Semen is a product of spermatozoa from the epididymis and secretions from the prostate, seminal vesicles, and bulbourethral glands.

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1 FERTILITY AND STERILITY Copyright e 993 The American Fertility Society Vol. 59, No., January 993 Printed on acid free paper in U.S.A. The role of aerobic and anaerobic semen cultures in asymptomatic couples undergoing in vitro fertilization: effects on fertilization and pregnancy rates Dale W. Stovall, M.D.t Linda E. Bailey, R.N. Luther M. Talbert, M.D. Department of Obstetrics and Gynecology, Division of Reproductive Endocrinology and Fertility, University of North Carolina School of Medicine, Chapel Hill, North Carolina Objective: To determine if routine semen culture is useful in asymptomatic couples undergoing in vitro fertilization and embryo transfer (IVF-ET). Design: Prospective data collection. Setting: All cultures and IVF cycles were performed at the University of North Carolina in Chapel Hill, North Carolina. Participants: All asymptomatic couples undergoing IVF-ET from January 989 through January 990. Interventions: Aerobic and anaerobic cultures were performed on semen samples obtained before IVF. Main Outcome Measures: Quantitative semen cultures were evaluated for both aerobic and anaerobic bacterial isolates. Fertilization and pregnancy rates (PRs) were compared in patients with positive and negative semen cultures. Results: Eighty percent of cultures contained at least one bacterial isolate. Three of the four most commonly isolated bacteria were normal skin flora. Positive culture results had no effect on either fertilization or PRs. Conclusions: Bacterial contamination is common with semen collection, yet routine semen cultures are not beneficial in asymptomatic couples undergoing IVF -ET. Fertil SterilI993;59:97-20 Key Words: In vitro fertilization, semen, and bacterial culture Semen is a product of spermatozoa from the epididymis and secretions from the prostate, seminal vesicles, and bulbourethral glands. Each of these re- Received June 5, 992; revised and accepted September 24, 992. * Presented at the Annual District IV Meeting of the American College of Obstetrics and Gynecology, Sulphur Springs, West Virginia, October 28 to 3, 990. t Reprint requests and present address: Dale W. Stovall, M.D., Department of Obstetrics and Gynecology, Division of Repro ductive Endocrinology and Infertility, University of Pittsburgh Magee Women's Hospital, 300 Forbes Avenue, Pittsburgh, Pennsylvania 523. gions are normally sterile, but cultures of seminal fluid from asymptomatic men commonly produce one or more aerobic and/or anaerobic bacteria (- 3). Positive semen cultures are more common in asymptomatic men with a prior history of genital tract infection. The number and concentration of bacterial isolates in semen are less common in healthy infertile men as compared with infertile men with a history of prostatitis or urethritis (2). The presence of bacteria in semen does not always signify infection; the concentration and type of bacteria are important. Bacteriospermia may represent contamination, colonization, or infection. Vol. 59, No., January 993 Stovall et al. Semen culture in IVF 97

2 A direct interaction between bacteria and spermatozoa has been documented. Bacteria can attach to the head, midpiece, and tail of spermatozoa (4-6). Although the presence of specific types of bacteria in semen has been correlated with male infertility in an infertility clinic setting (7), it is unclear whether bacteria have any direct effect on sperm function. If particular bacterial species impair the fertilizing ability of spermatozoa, this might have an impact on the success of in vitro fertilization (IVF). The purpose of this study was twofold. The first objective was to identify the concentration and types of aerobic and anaerobic bacteria contained in seminal fluid of asymptomatic couples undergoing IVF. Our second objective was to determine which, if any, of the bacteria isolated from semen have a detrimental effect on IVF or IVF pregnancy rates (PRs) when compared with couples whose husband's semen culture was negative. Patient Population MATERIALS AND METHODS Couples participating in the IVF program at the University of North Carolina between January 989 and March 990 were eligible for the study. A medical history and physical examination were obtained from each couple. A history of sexually transmitted diseases or any symptoms of urinary and prostatic infection such as urethral burning or discharge was obtained. Symptomatic couples and men with a history of genital tract infection were excluded from the study. Men with known varicoceles were also eliminated from the study population. Collection of Semen Semen samples were obtained from each husband within 4 weeks of their spouses' beginning medication for controlled ovarian hyperstimulation. The men were instructed to abstain from ejaculation for 3 days before obtaining a specimen. Each patient was given a hexachlorophene sponge (EZ Scrub; Becton Dickinson Acute Care, Franklin Lakes, NJ) and sterile field towel (Johnson and Johnson. Arlington, TX) and instructed on the procedure for cleaning his hands and penis before semen collection. All samples were collected by masturbation. The subjects were not allowed to use lubricants of any form. Samples were collected in a sterile specimen container (Fisher Scientific, Norcroff, GA) and delivered to the laboratory within 30 minutes. After liquefaction, semen analysis was performed on each semen sample. Only men who met the W orid Health Organizations criteria for normal semen analysis were candidates for the study (8). Semen Culture and Bacterial Identification After liquefication, a -ml aliquot of semen was immediately delivered to the University of North Carolina Hospital Microbiology Laboratory for quantitative bacteriologic culture. Semen cultures for both aerobic and anaerobic bacteria were performed. For aerobic culture, 0 JLL of semen were plated on Martin-Lewis, chocolate, V-selective, blood, MacConkey's, and Columbia colistin-nalidixic acid agar. For anaerobic culture, prereduced phenylethyl alcohol, blood, and anaerobic brainheart infusion agar were used. All agar preparations were preformed in the microbiology laboratory. Specific cultures were not performed for Chlamydia trachomatous, Mycoplasma hominis, or Urea plasma urealyticum. Colony and growth features, gram-stain characteristics, and specific biochemical kits were used to classify bacteria. In the case of anaerobic bacteria, a single kit (Innovative Diagnostic Systems, Atlanta, GA) was used. Numerous kits were used in the identification of aerobic bacteria depending on the genus. To determine bacterial concentrations, semen samples were first diluted with phosphate-buffered solution and vortexed to maintain even distribution of all organisms. The samples were then plated, and colony counts were performed. Using this technique, each colony represented 00 organisms/ml. In Vitro Fertilization Cycle and Outcome Controlled ovarian hyperstimulation consisted of midluteal phase down regulation with leuprolide acetate (Lupron; Tap Pharmaceutical, Inc., Chicago, IL) followed by human menopausal gonadotropins (Pergonal; Serono Laboratories, Inc., Randolph, MA). Oocyte retrieval was performed approximately 35 hours after the administration of human chorionic gonadotropin, 0,000 IV. All oocyte retrievals were performed with transvaginal ultrasound (US) guidance. The spermatozoa were washed in Ham's F-0 culture media (GIBCO Laboratories, Grand Island, NY) supplemented with bovine serum albumin (Sigma Chemical Co., St. Louis, MO). After collection, the ejaculate was centrifuged twice at 250 X g for 0 minutes. Each oocyte was inseminated with 50,000 motile spermatozoa. The embryo culture 98 Stovall et al. Semen culture in IVF Fertility and Sterility

3 , I media was supplemented with penicillin and streptomycin. Fertilization and PRs were recorded. Clinical pregnancy was defined as an intrauterine gestational sac documented by US. Data Analysis Fertilization and clinical PRs were the outcomes used for comparison. A X2 test was used to determine significant differences between couples whose husbands' semen culture were positive or negative. RESULTS Two hundred seven IVF couples and cycles were evaluated. All couples had at least year of infertility. The average age of the women in the study was 35.3 ± 3.5 years, and the average age of the men in the study was 35.9 ± 4.6 years. Two subsets of couples were isolated. The first group included men with positive semen cultures and the second men with negative semen cultures. Semen bacterial concentrations ranged from 0 2 organisms/ml to 0 6 organisms/ml. At least one bacterial isolate was identified in 67 (80.7%) of the cultures performed, with a mean of 2.85 bacterial isolates in each positive culture. The bacteria were divided into separate categories based on their mode of metabolism and gram-stain characteristics (9). The prevalence of each organism is presented in Table. The fertilization and PRs for patients with and without positive cultures are shown in Table 2. The number of eggs inseminated and the number of embryos transferred per cycle (patient) were similar in both groups. There were no differences in fertilization rates between patients with and without positive cultures: 60.0% and 62.9%, respectively (P> 0.05). There were no differences in clinical PRs between patients with and without positive semen cultures: 8. % and 8.9%, respectively (P> 0.05). Table 3 lists the fertilization rates for the four most common bacterial isolates. There was no correlation between the presence of any of these four bacteria and a decrease in either fertilization or clinical PRs as compared with patients with negative cultures. Insufficient data were available to evaluate more than the four most common bacteria. DISCUSSION Semen cultures commonly result in the isolation of numerous aerobic and anaerobic bacteria (-3). The first aim of this study was to determine the Table Prevalence of Bacterial Isolates in Semen of Asymptomatic Men Undergoing IVF Bacterial isolate* Aerobic and/or facultatively anaerobic grampositive cocci Micrococcus Staphylococcus (coagulase +) (coagulase -) Streptococcus (alpha hemolytic) (beta hemolytic) (group D) (gamma hemolytic) (nonhemolytic) Actinomyces Corynebacterium (Diptheroids) Gardenerella Facultatively anaerobic gram-negative bacilli Escherichia Klebsiella Enterobacter Proteus Morganii Gram-negative cocci and coccobacilli Neisseria Acinetobacter Asporogenous, gram-positive bacilli Lactobacillus Fusiform shaped gram-positive bacteria Hemophilus Anaerobic gram-negative bacilli Bacteroides Fusobacterium Anaerobic gram-positive cocci Peptococcus Peptostreptococcus Aerobic gram-negative cocci and bacilli Pseudomonas Eikenella Fungus Candida albicans Anaerobic gram-negative cocci Veillonella No. of positive culturest (5) 87 (42) 4 39 (9) (22) * Bacteria are categorized based on their mode of metabolism and gram-stain characteristics. t Percentages given for the most common isolates only (n = 207). incidence of positive semen cultures in an asymptomatic population of patients undergoing IVF and to characterize the bacteria isolated. Bacteria were cultured from the semen of 69% of these men. Of the four most commonly isolated bacteria, three (coagulase-negative staphylococci, alpha-hemolytic streptococci, and diptheroids) are common grampositive skin flora and the fourth (Enterococcus) is an inhabitant of the colon. As demonstrated by Mears (0), most gram-positive bacteria are considered commensals or normal flora of the distal Vol. 59, NO., January 993 Stovall et ai. Semen culture in IVF 99

4 Table 2 Comparison of IVF and PRs in Asymptomatic Patients With Positive and Negative Semen Cultures No. of Culture result couples inseminated fertilized No. of eggs inseminated per patient No. of embryos transferred per patient Clinical pregnancy % Positive culture 67,066 Negative culture (60.0)* 92 (62.9) 6.4 ± 3.t 7.6 ± 2.6t 3.3 ± 0.56t 3.7 ± 0.48t * Values in parentheses are percents. t Values are means ± SD. :/: Not significantly different. urethra, whereas most gram-negatives are considered pathogenic when recovered from semen specimens. If gram-positive bacteria are commensals of the distal urethra, this would help explain why meticulous washing did not eradicate these bacteria from seminal fluid. Gram-negative bacteria were not commonly isolated (4%). Interestingly, 9% of all specimens contained Enterococcus, which is clearly a pathogen in the reproductive tract. Enterococcus, Peptostreptococcus, and Escherichia coli are commonly found in semen cultures of men with nonacute prostatitis (). The principal objective of this study was to determine if fertilization and PRs were different in couples whose husbands had positive semen cultures compared with couples whose husbands had axenic semen. No differences in either the fertilization or PRs were found between these two groups. There was no correlation between the presence of any of the four most common bacteria isolated and a decrease in either fertilization or PRs. There are many possible explanations for these findings. Bacteria do interact with spermatozoa, and their attachment to the head, midpiece, and tail of spermatozoa has been documented (4-6). But routine sperm washing may eliminate bacteria from spermatozoa. Wong et al. (5) used repeat cultures and electron microscopy to Table 3 In Vitro Fertilization Rates Using Semen Contaminated by Four Common Bacterial Isolates Bacterial isolate inseminated Diptheroids 387 Coagulase negative staphylococcus 33 Enterococcus 7 a Hemolytic streptococcus 98 Negative cultures 305 fertilized 237 (6.2)* 22 (63.7)* 07 (62.6)* 8 (59.6)* 92 (62.9) * Not significantly different from patients with negative semen cultures; values in parentheses are percents. demonstrate the removal of bacteria from spermatozoa during sperm washing performed in preparation for intrauterine insemination. The presence of antibiotics (penicillin and streptomycin) used in our culture media may have eradicated bacteria not removed by washing, and finally, the addition of culture media may provide significant dilution to curtail bacterial growth. Other studies have evaluated the effects of bacterial contamination on spermatogenesis and sperm function. Some evidence supports an adverse effect of bacteria on semen parameters as well as fertilization rates. Swenson et al. (2) reported a decrease in sperm motility associated with bacteriospermia. Degeneration of hamster eggs fertilized by contaminated human spermatozoa has been reported, but two recent studies showed no harmful effects ofbacteria on semen parameters or the sperm penetration assay (3, 3). There is some evidence to suggest a positive correlation between bacterial contamination of semen and poor IVF outcome. In a study by Guillet-Rosso et al. (4), a significant difference was found in PRs, independent of the number of embryos transferred, between patients with negative cultures, 2% (48 pregnancies/232 cycles) and those with positive cultures, 2% (8 pregnancies/50 cycles, P < 0.05). Interestingly, there were no differences in fertilization rates between patients with positive and those with negative semen cultures. All of the patients with positive cultures were treated with the appropriate antibiotics (as determined by antibiotic sensitivities) before their IVF cycle. No information was given regarding the patient's symptomatology or history of genital infection. The authors concluded that zygote development was compromised by semen contamination and that routine semen culture was warranted before IVF. We performed both aerobic and anaerobic semen cultures. One might argue that any method used to recover anaerobic bacteria under aerobic conditions 200 Stovall et al. Semen culture in IVF Fertility and Sterility

5 might be insufficient to allow for an accurate representation of anaerobes. Instead of using an anaerobic transfer media, we simply transported each specimen to the microbiology laboratory as soon as the sample was collected. It seems reasonable to suspect that bodily fluids such as semen would allow anaerobic bacteria to maintain a reduced oxygen environment for some length of time. We predicted that all but the most fastidious organisms would be maintained using this methodology. Because we were able to culture several different anaerobic bacteria using this technique, our expectations were affirmed. In conclusion, we were unable to demonstrate a decrease in fertilization or PRs in the culture of men with contaminated semen, irrespective of the isolate. The finding of bacteria in semen does not signify infection and, if the concentration is low, will not affect the outcome of IVF. The data in this study do not support routine semen culture in preparation for IVF. REFERENCES. Rehewy MSE, Hafez ESE, Thomas A, Brown WJ. Aerobic and anaerobic bacterial flora in semen from fertile and infertile groups of men. Arch Androl 979;2: Toth A, Lesser ML. Asymptomatic bacteriospermia in fertile and infertile men. Andrologia 98;2: Hillier SL, Rabe LK, Muller CH, Zarutskie P, Kuzan FB, Stenchever MA. Relationship of bacteriologic characteristics to semen indicies in men attending an infertility clinic. Obstet Gynecol 990;75: Busolo F, Zanchetta R, Bertoloni G. Mycoplasmic localization patterns on spermatozoa from infertile men. Fertil Steril 984;42: Wong PC, Balmaceda JP, Blanco JD, Gibbs RS, Asch RH. Sperm washing and swim-up technique using antibiotics removes microbes from human semen. Fertil Steril 986;45: Toth A, O'Leary WM, Ledger W. Evidence for microbial transfer by spermatozoa. Obstet Gynecol 982;59: McGowan MP, Burger HG, Baker HWG, de Kretson DM, Kovasco G. The incidence of non-specific infection in the semen of fertile and subfertile males. Int J Androl 98;4: World Health Organization. WHO laboratory manual for the examination of human semen and semen-mucus interaction. 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge, 987: Holt JG, Krieg NR, editors. Bergey's manual of systematic bacteriology. Vol. and 2. Baltimore: Williams & Wilkins, Mears EM. Bacterial prostatitis versus "prostatosis": a clinical and bacteriological study. JAMA 975;224: Mobley DF. Semen cultures in the diagnosis of bacterial prostatitis. J Urol 975;4: Swenson CE, Toth A, O'Leary WM. Ureaplasma urealyticum and infertility: the effect of antibiotic therapy on semen quality. Fertil Steril 979;3: Naessens A, Foulon W, Debrucker P, Devroey P, Lauwers S. Recovery of microorganisms in semen and relationship to semen evaluation. Fertil Steril986;45: Guillet-Rosso F, Fari A, Taylor S, Forman R, Belaisch-Allart J, Testart J, et al. Systematic semen culture and its influence on IVF management. Br J Obstet GynaecoI987;94: Vol. 59, No., January 993 Stovall et al. Semen culture in IVF 20