ABSTRACT
Uncertainty exists concerning the type, adjunct, or dose of regimen to offer in frozen cycles in infertile women undergoing IVF/ICSI. Current systematic reviews have failed to identify one method of endometrial preparation as being more effective than another, whereas many IVF Units use variable and mixed protocols mainly based on their experience and convenience of use. Thus, we performed a four-center two-arm retrospective cohort study, encompassing 439 cycles in 311 women. The modalities analyzed were: Modified natural cycle without and with luteal support (Groups 1,2) and Hormone Replacement cycle (HRC) with and without GnRHa suppression (Groups 3,4). Various schemes of progesterone and estradiol were used and compared. χ2 tests for categorical data and t-tests for continuous data were employed, stratifying by exposure, along with univariate and multivariable Logistic Regression models and subgroup analyses, according to the number of embryos transferred (1 vs. ≥2) and day of transfer (d2 vs. d5). Group 3 presented with statistically significant higher live birth and miscarriage rates in comparison to Group 4 (RR = 5.87, 95%CI: 2.44–14.14 and RR = 0.19, 95%CI: 0.06–0.60, respectively), findings that persisted in subgroup analyses according to the day of transfer and the number of embryos transferred. Progesterone administration through the combination of vaginal tabs and gel was associated with lower clinical pregnancy rates when compared to tabs (RR = 0.19, 95%CI: 0.05–0.71). The stable estrogen protocol compared to increasing estrogen at day 5 was associated with a higher positive hCG test and clinical pregnancy rate, while the progesterone through vaginal tabs was linked with lower miscarriages compared either with gel or combinations. In conclusion, HRC with GnRHa appears to be superior to HRC without GnRHa, concerning live birth and miscarriage, especially when the number of embryos transferred are ≥2 and irrespective of day of transfer. The use of progesterone vaginal tabs compared to gel or combinations is associated with better outcomes. Age is a significant predictor of a negative hCG test and clinical pregnancy rates. A properly conducted RCT is needed to evaluate the optimal frozen embryo transfer preparation strategy.
Abbreviations: SD: standard deviation; BMI: body mass index; PCOS: polycystic ovarian syndrome; IQR: interquartile range; FSH: follicle-stimulating hormone; LH: luteinizing hormone; TSH: thyroid-stimulating hormone.
Introduction
There have been various trends on frozen-thawed embryo transfer (FET) cycles that have anecdotally been reported to be inferior to the fresh transfers (Davies et al. Citation1991; Muasher et al. Citation1991). However, the introduction of vitrification as a cryopreservation method has been a breakthrough and currently FET cycles are reported as exhibiting similar efficacy with regards to live birth rates, with the fresh transfers, also minimizing the risk of ovarian hyperstimulation syndrome (Shi et al. Citation2018). Likewise, they increase the cumulative pregnancy rate, reduce cost and can be carried out in a shorter time period than repeated fresh in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycles (Ghobara and Vandekekerckhove Citation2008; Ghobara et al. Citation2017).
A variety of strategies and regimens for FET have been used and proposed: spontaneous or modified natural cycles (NC); cycles in which the endometrium is artificially prepared by estrogen and progesterone with or without a gonadotrophin-releasing hormone agonist (GnRHa) (Lutjen et al. Citation1984; Jaroudi et al. Citation1991) and ovulation induction FET cycles (HRC). Reports have been plenty, but there is no existing consensus regarding the best protocol to choose in order to synchronize the endometrium, through a luteal phase ‘creation’ or replacement (Ghobara et al. Citation2017). Crucial parameters for the right choice encompass the control and flexibility of the timing of transfer, cost, and proper endometrial development, along with reasonable rates of clinical pregnancy, miscarriage, and live birth. Most HRC protocols try to mimic the natural cycle, replacing the natural variation of hormones; estrogens can be administered in the form of oral and vaginal tablets, transdermal patches and subcutaneous implants and progesterone in the form of oral and vaginal tablets or rings and intramuscular injections (Devroey and Pados Citation1998; Ghobara et al. Citation2017). Initial studies have reported similar pregnancy rates between NC and HRC, although there is evidence favoring the latter (Davies et al. Citation1991; Muasher et al. Citation1991); notably, a strong association of these cycles with miscarriage has been demonstrated (Lelaidier et al. Citation1995).
Currently, uncertainty exists concerning the type, adjunct, or dose of regimen to offer, while many IVF Units use variable and mixed protocols mainly based on their experience and convenience of use. Of note, systematic reviews, encompassing all published trials to date, have failed to identify one method of endometrial preparation in FET as being more effective than another (Groenewoud et al. Citation2013, Citation2017; Ghobara et al. Citation2017; Mackens et al. Citation2017). Furthermore, there is an augmenting tendency for a ‘freeze-all’ policy for a variety of clinical indications, including fertility preservation, repeated implantation failures, risk of OHSS, and inadequate endometrial development (Mizrachi et al. Citation2019).
In the absence of robust contemporary data, we performed a multicenter cohort study of both university and private IVF Units, to compare the different modalities used for pregnancy rates following FET treatment cycles in normoovulatory patients undergoing IVF/ICSI.
Results
From all four Units, complete data for 456 (148, 159, 22, and 127, respectively) cycles from 311 women was provided after application of the inclusion criteria. Following the exclusion criteria, 17 cycles were excluded due to older age (>43 years old), leaving a final total of 439 cycles in 311 women. Patient characteristics are presented in . Basal hormonal parameters and causes of infertility were similar between groups. In χ2 tests, mean age and ΒΜΙ, along with numbers of positive hCG tests, clinical pregnancies, and live births differed significantly among groups ().
Table 1. Descriptive characteristics of the study population [reported as total number (percentage) for counts]
In the univariate analysis, live birth was higher in Group 3 as compared to Group 4 (RR = 5.87, 95%CI: 2.44–14.14), and lower when progesterone administration through the combination of tabs and gel was compared to tabs only (RR = 0.10, 95%CI: 0.01–0.80) (); the former difference persisted in the multivariate analysis (RR = 5.77, 95%CI: 2.25–14.82) (). Miscarriage was lower in Group 3 compared to Group 4 (RR = 0.19, 95%CI: 0.06–0.60) and when progesterone administration through the combination of tabs and gel was compared to tabs (RR = 0.19, 95%CI: 0.05–0.71) (); the former difference persisted in the multivariate analysis (RR = 0.26, 95%CI: 0.07–0.95) ().
Table 2. Relative Risks for Exposures and Outcomes
Table 3. Adjusted relative risks for exposures and outcomes
Concerning the secondary outcomes, both a positive hCG test and clinical pregnancy were higher in Group 3 as compared to Group 4 (RR = 3.09, 95%CI: 1.93–4.94 and RR = 5.22, 95%CI: 2.82–9.63, respectively), while clinical pregnancy was lower when progesterone administration through the combination of tabs and gel was compared to tabs (RR = 0.19, 95%CI: 0.05–0.71) (). All differences persisted in the multivariate analysis (). All results remained stable when merging the Groups 1 and 2, while in all the others comparisons were not significant. In Group 1, women experienced zero events of clinical pregnancy, miscarriage, and live birth and thus were excluded from the analysis (, ).
In the subgroup analysis of day 3 embryo transfer, both live birth and clinical pregnancy were higher in Group 3 compared to Group 4 in both univariate and multivariable analyses (RR = 2.40, 95%CI: 0.63–9.12 and RR = 3.62, 95%CI: 0.95–13.75, respectively)Supplementary (, 5). In the subgroup analysis of day 5 embryo transfer, live birth was higher in Group 3 compared to Group 4 (RR = 4.83, 95%CI: 1.39–16.64), similar to positive hCG test and clinical pregnancy Supplementary (). The stable estrogen protocol was associated with higher positive hCG test and clinical pregnancy rates as compared to the increasing estrogen protocol, while the progesterone through tabs was linked with lower miscarriages compared either with gel or combinations (Supplementary Tables 6, 7). The rest of the comparisons yielded non-significant results Supplementary(–)8.
In the second subgroup analysis with ≥2 embryos transferred, both live birth and clinical pregnancies and number with a positive hCG test were higher in Group 3 compared to Group 4 (RR = 4.35, 95%CI: 1.76–10.79; RR = 4.41, 95%CI: 2.27–8.56 and RR = 2.33, 95%CI: 1.39–3.93, respectively); differences persisted in the multivariable analysis Supplementary (, 11). Positive hCG test rate was higher in the stable estrogen protocol compared to the increasing protocol (RR = 3.10, 95%CI: 1.04–9.22); clinical pregnancy was lower in the combination of progesterone regimens compared to tabs (RR = 0.16, 95%CI: 0.03–0.78); this difference persisted in the multivariable analysis Supplementary (, 9, 10).
Age was a significant predictor of negative hCG test and clinical pregnancy rates for women >40 years old in comparison to women <30 and 30–40 years old. This difference persisted in the comparison of women aged <30 years old vs. women 30–40 years old for positive hCG test, but not for clinical pregnancy (Supplementary –11).
Discussion
The purpose of this multicenter cohort study was to compare the different modalities used for frozen embryo transfer (FET) cycles in normoovulatory patients undergoing IVF/ICSI. The study encompassed four IVF Units and a total of 439 cycles in 311 women, from the initial cohort, after the application of the a priori set inclusion and exclusion criteria. Live birth was higher and miscarriage was lower in Hormone Replacement Cycles (HRC) with GnRH analogs (GnRHa) versus HRC without GnRHa, in both univariate and multivariate analyses. The same results were mirrored by positive hCG tests and clinical pregnancy rates. Clinical pregnancy was lower when progesterone administration through the combination of vaginal tabs and gel was compared to vaginal tabs. The direction of effects remained stable in subgroup analyses, according to the day of embryo transfer and the number of embryos transferred. Age was a significant predictor of negative hCG tests and clinical pregnancy rates for women >40 years old in comparison to women <30 and 30–40 years old. In the rest of the comparisons, e differences were non-significant or were lost in the multivariate analysis.
In our analysis, live birth was superior in HRC with GnRHa, when compared to HRC without GnRHa. Similar results were reported in a Cochrane review, including seven RCTs (Ghobara et al. Citation2017). In retrospective cohort studies, comparing HRC with GnRHa to NC, live birth rates were recorded as either similar (Alur-Gupta et al. Citation2018) or lower (Xiao et al. Citation2012). Miscarriage was lower in HRC with GnRHa, compared to HRC without. In contrast, in the Cochrane review, authors concluded that there was no evidence of a difference between groups studied in miscarriage rates (Ghobara et al. Citation2017). Our findings support the hypothesis of the positive role of the GnRHa in HRC cycles, by suppressing the premature LH rise, reversing the negative effect that this rise exerts on both follicular recruitment and endometrial development (Speroff and Vande Wiele Citation1971; Tesarik et al. Citation2003). Interestingly, the difference in live birth rates between HRC with and without GnRHa was persistent, both in day 3 and day 5 transfers, and when the embryos were ≥2, even when adjusting for age, BMI, and type of infertility in the multivariable analysis.
Concerning the secondary outcomes, in the univariate analysis, HRC with GnRHa was associated with a significant increase in a positive hCG test and clinical pregnancy, when compared with HRC without GnRHa. In contrast, in the Cochrane review, encompassing five RCTs and 872 participants, authors observed non-significant results, similar to those of nine non‐RCTs, referenced in the same review (Ghobara et al. Citation2017). Likewise, in a systematic review including three prospective studies with 631 cycles, authors reported no significant difference in clinical pregnancy rates and considerable heterogeneity among studies (Groenewoud et al. Citation2013). Notably, the findings in all the other comparisons of our study were similar to those of the referenced reviews.
Regarding the comparisons among endometrial preparation protocols, the stable estrogen protocol was associated with higher number of positive hCG tests and clinical pregnancy rates compared to the increasing one, only in embryo transfers at day 5 and when the number of embryos were ≥2. As for the progesterone protocol, the use of vaginal tabs was associated with higher live birth and lower miscarriage rates compared to both gel and combinations, in the univariate, but not in multivariate analysis, as well as with higher clinical pregnancy rates, persisted in both. In addition, vaginal tabs were associated with higher live birth, positive hCG test and clinical pregnancy rate when compared to both gel and combinations, when the embryos were ≥2 and with lower miscarriage rates, at day 5 embryo transfer. Unfortunately, there is not enough evidence in the literature for direct comparisons with our findings. A retrospective cohort, with approximately double the sample size of our study, reported no significant differences in the rates of live birth, implantation, and clinical pregnancy when various progesterone regimens were compared (Asoglu et al. Citation2019). Similarly, oral dydrogesterone demonstrated non-inferiority to micronized vaginal progesterone gel for clinical pregnancy (non-inferiority margin 10%) (Griesinger et al. Citation2018). As for the estrogen use, evidence shows no significant differences in endometrial thickness or clinical outcomes between transdermal and oral use (Kahraman et al. Citation2019). Interestingly, authors report the use of various regimens of doses when preparing the endometrium before and after the embryo transfer, and especially for the estrogen regimen (Simon et al. Citation1998; El-Toukhy et al. Citation2004).
The strength of the study lies in the fact that we involved four different IVF Units, two university and two private groups, from three different cities in Greece; we applied strict inclusion and exclusion criteria, removing studies where there was lack of any data; we performed direct comparisons and examined the most commonly used regimens in daily practice, also applying multivariable analyses; live birth and miscarriage constituted our primary endpoints.
Limitations of the study include its retrospective nature that is linked with known and unknown biases (Salas et al. Citation1999) and the small cohort size. Thus, some of the results in the univariate t were not persisted in the multivariate analysis, and vice versa: the latter possibly indicates that protocols and drug regimens are useful only in certain subgroups of participants and not generally. We also fully acknowledge the insurmountable fact that results reported have a high risk of bias and high heterogeneity, because of the wide range of infertility etiologies and the original data coming from four different IVF Units. Thus, differences observed should not be translated as frank effects of regimens, e.g. GnRHa, but rather as an inherent limitation of observational data to capture causal relations. We partly accounted for this in the multivariable analyses, where age, type of infertility and BMI variability were taken into account. Finally, we have not performed power analysis for the number of participants needed to include in this study: that was based on the number of patients that were employed in previous cohort studies on the same objective (Simon et al. Citation1998; Xiao et al. Citation2012; Mackens et al. Citation2017; Alur-Gupta et al. Citation2018).
In conclusion, HRC with GnRHa appears to be superior to HRC without GnRHa, concerning live birth and miscarriage, especially when the number of embryos transferred are ≥2 and irrespectively of the day of transfer. The use of progesterone vaginal tabs compared to gel or combinations is associated with better outcomes. The stable estrogen protocol is associated with higher positive hCG test and clinical pregnancy rate compared to increasing estrogen, only in embryo transfers at day 5 and when the number of embryos are ≥2. Age is a significant predictor of negative hCG tests and clinical pregnancy rates for women >40 years old when compared with women <30 and 30–40 years old. A properly conducted RCT, based on the reported weaknesses of the previous studies is needed to evaluate the optimal frozen embryo transfer preparation strategy, while other infertile populations should also be investigated.
Materials and methods
Patient population and study design
This is a four-center two-arm retrospective cohort study conducted at the Assisted Reproductive Units of the Third Department of Obstetrics and Gynecology, ‘Attikon Hospital’; Mitosis IVF Clinic, Athens; Embryolab, Thessaloniki; Department of Obstetrics and Gynecology, Medical School, Democritus University of Thrace, Alexandroupolis, Greece. The study protocol was registered in clinicaltrials.gov (NCT03965949); ethical and scientific approval was obtained from the Scientific committee of Embryolab Fertility Clinic (No 9/20-6-2018), while the Scientific Board and Bioethics Committee of the ‘Attikon’ Hospital was also notified (13/6/2018), before the start of the study; the collection of data lasted for 7 months (6–12/2009).
Following clinical evaluation along with previous medical and reproductive history, participants were categorized according to the infertility factor and demographic parameters were recorded along with the respective hormonal profiles. Inclusion criteria for the study entry were: age 25–42 years, BMI ≤ 35 and ≥19, and normo-ovulatory patients. Exclusion criteria included: poor ovarian response according to the Bologna criteria, PCOS patients according to the Rotterdam criteria, patients using donor oocytes and gestational carriers.
Written informed consents for the FET cycles were obtained during the fresh cycles, where both short and long 21 GnRH-analogs protocols were used. The type of the FET protocol was selected by the attending physician. All procedures and culture conditions, along with medical and paramedical personnel were the same for over 10 years of function of all Units, all of which were under standard operating procedures.
Oocyte retrieval was performed 36 hours post-hCG administration. Oocytes were incubated in Irvine Continuous Single Culture medium supplemented with HSA (Irvine CSC Complete) at 37 ° C, and 6% CO2 & 5% O2 for 2 hours before their denudation. The cumulus-corona complex was removed enzymatically using 80IU/ml hyaluronidase solution (FERTIPRO). On Day 0, ICSI was performed as described in details by Palermo G et al., 1992. Embryos were cultured in 25 μl Irvine Continuous Single Culture medium supplemented with HSA (Irvine CSC Complete), under oil at 37 0 C, 6% CO 2, and 5% O2 concentration. A monophasic approach was used as it concerned the culture system strategy. Therefore, embryos were cultured at the same drop up to blastocyst stage.
Embryo quality was assessed on days 2,3 and 5. For day-3 embryos, the number of cells, the appearance of blastomeres, and the presence of cytoplasm defects or fragmentation were evaluated (Veeck Citation1999). For the evaluation of day-5 embryos, we used the Gardner and Schoolcraft criteria (Gardner and Schoolcraft Citation1999) in which thin zona pellucida, smooth trophectoderm, equality and close adhesion of blastomeres, clearly visible blastocyst cavity, and well-developed inner cell mass with many closely aggregated cells are the most important parameters correlating to top blastocyst quality.
Embryos were cryopreserved using an open vitrification system. Briefly, embryos were initially exposed for 15 min in an Equilibration Solution (Irvine Vit Kit Freeze) which contained 7.5% Dimethyl Sulfoxide (DMSO) & 7.5% Ethylene Glycol (EG). Then, they were exposed for maximum 1 min in Vitrification Solution (Irvine Vit Kit Freeze), which contained 15% DMSO & 15% EG. The whole procedure took place in room temperature (~23oC). After their exposure to cryoprotectants, embryos by means of a single drop were placed in Cryotech open system devices before being plunged in liquid nitrogen.
Embryo transfers were conducted at days 3 or 5, while the maximum number of embryos transferred was less than three, as in accordance with the Hellenic legislation.
The following modalities were analyzed; representing the most commonly used in national IVF enters:
1. Modified natural cycle (NC), using urinary hCG (10,000 IU of Pregnyl®, Merck Sharp & Dohme, NJ, USA) or recombinant hCG (250 µg of Ovitrelle®, Merck Serono Pharmaceuticals), as ovulatory trigger, without luteal support (Group 1),
2. Modified NC, using hCG, with luteal support [progesterone (i. vaginal tablets ii. Vaginal gel iii. IM iiii. Combination (Utrogestan®, Besins, France) three times daily using 200 mg or in the form of vaginal gel (Vasclor 8% / Crinon 8%, 90 mg twice daily)] (Group 2)
3. Hormone Replacement cycle (HRC) [estrogens, i. Standard estradiol valerate (Cyclacur; Bayer Hellas A.B.E.E.), two tabs orally starting from day 2 and onwards or ii. Estradiol valerate increasing dose, starting at day 1 of cycle with 1 tab for 4 days, then 2 for 5 days, then 4 for 4], plus GnRHa suppression (starting at d21 of the previous cycle until the start of the luteal support (progesterone) (Group 3)
4. HRC without GnRHa suppression with luteal support (progesterone) (Group 4).
In cases of a positive pregnancy test, both progesterone and estrogen regimens were continued until 12 weeks of gestation. The primary outcome endpoints were live birth and miscarriage rates. Secondary endpoints included positive hCG test, clinical, multiple, and ectopic pregnancy rates; definitions used were according to those reported in Zegers-Hochschild et al. (Citation2017).
Statistical analysis
Continuous data are presented as mean ± SD or median [range] and categorical data are described by number of cases, including numerator, denominator, and percentages. Results for χ2 tests for categorical data and t-tests for continuous data are reported, stratifying by exposure. Significance level was set at 0.05. For the statistical analysis, R statistical software (R CORE TEAM Citation2020) and SAS were used.
Since there were patients with more than one measurement, univariate and multivariable Logistic Regression models using Generalized Estimating Equations (GEE) with an exchangeable working correlation structure, were employed to derive crude and adjusted relative risks (RRs with 95%CIs) for the differential impact of endometrial preparation and estrogen and progesterone administration modalities on positive hCG test and clinical pregnancy, miscarriage and live birth. The most commonly adopted modalities were used as references. Pre-specified parameters that were included in the models for potential confounding were the woman’s age (stratified in three subgroups: <30, 30–40, >40 years old), BMI [stratified in 4 subgroups: low (<19.9 kg/m2), normal (20–24.9 kg/m2), increased (25–29.9 kg/m2), obese (>30 kg/m2)] and type of infertility. Additionally, due to the fact that categories 1 and 4 of BMI contained very few patients, the variable was further stratified into two groups, low and normal (≤25 kg/m2), increased and obese (>25 kg/m2).
Subgroup analyses were performed according to the day of embryo transfer (day 3 vs. d5) and the number of embryos transferred (1 vs. ≥2). For each subgroup analysis, categorization for each protocol was applied. As specific groups with lower number of eligible women did not provide information across all confounders, three models were fitted, each adjusting for different confounders. Specifically, the models used were adjusted for age, BMI, and type of infertility, separately. Due to scarcity of observations in certain clinical outcomes, especially miscarriages, groups 1 and 2 in the endometrial preparation group were merged for both analyses.
Ethics approval
The study protocol was registered in clinicaltrials.gov (NCT03965949); ethical and scientific approval was obtained from the Scientific committee of Embryolab Fertility Clinic (No 9/20-6-2018), while the Scientific Board and Bioethics Committee of the ‘Attikon’ Hospital was also notified (13/6/2018), before the start of the study; collection of data lasted for 7 months (6–12/2009).
Authors’ contributions
Design of the study and writing of the paper: CS; major contribution to selection of samples, writing of the paper, interpretation of data for the work, gave their feedback of the final version to be published: AT, NC, GS, NK, GG; feedback of the final version for important intellectual content: NV; performed the statistical analysis: VK, DP.
Supplemental Material
Download MS Word (62.4 KB)Acknowledgments
Authors would like to thank the personnel of the above-involved Units for the collection of data.
Disclosure statement
The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
Supplemental data
Supplemental data for this article can be accessed here.
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