Administration of levofloxacin combined with metronidazole suppositories prior to in vitro fertilisation in women with chronic endometritis: a protocol of a single-centre, randomised, controlled clinical trial.

Chronic endometritis (CE) is a disease involving the disruption of microbial balance in the endometrium and the harmonious coexistence with the host immune system. 1 Since the majority of CE clinical symptoms in patients with atypical cases (no obvious symptoms or mild symptoms, such as abnormal uterine bleeding, chronic pelvic pain and increased vaginal secretions, etc) 2 lack specificity, and a blood test in the diagnosis of pathological changes is needed, at least an endometrial histopathological examination or invasive hysteroscopy examination invasive is needed to assist the diagnosis. In recent years, the impact of CE on adverse reproductive outcomes in women of childbearing age has garnered the attention of many scholars. CE is associated with unexplained infertility, repeated implantation failure and recurrent miscarriage. Kimura et al . summarised that the prevalence of CE in infertile women ranges from 2.8% to 56.8%, in women with recurrent implantation failure (RIF) from 14% to 67.5%, and in women with recurrent pregnancy loss from 9.3% to 67.6%, which significantly affects the reproductive health of women of childbearing age. 3 The aetiology of CE is still unclear, and it is predominantly believed to be related to pathogenic microorganism infection 1 4 ; common pathogens associated with endometritis include Chlamydia trachomatis , Streptococcus species, Enterococcus faecalis , Escherichia coli , Staphylococcus species, Klebsiella pneumoniae , Corynebacterium species, Ureaplasma and Mycoplasma species. Moreover, standardised antibiotic treatment for patients with endometritis can enhance their clinical pregnancy rate and live birth rate (LBR) and reduce the abortion rate. 5 6 Kitaya et al also demonstrated that the clinical pregnancy rate (45.7% vs 34.1%, p=0.032) and LBR (38.8% vs 27.9%, p=0.037) of the cumulative three in vitro fertilisation (IVF)-embryo transfer cycles were significantly higher than those of the non-CE group. 7 Cicinelli et al showed that after standard antibiotic treatment for patients with recurrent miscarriage and endometriosis, the LBR was higher in the effective hysteroscopic endometritis group than in the persistent hysteroscopic endometritis group. 8 McQueen et al observed an increase in LBR after antibiotic treatment in women with recurrent miscarriage 9 ; the treated endometritis group had a significantly higher LBR than the untreated endometritis group and the non-endometritis group (85%, 44%, and 40%, p=0.032). The rate of miscarriage was significantly lower in the treated group (15%, 56% and 60%, p=0.032). It has been reported in a retrospective study that the LBR in patients undergoing third-day embryo transfer after antibiotic treatment in RIF patients with CE was higher in the cured CE group than in the continued CE group (60.9% vs 13.3%). 10 Later, the other author also found similar results in women with unexplained infertility, with a higher LBR in the cured CE group (65.8%) than in the continued CE group (6.6%) and the non-CE group (4.8%). 11 McQueen et al showed that antibiotic treatment of CE improved single-pregnancy LBRs in women with CE and a history of recurrent miscarriage (7% before treatment and 56% after treatment). The cumulative LBR was 88% in the cured group of recurrent miscarriages with CE infection compared with 74% in the recurrent miscarriages without CE infection group. 12 Currently, the main treatment for CE is oral antibiotics. The most commonly used regimen in our hospital is levofloxacin tablets (500 mg once daily for 14 days) combined with metronidazole (400 mg two times a day for 14 days). However, oral antibiotics present issues such as resistance and low local drug concentration. Moreover, oral drugs are prone to cause gastrointestinal reactions like nausea, vomiting, diarrhoea and abdominal distension, as well as adverse reactions including rash, limb numbness and leucopenia, which adversely affect the effective rate of the treatment for CE. According to reports, the effective rate ranges from 27.9% to 75%. 8 13 A metronidazole suppository is mainly composed of metronidazole, which is used in the vagina. While currently indicated for the treatment of Trichomonas vaginalis infection, bacterial infection and other vaginitis caused by microorganisms, 14 15 metronidazole suppositories demonstrate considerable therapeutic potential for CE. Given the anatomical continuity between the vagina and uterine cavity, topical vaginal medication achieves direct proximity to the endometrium, resulting in high local drug concentrations. This route bypasses the systemic metabolism (eg, hepatic first-pass effect) inherent to oral administration, thereby significantly elevating drug concentrations at the target site. 16 Furthermore, vaginal suppositories facilitate localised release of active ingredients, preventing degradation by gastric acid and digestive enzymes associated with oral delivery, which ensures more stable therapeutic efficacy. 17 Additionally, by circumventing gastrointestinal absorption and systemic circulation, vaginal administration enables faster attainment of effective drug concentrations and more rapid onset of action. 16 Critically, minimal systemic absorption reduces adverse effects on hepatic and renal function as well as gut microbiota, diminishing systemic side effects and enhancing safety. 17 Finally, whereas oral therapy often induces patient reluctance due to side effects, topical vaginal administration demonstrates higher treatment compliance. 17 Consequently, adjunctive vaginal therapy may enhance the cure rate for CE, ultimately improving pregnancy outcomes. However, at present, there is no report of the metronidazole suppository in the treatment of CE. This study marks the first attempt to analyse the efficacy of oral levofloxacin combined with metronidazole suppositories in order to explore the treatment plan to improve the clinical pregnancy rate and LBR and reduce the abortion rate and the side effects caused by oral drugs.

We intend to carry out a single-centre randomised controlled clinical trial. The participating centre is Peking University Third Hospital (Beijing), and the detailed inclusion and exclusion criteria for participants are listed below. Inclusion criteria: Infertile patients aged 20–42 years. Hysteroscopy and endometrial biopsy are performed. The pathological diagnosis is CE. Serum follicle-stimulating hormone level on the second day of menstruation is ≤12 U/L. Has agreed and signed informed consent. Exclusion criteria: Patients with the following diagnoses: submucosal myoma, uterine malformation or previous surgery for correction of uterine deformity, intrauterine adhesions, previous history of pelvic tuberculosis or endometrial tuberculosis or the current pathology suggested endometrial tuberculosis, endometrial hyperplasia or endometrial cancer. Patients with a spouse or previous fetal or child chromosomal abnormalities who plan to undergo preimplantation genetic screening. Patients with a known history of allergy to the study drugs. Eligible women with CE will be invited to participate in the study if they meet the inclusion criteria. Researchers at each centre will explain the study protocol to potential participants. After signing informed consent, participants will be registered and randomly assigned to the oral levofloxacin plus metronidazole tablet group or the oral levofloxacin plus metronidazole suppository group. We will employ a computer-generated randomisation list in a 1:1 ratio, stratified by research centre with block sizes of four or six. The randomisation list will be prepared by independent biostatisticians not involved in recruitment and will not be provided to investigators at any centre. Randomisation will be implemented using sequentially numbered identical containers of the study drugs. Levofloxacin tablets will be manufactured by BEIJING JNOVA Pharmaceutical (China) Co. Metronidazole tablets will be manufactured by GrandPharma (China) Co. Metronidazole suppositories will be manufactured by Hubei TUNGSHUN Pharmaceutical Co. Due to the explicit use of the medications in this study, blinding is not involved. Participants in the intervention group will receive oral levofloxacin 0.5 g once daily+intravaginal metronidazole suppository 0.5 g once daily for 2 weeks. Participants in the control group will receive oral levofloxacin 0.5 g once daily+metronidazole 0.4 g two times a day orally for 2 weeks. The primary outcome is live births after embryo transfer, defined as the delivery of at least one baby after 22 weeks of gestation, exhibiting any sign of life. The following pregnancy-related outcomes after embryo transfer will be included in secondary outcomes: clinical pregnancy, ongoing pregnancy, biochemical pregnancy, miscarriage, cumulative live birth, birth weight, stillbirth, mode of delivery and adverse events (AEs) (including pre-eclampsia, gestational hypertension, gestational diabetes mellitus, premature rupture of membrane rate, preterm delivery, placenta previa and postpartum haemorrhage). To collect data, patients will be followed up via telephone for a period of 1 year. 1 At the first visit (pre-enrolment), medical history is collected to assess eligibility. During the second visit (enrolment), informed consent is obtained, and electronic randomisation is performed. The third visit (from treatment initiation until 2 weeks after medication cessation) evaluates compliance, adverse reactions and medical history updates. At the fourth visit (egg retrieval day), ovarian stimulation-related clinical data are recorded, while the fifth visit (embryo transfer day) documents transfer-related clinical data. At the sixth visit (14 days post-transfer), serum human chorionic gonadotropin is measured to determine pregnancy status, with negative results classified as non-pregnant and positive cases requiring further ultrasound confirmation. The seventh visit (28–30 days post-transfer) involves a gynaecological ultrasound to confirm clinical pregnancy. Finally, the eighth visit (1 year post-transfer) assesses long-term outcomes, including miscarriage, ectopic pregnancy or live birth. This systematic approach ensures comprehensive data collection for evaluating treatment efficacy and safety while adhering to ethical standards. Adverse reactions(AEs) and serious AEs (SAEs), will be closely monitored and recorded, including possible reactions to the study drugs and any complications during the IVF procedures. The standard IVF procedure, as outlined in our previous publication, 18 will be followed, using either long or short protocols of gonadotropin-releasing hormone analogues or antagonists. The starting dose of gonadotropins will be 75–225 international units, adjusted based on ovarian response to follicular development and serum oestradiol levels. IVF will be conducted 4–6 hours after oocyte retrieval, employing conventional IVF or intracytoplasmic sperm injection. Embryo transfer protocols will be based on local guidelines, with a maximum of two cleavage-stage embryos or one blastocyst transferred per cycle. Luteal support will begin on the day of oocyte retrieval, with embryo transfers conducted on day 3, 5 or 6 postretrieval. Detailed research visit schedules are shown in table 1 . Participants will be encouraged to record their daily intake of study drugs using online tools for compliance monitoring. Trial coordinators will regularly communicate with participants to ensure adherence to the study protocol. If serum HCG is negative, it is considered not pregnant. Positive results require further follow-up with gynaecological ultrasound results HCG, human chorionic gonadotropin. At Peking University Third Hospital, based on the previous results of our centre, the LBR after the first embryo transfer in each cycle for the control group is 33.6%. To demonstrate a 10% increase in the LBR in the experimental group (43.6%) compared with the control group, with a two-sided α of 0.05 and β of 0.2, and with a 1:1 allocation between the experimental and control groups, a total of 369 participants are required. Considering a possible protocol violation and a loss to follow-up rate of 5%, we plan to recruit 800 participants (400 in each group). The study will use SPSS V.25.0 (IBM Corp, New York, USA) for statistical analysis. Under the intention-to-treat (ITT) principle, the following three types of data can be considered for statistical analysis: Full analysis set (FAS): This includes almost all randomised subjects according to the ITT principle but excludes those who were excluded during the run-in period and had no follow-up data after enrolment from the FAS population. Per protocol set (PPS): This includes subjects who were compliant, did not violate the protocol and had complete baseline values of the primary outcome included in the PPS. Safety analysis set: This includes all randomised subjects who have received at least one treatment and at least one safety evaluation. Baseline analysis will be conducted using the FAS dataset, presenting normally distributed continuous data as mean±SD and comparing between groups using independent t-tests. Non-normally distributed data will be represented by the median (quartiles) and compared using non-parametric tests. Categorical data will be presented as frequencies and percentages, with between-group comparisons done using χ 2 tests or Fisher’s exact tests. The primary endpoint, LBR, will undergo FAS and PPS set analyses to assess result stability, with between-group comparisons of LBRs performed using χ 2 tests or Fisher’s exact tests. For secondary endpoint analysis, the FAS set will be used, applying similar methods as the baseline analysis. In the safety analysis, the rates of AEs and SAEs in both groups will be described, along with their relationship to the drug. Between-group comparisons will be conducted using χ 2 tests or Fisher’s exact tests. Logistic regression will be employed to identify factors influencing the primary endpoint (LBR) and further conduct subgroup analyses for the major influencing factors. Statistical significance will be set at two-sided p<0.05. The trial is expected to commence in October 2024, and recruitment will span 18 months. The first participant is recruited on 23 October 2024, and the last participant is anticipated to be recruited by December 2025. Patients and the public were not involved in formulating the research question or study design. They will not participate in recruitment, implementation or reporting of the study. Research findings will be disseminated to study participants via social media. An independent DSMC composed of at least two clinical doctors and one statistician will oversee the trial independently of the sponsor with no conflicts of interest. An interim analysis will be conducted after 6 months on the first 300 recruited participants. As the primary endpoint (live birth) may not be realised for most participants at the interim analysis, the DSMC will be asked to assess ongoing pregnancies. Based on the DSMC’s recommendations, the study may be terminated prematurely. AEs will be closely monitored during the study. If participants experience suspected severe adverse symptoms related to the drug, abnormal signs, abnormal liver or kidney function or abnormal blood test results, they will be advised to discontinue the trial drug. Researchers will continue observing symptoms and signs until they resolve or test results normalise. Rare AEs include allergic reactions. SAEs are defined as events occurring during the study and meeting one of the following criteria: death, life-threatening, causing serious or permanent disability, congenital anomaly or birth defect and prolonged hospitalisation. Currently, there are no reported SAEs related to the drug metronidazole. This study obtained approval from the Ethics Committee of Peking University Third Hospital on 28 June 2024 (reference number: IRB00006761-M2023857). Following the recruitment of the first participant, modifications to the protocol are not permitted unless necessary, requiring additional ethical approval. Written informed consent ( online supplemental file 1 ) is compulsory for all participants before their involvement in the trial. Data processing will be anonymous, with participant codes provided only in the central database. Personal information beyond the scope of the trial will not be collected, shared or retained about potential and enrolled participants before, during and after the trial to maintain confidentiality. Research findings will be submitted to scientific conferences on reproductive medicine and peer-reviewed journals.