Hypothalamic-pituitary-ovarian response to clomiphene citrate

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1 r FERTILITY AND STERILITY Vl. 59, N.3, March 1993 Cpyright (') 1993 The American Fertility Sciety Printed n acid-free paper in U.S.A. Hypthalamic-pituitary-varian respnse t clmiphene citrate in wmen with plycystic vary syndrme*t L. Michael Kettel, M.D.:j: Jseph F. Mrtla, M.D.1f Sctt J. Rseff, M.D. Samuel S. C. Yen, M.D., D.Sc.** Sarah L. Berga, M.D.II Department f Reprductive Medicine, University f Califrnia-San Dieg, La Jlla, Califrnia Objective: T examine the hypthalamic-pituitary sites f clmiphene citrate (CC) actin in wmen with plycystic varian syndrme (PCOS). Design: Prspective cntrlled trial. Patients, Participants: Seventeen wmen with PC OS and 9 nrmal-cycling wmen. Interventins: Subjects with PCOS received CC, 150 mg/d fr 5 days. Main Outcme Measures: Fllicle-stimulating hrmne (FSH) and luteinizing hrmne (LH) levels and LH pulse characteristics and their respnse t gnadtrpin-releasing hrmne (GnRH, 10 J.Lg) were examined befre and after 3 days f CC in PCOS subjects during a 12-hur frequent sampling study (n = 8). Daily urinary estrne glucurnide and pregnanedil glucurnide levels after CC were cmpared with cncentratins in nrmal-cycling wmen thrugh ne menstrual cycle. In anther nine PCOS subjects, pituitary and varian hrmnal cyclicity was mnitred by daily bld sampling. Results: Thirteen f 17 treated cycles were vulatry with nrmal luteal phases. In the vulatry cycles, serum LH, FSH, estradil (E 2), and estrne levels increased after CC. Luteinizing hrmne pulse frequency was unchanged, but LH pulse amplitude increased significantly after CC. Bth LH and FSH respnse t exgenus GnRH was significantly attenuated after CC treatment. In anvulatry cycles, serum LH, FSH, and E2 increased initially and then returned t baseline and remained unchanged fr the ensuing 40 days. Cnclusins: Clmiphene citrate-induced vulatin in wmen with PCOS is accmpanied by increased secretin f LH and FSH with enhanced estrgen secretin. The increased LH pulse amplitude after CC, tgether with decreased pituitary sensitivity t GnRH, suggests a hypthalamic effect. Fertil Steril 1993;59:532-8 Key Wrds: Clmiphene citrate, plycystic varian syndrme, vulatin inductin, LH pulses, hypthalamus, GnRH pulse generatr Received August 12, 1992; revised and accepted Nvember 5, * Supprted in part by Center grant HD frm the Natinal Institute f Child Health and Human Develpment and by General Clinical Research Center grant MOl frm the Natinal Institute f Health Divisin f Research Resurces, Bethesda, Maryland. The research was cnducted in part by Claytn Fundatin fr Research, Califrnia Divisin, La Jlla, Califrnia. t Presented in part at the 36th Annual Meeting f the Sciety fr Gyneclgic Investigatin, San Dieg, Califrnia, March 15 t 18, :j: Reprint requests: L. Michael Kettel, M.D., UCSD Medical Center (8433), 225 Dickinsn Street, San Dieg, Califrnia Kettel et ai. Clmiphene citrate in pca Clmiphene citrate (CC) is an estrgen agnistantagnist that is highly effective in initiating vulatin in wmen with plycystic varian syndrme (PCOS) and ther cnditins f chrnic anvulatin (1). Despite widespread use f CC during the past Present address: St. Barnabus Medical Center, Livingstn, New Jersey. II Present address: Department f Obstetrics and Gyneclgy, University f Pittsburgh, Magee-Wmen's Hspital, Pittsburgh, Pennsylvania. 1) Present address: Beth Israel Hspital, Bstn, Massachusetts. ** Claytn Fundatin Investigatr.

2 y' three decades, the mechanism f actin fr CC remains largely unknwn (2, 3). Previus studies in nrmal-cycling wmen perfrmed in the early fllicular phase (4) and luteal phase (5) f the menstrual cycle have identified a hypthalamic site f actin fr CC. In these studies, CC elicited an increase in luteinizing hrmne (LH) pulse frequency withut change in LH pulse amplitude. Althugh CC is ccasinally used in nrmal-cycling wmen, its primary clinical utility is in the anvulatry wman. Earlier studies in wmen with PCOS have described the vulatry pattern f gnadtrpin and varian sterid hrmnes in respnse t CC (6, 7), but assessments f hypthalamic-pituitary sites f actin have nt been made. Because wmen with PCOS exhibit chrnically high LH pulse frequency and amplitude (8-10), we reasned that the gnadtrpin-releasing hrmne (GnRH) pulse generatr may be perating at maximum capacity and that further acceleratin f pulse frequency in respnse t CC was unlikely t ccur. T test this hypthesis, LH pulsatility was assessed by frequent sampling studies perfrmed in eight wmen (9 cycles) with PCOS befre and during the administratin f CC, 150 mg/d fr 5 days. In additin, urinary estrne-3a-glucurnide and pregnanedil-3a-glucurnide levels were determined thrughut treatment cycles t discern vulatry respnses. In nine ther wmen with PCOS, pituitary-varian functin after treatment with CC was mnitred by daily LH, fllicle-stimulating hrmne (FSH), estradil (E 2), and prgesterne (P) levels. Subjects MATERIALS AND METHODS Seventeen wmen, ages 18 t 33 years, with clinical and bichemical evidence f PCOS were enrlled in this study. Subject selectin criteria included the fllwing: [1] irregular menses since menarche, [2] LH/FSH rati> 2.5:1, [3] serum prlactin < 25 Jlg/L, [4] serum dehydrepiandrsterne sulfate (DHEAS) < 11 nml/l and 17a-hydrxyprgesterne (17-0HP) < 6 nml/l r a nrmal adrencrtictrpic hrmne stimulatin test, and [5] hyperandrgenism with hirsutism and/r acne. Nne f the subjects had taken hrmnal medicatins r experienced vaginal bleeding fr 3 mnths befre the study. This prject was apprved by the Human Subjects Cmmittee f the University f Califrnia San Dieg and infrmed cnsent was btained frm each subject befre enrllment. Nine nrmal-cycling wmen between ages 18 and 32 years served as cntrls fr urinary sterid analysis. These wmen were regularly menstruating and vulatin was cnfirmed in a previus cycle by detectin f a midcycle LH surge using a urinary LH assay (Ovustick; Mnclnal Antibdies, Inc., Muntain View, CA). All cycling wmen were within 10% f ideal bdy weight and had nt used any hrmnal cntraceptin fr at least 6 mnths befre the study. Frequent Sampling Study Design (Grup 1) Eight wmen with PCOS were enrlled in this study. Each wman was admitted t the General Clinical Research Center (CRC) f the University f Califrnia-San Dieg Medical Center and studied befre and after treatment with CC. In this way, each subject served as her wn cntrl. Thrugh an indwelling intravenus catheter placed 1 hur befre the initiatin fthe study, bld was sampled at 10- minute intervals fr 10 hurs beginning at 8:00 A.M. At 6:00 P.M., a blus f GnRH (10 Jlg IV) was given and bld was sampled fr an additinal 2 hurs. After the baseline study, the wmen were instructed t take 150 mg f CC that evening and t cntinue t take the same dse each evening fr 5 days. They were readmitted t the CRC n the mrning after the third dse f CC fr a repeat identical study. Serum LH determinatins were made at each 10- minute sample, and serum FSH cncentratins were measured hurly. Serum E 2, estrne (E 1), teststerne (T), andrstenedine (A), P, and DHEAS cncentratins were determined n each study day at 8:00 A.M. Each subject cllected daily first-vid urine samples beginning 3 days befre the baseline study and cntinued until either a spntaneus menses ccurred r 40 days had elapsed. The nrmal-cycling wmen als cllected daily first-vid urine samples beginning n cycle day 1 and cntinued until menses ccurred. Daily urine samples were analyzed fr estrne glucurnide and pregnanedil glucurnide and were nrmalized t creatinine excretin. Daily Bld Sample Study Design (Grup 2) Nine wmen with PCOS were enrlled int this study. Each wman was given CC, 150 mg/d fr 5 days. Bld samples were btained daily beginning 3 days befre treatment and cntinuing until either a spntaneus menstrual cycle ccurred r 40 days had elapsed. Serum LH, FSH, E 2, and P cncentratins were determined in each sample. Vl. 59, N.3, March 1993 Kettel et al. Clmiphene citrate in pca 533

3 r Hrmne Assays Hrmne cncentratins were determined by previusly described radiimmunassay. All samples frm a given individual were measured in the same assay. At 50% binding, the intra-assay and interassay cefficient f variatin, respectively, fr each fthese hrmne cncentratins was as fllws: LH, 4.0% and 6.4%; FSH, 1.5% and 6.5%; E2, 8.0% and 12%; E 1, 9.5% and 19.4%; T, 5.8% and 9.4%; A, 6.7% and 9.7%; P, 5.9% and 9.2%; 17-0HP, 6.0% and 8.7%; DHEAS, 5.2% and 10%; estrne glucurnide, 6.5% and 8.4%; and pregnanedil glucurnide, 10.3% and 12%. LH Pulse Analysis Significant LH pulses were identified using the Cluster cmputer pulse detectin algrithm (11). A cluster cnfiguratin f 2 X 1 (2 values fr a nadir and 1 fr a peak) with a t-statistic f 2.1 fr bth upstrke and dwnstrke was chsen t limit the detectin f false-psitive and false-negative pulses t <5 %. Because the variance f a hrmne determinatin is a curvelinear functin fr the hrmne level, a secnd degree plynmial regressin analysis was perfrmed at tw ranges relating the SD t the hrmne cncentratin. This estimate f measurement errr was emplyed within the Cluster prgram t assess the significance f hrmne cncentratin changes. The resulting secnd degree cefficient, first degree cefficient, and intercept fr LH values between 0 and 70 lull were , , and , respectively, and fr LH values between 50 and 300 lull were , , and 1.53, respectively. Missing values were analyzed as the mean f the value immediately befre and after the missing data pint. The LH pulse frequency was defined as the number f pulses per unit f bservatin time (10 hurs). The pulse amplitude was defined as the difference in hrmne level between the cluster peak and the preceding nadir. Fr each individual the pulse amplitude used in analysis represents the mean f pulse amplitudes fr the 10-hur series. Statistical Analysis Statistically significant differences in mean hrmne cncentratins, LH pulse frequency, and LH pulse amplitude were assessed by paired t-testing. Differences in LH and FSH respnse t blus GnRH were assessed by paired t-testing n the integrated area under the respnse curve (AUC), calculated using the trapezid methd. Fr the subjects wh cllected daily urine samples, the benning f the luteal 534 Kettel et al. Clmiphene citrate in pca phase was identified using analysis f the urinary pregnanedil glucurnide curve. In this way, the mean ± SD f the early fllicular and midfllicular phase was determined, and the first pint where a significant increase in pregnanedil glucurnide ccurred was identified by a ne-tailed t-test. This pint has been shwn t crrespnd t the day f LH surge ± 1 day. Differences between grups in urinary estrne glucurnide and pregnanedil glucurnide curves thrughut the menstrual cycle were assessed by grup t-testing n the integrated AUC. In all analysis, P < 0.05 was cnsidered significant. RESULTS As determined by a sustained rise in serum P cncentratins' six f nine wmen in treatment grup 2 had an vulatry respnse t CC and three wmen failed t respnd. N differences in baseline LH, FSH, E2, El> T, A, r DHEAS were identified between grups. Hwever, bdy mass index was greater in the anvulatry grup (32.2 versus 24.8). In the vulatry grup, serum FSH increased (10.7 ± 1.6 t 15.4 ± 1.3 lull [mean ± SD], P < 0.01) during CC treatment with peak levels f FSH ccurring n treatment day 4 and declining thereafter until the midcycle surge. Crrespnding increases in serum E2 ccurred with nrmal midcycle and luteal phase hrmnal patterns (Fig. 1A). In the anvulatry grup, a similar rise in FSH ccurred during treatment (12.5 ± 1.7 t 16.0 ± 1.7 lull, P < 0.05, netailed) with an initial rise in serum E2 that persisted fr 3 days after treatment (Fig. 1B). Levels f all hrmnes then returned t baseline and remained cnstant thrughut the 40-day sampling interval. As determined by a sustained rise in urinary pregnanedil glucurnide, eight f nine cycles initiated with CC in grup 1 were vulatry. Using the first day f CC treatment as a reference pint, the average vulatry cycle length was 27.4 days (range, 25 t 29). The first significant rise in daily urinary pregnanedil glucurnide served as an indicatr f vulatin. The fllicular phase length f the CC-induced cycles was 12.5 ± 0.8 days and the luteal phase length was 14.8 ± 0.8 days. Cmparisns t cycles in nine untreated nrmal-cycling wmen reveal n difference in ttal cycle length, fllicular phase length, r luteal phase length. Ttal estrne glucurnide excretin, measured as the AUC, was significantly increased in the CC treated grup (P < 0.05), whereas pregnanedil glucurnide excretin was similar (Fig. 2).

4 yak 200 A B LH...J J 2 Figure 1 (A), Mean ± SE serum LH, FSH, E 2, and P levels in six vulatry cycles after CC, 150 mg/d fr 5 days, in wmen with PCDS. (B), Mean ± SE serum LH, FSH, E 2, and P levels in three anvulatry cycles after CC, 150 mg/d fr 5 days, in wmen with PCDS ::! w DAYS DAYS FROM LH PEAK DAYS "TJ ::> 3 r Analysis f the frequent sampling studies revealed the fllwing changes: there was a significant increase in transverse mean LH (37.6 ± 6.1 t 49.2 ± 8.1 lull, P < 0.01) and FSH (11.2 ± 1.1 t 12.8 ± 1.6 IU /L, P < 0.05) after 3 days f CC treatment. 100 NC...!5 PCO-<l CI 50 CI c: W DAYS Figure 2 Mean ± SE urinary estrne glucurnide (E1G) and pregnanedil glucurnide (PdG) levels in eight vulatry cycles in subjects with PCDS after treatment with CC and nine nrmalcycling wmen (NC). Data are nrmalized t urinary creatinine (CR) and centered arund the 1st day f (PdG) rise. LH pulse frequency was unaltered (9.8 ± 0.6 versus 9.0 ± 0.6 pulses/10 hurs) after CC. Hwever, there was a significant increase in mean LH pulse amplitude (11.6 ± 2.0 t 14.5 ± 2.0 lull, P < 0.05) (Fig. 3). T ascertain whether changes in LH pulsatility were secndary t an effect n the hypthalamus r pituitary, a blus f GnRH was given at the cnclusin f each frequent sampling study. There was a blunted LH and FSH respnse t GnRH after CC (Fig. 4). This was assciated with an attenuated maximal increment f bth LH (200.0 ± 92.7 versus ± 59.7 lull, P < 0.01) and FSH (9.0 ± 2.1 versus 6.4 ± 1.5 IU /L, P < 0.01) t GnRH after CC. DISCUSSION In this study, we describe changes in serum LH, FSH, E 2, and P cncentratins and LH pulsatile activity in wmen with PCOS after the administratin f CC. We were successful in achieving vulatin in 14 f 17 cycles. This success rate is cnsistent with published data (1, 12). The maximal dse f CC apprved by the Fd and Drug Administratin fr inductin f vulatin is 100 mg/ d fr 5 days. Hwever, there is widespread clinical experience with dses up t 250 mg/d fr 5 days. We chse a dse f 150 mg/d t induce vulatry cycles in as many subjects as pssible. Als, previusly published Vl. 59, N.3, March 1993 Kettel et al. Clmiphene citrate in pca 535

5 r :! pt6 * Befre CC After CC J:..J pt3 * 50 L! L- L- L L- L--L! CLOCK HOURS Figure 3 Serum LH cncentratins in tw PCOS subjects measured every 10 minutes fr 10 hurs befre and after CC treatment. Asterisks indicate LH pulses. studies explring the hypthalamic-pituitary actins f CC have used 150 mg/d and t cmpare gnadtrpin respnses, the same dse was used in the present study (4, 5). In the vulatry cycles induced with CC, the ttal estrne glucurnide excretin was significantly greater than in the cycles f nrmal wmen. This may reflect greater baseline El secretin in PCOS(13) and/r greater serum estrgen levels secndary t the multifllicular develpment frequently seen after CC-induced cycles. Interestingly, bth the vulatry and anvulatry cycles were assciated with increases in serum FSH. In the vulatry respnders, CC treatment was fllwed by increasing serum E2, decreasing levels f FSH and LH, and nrmal vulatry events in the midcycle and luteal phase. Hwever, in the anvulatry subjects, the changes in serum FSH, LH, and E2 were shrt lived and reverted t baseline after 3 days with n further changes in hrmne levels nted. Similar findings have been reprted previ- 0usly (12). One pssible explanatin fr the different respnses t CC is a preferential effect n the secretin f biactive FSH. It is pssible that CC induces a preferential increase in biactive serum FSH in cycles that ultimately result in vulatin. In men, CC, 100 mg/d fr 7 days, induced an increase in biactive FSH (14). Alternatively, ther factrs critical in the prcess f flliculgenesis such as insulin-like grwth factr (IGF) I and II, inhibin, activin, and the IGF -binding prteins may be invlved. 536 Kettel et ai. Clmiphene citrate in pca After 3 days f CC treatment, there was a significant increase in LH pulse amplitude withut a change in LH pulse frequency. Additinally, the LH and FSH respnse t exgenus GnRH after CC was significantly blunted. Increases in LH pulse amplitude may be explained by either a direct effect n pituitary gnadtrpe sensitivity t GnRH r t an increase in GnRH release at the time f each secretry burst with a subsequent increase in the incremental respnse f LH. If CC had increased pituitary gnadtrpe sensitivity, the respnse f LH and FSH t exgenus GnRH wuld have been augmented. In ur study, the increased LH pulse amplitude after CC, tgether with decreased pituitary sensitivity t GnRH, suggests an actin f CC at the hypthalamic level. This prpsitin is supprted by the ability f CC t enhance GnRH release frm medial basal hypthajamus in vitr (15) and the demnstratin f a significant dse-dependent respnse f LH release t exgenus GnRH in viv (16,17). We and thers have previusly investigated the hypthalamic-pituitary sites f CC actins in nrmal-cycling wmen. Early studies f the effects f CC n LH pulsatility were limited by shrt-sampling intervals (4 t 6 hurs) but reprted an increase in bth mean LH and LH pulse amplitude (18). Mre recent investigatins in nrmal wmen have examined LH pulsatile release using 1O-minute sampling frequency ver an extended perid and dem-

6 200 ;;;! J LH Befre CC'-" After CC MINUTES Figure 4 Abslute change ± SE frm baseline in serum LH and FSH respnse t GnRH (10 I'g IV) befre and after CC treatment. nstrated an increase in LH pulse frequency withut changes in amplitude (4, 5). Interestingly, the increase in LH pulse frequency, which suggests a direct effect f CC n the hypthalamic GnRH pulse generatr, ccurred in bth the fllicular phase (4) and luteal phase (5) f spntaneus menstrual cycles, situatins with very different hrmnal milieu. The changes we have shwn in LH pulse amplitude were accmpanied by an increase in serum E2 and E 1 Estrgen has been shwn t mdulate pituitary respnsiveness t GnRH (19, 20). Hwever, it is unlikely that the effects f CC n LH pulse amplitude are secndary t varian sterid hrmne feedback because increases in LH pulse amplitude d nt ccur during the prgressin f the nrmal fllicular phase f the cycle when levels f estrgens are increasing (21, 22). Previus investigatins have examined the respnse f LH and FSH t exgenus GnRH after treatment with CC. In men (23), CC (100 mg/d fr 5 days) induced a blunted LH and FSH respnse t GnRH (150,."g IV), similar t the findings in ur study, and in nrmal-cycling wmen in the early fllicular phase, CC (150 mg/d fr 3 days) did nt change LH and FSH respnses t GnRH (10,."g IV) (4). Thus, ur present findings in PCOS patients further enhances ur hypthesis that CC has a direct effect n hypthalamic release f GnRH. As stated previusly, CC increased LH pulse frequency in nrmal-cycling wmen when given in bth the fllicular and luteal phases f the menstrual cycle (4, 5). The inability f CC t elicit an increase in LH pulse frequency in the present study may be because the GnRH pulse generatr is already maximally expressed in PCOS. This is indirectly supprted by several studies demnstrating a high frequency f LH pulses in PCOS (8-10). When LH pulse frequency in PCOS is cmpared with LH pulse frequency in wmen during different phases f the nrmal menstrual cycle, the menpause, r with premature varian failure (24), there is n reprt f any faster frequency than that reprted fr PCOS. Climiphene citrate is ne f few pharmaclgic agents that have been reprted t increase LH pulse frequency. Thus, the absence f an increase in LH pulse frequency in wmen with PCOS treated with CC prvides additinal evidence that the GnRH pulse generatr is maximally perative in this syndrme. The mechanism(s) by which CC mediates all f its effects t initiate vulatin in the anvulatry wman remains unclear. Predictin f vulatry respnses t CC has been difficult. In previus studies, nly bdy weight was fund t have predictive value fr an vulatry respnse (25), and we nted a similar trend in that the subjects wh failed t vulate had a higher bdy mass index. Althugh ur data suggest that CC induces an increase in GnRH secretin at the level f the hypthalamus, the pssibility remains that there are ther effects n the pituitary r vary nt addressed in this study. Our results must be interpreted with cautin. We have nt cmpleted a widespread dse-respnse study f the hypthalamic-pituitary actins f CC in PCOS. It is pssible that different dses f CC wuld result in different respnses. Additinally, ur frequent sampling interval was restricted t day 3 f CC treatment and the results may have differed if an alternative day were chsen. In summary, we have studied the neurendcrine effects f CC in anvulatry wmen with PCOS. After CC, there was a significant increase in LH, FSH, and LH pulse amplitude but n change in LH pulse frequency. Pituitary sensitivity t GnRH was'diminished after CC. The increased LH pulse ampli- Vl. 59, N.3, March 1993 Kettel et al. Clmiphene citrate in pea 537

7 r= tude, tgether with decreased pituitary sensitivity t GnRH, suggests a hypthalamic effect f CC. In additin, the failure f CC t increase LH pulse frequency is cnsistent with the hypthesis that the GnRH pulse generatr is maximally expressed in PCOS. Acknwledgments. The authrs are grateful t the fllwing individuals frm the University f Califrnia-San Dieg: the staff fthe General Clinical Research Center and Pam Malcm, R.N., fr excellent clinical assistance; Shannn Petze, B.A., and Jeff Wng, B.A., fr technical assistance; and Allen Lein, Ph.D., fr advice and supprt. REFERENCES 1. Hammnd MG. Mnitring techniques fr imprved pregnancy rates during clmiphene vulatin inductin. Fertil Steril 1984;42: Adashi EY. Clmiphene citrate: mechanism(s) and site(s) f actin-a hypthesis revisited. Fertil Steril1984;42: Clark JH, Markaverich BM. The agnist-antagnistic prperties f clmiphene: a review. Pharmacl Ther 1982;15: Kerin JF, Liu JH, Phillipu G, Yen SSC. Evidence fr a hypthalamic site f actin f clmiphene citrate in wmen. J Clin Endcrinl Metab 1985;61: Maraucic M, Casper RF. The effect f luteal phase estrgen antagnism n luteinizing hrmne pulsatility and luteal functin in wmen. J Clin Endcrinl Metab 1987;64: Yen SSC, Vela P, Ryan KJ. Effect f clmiphene citrate in plycystic vary syndrme: relatinship between serum gnadtrpin and crpus luteum frmatin. J Clin Endcrinl Metab 1970;31: Jacbsn A, Marshall JR, Rss GT, Cargille CM. Plasma gnadtrpins during clmiphene induced vulatry cycles. Am J Obstet Gynecl 1968;102: Kazer RR, Kessel B, Yen SSC. Circulating luteinizing hrmne pulse frequency in wmen with plycystic vary syndrme. J Clin Endcrinl Metab 1987;65: Waldstreicher J, Santr N, Hall JE, Filicri M, Crwley WF. Hyperfunctin f the hypthalamic-pituitary axis in wmen with plycystic varian disease: indirect evidence fr partial gnadtrph desensitizatin. J Clin Endcrinl Metab 1988;66: Burger CW, Krsen T, van Kessel H, van Dp PA, Carn FJM, Schemaker J. Pulsatile luteinizing hrmne patterns in the fllicular phase f the menstrual cycle, plycystic varian disease (PCOSD) and nn-pcosd secndary amenrrhea. J Clin Endcrinl Metab 1985;61: Veldhuis JD, Jhnsn ML. Cluster analysis: a simple, versatile, and rbust algrithm fr endcrine pulse detectin. Am J Physil 1986;250:E Plsn DW, Kiddy DS, Masn HD, Franks S. Inductin f vulatin with clmiphene citrate in wmen with plycystic vary syndrme: the difference between respnders and nnrespnders. Fertil Steril 1989;51: Yen SSC. The plycystic vary syndrme. Clin Endcrinl (Oxf) 1980;12: Tenver JS, Dahl KD, Hsueh AJW, Lim P, Matsumt AM, Bremner WJ. Serum biactive and immunactive flliclestimulating hrmne levels and the respnse t clmiphene in healthy yung and elderly men. J Clin Endcrinl Metab 1987;64: Miyake A, Tasaka T, Sakumt T, Kawamura Y, Nagahara Y, An T. Clmiphene citrate induces luteinizing hrmne release thrugh hypthalamic luteinizing hrmne-releasing hrmne in vitr. Acta Endcrinl (Cpenh) 1983;103: Rebar R, Yen SSC, Vandenberg G, Naftlin F, Ehara Y, Engblm S, et al. Gnadtrpin respnses t synthetic LRF: dse-respnse relatinship in men. J Clin Endcrinl Metab 1973;36: Veldhuis JD, O'Dea LSL, Jhnsn ML. The nature f gnadtrpin-releasing hrmne stimulus-luteinizing hrmne secretry respnse f human gnadtrphs in viv. J Clin Endcrinl Metab 1989;68: Vandenberg G, Yen SSC. Effect f anti-estrgenic actin f clmiphene during the menstrual cycle: evidence fr a change in the feedback sensitivity. J Clin Endcrinl Metab 1973;37: Tsai CC, Yen SSC. Acute effects f intravenus infusin f 17p-estradil n gnadtrpin release in pre- and pstmenpausal wmen. J Clin Endcrinl Metab 1971;32: Yen SSC, Vandenberg G, Siler TM. Mdulatin f pituitary respnsiveness t LRF by estrgen. J Clin Endcrinl Metab 1974;39: Reame N, Sauder SE, Kelch RP, Marshall JC. Pulsatile gnadtrpin secretin during the human menstrual cycle: evidence fr altered frequency f gnadtrpin-releasing hrmne secretin. J Clin Endcrinl Metab 1984;59: Filicri M, Santr N, Merriam GR, Crwley WF. Characterizatin f the physilgical pattern f episdic gnadtrpin secretin thrughut the human menstrual cycle. J Clin Endcrinl Metab 1986;62: Wang CF, Lasley BL, Yen SSC. The rle f estrgen in the mdulatin f pituitary sensitivity t LRF (luteinizing hrmne-releasing factr) in men. J Clin Endcrinl Metab 1975;41: Rssmanith WG, Liu CH, Laughlin GA, Mrtla JF, Suh BY, Yen SSC. Relative changes in LH pulsatility during the menstrual cycle: using data frm hypgnadal wmen as a reference pint. Clin Endcrinl (Oxf) 1990;32: Lb RA, Gysler M, March CM, Gebelsmann U, Mishell DR Jr. Clinical and labratry predictrs f clmiphene respnse. Fertil Steril1982;37: Kettel et ai. Clmiphene citrate, in pca