New Proteomics Workflow Tracks Daily Reproductive Protein Changes

Researchers at the MELISA Institute have validated a proteomics workflow that enables the day-by-day tracking of thousands of proteins in the female reproductive tract. Published in Scientific Reports, a Nature Portfolio journal, the study demonstrates how a specific mass spectrometry technique can quantify protein abundance in cervicovaginal fluid with high precision. By analyzing samples collected daily from the same women, the team compared two distinct workflows to determine which method best detects changes in protein levels over time. This methodological advance provides a robust foundation for future investigations into fertility, the earliest stages of pregnancy, and potential biomarkers for gynecological diseases.

The study utilized samples from the EARLY-PREG biobank, a research platform built around a longitudinal preconception cohort. Women enroll in the study before pregnancy, and the biobank meticulously records when each specimen was collected in relation to the participant’s menstrual cycle. This temporal context is crucial for interpreting molecular data, as it allows researchers to place each measurement within its specific biological environment. The use of a urinary luteinizing hormone surge as a reference point for ovulation further enhances the ability to follow changes within each woman and compare measurements across participants at equivalent points in their cycles.

Cervicovaginal fluid is a rich source of biological information, containing proteins involved in a wide range of reproductive processes. The fluid can be collected using a minimally invasive procedure, making it a practical source for longitudinal studies. Measuring the proteome, or the complete set of proteins present in the fluid, reveals how the abundance of these molecules 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, spanning from the day of the urinary LH surge through the sixth day afterward. The researchers compared two workflows using the same samples: dia-PASEF and dda-FracOffline. Both methods were performed using a nanoElute liquid chromatography system coupled to a timsTOF Pro mass spectrometer, both from Bruker Daltonics, ensuring that the comparison was based on identical hardware and sample inputs.

The results showed a significant advantage for the dia-PASEF workflow. This method quantified 4,229 proteins, compared with 2,817 for the dda-FracOffline workflow, representing an increase of about 50% in proteome coverage. Beyond the sheer number of proteins detected, the measurements were more consistent with dia-PASEF. The method also detected more proteins whose abundance differed from the reference day, highlighting its superior sensitivity to subtle changes in protein levels. This consistency is critical for longitudinal studies, where distinguishing true biological changes from measurement noise is essential.

Mauricio Hernández, Head of Laboratory at MELISA Institute, emphasized the importance of measurement consistency in repeated-measures analysis. He noted that when comparing samples from the same woman on different days, researchers must determine whether a protein’s abundance changed or if the difference arose from the measurement process itself. With dia-PASEF, the measurements were more consistent, allowing the team to detect more proteins whose levels changed from day to day. This reliability is a key factor in the workflow’s validation for future use in the EARLY-PREG cohort.

In addition to improved protein detection and consistency, the dia-PASEF workflow offered a significant practical advantage in terms of time efficiency. Under the study conditions, instrument acquisition took approximately one hour per sample with dia-PASEF, compared with 16 hours for the dda-FracOffline method. This reduction in acquisition time could make larger longitudinal studies more feasible, particularly when each participant contributes multiple samples over several days.

The validation of this workflow lays the groundwork for future studies aiming to understand communication between the mother and embryo around the time of implantation. Dr. Elard Koch, Chairman of MELISA Institute, explained that each sample in the EARLY-PREG cohort comes with crucial information about its position in the cycle. By following women from before conception, the team can investigate in vivo questions that would otherwise be difficult to study, such as how the mother and embryo communicate in the days around implantation.

The team also tracked daily changes in CA-125 and HE4, proteins previously studied as biomarkers of ovarian and endometrial cancer. While the current study focused on methodological validation, the data provides a basis for future investigations into the clinical utility of these proteins. 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. The exploratory nature of these biological findings underscores the need for confirmation in larger cohorts and separate studies of diagnostic performance.

Based in Chile’s Biobío Region, the MELISA Institute brings together preconception follow-up, a longitudinal biobank, and molecular analysis to study women’s reproductive health. The authors declared no competing interests, and the proteomics data have been made available through ProteomeXchange/PRIDE.

Subject of Research: Validation of a dia-PASEF proteomics workflow for longitudinal analysis of cervicovaginal fluid in the EARLY-PREG cohort

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

Article References: EARLY-PREG enables day-by-day tracking of thousands of proteins in the female reproductive tract. Original publication (Bibliographic details unavailable.)

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DOI: Not provided

Keywords: proteomics, cervicovaginal fluid, EARLY-PREG, MELISA Institute, mass spectrometry, reproductive health, biomarkers, Scientific Reports, longitudinal study, dia-PASEF, enables, day-by-day