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Plasma testosterone levels in women in normal and pathological conditions
Plasma testosterone levels in women in normal and pathological conditions R. RUBENSand A. VERMEULEN Occasional discrepancies between plasma testosterone levels and clinical androgenicity disappear almost completely when the free, non-protein bound, testosterone is determined. The latter seems at the present time, the best single parameter of androgenicity. We suggest that the determination of the free testosterone concentration should be introduced into clinical routine.
SUMMARY Several parameters have been used in the past to evaluate androgenicity in the female; plasma testosterone levels seem to be by far the most reliable parameter applicable in the clinical routine. Using a very sensitive method we found a mean plasma testosterone level in the normal female of 35 ng & 15 ng/lOO ml; the latter shows a slight diurnal variation, with highest values in the morning, and a moderate cyclical variation with higher levels in the luteal phase of the menstrual cycle. During pregnancy the plasma testosterone increases and reaches a plateau of about 100 ng/ 100 ml from the second trimester on. Among the pharmacologic agents, A.C.T.H. causes a significant increase and corticoids a decrease of plasma testosterone levels, whereas gonadotrophins and clomiphene citrate increase slightly plasma testosterone levels. As far as pathological conditions are concerned, plasma testosterone levels are generally highly increased in congenital adrenal hyperplasia and in the adrenogenital syndrome; in virilizing ovarian tumors, the polycysticovarysyndrome, idiopathic hirsutism, acne and sebaceous alopecia as well as in hyperthyroidism a moderate increase is generally, although not always, observed.
KEYWORDS plasma testosterone; hirsutism; virilism; polycystic ovary syndrome; nonprotein-bound testosterone; dynamic exploration of virilism.
I. INTRODUCTION It is generally accepted that by far, the most important biologically active androgenic hormone is testosterone. Although other androgens are secreted by either the gonads or the adrenal cortex, they are probably only biologically active in as far as they are converted to testosterone (BARDIN and MAHOUDEAU, 1970). Testosterone was already isolated from biological fluids in 1935 (KILLINGER, 1970) but, until a few years ago the only, clinically useful, parameter for androgen secretion, was the determination of the urinary 17-ketosteroids by the Zimmermann reaction. The latter however
University of Ghent (Belgium), Akademisch Ziekenhuis and Metabolism.
Department of Medicine, Division ofEndocrinology
Europ. 3. Obstet. Gym. 6 (1971)
208
Rubens, Vermeulen m- 3SBng/lOOml
Test.
SD - 15.3ngllOOml
75_pg/lOOml
.
70. 65.
.
55. 50.
l .
45.
:
: . :
60.
.
t :
.
l
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9.
.
:
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l
l:
l
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t
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35.
. .
30.
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c
.
25.
.
20.
.
:
40.
.
*
.
.
.
l
l
15*
.
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t
l. l
.
10.
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i .
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5.
c
4
20
Fig. 1. Plasma testosterone
Plasma
30
40
levels in normal menstruating
SOY
females.
T.
80, ng/iooml. 70,
Fig. 2. Plasma testosterone
Europ. J. Obstet. Gynec. 6 (1971)
levels during menstrual
cycle.
plasma testosterone levels
does not only
measure
the
metabolites
of
testosterone but also the metabolites of moderately or weakly active androgens, secreted either by the adrenal cortex or the gonads. That the urinary 17-ketosteroids are a poor parameter of androgenicity is evident
from the fact that normal
adult females are within the range observed in normal adult males.
values in of those
During the last decade more specific methods for the estimation of androgen secretion have been developed and since 1966 we routinely determine the plasma testosterone levels as a parameter of androgenicity. The method which we developed in our laboratory is based on the electron capturing properties of the heptafluorbutyrate ester of testosterone and with minor modifications it has been used since in our laboratory (VERMEULEN, 1968). Methodology and statistical reliability have been reported in detail previously (VERMEULEN, 1968). The method has a sensitivity of 10 ng of testosterone per 100 ml, allowing the accurate determination of usual testosterone levels in female plasma. II. PHYSIOLOGY OF TESTOSTERONESECRETION IN FEMALES Plasma testosterone in normal females originates for two thirds from the adrenal cortex and for one third from the ovaries (KASE and KOWAL, 1962 ; SAVARD, MARSH and RICE, 1965; RYAN and SMITH, 1965; GRANT, 1968; HORTON, ROMANOFF and WALKER, 1966; BAIRD, UNO and MELBY, 1969) ; one third seems to be secreted as such, whereas two thirds originate from peripheral conversion of precursors, so called prehormones (BARDIN and MAHOUDEAU, 1970), mainly androstenedione. Plasma testosterone levels were deter-
mined between
209
8 and 10 a.m. in 100 normal
females, during reproductive life. As shown in Fig. 1 testosterone values vary between 10 and 70 ng/lOO ml (with a mean value of 35,8 ng/lOO ml), similar to the values reported by most authors (Table I). It is well known that in males plasma testosterone levels show a diurnal variation with highest values in the morning and lowest at about 8-9 p.m. (SOUTHREN, TOCHIMOTO, CARMODY and ISUGURI, 1965; DRAY, REINBERG and SEBAOUN, 1965 ; RESKO and EIKNES, 1966 ; LIPSETT, WILSON, K~RSCHNER, KORENMAN, FISHMAN, SARFATY and BARDIN, 1966). Although testosterone in females has a complex origin, the gonads being the minor source, a similar variation could nevertheless be expected, as it is well known that the adrenal cortical secretion shows a diurnal rhythm. The low plasma testosterone levels in females, close to the lower limit of detection of most methods used, were the reason that until recently this diurnal variation in females remained an open question; recently however we were able to demonstrate that 8 p.m. values are generally lower than 8 a.m. values by about one third. In order to determine whether there exists a cyclical variation in plasma testosterone levels, the latter were determined daily during one cycle in five normal females with regular periods. As shown in Fig. 2 there exists a small but significant cyclical variation in plasma testosterone levels, the values during the luteal phase being slightly higher than those during the follicular phase; our results however do not permit to decide whether there exists a preovulatory peak in plasma testosterone levels. Some authors reported increased testosterone levels around ovulation and during the luteal phase Europ.
3. Obstet. Gynec. 6 (1971)
210
Rubens, Vevmeulen
TABLE I. PLASMATESTOSTERONE LEVELSWITH DIFFERENT METHODS:RRSULTS. G.L.C. graphy. Double Isotope D: Double Isotope Derivative.
Author
Method
FINKELSTEIN et al. (1961) FORCHIELLIet al. (1963) RIONDELLet al. (1963) BROWNIEet al. (1964) BURGERet al. (1964) KIRSCHNERet al. (1965) LAMB et al. ( 1964) SEGRE et al. (1964) SURACE et al. (1966) VAN DER MOLEN et al. (1966) HUDSONet al. (1969) LOBOTSKYet al. (1964) BARDIN and LIPSETT(1967) SAROFF et al. (1967) RIVAROLA et al. (1966b) LLOYD et al. (1966) DRAY et al. (1968) KORRNMANet al. (1965) BARDINet al. (1968b) FURUYAMAet al. (1970) PRESENTSERIES
Enzymatic Enzymatic Double Isotope D G.L.C. Double Isotope D Double Isotope D Enzymatic Double Isotope D G.L.C. G.L.C. Double Isotope D Double Isotope D Double Isotope D Double Isotope D Double Isotope D Double Isotope D Double Isotope D Double Isotope D Double Isotope D Radioimmunassay G.L.C.
(LOBOTSKY,
WYSS,
ISMAIL,
HARKNESS
HUDSON,
COGHLAN,
SEGRE
and LLOYD,
1964;
LORAINE,
1965;
and
DULMANIS
and WINTOUR,
1964).
Although experiments performed by LIPal. (1966) suggested that posture might
SETT et
effect plasma testosterone levels, with higher values in the upright than in the recumbent position, we observed neither in males nor in females any effect of posture on plasma testosterone levels. Duringpregnancy, plasma testosterone levels increase progressively to reach a plateau, with a mean value of about 100 ng/lOO ml, from the end of the first trimester (Table II). Nevertheless plasma testosterone levels remain highly variable and even during the last trimester, values within the normal female range may be observed. All values lie however above the mean value for nonEurop. 3. Obstet. Gynec. 6 (1971)
=
Gas Liquid Ghromato-
Mean + S.D. (ng/lOO ml) 100 120 59 f 60 f 180 70 110 50 110 f 36
83 54f 37 f 68% 47 + 364 57 & 120 415 34 35 f
79 30
60
15 15 17 14,8 9 14 4 15
pregnant normal women. This variability of testosterone values during pregnancy should be compared to the rather wide range of normal values observed in non-pregnant females. Similar results were observed by other authors (DEMISCH, GRANT and BLACK, 1968; AUGUST, RIVAROLA,
and GRUMBACH, 1969 ; FOREST and MIGEON, 1968).
TKACHUK
TABLE II. APPARENT FREE TESTOSTERONE CONCENTR.4TIoN (A.F.T.~.) IN PREGNANCY
total plasma testosterone (ng/lOO ml n f S.D.) NON-PREGNANTFEMALES 70 FIRSTTRIMESTER PREGNANCY 7 SECONDTRIMESTER PREGNANCY 2 THIRD TRIMRSTRR PREGNANCY 14
36f
15
A.F. T.C. (ngllO0 ml f S.E.) 0,35 & 0,l
73&13
0,28 f
0,08
102&31
0,21 f
0,08
97*30
0,22 f
0,12
plasma
testosterone
levels
PHARMACOLOGICALFACTORS AFFECTING pituitary
III.
ne citrate
PLASMATESTOSTERONE IN FEMALES
211
disease do not respond to clomiphe(TAGATZ,
FIALKOW, SMITH and
SPADONI, 1970). A) Gonadotrophins As we considered it unwarranted to administer high doses of human chorionic
gonadotrophins to normal females in reproductive life, we have no personal experience of the influence of this drug upon plasma testosterone levels in normal females. Results in the literature are quite discordant: some authors (KIRSCHNER, LIPSETT and COLLINS, 1965) influence,
did not see any significant
whereas
others
(DIGNAM, PION,
HUDSON et al., 1964) observed a very small increase in plasma testosterone levels. In patients with hirsutism (Table VI) we observed a clearcut elevation of plasma testosterone levels under the influence of HCG during dexamethasone treatment. LAMB and
SIMMER, 1964;
B) Clomiphene Citrate, an anti-estrogen, is known to cause a release of LH and FSH (HELLER, ROWLEY and HELLER, 1969).
It
has been shown to cause a significant increase of
plasma testosterone
in
Ross and LIPSETT, 1971).
males
(BARDIN,
Recently
we ob-
served a slight increase of plasma testosterone in females after clomid@ treatment day for ten days). Women pogonadotropic gonadotropin
TABLE III. TOSTERONE
with anosmic hy-
hypogonadism
.Name
ANS. CHR. MASS.G. DE BRUY.C. Mos.
UPON
and Corticoids
(LLOYD, LOBOTSKY, SEGRE, KOBAYASHI, TAYMOR and BATT, 1966; BARDIN, HEMBREE and LIPSETT, 1968b).
D) Sex Hormones 1. Androgens In the view of the complex origin of plasma testosterone in females, it can hardly be expected that synthetic androgens will influence plasma testosterone to a significant extent. As administration of precursors of testosterone could possibly influence plasma testosterone levels, we administered dehydroepiandrosterone to females in a dose of 60 mg daily for 60 days; this did not have any clearcut influence upon plasma testosterone levels.
or impaired
secretion secondary to organic
EFFECTOF A.C.T.H. (I+00
(lOOmg/
C) A.C. T.H.-stimulation
As expected in the view of the adrenal origin of the major part of plasma testosterone in females, A.C.T.H. causes a moderate increase in plasma testosterone levels (Table III). On the other hand a decrease of 30 f 12 (S.D.) y0 of pl asma testosterone levels was observed after 8 days treatment with dexamethasone, 1 mg daily. Similar findings were reported in the literature by different authors
PLASMA
TES-
Id)
2. Progestogens GORDON,
SOUTHREN,
ALTMAN,
RAND
and
8 a.m.
F F F F
29 11 66 40
12 noon 32 15 75 51
4 p.m.
8 P.m.
40 20 82 47
26 17 84 51
OLIVO,
LEMBERGER
(1970)
recently showed that a potent progestogen (medroxyprogesterone
Sex
TOCHIMOTO,
small
acetate)
fall of plasma testosterone;
selves showed
that
progestogens
induces
a
we ourcause
a
decrease in the testosterone binding globulin (VERMEULEN, VERDONCK, VANDER STRAETEN and ORIE, 1969). Europ.
3 Obstet. Gynec. 6 (1971)
Rubens, Venneulen
212
3. Estrogens It is generally
TABLEIV.
admitted
that administration
of estrogens during the fertile period will cause anovulation and inhibition of ovarian function, which should result in a decrease in androgens secreted by the ovary. On the other hand, the increase in testosterone binding globulin tends to increase plasma testosterone levels (KIRSCHNER, BARDIN, HEMBREE and
estrogen variable.
Ross, 1970). administration
The final result of seems to be quite
4. Contraceptives As for estrogens the influence of combined (estrogen and progestogen containing) contraceptives is quite complex. While inhibition of LH release might decrease testosterone from ovarian origin the increase in binding protein (VERMEULEN and VERDONK, 1968 ; VERMEULEN et al., 1969) might result in an increase of testosterone levels. Hence the plasma testosterone values vary from low normal to definitely increased levels (Table
EFFECTOFCONTRACEPTIVES UPONPLASMA
TESToSTERoNE Name y& Li * ’ PENN.F. ’ LOOT.
r;;
R. T
VAN c. IL
BA. G.
Free T.
(%) 0.42 0.53 0.78 0.58 0.35 0.81 0.51
0.22
mean 0.53fO.O7(SE)
To.?& 1-. Free 1. (ng/lOO ml) (ng/lOOml) 99 95 82 101 30 130 72
75
96f9
0.42 0.50 0.64 0.59 0.11 1.05 0.37
0.17
0.49f0.10
B) Arrhenoblastoma Very high levels of plasma testosterone have been reported preoperatively in this very rare condition (DIGNAM et al., 1964; BRUCE, 1967). BRUCE (1967) observed a substantial fall in testosterone levels after operative removal of the tumor.
C) Hyperthyroidism DRAY, SEBAOUN, MOWSZOWICZ, DELZANT, DESGEZand DREYFUS(1967) were the first to report elevated plasma testosterone levels in IV). Recently, we and others hyperthyroidism. IV. PATHOLOGICAL CONDITIONS AFFECTING (VERMEULEN et al., 1969; SOUTHREN and GORDON, 1970) observed a similar increase PLASMATESTOSTERONE LEVELS in females, a consequence of an increased A) Adrenal Pathology In female patients suffering from Cushing binding of testosterone. syndrome slightly elevated plasma testosterone levels were found by BARDIN, LIPSETTand D) Virilism 1. Hirsutism FRENCH (1968a). The few observations we Plasma testosterone levels in idiopathic simwere able to make, are in accordance with ple hirsutism without other signs of virilisathese findings. In congenital adrenal hypertion are highly variable. Some patients have plasia we observed very high levels as has been reported in the literature (BURGER, normal levels whereas others have significantKENT and KELLIE, 1964; RIVAROLA, SAEZ ly increased values up to 150 ng/lOO ml. and MIGEON, 1967; HORTON and FRASIER, (LLOYD et al., 1966; DRAY, SEBAOUN, DELZANT, LEDRU and MOWSZO~ICZ, 1968; 1967). As could be expected, in female patients with Addison’s disease we had very low KORENMAN, KIRSCHNERand LIPSETT, 1965). levels of plasma testosterone (Table V). Increased urinary excretion of testosterone Europ. 3. Obstet. Gymx. 6 (1971)
plasma testosterone levels TABLE V. PLASMATESTOSTERONE IN ADRENAL 0Locx
(ng/lOO
A. Gushing’s syndrome PL.V. DEBEY. L. : WILL.
B.
s.
?
TABLE VI. PLASMA TESTOSTERONE LEVELSIN WOMEN WITH ACNE AND SEBACEOUSALOPECIA Plasma test. (ng/lOO ml)
28~. 43~.
H. H.
35 y.
H.
82 ng 73 ng 107 ng
PETR. ED.
53 y.
SPITA. L.
34 y.
116ng 40 ng
VINC. S.
23 y.
57 ng 117 ng
MISER.
21 y.
3
16 y.
182ng
BAEL.
29 y.
34 ng
661 ng
BAR. M.
20 y.
123 ng
Z
22 y. 53 y.
RUBB. L.
29 y. 61 y.
117 ng
Congenital adrenal hyperplasia
KIRCH.M. SEL. E. P. E. C.
PATH-
ml)
213
166 ng
DOND. M.
75 ng
Addison’s disease DB. H.
?
24 y.
5.3 ng
Co. M.
e
30 y.
11.5 ng
DESCH.L. S0.L.
$? ?
43 y. 33 y.
22.0ng 18.0ng
.H = hyperplasia glucuronides on the contrary seems to occur much more frequently (VERMEULEN, 1966; TUCKER, BISHOP and SOMMERVLLLE, 1969; MAHOUDEAU,DROSDOWSKY andJAYLE,l970); this could be a consequence of a difference in physical state of the hormone in the blood as will be discussed below. The variability of the testosterone level in hirsutism is not unexpected as evaluation of the condition is rather subjective and the intensity will be scored differently according to whether the hair is dark or fair. 2. Acne and sebaceous
alopecia
The sebaceous glands are probably target organs for the androgens (EBLING, 1970). We ,observed in 6 out of 8 patients a significantly increased level of plasma testosterone as acompared with a normal group (Table VI). This confirms earlier results concerning urinary testosterone excretion (VERMEULEN, 1966). 3. Polycystic ovary syndrome (P.C.O.) The P.C.O. syndrome remains an obscure chapter in female endocrinology and the
pathogenesis
is still controversial. Most authors (LLOYD et al., 1966 ; KORENMAN et al., 1965 ; HORTON and NEISSLER, 1968) observed increased plasma testosterone levels in the majority but not in all cases of P.C.O. The cases with normal levels however were clinically undistinguishable from those with higher levels. We ourselves studied five patients with P.C.O. and observed in all a significantly increased plasma testosterone value. V. DYNAMIC EXPLORATION OF VIRILISM IN FEMALES Considering the complex origin of testosterone in females, several authors (DIGNAM et al., 1964; LLOYD et al., 1966; BARDIN et al., 1968b) have emphasized the importance of the dynamic exploration of testosterone secretion in females. Different techniques have been used. They can be subdivided into two groups : - Tests
affecting
the adrenal
component
of
plasma testosterone namely dexamethasone suppression and ACTH stimulation. - Tests influencing the ovarian secretion of testosterone: H.C.G. stimulation tests or more frequently H.C.G. stimulation during dexamethasone suppression. An important decrease of elevated plasma testosterone levels during dexamethasone treatEurop.
3. Obstet. Gynec. 6 (1971)
214
Rubens, Vermeulen
VII.DYNAMICEXPLORATION
TABLE
OF VIRILISMIN FEMALES
Patient
Clinkat signs
A.M. V.L.J. C.J. M.J.
Hirsutism Acne + hirsutism Hirsutism + amenorrhea Hirsutism
Base line Tng/lOO ml
H.C.G. Stimulation** T nggllO0 ml
Dexamethasone Su#wession* T ng/lOO ml
63,8 70,7 56,9 68,5
59,5 76,3
27,7 43,7
i28,3
78,o 31,l
77,o
Diagnosis
Id. Hits. Id. Him. P.C.O. Id. Hirs.
*Dexamethasone suppression means determination of plasma testosterone level after five days dexamethasone 3 mg/day. **H.C.G. stimulation means determination of plasma testosterone after five days H.C.G. 1500 I.U. daily i.m. while under dexamethasone suppression 3 mg/day.
ment suggests an adrenal
origin of elevated testosterone levels; this origin will be confirmed if testost.erone levels increase excessively upon A.C.T.H. stimulation. Due to the long half life of the adrenal androgens, which are the main precursors of testosterone in the females, dexamethasone should be administered for at least five or perhaps better even ten days before the adrenal contribution can be evaluated. The dose of dexamethasone has furthermore to be rather high (3 mg a day) in order to block completely A.C.T.H. secretion. Theoretically the H.C.G.-stimulation test should be very useful to differentiate hirsutism from ovarian origin from hirsutism from adrenal origin. Indeed an important increase of plasma testosterone during H.C.G. treatment, or better during a combined dexamethasone - H.C.G. treatment wil indicate an ovarian origin. Such tests have originally been divised with urinary androgens as a parameter of androgenicity (NETTER,JAYLE, MUSSET,
LAMBERT
and
MAUVAIS-JARVIS,
tation of the results is difficult, the effects of dynamic exploration being ambiguous. Frequently an important decrease of plasma testosterone level upon dexamethasone inhibition is observed, while on the other hand H.C.G. stimulation causes a significant increase in testosterone levels, in which case the test does not allow to decide whether the adrenal or the ovary is the main source of the androgens (Table VII). This is in agreement with the results reported by LI,OYD et al., 1966; DIGNAM et al., 1964; KORENMAN et al., 1965 ; ETTINGER, VON WERDEN, THENAERS and FORSHAM, 197 1). VI.
FREE NON-PROTEINBOUND TESTOSTERONE
AND ANDROGENICITY From
personal
experience
testosterone determination, it can be considered
with
plasma
it appears
that
as an excellent para-
meter of androgenicity.
Nevertheless
there
are some circumstances where plasma testosterone levels do not correlate well with the clinical condition;
for example in pregnancy
1960 ; DESCOURT,JAYLE and MAUVIS-JARVIS,
and
1962 ; JAYLE, SCHOLLER, MAUVIS-JARVIS and
levels are increased without signs of virilisa-
SZPER, 1962;
HART, VAN DER MOLEN and
BAKKER, 1968 ; MAHOUDEAU
et al., 1970).
However, although occasionally those tests give useful information, often the interpreEurofi.
3.
Obstet. Gynec. 6 (1971)
hyperthyroidism,
plasma testosterone
tion, whereas in female hirsutism sometimes normal plasma testosterone levels are found. It is well known that plasma testosterone is largely
protein
bound,
and in analogy
to
plasma testosterone levels
cortisol and on the basis of some experimental evidence, it is generally accepted that only the
non-protein
bound
testosterone
215
TABLE VIII.
FREE TESTOSTERONE IN HIRSUTEFEMALES
Patient
Diapnosis*
Free T.
repre-
sents the biologically active fraction (VERMEULEN, STOICA and VERDONK, 197 1). The determination of this apparent free testosterone concentration (A.F.T.C.) by equilibrium dialysis as described by VERMEULEN et d. (1971) seems to give a much better parameter of androgenicity than total plasma testosterone. The mean free testosterone fraction in normal females was 0,96 f 0,07 (SE)% and the apparent free testosterone concentration varied between 0,2 and 0,75 ng/lOO ml against a mean free testosterone fraction of of 11,6 2,08 f 0,08 (SE)% and an A.F.T.C. -+ 0,7 (SE) ng/ 100 ml in males of the same age group. Whereas, as mentioned earlier, total testosterone increased during pregnancy, we found a normal or even slightly decreased apparent free testosterone concentration during the whole duration of pregnancy (Table II). Similarly in hyperthyroidism, notwhitstanding increased plasma testosterone levels, we observed a free testosterone concentration within the normal range. On the contrary we found an increased A.F.T.C. in 10 out of 13 cases of hirsutism, whereas the total plasma testosterone level was only increased in 7 out of 13 (Table VIII). Three patients with clinical hirsutism and normal total testosterone levels had a significantly increased free testosterone concentration. From our experience, it appears that the occasional discrepancy between the total plasma testosterone and clinical signs of androgenicity disappears almost completely when the apparent free concentration of testosterone is used as a parameter. Incidently, the A.F.T.C. discriminates much better between males and females than total
Tot.
(%)
T.
(4 100 ml)
DE KE. J, DE C. R. BERN. J. V. COP. R. v. LI. D. PR. J. D. C.
P.C.O. P.C.O. S.H. S.H. S.H. S.H. P.C.O. S.H. S.H. S.H. P.C.O. S.H. S.H.
C.D.R.
DEW. I. DE C. R. CHAV. COES. E. HUG. L. Mean
f
S.E
1.50 1.94 1.42 1.03 0.96 0.96 1.94
105 105 54 46 134 50
1.82
95 113 59 255 41 64
1.43 2.72 2.22 1.64 3.03 1.74hO.18
*SH=simple hirsutism P.C.O.=polycystic ovary
172
Free T.
(nbd 100 ml) 1.58 2.04 0.78 0.41 1.29 0.48
3.34 1.73 1.62 1.63 5.61 0.84 1.92
1.7950.38
syndrome
testosterone levels, as the ratio of the highest normal A.F.T.C. in females to the lowest A.F.T.C. in males is 7, whereas this ratio is only 4 for total testosterone levels. It appears to the authors, therefore, that the determination of the unbound testosterone fraction, which can be obtained at the expense of little extra work, should be performed whenever total plasma testosterone is determined, as it will perm.it, especially in the female, a more rational interpretation of total plasma testosterone levels. REFERENCES AUGUST, G. P., M. TKACHUK and M. H. GRUMBACH (1969) Plasma testosterone-binding affinity and testosterone in umbilical cord, plasma, late pregnancy, prepubertal children and adults. 3. clin.
Endocr. 29, 89 1. BAIRD, D. T., A. UNO and J. C. MELBY (1969) Adrenal secretion of androgens and estrogens. 3. Endocr. 45, 135. BARDIN, C. W. and M. B. LIPSETT (1967) Estimation of testosterone and androstenedione in human peripheral plasma. Steroids 9, 7 1. BARDIN, C. W., G. T. Ross and M. B. LIPSETT (1967) Site of action of clomiphene citrate in man: a study
Europ. j(. Obstet.
Gynec. 6 (1971)
216
Rubens,
of the pituitary-Leydig cell axis. 3. clin. Endocr. 27, 1558. BARDIN, C. W., M. B. LIPSETT and A. FRENCH ( 1968a) Testosterone and androstenedione production rates in patients with metastatic adrenal cortical carcinoma. 3. clin. Endocr. 28,215. BARDIN, C. W., W. C. HEMBREE and M. B. LIPSETT (1968b) Suppression of testosterone and androstenedione production rates with dexamethasone in women with idiopathic hirsutism and polycystic ovaries. 3. clin. Endocr. 28, 1300. BARDIN, C. W. and J. A. MAHOUDEAU (1970) Dynamics of androgens metabolism in women with hirsutism. Ann. clin. Res. 2, 25 1. BROWNIE, A. C., H. J. VAN DER MOLEN, E. E. NISHIWAZA and K. B. EIKNES (1964) Determination of testosterone in human peripheral blood using gas liquid chromatography with electron capture detection. Endocrinology 24, 109 1. BRUCE, J. E. F. (1967) An arrhenoblastoma with estimations of androgenic steroids. 3. Obstet. Gynaec. Brit. C&h. 74, 589. BURGER, H. G., J. R. KENT and A. E. KELLIE ( 1964) Determination of testosterone in human peripheral and adrenal venous plasma. 3. clin. Endocr. 24,432. DECOURT, J., M. F. JAYLE and P. MAUVAIS-JARVIS (1962) Exploration biochimique des syndromes virilisants para- et post-pubertaires chez la femme (tumeurs except&). Abstracts ofpapers I. Int. Congress on Hormonal Steroids, Milan. Excerpta med. I.C.S. 51, 100. DEMISCH, K., J. K. GRANT and W. BLACK (1968) Plasma testosterone in women in late pregnancy and after delivery. 3. Endow. 42, 477. DIGNAM, W. J., R. J. PION, E. J. LAMB and H. H. SIMMER (1964) Plasma androgens in women. II patients with polycystid ovaries. Acta endocr. (Kbh.) 45, 254. DRAY, F., A. REINBERG and J. C. R. SEBAOUN (1965) Rythme biologique de la testosterone libre chez l’homme adulte sain: existence d’une variation circadienne. C.R. Acad. Sci. (Paris) 261, 573. DRAY, F., J. SEBAOUN, I. MOWSZOWICZ, G. DELZANT, P. DESGEZ and G. DREYFUS (1967) Facteurs influencant les taux de la testosterone plasmatique chez I’homme; role des hormones thyroidiennes. C.R. Acad. Sci. (Paris) 264,2578. DRAY, F., J. SEBAOUN, G. DELZANT, M. J. LEDRU and I. Mowszow~cz (1968) Activitt de liaison de la testosterone dam le serum des femmes presentant un virilisme pilaire idiopathique. Rev. Franc. Etud. Clin. Biol. 13, 622. EBLING, F. J. (1970) Steroid Hormones and Sebaceous secretion, In: Advances in Steroid Biochemistry and Pharmacology, 2. M. H. BRIGGS,Ed., Academic Press, London/New York, p l-39.
Europ.
3.
Obstet.
Gynec. 6 (1971)
Vermeulen
ETTINGER, B., K. VON WERDER, G. C. THENAER~ and P. H. FORSHAM (1971) Plasma testosterone stimulation-suppression dynamics in hirsute women. Amer. 3. Med. 51, 170. FINKELSTEIN,M., E. FORCHIELLI and R. I. DORFMAN (1961) Estimation of testosterone in human plasma. 3. clin. Endocr. 21, 98. FORCHIELLI, E., G. SORCINI, M. S. NIGHTINGALE, N. BRUST R. DORFMAN, W. H. PERLOFP and G. JACOBSON ( 1963) Testosteronein human plasma. Analyt. Biochem. 5,416. FURUYAMA, S., D M. MAYES and C. A. NUGENT (1970) Aradioimmunoassayfor plasma-testosterone. Steroids 11,415. GORDON, G. G., A. L. SOUTHREN, S. TOCHIMOTO, J. OLIVO, K. ALTMAN, J. RAND and L. LEMBERGER (1970) Effect of medroxyprogesterone acetate (Provera) on the metabolism and biological activity of testosterone. 3. clin. Endocr. 30, 449. GRANT, J. K. (1968) The biosynthesis of adrenocortical steroids. 3. Endocr. 41, 111. HART, P. G., H. J. VAN DER MOLEN and J. H. H. BAKKER (1968) Heeft suppressie van de bijnierfunctie met dexamethason en stimulatie van de ovariumfunctie met Pregnyl (de “Proef van Netter”) betekenis voor het stellen van de indicatie: ovariumresectie? (Has suppression of the adrenal cortical function with dexamethasone and stimulation of ovarial function with HCG (Netter’s test) significance for the indication: wedge resection of the ovary?) Ned. 1. Verlosk. 68, 1. HELLER, G. G.: M. J. ROWLEY and G. V. HELLER (1969) Clomiphene citrate, a correlation of its effect on sperm concentration and morphology. Total gonadotrophins, ICSH, estrogen and testosterone excretion, and testicular cytology in normal men. 3. clin. Endow. 29, 638. HORTON, R., E. ROMANOFF and R. WALKER (1966) Androstenedione and testosterone in ovarian venous and peripheral plasma during ovariectomy for breast cancer. 3. clin. Endocr. 26, 1267. HORTON, R. and S. D. FRASIER (1967) Androstenedione and its conversion to plasma testosterone in congenital adrenal hyperplasia. 3. clin. Invest. 46, 1003. HORTON, R. and J. NEISLER (1968) Plasma androgens in patients with the polycystic ovary syndrome. 3. clin. Endocr. 28, 479. HUDSON B., COGHLAN J. P., DOLMANIS A. and M. WINTOUR (1964) The measurement of testosterone in biological fluids in the evaluation of androgen activity. Proceedings II Int. Congress Endocrinology, London. Excerpta med., I.C.S. 83, II, 1127. ISMAIL, A. A. A., R. A. HARKNESS and S. A. LORA~NE (1968) Observation on urinary testosterone excretion during the menstrual cycle and pregnancy.
plasma
testosterone levels
In : Testosterone. Proceedings of the workshop conference, J. Tamm, Ed., Georg Thieme Verlag, Stuttgart, p. 211. JAYLE, M. F., R. SCHOLLER, P. MAUVAIS-JARVIS and M. SZPER (1962) Exploration de la fonction ovarienne par les gonadotropines chorioniques assocites a la dexamtthasone. Application au diagnostic des virilismes ovariens. Clin. chim. Acta 7,322. KASE, N. and J. KOWAL (1962) In vitro production of testosterone in a human adrenal homogenate. 3. clin. Endocr. 22, 925. KILLINGER, D. W. (1970) Testosterone. Canad. med. Ass. 3. 103, 733. KIRSCHNER, M. A., M. B. LIPSETT and D. R. COLLINS (1965) Plasma ketosteroids and testosterone in man: A study of the pituitary-testicular axis. 3. clin. Invest. 44, 657. KIRSCHNER, M. A., C. W. BARDIN, W. C. HEMBREE and G. T. Ross (1970) Effect of estrogen administration on androgen production and plasma luteinizing hormone in hirsute women. 3. clin. Endocr. 30, 727. KORENMAN, S. G., M. A. KIRSCHNER and M. B. LIPSETT (1965) Testosterone production in normal and virilised women and in women with the SteinLeventhal syndrome or idiopathic hirsutism. 3. clin. Endocr. 25, 798. LAMB, E. J., W. J. DIGNAM, R. J. PION and H. H. SIMMER (1964) Plasma androgens in women. I. Normal and non hirsute females, oophorectomized and adrenalectomized patients. Acta Endocr. 45,243. LIPSETT, M. B., H. WILSON, M. A. KIRSCHNER S. G. KORENMAN, L. M. FISHMAN, G. A. SARFATY and C. W. BARDIN (1966) Studies on Leydig-cell physiology and pathology, secretion and metabolism of testosterone. Recent Progr. Hormone Res. 22,245. LLOYD, C. W., J. LOBOTSKY, E. .J. SEGRE, T. KOBAYASHI, M. L. TAYMOR and R. E. BATT (1966) Plasma testosterone and urinary 17-ketosteroids in women with hirsutism and polycystic ovaries. 3. clin. Endocr. 26, 314. LOBOTSKY, J., H. I. WYSS, E. J. SEGRE and C. W. LLOYD (1964) Plasma testosterone in normal women. 3. clin. Endocr. 24, 1261. MAHOUDEAU, J. A., M. A. DROSDOWSKYand M. F. JAYLE (1970) Dosage de la testosterone et de l’epitestosterone urinaires dam l’hypogonadisme male et dans l’hirsutisme. Ann. Endocr. (Paris) 31,585. NETTER, A., M. F. JAYLE, R. MUSSET, A. LAMBERT and P. MAUVAIS-JARVIS ( 1960) Les Cpreuves dynamiques dans le syndrome de Stein-Leventhal. Ann. Endocr. (Paris) 21, 590. RESKO, J. A. and K. B. EIKNES (1966) Diurnal testosterone levels in peripheral plasma of human male subjects. 3. clin. Endocr. 26, 573.
217
RIONDEL, V., .J. F. TAIT, M. GUT, S. A. TAIT, A. JOACHIM and B. LITTLE (1963) Estimation of testosterone in human peripheral blood using S-35 thiosemicarbazide. 3. clin. Endocr. 23, 620. RIVAROLA, M. A., J. M. SAEZ, W. J. MEYER, M. E. JENKINS and C. J. MIGEON (1966a) Metabolic clearance rate and blood production rate of testosterone and androst-4-ene-3,17-dione under basal conditions, A.C.T.H. and H.C.G. stimulation. Comparison with urinary production rate of testosterone. 3. clin. Endocr. 26, 1208. RIVAROLA, M. A. and C. J. MIGEON (196613) Determination of testosterone and androst-4-ene-3,17-dione concentration in human plasma. Steroids 7, 103. RIVAROLA, M. A., J. M. SAEZ and C. J. MIGEON (1967) Studies of androgens in patients with congenital adrenal hyperplasia 3. clin. Endocr. 27, 624. RIVAROLA, M. A., M. G. FOREST and C. J. MIGEON (1968) Testosterone, androstenedione, dehydroepiandrosterone in plasma during pregnancy and at delivery : concentration and protein binding. 3. clin. Endocr. 28, 34. RYAN, K. J. and 0. W SMITH (1965) Biogenesis of steroid hormones in the human ovary. Recent Progr. Hormone Res. 21,376. SAROFF, J., R. E. KEENAN, A. A. SANDBERG and W. R. SLAUNSWHITEJR. (1967) Determination of testosterone, androstendione and dehydroepiandrosterone in human plasma using bromine-82. Steroids 10, 15. SAVARD, K., J. M. MARSH and B. F. RICE (1965) Gonadotrophins and ovarian steroidogenesis. Recent Prop. Hormone Res. 21, 285. SEGRE, E. S., E. L. KLAIBER, J. LOBOTSKY and C. W. LLOYD (1964) Hirsutism and virilising syndromes. Ann. Rev. Med. 15, 315. SOUTHREN, A. L., S. TOCHIMOTO, N. C. CARMODY and K. ISUGURI (1965) Plasma production rates of testosterone in normal adult men and in patients with the syndrome of feminizing testes. 3. clin. Endocr. 25, 144 1. SOUTHREN,A. L. and G. G. GORDON (1970) Studies in androgen metabolism Mt. Sin. 3. Med. 37, 516. SURACE, M., M. Lum, V. MONETA, V. MARESCOTTI and F. POLVANI (1966) Plasma testosterone determination of women in normal and pathological conditions, using horizontal thin layer and gasliquid chromatography. Proceedings II. symposium on steroid hormones, Ghent, 1965. Excerpta med. I.C.S. 101, 16. TAGATZ, G., P. J. FIALKOW, D. SMITH and L. SPADONI (1970) Hypogonadotrofic hypogonadism associated with anosmia in the female. New Engl. 3. Med. 283, 1326. TUCKER, E. C., P. M. F. BISHOP and I. F. SOMMER-
Europ. 3.
Obstet.
Gynec. 6 (1971)
218
Rubens,
Vermeulen
VILLE (1969) Urinary testosterone and epitestobutyrate. In: Testosterone. Proceedings qf theWorkshop sterone glucuronide in hirsutism and certain Conference, J. TAMM, Ed., George Thieme Verlag, menstrual disorders. 3. Obstet. Gynaec. Brit. Cwlth. Stuttgart, p.13. 76, 1111. VERMEULEN, A. and L. VERDONCK(1968) Studies of VAN DER MOLEN, H. J., D. GROENand A. PETERSE the binding of testosterone to human plasma. (1966) Measurement of testosterone in plasma and Steroids 11,609. urine using gas-liquid chromatography. ProceedVERMEULEN, A., L. VERDONCK, M. VANDERSTRAETEN ings II. symposium on steroid hormones, Ghent, and N. ORIE (1969) Capacity of the testosterone 1965, Excerpta med., Z.C.S. 101,1. binding globulin in human plasma and influence VEMEULEN,A. (1966) Urinary excretion of testoof specific binding of testosterone on its metabolic sterone. Proceedings II. symposium on steroid horclearance rate. 3. clin. Endocr. 29, 1470. mones, Ghent, 1965. Excerpta med., Z.C.S. 101,71. VERMEULEN, A., T. STOICAand L. VERDONCK(1971) .VERMEULEN, A. (1968) Determination of testosterone The apparent free testosterone concentration in plasma by electron capture of the heptafluoroan index of androgenicity. 3. clin. Endocr. in press.
Euro&
3.
Obstet. G~nec. 6 (1971)
SUMMARY Several parameters have been used in the past to evaluate androgenicity in the female; plasma testosterone levels seem to be by far the most reliable parameter applicable in the clinical routine. Using a very sensitive method we found a mean plasma testosterone level in the normal female of 35 ng & 15 ng/lOO ml; the latter shows a slight diurnal variation, with highest values in the morning, and a moderate cyclical variation with higher levels in the luteal phase of the menstrual cycle. During pregnancy the plasma testosterone increases and reaches a plateau of about 100 ng/ 100 ml from the second trimester on. Among the pharmacologic agents, A.C.T.H. causes a significant increase and corticoids a decrease of plasma testosterone levels, whereas gonadotrophins and clomiphene citrate increase slightly plasma testosterone levels. As far as pathological conditions are concerned, plasma testosterone levels are generally highly increased in congenital adrenal hyperplasia and in the adrenogenital syndrome; in virilizing ovarian tumors, the polycysticovarysyndrome, idiopathic hirsutism, acne and sebaceous alopecia as well as in hyperthyroidism a moderate increase is generally, although not always, observed.
KEYWORDS plasma testosterone; hirsutism; virilism; polycystic ovary syndrome; nonprotein-bound testosterone; dynamic exploration of virilism.
I. INTRODUCTION It is generally accepted that by far, the most important biologically active androgenic hormone is testosterone. Although other androgens are secreted by either the gonads or the adrenal cortex, they are probably only biologically active in as far as they are converted to testosterone (BARDIN and MAHOUDEAU, 1970). Testosterone was already isolated from biological fluids in 1935 (KILLINGER, 1970) but, until a few years ago the only, clinically useful, parameter for androgen secretion, was the determination of the urinary 17-ketosteroids by the Zimmermann reaction. The latter however
University of Ghent (Belgium), Akademisch Ziekenhuis and Metabolism.
Department of Medicine, Division ofEndocrinology
Europ. 3. Obstet. Gym. 6 (1971)
208
Rubens, Vermeulen m- 3SBng/lOOml
Test.
SD - 15.3ngllOOml
75_pg/lOOml
.
70. 65.
.
55. 50.
l .
45.
:
: . :
60.
.
t :
.
l
.
9.
.
:
.
.
l
l:
l
.
.
t
.
35.
. .
30.
.s’
c
.
25.
.
20.
.
:
40.
.
*
.
.
.
l
l
15*
.
. .
t
l. l
.
10.
.
‘.
.
i .
. .
5.
c
4
20
Fig. 1. Plasma testosterone
Plasma
30
40
levels in normal menstruating
SOY
females.
T.
80, ng/iooml. 70,
Fig. 2. Plasma testosterone
Europ. J. Obstet. Gynec. 6 (1971)
levels during menstrual
cycle.
plasma testosterone levels
does not only
measure
the
metabolites
of
testosterone but also the metabolites of moderately or weakly active androgens, secreted either by the adrenal cortex or the gonads. That the urinary 17-ketosteroids are a poor parameter of androgenicity is evident
from the fact that normal
adult females are within the range observed in normal adult males.
values in of those
During the last decade more specific methods for the estimation of androgen secretion have been developed and since 1966 we routinely determine the plasma testosterone levels as a parameter of androgenicity. The method which we developed in our laboratory is based on the electron capturing properties of the heptafluorbutyrate ester of testosterone and with minor modifications it has been used since in our laboratory (VERMEULEN, 1968). Methodology and statistical reliability have been reported in detail previously (VERMEULEN, 1968). The method has a sensitivity of 10 ng of testosterone per 100 ml, allowing the accurate determination of usual testosterone levels in female plasma. II. PHYSIOLOGY OF TESTOSTERONESECRETION IN FEMALES Plasma testosterone in normal females originates for two thirds from the adrenal cortex and for one third from the ovaries (KASE and KOWAL, 1962 ; SAVARD, MARSH and RICE, 1965; RYAN and SMITH, 1965; GRANT, 1968; HORTON, ROMANOFF and WALKER, 1966; BAIRD, UNO and MELBY, 1969) ; one third seems to be secreted as such, whereas two thirds originate from peripheral conversion of precursors, so called prehormones (BARDIN and MAHOUDEAU, 1970), mainly androstenedione. Plasma testosterone levels were deter-
mined between
209
8 and 10 a.m. in 100 normal
females, during reproductive life. As shown in Fig. 1 testosterone values vary between 10 and 70 ng/lOO ml (with a mean value of 35,8 ng/lOO ml), similar to the values reported by most authors (Table I). It is well known that in males plasma testosterone levels show a diurnal variation with highest values in the morning and lowest at about 8-9 p.m. (SOUTHREN, TOCHIMOTO, CARMODY and ISUGURI, 1965; DRAY, REINBERG and SEBAOUN, 1965 ; RESKO and EIKNES, 1966 ; LIPSETT, WILSON, K~RSCHNER, KORENMAN, FISHMAN, SARFATY and BARDIN, 1966). Although testosterone in females has a complex origin, the gonads being the minor source, a similar variation could nevertheless be expected, as it is well known that the adrenal cortical secretion shows a diurnal rhythm. The low plasma testosterone levels in females, close to the lower limit of detection of most methods used, were the reason that until recently this diurnal variation in females remained an open question; recently however we were able to demonstrate that 8 p.m. values are generally lower than 8 a.m. values by about one third. In order to determine whether there exists a cyclical variation in plasma testosterone levels, the latter were determined daily during one cycle in five normal females with regular periods. As shown in Fig. 2 there exists a small but significant cyclical variation in plasma testosterone levels, the values during the luteal phase being slightly higher than those during the follicular phase; our results however do not permit to decide whether there exists a preovulatory peak in plasma testosterone levels. Some authors reported increased testosterone levels around ovulation and during the luteal phase Europ.
3. Obstet. Gynec. 6 (1971)
210
Rubens, Vevmeulen
TABLE I. PLASMATESTOSTERONE LEVELSWITH DIFFERENT METHODS:RRSULTS. G.L.C. graphy. Double Isotope D: Double Isotope Derivative.
Author
Method
FINKELSTEIN et al. (1961) FORCHIELLIet al. (1963) RIONDELLet al. (1963) BROWNIEet al. (1964) BURGERet al. (1964) KIRSCHNERet al. (1965) LAMB et al. ( 1964) SEGRE et al. (1964) SURACE et al. (1966) VAN DER MOLEN et al. (1966) HUDSONet al. (1969) LOBOTSKYet al. (1964) BARDIN and LIPSETT(1967) SAROFF et al. (1967) RIVAROLA et al. (1966b) LLOYD et al. (1966) DRAY et al. (1968) KORRNMANet al. (1965) BARDINet al. (1968b) FURUYAMAet al. (1970) PRESENTSERIES
Enzymatic Enzymatic Double Isotope D G.L.C. Double Isotope D Double Isotope D Enzymatic Double Isotope D G.L.C. G.L.C. Double Isotope D Double Isotope D Double Isotope D Double Isotope D Double Isotope D Double Isotope D Double Isotope D Double Isotope D Double Isotope D Radioimmunassay G.L.C.
(LOBOTSKY,
WYSS,
ISMAIL,
HARKNESS
HUDSON,
COGHLAN,
SEGRE
and LLOYD,
1964;
LORAINE,
1965;
and
DULMANIS
and WINTOUR,
1964).
Although experiments performed by LIPal. (1966) suggested that posture might
SETT et
effect plasma testosterone levels, with higher values in the upright than in the recumbent position, we observed neither in males nor in females any effect of posture on plasma testosterone levels. Duringpregnancy, plasma testosterone levels increase progressively to reach a plateau, with a mean value of about 100 ng/lOO ml, from the end of the first trimester (Table II). Nevertheless plasma testosterone levels remain highly variable and even during the last trimester, values within the normal female range may be observed. All values lie however above the mean value for nonEurop. 3. Obstet. Gynec. 6 (1971)
=
Gas Liquid Ghromato-
Mean + S.D. (ng/lOO ml) 100 120 59 f 60 f 180 70 110 50 110 f 36
83 54f 37 f 68% 47 + 364 57 & 120 415 34 35 f
79 30
60
15 15 17 14,8 9 14 4 15
pregnant normal women. This variability of testosterone values during pregnancy should be compared to the rather wide range of normal values observed in non-pregnant females. Similar results were observed by other authors (DEMISCH, GRANT and BLACK, 1968; AUGUST, RIVAROLA,
and GRUMBACH, 1969 ; FOREST and MIGEON, 1968).
TKACHUK
TABLE II. APPARENT FREE TESTOSTERONE CONCENTR.4TIoN (A.F.T.~.) IN PREGNANCY
total plasma testosterone (ng/lOO ml n f S.D.) NON-PREGNANTFEMALES 70 FIRSTTRIMESTER PREGNANCY 7 SECONDTRIMESTER PREGNANCY 2 THIRD TRIMRSTRR PREGNANCY 14
36f
15
A.F. T.C. (ngllO0 ml f S.E.) 0,35 & 0,l
73&13
0,28 f
0,08
102&31
0,21 f
0,08
97*30
0,22 f
0,12
plasma
testosterone
levels
PHARMACOLOGICALFACTORS AFFECTING pituitary
III.
ne citrate
PLASMATESTOSTERONE IN FEMALES
211
disease do not respond to clomiphe(TAGATZ,
FIALKOW, SMITH and
SPADONI, 1970). A) Gonadotrophins As we considered it unwarranted to administer high doses of human chorionic
gonadotrophins to normal females in reproductive life, we have no personal experience of the influence of this drug upon plasma testosterone levels in normal females. Results in the literature are quite discordant: some authors (KIRSCHNER, LIPSETT and COLLINS, 1965) influence,
did not see any significant
whereas
others
(DIGNAM, PION,
HUDSON et al., 1964) observed a very small increase in plasma testosterone levels. In patients with hirsutism (Table VI) we observed a clearcut elevation of plasma testosterone levels under the influence of HCG during dexamethasone treatment. LAMB and
SIMMER, 1964;
B) Clomiphene Citrate, an anti-estrogen, is known to cause a release of LH and FSH (HELLER, ROWLEY and HELLER, 1969).
It
has been shown to cause a significant increase of
plasma testosterone
in
Ross and LIPSETT, 1971).
males
(BARDIN,
Recently
we ob-
served a slight increase of plasma testosterone in females after clomid@ treatment day for ten days). Women pogonadotropic gonadotropin
TABLE III. TOSTERONE
with anosmic hy-
hypogonadism
.Name
ANS. CHR. MASS.G. DE BRUY.C. Mos.
UPON
and Corticoids
(LLOYD, LOBOTSKY, SEGRE, KOBAYASHI, TAYMOR and BATT, 1966; BARDIN, HEMBREE and LIPSETT, 1968b).
D) Sex Hormones 1. Androgens In the view of the complex origin of plasma testosterone in females, it can hardly be expected that synthetic androgens will influence plasma testosterone to a significant extent. As administration of precursors of testosterone could possibly influence plasma testosterone levels, we administered dehydroepiandrosterone to females in a dose of 60 mg daily for 60 days; this did not have any clearcut influence upon plasma testosterone levels.
or impaired
secretion secondary to organic
EFFECTOF A.C.T.H. (I+00
(lOOmg/
C) A.C. T.H.-stimulation
As expected in the view of the adrenal origin of the major part of plasma testosterone in females, A.C.T.H. causes a moderate increase in plasma testosterone levels (Table III). On the other hand a decrease of 30 f 12 (S.D.) y0 of pl asma testosterone levels was observed after 8 days treatment with dexamethasone, 1 mg daily. Similar findings were reported in the literature by different authors
PLASMA
TES-
Id)
2. Progestogens GORDON,
SOUTHREN,
ALTMAN,
RAND
and
8 a.m.
F F F F
29 11 66 40
12 noon 32 15 75 51
4 p.m.
8 P.m.
40 20 82 47
26 17 84 51
OLIVO,
LEMBERGER
(1970)
recently showed that a potent progestogen (medroxyprogesterone
Sex
TOCHIMOTO,
small
acetate)
fall of plasma testosterone;
selves showed
that
progestogens
induces
a
we ourcause
a
decrease in the testosterone binding globulin (VERMEULEN, VERDONCK, VANDER STRAETEN and ORIE, 1969). Europ.
3 Obstet. Gynec. 6 (1971)
Rubens, Venneulen
212
3. Estrogens It is generally
TABLEIV.
admitted
that administration
of estrogens during the fertile period will cause anovulation and inhibition of ovarian function, which should result in a decrease in androgens secreted by the ovary. On the other hand, the increase in testosterone binding globulin tends to increase plasma testosterone levels (KIRSCHNER, BARDIN, HEMBREE and
estrogen variable.
Ross, 1970). administration
The final result of seems to be quite
4. Contraceptives As for estrogens the influence of combined (estrogen and progestogen containing) contraceptives is quite complex. While inhibition of LH release might decrease testosterone from ovarian origin the increase in binding protein (VERMEULEN and VERDONK, 1968 ; VERMEULEN et al., 1969) might result in an increase of testosterone levels. Hence the plasma testosterone values vary from low normal to definitely increased levels (Table
EFFECTOFCONTRACEPTIVES UPONPLASMA
TESToSTERoNE Name y& Li * ’ PENN.F. ’ LOOT.
r;;
R. T
VAN c. IL
BA. G.
Free T.
(%) 0.42 0.53 0.78 0.58 0.35 0.81 0.51
0.22
mean 0.53fO.O7(SE)
To.?& 1-. Free 1. (ng/lOO ml) (ng/lOOml) 99 95 82 101 30 130 72
75
96f9
0.42 0.50 0.64 0.59 0.11 1.05 0.37
0.17
0.49f0.10
B) Arrhenoblastoma Very high levels of plasma testosterone have been reported preoperatively in this very rare condition (DIGNAM et al., 1964; BRUCE, 1967). BRUCE (1967) observed a substantial fall in testosterone levels after operative removal of the tumor.
C) Hyperthyroidism DRAY, SEBAOUN, MOWSZOWICZ, DELZANT, DESGEZand DREYFUS(1967) were the first to report elevated plasma testosterone levels in IV). Recently, we and others hyperthyroidism. IV. PATHOLOGICAL CONDITIONS AFFECTING (VERMEULEN et al., 1969; SOUTHREN and GORDON, 1970) observed a similar increase PLASMATESTOSTERONE LEVELS in females, a consequence of an increased A) Adrenal Pathology In female patients suffering from Cushing binding of testosterone. syndrome slightly elevated plasma testosterone levels were found by BARDIN, LIPSETTand D) Virilism 1. Hirsutism FRENCH (1968a). The few observations we Plasma testosterone levels in idiopathic simwere able to make, are in accordance with ple hirsutism without other signs of virilisathese findings. In congenital adrenal hypertion are highly variable. Some patients have plasia we observed very high levels as has been reported in the literature (BURGER, normal levels whereas others have significantKENT and KELLIE, 1964; RIVAROLA, SAEZ ly increased values up to 150 ng/lOO ml. and MIGEON, 1967; HORTON and FRASIER, (LLOYD et al., 1966; DRAY, SEBAOUN, DELZANT, LEDRU and MOWSZO~ICZ, 1968; 1967). As could be expected, in female patients with Addison’s disease we had very low KORENMAN, KIRSCHNERand LIPSETT, 1965). levels of plasma testosterone (Table V). Increased urinary excretion of testosterone Europ. 3. Obstet. Gymx. 6 (1971)
plasma testosterone levels TABLE V. PLASMATESTOSTERONE IN ADRENAL 0Locx
(ng/lOO
A. Gushing’s syndrome PL.V. DEBEY. L. : WILL.
B.
s.
?
TABLE VI. PLASMA TESTOSTERONE LEVELSIN WOMEN WITH ACNE AND SEBACEOUSALOPECIA Plasma test. (ng/lOO ml)
28~. 43~.
H. H.
35 y.
H.
82 ng 73 ng 107 ng
PETR. ED.
53 y.
SPITA. L.
34 y.
116ng 40 ng
VINC. S.
23 y.
57 ng 117 ng
MISER.
21 y.
3
16 y.
182ng
BAEL.
29 y.
34 ng
661 ng
BAR. M.
20 y.
123 ng
Z
22 y. 53 y.
RUBB. L.
29 y. 61 y.
117 ng
Congenital adrenal hyperplasia
KIRCH.M. SEL. E. P. E. C.
PATH-
ml)
213
166 ng
DOND. M.
75 ng
Addison’s disease DB. H.
?
24 y.
5.3 ng
Co. M.
e
30 y.
11.5 ng
DESCH.L. S0.L.
$? ?
43 y. 33 y.
22.0ng 18.0ng
.H = hyperplasia glucuronides on the contrary seems to occur much more frequently (VERMEULEN, 1966; TUCKER, BISHOP and SOMMERVLLLE, 1969; MAHOUDEAU,DROSDOWSKY andJAYLE,l970); this could be a consequence of a difference in physical state of the hormone in the blood as will be discussed below. The variability of the testosterone level in hirsutism is not unexpected as evaluation of the condition is rather subjective and the intensity will be scored differently according to whether the hair is dark or fair. 2. Acne and sebaceous
alopecia
The sebaceous glands are probably target organs for the androgens (EBLING, 1970). We ,observed in 6 out of 8 patients a significantly increased level of plasma testosterone as acompared with a normal group (Table VI). This confirms earlier results concerning urinary testosterone excretion (VERMEULEN, 1966). 3. Polycystic ovary syndrome (P.C.O.) The P.C.O. syndrome remains an obscure chapter in female endocrinology and the
pathogenesis
is still controversial. Most authors (LLOYD et al., 1966 ; KORENMAN et al., 1965 ; HORTON and NEISSLER, 1968) observed increased plasma testosterone levels in the majority but not in all cases of P.C.O. The cases with normal levels however were clinically undistinguishable from those with higher levels. We ourselves studied five patients with P.C.O. and observed in all a significantly increased plasma testosterone value. V. DYNAMIC EXPLORATION OF VIRILISM IN FEMALES Considering the complex origin of testosterone in females, several authors (DIGNAM et al., 1964; LLOYD et al., 1966; BARDIN et al., 1968b) have emphasized the importance of the dynamic exploration of testosterone secretion in females. Different techniques have been used. They can be subdivided into two groups : - Tests
affecting
the adrenal
component
of
plasma testosterone namely dexamethasone suppression and ACTH stimulation. - Tests influencing the ovarian secretion of testosterone: H.C.G. stimulation tests or more frequently H.C.G. stimulation during dexamethasone suppression. An important decrease of elevated plasma testosterone levels during dexamethasone treatEurop.
3. Obstet. Gynec. 6 (1971)
214
Rubens, Vermeulen
VII.DYNAMICEXPLORATION
TABLE
OF VIRILISMIN FEMALES
Patient
Clinkat signs
A.M. V.L.J. C.J. M.J.
Hirsutism Acne + hirsutism Hirsutism + amenorrhea Hirsutism
Base line Tng/lOO ml
H.C.G. Stimulation** T nggllO0 ml
Dexamethasone Su#wession* T ng/lOO ml
63,8 70,7 56,9 68,5
59,5 76,3
27,7 43,7
i28,3
78,o 31,l
77,o
Diagnosis
Id. Hits. Id. Him. P.C.O. Id. Hirs.
*Dexamethasone suppression means determination of plasma testosterone level after five days dexamethasone 3 mg/day. **H.C.G. stimulation means determination of plasma testosterone after five days H.C.G. 1500 I.U. daily i.m. while under dexamethasone suppression 3 mg/day.
ment suggests an adrenal
origin of elevated testosterone levels; this origin will be confirmed if testost.erone levels increase excessively upon A.C.T.H. stimulation. Due to the long half life of the adrenal androgens, which are the main precursors of testosterone in the females, dexamethasone should be administered for at least five or perhaps better even ten days before the adrenal contribution can be evaluated. The dose of dexamethasone has furthermore to be rather high (3 mg a day) in order to block completely A.C.T.H. secretion. Theoretically the H.C.G.-stimulation test should be very useful to differentiate hirsutism from ovarian origin from hirsutism from adrenal origin. Indeed an important increase of plasma testosterone during H.C.G. treatment, or better during a combined dexamethasone - H.C.G. treatment wil indicate an ovarian origin. Such tests have originally been divised with urinary androgens as a parameter of androgenicity (NETTER,JAYLE, MUSSET,
LAMBERT
and
MAUVAIS-JARVIS,
tation of the results is difficult, the effects of dynamic exploration being ambiguous. Frequently an important decrease of plasma testosterone level upon dexamethasone inhibition is observed, while on the other hand H.C.G. stimulation causes a significant increase in testosterone levels, in which case the test does not allow to decide whether the adrenal or the ovary is the main source of the androgens (Table VII). This is in agreement with the results reported by LI,OYD et al., 1966; DIGNAM et al., 1964; KORENMAN et al., 1965 ; ETTINGER, VON WERDEN, THENAERS and FORSHAM, 197 1). VI.
FREE NON-PROTEINBOUND TESTOSTERONE
AND ANDROGENICITY From
personal
experience
testosterone determination, it can be considered
with
plasma
it appears
that
as an excellent para-
meter of androgenicity.
Nevertheless
there
are some circumstances where plasma testosterone levels do not correlate well with the clinical condition;
for example in pregnancy
1960 ; DESCOURT,JAYLE and MAUVIS-JARVIS,
and
1962 ; JAYLE, SCHOLLER, MAUVIS-JARVIS and
levels are increased without signs of virilisa-
SZPER, 1962;
HART, VAN DER MOLEN and
BAKKER, 1968 ; MAHOUDEAU
et al., 1970).
However, although occasionally those tests give useful information, often the interpreEurofi.
3.
Obstet. Gynec. 6 (1971)
hyperthyroidism,
plasma testosterone
tion, whereas in female hirsutism sometimes normal plasma testosterone levels are found. It is well known that plasma testosterone is largely
protein
bound,
and in analogy
to
plasma testosterone levels
cortisol and on the basis of some experimental evidence, it is generally accepted that only the
non-protein
bound
testosterone
215
TABLE VIII.
FREE TESTOSTERONE IN HIRSUTEFEMALES
Patient
Diapnosis*
Free T.
repre-
sents the biologically active fraction (VERMEULEN, STOICA and VERDONK, 197 1). The determination of this apparent free testosterone concentration (A.F.T.C.) by equilibrium dialysis as described by VERMEULEN et d. (1971) seems to give a much better parameter of androgenicity than total plasma testosterone. The mean free testosterone fraction in normal females was 0,96 f 0,07 (SE)% and the apparent free testosterone concentration varied between 0,2 and 0,75 ng/lOO ml against a mean free testosterone fraction of of 11,6 2,08 f 0,08 (SE)% and an A.F.T.C. -+ 0,7 (SE) ng/ 100 ml in males of the same age group. Whereas, as mentioned earlier, total testosterone increased during pregnancy, we found a normal or even slightly decreased apparent free testosterone concentration during the whole duration of pregnancy (Table II). Similarly in hyperthyroidism, notwhitstanding increased plasma testosterone levels, we observed a free testosterone concentration within the normal range. On the contrary we found an increased A.F.T.C. in 10 out of 13 cases of hirsutism, whereas the total plasma testosterone level was only increased in 7 out of 13 (Table VIII). Three patients with clinical hirsutism and normal total testosterone levels had a significantly increased free testosterone concentration. From our experience, it appears that the occasional discrepancy between the total plasma testosterone and clinical signs of androgenicity disappears almost completely when the apparent free concentration of testosterone is used as a parameter. Incidently, the A.F.T.C. discriminates much better between males and females than total
Tot.
(%)
T.
(4 100 ml)
DE KE. J, DE C. R. BERN. J. V. COP. R. v. LI. D. PR. J. D. C.
P.C.O. P.C.O. S.H. S.H. S.H. S.H. P.C.O. S.H. S.H. S.H. P.C.O. S.H. S.H.
C.D.R.
DEW. I. DE C. R. CHAV. COES. E. HUG. L. Mean
f
S.E
1.50 1.94 1.42 1.03 0.96 0.96 1.94
105 105 54 46 134 50
1.82
95 113 59 255 41 64
1.43 2.72 2.22 1.64 3.03 1.74hO.18
*SH=simple hirsutism P.C.O.=polycystic ovary
172
Free T.
(nbd 100 ml) 1.58 2.04 0.78 0.41 1.29 0.48
3.34 1.73 1.62 1.63 5.61 0.84 1.92
1.7950.38
syndrome
testosterone levels, as the ratio of the highest normal A.F.T.C. in females to the lowest A.F.T.C. in males is 7, whereas this ratio is only 4 for total testosterone levels. It appears to the authors, therefore, that the determination of the unbound testosterone fraction, which can be obtained at the expense of little extra work, should be performed whenever total plasma testosterone is determined, as it will perm.it, especially in the female, a more rational interpretation of total plasma testosterone levels. REFERENCES AUGUST, G. P., M. TKACHUK and M. H. GRUMBACH (1969) Plasma testosterone-binding affinity and testosterone in umbilical cord, plasma, late pregnancy, prepubertal children and adults. 3. clin.
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