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The effects of three serotypes of Ureaplasma urealyticum on spermatozoal motility and penetration in vitro*†
FERTILITY AND STERILITY
Vol. 55, No. 1, January 1991
Printed on acid-free paper in U.S.A.
Copyright" 1991 The American Fertility Society
The effects of three serotypes of Ureaplasma urealyticum on spermatozoal motility and penetration in vitro*t
Deborah F. Talkington, Ph.D.:j:§ Jerry K. Davis, D.V.M., Ph.D.§ II Kay C. Canupp, M.S.§ Bonnie K. Garrett, M.S.§
Ken B. Waites, M.D.§~ Gertrude A. Huster, M.H.S.# Gail H. Cassell, Ph.D.**§II
The University of Alabama at Birmingham, Birmingham, Alabama
The effects of incubation of spermatozoa with three serotypes of Ureaplasma urealyticum on spermatozoal motility and penetration in vitro were investigated. Using computer-assisted videomicroscopy, three parameters of motility were determined: individual path lengths, individual vectorial distances, and percentage motility. Polyacrylamide gels were used as a medium for assessment of spermatozoal penetration. Ureaplasma-infected spermatozoa did have significantly greater path lengths and individual distances than did uninfected controls, but ureaplasma infection had no significant effect on percentage motility. Overall, there were no significant differences in penetration distances between ureaplasma-infected spermatozoa and their corresponding uninfected controls. Our conclusion is that the ureaplasmas did not adversely affect motility or penetration when spermatozoa were incubated with ureaplasmas for 45 minutes at ureaplasma:sperm ratios as high as 100:1. Fertil Steril55:170, 1991
Considerable disagreement exists concerning a role for Ureaplasma urealyticum in human infertility. Early epidemiologic studies indicated that U. urealyticum was linked to human reproductive fail-
Received April 26, 1990; revised and accepted August 15, 1990. *Supported by grant HD-16199 from the National Institute of Child Health and Human Development, Bethesda, Maryland. t Presented in part at the International Organization of Mycoplasmology International Symposium on Ureaplasmas of Humans: With Emphasis on Maternal and Neonatal Infections, Seattle, Washington, October 10 to 12, 1985. i Corresponding author. Present address: Deborah F. Talkington, Ph.D., Bldg. 5, Rm. 236, Division of Bacterial Diseases, Centers for Disease Control, 1600 Clifton Road, N.E., Atlanta, Georgia, 30333. (404-639-2415) § Department of Microbiology, School of Medicine. II Department of Comparative Medicine, School of Medicine. 1f Department of Pathology, School of Medicine. # Department of Epidemiology, School of Public Health. ** Reprint requests: Gail H. Cassell, Ph.D., Chairman, Department of Microbiology, Box 65 Volker Hall, UAB Station, University of Alabama at Birmingham, Birmingham, Alabama, 35294.
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ure on the basis of higher frequencies of isolation from infertile versus fertile couples and successful pregnancies in infertile couples after doxycycline therapy .1•2 However, subsequent investigators have questioned these findings because they did not discover significantly different isolation rates between fertile and infertile couples nor was the pregnancy rate increased after treatment with antibiotics. 3 •4 We have demonstrated that the incidence of U. urealyticum is significantly higher in those couples who have infertility associated with "male factor" than in other infertile couples. 5 However, there is little evidence that ureaplasmas actually cause male infertility. Several thorough review articles have been published6 •7 that highlight the controversy for and against a causal role for U. urealyticum in both the male and/or female partner. Ureaplasmas are frequently a part of the urethral flora of males and are known to infect and attach to spermatozoa,8 perhaps as they pass through the urethra during ejaculation. Although some investigators have been unable to correlate the presence of ureaplasmas with any alteration in Fertility and Sterility
semen characteristics,9·10 others have shown that in some cases ureaplasmas can affect semen parametersY·12 In one drug treatment trial, an increase in motility occurred after eradication of U. urealyticum. 13 Because swimming velocity of spermatozoa may be a primary factor in predicting fertility in the male partner/4 further work on motility ofureaplasma-infected semen is warranted. New techniques utilizing either multiple exposure photography 15 or computer-assisted videomicrography16 have been developed to give more accurate, objective measurements of motility. In an attempt to clarify the present controversy, we developed and standardized a computer-assisted video micrography system to evaluate the effect(s) of ureaplasma on spermatozoal motility. Another parameter of male fertility, sperm penetration, was also studied. Although human and bovine cervical mucus (CM) have been used as a test medium for sperm migration in vitro, 17 variations in mucus properties have made test standardization difficult. In this study, a polyacrylamide gel was used for the sperm penetration assay. Polyacrylamide gels have been reported to have sperm migration characteristics similar to CM and in experimental systems have the advantage of remaining uniform and stable for long time periods. 18 The purpose of this study was to investigate the effect(s) of three serotypes of U. urealyticum on (1) spermatozoal motility and percentage motility using computer-assisted videomicrography and (2) penetration of sperm into a polyacrylamide gel. MATERIALS AND METHODS
counts of 50 X 106 sperm/mL, at least 70% viable sperm by eosin-nigrosin staining, and a minimum volume of 3 mL. For each experiment, seminal fluid was allowed to liquefy at 37oC for 15 minutes and was used within 30 minutes of collection when sperm motility was optimal. The semen were washed to remove seminal plasma components. Ham's buffer (Gibco, Grand Island, NY), 20 pH 7.2, was added to the semen in a 3:1 ratio; after mixing, the solution was centrifuged for 10 minutes at 300 X g to sediment the spermatozoa. The supernatant fluid was removed, and the washed sperm were resuspended in Ham's buffer to a standard concentration of 40 X 106 sperm/mL. Ureaplasmas
U. urealyticum serotypes 1, 4, and 11 were tested individually to assess the effects of these laboratory strains on spermatozoal motility and penetration. Each serotype was cultured to log phase in lOB broth 21 without antibiotics and frozen at -70°C. Test aliquots of these stocks were grown to determine the number of colony-forming units (CFU) obtainable in a given incubation period at 3TC. Three days before each experiment, one serotype of ureaplasma was grown and passed at 24-hour intervals to a final volume of lliter. The organisms were centrifuged at 30,000 X g for 1 hour and the pellet resuspended in a volume of Ham's buffer to yield 40 X 108 CFU /mL. Tenfold dilutions of this mixture were made in Ham's buffer to give 40 X 107,40 X 106, and40 X 105 CFU/mL. All dilutions were plated onto A8 agar for plate counts, and these four ureaplasma dilutions were mixed with the washed spermatozoa in a dose-response experiment.
Specimens
Semen were collected by masturbation from a single donor into a sterile container after a minimum of 72 hours of sexual abstinence. The donor was screened for U. urealyticum and Mycoplasma hominis by urethral and semen cultures before the first donation; thereafter, semen were routinely cultured on A8 agar 19 at each collection to assure that semen were consistently ureaplasma- and mycoplasma-negative. Semen parameters were checked on repeat visits to determine if volume, spermatozoal counts, viability, and a subjective measurement of motility remained constant. The donor semen were checked to determine if seminal parameters were within the normal or average ranges of 60% to 70% motile sperm, minimal Vol. 55, No.1, January 1991
Dose-Response Experiments
Experiments were done for three serotypes in duplicate giving a total of 6 experiments. The procedure for one experiment for one serotype was as follows: a washed sperm sample containing 40 X 106 sperm/mL was divided into eight aliquots, four for mixing with the four ureaplasma dilutions (infected samples), four for concurrent mixing with an equal volume of Ham's buffer (uninfected controls). The ureaplasmas and washed spermatozoa were mixed in equal volumes to give the following ratios of ureaplasma to sperm: (A) 20 X 108 :20 X 106 (100:1); (B) 20 X 107:20 X 106 (10:1); (C) 20 X 106:20 X 106 (1:1); and (D) 20 X 105:20 X 106 (1:10). To allow adequate time for videotaping and Talkington et al.
In vitro effects of ureaplasmas on sperm
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gel scoring of each ratio individually, the remaining samples were maintained at 37oC and mixed appropriately with ureaplasmas or Ham's buffer (controls) at staggered intervals. At the same time one aliquot of sperm was mixed with an equal volume of ureaplasma dilution, another aliquot of sperm was mixed with an equal volume of Ham's buffer (uninfected control). After 45 minutes incubation at 37°C, sperm in infected and control samples were videotaped using the videomicrography apparatus for analysis of motility and were tested for penetration ability in polyacrylamide gels. Eight samples (1 infected and 1 control sample for each of the 4 ratios) were videotaped and analyzed for each experiment. This procedure (i.e., 1 experiment) was performed two times for each ofthe three serotypes for a total of six experiments. Penetration
Acrylamide, N,N'methylene his acrylamide (BIS), N, N, N', N'tetramethy !ethylenediamine (TEMED), ammonium persulfate (BioRad Laboratories, Richmond, CA), and 1% glucose in phosphate-buffered saline were used to prepare a polyacrylamide synthetic medium for the sperm penetration assay. The method of Lorton et al. 18 was followed with slight modifications. Gels were prepared in capillary tubes at pH 7.4 to yield medium with 1.8% acrylamide and 0.042% BIS. Duplicate capillary tube gels were placed horizontally into individual plastic reservoirs, each containing 90 J,tL of sperm sample. The tubes and reservoirs were incubated for 30 minutes in a humidified 37°C chamber. The capillary tubes were placed on a calibrated slide and examined at 200X magnification with a microscope. Tubes were scanned to determine the most distal site of penetration by five sperm and the distance penetrated by the vanguard spermatozoon. These distances, recorded in micrometers, represented penetration. Motility
Sperm velocity was measured using a modified video micrographic system. The equipment ineluded an MTI65 black and white camera, a Panasonic NV8950 VHS recorder, and a Video Timer VTG33 video timer (For-A, Los Angeles, CA), all interfaced to an Apple He computer (Apple Computer, Cupertino, CA) with monochrome monitor. The camera was adjusted so that the microscope and monitor images were parfocal. The sperm movements were recorded on the VHS recorder 172
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and later analyzed with a Bioquant II Image Analysis System with a HiP ad Digitizer pad (R & M Biometrics, Nashville, TN). Before experiments, amicrometer was taped to aid in standardizing later measurements. Each sample was removed from the incubator and immediately placed on a prewarmed hemocytometer chamber (American Optical, Buffalo, NY); six separate fields were videotaped for 1 minute each, giving 6 minutes of taped swimming time per sample. This was done for both infected and uninfected samples of each ratio after their respective incubation times. The date and time to a hundredth of a second were also recorded on the tape. Analysis
For each field, the percent motility for infected and uninfected samples was determined. The tape was stopped and every sperm in one frame was marked with the use of the Hipad Digitizer. The tape was then advanced for 3 seconds, and the sperm that had moved from their original position were marked. In this manner, the percent motility in each field was calculated. A specified length on the taped micrometer was measured on the digitizer pad to aid in standardizing measurements. The velocity of 60 sperm per sample was analyzed by choosing 10 sperm/field from the six recorded fields. Moving sperm were randomly chosen in each field as the videotape was advanced frame by frame. The paths of the sperm were traced for 3 seconds using the digitizer. The path length and individual distance (the vectorial straight line distance from the first to the last point) of each sperm path in micrometers were calculated by the computer and recorded to disk. In this manner, 60 sperm per infected sample and 60 sperm per uninfected control were analyzed for length of path and individual distance traveled. These analyses were performed for each of the four ratios, giving path length and distance data for 240 infected sperm and 240 uninfected control sperm (960 data points) per experiment. Statistical Analysis
For each experiment, a fully specified regression model was used for a fixed effect two-way ANOVA having one observation per cell, that observation being the difference between the infected group mean and the control group mean for motility measurements, and the difference between penetration distance for the vanguard sperm and the most disFertility and Sterility
Table 1
Overall Effect of Ureaplasma Infection on Average Path Lengths and Individual Distances of Motile Spermatozoa Serotype 4 Experiment no. 1"
Path length Infected Control Individual distances Infected Control
96 ± 6b 82 ± 5 1"
77 ± 7' 64±4
2" 87 ± 11 74± 8 2" 71 ± 9 58± 8
Serotype 1 Experiment no. 1" 73 ± 8 70±8 1" 58±2 53 ±3
2" 74± 5 56± 10 2" 59± 7 42 ± 7
Serotype 11 Experiment no. 1 88± 6 86± 5 1 71 ± 10 70± 4
2" 85±4 75 ± 5 2" 66±3 59± 2
a Statistically significant difference (P < 0.05) between infected and control path lengths or between infected and control individual vectorial distances. b Values are the average length traveled in Jtm in 3 seconds (240 spermatozoa per data point), ± SEM.
'Values are the average individual vectorial distance traveled in Jtm in 3 seconds (240 spermatozoa per data point) ± SEM.
tal site of penetration by five sperm and their corresponding control distances for gel migration measurements. Statistical tests were performed using an estimate of the variance from the raw data. These tests analyzed the significance of the effects of ratio, field, and ratio-field or gel-field interactions. Specific tests used for analyses were: 1. Partial F statistic to test for significant effects of ratio, field, and ratio-field (or gel-field) interaction. 2. Large sample approximation of the t statistic (i.e., z statistic) was used to test for differences between infected and control groups for motility, and the t-test only was used to test these differences for gel penetration. 3. Fisher's least significant difference was used to test for differences between ratio groups and, when appropriate, between ratio-field interaction groups. 4. Analysis of gel migration included correlation between the distances of the most distal site of penetration by five sperm and the vanguard sperm, and correlations between duplicate gels.
than did control sperm in both serotype 4 experiments (P < 0.001), both serotype 1 experiments (P < 0.05), and one serotype 11 experiment (P < 0.05). (See bottom portion of Table 1). No significant differences in sperm path lengths or vectorial distances traveled between infected and control sperm were found in the remaining experiments. When considering the ureaplasma:sperm ratios separately with their individual controls, infected sperm had significantly longer path lengths than controls as follows: ratios A, B, and C of both serotype 4 experiments (P < 0.10) and one serotype 1 experiment (P < 0.05); ratios A and B of one serotype 11 experiment (P < 0.10) and ratio A of the other serotype 1 experiment (P < 0.05). Infected sperm had significantly longer individual distances than controls as follows: ratios A, B, and C of both serotype 4 experiments (P < 0.10) and one serotype 1 experiment (P < 0.05); ratios A and C of the other serotype 1 experiment (P < 0.10); and ratio B of one serotype 11 experiment (P < 0.05). No significant differences between infected and control sperm path lengths or individual distances were found in the remaining ratios. Table 2 presents concise data for individual ratio comparisons. The difference in path length and individual vectorial distance between ureaplasma-infected and uninfected control spermatozoa is broken down by ureaplasma:spermatozoa ratio (ratios A to D). Numbers under the "Path" column are the mean path lengths of uninfected control spermatozoa subtracted from the mean path lengths of ureaplasma-infected spermatozoa at the indicated ureaplasma to spermatozoa ratios (in Jill). A positive number means that infected spermatozoa had longer mean path lengths, and the converse is true
RESULTS When considering all ureaplasma-infected spermatozoa (all 4 ureaplasma:sperm ratios together) with all uninfected control spermatozoa, infected sperm had significantly greater path lengths than uninfected control sperm in both serotype 4 experiments (P < 0.001), one serotype 1 experiment (P < 0.001), and one serotype 11 experiment (P < 0.01). (See top portion of Table 1). Infected sperm also traveled significantly longer vectorial individual distances from their starting points Vol. 55, No.1, January 1991
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Table 2 Differences in Mean Path Lengths and Mean Individual Vectorial Distances Between Ureaplasmalnfected and Control Spermatozoa Path/Distance Ratio
Serotype 4
Serotype 1
Serotype 11
A•
23/19b 14/13 15/18 02/03
35/23 28/12 12/10 -2/01
13/09 25/13 01/02 02/05
B
c
D
• A, 100:1 ureaplasma:spermatozoa ratio; B, 10:1 ratio; C, 1:1 ratio; D, 1:10 ratio. b The numbers represent the value obtained by subtracting the mean path length or vectorial distance of the control spermatozoa from those obtained from the ureaplasma-infected spermatozoa (in ,urn).
for a negative number. The same calculations appear under the "Distance" column for the actual distances traveled by uninfected and ureaplasmainfected spermatozoa (also in ~m). The data shown is only from those experiments shown in Table 1 in which a significant overall difference between infected and control spermatozoa was found; when a significant difference was obtained from both experiments for a given serotype, the data from both experiments were pooled (i.e., the serotype 4 path length and individual vectorial distance data, and the serotype 1 individual vectorial distance data; see Table 1). It is readily apparent that greatest differences between infected and control samples are at the higher ureaplasma to spermatozoa ratios. Overall, there were no significant differences in the percentages of motile spermatozoa between ureaplasma-infected and uninfected controls (P = 0.23) (Table 3). Also, there was no significant difference in motility between control or infected groups between experiments (P = 0.26), and there was no significant difference in percentage motility between the control groups of the various ratios (P = 0.74). The lack of a significant difference in percentage motility between ratio control groups within experiments verified that the percentage motility of sperm did not decrease significantly over the duration of an experiment. There were no overall significant differences between infected and control sperm penetration distances into the polyacrylamide gels nor were there significant differences in sperm penetration when considering individual ureaplasma:sperm ratios. There was excellent correlation between the distances traveled by the five distal sperm and the vanguard spermatozoan (r = 0.95). 174
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DISCUSSION
Ureaplasmas are often cited as having a possible role in human infertility based on epidemiologic evidence and data from antibiotic treatment trials, although specific mechanisms have not been described. Because the incidence of U. urealyticum has been found to be significantly higher in couples with "male factor" infertility,5 we chose to investigate the in vitro effects of three serotypes of U. urealyticum on spermatozoal motility (a primary indicator of male fertility) and on penetration. Overall in this study, there were no significant differences in penetration of polyacrylamide gels between ureaplasma-infected sperm and uninfected controls, and ureaplasma infection had no significant effect on percentage motility. However, in most experiments, ureaplasma-infected sperm were significantly more motile than uninfected controls. Because we could find no evidence that ureaplasma infection was detrimental to the spermatozoal parameters we studied, our general conclusion is that the ureaplasmas had no detrimental effect on spermatozoal motility or penetration in our experimental system. The motility data suggest that ureaplasma infection may even have had a stimulatory effect, but careful interpretation is warranted. The finding that ureaplasmas can enhance sperm motility is a novel one. It is possible that an increase in metabolic activity, and hence motility, followed exposure of the spermatozoa to the ureaplasmas, but this possibility is not consistent with our finding that ureaplasma infection did not affect the gel penetration ability of the spermatozoa. Another possible explanation that must be considered Table 3 Effect of U reaplasma Infection on Percentage Motility by Ratio Serotype 4
Serotype 1
Serotype 11
56± 09b 72 ± 25 46±05 77 ± 26 54± 11
58± 10 60 ± 19 42 ± 09 54± 11 57±08
60 ±09 77± 19 55± 20 49 ± 11 59± 09
Ratio
A•
B
c
D Control
a A, 100:1 ureaplasma:sperm ratio; B, 10:1 ratio; C, 1:1 ratio; D, 1:10 ratio. For each infected group (ratios A to D) an uninfected control group was also analyzed. The percentage motility of the control groups did not vary significantly between ratios in experiments or between experiments, so for purposes of comparison, the data for the uninfected control groups of ratios A to D of each serotype are averaged together (control). b Values are means ± SD of the number of motile spermatozoa expressed as a percentage of the spermatozoa examined.
Fertility and Sterility
is that the apparent stimulation of motility by ureaplasmas was an artifact caused by ureaplasmas adversely affecting only the motility of unhealthy sperm, leaving a more noticeable population of healthy, highly motile to be chosen for analysis. Our data are consistent with this possibility for three reasons. First, if the ureaplasmas affected only the unhealthy sperm, the greatest artifactual difference in motility between infected and control samples would have been found in the higher ureaplasma:sperm ratios (A and B versus C and D), and the magnitude of the motility difference would have decreased as the ureaplasma:sperm ratio decreased. In fact, significant differences in motility occurred most commonly in ratio A (9 times), then B (8 times), then C (7 times), and never in D. Second, although there were no significant differences in percent motility between infected and uninfected spermatozoa, the percent motility parameter does not necessarily reflect quantitative effects of swimming speed detectable with the sensitive video microscopic technique. Third, if ureaplasmas only affected unhealthy sperm, no difference in gel penetration ability between infected and control sperm would be expected since presumably the vanguard and distal sperm would be the healthiest sperm in both control and infected samples. Thus, our finding that ureaplasma infection enhanced motility may have been an artifact because of subtle detrimental effects of ureaplasmas on the less healthy sperm that caused the remaining more highly motile sperm to be picked for videomicroscopic analysis. Our results do not imply that ureaplasmas do not adversely affect semen parameters in vivo, only that significant detrimental effects are not seen in vitro after 45 minutes of incubation of ureaplasmas with healthy spermatozoa. It is certainly possible that sperm must be exposed to ureaplasmas or their soluble products for several hours before such effects can be detected in an entire population. It is noteworthy that of the three serotypes studied, the greatest increase in path length and individual distance traveled was found in spermatozoa infected with serotype 4 ureaplasmas. Serotype 4 ureaplasmas have been reported by some to be the most common serotype cultured from males with urethritis, 21 •22 and of eight serotypes examined for their ability to reduce the penetration of zona-free hamster eggs by human spermatozoa, only serotype 4 ureaplasmas were found to have a significant effect. 23 Serotype 4 ureaplasmas have also been reported as the most common type isolated from the Vol. 55, No.1, January 1991
cervix of women with pelvic inflammatory disease.24 When making physiological function comparisons in vitro, the distinction between in vitro and in vivo conditions must be appreciated; the influence of host factors was not examined in this study. The female reproductive tract environment is very complex and could easily influence interactions between infectious agents and spermatozoa that cannot presently be duplicated in vitro. Ureaplasmas could affect spermatozoal motility and penetration in vivo by interfering with the physiological and biochemical events that occur before fertilization. Ureaplasma antibodies could also be involved. Although the association of infertility and colonization of the endometrium with U. urealyticum is unclear, 25 it is entirely possible that ureaplasmas that colonize the endometrium may interfere with one or more steps in sperm maturation or capacitation. Final confirmation of the possible role of U. urealyticum in male sub fertility will require further well-controlled clinical and laboratory studies, perhaps involving fresh clinical isolates from infertile patients. Since in vivo interactions between spermatozoa and ureaplasmas may be difficult to simulate in vitro, the development of a well-defined animal model would be invaluable.
REFERENCES 1. Friberg J, Gnarpe H: Mycoplasma and human reproductive
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failure: III. Pregnancies in "infertile" couples treated with doxycycline forT mycoplasmas. Am J Obstet Gynecol116: 23, 1973 Gnarpe H, Friberg J: T-mycoplasmas as a possible cause for reproductive failure. Nature 242:120, 1973 Rehewey MSE, Thomas AJ, Hafez ESE, Brown WJ, Moghissi KS, Jaszczak S: Ureaplasma urealyticum {T mycoplasma) in seminal plasma and spermatozoa from infertile and fertile volunteers. Eur J Obstet Gynecol Reprod Biol 8/5:246, 1978 Matthews CD, Elmslie RG, Clapp KH, Svigos JM: The frequency of genital mycoplasma infection in human infertil· ity. Fertil Steril26:988, 1975 Cassell GH, Younger JB, Brown MB, Blackwell RE, Davis JK, Marriott P, Stagno S: Microbiologic study of infertile women at the time of diagnostic laparoscopy. N Engl J Med 308:502, 1983 Friberg J: Mycoplasmas and ureaplasmas in infertility and abortion. Fertil Steril 33:351, 1980 Styler M, Shapiro SS: Mollicutes {mycoplasma) in infertility. Fertil Steril44:1, 1985 Fowlkes DM, Dooher GB, O'Leary WM: Evidence by scanning electron microscopy for an association between sper· matozoa and T-mycoplasmas in men of infertile marriage. Fertil Steril26:1203, 1975 Upadhyaya M, Hibbard BM, Walker, SM: The effect of
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Ureaplasma urealyticum on semen characteristics. Fertil Steril41:304, 1984 Cintron RD, Wortham JWE, Jr, Acosta A: The association of semen factors with the recovery of Ureaplasma urealyticum. Fertil Steril 36:648, 1981 O'Leary WM, Frick J: The correlation of human male infertility with the presence of Mycoplasma T strains. Andrologia 7:309, 1975 Cracea E, Maicanescu-Georgescu M, Constantinescu S, Botez D: Genital mycoplasmas and chlamydiae in male infertility. Arch Roum Pathol Exp Microbiol T41:219, 1982 Swenson CE, Toth A, O'Leary WM: Ureaplasma urealyticum and human infertility: the effect of antibiotic therapy on semen quality. Fertil Steril31:660, 1979 Blasco L: Clinical tests of sperm fertilizing ability. Fertil Steril41:177, 1984 Makler A: Use of the elaborated multiple exposure photography (MEP) method in routine sperm motility analysis and for research purposes. Fertil Steril33:160, 1980 Katz DF, Overstreet JW: Sperm motility assessment by videomicrography. Fertil Steril35:188, 1981 Tredway DR, Wortham JWE, Jr, Condon-Mahony M, Baker D, Shane JM: Correlation of postcoital evaluation with in vitro sperm cervical mucus determinations and ureaplasma cultures. Fertil Steril43:286, 1985 Lorton SP, Kummerfeld HL, Foote RH: Polyacrylamide as
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a substitute for cervical mucus in sperm migration tests. Fertil Steril 35:222, 1981 Shepard MC: Culture media for ureaplasmas. In Methods in Mycoplasmology, Vol. I, Edited by S Razin, JG Tully. New York, Academic Press, 1983, p 137 Makler A, Fisher M, Murillo 0, Laufer N, DeCherney A, Naftolin F: Factors affecting sperm motility. IX. Survival of spermatozoa in various biological media and under different gaseous compositions. Fertil Steril41:428, 1984 Shepard MC, Lunceford CD: Serological typing of Ureaplasma urealyticum isolates from urethritis patients by an agar growth inhibition method. J Clin Microbiol 8:566, 1978 Cracea E, Constantinescu S, Lazar A: Serotypes of Ureaplasma urealyticum isolated from patients with nongonococcal urethritis and gonorrhoea and from symptomatic carriers. Sex Transm Dis 12:219, 1985 Busolo F, Zanchetta R: The effect of Mycoplasma hominis and Ureaplasma urealyticum on hamster egg in vitro penetration by human spermatozoa. Fertil Steril43:110, 1985 Cracea E, Boetz D, Constantinescu S, Georgescu-Braila M: Ureaplasma urealyticum serotypes isolated from cases of female sterility. Hyg I Abt Orig A252:535, 1982 Cassell GH, Davis JK, Waites KB, Rudd PT, Talkington DF, Crouse D, Horowitz SA: Pathogenesis and significance of urogenital mycoplasma infections. Adv Exp Med Bioi 224:93, 1987
Fertility and Sterility
Vol. 55, No. 1, January 1991
Printed on acid-free paper in U.S.A.
Copyright" 1991 The American Fertility Society
The effects of three serotypes of Ureaplasma urealyticum on spermatozoal motility and penetration in vitro*t
Deborah F. Talkington, Ph.D.:j:§ Jerry K. Davis, D.V.M., Ph.D.§ II Kay C. Canupp, M.S.§ Bonnie K. Garrett, M.S.§
Ken B. Waites, M.D.§~ Gertrude A. Huster, M.H.S.# Gail H. Cassell, Ph.D.**§II
The University of Alabama at Birmingham, Birmingham, Alabama
The effects of incubation of spermatozoa with three serotypes of Ureaplasma urealyticum on spermatozoal motility and penetration in vitro were investigated. Using computer-assisted videomicroscopy, three parameters of motility were determined: individual path lengths, individual vectorial distances, and percentage motility. Polyacrylamide gels were used as a medium for assessment of spermatozoal penetration. Ureaplasma-infected spermatozoa did have significantly greater path lengths and individual distances than did uninfected controls, but ureaplasma infection had no significant effect on percentage motility. Overall, there were no significant differences in penetration distances between ureaplasma-infected spermatozoa and their corresponding uninfected controls. Our conclusion is that the ureaplasmas did not adversely affect motility or penetration when spermatozoa were incubated with ureaplasmas for 45 minutes at ureaplasma:sperm ratios as high as 100:1. Fertil Steril55:170, 1991
Considerable disagreement exists concerning a role for Ureaplasma urealyticum in human infertility. Early epidemiologic studies indicated that U. urealyticum was linked to human reproductive fail-
Received April 26, 1990; revised and accepted August 15, 1990. *Supported by grant HD-16199 from the National Institute of Child Health and Human Development, Bethesda, Maryland. t Presented in part at the International Organization of Mycoplasmology International Symposium on Ureaplasmas of Humans: With Emphasis on Maternal and Neonatal Infections, Seattle, Washington, October 10 to 12, 1985. i Corresponding author. Present address: Deborah F. Talkington, Ph.D., Bldg. 5, Rm. 236, Division of Bacterial Diseases, Centers for Disease Control, 1600 Clifton Road, N.E., Atlanta, Georgia, 30333. (404-639-2415) § Department of Microbiology, School of Medicine. II Department of Comparative Medicine, School of Medicine. 1f Department of Pathology, School of Medicine. # Department of Epidemiology, School of Public Health. ** Reprint requests: Gail H. Cassell, Ph.D., Chairman, Department of Microbiology, Box 65 Volker Hall, UAB Station, University of Alabama at Birmingham, Birmingham, Alabama, 35294.
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In vitro effects of ureaplasmas on sperm
ure on the basis of higher frequencies of isolation from infertile versus fertile couples and successful pregnancies in infertile couples after doxycycline therapy .1•2 However, subsequent investigators have questioned these findings because they did not discover significantly different isolation rates between fertile and infertile couples nor was the pregnancy rate increased after treatment with antibiotics. 3 •4 We have demonstrated that the incidence of U. urealyticum is significantly higher in those couples who have infertility associated with "male factor" than in other infertile couples. 5 However, there is little evidence that ureaplasmas actually cause male infertility. Several thorough review articles have been published6 •7 that highlight the controversy for and against a causal role for U. urealyticum in both the male and/or female partner. Ureaplasmas are frequently a part of the urethral flora of males and are known to infect and attach to spermatozoa,8 perhaps as they pass through the urethra during ejaculation. Although some investigators have been unable to correlate the presence of ureaplasmas with any alteration in Fertility and Sterility
semen characteristics,9·10 others have shown that in some cases ureaplasmas can affect semen parametersY·12 In one drug treatment trial, an increase in motility occurred after eradication of U. urealyticum. 13 Because swimming velocity of spermatozoa may be a primary factor in predicting fertility in the male partner/4 further work on motility ofureaplasma-infected semen is warranted. New techniques utilizing either multiple exposure photography 15 or computer-assisted videomicrography16 have been developed to give more accurate, objective measurements of motility. In an attempt to clarify the present controversy, we developed and standardized a computer-assisted video micrography system to evaluate the effect(s) of ureaplasma on spermatozoal motility. Another parameter of male fertility, sperm penetration, was also studied. Although human and bovine cervical mucus (CM) have been used as a test medium for sperm migration in vitro, 17 variations in mucus properties have made test standardization difficult. In this study, a polyacrylamide gel was used for the sperm penetration assay. Polyacrylamide gels have been reported to have sperm migration characteristics similar to CM and in experimental systems have the advantage of remaining uniform and stable for long time periods. 18 The purpose of this study was to investigate the effect(s) of three serotypes of U. urealyticum on (1) spermatozoal motility and percentage motility using computer-assisted videomicrography and (2) penetration of sperm into a polyacrylamide gel. MATERIALS AND METHODS
counts of 50 X 106 sperm/mL, at least 70% viable sperm by eosin-nigrosin staining, and a minimum volume of 3 mL. For each experiment, seminal fluid was allowed to liquefy at 37oC for 15 minutes and was used within 30 minutes of collection when sperm motility was optimal. The semen were washed to remove seminal plasma components. Ham's buffer (Gibco, Grand Island, NY), 20 pH 7.2, was added to the semen in a 3:1 ratio; after mixing, the solution was centrifuged for 10 minutes at 300 X g to sediment the spermatozoa. The supernatant fluid was removed, and the washed sperm were resuspended in Ham's buffer to a standard concentration of 40 X 106 sperm/mL. Ureaplasmas
U. urealyticum serotypes 1, 4, and 11 were tested individually to assess the effects of these laboratory strains on spermatozoal motility and penetration. Each serotype was cultured to log phase in lOB broth 21 without antibiotics and frozen at -70°C. Test aliquots of these stocks were grown to determine the number of colony-forming units (CFU) obtainable in a given incubation period at 3TC. Three days before each experiment, one serotype of ureaplasma was grown and passed at 24-hour intervals to a final volume of lliter. The organisms were centrifuged at 30,000 X g for 1 hour and the pellet resuspended in a volume of Ham's buffer to yield 40 X 108 CFU /mL. Tenfold dilutions of this mixture were made in Ham's buffer to give 40 X 107,40 X 106, and40 X 105 CFU/mL. All dilutions were plated onto A8 agar for plate counts, and these four ureaplasma dilutions were mixed with the washed spermatozoa in a dose-response experiment.
Specimens
Semen were collected by masturbation from a single donor into a sterile container after a minimum of 72 hours of sexual abstinence. The donor was screened for U. urealyticum and Mycoplasma hominis by urethral and semen cultures before the first donation; thereafter, semen were routinely cultured on A8 agar 19 at each collection to assure that semen were consistently ureaplasma- and mycoplasma-negative. Semen parameters were checked on repeat visits to determine if volume, spermatozoal counts, viability, and a subjective measurement of motility remained constant. The donor semen were checked to determine if seminal parameters were within the normal or average ranges of 60% to 70% motile sperm, minimal Vol. 55, No.1, January 1991
Dose-Response Experiments
Experiments were done for three serotypes in duplicate giving a total of 6 experiments. The procedure for one experiment for one serotype was as follows: a washed sperm sample containing 40 X 106 sperm/mL was divided into eight aliquots, four for mixing with the four ureaplasma dilutions (infected samples), four for concurrent mixing with an equal volume of Ham's buffer (uninfected controls). The ureaplasmas and washed spermatozoa were mixed in equal volumes to give the following ratios of ureaplasma to sperm: (A) 20 X 108 :20 X 106 (100:1); (B) 20 X 107:20 X 106 (10:1); (C) 20 X 106:20 X 106 (1:1); and (D) 20 X 105:20 X 106 (1:10). To allow adequate time for videotaping and Talkington et al.
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gel scoring of each ratio individually, the remaining samples were maintained at 37oC and mixed appropriately with ureaplasmas or Ham's buffer (controls) at staggered intervals. At the same time one aliquot of sperm was mixed with an equal volume of ureaplasma dilution, another aliquot of sperm was mixed with an equal volume of Ham's buffer (uninfected control). After 45 minutes incubation at 37°C, sperm in infected and control samples were videotaped using the videomicrography apparatus for analysis of motility and were tested for penetration ability in polyacrylamide gels. Eight samples (1 infected and 1 control sample for each of the 4 ratios) were videotaped and analyzed for each experiment. This procedure (i.e., 1 experiment) was performed two times for each ofthe three serotypes for a total of six experiments. Penetration
Acrylamide, N,N'methylene his acrylamide (BIS), N, N, N', N'tetramethy !ethylenediamine (TEMED), ammonium persulfate (BioRad Laboratories, Richmond, CA), and 1% glucose in phosphate-buffered saline were used to prepare a polyacrylamide synthetic medium for the sperm penetration assay. The method of Lorton et al. 18 was followed with slight modifications. Gels were prepared in capillary tubes at pH 7.4 to yield medium with 1.8% acrylamide and 0.042% BIS. Duplicate capillary tube gels were placed horizontally into individual plastic reservoirs, each containing 90 J,tL of sperm sample. The tubes and reservoirs were incubated for 30 minutes in a humidified 37°C chamber. The capillary tubes were placed on a calibrated slide and examined at 200X magnification with a microscope. Tubes were scanned to determine the most distal site of penetration by five sperm and the distance penetrated by the vanguard spermatozoon. These distances, recorded in micrometers, represented penetration. Motility
Sperm velocity was measured using a modified video micrographic system. The equipment ineluded an MTI65 black and white camera, a Panasonic NV8950 VHS recorder, and a Video Timer VTG33 video timer (For-A, Los Angeles, CA), all interfaced to an Apple He computer (Apple Computer, Cupertino, CA) with monochrome monitor. The camera was adjusted so that the microscope and monitor images were parfocal. The sperm movements were recorded on the VHS recorder 172
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In vitro effects of ureaplasmas on sperm
and later analyzed with a Bioquant II Image Analysis System with a HiP ad Digitizer pad (R & M Biometrics, Nashville, TN). Before experiments, amicrometer was taped to aid in standardizing later measurements. Each sample was removed from the incubator and immediately placed on a prewarmed hemocytometer chamber (American Optical, Buffalo, NY); six separate fields were videotaped for 1 minute each, giving 6 minutes of taped swimming time per sample. This was done for both infected and uninfected samples of each ratio after their respective incubation times. The date and time to a hundredth of a second were also recorded on the tape. Analysis
For each field, the percent motility for infected and uninfected samples was determined. The tape was stopped and every sperm in one frame was marked with the use of the Hipad Digitizer. The tape was then advanced for 3 seconds, and the sperm that had moved from their original position were marked. In this manner, the percent motility in each field was calculated. A specified length on the taped micrometer was measured on the digitizer pad to aid in standardizing measurements. The velocity of 60 sperm per sample was analyzed by choosing 10 sperm/field from the six recorded fields. Moving sperm were randomly chosen in each field as the videotape was advanced frame by frame. The paths of the sperm were traced for 3 seconds using the digitizer. The path length and individual distance (the vectorial straight line distance from the first to the last point) of each sperm path in micrometers were calculated by the computer and recorded to disk. In this manner, 60 sperm per infected sample and 60 sperm per uninfected control were analyzed for length of path and individual distance traveled. These analyses were performed for each of the four ratios, giving path length and distance data for 240 infected sperm and 240 uninfected control sperm (960 data points) per experiment. Statistical Analysis
For each experiment, a fully specified regression model was used for a fixed effect two-way ANOVA having one observation per cell, that observation being the difference between the infected group mean and the control group mean for motility measurements, and the difference between penetration distance for the vanguard sperm and the most disFertility and Sterility
Table 1
Overall Effect of Ureaplasma Infection on Average Path Lengths and Individual Distances of Motile Spermatozoa Serotype 4 Experiment no. 1"
Path length Infected Control Individual distances Infected Control
96 ± 6b 82 ± 5 1"
77 ± 7' 64±4
2" 87 ± 11 74± 8 2" 71 ± 9 58± 8
Serotype 1 Experiment no. 1" 73 ± 8 70±8 1" 58±2 53 ±3
2" 74± 5 56± 10 2" 59± 7 42 ± 7
Serotype 11 Experiment no. 1 88± 6 86± 5 1 71 ± 10 70± 4
2" 85±4 75 ± 5 2" 66±3 59± 2
a Statistically significant difference (P < 0.05) between infected and control path lengths or between infected and control individual vectorial distances. b Values are the average length traveled in Jtm in 3 seconds (240 spermatozoa per data point), ± SEM.
'Values are the average individual vectorial distance traveled in Jtm in 3 seconds (240 spermatozoa per data point) ± SEM.
tal site of penetration by five sperm and their corresponding control distances for gel migration measurements. Statistical tests were performed using an estimate of the variance from the raw data. These tests analyzed the significance of the effects of ratio, field, and ratio-field or gel-field interactions. Specific tests used for analyses were: 1. Partial F statistic to test for significant effects of ratio, field, and ratio-field (or gel-field) interaction. 2. Large sample approximation of the t statistic (i.e., z statistic) was used to test for differences between infected and control groups for motility, and the t-test only was used to test these differences for gel penetration. 3. Fisher's least significant difference was used to test for differences between ratio groups and, when appropriate, between ratio-field interaction groups. 4. Analysis of gel migration included correlation between the distances of the most distal site of penetration by five sperm and the vanguard sperm, and correlations between duplicate gels.
than did control sperm in both serotype 4 experiments (P < 0.001), both serotype 1 experiments (P < 0.05), and one serotype 11 experiment (P < 0.05). (See bottom portion of Table 1). No significant differences in sperm path lengths or vectorial distances traveled between infected and control sperm were found in the remaining experiments. When considering the ureaplasma:sperm ratios separately with their individual controls, infected sperm had significantly longer path lengths than controls as follows: ratios A, B, and C of both serotype 4 experiments (P < 0.10) and one serotype 1 experiment (P < 0.05); ratios A and B of one serotype 11 experiment (P < 0.10) and ratio A of the other serotype 1 experiment (P < 0.05). Infected sperm had significantly longer individual distances than controls as follows: ratios A, B, and C of both serotype 4 experiments (P < 0.10) and one serotype 1 experiment (P < 0.05); ratios A and C of the other serotype 1 experiment (P < 0.10); and ratio B of one serotype 11 experiment (P < 0.05). No significant differences between infected and control sperm path lengths or individual distances were found in the remaining ratios. Table 2 presents concise data for individual ratio comparisons. The difference in path length and individual vectorial distance between ureaplasma-infected and uninfected control spermatozoa is broken down by ureaplasma:spermatozoa ratio (ratios A to D). Numbers under the "Path" column are the mean path lengths of uninfected control spermatozoa subtracted from the mean path lengths of ureaplasma-infected spermatozoa at the indicated ureaplasma to spermatozoa ratios (in Jill). A positive number means that infected spermatozoa had longer mean path lengths, and the converse is true
RESULTS When considering all ureaplasma-infected spermatozoa (all 4 ureaplasma:sperm ratios together) with all uninfected control spermatozoa, infected sperm had significantly greater path lengths than uninfected control sperm in both serotype 4 experiments (P < 0.001), one serotype 1 experiment (P < 0.001), and one serotype 11 experiment (P < 0.01). (See top portion of Table 1). Infected sperm also traveled significantly longer vectorial individual distances from their starting points Vol. 55, No.1, January 1991
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Table 2 Differences in Mean Path Lengths and Mean Individual Vectorial Distances Between Ureaplasmalnfected and Control Spermatozoa Path/Distance Ratio
Serotype 4
Serotype 1
Serotype 11
A•
23/19b 14/13 15/18 02/03
35/23 28/12 12/10 -2/01
13/09 25/13 01/02 02/05
B
c
D
• A, 100:1 ureaplasma:spermatozoa ratio; B, 10:1 ratio; C, 1:1 ratio; D, 1:10 ratio. b The numbers represent the value obtained by subtracting the mean path length or vectorial distance of the control spermatozoa from those obtained from the ureaplasma-infected spermatozoa (in ,urn).
for a negative number. The same calculations appear under the "Distance" column for the actual distances traveled by uninfected and ureaplasmainfected spermatozoa (also in ~m). The data shown is only from those experiments shown in Table 1 in which a significant overall difference between infected and control spermatozoa was found; when a significant difference was obtained from both experiments for a given serotype, the data from both experiments were pooled (i.e., the serotype 4 path length and individual vectorial distance data, and the serotype 1 individual vectorial distance data; see Table 1). It is readily apparent that greatest differences between infected and control samples are at the higher ureaplasma to spermatozoa ratios. Overall, there were no significant differences in the percentages of motile spermatozoa between ureaplasma-infected and uninfected controls (P = 0.23) (Table 3). Also, there was no significant difference in motility between control or infected groups between experiments (P = 0.26), and there was no significant difference in percentage motility between the control groups of the various ratios (P = 0.74). The lack of a significant difference in percentage motility between ratio control groups within experiments verified that the percentage motility of sperm did not decrease significantly over the duration of an experiment. There were no overall significant differences between infected and control sperm penetration distances into the polyacrylamide gels nor were there significant differences in sperm penetration when considering individual ureaplasma:sperm ratios. There was excellent correlation between the distances traveled by the five distal sperm and the vanguard spermatozoan (r = 0.95). 174
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DISCUSSION
Ureaplasmas are often cited as having a possible role in human infertility based on epidemiologic evidence and data from antibiotic treatment trials, although specific mechanisms have not been described. Because the incidence of U. urealyticum has been found to be significantly higher in couples with "male factor" infertility,5 we chose to investigate the in vitro effects of three serotypes of U. urealyticum on spermatozoal motility (a primary indicator of male fertility) and on penetration. Overall in this study, there were no significant differences in penetration of polyacrylamide gels between ureaplasma-infected sperm and uninfected controls, and ureaplasma infection had no significant effect on percentage motility. However, in most experiments, ureaplasma-infected sperm were significantly more motile than uninfected controls. Because we could find no evidence that ureaplasma infection was detrimental to the spermatozoal parameters we studied, our general conclusion is that the ureaplasmas had no detrimental effect on spermatozoal motility or penetration in our experimental system. The motility data suggest that ureaplasma infection may even have had a stimulatory effect, but careful interpretation is warranted. The finding that ureaplasmas can enhance sperm motility is a novel one. It is possible that an increase in metabolic activity, and hence motility, followed exposure of the spermatozoa to the ureaplasmas, but this possibility is not consistent with our finding that ureaplasma infection did not affect the gel penetration ability of the spermatozoa. Another possible explanation that must be considered Table 3 Effect of U reaplasma Infection on Percentage Motility by Ratio Serotype 4
Serotype 1
Serotype 11
56± 09b 72 ± 25 46±05 77 ± 26 54± 11
58± 10 60 ± 19 42 ± 09 54± 11 57±08
60 ±09 77± 19 55± 20 49 ± 11 59± 09
Ratio
A•
B
c
D Control
a A, 100:1 ureaplasma:sperm ratio; B, 10:1 ratio; C, 1:1 ratio; D, 1:10 ratio. For each infected group (ratios A to D) an uninfected control group was also analyzed. The percentage motility of the control groups did not vary significantly between ratios in experiments or between experiments, so for purposes of comparison, the data for the uninfected control groups of ratios A to D of each serotype are averaged together (control). b Values are means ± SD of the number of motile spermatozoa expressed as a percentage of the spermatozoa examined.
Fertility and Sterility
is that the apparent stimulation of motility by ureaplasmas was an artifact caused by ureaplasmas adversely affecting only the motility of unhealthy sperm, leaving a more noticeable population of healthy, highly motile to be chosen for analysis. Our data are consistent with this possibility for three reasons. First, if the ureaplasmas affected only the unhealthy sperm, the greatest artifactual difference in motility between infected and control samples would have been found in the higher ureaplasma:sperm ratios (A and B versus C and D), and the magnitude of the motility difference would have decreased as the ureaplasma:sperm ratio decreased. In fact, significant differences in motility occurred most commonly in ratio A (9 times), then B (8 times), then C (7 times), and never in D. Second, although there were no significant differences in percent motility between infected and uninfected spermatozoa, the percent motility parameter does not necessarily reflect quantitative effects of swimming speed detectable with the sensitive video microscopic technique. Third, if ureaplasmas only affected unhealthy sperm, no difference in gel penetration ability between infected and control sperm would be expected since presumably the vanguard and distal sperm would be the healthiest sperm in both control and infected samples. Thus, our finding that ureaplasma infection enhanced motility may have been an artifact because of subtle detrimental effects of ureaplasmas on the less healthy sperm that caused the remaining more highly motile sperm to be picked for videomicroscopic analysis. Our results do not imply that ureaplasmas do not adversely affect semen parameters in vivo, only that significant detrimental effects are not seen in vitro after 45 minutes of incubation of ureaplasmas with healthy spermatozoa. It is certainly possible that sperm must be exposed to ureaplasmas or their soluble products for several hours before such effects can be detected in an entire population. It is noteworthy that of the three serotypes studied, the greatest increase in path length and individual distance traveled was found in spermatozoa infected with serotype 4 ureaplasmas. Serotype 4 ureaplasmas have been reported by some to be the most common serotype cultured from males with urethritis, 21 •22 and of eight serotypes examined for their ability to reduce the penetration of zona-free hamster eggs by human spermatozoa, only serotype 4 ureaplasmas were found to have a significant effect. 23 Serotype 4 ureaplasmas have also been reported as the most common type isolated from the Vol. 55, No.1, January 1991
cervix of women with pelvic inflammatory disease.24 When making physiological function comparisons in vitro, the distinction between in vitro and in vivo conditions must be appreciated; the influence of host factors was not examined in this study. The female reproductive tract environment is very complex and could easily influence interactions between infectious agents and spermatozoa that cannot presently be duplicated in vitro. Ureaplasmas could affect spermatozoal motility and penetration in vivo by interfering with the physiological and biochemical events that occur before fertilization. Ureaplasma antibodies could also be involved. Although the association of infertility and colonization of the endometrium with U. urealyticum is unclear, 25 it is entirely possible that ureaplasmas that colonize the endometrium may interfere with one or more steps in sperm maturation or capacitation. Final confirmation of the possible role of U. urealyticum in male sub fertility will require further well-controlled clinical and laboratory studies, perhaps involving fresh clinical isolates from infertile patients. Since in vivo interactions between spermatozoa and ureaplasmas may be difficult to simulate in vitro, the development of a well-defined animal model would be invaluable.
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