The Endocannabinoid System in Pregnancy and Reproduction.

2009.05.11

Following the discovery of the endocannabinoids and cannabinoid receptors, research has focused on whether this system may be involved in the physiological regulation of pregnancy. Most studies examining the expression and role of cannabinoid receptors in the reproductive system have been carried out in the mouse. Das and associates used Northern blot hybridization and reverse transcriptase-polymerase chain reaction (RT-PCR) to demonstrate that CB1 but not CB2 mRNA is expressed in the mouse uterus. Both CB1 and CB2 mRNA have been identified in mouse pre implantation embryos. A recent study has shown that sex steroids control the expression of the CB1 gene in the anterior pituitary gland of both male and female rats, leading to the speculation that such a regulatory mechanism might be operational also in the reproductive organs. Paria et al. utilized cannabinoid receptor mutant mice to further investigate the role of CB1 and CB2 in pre implantation embryo development and in implantation. They found that the embryos recovered from CB1, CB2 mice were asynchronous with normal development. For example, on the fourth day following fertilization, about 98% of wild type embryos were blast cysts, whereas only about 61% of the double-knockout embryos were at the blast cyst stage (most of the mutant embryos were at the morula stage). Nevertheless, retarded embryo development had modest, if any, adverse effects on implantation. The mutant embryos were resistant to the effects of anandamide, and double-knockout mice were resistant to THC-induced implantation failure.

FAAH mRNA is also expressed in mouse pre implantation and implanted embryos, and uterine luminal and glandular epithelial cells. Furthermore, FAAH protein expression and activity was recently localized to these regions of mouse endometrial epithelium.

FAAH expression was demonstrated to fall from days 0 to 5.5 of pregnancy. Maccarrone and colleagues present two lines of evidence to suggest that FAAH modulation in the early stages of pregnancy are hormonally regulated and independent of the presence of embryos in the uterus. Firstly, pseudo pregnant mice undergo down regulation of FAAH expression and activity in the uterus; secondly, ovariectomised animal demonstrate less regulation of FAAH with pregnancy, however the down regulation is increased when these animals are treated with estrogen.

Until recently little information was available on cannabinoid receptor and FAAH distribution in human reproductive tissues and gestational tissues. RT-PCR studies have suggested that both CB1 and CB2 receptors are localized to the human myometrium. This method has also been used to demonstrate that human placenta expresses mRNA for both types of cannabinoid receptors; however it was unclear whether this study included placental membranes (amnion, chorio-decidua) or utilized solely placental villous tissue. Recently, we identified CB1 immune reactive labeling in most major cell types throughout all layers of the human placental membranes, as well as in the placental villous, suggesting that both cannabis and endocannabinoids could have an impact directly on placental tissues. Likewise FAAH activity has been demonstrated in human uterine epithelial cells, and in the epithelial layer and decidual layer of the human placenta. Human reproductive fluids, such as seminal plasma, mid-cycle oviduct fluid, follicular fluid and amniotic fluid have been reported to contain anandamide in the low nanomolar range.

Anandamide levels in the mouse uterus have been demonstrated to be inversely related to uterine receptivity for implantation; up regulation is correlated with uterine refractoriness to blast cyst implantation.

Furthermore, anandamide levels are highest in inter implantation sites and lowest at sites of implantation. The low levels of anandamide at the implantation sites correlated with high levels of both COX2 and FAAH levels in these regions, suggesting that one or both of these metabolizing enzymes may control the levels of anandamide. These studies correlate with the determination of blast cyst sensitivity to anandamide levels. Blastocysts exposed in culture to low levels (7 nM) of anandamide exhibit accelerated activity an trophoblast outgrowth, with an observed inhibition of differentiation at higher doses (28 nM). Liu et al. showed that low level of anandamide (14 nM) can significantly promote blast cyst attachment and outgrowth whereas high level (56nM) anandamide delay attachment and inhibits outgrowth of blast cysts. It was suggested that different culture conditions result in these discrepancies. Furthermore, anandamide and THC induce inhibition of embryo development and zonahatching of blast cysts most likely through a CB1 mediated pathway. Thus it is not surprising that increased cannabinoid levels may interfere with the implantation process. Infusion of the synthetic cannabinoid CP55, 940 via miniosmotic pumps during the pre implantation period prevents implantation in a CB1 receptor mediated mechanism, however infused THC does not produce this effect, except when administered with a cytochrome p450 inhibitor. This resulted in equivalent plasma levels, but an accumulation of THC in the uterus, suggesting that local metabolism of THC may protect against the deleterious effects of THC. When administered chronically anandamide prolongs the duration of pregnancy and increases the rate of still birth in rats, furthermore the postnatal development of the hypothalamic pituitary axis in the offspring of animals who receive anandamide during the pregnancy is temporarily inhibited particularly in males.

As was described for THC, anandamide decreases serum LH and prolactin levels in rats of both sexes. The effects are assumed to be due to hypothalamic regulatory centers, however, recently the CB1 receptor was identified in the anterior pituitary itself and receptor levels were demonstrated to be regulated by sex steroids allowing for the potential of a direct action. Intriguingly, hypothalamic levels of anandamide peak immediately before the onset of puberty in female rats, suggesting modulation of endocannabinoids and potentially FAAH in times of hormonal regulation aside from pregnancy.

In, perhaps, the most compelling study correlating the endocannabinoid system with pregnancy outcome, Maccarrone et al. reported the association between decreased levels of FAAH in maternal lymphocytes and early pregnancy loss in humans. This study also showed a clear regulation of FAAH expression and activity during the first trimester of normal pregnancy, with levels and activity peaking at 9–10 weeks, prior to dropping again by 12 weeks. No such increase was observed in the women who consequently miscarried. It was further shown that lymphocyte FAAH was stimulated by progesterone and Th2-type cytokines, which favor human fertility. Moreover, the addition of AEA to human lymphocytes in vitro inhibited the release of leukemia inhibiting factor, which is critical for implantation, and maintenance of the fetus in humans. More recently, Maccarrone et al. demonstrated low levels of FAAH in lymphocytes of in vitro fertilization-embryo transfer patients who failed to achieve an ongoing pregnancy than in those who become pregnant, and this was paralleled by a significant increase in blood AEA.

Interestingly, non-pregnant controls had the same FAAH activity and content as the subjects with normal gestation, suggesting that a down-regulation of FAAH occurred in lymphocytes of patients who failed to achieve pregnancy. Taken together these findings indicate that an active FAAH in maternal lymphocytes is needed for successful pregnancy, hence suggesting that the high levels of AEA that might follow the defective expression of FAAH could adversely affect gestation in humans. Indeed, approximately fourfold higher levels of blood AEA were observed in women experiencing miscarriage than in women with normal gestation (M. Maccarrone, V. Di Marzo, pers. comm.). Consistent with this proposal, defective leptin signalling, which causes sterility I leptin-deficient ob/ob mice has been recently associated with elevated levels of hypothalamic endocannabinoids in the same animals, whereas leptin treatment which restores fertility, reduces hypothalmic endocannabinoids.

In conclusion, recent studies have demonstrated that the endocannabinoid system is tightly modulated in gonadal tissues and during pregnancy. Marijuana, THC and other exogenous cannabinoids exert potent effects on this homeostasis. Furthermore these substances are modulated by and involved in the anterior pituitary and hypothalamic control of hormones and sex steroids. Thus these substances have the potential to have powerful effects on the reproductive health of females and males. Further studies into the roles of endocannabinoids in human hormone regulation and pregnancy will point towards the contribution of these compounds in normal and pathophysiology. Current understanding suggests that they may be critical in the areas of embryo implantation and miscarriage. For the time being it is clear that cannabis-based substances are contraindicated during pregnancy, as are compounds that might interact with endocannabinoid synthesis and metabolism.