Serum Levels of Follicle-Stimulating Hormone, Lutemizing Hormone, and Plasma Progestins Correlated with Human Ovulation*

Vol. 21, No.2, February 1970 Printed in U.S.A.

FERTILITY AND STERILITY

Copyright

© 1970 by The Williams & Wilkins Co.

SERUM LEVELS OF FOLLICLE-STIMULATING HORMONE, LUTEINIZING HORMONE, AND PLASMA PROGESTINS CORRELATED WITH HUMAN OVULATION* MARVIN A. YUSSMAN, M.D.,t MELVIN L. TAYMOR, M.D., JUN MIYATA, M.D.t CAROLYN PHETEPLACE, B.S.

AND

From the Department of Obstetrics and Gynecology, Harvard Medical School and the Department of Surgery (Gynecology), Peter Bent Brigham Hospital, Boston, Massachusetts

Since the first application of radioimmunoassay techniques to the measurement of pituitary gonadotropins in human plasma or serum in 1966,3, 12 a variety of reports have been published demonstrating serial levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in peripheral blood throughout the normal human menstrual cycle." R, 13. 14, 1", 20, 21 Among the causes of several discrepancies noted in these various reports may be the inconsistency in correlating the peaks of FSH and LH to some acceptable physiologic event, In an effort to relate the serial patterns of gonadotropin secretion to ovulation, data have been grouped either around the LH or FSH peaks that have been shown to occur close to midcycle, Others have utilized such presumptive evidence as the bibasal body temperature chart. This arbitrary and inconsistent grouping of pituitary gonadotropins has been necessary since ovulation in the human has been observed only with difficulty, In addition three recent papers relate plasma progesterone in the human to serum levels of the gonadotropins. 4 , 1R, 24 These authors presumed that the secretion of

progesterone reflected ovulation, since further corroboration of ovulation was unavailable, The present study was undertaken in an effort to relate accurately the gonadotropins and plasma progesterone to human ovulation as determined by biopsy of a fresh corpus luteum obtained at laparotomy.

"Supported by a grant from the Population Council. Inc. Patients on this study were hospitaliZf'd in the Clinical Center of thf' Peter Bent Brigham Hospital under National Institutes of Hf'alth Grant M01-FR0031. Presented at the 25th annual meeting of thf' American Fertilit~· Society. Miami Beach, Florida, April 26-28, 1969. .;. Present address: Universit~· of Louisyille School of Medicine, Louisville, Ky. t Fellow of the Population Council, Inc. 119

MATERIALS AND METHODS

Subjects and Procedure. Patients were chosen who demonstrated regularly occurring menstrual cycles and who were scheduled to undergo pelvic laparotomy for a variety of reasons unrelated to hormonal cycling. Eight such patients were scheduled for their elective surgical procedures as close to the time of their ovulation as could be predicted from previous basal body temperature measurements. Patients were admitted to the metabolic unit of the Peter Bent Brigham Hospital 4-5 days prior to their scheduled surgery, where blood was drawn at 8-hr, intervals, When the basal body temperature suggested that ovulation had occurred, the patients were taken to the operating room for their scheduled procedures, at which time a sample of endometrium was obtained by D&C, and a biopsy of the fresh corpus luteum was obtained, if present, Serum was assayed for FSH and LH and plasma assayed for progesterone and "total progestin," Of the 8 subjects studied, a fresh corpus luteum (less than 48 hr . old) was recovered in 5 subjects, A 3-dayold corpus luteum was found in 2 sub-

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YUSSMAN ET AL.

jects and no corpus luteum could be identified in the remaining subject. Dating of the Corpus Luteum. The criteria for the age of the corpus luteum were based on the studies of Corner.5 Estimations were made of perifollicular hemorrhage, engorgement of vessels, luteinization and vacuolization of theca interna, presence or absence of granulosal capillaries, bleeding into follicular cavity, increase in cytoplasmic volume, and radial arrangement of granulosal cells. These findings were kindly interpreted for the authors by Dr. Arthur Hertig and Dr. Hazel Gore, of the Department of Pathology, Harvard Medical School. When possible, age of the corpus luteum was confirmed by dating of the endometrium. Radioimmunoassay of FSH and LH. The radioimmunoassay technique for LH and FSH utilized in our laboratory is a modification of the double antibody technique described by Midgeley 12 and by Bagshawe et al,3 utilizing P25. The details of the methods have been previously published. 1 ,2 Competitive Protein-binding Analysis of Progesterone. This assay is based on the displacement of H3-labeled corticosterone by progesterone from binding sites on corticosterone binding globulin (CBG). Our method is a modification of the techniques first described by Murphy l5, 16 and by Neill et al. 18 Plasma utilized as the source of CBG was obtained from females under estrogen stimulation (0.1 mg. of ethanyl estradiol daily for 16 days). The binding capacity for such serum varied from 67 to 80%. Duplicate I-ml. samples of plasma to be assayed were extracted twice with 5 ml. of redistilled petroleum ether.* Extracts were dried at 45° C under nitrogen flow. A CBG-corticosterone-l, 2-H3 solution (CBG-BH3) was prepared using 0.5% plasma and 15 mp.g. % corticosterone-l,2-

* Ligroin. 30°_60° C., Mallinckrodt No. 4980,

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H3 (Specific Activity 48 c.jmmole).t The dried extracts were redissolved in 0.2 ml. of chloroform and spotted onto ethanolwashed silica gel Chroma gram paper.t The chromagram was developed with redistilled ether-benzene, 2: 1. Parallel lanes of 20 p.g. of standard progesterone solution were run simultaneously to identify the area of progesterone migration under shortwave ultraviolet light. This area was cut from the strip and eluted 3 times with 2 ml. of redistilled ethanol, following which it was evaporated to dryness. Of the CBG-BH3 solution, 0.5 ml. was added to each of the samples and to a series of standards ranging from 0.5 mp.g.-20 mp.g. The tubes were gently warmed and shaken to redissolve the extracts. Samples were allowed to incubate in an ice bath for a minimum of 1 hr. to allow for displacement of the labeled corticosterone and binding of the progesterone. Forty milligrams of washed, dried florisil were added to each tube and shaken on a horizontal shaker for 1 min. to adsorb the displaced, free BH3. Of the supernatant, 0.3 mI., containing the bound BH3, was transferred from each tube into 15 ml. of freshly prepared Bray's solution. The samples were cooled and then counted in a liquid scintillation counter without quench correction. The response curve of the standards was plotted as per cent difference in binding of each sample as compared to the total binding capacity of the sytem. Such a standard response curve is shown in Fig. 1. The per cent binding of each sample to be assayed was calculated and millimicrograms of progesterone read from the standard curve. A loss correction factor, obtained by a parallel run of H3-labeled progesterone, was used to adjust the final result. -r New England Nuclear Corporation, Boston, Mass. t No. 6060. Distillation Products Industries, Rochester, N. Y.

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FSH AND LH IN HUMAN OVULATION

A modification of the above procedure, utilizing simple petroleum ether extraction, was also carried out. Petroleum ether extracts progesterone, 17a-OR progesterone, 20a-OR progesterone, and progesterone and trace amounts of cortisol and corticosterone. 21 The particular petroleum ether utilized extracts 90% of the progesterone and 15-20% of the 17a-OR progesterone and 20a-OR progesterone. 14 The assay, then, without chromatographic separation is for progesterone contaminated with small amounts of 17a-OR and 20a-OR progesterone and is referred to, by common usage, as "total progestin." A comparison of plasma progesterone and plasma "progestin" is shown in Fig. 2. While the absolute levels show the expected variation, the pattern of response is essentially the same except for a slightly premature rise of the total progestin, possibly due the earlier appearance of 17a-OR progesterone, which has been demonstrated in Rhesus monkeys.13 Since the assay system without chromatography requires only 3 hr. to do multiple samples, it is more practical than the chromatographic separation of each sample. Therefore, total progestin was utilized for this study.

H.E. -

"PROGESTIN"

0---0

PROGESTERONE

O~~~~~~ -12

-56

-40

HOURS IN RELATION

-24

TO

-8

0

+8

OVULATION

FIG. 2. A comparison of plasma progestin levels (petroleum ether extraction) and progesterone levels (thin layer chromatography) in Subject

H.E.

SERUM

I

L H mIU/ml.

SERUM FSH mIU/ml.

I

PLASMA PROGESTIN m}olQ/ml.

25

BBT

DEVIATION FROM TIME OF OVULATION IN HOURS

20

FIG. 3. Serum levels of LH and FSH and of plasma progestin levels in Subject N. B. related to ovulation. 15

% CHANGE IN BINDING CAPACITY

RESULTS

10

BINDING CAPACITY 76.4% B'H

15 mpg %

PLASMA 0.5%

0~----~5~-----10~----~'5~----~20

PROGESTERONE (mpg)

FIG. 1. Standard curve of per cent change m binding capacity produced by progesterone.

Results of assays for FSR, LR, and total progestin from peripheral venous blood obtained at 8-hr. intervals in each of 6 patients are plotted in Figs. 3-8. The mean of these cycles is shown in Fig. 9. These deteminations are plotted in relation to the vertical line which marks the time of ovulation as determined by dating of the fresh corpus luteum. Results are plotted as ovulation (0) plus hours or as

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YUSSMAN ET AL.

described nadir of FSH prior to the rise of LH was not demonstrable in the present data. In each of the patients studied, a demonstrable rise of plasma progestin occurred from 24-40 hr. prior to ovulation. In all 5 of these cases, the rise was coincident with the rise of LH. In the 1 additional case, the rise preceded the rise in LH by 8 hr. Mean proliferative phase levels of progestin prior to the rise varied from 0.20.7 mp.g./ml. The mean level at the time of ovulation was 2.3 mp,g./ml.

SERUM I

LH mlU/mlol

SERUM

FSH mlU/ml.1

DISCUSSION

PLASMA PROGESTIN mpg/ml.

SST

DEVIATION FROM TIME OF OVULATION

IN

FIG. 4. Serum levels of LH and FSH and of plasma progestin levels in Subiect R. G. related to ovulation.

In recent reports from this laboratorY,21.22 utilizing radioimmunoassay, a well-defined midcycle peak of serum LH was demonstrated occurring around the time of midcycle. The FSH pattern, after an initial postmenstrual follicular rise, showed a gradual decrease to a well-defined nadir prior to the midcycle peak of LH, and a second rise that either coin-

o minus

hours. Since dating of the corpus luteum may be in error by as much as 12 hr., the hatched area on either side of the vertical line covers the 24-hr. range of time in which the ovulation may have occurred. In 5 of the 6 subjects, a well defined peak of LH occurred 16 hr. prior to the actual ovulation. In the sixth case, incorrect estimation of the time of ovulation prevented obtaining a sample at the correct time. In this patient (Fig. 8), the peak of LH had evidently occurred prior to ovulation also. In 5 of the 6 subj ects, a recognizable rise in LH occurred from 16-40 hr. prior to ovulation. Five cases demonstrate the return of plasma levels of LH to earlier proliferative phase levels by the time ovulation occurred. In 5 cases, a recognizable but less dramatic peak of FSH occurred simultaneously with the peak of LH and preceded ovulation by 16 hr. The previously

SERUM

LH mIU/ml.

SERUM

I

FSH mlU/mL

PLASMA PROGESTIN

mpg/rnl.

SST

DEVIATION FROM TIME OF OVULATION IN HOURS

FIG. 5. Serum levels of LH and FSH and of plasma progestin levels in Subiect H. E. related to ovulation.

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FSH AND LH IN HUMAN OVULATION

cided with or occurred shortly after the midcycle rise of LH. Subsequently, a number of other investigators 4. 14, 20 described the patterns of serum FSH and LH levels in the normal menstrual cycle. Within some degree of variation, the re:mlts were similar to our observations. In all studies, however, the exact relationship of these shifting FSH and LH patterns to each other and to ovulation itself remained in doubt because there had been no physiologic event such as ovulation to serve as a reference point for the values. We, along with others, had chosen the midcycle peak of LH in each cycle as our reference point. Cargille chose the peak of FSH in each cycle as his reference point. Interpretations that might be drawn from these studies have been limited by these arbitrary and variable reference points. The present study relates all observations to the time of ovulation as determined by dating of a corpus luteum biopsy. In addition, 8-hr. observations have fur-

SERUM

LH mIU/ml.r

SERUM

FS H mlU/ml.

PLASMA PROGESTIN

m)Jl]/ml.

BBT

-72

-56

DEVIATION

-40 FROM

-24 TIME OF QVULUATION IN HOURS

FIG. 7. Serum lenls of LH and FSH and of plasma progestin levels in Subject S. P. related to ovulation.

SERUM L H mIU/ml.

I

SERUM

L H I mJU/ml. I

SERUM FSH mlU/ml.

SERUM

FSH I mlU/ml.

PLASMA PROGESTIN mpg/ml.

I

BBT PLASMA PROGESTIN m)lo/ml. I

oL_ _~~~~_

BBT

98r------

DEVIATION FROM TIME OF OVULATION

IN HOURS

FIG. 6. Serum levels of LH and FSH and of plasma progestin levels in Subject T. B. related to ovulation.

DEVIATION FROM TIME

OF

OVULATION IN HOURS

FIG. 8. Serum lenls of LH and FSH and of plasma progestin levels in Subject G. R. related to Q\·ulation.

ther enhanced our ability to relate the changes to ovulation itself. As far as LH is concerned there appears

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YUSSMAN ET AL.

MEAN SERUM

LH

mlU/ml. I

MEAN I SERUM FSH mIU/ml.

MEAN PLASMA

P~~;~~~N

I

ol,--~:;::::;~~ DEVIATION FROM TIME

-8

0 +8

+ 24

OF OVULATION IN HOURS

FIG. 9. Mean levels of serum LH and FSH and 0.£ plasma progestin in 5 subjects related to ovulahon.

to be little doubt that the surge of LH precedes ovulation. This confirms the widely held concept that the surge of LH itself, is the significant driving force be~ hind ovulation. The demonstration that LH is significantly elevated 24 hr. prior to ovulation suggests that the prediction of imminent ovulation by means of a rapid LH assay would be feasible. The pyramidal midcycle pattern of LH demonstrated in this study was unexpected. Since the current concept of hypothalamic-pituitary relationship is that stored pituitary LH is released in response to hypothalamic LRF, a sharp rise and gradual decrease of LH would be the expected pattern. The pyramidal response would be more consistent with increased production of LH than with the release of stored LH. The data from the FSH levels are less dramatic. In most instances a rise occurred close to the rise of LH, and this was also seen in the mean values. In the present study, observations were initiated too late in the cycle to demonstrate the previously described nadir of FSH levels that occurs

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just prior to the midcycle peak of LH. The most significant observation concerning the FSH levels at this time is that these fluctuations are usually minor and never approximate those of the LH. Indeed, one might speculate that FSH may have a relatively insignificant role in the ovulatory process. A tonic level of FSH may be all that is necessary to produce or maintain follicles that will respond to the surge ofLH. The progesterone or "progestin" data provide additional fields for speculation. The rise in progesterone, or total progestin, 24-48 hr. prior to ovulation in the subjects reported in this study is the first in vivo demonstration in humans that the rise of this hormone is not exclusively dependent upon ovulation, but is also a product of the still unruptured but developing follicle or, perhaps, of other atretic follicles. In vivo ovine studies have demonstrated that perfusion of LH into the ovarian artery resulted in an increased secretion rate of progesterone by the ovary during the proliferative phase of the cycle. 6 The lack of a corpus luteum was also no hindrance to the increased progesterone secretion in rabbits when LH was exogenously administered. 9 Conversely, work in rats has demonstrated increased secretion of LH in response to a low dose of administered progesterone, indicating an important preovulatory function for the progesterone secreted by the unruptured follicle or by atretic follicles.17 The results of the present study suggest that in the human as well , progesterone may play a role in initiating the ovulatory process itself. This widening knowledge gained by improving methodology provides added insights into the process of ovulation and the promise of better control of this process both from the point of view of stimulation and inhibition. Acknowledgments. The authors are indebted to Doctors Arthur T. Hertig and Hazel Gore for

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FSH AND LH Il\" HUl\IAN OVULATION

iuterpretation of the pathologic material and to Elizabeth Hines and Roberta Todd for technical :l8sistance. HEFERENCES 1. Ao:'(o, T., GOLDSTEIX, D, P., TAYMOR, M. L., AND DOLCH, K. A radioimmunoassay method for human pituitary luteinizing hormone (LH) and human chorionic gonadotropin (HCG) using ""I-labeled LH. Amer J Obstet Gynec .')8 :996, 1967. 2. ,\0;0;0, T., AND TAYMOR, M. L. Radioimmunoassay for follicle-stimulating hormone (FSH) with '"5I-labeled FSH. Amer J Obstet Gynec 100:110,1968. 3. BAGSHAWE, K. K., WILDE, C. E., .\:'(D ORR, A. H. Radioimmunoassay for chorionic gonadotropin and luteinizing hormone. Lancet 1 :118, 1966. 4. CARGILLE, C, M., Ross, G. T. AND YOSHIMA, T. Daily variations in plasma follicle-stimulating hormone, luteinizing hormone and progesterone in the normal menstrual cycle. J Clin Endoer 29 :12, 1969. 5. CORNER, G. W. The histologic dating of the human corpus luteum of menstruation. Amer J Anat 98 :377, 1956. 6. DOMANSKI, E., SKRZECZKOWSKI, L., STUP:'>'ICKA, E., FITKO, R. AND DOBROWOLSKI, \V. Effect of gonadotropins on the secretion of progesterone and oestrogens by the sheep ovary perfused in situ. J Reprod FertiI14:365, 1967. 7. FADI.\:'>', C., A:'>'D RYA:'>', R. J. Radioimmunoassay for human follicle stimulating hormone. J Clin Endoer 27 :444,1967. 8. FAnlAN, C., AND RYAN, R. J. Radioimmunoassay for human luteinizing hormone. Proc Soc Exp Bioi ivleriI25:1130, 1967. 9. HILLARD. J., ARCHIBALD, D., A:'>'D SAWYER, C. H. Gonadotropic activation of preovulatory synthesis and release of progestin in the rabbit. Endocrinology 72 :59, 1963. 10. JOHANSSO:'>', E. D. B., NEILL, J. D. AND KNOBIL, E. Periovulatory progesterone concentration in the peripheral plasma of the rhesus monkey with a mt>thodologic note on the detection of o\'lilation. Endocrinology 82:143, 1968. 11. JOHAl\SSO:'(, E. D. B. Personal communication.

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12. MIDGELEY, .~. R. Radioimmunoassay: A method for human chorionic gonadotropin and human luteinizing hormone. Endocrinololgy 79 :10, 1966. 13. MIDGELEY, A. R. Radioimmunoassay for human follicle-stimulating hormone. J Clin Invest 27 :296,1967. 14. MIDGELEY, A. R., AND JAFFEE, R. B. Regulation of human gonadotropins. IV. Correlation of ,;erum concentrations of follicle-stimulation and luteinizing hormones during the menstrual cyel£'. J Clin Endocr 28 :1699, 1968. 15. MVRPHY, B. E. P. Application of th£' propert~· of protein-binding to the assay of minute quantities of hormones and other substances. Nal ure (London) 201 :679,1964. 16. MVRPHY, B. E. P. Some studies of the proteinbinding of steroids and their application to th£' routine micro and ultramicro measurement of \'ar'ious steroids in body fluids by competitive protein-binding radioassay. J Clin Endocr 27 :973, 1967. 17. NALLER, R., ANTwms-RoDRIQUEZ, J., AND McCANN, S. M. Effect of progesterone on the level of plasma luteinizing hormone (LH) in normal female rats. Endocrinology 29 :907, 1966. 18. XEILL, J. D .. JOHANSSOl\. E. D. B., DATTA, J. K., AND KNOBIL, E. Relationship between the plasma le\'els of luteinizing hormone and progesterone during the normal menstrual cycle. J Clin Endocr 27 :1167, 1967. 19. ODELL, W. N., Ross, G. T .. AND RAYFORD, P. L. Radioimmunoassay for luteinizing hormone in human plasma or serum: Physiologic studies. J. Clin Invest 46:248, 1967. 20. SAXE:'>'.\, B. B., DE:\WHA, H., GAl\DY, H. M., AND PETERSO:'>', R. E. Radioimmunoassay of human follicle-stimulating and luteinizing hormones in plasma. J. elin Endocr 28 :519, 1968. 21. TADIOII, M. L., Ao:'>'o, T .. AND PHETEPLACE, C. 111 Gonadotropins 1968, E. Rosenberg, Ed. Geron-X, Palo Alto, 1969, p. 349. 22. TADIOR, M. L., AONO, T., A:'>'D PHETEPLACE, C. Follicle-stimulating hormone and luteinizing hormone in serum during the menstrual cycle determined by radioimmunoassay. Acta Endoer 59 :298, 1968.