The novel incorporation of aromatase inhibitor in hormonal replacement therapy cycles: A randomized clinical trial

Open AccessPublished:December 19, 2021DOI:


      Research question

      Does the incorporation of the aromatase inhibitor (letrozole) in hormonal replacement therapy (HRT) improve the pregnancy outcome in vitrified – warmed blastocyst transfer cycles.


      A randomized clinical trial (registration number: NCT04507022). HRT used in all cycles. Exogenous estradiol (E2), 6 mg daily started on day 2 or 3 of cycle. Tri-laminar endometrium ≥ 9 mm was the targeted cutoff. Thereafter, participants were randomized into two groups. Group A (HRT plus letrozole), 2.5 mg oral letrozole was given twice daily for 5 days only with continuation of daily estradiol. Then, daily intramuscular (IM), progesterone (P) started, with continuation of E2. Group B (HRT only), daily IM P administered in addition to daily E2. In both groups, good quality day 5 blastocyst transfer was planned on 6th progesterone day with continuation of E2 and P. Ongoing pregnancy rate was the primary outcome.


      112 patients were randomized, 56 in each group. Three participants did not have good quality blastocyst after warming; one in group A and two in group B and excluded from the study. Group A and B included 55 and 54 participants, respectively. Ongoing pregnancy rate was significantly higher in group A than group B (RR 1.39; 95% CI: 1.04 to 1.86). Additionally, clinical pregnancy rate was significantly higher in group A (RR 1.31; 95% CI: 1.02 to 1.68).


      A new protocol of incorporating letrozole in HRT cycles appears to significantly increase probability of pregnancy, compared to HRT alone.


      Receptive endometrium and a good quality blastocyst are prerequisites for successful implantation. Blastocyst vitrification techniques have witnessed tremendous improvement in last years with remarkable embryo survival. Optimization of endometrial receptivity and implantation is however an everlasting challenge. The use of hormone replacement therapy (HRT) has been proven to be successful for preparing the endometrium to receive the vitrified warmed embryos (Mackens et al., 2017). Estradiol (E2) is given first to reach a satisfactory endometrial thickness, then followed by progesterone (P) to mimic the natural cycles. E2 is mostly given for 10 to 14 days, and its supplementation might be prolonged to reach a targeted endometrial thickness. Notably, assessment of endometrial thickness at the end of estrogen phase, with the use of ultrasound has been traditionally used to predict frozen embryo transfer (FET) cycle outcome. Clinical pregnancy rates (CPR) and live birth rates (LBR) were found to decrease for each millimetre of endometrial thickness below 7 mm (Liu et al., 2018). Nevertheless, the ongoing pregnancy rate (OPR) and LBR were reported to be higher among participants having endometrial thickness ≥ 9 mm at the end of estrogen phase (Haas et al., 2019, Pan et al., 2020). The cutoff 0.89 cm thickness was elicited to be among the major factors affecting LBR in 917 young women undergoing FET in a recent study (Pan et al., 2020).
      Regarding P supplementation, the ideal route and dose has been controversial. In comparison to IM injections, patients mostly prefer the vaginal route considering its easy and painless administration. However, in a randomized clinical trial (RCT) comparing 3 modes of luteal support (LS) in 645 HRT cycles, there was increased miscarriage and lower OPR in the vaginal-only group in comparison to the daily IM group and the group using daily vaginal in addition to IM P every third day. Randomization to the vaginal-only arm was terminated due to the worse outcome (Devine et al., 2018). Admittedly, there are rising number of studies addressing probable increased rates of early pregnancy loss with the vaginal route of P administration in HRT cycles (Alsbjerg et al., 2013, Labarta et al., 2017).
      Currently, there is a recent concept that endometrial compaction (decreased thickness) between the end of estrogen phase and the day of embryo transfer (ET) has favorable impact on the outcome of FET cycles. The investigators suggested that progesterone receptor deficiency or resistance could be responsible for the lack of compaction and the worse FET outcome. They proposed decreasing the dose of estrogen with the start of progesterone supplementation to change the estrogen- progesterone ratio and prevent further endometrial growth (Haas et al., 2019, Zilberberg et al., 2020).
      We thought of using aromatase inhibitors (AI) as an alternative to the decrease of estrogen dose. More notably, we considered incorporating AI owing to the many reports about its possible beneficial effect on implantation (Cortínez et al., 2005, Karaer et al., 2005, Miller et al., 2012). Miller et al., 2012 had reported that a lack of endometrial ανβ3 integrin expression was associated with poor implantation and a simple 5-day treatment of the aromatase inhibitor, letrozole 5 mg/day, given early in the cycle, was shown to increase the expression of this marker and improve the outcome (Miller et al., 2012). Therefore, it appeared interesting to bring together the convenient easy scheduled HRT protocol with letrozole to maximize the outcome of FET cycles. The most suitable time to incorporate letrozole seems to be after reaching the targeted endometrium thickness, with the absence of any pre-ovulatory follicle and before starting P. Our proposed protocol of incorporating letrozole 5 mg/day, for 5 days in a time relatively near implantation in HRT cycles has not been tested before. The aim of the present RCT is to evaluate the outcome of FET cycles using a new protocol of HRT plus letrozole versus HRT only in preparation of the endometrium for the transfer of good quality vitrified-warmed blastocyst. In both arms, we opted to target an endometrial thickness of ≥ 9 mm and use an intense luteal support to maximize the outcome in both groups.
      Materials and methods
      Study design
      This RCT was conducted in the period from the 12th of August 2020 to the 5th of February 2021 in a private center. Women undergoing Blastocyst FET, who fulfilled the following inclusion criteria, were considered eligible for enrollment: (i) Women aged from18 - 37 years old with either regular cycles or oligomenorrhea/amenorrhea; (ii) These participants should have at least one good quality blastocyst available for vitrification on day 5 (3 BB and more) and also for transfer after warming (Gardner and Schoolcraft 1999). (iii) Participants having tri-laminar endometrium of ≥ 9 mm after E2 preparation. Written informed consent was obtained from all participants. The study was self-funded and approved by the institutional review board of the private center (RFC041120190008) and Zagazig University (ZU-IRB #6327/9-7-2020) and its trial registration number is NCT04507022.
      Endometrial Preparation
      HRT was used in all the cycles. Exogenous estradiol was started on day 2 or 3 of the cycle. In all participants, 2mg oral estradiol valerate (cycloprogynova, Bayer, Germany), was given three times daily. Ultrasound evaluation of the endometrium was performed 10 to 12 days after the start of E2. Tri-laminar endometrium of ≥ 9 mm was the targeted cutoff. If not yet ready, E2 supplementation was continued with serial US assessment until the desired cutoff was achieved with absence of any pre-ovulatory follicle or luteinized endometrium. Cycles of patients not reaching the targeted endometrium were excluded from the current RCT. Upon reaching endometrial thickness ≥ 9 mm, our study participants were randomized into two groups. In group A (HRT plus letrozole), 2.5 mg oral letrozole tablets (femara, Novartis, Switzerland), was started, twice daily for 5 days only with continuation of the 6 mg daily E2 supplementation. Then, daily intramuscular (IM) progesterone in oil (100 mg IM progesterone, Prontogest, IBSA, Switzerland) was started, once per day with continuation of the 6 mg E2. In group B (HRT only), daily intramuscular (IM) progesterone in oil (100 mg IM progesterone) was administered in addition to the daily dose of oral 6 mg E2.
      Random assignment of recruited participants was performed in a ratio of 1: 1. In total, 112 sealed identical envelopes were prepared; 56 encompass ‘HRT plus letrozole group’ with all technical guidance, and the other 56 envelopes encompass ‘HRT only group’ with all instructions. Each participant was permitted to choose just one envelope to determine the group to which she was assigned. Randomization was done by an assistant who was not involved in endometrial preparation.
      Embryo transfer and continuation of luteal phase support
      In both groups, embryos were warmed and transferred on the 6th day of progesterone supplementation. Cases not having at least one good quality blastocyst available for transfer were excluded from the study. After embryo transfer, luteal support was continued with the daily 6 mg E2 and the daily 100 mg IM P. Additionally, oral natural progesterone 100 mg (Progest, Pharco, Egypt), three times daily was started from the day of ET and continued thereafter, in accordance with our unit protocol in cycles with artificially prepared endometrium. This luteal support modality was continued until a negative pregnancy test, or a clinical pregnancy was documented. Clinical pregnancy was defined as visualization of fetal cardiac pulsation by ultrasound at 6 weeks of gestation or later. Thereafter, vaginal progesterone in a dose of 100 mg three times daily (endometrin, Ferring) was given with continuation of the 6 mg oral E2 and the oral progesterone (100mg three times per day). The 100 mg IM progesterone was administered every third day. E2 was usually stopped at 10 weeks’ gestation. Meanwhile, progesterone supplementation as described continued till 12 weeks’ gestation. Ongoing pregnancy was defined as visualization of fetal cardiac pulsation by ultrasound at 12 weeks of gestation or later (Zegers-Hochschild et al., 2017).
      Endometrium thickness and hormonal evaluation
      The endometrium was measured by trans-vaginal ultrasound. The best image was taken, showing a longitudinal section of the endometrium with the entirety of the endometrial lining through to the end of cervical canal in view. The three investigators agreed on the exact measurement (E.E., A.A. and H.S.). Endometrial thickness (End Thick) was evaluated three times in group A and two times in group B. In group A, it was measured at the end of estrogen phase before starting AI (named End Thick IA), after the end of the 5 days of AI before starting progesterone (named End Thick IIA) and on the day of FET (named End Thick IIIA). In group B, it was measured at the end of estrogen phase immediately before starting progesterone and on the day of frozen embryo transfer. In group A, estradiol levels were to be measured in the three time points allocated for the evaluation of endometrium thickness, named E2 I, E2 II and E2 III respectively. Progesterone level was measured in all participants in both groups on the day of embryo transfer.
      Outcome variables
      Ongoing pregnancy rate was the primary outcome. Clinical pregnancy rate and the occurrence of endometrial compaction were the secondary outcomes.
      Statistical Analysis
      Data were statistically described in terms of mean ± standard deviation (± SD), median and range, or frequencies (number of cases) and percentages when appropriate. Numerical data were tested for the normal assumption using Kolmogorov Smirnov test. Comparison of numerical variables between the study groups was done using Student t test for independent samples in comparing 2 groups of normally distributed data and/or large enough samples, while Mann Whitney U test for independent samples was used if the data violated the normal assumption. Comparison of hormonal levels and their changes between the compaction states was done using Kruskal Wallis test. For comparing categorical data, Chi-square (χ2) test was performed. Exact test was used instead when the expected frequency is less than 5. Multivariate stepwise logistic regression was performed to test the difference in OPR between the 2 groups after adjusting for all important factors. Two-sided p values less than 0.05 was considered statistically significant. All statistical calculations were done using computer program IBM SPSS (Statistical Package for the Social Science; IBM Corp, Armonk, NY, USA) release 22 for Microsoft Windows.
      Sample Size Calculation
      Sample-size calculation was done using the ongoing pregnancy rate (OPR) as the primary outcome. Chi-squared test for independent samples was chosen to perform the calculation.
      In our preliminary pilot study upon 25 participants using HRT and letrozole, OPR was 80% (20/25). Meanwhile, OPR was 55% (11/20) in HRT cycles with the targeted endometrial thickness of ≥ 9 mm and the aforementioned protocol of intensive luteal support in FET cycles. For an alpha error of 0.05, and a power of 80%, the calculated size of each group was 54 cases. An expected cancelation rate of 4% was considered, owing to the probable absence of good quality blastocysts available for transfer. Therefore, the final size of each group was determined to be 56.
      A total of 218 women were potentially eligible for recruitment. Of these, 89 did not meet the a-priori inclusion criteria, fifteen women had refused to participate, and 2 participants did not reach the targeted endometrium thickness (Fig. 1). One hundred and twelve women were randomized at the end of estrogen phase into group A (HRT plus letrozole, n = 56) and group B (HRT only, n = 56). On the day of embryo transfer, three participants did not have good quality blastocyst available for transfer after warming; one in group A and two in group B and were excluded from the study. Therefore, group A and B included 55 and 54 participants, respectively.
      Figure 1
      Figure 1Flowchart of participants in the study
      No statistically significant differences were found between both groups regarding age, duration of infertility, basal AMH and the number of previous ART trials (Table I). The cause of infertility did not differ significantly between both groups (P = 0.695). The use of long or antagonist protocol in fresh cycles did not also differ significantly (P= 0.504). The duration of estradiol supplementation days to reach the desired endometrial thickness was comparable (named as the estrogen phase). Endometrial thickness at the end of estrogen phase, on the day of ET as well as the median of endometrium thickness change between these two timings were comparable between both groups (Table I).
      Table IParticipants’ characteristics in the two groups.
      Group AHRT+Letrozole(n = 55)Group BHRTonly(n = 54)P value
      Age (mean, ± SD, years)27.9 ± 5.128.2 ± 4.30.753
      Duration of infertility (median, range, years)4 (1 – 13)3.3 (1 – 11)0.325
      AMH (median, range, ug/ml)3.7 (0.6 – 18.8)2.9 (0.5 – 29)0.472
      Previous ART trials0 (0 – 3)1 (0 – 2)0.385
      Duration of E2 (mean, ± SD, days)12.9 ± 1.613.2 ± 2.30.447
      End Thick I (mean ± SD, mm)11 ± 1.511 ± 1.40.917
      End Thick III (mean ± SD, mm)11 ± 2.410.9 ± 2.10.763
      End Thick III-I change (median, range, mm)-0.5 (-3.5 – 5.0)0.1 (-4.5 – 4)0.675
      P (ET) (mean ± SD, ng/ml)36.3 ± 1035.7 ± 11.10.754
      ET (n)1.9 ± 0.341.8 ±0.380.565
      Group A: HRT+ letrozole; Group B: HRT only; AMH: Anti-Mullerian Hormone; End Thick I: Endometrial thickness at the end of estrogen phase; End Thick III: Endometrial thickness on embryo transfer day; P (ET): Progesterone level on embryo transfer day; ET (n): number of transferred embryos.
      Progesterone level on the day of FET was comparable between both groups as well as the number of transferred embryos (Table I).
      Clinical pregnancy rate was significantly higher in HRT plus letrozole group in comparison to HRT only group (RR 1.31, 95% CI 1.02 to 1.68). Ongoing pregnancy rate was also significantly higher in group A than group B (RR 1.39, 95% CI 1.04 to 1.86) (Table II).
      Table IIPregnancy outcome in the two groups.
      Group A(HRT + Letrozole)Group B(HRT only)P valueRR(95%CI)
      CPR44/55 (80.0%)33/54 (61.1%)0.0301.31 (1.02 to 1.68
      OPR41/55 (74.5%)29/54 (53.7%)0.0231.39 (1.04 to 1.86)
      CPR: Clinical pregnancy rate; OPR: Ongoing pregnancy rate.
      Endometrial compaction (decrease endometrium thickness on the day of ET in comparison to the thickness at the end of E2 phase) occurred in 29 participants in group A (52.7%) and in 27 participants in group B (50%) with no statistical significance (P = 0.776). In group A, endometrial thickness was measured along the 3 predetermined time points and participants had shown 3 patterns of change:
      15 participants had an increase in endometrial thickness between End Thick IA and End Thick IIA. Then, there was further increase between End Thick IIA and End Thick IIIA. This was named as the Non-Compaction group (27.3%).
      22 cases had a decrease in endometrial thickness between End Thick IA and End Thick IIA. Then, there was further decrease between End Thick IIA and End Thick IIIA. This was named as the Maintained-Compaction group (40%).
      18 participants had a decrease in endometrial thickness between End Thick IA and End Thick IIA. Then, there was increase in endometrial thickness between End Thick IIA and End Thick IIIA. This was named as the Regressed-Compaction group (32.7%).
      In group B, 27 participants had shown endometrial compaction with decreased endometrial thickness on the day of ET in comparison to the thickness at the end of E2 phase; meanwhile, the other 27 participants had shown non compaction with increased endometrial thickness on day of ET. The compaction state neither had an impact on CPR nor OPR in both group A and group B (Table III).
      Table IIIComparison of pregnancy events between cases who experienced endometrial compaction and those without compaction within each of the study groups.
      No compactionCompactionp value
      Group AClinical pregnancy19/26 (73.1%)25/29 (86.2%)0.224
      Ongoing pregnancy19/26 (73.1%)22/29 (75.9%)0.813
      Group BClinical pregnancy15/27 (55.6%)18/27 (59.3%)0.402
      Ongoing pregnancy13/27 (48.1%)16/27 (59.3%)0.413
      Group A: HRT+ letrozole; Group B: HRT only
      Progesterone level on the day of ET did not affect the compaction states neither in group A nor in group B (p = 0.670 and 0.634 respectively) (figure 2). This was the case for both pregnant and non-pregnant women. (P > 0.05).
      Figure 2
      Figure 2Median progesterone level (ng/ml) according to the compaction state in HRT plus letrozole group (figure A) and according to the occurrence of compaction in HRT only group (figure B)
      In group A, 40 participants agreed to undergo the E2 levels measurements at the 3 different timings. E2 levels declined significantly by a median of 26.6% between the end of E2 phase and after using AI (P < 0.001). Then, there was significant increase in E2 by about 12.6% on the day of FET (P = 0.015). A median 16% decline of E2 was elicited on the day of FET in comparison to the values at the end of E2 phase which was significant (P <0.001). Neither E2 levels nor their percent of changes at the three different timings had an impact on the compaction state (no compaction, regressed and maintained compaction), whether in pregnant or non-pregnant women (P > 0.05). Median E2 levels and their percent of change over the 3 measurement time points are presented in accordance with the compaction state in figure 3.
      Figure 3
      Figure 3Median estradiol levels (E2, pg/ml, figure A) and percent changes (figure B) over the measurement time points (E2 I, E2 II and E2 III) divided according to the compaction state
      In the current RCT, a new protocol of incorporating letrozole in HRT cycles was tested versus HRT only. In both groups, a targeted endometrial thickness of ≥ 9 mm, intense progesterone use and luteal support were applied in a trial to optimally prepare the endometrium and maximize the outcome of transferring good quality blastocysts. There was a statistically significant increase in the ongoing pregnancy rate upon letrozole addition to HRT in the aforementioned strategy.
      Estradiol supplementation days and endometrial thickness at the end of estrogen phase were comparable between both groups. Admittedly, an adequate estradiol priming of the endometrium appears necessary for optimal endometrial proliferation and subsequent induction of sufficient progesterone receptors. This comes out to be vital for permitting subsequent progesterone stimulation and inducing the endometrial receptivity (Paulson 2011). The competence of estradiol priming had been traditionally tested by the detection of an adequate endometrial luteinization using endometrial biopsy. However, an adequate endometrial thickness in HRT cycles was shown to predict in phase endometrial histology; meanwhile, women with abnormal biopsies had significantly thinner endometrium (Hofmann et al.,1996). Endometrium thickness appears to be a satisfactory predictor for an adequate E2 priming needed for optimum FET outcome. A targeted endometrium thickness of ≥ 7 mm has been the goal (Liu et al., 2018). Other investigators, however, reported higher OPR in participants having endometrial thickness > 8mm than those having 7-8 mm at the end of the estrogen phase in frozen cycles (Haas et al., 2019, Pan et al., 2020). In the current study, we opted to use 9 mm as a targeted endometrial thickness in both groups to maximize the outcome. Admittedly, the impact of the duration of (E2) treatment prior to frozen-blastocyst transfers has been controversial. In a study including 1,439 patients with freeze-only due to pre-implantation genetic testing (PGT), the variation in the duration of E2 before starting progesterone (P) supplementation did not impact the outcome of frozen euploid blastocyst embryo transfer (BET), where E2 duration ranged from 10 to 34 days (Sekhon et al., 2019). Another group of investigators, however, underscored that E2 exposure more than 28 days had an adverse impact on the LBR among 1377 frozen–thawed blastocyst transfers (Bourdon et al., 2018). In the current study, cases not reaching the desired endometrial thickness in 21 days were cancelled and underwent further endometrial evaluation.
      The concept of endometrial compaction and the impact of decreased endometrial thickness between the end of estrogen phase and the day of FET is a rising controversial issue. Two recent studies by almost the same team of investigators have reported favorable outcomes for the occurrence of endometrial compaction. A significant increase in OPR was reported with each decreasing quartile of change in endometrial thickness and increasing percentage of compaction in the progesterone phase in comparison with the estrogen phase. The decrease in thickness occurred in almost 42% (115/271) and 64% (144/225) in the first and second studies respectively (Haas et al., 2019, Zilberberg et al., 2020). On the contrary, Bu et al. reported that, patients with an increasing endometrium thickness on the day of FET had significantly higher CPR compared with those having non-increased endometrium (Bu et al., 2019). A major drawback in the compaction issue is the highly possible inter and even intra-observer variation. Strict criteria for measurement must be agreed upon before any reliable consideration.
      In the current study, endometrial compaction occurred in 52.7% of participants in group A and in 50% of participants in group B with no significant difference. In group A, endometrial thickness decreased in 40 participants following letrozole use; however, the thickness increased again in 18 of them on the day of embryo transfer. This appears to explain the comparable endometrial thickness between the two groups on the day of ET as well as the comparable median of endometrium thickness change between the end of estrogen phase and the day of ET. OPR and CPR were not affected by the compaction state in both groups. P level on the day of transfer had no impact on the compaction states in both groups, whether in pregnant or non-pregnant participants.
      Regarding estradiol level assessment prior to P supplementation, it was shown to have no impact on the outcome of HRT cycles in a study upon 468 patients (Celik et al., 2019). Therefore, in current study, E2 was evaluated only in group A with the use of AI, to investigate possible interrelationship between estradiol and compaction state with any probable impact on pregnancy. Haas et al. suggested that lowering the E2 supplementation dose with the start of P supplementation might promote compaction and enhance pregnancy (Haas et al., 2019). In the current study, we did not decrease the estradiol dose; however, the use of letrozole was partially for accomplishing this goal. There were detectable changes in estradiol levels, with significant decline after the use of letrozole, which was followed by a significant increase on the transfer day. A median of 16% significant decrease in estradiol was elicited on transfer day in comparison to the values at the end of E2 phase. Nevertheless, neither E2 levels nor their percent of changes along the three timings had an impact on the compaction state or pregnancy. These findings contradict the assumption that lowering estradiol levels might have correlation with the occurrence of compaction or pregnancy.
      In the current study, the use of letrozole was not only to decrease estradiol, but more importantly to enhance implantation, since there have been many reports about its possible beneficial impact (Cortínez et al., 2005, Karaer et al., 2005, Miller et al., 2012). B3-Integrin was reported to be one of the important biomarkers of uterine receptivity in humans and αvβ3 integrin has been specifically described as an important predictor of ART outcome (Donaghay and Lessey 2007, Massimiani et al., 2020). Others, however, doubted the functional significance of this marker (Creus et al., 2002). In an interesting study, significantly lower implantation and pregnancy rates were reported in women undergoing ART who had low integrin expression in comparison to those having normal expression. When these women with low expression received letrozole as a part of their ovarian stimulation for ART, the pregnancy rate was comparable to women having normal integrin expression. Letrozole, 5 mg on days 2-6 was given with gonadotrophins, beginning on day 3, in antagonist cycles (Miller et al., 2012). In the current study, the chosen dose of 5 mg letrozole for 5 days was in accordance with the aforementioned study; however, it was given at a different time in FET cycles.
      AI was shown to enhance the expression of TNF-α, LIF, extracellular matrix proteins laminin and collagen IV in mice ovarian stimulation cycles (Karaer et al., 2005). All these markers are known to have a significant role in the dynamic developmental events of implantation. However, the same beneficial effect of AI on these markers in humans are to be established. In a review addressing the molecular signaling controlling blastocyst-endometrium crosstalk in human, there was emphasis that factors secreted by both the embryo and endometrium such as LIF, integrins and their ligands are vital for the process of embryo adhesion and invasion (Donaghay and Lessey 2007).
      An important inquiry here is how AI can possibly enhance implantation given that its reported half-life is 48 hours: it might be that the drug accumulates in the endometrium and escapes clearance. Lossl and colleagues reported that the androgen level remained approximately twice as high in the follicular fluid - but not in plasma - in participants receiving AI in the early follicular phase of a flexible antagonist cycle. The investigators emphasized that the use of AI can significantly impact the local environment as far as 14 days after stopping treatment (Lossl et al., 2006). In accordance, in-phase histological dating of the endometrium was reported in a study assessing endometrial morphology during the implantation window in letrozole stimulated cycles (Cortínez et al., 2005).
      There have been many studies about the use of letrozole in frozen-thawed cycles. A meta-analysis and systematic review including 75 968 FET cycles had reported comparable CPR and LBR between letrozole, natural cycles, artificial cycles and artificial cycles with agonist suppression (Chen et al., 2020). Notably, there was no increased birth defect rate in the letrozole group in comparison to the other groups, which ensure this drug's safety. In a large dataset of 110 722 single FET cycles from the Japanese registry, Letrozole use (n= 2409 cases) was associated with significantly higher CPR and LBR as well as lower rate of miscarriage compared with natural (n= 41 470) and HRT (n= 66 843) cycles (Tatsumi et al., 2017). However, strict conclusions can't be drawn from this registry data due to the shortage of information concerning the number of previous ART failures, dose and duration of letrozole intake, embryo quality and the reasons for selecting the specified FET method. In all the reported studies using letrozole, it was given early in the cycle for inducing mild ovarian stimulation and preparing the endometrium for FET. It appeared as a safe and efficacious alternative to standard regimens in FET cycles. Our study is the first one to use this drug at an atypical time following E2 preparation and before the start of P supplementation in FET cycles. It could be questioned whether the use of this drug close to the time of ET might pose any risk. Two studies however, had also used letrozole relatively close to the ET timing in fresh cycles with no reported adverse effects. In these studies, 5 mg daily letrozole was given with gonadotropins, from the first day of ovarian stimulation until the trigger day in antagonist cycles in a trial to improve the ART outcome (Haas et al., 2017, Shapira et al., 2020). Nevertheless, follow up the neonatal outcome is vital for more reassurance.
      In the current study, the intramuscular modality of progesterone administration for luteal support was chosen to maximize the outcome as was previously suggested (Devine et al., 2018). Admittedly, there are rising number of studies addressing the need to rerevise the standard regimen of vaginal P used for luteal support in FET cycles. Labarta et al. had investigated the relationship between serum P on the day of FET and OPR in 244 patients undergoing HRT with the use of 400 mg vaginal P twice daily. Patients with serum P < 9.2 ng/ml on the day of ET had a significantly lower OPR than those with higher values and the investigators advised the use of backup LS or a change of modality in subsequent cycles (Labarta et al., 2017). Further, Vaginal P gel has been used once daily for long; however, there is a currently rising trend for increasing its frequency of administration (Alsbjerg et al., 2013, Alsbjerg et al., 2020). In a study by Alsbjerg and colleagues upon 346 patients, doubling the dose of vaginal P gel (Crinone 90mg) had resulted in a significantly lower early pregnancy loss and higher LBR in comparison to those using a single daily dose (Alsbjerg et al., 2013). The study, however, was retrospective and randomized controlled studies are needed to confirm these results.
      We measured P level on the day of FET to study whether it has an impact on the outcome and also to study its possible interrelationship with endometrial compaction. A P level of 20.6 ng/ml on the day of FET was reported as the optimal cut-off for predicting OPR in one study using IM P (Boynukalin et al., 2019); however, other investigators did not support these results (Kofinas et al., 2015). In the current study, P level was measured on the morning of FET, 1 to 2 hours before the daily injection to investigate the value and the steadiness of the blood level as well. Most of the cases had P levels exceeding 20 ng/ml and P level was comparable between pregnant and non-pregnant, whether for clinical and/or ongoing pregnancy. Nillius and Johansson had emphasized that P accumulation within fatty tissue after IM P administration might work as a reservoir with subsequent continuous and steady release. This might explain the steady and more sustained P concentration in plasma after IM administration in comparison to the rectal or vaginal route (Nillius and Johansson 1971).
      Following the transfer of blastocyst, intense P supplementation was strictly followed in according to our unit protocol. In HRT, there is no corpus luteum with neither endogenous P nor E2 production and most of the studies use hormonal replacement until 8- 12 weeks of gestation. There has been an emphasis on caution when using HRT, owing to the alarming rate of increased early pregnancy loss in some reports (Mackens et al., 2017). In a retrospective study of 4582 women undergoing HRT, a decreased pregnancy loss rate and increased LBR was reported in participants with day 16 serum progesterone concentrations >50 nmol/L (= 20 ng/ml) (Basnayake et al., 2018). There is no enough evidence that supplementation to 12 weeks is superior to 8 weeks support; however, we opted to give the intense LS till the maximally reported duration of 12 weeks’ gestation).
      The strength of our RCT is in its novel idea of testing the combination of the easy scheduled HRT protocol with letrozole versus HRT only, with promising results from the novel incorporation of letrozole with our suggested dose, duration and timing of administration. Limitations are that our conclusions apply only to participants meeting our inclusion criteria which limit the result generalization. The study was an open label owing to the non-availability of placebo and we do not yet have data of live births, which is the optimal outcome. Further, there is lack of precise information on the exact mechanism of action either on endometrial receptivity or factors positively impacting trophoblast development.
      In conclusion, a new protocol of incorporating letrozole in HRT cycles appears to significantly increase the probability of pregnancy, in comparison to HRT only in frozen transfer cycles.
      Alsbjerg B, Thomsen L, Elbaek HO, Laursen R, Povlsen BB, Haahr T, Humaidan P. 2020. Can combining vaginal and rectal progesterone achieve the optimum progesterone range required for implantation in the HRT-FET model? Reprod Biomed Online; 40:805-11.
      Alsbjerg B, Polyzos NP, Elbaek HO, Povlsen BB, Andersen CY, Humaidan P. 2013 Increasing vaginal progesterone gel supplementation after frozen-thawed embryo transfer significantly increases the delivery rate. Reprod Biomed Online; 26:133-7.
      Basnayake SK, Volovsky M, Rombauts L, Osianlis T, Vollenhoven B, Healey M. 2018. Progesterone concentrations and dosage with frozen embryo transfers – What's best? Australian and New Zealand Journal of Obstetrics and Gynaecology; 58:533-8
      Bourdon M, Santulli P, Kefelian F, Vienet-Legue L, Maignien C, Pocate-Cheriet Kh, de Mouzon J, Marcellin L, Chapron C. 2018. Prolonged estrogen (E2) treatment prior to frozen-blastocyst transfer decreases the live birth rate. Hum Reprod;33: 905–13.
      Boynukalin FK, Gultomruk M, Turgut E, Demir B, Findikli N, Serdarogullari M, Coban O, Yarkiner Z, Bahceci M. 2019. Measuring the serum progesterone level on the day of transfer can be an additional tool to maximize ongoing pregnancies in single euploid frozen blastocyst. Reprod Biol Endocrinol; 17: 102.
      Bu Z, Yang X, Song L, Kang B, Sun Y. 2019. The impact of endometrial thickness change after progesterone administration on pregnancy outcome in patients transferred with single frozen-thawed blastocyst. Reprod Biol Endocrinol; 25; 17:99.
      Celik C, Asoglu MR, Karakis LS, Findikli N, Gultomruk M, Cavkaytar S, Bahceci M. 2019. The impact of serum oestradiol concentration prior to progesterone administration on live birth rate in single vitrified-warmed blastocyst transfer cycles. Reprod Biomed Online; 39: 1026-33.
      Chen D, Shen X, Fu Y, Ding CH, Zhong Y, Zhou C. 2020. Pregnancy Outcomes Following Letrozole Use in Frozen-thawed Embryo Transfer Cycles: A Systematic Review and Meta-analysis. Geburtshilfe Frauenheilkd; 80: 820-33.
      Cortínez A, De Carvalho I, Vantman D, Gabler F, Iñiguez G, Vega M. 2005. Hormonal profile and endometrial morphology in letrozole-controlled ovarian hyperstimulation in ovulatory infertile patients. Fertil Steril; 83:110-5.
      Creus M, Ordi J, Fábregues F, Casamitjana R, Ferrer B, Coll E, Vanrell J A, Balasch J. 2002. alphavbeta3 integrin expression and pinopod formation in normal and out-of-phase endometria of fertile and infertile women. Hum Reprod; 17: 2279-86.
      Devine K, Richter KS, Widra EA, McKeeby JL. 2018. Vitrified blastocyst transfer cycles with the use of only vaginal progesterone replacement with Endometrin have inferior ongoing pregnancy rates: results from the planned interim analysis of a three-arm randomized controlled noninferiority trial. Fertil Steril; 109: 266-75.
      Donaghay M, Lessey BA. 2007. Uterine receptivity: alterations associated with benign gynecological disease. Semin Reprod Med; 25: 461-75.
      Gardner D K, Schoolcraft W B. 1999. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol; 11:307-11.
      Haas J, Smith R, Zilberberg E, Nayot D, Meriano J, Barzilay E, et al. 2019. Endometrial compaction (decreased thickness) in response to progesterone results in optimal pregnancy outcome in frozen-thawed embryo transfers. Fertil Steril; 112: 503-9.
      Haas J, Bassil R, Meriano J, Samara N, Barzilay E, Gonen N, Casper RF. 2017 . Does daily co-administration of letrozole and gonadotropins during ovarian stimulation improve IVF outcome? Reprod Biol and Endocrinol; 15: 70.
      Hofmann GE, Thie J, Scott Jr, Navot D. 1996. Endometrial thickness is predictive of histologic endometrial maturation in women undergoing hormone replacement for ovum donation. Fertil Steril; 66: 380-3.
      Karaer O, Vatansever HS, Oruç S, Ozbilgin K, Cilaker S, Koyuncu MF. 2005. The aromatase inhibitor anastrozole is associated with favorable embryo development and implantation markers in mice ovarian stimulation cycles. Fertil Steril; 83: 1797-806.
      Kofinas JD, Blakemore J, McCulloh DH, Grifo J. 2015. Serum progesterone levels greater than 20 ng/dl on day of embryo transfer are associated with lower live birth and higher pregnancy loss rates. J Assist Reprod Genet; 32: 1395-9.
      Labarta E, Mariani G, Holtmann N, Celada P, Remohí J, Bosch E. 2017. Low serum progesterone on the day of embryo transfer is associated with a diminished ongoing pregnancy rate in oocyte donation cycles after artificial endometrial preparation: a prospective study. Hum Reprod: 32: 2437–42.
      Liu KE, Hartman M, Hartman A, Luo ZC, Mahutte N. 2018. The impact of a thin endometrial lining on fresh and frozen-thaw IVF outcomes: an analysis of over 40 000 embryo transfers. Hum Reprod; 33: 1883-88.
      Lossl K, Andersen AN, Loft A, Freiesleben NLC, Bangsbøll S, C. Yding, Yding C, Andersen CY. 2006. Androgen priming using aromatase inhibitor and hCG during early-follicular-phase GnRH antagonist down-regulation in modified antagonist protocols. Hum Reprod; 21: 2593–600.
      Mackens S, Santos-Ribeiro S, van de Vijver A, Racca A, Van Landuyt L, Tournaye H, Blockeel C. 2017. Frozen embryo transfer: a review on the optimal endometrial preparation and timing. Hum Reprod; 32: 2234–42.
      Massimiani M, Lacconi V, La Civita F, Ticconi C, Rago R, Campagnolo L. 2020. Molecular Signaling Regulating Endometrium–Blastocyst Crosstal Int J Mol Sci;21: 23.
      Miller P, Parnell B A, Bushnell G, Tallman N, Forstein D A, Higdon H L, Kitawaki J B, Lessey B. 2012. Endometrial receptivity defects during IVF cycles with and without letrozole. Hum Reprod; 27: 881–8.
      Nillius SJ, Johansson EDB. 1971. Plasma levels of progesterone after vaginal, rectal, or intramuscular administration of progesterone. Am J Obstet Gynecol; 110:470–7.
      Pan Y , Hao G ,  Wang Q ,  Liu H ,  Wang Z ,  Jiang  Q,  Shi Y ,  Chen Z. 2020. Major Factors Affecting the Live Birth Rate After Frozen Embryo Transfer Among Young Women. Front Med (Lausanne).;7: 94.
      Paulson RJ. 2011. Hormonal induction of endometrial receptivity. Fertil Steril; 96: 530-5.
      Shapira M, Orvieto R, Lebovitz O, Nahum R, Aizer A, Segev-Zahav A, Haas J.2020. Does daily co administration of gonadotropins and letrozole during the ovarian stimulation improve IVF outcome for poor and sub optimal responders? Journal of Ovar Res; 13: 66.
      Sekhon L,  Feuerstein J ,  Pan S , Overbey J ,  Lee J A , Briton-Jones C , Flisser E , Stein D , Mukherjee T , Grunfeld L , Sandler B , Copperman AB. 2019. Endometrial preparation before the transfer of single, vitrified-warmed, euploid blastocysts: does the duration of estradiol treatment influence clinical outcome? Fertil Steril; 111: 1177-85.
      Tatsumi T, Jwa SC, Kuwahara A, Irahara M, Kubota T, Saito H. 2017. Pregnancy and neonatal outcomes following letrozole use in frozen-thawed single embryo transfer cycles. Hum Reprod; 32: 1244-48
      Zegers-Hochschild F, Adamson GD, Dyer S, Racowsky C, de Mouzon J, Sokol R, Rienzi L, Sunde A, Schmidt L, Cooke ID, Simpson JL, van der Poel S. 2017. The International Glossary on Infertility and Fertility Care, 2017. Hum Reprod; 32: 1786-801
      Zilberberg E, Smith R, Nayot D, Haas J, Meriano J, Barzilay E, Casper RF. 2020. Endometrial compaction before frozen euploid embryo transfer improves ongoing pregnancy rates. Fertil Steril; 113: 990-5.


      Dr Eman Elgindy obtained her MD in 2001 from Zagazig University and PhD in reproductive Medicine from Maastricht University in 2013. She is professor of Obstetrics and Gynecology, Zagazig University and Clinical Director of Rahem fertility center, Egypt. Dr Elgindy authored or co-authored many researches in national and international journals.
      Key Message
      Frozen embryo transfer has been increased worldwide, necessitating the need to improve the reproductive outcomes in these cycles. Letrozole incorporation in HRT cycles appeared to have a beneficial role in this respect, improving the ongoing and clinical pregnancy rates.