Fetuses of type 1 and 2 diabetic women experience higher incidences of malformations and fetal death as compared with nondiabetics, even when they achieve adequate glycemic control during the first trimester. We hypothesize that maternal diabetes adversely affects the earliest embryonic stage after fertilization and programs the fetus to experience these complications. To test this hypothesis, we transferred either one-cell mouse zygotes or blastocysts from either streptozotocin-induced diabetic or control mice into nondiabetic pseudopregnant female recipients. We then evaluated the fetuses at embryonic d 14.5 to assess fetal growth and the presence or absence of malformations. We found that fetuses from the diabetic mice transferred at the blastocyst stage but also as early as the one-cell zygote stage displayed significantly higher rates of malformations consistent with neural tube closure problems and abdominal wall and limb deformities. In addition, both these groups of fetuses were significantly growth retarded. To determine if this phenomenon was due to high glucose concentrations, two-cell embryos were cultured to a blastocyst stage in 52 mm D-glucose or L-glucose as an osmotic control, transferred into nondiabetic pseudopregnant mice, and examined at embryonic d 14.5. These embryos did not demonstrate any evidence of malformations, however, they did experience significantly higher rates of resorptions, lower implantation rates, and they were significantly smaller at embryonic d 14.5. In summary, exposure to maternal diabetes during oogenesis, fertilization, and the first 24 h was enough to program permanently the fetus to develop significant morphological changes.

Maternal diabetes is associated with an increased risk of miscarriages and congenital anomalies. Preovulatory oocytes in murine models also experience maturational delay and greater granulosa cell apoptosis. The objective of this study was to examine whether maternal diabetes influences preovulatory oocyte metabolism and impacts meiotic maturation. Streptozotocin-induced diabetic B6SJLF1 mice were superovulated, and oocytes were collected at 0, 2, and 6 h after human chorionic gonadotropin (hCG) injection. Individual oocyte concentrations of ATP, 5'-AMP, glycogen, and fructose-1,6-phosphate (FBP) and enzyme activities of glucose-6-phosphate dehydrogenase (G6PDH), adenylate kinase, hydroxyacyl-CoA dehydrogenase (Hadh2), and glutamic pyruvate transaminase (Gpt2) were measured. Protein levels of phosphorylated AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) were also measured. ATP levels were significantly lower in oocytes from diabetic mice, and the percent change in the AMP-to-ATP ratio was significantly higher in these oocytes. In contrast, activities of Hadh2 and Gpt2, two enzymes activated by AMPK, were significantly less in these oocytes. Additionally, glycogen and FBP levels, both endogenous inhibitors of AMPK, were elevated. Phosphorylated ACC, a downstream target of AMPK, and phosphorylated AMPK were both decreased in diabetic oocytes, thus confirming decreased AMPK activity. Finally, addition of the activator AICAR to the in vitro maturation assay restored AMPK activity and corrected the maturation defect experienced by the oocytes from diabetic mice. In conclusion, maternal diabetes adversely alters cellular metabolism leading to abnormal AMPK activity in murine oocytes. Increasing AMPK activity in these oocytes during the preovulatory phase reverses the metabolic changes and corrects delays in meiotic maturation.

Women with polycystic ovarian syndrome are at increased risk of miscarriage. Although evidence exists that metformin reduces this risk, the mechanism is unknown. This study tests the hypothesis that AMP kinase (AMPK) activation with metformin directly improves insulin signaling within the blastocyst, leading to improved pregnancy outcomes. Murine embryos were exposed to 200 nmol/l IGF-I, similar to the concentrations that can occur during polycystic ovary syndrome (PCOS). Resulting blastocysts were compared with embryos cocultured with excess IGF-I plus metformin and embryos cultured in control medium for the following: AMPK phosphorylation, insulin-stimulated glucose uptake, and apoptosis. Study and control blastocysts were also transferred into control animals. On embryonic day (E) 14.5, resulting fetuses were examined for size and rates of fetal implantation and resorption. Compared with control blastocysts, blastocysts exposed to high concentrations of IGF-I showed a decrease in AMPK activation and insulin-stimulated glucose uptake and an increase in the number of apoptotic nuclei. Blastocysts cocultured in metformin and excess IGF-I performed as well as controls in all studies. 5-Aminoimidazole-4-carboxamide 1-␤-D-ribofuranoside, another AMPK activator, also prevented the effects of excess IGF-I on blastocysts. Implantation rates and fetal size at day 14.5 were significantly lower among IGF-I-exposed embryos transferred into control mothers compared with control embryos transferred into control mothers. Both of these parameters were reversed by coincubation with metformin and IGF-I before transfer. Activation of embryonic AMPK may be the mechanism responsible for the improved pregnancy outcomes seen in PCOS patients taking metformin. Diabetes 56:2228-2234, 2007 W omen with polycystic ovary syndrome (PCOS) experience oligomenorrhea/anovulation, clinical or biochemical hyperandrogenism, and polycystic ovaries (1). The clinical presentation is variable, but many women with PCOS also exhibit obesity, hyperinsulinemia with insulin resistance, and infertility with recurrent pregnancy loss (2-4). The etiology of this recurrent pregnancy loss remains unclear. Both high concentrations of androgens and gonadotropins have been suggested, but only small, correlative clinical investigations have been conducted (5,6). Bioactive levels of the insulin-like growth factor, IGF-I, are also increased in PCOS patients because of an insulininduced decrease in the production of IGF binding protein-1 (IGFBP-1) (7,8).The preimplantation blastocyst stage embryo is an insulin-sensitive tissue (9,10). Prior studies have shown that the murine blastocyst responds to insulin or IGF-I by increasing glucose uptake and that this event occurs via the IGF-I receptor. IGF-I receptor signaling induces translocation of GLUT8 to the plasma membrane of the trophectoderm cells of the embryo (11). Expression of GLUT8 and translocation to the plasma membrane are critical for embryo survival (12,13). On exposure to high concentration of IGF-I or insul...

Pituitaries removed from ovariectomized adult rats were maintained for 18 weeks in organ culture using three different culture media. Gonadotropin secretion was assessed by RIA and was correlated with the histological features of the cultures. In medium favoring prolonged survival of the cultures, LH content of the medium fell to a low level within a few days. In the same cultures, FSH production initially decreased before increasing and leveling at a plateau which persisted until the end of the culture period. Cultures in medium unsuitable for long term survival of pituitary tissue displayed a similar decrease in LH production along with a gradual fall of FSH. It was concluded that contrary to LH, FSH may be secreted autonomously by pituitaries removed from hypothalamic control, provided that culture conditions are adequate for survival of gonadotropes.