Effect of different endometrial preparation protocols on pregnancy outcomes in frozen- embryo transfer cycles: a retrospective cohort study

Owing to the crucial role the endometrium plays in embryo implantation, the four main endometrial preparation protocols have become important factors in the study of pregnancy outcomes in the FET cycles. Previous studies have shown that the best of these four protocols remains controversial for women undergoing FET. A total of 10333 FET cycles from January 2018 to December 2018 were analyzed in this study. They were categorized into four groups according to endometrial preparation regimen: natural cycles (Group NC,n = 815), hormone replacement therapy cycles (Group HRT ,n = 6434), GnRH agonist articial cycles (Group GAC,n = 1392) and ovarian stimulation cycles (Group OC, n = 1692). All patients were followed up for at least 1 year. Pregnancy outcomes were compared between the four groups and multiple logistic regression models were used to adjust for the effects of confounding factors. *Comparisons were made by Pearson Chi-square test. **Comparisons were made by Kruskal-Wallis test.There was no statistically signicant difference between groups with the same corner letters, and a signicant difference between groups with different corner letters.NC, natural hormonal The results of this study also show the pregnancy outcomes for the four different endometrial preparation protocols of FET cycles (Table 2). In relation to pregnancy complications among the four groups, there was no statistical signicance between those who experienced ectopic pregnancy (P = 0.627) and miscarriage (P = 0.164). Biochemical pregnancy rate, clinical pregnancy rate and live birth rate in the NC group were also not statistically signicant compared to the other three groups. However, the HRT group had a higher biochemical pregnancy rate (64.6%) than the GAC (57.7%) and OC (59.5%) groups with p < 0.001.Similar results were found for clinical pregnancy and live birth rates in the HRT group compared to the GAC and OC groups (p < 0.001) .But there were no statistically signicant differences between Group OC and Group GAC in terms of biochemical pregnancy rates, clinical pregnancy rates, or live birth rates (p > 0.001). biochemical pregnancy, positive clinical pregnancy and live births the GAC and OC groups (p < 0.001), no signicant differences compared to the NC group. The ndings in previous also showed similar pregnancy outcomes between the natural cycle group and other endometrial preparation regimen groups. A retrospective cohort study analysed 214 NC cycles and 276 HRT cycles, with similar live birth rates (NC = 33.6% HRT = 29.3%, P = 0.47) and clinical pregnancy rates (NC = 40.2% HRT = 36.6%, P = 0.35) in the two groups(22). In addition, a prospective randomized controlled trial comparing clinical data from the NC group (n = and the OC group (n = 60) showed no signicant differences between the two groups in terms of positive biochemical pregnancy rate (NC = 34.0% OC = 23.1%, P = 0.22), clinical pregnancy rate (OC: 13/53 = 24.5%; NC: 12/52 = 23.1%; P = 0.86) and live birth rate (OC = 24.5% and NC = 23.1%, P = 0.86) (23). In this study also, there were no signicant differences in ectopic pregnancy rates and miscarriage rates between the four groups in our study. These ndings are also consistent with a prospective RCT trial which did not nd any difference in pregnancy outcome between the NC and GAC groups(24). Another study also corroborated that the NC and OC similar clinical frozen embryo transfer our Although, did not


Introduction
The frozen-thawed embryo transfer (FET) has increasingly been used in assisted reproductive technology (ART) compared to the fresh embryo transfer as a results of its higher live birth rate and lower risk of ovarian hyperstimulation syndrome (OHSS)(1-6).
Successful pregnancy is a result of dynamic crosstalk between a competent blastocyst and a receptive endometrium (7) .Synchronization of endometrial and embryonic development is an important factor which improves endometrial receptivity for a successful pregnancy(8-10). Owing to the crucial role the endometrium plays in embryo implantation, preparation of the endometrium involving endometrial thickness and morphology, uterine blood ow and other factors related to receptivity (11), has become an important factor in the study of pregnancy outcomes in the FET cycles (12,13).
There are four main endometrial preparation protocols for women undergoing FET cycles: natural cycle (NC),hormone replacement treatment cycle (HRT), GnRH agonist arti cial cycle (GAC) and ovulation stimulation cycle (OC).Although a retrospective cohort study and other studies comparing pregnancy outcomes in four endometrial preparation groups showed that the natural cycle was associated with higher live birth rates (14)(15)(16), studies by Groenewoud et al. (17) and Mounce et al.(18) showed no statistically signi cant difference in live birth rates, or clinical pregnancy rates between the natural and hormone replacement cycle groups. Other studies have also shown that the best of these four protocols remains controversial for women undergoing FET (19,20).
In the face of these controversial ndings, our aim in this study therefore was to explore the optimal endometrial preparation regimen by comparing the pregnancy outcomes of the four protocols, taking into account a number of factors known to in uence the pregnancy outcome of fresh embryo transfer.

Study Design and Patient Population
This was a retrospective cohort study comprising 10,333 cycles of frozen embryo transfers performed at Xinan Gynecological Hospital in Sichuan, China, from January 2018 to December 2018. All patients were followed up for at least 1 year, and the patients' clinical data were extracted from the electronic medical record system. Patients were excluded from the study if they met the following criteria: women who had a serious genetic, physical or mental illness that could affect their pregnancy; chromosomal abnormalities in either spouse; incomplete clinical and pregnancy data recording.
The study was approved by the Institutional Ethics Committee of Chongqing Medical University.
Endometrial Preparation natural cycle (Group NC). On day 2 to 3 of the menstrual cycle, veinous blood was taken to assess hormonal levels. Follicular development and endometrial thickness were monitored by ultrasound, and triggered with intramuscular injections of HCG (Shanghai ,Lizhu) 6000 U (not all HCG injections are necessarily given). When the dominant follicle has been ovulated and the endometrium was ≥ 8 mm, oral dydrogesterone 10 mg/dose (Abbott, Holland) twice a day was started (some use 20mg bid). When the dominant follicle has been ovulated and the endometrium is < 8mm, oral dydrogesterone combination tablets 20 mg/dose (Abbott, Holland) twice a day was given for 4 days. Blastocysts were thawed and resuscitated for transfer, or on day 2 if the embryos were in the cleavage stage.
hormone replacement treatment cycle (Group HRT). Ultrasound and hormonal levels including HCG were monitored on the second to third day of menstruation. When both ultrasound and hormones showed basal ovarian status (i.e. E2 < 183.5 pmol/L, endometrial thickness < 5 mm, after elimination of pregnancy), estradiol (Abbott, Holland) 2 mg/time, was administered one to twice a day for 10-12 days. After 10 days of continuous used, endometrial thickness was followed up and monitored. When the endometrial thickness was ≥ 8mm, luteal conversion was initiated, i.e. intramuscular progesterone (Zhejiang Xianju) 60 mg once daily or vaginal administration of Certolone (Merck, Switzerland) 90 mg/d, or vaginal administration of Angel (CyndeaPharma, France) 0.6 mg/d. Concurrently, estradiol administration was switched to complex packing estradiol tablets (Femoston, Abbott, Holland) 2-3 times daily. Thereafter, the frozen blastocysts were thawed for resuscitation transfer on day 6 of the luteal transition; for oocyte embryos, the frozen embryos were thawed for resuscitation transfer on day 4 of the luteal transition.
GnRH agonist arti cial cycle (Group GAC). After ruling out pregnancy on the second to third day of menstruation, a gonadotropin-releasing hormone agonist (GnRH-a) (Dalfylline, Beaufort-Epsom, France; or Dabigat, P zer, Germany) 3.75 mg was administered once intramuscularly. After 28-30 days, patients were followed by blood sampling and ultrasound to determine whether pituitary downregulation criteria were met (i.e. E2 < 183.5 pmol/L, FSH < 5U/L, LH < 5U/L, endometrial thickness < 5mm). If the criteria for down-regulation was not met, a further delay for about 1 to 2 weeks was allowed until the criteria was met and the endometrium was then prepared using an arti cial cycle protocol.
ovulation stimulation cycle (Group OC). Letrozole 2.5-5mg, or Tamoxifen 20-40mg, or Clomiphene 50mg was taken for 5 days on day 2-5 of menstruation. Monitored follicle development from day 10 of menstruation and decided whether to inject urinary sex hormones based on follicle development. When the follicle was greater than 18mm and the blood LH was 2-3 times the basal value, an intramuscular injection of 6,000-10,000 U of chlortetracycline HCG would be given. Post-ovulation medication was the same as in a natural cycle.
Embryo thawing, transfer and post-transfer corpus luteum support Transfer of embryos under ultrasound guidance according to conventional vitri cation and freezing methods. Oral estradiol was gradually reduced until discontinued at 8-9 weeks gestation. Intramuscular progesterone 60mg/d or vaginally administered progesterone (Certolone certolone 90mg/d or Angel 0.6mg/d) or oral progesterone (dydrogesterone) 30-40mg/d or oral complex packing estradiol tablets (Femoston, Abbott, Holland) 2 pieces/d was taken until 10-12 weeks gestation.

Pregnancy Con rmation
On 14 days after embryo transfer, pregnancy testing was done by measuring serum hCG level. Ultrasound was performed on day 28 after embryo transfer to determine the number of gestational sacs, and to monitor fetal heartbeats. All patients were closely followed up up to 1 year after embryo transfer.

Outcome parameters
The patients' baseline characteristics and clinical data were extracted from the medical record system: female age, female body mass index (BMI), male age, type of embryo transferred, endometrial thickness at one day before transfer, basal antral follicle count( AFC), basal anti-müllerian hormone(AMH) and basal follicle-stimulating hormone(FSH).
The primary outcomes were clinical pregnancy rates and live birth rates. Secondary outcomes were the incidence of biochemical pregnancy, miscarriage and ectopic pregnancy. Clinical pregnancy was de ned as the presence of at least one gestational sac in the uterine cavity con rmed by vaginal ultrasound at 28 days of gestation, with an embryonic bud and primordial heartbeat. Live birth was de ned as the birth of at least one live baby after 24 weeks of gestation. Biochemical pregnancy was de ned as positive serum β-HCG (β-HCG ≥ 5 U/L) at 14 days after transplantation. Ectopic pregnancy was de ned as a gestational sac observed by ultrasound outside the uterus.
Miscarriage was de ned as clinical pregnancy loss by the 24th week of gestation.

Statistical analysis
Non-normally distributed continuous variables were statistically described using median (25th-75th percentile) and comparisons between groups were made using a non-parametric test (Kruskal-Wallis rank sum test). For categorical variables, they were described in terms of number of cases (percentages) and comparisons between groups were made using the Pearson chi-square test. When overall rates between groups were statistically signi cant, further multiple comparisons were made using the partitions of χ²method, and p-values and corrected using the Boferroni method. Multiple logistic regression was used to analyse the pregnancy outcomes between the four endometrial preparation methods, adjusting for female age, female BMI, male age, type of embryo transferred, endometrial thickness at one day before transfer, basal AFC, basal All statistical analyses were carried out using the Statistical Package for Social Sciences (SPSS) version 25.0. Pvalue < 0.05 was considered to be statistically signi cant.

Results
A total of 10,333 FET cycles were included in our statistical analysis (Table 1). These FET cycles were divided into four groups: NC (n = 815), HRT (n = 6434), GAC (n = 1392) and OC (n = 1692) according to the endometrial preparation protocols. The results showed statistically signi cant differences in the general baseline characteristics of the four endometrial preparation groups (p < 0.05). The distribution of female age (< 35 or ≥ 35 years) was statistically signi cant between the four groups, with the HRT having the highest proportion of females aged < 35 years (73.8%). In terms of the body mass index (BMI), there was no statistical signi cance between the four groups in the proportion of people who were lean (BMI < 18.5 kg/m²) and obese (BMI ≥ 30.0 kg/m²). The results of the baseline characteristics also indicated that the HRT group had lower endometrial thickness at one day before transplantation (0.9cm ) compared to the NC, GAC and OC groups. Moreover, basal AFC and basal AMH were highest in the HRT group [basal AFC:15.4(9.0-23.0), basal AMH:3.8(1.9-6.4)ng/mL] but lowest in GAC group [basal AFC:9.0(5.0-15.0)), basal AMH:2.0(0.9-3.9)ng/mL]. Also, the NC group had a higher basal FSH than the HRT group and OC group [NC = 7.4(6.3-9.0)miu/L; HRT = 7.0(5.9-8.4)miu/L; OC = 7.1(6.0-8.6)miu/L, with p < 0.05], but this difference was not statistically signi cant when computed against the GAC group. The results of this study also show the pregnancy outcomes for the four different endometrial preparation protocols of FET cycles ( Table 2). In relation to pregnancy complications among the four groups, there was no statistical signi cance between those who experienced ectopic pregnancy (P = 0.627) and miscarriage (P = 0.164). Biochemical pregnancy rate, clinical pregnancy rate and live birth rate in the NC group were also not statistically signi cant compared to the other three groups. However, the HRT group had a higher biochemical pregnancy rate (64.6%) than the GAC (57.7%) and OC (59.5%) groups with p < 0.001.Similar results were found for clinical pregnancy and live birth rates in the HRT group compared to the GAC and OC groups (p < 0.001) .But there were no statistically signi cant differences between Group OC and Group GAC in terms of biochemical pregnancy rates, clinical pregnancy rates, or live birth rates (p > 0.001).

Discussion
Endometrial receptivity is widely considered as an important player in the success of pregnancy and so optimum treatment of the endometrium has been a key focus of interest for decades due to its potential clinical importance (21). In this study, we showed that in the overall population, the HRT group had higher rates of positive biochemical pregnancy, positive clinical pregnancy and live births than the GAC and OC groups (p < 0.001), with no signi cant differences compared to the NC group. The ndings in this study con rm previous studies that also showed similar pregnancy outcomes between the natural cycle group and other endometrial preparation regimen groups. A retrospective cohort study analysed 214 NC cycles and 276 HRT cycles, with similar live birth rates (NC = 33.6% HRT = 29.3%, P = 0.47) and clinical pregnancy rates (NC = 40.2% HRT = 36.6%, P = 0.35) in the two groups (22). In addition, a prospective randomized controlled trial comparing clinical data from the NC group (n = 59) and the OC group (n = 60) showed no signi cant differences between the two groups in terms of positive biochemical pregnancy rate (NC = 34.0% OC = 23.1%, P = 0.22), clinical pregnancy rate (OC: 13/53 = 24.5%; NC: 12/52 = 23.1%; P = 0.86) and live birth rate (OC = 24.5% and NC = 23.1%, P = 0.86) (23). In this study also, there were no signi cant differences in ectopic pregnancy rates and miscarriage rates between the four groups in our study. These ndings are also consistent with a prospective RCT trial which did not nd any difference in pregnancy outcome between the NC and GAC groups (24). Another study also corroborated that the NC and OC groups had similar clinical outcomes in frozen embryo transfer which is consistent with the results of our study (25). Although, we did not compute pregnancy outcome with age, a study showed that reduced pregnancy outcome has been seen to increase with increasing age of women, with the most occurring in women beyond 40 years of age. This implies that women below the age of 40 years tend to bene t from frozen embryo transfer(26).
After adjusting for confounding factors, there were no statistically signi cant differences in pregnancy outcomes in any of the three endometrial preparation groups compared to the NC group, which is consistent with the results of previous studies (22,27).Similarly, a retrospective cohort study primarily compared live birth rates between 923 GnRH agonist arti cial cycles and 105 natural cycles. After adjusting for female age, body mass index, diagnosis, preimplantation genetic screening/diagnosis, year and number of embryo transfers in a logistic regression model, the results showed no difference in any pregnancy outcome, with live birth rates aOR1.0,95%CI0.6-1.5(28). Compared to the other three groups, the HRT group having the lowest endometrial thickness at one day before transplantation. Two retrospective cohort studies also showed that the endometrial thickness was less in the HRT group than in the NC group (29,30).However, there was no statistical difference in the incidence of biochemical pregnancy, clinical pregnancy, miscarriage or live birth between the two groups (30), probably because endometrial thickness is not a decisive parameter for successful pregnancy (31). However, a study by Shaodi et al (2020) showed that to obtain optimal live birth rate, the endometrial thickness must remain within the range of 8.7-14.5mm as too thin or too thick endometrium would reduce the live birth live (32) .More studies are needed to address the effect of endometrial thickness on pregnancy outcome.
Patient speci city and acceptability need to be considered when deciding on the optimal endometrial preparation protocol for an individual. A study by Dancet and colleagues identi ed four treatment dimensions to be considered in the clinical advice given to patients during fertility treatment: burden, effectiveness, safety, and costs (33). Prato et al. also suggest that when two treatment options produce the same result, the less expensive option should be chosen (34). Natural cycles have a natural hormone-induced endometrial environment, avoid the use of exogenous hormones, avoid OHSS and are more conducive to embryo implantation, while being less costly and simple in terms of late luteal support, and are most acceptable to patients (35). Therefore, the natural cycle protocol could be the recommended option for endometrial preparation in the FET population. However, further research is needed in the future to determine the optimal endometrial preparation regimen in populations with different causes of infertility.
The strengths of this study include its relatively large sample size, limited exclusion criteria, and inclusion of women of all ages and infertility diagnoses. Furthermore, our study also took into consideration four endometrial preparation protocols commonly used in clinical practice and adjusted for other confounding factors using a multivariate logistic regression analysis model when exploring the effect of endometrial preparation protocols on pregnancy outcomes. However, the nature of the retrospective cohort study makes this study somewhat limited and more prospective cohort studies or RCT trial could be conducted in the future to explore pregnancy-related illnesses, fetal growth and development. Moreover, cost-effectiveness is an important factor to consider when developing a protocol for patients, and more research is needed in the future to investigate the optimal endometrial preparation protocol in the frozen-thawed embryo transfer population in relation to cost and different causes of infertility.

Conclusion
In conclusion, our study showed that natural cycles have similar pregnancy outcomes in terms of clinical pregnancy and live birth to the other three endometrial preparation options. Natural cycles have a natural hormone-induced endometrial environment, avoid the use of exogenous hormones, avoid OHSS, and are more conducive to embryo implantation.The natural cycles is also less costly and simple in terms of late luteal support.
The natural cycle protocol can therefore be the recommended option for endometrial preparation in the FET population.
Abbreviations FET, frozen-thawed embryo transfer; ART, assisted reproductive technology; OHSS, ovarian hyperstimulation syndrome; NC, natural cycle; HRT, hormone replacement treatment cycle; GAC, GnRH agonist arti cial cycle; OC, ovulation stimulation cycle; AFC, antral follicle count; AMH, anti-müllerian hormone; FSH , follicle-stimulating hormone Declarations Ethics approval and consent to participate The study was approved by the Institutional Ethics Committee of Chongqing Medical University.

Consent for publication
Not applicable.

Availability of data and materials
The datasets used during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.

Funding
The study was supported by the grants from Natural Science Foundation of Chongqing (nos:cstc2019jcyj-