THE JOURNAL OF UROLOGY Copyright© 1977 by The Williams & Wilkins Co.
Vol. 118, October Printed in U.S.A.
Review Article SEMEN CRYOPRESERVATION: AN UPDATE ROY WITHERINGTON, JOHN B. BLACK
AND
ARMAND M. KAROW, JR.*
From the Section of Urology and the Department of Pharmacology, The Medical College of Georgia School of Medicine, Augusta, Georgia
ABSTRACT
Cryopreservation of semen has a place in reproductive medicine. It is done best using liquid nitrogen as the refrigerant and the pre-freeze semen should be of good quality. High quality spermatozoa survive the freezing-thawing process and ordinarily result in good babies. Abnormal sperm generally do not survive the freezing-thawing process, which, consequently, results in a more viable union and outcome from the germ cells. Cryopreservation of semen can be used to preserve semen before medical or surgical sterilization, to augment the sperm count in patients with certain types of oligospermia and to manage the childless couple by donor semen artificial insemination in those instances in which the husband is infertile. Cryopreserved semen produces babies and perhaps thousands of humans have been the result of conception that resulted from cryopreserved spermatozoa. Frozen semen is recognized widely as an important component of modern medical practice. We shall endeavor to demonstrate the usefulness of semen cryopreservation before male sterilization, in the management of oligospermia and in the childless couple. CHARACTERISTICS OF FROZEN AND THAWED SEMEN
Thawed semen produces babies. Hundreds and perhaps thousands of humans have been born as the result of conception with sperm that have been frozen. Instances of conception occurring from semen preserved longer than 10 years have been recorded. 1 The frequency of insemination needed to achieve pregnancy with thawed semen has been reported to be as much as twice that necessary for fresh semen. 2 • 3 However, in our experience the frequency of pregnancy with thawed semen containing no fewer than 65,000,000 motile sperm per ml. is comparable to the frequency achieved with artificial insemination using fresh semen. Certain special attributes exist with frozen semen that must be understood. Ordinarily, a large number of motile sperm is important in fertility. Therefore, a potential semen donor must be carefully selected to obtain a high post-thaw motility. All sperm do not survive the freezing-thawing process equally and the procedure produces a 40 to 60 per cent attrition of motile spermatozoa. Also, cryopreservation decreases the fertility potential of semen from the majority of men. 2• 3 There is no absolute correlation between motility and fertility in cryopreserved and thawed sperm.4-7 The only absolute test of post-thaw fertility is pregnancy itself. The safety of thawed semen for clinical insemination exceeds that of fresh semen. The literature indicates that abnormal spermatozoa are killed by the freezing-thawing process. Thus, only the fit and healthy sperm survive. Consequently, the incidence of birth defects and of spontaneous abortion is much lower in pregnancies resulting from thawed semen than it is in the population at large. 1 • 2 In a series of 520 children Accepted for publication April 1, 1977. * Requests for reprints: Department of Pharmacology, Medical College of Georgia, Augusta, Georgia 30902. 510
resulting from artificial donor insemination using frozen semen 5 abnormal children were born (less than 1 per cent) and 41 spontaneous abortions occurred (less than 8 per cent). In the general population the occurrence rate of congenital abnormalities is 6 per cent and spontaneous abortions terminate 10 to 15 per cent of pregnancies. However, freezing has no discernible effect on the genes. Efforts to show that cryopreservation alters functional deoxyribonucleic acid of human sperm and other types of mammalian cells have been unsuccessful. 1, s---12 Similarly, experts in animal husbandry have been unable to attribute to cryopreservation any abnormalities found in the progeny of cattle or other animals in which thawed semen had been used for artificial insemination. Long-term stability of cryopreserved sperm is dependent upon storage in liquid nitrogen at minus 196C. The rate of semen deterioration in dry ice at minus 79C is appreciable during a 1 to 2-month period. Semen storage in conventional freezers at minus lOC is unacceptable for even short intervals. The use of liquid nitrogen as a refrigerant eliminates the need for any electrical machinery and, thereby, obviates the risk of thawing and loss of stored semen during a power failure. The hazard of premature thawing can be reduced further by storing each ejaculate provided by an individual donor in a separate freezer. Techniques that keep sperm alive in the frozen state also keep gonococci 13 and treponema 14 alive. Therefore, when a single cryogenic storage vessel is used to refrigerate semen from multiple donors it is important to place in quarantine each ejaculate until laboratory tests indicate that each specimen is free of these pathogens. A serologic test is adequate for the detection of syphilis and a culture is necessary to preclude gonorrhea. These tests must be done on each ejaculate from every donor. MALE STERILIZATION
Men who are candidates for a sterilizing procedure, whether it be chemotherapy, irradiation therapy or an operation, may benefit from semen cryopreservation. In addition, men whose profession has potentially sterilizing hazards, such as working with radioactive materials, might benefit from semen cryopreservation. Whether a man undergoes elective sterilization or sterilization as a consequence of therapy he should be
SEMEN CRYOPRESERV ATION
511
offered the benefits of semen cryopreservation. This choice is requires patience. In our experience less than 20 per cent of the important from a medicolegal standpoint since at least 1 pa- general population of men willing to be semen donors are tient who underwent vasectomy sued his physician for not satisfactory. Nevertheless, a panel of cryopreserved semen is informing him that sperm banking services were available. much more convenient and safer to use than non-frozen Although semen cryopreservation is the best available method lates. A cryopreserved semen panel, in effect, makes seto extend a man's fertile period beyond some sterilizing inci- men from many donors constantly available to the physician dent, this service should be considered as a hedge against the for use at any time regardless of when the female future rather than as fertility insurance. For the pre-vasec- ovulates. A semen repository obviates a frantic hunt for fresh tomy patient semen storage would seem to be a prudent hedge semen. Indeed, the physician can select carefully for his paagainst unforeseen family tragedy. tient suitable donor characteristics of somatotype, Candidates for semen cryopreservation should have a sperm history and blood type. Additionally, the physician elimiconcentration of at least 40 million per ml. and a minimum of nates the annoying problem of highly variable sperm counts 60 per cent spermatozoa motility. 10 • u; Observation of these that one observes in most fresh semen donors. With a standards will help make possible a reasonable number of donor panel the physician can be guaranteed a minimum motile post-thaw spermatozoa, which should improve the fer- number of motile sperm any time insemination is performed. tility potential. The physician can assure himself that the semen will be Patients may be told conservatively that fertile thawed free of transmissible venereal disease and the risk of birth semen is at least as safe in producing normal babies as is fresh defects and of spontaneous abortions will be reduced. semen. However, it must be emphasized that no one can ARTIFICIAL INSEMINATION WITH THAWED SEMEN guarantee or predict the fertility of a particular ejaculate when thawed. Indeed, in couples with normal fecundity conThe timing of ovulation for artificial insemination is crucial. ception depends upon normal day to day variability of the Of course, the best indicators of ovulation currently available physiologic status of the man and woman, which precludes are the shift in basal body temperature, ferning of cervical any guarantee ofresults. Patients also should know that while mucus and spinnbarkeit. In the near future a rapid chemical freezing itself immediately reduces fertility, storage beyond 3 assay to time ovulation may be available commercially. As years may decrease fertility even more. u; For these reasons, it many as 25 per cent of women who otherwise exhibit classical is advisable to do an annual post-thaw semen analysis. signs of ovulation before artificial insemination become The decision on the number of ejaculates to store depends ovulators with irregular cycles once the insemination process somewhat upon the volume of ejaculate and the number of is initiated. This is apparent particularly when donor insemianticipated inseminations. Only 0.5 ml. semen is usually re- nation is used. This difficulty in predicting ovulation in paquired for insemination and it is convenient to freeze semen in tients with irregular cycles has led to the use of drugs to 0.5 ml. aliquots. Thus, 1 ejaculate of3 ml. volume will usually induce ovulation. provide sufficient semen for 4 or 5 inseminations. The physiCryopreserved semen usually is available in 0.5 ml. cian performing the insemination may wish to use more than or straws. Each straw is about 13 cm. long and 3 mm. m 0.5 ml. each time or to perform several inseminations during diameter and it fits into disposable insemination devices. Most the ovulatory period. We believe that most men should store 3 physicians prefer to make 2 or 3 inseminations per cycle but a ejaculates. They should understand that 3 ejaculates usually few have experienced success with only 1. Insemination shouid means more than 3 inseminations but the actual number of be done by the technique with which the physician is most inseminations will depend upon many variables. experienced and comfortable. The vagina should be clean and excessive mucus should be removed from the cervix. However, OLIGOSPERMIA enough mucus should be present so that the cervix glistens. The use of cryopreservation techniques to store semen for Semen deposition at the cervical os is probably satisfactory. subsequent concentration and insemination has been proposed After deposition at the os the semen is painted over the surface frequently as a means of managing oligospermia. However, of the cervix. Endocervical insemination can be used but there the published results are conflicting. The technique certainly is some risk of intrauterine infection with this method. If may prove to be useful in the man whose only problem is an endocervical deposition is preferred, only a small amount of abnormally low concentration of normal spermatozoa. How- semen (0.1 to 0.2 ml.) should be injected into the canal. This ever, most men with oligospermia also have an exceptionally injection should be extended for a 5 to 10-minute period and high percentage of sperm with abnormal morphology. These the remaining semen from the straw can be applied to the deformed sperm seem to be especially susceptible to freeze- external surface of the cervix. induced death. To understand this situation better we are REFERENCES currently conducting research on the cryobiology of sperm from oligospermic men. 1. Sherman, J. K.: Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil. Steril., THE CHILDLESS COUPLE WITH INFERTILE HUSBAND
The chance to nurture children to maturity is almost without exception a positive motivating factor in marriage. This desire is so natural that a couple should not be forced to forego the experience. Rearing children adds a dimension of fulfillment to one's life and holds the immediate family together. The scarcity of adoptable babies makes artificial insemination a potentially attractive method to provide a child to a couple when the husband alone is infertile. Enlightened legislation makes artificial insemination with donor semen (AID) a sensible procedure. When the husband and wife consent in writing to artificial donor insemination statutes in many states recognize them as the legal parents of any progeny resulting from the procedure and gives the progeny all legal rights and protection that one might expect. The identification of suitable artificial insemination donors
24: 397, 1973. 2. Steinberger, E. and Smith, K. D.: Artificial insemination with
fresh or frozen semen. A comparative study. J.A.M.A., 223: 778, 1973.
3. Behrman, S. J. and Sawada, Y.: Heterologous and homologous inseminations with human semen stored in a liquid-nitrogen refrigerator. Fertil. Steril., 17: 457, 1966. 4. Sherman, J. K.: Long-term cryopreservation of motility and fertility of human spermatozoa. Cryobiology, 9: 332, 1972. 5. Friberg, J. and Gemzell, C.: Inseminations of human sperm after freezing in liquid nitrogen vapors with glycerol or egg-yolk-citrate as protective media. Amer. J. Obst. 116: 330, 1973.
6. Matheson, G. W., Carlborg, L. and Gemzell, C.: Frozen human semen for artificial insemination. Amer. J. Obst. Gynec., 104: 495, 1969. 7. Bunge, R. G. and Sherman, J. K.: Fertilizing capacity of frozen human spermatozoa. Nature, 172: 767, 1953.
512
WITHERINGTON, BLACK AND KAROW
8. Shikama, K.: Effect of freezing and thawing on the stability of double helix of DNA. Nature, 207: 529, 1965. 9. Stenchever, M.A., Hempel, J. M. and MacIntyre, M. N.: Maintenance of in vitro growth ability and chromosome integrity following the deep freezing of minced fetal tissue. Cryobiology, 1: 240, 1965. 10. Ackerman, D.R. and Sod-Moriah, U. A.: DNA content of human spermatozoa after storage at low temperatures. J. Reprod. Fertil., 17: 1, 1968. 11. Sherman, J. K. and Char, F.: Stability ofY chromosome fluorescence during freeze-thawing and frozen storage of human spermatozoa. Fertil. Steril., 25: 311, 1974. 12. Hauschka, T. S., Mitchell, J. T. and Niederpruem, D. J.: A
13. 14. 15. 16.
reliable frozen tissue bank: viability and stability of 82 neoplastic and normal cell types after prolonged storage at -78° C. Cancer Res., 19: 643, 1959. Sherman, J. K. and Rosenfield, J.: Importance of frozen"stored human semen in the spread of gonorrhea. Fertil. Steril., 26: 1043, 1975. Johnson, T. W.: Syphilis. In: Infectious Diseases. Edited by P. D. Hoeprich. New York: Harper & Row Publishers, p. 540, 1972. Tyler, E.: The clinical use of frozen semen banks. Fertil. Steril., 24: 413, 1973. Smith, K. D. and Steinberger, E.: Survival of spermatozoa in a human sperm bank. Effects of long-term storage in liquid nitrogen. J.A.M.A., 223: 774, 1973.
Vol. 118, October Printed in U.S.A.
Review Article SEMEN CRYOPRESERVATION: AN UPDATE ROY WITHERINGTON, JOHN B. BLACK
AND
ARMAND M. KAROW, JR.*
From the Section of Urology and the Department of Pharmacology, The Medical College of Georgia School of Medicine, Augusta, Georgia
ABSTRACT
Cryopreservation of semen has a place in reproductive medicine. It is done best using liquid nitrogen as the refrigerant and the pre-freeze semen should be of good quality. High quality spermatozoa survive the freezing-thawing process and ordinarily result in good babies. Abnormal sperm generally do not survive the freezing-thawing process, which, consequently, results in a more viable union and outcome from the germ cells. Cryopreservation of semen can be used to preserve semen before medical or surgical sterilization, to augment the sperm count in patients with certain types of oligospermia and to manage the childless couple by donor semen artificial insemination in those instances in which the husband is infertile. Cryopreserved semen produces babies and perhaps thousands of humans have been the result of conception that resulted from cryopreserved spermatozoa. Frozen semen is recognized widely as an important component of modern medical practice. We shall endeavor to demonstrate the usefulness of semen cryopreservation before male sterilization, in the management of oligospermia and in the childless couple. CHARACTERISTICS OF FROZEN AND THAWED SEMEN
Thawed semen produces babies. Hundreds and perhaps thousands of humans have been born as the result of conception with sperm that have been frozen. Instances of conception occurring from semen preserved longer than 10 years have been recorded. 1 The frequency of insemination needed to achieve pregnancy with thawed semen has been reported to be as much as twice that necessary for fresh semen. 2 • 3 However, in our experience the frequency of pregnancy with thawed semen containing no fewer than 65,000,000 motile sperm per ml. is comparable to the frequency achieved with artificial insemination using fresh semen. Certain special attributes exist with frozen semen that must be understood. Ordinarily, a large number of motile sperm is important in fertility. Therefore, a potential semen donor must be carefully selected to obtain a high post-thaw motility. All sperm do not survive the freezing-thawing process equally and the procedure produces a 40 to 60 per cent attrition of motile spermatozoa. Also, cryopreservation decreases the fertility potential of semen from the majority of men. 2• 3 There is no absolute correlation between motility and fertility in cryopreserved and thawed sperm.4-7 The only absolute test of post-thaw fertility is pregnancy itself. The safety of thawed semen for clinical insemination exceeds that of fresh semen. The literature indicates that abnormal spermatozoa are killed by the freezing-thawing process. Thus, only the fit and healthy sperm survive. Consequently, the incidence of birth defects and of spontaneous abortion is much lower in pregnancies resulting from thawed semen than it is in the population at large. 1 • 2 In a series of 520 children Accepted for publication April 1, 1977. * Requests for reprints: Department of Pharmacology, Medical College of Georgia, Augusta, Georgia 30902. 510
resulting from artificial donor insemination using frozen semen 5 abnormal children were born (less than 1 per cent) and 41 spontaneous abortions occurred (less than 8 per cent). In the general population the occurrence rate of congenital abnormalities is 6 per cent and spontaneous abortions terminate 10 to 15 per cent of pregnancies. However, freezing has no discernible effect on the genes. Efforts to show that cryopreservation alters functional deoxyribonucleic acid of human sperm and other types of mammalian cells have been unsuccessful. 1, s---12 Similarly, experts in animal husbandry have been unable to attribute to cryopreservation any abnormalities found in the progeny of cattle or other animals in which thawed semen had been used for artificial insemination. Long-term stability of cryopreserved sperm is dependent upon storage in liquid nitrogen at minus 196C. The rate of semen deterioration in dry ice at minus 79C is appreciable during a 1 to 2-month period. Semen storage in conventional freezers at minus lOC is unacceptable for even short intervals. The use of liquid nitrogen as a refrigerant eliminates the need for any electrical machinery and, thereby, obviates the risk of thawing and loss of stored semen during a power failure. The hazard of premature thawing can be reduced further by storing each ejaculate provided by an individual donor in a separate freezer. Techniques that keep sperm alive in the frozen state also keep gonococci 13 and treponema 14 alive. Therefore, when a single cryogenic storage vessel is used to refrigerate semen from multiple donors it is important to place in quarantine each ejaculate until laboratory tests indicate that each specimen is free of these pathogens. A serologic test is adequate for the detection of syphilis and a culture is necessary to preclude gonorrhea. These tests must be done on each ejaculate from every donor. MALE STERILIZATION
Men who are candidates for a sterilizing procedure, whether it be chemotherapy, irradiation therapy or an operation, may benefit from semen cryopreservation. In addition, men whose profession has potentially sterilizing hazards, such as working with radioactive materials, might benefit from semen cryopreservation. Whether a man undergoes elective sterilization or sterilization as a consequence of therapy he should be
SEMEN CRYOPRESERV ATION
511
offered the benefits of semen cryopreservation. This choice is requires patience. In our experience less than 20 per cent of the important from a medicolegal standpoint since at least 1 pa- general population of men willing to be semen donors are tient who underwent vasectomy sued his physician for not satisfactory. Nevertheless, a panel of cryopreserved semen is informing him that sperm banking services were available. much more convenient and safer to use than non-frozen Although semen cryopreservation is the best available method lates. A cryopreserved semen panel, in effect, makes seto extend a man's fertile period beyond some sterilizing inci- men from many donors constantly available to the physician dent, this service should be considered as a hedge against the for use at any time regardless of when the female future rather than as fertility insurance. For the pre-vasec- ovulates. A semen repository obviates a frantic hunt for fresh tomy patient semen storage would seem to be a prudent hedge semen. Indeed, the physician can select carefully for his paagainst unforeseen family tragedy. tient suitable donor characteristics of somatotype, Candidates for semen cryopreservation should have a sperm history and blood type. Additionally, the physician elimiconcentration of at least 40 million per ml. and a minimum of nates the annoying problem of highly variable sperm counts 60 per cent spermatozoa motility. 10 • u; Observation of these that one observes in most fresh semen donors. With a standards will help make possible a reasonable number of donor panel the physician can be guaranteed a minimum motile post-thaw spermatozoa, which should improve the fer- number of motile sperm any time insemination is performed. tility potential. The physician can assure himself that the semen will be Patients may be told conservatively that fertile thawed free of transmissible venereal disease and the risk of birth semen is at least as safe in producing normal babies as is fresh defects and of spontaneous abortions will be reduced. semen. However, it must be emphasized that no one can ARTIFICIAL INSEMINATION WITH THAWED SEMEN guarantee or predict the fertility of a particular ejaculate when thawed. Indeed, in couples with normal fecundity conThe timing of ovulation for artificial insemination is crucial. ception depends upon normal day to day variability of the Of course, the best indicators of ovulation currently available physiologic status of the man and woman, which precludes are the shift in basal body temperature, ferning of cervical any guarantee ofresults. Patients also should know that while mucus and spinnbarkeit. In the near future a rapid chemical freezing itself immediately reduces fertility, storage beyond 3 assay to time ovulation may be available commercially. As years may decrease fertility even more. u; For these reasons, it many as 25 per cent of women who otherwise exhibit classical is advisable to do an annual post-thaw semen analysis. signs of ovulation before artificial insemination become The decision on the number of ejaculates to store depends ovulators with irregular cycles once the insemination process somewhat upon the volume of ejaculate and the number of is initiated. This is apparent particularly when donor insemianticipated inseminations. Only 0.5 ml. semen is usually re- nation is used. This difficulty in predicting ovulation in paquired for insemination and it is convenient to freeze semen in tients with irregular cycles has led to the use of drugs to 0.5 ml. aliquots. Thus, 1 ejaculate of3 ml. volume will usually induce ovulation. provide sufficient semen for 4 or 5 inseminations. The physiCryopreserved semen usually is available in 0.5 ml. cian performing the insemination may wish to use more than or straws. Each straw is about 13 cm. long and 3 mm. m 0.5 ml. each time or to perform several inseminations during diameter and it fits into disposable insemination devices. Most the ovulatory period. We believe that most men should store 3 physicians prefer to make 2 or 3 inseminations per cycle but a ejaculates. They should understand that 3 ejaculates usually few have experienced success with only 1. Insemination shouid means more than 3 inseminations but the actual number of be done by the technique with which the physician is most inseminations will depend upon many variables. experienced and comfortable. The vagina should be clean and excessive mucus should be removed from the cervix. However, OLIGOSPERMIA enough mucus should be present so that the cervix glistens. The use of cryopreservation techniques to store semen for Semen deposition at the cervical os is probably satisfactory. subsequent concentration and insemination has been proposed After deposition at the os the semen is painted over the surface frequently as a means of managing oligospermia. However, of the cervix. Endocervical insemination can be used but there the published results are conflicting. The technique certainly is some risk of intrauterine infection with this method. If may prove to be useful in the man whose only problem is an endocervical deposition is preferred, only a small amount of abnormally low concentration of normal spermatozoa. How- semen (0.1 to 0.2 ml.) should be injected into the canal. This ever, most men with oligospermia also have an exceptionally injection should be extended for a 5 to 10-minute period and high percentage of sperm with abnormal morphology. These the remaining semen from the straw can be applied to the deformed sperm seem to be especially susceptible to freeze- external surface of the cervix. induced death. To understand this situation better we are REFERENCES currently conducting research on the cryobiology of sperm from oligospermic men. 1. Sherman, J. K.: Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil. Steril., THE CHILDLESS COUPLE WITH INFERTILE HUSBAND
The chance to nurture children to maturity is almost without exception a positive motivating factor in marriage. This desire is so natural that a couple should not be forced to forego the experience. Rearing children adds a dimension of fulfillment to one's life and holds the immediate family together. The scarcity of adoptable babies makes artificial insemination a potentially attractive method to provide a child to a couple when the husband alone is infertile. Enlightened legislation makes artificial insemination with donor semen (AID) a sensible procedure. When the husband and wife consent in writing to artificial donor insemination statutes in many states recognize them as the legal parents of any progeny resulting from the procedure and gives the progeny all legal rights and protection that one might expect. The identification of suitable artificial insemination donors
24: 397, 1973. 2. Steinberger, E. and Smith, K. D.: Artificial insemination with
fresh or frozen semen. A comparative study. J.A.M.A., 223: 778, 1973.
3. Behrman, S. J. and Sawada, Y.: Heterologous and homologous inseminations with human semen stored in a liquid-nitrogen refrigerator. Fertil. Steril., 17: 457, 1966. 4. Sherman, J. K.: Long-term cryopreservation of motility and fertility of human spermatozoa. Cryobiology, 9: 332, 1972. 5. Friberg, J. and Gemzell, C.: Inseminations of human sperm after freezing in liquid nitrogen vapors with glycerol or egg-yolk-citrate as protective media. Amer. J. Obst. 116: 330, 1973.
6. Matheson, G. W., Carlborg, L. and Gemzell, C.: Frozen human semen for artificial insemination. Amer. J. Obst. Gynec., 104: 495, 1969. 7. Bunge, R. G. and Sherman, J. K.: Fertilizing capacity of frozen human spermatozoa. Nature, 172: 767, 1953.
512
WITHERINGTON, BLACK AND KAROW
8. Shikama, K.: Effect of freezing and thawing on the stability of double helix of DNA. Nature, 207: 529, 1965. 9. Stenchever, M.A., Hempel, J. M. and MacIntyre, M. N.: Maintenance of in vitro growth ability and chromosome integrity following the deep freezing of minced fetal tissue. Cryobiology, 1: 240, 1965. 10. Ackerman, D.R. and Sod-Moriah, U. A.: DNA content of human spermatozoa after storage at low temperatures. J. Reprod. Fertil., 17: 1, 1968. 11. Sherman, J. K. and Char, F.: Stability ofY chromosome fluorescence during freeze-thawing and frozen storage of human spermatozoa. Fertil. Steril., 25: 311, 1974. 12. Hauschka, T. S., Mitchell, J. T. and Niederpruem, D. J.: A
13. 14. 15. 16.
reliable frozen tissue bank: viability and stability of 82 neoplastic and normal cell types after prolonged storage at -78° C. Cancer Res., 19: 643, 1959. Sherman, J. K. and Rosenfield, J.: Importance of frozen"stored human semen in the spread of gonorrhea. Fertil. Steril., 26: 1043, 1975. Johnson, T. W.: Syphilis. In: Infectious Diseases. Edited by P. D. Hoeprich. New York: Harper & Row Publishers, p. 540, 1972. Tyler, E.: The clinical use of frozen semen banks. Fertil. Steril., 24: 413, 1973. Smith, K. D. and Steinberger, E.: Survival of spermatozoa in a human sperm bank. Effects of long-term storage in liquid nitrogen. J.A.M.A., 223: 774, 1973.
