MODERN TRENDS. Associate Editor. Edward Wallach, M.D. ARTIFICIAL INSEMINATION WITH FROZEN SPERMATOZOA*

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1 MODERN TRENDS Edward Wallach, M.D. Associate Editor FERTILITY AND STERILITY Copyright 1978 The American Fertility Society VoL 29, No.4, April 1978 Printed in U.S.A. ARTIFICIAL INSEMINATION WITH FROZEN SPERMATOZOA* Rum ANSBACHER, M.D., M.S.t Department of Obstetrics and Gynecology, Letterman Army Medical Center, Presidio of San Francisco, California The use of frozen semen for artificial insemination was established successfully by the dairy industry over 50 years ago. Quality offspring, with increased milk production, sired by the same bull, became a reality. The techniques developed for storage of bull sperm have enabled cattlemen to utilize semen from a bull long after its death. Bunge and Sherman! first reported the use of frozen human semen for artificial insemination in This report stimulated other investigators to develop methods for freezing spermatozoa for subsequent use in artificial insemination. The potential uses and associated problems of insemination with frozen semen, a comparison of conception rates with fresh and frozen semen, and sperm banking are explored in this report. ARTIFICIAL INSEMINATION DONOR Behrman 2 stated that "the only true indication for artificial insemination is a psychologically normal female married to a psychologically healthy male who has, for whatever reason, total azoospermia." To this group should be added couples in which the man has necrospermia, severe oligospermia with infertility oflong duration, or demonstrable circulating antisperm antibodies; couples with undesirable genetic factors such as Rh incompatibility or other hereditary diseases; and possibly the unwed woman. For the proper work-up of the couple and the donor; a discussion of the legal, moral, religious, Received November 16, *The opinions or assertions contained herein are the private views of the author and are not to be construed as official or as representing the views of the Department of the Army or the Department of Defense. treprint requests: Rudi Ansbacher, M.D., Department of Obstetrics and Gynecology, Letterman Army Medical Center, P.O. Box 235, San Francisco, Calif and social aspects; and the techniques of artificial insemination the reader is referred to the chapter by Behrman 3 and the review by Beck. 4 INDICATIONS FOR FROZEN SEMEN Sherman 5 outlined the possible applications for frozen semen: 1. For oligospermic men, for whom multiple ejaculates are collected and stored, subsequently thawed, and used for insemination 2. To use in four or more consecutive daily attempts to inseminate 3. For storage of desired genetic characteristics 4. For fatherhood (in old age or after death) by men whose semen was stored many years previously Sherman5 also listed theoretical applications as they pertained to population control: 1. To evaluate genetics in man on an experimentally controlled basis from generation to generation 2. To store semen from men undergoing vasectomy 3. To preserve semen in case of radiation hazard 4. To cultivate both spermatogonia and oogonia on demand, to select sex or other desired genetic characteristics, and to study in vitro fertilization and development Other authors 6 8 expanded the above applications to include the preservation of spermatozoa from the man who faces permanent injury to spermatogenesis through surgery (prostatectomy or retroperitoneal lymphadenectomy), by irradiation (Hodgkin's disease and other testicular carcinomas), or by chemotherapy (leukemia and other cancers); the use of multiple inseminations for the woman who ovulates irregularly; the use of the same donor for subsequent pregnancies (the couple successfully using artificial insemination

2 376 ANSBACHER donor usually requests the same donor); and the availability of a pool of screened donors whose physical characteristics, genetic backgrounds, and blood types are known, acceptable, and compatible with those of the proposed recipients. There could be a variety of other biomedical uses, and sperm banking provides complete flexibility regarding the time and place for insemination.7 Beck4 has stated that semen banking provides immediate availability of potentially fertile semen which greatly increases the flexibility of any donor insemination program. It also provides insurance against the unavailability of fresh donor semen. One controversial use is the manipulation for eugenic purposes as proposed by Muller9 in PROBLEMS IN FREEZING HUMAN SEMEN As in animal husbandry, an individual's semen sample cannot always be frozen properly. Variations occur in the freezing capabilities of semen from different individuals and between samples obtained from the same person or animal. No ideal protective medium has been discovered to date. Initially, semen was pretreated with 10% glycerol by volume, placed in glass ampules, frozen and suspended in liquid nitrogen, and stored in the same container.10 Subsequently, an egg yolk-glycerol-citrate solution has been utilized as a protective medium. Aliquots of semen and this cryoprotective medium are mixed and sealed in glass ampules or plastic straws, frozen to -196 C in liquid nitrogen, and preserved in those containers. II For clinical use the glass ampule or plastic straw is allowed to thaw at room temperature for 30 minutes. When cells are frozen, the following factors must be considered7: (1) damage to the cellular integrity (thermal shock as freezing ensues); (2) the latent heat of crystallization (ice crystal formation when freezing occurs); (3) dehydration (fluid loss by the cell when ice forms); (4) increased salt concentration as the water freezes, resulting in damage to cellular membranes; (5) the zone of crystallization; and (6) the critical temperature at which the sample is stored. During the thawing process the same stages are reversed and may result in damage to the cell. The addition of glycerol seems to protect against the high salt concentration produced, whereas egg yolk seems to provide protection to the cellular membranes. April 1978 Pedersen and Lebech 12 studied semen from three healthy donors before and after freezing and thawing. Pertinent changes noted with electron microscopy included disruption and an irregular pattern to the outer membrane of the acrosomal region, a swelling of the anterior segment of the acrosome, and thinning of the content of the acrosomal area which gave the outer acrosomal membrane a tortuous and discontinuous appearance. They also reported a lower density of the matrix in the midpiece of the sperm. Alexander13 described membrane surface changes after various treatments of semen samples from donors whose frozen samples previously had been successfully utilized for artificial insemination. The fewest changes occurred after pretreatment of the semen samples with 10% glycerol, and motility counts were highest when 10% glycerol was used as the cryoprotective agent. Behrman and Ackerman14 stated that a serious difficulty in the use of frozen preserved human semen for donor insemination was the inability to predict without rather extensive clinical trials which specimens would be successful in producing conception and which would not. Fjallbrant and Ackerman15 used the cervical mucus-sperm penetration test of Kremer16 to compare prefrozen with post-thaw sperm motility after the semen specimens were treated with an egg yolk-glycerol preservative medium and cooled to storage temperature as described by Behrman and Sawada.17 These investigators demonstrated that spermatozoa which were frozen and thawed immediately suffered only slight damage, whereas those frozen for any prolonged period of time had severely reduced cervical mucus-penetrating ability. The motility of good semen samples was consistently reduced by about 20% after freezing and subsequent thawing in that study. Smith and Steinberger18 noted almost a 50% reduction of the original motility of spermatozoa frozen and subsequently thawed. They stated that specimens with initial sperm counts between 40 and 200 million/ml and motilities between 600/0 and 80% appeared to withstand long-term storage best. The major motility decreases appeared in specimens that had been stored in liquid nitrogen for up to 36 months, without a further significant loss in the mean motility rate with longer storage. As outlined by Sherman10 and further discussed by Ackerman and Behrman,7 the following studies should be included in a work-up pertaining to the use of frozen semen: (1) eosin and nigrosin

3 Vol. 29, No.4 ARTIFICIAL INSEMINATION WITH FROZEN SPERMATOZOA 377 (live-dead) staining; (2) motility studies; (3) metabolic studies (oxygen consumption; citric acid, fructose, and DNA analyses); (4) cervical mucussperm penetration tests; (5) electron and scanning electron microscopic studies; and (6) determination of fertility rates. Damage to the acrosome in the freeze-thaw process appears to allow leakage of enzymes from this area, and midpiece damage may result in the loss of adenosine triphosphate. 7 Obviously, the ideal cryoprotective medium has not yet been found. More research is needed concerning the proper timing through the various stages of freezing, the temperature of the initiation of freezing, the critical temperatures at which cells may be disrupted during freezing, the rate of temperature change, the supporting medium utilized, the duration and extent of supercooling, the type of crystal structure that occurs with freezing, the rate of thawing, and the nature of the freezethaw injury. More studies are needed concerning the physics of low temperature, biochemical changes, and the physiology of germ cells as these relate to cryobiology. Alexander13 concluded that better methods of cryopreservation are needed owing to the motility loss and the reduced numbers of spermatozoa with normal morphology after freezing and thawing; she stated that "cryopreservation is detrimental to spermatozoa and often causes considerable damage to the acrosome with a leakage of acrosomal contents." conception rate with 95% of pregnancies established within the first 6 months of therapy) and Koren and Lieberman23 (80% conception rate with most occurring in the first 4 months). Unfortunately, the conception rate with frozen semen remains about 50% to 60%.2 FREEZING SEMEN SAMPLES FROM OLIGOSPERMIC MEN The use of the split ejaculate from oligospermic men, with insemination of the better fraction, was championed by Amelar and Hotchkiss24 in The freezing of the better portion of the split ejaculate, with subsequent thawing, pooling, and use for insemination-despite the hopes generated5.8-has not been effective because of the loss of spermatozoal motility. Yussman25 noted that the ability of spermatozoa to survive the free~e-thaw process depended upon the number of motile spermatozoa prior to freezing as well as upon unexplained factors affecting spermatozoal survival. In addition, Beck and Silverstein26 reported a variability from individual to individual in motility recovery after the freeze-thaw process. The collection of oligospermic semen samples, the storage of these over a period of several months, and the thawing, cooling, and concentration of the samples by centrifugation with subsequent insemination into the wife have been "spectacularly unsuccessful," according to Behrman.3 RESULTS WITH FROZEN SEMEN FOR ARTIFICIAL INSEMINATION Sherman,19 in 1973, summarized the use of sperm banking to that time: 571 births were reported with a con.genital abnormality rate of 1%, and only 50 abortions occurred in the 621 pregnancies (8%). He noted that the congenital abnormality and abortion rates were less than would be expected in the general population. Through 1975 approximately 1000 births resulting from frozen semen insemination had been reported.20 Steinberger and Smith21 achieved a conception rate of 73% with the use of fresh semen (35 pregnancies in 48 attempts) as compared with a rate of 61% with the use of frozen semen (36 pregnancies in 59 attempts). Behrman2 has stated that a 70% to 75% success rate should be achieved within 3 to 4 months, with almost 90% of the conceptions occurring within 6 months, with the use of fresh donor semen. These rates are similar to those reported by Chong and Taymor22 (72% OTHER CONSIDERATIONS Risk of Gonorrhea Transfer. The importance of testing for Neisseria gonorrhoeae in any ejaculate used for artificial insemination was demonstrated by Jennings and his co-workers.27 These investigators collected the ejaculates from 13 men with N. gonorrhoeae on urethral smears. Portions of each ejaculate were immediately plated on Transgrow medium and repeat cultures were obtained after exposure to room air 2 hours later. Of the 13 infected ejaculates, N. gonorrhoeae was present in 10 on both the initial and the delayed cultures. Ejaculates utilized for sperm freezing should also be cultured, since this organism may withstand the freeze-thaw procedure. Mixing the Husband's Semen with Donor's Semen for Artificial Insemination. Quinlivan and Sullivan have shown that the mixing of husband's semen with that of the donor can be quite detrimental to conception, and therefore should not be performed unless the husband's

4 378 ANSBACHER semen is shown to have no effect on the donor's spermatozoa. Women receiving artificial insemination should be advised to abstain from sexual intercourse, or their husbands should be advised to use a condom, for 2 days prior to the artificial donor insemination. Although not tested, the above precautions should also be followed for those undergoing artificial insemination with frozen spermatozoa. SPERM BANKS The use of commercial sperm-freezing banks was advocated in the early 1970s. Idant Corporation established such units in New York, Baltimore, Ann Arbor, and Minneapolis. Publicity concerning sperm banks was widespread, as indicated by the feature article in the November 18, 1972, issue of the Chicago Tribune, "Sperm Banks: Fatherhood on Ice." The June 27, 1971, issue of the San Francisco Chronicle publicized the city's first frozen sperm bank. The July 18, 1975, issue of the same newspaper described its demise with an article entitled "Bad Seed: How Sperm Bank Lost Its Deposits." The reasons for the nonacceptance of sperm banking by both the public and physicians include lack of interest, the cost of maintaining the frozen semen samples, and, with the freezing techniques currently employed, the inadequate fertility rates produced by using these samples as compared with those achieved with fresh donor semen. CONCLUSIONS The early enthusiasm for using frozen semen has been tempered in the past 2 years, mainly as a result of the lowered conception rates achieved as compared with those achieved with fresh donor semen. The ideal method for freezing gametes has not yet been found, and the commercialization of sperm banking has not developed as previously publicized. Despite these failings, there is a place for the use of frozen semen, especially in selected clinical situations and for investigational purposes. Sperm banks are not reliable fertility insurance for men who elect to undergo vasectomy, since there is no assurance that an individual's semen sample can tolerate the freezing procedure and subsequent thaw. 30 Further refinements must be made for freezing gametes, and long-term studies on the offspring from frozen gametes must be April 1978 undertaken to assure the lack of subsequent genetic damage. REFERENCES 1. Bunge RG, Sherman JK: Fertilizing capacity of frozen human spermatozoa. Nature 172:767, Behrman SJ: Techniques of artificial insemination. In Progress in Infertility, First Edition, Edited by SJ Behrman, RW Kistner. Boston, Little, Brown and Co, 1968, p Behrman SJ: Artificial insemination. In Progress in Infertility, Second Edition, Edited by SJ Behrman, RW Kistner. Boston, Little, Brown and Co, 1975, p Beck WW Jr: A critical look at the legal, ethical, and technical aspects of artificial insemination. Fertil Steril 27:1, Kleegman S, Amelar RD, Sherman JK, Hirschhorn K, Pilpel H: Artificial donor insemination (roundtable). Med Aspects Hum Sexuality 4:85, Beck WW Jr: Artificial insemination and semen preservation. Clin Obstet Gynecol17:115, Ackerman DR, Behrman SJ: Artificial insemination and preservation of human semen. In Progress in Infertility, Second Edition, Edited by SJ Behrman, RW Kistner. Boston, Little, Brown and Co, 1975, p Finegold WJ: Artificial Insemination, Second Edition. Springfield Ill, Charles C Thomas, 1976, 141 pp 9. Muller HJ: Human genetic betterment. In The Control of Human Heredity and Evolution, Edited by TM Sonneborn. New York, The Macmillan Co, 1965, p Sherman JK: Practical applications and technical problems of preserving spermatozoa by freezing. Fed Proc 24(15)S:288, Ackerman DR, Behrman SJ: The freezing-preservation of human sperm. Yale Sci 41:(No 5):6, Pedersen H, Lebech PE: Ultrastructural changes in the human spermatozoon after freezing for artificial insemination. Fertil Steril 22:125, Alexander NJ: Surface structure of spermatozoa frozen for artificial insemination. Andrologia 9:155, Behrman SJ, Ackerman DR: Freeze preservation of human sperm. Am J Obstet Gynecol 103:654, Fjiillbrant B, Ackerman DR: Cervical mucus penetration in vitro by fresh and frozen-preserved human semen specimens. J Reprod Fertil 20:515, Kremer J: A simple sperm penetration test. Int J Fertil 10:209, Behrman SJ, Sawada Y: Heterologous and homologous inseminations with human semen frozen and stored in a liquid-nitrogen refrigerator. Fertil Steril 17:457, Smith KD, Steinberger E: Survival of spermatozoa in a human sperm bank. Effects oflong-term storage in liquid nitrogen. JAMA 223:774, Sherman JK: Synopsis of the use of frozen human semen since 1964: state of the art of human semen banking. Fertil Steril 24:397, Sherman JK: Clinical use of frozen human semen. Transplant Proc [Suppl1] 8:165, Steinberger E, Smith KD: Artificial insemination with fresh or frozen semen. A comparative study. JAMA 223: 778, Chong AP, Taymor ML: Sixteen years' experience with therapeutic donor insemination. Fertil Steril26:791, 1975

5 Vol. 29, No.4 ARTIFICIAL INSEMINATION WITH FROZEN SPERMATOZOA Koren Z, Lieberman R: Fifteen years' experience with artificial insemination. Int J Fertil 21:119, Amelar RD, Hotchkiss RS: The split ejaculate and its use in the management of male infertility. Fertil Steril 16: 46, Yussman MA: Principles and procedures of artificial insemination. Contemp Obstet Gynecol 5:107, Beck WW Jr, Silverstein I: Variable motility recovery of spermatozoa following freeze preservation. Fertil Steril 26:863, Jennings RT, Dixon RE, Nettles JB: The risks and prevention of Neisseria gonorrhoeae transfer in fresh ejaculate donor insemination. Fertil Steril 28:554, Quinlivan WLG, Sullivan H: The immunologic effects of husband's semen on donor spermatozoa during mixed insemination. Fertil Steril 28:448, Quinlivan WLG, Sullivan H: Spermatozoal antibodies in human seminal plasma as a cause of failed artificial donor insemination. Fertil Steril 28:1082, Ansbacher R: Answers to Questions: Value of "sperm banks"-are sperm banks totally reliable "fertility insurance" for men who elect to undergo vasectomy? Med Aspects Hum Sexuality 8:163,1974