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Study Question: What is the role of the endocannabinoid system (eCS) in the alterations of the endocrine system in a murine model of lipopolysaccharide (LPS)-induced miscarriage?, Summary Answer: In 7-days pregnant wild type, but not cannabinoid receptor type 1 knockout (CB1-KO) mice, LPS increased COX-2 expression and prostaglandin F 2α (PGF 2α ) production in the uterus leading to lower expression of prolactin receptor in the ovary and a marked regression of corpora lutea (CL), suggesting that the eCS mediates the deleterious effects of LPS on reproductive events., What Is Known Already: Appropriate systemic progesterone levels are critical for a successful pregnancy outcome. Precocious loss of luteal progesterone (P4) secretion leads to miscarriage in rodents. We have previously shown that LPS administration to pregnant mice induces embryonic resorption accompanied by a dramatic decrease in systemic progesterone levels in a murine model of inflammatory miscarriage, with the eCS mediating these LPS-induced deleterious effects., Study Design Samples/materials, Methods: CD1 wild-type (WT) and CB1-KO mice were randomly allocated to Vehicle (saline; i.p.) or LPS (0.5 μg/g body weight; i.p.) treated groups: (WT-Vehicle; WT-LPS; CB1-KO-Vehicle and CB1-KO-LPS). A single injection was given on day 7 of pregnancy and tissues (blood, ovary, uterus) were collected 6, 12, 24 and 48 h later. P4 and PGF2α plasma levels were determined by radioimmunoassay. Cyclooxygenase-2 (COX-2) mRNA (RT-PCR) and protein (Western blot) content in uterus was assayed. COX-2 and prolactin receptor (PrlR) mRNA levels in the ovary were assayed by RT-PCR. Tissue morphology of the CL was assessed by haematoxylin-eosin staining., Main Results and the Role of Chance: Treatment of 7-day pregnant WT mice with LPS induced a P4 withdrawal (p < 0.05), increased in uterine COX-2 mRNA and protein expression (p < 0.05) as well as an increase in uterine PGF 2α production (p < 0.05). These changes were absent in LPS-treated 7-day pregnant CB1-KO mice. In ovarian tissues, LPS treatment to 7-day pregnant WT mice induced a downregulation of PrlR mRNA expression (p < 0.05) together with an increase in COX-2 mRNA expression (p < 0.05) and PGF 2α content (p < 0.05). These effects were absent in the CB1-KO mice. Collectively, our results suggest a role for the eCS mediating LPS-induced deleterious effects on reproductive tissues., Limitations, Reasons for Caution: An important caveat of this study is the endocrine differences between mice and humans during pregnancy (e.g. P4 is produced by the CL throughout pregnancy in mice, whereas this is not the case in humans), which limits the extrapolation of the results presented here., Wider Implications of the Findings: Our findings provide new insights in the role of the endocannabinoid system in the physiopathology of reproduction as well as the role of this endogenous system as a mediator of LPS deleterious effects on reproductive tissues., Large Scale Data: None., Study Funding and Competing Interests: Dr Ana María Franchi was funded by Agencia Nacional para la Promoción Científica y Tecnológica (PICT 2010/0813 and PICT 2013/0097) and by Consejo Nacional de Investigaciones Científicas y Técnicas (PIP 2012/0061). The authors have no competing interests., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved.For Permissions, please email: journals.permissions@oup.com.)

Genital tract infections caused by Gram-negative bacteria induce miscarriage and are one of the most common complications of human pregnancy. LPS administration to 7-day pregnant mice induces embryo resorption after 24h, with nitric oxide playing a fundamental role in this process. We have previously shown that progesterone exerts protective effects on the embryo by modulating the inflammatory reaction triggered by LPS. Here we sought to investigate whether the in vivo administration of progesterone modulated the LPS-induced nitric oxide production from peripheral blood mononuclear cells from pregnant and non-pregnant mice. We found that progesterone downregulated LPS-induced nitric oxide production by a progesterone receptor-independent mechanism. Moreover, our results suggest a possible participation of glucocorticoid receptors in at least some of the anti-inflammatory effects of progesterone., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Genital tract infections are a common complication of human pregnancy that can result in miscarriage. We have previously shown that a lipopolysaccharide (LPS) induces embryonic resorption in a murine model of inflammatory miscarriage. This is accompanied by a dramatic decrease in systemic progesterone levels associated with a robust pro-inflammatory response that results in embryo resorption. Here, we tested the hypothesis that the endogenous cannabinoid system (eCS), through cannabinoid receptor 1 (CB1), plays a role in regulating progesterone levels and, therefore, the pro-inflammatory response. We show that LPS treatment in pregnant mice causes significant changes in the eCS ligands, which are reversed by progesterone treatment. We further show the CB1-KO mice maintain higher plasma progesterone levels after LPS treatment, which is associated with a feebler uterine inflammatory response and a significant drop in embryo resorption. These data suggest that manipulation of CB1 receptors and/or ligands is a potential therapeutic avenue to decrease infection-induced miscarriage., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Increased anandamide concentrations are associated with pregnancy failure. Anandamide levels are regulated by the fatty acid amide hydrolase (FAAH). The aim of the study was to investigate the role of progesterone (P) on FAAH modulation in murine peripheral blood mononuclear cells (PBMC) under septic conditions. We observed that in vivo administration of LPS to non-pregnant (NP) mice decreased FAAH activity of PBMC while in pregnant mice no changes in FAAH activity were observed. NP animals administered with P had a similar response to LPS as the pregnant animals. Also, NP mice injected with P antagonist and P showed that the effect of P on LPS-reduced FAAH activity was impaired. Furthermore, LPS produced a decrease in the ratio of PR-B/PR-A in NP animals. Our results showed that, in our model the endotoxin decreased PBMC's FAAH activity and this condition was reverted by P in a receptor-mediated fashion., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Lipopolysaccharide (LPS) administration to mice on day 7 of gestation led to 100% embryonic resorption after 24 h. In this model, nitric oxide is fundamental for the resorption process. Progesterone may be responsible, at least in part, for a Th2 switch in the feto-maternal interface, inducing active immune tolerance against fetal antigens. Th2 cells promote the development of T cells, producing leukemia inhibitory factor (LIF), which seems to be important due to its immunomodulatory action during early pregnancy. Our aim was to evaluate the involvement of progesterone in the mechanism of LPS-induced embryonic resorption, and whether LIF can mediate hormonal action. Using in vivo and in vitro models, we provide evidence that circulating progesterone is an important component of the process by which infection causes embryonic resorption in mice. Also, LIF seems to be a mediator of the progesterone effect under inflammatory conditions. We found that serum progesterone fell to very low levels after 24 h of LPS exposure. Moreover, progesterone supplementation prevented embryonic resorption and LPS-induced increase of uterine nitric oxide levels in vivo. Results show that LPS diminished the expression of the nuclear progesterone receptor in the uterus after 6 and 12 h of treatment. We investigated the expression of LIF in uterine tissue from pregnant mice and found that progesterone up-regulates LIF mRNA expression in vitro. We observed that LIF was able to modulate the levels of nitric oxide induced by LPS in vitro, suggesting that it could be a potential mediator of the inflammatory action of progesterone. Our observations support the view that progesterone plays a critical role in a successful pregnancy as an anti-inflammatory agent, and that it could have possible therapeutic applications in the prevention of early reproductive failure associated with inflammatory disorders.

Anandamide is an endocannabinoid known to participate in reproductive processes. This study observed that 17beta-oestradiol and progesterone modulated the production of anandamide and its metabolizing enzymes in the rat uterus. Anandamide production was highest at the oestrous stage and 17beta-oestradiol and progesterone stimulated its synthesis in ovariectomized rats. During early pregnancy, anandamide production remained constant on days 1-5 of gestation and diminished towards day 6. On day 6, implantation sites showed lower synthesis compared with interimplantation sites. In the delayed implantation model, 17beta-oestradiol inhibited anandamide synthesis compared with progesterone. During pseudopregnancy, anandamide production did not decrease towards day 6 as occurred during normal gestation. The administration of 17beta-oestradiol augmented anandamide production in rats on day 5 of pseudopregnancy; the treatment with mifepristone did not produce any change in anandamide synthesis. Anandamide-metabolizing enzymes were regulated by progesterone and 17beta-oestradiol. The effect of ovarian hormones on the synthesis of anandamide depends on different physiological conditions, oestrous cycle and early pregnancy, and on the presence of the activated blastocyst. Thus, ovarian hormones, as signals that emanate from the mother, operate in conjunction with the blastocyst intrinsic programme, regulating the synthesis of anandamide in a specific manner during crucial reproductive events that may compromise pregnancy outcome.

We attempted to explore possible mechanism(s) subserving the influence of oxytocin (O) and of progesterone (P) in the isolated rat uterus studying the action of these hormones on: the synthesis and release of prostaglandins (PGs), the metabolism of labelled arachidonic acid and the uptake of Ca2+ by the tissue from ovariectomized animals. The experiments were done with uterine preparations isolated from spayed rats treated or not with P prior to sacrifice and afterward incubated or not with O 'in vitro'. While uterine strips from untreated spayed rat uterus exhibited a basal release into the incubating medium of approximately the same amounts of PGF2 alpha, and PGE2, the 'in vitro' addition of O (50 mU/ml) increased significantly (p < 0.05) the output of PGF2 alpha without changing the release of PGE2. In tissue from rats injected with P prior to sacrifice the output of PGF2 alpha rose significantly (p < 0.01) as it did after the addition of O to preparations obtained from spayed rats treated with P in comparison to findings in uteri from spayed rats but not in comparison to uteri from spayed rats treated with P alone. Moreover, the 'in vitro' addition of O (50 mU/ml) only increased the formation of PGF2 alpha (p < 0.05) and of 5-HETE (p < 0.05); nevertheless the administration of P to spayed rats diminished significantly (p < 0.05) the formation of 6-keto-PGF1 alpha from uteri, but increased that of PGF2 alpha (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of progesterone in the solution suspending uterine preparations isolated from ovariectomized rats on the output of PGF2 infinity and PGE2-like material, were explored. Uterine incubation for one hour with progesterone (50 or 100 micrograms/ml, final concentration) did not alter PGF or PGE release into the bath medium, when compared with untreated controls. One hour after hormone removal the output of either prostaglandin, from uteri previously primed with progesterone at 50 micrograms/ml, was also similar to controls. However, preparations exposed to progesterone at 100 micrograms/ml for one hour and subjected afterwards to a second incubation interval of 60 minutes without the hormone, released into the suspending medium significantly more PGF, but not PGE, than controls. On the other hand, incubation with progesterone (at 100 micrograms/ml) for 120 minutes (not followed by a period of steroid removal) failed to change PGF and PGE uterine output. These findings show that there is a direct but complex effect of progesterone on rat uterine release of PGF, i.e., the positive correlation between the steroid and PGF required not only a priming period with a high concentration of progesterone, but also its subsequent withdrawal. Such peculiarity is discussed in terms of a possible influence of progesterone on enhancing intramural uterine levels of PGF and inhibiting prostaglandin release, and also in relation to the role of hormone withdrawal after corpora lutea regression on PGF concentration in blood from uterine veins during the sex cycle.

The effects of progesterone (P4) and of calcium-ionophore A-23187, on the release of prostaglandins (PGs) E2 and F2 alpha, in uteri isolated from ovariectomized rats and the influences of mepacrine and nifedipine, were explored. The metabolism of labelled arachidonic acid (AA) into different prostanoids (6-keto-PGF 1 alpha, PGE 2 and PGF2 alpha) in uterine segments from spayed rats, injected or not with P4, was also studied. In all cases ovariectomy was performed 20-25 days prior to sacrifice. One group of spayed rats were injected with 4.0 mg of P4 during two days and sacrificed 24 h after the last injection. The remaining spayed animals were considered as controls. Tissue samples from both groups were incubated for one hour in the absence or in the presence of either A-23187 (1.0 microgram/ml), mepacrine (10(-3) M) or nifedipine (10(-6) M), or a combination of A-23187 plus mepacrine. At the end of the incubating period PGs in the suspending solution were extracted, separated, identified (TLC) and quantitated. The metabolism of 14C-AA into different prostanoids was explored in uterine segments from spayed rats, injected or not with P4 prior to sacrifice. Tissue prepared from P4-injected rats as well as those from rats not receiving P4 but incubated with ionophore A-23187, generated and released significantly more PGF2 alpha into the incubating solution than basal controls, but failed to exhibit changes in the basal output of PGE.(ABSTRACT TRUNCATED AT 250 WORDS)

The present experiments report the effects of estradiol or of progesterone on the activity of 15-prostaglandin-dehydrogenase (PGDH) in the uterus of spayed rats. When the substrate was PGF2 alpha the treatment with progesterone (4 mg X day-1, two days) or with estradiol-17-beta (0.5 ug + 1 ug) did not show any effect on the activity of the enzyme. On the contrary, uteri from ovariectomized rats injected with a higher dose of estradiol-17-beta (0.5 ug + 50 ug) exhibited a significant increment. When the substrate was PGE2, progesterone failed again to modify the enzyme activity, whereas estradiol, both at a low and at a high doses, enhanced significantly the uterine PGDH activity. The possibility of two different PGDHs for each PG and the role of estradiol in enhancing PGE2 catabolism into 15-keto-PGE2 as a mechanism subserving the effect of estrogens on the output of this PG in the rat uterus, are discussed.

The output of prostaglandin (PG) F and PGE-like material into the solution bathing uterine horns isolated from adult ovariectomized and immature rats under the influence of progesterone, was studied. The injection of progesterone (0.5; 1.0; 2.0 or 4.0 mg) 6 hours prior to sacrifice enhanced significantly the release of PGF-like substance without modifying that of PGE. The augmentation of PGF was detected as early as one hour after injecting 4.0 mg of progesterone and remained elevated at 2,4,6, 12 and 24 hours, following the treatment. Puromycin (50 mg/Kg), injected 6 hours before killing, failed to alter the release of PGF-like substance but clearly blocked the stimulating effect of the hormone. In addition, progesterone also enhanced significantly the release of PGF-like material by horns isolated from immature animals. The results suggest that progesterone receptors do not appear to be important for the described effect of the hormone because the preparations employed in the present study have a very small content of these receptors. Alternatively, it can be hypothesized that only a reduced number of progesterone receptors are sufficient for the action of the hormone augmenting the output of PGF-like material from the uterus.

The accumulation and output of 3H -prostaglandins (PGs), E2 and F2 alpha, into and from uterine strips isolated from ovariectomized rats, either in presence or in absence of exogenous progesterone, were explored. Tissue-to-medium ratio of 3H-counts (T/M-ratio), was determined. The same was done in solutions containing 14C-sucrose. During a 60 min incubation period in a solution containing 3H -PGF2 alpha, a net accumulation of radioactivity was evident in control (no progesterone) uterine slices. The T/M-ratio for 3H-PGF2 alpha, increased with time, reaching maximal values at 45 min. Progesterone (100 ug.ml-1) attenuated the uptake process, as evidenced by stable values of T/M-ratio, as time progressed. On the other hand, control T/M-ratio for 3H-PGE2, although similar to that for 3H -PGF2 alpha, was not influenced by the presence of exogenous progesterone. Regarding labelled PG release from the tissue, it was observed that, during an experimental period of 60 min, most tritium from control slices was released within the first 30 min after incubation with 3H -PGF2 alpha, whereas, following the presence and subsequent removal of exogenous progesterone, the bulk of 3H -released took place at 6-70 min. On the other hand, the release of 3H after an incubation with 3H -PGE2, was also maximal as that for 3H -PGF2, alpha within the first 30 min and resulted not altered after a period of exposure and removal of progesterone. The foregoing results suggest an specific pharmacological effect of progesterone, attenuating the uptake and retarding the outflow of PGF2 alpha, but not that of PGE2, into and from uterine slices of ovariectomized rats. Findings reported herein are discussed in terms of progesterone priming and withdrawal, in relation to PGF2 alpha fluxes in the rat uterus during the sex cycle, as well as in relation to PG binding to tissue receptors.

The effects of injecting estradiol, progesterone or both hormones to immature rats, on the spontaneous contractions of isolated uterine horns and on prostaglandin (PG) E and F-like materials released into the bathing medium, were studied. The in vitro contractile decrement detected both in controls as well as in the group receiving progesterone (P4) alone, was only minor. On the other hand, preparations from rats treated with 17-beta-estradiol (E2) exhibited a significant and progressive reduction of spontaneous motility. Coincident with these smaller contractions a significant diminution of PGE-output accompanied by an enhancement of the release of PGF-like material into the suspending solution, were found. The initial (postisolation) isometric developed tension (IDT) of uterine preparations from immature rats injected with sex hormones was significantly higher than that of tissues from untreated control animals. Neither the initial contractile frequency (CF) nor its constancy changed following hormonal treatment (E2 or P4), except in the case of uteri from animals treatment (E2 or P4), except in the case of uteri from animals administered with P4, which showed a smaller decrement of CF than controls. Furthermore, tissue preparations obtained from animals injected with P4 released into the bathing media similar amounts of PGE-like material as controls, but significantly more PGF. It is interesting that the injection of E2 plus P4 increased the output of PGE-like material but did not modify that of PGE i. e., the depressive effect of estradiol on the release of PGE into the bathing media was abolished by the administration of P4. On the other hand, the addition of indomethacin or acetylsalicylic acid to the bath solution reduced significantly contractions of uterine horns isolated from immature animals. The foregoing results, supporting our previous findings on the effect of P4 on PGF release from the rat uterus, are in keeping with the hypothesis holding that better spontaneous motility correlates well with a greater release of PGE and that E2 injection coincides with a depression of both PGE output and myometrial contractions.