Bryan F. Mitchell

Inflammatory processes in preterm and term parturition

A role for the pro-inflammatory cytokines interleukin (IL)-1beta, IL-6, IL-8 and tumor necrosis factor alpha (TNF-alpha) is evident in term and preterm delivery, and this is independent of the presence of infection. All uterine tissues progress through a staged transformation near the end of pregnancy that leads from relative uterine quiescence and maintenance of pregnancy to the activation of the uterus that prepares it for the work of labour and production of stimulatory molecules that trigger the onset of labour and delivery. The uterus is activated by pro-inflammatory cytokines through stimulation of the expression and production of uterine activation proteins (UAPs). One of these actions is the stimulation of prostaglandin (PG) synthesis. Particularly important for labour is PGF(2alpha) and its receptor, PTGFR. In addition, pro-inflammatory cytokines are able to increase the synthesis of matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF) and the progesterone receptor C isoform, which leads to decreased tissue progesterone responsiveness. Some of these effects are replicated by PGF(2alpha), suggesting that it may act via its receptor to amplify the direct actions of cytokines. In turn, VEGF may enhance leukocyte recruitment to the uterus, and MMP-9 may promote activation of inactive pro-form cytokines. Pro-inflammatory cytokines also decrease the activity of 11beta-hydroxysteroid dehydrogenase, which likely increases intrauterine cortisol concentrations. In turn, cortisol may drive PG synthesis. Together these feed-forward mechanisms activate the uterus, trigger the production of uterine contractile stimulants and lead to labour and delivery.

Resistance exercise decreases the need for insulin in overweight women with gestational diabetes mellitus

OBJECTIVE This study examines the effects of circuit-type resistance training on the need for insulin in women with gestational diabetes mellitus. STUDY DESIGN Thirty-two patients with gestational diabetes mellitus were randomly assigned either to a group that was treated with diet alone or to a group that was treated with diet plus resistance exercise. RESULTS The number of women whose condition required insulin therapy was the same, regardless of treatment. However, a subgroup analysis that examined only overweight women (prepregnant body mass index, >25 kg/m(2)) showed a lower incidence of insulin use in the diet-plus-exercise group (P<.05). Women in the diet-plus-exercise group were prescribed less insulin (P<.05) and showed a longer delay from diagnosis to the initiation of insulin therapy (P<.05), compared with the diet-alone group. CONCLUSION Resistance exercise training may help to avoid insulin therapy for overweight women with gestational diabetes mellitus.

Relative importance of maternal constitutional factors and glucose intolerance of pregnancy in the development of newborn macrosomia.

The purpose of this case-control study was to determine the relative importance of various predictors of newborn macrosomia, with particular reference to maternal constitutional factors and glucose intolerance of pregnancy. Macrosomia was defined by both absolute birthweight > or = 4,000 g and birthweight > or = 90th centile for gestational age. One thousand mother/newborn pairs [209 macrosomic (cases) and 791 non-macrosomic newborns (controls)] were recruited. Mothers with pre-gestational diabetes mellitus were excluded. Data on pregnancy and pregnancy variables were collected by review of prenatal, labour, and delivery and newborn assessment records and interview with the mother. Predictors that entered the stepwise multiple regression model in order of significance were: previous history of macrosomia, increasing maternal weight, nonsmoking status, multiparity, male newborn gender, gestational age of 40-42 weeks, North American Aboriginal ethnicity, maternal birthweight > 4,000 g, maternal height and maternal age < 17 years. Glucose screen positive/100-g oral glucose tolerance test (GTT) negative status was a significant predictor for macrosomia as defined by birthweight greater than the 90th percentile for gestational age, but not for absolute birthweight over 4,000 g. It was the least significant of all the factors examined. Treated gestational diabetes was not a significant predictor. By multivariate analysis, maternal constitutional factors are more powerful predictors of newborn macrosomia than maternal mild glucose intolerance. Treatment of mothers with GDM may be masking the effect of more pronounced carbohydrate intolerance.

Transcriptional Regulation of Oxytocin Receptor by Interleukin-1β and Interleukin-6

The up-regulation of oxytocin (OT) receptors in late pregnancy results principally from increased synthesis of messenger RNA. The 5′-flanking region of the human OT receptor gene contains several putative binding sites for nuclear factor-interleukin-6 (NF-IL6), also known as CAAT/enhancer binding protein-β. This trans-acting factor modulates the expression of genes involved in acute inflammatory responses. Proinflammatory cytokines, such as IL-1β or IL-6, have been implicated as mediators in both preterm and term labor, particularly in association with intrauterine infection. We hypothesized that IL-1β and IL-6 induce OT receptor gene expression in human myometrial cells, and this is mediated by NF-IL6 and cognate response elements in the 5′-flanking region of the OT receptor gene. Contrary to the hypothesis, both IL-1β and IL-6 treatment resulted in a significant decrease in OT receptor messenger RNA measured by ribonuclease protection analysis. Using electrophoretic mobility shift assay, we have shown t...

Changes in 17β,20α-hydroxysteroid dehydrogenase activity supporting an increase in the estrogen/progesterone ratio of human fetal membranes at parturition

OBJECTIVE: Our purpose was to measure the activity of the reversible enzyme 17β,20α-hydroxysteroid dehydrogenase around parturition with estrogen and progestogen substrates. STUDY DESIGN: Classic kinetic studies and explant cultures were used to determine kinetic parameters and net enzyme activities in both oxidative and reductive directions for both sets of substrates. RESULTS: Affinity constant values for estrone, estradiol, and 20α-dihydroprogesterone were 1 to 8 µmol/L. Affinity constant for progesterone was 9 to 25 µmol/L. Maximal velocities for all substrates in the chorion were 20- to 70-fold higher than in amnion and severalfold higher for estrogen substrates compared with the progestins. Around parturition there was a significant change toward net formation of the stronger estrogen (estradiol) and the weaker progestin (20α-dihydroprogesterone), suggesting an increase in the local estrogen/progesterone ratio. CONCLUSION: The enzyme 17β,20α-hydroxysteroid dehydrogenase may be an important regulator of the local estrogen/progesterone ratio in fetal membranes around the time of parturition.

Oxytocin and its receptor in the process of parturition.

Objective: To review the potential roles of oxytocin (OT) and its receptor (OTR) in the regulation of parturition. Methods: We reviewed the literature to describe thhe molecular aspects of the mechanisms of action and the regulation of OT and its receptor, particularly in relation to changes that occur at parturition. Results: The literature provides strong support for a role for the OT/OTR system in teh regulation of human parturition. Paracrine rather than endocrine mechanisms might be more important, and the regulation of the receptor might be more important than the ligand. The system is regulated by a wide variety of chemical and physical factors, including sex steroids, orphan receptors, uterine stretch, and many others. There also might be important interactions with the immune system. Conclusion: The OT/OTR system provides a potentially important therapeutic target for regulating the timing of human birth.

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.

Relationship between gene expression and function of uterotonic systems in the rat during gestation, uterine activation and both term and preterm labour

We have documented gestation‐ and labour‐ (preterm and term) dependent changes in expression of genes encoding contraction associated proteins in the rat uterus and correlated these changes with various parameters of uterine contractility. The data demonstrate increased expression of contractile agonist systems concurrent with decreased expression of relaxant systems after gestational day 20. Significant increases in expression of oxytocin (OT), its receptor (OTR), prostaglandin (PG) H synthase isoform 1 (PGHS‐1) and PGF2α receptor (FP) occurred first, followed by increases in PGHS‐2, connexin‐43, endothelin‐1 (ET‐1) and the ET‐1 receptor isoform ETA. Expression of OTR and FP was significantly reduced during mid‐gestation compared to non‐pregnant animals. Expression of inducible nitric oxide synthase (iNOS) increased significantly during pregnancy then decreased concurrently with the increase in OTR and FP. Functional changes in uterine contractility accompany changes in gene expression. OT was the most potent contractile stimulant. Sensitivity of uterine strips to OT was reduced in early and mid‐pregnancy then increased at uterine activation. Progesterone antagonist‐induced preterm labour caused changes similar to those at normal term. Comparison of mRNA transcripts in separated endometrium and myometrium suggested that the endometrium is an important regulator of myometrial contractility, analogous to the relationship between endothelium and vascular smooth muscle. This novel combination of functional and genetic expression analyses provides new insight into the physiology of parturition.

The Interleukin 1beta-Induced Expression of Human Prostaglandin F2alpha Receptor Messenger RNA in Human Myometrial-Derived ULTR Cells Requires the Transcription Factor, NFkappaB

Abstract The molecular mechanisms that regulate the expression of genes involved in parturition are poorly understood. The mRNA expression of the prostaglandin F2alpha receptor (PTGFR), a uterine activating gene, is increased at labor and is required for uterine contractile activity in numerous animal models, although the signaling pathways responsible for this increased expression have not been identified. Proinflammatory cytokines have been proposed to regulate the expression of the uterine activating genes via activation of the nuclear transcription factor, NFkappaB, and initiate labor. However, it is uncertain whether uterine PTGFR is regulated this way. In this report, we demonstrate for the first time that treatment of immortalized human myometrial-derived ULTR cells with the proinflammatory cytokine IL1beta causes an increase in PTGFR mRNA levels. Furthermore, IL1beta treatment increased the nuclear levels of the RELA subunit of NFkappaB and increased binding of RELA to the NFkappaB DNA-binding site. Inhibition of NFkappaB activation with either the proteasome inhibitor MG132 or phenethyl caffeiate reduced PTGFR mRNA levels, which indicates that this transcription factor is important for basal transcription. Furthermore, this inhibition prevented IL1beta induction of PTGFR mRNA, which confirms that NFkappaB is required for the IL1beta-induced increase in PTGFR. These results are consistent with the proposal that proinflammatory cytokines directly regulate uterine activation genes and that the transcription factor NFkappaB is involved in both basal and IL1beta-stimulated transcription of the PTGFR gene.

Effects of LPS and IL-6 on oxytocin receptor in non-pregnant and pregnant rat uterus.

PROBLEM: Little is known regarding the regulation of the timing of parturition. Recent evidence suggests an interaction between the immune system and uterine contractility in late gestation.
 METHOD: Pregnant rats were treated with LPS in vivo in attempts to establish a model of premature parturition induced by the pro‐inflammatory response. Uterine explants were incubated in vitro to determine the effects of IL‐6 on uterine synthesis of oxytocin (OT) and its receptor (OTR).
 RESULTS: LPS injection was quite toxic to pregnant rats and gave extremely variable results. In animals that delivered, there was a marked increase in the uterine concentrations of OTR and OTR mRNA. There was no consistent effect regarding the timing of parturition. IL‐6 caused a significant increase in the concentration of OTR mRNA in uterine explants from pregnant rats but not in tissues from non‐pregnant animals.
 CONCLUSION: Rat uterine concentrations of OTR are regulated by IL‐6. Pro‐inflammatory cytokines may stimulate uterine contractility in late gestation rat uterine tissues through a mechanism involving stimulation of OTR.