Objective-An association between assisted reproductive technique (ART) and specific imprinting mutations, such as Beckwith-Wiedemann syndrome (BWS), has recently been documented. Based on experiments in farm animals that demonstrated an association between alterations in culture media during ART and large offspring syndrome, we hypothesized that the culture media could be implicated as a common factor among the children with BWS conceived after ART. Design-Retrospective case series.Setting-Registry from Academic Medical Center. Patient(s)-Nineteen children born after ART were identified within the registry.Main Outcome Measure(s)-Demographics of patients, type of ART, culture media, IVF parameters.Result(s)-Twelve of the 19 medical records from the reproductive endocrine centers were successfully obtained. Ten of 12 mothers of children with BWS had IVF, but no single, consistent culture media was used in this group. Half of the patients underwent IVF with intracytoplasmic sperm injection (ICSI; n = 5), whereas the other half had routine IVF. One child was conceived through clomiphene citrate (CC) stimulation and artificial insemination, whereas another patient conceived through gonadotropin stimulation with intrauterine insemination (IUI). The gonadotropin dosage and quantity of embryos transferred also varied significantly. The only consistent finding was that all 12 women received some type of ovarian stimulation medication. An association between assisted reproductive technology (ART) and congenital malformation syndromes associated with imprinting defects has recently been documented (4, 5). Three groups have reported an increased rate of ART conceptions among children with BWS (5-7). Only one of these studies prospectively determined the incidence of ART in children with BWS (5). Combined data from two studies suggest that approximately 4% of the children with BWS were born after ART (5, 7). HHS Public AccessAngelman syndrome, a congenital malformation syndrome characterized by mental retardation, motor defects, and lack of speech is associated with imprinting abnormalities and ART (MIM 105830) (4). Angelman syndrome occurs in children with imprinting abnormalities of a gene cluster on chromosome 15 resulting in a loss of function of the maternal allele of ubiquitin-protein ligase E3A (UBE3A) (MIM 601623) (8). Three children with Angelman syndrome have been reported who were conceived after IVF with intracytoplasmic sperm injection (ICSI) lending further support for the association between congenital malformation syndromes with imprinting mutations and ART (4, 9).Results from two embryo experiments conducted in cows and mice led to this proposed study. First, Thompson et al. (10) demonstrated that they could decrease the frequency of large offspring syndrome (LOS) in farm animals after ART by changing the protein supplementation in the culture medium from sera to albumin and amino acids. The LOS refers to a syndrome that occurs after ART in sheep and cows and is associated with macrosomia, polyhyd...

Maternal diabetes adversely affects preimplantation embryo development and pregnancy outcomes. The objective of this study was to determine whether diabetes has an impact at an earlier stage of development, the preovulatory oocyte. Models of both acute and chronic insulin-dependent diabetes were used. Acute hyperglycemia was induced by a single streptozotocin injection. Akita mice, which harbor an autosomal dominant mutation causing them to be chronically hypoinsulinemic and hyperglycemic, were used. In both models, preovulatory oocytes were markedly smaller when compared with control animals. A significantly greater number of control oocytes had progressed to meiotic maturation before diabetic oocytes. Both models were found to have smaller, less developed ovarian follicles with a greater number of apoptotic foci by histological evaluation as well as by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling staining. Immunohistochemistry displayed a greater amount of TNF-related apoptosis-inducing ligand (TRAIL) and KILLER, a key murine ligand and receptor involved in the extrinsic pathway, expressed in cumulus cells from hyperglycemic mice compared with controls, suggesting that this apoptotic pathway may be up-regulated under diabetic stress. Elevated KILLER expression was also confirmed through Western blotting. Connexin-43 expression was found to be lower by immunohistochemistry and Western blot analysis in the diabetic samples. Both models of maternal hyperglycemia and hypoinsulinemia may have a detrimental effect on oocyte maturation and development as detailed by the smaller sizes of oocytes and developing ovarian follicles, the lowered percentage reaching germinal vesicle breakdown, and the greater amount of apoptosis. In addition, there may be dysfunctional or decreased communication in diabetic oocytes, as demonstrated by lower expression of connexin-43.

Objective To determine if morbid obesity is associated with decreased pregnancy and live birth rates after IVF in women with PCOS Design Retrospective cohort study Setting University-based fertility center Patients 72 women with PCOS who completed their first IVF cycle between 2001 and 2007 Interventions IVF outcomes were compared between women with a BMI of less than 40 kg/m2 versus those women with a BMI of 40 kg/m2 or greater Main outcome measures Clinical pregnancy rate, live birth rate Results Morbidly obese women with PCOS (n=19) had significantly lower clinical pregnancy rates after IVF than PCOS patients who were not morbidly obese (n=53) (32% vs. 72%, RR 0.44, 95% confidence interval 0.22 to 0.87). Their live birth rates were lower too, although this difference was not statistically significant (32% vs. 60%, RR 0.52, 95% confidence interval 0.26 to 1.05). Conclusions Morbid obesity is associated with lower pregnancy rates in women with PCOS after IVF raising the question if weight loss may improve IVF success rates for morbidly obese PCOS patients.

We have previously shown that the adenosine analog 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR), an activator of AMP-activated protein kinase (AMPK), stimulates an increase in AMPK activity and induces meiotic resumption in mouse oocytes [Downs, S.M., Hudson, E.R., Hardie, D.G., 2002. A potential role for AMP-activated protein kinase in meiotic induction in mouse oocytes. Dev. Biol, 245, 200-212]. The present study was carried out to better define a causative role for AMPK in oocyte meiotic maturation. When microinjected with a constitutively active AMPK, about 20% of mouse oocytes maintained in meiotic arrest with dibutyryl cAMP (dbcAMP) were stimulated to undergo germinal vesicle breakdown (GVB), while there was no effect of catalytically dead kinase. Western blot analysis revealed that germinal vesicle (GV)-stage oocytes cultured in dbcAMP-containing medium plus AICAR possessed elevated levels of active AMPK, and this was confirmed by AMPK assays using a peptide substrate of AMPK to directly measure AMPK activity. AICAR-induced meiotic resumption and AMPK activation were blocked by compound C or adenine 9-beta-d-arabinofuranoside (araA, a precursor of araATP), both inhibitors of AMPK. Compound C failed to suppress adenosine uptake and phosphorylation, indicating that it did not block AICAR action by preventing its metabolism to the AMP analog, ZMP. 2'-deoxycoformycin (DCF), a potent adenosine deaminase inhibitor, reversed the inhibitory effect of adenosine on oocyte maturation by modulating intracellular AMP levels and activating AMPK. Rosiglitazone, an anti-diabetic agent, stimulated AMPK activation in oocytes and triggered meiotic resumption. In spontaneously maturing oocytes, GVB was preceded by AMPK activation and blocked by compound C. Collectively, these results support the proposition that active AMPK within mouse oocytes provides a potent meiosis-inducing signal in vitro.

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.