TallyHO obese female mice experience poor reproductive outcomes and abnormal blastocyst metabolism that is reversed by metformin

Abstract

Objective Obese women experience worse reproductive outcomes compared to normal weight women, specifically infertility, pregnancy loss, fetal malformations and developmental delay. The objective of this study was to use a genetic mouse model of obesity in order to recapitulate the human reproductive phenotype and further examine potential mechanisms and therapies. Methods New inbred, polygenic Type 2 diabetic TallyHO mice and age matched control C57BL/6 mice were superovulated to obtain morulae or blastocysts stage embryos which were cultured in human tubal fluid media. Deoxyglucose uptake was performed on insulin-stimulated individual blastocysts. Apoptosis was detected by confocal microscopy using TUNEL assay and Topro-3 nuclear dye. Embryos were scored for %TUNEL positive/total nuclei. AMPK activation, TNFα expression, and adiponectin expression were analyzed by western immunoblot and confocal immunofluorescent microscopy. Lipid accumulation was assayed by Bodipy. Finally all measured parameters were compared between TallyHO mice in morulaes cultured to blastocyst embryos in either human tubal fluid (HTF) media or HTF with 25ug/ml metformin added. Results TallyHo mice developed whole body abnormal insulin tolerance, decreased litter number and increased NEFA. Blastocysts demonstrated increased apoptosis, decreased insulin sensitivity, and decreased activation of AMP activated protein-kinase (AMPK). As a possible cause of the insulin resistance/abnormal P-AMPK, we found that Tumor necrosis Factor (TNFα) expression and lipid accumulation as detected by BODIPY were increased in TallyHO blastocysts and adiponectin was decreased. Culturing TallyHO morulae with the AMPK activator, metformin lead to a reversal of all abnormal findings, including increased p-AMPK, improved insulin-stimulated glucose uptake and normalization of lipid accumulation. Conclusions Women with obesity and insulin resistance experience poor pregnancy outcomes. Previously we have shown in mouse models of insulin resistance that AMPK activity is decreased and that activators of AMPK reverse the poor embryo outcomes. Here, we show for the first time using a genetically altered obese model, not a diet-induced model, that metformin reverses many of the adverse effects of obesity at the level of the blastocyst. Expanding on this we determine that activation of AMPK via metformin reduces lipid droplet accumulation, presumably by eliminating the inhibitory effects of TNFα, resulting in normalization of fatty acid oxidation and HADH2 activity. Metformin exposure in vitro was able to partially reversing these effects, at the level of the blastocyst and thus may be effective in preventing the adverse effects of obesity on pregnancy and reproductive outcomes.

Commonly used fertility drugs, a diet supplement, and stress force AMPK-dependent block of stemness and development in cultured mammalian embryos

“…Met improves maternal metabolism [6,[68][69][70][71][72][73][74][75][76] and ovulation [68,77] and is good for oocytes and embryos derived from females under obese and diabetic conditions [23][24][25]78], and thus, AMPK can improve compromised oocytes and embryos. CC alone increases Oct4, suggesting that Fig.…”

“…AMPK helps oocyte or blastocyst metabolism that is already compromised and helps IVM that has high levels of stress and has high oocyte or embryo loss rate due to diabetes [22][23][24][25]. Met or other AMPK agonists are often studied for their positive effects on maternal metabolism or when pathogenic insults are present.…”

“…However, in most studies, no metformin-alone control is done to test for possible toxicity in oocytes and embryos not under severe stress. We hypothesize that many AMPK activators occur in diets and drugs, and oocytes and embryos that are not under pathogenic stimuli may have a toxic AMPK activation levels that are too high [22,23,27,28].…”

Metformin in Reproductive Biology

“…The cell-to cell contact with trophectoderm was also altered because of an increase in tight junction permeability ( 55 ). In vivo , metformin reduced apoptosis in blastocysts of obese mice ( 56 ) possibly through an increase in NAMPT expression, but induced early bovine embryo arrest ( 57 ). Taken together, these studies demonstrate metformin's important contribution in the cross-talk between somatic cells and oocytes for the normal development of high-quality female germ cells and embryo developmental competence.…”

Metformin from mother to unborn child – Are there unwarranted effects?

“…Interestingly, when metformin is injected into new-born mice, whose organ maturation processes are still on-going, male mice seemed to have a prolonged lifespan [ 52 ]. Additionally, other in vitro rodent studies also showed that metformin can inhibit inflammatory reactions induced by various metabolic challenges such as: Angiotensin II (Ang-II) [ 39 ], lipopolysaccharides (LPS) 51 , cyclooxygenase (COX2) [ 53 ], maternal obesity [ 54 , 55 ] or cytokines [ 53 , 56 ]. Han et al showed that systemic inflammation induced by maternal hyperglycaemia can inhibit angiogenesis and blastocyst implantation, but metformin treatment can ameliorate these adverse effects [ 57 ].…”