Volume 157N um ber 6

of auditory function in ne wborn in fants re vealed by au­ditory bra instem poten tials. Ped iatri cs 1977 ;60:83 I.

12. Don M, Allen AR, Starr A. Effec t of dick rat e on thelatency of auditory bra in ste rn res ponses in h uman s. AnnO tol Rhinol Laryngo l 1977 ;86:186.

13. Prall H , Sohmer H . In tensity and rate functions ofcochlear an d brainstem evo ked responses to d ick stimuliin man. Arch O tol Rhi nol Laryngol 1976;2 12:85.

14. Prall H, Ben-David Y, Peled R, Podosh in L,Scharf B.

Fetal auditory brain stem response

Auditory bra in stern evoked potentials: d in icaI promiseof increasing stimulus ra te . Eleetroencc phalogr Clin Neu­rophys iol 1981 ;5 1:80.

15. StockardJE, StockardJJ, Coen RW. Auditory brain sternresponse variabili ty in in fants. Ear Hea r 1983;4:11.

16. Mattsse n J L, Fry WN, Boward CA, Miller E. Maiurationof the visual evo ked res ponse in new born min iature pigs.AmJ Vet Res 1978 ;39: 1279.

Pituitary responses to synthetic corticotropin-releasinghormone: Absence of modulatory effects by estrogenand progestin

J. H . Liu, M.n., D. n. Rasmussen, Ph.D.,* J. Rivier, Ph.D., W. Vale, Ph.n., andS. S. C. Yen, M.D., D.Se.

La Jolla, California

To determine il the activity of the adrenocorticotropic hormonal-adrenal axis is modulated in part byestrogens and progestins, we have compared pituitary and adrenal responses to corticotrophin-releasinghormone in normal women during the early follicular, late follicular, and midluteal phases of the menstrualcycle and in four women undergoing ovariectomy who received estradiol (E2l implants alone or incombination with oral medroxyprogesterone acetate administration. Basal adrenocorticotropic hormone andcortisol levels (9:00 AM) and responses to ovine CRH were unaffecled by variations in E2 andprogesterone during each phase of the menstrual cycle. In women undergoing ovariectomy who receivedE2 replacement therapy , basal concentrations 01 adrenocorticotropic hormone and cortisol (9:00 AM) andthe responses to human corticotrophin-releasing hormone were not altered by administration of E2 alone orE2 with medroxyprogesterone acetate. Under these experimental conditions, our findings suggest thatphysiologie changes in E2 and progesterone levels during the menstrual cycle and replacement doses ofestrogen and progestin commonly used clinically do not significantly influence basal adrenocort icotropichormone and cortisol levels or responsiveness to corticotrophin-releasing hormone. (AMJ OSSTET G VNECOL

1987;157 :1387-91 .)

Key words : Ad renocorticotropic horrno ne, co rt icotrophin-releasing hormone,me d roxyp rogeste ron e acetate , es trogen, p rogestin

Although it is widely recognized tha t there is an in­crease in the onset of d ep ressive syndrornes' and aber-

From the Department 0/Reproductiue M ediane, School 01 M edianeand the General Clin ical R esearch Centa , Un iversity 0/California ,San Diego, and Peptide B iology Laboratory, Salk Institute.

Supported in part by the Uni versity 01California, San Diego, GeneralClinical Research Center, National Ins titut es 0/Healt hlDivision0/ Research R esources Grant RR-0082 7, National Institute 0/Child H ealth and Human Deoelopment Population Center GrantHD-I2303 , and the M e/ion Foundation.

R esearch conducted in part by the Clayton. Foundati on [or R esearch,California Division.

Sponsored by the Society [o r Gynecologic In vestigali on .R eprint requests:j. H . Liu , M .D., Department 01R eproductiue Med­

icine T-002, School 0/ M edieine, Un iversity 01 California,San Diego, La Jolia, CA 92093.

*Reeipient 01 the Mellon Foundation Faculty Scholar Awa rd.

ra n t psychologie behavior" during the p remenstrualphase of the menstr ual cycle, th e role of ovarian ste­roi ds in th ese disorders rernains unclear, Since th epituita ry-adrenal ax is has bee n implica ted as a majorneuroen d ocrine modulator of psych ologie beh avio rand stress responses," p rel iminar y studies have focusedon per ipheral measurements of adrenocorticotropichormone (ACTH) and co rt isol output. T hese stud iessuggest that in normal wornen, basal levels of ACTHand cortisol are eIevated during the late follicular andmidcyde phases of the menstrual cycle, ' whereas in th epresence of high progestin levels, bas al co rt isol con­centrations have been reported to decrease." O n theother hand, other investigators have re ported thatthere is an enhanced adrenocortical responsiveness to

1387

1388 Liu et al.

stress during the luteal phase of the cycle." In view ofthese disparate findings, it seems nece ssary to examinefirst the modulatory role of ova rian steroids in ACT H­cortisol secretion at the pituitary and adrenal levels.To investigate these interactions, we have determinedthe pituitar y and adrenal responses to corticotropin­releasing hormone in normal women during the men­strual cycle and in women underoing ovariectomy re-ceiving hormone replacement therapy. .

Material and methods

Six normal women between the ages of 26 and43years, with menstrual cyd es be tween 28 and 32 days inlen gth , volunteered for this study. Four women un­dergoing ovariectomy between the ages of 40 and 60years were also studied. Each woman was within 10%of her ideal bod y weight.

In each experiment, subjects were ad mitted to theClinical Research Center at 8:00 AM after an ovemightfast . One hour after intravenous catheter placement,blood sam pies were collected at - 20,0,20,40,60, 80 ,100, 120, 150, and 180 minutes re lative to the injecti onof corticotrophin-releasing hormone. Each wom anwith anormal cycle received either saline solution orovine corticotrophin-releasing hormone, ( I /-tg/kg, in­travenous bolus) during the early follicular phase(da ys 1 through 5) of the first menstrual cycle. Duringsubsequent menstrual cycles, ovin e cor ticotrophin­releasing hormone was ad rniniste red during the latefollicular (days 11 to 14) and midluteal (6 to 8 da ysafter ovulation) phases. The timing of the late follicul arand midluteal phase experiments was determined bypelvic ultrasound monitoring.

Dur ing the 2 years of these experiments, humancorticotrophin-releasing hormone became available .Since both ovine corticotrophin-releasing hormoneand human cor ticotrophin-releasing hormone havesimilar biologie action ,? our laboratory switched ex­clus ively to human cor ticotrophin-releasing hormone.Thus this latter neuropeptide was used in our ex­periments in women undergoing ovariectomy. Eachwoman undergoing ovariectomy received a subcuta­neous 25 mg estradiol (E2) pellet for estrogen replace­me nt. This dos e of E2 maintains constant E2 levels inthe physiologie range (80 to 120 pg/rn l)." Tw o to 3months after E2 replacement, provocative testing withhuman cor ticotrophin-rcleas ing hormone ( I /-tg/kg, in­travenous bolus) was performed. Medroxyp rogester­one acetate, 10 mg by mouth, was th en ad ministe re dfor 10 da ys. On the ten th day of medroxyproges teroneacetate ad ministration, provocative tes ting with humancorticotrophin-releasing hormone was repeated. Dur­ing these experiments no discernible side effects wer enoted. This project was appro ved by the Human Sub-

Dece mber 1987Am J Obstet Gynecol

jects Committee of the University of California ,San Diego .

Synthetic ovine corticotrophin-releasing ho rmoneand human corticotrophin-releasing hormone wereprepared in an identical fashion. Each peptide was dis­solved in 5% mannitol (w/v) with 10-1mo1lL of ascorbicacid and ste rile water at a final concent ration of 200/-tg/ml. The solution was then sterilized by milliporefiltration, lyophilized , and stored in siliconized vials at- 700 C. Cort icotro pin-releasing factor was reconsti­tuted with sterile water immediatel y before each ex­periment." Blood sampies for ACTH were collected in2 ml polypropylene tubes containing ethylenediamine­tetraacetic acid (Sarsted t, West Ge rmany) and promptlycentrifuged at 40 C. Plasma was snap frozen in dryice/ethanol within 10 minutes after collecti on andstored at -700 C until assayed . Concomitant serumsam pies were obtained for luteinizing horrnone (LH),follide-stimulating hormone (FSH), E2 , progesterone4, and cortisol measurements.

ACTH was deterrnined in unextracted plasma by ra­dioimmunoassa y in duplicate. !" This assay uses high.pe rformance liquid chromatography purified iod ine125-ACT H ,_," tracer (Rad ioassay Systems Laboratory,In c., Carson, Ca lif.) and a commercially avai lable anti­serum from the IgG Corp. (Nashville, Tennessee). Se­rum cortisol , LH , FSH , E2 , and progesterone concen­trations were measured by previously established ra­dioirnmunoassays." 11 All sampIes from each subj eetwere measured in a single assay. The intraassay coef­ficient of variation for ACTH was 2.4 % to 5.8% at stan­dard doses between 3.3 and 34.6 pg/ml. The range ofintraassay coefficient of variation for cortisol was2.8% ro 6.2% between standard doses of 40.3 an d 394ng/m l. In terassay coefficients of vari an on were 11.8%and 6.5% for ACTH and cor tisol, respectively.

ACTH and cortisol responses were analyzed by ath ree-factor analysis of var iance with Newman-Keulstesting to examine for potential differences in re­sponses to cort ieotro phin-releasing hormone for eaehphase of the menstrual cycle . The integrated responseeurves were ealculated by the trapezoid method andanalyzed by the Student t test. All data are expressedas mean ± SE.

Results

Mean basal levels of LH, FSH, E2 , and progester­one during eaeh experiment are sumrnarized in T a­ble I. The LH, FSH , Sb and progesteron e concentra­tions during eaeh ph ase of the men strual eyde areeon sistent with previously reported valu es." In womenundergoin g ovariectomies, LH and FSH levels wereelevated in eompar ison with normal wornen, althoughE2 eoneentrations were maintained in the physiologie

Volurne 157Nurnber 6

Pituitary responses to synthetic corticotropin-releasing hormone 1389

o L..' --,1---,---,-_,---,---,-_",,---,---'--'

200

4800

4000>

3200 ~>

2400c:

1600 ~

'"800

0SAL EF ML

30000

24000>..1 8000~

12000i::;'"6000

0SAL EF LF ML40 sO 80 u;O 120 l~O 180 uio

MIHUTES

30

o . j ,-20 0 20

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....100CI:oo

Fig. 1. Left, Mean ( ± SEM) ACTH and eortisol coneen trat ions after the admin istration of salinesolution 0 1' ovine corticotrophin-releasing hormone (l fJ.g/kg, intravenous bolus) in six women d uringthe earl y follieular, late follieular, an d rnidlu teal phases of the menstrual cycle. Riglu, l ntegratedACTH and cortisol responses ( ::!:: SEM) to ovine corticotrophin-releasing ho rmone (l fJ.g/kg , int ra­veno usly) du ring the menstrual cycle.

~

30 [

'" E2 ........,20

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20 0

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20 00

1600 >..1200 '">

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Fig. 2. Left, Mean ( ::!:: SEM) ACTH and cortisol concernrations after hu man cort icou-ophin- releasinghorrn one adm inistration (I fJ.g/kg, intravenous bolus) in four women un dergoing ovariectomy re­ceiving subeutaneous E2 pellets 01' E. pellets and medroxyprogesterone acerate. Right, ln tegr atedACT H an d cortisol responses (± SEM) 1O hu man corti cotrophin -re leasing hormone (I fJ.g/kg, in­travenously) d uring E1 an d E. with med rox yprogesterone acetate.

ran ge. Despite the vari at ions in E. and progestin levelsdu ring eac h ex peri ment, basa l ACT H and cortisol con­cen tra tions were not significantly d ifferent withi n eachgro up . Althoug h basal ACTH levels were not d iffe re ntbetween the two groups, basal cortisol levels were ele­vated (p < 0.0 I) in wome n undergoing ovariectomy re ­ceiving E2 an d med roxyprogesterone ace ta te whencom pared with normal women in the late follicula rphase.

During saline solu tion administ ration in normalwornen , ACTH levels remained stable while cortisol

concentrations exhibited its characteristic midm orn­ing ded ine ( Fig. 1). T he ad minis tra tion of ovinecorti cotrophin-releas ing ho rmone elicited a promptrelease of ACT H that reached a peak inc rement at 40minutes . Peak ACTH levels were 27.3 :i:: 4.2, 26 .3 :i::

4.4 , an d 20 .7 :i:: 1.0 pg/ml for the early follicula r, latefollicular, an d midluteal ph ases, respectively. ACTHlevels then declined but remained elevated above base­line levels during the next 2 hours. Analysis of theACT H response curves and thc integrated re sponse foreach phase of the menstrual cyde did no t demoristrate

1390 Liu et al.

Table I. Mean ( ± SEM) basal hormone levels in women with normal cyd es and ovariectomies

December 1987Am J Obstet Gynccol

Normal toomen (n = 6) Ova riectomized tuomen (n = 4)

EF I LF I ML E 2 pellet I Ez + MPA

ACTH (pg/ml ) 13.3 :t 2.7 11.7 :t 2.9 11.5 ::!: 1.5 7.0 ::!: 0.5 7.7 :t 0.8Cort isol (ng/ml) 80 :t 14 70 ::!: 7 89 :t 14 8 1 :t 13 121 :t 10*LH (mIU/ml) 10.3 ::!: 0.5 19.7 ::!: 4.5 6.7 ::!: 0.8 69.6 :t 35.5 38:!: 16.3FSH (mIU/ml) 16.4 ::!: 0.9 15.9 :t 2 8.4 :t 0.7 45.3 :t 22.5 41 ::!: 19.5E. (pg/m l) 48 ::!: 7 265 :t 82 223 :t 83 139 :t 48 132 :t 40Progesterone (ng/ ml) 0.4 ::!: 0.1 1.2 ::!: 0.3 12.1 ::!: 2.6

EF = Early follicular ph ase; LF = late follicu lar ph ase; ML = midlu tea l phase: MPA = medroxyproges te ro ne acetate.*p < 0.01 ver sus late follicular phas e.

significant differences. Cortisol responses to ovinecorticotrophin-releasing hormone during each ph aseof the cyde were parallel to ACTH, with peak incre­ments observed at 80 minutes ( Fig. I ). No significantdi fferences in either pe ak 0 1' integrateel cortiso l re­sponses were evident between d ifferent phases o f thcmenstrual cyd e .

In women undergoing ovariectomy re ceiving E. re­placement, human cort icotro phi n- releas ing hormoneadministration elic ited a more rapid pituitary releaseof ACTH and achieved peak responses (19 .9 ± 2.1pg/ml) by 20 minutes ( Fig. 2). However, the dura­tion of ACTH release a fter ad mi nistration of humancorticotrophin-releasing hormone was much sho rterthan that of ovine corticotrophin-re leasi ng horrnone,with ACTH leve ls returning to basal level s by 100 min­utes. These differences are compatible with previousreports' anel probably reflect the Ionger half-life ofovine corticotrophin-releasing horrnone '" rather thanintrinsic differences in the pituitary-adrenal ax is be­tween the two gro ups. Serum cortisol concentrationsincreaseel 10 to 20 minutes after human corticotro phin­releasing hormone anel were parallel to ACTH ( Fig . 2).The combination of E2 and meelroxyprogesterone ac­etate treatrnent did not affect ACTH and cortisol re­sponses to human cor ticotrophin-releasing hormone.

Comment

1'0 demonstrate the potential modulatory effectsof gon adal steroids, we selected a dose of ovinecorticotrophin-relea sing hormone (I f.Lg/kg), whichrepresented a half-maximal stimulus." Similarly,the dose of human corticotrophin-rele asing hormone(l f.Lg/kg) used in this study has been repo rted to be

. submax imal." Under th ese experimental cond itions ,we have dcmonstrated that pituitary and ad re na l re­sponses to ovine con icotrophin-releasing hormonein women with normal cyd es were not altered byphysiologic fluctuations in ovarian steroid concentra­tions during different phases of th e menstrual cycle .In addition, ACTH a nd cortisol re sp onses to hu-

man cortico trophin-re leasing hormo ne women hav­ing ovariec to my were sirnilar during E. 01' E2 withmedrox yprogesterone acet a te administr ation . 1'0 th ebest of our knowledge , thi s is th e first study th at hasexamined the poten tial modulatory ro le of go na d­al ste roi ds o n pitu itary and aclre nal responses tocort icotrophin-releas ing ho rmo ne.

Basal ACTH and cortisol levels during each ph ase ofthe menstrual cyde werc also not sign ificantly different .These results are in ag reemen t with Au bert et al. ,11 bu tare at variance with findings re ported by Genazzan iet al ., who identified midcyde elevation s in ACTH andcortisol during daily morning determinations. SinceACTH and cortisol both elisplay acce nt ua ted episoelicsecretion during the morning ho urs," th is pul satile pat­tern could account fo r the re po r ted di fferences in basalhormone levels in th ese stud ies and the increased cor ­tisollevels observecl in women undergoing ovariectomyreceiving E? and medrox yprogesterone aceta te in ourstudy.

Our present results differ from previous reports inwhich pharmacologic ad min istratio n of estrogens a nelprogestins appeared to exe rt opposite influences onthe pituitary-adrenal axis. Estrogen administration hasbeen reported to increase peripheral cort isol levels,"which probabl y refl ects in pan a n estrogen-med iatedincrease in the hepatic produetion of corticoste roid­binding .globulin .'7 In con trast, proge stins appear to

suppress the pituitary-adrenal axis by vir tue of itsweak glucocorticoid properties. " Both glucocorticoidand progesterone receptor complexes have been re­por ted to share binding sites to the same regulatoryregion of deoxyribonuc!eic acid." When ad ministeredin pharmacologic doses, progestins effcctively inhibitcorticot rophin-releasing hormone-mediated ACT Hrelease in pituitary ceIl cu ltu res;" in hibit ACTH syn­thesis and release, and can cause ad rena l atrophy! ' Inour present study, however, the ad mi nistra tion ofsmaller replacement doses of LI and medroxyproges­terone acetate in wom en undergoing ovariectomy hadrelatively little effect on basal ACTH and cortisol le-

Volume 157Number 6

Pituitary responses to synthetic corticotropin-reJeasing hormone 1391

vels or pituitary responses to corticotrophin-reieasinghormone.

In sumrnary, a comparison of the pituitary andadrenal activity under different gonadal steroid en­vironments in women failed to demonstrate signifi­cant effects during basal or corticotrophin-reieasinghormone-stimulated conditions. In view of recent re­ports of alte red responses to corticotrophin-releasinghormone in various depressive disorders," these find­ings would indirectly support the notion that variationsin psychologie behavior and stress responses premen­strually reflect changes mediated primarily at a su­prapituitary level.

We are indebted to the nurses of the Clinical Re­search Center for their expert assistance in thesestud­ies, and the excellent technical assistance of CarlaLa Porte and Gail Laughlin.

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2. Reid R, Yen SSC. Prernenstrual syndrorne. AM] OBSTfTGYNECOL 1981;139:85.

3. Axelrod J,Reisine TD. Stress hormones: their interactionand regulation. Seienee 1984;224:452.

4. Genazzani AR, Lemarehand-Beraud TH, Auburt ML,Felber JP. Pattern of plasma ACTH, hGH, and corti­sol during the menstrual cycle. Clin Endoerinol Metab1975;41 :431.

5. Hellman L, Yoshida K, Barnett Z, et aI. The effeetof medroxyprogesterone on the pituitary adrenal axis.J Clin Endocrinol Metab 1976;42:912.

6. Marinari KT, Leshner AI, Doyle MP. Menstrual cycle sta­tus and adrenoeortieal reaetivity to psyehologieal stress.Psyehoneuroendoerinology 1976; 1:213.

7. Sehuermeyer TA, Tomai TP, Gold PW, et aI. Humaneortieotropin-releasing faetor in man: pharmaeokinetieproperties and dose-response of plasma adrenocortico­tropin and eortisol seeretion. J Clin Endocrmol Metab1984;59: 1103.

8. Liu j H, Yen SSC. Induetion of mideycle gonadotropinsurge by ovarian steroids in women: a eritieal evaluation.J Clin Endocrinol Metab 1983;57:797.

9. Liu JH, Muse K, Contreras P, et al. Augmentation ofACTH-releasing activity of synthetie eortieotropin releas-

ing factor (CRF) by vasopressin in women. j Clin Endo­erinol Metab 1983;57:108.

10. Nieholson WE, Davis DR, SherrelJ B], Orth DN. Rapidradioimmunoassay for corticotropin in unextraeted hu­man plasma. Clin Chem 1984;30:259.

1L Yen SSC, Martin PL, Burnier AM, et aI. Cireulating es­tradiol, estrone, and gonadotropin levels following theadministration of orally aetive 17 beta-estradiol in post­menopausal women. J Clin Endoerinol Metab 1975;40:518.

12. Ross GT, Cargille CM, Lipsett MG, et aI. Pituitaryarid gonadal hormones in women during spontaneousand indueed ovulatory cycles. Reeent Prog Horm Res1970;26: I.

13. Orth D, Jaekson R, Deeherney G, et aI. Effeet of syntheticovine eortieotropin-releasing factor: dose reponse ofplasma adrenocorticotropin funetion in man.j CIin Invest1983;71:587. .

14. Aubert ML, Lemarehand-Beraud T, Degullaume R, De­saulIes PA. Cortisol secretion du ring the normal menstrualcycle. Acta EndoerinoI (Copenh) 1971;155:78.

15. LiuJH, Kazer R, Rasmussen DD. Charaeterization ofthe24 hour seeretion pattern ofadrenocorticotropin and cor­tisol in normal women and patients with Cushing's dis­ease. J CEn Endoerinol Metab 1987;64: 1027.

16. Peterson RE, Nokes G, Chen PS, Blaek RL. Estrogensand adrenocortieal funetion in man. J Clin EndoreinolMetab 1960;20:495.

17. Sandberg AA, Slaunwhite WR. Transeortin: a eortieoste­roid binding prorein of plasma. 11. Levels in various con­ditions and the effects of estrogens. J Clin Invest1959;38: 1290.

18. Mathews JH, Abrams CAL, Morishima A. Pituitary­adrenal funetion in ten patients reeeiving medroxypro­gesterone acerate for true preeoeious puberty. J Clin En­docrinol Metab 1970;30:653.

19. Dietmar Von Der A, Janich S, Scheidereit C, et aI. Glu­eoeortoid andprogesterone reeeptors bind lO the samesires in two hormonally regulated promoters. Nature1985;313:706.

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