Combination of vitamin B12 active forms improved fetal growth in Wistar rats through up-regulation of placental miR-16 and miR-21 levels
Introduction
Fetal development and growth is a result of several factors comprising genetics, maternal nutrition, maternal metabolism and placental health and functions. In developing nations, suboptimal maternal nutrition compromises fetal growth and development. In India, vitamin B12 deficiency is commonly seen due to vegetarianism and lower consumption of milk and milk products, while the concentration of folate is adequate. A recent systematic review showed that vitamin B12 insufficiency among pregnant women across the world was common in all trimesters (20%–30%) [1]. The recommended dose of folic acid during pregnancy is 400 μg/day, however, the dosage prescribed in India is as high as 5 mg/day [2]. Although the DOHaD hypothesis began with fetal under nutrition, the role of maternal over nutrition is now recognized and a U-shaped relationship between birth weight and later health outcomes for the adult has been suggested [3], [4].
In our earlier report, we demonstrated that imbalance of folic acid and vitamin B12 due to higher doses of folate was associated with global DNA methylation and poor birth outcomes in Indian pregnant women [2], [5]. We also showed restoration of growth, development and function in trophoblastic cells viz. BeWo and JEG3 when supplemented with vitamin B12 in excess folate condition [6]. Vitamin B12 deficiency is also an independent risk factor leading to adverse gestational outcomes, neural tube defects and neurological complications [7], [8], [9]. Vitamin B12 deficiency in pregnancy is prevalent and has been associated with both lower birth weight (LBW), intrauterine retardation and preterm birth [10], [11].
Vitamin B12 deficiency is strongly associated with hyperhomocysteinemia, which predisposes to adverse pregnancy outcomes [12], [13]. It also increases the risk of developing preterm labor, intrauterine growth retardation (IUGR), preeclampsia and recurrent abortions [14], [15].
Several studies have recommended vitamin B12 supplementation during pregnancy although there is confusion on the use of forms of vitamin B12 viz. Cyanocobalamin (Cbl), Methylcobalamin (MeCbl), Adenosylcobalamin (AdCbl) and Hydroxycobalamin (HCbl) [16]. Thakkar et al., suggested giving a combination of the active forms of vitamin B12 (MeCbl + AdCbl) for treatment of vitamin B12 insufficiency [17]. Identifying the best vitamin B12 form for supplementation remains important.
We planned to study effect of vitamin B12 forms MeCbl, Cbl, HCbl, AdCbl and combination of active forms (MeCbl + AdCbl) along with recommended dose of folate (RFol: 400 μg/day) and high dose of folate (HFol: 5 mg/day) on gestational outcomes (in terms of birth weight (BW), placental weight (PW) and placental sufficiency (BW:PW)) in female Wistar rats. We measured plasma homocysteine levels in dams at gestational day 19 (GD19) as a probable marker associated with an imbalance in plasma folate and vitamin B12 in pregnancy.
Previous studies have shown the role of placental miRNAs involved in the development of preeclampsia, preterm labor, IUGR etc. [18], [19]. However, miRNA intermediated mechanism in placenta remains poorly explored. We examined the expression levels of two candidate miRNAs: miR-16 and miRNA-21 in placental tissue to study its association with differential maternal nutrition and fetal growth.
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Animal maintenance and ethics statement
The study protocol was carried out in compliance with the CPCSEA guidelines (Committee for the Purpose of Control and Supervision of Experiments on Animals) Government of India. This study was approved by the IAEC of National Toxicology Centre, Pune (Registration No.40/CPCSEA/1999 through RP No. 39/1415). We obtained 12–14 weeks old ninety-six virgin female Wistar rats from a single colony to decrease genetic variability. Female Wistar rats were housed in polypropylene cages and maintained at 22 ±
Effect of vitamin B12 forms on plasma vitamin B12 and folate levels
As expected plasmsa folate was significantly higher in HFol control group as compared to RFol control group, probably indicating non-metabolised folate in blood. Vitamin B12 levels were significantly higher in groups supplemented with vitamin B12 forms as compared to those in control group (Supplementary file: S2).
Effect of vitamin B12 forms on dams plasma homocysteine levels
In the present study, plasma homocysteine levels were significantly increased (p < 0.05) in HFol supplemented dams as compared to RFol dams at GD 19 (Fig. 2). Supplementation with Cbl
Discussion
Importance of vitamin B12 during pregnancy is well established. Very few RCTs have been conducted with inconclusive information on appropriate biomarkers of vitamin B12 status and also short and long-term effect of folate during pregnancy and its health outcomes.
Using rat model of pregnancy, we for the first time demonstrated the effect of various forms of vitamin B12 gestational outcomes in presence of recommended (RFol: 400 μg) and high folate (HFol: 5 mg) doses. The key findings of this study
Acknowledgements
We would like to thank Dr. Sunil Jalalpure, Director, Dr. Prabhakar Kore Basic Science Research Centre, KLE University, Belagavi and Dr. S.D. Lokhande, Principal, Sinhgad College of Engineering, SP Pune University, Pune for providing the research facilities to carry out the research work. We thank the staff of National Toxicology Centre and APT Research Foundation, Pune for their assistance at the animal house and Mr. Parandekar for his assistance during blood biochemistry.
Conflicts of interest
Authors declared that they have no conflict of interest.
Funding
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
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