LIE ASSAY OF GONADOTROPIN in human blood is one of the most important

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1 Changes in Human Serum FSH Levels During the Normal Menstrual Cycle MASAO IGARASHI, M.D., JUNJI KAMIOKA, M.D., YOICHI EHARA, M.D., and SEIICHI MATSUMOTO, M.D. LIE ASSAY OF GONADOTROPIN in human blood is one of the most important and challenging problems in reproductive physiology and gynecology. Despite some reported attempts,1-3, 9, 11, 12 the amount of gonadotropin in human blood during the normal menstrual cycle is too low to be assayed without special extraction from a lot of pooled plasma. Recently, cyclic change in human serum LH levels during the normal menstrual cycle was demonstrated/ii but little is known about the change in blood FSH during the cycle. This study was undertaken to elucidate the change in human blood FSH during the normal menstrual cycle using the sensitive bioassay method for FSH devised recently.s MATERIALS AND METHODS About 20 ml. of blood was obtained from 7 to 10 P.M. every night from a 35-year-old para 3 showing normal biphasic basal body-temperature curve during 1 menstrual cycle. The serum was separated by a centrifuge, and kept frozen until FSH bioassay. This woman did not receive any hormones or drugs during the experiment. FSH in serum was assayed according to the Igarashi-McCann FSH bioassay method. Unextracted serum was added and mixed with HCG (Primogonyl) and saline solution (0.25 LU. HCG per 0.5 ml. saline) at the ratio of 1.5 ml. of serum to 0.5 ml. of HCG-saline solution just before bioassay. Each 2.0 ml. of the serum-hcg From the Department of Obstetrics and Gynecology, School of Medicine, Gunma University, Maebashi, Japan. Supported in part by Research Grant M66.12 from the Population Council, Inc., New York, N.Y. The authors wish to express their sincere thanks to Mrs. Yoshiko Igarashi for the daily use of her blood during the study. We are also indebted to the Endocrinology Study Section, National Institutes of Health, U. S. Public Health Service, for the generous supply of NIH FSH-S2 they furnished, and to the Schering A. G. Company, Berlin, Germany, for the supply of Primogonyl they made available. 672

2 VOL. 18, No.5, 1967 SERUM FSH IN MENSTRUAL CYCLE 673 solution was subcutaneously injected once daily for 3 days into an immature female Swiss albino mouse, weighing gm. Five to six assay animals were used for each day's sample. On the first and second assay days 0.7 ml. of the serum-hcg solution was given, and 0.6 ml. was given on the third day. The mice were sacrificed and uterine weights measured 24 hr. after the last injection was given. Any mouse showing a reduction of body weight during assay was discarded. Special care was exerted to avoid intraperitoneal injection and to avoid leakage from the injection site. A design was used against NIH -FSH -S2 standard. The index of precision (A) and the relative potency were calculated by the method recommended by Gaddum. RESULTS FSH activity in 1.5 ml. of serum was detectable in 23 of the total 27 assays as shown in Fig. 1. During menstruation, serum FSH levels were relatively high. Then, peaks of serum FSH levels appeared on Day 8 and Day 11. After Day 12, serum FSH levels decreased gradually. The thermal shift of basal body temperature (BBT) induced by ovulation and corpus luteum formation was observed from Day 22 to Day 23. Serum FSH showed relatively low levels in the preovulatory and the ovulatory phase from Day 14 to Day 24. Then, FSH levels in at least 1.5 ml. of serum were undetectable on Days 20, 27, 29, and 32 in the premenstrual phase. DISCUSSION Few reports on quantitative estimation of gonadotropic activity in human blood have been published. Apostolakis and Apostolakis and Voight proposed an extraction method for human plasma gonadotropin and reported that total gonadotropin activity, extracted from 50 to 60 ml. of plasma from menstruating women, was detectable only at the mid cycle, although plasma FSH was detectable in almost all of 54 postmenopausal women. They also demonstrated that FSH and ICSH activity are present in human blood, but quantitative estimation in human blood FSH and ICSH was difficult using their method. Antoniades et al. and McArthur and coworkersll 12 studied extensively the purification of plasma gonadotropin and recently published the results of assayed FSH and LH content of pooled human menopausal plasma and of subfractions prepared by Cohn methods 6 and 9. Quite recently a rapid and simple method for estimation of total gonadotropin activity in human plasma was devised by Keller and "Rosemberg, but plasma FSH levels in male and female subjects in the re~

3 674 IGARASHI ET AI.. FERTILITY & STERILITY Mens E o 'S. (\J (/) ::c (/) ll... ::c Z :> '" CI' Q) CI :::I. 200 Fig. 1. Human serum FSH levels (shaded areas) during normal menstrual cycle. Relatively high levels in menstruation, peaking on Days 8 and 11. Gradual decrease after Day 12; relatively low levels in preovulatory and ovulatory phase (Days 14-24); undetectable levels on Days 20, 27, 29, and 32 in premenstrual phase. Shift in BBT (curve at top), induced by ovulation and corpus luteum formation (Days 22-23).

4 VOL.lB, No.5, 1967 SERUM FSH IN MENSTRUAL CYCLE 675 productive years cannot be detected, even by their new method, without pooling a great deal of plasma. The data presented here demonstrated that FSH in just 1.5 ml. of unextracted plasma was detectable with the Igarashi-McCann FSH bioassay method. The precision index of this bioassay was over 0.2, but the sensitivity was excellent. Consequently this method does not require any extraction of FSH from the blood even in menstruating women. As shown in the original report, the results in the Igarashi-McCann FSH bioassay are specific for FSH, unless the contaminated LH is above 10.0 flg equivalent to NIH-LH-S1. The mice used in this investigation were of the same strain as those used in the original work, so they should have shown the same sensitivity, specificity, and accuracy as shown in the original report. Yokota et al. demonstrated that the range of human serum LH levels are 31 to 56 flg.j100 ml. equivalent to NIH-LH-S1 (mean: 43.2 ± 4.3) in the early follicular phase, 26 to 98 (mean: ;- 8.4) in the late follicular phase, 87 to 216 (mean: ± 9.3) in the preovulatory and ovulatory phase, 36 to 72 (mean: ;- 7.1) in the early luteal phase, and 29 to 44 (mean: ;- 2.1) in the late luteal phase. Consequently the amount of LH contained in the 1.5 ml. of human serum is calculated to be from 0.47 to 0.84 flg. in the early follicular phase, 0.39 to 1.47 flg. in the late follicular phase, 1.31 to 3.24 JLg. in the preovulatory and ovulatory phase, 0.54 to 1.08 flg. in the early luteal phase, and from 0.44 to 0.66 JLg. in the late luteal phase. All the above-mentioned LH levels are far below the critical value of 10.0 flg. that interferes with FSH assay. Human serum LH levels are highest at the mid cycle and lowest at the follicular and luteal phases as reported in the previous paper. 15 Consequently, clear dissociation in FSH and LH secretion from the pituitary during the normal menstrual cycle was demonstrated. The donor whose blood was used in this study showed slight anemia in the luteal premenstrual phase due to daily blood collection. However, the low FSH levels in the luteal phase were not attributed to the anemia, because serum concentration of hormones cannot be reduced by slight anemia. There is a lack of agreement among the reports 4-6, 10, 13, 14 about the detailed patterns of urinary excretion of FSH and LH during the menstrual cycle. However, the tendency of the highest FSHjLH ratio in the follicular phase and of the lowest ratio in the ovulatory phase in serum corresponds well with the same cyclic changes of FSHjLH ratio in urine as reported in most of the references.4, 5, 10, 14

5 676 IGARASHI ET AL. FERTILITY & STERILITY SUMMARY Cyclic changes in human serum FSH levels during the normal menstrual cycle were studied in a 35-year-old woman. About 20 m!. of blood was collected every day during 1 menstrual cycle, and the results were compared with BBT. FSH activity in 1.5 m!. of unextracted serum was assayed by the Igarashi-McCann FSH bioassay method. Serum FSH levels were high in the follicular phase, lower in the ovulatory phase, and undetectable in the late luteal phase. The peak on the eighth day of the cycle showed {-tg., equivalent to NIH-FSH-S2 per 100 m!. of serum. Clear dissociation was demonstrated between the cyclic change in serum FSH levels reported here and the cyclic change in serum LH levels reported previously15 during the normal menstrual cycle. Department of Obstetrics and Gynecology Gunma University School of Medicine Maebashi, Japan REFERENCES 1. ANrONIADES, H. N., PENNELL, R. B., McARTHUR, J. W., INGERSOLL, F. M., ULFEL DER, H., and ONCLEY, J. L. Preparation and concentration of the pituitary gonadotropins from human plasma. J Biol Chern 228:863, ApOSTOLAKIS, M. Detection and estimation of pituitary gonadotrophins in human plasma. J Endocr 19:377, ApOSTOLAKIS, M., and VOIGT, K. D. Gonadotropine. Thieme, Stuttgart, BECKER, K. L., and ALBERT, A. Urinary excretion of follicle-stimulating and luteinizing hormones. J Clin Endocr 25:962, BROWN, P. S. Human urinary gonadotrophins. II. In relation to the menstrual cycle, secondary amenorrhea and the response to estrogen. J Endocr 18:46, 1959'. 6. FUX:USIDMA, M., STEVENS, V. C., GANTT, C. L., and VORYS, N. Urinary FSH and LH excretion during the normal menstrual cycle. J Clin Endocr 24:205, GADDUM, J. H. Bioassays and mathematics. Pharmacol Rev 5:87, IGARASID, M., and MCCANN, S. M. A new sensitive bioassay for follicle-stimulating hormone (FSH). Endocrinology 74:440, KELLER, P. J., and ROSEMBERG, E. Estimation of pituitary gonadotropins in human plasma. J Clin Endocr 25: 1050, McARTHUR, J. W., WORCESTER, J., and INGERSOLL, F. M. The urinary excretion of interstitial-cell and follicle-stimulating hormone activity during the normal menstrual cycle. J Clin Endocr 18: 1186, McARTHUR, J. 'V., and ANTONIADES, H. N. "Purification of Plasma Gonadotropin Concentrates by Tetrametaphosphate Precipitation." In Human Pituitary Gonadotropins: A Workshop Conference, Albert, A., Ed. Thomas, Springfield, III., 1961, p McARTHUR, J. W., ANTONIADES, H. N., LARSON, L. R., PENNELL, R. B., INGER SOLL, F. M., and ULFELDER, H. Follicle-stimulating hormone and luteinizing hormone content of pooled human menopausal plasma and of subfractions prepared by Cohn methods 6 and 9. J Clin Endocr 24:425, 1964.

6 VOL. 18, No.5, 1967 SERUM FSH IN MENSTRUAL CYCLE ROSEMBERG, E., and KELLER, P. J. Studies on the urinary excretion of folliclestimulating and luteinizing hormone activity during the normal menstrual cycle. J Glin Endacr 21:1262, TAYMOR, M. L. Excretion of follicle stimulating hormone and interstitial-cell stimulating hormone in different phases of the normal menstrual cycle. J Glin Endacr 21:976, YOKOTA, N., IGARASID, M., and MATSUMOTO, S. Human serum luteinizing hormone (LH) levels during the normal menstrual cycle and after ovariectomy. Endacr Japan 12:92, Postgraduate Course in Endocrinology Lenox Hill Hospital is offering a 5-day, full-time postgraduate course in human endocrinology, with emphasis on gynecological and medical aspects. The course will be given from Oct. 23 to 27, 1967, under the direction of Drs. Herbert S. Kupperman and Iven S. Young. The tuition fee is $150. This will be a practical, didactic, and clinical presentation, with emphasis on the therapeutic management of endocrine disorders in the human. It will include a discussion of the diagnosis and management of intersex. Endocrine therapy for menstrual abnormalities and ovulatory defects will be presented. Adrenocortical, thyroid, parathyroid, ovarian, testicular, and pituitary abnormalities will be emphasized. There will be descriptions and demonstrations of practical diagnostic tests, including the most recent procedures, and discussions of the therapeutic uses of the newer progestional, gonadal, and corticoid steroids as well as of the available pituitary preparations. Some pertinent aspects of male and pediatric endocrinology will be included, and the diagnostic importance of chromosomal analysis will be elaborated upon. For further information, write to Dr. WILLIAM D. SrCHER, Coordinator, Medical Education, Lenox Hill Hospital, 100 E. 77 St., New York, N. Y

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