Introduction Pregnancy loss occurs in 25% of all pregnancies. While 50-60% of the pregnancy losses are due to fetal chromosomal and structural abnormalities, incompatible with life, some of the remaining euploid pregnancy losses are likely caused by maternal conditions. At least 2% of all women suffer ≥3 consecutive pregnancy losses, defined as recurrent pregnancy loss (RPL). A hyperactive maternal immune system has been suspected to be responsible for rejection of healthy fetuses and be the underlying cause for unexplained recurrent pregnancy loss. However, very sparsely evidence-based treatment is available. The anti-inflammatory immune modulatory effects of hydroxychloroquine (HCQ) and casuistic reports of live births by women with previous RPL after HCQ-treatment, have given rise to several hypothesizes for HCQs impact on RPL. In this multicenter randomized, double-blinded, placebo-controlled trial (db-RCT) we aim to uncover whether HCQ can increase the live birth rate (LBR) in women with RPL, thus laying the foundation for an evidence-based treatment to make a real difference for a much-overlooked group of patients. Methods and analysis We will assess HCQ treatment in women with a minimum of four consecutive unexplained pregnancy losses (or three pregnancy losses including a second trimester pregnancy loss), and monitor a potential difference in LBR among women allocated treatment with HCQ and placebo, without exclusion of any included patients (Intention to treat analysis) and with exclusion of those with ectopic pregnancy, pregnancy loss due to chromosome abnormalities, neglect of treatment, induced abortion or those who withdraw from the treatment earlier than the protocol dictates (per protocol analysis). Secondary outcome measures include comparison of the rate of perinatal complications in the intervention group to the placebo group and examination of inflammatory and immunological mechanisms in peripheral blood. We aim to include 186 patients in the db-RCT. In addition, we will in a sub-study with 10 women monitor metabolic activity measured by ultra-low dose FDG-PET/CT scans before and during pregnancy. Metabolic activity correlates with grade of inflammation and will be measured in the uterus and immune-related tissue such as bone marrow, spleen, thymus, and lymph nodes. Ethics and Dissemination The db-RCT including the PET/CT sub-study has been approved by The Regional Committee on Health Research Ethics, The Danish Medicines Agency, The Danish Data Protection Agency, and transferred to The Center for Data handling Capital Region of Denmark in May 2024. The trial conforms to good clinical practice guidelines (GCP) and has been registered at ClinicalTrials.gov (ClinicalTrials.gov ID NCT03305263). Findings will be disseminated through peer-reviewed journals and at professional conferences. Strengths and limitations of this study - Assessment of HCQ treatment in women with RPL to potentially increase LBR. - Assessment of the rate of perinatal outcomes associability with HCQ treatment. - RPL trials so far have struggled to obtain pregnancy tissue from PL and if obtained been severely contaminated with maternal tissue. We will use circulating cell-free fetal DNA (cffDNA), isolated from maternal peripheral blood for fetal genetic diagnostics as we demonstrated feasible in our recent Lancet publication. - Non-invasive monitoring of inflammation in the uterus before and during pregnancy by the means of ultra-low dose PET/CT scans.

The authors have declared no competing interest.

NCT03305263

The trial is supported by a grant of 491.500 DKK by the Regional Medicine Foundation in Denmark. The patients are covered by patient insurance

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The Scientific Ethics Committees for the Capital Region of Denmark gave ethical approval for this work (H-17001899)

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