The placenta, essential for normal fetal development by providing adequate nutrients to allow appropriate growth and maturation of the fetus in preparation for birth, is also a ‘protective’ barrier in the sense that it prevents entry into the fetal circulation of substances that are either toxic, or that drive fetal growth at inappropriate rates. An important aspect of this ‘filtering’ function of the placenta is limiting the entry of glucocorticoids of maternal origin into the fetal compartment. This is achieved by the presence of enzymes, transporters and receptors collectively termed the ‘placental glucocorticoid barrier’ [1-4].
Antenatal glucocorticoids are routinely administered to the mother for the treatment of a variety of pregnancy and fetal complications. Asthmatic women often experience an increase in severity of their symptoms during pregnancy leading to increased use of glucocorticoids. The threat of preterm birth results in administration of the synthetic glucocorticoid, betamethasone, to rapidly mature fetal organs (especially, the lungs) to promote survival. Further, stressful events during pregnancy such as natural disasters and famines for example, expose fetuses to higher than normal levels of maternally secreted glucocorticoids.
The effects of exposure to high levels of glucocorticoids during fetal development have now been well described [1, 5-9], and the advantages (e.g., maturation of lung surfactant production and increased hepatic glycogen deposition) are offset by effects that limit fetal growth and induce perturbations of brain growth and perfusion [1, 10-11]. However, while fetal/neonatal effects have been intensively investigated, the consequences of glucocorticoid excess on placental structure and function has received little attention to date. The knowledge that male fetuses are more likely to be affected negatively following events that usually increase fetal glucocorticoid exposure, has alerted researchers to the possibility that such sex-related effects could arise in the placenta. This chapter will describe the differences that exist between a male and female placenta with respect to the glucocorticoid barrier, and summarise current human clinical and experimental animal work that has explored the differential response of the placenta of a male and female fetus to glucocorticoid exposure.
2. The placental glucocorticoid barrier
While glucocorticoids are essential for the development of many organs, during pregnancy, the placenta acts as a barrier to prevent excess entry of maternal glucocorticoids into the fetal compartment [12-14]. This placental barrier to glucocorticoids is achieved predominantly by the presence of 11β-hydroxysteriod dehydrogenase type 2 (11βHSD2), which converts the biologically active glucocorticoid (cortisol in humans, corticosterone in mice and rats) to its physiologically inert form . The placental ‘barrier’ is not complete, and under normal conditions a proportion (~10-15%) of maternal glucocorticoids reaches the fetal circulation . While 11βHSD2 is the major component of the placental glucocorticoid barrier, other proteins contained within the placenta may also help to limit the transfer of maternal glucocorticoid to the fetus. The multi-drug resistance P-glycoprotein (ABCB1) is a membrane-bound protein, which mediates the efflux of glucocorticoids out of the placenta back into the maternal circulation, thus reducing the amount of glucocorticoids able to diffuse down the concentration gradient into the fetal circulation [15-17].
The response of the placenta itself to glucocorticoids is mediated by the glucocorticoid (GR) and mineralocorticoid receptor . The most prominent isoform of the GR, both in the placenta and throughout the whole body, is GRα. This isoform mediates the biological effects of glucocorticoids, which include cell growth, proliferation and differentiation . The placenta has not generally been considered a mineralocorticoid target tissue, however work by Driver et al  has suggested that placental trophoblast cells express a functional mineralocorticoid receptor, which is in part responsible for the transport of sodium across the placenta . Because of the limited data on the role of the mineralocorticoid receptor in the placenta, our discussion will focus primarily on GR mediated effects.
3. Causes of elevated glucocorticoids during pregnancy
There are many circumstances during pregnancy in which the circulating levels of maternal glucocorticoids are elevated, resulting in placental and fetal exposure to excess glucocorticoids. The glucocorticoids within the maternal system can either be endogenous, originating from within the mother; or exogenous, where the glucocorticoid has been administered to the mother as a drug or treatment. Exogenous glucocorticoids are generally synthetic, such as betamethasone, dexamethasone or prednisone. The period of time when maternal, and therefore fetal and placental glucocorticoid levels, are elevated will vary considerably depending on the clinical circumstance, and effects arising from either acute or chronic exposures have been identified. Thus, the type of glucocorticoid, duration of exposure, and time in gestation need to be taken into account when determining the consequences for the fetus and placenta.
3.1. Exposure to natural glucocorticoids
Periods of stress, both physical (illness, excess exercise, famine/under nutrition) or psychological (anxiety) in origin, result in the elevation of endogenous glucocorticoids . While cortisol can cross the placenta, it is a good substrate for 11βHSD2, and is readily catalysed by this enzyme under normal levels. However, when levels of cortisol are elevated, the barrier is overwhelmed and more cortisol is able to cross the placenta into the fetal circulation . Deleterious effects of excess endogenous glucocorticoids on the fetus and newborn have been well documented [3, 6, 22-25]. These effects are greater for male fetuses. For example, males have been shown to have greater instances of
3.2. Exposure to synthetic glucocorticoids
Antenatal glucocorticoids are routinely administered to the mother for the treatment of a variety of pregnancy and fetal complications. Women who suffer from asthma are required to continue their glucocorticoid medication for the ongoing treatment/prevention of their symptoms, which in 33% of cases worsen during pregnancy [30-31]. Women whose babies are at risk of congenital adrenal hyperplasia are administered antenatal glucocorticoid treatment to return fetal adrenal hormone levels to normal and thus virilisation (the abnormal development of male sexual characteristics in a female) and fertility problems are prevented . Further, pregnant mothers threatening preterm birth (~7-10% of all pregnancies), receive antenatal glucocorticoids, to mature the lungs of the fetus prior to birth to reduce neonatal morbidity and mortality . As for cortisol, synthetic glucocorticoids can be catalysed by 11βHSD2, however they are a poor substrate for the enzyme, and more freely cross the placenta than cortisol . The presence of excess maternal glucocorticoids can have positive effects on fetal development and maternal health and in many situations cannot be avoided. The National Institute of Health recommend treatment of all women at risk of preterm delivery, between 24 and 34 weeks of gestation, with synthetic glucocorticoids to prematurely mature fetal organs, primarily the lung, to improve neonatal survival . Therefore a large proportion of this population of babies, are exposed to single, and sometimes multiple courses of synthetic glucocorticoids in the period leading up to birth . While antenatal glucocorticoids are the most effective treatment for improving preterm birth survival rates, the scientific community continues to question whether the use of glucocorticoids to reduce the morbidity and mortality associated with preterm birth, is worth the risk of the potential negative outcomes on metabolism and neurodevelopment seen within these babies during childhood and into adulthood . While the consensus is currently ‘yes’, the guidelines for women threatening preterm birth state that only a single, and not multiple doses of glucocorticoids, should be given until more convincing data of the benefits of multiple doses are obtained . Much work is examining the outcomes of antenatal glucocorticoids for the fetus, however the effect of glucocorticoids on the placenta, including the potential sex-specific effects, need to be considered as these may contribute to, or compound, the fetal outcomes.
4. Excess glucocorticoids and the programming of disease
The Barker Hypothesis of Developmental Origins of Health and Disease (DoHaD) states that diseases, such as coronary heart disease, hypertension and diabetes, may be consequences of
Recently, a role for the placenta in mediating developmental programming of excess glucocorticoids and other
5. Susceptibility of the placenta to negative outcomes from glucocorticoid exposure
There are several reasons for the susceptible of the placenta to adverse outcomes caused by excess glucocorticoid exposure. I.) The placenta is in direct contact with the maternal circulation and thus is directly bathed in circulating maternal glucocorticoids. II.) One of the main roles of the placenta, as described above, is as a barrier to prevent fetal exposure to excess glucocorticoids; therefore the placenta may be directly altered by the glucocorticoid exposure
6. Sex-specific placental regulation of glucocorticoids
The placental is primarily derived from embryonic tissue and therefore has the same genetic content as the fetus. In recent studies examining both human and animal models, a number of fundamental differences between the placenta of a male and female fetus have been uncovered. Differences in placental proportions  and surface area  have been noted in placentas of males and females. Specifically, females have been shown to have a greater exchange region of the placenta compared to males , and within this exchange region, females have been reported to have a larger surface area . Expression of genes and proteins known to have fundamental roles in controlling placental development , nutrient transfer, and other placental functions [68-71] differ between a male and female placenta. Specifically, placentas of male fetuses have been reported to have higher levels of the glucose transporter , higher levels of epidermal growth factor binding protein at term , but lower levels of activity of the sodium-hydrogen exchanger . Levels of pregnancy hormones, produced by the placenta, differ for a male and female. For example, maternal serum human chorionic gonadotrophin levels are significantly higher in pregnancies carrying a female fetus from as early as 3 weeks of pregnancy . Placental levels of progesterone also differ for a male and female fetus in many species, with a study in the gray seal, for example, showing higher levels in females than males . Because progesterone is primarily of placental origin, these differences provide further evidence of the fundamental differences that exist in the placenta of a male and female fetus. Further, the placenta of a male and female fetus have also been shown to respond differently to adverse
Evidence is beginning to emerge from studies in the clinical setting demonstrating that human placentas are sexually dimorphic in their regulation of normal glucocorticoid levels and these differences are exacerbated in response to excess maternal glucocorticoids. Much of this clinical evidence is arising from the work of Clifton and colleagues, who focus on identifying the effect of glucocorticoids on fetal and placental development, by studying mothers who suffer from asthma and thus use inhaled glucocorticoid treatments throughout their pregnancy. Asthma affects between 3% and 12% of pregnant women worldwide and the prevalence among pregnant women is rising . It is well recognised that women (and their babies) with asthma are at increased risk of poor pregnancy outcomes . Clifton and colleagues have also examined preterm babies and the consequences of excess glucocorticoid exposure on their placentas.
6.1. Effect of glucocorticoids on placental development and other pathways
Female babies born to asthmatic mothers, who utilised inhaled glucocorticoid treatments to manage their symptoms, were found to be growth-reduced unlike male babies born to asthmatic mothers, who were normally grown, despite similar cord blood cortisol levels . Placentas of male and female babies born to these mothers, had reduced vascularisation within the placental villi, resulting in reduced absolute fetal capillary volume , although this was most striking in placentas of male fetuses. Further, placentas of glucocorticoid-exposed males also had a reduced fetal capillary length , which was not observed in placentas of female fetuses. The authors speculate that glucocorticoid treatment may adversely affect placental vasculogenesis and/or angiogenesis by causing endothelial cell rounding and capillary regression, an observation made in other tissues after glucocorticoid exposure [95-97]. These effects may be mediated by members of the vascular endothelial growth factor family or inflammatory cytokines, both of which play a key role in placental vasculogenesis and angiogenesis . The observed changes in placental morphometry in male placentas would be expected to affect placental heamodynamics, however the absence of these changes in the female placenta do not adequately explain the reduced fetal growth of the female fetus in this high glucocorticoid environment.
A study by Stark et al , examined the placental pro-: anti-oxidant balance in response to antenatal betamethasone in placentas of preterm babies. Glucocorticoids have previously been shown to influence fetal reactive oxygen species production and antioxidant defences [100-101]. These pathways are involved in preparing the fetus for the increase in free oxygen radical generation which is experienced during the fetal to neonatal transition . Stark and colleagues observed that a pro-oxidant state was present in placentas of male fetuses, but not females following glucocorticoid exposure. Specifically, they reported that males had higher levels of the oxidative stress marker, protein carbonyl and a decreased level of the anti-oxidant enzyme, glutathione peroxidase. The authors suggested that these findings could contribute to the patho-physiologic processes underlying oxygen radical diseases of the newborn ; conditions known to exhibit a male excess .
6.2. Sex-specific effects of excess glucocorticoids on the placental glucocorticoid barrier
As the placental glucocorticoid barrier demonstrates sexually dimorphic regulation under normal conditions, and the placental response to glucocorticoids is crucial in determining fetal growth outcomes, the effect of excess maternal glucocorticoids on this barrier have been investigated. The expression of the GR within the placenta of male and female fetuses is reported to be sexually dimorphic under normal conditions (females having higher expression levels), whereas the response of GR to excess glucocorticoids is similar between the sexes .
In preterm babies, whose mothers received antenatal betamethasone, the activity of placental 11βHSD2 (predominant component of the glucocorticoid barrier) was reduced in placentas of male fetuses only . This reduction in 11βHSD2 activity would be expected to compound the already increased exposure of the male fetus to cortisol brought about by the decreased term 11βHSD2 activity within male placentas during a normal pregnancy . This may further compound the reduced immune function in male fetuses, thus increasing their susceptibility to disease. Further, glucocorticoids are important for fetal adrenal development . Male preterm babies exposed to excess glucocorticoids
Animal models of glucocorticoid exposure
The effects of excess maternal glucocorticoid exposure, on placental growth and development, has been investigated using a range of animal models including the sheep [105-106], rat , mouse  and spiny mouse . Most of these studies have utilised synthetic glucocorticoids (namely dexamethasone or betamethasone) and been designed to mimic the level of exposure experienced by the preterm infant. However, there are also a large number of studies using glucocorticoids at other developmental time points including very early in gestation. When considering the data generated from animal models, it is important to take into consideration not only the timing of glucocorticoid exposure but also the timing of placental and fetal development in the species being used, as there is considerable variation in placental development and overall structure between species. Many of these studies, particularly those in the sheep and rat, have not analysed data according to fetal sex. However a couple of recent studies in the mouse and spiny mouse have demonstrated markedly different outcomes in placental development and gene expression in placentas of males and females suggesting that alterations occurring within the placenta, following glucocorticoid exposure, are dependent upon fetal sex.
The sheep placenta is made up of 60-70 individual placentomes called cotyledons, which are cup shaped structures with fetal tissue surrounded by maternal tissue. Administration of dexamethasone for 48 hours between 64-66 days of gestation (term=145-150 days) resulted in generally larger cotyledons with overgrowth of the fetal tissue when the placenta was examined at completion of the infusion . However, this was not observed in other studies using betamethasone later in gestation (around 100 days of gestation) . Unfortunately, neither of these studies separated data according to fetal sex. In another study, pregnant ewes received intramuscular injections of dexamethasone on day 40 and 41 of gestation and the placentas were examined at day 50, 100 or 140 days of gestation. In this case, data was analysed separately for males and females and whilst dexamethasone exposure significantly increased placental
Dexamethasone exposure during late pregnancy has been shown to significantly reduce placental weight in the rat [91, 109]. This was associated with reduced expression of vascular endothelial growth factors (
Given the extensive use of the mouse for development studies, it is somewhat surprising that there has been little research of the effects of glucocorticoids on the mouse placenta. We have recently shown that dexamethasone exposure for 2 days around mid-gestation (day 12.5-14.5 of gestation, term=20 days) caused decreases in fetal body weight at day 14.5, but placental weight was only reduced in placentas from female fetuses . These changes in placental growth were associated with sex-specific changes in placental gene and protein expression: at day 14.5, the placentas from female fetuses had higher mRNA levels of expression of
We have also utilised a precocial rodent, the spiny mouse (
There is now a growing body of evidence to suggest that the placenta of a male and female differs and that this may underlie the greater vulnerability of males to stressors that occur during pregnancy. Here we provided evidence that the placental response to changes in maternal glucocorticoid status differs depending on the sex of the fetus and raises the important question: are differences in fetal outcomes driven by the fetus itself or the placenta. We suggest that the placenta should become an organ of greater interest to clinical obstetrics and perinatology, particularly with respect to how the placenta may function differently for a male and female fetus during periods of high glucocorticoid exposure.
With respect to the clinical use of glucocorticoids, the different response of a male and female to even a small dose of synthetic glucocorticoids must be followed up in a large clinical based study. At least from experimental data, the question has been raised, “Should the sex of the fetus be taken into consideration when synthetic glucocorticoids are administered during pregnancy”?