IN VITRO FERTILIZING CAPACITY OF FRESH AND CRYOPRESERVED HUMAN SPERMATOZOA: A COMPARATIVE STUDY OF FREEZING AND THAWING PROCEDURES

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1 FERTLTY ND STERLTY Copyright c 1981 The merican Fertility Society Vol. 36, No.3, September 1981 Printed in U.S. N VTRO FERTLZNG CPCTY OF FRESH ND CRYOPRESERVED HUMN SPERMTOZO: COMPRTVE STUDY OF FREEZNG ND THWNG PROCEDURES JCQUES COHEN, M.Sc.* PUL FELTENt GERRD H. ZELM\ER, PH.D. Department of Endocrinology, Growth, and Reproduction, Medical Faculty, Erasmus University, Rotterdam, The Netherlands The capacity of human spermatozoa to penetrate zona-free hamster ova was used to investigate several methods of freezing and thawing sperm. No differences in postthaw sperm viability were obtained after cooling spermatozoa either with a complex egg yolk cryoprotective medium or glycerol. The in vitro fertilizing capacity of postthaw spermatozoa frozen with a moderate freezing rate was similar to that of spermatozoa frozen with a slow freezing rate. Significantly higher in vitro fertilization percentages were found when the spermatozoa were thawed at an environmental temperature of 22 C (mean 51%) or 37 C (mean 52%), as compared with spermatozoa thawed at 4 C (mean 37%). ndividual differences of in vitro fertilization percentages are discussed in relation to changes in motility. t is demonstrated that a high prefreeze in vitro fertilization percentage is no guarantee of a high postthaw fertilizing ability. Fertil Steril36:356, 1981 The use of cryopreserved human semen in artificial insemination offers numerous advantages over nonfrozen semen. The freezing of human sperm has been claimed to be less successful; that is, the pregnancy rate with fresh semen was shown to be higher.1-4 One of the basic problems encountered in human sperm cryopreservation is the unpredictability of postthaw sperm viability. Different sperm viability assays have been applied in the field of cryopreservation. The most commonly used viability assay is the determination of prefreeze sperm motility. Many investigators have demonstrated that the motility prior to freezing is not Received February 13, 1981; revised and accepted pril 24, *Reprint requests: Jacques Cohen, M.Sc., Department of Endocrinology, Growth, and Reproduction, Medical Faculty, Erasmus University, P.O. Box 1738, Rotterdam, The Netherlands. tduring a training period for a course in technical zoology. related to postthaw motility.5, 0 Others, however, have found a high correlation between prefreeze and postthaw motility.7 Other viability assays are, in essence, modified motility assays: cervical mucus penetration tests in vitro,8 bovine mucus penetration tests with standard freeze-stored mu CUS,9 24-hour survival rates in cervical mucus,lo and motility longevity tests.ll Repeated freezethawing of specimens is also used to study the viability of individual sperm samples.12 The evaluation of different methods of freezing and thawing, including the use of different cryoprotective media, is dependent on these viability assays.10,13 n the present study several methods employed in the cryoprotection of human semen were compared by means of the zona-free hamster ova test system.14, 15 Two rates of controlled liquid nitrogen vapor freezing and three rates of thawing were evaluated. The two semen protective diluents studied were glycerol and an egg yolk-glu- 356

2 Vol. 36, No.3 N VTRO FERTLZNG CPCTY OF FREEZE-THWED SPERM 357 cose-citrate extender. n important aspect of this study is the use of individual sperm samples, which were subjected to different procedures. The relationship between prefreezelpostthaw in vitro fertilization rates and prefreeze/postthaw motility will also be discussed. Sperm motility was assayed by subjective estimation of the percentage of motility or by an objective semiautomatic determination of sperm tracks visible on multiple-exposure photographs.16, 17 MTERLS ND METHODS Collection and Examination of Semen Fresh semen specimens were obtained from young volunteers and allowed to liquify. n aliquot was removed for routine analysis, including the determination of the percentage of motility, grading of motility, and concentration. Throughout this study the analyses were performed by the same investigator, using a Makler counting chamber (EOp, Electro-Optical, Rehovoth, srael). For evaluation of the three thawing rates the specimens were, moreover, subjected to multiple exposure photography.16, 17 semiautomatic motility analyzing system was used in which the coordinates of sperm tracks visible on photographs (Kodak plus X-pan) or slides (160 S Kodak Ektachrome) are stored by a digitizer-unit into a computer system.17 The percentage of motility and the average velocity of the semen samples were obtained on a printout sheet. Diluents and Freezing Procedures Two different protective semen diluents were used. The first was analytical grade glycerol, which was added to the semen in five steps in the course of 5 minutes with a sterile graduated syringe. The solutions were gently mixed between the additions until a 10% volume concentration was achieved. The second diluent was a more complex cryoprotective medium freshly prepared for each experiment. This medium consisted of fresh hen's egg yolk, glycerol, glucose, sodium citrate, glycine, and erythromycin.18 The mixture was inactivated, and the ph was adjusted to 7.4. The complex cryoprotective medium thus obtained was added slowly, drop by drop, to the semen and was mixed gently until a 1:1 ratio was achieved. The semen diluent mixtures were pipetted into 2-ml glass ampules and placed in an ice-water bath for 10 minutes. The open ampules were transferred to a liquid nitrogen vapor chamber (Cryoson, biological freezer) connected with a cooling speed controlling device (tram 122, Cryoson) and frozen according to one of two different trajectories. With cooling procedure a temperature of C was reached within 15 minutes (moderate cooling speed); with cooling procedure B a temperature of -100 C was reached in 57 minutes (slow cooling speed). mpules frozen with trajectory were initially cooled at a rate of 1 C/min. Between C and - 20 C the samples were cooled with a rate of 7 C per minute. mpules frozen with trajectory B were cooled to - 5 C with a freezing rate of 1 C per 4.5 minutes. Until - 42 C the rate slowly increased to 0.6 C per minute. The ampules were stored for two days in liquid nitrogen and thawed in a water bath at 37 C for 5 minutes (experiments 1 and 2) or in another way (experiment 3). n Vitro Fertilization with Zona-Free Hamster Ova Fresh or freeze-thawed semen was pipetted into plastic sterile tubes. For each 0.5 ml of semen one tube was used. Two milliliters of an embryo culture medium, TMP with 0.3% bovine serum albumin (BS),17 was carefully pipetted on the semen layer, in order to obtain a two-layer spermpurifying system as described by Hellema 19 and Lopata et a1. 20 We placed the tubes at 37 C for 1 hour to allow the motile spermatozoa to migrate into the upper layer of sperm-free medium. pproximately 1.5 ml of the upper layer was removed and pipetted into another sterile tube. This sperm fraction contained spermatozoa of high motility (50% to 100%). Embryo culture medium was added to a final volume of 15 ml, and the tubes were centrifuged for 5 minutes at 400 x g. The supernatants were removed, and the pellets were washed once again. The final pellets were suspended in TMP medium with 3% BS, and the motility of the spermatozoa was determined. The specimens were then diluted until the final concentration ranged from 1 to 3 X 106 motile spermatozoa per milliliter. 0.2-ml aliquot of this final dilution was incubated at 37 C under mineral oil (Squibb) in air for 5 hours. Female golden hamsters 3 to 5 months old were given 30 U pregnant mare serum (PMS) (Gestyl, Organon) on the day of the postoestrous discharge. Forty-eight hours later they received 20 to 25 U human chorionic gonadotropin (hcg) (Pregnyl, Organon). The animals were killed 15 to 16 hours after the last injection, and the oviducts were dissected and placed in TMP medi-

3 358 COHEN ET L. um containing 0.1 % hyaluronidase. The cumulus clots were teased out of the oviduct, and the ova were washed four times and transferred to a drop of 0.1% trypsin for 1 minute at room temperature. The zona-free ova were washed carefully in fresh medium and brought into the droplets containing the incubating spermatozoa. fter 2 hours the ova were removed and washed two or three times. The ova were examined with a phase-contrast microscope after fixation and staining with 0.25% acetocarmine. The presence of a swelling sperm head was considered to be the criterion for fertilization. The number of fertilizing spermatozoa was counted in each ovum. binding score was determined for each ovum in the following manner: no spermatozoa attached to the ovum surface, binding score 0; 1 to 5 spermatozoa, binding score 1; 6 to 10 spermatozoa, binding score 2; 11 to 15 spermatozoa, binding score 3; 16 to 20 spermatozoa, binding score 4; and 21 or more spermatozoa, binding score 5. The average binding score, or binding index, was calculated. Experiment 1: Comparison of Two Cryoprotective Media Each semen sample was divided into three parts. One part was used for studying the in vitro fertilizing capacity of the fresh ejaculate, and the two other parts were diluted with either glycerol or egg yolk extender. Both samples were frozen (slow freezing speed, program B) and thawed in a water bath at 37 C. Experiment 2: Comparison of Two Cooling Rates Donors were asked to volunteer twice. ll ejaculates were divided into two parts: one part was processed in the same way as described above, while the other part was frozen with a slow speed (first ejaculate) or with a moderate speed (second ejaculate). Experiment 3: Effects of Three Different Thawing Procedures From each semen sample an aliquot was used for photography (MEP) and determination of prefreeze fertilizing capacity. The remainder was diluted with egg yolk extender. The mixture was frozen (trajectory B) in three ampules after an aliquot had been photographed for motility analysis. The samples were thawed at the same time in three different ways: in a water bath at 37 C for 5 minutes, in air at room temperature (22 C) September 1981 for 15 minutes, and in a 5 C alcohol bath for 15 minutes. Before the samples were processed for the study of the in vitro fertilizing capacity, an aliquot was photographed for motility analysis. Statistical nalyses Data were subjected to analyses of variance. The individual groups were analyzed with the LSD test only after an overall statistical significance was obtained. The data of experiment 2 were subjected to analysis of variance with a twofactorial block design. RESULTS Experiment 1: Comparison of Two Cryoprotective Media Data evaluating the technique of sperm purification by spontaneous migration of spermatozoa into medium are presented in Table 1. The in vitro fertilizing capacity of the sperm that passed actively into the upper medium layer is compared with the nonmigrating sperm remaining in the lower layer. Only the seminal plasma layers with a concentration of more than 1 x 10 6 motile spermatozoa per milliliter after washing were studied. Spermatozoa which passed into the upper medium layer fertilized more zona-free hamster ova than spermatozoa that did not migrate into the upper layer (Table 1). This was the case in all prefreeze and 5 out of 6 postthaw suspensions. Prefreeze and postthaw in vitro fertilization percentages and motility of nine sperm samples are presented in Table 2. For each sperm sample the number of ova fertilized and the number of ova incubated are indicated. n average of26 ova was used. The in vitro fertilizing capacity offresh sperm ranged from 29% to 100% (average 83%). n most cases there was a marked decrease of the in vitro fertilizing capacity of freeze-thawed spermatozoa (Table 2). No significant differences were found between the in vitro fertilizing capacity of samples frozen with glycerol or egg yolk extender. However, the influence of the cryoprotectives was different in individual cases. The in vitro fertilization percentage of two semen samples did not decrease after freezing (donors 1 and 4), while in all cases a loss in motility could be observed (Table 2). The individual freeze-thaw viability can be compared by using the recovery rate: prefreeze motility percentage (in vitro fertilization percentage) x 100/postthaw motility percentage (in vitro fertilization percentage). Comparison of the

4 Vol. 36, No.3 N VTRO FERTLZNG CPCTY OF FREEZE-THWED SPERM 359 TBLE 1. n Vitro Fertilization Percentages of Spermatozoa Derived from the Upper Medium Layer and Lower Seminal Plasma Layer of a Simple Sperm Purifying System, in Which the Spermatozoa Migrate into the Upper Diluent Prefreeze in vitro fertilization (%) Postthaw in vitro fertilization (%) Spermatozoa from Upper layer Lower layer Glycerol Complex cryoprotective Upper Lower Upper Lower /24 (100) 22/22 (100) 29/29 (100) 32/32 (100) 32/33 (97) 27/33 (82) (64) 8/14 (54) 19/24 (75) 27/28 (97) 19/24 (80) 17/26 (65) 8/23 (35) 11/30 (37) 19/22 (86) 9/16 (56) 4/21 (19) 25/26 (96) 13/23 (56) 0/22 (0) 12/15 (80) 6/13 (56) recovery rates presented in Table 2 shows that a high motility does not correlate with a high in vitro fertilization recovery rate (donor 7, cryoprotective medium, Table 2). On the other hand, sperm with a moderate motility recovery rate (more or less 40%) can have a high in vitro fertilization recovery rate (donors 1 and 4, egg yolk extender, and donor 4, glycerol). Sperm with comparable moderate motility recovery rates sometimes had low in vitro fertilization rates, for example, sperm from donor 6 frozen with egg yolk extender and sperm from donor 7 frozen with glycerol. Experiment 2: Comparison of Two Cooling Rates No significant differences were found between the postthaw in vitro fertilizing capacity of sperm frozen at two different speeds (moderate and slow). n order to investigate the influence of two cooling rates, fresh sperm were obtained twice from each donor. The in vitro fertilization percentages were remarkably similar between cryopreserved sperm frozen at both speeds (Table 3). Experiment 3: Effects of Three Different Thawing Procedures No differences in motility (MEP) were found between fresh and cryopreserved prefreeze sperm. The percentage motility as well as the average velocity did not change significantly after addition of egg yolk extender (Table 4). The influence of freezing and thawing on the in vitro fertilizing capacity of spermatozoa was studied with sperm from 15 different donors. The prefreeze in vitro fertilization rates were significantly higher than the postthaw in vitro fertilization percentages. n 7 out of 45 postthaw sperm Donor TBLE 2. Effects of Freezing with Two Cryoprotectives, an Egg Yolk-Glucose-Citrate Extender () and 10% Glycerol (B), on the n Vitro Fertilizing Capacity and Motility of Human Spermatozoa Cryoprotective n vitro fertilization Percentage motility Recovery rate (%) medium Prefreeze" Poetthaw Prefreeze b Postthaw n vitro fer- Percentage tilization motility af = 14.6, df 2 and 16, P < bf = 55.4, df 2 and 14, P < (44%) 19/65 (44%) B 1115 (7%) /24 (100%) 4/21 (19%) B 8/21 (38%) /22 (100%) 2/32 (6%) B 3/15 (20%) /29 (100%) 25/26 (96%) B (86%) /32 (100%) 13/23 (56%) 56 B 19/22 (86%) 86 32/33 (97%) 2/17 (12%) B 3/23 (13%) /33 (82%) 3/14 (22%) B 1118 (6%) (64%) 2/15 (15%) B 0/20 (0%) /27 (11%).36/51 (71%) B 4/28 (14%)

5 360 COHEN ET L. TBLE 3. n Vitro Fertilization Percentages of 10 Semen Samples from Five Donors (Experiment 2r Slow freezing speed Moderate freezing speed Prefreeze b Postthaw b 140/142 (99 ± 8)C 44/160 (37 ± 18)d 106/129(93 ± 7)" (35 ± 12) adata subjected to a two-factorial block design analysis of variance. bmean ± standard error of the mean. cf = for P < Significant difference between prefreeze and postthaw data, df 1 and 4. df = p.325, no interaction between the two freezing procedures, df 1 and 4. samples the in vitro fertilization recovery rates were 100%. The motility recovery rates were less than 100% in all cases. Freezing did not change the average velocity of the sperm significantly (Table 5). The number of spermatozoa attached to the surface of the egg was the same in prefreeze and postthaw samples. The in vitro fertilizing capacity of sperm thawed at an environmental temperature of 4 C was significantly lower than the in vitro fertilizing ca pacity of sperm thawed at 22 or 37 C. n five cases a clear difference between in vitro fertilization percentages was found (higher than 25%). n six cases the differences were smaller than 20%. n four cases no differences between in vitro fertilization percentages of differently thawed samples could be demonstrated (Table 5). The relation between prefreeze and postthaw in vitro fertilization percentages of seven individual donors.is presented in Figure 1. The in vitro fertilizing capacity of sperm from three donors with high prefreeze in vitro fertilization percentages (higher than 50%) decreased significantly after thawing. One sample of more than 50% (R) declined to less than 10%, while another of 33% (L) did not decrease after thawing (Fig. 1). DSCUSSON The possibility of evaluating in a comparative study the different freezing methods for human spermatozoa is limited. One frozen ejaculate can be used for inseminations of only one or two women. With the present method it is possible to com- September 1981 pare the in vitro fertilizing capacity of fresh and frozen sperm samples from one donor. The fertilizing capacity of freeze-thawed sperm is generally found to be lower than that of fresh sperm in human insemination. -4 n this study maximal in vitro fertilization of human spermatozoa using zona-free hamster ova was more frequently observed when fresh sperm was compared with freeze-thawed sperm. n some cases the in vitro fertilizing capacity of freeze-thawed sperm was comparable to that of fresh sperm. These results differ from the observations of Binor et al.,21 who noticed that the percentage of fertilized eggs after exposure to fresh sperm was always higher than that following exposure to freeze-thawed spermatozoa. t is obvious from the present data that both fresh and freeze-thawed spermatozoa have a. similar capacity to bind to the surface of the denuded hamster egg. The different specimens studied here have been compared, with the use of similar motile sperm concentrations and a similar capacitation time prior to incubation with the eggs. Others have demonstrated that the concentration of motile spermatozoa present in the incubating mixture should be constant and 1 x 106/ml or higherl4, 21 in order to obtain repeatable results. The effectiveness of different diluents to protect spermatozoa against freeze damage is difficult to evaluate, since criteria for the measurement of pregnancy rates and the clinical follow-up of the inseminated women differ considerably. Many observers have suggested that glycerol can be used as a cryoprotective,22,23 while others have preferred more complex cryoprotective media containing egg yolk, citrate, fructose, and glycerol. 2, 6, 18 Most of the pregnancy rates obtained in these studies, ranged between 30% and 50%. n one trial with 83 patientslo both cryoprotective additions were compared. Out of 26 women who became pregnant, 16 women conceived after use of the more complex medium and 10 conceived with sperm frozen with glycerol. This difference was not significant, and it was therefore concludedthat the two protective diluents were similarly effective.1o Recently the two cryoprotective additions were compared in a pilot study.13 Morphologic studies suggested that the complex cryopro- TBLE 4. Effect of Dilution of Sperm with Cryoprotective Medium on the Motility (MEP) of Spermatozoa from 14 Donors Fresh semen Fresh semen diluted with cryoprotective medium F df Percentage motility verage velocity (/Lmlsec) 38 ± 6 29 ± 2 34 ± 5 29 ± NS 0.03 NS 1 and 13 1 and 13

6 Vol. 36, No.3 N VTRO FERTLZNG CPCTY OF FREEZE,THWED SPERM 361 TBLE 5. Effects of Different Thawing Temperatures on the n Vitro Fertilization and Motility (MEP) of Spermatozoa Postthaw thawing procedure at Prefreeze F Significance n 4 C. 22 C 37 C Percentage motility 34 ± 4a 12 ± 2 11 ±2 10 ± S P < 0.Ql 19 3 and 54 verage velocity 29 ± ± 1 26 ±.2 26 ± NS 19 3 and 54 (f.lmlsec) n vitro fertiliza- 75 ± sa 37 ± 7 b 51 ± S 52 ± P < and 42 tion (%) Binding index 3.4 ± S ± ± ± NS 12 3 and 33 aprefreeze data significantly higher than postthaw data for P < bsignificantdifferences between 4 0 C and 22/37 0 C for P < tective medium protected the spermatozoa better than glycerol alone. Moreover, these observers showed that the egg yolk extender affected motility less than glycerol. 13 Nevertheless, the results of the present experiment do not indicate such differences in postthaw motility. The in vitro fertilizing capacities are comparable to the pregnancy rates of different sperm banks.2, 6,18,22-24 The freezing rates used in this study are comparable to the slow-freezing speed method reported by Behrman and Sawada and the dry ice vapor method with a moderate freezing speed.6 The in vitro fertilizing capacity of freeze-thawed spermatozoa was affected by both methods in a comparable way. The individual variations observed in the present study are in agreement with the findings of Friberg and Gemzell,25 who noticed a marked individual variation rather than differences between methods. 5 (; N c 100%..,.,...,...,.,~-r----_eN (12/12) 50% - J ~4(371 ~ (4/16)Ko ~--- _ --- (5/30) GO :r:: -... (9/19) (8/18) (7/17)....c(4/19) 0% ~ ~~(~0~/~16~)-- pre-freeze post-thaw FG. 1. nfluence of freezing and thawing (22 C) on the in 0 vitro fertilizing capacity of seven donors with relatively high (e e) and relatively low ( ) prefreeze in vitro fertilization percentages. The number of eggs penetrated and the total number of eggs used are indicated. Sawada et al. 26 have compared four different thawing methods. Three of these were used in the present study. The time required to. reach 0 C is very short when the ampules are placed in a water bath of 37 C. This duration increases when a lower environmental temperature is used.26 Matheson et al.18 noticed that the motility recovery of sperm thawed at room temperature (51%) was higher than the motility recovery after the samples were thawed at 4 or 37 C. The motility recovery rates in the present study were determined by multiple exposure photography. ccording to these analyses a difference in thawing temperature does not result in a different motility. However, the fertilizing capacity of spermatozoa thawed at 4 C was significantly lower than the in vitro fertilizing capacity of spermatozoa thawed at.higher temperatures. t is well known that a high prefreeze motility is no guarantee for a high postthaw viability.5, 6,12 One of the aims of the present report was study of the influence of freezing on individual sperm samples with different prefreeze in vitro fertilization percentages. Obviously sperm with a high prefreeze in vitro fertilizing capacity do not necessarily have a high postthaw in vitro fertilization percentage. However, sperm with a moderate (30% to 50%) prefreeze in vitro fertilization capacity did not always lose this capacity after thawing. The postthaw in vitro fertilization percentages from sperm with a moderate prefreeze in vitro fertilizing capacity were sometimes higher than or comparable to those of freezethawed sperm originating from fresh sperm with a high in vitro fertilization percentage. Sperm of donors with a moderate prefreeze in vitro fertilization percentage that does not diminish by freezing may be useful for insemination. cknowledgments. Prof; v. d. Werff ten Bosch, Maarten Mooyaart, and Peter Kouwenhoven, Physiology and utomatic Signal Processing, Medical Faculty, Rotterdam, are acknowledged for their advice and support. Dr. J. H. afjes, Physiology, Erasmus University, is acknowledged for his critical reading of the manuscript. df

7 362 COHEN ET L. REFERENCES 1. nsbacher R: rtificial insemination with frozen spermatozoa. Fertil Steril 29:375, Behrman SJ, ckerman DR: Freeze preservation of human sperm. m J Obstet Gynecol 103:654, Sawada Y, ckerman DR, Behrman SJ: Motility and respiration of human spermatozoa after cooling to various low temperatures. Fertil Steril 18:775, Sherman JK: Synopsis of the use of frozen human semen since State of the art of human semen banking. Fertil Steril 24:397, melar RD, Dubin L:.For donor insemination do you prefer fresh or frozen-thawed semen? What are the bases for your preferences? nt J Fertil 25:4, Behrman SH, Sawada Y: Heterologous and homologous inseminations with human semen frozen and stored in a liquid-nitrogen refrigerator. Fertil Steril17:457, Keel B, Karow M: Motility characteristics of human sperm, nonfrozen and cryopreserved. rch ndrol 4:205, Fjallbrant B, ckerman DR: Cervical mucus penetration in vitro by fresh and frozen-preserved human semen specimens. J Reprod Fertil 20:515, Zavos PM, Cohen MR: Bovine mucus penetration test: an assay for fresh and cryopreserved human spermatozoa. Fertil Steril 34:175, Friberg J, Gemzell C: nseminations of human sperm after freezing in liquid nitrogen vapors with glycerol or glycerol egg-yolk citrate as protective media. m J Obstet Gynecol 116:330, Keel B, Karow M: Reduced motility longevity in thawed human spermatozoa. rch ndrol 4:213, nsari H: Repeated freeze-thawing for assessment of semen freezability. Fertil Steril 27:213, Harrison RF, Sheppard BL: comparative study in methods of cryoprotection for human semen. Cryobiology 17: 25, Overstreet JW, Yanagimachi R, Katz DF, Hayashi K, Hansou FW: Penetration of human spermatozoa into the human zona pellucida and the zona-free hamster egg: a study of fertile donors and infertile patients. Fertil Steril 33:534, 1980 September Yanagimachi R, Yanagimachi H, Rogers BJ: The use of zona-free animal ova as a test-system for the assessment of the fertilizing capacity of human spermatozoa. Bio Reprod 15:471, Makler : new multiple exposure photography method for objective human spermatozoal motility determination. Fertil Steril 30:192, Cohen J, Mooyaart M, Vreeburg JTM, Yanagimachi R, Zeilmaker GH: Fertilizing ability and motility of spermatozoa from fertile and infertile men after exposure to heterologous seminal plasma. n nstrumental nsemination. Clinics in ndrology, Edited by ESE Hafez. The Hague, Martinus Nyhoff, n press, Matheson GW, Carlborg L, Gemzell C: Frozen human semen for artificial insemination. m J Obstet Gynecol 104:495, Hellema HWJ, Riimke Ph: The micro-sperm immobilization test: the use of only motile spermatozoa and studies of complement. Clin Exp mmunol 31:1, Lopata, Patullo JP, Chang, James B: method for collecting motile spermatozoa from human semen. Fertil Steril 27:677, Binor Z, Sokoloski JE, Wolf DP: Penetration of the zonafree hamster egg by human sperm. Fertil Steril 33:321, Keetel WC, Bunge RG, Bradbury JT, Nelson WO: Report of pregnancies with frozen semen in infertile couples. JM 160:102, Tyler ET: The clinical use of frozen semen banks. Fertil Steril 24:413, Carl borg L: Some problems involved in freezing and insemination with human sperm. Current Problems in Fertility, Edited by ngelman-sundbeg, NO Lunnell. New York, Plenum Press, 1971, p Friberg J, Gemzell C: Sperm freezing and donor insemination. nt J Fertil 22:148, Sawada Y, ckerman DR, Behrman SJ: Motility and respiration of human spermatozoa after cooling to various low temperatures. Fertil Steril 18:775, 1967