Relationship of Seminal Plasma Testosterone and Dihydrotestosterone to Sperm Count and Motility in Man
Syst Biol Reprod Med Downloaded from informahealthcare.com by Osaka University on 02/10/15 For personal use only.
J. BAIN,M. DUTHIE,and J. KEENE A radioimmunoassay previously described for serum T and DHT was adapted for use in the measurement of these hormones in seminal plasma. The seminal plasmas of 273 semen specimens were assayed for T and DHT concentration. Mean levels of these hormones for various sperm count and sperm motility categories were determined. We found that: (1) the DHT level of the azoospermic group was significantly lower than all other groups; (2) the T level of the group whose mean sperm count exceeded 40 x 106/ml was higher than that of all other groups; (3) the DHT level of the group with absent sperm motility was lower than the level of all other groups; and (4) changes in sperm motility were not accompanied by changes in T levels. We concluded that idiopathic male subfertility as evidenced by oligospermia, azoospermia, and decreased sperm motility may be related to insufficient androgen production due to a primary intratesticular defect.
Key Words: Male infertility, Oligospermia;Azoospermia; Sperm motility; Seminal plasma; Testosterone; Dihydrotestosterone.
INTRODUCTION Little is known about pathophysiologic mechanisms in most cases of male infertility or subfertility, although serum hormonal interrelationships have been reasonably well established. Androgens are essential in the production of an adequate number of normal sperm with motility sufficiently forceful to allow some sperm to reach and one sperm to penetrate an ovum in the fallopian tube. An assessment of intratesticular androgen activity in man is very difficult. Testicular biopsy usually yields an inadequate amount of material for detailed studies. Although there may be a variety of sources for seminal plasma androgens, fluctuations in androgens in this fluid might be related to variations in sperm count and motility. Not only can steroid hormones be measured in seminal plasma, but dihydrotestosterone (DHT) levels are significantly decreased in azoospermic patients while testosterone (T) levels remain unchanged [ 6 ] . It has also been demonstrated that DHT and not T concentrations are significantly suppressed in the seminal plasma of azoospermic subjects [a]. The relationship of seminal androgens (T and DHT) to sperm count and motility Received December 1977. From the Departments of Medicine and Obstetrics and Gynecology, University of Toronto and Mount Sinai Hospital, Reproductive Biology Unit, Mount Sinai Hospital, and the Research Institute, Hospital for Sick Children, Toronto, Canada. Address reprint requests to: J. Bain, Mount Sinai Hospital, 600 University Avenue, Suite 639-640, Toronto, Ontario, Canada, M5G 1x5.
35 8 Elsevier North Holland, Inc., ARCHIVES
OF ANDROLOGY 2, 35-39 (1979)
OI4&5016/79/01W35+05$02.25
36
J. Bain, M. Duthrie, and J. Keene
has received relatively little attention. We undertook to assay seminal plasmaT and DHT in a large number of human specimens with a range of sperm count and motility. Finding a correlation among these parameters could result in new insights into the pathophysiologic basis of male subfertility. MATERIALS AND METHODS
Syst Biol Reprod Med Downloaded from informahealthcare.com by Osaka University on 02/10/15 For personal use only.
Clinical Studies
Men presented for a fertility investigation to either an individual physician or the Reproductive Biology Unit of the Mount Sinai Hospital. The seminal plasma from their semen specimens analyzed by the Division of Cytology at the hospital was stored and kept frozen at -20°C until ready for assay. Two hundred and seventy-three individual seminal plasmas were collected. and the T and DHT content was measured by radioimmunoassay as described above. The semen samples were categorized according to sperm count and sperm motility found for each specimen. The mean T and DHT values for each category were determined and statistically significant differences were determined by the t test. Radioimmunoassay of T and DHT
Radioimmunoassays of T and DHT in human serum were adapted to the measurement of T and DHT in human seminal plasma [ 2 . 31. A pool of seminal plasma was prepared from semen samples collected from men having varying sperm counts. Sperm were separated by centrifugation and the various plasmas mixed to create a control pool. Several control samples were created by either diluting the pool or adding known amounts of T or DHT. To aliquots of a control sample in which T and D H T had previously been measured after extraction and separation on a celite column [ 2 , 31 were added known and increasing amounts of T and DHT. The concentrations of T and DHT measured were compared to those expected and the correlation coefficient determined. The interassay variability for both hormones in three different seminal plasma pools was determined. The intraassay variability for a fourth pool was measured.
RESULTS Radioimmunoassay Characteristics
Table 1 demonstrates the results of a recovery study in which known amounts of T and DHT were added to a seminal plasma pool, extracted, separated by celite column chromatography, and measured by radioimmunoassay. Three seminal plasma pools had mean T concentrations of 8.1 ngidl, 12.5 ngidl, and 18.7 ng/dl. The interassay coefficient of variation for T in each of these pools is 9.5%,6.2%, and 7.8%, respectively. For DHT the mean levels in each of these pools was 21.8 ngidl, 30.6 ng/dl, and 46.8 ngidl. The interassay coefficient of variation was 10.7%,9.1%, and 11.2%, respectively. Table 2 gives the intraassay variability for both T and DHT in a seminal plasma pool. The coefficient of variation of T is 3.8% and DHT, 4.0%. Clinical Studies
The various sperm-count categories and the associated mean T and DHT levels are illustrated in Figure 1. When the r test was applied, it was found that the T level of the azoospermic group (sperm count = O), 10.6 ngidl was significantly lower than the values of all other sperm-count groups.
Testosterone and Sperm Characteristics
37
TABLE 1 Recovery Study of T and DHT Added to Seminal Plasma"
Syst Biol Reprod Med Downloaded from informahealthcare.com by Osaka University on 02/10/15 For personal use only.
AMOUNT T OR DHT ADDED(PG)
50 100 250 500 lo00 2500 5000
PERCENT
PERCENT
T
DHT
RECOVERED
RECOVERED
103 99 94 101 91 91 99
103 113 110 104 106 102 106
r Value (total expected vs. total found): for T, 0.9997; for DHT, 0.9996 (p < 0.001).
When the androgen levels of the >40 x 106/mlgroup were compared with the other groups, it was found that for T all other groups were significantly different (except, inexplicably, the >0-10 x 106/mlgroup). For DHT the >40 x 106/ml value of 37.2 nddl was not different from the 30-40 x 106/mlor 20-30 x 106/mlgroups, but it was higher than the values found for the other oligospermic or azoospermic groups (with oligospermia defined as a sperm count 50% group (36.2 ng/dl) was compared to the levels found for each of the other groups, it was found to be significantly higher than only the azoospermic level of 22.6 ng/dl. DISCUSSION
In seminal plasma, the following conditions are observed: (1) DHT is present in higher concentrations than T; (2) the DHT level for azoospermic subjects is significantly lower than for all other sperm-count categories; (3) the mean T for subjects with sperm counts greater than 40 x 106/ml is higher than for all other groups; (4) the T levels for all groups with sperm counts less than 20 x 106/mlare lower than the T level of TABLE 2 Intraassay Variability of T and DHT in Seminal Plasma
Number of samples Mean level (ng/dl) S.E.M. Coefficient of variation (%)
T
DHT
13 26.2 0.28 3.8
13 41.7 0.46 4.0
38
J. Bain, M. Duthrie, and J. Keene 40
Syst Biol Reprod Med Downloaded from informahealthcare.com by Osaka University on 02/10/15 For personal use only.
a 15
c 10
~
0-10
~
0-20
20-30
Sperm Count X l O l m l
FIGURE 1.
Seminal plasma T and DHT related to sperm count.
the >40 x 106/mlgroup; ( 5 ) the T concentration does not vary with degree of sperm motility; (6) in the group with absent sperm motility the DHT level is lower than in all other groups; and (7) in the group with sperm motility in excess of 50% the DHT level is only greater than the level of the nonmotile group. Few studies relating seminal plasma T and DHT levels to sperm quantity or quality have been reported. Twenty-one men with sperm counts in excess of 40 x 106/ml had mean seminal plasma T and DHT levels of 17.6 ng/dl and 39.6 ng/dl, respectively [ 5 , 71. In 99 men we found mean values of 13.7 ng/dl for T and 37.2 ng/dl for DHT. Seminal plasma T levels were unaffected by changes in sperm count, but the mean DHT level was significantly reduced when the sperm count was either less than 40 x 106/ml (25.3 ng/dl, N = 10) or zero (6.9 ng/dl, N = 11) [5, 71. In 47 specimens where the sperm count was less than 10 x 1O6/m1, we found the mean DHT to be 27.8 ng/dl and in 27 azoospermic specimens, 19.7 ng/dl.
c
II 1
30
1
T
0.20
1.1
T
>50
Sperm Motility l b
FIGURE 2.
Seminal plasma T and DHT related to sperm motility.
Syst Biol Reprod Med Downloaded from informahealthcare.com by Osaka University on 02/10/15 For personal use only.
Testosterone and Sperm Characteristics
39
Although there is not complete agreement between our findings and these reported studies, the same general trends emerge. Decreases in seminal plasma androgens, particularly DHT, are related to decreases in both sperm production and sperm motility. The source of seminal plasma androgens remains uncertain, but it is reasonable to postulate that a reduction of androgen concentration within the seminiferous tubules (and hence decreased androgens appearing in seminal plasma) is a direct cause of decreased sperm production. The reasons for decreased androgen production have yet to be elucidated. Male subfertility, as evidenced by oligospermia, azoospermia, or decreased sperm motility, is a pathologic process that originates within the testis rather than being secondary to extratesticular influences such as hypothalamic-pituitary insufficiency. In a study of 215 men, mean L H rose as sperm count fell below 5 x 106/ml, and mean FSH rose with sperm counts less than 10 X 106/ml [l]. No changes in serum T or DHT levels with changes in sperm count could be elicited, although mean serum T levels in the azoospermic group did tend to be lower than in other groups. A significant lowering of blood levels of androgens in human male oligospermia has been reported previously [4]. The nature of the intratesticular defect in idiopathic male subfertility not only remains unknown, but defies more direct study because of ethical considerations. Until a suitable animal model is found or new diagnostic tools for safe in vivo human studies are created, investigators will have to content themselves with studying those human tissues and fluids that are readily accessible. Acknowledgment: The authors would like to acknowledge the secretarial assistance of Morag Smith and Edith Sohn. We are also grateful to Ingrid Foldes and her staff within the Division of Cytology at Mount Sinai Hospital for the semen analyses. This study was supported in part by the Medical Research Council of Canada and the Research Institute, Mount Sinai Hospital, Toronto, Canada. REFERENCES Bain J (1978): Neuroendocrine parameters of male fertility and infertility. In Bain J, Hafez ESE, Barwin BN eds: Progress in Reproductive Biology, vol 3: Andrology: Basic and Clinical Aspects of Male Reproduction and Infertility. Basel: Karger, pp 33-45 Bain J, Grover PK, Swerdloff RS, et a1 (1974): One column chromatography and simultaneous radioimmunoassay (RIA) of testosterone and dihydrotestosterone (DHT). J Steroid Biochem 5: 304 (abstr 40) Ode11 WD, Swerdloff RS, Bain J, et a1 (1974): The effect of sexual maturation on testicular response to LH stimulation of testosterone secretion in the intact rat. Endocrinology 95: 13801384 Purvis K, Brenner PF, Landgren B-M, et a1 (1975): Indices of gonadal function in the human male. I. Plasma levels of unconjugated steroids and gonadotrophins under normal and pathological conditions. Clin Endocrinol 4: 237-246
5 . Purvis K, Diczfalusy E (1976): Steroid pattern
of human seminal plasma under normal and pathological conditions. In Hubinont PO, L’Hermite, M eds: Fifth International Seminar on Reproductive Physiology and Sexual Endocrinology: Sperm Action. Basel: Karger, pp 89- 106 6. Purvis K, Landgren B-M, Cekan 2 , et al(l974): Steroids in human seminal plasma and circulating blood. J Steroid Biochem 5: 382 (abstr 361) 7. Purvis K, Landgren B-M, Cekan 2 , et al(1975): Indices of gonadal function in the human male. 11. Seminal plasma levels of steroids in normal and pathological conditions. Clin Endocrinol 4: 247-258 8. Tea NT, Grenier J and Scholler R (1976): Steroid hormones in human semen. In: Fifth International Seminar on Reproductive Physiology and Sexual Endocrinology: Sperm Action. PO Hubinont and M L’Hermite (eds) S Karger, Basel, pp 107- 114
Syst Biol Reprod Med Downloaded from informahealthcare.com by Osaka University on 02/10/15 For personal use only.
J. BAIN,M. DUTHIE,and J. KEENE A radioimmunoassay previously described for serum T and DHT was adapted for use in the measurement of these hormones in seminal plasma. The seminal plasmas of 273 semen specimens were assayed for T and DHT concentration. Mean levels of these hormones for various sperm count and sperm motility categories were determined. We found that: (1) the DHT level of the azoospermic group was significantly lower than all other groups; (2) the T level of the group whose mean sperm count exceeded 40 x 106/ml was higher than that of all other groups; (3) the DHT level of the group with absent sperm motility was lower than the level of all other groups; and (4) changes in sperm motility were not accompanied by changes in T levels. We concluded that idiopathic male subfertility as evidenced by oligospermia, azoospermia, and decreased sperm motility may be related to insufficient androgen production due to a primary intratesticular defect.
Key Words: Male infertility, Oligospermia;Azoospermia; Sperm motility; Seminal plasma; Testosterone; Dihydrotestosterone.
INTRODUCTION Little is known about pathophysiologic mechanisms in most cases of male infertility or subfertility, although serum hormonal interrelationships have been reasonably well established. Androgens are essential in the production of an adequate number of normal sperm with motility sufficiently forceful to allow some sperm to reach and one sperm to penetrate an ovum in the fallopian tube. An assessment of intratesticular androgen activity in man is very difficult. Testicular biopsy usually yields an inadequate amount of material for detailed studies. Although there may be a variety of sources for seminal plasma androgens, fluctuations in androgens in this fluid might be related to variations in sperm count and motility. Not only can steroid hormones be measured in seminal plasma, but dihydrotestosterone (DHT) levels are significantly decreased in azoospermic patients while testosterone (T) levels remain unchanged [ 6 ] . It has also been demonstrated that DHT and not T concentrations are significantly suppressed in the seminal plasma of azoospermic subjects [a]. The relationship of seminal androgens (T and DHT) to sperm count and motility Received December 1977. From the Departments of Medicine and Obstetrics and Gynecology, University of Toronto and Mount Sinai Hospital, Reproductive Biology Unit, Mount Sinai Hospital, and the Research Institute, Hospital for Sick Children, Toronto, Canada. Address reprint requests to: J. Bain, Mount Sinai Hospital, 600 University Avenue, Suite 639-640, Toronto, Ontario, Canada, M5G 1x5.
35 8 Elsevier North Holland, Inc., ARCHIVES
OF ANDROLOGY 2, 35-39 (1979)
OI4&5016/79/01W35+05$02.25
36
J. Bain, M. Duthrie, and J. Keene
has received relatively little attention. We undertook to assay seminal plasmaT and DHT in a large number of human specimens with a range of sperm count and motility. Finding a correlation among these parameters could result in new insights into the pathophysiologic basis of male subfertility. MATERIALS AND METHODS
Syst Biol Reprod Med Downloaded from informahealthcare.com by Osaka University on 02/10/15 For personal use only.
Clinical Studies
Men presented for a fertility investigation to either an individual physician or the Reproductive Biology Unit of the Mount Sinai Hospital. The seminal plasma from their semen specimens analyzed by the Division of Cytology at the hospital was stored and kept frozen at -20°C until ready for assay. Two hundred and seventy-three individual seminal plasmas were collected. and the T and DHT content was measured by radioimmunoassay as described above. The semen samples were categorized according to sperm count and sperm motility found for each specimen. The mean T and DHT values for each category were determined and statistically significant differences were determined by the t test. Radioimmunoassay of T and DHT
Radioimmunoassays of T and DHT in human serum were adapted to the measurement of T and DHT in human seminal plasma [ 2 . 31. A pool of seminal plasma was prepared from semen samples collected from men having varying sperm counts. Sperm were separated by centrifugation and the various plasmas mixed to create a control pool. Several control samples were created by either diluting the pool or adding known amounts of T or DHT. To aliquots of a control sample in which T and D H T had previously been measured after extraction and separation on a celite column [ 2 , 31 were added known and increasing amounts of T and DHT. The concentrations of T and DHT measured were compared to those expected and the correlation coefficient determined. The interassay variability for both hormones in three different seminal plasma pools was determined. The intraassay variability for a fourth pool was measured.
RESULTS Radioimmunoassay Characteristics
Table 1 demonstrates the results of a recovery study in which known amounts of T and DHT were added to a seminal plasma pool, extracted, separated by celite column chromatography, and measured by radioimmunoassay. Three seminal plasma pools had mean T concentrations of 8.1 ngidl, 12.5 ngidl, and 18.7 ng/dl. The interassay coefficient of variation for T in each of these pools is 9.5%,6.2%, and 7.8%, respectively. For DHT the mean levels in each of these pools was 21.8 ngidl, 30.6 ng/dl, and 46.8 ngidl. The interassay coefficient of variation was 10.7%,9.1%, and 11.2%, respectively. Table 2 gives the intraassay variability for both T and DHT in a seminal plasma pool. The coefficient of variation of T is 3.8% and DHT, 4.0%. Clinical Studies
The various sperm-count categories and the associated mean T and DHT levels are illustrated in Figure 1. When the r test was applied, it was found that the T level of the azoospermic group (sperm count = O), 10.6 ngidl was significantly lower than the values of all other sperm-count groups.
Testosterone and Sperm Characteristics
37
TABLE 1 Recovery Study of T and DHT Added to Seminal Plasma"
Syst Biol Reprod Med Downloaded from informahealthcare.com by Osaka University on 02/10/15 For personal use only.
AMOUNT T OR DHT ADDED(PG)
50 100 250 500 lo00 2500 5000
PERCENT
PERCENT
T
DHT
RECOVERED
RECOVERED
103 99 94 101 91 91 99
103 113 110 104 106 102 106
r Value (total expected vs. total found): for T, 0.9997; for DHT, 0.9996 (p < 0.001).
When the androgen levels of the >40 x 106/mlgroup were compared with the other groups, it was found that for T all other groups were significantly different (except, inexplicably, the >0-10 x 106/mlgroup). For DHT the >40 x 106/ml value of 37.2 nddl was not different from the 30-40 x 106/mlor 20-30 x 106/mlgroups, but it was higher than the values found for the other oligospermic or azoospermic groups (with oligospermia defined as a sperm count 50% group (36.2 ng/dl) was compared to the levels found for each of the other groups, it was found to be significantly higher than only the azoospermic level of 22.6 ng/dl. DISCUSSION
In seminal plasma, the following conditions are observed: (1) DHT is present in higher concentrations than T; (2) the DHT level for azoospermic subjects is significantly lower than for all other sperm-count categories; (3) the mean T for subjects with sperm counts greater than 40 x 106/ml is higher than for all other groups; (4) the T levels for all groups with sperm counts less than 20 x 106/mlare lower than the T level of TABLE 2 Intraassay Variability of T and DHT in Seminal Plasma
Number of samples Mean level (ng/dl) S.E.M. Coefficient of variation (%)
T
DHT
13 26.2 0.28 3.8
13 41.7 0.46 4.0
38
J. Bain, M. Duthrie, and J. Keene 40
Syst Biol Reprod Med Downloaded from informahealthcare.com by Osaka University on 02/10/15 For personal use only.
a 15
c 10
~
0-10
~
0-20
20-30
Sperm Count X l O l m l
FIGURE 1.
Seminal plasma T and DHT related to sperm count.
the >40 x 106/mlgroup; ( 5 ) the T concentration does not vary with degree of sperm motility; (6) in the group with absent sperm motility the DHT level is lower than in all other groups; and (7) in the group with sperm motility in excess of 50% the DHT level is only greater than the level of the nonmotile group. Few studies relating seminal plasma T and DHT levels to sperm quantity or quality have been reported. Twenty-one men with sperm counts in excess of 40 x 106/ml had mean seminal plasma T and DHT levels of 17.6 ng/dl and 39.6 ng/dl, respectively [ 5 , 71. In 99 men we found mean values of 13.7 ng/dl for T and 37.2 ng/dl for DHT. Seminal plasma T levels were unaffected by changes in sperm count, but the mean DHT level was significantly reduced when the sperm count was either less than 40 x 106/ml (25.3 ng/dl, N = 10) or zero (6.9 ng/dl, N = 11) [5, 71. In 47 specimens where the sperm count was less than 10 x 1O6/m1, we found the mean DHT to be 27.8 ng/dl and in 27 azoospermic specimens, 19.7 ng/dl.
c
II 1
30
1
T
0.20
1.1
T
>50
Sperm Motility l b
FIGURE 2.
Seminal plasma T and DHT related to sperm motility.
Syst Biol Reprod Med Downloaded from informahealthcare.com by Osaka University on 02/10/15 For personal use only.
Testosterone and Sperm Characteristics
39
Although there is not complete agreement between our findings and these reported studies, the same general trends emerge. Decreases in seminal plasma androgens, particularly DHT, are related to decreases in both sperm production and sperm motility. The source of seminal plasma androgens remains uncertain, but it is reasonable to postulate that a reduction of androgen concentration within the seminiferous tubules (and hence decreased androgens appearing in seminal plasma) is a direct cause of decreased sperm production. The reasons for decreased androgen production have yet to be elucidated. Male subfertility, as evidenced by oligospermia, azoospermia, or decreased sperm motility, is a pathologic process that originates within the testis rather than being secondary to extratesticular influences such as hypothalamic-pituitary insufficiency. In a study of 215 men, mean L H rose as sperm count fell below 5 x 106/ml, and mean FSH rose with sperm counts less than 10 X 106/ml [l]. No changes in serum T or DHT levels with changes in sperm count could be elicited, although mean serum T levels in the azoospermic group did tend to be lower than in other groups. A significant lowering of blood levels of androgens in human male oligospermia has been reported previously [4]. The nature of the intratesticular defect in idiopathic male subfertility not only remains unknown, but defies more direct study because of ethical considerations. Until a suitable animal model is found or new diagnostic tools for safe in vivo human studies are created, investigators will have to content themselves with studying those human tissues and fluids that are readily accessible. Acknowledgment: The authors would like to acknowledge the secretarial assistance of Morag Smith and Edith Sohn. We are also grateful to Ingrid Foldes and her staff within the Division of Cytology at Mount Sinai Hospital for the semen analyses. This study was supported in part by the Medical Research Council of Canada and the Research Institute, Mount Sinai Hospital, Toronto, Canada. REFERENCES Bain J (1978): Neuroendocrine parameters of male fertility and infertility. In Bain J, Hafez ESE, Barwin BN eds: Progress in Reproductive Biology, vol 3: Andrology: Basic and Clinical Aspects of Male Reproduction and Infertility. Basel: Karger, pp 33-45 Bain J, Grover PK, Swerdloff RS, et a1 (1974): One column chromatography and simultaneous radioimmunoassay (RIA) of testosterone and dihydrotestosterone (DHT). J Steroid Biochem 5: 304 (abstr 40) Ode11 WD, Swerdloff RS, Bain J, et a1 (1974): The effect of sexual maturation on testicular response to LH stimulation of testosterone secretion in the intact rat. Endocrinology 95: 13801384 Purvis K, Brenner PF, Landgren B-M, et a1 (1975): Indices of gonadal function in the human male. I. Plasma levels of unconjugated steroids and gonadotrophins under normal and pathological conditions. Clin Endocrinol 4: 237-246
5 . Purvis K, Diczfalusy E (1976): Steroid pattern
of human seminal plasma under normal and pathological conditions. In Hubinont PO, L’Hermite, M eds: Fifth International Seminar on Reproductive Physiology and Sexual Endocrinology: Sperm Action. Basel: Karger, pp 89- 106 6. Purvis K, Landgren B-M, Cekan 2 , et al(l974): Steroids in human seminal plasma and circulating blood. J Steroid Biochem 5: 382 (abstr 361) 7. Purvis K, Landgren B-M, Cekan 2 , et al(1975): Indices of gonadal function in the human male. 11. Seminal plasma levels of steroids in normal and pathological conditions. Clin Endocrinol 4: 247-258 8. Tea NT, Grenier J and Scholler R (1976): Steroid hormones in human semen. In: Fifth International Seminar on Reproductive Physiology and Sexual Endocrinology: Sperm Action. PO Hubinont and M L’Hermite (eds) S Karger, Basel, pp 107- 114
