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The human fetal adrenal produces cortisol but no detectable aldosterone throughout the second trimester Johnston, Zoe C; Bellingham, Michelle; Filis, Panagiotis; Soffientini, Ugo; Hough, Denise; Bhattacharya, Siladitya; Simard, Marc; Hammond, Geoffrey L; King, Peter; O’Shaughnessy, Peter J; Fowler, Paul A Feb 12, 2018

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RESEARCH ARTICLE Open AccessThe human fetal adrenal produces cortisolbut no detectable aldosterone throughoutthe second trimesterZoe C. Johnston1, Michelle Bellingham1, Panagiotis Filis2, Ugo Soffientini1, Denise Hough1, Siladitya Bhattacharya3,Marc Simard4, Geoffrey L. Hammond4, Peter King5, Peter J. O’Shaughnessy1 and Paul A. Fowler2*AbstractBackground: Human fetal adrenal glands are highly active and, with the placenta, regulate circulating progesterone,estrogen and corticosteroids in the fetus. At birth the adrenals are essential for neonate salt retention throughsecretion of aldosterone, while adequate glucocorticoids are required to prevent adrenal insufficiency. The objectiveof this study was to carry out the first comprehensive analysis of adrenal steroid levels and steroidogenic enzymeexpression in normal second trimester human fetuses.Methods: This was an observational study of steroids, messenger RNA transcripts and proteins in adrenals from up to109 second trimester fetuses (11 weeks to 21 weeks) at the Universities of Aberdeen and Glasgow. The study designwas balanced to show effects of maternal smoking.Results: Concentrations of 19 intra-adrenal steroids were quantified using liquid chromatography and massspectrometry. Pregnenolone was the most abundant steroid while levels of 17α-hydroxyprogesterone,dehydroepiandrosterone sulphate (DHEAS) and progesterone were also high. Cortisol was present in alladrenals, but aldosterone was undetected and Δ4 androgens were low/undetected. CYP17A1, CYP21A2 andCYP11A1 were all highly expressed and the proteins localized to the adrenal fetal zone. There was low-levelexpression of HSD3B and CYP11B2, with HSD3B located mainly in the definitive zone. Maternal smoking altered fetalplasma adrenocorticotropic hormone (ACTH) (P = 0.052) and intra-adrenal progesterone, 17α-hydroxyprogesterone and16α-hydroxyprogesterone, but not plasma or intra-adrenal cortisol, or intra-adrenal DHEAS. Fetal adrenal GATA6 andNR5A1 were increased by maternal smoking.Conclusions: The human fetal adrenal gland produces cortisol but very low levels of Δ4 androgens and no detectablealdosterone throughout the second trimester. The presence of cortisol in fetal adrenals suggests that adrenalregulation of circulating fetal ACTH remains a factor in development of congenital adrenal hyperplasia duringthe second trimester, while a relative lack of aldosterone explains the salt-wasting disorders frequently seen inextreme pre-term neonates. Finally, maternal smoking may alter fetal adrenal sensitivity to ACTH, which couldhave knock-on effects on post-natal health.Keywords: Human, Adrenal, Fetus, Steroid, Maternal smoking* Correspondence: p.a.fowler@abdn.ac.uk2Institute of Medical Sciences, School of Medicine, Medical Sciences &Nutrition, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UKFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Johnston et al. BMC Medicine  (2018) 16:23 DOI 10.1186/s12916-018-1009-7BackgroundThe human fetal adrenal gland develops initially as partof the adrenogonadal primordium and is distinct in thehuman by 7–8 weeks of gestation. The adrenals secretecortisol in response to adrenocorticotropic hormone(ACTH) as early as week 8 of gestation [1], although themain steroids produced in fetal life are dehydroepian-drosterone (DHEA) and its sulphate (DHEAS), whichact as substrates for placental estrogen production [2].Together, the fetal adrenal glands and placenta dominatehuman fetal steroid endocrinology in a manner seenonly in higher primates. Normal development and func-tion of the fetal adrenals is also essential for several pro-cesses that can affect the fetus itself or the health of theneonate. For example, disruption of the fetal adrenalscan lead to disorders of sex development [3], while fetalmisprogramming of the stress axis, through altered fetalcortisol secretion, may predispose to diseases in later life[4]. The adrenal glands are also essential for survivalearly in post-natal life through the secretion of aldoster-one, which prevents salt-wasting disorders [5], whileadequate cortisol is required to prevent adrenal insuffi-ciency in the newborn [6]. Interestingly, pre-term neo-nates are prone to salt-wasting disorders, which mayindicate a failure of aldosterone synthesis in fetal life oraltered sensitivity to the steroid [5]. Despite the import-ance of the adrenal glands for fetal and post-natal health,however, their development during fetal life in the hu-man is not well described or understood. Animal modelsare of limited relevance due to significant species differ-ences in fetal adrenal structure, steroidogenic pathwaysand endocrinology of pregnancy. However, only a limitednumber of studies have examined the human fetal adrenaldirectly ([1, 7–11] and in review [12, 13]). Consequently,we have examined human fetal adrenal steroidogenesisduring the second trimester in a large number of well-documented, normal, human fetuses. This includes thefirst comprehensive liquid chromatography (LC)/massspectrometry (MS) analysis of human fetal adrenal steroidlevels during the second trimester.Maternal smoking during pregnancy remains a signifi-cant public health issue, as it can disrupt normal fetal pro-gramming and can have irreversible effects on the post-natal life of the offspring [14]. Cigarette smoke containsmany potential toxicants (e.g. heavy metals, aldehydes, ni-trosamines and polycyclic aromatic hydrocarbons) [15]that can cross the placental barrier to reach the fetus.However, mechanisms behind the long-term effects ofsmoking on the human fetus remain largely unknown.Maternal smoking can affect fetal adrenal function anddevelopment of the hypothalamo-pituitary-adrenal (HPA)axis [16], which may be one mechanism by which smok-ing programmes later health deficits of the offspring [17,18]. We have, therefore, extended our developmentalstudy to examine the effects of maternal smoking on hu-man fetal adrenal steroidogenesis.MethodsSample collectionThe collection of fetal material involved in the adrenalstudies was approved by the National Health Service(NHS) Grampian Research Ethics Committees (REC 04/S0802/21). Human fetal kidneys were collected under aseparate, newer ethics permission as part of the ScottishAdvanced Fetal Research (SAFeR) Study. This was ap-proved by NHS Grampian Research Ethics Committees(REC 15/NS/0123). In all cases, women seeking electiveterminations of pregnancy were recruited with full writ-ten, informed consent by nurses working independentlyof the study at the Aberdeen Pregnancy Counselling Ser-vice. Maternal data, medications used and self-reportednumber of cigarettes smoked per day were recorded.Only fetuses from normally progressing pregnancies (de-termined at ultrasound scan prior to termination) fromwomen over 16 years of age and between 11 and21 weeks of gestation (7 and 20 weeks for fetal kidneys)were collected following termination by RU486 (mife-pristone) treatment (200 mg) and prostaglandin-induceddelivery, as detailed previously [19]. Fetuses were trans-ported to the laboratory within 30 min of delivery,weighed, sexed and the crown-rump length recorded.Blood samples were collected by cardiac puncture exvivo and plasma prepared in heparin-coated tubes wasstored at –80 °C. Fetal tissues were snap-frozen in liquidnitrogen and stored at –80 °C or fixed in 10% neutralbuffered formalin. Adrenal samples were analysed infour groups: control female, smoke-exposed female, con-trol male and smoke-exposed male, with groups bal-anced as far as possible for gestational age (Table 1).Maternal smoking status was confirmed by measure-ment of fetal plasma cotinine using a commercially avail-able kit (Cozart Plc, Abingdon, Kent, UK). Fifty-six fetalkidneys were analysed as a single group and as fourgroups: control female, smoke-exposed female, controlmale and smoke-exposed male.Plasma measurements of hormones and binding globulinsAssays were performed using plasma samples from 60 sec-ond trimester fetuses. Plasma ACTH levels were measured(25 μL/fetus) using a single Milliplex® MAP Human Pituit-ary Magnetic Bead Panel 1 kit (ACTH, growth hormone(GH), thyroid-stimulating hormone (TSH), ciliary neuro-trophic factor (CNTF), agouti-related protein (AGRP);Millipore Limited, Watford, UK) and analysed using a Bio-Plex200 system (Bio-Rad Laboratories Ltd, Hemel Hemp-stead, UK). Intra- and inter-assay coefficients of variationwere < 10% and < 15% respectively, sensitivity was 0.91 pg/mL and cross-reactivity was negligible. Cortisol wasJohnston et al. BMC Medicine  (2018) 16:23 Page 2 of 16measured in 50 μL of plasma/fetus using a DetectX® Corti-sol Enzyme Immunoassay kit (Arbor Assays, Ann Arbor,MI, USA). Cross-reactivity with other steroids was < 8%,sensitivity was 17.3 pg/mL and inter- and intra-assay coeffi-cients of variation were 5.4% and 8.1% respectively.Corticosteroid-binding globulin (CBG) concentrations weremeasured by an established ligand-saturation assay [20].Protein, DNA and RNA extractionsTo avoid sampling error due to heterogeneous morph-ology of the adrenal gland, only whole human fetal ad-renal glands were homogenized in the presence ofprotease inhibitors (Protease Inhibitor Cocktail, Sigma-Aldrich Company Ltd, Gillingham, UK). Protein, DNAand RNA were extracted from total tissue homogenateswith Qiagen AllPrep DNA/RNA/Protein mini kits(Qiagen Ltd, Crawley, UK). RNA was extracted fromwhole human fetal kidneys in the same way.Intra-adrenal steroid quantification with LC/MSSteroids were extracted from the unused, combined,flow-through fractions of the Qiagen AllPrep DNA/RNA/protein extraction protocol and quantified by LC/MS. This approach was taken to maximize the datagathered from as many fetuses as possible and reflectsthe unique nature and relative scarcity of the humanfetal samples.Steroid extractionsInitially, we determined whether intra-adrenal steroidlevels could be accurately measured from the unusedflow-through fractions of the Qiagen AllPrep DNA/RNA/protein extraction protocol. The first studies, usingTable 1 Morphological data for the mothers and fetuses involved in adrenal studiesPopulation Characteristic Female fetuses Male fetusesControl Smoke-exposed Control Smoke-exposedTotal adrenal samples N 29 19 31 30Maternal indices Age (years) 25.5 ± 1.2 23 ± 1.2 23.8 ± 1.06 26.2 ± 1Body mass index (BMI, kg/m2) 24.1 ± 0.8 26 ± 1.3 26 ± 0.95 25.8 ± 1.2Cigarettes/day 0 ± 0 9.3 ± 0.7 0.2 ± 0.2 10.2 ± 1Fetal indices Age (weeks) 14.6 ± 0.4 14.3 ± 0.8 14.6 ± 0.6 14.6 ± 0.5Weight (g) 77 ± 13.3 75.4 ± 14.1 83.4 ± 12 91.8 ± 12.9Crown-rump length (CRL, mm) 98.7 ± 5.2 95.1 ± 5 101.4 ± 4.3 105.5 ± 5.5Ponderal index (weight, g/[CRL, cm3]) 70.2 ± 6.7 72.8 ± 5.4 68.8 ± 3.3 63.2 ± 1.8Plasma cotinine (ng/mL) 6.8 ± 3.3 30.2 ± 5.4 5.3 ± 1.2 41.6 ± 6.4Sub-population: plasma steroidsa N 14 16 13 17Maternal indices Age (years) 24.3 ± 1.7 23.2 ± 1.3 24.5 ± 1.8 24.7 ± 1.2BMI (kg/m2) 23.5 ± 1.2 23.8 ± 1.3 26.2 ± 1.4 25.3 ± 1.4Cigarettes/day 0 ± 0 11 ± 1.3 0 ± 0 12.5 ± 1.1Fetal indices Age (weeks) 15.6 ± 0.6 15.2 ± 0.6 15.2 ± 0.6 15.1 ± 0.6Weight (g) 104.8 ± 19.9 93.6 ± 16.7 101.7 ± 22.5 97.1 ± 19.3CRL (mm) 109.3 ± 7.7 106.9 ± 5.7 108 ± 7.4 107.2 ± 7.4Ponderal index (weight, g/[CRL, cm3]) 64.9 ± 2.6 64 ± 2.6 73.9 ± 4.7 65 ± 2Plasma cotinine (ng/mL) 3.3 ± 1.2 41.6 ± 2.8 2.6 ± 0.9 48.6 ± 2.2Sub-population: mRNA/protein/steroidsa N 15 15 15 15Maternal indices Age (years) 24.7 ± 1.6 23.2 ± 1.4 22.9 ± 1.3 26.6 ± 1.4BMI (kg/m2) 24.2 ± 0.8 26.5 ± 1.4 26.23 ± 1.5 24.6 ± 1.6Cigarettes/day 0 ± 0 8.8 ± 0.8 0 ± 0 12 ± 1.3Fetal indices Age (weeks) 15.3 ± 0.6 15.1 ± 0.6 15.2 ± 1.2 15.2 ± 0.6Weight (g) 97 ± 19.5 84.8 ± 17 94.9 ± 18.8 94.7 ± 19.9CRL (mm) 106.4 ± 6.8 97.4 ± 6.1 104.3 ± 6.9 102.3 ± 8.7Ponderal index (weight, g/[CRL, cm3]) 66.5 ± 2.3 75.5 ± 6.7 72 ± 4.6 67 ± 3.1Plasma cotinine (ng/mL) 7.6 ± 4.6 30.2 ± 5.4 4 ± 1.5 50.9 ± 5.7aAlthough there is some overlap between the two study sub-populations, not all plasma samples were from fetuses where the adrenal glands were collected.Therefore, the two populations were not analysed togetherJohnston et al. BMC Medicine  (2018) 16:23 Page 3 of 16a [3H]-testosterone recovery standard with mouse ad-renal tissue homogenate, showed that 72% of testoster-one was recovered in the discardable flow-throughfraction from the protein precipitation step (step 15 ofthe Qiagen manual) with 89.6% total recovery from thetotal combined waste fractions. In further preliminarystudies using LC/MS, the recovery of intra-adrenal ste-roids (from human fetal adrenals) was measured bycomparison of steroid levels in the initial tissue lysatewith levels in recovered fractions after RNA/DNA/pro-tein extraction. Steroids were recovered primarily in theprotein precipitation step (step 15 in the Qiagen pro-tein/DNA/RNA extraction protocol), with the exceptionof DHEAS, which was recovered in all fractions. Esti-mates of steroid recovery efficiencies from combinedfractions were as follows: cortisol 87%, androstenedione122%, 17α-hydroxyprogesterone 113%, 21-deoxycortisol87%, 11-deoxycortisol 111% and DHEAS 56%. Thesedata show that most steroids could be recovered fromtissues with high efficiency during Qiagen extraction.For subsequent studies all of the residue fractions fromthe Qiagen extraction process (steps 8, 15, 21 and 22)were combined and extracted.Three deuterated steroids (15 ng) were added as in-ternal standards (ISs) prior to steroid extraction, as de-scribed below. Steroids in the combined residuefractions were extracted twice using ethyl acetate (4:1 ra-tio to sample volume), subjected to a solid-phase extrac-tion using 1 mL Strata-X, 33 μm polymeric reversephase cartridges (Phenomenex, Torrance, CA, USA) anddissolved in a final volume of 150 μL methanol. The per-centage recovery of all steroids through ethyl acetateand solid phase extractions was > 82%, and the percentrelative standard deviation (RSD; n = 6) of all steroidmeasurements was between 1.82 and 8.75%.The standards prepared in methanol were not signifi-cantly different than those prepared using the extractionmatrix, while recovery and percent RSD showed negli-gible differences. We therefore used standards in metha-nol for further experiments.Intra-adrenal steroid quantification with LC/MSSteroids were separated by reversed phase LC on an Ul-tiMate 3000 RSLCnano (Thermo Fisher Scientific, Wal-tham, MA, USA) separation system, using an Accucore™PFP LC column with trimethyl silane (TMS) endcapping(50 mm, 2.1 mm, 2.6 μm, Thermo Fisher). For both posi-tive and negative modes, the mobile phase was a mixtureof solvents A (1% formic acid in water) and B (49:49:2methanol:acetonitrile:isopropanol). Steroids were elutedat a flow rate of 0.4 mL/min, using a linear gradientfrom 24% B to 47% B in 2.0 min, followed by a lineargradient to 66% B in 2.7 min and a subsequent lineargradient to 100% B in 0.1 min. Elution at 100% B wasmaintained for 0.9 min before lowering it to 24% B over0.1 min and equilibrating the column for 2.5 min. Thetotal run time was 8.3 min and the injection volume was5 μL.Steroids were detected with a Q Exactive Orbitrap(Thermo Fisher) mass spectrometer, using full-scan de-tection (250–500 m/z). Table 2 contains a summary ofthe limits of quantification for all steroids in this study.For each sample, one MS run was completed at full scanin positive mode and another MS run at full scan innegative mode. MS mode was found to be more suitedfor steroid profiling with the Orbitrap MS system, andthe sensitivities were similar to those achieved using LC-MS/MS with a triple quadrupole MS system [21].All instruments were controlled by Chromeleon soft-ware (Thermo Fisher), and data acquisition, peak integra-tion and quantification were performed using Xcalibur 2.2software (Thermo Fisher). Calibration curves were used toquantify the steroids, using the ratio of the steroid peakarea relative to the peak area of a specific deuterated ISthat had similar elution time and/or chemical properties.Deuterated cortisol (4-pregnen-11β,17,21-triol-3,20-dione-9,11,12,12-d4; Steraloids, Newport, RI, USA) was usedas the IS for aldosterone, cortisol, cortisone, cortico-sterone, 11-deoxycortisol, cortisone sulphate, cortico-sterone sulphate, 11-dehydrocorticosterone and Δ5-androstenediol. Deuterated progesterone (4-pregnen-3,20-dione-2,2,4,6,6,17α,21,21,21-d9; Steraloids) was usedas the IS for DHEA, 17α-hydroxypregnenolone, 17α-hydroxyprogesterone, 16α-hydroxyprogesterone, testoster-one, deoxycorticosterone, progesterone, pregnenolone andΔ4-androstenedione. Deuterated DHEAS (5-androsten-3β-ol-17-one-16,16-d2, sulphate, sodium salt; Steraloids)was used as the IS for DHEAS. Calibration curves wereconstructed from the LC/MS analyses of six to nine cali-brator samples in the range of 1–2000 ng/mL. Calibratorsamples contained all three deuterated ISs at a concentra-tion of 100 ng/mL, as well as steroid standards at relevantconcentrations from a dilution series in methanol. Ster-oid standard stocks, from which the calibrator dilutionseries were made, were all sourced from Sigma-Aldrichor Steraloids.To determine limits of detection (LODs) and limits ofquantification (LOQs), calibrator samples (n = 13) wereprepared at concentrations ranging from 0.01 ng/mL to10,000 ng/mL, containing all of the steroids and deuter-ated ISs at a fixed concentration of 100 ng/mL. Thesewere prepared in both methanol and Qiagen buffer mix-ture. Calibration curves were generated by performingleast-squares regression analysis on peak area ratios rela-tive to the IS at different concentrations, within the sensi-tivity range of each steroid. The LOD and LOQ weredefined as the lowest steroid concentration with a signal-to-noise ratio (S/N) larger than 3 and 10 respectively.Johnston et al. BMC Medicine  (2018) 16:23 Page 4 of 16Table2Intra-adrenalsteroidsmeasuredbyLC/MS TotalControlfemaleSmoke-exposedfemaleControlmaleSmoke-exposedmaleSteroidLOQ(ng/mLMeOH)LOQ(adjusted)aQSb(n=60)Mean±SEM(ng/mgtissue)QS(n=15)Mean±SEM(ng/mgtissue)QS(n=15)Mean±SEM(ng/mgtissue)QS(n=15)Mean±SEM(ng/mgtissue)QS(n=15)Mean±SEM(ng/mgtissue)Pregnenolone100.05585.019±0.49154.619±0.79134.299±0.927154.073±0.828157.087±1.13517α-Hydroxyprogesterone10.005601.291±0.182151.086±0.151151.115±0.189150.878±0.159152.087±0.624DHEAS0.10.0005601.153±0.178151.426±0.412150.782±0.16150.966±0.271151.437±0.468Progesterone10.005601.089±0.185150.993±0.194150.95±0.278151.066±0.504151.347±0.415Cortisol0.10.0005560.626±0.087140.713±0.198140.498±0.127130.471±0.142150.824±0.198Corticosterone0.10.0005570.354±0.075130.265±0.073140.213±0.08150.345±0.153150.593±0.22416α-Hydroxyprogesterone0.10.0005590.259±0.035150.259±0.036140.18±0.04150.192±0.038150.407±0.11611-Deoxycortisol0.10.0005600.119±0.017150.148±0.039150.1±0.03150.082±0.02150.146±0.042Cortisone10.005370.108±0.01890.15±0.04790.096±0.03490.075±0.023100.109±0.037Testosterone0.10.0005320.032±0.00790.062±0.0280.028±0.01370.017±0.00880.023±0.008Deoxycorticosterone0.10.0005390.023±0.003120.03±0.006100.018±0.003100.019±0.00470.025±0.009Corticosteronesulphate100.05100.018±0.0130.022±0.01920.003±0.00210.001±0.00140.045±0.03211-Dehydrocorticosterone0.10.0005120.003±0.00140.005±0.00430.001±020.002±0.00230.005±0.003Aldosterone10.0050<LOQ0<LOQ0<LOQ0<LOQ0<LOQDHEA100.050<LOQ0<LOQ0<LOQ0<LOQ0<LOQΔ4-Androstenedione100.050<LOQ0<LOQ0<LOQ0<LOQ0<LOQΔ5-Androstenediol10.0050<LOQ0<LOQ0<LOQ0<LOQ0<LOQ17α-Hydroxypregnenolone1000.50<LOQ0<LOQ0<LOQ0<LOQ0<LOQCortisonesulphate100.050<LOQ1<LOQ1<LOQ1<LOQ1<LOQa Equivalenttong/mgoftissueLOQbQuantifiablesamplesoutofnsamplesasstatedinbracketsLOQlimitofquantitation,QSquorumsensing,SEMstandarderrorofthemeanJohnston et al. BMC Medicine  (2018) 16:23 Page 5 of 16Real-time reverse transcription polymerase chain reaction(RT-PCR)Messenger RNA (mRNA) from 60 human fetal adrenalswas reversed transcribed using random hexamers andSuperscript III (Life Technologies, Paisley, UK) [19, 22,23]. Real-time RT-PCR was performed using Brilliant IISYBR Green Master Mix (Agilent Technologies, SantaClara, CA, USA) and an MX3000 cycler (Stratagene,Amsterdam, the Netherlands). mRNA levels wereexpressed relative to two housekeeping genes (HKGs),TATA box binding protein (TBP) and Phosphomannomu-tase 1 (PMM1), which were identified as the optimalHKGs from a total of six HKGs tested using NormFin-der [24]. Data are expressed as the geomean of valuesrelative to each HKG multiplied by 103. NR3C2 tran-script levels in human fetal kidneys were measured byreal-time PCR using LightCycler 480 SYBR Green IMastermix (Roche, Basel, Switzerland) and a RocheLightCycler 480 and were expressed relative to ACTB.All primers were designed as previously described [22];the sequences are reported in Table 3. Primers designedto amplify HSD3B recognized both type 1 and type 2isoforms of the enzyme.ImmunohistochemistryAdrenal sections (5 μm) were dewaxed and rehydrated,and antigen retrieval was carried out in citrate buffer(PT Module Buffer 1; Thermo Fisher) using a bench topautoclave. Endogenous peroxidase activity was quenchedusing DAKO Real™ peroxidase block (Agilent Technolo-gies), and 20% Normal Goat Serum (Vector Laboratories,Burlingame, CA, USA) was used to block non-specificbinding sites. The primary antibodies were for detectionof CYP11A1 (non-commercial rabbit polyclonal antibody(gift from A.H. Payne)), CYP17A1 (non-commercial rabbitpolyclonal antibody (gift from I. Mason)), CYP21A2 (poly-clonal rabbit antibody (Sigma;HPA048979)) and HSD3B(non-commercial rabbit antibody which recognizes bothHSD3B1 and HSD3B2 (gift from I. Mason)). The second-ary antibody used was Polyclonal Goat anti-Rabbit Im-munoglobulin Horseradish Peroxidase (HRP) (DAKO;Agilent Technologies). Signal amplification was necessaryonly for HSD3B detection and was carried out using theTSA Plus DNP (HRP) System (PerkinElmer, Waltham,MA, USA). Visualization was by 3,3’-diaminobenzidine(DAB) (DAKO Real™, Agilent Technologies) and sectionswere counterstained with hematoxylin. For double im-munofluorescence of CYP11A1 and CYP21A2, sectionswere dewaxed, antigen-retrieved, blocked and probed withCYP11A1 primary antibody as above. Antibody detectionwas carried out with Goat anti-rabbit HRP (DAKO(P0450); 1:1000 in phosphate-buffered saline (PBS))followed by use of the Tyramide-TSA™ Plus Fluoresceinkit (PerkinElmer). Antigen retrieval was repeated in orderto denature the first set of antibodies. Sections were thenre-probed with CYP21A2 primary antibody as above withdetection by Alexa Fluor 594 (red) goat anti-rabbit (1:1000in TBS) and counterstained with ToPro®-3-iodide (Invitro-gen; Select FX™ Nuclear Labeling Kit; S33025). Confocalimages were captured using a Zeiss LSM 710 microscopeand ZEN software (Carl Zeiss Microimaging, LLC, Thorn-wood, NY, USA).Western blottingProteins were separated by sodium dodecyl sulphate(SDS) gel electrophoresis using a XCell4 SureLock™Midi-Cell apparatus (Life Technologies). Protein samples(30 μg) in NuPAGE® LDS Sample Buffer, containingNuPAGE® Sample Reducing Agent, were loaded to aNuPAGE® 4–12% Bis-Tris Midi Protein Gel (Life Tech-nologies). Electrophoresis was carried out in NuPAGE®MOPS SDS Running Buffer, and the separated proteinswere transferred to nitrocellulose using the InvitrogeniBlot® system and iBlot® Transfer Stacks (Life Technolo-gies). Blocking was carried out using Odessey® BlockingBuffer (PBS; LI-COR, Cambridge, UK). Membranes wereprobed overnight with one of a number of primary anti-bodies diluted in Odyssey buffer. The primary antibodiesused were anti-CYP11A1, anti-HSD3B, anti-CYP17A1and anti-CYP21A2, as used for immunohistochemistry,and anti-β-actin (monoclonal mouse antibody (ab8226;Table 3 Primers used for RT-qPCRForward ReverseCYP11A1 tttttgcccctgttggatgca ccctggcgctccccaaaaatCYP11B1 tgtgtgatgctgccggagga cgcaatcggttgaagcgccCYP11B2 aggtggacagcctgcatccctg gcacatctgggttcagccgcCYP17A1 ccatttcctgaacgcaccgg agagaggccaaggaaacagggctCYP21A2 cggacctgtccttgggagactactcc ctgggctctcatgcgctcacaDAX1(NR0B1)cctcccaggtccaagccatcaa tgagttccccactggagtccctGATA6 aataattccattcccatgactccaacttc aatacttgagctcgctgttctcgggHSD3Ba ccacaccgcctgtatcattgatgtct taggagttgggcccggctacctMC2R tcttccacgcactgcggtacc catcagcgggaacagcgacgMRAP cacagacatggccaacgggac agcaccacgaaggcagccagPMM1 aacatctcgcccatcggcc tcaaagctgatcatgcctcctcgPOR ttttcagcatgacggacatgattctgt tttcttcatcttttccacaaagctgctcSF-1(NR5A1)tcccttctgccgcttccagaaat tgaagccattggcccgaatctSTAR ggctggcatggccacagact ttgggcagccaccccttgaSULT2A1 caagatgtccaattattccctcctgagtgt tctcgaggaagatctgccatcttctcTBP aggaaaaaattgaatagtgagacgagttcca tggactaaagatagggattccgggagtNR3C2 tggcctggatgtggttggatttagg agaaacttgaccccaccgtctttccACTB ttcctgggcatggagtcctgtg ttgatcttcattgtgctgggtgccaAmplifies both HSD3B1 and HSD3B2Johnston et al. BMC Medicine  (2018) 16:23 Page 6 of 16Abcam, Cambridge, UK)). Bound antibody was detectedwith fluorescent secondary antibody (Cross Adsorbed,DyLight conjugated secondary antibodies; ThermoFisher) and scanned using an Odyssey CLx-0565 Imager(LI-COR). Bands were quantified using Image Studiosoftware (LI-COR).Statistical analysisA general linear model was used to examine the effects ofage, sex, smoking and any interactions between these fac-tors on mRNA transcript, protein and steroid levels. Thedata were modelled by a quasi-Poisson distribution to ac-count for over-dispersion. The minimal adequate modelwas used followed by Tukey’s post hoc analysis for factor-ial variables. Levene’s test was used to compare variabilitybetween groups. Statistical analysis was performed usingR Studio (Boston, MA, USA). For statistical analysis of ad-renal steroids, measurements below the LOQ for eachsteroid were substituted with a value of 0.ResultsFetal plasma levels of adrenal-related hormonesACTH, cortisol and CBG were detectable in fetal plasma(using a sub-population of samples, see Table 1)throughout the second trimester (Fig. 1). Levels ofACTH and cortisol did not change significantly duringthis period, but CBG showed a significant decrease be-tween 12 and 20 weeks of gestation (Fig. 1c; P = 0.038).Hormone levels did not differ significantly according tofetal sex. Maternal smoking was associated with alteredlevels of ACTH (Fig. 1a; interaction between age andsmoking, P = 0.052) with levels generally higher towardsthe start of the second trimester. Levels of cortisol(Fig. 1b) and CBG (Fig. 1c) were similar between controland smoke-exposed fetuses.Fetal adrenal steroid contentOverall fetal adrenal weight increased exponentially dur-ing the second trimester (P < 0.001) with no differencebetween sexes or due to maternal smoking (Fig. 1d).Intra-adrenal steroid concentrations (measured in a sub-population of the total, see Table 1) during the sameperiod are shown in Fig. 2, arranged within relevant ste-roidogenic pathways. Pregnenolone was the most abun-dant steroid found in the human fetal adrenal (Fig. 2)with high levels of 17α-hydroxyprogesterone, DHEASand progesterone also present. Cortisol levels were vari-able but were present in all samples. In contrast, aldos-terone was not detectable in any samples, althoughdeoxycorticosterone and corticosterone were present.Androstenedione was also undetectable in all samples,although this may be related to a relatively high LOQ(Table 2). Testosterone was detected at low levels in 32out of 60 samples (detailed in Table 2). Most adrenalsteroids showed no significant change in concentra-tion over the course of the second trimester (Fig. 3).The exceptions were pregnenolone (P = 0.004), 17α-hydroxyprogesterone (P < 0.001), 16α-hydroxyprogesterone(P = 0.02) and corticosterone (P < 0.001), which decreasedsignificantly over the same period (Fig. 3). Since adrenalFig. 1 Fetal plasma ACTH, cortisol and CBG and adrenal weight during the second trimester. Data points from individual fetuses are shown(circles for control and triangles for smoke-exposed, where groups are separated). Black lines indicate generalized linear regression, and grey filldenotes the confidence interval (0.95) around an individual regression. There was no effect of fetal sex on levels of ACTH, cortisol or CBG (n = 60).The interaction between fetal smoke exposure and gestational age on ACTH levels (a) approached significance, however (P = 0.052). Levels ofcortisol (b) and CBG (c) were unaffected by age or maternal smoking. Levels of CBG decreased between 12 and 20 weeks (c; P = 0.038). Combinedfetal adrenal weights increased between 12 and 20 weeks of gestation (d; P < 0.001; n = 109) with no effect of fetal sex or maternal smokingJohnston et al. BMC Medicine  (2018) 16:23 Page 7 of 16weight increases markedly during the second trimester, thetotal adrenal content of measured, detectable steroids in-creased over this period with the exception of cortico-sterone which was unchanged (Additional file 1: Figure S1).No differences between control male and female fetuseswere seen.Maternal smoking was associated with altered intra-adrenallevels of progesterone (P= 0.02), 17α-hydroxyprogesterone(P = 0.02) and 16α-hydroxyprogesterone (P = 0.04) (Fig. 4;expressed per adrenal pair). Each of these steroids showeda significant interaction between smoking and gestationalage across the second trimester.Tissue transcript and protein expressionmRNA transcriptsMost enzymes in the adrenal steroidogenic pathwayshowed high levels of transcript expression throughoutthe second trimester (Fig. 5a). Levels of CYP17A1, STAR,CYP21A2 and CYP11A1, all of which are all involved inthe initial steps of steroid synthesis, were particularlyhigh. In contrast, levels of HSD3B and CYP11B2 tran-scripts were low but detectable. Transcripts encodingthe ACTH receptor (MC2R), its accessory proteinMRAP and the key steroidogenic transcription factorsNR5A1 and GATA6 were also detectable in all adrenalsFig. 2 Intra-adrenal steroid levels in the human fetal adrenal during the second trimester. Steroid levels, expressed as ng/mg of tissue, are shownfor each steroid measured (n = 60) and are arranged within the canonical steroidogenic pathways. The enzymes responsible for each conversionare shown in grey boxes. Data points from individual fetuses are shown, and the limit of quantitation for each steroid is shown as a horizontal line.Aldosterone, 17α-hydroxypregnenolone, DHEA and androstenedione were not detectable in any of the 60 samples. *The conversion of11-deoxycorticosterone to aldosterone by CYP11B2 occurs via corticosterone and 18-hydroxycorticosterone as intermediatesJohnston et al. BMC Medicine  (2018) 16:23 Page 8 of 16investigated. Most transcripts showed no significantchange in expression during the second trimester (Add-itional file 2: Figure S2) with the exceptions of CYP11B2(P = 0.003) and HSD3B (P = 0.004), which both increasedduring this period (Fig. 5b), and POR (P = 0.03), whichincreased during the initial part of the second trimester(Fig. 5b). Similarly, there were no sex differences in anyof the transcript species measured apart from the keysulphation enzyme SULT2A1, which was significantlyhigher in males than in females (P = 0.04).Maternal smoking was associated with increased ex-pression of GATA6 and NR5A1 (Fig. 5b) in both sexes(P < 0.001). Furthermore, maternal smoking was also as-sociated with an increase in the variability (Additionalfile 3: Figure S3) of STAR (P = 0.004) and CYP17A1 (P =0.02) in males.Aldosterone acts primarily through the NR3C2 recep-tor in the kidney, and to determine whether the receptoris expressed during the second trimester, NR3C2 levelswere measured in human fetal kidneys. Transcript levelswere variable but were detectable in all 56 human fetalsamples tested (Fig. 5c). There were no significant differ-ences in transcript levels due to gestational age, fetal sexor maternal smoking status.ProteinsCYP11A1 (Fig. 6a and d) was primarily localized in theadrenal fetal zone and transitional zone, with some im-munoreactive cells also present in the definitive zone.CYP17A1 was localized to the fetal zone and transitionzone (Fig. 6b), while CYP21A2 was most prominent inthe fetal zone although also detectable in the definitivezone (Fig. 6c and d). CYP11A1 and CYP21A2 were co-localized in some cells in the fetal zone, although manycells expressed CYP21A2 alone while a few expressedonly CYP11A1 (Fig. 6d).HSD3B was present primarily in cells of the adrenaldefinitive zone (Fig. 6e), although a small number ofcells in the fetal zone in younger fetuses (12–13 weeks;Fig. 6f ) also expressed the protein. These cells werecomparable in size to the other cells of the fetal zoneand were larger than the densely packed cells of the de-finitive zone. HSD3B-expressing cells in the fetal zonewere not observed in the adrenals of fetuses older than13 weeks.Adrenal levels of CYP11A1, CYP17A1 and CYP21A2protein, measured using Western blotting (Fig. 6g), weresimilar between males and females, and no significantdifferences were observed between control and smoke-Fig. 3 Changes in intra-adrenal steroid levels (ng/mg of tissue) during the second trimester. Data points from individual fetuses are shown(n = 60), and steroids which show a significant change with gestational age are indicated with an asterisk. Levels of pregnenolone (P = 0.004),17α-hydroxyprogesterone (P < 0.001), 16α-hydroxyprogesterone (P = 0.02) and corticosterone (P < 0.001) decreased from 12 to 19 weeks. Generalizedlinear regressions are shown as black lines with the corresponding confidence intervals (0.95) in greyJohnston et al. BMC Medicine  (2018) 16:23 Page 9 of 16exposed groups. Levels of CYP11A1 were not signifi-cantly altered by gestational age (Fig. 6h), whereas con-centrations of both CYP17A1 (Fig. 6i) and CYP21A2(Fig. 6j) increased significantly between 12 and 19 weeksof gestation, regardless of sex or smoke exposure (P =0.04, and P = 0.004 respectively). HSD3B was barely de-tectable by Western blotting and could not be reliablyquantified (not shown).DiscussionThe human fetal adrenal cortex and the placenta regu-late most aspects of fetal steroid endocrinology, togetherwith the fetal liver and testes [25]. Normal developmentof the adrenal gland is therefore essential in maintainingfetal levels of glucocorticoids, mineralocorticoids and es-trogens. Failure of normal adrenal development can leadto congenital adrenal hyperplasia (most commonlythrough loss of 21-hydroxylase activity and leading todisorders of sex development [26, 27]), salt-wasting dis-orders and adrenal insufficiency in the newborn [3, 12,28, 29]. In this study, we have used a large cohort (up to109 fetuses) of well-characterized, normal, human fetalsamples to detail changes in fetal adrenal steroidogenesisduring the second trimester, a critical time for fetalgrowth, development and sex differentiation. We showthat the fetal adrenal produces cortisol throughout thesecond trimester, possibly from placental progesterone[27]. However, we also show that aldosterone is not syn-thesized in detectable amounts during this period, whichmay be of importance in understanding pre-term neo-natal salt wasting. Maternal smoking changes fetalACTH levels, but this is not accompanied by down-stream effects on the primary adrenal steroids.Together, the concentrations of intra-adrenal steroidsand expression of steroidogenic enzymes indicate thatthe fetal adrenal is highly active throughout the secondtrimester in the human. In our studies, the enzymes ne-cessary for adrenal steroid synthesis were expressed byweek 12 of gestation and mostly at relatively high levelsapart from HSD3B and CYP11B2, which were low at thisstage. Low levels of HSD3B and CYP11B2 transcripts inthe late second/early third trimester have also been re-ported previously [9, 30]. Despite low HSD3B transcriptand protein expression, however, the Δ4 steroids proges-terone, 17α-hydroxyprogesterone and cortisol werepresent in significant amounts throughout the secondtrimester. The fetal adrenals also generated significantamounts of 16α-hydroxyprogesterone, as reported previ-ously [31, 32], due to the relatively high 16α-hydroxylation activity of human CYP17A1 [33]. Thepresence of these Δ4 steroids in such high amounts mayoccur because HSD3B enzyme activity does not reflecttranscript or protein levels or because placental proges-terone, derived from the circulation, is being convertedin the fetal adrenals as previously suggested [13, 34]. Ele-vated amniotic fluid levels of 17α-hydroxyprogesteronehave been reported to be a reasonable indicator of 21-hydroxylase deficiency and congenital adrenal hyperpla-sia in the mid-gestation fetus [35, 36]. While molecularmethods are now more commonly used to diagnose con-genital adrenal hyperplasia [37], our results show thatfetal adrenal steroid concentrations do not change mark-edly after 12 weeks. It is, therefore, possible that amni-otic fluid from early second trimester fetuses could beused to confirm diagnosis of 21-hydroxylase deficiency.Salt-wasting disorders frequently occur in extremepre-term neonates [38], and it has been suggested thatthis may be due to low expression of the mineralocortic-oid receptor (NR3C2) in the pre-term kidney [39]. Theresults described here suggest, however, that neonatalsalt-wasting syndrome in pre-term infants may occur be-cause of a limited capacity of the fetal adrenal to gener-ate aldosterone. This is consistent with earlier studiesshowing that tissue from second trimester human adre-nals has little or no capability to produce aldosterone invitro from tritiated substrate [40, 41]. It is also consistentFig. 4 The effects of maternal smoking on intra-adrenal steroidlevels during the second trimester. Measurements from individualfetuses are shown. Steroids are expressed as ng per total combinedadrenal weight (n = 60). Maternal smoking was associated with alteredintra-adrenal levels of three of the 19 steroids measured during thesecond trimester: progesterone (P = 0.02), 17α-hydroxyprogesterone (P= 0.02) and 16α-hydroxyprogesterone (P = 0.04) with P values corre-sponding to the interaction between smoke exposure and gesta-tional age. Black lines show generalized linear regressions, and grey fillshows the confidence interval (0.95) around the regressionJohnston et al. BMC Medicine  (2018) 16:23 Page 10 of 16with reports that pre-term neonates (26–32 weeks gesta-tion) have normal sensitivity to aldosterone but reducedcapacity to secrete the steroid [5] and with the demon-stration (Fig. 5c) that the human fetal kidney consist-ently expresses NR3C2 during the late first and thesecond trimester. Both CYP11B2 (aldosterone synthase)and CYP11B1 can catalyse the conversion of 11-deoxycorticosterone to corticosterone, although furtherconversion to aldosterone, via the intermediate 18-hydroxycorticosterone, is only performed by CYP11B2Fig. 5 Expression levels of key mRNA transcripts in the human fetal adrenal and fetal kidney during the second trimester. a Overall adrenal transcriptlevels (relative to housekeeping genes (HKGs)) during the second trimester are shown on a log scale. Data points represent mRNA levels in individualfetuses. b Changes in adrenal transcript levels during the second trimester, due to fetal sex and/or maternal smoking. Expression of CYP11B2(P = 0.003, n = 56) and HSD3B (P = 0.004, n = 58) increased between 12 and 19 weeks of gestation, while POR (P = 0.03, n = 60) increased duringthe initial phase of the trimester. Other transcripts did not change significantly during the second trimester. Black lines show the generalizedlinear regression, and grey fill denotes the confidence interval (0.95) around the regression. Levels of SULT2A1 were significantly different betweenmale and female fetuses, P = 0.04 (n = 58), but there was no effect of maternal smoking (not shown). Maternal smoking was associated with increasedlevels of the transcription factor GATA6 (P < 0.001; n = 56) in both male and female fetuses and increased levels of NR5A1 (P = 0.04; n = 58) in malefetuses compared to control males. There were no other effects of age or smoking on transcript levels. CF control females, SF smoke-exposed females,CM control males, SM smoke-exposed males. c Transcript levels of the mineralocorticoid receptor NR3C2 in the human fetal kidney (n = 56; from 7 to20 weeks of gestation). NR3C2 transcript expression did not differ significantly due to fetal age or sex, or maternal smoking. In all datasets, thehorizontal line corresponds to the mean expression, and each data point corresponds to an individual fetusJohnston et al. BMC Medicine  (2018) 16:23 Page 11 of 16[42–44]. Therefore, the absence of detectable fetal adrenalaldosterone in this study is likely a reflection of the lowexpression of CYP11B2 as previously predicted [13].Human fetal adrenal glucocorticoid and androgen pro-duction is dependent on ACTH released by the fetal pi-tuitary during the second trimester [13]. It has also beenproposed that expression of the enzyme HSD3B andinteraction with fetal ACTH (through altered negativefeedback) is key to understanding the regulation of fetaladrenal steroid production [1]. Goto et al. [1] have re-ported that transiently elevated HSD3B expression occurstowards the end of the first trimester, leading to early cor-tisol biosynthesis. They suggest that this has a negativefeedback effect on the fetal pituitary, reducing ACTHstimulation of fetal adrenal androgen secretion andthereby protecting female fetuses from masculinization.Our results extend the work of Goto et al [1] into the sec-ond trimester and show that the fetal adrenal continues tomake cortisol throughout this period, despite the loss ofHSD3B activity. This means that ACTH will remain undernegative feedback control during the second trimester.One complication of the hypothesis of Goto et al. [1] isFig. 6 Steroidogenic enzyme localization and expression in the fetal adrenal. a CYP11A1 (brown) was localized primarily in the inner fetal zone(yellow arrow) with some cells in the definitive zone (black arrow) exhibiting weak immunostaining. b CYP17A1 (brown) was expressed throughout theadrenal, while (c) CYP21A2 (brown) was localized primarily in the fetal zone (yellow arrow), although protein expression was also seen in the definitivezone (black arrows, inset). d CYP11A1 (cyan) and CYP21A2 (magenta) co-localize in some cells of the fetal zone (yellow arrow), but most cells onlyexpress one of these enzymes with CYP21A2 the predominant enzyme. e HSD3B was predominantly localized to the outer definitive zone (black arrow),although it was also present in some cells within the fetal zone of adrenals at 12 weeks of gestation (f; yellow arrow). These HSD3B-positive cells in thefetal zone were not observed after 13 weeks of gestation. g Representative Western blot showing CYP11A1, CYP17A1, CYP21A2 and the loading controlβ-actin. Molecular weight markers are labelled on the left of the image. h–j Expression levels of CYP11A1, CYP17A1 and CYP21A2 protein in the fetaladrenal during the second trimester. Data points from individual fetuses are shown. Black lines show the generalized linear regression, and grey filldenotes the confidence interval (0.95) around the regression. Expression of CYP11A1 was unaffected by gestational age, while both CYP17A1 (P = 0.004)and CYP21A2 (P = 0.04) showed increased expression between 12 and 19 weeksJohnston et al. BMC Medicine  (2018) 16:23 Page 12 of 16that higher HSD3B activity during the first trimester willtend to favour androgen production, which will then be ina balance between enzyme activity and altered negativefeedback. This is particularly pertinent as humanCYP17A1 has poor C17-20 lyase activity with 17α-hydroxyprogesterone as substrate [44], meaning that ad-renal Δ4 androgens are only likely to be generated de novo(under ACTH regulation) rather than from placental pro-gesterone. Adrenal androstenedione is present at the endof the first trimester at relatively high levels [1, 11] but notin the second trimester. This suggests that adrenal Δ4 an-drogen production, by both sexes, may be higher duringthe first trimester, reflecting HSD3B activity rather thanaltered negative feedback.The presence of adrenal cortisol throughout thesecond trimester also has implications for treatment ofcongenital adrenal hyperplasia. This condition arisesthrough deficiency of one of the enzymes involved incortisol synthesis, commonly 21-hydroxylase. In turn,this leads to increased ACTH levels through impairednegative feedback and to increased adrenal androgenproduction. The condition is normally treated throughadministration of dexamethasone from the first trimesteruntil birth. It has been suggested, however, that dexa-methasone treatment in these cases may only be neces-sary in the first half of pregnancy [45]. One proposedreason is that fetal cortisol is absent (based on HSD3Bexpression) during the second trimester and that fetalACTH would not normally be under negative feedbackcontrol during this period. The demonstration here thatthe fetal adrenal produces cortisol throughout the sec-ond trimester is of direct relevance, therefore, and sug-gests that normal homeostatic feedback regulationremains in place at all times after the first trimester.One possible confounding factor in these studies is theeffect of the termination regime on fetal ACTH and ster-oid levels. RU486 acts as an antagonist at both the proges-terone and glucocorticoid receptors, and in non-pregnantsubjects daily administration will increase ACTH andcortisol over a 7-day period [46]. The dose of RU486 usedclinically in this study, however, was 200 mg/patient (aver-age 2.7 mg/kg), which is less than the dose required foranti-glucocorticoid action (4.5 mg/kg) [47]. Furthermore,while RU486 transfers readily across the placenta, fetalplasma concentrations of the drug are 0.1–10% of thosefound in the maternal circulation [48, 49], and it is highlyunlikely that fetal drug concentrations will reach thethreshold levels required to affect the fetal HPA axis. Thisis borne out by an earlier study which reported that use ofRU486 during terminations (at 600 mg, three times thedose used in this study) had no effect on fetal or maternalplasma cortisol concentrations [48]. Similarly, RU486 isreported to have no effect on placental ACTH during firstor second trimester termination [50].The adrenal gland is highly zonated both morpho-logically and functionally. Earlier studies by Jaffe andcolleagues [13, 51–53] developed the concept, basedon spatial distribution of steroidogenic enzymes, thatthe human fetal adrenal in the third trimester is com-posed of three functional zones which are analogousto the adult cortical zones. They proposed that thedefinitive, transitional and fetal zones are analogousto the zona glomerulosa, the zona fasciculata and thezona reticularis respectively. Our immunolocalizationresults generally agree with these and other studies[7, 9, 10, 13, 51–53] and suggest that functional zon-ation is present from as early as 12 weeks. These im-munolocalization studies show that the fetal zone isthe main site of CYP11A1, CYP17A1 and CYP21A2expression during the second trimester. This meansthat the fetal zone is likely to be the main site ofDHEA production and of de novo steroid synthesis.Synthesis of other adrenal steroids requires HSD3Bactivity, however, which is localized primarily in thedefinitive zone. This means either that steroid intermedi-ates move between the adrenal zones to facilitate de novosynthesis or that Δ4 steroid synthesis depends largely onplacental progesterone as described above. Given the lowlevels of adrenal HSD3B during the second trimester andthe intense vascularization of the fetal zone, which wouldfavour delivery of steroid substrate from the circulation,use of placental progesterone appears the more likelyroute [34]. The presence of all components of thesteroidogenic pathway in the adrenal also means, however,that de novo synthesis is likely to contribute to overallsteroid levels and to androgen levels in particular, asdiscussed above. Our co-localization studies show that,within the fetal zone, different cells express differentcombinations of steroidogenic enzymes. This may bebecause they represent different populations of cells, or itpossibly may be due to the local environment of the cell.The effect of maternal smoking on fetal adrenal func-tion and HPA axis development is most clearly seen inthe association between maternal smoking and suddeninfant death syndrome [54], which may be linked to ad-renal dysfunction [55]. Our results show that maternalsmoking is associated with altered circulating fetalACTH (P = 0.052), although there was no effect onplasma cortisol levels. Intra-adrenal fetal cortisol con-centrations were also unaffected by smoke exposure,which suggests that there is a reduced sensitivity of theadrenal to ACTH through down-regulation of either thereceptor or second messenger systems. These data areconsistent with a previous study showing increasedACTH, but unchanged cortisol, at birth following expos-ure to maternal smoking in utero [16]. These pro-grammed changes in adrenal sensitivity to ACTHinduced by maternal smoking may be of importanceJohnston et al. BMC Medicine  (2018) 16:23 Page 13 of 16after birth if ACTH levels return to normal in exposedneonates but adrenal sensitivity remains reduced. Con-sistent with this hypothesis, neonatal cortisol levels andcortisol response to stress are reported to be significantlyreduced in the first month after birth following exposureto maternal smoking in utero [56].Maternal smoking is also associated with altered whole-adrenal levels (nanograms/adrenal pair) of progesterone,17α-hydroxyprogesterone and 16α-hydroxyprogesteroneacross the second trimester. As discussed above, it is likelythat Δ4 steroids in the fetal adrenal are derived, at least inpart, from placental progesterone, and so these changesmay reflect alterations in placental activity in response tomaternal smoking. A number of studies have shown thatmaternal smoking can alter placental function and may re-duce placental progesterone concentrations [57]. We havereported that circulating maternal progesterone is notclearly affected by maternal smoking [58]. However, ifplasma concentrations in the fetus more closely representplacental production, then this may have a knock-on effecton total adrenal steroid levels.The most marked effects of maternal smoking on thefetal adrenal were increased levels of NR5A1 and GATA6transcripts. NR5A1 encodes steroidogenic factor 1 (SF-1), which is a nuclear receptor transcription factor es-sential for development and function of the adrenals andgonads [8]. Disruption of SF-1, unless severe, has greaterclinical effects on the gonads than the adrenals [59], butit is unclear what effects overexpression of SF-1 in uterowill have on adrenal development in the human. Overex-pression of SF-1 in mice leads to an increase in adrenalsize through increased commitment of precursors to thesteroidogenic lineage [14, 60], although maternal smok-ing had no significant effects on human fetal adrenalweight in our study. It remains to be seen, however,whether the morphological and cellular composition ofthe fetal adrenals is affected by smoking. GATA6 is azinc finger transcription factor which works in concertwith SF-1 [61] to activate steroidogenic enzyme expres-sion in general and HSD3B in particular [62–64]. Whileno significant changes in steroidogenic enzyme expres-sion were seen in the smoke-exposed group, the in-creased variability in STAR and CYP17A1 expressionmay be related to altered transcription factor expression.ConclusionsAlong with the placenta, the fetal adrenal acts to regu-late fetal steroid endocrinology, and normal fetal devel-opment of the adrenals is essential for post-natal health.Mapping the interactions between placenta, adrenalsand other endocrine organs (e.g. the testes) is essential,therefore, to understand how these processes can bedysregulated. This study is the first to comprehensivelydocument the changes in adrenal steroid levels andsteroidogenic enzyme expression throughout the secondtrimester and shows that a combination of substrateavailability, enzyme expression and enzyme specificityregulates adrenal steroid production. Critically, thisstudy shows that the fetal adrenals do not synthesize de-tectable levels of aldosterone, which is likely to explainpre-term salt-wasting conditions. They do, however,synthesize cortisol throughout the second trimester,which indicates that fetal ACTH secretion is under in-hibitory regulation by the adrenal during this periodwith implications for our understanding and treatmentof congenital adrenal hyperplasia. Maternal smoking didnot have marked effects on fetal adrenal steroidogenicfunction, but it did alter fetal ACTH levels, which maytrigger homeostatic mechanisms in the adrenal leadingto knock-on effects in the neonate.Additional filesAdditional file 1: Figure S1. Changes in whole intra-adrenal steroidlevels (ng/adrenal pair) during the second trimester. Data points fromindividual fetuses are shown (n = 60). All detectable steroid levels increasesignificantly with gestational age (P < 0.001) with the exception ofcorticosterone. Generalized linear regressions are shown as black lineswith the corresponding confidence intervals (0.95) in grey. (TIF 80 kb)Additional file 2: Figure S2. Age-dependent changes in adrenalexpression of mRNA transcripts encoding factors involved in steroidsynthesis. CYP11B2, HSD3B and POR are also shown in Fig. 5 and havebeen included here for completeness. Transcript levels are plottedrelative to housekeeping genes (HKGs), and each point represents datafrom an individual fetus. Black lines denote a generalized linearregression, and grey fill denotes the confidence interval (0.95) around theregression. (TIF 310 kb)Additional file 3: Figure S3. Effect of maternal smoking on STAR andCYP17A1 transcript levels in the fetal adrenal during the second trimester.Maternal smoking was associated with an increase in the variability(Levene’s test) of transcript expression of STAR (P = 0.004) and CYP17A1(P = 0.02), in male smoke-exposed (SE) fetuses compared to male controls(C). Each point represents data from an individual fetus and transcriptlevels are expressed relative to HKGs. Black lines denote a generalizedlinear regression, and grey fill denotes the confidence interval (0.95)around the regression. Data points for females are shown in the left ofeach panel and males on the right. Data points for controls are shown onthe top of each panel and smoke-exposed on the bottom. (TIF 339 kb)AbbreviationsACTH: Adrenocorticotropic hormone; CBG: Corticosteroid-binding globulin;CRL: Crown-rump length; DHEA: Dehydroepiandrosterone;DHEAS: Dehydroepiandrosterone sulphate; HPA: Hypothalamo-pituitary-adrenal;IS: Internal standard; LOD: Limit of detection; LOQ: Limit of quantitation;PMM1: Phosphomannomutase 1; RSD: Relative standard deviation; S/N: Signal-to-noise ratio; SEM: Standard error of the mean; SF-1: Steroidogenic factor 1;TBP: TATA box binding proteinAcknowledgementsWe are grateful to Margaret Fraser, Linda Robertson, Ana Monteiro, LynneFleming and Sam Flannigan for expert technical assistance and to the IanFraser Cytometry Centre, University of Aberdeen, for the multiplex assay. Wethank Karl Burgess for advice in developing the steroid LC/MS system andIan Mason for provision of antibodies. The staff at Grampian NHS PregnancyCounselling Service were essential for fetal collection. We also thank SerenaBanh and Natasha Walker (University of Aberdeen) for the human fetalkidney data shown in Fig. 5c.Johnston et al. BMC Medicine  (2018) 16:23 Page 14 of 16FundingThis study was supported by grants from the Medical Research Council(MR/L010011/1 to PAF and PJOS and MR/K501335/1 to MB, PAF and PJOS),the Canadian Institutes of Health Research (MOP-111102 to GLH), a CanadaResearch Chair in Reproductive Health (to GLH), and a post-doctoral fellowshipfrom the Fonds de Recherche du Québec en Santé and the Michael SmithFoundation for Health Research (to MS).Availability of data and materialsThe datasets generated and/or analysed during the current study are allpresented within the manuscript and its additional files.Authors’ contributionsPAF and PJOS conceived and designed the study, analysed and interpretedthe data and drafted and revised the article. ZCJ conceived and designedthe study, acquired, analysed and interpreted the data and drafted andrevised the article. MB and SB conceived and designed the study andanalysed and interpreted the data. PF, US, DH, MS, GLH and PK acquired andinterpreted the data. All authors read and approved the final manuscript.Ethics approval and consent to participateThe collection of fetal material was approved by the NHS Grampian ResearchEthics Committee (REC 04/S0802/21 and REC 15/NS/0123).Consent for publicationNot applicable.Competing interestsThe authors declare that they have no competing interests.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Institute of Biodiversity, Animal Health & Comparative Medicine, College ofMedical, Veterinary & Life Sciences, University of Glasgow, Glasgow G61 1QH,UK. 2Institute of Medical Sciences, School of Medicine, Medical Sciences &Nutrition, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.3Institute of Applied Health Sciences, School of Medicine, Medical Sciences &Nutrition, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.4Department of Cellular and Physiological Sciences, Life Sciences Institute,University of British Columbia, British Columbia V6T 1Z3, Canada. 5Centre forEndocrinology, William Harvey Research Institute, Barts and the London,Queen Mary University of London, Charterhouse Square, London EC1M 6BQ,UK.Received: 7 August 2017 Accepted: 18 January 2018References1. 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