THE ASSOCIATION OF SEMEN FACTORS WITH THE RECOVERY OF UREAPLASMA UREALYTICUM*

Transcription

1 FERTILITY AND STERILITY Copyright <> 1981 The American Fertility Society Vol. 36, No. 5, November 1981 Printed in U.S A. THE ASSOCIATION OF SEMEN FACTORS WITH THE RECOVERY OF UREAPLASMA UREALYTICUM* REBECCA D. CINTRON, M.S. J. W. EDWARD WORTHAM, JR., PH.D.t ANIBAL ACOSTA, M.D., PH.D. Department of Biological Sciences, Old Dominion University, Norfolk, Virginia 23508, and Department of Obstetrics and Gynecology, Eastern Virginia Medical School, Norfolk, Virginia Ninety-six semen samples from males under examination for suspected subfertility were examined to determine if alterations in specific semen parameters could be associated with the presence or absence of Ureaplasma urealyticum. Forty-three (44.8%) of the specimens cultured positive for U. urealyticum. Both positive and negative specimens have a normal average motility rating. Sperm counts (millions per milliliter) were slightly higher in U. urealyticum positive samples. Average ejaculate volumes were identical between the two groups. Morphologic abnormalities were not found more frequently in U. urealyticum-positive samples. No statistically significant difference between seminal parameters of positive and negative samples were noted with the one-way analysis of variance test. Nonparametric analysis produced similar nonsignificant results. Fertil Steril 36:648, 1981 Tiny mycoplasmas, morphologically distinct from any previously described cultured PPLO, were initially reported by Shepard, 1 who had examined agar cultures of urethral exudates from males with nongonococcal urethritis. The microorganism was later renamed Ureaplasma urealyticum,2 primarily because of its unique capacity, within the class Mollicutes, to produce urease. In recent years, the potential role of U. urealyticum in the production of genitourinary disease, diseases of the newborn, and various instances of reproductive wastage remains a controversial issue. Of particular interest to this laboratory is the association of U. urealyticum with subfertile couples or those with primary infertility. Whether Received March 19, 1981; revised and accepted July 10, *Presented at the Thirty-Seventh Annual Meeting of the American Fertility Society, March 14 to 18, 1981, Atlanta, Georgia. treprint requests: Dr. J. W. Edward Wortham, Jr., Department of Biological Sciences, Old Dominion University, Norfolk, Virginia the organism is a major contributor to infertility or an opportunist remains unresolved without controlled studies. Investigations involving the potential effects of U. urealyticum on various aspects of semen quality have been considered less frequently. Several studies determined that positive U. urealyticum cultures in cases of idiopathic infertility primarily affected sperm motility. 3 4 Scanning electron microscopy by Gnarpe and Friberg 5 demonstrated that U. urealyticum is associated with the head or midpiece of the spermatozoon. Sphere-shaped particles approximately 200 nm in diameter were present on sperm cells and were connected to one another by filamentous material in U. urealyticum positive samples. 6 Coil-tailed sperm have been reported to be more frequent in U. urealyticum positive samples. 6 Toth et al. 7 noted fuzzy granular coatings along the midpiece and tail of the spermatozoon and used this observation and the humber of coiled tails to predict infection with U. urealyticum. Swenson et al. 8 analyzed changes in semen parameters between the initial and follow-up visit. A definitive improvement was no-

2 Vol. 36, No.5 UREAPLASMA UREALYTICUM 649 ticed in the cell motility where U. urealyticum had been eradicated from the semen. The primary objective of the research reported herein was to compare the semen parameters of male partners from suspected subfertile marriages and attempt to correlate any observed alterations in specific sperm characters with the presence or absence of U. urealyticum. MATERIALS AND METHODS A total of 96 semen specimens from males of couples under examination for suspected infertility were used in this study. Seventeen semen samples from males known to be fertile served as a control group. Patients were instructed to collect the specimen by masturbation after a minimum of 3 days' abstinence from sexual intercourse. The specimen was processed by the Old Dominion University Andrology Laboratory within 2 to 3 hours after emission. The analysis consisted of a determination of ejaculate volume, sperm cell count and differential morphology, motility rating, U. urealyticum culture, and, in several specimens, the detection of sperm-immobilizing and sperm-agglutinating antibodies. The volume was approximated with the use of the graduated collection container. To determine spermatozoan motility, we immediately placed a drop of mixed semen on slides and examined ten high power fields for quick progressive sperm (very active motility), sluggish progressive sperm (slow but deliberate movement), and nonmotile sperm. The percentages were calculated from the total number of cells observed. Eosin-stained preparations were used for the live/dead cell differential, and we counted only nonmotile cells in ten fields. Dead cells are permeable to eosin, allowing for easy recognition by a pink color. Appropriate percentages were calculated from total numbers observed. The sperm count (millions per milliliter) was performed with the use of a hemocytometer and two different dilutions. The count was repeated if the agreement between the two dilutions was not within 10%. Two air-dried and two fixed slides (one/one 95% EtOH and ether) were prepared from each sample. The Papanicolaou (PAP) staining technique was performed, and morphologic characteristics were evaluated by a single observer (J. W. E. Wortham, Jr.) on the basis of criteria established by the International Fertility Association. 9 For U. urealyticum culture, 0.1 ml semen from each specimen was inoculated into 3.6 ml of U9B Urease broth, 10 which had been modified by increasing the concentration of potassium phosphate (to 0.03 gm/100 ml) and the addition of 2% L-cysteine-HCl (0.5 ml/100 ml). U9B broth was checked for bacterial sterility as well as its ability to support the growth of U. urealyticum. Another 0.1 ml semen was distributed over the agar surface of an A 7 plate. 11 Blood agar and eosin methylene blue (EMB) agar were also inoculated and streaked for isolation. All material was incubated at 36 C; A7 agar was incubated in an anaerobic jar with a Gas Pak hydrogen/carbon dioxide envelope (Baltimore Biological Laboratories, Cockeysville, Md.). Quality control medium was inocu-. lated concurrently and incubated with the samples. The strain of U. urealyticum designated as 960, serotype Vill, obtained from M. C. Shepard, served as the positive control. 2 Mycoplasma hominis, strain PG 21, obtained from the National Institute of Allergy and Infectious Diseases, served as the negative control. The identification of U. urealyticum was based on its unique capacity to hydrolyze urea. Phenol red indicator in U9B media changes color from yellow to bright pink, indicating the accumulation of ammonia resulting from urea degradation. At the first sign of a color change, the broth was subcultured to a second vial ofu9b and incubated at 35 C. Differential agar medium, A7, contains manganese sulfate, which detects the by-products of urea hydrolysis directly in or on the suspected U. urealyticum colonies. Clinical isolates were of variable size on A 7 agar and appeared dark brown in color when viewed under low power. Cultures with a mixture of U. urealyticum and the classical Mycoplasma sp. were treated with Dienes' stain. 12 Mycoplasma sp. appeared royal blue, whereas U. urealyticum colonies maintained the characteristic dark brown color. U9B broth which remained yellow for 7 days. was considered a negative test. Plates are initially observed after 48 hours' incubation and held for a total of7 days before reported as negative. A semen sample was not considered positive for U. urealyticum unless both vials of U9B broth turned clear pink and A7 agar unequivocally demonstrated the presence of the organism. Blood agar and EMB agar were examined within 48 hours for potential contaminants which could falsify color change reactions in U9B broth and A 7 agar. Differences in semen parameters between U. urealyticum positive and negative samples were statistically compared by a one-way analysis of

3 650 CINTRON ET AL. November PERCENT TOTAL MOTILE CELLS 25.& Z FIG. 1. The relative frequency distribution of the percentage of total motile cells in the U. urealyticum-positive group (black bar, n = 43) and U. urealyticum-negative group (white bar, n = 53). variance test. Nonparametric statistical analysis included the Mann-Whitney U test and the Kolomogorov-Smimov goodness of fit test with a sharp angulation between the head and midpiece ("bent" forms) and those microcephalic forms were also specifically recorded. An examination of the data indicated that over 80% of both U. urealyticum-positive and -negative patients had less than 10% coiled tails in their semen. Other individual abnormal categories did not demonstrate any significant differences between the mean values for the positive and negative groups. The average cell count ( x 10 6 /ml) was 76.1 for positive samples and 72.2 for negative samples. Average ejaculate volumes were identical between the two groups. Statistical analysis has revealed no significant difference in the mean values for sperm count or volume between the two groups (Table 1). RESULTS A total of 96 semen samples were examined as part of a comprehensive fertility investigation. Of these, 43 (44.8%) cultured U. urealyticum positive. A comparison of total progressive sperm in the positive and negative U. urealyticum groups is represented in Figure 1. The range of progressive sperm in the positive group was from 22% to 83% with an average value of 58.3%. Seventyeight percent of all U. urealyticum-positive specimens had sperm motilities that met the minimum value established for "normal" populations (50% forward progression). 13 The negative U. urealyticum group ranged from 4% to 78% progressive cells, with an average value of 53.7%. Sixtyeight percent of the negative samples also showed sperm motility within normal limits. The oneway analysis of variance indicated no significant difference between the mean values for motility and the presence or absence of U. urealyticum (significance level P ::!S 0.05). The relative frequency distribution of the percentage of normal spermatozoa between the two groups is illustrated in Figure 2. The average number of normal sperm (a minimum of 60% morphologically normal has been determined to be the reference point for overall evaluation of semen cytology) in the U. urealyticum negative group was 57.8% and 55.5% in U. urealyticum-positive samples. Several abnormal categories were evaluated in addition to the abnormal group as a whole. Coil-tailed spermatozoa were of particular interest because of their use by others as an indicator of semen cytology in the prediction of U. urealyticum infection. 7 Sperm DISCUSSION The results reported herein indicate that males under investigation for infertility with U. urealyticum infections do not have an overall decrease in semen quality when compared with those lacking this organism. Positive specimens had an average motility rating of~ 50% progressive cells. This is in disagreement with the work of several researchers 3 ' 4 ' 8 who presented data indicating motility as the major semen parameter influenced by the presence of U. urealyticum. The difference in both the sperm cell count and volume were not found to be statistically significant. The breakdown of abnormal forms into coiled-tail, bent, and microcephalic forms also was not found to be significantly higher in U. urealyticum-positive samples. Other investigators have reported various morphologic anomalies that may be associated with the presence of U. urealyticum. 6 ' 7, 14 The population sampled by our laboratory represented a group of individuals seeking counsel UI... PERCENT NORMAL MORPHOLOGY FIG. 2. The rela~ive frequency distribution of the percentage of ~~rpholog1cally normal sperm in the U. urealyticum-posttlve group (black bar, n = 41) and U. urealyticum-negative group (white bar, n = 53) '

4 .. Vol. 36, No.5 UREAPLASMA UREALYTICUM 651 TABLE 1. Comparison of Sperm Count and Ejaculate Volume Between U. urealyticum Negative and Positive Semen Samples Number tested Mean Count Sign" Total mean Negative Positive Negative Positive x 10 6 /ml NS 73.9 Total cells ( x 10 6 ) NS Volume NS 4.3 anot significant, one-way ANOV A. Significance level P,;;; for infertility. The comparison of mean values between the two groups by one-way. analysis of variance did not indicate that the presence of U. urealyticum in semen had any effect on the tested seminal parameters within this population. All nonparametric analyses produced similar nonsignificant results. Of course, these reported results do not rule out the possibility of some other biologic effect that is undetectable by our methods. The mechanism of U. urealyticum involvement in expressed disease states must be investigated further. Although we were unable to detect any effect on semen parameters, many questions remain unanswered. Only recently has U. urealyticum been isolated from the testes of infertile men. 15 The attachment of this organism to sperm cells has been documented. 5 6 Whether attachment occurs while spermatozoa are stored in the cauda region of the epididymis during the mixing of cells with fluid from accessory secretory glands or even after sperm cells are deposited in the female genital tract is uncertain. The average volume of ejaculate calculated from our U. urealyticum-positive group seems to indicate that if the organism is present in the accessory glands, it is not causing a hyperproduction of prostatic or seminal vesical fluids. Apparently a minimum of eight serotypes of human U. urealyticum are well recognized. 16 A possible ninth strain (Vancouver) has been recently detected. 17 Serotyping of clinical isolates from infertile couples may indicate whether one or more of these strains can be associated with various reproductive anomalies. The high incidence of U. urealyticum in asymptomatic individuals may then be more effectively evaluated. Should treatment of U. ureaiyticum infections with the tetracyclines eventually be indicated, serotyping of clinical isolates would become especially important, since tetracycline-resistant isolates of U. urealyticum have already been reported This laboratory is currently engaged in more comprehensive studies in an attempt to assess certain situations where colonization with U. urealyticum may have an influencing effect on male infertility. Semen from males who have had an appropriate course of antibiotic therapy to eradicate U. urealyticum infection are being evaluated to detect any statistically significant shift in their semen character. Quantitation of numbers of infecting ureaplasmas in semen is being done to deterinine whether disease may be related to the titer of infecting organisms or to a possible interaction between U. urealyticum and other microbes that may colonize the reproductive tract. Acknowledgments. The authors are grateful for the generous contributions of Dr. M. C. Shepard, Dr. J. F. Matta, and the members of the Old Dominion University Andrology Laboratory toward the completion of this research. REFERENCES 1. Shepard MC: The recovery of pleuropneumonia-like organisms from negro men with and without nongonococcal urethritis. Am J Syph Gonor Vener Dis 28:113, Shepard MC, Lunceford CD, Ford DK, Purcell RH, Taylor-Robinson D, Razin S, Black ET: Ureaplasma urealyticum gen. nov., sp. nov: Proposed nomenclature for the human T (T strain) mycoplasmas. Int J Syst Bacterial 24:160, O'Leary WM, FrickJ: Correlation ofhuman male infertility with the presence of Mycoplasma T-strains. Andrologia 7:309, Fowlkes DM, MacLeod J, O'Leary WM: T -mycoplasmas and human infertility: correlation of infection with alterations in seminal parameters. Fertil Steril 26:1212, Gnarpe H, Friberg J: T-mycoplasmas on spermatozoa and infertility. Nature 245:97, Fowlkes DM, Dooher GB, O'Leary WM: Evidence by scanning electron microscopy for an association between spermatozoa and T-mycoplasmas in men of infertile marriage. Fertil Steril 26:1203, Toth ~.Swenson CE, O'Leary WM: Light microscopy as an aid in predicting Ureaplasma infection in human semen. Fertil Steril 30:586, Swenson CE, Toth A, O'Leary WM: Ureaplasma urealyticum and human infertility: the effect of antibiotic therapy on semen quality. Fertil Steril 31:660, Freund M: Standards for the rating of human sperm morphology-a cooperative study. Int J Fertill1:97, Shepard MC, Lunceford CD: Urease color test medium U9 for the detection and identification oft-mycoplasmas in clinical material. Appl Microbial 20:539, 1970

5 652 CINTRON ET AL. 11. Shepard MC, Lunceford CD: Differential agar medium (A7) for identification of Ureaplasma urealyticum (human T-mycoplasmas) in primary cultures of clinical material. J Clin Microbiol 3:613, Madoff S: Isolation and identification of PPLO. Ann NY Acad Sci 79:383; Freund M, Peterson RM: Semen evaluation and fertility. In Human Semen and Fertility Regulation in Men, Edited by ESE Hafez. St; Louis, C. V. Mosby Co, 1976, p Grossgebauer K, Hennig A, Hartmann D: Mycoplasma induced damage of sperm head in infertile men. Haufarzt 28:299, Engel VS, Baumann.B: Das Vorkommen von Mykoplasmen in Hodengewebe. Dermatol Monatsschr 165:593, 1979 November Shepard MC, Lunceford CD: Serological typing of Ureaplasma urealyticum isolates from urethritis patients by an agar growth inhibition method. J Clin Microbiol8:566, Robertson JA, Stemke GW: Modified metabolic inhibition test for serotyping strains of Ureaplasma urealyticum (Tstrain Mycoplasma). J Clin Microbiol 9:673, Spaepen MS, Kundsin RB, Horne HW: Tetracycline resistant T-mycoplasmas (Ureaplasma urealyticum) from patients with a history of reproductive failure. Antimicrob Agents Chemother 9:1012, Evans E, Taylor-Robinson D: The incidence of tetracycline resistant strains of Ureaplasma urealyticum. J Antimicrob Chemother 4:57, 1978