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Placental proteins (human chorionic gonadotropin, human placental lactogen, pregnancy-specific β1-glycoprotein, and placental protein 5) in seminal plasma of normal men and patients with infertility
FERTILITY AND STERILITY Copyright © 1983 The American Fertility Society
Vol. 39, No.5, May 1983 Printed in U.SA.
Placental proteins (human chorionic gonadotropin, human placental lactogen, pregnancy-specific f31-glycoprotein, and placental protein 5) in seminal plasma of normal men and patients with infertility
Jau-Nan Lee, M.D., Ph.D.* Jong-Da Lian, M.D.t Jau-Hong Lee, M.D.:J: Tim Chard, M.D., F.R.C.O.G.§ Kaohsiung Medical College, Taichung Veterans General Hospital, China Medical College, Taiwan, Republic of China, and St. Bartholomew's Hospital Medical College, London, England
Placental proteins, including human chorionic gonadotropin (hCG), human placental lactogen (hPL), pregnancy-specific I3rglycoprotein (SP 1), and placental protein 5 (PP5) have been detected in human seminal plasma of 20 normal men and 42 patients with infertility. Levels of hPL, SP1 , and PP5 were similar in these groups. There were significantly higher levels of hCG in subjects with normal sperm counts than in those with oligospermia or azoospermia. The levels of PP5 in seminal plasma showed an association with sperm motility, suggesting that PP5 may have a significant biologic function in the maintenance of sperm motility. Fertil Steril 39:704, 1983
Human chorionic gonadotropin (hCG), human placental lactogen (hPL) , pregnancy-specific 131glycoprotein (SP 1 ), and placental protein 5 (PP5) are normal products of the placental trophoblast. However, there is evidence that they are not produced exclusively by the placenta. For example, hCG has been identified in normal testes. 1, 2 Fur-
thermore, it has recently been shown that material apparently identical to PP5 can be found in human seminal plasma. 3 In the present study, we measured the concentrations of hCG, hPL, SP b and PP5, using radioimmunoassays (RIAs), in the seminal plasma of normal men and of patients with defects of sperm production. MATERIALS AND METHODS
Received October 18, 1982; revised and accepted December 14, 1982. *Reprint requests: Dr. Jau-Nan Lee, Department of Obstetrics and Gynaecology, Kaohsiung Medical College, Kaohsiung, Taiwan, Republic of China. tDepartment of Internal Medicine, Taichung Veterans General Hospital. :j:Department of Surgery, China Medical College. §Department of Reproductive Physiology, St. Bartholomew's Hospital Medical College, London, England. 704
Lee et al.
hCG, hPL, SP I , PP5 in seminal plasma
Seminal fluid samples were obtained from 20 control subjects with normal sperm counts, 21 patients with oligospermia (counts < 20 x 106/m I), and 21 patients with azoospermia. The subjects ranged in age from 23 to 40 years, and the median of age in each group was 26, 28, and 29 years, respectively. The specimens were collected by masturbation and allowed to liquefy. A small portion of the sample was used for routine Fertility and Sterility
Table 1. Concentrations of hCG, hPL, -SPI , and PP5 in Normal Men and in Patients with Oligospermia and Azoospermia Placental protein
Concentration" Normospermic (rJ = 20)
heG hPL
SPI PP5
5.2 18.8 4.6 802.0
± 0.9 ± 0.8 ± 1.1 ± 105.3
O~08permic = 21)
3.2 21.4 4.6 846.4
± 0.3 b ± 1.0 ± 0.6 ± 116.8
Azoospermic (rJ = 21)
2.6 19.2 4.2 800.2
± ± ± ±
0.4· 0.8 0.5 112.0
aValues are micrograms per liter (mean ± standard error of the mean). bProbability of difference from normospermic group, P < 0.05. cProbability of difference from normospermic group, P < 0.01.
semen analysis. The remainder was centrifuged at 3000 RPM for 40 minutes at room temperature. The seminal plasma was decanted, aliquoted, and stored at - 20° C until assayed. Seminal plasma hCG, hPL, SP!> and PP5 were measured by RIA as described previously4-7; the minimum detection limits of each assay were 1, 5, 2, and 4 Jl.gll, respectively. In the case of hCG, a specific ~-chain antiserum (AB485-6) was employed for RIA. 4 The RIA measurement of SP1 was predominantly SPl~.8
seminal plasma were related to the sperm counts. It is known that hCG can stimulate testosterone secretion by the testes both in viV0 10. 11 and in vitro.12 In tum, testosterone plays a role in spermatogenesis: it can prevent regression of seminiferous tubules after hypophysectomyl3. 14 and reinitiate spermatogenesis once atrophy has occurred. 15 On the basis of the present results, therefore, it is possible to speculate that endogenous hCG in the normal man may play a role in spermatogenesis. Similarly, we have also shown that the levels of PP5 in seminal plasma~late to sperm motility, although not to the absolute number of motile forms. A previous report did not find this relationship, although it should be noted that the study was confined to 25 normal men. 3 PP5 is known to be associated with the coagulation and fibrinolytic systems and has antiplasmin activity.16.17 It is also known that seminal plasma contains activators of plasminogen18 and inhibitors of trypsin,19-21 which may inhibit sperm acrosomal proteases. 22 Thus, we suggest that the relatively high concentration of PP5 in human seminal plasma may have a significant biologic function in the maintenance of sperm motility.
RESULTS
Materials with the immunochemical properties ofhCG, hPL, SP!> and PP5 were detected in human seminal plasma. The concentrations of PP5 (up to 816.2 Jl.g/l) were greater than those in maternal blood in late pregnancy. 9 The levels of hPL, SP!> and PP5 in seminal plasma were similar in all clinical groups. However, the levels of hCG were below the normal in both the oligospermic and the azoospermic groups (Table 1). The levels of PP5 did not vary with sperm count, but the highest levels were associated with the highest motility at 2 to 3 hours (Fig. 1). DISCUSSION
Our results show that human seminal plasma contains substances that are immunochemically similar to materials normally considered "specific" to the placenta. The precise cellular origin has not been determined but may not necessarily be the testis, because a previous study on PP5 showed similar levels in vasectomized and nonvasectomized subjects. 3 The functional significance is also uncertain. However, the levels of hCG in Vol. 39, No.5, May 1983
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40-70;(
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Sperm motility Figure 1 The relationship between the concentration of PP5 in seminal plasma and sperm motility 2 to 3 hours after ejaculation. The bar indicates the median level of PP5.
Lee et al. kCG, hPL, SPI , PP5 in seminal plasma
705
REFERENCES 1. Braunstein GD, Rasor J, Wade WE: Presence in normal human testes of a chorionic-gonadotropin-like substance distinct from human luteinizing hormone. N Engl J Med 293:1339, 1975 2. Chowdhury M, Steinberger E: Effect of age and estrogen therapy on the immunoreactive human chorionic gonadotropin content of testes. Fertil Steril 31:328, 1979 3. Ranta T, Siiteri JE, Koistinen HT, Salem HT, Bohn H, Koskimies AI, Seppala M: Human seminal plasma contains a protein that shares physicochemical and immunochemical properties with placental protein 5 from the human placenta. J Clin Endocrinol Metab 53:1087, 1981 4. Lenton EA, Grudzinskas JG, Neal LM, Chard T, Cooke ID: Chorionic gonadotropin concentration in early human pregnancy: comparison of specific and nonspecific assays. Fertil Steril 35:40, 1981 5. Niven PAR, Landon J, Chard T: Placental lactogen levels as a guide to outcome of threatened abortion. Br Med J 3:799,1972 6. Grudzinskas JG, Gordon YB, Jeffrey D, Chard T: Concentrations of pregnancy specific beta-1-glycoprotein in maternal blood in normal pregnancy and in intrauterine growth retardation. Lancet 1:333, 1977 7. Obiekwe BC, Pendlebury DJ, Gordon YB, Grudzinskas JG, Chard T, Bohn H: The radioimmunoassay of placental protein 5 and circulating levels of maternal blood in the third trimester of normal pregnancy. Clin Chim Acta 95:509, 1979 8. Teisner B, Grudzinskas JG, Hindersson P, AI-Ani ATM, Westergaard JG, Chard T: Molecular heterogeneity of pregnancy specific glycoprotein: the effect on measurement by radioimmunoassay and electroimmunoassay. J Immunol Methods 31:141, 1979 9. Lee J-N, Salem HT, Huang SC, Ouyang PC, Seppala M, Chard T: Placental protein 5 (PP5) in severe pre-eclampsia and eclampsia. Int J Gynaecol Obstet 19:65, 1981 10. Brinck-Johnsen T, Eik-Nes KB: Effect of human chorionic gonadotropin on the secretion of testosterone and 4-androsterone-3,17-dione by the canine testis. Endocrinology 61:676, 1957
706
Lee et al.
heG, hPL, SPI> PP5 in seminal plasma
11. Saez JM, Bertrand J: Studies on testicular function in children: plasma concentrations of testosterone, dehydroepiandrosterone and its sulfate before and after stimulation with human chorionic gonadotropin. Steroids 12:749, 1968 12. Van Damme MP, Robertson DM, Diczfalusy E: An improved in vitro bioassay method for measuring luteinizing hormone (LH) activity using mouse Leydig cell preparations. Acta Endocrinol (Copenh) 77:655, 1974 13. Walsh EL, Cuyler WK, McCullagh DR: Physiologic maintenance of male sex glands: effect of androtin on hypophysectomized rats. Am J Physiol 107:508, 1934 14. Clermont Y, Harvey SC: Effects of hormones on spermatogenesis in the rat. Ciba Collog Endocrinol 16:173, 1967 15. Smith PE: Maintenance and restoration of spermatogenesis in hypophysectomized rhesus monkeys by androgen administration. Yale J BioI Med 17:281, 1944 16. Salem HT, Seppala M, Ranta T, Bohn H, Chard T: The effects of protamine on serum levels of placental protein 5 (PP5) in normal and abnormal pregnancy: a possible relation to coagulation abnormalities. Br J Obstet Gynaecol 88:367, 1981 17. Salem HT, Seppala M, Chard T: The effect of thrombin on serum placental protein 5 (PP5): is PP5 the naturally occurring antithrombin III of the human placenta? Placenta 2:205, 1981 18. Liedholm P, Astedt B, Kullander S: Passage oftranexamic acid (AMCA) to semen in man and its effect on the fibrinolytic activity and on migration of spermatozoa. Fertil Steril 24:517, 1973 19. Zaneveld LJD, Robertson RT, Kessler M, Williams WL: Inhibition of fertilization in vivo by pancreatic and seminal plasma trypsin inhibitors. J Reprod Fertil 25:387, 1971 20. Hirschhauser C, Kionke M: Properties of a human seminal plasma inhibitor for trypsin. Fertil Steril22:360, 1971 21. Liedholm P, Astedt B: Fibrinolytic inhibitors in human seminal plasma. Experientia 10:1113, 1974 22. Zaneveld LJD, Dragoje BM, Schumacher GFB: Acrosomal proteinase and proteinase inhibitor of human spermatozoa. Science 177:702, 1972
Fertility and Sterility
Vol. 39, No.5, May 1983 Printed in U.SA.
Placental proteins (human chorionic gonadotropin, human placental lactogen, pregnancy-specific f31-glycoprotein, and placental protein 5) in seminal plasma of normal men and patients with infertility
Jau-Nan Lee, M.D., Ph.D.* Jong-Da Lian, M.D.t Jau-Hong Lee, M.D.:J: Tim Chard, M.D., F.R.C.O.G.§ Kaohsiung Medical College, Taichung Veterans General Hospital, China Medical College, Taiwan, Republic of China, and St. Bartholomew's Hospital Medical College, London, England
Placental proteins, including human chorionic gonadotropin (hCG), human placental lactogen (hPL), pregnancy-specific I3rglycoprotein (SP 1), and placental protein 5 (PP5) have been detected in human seminal plasma of 20 normal men and 42 patients with infertility. Levels of hPL, SP1 , and PP5 were similar in these groups. There were significantly higher levels of hCG in subjects with normal sperm counts than in those with oligospermia or azoospermia. The levels of PP5 in seminal plasma showed an association with sperm motility, suggesting that PP5 may have a significant biologic function in the maintenance of sperm motility. Fertil Steril 39:704, 1983
Human chorionic gonadotropin (hCG), human placental lactogen (hPL) , pregnancy-specific 131glycoprotein (SP 1 ), and placental protein 5 (PP5) are normal products of the placental trophoblast. However, there is evidence that they are not produced exclusively by the placenta. For example, hCG has been identified in normal testes. 1, 2 Fur-
thermore, it has recently been shown that material apparently identical to PP5 can be found in human seminal plasma. 3 In the present study, we measured the concentrations of hCG, hPL, SP b and PP5, using radioimmunoassays (RIAs), in the seminal plasma of normal men and of patients with defects of sperm production. MATERIALS AND METHODS
Received October 18, 1982; revised and accepted December 14, 1982. *Reprint requests: Dr. Jau-Nan Lee, Department of Obstetrics and Gynaecology, Kaohsiung Medical College, Kaohsiung, Taiwan, Republic of China. tDepartment of Internal Medicine, Taichung Veterans General Hospital. :j:Department of Surgery, China Medical College. §Department of Reproductive Physiology, St. Bartholomew's Hospital Medical College, London, England. 704
Lee et al.
hCG, hPL, SP I , PP5 in seminal plasma
Seminal fluid samples were obtained from 20 control subjects with normal sperm counts, 21 patients with oligospermia (counts < 20 x 106/m I), and 21 patients with azoospermia. The subjects ranged in age from 23 to 40 years, and the median of age in each group was 26, 28, and 29 years, respectively. The specimens were collected by masturbation and allowed to liquefy. A small portion of the sample was used for routine Fertility and Sterility
Table 1. Concentrations of hCG, hPL, -SPI , and PP5 in Normal Men and in Patients with Oligospermia and Azoospermia Placental protein
Concentration" Normospermic (rJ = 20)
heG hPL
SPI PP5
5.2 18.8 4.6 802.0
± 0.9 ± 0.8 ± 1.1 ± 105.3
O~08permic = 21)
3.2 21.4 4.6 846.4
± 0.3 b ± 1.0 ± 0.6 ± 116.8
Azoospermic (rJ = 21)
2.6 19.2 4.2 800.2
± ± ± ±
0.4· 0.8 0.5 112.0
aValues are micrograms per liter (mean ± standard error of the mean). bProbability of difference from normospermic group, P < 0.05. cProbability of difference from normospermic group, P < 0.01.
semen analysis. The remainder was centrifuged at 3000 RPM for 40 minutes at room temperature. The seminal plasma was decanted, aliquoted, and stored at - 20° C until assayed. Seminal plasma hCG, hPL, SP!> and PP5 were measured by RIA as described previously4-7; the minimum detection limits of each assay were 1, 5, 2, and 4 Jl.gll, respectively. In the case of hCG, a specific ~-chain antiserum (AB485-6) was employed for RIA. 4 The RIA measurement of SP1 was predominantly SPl~.8
seminal plasma were related to the sperm counts. It is known that hCG can stimulate testosterone secretion by the testes both in viV0 10. 11 and in vitro.12 In tum, testosterone plays a role in spermatogenesis: it can prevent regression of seminiferous tubules after hypophysectomyl3. 14 and reinitiate spermatogenesis once atrophy has occurred. 15 On the basis of the present results, therefore, it is possible to speculate that endogenous hCG in the normal man may play a role in spermatogenesis. Similarly, we have also shown that the levels of PP5 in seminal plasma~late to sperm motility, although not to the absolute number of motile forms. A previous report did not find this relationship, although it should be noted that the study was confined to 25 normal men. 3 PP5 is known to be associated with the coagulation and fibrinolytic systems and has antiplasmin activity.16.17 It is also known that seminal plasma contains activators of plasminogen18 and inhibitors of trypsin,19-21 which may inhibit sperm acrosomal proteases. 22 Thus, we suggest that the relatively high concentration of PP5 in human seminal plasma may have a significant biologic function in the maintenance of sperm motility.
RESULTS
Materials with the immunochemical properties ofhCG, hPL, SP!> and PP5 were detected in human seminal plasma. The concentrations of PP5 (up to 816.2 Jl.g/l) were greater than those in maternal blood in late pregnancy. 9 The levels of hPL, SP!> and PP5 in seminal plasma were similar in all clinical groups. However, the levels of hCG were below the normal in both the oligospermic and the azoospermic groups (Table 1). The levels of PP5 did not vary with sperm count, but the highest levels were associated with the highest motility at 2 to 3 hours (Fig. 1). DISCUSSION
Our results show that human seminal plasma contains substances that are immunochemically similar to materials normally considered "specific" to the placenta. The precise cellular origin has not been determined but may not necessarily be the testis, because a previous study on PP5 showed similar levels in vasectomized and nonvasectomized subjects. 3 The functional significance is also uncertain. However, the levels of hCG in Vol. 39, No.5, May 1983
2.2
•
_ 1.8
~ E
It)
Go Go
-
•
1.4
0
c
•
• • •
-.-
-I-••
-Is•
..
0
..
:; 1.0
c
•uc
S 0.6
..• .. •.. •
0.2
• <40;(
40-70;(
• • •• ••
>70;(
Sperm motility Figure 1 The relationship between the concentration of PP5 in seminal plasma and sperm motility 2 to 3 hours after ejaculation. The bar indicates the median level of PP5.
Lee et al. kCG, hPL, SPI , PP5 in seminal plasma
705
REFERENCES 1. Braunstein GD, Rasor J, Wade WE: Presence in normal human testes of a chorionic-gonadotropin-like substance distinct from human luteinizing hormone. N Engl J Med 293:1339, 1975 2. Chowdhury M, Steinberger E: Effect of age and estrogen therapy on the immunoreactive human chorionic gonadotropin content of testes. Fertil Steril 31:328, 1979 3. Ranta T, Siiteri JE, Koistinen HT, Salem HT, Bohn H, Koskimies AI, Seppala M: Human seminal plasma contains a protein that shares physicochemical and immunochemical properties with placental protein 5 from the human placenta. J Clin Endocrinol Metab 53:1087, 1981 4. Lenton EA, Grudzinskas JG, Neal LM, Chard T, Cooke ID: Chorionic gonadotropin concentration in early human pregnancy: comparison of specific and nonspecific assays. Fertil Steril 35:40, 1981 5. Niven PAR, Landon J, Chard T: Placental lactogen levels as a guide to outcome of threatened abortion. Br Med J 3:799,1972 6. Grudzinskas JG, Gordon YB, Jeffrey D, Chard T: Concentrations of pregnancy specific beta-1-glycoprotein in maternal blood in normal pregnancy and in intrauterine growth retardation. Lancet 1:333, 1977 7. Obiekwe BC, Pendlebury DJ, Gordon YB, Grudzinskas JG, Chard T, Bohn H: The radioimmunoassay of placental protein 5 and circulating levels of maternal blood in the third trimester of normal pregnancy. Clin Chim Acta 95:509, 1979 8. Teisner B, Grudzinskas JG, Hindersson P, AI-Ani ATM, Westergaard JG, Chard T: Molecular heterogeneity of pregnancy specific glycoprotein: the effect on measurement by radioimmunoassay and electroimmunoassay. J Immunol Methods 31:141, 1979 9. Lee J-N, Salem HT, Huang SC, Ouyang PC, Seppala M, Chard T: Placental protein 5 (PP5) in severe pre-eclampsia and eclampsia. Int J Gynaecol Obstet 19:65, 1981 10. Brinck-Johnsen T, Eik-Nes KB: Effect of human chorionic gonadotropin on the secretion of testosterone and 4-androsterone-3,17-dione by the canine testis. Endocrinology 61:676, 1957
706
Lee et al.
heG, hPL, SPI> PP5 in seminal plasma
11. Saez JM, Bertrand J: Studies on testicular function in children: plasma concentrations of testosterone, dehydroepiandrosterone and its sulfate before and after stimulation with human chorionic gonadotropin. Steroids 12:749, 1968 12. Van Damme MP, Robertson DM, Diczfalusy E: An improved in vitro bioassay method for measuring luteinizing hormone (LH) activity using mouse Leydig cell preparations. Acta Endocrinol (Copenh) 77:655, 1974 13. Walsh EL, Cuyler WK, McCullagh DR: Physiologic maintenance of male sex glands: effect of androtin on hypophysectomized rats. Am J Physiol 107:508, 1934 14. Clermont Y, Harvey SC: Effects of hormones on spermatogenesis in the rat. Ciba Collog Endocrinol 16:173, 1967 15. Smith PE: Maintenance and restoration of spermatogenesis in hypophysectomized rhesus monkeys by androgen administration. Yale J BioI Med 17:281, 1944 16. Salem HT, Seppala M, Ranta T, Bohn H, Chard T: The effects of protamine on serum levels of placental protein 5 (PP5) in normal and abnormal pregnancy: a possible relation to coagulation abnormalities. Br J Obstet Gynaecol 88:367, 1981 17. Salem HT, Seppala M, Chard T: The effect of thrombin on serum placental protein 5 (PP5): is PP5 the naturally occurring antithrombin III of the human placenta? Placenta 2:205, 1981 18. Liedholm P, Astedt B, Kullander S: Passage oftranexamic acid (AMCA) to semen in man and its effect on the fibrinolytic activity and on migration of spermatozoa. Fertil Steril 24:517, 1973 19. Zaneveld LJD, Robertson RT, Kessler M, Williams WL: Inhibition of fertilization in vivo by pancreatic and seminal plasma trypsin inhibitors. J Reprod Fertil 25:387, 1971 20. Hirschhauser C, Kionke M: Properties of a human seminal plasma inhibitor for trypsin. Fertil Steril22:360, 1971 21. Liedholm P, Astedt B: Fibrinolytic inhibitors in human seminal plasma. Experientia 10:1113, 1974 22. Zaneveld LJD, Dragoje BM, Schumacher GFB: Acrosomal proteinase and proteinase inhibitor of human spermatozoa. Science 177:702, 1972
Fertility and Sterility