The impact of metformin on placental ageing in humans and mice
Placental ageing refers to the physiological accumulation of a senescent phenotype over healthy pregnancy. In pregnancies affected by complications such as pre-eclampsia and fetal growth restriction, placental ageing is notably accelerated and observed at an earlier gestational age. Metformin is used during pregnancy for an increasing variety of indications, including treatment of gestational diabetes and may have a role in slowing cellular ageing. It is therefore essential to understand the potential impact of metformin on placental ageing. Placental samples (n=105) were obtained from women with BMI =30kg/m2 randomised to treatment with metformin or placebo during pregnancy. Ageing was assessed by measuring telomere length, histological examination, and using array-based technologies to investigate gene expression and methylation. Results were validated using isolated human trophoblasts treated in vitro with metformin, and in a complementary mouse model. There were no differences between metformin-exposed and control placentas in terms of telomere length, fibrosis, or calcification. There were no differences in placental gene expression or methylation patterns by metformin status. In our mouse model, no genes classically associated with cellular ageing were differentially expressed and no senescence pathway showed evidence of enrichment with metformin treatment. There was no evidence that metformin either slows or accelerates placental ageing pathways in the complementary models that we investigated. Our findings are reassuring with regards to the safety of metformin used to treat gestational diabetes, but do not support a role for metformin in the prevention of adverse pregnancy outcomes in non-diabetic women. Overall design: Wild-type C57BL/6J mice obtained from Charles River Laboratories were bred in house. Female mice were ad libitum fed a well-established obesogenic diet (no. 824053, Special Dietary Services) (Fernandez-Twinn et al., 2012) supplemented with condensed milk (no. 12029969, Nestlé) from weaning (3 weeks of age). A first pregnancy was commenced at 6 weeks of age, and mating for the second pregnancy was commenced when the mice achieved a body weight of =35g (~18 weeks of age). The day of the plug was considered embryonic day 0.5 (E0.5) and fetuses were culled at E18.5. Mice were randomized to receive metformin (0215169-CF, MP Biomedicals) in the condensed milk one week before mating and throughout the subsequent pregnancy to a dose of 300mg/kg/day (equivalent to 1.7g in a 70kg man) (Salomäki et al., 2014). We calculated that a sample size of 12 per group would be sufficiently powered to detect differential gene expression using a log 2-fold change of 1, mean coefficient of variation of 0.4 and mean depth of coverage of 50. 35-45mg of placental tissue was isolated from 12 control and 12 metformin treated placentas. RNA was extracted using the miRNeasy micro kit (QIAGEN), with DNA contamination removed using the DNAse kit (QIAGEN). Both steps were performed according to the manufacturer's instructions. RNA quality and concentration was assessed by nanodrop analysis. RNA integrity (RIN) of > 8 for all samples was confirmed by analysis with the Agilent Bioanalyser 2100 system (Agilent RNA 6000 Nano Kit). 400ng of total RNA was used for library construction (TruSeq Stranded mRNA Library Pre Kit; Illumina). Indexed libraries were normalized, pooled and sequenced on a NovaSeq 6000 (50bp paired-end).