Maternal serum proteome changes between the first and third trimester of pregnancy in rural southern Nepal

Abstract

Characterization of normal changes in the serum proteome during pregnancy may enhance understanding of maternal physiology and lead to the development of new gestational biomarkers. In 23 Nepalese pregnant women who delivered at term, two-dimensional difference in-gel electrophoresis (DIGE) was used to assess changes in relative protein abundance between paired serum samples collected in the first and third trimesters. One-hundred and forty-five of over 700 protein spots in DIGE gels (pI 4.2-6.8) exhibited nominally significant (p < 0.05) differences in abundance across trimesters. Additional filtering using a Bonferroni correction reduced the number of significant (p < 0.00019) spots to 61. Mass spectrometric analysis detected 38 proteins associated with gestational age, cytoskeletal remodeling, blood pressure regulation, lipid and nutrient transport, and inflammation. One new protein, pregnancy-specific β-glycoprotein 4 was detected. A follow-up isotope tagging for relative and absolute quantitation (iTRAQ) experiment of six mothers from the DIGE study revealed 111 proteins, of which 11 exhibited significant (p < 0.05) differences between trimesters. Four of these proteins: gelsolin, complement C1r subcomponent, α-1-acid glycoprotein, and α-1B-glycoprotein also changed in the DIGE analysis. Although not previously associated with normal pregnancy, gelsolin decreased in abundance by the third trimester (p < 0.01) in DIGE, iTRAQ and Western analyses. Changes in abundance of proteins in serum that are associated with syncytiotrophoblasts (gelsolin, pregnancy-specific β-1 glycoprotein 1 and β-2-glycoprotein I) probably reflect dynamics of a placental proteome shed into maternal circulation during pregnancy. Measurement of changes in the maternal serum proteome, when linked with birth outcomes, may yield biomarkers for tracking reproductive health in resource poor settings in future studies.

Fig. 1

Peptides located at numbered positions in this silver stained 2D gel image were identified by LC-MS/MS to detect proteins in depleted maternal serum that exhibit differences in relative abundance measured by DIGE between the first and third trimesters of pregnancy. These arbitrary numbers are used to indicate proteins detected in the corresponding gel spots in Table I. A Precision Plus Protein Standard Ladder (BioRad 161-0363) was used.

Fig. 2

The log standardized abundance of DIGE spot 86 (gelsolin) for 23 women, stratified by nutrient subgroup status, exhibits marked decreases in relative abundance in the third trimester. The designations “F” and “P” indicate membership in daily folate or placebo allocation groups as described in the methods. The designations “1” and “3” respectively indicate folate insufficiency (serum <7.8 n moles/L) or sufficiency (serum folate >22.5 n moles/L) at the time of blood sample collection, while the order of appearance indicates the trimester (first or third).

Fig. 3

Three technical replicates of non-depleted serum (1.5 µL serum per lane), respectively pooled (n=23) from the first and third trimester, were analyzed by Western blotting to detect differences in protein abundance. The average relative intensities of gelsolin, Apo A-I and α-1-acid glycoprotein are significantly different (0.05 A.) A2M (~161 kDa). B.) ceruloplasmin (~120 kDa). C.) gelsolin (~83 kDa). D.) Apo A-I (~28 kDa). E.) α-1-acid glycoprotein (~22 kDa).