The impact of vitamin C supplementation in pregnancy and in vitro upon fetal membrane strength and remodeling

Abstract

Generation of reactive oxygen species (ROS) has been suggested as a mechanism of fetal membrane (FM) weakening leading to rupture, particularly with preterm premature rupture of the fetal membranes (PROM). In vitro, FM incubation with tumor necrosis factor (TNF) mimics physiological FM weakening, concomitant with generation of ROS and collagen remodeling. Proinflammatory cytokines are also postulated to have a role in the development of the FM physiological weak zone where rupture normally initiates in-term gestations. We hypothesized that antioxidant treatment may block ROS development and resultant FM weakening. Two studies examining antioxidant effects upon FM strength were conducted, one in vivo and the other in vitro. Fetal membrane of patients enrolled in a multicenter placebo-controlled trial to determine the effect of vitamin C (1 g/day) and vitamin E (400 IU/day) upon complications of pre-eclampsia were examined for FM biomechanical properties and biochemical remodeling at birth. Separately, biomechanics and biochemical markers of remodeling were determined in FM fragments incubated with TNF with or without vitamin C preincubation. Supplemental dietary vitamin C in combination with vitamin E did not modify rupture strength, work to rupture, or matrix metalloproteinase-9 (MMP9; protein or activity) either within or outside the term FM physiological weak zone. In vitro, TNF decreased FM rupture strength by 50% while increasing MMP9 protein. Vitamin C did not inhibit these TNF-induced effects. Vitamin C alone had a weakening effect on FM in vitro. We speculate that vitamin C supplementation during pregnancy will not be useful in the prevention of preterm PROM.

Figure 1. In Vivo Vitamin C and Vitamin E effect upon FM Strength

(A) Rupture Force and (B) Work to Rupture are shown for FM of vitamin treated (N = 13) and placebo control (N = 14) patients. Results from FM fragments cut from the weak zone and other areas of each FM are s displayed separately. Data are shown as mean ± SD. (Panel A: + p=.09, ++ p=.71, * p < .001; Panel B: + p=.14, ++ p=.75, * p <.001)

Figure 2. In Vivo Vitamin C and Vitamin E effect upon FM MMP9

(A) MMP9 immunoreactivity determined by densitometry of Western blots and (B) MMP9 Activity determined by densitometry of Gel zymographs are displayed for FM of vitamin treated (N = 13) and placebo control (N = 14) patients. In each case results from FM fragments cut from the weak zone and other areas of each FM are displayed separately. Data are shown as mean ± SD. (Panel A: + p=.27, ++ p=.17, * p < .001; Panel B: + p=.13, ++ p=.10, * p <.001)

Figure 3. In Vitro Vitamin C does not inhibit TNF induced FM weakening

TNF incubation resulted in significant FM weakening that was not inhibited by pretreatment with Vitamin C. Rupture Force for four treatment groups are displayed: Control, Vitamin C pre-treatment, TNF, Vitamin C followed by TNF (see Methods for details). Data points represent pooled results of six experiments using three FM fragments per treatment group in each experiment (N=18). Data are presented as mean ± SD. (* p=.04, + p=.02, ++ p=.86)

Figure 4. In Vitro Vitamin C does not inhibit TNF induced changes in MMP9 and TIMP3

Western Blots for a representative FM. Treatment groups include: Control, Vitamin C pre-treatment, TNF, Vitamin C followed by TNF (see Methods for details). Data are presented as mean ± SD.

Figure 5. In Vitro Vitamin C does not inhibit TNF induced changes in MMP9 and TIMP3

Densitometry for pooled results of five experiments using three FM fragments per treatment group in each experiment (N=15) are displayed. A. MMP9; B. TIMP3. Treatment groups include: Control, Vitamin C pre-treatment, TNF, Vitamin C followed by TNF (see Methods for details). (* p <.001, + p < .05, both versus control untreated)