Ovulatory cycle frozen embryo transfer yields similar pregnancy outcomes to programmed transfer while avoiding painful injections.

Purpose Optimal endometrial preparation is essential for the transfer of a euploid embryo. Recent works have shown advantages of ovulatory cycle frozen-thawed embryo transfer (Ovu-FET) for simplicity, implantation rate, and reduction of hypertensive disorders of pregnancy; however, few women over age 40 years were included in these studies. This study compared pregnancy outcomes in patients aged 40–45 years between Ovu-FET and programmed cycle FET (PC-FET).

Retrospective cohort study in a university-affiliated fertility center. Two hundred eighty-seven FET cycles with euploid embryo transfer between 2015 and 2022 were included, with 116 cycles utilizing the PC-FET and 171 the Ovu-FET protocol. The use of letrozole was permitted for patients with irregular cycles. The primary outcome was ongoing pregnancy rate with secondary outcomes including pregnancy rate, clinical pregnancy rate, biochemical rate, and clinical loss rate. Multivariate regression was utilized to adjust for potential covariates.

The ongoing pregnancy rates were similar between the Ovu-FET versus PC-FET groups (62.6% vs. 58.6%, P = 0.50). Pregnancy rates (77.2% vs. 82.8%, P = 0.25) and clinical pregnancy rates (69.5% vs. 68.1%, P = 0.79) were also similar. Clinical loss rates were similar between the two groups (7.0% vs. 9.5%, P = 0.41). Biochemical loss rates were slightly lower for the Ovu-FET versus PC-FET groups (7.6% vs. 14.7%, P = 0.06) without reaching statistical significance.

Our study supports the use of Ovu-FET in women aged 40 years and older undergoing euploid embryo transfers. The results should strengthen confidence in the use of Ovu-FET for older patients.

Frozen embryo transfer, Endometrial preparation, Age, Ovulatory FET, Natural cycle FET, Letrozole FET

Optimal endometrial preparation is paramount when transferring a euploid embryo. There is abundant historical support for the programmed cycle, with sustained implantation rates in the first three transfer cycles remaining stable [1]. The disadvantages of the programmed cycle are also clear, including painful daily injections of intramuscular progesterone-in-oil for weeks to months as used in many programs in the USA [2] and potentially a higher risk of hypertensive disorders of pregnancy (HDP) [3–7]. Although Ovu-FET requires daily monitoring forseveral days to detect the dominant follicle and the LH surge, it offers greater comfort with far fewer needle sticks, and data show pregnancy rates at least comparable to programmed cycle frozen embryo transfers (PC-FET) in women with regular, ovulatory cycles [8–13]. Other studies have shown that for many women with anovulatory cycles, ovulation can be achieved using letrozole, allowing for Ovu-FET [14, 15]. Benefits of Ovu-FET over PC-FET include freedom from intramuscular injections and lower rates of hypertensive disorders of pregnancy, postpartum hemorrhage, and cesarean section; thus, the choice of FET protocol could have important implications for maternal and perinatal health [16, 17]. Much of the maternal benefit is hypothesized to be due to the presence of the corpus luteum as outlined by studies by von Versen-Hoynck et al. [4, 5]. Past studies comparing Ovu-FET to PC-FET have largely excluded older patients, with many studies restricting patients to those under 40 years of age at the time of the embryo transfer [3, 6, 9, 10, 18, 19]. Among studies that included women over 40, the numbers of patients undergoing Ovu-FET were very low. We are not aware of any past study that exclusively compared the outcomes of Ovu-FET and PC-FET in women over 40. Some have questioned if changes in menstrual cycle length that occurs with advancing maternal age (AMA) hamper successful and sustained implantation of a euploid embryo in an Ovu-FET. Although many women continue to have regular, ovulatory cycles until perimenopause, more subtle changes occur during the follicular phase. Klein et al. compared characteristics of the ovarian cycle in women aged 20–25 years to those aged 40–45 years [20]. They found that the follicular phase length was shorter in the older cohort by nearly 2 days, while the luteal phase length was similar. The LH surge was earlier in the older cohort, attributed to earlier selection of the dominant follicle. Similar findings were observed in two recent studies using electronic monitoring devices [21, 22]. Given the shortened follicular phase and cycle irregularity that sometimes occur with older patients, and the reluctance of many fertility centers to use Ovu-FET in this older population, our study seeks to determine if the menstrual changes associated with reproductive aging adversely affect Ovu-FET outcomes in the older patients. Specifically, our objective is to determine if pregnancy rates in patients aged 40–45 years differ between Ovu-FET and PC-FET after autologous euploid FET. For the purposes of this study, all ovulatory FET cycles—including those performed during a true natural cycle and those performed during a letrozole stimulated cycle—were classified, as Ovu-FET. This classification is supported by a prior study from our group comparing natural cycles to letrozole-stimulated cycles in over 1,700 FET [15]. In the referenced study, there were no significant differences between natural FET and letrozole-stimulated FET cycles regarding implantation, clinical pregnancy, ongoing pregnancy/livebirth, biochemical loss, or clinical loss rates. Furthermore, crude and adjusted risk ratios for all pregnancy outcomes for both ovulatory and anovulatory patients were similar.

Study design This was a retrospective cohort study in a single university-affiliated fertility center. Inclusion criteria consisted of patients aged 40–45 years undergoing their first euploid FET cycle between January 2015 and December 2022. All embryos were cryopreserved using vitrification at the blastocyst stage after blastomere biopsy for PGT-A. Only autologous FET cycles were included. All patients had a normal uterine cavity evaluation by sonohysterogram or hysteroscopy within 12 months before embryo transfer. This study was approved by the affiliated university’s institutional review board (UConn Health IRB number 23X-028–2). Treatment protocol Protocols for oocyte hyperstimulation in oocyte retrieval cycles were selected based on patient factors and physician preference, as described in a few of our prior studies [15, 23–25]. Gonadotropin dosing was determined based on patient’s ovarian reserve parameters, body mass index (BMI), and prior response to ovarian stimulation. The patients were triggered for final oocyte maturation when lead follicles reached at least 17 mm in mean diameter using either hCG 3,300–10,000 IU subcutaneously (PREGNYL; Merck, Kenilworth, NJ; NOVAREL; Ferring Pharmaceuticals, Parsippany, NJ), gonadotropin-releasing hormone (GnRH) agonist 1 mg (leuprolide acetate, Abbott Laboratories, Chicago, IL), or a combination of the two medications. Transvaginal ultrasound-guided oocyte retrieval was performed 35 hours after the trigger injection. Oocytes were fertilized via either intracytoplasmic sperm injection (ICSI) or conventional insemination and were cultured to the blastocyst stage. The zona pellucida of any high-quality (3 [22] or higher) blastocysts was breached and a trophectoderm biopsy was performed for PGT-A before embryo vitrification. PGT-A was performed primarily by CooperGenomics (Livingston, NJ) or Natera (Austin, TX, and San Carlos, CA) by physician preference or as requested by the third-party payor. Only patients with at least one euploid embryo were included in this study. In the FET cycle, the endometrium was prepared for Ovu-FET or PC-FET according to physician preference and the patient’s ovulatory status, as described in detail [15]. PC-FET cycles began with downregulation using a GnRH agonist in the luteal phase of the preceding cycle. PC-FET cycles were monitored with a baseline ultrasound and serum hCG, estradiol, and progesterone levels at the onset of menses. Increasing doses of oral estradiol (Estrace, Allergan, Madison, NJ) or transdermal estradiol (Vivelle-Dot, Novartis, Cambridge, MA) were administered to simulate rising estradiol levels of the follicular phase. Serum estradiol levels were measured on days 4, 8, and 12. An ultrasound was performed on cycle day 12 to assess the endometrial thickness and to confirm the absence of developing follicles. When the endometrial thickness reached 7 mm or more, intramuscular progesterone in oil 75 mg daily (Watson Pharmaceuticals, Copiague, NY) was started. Frozen-thawed embryo transfer was performed on the 6th day of intramuscular progesterone using transabdominal ultrasound guidance. Serum estradiol and progesterone levels were drawn on post-transfer days 5 and 10. Additional oral or transdermal estradiol and/or intramuscular or vaginal progesterone was administered as needed to achieve serum estradiol near 200 pg/mL or serum progesterone levels near 20 ng/mL. A serum hCG level was drawn 9 days post-transfer. Patients undergoing Ovu-FET were prepared as follows. Letrozole-stimulated FET cycles followed the same protocol as natural FET cycles, except that in letrozole cycles, oral letrozole 5 mg daily on cycle days 3–7 was administered to stimulate follicular recruitment. Letrozole was added to the FET protocol for patients with a history of irregular cycles, PCOS, or an elevated AMH level at the discretion of their treating physician. In both natural and letrozole FET cycles, transvaginal ultrasound was performed on cycle day 10 to ensure that a follicle had been recruited and that the endometrial stripe measured at least 7 mm. Patients were monitored with daily blood tests starting on cycle day 10 until the luteal hormone (LH) surge (defined as 20 IU/L or higher) was detected [24]. For patients in whom the day of the LH surge was not clear, labs were drawn the next day. LH surge was identified when the LH level exceeded 20 IU/L with a fall of estradiol by 20% on the following day [25, 26]. Two days after the LH surge, vaginal progesterone (Crinone 90 mg daily, Merck, Kenilworth, NJ, or ENDOMETRIN 100 mg twice daily, Ferring Pharmaceuticals, Parsipanny, NJ) was started. Embryo transfer was performed 6 days after the LH surge using transabdominal ultrasound guidance. The initial pregnancy test was performed 9 days after blastocyst embryo transfer, and an ultrasound was performed to confirm intrauterine pregnancy at 6–8 weeks of gestation. Progesterone supplementation was stopped at 7–9 weeks following natural and letrozole-stimulated FET cycles and at 10–12 weeks following programmed FET cycles. For this study, letrozole-stimulated and natural cycles were grouped together as Ovu-FET. Outcome measures The primary outcome was the ongoing pregnancy rate (OPR). Secondary outcomes included pregnancy rate (PR), clinical pregnancy rate (CPR), biochemical loss rate (BLR), and clinical loss rate (CLR). Pregnancy was defined as a positive serum hCG level 9 days after FET. Clinical pregnancy was defined as a positive serum hCG level with ultrasound evidence of an intrauterine gestational sac at 7 weeks of gestation. Biochemical loss was defined as a positive serum hCG level followed by declining hCG levels without evidence of a gestational sac on ultrasound. Clinical loss was defined as pregnancy losses among those with a clinical pregnancy. Ongoing pregnancy was defined as a viable pregnancy beyond 10 week of gestation. Statistical analysis Discovering JMP(R)17 (Copyright © 2022–2023, JMP Statistical Discovery LLC, Cary, NC, USA) was used for statistical analysis. Student’s t-test was used to compare continuous variables between two groups and one-way ANOVA for continuous variables when there are more than two groups. The chi-squared test was used for categorical variables. Continuous variables were presented as mean ± standard deviation (SD), and categorical variables were presented as count and percentage (%). For multiple regression analysis, SPSS Statistics version 27 (IBM, Armonk, NY) was utilized to assess the association between the FET protocols and the pregnancy outcomes while adjusting for potential covariates. These covariates included FET type, age at embryo transfer, body mass index (BMI), anti-Müllerian hormone (AMH) level, and primary infertility diagnosis.

Baseline characteristics A total of 287 euploid FET cycles (287 patients) were included with only each patient’s first transfer analyzed. There were 116 PC-FET and 171 Ovu-FET, including 45 Ovu-FET cycles that utilized letrozole stimulation in the follicular phase. The baseline characteristics are presented in Table 1. Patients undergoing Ovu-FET were on average 8 months younger than those utilizing PC-FET (P < 0.05). Other baseline characteristics between the Ovu-FET and PC-FET groups were similar. Table 1. | Baseline characteristics | Ovu-FET (N = 171) | PC-FET (N = 116) | P-value | |---|---|---|---| | Cycle age (y), mean ± SD | 41.5 ± 1.3 | 42.2 ± 1.6 | < 0.0001 | | BMI (kg/m2), mean ± SD | 26.9 ± 5.8 | 27.9 ± 5.5 | 0.16 | | Baseline AMH (ng/mL), mean ± SD | 2.5 ± 2.6 | 2.3 ± 2.8 | 0.63 | | Baseline FSH (IU/L), mean ± SD | 8.0 ± 2.9 | 8.1 ± 3.1 | 0.92 | | Parous, % (n) | 19.9 (34) | 21.5 (25) | 0.77 | | Smoking, % (n) | 0 (0) | 1.7 (3) | 0.08 | | Primary diagnosis, % (n) | 0.15 | || | Unexplained | 45 (77) | 44.7 (68) | | | Anovulation | 5.8 (10) | 9.2 (14) | | | Male factor | 22 (37) | 22.3 (30) | | | Diminished ovarian reserve | 0 (0) | 0.6 (1) | | | Tubal factor | 3.5 (6) | 2.6 (4) | | | Endometriosis | 1.7 (3) | 7.9 (12) | | | Current pregnancy loss | 16 (27) | 7.2 (11) | | | Fibroids | 4.1 (7) | 4.0 (6) | | | Other | 2.3 (4) | 1.4 (2) | | | Number of embryos transferred, mean ± SD | 1.1 ± 0.27 | 1.1 ± 0.31 | 0.90 | | PGT-A, % (n) | 100 (171) | 100 (116) | Overall clinical outcomes The overall clinical outcomes are summarized in Table 2. The ongoing pregnancy rates (OPR) were similar between the Ovu-FET versus PC-FET groups (62.6% vs. 58.6%, P = 0.50). Likewise, the pregnancy rates (PR) (77.2% vs. 82.8%, P = 0.25) and the clinical pregnancy rates (CPR) (69.5% vs. 68.1%, P = 0.79) were similar between the groups. The biochemical loss rate (BLR) was lower in the Ovu-FET group (7.6% vs. 14.7, P = 0.06), although statistical significance was not reached and this study was not powered to evaluate differences in the loss rates. The clinical loss rates (CLR) were similar between the two groups (7.0% vs. 9.5%, P = 0.41). Table 2. | FET Type | Ovu-FET (n = 171) | PC-FET (n = 116) | P-value | |---|---|---|---| | Pregnancy rate, % (n) | 77.2 (132) | 82.8 (96) | 0.25 | | Clinical pregnancy rate, % (n) | 69.5 (119) | 68.1 (79) | 0.79 | | Ongoing pregnancy rate, % (n) | 62.6 (107) | 58.6 (68) | 0.50 | | Biochemical loss rate, % (n) | 7.6 (13) | 14.7 (17) | 0.06 | | Clinical loss rate, % (n) | 7.0 (12) | 9.5 (11) | 0.41 | As there was a difference in the mean patient age at the time of transfer between the groups and a trend toward more anovulatory patients in the PC-FET group, a multiple regression analysis was performed to assess the influence of potential covariates. There was no significant association between ongoing pregnancy rate and FET type (P = 0.58), cycle age (P = 0.73), BMI (P = 0.99), primary diagnosis of anovulation (P = 0.73), number of embryos transferred (P = 0.9), or AMH (P = 0.13). A sub-analysis was performed for each patient year of age to look for potential differences among OPR in older years and is reported in Fig. 1. The OPRs Ovu-FET versus PC-FET, for each year of age at the time of the FET were as follows: 59.5% versus 60.0% for 40-41 years, 63.2% versus 55.2% for 41-42 years, 70.5% versus 70.8% for 42-43 years, and 65.2% versus 51.2% for 43 years and older. Advancing maternal age did not adversely affect OPR in Ovu-FET cycles. There were no statistically significant differences within any of these age strata for PR, CPR, OPR, or miscarriage rates. When comparing the letrozole-stimulated cycle FET outcomes to the natural cycle FET outcomes, there were no significant differences in PR, CPR, OPR, and miscarriage rates either. All P-values were > 0.05. The day of LH surge on average was slightly later in cycles that used letrozole compared to the true natural cycles, though this difference did not reach statistical significance. The serum estradiol level on the day of surge was higher in cycles thate used letrozole (P < 0.05), as shown in Table 3. There were no statistically significant differences in the ongoing pregnancy rates between these groups. Table 3. | Age group | All 40 + | 40–42 | 43 + | ||| |---|---|---|---|---|---|---| | FET Cycle Protocol | Natural (N = 126) | Letrozole (N = 45) | Natural (N = 94) | Letrozole (N = 33) | Natural (N = 32) | Letrozole (N = 12) | | OPR % (N) | 64.3 (81) | 57.8 (26) | 64.9 (61) | 51.5 (17) | 62.5 (20) | 75 (9) | | LH surge daye, mean ± SD | 13.9 ± 2.6 | 14.8 ± 3.2 | 14.0 ± 2.7 | 14.6 ± 3.3 | 14.2 ± 2.6 | 14.7 ± 1.8 | | Estradiol on day of surge in pg/mL, mean ± SD | 297 ± 125 | 358 ± 169* | 300 ± 125 | 371 ± 175 | 299 ± 114 | 291 ± 145 |

In this study comparing cycle protocol for patients 40 to 45 years of age undergoing their first euploid frozen embryo transfer, we found that pregnancy rates were similar between those using an Ovu-FET and those using a PC-FET. There was a trend toward a higher biochemical loss rate in PC-FET compared to with Ovu-FET, though statistical significance was not reached. However, there were no statistically significant differences between the groups in OPRs at 62.6% and 58.6% for Ovu-FET and PC-FET, respectively. Given the convenience and safety of Ovu-FET, we believe these findings provide reassuring evidence supporting its more extensive use for euploid embryo transfer in older patients. To our knowledge, this is the first study focusing on women 40 years and older, comparing pregnancy outcomes of Ovu-FET and PC-FET for euploid embryo transfer. The variation in menstrual cycle length with advancing maternal age has previously been outlined by Klein et al. [20]. The follicular phase length is shorter in women aged 40–45 years compared to women aged 20–25. Notably, the older cohort had a higher basal FSH and a shortened follicular phase length (12.9 days vs. 14.8 days) with an earlier LH surge. The authors concluded that the shortened follicular phase was due to earlier selection of the dominant follicle. More recently, the Apple Women’s Health Study used electronic devices to track over 165,000 menstrual cycles in 12,608 participants. Using age 35–39 years as the reference group, women aged 20–24 years had cycles that were 1.49 days longer, while women aged 40–44 years had cycles that were 0.49 days shorter [22]. Similarly, a study of Japanese women wearing basal body temperature recording devices examined data from over 9 million menstrual cycles in 80,000 participants. The follicular phase lasted 18 days for women aged 20–29 and only 15.4 days in those over 40 years (P < 0.0001). The luteal phase length however did not differby age [21]. With this information, one might question whether a shortened follicular phase would negatively impact pregnancy outcomes in an Ovu-FET cycle. Our current study makes a convincing argument that in women aged 40-45 years undergoing euploid embryo transfers, the slight shortening of the follicular phase does not adversely impact pregnancy outcomes compared to PC-FET. There are many benefits to using an Ovu-FET related to both maternal and neonatal outcomes. One of the most studied maternal morbidities associated with IVF is the spectrum of hypertensive disorders of pregnancy (HDP). The risk of HDP after FET has been reported to be as high as 15.3% [5, 27–29]. A lower risk of HDP has been found in pregnancies with a corpus luteum, such as those conceived in a natural and letrozole-stimulated FET cycle [8]. A recent meta-analysis of 85 studies comparing the risk of HDP with advanced reproductive technology (ART) demonstrated that FET was associated with a 1.73 fold increased risk of hypertensive disorders compared to patients after spontaneous conception [27]. Furthermore, compared to young patients, patients past the 40th birthdays had a 1.63 fold increased risk of HDP compared to younger patients [30]. Given the potential protective effect of the corpus luteum in preventing HDP, use of Ovu-FET should be more strongly considered in the older patients. The strength of this paper lies in the elimination of many variables present in past studies. All patients were treated in the same center using standardized programmed FET cycles with GnRH-a suppression and luteal support with daily intramuscular progesterone. Estrogen and progesterone levels were measured and optimized as needed. Endometrial thickness greater than 7 mm was verified. Likewise, all Ovu-FET patients were treated in a standardized fashion, with the day of surge determined with serum LH measurements, without the use of adjunctive hCG, and with vaginal progesterone supplementation. Many confounding variables were minimized by including only the first euploid embryo transfers. Our study was not without limitations, one of which being a small sample size. As this study focused on the first euploid transfer for each patient, we excluded all transfers of untested embryos and all subsequent transfers for the same patient. Patients’ allocation to a particular protocol was based on physician preference and clinical judgment. This likely introduced confounding bias, as a higher proportion of women in the PC-FET group met the diagnosis of anovulation than the natural FET group. Due to the retrospective nature of this study, temporal bias may be present. For instance, as the use of letrozole for anovulatory patients became more common in our clinic, this may have influenced protocol selection over time. Some of the reluctance to use the natural LH surge to time embryo transfer in an Ovu-FET cycle comes from a lack of confidence in the reliability of the natural LH surge. A standardized definition of the LH surge has been lacking, as the amplitude and duration of the LH surge as well as the time to ovulation from the onset of the surge, varies between individuals [31]. This has led many centers to initiate ovulation or augment the LH surge with exogenous hCG [12, 32]. Nonetheless, in this study, we relied solely on the spontaneous LH surge without additional hCG, and we supplemented with vaginal progesterone 2 days after an AM blood draw showing an LH surge > 20 IU/mL. If the LH surge was deemed borderline or equivocal, serum was drawn the following day, and the surge was defined as the first day LH exceeded 20 IU/mL with a 20% drop in estradiol on the following day [26]. Blastocyst transfer occurred 6 days following the surge. This method has been effective, and the implantation rates appear similar with this minor variability in transfer day [25]. There is some debate in the literature regarding the necessity of luteal phase support (LPS) in natural or letrozole-stimulated FET cycles. Our center continues this luteal phase support until 7–9 weeks of gestation based on individual physician preference. A recent meta-analysis addressed this question with a review of 6 studies, which found a significant advantage in favor of LPS with progesterone in natural FET cycles [33]. However, randomized controlled trials assessing the need for LPS in letrozole-stimulated FET cycles remain limited. In past studies of older reproductive age women, altered ovarian function has be observed as early as age 43, includes hyperestrogenism, hypergonadotropinism, and decreased luteal phase progesterone excretion [34]. For this reason, continuing supplemental progesterone until 7 weeks is likely prudent during Ovu-FET in the older patient.

In conclusion, for women aged 40 years or older undergoing euploid embryo transfer, the pregnancy rates were similar between Ovu-FET and PC-FET cycles. Given the increased risk of hypertensive disorders of pregnancy (HDP) with increasing maternal age, as well as the higher rates of HDP observed in patients undergoing PC-FET, expanding the use of Ovu-FET in this population may offer clinical benefits. Our study demonstrates that when the LH surge is appropriately identified and adequate luteal phase support is provided, Ovu-FET is a reliable and effective option for women aged 40–45 undergoing euploid embryo transfers. These findings should help instill greater confidence in the use of Ovu-FET for older patients. Author contribution All authors contributed to the study conception and design. Material preparation and data collection were performed by KW, DG, and JC. Data analysis was performed by KW, DG, JC, and LE. The first draft of the manuscript was written by KW, DG, and JC. All authors provided feedback, revised, and edited previous versions of the manuscript. All authors read and approved the final manuscript. Availability of data and materials Data supporting Fig. 1 and Tables 1, 2 and 3 are not publicly available in order to protect patient privacy. Declarations Ethics approval This was a retrospective cohort study in a single university-affiliated fertility center. This study was approved by the affiliated university’s institutional review board (UConn Health IRB number 23X-028–2). Conflict of interest 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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