In the first trimester of pregnancy, placental development involves a wide range of cellular processes. These include trophoblast proliferation, fusion, and differentiation, which are dependent on tight cell cycle control. The intrauterine environment affects placental development, which also includes the trophoblast cell cycle. In this work, we focus on maternal obesity to assess whether an altered intrauterine milieu modulates expression and protein levels of placental cell cycle regulators in early human pregnancy. For this purpose, we use first trimester placental tissue from lean and obese women (gestational week 5+0–11+6, n = 58). Using a PCR panel, a cell cycle protein array, and STRING database analysis, we identify a network of cell cycle regulators increased by maternal obesity in which breast cancer 1 (BRCA1) is a central player. Immunostaining localizes BRCA1 predominantly to the villous and the extravillous cytotrophoblast. Obesity-driven BRCA1 upregulation is not able to be explained by DNA methylation (EPIC array) or by short-term treatment of chorionic villous explants at 2.5% oxygen with tumor necrosis factor α (TNF-α) (50 mg/mL), leptin (100 mg/mL), interleukin 6 (IL-6) (100 mg/mL), or high glucose (25 nM). Oxygen tension rises during the first trimester, but this change in vitro has no effect on BRCA1 (2.5% and 6.5% O2). We conclude that maternal obesity affects placental cell cycle regulation and speculate this may alter placental development.

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Endoplasmic reticulum (ER)-stress activates the unfolded protein response (UPR), which plays a (patho)physiological role in the placenta. Oxygen and hyperinsulinemia are major regulators of placental development. Thus, we hypothesized that oxygen, insulin and their interplay modulate ER-stress in early pregnancy. Using the human first-trimester trophoblast cell line ACH-3P, we quantified mRNA and protein of several members of UPR by RT-qPCR and Western blotting, respectively. ER-stress induction using tunicamycin and brefeldin A resulted in increased CHOP (4.6-fold change; P ≤ 0.001), XBP1 expression (1.7- and 1.3-fold change, respectively; P ≤ 0.001 and P < 0.05) and XBP1 splicing (7.9- and 12.8-fold change, respectively; P ≤ 0.001). We subsequently analyzed the effect of oxygen (6.5%, 2.5%), insulin (0.1–10 nM) and their interaction using ANCOVA adjusted for cell passage as co-variate. Although GRP78 protein remained unaffected, low oxygen (2.5% O2) increased IRE1α phosphorylation (+52%; P < 0.05) and XBP1 splicing (1.8-fold change; P ≤ 0.001) after 24 h, while eIF2α protein and CHOP expression were downregulated (−28%; P < 0.05 and −24%; P ≤ 0.001; respectively). eIF2α phosphorylation was also reduced after 48 h by low oxygen (−61%; P < 0.05) but increased in the presence of insulin (+46%; P ≤ 0.01). These changes were not PERK-mediated, since PERK phosphorylation and total protein were not altered. Overall, our results suggest that IRE1α and eIF2α UPR-pathways are differentially regulated by oxygen and insulin in early pregnancy.

Introduction PCOS in adolescent girls and young women is nowadays thought to be, in essence, an epiphenomenon of ectopic fat accumulation. By definition, "adolescent PCOS" is characterized by androgen excess (as well clinical as biochemical) and oligo-anovulation (often judged by oligo-amenorrhea) (1) presenting between 2 and 8 years after menarche. There is no FDA-approved treatment for "adolescent PCOS". In SPIOMET4HEALTH (a project funded by the European Commission under Grant Agreement 899671), we aim at reducing ectopic fat in an early phase of PCOS, with a standardized lifestyle intervention plus a pharmacological addendum consisting of either placebo, or PIO, or SPIO, or SPIOMET. In "adolescent PCOS", low-dose pioglitazone (7.5 mg/d) may exert insulin-sensitizing and gonadotropin-normalizing effects, in part by raising the circulating concentrations of high-molecular-weight adiponectin; low-dose spironolactone (50 mg/d) may not only act as an anti-androgen but also exert anti-mineralocorticoid effects that raise energy expenditure by activating brown adipose tissue; low-dose metformin (850 mg/d) is known to act through multiple mechanisms and was recently shown to be capable of changing the relative deficit of GDF15 into an abundance that is thought to contribute to reduce liver fat. Subjects & Methods In the SPIOMET4HEALTH trial, patients with "adolescent PCOS" (age range 12. 0–23.9 years; BMI &lt;35 kg/m 2) will be recruited in seven centers across Europe. A total of 364 patients are expected to engage into a lifestyle intervention, and to receive either placebo, or PIO, or SPIO, or SPIOMET once daily (1: 1: 1: 1 randomization; single tablets; double blinding) for 12 months. Post-treatment follow-up will span 6 months. The primary endpoint is ovulation rate, as judged by a combination of menstrual data and progesterone concentrations in saliva; the analysis will start by testing for superiority between placebo and SPIOMET. Secondary endpoints include pre-treatment, on-treatment and post-treatment measures of androgen excess, body composition and insulin sensitivity, as well as measures of quality of life, and of adherence to treatment. The design of this trial has been endorsed by the European Medicines Agency, as part of a "Paediatric Investigation Plan". Expected Results/Discussion The SPIOMET4HEALTH project is expected to deliver the first results of an international, randomized, double-blind, active-controlled/placebo-controlled trial evaluating the safety, efficacy, and tolerability of the fixed dose combination SPIOMET in adolescent girls and young women with PCOS. Favorable results of this Phase 2 trial may advance SPIOMET into Phase 3. Reference: Ibáñez L, et al. An International Consortium Update: pathophysiology, diagnosis, and treatment of polycystic ovarian syndrome in adolescence. Horm Res Paediatr 2017;88: 371-395. Presentation: No date and time listed

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