High-throughput longitudinal proteomics of cervicovaginal fluid during consecutive days of women’s menstrual cycles: a new robust, sensitive, and efficient method

Proteomic studies of the female reproductive system are expected to significantly impact women’s reproductive health and the ability to address fertility problems. Cervicovaginal fluid (CVF), a complex combination of uterine, cervical, and vaginal secretions, represents an important source of marker molecules to understand different pathophysiological processes of the reproductive tract. However, reliable biomarker discovery on this fluid remains challenging due to limitations in the sensitivity and reproducibility of current mass spectrometry techniques for large-scale longitudinal analyses. To address these challenges, this study compared dia-PASEF with the conventional DDA-FracOffline approach in a longitudinal proteomic study of CVF samples from the EARLY-PREG preconception open cohort biorepository. The study included 7-day menstrual cycles with day-by-day CVF samples since ovulation. Regarding the number of identified and quantified proteins over the entire period, DDA-FracOffline detected 2,817 quantifiable proteins versus 4,229 for dia-PASEF. The dia-PASEF approach performed 33% better than DDA for detecting quantifiable proteins in CVF with a coefficient of variation as low as 5%. The data-independent approach overcomes the limitations of DDA for longitudinal prospective studies with repeated measurements, enabling the quantification of dynamic changes over time in a panel of biomarkers associated with key biological pathways and pathophysiological processes of the female reproductive system. Thus, the dia-PASEF method emerges as a robust, sensitive, and efficient method for longitudinal proteomic studies of CVF in women’s health research and diagnosis.