Hydroxychloroquine Sulfate Tablets for Patients with Recurrent Implantation Failure: A Double-Blind, randomized, placebo-controlled trial

Hydroxychloroquine Sulfate Tablets for Patients with Recurrent Implantation Failure: A Double-Blind, randomized, placebo-controlled trial | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hydroxychloroquine Sulfate Tablets for Patients with Recurrent Implantation Failure: A Double-Blind, randomized, placebo-controlled trial Dong-Lin Han, Li Li, Jing Shi, Yi-Meng Ge, Shu-Lin Yang, Yi-Fan Chu, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7141881/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Jan, 2026 Read the published version in Trials → Version 1 posted 5 You are reading this latest preprint version Abstract Background Recurrent implant failure (RIF) remains clinically unresolved at this stage. Hydroxychloroquine, as an immunomodulator, is still lacking clinical evidence, but it is being used by increasing numbers of reproductive centers and physicians worldwide, so a well-designed randomized controlled trial (RCT) is urgently needed to elucidate whether hydroxychloroquine can improve pregnancy outcomes in patients with RIF. Methods and Analysis In this study, we plan to recruit 686 volunteers who will undergo IVF/ICSI at 5 reproductive centers from 6 December 2022. Participants will be randomized to two parallel groups and treated with hydroxychloroquine sulfate tablets or placebo from the start of endometrial preparation to 14 days after frozen embryo transfer (if not pregnant) or to 12 weeks of pregnancy (if pregnant).The primary outcome is live birth rate, and the secondary outcomes include biochemical pregnancy rate ,clinical pregnancy rate, embryo attachment rate ,first trimester abortion rate and ongoing pregnancy rate ,birth weight ,pregnancy and perinatal complications ,congenital anomaly and other adverse events. Discussion This study aims to evaluate whether hydroxychloroquine (HCQ) improves pregnancy outcomes in patients with recurrent implantation failure (RIF). Secondary objectives include comparative analysis of gestational complications between the intervention and control groups. Trial registration ChiCTR2100047584 [Chinese Clinical Trial Registry (ChiCTR): registered on 20 June 2021] LM2021267 [Ethics Committee of Peking University Third Hospital] Hydroxychloroquine RIF IVF/ICSI Endometrial receptivity Reproductive immunity Figures Figure 1 Introduction Background and rationale {6a} There are about 186 million people and 8-12% of couples of childbearing worldwide suffering from infertility. The rate of success in Vitro Fertilization and Embryo Transfer (IVF-ET) is about 40%-60% in recent 10 years. It is difficult to improve the success rate, although there were some effective and auxiliary medical techniques, including assisted hatching, preimplantation genetic testing, intrauterine drug perfusion and frozen embryo transfer in recent years. The 8% -10% of patients still experience Repeated Implantation Failure (RIF) and fail to achieve successful pregnancy[1]. Adverse pregnancy outcomes pose great challenges to clinicians and cause deep frustration to patients. The factors influencing implantation are complex and unclear. It is reported 50% – 60% may be related to local immunity of the endometrium, and abnormalities in local immunity decrease the receptivity of endometrium [2, 3]. Despite the lack of adequate evidence-based medical evidence, immunomodulators are applied clinically as the most common intervention to improve pregnancy outcomes in women with RIF[4]. Hydroxychloroquine, as an immunomodulator, first applied in clinical practice as an antimalarial in 1946, after that it was routinely used to treat autoimmune diseases[5]. In addition, hydroxychloroquine has the effect of preventing venous thrombosis for patients with systemic lupus erythematosus (SLE) and antiphospholipid antibody syndrome[6, 7]. Several clinical studies have confirmed that hydroxychloroquine can be safely and effectively used in autoimmune diseases such as SLE and rheumatoid arthritis during pregnancy without significant adverse effects on placental and fetal development, so it is the first choice of immunomodulators during pregnancy and lactation [8, 9]. Animal experiments have confirmed that in primary antiphospholipid antibody syndrome, hydroxychloroquine can significantly reduce the thrombus size after vascular injury in mice compared with oral anticoagulants alone[10]. And several studies have suggested that hydroxychloroquine has the abilities of anti-diabetic[11], lipid-lowering[12] and endothelial protection of antioxidation[13]. In recent years, some studies indicated that hydroxychloroquine may has the potential to facilitate embryo implantation and improve IVF outcomes by effecting the change of inflammatory factor of Th1/Th2 balance[14]. Although some trials have shown that hydroxychloroquine may improve pregnancy outcomes, these trials are small or non-randomized studies or combination regimens and are insufficient to draw the conclusion[15, 16]. Evidence-based medical evidence is currently lacking, a well-designed and adequately powered randomized controlled trial (RCT) is therefore urgently needed to clarify whether hydroxychloroquine can improve pregnancy outcomes in RIF patients. For this purpose, we designed the double-blind, randomized, placebo-controlled clinical trial. Background and rationale: choice of comparators{6b} The investigational product in this study is hydroxychloroquine sulfate tablets. Both hydroxychloroquine and placebo were manufactured by Shanghai Shangyao Zhongxi Pharmaceutical Co., Ltd. The placebo is identical to hydroxychloroquine in all aspects including excipients, appearance, and odor, except for the absence of active ingredient. Study medications (hydroxychloroquine and placebo) were labeled according to the randomization sequence. The packaging and tablets of hydroxychloroquine and placebo are visually indistinguishable. Placebo quality, including composition and bacterial contamination, was strictly controlled in compliance with Good Manufacturing Practice (GMP) standards. Objectives {7} This trial primarily aims to evaluate the efficacy and safety of hydroxychloroquine sulfate tablets in patients with recurrent implantation failure (RIF), assessing pregnancy outcomes and gestational complications. We hypothesize that hydroxychloroquine treatment will increase live birth and clinical pregnancy rates while reducing adverse pregnancy events in patients with recurrent implantation failure (RIF). Trial design {8} This is a prospective, randomized, double-blind, controlled clinical trail of hydroxychloroquine vs placebo (The ratio of the two is 1:1) to assess the efficacy and safety of hydroxychloroquine sulfate tablets in patients with recurrent implantation failure (RIF). This study will be conducted at five clinical hospitals. The primary outcome is live birth rate, and the secondary outcomes include biochemical pregnancy rate, clinical pregnancy rate, embryo attachment rate, first trimester abortion rate and ongoing pregnancy rate, birth weight, pregnancy and perinatal complications, congenital anomaly and other adverse events. Methods: participants, interventions, and outcomes Study setting {9} This multicenter clinical trial will be conducted at five leading reproductive medical centers in China: Peking University Third Hospital, the First Affiliated Hospital of Anhui Medical University, the Second Hospital of Hebei Medical University, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, and Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology. The study will strictly adhere to the principles of the Declaration of Helsinki. All participating institutions are nationally recognized as top-tier reproductive medicine centers with comprehensive clinical research infrastructure, ensuring robust data collection. Eligible patients meeting the inclusion criteria outlined below will be enrolled in the study. The screening flowchart (inclusion/exclusion criteria) is presented in [ Figure 1 ] . Eligibility criteria {10} I nclusion criteria 1. Infertile patients with recurrent implantation failure (RIF) and undergoing frozen-thawed embryo transfer (FET) after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), or infertile patients with RIF undergoing preimplantation genetic testing for aneuploidy (PGT-A). 2. Married women aged 18 to 40 years (Excluding cut-off). 3. Married women with 2 or more failed embryo transfer cycles. 4. Able to understand the informed consent form, voluntarily participate in this clinical trial and sign the informed consent form. 5. Able to comply with the study protocol and complete the trial. E xclusion criteria 1. Women currently receiving any corticosteroid or immunosuppressive therapy, such as prednisone, cyclosporine, and azathioprine, or patients receiving these medications who did not have a 2-month washout period prior to screening. 2. Women with known autoimmune diseases such as Systemic Lupus Erythematosus (SLE), antiphospholipid syndrome, Sjogren syndrome, and scleroderma. 3. Women diagnosed with diseases affecting the uterine cavity, such as uterine malformation and submucous fibroids. 4. Women or their partner with karyotypic abnormalities (excluding chromosomal polymorphisms). 5. Women with recurrent pregnancy loss, defined as two or more clinical pregnancy failures confirmed by ultrasonography or histopathology. 6. Women with medical contraindications to assisted reproductive technology and/or pregnancy. 7. Women with thin endometrium (< 6 mm before transfer) and is insensitive or unresponsive to hormonal drug therapy for endometrial preparation. 8. Women with known allergy to quinolines or history of relevant allergy. 9. Women who are receiving treatment with aspirin, Dalteparin Sodium Injection, Clexane and Nadroparin Calcium Injection in addition to the conventional drugs for transplantation. Who will take informed consent? {26a} Patients meeting the diagnostic criteria for recurrent implantation failure (RIF) who are undergoing frozen embryo transfer (FET) following in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), or those with RIF indicated for preimplantation genetic testing for aneuploidy (PGT-A), will be screened according to the aforementioned criteria. After being assessed as eligible by the attending physician at the reproductive center, the investigator will obtain preliminary study materials and provide comprehensive explanations regarding the trial objectives, procedures, potential benefits, and risks to the patients. Qualified participants expressing interest will receive a copy of the informed consent form for review. The formal study will commence only after the signed informed consent is obtained. Interventions: description{11a} Screening period The trial and study plan will be announced to all participants and eligible couples will sign the informed consent. Check the inclusion and exclusion criteria to determine the eligible participants. Screen-eligible subjects were randomized equally to two parallel treatment groups: A) hydroxychloroquine group: 0.2 g bid, orally; B) placebo group: 0.2 g bid, orally. The study stuff will dispense the first bottle (100 tablets/bottle) of study drug (hydroxychloroquine/placebo) corresponding to the drug number and instruct the patients on how to take it. Manual cycle + thawing transplant period The trial medication will be taken at the same time as the initiation of a hormone replacement cycle regimen. Subjects will take 2 tablets every morning and evening. The endometrium will be prepared with a hormone replacement cycle regimen. Estradiol valerate (Progynova, Delpharm Lille SAS, France) and/or estradiol tablets (Femoston, Abbott Biologicals B.V., the Netherlands) (2-8mg daily) will be started on days 2-5 of the menstrual cycle or 28-35 days after long-acting GnRH-agonist suppression. When the endometrial thickness is sufficient (≥ 6 mm), luteal phase support will be added with oral dydrogesterone (Daphnetone, Abbott) 10-20 mg twice daily and vaginal progesterone gel (Xernox, Merck Serono) 90 mg daily. After endometrial preparation, participants will undergo frozen-thawed embryo transfer (FET). Each participant will be transferred with one blastocyst or two cleaved embryos. All embryos transferred will be of good quality. 2 weeks after transfer, the serum hCG test will be performed. Post-transplant Follow-up Period Participants will undergo serum hCG testing 12-14 days after transplant. If the participant is confirmed to be pregnant, a second bottle of trail medication with the corresponding number will be dispensed on the day of the pregnancy test (12-15 days after transplantation) and continue medication as well as luteal phase support until the end of the first trimester (12 weeks of pregnancy). Then, continue follow-up the patients until the end of pregnancy. The drug will be discontinued if the transplantation fails or the pregnancy is lost, the remaining medication will be returned to the study stuff. Discontinuation criteria include but not limited to the following points: 1. Spontaneous pregnancy. 2. Serious complications or side effects. 3. Participants using prohibited medication, such as other glucocorticoids or immunosuppressive medication. 4. Thin endometrium (< 6 mm before metastasis). 5. Participants who want to withdraw from the trial. Interventions: adherence {11c} Both hydroxychloroquine and placebo used in this trial are tablet formulations with simple administration procedures, neutral odor, and minimal reported adverse reactions, ensuring high protocol adherence. The following measures will be implemented to further enhance compliance: 1. Maintain open communication channels between investigators and participants to facilitate immediate reporting of medication loss. 2. Conduct adherence education sessions for participants and their families, emphasizing therapeutic benefits and engaging family supervision. 3. Provide comprehensive counseling on investigational product mechanisms, placebo effects, and adherence significance while encouraging active discussion of treatment-related concerns. 4. Instruct participants to report all adverse/unexpected drug reactions without self-discontinuation, coupled with healthcare guidance to establish trusting investigator-participant relationships. Outcomes {12} Primary outcomes Live birth rate: Live birth after frozen-thawed embryo transfer, defined as delivery of any newborns ≥ 28 weeks of gestation with vital signs. Live birth rate was calculated as number of live birth/ number of transplantation. Secondary outcomes 1. Biochemical pregnancy rate: biochemical pregnancy is defined as serum β-hCG ≥ 30 mIU/mL at 12-15 days after embryo transfer, biochemical pregnancy rate = biochemical pregnancy cycle/transfer cycle. 2. Clinical pregnancy rate: Clinical pregnancy is defined as ultrasound detection of intrauterine gestational sac or clinical diagnosis of ectopic pregnancy at 28 ~ 30 days after embryo transfer, clinical pregnancy rate = clinical pregnancy cycle/transfer cycle. 3. Embryo attachment rate: embryo implantation rate = number of gestational sacs with heart tube pulsation/number of embryos transferred. 4. First trimester abortion rate and ongoing pregnancy rate: abortion refers to termination of pregnancy without reaching 28 weeks and fetal weight less than 1000 g; early abortion refers to fetal loss before 12 weeks of gestation, early abortion rate = early abortion cycle/clinical pregnancy cycle; ongoing pregnancy is a live intrauterine pregnancy continuing until 10 weeks of gestation, ongoing pregnancy rate = ongoing pregnancy cycle/transplantation cycle. 5. Birth weight. 6. Pregnancy and perinatal complications. 7. Congenital anomaly. 8. Other Adverse Events. Participant timeline {13} This study employs standardized timelines for participant enrollment, interventions, assessments, and follow-up visits to ensure operational consistency across all research sites [ Table 1 ] . For participants achieving successful post-transfer pregnancy, study medication will be maintained until 12 weeks of gestation (approximately 16 weeks), with follow-up continuing until 6 weeks postpartum (approximately 47 weeks). Participants with unsuccessful pregnancy will receive medication for approximately 4 weeks, followed by monitoring until 30 days post-transfer (approximately 8 weeks). Participants retain the right to withdraw from the study at any time without forfeiting any entitled benefits, and safety evaluations will be conducted upon withdrawal to assess participant status. All follow-up data will be collected in strict accordance with the study protocol. Sample size {14} According to the meta-analysis published in 2013, the live birth rate was estimated to be 16.1% in women who failed three or more embryo transfer cycles[17]. And live birth rates range from 15.5% to 29% in women who have failed two or more embryo transfer cycles[18]. It has also reported that the combination of prednisone with low molecular weight heparin or aspirin can increase the live birth rate by 10.8 – 14%[19, 20]. In present study, we plan to test the primary hypothesis of a difference of 10% in the live birth rate between the two randomization arms. In patients with RIF, we assume that the live birth rate will be 30% in the hydroxychloroquine group and 20% in the placebo group. For the sample size calculation, the two-side significance level will be set at α = 0.05 and the statistical power will be calculated as 1-β = 0.80. The ratio between groups is 1:1. The minimum sample size for each group was 291, with a total of 582 participants. Considering the dropout rate is 15%, we expect to a final total of 686 subjects. Assignment of interventions (for controlled trials) Allocation: sequence generation {16a} Participants will be randomly allocated to two parallel groups to receive either hydroxychloroquine sulfate tablets or placebo treatment from endometrial preparation initiation through the first trimester of pregnancy (if conception occurs). All eligible participants will be randomized to one of the two study arms using a computer-generated randomization sequence prepared by the Data Coordinating Center (DCC) with SAS version 9.2 (SAS Institute Inc., Cary, NC), stratified by embryo stage (cleavage-stage or blastocyst). Blinding (masking) {17a} This clinical study is a double-blind trial. The randomization code list was generated by the statistical unit, with sealed blinding envelopes maintained in duplicate copies stored separately at the leading institution, the National Drug Clinical Trial Institution, and the sponsor. The entire medication coding process was documented by the blinding officer as blinding documentation, preserved as part of the clinical trial records. The randomization schedule will be maintained under strict confidentiality by Data Coordinating Center (DCC) personnel, with registration staff having no access to allocation information. Investigators will remain blinded to treatment group assignments throughout the trial. Blinding (masking): emergency unblinding {17b} Each coded investigational product has a corresponding emergency envelope disclosing its assigned treatment group. These envelopes are securely maintained at participating sites, with unauthorized opening strictly prohibited. Emergency envelopes are distributed to clinical trial sites alongside correspondingly numbered medications and will be uniformly retrieved post-trial completion. Emergency unblinding is permitted exclusively for critical situations (e.g., serious adverse events) or when life-saving interventions require treatment knowledge. Unblinding must be authorized by the site's principal investigator, accompanied by detailed documentation including reason, timestamp, location, and investigator signature. Immediate notification must be sent to the lead institution and clinical monitors following unblinding, with complete preservation of case records. Data collection, management, and analysis Data collection plan {18a} All data collection (including baseline and follow-up measures) will be conducted in accordance with ICH-GCP guidelines using a blinded management process: Certified clinical research coordinators (CRCs), who remain blinded to subject group allocation, will perform data collection after completing standardized data collection protocol (DCP-01) training and signing confidentiality agreements. Baseline data will be automatically extracted through the electronic data capture (EDC) system integrated with hospital electronic medical records (EMR), while pregnancy outcomes, complications and adverse event data will be recorded in real-time via telephone follow-up. A permission segregation mechanism is implemented for data entry and modification, where only authorized investigators and designated data managers have system operation rights, with monitors retaining only data verification privileges. Case report form (CRF) data will be transferred to the central database after entry, with all data changes recording the operator, timestamp and revision rationale. Data collection plan: retention {18b} This study implements two key measures to enhance participant compliance: First, flexible scheduling of follow-up visits (with a ±3-day adjustment window) to accommodate participants' availability and minimize missed appointments. Second, a three-stage progressive reminder system (via phone call, text message, and email) prior to each scheduled follow-up visit. Data management {19} This study implements a closed-loop data management system: After double-entry verification of CRF data, the data manager generates a comprehensive data review report including study completion status (with subject dropout list), eligibility criteria verification, completeness check, logical consistency validation, outlier detection, time window assessment, concomitant medication review, and adverse event evaluation. The sponsor's monitors conduct source data verification (SDV). Throughout the study and post-completion, both investigational sites and the sponsor must maintain all study documents (CRFs, medical records, lab results, informed consent forms, drug dispensing/disposal records, safety reports, terminated participant information, etc.) in compliance with Good Clinical Practice (GCP) requirements. The sponsor must notify investigators regarding documents eligible for disposal, and no documents may be destroyed/relocated without prior written sponsor approval. Confidentiality {27} This study assigns unique de-identified codes to each participant for data archiving, with master key access strictly limited to authorized research team members during the active study phase. Upon study completion, the master key file will be physically encrypted and stored separately by the principal investigator according to predefined data security protocols. During publication, we rigorously adhere to biomedical data sharing ethics guidelines, systematically anonymizing potentially identifiable parameters (such as rare medical histories or specific spatiotemporal events) to ensure zero risk of individual privacy disclosure. Plans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in this trial/future use {33} No biological specimens will be collected in this trial or for future use. Statistical methods Statistical methods for primary and secondary outcomes {20a} SPSS 21.0 (SPSS Inc., Chicago, IL, USA) will be used to analyze the data. Normally distributed continuous variables will be expressed as mean ± standard deviation, student's test is used to test between-group differences. Continuous variables that are not normally distributed will be presented as medians and ranges, Wilcoxon rank-sum test is used for between-group differences. Categorical data are presented as frequency and percentage. Pearson's chi-square test was used to assess differences in these measures, and Fisher's exact test will be used if the expected frequencies was less than 5, P < 0.05 will be considered significant. The analysis will follow the intention-to-treat principle. Pearson's chi-square test will be used to compare the primary outcome live birth rates between the two groups. Secondary outcomes, such as pregnancy and implantation rates, will be also analyzed by Pearson's chi-square test. Per-protocol analysis will be performed based on actual participants who complete the entire trial. As a secondary analysis, we will fit a generalized linear mixed effects model with logit link to compare treatment groups for the primary outcome of live birth and binary secondary outcomes (such as pregnancy rate and implantation), adjusting for factors such as randomization strata of embryo stage and other explanatory variables. A random intercept will be included to adjust for the correlation between patients within sites. Interim analyses {21b} This study does not plan to conduct an interim analysis. Methods for additional analyses (e.g., subgroup analyses) {20b} This study does not plan to conduct subgroup analyses. Methods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c} Through the appropriate measures described previously, missing data will be minimized, with no imputation applied. Illogical data must be confirmed by clinical investigators, and unused data shall be archived and managed alongside trial data in compliance with applicable regulations. Plans to give access to the full protocol, participant-level data, and statistical code {31c} In accordance with international guidelines for clinical trial data sharing, this study implements a standardized data disclosure mechanism: Following ethics review, de-identified datasets may be disclosed upon authorization by the study steering committee after compliance review conducted jointly by the Sponsor (Reproductive Medicine Center of Peking University Third Hospital) and site principal investigators from participating centers in response to legitimate academic requests. The data sharing procedures execute standardized confidentiality management protocols: 1) Processing of identifiable information strictly adheres to institutional and national regulations, removing direct identifiers (including name, national ID number, etc.) and potential identifiers (e.g., disease subtypes, extreme age values among demographic characteristics); 2) A data integrity verification mechanism is implemented utilizing hash algorithms to ensure data consistency during transmission. Oversight and monitoring Composition of the coordinating centre and trial steering committee {5d} This multicenter, double-blind, prospective randomized controlled trial (pRCT) employed a protocol developed and led by the Reproductive Medicine Center of Peking University Third Hospital. Five participating hospitals collaboratively executed core operational processes: subject recruitment/screening, blinded follow-up management, standardized data collection, and intention-to-treat (ITT)-based statistical analyses. The project office conducted biweekly cross-site quality evaluation meetings via videoconference, employing Good Clinical Practice (GCP)-compliant auditing protocols to monitor protocol deviations while dynamically executing risk mitigation measures. Systematic review of trial progress and necessary adjustments were coordinated within this framework. Composition of the data monitoring committee, its role and reporting structure {21a} The Data Monitoring Committee (DMC) for this study comprises one epidemiology expert from Peking University Third Hospital, one epidemiology expert from the Robinson Research Institute and Adelaide Medical School, Adelaide, South Australia, Australia, and one clinical research methodology expert from Queen Mary Hospital of The University of Hong Kong. All members remain independent from the trial design and implementation process, with no financial interests related to the sponsor. The committee primarily conducts dynamic risk assessment and data quality control through the following mechanisms. First, real-time screening of safety signals (including but not limited to pregnancy loss rate threshold alerts and spatiotemporal cluster analysis of serious adverse events), with quarterly risk-benefit evaluations based on cumulative data; Second, supervision of protocol compliance (encompassing blinded status verification and critical procedural timeline adherence reviews). Adverse event reporting and harms {22} An adverse event (AE) refers to any untoward medical occurrence (including clinically significant abnormal laboratory findings), symptom, or disease temporally associated with the use of the investigational product, regardless of suspected causal relationship. Pre-existing conditions identified during screening should not be recorded as AEs but documented as medical history/concomitant conditions. A serious adverse event (SAE) is defined as any adverse medical event occurring after administration of the investigational product that results in death, is life-threatening, causes persistent or significant disability/incapacity, requires or prolongs hospitalization, or leads to congenital anomalies/birth defects. All AEs, irrespective of suspected relationship to the investigational product, must be fully documented in the subject's case report form (CRF) throughout the study period, including precise onset/cessation dates and times, detailed signs/symptoms, severity grading, causal relationship assessment, action taken, outcome, SAE determination, and whether the event led to study discontinuation. Frequency and plans for auditing trial conduct {23} The study will establish an independent audit team to conduct systematic audits at each participating site every six months, focusing on verification of trial documentation integrity, source data traceability, and protocol compliance. All auditing procedures must adhere strictly to Good Clinical Practice (GCP) standards using standardized operating procedures, with objectivity ensured through a conflict of interest declaration process. Audit records will be permanently archived in the trial master file (TMF) as per regulatory requirements. Plans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25} Any modification to the protocol, whether constituting a substantive amendment or administrative change to any section, shall be considered a protocol amendment. During the study conduct, if protocol amendments are required, they must undergo thorough discussion between the investigator and sponsor. Following mutual agreement on the necessity of amendments, the sponsor shall revise the protocol accordingly. The amended protocol must be resubmitted to the ethics committee for review and approval. Except for administrative amendments (e.g., typographical corrections), investigators must await formal ethics committee approval prior to implementing revised protocol versions. All approved amendments shall be documented in the trial master file (TMF) with version control and effective dates clearly specified. Dissemination plans {31a} The study results will be fully disclosed in international peer-reviewed journals, reporting both positive and negative findings. Participants opting to receive study-level outcomes will be provided with lay summaries compliant with scientific communication standards. Discussion This is a prospective, randomized, double-blind, controlled clinical trail of hydroxychloroquine vs placebo( The ratio of the two is 1:1) to assess the efficacy and safety of hydroxychloroquine sulfate tablets in patients with recurrent implantation failure(RIF).The primary outcome is live birth rate, and the secondary outcomes include biochemical pregnancy rate ,clinical pregnancy rate ,embryo attachment rate ,first trimester abortion rate and ongoing pregnancy rate ,birth weight ,pregnancy and perinatal complications ,congenital anomaly and other adverse events. So far, there is no uniform diagnostic criteria for RIF. The widely accepted definition in China is that women < 40 years of age who fail to achieve a clinical pregnancy with 2 or more fresh or frozen-thawed embryo transfer cycles and a cumulative transfer of at least 3 high-quality embryos are RIF. C Coughlan et al suggested RIF to be defined as the failure after transfer of at least 4 good-quality embryos in a minimum of three fresh or frozen cycles [ 21 ]. Implantation failure may be caused by a number of different factors, including the maternal immune system, embryo and parental genetics, anatomical factors, hematologic factors, reproductive tract microbiome, and endocrine environment[ 22 ]. Of these, 50–60% may be associated with local immunity of the endometrium[ 2 , 3 ]. Despite the lack of adequate evidence-based medical evidence in various therapeutic interventions, clinicians may attempt to “do something different” after RIF due to feel pressure. Immunomodulators are applied clinically as the most common intervention to improve pregnancy outcomes in women with RIF[ 4 ]. In recent years, hydroxychloroquine is being used by an increasing number of IVF centers and reproductive physicians worldwide beacuse its safety and validity during pregnancy and lactation. In terms of immune regulation, hydroxychloroquine can inhibit the proliferation of T cells, reduce the production of inflammatory cytokines like INF-γ, TNF, IL-1, IL-6 and IL-2, increase IL-10 and IL-4 levels, and can also prevent the interaction of Toll-like receptors (TLR) with nucleic acid ligands[ 23 ]. In addition, hydroxychloroquine has an anti-infective effect[ 24 ], which can alkalinize acidic vesicles from cells and inhibit the growth of microorganisms[ 25 ]. So it may play a role in chronic endometritis, which is another possible mechanism leading to repeated implantation failure. Some studies exist on the use of hydroxychloroquine in patients with RIF. Sonia Sadeghpour et al found that HCQ administration in RIF women with immune cell disease during pregnancy affected Th17/Treg ratio, and increased Treg and reduced Th17 responses, which may be associated with successful outcome of pregnancy[ 26 ]. In addition, hydroxychloroquine use in RIF patients with a high Th1/Th2 ratio during the implantation window could reduce this ratio and via a shift in Th2 responses[ 14 ]. These results suggest that hydroxychloroquine can change endometrial receptivity by regulating cellular immunity. At the same time, hydroxychloroquine has an anti-infective effect[ 24 ] and can alkalinize acidic vesicles in cells and inhibit the growth of microorganisms[ 25 ], i.e. It may play a role in chronic endometritis, which is another possible mechanism leading to recurrent implantation failure. However, clinical trials to assess the efficacy of hydroxychloroquine in patients with RIF are limited, and although evidence-based medical evidence for the use of hydroxychloroquine in patients with RIF is lacking, hydroxychloroquine is being used by an increasing number of in vitro fertilization centers and reproductive physicians worldwide. Therefore, we designed this randomized controlled trial (RCT) to clarify whether hydroxychloroquine could improve pregnancy outcomes in patients with RIF. Our hypothesis is that hydroxychloroquine will improve pregnancy outcomes and endometrial receptivity in RIF patients undergoing fresh embryo transfer, providing direct evidence for clinical treatment, and if this hypothesis is confirmed, a larger multicenter RCT will be conducted in the future to further investigate the optimal dose and duration of hydroxychloroquine to improve pregnancy outcomes in RIF patients and provide more solid evidence for clinical treatment. Strengths and Limitations Our study has several strengths and our randomized controlled trial design enables a more direct description of the treatment effect of hydroxychloroquine in patients with recurrent implantation failure and provides a more robust causal relationship. In addition, this study is a multicenter clinical trial, which is more representative and authoritative and can provide feasible methods for the clinical treatment of patients with recurrent implantation failure and provide improved data for guidelines. Finally, our experiments are designed and participated by authoritative statistical experts, obstetricians and gynecologists, scientists, etc., ensuring the quality of the study. The limitation of this study refers to its RCT nature, which the inclusion and exclusion criteria of studies will lead to certain limitations on the representativeness and external authenticity of research results. Abbreviations RIF Recurrent Implantation Failure RCT Randomized Controlled Trial IVF In Vitro Fertilization ICSI Intracytoplasmic Sperm Injection HCQ Hydroxychloroquine IVF-ET In Vitro Fertilization and Embryo Transfer SLE Systemic Lupus Erythematosus GMP Good Manufacturing Practice PGT-A Preimplantation Genetic Testing for Aneuploidy hCG Human Chorionic Gonadotropin DCC Data Coordinating Center CRCs Certified Clinical Research Coordinators EDC Electronic Data Capture EMR Electronic Medical Records SDV Source Data Verification ITT Intention-To-Treat ICH-GCP International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use - Good Clinical Practice GCP Good Clinical Practice IDMC Independent Data Monitoring Committee PPI Patient and Public Involvement SAP Statistical Analysis Plan AE Adverse Event CRF Case Report Form TMF Trial Master File TLR Toll-Like Receptors INF-γ Interferon Gamma Declarations Trial status Research Protocol Version 1.5. Recruitment began on November 16, 2021. As of June 24, 2025, 394 participants have been enrolled. Recruitment is currently ongoing and is expected to be completed by December 2027. Availability of data and materials {29} The datasets utilized and/or analyzed in this study are available from the corresponding author upon reasonable request. Acknowledgements We thank all the patients who participated in this project and SPH Zhongxi Pharmaceutical Co., Ltd for their support. Funding Both the applicant and the main implementer have the National Natural Science Foundation of China (Fund No. 82271863), and fund from National Clinical Research Center for Obstetrics and Gynecology (Fund No. BYSYSZKF2022008). Author information Authors and Affiliations Center for Reproductive Medicine, Department of Obstetrics and Gynecology, Third Hospital, Peking University Third Hospital, Beijing, China Dong-Lin Han, Li Li, Yi-Meng Ge, Jie Zhao and Rong Li. National Clinical Research Center for Obstetrics and Gynecology, Beijing, China Li Li, Yi-Meng Ge, Jie Zhao and Rong Li. Key Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China Dong-Lin Han, Li Li, Jie Zhao and Rong Li. Beijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China Li Li, Jie Zhao and Rong Li. State Key Laboratory of Female Fertility Promotion, Department of Obstetrics and Gynecology, Peking University, Third Hospital, Beijing, China Li Li, Jie Zhao and Rong Li. Department of Pharmacy, Peking University Third Hospital, Beijing, China Jing Shi Reproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Shu-Lin Yang and Yi-Fan Chu. Reproductive Medicine Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People's Republic of China Zhuo-Yao Mai and Hui Chen. Department of Reproductive Medicine, The Second Hospital of Hebei Medical University Yi-Wen Zhang and Gui-Min He. Reproductive Medicine Center, Tongji Hospital, Tongji Medicine College, Huazhong University of Science and Technology, Wuhan, People's Republic of China. Jing Yue Reproductive Medicine Center, Department of Obstetrics and Gynecology, the First Affiliated Hospital of Anhui Medical University, No 218 Jixi Road, Hefei 230022, Anhui, China Hui-Fen Xiang Contributions Dong-Lin Han and Li Li contributed equally to this work. Dong-Lin Han: Manuscript writing, methodology, conception. Li Li: Write the manuscript, supervise and review it. Jie Zhao: Concepts, methods, writing review and editing. Rong Li: Resources, concepts, methods, project management and capital acquisition. Jing Yue, Jing Shi and Yi-Meng Ge provide expertise for data collection and analysis. Jing Shi, Shu-Lin Yang, Yi-Fan Chu, Zhuo-Yao Mai, Yi-Wen Zhang, Hui Chen, Gui-Min He and Hui-Fen Xiang provide resources for project data collection. All authors contributed to the article and approved the submitted version. Corresponding author Correspondence to Rong Li and Jie Zhao. Ethics declarations Ethics approval and consent to participate {24} This project was approved by the Ethics Committee of Peking University Third Hospital [Acceptance No.: M2021169 Project No.: LM2021267]. All participants must provide written informed consent prior to study participation. Consent for publication {32} Not applicable. Competing interests {28} All authors declare no competing interests. References Mak JSM, Chung CHS, Chung JPW, Kong GWS, Saravelos SH, Cheung LP, Li TC. The effect of endometrial scratch on natural-cycle cryopreserved embryo transfer outcomes: a randomized controlled study. Reprod Biomed Online. 2017;35:28–36. Bashiri A, Halper KI, Orvieto R. Recurrent Implantation Failure-update overview on etiology, diagnosis, treatment and future directions. Reprod Biol Endocrinol. 2018;16:121. Li Y, Yu S, Huang C, Lian R, Chen C, Liu S, Li L, Diao L, Markert UR, Zeng Y. Evaluation of peripheral and uterine immune status of chronic endometritis in patients with recurrent reproductive failure. Fertil Steril. 2020;113:187–e196181. Abdolmohammadi-Vahid S, Danaii S, Hamdi K, Jadidi-Niaragh F, Ahmadi M, Yousefi M. Novel immunotherapeutic approaches for treatment of infertility. Biomed Pharmacother. 2016;84:1449–59. Plantone D, Koudriavtseva T. Current and Future Use of Chloroquine and Hydroxychloroquine in Infectious, Immune, Neoplastic, and Neurological Diseases: A Mini-Review. Clin Drug Investig. 2018;38:653–71. Tektonidou MG, Laskari K, Panagiotakos DB, Moutsopoulos HM. Risk factors for thrombosis and primary thrombosis prevention in patients with systemic lupus erythematosus with or without antiphospholipid antibodies. Arthritis Rheum. 2009;61:29–36. Ruiz-Irastorza G, Egurbide MV, Pijoan JI, Garmendia M, Villar I, Martinez-Berriotxoa A, Erdozain JG, Aguirre C. Effect of antimalarials on thrombosis and survival in patients with systemic lupus erythematosus. Lupus. 2006;15:577–83. Ahmadi M, Ghaebi M, Abdolmohammadi-Vahid S, Abbaspour-Aghdam S, Hamdi K, Abdollahi-Fard S, Danaii S, Mosapour P, Koushaeian L, Dolati S, et al. NK cell frequency and cytotoxicity in correlation to pregnancy outcome and response to IVIG therapy among women with recurrent pregnancy loss. J Cell Physiol. 2019;234:9428–37. Hviid MM, Macklon N. Immune modulation treatments-where is the evidence? Fertil Steril. 2017;107:1284–93. Edwards MH, Pierangeli S, Liu X, Barker JH, Anderson G, Harris EN. Hydroxychloroquine reverses thrombogenic properties of antiphospholipid antibodies in mice. Circulation. 1997;96:4380–4. Gerstein HC, Thorpe KE, Taylor DW, Haynes RB. The effectiveness of hydroxychloroquine in patients with type 2 diabetes mellitus who are refractory to sulfonylureas–a randomized trial. Diabetes Res Clin Pract. 2002;55:209–19. Petri M, Lakatta C, Magder L, Goldman D. Effect of prednisone and hydroxychloroquine on coronary artery disease risk factors in systemic lupus erythematosus: a longitudinal data analysis. Am J Med. 1994;96:254–9. Virdis A, Tani C, Duranti E, Vagnani S, Carli L, Kühl AA, Solini A, Baldini C, Talarico R, Bombardieri S, Taddei S, Mosca M. Early treatment with hydroxychloroquine prevents the development of endothelial dysfunction in a murine model of systemic lupus erythematosus. Arthritis Res Ther. 2015;17:277. Ghasemnejad-Berenji H, Ghaffari Novin M, Hajshafiha M, Nazarian H, Hashemi SM, Ilkhanizadeh B, Ghasemnejad T, Sadeghpour S, Ghasemnejad-Berenji M. Immunomodulatory effects of hydroxychloroquine on Th1/Th2 balance in women with repeated implantation failure. Biomed Pharmacother. 2018;107:1277–85. Pasquier E, de Saint-Martin L, Marhic G, Chauleur C, Bohec C, Bretelle F, Lejeune-Saada V, Hannigsberg J, Plu-Bureau G, Cogulet V, Merviel P, Mottier D. Hydroxychloroquine for prevention of recurrent miscarriage: study protocol for a multicentre randomised placebo-controlled trial BBQ study. BMJ Open. 2019;9:e025649. de Moreuil C, Alavi Z, Pasquier E. Hydroxychloroquine may be beneficial in preeclampsia and recurrent miscarriage. Br J Clin Pharmacol. 2020;86:39–49. Potdar N, Gelbaya TA, Konje JC, Nardo LG. Adjunct low-molecular-weight heparin to improve live birth rate after recurrent implantation failure: a systematic review and meta-analysis. Hum Reprod Update. 2013;19:674–84. El-Toukhy T, Campo R, Khalaf Y, Tabanelli C, Gianaroli L, Gordts SS, Gordts S, Mestdagh G, Mardesic T, Voboril J, et al. Hysteroscopy in recurrent in-vitro fertilisation failure (TROPHY): a multicentre, randomised controlled trial. Lancet. 2016;387:2614–21. Fawzy M, El-Refaeey AA. Does combined prednisolone and low molecular weight heparin have a role in unexplained implantation failure? Arch Gynecol Obstet. 2014;289:677–80. Siristatidis C, Dafopoulos K, El-Khayat W, Salamalekis G, Anifandis G, Vrantza T, Elsadek M, Papantoniou N. Administration of prednisolone and low molecular weight heparin in patients with repeated implantation failures: a cohort study. Gynecol Endocrinol. 2018;34:136–9. Coughlan C, Ledger W, Wang Q, Liu F, Demirol A, Gurgan T, Cutting R, Ong K, Sallam H, Li TC. Recurrent implantation failure: definition and management. Reprod Biomed Online. 2014;28:14–38. Franasiak JM, Alecsandru D, Forman EJ, Gemmell LC, Goldberg JM, Llarena N, Margolis C, Laven J, Schoenmakers S, Seli E. A review of the pathophysiology of recurrent implantation failure. Fertil Steril. 2021;116:1436–48. Robertson SA, Jin M, Yu D, Moldenhauer LM, Davies MJ, Hull ML, Norman RJ. Corticosteroid therapy in assisted reproduction - immune suppression is a faulty premise. Hum Reprod. 2016;31:2164–73. Richard SA, Kampo S, Hechavarria ME, Sackey M, Buunaaim ADB, Kuugbee ED, Anabah TW. Elucidating the Pivotal Immunomodulatory and Anti-Inflammatory Potentials of Chloroquine and Hydroxychloroquine. J Immunol Res. 2020;2020:4582612. Chandler LC, Yusuf IH, McClements ME, Barnard AR, MacLaren RE, Xue K. Immunomodulatory Effects of Hydroxychloroquine and Chloroquine in Viral Infections and Their Potential Application in Retinal Gene Therapy. Int J Mol Sci 2020, 21. Sadeghpour S, Ghasemnejad Berenji M, Nazarian H, Ghasemnejad T, Nematollahi MH, Abroon S, Paktinat S, Heidari Khoei H, Ghasemnejad Berenji H, Ghaffari Novin M. Effects of treatment with hydroxychloroquine on the modulation of Th17/Treg ratio and pregnancy outcomes in women with recurrent implantation failure: clinical trial. Immunopharmacol Immunotoxicol. 2020;42:632–42. Tables Table 1 is available in the Supplementary Files section. Supplementary Files Table1.docx SPIRITchecklist.docx Cite Share Download PDF Status: Published Journal Publication published 30 Jan, 2026 Read the published version in Trials → Version 1 posted Editorial decision: Accept 14 Jan, 2026 Reviewers agreed at journal 02 Oct, 2025 Reviewers invited by journal 02 Oct, 2025 Editor assigned by journal 17 Aug, 2025 First submitted to journal 13 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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{6a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThere are about 186 million people and 8-12% of couples of childbearing worldwide suffering from infertility. The rate of success in Vitro Fertilization and Embryo Transfer (IVF-ET) is about 40%-60% in recent 10 years. It is difficult to improve the success rate, although there were some effective and auxiliary medical techniques, including assisted hatching, preimplantation genetic testing, intrauterine drug perfusion and frozen embryo transfer in recent years. The 8% -10% of patients still experience Repeated Implantation Failure (RIF) and fail to achieve successful pregnancy[1]. Adverse pregnancy outcomes pose great challenges to clinicians and cause deep frustration to patients.\u003c/p\u003e\n\u003cp\u003eThe factors influencing implantation are complex and unclear. It is reported 50% \u0026ndash; 60% may be related to local immunity of the endometrium, and abnormalities in local immunity decrease the receptivity of endometrium [2, 3]. Despite the lack of adequate evidence-based medical evidence, immunomodulators are applied clinically as the most common intervention to improve pregnancy outcomes in women with RIF[4].\u003c/p\u003e\n\u003cp\u003eHydroxychloroquine, as an immunomodulator, first applied in clinical practice as an antimalarial in 1946, after that it was routinely used to treat autoimmune diseases[5]. In addition, hydroxychloroquine has the effect of preventing venous thrombosis for patients with systemic lupus erythematosus (SLE) and antiphospholipid antibody syndrome[6, 7]. Several clinical studies have confirmed that hydroxychloroquine can be safely and effectively used in autoimmune diseases such as SLE and rheumatoid arthritis during pregnancy without significant adverse effects on placental and fetal development, so it is the first choice of immunomodulators during pregnancy and lactation [8, 9].\u003c/p\u003e\n\u003cp\u003eAnimal experiments have confirmed that in primary antiphospholipid antibody syndrome, hydroxychloroquine can significantly reduce the thrombus size after vascular injury in mice compared with oral anticoagulants alone[10]. And several studies have suggested that hydroxychloroquine has the abilities of anti-diabetic[11], \u0026nbsp;lipid-lowering[12] and endothelial protection of antioxidation[13]. In recent years, some studies indicated that hydroxychloroquine may has the potential to facilitate embryo implantation and improve IVF outcomes by effecting the change of inflammatory factor of Th1/Th2 balance[14]. Although some trials have shown that hydroxychloroquine may improve pregnancy outcomes, these trials are small or non-randomized studies or combination regimens and are insufficient to draw the conclusion[15, 16]. Evidence-based medical evidence is currently lacking, a well-designed and adequately powered randomized controlled trial (RCT) is therefore urgently needed to clarify whether hydroxychloroquine can improve pregnancy outcomes in RIF patients. For this purpose, we designed the double-blind, randomized, placebo-controlled clinical trial.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBackground and rationale: choice of comparators{6b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe investigational product in this study is hydroxychloroquine sulfate tablets. Both hydroxychloroquine and placebo were manufactured by Shanghai Shangyao Zhongxi Pharmaceutical Co., Ltd. The placebo is identical to hydroxychloroquine in all aspects including excipients, appearance, and odor, except for the absence of active ingredient. Study medications (hydroxychloroquine and placebo) were labeled according to the randomization sequence. The packaging and tablets of hydroxychloroquine and placebo are visually indistinguishable. Placebo quality, including composition and bacterial contamination, was strictly controlled in compliance with Good Manufacturing Practice (GMP) standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eObjectives {7}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis trial primarily aims to evaluate the efficacy and safety of hydroxychloroquine sulfate tablets in patients with recurrent implantation failure (RIF), assessing pregnancy outcomes and gestational complications. We hypothesize that hydroxychloroquine treatment will increase live birth and clinical pregnancy rates while reducing adverse pregnancy events in patients with recurrent implantation failure (RIF).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial design {8}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis is a prospective, randomized, double-blind, controlled clinical trail of hydroxychloroquine vs placebo (The ratio of the two is 1:1) to assess the efficacy and safety of hydroxychloroquine sulfate tablets in patients with recurrent implantation failure (RIF). This study will be conducted at five clinical hospitals. The primary outcome is live birth rate, and the secondary outcomes include biochemical pregnancy rate, clinical pregnancy rate, embryo attachment rate, first trimester abortion rate and ongoing pregnancy rate, birth weight, pregnancy and perinatal complications, congenital anomaly and other adverse events.\u003c/p\u003e"},{"header":"Methods: participants, interventions, and outcomes","content":"\u003cp\u003e\u003cstrong\u003eStudy setting {9}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis multicenter clinical trial will be conducted at five leading reproductive medical centers in China: Peking University Third Hospital, the First Affiliated Hospital of Anhui Medical University, the Second Hospital of Hebei Medical University, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, and Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology. The study will strictly adhere to the principles of the Declaration of Helsinki.\u003c/p\u003e\n\u003cp\u003eAll participating institutions are nationally recognized as top-tier reproductive medicine centers with comprehensive clinical research infrastructure, ensuring robust data collection. Eligible patients meeting the inclusion criteria outlined below will be enrolled in the study. The screening flowchart (inclusion/exclusion criteria) is presented in\u0026nbsp;\u003cstrong\u003e[\u003c/strong\u003e\u003cstrong\u003eFigure 1\u003c/strong\u003e\u003cstrong\u003e]\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEligibility criteria {10}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eI\u003c/strong\u003e\u003cstrong\u003enclusion criteria\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Infertile patients with recurrent implantation failure (RIF) and undergoing frozen-thawed embryo transfer (FET) after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), or infertile patients with RIF undergoing preimplantation genetic testing for aneuploidy (PGT-A).\u003c/p\u003e\n\u003cp\u003e2. Married women aged 18 to 40 years (Excluding cut-off).\u003c/p\u003e\n\u003cp\u003e3. Married women with 2 or more failed embryo transfer cycles.\u003c/p\u003e\n\u003cp\u003e4. Able to understand the informed consent form, voluntarily participate in this clinical trial and sign the informed consent form.\u003c/p\u003e\n\u003cp\u003e5. Able to comply with the study protocol and complete the trial.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eE\u003c/strong\u003e\u003cstrong\u003exclusion criteria\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Women currently receiving any corticosteroid or immunosuppressive therapy, such as prednisone, cyclosporine, and azathioprine, or patients receiving these medications who did not have a 2-month washout period prior to screening.\u003c/p\u003e\n\u003cp\u003e2. Women with known autoimmune diseases such as Systemic Lupus Erythematosus (SLE), antiphospholipid syndrome, Sjogren syndrome, and scleroderma.\u003c/p\u003e\n\u003cp\u003e3. Women diagnosed with diseases affecting the uterine cavity, such as uterine malformation and submucous fibroids.\u003c/p\u003e\n\u003cp\u003e4. Women or their partner with karyotypic abnormalities (excluding chromosomal polymorphisms).\u003c/p\u003e\n\u003cp\u003e5. Women with recurrent pregnancy loss, defined as two or more clinical pregnancy failures confirmed by ultrasonography or histopathology.\u003c/p\u003e\n\u003cp\u003e6. Women with medical contraindications to assisted reproductive technology and/or pregnancy.\u003c/p\u003e\n\u003cp\u003e7. Women with thin endometrium (\u0026lt; 6 mm before transfer) and is insensitive or unresponsive to hormonal drug therapy for endometrial preparation.\u003c/p\u003e\n\u003cp\u003e8. Women with known allergy to quinolines or history of relevant allergy.\u003c/p\u003e\n\u003cp\u003e9. Women who are receiving treatment with aspirin, Dalteparin Sodium Injection, Clexane and Nadroparin Calcium Injection in addition to the conventional drugs for transplantation.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWho will take informed consent? {26a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePatients meeting the diagnostic criteria for recurrent implantation failure (RIF) who are undergoing frozen embryo transfer (FET) following in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), or those with RIF indicated for preimplantation genetic testing for aneuploidy (PGT-A), will be screened according to the aforementioned criteria. After being assessed as eligible by the attending physician at the reproductive center, the investigator will obtain preliminary study materials and provide comprehensive explanations regarding the trial objectives, procedures, potential benefits, and risks to the patients. Qualified participants expressing interest will receive a copy of the informed consent form for review. The formal study will commence only after the signed informed consent is obtained.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterventions: description{11a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScreening period\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trial and study plan will be announced to all participants and eligible couples will sign the informed consent. Check the inclusion and exclusion criteria to determine the eligible participants. Screen-eligible subjects were randomized equally to two parallel treatment groups: A) hydroxychloroquine group: 0.2 g bid, orally; B) placebo group: 0.2 g bid, orally. The study stuff will dispense the first bottle (100 tablets/bottle) of study drug (hydroxychloroquine/placebo) corresponding to the drug number and instruct the patients on how to take it.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eManual cycle + thawing transplant period\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe trial medication will be taken at the same time as the initiation of a hormone replacement cycle regimen. Subjects will take 2 tablets every morning and evening.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe endometrium will be prepared with a hormone replacement cycle regimen. Estradiol valerate (Progynova, Delpharm Lille SAS, France) and/or estradiol tablets (Femoston, Abbott Biologicals B.V., the Netherlands) (2-8mg daily) will be started on days 2-5 of the menstrual cycle or 28-35 days after long-acting GnRH-agonist suppression.\u003c/p\u003e\n\u003cp\u003eWhen the endometrial thickness is sufficient (\u0026ge;\u0026nbsp;6 mm), luteal phase support will be added with oral dydrogesterone (Daphnetone, Abbott) 10-20 mg twice daily and vaginal progesterone gel (Xernox, Merck Serono) 90 mg daily.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAfter endometrial preparation, participants will undergo frozen-thawed embryo transfer (FET). Each participant will be transferred with one blastocyst or two cleaved embryos. All embryos transferred will be of good quality. 2 weeks after transfer, the serum hCG test will be performed.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePost-transplant Follow-up Period\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants will undergo serum hCG testing 12-14 days after transplant. If the participant is confirmed to be pregnant, a second bottle of trail medication with the corresponding number will be dispensed on the day of the pregnancy test (12-15 days after transplantation) and continue medication as well as luteal phase support until the end of the first trimester (12 weeks of pregnancy). Then, continue follow-up the patients until the end of pregnancy. The drug will be discontinued if the transplantation fails or the pregnancy is lost, the remaining medication will be returned to the study stuff.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eDiscontinuation criteria include but not limited to the following points:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e1. Spontaneous pregnancy.\u003c/p\u003e\n\u003cp\u003e2. Serious complications or side effects.\u003c/p\u003e\n\u003cp\u003e3. Participants using prohibited medication, such as other glucocorticoids or immunosuppressive medication.\u003c/p\u003e\n\u003cp\u003e4. Thin endometrium (\u0026lt; 6 mm before metastasis).\u003c/p\u003e\n\u003cp\u003e5. Participants who want to withdraw from the trial.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterventions: adherence\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;{11c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth hydroxychloroquine and placebo used in this trial are tablet formulations with simple administration procedures, neutral odor, and minimal reported adverse reactions, ensuring high protocol adherence. The following measures will be implemented to further enhance compliance:\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e1. Maintain open communication channels between investigators and participants to facilitate immediate reporting of medication loss.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e2. Conduct adherence education sessions for participants and their families, emphasizing therapeutic benefits and engaging family supervision.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. Provide comprehensive counseling on investigational product mechanisms, placebo effects, and adherence significance while encouraging active discussion of treatment-related concerns.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4. Instruct participants to report all adverse/unexpected drug reactions without self-discontinuation, coupled with healthcare guidance to establish trusting investigator-participant relationships.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOutcomes {12}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePrimary outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLive birth rate: Live birth after frozen-thawed embryo transfer, defined as delivery of any newborns \u0026ge; 28 weeks of gestation with vital signs. Live birth rate was calculated as number of live birth/ number of transplantation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSecondary outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e1. Biochemical pregnancy rate: biochemical pregnancy is defined as serum\u0026nbsp;\u0026beta;-hCG\u0026nbsp;\u0026ge;\u0026nbsp;30 mIU/mL at 12-15 days after embryo transfer, biochemical pregnancy rate = biochemical pregnancy cycle/transfer cycle.\u003c/p\u003e\n\u003cp\u003e2. Clinical pregnancy rate: Clinical pregnancy is defined as ultrasound detection of intrauterine gestational sac or clinical diagnosis of ectopic pregnancy at 28 ~ 30 days after embryo transfer, clinical pregnancy rate = clinical pregnancy cycle/transfer cycle.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e3. Embryo attachment rate: embryo implantation rate = number of gestational sacs with heart tube pulsation/number of embryos transferred.\u003c/p\u003e\n\u003cp\u003e4. \u0026nbsp;First trimester abortion rate and ongoing pregnancy rate: abortion refers to termination of pregnancy without reaching 28 weeks and fetal weight less than 1000 g; early abortion refers to fetal loss before 12 weeks of gestation, early abortion rate = early abortion cycle/clinical pregnancy cycle; ongoing pregnancy is a live intrauterine pregnancy continuing until 10 weeks of gestation, ongoing pregnancy rate = ongoing pregnancy cycle/transplantation cycle.\u003c/p\u003e\n\u003cp\u003e5. Birth weight.\u003c/p\u003e\n\u003cp\u003e6. \u0026nbsp;Pregnancy and perinatal complications.\u003c/p\u003e\n\u003cp\u003e7. Congenital anomaly.\u003c/p\u003e\n\u003cp\u003e8. Other Adverse Events.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eParticipant timeline {13}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study employs standardized timelines for participant enrollment, interventions, assessments, and follow-up visits to ensure operational consistency across all research sites \u003cstrong\u003e[\u003c/strong\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e\u003cstrong\u003e]\u003c/strong\u003e. For participants achieving successful post-transfer pregnancy, study medication will be maintained until 12 weeks of gestation (approximately 16 weeks), with follow-up continuing until 6 weeks postpartum (approximately 47 weeks). Participants with unsuccessful pregnancy will receive medication for approximately 4 weeks, followed by monitoring until 30 days post-transfer (approximately 8 weeks). Participants retain the right to withdraw from the study at any time without forfeiting any entitled benefits, and safety evaluations will be conducted upon withdrawal to assess participant status. All follow-up data will be collected in strict accordance with the study protocol.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSample size {14}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAccording to the meta-analysis published in 2013, the live birth rate was estimated to be 16.1% in women who failed three or more embryo transfer cycles[17]. And live birth rates range from 15.5% to 29% in women who have failed two or more embryo transfer cycles[18]. It has also reported that the combination of prednisone with low molecular weight heparin or aspirin can increase the live birth rate by 10.8 \u0026ndash; 14%[19, 20].\u003c/p\u003e\n\u003cp\u003eIn present study, we plan to test the primary hypothesis of a difference of 10% in the live birth rate between the two randomization arms. In patients with RIF, we assume that the live birth rate will be 30% in the hydroxychloroquine group and 20% in the placebo group. For the sample size calculation, the two-side significance level will be set at \u0026alpha; = 0.05 and the statistical power will be calculated as 1-\u0026beta; = 0.80. The ratio between groups is 1:1. The minimum sample size for each group was 291, with a total of 582 participants. Considering the dropout rate is 15%, we expect to a final total of 686 subjects.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAssignment of interventions (for controlled trials)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAllocation: sequence generation\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;{16a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eParticipants will be randomly allocated to two parallel groups to receive either hydroxychloroquine sulfate tablets or placebo treatment from endometrial preparation initiation through the first trimester of pregnancy (if conception occurs). All eligible participants will be randomized to one of the two study arms using a computer-generated randomization sequence prepared by the Data Coordinating Center (DCC) with SAS version 9.2 (SAS Institute Inc., Cary, NC), stratified by embryo stage (cleavage-stage or blastocyst).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlinding (masking)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;{17a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis clinical study is a double-blind trial. The randomization code list was generated by the statistical unit, with sealed blinding envelopes maintained in duplicate copies stored separately at the leading institution, the National Drug Clinical Trial Institution, and the sponsor. The entire medication coding process was documented by the blinding officer as blinding documentation, preserved as part of the clinical trial records. The randomization schedule will be maintained under strict confidentiality by Data Coordinating Center (DCC) personnel, with registration staff having no access to allocation information. Investigators will remain blinded to treatment group assignments throughout the trial.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBlinding (masking): emergency unblinding {17b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEach coded investigational product has a corresponding emergency envelope disclosing its assigned treatment group. These envelopes are securely maintained at participating sites, with unauthorized opening strictly prohibited. Emergency envelopes are distributed to clinical trial sites alongside correspondingly numbered medications and will be uniformly retrieved post-trial completion. Emergency unblinding is permitted exclusively for critical situations (e.g., serious adverse events) or when life-saving interventions require treatment knowledge. Unblinding must be authorized by the site\u0026apos;s principal investigator, accompanied by detailed documentation including reason, timestamp, location, and investigator signature. Immediate notification must be sent to the lead institution and clinical monitors following unblinding, with complete preservation of case records.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection, management, and analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection plan\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;{18a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data collection (including baseline and follow-up measures) will be conducted in accordance with ICH-GCP guidelines using a blinded management process: Certified clinical research coordinators (CRCs), who remain blinded to subject group allocation, will perform data collection after completing standardized data collection protocol (DCP-01) training and signing confidentiality agreements. Baseline data will be automatically extracted through the electronic data capture (EDC) system integrated with hospital electronic medical records (EMR), while pregnancy outcomes, complications and adverse event data will be recorded in real-time via telephone follow-up. A permission segregation mechanism is implemented for data entry and modification, where only authorized investigators and designated data managers have system operation rights, with monitors retaining only data verification privileges. Case report form (CRF) data will be transferred to the central database after entry, with all data changes recording the operator, timestamp and revision rationale.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData collection plan: retention\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;{18b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study implements two key measures to enhance participant compliance: First, flexible scheduling of follow-up visits (with a\u0026nbsp;\u0026plusmn;3-day adjustment window) to accommodate participants\u0026apos; availability and minimize missed appointments. Second, a three-stage progressive reminder system (via phone call, text message, and email) prior to each scheduled follow-up visit.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData management {19}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study implements a closed-loop data management system: After double-entry verification of CRF data, the data manager generates a comprehensive data review report including study completion status (with subject dropout list), eligibility criteria verification, completeness check, logical consistency validation, outlier detection, time window assessment, concomitant medication review, and adverse event evaluation. The sponsor\u0026apos;s monitors conduct source data verification (SDV). Throughout the study and post-completion, both investigational sites and the sponsor must maintain all study documents (CRFs, medical records, lab results, informed consent forms, drug dispensing/disposal records, safety reports, terminated participant information, etc.) in compliance with Good Clinical Practice (GCP) requirements. The sponsor must notify investigators regarding documents eligible for disposal, and no documents may be destroyed/relocated without prior written sponsor approval.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConfidentiality {27}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study assigns unique de-identified codes to each participant for data archiving, with master key access strictly limited to authorized research team members during the active study phase. Upon study completion, the master key file will be physically encrypted and stored separately by the principal investigator according to predefined data security protocols. During publication, we rigorously adhere to biomedical data sharing ethics guidelines, systematically anonymizing potentially identifiable parameters (such as rare medical histories or specific spatiotemporal events) to ensure zero risk of individual privacy disclosure.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans for collection, laboratory evaluation, and storage of biological specimens for genetic or molecular analysis in this trial/future use {33}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo biological specimens will be collected in this trial or for future use.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical methods\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical methods for primary and secondary outcomes {20a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSPSS 21.0 (SPSS Inc., Chicago, IL, USA) will be used to analyze the data. Normally distributed continuous variables will be expressed as mean \u0026plusmn; standard deviation, student\u0026apos;s test is used to test between-group differences. Continuous variables that are not normally distributed will be presented as medians and ranges, Wilcoxon rank-sum test is used for between-group differences. Categorical data are presented as frequency and percentage. Pearson\u0026apos;s chi-square test was used to assess differences in these measures, and Fisher\u0026apos;s exact test will be used if the expected frequencies was less than 5, P \u0026lt; 0.05 will be considered significant.\u003c/p\u003e\n\u003cp\u003eThe analysis will follow the intention-to-treat principle. Pearson\u0026apos;s chi-square test will be used to compare the primary outcome live birth rates between the two groups. Secondary outcomes, such as pregnancy and implantation rates, will be also analyzed by Pearson\u0026apos;s chi-square test. Per-protocol analysis will be performed based on actual participants who complete the entire trial. As a secondary analysis, we will fit a generalized linear mixed effects model with logit link to compare treatment groups for the primary outcome of live birth and binary secondary outcomes (such as pregnancy rate and implantation), adjusting for factors such as randomization strata of embryo stage and other explanatory variables. A random intercept will be included to adjust for the correlation between patients within sites.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInterim analyses {21b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not plan to conduct an interim analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods for additional analyses (e.g., subgroup analyses) {20b}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study does not plan to conduct subgroup analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods in analysis to handle protocol non-adherence and any statistical methods to handle missing data {20c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThrough the appropriate measures described previously, missing data will be minimized, with no imputation applied. Illogical data must be confirmed by clinical investigators, and unused data shall be archived and managed alongside trial data in compliance with applicable regulations.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans to give access to the full protocol, participant-level data, and statistical code {31c}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn accordance with international guidelines for clinical trial data sharing, this study implements a standardized data disclosure mechanism: Following ethics review, de-identified datasets may be disclosed upon authorization by the study steering committee after compliance review conducted jointly by the Sponsor (Reproductive Medicine Center of Peking University Third Hospital) and site principal investigators from participating centers in response to legitimate academic requests.\u003c/p\u003e\n\u003cp\u003eThe data sharing procedures execute standardized confidentiality management protocols: 1) Processing of identifiable information strictly adheres to institutional and national regulations, removing direct identifiers (including name, national ID number, etc.) and potential identifiers (e.g., disease subtypes, extreme age values among demographic characteristics); 2) A data integrity verification mechanism is implemented utilizing hash algorithms to ensure data consistency during transmission.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eOversight and monitoring\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComposition of the coordinating centre and trial steering committee {5d}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis multicenter, double-blind, prospective randomized controlled trial (pRCT) employed a protocol developed and led by the Reproductive Medicine Center of Peking University Third Hospital. Five participating hospitals collaboratively executed core operational processes: subject recruitment/screening, blinded follow-up management, standardized data collection, and intention-to-treat (ITT)-based statistical analyses.\u003c/p\u003e\n\u003cp\u003eThe project office conducted biweekly cross-site quality evaluation meetings via videoconference, employing Good Clinical Practice (GCP)-compliant auditing protocols to monitor protocol deviations while dynamically executing risk mitigation measures. Systematic review of trial progress and necessary adjustments were coordinated within this framework.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eComposition of the data monitoring committee, its role and reporting structure {21a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Data Monitoring Committee (DMC) for this study comprises one epidemiology expert from Peking University Third Hospital, one epidemiology expert from the Robinson Research Institute and Adelaide Medical School, Adelaide, South Australia, Australia, and one clinical research methodology expert from Queen Mary Hospital of The University of Hong Kong. All members remain independent from the trial design and implementation process, with no financial interests related to the sponsor. The committee primarily conducts dynamic risk assessment and data quality control through the following mechanisms. First, real-time screening of safety signals (including but not limited to pregnancy loss rate threshold alerts and spatiotemporal cluster analysis of serious adverse events), with quarterly risk-benefit evaluations based on cumulative data; Second, supervision of protocol compliance (encompassing blinded status verification and critical procedural timeline adherence reviews).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAdverse event reporting and harms {22}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn adverse event (AE) refers to any untoward medical occurrence (including clinically significant abnormal laboratory findings), symptom, or disease temporally associated with the use of the investigational product, regardless of suspected causal relationship. Pre-existing conditions identified during screening should not be recorded as AEs but documented as medical history/concomitant conditions. A serious adverse event (SAE) is defined as any adverse medical event occurring after administration of the investigational product that results in death, is life-threatening, causes persistent or significant disability/incapacity, requires or prolongs hospitalization, or leads to congenital anomalies/birth defects. All AEs, irrespective of suspected relationship to the investigational product, must be fully documented in the subject\u0026apos;s case report form (CRF) throughout the study period, including precise onset/cessation dates and times, detailed signs/symptoms, severity grading, causal relationship assessment, action taken, outcome, SAE determination, and whether the event led to study discontinuation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFrequency and plans for auditing trial conduct {23}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study will establish an independent audit team to conduct systematic audits at each participating site every six months, focusing on verification of trial documentation integrity, source data traceability, and protocol compliance. All auditing procedures must adhere strictly to Good Clinical Practice (GCP) standards using standardized operating procedures, with objectivity ensured through a conflict of interest declaration process. Audit records will be permanently archived in the trial master file (TMF) as per regulatory requirements.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePlans for communicating important protocol amendments to relevant parties (e.g. trial participants, ethical committees) {25}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAny modification to the protocol, whether constituting a substantive amendment or administrative change to any section, shall be considered a protocol amendment. During the study conduct, if protocol amendments are required, they must undergo thorough discussion between the investigator and sponsor. Following mutual agreement on the necessity of amendments, the sponsor shall revise the protocol accordingly. The amended protocol must be resubmitted to the ethics committee for review and approval. Except for administrative amendments (e.g., typographical corrections), investigators must await formal ethics committee approval prior to implementing revised protocol versions. All approved amendments shall be documented in the trial master file (TMF) with version control and effective dates clearly specified.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDissemination plans {31a}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study results will be fully disclosed in international peer-reviewed journals, reporting both positive and negative findings. Participants opting to receive study-level outcomes will be provided with lay summaries compliant with scientific communication standards.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis is a prospective, randomized, double-blind, controlled clinical trail of hydroxychloroquine vs placebo( The ratio of the two is 1:1) to assess the efficacy and safety of hydroxychloroquine sulfate tablets in patients with recurrent implantation failure(RIF).The primary outcome is live birth rate, and the secondary outcomes include biochemical pregnancy rate ,clinical pregnancy rate ,embryo attachment rate ,first trimester abortion rate and ongoing pregnancy rate ,birth weight ,pregnancy and perinatal complications ,congenital anomaly and other adverse events.\u003c/p\u003e\u003cp\u003eSo far, there is no uniform diagnostic criteria for RIF. The widely accepted definition in China is that women\u0026thinsp;\u0026lt;\u0026thinsp;40 years of age who fail to achieve a clinical pregnancy with 2 or more fresh or frozen-thawed embryo transfer cycles and a cumulative transfer of at least 3 high-quality embryos are RIF. C Coughlan et al suggested RIF to be defined as the failure after transfer of at least 4 good-quality embryos in a minimum of three fresh or frozen cycles [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Implantation failure may be caused by a number of different factors, including the maternal immune system, embryo and parental genetics, anatomical factors, hematologic factors, reproductive tract microbiome, and endocrine environment[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Of these, 50\u0026ndash;60% may be associated with local immunity of the endometrium[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Despite the lack of adequate evidence-based medical evidence in various therapeutic interventions, clinicians may attempt to \u0026ldquo;do something different\u0026rdquo; after RIF due to feel pressure.\u003c/p\u003e\u003cp\u003eImmunomodulators are applied clinically as the most common intervention to improve pregnancy outcomes in women with RIF[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In recent years, hydroxychloroquine is being used by an increasing number of IVF centers and reproductive physicians worldwide beacuse its safety and validity during pregnancy and lactation. In terms of immune regulation, hydroxychloroquine can inhibit the proliferation of T cells, reduce the production of inflammatory cytokines like INF-γ, TNF, IL-1, IL-6 and IL-2, increase IL-10 and IL-4 levels, and can also prevent the interaction of Toll-like receptors (TLR) with nucleic acid ligands[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. In addition, hydroxychloroquine has an anti-infective effect[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], which can alkalinize acidic vesicles from cells and inhibit the growth of microorganisms[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. So it may play a role in chronic endometritis, which is another possible mechanism leading to repeated implantation failure.\u003c/p\u003e\u003cp\u003eSome studies exist on the use of hydroxychloroquine in patients with RIF. Sonia Sadeghpour et al found that HCQ administration in RIF women with immune cell disease during pregnancy affected Th17/Treg ratio, and increased Treg and reduced Th17 responses, which may be associated with successful outcome of pregnancy[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In addition, hydroxychloroquine use in RIF patients with a high Th1/Th2 ratio during the implantation window could reduce this ratio and via a shift in Th2 responses[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. These results suggest that hydroxychloroquine can change endometrial receptivity by regulating cellular immunity. At the same time, hydroxychloroquine has an anti-infective effect[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and can alkalinize acidic vesicles in cells and inhibit the growth of microorganisms[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], i.e. It may play a role in chronic endometritis, which is another possible mechanism leading to recurrent implantation failure. However, clinical trials to assess the efficacy of hydroxychloroquine in patients with RIF are limited, and although evidence-based medical evidence for the use of hydroxychloroquine in patients with RIF is lacking, hydroxychloroquine is being used by an increasing number of in vitro fertilization centers and reproductive physicians worldwide. Therefore, we designed this randomized controlled trial (RCT) to clarify whether hydroxychloroquine could improve pregnancy outcomes in patients with RIF.\u003c/p\u003e\u003cp\u003eOur hypothesis is that hydroxychloroquine will improve pregnancy outcomes and endometrial receptivity in RIF patients undergoing fresh embryo transfer, providing direct evidence for clinical treatment, and if this hypothesis is confirmed, a larger multicenter RCT will be conducted in the future to further investigate the optimal dose and duration of hydroxychloroquine to improve pregnancy outcomes in RIF patients and provide more solid evidence for clinical treatment.\u003c/p\u003e\u003cdiv id=\"Sec39\" class=\"Section2\"\u003e\u003ch2\u003eStrengths and Limitations\u003c/h2\u003e\u003cp\u003eOur study has several strengths and our randomized controlled trial design enables a more direct description of the treatment effect of hydroxychloroquine in patients with recurrent implantation failure and provides a more robust causal relationship. In addition, this study is a multicenter clinical trial, which is more representative and authoritative and can provide feasible methods for the clinical treatment of patients with recurrent implantation failure and provide improved data for guidelines. Finally, our experiments are designed and participated by authoritative statistical experts, obstetricians and gynecologists, scientists, etc., ensuring the quality of the study. The limitation of this study refers to its RCT nature, which the inclusion and exclusion criteria of studies will lead to certain limitations on the representativeness and external authenticity of research results.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRIF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRecurrent Implantation Failure\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRCT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRandomized Controlled Trial\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIVF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIn Vitro Fertilization\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eICSI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIntracytoplasmic Sperm Injection\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHCQ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHydroxychloroquine\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIVF-ET\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIn Vitro Fertilization and Embryo Transfer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSLE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSystemic Lupus Erythematosus\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGMP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGood Manufacturing Practice\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePGT-A\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePreimplantation Genetic Testing for Aneuploidy\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ehCG\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHuman Chorionic Gonadotropin\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDCC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eData Coordinating Center\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCRCs\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCertified Clinical Research Coordinators\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEDC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eElectronic Data Capture\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eEMR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eElectronic Medical Records\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSDV\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSource Data Verification\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eITT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIntention-To-Treat\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eICH-GCP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInternational Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use - Good Clinical Practice\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGCP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGood Clinical Practice\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eIDMC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eIndependent Data Monitoring Committee\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePPI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePatient and Public Involvement\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSAP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eStatistical Analysis Plan\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eAE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAdverse Event\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCRF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCase Report Form\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTMF\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTrial Master File\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTLR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eToll-Like Receptors\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eINF-γ\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eInterferon Gamma\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cdiv id=\"Sec40\" class=\"Section3\"\u003e\u003ch2\u003eTrial status\u003c/h2\u003e\u003cp\u003eResearch Protocol Version 1.5. Recruitment began on November 16, 2021. As of June 24, 2025, 394 participants have been enrolled. Recruitment is currently ongoing and is expected to be completed by December 2027.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\n\u003ch3\u003eAvailability of data and materials {29}\u003c/h3\u003e\n\u003cp\u003eThe datasets utilized and/or analyzed in this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all the patients who participated in this project and SPH Zhongxi Pharmaceutical Co., Ltd for their support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBoth the applicant and the main implementer have the National Natural Science Foundation of China (Fund No. 82271863), and fund from National Clinical Research Center for Obstetrics and Gynecology (Fund No. BYSYSZKF2022008).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors and Affiliations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCenter for Reproductive Medicine, Department of Obstetrics and Gynecology, Third Hospital, Peking University Third Hospital, Beijing, China\u003c/p\u003e\n\u003cp\u003eDong-Lin Han, Li Li, Yi-Meng Ge, Jie Zhao and Rong Li.\u003c/p\u003e\n\u003cp\u003eNational Clinical Research Center for Obstetrics and Gynecology, Beijing, China\u003c/p\u003e\n\u003cp\u003eLi Li, Yi-Meng Ge, Jie Zhao and Rong Li.\u003c/p\u003e\n\u003cp\u003eKey Laboratory of Assisted Reproduction (Peking University), Ministry of Education, Beijing, China\u003c/p\u003e\n\u003cp\u003eDong-Lin Han, Li Li, Jie Zhao and Rong Li.\u003c/p\u003e\n\u003cp\u003eBeijing Key Laboratory of Reproductive Endocrinology and Assisted Reproductive Technology, Beijing, China\u003c/p\u003e\n\u003cp\u003eLi Li, Jie Zhao and Rong Li.\u003c/p\u003e\n\u003cp\u003eState Key Laboratory of Female Fertility Promotion, Department of Obstetrics and Gynecology, Peking University, Third Hospital, Beijing, China\u003c/p\u003e\n\u003cp\u003eLi Li, Jie Zhao and Rong Li.\u003c/p\u003e\n\u003cp\u003eDepartment of Pharmacy, Peking University Third Hospital, Beijing, China\u003c/p\u003e\n\u003cp\u003eJing Shi\u003c/p\u003e\n\u003cp\u003eReproductive Medicine Center, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China\u003c/p\u003e\n\u003cp\u003eShu-Lin Yang and Yi-Fan Chu.\u003c/p\u003e\n\u003cp\u003eReproductive Medicine Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, People\u0026apos;s Republic of China\u003c/p\u003e\n\u003cp\u003eZhuo-Yao Mai and Hui Chen.\u003c/p\u003e\n\u003cp\u003eDepartment of Reproductive Medicine, The Second Hospital of Hebei Medical University\u003c/p\u003e\n\u003cp\u003eYi-Wen Zhang and Gui-Min He.\u003c/p\u003e\n\u003cp\u003eReproductive Medicine Center, Tongji Hospital, Tongji Medicine College, Huazhong University of Science and Technology, Wuhan, People\u0026apos;s Republic of China.\u003c/p\u003e\n\u003cp\u003eJing Yue\u003c/p\u003e\n\u003cp\u003eReproductive Medicine Center, Department of Obstetrics and Gynecology, the First Affiliated Hospital of Anhui Medical University, No 218 Jixi Road, Hefei 230022, Anhui, China\u003c/p\u003e\n\u003cp\u003eHui-Fen Xiang\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eContributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDong-Lin Han and Li Li contributed equally to this work. Dong-Lin Han: Manuscript writing, methodology, conception. Li Li: Write the manuscript, supervise and review it. Jie Zhao: Concepts, methods, writing review and editing. Rong Li: Resources, concepts, methods, project management and capital acquisition. Jing Yue, Jing Shi and Yi-Meng Ge provide expertise for data collection and analysis. Jing Shi, Shu-Lin Yang, Yi-Fan Chu, Zhuo-Yao Mai, Yi-Wen Zhang, Hui Chen, Gui-Min He and Hui-Fen Xiang provide resources for project data collection. All authors contributed to the article and approved the submitted version.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding author\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCorrespondence to\u0026nbsp;Rong Li and Jie Zhao.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics declarations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate {24}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis project was approved by the Ethics Committee of Peking University Third Hospital [Acceptance No.: M2021169 Project No.: LM2021267].\u0026nbsp;All participants must provide written informed consent prior to study participation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication {32}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests {28}\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMak JSM, Chung CHS, Chung JPW, Kong GWS, Saravelos SH, Cheung LP, Li TC. 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Biomed Pharmacother. 2016;84:1449\u0026ndash;59.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePlantone D, Koudriavtseva T. Current and Future Use of Chloroquine and Hydroxychloroquine in Infectious, Immune, Neoplastic, and Neurological Diseases: A Mini-Review. Clin Drug Investig. 2018;38:653\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eTektonidou MG, Laskari K, Panagiotakos DB, Moutsopoulos HM. Risk factors for thrombosis and primary thrombosis prevention in patients with systemic lupus erythematosus with or without antiphospholipid antibodies. Arthritis Rheum. 2009;61:29\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRuiz-Irastorza G, Egurbide MV, Pijoan JI, Garmendia M, Villar I, Martinez-Berriotxoa A, Erdozain JG, Aguirre C. Effect of antimalarials on thrombosis and survival in patients with systemic lupus erythematosus. Lupus. 2006;15:577\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eAhmadi M, Ghaebi M, Abdolmohammadi-Vahid S, Abbaspour-Aghdam S, Hamdi K, Abdollahi-Fard S, Danaii S, Mosapour P, Koushaeian L, Dolati S, et al. NK cell frequency and cytotoxicity in correlation to pregnancy outcome and response to IVIG therapy among women with recurrent pregnancy loss. J Cell Physiol. 2019;234:9428\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eHviid MM, Macklon N. Immune modulation treatments-where is the evidence? Fertil Steril. 2017;107:1284\u0026ndash;93.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEdwards MH, Pierangeli S, Liu X, Barker JH, Anderson G, Harris EN. Hydroxychloroquine reverses thrombogenic properties of antiphospholipid antibodies in mice. Circulation. 1997;96:4380\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGerstein HC, Thorpe KE, Taylor DW, Haynes RB. The effectiveness of hydroxychloroquine in patients with type 2 diabetes mellitus who are refractory to sulfonylureas\u0026ndash;a randomized trial. Diabetes Res Clin Pract. 2002;55:209\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePetri M, Lakatta C, Magder L, Goldman D. Effect of prednisone and hydroxychloroquine on coronary artery disease risk factors in systemic lupus erythematosus: a longitudinal data analysis. Am J Med. 1994;96:254\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eVirdis A, Tani C, Duranti E, Vagnani S, Carli L, K\u0026uuml;hl AA, Solini A, Baldini C, Talarico R, Bombardieri S, Taddei S, Mosca M. Early treatment with hydroxychloroquine prevents the development of endothelial dysfunction in a murine model of systemic lupus erythematosus. Arthritis Res Ther. 2015;17:277.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eGhasemnejad-Berenji H, Ghaffari Novin M, Hajshafiha M, Nazarian H, Hashemi SM, Ilkhanizadeh B, Ghasemnejad T, Sadeghpour S, Ghasemnejad-Berenji M. Immunomodulatory effects of hydroxychloroquine on Th1/Th2 balance in women with repeated implantation failure. Biomed Pharmacother. 2018;107:1277\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePasquier E, de Saint-Martin L, Marhic G, Chauleur C, Bohec C, Bretelle F, Lejeune-Saada V, Hannigsberg J, Plu-Bureau G, Cogulet V, Merviel P, Mottier D. Hydroxychloroquine for prevention of recurrent miscarriage: study protocol for a multicentre randomised placebo-controlled trial BBQ study. BMJ Open. 2019;9:e025649.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ede Moreuil C, Alavi Z, Pasquier E. Hydroxychloroquine may be beneficial in preeclampsia and recurrent miscarriage. Br J Clin Pharmacol. 2020;86:39\u0026ndash;49.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003ePotdar N, Gelbaya TA, Konje JC, Nardo LG. Adjunct low-molecular-weight heparin to improve live birth rate after recurrent implantation failure: a systematic review and meta-analysis. Hum Reprod Update. 2013;19:674\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eEl-Toukhy T, Campo R, Khalaf Y, Tabanelli C, Gianaroli L, Gordts SS, Gordts S, Mestdagh G, Mardesic T, Voboril J, et al. Hysteroscopy in recurrent in-vitro fertilisation failure (TROPHY): a multicentre, randomised controlled trial. Lancet. 2016;387:2614\u0026ndash;21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFawzy M, El-Refaeey AA. Does combined prednisolone and low molecular weight heparin have a role in unexplained implantation failure? Arch Gynecol Obstet. 2014;289:677\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSiristatidis C, Dafopoulos K, El-Khayat W, Salamalekis G, Anifandis G, Vrantza T, Elsadek M, Papantoniou N. Administration of prednisolone and low molecular weight heparin in patients with repeated implantation failures: a cohort study. Gynecol Endocrinol. 2018;34:136\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCoughlan C, Ledger W, Wang Q, Liu F, Demirol A, Gurgan T, Cutting R, Ong K, Sallam H, Li TC. Recurrent implantation failure: definition and management. Reprod Biomed Online. 2014;28:14\u0026ndash;38.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eFranasiak JM, Alecsandru D, Forman EJ, Gemmell LC, Goldberg JM, Llarena N, Margolis C, Laven J, Schoenmakers S, Seli E. A review of the pathophysiology of recurrent implantation failure. Fertil Steril. 2021;116:1436\u0026ndash;48.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRobertson SA, Jin M, Yu D, Moldenhauer LM, Davies MJ, Hull ML, Norman RJ. Corticosteroid therapy in assisted reproduction - immune suppression is a faulty premise. Hum Reprod. 2016;31:2164\u0026ndash;73.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRichard SA, Kampo S, Hechavarria ME, Sackey M, Buunaaim ADB, Kuugbee ED, Anabah TW. Elucidating the Pivotal Immunomodulatory and Anti-Inflammatory Potentials of Chloroquine and Hydroxychloroquine. J Immunol Res. 2020;2020:4582612.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eChandler LC, Yusuf IH, McClements ME, Barnard AR, MacLaren RE, Xue K. Immunomodulatory Effects of Hydroxychloroquine and Chloroquine in Viral Infections and Their Potential Application in Retinal Gene Therapy. Int J Mol Sci 2020, 21.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eSadeghpour S, Ghasemnejad Berenji M, Nazarian H, Ghasemnejad T, Nematollahi MH, Abroon S, Paktinat S, Heidari Khoei H, Ghasemnejad Berenji H, Ghaffari Novin M. Effects of treatment with hydroxychloroquine on the modulation of Th17/Treg ratio and pregnancy outcomes in women with recurrent implantation failure: clinical trial. Immunopharmacol Immunotoxicol. 2020;42:632\u0026ndash;42.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"trials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trls","sideBox":"Learn more about [Trials](http://trialsjournal.biomedcentral.com/)","snPcode":"13063","submissionUrl":"https://www.editorialmanager.com/trls","title":"Trials","twitterHandle":"MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hydroxychloroquine, RIF, IVF/ICSI, Endometrial receptivity, Reproductive immunity","lastPublishedDoi":"10.21203/rs.3.rs-7141881/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7141881/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRecurrent implant failure (RIF) remains clinically unresolved at this stage. Hydroxychloroquine, as an immunomodulator, is still lacking clinical evidence, but it is being used by increasing numbers of reproductive centers and physicians worldwide, so a well-designed randomized controlled trial (RCT) is urgently needed to elucidate whether hydroxychloroquine can improve pregnancy outcomes in patients with RIF.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods and Analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn this study, we plan to recruit 686 volunteers who will undergo IVF/ICSI at 5 reproductive centers from 6 December 2022. Participants will be randomized to two parallel groups and treated with hydroxychloroquine sulfate tablets or placebo from the start of endometrial preparation to 14 days after frozen embryo transfer (if not pregnant) or to 12 weeks of pregnancy (if pregnant).The primary outcome is live birth rate, and the secondary outcomes include biochemical pregnancy rate ,clinical pregnancy rate, embryo attachment rate ,first trimester abortion rate and ongoing pregnancy rate ,birth weight ,pregnancy and perinatal complications ,congenital anomaly and other adverse events.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDiscussion\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study aims to evaluate whether hydroxychloroquine (HCQ) improves pregnancy outcomes in patients with recurrent implantation failure (RIF). Secondary objectives include comparative analysis of gestational complications between the intervention and control groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTrial registration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eChiCTR2100047584 [Chinese Clinical Trial Registry (ChiCTR): registered on 20 June 2021]\u003c/p\u003e\n\u003cp\u003eLM2021267 [Ethics Committee of Peking University Third Hospital]\u003c/p\u003e","manuscriptTitle":"Hydroxychloroquine Sulfate Tablets for Patients with Recurrent Implantation Failure: A Double-Blind, randomized, placebo-controlled trial","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-15 16:14:41","doi":"10.21203/rs.3.rs-7141881/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Accept","date":"2026-01-14T14:42:04+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-10-02T15:13:34+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-02T13:55:35+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-17T09:09:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"Trials","date":"2025-08-13T11:37:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"trials","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"trls","sideBox":"Learn more about [Trials](http://trialsjournal.biomedcentral.com/)","snPcode":"13063","submissionUrl":"https://www.editorialmanager.com/trls","title":"Trials","twitterHandle":"MedicalEvidence","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"41d99f80-837d-4a67-9257-49126fdba604","owner":[],"postedDate":"October 15th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-02T16:01:27+00:00","versionOfRecord":{"articleIdentity":"rs-7141881","link":"https://doi.org/10.1186/s13063-026-09470-4","journal":{"identity":"trials","isVorOnly":false,"title":"Trials"},"publishedOn":"2026-01-30 15:58:22","publishedOnDateReadable":"January 30th, 2026"},"versionCreatedAt":"2025-10-15 16:14:41","video":"","vorDoi":"10.1186/s13063-026-09470-4","vorDoiUrl":"https://doi.org/10.1186/s13063-026-09470-4","workflowStages":[]},"version":"v1","identity":"rs-7141881","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7141881","identity":"rs-7141881","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}