In Brief OBJECTIVE: Gestational diabetes mellitus (GDM) is a frequent complication of pregnancy. Epidemiologic and pathophysiologic data suggest a close link of this disease to non-insulin-dependent diabetes mellitus. Within the calpain-10 gene various single-nucleotide polymorphisms have been identified that increased the risk for non-insulin-dependent diabetes mellitus. Therefore, we examined single-nucleotide exchanges of this gene in women with GDM. METHODS: A total of 875 unselected women were prospectively screened for GDM. Eighty women of this population, 40 patients with an abnormal oral glucose tolerance test and 40 normal controls, were randomly selected. DNA samples isolated from sera of the control and study groups were analyzed with respect to single-nucleotide polymorphisms of the calpain-10 gene at positions 43, 19, and 63 using polymerase chain reaction amplification and restriction analysis. RESULTS: Women with GDM were more likely to be homozygous for the allele 1 of single-nucleotide polymorphism 63 (P = .02 by χ2 test). With respect to single-nucleotide polymorphisms 19 and 43, no significant differences in allele distribution were detected between controls and women with GDM. When comparing the different haplotypes for calpain-10 (single-nucleotide polymorphisms 43, 19, and 63), all women with the haplotype combination 121/221 (n = 8) had gestational diabetes (P = .005 by Fisher exact test). CONCLUSION: Our results indicate that the haplotype 121/221 of the calpain-10 gene may be associated with disturbances of glucose metabolism during pregnancy. LEVEL OF EVIDENCE: II-1 Genetic variation in the calpain-10 gene is associated with alterations in glucose metabolism in pregnant women.
Summary Pre-eclampsia (PE) is a syndrome that affects multiple organ systems and is the most severe hypertensive disorder in pregnancy. It frequently leads to preterm delivery, maternal and fetal morbidity and mortality and life-long complications 1 . We currently lack efficient screening tools 2, 3 and early therapies 4, 5 to address PE. To investigate the early stages of early onset PE, and identify candidate markers and pathways, we performed spatio-temporal multi-omics profiling of human PE placentae and healthy controls and validated targets in early gestation in a longitudinal clinical cohort. We used a single-nuclei RNA-seq approach combined with spatial proteo- and transcriptomics and mechanistic in vitro signalling analyses to bridge the gap from late pregnancy disease to early pregnancy pathomechanisms. We discovered a key disruption in villous trophoblast differentiation, which is driven by the increase of transcriptional coactivator p300, that ultimately ends with a senescence-associated secretory phenotype (SASP) of trophoblasts. We found a significant increase in the senescence marker activin A in preeclamptic maternal serum in early gestation, before the development of clinical symptoms, indicating a translation of the placental syndrome to the maternal side. Our work describes a new disease progression, starting with a disturbed transition in villous trophoblast differentiation. Our study identifies potential pathophysiology-relevant biomarkers for the early diagnosis of the disease as well as possible targets for interventions, which would be crucial steps toward protecting the mother and child from gestational mortality and morbidity and an increased risk of cardiovascular disease later in life.
Genome amplification and cellular senescence are commonly associated with pathological processes. While physiological roles for polyploidization and senescence have been described in mouse development, controversy exists over their significance in humans. Here, we describe tetraploidization and senescence as phenomena of normal human placenta development. During pregnancy, placental extravillous trophoblasts (EVTs) invade the pregnant endometrium, termed decidua, to establish an adapted microenvironment required for the developing embryo. This process is critically dependent on continuous cell proliferation and differentiation, which is thought to follow the classical model of cell cycle arrest prior to terminal differentiation. Strikingly, flow cytometry and DNAseq revealed that EVT formation is accompanied with a genome-wide polyploidization, independent of mitotic cycles. DNA replication in these cells was analysed by a fluorescent cell-cycle indicator reporter system, cell cycle marker expression and EdU incorporation. Upon invasion into the decidua, EVTs widely lose their replicative potential and enter a senescent state characterized by high senescence-associated (SA) β-galactosidase activity, induction of a SA secretory phenotype as well as typical metabolic alterations. Furthermore, we show that the shift from endocycle-dependent genome amplification to growth arrest is disturbed in androgenic complete hydatidiform moles (CHM), a hyperplastic pregnancy disorder associated with increased risk of developing choriocarinoma. Senescence is decreased in CHM-EVTs, accompanied by exacerbated endoreduplication and hyperploidy. We propose induction of cellular senescence as a ploidy-limiting mechanism during normal human placentation and unravel a link between excessive polyploidization and reduced senescence in CHM.
Human placenta and trophoblasts express WNT ligands and WNT receptors suggesting a role for WNT-signalling in placental development. Indeed, a recombinant WNT ligand was recently shown to promote trophoblast migration/invasion, however, the involved signalling cascades and their target genes have not been elucidated. The aim was to investigate signal transduction via canonical WNT-signalling or phosphatidylinositide 3-kinase (PI3K)/AKT-signalling, their cross-talk as well as trophoblast-specific protease expression in trophoblastic SGHPL-5 cells and primary 1st trimester extravillous trophoblasts (EVT). WNT3A-dependent activation/phosphorylation of AKT (pAKT) and the down-stream kinaseglycogen synthase kinase (GSK)-3β were determined by Western blotting (WB). WNT3A-induced canonical WNT-signalling was analysed by luciferase reporter assay (TOPFlash) and nuclear recruitment of β-catenin. Trophoblast migration was studied using transwell assays and villous explant cultures. MMP2 expression/activation was investigated by qRT-PCR and WB/gelatine-zymography of supernatants. All experiments were performed +/– inhibitors of AKT-signalling or canonical WNT-signalling using LY294002 (PI3K inhibitor) or recombinant Dickkopf-1 (DKK1). WNT3A induced pAKT, luciferase expression of the canonical WNT reporter (P < 0.05) as well as accumulation of nuclear β-catenin. Inhibition of PI3K abolished WNT-dependent pAKT, pGSK-3β and cell migration, but did not affect TOPFlash activity or appearance of nuclear β-catenin. Inhibition through DKK1 did not influence pAKT and pGSK-3β, but decreased WNT reporter activity, nuclear β-catenin and cell migration. Both inhibitors decreased WNT3A-induced MMP2 expression in SGHPL-5 cells and pure EVTs (P < 0.05). WNT3A activates PI3K/AKT as well as canonical WNT-signalling through distinct receptors in invasive trophoblasts, since DKK1 did not activate the particular kinase. Although cross-talk between PI3K/AKT and canonical WNT-signalling has been observed in some cell types, these pathways seem to act independently in trophoblasts. However, both pathways promote Wnt-dependent migration and expression of MMP2. This is the 1st study identifying MMP2 as a novel target gene of canonical WNT-signalling in trophoblast.
Decidualization, the differentiation process of maternal uterine stromal cells into secretory decidual cells, is a prerequisite for successful implantation and progression of pregnancy. For in vitro differentiation mostly primary human endometrial stromal cells (HESC) isolated from uterine samples after hysterectomy for benign gynaecological diseases are utilised. However, a continuous supply of endometrial tissue is often lacking. Hence, we analysed whether cultivated human decidual stromal cells (HDSC) prepared from first trimester pregnancy terminations may represent an alternative model system for in vitro decidualization. Moreover, based on the expression of critical marker genes these cells were compared to a previously established endometrial stromal cell line during in vitro differentiation.HDSC isolated from decidual tissue attached to first trimester placentae, and telomerase-transformed human endometrial stromal cells (THESC) were characterised by immunofluorescence and differentiated in vitro using either cyclic adenosine monophosphate (cAMP) and/or estrogen (E2)/progesterone (P4). Proliferation was measured by analyzing cumulative cell numbers. Expression of mRNAs encoding progesterone receptor (PR), prolactin (PRL), insulin-like growth factor binding protein-1 (IGFBP1), and Dickkopf-1 (DKK1) was evaluated using quantitative PCR after 3, 6, 9 and 12 days of in vitro differentiation. PRL and IGFBP-1 protein expression was investigated by enzyme-linked immunosorbent assay (ELISA) and Western blotting, respectively. Furthermore, forkhead box O1A (FOXO1A), a critical transcription factor in decidualization, was analysed by immunofluorescence and Western blotting at two different time points of differentiation.Treatment with cAMP provoked morphological changes and growth arrest of THESC and HDSC, the latter showing loss of cells after 6 days of treatment. E2P4 stimulation did neither affect cell morphology nor proliferation of THESC and HDSC. Upon cAMP stimulation PR mRNA was suppressed in HDSC but not in THESC, whereas E2P4 did not alter transcript levels in both cell types. Protein expression of PR-A and PR-B was detectable in HDSC and diminished under cAMP, whereas THESC failed to produce the nuclear receptors. Supplementation of cAMP induced mRNA and protein expression of PRL and IGFBP-1 in both cell types at day 3, 6, 9, and 12 of treatment. In HDSC stimulation with E2P4 increased PRL and IGFBP-1 mRNA and protein production, whereas hormone treatment did not induce the two factors in THESC. E2P4 increased DKK1 mRNA at all time points in HDSC and cAMP provoked induction at day 9 and 12 of differentiation. In contrast, cAMP suppressed DKK1 mRNA in THESC, whereas E2P4 was ineffective. In both cell types combined treatments with cAMP and E2P4 provoked higher expression levels of PRL and IGFBP1 mRNA and protein as compared to cAMP stimulation alone. FOXO1A protein and its nuclear abundance were increased by cAMP in both cell types. However, reduction of its nuclear localisation upon E2P4 treatment could only be observed in HDSC.Both HDSC and THESC may represent suitable model systems for cAMP-dependent in vitro decidualization. Since cAMP decreases cell viability of HDSC after 6 days of incubation, this substance should be preferentially used in short-term experiments. Progesterone treatment of THESC might not be applicable since these cells lack progesterone response and PR protein. In contrast, stimulation of PR-expressing HDSC with E2P4 or cAMP/E2P4 may represent an appropriate protocol for human in vitro decidualization inducing and maintaining expression of critical marker genes in a time-dependent manner.
Up to 20% of women receiving epidural analgesia experience epidural-related maternal fever (ERMF) [1,2]. Importantly, intrapartum maternal fever is associated with increased perinatal mortality and neonatal morbidity [3]. This cell culture study investigates the dose-dependant effect of ropivacaine and possible alleviating impact of dexamethason on human umbilical vein endothelial cells (HUVECs) and human placental trophoblasts (TBs).
