Introduction
Dysregulation of the hypothalamic–pituitary–adrenal (HPA) axis has been proposed as a key mechanism underlying the link between early-life development and later-life disease.Reference Edwards, Benediktsson, Lindsay and Seckl 1 , Reference Reynolds 2 Low birth weight, a surrogate marker of an adverse prenatal environment, is associated with higher fasting plasma cortisol,Reference van Montfoort, Finken, le Cessie, Dekker and Wit 3 increased reactivity of the HPA axisReference Reynolds, Walker and Syddall 4 , Reference Wust, Entringer, Federenko, Schlotz and Hellhammer 5 and with cardiometabolic disease, mood disorders and accelerated cognitive decline.Reference Reynolds 2 A number of studies have suggested that there may be sex differences in this response, but to our knowledge there has been no systematic review to establish whether this finding is consistent across all published studies. This may be an important consideration in the aetiology of later-life diseases, including depression and cardiometabolic disease, which show marked sex differences in presentation and prevalence. This systematic review of published observational studies aimed to determine whether there were sex differences in the HPA axis responses to prenatal stress in humans. Specifically, we aimed to determine whether there were sex-specific differences in placental handling of glucocorticoid hormones and thus fetal glucocorticoid exposure and in HPA axis outcomes in the offspring at birth and across the lifespan.
Methods
Data sources
The Meta-Analysis of Observational Studies in Epidemiology guidelines were followed for conductReference Stroup, Berlin and Morton 6 and the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines for reportingReference Liberati, Altman and Tetzlaff 7 of this systematic review. The literature search was conducted using Embase, MEDLINE and Web of Science from inception to 31 October 2016. The searches were limited to human studies and used terms as both keywords and indexing terms (Medical Subject Headings, MeSH) to identify studies related to sex differences, prenatal exposures and the HPA axis, for example, ‘sex adj2 difference*’, ‘prenatal’, ‘cortisol’ (for a full list, see Supplementary Table S1: ‘Search Terms’).
Study selection
All identified abstracts were screened for relevance by one reviewer (T.C.) and those that remained after screening were assessed by two reviewers (T.C. and S.M.G.) to establish whether they met the inclusion criteria. Any discrepancies in selection were discussed by all researchers until a consensus was reached. Studies were included if they investigated the prenatal environment, had a biological outcome that was related to the HPA axis (including placental handling of glucocorticoid hormones) and compared males and females. Studies were excluded if they investigated specific disease states where the disease does not reflect normal physiology (e.g. congenital adrenal hyperplasia, Cushing’s disease). There were no exclusions related to study design.
Data extraction
Two independent reviewers (T.C. and S.M.G.) extracted relevant information on study design, characteristics, nature of prenatal exposure, methodology and outcome measures using a pre-specified data extraction form. Any disagreements were discussed and resolved by consensus.
Quality assessment
Studies were assessed for quality and risk of bias by the two reviewers (T.C. and S.M.G. or T.C. and R.M.R.) independently using a systematic scoring system 8 with objective criteria relating to the clarity of the research question, participant recruitment and retention, reliability of exposure and outcome measures, and consideration of confounding variables (Supplementary Table S2: ‘Quality Scoring’). A paper could attain a rating of ‘high’, ‘intermediate’ or ‘low’ with 80, 60 or <60%, respectively, of the 14 applicable criteria being satisfied.
Data analysis and synthesis
Due to considerable heterogeneity in both clinical characteristics, study design and data, a descriptive synthesis of results was conducted. This included a description of the study design and sample studied, details of the prenatal exposure, biological outcome measurement and whether there were sex differences in this outcome. Studies were grouped according to whether they (a) measured changes in genes regulating fetal glucocorticoid exposure in the placenta or (b) investigated HPA axis outcomes in the offspring at birth or longer-term follow-up. For presentation purposes we grouped the prenatal environmental exposures into maternal asthma, inhaled corticosteroids, antenatal corticosteroids, preterm birth, low birth weight and other forms of antenatal stress (e.g. maternal cortisol, subjective stress, significant life events).
Results
Study design and participants
From 173 titles and abstracts, 39 full-text articles were assessed, of which 23 were included in the final data synthesis, including data on 3739 participants (Fig. 1). The studies included 12 prospective cohort studies, nine cross-sectional studies and two case–control studies, and were conducted in Europe (n=10), Australia (n=7), United States (n=3), South America (n=2) and the Philippines (n=1). Sample characteristics are detailed in Table 1.
Fig. 1 Flow (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) diagram of included studies. HPA, hypothalamic–pituitary–adrenal.
Table 1 Descriptive table of all included articles
SGA, small for gestational age; AGA, average size for gestational age; LGA, large for gestational age; GC, glucocorticoid medication, for example, betamethasone, dexamethasone; BMI, body mass index.
Articles ordered by year of publication.
a Asthma severity rated according to Australian Asthma Management guidelines.
b Asthma severity rated according to Asthma Control Questionnaire (taking into account night waking, morning asthma symptoms, activity limitation, shortness of breath, wheezing, short-acting β2-agonist use).Reference Juniper, O’Byrne, Guyatt, Ferrie and King 32
Studies investigating sex differences in placental glucocorticoid handling
Eight studies investigated whether there were sex differences in genes regulating placental glucocorticoid handling and thus fetal glucocorticoid exposure (Table 2). The sample size varied from 43Reference Stark, Wright and Clifton 16 to 244Reference Demendi, Borzsonyi and Pajor 20 placentas with exposures including maternal asthma, prenatal corticosteroids and preterm birth. Four studies investigated the effects of maternal glucocorticoid exposure on placental glucocorticoid receptor (GR) expression.Reference Murphy, Gibson and Giles 12 , Reference Hodyl, Wyper and Osei-Kumah 17 , Reference Saif, Hodyl and Hobbs 25 , Reference Saif, Hodyl and Stark 28 Studies that investigated maternal asthma as a stressor identified sex differences, with females showing a decrease in GR messenger RNA compared with malesReference Hodyl, Wyper and Osei-Kumah 17 and a different expression pattern of GR isoforms.Reference Saif, Hodyl and Hobbs 25 One study found no sex-specific effect of inhaled forms of corticosteroids, except that males in the treatment group and the control group showed a positive correlation between placental GR heterogeneous nuclear RNA and cord blood cortisol, but that there was no such association where the mothers had untreated asthma or the offspring was female.Reference Hodyl, Wyper and Osei-Kumah 17 One study investigated the expression of GR isoforms in preterm birth, finding no sex differences in the total expression of GR apart from an increased level of GRα D2 in males.Reference Saif, Hodyl and Stark 28 It did however find differences in GR localization, with preterm male placentas showing higher cytoplasmic concentrations of GRα C and GRα A compared with full-term male placentas, with no such relationship in females.Reference Saif, Hodyl and Stark 28
Table 2 Studies assessing placental outcomes
mRNA, messenger RNA; GR, glucocorticoid receptor; SGA, small for gestational age; AGA, average size for gestational age; LGA, large for gestational age; GC, glucocorticoid medication; 11β-HSD, 11β-hydroxysteroid dehydrogenase; BMI, body mass index; IQR, interquartile range.
Articles ordered according to outcome axis measure (placental GR then 11β-HSD enzymes), then by year of publication.
a Asthma severity assessed by Juniper asthma questionnaire.Reference Juniper, O’Byrne, Ferrie, King and Roberts 33
b Data represented as median (IQR) relative protein expression.
c Asthma severity rated according to Australian Asthma Management guidelines.
Four studies investigated effects on the expression and activity of 11β-hydroxysteroid dehydrogenase (11β-HSD) enzymes, with three of them finding a sex difference. With the exception of two studies, females showed changes that could lead to lower metabolism of cortisol by the placenta compared with males. This included reduced activity of placental 11β-HSD2 with maternal asthmaReference Murphy, Gibson and Giles 12 and prenatal corticosteroids, and with offspring reduced size for gestational ageReference Mericq, Medina and Kakarieka 15 as well as increased activity of 11β-HSD1 with increased maternal cortisol.Reference Mina, Raikkonen, Riley, Norman and Reynolds 27 In contrast, one study found that 11β-HSD2 was increased in females following betamethasone exposure >72 h before delivery.Reference Stark, Wright and Clifton 16 Another study found reduced placental 11β-HSD2 in both male and female preterm infants, with no sex difference.Reference Demendi, Borzsonyi and Pajor 20
Studies investigating sex differences in HPA axis outcomes in the offspring
Cord blood cortisol concentrations
Six studies examined sex differences in umbilical cord blood cortisol concentrations with data from n=527 participants and prenatal exposures of maternal asthmaReference Murphy, Gibson and Giles 12 , Reference Hodyl, Wyper and Osei-Kumah 17 , Reference Saif, Hodyl and Hobbs 25 and antenatal betamethasone for preterm birthReference Ballard, Ballard and Granberg 9 , Reference Stark, Wright and Clifton 16 , Reference Hodyl, Stark, Butler and Clifton 22 (Table 3). Two studies found evidence of sex differences, with higher umbilical blood cortisol in females whose mothers had moderate or severe asthma compared with mild asthmaReference Hodyl, Wyper and Osei-Kumah 17 and whose mothers were treated with betamethasone <72 h before delivery.Reference Stark, Wright and Clifton 16 Two studies with maternal asthmaReference Murphy, Gibson and Giles 12 , Reference Saif, Hodyl and Hobbs 25 and two with betamethasoneReference Ballard, Ballard and Granberg 9 , Reference Hodyl, Stark, Butler and Clifton 22 found no sex differences in cord cortisol concentrations.
Table 3 Offspring outcomes
HPA, hypothalamic–pituitary–adrenal; SLE, significant life event; ELBW, extremely low birth weight; GC, glucocorticoid medication, for example, betamethasone, dexamethasone; VLBW, very low birth weight; ACTH, adrenocorticotropic hormone; CI, confidence interval; .
Articles ordered according to HPA axis measure (static then dynamic), then by age at follow-up, then by year of publication. Where an article refers to both static and dynamic HPA axis measures, it is placed with the others that refer to dynamic measures.
a Asthma severity rated according to Australian Asthma Management guidelines.
b Asthma severity rated according to Asthma Control Questionnaire (taking into account night waking, morning asthma symptoms, activity limitation, shortness of breath, wheezing, short-acting β2-agonist use).Reference Juniper, O’Byrne, Guyatt, Ferrie and King 32
Static HPA axis measures
Five studies investigated plasma/serum cortisol concentrations (either in the morning or diurnal profiles), three with low birth weightReference Szathmari, Vasarhelyi and Tulassay 10 , Reference Fall, Dennison and Cooper 11 , Reference Kaseva, Pyhala and Pesonen 30 , and two with preterm birthReference Lee, Fried, Thayer and Kuzawa 23 , Reference Quesada, Tristao, Pratesi and Wolf 24 , as the exposure (Table 3). The age at follow-up varied from 5 months to 72 years. One found that in childhood females born preterm had higher morning salivary cortisol than full-term females, as well as preterm and full-term males.Reference Quesada, Tristao, Pratesi and Wolf 24 One study in young adults found no difference in cortisol awakening response or diurnal cortisol secretion between individuals with very low birth weight and controls, with no sex differences.Reference Kaseva, Pyhala and Pesonen 30 Another study in young adults found that males with preterm birth had decreased waking cortisol compared with males with term birth, and a shallower slope of diurnal cortisol decline, but found no such relationships among females.Reference Lee, Fried, Thayer and Kuzawa 23 In contrast, a study in older adultsReference Fall, Dennison and Cooper 11 found higher morning serum cortisol concentrations in males and females of low birth weight than in males and females of higher birth weight but no formal statistical comparison was conducted. Two studies found increased morning salivary or serum cortisol in offspring exposed to neonatal corticosteroids or low birth weight, but found no sex differences in this effect either in early adulthoodReference Szathmari, Vasarhelyi and Tulassay 10 or adolescence.Reference ter Wolbeek, Kavelaars and de Vries 34 Another study investigated urinary steroid metabolites in extremely low birth weight children, with extremely low birth weight males and females having increased cortisol production compared with controls, but did not test whether there was any sex difference.Reference Gohlke, Wudy and Stutte 26 A study investigating GR gene methylation in DNA extracted from buccal cells collected in infancy found that females whose mothers had experienced a significant life event during pregnancy had increased methylation of NR3C1 compared with control females, finding no such relationship in males.Reference Ostlund, Conradt and Crowell 31
Dynamic HPA axis measures
Six studies used a stress challenge to attempt to elicit differences in HPA axis reactivity (Table 3). Two used versions of the Trier Social Stress Test (TSST) were conducted in children and adolescents.Reference Alexander, Rosenlocher and Stalder 19 , Reference Quesada, Tristao, Pratesi and Wolf 24 , Reference ter Wolbeek, Kavelaars and de Vries 34 Although the prenatal exposure differed (preterm birth;Reference Quesada, Tristao, Pratesi and Wolf 24 antenatal glucocorticoidsReference Alexander, Rosenlocher and Stalder 19 ), both found a sex difference in HPA responses with a greater response in peak salivary cortisol in females. Two studies used other stress-inducing tasks. One study among toddlers involved a brief period of separation from the child’s mother and found that females had an increase in post-stressor cortisol and males did not, as well as a greater increase in cortisol in females whose mothers reported greater subjective stress during pregnancy, with no such correlation in males.Reference Yong Ping, Laplante and Elgbeili 29 Another studyReference de Bruijn, van Bakel, Wijnen, Pop and van Baar 14 used a different stress-inducing task with 4-year-old children (confronting the child with a transparent box from which it was impossible to extract a toy), and found no difference in cortisol between males and females who had been exposed to prenatal maternal anxiety. One study used a physiological stressor – a breath of 35% inhaled CO2 gas – in offspring exposed to maternal depression and found no sex differences in peak salivary cortisol.Reference Vedhara, Metcalfe and Brant 21
Neither of the studies testing HPA axis reactivity or central negative feedback [by adrenocorticotropic hormone (ACTH) stimulation or dexamethasone suppression, respectively] found sex differences in HPA axis responses to exposures of low birth weightReference Reynolds, Walker and Syddall 13 or very low birth weight.Reference Kaseva, Pyhala and Pesonen 30
Quality assessment
Most studies were determined to be of intermediate quality, scoring 60–80%, with three being classified as low quality, scoring under 60%. Studies scored as low quality either did not statistically adjust for confounding variables, did not report loss to follow-up, reported a loss to follow-up rate of over 20% or did not assess exposures more than once over time where it would have been applicable (Supplementary Table S2: ‘Quality Assessment’).
Discussion
This systematic review including 23 studies and data on n=3739 participants showed evidence of sex differences in HPA responses to prenatal stressors with increased vulnerability of the female HPA axis at a number of different levels and in response to a number of different prenatal stressors.
The placenta plays a key role in regulating fetal glucocorticoid exposure through the activity of the enzymes such as 11β-HSD2, which deactivates cortisol; 11β-HSD1, which regenerates cortisol; and the levels of GR, which signal glucocorticoid sensitivity. Overall, the findings suggested that in comparison with males, the female placenta increases its permeability to maternal glucocorticoids following maternal stress. This occurred through sex-specific lower expression of 11β-HSD2Reference Murphy, Gibson and Giles 12 or increased expression of 11β-HSD1Reference Mina, Raikkonen, Riley, Norman and Reynolds 27 and by changes in GR expression and localization,Reference Hodyl, Wyper and Osei-Kumah 17 , Reference Saif, Hodyl and Hobbs 25 , Reference Saif, Hodyl and Stark 28 consistent with increased female fetal exposure to glucocorticoids. Having said this, one relatively large study that examined the association with preterm birth found a reduction in 11β-HSD2 in both sexes, with no sex difference,Reference Demendi, Borzsonyi and Pajor 20 , Reference Johnstone, Bocking, Unlugedik and Challis 35 suggesting that in preterm birth at least, the nature of any sex difference is more complex. However, it may be the case that a dysfunctional placental 11β-HSD2 barrier is a causative factor in preterm birth itself,Reference Demendi, Borzsonyi and Pajor 20 , Reference Johnstone, Bocking, Unlugedik and Challis 35 and thus it is to be expected that preterm infants of both sexes show reduced placental 11β-HSD2.
When examining sex differences in HPA axis outcomes in the offspring, we identified studies using cord blood as an early measure of the infant HPA axis, and a number of studies measuring HPA axis activity in children and adults using both static and dynamic measures. Data on cord cortisol concentrations are difficult to interpret as they may represent either the basal level of secretion in the fetal HPA axis, the HPA axis response of the fetus to the stress of birth or short-term suppression of the fetal HPA axis by glucocorticoid medications. Two of the six identified studiesReference Stark, Wright and Clifton 16 , Reference Hodyl, Wyper and Osei-Kumah 17 reported higher cortisol concentrations in blood measured in female offspring, consistent with increased fetal glucocorticoid exposure in females. Findings from later in life were also not consistent. Studies on diurnal cortisol secretion either did not find a sex difference,Reference Szathmari, Vasarhelyi and Tulassay 10 , Reference Gohlke, Wudy and Stutte 26 , Reference Kaseva, Pyhala and Pesonen 30 found altered secretion in malesReference Lee, Fried, Thayer and Kuzawa 23 or in females.Reference Quesada, Tristao, Pratesi and Wolf 24 Although the findings of HPA axis measures in the studies conducted in children and adults were not consistent, the only sex difference in HPA axis reactivity reported was consistent with increased reactivity in females.Reference Alexander, Rosenlocher and Stalder 19 , Reference Quesada, Tristao, Pratesi and Wolf 24 , Reference Yong Ping, Laplante and Elgbeili 29 Of note, both of these studies were conducted using the TSSTReference Alexander, Rosenlocher and Stalder 19 , Reference Quesada, Tristao, Pratesi and Wolf 24 and a maternal separation event,Reference Yong Ping, Laplante and Elgbeili 29 whereas other stress tests and hormone challenges failed to show any sex differences.Reference Reynolds, Walker and Syddall 13 , Reference de Bruijn, van Bakel, Wijnen, Pop and van Baar 14 , Reference Vedhara, Metcalfe and Brant 21 , Reference Kaseva, Pyhala and Pesonen 30 The absence of findings from dexamethasone or ACTH administration suggests that the mechanism of sex-specific programming may be to do with higher neural inputs into the HPA axis rather than its sensitivity to its own components. However, one study suggested that sensitivity of the female physiology in general to glucocorticoids may be increased in situations of maternal stress.Reference Ostlund, Conradt and Crowell 31
We included a broad definition of prenatal stressors including maternal inflammation, pharmacological intervention, psychological stressors and low birth weight/preterm birth. Low birth weight and preterm birth are considered the outcome of growth-restricting stressors during pregnancy and small for gestational age was associated with reduced activity of prenatal 11β-HSD2 and increased activity of 11β-HSD1 in females. These changes would allow the delivery of more maternal cortisol to the fetus, and may indeed be a pathway contributing to growth restriction. Females with low birth weight or preterm birth had higher morning cortisol in one study,Reference Quesada, Tristao, Pratesi and Wolf 24 but not all.Reference Szathmari, Vasarhelyi and Tulassay 10 , Reference Fall, Dennison and Cooper 11 , Reference Lee, Fried, Thayer and Kuzawa 23 , Reference Kaseva, Pyhala and Pesonen 30 There was also evidence that females born preterm had increased HPA axis reactivity to stressful stimuli.Reference Quesada, Tristao, Pratesi and Wolf 24 Whether these changes are a cause or consequence of preterm birth is unknown. Nevertheless, if low birth weight and preterm birth are considered as a marker of prenatal stress, these findings support the argument that females are more vulnerable to the programming effects of prenatal stress in terms of HPA axis reactivity, possibly not in terms of diurnal secretion.
A number of studies considered maternal asthma as a prenatal stressor. Maternal asthma was found to decrease the placental barrier to glucocorticoids in terms of 11β-HSD2Reference Murphy, Gibson and Giles 12 and, putatively, GR.Reference Hodyl, Wyper and Osei-Kumah 17 , Reference Saif, Hodyl and Hobbs 25 In one study, treatment of asthma with inhaled betamethasone ameliorated the effect,Reference Murphy, Gibson and Giles 12 which suggests that it is the presence of the asthma itself rather than any associated factors in lifestyle or treatment that is responsible for the programming. However, it is possible that factors relating to treatment administration and compliance confound these results. No studies examined the association between maternal asthma and offspring later-life HPA axis outcomes, though these might be anticipated due to the observed alteration of the placental glucocorticoid barrier. A large follow-up study of clinical outcomes of offspring exposed to asthma in utero also did not examine whether there were sex differences in outcomes.Reference Tegethoff, Olsen, Schaffner and Meinlschmidt 36
Although use of prenatal glucocorticoids was associated with increased activity of placental 11β-HSD2 in females, it was also associated with an increase in cord blood cortisol in females when given within 72 h of delivery.Reference Stark, Wright and Clifton 16 Prenatal glucocorticoids were also associated with differential later-life outcomes, with females showing greater HPA axis reactivity to stress in terms of cortisol release.Reference Alexander, Rosenlocher and Stalder 19 Overall, glucocorticoid medication appeared to induce offspring, although not placental, changes that were similar to those observed with inflammatory stress and low birth weight.
Our review identified studies investigating a variety of psychological stressors including maternal mood,Reference Vedhara, Metcalfe and Brant 21 emotional complaints,Reference de Bruijn, van Bakel, Wijnen, Pop and van Baar 14 subjective stressReference Yong Ping, Laplante and Elgbeili 29 and presence of psychopathology,Reference Alexander, Rosenlocher and Stalder 19 but sex differences in the offspring HPA axis were only apparent in one study.Reference Yong Ping, Laplante and Elgbeili 29 This suggests that either the psychological measures studied were not severe enough to reliably induce a programming effect or that a biological/hormonal response to stress – or its pharmacological mimic – is required to induce sex-specific prenatal programming. The one study that found a sex differenceReference Yong Ping, Laplante and Elgbeili 29 investigated mothers who had all been affected by a single major stressor – the Iowa flood of 2008 – suggesting that other studies investigated stressors whose nature was too broad, or not sufficiently severe. No studies investigated the association between maternal psychological measures and placental outcomes.
Mechanisms of the sex differences in the HPA axis responses to stress are unknown, but it is conceivable that they exist to effect the vulnerability–viability trade-off that is made by male and female fetuses in response to stress. It has been observed that males exposed to early environmental adversity suffer a risk to their viability. Although females suffer no such cost to their viability, they show increased vulnerability to adversity later in life.Reference Sandman, Glynn and Davis 37
A strength of our study is that a systematic and comprehensive review process, devised with an experienced librarian, reported in line with PRISMA guidelines, was followed for this review. Two reviewers independently assessed eligibility of the titles, abstracts and full-text studies. Studies only including specific clinical populations with altered HPA physiology, for example, congenital adrenal hyperplasia, were excluded to ensure that our results were generalizable to the general population. However, there are some potential limitations to our study. The search terms were broad, and it is possible we have missed some potentially eligible studies. The studies were heterogeneous in terms of size, countries, ethnicities, age groups, methodology and reporting of statistical analysis. Most studies were graded as intermediate quality because they did not report sample size justification, participation rate of eligible persons, experimenter blinding or loss to follow-up.
In conclusion, although there was heterogeneity in study type and the nature of the findings, this systematic review demonstrated some evidence of increased programmed vulnerability to maternal stressors of the HPA axis in females compared with males, particularly in terms of HPA axis reactivity. Although there is evidence that males are not unaffected, any effect appears to be manifested in altered diurnal cortisol secretion. The limited evidence from diverse, moderate quality studies suggests that further research is required in order to determine whether this is a mechanism underlying differences in later-life diseases.
Acknowledgements
The authors thank Sheila Fisken, academic support librarian, for her help in designing the literature searches.
Financial Support
T.C. was supported by a College of Medicine and Veterinary Medicine summer vacation project bursary from the University of Edinburgh. R.M.R. acknowledges the support of Tommy’s and the British Heart Foundation.
Conflicts of Interest
None.
Supplementary material
To view supplementary material for this article, please visit https://doi.org/10.1017/S204017441600074X
