Intensive luteal phase support in hormone replacement and modified natural cycle frozen embryo transfers in ovulatory patients: A propensity score-matched study.

Frozen embryo transfer (FET), which is fueled by significant advancements in vitrification techniques, coupled with its expanding indications, has emerged as an integral component of assisted reproductive technology (ART) [ 1 , 2 ]. The “freeze-all” strategy can reduce the risk of ovarian hyperstimulation syndrome, enhance endometrial receptivity, and allow for preimplantation genetic testing, all of which have contributed to the rise of FET [ 3 – 6 ]. However, the successful of FET depends on the precise synchronization between embryo developmental stage and endometrial receptivity, which depends on choosing the optimal endometrial preparation protocol [ 7 ]. The predominant methods for endometrial preparation in current practice include the natural cycle (NC), modified natural cycle (mNC), and artificial cycle with hormone replacement therapy (HRT). Both the NC and the mNC protocols rely on endogenous hormone production and corpus luteum formation. The mNC approach is considered more flexible due to the active intervention that to promotes the maturation of the dominant follicle. However, the NC and the mNC protocols may lead to a relatively high cycle cancellation rate, up to 20%, reported in some studies, due to the absence of the dominant follicle [ 8 ]. It is worth emphasizing that the HRT protocol provides better control over endometrial development by administering estradiol and progesterone externally, it may increase the risks of obstetric and perinatal complications due to the lack of a corpus luteum [ 9 – 11 ]. Despite numerous randomized controlled trials (RCT) comparing the pregnancy outcomes and maternal-fetal safety of these endometrial preparation methods, the superiority of one approach over another remains elusive, mainly due to the heterogeneity in study designs, specifically in monitoring protocols, use of ovulation triggers, and luteal phase support (LPS). A recent systematic review and meta-analysis assessing obstetric and neonatal outcomes following NC and HRT endometrial preparation, encompassing 30 studies with over 110,000 patients, revealed that the NC group receiving LPS had a lower risk of preterm birth compared to the HRT group while NC without LPS did not. However, the interpretation of these findings could not lead to a definite conclusion due to the substantial heterogeneity across studies and the low to moderate quality of evidence [ 12 ]. In HRT cycles, the sole reliance on vaginal micronized progesterone may result in suboptimal absorption and lower serum progesterone concentrations in a subset of patients, with detrimental effects on pregnancy outcomes following FET [ 13 , 14 ]. To circumvent this drawback, it has been advocated to monitor serum progesterone prior to embryo transfer. Rescue protocols involving additional progesterone administration in patients with low levels has been shown to restore pregnancy rates similar to those of patients with adequate progesterone levels [ 15 , 16 ]. Concurrently, some evidence came from mNC cycles that clinical pregnancy rates could be improved by a supplementation with exogenous progesterone [ 17 , 18 ]. Given the potential role of intensive LPS in optimizing the endometrial milieu and pregnancy outcomes, the question arises whether the clinical effectiveness of HRT and mNC protocols would still differ significantly if both incorporated vaginal micronized progesterone and oral dydrogesterone for LPS. To address this issue, we conducted a retrospective cohort study to compare the pregnancy outcomes of HRT and mNC FET cycles in ovulatory women who received intensive LPS.

We collected a total of 2365 autologous FET cycles, comprising 1892 HRT and 473 mNC cycles, among which 1419 HRT and 473 mNC cycles were included in the final analysis after the propensity score matching. The patient demographic and cycle characteristics before and after propensity score matching are presented in Table 1 . Prior to matching, there were significant differences between the two groups in terms of BMI, and the history of cesarean section and endometrial thickness. After matching, the baseline covariates were well balanced, as evidenced by the lack of remarkable differences between the matched groups and the standardized mean differences falling below 0.1 for all variables ( Table 1 and S1 - S2 Figures ). Data are presented as mean (SD) for continuous variables and n (%) for categorical variables. BMI: body mass index; PGT-A: preimplantation genetic testing for aneuploidy. Table 2 summarizes the pregnancy outcomes of the propensity-matched cohorts. The live birth rate was similar between the mNC and HRT groups (34.7% and 34.8%, respectively; adjusted RR 1.02, 95% CI 0.80–1.29, p = 1.00). Also, there were no significant differences in the secondary outcomes including positive hCG (54.3% vs 51.3%, p = 0.276), clinical pregnancy (42.9% vs 42.0%, p = 0.767), ongoing pregnancy (35.5% vs 35.7%, p = 1.000), implantation (35.2% vs 35.6%, p = 0.859), biochemical pregnancy (11.4% vs 9.3%, p = 0.212), miscarriage (7.8% vs 7.1%, P = 0.683), and multiple pregnancy (4.0% vs 2.7%, p = 0.218) rates. Data are presented as n (%) for categorical variables, mean (SD) for continuous variables, and fractions (%) for rates. The incidence of major congenital anomalies was low and similar in both arms, with 1 case (<1%) in the mNC group and 2 cases (<1%) in the HRT group. Reported birth defects included a solitary testicle in the mNC group and 2 cardiac malformations (1 pulmonary artery anomaly and 1 case of mitral valve insufficiency) in the HRT group. The frequencies of preterm birth were also low and comparable between the two protocols for both singleton and twin pregnancies ( Table 2 ). Mean birth weights were in the normal range and did not differ significantly for singletons (3159 gram vs 3171 gram) or twins (2458 gram vs 2432 gram) in the mNC and HRT groups, respectively. There was no significant difference in pregnancy outcomes between the mNC and HRT groups, both before and after matching. In the unadjusted analysis, the mNC protocol had a relative risk of 1.00 (95% CI 0.86–1.15) for live birth, 1.02 (95% CI 0.91–1.15) for clinical pregnancy, 1.00 (95% CI 0.87–1.15) for ongoing pregnancy, and 1.02 (95% CI 0.90–1.16) for implantation relative to the HRT protocol. After matching, the corresponding adjusted relative risks remained virtually unchanged at 1.02 (95% CI 0.80–1.29) for live birth, 1.07 (95% CI 0.85–1.35) for clinical pregnancy, 1.02 (95% CI 0.80–1.29) for ongoing pregnancy, and 1.04 (95% CI 0.93–1.17) for implantation ( Table 3 ).

Our results suggest that in patients receiving intensive LPS with a combination of vaginal micronized progesterone and oral dydrogesterone, the clinical effectiveness of FET with HRT and mNC protocols is equivalent. The choice of endometrial preparation can be based on individual patient characteristics and preferences.

A retrospective analysis of all autologous FET cycles performed between January 2022 and September 2023 was carried out at our academic fertility center. Women aged 20–45 years with regular ovulatory cycles, defined as a menstrual cycle length of 24–38 days, who underwent endometrial preparation with either an HRT or mNC protocol were eligible for inclusion. The exclusion criteria were: oocyte vitrification, adenomyosis, submucosal fibroids, surgically retrieved sperm, hydrosalpinx documented on hysterosalpingography, and endometrial thickness <7mm at the final ultrasound assessment prior to progesterone initiation. Patients’ baseline characteristics, treatment parameters, and pregnancy outcomes were extracted from the electronic medical records. For patients receiving prenatal care and delivery at outside facilities, obstetric and neonatal outcomes were obtained through standardized telephone interviews. The study was approved by the Institutional Review Board on August 29, 2024 (IRB reference number: IRB.TAHN.071). Data were accessed for research purposes on August 30, 2024. All patient data were anonymized prior to analysis, and the authors did not have access to any identifying information during or after data collection. Following baseline transvaginal ultrasound on cycle days 2–4 and counseling regarding endometrial preparation options, the selection of the HRT or mNC protocol was determined through a shared decision-making process involving both the patient and the treating physician, rather than being based on rigid, pre-defined clinical criteria. This decision was primarily guided by non-clinical factors and specific patient preferences. Key considerations encompassed logistical aspects, such as patient convenience related to the frequency and feasibility of monitoring visits (particularly relevant for mNC cycles), patient desire concerning the extent of exogenous hormone administration, and individual acceptance of the potential risk for cycle cancellation inherent to the mNC protocol. In the mNC protocol, ultrasound monitoring was started on cycle day 7, with subsequent visits scheduled according to follicular growth. When the leading follicle reached a mean diameter of ≥16 mm with an endometrial thickness ≥7 mm and serum progesterone level <1.5 ng/ml, final oocyte maturation was triggered with 5000 IU of human chorionic gonadotropin (hCG, IVF-C, LG Chem). Progesterone supplementation was initiated the following day. Cycles were cancelled if no dominant follicle developed by day 21, premature ovulation occurred, or serum progesterone level was ≥ 1.5 ng/ml. In the HRT protocol, oral estradiol valerate 6 mg daily (Progynova, Bayer) was started on cycle day 2–4. An ultrasound follow-up was performed on day 10 to assess endometrial development, with dose adjustments if needed. Progesterone supplementation was initiated on day 14 if the endometrium reached a minimum thickness of 7 mm. Cycles were cancelled if the endometrial thickness remained below 7 mm despite extending estrogen administration to 21 days. In pregnant patients, estrogen was continued until 7 weeks of gestation. Two groups received an identical intensive LPS regimen including vaginal micronized progesterone 800 mg daily (Cyclogest, Actavis) and oral dydrogesterone 30 mg daily (Duphaston, Abbott). Serum progesterone and estradiol levels were not routinely monitored during the luteal phase in either protocol. This approach was based on the fixed, high-dose nature of our combined LPS regimen, which aimed to provide sufficient support without the need for individualized hormone level adjustments or rescue protocols, consistent with recent perspectives suggesting that such monitoring may be unnecessary with adequate LPS [ 19 ]. Cleavage-stage embryos were transferred on the morning of progesterone day 4, while blastocysts were transferred on the morning of progesterone day 6. Serum beta-hCG levels were checked 10–12 days after transfer, followed by transvaginal ultrasound 2 weeks later to confirm fetal viability. LPS was maintained until 12 weeks of gestation in ongoing pregnancies. Following oocyte retrieval, oocyte-cumulus complexes were cultured for 2–4 hours, then denuded and assessed for maturity. Metaphase II oocytes underwent ICSI, with the injected oocytes cultured in a continuous single culture medium (Fujifilm Irvine Scientific) at 37°C, 5% O2, and 6% CO2. Embryo morphology was evaluated on day 3 (67–69 hours post-ICSI), with good-quality cleavage-stage embryos defined as having ≥6 cells, < 25% fragmentation, stage-specific cell sizes, and/or evidence of compaction [ 20 ]. Blastocysts were graded on day 5 (114–116 hours post-ICSI) using the Gardner system based on expansion, inner cell mass, and trophectoderm. Blastocysts graded ≥3BB were considered as good quality [ 21 ]. Slower growing embryos were cultured until day 6. Embryos were cryopreserved at either cleavage or blastocyst stage using vitrification (Cryotech, Japan). The number of embryos transferred followed the American Society for Reproductive Medicine guidelines [ 22 ], considering factors like age, embryo quality, and previous IVF outcomes, with a maximum of 2 embryos transferred per cycle. The primary outcome was the live birth rate, defined by the number of deliveries of at least one live neonate beyond 23 weeks of gestation per embryo transfer cycle. Secondary outcomes included the rates of positive hCG (serum beta-hCG > 5 IU/L at 10–12 days post-transfer), clinical pregnancy (gestational sac on ultrasound), ongoing pregnancy (fetal cardiac activity at 12 weeks), implantation (number of gestational sacs divided by the number of embryos transferred), biochemical pregnancy loss (positive hCG that failed to progress to clinical pregnancy), miscarriage (pregnancy loss after clinical confirmation up to 22 weeks of gestation), and multiple pregnancy (≥2 gestational sacs visualized). The preterm birth rate was defined as the number of deliveries before 37 weeks of gestational age divided by the total number of live births (excluding 5 cases due to missing date of birth information). Other pertinent perinatal outcomes included gestational age at birth, birthweight, and the incidence of major congenital abnormalities. Baseline characteristics and outcomes were compared between participants who received the HRT and mNC protocol, with categorical variables presented as number (percentage) and quantitative variables presented as means [standard deviation (SD)]. We utilized the propensity score methods to create matched data that achieved balance between the two groups. The propensity score was estimated using a multivariable logistic regression on the baseline characteristics of the participants, including age, body mass index (BMI) group (underweight: < 18, normal BMI: 18–  25 kg/m2), history of cesarean section, number of consecutive failed embryo transfers, duration and type of infertility, number, stage, and quality of embryos transferred. Then we used the MatchIt package for 1:3 matching, considering both optimal and nearest neighbor methods with a caliper of 0.1. The greater reduction in the absolute standardized mean differences (SMD) of the covariates before and after adjustment was achieved with the optimal method ( S1 - S2 Figures ). The treatment effect of using mNC compared to HRT on clinical outcomes was estimated both unadjusted and adjusted for all covariates that we used in the logistic (propensity scores) model, to clean up possible residual confounding and improve the precision of the estimation. We performed analyses on pregnancy, biochemical pregnancy, clinical pregnancy, ongoing pregnancy, live birth, multiple pregnancy, and implantation rate. For the adjusted analyses, we used the multiple regression model (log-binomial regression to estimate the risk ratio, and the Poisson regression for implantation rate to estimate the mean ratio. All statistical tests were two-tailed, with P < 0.05 denoting significance. All analyses were performed using the R language version 4.3.1 (R Foundation for Statistical Computing, Vienna, Austria).

The propensity scores, which represent the probability of being treated with the modified natural cycle (mNC) protocol for each embryo transfer cycle, were estimated using a multivariable logistic regression model. This model predicted the likelihood of selecting the mNC protocol based on patient and cycle characteristics, including age, body mass index, history of cesarean section, number of consecutive failed embryo transfers, duration and type of infertility, number, stage, and quality of embryos transferred. The plot shows the distribution of propensity scores in the hormone replacement therapy (HRT) group and the mNC group before and after propensity score matching. (TIF) The plot compares the absolute standardized mean differences of covariates between the modified natural cycle (mNC) and hormone replacement therapy (HRT) groups after propensity score matching using two different methods: optimal and nearest neighbor (with a caliper of 0.1). Each dot represents a covariate. The optimal matching method (red dots) achieved better balance compared to the nearest neighbor method (green dots), as evidenced by the smaller absolute standardized mean differences across all covariates. The dashed vertical lines indicate the recommended thresholds for acceptable balance (0.1 and 0.2). (TIF)