ResearchPad - basic-mechanisms-in-reproduction:-from-beginning-to-end Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[OR20-03 Transcriptional Changes in Lipid Metabolism of Adipocytes Derived from Subcutaneous Abdominal Adipose Stem Cells of Normal-Weight Polycystic Ovary Syndrome Women]]> Normal-weight polycystic ovary syndrome (PCOS) women exhibit increased adipose insulin resistance in vivo (1) accompanying enhanced subcutaneous (SC) abdominal adipose stem cell (ASC) development to adipocytes with greater lipid accumulation per cell in vitro (2). To determine whether this phenomenon is associated with abnormal adipogenic gene transcription during ASC differentiation into adipocytes, SC abdominal ASCs isolated from three non-Hispanic Caucasian normal-weight PCOS women and three age- and BMI-matched controls were cultured in adipogenic differentiation medium for 3–12 days. After RNA isolation, gene expression levels were determined by RNA sequencing at days 3, 7, and 12. Differentially expressed genes were filtered for significance (padj<0.05) and fold change (>2-fold); upstream regulator genes and gene ontology (GO) functions were determined using Ingenuity Pathway Analysis. Gene set enrichment analysis (GSEA) also was used to identify enriched cellular processes (3). Differentially expressed genes in PCOS vs. control cells were either upregulated (466, 768 and 441 genes on days 3, 7 and 12, respectively) or downregulated (742, 974 and 605 genes on days 3, 7 and 12, respectively) over time, with critical genes governing adipocyte cell differentiation in PCOS cells increased 2–6 fold at days 3, 7 and 12 (PPARγ, CEBPα, ADIPOQ, AGPAT2, FABP4, LPL, PLIN1). The predicted upstream regulator genes TGFβ1 (an adipogenic inhibitor) and TNF (a pro-inflammatory adipokine) were significantly reduced in PCOS relative to control cells at all time points. The GO functions lipid oxidation and free fatty acid (FFA) beta-oxidation were enriched amongst upregulated genes in PCOS cells across all time points, while acylglycerol synthesis was increased at days 7 and 12 alone (z>2, p<0.05, all GO functions). In parallel, GSEA showed in PCOS cells significantly increased transcripts related to oxidative phosphorylation, peroxisome activity and adipogenesis at all time points (p<0.05). Thus, adipocytes derived from SC abdominal ASCs of normal-weight PCOS women exhibit early activation of adipogenic genes, potentially underlying their exaggerated lipid accumulation in vitro, as previously described (2). These PCOS-related changes in gene expression involve an increase in both oxidative phosphorylation and FFA beta oxidation, which could disrupt the balance between energy production and lipid storage, particularly when caloric intake exceeds energy utilization. References: (1) Dumesic DA, et al JCEM 2019;104(6):2171–83; (2) Leung KL, et al. JES 2019;3:Supplement 1, SUN-107 (3) Subhramanian A, et al. PNAS 2005;102:43

<![CDATA[OR20-06 Kisspeptin as a Biomarker for Pregnancy Complications]]> Background:

Placentation (invasion of the placenta into the maternal endometrium) is hypothesised to be critical for healthy placental function and is abnormal in two thirds of miscarriage. Kisspeptin has emerged as a putative regulator of physiological placentation; it is highly expressed in placental syncitio-trophoblasts, whereas its receptor is expressed in both syncitio- and cyto-trophoblasts, such that kisspeptin is hypothesized to play an important paracrine role to regulate placentation. Circulating kisspeptin levels are considerably raised during healthy pregnancy and are reduced in women with miscarriage.


We aimed to investigate the utility of circulating kisspeptin concentrations in the assessment of pregnancy complications and assess whether kisspeptin provides additional diagnostic information compared to beta human chorionic gonadotropin (βhCG) alone.


This study was performed in collaboration with the Early Pregnancy Outcome Study (EPOS), which aims to identify novel pregnancy biomarkers. Women were invited to attend every fortnight for blood-sampling, clinical and ultrasound assessment during the first trimester, and repeated during the second and third trimesters. Asymptomatic women with healthy pregnancy (n=265) provided 960 blood-samples. Women with pregnancy complications including miscarriage (n=95), pre-eclampsia (PET; n=24), pregnancy induced hypertension (PIH; n=14), gestational diabetes (GDM; n=41), preterm birth (PTB; n=14) and intrauterine growth restriction (IUGR; n=24) provided 569 blood-samples.


Gestation-adjusted circulating kisspeptin and βhCG levels were lower, by 66% and 57%, respectively, in women with miscarriage compared to healthy pregnant controls (p<0.0001). Area under ROC curve for diagnosis of miscarriage was greater for the combination of both kisspeptin and βhCG together (0.92) than for either measure alone (βhCG 0.859, kisspeptin 0.874). An adjusted logistic regression model revealed that an 100pmol/L increase in plasma kisspeptin reduced the odds of miscarriage by 42%. Gestation-adjusted kisspeptin levels were lower in women with GDM (P=0.002), or IUGR (P<0.0001), and higher in women with PTB (P=0.004). Kisspeptin increased with gestation greater in PET (P=0.008) and PIH (P<0.0001) than in healthy controls.


Plasma kisspeptin is a promising biomarker for pregnancy complications and provides additional diagnostic capability over that provided by βhCG alone.

<![CDATA[OR20-05 Regulation of Mouse Uterine Contractility by Regulator of G-Protein Signalling 2]]> INTRODUCTION: Uterine contractions in labour are produced by activation of receptors such as FP (PGF) and OXTR (oxytocin). Such receptors signal via Gαq proteins that can be selectively turned off by the regulator of G-protein signaling 2 (RGS2). RGS2 expression can be upregulated by Gαs-coupled receptor signaling. PGE2 elicits various uterine effects, some via activation of Gαs-coupled receptors. In primary human myometrial cells, PGE2 enhances RGS2 expression that attenuates subsequent oxytocin-stimulated calcium responses associated with contraction. RGS2 expression is higher in preterm non-labour versus labour human myometrium, suggesting that RGS2 promotes quiescence in the pregnant uterus. To directly study whether RGS2 dampens uterine contraction, we analyzed the ex vivo contractility of pregnant mouse uteri. We hypothesize that loss of RGS2 expression enhances oxytocin and PGF-stimulated uterine contractility.

METHODS: Wildtype (WT) and RGS2 knockout (KO) female mice (8-14 weeks) were time-mated and euthanized on days 15-20 of pregnancy or during active labour (n=5-11 per group). Uteri were snap frozen for RNA/protein expression analysis by qPCR and western blot. Uterine sections from d18-19 were dissected into longitudinal strips for ex vivo contractility measurement. Tissues were submerged in Krebs-Henseleit buffer and tied to force transducers to measure isometric force development. Tissues were treated with or without 1μM PGE2 for 2 hours. In parallel, pretreated and non-pretreated tissues were exposed to increasing concentrations of oxytocin or PGF (0.1-10uM). All tissues were treated with 100mM KCl to determine the maximum contraction at the end of the experiment. Data were analyzed by ANOVA (Bonferroni post-hoc), or Student’s t-test, as appropriate.

RESULTS: RGS2 expression in WT uteri sharply decreases at day 19 (p<0.05), remaining low during labour. OXTR and FP receptor expression increase at day 19 (p<0.01 and p<0.05, respectively) and are sustained through labour. Compared to WT, uteri from KO mice produced larger oxytocin-stimulated contractile amplitudes (p<0.05), and more frequent PGF-stimulated contractions (p<0.05). PGE2 pretreatment enhanced RGS2 expression in WT uteri, which reduced the peak amplitudes and integrals elicited by subsequent oxytocin treatment, compared to non-pretreated uteri (p<0.05).

CONCLUSION: RGS2 may play a pro-quiescent role in pregnancy, since RGS2 loss enhances uterine contractility. Concomitant upregulation of pro-contractile receptors and RGS2 downregulation may facilitate labour activation. This mechanisms may be involved in the etiology of spontaneous preterm labour; loss of RGS2 expression observed in human preterm labour may allow for early contractile activation. Understanding RGS2’s role in quiescence is important to developing more effective strategies for managing preterm labour.

<![CDATA[OR20-07 Placentas from Obese Women Are Resistant to the Effect of Insulin on Triglyceride Content Ex Vivo]]> Background: Obesity affects 25% of pregnant women and is associated with a higher risk of neonatal complications, such as macrosomia and increased adiposity. The placenta may contribute to neonatal adiposity by accumulating and transferring excess lipid in response to maternal hyperinsulinemia. We previously found that insulin promotes a 3-fold increase in placental triglyceride (TG) content in lean women. We hypothesized that obese women have higher placental insulin resistance compared to lean women[FC1] with respect to TG content. Methods: Healthy, lean women (n=12; mean age 34±1 yrs; BMI 22±0.4 kg/m2) and non-diabetic, obese women (n=9; mean age 32±2 yrs; BMI 33±0.4 kg/m2, p<0.0001) consented for placenta collection at elective c-section under fasting conditions. Placental villous explants were immediately flash frozen or cultured for 24 hours, starved, then treated for 48 hours with 0.1nM, 1nM, 10nM, or 100nM of insulin, or vehicle. Lipids were extracted from basal and treated explants using a chloroform-methanol separation protocol. TG content was quantified by spectrophotometer and normalized to weight. Data were analyzed by two-way ANOVA. Results: Basal placenta tissue from obese women contained a 1.5-fold higher level of TG compared to lean women (9.4±0.5 vs 5.7±0.5 mcg/mg, p=0.001). Placental response to insulin in lean women peaked at 1nM insulin (20.2±3.3 mcg/mg), and plateaued at higher doses of 10nM (18.6±3.3 mcg/mg) and 100nM (22.8±2.8 mcg/mg, p=NS respectively). In contrast, placenta explants from obese women required the highest insulin dose of 100 nM for maximal response (23.6±3.2 mcg/mg), and showed a gradual dose response from 0.1 nM insulin (9.5±2), 1nM (14.8±2), 10 nM (16.9±3). At 100nM insulin, the difference in TG content was variable, but on average was 2-fold higher than vehicle treated placenta (vs 11.8±2.5[FC2] [AA3] mcg/mg, p=0.002). Conclusion: Our findings indicate that placenta from obese women develop insulin resistance similar to peripheral tissues, which can be overcome by high insulin doses. This placental insulin resistance likely occurs in response to chronic hyperinsulinemia, leading to interference of insulin signaling pathways, and may protect the neonate from excessive nutrient flux.

<![CDATA[OR20-04 Modeling Uterine Disorders Utilizing Adult Uterine Stem Cells]]> <![CDATA[OR20-01 AMH Preserves Fertility in Chemotherapy-Treated Mice Through a Mechanism Involving Autophagy]]> <![CDATA[OR20-02 Maternal Adiponectin Prevents Against Metabolic Dysfunction in Prenatally Androgenized PCOS-Like Mice]]>