https://orcid.org/0000-0003-0413-9045
Abstract
Objective
To investigate whether different endometrial preparation methods lead to different results.
Design
Retrospective cohort study.
Patients
Women with recurrent pregnancy loss undergoing frozen embryo transfer (FET).
Interventions
Natural cycle (NC) protocol (n = 111) with no drug or human chorionic gonadotropin (HCG) used for endometrial preparation, vs. the hormone replacement therapy (HRT) protocol (n = 797) with estrogen or gonadotropin releasing hormone agonist (GnRH-a) plus estrogen used for endometrial preparation.
Main outcome measures
Miscarriage rate and live birth rate (LBR).
Results
Compared to women in the HRT protocol, women undergoing NCs had fewer previous FET cycles, lower antral follicle counts (AFCs), fewer oocytes retrieved and a thicker endometrium on the day of progesterone administration. Women in the HRT group had a higher miscarriage rate (29.4% vs. 17.2%) and a lower LBR (37% vs. 46.9%) than the rates of women in the NC group. Univariate analysis showed that female age also had a negative association with the miscarriage rate. Logistic regression indicated that endometrial preparation using the NC protocol was linked to a decreased likelihood of miscarriage.
Conclusions
The NC protocol decreased the miscarriage rate and increased the LBR for patients with recurrent pregnancy loss compared with the HRT protocol.
Introduction
With the development of cryopreservation techniques, frozen embryo transfer (FET) has been extensively used in clinical treatment. Data from the International Committee for Monitoring Assisted Reproductive Technologies (ICMART) indicated that the number of reported FET cycles increased by 16% from 2011 to 2012 [Citation1], and 16.4% from 2012 to 2013 [Citation2]. The results generated from the data reporting system of the Chinese Society of Reproductive Medicine (CSRM) showed that FET cycles increased by 46.1% from 2013 to 2016 [Citation3]. Compared with fresh embryo transfer, FET cycles have several advantages, including better implantation rates [Citation4], a higher live birth rate (LBR) [Citation5], a decreased risk of miscarriage [Citation5], a lower small for gestational age rate, a lower low birth weight rate, a lower preterm delivery rate [Citation6], and a lower incidence of ectopic pregnancy [Citation7].
There are three main protocols for preparing the endometrium for reception: natural cycles (NCs) that involve monitoring the spontaneous luteinizing hormone (LH) rise and ovulation; hormonal replacement therapy (HRT) cycles with estrogen used for endometrial preparation or pretreatment with (gonadotropin releasing hormone agonist (GnRH-a) prior to estrogen administration; and ovarian stimulation (OS) cycles with clomiphene citrate (CC), letrozole, or gonadotropins for follicle growth and ultimately endometrial preparation [Citation8]. Previous studies compared different protocols for endometrial preparation but showed inconsistent results [Citation9–12].
As a distressing obstetric complication, pregnancy loss affects 7.5–10.7% of all women attempting to conceive [Citation13]. Recurrent pregnancy loss, defined as two or more pregnancy losses prior to 20 weeks, affects 1–3% of women during their reproductive period [Citation14]. Decreasing pregnancy loss is vital for these women undergoing IVF procedures. It has been reported that NCs of FET have a lower miscarriage rate than HRT cycles [Citation15–17]. However, these studies included all kinds of women trying to undergo FET. It continues to be unknown which protocol is better for women with recurrent pregnancy loss history. Therefore, our study retrospectively analyzed single-center data over five years; and compared the clinical outcomes of three protocols for endometrial preparation in women with a history of recurrent pregnancy loss to investigate whether different endometrial preparation methods lead to different results.
Materials and methods
Patients and study design
A total of 908 FET cycles, performed in the Center for Assisted Reproductive Technology of Northwest Women’s and Children’s Hospital, from January 1 2016 to December 30 2020, were analyzed. All patients were followed up until they gave birth. The data were derived from the medical records of our electronic database and were approved by the Ethics Committee of the Clinical Application of Human Assisted Reproductive Technology of Northwest Women’s and Children’s Hospital (IRB number: 2021002). As all data were retrospectively collected and anonymized, informed consent was not required.
The inclusion criteria for patients were as follows: (1) women who had a history of more than two prior pregnancy losses; (2) embryo cryopreservation after previous IVF cycles; (3) complete follow-up data; and (4) had undergone evaluation for parents’ karyotype, female uterus, thyroid function, fasting blood glucose, antiphospholipid antibody, autoimmune antibody, protein S, protein C, and male sperm. For patients with abnormal karyotype, PGT was used for treatment after fully discussed with experience genetics. Drugs were used for endocrine disorder. Aspirin and low molecular heparin were used for antiphospholipid antibody syndrome. Rheumatologist would give some treatment advice for women with autoimmune disease. Anti-thrombophilia therapy would be performed for women with thrombophilia. The exclusion criteria were: (1) untreated uterine malformation; and (2) no embryo survival after thawing.
In order to exclude the effect of aneuploid embryos on miscarriage, we performed a subgroup analysis. In the subgroup group, we chose women who underwent PGT cycle to assess the effect of the endometrium preparation protocol to miscarriage rate.
Endometrium preparation
All the patients were non-selectively allocated to two different protocols of endometrium preparation, according to their desire, characteristics, and the physician’s judgment. Generally, patients with regular ovulation or who preferred a NC were chosen for the NC protocol. Patients who were proven to be in anovulation or refused to monitor too often were allocated to the HRT protocol.
Nature cycle protocol
In the NC, women started their follicle growth monitoring with vaginal ultrasound on days 8–10 of the menstrual cycle. When the main follicle reached a mean diameter of 17 mm, serum LH was tested. If LH ≥20 IU/L, vaginal ultrasound was performed daily until ovulation. If LH <20 IU/L, 10,000 U of human chorionic gonadotropin (HCG) was applied for the final ovulation. From the day of ovulation, 60 mg of intramuscular progesterone or 600 mg of micronized progesterone was initiated daily until the day of embryo transfer. Three days later or five days later, frozen thawed embryo transfer was scheduled for cleavage-stage embryos and blastocyst-stage embryos, respectively.
Hormone replacement therapy cycle protocol
On the 5th day of the menstrual cycle, patients started to use a daily dose of 4–6 mg oral estradiol valerate (Progynova; Bayer Schering Pharma AG, Berlin, Germany) for 10–15 days. When the thickness of the endometrium was reached to 7 mm or more and the serum P was <1.2 ng/mL, 60 mg of intramuscular progesterone or 600 mg of micronized progesterone was applied daily until the day of embryo transfer. Four days later or six days later, frozen-thawed embryo transfer was arranged for cleavage-stage embryos and blastocyst-stage embryos, respectively.
Luteal phase support
From the day of embryo transfer, 60 mg intramuscular progesterone plus 20 mg of dydrogesterone and 4–6 mg of oral estradiol valerate daily were administered for luteal support. Sometimes, we would choose to use 600 mg of micronized progesterone plus 20 mg of dydrogesterone and 4–6 mg of oral estradiol valerate daily, or 90 mg of progesterone vaginal gel plus 20 mg of dydrogesterone and 4–6 mg of oral estradiol valerate daily for luteal support.
Pregnancy confirmation
Pregnancy was confirmed 12 or 14 days after embryo transfer according to serum β-HCG level. If the β-HCG level was ≥50 IU/L, luteal phased support would continue until 10 weeks. Four weeks after pregnancy confirmation, ultrasound was performed to determine fetal viability and the number of gestational sacs. The biochemical pregnancy rate was defined as pregnancy identified by serum β-HCG >7 IU/L. Clinical pregnancy was defined after ultrasonographic visualization of gestational sacs. Miscarriage was defined as a spontaneous loss before 28 weeks of gestational age. The LBR was defined as the live-birth born after 28 weeks of gestational age per woman.
Statistical analysis
Data are expressed as means and standard deviations (SDs) (for continuous variables) or counts and percentages (for categorical variables). Two group comparisons were performed by two-tailed Student’s t-test or the Mann–Whitney U-tests (continuous variables) or the Chi-square (χ2) test (categorical variables). Student’s t-test was used to analyze parametric data and the M–W U-test was used to analyze non-parametric data. We have checked the distribution of the data and used a two-tailed Student’s t-test or the Mann–Whitney U-tests separately.
Univariate analysis was utilized to analyze the association between variables and miscarriage rate. Stepwise binary logistic analysis was used to analyze the association between the endometrial preparation protocols and the miscarriage rate. Crude and adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were computed. Covariates were chosen based on the studies published in recent years and the statistically significant variables in our study. The included covariates were as follows: woman’s age at embryo transfer, previous number of miscarriages, endometrial preparation protocol, number of previous frozen cycles, antral follicle count (AFC), number of oocytes retrieved and endometrial thickness on the day of progesterone administration.
In adjusted model I, we adjusted for the woman’s age at embryo transfer and number of previous miscarriages. In adjusted model II, we adjusted for the woman’s age at embryo transfer, previous number of miscarriages, number of previous frozen cycles, AFC, number of oocytes retrieved and endometrial thickness on the day of progesterone administration. Data were analyzed using Statistical Package for the Social Science (SPSS) 22.0 software (SPSS Inc., Chicago, IL). For all statistical tests, p < .05 defined statistical significance.
Results
A total of 908 patients between January 2016 and December 2020 were included in our study, of whom, 797 underwent HRT cycles, while 111 underwent NCs. The baseline characteristic of the two groups is shown in . Compared with the HRT protocol, women in the NC protocol underwent fewer previous FET cycles and had a lower basal AFC. There were no significant differences in the other covariates.
Table 1. Baseline characteristics of patients between two endometrial preparation protocols.
The clinical outcomes are presented in . Women in the HRT group had more oocytes retrieved, a thinner endometrium on the day of progesterone administration, a higher chance of miscarriage, and a lower LBR than those of women in the NC group. The type of embryo transferred, biochemical pregnancy rate, clinical pregnancy rate (CPR), preterm birth rate, and ectopic pregnancy rate were comparable between the two groups.
Table 2. Clinical outcomes of patients between two endometrial preparation protocols.
Univariate analysis was applied to evaluate the effect of each variable on the miscarriage rate (). Women’s age, both at oocyte retrieve and embryo transfer, had a negative association with the miscarriage rate. Women’s BMI, number of previous FET cycles, previous number of miscarriages, basal AFC, number of oocytes retrieved, endometrial thickness on the day of P administration and number of good quality transferred embryos had no effect on the miscarriage rate.
Table 3. Univariate analysis for the miscarriage rate.
The association between endometrial preparation protocols and the miscarriage rate was analyzed using a logistic regression model with crude and adjusted models (). Compared with the HRT protocol, endometrial preparation using the NC protocol was associated with a decreased chance of miscarriage in the three regression models.
Table 4. Relationship between endometrial preparation protocols and the miscarriage rate.
Discussion
To the best of our knowledge, our study has the largest sample size among studies analyzing the association between endometrial preparation protocols and the miscarriage rate in patients with recurrent pregnancy loss. Our study suggested that compared with the HRT protocol, endometrial preparation with NCs had a lower miscarriage rate and a higher liver birth rate.
With the development of FET, an increasing number of studies have evaluated different endometrial preparation protocols for FET, but the results are conflicting. Most studies have concentrated on the CPR or LBR of different protocols [Citation18–20]. Fewer studies have reported miscarriage rates between different protocols. In 2020, one systematic review in the Cochrane database including six studies comparing programmed cycles with NCs revealed comparable results regarding the miscarriage rate between the NC and HRT protocols [Citation12]. Another systematic review and network analysis with 26 RCTs and 113 cohort studies showed that there was no evidence of any significant difference regarding the miscarriage rate in comparisons of different endometrial preparation protocols [Citation21]. A retrospective study including 12,950 FET cycles found that the chance of early pregnancy loss was higher for HRT protocols than for the NC protocol [Citation10]. Our study focused on patients with recurrent pregnancy loss and observed that the NC protocol could be related to a lower incidence of miscarriage, which was consistent with the findings of Liu et al. [Citation22]. Notably, our study included the largest sample sizes of Chinese patients with recurrent pregnancy loss.
The reasons for recurrent pregnancy loss are still unclear. It was reported that the parents’ age, genetic factors, anatomical factors, immune disorders, thrombophilia, male sperm quality and even unhealthy lifestyle and history of miscarriage were associated with pregnancy loss [Citation14,Citation23–30]. As the first step, successful implantation requires the synchronization of the embryo and a receptive endometrium, which is called the implantation window [Citation31]. Abnormal endometrial receptivity was suggested to contribute to recurrent pregnancy loss [Citation32,Citation33]. In HRT cycles, the supplementation of estrogen can cause nonphysiological serum estrogen levels, which increased the resistance of feto-placental blood flow [Citation34], and damaged endometrial receptivity [Citation8]. A study performed in baboons indicated that prematurely elevating serum estradiol disturbed the invasion of the trophoblasts and suppressed uterine artery remodeling [Citation35]. Moreover, HRT cycles would form a hypothalamic-pituitary suppression environment that leads to an absence of the corpus luteum (CL), which in turn increases pregnancy loss [Citation36].
Moreover, our study found that the age of women at embryo transfer was significantly associated with pregnancy loss after logistic regression analysis. This result was in accordance with the study of Sauer [Citation37]. A possible explanation is that advanced maternal age increases chromosome segregation errors during meiosis, giving rise to an aneuploid embryo that would result in a spontaneous abortion, as mentioned by Mikwar et al. [Citation38].
There was some strength of our present work. First, our study had a relatively large sample size of patients with recurrent pregnancy loss, which guaranteed statistical power. Second, we used univariate analysis and logistic regression to make the results more reliable. Third, we have completed the follow-up procedure until their delivery for all the women so that we could provide reliable information about the LBR.
The study also had some limitations. First, as a retrospective study, we could not eliminate some confounders affecting pregnancy loss, including exercise, nutrition intake, other drugs used or the psychological status of the women. Meanwhile, the number of two groups was not comparable and there were really some differences between two groups, including the numbers of previous FET cycles, endometrium thickness on the progesterone administration day, basal AFCs, number of oocytes retrieval and more patients accompanies with anovulation in HRT group, which may cause some selection bias. However, we used the univariate analysis and logistic regression model (including crude and adjusted model) to minimize the bias. Furthermore, not all the women in our research underwent preimplantation genetic testing (PGT), we could not exclude the effect of aneuploid embryos on miscarriage. However, in the subgroup analysis, we chose women with PGT data. The results also showed that the NC protocol had a decreased miscarriage rate compared with the HRT protocol in the PGT subgroup (10% for the NC protocol vs. 21.28% for the HRT protocol).
In conclusion, our findings showed that the NC protocol could decrease the miscarriage rate and improve the LBR for patients with recurrent pregnancy loss. A prospective, multi-center, randomized controlled clinical trial is still required to confirm our results. This study can provide some consulting advice for women experiencing recurrent pregnancy loss in the IVF procedure.
Author contributions
XM and JZS: co-wrote and completed the manuscript based on all the coauthor’s feedback. XTL: contributed to the project development and revision of manuscript. XM and HYZ: contributed to the project development and the statistical analysis. JZS and HYZ: co-contributed to the conception of the study. All coauthors reviewed the drafts of the manuscript and provided the idea about the intellectual content. All authors approved the final version of the article.
Ethical approval
The study received approval by the Ethics Committee for the Clinical Application of Human Assisted Reproductive Technology of Northwest Women’s and Children’s Hospital. Informed consent was obtained from all individual participants included in the study. We declare that all methods of this study were performed in accordance with the relevant guidelines and regulations (Declaration of Helsinki).
Acknowledgements
We thank all the clinicians, scientists, and embryologists in the Northwest Women and Children’s Hospital for their assistance with data collection, and all the patients for their contribution to this study.
Disclosure statement
The authors declare that they have no competing interests.
Data availability statement
The datasets analyzed during the current study are available from the corresponding author on reasonable request.
Additional information
Funding
References
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