repeated use of betamethasone in rabbits: effects of treatment variation on adrenal suppression,...

995

Repeated use of betamethasone in rabbits: Effects of treatmentvariation on adrenal suppression, pulmonary maturation, andpregnancy outcome

Leslie Pratt, MD,a, b Ronald R. Magness, PhD,a, c, d Terry Phernetton, BS,a Susan K. Hendricks,MD,b, c David H. Abbott, PhD,c, e and Ian M. Bird, PhDa, c

Madison, Wisconsin

OBJECTIVE: The aim of the study was to determine whether reduced birth weight, adrenal suppression,and lung maturation occur in parallel and are cumulative with increasing courses of betamethasone.STUDY DESIGN: Time-bred rabbits were assigned to a control group or to receive saline solution or 1, 2, or3 courses of betamethasone (early treatment, beginning day 19). Two additional groups (n = 5 per group)were given 1 or 2 late courses (late treatment). Birth weight, serum cortisol, adrenal 17α-hydroxylase(P450c17) messenger ribonucleic acid and fetal lung surfactant proteins A and B were quantified on day 27.RESULTS: Fetal weight was inversely proportional to the number of courses, with late treatment having agreater effect. Maternal cortisol and P450c17 levels were progressively suppressed with each early course,but fetal cortisol and P450c17 levels were only suppressed after 3 courses. A single late treatment pro-foundly suppressed both maternal and fetal cortisol and P450c17 messenger ribonucleic acid levels. In con-trast, fetal lung surfactant proteins A and B increased progressively with betamethasone courses, regardlessof timing.CONCLUSIONS: Time from last injection to delivery determined adrenal suppression, whereas total be-tamethasone courses determined surfactant protein production. Lower birth weight was dependent on thenumber of courses and was greater with late treatment. (Am J Obstet Gynecol 1999;180:995-1005.)

Key words: Corticosteroids, fetal organ maturity, fetal rabbit, pulmonary, respiratory distress syn-drome, surfactants

Since Liggins1 first reported the beneficial pulmonaryeffects of maternally administered corticosteroids onfetal sheep in 1969, many subsequent studies havedemonstrated the benefits of antenatal corticosteroids inpreterm human neonates. Because these effects areknown to be maximal at 48 hours after administration,

and to wane by between 7 and 10 days after administra-tion,2 many clinicians routinely use repeated courses ofbetamethasone on a weekly basis when the risk ofpreterm birth persists. Although the 1994 NationalInstitutes of Health consensus panel strongly recom-mended the use of betamethasone for pregnancies atrisk for preterm delivery,3 the advisability of repeated useremains uncertain.

Few animal or human studies published to date sup-port the safety4 or necessity5 of repeated weekly coursesof betamethasone. Animal studies in the 1970s showeddiminished fetal growth after antenatal corticosteroiduse.6-8 Subsequent observational human studies have notshown any effects on fetal growth after a single antenatalcorticosteroid course. The possible effects of repeatedcorticosteroid exposure are unknown. A single course ofbetamethasone has been shown to cause suppression ofthe human fetal adrenal gland for approximately 4 dayswith resolution of suppression by 7 days after treatment2;however, it is unknown whether the same effect would bepresent after multiple weekly courses of corticosteroids.

From the Perinatal Research Labsa and the Division of Maternal-FetalMedicine,b Department of Obstetrics and Gynecology,c the Department ofMeat and Animal Science,d and the Wisconsin Regional PrimateResearch Center,e University of Wisconsin-Madison.Supported in part by The Wisconsin Perinatal Foundation, Madison,and by grant awards NIH HL49210, NIH HD33255, NIH HL57653,NIH HL56702, AHA(WI) 95-GB-41, and NIH RR00167.Presented in part at the Sixteenth Annual Meeting of the Society ofPerinatal Obstetricians, Kamuela, Hawaii, February 4-10, 1996, andat the Forty-third Annual Meeting of the Society for GynecologicInvestigation, Philadelphia, Pennsylvania, March 20-23, 1996.Received for publication May 22, 1998; revised November 9, 1998; ac-cepted November 23, 1998.Reprint requests: Ian M. Bird, PhD, 7E Meriter Hospital, 202 S ParkSt, Madison, WI 53715.Copyright © 1999 by Mosby, Inc.0002-9378/99 $8.00 + 0 6/1/96053

Fetus-Placenta-Newborn

Of concern, Bradley et al9 has reported the case of an in-fant born with cushingoid features after exposure to 7weekly courses of betamethasone.

The purpose of this study was to determine whether re-peated courses of betamethasone would result in sup-pression of fetal or maternal adrenal function and in ac-celeration of fetal lung maturity in rabbits. The study wasfurther intended to determine whether a treatmentschedule could be found that would result in fetal lungmaturity without causing either fetal or maternal adrenalsuppression or decreased fetal birth weight.

Methods

Animals. Thirty-five New Zealand White rabbits(Hazelton [Covance] Research Facility, Kalamazoo,Mich) were time bred (day 0) and randomly assigned to5 treatment groups. Seven batches (n = 5 animals each)were treated. Each batch consisted of an animal fromevery treatment group, resulting in a randomized blockdesign. All rabbits were housed in individual cages withinthe same environmentally controlled room, were fed astandard diet, and were taken from their cages andweighed at gestational ages of 19, 22, and 25 days. Thisstudy was approved by the University ofWisconsin–Madison Medical School Animal Care andUse Committee.

Treatment protocols. Treatment schedules are outlinedin Table I. A control group of rabbits (n = 7) was handledonly for weighing and received no injections. To ascer-tain the magnitude of any injection effect, an additionalgroup received only intramuscular injections of 0.2 mLnormal saline solution (n = 7) on the same schedule asthe betamethasone-treated groups. Animals receiving be-tamethasone (Celestone Soluspan; Schering Corp,Kenilworth, NJ) beginning on gestational day 19 (of aterm of 31 days) were given 2 intramuscular injections of0.1 mg/kg betamethasone 24 hours apart either 1, 2, or 3

times. These groups (n = 7 per group) are referred to as1BΕΤΑ, 2BΕΤΑ, and 3BΕΤΑ, respectively. To normalizebetween groups any stressor effects from injections, theseanimals received 0.2 mL normal saline solution intra-muscularly when not scheduled to receive betametha-sone, such that each rabbit received a total of 6 injec-tions.

To determine any additional effect related to intervalfrom the last steroid injection until death, 2 furthergroups of rabbits (n = 5 per group) were given 3 sets ofinjections as before, including 1 (day 25-26) and 2 (day22-23 and day 25-26) late courses of betamethasone, re-spectively, with the last injection occurring 24 hours be-fore the animals were killed (in reverse order of the earlytreatment groups, Table I). These latter 2 groups are re-ferred to as the late treatment groups. Additional ani-mals treated with saline solution on days 19 and 20, 22and 23, and 25 and 26 (n = 2) were included for controlpurposes with each late treatment group to check forbatch variation, which was not observed.

Sample collection. All does were killed on gestationalday 27 (term is 31 days) by injection of pentobarbitalsodium (Beuthanasia-D Special; Schering-Plough AnimalHealth) into an ear vein. Immediately after death a mid-ventral laparotomy incision was made and maternalblood was collected from the inferior vena cava andplaced immediately on ice. Uteri were removed and im-mediately opened; fetuses and placentas (with mem-branes removed) were weighed. The numbers of live-born and stillborn fetuses were noted. Fetal trunk bloodwas collected, pooled for each litter, and immediatelyplaced on ice. Fetal lungs were removed and weighed.Representative sections of maternal and the right lowerand middle lobes of fetal lungs were snap-frozen in liquidnitrogen, with the remainder of the lung tissue placed in4% formaldehyde in sodium cacodylate buffer (0.1mol/L, pH 7.4). Similarly, 1 maternal and 1 fetal adrenal

996 Pratt et al April 1999Am J Obstet Gynecol

Table I. Betamethasone treatment schedules

Day of gestation

Treatment 19 20 21 22 23

Early treatment groups

1 Control (n = 7) None None — None None2 Saline solution (n = 7) Saline solution Saline solution — Saline solution Saline solution3 1ΒΕΤΑ (n = 7) Betamethasone Betamethasone — Saline solution Saline solution4 2ΒΕΤΑ (n = 6) Betamethasone Betamethasone — Betamethasone Betamethasone5 3ΒΕΤΑ (n = 6) Betamethasone Betamethasone — Betamethasone Betamethasone

Late treatmentgroups

2 Saline solution (n = 2) Saline solution Saline solution — Saline solution Saline solution3 1ΒΕΤΑ (n = 5) Saline solution Saline solution — Saline solution Saline solution4 2ΒΕΤΑ (n = 5) Saline solution Saline solution — Betamethasone Betamethasone

Doses of either 0.1 mg/kg betamethasone or 0.2 mL normal saline solution, or both, were given intramuscularly at varying days of gestation to 5 early treatment groups and 3 late treatment groups of does.

gland were removed and placed in liquid nitrogen, withcorresponding pairs placed in fixative. All blood was col-lected into a red-top Vacutainer tube (Becton Dickinson,Franklin Lakes, NJ) and placed immediately on ice.Serum samples were separated by centrifugation andstored at –20°C. Tissues placed in liquid nitrogen werestored at –70°C. Tissues placed in fixative overnight wereembedded in paraffin, cut into sections (6 µm), andmounted on polylysine-coated glass slides for deparaf-finization and hematoxylin-eosin staining.

Steroid assay proceduresCortisol. All serum samples were assayed for cortisol

concentration by an enzyme immunoassay modifiedfrom that used by Ziegler et al.10 Microtiter plates (Nunc-Immuno Plate Maxisorb F96 certified; VWR Scientific,Chicago, Ill) were coated with 100 µL cortisol antibodysolution per well (R4866, anticortisol–bovine serum albu-min, diluted to 1:22,000 with 50 mmol/L bicarbonatebuffer, pH 9.6) before immediate storage in a humidifiedchamber at room temperature for 6 hours followed by in-cubation at 4°C for 2 days. The antibody was then re-placed with phosphate buffer (150 µL phosphate-buffered saline solution, 0.1 mol/L, pH 7.0, 0.1% bovineserum albumin) and stored at –20°C. On the day of assayaliquots of between 20 and 100 µL rabbit serum were ex-tracted with 5 mL ethyl ether and evaporated to drynessunder filtered air in a 40°C water bath. Samples were re-constituted in 50 µL phosphate buffer. A 50-µL aliquot ofreference preparation or a 50-µL aliquot of reconstitutedsample was mixed with 250 µL cortisol and horseradishperoxidase (diluted 1:62,500 in phosphate buffer) andapplied as 100 µL per duplicate well.

The reference preparation used 11β,17α,21-trihydroxy-pregn-4-ene-3,20-dione (cortisol; Sigma Chemical Co, StLouis, Mo) ranging from 3.16 to 1000 pg. The referencepreparation, samples, and horseradish peroxidase conju-gate were allowed to incubate in a humidified chamber

for 2 hours at room temperature. After the cortisol andhorseradish peroxidase not bound to the antibody-coated well were removed by washing the plate 5 times,100 µL 40 nmol/L 2,2´-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) and 0.5 mol/L hydrogen peroxide in cit-rate buffer, pH 4.0, was added to each well and allowed toincubate for 1 hour at room temperature in a humidifiedchamber. The reaction was stopped with 0.15 mol/L hy-drofluoric acid, 6.0 nmol/L sodium hydroxide, and 1.0mol/L ethylenediaminetetraacetic acid (EDTA).Absorbance was measured at 410 nm on a DynatechMR5000 Microelisa Plate Reader (Dynatech LaboratoriesChantilly, Va).

At 50% binding the cortisol antibody cross-reacted60% with cortisone, 2.5% with corticosterone, and <1%with the following steroids: deoxycorticosterone, 11α-hy-droxyprogesterone, 11β-hydroxyprogesterone, 17α-hydroxy-progesterone, 20β-hydroxyprogesterone, 20α-hydroxypro-gesterone, progesterone, 17α-hydroxypregnenolone,testosterone, aldosterone, dehydroandrosterone, estradiol,and cholesterol. The antibody also cross-reacted 96%with prednisolone, 66% with prednisone, <0.5% withdexamethasone, and <0.1% with betamethasone. Serialdilutions of pooled rabbit maternal and fetal serum sam-ples were parallel to the cortisol reference preparation(P > .05, not significant). Accuracy for added rabbitserum to dilutions of the reference preparation was104.3% ± 0.9% (mean ± SEM; n = 6). Assay sensitivity was3 pg/well. Intra-assay coefficients of variation for low andhigh pools were 2.2% and 2.8%, respectively, and interas-say coefficients of variation for the same pools were 4.9%and 4.3%, respectively.

Aldosterone. Total aldosterone concentrations weremeasured in rabbit serum (100 µL) with an antibody-coated tube radioimmunoassay kit, Active Aldosterone(DSL-8600; Diagnostic Systems Laboratories, Inc,Webster, Tex). The aldosterone reference preparationused (11β,21-dihydroxy-3,20-dioxo-4-pregnen-18-al;Sigma) ranged from 2.5 to 160 pg. At 50% binding the al-dosterone antibody cross-reacted 0.03% with corticos-terone and <0.05% with the following steroids: 18-hy-droxycorticosterone, cortisol, and deoxycorticosterone.The antibody also cross-reacted <0.01% with pred-nisolone, dexamethasone, betamethasone, and spirono-lactone. Serial dilutions of pooled maternal rabbit serumsamples (2-132 µL) were parallel to the aldosterone ref-erence preparation (P > .05, not significant). Accuracyfor added rabbit serum to dilutions of the referencepreparation was 107.2% ± 2.4% (mean ± SEM; n = 6).Assay sensitivity was 1.6 pg/well. Intra-assay coefficientsof variation for low and high pools were 6.5% and 4.4%,respectively, and interassay coefficients of variation forthe same pools were 9.3% and 5.1%, respectively.

Messenger ribonucleic acid (RNA) analysis (P450c17,3β-hydroxysteroid dehydrogenase) RNA extraction.

Volume 180, Number 4 Pratt et al 997Am J Obstet Gynecol

Day of gestation

24 25 26 27

— None None Killed— Saline solution Saline solution Killed— Saline solution Saline solution Killed — Saline solution Saline solution Killed— Betamethasone Betamethasone Killed

— Saline solution Saline solution Killed— Betamethasone Betamethasone Killed— Betamethasone Betamethasone Killed

Maternal adrenal glands or pooled fetal adrenal glandswere homogenized with a Powergen 700 Polytron (FisherScientific, Pittsburgh, Pa) at 4°C into RNAzol B solution(Cinna Biotecx, Houston, Tex) before transfer in 1-mLvolumes to microcentrifuge tubes. Phase separation wasachieved by mixing with 0.15 mL trichloromethane, in-cubation at 4°C for 5 minutes, and centrifugation at12,000g for 20 minutes at 4°C. The recovered upperphase (0.6 mL) was extracted twice with a mixture ofphenol, chloroform, and isoamyl alcohol in the presenceof heavy-grade phase-lock gel (5 Prime→3; Prime, Inc,Boulder, Colo). Total RNA was then precipitated by theaddition of isopropanol for 1 hour at –20°C, recoveredby centrifugation for 30 minutes at 12,000g at 4°C, andwashed in 75% ethanol (1.0 mL) before dissolution inmolecular biology grade water (0.1 mL). Recovery andpurity were determined by absorbances at 260 and 280nm, and samples were stored at –70°C before analysis.

Northern blot analysis. To verify specificity of thehuman-based probes for rabbit messenger RNA, samplesfrom 1 batch of rabbits were separated by electrophoresison gels containing 1.1% agarose in the presence offormaldehyde. The presence and integrity of the majorRNA species were examined under ultraviolet light to en-

sure consistency between lanes. RNA was transferred to aMagna NT membrane (Molecular Separation Inc,Westborough, Mass) by vacuum blotting at 7 psi for 1.5hours (Bio-Rad Laboratories, Hercules, Calif) and cross-linked under ultraviolet light. Prehybridization was car-ried out at 42°C overnight in a final buffer compositionof 50% formamide, 5× saline–sodium citrate (SSC), 1×phosphate-EDTA (PE), and 50 mg/mL transfer RNA(20× SSC contains 3.0 mol/L sodium chloride and 0.3mol/L trisodium citrate, pH 7.0; 5× PE contains 250mmol/L tris(hydroxymethyl)aminomethane (Tris) hy-drochloride, pH 7.5, 0.5% sodium pyrophosphate, 5%sodium dodecyl sulfate [SDS], 1% polyvinylpyrrolidone,1% ficoll, 25 mmol/L EDTA, and 1% bovine serum albu-min). Hybridizations were performed sequentially in thesame buffer at 42°C for 16 to 24 hours with antisenseprobes to human P450c17 and 3β-hydroxysteroid dehy-drogenase (3βHSD). Each antisense probe was labeledwith phosphorus 32 by asymmetric polymerase chain re-action in the presence of deoxycytidine triphosphatetagged with phosphorus 32 (Amersham Corp, ArlingtonHeights, Ill).11 The blots were then washed in 2× SSCcontaining 0.1% SDS at room temperature for 15 min-utes and twice in 0.1× SSC containing 0.1% SDS at roomtemperature for 30 minutes before direct radioimagingquantification of bound probe (Bio-Rad model GS-250Phosphorimager) with BI Screen for 4 hours and subse-quent exposure to film (Hyperfilm; Amersham)overnight. Blots were subsequently stripped by repeatedwashing in 0.1× SSC and 0.5% SDS at 65°C for 1 hourand checked for lack of radioactivity before reprobing.Finally, all blots were probed for glyceraldehyde 3-phos-phate dehydrogenase messenger RNA with an antisenseprobe generated by asymmetric polymerase chain reac-tion against bases 39 through 900 of the human comple-mentary deoxyribonucleic acid, and bound probe wasquantified as described previously. Binding of glyceralde-


Table II. Fetal lung and placental weights

Fetal lung weight Placental weight

Proportion ProportionAbsolute of birth weight Absolute of birth weight

Treatment group (mean ± SE, g) (mean ± SE, %) (± SE, g) (mean ± SE, %)

Control 1.00 ± 0.02 3.27 ± 0.04 4.69 ± 0.10 16.14 ± 0.25Saline solution 1.07 ± 0.03 3.46 ± 0.60 5.43 ± 0.14 16.96 ± 0.351ΒΕΤΑ (early) 0.92 ± 0.02* 3.08 ± 0.04* 5.14 ± 0.10 17.03 ± 0.291ΒΕΤΑ (late) 0.83 ± 0.03† 3.08 ± 0.07* 4.37 ± 0.11‡ 16.02 ± 0.332ΒΕΤΑ (early) 0.78 ± 0.03‡ 2.89 ± 0.05† 4.68 ± 0.12* 17.23 ± 0.342ΒΕΤΑ (late) 0.60 ± 0.02§ 2.82 ± 0.06† 3.50 ± 0.10§ 16.40 ± 0.503ΒΕΤΑ 0.60 ± 0.02§ 2.99 ± 0.07† 3.75 ± 0.12§ 19.96 ± 0.89ll

*P < .05 (vs saline solution).†P < .05 (vs control, saline solution).‡P < .05 (vs control, saline solution, early treatment 1ΒΕΤΑ).§P < .05 (vs all other groups except 2ΒΕΤΑ late treatment).llP < .05 (vs control, 1ΒΕΤΑ late treatment, 2ΒΕΤΑ late treatment), all by Kruskal-Wallis analysis of variance.

Table III. Maternal aldosterone levels

Treatment group Mean ± SE (pg/mL)

Control 220.8 ± 50.2Saline solution 316.5 ± 88.71ΒΕΤΑ (early) 194.5 ± 70.92ΒΕΤΑ (early) 238.6 ± 31.63ΒΕΤΑ 163.1 ± 109.01ΒΕΤΑ (late) 56.6 ± 3.1*2ΒΕΤΑ (late) 109.5 ± 34.3

*P < .05 (vs saline solution, early treatment 2ΒΕΤΑ) by Kruskal-Wallis analysis of variance with Dunn multiple comparisonmethod.

hyde 3-phosphate dehydrogenase probe per lane wasthen used to normalize data for P450c17 and 3βHSDmessenger RNA against minor variations in lane loading.P450c17 and 3βHSD messenger RNA levels were ex-pressed as a percentage of the maternal normal saline so-lution control value from the same slot blot.

Slot blot analysis. Having verified the specificity of theprobes for the corresponding rabbit messenger RNA,quantification of the changes in expression of P450c17and 3βHSD were performed en mass by slot blotting,which has the advantage of enhanced throughput effi-ciency and fully minimized variation between mem-branes. Samples of total RNA (10 µg) were diluted to100-µL volume before precipitation with 10 µL 3 mol/Lsodium acetate (pH 5.2) and 500 µL ethanol. After re-covery by centrifugation, RNA was dissolved in 100 µLTris-EDTA (TE) buffer, pH 7.5, denatured in the pres-ence of 40 µL formaldehyde and 70 µL 20× SSC for 15minutes at 65°C, and finally applied directly to MagnaNT membrane under 1 psi vacuum with a Slot Blotter(Bio-Rad). Sample wells were rinsed with 500 µL 20×SSC to ensure efficient loading. Membranes were thenremoved, cross-linked, and hybridized as previously de-scribed.

Analysis of surfactant proteins A and BSample processing. Surfactant proteins A (SP-A) and B

(SP-B) were analyzed in maternal and fetal lung tissuewith procedures that were based on those described pre-viously.12 The right lower and middle lobes of all fetallungs were homogenized in a Tris buffer with protease in-hibitors—composed of 150 mmol/L sodium chloride, 50mmol/L Tris hydrochloride, 10 mmol/L EDTA (pH 7.4),0.1% polysorbate 20, 0.1% β-mercaptoethanol, 0.1

mmol/L phenylmethylsulfonylfluoride, 5 µg/mL leu-peptin, and 5 µg/mL aprotinin—with a glass pestle, fol-lowed by brief centrifugation to remove unsolubilized de-bris and sonication of the supernatant (Sonifier CellDisruptor model W185; Heat Systems-Ultrasonics, Inc,Plainview, NY). Protein content of solubilized tissue wasdetermined with the Bio-Rad Protein MicroAssay withquantification on a Bio-Rad 3550 microplate reader.Pooled samples for each litter were created by takingequal amounts of protein from each individual fetal lungsample. Maternal lung samples were homogenized andprotein was quantified in an identical fashion. Maternalstandards were created by pooling equal amounts of pro-tein from all maternal control does and salinesolution–treated does. These standards were then usedon all gels as normalization control preparations.

Western blot analysis. For analysis of SP-A, 20 µg proteinfrom each sample was separated on a 12% SDS polyacryl-amide gel with the Bio-Rad MiniProtean apparatus andthen transferred to an Immobilon-P membrane(Millipore Corp, Bedford, Mass). Immunoblotting wasperformed at room temperature with a polyclonal an-tirabbit SP-A antibody (1:5000 dilution, 2 hours) raisedin a guinea pig (antibody was generously provided by DrCarole R. Mendelson, University of Texas SouthwesternMedical Center, Dallas, Texas), together with ananti–guinea pig second antibody conjugated to horserad-ish peroxidase (1:8000 dilution, 1 hour; Sigma). Resultswere detected by enhanced chemiluminescence detec-


Fig 1. Fetal loss rates for treatment groups expressed as percent-age of fetuses per litter (mean ± SE). Striped bars, Early treatmentgroups; filled bars, late treatment groups. P = .16, Kruskal-Wallis1-way analysis of variance on ranks (all groups); P = .003, χ2 fortrend (early treatment groups).

Fig 2. Birth weights (mean ± SE) of all live-born pups are re-ported for each treatment group. Striped bars, Early treatmentgroups; filled bars, late treatment groups. Asterisk, P < .05 (vssaline solution group); two asterisks, P < .05 (vs saline solution,control, and early treatment 1ΒΕΤΑ groups); plus sign, P < 0.05(vs saline solution, control, early treatment 1ΒΕΤΑ, early treat-ment 2ΒΕΤΑ, and late treatment 1ΒΕΤΑ groups); two plus signs,P < .05 (vs all other groups). All tests were by Kruskal-Wallisanalysis of variance with Dunn multiple comparison method.

tion (ECL; Amersham), and the 35-kd band (matureform)12 was quantified by scanning transmission densito-metry (Bio-Rad Imaging Densitometer model GS-670).SP-B analysis was performed on the same samples with 25µg protein separated on a 10% SDS reducing polyacryl-amide gel. Immunoblotting was performed with a mono-clonal antipig SP-B antibody (1:8000 dilution, 2 hours)raised in a mouse (antibody was generously provided byDr Yasuhiro Suzuki, University of Kyoto, Kyoto, Japan) to-gether with antimouse antibody conjugated to horserad-ish peroxidase (1:3000 dilution, 1 hour; Bio-Rad). Theantipig SP-B antibody has previously been shown to cross-react with rabbit SP-B.13 The 8-kd band was quantified.All values were then expressed as percentages of the ma-ternal normal saline solution standard run on the samegel.

Statistics. Statistical analysis was performed with 1-wayanalysis of variance and the Student-Newman-Keuls posthoc test for parametric data with normal distribution.Data with nonnormal distribution were analyzed with theKruskall-Wallis analysis of variance on ranks with theDunn or Dunnett post hoc methods. A χ2 test for trendwas used for nonparametric data (fetal loss rates).Significance was defined as P < .05. Data are expressed asmean ± SEM. Data analysis was performed with SigmaStatfor Windows version 1.0 (Jandel Corp, San Rafael, Calif)and StatMost v3. 0 (DataMost Corp, Sandy, Utah) soft-ware.

One of the 3ΒΕΤΑ group does had premature sponta-neous delivery on day 27 before being killed. This doe

was retained in the maternal data. Two other does (n = 1each in the 2ΒΕΤΑ and 3ΒΕΤΑ groups) had spontaneousloss of all conceptuses on days 22 and 23, respectively,and were not included in subsequent data analysis. Asixth doe was added to the 3ΒΕΤΑ group to maintain abalanced study. All does in both the early (n = 7) and late(n = 2) saline solution groups were combined for dataanalysis.

Results

At death on day 27, litter sizes (all fetuses) rangedfrom 4 to 14. The fetal loss rate (percentage of fetal re-sorptions and stillbirths) of all does is shown in Fig 1. A


Fig 3. Serum cortisol levels for all does and pooled fetal serumlevels for each litter (mean ± SE). Stippled bars (a), Maternal earlytreatment groups; filled bars (b), maternal late treatment groups;striped bars (c), fetal early treatment groups; gray bars (d), fetal latetreatment groups. Asterisk, P < .05 (vs maternal control, saline so-lution, and early treatment 1ΒΕΤΑ groups); plus sign, P < .05 (vsfetal control, saline solution, early treatment 1ΒΕΤΑ, and earlytreatment 2ΒΕΤΑ groups). All tests were by Kruskal-Wallis analy-sis of variance with Dunn multiple comparison method.

Fig 4. Northern blot analysis of changes in maternal and fetaladrenal P450c17 and 3βHSD messenger RNA levels after re-peated betamethasone treatment. Representative data obtainedby Northern blot analysis of maternal and fetal adrenal messen-ger RNA (25 µg/lane) are shown. Blot was sequentially probedfor P450c17, 3βHSD, and glyceraldehyde 3-phosphate dehydro-genase (GAPDH) messenger RNA, as described. Positions of spe-cific signal are indicated by arrows. MW, Molecular weight (inkilobases); Cn, control group; NS, saline solution group; 1Β,group 1ΒΕΤΑ; 2Β, group 2ΒΕΤΑ; 3Β, group 3ΒΕΤΑ (all earlytreatment groups).

significant increase in fetal loss was associated with 2 and3 courses of betamethasone in the early treatment butnot the late treatment groups.

Fetal birth weight (Fig 2) showed a significant differ-ence between groups (overall P < .0001 by Kruskal-Wallis1-way analysis of variance). Whereas normal saline solu-tion did not result in a difference from the controlgroup, increasing numbers of courses of betamethasoneresulted in progressive decreases in birth weight. Latetreatments resulted in a greater decline in birth weightthan did the same number of courses given at an earliergestational age. A single course given on days 25 and 26had the same effect as did 2 courses given between days19 and 23. A similar decrease was seen in fetal lungweight (P < .05 by Kruskal-Wallis 1-way analysis of vari-ance, Table II). When expressed as a percentage of birthweight, betamethasone was found to result in a greaterproportional decrease in fetal lung weight with 2 and 3

courses of betamethasone (overall P < .0001 by Kruskal-Wallis 1-way analysis of variance, Table II). Placentalweights decreased with increasing courses of betametha-sone but did not show as marked a decrease (Table II).

Maternal serum cortisol level showed a progressive de-cline in the early treatment groups with increasing num-bers of courses of betamethasone (Fig 3; P < .001 byKruskal-Wallis 1-way analysis of variance). A significantdecline in cortisol level was not seen with the saline solu-tion injections alone. A positive correlation was seen withlinear regression analysis of the saline solution and earlytreatment groups (r2 = 0.323, P = .002). In contrast, the1ΒΕΤΑ and 2ΒΕΤΑ late treatment regimens resulted incomplete suppression of maternal cortisol. In the earlytreatment groups fetal serum cortisol level showed alesser decline than that observed for maternal cortisollevel. The fetal serum cortisol decline only became sig-nificant at 3 courses (3BETA, Fig 3; P < .0001 by Kruskal-


Fig 5. Quantification of adrenal messenger RNA by slot blot hybridization analysis. Changes in levels of P450c17 (A)and 3βHSD (B) messenger RNA from maternal and fetal adrenal tissue were assessed by slot blot hybridization analysis(10 µg/well) as described. All data were normalized to glyceraldehyde 3-phosphate dehydrogenase expression in sameslot. Ratio of P450c17/3βHSD is shown in C. Stippled bars (a), Maternal early treatment groups; filled bars (b), maternallate treatment groups; striped bars (c), fetal early treatment groups; gray bars (d), fetal late treatment groups. Asterisk, P <.05 (vs control maternal value); two asterisks, P < .05 (vs control, saline solution, and early treatment 1BETA maternalvalues); caret, P < .05 (vs control, saline solution, early treatment 1BETA, and early treatment 2BETA maternal values);pound sign, P < .05 (vs all early treatment values except 3BETA maternal value); underscored asterisk, P < .05 (vs all othertreatment group maternal values); plus sign, P < .05 (vs control, saline solution, early treatment 1BETA, and early treat-ment 2BETA fetal values); two plus signs, P < .05 (vs all other treatment fetal values). All tests were by Kruskal-Wallisanalysis of variance with Dunn multiple comparison method.

Wallis 1-way analysis of variance). With late treatment,however, both 1ΒΕΤΑ and 2ΒΕΤΑ courses completelysuppressed fetal cortisol levels. The 3ΒΕΤΑ treatmentsresulted in a complete suppression of both maternal andfetal cortisol.

Maternal serum aldosterone levels were found to beunchanged in the early treatment groups (Table III).Although the late treatment groups showed a decrease inaldosterone level, this was only significant in the 1ΒΕΤΑgroup. Preliminary analysis showed most fetal aldos-terone levels (75%) to be below the limits of sensitivity(16 pg/mL) of the assay used (data not shown).

Northern blot analysis of P450c17 and 3βHSD messen-ger RNA in rabbit adrenal glands with human comple-mentary deoxyribonucleic acid–based probes showedthat the signal was cleanly detected as a single band at theexpected molecular weight in both maternal and fetalRNA preparations (Fig 4). In subsequent analysis of RNAsamples en mass by slot blot hybridization, results wereexpressed as a percentage of the level in the maternalnormal saline solution control group with no change inglyceraldehyde 3-phosphate dehydrogenase expressionobserved. However, 3ΒΕΤΑ resulted in a dramatic andsignificant decline in both maternal P450c17 messengerRNA levels (vs control, saline solution, and 1ΒΕΤΑ earlytreatment groups) and fetal P450c17 messenger RNA lev-els (vs all other early treatment groups). With late treat-ment a 1ΒΕΤΑ course resulted in decline of P450c17messenger RNA similar to that in both the maternal andfetal samples in the 3ΒΕΤΑ group (Fig 5, A). Maternaladrenal tissue showed a small but significant increase in3βHSD messenger RNA levels only with late treatment. Asignificant increase was also seen in fetal adrenal tissueonly with the 1ΒΕΤΑ late treatment group (Fig 5, B). Theratio of P450c17 to 3βHSD has been shown to be the true

determinant of cortisol secretion.14 Analysis of this ratiowas therefore undertaken and showed a significant dif-ference between groups in both maternal and fetal ratios(overall P < .0001 by Kruskal-Wallis 1-way analysis of vari-ance). This ratio showed changes similar to those seenfor P450c17 alone with increasing courses of betametha-sone (Fig 5, C).

Maternal adrenal weights were not different betweengroups (data not shown). Histologic examination of ma-ternal adrenal tissue revealed no apparent change in rel-ative adrenocortical zonation with betamethasone ad-ministration (data not shown).

A representative Western blot of SP-A and SP-B isshown in Fig 6. Although no significant change from thecontrol group was seen with saline solution in fetal SP-Aor SP-B levels, a significant increase was observed forboth SP-A and SP-B in the 3ΒΕΤΑ treatment groups (Fig7). Linear regression analysis of SP-A and SP-B in theearly treatment groups showed a significant correlationbetween the number of courses of betamethasone andthe level of surfactant protein. (SP-A r2 = 0.248, P < .01;SP-B r2 = 0.363, P < .05). Late treatment with either 1 or 2courses of betamethasone showed increases in both SP-Aand SP-B that did not reach significance, similar to theearly treatment groups 1ΒΕΤΑ and 2ΒΕΤΑ. Histologicsections of fetal lung demonstrated significant matura-tion after 3 doses of betamethasone, with pulmonary ar-chitecture appearing similar to that in the mature doe(data not shown).

Comment

The benefits of antenatal maternal administration ofcorticosteroids for the prevention of respiratory distresssyndrome, prevention of intraventricular hemorrhage,and improvement of survival in the preterm neonate are


Fig 6. Western blot analysis of SP-A and SP-B. Representative Western blot radiographs labeled for SP-A (20 µg pro-tein/lane) and SP-B (25 µg protein/lane) are shown. SP-A (29-kd and 36-kd bands) and SP-B (8-kd band) are labeled.Mat. Control, Pooled maternal control standard; Mat. Saline, pooled maternal saline solution standard; Fet. Control, fetalcontrol; Fet. Saline, fetal saline solution; 1 Beta, 2 Beta, and 3 Beta, all fetal samples pooled for each litter (all early treat-ment groups).

well established.3 One of the remaining unansweredquestions regards the necessity or advisability of repeateddoses of antenatal corticosteroids. This study was de-signed to compare the extent of the known adverse ef-fects of repeated courses of betamethasone with the ef-fectiveness of repeated courses on lung maturation andto develop a protocol that would minimize the adverse ef-fects while maximizing the benefits for fetal lung devel-opment.

Studies from the 1970s demonstrated decreased fetalgrowth in rat6, 7 and rabbit15 models after antenatal corti-costeroid use. Our results support the previous concernsregarding corticosteroid exposure and decreased birthweight in the rabbit model. Conceptuses exposed toequivalent doses later in gestation (days 25 and 26 vs days19 and 20) showed a significantly greater decline ingrowth. This finding suggests that the steroid effect onfetal growth is dependent on both timing of administra-tion and total number of courses. The decrease in fetallung growth with increased time of exposure to cortico-steroids was less pronounced than was the decrease seenin fetal weight, although it was still apparent. This is con-sistent with the study by Johnson et al,16 in which re-peated intramuscular betamethasone treatments of rhe-sus monkeys for 13 days at 2 mg/d resulted in smallerlungs and other organs, including the brain.

It is noteworthy that neither our own study nor otherstudies17, 18 were able to define a corticosteroid dose ortreatment regimen that improved neonatal pulmonaryfunction without causing intrauterine growth restriction.Conversely, both our study and that of Sun et al18 showeddoses of corticosteroid capable of causing growth restric-tion without improving measures of pulmonary maturity.Ikegami et al19 recently reported decreased fetal growthin the sheep model after repeated use of betamethasone.This point may be of clinical significance.

In this rabbit study administration of corticosteroidearly in the last trimester was associated with fetal loss.More than 25 years ago Wellmann et al20 showed that therate of fetal resorption in the rabbit increased propor-tionally to the total amount of cortisone exposure inutero, although they did not note the gestational age atwhich steroid administration was initiated. It is unknownwhether this increased fetal loss is of clinical concern.

In this study prolonged corticosteroid use was associ-ated with significant maternal and fetal adrenal suppres-sion. A single course of betamethasone has been associ-ated with transient cortisol suppression in fetal cordblood.21, 22 Repeated use of betamethasone may result insimilar chronic suppression of the maternal and fetal pi-tuitary-adrenal corticotropin-cortisol axis, with potentialadverse effects. A case report by Bradley et al9 confirmedthe occurrence of chronic adrenal suppression in boththe mother and infant with prolonged use of betametha-sone. Conversely, Terrone et al4 recently reported no as-sociation between total number or doses of cortico-

steroid in a series of 79 infants receiving varying amountsof betamethasone before delivery. Parker et al23 reportedthat cord blood levels of cortisol in human neonates ex-posed to antenatal corticosteroids remained significantlylower than in control infants through 4 days after initialtreatment. In their study of cholesterol levels (a precur-sor to steroidogenesis) they found that serum levels re-mained elevated 7 days after steroid treatment, suggest-ing a prolonged alteration in adrenal steroidogenesis.Long-term weekly steroid use in utero may have a signifi-cant effect on lipid metabolism that has not yet been fullyexplored.

We studied maternal and fetal adrenocortical functionby determining levels of serum cortisol and adrenal tis-


Fig 7. Fetal SP-A and SP-B levels. Quantification of SP-A levels(30 to 38-kd band, A) and SP-B levels (8-kd band, B) are ex-pressed as percentage (mean ± SE) of maternal saline solutionstandard run on same gel. Striped bars, Early treatment groups;filled bars, late treatment groups. Asterisk, P = .02 (SP-A); two aster-isks, P = .007 (SP-B). All tests were by Kruskal-Wallis analysis ofvariance with Dunn multiple comparison method.

sue messenger RNA for P450c17. P450c17 messengerRNA codes for 17α-hydroxylase, the rate-limiting enzymefor cortisol production, whose induction is corticotropindependent.14 Specificity of suppression of adrenal corti-sol secretion and P450c17 messenger RNA expressionwas studied by determination of both serum aldosteronelevels (as a monitor of zona glomerulosa function, whichdoes not express P450c17) and adrenal messenger RNAfor 3β-HSD (expressed throughout the adrenal gland ina manner not solely dependent on pituitary corti-cotropin),14 as well as monitoring of adrenal weight. Ourresults confirm that betamethasone use causes adrenalsuppression of cortisol biosynthesis, as predicted by thework of Ballard et al.8 We have further shown that sup-pression of adrenal glucocorticoid production is princi-pally due to loss of P450c17, resulting in a dramatic fall inthe P450c17-to-3βHSD ratio. As expected, maternal al-dosterone levels showed no systematic change in thisstudy, consistent with the lack of expression of P450c17in the zona glomerulosa. Maternal 3βHSD messengerRNA levels increased slightly with late treatment only. Asimilar effect was seen in fetal adrenal for the 1ΒΕΤΑ latetreatment group. It is unlikely this was the result of an in-direct increase in circulating angiotensin II, becauseBerry et al24 found a single dose of fetal betamethasoneresulted in a dose-dependent suppression of the fetalrenin-angiotensin-aldosterone axis in premature new-born lambs.

We found that overall maternal adrenal weights and ar-chitecture remained unchanged. This is consistent withthe theory that adrenal suppression is principally causedby loss of corticotropin-induced P450c17 expressionrather than permanent alterations in adrenal architec-ture. Similarly, Torres and First25 reported that dexa-methasone given daily as late as 10 days before deliverydid not alter maternal adrenal weight. In contrast,Challis et al26 showed that high doses of dexamethasonegiven daily for 16 to 26 days to pregnant rhesus monkeysresulted in marked atrophy of the fetal adrenal glands,with appreciable regression of the fetal zone noted. Thedose used in our study is comparable to a 120-kg womanreceiving the standard clinical dose of 12 mg twice, 24hours apart. Higher doses might result in more pro-nounced adrenal suppression.

The most interesting aspect of the observed adrenalsuppression was the comparison of the effects of earlyand late treatment, which suggested that the suppressionof P450c17 expression, although acute and severe, wasrapidly reversible. Thus the extent of adrenal suppres-sion at the time of delivery may be more closely related tothe interval from the last administered dose than to thetotal amount of betamethasone administered. This find-ing is consistent with the observation of Ballard et al thatafter a single course of betamethasone human cordblood serum cortisol levels were suppressed only if the

last injection was within 4 days of delivery. Ikegami et al27

reported a similar effect in sheep. They showed that be-tamethasone suppressed the normal postnatal increasein plasma cortisol level when given 2 and 4 days beforeparturition but not when given 7 days before parturition.However, there may be a limit beyond which adrenal ef-fects do not resolve so quickly. Certainly the cushingoidinfant born after 7 weeks of betamethasone described byBradley et al9 is of concern. Jobe et al28 showed a dose-de-pendent suppression of the postnatal cortisol surge inlambs who had received direct injections of cortico-steroid in utero. Although fetal treatment did not altercord cortisol levels, a recent report by Ikegami et al19 hasshown adrenal suppression in newborn lambs exposed tohigher doses (0.5 mg/kg) repeated weekly for as long as3 weeks before delivery.

Our results suggest that, in contrast to the finding onadrenal suppression, the total amount of betamethasoneis more important than the timing of administration insignificantly increasing SP-A and SP-B production.Higher doses may have a different effect. Postnatal lungfunctional responses to betamethasone have been re-ported to persist for ≥7 days by many authors.3 Ikegami etal27 studied the effects of interval from a single corticos-teroid treatment to delivery on several other indices ofpostnatal lung function in preterm lambs and reportedthat the pulmonary maturation effect lasted 7 days. Theysuggested that a re-treatment strategy might be helpfulbecause of the lack of progressive maturation after fetalbetamethasone treatment. Whether the effect of be-tamethasone on SP-A and SP-B production continuespast 7 to 10 days, however, remains unknown.

The effect of repetitive dosing on pulmonary matura-tion was recently addressed by Polk et al5 in a study ofpreterm lambs. Their dosage schedule corresponded toour early treatment schedule for 1 and 2 courses of be-tamethasone (1ΒΕΤΑ and 2ΒΕΤΑ). Betamethasone in-creased messenger RNA for SP-A and SP-C with a singledose but subsequently decreased these levels with 2doses. Conversely, SP-B continued to increase with thesecond dose. They concluded that re-treatment did notaugment postnatal lung function. A later publication bythe same group19 reported that repetitive 7-day intervalexposures progressively enhanced postnatal lung func-tion as measured by lung compliance, ventilation effi-ciency, and lung volume. However, resultant growth andendocrine abnormalities were noted. Betamethasone ad-ministration in the rabbit model used in these studies re-sulted in increased SP-A and SP-B production but notwithout significant growth restriction in the fetus andtemporary adrenal suppression in both mother andfetus. Subsequent studies should concentrate on devel-oping a dosage schedule that could increase SP-B andpulmonary function while minimizing adverse effects.Sun et al18 previously showed greater growth restriction


with a single dose than with the same total amount of be-tamethasone in 2 divided doses 24 hours apart. A recentarticle by Engle et al29 reported the use of single- versussplit-dose dexamethasone treatment in the monkey andfound greater pulmonary maturity and less adrenal sup-pression with multiple low doses than with the same totalamount of dexamethasone given in a single larger dose.

This rabbit model shows optimal timing for delivery tobe ≥4 days after betamethasone administration, regard-less of the total amount of betamethasone administered.This interval results in decreased adrenal suppressionwhile maintaining pulmonary maturation effects. Theremay, however, be a total amount of corticosteroid admin-istered beyond which adrenal suppression does not abateso quickly, as evidenced by the report by Bradley et al.9

Future studies need to investigate the possible benefits ofmore frequently repeated low-dose administration,rather than weekly bolus injections, on postnatal out-come. Until such studies are completed we recommendrepeating betamethasone treatment on a weekly basisonly in cases at serious risk for delivery within the ensu-ing week. The clinician should remain cognizant ofadrenal suppression and the possible risk of cushingoidsymptoms in both the mother and fetus.

We thank Dr Carole Mendelson, Dallas, Texas, and DrYasuhiro Suzuki, Kyoto, Japan, for providing the SP-Aand SP-B antibodies, respectively.

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