menstrual cycle*†

Vol. 43, No.3, March 1985 Printed in U.S.A.

FERTILITY AND STERILITY Copyright ' 1985 The American Fertility Society

A comprehensive dose-response study of clomiphene citrate for enhancement of the primate ovarian/menstrual cycle*t

Burt A, Littman, M.D.:j:§ Gary D. Hodgen, Ph.D.11 Pregnancy Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland

Despite the increasing use of clomiphene citrate (CC) in normally cycling women undergoing in vitro fertilization, comprehensive data on the dose-response effects of the drug are unavailable. Twenty-four adult, normally cycling monkeys were given CC on cycle days 5 through 9 in doses ranging from 1 mg/kg to 10 mg/kg. Serum estradiol (E2 ), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and progesterone were measured daily. CC stimulated an early, attenuated rise in serum LH which correlated with subsequent follicle development. Concurrently, serum E2 rose to preovulatory levels. No significant increase in serum FSH concentration was seen as a result ofCC therapy. No correlation was seen between CC dose and peak levels of E 2 , LH, or FSH. The ovulatory status of the treatment cycles was also independent of dose. The factors that modulate these hormonal changes may involve direct effects of CC, estrogen feedback, and/or ovarian factors as yet uncharacterized. The individual variation of ovarian response, however, appears to be independent of the dose ofGG. Fertil Steril43:463, 1985

Clomiphene citrate (CC) is finding increased use in women with normal, spontaneous menstrual cycles to promote the development of multiple ovarian follicles as a prelude to in vitro ferReceived September 25, 1984; revised and accepted November 28, 1984. *Presented in part at the Fortieth Annual Meeting of The American Fertility Society, April 2 to 7, 1984, New Orleans, Louisiana. tSupported in part by Ford Foundation grant 810-0293. :j:Recipient of the National Fellowship in Reproductive Medicine. §Present address and reprint requests: Burt A. Littman, M.D., Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Fertility, The George Washington University Medical Center, 2150 Pennsylvania Avenue, N.W., Washington, D.C. 20037. IIPresent address: Scientific Director, The Jones Institute for Reproductive Medicine, Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia 23501. Vol. 43, No.3, March 1985

tilization. Doses of 50 to 150 mg daily have been used, either alone or in combination with human menopausal gonadotropin in an attempt to optimize follicle growth. I, 2 CC, an antiestrogen, is thought to stimulate the ovary indirectly, via its action on the hypothalamus and/or pituitary, leading to increased gonadotropin secretion. 3 , 4 While several studies have examined the hormonal response to CC in both patients with ovulatory dysfunction 5 - 7 and with normal menstrual cycles,8 it remains unclear as to whether a correlation exists between the dose of CC employed and the levels of gonadotropins achieved in peripheral circulation. Similarly, no study has examined directly the relationship between CC dose and follicle development as manifested by estrogen production. Here, using the monkey model, we examined the hormonal response to CC administered in daily doses equivalent, on a body weight basis in adult womLittman and Hodgen Clomiphene citrate dose response

463

Table 1. Relationship Between CC Dose and Frequency of Response Pattern CC dose 2 mg

3 9 15 30

Response patterna 3 4 5

1 0 1 0

1 1 0 0

1 2 0 3

1 0 2 0

RESULTS

6

no. Df cycles

2 2 2 3

Statistical comparisons were made with a twoway analysis of variance.

0 1 1 0

aSee text and Figures 1 to 6 for full description of response patterns.

en, to 50, 150, 250, and 500 mg. We asked: (1) Would increasing doses of CC result in a proportional rise in estrogen and/or gonadotropin secretion? (2) Would the estrogen response to CC correlate with the degree of endogenous gonadotropin stimulation resulting from administration of the drug?

The average weight of the monkeys did not differ among treatment groups. Overall, the mean weight of the monkeys under study was 2.96 ± 0.8 kg. Thus, the doses of CC administered by weight were approximately 1, 3, 5, and 10 mglkg, respectively, for each of the four treatment groups. CLOMIPHENE CITRATE RESPONSE PATTERNS

Six distinct hormonal response patterns were noted irrespective of CC dose. They are depicted in Figures 1 through 6 and described in detail below. The lack of association between CC dose and hormonal response pattern is presented in Table 1. OVULATORY MIDCYCLE LH SURGE N= 9

MATERIALS AND METHODS

Regularly cycling cynomolgus monkeys (Macaca fascicularis) (n = 24) were selected on day 1 of menses. The care and maintenance of these animals has been previously described. 9 CC (Serophene, Serono Laboratories, Inc., Randolph, MA) was prepared for oral administration in gelatin capsules in doses of 3, 9, 15, and 30 mg. Each dose was administered to six monkeys daily from days 5 through 9 of the menstrual cycle by placement within the posterior pharynx under ketamine hydrochloride anesthesia (Parke-Davis, Morris Plains, NJ) (40 mg intramuscularly). Daily blood samples were obtained by femoral puncture from cycle day 2 through cycle day 40 or until a subsequent menstrual period. Samples were obtained under ketamine anesthesia and allowed to clot. Sera were harvested after centrifugation and stored frozen (-lOOC). Twelve untreated adult monkeys with normal ovulatory menstrual cycles served as controls. These data have been published previously.lO Serum estradiol (E 2 ), progesterone (P), folliclestimulating hormone (FSH), and luteinizing hormone (LH) were measured by radioimmunoassay as previously described. 9 , 11 Cross-reaction of CC within the E2 assay was tested and found to be negligible. All reagents and protocols were obtained from the National Pituitary Agency under the aegis of the Hormone Distribution Officer, National Institute of Arthritis, Diabetes, Digestive and Kidney Diseases. 464

Littman and Hodgen Clomiphene citrate dose response

E,

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p. ng/ml

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Clomiphene' 1 Control

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28

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DAYS

Figure 1 Mean hormone levels (mean ± SEM) for those monkeys treated with CC on cycle days 5 through 9, demonstrating a midcycle gonadotropin surge (n = 9). Data are normalized to the early attenuated LH rise (cycle day 8.0 ± 0.5) and to the midcycle LH surge (cycle day 15.6 ± 0.4). The shaded area represents the hormone levels of untreated control animals (mean ± SEM) (n = 12) normalized to the midcycle LH surge (cycle day 12.3 ± 0.6).

Fertility and Sterility

OVULATORY DELAYED LH SURGE N =2

E

-~

300

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.,,:.:~ LH

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Figure 2 Individual hormone levels for those monkeys treated with CC on cycle days 5 through 9, demonstrating a delayed gonadotropin surge followed by P secretion indicative of ovulation (n = 2).

line value of these monkeys (group mean of cycle days 2 to 5) and with the comparable mean FSH ofthe control group (day 7). The respective values of5.3 ± 0.7,4.7 ± 0.5, and 4.3 ± 0.9 J-Lg/ml were similar (P > 0.05), again demonstrating no significant stimulation of peripheral FSH levels by CC administration. The CC-treated animals displayed a second, higher peak ofLH (199 ± 41 ng/ml) on cycle day 15.6 ± 0.4 (range, cycle days 14 to 18) in conjunction with peak E2 production. This LH peak was similar to the midcycle LH surge of the control group (143 ± 16 ng/ml), which occurred on cycle day 12.3 ± 0.6 (range, cycle days 8 to 14). The midcycle LH surge was significantly delayed (P < 0.01) by CC administration when compared with the control group. Of note is that although the CC-treated group achieved E2 levels of ~ 200 pg/ ml by cycle day 8 on the average, the midcycle LH surge did not occur in these animals until 7 days later. In contrast, the control group manifested a midcycle LH surge approximately 48 hours after achieving an E2 level of 200 pg/ml. OVULATORY EARLY LH SURGE

N=2

Figure 1 depicts an apparently normal midcycle ovulatory pattern. This was the most common pattern seen (n = 9). Data have been normalized to the initial attenuated CC-associated LH peak and to the midcycle LH surge, which resulted in subsequent P secretion. Although the peak serum E2 concentration achieved by the CC-treated group (436 ± 32 pg/ ml) (mean ± standard error of the mean [SEM]) was not significantly different from that achieved by the control group (357 ± 56 pg/ml), the rate of rise of peripheral E2 induced by CC was more rapid. Accompanying this rapid rise in E2 was a transient rise of serum LH to a peak value of 32 ± 3 ng/ml on cycle day 8.0 ± 0.5 (range, cycle days 6 to 10). This increase in LH concentration was significantly higher (P < 0.001) than the mean LH value for the corresponding day in the control cycle. Despite the obvious increases in serum E2 and LH, no corresponding rise was seen for FSH, as shown in Figure 1. To further investigate the peripheral FSH response to CC, peak FSH levels associated with CC administration (mean, cycle day 7.0 ± 0.4; range, cycle days 6 to 10) were grouped and compared with the mean FSH baseVol. 43, No.3, March 1985

-131M 0--0 883M

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I'II/ml

2IIr

10 5

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Figure 3 Individual hormone levels for those monkeys treated with CC on cycle days 5 through 9, demonstrating an ovulatory response to the early attenuated LH rise seen in association with CC administration (n = 2).

Littman and Hodgen Clomiphene citrate dose response

465

ANOVULATORY SINGLE LH PEAK N= 6

E2 to peak values of 365 and 265 pg/ml were

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achieved on cycle days 22 and 23, respectively. These were accompanied by LH surges to 125 and 226 ng/ml and FSH peaks to 5.4 and 6.2 fJ..g/ml. The ensuing luteal phases measured 16 days and 13 days, with peak P levels of 5.7 and 8.0 ng/ml, respectively. Figure 3 depicts the two animals that demonstrated an ovulatory pattern characterized by a single LH peak. E2 rose rapidly to peak levels of 418 and 464 pg/ml on cycle day 10. LH rose to peak values of 48 and 33 ng/ml on cycle days 10 and 11, and FSH demonstrated peak values of 3.4 and 5.4 fJ..g/ml on days 11 and 12, respectively. The ensuing luteal phases were each of 20 days duration, with peak P levels of 28 and 13 ng/ml, respectively. Of note is that in monkey 883M the LH peak occurred 5 days after achieving a serum E2 level of > 200 pg/ml, despite a relatively rapid

+21 +30

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2468101214161820222421213032343131

ANOVULATORY MULTIPLE LH PEAKS

DAYS

N=3

Figure 4 Mean hormone levels (mean ± SEM) for those monkeys treated with CC on cycle days 5 through 9, demonstrating an early attenuated rise in LH followed by follicle atresia and anovulation (n = 6). Data are normalized to the peak LH concentration.

A midcycle surge of FSH was seen to accompany the midcycle LH surge. The peak FSH level achieved by the CC-treated group (9.3 ± 1.4 fJ..g/ml) was similar to that seen in the control group (10.0 ± 1.3 fJ..g/ml). P levels increased in both treatment and control groups subsequent to the mid cycle LH surge. The luteal phase in each case was similar both in duration and in level of P production. Total cycle length as measured to the onset of the subsequent menstrual period in the CC-treated group was 31.2 ± 0.9 days, which was significantly longer (P < 0.01) than the total cycle length of the control group (27.0 ± 1.1 days). This difference relates primarily to the prolonged follicular phase seen following CC treatment. Figure 2 depicts the two monkeys that demonstrated an ovulatory pattern characterized by a delayed LH surge. Both monkeys showed a CC-induced rapid rise in serum E2 to peak levels of 370 and 312 pg/ml on cycle days 7 and 9, respectively. This initial rise in E2 was accompanied in each case by a transient increase of LH to 21 and 27 ng/ml and a transient increase of FSH to 4.4 and 5.6 fJ..g/ml, respectively. Secondary rises in serum 466

Littman and Hodgen Clomiphene citrate dose response

P, ngiml

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11

13

15

17

19

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23·25

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29

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DAYS

Figure 5 Mean hormone levels (mean ± SEM) for those monkeys treated with CC on cycle days 5 through 9, demonstrating multiple LH spikes in association with repetitive attempts at renewed follicle development (n = 3). Two monkeys remained anovulatory throughout the observation period of 40 days. One monkey demonstrated a rise in P following an LH peak on cycle day 32, consistent with ovulation (data not shown). Fertility and Sterility

ANOVULATORY NO RESPONSE N =2

LH 100 75 ng/ml 50

~

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2O~ 16

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Figure 6 Individual hormone levels for those monkeys treated with CC on cycle days 5 through 9, demonstrating no response throughout the observation period (n = 2).

rise in E 2 levels. In contrast, monkey 883M manifested an LH peak within 24 hours of achieving an E2 level of 200 pg/ml. Figure 4 depicts the mean hormone levels for the most common anovulatory pattern seen (n = 6). Data are normalized to the CC-induced LH peak. Although CC administration resulted in a rapid rise in serum E2 levels to an average peak value of 255 ± 52 pg/ml, these levels fell quickly and remained at baseline for the remaining period of observation. A transient rise in serum LH to an average peak value of 47 ± 4 ng/ml occurred on day 8.5 ± 0,6 (range, days 7 to 10), This level of LH secretion is significantly higher than the LH peak value seen in the group manifesting a normal ovulatory pattern (Fig. 1) (P < 0,01). Further, the decline in serum E2 occurred 48 hours following this LH peak. No significant rises in FSH or in P were seen during the observation period. Figure 5 shows the initial response (days 3 to 32) of those monkeys (n = 3) that demonstrated a hormonal pattern characterized by multiple rises in serum E2 in conjunction with multiple LH spikes. The data are presented as a composite, normalized to each LH peak. A rapid, CC-induced rise in serum E2 to an average peak of 299 ± 138 pg/ml occurred on cycle day 6.3 ± 0.3. Two addiVol. 43, No.3, March 1985

tional transient elevations in serum E2 occurred at approximately ll-day intervals during the observation period. Multiple LH peaks occurred throughout the cycle. Mean LH levels of 50 ng/ml or greater were associated with a subsequent decline in serum E2 levels. No meaningful elevations were seen in peripheral FSH levels. Two of the three monkeys demonstrated no rise in P production throughout the 40-day observation period, despite the multiple LH spikes, One monkey, however, did develop a rise in P in conjunction with an LH peak of 100 ng/ml on day 32 of the cycle. Peak P values of 8 ng/ml were achieved over the ensuing 8 days prior to discontinuation of the protocol. Figure 6 shows the two monkeys that demonstrated no response of serum gonadotropins or serum E2 to CC administration. DOSE RESPONSE

Table 2 presents the peak values of LH, FSH, and E2 which occurred in association with CC administration grouped by CC dose, The mean values for both gonadotropins and E2 were similar for each of the four doses of CC utilized in this study, suggesting the absence of any dose-response relationship for these parameters. DISCUSSION

Clomiphene citrate is usually administered in a sequentially increasing dosage regimen based on the response in the previous cycle. 12 There is concern, however, about the possible deleterious effects of CC, an antiestrogen, on cervical mucus, uterine histology, and ovarian folliculogeneSis. 13 , 14 Thus, for the ovarian stimulation of normally cycling women undergoing in vitro fertilization, CC has been used in doses ranging from 50 to 150 mg in an attempt to balance the positive effects of the drug on follicle growth with potential negative effects on other target orTable 2. Relationship Between CC Dose and Peak Hormone Response (Cycle Days 6 to lO)a

E.

CC dose mg

3 9 15 30

LH

pglml

309 300 298 354

± ± ± ±

FSH

nglml

27 75 87 75

35.0 32.7 38.1 40.3

± ± ± ±

I'/!Iml

5.8 3.1 8.6 5.6

6.0 5.0 4.5 5.1

± ± ± ±

1.1 0.7 0.3 0.5

aMean ± SEM.

Littman and Hodgen Clomiphene citrate dose response

467

gans. 1, 2 The inherent assumption of these treatment protocols is that the response to CC is dosedependent. The present study suggests that there is in fact no correlation between the dose of CC and the ensuing peripheral concentration of estrogen or gonadotropins in normally cycling monkeys. Further, the ovulatory status ofthese animals subsequent to CC administration appears to be independent of dose, suggesting that it may be the neuroendocrine milieu inherent to a given cycle or individual which determines the outcome of a CC-stimulated cycle rather than the dose administered. Several studies have documented a CC-induced, early, transient increase in peripheral LH concentration in women with ovulatory dysfunction 5 - 7 and in women with normal menstrual cycles. s In the present study, 22 of the 24 monkeys undergoing CC stimulation demonstrated this attenuated rise in LH between cycle days 6 and 11. The magnitude and timing of the peak level of LH achieved in response to CC stimulation appears to dictate, at least in part, the subsequent fate of the dominant follicle. Peak LH levels of 50 ng/ml which occurred between cycle days 7 and 10 (n = 9) were associated with a subsequent fall in peripheral estrogen levels, which suggests follicle atresia. In six of these animals, no follicle growth ensued over the remainder of the study period. Three animals, however, did show later preovulatory rises in estrogen concentration which were terminated by repetitive LH peaks of 50 ng/ml. Two additional monkeys achieved peak LH levels of 48 and 33 ng/ml on cycle days 10 and 11, respectively, which resulted in apparent ovulation. The mechanism by which the elevated peripheral levels of LH result in follicle atresia cannot be determined from the present study. We have previously shown that human chorionic gonadotropin (hCG), in pharmacologic doses, is capable of inducing follicle atresia when administered prior to the endogenous midcycle gonadotropin surge. 15 When administered in the peri ovulatory interval, however, hCG was without effect. Elevated levels of LHIhCG may result in receptor down-regulation within the follicle. 16 Alternatively, increased androgen production by the theca cell in response to elevated levels of LHIhCG may promote follicle atresia. Indeed, CC administration has been shown to increase peripheral levels of androstenedione and testosterone in women.17 468

Littman and Hodgen Clomiphene citrate dose response

The factors which modulate this early attenuated rise in LH are unclear. CC may act at the level of the hypothalamus to increase the secretion of gonadotropin-releasing hormone (GnRH).3 Alternatively, CC may act at the level of the pituitary gland to enhance pituitary sensitivity to GnRH4 or to directly stimulate the release of gonadotropins. 1s It is difficult, however, to separate the direct hypothalamic/pituitary effects of CC administration on LH secretion in vivo from the feedback effects of the rising concentration of peripheral estrogen. Indeed, there is evidence that CC may directly stimulate estrogen production at the level of the ovary. 19, 20 CC may inhibit the negative feedback effect of estrogen on LH secretion. 21 The present study and others,14 suggest that CC inhibits the positive feedback effect of estrogen as well. Estrogen levels maintained> 200 pg/ml usually result in a spontaneous midcycle LH surge within 48 hours.22 Here, those animals demonstrating a midcycle LH surge (n = 9) maintained estrogen levels at or > 200 pg/ml for approximately 7 days prior to the onset of the midcycle LH increase. Thus, the early transient rise in LH secretion may be a response to estrogen-positive feedback partially modulated by the antiestrogenic effect of CC at the pituitarylhypothalamus. An additional negative modulating influence on the early attenuated LH rise may derive from the CC-stimulated ovary. We have previously demonstrated the presence of a highly transient ovarian product designated gonadotropin surgeinhibiting factor (GnSIF).lO GnSIF activity, although not characterized chemically, can be stimulated by the administration of human menopausal gonadotropin and appears to block the action of GnRH on the pituitary, resulting in impairment of normal estrogen-positive feedback for the LH surge. It is unclear, however, whether ovarian stimulation by CC results in an increased production of GnSIF. Whereas CC administration resulted in a definite increase in peripheral LH concentration, there was no associated significant rise in peripheral FSH when compared with pretreatment levels or with the concentration on the corresponding cycle day of untreated control subjects. Previous studies have suggested an early increase in peripheral FSH concentration in response to CC stimulation in women with normal menstrual cycless and in women with ovulatory dysfunction. 5 - 7 Fertility and Sterility

Some of these studies, however, did not make use of statistical comparisons. 5 - 7 Indeed, one such study presented case reports suggesting the response of FSH to CC to be quite variable and iIi some cases absent. 6 Statistically significant increases in FSH have been demonstrated in women by the third day ofCC stimulation. 8 Comparisons were made only with the mean value for cycle day 1 prior to treatment. No comparison was presented with the appropriate cycle day of untreated control women. The disparity in FSH response may be due to ovulatory status, species differences, or differences in assay sensitivity. However, the same study which demonstrated a significant rise in peripheral FSH concentration showed no significant increase in FSH pulsatility in response to CC, which further suggests a minimal effect of CC on FSH secretion. 8 The lack of increase in FSH secretion in the face of rising LH secretion in response to CC, a situation not unlike that seen in polycystic ovarian disease, may derive from differential feedback effects of the rising estrogen concentrations. CC, however, is capable of blocking the negative feedback effect of estrogen on FSH secretion. 21 Recently, an increase in follicular fluid inhibin levels has been demonstrated following CC administration, as compared with untreated control subjects. 23 The dramatic rise in peripheral estrogen concentration in the absence of a significant increase in peripheral FSH levels may provide further evidence for a direct stimulatory effect of CC upon the aromatase system within the follicle. Indeed, the transient rise in peripheral LH seen during the follicular phase may provide for the increased availability of androgen substrate. 24 Alternatively, there is in vitro evidence that LH alone, in the presence of basal levels of FSH, may act as a direct stimulatory influence on the aromatase enzyme. 25 Acknowledgments. We express our appreciation to Donald Barber, Adrian Coleman, Dick Greenawalt, Almorris Lynch, and Barbara Murphy for their technical assistance. In addition, we gratefully acknowledge the generous gift of clomiphene citrate (Serophene) by Serono Laboratories, Inc., Randolph, MA. REFERENCES 1. Lopata A: Concepts in human in vitro fertilization and embryo transfer. Fertil Steril 40:289, 1983 2. Quigley MM, Maklad NF, Wolf DP: Comparison of two clomiphene citrate dosage regimens for follicular recruitment in an in vitro fertilization program. Fertil Steril 40:178,1983 Vol. 43, No.3, March 1985

3. Miyake A, Aono T, Minagawa J, Kawamura Y, Kurachi K: Changes in plasma luteinizing hormone-releasing hormone during clomiphene-induced ovulatory cycles. Fertil Steril 34: 172, 1980 4. Adashi EY, Hsueh AJW, Bambino TH, Yen SSC: Disparate effect of clomiphene and tamoxifen on pituitary gonadotropin release in vitro. Am J Physiol 240:E125, 1981 5. Ross GT, Cargille CM, Lipsett MB, Rayford PL, Marshall JR, Strott CA, Rodbard D: Pituitary and gonadal hormones in women during spontaneous and induced ovulatory cycles. In Recent Progress in Hormone Research, Edited by EB Astwood. New York, Academic Press, 1970, pI 6. Wu CH: Plasma hormones in clomiphene citrate therapy. Obstet Gynecol 49:443, 1977 7. Jacobson A, Marshall JR, Ross GT, Cargille CM: Plasma gonadotropins during clomiphene-induced ovulatory cycles. Am J Obstet Gynecol 102:284, 1968 8. Vandenberg G, Yen SSC: Effect of anti-estrogenic action of clomiphene during the menstrual cycle: evidence for a change in the feedback sensitivity. J Clin Endocrinol Metab 37:356, 1973 9. Goodman AL, Nixon WE, Johnson DK, Hodgen GD: Regulation of folliculogenesis in the cycling rhesus monkey: selection of the dominant follicle. Endocrinology 100:155, 1977 10. Littman BA, Hodgen GD: Human menopausal gonadotropin stimulation in monkeys: blockade of the luteinizing hormone surge by a highly transient ovarian factor. Fertil Steril 41:440, 1984 11. Peckham WD, Tontala FJ: A new radioimmunoassay for monkey luteinizing hormone. BioI Reprod (Suppl 1) 24:119A, 1981 12. Rust LA, Israel R, Mishell DR: An individualized graduated therapeutic regimen for clomiphene citrate. Am J Obstet Gynecol 120:785, 1974 13. Van Campenhout J, Simard R, Leduc B: Antiestrogenic effect of clomiphene in the human being. Fertil Steril 19:700, 1968 14. Marut EL, Hodgen GD: Antiestrogenic action of high-dose clomiphene in primates: pituitary augmentation but with ovarian attenuation. Fertil Steril 38:100, 1982 15. Williams RF, Hodgen GD: Disparate effects of human chorionic gonadotropin during the late follicular phase in monkeys: normal ovulation, follicular atresia, ovarian acyclicity, and hypersecretion of follicle-stimulating hormone. Fertil Steril 33:64, 1980 16. Schwall RH, Erickson GF: A new in vitro model system for the study of luteinizing hormone receptor down regulation. J BioI Chem 258:3442, 1983 17. Dupon C, Rosenfield RL, Cleary RE: Sequential changes in total and free testosterone and androstenedione in plasma during spontaneous and Clomid-induced ovulatory cycles. Am J Obstet GynecoI115:478, 1973 18. Docke F: Ovulation-inducing action of clomiphene citrate in the rat. J Reprod Fertil 18:135, 1969 19. Zhuang L, Adashi EY, Hsueh AJW: Direct enhancement of gonadotropin-stimulated ovarian estrogen biosynthesis by estrogen and clomiphene citrate. Endocrinology 110:2219, 1982 20. Engels JA, Friedlander RL, Eik-Nes KB: An effect in vitro of clomiphene on the rate of conversion of androstenedione-C14 to estrone-C14 and E-C14 by the canine ovary. Metabolism 17:189, 1968

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21. Vaitukaitis JL, Bermudez JA, Cargille CM, Lipsett MD, Ross GT: New evidence for an anti-estrogenic action of clomiphene citrate in women. J Clin Endocrinol Metab 32:503, 1971 22. Karsch FJ, Weick RF, Butler WR, Dierschke DJ, Krey LC, Weiss G, Hotchkiss J, Yamaji T, Knobil E: Induced LH surges in the rhesus monkey: strength-duration characteristics of the estrogen stimulus. Endocrinology 92:1740,1973 23. Marrs RP, Lobo R, Campeau JD, Nakamura RM, Brown J, Ujita EL, diZerega GS: Correlation of human follicular fluid inhibin activity with spontaneous and induced folli" cle maturation. J Clin Endocrinol Metab 58:704, 1984

470

24. Tsang BK, Armstrong DT, Whitfield JF: Steroid biosynthesis by isolated human ovarian follicle cells in vitro. J Clin Endocrinol Metab 51:1407, 1980 25. Wang G, Hsueh AJW, Erickson GF: LH stimulation of estrogen secretion in cultured granulosa cells. Mol Cell Endocrinol 24:17, 1981

Littman and Hodgen Clomiphene citrate dose response

Fertility and Sterility