The embryonic and fetal developments are susceptible to environmental factors, and any sub-optimal conditions during the development stages of gametes, embryos, and the fetus could affect the health of the offspring after birth, which is known as the “embryo-fetal origin of disease” theory . A vast body of evidence suggests that children conceived by IVF-ET may be at increased risk of chronic diseases [8, 10]. In this study, increased body weight, abnormal glucose metabolism and lipid metabolism, particularly abnormal liver lipid deposition, were observed in IVF-ET male mice offspring. Sex-specific phenotypes, such as male offspring exhibiting more severely impaired glucose metabolism, insulin resistance, abnormal pyruvate tolerance, and earlier weight gain than females, were found in our study. It was evident that there may be gender differences in the effects of IVF-ET on offspring. To date, various metabolic phenotypes of IVF-ET offspring have been reported. Lower birth weight  and catch-up growth [15, 29], normal birth weight , and higher body weight as found in our study, were observed. IVF-ET offspring could present with impaired glucose metabolism, insulin resistance, and abnormal pyruvate tolerance after receiving a high-fat diet ; Severe glucose metabolism dysfunction sometimes occurs only in female offspring and sometimes only in male offspring [15, 16, 18, 29]. We presumed that the sex-specific phenotypes might be caused by different mouse strains, culture media, breeding conditions , as well as female estrogenic protection and epigenetic modifications of imprinted genes .
Moreover, we found increased body fat and liver weight, abnormal liver steatosis and lipid deposition, higher TG/LDL-C, and lower HDL-C in the male IVF-ET offspring at 22 weeks. These findings suggest abnormal lipid metabolism in IVF-ET male mice. It is well known that hepatic lipid metabolism plays a key role with essential functions in the de novo synthesis of fatty acids and ketogenesis . Therefore, we have sequenced the liver transcriptome and carried out an in-depth study of the possible mechanisms underlying the abnormalities in lipid metabolism. GO, and KEGG enrichment analysis results revealed an upregulated transcriptional status of genes involved in the primary synthesis of hepatic lipids in the livers of IVF-ET mice compared to CON mice. It suggests that the mechanism of increased hepatic lipid deposition in IVF-ET male offspring may be related to abnormalities in pathways related to lipid synthesis. It is well known that hyperglycemia, insulin resistance, hypertriglyceridemia, and abnormal fatty deposits in the liver are the main features of Non-alcoholic fatty liver disease (NAFLD) and other metabolic syndromes. Therefore, we speculate that IVF-ET conceived male mice might be susceptible to metabolic disorders relative to NAFLD.
Previous studies have shown that increased phosphorylation of insulin receptors AKT and ERK1/2 contributes to the regulation of hepatic mitochondrial function and energy metabolism [32–35]. Growth differentiation factor 15 (GDF15), a member of the transforming growth factor-β superfamily, is abundantly expressed in placental trophoblast cells during gestation and is synthesized and secreted mainly in the liver, where it is involved in various biological processes, including energy balance, body weight regulation and cachexia due to cancer and chronic diseases [36, 37]. It can promote fatty acid oxidation and inhibit lipid de novo synthesis in hepatocytes by binding to the GFRAL-RET receptor on the surface of target cells, leading to activation of the downstream AKT and ERK1/2 pathways . G protein signaling regulator 16 (RGS16) was initially cloned from the retina , which expressions in the heart, liver, hematopoietic cells, and brain [40, 41]. RGS16 is involved in adaptive immunity, platelet migration, and glucose or fat metabolism. High expression of RGS16 leads to hepatic steatosis, reduced blood glucose and β-ketone levels, and decreased gene expression for fatty acid oxidation in the liver; In contrast, Rgs16 KO mice exhibited the opposite phenotype as elevated expression of fatty acid oxidation genes in the liver, higher rates of fatty acid oxidation in liver extracts, and higher plasma β-ketone levels [26, 42]. In IVF-ET mice, we found increased expression of RGS16, downregulation of GDF15, p-ERK1/2, and p-AKT, and increased expression of lipogenic genes in the livers of IVF-ET male offspring. We, therefore, deduced that abnormal lipid metabolism in IVF-ET male offspring might be associated with abnormal GDF15-RGS16-p-ERK1/2/p-AKT pathway. Combining the known opposite functions reported with GDF15/RGS16 and the opposite expression found in our studies, we presumed that there might be a negative relationship between these two genes. However, no studies have shown an association between GDF15 and RGS16.
In vitro in HepG2 cells, knockdown of GDF15 resulted in abnormal upregulated expression of RGS16, inhibition of the phosphorylation of ERK1/2 and AKT, elevated expression of lipogenic genes, decreased expression of fatty acid oxidation genes, and increased cellular lipid deposition. Overexpression of GDF15 reduced the expression of RGS16, and subsequently increased the phosphorylation of ERK1/2 and AKT, reduced the expression of lipogenic genes, and increased the expression of fatty acid oxidation genes. These findings suggest that there may be a negative regulation between GDF15 and RGS16. Therefore, we speculate that RGS16 negatively regulated by GDF15 could be a possible mechanism for the abnormal lipid metabolism phenotype in IVF-ET male mice and may be an effective way to improve the metabolic abnormalities in IVF-ET offspring. Nevertheless, this is an in vitro cell experiment and might not fully explain the lipid metabolism pathway of IVF-ET offspring.
However, the factors affecting the reduced hepatic GDF15 expression in their offspring remain to be investigated, and epigenetic changes or intergenerational inheritance may be a potential and valid explanation [43, 44]. When genome-wide epigenetic reprogramming occurs, the fertilization and pre-implantation stages are critical periods of development. Manipulations during in vitro fertilization and in vitro culture of fertilized eggs may lead to oxidative stress in fertilized egg cells, affecting cell division, among other things . ROS (reactive oxygen species, superoxide, and hydrogen peroxide) produced by oxidative stress have been shown to regulate major epigenetic processes, DNA methylation, and histone acetylation . The abnormal epigenetic modifications may also be associated with direct, intergenerational, and transgenerational effects in offspring .
In summary, the IVF-ET model in this study eliminates the genetic diversity and complex environmental background of humans and is used to demonstrate that in vitro fertilization-embryo transfer can have long-term effects on the growth and glucolipid metabolism in offspring. Inhibiting RGS16 expression could reverse the abnormal lipid metabolism caused by GDF15 under-expression, which may be an effective way to alleviate abnormal lipid metabolism in the offspring of IVF-ET males. However, our findings may not be directly applicable to humans due to the complex human genetic background, hormonal environment, and application of various ovulation drugs in humans. Still, it provides valuable information for prevention and clinical decision-making.