CONTRACEPTION

PROGESTERONE METABOLISM BY HUMAN PROLIFERATIVE

AND SECRETORY ENDOMETRIA

P. Garzon, R. Aznar, E. Olivera, and A. J. Gallegos

Seccidn de Biologia Molecular, DivisiBn de Biologia de la Reproduccick,
Instituto Mexican0 de1 Seguro Social, Apartado Postal 12-1184
Mexico 12, D. F. - Mexico

ABSTRACT

Human proliferative and secretory endometria biotransformed progesterone-

6,7-3H into the following products: 6B-hydroxy-4-pregnen-3,20_dione,
20a-hydroxy-4-pregnen-3-one, and 20B-hydroxy-4-pregnen-3-one, 5a-pregnane-
3,20-dione, and 5B-pregnane-3,20-dione.

Progesterorle-6,7-3H biotransformation into the above metabolites occurred

in absence (control) and in presence of 10 and 100 ug/ml of unlabelled
progesterone. Incubation periods of 6, 24, 48, and 72 hours were chosen
to trace the biotransformation of the radioactive precursor.

A decrease of TM was observed at increasing dosages of labelled progester-

one and longer incubation periods. Linearity of the progesterone 6,7-3H
biotransforming process is only observed in controls of proliferative
endometria. Proportionately greater concentrations of highly polar com-
pounds were formed by proliferative endometria than by secretory ones.
However, lesser amounts of pregnanediones were found to be formed by the
former. The C-20 reduced proqesterone derivatives did not chanae under
any conditions. Further investigation will reveal whether the in vitro
endometrial capabilities are similar in tissue exposed to bioacEvpro-
gesterone-releasing intrauterine devices.

Accepted for publication April 26, 1977

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INTRODUCTION

A few years ago Scommegna et al. demonstrated, first in animals (1) and
--
later in humans (2), that the intrauterine administration of proqesterone
is a suitable approach to contraception. This basic concept was improved
by engineering a precise releasing membrane for progesterone (3). Several
authors (4-7) have shown that the constant intrauterine release of 65 pg
of progesterone through a T-shaped device constitutes a suitable alternative
to other bioactive IUDs (8). Additionally, it has been documented that
the human endometrium is capable of a series of steroid biotransformations,
and a variety of metabolites have been identified (9). Pregnenolone has
been demonstrated to be transformed into progesterone (10) which in turn
may also be transformed into at least six clearly distinguishable products
(11) whose uterine biological activities are still largely unknown. Inter-
conversions of steroids such as androstenedione to testosterone and estradiol
to estrone all have been shown to occur in this tissue (12). Most of the
above results have been obtained during brief incubations of human endo-
metria in the presence of NADP- and NADPH-generating enzyme systems.

Among the usually isolated progesterone metabolites, predominance of 5~

pregnane-3,2D-dione and 20a-hydroxy-4-pregnen-3-one has been reported to
occur in proliferative and secretory endometrial samples (13, 14).

Since it was known that 5a-reductase and 20cc-hydroxysteroid dehydrogenase

(20a-HSD) activities differ throughout the menstrual cycle, the purpose
of the present study was to establish the degrees by which addition of
progesterone to long-term incubations of human endometria from different
stages of the menstrual cycle might modify these enzymatic processes. Thus,
a study with human endometria previously exposed to steroid releasing IUDs
might indicate whether or not steroid biotransformation capacity is altered.

EXPERIMENTAL PROCEDURE

Materials. Thin-layer and paper chromatographic chambers were kept saturated

with analytical grade solvent which was distilled prior to use. Steroid
carrier purity was assessed by crystallizations and melting point measurements.
Progesterone 6,7-3H (specific activity 50.3 Ci/mmol) was purchased from New
England Nuclear Corp. and purified by paper chromatography in hexane-formamide
for 2 hours with overrun. Carrier location on paper strips was determined
bv the use of an Actiqraoh III (Nuclear-Chicaqo). Radioactivitv countinq
was oerformed in a Mark II liquid scintillation spectrometer (Nuclear-Chicago).
The presence of carrier steroid with a double bond at C-4 of ring A became
evident on the parallel paper chromatograms when an ultraviolet lamp was used
at 240 nm.

Human oroliferatvie and secretorv endometria were obtained from normal men-
struating women by curettage using the Novak type of curette. All biopsies
were immediatelv immersed in warm Eaqle's culture media (15) in order to
preserve the tissue viability. After thoroughly rinsing the endometrial
tissue with the culture media and removing the excess fluid by blotting with
sterile gauze, each biopsy was divided into three parts (of approximately
50 mg) and placed in the pre-weighed tissue culture flasks. The control
culture contained 10 ml of Eagle's minimum essential medium supplemented

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with penicillin 100 U/ml, streptomycin sulfate 100 ug/ml, 10 mM glutamine

and 11 uCi of pure (purified before use by paper chromatography) progester-
one-6,7-3H. Treated cultures included the same content as above but with
the addition of 10 or 100 ug/ml of unlabelled progesterone. All incubations
were performed at 37 + 1°C in an atmosphere of 95% O2 and 5% CO2 with oc-
casional gentle shaking.

Aliquots of 1 ml each were taken from the incubation flasks and placed in
other flasks containing warm acetone at 6, 24, 48 and 72 hours after
initiation of the incubation process. Steroid extraction was performed
three times with v/v of warm acetone (40°C) which was then filtered three
times and rinsed with ethyl alcohol. The pooled acetone and ethanolic
extracts were evaporated by applying negative pressure in a flash-evap-
orator (Buchler). A further exhaustive chloroform partition of the culture
media was performed. The organic extracts thus obtained were concentrated
in a flash-evaporator and the residue resuspended in 10 ml of chloroform-
methanol (1:l). Paper chromatography of the organic extracts was carried
out using a hexane-formamide system for 45 minutes with overrun (16).
Separation of 20a-hydroxy-4-pregnen-3-one from its isomer 20B-hydroxy-4-
pregnen-3-one was accomplished in hexane-formamide for 2 hours with over-
run. Subsequent oxidation with Cr03 and acetylation with acetic anhydride
was expected to form proqesterone and the 2Oa or 20B acetylated derivatives,
respectively (17). Mixtures of 5a and 56-pregnanediones were separated by
thin-layer chromatoqraphv usinq silica qel F-254 (Merck) in the system
petroleum ether:diethyl ether (60:40). -All radioactive compounds-eluted
from the origin of the hexane-formamide paper chromatograms were further
partitioned in a hexane-benzene (1:l) system on parallel arrayed paper
strips. Only 6!$hydroxy-4-pregnen-3,20_dione was identified from the origin.
This metabolite was oxidized, acetylated and purified in hexane-benzene and
heptane-formamide systems, respectively. Zimmerman's color reaction allowed
paper chromatogram location of steroid carriers with ketone functional groups
at C-3 and C-20 positions of the steroid molecule. Crystallizations to
constant specific activities were performed as described elsewhere (18).

RESULTS

Highly polar metabolites such as 6B-hydroxy-4-pregnen-3,20-dione had an Rf

value in hexane-benzene of 0.34. Upon oxidation to 4-pregnen-3,6,20-trione,
they revealed an Rf value of 0.68. The acetylated derivative in heptane-
formamide had an Rf of 0.20. Compounds of fractions II and III presented
Properties similar to those of identical metabolites isolated from human
cornea exposed to progesterone-4-'4C. Thin-layer chromatography was used
to separate 5a-pregnane-3,20-dione from its isomer 56-pregnane-3,20-dione.
Identical mobility to unlabelled carriers was observed. Rf values of 0.49
and 0.38 were recorded.

An average of 90% of the initial radioactivity was recovered from culture

media incubated with progesterone-6,7-3H. The radioactive areas detected
in the hexane-formamide paper chromatograms corresponded to the following
carriers:

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I Origin (Unidentified except for 66-hydroxy-4-

pregnen-3,20-dione)

II ZOa-hydroxy-4-pregnen-3-one

III 206-hydroxy-4-pregnen-3-one

IV 4-pregnen-3,20-dione

V Mixture of 5a and 56-pregnane-3,20-dione.

Total metabolites (TM) are considered as the sum of radioactivity found

with chromatographic behavior different from that exhibited by the original
incubated progesterone. So far, we have identified progesterone reduced
compounds II, III and V. In addition, a number of highly polar compounds
remaining at the origin of several chromatographic separations are in the
final process of identification.

Figure 1 represents the percentage of total metabolites formed at different

times by proliferative and secretory endometria. It can be seen that the
addition of 10 and 100 ug/ml of progesterone to the incubation media de-
creases the amount of TM formed. This effect is more apparent in the
presence of 100 ug/ml of progesterone. In general terms, progesterone is
biotransformed in greater quantities by secretory than by proliferative
endometria although those differences tend to be less apparent with the
addition of 100 ug/ml of exogenous progesterone.

Figure 1. Represents the percentage of total metabolites formed at

different times by (A) proliferative and (B) secretory endo-
metria incubated with progesterone-6,7-3H. Control (e-s);
10 ug (A-A) and 100 ug (o-o) of added unlabelled pro-
gesterone per ml of incubation media.

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In Figure 2 results are expressed as percentages of recovered radio-

activity at the origin relative to TM after hexane-formamide chromato-
graphic procedures. It can be seen that in all cases incubations per-
formed with proliferative endometria biotransformed more progesterone
to polar compounds than those carried out with secretory endometria.
There appear to be fewer polar compounds formed in tissue samples incu-
bated with exogenous progesterone (10 or 100 pg/ml).

Figure 3 represents the percentages that 20~ and POB-reduced compounds

constitute of the total metabolite fraction. There is no difference in
the formation of these reduced compounds between secretory and prolif-
erative endometria. However, the addition of 100 pg/ml of exogenous
progesterone to secretory incubations seems to increase the production
of these metabolites.

Figure 4 represents the percentages of 5c4- and 5B-oregnane-3,20-dione

metabolites formed at different times under different experimental con-
ditions. It clearly shows that secretory endometria biotransform greater
amounts of progesterone than proliferative ones. Formation of these
reduced compounds, and that of polar metabolites remaining at the origin,
comprise the majority of biotransformed incubated radioactive progesterone.

Figure 2. Represents the percentaqe of hiqhlv polar metabolites eluted

from the originof the hexane-formamide paper chromatograms
after 45 minutes overflow. (A) Secretory and (B) proliferative
endometria incubated with progesterone-6,7-3H. (a) Control;
(b) 10 pg and (c) 100 ug of added unlabelled progesterone per
ml of culture media.

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Figure 3. Represents the percentage of ZOn-hydroxy-4-pregnen -3-one and

20f3-hydroxy-4-pregnen -3-one as a fraction of the total metab-
olites obtained from (A) secretory and (B) proliferative endo-
metria incubated with progesterone-6,7-3H. (a) Control; (b)
10 ug and (c) 100 1-19of added unlabelled progesterone per ml
of culture media.

Figure 4. Represents the percentage of 5n-pregnane-3,20-dione and 5B-

pregnane3,20_dione as a fraction of the total metabolites
obtained from (A) secretory and (B) proliferative endometria
incubated with progesterone-6,7-3H. (a) Control; (b) 10 ug
and (c) 100 ug of unlabelled progesterone per ml of culture
media.

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TABLE 1

Crystallizations to constant specific activities

(Values represented in dpm/'ng)
-__-__ I_- ._____.~
I II III IV V
-____ ___ _~____

6,2-hydr2xy-4-pregnen -3,20-dione 5820 6703 6791 5732

.__-__ __-- J_al_ja)__b':__.OP___.

2&j-hydroxy-4-pregnen -3-one 848 855 855 850

_____ __-____. _~. (a) _ (a)_ ._k__~__O_~__
2Dt-hydroxy-4-pregnen -3-one 877 692 592 531 572
__.___~_._ (a) (a) (ci Cc) (al

5 I-pregnarre-3,20-dione 5296 3470 3960 3559

-~__---__ (a) ._W (e) (e) _._~

53-pregnane-3,20-dione 660 626 630 635

________ ._~~___. (f)
__.-- if)__.&_ (b) -.___
Solvents: (a) 70% MeOH (b) 50% MeOH (c) Hexane (d) Acetone-Hexane
(e) Heptane (f) Diethyl Ether-MeOH

Table I represents crystallizations to constant specific activities of

isolated progesterone metabolites. Only 6B-hydroxy-4-pregnene-3,20-dione
has been characterized from the most polar reg-;on after hexane-benzene
paper chromatography.

DISCUSSION

The amounts of total metabolites formed by the control secretory endo-

metria at 6 hours are somewhat similar to those formed during 72-hour
incubations by the control proliferative endometria. The temporary pat-
terns of these reactions are also different. The total metabolites formed
by proliferative endometria shown in Figure 1 suggest a constant increase
in total metabolite formation from 26% at 6 hours to 41% at 72 hours. In
contrast, a plateau is reached by secretory endometria at 6 hours and the
value remains constant at 45 ? 3% throughout the experimental period.

The addition of 10 or 100 ug/ml of exogenous progesterone to the incuba-

tion media decreases the amount of total metabolites formed by both pro-
liferative and secretory endometria. The greater amounts of exogenous
progesterone tend to produce greater decreases in all experimental condi-
tions. A larger reduction in the biosynthesis of total metabolites was
observed in secretory than in proliferative endometria. An average re-
duction of approximately 15% in total metabolite formation was noted in

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the presence of 10 pg of progesterone in the incubation media, while the

addition of 100 pg decreased by approximately 50% the amount of total
metabolites formed throughout the experimental period by secretory endo-
metria. This effect is less marked with proliferative endometria. As
shown in Figure 2, the percentage of polar compounds formed by prolif-
erative endometria is greater than that produced by secretory endometria.

The addition of exogenous progesterone to the incubation media of prolif-

erative endometria also tends to produce a slight decrease in the forma-
tion of polar metabolites by this tissue. In contrast, the amount of
polar metabolites formed by secretory endometrium in the presence of
exogenous progesterone seems to remain unchanged.

Short-term endometrial incubations with radioactive progesterone have

demonstrated that the PO-HSD activity is greater in secretory than in
proliferative endometria (19). However, the reverse situation is ob-
served for 5a-reductase activity. Neither observation was confirmed
under present long-term experimental conditions. The long-term prolif-
erative and secretory endometrial incubation periods with radioactive
progesterone alone or with added unlabelled progesterone may have allowed
increasing amounts of hydrophilic metabolites to be formed. The C-5
reduced metabolites in secretory endometria were found in quantities
double those of proliferative endometria. The presence of 10 and 100
ug/ml of exogenous progesterone does not seem to influence the formation
of C-5 reduced metabolites and highly polar compounds. However, as
shown in Figure 1, the addition of small quantities of unlabelled pro-
gesterone to either secretory or proliferative endometria produces a de-
crease in total steroid biotransformation capacity.

The biological significance of these results might be assessed by per-

forming similar studies with human endometria obtained from patients
wearing progesterone-releasing IUDs.

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Intrauterine administration of progesterone by a slow releasing
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2. Scommegna, A., Avila, T., Luna, M., Rao, R. & Dmowski, W.P.:
Fertility control by intrauterine release of progesterone. Obstet.
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3. Zaffaroni, A.: Special requirements for hormone releasing intra-

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