HIGH DOSES OF ESTROGENS DO NOT INTERFERE WITH THE OVULATION-INDUCING EFFECT OF CLOMIPHENE CITRATE

FERTILITY AND STERILITY Copyrght 1976 The Amercan Fertlty Socety Vol. 27, No.4, Aprl 1976 Prnted n U.S.A. HIGH DOSES OF ESTROGENS DO NOT INTERFERE WITH THE OVULATION-INDUCING EFFECT OF CLOMIPHENE CITRATE HANS-D. TAUBERT AND JEANNE S. E. DERICKS-TAN Abtelung fur Gynakologsclre Endokrnologe, Zentrum der Frauenhelkunde und Geburtshlfe, J. W. Goetlre Unverstat, Frankfurt am Man, Federal Republc of Germany A sequental regmen of 100 mg of clomphene ctrate/day (days 5 to 9) and 180 f.l-g of ethynyl estradol or 7.5 mg of conjugated estrogens (days 10 to 16 or 17) was gven to 29 nfertle patents. Eleven patents conceved. Four pregnances termnated as early abortons; the remag seven went to term. In four cases an ovaran hyperstmulaton syndrome developed. Seral measurements of lutenzng hormone (LH), follcle-stmulatng hormone (FSH), 17{3-estradol (E,), and progesterone (P) were carred out n four women takng clomphene, fve beng treated wth clomphene and ethynyl estradol, and four recevng clomphene and conjugated estrogens. In all ovulatory cycles, preovulatory E 2 peaks that were sgnfcantly hgher than those n normal cycles were noted. The hghest E 2 levels were found n women recevng only clomphene. The admnstraton of hgh doses of estrogens dd not affect the serum levels of FSH, LH, and P to any dscernble degree, and the length of the luteal phase was normal. In women who seem to ovulate but fal to conceve when treated wth clomphene ctrate [l-p-(,b-dethyl-amnoethoxy) phenyl-1,2-dphenyl-2-chloroethylene I, t s not uncommon to fnd pror to ovulaton a hghly vscous cervcal mucus exhbtng a weak ferg pattern and decreased spbarkhet.l The antestrogenc effect of clomphene ctrate (C) has been mplemented as the most lkely cause of ths phenomenon, whch may lead to decreased sperm penetraton nto the cervcal mucus and thus could contrbute to a certan extent to the stll relatvely low pregnancy rate obtaned wth ths compound. 1. 3 Estrol has been used as a means of counteractng ths undesrable sde effect of C therapy, but ts estrogenc potental does not always seem to suffce. Snce the applcaton of more potent estrogens could nterfere wth Receved September 11, 1975. 375 the ovulaton-nducng effect ofc, the effcacy of a sequental regmen of C followed by ethynyl estradol (EE2) or conjugated estrogens (CE) was studed. MATERIALS AND METHODS Patents. Twenty-e patents, twenty-fve wth prmary nfertlty and four wth secondary nfertlty, whose ages ranged from 29 to 40 years (mean age, 30.9) were studed. They were treated from the 5th through the 9th day of the cycle wth 100 mg of C/day. Thereafter, they receved three tmes daly ether 60 p.,g of EE2 or 2.5 mg of CE from day 10 through day 16 or 17. Seral measurements of follcle-stmulatng hormone (FSH), lutenzng hormone (LH), 17,B-estradol (E2), and progesterone (P) levels were performed throughout one cycle n fve of the women recevng C + EE2 and n four of the group recev-

376 TAUBERT AND DERICKS-TAN Aprl 1976 10 10 100 Conjugated estrogens 1000 10000 100000 test tube FIG. 1. Cross-reactvty of EE2 and CE wth an ant-17{3-estradol serum. ng C + CEo These results were compared wth those obtaned from four other women who had been treated wth C only. Serum LH, FSH, and E2 levels were also determned n another group of anovulatory women, begng 2 days before and endng 2 days after admnstraton of 100 mg of C/day for 5 days (Fg. 6). Altogether, E2 levels were determned daly n seven anovulatory women and compared wth results obtaned from eleven normal, ovulatory cycles (Fg. 5). Drugs. EE2 (Progynon C) was obtaned from Scherng A.G., Berln, Germany; CE (Presomen) was suppled by Kal Cheme, Haover, Germany; and C (Dynerc) was suppled by Merrel GmbH, Gro-Gerau, Germany. Hormone Assays. Serum LH and FSH levels were measured by sold-phase, double-antbody radommunoassay, wth the kt obtaned from CIS (CEA IRE-Sorn, Fleurus, Belgum). Human ptutary LH and FSH were used as standards. One mllgram of LH represented a bologc actvty of 3500 IV of Second Internatonal Reference Preparaton of human gonadotropns (2nd IRP) and a radommunologc actvty of 2800 IU of 68/39 Medcal Research Councl preparaton (MRC) (Mll Hll, England). One mllgram of FSH represented a bologc actvty of 00 IU of 2nd IRP and a radommunologc actvty of 2150 IU of 68/40 MRC. P and E2 were measured wth the radommunoassay kt (dextran-charcoal method) obtaned from CIS. Cross-Reacton ofee 2 and CE wth Ant E2 Serum. Inasmuch as the admnstraton of EE2 and CE could nterfere wth measurement of endogenous E2, varous amounts of EE2 and CE were ncubated wth 5 nc of 17,B-2,4,6,7-3 H-estradol (specfc actvty, 250 p.,c/p.,g; IDW, Sprendlngen, Germany) and an ant- 17,B-estradol serum. There was no crossreacton when 2,000 to 40,000 pg of EE2 were added/test tube (Fg. 1). The CE showed a 0.5% cross-reacton compared wth E2 n the dosage range of 1,000 to 100,000 pg/test tube. These data clearly showed that the measurement of endogenous E2 would not be notceably affected by the admnstraton of 180 p.,g of EE2 or 7.5 mg of CE/day. RESULTS Effect of EE 2 and CE on Ovulaton I nducton by C. FSH, LH, E, and P levels were measured n four women who had receved only 100 mg of C from days 5 through 9 of the cycle. In three of these women, a rather marked rse n serum E2, to maxmal values exceedng 700 pg/ml, was followed by mdcycle LH peaks of more than 30 ng/ml, and a rse n serum P above 10 ng/ml (Fg. 2). In the fourth patent (patent B. L.) ovulaton apparently faled to occur, although smlar rses n LH and E2 were observed. In contrast, n patent B. B. a relatvely low mdcycle LH peak was followed by P levels compatble wth the occurrence of ovulaton. In four of fve patents recevng the sequental regmen of C + EE2, LH peaks were observed that were wthn the nor-

Vol. 27, No.4 EFFECT OF ESTROGENS ON CLOMIPHENE EFFECT 377 C C C C 9 n MA n D.S. n S.L n SS 16 :r: Ul 7 LL. o 12 ;.. <>----""<\ : \ 1. bo I', 5! : : o, 'J 1\ E!\ o ' \ B ', \ '0 0 cr. 0-' I rp \ /'J\ c 3 :r: h I -' 4 10 15 5 10 15 10 15 C BOO C c E n n n 8.' 600 30 N UJ. E 400 cr. c tl, tt 0 1 : J P- o,. \\,. DDqooooqad, "0 a";--- ""'---<>-j-"""'..i, 5 10 15 5 10 15 5 10 15 FIG. 2, Effect of C on serum LH, FSH, E2, and P levels n four anovulatory women, The numbers on the abscssa represent days of the cycle. l -. mal lmts of our laboratory (17.0 ± 8.1 ng/ml; n = 17) (Fg. 3). The LH peaks were preceded n three cases by rses n E2 levels, to more than 0 pg/ml, and were followed by P levels consstent wth a normal luteal phase. In patents G. D. and K. B., the serum E2 level dd not ncrease to the same extent, and ovulaton obvously faled to occur, even though LH levels rose to 13.5 and 8.5 ng/ml, respectvely. It should be emphaszed that both women had rather hgh FSH levels throughout the perod of observaton, and the mdcycle peaks of FSH were hgher than those normally found (6.7 ± 2.2 ng/ml; n = 13). Two of four women treated wth C + CE also showed rses n serum E2 levels, to peak values n excess of 400 to 800 pg/ml, respectvely, on the 13th and 15th days of the treatment cycle; these rses were followed by LH peaks and corpus luteum formaton, as shown by an elevaton of serum P levels. The remaig two women apparently dd not ovulate even though a normal LH peak was found for patent C. S., whle n patent M. K. the LH level rose just to the lower lmt of the normal range. The latter patent had unusually hgh serum FSH levels, smlar to those found for the two women who faled to ovulate wth C + EE2 (Fg. 4). Effect of C on Serum E 2, LH, and FSH Levels. Snce serum E2 appeared to reach much hgher levels when ovulaton was nduced wth C, C + EE 2, or C + CE (Fgs. 2 to 4), compared wth those of the normal OVUlatory cycle, seral measurements of E2 obtaned from the 10 patents who apparently ovulated under C therapy were compared wth those of seven normal cycles. As s shown n Fgure 5, C therapy resulted n serum E2 levels sgnfcantly hgher than those measured n normal OVUlatory cycles. The

378 TAUBERT AND DERICKS-TAN Aprl 1976 E I <J) '; : G.L E. N W 800 I[) E Or c 600 30 e C; I[)Q 0 10 51015 5 10.15 25 C EE2 C EE2. '. MJ\, '0 q.,/ I, 51015 5 10 15 51015 5101525 FIG. 3. Effect of a sequental regmen of C + EE2 on serum LH, FSH, E2, and P levels n fve women. The numbers on the abscssa represent days of the cycle. dfference between the mean serum E2 levels (± standard devaton) of treatment cycles (upper lne) and the respectve values for normal cycles (lower lne) was sgnfcant by the Wlcoxon test at the C CE C CE t E U.H. AW C.S. en 9 c :I: 16 <J) u.. 7 12 5 E 8 :I:...J 4 3 800 40! -I -I -, :' 51015 5 C CE 5% level for the perod between day - 9 and day-1. Because these values were centered around the day of the LH peak, the relatonshp between the E2 rse and the C CE,, 5 10 15 C CE 912.5 C 1'- CE f!1n I,- 1, - - 1 1 1 1 1,, 1 1 'til I I f l' 'K I 51015 C CE M.K. 51015 5 10 15 51015 FIG. 4. Effect of a sequental regmen of C + CE on serum LH, FSH, E 2, and P levels n four nfertle patents. The numbers on the abscssa represent the days of the cycle.

Vol. 27, No.4 EFFECT OF ESTROGENS ON CLOMIPHENE EFFECT 379 Estradol pg/ml 600 4 0 LH ng/ml 7 6 5 4 3 2!CIOrrPhene 5xl00mg 1 + + + 0 2 4 6 8 m LH... Peak Day of cycle FIG. 5. Serum E2 levels n normal ovulatory cycles (.) (n = 7) and n clomphene-treated cycles (0) (n = 10). tme of C treatment was not readly apparent. Therefore serum E2 levels were determned n a larger number of women 2 days before, durng, and 2 days after C admnstraton. The women were treated as a sngle group rrespectve of whether they ovulated durng a partcular treatment cycle. However, all had experenced wthdrawal bleedng followng the admnstraton of a progestogen. The results are summarzed and correlated wth the rses n serum LH and FSH n Fgure 6. Day 0 represents the 4th day after the onset of the last vagnal bleedng. There was a hghly sgnfcant ncrease n serum LH levels, from 1.9 ± 1.0 ng/ml on day -1 to a maxmum of 4.0 ± 1.7 ng/ml on day 6 (r = 0.446 between day 0 and day 6, P < 0.01), and n E2 levels, from 48 pg/ ml on day - 1 to 294 pglml on day 5 (r = 0.6089, P < 0.01). FSH levels also rose sgnfcantly (r = 0.4156 between day 0 and day 4, P < 0.01), but decreased agan after havng reached a maxmum on day 4 of treatment (r = 0.4099 between day 4 and day 7,P < 0.01). Clncal Results. The effcacy of any method for treatng anovulaton should be judged not only by the ovulaton rate, but more so by the number of conceptons obtaned and pregnances carred to term. In vew of ths, patents FSH ng/ml 7 6 5 4 3 2 Estradol pg/ml 400 0 n" 8 15 19 19 22 18 16-1 0 23456 7 n " 8 14 19 19 18 21 19 17-1 0 2 3 4 5 6 7 days n " 9 12! Clomphene 5.100 mg 1 12 1 tt 15 13 r -1 0 2 3 4 5 6 7 days FIG. 6. Effect of C on serum E2, LH, and FSH levels n anovulatory women. The values represent means ± standard devaton. were treated durng 38 cycles wth C + EE2, and 9 durng 15 cycles wth C + CEo The dagnoss ofluteal phase nsuffcency was made accordng to defntons gven n a prevous report. 4 As judged by the rse n basal body temperature, ovulaton apparently occurred n 31 of 38 cycles after treatment wth C + EE2 and n 13 of 15 cycles after admnstraton of C + CEo The mean day of ovulaton n the frst group

380 TAUBERT AND DERICKS-TAN Aprl 1976 Dagnoss TABLE 1. Results of C + EE2 and C + CE Therapy n 29 Infertle Patents Polycystc ovares 2 0 Luteal phase nsuftlcency 19 5 Anovulaton 7 4 Post-pll amenorrhea 1 0 Total 29 9 No. Pregnances Abortons OverstmulatoDs of patents C+ EE, C + CE C+ EE, C+ CE C+ EE, C+ CE 0 0 0 1 0 2 1 1 2 0 0 2 0 0 1 0 0 0 0 0 2 3 1 3 1 was 19.9 ± 6.9, n the latter 17.0 ± 2.7. The length of the luteal phase was 12.7 ± 2.0 days. Ths dfference was not statstcally sgnfcant. The relatvely large standard devaton for cycles after treatment wth C + EE2 was manly due to delayed ovulaton n two cycles (days 37 and 38, respectvely). In 24 of 31 C + EE2 treatment cycles and n 11 of 13 C + CE cycles, however, ovulaton occurred before day 21. The postcotal test could be performed n only 12 women, 5 of whom showed mprovement under therapy. Pregnances. Eleven patents conceved (37.0%), e after treatment wth C + EE2, and two after recevng C + CEo In fve cases, concepton occurred durng the frst treatment cycle, n four cases durng the second, and n one case each durng the thrd and fourth cycles (Table 1). Four of the eleven pregnances termnated as abortons durng the frst 8 weeks of gestaton. In two of these, the dagnoss of pregnancy was based on the measurement of radommunoassayable choronc gonadotropn n the serum before the mssed menses, as the conventonal mmunochemcal pregnancy tests never became postve. The hstologc fndngs of the materal obtaned at curettage, however, were consstent wth the dagnoss of mnaborton. The remag seven patents delvered healthy chldren at term, ncludng one set of twns. Complcatons. In three patents recevng C + EE2 and n one patent treated wth C + CE, sgns of ovaran hyper- stmulaton developed durng the luteal phase. In one patent who conceved, an ovaran cyst of 8 cm dameter quckly regressed after 150 ml of serous flud had been wthdrawn by paracentess, and the pregnancy went to term. A rght adnexal mass consstng of two separate ovaran cysts was found n another patent durng the 7th week of pregnancy. The pregnancy termnated 1 week later n mscarrage. Ffteen days after the presumed date of ovulaton, a thrd patent developed a left ovaran cyst of 7 cm dameter and showed sgns of an acute abdomen. The dagnoss of pregnancy could not be establshed; the symptoms regressed wth bed rest. In the fourth patent, a luten cyst was found durng an emergency appendectomy (Table 1). DISCUSSION The admnstraton of 180 ILg of EE2 and 7.5 mg of CE/day for 6 or 7 days followng a course of 100 mg of C from day 5 to day 9 of the cycle was not found to nterfere wth the ovulaton-nducng effect of C. Ths s amply documented by the observaton that 11 of 29 nfertle patents conceved after these two sequental regmens and that longtudnal studes of the serum levels of LH, FSH, E2, and P revealed profles consstent wth ovulaton. The average day of ovulaton dd not occur later than commonly seen after treatment wth C alone,s and the duraton of the luteal phase dd not dffer from the norm of 12.7 ± 1.7 days reported by Dorng. 6. These results also corroborate an earler observaton

Vol. 27, No.4 EFFECT OF ESTROGENS ON CLOMIPHENE EFFECT 381 made by Roy et au showng that 100 p,g of EE2 dd not nhbt ovulaton n two women when gven n conjuncton wth C. Smlarly, Vatukats et ai.b reported that C abolshed the gonadotropn-suppressng effect of 100 p,g of EE2 gven to normally menstruatng women durng the early follcular phase. The dose of CE used n the present study would also be expected to have a suppressve effect snce the admnstraton of 7.5 mg/day for 14 days was found to mpede the release of LH and FSH by the ptutary n response to LH-releasng hormone. 9 There was lttle dfference n the cyclc patterns of LH, FSH, E2, and P n the fve women treated wth C + EE2 and the four women gven C + CE, compared wth those recevng only C. However, n the latter cases the serum E2 levels were hgher. In three of four women not showng a rse n P to luteal phase levels after treatment (Fgs. 2 to 4), serum E2 levels remaned below 0 pg/ml. Ths could be consdered normal for the preovulatory E2 peak n normal cycles (217 ± 125 pg/ml) but s much lower than values observed after C treatment (Fg. 5). Therefore, t must be assumed that the excessve rse n serum E2, whch has also been observed by Vandenberg and Yen, 10 s a prerequste for ovulaton after treatment wth C. As s shown n Fgure 6, ths rse begns concomtantly wth the ncrease n serum LH and FSH levels descrbed prevously by varous observers.n,13 A possble explanaton for the absence of an nhbtng effect of EE2 and CE upon C-nduced ovulaton could be derved from results reported by Vandenberg and Yen lo and Carglle et al. 14 Vandenberg and Yen lo showed that, n women wth normal ovulatory cycles, C nduces a sgnfcant rse n the crculatng LH level when gven durng the follcular phase but not when gven durng the luteal phase. The rse n LH was greater durng the late luteal phase than durng the early luteal phase; the reverse was found for FSH. Ths dsparty was beleved to be due to a preferental nhbton of FSH by E2.15 These observatons were nterpreted to mean that the postve feedback for E2 s ncreased by C durng the late follcular phase, whereas the negatve feedback s decreased. Carglle et al. 14 demonstrated that the reacton of plasma LH and FSH to the admnstraton of 100 p,g of EE2 depends to a great extent upon the tme of admnstraton. When EE2 was gven between cycle days 1 and 7, both LH and FSH were suppressed. Admnstraton between cycle days 9 and 18 of the cycle, however, resulted n a transent rse n plasma FSH levels followed by a declne, whle LH levels showed a sustaned elevaton, wth a maxmum near mdcycle. Ths type of reacton was not seen durng the admnstraton of EE2 and CE after C n the present study. The cyclc pattern of LH resembled closely that of a normal cycle, whle the pattern of FSH showed some erratc spkes, partcularly n women who faled to show normal P secreton (Fg. 4, patents C. S. and M. K.). As a consequence, t must be assumed that C ntates a change n the processes medatng the feedback mechansms at the ptutary or hypothalamc level, resultng n a consderable rse n the threshold for the negatve feedback. Even hgh doses of EE2, therefore, wll not nterfere wth the preovulatory surge of LH. It was not the prmary objectve of ths study to evaluate the effect of the sequental regmen on the qualty of the cervcal mucus and the sperm penetraton test. Only fve of twelve women showed mprovement, yet two of the patents who faled to show mprovement conceved. Conversely, Sek et al.16 reported that only two of eght patents recevng C and 50 p,g of qunestrol, and showng mprovement by the Huhner test, conceved. A much larger number

382 TAUBERT AND DERICKS-TAN Aprl 1976 of cases wll be requred to demonstrate conclusvely that the sequental regmen of C plus EE2 or CE brngs about an mprovement n the qualty of cervcal mucus that correlates wth the number of pregnances acheved. We conclude that even hgh doses of estrogens do not nterfere wth the ovulaton-nducng effect of C when t s gven n a sequental regmen, and that ths form of therapy mght be of use n certan cases of otherwse therapy-resstant nfertlty. Acknowledgments. We thank Dr. V. Insler for hs advce n ntatng ths study. The techncal assstance of Mrs. I. Kemma, Mrs. G. Langer Gerhards, Mrs. N. Dolack, and Mr. H. Sauer s gratefully acknowledged. REFERENCES 1. Graff M: Suppresson of cervcal mucus durng clomphene therapy. Fertl Sterl 22:9, 1971 2. Sharf M, Graff M, Kuzmnsky T: Combned therapy wth qunestrol and clomphene n functonal sterlty. Obstet Gynecol 37:260, 1971 3. Whtelaw MJ, Kalman CF, Grams LR: The sgnfcance of the hgh ovulaton rate versus the low pregnancy rate wth Clomd. Am J Obstet Gynecol107:865, 1970 4. Taubert H-D, Jrgensen 0, Becker H: Clncal observatons on luteal phase nadequacy. Bull Swss Acad Med Sc 25:586, 1969 5. Taubert H-D: De medkamentose Auslosung der Ovulaton mt Clomphen. Gynaekologe 1:139,1969 6. Dorng GK: Zahlen zur Physologe des Zyklus. Fortschr Med 84:694,1966 7. Roy S, Greenblatt RB, Mahesh VB, Jungck EC: Clomphene ctrate: further observatons on ts use n nducton of ovulaton n the human and on ts mode of acton. Fertl Sterl14:575, 1963 8. Vatukats J, Bermudez JA, Carglle CM, Lpsett MB, Ross GT: New evdence for an ant-estrogenc acton of clomphene ctrate n women. J Cln Endocrnol Metab 32:503, 1971 9. Taubert H-D, Dercks-Tan JSE: Unpublshed data 10. Vandenberg G, Yen SSC: Effect of ant-estrogenc acton of clomphene durng the menstrual cycle: evdence for a change n the feedback senstvty. J Cln Endocrnol Metab 37:356, 1973 11. Boon B, Schalch C, Lee DS, Rechln S, Parlow AF': Presented at the Aual Meetng of the Amercan College of Obstetrcs and Gynecology, Washngton DC, Aprl 17 to, 1967 12. Bardn CW, Ross GT, Lpsett MB: Ste of acton of clomphene ctrate n men: a study of the ptutary-leydg cell axs. J Cln Endocrnol Metab 27:1558, 1967 13. Jacobson A, Marshall JR, Ross GT, Carglle CM: Plasma gonadotropns durng clomphene nduced ovulatory cycles. Am J Obstet Gynecol 102:284, 1968 14. Carglle CM, Vatukats J, Bermudez JA, Ross GT: A dfferental effect of ethnyl estradol upon plasma FSH and LH relatng to tme of admnstraton n the menstrual cycle. J Cln Endocrnol Metab 36:87, 1973 15. Yen SSC, Tsa CC: The bphasc pattern n the feedback acton of ethnyl estradol on the release of ptutary FSH and LH. J Cln Endocrnol Metab 33:882, 1971 16. Sek M, Tajma C, Maed HR, Sek K, Yoshhara T: Effect of qunestrol admnstered wth clomphene ctrate on serum follcle-stmulatng-hormone and lutenzng-hormone and on other clncal fndngs. Am J Obstet Gynecol 116:388, 1973