FACTORS AFFECTING SPERM MOTILITY. VI. SPERM VIABILITY UNDER THE INFLUENCE OF BACTERIAL GROWTH IN HUMAN EJACULATES

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1 FERTILITY AND STERILITY Copyright c 191 The American Fertility Society Vol. 3, No.6, June 191 Printed in U.SA. FACTORS AFFECTING SPERM MOTILITY. VI. SPERM VIABILITY UNDER THE INFLUENCE OF BACTERIAL GROWTH IN HUMAN EJACULATES AMNON MAKLER, M.D.* YAKOV URBACH, M.D. ELI LEFLER, D.Sc. DAVID MERZBACH, PH.D. Infertility Institute and Department of Microbiology, Technion Faculty of Medicine, Rambam Medical Center, Haifa, Israel The influence of bacterial growth on human sperm motility and viability was evaluated objectively with the multiple-exposure photography method. Experimental semen specimens, obtained from normal donors by nonaseptic means of masturbation, were incubated with antibiotics at room temperature or body temperature for 2 hours. Although bacteria, grew in control specimens, were totally eradicated in all antibiotic-treated specimens, no significant difference was found between these groups with regard to sperm motility throughout the time of incubation. Sperm survival was not inhibited, nor was it extended as a result of suppression of bacterial growth. In both groups, survival time was much shorter in specimens incubated at body temperature than in those kept at room temperature. Sperm motility was not affected after 2 hours of incubation of fresh specimens with high concentrations of various pathogenic bacteria similar to those found in severe prostatitis. The question of whether the use of antibiotics in the treatment of asthenospermia per se has a prognostic value is discussed. Fertil Steril 3:666, 191 The survival of spermatozoa after ejaculation is dependent on the environmental conditio]ls under which they are kept. In the female genital tract they may remain active for several days, especially within the mucus of the cervical crypts. 1 On the contrary, their activity is of much shorter duration if they remain in the seminal fluid outside the body.2,3 Most investigators agree that the majority of spermatozoa cease to move within the first 2 hours, as was recently confirmed by our objective determination. It was assumed there that this relatively short life-span in vitro was due to bacterial growth within artificially contaminated seminal fluid. The presence of bacteria may lead to a change in hydrogen ion concentration or depletion of carbohydrates which are essential for spermatozoal activity.6 The excretion of bacterial toxic products may also be a factor in the short spermatozoal life-span in vitro. 7 If this assumption is correct, suppression of bacterial growth by added antibiotics should extend in vitro sperm survival and may justify antibacterial treatment to improve sperm motility in asthenospermic patients when male accessory gland infection is suspected. In the present study, sperm motility and viability as affected by bacterial growth and suppression in human ejaculates was objectively evaluated with the aid of the multiple-exposure photography method. MATERIALS AND METHODS Received October 1, 190; revised and accepted January, 191. *Head of Infertility Institute. To whom reprint requests should be addressed. Ejaculates from healthy fertile donors between 21 and years of age were collected in clean glass vials by nonaseptic means of masturbation. Thirty-seven specimens that fulfilled our criteria 666

2 Vol. 3, No.6 FACTORS AFFECTING SPERM MOTILITY. VI 667 TABLE 1. Isolated Microorganisms and Their Occurrence in Twenty-Four Specimens Microorganism Staphylococcus epidermidis Diphtheroids Streptococcus faecalis a-hemolytic streptococcus Non-hemolytic streptococcus Escherichia coli Staphylococcus citreus Alcaligenes faecalis Pseudomonas aeruginosa Klebsiella aerogenes Staphylococcus aureus Candida species Neisseria species % Occurrence in 2 specimens as normospermic, namely, concentration above 20 million/ml and above % motile spermatozoa were incorporated in the study. Specimens from eight donors that were found to contain leukocytes were also included despite the fact that none of the donors complained of any symptoms related to prostatitis. All specimens were used within 1 hour in the following experiments. Experiment 1. Each of the first 2 specimens was divided into 0.9-ml portions and transferred into glass test tubes. Aliquots (0.1 ml) from a stock solution containing sodium penicillin-g and streptomycin sulfate dissolved in normal saline were added to each experimental test tube, by which a concentration of 1000 units and 00 f.lg/ ml, respectively, as recommended elsewhere, was provided. The same amount of normal saline only was added to each control test tube. All test tubes were sealed with parafilm to prevent evapo ration. Pairs of experimental and control specimens were kept at room temperature (20 to 23 C) or incubated at body temperature (37 C). Samples (0.0 ml) were taken by aseptic means from all specimens at 2-hour intervals throughout the first 12 hours and finally after 2 hours, and were used for bacterial identification and quantitative assay. At the same time ph and sperm motility were also determined in all test tul;>es. Experiment 2. Thirteen specimens were divided and prepared as described previously. A sample (0.1 ml) from a culture medium containing 107 of one of the following bacteria was added to the antibiotic-free portions of each specimen: Escherichia coli, Streptococcus faecalis (enterococcus), Proteus morganii, a-hemolytic streptococcus, and Staphylococcus epidermidis (S. albus). These species and the mentioned concentration were selected according to reported findings in patients with prostatitis or prostatosis, where the bacterial concentration in seminal fluid does not exceed 106/m Sperm motility was determined in all specimens before and after 2 hours of incubation at 37 C. Microbiologic Studies. Samples taken periodically from each specimen, were spread on blood and MacConkey's agar plates. Bacteria were identified after hours of incubation at 37 C according to standard microbiologic techniques. 13 A quantitative bacterial assay was performed by preparing 10-fold dilutions and spreading 0.1 ml of the dilutant on proper agar plates, with a glass spreaderl; ph was determined with a glass electrode digital ph meter (model PBS720, Kibbutz EI-Hama, Israel). Sperm motility was determined with the aid of the multiple-exposure photography method described in detail elsewhere.1, 16 Each of two to three drops from a well-mixed specimen was placed in the improved 10-f.Lm chamber17 (manufactured by EI-Op, Israel Electro-Optical Industry, Rehovoth, Israel), and to 12 fields containing 200 to 00 spermatozoa/specimen were photographed at a predetermined location. Each film was exposed for 1 second during which time the sample was illuminated by six light pulses. Images of photographed sperm were projected onto sheets of paper from which the percentage of motile sperm and their velocity were calculated with the specially designed microcomputer. 1 The results from all experiments were statistically analyzed, and significance was determined by Student's paired t-test. RESULTS Thirteen species of microorganisms were isolated from the 2 specimens that were collected by masturbation. Each specimen was found to contain more than one type of bacteria (Tables 1 and 2), and most of these microorganisms were isolated several times from different specimens. In contrast, bacteria were completely eradicated in all test tubes containing antibiotics, whether incubated at room temperature or body temperature. Figures 1 and 2 demonstrate relative TABLE 2. Number of Isolated Bacterial Species per Specimen Isolated species per specimen No. of specimens 3 1 2

3 66 MAKLER ET AL. June 191 CONTROl.-- ANTIBIOTICS C CONTROl.-- ANTIBIOTICS-- 37 C PERCENT MOTILE PERCENT MOTILE 0 ::l :3 or z "'0 AV VELOCITY z a:: AV VELOCITY O ,---- ol ph VALUE.0 =-_""""'=========-""""::::::: ph VALUE.0-===:::::====:::::::::"""""=:::::::::----_l 7.0 i i c: J 7 6 BACTERIAL GROWTH HOURS FIG. 1. Mean change in rates of bacterial growth, ph values, and relative sperm motility in 2 specimens incubated at 23 C for 2 hours. HOURS FIG. 2. Mean change in rates of bacterial growth, ph values, and relative sperm motility in 2 specimens incubated at 37 C for 2 hours. change in sperm velocity and percentage of motility throughout the 2 hours at either temperature of incubation. The mean ph values in all specimens and the rate of bacterial growth in the antibiotic-free specimens are also shown. Changes in sperm motility with time were almost identical in experimental and antibiotic-free specimens. The steady growth of bacteria essentially did not affect sperm motility, nor did the presence of antibiotics induce any harmful effect on their motility at any temperature of incubation. However, survival of spermatozoa incubated at room temperature differed significantly from that of spermatozoa incubated at body temperature. After 2 hours, motile spermatozoa were still present in many specimens kept at room temperature, unlike' those incubated at body temperature. In the latter, no motile sperm could be found after 12 hours, regardless of the presence or absence of bacteria. The steady growth of bacteria in all antibiotic-free specimens was more remarkable at body temperature. ph values remained unchanged in all specimens containing antibiotics throughout the 2 hours; the same was true in control specimens for the first to 12 hours. Only after hours in specimens incubated at 37 C or after 12 hours in those kept at room temperature did ph values decrease significantly, after a phase of extensive bacterial multiplication. The histograms shown in Figure 3 describe mean values of sperm motility in specimens after incubation with various pathogenic bacteria at 37 C for 2 hours. No significant decrease in sperm velocity or in percentage of motility was induced by the presence of any of these bacteria, as compared with controls containing antibiotics.

4 Vol. 3, No.6 FACTORS AFFECTING SPERM MOTILITY. VI 669 Specimens containing leukocytes did not differ from leukocyte-free specimens with regard to sperm velocity or survival rate. AV Velocity m/sec DISCUSSION Isolation of bacteria from human semen specimens and contamination of ejaculates by bacteria even after delivery by aseptic means have been described by others.9, 10, 12, 19 Therefore, it was not surprising that bacteria were isolated from all specimens collected by nonaseptic means into nonsterile containers as is generally done. The steady growth of microorganisms in our antibiotic-free specimens excludes the possibility that the antibacterial factor in human semen, described by others,20, 21 had any significant bacteriostatic effect. Practically no suppression of bacteria was demonstrated by us, and the curves of their growth within ejaculates resembled those in ordinary culture media under optimal conditions. However, in contrast to other reports,22, 23 our results clearly demonstrated that the presence of bacteria in seminal fluid, of whatever origin, and in high concentration, does not interfere with normal sperm motility and viability. Moreover, suppression of bacterial growth in ejaculates could not extend sperm survival beyond what was expected in intact specimens. The shorter lifespan of spermatozoa incubated at body temperature was not due to extensive bacterial growth or to change in ph as has been suggested by others.,2 The survival in control specimens was basically the same as that in experimental specimens in which bacterial growth was completely suppressed at 37 C. Sperm became immotile regardless of changes in ph and much before the ph became significantly lower. It is still unclear why sperm survival in vitro is much less at 37 C than at room temperature, whereas it il'l considerably longer in the female genital tract. 1 We could not extend this life-span by adding various kinds of carbohydrates, and it would seem that consumption of intrinsic metabolic sources and their wastage are more rapid in spermatozoa incubated at body temperature. Adding pathogenic bacteria to fresh specimens in amounts similar to, or higher than, those found in prostatic fluid of patients with prostatitis9, 10, 12 did not induce any deleterious effect on sperm motility. This finding is in disagreement with a study in which E. coli was shown to immobilize human sperm.21 However, in the latter the concentration of bacteria was 1013, which is FIG. 3. Mean values of sperm velocity and percentage of motility in 13 specimens after 2-hour incubations at 37 C with various pathogenic bacteria (l06/ml) as compared with controls. about 1 million times that in our study and therefore without any clinical implication. From the quantities used in our experiments, although based on in vitro studies, important clinical and practical conclusions may be drawn: for asthenospermic patients, treatment of symptomatic or asymptomatic prostatitis in an effort to improve sperm motility per se seems to be of a questionable value. Naturally, spermatozoa are mixed with seminal fluid just after ejaculation and remain in this medium only for a short while before they penetrate the cervical mucus. During this short time, the presence of bacteria within seminal fluid may not interfere with spermatozoal motility. It is therefore doubtful whether infected seminal fluid is a cause of asthenospermia, although many investigators believe that it is, and maintain that sperm motility improves after such patients are treated with antibiotics.2, 26 Our results support the idea that no relationship exists between semen infection and poor sperm motility27; the latter seems to be due to other environmental or intrinsic factors that remain to be investigated. Whether or not the presence of bacterial growth may affect fertility is currently being investigated by us. REFERENCES 1. Hafez ESE: Physiology of spermatozoal insemination in the female reproductive tract. In Homologous Artificial Insemination, Edited by JC Emperaire, A Audebent, ESE Hafez. The Hague, Mastinus Nijhoff Publishers, 190, p 1 2. Falk HC, Kaufman SA: What constitutes normal semen? Fertil Steril1:9, Freund M: Interrelationship among the characteristics of human semen and factors affecting semen specimen quality. J Reprod Fertil :2, Makler A, Zaidise J, Paldi E, Brandes JM: Factors affecting sperm motility. I. In vitro change in motility with time after ejaculation. Fertil Steril 31:17, 1979

5 670 MAKLER ET AL. June 191. Appel RA, Evans PR: The effect of temperature on sperm motility. II. Is bacterial growth a factor? Fertil Steril 30:36, Pomerol JM, Marina S: Urological aspects of the treatment of infertile males. In Male Fertility and Sterility, Edited by RE Mancini, L Martini. London, Academic Press, 197, p Paulson JD, Polakoski KJ: Isolation of a spermatozoal immobilization factor from Escherichia coli filtrates. Fertil Steril 2:12, Ackerman DR, Behrman SJ: Artificial insemination and preservation of human semen. In Progress in Infertility, Edited by SJ Behrman, RW Kistner. Boston, Little Brown and Co, 197, p Meares EM: Bacterial prostatitis versus prostatosis; A clinical and bacteriological study. JAMA 22:1372, Enroth P, Ljungh-Wadstrom A, Moberg PJ, Nor-ol CE: Studies on bacterial flora in semen from males in infertile relations. Int J Androl 1:10, Ulstein M,Capell P, Holmes KK, Paulsen CA: NonBymptomatic genital tract infection and male infertility. In Human Semen and Fertility Regulation in Men, Edited by ESE Hafez. St Louis, CV Mosby Co, 1976, p Comhaire F, Verschaegen G, Vermulen L: Diagnosis of accessory gland infection and its possible role in male infertility. Int J Androl 3:32, Cowan ST, Steel KJ: Manual for the Identification ofmedical Bacteria, Edited by ST Cowan. Cambridge, Cambridge University Press, Wilson GS, Miles AA". Topley and Wilson's Principles of Bacteriology and Immunity. Edward Arnold Ltd., London, 19, p Makler A: A new multiple exposure photography (MEP) method for objective sperm motility determination. Fertil Steril 30:192, Makler A: Use of the elaborated multiple-exposure photography method in routine semen analysis and for research purposes. Fertil Steril 33:160, Makler A: The improved 10-. m chamber for rapid semen analysis. Fertil Steril33:337, Makler A: Use of microcomputer in combination with the MEP method for sperm motility determination. J Urol 12:372, MobergPJ, EnrothJ, LjungA, Nord.CE: Bacterial flora in semen before and after doxycycline treatment of infertile couples. Int J Androl 3:6, Rozansky R, Gurevitch J, Brzezinsky A, Eckerling B: Inhibition of growth of Staphylococcus aureus by human semen. J Lab Clin Med 3:126, Mardh PA, Colleen S: Antimicrobial activity of human seminal fluid, Stand J Urol Nephrol 9:17, Teague NS, Boyarsky S, Glenn JF: Interferep.ce of human spermatoz(l motility by Escherichia coli. Fertil Steril 22:21, Del Porto GB; Derrick FC, Bannister ER: Bacterial effect on sperm motility. Urology :63, Appell RA, Evans PR: The effect of temperature on sperm motility and' viability. Fertil Steril2:1329, Hommonai TZ, Sasson S, Paz G,. Kraicer PF: Improvement of fertility and semen quality in men treated with a combination of anticongestive and antibiotics drugs. Int J Fertil 20:, Derrick JR, Dahlberg B: Male genital tract infection and sperm viability. In Human Semen and Fertility Regulation in Men, Edited by ESE Hafez; St Louis, CV Mosby Co, 1976, p Eliasson R, Arver S, Johnson 0, Kvist U, Lindholmer C: Some effects of human seminal plasma on spermatozoa. In Recent Progress in Andrology, Edited by A Fabrini, E Steinberger. London, Academic Press, 197, p 2