Quantitative proteomic profiling of Cervicovaginal fluid from pregnant women with term and preterm birth

Young Eun Kim,Sung Ki Lee,Kwonseong Kim,Dukjin Kang,Han Bin Oh

doi:10.1186/s12953-021-00171-1

Young Eun Kim, Sung Ki Lee + Show 3 more

Open Access

https://doi.org/10.1186/s12953-021-00171-1

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Abstract

BackgroundPreterm birth (PTB) is one of major causes of perinatal mortality and neonatal morbidity, but knowledge of its complex etiology is still limited. Here we present cervicovaginal fluid (CVF) protein profiles of pregnant women who subsequently delivered at spontaneous preterm or term, aiming to identify differentially expressed CVF proteins in PTB and term birth.MethodsThe CVF proteome of women who sequentially delivered at preterm and term was analyzed using isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional nanoflow liquid chromatography-tandem mass spectrometry (2D-nLC-MS/MS). We compared the CVF proteome of PTB (n = 5) and control subjects (term birth, n = 7) using pooled control CVF (term birth, n = 20) as spike-in standard.ResultsWe identified 1294 CVF proteins, of which 605 were newly identified proteins. Of 990 proteins quantified in both PTB and term birth, 52 proteins were significantly up/down-regulated in PTB compared to term birth. The differentially expressed proteins were functionally associated to immune response, endopeptidase inhibitors and structural constituent of cytoskeleton. Finally, we confirm the down-regulation of SERPINB7 (a serine-type protease inhibitor) in PTB compared to control by Western blot.ConclusionsTaken together, our study provide quantitative CVF proteome profiles of pregnant women who ultimately delivered at preterm and term. These promising results could help to improve the understanding of PTB etiology and to discover biomarkers for asymptomatic PTB.

Highlights

Preterm birth (PTB) is one of major causes of perinatal mortality and neonatal morbidity, but knowledge of its complex etiology is still limited
The pooled cervicovaginal fluid (CVF) sample obtained from 20 individual term birth subjects was used as a spike-in reference standard for both Experiment 1 and 2
Study design A total of 62 pregnant women participated in the study and CVF samples were collected at 14–20 weeks of gestation

Summary

Methods

Subject recruitment This prospective observational study was approved by local research ethics committee and all participants signed informed consent before enrollment. Tryptic digestion and iTRAQ labeling procedure The pooled control CVF sample as a reference standard was prepared from 20 term birth individual subjects by taking of equal amount of CVF protein in each sample. We performed two independent iTRAQ experiments to compare the CVF proteome of individual PTB and individual control (term birth) subjects (Fig. 1b). The iTRAQ-labeled peptides were loaded on biphasic trapping column and eluted with following 12-step salt gradients: (1) 0 mM (2) 20 mM (3) 24 mM (4) 26 mM (5) 28 mM (6) 30 mM (7) 35 mM (8) 40 mM (9) 60 mM (10) 100 mM (11) 200 mM (12) 1000 mM ammonium bicarbonate in 0.1% formic acid. Data analysis and bioinformatics The acquired raw files were searched using MaxQuant search engine 1.6.1.0 against the uniprot human database (Jan 3, 2018 release; 71,585 entries) for protein identification and iTRAQ quantification [30]. The intensities of protein bands were analyzed using the ImageJ program (National Institutes of Health, Bethesda, Maryland)

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