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.

As a senior scientist, ESK 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.

This work was funded by FISAR, Human Reproduction Grant ″Embryo–Mother Crosstalk″ REH042024–01, and Grant MEL205062018 from FISAR Foundation. https://www.fisarchile.org/.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

Yes

The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

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

I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals.

Yes

I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).

Yes

I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable.

Yes

All data produced in the present study are available upon reasonable request to the authors