Journal of in Vitro Fertilization and Embryo Transfer, Vol. 8, No. 5, 1991
Microbial Flora in Semen During in Vitro Fertilization C. HUYSER, 1'4 F. LE R. FOURIE, 1 M. OOSTHUIZEN, 2 and A. NEETHLING 3
Submitted: February 10, 1991 Accepted: June 4, 1991
gram studies, which is then administered to the husband (3,4). It has been stated (5) that routine supplementation of penicillin and streptomycin to culture media significantly reduces bacterial concentration in semen samples. Repeated washing of contaminated ejaculates also improves the quality of sperm and reduces the concentration of microbes present after swim-up. The fertilization capabilities of infected spermatozoa are probably not affected adversely after completing this procedure. The aim of this retrospective study was to determine the frequency of bacterial and fungal infections in semen of patients attending an IVF clinic. The effectiveness of antibiotic treatment as well as the wash and swim-up method used during in vitro fertilization and embryo transfer was evaluated.
Semen samples from 183 consecutive unselected men participating in an in vitro fertilization program were retrospectively studied to determine the bacterial and fungal contamination rate before and after antibiotic treatment. To ascertain the influence of semen preparation (wash and swim-up method) on the incidence of microorganisms, semen from 102 male patients was studied before and after swimup. Antimicrobial treatment by prescription of antibiotics decreased the incidence of pathogens by 16.3% (P < 0.0001). Semen processing was more effective by ridding 57.4% of semen samples of microbial contaminants (P < 0.0001). When infection of culture media was observed during routine microscopy, all infected oocytes were degenerated, without evidence of fertilization or pronuclei. KEY WORDS: in vitro fertilization; sperm/semen bacteriology;
antibiotic treatment.
MATERIALS AND METHODS
INTRODUCTION
One hundred eighty-three couples attended the Pretoria infertility unit at the H. F. Verwoerd hospital and the males were incorporated into this study. Approximately 2 weeks before in vitro fertilization treatment (between day 1 and day 5 of the patient's menstrual cycle), the husband's semen sample (day 1 sample) was collected by masturbation into a sterile screw-capped plastic (Protea, Promex plastics) specimen container. Patients were requested to adhere to a 3-day abstinence period. Prior to masturbation, patients had to urinate, then wash their hands thoroughly, and were further requested to prevent contamination of the sperm pots issued. After liquefaction at room temperature, the quality of semen was evaluated and 0.2 ml subjected to bacteriological examination. Identification of aerobic and anaerobic bacteria, as well as fungal growth, was done by Gram staining, colony and growth characteristics, and biochemical sugar reactions (6-9). Antimicrobial sus-
Bacteriologic assessment of semen in the weeks preceding an in vitro fertilization (IVF) treatment cycle is often requested in an effort to limit oocyte fertilization failures. Contaminated semen has been documented to be causative of suboptimal fertilization rates during IVF treatment (1,2). A detailed bacteriological diagnosis of the male's sperm is therefore obtained in our program and the appropriate antibiotic identified according to antibiotic bio1 Department of Obstetrics and Gynaecology, University of Pretoria, Pretoria, RSA. z Department of Microbiology, University of Pretoria, Pretoria, RSA. 3 Institute for Biostatistics, Medical Research Council, Pretoria, RSA. 4 To whom correspondence should be addressed at Reproductive Biology Research Unit, Department of Obstetrics and Gynaecology, University of Pretoria, P.O. Box 667, Pretoria 0002, RSA. 0740-7769/91/1000-0260506.50/09 1991PlenumPublishingCorporation
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ceptibility biograms utilizing a variety of antimicrobials were also performed. Appropriate antibiotics were prescribed for 1 week prior to oocyte recovery as determined by sensitivity studies. On the morning of laparoscopy (day 14 of the menstrual cycle), semen of each patient was collected in a similar fashion as described above and analyzed for microbial presence (day 14 sample). The concentration of motile spermatozoa for insemination of oocytes was prepared by the technique of centrifugation and swim-up as follows: 1.0 ml semen was diluted with 2.0 ml Ham's F-10 medium (Flow Laboratories, S.A. Scientific, Cat. No. 10411-22) supplemented with 10% patient serum and potassium penicillin-G (0.05 g/L; Sigma, Cat. No. Pen-K). This was thoroughly mixed in a 16 x 125mm sterile Falcon tissue culture test tube (Lasec, Cat. No. 3033) and thereafter centrifuged at 500g for l0 min. The supernatant was discarded and the pellet was resuspended in 2.0 ml of fresh Ham's F-10 medium and again centrifuged. After discarding the supernatant, the pellet was gently overlaid with 1.0 ml of fresh Ham's F-10 medium. The test tube was thereafter placed in an incubator at a gas phase of 5% CO2 in air at 37~ for 30 min. This allowed motile sperm to migrate from the pellet into the medium above. After 30 rain, 1.0 ml of the supernatant was transferred into a sterile test tube. This sperm sample was evaluated and used for insemination of oocytes (10). All oocytes were incubated at 37~ in 5% CO2 in air. After a 12- to 18-hr period the occurrence of pronuclei was microscopically checked and the oocytes transferred to fresh culture medium (11). One hundred two spermatozoal aliquots used for insemination were submitted after a 24-hr incubation period for further bacteriological cultures. If infection was suspected by laboratory personnel during microscopical evaluation, an aliquot of culture medium was also assessed for possible microbial contamination. Data were analyzed for significant differences with either Mcnemar's test of symmetry or Student's t test.
RESULTS The microbial patterns before (day 1) and after specific antimicrobial therapy (day 14) are summarized in Table I. A total of 26 microorganisms was isolated from a total of 183 semen samples. The
Table I. Incidence of Microorganisms in Semen Samples Before (Day 1) and After Specific Antimicrobial Therapy (Day 14) (n = 183) Species of microorganisms Commensal flora Staphylococcus epidermidis Nonhemolytic streptococci Alpa-hemolytic streptococci Diphtheroid organisms LactobaciUus sp. Micrococcus sp. Bacillus sp. Beta-hemolytic streptococci (not Lancefield groups A, B, C, G) Haemophilus parainfluenzae Neisseria lactamica Pathogenic flora Enterococcus faecalis Escherichia coli Staphylococcus aureus Beta-hemolytic streptocci (Lancefield groups A, B, C, G) Klebsiella pneumoniae Peptostreptococcus sp. Candida albieans Enterobacter agglomerans Proteus mirabilis Klebsiella oxytoca Enterobacter cloacae Torulopsis glabrata Yeast sp. Enterobacter sp. Citrobacter freundii Serratia marcescens Specimens totally free of microorganisms
Day 1 (%)
Day 14 (%)
49.0 23.0 16.9 4.4 1.1 1.1 0.5
50.3 4.9 4.9 6.0 1.6 1.1 1.1
1.1 0 0
0 0.5 0.5
16.0 7.7 4.9
3.8 (c) ~ 6.6 (a) 2.2
3.8 0.5 2.2 0.5 1.6 1.1 0.5 0 0 0 0.5 0 0
2.7 2.7 (d) 1.1 1.6 (e) 0 0.5 1.1 (b) 1.1 1.1 0.5 0.5 0.5 0.5
7.7
24.0*
a Microscopically observed infection of culture media: (a) 2.7%; (b) 1.1%; (c) 0.5%; (d) 0.5%; (e) 0.5%. * P < 0.0001, Mcnemar's test of symmetry.
antibiotic treatment of patients reduced the contamination rate from 92.3% on day 1 to 76.0% on day 14 (P < 0.000l). This implies that 7.7% of semen samples on day 1 and 24.0% on day 14 were uncontaminated. Commensal flora contributed largely to the contaminated status and commensal organisms cultured most frequently on day 1 were Staphylococcus epidermidis (49.0%), nonhemolytic streptococci (23.0%), and alpha-hemolytic streptococci (16.9%). Although S. epidermidis showed no significant change in incidence after antimicrobial treatment (day 14, 50.3%), nonhemolytic streptococci and alpha-hemolytic streptococci decreased (day 14, both 4.9%) after antimicrobial treatment. Enterococcus faecalis (a potentially pathogenic microorganism) decreased from 16.0% on day 1 to 3.8% on day 14. Escherichia coli tended to be more persistent (7.7% on day 1 and 6.6% on day 14).
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The occurrence of new potential pathogenic contaminants, which were not present on day 1 but cultured on day 14 after antimicrobial treatment, comprised E. coli, Enterococcus faecalis, betahemolytic streptococci, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Candida albicans, Torulopsis glabrata, yeast sp., Citrobacter freundii, and Serratia marcescens. These occurred in 10.9% of cases. Microscopically observed infection of oocyte culture media was caused by the pathogens E. coli (2.7%), Klebsiella oxytoca (1.1%), Enterococcus faecalis (0.5%), Klebsiella pneumoniae (0.5%), and Candida albicans (0.5%) (Table I). Oocytes cultured in infected media were found to be degenerative, with no evidence of pronuclei. In this study the following microorganisms recurred in semen samples (n = 156) before and after antimicrobial treatment: S. epidermidis, which was repeatedly isolated in 52 (28.4%) semen samples, and E. coli, in 4 (2.2%). Antibiotics prescribed were broad-spectrum antibiotics (58.3%), sulfonamides (29.5%), tetracyclines (8.3%), erythromycin (4.5%), cephalosporins (1.9%), and metronidazole (0.6%) (Table II). Treatment with broad-spectrum penicillins and sulfonamides significantly decreased the occurrence of organisms (P = 0.0001 and P = 0.0075, respectively). From a total of 183 patients, 14.8% did not receive any antimicrobial treatment due to the occurrence of negligible numbers of commensal organisms on day I. On day 14, however, 11.1% of these patients presented with potential pathogenic organisms. Table II. Antimicrobial Medication Prescribed (Based on Susceptibility Testing) and the Occurrence of Pathogenic Microorganisms Thereafter (n = 156) % of patients Medication
Treated
With pathogens after treatment
Broad-spectrum penicillins* Sulfonamides** Tetracyclines Erythromycin Cephalosporins Metronidazole (Flagyl) No medication used
58.3 29.5 8.3 4.5 1.9 0.6 14.8
29.7 21.7 7.7 0 33.3 0 11.1
* P = 0.0001, effective decrease in microorganisms after treatment (Student's t test). ** P = 0.0075, effective decrease in microorganisms after treatment (Student's t test).
Table III summarizes the incidence of microbial colonization before and after specific antimicrobial treatment and sperm wash. The administration of antibiotics increased the incidence of microbial-free samples significantly, from 7.7 to 24.0% (P < 0.0001). The prevalence of commensal and potential pathogenic organisms is illustrated in Table III; both types of organisms were slightly suppressed by the antibiotic action. Sperm wash and swim-up proved to be the most effective procedure, as it decreased the incidence of microbial presence by 57.4% (P < 0.0001). The total number of microorganisms per semen sample (of patients receiving antimicrobial therapy) was significantly reduced, from 1.5 on day 1 to 0.9 on day 14 (P = 0.0001). Semen processing further decreased the number of microorganisms to 0.18 per semen sample (P = 0.0001).
DISCUSSION In this study it was again confirmed that bacterial and fungal contaminants occur commonly in semen of patients attending an IVF clinic. This is in accordance with the results reported by Busolo et al. (12) and Swenson et al. (13), where a high bacterial isolation rate was found. To what extent these organisms are part of the normal seminal flora or are indicative of an infectious state is still debatable. According to Meares (14) most Gram-positive isolates could be considered commensal or normal flora of the urethra. The universal presence of organisms such as S. epidermidis, the alpha-hemolytic streptococci group, and diphtheroids in seminal plasma could indicate a noninterference situation with regard to sperm performance. The latter statement, however, has not been substantiated and is hence part of controversial speculation. The studies by Rehewy et al. (15) and Toth and Lessel (16) have demonstrated a higher prevalence of infected semen in a population of infertile men. The results of the present investigation clearly indicate that a large number of potentially pathogenic microorganisms were still present after antimicrobial therapy (based on susceptibility testing). The occurrence of new contaminants not present on day 1 but cultured on day 14 after antimicrobial treatment was also illustrated in this study. According to Stiver et al. (17) and Eggert-Kruse et al. (18), the effect of prophylactic antibiotics, such as broadspectrum antibiotics, could result in the selection of
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Table III. Subdivision of Microorganisms Before and After Specific Antimicrobial Treatment and Sperm Wash (n = 183) Microorganisms (%)
Day 1, semen (before antimicrobial treatment) Day 14, semen (after antimicrobial treatment) Day 14 after 24 hr of incubation (sperm wash)
(A) None
(B) Commensal
(C) Pathogenic
(D) Commensal & pathogenic
E ( B + C + D) Total occurrence
7.7
55.7
22.4
14.2
92.3
24.0*
54.6
15.3
7.1
77.0
80.4*
7.8
11.8
0
19.6
resistant organisms and therefore cause a disturbance in the microbial equilibrium of the genital tract. Variations in the composition of seminal bacterial flora before and after antimicrobial treatment were also reported by Moberg et al. (19). EggertKruse et al. (18), however, pointed out that spontaneous variations of microbial flora could also occur. It should further be borne in mind that the process of masturbation and semen collection is far from ideal if sterility of the semen sample is considered. It is of significance that the process of sperm wash and swim-up appeared to be highly effective in removing organisms in more than 50% of known contaminated semen samples. This is in accordance with the findings of Forman et al. (1) and Wong et al. (20). In this study the physical treatment of sperm proved to be more effective than the prescription of various antibiotics. Although the supplementation of penicillin to the culture media would probably contribute to the suppression of microbial infections, these results indicate that the use of antibiotics could possibly be lessened in an IVF program. Functional disturbances of the accessory sex glands in patients with infectious diseases can be diagnosed by measurement of several biochemical markers (21). These measurements could indicate whether infection of the accessory sex glands is likely, thereby necessitating antibiotic treatment. If these organs are not infected, the use of antibiotics during IVF would be unnecessary. To conclude, this study has illustrated the general microbial contamination of semen samples of men participating in an IVF program. Specific antibiotic treatment was effective, but not as effective as sperm wash and swim-up, in clearing the semen sample of microorganisms. The prescription and possible detrimental effect of antibiotics on the normal reproductive tract flora are questioned.
REFERENCES l. Forman R, Guillet-Rosso F, Fari A, Volante R, Frydman R, Testart J: Importance of semen preparation in avoidance of reduced in vitro fertilization results attributable to bacteria. Fertil Steril 1987;47:527-530 2. Guillet-Rosso F, Fari A, Taylor S, Forman R, BelaischAllart J, Testart J, Frydman R: Systematic semen culture and its influence on IVF management. Br J Obstet Gynaecol 1987;94:543-547 3. Riedel HH, Baukloh V, Mettler L: Minimal requirements for ejaculates used for in vitro fertilization. Arch Androl 1984;12:69-77 4. Serum K, Mettler L, Michelmann HW: Influence of microbial contamination on fertility rate after in vitro fertilization and embryo transfer in men. Dtsch Tierarztl Wochenschr 1987;94:503-506 5. Riedel HH, Langenbucher H, Mettler L: Significance of sperm bacteriology for the in vitro fertilization of human and mouse oocytes. J Reprod Med 1986;31:605-608 6. Coyle MB, Morello JA, Smith PB: Aerobic Bacteria. In Manual of Clinical Microbiology, 4th ed., EH Lenette, A Balows, WJ Hausler, HJ Shadomy (eds). Washington DC, American Society, 1985, pp 143-411 7. Allen SD: Anaerobic bacteria. In Manual of Clinical Microbiology, 4th ed., EH Lenette, A Balows, WJ Hausler, HJ Shadomy (eds). Washington DC, American Society, 1985, pp 413-472 8. Cooper BH: Fungi. In Manual of Clinical Microbiology, 4tb ed., EH Lenette, A Balows, WJ Hausler, HJ Shadomy (eds). Washington DC, American Society, 1985, pp 496-594 9. Cruickshank R, Duguid JP, Marmion BB, Swain RHA: Medical Microbiology, 12th Ed., Vol 2, Part 1, Part 2. London, Churchill Livingstone, 1975, pp 170-189, 353-493 10. Van der Merwe JV, Irsigler UM, De Milander C, Wimmers MSE, Botes ADE, Snyman E, Von Prince MS, Koch Z, Hurter P: In vitro fertilization and embryo transfer in Pretoria. S Afr Med J 1984;66:641-644 l 1. Van der Merwe JV, Truter P, Sevenster C, Grace A, Botes A, Wimmers M, De Millander C, Snyman E: Phase II of a successful university-based in vitro fertilisation programme. S Afr Med J 1987;71:223-226 12. Busolo F, Zanchetta R, Lanzone E, Cusinato R: Microbial flora in semen of asymptomatic infertile men. Andrologia 1984;16:26%275 13. Swenson ChE, Toth A, Toth CI, Wolfgruber L, O'Leary
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14. 15.
16. 17.
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WM: Asymptomatic bacteriospermia in infertile men. Andrologia 1980;12:%11 Meares EM: Bacterial prostatitis vs "prostatosis." A clinical and bacteriological study. JAMA 1973 ;224:1372-1375 Rehewy MSE, Hafez ESE, Thomas A, Brown WJ: Aerobic and anaerobic bacterial flora in semen from fertile and infertile groups of men. Arch Androl 1979;2:263-266 Toth A, Lessel ML: Asymptomatic bacteriospermia in infertile men. Fertil Steril 1981 ;36:88-91 Stiver HG, Forward KR, Tyrrell DL, Krip G, Livingstone RA, Fugere P, Lemay M, Verschelden G, Hunter JD, Carson GD: Comparative cervical microflora shifts after cefoxitin or cefazolin prophylaxis against infection following cesarean section. Am J Obstet Gynecol 1984;149:718--721
18. Eggert-Kruse W, Hofmann H, Gerhard I, Bilke A, Runnebaum B, Petzoldt D: Effect of antimicrobial therapy on sperm-mucus interaction. Hum Reprod 1988;3:861-869 19. Moberg PJ, Enroth P, Lung A, Nord C-E: Bacterial flora in semen before and after doxycycline treatment of infertile couples. Int J Androl 1980;3:46-58 20. 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 1986; 45:97-100 21. Comhaire FH, Vermeulen L, Pieters O: Study of the accuracy of physical and biochemical markers in semen to detect infectious dysfunction of the accessory glands. J Androl 1989;10:50-53
Journal of in Vitro Fertilization and Embryo Transfer, Vol. 8, No. 5, 1991
Microbial Flora in Semen During in Vitro Fertilization C. HUYSER, 1'4 F. LE R. FOURIE, 1 M. OOSTHUIZEN, 2 and A. NEETHLING 3
Submitted: February 10, 1991 Accepted: June 4, 1991
gram studies, which is then administered to the husband (3,4). It has been stated (5) that routine supplementation of penicillin and streptomycin to culture media significantly reduces bacterial concentration in semen samples. Repeated washing of contaminated ejaculates also improves the quality of sperm and reduces the concentration of microbes present after swim-up. The fertilization capabilities of infected spermatozoa are probably not affected adversely after completing this procedure. The aim of this retrospective study was to determine the frequency of bacterial and fungal infections in semen of patients attending an IVF clinic. The effectiveness of antibiotic treatment as well as the wash and swim-up method used during in vitro fertilization and embryo transfer was evaluated.
Semen samples from 183 consecutive unselected men participating in an in vitro fertilization program were retrospectively studied to determine the bacterial and fungal contamination rate before and after antibiotic treatment. To ascertain the influence of semen preparation (wash and swim-up method) on the incidence of microorganisms, semen from 102 male patients was studied before and after swimup. Antimicrobial treatment by prescription of antibiotics decreased the incidence of pathogens by 16.3% (P < 0.0001). Semen processing was more effective by ridding 57.4% of semen samples of microbial contaminants (P < 0.0001). When infection of culture media was observed during routine microscopy, all infected oocytes were degenerated, without evidence of fertilization or pronuclei. KEY WORDS: in vitro fertilization; sperm/semen bacteriology;
antibiotic treatment.
MATERIALS AND METHODS
INTRODUCTION
One hundred eighty-three couples attended the Pretoria infertility unit at the H. F. Verwoerd hospital and the males were incorporated into this study. Approximately 2 weeks before in vitro fertilization treatment (between day 1 and day 5 of the patient's menstrual cycle), the husband's semen sample (day 1 sample) was collected by masturbation into a sterile screw-capped plastic (Protea, Promex plastics) specimen container. Patients were requested to adhere to a 3-day abstinence period. Prior to masturbation, patients had to urinate, then wash their hands thoroughly, and were further requested to prevent contamination of the sperm pots issued. After liquefaction at room temperature, the quality of semen was evaluated and 0.2 ml subjected to bacteriological examination. Identification of aerobic and anaerobic bacteria, as well as fungal growth, was done by Gram staining, colony and growth characteristics, and biochemical sugar reactions (6-9). Antimicrobial sus-
Bacteriologic assessment of semen in the weeks preceding an in vitro fertilization (IVF) treatment cycle is often requested in an effort to limit oocyte fertilization failures. Contaminated semen has been documented to be causative of suboptimal fertilization rates during IVF treatment (1,2). A detailed bacteriological diagnosis of the male's sperm is therefore obtained in our program and the appropriate antibiotic identified according to antibiotic bio1 Department of Obstetrics and Gynaecology, University of Pretoria, Pretoria, RSA. z Department of Microbiology, University of Pretoria, Pretoria, RSA. 3 Institute for Biostatistics, Medical Research Council, Pretoria, RSA. 4 To whom correspondence should be addressed at Reproductive Biology Research Unit, Department of Obstetrics and Gynaecology, University of Pretoria, P.O. Box 667, Pretoria 0002, RSA. 0740-7769/91/1000-0260506.50/09 1991PlenumPublishingCorporation
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MICROBIAL FLORA IN SEMEN DURING IVF
ceptibility biograms utilizing a variety of antimicrobials were also performed. Appropriate antibiotics were prescribed for 1 week prior to oocyte recovery as determined by sensitivity studies. On the morning of laparoscopy (day 14 of the menstrual cycle), semen of each patient was collected in a similar fashion as described above and analyzed for microbial presence (day 14 sample). The concentration of motile spermatozoa for insemination of oocytes was prepared by the technique of centrifugation and swim-up as follows: 1.0 ml semen was diluted with 2.0 ml Ham's F-10 medium (Flow Laboratories, S.A. Scientific, Cat. No. 10411-22) supplemented with 10% patient serum and potassium penicillin-G (0.05 g/L; Sigma, Cat. No. Pen-K). This was thoroughly mixed in a 16 x 125mm sterile Falcon tissue culture test tube (Lasec, Cat. No. 3033) and thereafter centrifuged at 500g for l0 min. The supernatant was discarded and the pellet was resuspended in 2.0 ml of fresh Ham's F-10 medium and again centrifuged. After discarding the supernatant, the pellet was gently overlaid with 1.0 ml of fresh Ham's F-10 medium. The test tube was thereafter placed in an incubator at a gas phase of 5% CO2 in air at 37~ for 30 min. This allowed motile sperm to migrate from the pellet into the medium above. After 30 rain, 1.0 ml of the supernatant was transferred into a sterile test tube. This sperm sample was evaluated and used for insemination of oocytes (10). All oocytes were incubated at 37~ in 5% CO2 in air. After a 12- to 18-hr period the occurrence of pronuclei was microscopically checked and the oocytes transferred to fresh culture medium (11). One hundred two spermatozoal aliquots used for insemination were submitted after a 24-hr incubation period for further bacteriological cultures. If infection was suspected by laboratory personnel during microscopical evaluation, an aliquot of culture medium was also assessed for possible microbial contamination. Data were analyzed for significant differences with either Mcnemar's test of symmetry or Student's t test.
RESULTS The microbial patterns before (day 1) and after specific antimicrobial therapy (day 14) are summarized in Table I. A total of 26 microorganisms was isolated from a total of 183 semen samples. The
Table I. Incidence of Microorganisms in Semen Samples Before (Day 1) and After Specific Antimicrobial Therapy (Day 14) (n = 183) Species of microorganisms Commensal flora Staphylococcus epidermidis Nonhemolytic streptococci Alpa-hemolytic streptococci Diphtheroid organisms LactobaciUus sp. Micrococcus sp. Bacillus sp. Beta-hemolytic streptococci (not Lancefield groups A, B, C, G) Haemophilus parainfluenzae Neisseria lactamica Pathogenic flora Enterococcus faecalis Escherichia coli Staphylococcus aureus Beta-hemolytic streptocci (Lancefield groups A, B, C, G) Klebsiella pneumoniae Peptostreptococcus sp. Candida albieans Enterobacter agglomerans Proteus mirabilis Klebsiella oxytoca Enterobacter cloacae Torulopsis glabrata Yeast sp. Enterobacter sp. Citrobacter freundii Serratia marcescens Specimens totally free of microorganisms
Day 1 (%)
Day 14 (%)
49.0 23.0 16.9 4.4 1.1 1.1 0.5
50.3 4.9 4.9 6.0 1.6 1.1 1.1
1.1 0 0
0 0.5 0.5
16.0 7.7 4.9
3.8 (c) ~ 6.6 (a) 2.2
3.8 0.5 2.2 0.5 1.6 1.1 0.5 0 0 0 0.5 0 0
2.7 2.7 (d) 1.1 1.6 (e) 0 0.5 1.1 (b) 1.1 1.1 0.5 0.5 0.5 0.5
7.7
24.0*
a Microscopically observed infection of culture media: (a) 2.7%; (b) 1.1%; (c) 0.5%; (d) 0.5%; (e) 0.5%. * P < 0.0001, Mcnemar's test of symmetry.
antibiotic treatment of patients reduced the contamination rate from 92.3% on day 1 to 76.0% on day 14 (P < 0.000l). This implies that 7.7% of semen samples on day 1 and 24.0% on day 14 were uncontaminated. Commensal flora contributed largely to the contaminated status and commensal organisms cultured most frequently on day 1 were Staphylococcus epidermidis (49.0%), nonhemolytic streptococci (23.0%), and alpha-hemolytic streptococci (16.9%). Although S. epidermidis showed no significant change in incidence after antimicrobial treatment (day 14, 50.3%), nonhemolytic streptococci and alpha-hemolytic streptococci decreased (day 14, both 4.9%) after antimicrobial treatment. Enterococcus faecalis (a potentially pathogenic microorganism) decreased from 16.0% on day 1 to 3.8% on day 14. Escherichia coli tended to be more persistent (7.7% on day 1 and 6.6% on day 14).
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The occurrence of new potential pathogenic contaminants, which were not present on day 1 but cultured on day 14 after antimicrobial treatment, comprised E. coli, Enterococcus faecalis, betahemolytic streptococci, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Candida albicans, Torulopsis glabrata, yeast sp., Citrobacter freundii, and Serratia marcescens. These occurred in 10.9% of cases. Microscopically observed infection of oocyte culture media was caused by the pathogens E. coli (2.7%), Klebsiella oxytoca (1.1%), Enterococcus faecalis (0.5%), Klebsiella pneumoniae (0.5%), and Candida albicans (0.5%) (Table I). Oocytes cultured in infected media were found to be degenerative, with no evidence of pronuclei. In this study the following microorganisms recurred in semen samples (n = 156) before and after antimicrobial treatment: S. epidermidis, which was repeatedly isolated in 52 (28.4%) semen samples, and E. coli, in 4 (2.2%). Antibiotics prescribed were broad-spectrum antibiotics (58.3%), sulfonamides (29.5%), tetracyclines (8.3%), erythromycin (4.5%), cephalosporins (1.9%), and metronidazole (0.6%) (Table II). Treatment with broad-spectrum penicillins and sulfonamides significantly decreased the occurrence of organisms (P = 0.0001 and P = 0.0075, respectively). From a total of 183 patients, 14.8% did not receive any antimicrobial treatment due to the occurrence of negligible numbers of commensal organisms on day I. On day 14, however, 11.1% of these patients presented with potential pathogenic organisms. Table II. Antimicrobial Medication Prescribed (Based on Susceptibility Testing) and the Occurrence of Pathogenic Microorganisms Thereafter (n = 156) % of patients Medication
Treated
With pathogens after treatment
Broad-spectrum penicillins* Sulfonamides** Tetracyclines Erythromycin Cephalosporins Metronidazole (Flagyl) No medication used
58.3 29.5 8.3 4.5 1.9 0.6 14.8
29.7 21.7 7.7 0 33.3 0 11.1
* P = 0.0001, effective decrease in microorganisms after treatment (Student's t test). ** P = 0.0075, effective decrease in microorganisms after treatment (Student's t test).
Table III summarizes the incidence of microbial colonization before and after specific antimicrobial treatment and sperm wash. The administration of antibiotics increased the incidence of microbial-free samples significantly, from 7.7 to 24.0% (P < 0.0001). The prevalence of commensal and potential pathogenic organisms is illustrated in Table III; both types of organisms were slightly suppressed by the antibiotic action. Sperm wash and swim-up proved to be the most effective procedure, as it decreased the incidence of microbial presence by 57.4% (P < 0.0001). The total number of microorganisms per semen sample (of patients receiving antimicrobial therapy) was significantly reduced, from 1.5 on day 1 to 0.9 on day 14 (P = 0.0001). Semen processing further decreased the number of microorganisms to 0.18 per semen sample (P = 0.0001).
DISCUSSION In this study it was again confirmed that bacterial and fungal contaminants occur commonly in semen of patients attending an IVF clinic. This is in accordance with the results reported by Busolo et al. (12) and Swenson et al. (13), where a high bacterial isolation rate was found. To what extent these organisms are part of the normal seminal flora or are indicative of an infectious state is still debatable. According to Meares (14) most Gram-positive isolates could be considered commensal or normal flora of the urethra. The universal presence of organisms such as S. epidermidis, the alpha-hemolytic streptococci group, and diphtheroids in seminal plasma could indicate a noninterference situation with regard to sperm performance. The latter statement, however, has not been substantiated and is hence part of controversial speculation. The studies by Rehewy et al. (15) and Toth and Lessel (16) have demonstrated a higher prevalence of infected semen in a population of infertile men. The results of the present investigation clearly indicate that a large number of potentially pathogenic microorganisms were still present after antimicrobial therapy (based on susceptibility testing). The occurrence of new contaminants not present on day 1 but cultured on day 14 after antimicrobial treatment was also illustrated in this study. According to Stiver et al. (17) and Eggert-Kruse et al. (18), the effect of prophylactic antibiotics, such as broadspectrum antibiotics, could result in the selection of
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Table III. Subdivision of Microorganisms Before and After Specific Antimicrobial Treatment and Sperm Wash (n = 183) Microorganisms (%)
Day 1, semen (before antimicrobial treatment) Day 14, semen (after antimicrobial treatment) Day 14 after 24 hr of incubation (sperm wash)
(A) None
(B) Commensal
(C) Pathogenic
(D) Commensal & pathogenic
E ( B + C + D) Total occurrence
7.7
55.7
22.4
14.2
92.3
24.0*
54.6
15.3
7.1
77.0
80.4*
7.8
11.8
0
19.6
resistant organisms and therefore cause a disturbance in the microbial equilibrium of the genital tract. Variations in the composition of seminal bacterial flora before and after antimicrobial treatment were also reported by Moberg et al. (19). EggertKruse et al. (18), however, pointed out that spontaneous variations of microbial flora could also occur. It should further be borne in mind that the process of masturbation and semen collection is far from ideal if sterility of the semen sample is considered. It is of significance that the process of sperm wash and swim-up appeared to be highly effective in removing organisms in more than 50% of known contaminated semen samples. This is in accordance with the findings of Forman et al. (1) and Wong et al. (20). In this study the physical treatment of sperm proved to be more effective than the prescription of various antibiotics. Although the supplementation of penicillin to the culture media would probably contribute to the suppression of microbial infections, these results indicate that the use of antibiotics could possibly be lessened in an IVF program. Functional disturbances of the accessory sex glands in patients with infectious diseases can be diagnosed by measurement of several biochemical markers (21). These measurements could indicate whether infection of the accessory sex glands is likely, thereby necessitating antibiotic treatment. If these organs are not infected, the use of antibiotics during IVF would be unnecessary. To conclude, this study has illustrated the general microbial contamination of semen samples of men participating in an IVF program. Specific antibiotic treatment was effective, but not as effective as sperm wash and swim-up, in clearing the semen sample of microorganisms. The prescription and possible detrimental effect of antibiotics on the normal reproductive tract flora are questioned.
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18. Eggert-Kruse W, Hofmann H, Gerhard I, Bilke A, Runnebaum B, Petzoldt D: Effect of antimicrobial therapy on sperm-mucus interaction. Hum Reprod 1988;3:861-869 19. Moberg PJ, Enroth P, Lung A, Nord C-E: Bacterial flora in semen before and after doxycycline treatment of infertile couples. Int J Androl 1980;3:46-58 20. 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 1986; 45:97-100 21. Comhaire FH, Vermeulen L, Pieters O: Study of the accuracy of physical and biochemical markers in semen to detect infectious dysfunction of the accessory glands. J Androl 1989;10:50-53
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