The myometrial effects of progesterone.
MD thesis, University of Glasgow.
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Introduction: Preterm birth is the leading cause of perinatal morbidity and mortality and rates are rising. The UK now has the highest rate of premature birth in Europe with 5.3% of overall births in Scotland occurring spontaneously before 37 weeks gestation (1, 2) .Preterm babies have higher rates of perinatal mortality and morbidity and those that survive are at risk of multiple conditions including respiratory distress syndrome, central nervous system abnormalities, necrotising enterocolitis and sepsis. The mechanisms of preterm birth are poorly understood. Preterm birth can be spontaneous or induced and spontaneous preterm labour has multiple aetiologies. Current evidence suggests that prolonged treatment with progesterone and 17 α-hydroxyprogesterone caproate (17OHPC) may reduce the incidence of premature delivery in high risk patients with a history of spontaneous preterm birth (3) or with a short cervix. However, progesterone is not uniformly effective in preventing preterm labour and at present its principal mode of action on myometrium is unknown. I aimed to determine some of the specific mechanisms of action of progesterone.
Aims: I hypothesised that progesterone has a direct inhibitory effect on spontaneous myometrial contractility, induces increased sensitivity to tocolytic agents and decreases sensitivity to contractile agonists such as oxytocin. I also hypothesised that progesterone has inhibitory effects on endogenous uterine stimulants, stimulatory effects on endogenous uterine relaxants, induces upregulation of endogenous receptors that inhibit uterine contractions and inhibits contraction associated proteins both in vitro and in vivo.
Methods: I recruited women already enrolled in the STOPPIT (a double blind randomised placebo controlled study of progesterone for the prevention of preterm birth in twins) who were given vaginal progesterone, or placebo and who were scheduled for caesarean section. I also recruited women with healthy twin or singleton pregnancies undergoing elective caesarean section. Myometrial biopsies were obtained from the upper border of the lower uterine segment incision during caesarean section. Samples were divided and used for contractility measurements, or subsequent mRNA, protein and immunohistochemical analysis.
Myometrial strips were cut and suspended under resting tension within organ baths. Concentration-response curves were carried out in response to oxytocin, levcromakalim, nifedipine and ritodrine to ascertain any reduction in effect by progesterone on oxytocics or enhancement of tocolytic effects. I also carried out concentration-response curves to progesterone alone and in the presence of potassium channel blocking agents. I then assessed ex vivo, the inherent contractility of the placebo versus progesterone groups from myometrium sampled from the STOPPIT cohort of patients.
I carried out cell culture experiments on myometrium from healthy singleton women who were not in labour at the time of sampling. Myometrial explants were placed in cell culture medium, cultured for 1, 4 and 24 hours, and the supernatants were then analysed using Bio-Plex array technology to ascertain cytokine release. I selected time points and concentrations conditions to incubate myometrial tissue using progesterone and 17OHPC and was able to assess cytokine release.The myometrial explants were used for subsequent molecular studies. I performed real time-polymerase chain reaction (RT-PCR) (Abi,Taqman) to quantitate endogenous inhibitors of uterine contractility (cGRPR, EP2,NOS), cytokines (interleukins- IL6, IL8, IL1β), uterine stimulants COX-2 and gap junction components ( connexin 26 and connexin 43) expressed relative to housekeeping gene 18s.
Lastly, I analysed the STOPPIT cohort of myometrial samples for to determine the in vivo effect of progesterone. We carried out RT-PCR (Abi,Taqman) to quantitate endogenous inhibitors of uterine contractility (cGRPR, EP2,NOS, PGDH), cytokines (IL6, IL8, IL1β) and gap junction components (connexin 26 and 43).I performed immunohistochemistry, staining for localisation of pro-inflammatory cytokines. I then carried out protein expression analysis using Western blot for contraction associate protein, connexin 43.
The project was approved by North Glasgow University Hospitals Research Ethics Committee ref no. 05/S0705/18. All patients gave written informed consent to participate.
Results: I found that progesterone, exerted consistent, rapid and sustained inhibition of the amplitude of spontaneous myometrial contractions in vitro at high concentrations however, this affect was not achieved through the principal potassium channels. Levcromakalim, a KATP channel opener, produced a concentration-dependent inhibition of the amplitude and frequency of spontaneous contractions. These effects were antagonised by the KATP channel blocker, glibenclamide. In contrast, glibenclamide had no effect on the progesterone-induced inhibition of myometrial contractility. Charybdotoxin 10 nM (which blocks IKCa, BKCa and Kv channels), iberiotoxin 100 nM (which blocks BKCa channels) and apamin 100 nM (which blocks SKCa channels) failed to affect the ability of progesterone to inhibit myometrial contractility.
In contrast, 17OHPC did not exert any inhibitory effect on myometrial activity in vitro. Results indicated, at the selected pharmacological doses used in vitro that progesterone did not increase sensitivity to tocolytic agents tested. There was no decrease in sensitivity to the uterotonin oxytocin. Lastly, from our STOPPIT patient cohort I demonstrated no difference between the progesterone and placebo groups in either spontaneous contractility, response to tocolytics as above or response to oxytocin. One main conclusion of this arm of the study is that in vivo progesterone therapy to prevent pre-term labour does not appear to modify contractility ex vivo.
I demonstrated that administration of progesterone but not 17OHPC for up to 24 hours in vitro does not appear to modify mRNA expression of uterine stimulants such as cytokines, COX-2 or endogenous uterine relaxants such as NOS and PGDH. Progesterone but not 17OHPC inhibited production of gap junction component connexin 43. This modification of contraction associated protein is in agreement with other literature presented on human myometrial data in vitro (4) .
I used STOPPIT patients as a potential example of the myometrial effects of progesterone in vivo with a placebo treated control group. Prolonged maternal administration of progesterone appeared to inhibit expression of gap junction components connexin 26 and 43 in myometrium. Connexin 43 importantly, was also modified in vitro within the progesterone treated arm. However, ex-vivo assessment of the functional impact on human myometrium does not demonstrate a long-term inhibitory impact on myometrial function with down regulation of endogenous contractile inhibitors such as eNOS and EP2. The connexins play an essential role in regulating synchronous myometrial contractions. If progesterone has been of benefit in those at risk of preterm labour with a history of spontaneous preterm birth, it is possible therefore that this is by reducing connexin expression, which prevents the development of these synchronous contractions whilst on progesterone therapy.
In summary, I have demonstrated putative mechanisms by which progesterone (and its analogue 17OHPC) might prevent preterm birth. Further studies characterising these pathways might inform the design of other agents which could provide additional efficacy in preventing preterm delivery.
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