Effects of tamoxifen on corpus luteum function and luteal phase length in cynomolgus monkeys*

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1 FERTILITY AND STERILITY Copyright Q 1990 The American Fertility Society Printed on acid-free paper in U.S.A. Effects of tamoxifen on corpus luteum function and luteal phase length in cynomolgus monkeys* David L. Olive, M.D.t Natalie Schultz, M.D. Robert M. Riehl, Ph.D. Terry R. Groff, M.D. Robert S. Schenken, M.D. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Infertility, The University of Texas Health Science Center at San Antonio, San Antonio, Texas Previous data in nonhuman primates have demonstrated that tamoxifen prolongs the luteal phase without altering reproductive hormone levels. A small study in humans found no effect on menstrual cycle length, but an increase in luteal ovarian steroid levels. In view ofthese conflicting results, we studied the effect oftamoxifen on corpus luteum (CL) function in monkeys (n = 20). Blood was obtained daily beginning cycle day 8, and sera assayed for estradiol (E 2), progesterone (P), luteinizing hormone, and follicle-stimulating hormone. Four days after the midcycle E2 peak, laparoscopy confirmed CL formation, and the animals were administered (1) lactose (n = 7), (2) tamoxifen, 0.5 mg.kg-1.d-1 (n = 6), or (3) tamoxifen, 3.0 mg.kg-1.d-1 (n = 7) for 12 consecutive days. Serum collection continued until cycle day 50 or menses, whichever came first. Results indicate a biphasic response among tamoxifen-treated animals, with 7 of 13 developing prolonged luteal phases. There was, however, no significant difference in luteal phase length among the three groups, although when the two groups given tamoxifen were combined, the difference in luteal phase length versus controls approached significance. No differences were found among peak P levels, mean luteal phase P levels, or mean luteal phase gonadotropin levels. No variables were found to correlate significantly with luteal phase length. These results suggest that luteal phase administration of the antiestrogen tamoxifen does not alter pituitary gonadotropin secretion or CL function. However, tamoxifen does prolong luteal phase length in a subset of monkeys, perhaps via a direct effect on the endometrium. Fertil Steril54:333, 1990 Hoffman! and Knobil 2 originally proposed that endogenous estrogen (E) causes spontaneous corpus luteum (CL) regression in nonconception cycles. This hypothesis was supported by data in rhesus monkeys,3 baboons,4 and women 5 demonstrating that premature luteolysis followed admin- Received October 23, 1989; revised and accepted April 5, * Supported by grant from the Special Programme of Research, Development, and Research Training in Human Reproduction, World Health Organization, Geneva, Switzerland. t Reprint requests: David L. Olive, M.D., Department of Obstetrics and Gynecology, The University of Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, Texas istration of exogenous E. In vitro studies have also shown that E decreases progesterone (P) production by isolated monkey6 and human 7 luteal cells. Further, the administration of anti-es in the luteal phase has been shown to attenuate this luteolytic effect of exogenous E. 4,8 Collectively, these studies suggest that endogenous E hastens luteolysis in primates. The role of E as a naturalluteolytic agent, however, has been questioned. In monkeys, anti-e administration failed to impair luteal function in natural cycles. 8 Additionally, luteal function was unaltered in monkeys after significant lowering of endogenous E by inhibition of aroniatase. 9 Thus, it has been suggested that the mechanisms of Olive et al. Tamoxifen and the CL 333

2 E-induced luteolysis and spontaneous luteolysis differ. Two reports have spurred interest in the effect of the anti-e tamoxifen on luteal phase function and length. Tajima,IO in a study of infertile women, found enhancement of luteal P and estradiol (E2) secretion after tamoxifen administration. Ravindranath and Moudgal ll reported that tamoxifen prolonged luteal phase length in the bonnet monkey. The present investigation was designed to corroborate and extend these observations by determining the effect of two different doses of tamoxifen on CL function and luteal phase length. MATERIALS AND METHODS Animal Husbandry Twenty-four adult female cynomolgus monkeys (Macaca fascicularis) weighing 3.0 to 4.0 kg and having regular menstrual cycles were maintained as previously described.12 For 2 to 3 months the monkeys were checked for menses but no additional procedures were performed. Monkeys were entered in the protocol after two spontaneous menstrual cycles of normal duration. A 3.5-mL blood sample was collected daily at 9: 00 A.M. from cycle day 8 until menses or cycle day 50, whichever came first. Samples were collected by femoral venipuncture, within 10 minutes of ketamine HCI-induced (15 mgjkg, Vetalar; Parke Davis, Detroit, MI) anesthesia. Each sample was immediately centrifuged at 2,000 X g for 10 minutes, and the sera stored at -20"C. Confirmation of Ovulation Laparoscopy was performed using ketamine HCI-induced anesthesia 4 days after the midcycle E2 peak to document the presence of a CL. Only monkeys demonstrating a midcycle E2 peak and a CL (n = 20) were continued on the protocol. Hormone Treatment Monkeys meeting the above criteria were randomized into three groups: (1) controls (n = 7), (2) low-dose tamoxifen (n = 6), and (3) high-dose tamoxifen (n = 7). Beginning 4 days after the midcycle E2 peak, control monkeys received lactose by gavage daily for 12 days. The low-dose tamoxifen and high-dose tamoxifen groups received 0.5 mg kg-1.d-1 and 3.0 mg.kg-1.d-l, respectively, by ga- vage for 12 days, beginning 4 days after the midcycle E2 peak. All animals were treated at 8:00 A.M. in the fasting state. Hormone Assays The following reagents were obtained from the National Hormone and Pituitary Program (University of Maryland School of Medicine, Baltimore, MD) of the National Institute of Diabetes, Digestive, and Kidney Diseases: human folliclestimulating hormone (FSH, AFP-4822B), monkey luteinizing hormone (LH, LER ) and FSH (LER ), ovine LH (LER 1045-C2), and antiserum to FSH (batch #5). Iodination of LH and FSH (AFP-9900) was performed by a modification13 of the method of Greenwood et al. 14 Ovine antirabbit gamma globulin (second antibody) was obtained from Endocrine Sciences (Oxnard, CA). Coat-A-Count E2 radioimmunoassay kits were obtained from Diagnostic Products Corporation (Los Angeles, CA). Gonadotropins were measured by a double-antibody method previously described.15 Diluent for all assays consisted of 0.01 M phosphate-buffered saline, ph 7.6, supplemented with 0.05 Methylene diamine tetra-acetic acid and 5 mgjml bovine serum albumin (Fraction V; Sigma, St. Louis, MO). Monkey plasma samples (0.1 ml), control plasma containing a known amount of hormone (0.1 ml), and hormone standards (0.1 ml) were incubated in the presence of the first antibody at dilutions of 1: 5,000 (FSH) and 1:25,000 (LH) for 72 hours (FSH) and 24 hours (LH) at 4 DC. Iodinated hormones (20,000 cpmjtube) containing normal rabbit serum (0.5% vol/vol) were added and incubation at 4 DC continued for 48 hours (FSH) and 24 hours (LH). Second antibody at a dilution of 1:360 was added and assays incubated at 4 DC for 24 hours. Subsequently, 3 ml of buffer was added and the tubes centrifuged at 4,000 X g for 30 minutes at 4 DC. The supernatant was removed and the amount of 1251 in the pellet determined, using a Pachard model PGCD504 gamma counter (Pachard Instruments, Laguna Hills, CA). Values for unknown samples were determined from a log-logit transformation of the binding values for the standards of each assay. The sensitivity of the assays was 27.2 ngjtube for LH, and 410 ngjtube for FSH. Intra-assay coefficients of variation (CV) were 6.3% and 8.1 % for FSH and LH, respectively. Interassay CVs were 4.3% and 2.1 % for FSH and LH, respectively. 334 Olive et al. Tamoxifen and the CL Fertility and Sterility

3 luteal Phase (Days) IS Contral lowdownx HighDowTMX Figure 1 Length of the luteal phase in monkeys treated as controls and with low-dose (0.5 mg.kg-1.d-1) and high-dose (3.0 mg.kg-1.d-1) tamoxifen. Each symbol indicates the luteal phase length of a single animal. TMX, tamoxifen. Plasma E2 levels were measured according to the protocol provided by the manufacturer, using an incubation period of 3 hours at 22 C. Assay sensitivity was 5 pg/ml. Intra-assay and interassay CV s were 10.8% and 3.7%, respectively. Plasma P levels were measured by the double-antibody method previously described. 15 Assay sensitivity was 100 pg/ml. Intra-assay and interassay CV s were 15.0% and 7.0%, respectively. Statistical Analysis Comparison of the groups was performed using one-way analysis of variance. Comparison of controls versus pooled tamoxifen-treated animals was performed using Student's t-test. Non-normally distributed data were analyzed by nonparametric testing, using the Mann-Whitney V-test and the Kruskal-Wallis test for two- and three-sample comparisons, respectively. Correlations were performed utilizing the Spearman rank order correlation test. Forward stepwise multiple linear regression was performed using inclusion and exclusion criteria with a threshold of P = The total area circumscribed by hormone response curve (area under curve [AVC]) was calculated by summing the areas of triangles and trapezoids formed by the daily hormonal values. Calculations were carried out using Abstat release 4.13 (Anderson Bell, Parker, CO) on an IBM (International Business Machines, Inc., Boca Rotan, FL) personal computer. Cycle Length RESULTS Follicular phase length was defined as the number of days from onset of menses until LH surge. The luteal phase was defined as the time from the day after LH surge until the day before onset of subsequent menses. No differences were seen among the three groups for length of the follicular phase. The luteal phase, however, produced highly variable results. Only one of the seven control animals had a luteal phase longer than 17 days, whereas 6 of 13 tamoxifen-treated animals had prolongation ofthe luteal phase (Fig. 1). Mean luteal phase length did not differ significantly among the three groups (P = 0.19). However, comparison of controls versus low-dose tamoxifen suggested a notable difference (P = 0.04), and control monkeys demonstrated a nonsignificant trend toward a shorter luteal phase when compared with the pooled group of tamoxifen-treated animals (P = 0.07). Basal serum P concentration was arbitrarily defined as 3 consecutive days < 0.2 ng/ml. In assessing the length of the luteal phase before reaching basal P levels, there were no differences among the three groups (Table 1). Hormonal Parameters No differences were noted among the three groups when comparing peak luteal phase P, mean luteal phase P, or P AVC. Similarly, there were no differences in LH AVC, FSH AVC, mean LH, or mean FSH in the luteal phase among the three groups. Finally, peak E2 levels did not differ among the groups, nor did mean luteal E2 or E2 AVC differ (Table 2). Interaction Between Cycle Length and Hormonal Parameters In an attempt to determine which hormonal variables might influence luteal phase length, rank Table 1 Menstrual Cycle Length in Tamoxifen-treated Monkeys and Controls' Luteal Follicular Luteal phase P Group No. phase phase elevation Control ± ± ± 1.5 Low-dose tamoxifenc ± ± ± 0.9 High-dose tamoxifen d ± ± ± 1.0 Values are means ± SEM. b Number of days from LH surge until basal P levels are obtained (P ::s;; 0.2 ng/ml for 3 consecutive days).- c 0.5 mg.kg-1.d-1 tamoxifen orally for 12 days. d 3.0 mg.kg-1.d-1 tamoxifen orally for 12 days. Olive et ai. Tamoxifen and the CL 335

4 Table 2 Hormonal Characteristics of Tamoxifen-treated Monkeys and Controls Group Control (n = 7) Low-dose tamoxifen a (n = 6) High-dose tamoxifen b (n = 7) Peak P (ng/ml) Mean P (ng/ml) PAVC Mean LH (miu/ml) LHAVC Mean FSH (miu /ml) FSHAVC Peak E2 (pg/ml) Mean luteal E2 (pg/ml) Luteal E2 AVC 6.59 ± ± ± ± ,955.0 ± 1, ± ± ± ± ,269.5 ± ± ± ± ± ,448.0 ± 1, ± ± ± ± ,264.0 ± ± ± ± ± ,597.0 ± ± ± ± ± ,741.8 ± a 0.5 mg. kg-l. d-l tamoxifen orally for 12 days. b 3.0 mg. kg-l. d-' tamoxifen orally for 12 days. order correlation was performed between luteal phase length and the following: mean luteal phase LH, mean luteal phase FSH, height of the LH surge, height of the E2 peak, E2 peak to LH surge interval, mean luteal phase E 2, and follicular phase length. No significant correlations were observed among these variables as related to luteal phase length, although mean luteal phase E2 approached significance (0.10 > P > 0.05). Similarly, multiple linear regression failed to discern any significant predictor variables for the outcome variable luteal phase length. DISCUSSION Tamoxifen, an amnio-ether derivative ofpolycyclic phenol, has been used extensively as an E antagonist. The transisomer of the compound has potent antiestrogenic properties and acts by blocking E receptors16; although agonistic activity is noted in some species, this effect is minimal and of little consequence. An extensive literature exists regarding the effects of tamoxifen on the follicular phase in its role as an ovulation induction agent. However, relatively little work has been performed investigating the effects of luteal phase administration of tamoxifen. The luteolytic effect of exogenous E has been demonstrated in humans,5.17 rhesus monkeys,3 baboons,4 and cynomolgus monkeys.8 Furthermore, luteal phase administration of another anti-e (clomiphene citrate [CC]) has been shown to maintain normal luteal function in the presence of luteolytic exogenous E2 levels.4.8 These results suggest E2 may act as a self-regulating luteolytic agent produced by the CL. Despite the aforementioned data, many remain unconvinced of the role of E2 in spontaneous luteolysis. Utilizing otherwise untreated cynomolgus monkeys, Westfahl and Resko8 were unable to demonstrate a difference in luteal function between CC-treated animals and those treated with vehicle alone. Similar data have been obtained in humans18 and baboons/ 9 in which the E antagonists tamoxifen and MER25 failed to affect luteal function when administered throughout the luteal phase. Although it could be argued that levels were insufficient to antagonize locally elevated E2 levels, the observation that luteal function is unaltered in the cynomolgus macaque after reduction of endogenous E2 by an aromatase inhibitor 9 makes this caveat unlikely. Rather, the data suggest that spontaneous luteal regression is unrelated to E2 levels. If this latter conclusion is correct, the administration of luteal phase tamoxifen would be expected to produce little, if any, effect on luteal function. However, preliminary data have suggested otherwise. In a study of five infertile women, Tajima10 reported a significant increase in luteal P and E 2levels after tamoxifen 10 mg/d for 5 days in the luteal phase. However, it is unclear from the report to what degree ovulation dysfunction may have been initially present; in addition, tamoxifen was not given consistently over the same 5-day period to all subjects. Recently, Ravindranath and Moudgal ll administered tamoxifen, 15 mg/d for 13 days, in the luteal phase in the bonnet monkey. They noted a significant prolongation of luteal phase length, as well as a suggestion of altered luteal function, as evidenced by a change in the P profile. However, no statistical analysis of P or E2 levels was provided, and no additional hormonal data were assessed. Our study indicates that, in the cynomolgus 336 Olive et ai. Tamoxifen and the CL Fertility and Sterility

5 monkey, tamoxifen in either low- or high-dose formulation does not affect spontaneous luteal function. Tamoxifen had no effect on pituitary gonadotropin secretion because no differences in gonadotropin levels were observed and had no direct ovarian effect as E2 and P levels were unaltered after tamoxifen treatment. The uniformity of treatment schedule, increased sample size, and utilization of two dosage schemes support the contention that spontaneous luteal regression is not E2-induced and therefore unaffected by the E2 antagonist, tamoxifen. Interestingly, despite an absence of hormonal alteration, a large subset of tamoxifen -treated monkeys did experience prolongation of the luteal phase. This suggests that, in some animals, tamoxifen is acting directly at the level of the endometrium to produce inhibition of menses. Such a suggestion is not unprecedented. Sherman et a1. 18 found continuous tamoxifen to cause a delay in luteal phase endometrial development. Tajima and associates20 also reported reduced endometrial glandular development, despite a failure oftamoxifen to block ovulation. Manni et a1.21 noted irregular cycles in women undergoing continuous tamoxifen treatment at a dose of approximately 2 mg kg-i. d-1.21 Lunan and Green22 provide explanation for this phenomenon by demonstrating inhibition of uptake of radiolabled E2 by human endometrium after tamoxifen exposure.22 Finally, Fritz and colleagues23 have shown a delay in endometrial histological development, as well as a reduction in endometrial E and P receptors after the luteal administration of CC. A direct endometrial effect by an anti -E may be significant as a modulator of pregnancy maintenance. Progesterone results in diminution of its own receptors. Data from the aforementioned studies suggest that the role of luteal phase E is to replenish and maintain a requisite level of P receptor to provide a continued endometrial progestational response.23 Interference with this action would be expected to sufficiently alter endometrial development and inhibit successful implantation, an observation recently confirmed in the bonnet monkey.24 Future studies should be directed at defining a dose and window of antifertility activity by this agent when administered in the luteal phase. Such investigations will not only clarify the role of luteal phase E in the reproductive process but also may aid in the development of tamoxifen as an effective postovulatory contraceptive agent for fertility regulation. Acknowledgments. We thank the World Health Organization, Special Programme of Research, Development, and Research Training in Human Reproduction for their support and advice. Antiserum to ovine LH was kindly provided by Gordon Niswander, Ph.D., of Colorado State University, Fort Collins, Colorado. We also acknowledge the technical assistance of Mr. Arturo Moreno, Animal Surgery Facility Supervisor; Ms. Wendy Anderson, Research Associate; and Ms. Donna Johnson, Research Associate, as well as the assistance of Ms. Irma Garcia, Administrative Secretary, and Ms. Gretta Small, Editor, in the preparation of this manuscript. REFERENCES 1. Hoffman F: Untersuchungen liber die hormonale Beeinfiussung der Lebensdauer des Corpus Luteum in Zyklus der Frau. Geburtshilfe Frauenheilkd 20:1153, Knobil E: On the regulation of the primate corpus luteum. BioI Reprod 8:246, Karsch FJ, Krey LC, Weick RF, Dierschke DJ, Knobil E: Functionalluteolysis in the rhesus monkey: the role of estrogen. Endocrinology 92:1148, Westfahl PK, Kling OR: Relationship of estradiol to luteal function in the cycling baboon. 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