Investigation of subfertility in the female Nsmf knockout mouse.

OBJECTIVE: To study if a pituitary or ovarian defect contributes to subfertility of the female Nsmf knockout (KO) mouse, an animal model of the hypogonadotropic hypogonadism gene NSMF. DESIGN: Analysis of hypothalamic, pituitary and ovarian gene expression at baseline, serum gonadotropin levels before and after gonadotropin-releasing hormone (GnRH) stimulation, ovarian response and implantation after superovulation, gonadotropin effects after ovariectomy, and ovarian NSMF protein expression. SETTING: University research laboratory. PATIENTS: None; mice were used. INTERVENTIONS: Gonadotropin-releasing hormone stimulation, superovulation, and ovariectomy in separate experiments. MAIN OUTCOME MEASURES: Gene expression in the hypothalamus, pituitary, and ovary; ovarian response and implantation after superovulation; serum gonadotropins after GnRH stimulation and ovariectomy; Western blot to measure ovarian NSMF expression. RESULTS: We found increased hypothalamic Kiss1, Gnrh1, and Jak2 mRNA expression in female Nsmf KO vs. wild type (WT) mice. However, pituitary gonadotropin, and GnRH receptor gene expression was not affected, and serum gonadotropin levels were normal. Gonadotropins increased after ovariectomy for both groups. Baseline Kiss1, Fshr, Prkaca, Prkar1a, and Gdf9 ovarian mRNA expression was increased and Cyp19a1 expression was decreased in Nsmf KO mice, while superovulated Nsmf KO mice had reduced ovarian Kiss1r, Prkar1a, and Fshr mRNA expression, 50% less oocytes, and normal implantation. Western blot demonstrated NSMF protein expression in the ovary of WT mice. CONCLUSIONS: Altered hypothalamic and ovarian gene expression was demonstrated in female Nsmf KO mice. It is possible that increased hypothalamic Gnrh1 and Kiss1 mRNA expression could compensate for reduced NSMF enabling a normal pituitary gonadotropin response. Impaired superovulation response, altered ovarian gene expression, and decreased number of oocytes indicate ovarian dysfunction, but a uterine factor cannot be excluded. These findings provide an anatomic basis for future mechanistic studies of subfertility in female Nsmf KO mice.

Genetics of hypogonadotropic Hypogonadism-Human and mouse genes, inheritance, oligogenicity, and genetic counseling.

Hypogonadotropic hypogonadism, which may be normosmic (nHH) or anosmic/hyposmic, known as Kallmann syndrome (KS), is due to gonadotropin-releasing hormone deficiency, which results in absent puberty and infertility. Investigation of the genetic basis of nHH/KS over the past 35 years has yielded a substantial increase in our understanding, as variants in 44 genes in OMIM account for ~50% of cases. The first genes for KS (ANOS1) and nHH (GNRHR) were followed by the discovery that FGFR1 variants may cause either nHH or KS. Associated anomalies include midline facial defects, neurologic deficits, cardiac anomalies, and renal agenesis, among others. Mouse models for all but one gene (ANOS1) generally support findings in humans. About half of the known genes implicated in nHH/KS are inherited as autosomal dominant and half are autosomal recessive, whereas only 7% are X-linked recessive. Digenic and oligogenic inheritance has been reported in 2-20% of patients, most commonly with variants in genes that may result in either nHH or KS inherited in an autosomal dominant fashion. In vitro analyses have only been conducted for both gene variants in eight cases and for one gene variant in 20 cases. Rigorous confirmation that two gene variants in the same individual cause the nHH/KS phenotype is lacking for most. Clinical diagnosis is probably best accomplished by targeted next generation sequencing of the known candidate genes with confirmation by Sanger sequencing. Elucidation of the genetic basis of nHH/KS has resulted in an enhanced understanding of this disorder, as well as normal puberty, which makes genetic diagnosis clinically relevant.

Rare Müllerian Anomaly: Complete Septate Uterus with Simultaneous Longitudinal and Transverse Vaginal Septa.

BACKGROUND: We present a patient with primary amenorrhea and a rare combination of anomalies. She was found to have a septate uterus, double cervix, and a longitudinal and a low transverse vaginal septum. CASE: An 18-year-old girl with primary amenorrhea presented with severe monthly pelvic pain. Examination and imaging revealed a thin transverse vaginal septum, complete septate uterus, double cervix, and a longitudinal vaginal septum. The transverse and longitudinal vaginal septa were excised and repaired. SUMMARY AND CONCLUSION: Although repetitive pregnancy loss and preterm birth are associated with various Müllerian duct anomalies, clinicians should also be suspicious of the presented anomaly in cases of primary amenorrhea and cyclic pelvic pain. To our knowledge, this is the only case of simultaneous septate uterus with longitudinal and transverse vaginal septum and the second case of combined longitudinal and transverse septum, which caused primary amenorrhea. This rare anomaly further supports the bidirectional regression theory of Müllerian development.

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

Obese women experience worse reproductive outcomes than normal weight women, specifically infertility, pregnancy loss, fetal malformations and developmental delay of offspring. The aim of the present study was to use a genetic mouse model of obesity to recapitulate the human reproductive phenotype and further examine potential mechanisms and therapies. New inbred, polygenic Type 2 diabetic TallyHO mice and age-matched control C57BL/6 mice were superovulated to obtain morula or blastocyst stage embryos that were cultured in human tubal fluid (HTF) medium. Deoxyglucose uptake was determined for individual insulin-stimulated blastocysts. Apoptosis was detected by confocal microscopy using the terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) assay and Topro-3 nuclear dye. Embryos were scored for TUNEL-positive as a percentage of total nuclei. AMP-activated protein kinase (AMPK) activation, tumour necrosis factor (TNF)-α expression and adiponectin expression were analysed by western immunoblot and confocal immunofluorescent microscopy. Lipid accumulation was assayed by BODIPY. Comparisons were made between TallyHO morulae cultured to blastocyst embryos in either HTF medium or HTF medium with 25 µg mL(-1) metformin. TallyHO mice developed whole body abnormal insulin tolerance, had decreased litter sizes and increased non-esterified fatty acid levels. Blastocysts from TallyHO mice exhibited increased apoptosis, decreased insulin sensitivity and decreased AMPK. A possible cause for the insulin resistance and abnormal AMPK phosphorylation was the increased TNF-α expression and lipid accumulation, as detected by BODIPY, in TallyHO blastocysts and decreased adiponectin. Culturing TallyHO morulae with the AMPK activator metformin led to a reversal of all the abnormal findings, including increased AMPK phosphorylation, improved insulin-stimulated glucose uptake and normalisation of lipid accumulation. 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 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 TNF-α, resulting in normalisation of fatty acid oxidation and HADH2 (hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase/enoyl-CoA hydratase (trifunctional protein), alpha subunit) activity. Metformin exposure in vitro was able to partially reverse these effects, at the level of the blastocyst, and may thus be effective in preventing the adverse effects of obesity on pregnancy and reproductive outcomes.

Preimplantation exposure of mouse embryos to palmitic acid results in fetal growth restriction followed by catch-up growth in the offspring.

Free fatty acids (FFAs) are energy substrates for many cell types, but in excess, some FFAs can accumulate in nonadipose cells, inducing apoptosis. Also known as lipotoxicity, this phenomenon may play a role in the development of obesity-related disease. Obesity is common among reproductive age women and is associated with adverse pregnancy and fetal outcomes; however, little is known about the effects of excess FFAs on embryos and subsequent fetal development. To address this knowledge gap, murine blastocysts were cultured in excess palmitic acid (PA), the most abundant saturated FFA in human serum, and ovarian follicular fluid. Targets susceptible to aberrations in maternal physiology, including embryonic IGF1 receptor (IGF1R) expression, glutamic pyruvate transaminase (GPT2) activity, and nuclei count, were measured. PA-exposed blastocysts demonstrated altered IGF1R expression, increased GPT2 activity, and decreased nuclei count. Trophoblast stem cells derived from preimplantation embryos were also cultured in PA. Cells exposed to increasing doses of PA demonstrated increased apoptosis and decreased proliferation. To demonstrate long-term effects of brief PA exposure, blastocysts cultured for 30 h in PA were transferred into foster mice, and pregnancies followed through Embryonic Day (ED)14.5 or delivery. Fetuses resulting from PA-exposed blastocysts were smaller than controls at ED14.5. Delivered pups were also smaller but demonstrated catch-up growth and ultimately surpassed control pups in weight. Altogether, our data suggest brief PA exposure results in altered embryonic metabolism and growth, with lasting adverse effects on offspring, providing further insight into the pathophysiology of maternal obesity.

Diet-induced obesity model: abnormal oocytes and persistent growth abnormalities in the offspring.

Associations between maternal obesity and adverse fetal outcomes are well documented, but the mechanisms involved are largely unknown. Most previous work has focused on postconceptional events, however, our laboratory has shown pre- and periconceptional aberrations in maternal glucose metabolism have adverse effects on oocytes and embryos that carry on to the fetus. To demonstrate effects of maternal obesity in the pre- and periconceptional periods, we compared reproductive tissues from diet-induced obese female mice to those of control mice. Ovaries were either stained for follicular apoptosis or dissected and evaluated for oocyte size and meiotic maturation. Mice were also mated and followed for reproductive outcomes including preimplantation embryonic IGF-I receptor (IGF-IR) immunostaining, midgestation fetal growth, and midgestational placental IGF receptor 2 (Igf2r) mRNA. Delivered pups were followed for growth and development of markers of metabolic syndrome. Compared with controls, obese mice had significantly more apoptotic ovarian follicles, smaller and fewer mature oocytes, decreased embryonic IGF-IR staining, smaller fetuses, increased placental Igf2r mRNA, and smaller pups. All weaned pups were fed a regular diet. At 13 wk pups delivered from obese mice were significantly larger, and these pups demonstrated glucose intolerance and increased cholesterol and body fat suggesting early development of a metabolic-type syndrome. Together, our findings suggest maternal obesity has adverse effects as early as the oocyte and preimplantation embryo stage and that these effects may contribute to lasting morbidity in offspring, underscoring the importance of optimal maternal weight and nutrition before conception.