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Invited critical review
Endocannabinoids and pregnancy
Anthony H. Taylor, Akwasi A. Amoako, Katerina Bambang, Tulay Karasu, Alpha Gebeh, Patricia M.W. Lam,
Timothy H. Marzcylo, Justin C. Konje ⁎
Endocannabinoid Research Group, Reproductive Sciences Section, University of Leicester, Leicester, United Kingdom
Received 18 December 2009
Received in revised form 9 March 2010
Accepted 9 March 2010
Available online 17 March 2010
Acylethanolamides such as anandamide (AEA), and monoacylglycerols like 2-arachidonoylglycerol are
endocannabinoids that bind to cannabinoid, vanilloid and peroxisome proliferator-activated receptors. These
compounds, their various receptors, the purported membrane transporter(s), and related enzymes that
synthesize and degrade them are collectively referred to as the “endocannabinoid system (ECS)”. Poorly
deﬁned cellular and molecular mechanisms control the biological actions of the ECS. Over the last decade
evidence has been emerging to suggest that the ECS plays a signiﬁcant role in various aspects of human
reproduction. In this review, we summarize our current understanding of this role especially the
involvement of AEA and related ECS elements in regulating oogenesis, embryo oviductal transport,
blastocyst implantation, placental development and pregnancy outcomes, and sperm survival, motility,
capacitation and acrosome reaction. Additionally, the possibility that plasma and tissue AEA and other
cannabinoids may represent reliable diagnostic markers of natural and assisted reproduction and pregnancy
outcomes in women will be discussed.
© 2010 Elsevier B.V. All rights reserved.
1. Introduction . . . . . . . . . . .................................................. 921
2. The endocannabinoid system (ECS).................................................. 922
3. Endocannabinoids and gametogenesis . . . . . ........................................... 923
3.1. Oogenesis . . . . . . . . .................................................. 923
3.2. Spermatogenesis . . . . . .................................................. 924
4. Endocannabinoids, early embryo-blastocyst development and oviductal transport . . . . . . . ..................... 924
4.1. Endocannabinoids and early embryo development . . . . . . . . . . . . . . . . . . ..................... 924
4.2. Endocannabinoids and transport through the Oviduct . . . . . . . . . . . . . . . . . ..................... 924
5. Endocannabinoids and implantation ................................................. 925
6. Endocannabinoids in early pregnancy................................................. 925
7. Endocannabinoids in the maintenance of pregnancy and labour. . . . . . . . . . . . . . . . ..................... 925
8. Feto-placental changes in endocannabinoid levels ........................................... 926
9. Possible “immunocannabinoid”system actions in pregnancy . . . . . . . . . . . . . . . . . ..................... 926
10. Conclusions . . . . . . . . . . .................................................. 927
Conﬂicts of interest . . . . . . . . . . .................................................. 927
References . . .............................................................. 927
Endocannabinoids are endogenous ligands that bind to the same
receptors as the most psychoactive agent in marijuana, delta-9-
-THC). The use of marijuana, an illicit drug
in the UK and USA [1,2], appears to be increasing [3,4]. Marijuana use
is associated with infertility in males and females, spontaneous
miscarriages, placental abruption, preterm birth, stillbirths [5–7] and
Clinica Chimica Acta 411 (2010) 921–930
⁎Corresponding author. Endocannabinoid Research Group, Reproductive Sciences
Section, Department of Cancer Studies and Molecular Medicine, University of Leicester,
Robert Kilpatrick Clinical Sciences Building, Leicester Royal Inﬁrmary, Leicester,
Leicestershire, LE2 7LX UnitedKingdom. Tel.: +44 116 252 5826;fax: + 44 116252 5824.
E-mail address: email@example.com (J.C. Konje).
0009-8981/$ –see front matter © 2010 Elsevier B.V. All rights reserved.
Contents lists available at ScienceDirect
Clinica Chimica Acta
journal homepage: www.elsevier.com/locate/clinchim
Author's personal copy
delivery of low birth weight babies . The fact that marijuana has
severe consequences on pregnancy, would suggest that the endoge-
nous ligands of the receptors are involved in the modulation of
reproduction. Recently, comprehensive studies by us and others have
indicated that endocannabinoids (especially the prototypical endo-
cannabinoid, N-acyl arachidonoylethanolamide [anandamide, AEA];
Fig. 1), are actively involved in the process of reproduction and
pregnancy. This review will examine the role of cannabinoids in
pregnancy, from gametogenesis through to birth with special focus
upon the effects of endocannabinoids and the endocannabinoid
system on these processes.
2. The endocannabinoid system (ECS)
The isolation of a speciﬁc cannabinoid receptor in the porcine brain
), that is responsible for mediating the pharmacological
effects of Δ
-THC , initiated the search for the endogenous ligands.
The ﬁrst ligand identiﬁed that could imitate the pharmacology of Δ
THC at the CB
receptor was anandamide  (Fig. 1). Subsequently,
isolation of a second cannabinoid receptor (CB
immune system  preceded the identiﬁcation of a second major
endocannabinoid, 2-arachidonoylglycerol (2-AG) (Fig. 1).Both AEA and
2-AG have afﬁnity for CB
, although with differentavidities.
More recently, other bioactive endocannabinoids have been identiﬁed
including O-arachidonoylethanolamine (virodhamine) ,N-arachi-
donoyl dopamine  and most recently N-acyl taurines, such as N-
arachidonoyl taurine . In addition, several endocannabinoid
congeners have been identiﬁed which have greatly reduced afﬁnity
yet elicit endocannabinoid-like activities. These include
oleoylethanolamide (OEA) and palmitoylethanolamide (PEA) (Fig. 1),
which are thought to elicit their endocannabinoid-like activities
through the so-called ‘entourage effect’, by either inhibition of
endocannabinoid catabolism , or reducing cellular uptake of AEA
thus leading to increased AEA concentrations [17,18]. Of all the possible
endocannabinoids, 2-AG and AEA are the most frequently studied, with
quantiﬁed levels in human tissues for 2-AG being consistently 10–100
times higher than those of AEA [19–21]. AEA, the entourage lipids OEA
and PEA, and to a lesser extent 2-AG have been quantiﬁed in human
biological ﬂuids including plasma and serum, seminal plasma, cerebro-
spinal ﬂuid, follicular ﬂuid, oviductal ﬂuid, amniotic ﬂuid, ovarian cyst
ﬂuid, milk and peritoneal ﬂuid, but seem to be absent from urine and
The ligands only comprise part of the endocannabinoid system;
the enzymes concerned with endocannabinoid synthesis and degra-
dation, a putative membrane transport system and the receptors
through which they elicit their physiological effects complete the
various components (Fig. 2). Endocannabinoids are synthesised on
demand from membrane phospholipids precursors and are not stored
In most tissues, the main rate-limiting step in AEA synthesis is the
conversion of N-arachidonoyl phosphatidylethanolamine (NAPE) into
AEA by a speciﬁc phospholipase D enzyme (NAPE-PLD) [30,31].
However, the continued synthesis of AEA in NAPE-PLD knockout mice
highlighted alternative pathways for the formation of AEA . These
pathways involve either hydrolysis of NAPE by phospholipase C to
yield phosphoAEA which is dephosphorylated by a protein tyrosine
phosphatase  or sequential acyl hydrolysis of NAPE and lysoNAPE
by abhydrolase domain containing 4 (Abh4) to form glycerophospho-
NAPE, which is then hydrolysed to AEA by a phosphodiesterase .
The synthesis of other endocannabinoids, especially the monoacyl
glycerol, 2-AG, is less well-deﬁned, but also thought to occur on
demand. For example, 2-AG is produced via diacylglycerol from
arachidonic acid-rich phospholipid precursors by the sequential
actions of phospholipase C and diacylglycerol lipase .
Degradation of AEA and 2-AG to free arachidonic acid is primarily
by the serine hydrolases fatty acid amide hydrolase (FAAH)  and
monoacylglycerol lipase (MAGL) [37–39], respectively. In addition to
hydrolysis, AEA has been shown to be metabolised by cyclooxygenase
2 to prostamides, ethanolamide derivatives of prostaglandins ,
and by the cytochrome P450 isozyme CYP2D6 to several epoxide
metabolites , which are thought to have their own physiological
actions. Endocannabinoid degradation requires transport of the
endocannabinoids into cells and how these cross the plasma
membrane is still open to debate [42,43]. Current theories include
carrier protein-mediated transport , diffusion driven by metab-
olism by FAAH , diffusion driven by compartmentalization of
intracellular AEA  and endocytosis .
Endocannabinoids predominantly exert their effects through bind-
ing to the cannabinoid receptors (CB
and GPR55). These seven-
transmembrane G-protein coupled receptors differ signiﬁcantly in their
distributions. Although CB
was initially localized in the nervous system
in the spleen,these receptors are now knownto be more widely
distributed throughoutthe body [48–58]. These receptors seem to signal
ligand occupancy by coupling inhibitory Gα
proteins which inhibit
adenylate cyclase activity, thus reducing intracellular cAMP concentra-
tions . In addition, agonist binding to CB receptors also leads to
activation of MAP kinases, which may partly explain the extensive
pathophysiological roles ascribed to endocannabinoids such as cell cycle
progression, apoptosis, cell differentiation and oncogenesis [60,61].
Stimulation of CB receptors can also cause rapid, transient elevation of
concentrations via phospholipase C, the inhibition of
channelsand activation of K
may not be the only effects endocannabinoids exert, as they bind to and
activate other types of receptors. For example, the transient receptor
potential vanilloid type 1 (TRPV1), a ligand-gated non-selective cation
channel that can be activated by a number of stimuli including heat and
pH and is involved in the transmission and modulation of pain, also
binds endocannabinoids, especially AEA and N-arachidonoyl dopamine
. Similarly, both endocannabinoids and entourage compounds bind
to and activate the G-protein coupled receptor GPR55, which is
considered a putative cannabinoid receptor  because compounds
Fig. 1. Chemical structures of the most psychoactive component of marijuana (Δ
tetrahydrocannabinol, the major endocannabinoids, and the “entourage compounds”.
922 A.H. Taylor et al. / Clinica Chimica Acta 411 (2010) 921–930
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other than endocannabinoids also bind to this receptor. The so-called,
‘entourage endocannabinoids,’OEA and PEA, also bind to peroxisome
proliferator-activated receptors (PPAR). In particular, they activate the
PPARαisoform to modulate cell differentiation, lipid metabolism and
regulate food intake .
3. Endocannabinoids and gametogenesis
The recent demonstration of the endocannabinoid system in the
human ovary has attracted a growing interest into the exact role of the
endocannabinoids in mammalian folliculogenesis .CB
receptors have been localized in oocytes at all stages of maturation
and the AEA synthetic enzyme NAPE-PLD and the hydrolytic enzyme
FAAH have also been localized in the growing secondary and tertiary
follicles and corpora lutea and albicantes . In addition, AEA has
been quantiﬁed in human follicular ﬂuid  and its concentration
found to be strongly correlated to oocyte maturation and quality in
women undergoing infertility treatment . The exact molecular
mechanism by which endocannabinoids modulate ovarian folliculo-
genesis remains speculative, but it is believed that they act through a
combination of central and peripheral mechanisms related to the
actions of gonadotrophins. Evidence for this comes from the now
dated studies showing that Δ
-THC exerted an adverse effect on
oocyte development and ovulation  in which Δ
-THC was shown
to interrupt the hypothalamic-pituitary-ovarian axis [68–70] and
suppress plasma follicle-stimulating hormone (FSH) and pre-ovula-
tory luteinizing hormone (LH) surge [71–73], causing an ovulation in
humans ,rats, rabbits and rhesus monkeys .The
reduction in gonadotrophin production results in poor quality oocytes
being retrieved during in-vitro fertilization . Since it is known that
cAMP accumulation is required for normal follicular development,
maturation and ovulation  and Δ
-THC acting through the CB
receptor normally has an inhibitory effect on cAMP levels, then the
inhibitory effect of Δ
-THC on folliculogenesis may be mediated via its
inhibition of adenylate cyclase, thereby reducing tissue concentra-
tions of cAMP, as has recently been shown in cultured rat granulosa
Other evidence also suggests that the endocannabinoid system,
which regulates energy balance by modulating appetite, food intake
and glucose metabolism [81–84] could interact with gametogenesis
through nutritional control. Obesity is commonly associated with
menstrual irregularities, chronic oligo-anovulation and infertility 
and regular ovulation is restored after simple management strategies
aimed at weight reduction leading to improved natural conception
[86,87]. Simultaneously, insulin resistance is a cardinal feature of
obesity-associated ovulatory dysfunction in the presence or absence
of polycystic ovary syndrome  and androgen hypersecretion, as a
result of hyperinsulinaemia, leads to altered ovarian physiology and
ovulatory dysfunction . Activation of CB
receptors in pancreatic
beta cells by AEA induces glucose intolerance, insulin hypersecretion
and insulin resistance in rats [90,91], suggesting that activation of the
ECS in the pancreas might explain some forms of female infertility.
The use of CB
antagonists (such as Accomplia®; rimonabant) for the
treatment of obesity, and its metabolic consequences, has been shown
to be very effective in reducing body weight or controlling leptin
concentrations [92,93] although with some serious adverse side
effects that have led to its discontinuance . It is therefore clear that
oogenesis could be disrupted through perturbations of the complex
interplay that exists between the endocannabinoids, leptin produc-
tion and obesity. For example, sustained stimulation of CB
hypothalamus by AEA or 2-AG has been observed in leptin-deﬁcient
mice, with elevation of hypothalamic levels of AEA and 2-AG which
Fig. 2. Components of the endocannabinoid system: The ligands 2-acylglycerol (2AG) and anandamide (AEA) are show as blue and red spheres respectively. The putative, but not yet
identiﬁed endocannabinoid membrane transporter (EMT) is shown as a bi-directional regulator. The enzymes responsible for the synthesis and degradation of AEA (N-acyl
transferase (NAT) and N-arachidonoyl phosphatidylethanolamine phospholipase D (NAPE-PLD) and fatty acid amide hydrolase (FAAH-1/2)) and of 2-AG (Di-acyl glycerol lipase
(DAG) and monoacyl glycerol lipase (MAGL)), convert these two endocannabinoids into arachidonic acid (AA; green spheres) and ethanolamine (silver spheres) or AA and glycerol
(yellow spheres), respectively. Cyclooxygenase 2 (COX-2) converts AEA into prostaglandin ethanolamines. Glycerol is acted upon by phospholipase C to produce diacylglycerols that
are reconverted into 2AG by DAGL. AEA once internalised may interact with the cytoplasmic domain of the transient vanilloid receptor type 1 (TRPV1) channel protein to alter
intracellular calcium levels. To aid clarity the possible signalling pathways of cannabinoid receptor (CBR) signalling have been omitted.
923A.H. Taylor et al. / Clinica Chimica Acta 411 (2010) 921–930
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was reversed after leptin treatment . Since FAAH is regulated in
some tissues by leptin [96–101] then these data are highly suggestive
of regulatory networks between leptin, FAAH and endocannabinoids
existing in the hypothalamus that are responsible for the exquisite
control of folliculogenesis. Nevertheless, this area of research is at an
early stage and further work is required to understand the complex
network of central and peripheral actions of endocannabinoids with
regard to ovarian function.
Accumulating evidence suggest that endocannabinoids may also
modulate male reproductive function . The ﬁrst indication for
this comes from studies of chronic marijuana users, where transient
adverse reproductive toxicity occurs and Δ
-THC directly inhibits
sperm motility and viability in vitro . In experimental animals,
-THC induces impotence in rats , decreases rat testicular
weight [105,106], decreases testosterone production which impairs
spermatogenesis and sperm motility [107,108] and in mice similarly
reduces the production of spermatozoa that tend to have abnormal
The exact mechanism by which Δ
-THC inhibits spermatogenesis
remains speculative but may involve a complex cross-talk between
FSH, LH and cannabinoids .Δ
-THC inhibits the release of FSH
and LH from the pituitary gland and consequently [111–114] thereby
inhibits spermatogenesis and testosterone production. By contrast, 2-
AG and to a greater extent AEA, mimic Δ
-THC by inhibiting FSH, LH
and prolactin release from the pituitary gland [115–120] suggesting a
common mechanism of action in both the hypothalamus and
receptors have been demonstrated in
gonadotrophs within the hypothalamus  whilst the anterior
pituitary gland expresses CB
and TRPV1 receptors [115–117]
supporting a direct endocannabinoid effect on gonadotrophin
production in the male, similar to that observed in the female.
There is also some evidence of a direct effect of cannabinoids on
the testis [122–126]. Recent interest in the localization of the
endocannabinoid system within male reproductive tissues and
measurement of endocannabinoids in ﬂuids of several mammalian
species has been the subject of intense interest, as well as provoking
controversy. For example, spermatogenic output depends on a
balance between cell proliferation and apoptosis and although,
has been identiﬁed in rodent Sertoli and Leydig cells,
suggesting that endocannabinoids are produced by the testis and may
play a crucial role in spermatogenesis and testosterone production
[127,128], the predominant cannabinoid receptor found in mamma-
lian Sertoli cells is CB
, which has anti-apoptotic effects and counter-
acts the pro-apoptotic effects that occur through the TRPV1 receptor
(also present in the Sertoli cell), thereby protecting the germinal
epithelium and germ cells from apoptosis, promoting meiotic
progression  and ensuring increased spermatogenic output
[126,128–131]. The recent demonstration and cloning of testis-
speciﬁc functional isoforms of the CB
receptor that have
differential ligand binding properties [132,133] may help to resolve
some of these controversies.
4. Endocannabinoids, early embryo-blastocyst development and
The oviduct is central to the process of human reproduction and
plays a critical role in oocyte fertilization and pre-implantation
embryo development during its passage to the uterus . Events
occurring during oviductal transport must be co-ordinated such that
the blastocyst is ready for implantation when it arrives in the uterine
cavity and the latter is ready to receive the blastocyst, usually 6 days
post-fertilization . These critical events are synchronised and
regulated by several endocrine, paracrine and autocrine factors
[96,135] of which the endocannabinoid system has been identiﬁed
as one of the key hormone–cytokine/receptor signalling systems
4.1. Endocannabinoids and early embryo development
In the mouse, FAAH is expressed at the 2-cell stage of the early pre-
implantation embryo [136,141,142] while NAPE-PLD, CB
expressed at later stages. As development proceeds to the blastocyst
stage FAAH levels are signiﬁcantly up regulated. In homozygous FAAH
knockout mice, embryos recovered from oviducts on day 3 of
pregnancy showed delayed and asynchronous development com-
pared to wild type mice  suggesting that low levels of AEA are
beneﬁcial and high levels detrimental to blastocyst development
[136,141]. Furthermore, in vitro culture of mouse blastocysts with AEA
indicated that 7nM concentrations of AEA allowed blastocyst
hatching, and 28nM concentrations prevented blastocyst hatching
and induced apoptosis . These data suggest that AEA synthesis by
NAPE-PLD and its degradation by FAAH in embryos and within
oviducts has to be well synchronised such that a locally appropriate
“anandamide tone”is created to permit normal embryo development
and oviductal transport . Furthermore, studies with CB receptor
knockout mice suggest that CB receptor signalling is important in
oviductal transport and blastocyst development. For example, on day
4 of pregnancy, only 60–70% of CB
knockout and CB
double knockout embryos were blastocysts, whereas most of the
wild type embryos were blastocysts . Data on the effects of
endocannabinoids on human embryo/blastocyst development are,
however, lacking. Nevertheless, the observation that low plasma
levels of AEA and high levels of FAAH in peripheral mononuclear cells
observed during the putative “implantation window”in humans
[66,144,145] suggests that the ECS also plays a similar role in human
4.2. Endocannabinoids and transport through the Oviduct
signalling has been shown to play a crucial role in oviductal
transport of embryos. Studies on CB
knockout mice showed that only those lacking CB
protein had a
large number of embryos in the morula or blastocyst stage retained in
their oviducts on day 4 of pregnancy whereas CB2 knockout and wild
type mice had none retained, providing evidence that oviductal
transport is delayed in CB
deﬁcient mice [140,143] and that CB
probably not involved in oviductal transport. This was conﬁrmed by
studies using wild type mice treated with the CB
(SR141716) with retention of embryos in the oviducts but not when
antagonist (SR144528) was used .
The mechanism whereby endocannabinoids might affect normal
transport is currently unclear. Transport of embryos through the
oviduct is aided by a peristaltic wave of oviductal smooth muscle
movement regulated by the sympathetic nervous system . The
demonstration of CB
co-localization with adrenergic receptors in
rodent oviduct, suggests that endocannabinoids could affect cate-
cholamine function . Indeed, excess sympathetic stimulation has
been shown to lead to increased smooth muscle contraction and
impaired embryo transport at the isthmus-uterine junction . The
overstimulation of the oviductal smooth muscle with potassium
chloride led to a 33% overﬂow of radio-labelled noradrenaline into the
oviducts of CB
knockout mice, an effect that was signiﬁcantly higher
compared to that in CB
knockout and wild-type oviducts ,
suggesting that CB
signalling negatively regulates neuronal nor-
adrenaline release to directly modulate tubal motility. The authors
suggest that aberrations in the endocannabinoid system could
therefore form a mechanistic basis for ectopic pregnancy, where
there is no obvious tubal damage.
924 A.H. Taylor et al. / Clinica Chimica Acta 411 (2010) 921–930
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In humans, CB
has been localized in the Fallopian tube smooth
muscle wall and CB
transcript levels were found to be signiﬁcantly
lower in the Fallopian tubes of patients with ectopic pregnancy
compared to non pregnant controls . Furthermore, that study
examined 2 polymorphisms of the CB
gene and found a possible
association between the 1359G/A polymorphism and ectopic preg-
nancy. The small numbers involved make a deﬁnitive association
difﬁcult, but if larger studies support a possible genetic predisposition
to ectopic pregnancy due to CB
dysfunction, then genetic screening of
women could lead to the identiﬁcation or directed therapies for
women at risk of this life-threatening condition .
5. Endocannabinoids and implantation
It is now acknowledged that the endocannabinoid system is
critical in the process of implantation [66,79,110,139,147–154].
Successful implantation depends on an accurate interaction between
the endometrium and the implanting blastocyst. Firstly, it requires a
receptive uterine environment and secondly, an activated blastocyst,
capable of invasion and subsequent implantation [155–157].
Implantation occurs about six to seven days after fertilization and
in order for the process to begin, the blastocyst needs to emerge from
the zona pellucida (‘blastocyst hatching’) thereby exposing a
trophoblast capable of invasion [152,155], followed by apposition
with the endometrium, adhesion and ﬁnally, trophoblast invasion
. Animal studies have established that AEA regulates the
‘window’during which the uterus is receptive [29,79,139,157].As
the blastocyst enters the uterus, it is differentially affected by local
physiological AEA concentrations. At low concentrations, AEA acti-
vates ERK signalling in dormant mouse blastocysts, whereas high
levels of AEA inhibit calcium mobilisation . Furthermore, low
AEA levels promote blastocyst growth. Studies by Paria et al.have
shown that trophoblasts exposed to low levels of endocannabinoids
show accelerated growth and differentiation as well as increased rates
of zona hatching [137,158,159]. These effects are mediated via CB
receptors. Trophoblasts also expresses CB
receptors whose function
in this tissue is currently unknown [158,160]. At the same time, lower
AEA levels and elevated levels of FAAH occur in the uterus at
implantation sites  when compared to the inter-implantation
sites, which have much higher levels of AEA and lower levels of FAAH.
There is indirect evidence that during apposition, the mouse
blastocyst produces an unidentiﬁed lipid  that induces the
changes in FAAH and NAPE-PLD enzyme expression conducive for
attachment and initial invasion and thus the changes in localized AEA
concentrations at the implantation site [157,162]. Evidence that local
AEA concentrations are critical to implantation comes from studies
demonstrating that embryos exposed to high levels of AEA in the
uterus suffer embryotoxicity, reduced trophoblast proliferation and
implantation failure [158,160,162].
Similarly, women in an in vitro fertilization programme who have
successful implantation, have low serum AEA levels associated with
elevated levels of FAAH in their peripheral lymphocytes at 6 weeks
gestation [163,164]. The process is likely being controlled by the
expression and activity of lymphocytic FAAH. Indeed, it has been
suggested that the decreased activity and expression of FAAH in
peripheral lymphocytes could be used as an early marker for ﬁrst
trimester miscarriage . These data are mirrored by the studies of
Trabucco et al.who showed that placental tissue from women
following spontaneous miscarriage had very low levels of FAAH and
increased expression of the CB
receptor . These ﬁndings are
corroborated by observations that higher plasma AEA levels in women
with threatened miscarriage are associated with a subsequent
spontaneous miscarriage .
Plasma AEA levels ﬂuctuate during the menstrual cycle
[23,24,144,145]. Recently our group showed that plasma AEA levels
declined during the implantation window in women with a successful
pregnancy after IVF treatment and in women with normal menstrual
cycles [66,144,145]. During this period, FAAH has been shown to have
the highest activity, whereas there is no change in CB
AMT and NAPE-PLD activity . Interestingly, FAAH and proges-
terone show the same ﬂuctuations during the menstrual cycle .
This is conﬁrmed by the ﬁnding that progesterone up-regulates the
FAAH gene [166,167], but contradicted by the ﬁnding that serum
progesterone was not correlated with plasma AEA levels during the
menstrual cycle or in women with positive implantation in an IVF
treatment programme . There were, however, positive correla-
tions between the levels of 17β-estradiol, FSH and LH, suggesting that
implantation could be regulated by changes in the levels of these
hormones and their inﬂuence of AEA levels in the uterus .
Research so far indicates that the endocannabinoid system is an
important regulator for implantation and successful pregnancy.
Endocannabinoid signalling is under tight control and any disruption
of this balance may lead to reproductive failure. The delicate
intricacies related to how endocannabinoids regulate implantation
and whether this is a direct inﬂuence or via T lymphocyte function
remains to be elucidated.
6. Endocannabinoids in early pregnancy
-THC is known to cross the placenta and affect human
pregnancies, it was not known if the effect was via actions on
trophoblast activity. Recently, however, we demonstrated that Δ
THC acts directly on the human BeWo cytotrophoblast cell line to
inhibit cell growth and the transcription of genes involved in growth
and apoptosis , via the relatively unstudied CB
Indeed, using human cell line models of the 1st and 3rd trimester
trophoblast and the pregnant rat model, we have shown that in
human cells AEA inhibits cell proliferation via the CB
receptor by the
differential modulation of histone deactylase 3 (HDAC3) expres-
sion . AEA at concentrations in excess of 15 μM stimulate HDAC3
expression in ﬁrst trimester trophoblast cells and inhibit HDAC3
expression in third trimester trophoblasts. These data support the
observations that Δ
-THC has differential effects on the ﬁrst and third
trimester trophoblast [171,172], and that differential CB receptor
expression in the rat placenta occurs during gestation . Indeed,
treatment of rats with varying doses of AEA in excess of 10 μM induces
trophoblast cell apoptosis via the CB
receptor . These data
suggest that there may be subtle differences in the actions of exo- and
endo-cannabinoids in the placenta in the human and rat and that a
complex interplay between AEA concentrations and implantation of
the blastocyst and subsequent placentation is important during early
pregnancy. Indeed, in studies of IVF-ET women, where the introduc-
tion of the embryo can precisely be timed and implantation can be
closely followed , plasma AEA levels initially increase, presumably
to aid blastocyst hatching and implantation, but then subsequently
fall, presumably to prevent trophoblast apoptosis and so aid retention
of the developing embryo. It therefore seems that a critical balance in
endocannabinoid levels, which has been shown to be vital to
successful pregnancy, is required for early trophoblast development
and subsequently that of the placenta.
7. Endocannabinoids in the maintenance of pregnancy and labour
The fact that the use of marijuana is associated with preterm
labour, prolonged pregnancy, fetal growth restriction, stillbirth and
placental abruption suggest that the endocannabinoid system is
involved in late pregnancy and labour. We have quantiﬁed AEA levels
during pregnancy and found that the levels are low in the ﬁrst
trimester (similar to the levels measured in the luteal phase of the
menstrual cycle) and are maintained at these low levels until the
beginning of the 3rd trimester when they start to rise. They then rise
by almost 2- to 4-fold with the onset of labour (Table 1;[23,24,174]).
925A.H. Taylor et al. / Clinica Chimica Acta 411 (2010) 921–930
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In the rat, CB1, CB
and TRPV1 expression ﬂuctuates in the decidua
in a predictable pattern from a high level on days 10–12 of pregnancy
to a lower level towards the end of pregnancy . Simultaneously,
plasma AEA levels are not signiﬁcantly altered, but local tissue levels
of AEA and PEA, but not OEA are increased at two points through
pregnancy; on day 10, when the placenta and decidua regress to make
room for the developing embryo and on day 19 when the activation of
the myometrium is initiated in preparation for parturition on day 21
. These observations suggest that local production of endocan-
nabinoids may be associated with the apoptosis-induced regression of
the decidua in the rat and the preparation of parturition events. These
rodent data together with the aforementioned increase of plasma AEA
levels indicated in the 3rd trimester of pregnancy suggest that plasma
AEA levels could be elevated in women with incipient preterm labour,
preterm premature rupture of membranes or cervical insufﬁciency,
obstetrical problems that are almost impossible to predict. Data
emerging from our laboratory support this suggestion, as women at
high risk of preterm birth, or presenting with abdominal pain
consistent with the Braxton Hicks myometrial contractions indicative
of impending preterm labour, that subsequently went on to deliver
preterm have higher levels of plasma AEA than those that continue to
term . Indeed, those who delivered quicker (less than 6 days)
have even higher plasma AEA levels than those that took longer to
deliver but still delivered prematurely. These data are consistent with
studies performed in the CB
knockout mouse, in which delivery
occurs preterm (i.e. 1 day early) and could be prevented by changing
cortisol and progesterone levels, suggesting that the entire endocan-
nabinoid system, (i.e. not only endocannabinoid levels), is involved in
the timing of parturition . The alternative observation that other
endocannabinoids are present in the uterus at this important time
during pregnancy, and the apparent increase in plasma AEA levels in
women in labour, even if they had been induced , suggest that
the ‘entourage effect’is in action and that FAAH activity is maximised
at this point and that FAAH is dealing with the other endocannabi-
noids that exist at higher levels than AEA [19–21,28]. This might
explain the apparent discrepancy between increasing lymphocyte
numbers during the latter stages of pregnancy  and the increased
levels of plasma AEA during labour. The obvious extension of these
observations is that CB
receptor mutations  and CB
or FAAH mutations or activities may be important in the timing of
birth and hence an important area of study for perinatologists. In our
opinion, this understudied area of endocannabinoid research needs
It is not clear what elevated plasma AEA levels mean in relation to
parturition. Three main tissue-speciﬁc events occur during the
parturition process; myometrial activation, cervical ripening and
fetal membrane rupture. The actions of endocannabinoids in
myometrial activation, cervical ripening and fetal membrane are
unclear due to the paucity of data. Studies on oxytocin-stimulated
myometrial muscle strips demonstrated a relaxant effect of both Δ
THC and AEA through CB
. Recently, we demonstrated that AEA
increased the production of phosphorylated ERK in primary human
myometrial cells and a human myometrial cell line, without changes
in intracellular calcium levels . Such changes in the myometrial
cell should result in myometrial relaxation as was suggested by
Dennedy and colleagues . The data suggest that the myometrium
is a possible endocannabinoid target during the latter stages of
pregnancy. The fetal membranes and placenta at term are also obvious
targets as these tissues express the cannabinoid receptors  and
contain signiﬁcant amounts of AEA (Table 1).
8. Feto-placental changes in endocannabinoid levels
Levels of FAAH in both the human placenta and the maternal
circulation increase towards the end of the ﬁrst trimester of
pregnancy, before declining by the early second trimester [163,182].
Furthermore, high levels of FAAH have been observed in the villous
cytotrophoblast . Its expression in the syncytiotrophoblast
suggest that FAAH in these cells help prevent the transfer of AEA
from maternal blood , which, to some degree, has been
suggested as a protective mechanism for the developing fetus .
Previous studies have suggested that circulating FAAH and AEA levels
may be critical to the outcome of early pregnancy [163,164]. As stated
earlier, decreased expression and activity of FAAH in peripheral blood
lymphocytes maybe an early marker of spontaneous miscarriage
. The importance of FAAH in early placental development has
been supported by a recent study that demonstrated the expression of
FAAH and CB
(rather than CB
) receptors in the human ﬁrst trimester
placenta. This study also provided evidence that the endocannabinoid
regulation within placental tissue is independent of the maternal
immune system . We recently demonstrated the presence of
receptors and FAAH immunoreactive protein in ﬁrst
trimester trophoblast which was supported by the presence of
transcripts for these genes in the tissue . The apparent
discrepancy between these two studies could be related to the
antibodies used, as has been documented . Nevertheless,
cannabinoid receptors are expressed by placental tissue from the
early ﬁrst trimester right through to term [181–183]. Interestingly,
levels of AEA fall progressively during pregnancy [23,24], supporting
other evidence that low systemic levels are required for normal
pregnancy progression . Therefore, exposure to the exocanna-
-THC could lead to inappropriate activation of the CB
mediated pathways in the placental trophoblast.
9. Possible “immunocannabinoid”system actions in pregnancy
Although the involvement of the endocannabinoid system in
reproduction may be direct, it may also be through modulation of the
immune system. Despite a great interest in the role of cannabinoids
within the immune system [57,185–189], the possible relationship
between endocannabinoids and immunity, especially in relation to
reproductive events, remains to be clearly determined. For successful
implantation and maintenance of pregnancy, the embryonic/fetal
allograft [190,191] has to be tolerated by the maternal immune
Anandamide concentrations in different human biomatrices related to reproduction
and the reproductive tract.
Biomatrix/Tissue AEA (nM)
Follicular ﬂuid Immature oocyte 0.36–1.96 
Mature oocyte 0.25–2.78 
Plasma (menstrual cycle) Follicular Phase 1.45–1.68 [23,24,143]
Ovulation 1.26–4.04 
Luteal Phase 0.77–0.87 [23,24,143]
Plasma (pregnancy) 1st trimester 0.89–0.91 [23,24]
2nd trimester 0.44–0.91 [23,24]
3rd trimester 0.44–1.14 [23,24]
Term not in labour 0.68–1.27 [23,24]
Term in active labour 2.30–2.50 [23,24]
Amniotic ﬂuid 0.01–0.18 [26,215]
Umbilical artery plasma 0.32–1.53 [26,215]
Umbilical vein plasma 0.38–1.84 [26,215]
Maternal Plasma at term 0.59–1.91 [26,215]
AEA = anandamide;
in pmol/g wet weight;
only a single human study performed;
? = data unknown.
926 A.H. Taylor et al. / Clinica Chimica Acta 411 (2010) 921–930
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system through mechanisms that might involve the endocannabinoid
system . For example, arachidonic acid (AA), a local immuno-
modulatory molecule , is released from cultured mouse
macrophage cells by Δ
-THC and AEA through a CB
mechanism and by nitric oxide (without raising AEA levels) indicating
that both CB receptor-dependent and -independent mechanisms are
involved in this possible immunomodulatory pathway [193,194].
Maccarrone et al.  also demonstrated that bacterial lipopolysac-
charide down regulates FAAH expression in human peripheral
lymphyocytes, an effect that was not inﬂuence by AEA-induced
activation of the cannabinoid receptors, whilst mast cells absorb AEA
in a saturable process that activates intracellular FAAH and AEA
degradation . Additionally, the physiological protection of the
fetus from rejection is believed to be dependent on a type 2 T-helper
(Th2) cell immune response at the fetal–maternal interface . The
initial state of pregnancy is nevertheless characterised by immuno-
suppression after an early inﬂammatory state during implantation
. Indeed, the demonstration that monocytes  and leuko-
cyte/macrophages [200,201] numbers increase towards the end of
pregnancy suggest an inﬂammation–immunosuppression–inﬂamma-
tion phenotype during pregnancy . The production of plasma
AEA mimics this inﬂammation–immunosuppression–inﬂammation
pattern during implantation, the 2nd and 3rd trimesters of pregnancy
and just before labour [23,24,66]. Such data suggest that immune cells
may take part in regulating the peripheral endocannabinoid system or
local endocannabinoid homeostasis and vice versa.
Many studies have demonstrated that, in the main, endocannabi-
noids suppress the production of inﬂammatory cytokines in innate
and adaptive immune responses both in animal models and in human
cell cultures [186,203,204]. Immunosuppression is one of the most
important steps involved in early pregnancy  and evidence of a
reduction in interleukin-1β, -10, and -12 levels in animal models of
inﬂammatory disease [206–208] suggests that similar reductions in
the levels of these cytokines in reproductive tissues may occur in
response to local endocannabinoid production, although this is yet to
be directly determined. However, cannabinoids have also been
demonstrated to increase the production of IL-1, -4, -6 and -10
cytokines either in the presence of bacteria [204,207,209] or alone
[210,211], suggesting that in vivo, cannabinoids may either suppress
or enhance the production of pro-inﬂammatory agents, depending on
either the nature of the pro-inﬂammatory stimulus or the type of
cannabinoid present. These changes may arise from the predominant
effect of cannabinoids on helper T-cell (Th) biasing [186,203] whereby
Th-1 cell activity is suppressed and Th-2 cell activity is increased
[208,212]. This may change the levels of leukaemia inhibitory factor,
which is known to be essential for successful invasion and migration
during placental development [213,214].
Taken together these data suggest that endocannabinoids have a
key role, not only on the cells and tissues of the fetal–maternal
interface but also on their interaction with the innate and adaptive
immune system. This under-explored area of possible interaction
between the immunocannabinoid system  and reproduction is
likely to be a focus of intense research in the near future.
The endocannabinoid system (ECS) is involved in various aspects
of human reproduction. It appears to be an important cog in the
complex mechanisms (wheel) of gametogenesis. With respect to
spermatogenesis, 2-AG appears to promote meiotic progression in the
germ cell by activating type-2 cannabinoid receptors . Levels of
AEA also appear to be related to follicular size and quality of the ova
produced during oogenesis. This system is involved in the regulation
of fertilization through actions that result in spermatozoa activation
and thereafter inﬂuence hatching of the embryo, blastocyst matura-
tion, oviductal transport, implantation and early pregnancy mainte-
nance. Changes in plasma levels of some of the cannabinoid ligands
(e.g. anandamide) provide evidence for a tight regulation of early
pregnancy by this group of compounds. Changes in pregnancy and
labour also suggest an involvement in labour onset and maintenance.
Although this review has highlighted areas where the evidence is
coherent and strong, so many questions remain unanswered and only
through collaborative and international studies will these be
adequately addressed. Such collaboration may initially focus on the
changes of the cannabinoid receptors, FAAH, NAPE-PLD, and AEA
which have been thoroughly investigated in animals and partly in
humans, to determine the translational potential of the ECS in
establishing and maintaining a healthy pregnancy.
Conﬂicts of interest
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