Frontiers in Neuroendocrinology 31 (2010) 359–376

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Frontiers in Neuroendocrinology

journal homepage: www.elsevier .com/locate /yfrne

Review

Baby on board: Do responses to stress in the maternal brain mediate adversepregnancy outcome?

Alison J. Douglas *

Laboratory of Neuroendocrinology, Centre for Integrative Physiology, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh EH8 9XD, United Kingdom

a r t i c l e i n f o a b s t r a c t

Article history:Available online 31 May 2010

Keywords:CRHCytokinesDopamineGlucocorticoidshCGProgesteroneProlactinPregnancy failurePreterm laborStress

0091-3022/$ - see front matter � 2010 Elsevier Inc. Adoi:10.1016/j.yfrne.2010.05.002

* Fax: +44 131 650 2872.E-mail address: alison.j.douglas@ed.ac.uk

Stress and adverse environmental surroundings result in suboptimal conditions in a pregnant mothersuch that she may experience poor pregnancy outcome including complete pregnancy failure and pre-term labor. Furthermore her developing baby is at risk of adverse programming, which confers suscepti-bility to long term ill health. While some mechanisms at the feto-maternal interface underlying theseconditions are understood, the underlying cause for their adverse adaptation is often not clear. Progester-one plays a key role at many levels, including control of neuroendocrine responses to stress, procuring therequired immune balance and controlling placental and decidual function, and lack of progesterone canexplain many of the unwanted consequences of stress. How stress that is perceived by the mother inhib-its progesterone secretion and action is beginning to be investigated. This overview of maternal neuro-endocrine responses to stress throughout pregnancy analyses how they interact to compromiseprogesterone secretion and precipitate undesirable effects in mother and offspring.

� 2010 Elsevier Inc. All rights reserved.

1. Introduction

‘Baby on Board’ warns us to drive carefully as the consequencesof not doing so may be undesirable. Indeed, any trauma experi-enced by a baby or child might have long term physical and psy-chological consequences since their brains and other systems arehighly susceptible to any adverse conditions during the develop-mental period. These ‘Baby on Board’ car signs typically appearafter the birth of the child, but there is now wide appreciation thatduring pregnancy itself, when the developing embryo and fetus are‘on board’ the mother, they are highly vulnerable to maternal trau-ma, needing protection through even the most primitive stages.Three major adverse outcomes are possible when the mother expe-riences a range of suboptimal situations during gestation: preg-nancy failure, preterm labor and adverse fetal programming, eachwith its own long term consequences for mother and offspring.Each depends upon the timing of the experience and other moresubtle, perhaps unknown, factors that may or may not be avoid-able. This review aims to address how responses of the mother’sneuroendocrine system to the environment determine pregnancyoutcome. Adaptations and responses to the environment in humanpregnancy will be outlined where available, supplemented by evi-dence from key experimental animal models.

ll rights reserved.

2. Neuroendocrine systems in pregnancy

Although offspring are located and develop within the uterus,the preparation and control of uterine function, and ultimatelyreproduction, is the responsibility of the maternal brain. Forwarddrive from the brain to the reproductive organs comprises key neu-roendocrine systems. These tightly regulate the hormones thatplay major roles in providing the appropriate environment forestablishing and maintaining pregnancy. They signal mating andimplantation, sustain an optimally-balanced hormonal and im-mune milieu, cause adaptation of maternal organs (including thebrain) to support conceptus development, and prepare motherand fetus for birth and subsequent life. Most, if not all, neuroendo-crine hormones and their central regulators are involved in thistask, underlying profound changes in many maternal physiologicalsystems including appetite and metabolism, osmoregulation,stress coping, emotion, fertility and behaviors [26,159]. All aspectsof the maternal environment impact on the performance and adap-tation of these systems. We will analyze the influence of the envi-ronment, and particularly stress, on the key neuroendocrine axesestablishing and maintaining pregnancy and discuss how their re-sponses could be detrimental to mother and offspring.

2.1. Neuroendocrine systems establishing pregnancy (Fig. 1)

The neuroendocrine system controlling female reproductive cy-cles is the hypothalamo–pituitary–gonadal (HPG) axis. In cycling,

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non-pregnant females the HPG axis signals development of theovarian follicles. This requires drive from the hypothalamus bygonadotrophin-releasing hormone (GnRH) to stimulate gonadotro-phin secretion from the anterior pituitary, which causes sex steroid(estrogen and progesterone) secretion by the ovarian follicle andoocyte maturation. The process culminates in ovulation, whenthe fertilizable egg is released, and then conversion of the follicleto a corpus luteum. Progesterone secretion from the corpus luteumcontinues under the brief control of a key pituitary gonadotrophin,luteinising hormone (LH), and prepares the endometrium to re-ceive a fertilized egg. If a fertilized egg does not implant in theendometrium the corpora lutea die a week to 10 days later.

When an oocyte is fertilized in women, the early conceptusstarts to produce chorionic gonadotrophin (hCG), even beforeimplantation. The a subunit of this glycoprotein is identical to LHand binds LH receptors. As LH receptors are expressed in the endo-metrium, initially the conceptus is able to communicate with adja-cent maternal tissues to facilitate its own implantation [18,141]. Amajor role of hCG in the endometrium is to promote angiogenesis,and it does this in part by increasing vascular epithelial growth fac-tor (VEGF; [18]). As implantation proceeds and the conceptus, nowwith extensive surrounding trophoblast layers, produces increas-ing amounts of hCG, secretion increases exponentially into thematernal tissue and bloodstream, and acts as a signal to rescuethe corpus luteum and sustain progesterone secretion. This proges-terone is essential for pregnancy establishment and maintenance,and starts to provide the appropriate endometrial environmentfor implantation, including changes in blood flow, glandular secre-tion and immune balance.

The processes are essentially similar in rodents except that theircorpora lutea die rapidly within a day of ovulation so an additionalprocess is required to maintain them after mating. This only occurswith copulation so, whether or not eggs become fertilized, a mat-ing-induced neurogenic reflex is initiated. This physical stimula-tion-dependent reflex drives increased LH secretion which wasinitially thought to be associated with implantation [66]. This LHmight, very early after mating, have the same basic function tomaintain corpus luteum progesterone secretion as hCG does in wo-men. Additionally, in rodents as in women, LH is produced by theconceptus and facilitates endometrial angiogenesis through VEGF(S. Perrier d’Hauterive, personal communication). Other pituitaryhormones also facilitate corpus luteum progesterone secretion,notably prolactin, and can be considered under the ‘gonadotrophin’umbrella.

Prolactin secretion from anterior pituitary lactotrophs is con-trolled by hypothalamic neuroendocrine neurons. Typically prolac-tin is under strong inhibitory control by dopamine from thetuberinfundibular dopamine (TIDA) neurons in the arcuate nu-cleus, and a circadian rhythm is described (in women and prima-tes: [17], in rodents at proestrous, [63]). In pregnancy increasingprolactin secretion is normal and important for several aspects ofpregnancy maintenance. In women increasing prolactin is reportedin early gestation, from at least week 6 [17], although whether thecircadian rhythm is maintained or not is unknown. Prolactin isimportant because it facilitates the formation of the corpus luteum[140]. It also drives progesterone secretion by both interactionwith the LH receptor to promote its role in steroidogenesis andby inhibiting the 20 alpha hydroxysteroid dehydrogenase(20aHSD) enzyme, which degrades progesterone [63]. The tropho-blast also secretes placental lactogen (somatomammotrophin)which acts at prolactin receptors and has a variety of growth-re-lated functions in maternal tissues. In rodents the prolactin secre-tory profile is different from women- basal prolactin increases andis also secreted in superimposed peaks in early gestation. These arenow thought to be the essential mechanism rescuing the corpusluteum in rodent pregnancy; e.g. transgenic mice lacking prolactin

are unable to achieve pregnancy [11,63]. Rats clearly have bi-dailypeaks, one in the afternoon (later light phase) and one in the middark phase [71], although mice are reported to only have one slowpeak (more of a surge) shortly after lights off [202] which resem-bles a circadian rhythm. Replacement of LH or hCG does not rescuepregnancy in prolactin null mice since they are unable to inhibitthe 20aHSD enzyme [11,134]. As pregnancy proceeds rodents alsosecrete placental lactogen similar to women, except that two typescan be detected, at least in rats.

The effects of prolactin occur not only in the corpus luteum butadditionally at the fetal-maternal interface, i.e. the decidua. Wherethe trophoblast invades, the endometrium responds by transform-ing into the decidua, which is a syncytium-like structure that ishighly vascularized. In women prolactin is essential for adequatedecidualisation, and this is similar according to some studies in ro-dents [12,17,77,81,88,100,105]. Indeed, in both women and ro-dents prolactin and a number of prolactin-like peptides that aresynthesized and secreted within the decidua also contribute tomultiple pregnancy-supporting functions such as steroidogenesisand angiogenesis to optimize implantation and placenta formation[1,12,88,100]. Although these peptides and their receptors are syn-thesized in the decidua, circulating prolactin is also expected to ac-cess this important barrier tissue to at least initially cause theabove effects [45], as well as later enhance the endogenous localsystem. Thus, prolactin and placental lactogen(s) are key hormonesin driving the functions of the corpus luteum, decidua, and a vari-ety of other maternal tissues [71], and so, together with the HPGaxis, are responsible for pregnancy establishment.

Since prolactin secretion increases during pregnancy, theremust be a change in TIDA neuron activity and dopamine secretion.How prolactin feedback to TIDA neurons changes in women to al-low this increase is not investigated. However, many papers haveaddressed mechanisms underlying the maintenance of the prolac-tin peaks in pregnant rodents. Evidence shows in rats that dopa-mine closely controls the prolactin peaks; for example, dopaminecontent in the mediobasal hypothalamus changes in anti-phasewith the prolactin peaks [71], showing that inhibition of dopamineunderlies them. What drives the repeated peak pattern is not com-pletely characterized yet but hypothalamic mu- and kappa-opioidscontribute to the inhibition of TIDA neurons [71], while oxytocin isestablished as a prolactin-releasing factor in early pregnancy. Oxy-tocin contributes to initiation of the rise in prolactin secretion ateach peak [71,110], since evidence shows that during early preg-nancy oxytocin antagonist treatment blunts and alters prolactinpeak timing and median eminence dopamine content [19,110].However, oxytocin is not essential since transgenic mice lackingoxytocin or oxytocin receptor still mate, attain pregnancy and givebirth to live young [130,184]. Activation of central noradrenalinepathways is also associated with prolactin secretion since nor-adrenaline release in the preoptic area occurs at the same timeas prolactin surges in the blood [181]; however, a causative linkin pregnancy is not reported. Prolactin further controls its ownsecretion by negatively feeding back to control TIDA neurons,and in early pregnancy this contributes to maintenance of thesecretory profile [71,75].

2.2. Immune balance in early pregnancy and interaction withneuroendocrine systems

Along with gonadotrophins and progesterone, it could be ar-gued that procurement and maintenance of the appropriate im-mune balance in very early gestation is the critical physiologicaladaptation the mother’s body must make for pregnancy to proceed.The optimal tolerogenic immune environment appears to be a biastowards cytokines from a particular subset of immune cells in thedecidua: the T helper (h) 2-type cells. These cells secrete anti-

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inflammatory cytokines. In contrast, Th1-type cells secrete pro-inflammatory cytokines and these decrease in pregnancy. In wo-men, this Th2-cytokine predominance is required for trophoblastsecretion of hCG and placental lactogen ([53]; and vice versa,hCG contributes directly to immune balance, at least in part by di-rectly enhancing LIF activity and inhibiting interleukin (IL)6 activ-ity in the endometrium/decidua [18].

There is therefore an increasing Th2:Th1 ratio that is beneficialto implantation and pregnancy establishment and maintenance.This preferential ratio seems to be driven by increasing progester-one secretion; progesterone is known as an immuno-steroid andfacilitates Th2 cytokine expression and activity. It does this by pro-moting secretion of a particular protein called progesterone-in-duced blocking factor (PIBF; [3]) from lymphocytes and decidualCD56+ cells. PIBF inhibits T-cell lymphopoiesis and reduces naturalkiller (NK) cell activity which alters the proportion of Th2 cyto-kines (e.g. IL10, IL4, LIF) to Th1 cytokines (e.g. IL 12, IL6, TNFa,IFNc) [182,183]. Therefore progesterone is not only required forappropriate endometrial development and secretion but also forprocurement of the optimal immune balance.

However, progesterone is not the only pregnancy hormone tomoderate immune balance. Prolactin and prolactin-like peptides,including placental lactogens, also have important immunomodu-latory roles and a key role for prolactin peptides locally in the de-cidua is in immune regulation [77]. For example, prolactinenhances production of IL10 in the decidua [105]. Indeed prolactinand its related peptides are cytokines, acting via a janus kinase(JAK)/Signal Transducers and Activators of Transcription (STAT)receptor signaling pathway. Th2 cytokines are required for secre-tion of all the important pregnancy hormones, as part of the posi-tive mechanism sustaining pregnancy. Therefore, co-ordinatedsecretion and action of the gonadotrophins and progesterone causedevelopment of the endometrium/decidua and also helps preventmaternal immune rejection of the fetal allograft.

2.3. Neuroendocrine and placental hormones maintaining pregnancy

After the initial compete reorganization of the maternal hor-monal environment, ensuring that pregnancy becomes established,mechanisms come into play that maintain the important functionsfor ongoing gestation and support of the developing fetus. Here abrief overview of these will be given as a basis for the next sectionon the effects of stress. Progesterone secretion and action contin-ues to be crucial to pregnancy survival and in both women and ro-dents the mechanisms sustaining this change after about a third toa half of the gestation period. In women after the first trimester theplacenta takes over from the corpus luteum as the major source ofsex steroid synthesis and secretion. Trophoblast factors presum-ably continue to be important in driving progesterone secretion,and include LH, placental lactogen and corticotrophin-releasinghormone (CRH); pituitary prolactin secretion gradually increasesand also continues to facilitate progesterone secretion by inhibit-ing its degradation in the placenta and decidua. In rodents the cru-cial pituitary prolactin surges of early pregnancy are replaced byplacental lactogens which then provide the drive for corpus luteumprogesterone secretion [71], acting in a manner analogous to hGCin first trimester women, although the rodent placentas and decid-uas do produce some progesterone. So in both women and rodents,although the underlying mechanisms differ substantially andchange from early pregnancy, progesterone continues to be the pri-mary hormone maintaining pregnancy via its endocrine-immuneroles.

In late pregnancy progesterone and prolactin/placental lactogencontinue to be important hormones, driving maternal adaptationsand therefore fetal growth and development appropriately untilbirth. They play a prominent role in the development and adapta-

tion of maternal organs needed at birth and in lactation. Progester-one is particularly important in maintaining uterine quiescenceand in developing and maturing mammary gland acinar cells,while prolactin and the lactogens stimulate mammary gland geneexpression (e.g. of casein, [99]) in preparation for milk production.Both progesterone and prolactin are also important contributors tothe brain adaptations needed for initiation of maternal behaviorswhich appear perinatally [26,70,158]. As term pregnancy ap-proaches, a real or ‘functional’ progesterone withdrawal occurs[174] which enhances myometrial sensitivity to various uterotonichormones and leads to labor. The complex hormones and their cas-cades of action that underlie parturition are not within the scope ofthis review so will not be further considered in detail.

2.4. Neuroendocrine predictors of poor pregnancy outcome

Implantation and pregnancy proceed when the above adapta-tions and mechanisms are recruited and co-ordinated. However,even assuming genetic normality of the conceptus, in many casesthey do not optimally emerge due to factors affecting maternalphysiological or psychological homeostasis such as adverse envi-ronmental conditions. Altered patterns of secretion of all of the fac-tors mentioned above have been investigated for their roles asmarkers of abnormal pregnancy outcome.

2.4.1. Early pregnancyIn early pregnancy major risks include complete pregnancy fail-

ure, or miscarriage. When the embryo does not implant sufficientlythe trophoblast-maternal interface is not able to be maintainedand the embryo is shed in women, although for rodents the em-bryo is typically resorbed. The great majority of miscarriages aredue to genetic/chromosomal abnormalities of the conceptus,which are independent of neuroendocrine abnormalities. Never-theless, some are due to environmental conditions which may im-pact on the neuroendocrine mechanisms establishing pregnancy.

It was known for many years that low circulating progesteroneconcentrations correlated with the occurrence of miscarriage. Re-cently we have established that in the early post-implantation per-iod progesterone concentrations below a cut-off level can predictlater miscarriage in women [6], powerfully revealing that proges-terone does not just decrease at the time of abortion or extant cor-pus luteum failure. Another major risk in early pregnancy is nowbecoming recognized. Adverse maternal conditions lead to pro-found alterations in embryo development such that the fetusmay be born small and/or acquire susceptibility to disorders or dis-eases later in adulthood, such as depression, obesity and cardiovas-cular disease. This unwanted phenomenon has become known asfetal programming, and is a large and expanding area of researchcomprising multi-dimensional characteristics [36,106,112]. Theseare too numerous to mention in this focused review but includekey aspects that are highly relevant, and neuroendocrine systemsare inherently implicated in their etiology. In early gestation, a ma-jor emerging part of this is the realization that abnormal progester-one levels contribute to adverse programming. Thus, lowprogesterone concentrations in early gestation appear to underlieundesirable childhood conditions including atopic dermatitis andsusceptibility to asthma [143,144]. This further indicates thatmechanisms controlling progesterone secretion in gestation maybe faulty early on and could be measurable as predictive markersin pregnant women, allowing potential timely intervention.

As a major controller of progesterone secretion in women, itmight be expected that lack of hCG would underlie lack of proges-terone in association with pregnancy failure. However, hCG levelsare not good predictors or measures of pregnancy failure, oftenbeing within the acceptable range even as abortion occurs [64].However, a range of hCG gene splice variants have been identified

wk4 5 6 7 8 9 10 11

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Fig. 2. Prolactin concentrations in early pregnant women- relationship withmiscarriage. Serum collected from women at their first GP visit (same study as[6]) was analyzed for prolactin and showed that increasing prolactin concentrationcorrelated with increasing gestation week (wk) in successful pregnancies (greenline) but not in pregnancies later ending in miscarriage (red line). r = correlationcoefficient between prolactin concentration and week of gestation, p = probabilitythat r is significant.

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in women, some of which seem to confer susceptibility to recur-rent miscarriage [156]. Although hCG/LH play important roles inthe endometrium, decidua and corpus luteum, overall, there isnot an obvious close correlation between plasma hCG and proges-terone concentrations suggesting that other factors play prominentroles too. Some evidence is beginning to emerge that miscontrol ofprolactin is associated with poor prognosis. Firstly it has been rec-ognized that in non-pregnant women prolonged hyperprolactine-mia causes infertility [190,196], and this is likely to be becauselong term exposure to prolactin inhibits the HPG axis at severallevels. The critical role of prolactin in pregnant women is less clear,but we have preliminary data indicating that low circulating pro-lactin seems to be associated with miscarriage in women (Fig. 2).As this was observed in the same women who exhibit low proges-terone concentration [6], some limited evidence for a correlationbetween these two key systems exists in human pregnancy. Thesefindings are reinforced by rodent studies. In particular prolactinnull mice cannot proceed into pregnancy and LH is able to preventpregnancy failure since prolactin seems to be necessary to preventprogesterone degradation [11]. Furthermore, low prolactin duringearly gestation in mice negatively affects offspring development[94]. The underlying mechanisms for this remain to be investigatedbut potentially include all those factors already mentioned, such aspoor angiogenesis and immune imbalance and therefore poor feto-maternal interface dynamics.

Fig. 1. Control of progesterone secretion in early pregnancy. Initially, at ovulation andcorpus luteum. In women, the blastocyst secretes chorionic gonadotrophin (hCG), wappropriate immune balance and then, after implantation, with the corpus luteum to sclearly a mating-induced neurogenic reflex activates supraoptic/paraventricular nuclei (Sto set up prolactin surges which induce corpus luteum formation and progesterone secr20aHSD activity which would otherwise degrade progesterone. Prolactin is also expressimmune balance and inhibiting 20aHSD. Placental lactogen (PL), acting via prolactin receparticularly in rodents, in a manner analogous to hCG. hCG and PL also maintain a fprolactins, sustain immune balance and appropriate levels of angiogenesis. The resultisuccess. Constructed from information taken from [14,18,63,71,77,140].

The progesterone and prolactin requirements seem inextricablylinked to the immune system. Studies show that miscarriage inwomen is associated with increased Th1-cytokines [39,149,182]

copulation, LH from the anterior pituitary drives progesterone secretion from thehich firstly communicates with the endometrium to promote angiogenesis andustain progesterone secretion. In rodents LH increases in early gestation, althoughON/PVN) oxytocin (OXT) neurons and inhibits arcuate nucleus (ARC) TIDA neuronsetion. Prolactin in women and rodents is important for LH signaling and inhibitinged and released in the developing decidua and is important there for angiogenesis,ptors, also facilitates corpus luteum progesterone secretion, among other functions,

eto-maternal dialogue between the trophoblast-decidua and, along with decidualng sustained progesterone secretion is essential for pregnancy establishment and

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and rejection of the fetal-allograft. However, studies directly link-ing progesterone and/or prolactin with immune imbalance in earlypregnancy in women are not apparent in the literature.

2.4.2. Late pregnancyDisorders of late gestation include pre-eclampsia and preterm

birth, both of which confer risk to the mother and poor prognosisfor the offspring, which are likely to be born before gestationaldevelopment is complete and to have low birth weight. Pre-eclampsia is a major condition where the mother acquires hyper-tension, leading to high blood pressure and edema that impacton many maternal organs causing damage. However, the patho-genesis of pre-eclampsia is unclear and may or may not be relatedto inadequate implantation of the trophoblast. Regardless of thecause, pre-eclampsia results in altered immune signaling and hy-poxia at the feto-maternal interface.

Preterm labor and birth occur when the uterus becomes prema-turely sensitive to uterotonic factors and may arise from variousconditions such as infection [186]. Once the uterus and its positivefeedback to oxytocin neurons becomes sensitized subsequent birthis hard to delay for any appreciable length of time. Several drugsare available for delaying birth but have undesirable side effects,although oxytocin antagonist treatment proves effective in theshort term [186]. The main concern for clinicians is to delay birthlong enough so that drugs can be administered to accelerate fetaldevelopment and to promote neonatal survival after birth. Suchdrugs often include synthetic glucocorticoids which are effectivein maturing fetal organs sufficiently. Many studies have tried todiscover markers for predicting preterm labor and have investi-gated whether they are reliable, including the key pregnancyhormones already described. Just as progesterone or the progester-one:estrogen ratio decrease near term it is possible that mecha-nisms underlying preterm labor include low progesterone. Somestudies correlate low progesterone or a low progesterone:estriolratio with preterm birth [154], but insufficient evidence points toabrogation of preterm labor by progestagens [178]. If progesteronesecretion is low then it is likely that gonadotrophins and/or factorscontrolling the enzymes in the progesterone synthesis and degra-dation pathway are also compromised. One such hormone is hCGand, perhaps surprisingly, it could be a predictor of preterm labor[207]. On the other hand, although prolactin inhibits the progester-one degrading enzyme, 20aHSD, and plays an important role at thefeto-maternal interface, evidence shows that it is unlikely to be afactor underlying preterm labor [73,9,107,108].

3. Overview of the effect of stress on neuroendocrine systems

Many unwelcome environmental conditions influence the neu-roendocrine systems establishing and maintaining pregnancy. Theexperience of stress encompasses some of these conditions andmuch effort has gone into research on its effects on the neuroendo-crine hormonal systems driving reproduction and their role incausing adverse pregnancy outcomes.

Stress may be experienced due to the acute or sustained appli-cation of unexpected or undesirable conditions. Such conditionsencompass a huge range of potential factors. Psychological stressmay be mild such as sustained noise, novel environment or crowd-ing, or may be severe such as social stress or physical or emotionalabuse, or repeated pressurized environment (e.g. high work de-mands). Physical stress may include hunger (as well as poor diet)or water deprivation, fever or immune stress and physical harm.When experienced briefly, stress causes acute and transient symp-toms and appropriate mechanisms are initiated to restore homeo-stasis, often avoiding any further effects or damage. On the otherhand, evidence seems to suggest that sustained or chronic stress

exposure causes long term adverse effects on the mother and/oroffspring. The impact of stress on pregnancy depends upon multi-variate and multidimensional physiological responses. These re-sponses comprise direct effects on the hormones establishingpregnancy as indicated above, activation of the hypothalamo–pitu-itary–adrenal (HPA) axis and sympathetic system, and direct andindirect effects on immune balance.

3.1. Gonadotrophin, prolactin and progesterone responses to stress

Stress is well known as a disrupter of reproduction, seriouslyreducing fertility to the extent that estrous cycles may stop in wo-men. This may occur with repeated acute, prolonged or chronicstress, intensive exercise, undernutrition or other environmentalfactors [125,153,188]. Limited studies in non-pregnant womenand monkeys suggest that CRH inhibits gonadotrophin secretion[13,133] but this is supported by multiple reports in rodents thatacute stress readily disrupts gonadotrophin secretion, suppressingLH pulsatility [98,152] and increasing prolactin secretion [65,145].When sustained this leads to infertility due to lack of gonadotro-phin-induced stimulation of follicle maturation. Stress-inducedactivation of CRH neurons in the hypothalamus and limbic system(e.g. amygdala) play a key role in inhibiting pituitary gonadotro-phin secretion in cycling rats via projections to the locus coeruleusin the brain stem [30,98,117,192]. These brainstem noradrenergicneurons project to the preoptic area and have been shown to inhi-bit GnRH neurons via GABA [117] and/or calcitonin-gene related-peptide (CGRP, [97]). However, although increased noradrenalineconcentrations in the preoptic area are also linked with prolactinsurges, brainstem noradrenergic neurons do not appear to mediatestress-induced increases in prolactin secretion [145]. In fact, pro-lactin secretory responses to stress are bi-modal, increasing whenbasal levels are low and decreasing under certain circumstanceswhen prolactin levels are elevated, such as at the proestrous surge[65,145], and though the underlying difference between the re-sponses is not understood, it seems to be associated with highsex steroid levels since estradiol plus progesterone treatment in-duces a surge of prolactin secretion [145]. Stress also typically in-creases progesterone secretion in cycling female mammals [124]which could be due to the accompanying increased prolactin secre-tion. Together these hormonal responses prevent follicular devel-opment and ovulation.

3.2. Hypothalamo–pituitary–adrenal axis responses to stress

Another major neuroendocrine axis responds to stress and hasbecome a defining hormonal framework when considering the ef-fects of stress on the body since the hormones act on multiplebrain and body targets. Glucocorticoids are steroid hormones se-creted in response to activation of the HPA axis by stress. Physicalstressors such as hunger and cytokines recruit hypothalamic path-ways via vagal/brainstem afferents, while psychological stressorssuch as noise or novel environment are perceived by higher centerswhich then project to the hypothalamus, partly via the brainstemafferents mentioned above. Such inputs rapidly stimulate CRHand vasopressin neurons in the hypothalamic paraventricular nu-cleus (PVN). CRH and vasopressin secretion within the medianeminence together drive anterior pituitary adrenocorticotrophichormone (ACTH) secretion into the periphery which enhancesadrenal cortex glucocorticoid secretion. Additionally, stress acutelyincreases PVN neuron mRNA expression, including that of CRH andvasopressin, as well as immediate early genes like NGFI-B (syn.nur77) and c-fos [47], providing a marker for neuron activity. Glu-cocorticoid secretion peaks 15–30 min following the start of thestress exposure and acts via mineralocorticoid and glucocorticoidreceptors. Typically glucocorticoids bind to the higher affinity min-

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eralocorticoid receptor unless their concentration is elevated,when they then bind also to the glucocorticoid receptor. Glucocor-ticoids control tissue glucose availability and metabolism, as wellas maturing developing and dividing cells. In part their action onany target is determined also by enzymes (11 beta hydroxysteroiddehydrogenase enzyme type 1 or 2; 11bHSD I or II) that reactivateor degrade it, respectively, providing control of local access to thereceptors. Glucocorticoid negative feedback then typically inhibitshypothalamic and pituitary activation helping to quickly restorehomeostasis. More prolonged or repeated stress generally resultsin sustained high glucocorticoid secretion and altered feedbackleading to altered responsiveness of the HPA axis, with elevatedvasopressin mRNA expression- and vasopressin alone, rather thanin synergy with CRH, mediates activation of subsequent acute HPAresponses to stress. This is known as a state of chronic stress. Bothacute and chronic stress affect the other neuroendocrine systemsand therefore have an impact on reproduction.

3.3. Catecholamine responses to stress

Sympathetic nervous system responses to stress also impact onthe HPG axis and the uterus with the potential for disruption of ex-pected physiology. Typically, stress transiently and rapidly (withinseconds) induces increased adrenaline and noradrenaline secretionfrom the adrenal medulla and sympathetic nerve endings. As suchthey confer a more rapid response to stress than progesterone orglucocorticoids can. The catecholaminergic hormones provide animmediate energy source by mobilizing glucose, redirecting bloodflow from less essential processes to the skeletal muscles to enableflight if appropriate and facilitating stress coping. As well as theperipheral activation and secretion, central sympathetic/auto-nomic responses to stress lead to activation of central noradrener-gic pathways from the brainstem to the limbic system andhypothalamus [51]. There they interact with various neuroendo-crine systems and mediate behavioral responses to stress such asanxiety, fear and aggression.

3.4. Immune responses to stress

Stress, whether immune or non-immune, increases cytokineexpression and activity from immune cells. Most evidence for thiscomes from animal models and typically shows that stress partic-ularly increases Th1-type pro-inflammatory cytokine expression,

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Fig. 3. Effect of stress on progesterone and prolactin secretion in early pregnancy. a. Effecstress on morning plasma progesterone concentration in day 5.5 pregnant C57/Bl6 mprogesterone RIA. Data are % of control (vehicle- or un-treated) pregnant mice. *p < 0.05 vor 24 h fast (10 am–10 am) stress on morning plasma prolactin concentration in day 5.using an in-house prolactin ELISA. Data are % of control (vehicle- or un-treated) pregnaaverage after 2–4 h) or 24 h fast (10 am–10 am) stress on TIDA neuron activationimmunocytochemistry for Fos and tyrosine hydroxylase (TH, enzyme in dopamine syntheor un-treated) pregnant mice. *p < 0.05 vs. pregnant control.

including TNFa, IL1 and IL6 [113,205]. Stress also induces expres-sion and activity of pro-inflammatory cytokines from various othercell types including decidual cells [21,78] anterior pituitary cells[79,83,84,131,176], adrenal gland cells [82,193] and in the brain[131]. Since there is a complex interaction between cytokinesand the neuroendocrine system, this has consequences for secre-tion and effects on the key pregnancy hormones.

4. Impact of stress in pregnancy

Stress has been implicated in many problems encountered dur-ing pregnancy, including complete pregnancy failure (miscarriage),pre-eclampsia, preterm labor and fetal programming [67,93,95,96,135,162]. However, how stress impacts on each of these conditionsis sometimes less clear. Each hormone system responding to stressas described above has a different response profile to the othersand often is also different compared to that in non-pregnant fe-males, depending upon the day or stage of pregnancy. The impactof stress on each neuroendocrine system its own way adversely af-fects the implantation procedure and/or the developing embryoinitially, or fetus later. Furthermore, crosstalk between these sys-tems compounds the likelihood of a damaging outcome. Each ofthese maternal responses will be considered for their effect onpregnancy.

5. Stress responses in early pregnancy

5.1. Placental and pituitary gonadotrophin and prolactin responses tostress in early pregnancy

Progesterone is the pregnancy-maintaining hormone and so theeffect of stress on progesterone secretion is key to understandingits impact on pregnancy progression. Unfortunately, progesteroneconcentration after stress has rarely been analyzed in pregnantwomen. However, an emerging theme in early pregnancy is theattenuation of progesterone secretion by stressful conditions,shown by limited but insightful studies in mice [21,57,78]. Stressdecreases progesterone secretion in the immediate post-implanta-tion period in mice. Various stressors including 24 h noise andimmune stress result in reduced circulating progesterone concen-tration [21,57,78], and we have recently confirmed the inhibitionusing other stressors (Fig. 3a). This is in stark contrast to itsreported stimulation of progesterone secretion in non-pregnant

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in day 5.5 pregnant C57/Bl6 mice. Brain sections were processed by doublesis) and % of TH cells co-labeled for Fos were counted. Data are % of control (vehicle-

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females, as discussed above. We can tentatively presume that de-creased progesterone secretion reflects lack of gonadotrophinsecretion and action, and this has been investigated also, but onlysuperficially so far.

As for progesterone, responses of gonadotrophins to acute orprolonged stress during early pregnancy are different to those innon-pregnant females. In women we should consider the majorgonadotrophin hCG, but also pituitary prolactin and other placen-tal and decidual factors. There are mixed reports of the effects ofstress on hCG secretion. Circulating hCG has variously been shownto decrease [64], to not change [115], or to increase [8,9,172] as aresult of stressful situations. Increased hCG secretion seems coun-terintuitive, but perhaps occurs in an attempt to compensate forthe stress by driving higher progesterone secretion. For example,oxidative stress in the trophoblast clearly increases hCG secretion,and in culture various stress hormones also increase hCG secretion[9,172,185]. This indicates that direct threat due to poor perfusionby oxygenated blood, e.g. when there is inadequate angiogenesis,attempts to overcome the adverse signal by increasing progester-one and estrogen. Such a response may not be successful in main-taining gestation if the trophoblast and/or decidua remaininadequate and sufficient implantation is not achieved. OverallhCG cannot be a reliable marker or predictor of pregnancy failure,though whether the hCG gene splice variants associated with re-peated miscarriage [155,156] confer susceptibility to stress is notreported. Other than hCG studies like this, relevant pituitary andplacental responses to stress are not well investigated in pregnantwomen. Stress effects on LH or prolactin are not reported in the lit-erature to our knowledge. The effect of stress on placental lacto-gen, decidual prolactin and prolactin-like peptides is uncertain;heat stress and smoking decrease placental hormone secretiongenerally [150,194] but a clear description of responses in otherstress paradigms seems unavailable.

In mice there are some reports that lend insight into gonadotro-phin responses to stress in pregnancy. Typically LH secretion isinhibited anyway by the increasing luteal progesterone and estro-gen, via negative feedback mechanisms acting both centrally andat the pituitary (reviewed in [158]), so it is possible that prolongedstress which decreases progesterone secretion removes the feed-back allowing LH secretion to increase. One mouse study shows in-creased LH secretion during immune stress [57], supporting thisassertion. However, the same mice also exhibit decreased ovarianLH receptor expression, which would decrease LH-induced steroi-dogenesis [57]. LH responses to other stressors in early pregnancyare unknown. However, importantly, we believe that stress doesinhibit pituitary prolactin secretion at this time. We have reportedthat physical and psychological stressors applied to early pregnantmouse models leads to prolonged decrease in basal prolactin secre-tion. In our hands, immune stress induced by lipopolysaccharide(LPS) injection, hunger (after 24 h fast) or 24 h noise stress reducesperipheral prolactin concentration (Fig. 3b). This arises from anexaggerated hypothalamic response to stress such that TIDA neu-rons are highly activated compared to pregnant or virgin controlmice (Fig. 3c). Therefore, although effects on the rodent prolactinpeaks are currently unknown, even the elevated basal prolactinconcentration of early pregnancy is likely to play an important rolein pregnancy establishment. Furthermore, this means that brainperception and processing of the stressor contributes to the effectand it is not simply direct attenuation of pituitary lactotroph cellsby peripheral blood-borne signal(s) generated by the stress, suchas cytokines. How TIDA neuron responsiveness to the stress signalbecomes enhanced in pregnancy is uncertain as little is reported,but may be due to the previous exposure to prolonged high proges-terone [145]. As well as affecting hypothalamic stress responses,some stressors (particularly pro-inflammatory cytokines) havebeen reported to directly inhibit pituitary gonadotrophin secretion

[37,79,131] and gonadotrophin signaling in the corpus luteum iscompromised [57], affecting the pregnancy that way. The impor-tance of prolactin on pregnancy maintenance is reinforced by re-ports that prolactin replacement successfully rescues stress-threatened pregnancy by preventing progesterone degradation inboth the ovary and placenta/decidua [11,57]. Currently it is hardto find any literature on the effects of stress on the secretion of pla-cental or decidual hormones in early pregnant rodents.

5.2. HPA axis responses to stress in early pregnancy

As might be expected, maternal HPA basal activity and re-sponses to stress change dramatically during pregnancy. Depend-ing upon the species, this is in part due to the sex steroids,particularly progesterone, and in part due to new sources of HPAhormones, including CRH, ACTH and glucocorticoids, i.e. from theembryo/fetus, placenta and decidua. Many of the HPA axis adapta-tions in pregnancy have been extensively reviewed but merit fur-ther discussion here in the context of interactions with the HPGaxis, prolactin, and pregnancy failure.

Even in early gestation basal HPA axis activity is changed – with asuppressed circadian rhythm reported in women (see [27]). The pri-mary attention on the HPA axis in early pregnancy has been turnedtowards the glucocorticoids (i.e. cortisol). It seems that low salivarycortisol concentrations in women during the peri-implantation per-iod are optimal since elevated cortisol at 2–3 weeks correlates withlater miscarriage [126]. The few studies that have directly measuredcortisol in women later in the first trimester of gestation do notconfirm this association [27,125,132,138], so perhaps the peri-implantation period is a particularly sensitive time window for glu-cocorticoid action. Other HPA axis hormones are also associatedwith pregnancy failure in the first trimester, including increased cir-culating CRH [6]. However, the measured CRH is unlikely to be se-creted from the hypothalamus, but to instead come from theendometrium and developing placenta [72]. The role of intra-uterine sources of CRH will be discussed later, after the effects ofstress on the HPA axis in early pregnancy are reviewed.

Glucocorticoid secretory responses to stress in early pregnancyare only sparsely reported for women. Studies in women have uti-lized acute stressors such as the Trier Social Stress test (publicspeaking to a hostile audience) in first trimester pregnancy andshown that cortisol concentrations strongly increase [89,129].However, the effects of prolonged stress in early gestation on glu-cocorticoids are not clear. In women where reported perceivedstress or experience of bereavement or abuse is recorded there isno clear long term measurable effect on cortisol concentrations[132], although pregnancy outcome was not a reported parameterin this study and it is not clear from the literature that elevatedcortisol levels always result in pregnancy failure [125].

Similarly to women, the basal HPA axis activity is suppressedbut responses to acute stress are robust in rodents at this stageof pregnancy, and the response is similar to that seen in virgin fe-males (reviewed in [27,138]; Fig 4a). Overall the evidence indicatesno adaptation in glucocorticoid secretory responses in pregnant vs.non-pregnant females. Thus, transient high glucocorticoid concen-tration prevails during stress suggesting that this is not acutelyharmful to pregnancy maintenance. Rapid negative feedback en-ables rapid restoration of homeostasis, including for mother andoffspring metabolism, and/or for promoting tissue developmentand maturation in the face of inhibited prolactin and progesteronesecretion as detailed above. Prolonged stress that precipitatespregnancy failure in rodents does not appear to result in sustainedelevated corticosterone concentrations (Fig. 5a), as in women, sohigh corticosterone levels do not accompany pregnancytermination.

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Fig. 4. Acute effect of LPS on HPA axis activity in early pregnancy. (a) Effect of i.p. LPS (12 lg/mouse) on plasma corticosterone concentration in virgin, day 5.5 and day 10.5pregnant C57/Bl6 mice data are mean ± sem concentration at 90 min after injection, n = 4–9/group. Mice were decapitated and blood collected and analyzed using acommercial corticosterone RIA kit. (b) Acute effect of i.p. LPS on parvocellular PVN CRH expression in the same virgin, day 5.5 and day 10.5 pregnant mice as in (a). Brainswere collected, cryostat sectioned at 15 lm and processed by in situ hybridization for CRH mRNA using an 35S dATP-labeled oligonucleotide probe as previously described[144]; data are mean ± sem total grain area per PVN profile calculated from average grain area per cell � number of positive cells per profile. *p < 0.05 vs. vehicle (data notshown); #p < 0.05 vs. virgin.

366 A.J. Douglas / Frontiers in Neuroendocrinology 31 (2010) 359–376

One might anticipate that the hypothalamic elements of theHPA axis in early pregnancy underlie the changes in basal andstress-induced glucocorticoid secretion, with stimulated geneexpression of CRH and vasopressin and their increased secretion.All reports available indicate that basal CRH and vasopressincontent and expression remain stable during early pregnancy

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Fig. 5. Prolonged effect of stress on HPA axis activity in early pregnancy. (a) Corticexperiencing pregnancy failure; mice were exposed to 24 h noise stress on day 5.5 of pregstatus and serum was analyzed for corticosterone using a commercial RIA kit. Data are mand c) Effect of stress at day 5.5 pregnancy on parvocellular PVN CRH and vasopressin (AVvehicle as a control (Con) or were subjected to 24 h noise stress (Stress) or given LPS (i.p35S dATP-labeled oligonucleotide probes as previously described [144]. Data are mean ± sof 24 h noise stress at day 5.5 pregnancy on parvocellular PVN CRH and vasopressin (AVwere either given vehicle (sesame oil, 200 ll) and left undisturbed as a control or were(Dydro, progesterone substitute, i.p. 1.25 mg/200 ll). Brains were collected on day 7.5, 935S dATP-labeled oligonucleotide probes as previously described [144]. Data are mean ±

[80,101,124]. However, we have started to investigate their re-sponses to stress since, intriguingly, Nakamura [124] showed thatmedian eminence CRH content does not decrease with acute re-straint stress in early pregnancy, suggesting that CRH is not re-leased. We approached this by analyzing mRNA responses in thePVN and unexpectedly found that although immune stress acutely

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osterone concentration in stress-sensitive mice (DBA-mated CBA females) micenancy and killed on the morning of day 13.5 when they were checked for pregnancyean ± sem corticosterone concentration non-stressed and stressed pregnant mice. (bP) mRNA expression on day 6.5 pregnancy. DBA-mated CBA mice were either given

. 2 lg/mouse). Brains were cryostat cut and processed by in situ hybridisation usingem grain area per cell, n = 7–9/group. *p < 0.05 vs. control. (d and e) Prolonged effectP) mRNA expression 1–4 days later. On day 5.5 of pregnancy DBA-mated CBA micegiven vehicle and subjected to 24 h noise stress (Stress) or given Dydrogesterone

.5 or 10.5 of pregnancy and cryostat cut and processed by in situ hybridisation usingsem grain area per cell, n = 4–5/group. *p < 0.05 vs. control, #p < 0.05 vs. Stress.

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stimulates CRH mRNA expression in virgin mice, CRH mRNA re-sponses are strongly attenuated in early pregnant mice (Fig. 4b).It seems that, unlike the arcuate TIDA neurons, PVN neuron re-sponses to stress are weak in early pregnancy. So far no studieshave investigated the underlying reasons for attenuated PVN re-sponses in early gestation, or why they are dissociated from thestrong pituitary and adrenal cortex responses. It may be that theimmune stress directly stimulated the pituitary corticotrophs,since cytokines directly increase ACTH secretion [10,20,79], andadrenal cortex glucocorticoid secretion [205], so potentiallybypassing the hypothalamic level of control. Other non-immunestressors also induce cytokine activity [21,78,79,131], which coulddirectly stimulate ACTH and/or glucocorticoid secretion, so in thatway stress generally may under certain circumstances drive thepituitary–adrenal axis independently from the hypothalamic re-sponse. Alternatively, parvocellular PVN vasopressin neuronsmay still respond to acute stress in early pregnancy and couldalone drive pituitary ACTH secretion as it does in chronic stress,but vasopressin mRNA or secretory responses to acute stress at thistime are not yet reported.

The effects of prolonged or chronic stress on hypothalamic acti-vation in early pregnancy are also just beginning to be analyzed inrodent models. Using an abortion-prone mouse model, the DBA-mated CBA female that exhibits increased pregnancy failure espe-cially after stress in association with cytokine imbalance [4], wehave measured HPA axis activity at a range of days post-stress.Compared to non-stressed controls a low dose of LPS slightly butsignificantly increased both CRH and vasopressin mRNA in thePVN after 24 h, while 24 h noise stress also slightly increased vaso-pressin mRNA (Fig. 5b and c). In the same mice there was little orno change in serum corticosterone concentration, but as expected asubstantial decrease in progesterone concentration (personal com-munication, Friebe A, Arck PC). At later times, e.g. 2–5 days afterthe end of 24 h noise stress, we have observed no long termstress-activation but vasopressin mRNA progressively rises instressed mothers compared to the day after stress and comparedto unstressed controls (Fig. 5d and e). This suggests that there isno activation of the PVN until later and even then only vasopressinexpression responds. This could be due to an emerging chronicphysiological stress state, as it is known that basal vasopressinmRNA increases in such conditions. It could also be due to the re-lease of other factors relating to the emerging pregnancy failure,for example pro-inflammatory cytokines from the degrading tro-phoblast and decidua, or altered hemodynamics/osmoregulation,also resulting from the lack of sex steroids following stress in thistime frame [21]. It is interesting to speculate that progesteronehelps to restrain PVN neuron activation throughout pregnancysince progesterone replacement with Dydrogesterone during stressattenuates the stress-induced increase in vasopressin mRNA(Fig. 5e). On the other hand the Dydrogesterone treatment effec-tively prevents pregnancy failure [78], so its ultimate effect onvasopressin mRNA could be indirect. Also, in these mice corticoste-rone concentrations are similar regardless of day or treatment,and this matches more closely the lack of CRH mRNA response,further reinforcing our tentative conclusion that pregnancy failureis not associated directly with activation of the HPA axis or ele-vated glucocorticoids, at least across this early-midpregnancyperiod.

Intra-uterine CRH: CRH is not only expressed in the PVN, but isexpressed in peripheral reproductively-related tissues; this localsource and site of action is known to be important for pregnancy,particularly in women. CRH and related peptides – urocortins –are particularly expressed in the endometrium and decidua in earlypregnancy, and act via CRH receptor types 1 or 2 within thesestructures or are secreted into the blood. Endometrial CRH is impor-tant for optimal implantation by moderating early maternal im-

mune tolerance. CRH locally enhances decidualisation, at leastpartly via IL1 and IL6 (reviewed in [72]), so appropriate control ofits expression and action is also required in pregnancy. Progester-one induces endometrial CRH expression, so in part progesteroneacts via CRH within these tissues. Estrogens and glucocorticoidsboth suppress CRH expression, and overall the balance betweenthese steroids will determine any CRH effects locally within theuterus [104]. Despite the positive effect of CRH on implantation,elevated CRH (and urocortins) in the endometrium is associatedwith repeated spontaneous abortion [102,116]. The effect of stresson endometrial CRH expression or actions in early pregnancy is un-clear from the literature, though stress-related CRH evidently com-promises utero-placental circulation [74]. Although rodents doexpress CRH within the uterus, its roles and actions are less wellunderstood than for women. The effects of stress in such modelsare also unclear, but one report mentions that stress-induced im-mune responses are not mediated by peripheral CRH [123].

Glucocorticoids also exert a multi-level impact particularlywithin the uterus, with proven adverse effects causing adverse fe-tal programming [119,167]. As steroids, glucocorticoids can readilycross the placenta to access the embryo. As indicated above, gluco-corticoid action is determined locally by enzymes and the placentaexpresses 11bHSD II which degrades the steroid. However, in earlygestation this protective barrier is only emerging and weakly con-trols glucocorticoids before the placental 11bHSD II expression isoptimal in midpregnancy [7,76]. Therefore, the embryo is particu-larly vulnerable to stress in early pregnancy. This has been shownin paradigms using maternal protein deprivation which, even priorto implantation, confers susceptibility to anxiety and cardiovascu-lar disease in the offspring in later life [197]. Also maternal stressin the first week of gestation in mice compromises placental11bHSD II expression leading to low birth weight and altered en-ergy homeostasis in offspring [137]. Together these studies showthat the embryo-maternal interface is readily compromised evenin the very early stages of pregnancy, including before decidua orplacenta formation. Overall, the combination of robust maternalresponses to acute stress and incomplete breakdown of glucocorti-coids by the placenta equate with high risk of embryo program-ming at this time.

5.3. Sympathetic nervous system responses to stress in earlypregnancy

The uterus is extensively innervated by the sympathetic ner-vous system and expresses a- and b-adrenoreceptors. It was recog-nized more than 30 years ago that myometrial sympatheticinnervation adapts in pregnancy, with adrenergic innervationreceding from the uterus in early pregnancy in women as part ofits inherent remodeling [187,206]. The resulting reduction in sym-pathetic nerve activity could help to prevent inadvertent inductionof myometrial contraction through pregnancy. However, like HPAaxis hormone secretion, adrenaline and noradrenaline secretory re-sponses into the circulation are strong (data from early second tri-mester; [89,129]), raising the possibility that they could still affectimplantation, decidualisation and embryo programming especiallyif secretion is repeated or sustained with stress. Rodent studieshave given further insight into mechanisms underlying altereduterine innervation. For example, the sympathetic innervationseems to become mainly confined to the outer myometrium[171]. This denervation is accompanied by altered adrenoreceptorexpression and signaling [114] and increased monoamine trans-porter activity [22]. The change in catecholaminergic innervationcould in part be mediated by nitric oxide [118] and, importantly,the sex steroids [55,56]. However, whether circulating catechola-mines directly affect decidual or placental function in women orrodents at this stage of pregnancy is unclear at present.

Table 1Summary of hormonal secretory responses to stress in early pregnancy.

Hormone Type of stress Stress effect on secretion Notes Reference

hCG Pregnancy worries Decrease Women Gagnon et al. (2008)IVF anxiety No change Milad et al. (1998)Hypertension Increase Sitras et al. (2009), Kharfi Aris et al. (2007)

Prolactin Immune, fast, noise Decrease Mice Fig. 2a and b, Parker and Douglas (2010)LH Immune Increasec Mice Erlebacher et al. (2003)Progesterone Immune, fast, noise Decrease Mice Fig. 2a

Women Milad et al. (1998)Cortisol Trier social stress

Perceived stressaIncreaseNo change

Women Nierop et al. (2008), Klinkenberg et al. (2009)Obel et al. (2005), Nakamura et al. (2008)Fig. 3a, Brunton et al. (2008), Parker and Douglas (2010)

Corticosterone Immune, fast, noiseChronic stressb

IncreaseNo change

Mice

Catecholamines Trier social stress Increase Women Nierop et al. (2008), Klinkenberg et al. (2009)Placental lactogen (heat, smoking) (decrease)c Women Regnault et al. (1999), Varvarigou et al. (2009)Decidual prolactin/prolactin-like peptides (heat, smoking) (decrease)c Women Regnault et al. (1999), Varvarigou et al. (2009)Cytokines* Fever Th1 Th1 Women Chrousos (1995)

Perceived stressa Th2c Arck et al. (2002)24 h noise Th1 increase Mice Joachim et al. (2003), Blois et al. (2004)24 h noise Th2 decrease Joachim et al. (2003), Blois et al. (2004)

Overview of hormonal secretion and cytokine activity (*evidence mainly from decidua rather than circulating cytokines) in response to acute or prolonged stress in earlygestation.

a Perceived stress is the reported subject perception of their stress environment assessed by questionnaire.b Chronic stress data derived from mice exposed to stress for 24 h at day 5.5 of pregnancy and analyzed 7 days later.c Response not clear from published data and/or from very limited reports.

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5.4. Immune responses to stress in early pregnancy and interactionwith neuroendocrine hormones

As indicated above, immune balance is critical for pregnancyestablishment. In miscarrying women perceived high stress scorescorrelate with increased Th1-type pro-inflammatory cytokines inparticular [4,5]. Since there is a complex interaction between cyto-kines and neuroendocrine systems, this has consequences for secre-tion and effects of the key pregnancy hormones. The pregnancycytokine balance is established due to the increasing progesteronesecretion and its action via PIBF on cytokine expression and activity[148]. Although not reported in women, it has been shown in ro-dents that stress-induced pregnancy failure is accompanied by de-creased PIBF and that the decreased Th2:Th1 cytokine ratio is due tothe reduced progesterone secretion. Progesterone replacement canprevent stress-induced cytokine imbalance [21,78] and so, since itcan target multiple mechanisms that maintain pregnancy in rodentmodels, could prove be a useful protective therapy in identified wo-men at risk of stress-induced pregnancy failure. Likewise, stress-in-duced low prolactin, as well as compromising progesteronesecretion, is expected to directly compromise pregnancy immunebalance, and synergism between all these mechanisms is obviouslynecessary for the optimal pregnancy hormonal environment.

This synergism includes glucocorticoids. To our knowledge littleis understood of glucocorticoid actions in the uterus in early preg-nant women but rodent studies indicate that the local decidual re-sponse to the invading trophoblast requires NFjB [122], so fromone angle glucocorticoids might seem harmful since they typicallyinhibit pro-inflammatory mechanisms via NFjB. Alongside this,the HPA axis, including increased central CRH and peripheral gluco-corticoid secretion, inhibits the HPG axis [38]. So stress-inducedglucocorticoids are likely to restrain local mechanisms and risk poorimplantation. On the other hand, glucocorticoids inhibit undesir-able circulating pro-inflammatory cytokine activity and reduce cir-culating dendritic immune cells and NK cells [123,179], so may havesome beneficial role in limiting fever or general immune responses.However, any cytokines produced will stimulate the HPA axis viavagal and brainstem mechanisms [27], resulting in a feedback loopbetween immune factors and HPA hormones. From the availableinformation, whether glucocorticoids alone directly or indirectly

elevate risk of early pregnancy failure by interaction with cytokinesis still debatable and requires more thorough investigation.

Overall, early pregnancy is a risky time with potential for failureor adverse programming as a result of stress (Table 1). Clear evi-dence links stress-induced low prolactin and/or progesterone withpregnancy failure, at least partly mediated by cytokine imbalance.

6. Stress responses in midpregnancy

Mid gestation represents a transition between mechanismsestablishing pregnancy and those maintaining pregnancy and de-pends upon continued progesterone secretion and its underlyingdrive, as discussed above. However, to our knowledge, the effectsof stress on prolactin and placental lactogens or even progesteronesecretion remain unreported at this time. Complete pregnancy fail-ure itself is less likely around this time, since by now a large mater-nal metabolic, physical and psychological investment has beenmade. More likely outcomes now are pre-eclampsia and pretermlabor, of which preterm labor is evidently most susceptible tostress, and fetal programming, which is still a highly relevant risk.

In women progesterone and placental lactogen secretion fromthe placenta predominate in midpregnancy and prolactin secretionis also high. Although their responses to stress are largely unknown,they are unlikely to be as responsive as the hypothalamus/pituitaryaxes. In rodents, but not women, the high placental lactogen secre-tion at this time inhibits pituitary prolactin secretion and action,but will itself, and with other prolactin-like hormones, directly con-tinue to facilitate corpus luteum progesterone secretion [175].

HPA axis and catecholamine responses to stress exposure inmidpregnancy are reported in women and are similar to those inearly pregnancy, with robust ACTH, glucocorticoid, adrenaline andnoradrenaline secretion regardless of stressor [129] used includingimmune, psychological or other physical stressors. HPA axis but notcatecholamine secretory responses in midpregnancy are reportedin rodent models and generally mirror those in women [27].

Nevertheless, it is known that various neuroendocrine systems,including their responses to stress, adapt in midpregnancy. Forexample, opioid inhibition develops on oxytocin neurons frommidpregnancy onwards [159]. This is indicative of the start of

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many overt maternal neuroendocrine adaptations that emergethrough the second half of gestation, and their responses to stressare particularly well investigated.

7. Stress responses in late pregnancy

Despite the evidence for acute, prolonged or chronic stressimpacting on the HPG axis and prolactin secretion in early preg-nancy and the role of stress during late pregnancy in precipitatingconditions like preterm labor (reviewed in [67,93,95,96,135,162]),again, no studies seem to have reported the effects of stress on thesecretion of gonadotrophins or progesterone. In contrast, much isnow understood about adaptations in the HPA axis and the mech-anisms that underlie them, and these are addressed in the next sec-tion. However, some insights into the role of progesterone withinthe brain and uterus at this time have been defined and the evi-dence for this and the involvement of other key pregnancy hor-mones in the HPA axis adaptations and other uterine andplacental mechanisms will discussed in parallel.

7.1. HPA axis adaptations in late pregnancy

The changes in the HPA axis in late pregnancy have been exten-sively reviewed recently (e.g. [27,46,52,157]) so here, instead ofanother detailed review, we will aim to analyze whether and towhat extent the HPA axis contributes to adverse outcomes. Thesection will start off with a summary of the adaptations reportedalongside mechanisms driving them and their contribution topre-eclampsia and preterm labor, then will discuss the impact ofstress on the feto-maternal interface, before finally consideringsympathetic responses and their effects on pregnancy outcome.

Overall, stress-induced activity of the HPA axis becomesstrongly attenuated by late gestation in women and most relevantanimal models, and this appears to be a global phenomenon sincealmost any stress applied reveals similar results [27,86,165]. Fromrodent studies we know that the central hypothalamic elementsare inhibited, thus both CRH and vasopressin mRNA in the parvo-cellular PVN and secretion from the median eminence are reduced([24,27,42,43,101], and they are less sensitive to incoming signals[46].

Many higher centers and regions projecting to the PVN, such asthe amygdala, septum and cingulate cortex, also exhibit attenuatedresponses to stress [42], suggesting that stressor perception and/ortransmission of stress signals to the PVN is decreased, at least insome aspects. Unexpectedly, although brainstem noradrenergicpathways are a major relay of stress signals to the hypothalamus[46], they still become robustly activated by stress at this time[25,42] However, we have found that although their expressionof Fos is induced (an immediate early gene indicator of neuronalactivation), the release of noradrenaline from terminals locallywithin the PVN is strongly restrained. Evidence indicates that co-expressed opioids, which increase in late pregnancy, are co-re-leased at noradrenaline terminals in the PVN during stress and pre-synaptically inhibit the local noradrenaline release [25,50,51], thusreducing a primary stimulatory signal. This might be able to ex-plain the attenuated responses of PVN neurons to stress. Interest-ingly, although little is reported on the effect of stress onprogesterone secretion in late pregnancy, it is progesterone thatis emerging as the underlying controller of these HPA axis re-sponses. Mimicking pregnancy progesterone and estrogen levelsinduces strong inhibition on some neuroendocrine systems suchas the oxytocin neurons, including their responses to stress [49].But, progesterone’s metabolite, allopregnanolone, is emerging asa more potent inhibitor of the HPA axis and has been shown tobe an underlying cause of the pregnancy-attenuated HPA re-

sponses [24]. Thus, blocking enzymatic conversion of progesteroneto allopregnanolone with finasteride prevents the HPA axis hypo-responsiveness to stress. It does this at least partly by inducingbrainstem (nucleus tractus solitarius) opioid and opioid receptorexpression, and consequently inhibits noradrenaline release whichactivates CRH/vasopressin neurons less. There is also a potentialrole for allopregnanolone or other neurosteroid metabolites di-rectly on PVN neurons where they might be expected to enhanceGABA inhibition by facilitating GABAA receptor signaling, as shownfor other neuroendocrine (i.e. oxytocin) neurons in late gestation[28,139]. Thus, progesterone and progesterone-related action onselected populations of neurons in the brain explains the late preg-nancy phenomenon of HPA axis hyporesponsiveness.

The lack of PVN activity seems to now underlie reduced pitui-tary and adrenal cortex responses [101], in contrast to early preg-nancy. Nevertheless, elevated basal maternal glucocorticoidsecretion is observed in women, sheep, mice and rats in late gesta-tion, although in rats is only evident on the last day or two ofgestation [27,128]. This reflects the importance of maternal gluco-corticoids in the facilitation of fetal organ development in the lastfew days before birth. However, high glucocorticoid concentrationsare not solely due to increased HPA activation since basal andstimulated ACTH and corticosterone concentrations become disso-ciated in late pregnancy. In some species this results from increas-ing fetal secretion of glucocorticoids (i.e. sheep, and facilitatingonset of birth), and in others from altered sensitivity of the adrenalcortex to ACTH [31].

Because of the high glucocorticoid secretion near term, it mightbe argued that late pregnancy is ‘stressful’. If that were the casesigns of chronic stress might be expected, including attenuatedCRH but retained vasopressin mRNA/secretory responses to stress,increased glucocorticoid negative feedback, and anxiety or depres-sion. In pregnant women reported anxiety and depressive-likesymptoms are mainly associated with adverse environmental con-ditions [92]; although not investigated in pregnancy, depressivesymptoms in humans are associated with a sustained increase inplasma CRH [87,151]. Other measures are not investigated in preg-nant women but in rodents evidence suggests that under basalconditions pregnancy is not a state of chronic stress. Taking thePVN responses first, although basal and stimulated CRH activityare reduced similar to that during chronic stress, basal and stimu-lated vasopressin activity are also reduced, which is not a conse-quence of chronic stress. Therefore the key hypothalamicmarkers do not match a chronic stress state. Some analysis hasbeen made of glucocorticoid feedback to the maternal brain. Thereis increased 11bHSD type I activity in the PVN and pituitary whichmight indicate increased local generation of glucocorticoids, butthere is little or no change in glucocorticoid receptor or mineralo-corticoid receptor expression in the hippocampus as might be ex-pected with increased feedback. In vivo studies that test thephysiological responses as a whole do not reveal altered feedbackmechanisms [80]. Furthermore, anxiety is decreased rather thanincreased [128,173]. So there does not seem to be a typical chronicstress phenotype despite the increased glucocorticoid secretion fora few days perinatally, and the natural increase in glucocorticoidsin the maternal circulation towards birth suggests that they are notalone detrimental to mother or fetus. Despite this, stress continuesto be reported as a risk factor for various maternal and fetal condi-tions. These conditions appear to be often related directly to thefeto-maternal interface, so the next section will focus on that andits ability to be compromised by stress.

7.2. Stress and the feto-maternal interface

The feto-maternal interface continues to be important in lategestation for several reasons, including protection of the fetus

370 A.J. Douglas / Frontiers in Neuroendocrinology 31 (2010) 359–376

and production of peptides thought to regulate birth. Placentalprotection of the fetus from harmful maternal factors, includingglucocorticoids, continues to strengthen in late gestation. Indeed,the concept of fetal programming and its relationship to glucocor-ticoids was first identified in late gestation and is now establishedas an important mediator of poor long term health and wellbeingin humans [166]. As already indicated above, maternal mecha-nisms for restricting glucocorticoid access to the fetus at this stagehave been clarified in various rodent models (reviewed in [26,27]).The primary mechanism is perhaps the prevention of excessiveglucocorticoid secretion in response to the environment in the firstplace. Secondly, cortisol/corticosterone is carried in the circulationby corticosteroid binding globulin (CBG, transcortin) which is se-creted by the liver. This substantially reduces free glucocorticoidlevels in pregnancy, which is sometimes not clear from data re-ported in the literature [48]. Thus, in mid-late pregnancy, whenCBG secretion is increased, the majority of glucocorticoid is notbioactive and the fetus is not overly exposed to it regardless ofwhether the mother experiences stress or not. Thirdly, but argu-ably most importantly, the placenta expresses high levels of11bHSD type II enzyme which inactivates glucocorticoids and pre-vents their transfer across the maternal-fetal barrier, so protectingthe offspring [167]. Generally this appears to be highly effectivedespite high basal glucocorticoid secretion. On the other hand,clinically there is a problem. Any glucocorticoid not susceptibleto enzymatic degradation, such as the synthetic steroid dexameth-asone, escapes the barrier and so readily causes adverse fetal ef-fects [23,167]. Therefore, the use of such drugs at preterm labor/birth to facilitate fetal tissue development, such as lung matura-tion, inevitably is inadvisable. The reason stress has such a pro-found impact on fetal development in the perinatal period isgenerally not because it increases maternal glucocorticoid secre-tion but because it compromises placental expression and activityof 11bHSD II [33,35,54,103,111,120,169,198,199], thus breakingthe barrier and allowing placental glucocorticoid transfer [201].How stress causes this seemingly minor but destructive changein a placental enzyme is not well understood but some potentialmechanisms can be discussed.

The pregnancy steroids are an obvious potential regulator of11bHSD II. Estrogen increases 11bHSD II activity to facilitate pri-mate fetal-placental development [2], although inhibition has beenshown in cultured human placenta [180]. Expression patterns of11bHSD II, glucocorticoid and mineralocorticoid receptor in thehuman endometrium and decidua also indicate a role for proges-terone [109]. Progesterone does the opposite to estrogen, inhibit-ing 11bHSD II mRNA and activity in various tissues including theall the tissues comprising the feto-maternal interface in humans,primates and rodents [146,147,180]. Interestingly, progesteroneis also susceptible to degradation by 11bHSD II in some human tis-sues [146] so, if true also for the placenta (as in rodents [29]), couldthen regulate local concentration of this pregnancy steroid- andmight be important in optimizing the intra-uterine/decidualenvironment.

Rodent models show that progesterone secretion and placental11bHSD II both increase from early gestation it may be that proges-terone’s inhibitory effect emerges only at high circulating levels,and this correlates with the reported natural decrease in 11bHSDII activity at term pregnancy [40,41]. Therefore, the barrier may al-ready be weakened at this time even in the absence of stress. Inter-estingly as well as glucocorticoids, progesterone binds to themineralocorticoid receptor, and has antagonist-like action so thisway may protectively prevent glucocorticoid binding under nor-mal conditions. On the flip side, if progesterone decreases as it doesnear term (to facilitate myometrial contractility and birth) andwith stress, then that may allow glucocorticoids to exert their det-rimental effects more. This may be partly how low progesterone

contributes to fetal programming- evidence for this includes datashowing that blocking of the progesterone/mineralocorticoidreceptor by RU486 perinatally compromises sex differentiation inthe male brain [195].

The other placental factors of interest here are CRH and the uro-cortins, which in women are particularly involved in the onset oflabor. CRH and urocortin secretion by the placenta gradually in-crease in late pregnant women [68,142,168,174]. They act viaCRH 1 and 2 receptors in the endometrium and myometrium andare thought to trigger a switch in their progesterone receptor A/Bsubtype balance [174]. This facilitates onset of uterine contractionsand subsequent delivery [191]. They also evidently inhibit placen-tal progesterone production in vitro [177,204], so accelerating theprogress towards birth as their secretion increases. ThereforeCRH and urocortin levels correlate with birth timing, even beingelevated in women with preterm birth [62,91,174]. As such theyhave been proposed as a possible predictive factor for imminentbirth, but there is uncertainty as to whether they actually cause on-set of labor [69]. There is also a debate whether CRH and urocortinsare markers for fetal/placental stress.

Stress effects on placental CRH and urocortin expression are notwell described and their effects on 11bHSD II expression or activityare not reported yet, to our knowledge, so their role in stress-in-duced fetal programming is currently unclear. However, some use-ful information is available in limited studies. For example,hypertension, which is a marker for pre-eclampsia – reflecting fe-tal/placental distress, increases fetal secretion of urocortin [60,61].In parallel, higher plasma CRH correlates with higher placentalresistance [74]. However, whether these responses affect fetalhealth is unclear. CRH also exerts inflammatory actions, being se-creted at inflammatory sites [85]. Since cytokine balance is funda-mentally important for maintaining gestation, particularly at thefeto-maternal interface, increasing CRH could contribute in thisway to pre-eclampsia or infection-related preterm labor. It isknown that pro-inflammatory cytokines decrease 11bHSD II activ-ity in smooth muscle cells and placenta [90,189], correlated withsuboptimal pregnancy status. Conditions such as intra-uterinegrowth retardation, pre-eclampsia or preterm labor, as well asinfection, are associated with elevated cytokines such as IL1b, IL6and TNFa [59,121,203] so perhaps it is not surprising that theyare accompanied by low birth weight and increased neonatal andchildhood morbidity. Evidently there is a complex interaction be-tween placental peptides and steroids that normally sustains ges-tation until term, but pathophysiological conditions and stressseverely affect their inter-communication with potentially seri-ously and/or long term consequences.

7.3. Catecholamine responses to stress in late pregnancy

As well as the HPA axis responses to stress, it is also importantto consider the sympathetic hormones in late pregnancy. The ma-ture human placenta is rich in both a- and b-adrenoceptors[58,136,163,164] and although sympathetic innervation recedesthrough early-midpregnancy, as noted above, elevated catechola-mines still result in fetal growth retardation such as that observedwith fetal programming [160]. This may be because in late preg-nancy the endometrium/myometrium becomes re-innervated[170] allowing adrenergic action. Psychosocial stress also inducesrobust catecholamine secretory responses in second and third tri-mester as measured by heart rate or salivary a amylase [89,129],and furthermore, others have reported more enhanced noradrena-line levels when women are fearful of birth [161]. Therefore, ad-verse fetal programming may also arise due to the circulatinghormones secreted in response to stress that bypass the sympa-thetic nervous system. Rodent studies indicate that b-receptor sen-sitivity increases as progesterone decreases near term. Indeed,

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Fig. 6. Effect of stress on catecholamine secretion in late pregnant rats. Blood samples were collected from virgin and 21 day pregnant rats with an indwelling jugular cannulabefore and after exposure to airpuff startle (a–c): five blasts of air directed at the nose repeated three times [127]; or forced swim (d–f, 19 �C water for 90 s) (grey bars);controls were undisturbed (white bars). Plasma was analyzed for ACTH, adrenaline and noradrenaline using commercially-available RIA kits. Data are mean ± sem deltahormone concentration at 2.5 min (catecholamines) or 10 min (ACTH) after stress compared to before, n = 3–7/group. *p < 0.05 vs. control, #p < 0.05 vs. virgin stressed group.

A.J. Douglas / Frontiers in Neuroendocrinology 31 (2010) 359–376 371

artificially decreasing progesterone also increases b-receptorexpression [56] so any stress-induced change in progesterone willimpact on uterine sensitivity to catecholamines. We have discov-ered that unlike HPA axis responses to either mild airpuff or stron-ger forced swim stress, catecholamine secretory responses areweakly or not attenuated in the late pregnant rat (Fig. 6), particu-larly for noradrenaline. This is in line with our data showing thatstress also strongly activates centrally-projecting brainstem norad-renergic neurons in the A2 region in late pregnancy [25]. As stressstrongly induces adrenaline and noradrenaline secretion in latepregnancy then these could mediate fetal programming via theupregulated b-receptors.

Interestingly, there is clear in vitro evidence that catechola-mines potently attenuate 11b-HSD II expression in human placen-tal cells [163], so stress-induced catecholamines could also readilycompromise placental function and underlie adverse fetal pro-gramming that way. In parallel with 11bHSD II regulation of localglucocorticoid concentration, another enzyme regulates local cate-cholamine concentration. Catecholamine action in the placenta ishighly dependant upon the activity of monoamine oxidase (MAO)type A [200], which is abundantly expressed [32]. This would beprotective for the fetus, but previous studies have reported adown-regulation of placental MAO activity in various pathologicalstates of pregnancy. For example, MOA activity is reduced in pre-eclampsia and toxemia [15,15,16,32], conditions that are charac-terized by elevated levels of plasma catecholamines, and in vitrostudies of term placenta show that MAO activity is attenuated un-der anoxic conditions [34]. It is important to note that high levelsof progesterone have been shown to increase MAO-A activity inuterine tissues [44], so this represents another role for progester-one in establishing and maintaining viability of the feto-maternalinterface. Although it is not reported whether psychological stressimpacts on MAO-A expression or activity, the enzyme is sensitive

and mediates adverse conditions and has an important role inthe protection of the fetus.

Therefore, there is multi-level control of steroid mechanisms inthe placenta to protect the fetus, and since catecholamines interactwith them and respond to stress, they pose a risk to the pregnancy.Other factors inevitably also interact with the catecholamines,including cytokines and prolactin/placental lactogen. Furtherinvestigation of stress effects on the brainstem noradrenergic neu-rons and pre-motor sympathetic neurons in late pregnancy [25,27],will be key to further understanding maternal-fetal interface integ-rity more fully.

8. Conclusion

Stress has a negative impact on pregnancy maintenance and/oron the embryo and fetus itself, and the mechanisms by which itpotentially mediates the adverse outcomes are multiple and com-plex. Key roles are identified for each of progesterone, prolactin,glucocorticoids and cytokines. However, in responding to stressthe overall conclusion is that the maternal brain mediates the mostprofound consequences, causing decreased secretion of importantpregnancy-maintaining hormones such as prolactin and progester-one (Fig. 7). New evidence indicates that in early pregnancy this islikely to be directly by compromised secretion of hypothalamicneuroendocrine factors such as dopamine and oxytocin, in con-junction with indirect attenuation of corpus luteum function, andso fails to procure the appropriate intra-uterine environment. Itmay emerge that neuroendocrine systems also compromise theexpression and action of multiple decidual and placental factorsat this time and it might be predicted that cytokines, placental lac-togen and decidual prolactin and LH would be important targets.Later, when factors maintaining pregnancy are primarily secreted

Fig. 7. Summary of key neuroendocrine factors determining pregnancy maintenance during stress. Stress-induced inhibition of secretion of key pregnancy hormonesincludes impact on progesterone and prolactin concentrations. These would seriously compromise the appropriate intra-uterine environment, whereby key placental anddecidual factors could be inadequately secured at the necessary time during pregnancy. *Including placental lactogens and other prolactin-like peptides; ? = includingmechanisms moderating glucocorticoid action such as MR/GR expression and 11bHSD II expression and activity.

372 A.J. Douglas / Frontiers in Neuroendocrinology 31 (2010) 359–376

from the placenta/decidua, neuroendocrine systems may play lessof a role in mediating acute stress responses. It is interesting tospeculate that, nonetheless, factors such as progesterone and pro-lactin/prolactin-like peptides, still decrease under stressful envi-ronmental conditions, and so contribute to suboptimal pregnancyoutcome then too. How they might do this is unclear, but theymay react to the (sometimes overlooked) sympathetic systemwhich retains robust secretory responses regardless of pregnancystage. Therefore, the maternal brain needs to be more deeplyprobed in both women and appropriate rodent models while ithas a ‘Baby on Board’.

Acknowledgments

Thanks go to my PhD student Victoria J. Parker for prolactin andmRNA analysis, and to Petra Arck, Sandra Blois, Astrid Friebe andMiake Pincus for ongoing collaboration using the DBA-mated CBAmouse model and a cohort of pregnant women in Germany. Thanksalso to Mike Ludwig for comments on an early draft of the manu-script. AJD was a partner in the EU Network of Excellence on Em-bryo Implantation Control (EMBIC), and was funded by the MRC,The Wellcome Trust, and The Society for Endocrinology.

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