Maternal Multiple Micronutrient Supplementation Stabilizes Mitochondrial DNA Copy Number in Pregnant Women in Lombok, Indonesia

Abstract

Background: The Supplementation with Multiple Micronutrients Intervention Trial (SUMMIT) in Lombok, Indonesia showed that maternal multiple micronutrients (MMN), as compared with iron and folic acid (IFA), reduced fetal loss, early infant mortality, and low birth weight. Mitochondria play a key role during pregnancy by providing maternal metabolic energy for fetal development, but the effects of maternal supplementation during pregnancy on mitochondria are not fully understood.

Objective: The aim of this study was to assess the impact of MMN supplementation on maternal mitochondrial DNA copy number (mtDNA-CN).

Methods: We used archived venous blood specimens from pregnant women enrolled in the SUMMIT study. SUMMIT was a cluster-randomized double-blind controlled trial in which midwives were randomly assigned to distribute MMN or IFA to pregnant women. In this study, we selected 108 sets of paired baseline and postsupplementation samples (MMN = 54 and IFA = 54). Maternal mtDNA-CN was determined by real-time quantitative polymerase chain reaction in baseline and postsupplementation specimens. The association between supplementation type and change in mtDNA-CN was performed using rank-based estimation for linear models.

Results: In both groups, maternal mtDNA-CN at postsupplementation was significantly elevated compared with baseline (P < 0.001). The regression revealed that the MMN group had lower postsupplementation mtDNA-CN than the IFA group (β = -4.63, P = 0.003), especially for women with mtDNA-CN levels above the median at baseline (β = -7.49, P = 0.007). This effect was rapid, and observed within 33 d of initiation of supplementation (β = -7.39, P = 0.017).

Conclusion: Maternal MMN supplementation rapidly stabilized mtDNA-CN in pregnant women who participated in SUMMIT, indicating improved mitochondrial efficiency. The data provide a mechanistic basis for the beneficial effects of MMN on fetal growth and survival, and support the transition from routine IFA to MMN supplementation.This trial was registered at www.isrctn.com as ISRCTN34151616.

Keywords: iron and folic acid; mitochondrial DNA copy number; mtDNA-CN; multiple micronutrients; oxidative stress; pregnancy; supplementation.

FIGURE 1

Flowchart for mtDNA-CN assessment of 108 pregnant women enrolled in the SUMMIT study. The SUMMIT study enrolled 31,290 pregnant women in the primary birth cohort and 28,426 children were born alive. Blood samples were drawn before (baseline) and postsupplementation in a random subsample of 2369 participants. The postsupplementation blood collection was at 1 of 4 points: 1 mo after enrollment, 36 weeks of gestation, 1 wk postpartum, or 12 wk postpartum. We purposively and blindly elected 160 sets of paired baseline and postsupplementation samples. Of these samples, we selected those in which the postsupplementation blood draw was performed either 1 mo after initiation of supplementation or at ∼36 weeks of gestation (n = 108), and postpartum sets were excluded (n = 52). These 108 women were given MMN (n = 54) or IFA (n = 54) supplementation. Further, these samples were categorized as below (<33) or above (≥33) the median days of supplementation, and below (<14.1) or above (≥14.1) the median of baseline mtDNA-CN. *Low baseline < 14.1, and high baseline ≥ 14.1. ^#Short duration of supplementation <33 d, and long ≥33 d. IFA, iron and folic acid; MMN, multiple micronutrient; mtDNA-CN, mitochondrial DNA copy number; SUMMIT, Supplementation with Multiple Micronutrients Intervention Trial.

FIGURE 2

Paired plot between baseline and postsupplementation mtDNA-CN of 108 pregnant women enrolled in the Supplementation with Multiple Micronutrients Intervention Trial study. MtDNA-CN significantly increased in the postsupplementation sample (P  < 0.001). MtDNA-CN data are shown as median (IQR). Comparison between baseline and postsupplementation was performed using Wilcoxon's Signed Rank test. Shown below the bars are medians (IQRs). ***Statistically significant difference between mtDNA-CN at baseline and postsupplementation, P < 0.001. IFA, iron and folic acid; MMN, multiple micronutrient; mtDNA-CN, mitochondrial DNA copy number.

FIGURE 3

Paired plot comparison of the MMN group with the IFA group for low baseline mtDNA-CN (A), high baseline mtDNA-CN (B), supplementation duration <33 d (C), and supplementation duration ≥33 d (D) among 108 pregnant women enrolled in the Supplementation with Multiple Micronutrients Intervention Trial study. High baseline mtDNA-CN ≥ 14.1; low baseline mtDNA-CN < 14.1. IFA, iron and folic acid; MMN, multiple micronutrient; mtDNA-CN, mitochondrial DNA copy number.