Changes of Hypophysio-Ovarian Endocrinological Function by Aging in Women

Tohoku J. exp. Med., 1977, 121, 231-238 Changes of Hypophysio-Ovarian Endocrinological Function by Aging in Women NOBUAKI FURUHASHI, MASAKUNI SUZUKI, TETSURO ABE, YOSHIHIRO YAMAY A and KATSUYUKI TAKAHASHI Department of Obstetrics and Gynecology, Tohoku University S chool of Medicin.e, Sendlai FuavuAsxz, N., Suzuxr M., ABE, T., YAMAYA, Y. and TAKAHASM, K. Changes of Hypophysio-Ovarian Endocrinological Function by Aging in Women. Tohoku J. exp. Med., 1977, 121 (3), 231-238 \Serum estradiol, progesterone, LH and FSH were concomitantly measured by radioimmunoassay in pre- and post-menopausal women in an attempt to study hormonal changes occurring in menopause. The level of serum estradiol in post-menopausal women was maintained at the same level as in pre-menopausal stage within one year after menopause and thereafter began to decrease to the senile minimal level. On the other hand, the mean serum levels of FSH and LH were elevated gradually after menopause. At the conclusion of this study, it was suggested that the key point of the mechanism of menopause may exist not only in the depletion of ovarian function but in the change of the regulatory mechanism of LH and FSH secretion by aging. \ estradiol ; progesterone ; LH ; FSH ; post-menopause Studies published concerning urinary and serum hormone levels in women at various stages from sexual maturity to post-menopausal periods are numerous, but reports of parallel assays of the serum of individuals to investigate the changes of these hormone levels with the advancing age, as well as their interrelationship, are few. The authors performed this study to elucidate the mechanism of menopause through determination of the serum concentrations of these hormones in women at the stages of sexual maturity, climacterium and senility, and evaluation of the changes associated with aging and their interrelations. Subjects MATERIALS AND METHODS Eighty-two women, 20-49 years old with normal, regular menstruation for at least three months immediately preceding the study, and 70 women in post-menopausal stage were studied. The mean age of the post-menopausal group was 52.4 }7.0 years (mean }s.d.). Termination of reproductive life and cessation of menstruation for more than four months prior to the study were prerequisites for admission to the post-menopuasal group. The women with normal menstrual cycle were divided by decades into three groups and further classified according to collection time of blood samples in relation to the menstrual cycle, i.e., the first, second or third ten-day period of the cycle. Received for publication, June 7, 1976. Reprint requests: Dr. Tetsuro Abe, Dept. of Ob/Gyn., Tohoku University School of Medicine, 1-1 Seiryo-machi, Sendai 980, Japan 231

232 N. Furuhashi et al. Collection of serum samples Subjects were confirmed to have received no drug medication for at least the preceding month. Samples of venous blood were drawn between 01:00 and 03:00 p.m. from which sera were separated and frozen to be stored at -20 Ž until assays. Radioimmunoassa,u The serum estradiol-17ƒà level was determined by the radioimmunoassay method of Wu and Lundy (1971), using antiserum against estradiol-6-bsa and a column of Sephadex LH-20 for chromatographic separation. The radioimmunoassay for serum levels of progesterone was carried out without the chromatographic procedure, using antiserum against progesterone-3-bsa. The double-antibody method of Monroe et al. (1968) was employed in the radioim munoassay of serum LH and FSH, which were expressed in ng/ml. LER-907 (supplied from NIAMDD) was used as hlh and hfsh reference standards. In the statistical data analysis, Student's t-test was used to assess the significance of differences of mean values. TABLE 1. Levels of serumn estradiol in women with normal menstrual cycle and in post-menopausal women Women with normal menstrual cycle Post-menopausal women The values are expressed as mean }S.E. (pg/ml). The number of determinations is given in the square bracket.

Changes of Endocrinological Function by Aging in Women 233 RESULTS Serum estradiol Table 1 shows mean ( }S.E.) levels of serum estradiol of menstruating women from age 20 through 49 at various stages of the menstrual cycle, and post-menopausal women at various periods after the termination of menstruation. Women in their 20's and 30's exhibited prominent peaks in the middle stage of the menstrual cycle, from the 11th through the 20th days. The mean serum concentration of estradiol of women in the postmenopausal stage for less than two years did not significantly differ from the resting level in normal menstruating women, whereas women in the post-menopausal stage for more than two years displayed signifi cantly lower average levels than these counterparts for less than two years (p<0.05). Serum progesterone (Table 2) The mean value of serum progesterone during the last 10 days of the normal menstrual cycle was significantly more depressed in the women aged 30 to 49 than in the postmenopausal groups, but the serum levels of this hormone were markedly lower in the post-menopausal group than in any group of women with normal menstrual cycle (p<0.005). TABLE 2. Levels of serum progesterone in women. with normal menstrua l cycle and in post-menopausal women Women.with normal menstrual cycle Post-menopausal women The values are expressed as mean }s.e. (pg/ml). The number of determinations is given in the square bracket.

234 N. Furuhashi et al. TABLE 3. Levels of serum LH in women with normal menstrual cycle and in post-menopausal women. Women with normal menstrual cycle Post-menopausal women The values are expressed as mean+s.e. (ng/ml) The number of determinations is given in the square bracket. Fig. 1. Correlation between levels of serum LH or FSH and levels of serum estradiol in women of 20-29 years of age. n: Number of determinations.

Changes of Endocrinological Function by Aging in Women 235 TABLE 4. Women with normal menstrual cycle Levels of serum FSH in women with. normal menstrual cycle and post-menopausal women The values are expressed as mean }S.E. (ng/ml). The number of determinations is given in the square bracket. Serum LH (Table 3) and FSH (Table 4) The post-menopausal groups showed basal levels of serum LH and FSH higher by five to ten-fold and ten-fold, respectively, than menstruating women from age 20 to 30 years. Compared with the group within the first year of post - menopause, the group one to three years after menopause showed significantly elevated serum levels of these hormones (p<0.005) Correlation among serum levels of estradiol, progesterone, LH and FSH There were positive correlations in women in the age group from 20 to 29 (Fig. 1) and a negative correlation in postrnenopausal groups (Fig. 2) between the serum estradiol and LH concentrations and also between serum levels of estradiol and FSH. No significant correlations were demonstrated between serum levels of progesterone and LH or FSH in any premenopausal women from age 20 to 49 and in post-menopausal women at any period following menopause. DISCUSSION Many investigations have demonstrated that blood levels of LH and FSH in post-menopausal women are markedly elevated and remain high for many years as compared with normally menstruating women (Odell et al. 1967, 1968; Wide et al. 1973). However, in these reports only serum levels of LH and FSH associated

236 N. Furuhashi et al. Fig. 2. Correlation between levels of serum LH or FSH and levels of serum estradiol in post-menopausal women. n: Number of determinations. with age-dependent changes were elucidated and neither information concerning simultaneous changes in serum concentrations of these hormones in relation to changes in steroid hormones following menopause was provided nor did they present correlation of serum levels of LH, FSH and sex steroid hormones in the same individual. Post-menopausal elevation of serum levels of LH and FSH in this study revealed indications that they do not sharply increase but become progressively elevated to reach their peaks one to three years after menopause, remaining almost consistently high for at least the ensuing 10 years. Wide et al. (1973) reported the highest FSH and LH levels in women aged 51 to 60 years, and significantly lower levels after 60 years of age, suggesting that senescence of pituitary function occurs as a part of systemic aging. In the present investigation serum estradiol was not found to decrease to an extremely low level after the cessation of menstruation but declined gradually over many years. No significant difference was observed between the mean basal level of serum estradiol in normally menstruating women in the age group from 20-39 years and the mean values of those in the post-menopausal stage for less than two years. These findings support the report of no morphological evidence of

Changes of Endocrinological Function by Aging in Women '137 prominent changes in the ovaries for some time after menopause (Procope 1969). In a study on the ovarian function in women close to and in the stage of menopause, Furuhjelm (1966) determined the total gonadotrophin, estrogen, 17-KS and pregnanediol concentrations in the urine in a series of 273 subjects ranging in age from 20 to 78 years and thereby noted that the menopausal process might be divided into three successive stages; i.e., (1) depression of Iuteal function, (2) oligomenorrhoea and (3) menopause. The present investigation has disclosed that statistically significant decline of serum progesterone concentration with advancing age precedes that of serum estradiol level, and this finding eventually furnishes an explanation for the view put forth by Furuhjelm (1966). The positive correlation noted, in this study, to exist between the seruin estradiol concentration and the serum LH or FSH levels in the age groups 20-29 years provides evidence for the presence of ovulation (Vande Wide et al. 1970). The serum LH or FSH level did not correlate significantly with the serum estradiol concentration in women from 30-49 years of age. Several reasons tray be adduced to explain these findings as follows: even if these women have menstrual cycle, (1) the proportion of women with anovulatory menstrual cycle increases with advancing age; (2) changes of endocrinological correlation among the hypothalamus, hypophysis and ovary proceed with aging (Adaanopoulos et al. 1971); (3) depression of ovarian response to gonadotropins associated with aging: (4) a transient hyperfunction of the hypothalatno-hypophysial system proceed with aging due to changes of reactivity of the system to the sex hormones secreted from thee ovary; and (5) others. A negative correlation was observed to exist between the serum LH or FSH levels and the estradiol concentration in the post-menopausal women. Reports by Seyler and Reichlin (1973), Siler and Yen (1973) and Yen et al. (1974) showed that estradiol directly inhibits the sensitivity of LH and FSH to LRF. These facts seem to indicate that the negative feedback menchanism would be maintained for at least 10 years even following menopause; hence the decline of estradiol due to aging may lead to the consequent elevation of serum levels of hypophysial LH and FSH. Neither women with normal menstrual cycle nor postrnenopausal women were found to show any significant correlation of serum progesterone level with the,serum LH and FSH concentrations. This is reasonable in the simultaneous assays on the same subjects since the surge of serum LH or FSH elevation occurs at the midcycle period preceding the sharp increase of progesterone secretion. The results obtained in the present study, moreover, clarified what had been inferred from morphological observations, urinary hormone assays and determina tions of serum LH and FSH as to physiological changes in the hypophysio-ovarian system associated with aging. Acknowledgment We wish to express gratitude to Drs. Akira Kanbegawa and Hiroo Makino, Teikoku- Zoki Co., Ltd., for guidance and helpful advice concerning radioimmunoassay for sex steroid

238 N. Furuhashi et al. hormones in this investigation. The supply of HLH and HFSH and their specific antibody preparations from NIAMDD Inc. is also gratefully acknowledged. References 1) Adamopoulos, D.A., Loraine, J.A. & Dove, G.A. (1971) Endocrinological studies in women approaching the menopause. J. Obstet. Gynaec. Brit. commonw., 78, 62-79. 2) Furuhjelm, M. (1966) Urinary excretion of hormones during the climacteric. Acta obstet. gynec. scand.. 45, 352-365. 3) Monroe, S.E., Parlow, A.F. & Midgley, A.R., Jr. (1968) Radio immunoassay for rat luteinizing hormone. Endocrinology, 83, 1004-1012. 4) Ode11, W.D., Ross, G.T. & Rayford, F.L. (1967) Radioimmunoassay for LH in human plasma and serum. J. clin. Invest., 46, 248-255. 5) Odell, W.D., Parlow, A.F., Cargille, C.M. & Ross, G.T. (1968) Radioimmunoassay human follicle stimulating hormone: physiological studies. J. clin. Invest., 47, 2551 2562. 6) Procope, B.J. (1969) Studies on the urinary excretion, biological effects and origin of estrogens in post-menopausal women. Arta endocr. (Kbh.), Suppl. 135, 9-83. 7) Seyler, L.E. &. Reichlin. S. (1973) L14-RH in human plasma of post-menopasual women. J. clin Endocr., 37, 197-203. 8) Siler, T.M. & Yen. S.S.C. (1973) Augmented gonadotropin response to synthetic LRF in hypogonadal state. J. din. Endocr., 37, 491-494. 9) Vande Wiele, R.L., Bogumil, J., Dyrenfurth, I.. Ferin, M., Jewelewicz, R., Warren, M., Rizkallach, T. & Mikhail, G. (1970) Mechanisms regulating the menstrual cycle in men. Recent Progr. Hormone Res., 26, 63-103. 10) Wide, L., Nillius, S.T., Gemzell, C. & Ross, P. (1973) Serum levels and urinary excretion of FSH and LH in healthy men and women. Acta endocr. (Kbh.), Suppl. 174, 41-58. 11) Wu, C.H. & Lundy, L.E. (1971) Radioimmunoassay of plasma estrogens. Steroids, 18, 91-111. 12) Yen, S.S.C., V'andenberg. R. & Siler, T.M. (1974) Modulation of pituitary responsi veness to LRF by estrogen. J. clin. Eadocr., 39, 170-177.