Endometrial preparation protocols did not impact outcomes after frozen-thaw embryo transfer for chronic endometritis treatment

Abstract

Background

Intended to investigate the potential effects of endometrial preparation procedures on the results of subsequent frozen embryo transfer (FET) cycles in infertile women with chronic endometritis (CE) following a single course of antibiotic therapy.

Methods

From July 2020 to December 2023, 773 infertile patients participated in a retrospective research. CD138 was immunostained in endometrial tissues taken during the proliferative phase. CE was suggested by the presence of CD138-positive cells in the stromal cells. Oral antibiotics were administered to every patient with a CE diagnosis. After one course of drug treatment, patients had endometrial preparation and frozen embryo transfers. This study compared the impact of different endometrial preparation strategies on pregnancy outcomes.

Results

Four regimens, hormone replacement treatment (HRT), ovulation induction, natural cycle, and gonadotrophin-releasing hormone agonist-HRT (GnRH agonist-HRT), did not significantly alter the outcome of pregnancy in patients with CE. Compared to other groups, the ovulation induction cycle group had a higher clinical pregnancy rate and embryo implantation rate. In addition, the miscarriage rate is lower compared to other populations. Patients with treated CE who received HRT, GnRH agonist-HRT, ovulation induction cycle, and natural cycle did not differ substantially (P > 0.05).

Conclusion

Patients with treated CE did not see any change in pregnancy outcomes as a result of endometrial preparation procedures.

Introduction

The ongoing inflammatory state of the endometrium is the hallmark of chronic endometritis (CE), a chronic infectious illness that frequently manifests as either no symptoms or mild clinical abnormalities. Prolonged leukorrhea, pelvic discomfort, dyspareunia, and irregular uterine flow are some examples of these disruptions [1]. However, the endometrium with CE may exhibit focal or diffuse endometrial hyperemia, mucosal edema, and endometrial micro-polyps (usually 1–2 mm in diameter) with hysteroscopy [2]. Several studies have documented correlations between CE and unfavorable reproductive outcomes, including infertility, recurrent implantation failure (RIF), and recurrent pregnancy loss (RPL) [3–5]. Additionally, research showed a link between a higher frequency of CE and occlusion of the fallopian tubes and hydrosalpinx [6, 7].

For CE, there are no accepted, defined criteria or diagnostic standards. The presence of plasma cell infiltration within the endometrial stroma serves as the basis for the histological diagnosis of CE. The main diagnostic technique for CD138 is immunohistochemical (IHC) testing [6]. A transmembrane heparan sulfate proteoglycan that is expressed on the surface of plasma cells is called CD138 [8]. Hysteroscopy, a biopsy method, is used to help diagnose intrauterine abnormalities when endometrial micro-polyps (usually 1–2 mm in diameter), focal or widespread hyperemia, and stromal edema are observed [9]. To lessen or completely eradicate plasmacyte infiltration in the endometrial stroma, an adequate antibiotic therapy was empirically provided [5, 10], despite the fact that the majority of treatments were not predicated on endometrial culture results. Doxycycline’s wide range of antibacterial activity made it the first-line therapy [11], followed by second-line antibiotic therapies such as ciprofloxacin or metronidazole. Research has indicated that women who undergo antibiotic therapy have a higher pregnancy rate than those who do not [12].

To our best knowledge, few studies have investigated whether endometrial preparation protocols impact outcomes after FET in the context of CE treatment. The aim in this study was to determine whether endometrial preparation procedures could affect the result of a pregnancy in infertile women with CE who had received one round of antibiotic therapy. We specifically looked at how these procedures affected the outcome of subsequent FET cycles.

Method

Study design

From July 2020 to December 2023, we carried out a retrospective analysis at Bei Jing Chao-Yang Hospital, Capital Medical University. As standard procedures for histopathological investigation, women had endometrial biopsies and hysteroscopies, specifically for CD138 staining to detect CE. Infertility, RSA, ET failure, a history of tuberculosis (TB), and atypical uterine findings, including endometrial polyps, intrauterine adhesions (IUA), and congenital uterine deformities, were among the conditions for which hysteroscopy was conducted. The Chao-Yang Hospital’s medical ethics committee gave its approval for this investigation.

Study population

We then included individuals who had a FET cycle within 12 months of a hysteroscopy in order to evaluate the effect of CE on reproductive outcomes after antibiotic treatment. The study consisted of 924 women who were referred for endometrial biopsy, hysteroscopy, and CD138 IHC investigation to try to diagnose CE. Individuals with incomplete medical history data, couples with chromosomal abnormalities, patients who had not undergone In Vitro Fertilization-Embryo Transfer (IVF-ET) at our department, patients who had received antibiotic treatment within three months prior to hysteroscopy, and patients with incomplete cycle data or incomplete follow-up information were all eliminated. 773 women had been included in the analysis once these criteria were applied. Initially, the initial analysis to figure out risk variables for CE included all patients. Patients with CE were further separated into four groups: GnRH agonist-HRT (n = 218), HRT (n = 182), ovulation induction cycle (n = 207), and natural cycle (n = 166). Retrospective analysis was performed on 923 FET cycles. Figure 1 shows the flowchart for the study.

Fig. 1.

Flow diagram of the study and distribution of the population investigated

Data collection

We conducted a search of the electronic medical database to retrieve data, which encompassed the following categories: (1) Maternal characteristics, including maternal age, body mass index (BMI), gravidity, parity, primary cause of infertility, and duration of infertility; (2) Treatments and pregnancy outcomes associated with FET, such as the number of embryo transfer (ET) procedures, the number of good quality embryos transferred, blastocyst rate, insemination methods, and stimulation protocols for FET; (3) gynecologic medical histories, including dysmenorrhea, hydrosalpinx, endometriosis, polycystic ovary syndrome (PCOS), intrauterine device (IUD) use, pelvic inflammatory disease, endometrial polyps, intrauterine adhesions (IUA), tuberculosis, uterine cavity surgery and cervical surgery.

Sample collection

Two skilled professionals conducted hysteroscopy during the follicular phase (3–7 days after menstruation) of the menstrual cycle using a 30° rigid hysteroscope (OLYMPUS, Japan) with an outer diameter of 5 mm. The uterine cavity was expanded with saline (0.9%) to a pressure of 80–100 mmHg. In addition to various intrauterine manifestations (intrauterine adhesion, endometrial polyps, submucosal myoma, and uterine abnormalities), hysteroscopic characteristics of CE, including endometrial micro-polyps, hyperemia, and edema, were also seen. Endometrial biopsy specimens were taken from the endometrial congestion using a curette as soon as the hysteroscopy was completed. The biopsy sample was taken in the vicinity of the anterior endometrium in patients with a normal uterine cavity. After being preserved in 4% paraformaldehyde, the endometrial samples were transported to the lab for CD138 IHC and histological examination. Experienced senior physicians performed hysteroscopy during the proliferative period (3–7 days after menstruation), and histological investigation verified the endometrial phase. The uterine cavity was thoroughly examined during hysteroscopy. During hysteroscopy, biopsy forceps were used for collecting endometrial biopsy samples.

Diagnosis of CE

According to the Delphi poll reported previously [13], in order to use hysteroscopy to diagnose CE, at least one of the following requirements have to be fulfilled: (1) localized or diffuse endometrial hyperemia; (2) micro-polyps (less than 1 mm in size with a definite connective-vascular axis); (3) stromal edema; and (4) a “strawberry” appear which is defined by extensive areas of hyperemia with white central spots. Two skilled gynecologic pathologists from Chao-Yang Hospital Department of Pathology examined each endometrial biopsy specimen. In the study, the plasma cell membrane displayed significant positive CD138 staining, but the cytoplasm displayed mild positive staining. The nuclei were spherical and orientated to one side, and the thick chromatin was arranged in a wheel-like pattern radially along the nuclear membrane. After scanning the entire section at a lower magnification, the number of plasma cells was counted at 400× magnification, focusing on the area with the highest cell density. This was carried out in compliance with the guidelines established [3, 14], using CD138 IHC categorizing, the gold standard for identifying CE is the presence of one or more plasma cells in the ovarian stroma per 10 high-power fields (HPF).

Treatment and follow-up of CE

Oral doxycycline (100 mg twice daily for 14 days) or oral levofloxacin (500 mg twice daily for 14 days) in combination with oral metronidazole (200 mg twice daily for 14 days) were given at random to women whose CD138 IHC staining revealed CE. On a case-by-case basis or in accordance with the patient’s wishes, the doctor decided whether to treat women who tested positive on hysteroscopy but negative for CE on CD138 IHC staining with antibiotics. Women who tested negative for CE on hysteroscopy and CD138 IHC staining were not treated with antibiotics.

FET protocol and luteal phase support (LPS)

The endometrial preparation approach for FET was customized to each patient’s unique traits and preferences and included the natural cycle, ovulation induction cycle, HRT, and GnRH agonist-HRT. Follicle size and endometrial thickness were measured using transvaginal ultrasonography during days 8–10 of the menstrual cycle as part of the natural cycle protocol. When the follicular diameter was at least 18 mm, the progesterone concentration was no more than 1.5 ng/ml, and the endometrial thickness was at least 7 mm, 250 µg of recombinant human chorionic gonadotropin (hCG; Merck, Europe B.V.) was injected intramuscularly. 24 to 48 h following the hCG treatment, ovulation was seen. During the ovulation induction cycle, oral dysdrogesterone (10 mg three times a day; Abbott Biologicals B.V., the Netherlands) was then given beginning one day after ovulation and continuing for 14 days after FET. Letrozole 2.5 mg/day was given orally for four days from the second to the fourth day of menstruation. In addition, injections of HMG (Li Zhu, China) ranging from 37.5 IU to 75 IU were administered; the dosage was modified based on follicular development. 250 µg of recombinant human chorionic gonadotropin (hCG; Merck, Europe B.V.) was injected intramuscularly once the follicular diameter was at least 18 mm, the progesterone concentration was no more than 1.5 ng/mL, and the endometrial thickness was at least 7 mm. As part of the hormone replacement therapy regimen, patients received 4 or 6 mg of oral estrogen (estradiol valerate [Progynova]; Bayer, France) every day for one week, starting on the second or third day of the menstrual cycle. On days two through four of the menstrual cycle, patients in the GnRH agonist-HRT protocol were administered a gonadotropin-releasing hormone agonist (triptorelin acetate [Diphereline]; Li Zhu, China). When the endometrial thickness was less than 5 mm, the concentration of LH and FSH was below 5 IU/L, and the estradiol level was less than 50 pg/mL, treatment with 4 to 6 mg of oral estrogen was started. Women were prescribed a combination of oral dydrogesterone (10 mg three times per day) and progesterone vaginal sustained-release gel (Crinone; Merck Serono, UK) at a dosage of 90 mg once per day once the endometrial thickness reached 7 mm, the serum progesterone concentration was below 1.5 ng/mL, and the serum estradiol concentration peaked at 200 pg/mL. The embryos were stored in cryogenic and then thawed using the vitrification method. On the fourth day (P + 3) after progesterone exposure, cleavage stage embryos were transplanted, and on the sixth day (P + 5), blastocysts were transferred. Embryos were vitrified and warmed in compliance with previously established procedures [15]. If a cleavage stage embryo had at least six blastomeres, a fragmentation rate of less than 20%, and no discernible morphological defects, it was considered acceptable quality. Grades 3–6 AA/AB/BA/BB were assigned to good quality blastocysts since they were larger than 3BB and had a cavity expansion level, inner cell mass, and trophectoderm quality. The patient’s age, BMI, embryo quality, uterine condition (e.g., scarred uterus, uterine deformities), and personal preferences were taken into consideration when deciding how many frozen embryos to transfer—one or two. Exogenous progesterone and estrogen were administered until 10–12 weeks of gestation, if pregnancy was successful.

Outcomes and definitions

Two weeks following FET, the serum β-hCG test was performed. Until patients who had a positive pregnancy test, LPS was maintained until 10–12 weeks of gestation. The existence of an intrauterine gestational sac, with or without a fetal heartbeat, as seen by transvaginal ultrasonography after six weeks of gestation was considered clinical pregnancy. The number of gestational sacs divided by the number of transplanted embryos was used to determine the implantation rate. Pregnancy loss before 12 weeks of gestation was referred to as an early miscarriage. Pregnancy loss that happens after 12 weeks but before 28 weeks of gestation is referred to as a late miscarriage. The failure to obtain a clinical pregnancy after transferring at least four high-quality embryos during at least three fresh or frozen IVF cycles was known as RIF [16].

Statistical analysis

SPSS 26.0 software was used for all statistical analyses and graphical representations (SPSS Inc., Chicago, IL). The Student’s t-test was used to evaluate continuous data, which was shown as mean ± SD. The chi-square test or Fisher’s exact test was used when necessary, and categorical variables were represented as percentages of the total. Statistical significance was defined as a two-sided P-value of less than 0.05.

Results

Table 1 displays the clinical features of the ovulation induction cycle, natural cycle, HRT, and GnRH agonist-HRT regimens in patients with treated CE. The GnRH-agonist-HRT group was significantly larger than the natural cycle group among the 773 study participants. The four groups of patients were similar in age and were all over 35 years old. Compared to women in the natural cycle, ovulation induction cycle, and HRT groups, females in the GnRH agonist-HRT group experienced infertility for a longer period of time (3.33 years versus 3.09 years versus 3.03 years versus 2.76 years, respectively; P = 0.012). Furthermore, there was a statistically significant difference (P = 0.001) in the number of cleavage stage embryos transferred in the GnRH agonist-HRT group (75.77%) as compared to the natural cycle (18.89%), ovulation induction cycle (18.60%), and HRT groups (53.08%). Statistically significant differences were seen between patients in the GnRH agonist-HRT group and those in the other three endometrial preparation procedures, with lower basal AFC and higher basal FSH levels (P = 0.026 for basal AFC and P = 0.015 for basal FSH).The pregnancy results of HRT, GnRH agonist-HRT, ovulation induction cycle, and natural cycle in individuals with CE are shown in Table 2. Therefore, it can be said that among patients with treated CE, there were virtually no differences in the outcomes of pregnancy amongst the four endometrial preparation methods. In particular, there was no statistically significant difference in the embryo implantation rates for the four endometrial preparation regimens among patients with CE (P = 0.132). These rates were 58.85%, 64.49%, 60.28%, and 56.99%, respectively. These insignificant differences continued even after adjusting for confounding variables like maternal age, the duration of infertility, BMI, basal AFC, the number of transferred embryos, the number of high-quality embryos, the stage of embryo transfers, the type of infertility, and the aetiology of infertility. The group that underwent ovulation induction cycle had a higher rate of clinical pregnancy and embryo implantation than the other groups. But there was a lower miscarriage rate than in other groups.

Table 1.

General data analysis of the FET cycle after hysteroscopy in post-treatment CE patients

	Natural cycle (n = 166)	Ovulation induction cycle (n = 207)	HRT (n = 182)	GnRH agonist-HRT (n = 218)	P
Age (years)	35.37 ± 4.67	35.22 ± 5.15	35.11 ± 4.66	35.44 ± 4.56	0.751
BMI (kg/m2)	22.64 ± 3.54	22.95 ± 3.69	22.77 ± 3.67	23.28 ± 3.59	0.464
Basal FSH (IU/L)	6.32 ± 2.42	5.78 ± 2.25	6.6 ± 3.78	6.59 ± 3.21	0.026a
Basal AFC	10.92 ± 9.12	11.83 ± 10.03	8.55 ± 8.69	9.5 ± 9.10	0.015b
Cause of infertility(%)
Women	61.45%(102/166)	68.12%(141/207)	64.29%(118/182)	68.81%(150/218)	0.403
Male factor	9.64%(16/166)	13.53%(28/207)	17.03%(31/182)	15.60%(34/218)	0.410
Both	15.66%(26/166)	8.70%(18/207)	6.04%(11/182)	4.59%(10/218)	0.134
Unknown	13.25%(22/166)	9.65%(20/207)	12.64%(23/182)	11.00%(24/218)	0.477
Duration of infertility (year)	3.09 ± 2.63	3.03 ± 2.52	2.76 ± 2.35	3.33 ± 2.54	0.012c
No. of embryos transferred	1.12	1.21	1.57	1.61	0.065
Type of embryo transfer
Cleavage-stage embryo(%)	18.89%(41/217)	18.60%(56/301)	53.08%(181/341)	75.77%(344/454)	0.001d
Blastocyst(%)	81.11%(176/217)	81.40%(245/301)	46.92%(160/341)	24.23%(110/454)	0.027e
Insemination method
IVF(%)	76.56%(147/192)	66.53%(163/245)	65.89%(141/214)	68.01%(185/272)	0.228
ICSI(%)	23.44%(45/192)	33.47%(82/245)	34.11%(73/214)	31.99%(87/272)	0.574

Superscripts indicate significant differences between

a b c GnRH- agonist-HRT group and other three endometrial preparation protocols

d e Characterized by grade 1 2 cleavage stage embryos or grade 1 blastocysts

HRT, hormone replacement therapy; GnRH agonist, gonadotrophin-releasing hormone agonist; FET, frozen embryo transfer; BMI, body mass index; AMH, anti-Mullerian hormone

Table 2.

Analysis of pregnancy outcomes during FET cycle after hysteroscopy in post-treatment CE patients

	Natural cycle (n = 192)	Ovulation induction cycle (n = 245)	HRT (n = 214)	GnRH agonist-HRT (n = 272)	P
Embryo implantation rate (%)	58.85(113/192)	64.49%(157/245)	60.28%(129/214)	56.99%(155/272)	0.132
Clinical pregnancy rate (%)	29.17%(56/192)	32.65%(80/245)	32.71%(70/214)	27.94%(76/272)	0.357
Miscarriage rate (%)	11.46%(22/192)	6.83%(17/249)	9.81%(21/214)	9.19%(25/272)	0.126

Discussion

This study aimed to assess the impact of endometrial preparation practices on pregnancy outcomes in individuals with a history of treated CE during FET cycles. According to our findings, none of the pregnancy outcomes including the rate of live birth were significantly impacted by the endometrial preparation program. When comparing the natural cycle, ovulation induction cycle, HRT, and GnRH agonist-HRT protocols for patients with CE, the comparative analysis found no appreciable differences in almost all pregnancy outcomes. Reproductive medicine contends that the main variables affecting the success of FET are the number and quality of embryos, the endometrium’s thickness and receptivity, and the timing of endometrial growth [17]. This is in line with the fact that there is not enough data to conclusively say which endometrial preparation method is better for infertile women having FET [18]. Consistent results from a cochrane study that looked at 18 randomized controlled trials support the previously indicated conclusions [19]. Additionally, women undergoing HRT FET did not have better reproductive results after pretreatment with GnRH agonists [20, 21]. This study then attempted to investigate any possible differences in pregnancy outcomes between individuals who underwent the difference endometrial preparation technique but had cured CE. This result is in line with earlier research [22]. Xiong et al. discovered no significant differences between the treated CE group and the control group (52.1% and 52.6% live birth rates, respectively) when baseline statistics, such as the percentage of endometrial preparation procedures [23]. The clinical pregnancy rate did not differ significantly between patients with and without CE(OR 0.39, 95% CI 0.09 1.61), according to Brecht et al. [24]. In individuals with resolved CE, prompt removal of harmful bacteria from the endometrial tissue with antibiotic therapy may improve endometrial receptivity and aid in the restoration of endometrial immunological homeostasis. According to this, the endometrial immune milieu in these individuals might return to normal, which could make the endometrial preparation procedure just as effective as it is in patients who have never had CE. It’s crucial to know that the current results contradict some previous studies. According to Keiji et al., patients with treated CE had a much lower clinical pregnancy rate than those without the disease [25]. Additionally, In the study by Duan et al., patients with treated CE had a considerably higher rate of spontaneous abortions than patients without the condition [26]. In the setting of FET cycles, there was no discernible difference in the miscarriage rate between patients with and without CE. These discrepancies could result from differences in treatment concept, embryo transfer procedures, or diagnostic standards for CE amongst studies. There are various restrictions on this study. First of all, there are no standard diagnostic standards for endometritis, and many studies have used disparate standards, leading to varying findings. To get more precise results, these patients should be examined in the future using the most recent definitions. Furthermore, because it was a retrospective analysis, some participants’ clinical and demographic information was lacking and excluded, which could have affected the findings. Properly planned prospective studies are required for additional validation. And then, the findings derived from patients with CE, and the conclusion is not suitable to be extended to other assisted reproductive populations. Finally, most of all patients was not performed hysteroscopy after drug treatment, so it is hard to know whether the pathological turned negative. To get more solid and compelling data about the diagnosis and treatment of infertility patients with CE, prospective randomized controlled studies or multicenter clinical trials are required. According to the study, there is no connection between the endometrial preparation procedures and the results of pregnancies in people with treated CE. These results underscore the significance of choosing a suitable endometrial preparation procedure according to the patient’s endometrial and ovulatory condition, irrespective of the patient’s chronic endometritis history. Clinical management in these situations may benefit greatly from this information.

Abbreviations

FET

Frozen Embryo Transfer

CE

Chronic Endometritis

HRT

Hormone Replacement Treatment

GnRH

Gonadotrophin-Releasing Hormone

RPL

Recurrent Pregnancy Loss

IHC

Immunohistochemical

TB

Tuberculosis

IVF-ET

In Vitro Fertilization-Embryo Transfer

BMI

Body Mass Index

PCOS

Polycystic Ovary Syndrome

IUD

Intrauterine Device

IUA

Intrauterine Adhesions

HPF

High-Power Fields

AMH

Anti-Mullerian Hormone

hCG

Human Chorionic Gonadotropin

Author contributions

X.S analysed datas and prepared all figures and tables, SYZ wrote the main manuscript text.

Funding

Not applicable.

Data availability

The data used in this paper will not be openly shared to protect the included informants’ privacy and due to general security concerns.

Declarations

Ethics approval and consent to participate

The study was approved in advance by the ethical committee. Consent was given in accordance with the laws governing privacy in research.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.