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Effect of oral contraceptives or dexamethasone on plasma β-endorphin during the menstrual cycle*
FERTILITY AND STERILITY
Vol. 51, No.1, January 1989
Printed in U.S.A.
Copyright<> 1989 The American Fertility Society
Effect of oral contraceptives or dexamethasone on plasma P-endorphin during the menstrual cycle* Giuseppina Comitini, M.D. Felice Petraglia, M.D.t Fabio Facchinetti, M.D.
Michele Monaco, M.D. Annibale Volpe, M.D. Andrea Riccardo Genazzani, M.D.
Department of Obstetrics and Gynecology, University of Modena, Modena, Italy
Several studies have showed a significant increase of plasma {3-endorphin levels during the periovulatory days of the menstrual cycle. The aim of the present study was to investigate the origin of the periovulatory changes of plasma {3-endorphin, trying to discriminate between a possible ovarian and/or pituitary origin. Daily plasma {3-endorphin, luteinizing hormone (LH), and cortisol levels were measured from the 8th to the 20th day of the menstrual cycle in healthy normal-cycling women (10 cases) before and during dexamethasone (DEX; 6 cases) or estroprogestinic treatment with monophasic (5 cases) or triphasic (5 cases) pill. In the control menstrual cycle, during the preovulatory days, a significant increase of plasma {3-endorphin was found. While oral contraceptives abolished the midcycle increase of plasma {3-endorphin, the periovulatory plasma {3-endorphin peak was present during DEX treatment. Plasma cortisol levels did not show any significant change throughout the control menstrual cycle, while they were significantly lowered by the DEX administration and significantly increased during estroprogestinic treatment. These results suggest that the increase of plasma {3-endorphin during the periovulatory days is related to the ovulatory function, and suggest a possible ovarian origin. Fertil Steril51: 46,1989
Several studies have showed that endogenous opioid peptides play a role in the control of the reproductive function. {3-endorphin, a potent endogenous opioid peptides, is independently synthesized in brain, pituitary, and gonads. 1 Hypothalamic {3endorphin inhibits luteinizing hormone (LH) secretion, acting on the pulsatile release of gonadotropin-releasing hormone (GnRH) from the mediobasal hypothalamus, 2-5 and this central activity is gonadal steroid-modulated. 6 •7 Circulating {3-endorphin does not seem to play a role in the mechanisms of control of LH secretion, and its target is still unknown. However, a possible role of plasma {3-endorphin in reproductive function is suggested Received March 7, 1988; revised and accepted September 2, 1988. *Supported in part by the Consiglio Nazionale delle Ricerche, gruppo Endocrinologia. t Reprint requests: Felice Petraglia, M.D., Department of Obstetrics and Gynecology, University of Modena, Via del Pozzo 71, 41100 Modena, Italy.
46
Comitini et al.
{3-endorphin and menstrual cycle
by the evidence that gonadal steroids influence plasma {3-endorphin levels. Indeed, plasma {3-endorphin levels in prepubertal girls8 and in postmenopausal women are lower than in adult subjects.9·10 The evidence that, in healthy women, plasma {3-endorphin levels change during the menstrual cycle, with the highest values during the periovulatory phase, 11 •12 further supports the relation between gonads and plasma {3-endorphin. No changes of plasma {3-endorphin are found in anovulatory patients, 12 but a midcycle peak is restored after induction of ovulation (with pulsatile GnRH or gonadotropins)P However, the mechanisms causing this periovulatory peak of plasma {3-endorphin are unknown. Pituitary corticotropes are the major sources of plasma {3-endorphin in humans 13; and actually, plasma {3-endorphin results in hypothalamus-pituitary-adrenal axis hormone. Indeed, corticotropin-releasing factor increases and glucocorticoids decrease plasma {3-endorphin, and circadian changes. of plasma {3-endorphin are similar to Fertility and Sterility
those of hypothalamus-pituitary-adrenal axis hormones.14-16 The recent findings that [j-endorphin concentrations in ovarian follicular fluid are 10 to 15 times higher than in plasma/ 7- 19 and that LH stimulates messenger rybonucleic acid (mRNA) for [j-endorphin in the ovary, 20 suggest that the ovary may contribute to circulating the [j-endorphin pool. However, in view of the stimulatory effect of estrogen on plasma [j-endorphin, 21- 23 a possible cause of plasma [j-endorphin changes during periovulatory phase may also be the typical midcycle increase of the plasma estradiol (E 2) levels. The aim of this study was to investigate the origin of periovulatory changes of plasma [j-endorphin, discriminating between the possible contributions of the pituitary gland and/or the mature ovarian follicles. With this purpose, we measured plasma [j-endorphin levels in healthy women during the menstrual cycle, and before and during DEX or oral estroprogestinic contraceptive (monophasic and triphasic pill) treatments. MATERIALS AND METHODS Subjects
Ten healthy women (age range, 18 to 24 years) participated in the present study. They were graduate students or technicians at our Medical Center, and were requested to give an informal written consent. The diagnosis of ovulatory cycles was previously made using basal body temperature (BBT) curves and echographic monitoring of folliculogenesis. Protocol
A control menstrual cycle was studied in all subjects, collecting samples from the 8th to the 20th day of the menstrual cycle. The next month, six of these women were studied during treatment with dexamethasone (DEX, Decadron, Merck Sharp & Dohme, B. V. Haarlem, The Netherlands; 1 mg/ day by mouth at 8 A.M. and 11 P.M., from the 8th to the 18th day of the menstrual cycle). Blood samples were collected with the same schedule as in the control menstrual cycle. After 3 more months, subjects were subdivided in two groups of five women, and treated with two different oral contraceptives (OCs): monophasic pill (Planum, Menarini, Florence, Italy; 30 J.Lg of ethinyl~estradiol [EE2] and 150 .J.Lg of desogestrel) or triphasic pill (Trigynon, Schering, Berlin, FRG; 6 days: 30 J.Lg of EE 2 and 50 J.Lg of levonorgestrel; 5 days: 40 J.Lg of EE 2 and 75 J.Lg Vol. 51, No.1, January 1989
of levonorgestrel; 10 days: 30 J.Lg EE 2 and 125 J.Lg of levonorgestrel). During the 2nd month of treatment, daily blood samples were collected from the 8th to the 20th day of the menstrual cycle. All blood samples were collected between 8:00 A.M. and 8:30A.M., after 15 minutes from the cannulation of the forearm vein. Blood was collected in plastic tubes containing heparin and trasylol (1000 IKU /ml), immediately centrifuged, and the plasma stored in aliquots at -20oC until assay. Radioimmunoassays
Each plasma sample was assayed by double antibody radioimmunoassay (RIA) for [j-endorphin, LH, and cortisol. Plasma [j-endorphin levels were measured after silicic acid extraction and Sephadex G-75 column chromatography, as previously described. 8 Anti-C-terminal [j-endorphin serum (1 to 30K final dilution) was employed. It was 100% crossreactive with human [j-endorphin, 97% with human [j-lipotropin, and 2% with a- and )'-endorphin, and did not cross-react with met-enkephalin, adrenocorticotropic hormone (ACTH), leucine-enkephalin, or 'Y-lipotropin. Synthetic [j-endorphin (Organon, Oss, The Netherlands; [j-lipotropin, 61 to 91) was used in the preparation of the standard curve and for labeling with 125I (chloramine-T method). Double-antibody RIA were performed with the antisera (100 J.Ll), followed by an 18-hour incubation with tracer (100 J.Ll} and an additional 12 hours with the precipitating rabbit gamma globulin serum (1 to 15). The sensitivity of the assay was 2.5 fmoljtube for [j-endorphin. The interassay and intra-assay coefficients of variation at 50% binding (evaluated on 7 different samples) were 9.5 ± 1.1% and 6.6 ± 0.8%, respectively. Plasma LH and cortisol levels were measured by double-antibody RIA, using materials provided by Radim (Rome, Italy). [j-endorphin concentrations are reported as fmol/ml, whereas LH and cortisol levels are expressed as miU /ml and ng/ml, respectively. Statistical Analysis
Statistical analysis of results were performed using the analysis of variance and the Duncan test for multiple comparison. RESULTS
In the control menstrual cycle, all subjects showed the typical midcycle surge of plasma LH Comitini et al.
{3-endorphin and menstrual cycle
47
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were significantly lower in comparison with those found in the control menstrual cycle (P < 0.01), and the midcycle LH surge was absent (Fig. 4). No significant difference in basal plasma {3-endorphin levels between the control and the oral contraceptive menstrual cycle were observed. However, during the cycle of treatment with monophasic or triphasic pill, no significant midcycle increase of plasma {3-endorphin was found. During OC cycle treatments, plasma cortisol levels were significantly higher than those found during the control cycle. Plasma cortisol ranged between 24.2 ± 2.3 and 29.6 ± 3. 7 during the cycle with monophasic pill and between 22.0 ± 6.3 and 29.0 ± 7.5 during the cycle with the triphasic pill. DISCUSSION
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Figure 1 LH (miU/ml, upper panel) andP-endorphin (fmol/ ml, lower panel) (mean ± SE) plasma levels in control healthy women (n = 10) during the menstrual cycle with reference to midcycle surge of plasma LH (day 0).
levels (Figs. 1 and 2). Mean± standard error (SE) of plasma {3-endorphin levels during the menstrual cycle ranged between 6.1 ± 1.5 and 10.0 ± 3.2 fmolj ml, and 2 days before the plasma LH rise, {3-endorphin levels significantly increased (28.3 ± 7.8; P < 0.01 versus midfollicular and early and midluteal phase; Fig. 1). No significant changes of plasma cortisol levels (range, 10.5 ± 0.5 and 18.6 ± 2.9 ng/ ml) were observed during the control menstrual cycle. During DEX treatment, a normal midcycle LH surge was observed and no changes of basal plasma levels were found, in comparison with the control cycle (Fig. 2). Dexamethasone treatment significantly decreased mean basal plasma {3-endorphin levels since the 2nd day oftreatment (P < 0.01) and the preovulatory {3-endorphin peak (P < 0.01; Fig. 2). In particular, the plasma {3-endorphin peak was observed in five of six subjects, a lower result than that found in the control menstrual cycle (Fig. 3). Plasma cortisol levels were significantly lowered by the DEX administration and ranged between 1.9 ± 0.3 and 2.9 ± 0.8 ng/ml. During estroprogestinic treatment with both monophasic or triphasic pill, plasma LH levels 48
Comitini et al.
The present study showed that, while OCs abolished the midcycle increase of plasma {3-endorphin in healthy women, a periovulatory plasma {3-endorphin peak was present during DEX treatment. The evidence that the blockade of ovulation by contraceptive pill is associated with the lack of both plasma LH and {3-endorphin surge suggests a relationship between the midcycle surges of these hormones. It is known that contraceptive steroids in-
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Figure 2 LH (miU/ml, upper panel) and P-endorphin (fmol/ ml, lower panel) (mean ± SE) plasma levels in control healthy women (n = 6) during the menstrual cycle (dashed line) and during DEX treatment (solid line) with reference to midcycle surge of plasma LH (day 0).
P-endorphin and menstrual cycle Fertility and Sterility
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tween the increase of !3-endorphin and cyclic changes of gonadal steroids. Indeed, in normal condition, during the menstrual cycle, gonadal steroids change with a time pattern that may not be mimicked by the pharmacologic treatment. Therefore, in physiologic conditions, the gonadal steroids may have a role in determining the midcycle increase of plasma !3-endorphin. Conversely, the DEX-induced decrease of basal plasma !3-endorphin and cortisol confirms that plasma !3-endorphin is a part of the hypothalamuspituitary-adrenal axis. However, a midcycle plasma !3-endorphin peak was observed in five of six subjects treated with DEX, though the rise was lower than that found during the control menstrual cycle. Therefore, it may be suggested that the midcycle surge of !3-endorphin originated from a nonDEX-sensitive source. Among the possible other sources of plasma !3endorphin in humans, the intermediate lobe of pituitary gland, the lymphocytes, or the gonads have been reported. 1 •13 The present data, showing the close correlation between ovulation and plasma !3endorphin during the preovulatory days, suggest an ovarian origin of this peak. There are no data on the glucocorticoid effect on ovarian !3-endorphin
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Figure 3 LH (miU/ml, left panel) and !3-endorphin (fmol/ ml, right panel) (mean ± SE) plasma levels in control healthy women (n = 6) during the menstrual cycle (dashed line) and in subjects treated with DEX (solid line) from the 11th to the 18th day of the menstrual cycle.
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hibit the ovulation acting on the hypothalamus-pituitary-ovary axis. 24 Recent studies have shown that the chronic administration of OCs decreased pituitary !3-endorphin concentrations25 and hypothalamic GnRH and plasma LH levels in rat. 24•25 Therefore, the present results correlate the presence of plasma !3-endorphin rise with ovulation. Moreover, the evidence that both monophasic and triphasic pill blocked the midcycle !3-endorphin surge, may exclude indirectly a single relation beVol. 51, No.1, January 1989
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Figure 4 LH (miU/ml, upper panel) and !3-endorphin (fmol/ ml, lower panel) (mean ± SE) plasma levels in control healthy women during the menstrual cycle (circles) and during estroprogestinic treatment with monophasic (n = 5, squares) and triphasic (n = 5, asterisks) pill. Comitini et al.
!3-endorphin and menstrual cycle
49
secretion. Indeed, recent findings have shown that {j-endorphin is present in ovarian granulosa cells and that LH stimulates mRNA for {j-endorphin in the ovary. 20•25 The evidence that ovarian follicular fluid contains {j-endorphin concentrations higher than in peripheral blood supports a local secretion of ovarian {j-endorphin. 17 Moreover, the presence of the highest follicular fluid {j-endorphin concentrations during the preovulatory days 18 indicates that {j-endorphin secretion increases during these days, and this might be hypothesized to contribute to plasma {j-endorphin pool. Moreover, the association between the ovulation, the LH surge, and the increase of plasma {j-endorphin has a physiologic significance to be elucidated. In conclusion, the present study, showing that the treatment with OCs, but not with DEX, blocks the preovulatory surge of plasma {j-endorphin, suggests that in women, during menstrual cycles, the ovulation process determines the midcycle increase of plasma {j-endorphin.
16.
Acknowledgment. Cho H. Li, Ph.D. kindly supplied the antiserum to {3-endorphin.
17.
10.
11.
12.
13.
14.
15.
REFERENCES 1. Facchinetti F, Petraglia P, Genazzani AR: Localization and expression of the three opioid systems. Semin Reprod Endocrinol5:104, 1987 2. Ferin M, Van Vught D, Wardlaw S: The hypothalamic control of the menstrual cycle and role of endogenous opioid peptides. Rec Prog Horm Res 40:441, 1984 3. Morley JE: Neuroendocrine effects of endogenous opioid peptides in human subjects: a review. Psychoneuroendocrinology 39:361, 1983 4. Yen SSC, Quigley ME, ReidRL, RopertJF, CetelNS: Neuroendocrinology of opioid peptides and their role in the control of gonadotropin and prolactin secretion. Am J Obstet Gynecol15:485, 1985 5. Petraglia F, Porro C, Facchinetti F, Cicoli C, Bertellini E, Volpe A, Barbieri GC, Genazzani AR: Opioid control of LH secretion in humans: menstrual cycle, menopause and aging reduce effect of naloxone but not morphine. Life Sci 38:2103, 1986 6. Quigley ME, Yen SSC: The role of endogenous opiates on LH secretion during the menstrual cycle. J Clin Endocrinol Metab 51:179, 1980 7. Petraglia F, D'Ambrogio G, Comitini G, Facchinetti F, Volpe A, Genazzani AR: Impairment of opioid control of luteinizing hormone secretion in menstrual disorders. Fertil Steril 43:534, 1985 8. Genazzani AR, Facchinetti F, Pintor C, Parrini D, Petraglia F, Bagnoli F, Corda R: Proopiocortin-related peptide plasma levels throughout prepuberty and puberty. J Clin Endocrinol Metab 57:56, 1983 9. Genazzani AR, Facchinetti F, Ricci-Danero G, Parrini D,
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{3-endorphin and menstrual cycle
18.
19. 20.
21.
22.
23.
24.
Petraglia F, La Rosa R, D'Antona N: Beta-lipotropin and beta-endorphin in physiological and surgical menopause. J Endocrinol Invest 4:375, 1981 Aleem FA, Mcintosh TK: Menopausal syndrome: plasma levels of beta-endorphin in post-menopausal women measured by a specific radioimmunoassay. Maturitas 7:329, 1985 Vrbicky KW, Baumstark JS, Wells IC, Hilgers TW, Kable WT, Elias CJ: Evidence for the involvement of {3-endorphin in the human menstrual cycle. Fertil Steril38:701, 1982 Petraglia F, DiMeo G, De Leo V, Nappi C, Facchinetti F, Genazzani AR: Plasma {3-endorphin levels in anovulatory states: changes after treatments for the induction of ovulation. Fertil Steril45:185, 1986 Krieger DT, Liotta AS, Brownstein MJ, Zimmermann EA: ACTH, {3-lipotropin and related peptides in brain, pituitary and blood. Rec Prog Horm Res 36:377, 1980 McLoughlin L, Tomlin S, Grossman A, Lytras N, Schally AV, Coy D, Besser GM, Rees LH: CRF-41 stimulates the release of {3-lipotropin and {3-endorphin in normal human subjects. Neuroendocrinology 38:282, 1984 Nakao F, Oki S, Tanaka I, Nakai Y, Himura H: Concomitant secretion of {3-MSH with ACTH and {3-Endorphin in humans. J Clin Endocrinol Metab 51:1205, 1980 Petraglia F, Facchinetti F, Parrini D, Miceli G, DeLuca S, Genazzani AR: Simultaneous circadian variations of plasma ACTH, {3-lipotropin, {3-endorphin and cortisol. Horm Res 17:14 7, 1983 Petraglia F, Segre A, Facchinetti F, Campanini D, Ruspa M, Genazzani AR: {3-endorphin and met-enkephalin in peritoneal and ovarian fluids of fertile and postmenopausal women. Fertil Steril 44:615, 1985 Petraglia F, DiMeo G, Storchi R, Segre A, Facchinetti F, Szalay S, Genazzani AR: Proopiomelanocortin-related peptides and methionine enkephalin in human follicular fluid: changes during the menstrual cycle. Am J Obstet Gynecol157:142, 1987 Aleem FA, Omar RA, El Tabbakh GH: Immunoreactive {3endorphin in human ovaries. Fertil Steril 45:507, 1986 Meiner MH, Young SL, Czerwiec FS, Lyn D, Puell D, Roberts JL, Koos RD: The regulation of granulosa cell proopiomelanocortin in menenger ribonucleic acid by androgens and gonadotropins. Endocrinology 119:2082, 1986 Petraglia F, Penalva A, Locatelli V, Cocchi D, Panerai AE, Genazzani AR, Muller EE: Effect of nonadectomy and gonadal steroid replacement on pituitary and plasma {3-endorphin levels in the rat. Endocrinology 111:1224, 1982 Forman LJ, Sonntan VW, Meites J: Immunoreactive betaendorphin in the plasma, pituitary and hypothalamus of young female rats on the day of estrous and intact and chronically castrated old constant female rats. Life Sci 33: 993,1983 Aleem FA, Mcintosh T: Elevated plasma levels of {3-endorphin in a group of women with polycystic ovarian disease. Fertil Steril 42:686, 1984 Henzl MR: Contraceptive hormones and their clinical use. In Reproductive Endocrinology, Edited by SSC Yen, RB Yaffe. Philadelphia, W. B. Saunders Company, 1986, p 643
25. Tejwani GA, Vaswani KK, Barbacci JC: Effect of oral contraceptives on the rat brain and pituitary opioid peptides. Peptides 6:555, 1985
Fertility and Sterility
Vol. 51, No.1, January 1989
Printed in U.S.A.
Copyright<> 1989 The American Fertility Society
Effect of oral contraceptives or dexamethasone on plasma P-endorphin during the menstrual cycle* Giuseppina Comitini, M.D. Felice Petraglia, M.D.t Fabio Facchinetti, M.D.
Michele Monaco, M.D. Annibale Volpe, M.D. Andrea Riccardo Genazzani, M.D.
Department of Obstetrics and Gynecology, University of Modena, Modena, Italy
Several studies have showed a significant increase of plasma {3-endorphin levels during the periovulatory days of the menstrual cycle. The aim of the present study was to investigate the origin of the periovulatory changes of plasma {3-endorphin, trying to discriminate between a possible ovarian and/or pituitary origin. Daily plasma {3-endorphin, luteinizing hormone (LH), and cortisol levels were measured from the 8th to the 20th day of the menstrual cycle in healthy normal-cycling women (10 cases) before and during dexamethasone (DEX; 6 cases) or estroprogestinic treatment with monophasic (5 cases) or triphasic (5 cases) pill. In the control menstrual cycle, during the preovulatory days, a significant increase of plasma {3-endorphin was found. While oral contraceptives abolished the midcycle increase of plasma {3-endorphin, the periovulatory plasma {3-endorphin peak was present during DEX treatment. Plasma cortisol levels did not show any significant change throughout the control menstrual cycle, while they were significantly lowered by the DEX administration and significantly increased during estroprogestinic treatment. These results suggest that the increase of plasma {3-endorphin during the periovulatory days is related to the ovulatory function, and suggest a possible ovarian origin. Fertil Steril51: 46,1989
Several studies have showed that endogenous opioid peptides play a role in the control of the reproductive function. {3-endorphin, a potent endogenous opioid peptides, is independently synthesized in brain, pituitary, and gonads. 1 Hypothalamic {3endorphin inhibits luteinizing hormone (LH) secretion, acting on the pulsatile release of gonadotropin-releasing hormone (GnRH) from the mediobasal hypothalamus, 2-5 and this central activity is gonadal steroid-modulated. 6 •7 Circulating {3-endorphin does not seem to play a role in the mechanisms of control of LH secretion, and its target is still unknown. However, a possible role of plasma {3-endorphin in reproductive function is suggested Received March 7, 1988; revised and accepted September 2, 1988. *Supported in part by the Consiglio Nazionale delle Ricerche, gruppo Endocrinologia. t Reprint requests: Felice Petraglia, M.D., Department of Obstetrics and Gynecology, University of Modena, Via del Pozzo 71, 41100 Modena, Italy.
46
Comitini et al.
{3-endorphin and menstrual cycle
by the evidence that gonadal steroids influence plasma {3-endorphin levels. Indeed, plasma {3-endorphin levels in prepubertal girls8 and in postmenopausal women are lower than in adult subjects.9·10 The evidence that, in healthy women, plasma {3-endorphin levels change during the menstrual cycle, with the highest values during the periovulatory phase, 11 •12 further supports the relation between gonads and plasma {3-endorphin. No changes of plasma {3-endorphin are found in anovulatory patients, 12 but a midcycle peak is restored after induction of ovulation (with pulsatile GnRH or gonadotropins)P However, the mechanisms causing this periovulatory peak of plasma {3-endorphin are unknown. Pituitary corticotropes are the major sources of plasma {3-endorphin in humans 13; and actually, plasma {3-endorphin results in hypothalamus-pituitary-adrenal axis hormone. Indeed, corticotropin-releasing factor increases and glucocorticoids decrease plasma {3-endorphin, and circadian changes. of plasma {3-endorphin are similar to Fertility and Sterility
those of hypothalamus-pituitary-adrenal axis hormones.14-16 The recent findings that [j-endorphin concentrations in ovarian follicular fluid are 10 to 15 times higher than in plasma/ 7- 19 and that LH stimulates messenger rybonucleic acid (mRNA) for [j-endorphin in the ovary, 20 suggest that the ovary may contribute to circulating the [j-endorphin pool. However, in view of the stimulatory effect of estrogen on plasma [j-endorphin, 21- 23 a possible cause of plasma [j-endorphin changes during periovulatory phase may also be the typical midcycle increase of the plasma estradiol (E 2) levels. The aim of this study was to investigate the origin of periovulatory changes of plasma [j-endorphin, discriminating between the possible contributions of the pituitary gland and/or the mature ovarian follicles. With this purpose, we measured plasma [j-endorphin levels in healthy women during the menstrual cycle, and before and during DEX or oral estroprogestinic contraceptive (monophasic and triphasic pill) treatments. MATERIALS AND METHODS Subjects
Ten healthy women (age range, 18 to 24 years) participated in the present study. They were graduate students or technicians at our Medical Center, and were requested to give an informal written consent. The diagnosis of ovulatory cycles was previously made using basal body temperature (BBT) curves and echographic monitoring of folliculogenesis. Protocol
A control menstrual cycle was studied in all subjects, collecting samples from the 8th to the 20th day of the menstrual cycle. The next month, six of these women were studied during treatment with dexamethasone (DEX, Decadron, Merck Sharp & Dohme, B. V. Haarlem, The Netherlands; 1 mg/ day by mouth at 8 A.M. and 11 P.M., from the 8th to the 18th day of the menstrual cycle). Blood samples were collected with the same schedule as in the control menstrual cycle. After 3 more months, subjects were subdivided in two groups of five women, and treated with two different oral contraceptives (OCs): monophasic pill (Planum, Menarini, Florence, Italy; 30 J.Lg of ethinyl~estradiol [EE2] and 150 .J.Lg of desogestrel) or triphasic pill (Trigynon, Schering, Berlin, FRG; 6 days: 30 J.Lg of EE 2 and 50 J.Lg of levonorgestrel; 5 days: 40 J.Lg of EE 2 and 75 J.Lg Vol. 51, No.1, January 1989
of levonorgestrel; 10 days: 30 J.Lg EE 2 and 125 J.Lg of levonorgestrel). During the 2nd month of treatment, daily blood samples were collected from the 8th to the 20th day of the menstrual cycle. All blood samples were collected between 8:00 A.M. and 8:30A.M., after 15 minutes from the cannulation of the forearm vein. Blood was collected in plastic tubes containing heparin and trasylol (1000 IKU /ml), immediately centrifuged, and the plasma stored in aliquots at -20oC until assay. Radioimmunoassays
Each plasma sample was assayed by double antibody radioimmunoassay (RIA) for [j-endorphin, LH, and cortisol. Plasma [j-endorphin levels were measured after silicic acid extraction and Sephadex G-75 column chromatography, as previously described. 8 Anti-C-terminal [j-endorphin serum (1 to 30K final dilution) was employed. It was 100% crossreactive with human [j-endorphin, 97% with human [j-lipotropin, and 2% with a- and )'-endorphin, and did not cross-react with met-enkephalin, adrenocorticotropic hormone (ACTH), leucine-enkephalin, or 'Y-lipotropin. Synthetic [j-endorphin (Organon, Oss, The Netherlands; [j-lipotropin, 61 to 91) was used in the preparation of the standard curve and for labeling with 125I (chloramine-T method). Double-antibody RIA were performed with the antisera (100 J.Ll), followed by an 18-hour incubation with tracer (100 J.Ll} and an additional 12 hours with the precipitating rabbit gamma globulin serum (1 to 15). The sensitivity of the assay was 2.5 fmoljtube for [j-endorphin. The interassay and intra-assay coefficients of variation at 50% binding (evaluated on 7 different samples) were 9.5 ± 1.1% and 6.6 ± 0.8%, respectively. Plasma LH and cortisol levels were measured by double-antibody RIA, using materials provided by Radim (Rome, Italy). [j-endorphin concentrations are reported as fmol/ml, whereas LH and cortisol levels are expressed as miU /ml and ng/ml, respectively. Statistical Analysis
Statistical analysis of results were performed using the analysis of variance and the Duncan test for multiple comparison. RESULTS
In the control menstrual cycle, all subjects showed the typical midcycle surge of plasma LH Comitini et al.
{3-endorphin and menstrual cycle
47
-4
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0
+I
+2
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were significantly lower in comparison with those found in the control menstrual cycle (P < 0.01), and the midcycle LH surge was absent (Fig. 4). No significant difference in basal plasma {3-endorphin levels between the control and the oral contraceptive menstrual cycle were observed. However, during the cycle of treatment with monophasic or triphasic pill, no significant midcycle increase of plasma {3-endorphin was found. During OC cycle treatments, plasma cortisol levels were significantly higher than those found during the control cycle. Plasma cortisol ranged between 24.2 ± 2.3 and 29.6 ± 3. 7 during the cycle with monophasic pill and between 22.0 ± 6.3 and 29.0 ± 7.5 during the cycle with the triphasic pill. DISCUSSION
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day•
Figure 1 LH (miU/ml, upper panel) andP-endorphin (fmol/ ml, lower panel) (mean ± SE) plasma levels in control healthy women (n = 10) during the menstrual cycle with reference to midcycle surge of plasma LH (day 0).
levels (Figs. 1 and 2). Mean± standard error (SE) of plasma {3-endorphin levels during the menstrual cycle ranged between 6.1 ± 1.5 and 10.0 ± 3.2 fmolj ml, and 2 days before the plasma LH rise, {3-endorphin levels significantly increased (28.3 ± 7.8; P < 0.01 versus midfollicular and early and midluteal phase; Fig. 1). No significant changes of plasma cortisol levels (range, 10.5 ± 0.5 and 18.6 ± 2.9 ng/ ml) were observed during the control menstrual cycle. During DEX treatment, a normal midcycle LH surge was observed and no changes of basal plasma levels were found, in comparison with the control cycle (Fig. 2). Dexamethasone treatment significantly decreased mean basal plasma {3-endorphin levels since the 2nd day oftreatment (P < 0.01) and the preovulatory {3-endorphin peak (P < 0.01; Fig. 2). In particular, the plasma {3-endorphin peak was observed in five of six subjects, a lower result than that found in the control menstrual cycle (Fig. 3). Plasma cortisol levels were significantly lowered by the DEX administration and ranged between 1.9 ± 0.3 and 2.9 ± 0.8 ng/ml. During estroprogestinic treatment with both monophasic or triphasic pill, plasma LH levels 48
Comitini et al.
The present study showed that, while OCs abolished the midcycle increase of plasma {3-endorphin in healthy women, a periovulatory plasma {3-endorphin peak was present during DEX treatment. The evidence that the blockade of ovulation by contraceptive pill is associated with the lack of both plasma LH and {3-endorphin surge suggests a relationship between the midcycle surges of these hormones. It is known that contraceptive steroids in-
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Figure 2 LH (miU/ml, upper panel) and P-endorphin (fmol/ ml, lower panel) (mean ± SE) plasma levels in control healthy women (n = 6) during the menstrual cycle (dashed line) and during DEX treatment (solid line) with reference to midcycle surge of plasma LH (day 0).
P-endorphin and menstrual cycle Fertility and Sterility
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tween the increase of !3-endorphin and cyclic changes of gonadal steroids. Indeed, in normal condition, during the menstrual cycle, gonadal steroids change with a time pattern that may not be mimicked by the pharmacologic treatment. Therefore, in physiologic conditions, the gonadal steroids may have a role in determining the midcycle increase of plasma !3-endorphin. Conversely, the DEX-induced decrease of basal plasma !3-endorphin and cortisol confirms that plasma !3-endorphin is a part of the hypothalamuspituitary-adrenal axis. However, a midcycle plasma !3-endorphin peak was observed in five of six subjects treated with DEX, though the rise was lower than that found during the control menstrual cycle. Therefore, it may be suggested that the midcycle surge of !3-endorphin originated from a nonDEX-sensitive source. Among the possible other sources of plasma !3endorphin in humans, the intermediate lobe of pituitary gland, the lymphocytes, or the gonads have been reported. 1 •13 The present data, showing the close correlation between ovulation and plasma !3endorphin during the preovulatory days, suggest an ovarian origin of this peak. There are no data on the glucocorticoid effect on ovarian !3-endorphin
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Figure 3 LH (miU/ml, left panel) and !3-endorphin (fmol/ ml, right panel) (mean ± SE) plasma levels in control healthy women (n = 6) during the menstrual cycle (dashed line) and in subjects treated with DEX (solid line) from the 11th to the 18th day of the menstrual cycle.
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hibit the ovulation acting on the hypothalamus-pituitary-ovary axis. 24 Recent studies have shown that the chronic administration of OCs decreased pituitary !3-endorphin concentrations25 and hypothalamic GnRH and plasma LH levels in rat. 24•25 Therefore, the present results correlate the presence of plasma !3-endorphin rise with ovulation. Moreover, the evidence that both monophasic and triphasic pill blocked the midcycle !3-endorphin surge, may exclude indirectly a single relation beVol. 51, No.1, January 1989
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Figure 4 LH (miU/ml, upper panel) and !3-endorphin (fmol/ ml, lower panel) (mean ± SE) plasma levels in control healthy women during the menstrual cycle (circles) and during estroprogestinic treatment with monophasic (n = 5, squares) and triphasic (n = 5, asterisks) pill. Comitini et al.
!3-endorphin and menstrual cycle
49
secretion. Indeed, recent findings have shown that {j-endorphin is present in ovarian granulosa cells and that LH stimulates mRNA for {j-endorphin in the ovary. 20•25 The evidence that ovarian follicular fluid contains {j-endorphin concentrations higher than in peripheral blood supports a local secretion of ovarian {j-endorphin. 17 Moreover, the presence of the highest follicular fluid {j-endorphin concentrations during the preovulatory days 18 indicates that {j-endorphin secretion increases during these days, and this might be hypothesized to contribute to plasma {j-endorphin pool. Moreover, the association between the ovulation, the LH surge, and the increase of plasma {j-endorphin has a physiologic significance to be elucidated. In conclusion, the present study, showing that the treatment with OCs, but not with DEX, blocks the preovulatory surge of plasma {j-endorphin, suggests that in women, during menstrual cycles, the ovulation process determines the midcycle increase of plasma {j-endorphin.
16.
Acknowledgment. Cho H. Li, Ph.D. kindly supplied the antiserum to {3-endorphin.
17.
10.
11.
12.
13.
14.
15.
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25. Tejwani GA, Vaswani KK, Barbacci JC: Effect of oral contraceptives on the rat brain and pituitary opioid peptides. Peptides 6:555, 1985
Fertility and Sterility