Longitudinal cervicovaginal fluid proteomics reveals the molecular landscape of early pregnancy

Early pregnancy requires tightly regulated molecular interactions between the maternal reproductive tract and the developing embryo. These interactions involve dynamic proteomic changes that coordinate epithelial remodeling, immune modulation, and preparation for implantation. The cervicovaginal fluid (CVF)-comprising vaginal, cervical, endometrial, and oviductal secretions-offers a non-invasive window into these processes and serves as a valuable source of protein biomarkers due to its role in maintaining reproductive tract homeostasis and immunity. Despite this potential, the temporal proteomic landscape during the peri-implantation window remains poorly characterized, limiting our understanding of maternal-embryonic communication and the discovery of early biomarkers of reproductive success. We aimed to map longitudinal proteomic dynamics in conception and non-conception cycles, identifying proteins and pathways involved in early pregnancy establishment.

We enrolled five women in a preconception, longitudinal, bidirectional, counterfactual cohort. Each contributed a conception cycle (positive pregnancy outcome) and a non-conception cycle. We collected daily samples beginning at the luteinizing hormone (LH) surge (LH0) to day fourteen (LH14), confirming ovulation by LH peak. We processed samples in triplicate, totaling 444 acquisitions. Proteins were profiled using dia-PASEF on a timsTOF mass spectrometer and analyzed in DIA-NN against a spectral library of CVF and female tissues generated from our cohort. Batch effects were corrected with ComBat, data were log₂-transformed and normalized, and temporal protein trajectories were modeled using MEFISTO. Factor decomposition revealed distinct temporal proteomic signatures that clearly separated cycle types in multivariate space.

Across the experiment, we identified 4,710 proteins, of which 697 showed statistically significant (p < 0.05) differences in temporal trajectories between conception and non-conception cycles. In conception cycles, proteins involved in epithelial adhesion, cytoskeletal organization, and cytokine-mediated signaling increased in abundance between LH5 and LH10, coinciding with the implantation window. Functional enrichment analysis revealed an over-representation of pathways related to cell adhesion, immune response, and glycosylation. Immune-associated proteins, including complement components and cytokine regulators, exhibited temporal patterns consistent with maternal-embryonic dialogue. Members of the mucin family, notably MUC4 and MUC16, showed progressive up-regulation, suggesting roles in modulating the epithelial interface during implantation.

We present the first high-resolution dia-PASEF-based longitudinal proteomic map of early luteal phase cycles with confirmed conception or non-conception. The data reveal outcome-specific molecular programs enriched in adhesion, immune, and glycosylation pathways, underscoring the fine regulation of the maternal reproductive tract during implantation. These insights enhance understanding of early pregnancy biology and support the potential of temporal proteomics to identify non-invasive biomarkers of reproductive success.