EARLY-PREG: Preconception, longitudinal, bidirectional, and counterfactual open cohort of women trying to conceive for the characterization of maternal-embryonic molecular crosstalk during the first weeks after conception.

BACKGROUNDMaternal-embryonic signalling involves intense and complex molecular exchange between the maternal reproductive system and the early embryo. This interaction begins after fertilisation but is poorly understood before implantation. EARLY-PREG is structured as a bidirectional prospective-retrospective preconception open cohort designed to characterise time-dependent molecular signatures of maternal-embryonic communication during the earliest stage of pregnancy. The cohort supports a dynamic and structured longitudinal biobank of maternal biological fluids, cells and tissues-derived samples collected during the peri-implantation window. STUDY DESIGNParticipants were recruited in three rolling waves from 2017 to 2024 so far, in Concepcion, Chile. Healthy women trying to conceive were enrolled and completed a survey with health, lifestyle, and sociodemographic data. Menstrual cycles were prospectively monitored with ovulation and fertile window ascertainment (ultrasound, fertility monitor, and/or LH strips) and followed for a maximum of six consecutive cycles or until implantation of a viable embryo exposure occurred. Each cycle was synchronised within a longitudinal repeated-measures design incorporating systematic day-by-day sampling anchored to ovulation (day 0). Biological samples include cervicovaginal fluid (CVF), urine, saliva, and blood. In addition, cervical brushings were collected during the peri-implantation window (days 12-14 post-ovulation) and on day 21 post-ovulation. Retrospective temporal alignment of ovulation and defined physiological windows was performed using hormonal curves (LH, oestradiol, progesterone and beta-hCG) measured in stored blood and urine samples. RESULTS TO DATEAt present, 1,183 women have been contacted, of whom 223 met all eligibility criteria. Among participants trying to conceive, 129 completed at least one full longitudinal repeated-measures cycle; 35 achieved full-term pregnancies and 17 experienced early pregnancy loss (ELP). In addition, 40 abstinent and 5 sterilised women completed the protocol. To date, 292 menstrual cycles have been fully documented and sampled. 52 cycles correspond to conception cycles and 240 cycles to the absence of embryo implantation. Among the latter, 31 cycles were classified as non-conception counterfactual cycles and 209 to non-counterfactual cycles. The biorepository encompasses maternal biological samples collected during the first 2 weeks after ovulation across all documented cycles. Biospecimen collection compliance exceeded predefined protocol thresholds for most sample types. CURRENT AND FUTURE DIRECTIONSThe cohort currently supports longitudinal proteomic analyses within a within-individual counterfactual framework, comparing the exposure to a viable embryo implantation (conception cycle) with the absence of embryo implantation (non-conception cycle) to characterise time-dependent molecular signatures of early pregnancy. Additional outcomes of the cohort include implantation failure and early pregnancy loss. A fourth rolling recruitment wave is planned for 2026 to characterise time-dependent immunophenotypic variation in maternal peripheral blood mononuclear cells (PBMCs) during early pregnancy stages. STUDY FUNDINGThis work forms part of the EARLY-PREG preconception open cohort and has been supported by research grants awarded by Fundacion de Investigacion San Ramon (FISAR), Chile. The pilot study and first recruitment wave were supported by grants #MEL109112011 and #MEL109112011R4 awarded to E.S.K., C.V., and J.F.S. The second recruitment wave was supported by grants #MEL109112011R5 and #MEL131032017R1 awarded to E.S.K. The third wave was supported by grant #MEL205062018 awarded to E.S.K. and M.H. Current funding for the fourth recruitment wave and mass spectrometry research on maternal CVF is supported by grant REH042024-01 awarded to M.H., G.N., and E.S.K. TRIAL REGISTRATION NUMBERNCT07358026.