EARLY-PREG enables day-by-day tracking of thousands of proteins in the female reproductive tract

In a pioneering study of cervicovaginal fluid samples from the EARLY-PREG biobank, researchers at MELISA Institute validated a proteomics workflow that quantified about 50% more proteins than the comparison workflow. By collecting samples from each woman on consecutive days, researchers can track how protein levels change from one day to the next. The method opens up new ways to study fertility, the earliest stages of pregnancy and biomarkers of clinical interest in the EARLY-PREG preconception cohort.

A study by MELISA Institute, published in Scientific Reports, a Nature Portfolio journal, shows how researchers can track thousands of proteins in cervicovaginal fluid over seven consecutive days. By analyzing samples collected daily from the same women, the team could compare how well two workflows detected changes in protein abundance.

In a pioneering study of cervicovaginal fluid samples from the EARLY-PREG biobank, researchers at MELISA Institute validated a proteomics workflow that quantified about 50% more proteins than the comparison workflow. By collecting samples from each woman on consecutive days, researchers can track how protein levels change from one day to the next. The method opens up new ways to study fertility, the earliest stages of pregnancy and biomarkers of clinical interest in the EARLY-PREG preconception cohort.

EARLY-PREG is a research platform built around a longitudinal preconception cohort. Women enroll before pregnancy, and the biobank records when each specimen was collected in relation to the participant’s menstrual cycle. Researchers can therefore place each molecular measurement in its biological context.

The team uses the urinary luteinizing hormone (LH) surge as a reference point for ovulation. This allows researchers to follow changes within each woman and compare measurements across women at equivalent points in their cycles.

A molecular view of the menstrual cycle

Cervicovaginal fluid can be collected with a minimally invasive procedure. It contains proteins involved in a range of reproductive processes. Measuring its proteome—the set of proteins present in the fluid—reveals how their abundance changes over time.

For the methodological validation, the team selected 42 biobank samples from six women, all collected during cycles without conception. Each woman contributed one sample per day, from the day of the urinary LH surge (d0) through the sixth day afterward (d6). The researchers compared both workflows using the same samples. They used a nanoElute liquid chromatography system coupled to a timsTOF Pro mass spectrometer, both from Bruker Daltonics.

The dia-PASEF workflow quantified 4,229 proteins, compared with 2,817 for dda-FracOffline—about 50% more. Measurements were more consistent with dia-PASEF, which also detected more proteins whose abundance differed from the reference day.

“When we compare samples from the same woman on two different days, we need to know whether a protein’s abundance changed or whether the difference came from the measurement itself. With dia-PASEF, our measurements were more consistent, and we detected more proteins whose levels changed from day to day,” explains Mauricio Hernández, Head of Laboratory at MELISA Institute.

Under the study conditions, instrument acquisition took approximately one hour per sample with dia-PASEF, compared with 16 hours with dda-FracOffline. Shorter acquisition times could make larger longitudinal studies more feasible when each participant contributes multiple samples.

A platform for studying fertility and the earliest stages of pregnancy

One goal of EARLY-PREG is to study communication between mother and embryo around the time of implantation. This validation lays the groundwork for future studies comparing changes in protein abundance in cycles with and without conception.

“At EARLY-PREG, each sample comes with a crucial piece of information: when in the cycle it was collected. We follow women from before conception because we want to investigate a question in vivo that would otherwise be difficult to study: How do mother and embryo communicate in the days around implantation?” says Dr. Elard Koch, Chairman of MELISA Institute.

The team also tracked daily changes in CA-125 and HE4, proteins previously studied as biomarkers of ovarian and endometrial cancer. Future studies in clinical populations could examine whether changes in these proteins over time are informative for diagnosis or for monitoring disease progression and response to treatment.

This study validated a measurement workflow. The biological findings are exploratory and require confirmation in larger cohorts. Clinical applications would require separate studies of diagnostic performance and clinical utility.

Based in Chile’s Biobío Region, MELISA Institute brings together preconception follow-up, a longitudinal biobank and molecular analysis to study women’s reproductive health.

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Article: Hernandez M. et al. A dia-PASEF workflow improves proteome coverage and quantitative performance in repeated-measures analysis of cervicovaginal fluid from a preconception cohort (EARLY-PREG). Scientific Reports (2026). DOI: 10.1038/s41598-026-75386-3.
EARLY-PREG registration: ClinicalTrials.gov, NCT07358026. Proteomics data: ProteomeXchange/PRIDE, PXD071350.
Funding: Fundación de Investigación San Ramón (FISAR). Support for the proteomics research includes grant REH042024-01.
Scientific contact: Dr. Elard Koch · ekoch@melisainstitute.org