STUDY QUESTIONS Embryo‒maternal crosstalk involves intense and complex molecular exchange between the early embryo and the mother. This interaction begins after fertilization but is poorly understood before implantation. EARLY-PREG is a preconception open cohort that aims to research the proteome signature of maternal–embryonic communication interrogating a growing biorepository of maternal fluids and tissues collected during the first two weeks of a natural conception.

WHAT IS KNOWN ALREADY To understand the mother-embryo communication in humans, in vivo, ex vivo, and in vitro biological models have been developed to simulate certain phases of the implantation process and its related events. Mass spectrometry for proteomic profiling in longitudinal cohort studies has emerged as a modern method for understanding complex biological processes directly in vivo. Proteomic profiles have been investigated during pregnancy in women from the 8th week of gestation, but not before.

STUDY DESIGN, SIZE, DURATION In three waves of recruitment from 2017 to 2024 so far, healthy women seeking to conceive have been recruited in Concepcion, Chile. Participants completed a survey with health, lifestyle, and sociodemographic data and their menstrual cycles with ovulation and fertile window ascertainment (ultrasound, fertility monitor, and/or LH strips) were followed prospectively until pregnancy was achieved or for a maximum of six consecutive cycles. The follow-up of every cycle includes systematic day-by-day sampling of cervicovaginal fluid (CVF), urine, saliva, and blood. In addition, a cervicovaginal brushing between days 12th and 14th post-ovulation is collected. The day of ovulation and other time windows of interest in each cycle is retrospectively corrected by hormonal curves (LH, oestradiol, progesterone and beta-hCG) on stored urine samples. Blood samples and cervical brushings are collected on day 21 post-ovulation and in each trimester of pregnancy.

PARTICIPANTS, MATERIALS, SETTING, METHODS At the present, 1,183 women has been contacted, of whom 223 met all eligibility requirements; 129 participants who were trying to conceive completed the protocol for at least one complete cycle, of whom 35 participants achieved full-term pregnancies and 20 had early pregnancy losses; 40 abstinent and 5 sterilized women entered and completed the protocol. A total of 292 menstrual cycles have been fully documented and sampled; 238 cycles have been classified as non-conception cycles, and 54 as conception cycles. The biorepository comprehends maternal fluids and tissues throughout the first 2 weeks after ovulation and during early pregnancy for all 292 cycles so far. Biospecimen collection compliance has been high. The study is currently analysing the proteome of CVF samples from women who conceived and their counterfactual non-conception cycles to characterize the proteome signature of early pregnancy in this maternal fluid. A fourth recruitment wave to characterize changes of the immunophenotype in maternal peripheral blood mononuclear cells (PBMC) during ultra-early pregnancy is planned to begin during 2025.

STUDY FUNDING/COMPETING INTEREST The EARLY-PREG preconception open cohort has been supported by multiple research grants awarded by the FISAR Foundation (www.fisachile.org). The pilot study and the first wave of recruitment was supported by grants #MEL109112011 and #MEL109112011R4 awarded to E.S.K, C.V. and J.F.S. The second wave of recruitment was supported by supplemental 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 design of the fourth recruitment wave and mass spectrometry research on maternal CVF is supported by grant No. REH042024-01 awarded to M.H., G.N., and E.S.K. As a senior scientist, E.S.K. has served as an honorary research consultant and/or reviewer on research applications for the FISAR Foundation since 2015. No other conflicts of interest are reported.

TRIAL REGISTRATION NUMBER Not applicable.

TRIAL REGISTRATION DATE Not applicable.

DATE OF FIRST PATIENT’S ENROLMENT Not applicable.

WHAT DOES THIS MEAN FOR PATIENTS?Previous studies have shown that a successful pregnancy relies on an exchange of signals between the embryo and the mother. This complex communication begins just after the sperm meets the egg—before the embryo attaches to the uterus—during a stage called the preimplantation period. Although it plays a critical role in preparing the maternal body for pregnancy, the mechanisms behind this communication are still not fully understood.

The unique “dialogue” between the embryo and the mother is a two-way exchange known as embryo–maternal crosstalk. Research has suggested that this interaction, mediated by molecules in the uterine fluid, helps the embryo implant properly and modulates the maternal immune system to ensure acceptance of the embryo.

To date, most of what we know about this process comes from animal studies and laboratory experiments, which may not accurately reflect what happens in spontaneous human pregnancies. To better understand this embryo–maternal crosstalk, we designed the EARLY-PREG preconception open cohort. This study follows healthy women who are trying to become pregnant, following them from before conception through the first two weeks of a natural conception and continuing into pregnancy and childbirth. It includes the daily collection of maternal body fluid samples—such as saliva, urine, blood, and cervicovaginal fluid—during key phases of the menstrual cycle. The samples are processed, preserved and stored in a biorepository for research with omic techniques.

Using advanced techniques to study proteins through mass spectrometry in these fluids, EARLY-PREG aims to explain how pregnancy begins and why some pregnancies succeed while others do not. This knowledge may reveal key biological clues to improve fertility care, assisted reproduction and support early pregnancy health, and deepen our understanding of how life begins.

As a senior scientist, E.S.K has served as an honorary research advisor and/or reviewer on research applications for the FISAR Foundation since 2015. No other conflicts of interest are reported.

The EARLY-PREG preconception open cohort has been supported by multiple research grants awarded by the FISAR Foundation (www.fisarchile.org). The pilot study and the first wave of recruitment was supported by grants #MEL109112011 and #MEL109112011R4 awarded to E.S.K, C.V. and J.F.S. The second wave of recruitment was supported by supplemental 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 design of the fourth recruitment wave and mass spectrometry research on maternal CVF is supported by grant No. REH042024-01 awarded to M.H, G.N., and E.S.K.

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The ethics committee of the Servicio de Salud Concepcion, Biobio region, Chile gave ethical approval for this work (CODE: 17-03-06).

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