Mycoplasmic localization patterns on spermatozoa from infertile men

FERTILITY AND STERILITY Copyright

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Vol. 42, No.3, September 1984 Prinred in U.SA.

1984 The American Fertility Society

Mycoplasmic localization patterns on spermatozoa from infertile men

Franco Busolo, Ph.D. *t Renato Zanchetta, M.DJ Giulio Bertoloni, Ph.D.* University of Padua Medical School, Padua, Italy

Two mycoplasmas have been observed with increasing frequency in patients with genitourinary disorders: Mycoplasma hominis and Ureaplasma urealyticum. Mycoplasma cells of both these species have been demonstrated to be capable of attaching to human spermatozoa of infertile patients. The mechanisms for the association of infertility and mycoplasma infection have not been established. The main objective of this article was to explain the significance of some morphologic features of spermatozoa of patients with unexplained infertility using light and electron microscopy. These studies and quantitative analysis of ureaplasmas in the semen indicate that at least two patterns can be seen. Frequently, sphere-shaped particles adhering mainly to the midpiece of spermatozoa were detected. In a second, more complex pattern ureaplasmas were seen inside a swollen zone on the midpiece, which suggests that the infection does not occur in the urethra, but at another unknown site. Furthermore, the sphere-shaped particles cannot be associated with ureaplasmas because their titers in the semen of infertile patients were much lower than those expected. Fertil Steril 42:412, 1984

Five mycoplasma species are known to be present in the human genitourinary tract: Mycoplasma hominis, Ureaplasma urealyticum, Mycoplasma fermentans, Mycoplasma primatum, and Mycoplasma genitalium. The latter three species are uncommon organisms that are rarely isolated. M. hominis and U. urealyticum are involved in a variety of genitourinary disorders. 1 Epidemiologic studies on the role of these microorganisms in human infertility have given conflicting results. Gnarpe and Friberg2 demonstrated that U. urealyticum could play a role in human reproductive Received January 24, 1984; revised and accepted May 4, 1984. *Institute of Microbiology. tReprint requests: Franco Busolo, Ph.D., Istituto di Microbiologia, Via Gabelli 63, 35100 Padova, Italy. tInstitute of Anatomy. 412

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failure, and Horne et al.a showed a significant association between ureaplasmas and subclinical endometrial inflammation. On the contrary, De Lavois et al.,4 Ulstein et al.,5 and Matthews et al. 6 found no differences in frequency of ureaplasmas from the genital tract of infertile men and fertile control subjects. Other studies showed that ureaplasmas may adversely affect spermatozoal motility 7,8 and morphology.9,10 Fowlkes et al. 9 noted two morphologic features of spermatozoa from ureaplasrna-positive patients by scanning electron microscopy (SEM): the presence of clusters of small sphere-shaped particles adherent to the cells and a large number of coiled tails. Toth et al. lO were able to predict the presence of ureaplasmas in the semen using light microscopy, and Swenson et al. 11 showed that the eradication of ureaplasmas from the semen improved sperm motility. Fertility and Sterility

The purpose of this article is to present the results of morphologic features of spermatozoa studied by light, SEM, and transmission electron microscopy (TEM) and the relation of these features to ureaplasma titers in the semen of patients with unexplained infertility. MATERIALS AND METHODS PATIENTS

The present study was carried out on 116 male patients that were under treatment at the Urology Institute and the Institute of Semeiotica Medica at the University of Padua. All the patients showing hormones in the normal range were affected by unexplained infertility without urinary diseases, varicocele, and antispermatozoal antibodies. Their partners were ovulating regularly and had patent fallopian tubes as judged by hysterosalpingography. Seminal specimens of 30 healthy control subjects with proven fertility were examined only for the presence of mycoplasmas. SPECIMENS

Semen was collected by masturbation into a sterile container after 3 days of sexual abstinence. Aliquots were cultured, fixed, and stained within 2 hours for detection of mycoplasma. ROUTINE SEMEN ANALYSIS

The semen analysis was performed according to Eliasson 12 with some minor modifications. We used as minimal criteria for judging the fertility 30 million spermatozoa/ml, at least 60% of good motility after 2 hours, and 75% of spermatozoa with normal forms. ISOLATION AND IDENTIFICATION OF MYCOPLASMAS

The media used for the isolation of mycoplasmas were essentially the same as described by McCormack et al. 13 Viable ureaplasmas were counted as color changing units (CCUs) using the tube dilution method described by Masover et al. 14 The large-colony mycoplasmas were identified according to the method of Clyde. 15 U. urealyticum was identified by hydrolysis of urea to ammonia resulting in a rise in pH and a visible change in the indicator. If a change occurred, a sample was subcultured on solid and liquid meVol. 42, No.3, September 1984

dia. The specimen was considered positive only when the secondary broth changed color and tiny colonies of typical morphology were observed on relative agar medium. LIGHT MICROSCOPY

After liquefaction of fresh seminal fluid, spermatozoa were washed three times in phosphatebuffered saline, and a drop was examined at 250 x and 400 x using phase-contrast interference microscopy (Leitz Ortholux II, Ernst Leitz GmbH, Wetzlar, West Germany). The indirect immunofluorescence technique was performed on previously washed spermatozoa which were positive for M. hominis. As a negative control, a smear treated only with fluorescein-conjugated goat anti-rabbit IgG (Hyland, Costa Mesa, CA) was used. Unfortunately, the same procedure was not carried out for the detection of U. urealyticum because commercial antisera were not available. TRANSMISSION ELECTRON MICROSCOPY

The spermatozoa were washed with 0.1 M phosphate buffer (pH 7.2), fixed in 3% (vol/vol) glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) for 120 minutes at 4°C. Then they were washed with· cacodylate buffer, postfixed in 1% (wt/vol) osmium tetroxide in 0.1 M phosphate buffer (pH 7.2) for 90 minutes at 4°C and washed again. After centrifugation at 2000 x g for 10 minutes, the pellet was embedded in 2% (wt/vol) purified agar in 0.1 M phosphate buffer and allowed to solidify at room temperature. Agar blocks (1 x 1 x 1 mm) were cut and dehydrated through a graded series of ethanol and embedded in epoxy resin. 16 Thin sections were cut with an LKB 8800A ultratome III microtome (LKB-Produkter, Bromma, Sweden) equipped with a glass knife, placed on carbon-coated 300-mesh copper grids, and stained with uranyl acetate and lead citrate. 17 The specimens were carbon-coated in an evaporator and examined by Hitachi HS 9 electron microscope (Hitachi Ltd., Tokyo, Japan). SCANNING ELECTRON MICROSCOPY

Semen samples were centrifuged in 0.1 M phosphate buffer (pH 7.2) for 3 minutes at 500 x g. The spermatozoa, washed twice, were then fixed in 2.5% solution of glutaraldehyde in phosphate buffer. After fixation, the spermatozoa were allowed to sediment, and the supernatant glutaralBU801o et al. Mycoplasmas on human spermatozoa

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sperm.tozo. million/ml

titer of

ur.apla.ma. (c.c.u.lml)

Figure 1 Correlation between the CCUs of U. urealyticum and the number of spermatozoa in infertile men. The number inside the columns indicates the number of patients examined. The bar represents the standard error.

dehyde was gently replaced by distilled water. Then the samples were dehydrated in 50%, 70%, 90%, 95%, and 100% acetone. Two drops of acetone containing spermatozoa were placed on coverslips glued to the SEM specimen tubes. The specimens were coated with gold-palladium. A negative potential was applied to the gold cathode, which was enclosed in the process chamber at a pressure of 50 to 5000 millitor. The specimens were observed using a Cambridge Stereoscan 250 microscope (Cambridge Ltd., Cambridge, England) equipped with a 250-ILm aperture and operated with an accelerating voltage of 25 k V.

RESULTS Mycoplasmas were isolated in 56 (48.3%) of the 116 examined samples. U. urealyticum was pres-

ent in 49 (42.2%), M. hominis in 3 (2.6%), and both species in 4 (3.5%) samples of seminal fluid. U. urealyticum was found in 10% of healthy control subjects, and the titer ranged from 10 to 103 CCU/ml. Viable ureaplasma counts showed that 41 of the 56 ureaplasma-positive patients (73.2%) gave a titer ranging from 102 to 104 CCU/ml. Only one patient had 107 CCU/ml and two had 106 CCUI ml. Figure 1 shows that there was no relationship between the concentration of spermatozoa and the titer of ureaplasmas. Indeed, nine ureaplasrna-positive patients having a titer of 104 CCU/ml had a concentration of spermatozoa higher than the five patients with a titer of 10 CCU/ml. These data suggest that U. urealyticum does not affect the quantity of spermatozoa produced during spermatogenesis. In 41 patients, cited above, the ratio between CCU per milliliter and the number of spermatozoa in 1 ml of seminal fluid ranged from 1:3400 to 1:295,000. PHASE.CONTRAST INTERFERENCE MICROSCOPY

Every mycoplasma-positive semen sample showed sphere-shaped particles adherent mainly to the midpiece of the tail of the sperm. In most of the cases they were present in 50% to 60% of the spermatozoa, determined as a mean of at least ten microscopic fields observed (Fig. 2a). The same pattern was sometimes observed in mycoplasmanegative samples, although in a lower percentage than in the mycoplasma-positive samples. FLUORESCENCE MICROSCOPY

Figure 2b shows a spermatozoon from an M. hominis-positive sample with a specific fluores-

Figure 2 Spermatozoa from infertile men. (a), Phase-contrast interference micrograph showing sphere-shaped particles on the midpiece of spermatozoa. Notice the spermatozoon free of sphere-shaped particles. (b), Demonstration of M. hominis on the midpiece with the immunofluorescence technique.

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Fertility and Sterility

Figure 3 Scanning (a) and transmission (b, c, and d) electron micrographs of spermatozoa from patients with unexplained infertility. (a), Spermatozoa with sphereshaped particles on the midpiece and postacrosomal cap (arrows). (b), Mycoplasmalike particles (arrows) on the mid piece of the spermato-' zoon. (c), Mycoplasma cells inside the swollen zone. (d), Trilaminar membrane (ar-· rows) of mycoplasma cells in a higher magnification of c. Bar markers represent 1 ILm (a, b, and c) and 0.1 ILm (d).

cence on the midpiece. However, not all the spermatozoa with the sphere-shaped particles showed specific fluorescence. Negative control samples did not have a fluorescent pattern. SCANNING ELECTRON MICROSCOPY AND TRANSMISSION ELECTRON MICROSCOPY

We examined four positive (three for U. urealyticum and one for M. hominis) and four negative samples to obtain more evidence that sphereshaped particles could be considered mycoplasmas. Using SEM, according to Fowlkes et al.,9 we found small projecting structures in the midpiece and sphere-shaped particles on the anterior part of the coiled tail (Fig. 3a). These structures were present in only one mycoplasma-negative ejaculate. These data confirmed the patterns observed with light microscopy. TEM revealed two patterns in the same specimen. In the first pattern, the more evident characteristic was the presence of a swollen zone on the midpiece delimited by a Vol. 42, No.3, September 1984

membrane. Also, some moderately electron-dense sphere-shaped particles with a finely granular matrix were seen. Their diameters ranged from 100 to 300 nm (Fig. 3b). The second pattern was seen only in a few spermatozoa. Mycoplasma cells were delimited by a membrane, and the size of these cells ranged from 220 to 1600 nm (Fig. 3c). Their matrix was finely and uniformly granular and surrounded by a typical trilaminar membrane (Fig. 3d). The two patterns described above were not found in mycoplasma-negative ejaculates. DISCUSSION

Some morphologic characteristics of spermatozoa could indicate the presence of a mycoplasma infection. With the Papanicolaou stain, a fine greenish granularity on the tail and fine interlacing filaments can be observed, particularly on a localized area of the spermatozoa. 10 Small outBU80lo et al. Mycoplasmas on human spermatozoa

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growths were seen on the midpiece of the sperm cells from ureaplasma-positive ejaculates with SEM. 18 Thes~ were recognized as sphere-shaped particles adhering to the midpiece of the tail. 9 Phase-contrast interference microscopy showed a high percentage of spermatozoa from M. homin is-positive samples with sphere-shaped particles adhering to the midpiece of the tail. Instead, immunofluorescence technique showed on the same samples only a low percentage to be fluorescent, which indicated that not all the sphereshaped particles were M. hominis. Examination by TEM revealed the presence of probable ureaplasma-like particles in only one of the three samples from ureaplasma-positive patients. But no relationship was seen between these ureaplasma-like particles and ureaplasmas, because in all three samples the sphere-shaped particles were seen with phase-contrast interference microscopy. This observation was confirmed by quantitative analysis of ureaplasmas in the semen. In most of our cases viable ureaplasmas were present in the seminal fluid but only at very low concentration. In fact, 41 of 56 patients had a· titer ranging from 10 to 104 CCU/mi. The ratio between the concentration of viable ureaplasmas and the number of spermatozoa was very low, ranging from 1:3400 to 1:295,000. Such a ratio was ten times lower than that reported by TaylorRobinson and McCormack. 1 Previous researchers reported that the genital organs were aseptic in healthy men (with the exception of the urethra)19 and never showed mycoplasmas in the semen from the vas deferens of vasectomized patients, despite their isolation from the voided urine. This fact suggests a mycoplasma contamination of semen with a possible attachment of mycoplasma cells on spermatozoa during the passage through the urethra. By using TEM we found such a complex pattern inside the swollen zone containing elements surrounded by a typical trilaminar membrane and ribosomal texture that probably required a longer time than the normal time of passage of sperm through the urethra. These elements were very similar to those described in a previous article on the morphology of mycoplasma cells20 ; and so we considered only these as mycoplasmas. On the other hand, there was no evidence that infections of the genital tract involved multiple sites or were limited to a specific organ. Another more frequent pattern was similar to that reported by Grossgebauer et al.,21 but this 416

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did not fully convince us that these particles were mycoplasmas. Furthermore, we found a significant difference in motility of spermatozoa from ureaplasma-positive and -negative patients in our epidemiologic investigations, even though no effect was seen on motility and morphology of spermatozoa incubated overnight with ureaplasmas in experiments carried out in vitro.22 Moreover, the same patterns (sphere-shaped particles and coiled tails) observed in pathologic specimens from naturally infected patients were never found in artificial ureaplasma overcontamination of washed normal spermatozoa. These data might indicate that the effects of ureaplasmas on spermatozoa in vitro are different from those occurring in vivo. In conclusion, our data showed the following: (1) The light-microscopic observation of sphereshaped particles does not allow us to discern the presence of mycoplasmas in semen and therefore can never replace the cultures. (2) The complexity of the morphologic patterns observed with TEM suggested that a mere contamination of sperm was not always present. Further studies are needed to illuminate the anatomic extent of my coplasmal colonization in male patients as well as the. relationship between these microorganisms and infertility.

REFERENCES 1. Taylor-Robinson D, McCormack WM: The genital myco-

plasmas. N Engl J Med 302:1003, 1980 2. Gnarpe H, Friberg J: Mycoplasma and human reproductive failure. I. The occurrence of different mycoplasmas in couples with reproductive failure. Am J Obstet Gynecol 114:727, 1972 3. Horne HW, Hertig AT, Kundsin RB, Kosasa TS: Subclinical endometrial inflammation and T-mycoplasma: a possible cause of human reproductive failure. Int J Fertil 18:226, 1973 4. De Lavois J, Harrison RF, Blades M, Hurley R, Stanley VC: Frequency of mycoplasma in fertile and infertile couples. Lancet 1:1073, 1974 5. Ulstein M, Capell P, Holmes KK, Paulsen CA: Nonsymptomatic genital tract infection and male infertility. In Human Semen and Fertility Regulation in Men, Edited by ESE Hafez. St. Louis, C. V. Mosby, 1976, p 355 6. Matthews CD, Elmslie RG, Clapp KH, Svigos JM: The frequency of genital Mycoplasma infection in human fertility. Fertil Steril 26:988, 1975 7. O'Leary WM, Frick J: The correlation of human male infertility with the presence of mycoplasma T-strain. Andrologia 7:309, 1975 Fertility and Sterility

8. Fowlkes DM, MacLeod J, O'Leary WM: T-mycoplasmas and human infertility: correlation of infection with alterations in seminal parameters. Fertil Steril 26:1212, 1975 9. 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, 1975 10. Toth A, Swenson CE, O'Leary WM: Light microscopy as an aid in predicting Ureaplasma infection in human semen. Fertil Steril 30:586, 1978 11. Swenson CE, Toth A, O'Leary WM: Ureaplasma urealyticum and human infertility: the effect of antibiotic therapy on semen quality. Fertil Steril 31:660, 1979 12. Eliasson R: Analysis of semen. In The Testis, Edited by H Burger, D de Kretser. New York, Raven Press, 1981, p 381 13. McCormack WM, Rosner B, Lee Y-H: Colonization with genital mycoplasmas in women. Am J Epidemiol 97:240, 1973 14. Masover GK, Catlin J, Hayflick L: The effect of growth and urea concentration on ammonia production by ureahydrolising Mycoplasma (Ureaplasma urealyticum). J Gen Microbiol 98:587, 1977

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15. Clyde WA Jr: Mycoplasma identification based upon growth inhibition by specific antisera. J Immunol 92:958, 1964 16. Locwood WR: A reliable and easily sectioned epoxy embedding medium. Anat Rec 50:129, 1964 17. Reynolds EW: The use of lead citrate and high pH as an electron-opaque stain in electron microscopy. J Cell Bioi 17:208, 1973 18. Gnarpe H, Friberg J: T-mycoplasmas on spermatozoa and infertility. Nature 245:97, 1973 19. Berger RE, Alexander ER, Monda ED, Ansell J, McCormick G, Holmes KK: Chlamydia trachomatis as a cause of acute "idiopathic" epididymitis. N Engl J Med 298:301, 1978 20. Meloni GA, Bertoloni G, Busolo F, Conventi L: Colony morphology, ultrastructure and morphogenesis in M. hominis, A. laidlawii and U. urealyticum. J Gen Microbiol 116:435, 1980 21. Grossgebauer K, Henning A, Hartmann D: Mykoplasmenbedingte spermatozoenkopfschiiden bei infertilen miinnern. Hautarzt 28:299, 1977 22. Busolo F, Zanchetta R: UnpUblished data

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