J. Endocrinol. Invest. 13: 127-132, 1990
Effect of pulsatile luteinizing hormone - releasing hormone administration on pituitary - gonadal function in elderly man M. Giusti*, G. Marini**, L. Traverso*, P. Cavagnaro*, L. Granziera***, and G. Giordano*. *I.S.M.I. Cattedra di Endocrinologia, Universita di Genova, **Casa di Riposo di Genova, and ***Ricerca e Sviluppo, Medical Systems, 16100 Genova, Italy administration (p < 0,01). LH pulses were identified in 5 out of 8 subjects on day O. On day 14, all the exogenous LHRH pulses were followed by significant LH bursts. There was not a significant decrease in the pituitary LH responsiveness to LHRH test from day 0 to day 14. Our study seems to indicate that pituitary - gonadal unit in normal elderly men can be modulated by pulsatile administration of LHRH. A pulse frequency of LHRH which is probably similar to the physiological one, could induce a slight increase in T levels via qualitative changes in LH activity. We can assume that clinical changes in gondadal activity might also be connected to some disturbances in endogenous LHRH pulsar.
ABSTRACT. The effect of short-term pulsatile LHRH administration was studied in 8 healthy subjects ranging from 60 to 81 yr to see if the decrease of pituitary gonadal function could be in part due to changes in the discharge of LHRH from the hypothalamus. Gonadotropin and testosterone (T) secretion was evaluated two weeks before and during LHRH (122-160 ng/kg bw every 120 min sc) infusion. In addition, a bolus dosage of LHRH (50 IJ. iv) was given both at the beginning and at the end of pulsatile LHRH administration in order to test gonadotrophs sensitivity. A significant inGrease in gonadotropin levels from day 0 to day 4 was found, and was followed by a subsequent decrease from day 7 to day 14. A slight significant increase in T levels was observed during LHRH
INTRODUCTION There are aging changes in the hypothalamic pituitary - gonadal axis in many mammalian species. The aging male rat exhibits decreased fertility and sexual activity (1 , 2) which seem to partially attribute to the decrease of the functional activity of LHRH neurons and/ or neurotransmitters involved in the LHRH control (3). In fact, age-related changes were noted in synaptic organization of LHRH neurons (3) and, even if the number or size of LHRH neurons did not change with aging (3), several studies showed that the LHRH content of the median eminentia was found to be reduced by radioimmunoassay (4) and immunochemistry (5) in aged male rats. A further suggestion can be raised from experimental studies, with regards to central mechanisms of pituitary-gonadal activity in old rats, in which fetal
hypothalamic graft increases LH and testosterone (T) release and restores neuroendocrine reproductive function in impotent aged male rats (6). Several data indicate that pituitary - gonadal function in man is also modified by aging. A decrease in testicular responsiveness to human chorionic gonadotropin (7) and a drop in the Leydig cell number (8) seems to underline a primary gonadal damage with aging. On the other hand, age-related changes at the hypothalamic - pituitary level have been found in man and they could suggest an impairment in neuroendocrine control of LHRH LH unit. In fact, LH as well as FSH and a"subunit response after LHRH is generally blunted or delayed (9-11). The opiate receptor antagonist naloxone doses not increase LH release in men over 60 yr with normal T levels (12). Whereas clomiphene citrate, which probably acts via the hypothalamic target site (13), does not seem to restore LH (7) and LH-related gonadal function (14). Spontaneous pulsatile LH secretion has been extensively studied in aging subjects (14-18) and sev-
Key-wordS: LHRH, pulsatile administration, LH, testosterone. elderly man. Correspondence: Massimo Giusti. M.D., I.S.M.I., Cattedra di Endocrinologia. Viale Benedetto XV, 6, 1-16100 Genoa. Italy.
Received April 14, 1989; accepted November 6, 1989.
127
M. Giusti, G. Marini, L. Traverso, et al.
eral studies reported a qecrease, both in frequency (15, 16) and amplitude (18) of the LH pulses in elderly men compared to that in young men. Together with an increase in LH response to sex hormone feed-back (16, 17), this pattern may contribute to important changes in hypothalamic - pituitary function. The pulsatile nature of LHRH-LH release is a physiological property due both to the activity of the endogenous pulse generator and to the responsiveness of the gonadotrophs (19). In addition, pulsatile, but not continuous exogenous LHRH administration, can restore normal gonadal function in hypogonadotropin individuals (20, 31). The aim of the study was to evaluate the effect of a two-week exogenous pulsatile LHRH administration in a group of normal elderly men. Our working hypothesis was to verify if a physiological like pattern of LHRH short-term, given by a portable pump, could improve pituitary - gonadal function with aging.
at weekly intervals and the injection site was changed at least every 4 days. Dosages of LHRH varied from 122-160 ng/kg bw per pulse. The pump was programmed to give a pulse which lasted one minute every 120 min. During the tests performed on day 7 and 14, each pulse of exogenous LHRH was identified and specimens were collected at the actual moment of drug delivery. Fifty /lg of LHRH (Biodata, Milan, Italy) were given iv in bolus in all but one subject at the end of the test on day 0 and 14. In case 3, the latter LHRH test was performed on day 7. Gonadotropin response to LHRH test was evaluated only 60 min after peptide administration. The protocol was chosen in order to slightly modify the subjects habitual way of life and was approved by the Ethical Committees of the Geriatric Institute. Hormone assays All serum samples from an individual subject were run in duplicate in the same assay. LH and FSH were measured by double antibody radioimmunoassay using reagents provided by Biodata. Gonadotropin values were expressed in mlU/ml as equivalent of the 2nd - IRP - hMG. The sensitivity of the method was 1 mlU/ml for both hormones. The intraassay coefficient of variation (CV) was 5.3% and 6.8% at level of 8.4 mlU/ml for LH and 9.7 mlU/ml for FSH, respectively. Serum T was measured without extraction using reagents obtained from DPC (Los Angeles, CA). Sensitivity was 15 ng/ dl. The intraassay CV was 4.0% at a level of 730 ng / dl. In our laboratory, normal levels for adult male subjects were found to be 5-20 mlU/ml and 350-1000 ng/ dl for both gonadotropins and T, respectively.
MATERIALS AND METHODS Subjects Eight elderly subjects ranging from 60 to 81 yr were enrolled in this study after giving their consent. They were housed in the Social Service Section of the Geriatric Institute in Genoa. A complete history and physical examination was performed on all subjects. They were non-smokers and their intake of alcohol was less than 40 g/day. According to Mini Mental State (MMS) and Ischemic Score (IS) of Haschinski, cognitive brain function was evaluated. All subjects had a score greater than 24 for MMS and less than 3 for IS. Body mass index (kg/m2) ranged from 21.5 to 26.0. Apart from stabilized cardiovascular diseases no other acute or chronic illnesses were found. The subjects were not obliged to stay in bed and a normal life was conducted. A balanced diet was given at 11 :30 and 19:30 h in the institute refectory.
Oata analysis All values are expressed as mean ± SE. Subsequent individual LH series were analyzed for significant LH pulses using the Santen and Bardin method (22), adjusted to a threshold requirement of 4 times individual intraassay CV according to Veldhuis (23). The LH pulse was identified as increment from nadir (mean of 2 consecutive samples) to peak (following sample) of at least 4 times intraassay CV. In the case of the first 2 samples of an experiment the basal value was taken as nadir, if necessary. Moreover, the reputed peak had to be followed by 2 consecutive decreasing values and the amplitude had to be greater than the sensitivity of the assay method. The amplitude of a pulse was defined as the difference between the peak and the preceding
Protocol LH, FSH and T secretion was evaluated in rest condition on day 0, 1, 4, 7, 10 and 14 of the study. Every 15 min, multiple blood samples, were collected from 08:00 to 11 :00 h on day 0, 7 and 14, while morning basal samples were drawn on day 1, 4 and 10. LHRH (Lutrelef, Ferring, Kiel, Germany) was sc administered in a pulsatile manner by portable pump (Zyklomat VAR Franconia, Ferring) from 12:00 h of day 0 to 11 :00 h of day 14. Pumps were reloaded
128
Aging and LHRH pulsatile administration
nadir. During the pulsatile LHRH administration, individual LH pulses each pump activation were ananalyzed as previously reported (24). Nadir was defined as the mean value of 2 consecutive samples obtained before exogenous LHRH pulse. Peak was defined as the highest value found after LHRH pulse. The amplitude was measured by subtracting the nadir from the peak value. LH increase after LHRH pulse was considered significant if its amplitude was gre'ater than 4 times the individual intraassay CV. Results were analyzed using two-tailed paired t test on log-transformed data. P values less than 0.05 were considered statistically significant.
case DAY 0
1 600
10 400 5
200
0
0
E
""'=>-
~
20
600 g'
E 15
400 w
z
200
10
I
ffi
f-
(J)
...J
0
RESULTS
0
f-
(J)
w
20
All but one patient tolerated the mini-pump and the LHRH pulsatile infusion. Case 3 showed a bad compliance to the delivery system and stopped the pulsatile LHRH infusion on day 7 after a basal blood sample. Mean LH, FSH and T levels were 21.0 ± 6.3 mIU/ml, 19.8 ± 9.5 mlU/ml and 318 ± 22 ng/dl before LHRH administration (day 0), respectively. Significant LH pulses were identified in 5 out of 8 subjects. In these 5 subjects pulse amplitude of LH ranging from 2.5 to 5.8 mlU/ml (Table 1, Fig. 1). During short-term pulsatile LHRH administration a significant increase in LH and FSH levels from day 0 to day 4 were found, followed by a subsequent decrease from day 7 to day 14 (Fig. 2). A slight but significant increase in T levels was also observed (Fig. 2). Higher levels of T were found in 71 % of subjects on day 1 , 4, 10 and 14 and
0
f-
600 15
400 200
10
o
o 30
60
90
120
150
180 min
Fig. 1 - LH (.........) and T (0-0) behavior at the day 0, 7 and 14 in case 1. Arrows represent both the significant LH pulses on day 0 and the moment of LHRH infusion day 7 and 14.
in 87% of subjects on day 7 (Fig. 2). Mean T values during pulsatile LHRH administration were 391 ± 50 ng/ dl (p < 0.Q1 from day 0). When the pump was switched-on significant LH LHRH - induced bursts were observed following 17 day 7 out of 20 LHRH pulses (Table 1, Fig. 2).
On
Table 1 - Number and amplitude (mIU/ml) of spontaneous LH
pulses or LH pulses induced by pulsatile LHRH administration. See also methods for the definition of LH pulse. Case (no.)
Day 14 Day 7 Day 0 on LHRH on LHRH before LHRH Mean Mean Pulses1 Mean Pulses 1 Pulses amplitude amplitude amplitude
1
4.6
1 (1)
6.0
2 (2)
6.1
0(1)
n.e. 2
1 (1)
14.1
2
1
5.8
3
2
4.1
4
2.5
1 (1)
5.2
1 (1)
1.5
5
0
n.e. 2
0(2)
n.e. 2
1 (1)
3.7
6
0
n.e. 2
2 (2)
11.6
2 (2)
6.3
7 8
0
n.e. 2
3.2
2 (2)
4.2
2 (2)
3.2
1 (1)
10.3
1 (1)
8.7
600 _
70
"0
~60
500
g;
E 50
400
il340 u.
g....
300-:;;
::J
Q)
Q)
....0
: 30
200
I20 --J
I
100 10 0
o1
4
7
10
0 14 days
(/J
Q)
f-
r
Fig. 2 - Mean LH, FSH and T levels before and during pulsatile LHRH administration in normal elderly subjects (A p < 0.05 and AA p < 0.01 vs day 0).
, In brackets number of LHRH pulses during the sampling period. 2 n.e. = not evaluable.
129
M. Giusti, G. Marini, L. Traverso, et al.
the inabilty of gonadotrophic cell responsiveness to LHRH itself. Our study shows that in healthy elderly men shortterm pulsatile LHRH infusion discloses a rapid increase in gonadotropin secretion which is coupled with T enhancement as was observed in patients with idiopathic hypogonadotropic hypogonadism using identical protocols of peptide administration (20, 21). This rapid response of pituitary to exogenous LHRH given in a pulsatile way would tend to minimize the possibility that the impairment of pituitary gondadal secretion in elderly men can be due to a reduced sensitivity of gonadotropin-secreting cells. Aging-related biophysical changes in the endogenous LH molecule have been reported. in old rats (30). In elderly men update findings seem to be scanty and controversial, after having discovered that the biological LH activity was both unchanged (14) and reduced (28, 29). Moreover, it is well known that the rhythm in edogenous LHRH secretion is an important modulator of LH molecule discharge which is enriched in biological activity (31 ). We can hypothesize that an impaired secretory reserve in bio!ogical LH in aging can be linked to a disturbance of LHRH pulsar. We have only preliminary data regards to the increase of biological to immunological LH ratio during pulsatile LHRH administration in elderly humans (32), but present work indirectly strengthens this hypotesis. In fact, the increase in T levels observed in our subjects even after the quantitative decline in LH secretion could be due to a preferential release inLH with enhanced bioactivity during pulsatile LHRH administration. Besides, present data seem to conflict with the opinion of a decline in T levels with aging, as an indication of decreasing sensitivity of gonadal target (14, 15). In physiological conditions there is no uniformity of LH pulse properties which are likely to express the lack of regularity of the oscillator controlling LHRH release (23) and our choice of a fixed LHRH regimen could be considered arbitrary. However, injection of LHRH pulses every 2 h resulted for 3-4 days in increased plasma LH concentration in adult (33) or elderly (present study) normal men. This was presumably due to increased pituitary exposure to LHRH from the additive effects of exogenous LHRH pulses and endogenous LHRH secretion (33). LH pulses were identified following the majority of LHRH pulses recorded (100% of LHRH pulses recorded on day 14) and LH responsiveness to testing dosage of LHRH was found to be slightly (not sig-
after LH LHRH - induced significant pulses were found in 5 out of 7 subjects and in all subjects studied on day 14 (Table 1). Nadir to peak amplitude ranged from 4.2 to 11.6 mlU/ml and from 1.5 to 14.1 mIU/ml,7 and 14 days after LHRH administration, respectively. Pituitary LH responsiveness to LHRH given in a bolus fashion decreased in non-significant way from the former (69.7 ± 21.7 mIU/ml) and the latter (39.6 ± 7.2 mIU/ml) study. DISCUSSION Unlike that observed in vivo and in vitro experimented in rats (1-6), the effect of aging on hypothalamus - pituitary - gonadal axis in man still seems to be unclear. For instance, T levels have often been (14, 15, 17, 25) but not always (12, 26, 27) found to be reduced and it has not been established whether or not central (10, 12, 15, 17, 18,28) and/ or peripheral (7, 8, 14, 15, 18, 20, 25) mechanisms are involved in this phenomenon. Consequently, some variations found during aging progress are thought to be connected with the experimental model employed and the concurrence of unconsidered chronic illnesses (29). In a group of selected healthy elderly men we performed the present study in order to rule out some factors which are able to modify results such as obesity, heavy alcohol intake, concomitant drugs and smoking. On the other hand, we chose a protocol which has not been excessively demanding on the subjects in order not to change their normal way of life. On average, a slight increase in basal gonadotropin levels and a more evident reduction of T levels have been observed in our subjects. Moreover, the levels of gonadotropins were not dramatically increased in spite of the decrease in T levels. This phenomenon gives the impression that a central impairment due to a greater feed-back responsiveness of the hypothalamus pituitary complex takes place in the elderly men (15, 17). It could also suggest that in normal elderly men a complex central alteration of central nervous system - LHRH pulse generator is present with aging. Besides, available data do not clearly show if the decrease in amplitude or frequency of LH bursts found in spontaneous conditions (15, 16, 18) and after dihydrotestosterone infusion (17) could be due both to anomalous LHRH discharge in portal flow and to
130
Aging and LHRH pulsatile administration
nificantly) reduced in our subjects. If a down regulation phenomenon seems to be unlikely, we cannot exclude a certain degree of reduction of LH pool ready - releasable and/ or an increase in the negative feed-back of T at pituitary level. In conclusion, according to some other authors (15, 17, 18,28) and to experimental data in rats (36) our study indicates that functional capacity of the central nervous system - LHRH pulse generator becomes impaired with the process of aging. The increase in T levels might be due to a physiological pulse frequency of LHRH inducing qualitative changes in LH activity. Endogenous LHRH pulsatility seems to be a multifactorial phenomenon and several neurotrasmitters and neuropeptides can modulate LHRH discharge, on which the feed-back mechanisms of gonadal hormones operate (19). Altered intracellular androgen processing in the hypothalamus and/or changes in neuromodulator systems (4, 34) which may be connected to disturbances in endogenous LHRH pulsar is still unclear.
7. Rubens R., Dhont M., Vermeulen V. Further stUdies on Leydig cell function in old age. J. Clin. Endocrinol. Metab. 39: 40, 1974. 8. Neaves W.B., Johnson L., Porter J.C., Parker C.R. Jr., Petty C.S. Leydig cell numbers, daily sperm production, and serum gonadotropin levels in aging men. J. Clin. Endocrinol. Metab. 55: 756, 1984. 9. Blackman M.R., Tsitouras p.o., Harman S.M. Reproductive hormones in aging men III: Basal and LH-RH stimulated serum concentrations of the common alpha-subunit of the glycoprotein hormones. J. Gerontol. 42: 476, 1987. 10. Harman S.M., Tsitouras p.o., Costa PT., Blackman M.R. Reproductive hormones in aging men II: Basal pituitary gonadotropins and gonadotropin responses to luteinizing hormone releasing hormone. J. Clin. Endocrinol. Metab. 54: 547, 1982. 11. Snayder p.o., Reitano J.F., Utiger R.o. Serum LH and FSH responses to synthetic gonadotropin-releasing hormone in normal men. J. Clin. Endocrinol. Metab. 41: 938, 1975. 12. Petraglia F., Porro C., Facchinetti F., Cicoli C., Bertellini E., Volpe A., Berbieri G.C., Genazzani A.R. Opioid control of LH secretion in humans: menstrual cycle, menopause and aging reduce effect of naloxone but not of morphine. Life Sci. 38: 2103, 1986.
REFERENCES 1. Gray G.o., Smith E.R., Dorsa D.M., Davidson J.M. Sexual behaviour and testosterone in middle-age rats. Endocrinology 109: 1597, 1981.
13. Kerin J.F., Liu J.H., Phillipou G., Yen S.S.C. Evidence for a hypothalamic site of action of clomiphene citrate in women. J. Clin. Endocrinol. Metab. 61: 265, 1985.
2. Larsson K. Sexual activity in senile male rats. J. Gerontol. 13: 136, 1958. 3. Witkin J.w. Aging changes in synaptology of luteinizing hormone - releasing hormone neurons in male rat preoptic area. Neuroscience 22: 1003, 1987.
14. Tenover J.S., Matsumoto A.M., Phymate S.R., Bremner W.J. The effects of aging in normal men on bioavailable testosterone and luteinizing hormone secretion: response to clomiphene citrate. J. Clin. Endocrinol. Metab. 65: 1118, 1987.
4. Simpkin JW., Estes K.S., Kalra B.S., Kalra S.P. Alterations in hypothalamic neurotransmitters contribute to age-related decline in reproductive function in the male rat. In: Negro-Vilar A. (Ed.), Male Reproduction and Fertility. Raven Press, New York, 1983, p. 95.
15. Deslypere J.P., Kaufman J.M., Vermeulen T., Vogelaers D., Vandelem J.L., Vermeulen A. Influence of age on pulsatile luteinizing hormone release and responsiveness of the gonadotrophs to sex hormone feed-back in man. J. Clin. Endocrinol. Metab. 64: 68, 1987.
5. Hoffman G.E., Sladek J.R. Jr. Age-related changes in dopamine, LHRH and somatostatin in the rat hypothalamus. Neurobiol. Aging 1: 27, 1980.
16. McFadyen I.J., Volton A.E., Cameron E.H.o., Hunter W.M., Raab J., Forrest A.P.M. Gonadal-pituitary hormone levels in gynaecomastia. Clin. Endocrinology 13: 77, 1980.
6. Huang H.H., Kissane J.Q., Hawrylewicz E.J. Restoration of sexual function and fertility by fetal hypothalamic transplant in imponent aged male rats. Neurobiol. Aging 8: 465, 1987.
17. Winters S.J., Sherins R.J., Troen P. The gonadotropin - suppressive activity of androgen is increased in elderly men. Metabolism 33: 1052, 1984.
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18. Winters S.J., Troen P. Episodic luteinizing hormone (LH) secretion and the response of LH and follicle - stimulating hormone to LH - releasing hormone in aged men: Evidence for coexistent primary testicular insufficiency and an impairment in gonadotropin secretion. J. Clin. Endocrino!. Metab. 55: 560, 1982.
26. Nankin H.A., Calkins J.H. Decreased bioavailable testosterone in aging normal and impotent men. J. Clin. Endocrino!. Metab. 63: 1418, 1986. 27. Neischlag E., Lammers U., Freischem CW., Langer K., Wickings E.J. Reproductive functions in young fathers and grandfathers. J. Clin. Endocrino!. Metab. 55: 676, 1982.
19. Rasmussen D.D. New concepts in the regulation of hypothalamic gonadotropin - releasing hormone (GnRH) secretion. J. Endocrino!. Invest. 9: 427,1986.
28. Urban R.D., Veldhuis J.D., Blizzard A.M., Dufau M.L. Attenuated release of biologically active luteinizing hormone in healthy aging men. J. Clin. Invest. 81: 1020, 1988.
20. Crowley W.F., McArthur J.M. Stimulation of the normal menstrual cycle in KalIman's syndrome by pulsatile administration of luteinizing hormone - releasing hormone (LHRH). J. Clin. Endocrino!. Metab. 51: 173, 1981.
29. Warner BA, Dufau M.L., Santen A.J. Effect of aging and illness on the pituitary testicular axis in men: qualitative as well as quantitative changes in luteinizing hormone. J. Clin. Endocrino!. Metab. 60: 263, 1985.
21. Furr B.J.A., Woodburn J.A. Luteinizing hormone - releasing hormone and its analogues: a review of biological and clinical uses. J. Endocrino!. Invest. 11: 535, 1988.
30. Conn P.M., Cooper R., Mc Namara C., Rogers D.C., Sho.enhardt L. Qualitative change in gonadotropin during normal aging in the male rat. Endocrinology 106: 1549, 1980.
22. Santen A.J., Bardin CW. Episodic luteinizing hormone secretion in man. Pulse analysis, clinical interpretation, physiologic mechanisms. J. Clin. Invest. 52: 2617, 1973.
31. Veldhuis J.D., Johnson M.L., Dufau M.L. Preferential release of bioactive luteinizing hormone in response to endogenous and low-dose exogenous gonadotropin releasing hormone (GNRH) pulses in man. J. Clin. Endocrino!. Metab. 64: 1275, 1987.
23. Veldhuis J.D., Evans W.S., Johnson M.L., Wills M.A., Rogol A.D. Physiological properties of the luteinizing hormone pulse signal: impact of intensive and extended venous sampling paradigms on its characterization in healthy men and women. J. Clin. Endocrinol. Metab. 62 881, 1986.
32. Giusti M., Marini G., Traverso L., Cavagnaro P., Peluffo L., Giordano G. Biological LH activity in the elderly: effect of shortterm pulsatile LHRH administration. XIV Int. Congress of Gerontology, 19-23 June, 1989, Acapulco. Book of Abstracs, p. 430. 33. Sauder S.E., Frager M.S., Case G.D., Kelch R.P., Marshall J.C. Effect of changing gonadotrophin - releasing hormone pulse frequency on gonadotrophin secretion in men. Clin. Endocrino!. (Oxf.) 28: 647,1988.
24. Giusti M., Torre A., Traverso L., Cavagnaro P., Attanasio A., Giordano G. Endogenous opioid blockade and gonadotropin secretion: role of pulsatile luteinizing hormone administration in anorexia nervosa and weight loss amenorrhea. Ferti!. Steri!. 49: 797, 1988. 25. Baker HW.E., Burger H.G., De Kretser D.M., Hudoson B., O'Connor S., Wang C., Mirovics A., Court J., Dunlop M., Rennie G.C. Changes in the pituitary - testicular system with age. Clin. Endocrino!. (Oxf.) 5: 349, 1976.
34. Simpkins J.w., Millard JW. Influence of age on neurotransmitter function. Endocrino!. Metab. Clin. North Am. 16: 947, 1987.
132
Effect of pulsatile luteinizing hormone - releasing hormone administration on pituitary - gonadal function in elderly man M. Giusti*, G. Marini**, L. Traverso*, P. Cavagnaro*, L. Granziera***, and G. Giordano*. *I.S.M.I. Cattedra di Endocrinologia, Universita di Genova, **Casa di Riposo di Genova, and ***Ricerca e Sviluppo, Medical Systems, 16100 Genova, Italy administration (p < 0,01). LH pulses were identified in 5 out of 8 subjects on day O. On day 14, all the exogenous LHRH pulses were followed by significant LH bursts. There was not a significant decrease in the pituitary LH responsiveness to LHRH test from day 0 to day 14. Our study seems to indicate that pituitary - gonadal unit in normal elderly men can be modulated by pulsatile administration of LHRH. A pulse frequency of LHRH which is probably similar to the physiological one, could induce a slight increase in T levels via qualitative changes in LH activity. We can assume that clinical changes in gondadal activity might also be connected to some disturbances in endogenous LHRH pulsar.
ABSTRACT. The effect of short-term pulsatile LHRH administration was studied in 8 healthy subjects ranging from 60 to 81 yr to see if the decrease of pituitary gonadal function could be in part due to changes in the discharge of LHRH from the hypothalamus. Gonadotropin and testosterone (T) secretion was evaluated two weeks before and during LHRH (122-160 ng/kg bw every 120 min sc) infusion. In addition, a bolus dosage of LHRH (50 IJ. iv) was given both at the beginning and at the end of pulsatile LHRH administration in order to test gonadotrophs sensitivity. A significant inGrease in gonadotropin levels from day 0 to day 4 was found, and was followed by a subsequent decrease from day 7 to day 14. A slight significant increase in T levels was observed during LHRH
INTRODUCTION There are aging changes in the hypothalamic pituitary - gonadal axis in many mammalian species. The aging male rat exhibits decreased fertility and sexual activity (1 , 2) which seem to partially attribute to the decrease of the functional activity of LHRH neurons and/ or neurotransmitters involved in the LHRH control (3). In fact, age-related changes were noted in synaptic organization of LHRH neurons (3) and, even if the number or size of LHRH neurons did not change with aging (3), several studies showed that the LHRH content of the median eminentia was found to be reduced by radioimmunoassay (4) and immunochemistry (5) in aged male rats. A further suggestion can be raised from experimental studies, with regards to central mechanisms of pituitary-gonadal activity in old rats, in which fetal
hypothalamic graft increases LH and testosterone (T) release and restores neuroendocrine reproductive function in impotent aged male rats (6). Several data indicate that pituitary - gonadal function in man is also modified by aging. A decrease in testicular responsiveness to human chorionic gonadotropin (7) and a drop in the Leydig cell number (8) seems to underline a primary gonadal damage with aging. On the other hand, age-related changes at the hypothalamic - pituitary level have been found in man and they could suggest an impairment in neuroendocrine control of LHRH LH unit. In fact, LH as well as FSH and a"subunit response after LHRH is generally blunted or delayed (9-11). The opiate receptor antagonist naloxone doses not increase LH release in men over 60 yr with normal T levels (12). Whereas clomiphene citrate, which probably acts via the hypothalamic target site (13), does not seem to restore LH (7) and LH-related gonadal function (14). Spontaneous pulsatile LH secretion has been extensively studied in aging subjects (14-18) and sev-
Key-wordS: LHRH, pulsatile administration, LH, testosterone. elderly man. Correspondence: Massimo Giusti. M.D., I.S.M.I., Cattedra di Endocrinologia. Viale Benedetto XV, 6, 1-16100 Genoa. Italy.
Received April 14, 1989; accepted November 6, 1989.
127
M. Giusti, G. Marini, L. Traverso, et al.
eral studies reported a qecrease, both in frequency (15, 16) and amplitude (18) of the LH pulses in elderly men compared to that in young men. Together with an increase in LH response to sex hormone feed-back (16, 17), this pattern may contribute to important changes in hypothalamic - pituitary function. The pulsatile nature of LHRH-LH release is a physiological property due both to the activity of the endogenous pulse generator and to the responsiveness of the gonadotrophs (19). In addition, pulsatile, but not continuous exogenous LHRH administration, can restore normal gonadal function in hypogonadotropin individuals (20, 31). The aim of the study was to evaluate the effect of a two-week exogenous pulsatile LHRH administration in a group of normal elderly men. Our working hypothesis was to verify if a physiological like pattern of LHRH short-term, given by a portable pump, could improve pituitary - gonadal function with aging.
at weekly intervals and the injection site was changed at least every 4 days. Dosages of LHRH varied from 122-160 ng/kg bw per pulse. The pump was programmed to give a pulse which lasted one minute every 120 min. During the tests performed on day 7 and 14, each pulse of exogenous LHRH was identified and specimens were collected at the actual moment of drug delivery. Fifty /lg of LHRH (Biodata, Milan, Italy) were given iv in bolus in all but one subject at the end of the test on day 0 and 14. In case 3, the latter LHRH test was performed on day 7. Gonadotropin response to LHRH test was evaluated only 60 min after peptide administration. The protocol was chosen in order to slightly modify the subjects habitual way of life and was approved by the Ethical Committees of the Geriatric Institute. Hormone assays All serum samples from an individual subject were run in duplicate in the same assay. LH and FSH were measured by double antibody radioimmunoassay using reagents provided by Biodata. Gonadotropin values were expressed in mlU/ml as equivalent of the 2nd - IRP - hMG. The sensitivity of the method was 1 mlU/ml for both hormones. The intraassay coefficient of variation (CV) was 5.3% and 6.8% at level of 8.4 mlU/ml for LH and 9.7 mlU/ml for FSH, respectively. Serum T was measured without extraction using reagents obtained from DPC (Los Angeles, CA). Sensitivity was 15 ng/ dl. The intraassay CV was 4.0% at a level of 730 ng / dl. In our laboratory, normal levels for adult male subjects were found to be 5-20 mlU/ml and 350-1000 ng/ dl for both gonadotropins and T, respectively.
MATERIALS AND METHODS Subjects Eight elderly subjects ranging from 60 to 81 yr were enrolled in this study after giving their consent. They were housed in the Social Service Section of the Geriatric Institute in Genoa. A complete history and physical examination was performed on all subjects. They were non-smokers and their intake of alcohol was less than 40 g/day. According to Mini Mental State (MMS) and Ischemic Score (IS) of Haschinski, cognitive brain function was evaluated. All subjects had a score greater than 24 for MMS and less than 3 for IS. Body mass index (kg/m2) ranged from 21.5 to 26.0. Apart from stabilized cardiovascular diseases no other acute or chronic illnesses were found. The subjects were not obliged to stay in bed and a normal life was conducted. A balanced diet was given at 11 :30 and 19:30 h in the institute refectory.
Oata analysis All values are expressed as mean ± SE. Subsequent individual LH series were analyzed for significant LH pulses using the Santen and Bardin method (22), adjusted to a threshold requirement of 4 times individual intraassay CV according to Veldhuis (23). The LH pulse was identified as increment from nadir (mean of 2 consecutive samples) to peak (following sample) of at least 4 times intraassay CV. In the case of the first 2 samples of an experiment the basal value was taken as nadir, if necessary. Moreover, the reputed peak had to be followed by 2 consecutive decreasing values and the amplitude had to be greater than the sensitivity of the assay method. The amplitude of a pulse was defined as the difference between the peak and the preceding
Protocol LH, FSH and T secretion was evaluated in rest condition on day 0, 1, 4, 7, 10 and 14 of the study. Every 15 min, multiple blood samples, were collected from 08:00 to 11 :00 h on day 0, 7 and 14, while morning basal samples were drawn on day 1, 4 and 10. LHRH (Lutrelef, Ferring, Kiel, Germany) was sc administered in a pulsatile manner by portable pump (Zyklomat VAR Franconia, Ferring) from 12:00 h of day 0 to 11 :00 h of day 14. Pumps were reloaded
128
Aging and LHRH pulsatile administration
nadir. During the pulsatile LHRH administration, individual LH pulses each pump activation were ananalyzed as previously reported (24). Nadir was defined as the mean value of 2 consecutive samples obtained before exogenous LHRH pulse. Peak was defined as the highest value found after LHRH pulse. The amplitude was measured by subtracting the nadir from the peak value. LH increase after LHRH pulse was considered significant if its amplitude was gre'ater than 4 times the individual intraassay CV. Results were analyzed using two-tailed paired t test on log-transformed data. P values less than 0.05 were considered statistically significant.
case DAY 0
1 600
10 400 5
200
0
0
E
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~
20
600 g'
E 15
400 w
z
200
10
I
ffi
f-
(J)
...J
0
RESULTS
0
f-
(J)
w
20
All but one patient tolerated the mini-pump and the LHRH pulsatile infusion. Case 3 showed a bad compliance to the delivery system and stopped the pulsatile LHRH infusion on day 7 after a basal blood sample. Mean LH, FSH and T levels were 21.0 ± 6.3 mIU/ml, 19.8 ± 9.5 mlU/ml and 318 ± 22 ng/dl before LHRH administration (day 0), respectively. Significant LH pulses were identified in 5 out of 8 subjects. In these 5 subjects pulse amplitude of LH ranging from 2.5 to 5.8 mlU/ml (Table 1, Fig. 1). During short-term pulsatile LHRH administration a significant increase in LH and FSH levels from day 0 to day 4 were found, followed by a subsequent decrease from day 7 to day 14 (Fig. 2). A slight but significant increase in T levels was also observed (Fig. 2). Higher levels of T were found in 71 % of subjects on day 1 , 4, 10 and 14 and
0
f-
600 15
400 200
10
o
o 30
60
90
120
150
180 min
Fig. 1 - LH (.........) and T (0-0) behavior at the day 0, 7 and 14 in case 1. Arrows represent both the significant LH pulses on day 0 and the moment of LHRH infusion day 7 and 14.
in 87% of subjects on day 7 (Fig. 2). Mean T values during pulsatile LHRH administration were 391 ± 50 ng/ dl (p < 0.Q1 from day 0). When the pump was switched-on significant LH LHRH - induced bursts were observed following 17 day 7 out of 20 LHRH pulses (Table 1, Fig. 2).
On
Table 1 - Number and amplitude (mIU/ml) of spontaneous LH
pulses or LH pulses induced by pulsatile LHRH administration. See also methods for the definition of LH pulse. Case (no.)
Day 14 Day 7 Day 0 on LHRH on LHRH before LHRH Mean Mean Pulses1 Mean Pulses 1 Pulses amplitude amplitude amplitude
1
4.6
1 (1)
6.0
2 (2)
6.1
0(1)
n.e. 2
1 (1)
14.1
2
1
5.8
3
2
4.1
4
2.5
1 (1)
5.2
1 (1)
1.5
5
0
n.e. 2
0(2)
n.e. 2
1 (1)
3.7
6
0
n.e. 2
2 (2)
11.6
2 (2)
6.3
7 8
0
n.e. 2
3.2
2 (2)
4.2
2 (2)
3.2
1 (1)
10.3
1 (1)
8.7
600 _
70
"0
~60
500
g;
E 50
400
il340 u.
g....
300-:;;
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Q)
Q)
....0
: 30
200
I20 --J
I
100 10 0
o1
4
7
10
0 14 days
(/J
Q)
f-
r
Fig. 2 - Mean LH, FSH and T levels before and during pulsatile LHRH administration in normal elderly subjects (A p < 0.05 and AA p < 0.01 vs day 0).
, In brackets number of LHRH pulses during the sampling period. 2 n.e. = not evaluable.
129
M. Giusti, G. Marini, L. Traverso, et al.
the inabilty of gonadotrophic cell responsiveness to LHRH itself. Our study shows that in healthy elderly men shortterm pulsatile LHRH infusion discloses a rapid increase in gonadotropin secretion which is coupled with T enhancement as was observed in patients with idiopathic hypogonadotropic hypogonadism using identical protocols of peptide administration (20, 21). This rapid response of pituitary to exogenous LHRH given in a pulsatile way would tend to minimize the possibility that the impairment of pituitary gondadal secretion in elderly men can be due to a reduced sensitivity of gonadotropin-secreting cells. Aging-related biophysical changes in the endogenous LH molecule have been reported. in old rats (30). In elderly men update findings seem to be scanty and controversial, after having discovered that the biological LH activity was both unchanged (14) and reduced (28, 29). Moreover, it is well known that the rhythm in edogenous LHRH secretion is an important modulator of LH molecule discharge which is enriched in biological activity (31 ). We can hypothesize that an impaired secretory reserve in bio!ogical LH in aging can be linked to a disturbance of LHRH pulsar. We have only preliminary data regards to the increase of biological to immunological LH ratio during pulsatile LHRH administration in elderly humans (32), but present work indirectly strengthens this hypotesis. In fact, the increase in T levels observed in our subjects even after the quantitative decline in LH secretion could be due to a preferential release inLH with enhanced bioactivity during pulsatile LHRH administration. Besides, present data seem to conflict with the opinion of a decline in T levels with aging, as an indication of decreasing sensitivity of gonadal target (14, 15). In physiological conditions there is no uniformity of LH pulse properties which are likely to express the lack of regularity of the oscillator controlling LHRH release (23) and our choice of a fixed LHRH regimen could be considered arbitrary. However, injection of LHRH pulses every 2 h resulted for 3-4 days in increased plasma LH concentration in adult (33) or elderly (present study) normal men. This was presumably due to increased pituitary exposure to LHRH from the additive effects of exogenous LHRH pulses and endogenous LHRH secretion (33). LH pulses were identified following the majority of LHRH pulses recorded (100% of LHRH pulses recorded on day 14) and LH responsiveness to testing dosage of LHRH was found to be slightly (not sig-
after LH LHRH - induced significant pulses were found in 5 out of 7 subjects and in all subjects studied on day 14 (Table 1). Nadir to peak amplitude ranged from 4.2 to 11.6 mlU/ml and from 1.5 to 14.1 mIU/ml,7 and 14 days after LHRH administration, respectively. Pituitary LH responsiveness to LHRH given in a bolus fashion decreased in non-significant way from the former (69.7 ± 21.7 mIU/ml) and the latter (39.6 ± 7.2 mIU/ml) study. DISCUSSION Unlike that observed in vivo and in vitro experimented in rats (1-6), the effect of aging on hypothalamus - pituitary - gonadal axis in man still seems to be unclear. For instance, T levels have often been (14, 15, 17, 25) but not always (12, 26, 27) found to be reduced and it has not been established whether or not central (10, 12, 15, 17, 18,28) and/ or peripheral (7, 8, 14, 15, 18, 20, 25) mechanisms are involved in this phenomenon. Consequently, some variations found during aging progress are thought to be connected with the experimental model employed and the concurrence of unconsidered chronic illnesses (29). In a group of selected healthy elderly men we performed the present study in order to rule out some factors which are able to modify results such as obesity, heavy alcohol intake, concomitant drugs and smoking. On the other hand, we chose a protocol which has not been excessively demanding on the subjects in order not to change their normal way of life. On average, a slight increase in basal gonadotropin levels and a more evident reduction of T levels have been observed in our subjects. Moreover, the levels of gonadotropins were not dramatically increased in spite of the decrease in T levels. This phenomenon gives the impression that a central impairment due to a greater feed-back responsiveness of the hypothalamus pituitary complex takes place in the elderly men (15, 17). It could also suggest that in normal elderly men a complex central alteration of central nervous system - LHRH pulse generator is present with aging. Besides, available data do not clearly show if the decrease in amplitude or frequency of LH bursts found in spontaneous conditions (15, 16, 18) and after dihydrotestosterone infusion (17) could be due both to anomalous LHRH discharge in portal flow and to
130
Aging and LHRH pulsatile administration
nificantly) reduced in our subjects. If a down regulation phenomenon seems to be unlikely, we cannot exclude a certain degree of reduction of LH pool ready - releasable and/ or an increase in the negative feed-back of T at pituitary level. In conclusion, according to some other authors (15, 17, 18,28) and to experimental data in rats (36) our study indicates that functional capacity of the central nervous system - LHRH pulse generator becomes impaired with the process of aging. The increase in T levels might be due to a physiological pulse frequency of LHRH inducing qualitative changes in LH activity. Endogenous LHRH pulsatility seems to be a multifactorial phenomenon and several neurotrasmitters and neuropeptides can modulate LHRH discharge, on which the feed-back mechanisms of gonadal hormones operate (19). Altered intracellular androgen processing in the hypothalamus and/or changes in neuromodulator systems (4, 34) which may be connected to disturbances in endogenous LHRH pulsar is still unclear.
7. Rubens R., Dhont M., Vermeulen V. Further stUdies on Leydig cell function in old age. J. Clin. Endocrinol. Metab. 39: 40, 1974. 8. Neaves W.B., Johnson L., Porter J.C., Parker C.R. Jr., Petty C.S. Leydig cell numbers, daily sperm production, and serum gonadotropin levels in aging men. J. Clin. Endocrinol. Metab. 55: 756, 1984. 9. Blackman M.R., Tsitouras p.o., Harman S.M. Reproductive hormones in aging men III: Basal and LH-RH stimulated serum concentrations of the common alpha-subunit of the glycoprotein hormones. J. Gerontol. 42: 476, 1987. 10. Harman S.M., Tsitouras p.o., Costa PT., Blackman M.R. Reproductive hormones in aging men II: Basal pituitary gonadotropins and gonadotropin responses to luteinizing hormone releasing hormone. J. Clin. Endocrinol. Metab. 54: 547, 1982. 11. Snayder p.o., Reitano J.F., Utiger R.o. Serum LH and FSH responses to synthetic gonadotropin-releasing hormone in normal men. J. Clin. Endocrinol. Metab. 41: 938, 1975. 12. Petraglia F., Porro C., Facchinetti F., Cicoli C., Bertellini E., Volpe A., Berbieri G.C., Genazzani A.R. Opioid control of LH secretion in humans: menstrual cycle, menopause and aging reduce effect of naloxone but not of morphine. Life Sci. 38: 2103, 1986.
REFERENCES 1. Gray G.o., Smith E.R., Dorsa D.M., Davidson J.M. Sexual behaviour and testosterone in middle-age rats. Endocrinology 109: 1597, 1981.
13. Kerin J.F., Liu J.H., Phillipou G., Yen S.S.C. Evidence for a hypothalamic site of action of clomiphene citrate in women. J. Clin. Endocrinol. Metab. 61: 265, 1985.
2. Larsson K. Sexual activity in senile male rats. J. Gerontol. 13: 136, 1958. 3. Witkin J.w. Aging changes in synaptology of luteinizing hormone - releasing hormone neurons in male rat preoptic area. Neuroscience 22: 1003, 1987.
14. Tenover J.S., Matsumoto A.M., Phymate S.R., Bremner W.J. The effects of aging in normal men on bioavailable testosterone and luteinizing hormone secretion: response to clomiphene citrate. J. Clin. Endocrinol. Metab. 65: 1118, 1987.
4. Simpkin JW., Estes K.S., Kalra B.S., Kalra S.P. Alterations in hypothalamic neurotransmitters contribute to age-related decline in reproductive function in the male rat. In: Negro-Vilar A. (Ed.), Male Reproduction and Fertility. Raven Press, New York, 1983, p. 95.
15. Deslypere J.P., Kaufman J.M., Vermeulen T., Vogelaers D., Vandelem J.L., Vermeulen A. Influence of age on pulsatile luteinizing hormone release and responsiveness of the gonadotrophs to sex hormone feed-back in man. J. Clin. Endocrinol. Metab. 64: 68, 1987.
5. Hoffman G.E., Sladek J.R. Jr. Age-related changes in dopamine, LHRH and somatostatin in the rat hypothalamus. Neurobiol. Aging 1: 27, 1980.
16. McFadyen I.J., Volton A.E., Cameron E.H.o., Hunter W.M., Raab J., Forrest A.P.M. Gonadal-pituitary hormone levels in gynaecomastia. Clin. Endocrinology 13: 77, 1980.
6. Huang H.H., Kissane J.Q., Hawrylewicz E.J. Restoration of sexual function and fertility by fetal hypothalamic transplant in imponent aged male rats. Neurobiol. Aging 8: 465, 1987.
17. Winters S.J., Sherins R.J., Troen P. The gonadotropin - suppressive activity of androgen is increased in elderly men. Metabolism 33: 1052, 1984.
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M. Giusti, G. Marini, L. Traverso, et al.
18. Winters S.J., Troen P. Episodic luteinizing hormone (LH) secretion and the response of LH and follicle - stimulating hormone to LH - releasing hormone in aged men: Evidence for coexistent primary testicular insufficiency and an impairment in gonadotropin secretion. J. Clin. Endocrino!. Metab. 55: 560, 1982.
26. Nankin H.A., Calkins J.H. Decreased bioavailable testosterone in aging normal and impotent men. J. Clin. Endocrino!. Metab. 63: 1418, 1986. 27. Neischlag E., Lammers U., Freischem CW., Langer K., Wickings E.J. Reproductive functions in young fathers and grandfathers. J. Clin. Endocrino!. Metab. 55: 676, 1982.
19. Rasmussen D.D. New concepts in the regulation of hypothalamic gonadotropin - releasing hormone (GnRH) secretion. J. Endocrino!. Invest. 9: 427,1986.
28. Urban R.D., Veldhuis J.D., Blizzard A.M., Dufau M.L. Attenuated release of biologically active luteinizing hormone in healthy aging men. J. Clin. Invest. 81: 1020, 1988.
20. Crowley W.F., McArthur J.M. Stimulation of the normal menstrual cycle in KalIman's syndrome by pulsatile administration of luteinizing hormone - releasing hormone (LHRH). J. Clin. Endocrino!. Metab. 51: 173, 1981.
29. Warner BA, Dufau M.L., Santen A.J. Effect of aging and illness on the pituitary testicular axis in men: qualitative as well as quantitative changes in luteinizing hormone. J. Clin. Endocrino!. Metab. 60: 263, 1985.
21. Furr B.J.A., Woodburn J.A. Luteinizing hormone - releasing hormone and its analogues: a review of biological and clinical uses. J. Endocrino!. Invest. 11: 535, 1988.
30. Conn P.M., Cooper R., Mc Namara C., Rogers D.C., Sho.enhardt L. Qualitative change in gonadotropin during normal aging in the male rat. Endocrinology 106: 1549, 1980.
22. Santen A.J., Bardin CW. Episodic luteinizing hormone secretion in man. Pulse analysis, clinical interpretation, physiologic mechanisms. J. Clin. Invest. 52: 2617, 1973.
31. Veldhuis J.D., Johnson M.L., Dufau M.L. Preferential release of bioactive luteinizing hormone in response to endogenous and low-dose exogenous gonadotropin releasing hormone (GNRH) pulses in man. J. Clin. Endocrino!. Metab. 64: 1275, 1987.
23. Veldhuis J.D., Evans W.S., Johnson M.L., Wills M.A., Rogol A.D. Physiological properties of the luteinizing hormone pulse signal: impact of intensive and extended venous sampling paradigms on its characterization in healthy men and women. J. Clin. Endocrinol. Metab. 62 881, 1986.
32. Giusti M., Marini G., Traverso L., Cavagnaro P., Peluffo L., Giordano G. Biological LH activity in the elderly: effect of shortterm pulsatile LHRH administration. XIV Int. Congress of Gerontology, 19-23 June, 1989, Acapulco. Book of Abstracs, p. 430. 33. Sauder S.E., Frager M.S., Case G.D., Kelch R.P., Marshall J.C. Effect of changing gonadotrophin - releasing hormone pulse frequency on gonadotrophin secretion in men. Clin. Endocrino!. (Oxf.) 28: 647,1988.
24. Giusti M., Torre A., Traverso L., Cavagnaro P., Attanasio A., Giordano G. Endogenous opioid blockade and gonadotropin secretion: role of pulsatile luteinizing hormone administration in anorexia nervosa and weight loss amenorrhea. Ferti!. Steri!. 49: 797, 1988. 25. Baker HW.E., Burger H.G., De Kretser D.M., Hudoson B., O'Connor S., Wang C., Mirovics A., Court J., Dunlop M., Rennie G.C. Changes in the pituitary - testicular system with age. Clin. Endocrino!. (Oxf.) 5: 349, 1976.
34. Simpkins J.w., Millard JW. Influence of age on neurotransmitter function. Endocrino!. Metab. Clin. North Am. 16: 947, 1987.
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