SUPPLEMENTATION OF CULTURE MEDIUM WITH MELATONIN IMPROVES BLASTOCYST DEVELOPMENT AND REVERSES GLUCOSE INTOLERANCE IN IVF MICE

Abstract

This study aimed to determine if the adverse effects of IVF on blastocyst and offspring could be reversed by supplementation of culture medium with melatonin in mice. A cross-sectional mouse model was utilized, wherein blastocysts were generated by natural mating (control group) or IVF with or without melatonin (10-6M) (mIVF and IVF group respectively) in clinical grade fertilization and culture media. Blastocysts were transferred to pseudo-pregnant ICR females. Cell lineage allocation was assessed for 30-40 blastocysts from each group. Body weight, glucose tolerance, energy expenditure, hepatic gene expression was measured in 6-10 mice from each group. C57BL/6 female mice and DBA2 male mice were used to generate blastocysts by natural mating (control group) or IVF with or without melatonin (10-6M) (mIVF and IVF group respectively) in clinical grade fertilization and culture media. Blastocysts were transferred to pseudo-pregnant recipients. Embryo development was recorded for IVF groups. Cell numbers of inner cell mass (SOX2) or trophectoderm (Cdx2+) were determined by immunohistochemistry. Males were weaned at 3 weeks of age onto a chow diet or a high-fat diet (60% fat) for 8 weeks. Glucose tolerance was assessed by an intraperitoneal glucose tolerance test (2g/Kg). Energy metabolism was examined in metabolic cages. Hepatic gene expression was measured by RNA-Seq and validated by real-time quantitative PCR. Blastocyst rates were similar in the two IVF groups. Reduced inner cell mass, trophectoderm and total cell number were observed in IVF blastocysts, compared with the control group (P<0.05). IVF pups had significantly lower birth weight and body weight before weaning, but exhibited increased body weight and liver weight at 11 weeks of age compared with controls (P<0.05), independently of diet. IVF mice were glucose intolerant shown as an increased glucose area under the curve, and had decreased energy expenditure as well as a large number of differentially expressed genes related to metabolic pathways in the liver tissue compared with the control group (P<0.05), independently of diet. The mIVF group showed intermediate cell numbers in blastocysts, body weight, liver weight and differentially expressed hepatic genes compared with the IVF group and control group, and normalized glucose tolerance and energy expenditure compared with the IVF group (P<0.05), independently of diet. Importantly, there was no significant difference in glucose tolerance and energy expenditure between the mIVF group and the control group. The data suggests that supplementation of culture medium with melatonin improved IVF blastocyst differentiation, reduced excessive weight gain after birth and normalized glucose intolerance in adult male offspring of IVF mice.