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A Study of Hyaluronidase in the Semen of the Husband in Infertile Marriages1
THE JOURNAL OF UROLOGY
Vol. 61, No. 4, April 1949 Printed in U.S.A.
A STUDY OF HYALURONIDASE IN THE SEMEN OF THE HUSBAND IN INFERTILE MARRIAGES 1 LEWIS MICHELSON, JOHN 0. HAMAN
AND
PETER KOETS 2
From the Department of Obstetrics and Gynecology, Stanford University School of Medicine, San Francisco, Calif.
The hyaluronidase concentration of semen in relation to the sperm count has been the subject of reports by Joel and Eichenberger and by Kurzrok, Leonard and Conrad. In the first publication a sustained increase of hyaluronidase content with increasing sperm count is apparent, no hyaluronidase is found in cases of absence of spermatozoa and very low values in hypo- and oligospermia. In the second publication no such smooth relationship between hyaluronidase concentration and sperm count is found and cases of high sperm counts with no hyaluronidase are numerous. For the following study semen specimens of the husbands of 233 infertile marriages were selected, together with those of 18 donors of recently proven fertility as controls. The patients' wives had been examined and were apparently fertile. The length of time of the barren marriages varied from 1½ to 17 years. Thirty-one, or 13.3 per cent, of the patients' semens contained no sperm and were absolutely sterile. One hundred and seven, or 45.9 per cent, had counts up to 59 million per cubic centimeter. The majority of investigators believe that any number under 60 million indicates reduced fertility. There were 95, or 40.8 per cent, specimens containing 60 million or more spermatozoa per cubic centimeter, indicating normal fertility. The hyaluronidase concentration of the semen specimens was measured by determining the rate of hydrolysis of hyaluronic acid as expressed in the decrease of its viscosity in solution. The viscosimetric apparatus was constructed along the lines as described by Werthessen, Berman, Greenberg and GargilL The viscosimeter had a bulb capacity of 0.5 cc, a capillary lumen of 0.3 mm. and a capillary length of 175 mm. The following amounts were used in each measurement: cc
Semen................................................................... Hayem's solution........................................................ Citrate buffer pH 4.6..................................................... Hyaluronic acid solution................................................
.25 .25 1.0 1.0
The quantity of semen to be used for viscosimetric analysis was diluted as soon as possible after receipt with an equal volume of Hayem's solution. The mixture was centrifuged before viscosity measurements were made. Hyaluronic acid was prepared by extracting clean umbilical cords with cold water, followed by precipitation by cold alcohol and potassium acetate, without the use of acid. The hyaluronic acid was stored in dry form and made up into 0.1 per cent solution as required. 1 Read at meeting of Western Section of American Society for Study of Sterility, San Francisco, Calif., February 7-8, 1948. 2 Agnes Lemme Schilling Fellow in Obstetrics and Gynecology.
799
800
LEWIS MICHELSON, JOHN O. HAMAN AND PETER KOETS
During the measurement of the viscosity the temperature were maintained at 30° C.
The reaction constant k for the hydrolysis process was calculated by plotting the logarithm of the time of flow against the total time elapsed after mixing semen specimen and hyaluronic acid. This is on the assumption that the reaction is a monomolecular one, which seems to hold for the initial part of the hydrolysis process. The constant k was calculated similarly for the hydrolysis of hyaluronic acid under identical conditions by three concentrations of purified hyaluronidase (enzyme preparation of Ortho Research Foundation) 3• The concentration of the semen specimen was then expressed in milligrams of the dry enzyme preparation.
l
HYALURONIDASE
mg/ cc .500
,400 ,/ ,/
. ,/ . . tt:-- /
• 3-00
/
_,,, . + •, /. ....'"!'---··
-j,/ '/
,.
+. /
/
,/
.\_>f1' ::; / .
• 200
.. ·.:.· ··. _:._;.. _: ..
/
•
• I 00:
.
;.··.:~_,.;. ~~
..
/
,"'
•
.....
f o~•_ _ _5_0_ _ _ ,o_o_ _ _,s_o_ _ _2-00_ _ _2_so---,-o-o---,s-0---.0-0---,-,--,50,----SPERM
COU~T
mllllon / cc
FIG. 1
The use of the reaction constant permits 21, more exact estimation of hyaluronidase in very low concentrations than does the "half-time" viscosity method. In figure 1 the hyaluronidase concentrations of 233 semen specimens of husbands of infertile marriages in relation to the sperm counts of the specimens are represented by full dots. In addition, the same data for 18 fertile controls are represented by crosses. The standard deviation for this series of controls was calculated to be 0.0352 mg. The broken lines in figure 1 represent the average through the control values, and the limits for these values plus and minus twice the standard deviation. The full drawn line represents the mean values for the semen specimens of the patients after the data have been averaged with class intervals of 10 million per cubic centimeter .4 3 We wish to thank Dr. C. E. Folsome of the Ortho Research Foundation for his cooperation in supplying us with the purified enzyme. 4 The advice of Dr. H. Gray with regard to the statistical calculations is gratefully acknowledged.
801
HYALURONIDASE IN SEMEN OF HUSBAND
Two facts are apparent from the graph: 1. The hyaluronidase concentration of the semen specimens does not become zero at a sperm count of zero, but reaches an average value of 0.055 milligram per cubic centimeter, indicating that this amount of enzyme is present in the semen of some patients even in the absence of spermatozoa in the ejaculate. At
,2 .22
I
,20
I
. IB
I
;·
, IG
I I
• 14
/,;·
• 12
I ,lO-
1
.o,
.02
/ I/
l/ /
_1// / /I /
I
,OG
1
/~//
// I I
./'' :
,oo
200
400
FIG. 2 TABLE
~
1
CONTROLS
PATIENTS
Y2/x
y'/x
Y'fx.
y'/x
75 150 250 400
.0057 .0059 .0061 -
.00012 .00018 .00025 -
.0052 .0053 .0054
.00015 .00020 .00025
increase
7%
108%
4%
67%
-
the intersection of the axes 13 points are located, representing specimens of azoospermia cases with no hyaluronidase. The diagnostic importance of the presence or absence of hyaluronidase in the ejaculated semen of azoospermia cases in determining the patency of the ductal systems of and from the testis will be discussed in another paper. 2. Of 95 patients with sperm counts of 60 million per cubic centimeter and higher, 21 or 22.1 per cent, show hyaluronidase concentrations below the lower
802
LEWIS MICHELSON, JOHN 0. HAMAN AND PETER KOETS
limit of the zone of controls. These abnormally low values are not experimental errors, since specimens of such patients repeated after intervals of four months maintained the same ratio between sperm count and hyaluronidase concentration. It is evident that the average of the patients' semen specimens is lowered markedly by this group. When the square of the hyaluronidase concentration (y)is plotted against the sperm count (x) the curves, representing the average values for the control semens and the patients' semens, become very nearly straight lines for sperm counts over 60 million per cubic centimeter (figure 2), indicating that the relation between hyaluronidase concentration and sperm count satisfies an equation: y2
= Cx
In table 1 the value of C is calculated both for y2/x and for y 8/x. It is clear that C is very nearly constant for y 2 = Cx, but not for y 8 = Cx. A relationship between hyaluronidase concentration and sperm count of the type y 2 = Cx may have its origin (as was suggested to us by Dr. A. E. Lewis) in the conditions of the production of the enzyme and the sperm protein. If in the synthesis of a molecule of sperm protein one molecule of a substance A participates (for instance, an amino acid), and in that of a molecule of hyaluronidase two molecules of the same substance, the amounts of hyaluronidase and of sperm protein produced in a unit of time will be in the ratio[A]2/[A], and the final concentrations will follow an equation of the type y 2 = Cx. The deviation from the linear relationship for sperm counts of less than 60 million per cc can then be looked upon as originating in a faulty synthesis of either of these proteins. SUMMARY
The semen of 233 husbands of infertile marriages and of 18 controls of recently proven fertility have been analyzed by a viscosimetric method to determine the hyaluronidase concentration per cubic centimeter. The relation between hyaluronidase concentration y (in milligrams per cc) and the sperm count x (in millions per cc) can be expressed by the equation: y2
=
.0053
X
for the patients' semen specimens, and by y 2 = .0059 X for the controls. For sperm counts lower than 60 million per cubic centimeter the hyaluronidase concentration is on the average higher than calculated by the equation. An average of .055 milligram hyaluronidase per cc is found in the semen of a number of patients in the absence of spermatozoa in the ejaculate.
490 Post St., San Francisco, Calif. REFERENCES JoEL, C. A. AND EICHENBERGER, E.: Schweiz. med. Wchnschr., 26: 601, 1945. KuRZROK, R., LEONARD, S. L. AND CONRAD, H.: Am. J. Med., 1: 491, 1946. WERTHESSEN, N. T., BERMAN, S., GREENBERG, B. E. AND GARGILL, S. L.: J. Urol., 64: 565, 1945.
Vol. 61, No. 4, April 1949 Printed in U.S.A.
A STUDY OF HYALURONIDASE IN THE SEMEN OF THE HUSBAND IN INFERTILE MARRIAGES 1 LEWIS MICHELSON, JOHN 0. HAMAN
AND
PETER KOETS 2
From the Department of Obstetrics and Gynecology, Stanford University School of Medicine, San Francisco, Calif.
The hyaluronidase concentration of semen in relation to the sperm count has been the subject of reports by Joel and Eichenberger and by Kurzrok, Leonard and Conrad. In the first publication a sustained increase of hyaluronidase content with increasing sperm count is apparent, no hyaluronidase is found in cases of absence of spermatozoa and very low values in hypo- and oligospermia. In the second publication no such smooth relationship between hyaluronidase concentration and sperm count is found and cases of high sperm counts with no hyaluronidase are numerous. For the following study semen specimens of the husbands of 233 infertile marriages were selected, together with those of 18 donors of recently proven fertility as controls. The patients' wives had been examined and were apparently fertile. The length of time of the barren marriages varied from 1½ to 17 years. Thirty-one, or 13.3 per cent, of the patients' semens contained no sperm and were absolutely sterile. One hundred and seven, or 45.9 per cent, had counts up to 59 million per cubic centimeter. The majority of investigators believe that any number under 60 million indicates reduced fertility. There were 95, or 40.8 per cent, specimens containing 60 million or more spermatozoa per cubic centimeter, indicating normal fertility. The hyaluronidase concentration of the semen specimens was measured by determining the rate of hydrolysis of hyaluronic acid as expressed in the decrease of its viscosity in solution. The viscosimetric apparatus was constructed along the lines as described by Werthessen, Berman, Greenberg and GargilL The viscosimeter had a bulb capacity of 0.5 cc, a capillary lumen of 0.3 mm. and a capillary length of 175 mm. The following amounts were used in each measurement: cc
Semen................................................................... Hayem's solution........................................................ Citrate buffer pH 4.6..................................................... Hyaluronic acid solution................................................
.25 .25 1.0 1.0
The quantity of semen to be used for viscosimetric analysis was diluted as soon as possible after receipt with an equal volume of Hayem's solution. The mixture was centrifuged before viscosity measurements were made. Hyaluronic acid was prepared by extracting clean umbilical cords with cold water, followed by precipitation by cold alcohol and potassium acetate, without the use of acid. The hyaluronic acid was stored in dry form and made up into 0.1 per cent solution as required. 1 Read at meeting of Western Section of American Society for Study of Sterility, San Francisco, Calif., February 7-8, 1948. 2 Agnes Lemme Schilling Fellow in Obstetrics and Gynecology.
799
800
LEWIS MICHELSON, JOHN O. HAMAN AND PETER KOETS
During the measurement of the viscosity the temperature were maintained at 30° C.
The reaction constant k for the hydrolysis process was calculated by plotting the logarithm of the time of flow against the total time elapsed after mixing semen specimen and hyaluronic acid. This is on the assumption that the reaction is a monomolecular one, which seems to hold for the initial part of the hydrolysis process. The constant k was calculated similarly for the hydrolysis of hyaluronic acid under identical conditions by three concentrations of purified hyaluronidase (enzyme preparation of Ortho Research Foundation) 3• The concentration of the semen specimen was then expressed in milligrams of the dry enzyme preparation.
l
HYALURONIDASE
mg/ cc .500
,400 ,/ ,/
. ,/ . . tt:-- /
• 3-00
/
_,,, . + •, /. ....'"!'---··
-j,/ '/
,.
+. /
/
,/
.\_>f1' ::; / .
• 200
.. ·.:.· ··. _:._;.. _: ..
/
•
• I 00:
.
;.··.:~_,.;. ~~
..
/
,"'
•
.....
f o~•_ _ _5_0_ _ _ ,o_o_ _ _,s_o_ _ _2-00_ _ _2_so---,-o-o---,s-0---.0-0---,-,--,50,----SPERM
COU~T
mllllon / cc
FIG. 1
The use of the reaction constant permits 21, more exact estimation of hyaluronidase in very low concentrations than does the "half-time" viscosity method. In figure 1 the hyaluronidase concentrations of 233 semen specimens of husbands of infertile marriages in relation to the sperm counts of the specimens are represented by full dots. In addition, the same data for 18 fertile controls are represented by crosses. The standard deviation for this series of controls was calculated to be 0.0352 mg. The broken lines in figure 1 represent the average through the control values, and the limits for these values plus and minus twice the standard deviation. The full drawn line represents the mean values for the semen specimens of the patients after the data have been averaged with class intervals of 10 million per cubic centimeter .4 3 We wish to thank Dr. C. E. Folsome of the Ortho Research Foundation for his cooperation in supplying us with the purified enzyme. 4 The advice of Dr. H. Gray with regard to the statistical calculations is gratefully acknowledged.
801
HYALURONIDASE IN SEMEN OF HUSBAND
Two facts are apparent from the graph: 1. The hyaluronidase concentration of the semen specimens does not become zero at a sperm count of zero, but reaches an average value of 0.055 milligram per cubic centimeter, indicating that this amount of enzyme is present in the semen of some patients even in the absence of spermatozoa in the ejaculate. At
,2 .22
I
,20
I
. IB
I
;·
, IG
I I
• 14
/,;·
• 12
I ,lO-
1
.o,
.02
/ I/
l/ /
_1// / /I /
I
,OG
1
/~//
// I I
./'' :
,oo
200
400
FIG. 2 TABLE
~
1
CONTROLS
PATIENTS
Y2/x
y'/x
Y'fx.
y'/x
75 150 250 400
.0057 .0059 .0061 -
.00012 .00018 .00025 -
.0052 .0053 .0054
.00015 .00020 .00025
increase
7%
108%
4%
67%
-
the intersection of the axes 13 points are located, representing specimens of azoospermia cases with no hyaluronidase. The diagnostic importance of the presence or absence of hyaluronidase in the ejaculated semen of azoospermia cases in determining the patency of the ductal systems of and from the testis will be discussed in another paper. 2. Of 95 patients with sperm counts of 60 million per cubic centimeter and higher, 21 or 22.1 per cent, show hyaluronidase concentrations below the lower
802
LEWIS MICHELSON, JOHN 0. HAMAN AND PETER KOETS
limit of the zone of controls. These abnormally low values are not experimental errors, since specimens of such patients repeated after intervals of four months maintained the same ratio between sperm count and hyaluronidase concentration. It is evident that the average of the patients' semen specimens is lowered markedly by this group. When the square of the hyaluronidase concentration (y)is plotted against the sperm count (x) the curves, representing the average values for the control semens and the patients' semens, become very nearly straight lines for sperm counts over 60 million per cubic centimeter (figure 2), indicating that the relation between hyaluronidase concentration and sperm count satisfies an equation: y2
= Cx
In table 1 the value of C is calculated both for y2/x and for y 8/x. It is clear that C is very nearly constant for y 2 = Cx, but not for y 8 = Cx. A relationship between hyaluronidase concentration and sperm count of the type y 2 = Cx may have its origin (as was suggested to us by Dr. A. E. Lewis) in the conditions of the production of the enzyme and the sperm protein. If in the synthesis of a molecule of sperm protein one molecule of a substance A participates (for instance, an amino acid), and in that of a molecule of hyaluronidase two molecules of the same substance, the amounts of hyaluronidase and of sperm protein produced in a unit of time will be in the ratio[A]2/[A], and the final concentrations will follow an equation of the type y 2 = Cx. The deviation from the linear relationship for sperm counts of less than 60 million per cc can then be looked upon as originating in a faulty synthesis of either of these proteins. SUMMARY
The semen of 233 husbands of infertile marriages and of 18 controls of recently proven fertility have been analyzed by a viscosimetric method to determine the hyaluronidase concentration per cubic centimeter. The relation between hyaluronidase concentration y (in milligrams per cc) and the sperm count x (in millions per cc) can be expressed by the equation: y2
=
.0053
X
for the patients' semen specimens, and by y 2 = .0059 X for the controls. For sperm counts lower than 60 million per cubic centimeter the hyaluronidase concentration is on the average higher than calculated by the equation. An average of .055 milligram hyaluronidase per cc is found in the semen of a number of patients in the absence of spermatozoa in the ejaculate.
490 Post St., San Francisco, Calif. REFERENCES JoEL, C. A. AND EICHENBERGER, E.: Schweiz. med. Wchnschr., 26: 601, 1945. KuRZROK, R., LEONARD, S. L. AND CONRAD, H.: Am. J. Med., 1: 491, 1946. WERTHESSEN, N. T., BERMAN, S., GREENBERG, B. E. AND GARGILL, S. L.: J. Urol., 64: 565, 1945.