frozen embryo transfer. The preparation method and endometrial thickness on the day of transfer were recorded.
Statistical analysis
Statistical analyses were conducted using SPSS version 25 (IBM Corp., Armonk, NY, USA). Data normality was assessed with the Kolmogorov–Smirnov test. Normally distributed variables were presented as mean ± standard deviation, and non-normally distributed variables as median. The use of median (IQR) was preferred over minimum–maximum values to provide a more robust representation of central tendency in skewed distributions.
Comparisons between PPOS and GnRH antagonist groups were made using Pearson’s chi-square test or Fisher’s exact test for categorical variables, and the Mann–Whitney U test for continuous non-normally distributed variables.
Multivariable logistic regression analysis was performed to identify independent predictors of clinical pregnancy, defined as the presence of a gestational sac with fetal heartbeat. Clinically relevant variables were included in the model. Model fit was evaluated using the Hosmer–Lemeshow test, and overall significance with the Omnibus test. A p-value < 0.05 was considered statistically significant.
Results
After applying the exclusion criteria, 173 of the initial 2,016 PGT-A cycles were excluded due to missing or incomplete data and cycle cancellations. The remaining 1,843 cycles were included in the analysis and divided into two groups according to stimulation protocol (Fig. 2) : the PPOS group (n = 666) and the GnRH antagonist group (n = 1,177). Baseline demographic and clinical characteristics—including age, BMI, AMH, FSH, LH, E2 levels, AFC, smoking status, and indications for IVF or PGT-A were comparable between groups (p > 0.05; Table 1).
Fig. 2.
Study flow of the PGT-A cohort by stimulation protocol. Diagram shows disposition of cycles and embryos according to ovarian stimulation regimen. Of 1,843 PGT-A cycles, 666 were managed with PPOS and 1,177 with a GnRH-antagonist protocol. Downstream counts are shown for each arm: blastocysts cultured (PPOS 4,321; GnRH-ant 7,264), biopsied blastocysts (PPOS 2,313; GnRH-ant 4,178), and thawed and transferred cycles (PPOS 317; GnRH-ant 514). All transfers were frozen embryo transfers following vitrification
Table 1.
Comparison of demographic and clinical characteristics
| PPOS(n=666) | GnRH antagonist(n=1177) | P Value | |
|---|---|---|---|
| Maternal age | 38 (35-41) | 38 (35-40) | 0.627 |
| <37 (%) | 37.4 (249/666) | 37.3 (439/1177) | 0.970 |
| ≥37 (%) | 62.6 (417/666) | 62.7 (738/1177) | |
| BMI (kg/m2) | 23.50 (21.60-26.35) | 24.10 (22.30-26.00) | 0.061 |
| AMH (ng/ml) | 1.20 (0.94-1.24) | 1.10 (0.82-1.15) | 0.683 |
| FSH (mIU/ml) | 6.70 (4.70-9.88) | 7.47 (5.44-9.24) | 0.118 |
| LH (mIU/ml) | 5.80 (3.65-8.23) | 6.04 (4.33-7.55) | 0.290 |
| E2 (pg/ml) | 53 (43-74) | 52 (37-84) | 0.124 |
| AFC | 10 (9-15) | 10 (8-15) | 0.499 |
| Indications of IVF | |||
| DOR (%,n) | 32.1 (214/666) | 33.4 (393/1177) | 0.817 |
| Male factor (%,n) | 29.0 (193/666) | 28.4 (334/1177) | |
| Unexplained infertility (%,n) | 14.4 (96/666) | 13.3 (157/1177) | |
| PCOS (%,n) | 14.4 (96/666) | 13.5 (159/1177) | |
| Tubal factor (%,n) | 10.1 (67/666) | 11.4 (134/1177) | |
| Smoking (%,n) | 19.1 (127/666) | 22.7 (267/1177) | 0.069 |
| Indications of PGT-A | |||
| Advanced maternal age (%,n) | 43.2 (288/666) | 40.0 (471/1177) | 0.535 |
| Repeated implantation failure (%,n) | 14.9 (99/666) | 16.7 (196/1177) | |
| Recurrent spontaneous miscarriage (%,n) | 16.1 (107/666) | 17.0 (200/1177) | |
| Mixed (%,n) | 25.8 (172/666) | 26.3 (310/1177) | |
Data are presented as median (IQR) or frequency (%)
PPOS Progestin-primed ovarian stimulation, GnRH Gonadotropin-releasing hormone, BMI Body mass index, AMH Anti müllerian hormone, FSH Follicle stimulating hormone, LH Luteinizing hormone, E2 Estradiol, AFC Antral follicle count, DOR Diminished ovarian reserve, PCOS Polycystic ovary syndrome
p≤0.05 Statistical Significance
* Mann-Whitney U Test
The median stimulation duration was significantly shorter in the PPOS group compared to the antagonist group (8 vs. 9 days, p = 0.001), whereas the total gonadotropin dose and incidence of premature ovulation were similar between groups (Table 2). There were no significant differences in the number of retrieved oocytes, mature (MII) oocytes, fertilized (2PN) embryos, or biopsied embryos per cycle. A total of 4,321 embryos in the PPOS group and 7,264 embryos in the antagonist group reached the blastocyst stage, of which 2,313 (53.5%) and 4,178 (57.5%), respectively, underwent PGT-A biopsy.
Table 2.
Outcomes of ovarian stimulation and genetic results between the two groups
| PPOS(n=666) | GnRH antagonist(n=1177) | P Value | |
|---|---|---|---|
| Days of stimulation | 8 (8-9) | 9 (8-10) | 0.001* |
| Total gonadotropin dose (IU) | 3300 (2700-3900) | 3300 (2400-3900) | 0.389 |
| Dual trigger (%) | 92.2 (614/666) | 91.6 (1078/1177) | 0.650 |
| Premature ovulation (%) | 0.6 | 0.8 | 0.554 |
| Number of retrieved oocytes | 11 (6-16) | 11 (7-17) | 0.194 |
| Number of M2 oocytes | 10 (5-15) | 10 (6-15) | 0.900 |
| Number of 2PN embryos perPGT-A cycle | 7 (4-12) | 8 (5-12) | 0.974 |
| Number of D5 embryo (n) | 2944 | 4893 | NA |
| Number of D5 embryo per PGT-A cycle | 4 (2-7) | 4 (2-6) | 0.064 |
| Number of embryo biopsy | 3 (2-5) | 3 (2-5) | 0.254 |
| D5 embryo (%) | 68.1 (2944/4321) | 67.3 (4893/7264) | 0.198 |
| D6 embryo (%) | 31.8 (1377/4321) | 32.6 (2371/7264) | 0.197 |
| High quality blastocyst rate (%) | 46.6 (2015/4321) | 48.0 (3488/7264) | 0.215 |
| Total number of biopsied blastocysts (n) | 2313 | 4178 | NA |
| PGT-A results of embryos (n,%) | |||
| Euploidy | 32.6 (755/2313) | 32.1 (1341/4178) | 0.653 |
| Mosaicism | 13.5 (313/2313) | 15.1 (632/4178) | 0.081 |
| Aneuploidy | 55.9 (1293/2313) | 56.3 (2352/4178) | 0.751 |
| Aneuploidy status of subgroup based on maternal age (%) | |||
| <37 | 35.5 (459/1293) | 31.1 (731/2352) | 0.152 |
| ≥37 | 64.5 (834/1293) | 68.9 (1621/2352) | |
| Aneuploidy status of subgroup based on ovarian reserve (%) | |||
| DOR | 43.6 (564/1293) | 39.4 (927/2352) | 0.185 |
| Non-DOR | 56.4 (729/1293) | 60.6 (1425/2352) | |
Data are presented as median (IQR) or frequency (%).Total embryo counts are presented as aggregate numbers. Per-cycle embryo parameters are presented as median (IQR)
PPOS Progestin-primed ovarian stimulation, GnRH Gonadotropin-releasing hormone
p≤0.05 Statistical Significance
*Mann-Whitney U Test, **Chi-Square Test
Embryo development outcomes were similar: the proportion of day-5 blastocysts (68.1% vs. 67.3%, P = 0.198), high-quality blastocysts (46.6% vs. 48.0%, P = 0.215), and the number of biopsied blastocysts per cycle (p = 0.254) did not differ significantly. PGT-A results revealed no statistical differences in the rates of euploid (32.6% vs. 32.1%, p = 0.653), mosaic (13.5% vs. 15.1%, p = 0.081), or aneuploid embryos (55.9% vs. 56.3%, p = 0.751). Subgroup analysis by maternal age and ovarian reserve also showed no significant differences in aneuploidy rates.
Frozen–thawed embryo transfers were performed in 317 cycles in the PPOS group and 514 cycles in the antagonist group. The type of endometrial preparation and endometrial thickness on the day of transfer were similar between groups. Biochemical pregnancy rates (67.2% vs. 67.3%, p = 0.971), clinical pregnancy rates (61.5% vs. 62.5%, p = 0.787), miscarriage rates (11.3% vs. 13.1%, p = 0.547), and live birth rates per transfer (54.6% vs. 54.3%, p = 0.934) were all comparable. Cumulative live birth rate per initiated PGT-A cycle was also similar (33.7% vs. 30.1%, p = 0.100). Subgroup analysis by maternal age and ovarian reserve revealed no significant differences in clinical pregnancy rates (Table 3).
Table 3.
Pregnancy outcomes of the two groups
| PPOS(n=317) | GnRH antagonist(n=514) | P Value | |
|---|---|---|---|
| Freeze- Thawed and transferred cycle | 47.6 (317/666) | 43.7 (514/1177) | 0.104 |
| Endometrial preparation | |||
| HRT (%) | 10.7 (34/317) | 12.1 (62/514) | 0.828 |
| GnRH agonist HRT (%) | 82.0 (260/317) | 81.1 (417/514) | |
| Natural cycle (%) | 7.3 (23/317) | 6.8 (35/514) | |
| Endometrial thickness (mm) | 9 (8.60-9.90) | 9 (8.50-9.70) | 0.385 |
| Biochemical pregnancy (n, %) | 67.2 (213/317) | 67.3 (346/514) | 0.971 |
| Clinical pregnancy rate (n, %) | 61.5 (195/317) | 62.5 (321/514) | 0.787 |
| Subgroup based on maternal age | |||
| <37 | 60.5 (118/195) | 60.1 (193/321) | 0.930 |
| ≥37 | 39.5 (77/195) | 39.9 (128/321) | |
| Subgroup based on ovarian reserve | |||
| DOR | 27.2 (53/195) | 29.9 (96/321) | 0.507 |
| Non-DOR | 72.8 (142/195) | 70.1 (225/321) | |
| Miscarriage rate (n,%) | 11.3 (22/195) | 13.1 (42/321) | 0.547 |
| Live Birth Rate per transfer (n,%) | 54.6 (173/317) | 54.3 (279/514) | 0.934 |
| Cumulative live birth rate per initiated PGT-A cycle (%) | 33.7 (225/666) | 30.1 (355/1177 ) | 0.100 |
Data are presented as median (IQR) or frequency (%)
PPOS Progestin-primed ovarian stimulation, GnRH Gonadotropin-releasing hormone, HRT Hormone replacement therapy
p≤0.05 Statistical Significance
*Mann-Whitney U Test, **Chi-Square Test
Multivariable logistic regression analysis for clinical pregnancy demonstrated that the model was statistically significant (χ² = 55.855, p < 0.001) with acceptable goodness-of-fit (Hosmer–Lemeshow test, p = 0.075). The model explained 8.8% of the variance in clinical pregnancy (Nagelkerke R² = 0.088). Endometrial thickness was identified as the strongest independent predictor of clinical pregnancy; each 1 mm increase in thickness was associated with a 1.676-fold increase in pregnancy odds (OR = 1.676; 95% CI: 1.422–1.976; p < 0.001). Other demographic and clinical variables—including female age, BMI, AMH, AFC, smoking status, and stimulation duration—were not significantly associated with clinical pregnancy (p > 0.05) (Table 4).
Table 4.
Adjusted logistic regression analysis for clinical pregnancy per first frozen-thawed embryo transfer cycle
| Factors | OR | 95% CI | P |
|---|---|---|---|
| Age | 0.997 | 0.963-1.032 | 0.869 |
| BMI | 0.973 | 0.929-1.108 | 0.235 |
| AFC | 1.010 | 0.989-1.031 | 0.368 |
| AMH | 1.001 | 0.919-1.091 | 0.975 |
| Smoking | 1.220 | 0.820-1.816 | 0.326 |
| Indications of PGT-A | |||
| Mixed | Reference | ||
| Advanced maternal age | 1.441 | 0.949-2.187 | 0.087 |
| Repeated implantation failure | 1.136 | 0.742-1.738 | 0.558 |
| Recurrent spontaneous miscarriage | 1.487 | 0.953-2.320 | 0.081 |
| Day of stimulation | 1.061 | 0.955-1.178 | 0.272 |
| Ovulation Trigger | |||
| GnRH agonist | Reference | ||
| HCG | 0.868 | 0.433-1.736 | 0.848 |
| Dual | 0.940 | 0.500-1.767 | 0.688 |
| Endometrial preparation | |||
| HRT | Reference | ||
| GnRH agonist HRT | 1.277 | 0.833-1.958 | 0.262 |
| Natural cycle | 0.873 | 0.475-1.603 | 0.661 |
| Endometrial thickness | 1.676 | 1.422-1.976 | 0.001* |
| Ovarian stimulation protocol (PPOS/GnRH antagonist) | 0.968 | 0.704-1.330 | 0.840 |
AFC Antral follicle count, AMH Anti müllerian hormone, PPOS Progestin-primed ovarian stimulation, GnRH Gonadotropin-releasing hormone, HCG Human chorionic gonadotropin, HRT Hormone replacement therapy
Model χ2=55.855, p<0.001Nagelkerke R2=0.088Hosmer ve Lemeshow χ2=14.267, p=0.075
Discussion
In this large-scale retrospective study, we compared the embryological and clinical outcomes of PPOS and GnRH antagonist protocols in PGT-A cycles. Our findings demonstrated no statistically significant differences between the two stimulation regimens in terms of blastocyst euploidy rates, clinical pregnancy, or live birth outcomes. These results support the clinical equivalence of PPOS as a valid alternative to GnRH antagonist protocols in PGT-A cycles where fresh embryo transfer is not planned.
PPOS has been described as an effective and practical alternative for preventing premature LH surge during the follicular phase through oral medroxyprogesterone acetate administration. Compared with GnRH antagonist protocols, PPOS offers practical advantages such as reduced injection burden, lower medication costs, and greater scheduling flexibility, without adversely affecting the number or quality of mature oocytes [5]. Its main limitation—potential impairment of endometrial receptivity—is irrelevant in PGT-A cycles, as all embryos are vitrified before transfer. Furthermore, our study shows that PPOS is equally effective as antagonists in preventing premature ovulation.
One theoretical concern regarding PPOS is that supraphysiological progesterone levels in the follicular phase may modulate granulosa cell signaling pathways via membrane-associated progesterone receptor component 1, influencing PI3K/AKT and MAPK signaling, and potentially impairing oocyte competence and embryo development [8, 9]. Some clinical studies have reported reduced cumulative live birth rates [10] or lower euploidy rates, particularly in older women, associated with PPOS [11, 12]. For example, Pai et al. [12] reported that PPOS use in women over 38 years was linked to lower euploidy rates, while Wan et al. [11] observed potential protocol-related differences in aneuploidy rates by age. Conversely, other robust investigations, including randomized controlled trials, have found no significant differences in blastocyst euploidy rates between PPOS and GnRH antagonist protocols [6, 14, 17–20]. La Marca et al. [14] observed similar embryological and chromosomal outcomes between the two regimens, and these results have been confirmed by well-matched PGT-A cohorts from Tan et al. [19] and Wang et al. [20]. In line with this evidence, our study demonstrated comparable euploid and aneuploid rates across groups, with no significant differences detected in subgroup analyses stratified by maternal age or ovarian reserve. The lack of significant differences in aneuploidy rates among subgroups should be interpreted not as suggesting that maternal age or ovarian reserve does not affect aneuploidy status, but rather as protocol equivalence at similar ages and ovarian reserves. But it should be added that our comparatively modest euploid yield is biologically plausible given cohort composition: diminished ovarian reserve reduces blastocyst euploidy independent of age [21], while male-factor parameters are associated with increased aneuploidy and mosaicism [22, 23]. Age-related decline and embryo-level features (morphology/kinetics) further modulate expected euploidy in this age range [24]. The euploidy rate and cumulative live birth rate per PGT-A cycle reported in our study are within the generally accepted ranges for PGT-A application, considering that our study population predominantly included indications for DOR and male factor, along with the age factor. Another factor to consider is that outcomes were analyzed at the cycle level rather than the individual patient level, which may limit interpretation of cumulative live birth rates per patient.
Our results also confirm that PPOS does not adversely affect oocyte yield, fertilization rate, or blastocyst development compared with GnRH antagonist protocols. High-quality blastocyst rates were similar, and no differences were found in euploidy rates, implantation rates, miscarriage rates, or live birth rates per transfer. This is biologically consistent with the notion that stimulation protocol choice does not significantly alter chromosomal segregation fidelity during oocyte meiosis or early embryonic cleavage. In addition, differences in gonadotropin formulations, such as recombinant FSH versus HMG, may affect follicular endocrine dynamics and oocyte developmental capacity [25]. While individualized dosing strategies and standardized laboratory conditions at our center likely minimized these effects, further evaluation of the gonadotropin type-independent effect is needed in prospective studies specifically designed to address this question.
In our multivariable logistic regression analysis, endometrial thickness emerged as the strongest independent predictor of clinical pregnancy. Each 1 mm increase in endometrial thickness was associated with a 1.676-fold increase in the likelihood of clinical pregnancy. This finding underscores the critical role of optimal endometrial development in frozen embryo transfer cycles and is consistent with previous research [26]. Similarly, the large-scale analysis by Shaodi et al. reported a positive association between increasing endometrial thickness and improved clinical pregnancy outcomes in hormone replacement therapy–based FET cycles, suggesting that endometrial thickness may reflect underlying receptivity rather than acting as an isolated causal factor [27]. Other variables, including protocol type, maternal age, and ovarian reserve parameters, were not significantly associated with clinical pregnancy, suggesting that in vitrified transfer cycles, reproductive success may be more dependent on endometrial receptivity than on the ovarian stimulation protocol itself. Although maternal age is a well-established determinant of reproductive outcomes, the inclusion of only single euploid embryo transfers may attenuate the independent predictive effect of age on clinical pregnancy, as the primary biological pathway linking age to implantation—embryo aneuploidy—has already been partially controlled [28]. Although the multivariable logistic regression model was statistically significant, its explanatory power was modest, as reflected by a Nagelkerke R² value of 0.088. Therefore, this model should not be interpreted or used as a predictive tool for clinical pregnancy. Its primary value is to identify factors associated with clinical pregnancy within the present cohort. The low explanatory power also underscores the multifactorial and incompletely understood nature of implantation, which is likely influenced by complex biological and procedural factors not captured in this dataset, including embryo-endometrial synchrony, embryo-level characteristics beyond ploidy, uterine receptivity, microbiome-related factors, and technical aspects of embryo transfer.
From a practical perspective, PPOS offers several workflow and patient-centered advantages in PGT-A cycles. As observed in our study and others [29], stimulation duration may be slightly shorter with PPOS, which can enhance flexibility in clinical scheduling. However, this difference may have occurred due to the adoption of a flexible protocol in the antagonist group and the cycle may have been prolonged. Furthermore, the use of oral agents eliminates the need for daily antagonist injections, improving patient comfort. When combined with equivalent reproductive outcomes, these features make PPOS an attractive option for clinics aiming to optimize efficiency and patient satisfaction.
Strengths of our study include its large sample size, the use of standardized laboratory protocols, and the inclusion of only single, high-quality blastocyst transfers, which minimized variability. The comprehensive evaluation of both embryological and clinical outcomes allowed for a thorough comparison between protocols. Main limitations include its retrospective design, single-center nature. Another important limitation is the absence of long-term neonatal and developmental follow-up. Although live birth is a clinically meaningful endpoint, it does not fully address the safety profile of PPOS with respect to perinatal outcomes, congenital anomalies, neonatal morbidity, or longer-term child development. This limitation is particularly relevant because PPOS exposes developing follicles to a different hormonal milieu during ovarian stimulation. Therefore, future prospective studies should incorporate standardized neonatal and developmental follow-up to more comprehensively evaluate the safety of PPOS beyond its efficacy in achieving pregnancy and live birth. Although baseline characteristics were comparable between groups, the retrospective design remains susceptible to residual and unmeasured confounding, including physician preference in protocol selection, prior ovarian response history, patient-specific clinical decision-making, and lifestyle-related factors not captured in the dataset. Therefore, the present findings should be interpreted as demonstrating comparable outcomes within this retrospective cohort rather than definitive causal equivalence between protocols. Future prospective randomized controlled trials are needed to validate these findings; alternatively, well-designed propensity score-matched or propensity score-adjusted analyses may provide more robust control for confounding when retrospective data are used. The retrospective design limits causal inference, and future prospective or propensity-adjusted studies are warranted to further validate these findings. While euploidy rate is a robust surrogate endpoint, potential differences in post-implantation developmental competence remain outside the scope of this study. These results should be interpreted in light of potential heterogeneity in embryo transfer and endometrial preparation. Finally, mechanistic effects of progestin exposure on follicular development, oocyte competence, and embryo developmental potential could not be directly evaluated in this retrospective clinical study. As the study was conducted in a single high-volume tertiary center with specific clinical workflows, extrapolation to different populations or laboratory environments should be approached cautiously. Our program conducts a substantially higher proportion of freeze–thaw cycles than fresh transfers, and while GnRH agonist–based HRT is our default regimen, alternative preparations are occasionally used. Despite institutional standards for endometrial preparation, transfer technique, and luteal-phase support, the retrospective design may have introduced subtle, patient-specific variation. The retrospective single-center design may have introduced selection bias and may limit the generalizability of our findings to other IVF settings with different patient populations, laboratory conditions, or clinical workflows Prospective randomized controlled trials incorporating broader biological, embryological, and long-term clinical outcomes are needed to further validate these findings.
In conclusion, PPOS and GnRH antagonist protocols yield equivalent blastocyst euploidy, pregnancy, and live birth rates in PGT-A cycles. The absence of adverse effects on pregnancy rates in high-risk subgroups, such as women of advanced maternal age and those with diminished ovarian reserve, suggests that PPOS can be safely implemented in these populations. Given its practical advantages, PPOS represents a valid alternative to GnRH antagonist protocols in PGT-A cycles where fresh transfer is not planned. Because this study was conducted at a single high-volume tertiary IVF center with specific clinical workflows and standardized laboratory conditions, extrapolation of these findings to other IVF settings with different patient populations, stimulation practices, laboratory procedures, PGT-A platforms, or embryo transfer policies should be approached with caution. Nonetheless, multicenter randomized controlled trials using standardized PGT-A methodologies across diverse patient populations are warranted to strengthen the evidence base for this clinical practice.
Authors' contributions
Dr Elmas and Prof Tiras are the principal investigator of the study. They contributed at all levels of the study. Dr Yuceturk, Dr Karaosmanoglu and Dr Peker contributed to the design, methodology, writing, editing and supervision of the study. Dr Aksu Turan and Dr Berkil contributed to the data collection. Dr Albayrak and Dr Ozer Aslan contributed to the methodology of the study and the processing and analysis of the data for this study.
Funding
None.
Data availability
The datasets generated and/or analyzed during the current study are not publicly available due to patient confidentiality and institutional ethical restrictions but are available from the corresponding author on reasonable request.
Declarations
Ethics approval and consent to participate
This retrospective study was approved by the Institutional Review Board of Acibadem Mehmet Ali Aydinlar University (Approval No: 2025-10/382, Date: 26.06.2025). The study was conducted in accordance with the Declaration of Helsinki and relevant national regulations. Written informed consent for treatment was obtained from all participants at the time of their clinical procedures. Due to the retrospective design of the study and the use of fully anonymized data, additional informed consent for participation in this research was waived by the Ethics Committee. Patient confidentiality was strictly maintained, and all data were analyzed anonymously with institutional permission.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets generated and/or analyzed during the current study are not publicly available due to patient confidentiality and institutional ethical restrictions but are available from the corresponding author on reasonable request.