Elevated bioactive luteinizing hormone in women with the polycystic ovary syndrome*

Vol. 39, No.5, May 1983

FERTILITY AND STERILITY Copyright © 1983 The American Fertility Society

Printed in U.SA.

Elevated bioactive luteinizing hormone in women with the polycystic o'VRry syndrome*

Rogerio A. L~bo, M.D. t Oscar A. Kletzky, M.D. Joseph D. Campeau, M.S. Gere S. diZerega, M.D. Department of Obstetrics and Gynecology, University of Southern California School of Medicine, Los Angeles County-University of Southern California Medical Center, Women's Hospital, Los Angeles, California

Serum measurements of bioactive (bio) luteinizing hormone (LH), immunoreactive (i) LH, iLHlfollicle-stimulating hormone (FSH) ratios, serum androgens and estradiol (E~ were determined in 20 women with the clinical diagnosis of the polycystic ovary syndrome (PCa), and compared with the levels of 10 women with chronic anovulation (CA) and 10 control subjects in the early follicular phase. Women with CA and control subjects had similar levels of E 2 , androgens, bioLH, iLH, and iLHI FSH ratios. Fourteen of 20 women with pca had levels of iLH exceeding 3 standard deviations (SD) of the levels of control women (21 mIUlml), and 13 of20 had iLHI FSH ratios above 32 (3 SD of control levels). Nineteen of 20 women, however, had bioLH levels above 70 mIUlml (3 SD of control levels). Mean levels for bioLH were 131 ± 18 in pca, 39 ± 3 in control subjects, and 40 ± 3 in women with CA. The ratio of bioLHliLH was 3.5 ± 0.4 in control subjects and 3.2 ± 0.3 in women with CA but significantly elevated in pca (4.6 ± 0.4, P < 0.05). There was, however, a significant positive correlation between bioLH and iLH values in pca (r = 0.64, P < 0.01). A significant correlation was found between bioLH and serum testosterone as well as between bioLH and serum dehydroepiandrosterone sulfate (DHEA-S) (P < 0.05), although no correlation was found between iLH and serum DHEA-S. Weight and obesity also did not correlate with either iLH or bioLH in women with pca and CA. These data suggest that bioLH may be an important hormonal marker in the clinical diagnosis of pca. Fertil Steril39:674, 1983

The polycystic ovary syndrome (PCO) has been well characterized by a constellation of clinical symptoms and signs, and by laboratory studies. 1 In recent years, the mere presence or absence of

Received September 13, 1982; revised and accepted December 9,1982. *Supported in part by grants from the Ford Foundation (690-0650A) and the National Institute of Child Health and Development (HD-00401). tReprint requests: Rogerio A. Lobo, M.D., Women's Hospital, Room 1M2, 1240 North Mission Road, Los Angeles, California 90033. 674

Lobo et al. Bioactive LH in PCO

enlarged cystic ovaries has become of less importance in making the diagnosis ofPCO. 1- 4 Perhaps the most striking biochemical abnormality in PCO has been the finding of inappropriate gonadotropin secretion, with patients exhibiting high levels of serum luteinizing hormone (LH).5 Some authors 3 , 6 have subdivided patients with PCO on the basis of whether or not LH levels were in· creased. As women with PCO may have lower levels of serum follicle-stimulating hormone (FSH), the ratio of LH/FSH has also been used as a useful laboratory indicator of inappropriate gonadotropin secretion for the diagnosis ofPCO. 7 In Fertility and Sterility

this study, we assessed the levels ofbioactive LH (bioLH) and correlated these findings with the measurements of immunoreactive LH (iLH), the LH/FSH ratio, and the androgen and estrogen levels of women with PCO. MATERIALS AND METHODS

Twenty consecutive female patients who had clinical diagnoses ofPCO were studied. The clinical diagnosis was based upon the careful history taking and examination by an experienced clinician without regard to hormonal data. These 20 women were diagnosed to have PCO based upon strict clinical criteria as described below. I - 7 Women diagnosed to have PCO were 18 to 26 years of age and had oligomenorrhea since menarche at 12 to 14.5 years of age. These women reported never having had cyclic menses during their lives and, in addition, were overweight and presented with the complaints of infertility and hirsutism. Hirsutism was considered to be mild to moderate in these women, and no woman exhibited virilization. All women were considered overweight or obese as defined by a Ponderal Index (p1)8 of less than 12. Ten of these women had enlarged ovaries noted at the time of initial pelvic examination. Ten women with a history of chronic anovulation (CA) who were not considered to have PCO were also studied. These women, 18 to 38 years of age, had cyclic menses in the past but presented with oligomenorrhea. Hirsutism was absent in these women. Women with CA had positive withdrawal bleeding in response to intramuscular progesterone and had previously been evaluated having the findings of normal follicular phase levels of serum estradiol (E 2 ) androstenedione (a 4 A), testosterone (T), and prolactin (PRL). These women were given the diagnosis of CA due to hypothalamic dysfunction and were candidates for clomiphene therapy. As a group, women with CA were also overweight, and their PIs were similar to those of women with PCO (mean, PI 11.8 ± 0.3 in CA and 11.3 ± 0.15 in PCO). Ten women, 20 to 33 years of age, who were studied in the early follicular phase (days 2 to 7) served as control subjects. The control women were considered to be ovulatory, as determined from histories of cyclic menses of 27 to 31 days' duration with 3 to 5 days of normal menstrual flow and molimina. Luteal phase levels of progesterone were> 3 ng/ml, as determined in previous studies of these women. They were taking no medications and had no symptoms or signs of anVol. 39, No.5, May 1983

drogen excess. None of these women were considered o'verweight, and the mean PI of these women was 12.4 ± 0.15. Fasting venous blood was obtained on at least 2 different days in each subject prior to any treatment and pooled prior to the time of analysis. Sera were separated and stored at - 20° C until the time of analysis for LH, FSH, PRL, T, unbound T, E 2 , unbound E 2 , a 4 A, androst-5ene-3~,17~-diol (A 2 ), dehydroepiandrosterone (DHEA), and its sulfate (DHEA_S).9-15 BioLH was measured by the technique described by Van Damme et al. 16 and as validated for measurement in female plasma. 17 In this assay we used the interstitial cells of 10-week-old Swiss-Webster mice. One mouse was used for approximately 50 assay tubes. The testes were expressed free from their tunica and placed in 30 ml of minimum essential medium, Dulbecco's modification of Eagle's medium without glutamine, Bio fluids #104. After stirring for 10 minutes, the mixture was funneled through nylon mesh (80 ILl) into a water bath for 60 minutes at 30° C. This incubation was carried out at 80 oscillations per minute in an atmosphere of 95% O2 and 5% CO 2 , The interstitial cells were obtained after centrifugation (400 x g for 10 minutes) and were resuspended in 5 ml of fresh culture medium and then placed on ice. Trypan blue, 100 ILl, was added to 100 ILl of the cell suspension for a check for viability, and the cells were counted on a hemocytometer. The resuspended cells were diluted to a concentration of 6 x 106 cells/ml, and 100 ILl of these diluted cells were added to each tube. Standards ofLH (2nd IRP), 1000 ng/ml to 3 ng/ml, and human chorionic gonadotropin (hCG) (Organon Pharmaceuticals, West Orange, NJ), 10 mIU/ml to 1 mIU/ml, were added to culture tubes. Unknowns were run at the equivalent of 1 ILl and 5 ILl of whole serum in duplicate. Media were used for all dilutions, and the final volume added to each tube containing the cells was 100 ILL Incubations of unknowns or standards and interstitial cells were carried out in a shaker bath at 34° C in an atmosphere of 95% O2 and 5% CO 2 for 3 hours. After 3 hours, the tubes were placed in a 60° C water bath for 30 minutes. At the end of this time, the tubes were frozen at - 20° C, and T was assayed the following day. T was measured by radioimmunoassay (RIA) by the use of a direct assay kit purchased from Partex, Santa Monica, CA. The T concentrations as determined by RIA were used to generate standard curves by linear regression with the use of both hCG and LH stanLobo et al. Bioactive LH in peD

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Figure 1 Serum measurement of iLH, LH/FSH ratios, and bioLH in control subjects, women with CA and women with PCO. Closed circles for women with PCO indicate values exceeding 3 SD of mean control levels.

dards. A requirement for validation was that the regression lines using both of these standards were parallel. This occurred in each assay. In addition, parallelism also existed between the dilutions of serum (1 1-11 and 5 1-11). The results were reproducible and accurate as assessed by replication of known values in different assays. The interassay coefficient of variation was 9.5%, and the sensitivity of the assay was 0.07 mIU per tube. The immunoreactive LH assay has been described previously9 and is a conventional direct double-antibody assay utilizing the 2nd IRP LH standard. Student's t-test and the linear regression test by the method of least squares were used for statistical analysis.

The bioLH/FSH ratios were similar in control subjects and women with CA (Fig. 2). Women with PCO had a mean bioLH/iLH ratio of 4.55 ± 0.42, which was significantly higher than the ratios of control subjects and women with CA (P < 0.05). There was a significant positive correlation between bioLH and iLH. In women with PCO, this correlation coefficient was 0.64 (P < 0.01). Neither weight nor the PI were correlated with the iLH or bioLH in women with PCO and CA. The androgen levels in the women with PCO were all significantly elevated, compared with control levels (P < 0.01); whereas women with CA had normal levels (Table 1). Unbound E2 was also significantly elevated (46.9 ± 5.8 pg/ml in women with PCO and 18 ± 2 pg/ml in control subjects (P < 0.005). Unbound E2 was 26 ± 5 pg/ml in women with CA, which was not significantly different from levels in control subjects. Among the androgen levels depicted in Table 1, only T showed a significant positive correlation with iLH (r = 0.66, P < 0.01). Similarly, there was a significant positive correlation between bioLH and T (r = 0.52, P < 0.01) but no significant correlation between bioLH and A4 A, A2 , or

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RESULTS The mean iLH levels, iLH/FSH ratios, and bioLH levels of the control subjects and women with CA were similar (Fig. 1). However, women with PCO had significantly higher levels of all three measurements, compared with control subjects and women with CA (P < 0.01); whereas all women had normal PRL levels. Seventeen women with PCO (70%) had iLH levels above 21 mIU/ml. This value was 3 standard deviations (SDs) from the mean of control women. Thirteen women (65%) had iLH/FSH ratios > 3.2, which exceeded 3 SD of control levels. However, 19 of the 20 women (95%) had elevated bioLH levels > 70 mIU/ml. This was also 3 SD from control levels. 676

Lobo et al. Bioactive LH in pca

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Table 1. Mean ± Standard Error of Serum T, Unbound T, CA andPCa

Control (n = 10) CA (n = 10) PCO (n = 20)

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0.37 ± 0.03 0.22 ± 0.06 0.53 ± 0.04

3.2 ± 0.4 3.3 ± 0.5 15.5 ± 3.2

1.2 ± 0.3 0.6 ± 0.2 2.2 ± 0.3

4.4 ± 0.6 3.1 ± 1.6 9.1 ± 1.8

1.7 ± 0.1 2.0 ± 0.5 3.3 ± 0.7

0.50 ± 0.08 0.60 ± 0.15 0.90 ± 0.10

DHEA. Unbound E2 levels in women with pca did not correlate significantly with either bioLH (r = 0.20) or the bioLH/iLH ratio (r = 0.01). There was a significant positive correlation between bioLH and serum DHEA-S in women with pca (r = 0.42, P < 0.05) but not in control subjects. Figure 3 depicts the correlation of bioLH and serum DHEA-S in normal women (open circles) and those with pca (closed circles) (r = 0.57, P < 0.01). In the ten women with CA, bioLH levels (28 to 59 mIU/ml) also correlated significantly with serum DHEA-S levels (0.21 to 4.0 f.Lg/ml) (r = 0.66, P < 0.05). Although bioLH correlated significantly with DHEA-S, there was no such correlation between iLH and DHEA-S (r = 0.22). DISCUSSION

This study demonstrates that when the clinical diagnosis of pca is made, the measurement of serum LH and the LHIFSH ratio confirms this diagnosis in no more than 70% of the cases. However, utilizing the measurement ofbioLH, 95% of women may be found to have an elevated level, indicating that bioLH may be a more usefullaboratory marker of pca in women who have suggestive clinical signs and symptoms. Women with pca have elevated serum levels of both ovarian and adrenal androgens. It has been shown that these levels of androgens are closely correlated with decreases in sex-hormonebinding globulin and increases in the unbound fraction ofT, A 2, and E 2.I8 Although the elevated iLH may help to explain the increased T levels, probably due to ovarian stromal production, the increased levels of adrenal androgens have remained largely unexplained. Adrenocorticotropic hormone levels are normal in pca and therefore do not explain the elevated serum DHEA-S levels. 19, 20 We report here that bioLH positively correlates with serum DHEA-S, which is quantitatively the most important circulating C19 steroid of primarily adrenal origin. 21 The positive correVol. 39, No.5, May 1983 I

DHEA, DHEA-S, and A2 in Control Subjects and Women with

lation between bioLH and serum DHEA-S represents a novel observation, especially because iLH showed no correlation. This finding may help explain in part the elevated adrenal androgen levels in women with pca. There is no evidence that the normal ovary secretes DHEA-S.2I However, studies with the human fetal adrenal have also failed to show a stimulatory effect with exogenously administered LH or hCG. 22 These findings suggest that the characteristics of endogenous bioLH may be different, or that the conditions in which bioLH is secreted may also be contributory to the findings of elevated adrenal androgen secretion. The increased ratios of bioLH/iLH in pca as compared with control levels and levels in women with CA suggest that women with pca in part may secrete an LH molecule with different biologic activity. Similar observations have been made in menopausal women. 23 Using slightly different techniques, Dufau et al. 24, 25 determined the biologic activity of LH in rhesus monkeys, normal premenopausal women during the menstrual cycle, castrated women, and normal men.

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Lobo et a1. Bioactive LH in pca

677

Whereas the ratio of bioLH to iLH was close to unity in normal women, with a slight rise noted at midcycle, this ratio was 2 to 3 in men and postmenopausal or castrated women. Furthermore, LH-releasing horIlione stimulation resulted in increased ratios of bioLH to iLH in the late follicular and luteal phases but not in the early follicular phase. When taken together, our results suggest that an elevated ratio of bioLH to iLH may occur during states of increased biosynthesis and release of gonadotropins such as in PCO. Data such as these may lead to further understanding of the pathophysiology of PCO. REFERENCES 1. Yen SSC: The polycystic ovary syndrome. Clin Endocrinol (Oxf) 12:177, 1980 2. Goldzieher JW: Polycystic ovarian disease. Fertil Steril 35:371, 1981 3. Vejlsted H, Albrechtsen R: Biochemical and clinical effect of ovarian wedge resection in polycystic ovary syndrome. Obstet Gynecol 47:575, 1976 4. Kim MH, Rosenfield RL, Hosseinian AH, Schneir HG: Ovarian hyperandrogenism with normal and abnormal histologic findings of the ovaries. Am J Obstet Gynecol 134:455, 1979 5. Rebar R, Judd HL, Yen SSC, Rakoff J, Vandenberg G, Naftolin F: Characterization of the inappropriate gonadotropin secretion in polycystic ovary syndrome. J Clin Invest 57:1320, 1976 6. Berger MJ, Taymor ML, Patton WC: Gonadotropin levels and secretory patterns in patients with typical and atypical polycystic ovarian disease. Fertil Steril 26:619, 1975 7. Lobo RA, Granger L, Goebelsmann D, Mishell DR Jr: Elevations in unbound serum estradiol as a possible mechanism for inappropriate gonadotropin secretion in women with PCO. J Clin Endocrinol Metab 52:156,1981 8. Seltzer CC: Some re-evaluations of the build and blood pressure study, 1959, as related to ponderal index somatotype and mortality. N Engl J Med 274:245, 1966 9. Kletzky OA, Davajan V, Nakamura RM, Mishell DR Jr: Classification of secondary amenorrhea based on distinct hormonal patterns. J Clin Endocrinol Metab 41:660, 1975 10. Barberia JM, Abu-Fadil S, Kletzky OA, Nakamura RM, Mishell DR Jr: Serum prolactin patterns in early human gestation. Am J Obstet GynecoI121:1107, 1975 11. Stumpf P, Nakamura RM, Mishell DR Jr: Changes in the physiologically free pools of estradiol and testosterone during exposure to levonorgestrel. J Clin Endocrinol Metab 52:138, 1981

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Lobo et al. Bioactive LH in pea

12. Goebelsmann D, Bernstein GS, Gale 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 Teprow, R Crozier. New York, Aca· demic Press, 1979, p 165 13. Thorneycroft IH, Ribeiro WO, Stone S, Tillson SA: A radioimmunoassay of androstenedione. Steroids 21:111, 1973 14. Lobo RA, Goebelsmann D: Evidence for reduced 3(3-01hydroxysteroid dehydrogenase activity in some hirsute women thought to have polycystic ovary syndrome. J Clin Endocrinol Metab 53:394,1981 15. Lobo RA, Kletzky OA, Kaptein EM, Goebelsmann D: Prolactin modulation of dehydroepiandrosterone sulfate secretion. Am J Obstet Gynecol 138:632, 1980 16. Van Damme MP, Robertson DM, Diczfalusy E: An improved in vitro bioassay method for measuring luteinizing hormone (LH) activity using mouse Leydig cell preparations. Acta Endocrinol (Copenh) 77:655, 1974 17. Romani P, Robertson DM, Diczfalusy E: Biologically active luteinizing hormone (LH) in plasma. Validation of the in vitro bioassay when applied to plasma of women. Acta Endocrinol (Copenh) 83:454, 1976 18. Lobo RA, Goebelsmann D: Effect of androgen excess on inappropriate gonadotropin secretion (IGS) as found in the polycystic ovary syndrome (PCO). Am J Obstet Gynecol 42:394, 1982 19. Chang RJ, Mandel FP, Wolfsen AR, Judd HL: Circulating levels of plasma adrenocorticotropin in Polycystic Ovary Disease. J Clin Endocrinol Metab 54:1265, 1982 20. Lobo RA, Granger LR, Paul WL, Goebelsmann D, Mishell DR Jr: The relationship between psychological stress, neurotransmitters and androgen secretion in women with PCO. Fertil Steril (Abstr) 36:425, 1981 21. Lobo RA, Paul WL, Goebelsmann D: Dehydroepiandrosterone sulfate as an indicator of adrenal androgen function. Obstet Gynecol 57:69, 1981 22. Jujieda K, Faiman C, Reyes FI, Winter JSD: The control of steroidogenesis by human fetal !ldrenal cells in tissue culture. III. The effect of various hormonal peptides. J Clin Endocrinol Metab 52:690,1981 23. Sileva de Sa MF, Rebar RW: Altered forms ofimmunoreactive urinary FSH and LH in premature ovarian failure (POF). Presented at the Annual Meeting of the Society for Gynecologic Investigation, St. Louis, Missouri, March 19, 1981. Abstract 11 24. Dufau ML, Beitius IZ, McArthur JW, Catt KJ: Effects of luteinizing hormone releasing hormone (LHRH) upon bioactive and immunoreactive serum LH levels in normal subjects. J Clin Endocrinol Metab 43:658, 1975 25. Dufau ML, Hodgen GD, Goodman AL, Catt KJ: Bioassay of circulating luteinizing hormone in the rhesus monkey: comparison with radioimmunoassay during physiological changes. Endocrinology 100:1557, 1976

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