Europ.J.clin.1nvest.
6,
249-254
(1976)
Luteinizing Hormone and Follicle Stimulating Hormone and the Response to Luteinizing Hormone Releasing Hormone in Relation to Sex and Age J. Lindholm, 0. Korsgaard, P. Rasmussen, and S . Micic
Department of Neurosurgery, Division Of Neuroendocrinology, Rigshospitalet, University Clinic, Copenhagen and Hormone Department, Statens Seruminstitut, Copenhagen, Denmark Received: July 1 , 1975, and in revised form: February 2 , 1 9 7 6 Abstract
Serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) before and after intravenous injection of luteinizing hormone releasing hormone (LHRH) were studied in 7 1 male and female subjects of various ages. Mean basal FSH and LH levels were not significantly different in the male groups, except that FSH was significantly higher in the very old subjects. Postmenopausal female subjects had much higher concentrations of both hormones than had premenopausal women. LH was higher in female subjects shortly after the menopause than in very old female subjects. The serum concentrations of both FSH and LH 30 min. after intravenous injection of 200 pg LHRH were not different from the 60 min. values. There was no significant difference in the response of LH in the male groups. The peak FSH concentration was higher in the very old male subjects. Postmenopausal women had a much higher peak concentration of both FSH and LH than had younger subjects. The increment of LH, but not of FSH, was larger in female subjects shortly after the menopause than in very old female subjects. In both sexes there was a significant correlation between the basal FSH/LH ratio and age. In younger male subjects there was a close positive correlation between basal LH and serum testosterone, in older male subjects this correlation was negative and significant. Key words
Follicle stimulating hormone, Lutenizing hormone, Pituitary gonadotropins, Luteinizing hormone releasing hormone, Pituitary function, Testicular function.
introduction
This paper presents the results of studies on pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) before and after intravenous (iv) injection of a synthetic luteinizing hormone releasing hormone (LHFW) in men and women in various age groups. The relationship between serum concentrations of gonadotropins and testosterone in men was also studied. Materials and Methods
Subjects
7 1 subjects were studied. All were apparently in good health with no signs of
endocrine, renal or hepatic disease. Male and female subjects were each divided into four groups according to age (Table 1 ) . Most subjects were hospital staff (nurses, secretaries, physicians) except those above 67 years old, who were patients recovering from minor coronary infarctions. All women over 50 years old were postmenopausal. Procedures
All tests were begun between 0800 and 0830 h. A catheter was inserted into an arm vein and samples were drawn at - 10, 0, 20, 30 and 60 minutes after a rapid iv injection of 200 p g LHRH Hoechst. Blood for testosterone determination was also obtained at zero time.
J. Lindholm et al.: L u t e i n i z i n g Hormone and F o l l i c l e S t i m u l a t i n g H o r m o n e
250
Table 1. Serum LH and F S H ( i u / l i t r e ) r e s p o n s e t o LHRH ( 2 0 0 pg i v ) i n
normal men and women LH 0 min.
mean ( r a n g e )
3 0 min. mean ( r a n g e )
A LH mean ( r a n g e )
Men
age (years)
N
I
20-29
8
9 ( 7-17)
50 ( 2 4 - 90)
4 0 (15-
I1
30-49
9
10 ( 4-21)
46 ( 1 6 - 96)
36 (12- 7 5 )
I11
50-67
8
9 ( 4-20)
46 (27- 95)
38 (22- 73)
IV
79-91
8
1 8 ( 3-49)
43
(15- 74)
24 (12- 47)
I
20-29
12
10 ( 4-18)
35 (18- 52)
24 (13- 4 2 )
I1
30-49
10
11 ( 8-17)
37
26 (12-
111
50-69
8
65 (26-98)
197 (76-464)
132 (21-366)
IV
79-91
8
26 ( 4-46)
68 (12-105)
42 ( 8- 59)
~~
~
~~~
~
64)
Women (20-
55)
38)
FSH
Men
0 min. mean ( r a n g e )
3 0 min. mean ( r a n g e )
A FSH mean ( r a n g e )
Basal
FSH
(range)
LH
I1
15 ( 6- 36)
20 ( 7- 46)
5 (1-
12)
1.5
1x1
1 3 ( 5-
32)
1 9 ( 6- 40)
6 (1- 15)
1.9
(1.0
-
IV
33 ( 8- 81)
38 (11- 90)
6 (3-
13)
2.2
(1.3
-
3.7)
I
14 ( 8- 26)
2 0 ( 8- 27)
6 (1- 1 3 )
1 .5
(0.9
-
2.7)
I1
21 ( 8-
53)
26 ( 1 3 - 6 1 )
5 (1-
8)
1.8
(1.0
111
1 6 0 (33-295)
200 (99-3421
4 0 ( 9 - 86)
2.6
(0.6
I V
138 (13-267)
1 8 0 (16-380)
44 (3-113)
6.2
( 1 . 3 -12.4)
10 ( 6-
I
15)
17 ( 8-
25)
7 (2- 15)
1.1
(0.6 (0.7
2.0) 2.8) 2.8)
Women
2.
All fertile women were studied on the - 7 . day of their menstrual cycle.
Hormone Assays
Serum L H and FSH were measured by a specific double antibody radio-immunoassay ( 1 , 2 ) . The details and modifications have been described elsewhere ( 3 , 4 ) . The human pituitary gonadotropin preparation LER 9 0 7 (NIH) was used as primary standard in both assays. In our assays 1 mg of this standard is equivalent to 200 international units (iu.) of the 2 . IRP-HMG for L H and to 3 3 iu of the 2 . IRP-HMG for FSH. The results are given in terms of the 2 . IRP-HMG which was used
-
4.4) 4.4)
as the laboratory standard. The intraassay coefficient of variation was 6 per cent for both assays and the inter-assaycoefficient was 1 1 and 1 3 per cent for the LH and F S H assay, respectively. Percent of tracer bound to antibody in the absence of standard (zero standard) was 20.1 k 2.7 and 27.4 k 4 . 1 (mean c inter-assay SD) in the F S H and L H assays respectively. Intra-assay SD ( 3 replicates) 0 . 9 8 for FSH and 1 . 1 2 for LH
.
The concentration of LER 90) required to give 2 0 and 80 per cent inhibition was 4 8 . 3 k 11.7 and 8 1 8 k 8 6 ng/ml of incubate for FSH and 8.8 2 0 . 8 and 1 2 1 . 6 C 1 4 . 7 for L H (mean ? inter-assay SD) .
J . Lindholm e t a l . : L u t e i n i z i n g Hormone and Follicle S t i m u l a t i n g Hormone
These data are derived from 1 0 consecutive assays over a period of 6 months. In both assays 0.5 ml of serum could be incorporated into 1 ml of assay incubate without any non-specific effects. Serum testosterone was measured by radio-immunoassay as described by Coyotupa e t a l . ( 5 ) . The inter-assay coeff icient of variation for this assay was 1 2 per cent. Statistics
All differences were analysed statistically with the Mann-Whitney rank sum test. Correlation was calculated as Spearman's coefficient of ,rank correlation. Results
The mean basal LH and FSH concentrations are shown in Table 1 for each of the 8 groups. In no case was the concentration of either LH or FSH statistically different at - 10 min. and at zero time. The mean basal levels of the gonadotropins were not statistically different in the male groups I, I1 and 111, whereas the oldest group had higher concentrations of both LH and FSH. However, only the difference of FSH was statistically significant (p < 0.05). The two older groups of female subjects had significantly higher mean basal concentrations of both LH and FSH (p < 0 . 0 5 ) than had the two younger groups. Group I11 had higher levels of LH, but not of FSH, than had group IV (p < 0.01). There was no significant difference between groups I and 11. In all subjects a prompt increase of LH occurred after injection of LHRH. The rise of FSH was somewhat less pronounced but was still Statistically significant (Table 1 ) . In no group was there a signkficant difference between the 30 and 6 0 min. concentrations of LH and FSH. However, in younger subjects LH tended to be slightly higher at 30 min. than at 6 0 min. Otherwise the maximum response occurred at 6 0 min. The response of LH and FSH did not differ significantly in the male groups except that the mean 30 min. concentration of FSH was significantly higher in group IV (p < 0 . 0 5 ) . Female subjects in groups I and I1 responded identically to LHRH, whereas there was a much higher mean 30 min. LH concentration in postmenopausal women, the peak value being significantly (p < 0 . 0 1 ) higher in group 111. The mean A LH (30 min. - 0 min. concentration) was
25 1
also larger in group 111 than in any other group. Older female subjects (groups 111 and IV) had higher FSH concentration at 3 0 min. than younger subjects (p < 0 . 0 1 ) . There was, however, no significant difference between groups I11 and IV. In postmenopausal women there was a very marked between-subject variation of both LH and FSH. The FSH/LH ratio at 0 min. is seen in Table 1. In the male subjects this ratio increased with increasing age because the FSHs level rose relatively more than did the LH levels. The same was seen in the female subjects in whom the FSH/LH ratio changed even more markedly with increasing.age. There was a significant correlation between the FSH/ LH ratio and age, in male subjects R = 0.58 (p < 0.01) and in female subjects R = 0 . 6 4 (p < 0.001). The correlation between basal LH and peak LH (expressed as A LH) in male subjects aged 23 67 years was significant, R = 0 . 7 0 (Fig. 1 A). It should, however, be noted that if the 4 upper points are arbitrarily excluded this correlation is no longer statistical significant, R now = 0.20. There was no correlation between basal and peak LH in premenopausal women (Fig. 1 B) and no correlation was observed between basal and peak FSH in any group. In 1 6 male subjects chosen at random serum testosterone was measured during the LHRH test. There was no measurable change within 6 0 min. after injection of LHRH in any of these subjects. The relationship between age, basal LH and FSH and testosterone was studied in young and old men. The results in the 1 3 youngest subjects were arbitrarily chosen. As the number of very old men studied was considered too small 5 additonal healthy old men had their FSH, LH and testosterone measured. There was no correlation between peak LH values and serum testosterone in either group. There was, however, a very close correlation between basal LH and testosterone in the young men ( R = 0.84, p < 0 . 0 1 ) and a negative correlation could be found in the old men ( R = - 0 . 7 5 , p < 0.01) (Fig. 1 C and 1 D). A similar although less marked, negative correlation between basal FSH and testosterone was found in the elderly men ( R = - 0.65, p < d.051, but not in the younger men (R = 0 . 5 1 , p < 0.05). The mean serum concentration of testosterone in the 1 3 younger men was 6.6 pg/litre (range 3 . 8 - 1 1 . 7 ) and in the 1 3 older men it was 3.3 ug/litre (range 0..6 - 6 . 5 ) . This difference is significant (p < 0 . 0 1 ) .
-
J. Lindholm et al.: Luteinizing Hormone and Follicle Stimulating Hormone
252
4
75 ..
iu/litre
.*
d L H iu/litre
50 '-
.
..
. . . . 1.
..
0
4
t
'
25-
0 0
0
10
20 Basal L H iu/litre
Fig. 1A. Relationship between basal and peak serum LH (ALH) i n men
I 20
.*
0
.
.. . 20 Basal L H i u/litre
10
Fig. 1B. Relationship between basal and peak LH (ALH) in premenopausal women
t Basal L H iu/l
'.
. 10 ..
. ... 0
0
5.0
0
20 .-
.
lo '.
10.0S-testosterone pg/I it re
. ..
4 ' " 0
. 5.0
+
10.0 S-testosterone @/litre
Fig. 1C. Relationship between serum basal LH and testosterone in young men (age = 23 - 47 years, N = 13)
Fig. 1D. Relationship between serum LH and testosterone in old men (age = 79 - 91 years, N = 13)
Discussion
ations in the response to LHRH w o u M also be eliminated ( 1 0 ) . The results found in this study are in good agreement with those published by Hashimoto et al. (1 1 ) and by Wide et al. ( 1 2) who found a significant increase of basal FSH with age in male subjects, whereas LH did not change. Nieschlag et al. ( 1 3 ) found an increase of only LH, Isurugi et al. ( 1 4 ) an increase of both hermones and Haug et al. (7) of neither hormone with increasing age. Fertile women in the follicular phase of the menstrual cycle had basal LH and FSH values identical with those found in younger male subjects. Postmenopausal
In these studies a dose of 200 pg LHRH was administered. It has been established that doses of LHRH above 200 ug will produce no nor only a slightly higher response of LH and FSH (6, 7). Fertile women were studied early in the follicular phase of the menstrual cycle when the response to LHRH is at a nadir (8, 9 ) .
All tests were performed early in the morning to exclude the effects of any circadian variation of the gonadotropins in serum. Any diurnal fluctu-
J. Lindholm et a l . : Luteinizing Hormone and Follicle Stimulating Hormone
253
women, however, had much higher levels of LH and in particular of FSH - the mean concentration of both hormones being higher in women aged 50 6 9 years than in very old subjects. Only the difference for LH was statistically different. Wide e t a l . ( 1 2 ) found the same decrement in very old women. Hashimoto e t al. (1 1 ) have reported on the effects of age and sex on the response to LHRH. The results presented here are in good agreement with those given by Hashimoto e t al. Thus we found that both A LH and A FSH were unchanged in men of increasing age whereas mean peak FSH concentration was significantly higher in older men. Mean peak LH did not vary significantly. The present study demonstrates that in women the mean 30 min. LH and FSH values were significantly higher in the postmenopausal groups. Again there was a difference between the age groups 5 0 - 69 and 7 9 91 years, the latter age-group having a distinctly lower mean 3 0 min. and a lower A value of LH but not of FSH. The increase of LH in men has been found to be related to the basal LH concentration and this is similar to the relationship between basal thyrotrophic hormone (TSH) and TSH after iv administration of thyrotrophin releasing hormone (TRH) ( 1 5 ) . In women this correlation does not seem to be present. No relationship between basal and peak FSH was observed in either sex. The present finding that serum testosterone does not rise following injection of LHRH agrees with the observations of Besser et al. (16 ) and of Kley et al. ( 1 7 ) . However, it is contrary to the report by Judd et al. (18) who observed a rise of serum testosterone within 3 0 60 min. after injection of LHRH and by Schwarzstein e t al. ( 1 0 ) who observed a prompt increase of testosterone 8 - 16 min. after administration of LHRH. Mean basal serum testosterone concentration was significantly lower in older than in younger men, which is in agreement with previous reports ( 7 , 13 , 1 9 ,
In old male subjects the negative correlation between LH and testosterone probably reflects a compensatory rise of LH secondary to the age dependent failure of Leydig-cell function.
20).
9.
-
-
As the concentration of LH does not seem to be reduced in old age, it seems most logical to ascribe the age dependent reduction of serum testosterone to a diminished function of the Leydig-cells. There is, however, no agreement on this point and the view that the reduction of testicular function is secondary to a lowered pituitary gonadotropic stimulation is still put forward ( 7 ) . In young men serum testosterone was very closely correlated to basal LH, but there was no correlation between the 3 0 min. LH and basal testosterone concentration.
Acknowledgment. We are indepted to E.P. Bennett,
M.Sci., Hormone Department, Statens Seruminstitut, Copenhagen, for the testosterone determinations.
References 1. Midgley, A.R.: Radioimmunoassay: A method for
human chorionic gonadotropin and human luteinizing hormone. Endocrinology 79, 10 - 1 8 (1966) 2. Midgley, A.R.:
Radioimmunoassay for human follicle stimulating hormone. J. din. Endocr.
-
27, 2 9 5 299 (1967) 3. Pedersen, A.H., Larsen, J.F., Micic, S.,
ROOS, J., Sele, V.: The effect of intravenous synthetic luteinizing hormone - releasing hormone (LEI - RH) in infertile women with anovulation. Acta obstet. gynec. scand. Suppl. 29, 89 - 9 5 (1974) 4. ROOS, J., Micic, S.: Radioimmunological de-
termination of the hypophyseal gonadotropins LH and FSH in human serum. Ugeskr. Lag. 1 3 7 , 111 - 1 1 7 (1975) 5. Coyatupa, J., Parlow, A.F., Abraham, J.E.:
6.
7.
8.
10.
Simultaneous radioimmunoassay of plasma testosterone and dihydrotestosterone. Analyt. Lett. 5 , 329 - 3 4 0 ( 1 9 7 2 ) Rebar, R., Yen, S.S.C., Vandenberg, G., Naftolin, F., Ehara, Y., Engblom, S., Ryan, K.J., Rivier, J., Amoss, M., Guillemin, R.: Gonadotropin responses to synthetic LRF: Doseresponse relationship in men. J. clin. EndoCK. 36, 1 0 - 1 6 ( 1 9 7 3 ) Haug, E., Askvaag, A., Sand, T., Torjesen, P.A.: The gonadotropin response to synthetic gonadotropin-releasing hormone in males in relation to age, dose and basal serum levels of testosterone, oestradiol and gonadotropins. Acta endocr. (Kbh.) 77, 6 2 5 - 635 (1974) Nillius, S.J., Wide, L.: Variation in LH and FSB response to LH-releasing hormone during the menstrual cycle. J. Obstet. Gynaec. Brit. Cwlth. 79, 8 6 5 - 8 7 3 (1972) Yen, S.S.C., Vandenberg, G., Rebar, R., Ehara, Y.: Variation of pituitary responsiveness to synthetic LRF during different phases of the menstrual cycle. J. clin. Endocr. 35, 9 3 1 - 9 3 4 (1972) Schwarzstein, L., de Laborde, N.P., Aparicio, N.J., Turner, D., Mirkin, A., Lhullier, F. R., Rosner, J.M.: Daily variations of FSH, LH and testosterone response to intravenous luteinizing hormone-releasing factor (LRF) in normal men. J. clin. Endocr. 40, 3 1 3 -
317 (1975) 11. Hashimoto, T., Miyai, K., Izumi, K., Kume-
hera, Y.: Gonadotropin response to synthetic LH-RH in normal subjects: correlation
254
J. Lindholm et al.: Luteinizing Hormone and Follicle Stimulating Hormone
between LH and FSH. J. clin. Endocr. 3 7 , 910
-
916 (1974)
12. Wide, L., Nillius, S.J., Gemzell, C., ROOS,
P.: Radioimmunosorbent assay of follicle stimulating hormone and luteinizing hormone in serum and urine from men and women. Acta endocr. (Kbh.), Suppl. 1 7 3 ( 1 9 7 3 ) 13. Nieschlag, E., Kley, H.K., Wiegelmann, W., Solbach, H., Krfiskemper, H.L.: Lebensalter und endokrine Funktion des Testes des erwachsenen Mannes. Dtsch. med. Wschr. 9 8 , 1 2 8 1 - 1284 (1973) 1 4 . Isurugi, K . , Fukutani, K., Takayashu, H.,
Wakabayashi, K., Tamaoki, B.I.: Age-related changes in serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in normal men. J. clin. Endocr. 3 9 , 955 - 957 (1974) 1 5 . Weeke, J.: Thyrotropin response to thyro-
tropin releasing hormone in normal subjects. Europ. J. clin. Invest. 4, 2 9 - 3 2 ( 1 9 7 4 ) 16. Besser, G.M., McNeilly, A . S . , Anderson, D. C., Marshall, J.C., Harsonlis, P., Hall, R., Ormston, B.J., Alexander, L., Collins, W.P.: Hormonal responses to synthetic luteinizing hormone and follicle stimulating hormonereleasing hormone in man. Brit. med. J. 3, 267
-
271 (1972)
1 7 . Kley, H.K., Wiegelmann, W., Nieschlag, E.,
Solbach, H.G., Zimmermann, H., Kriiskemper, H.L.: LH, FSH and testosterone in plasma following LH-RH infusion: a combined test for pituitary and Leydig-cell function. Acta endocr. ( a h . ) 75, 417 - 427 ( 1 9 7 4 ) 1 8 . Judd, H.L., Rebar, R., Vandenberg, G., Yen, S.S.C.: Effect of luteinizing hormone-releasing factor in Leydig-cell function. J. clin. Endocr. 38, 8 - 1 3 ( 1 9 7 4 ) 19. Vermeulen, A., Rubens, R., Verdonck, L.: Testosterone secretion and metabolism in males senescene. J. clin. Endocr. 34, 7 3 0 735 20.
(1972)
Pirke, K.M., Doerr, P.: Age-related changes and interrelationships between plasma testosterone, oestradiol and testosterone-binding globulin in normal adult males. Acta endocr. (Kbh.) 74, 7 9 2
-
8 0 0 (1973)
Dr. J. Lindholm Department of Neurosurgery Division of Neuroendocrinology Rigshospitalet 2092 Copenhagen 0 2 1 0 0 Denmark
6,
249-254
(1976)
Luteinizing Hormone and Follicle Stimulating Hormone and the Response to Luteinizing Hormone Releasing Hormone in Relation to Sex and Age J. Lindholm, 0. Korsgaard, P. Rasmussen, and S . Micic
Department of Neurosurgery, Division Of Neuroendocrinology, Rigshospitalet, University Clinic, Copenhagen and Hormone Department, Statens Seruminstitut, Copenhagen, Denmark Received: July 1 , 1975, and in revised form: February 2 , 1 9 7 6 Abstract
Serum follicle stimulating hormone (FSH) and luteinizing hormone (LH) before and after intravenous injection of luteinizing hormone releasing hormone (LHRH) were studied in 7 1 male and female subjects of various ages. Mean basal FSH and LH levels were not significantly different in the male groups, except that FSH was significantly higher in the very old subjects. Postmenopausal female subjects had much higher concentrations of both hormones than had premenopausal women. LH was higher in female subjects shortly after the menopause than in very old female subjects. The serum concentrations of both FSH and LH 30 min. after intravenous injection of 200 pg LHRH were not different from the 60 min. values. There was no significant difference in the response of LH in the male groups. The peak FSH concentration was higher in the very old male subjects. Postmenopausal women had a much higher peak concentration of both FSH and LH than had younger subjects. The increment of LH, but not of FSH, was larger in female subjects shortly after the menopause than in very old female subjects. In both sexes there was a significant correlation between the basal FSH/LH ratio and age. In younger male subjects there was a close positive correlation between basal LH and serum testosterone, in older male subjects this correlation was negative and significant. Key words
Follicle stimulating hormone, Lutenizing hormone, Pituitary gonadotropins, Luteinizing hormone releasing hormone, Pituitary function, Testicular function.
introduction
This paper presents the results of studies on pituitary luteinizing hormone (LH) and follicle stimulating hormone (FSH) before and after intravenous (iv) injection of a synthetic luteinizing hormone releasing hormone (LHFW) in men and women in various age groups. The relationship between serum concentrations of gonadotropins and testosterone in men was also studied. Materials and Methods
Subjects
7 1 subjects were studied. All were apparently in good health with no signs of
endocrine, renal or hepatic disease. Male and female subjects were each divided into four groups according to age (Table 1 ) . Most subjects were hospital staff (nurses, secretaries, physicians) except those above 67 years old, who were patients recovering from minor coronary infarctions. All women over 50 years old were postmenopausal. Procedures
All tests were begun between 0800 and 0830 h. A catheter was inserted into an arm vein and samples were drawn at - 10, 0, 20, 30 and 60 minutes after a rapid iv injection of 200 p g LHRH Hoechst. Blood for testosterone determination was also obtained at zero time.
J. Lindholm et al.: L u t e i n i z i n g Hormone and F o l l i c l e S t i m u l a t i n g H o r m o n e
250
Table 1. Serum LH and F S H ( i u / l i t r e ) r e s p o n s e t o LHRH ( 2 0 0 pg i v ) i n
normal men and women LH 0 min.
mean ( r a n g e )
3 0 min. mean ( r a n g e )
A LH mean ( r a n g e )
Men
age (years)
N
I
20-29
8
9 ( 7-17)
50 ( 2 4 - 90)
4 0 (15-
I1
30-49
9
10 ( 4-21)
46 ( 1 6 - 96)
36 (12- 7 5 )
I11
50-67
8
9 ( 4-20)
46 (27- 95)
38 (22- 73)
IV
79-91
8
1 8 ( 3-49)
43
(15- 74)
24 (12- 47)
I
20-29
12
10 ( 4-18)
35 (18- 52)
24 (13- 4 2 )
I1
30-49
10
11 ( 8-17)
37
26 (12-
111
50-69
8
65 (26-98)
197 (76-464)
132 (21-366)
IV
79-91
8
26 ( 4-46)
68 (12-105)
42 ( 8- 59)
~~
~
~~~
~
64)
Women (20-
55)
38)
FSH
Men
0 min. mean ( r a n g e )
3 0 min. mean ( r a n g e )
A FSH mean ( r a n g e )
Basal
FSH
(range)
LH
I1
15 ( 6- 36)
20 ( 7- 46)
5 (1-
12)
1.5
1x1
1 3 ( 5-
32)
1 9 ( 6- 40)
6 (1- 15)
1.9
(1.0
-
IV
33 ( 8- 81)
38 (11- 90)
6 (3-
13)
2.2
(1.3
-
3.7)
I
14 ( 8- 26)
2 0 ( 8- 27)
6 (1- 1 3 )
1 .5
(0.9
-
2.7)
I1
21 ( 8-
53)
26 ( 1 3 - 6 1 )
5 (1-
8)
1.8
(1.0
111
1 6 0 (33-295)
200 (99-3421
4 0 ( 9 - 86)
2.6
(0.6
I V
138 (13-267)
1 8 0 (16-380)
44 (3-113)
6.2
( 1 . 3 -12.4)
10 ( 6-
I
15)
17 ( 8-
25)
7 (2- 15)
1.1
(0.6 (0.7
2.0) 2.8) 2.8)
Women
2.
All fertile women were studied on the - 7 . day of their menstrual cycle.
Hormone Assays
Serum L H and FSH were measured by a specific double antibody radio-immunoassay ( 1 , 2 ) . The details and modifications have been described elsewhere ( 3 , 4 ) . The human pituitary gonadotropin preparation LER 9 0 7 (NIH) was used as primary standard in both assays. In our assays 1 mg of this standard is equivalent to 200 international units (iu.) of the 2 . IRP-HMG for L H and to 3 3 iu of the 2 . IRP-HMG for FSH. The results are given in terms of the 2 . IRP-HMG which was used
-
4.4) 4.4)
as the laboratory standard. The intraassay coefficient of variation was 6 per cent for both assays and the inter-assaycoefficient was 1 1 and 1 3 per cent for the LH and F S H assay, respectively. Percent of tracer bound to antibody in the absence of standard (zero standard) was 20.1 k 2.7 and 27.4 k 4 . 1 (mean c inter-assay SD) in the F S H and L H assays respectively. Intra-assay SD ( 3 replicates) 0 . 9 8 for FSH and 1 . 1 2 for LH
.
The concentration of LER 90) required to give 2 0 and 80 per cent inhibition was 4 8 . 3 k 11.7 and 8 1 8 k 8 6 ng/ml of incubate for FSH and 8.8 2 0 . 8 and 1 2 1 . 6 C 1 4 . 7 for L H (mean ? inter-assay SD) .
J . Lindholm e t a l . : L u t e i n i z i n g Hormone and Follicle S t i m u l a t i n g Hormone
These data are derived from 1 0 consecutive assays over a period of 6 months. In both assays 0.5 ml of serum could be incorporated into 1 ml of assay incubate without any non-specific effects. Serum testosterone was measured by radio-immunoassay as described by Coyotupa e t a l . ( 5 ) . The inter-assay coeff icient of variation for this assay was 1 2 per cent. Statistics
All differences were analysed statistically with the Mann-Whitney rank sum test. Correlation was calculated as Spearman's coefficient of ,rank correlation. Results
The mean basal LH and FSH concentrations are shown in Table 1 for each of the 8 groups. In no case was the concentration of either LH or FSH statistically different at - 10 min. and at zero time. The mean basal levels of the gonadotropins were not statistically different in the male groups I, I1 and 111, whereas the oldest group had higher concentrations of both LH and FSH. However, only the difference of FSH was statistically significant (p < 0.05). The two older groups of female subjects had significantly higher mean basal concentrations of both LH and FSH (p < 0 . 0 5 ) than had the two younger groups. Group I11 had higher levels of LH, but not of FSH, than had group IV (p < 0.01). There was no significant difference between groups I and 11. In all subjects a prompt increase of LH occurred after injection of LHRH. The rise of FSH was somewhat less pronounced but was still Statistically significant (Table 1 ) . In no group was there a signkficant difference between the 30 and 6 0 min. concentrations of LH and FSH. However, in younger subjects LH tended to be slightly higher at 30 min. than at 6 0 min. Otherwise the maximum response occurred at 6 0 min. The response of LH and FSH did not differ significantly in the male groups except that the mean 30 min. concentration of FSH was significantly higher in group IV (p < 0 . 0 5 ) . Female subjects in groups I and I1 responded identically to LHRH, whereas there was a much higher mean 30 min. LH concentration in postmenopausal women, the peak value being significantly (p < 0 . 0 1 ) higher in group 111. The mean A LH (30 min. - 0 min. concentration) was
25 1
also larger in group 111 than in any other group. Older female subjects (groups 111 and IV) had higher FSH concentration at 3 0 min. than younger subjects (p < 0 . 0 1 ) . There was, however, no significant difference between groups I11 and IV. In postmenopausal women there was a very marked between-subject variation of both LH and FSH. The FSH/LH ratio at 0 min. is seen in Table 1. In the male subjects this ratio increased with increasing age because the FSHs level rose relatively more than did the LH levels. The same was seen in the female subjects in whom the FSH/LH ratio changed even more markedly with increasing.age. There was a significant correlation between the FSH/ LH ratio and age, in male subjects R = 0.58 (p < 0.01) and in female subjects R = 0 . 6 4 (p < 0.001). The correlation between basal LH and peak LH (expressed as A LH) in male subjects aged 23 67 years was significant, R = 0 . 7 0 (Fig. 1 A). It should, however, be noted that if the 4 upper points are arbitrarily excluded this correlation is no longer statistical significant, R now = 0.20. There was no correlation between basal and peak LH in premenopausal women (Fig. 1 B) and no correlation was observed between basal and peak FSH in any group. In 1 6 male subjects chosen at random serum testosterone was measured during the LHRH test. There was no measurable change within 6 0 min. after injection of LHRH in any of these subjects. The relationship between age, basal LH and FSH and testosterone was studied in young and old men. The results in the 1 3 youngest subjects were arbitrarily chosen. As the number of very old men studied was considered too small 5 additonal healthy old men had their FSH, LH and testosterone measured. There was no correlation between peak LH values and serum testosterone in either group. There was, however, a very close correlation between basal LH and testosterone in the young men ( R = 0.84, p < 0 . 0 1 ) and a negative correlation could be found in the old men ( R = - 0 . 7 5 , p < 0.01) (Fig. 1 C and 1 D). A similar although less marked, negative correlation between basal FSH and testosterone was found in the elderly men ( R = - 0.65, p < d.051, but not in the younger men (R = 0 . 5 1 , p < 0.05). The mean serum concentration of testosterone in the 1 3 younger men was 6.6 pg/litre (range 3 . 8 - 1 1 . 7 ) and in the 1 3 older men it was 3.3 ug/litre (range 0..6 - 6 . 5 ) . This difference is significant (p < 0 . 0 1 ) .
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J. Lindholm et al.: Luteinizing Hormone and Follicle Stimulating Hormone
252
4
75 ..
iu/litre
.*
d L H iu/litre
50 '-
.
..
. . . . 1.
..
0
4
t
'
25-
0 0
0
10
20 Basal L H iu/litre
Fig. 1A. Relationship between basal and peak serum LH (ALH) i n men
I 20
.*
0
.
.. . 20 Basal L H i u/litre
10
Fig. 1B. Relationship between basal and peak LH (ALH) in premenopausal women
t Basal L H iu/l
'.
. 10 ..
. ... 0
0
5.0
0
20 .-
.
lo '.
10.0S-testosterone pg/I it re
. ..
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. 5.0
+
10.0 S-testosterone @/litre
Fig. 1C. Relationship between serum basal LH and testosterone in young men (age = 23 - 47 years, N = 13)
Fig. 1D. Relationship between serum LH and testosterone in old men (age = 79 - 91 years, N = 13)
Discussion
ations in the response to LHRH w o u M also be eliminated ( 1 0 ) . The results found in this study are in good agreement with those published by Hashimoto et al. (1 1 ) and by Wide et al. ( 1 2) who found a significant increase of basal FSH with age in male subjects, whereas LH did not change. Nieschlag et al. ( 1 3 ) found an increase of only LH, Isurugi et al. ( 1 4 ) an increase of both hermones and Haug et al. (7) of neither hormone with increasing age. Fertile women in the follicular phase of the menstrual cycle had basal LH and FSH values identical with those found in younger male subjects. Postmenopausal
In these studies a dose of 200 pg LHRH was administered. It has been established that doses of LHRH above 200 ug will produce no nor only a slightly higher response of LH and FSH (6, 7). Fertile women were studied early in the follicular phase of the menstrual cycle when the response to LHRH is at a nadir (8, 9 ) .
All tests were performed early in the morning to exclude the effects of any circadian variation of the gonadotropins in serum. Any diurnal fluctu-
J. Lindholm et a l . : Luteinizing Hormone and Follicle Stimulating Hormone
253
women, however, had much higher levels of LH and in particular of FSH - the mean concentration of both hormones being higher in women aged 50 6 9 years than in very old subjects. Only the difference for LH was statistically different. Wide e t a l . ( 1 2 ) found the same decrement in very old women. Hashimoto e t al. (1 1 ) have reported on the effects of age and sex on the response to LHRH. The results presented here are in good agreement with those given by Hashimoto e t al. Thus we found that both A LH and A FSH were unchanged in men of increasing age whereas mean peak FSH concentration was significantly higher in older men. Mean peak LH did not vary significantly. The present study demonstrates that in women the mean 30 min. LH and FSH values were significantly higher in the postmenopausal groups. Again there was a difference between the age groups 5 0 - 69 and 7 9 91 years, the latter age-group having a distinctly lower mean 3 0 min. and a lower A value of LH but not of FSH. The increase of LH in men has been found to be related to the basal LH concentration and this is similar to the relationship between basal thyrotrophic hormone (TSH) and TSH after iv administration of thyrotrophin releasing hormone (TRH) ( 1 5 ) . In women this correlation does not seem to be present. No relationship between basal and peak FSH was observed in either sex. The present finding that serum testosterone does not rise following injection of LHRH agrees with the observations of Besser et al. (16 ) and of Kley et al. ( 1 7 ) . However, it is contrary to the report by Judd et al. (18) who observed a rise of serum testosterone within 3 0 60 min. after injection of LHRH and by Schwarzstein e t al. ( 1 0 ) who observed a prompt increase of testosterone 8 - 16 min. after administration of LHRH. Mean basal serum testosterone concentration was significantly lower in older than in younger men, which is in agreement with previous reports ( 7 , 13 , 1 9 ,
In old male subjects the negative correlation between LH and testosterone probably reflects a compensatory rise of LH secondary to the age dependent failure of Leydig-cell function.
20).
9.
-
-
As the concentration of LH does not seem to be reduced in old age, it seems most logical to ascribe the age dependent reduction of serum testosterone to a diminished function of the Leydig-cells. There is, however, no agreement on this point and the view that the reduction of testicular function is secondary to a lowered pituitary gonadotropic stimulation is still put forward ( 7 ) . In young men serum testosterone was very closely correlated to basal LH, but there was no correlation between the 3 0 min. LH and basal testosterone concentration.
Acknowledgment. We are indepted to E.P. Bennett,
M.Sci., Hormone Department, Statens Seruminstitut, Copenhagen, for the testosterone determinations.
References 1. Midgley, A.R.: Radioimmunoassay: A method for
human chorionic gonadotropin and human luteinizing hormone. Endocrinology 79, 10 - 1 8 (1966) 2. Midgley, A.R.:
Radioimmunoassay for human follicle stimulating hormone. J. din. Endocr.
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27, 2 9 5 299 (1967) 3. Pedersen, A.H., Larsen, J.F., Micic, S.,
ROOS, J., Sele, V.: The effect of intravenous synthetic luteinizing hormone - releasing hormone (LEI - RH) in infertile women with anovulation. Acta obstet. gynec. scand. Suppl. 29, 89 - 9 5 (1974) 4. ROOS, J., Micic, S.: Radioimmunological de-
termination of the hypophyseal gonadotropins LH and FSH in human serum. Ugeskr. Lag. 1 3 7 , 111 - 1 1 7 (1975) 5. Coyatupa, J., Parlow, A.F., Abraham, J.E.:
6.
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Simultaneous radioimmunoassay of plasma testosterone and dihydrotestosterone. Analyt. Lett. 5 , 329 - 3 4 0 ( 1 9 7 2 ) Rebar, R., Yen, S.S.C., Vandenberg, G., Naftolin, F., Ehara, Y., Engblom, S., Ryan, K.J., Rivier, J., Amoss, M., Guillemin, R.: Gonadotropin responses to synthetic LRF: Doseresponse relationship in men. J. clin. EndoCK. 36, 1 0 - 1 6 ( 1 9 7 3 ) Haug, E., Askvaag, A., Sand, T., Torjesen, P.A.: The gonadotropin response to synthetic gonadotropin-releasing hormone in males in relation to age, dose and basal serum levels of testosterone, oestradiol and gonadotropins. Acta endocr. (Kbh.) 77, 6 2 5 - 635 (1974) Nillius, S.J., Wide, L.: Variation in LH and FSB response to LH-releasing hormone during the menstrual cycle. J. Obstet. Gynaec. Brit. Cwlth. 79, 8 6 5 - 8 7 3 (1972) Yen, S.S.C., Vandenberg, G., Rebar, R., Ehara, Y.: Variation of pituitary responsiveness to synthetic LRF during different phases of the menstrual cycle. J. clin. Endocr. 35, 9 3 1 - 9 3 4 (1972) Schwarzstein, L., de Laborde, N.P., Aparicio, N.J., Turner, D., Mirkin, A., Lhullier, F. R., Rosner, J.M.: Daily variations of FSH, LH and testosterone response to intravenous luteinizing hormone-releasing factor (LRF) in normal men. J. clin. Endocr. 40, 3 1 3 -
317 (1975) 11. Hashimoto, T., Miyai, K., Izumi, K., Kume-
hera, Y.: Gonadotropin response to synthetic LH-RH in normal subjects: correlation
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12. Wide, L., Nillius, S.J., Gemzell, C., ROOS,
P.: Radioimmunosorbent assay of follicle stimulating hormone and luteinizing hormone in serum and urine from men and women. Acta endocr. (Kbh.), Suppl. 1 7 3 ( 1 9 7 3 ) 13. Nieschlag, E., Kley, H.K., Wiegelmann, W., Solbach, H., Krfiskemper, H.L.: Lebensalter und endokrine Funktion des Testes des erwachsenen Mannes. Dtsch. med. Wschr. 9 8 , 1 2 8 1 - 1284 (1973) 1 4 . Isurugi, K . , Fukutani, K., Takayashu, H.,
Wakabayashi, K., Tamaoki, B.I.: Age-related changes in serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels in normal men. J. clin. Endocr. 3 9 , 955 - 957 (1974) 1 5 . Weeke, J.: Thyrotropin response to thyro-
tropin releasing hormone in normal subjects. Europ. J. clin. Invest. 4, 2 9 - 3 2 ( 1 9 7 4 ) 16. Besser, G.M., McNeilly, A . S . , Anderson, D. C., Marshall, J.C., Harsonlis, P., Hall, R., Ormston, B.J., Alexander, L., Collins, W.P.: Hormonal responses to synthetic luteinizing hormone and follicle stimulating hormonereleasing hormone in man. Brit. med. J. 3, 267
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1 7 . Kley, H.K., Wiegelmann, W., Nieschlag, E.,
Solbach, H.G., Zimmermann, H., Kriiskemper, H.L.: LH, FSH and testosterone in plasma following LH-RH infusion: a combined test for pituitary and Leydig-cell function. Acta endocr. ( a h . ) 75, 417 - 427 ( 1 9 7 4 ) 1 8 . Judd, H.L., Rebar, R., Vandenberg, G., Yen, S.S.C.: Effect of luteinizing hormone-releasing factor in Leydig-cell function. J. clin. Endocr. 38, 8 - 1 3 ( 1 9 7 4 ) 19. Vermeulen, A., Rubens, R., Verdonck, L.: Testosterone secretion and metabolism in males senescene. J. clin. Endocr. 34, 7 3 0 735 20.
(1972)
Pirke, K.M., Doerr, P.: Age-related changes and interrelationships between plasma testosterone, oestradiol and testosterone-binding globulin in normal adult males. Acta endocr. (Kbh.) 74, 7 9 2
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8 0 0 (1973)
Dr. J. Lindholm Department of Neurosurgery Division of Neuroendocrinology Rigshospitalet 2092 Copenhagen 0 2 1 0 0 Denmark
