The concurrent in vitro and in vivo release of PGE2 from a controlled-release hydrogel polymer pessary for cervical ripening

Twenty five patients booked for induction of labour, at 38 weeks or more gestation, were administered a controlled release vaginal polymer pessary containing 10 mg prostaglandin E2 (PGE2), designed to release 0.6 mg per hour in vivo. The release profile from the polymer was linear throughout the eight hour observation period with a correlation coefficient of 0.81, and regression slope of 0.93 mg/hr. with 95% confidence intervals of 0.63 mg/hr. to 1.23 mg/hr. This compared with a concomitant release profile in vitro which was uniform with time for the first five hours, but then continued at a decreasing rate with a correlation coefficient of 0.98. The relationship between PGE2 release and cervical score change was linear, with a correlation coefficient of 0.65. The results show that PGE2 release from the pessary in vivo is predictable, and suggest that the controlled release pessary offers the advantages of greater control of cervical ripening than alternative vehicles currently available.     

Prostaglandin release from preparations used vaginally for the induction of labour

The release and absorption profiles from the vagina of PGE2 in different vehicles used for cervical ripening and labour induction have been studied observing changes in concentrations of PGE metabolite (PGEM) and PGF metabolite (PGFM). In all groups a rise in PGEM concentration occurred over the 6 hour observation time but with wide variation. The profiles obtained differed markedly between the preparations under investigation correlating with the uterine contractions generated. PGFM generally showed little change. The model used could be explored further to enable modification of the vehicles used for PGE2 incorporation to achieve improved clinical results.     

Physiological involvement of placental endothelin-1 and prostaglandin F2alpha in uteroplacental circulatory disturbance in pregnant rats exposed to heat stress

Several studies suggest that heat stress affects placental functions including uteroplacental circulation, subsequently leading to pregnancy failure and birth weight reduction. To clarify the involvement of endothelin and placental prostaglandin (PG) systems in the uteroplacental circulation during heat stress, we examined the effects of i.v. administration of the endothelin receptor antagonist bosentan and the cyclooxygenase inhibitor indomethacin on uteroplacental blood flow and on placental PGE2 and PGF2alpha levels and their 13,14-dyhydro-15-keto-metabolites (PGEM and PGFM, respectively) in heat-exposed or non-heat-exposed pregnant rats. The administration of bosentan or indomethacin did not change uteroplacental blood flow in non-heat-exposed pregnant rats. In contrast, heat reduced uteroplacental blood flow in pregnant rats, but the reduction was reversed by the administration of bosentan or indomethacin before heat exposure. Heat did not change placental PGE2 or PGEM levels, but in pregnant rats it increased placental PGF2alpha and PGFM levels, which were reversed by bosentan or indomethacin. Our results suggest that the activation of placental endothelin receptor and PGF2alpha systems are involved in the uteroplacental circulatory disturbances produced by heat. PGF2alpha systems activated by heat may be involved in the vasoconstricting effects of endothelin-A and -B receptors during heat exposure.     

Improvement of radioimmunoassays for prostaglandins in bovine blood plasma and their application to monitor reproductive functions

Efficient RIA procedures are required for determination of prostaglandins (PGF(2alpha), PGE(2), PGI(2) and their metabolites) in bovine blood plasma to elucidate their significance in reproductive endocrinology. A new rapid efficient prepurification was developed using commercial octadecyl silicagel cartridges. Prepurification is especially necessary for the determination of 13,14-dihydro-15-keto-PGE(2) (PGEM). After prepurification, PGEM was first converted into the more stable 13,14-dihydro-15-keto-PGA(2) (PGAM) and measured in a RIA-system for PGAM. For PGF(2alpha), 13,14-dihydro-15-keto-PGF(2alpha) (PGFM), PGE(2) and 6-keto-PGF(1alpha) direct tests using 50 mul plasma per tube were elaborated. The validity of the tests was monitored by high performance liquid chromatography radioimmunoassay (HPLC RIA ). Infusion studies using PGF(2alpha) and PGE(2) showed that about 10% of these hormones remained unmetabolized after the first passage through the lungs. The biological half life of the metabolites PGFM and PGEM in bovines was estimated to be 4 min. Thus, PGFM and PGEM measurements in the peripheral circulation reflect even short-term secretory changes of PGF(2alpha) and PGE(2). During the infusion of PGF(2alpha) the levels of progesterone decreased but were not affected by PGE(2). Both prostaglandins caused increased oxytocin secretion. In the cow peripartum first PGEM elevations were measured 5 to 8 d ante partum, whereas PGFM increased 1 to 2 d ante partum. Then both prostaglandins increased simultaneously until parturition. In the postpartal phase PGFM was higher than PGEM, and both prostaglandins remained elevated for several days. Prostacyclin levels remained unchanged during the peripartal period.     

Plasma concentration of 13,14-dihydro-15-keto-PGE2 (PGEM) after various ways of cervix ripening with PGE2

PGEM concentration was determined radioimmunologically in a non-pregnant woman, in whom PGE2 was infused intravenously at increasing rates and in women, in whom labor was induced by various methods for local application of PGE2. There was excellent correlation between the amount of PGE2 infused intravenously and the levels of PGEM determined in the peripheral plasma. The following methods of local application of PGE2 were included in the study: 0.4 mg PGE2 gel placed retroamnially by means of a balloon catheter, 0.4 and 0.5 mg PGE2 applied endocervically and 3 mg PGE2 placed intravaginally in form of a single vaginal tablet; also included was a control-group, where only vaginal examination was performed. Bloods were drawn before, 30 minutes, 1, 2 and 3 hours after PGE2 administration. Mean levels of PGEM in the maternal peripheral plasma did not change neither within nor between the various groups. It is concluded from the present study, that local application of doses currently used to soften the cervix and/or induce labor at term do not lead to the same PGEM-concentration in the maternal blood as after intravenous infusion of PGE2 in doses normally used to induce labor.     

The concurrent in vitro and in vivo release of PGE2 from controlled-release hydrogel polymer pessary for cervical ripening

Twenty five patients booked for induction of labour, at 38 weeks or more gestation, were administered a controlled release vaginal polymer pressary containing 10mg prostaglandin E₂(PGE₂), designed to release 0.6mg per hour in vivo. The release profile from the polymer was linear throughout the eight hour observation period with a correlation coefficient of 0.81, and regression slope of 0.93 mg/hr. with 95% confidence intervals of 0.63mg/hr. to 1.23 mg/hr. This compared with a concomitatnt release profile in vitro which was uniform with time for the first five hours, but then continued at a decreasing rate with a correlation coefficient of 0.98. The relationship between PGE₂ release and cervical score change was linear, with a correlation coefficient of 0.65. The results show that PGE₂ release from the pessary in vivo is predictable, and suggest that the controlled release pessary offers the advantages of greater control of cervical ripening than alternative vehicles currently available.     

A dystocia and caesarean section model to characterize uteroplacental prostaglandin concentrations associated with retained placenta in dairy cattle

Plasma concentrations of prostaglandin F(2a) (PGF(2a), 13, 14-dihydro-15-keto-prostaglandin F(2a) (PGFM), prostaglandin E(2) (PGE(2)) and 13,14-dihydro-15-keto-prostaglandin E(2) (PGEM) were determined by RIA in blood samples taken from the jugular vein and the uteroplacental circulation (umbilical vein, umbilical artery and uterine vein) of 13 Holstein Friesian cows during caesarean section. According to discharge of placenta cows were divided in 2 groups. Group I (shedding of placenta within 12 hours, NRP, n=8) and Group II (retained placenta, RP, n=5). In blood samples taken from the jugular vein before surgery, no significant differences existed between groups regarding PGF(2a), PGFM, PGE(2) and PGEM. Concentrations of PGF(2a) and PGFM in the uteroplacental circulation of NRP cows were significantly higher than those of RP cows (except for PGFM in the umbilical vein). For all sampling sites except the jugular vein before surgery, PGE(2) and PGEM levels of NRP cows were significantly higher compared to RP cows.     

Uteroplacental production of eicosanoids in ovine pregnancy

Dramatic cardiovascular alterations occur during normal ovine pregnancy which may be associated with increased prostaglandin production, especially of uteroplacental origin. To study this, we examined (Exp 1) the relationships between cardiovascular alterations, e.g., the rise in uterine blood flow and fall in systemic vascular resistance, and arterial concentrations of prostaglandin metabolites (PGEM, PGFM and 6-keto-PGF1 alpha) in nonpregnant (n = 4) and pregnant (n = 8) ewes. To determine the potential utero-placental contribution of these eicosanoids in pregnancy, we also studied (Exp 2) the relationship between uterine blood flow and the uterine venous-arterial concentration differences of PGE2, PGF2 alpha, PGFM, 6-keto-PGF1 alpha, and TxB2 in twelve additional late pregnant ewes. Pregnancy was associated with a 37-fold increase in uterine blood flow and a proportionate (27-fold) fall in uterine vascular resistance (p less than 0.01). Arterial concentrations of PGEM were similar in nonpregnant and pregnant ewes (316 +/- 19 and 245 +/- 38 pg/ml), while levels of PGFM and PGI2 metabolite 6-keto-PGF1 alpha were elevated 23-fold (31 +/- 14 to 708 +/- 244 pg/ml) and 14-fold (12 +/- 4 to 163 +/- 78 pg/ml), respectively (p less than 0.01). Higher uterine venous versus uterine arterial concentrations were observed for PGE2 (397 +/- 36 and 293 +/- 22 pg/ml) and 6-keto-PGF1 alpha (269 +/- 32 and 204 +/- 32 pg/ml), p less than 0.05, but not PGF2 alpha or TxB2. Although PGFM concentrations appeared to be greater in uterine venous (1197 +/- 225 pg/ml) as compared to uterine arterial (738 +/- 150 pg/ml) plasma, this did not reach significance (0.05 less than p less than 0.1). In normal ovine pregnancy arterial levels of PGI2 are increased, which may in part reflect increased uteroplacental production. Moreover the gravid ovine uterus also appears to produce PGE2 and metabolize PGF2 alpha.     

Prospective study of different methods and routes of administration of prostaglandin E2 to improve the unripe cervix.

Methods of vaginal and extra-amniotic prostaglandin administration to achieve ripening of the cervix as a preliminary to induction of labour are described. Three groups of twenty patients with unfavourable induction features were studied, each receiving prostaglandin E2 the evening prior to planned induction. One group received PGE2 500 micrograms suspended in a viscous medium extra-amniotically. One group received PGE2 3 mg suspended in a viscous medium into the vaginal vault. A third group received a 3 mg PGE2 vaginal pessary to the posterior fornix. Improvement in cervical status at time of induction occurred in all groups but no single group had a significant advantage when regarding mean improvement, the induction-delivery interval or the number of patients in whom labour began before formal induction. However, with regard to relative cost, ease of preparation and storage, as well as patient and medical staff convenience, Prostaglandin E2 in pessary form is a superior form of administration.     

Pre-parturition profile of steroids and prostaglandin in cows with or without foetal membrane retention

Retained foetal membranes in cattle is one of the most common complications associated to the reduction in milk yield and impaired fertility in dairy cattle. In order to determine some endocrine mechanisms controlling parturition and delivery of foetal membranes, plasma concentrations of steroids and prostanoids were determined in 20 healthy Holstein cows. Samples were taken within the interval of 5 days pre-parturition to 12h after calving. Progesterone (P4) levels were similar in cows with (PR) and without (NPR) placental retention. While the estradiol-17beta (E2) peak at parturition was lower in PR than in NPR cows, cortisol levels were greater in PR cows 12 and 24h pre-parturition. The Prostaglandin F2alpha metabolite (PGFM) levels were higher at parturition in NPR compared with the PR group, but 12h later, these levels in the PR group increased so that concentrations were greater as compared with NPR cows. The Prostaglandin E2 metabolite (PGEM), 24, 48 and 72 h pre-parturition, were higher in PR cows. However, the PGFM:PGEM ratio was greater in cows up NPR at all time when included, indicating the importance of higher levels of Prostaglandin F2alpha (PGF2alpha) than Prostaglandin E2 (PGE2) for normal placental delivery. In conclusion, placental retention was related to both estrogen and PGF2alpha deficiency, which may be a consequence of metabolic stress leading to PGE2 and maternal cortisol synthesis before parturition.     

Uterine activity and prostaglandin production following intraamniotic hyperosmolar urea

The relationship between endogenous prostaglandin (PG) production and uterine activity was studied in hyperosmolar urea induced abortion patients. Polygraphic recordings of intraamniotic pressure were obtained at periodic intervals following intraamniotic injection of 80 gm urea. At 0, 0.25, 1, 4 and 8 hours amniotic fluid and blood samples were obtained for PGE, PGF and 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) analysis by radioimmunoassay. Blood was also sampled at time of absorption. In eight patients studies, uterine tone was elevated by 0.25 hour although no rhythmic contractions were observed by 1 hour. At 4 hours, amniotic fluid PGF concentration increased significantly (P < .01) over the pre-injection value and continued to increase at 8 hours. Amniotic fluid PGE, PGFM and all plasma PG's showed no change during the 8 hour period following urea administration. At time of abortion the plasma PGFM concentration was significantly greater than at the time of injection (238 ± 54.4 vs. 86.7 ± 7.3 pg/ml). There was no significant differences between pre-injection and absorption plasma PGF or PGE concentrations. In the present study, there is no evidence that increased prostaglandin production precedes urea induced contractions. The possible role of PG's in uterine contractions is discussed.     

The radioimmunological estimation of 13, 14-dihydro-15-ketoprostaglandin E2 in plasma and cervical mucus

Highly specific antibodies to 13,14-dihydro-15-ketoprostaglandin E2 (PGEM) were raised in rabbits. The animals were immunized with PGEM-bovine serum albumin (BSA)-conjugates. The metabolites were extracted with dichloromethane followed by column chromatography. The final antisera dilution was 1:15000 and the cross-reactivity towards prostaglandin A2, F2 alpha, I2, 13,14-dihydro-15-ketoprostaglandin F2 alpha was less than 0.1%. The limit of detection was 7.8 +/- 4.7 pg/ml plasma at the standard range of 3.9 to 500 pg/ml. The intra- and inter-assay variations were 5 and 12%, respectively. PGEM was measured throughout the menstrual cycle in female volunteers. In normal ovulatory women (n = 6) plasma concentrations of PGEM varied between 0.94 to 2.19 ng/ml. A significant increase of plasma PGEM was detected in the preovulatory phase of the cycle (P less than 0.01) over basal levels. In three of these volunteers cervical mucus was analyzed on PGEM and PGFM concentrations showing a fluctuation from 2 pg to 109 pg for PGEM and 0.05 pg to 2.4 pg for PGFM per ml of cervical mucus. The lowest concentrations have been found at the time of ovulation. The application of the radioimmunological method to the measurement of PGEM in addition to the measurement of prostaglandin E2 may be useful for estimating the turnover rates of this fatty acid.     

Cervical ripening and plasma prostaglandin levels. Comparison of endocervical and extra-amniotic PGE2

Two modes of cervical application of a gel containing PGE2 have been compared in a total of 30 patients with indication for induction of labor and unripe cervix. Fifteen patients had gel injected endocervically; in 10 patients the gel contained 400 micrograms PGE2, in 5 controls the gel was inactive. Fifteen subjects had a 15 ml Foley catheter passed through the cervix and placed extra-amniotically; in 10 of them 3 ml gel with 400 or 800 micrograms PGE2 was injected, while 5 controls received inactive gel. Plasma levels of 13,14-dihydro-15-keto-PGE2 alpha (PGFM) were measured in blood samples drawn before and 1/2, 1, 2, 4, 6, and 8 hours after gel application. Neither the Foley catheter nor the application of inactive gel caused significant changes in the cervical scores or the PGFM levels. PGE2 in the endocervix increased cervical scores without altering plasma PGFM levels. Extra-amniotic PGE2 caused a more rapid increase of the cervical scores and a progressive rise in PGFM levels. The plasma (PGFM) levels were found to be related to the degree and to the rate of cervical dilatation. The correlation with cervical dilatation was highly significant. Labor began spontaneously or after artificial rupture of the membranes in 80% of the extra-amniotic, and 50% of the endocervical PGE2-group, but in none of the controls. These data indicate that increased uterine PGF2 alpha production is not necessary for the early stages of cervical ripening, whereas dilatation beyond 4 cm does not proceed without such increase.     

The measurement of the main PGE metabolite, 13,14-dihydro-15-keto prostaglandin E by radioimmunoassay using methyl oxime stabilization.

The measurement of 13,14-dihydro-15-keto prostaglandin E2 [PGEM] is complicated by the artefactual formation of compounds of the corresponding A series which are reactive towards protein. Existing methods of assay depend on the deliberate dehydration to the 'A' form followed by cyclization in alkaline solution to a bicyclic derivative which is stable and can be measured by radioimmunoassay. We report an alternative approach using methyl oximation of the 9- and 15-keto groups which confer stability on the molecule. This derivatization is simple and does not involve an active intermediate such as those of the PGA series. The antiserum for radioimmunoassay is raised against the methyl oxime form. The label is the methyl oxime of PGEM coupled to a tripeptide Pro-gly-tyr through the nitrogen in the proline ring. This is a linkage distinct from that used to raise the antiserum and thus is not preferentially recognized over the endogenous analyte; this results in a high sensitivity assay. The results correlated well with those from the bicyclic assay when both assays were used to measure the same samples of peripheral blood from women receiving a sustained release PGE pessary for ripening the cervix. The technique provides a rapid and reliable method for determining prostaglandin E metabolites.     

Dynamics of prostaglandin secretion, intrauterine fluid and uterine clearance in reproductively normal mares and mares with delayed uterine clearance

Two experiments were performed to investigate relationships between oxytocin, prostaglandin release, uterine emptying and fluid accumulation in the uterus. In Experiment 1, the effect of oxytocin on the pattern of prostaglandin release during uterine clearance of radiocolloid was measured in 5 normal mares and 5 mares with delayed uterine clearance. Uterine clearance was measured during estrus by scintigraphy at 0, 60 and 120 min after colloid infusion. After the 120-min reading, 20 IU, i.v., oxytocin were given, and the amount of colloid cleared was measured at 135, 150 and 180 min. Plasma was obtained prior to and during scintigraphy at 5- and 15-min intervals to measure concentrations of 15-keto-13,14-dihydro-PGF2 alpha metabolite (PGFM) by RIA. In Experiment 2, plasma PGFM levels were compared after administration of oxytocin in 8 normal mares and 6 mares with delayed uterine clearance to determine if intrauterine fluid stimulated prostaglandin release. Mares received 2 treatments in a cross-over design. Treatment 1 consisted of 20 IU, i.v., oxytocin during estrus. Treatment 2 consisted of an infusion of 10 mL, i.u., saline 15 min prior to oxytocin administration. Treatments were performed 4 to 6 h apart. Blood was collected and PGFM was measured as in experiment 1. Data were analyzed by least squares analysis of variance. In Experiment 1, regression analysis of scintigraphy and PGFM profiles indicated that time response curves differed between groups (P < 0.01). At 120 min, normal mares retained 40.4 +/- 4.9% (mean +/- SEM) of the radiocolloid while mares with delayed clearance retained 88 +/- 5%. Fifteen minutes after oxytocin administration (135 min), all normal mares and 4 of 5 mares with delayed clearance retained only < 6% of the colloid. During the first 120 min, plasma PGFM concentrations did not differ between the 2 groups. After oxytocin was given, plasma PGFM concentrations increased in 4 of 5 mares with delayed uterine clearance (80 to 3,096 pg/mL) but not in normal mares (13 to 46 pg/mL). In Experiment 2, plasma PGFM concentrations did not rise in normal mares but rose in 3 of 6 mares with delayed clearance (135 to 483 pg/mL) independent of treatment or period. The results suggest that intrauterine clearance of radiocolloid after oxytocin administration appears to be independent of PGF2 alpha release in normal mares during estrus. The difference in prostaglandin release response after oxytocin administration between the 2 groups was unrelated to the presence of intrauterine fluid.     

The systemic absorption from the vagina of prostaglandin E2 administered for the induction of labour

The absorption rates of PGE2 released from tablets, gel and pessary bases inserted into the vagina for the induction of labour in a total of 27 patients have been measured by means of serial plasma 15-keto-PGE2 levels. The results are discussed with a view to obtaining both the optimum dose of PGE2 and vehicle of release.     

Fetal responses to maternal and intra-amniotic lipopolysaccharide administration in sheep

A link between intrauterine infection and premature labor is widely accepted, yet the fetal inflammatory responses to such infections are not well understood. Our aim was to use a sheep model in which an inflammatory state was induced by lipopolysaccharide (LPS) administration during pregnancy to the maternal systemic, intra-amniotic or extra-amniotic compartments. Fetal and maternal blood gases and uterine electromyographic activity along with fetal and maternal circulating concentrations of prostaglandins PGE2 and PGFM, cortisol, and interleukin-6 were determined. Maternal systemic LPS treatment resulted in mild maternal hypoxemia, a rise in temperature, greater fetal hypoxemia, and a marked rise in fetal cortisol and PGE2 concentrations that persisted for 48 h. Intra-amniotic administration of LPS at doses higher than those used systemically caused an increase in fetal cortisol and PGE2 concentrations as well as a rise in uterine activity, but these were lesser in magnitude. Extra-amniotic LPS administration caused no overt fetal or maternal inflammatory responses. We conclude that maternal LPS treatment markedly elevated fetal cortisol and PGE2 concentrations. This may be a potential protective mechanism that aids the fetus in the event of premature delivery. The attenuated fetal response to intra-amniotic LPS treatment, despite the much higher dose used, may support a role for the amniotic fluid in protecting the fetus from endotoxin exposure during pregnancy.     

Specific production of prostaglandin E by human amnion in vitro

Prostaglandin production by intra-uterine human tissues has been investigated using a method of tissue superfusion. Tissues were obtained at elective Caesarean section and after spontaneous vaginal delivery. It was found that all the tissues studied (amnion, chorion, decidua and placenta) produced more prostaglandin E (PGE) and 13,14-dihydro-15-keto-prostaglandin F (PGFM — the major circulating metabolite of prostaglandin F) than prostaglandin F (PGF). Amnion produced significantly more PGE (but not PGF or PGFM) than any other tissue. Prostaglandin production by each tissue was similar whether it was taken at elective Caesarean section or after spontaneous vaginal delivery.     

Control of luteolysis in the one-humped camel (Camelus dromedarius)

Blood plasma concentrations of 13,14-dihydro-15-keto PGF2 alpha (PGFM) were measured in groups of mature non-pregnant and pregnant camels to study PGF2 alpha release patterns around the time of luteolysis and the timing of the signal for pregnancy recognition. Injection of each of four camels with 10 and 50 mg of PGF2 alpha showed clearly that five times the dose of exogenous hormone produced five times the amount of PGFM in peripheral plasma, thereby indicating that, as in other animal species, PGFM is the principal metabolite of PGF2 alpha in the camel. Serial sampling of three non-pregnant camels on each of days 8, 10 and 12, and three pregnant camels on day 10, after ovulation for 8 h showed a significant (P < 0.05) rise in mean plasma PGFM concentrations only on day 10 in the non-pregnant, but not the pregnant, animals. A single intravenous injection of 20, 50 or 100 iu oxytocin given to three groups of three non-pregnant camels on day 10 after ovulation did not increase their basal serum PGFM concentrations. However, daily treatment of six non-pregnant camels between days 6 and 15 (n = 3) or 20 (n = 3) after ovulation with 1-2 g of the prostaglandin synthetase inhibitor, meclofenamic acid, inhibited PGF2 alpha release and thereby resulted in continued progesterone secretion throughout the period of meclofenamic acid administration. These results showed that, as in other large domestic animal species, release of PGF2 alpha from, presumably, the endometrium controls luteolysis in the dromedary camel. Furthermore, reduction in the amount of PGF2 alpha released is associated with luteal maintenance and the embryonic signal for maternal recognition of pregnancy must be transmitted before day 10 after ovulation if luteostasis is to be achieved. However, the results also indicate that, in contrast to ruminants, the release of endometrial PGF2 alpha in the non-pregnant camel may not be controlled by the release of oxytocin.     

Prostaglandin concentrations in intra-uterine tissues from late-pregnant sheep before and after labour

Prostaglandin E (PGE), prostaglandin F (PGF) and 13,14-dihydro-15-keto-prostaglandin F (PGFM) have been measured in cotyledons and myometrium from sheep before and after labour. Fetal cotyledons contained more PGE than maternal cotyledons which in turn contained more than myometriu. The maternal cotyledon contained the highest concentrations of PGF, but the fetal cotyledon was the only tissue exhibiting a statistically significant rise in the concentration of PGF following labour. Concentrations of PGFM were closely correlated with (although usually lower than) those of PGF.