Donna Slater

Development and validation of primary human myometrial cell culture models to study pregnancy and labour

BackgroundThe development of the in vitro cell culture model has greatly facilitated the ability to study gene expression and regulation within human tissues. Within the human uterus, the upper (fundal) segment and the lower segment may provide distinct functions throughout pregnancy and during labour. We have established primary cultured human myometrial cells, isolated from both upper and lower segment regions of the pregnant human uterus, and validated them for the purpose of studying human pregnancy and labour. The specific objectives of this study were to monitor the viability and characterize the expression profile using selected cellular, contractile and pregnancy associated markers in the primary cultured human myometrial cells. Labour has been described as an inflammatory process; therefore, the ability of these cells to respond to an inflammatory stimulus was also investigated.MethodsMyometrial cells isolated from paired upper segment (US) and lower segment (LS) biopsies, obtained from women undergoing Caesarean section deliveries at term prior to the onset of labour, were used to identify expression of; α smooth muscle actin, calponin, caldesmon, connexin 43, cyclo-oxygenase-2 (COX-2), oxytocin receptor, tropomyosin and vimentin, by RT-PCR and/or immunocytochemistry. Interleukin (IL)-1β was used to treat cells, subsequently expression of COX-2 mRNA and release of interleukin-8 (CXCL8), were measured. ANOVA followed by Bonferroni’s multiple comparisons test was performed.ResultsWe demonstrate that US and LS human myometrial cells stably express all markers examined to at least passage ten (p10). Connexin 43, COX-2 and vimentin mRNA expression were significantly higher in LS cells compared to US cells. Both cell populations respond to IL-1β, demonstrated by a robust release of CXCL8 and increased expression of COX-2 mRNA from passage one (p1) through to p10.ConclusionsIsolated primary myometrial cells maintain expression of smooth muscle and pregnancy-associated markers and retain their ability to respond to an inflammatory stimulus. These distinct myometrial cell models will provide a useful tool to investigate mechanisms underlying the process of human labour and the concept of functional regionalization of the pregnant uterus.

Pre-pregnancy Body Mass Index (BMI) and delivery outcomes in a Canadian population

BackgroundWorldwide there has been a dramatic increase in the prevalence of overweight and obesity in women of childbearing age. Growing evidence suggests that maternal overweight and obesity is associated with poor maternal and perinatal outcomes. This study evaluated the impact of maternal pre-pregnancy overweight and obesity on pregnancy, labour and delivery outcomes in a cohort of women with term, singleton pregnancies cared for by family physicians in community based practices.MethodsThis study is a secondary analysis of the All Our Babies Cohort, a prospective, community-based pregnancy cohort in Calgary, Alberta. Maternal self-reported data on height and pre-pregnancy weight from term, singleton, cephalic pregnancies (n = 1996) were linked to clinical data on pregnancy and birth events retrieved from electronic health records. Descriptive and bivariate regression analysis were used to compare pregnancy and birth outcomes between women categorized as normal weight, overweight and obese based on the pre-pregnancy BMI. Multinomial regression analysis stratified by type of labour onset examined the association between pre-pregnancy BMI and mode of delivery controlling for maternal age, pre-existent health conditions, parity, fertility treatments, history of C-section and pregnancy complications.ResultsThe cohort consisted of 65.8% normal weight, 23.6% overweight and 10.6% obese women. Women with increased pre-pregnancy BMI were more likely to develop pregnancy complications such as preeclampsia (OR 3.5, CI 2.0-4.6 for overweight; OR 5.3, CI 3.3-8.5 for obese) and gestational diabetes (OR 3.0, CI 1.8-5.0 for overweight; OR 6.5, CI 3.7-11.2 for obese) than normal weight women. Spontaneous onset of labour was recorded in 71.2% of women with normal pre-pregnancy BMI, whereas 39.3% of overweight and 49% of obese women had their labour induced. For women with spontaneous labour, pre-pregnancy BMI was not a significant risk factor for mode of delivery, controlling for covariates. Among women with induced labor, obesity was a significant risk factor for delivery by C-section (adjusted OR 2.2; CI 1.2-4.1).ConclusionsEven among women with term, singleton pregnancies obtaining prenatal care in community-based settings, obese women who undergo labour induction are at increased risk of obstetrical interventions at delivery. These findings highlight the importance of tailored maternal care in pregnancy and at delivery of pregnant women with increased BMI in order to improve the outcomes and wellbeing of these women and their children.

Cyclooxygenases 1 and 2 Differentially Regulate Blood Pressure and Cerebrovascular Responses to Acute and Chronic Intermittent Hypoxia: Implications for Sleep Apnea

Background Obstructive sleep apnea (OSA) is associated with increased risk of cardiovascular and cerebrovascular disease resulting from intermittent hypoxia (IH)‐induced inflammation. Cyclooxygenase (COX)‐formed prostanoids mediate the inflammatory response, and regulate blood pressure and cerebral blood flow (CBF), but their role in blood pressure and CBF responses to IH is unknown. Therefore, this studys objective was to determine the role of prostanoids in cardiovascular and cerebrovascular responses to IH. Methods and Results Twelve healthy, male participants underwent three, 6‐hour IH exposures. For 4 days before each IH exposure, participants ingested a placebo, indomethacin (nonselective COX inhibitor), or Celebrex® (selective COX‐2 inhibitor) in a double‐blind, randomized, crossover study design. Pre‐ and post‐IH blood pressure, CBF, and urinary prostanoids were assessed. Additionally, blood pressure and urinary prostanoids were assessed in newly diagnosed, untreated OSA patients (n=33). Nonselective COX inhibition increased pre‐IH blood pressure (P≤0.04) and decreased pre‐IH CBF (P=0.04) while neither physiological variable was affected by COX‐2 inhibition (P≥0.90). Post‐IH, MAP was elevated (P≤0.05) and CBF was unchanged with placebo and nonselective COX inhibition. Selective COX‐2 inhibition abrogated the IH‐induced MAP increase (P=0.19), but resulted in lower post‐IH CBF (P=0.01). Prostanoids were unaffected by IH, except prostaglandin E2 was elevated with the placebo (P=0.02). Finally, OSA patients had elevated blood pressure (P≤0.4) and COX‐1 formed thromboxane A2 concentrations (P=0.02). Conclusions COX‐2 and COX‐1 have divergent roles in modulating vascular responses to acute and chronic IH. Moreover, COX‐1 inhibition may mitigate cardiovascular and cerebrovascular morbidity in OSA. Clinical Trial Registration URL: www.clinicaltrials.gov. Unique identifier: NCT01280006

Effects of PGF2α on the Expression of Uterine Activation Proteins in Pregnant Human Myometrial Cells From Upper and Lower Segment

CONTEXT The lower and upper segments of the uterus may play different roles in the process of parturition. The switch from pregnancy to delivery involves changes in expression of uterine activation proteins (UAPs). Prostaglandin (PG) F2α has multiple and complex roles in the birth process in addition to its vital contractile role. OBJECTIVE The purpose of this study was to investigate whether PGF2α regulates the expression of UAPs in human myometrium and to compare PGF2α actions in lower and upper segments. DESIGN Cultured human myometrial cells from upper and lower segments were treated with PGF2α. Western blotting was used to determine the levels of connexin 43 (CX-43), prostaglandin endoperoxide synthase-2 (PTGS-2; cyclooxygenase-2), oxytocin receptor (OTR), and PGF2α receptor (PTGFR) in the cells. The small interfering RNA approach was used to knock down PTGFR. RESULTS PGF2α dose dependently increased CX-43 and PTGS-2 while decreasing PTGFR in upper and lower segments. PGF2α increased OTR in the lower segment while decreasing it in the upper segment. PGF2α lost its effects on PTGS-2 and OTR in PTGFR knockdown cells, but its effect on CX-43 remained. AL8810, a specific antagonist of PTGFR, reversed the actions of PGF2α on UAPs except for CX-43 in the lower segment. Indomethacin reversed the PGF2α-induced effects on CX-43 and PTGS-2, but it did not alter PGF2α-induced PTGFR and OTR expression. The stimulatory effects of PGF2α were enhanced in the presence of IL-1β, which reversed the inhibitory effect of PGF2α on PTGFR. CONCLUSION PGF2α regulates UAPs in both upper and lower segment cells through either direct or indirect pathways, indicating that PGF2α uniquely participates in uterine preparation for the onset of labor.

Proliferative Action of the Androgen Receptor in Human Uterine Myometrial Cells—A Key Regulator for Myometrium Phenotype Programming

CONTEXT During pregnancy, the myometrium undergoes a phenotype programming starting from an early proliferative stage, to an intermediate synthetic stage, to a late contractile stage, after which the cells commit to labor. Steroid receptors play important roles in regulating myometrial cell phenotype during pregnancy, although detailed mechanisms are not fully defined. OBJECTIVE The aim of the study was to investigate the expression and function of the androgen receptor (AR) in myometrial cells during pregnancy. DESIGN AND SETTING Human primary myometrial cells, immortalized myometrial cells, rat pregnant and tubal ligation models were used. Immunohistochemistry, Western blot and real-time PCR, cell proliferation, and flow cytometry assays were applied. RESULTS The AR is highly expressed in the proliferative stage of pregnancy, starts to decrease in the synthetic stage, and reaches the lowest levels in the contractile stage. Both the mechanical stretch by the growing fetus and the decreased ratio of progestin:estrogen are responsible for AR protein reduction. AR regulates myometrial cell proliferation ligand-independently. Decreased AR expression delays the G(1)-S phase transition of human myometrial cell cycling and reduces expression of several cyclins. These AR actions are mediated through reducing IGF-I receptor protein stability, thus weakening PI3K/Akt signal cascade downstream of IGF-I. AR is required for IGF-I receptor protein stability by preventing the IGF-I receptor from ubiquitylation and protein degradation through both proteosomal and lysosomal pathways. CONCLUSION AR is a key regulator for myometrial cell proliferation, suggesting its critical role in myometrium phenotype programming during pregnancy.

Perinatal outcomes of maternal overweight and obesity in term infants: a population-based cohort study in Canada

The objective of this study was to assess the impact of increased pre-pregnancy maternal body mass index (BMI) on perinatal outcomes in term, singleton pregnancies who received prenatal care in community-based practices. The sample of 1996 infants included in the study was drawn from the All Our Babies Study, a prospective pregnancy cohort from Calgary. Multivariable logistic regression explored the relationship between the main outcomes, infant birth weight, Apgar score, admission to neonatal intensive care (NICU) and newborn duration of hospitalization, and BMI prior to pregnancy. Approximately 10% of the infants were macrosoms, 1.5% had a low Apgar score (<7 at 5 min), 6% were admitted to intensive care and 96% were discharged within 48 h after delivery. Although the infants of overweight and obese women were more likely to have increased birth weight as compared to infants of normal weight women, there were no differences in Apgar score, admission to NICU, or length of postnatal hospital stay among groups. This study suggests that in otherwise healthy term, singleton pregnancies, obesity does not seem to increase the risk of severe fetal impairment, neonatal admission to intensive care or duration of postnatal hospitalization.

An inhaled dose of budesonide induces genes involved in transcription and signaling in the human airways: enhancement of anti‐ and proinflammatory effector genes

Although inhaled glucocorticoids, or corticosteroids (ICS), are generally effective in asthma, understanding their anti‐inflammatory actions in vivo remains incomplete. To characterize glucocorticoid‐induced modulation of gene expression in the human airways, we performed a randomized placebo‐controlled crossover study in healthy male volunteers. Six hours after placebo or budesonide inhalation, whole blood, bronchial brushings, and endobronchial biopsies were collected. Microarray analysis of biopsy RNA, using stringent (≥2‐fold, 5% false discovery rate) or less stringent (≥1.25‐fold, P ≤ 0.05) criteria, identified 46 and 588 budesonide‐induced genes, respectively. Approximately two third of these genes are transcriptional regulators (KLF9, PER1, TSC22D3, ZBTB16), receptors (CD163, CNR1, CXCR4, LIFR, TLR2), or signaling genes (DUSP1, NFKBIA, RGS1, RGS2, ZFP36). Listed genes were qPCR verified. Expression of anti‐inflammatory and other potentially beneficial genes is therefore confirmed and consistent with gene ontology (GO) terms for negative regulation of transcription and gene expression. However, GO terms for transcription, signaling, metabolism, proliferation, inflammatory responses, and cell movement were also associated with the budesonide‐induced genes. The most enriched functional cluster indicates positive regulation of proliferation, locomotion, movement, and migration. Moreover, comparison with the budesonide‐induced expression profile in primary human airway epithelial cells shows considerable cell type specificity. In conclusion, increased expression of multiple genes, including the transcriptional repressor, ZBTB16, that reduce inflammatory signaling and gene expression, occurs in the airways and blood and may contribute to the therapeutic efficacy of ICS. This provides a previously lacking insight into the in vivo effects of ICS and should promote strategies to improve glucocorticoid efficacy in inflammatory diseases.

CRH Acts on CRH-R1 and -R2 to Differentially Modulate the Expression of Large-Conductance Calcium-Activated Potassium Channels in Human Pregnant Myometrium

CRH has been implicated to play a key role in the control of human pregnancy and parturition. Large-conductance potassium channels (BKCa) play a pivotal role in the modulation of uterine contractility during pregnancy. The objectives of the present study were to investigate the effect of CRH on BKCa expression in human pregnant myometrial cells. Myometrial tissues were collected at cesarean section from pregnant women not-in-labor (TNL) or in-labor (TL) at term, and myocytes were isolated and cultured. CRH was identified in human pregnant myometrium and mainly expressed in myometrial myocytes. Cultured myometrial cells were able to secrete CRH. In TNL myometrial cells, CRH treatment increased the expression of BKCa α- and β-subunits. CRH receptor type 1 (CRH-R1) antagonist, antalarmin, decreased whereas CRH receptor type 2 (CRH-R2) antagonist, astressin2b, increased the expression of BKCa. CRH-R2 small interfering RNA (siRNA) caused an increase, but CRH-R1 siRNA resulted in a decrease, in BKCa expression. In contrast to TNL cells, CRH exhibited an opposite effect on BKCa expression in TL myometrial cells, i.e. decreased BKCa expression. Antalarmin enhanced but astressin2b reduced BKCa expression. CRH-R2 siRNA decreased whereas CRH-R1 siRNA increased BKCa expression. 1,3-Dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one significantly inhibited the frequency of spontaneous contractions of myometrial strips, and this effect was significantly decreased in TL strips compared with TNL ones. Our data suggest that CRH-R1 and CRH-R2 show differential regulation of BKCa expression. These effects mediated by CRH-R1 and CRH-R2 are changed after the onset of labor. This leads us to suggest that CRH may fine-tune myometrial contractility by modulating the expression of BKCa during pregnancy and labor.

Labour is associated with decreased expression of the PGF2α receptor (PTGFR) and a novel PTGFR splice variant in human myometrium but not decidua

The prostaglandin F(2α) receptor (PTGFR) is believed to play a role in the process of parturition. The main support for this comes from animal studies; however, in humans, the evidence is less clear. The gene coding for PTGFR may be subject to alternative splicing to generate alternate variants with different signalling pathways. We have determined regional (upper versus lower segment) and labour-associated expression of PTGFR mRNA and a recently identified splice variant of PTGFR in human myometrium and decidua. We also examined the effect of the inflammatory cytokine interleukin-1β (IL-1β) on PTGFR mRNA expression in a model of cultured human myometrial smooth muscle cells. We identified a PTGFR transcript variant 2 (PTGFR-v2) generated by alternate splicing in human myometrium and decidua. The PTGFR-v2 contains an additional 71 base pair exon, which results in a truncated protein at 297 amino acids compared with the PTGFR transcript variant 1 (PTGFR-v1) at 359 amino acids. In contrast to our hypothesis, we demonstrate that PTGFR-v1 and PTGFR-v2 mRNA expression is not significantly higher in upper segment compared with lower segment paired samples. We also show a labour-associated decrease in PTGFR-v1 and PTGFR-v2 mRNA expression in lower segment myometrial samples. IL-1β-stimulated mRNA expression of both PTGFR variants in a distinct time-dependent manner in myometrial cell cultures. We suggest that the role of the PTGFR in the human uterus requires further validation prior to pursuing it as a target for the treatment of preterm labour. In addition, the presence of distinct variants suggests further levels of gene regulation within the pregnant uterus.

Maternal Whole Blood Gene Expression at 18 and 28 Weeks of Gestation Associated with Spontaneous Preterm Birth in Asymptomatic Women

The heterogeneity of spontaneous preterm birth (SPTB) requires an interdisciplinary approach to determine potential predictive risk factors of early delivery. The aim of this study was to investigate maternal whole blood gene expression profiles associated with spontaneous preterm birth (SPTB, <37 weeks) in asymptomatic pregnant women. The study population was a matched subgroup of women (51 SPTBs, 114 term delivery controls) who participated in the All Our Babies community based cohort in Calgary (n = 1878). Maternal blood at 17–23 (sampling time point 1, T1) and 27–33 weeks of gestation (T2) were collected. Total RNA was extracted and microarray was performed on 326 samples (165 women). Univariate analyses determined significant clinical factors and differential gene expression associated with SPTB. Thirteen genes were validated using qRT-PCR. Three multivariate logistic models were constructed to identify gene expression at T1 (Model A), T2 (Model B), and gene expression fold change from T1 to T2 (Model C) associated with SPTB. All models were adjusted for clinical factors. Model C can predict SPTB with 65% sensitivity and 88% specificity in asymptomatic women after adjusting for history of abortion and anaemia (occurring before T2). Clinical data enhanced the sensitivity of the Models to predict SPTB. In conclusion, clinical factors and whole blood gene expression are associated with SPTB in asymptomatic women. An effective screening tool for SPTB during pregnancy would enable targeted preventive approaches and personalised antenatal care.