Effect of continuous oral administration of estriol on corpus luteum function

EFFECT

OF

CONTINUOUS

ESTRIOL

ON

CORPUS

1

J. Vdhapassi 1 2

Middle

Department

ORAL

Finland

LUTEUM

and H.

Central

ADMINISTRATION

of Clinical Chemistry, SF -00290 Helsinki

FUNCTION*

Adlercreutz

Hospital.

OF

2

Jyvaskyla

University 29, Finland

,

of Helsinki,

ABSTRACT Estriol, 6 mg per day, administered orally to six normal women, caused a statistically significant reduction in plasma LH levels in the follicular phase and during and immediately after ovulation as compared to control cycles. In the luteal phase,,a highly significant increase in estradiol production and a reduction in progesterone production, as judged from the plasma levels of these hormones ? was observed. The mean length of the menstrual cycle was not significantly altered and no pregnancy occurred in this small group of women. It is concluded that estriol administration significantly influences corpus luteum function perhaps by affecting steroid biosynthesis. It is suggested that the remarkable increase in plasma estradiol levels which results from estriol administration cause a reduction in the functional life-span of the corpus luteum. However, the significantly lower plasma LH level seen during and immediately after ovulation may also be partly responsible for the luteolytic effect observed.

*

The results were on Contraceptive

Accepted

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for publication

1975

VOL.

reported Research January

11 NO. 4

to the International (ICCR) in March 21,

Committe 1974.

1975

427

CONTRACEPTION

INTRODUCTION The mechanism by which postcoitally administered estrogens interrupt pregnancy is not yet known (l), but it has been suggested that the contraceptive effect is due to luteolysis. Various types of estrogens cause luteolysis in the hamster (2)) guinea pig (3,4), sheep (5,6), cow (7)) monkey (8) and in humans (9). Administration of estriol to rats, rabbits and hamsters is effective in preventing pregnancy (lo-131 and the implantation of silastic capsules containing estradiol into Rhesus monkeys causes a comparatively high level of plasma estradiol in the luteal phase and results in a premature decline in the plasma progesterone concentration and a 43 % reduction in the functional life-span of the corpus luteum (14). The present investigation was undertaken in order to study the effect of orally administered estriol on corpus luteum function in normal women. In addition to the determination of LH, progesterone and estradiol in plasma, the plasma levels of unconjugated and conjugated estriol were followed in some subjects by a specific mass fragmentographic procedure. The results indicate that daily administration of 6 mg of estriol significantly decreases plasma LH during the follicular phase and during and immediately after ovulation and causes a significant increase in plasma estradiol with a concomitant decrease in the plasma progesterone level in the luteal phase of the menstrual cycle.

MATERIALS

AND

METHODS

Ovulatory function was monitored in six normally menstruating women by assaying plasma estradiol, progesterone and LH every 2nd During the next cycle, or every 3rd day during one control cycle. 6 mg of estriol was administered orally every morning in one single dose and the same measurements made. In two of the subjects plasma conjugated and/or unconjugated estriol levels were also determined Basal in samples drawn in the morning before the next estriol dose. body temperature was measured daily. An additional two women were followed in the same way throughout a control cycle, but during the second cycle, 20 mg of estriol was administered twice daily during 7 days of the luteal phase. On the 5000 IU of human chorionic fifth day of estriol administration, gonadotrophin (HCG) (Pregnyl, Organon) was given by injection. Plasma estradiol, progesterone and LH were assayed during both cyPlasma unconjugated and conjugated estriol was measured in cles. three samples taken from each patient during estriol treatment. Further, unconjugated and conjugated estriol were estimated 14 times in the same way as described above in one subject who was given 20 mg estriol every morning in one single dose during one menstrual cycle.

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Plasma Luteinizinp Hormone (LH) was determined using a double antibody solid phase (DASP) technique, for the most part according to and purified human Den Hollander and Schuurs (15). The antiserum LH were kindly supplied by the National Institute of Arthritis, Metabolism and Digestive Diseases (Bethesda, U. S . A.). The radioiodination of LH with 1251was carried out by coupling the lactoperoxidase to a cross-linked copolymer of maleic anhydrtde and butanediol divinyl ether (E. Merck, Darmstadt, Germany) and using the coupled enzyme for lodinatton (16). Human Pttuitary Luteinizing Hormone (68/40) (National Institute for Medlcal Research, London, England) was used for the construction of the standard curves (assuming a value of 40 U /ampoule). Antigen (unknown or standard), labelled antigen and first antibody were incubated for a period of 48 h in a volume of 550 ul. Insolubilized second antibody [ sheep antt(rabbit y -globulin) immunosorbent , 5.4 ml diluted in 50 ml of 0.02 M sodium phosphate buffer, pH 7, containing 0.02 IM NaCl, 0.005 IM Na-EDTA and 0.1 % w/v merthiolate] was then added (500 111) and the tubes were rotated at room temperature for 16h before centrifugation, after which the supernatant was discarded and the solid phase washed and counted with an LKB-Wallac gamma counter. All assays were carrted out in duplicate. The interassay coefficient of variation as calc.ulated from assays carried out on a pooled sample 17 times during 18 months IS 14.2 X. In addition, 67 analyses were carried out on the hLH reference preparatton LER-907 during a 2-year period by three different persons revealing a coefficient of variation of 9.6 %. Plasma estradiol was determined according to the method of Ontkki and Adlercreutz (17). The estradiol LS precipitated with the P-globulin fraction leavtng most other estrogens 111solution. It is then extracted from the precipttate and RIA carrted out in the usual way using dextran-coated charcoal for the separation of bound and unbound steroid. The antibody (anti-17@-estradiol-6-bovine serum albumin) used was purchased from Searle Dtagnostlc (High Wycombe, England). The specLftctty of the method LS good but the sensttivlty of the procedure 1s slightly less than when direct methods are used because of the lower recovery. When calculatmg the results, a few of the values falling helow the limit of detection (lo-21 pg/ml depending on the labelled steroid used) were not Included as zero values but were given the value of the limit of detection. This means that the mean values cited for the follicular phase of the menstrual cycle may be sltghtly higher than the actual values. iMost assays were carried out in duplicate. Plasma progesterone was determtned by a direct method uttltzing an anti-progesterone-ll-succinate bovtne serum albumin, purchased from Endocrine Sciences (Callfornla , U. S . A. >. The cross-reactlon with other naturally occurrlng sterotds is small and an addttional chromatographic step did not change the values obtained. Extraction of progesterone is carrted out twice with 3 volumes of petroleum ether (b.p. 30-GOOC, iMalltnckrodt, St. LOULS, MO. ? U.S.A.). After evaporatton of the solvent. the residue IS dtssolved

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in borate buffer, pH 8 .O, and the assay carried out as described by Haukkamaa (18). In this method the separation of bound and unbound steroid 1s carried out using deutran-coated charcoal. All determinations were carried out in duplicates, the lnterassay coefficient of variation being 5.1 to 13.6 % depending on the concentration level. Plasma conlugated and unconjugated estriol was determined by a mass fragmentographic technique recently developed in this laboraFollowing extraction of unconjugated estrlol, the conjutory (19). gates are hydrolyzed with an extract of Helix pomatia and the liberated steroids extracted. Both fractions are purified by solvent partition, methylation and chromatography on partially deactivated alumina. After silylation , the ditrimethylsilyl ether derivative (TM.51 of the methylated estriol is determined by mass fragmentography using the deuterated TMS derivative of methylated estriol as internal standard. The method is highly specific for estriol. Statistical treatment of the results: Every cycle was divided into periods of three days starting from the ovulation period (day 0 The “ovulation” day was chosen If most of the folin the figures). lowing criteria were fulfilled: The plasma LH level is high (usually highest) on that day and in that and in the previous plasma sample the estradiol value 1s high. The next sample should have a significantly increased plasma progesterone value (most Important criterion) and the basal body temperature should be elevated. Every point in Figures l-3 represent the mean value of all values obtained during three days, e .g . day +3 represents days +2 to +4 and all values obtained for these days were used in the calculation of the mean value. It is likely that ovulation occurred in some subjects closer to the time of the previous sample. However, because all subjects were treated in the same way, an error of one or two days has no Lnfluence on the conclusions drawn.

using

The statistical treatment of the grouped the t-test according to de Jonge (20).

results

was

carried

out

RESULTS Length of menstrual cycle: Administration of 6 mg of estrlol daily did not change the mean length of the menstrual cycle (6subjects), and no pregnancies occurred. All subjects ovulated in both cycles. In three subjects the cycle was shorter and in two subjects it was longer and in one subject the cycle length remained unchanged. Effect of estriol on plasma LH: Mean plasma LH levels decreased to a highly significant degree (p< 0.001) during the entire follicular phase and during and immediately after ovulation but remained the same in the luteal phase as during the control cycle (Figure 1).

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LH mlU/ml

I

I

I

-16 -12 0-0

x------x

Figure

1.

-9

I

-6

I

-3

Control 6 mg estriol/day

I

I

0

+3

I

+6

I

I

+9

+12

Days

Mean plasma LH in SLX normal women during one control cycle and one cycle during which 6 mg per day of estriol The significance of differences was administered orally. between mean values are indicated by stars (* = p
Effect of estriol on plasma estradlol: Mean plasma estradlol levels did not chance sienificantlv in the follicular chase of the menstrual cycle or duryng a\d immed;ately after ovulati&on when estriol was administered continuously. However, a remarkable mean increase in the plasma level of estradiol was found in the luteal phase; if 5 of the 6 subjects treated, the mean peak level (all subjects) exceeded the peak level seen at the time of ovulation. This mean rise was highly significant (p< 0 .OOl> during the whole period between the 5th and the 13th postovulatory days as compared to the control cycle (Figure 2).

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ESTRADIOL pg/ml 3OOr

I

-15

-12

0-a

x-----x

Figure

2.

I

-9

I

I

-6

-3

I

I

0

+3

+6

Control 6 mg estriol/day

+9

+12

Days

Mean plasma estradiol in six normal women during one menstrual cycle before and during oral administration of See also legend to Figure 1. 6 mg per day of estriol.

If all plasma progesEffect of estriol on plasma progesterone: terone values durine the luteal chase were treated as one Y eroun1 then. a highly significant decrease as compared to the control cycle, The maximum progesterone level was (p could be observed. the same, but the peak was of shorter duration. Highly significant (p
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Another highly signlftcant difference was seen tn the beginning of the menstrual cycle, here the plasma progesterone level was stgnificantThe origin ly (p increased in the estrlol-treated subjects. of this slight but clearly increased productton of progesterone remains obscure.

PROGESTERONE ng/ml 8

6

-• Control x------x 6 mg estriol/day l

Figure

3.

Mean plasma progesterone during one menstrual cycle administration of 6 mg per legend to Figure 1.

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Days

level tn SIX normal women before and during oral day of estrtol. See also

433

CONTRACEPTION

results

Plasma levels of unconjugated and conjugated obtained are presented in the Table.

estriol:

The

Table,

Plasma levels of unconjugated and conjugated estriol following oral admlnistration of estriol. Determinations were carried out by mass fragmentography.

Subject

No. of assays

Treatment Est riol/day

Unconjugated estriol

Conjugated estriol

pg/l

(+ - SD> -+ 0.83

irgil

(+_SD>

H.H.

10

6 mg

2.15

not determined

N.M.

12

6 mg

I .75 -+ 0.33

11.6

-+

M.E.

lh

20 mg

1.58 -+ 0.27

29.7

-+ 18.1

3.3

J. R.

3

40 mg

2.10

-+ 0.70

25.9 -+ 10.4

V.K.

3

LO mg

2.14

t 0.66 -

68.8

+ 31.4 -

-

The Increasing conjugates

plasma level of unconjugated estriol is not influenced However, the the amount of estriol admtnistered. clearly increase with increased dosage.

by estrlol

Effect of estriol and HCG in the luteal phase of the menstrual cycle: Only two subjects have been investigated. However, the administration of.5000 1U of HCG following 5 days treatment with 2 x 20 mg estrlol orally/day did not cause any large Increase in In both subjects the peak plasma progesterone plasma progesterone. levels were higher in the control cycles. In one of the two subjects, a slight prolongation of the luteal phase progesterone peak was observed after HCG administration and the plasma estradiol showed a remarkable increase to almost the same level as prior to ovulation no prolongation of the luteal phase (Figure 4). In the other subject, was observed, the plasma level of progesterone remained lower than the estradiol level was higher during the control cycle. However, during the luteal phase (mean of all five postovulatory values was 251 pg/ml as compared to 58 pg/ml for the control cycle).

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Ovulation

Progesterone nglml

Estradiol

1

w/ml

1 8 :

Day of menstrual

cycle Estradiol w/ml -300

- 200

- 100

-0 Day of menstrual

Figure

L.

APRIL

1975

cycle

Plasma estradiol and progesterone levels in one normal woman in a control cycle (upper panel) and in a cycle in which 40 mg estriol was administered daily postovulatory for six days and HCG was administered on the 5th day of estrlol treatment.

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DISCUSSION The results obtained seem to indicate that daily oral administration of 6 mg of estriol inhibits hypophyseal release of LH to some degree as judged from the lowered plasma LH levels. Despite the fact that this effect could be observed during the whole follicular phase and during and immediately after ovulation. ovulation was not inhibited by this weak estrogen. The decreased level of LH seen could not be due to increased plasma estradiol levels, because the estradiol level was the same as in the control cycles during the period in question. There was no change in the mean length of the menstrual cycle, because it was shortened in three subjects and increased in length in 2 of the other subjects No pregnancies occurred in this small group and subjectively the women felt very well. The effect of estriol on plasma steroid hormone levels was not the same in all subjects; the increase of estradiol in the luteal phase was seen in 5 of the 6 women and the decrease in progesterone levels was clear in 4 of the 6 subjects. However, all values obtained were used in the statistical treatment of the results and the conclusions drawn are based on the mean values obtained. The remarkably increased plasma estradiol values in the luteal phase and the shorter progesterone peak indicate that the function of the corpus luteum was affected. Because of the shorter progesterone peak,the effect can be regarded as “luteolytic” .% If all LH-values in the luteal phase were included in the calculations, no change could be observed, and it is therefore suggested that the increased estradiol production in the ovaries is caused by a direct effect of estriol on estradiol biosynthesis, which seems directed more towards estrogen than towards progesterone production. However, the stimulation of estradiol production in the luteal phase may lead secondarily to luteolysis as observed after implantation of estradiol-containing sllastic capsules into monkeys (14). The luteolytic effect of estriol is therefore perhaps mediated via the increased estradiol production. The method used for estradiol determination (17) involves extraction of estradiol from plasma after coprecipitation of the estradiol with the P-globulin fraction. The recovery of the radioactive internal standard therefore .eives an estimate of the percentage estradlol bound to sex hormone binding globulin at + 4OC in &vitro in the presence of endogenous steroids. The values obtained in the follicular and luteal phases and before and after treatment were compared after statistical treatment of the results. There was no significant difference in the percentage of estradiol bound in the two phases of the menstrual cycle under control or treatment conditions. However, the mean percentage decreased significantly from 54.7 to 42.7 % (p< O.OOj;) when the control cycle and the treatment cycle were These results seem to indicate that administration of6 mg compared. of estriol/day does not cause any significant increase in sex hormone

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binding globulin, but probably displaced some estradiol from the binding sites. If such displacement also occurs in viva cannot be -However. if occurring. it would elucidated from these expertments. not account for the considerable increase tn plasma estradiol seen during the luteal phase of the menstrual cycle, because the effect was the same in the follicular phase, where no estradiol increase was The relatively higher amount of estradiol unbound may be one noted. reason for the lower plasma LH during the follicular phase and dut-1 :.I$ However. and after ovulation and may ccntribute to the luteolysis. these data do not contradict our view that there LS an increased estradiol production during the luteal phase, which is more ltkely to be the matn cause of the luteolytic effect observed. Administration of large doses of estrtol (40 mg/day) postovulatory also seemed to depress the production of progesterone in the luteal phase and the response of plasma progesterone to the administration of HCG was very slight in one subject and undetectable in the other. However. in the subject with the slight prolongation of the progesterone peak after adminis:ration of HCG in particular, the estradiol level increased lo-fold from the value observed at the time of the HCG administration and in the other subject the level was significantly elevated. These results also suggest that estriol administration affects ovarian steroid biosynthesis and that this results in increased estradiol and decreased progesterone production. There is some evidence which suggests that the luteolytic effect of estrogens is mediated through prostaglandtns . Blatchley et al --. (21) demonstrated conclusively an endo.genous secretion of prostaglandin F2J, a substance known to be luteolyttc, into the uteroovarian vein of estrogen-treated guinea-pigs. In the macaque monkey estrogen administration during the luteal phase of the menstrual cycle caused luteolysis and in more than half of the animals the plasma prostaglandin F level rose. The demonstration tn the present study that a comparative1.y weak natural estrogen tn doses far below those used by Gore -et al.. (9) (30 mg diethylstilbestrol dtphosphate daily for 5 days) has luteolytic effects, suggests that estrogens produced by the ovary during the luteal phase may contribute to the regulation on the life-span of the corpus luteum. It was of interest to observe that the plasma unconjugated estriol level did not increase when the dose of estriol was increased from 6 to 40 mg/day despite the fact that there was a clear increase in the level of conjugated estriol. It seems that this phenomenon needs further investigation. In such studies the use of a highly specific mass fragmentographic procedure is specially advantageous because other estrogens do not influence the results at all. As the effect of estriol on corpus luteal function was not the same in all subjects, further investigations using other doses of estriol are planned in order to find out, at which dose level luteolysis occurs without inhibition of ovulation. In those studies, plasma measurements of unconjugated and conjugated estriol may also be of value.

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ACKNOWLEDGMENTS The sktliful technical asststance of Mrs. Slrkka Tlalnen, Mrs. Anja Manner, Mrs. Rauni Lehtola, Mrs. Helena Ltndgren and Miss Inga Wiik is greatly appreciated. We thank also the Nattonal Pituitary Agency (University of Maryland f School of Medicine). National Institute of Arthritis, Metabolism and Digestive Diseases, Bethesda, U. S. A. , for supplying the human LH and anti-human LH serum and the National Institute for Medical Research, London: England, for supplying the human ICSH used as standard in LH determinattons . This work was supported by the ICCR of the Population Council and the Ford Foundation, New York.

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Scublinsky, A. and Wotiz , H.H. The contraceptive action of III. Oral activity of oestriol and its impeding oestrogens. 18-homo analogue in the rat. J Reprod Fert 26: 233-236 (1371)

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Scublmsky, A. and Wotiz, H.H. The contraceptive Effectiveness of oestriol VII. impeding oestrogens. J Reprod Fert 26: 365-367 rabbit and the hamster.

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Karsch, F.J. 7 Krey, L.C., Weick, R.F., Dierschke, Functional luteolysis in the Rhesus monkey: Knobil, E. role of estrogen. Endocrinology 92: 1148-1152 (1973)

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Den Hollander, F.C. and Schuurs, A.H. W .IM. assay Methods (K. E. Kirkham and W. M. Hunter, Livingstone, Edinburgh, 1971, p. 419-422

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Onikki , S. and Adlercreutz tion of plasma estradiol-17P globulin fraction. J Steroid

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Haukkamaa , M. Binding of progesterone by rat myometrium during pregnancy and by human myometrium in late pregnancy. J Steroid Biochem 5: 73-79 (1974)

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Adlercreutz , H. , Nylander , P. and Hunneman, D. H. on the mass fragmentographic determination of plasma Biomedical Mass Spectrometry (In press)

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Inleiding tot de Medische Statistiek. Dee1 2. De Jonge , H. Verhandeling van het Nederlands Instituut voor Praeventieve Geneeskunde , Leiden , 1964, p. 486

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Blatchley , F. R. , Donovan, B T. , Poyser, N. L. , Horton, E. W. Thompson, C.J. and Los, M. Identification of prostaglandin F2u in the utero-ovarian blood of guinea-pig after treatment with oestrogen. Nature 230: 243-244 (1971)

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, H. Radioimmunological determinausing precipitation of the plasma Biochem 4: 633-639 (1973)

Studies estriol.

,

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,