CRYOBIOLOGY
28, 246-250 (1991)
Cryosurvival
of Trichomonas
J. K. SHERMAN,* *Department
of Anatomy
Vagina/is during Human Semen
T. L. HOSTETLER,‘t
Cryopreservation
K. MCHENRY,t
and TDepartment of Microbiology for Medical Sciences, Little Rock,
and Immunology, Arkansas 72205
of
AND J. J. DALYt University
of Arkansas
Despite a 90% cryosurvival of Trichomonas vaginalis in their growth medium trypticase yeast maltose (TYM) with DMSO, none of these parasites have previously been observed to survive during cryopreservation of infected human semen with glycerol (Andrologia 18, 323 (1986)). This could have been due to the failure of the culture method used to detect low numbers of survivors. The prospects of possible transmission of T. vaginalis by artificial insemination with cryobanked (- 196°C) semen prompted an investigation of the cryosurvival of this parasite in the presence of semen with the cryoprotectant glycerol, using a more sensitive culture method for viability evaluation. Semen and seminal fluid from the same 23 ejaculates, as well as culture medium, were inoculated with small clinical numbers of T. vaginalis and evaluated as to their survival before and after cryopreservation. Results indicated: (i) The highest cryosurvival of T. vaginalis (4.5%) was in cryobanked (glycerolated) semen, (ii) semen, as well as glycerol, shows cryoprotective action, and (iii) glycerol reduced survival of parasites in semen, seminal fluid, and TYM medium during exposure prior to freezing. Clinical information on infectivity of small numbers of T. vaginalis and the data presented here suggests that these organisms could be transmitted by artificial insemination with infected cryobanked human semen. B 1991 Academic Press, Inc.
The exclusive use of cryopreserved semen has been recommended for use in artificial insemination because of advantages in screening for AIDS (1, 18). Populations of T. vaginalis show up to 90% cryosurvival during cryopreservation at - 196°C with the cryoprotectant DMSO in growth medium such as trypticase yeast maltose (TYM) (10). In the only observations with semen as the medium and with the commonly used cryoprotectant glycerol, however, it was found that no organisms survived during cryopreservation of infected human semen for cryobanking (7). It is possible that the lack of cryosurvival observed was due to the culture techniques employed which could not detect the viability of small numbers of the surviving parasites. In order to assess the potential for transmission of T. vaginalis with cryopreserved semen, we have evaluated the cryosurvival of the organism in semen processed with standard cryobanking methods, using a
Trichomonas vaginalis is a sexually transmitted protozoan that is a common cause of vaginitis and cervicitis. Recently, populations of this organism have been shown to survive and even grow in human semen and seminal fluid (2,4). Thus, it may be possible that T. vaginalis can be transmitted from an infected male donor to a female recipient by artificial insemination procedures not only when fresh (14), but also when cryopreserved semen is used. The effective drug of choice in treating trichomoniasis, metronidazole, is not recommended for use during pregnancy because of potential teratogenic and carcinogenic effects (11). An infected woman, therefore, may face an uncomfortable pregnancy as well as the possible complication of neonatal trichomonad pneumonitis in her newborn (8).
Received March 12,199O; accepted August 30,199O. 246 OOll-2240191 $3.00 Copyright 8 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
CRYOSURVIVAL
OF
Trichomonas
proven, sensitive solid medium culture method. Trichomonads were inoculated into fresh semen, seminal fluid, and TYM medium prior to cryopreservation. These thawed preparations were then evaluated for numbers of surviving organisms by a colony count technique. Relatively small numbers of trichomonads were inoculated into the semen samples to approximate the size of their populations in the clinical situation. MATERIALS
AND
METHODS
Organisms
Isolates of T. vaginalis were obtained from infected patients at the University of Arkansas for Medical Sciences Hospital. The trichomonads were maintained at 37°C in standard TYM medium supplemented with 5% inactivated human serum (5). Human
Semen and Seminal Fluid
Semen was obtained from individuals who had been previously screened as donors in the University Human Semen Cryobank. The semen samples were prepared for processing as soon as possible following ejaculation, liquefaction, and analysis. Seminal fluid was obtained, as the supernatant, following centrifugation of semen at 7000 g, for 20 min, leaving the spermatozoa in the pellet. Preparation
of Trichomonads
Late log and early stationary phase cultures of T. vaginalis were centrifuged and washed twice with phosphate-buffered saline (PBS) and resuspended in fresh PBS. The suspension was adjusted so as to contain from 2 to 4 million cells/ml. Ten microliters of this suspension were added to 1 ml of whole semen, TYM medium, and seminal fluid, with and also without the cryoprotective agent glycerol, for comparison of the cryosurvival of the organisms following freezing and thawing. The final concen-
vaginalis
247
IN SEMEN
tration of trichomonads in the samples was from 2-4 X lo4 cells/ml. Glycerolation
and Freezing
A standard proven method of cryopreservation, with rates of cooling and rewarming which provide for successful cryobanking, was employed (16, 18). Glycerol was added to each appropriate preparation by stepwise addition and mixing to a final concentration of 10% by volume. Suitably labeled plastic straws were then tilled with 0.3 ml of the glycerolated samples to provide the aliquots for treatment and evaluation of control (unfrozen) and for experimental (frozen and thawed) samples. Samples in straws, sealed with nylon plugs, in the series to be frozen, were exposed for 1 h to the vapor of liquid nitrogen in perforated aluminum tubes. Tubes were on metal canes within a sealed metal canister, which was surrounded and almost completely immersed in the liquid nitrogen (- 196”Q in a refrigerator container. Following the freezing cycle, straws in the tubes were fully immersed in liquid nitrogen in an open-ended (mesh) canister in the same refrigerator container, until removed for thawing and measurement of the cryosurvival. Samples of each preparation were obtained for evaluation of trichomonad survival within 15 min after(i) inoculation with trichomonads, (ii) final addition of glycerol, and (iii) freezing and thawing, as well as after periods of up to 9 weeks of storage in liquid nitrogen. Survival
of Trichomonas
vaginalis
The colony count technique was used to measure viability of trichomonads inoculated into semen, seminal fluid, and TYM medium before and after treatments. This technique allows for both detection of small numbers of surviving organisms and the use of small amounts of the human genital secretions or medium in evaluation. Preparation of inoculated agar plates and colony counts followed the procedures of Ivey (9),
248
SHERMAN
Matthews and Daly (IS), and Daly et al. (3). Three tubes with 10 ml of liquefied TYM medium containing 0.45% Noble agar were inoculated with aliquots from each treatment. The contents of each tube were then poured into a petri dish and allowed to harden. The plates were then placed into Gas Pak jars (BBL; Cockeysville, MD) under anaerobic conditions created by using CO, and H, generator envelopes. The jars were incubated for 3-4 days at 37”C, at which time the plates were removed, and the developed colonies counted. Data were expressed as the percentage of survival relative to the population of trichomonads in the TYM unfrozen (control) samples. Statistical tests of significance were performed using ANOVA (SAS Institute Inc., Cary, NC). Cryosurvival of spermatozoa was also observed, as is routine in semen cryobanking. RESULTS
In 18 experiments utilizing a successful technique in cryobanking of semen for artificial insemination, 4.5% of the original inoculum of T. vaginalis was found to survive freezing and thawing (Table 1). Trichomonads survived better in glycerolated semen than they did in the growth and maintenance medium (TYM) which also contained glycerol (P = 0.001). Interestingly, survival was found to be as good in semen without glycerol as it was in the TYM with glycerol. TABLE 1 Survival of Trichomonas vaginalis in Unfrozen and Frozen Human Semen and TYM Medium Treatment TYM medium TYM medium + 10% glycerol Semen Semen + 10% glycerol
Unfrozen
Post-thaw
100
0.01 2 0.01
42 k 8 61 + 8
0.15 2 0.10 0.23 + 0.11
48 k 8
4.50 k 1.30
Note. Mean percentage ? SEM is relative to viable cells in unfrozen TYM medium as 100%. Number of experiments = 18.
ET
AL.
Both of these findings indicate that semen exhibits cryoprotective qualities with trichomonads and helps explain the increased cryosurvival of T. vaginalis in semen with glycerol. Very poor cryosurvival was observed, however, in TYM medium without the addition of glycerol. Decreased survival of trichomonads was noted in unfrozen preparations in semen alone, semen + glycerol, and in TYM + glycerol (P = O.OOOl), compared with that in TYM without glycerol. An average of 72% of the spermatozoa survived the standard cryopreservation methods with glycerol, as expected. In 9 experiments, 3.6% of the original inoculum survived after initial freezing and thawing in semen with glycerol with no statistically significant loss after 9 weeks of storage (Table 2). It was decided to examine further the effect of semen with and without glycerol on the reduction of survival of unfrozen populations of T. vaginalis in a clinical isolate from another patient. Seminal fluid was also tested for its effect on initial prefreeze survival of trichomonads. In 5 experiments with unfrozen preparations (Table 3) no statistically significant difference was found between the number of surviving organisms in semen (P = 0.079) and seminal fluid (P = 0.051) when compared with the numbers of cells in the TYM medium. However, addition of glycerol again was noted to cause a reduction of surviving organisms in semen (P = O.OOOl), seminal fluid (P = TABLE 2 Cryosurvival of Trichomonas vaginalis in Glycerolated Human Semen Treatment
Unfrozen
Oh Post-thaw
9 Weeks Post-thaw
Semen + 10% glycerol
43 ?I 15
3.6 k 1.6
2.4 + 0.90
Note. Mean percentage + SEM is relative to viable cells in unfrozen TYM medium as 100%. ANOVA: No significant differences between post-thaws, with P > 0.05. Number of experiments = 9.
CRYOSURVIVAL
TABLE
OF
Trichomonas
3
Survival of Trichomonas vaginalis in Unfrozen Semen, Seminal Fluid, and TYM Medium Treatment
Unfrozen survival
TYM medium (T) TYM medium + 10% glycerol (T’G) Semen (S) Semen + 10% glycerol (SG) Seminal fluid (SF) Seminal fluid + 10% glycerol (SFG)
100 26 12 17 70
k f k *
12 14 9 13
19 f 8
Note. Mean percentage * SEM is relative to viable cells in unfrozen TYM medium as 100%. ANOVA: Not significant (P > 0.05) for T:S, T:SF, S:SF, SG:TG, SG:SFG. All others significant with P < 0.05. Number of experiments = 5.
O.OOOl),
and the TYM
medium
(P =
0.0003). DISCUSSION
Cryopreservation of trichomonad strains is routine, but not with glycerol as the cryoprotectant (13). The present work demonstrates that glycerol has an initial adverse affect, during contact prior to freezing, on the survival of T. vaginalis in TYM, semen, and seminal fluid which may, in part, account for the poor cryosurvival with glycerol as a cryoprotectant. There is the possibility of osmotic shock in spite of glycerol’s stepwise addition, as well as during dilution in plating after freezing and thawing. Glycerol is also known to be a metabolic end product of T. vaginalis (19) and in large quantities may be toxic to the organisms. In early attempts, using colonycounts as the criterion for viability, Ivey reported that cryosurvival of T. vaginalis in glycerolated TYM medium was approximately 0.2% (10). This low recovery is compatible with that in the data on TYM presented here. Use of dimethyl sulfoxide (DMSO), however, is the cryopreservative of choice for T. vaginalis strains, effecting over 90% cryosurvival (6, 10). Moreover, such cryopreserved organisms retain their
vaginalis
IN
SEMEN
249
pathogenicity, as indicated by a mousevirulence assay (6, 12). In the present study with glycerol, cryosurvival of trichomonads was enhanced by human semen when compared with TYM medium. Also, in semen without glycerol, cryosurvival was greater than in the TYM medium with this cryoprotectant. Semen, therefore, appears to aid in the cryoprotection of T. vaginalis and thus, in this application at least, can be considered a cryoprotectant. The data regarding the decrease in the numbers of trichomonads in unfrozen semen and seminal fluid were not entirely in statistical agreement, showing significant differences with one strain, but not another. However, the probability values obtained in these experiments still indicate a trend toward a reduction of viable trichomonads in both unfrozen semen and seminal fluid samples when compared to viable numbers in the TYM control medium (unfrozen). Such an initial reduction of viable cells may be due to a change in medium conditions or a response possibly to inhibitory substances in semen. Presence of inhibitory substances is not supported, however, by the finding that populations of T. vaginalis grow and survive in semen at 37°C for up to 24 h (2, 4). It is not known whether there will normally be enough viable trichomonads in cryobanked insemination units of infected ejaculates to establish infection by T. vaginalis in the recipient. The absence of an animal model for such studies with T. vaginalis precludes experimental assessment of the numbers of trichomonads required to establish themselves in the vagina. However, the relatively low numbers or organisms associated with males who lack symptoms, combined with the epidemiological evidence that these males can transmit the organism (ll), suggests that the numbers needed for establishment may be fairly small. Therefore, in the absence of facts to the contrary and along with the cryosurviva1 data presented in this report, T. va-
250
SHERMAN
gin& seems capable of being transmitted by artificial insemination with cryobanked human semen. REFERENCES
1. Centers for Disease Control. Semen banking, organ and tissue transplantation and HIV antibody testing. MMWR 37, 57 (1988). 2. Daly, J. J., Sherman, J. K., Green, L., and Hostetler, T. L. Survival of Trichomonas vaginalis in human semen. Genitourin. Med. 65, ltkXO8 (1989). 3. Daly, J. J., Baker, M. L., and Burton, S. B. The sensitivity of Trichomonas vaginalis and Trichomonas gallinae to ultraviolet radiation. Photochem. Photobiol. 33, 191-196 (1981). 4. Daly, J. J., Sherman, J. K., Haley, T., and Hostetler, T. L. Differences in the effect of dog seminal fluid and human seminal fluid and semen on In vitro survival of Trichomonas vaginalis. Sex. Transm. Dis. 17, 106109 (1990). 5. Diamond, L. S. The establishment of various trichomonads of animals and man in axenic culture. J. Parasitol. 43, 48U90 (1957). 6. Diamond, L. S., Bartges, I. L., and Reardon, L. V. Virulence of Trichomonas vaginalis after freeze-preservation for 2 years in liquid nitrogen vapor. Cryobiology 1, 295-297 (1965). 7. Glander, H. J., Rytter, M., Baumann, L., and Schonbom, C. Risk of transmission of sexually transmitted diseases by cryopreserved semen. Andrologia 18, 323-326 (1986). 8. Honigberg, B. M. Trichomonads found outside the urogenital tract of humans. In “Trichomonads Parasitic in Humans” (B. Honigberg, Ed.), pp. 342-393. Springer-Verlag, New York, 1990. 9. Ivey, M. H. Growth characteristics of clones of
ET AL. Trichomonas vaginalis in solid asitol. 47, 539-544 (1961).
medium. J. Par-
10. Ivey, M. H. Use of solid medium techniques to evaluate factors affecting the ability of Trichomonas vaginalis to survive freezing. J. Parasitol. 61, 1101-1103 (1975). 11. Krieger, J. N. Urologic aspects of trichomoniasis. Invest. Ural. 18, 411417 (1981). 12. Lindgren, R. D., and Ivey, M. H. The effect of cultivation and freezing on the virulence of Trichomonas vaginalis for mice. J. Parasitol. 50, 226-229 (1964). 13. Linstead, D. Cultivation of trichomonads parasitic in humans. In “Trichomonads Parasitic in Humans” (B. Honigberg, Ed.), pp. 91-111. Springer-Verlag, New York 1990. 14. Mascola, L., and Guinan, M. E. Screening to reduce transmission of sexually transmitted diseases in semen used for artificial insemination. N. Engl. J. Med. 314, 1354-1359 (1986). 15. Matthews, R. S., and Daly, J. J. Trichomonas gallinae: Use of solid medium to test survival under various environmental conditions. Exp. Parasitol. 36, 288-298 (1974). 16. Sherman, J. K. Cryopreservation of human semen. In “Techniques of Human Andrology” (E. Hafez, Ed.), pp. 395c420. Elsevier NorthHolland, New York/Amsterdam, 1977. 17. Sherman, J. K. Frozen semen: Efficiency in artificial insemination and advantage in testing for acquired immune deficiency syndrome. Fertil. Steril. 47, 19-21 (1987). 18. Sherman, J. K. Cryopreservation of human semen. In “Handbook of the Laboratory Diagnosis and Treatment of Infertility” (B. A. Keel and B. W. Webster, Eds.), pp. 229-259. CRC Press, Boca Raton, 1990. 19. Steinbuchel, A., and Miiller, M. Glycerol, a metabolic end product of Trichomonas vaginalis and Tritrichomonas foetus. Mol. Biochem. Parasitol. 20, 45-55 (1986).
28, 246-250 (1991)
Cryosurvival
of Trichomonas
J. K. SHERMAN,* *Department
of Anatomy
Vagina/is during Human Semen
T. L. HOSTETLER,‘t
Cryopreservation
K. MCHENRY,t
and TDepartment of Microbiology for Medical Sciences, Little Rock,
and Immunology, Arkansas 72205
of
AND J. J. DALYt University
of Arkansas
Despite a 90% cryosurvival of Trichomonas vaginalis in their growth medium trypticase yeast maltose (TYM) with DMSO, none of these parasites have previously been observed to survive during cryopreservation of infected human semen with glycerol (Andrologia 18, 323 (1986)). This could have been due to the failure of the culture method used to detect low numbers of survivors. The prospects of possible transmission of T. vaginalis by artificial insemination with cryobanked (- 196°C) semen prompted an investigation of the cryosurvival of this parasite in the presence of semen with the cryoprotectant glycerol, using a more sensitive culture method for viability evaluation. Semen and seminal fluid from the same 23 ejaculates, as well as culture medium, were inoculated with small clinical numbers of T. vaginalis and evaluated as to their survival before and after cryopreservation. Results indicated: (i) The highest cryosurvival of T. vaginalis (4.5%) was in cryobanked (glycerolated) semen, (ii) semen, as well as glycerol, shows cryoprotective action, and (iii) glycerol reduced survival of parasites in semen, seminal fluid, and TYM medium during exposure prior to freezing. Clinical information on infectivity of small numbers of T. vaginalis and the data presented here suggests that these organisms could be transmitted by artificial insemination with infected cryobanked human semen. B 1991 Academic Press, Inc.
The exclusive use of cryopreserved semen has been recommended for use in artificial insemination because of advantages in screening for AIDS (1, 18). Populations of T. vaginalis show up to 90% cryosurvival during cryopreservation at - 196°C with the cryoprotectant DMSO in growth medium such as trypticase yeast maltose (TYM) (10). In the only observations with semen as the medium and with the commonly used cryoprotectant glycerol, however, it was found that no organisms survived during cryopreservation of infected human semen for cryobanking (7). It is possible that the lack of cryosurvival observed was due to the culture techniques employed which could not detect the viability of small numbers of the surviving parasites. In order to assess the potential for transmission of T. vaginalis with cryopreserved semen, we have evaluated the cryosurvival of the organism in semen processed with standard cryobanking methods, using a
Trichomonas vaginalis is a sexually transmitted protozoan that is a common cause of vaginitis and cervicitis. Recently, populations of this organism have been shown to survive and even grow in human semen and seminal fluid (2,4). Thus, it may be possible that T. vaginalis can be transmitted from an infected male donor to a female recipient by artificial insemination procedures not only when fresh (14), but also when cryopreserved semen is used. The effective drug of choice in treating trichomoniasis, metronidazole, is not recommended for use during pregnancy because of potential teratogenic and carcinogenic effects (11). An infected woman, therefore, may face an uncomfortable pregnancy as well as the possible complication of neonatal trichomonad pneumonitis in her newborn (8).
Received March 12,199O; accepted August 30,199O. 246 OOll-2240191 $3.00 Copyright 8 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
CRYOSURVIVAL
OF
Trichomonas
proven, sensitive solid medium culture method. Trichomonads were inoculated into fresh semen, seminal fluid, and TYM medium prior to cryopreservation. These thawed preparations were then evaluated for numbers of surviving organisms by a colony count technique. Relatively small numbers of trichomonads were inoculated into the semen samples to approximate the size of their populations in the clinical situation. MATERIALS
AND
METHODS
Organisms
Isolates of T. vaginalis were obtained from infected patients at the University of Arkansas for Medical Sciences Hospital. The trichomonads were maintained at 37°C in standard TYM medium supplemented with 5% inactivated human serum (5). Human
Semen and Seminal Fluid
Semen was obtained from individuals who had been previously screened as donors in the University Human Semen Cryobank. The semen samples were prepared for processing as soon as possible following ejaculation, liquefaction, and analysis. Seminal fluid was obtained, as the supernatant, following centrifugation of semen at 7000 g, for 20 min, leaving the spermatozoa in the pellet. Preparation
of Trichomonads
Late log and early stationary phase cultures of T. vaginalis were centrifuged and washed twice with phosphate-buffered saline (PBS) and resuspended in fresh PBS. The suspension was adjusted so as to contain from 2 to 4 million cells/ml. Ten microliters of this suspension were added to 1 ml of whole semen, TYM medium, and seminal fluid, with and also without the cryoprotective agent glycerol, for comparison of the cryosurvival of the organisms following freezing and thawing. The final concen-
vaginalis
247
IN SEMEN
tration of trichomonads in the samples was from 2-4 X lo4 cells/ml. Glycerolation
and Freezing
A standard proven method of cryopreservation, with rates of cooling and rewarming which provide for successful cryobanking, was employed (16, 18). Glycerol was added to each appropriate preparation by stepwise addition and mixing to a final concentration of 10% by volume. Suitably labeled plastic straws were then tilled with 0.3 ml of the glycerolated samples to provide the aliquots for treatment and evaluation of control (unfrozen) and for experimental (frozen and thawed) samples. Samples in straws, sealed with nylon plugs, in the series to be frozen, were exposed for 1 h to the vapor of liquid nitrogen in perforated aluminum tubes. Tubes were on metal canes within a sealed metal canister, which was surrounded and almost completely immersed in the liquid nitrogen (- 196”Q in a refrigerator container. Following the freezing cycle, straws in the tubes were fully immersed in liquid nitrogen in an open-ended (mesh) canister in the same refrigerator container, until removed for thawing and measurement of the cryosurvival. Samples of each preparation were obtained for evaluation of trichomonad survival within 15 min after(i) inoculation with trichomonads, (ii) final addition of glycerol, and (iii) freezing and thawing, as well as after periods of up to 9 weeks of storage in liquid nitrogen. Survival
of Trichomonas
vaginalis
The colony count technique was used to measure viability of trichomonads inoculated into semen, seminal fluid, and TYM medium before and after treatments. This technique allows for both detection of small numbers of surviving organisms and the use of small amounts of the human genital secretions or medium in evaluation. Preparation of inoculated agar plates and colony counts followed the procedures of Ivey (9),
248
SHERMAN
Matthews and Daly (IS), and Daly et al. (3). Three tubes with 10 ml of liquefied TYM medium containing 0.45% Noble agar were inoculated with aliquots from each treatment. The contents of each tube were then poured into a petri dish and allowed to harden. The plates were then placed into Gas Pak jars (BBL; Cockeysville, MD) under anaerobic conditions created by using CO, and H, generator envelopes. The jars were incubated for 3-4 days at 37”C, at which time the plates were removed, and the developed colonies counted. Data were expressed as the percentage of survival relative to the population of trichomonads in the TYM unfrozen (control) samples. Statistical tests of significance were performed using ANOVA (SAS Institute Inc., Cary, NC). Cryosurvival of spermatozoa was also observed, as is routine in semen cryobanking. RESULTS
In 18 experiments utilizing a successful technique in cryobanking of semen for artificial insemination, 4.5% of the original inoculum of T. vaginalis was found to survive freezing and thawing (Table 1). Trichomonads survived better in glycerolated semen than they did in the growth and maintenance medium (TYM) which also contained glycerol (P = 0.001). Interestingly, survival was found to be as good in semen without glycerol as it was in the TYM with glycerol. TABLE 1 Survival of Trichomonas vaginalis in Unfrozen and Frozen Human Semen and TYM Medium Treatment TYM medium TYM medium + 10% glycerol Semen Semen + 10% glycerol
Unfrozen
Post-thaw
100
0.01 2 0.01
42 k 8 61 + 8
0.15 2 0.10 0.23 + 0.11
48 k 8
4.50 k 1.30
Note. Mean percentage ? SEM is relative to viable cells in unfrozen TYM medium as 100%. Number of experiments = 18.
ET
AL.
Both of these findings indicate that semen exhibits cryoprotective qualities with trichomonads and helps explain the increased cryosurvival of T. vaginalis in semen with glycerol. Very poor cryosurvival was observed, however, in TYM medium without the addition of glycerol. Decreased survival of trichomonads was noted in unfrozen preparations in semen alone, semen + glycerol, and in TYM + glycerol (P = O.OOOl), compared with that in TYM without glycerol. An average of 72% of the spermatozoa survived the standard cryopreservation methods with glycerol, as expected. In 9 experiments, 3.6% of the original inoculum survived after initial freezing and thawing in semen with glycerol with no statistically significant loss after 9 weeks of storage (Table 2). It was decided to examine further the effect of semen with and without glycerol on the reduction of survival of unfrozen populations of T. vaginalis in a clinical isolate from another patient. Seminal fluid was also tested for its effect on initial prefreeze survival of trichomonads. In 5 experiments with unfrozen preparations (Table 3) no statistically significant difference was found between the number of surviving organisms in semen (P = 0.079) and seminal fluid (P = 0.051) when compared with the numbers of cells in the TYM medium. However, addition of glycerol again was noted to cause a reduction of surviving organisms in semen (P = O.OOOl), seminal fluid (P = TABLE 2 Cryosurvival of Trichomonas vaginalis in Glycerolated Human Semen Treatment
Unfrozen
Oh Post-thaw
9 Weeks Post-thaw
Semen + 10% glycerol
43 ?I 15
3.6 k 1.6
2.4 + 0.90
Note. Mean percentage + SEM is relative to viable cells in unfrozen TYM medium as 100%. ANOVA: No significant differences between post-thaws, with P > 0.05. Number of experiments = 9.
CRYOSURVIVAL
TABLE
OF
Trichomonas
3
Survival of Trichomonas vaginalis in Unfrozen Semen, Seminal Fluid, and TYM Medium Treatment
Unfrozen survival
TYM medium (T) TYM medium + 10% glycerol (T’G) Semen (S) Semen + 10% glycerol (SG) Seminal fluid (SF) Seminal fluid + 10% glycerol (SFG)
100 26 12 17 70
k f k *
12 14 9 13
19 f 8
Note. Mean percentage * SEM is relative to viable cells in unfrozen TYM medium as 100%. ANOVA: Not significant (P > 0.05) for T:S, T:SF, S:SF, SG:TG, SG:SFG. All others significant with P < 0.05. Number of experiments = 5.
O.OOOl),
and the TYM
medium
(P =
0.0003). DISCUSSION
Cryopreservation of trichomonad strains is routine, but not with glycerol as the cryoprotectant (13). The present work demonstrates that glycerol has an initial adverse affect, during contact prior to freezing, on the survival of T. vaginalis in TYM, semen, and seminal fluid which may, in part, account for the poor cryosurvival with glycerol as a cryoprotectant. There is the possibility of osmotic shock in spite of glycerol’s stepwise addition, as well as during dilution in plating after freezing and thawing. Glycerol is also known to be a metabolic end product of T. vaginalis (19) and in large quantities may be toxic to the organisms. In early attempts, using colonycounts as the criterion for viability, Ivey reported that cryosurvival of T. vaginalis in glycerolated TYM medium was approximately 0.2% (10). This low recovery is compatible with that in the data on TYM presented here. Use of dimethyl sulfoxide (DMSO), however, is the cryopreservative of choice for T. vaginalis strains, effecting over 90% cryosurvival (6, 10). Moreover, such cryopreserved organisms retain their
vaginalis
IN
SEMEN
249
pathogenicity, as indicated by a mousevirulence assay (6, 12). In the present study with glycerol, cryosurvival of trichomonads was enhanced by human semen when compared with TYM medium. Also, in semen without glycerol, cryosurvival was greater than in the TYM medium with this cryoprotectant. Semen, therefore, appears to aid in the cryoprotection of T. vaginalis and thus, in this application at least, can be considered a cryoprotectant. The data regarding the decrease in the numbers of trichomonads in unfrozen semen and seminal fluid were not entirely in statistical agreement, showing significant differences with one strain, but not another. However, the probability values obtained in these experiments still indicate a trend toward a reduction of viable trichomonads in both unfrozen semen and seminal fluid samples when compared to viable numbers in the TYM control medium (unfrozen). Such an initial reduction of viable cells may be due to a change in medium conditions or a response possibly to inhibitory substances in semen. Presence of inhibitory substances is not supported, however, by the finding that populations of T. vaginalis grow and survive in semen at 37°C for up to 24 h (2, 4). It is not known whether there will normally be enough viable trichomonads in cryobanked insemination units of infected ejaculates to establish infection by T. vaginalis in the recipient. The absence of an animal model for such studies with T. vaginalis precludes experimental assessment of the numbers of trichomonads required to establish themselves in the vagina. However, the relatively low numbers or organisms associated with males who lack symptoms, combined with the epidemiological evidence that these males can transmit the organism (ll), suggests that the numbers needed for establishment may be fairly small. Therefore, in the absence of facts to the contrary and along with the cryosurviva1 data presented in this report, T. va-
250
SHERMAN
gin& seems capable of being transmitted by artificial insemination with cryobanked human semen. REFERENCES
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