Effects of norethindrone on gonadotropin and ovarian steroid secretion when used for cycle programming during in vitro fertilization

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1 FERTILITY AND STERILITY Copyright 1990 The American Fertility Society Printed on acid-free paper in U.S.A Effects of norethindrone on gonadotropin and ovarian steroid secretion when used for cycle programming during in vitro fertilization Robert E. Anderson, M.D.* Andrea L. Stein, M.D.t RichardJ. Paulson, M.D. Frank Z. Stanczyk, Ph.D. Ariel G. Vijod, M.S. Rogerio A. Lobo, M.D.t Department of Obstetrics and Gynecology, University of Southern California School of Medicine Women's Hospital, Los Angeles, California Norethindrone (NET) has been used for cycle programming and may result in attenuated responses to controlled ovarian hyperstimulation. The effects of NET on gonadotropin secretion, its bioavailability to the ovary, and its effect on ovarian steroidogenesis in vivo and in vitro were assessed. Endogenous secretion of luteinizing hormone and folliclestimulating hormone wa,s attenuated by 59% and 50%, respectively, after 2 weeks of orally administered NET. Twelve hours after a single 10-mg oral dose, significant levels of NET were measured in samples of peripheral (8.8 ± 1.9 ng/ml) and ovarian venous blood (10.5 ± 3.1 ng/ml), follicular fluid (7.1 ± 2.1 ng/ml), and homogenates of ovarian tissue (8.0 ± 0.6 ng/g). Furthermore, NET was detectable in follicular fluid 2 weeks after its withdrawal (863 ± 149 pg/ml). However, there were no effects of NET on follicular fluid levels of estradiol and progesterone in vivo or on luteinized granulosa cell steroidogenesis in vitro. We conclude that when used for cycle programming in in vitro fertilization, NET does not inhibit ovarian steroidogenesis but does affect the hypothalamic-pituitary axis. Fertil Steril54:96, 1990 Programmed oocyte retrieval is a means of simplifying controlled ovarian hyperstimulation for in vitro fertilization (IVF).1,2 Programmed oocyte retrieval involves daily administration of an oral contraceptive or progestin, such as norethindrone (NET), starting in the late luteal phase ofthe cycle preceding controlled ovarian hyperstimulation. Subsequent controlled ovarian hyperstimulation Received August 14, 1989; revised and accepted March 21, * Present address: Hoag Fertility Services, Hoag Memorial Hospital Presbyterian, Newport Beach, California. t Present address: th Street, Suite 220, Santa Monica, California. * Reprint requests: Rogerio A. Lobo, M.D., Women's Hospital, Room 1M2, Los Angeles County and University of Southern California Medical Center, 1240 North Mission Road, Los Angeles, California Anderson et al. Bioavailability and effects of NET and oocyte retrieval are then performed in accordance with a predetermined fixed schedule. Although preliminary reports indicate that pregnancy rates achieved with programmed oocyte retrieval are similar to those obtained from conventional stimulation regimens, notable differences have been detected. 1,2 Serum estradiol (E2) levels and the number of follicles and oocytes per patient are significantly decreased in patients treated with programmed oocyte retrieval. 3 Furthermore, compared with 2.5 mg/d, 10 mg of NET lowered to a greater degree follicular phase serum estrogen levels and levels of urinary pregnanediol after oocyte retrieval. 4 Our own experience with 10 mg of NET to program the start of controlled ovarian hyperstimulation for IVF is that a protracted response to controlled ovarian hyperstimulation results is similar to that observed in patients pretreated Fertility and Sterility

2 with a gonadotropin-releasing hormone analog (GnRH-a).5 Little data are available concerning the effects of progestins on gonadotropin secretion or on ovarian steroidogenesis. In nonhuman primates, the administration of NET has been shown to inhibit the preovulatory gonadotropin surge. 6 However, NET also lowered E2 levels before any effect on luteinizing hormone (LH) and follicle-stimulating hormone (FSH) was demonstrable, thus suggesting a direct ovarian effect. Norethindrone, therefore, by inhibiting the hypothalamic-pituitary-ovarian axis at one or more sites, could explain the findings observed during programmed oocyte retrieval. The purpose of this study was to understand more fully the effects of NET on the hypothalamicpituitary-ovarian axis when used for programmed oocyte retrieval. We determined the levels of NET and its disappearance from the circulation after oral ingestion of a 10-mg dose. The effect of this dose of NET on LH and FSH secretion was also evaluated. In addition, we studied the bioavailability of NET to the ovary and whether or not NET has an acute direct effect on ovarian steroidogenesis. MATERIALS AND METHODS Hypothalamic-Pituitary Effects Ten normal ovulatory women (ages 20 to 34 years, mean 30.6 years) and 7 infertile women (ages 28 to 35 years, mean 32.3 years) were studied. All women were within 15% oftheir ideal body weight. Four of the 7 infertile women were anovulatory and received NET (Norlutin; Parke-Davis, Morris Plains, NJ) 10 mg/d by mouth for 2 weeks. Blood was obtained before the first dose, then after 1 and 2 weeks of NET. The other 3 infertile women were ovulatory and also received NET 10 mg/d for 2 weeks, but beginning on day 21 of a normal cycle. In these 3 women, we assessed the disappearance of NET from the circulation after 2 weeks of administration by obtaining blood samples every 12 hours for 48 hours. To compare the suppressive effects of NET on the hypothalamic-pituitary axis, we compared serum LH and FSH levels after NET with the gonadotropin levels obtained from the 10 normal ovulatory women on day 3 of their menstrual cycles. All venous blood samples for these studies were allowed to clot at room temperature and the serum was separated and stored at -20 C until assayed for NET, LH, and FSH. Ovarian Effects: Ovarian Bioavailability To determine the ovarian bioavailability of NET, three women (ages 33, 38, and 40 years, mean weight 61.5 kg) undergoing elective hysterectomy and oophorectomy, were studied. This study was approved by the Institutional Review Board of our medical center and each patient gave written informed consent before participation. All procedures were performed in the late follicular phase of the cycle. A single 10-mg oral dose of NET was given 12 hours before surgery. During hysterectomy, samples of peripheral and ovarian venous blood were obtained, as was follicular fluid, from the largest single ovarian follicle by direct puncture with a 21-gauge needle. After oophorectomy, 250- mg wedges of ovarian tissue, not containing large follicles, were excised. These samples were stored at 20 C until homogenized and assayed for NET. Follicular Fluid Levels The potential effect of NET on ovarian steroidogenesis was studied in vivo by comparing follicular fluid concentrations of E2 and progesterone (P) at the time of oocyte retrieval in women undergoing IVF. Women pretreated with NET (n = 3) were compared with eight others not pretreated. Three women were given NET 10 mg/d for 2 weeks, starting on day 21 of their cycles. They were then stimulated with human menopausal gonadotropins (hmg) using the same protocol as used for the eight women not pretreated with NET. Daily transvaginal ultrasound examinations were performed and human chorionic gonadotropin (hcg) was given when the lead follicle reached 16 to 17 mm in diameter. During oocyte retrieval, pure follicular fluid was obtained and stored at -20 C until assayed for NET, E2, and P. Granulosa Cell Function Norethindrone effects on acute in vitro ovarian steroidogenesis were assessed by establishing primary luteinized granulosa cell cultures. Pooled granulosa cells were obtained from follicular fluid aspirated during oocyte retrieval from patients stimulated with hmg and/or hcg, without prior exposure to NET. Granulosa cells from 10 to 15 mature follicles were obtained and pooled. The cells were washed three times with Media 199 Anderson et al. Biaavailability and effects af NET 97

3 mlufml _ SERUM FSH _ SERUM lh assay CVs were 8.7% and 7.0%, respectively. Estradiol was measured by a previously validated RIA after diethyl ether extraction and celite column chromatography.b Progesterone was measured by a previously validated RIA after diethyl ether extraction. 9 PRE 1 WK 2 WK LENGTH OF TREATMENT Figure 1 Levels of serum LH and FSH before and after 1 and 2 weeks of oral NET (10 mg/d) in four anovulatory women. Significant decreases were detected after 2 weeks for LH and after 1 and 2 weeks for FSH. * P < 0.05 compared with pretreatment levels. (Gibco Laboratories, Grand Island, NY) before quantitation by Trypan blue (Sigma Chemical Company, St_ Louis, MO) exclusion. One hundred thousand cells per well were grown for 48 hours to Media 199 with 10% fetal calf serum (FCS). The cells were then washed again and grown for an additional 24 hours in the presence of NET. Norethindrone, in concentrations of 0.1 ng/ml, 1.0 ng/ml, 10 ng/ml, and 100 ng/ml was added along with Media 199, 10% FCS, and hcg 30 ng/ml (obtaining a total volume of 1.0 ml/well). The incubation of cells with each concentration of NET was carried out in triplicate. Control preparations consisted of Media 199 with 10% FCS and hcg 30 ng/ml. After 24 hours, media from each well was aspirated and stored at 20 C until assayed fore2 and P. Cells were removed from the wells by gentle scraping and placed in phosphate-buffered saline. Protein content was determined by the Bicinchoninic acid protein assay (Pierce Chemical Company, Rockford, IL). Assays Luteinizing hormone and FSH were measured with double-antibody kits (Rapid LH and Rapid FSH; Serono Diagnostics, Norwell, MA). The sensitivity of both Rapid LH and Rapid FSH assays is 1.0 miu/ml of serum. Interassay and intra-assay coefficients of variation (CVs) were 9.5% and 5.5% for LH and 6.4% and 4.2% for FSH, respectively. Norethindrone was measured by a previously validated radioimmunoassay (RIA) after hexane extraction. There is no cross-reactivity with any other steroids in this assay. The sensitivity of the assay is 15 pg/ml of serum. 7 Interassay and intra- 98 Anderson et al. Bioavailability and effects of NET Statistics The data were analyzed by paired and unpaired t-tests, repeated measures analysis of variance (NET, LH, and FSH levels), and one-way analysis of variance (E 2 and P concentrations). RESULTS Hypothalamic-Pituitary Effects Norethindrone, when given daily to the four anovulatory women, significantly lowered both serum LH and FSH (Fig. 1). Luteinizing hormone decreased from 18.8 ± 4.4 miu /ml to 12.4 ± 2.9 miu/ml after 1 week, then to 7.4 ± 1.6 miu/ml (P < 0.05) after 2 weeks. Follicle-stimulating hormone fell from 7.9 ± 0.5 miu/ml to 4.8 ± 0.8 miu/ml (P < 0.05) after 1 week, then to 3.9 ± 0.3 miu/ml (P < 0.05) after 2 weeks. After 2 weeks of daily administration, NET was r~ TIME AFTER NET WITHDRAWAL (HAS) TIME AFTER NETWITHDRAWAL(HRS) Figure 2 Levels of serum NET (top panel) and gonadotropins (lower panel) after withdrawal of NET after 2 weeks of oral administration (10 mg/d) in 3 ovulatory infertile women. Levels of LH and FSH at 12, 24, 36, and 48 hours after withdrawal of NET were significantly lower than those of 10 ovulatory controls on cycle day 3. * P < 0.05 compared with cycle day 3 control levels. Fertility and Sterility

4 NET (NG/ML) NET (NG/GM) 16~ ~~~10 PERIPHERAL OVARIAN FOLLICULAR VEIN VEIN FLUID SOURCE o OVARIAN HOMOGENATE Figure 3 Levels of NET in peripheral blood, ovarian venous effluent, follicular fluid, and homogenates of ovarian tissue, 12 hours after oral ingestion of a 10-mg dose, in three women undergoing hysterectomy and oophorectomy. detected in serum up to 48 hours (1.3 ± 0.6 ng/ml) after the last dose (Fig. 2). The suppressed levels of LH and FSH were noted to persist during these 48 hours and, when compared with gonadotropin levels on day 3 of the natural cycle of control subjects, were significantly suppressed (5.9 ± 1.1 miu/ml versus 11.9 ± 1.7 miu/ml, P < 0.05, for LH; 3.9 ± 1.0 miu/ml versus 9.3 ± 2.1 miu/ml, P < 0.05, for FSH). Ovarian Effects A single 10-mg oral dose of NET, given 12 hours before elective hysterectomy and oophorectomy, resulted in levels of NET in peripheral venous blood (8.8 ± 1.9 ng/ml) that were similar to those of the infertile women 12 hours after ingesting (Fig. 2). Norethindrone levels were similar in ovarian venous effluents (10.5 ± 3.1 ng/ml), follicular fluid (7.1 ± 2.1 ng/ml), and in homogenates of ovarian tissue was 8.0 ± 0.6 ng/g (Fig. 3). Norethindrone was measured in the follicular fluid of the three women who underwent IVF after cycle programming with NET and compared with controls having comparable oocyte maturational status. Levels of 0.68 to 1.3 ng/ml were measured at the time of oocyte retrieval, which occurred up to 14 days after receiving the last dose of NET (Table I). Levels of E2 and P-in the follicular fluid of these patients fell within the 95% confidence limits of follicular fluid levels of control women not pretreated with NET.lO Production of E2 and P by granulosa cells was assessed with and without various doses of NET (Fig. 4). None of the doses of NET studied affected levels of P or E2 in conditioned media. Table 1 Follicular Fluid Steroid Concentrations From Programmed Oocyte Retrieval and Control IVF Cycles Steroid NET (pg/ml) E2 (ng/ml) P (pg/ml) "See reference 10. b ND, not done. Control" Programmed oocyte retrieval ND b 682 1, ± ± DISCUSSION Our data, although obtained in only a few subjects, suggest that NET, as used for programmed oocyte retrieval, inhibits endogenous gonadotropin secretion and does not appear to have a direct effect on ovarian steroidogenesis. This latter suggestion is made in spite of our findings that NET appears to be freely available to the ovary and persists in follicular fluid for at least 2 weeks. Twelve hours after a single, 10-mg oral dose of NET, levels were measured in follicular fluid (7.1 ± 2.1 ng/ml) and homogenates of ovarian tissue (8.0 ± 0.6 ng/ml). Norethindrone was still detect- CONT 0.1 to 10 NET lng/mil CXlNT to >00 NET (ng/mll Figure 4 Production of P (upper panel) and E2 (lower panel) by primary cultures of granulosa cells in untreated control preparations and after exposure to several concentrations of NET expressed as Itg/mL and pg/g of protein, respectively. There was no significant decrease in the production of either steroid at any concentration of NET compared with control preparations. Anderson et at. Bioavailability and effects of NET 99

5 T able in follicular fluid (863 ± 149 pg/ml) 2 weeks after its withdrawal. However, despite its presence in the ovary for prolonged periods, follicular fluid levels of E2 and P did not appear to be affected. The in vitro study of luteinized granulosa cells confirmed that NET in pharmacological concentrations was unable to acutely suppress the secretion of E2 and P. Since NET does not undergo bioactivation in vivo, this lack of suppression could not be explained on the basis of the absence of some required metabolic change in the NET molecule in vitro. A clearly inhibitory effect on endogenous gonadotropin secretion was demonstrable. With a 10-mg dose, we were able to measure significant levels of NET up to 48 hours after the last dose was administered. After daily administration of 2 weeks, endogenous secretion of both LH and FSH were inhibited and remained low up to 48 hours after the last dose. Whether this inhibition resulted from decreased GnRH -a pulsatility or a direct pituitary inhibition cannot be determined from this study. However, other recent data from our laboratory has demonstrated an inhibition of LH pulse frequency as well as the response of LH to GnRH-a with 10 mg of NET when administered to normal ovulatory women.ll Our data demonstrate that circulating NET was freely available to the ovary as evidenced by the absence of a gradient between ovarian venous effluent and peripheral serum levels. Yet although NET was not present in serum after 2 weeks, follicular fluid levels were still measurable. In fact, the eightfold decrease in follicular fluid concentration could be entirely accounted for by the dilutional effect of increased follicular fluid volume. This suggests that although NET readily gains access to the intrafollicular space, some unknown mechanism prevents later egress back into the peripheral circulation. This finding is consistent with recent studies demonstrating steroid sequestration by follicles with high intrafollicular E2 levels.12 Although we have only studied a few subjects, our data suggest that through an inhibitory effect on the hypothalamic-pituitary axis, NET indirectly induces ovarian quiescence. This in turn leads to prolongation of the stimulation phase during programmed oocyte retrieval cycles. Specifically, we hypothesize that attenuation of endogenous gonadotropin secretion induced by NET treatment during the late luteal phase disrupts the recruitment of a new cohort of follicles that would take place in the presence of a normal gonadotro- pin milieu. Compared with the ovary in a natural cycle, the ovary after suppression with NET would contain only small primordial follicles and would not possess the normal complement of secondary preantral follicles characteristically found in the early follicular phase. Therefore, compared with the ovary in a natural cycle, recruitment of follicles from an ovary after NET treatment would be relatively less efficient. More gonadotropin stimulation would be required over a longer period of time to enable follicles to attain preovulatory status. Since the daily amount of gonadotropins administered at the beginning of stimulation during the recruitment stage is the same as in conventional controlled ovarian hyperstimulation, an additional result would be that a lesser number of follicles is recruited. The observed reduction in steroidogenesis during programmed oocyte retrieval is likely due, therefore, to this lesser number of follicles rather than to a direct effect of NET on steroidogenesis. We cannot, however, rule out the possibility that NET may exert other inhibitory effects on the ovary or on other parts ofthe reproductive system. Although preliminary data suggest no adverse clinical effects,1,2,13 only long-term data will be able to confirm this. REFERENCES 1. Templeton A, Van Look P, Lumsden MA, Angell R, Aitken J, Duncan AW, Baird DT: The recovery of pre-ovulatory oocytes using a fixed schedule of ovulation induction and follicle aspiration. Br J Obstet GynaecoI91:148, Frydman R, Forman R, Rainhorn J-D, Belaisch-Allart J, Hazout A, Testart J: A new approach to follicular stimulation for in vitro fertilization: programmed oocyte retrieval. Fertil Steril46:657, Frydman R, Rainhorn J-D, Forman R, Belaisch-Allart J, Fernandez H, Lassalle B, Testart J: Programmed oocyte retrieval during routine laparoscopy and embryo cryopreservation for later transfer. Am J Obstet Gynecol 155:112, Thatcher SS, Boyle HP, Glasier AF, Hillier SG, Baird DT: A comparison of dosages of norethisterone for synchronization of cycles in a fixed regimen of follicular augmentation and in vitro fertilization. Fertil SteriI49:848, Anderson RE, Paulson RJ, Sauer MV, Lobo RA: Evidence supporting the routine use of a GnRH agonist (GnRH-a) during ovarian stimulation for in vitro fertilization (lvf). (Abstr. P149) Presented at the 44th Annual Meeting of The American Fertility Society, Atlanta, Georgia, October 8 to 13, Published in the Scientific Program and Abstracts by The American Fertility Society, 1988, p S93 6. Moudgal RN, Rao AJ, Murthy GSRC, Neelakanta R, Banavar SR, Kotagi SG, Kumar TCA: Effect of intranasal administration of norethisterone and progesterone on pituitary and gonadal function in adult male and female bonnet monkeys (Macaca radiata). Fertil Steril44:120, Anderson et al. Bioavailability and effects of NET Fertility and Sterility

6 7. Stanczyk FZ, Brenner PF, Mishell DR, Jr, Ortiz A, Gentzschein EKE, Goebelsmann U: A radioimmunoassay for norethindrone (NET): measurement of serum NET concentrations following ingestion of NET -containing oral contraceptive steroids. Contraception 18:615, Goebelsmann U, Bernstein GS, G(ile JA, Kletzky OA, Nakamura RM, Coulson AH, Korelitz JJ: Serum gonadotropin, testosterone, estradiol and estrone levels prior to and following bilateral vasectomy. In Vasectomy: Immunologic and Pathophysiologic Effects in Animals and Man, Edited by IH Leprow, R Crozier. New York, Academic Press, 1979, p Scott JZ, Stanczyk FZ, Goebelsmann U, Mishell DR, Jr: A double-antibody radioimmunoassay for serum progesterone using progesterone-3-(o-carboxymethyl)oximino [ 125 Il-iodohistamine as radioligand. Steroids 31:393, 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 follicle maturation. J Clin Endocrinol Metab 58:704, Levin JH, Anderson RE, Stanczyk FZ, Lobo RA: Characteristics of progestin (P) inhibition of gonadotropin secretion in normal women. (Abstr. 419) Presented at the 36th Annual Meeting of the Society For Gynecologic Investigation, San Diego, California, March 15 to 18, Published in the Scientific Program and Abstracts by the Society For Gynecologic Investigation, 1989, p Zimmermann RC, Westhof G, Hoedemaker M, Grunert E, Braendle W: Is follicular fluid steroid content representative of follicular steroid secretion? Hum Reprod 4:29, Gerli S, Remohi J, Partrizio P, Borrero C, Balmaceda JP, Silber SJ, Asch RH: Programming of ovarian stimulation with norethindrone acetate in IVF/GIFT cycles. Hum Reprod 4:746, 1989 Anderson et al. Bioavailability and effects of NET 101