Metformin is a widely prescribed medication whose mechanism of action is not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing the risk of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformin’s impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200 µM (therapeutic range) and 2000 µM (supra-therapeutic range) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance were detected between vehicle and 200 µM metformin-treated cells, 2000 µM metformin impaired oxidative metabolism and increased the abundance of lactate and TCA cycle intermediates, α-ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000 μM, but not 200 µM metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-therapeutic concentrations of metformin impair trophoblast metabolism and differentiation whereas metformin concentrations in the therapeutic range do not strongly impact these processes.

Metformin is a widely prescribed medication whose mechanism of action in not completely defined and whose role in gestational diabetes management remains controversial. In addition to increasing risks of fetal growth abnormalities and preeclampsia, gestational diabetes is associated with abnormalities in placental development including impairments in trophoblast differentiation. Given that metformin impacts cellular differentiation events in other systems, we assessed metformin's impact on trophoblast metabolism and differentiation. Using established cell culture models of trophoblast differentiation, oxygen consumption rates and relative metabolite abundance were determined following 200 μM (near-physiologic) and 2000 μM (supra-physiologic) metformin treatment using Seahorse and mass-spectrometry approaches. While no differences in oxygen consumption rates or relative metabolite abundance was detected between vehicle and 200 μM metformin treated cells, 2000 μM metformin impaired oxidative metabolism and increased abundance of lactate and TCA cycle intermediates, α-ketoglutarate, succinate, and malate. Examining differentiation, treatment with 2000 μM, but not 200 μM metformin, impaired HCG production and expression of multiple trophoblast differentiation markers. Overall, this work suggests that supra-physiologic concentrations of metformin impairs trophoblast metabolism and differentiation whereas physiologic concentrations of metformin do not strongly impact these processes.

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Cytotrophoblasts fuse to form and renew syncytiotrophoblasts necessary to maintain placental health throughout gestation. During cytotrophoblast to syncytiotrophoblast differentiation, cells undergo regulated metabolic and transcriptional reprogramming. Mitochondria play a critical role in differentiation events in cellular systems, thus we hypothesized that mitochondrial metabolism played a central role in trophoblast differentiation. In this work, we employed static and stable isotope tracing untargeted metabolomics methods along with gene expression and histone acetylation studies in an established cell culture model of trophoblast differentiation. Trophoblast differentiation was associated with increased abundance of the TCA cycle intermediates citrate and α-ketoglutarate. Citrate was preferentially exported from mitochondria in the undifferentiated state but was retained to a larger extent within mitochondria upon differentiation. Correspondingly, differentiation was associated with decreased expression of the mitochondrial citrate transporter (CIC). CRISPR/Cas9 disruption of the mitochondrial citrate carrier showed that CIC is required for biochemical differentiation of trophoblasts. Loss of CIC resulted in broad alterations in gene expression and histone acetylation. These gene expression changes were partially rescued through acetate supplementation. Taken together, these results highlight a central role for mitochondrial citrate metabolism in orchestrating histone acetylation and gene expression during trophoblast differentiation.