Daily plasma progesterone levels during the menstrual cycle

volume 85

number 8

April 15, 1963

American Journal of

Obstetrics and Gynecology

GYNECOLOGY Daily plasma progesterone levels during the menstrual cycle C.

A.

WOOLEVER,

M.D.

Denver, Colorado

Before the isolation of progesterone from human placental tissue in 1952, 9 - 11 most authors referred to the results of their assays in terms of progestational activity rather than as specific units of progesterone. In recent years, physicochemical methods have allowed more precise measurements of the hormone itself. Yet, these methods have not been sufficiently sensitive to allow repeated daily blood studies to be carried out in the same patient, and only selective values as determined by these techniques have been reported.1"-';; [n our laboratory, development of a sensitive and specific technique for the determination of progesterone in plasma16 ' 17 has permitted daily studies throughout the menstrual cycle. The results of these investigations are reported in this paper.

THE corpus luteum of the ovary was described by de Graaf' in 1672, but it was not until 1898~more than two hundred years later~~that its function as an organ of internal secretion was suggested.~ Investigation of the chemical nature of the corpus luteum hormone was stimulated by the production of a crude but physiologically active extract by Corner and Allen. 3 This opened the way to a period of intensive study which, in 1934, resulted in the finding of a pure hormone of the corpus luteum, progesterone, by four independent laboratories."-'

From the Department of Obstetrics and Gynecology University of Colorado Medical Center. This work was supported by Public Health Service Research Grant AM-03990-03 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service; and by grants from the Fluid Research Committee of the University of Colorado Medical Center and from the Ayerst Laboratories.

Materials and methods

The subjects used in this study were healthy, adult females ranging in age from 981

April 15, 196:) Am.]. Obst. & Gyncc.

982 Woolever

19 to 43 years. They were chosen because of the regularity of their menstrual cycles and the absence of general or gynecologic disease. Each donor carried out her usual daily activities and kept a basal body temperature record which was correlated with the results of the plasma assays. At approximately the same time each morning, 10 to 15 mi. of blood was taken from the antecubital vein into heparinized centrifuge tubes. The plasma was separated and the extraction was begun immediately except in a few instances when it was necessary to freeze the plasma until analysis could be done. The period of storage varied up to thirty days. All determinations were done on 5 to 8 mi. of plasma using a double isotope derivati\'e method which has been described elsewhere.1';, 17 Results Figs. 1 through 4 show the oral temperature (broken line) and daily plasma progesterone levels (solid line) of 5 subjects during their menstrual cycles. The temperature graph of one of these (L. D.) would appear to indicate an anovulatory cycle. The preovulatory plasma progesterone values range from 0 to 0.53 lAg per cent with a mean value of 0.17 11g per cent. The concentration found in the plasma of the anondatory subject (L. D.) is somewhat higher (0.18 to 0.93 ftg per cent) than that found in the preont!atory phase of the other subjects. The mean (0.43 fLg per cent) of all determinations done on this donor approaches that found for a pool of male plasma (0.66 N?; per cent) .11'' 17 Progesterone content of the plasma in the second half of the cycle is much higher, 0.6 to 2.1 ftg per cent (mean 1.08 lAg per cent) . The average of the plasma values during this phase was somewhat lower in the two older donors (L.G. . 0.96;N.H., 1.03) thaninthc younger girls ( 1.15 and 1.12) and this phase of the cycle was shorter in the former subjects. Although no Ruch relationship appears to be present in the first half of the cycle, it was only in the oldest of the do-

nors that a value of 0 was found at any time. The subject L. D. did not exhibit any significant increase in plasma progesterone during the second half of the cycle. Beginning 2 to 4 days before the major ovulatory rise, there appears to be another, smaller peak in plasma progesterone concentration. In subject J. W., two such elevations can be noted-one occurring just prior to the major ovulatory peak on days 30 to 32 and the other on days 15 and 16. Just after the first rninor peak, she experienced 4 days of slight bleeding. From the onset of this episode to the first day of her next period, the time interval \vas 30 days which is the average duration of her usual menstrual cycle. Two days of light spotting preceded the onset of the menstrual period occurring at the t>nd of the cycle studied in the subject L. G. This was in contrast to the abrupt onset of flow which characterized the menstrual periods of the other subjects. It will be noted that the second half cle\·ation of plasma progesterone in this donor was prolonged into the period of bleeding, in contrast to the premenstrual fall seen in the others. Comment

In 1950, Forbes 1 ' reported serial plasma progesterone levels, determined by bioassay, of 4 women during the menstrual cycle. Comparison of these data with ours reveals interesting qualitative similarities and quantitative differences. The bioassay did not consistently demonstrate the presence of progesterone in all samples taken during the cycles studied. This absence was most notable in the one patient having an anovulatory cycle and during the preondatory phase of the others. Yet, in those subjects who had a biphasic temperature curve, the form of the graph drawn from the plasma progesterone concentrations found by either method is almost identical. Both show an abrupt rise beginning about midcycle and a rapid fall starting 5 to 7 days before the onset of menstruation (except in subject L. G.). Each report demonstrates a smaller peak in progesterone levels im-

Volume 85 Number 8

Plasma progesterone levels during menstrual cycle

mediately prior to the ovulatory phase of the cycle. The quantitative differences between the results of the two techniques of assay are quite marked. The bioassay method shows plasma progesterone concentrations varying

983

from 170 to 520 ~tg per cent during the ovulatory phase of the cycle. In contrast to these values, the range found by the present method is only 0.17 to 2.09 p,g per cent during this phase. In the preovulatory phase, the physicochemical technique demonstrated

38 WSF Po 13r.o

I

J

I

,,'

A

I

I\

\

'

I

\ \

Fig. 1. Plasma progesterone levels in the menstrual cycle. Shaded area indicates menstrual period. Broken line, temperature: solid line, plasma progesterone. Ovulatory cyde.

2..5·-

MENSTRUATION

MENSTRUATION ~

MENSTRUATION

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/"'--....

' ...____ , I l

I

I I I r

v

4S

I

2

30

Fig. 2. Plasma progesterone levels in the menstrual cycle. Prolonged ovulatory cycle. Shaded areas indicate menstrual period. Broken line, temperature; solid line, plasma progesterone.

984

April 15, !96l

Woolever

~~;;;,;---------~-

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Fig. 3. Plasma progesterone in menstrual cycle. Ovulatory cycles. LG, irregular shedding (?). Shaded areas indicate memtruation. Broken line, temperature: solid line, plasma progesterone.

the presence of small amounts of progesterone in all but two samples taken from one patient. During that part of the cycle extending from the end of menstruation to just before ovulation, the bioassay showed the presence of progesterone in only one of all of the samples taken from all of the donors. During menstruation, the latter technique gave a positive value more frequently. The quantitative differences are still more evident in a comparison of the anovulatory cycles studied in the two reports. The bioassay failed to reveal the presence of progesterone in any of these samples, whereas the present method yielded values higher than those found in the first half of the ovulatory cycle. The divergence of the quantitative results derived from the two methods is not difficult to reconcile if one keeps in mind the difference between the techniques used in the

J.

OIJ>t. & Gyn<·c.

analysis. The present method has been shown to be specific for the measurement of the hormone progesterone 10 • 17 and the results obtained agree with those found by other physicochemical techniques. 1 "- 15 The bioassay is a measure of the "progestational activity" of the sample. Forbesw, "0 has pointed out that it is subject to synergistic and antagonistic effects of other hormones. Variations in the ratio of progesterone to estrogens and to other progestational agents can nullify or greatly augment the responst• to a given test dose of progesterone. This difference in the techniques would explain the divergence of the absolute values found in the two studies. The absence of progestational changes in the endometrium of the preovulatory phase of the cycle and in the anovulatory cycle, in the presence of circulating progesterone, may be accounted for in a similar manner. Further information on this subject must await the development of techniques which will allow the serial rneasurement of the plasma proge:>terone and estrogen levels in the same menstrual cycle. Such a method is now under investigation in our laboratory. The peak in plasma progesterone concentration occurring in the second half of the cycle coincides with the development of the corpus luteum. The presence of significant amounts of progesterone in the ripe graafian follicle, prior to ovulation, has been shown by Zander." 1 This would appear to correlate well with the smaller rise in plasma content shown just before the beginning of the major peak in both studies. The slight fall between these two peaks may represent an interruption of secretion by the event of ovulation. Support for this concept is gained from the sequence of events in one subject, J. W. The usual duration of the menstrual cycle in this girl is 29 to 30 days. If one assumes that ovulation occurs 14 clays prior to menstruation and that the ripe follicle contains and secretes significant amounts of progesterone for the 2 to 3 days prior to ovulation, there should be a transient rise in plasma progesterone on days 14 to 16 and a fall on days 16 or 17 of the cycle of this subject. Exam-

Volume 85 Number 8

Plasma progesterone levels during menstrual cycle

ination of the results of the daily assays does show such a rise occurring on days 15 and 16 with a drop on day 17. However, instead of being followed by the major ovulatory peak. the plasma progesterone levels returned to the preovulatory range. This was accompanied by 4 days of light uterine bleeding. If this can be interpreted as regression of the follicle and failure of development of the corpus luteum, perhaps due to excessive bleeding into the follicle at the time of ovulation, then we may assume that a new cycle began with the onset of the bleeding. Again it can be expected that there will be a transient rise in plasma progesterone levels Jj to 16 days later and a slight fall On day 16 or 17. The chart does show a rise on days 30 to 32 with a small drop on day 33. This is followed by the major ovulatory peak and a normal menstrual period began 30 days after the onset of the previous bleeding episode. One donor (L. G.) had 2 days of spotting before the onset of the full menstrual flow. Her plasma progesterone levels demonstrate the preovulatory rise and dip and the major ovulatory rise but fail to show the rapid premenstrual fall seen in the other ovulatory patients. McKelvey and Samuels 22 have shown that in irregular shedding of the endometrium there is a persistence of urinary pregnanediol into the bleeding phase of the cycle. This patient may represent an example of this syndrome. It is of interest to note that this subject had the lowest preovulatory levels, the lowest ovulatory peak, and the shortest ovulatory phase of any of those studied. She is also the oldest of the group and the only one in whom pmgesterone was absent from any sample. The presence of progesterone in peripheral plasma during the preovulatory phase of the cycle, in the anovulatory cycle, and in the male'"' 17 indicates a source other than the corpus luteum. The identification of progesterone in the adrenal venous blood of the human" 3 and the increase in urinary pregnanediol following the m JCction of ACTH 2 ' ' 25 would suggest that some part of

985

Fig. 4. Plasma prog<:sterone in menstrual cycle. Anovulatory cycle. Shaded area indicates menstrual period. Broken line, temperature; solid line, plasma progesterone.

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JA 39 W.WF P4G,, 2Bd.i

1.0

Fig. 5. Plasma progesterone levels following stimulation with chorionic gonadotropin and with ACTH. Small graph with shaded area indicates basal body temperature and days of cycle in which experiment was done.

986

Woolever

the plasma level IS contributed by the adrenal. The narrow range of values found in the first half of the ondatory cycles suggests a rdatin+v stable rate of secretion from sonw source or sources (adrenal and/or ovary\ during this time. The only apparent difference between these subjects and the one donor who did not onilate is in the function of t!w mary. Yet this latter subject had plasma progesterone \'alucs considerably hig·lwr than those found in the preovulatory phase of the other cycle' studied. This might imply that the ovary can sc•crete progesterone in the absence of the corpus luteum. The still hig}wr value found for the male plasma pool may indicate a similar role for the testis in tlw mak. Thes<· comparisons do not establish that the testis or tlw oyary. in the absenct" of a corpus luteum, do secrete progt"slerone. Preliminary studies have bet"n started to investi!!,ate this question further. Three subjerts were g;iven infusions of chorionic gonadotropin" and adrenocorticotropin and the changes in plasma progesterone were observed (Fig. 5) . The study period extended over the first :{ days of the ovulatory phase of thr cycle as jttdged bv basal temperatun· rf'cords. The expected rise in plasma progesterone lew! with chorionic gonadotropin infusion was noted. However, during the infusion of adrenocorticotropin there was a fall in plasma progesterone levels. This failure to obtain an increase in the level might suggest that the adrenal cortex docs not contribute a ~il;nificant amount to the plasma progesterone- at least during the ovulatory phase of the cycle. Further studies will he necessary to evaluate the significance of this f-inding, but the implication of these experiments would seem to be that the gonad IS the major souree of plasma progesterone. Summary

Plasma progesterone levels during the menstrual cycle vary from 0 to 0.53 p.g per cent in the first half and from 0.6 to 2.1 J-Lg per cent in the postovulatory phase. Values

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&

1

found in an anovulatory cycle were somewhat higber ( 0.18-0.93 l'f:!. per cent than 1 hose found in the lirst half of the O\ ulatory nTle. Certain similarities to and differences between the results found bv the bioassay and physicochemical methods have het~n discusst"d. It i~ suggested that the gonad i~ the major source of the progesterone found m the plasma in the abst·nce of pregnam''

Addendum. Tlw editors of the JouR'-i.\J have pointed out !hat the temperature nTord in Fig. I is not consistPnt with tht• usual ovulatorv cydc. Thi' ohs<'rvation ha.< lr·d H> to re-Pxaminr· all of rlw tt·mperatnrc rr·cords and fO make an imragroup comparison of temperatHrP changes :1nd plasma progcstermw changpq :Tahir~ I '. From an examination of the data pr,•;,,•med in this numwr, the following obsen·atinn' can lw mand in either the temperature or the progest<'l'Oil<' level~. This is in kt•Pping with current conc<·pt> of the anovulatory nck. :!. In ;til of thr: n·!uaining patient,, positi\·t· changes in basal tt'tnp<·rature can b·· , orrelatcd vdth positiv•• changes in plasma progestcron<' for rfw corrPspouding time p!"riods wh<'rc thest' data are a\·ailahk. In :mbjt•ns J. B. and L (;., thi.~ is dearly rdlectcd in the shape of tlw temperature curw. In patient J. \V., this trl'nd is less obvious dtit' in part to i ht• lnng tir~t phase and in pan tn thf' lnw temperatures recorded in days :1:1 to 37. In subject N. H., tht: correlations an· still mon· obscure on inspection of tht:' l<"lllpcrature cun·1· alorH>, Whether or not tfw phvsical condition of tht· patinll on days II to J :) contributr•s to this is difficult to determine. Iu spite of this, thi•re is a small increase in rempNmun· during the time of the prog<'sterone measurements takm in this cycle. Tlw shape of thP plasma progesterone curve and the magnitude of the progesterone values during this time an· compatible with thost' obtained in the same phasP of tlw cycle of patients L. n .. ]. \V,, and J B. There is a distinct diffPrence in both the shape of thf' cun-e, the magnitude of the valurs, and the correlation with temperature changes when compared with thosr of subject L. D.

Volume 85 Number 8

Plasma progesterone levels during menstrual cycle

987

Table I Mean preovulatory values

Mean difference Mean

Patient

Days

Plasma progesterone (p.g per Temperature (degrees F.) cent)

Days

"~

,/

values

Plasma Plasma progesterone progesterone Temperature (!-Lg per Temperature (!-Lg per (degrees F.) cent) (degrees F.) cent)

L. D.*

2 to 11

97.8

0.47

12 to 24

97.6

0.45

-0.2

-0,02

J, B.

I to 14

97.7

0.19

15 to 28

98.4

1.15

+0.7

+0.96

L. G.

2 to 15

97.4

0.15

16 to 23

97.9

1.03

+0.5

+0.88

J. w.

2 to 32 17 to 32t

97.4 97.3

0.17 0.14

33 to 47

97.8

1.12

+0.4 +0.5

+0.95 +0.98

98.0 97.8

0.18

16 to 27

98.2

0.96

+0.2 0+.4

+0.78

N. H.

6 to 1 to

*The cycle has been broken arbitrarily after day 11 for purposes of comparison. tDays 17 to 32 represent the first half of a new cycle according to our interpretation of the sequence in this subject. tDays 6 to 15 of the first cycle for which plasma progesterone vaJucs are not available. During days 11 to 15, this patient had signs and symptoms of a cold. §Days I to 14 of the sec.ond cycle during which plasma progesterone values were obtained.

Comparison of the last half of the first cycle (days 16 to 27) with the first half of the next cycle (days 1 to 14) shows the same progesterone-temperature correlation seen in the ovulatory cycles of the other individuals. Although it is dangerous to draw any conclusions from one cycle relative to another, this comparison would seem to suggest further that the first cycle is ovulatory. It is for these reasons that we have included patient N. H. in the ovulatory group. The correlation of the temperature change and the plasma progesterone change during the menstrual cycle does suggest a cause and efiect relationship. However, it will be noted that the nature of the temperature increase is such that there is a rather abrupt: rise, a plateau, and then a rapid fall. This is distinctly different from the usual trend shown by the more gradual rise, peaking, and prolonged fall of the plasma progesterone level. This is evident in both the

physicochemical assay and in the bioassay of Forhes. 18 It has been shown, as previously noted, that the progesterone effect on the endometrium is influenced by other progestogens and by estrogens. Perhaps there is a similar interhormonal relationship in regard to the temperature effect of progesterone. Also, it may well be that the cornpound or compounds that may influence the temperature effects are not the same, either qualitatively or quantitatively, as those which influence the endometrial response. Such a difference may help to explain why the infertile couple, in whom all investigative results (including temperature records) are normal, fails to accomplish pregnancy. This does not mean that temperature graphs are of no value in the assessment of the infertile couple. It means only that our interpretation, relative to the hormonal conditions of the menstrual cycle, may not always be correct.

REFERENCES

1. de Graaf, Regner: De mulierum organis genera tioni inservien tibus, 16 72. 2. Prenant, L. A.: Rev. Gen. Sc. 9: 646, 1898. 3. Corner, G. W., and Allen, W. M.: Am. J. Physiol. 88: 326, 1929. 4. Butenandt, A., Westphal, A., and Hohlivig, W.: Ztschr. physiol. Chern. 227: 84, 1934. 5. Slotta, K. H., Ruschig, H., and Fels, E.: Chern. Berichte. 67: 1270, 1934. 6. Allen, W. M., and Wintersteiner, 0.: Science 80: 190. 1934.

7. Wintersteiner, 0., and Allen, W. M.: Bioi. Chern. 107: 321, 1934. 8. Hartman, M., and Wettstein, A.: Helvet. chim. acta 17: 1365, 1934. 9. Salhanick, H. A., NoaH, M. W., Zarrow, M. X., and Samuels, L. T.: Science 115: 708, 1952. 10. Diczfaluzy, E.: Acta endocrinoL 10: 373, 1952. 11. Pearlman, W. H., and Cerceo, E.: J. Clin. Endocrinol. 12: 916, 1952.

988 Woolever

I:!. Zander, J., and Simmer, H.: Klin. Wchnsrhr. 32: 529, 1954. 1:J. Z:mdc·r. .J , and \'On Miinstrrmann, :\. :\L: Klin. Wchnschr. 34: 494, 1956. 1-l·. Short, R. V.: J. Endocrinology 16: 415, 1958. 15. Oertel, G. W., Weiss, S. P., and Eik-Kcs, K. B.: J. Clin. EndocrinoL 19: 213, 1959. lfi. \Voolen·r, C. A., and Goldfien. A.: Intcrnat. J_.\ppl. Radiat. (In press.) 17. Wooh:-ver, C. A .. and Goldficn, A.: Proc. I nternat. Con gr. Hormonal Steroids. 1 In pn•s,.'

Pl. Forbes. T. R.: AM. J. OB,;T. & GYNF.C. 60: 180. 1950. 19. Forbes, T. R., Coulornbrl', A. J., and Coulombrc, J.: Endocrinology 68: 858, 1961,

.\nL

J.

.\p: ~.-,_]'ltd Oh;.,r, & Gynee.

20. Forbes, T. R.: Brook Lodge Symposium, Progesterone, Brook Lodge Press, 1961, p. 71. :!1 Z;Jndrr. J.. Forbes. T. R .. \'Oll Miinsterrnann. A. M., and Neh<'l', R.: J. Clin. Endocrinol. 18: :1:)7, 1958. .:2. McK(·Iwy, J L. and Samw·ls, L. .\'.r. J. Omn. & Gv:--:Ec. 64: 988, 195:2. 23. Short, R. V.: Biochemical Sod,•ty !:iymposia: Biosynthesis and St>cn:tion of Adrenocortical Steroids. 1\o. 18, p. 59, London, I %0. 2·1·. Klopper, A., Strong, J. A., and Cook, L R.: J. Endo