Study on the impact of antibiotics on the outcome of repeated frozen-thawed embryo transfer in women with initial implantation failure and chronic endometritis. Chronic endometritis is a chronic inflammation of the endometrium that occurs when the endometrium is infected by patho

Study on the effect of antibiotics on the outcome of repeated frozen-thawed embryo transfer in women with first-time implantation failure and chronic endometritis

Source of this article: Wei Longlong, Zhang Cuilian. Study on the impact of fusion embryo transfer outcomes [J]. Chinese General Medicine, 2022, 25(21): 2605-2609.

Chronic endometritis (CE) is a kind of endometrium infected by pathogenic microorganisms The chronic inflammation that occurs is a persistent and latent infection characterized by endometrial stromal plasma cell infiltration [1]. CE can have adverse effects on female fertility. The prevalence of CE in infertile people is 40.8%~57.0%, and it can reach 14.0% in patients with repeated implantation failure (RIF) in in vitro fertilization and embryo transfer (IVF-ET). ~57.6%[2,3]. However, there are few studies on the incidence of CE in people with first-time IVF-ET implant failure. The main cause of CE is pathogenic microbial infection. The currently recognized treatment method at home and abroad is antibiotic treatment, among which doxycycline is listed as the first-line treatment. Numerous studies have shown that standardized application of antibiotics can effectively treat CE and improve the reproductive prognosis of patients [4]. This article aims to study the prevalence of CE among patients with first-time implantation failure and the impact of antibiotic treatment on the outcome of re-frozen-thaw embryo transfer, in order to discover the cause in advance and intervene as early as possible.

1 Objects and methods

1.1 Research subjects

381 patients who failed the first IVF-ET implantation at Henan Provincial People's Hospital from May 2017 to May 2021 were selected as the research subjects. Inclusion criteria: (1) Age 40 years old; (2) At least one high-scoring cleavage stage embryo or one high-scoring blastocyst was transplanted for the first time; (3) Endometrial thickness on the day of transplantation was between 8 and 14 mm. The embryo scoring standards are: (1) High-scoring cleavage stage embryos: ① 7 to 9 blastomeres on day 3; ② The proportion of embryo fragments is ≤5%; ③ No multinucleation in the pronuclear stage. (2) High-scoring blastocysts : Blastocysts that do not contain C in scores of 3BB, 4BB and above [5]. Exclusion criteria: (1) Those combined with other uterine lesions, such as endometrial polyps, intrauterine adhesions, uterine septum, uterine fibroids, etc.; (2) Those combined with hydrosalpinx; (3) One spouse Or those with chromosomal abnormalities in both sides; (4) Those with low ovarian function; (5) Those with clearly diagnosed immune-related diseases, such as systemic lupus erythematosus, ankylosing spondylitis, etc.; (6) those who have experienced Ultrasound examination revealed endometriosis ; (7) Those with reproductive tract inflammation, fever, cough and related surgical contraindications before surgery. This study was approved by the Ethics Committee of Henan Provincial People's Hospital (SYSZ-LL-2021091501).

1.2 Research methods

collected patients’ clinical data [age, body mass index (BMI), years of infertility, endometrial thickness on the day of first transplantation, number of embryos for first transplantation, type of infertility (primary infertility, secondary infertility) ) and the pregnancy outcomes (implantation rate, pregnancy rate, miscarriage rate) of patients undergoing re-frozen-thaw embryo transfer.

1.2.1 Hysteroscopy and endometrial biopsy

All patients completed various preoperative examinations and underwent hysteroscopy after eliminating obvious surgical contraindications. The manifestations of CE under hysteroscopy are [3]: (1) Congestion: the vascular network bulges, which is more obvious around the glands; (2) Interstitial edema: the follicular phase the endometrium remains the same in the proliferative phase Pale and thickened; (3) Focal or diffuse micropolyps: There are pedicle-like protrusions less than 1 mm on the endometrial surface. For patients with CE characteristic manifestations under hysteroscopy, comprehensive curettage of the uterine cavity was performed after surgery, and endometrial tissue was collected for routine pathological examination and immunohistochemical staining. Hysteroscopy is completed by 1 to 2 fixed deputy chief physicians. see picture 1.

(1) For the first time, this study focuses on the target group of people who fail to implant for the first time in in vitro fertilization-embryo transfer (IVF-ET), in order to find the possible causes of implantation failure in patients early and quickly, and carry out intervention and treatment to reduce this type of disease. The financial and psychological burden on patients.(2) This study found that chronic endometritis (CE) occurs in a certain proportion among people who failed IVF-ET for the first time, and confirmed that oral antibiotic treatment has positive significance in improving the outcome of repeated assisted pregnancy for this type of patients. The conclusion has great guiding significance for clinical work.

This study is a retrospective study with a limited sample size and large differences between groups, which may have adverse effects on the statistical results; (2) This study is a single-center study, and the experience and pathological diagnosis level of the surgeon may also differ from other research units. Inconsistent standards; (3) The current CE diagnostic standards are not unified, which is not conducive to the establishment of inclusion criteria for the research population. At the same time, it is difficult to compare and analyze different studies with each other.

1.2.2　Diagnostic criteria for CE

All intimal sections taken were subjected to routine pathological examination and hematoxylin-eosin staining (HE) to look for plasma cells . The criteria for judging plasma cells under the microscope are large cell body, high nuclear-cytoplasmic ratio, alkaline cytoplasm and "spoke-like" radial arrangement of heterochromatin in the nucleus. At the same time, transmembrane heparan sulfate proteoglycan (CD138) was used as a specific marker of plasma cells for immunohistochemical staining [6]. Diagnostic criteria for CE: CE is diagnosed when the characteristic manifestations of CE under hysteroscopy combined with CD138 positivity are present.

1.2.3　Therapeutic strategy of CE

After surgery, all patients received routine anti-infective treatment, oral cefaclor (Eli Lilly Suzhou Pharmaceutical Co., Ltd., National Drug Approval No. H20020571) 2 times/d, 0.25 g/time, a total of 3 d. At the same time, patients with CE are instructed to take doxycycline hydrochloride enteric-coated capsules (Yongxin Pharmaceutical Industry Co., Ltd., national drug approval number H20030627), 2 times/d, 100 mg/time, combined with metronidazole tablets (Sichuan Kelun Pharmaceutical Co., Ltd., National Drug Approval No. H20057236), 2 times/d, 400 mg/time, a total of 14 days is a course of treatment.

1.3 Group situation

After the treatment, hysteroscopy was reviewed again, and endometrial tissue was taken during the operation for CD138 immunohistochemical staining to determine the treatment effect. According to the CE outcome of patients after antibiotic treatment, they are divided into CE negative group (n=37) and CE persistent positive group (n=11). There is no CE performance under hysteroscopy (n=27) and pathological examination excludes CE (n =306) is divided into the non-CE group (n=333). Re-embryo transfer is performed according to the patient's needs. The grouping process is shown in Figure 2.

1.4 Frozen-thaw embryo transplantation

The embryos are thawed and resuscitated according to the vitrification and thawing routine. The embryo transfer procedure is performed under ultrasound guidance. No more than 2 cleavage stage embryos can be transferred at a time. Blastocysts are given priority and single blastocyst transfer is recommended. Postoperative luteal support was routinely performed.

1.5 Observation of indicators related to pregnancy outcome of re-frozen-thaw embryo transfer

After treatment, patients were arranged to undergo embryo implantation surgery. Pregnancy testing was performed 14 days after the operation, and vaginal ultrasound examination was performed on the 28th and 35th days after the operation. After confirmation of pregnancy, close follow-up will be conducted throughout the pregnancy, and delivery and birth status of the newborn will be registered in a timely manner.

Embryo implantation rate = number of gestational sacs/total number of transplanted embryos × 100%; clinical pregnancy rate = number of clinically pregnant patients/total number of transplanted patients × 100%; early abortion rate = number of spontaneous abortion patients/number of clinically pregnant patients × 100% . The spontaneous abortions recorded in this study all occurred before 12 weeks of pregnancy, which were early abortions .

1.6 Statistical methods

used SPSS 22.0 statistical software for data analysis. The measurement data of that conforms to the normal distribution of are expressed as (x±s), and the comparison between multiple groups uses one-way analysis of variance ; the measurement data of that is not normally distributed in is expressed as M (P25, P75), and the comparison between multiple groups is The Kruskal-Wallis test was used; count data were expressed as relative numbers, and the χ2 test was used for comparisons between multiple groups. The difference is statistically significant with P0.05.

2 Results

2.1 Comparison of clinical data between three groups

CE negative group, CE persistent positive group and non-CE group age, BMI, endometrial thickness on the day of transplantation, years of infertility, number of embryos transferred, and infertility types, there was no statistical difference. Meaning (P0.05), see Table 1.

2.2 Comparison of pregnancy outcomes among three groups

Comparison of embryo implantation rate and clinical pregnancy rate between CE negative group, CE persistent positive group and non-CE group, the difference is statistically significant (P0.05); among them, non-CE group, CE negative group The embryo implantation rate and clinical pregnancy rate were both higher than those in the CE continuously positive group, and the difference was statistically significant (P0.05). There was no statistically significant difference in the embryo implantation rate and clinical pregnancy rate between the CE negative conversion group and the non-CE group (P0.05); there was no statistically significant difference in the early miscarriage rate between the three groups (P0.05), see Table 2.

3 Discussion

CE refers to persistent chronic inflammation limited to the endometrium, and its pathological is characterized by endometrial stromal infiltration of plasma cells [7]. In recent years, with the popularization of hysteroscopy and the continuous improvement of pathological diagnosis technology, CE has been found to be related to a variety of female reproductive diseases, including infertility, recurrent miscarriage , RIF, endometrial polyps and Endometriosis, etc.[3,4]. Among them, RIF, as a hot and difficult issue in the field of assisted reproduction, has attracted widespread attention from the medical community. A large number of studies have shown that the prevalence of CE in patients with RIF is 7.7% to 66.0%, and active antibiotic treatment can significantly improve the pregnancy outcomes of CE patients [3, 8, 9, 10]. However, no relevant research has been found on the incidence of CE in patients with initial IVF-ET implantation failure and whether antibiotic treatment is beneficial to the outcomes of repeated frozen-thaw embryo transfer in such patients. As we all know, the cost of assisted reproductive technology is high, the cycle is long, and the number of follow-up visits is high, which imposes a large financial burden on patients; in addition, long periods of infertility and multiple failed transplants will further increase the psychological burden on patients, and Anxiety and nervousness, in turn, can adversely affect reproductive prognosis, creating a vicious cycle. Therefore, it is of great significance to find possible causes of implant failure in patients early and quickly, and to carry out intervention and treatment. The clinical manifestations of

CE are mild and lack specificity, and are easily overlooked. Hysteroscopy is the most intuitive way to detect CE, but most studies do not recommend a single hysteroscopy as the standard for confirming CE [11]. Currently, the diagnosis of CE is mainly based on the discovery of plasma cell infiltration in the pathological examination of the endometrium. HE staining is the most commonly used method, but it also has certain limitations, such as observation errors caused by patients being in different stages of the menstrual cycle when sampling and the difficulty in identifying plasma cells in the tissue [12]. In recent years, more and more studies have proposed using CD138, a specific marker of plasma cells, to assist in the diagnosis of CE. It specifically marks plasma cells in the intimal stroma through immunohistochemical staining, which can significantly improve the efficiency of diagnosis. [13]. Reviewing domestic and foreign literature, the overall number of plasma cells in each section, the average number of plasma cells per 1 or 10 high-power fields (HPF), and the number of plasma cells in multiple high-power fields can be used as the judgment criteria. The number of plasma cells used to diagnose CE is also inconsistent in different studies. Some scholars consider ≥5 CD138-positive plasma cells as positive; in addition, some scholars use plasma cell density (PCD) as the standard for diagnosing CE [8, 14,15]. CICINELLI et al. [16] studied 1 to 4 plasma cells/HPF as mild CE, and ≥5 plasma cells/HPF as severe CE, and found that hysteroscopy combined with CD138 immunohistochemical examination can improve the quality of patients with mild CE. Diagnostic accuracy. Therefore, this study proposed the presence of CE characteristic manifestations under hysteroscopy combined with CD138 positivity as the diagnostic criteria for CE, without emphasizing the specific CD138 count, in order to improve the detection rate of CE.

The target population included in this study is women under 40 years old, with normal ovarian reserve and function. The endometrial thickness on the day of transplantation is between 8 and 14 mm, and all of them have experienced first-time embryo implantation failure; at the same time, it excludes possible effects on embryo implantation. Factors such as hydrosalpinx, congenital and acquired uterine anatomical and functional abnormalities, endometriosis, etc. This study found that among women whose first IVF-ET implantation failed, the incidence rate of CE reached 12.6%.The possible mechanisms by which CE causes embryo implantation failure include: CE can cause abnormal expression of cytokine , growth factor , and apoptosis-related proteins, thereby reducing the receptivity of the endometrium, leading to implantation failure; at the same time, CE It can induce changes in the local immune microenvironment and inflammatory microenvironment of the endometrium, hindering the process of endometrial decidualization, embryo adhesion and penetration, and the invasion process of trophoblasts [17,18]. The main cause of

CE is infection by pathogenic microorganisms. Common pathogenic bacteria include Streptococcus , Escherichia coli , Enterococcus faecalis , Staphylococcus , Proteus , and genital tract Mycoplasma and Chlamydia et al [19]. Currently, the accepted treatment method for CE is standardized oral antibiotics. The first-line drug is the broad-spectrum antibiotic doxycycline (100 mg/time, 2 times/d, 14 days). The antibacterial spectrum of doxycycline makes it effective against both bacteria and mycoplasma; while metronidazole is mainly used In view of the infection of by anaerobic bacteria, combined with the current actual clinical work, this study uses a combination of doxycycline and metronidazole to treat CE. Multiple studies have confirmed the therapeutic effect of oral antibiotics on CE and their improvement in reproductive prognosis. KITAYA et al. [19] found that the cure rate of CE with doxycycline was 92.3%, and the cumulative total cure rate was 99.1%. JOHNSTON-MACANANNY et al. [18] found that after treatment with doxycycline, the cure rate reached 70%. Some patients who were insensitive to doxycycline were re-examined after combined medication. Intimal biopsy showed that the plasma cell clearance effect was good. In this study, patients in the CE group were re-examined after receiving adequate and standardized oral antibiotic treatment. The CE cure rate It was 77.08% (37/48), which is consistent with the above research results and once again proves the significant role of standardizing antibiotics in the treatment of CE. In this study, after another frozen-thawed embryo transfer, the embryo implantation rate and clinical pregnancy rate of patients in the CE-negative group were significantly higher than those in the CE-continuously positive group. This conclusion is consistent with the results of domestic and foreign studies [3, 19]. It is worth noting that the results of this study show that the implantation rate and clinical pregnancy rate of patients in the CE negative conversion group are higher than those in the non-CE group, but the difference is not statistically significant. This result has also been supported in other studies of the same type [ 3, 19]. The reasons for the above results may be: (1) Hysteroscopy also has a certain positive effect on embryo implantation, which may be related to the fact that the uterine distending fluid flushes the uterine cavity and stimulates the secretion of implantation factors; (2) Multiple studies support that endometriosis Endometrial stimulation and diagnosis and curettage are of great help to implantation failure. Many reproductive centers use endometrial diagnosis and curettage as an important measure in the treatment of RIF [20, 21, 22]. The results of this study show that the rational use of antibiotics can not only effectively cure CE, but also significantly improve the reproductive prognosis of patients with failed IVF-ET implantation for the first time, which has very important clinical significance.

In summary, the incidence of CE is high in patients with first-time implant failure. It is recommended to actively perform hysteroscopy to find possible causes. Patients diagnosed with CE should be treated with standard antibiotics. Uncured CE will be a serious risk for recurrence. Impact on outcomes of frozen-thaw embryo transfer. In future research, multi-center, large-sample studies should be conducted as soon as possible to determine unified and standardized CE diagnostic standards to avoid missed diagnosis and over-treatment. At the same time, embryo implantation is a complex process with multiple factors and steps, and CE is only one of its influencing factors. Comprehensive consideration and intervention are required in clinical work in order to provide more effective advice and individual treatment for this type of patients. treatment options.

There is no conflict of interest in this article. The references of

are omitted.