Catholic University School of Medicine, Institute of Internal Medicine, Division of Endocrinology, Largo A. Gemelli 8, 00168 Rome
KLINEFELTER'S SYNDROME: EFFECTS OF OESTROGEN ON GROWTH HORMONE, PROLACTIN AND THYROTROPHIN RELEASE, AND ON THYROTROPHIN AND PROLACTIN RESPONSES TO THYROTROPHIN-RELEASING HORMONE
By Antonino Barbarino and Laura De Marinis
ABSTRACT
In 4 normal men and 8 patients with Klinefelter's syndrome the effects of oestradiol-17 \g=b\(15\g=m\g/kg daily for 8\p=n-\13days) on serum levels of pituitary hormones were investigated. Control levels of gonadotrophins in the Klinefelter patients were significantly higher than in the normal males, whereas serum testosterone (T) levels were lower. Oestradiol induced a decrease in serum gonadotrophin concentrations in both the control subjects and the Klinefelter patients, whereas a testosterone suppression was observed in the normal subjects, but not in the Klinefelter patients. Control serum growth hormone (hGH), and prolactin (hPRL) levels were of comparable magnitude in both groups and significantly increased during oestradiol administration. Serum thyrotrophin (TSH) levels were normal before and during oestrogen treatment. Basal serum oestradiol levels were within the normal male range, and were increased during treatment. Prolactin and thyrotrophin responsiveness to TRH stimulation was examined in Klinefelter patients before and during oestrogen administration. Before treatment, hPRL responses to TRH were higher than those observed in normal men. During oestrogen treatment hPRL responses to TRH were significantly increased when compared to those observed before treatment. TSH responses to TRH were normal both before and
during
treatment.
These studies indicate that in patients with Klinefelter's syndrome macological doses of oestrogen induce different effects on the
pharhypo-
Reprints: M.D., Institute of Internal Medicine, Division of Endocrinology, Catholic School of Medicine, Largo A. Gemelli 8, 00168 Rome, Italy
A. Barbarino
University
thalamic-pituitary axis, lactin,
thyrotrophin,
and
regards the release of gonadotrophins, progrowth hormone, similar to those observed in
as
normal men. This fact supports the conclusion that in Klinefelter's syndrome the abnormality in the pituitary-gonadal feedback mechanism is selectively confined to the testosterone feedback control of gonadotrophin secretion. Finally, in patients with Klinefelter's syndrome, oestrogen is capable of inducing a significant increase of the hPRL response to TRH stimulation.
Oestrogen administration has been shown to increase plasma growth hormone (hGH) levels in men (Wiedemaim el al. 1976; Frantz 8c Rabkin 1965), post¬ menopausal women (Frantz & Rabkin 1965), and women taking oestrogen-con¬ taining oral contraceptives (Spellacy et al. 1967; Yen 8c Vela 1968). Basal plas¬ ma prolactin (hPRL) levels were significantly increased by chronic oestrogen administration in post-menopausal women (Frantz et al. 1972; Yen et al. 1974), and normal men (Carlson et al. 1973). In addition, augmentation of the PRL response to TRH stimulation after pre-treatment with diethylstilboestrol has been demonstrated in normal men (Carlson et al. 1973). Although patients with Klinefelter's syndrome have a primary involvement of the reproductive sys¬ tem, other abnormalities have occasionally been reported. A decreased TSH in response to TRH administration has been recently noted in Kline(Smals el al. ¡977). On the contrary, an increased PRL respon¬ siveness to TRH stimulation has been found in patients with this syndrome (Burman et al. 1975). Suppression of the elevated serum gonadotrophin values to normal levels or below in patients with Klinefelter's syndrome requires fre¬ quent and prolonged administration of testosterone compared to that needed in normal subjects (Fukutani et al. 1974; Capell et al. 1973; Stewart-Bentley et al. 1974). These data imply that in Klinefelter patients the hypothalamic-pituitary mechanisms are less sensitive to testosterone than in normal men. However, the response of the hypothalamic-pituitary unit to oestradiol administration has not been investigated in these patients. Recently, we have studied and reported the effects of oestradiol administration on gonadotrophin release in patients with Klinefelter's syndrome (Barbarino et al. 1979). We will report here the effects of oestradiol on GH, PRL, and TSH release and on the TSH and PRL response to TRH administration in our patients. reserve
felter patients
Subjects
SUBJECTS
AND METHODS
Four normal men, age range 28 to 46, and 8 patients with Klinefelter's syndrome age range 19 to 38 years, were investigated'1. All patients exhibited the classical fo nil of Klinefelter's syndrome, with a characteristic clinical appearance, elevated serum
1) Informed
consent was obtained from all
subjects
involved.
low testosterone concentrations, and a 47-XXY leukocyte karyopatients had received testosterone therapy prior to this study, but the type. therapy was withheld for at least 40 days before the investigation. The remaining 5 patients had never received testosterone therapy. Blood samples were collected daily at 08.00 h for 3 days before oestradiol administration. After obtaining these samples, 15 /¿g/kg body weight of oestradiol-i7 ß (Oe¿) in oil suspension (Progynon®, Schering) was administered im daily for 8 to 13 days to each subject. Since daily blood samples were obtained from each subject before receiving oestradiol, the serum Oe2 values reported represent the minimum circulating blood concentration. In order to determine the fluctuation in the blood level of LH, hPRL, TSH and hGH, blood samples were collected every 15 min for a 3-h period before and at the end of treatment in all subjects. Prolactin and TSH were also measured under basal conditions and at 20, 45, and 60 min after iv injection of 200 iig TRH before and during oestrogen treatment in Klinefelter patients.
gonadotrophin levels, Three
Assay procedures Blood samples were centrifuged within 2 h of collection and separate serum aliquots for hormone determination were stored at 20°C until assayed. Serum FSH and LH were measured by specific radioimmunoassay using the doubleantibody technique (Serra et al. 1974). The results are expressed as equivalent of the 2nd IRP-HMG in mIU/ml of serum. The lower limit of sensitivity, calculated as the hormone concentration which cor¬ responded to the lower 95 °/o confidence limit of the buffer control tubes run in each assay, averaged 2.5 mlU 2nd IRP-HMG/ml lor LH and 2.2 mlU 2nd IRP-HMG/ml for FSH. Serum prolactin levels were measured using a homologous double-antibody radio¬ immunoassay (McNeilly 1973). The sensitivity of assay was 1.5 ng/ml, the intra-assay variation was 4 °/o and the inter-assay variation 7 %. Serum hGH was measured by tadioimmunoassay as described previously (Barbarino et al. 1973). Thyrotrophin was determined in serum by RIA, using polyethylene glycol (PEG) to separate the bound and free hormone (Desbuquois Se Aurbach 1971). The lower limit of sensitivity was 0.2 ¿iU/ml. Concentrations of oestradiol-17 ß and testosterone in serum were measured by specific radioimmunoassays, with the use of Dextran coated charcoal to separate free and bound hormone (De Hertogh et al. 1975; Horion et al. 1967). The procedure of these assays involved a single diethylether extraction of the samples (0.5-1.0 ml) followed by direct assay of the dried extract without preparatory chromatography. Sensitivity of the method, defined as the amount of unlabelled steroid required to displace 5 "la of the tracer bound in buffer control tubes, was 3.2 ± 1.2 (sd) pg for oestradiol and 3.8 ± 1.5 (sd) pg for testosterone. The precision of both these assays expressed as the coefficient of variation was 10 °/o within and 16% between assays. The normal male range for GH, LH, FSH, TSH, PRL. and Oca is indicated in Table 1. All samples were assayed in duplicate. To avoid inter-assay variations all samples from a serial study were measured in a single assay. -
-
Analysis of
-
data
Transverse mean values for GH, PRL, and TSH during chronic oestrogen admi¬ nistration and control periods were compared by the unpaired ¿-test. The ¿-test for paired data was employed to compare PRL and TSH responses to TRH obtained before and during oestrogen treatment, while the unpaired t-test was employed to compare the response of patients with that in normal subjects.
Hormone measurements in 8
patients
Table 1. with Klinefelter's
oestrogen
syndrome before
Oestrogen treatment
Normal values*
Su.
and
during
treatment.
S5S fin
¿It ) r/3
+ " O »ó Oh
cm
-c
g »o
c
w
BE.
before
m;
155
during
131
KIN
60 12
2.1 2.(1
¡.52 5.80
11.11 22.33
45 235
before
141
60
40
1.7
during
143
is
1.7
0.90 2.72
5.64 21.50
30 230
before
371 276
75
45
I.I
¡8
8
1.5
2.90 10.60
¡0.24 22.75
40 300
211 157
33 18
20
0.6 0.8
2.10 3.53
5.28 6.93
35 225
before
230
2.1
210
30 12
¡8
during
6
2.2
0.58 1.76
before
70 90
32 ¡0
20 4
1.6
0.99
1.6
230 198
120 40
60 10
300 290
30 10
20 6
1.4
R.I.
C.I.
during D.E.
before
during
4
I..O. 7.8
12.8
50 ¡45
1.95
4.2 11.3
20 275
1.8 1.7
¡.20 4.70
15.60
50 250
1.3
2.60 5.20
6.20 16.20
25 200
T.A.
during GR.
during before
8.40
B.O.
before
during before oestrogen mean
se
207
67
35
1.6
1.60
7.35
37
±35
±¡7
±6
±0.2
±0.30
±0.87
±4
8
1.6
4.53
16.17
±¡.7 ±0.1
±1.01
±2.03
232 ±16
< 0.01
< 0.005
< 0.001
during oestrogen mean se
paired /-test
186
31
±25
±12
NS < 0.005 < 0.005
NS
: Transverse mean of values in samples obtained at ¡5 min intervals for 3 h. *: Normal range for adult males.
+
RESULTS
Effects of chronic oestrogen administration
on
hypophyseal trophic
hormone release
1) Klinefeller patients. The transverse mean of serum hGH, hPRL, and TSH concentrations obtained at 15 min intervals during 3 h periods is reported in Table 1. Before treatment, serum hGH and hPRL levels in this group of patients were comparable to values found in normal men (Table 1). Basal se¬ rum levels of both hGH and hPRL rose significantly in 7 of the 8 patients during oestrogen treatment. One patient, subject D.E., showed no significant response of either hGH and hPRL to oestrogen; this patient showed the lowest LH peak during positive feedback, as reported previously (Barbarino el al. -
Hormone measurements in 4 normal
Table 2. subjects before and
Oestrogen
i¡3 5
treatment
co
ti.
i-g
tC^) c/o
H s.
during oestrogen
treatment.
tJ "a
O ib -c
c
Pi
ta
.bo
g O
a.
S.A. before
during
680 360
7 3
1.8
800 320
in
1.5
5
1.4
520 210
I 1
1.9
4
1.6
640 280
15
i.i.
1.2 6.2
6.8 10.6
20 170
0.9 3.4
4.4 9.8
15 215
2.3 5.2
7.4 12.6
30 280
0.6
6.2
4.8
I 1.8
18 330
A.L.
before
during P.R.
before
during P.A.
before
during
3
2.3 2.2
10.7
1.8
1.2
6.2
20.8
± 1.4 ± 0.2
± 0.4
±0.6
±3.3
4.9 0.6
11.2
248.7
h 0.6
± 35.3
< 0.01
< 0.005
< 0.01
m
before Oe-> mean -
SE
660
II
±57
± 1.7
-
during Oej -mean se
292
4.5
±32
± 0.6
1.5
1.8
± 4.3 ± 0.2
±
•
paired ¿-test Transverse
< 0.005
mean
of values in
< 0.02 < 0.005
samples
NS
obtained at 15 min intervals for 3 h.
Prolactin response
to
TRH in 8
Table 3. patients with Klinefelter's
oestrogen
syndrome before
and
during
treatment.
hPRL
(ng/ml)
in after TRH administration
Treatment -30
60
2d
B.E. before
12
m
during
22
24
before
4 22
14 ¡8
43 47
35 43
5 23
33
22
7
44
311
26
26 37
¡4 22
R.I.
during C.I. before
I I
m
during
33
35
38 74
before
5 7
6
¡2 14
before
9
Hi
during
13
D.E.
during
9
6 4
54 90
42
¡8 34
III
LO.
T.A. before
during
12
4 14
33 84
32 48
¡7 30
before
7 12
42 ¡4
68
23 30
21
6 12
38 56
16 33
¡0 2¡
¡2 ± ¡.5
G.R.
during
¡2
BO.
before
7
during
I I
before oestrogen 7.5
7.3
37
25
1
±0.8
±4
±3.4
¡6.8
±2.9
17.7 ± 3.2
±9
< 0.01
< 0.0¡
< 0.005
6.6
6.5 ±1.0 NS
23
17
¡0
±2.0
±2.0
< 0.0¡
< 0.05
± ¡.5 NS
mean
±
SE
during oestrogen mean si:
paired Z-test
+
normal males
± 1.0 NS
unpaired /-test Paired /-test
comparing
mean
< 0.005
< 0.005
PRL concentration before and
treatment. + + :
22
±3.2
=
SE
:
34
± 4.3
(N 12) mean
+
59.6
Unpaired
/-test
comparing
with KS before treatment.
mean
during oestrogen
PRL concentration in normal males and
patients
Table 4. response to TRH in 8
Thyrotrophin
patients with Klinefelter's syndrome before and
during oestrogen
treatment.
TSH
(.«U/ml)
rin after TRH
Treatment -30
20
60
45
Patients with KS before oestrogen mean SE
1.5
¡.6
¡8
±0.2
±0.2
±3
12
t 3.3
7 1.7
during oestrogen mean
SE
paired /-test
+
normal males
17
±3
NS
NS
¡2
6
i 2.6
± 1.5
NS
NS
NS
(N 8) SE
unpaired /-test :
¡.6
±0.1
=
mean
+
1.6
±0.1
+
Paired /-test
comparing
1.8
1.7
¡0.5
±0.1
±0.1
± 1.5
NS
NS
NS
mean
8.0
b
4.3
± 0.3 NS
TSH concentration before and
NS
during oestrogen
treatment. + +
:
Unpaired
/-test
comparing
with KS before treatment.
mean
TSH concentration in normal males and
patients
1979). A significant elevation in the mean PRL level was noted 72 h after initiation of oestrogen treatment, and hPRL concentrations reached maximum levels at the end of the treatment. The quantitative change was similar to that found in normal men (Table 2). Before treatment, serum TSH. T,, and T3 levels were normal in all patients. Basal serum TSH concentrations did not change significantly during oestrogen treatment (Table 1). The pattern of go¬ nadotrophin release in response to oestrogen administration has been reported elsewhere (Barbarino el al. 1979). It should be noted, however, that the basal gonadotrophin concentrations were elevated before treatment in all patients and were significantly decreased at the end of oestrogen administration (Ta¬ ble 1). Before treatment all patients had significantly lower testosterone con¬ centrations than normal men, and the levels did not change significantly dur¬ ing treatment (Table 1). Basal serum oestradiol levels were within the normal
male range.
treatment, oestradiol concentrations increased to levels those seen during the late follicular phase of the normal female
During
comparable to cycle (Table 1).
menstrual
2) Normal subjects. The transverse mean of serum hGH, hPRL, and TSH concentrations obtained at 15 min intervals during 3 h periods is reported in Table 2. Basal serum concentrations of T, hFSH ond Oe., are also reported in Table 2. Basal serum levels of hGH and hPRL rose significantly during oestro¬ gen administration in all subjects. Serum TSH levels did not change signifi¬ cantly during treatment. On the contrary, serum gonadotrophin and testoste¬ rone concentrations were significantly decreased at the end of the oestrogen treatment, whereas circulating Oe., concentrations increased as expected. -
Prolactin and
thyrotrophin response
to
TRH
(Tables
2 and
3)
Before oestrogen treatment, the mean hPRL response to TRH was higher than that observed in normal men at 20 and 45 min. Despite the fact that basal hPRL levels were elevated when the patients were tested during oestrogen treatment, the mean hPRL response to TRH was significantly higher (P < 0.05) than that observed before treatment at all time intervals studied. In fact every patient, except subject D.E., had an increased basal hPRL concentration and serum hPRL response to TRH when re-tested. The mean peak TSH concentra¬ tion after TRH administration was 18 ± 3 /yU/ml before and 17 ± 3 //U/ml during oestrogen. TSH responses to TRH were normal at all time intervals studied during both examinations.
DISCUSSION We have previously demonstrated that oestrogen exerts both negative and posi¬ tive feedback effects on LH release in patients with Klinefelters' syndrome
(Barbarino
et
al. 1979).
In the present study we have shown significant effects of oestrogen upon serum hGH and hPRL secretion in these patients, and the effects were similar to those observed in normal men. A stimulatory effect of oestrogen on GH secretion has been previously docu¬ mented in normal men (Wiedemann et al. 1976) and post-menopausal women (Frantz 8c Rabkin 1965). Our results clearly show that chronic oestrogen adminis¬ tration also elevates basal hGH levels in patients with Klinefelters syndrome. The only patient in our study showing no significant GH response to oestrogen administration, failed to show an increase in hPRL levels and exhibited the lowest LH peak during positive feedback (Barbarino et al. 1979). One can only specu-
whether the lack of hypophyseal trophic hormone response to oestro¬ gen of this patient indicates a more profound alteration of the hypothalamicpituitary axis. However, the clinical features and the genetic and hormonal findings were similar in this group of patients with Klinefelter's syndrome. Basal serum PRL levels were normal in patients with Klinefelter's syndrome and were significantly increased by chronic oestrogen administration in the 7 subjects who showed a hGH response to oestrogen. The stimulatory effect of oestrogen on pituitary PRL secretion has been well documented in hypogonadal women (Yen et al. 1974). On the contrary, data concerning oestrogen effects on basal hPRL secretion in men are conflicting. A significantly increased mean hPRL level was observed in a group of men on chronic oestrogen therapy for carcinoma of the prostate, and a rise of plas¬ ma PRL was observed after one week in 3 of 5 men treated with 15 to 50 mg diethylstilboestrol (DES) per day (Frantz el al. 1972). On the contrary, 5 mg DES per day for 5 days or 10 mg DES per day for 3 days failed to produce a significant rise of basal PRL in normal men (Carlson et al. 1973). This dis¬ crepancy may be due to the oestrogen dose, since it has been demonstrated that oestrogen exerts a dose-related enhancement of pituitary hPRL release in hu¬ man subjects (Ehara et al. 1976). In previous reports, basal plasma hPRL concentrations have been noted to be normal in patients with Klinefelter's syndrome: however, Barman et al. (1975) observed that PRL response to TRH stimulation was exaggerated com¬ pared to that of normal men. While our studies confirmed this observation it is of some interest to note that during oestrogen administration, in spite of elevated PRL levels, serum prolactin rose to peak levels significantly higher than those observed before treatment. Thus, oestrogen treatment appears to increase basal hPRL levels and potentiate PRL response to TRH stimulation in normal men (Carlson et al. 1973), as well as in patients with Klinefelter's syn¬ drome. In our patients basal TSH levels and the TSH response to TRH stimulation were normal before treatment and did not change during oestrogen administra¬ tion. These results are in agreement with the findings of Burman et al. (1975). However they are somewhat different from those of Srnals et al. (1977) who reported a decreased TSH reserve in Klinefelter patients, both on or off testo¬ sterone treatment, in comparison to euthyroidal eugonadal male controls. The same investigators suggested that this blunted TSH response to TRH might be caused by the concomitant hypergonadotrophism, through a lack of available subunits. This hypothesis is not supported by our finding that oestrogen treated Klinefelter patients did not show an increase of TSH response to TRH stimu¬ lation in the presence of decreased gonadotrophin concentration. The effects late
as
to
of oestrogen treatment on basal TSH levels and TSH responses to TRH stimu¬ lation in normal men are also quite contradictory; increased basal TSH levels and enhanced TSH responses to TRH have been reported by some investi¬ gators (Hall et al. 1971; Mortimer ei al. 1974). On the other hand, no change in these parameters have been noted by other investigators (Carlson el al. 1973; Guai et al. 1972). The discrepancies may well relate to the oestrogen dose and preparation, as well as the difference in TRH dose. Basal gonadotrophin levels were elevated before treatment in all patients. Although oestrogen had a biphasic effect on LH release (Barbarino el at. 1979), gonadotrophins were significantly decreased at the end of oestrogen treatment. These results clearly show that in patients with Klinefelter's syndrome, oestra¬ diol is capable of decreasing gonadotrophin release as it does in normal men (Sanlen 1975; Sherins Sc Loriaux 1973; Kulin & Reiter 1972). While the hor¬ mone measurements confirmed the existence of testosterone deficiency and gonadotrophin excess in patients with Klinefelter's syndrome, pharmacolo¬ gical doses of oestradiol appeared to produce different effects on the hypo¬ thalamic-pituitary axis, with respect to the release of gonadotrophins, thyrotrophin, hGH, and hPRL. The observation that these effects are similar to those found in normal men supports the conclusion that in Klinefelter's syndrome the abnormality in the pituitary-gonadal feedback mechanism is selectively confined to the testoste¬ rone feedback control of gonadotrophin secretion.
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(1973) 752. Carlson H. E.. Jacobs L. S. Se Daughaday W. H.: J. clin. Endocr. 37 (1973) 488. De Hertogh R.. van der Heydcn I. Se Ekka E.: J. Steroid Biochem. 6 (1975) 1333. Desbuquois B. Se Aurbach G. D.: J. clin. Endocr. 33 (1971) 732. Ehara Y., Suer T. M. Se Yen S. S. C: Amer. J. Obstet. Gynec. 726' (1976) 455. Frantz A. G., Kleinberg D. L. Se Noci G. L.: Recent Progr. Hormone Res. 28 (1972) 527. Frantz A. G. Se Rabkin M. T.: J. clin. Endocr. 25 (1965) 1470. Fukulani K.. Usurugi K., Takayasu H., Wakabayashi K. Sc Tamaoki B.: J. clin. Endocr. 39
(1974)
856.
Guai C. Kastin A. J. V Schally A. V.: Recent Progr. Hormone Res. 28 Hall R.. Amos J. & Omis/on J. B.: Brit. med. J. / (1971) 582. Horion R.. Kalo T. & Sherins R.: Steroids 10 (1967) 245. Kulin H. E. Se Reilcr E. ().: J. clin. Endocr. 35 (1972) 836.
(1972)
173.
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McNeilly A. S.: 3
(1974)
J.
&
McNeilly
A. S.: Clin. Endocr.
97.
Santen R. J.: J. clin. Invest. 56 (1975) 1555. Serra G. B. Coniglia R. 8- Barile G.: Workshop Conference
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Received
on
November 2nd, 1978.
KLINEFELTER'S SYNDROME: EFFECTS OF OESTROGEN ON GROWTH HORMONE, PROLACTIN AND THYROTROPHIN RELEASE, AND ON THYROTROPHIN AND PROLACTIN RESPONSES TO THYROTROPHIN-RELEASING HORMONE
By Antonino Barbarino and Laura De Marinis
ABSTRACT
In 4 normal men and 8 patients with Klinefelter's syndrome the effects of oestradiol-17 \g=b\(15\g=m\g/kg daily for 8\p=n-\13days) on serum levels of pituitary hormones were investigated. Control levels of gonadotrophins in the Klinefelter patients were significantly higher than in the normal males, whereas serum testosterone (T) levels were lower. Oestradiol induced a decrease in serum gonadotrophin concentrations in both the control subjects and the Klinefelter patients, whereas a testosterone suppression was observed in the normal subjects, but not in the Klinefelter patients. Control serum growth hormone (hGH), and prolactin (hPRL) levels were of comparable magnitude in both groups and significantly increased during oestradiol administration. Serum thyrotrophin (TSH) levels were normal before and during oestrogen treatment. Basal serum oestradiol levels were within the normal male range, and were increased during treatment. Prolactin and thyrotrophin responsiveness to TRH stimulation was examined in Klinefelter patients before and during oestrogen administration. Before treatment, hPRL responses to TRH were higher than those observed in normal men. During oestrogen treatment hPRL responses to TRH were significantly increased when compared to those observed before treatment. TSH responses to TRH were normal both before and
during
treatment.
These studies indicate that in patients with Klinefelter's syndrome macological doses of oestrogen induce different effects on the
pharhypo-
Reprints: M.D., Institute of Internal Medicine, Division of Endocrinology, Catholic School of Medicine, Largo A. Gemelli 8, 00168 Rome, Italy
A. Barbarino
University
thalamic-pituitary axis, lactin,
thyrotrophin,
and
regards the release of gonadotrophins, progrowth hormone, similar to those observed in
as
normal men. This fact supports the conclusion that in Klinefelter's syndrome the abnormality in the pituitary-gonadal feedback mechanism is selectively confined to the testosterone feedback control of gonadotrophin secretion. Finally, in patients with Klinefelter's syndrome, oestrogen is capable of inducing a significant increase of the hPRL response to TRH stimulation.
Oestrogen administration has been shown to increase plasma growth hormone (hGH) levels in men (Wiedemaim el al. 1976; Frantz 8c Rabkin 1965), post¬ menopausal women (Frantz & Rabkin 1965), and women taking oestrogen-con¬ taining oral contraceptives (Spellacy et al. 1967; Yen 8c Vela 1968). Basal plas¬ ma prolactin (hPRL) levels were significantly increased by chronic oestrogen administration in post-menopausal women (Frantz et al. 1972; Yen et al. 1974), and normal men (Carlson et al. 1973). In addition, augmentation of the PRL response to TRH stimulation after pre-treatment with diethylstilboestrol has been demonstrated in normal men (Carlson et al. 1973). Although patients with Klinefelter's syndrome have a primary involvement of the reproductive sys¬ tem, other abnormalities have occasionally been reported. A decreased TSH in response to TRH administration has been recently noted in Kline(Smals el al. ¡977). On the contrary, an increased PRL respon¬ siveness to TRH stimulation has been found in patients with this syndrome (Burman et al. 1975). Suppression of the elevated serum gonadotrophin values to normal levels or below in patients with Klinefelter's syndrome requires fre¬ quent and prolonged administration of testosterone compared to that needed in normal subjects (Fukutani et al. 1974; Capell et al. 1973; Stewart-Bentley et al. 1974). These data imply that in Klinefelter patients the hypothalamic-pituitary mechanisms are less sensitive to testosterone than in normal men. However, the response of the hypothalamic-pituitary unit to oestradiol administration has not been investigated in these patients. Recently, we have studied and reported the effects of oestradiol administration on gonadotrophin release in patients with Klinefelter's syndrome (Barbarino et al. 1979). We will report here the effects of oestradiol on GH, PRL, and TSH release and on the TSH and PRL response to TRH administration in our patients. reserve
felter patients
Subjects
SUBJECTS
AND METHODS
Four normal men, age range 28 to 46, and 8 patients with Klinefelter's syndrome age range 19 to 38 years, were investigated'1. All patients exhibited the classical fo nil of Klinefelter's syndrome, with a characteristic clinical appearance, elevated serum
1) Informed
consent was obtained from all
subjects
involved.
low testosterone concentrations, and a 47-XXY leukocyte karyopatients had received testosterone therapy prior to this study, but the type. therapy was withheld for at least 40 days before the investigation. The remaining 5 patients had never received testosterone therapy. Blood samples were collected daily at 08.00 h for 3 days before oestradiol administration. After obtaining these samples, 15 /¿g/kg body weight of oestradiol-i7 ß (Oe¿) in oil suspension (Progynon®, Schering) was administered im daily for 8 to 13 days to each subject. Since daily blood samples were obtained from each subject before receiving oestradiol, the serum Oe2 values reported represent the minimum circulating blood concentration. In order to determine the fluctuation in the blood level of LH, hPRL, TSH and hGH, blood samples were collected every 15 min for a 3-h period before and at the end of treatment in all subjects. Prolactin and TSH were also measured under basal conditions and at 20, 45, and 60 min after iv injection of 200 iig TRH before and during oestrogen treatment in Klinefelter patients.
gonadotrophin levels, Three
Assay procedures Blood samples were centrifuged within 2 h of collection and separate serum aliquots for hormone determination were stored at 20°C until assayed. Serum FSH and LH were measured by specific radioimmunoassay using the doubleantibody technique (Serra et al. 1974). The results are expressed as equivalent of the 2nd IRP-HMG in mIU/ml of serum. The lower limit of sensitivity, calculated as the hormone concentration which cor¬ responded to the lower 95 °/o confidence limit of the buffer control tubes run in each assay, averaged 2.5 mlU 2nd IRP-HMG/ml lor LH and 2.2 mlU 2nd IRP-HMG/ml for FSH. Serum prolactin levels were measured using a homologous double-antibody radio¬ immunoassay (McNeilly 1973). The sensitivity of assay was 1.5 ng/ml, the intra-assay variation was 4 °/o and the inter-assay variation 7 %. Serum hGH was measured by tadioimmunoassay as described previously (Barbarino et al. 1973). Thyrotrophin was determined in serum by RIA, using polyethylene glycol (PEG) to separate the bound and free hormone (Desbuquois Se Aurbach 1971). The lower limit of sensitivity was 0.2 ¿iU/ml. Concentrations of oestradiol-17 ß and testosterone in serum were measured by specific radioimmunoassays, with the use of Dextran coated charcoal to separate free and bound hormone (De Hertogh et al. 1975; Horion et al. 1967). The procedure of these assays involved a single diethylether extraction of the samples (0.5-1.0 ml) followed by direct assay of the dried extract without preparatory chromatography. Sensitivity of the method, defined as the amount of unlabelled steroid required to displace 5 "la of the tracer bound in buffer control tubes, was 3.2 ± 1.2 (sd) pg for oestradiol and 3.8 ± 1.5 (sd) pg for testosterone. The precision of both these assays expressed as the coefficient of variation was 10 °/o within and 16% between assays. The normal male range for GH, LH, FSH, TSH, PRL. and Oca is indicated in Table 1. All samples were assayed in duplicate. To avoid inter-assay variations all samples from a serial study were measured in a single assay. -
-
Analysis of
-
data
Transverse mean values for GH, PRL, and TSH during chronic oestrogen admi¬ nistration and control periods were compared by the unpaired ¿-test. The ¿-test for paired data was employed to compare PRL and TSH responses to TRH obtained before and during oestrogen treatment, while the unpaired t-test was employed to compare the response of patients with that in normal subjects.
Hormone measurements in 8
patients
Table 1. with Klinefelter's
oestrogen
syndrome before
Oestrogen treatment
Normal values*
Su.
and
during
treatment.
S5S fin
¿It ) r/3
+ " O »ó Oh
cm
-c
g »o
c
w
BE.
before
m;
155
during
131
KIN
60 12
2.1 2.(1
¡.52 5.80
11.11 22.33
45 235
before
141
60
40
1.7
during
143
is
1.7
0.90 2.72
5.64 21.50
30 230
before
371 276
75
45
I.I
¡8
8
1.5
2.90 10.60
¡0.24 22.75
40 300
211 157
33 18
20
0.6 0.8
2.10 3.53
5.28 6.93
35 225
before
230
2.1
210
30 12
¡8
during
6
2.2
0.58 1.76
before
70 90
32 ¡0
20 4
1.6
0.99
1.6
230 198
120 40
60 10
300 290
30 10
20 6
1.4
R.I.
C.I.
during D.E.
before
during
4
I..O. 7.8
12.8
50 ¡45
1.95
4.2 11.3
20 275
1.8 1.7
¡.20 4.70
15.60
50 250
1.3
2.60 5.20
6.20 16.20
25 200
T.A.
during GR.
during before
8.40
B.O.
before
during before oestrogen mean
se
207
67
35
1.6
1.60
7.35
37
±35
±¡7
±6
±0.2
±0.30
±0.87
±4
8
1.6
4.53
16.17
±¡.7 ±0.1
±1.01
±2.03
232 ±16
< 0.01
< 0.005
< 0.001
during oestrogen mean se
paired /-test
186
31
±25
±12
NS < 0.005 < 0.005
NS
: Transverse mean of values in samples obtained at ¡5 min intervals for 3 h. *: Normal range for adult males.
+
RESULTS
Effects of chronic oestrogen administration
on
hypophyseal trophic
hormone release
1) Klinefeller patients. The transverse mean of serum hGH, hPRL, and TSH concentrations obtained at 15 min intervals during 3 h periods is reported in Table 1. Before treatment, serum hGH and hPRL levels in this group of patients were comparable to values found in normal men (Table 1). Basal se¬ rum levels of both hGH and hPRL rose significantly in 7 of the 8 patients during oestrogen treatment. One patient, subject D.E., showed no significant response of either hGH and hPRL to oestrogen; this patient showed the lowest LH peak during positive feedback, as reported previously (Barbarino el al. -
Hormone measurements in 4 normal
Table 2. subjects before and
Oestrogen
i¡3 5
treatment
co
ti.
i-g
tC^) c/o
H s.
during oestrogen
treatment.
tJ "a
O ib -c
c
Pi
ta
.bo
g O
a.
S.A. before
during
680 360
7 3
1.8
800 320
in
1.5
5
1.4
520 210
I 1
1.9
4
1.6
640 280
15
i.i.
1.2 6.2
6.8 10.6
20 170
0.9 3.4
4.4 9.8
15 215
2.3 5.2
7.4 12.6
30 280
0.6
6.2
4.8
I 1.8
18 330
A.L.
before
during P.R.
before
during P.A.
before
during
3
2.3 2.2
10.7
1.8
1.2
6.2
20.8
± 1.4 ± 0.2
± 0.4
±0.6
±3.3
4.9 0.6
11.2
248.7
h 0.6
± 35.3
< 0.01
< 0.005
< 0.01
m
before Oe-> mean -
SE
660
II
±57
± 1.7
-
during Oej -mean se
292
4.5
±32
± 0.6
1.5
1.8
± 4.3 ± 0.2
±
•
paired ¿-test Transverse
< 0.005
mean
of values in
< 0.02 < 0.005
samples
NS
obtained at 15 min intervals for 3 h.
Prolactin response
to
TRH in 8
Table 3. patients with Klinefelter's
oestrogen
syndrome before
and
during
treatment.
hPRL
(ng/ml)
in after TRH administration
Treatment -30
60
2d
B.E. before
12
m
during
22
24
before
4 22
14 ¡8
43 47
35 43
5 23
33
22
7
44
311
26
26 37
¡4 22
R.I.
during C.I. before
I I
m
during
33
35
38 74
before
5 7
6
¡2 14
before
9
Hi
during
13
D.E.
during
9
6 4
54 90
42
¡8 34
III
LO.
T.A. before
during
12
4 14
33 84
32 48
¡7 30
before
7 12
42 ¡4
68
23 30
21
6 12
38 56
16 33
¡0 2¡
¡2 ± ¡.5
G.R.
during
¡2
BO.
before
7
during
I I
before oestrogen 7.5
7.3
37
25
1
±0.8
±4
±3.4
¡6.8
±2.9
17.7 ± 3.2
±9
< 0.01
< 0.0¡
< 0.005
6.6
6.5 ±1.0 NS
23
17
¡0
±2.0
±2.0
< 0.0¡
< 0.05
± ¡.5 NS
mean
±
SE
during oestrogen mean si:
paired Z-test
+
normal males
± 1.0 NS
unpaired /-test Paired /-test
comparing
mean
< 0.005
< 0.005
PRL concentration before and
treatment. + + :
22
±3.2
=
SE
:
34
± 4.3
(N 12) mean
+
59.6
Unpaired
/-test
comparing
with KS before treatment.
mean
during oestrogen
PRL concentration in normal males and
patients
Table 4. response to TRH in 8
Thyrotrophin
patients with Klinefelter's syndrome before and
during oestrogen
treatment.
TSH
(.«U/ml)
rin after TRH
Treatment -30
20
60
45
Patients with KS before oestrogen mean SE
1.5
¡.6
¡8
±0.2
±0.2
±3
12
t 3.3
7 1.7
during oestrogen mean
SE
paired /-test
+
normal males
17
±3
NS
NS
¡2
6
i 2.6
± 1.5
NS
NS
NS
(N 8) SE
unpaired /-test :
¡.6
±0.1
=
mean
+
1.6
±0.1
+
Paired /-test
comparing
1.8
1.7
¡0.5
±0.1
±0.1
± 1.5
NS
NS
NS
mean
8.0
b
4.3
± 0.3 NS
TSH concentration before and
NS
during oestrogen
treatment. + +
:
Unpaired
/-test
comparing
with KS before treatment.
mean
TSH concentration in normal males and
patients
1979). A significant elevation in the mean PRL level was noted 72 h after initiation of oestrogen treatment, and hPRL concentrations reached maximum levels at the end of the treatment. The quantitative change was similar to that found in normal men (Table 2). Before treatment, serum TSH. T,, and T3 levels were normal in all patients. Basal serum TSH concentrations did not change significantly during oestrogen treatment (Table 1). The pattern of go¬ nadotrophin release in response to oestrogen administration has been reported elsewhere (Barbarino el al. 1979). It should be noted, however, that the basal gonadotrophin concentrations were elevated before treatment in all patients and were significantly decreased at the end of oestrogen administration (Ta¬ ble 1). Before treatment all patients had significantly lower testosterone con¬ centrations than normal men, and the levels did not change significantly dur¬ ing treatment (Table 1). Basal serum oestradiol levels were within the normal
male range.
treatment, oestradiol concentrations increased to levels those seen during the late follicular phase of the normal female
During
comparable to cycle (Table 1).
menstrual
2) Normal subjects. The transverse mean of serum hGH, hPRL, and TSH concentrations obtained at 15 min intervals during 3 h periods is reported in Table 2. Basal serum concentrations of T, hFSH ond Oe., are also reported in Table 2. Basal serum levels of hGH and hPRL rose significantly during oestro¬ gen administration in all subjects. Serum TSH levels did not change signifi¬ cantly during treatment. On the contrary, serum gonadotrophin and testoste¬ rone concentrations were significantly decreased at the end of the oestrogen treatment, whereas circulating Oe., concentrations increased as expected. -
Prolactin and
thyrotrophin response
to
TRH
(Tables
2 and
3)
Before oestrogen treatment, the mean hPRL response to TRH was higher than that observed in normal men at 20 and 45 min. Despite the fact that basal hPRL levels were elevated when the patients were tested during oestrogen treatment, the mean hPRL response to TRH was significantly higher (P < 0.05) than that observed before treatment at all time intervals studied. In fact every patient, except subject D.E., had an increased basal hPRL concentration and serum hPRL response to TRH when re-tested. The mean peak TSH concentra¬ tion after TRH administration was 18 ± 3 /yU/ml before and 17 ± 3 //U/ml during oestrogen. TSH responses to TRH were normal at all time intervals studied during both examinations.
DISCUSSION We have previously demonstrated that oestrogen exerts both negative and posi¬ tive feedback effects on LH release in patients with Klinefelters' syndrome
(Barbarino
et
al. 1979).
In the present study we have shown significant effects of oestrogen upon serum hGH and hPRL secretion in these patients, and the effects were similar to those observed in normal men. A stimulatory effect of oestrogen on GH secretion has been previously docu¬ mented in normal men (Wiedemann et al. 1976) and post-menopausal women (Frantz 8c Rabkin 1965). Our results clearly show that chronic oestrogen adminis¬ tration also elevates basal hGH levels in patients with Klinefelters syndrome. The only patient in our study showing no significant GH response to oestrogen administration, failed to show an increase in hPRL levels and exhibited the lowest LH peak during positive feedback (Barbarino et al. 1979). One can only specu-
whether the lack of hypophyseal trophic hormone response to oestro¬ gen of this patient indicates a more profound alteration of the hypothalamicpituitary axis. However, the clinical features and the genetic and hormonal findings were similar in this group of patients with Klinefelter's syndrome. Basal serum PRL levels were normal in patients with Klinefelter's syndrome and were significantly increased by chronic oestrogen administration in the 7 subjects who showed a hGH response to oestrogen. The stimulatory effect of oestrogen on pituitary PRL secretion has been well documented in hypogonadal women (Yen et al. 1974). On the contrary, data concerning oestrogen effects on basal hPRL secretion in men are conflicting. A significantly increased mean hPRL level was observed in a group of men on chronic oestrogen therapy for carcinoma of the prostate, and a rise of plas¬ ma PRL was observed after one week in 3 of 5 men treated with 15 to 50 mg diethylstilboestrol (DES) per day (Frantz el al. 1972). On the contrary, 5 mg DES per day for 5 days or 10 mg DES per day for 3 days failed to produce a significant rise of basal PRL in normal men (Carlson et al. 1973). This dis¬ crepancy may be due to the oestrogen dose, since it has been demonstrated that oestrogen exerts a dose-related enhancement of pituitary hPRL release in hu¬ man subjects (Ehara et al. 1976). In previous reports, basal plasma hPRL concentrations have been noted to be normal in patients with Klinefelter's syndrome: however, Barman et al. (1975) observed that PRL response to TRH stimulation was exaggerated com¬ pared to that of normal men. While our studies confirmed this observation it is of some interest to note that during oestrogen administration, in spite of elevated PRL levels, serum prolactin rose to peak levels significantly higher than those observed before treatment. Thus, oestrogen treatment appears to increase basal hPRL levels and potentiate PRL response to TRH stimulation in normal men (Carlson et al. 1973), as well as in patients with Klinefelter's syn¬ drome. In our patients basal TSH levels and the TSH response to TRH stimulation were normal before treatment and did not change during oestrogen administra¬ tion. These results are in agreement with the findings of Burman et al. (1975). However they are somewhat different from those of Srnals et al. (1977) who reported a decreased TSH reserve in Klinefelter patients, both on or off testo¬ sterone treatment, in comparison to euthyroidal eugonadal male controls. The same investigators suggested that this blunted TSH response to TRH might be caused by the concomitant hypergonadotrophism, through a lack of available subunits. This hypothesis is not supported by our finding that oestrogen treated Klinefelter patients did not show an increase of TSH response to TRH stimu¬ lation in the presence of decreased gonadotrophin concentration. The effects late
as
to
of oestrogen treatment on basal TSH levels and TSH responses to TRH stimu¬ lation in normal men are also quite contradictory; increased basal TSH levels and enhanced TSH responses to TRH have been reported by some investi¬ gators (Hall et al. 1971; Mortimer ei al. 1974). On the other hand, no change in these parameters have been noted by other investigators (Carlson el al. 1973; Guai et al. 1972). The discrepancies may well relate to the oestrogen dose and preparation, as well as the difference in TRH dose. Basal gonadotrophin levels were elevated before treatment in all patients. Although oestrogen had a biphasic effect on LH release (Barbarino el at. 1979), gonadotrophins were significantly decreased at the end of oestrogen treatment. These results clearly show that in patients with Klinefelter's syndrome, oestra¬ diol is capable of decreasing gonadotrophin release as it does in normal men (Sanlen 1975; Sherins Sc Loriaux 1973; Kulin & Reiter 1972). While the hor¬ mone measurements confirmed the existence of testosterone deficiency and gonadotrophin excess in patients with Klinefelter's syndrome, pharmacolo¬ gical doses of oestradiol appeared to produce different effects on the hypo¬ thalamic-pituitary axis, with respect to the release of gonadotrophins, thyrotrophin, hGH, and hPRL. The observation that these effects are similar to those found in normal men supports the conclusion that in Klinefelter's syndrome the abnormality in the pituitary-gonadal feedback mechanism is selectively confined to the testoste¬ rone feedback control of gonadotrophin secretion.
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Received
on
November 2nd, 1978.
