CONTRACEPTION

ESTROGEN-PROGESTOGEN ONCE-A-MONTH INJECTABLE

CONTRACEPTIVES AND SERUN PROLACTIN

J. Garza-Flores, V.M. Alba, M.C. Cravioto, L. Hernandez, G. Perez-Palacios

Departamento de Biologia de la Reproduccibn Instituto National de la Nutricibn S. Zubirgn

Mexico City, Mexico

G. Alvarado, R. Rivera

Instituto de Investigacibn Cientifica Universidad Jugrez de1 Estado de Durango

Durango, Mexico

R. Recio, S. Basso1

Departamento de Biologia de la Reproducci& Hospital Universitario

Universidad Autonoma de Coahuila Torreen, Coahuila, Mexico

Abstract

To assess the effect of hormonal monthly injectable contraceptives upon the serum values of immunoreactive prolactin (Prl), three groups of women of reproductive age exposed to different estrogen-progestogen injectable formulation for a minimum of one year were studied. The first group (n=lO) received dihydroxyprogesterone acetophenide 150 mg and estradiol enanthate 10 mg (DHPA/E2- EN), Group 2 (n=21) received medroxyprogesterone acetate 25 mg and estradiol cypionate 5 mg (MPA/E2-C) and Group 3 (n=19) was exposed to norethisterone enanthate 50 mg and

Reprint requests and address for correspondence:

J. Garza-Flores; Department of Reproductive Biology, National Institute of Nutrition S. Zubiran; Vasco de Quiroga No. 15 Tlalpan, 14000 Mexico City, Mexico

Submitted for publication February 13, 1989 Accepted for publication February 24, 1989

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estradiol valerate 5 mg (NET-EN/E2-V). A group of IUD users (n=16) served as the control group. Serum Prl and 17p- estradiol (E2) concentration were determined in blood samples (0 and 15 min.) on days 0 (day of last injection), 10, 20 and 30 after last contraceptive injection. The results demonstrated a slight though not significant increase (p ~0.05) in serum Prl in the three experimental groups as compared with the IUD control group. This increase in Prl levels observed on day 10 post-last injection never exceeded the upper limits of the normal range (20 ng/ml). Overall, the data demonstrated that the chronic administration of these estrogen/progestogen once-a- month injectable contraceptives does not affect the Prl baseline secretion in women.

Introduction

It is well known that estrogen administration results in significant increase in prolactin (Prl) secretion in different mammalian species including rodents (1,2), bulls and non-human primates (3-6). The studies carried out in normal women using estrogen-containing contraceptive agents have produced conflicting results; thus, some reports have suggested significant increases in serum Prl concentrations (7,8) and modified episodic pattern of Prl release in women during hormonal oral contraceptive administration (9).

Other reports, however, show normal Prl levels in subjects taking the same preparation (lO,ll), and no hyperprolactinemia in women with normal menstrual cycles after discontinuing the use of combined oral contraceptives has been found (12,13); this could be related to the antiestrogenic activity of progestins contained in the pill (14).

The change in prolactin levels during the menstrual cycle has been investigated, but the results are controversial since some revealed increased Prl levels at midcycle and during the luteal phase (15), while other studies did not reveal any cycle-related variations (16).

The apparent lack of correlation between the data obtained from animal studies as compared with the effect in women and the fact that there are countries where, in addition to the pill, once-a-month injectable contraceptives have been used for a number of years (17), it was decided to undertake a study aimed at assessing the effects of combined once-a-month injectable contraceptives on Prl serum levels in chronically exposed women.

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subjects and Methods

Fifty healthy women of reproductive age entered this comparative study after being informed and written consent obtained. They had been receiving combined once-a-month injectable contraceptives for more than one year. Medical history was taken on admission and a complete gynecological examination including Papanicolaou smear was performed.

Women were grouped according to their previous use of monthly injectables or CUT 200 intrauterine device (IUD):

Group 1. Dihydroxyprogesterone acetophenide (DHPA) 150 mg + estradiol enanthate (EZ-EN) 10 mg (n=lO)

Group 2. Medroxyprogesterone acetate (MPA) 25 mg + estradiol cypionate (E2-C) 5 mg (n=21)

Group 3. Norethisterone enanthate (NET-EN) 50 mg + estradiol valerate (E2-V) 5 mg (n-19)

Group 4. IUD users (n=16)

Additionally, a control experiment was performed in 9 subjects on the effect of stress induced by direct venepuncture or through a cannula inserted 30 minutes prior on Prl levels. Subjects were randomly allocated and samples were taken on days 5 and 6 of a menstrual cycle.

In all women from the experimental groups, two blood samples were obtained from an antecubital vein with a 15- minute interval on days 0 (day of injection), 10, 20 and 30. In the control group, blood samples were taken on days 5, 15 and 25 of the menstrual cycle. The blood was allowed to clot and centrifuged, the stored at -2oOc

plasma separated and until analyzed. The Prl radioimmunoassay was

undertaken following the protocol provided by the Matched Reagent Programme of the World Health Organization (18). The antiserum was obtained from Dr. A.F. Parlow through the WHO. THe cross-reactivity of antiserum with growth hormone was 1.5%. The Prl standard used was IRP 751504. The separation of the bound and free fractions was performed by the second antibody technique. The sensitivity of the Prl assay was 1.1 ng/ml (37 mIU/l). Within-batch and between- batch coefficients of variation were 4.5 and 6.3%, respectively, at the dose of 12 ng/ml. The normal values for Prl, established in our laboratory, are from 3 to 19 ng/ml (97 to 617 mIU/l). The determination of 17l3-estradiol was carried out following the method and with reagents kindly provided by the WHO Matched Reagent Programme (18). The sensitivity of the E2 assay was 13 pg/ml (47.6 fmol/tube) . The intra- and interassay coefficients of

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variation were 7.8 and 11.3%, respectively. The results are expressed in ng/ml for Prl and pg/ml for estradiol. Since it had been noticed that Prl levels in women were not normally distributed (lo), the results were expressed as the geometric mean with 95% confidence limits. The differences within each group among sampling days were calculated by analysis of variance followed by the Wilcoxon test.

Description of the Preparations:

Perlutal (Promeco, Mexico City) containing 150 mg dihydroxyprogesterone acetophenide (DAPA) (2$-methyl-2a- phenyl-16a,l7a-dioxolane-pregn-4-en-3,20 dione) and 10 mg estradiol enanthate (EZ-EN) (3,178-dihydroxyestra-1,3,5(10)- trien-17B-yl-heptanoate) dissolved in 1 ml benzyl benzoate.

Cycloprovera (Upjohn Company, Crawley, U.K.) containing 25 mg medroxyprogesterone acetate (MPA) (6a-methyl-3,20- dioxo-4-pregn-17a-yl-acetate) and 5 mg estradiol cypionate (E2-C) (3,17~-dihydroxy-1,3,5(lO)-trien-l7$-yl-(3-cyclo- pentyl)-propionate; manufactured as a 0.5 ml aqueous microcrystalline suspension.

HRP102 (Shering AG, Berlin) containing 50 mg norethisterone enanthate (NET-EN) (17a-ethynyl-178-hydroxy- 4-estren-3-one-178-yl-heptanoate) and 5 mg estradiol valerate (E2-V) (3-hydroxy-1,3,5(10)-trien-178-yl-valerate); manufactured as 1 ml oily solution.

The admission physical data of participating women are shown in Table I. Women from Groups 1 and 4 were older than those of Groups 2 and 3. The mean duration of usage in Group 1 was more than 3 years, whereas mean exposure in Groups 2 and 3 was 1 year.

Results

The results of Prl in normal women of reproductive age not exposed to contraception are shown in Figure 1. There were no significant differences between the women whose blood samples were taken with a syringe as compared with prior insertion of an intravenous catheter on day 5 or 6 of a menstrual cycle.

The Prl levels obtained before the last injection (day 0) of the once-a-month contraceptive groups were found within normal limits (3-19 ng/ml). All preparations tested were able to induce a moderate elevation in serum Prl, with maximum Prl levels observed 10 days after injection.

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Table I. Physical characteristics of participating women (mean + s.d.)

Group We Weight Height Q.I. Months

(years) (kg) (m) (W/H? of use

1 (IFlO) DHPA/E2-EN 30.2 56.3 1.52 24.3 43.0

s.d. (8.0) (7.9) (0.01) (0.3)

2 (n=21) MPh/EZ-C 24.9 60.0 1.57 24.1 14.8

s.d. (4.2) (11.1) (0.04) (0.41

3 (n=21) NET-EN/E2-V 24.0 55.5 1.54 23.4 15.1

S.d. (3.9) (7.9) (0.05) (0.3)

4 (n=16) Control IUD group 27.6 59.4 1.57 23.6

s.d. (5.5) (7.7) (0.04) (0.3)

A

16

8

Figure 1.

(n=9)

Syringe Catheter

1 I-L +15’

Comparative prolactin levels in normal women in whom blood samples were obtained either by direct venepuncture or through a catheter inserted 30 minutes prior. Geometric mean and 95% confidence limits are shown.

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Figure 2 shows the Prl and E2 serum levels after injection of 150 mg DHPA plus 10 mg EZ-EN. A small though not significant (P >0.05) increase in serum Prl was observed. The Prl levels returned to pre-treatment values 30 days after injection. Serum Prl elevations were parallel to the elevation of E2 observed in the sample obtained 10 days after injection.

The results obtained after 25 mg MPA plus 5 mg E2-C are shown in Figure 3. As observed, only a slight increase in Prl serum levels was noted during the treatment period.

Similarly, the Prl levels after 50 mg NET-EN plus 5 mg E2-V did not exhibit significant variations (Figure 4).

The Prl and E2 serum levels obtained in the IUD control group are presented in Figure 5. No major variations were noted in serum Prl throughout the observation interval in this group.

Discussion

Previous studies in rodents have shown that estradiol is a potent stimulator of Prl secretion (1,2). However, in non-human primates and in humans, the role of estrogens to induce Prl release is still controversial (3,5,7-10). The increase in endogenous estrogens during the menstrual cycle has been found to have little influence on Prl secretion. However, it has been noted that Prl to TRH response is exaggerated during the preovulatory period (11,15,16).

In women the administration of pharmacological doses of estrogen causes a significant increase in serum Prl (7,8), but conflicting data have been published on the effects of progestins alone on Prl secretion both in experimental animals and humans (1,2,14). Recent experimental evidence has suggested a possible estrogen-progestin synergism in inducing hyperprolactinemia in non-human primates (4,5). The present study included women who were exposed to three different estrogen-progestin combinations used as once-a- month injectables for a minimum of 1 year. Although in this study there was a tendency for a transient increase in serum Prl, which occurred 10 days after combined injectable contraceptives, it never exceeded the upper limit of normal for serum Prl established in our laboratory.

The increase in Prl levels which occurred after injection was more evident after the DHPA/EZ-EN combination which contains twice the dose of estrogen (10 mg) than the other two formulations and the time of exposure to DHPA/E2- EN was longer (Table I).

Since it has been previously reported that venepuncture sampling might alter Prl secretion (19,20), it was decided

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(n=lO) I

A A

z

5 a. V

Day 0 Day 10 Day 20 Day 30 ci

Figure 2. Prolactin and estradiol levels in serum after injection of DHPA/EZ-EN (Group 1); geometric mean plus 95% confidence limits.

P (n=21) 0

20- (372) -300

-250

Day 0 Day 10 Day 20 Day 30

Figure 3. Prolactin and estradiol serum levels after injection of MPA/E2-C (Group 2); geometric mean plus 95% confidence limits.

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P (n=19)

T

d -(440) I

20 - -300

;

ii 2 ‘250 z

V -200 i V

l E lo- -150

i

‘ij ._

-. .,

e ~ --__ --__ -100

P *

; oi I * 7:

0 -_

50 2

0 5

91 0’ +15’ 0’ +15’ 0’ +15’ 0’ +15’ - r. Ji

Figure 4

Day 0 Day 10 Day 20 Day 30

Prolactin levels and estradiol serum levels after injection of NET-EN/E2-V (Group 3): geometric mean plus 95% confidence limits.

(n=l6)

(309) -

5 0

i 0’ +15’ 0’ +15’ 0’ +15’ 0’ +15’

Day 5 Day 15 Day 25 Day 5

? I

I

300 & A

250 z

5 200 5

150 5 ‘e

100 ;

50 ii

0 4

i

Figure 5. Prolactin and estradiol serum levels in IUD users (Group 4); geometric mean plus 95% confidence limits.

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to compare the effects of direct venepuncture and intravenous catheter on Prl levels in a group of non- contraceptive users. The results obtained did not show major differences in the two forms of blood collection and therefore, for the purposes of this study, direct venepuncture was chosen. Since diurnal variations in Prl secretion have been reported (10,16,21,22), in this study sampling time was confined from 10:00 a.m. to 12:00 p.m.

Assessment of the safety of existing as well as new once-a-month estrogen-progestin formulations has been considered important in view of the wide acceptance by some cultures of this method of contraception (17,23-26).

Overall, the data presented herein indicate that there is no tendency to hyperprolactinemia in normal women chronically exposed to combined once-a-month injectable contraceptives. These results underscore the safety of this method of fertility regulation in women.

Acknowledgements

This work was supported by the Special Programme of Research Training in Human Reproduction (Geneva, Switzerland). The assistance of Miss Guadalupe Alarcon is appreciated.

1.

2.

3.

4.

5.

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