Efficacy of treatment and recurrence rate of leukocytospermia infertile men with prostatitis*

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1 ,, ',,' -1,,,i; -; ::0 urf)~ogy-androi(jgy FERTILITY AND STERILITY Copyright 1994 The American Fertility Society VoL 62, No.3, September 1994 Printed on acid-free paper in U. S. A. Efficacy of treatment and recurrence rate of leukocytospermia infertile men with prostatitis*. 1n Emmett F. Branigan, M.D.t Charles H. Muller, Ph.D.:j: University of Washington School of Medicine, Seattle, Washington Objective: To identify men with leukocytospermia and prostatitis in an infertility practice and evaluate the effect of various treatments and recurrence rates after treatment. Design: A prospective randomized trial of men with leukocytospermia. Setting: Academic tertiary infertility clinic. Patients: One hundred two men with leukocytospermia identified on smear of semen using Bryan-Leishman stain and in expressed prostatic secretion. Interventions: Treatment groups were no treatment group; antibiotic treatment alone group; frequent ejaculation alone group; and antibiotic treatment with frequent ejaculation group. Main Outcome Measure: Resolution of leukocytospermia on semen smear. Results: Significant resolution of leukocytospermia occurred in all treatment groups at 1 month compared with no treatment. The resolution was sustained at 2 and 3 months only in those who took antibiotics and frequently ejaculated. Conclusions: Antibiotic treatment, frequent ejaculation, and antibiotic treatment with frequent ejaculation effectively treat leukocytospermia immediately after the treatment phase. However, only antibiotic treatment coupled with frequent ejaculation is effective 3 months after treatment. Fertil Steril 1994;62: Key Words: Leukocytospermia, doxycycline, Bryan-Leishman stain, ejaculation, prostatitis Leukocytospermia (> 1.0 X 10 6 white blood cells [WBC]/mL of semen) (1) has been associated with significant adverse effects on semen parameters and sperm function. Wolff and Anderson (2) and Barratt et al. (3), using monoclonal antibodies to detect leukocytes in semen, showed significantly increased leukocyte numbers in the semen of infertility patients compared with fertile donors. Men with leukocytospermia were found to have signifi- Received November 16, 1993; revised and accepted April 19, * Presented in part at the 41st Annual Meeting of the Pacific Coast Fertility Society, Palms Springs, California, April 14 to 18, t Reprint requests: Emmett F. Branigan, M.D., Division of Reproductive Endocrinology and Infertility, University of Washington, 4225 Roosevelt Way N.E., Suite 305, Seattle, Washington (FAX: ). :j: Department of Urology. cant decreases in total sperm number, total motile sperm, and sperm velocity (4). Both the presence of WBCs and leukocyte products have been shown to affect fertilization adversely in the hamster sperm penetration assay (SPA) (5, 6). Two groups have shown reduced fertilization in human IVF when increased numbers of leukocytes are present in semen (7, 8). Traditionally, the presence of increased numbers of leukocytes has been presumed to be a clinical sign of genital tract infection. However, leukocytospermia has a heterogeneous etiology, including infection, inflammation, and autoimmunity (9). The majority of men with leukocytospermia have semen cultures that fail to show active genital tract infection (10). The semen cultures are difficult to obtain and evaluate because of the unclear demarcation between pathogenic and nonpathogenic bacteria. Berger et al. (11) showed that half of infertile men 580 Branigan and Muller Treatment of leukocytospermia

2 with abnormal SPAs and high number of leukocytes treated with doxycycline had normal SPA results and markedly improved pregnancy rates after treatment. Two studies of doxycycline treatment of couples with unexplained infertility showed no improvement in pregnancy rates, but leukocytospermia was not identified in these couples (12, 13). The aims of this study are [1] to identify rigorously men with leukocytospermia and prostatitis in an infertility population; [2] to evaluate the efficacy of antibiotic treatment, frequent ejaculation, and antibiotic treatment with frequent ejaculation in men with leukocytospermia; and [3] to assess the time course of treatment and recurrence rate of leukocytospermia. MATERIALS AND METHODS Seven hundred fifty-five men seen at the Fertility and Endocrine Center of the University of Washington were screened for the presence of leukocytospermia. This evaluation consisted of a history and physical examination, prostatic massage, and semen analysis. A morphology slide was made at the time of semen analysis and stained by the Bryan Leishman method of Couture et al. (14) (This method allows discrimination of leukocytes from immature sperm forms.). We have found <5% variation in our laboratory using this staining and counting method when compared with monoclonal antibody HLe-1 (panleukocyte)-stained slides. The same technician was used for all analyses. Men with ~6 WBCs/100 sperm by this method were designated leukocytospermic on semen smear if their sperm concentration was ~20 X 10 6 sperm/ml. A wet prep of expressed prostatic fluid was analyzed for number of WBCs and presence of leukocyte clumping. The presence of ~20 WBCs per high power field and/or leukocyte clumping on expressed prostatic secretion was required for a leukocytospermia diagnosis. Only men with ~6 WBCs/100 sperm on their semen smears and ~20 WBCs per high power field and/or clumping on expressed prostatic secretion were entered into the study. Semen cultures were not done on any of the men. One hundred two men who met both criteria were randomized into the treatment phase. Using random permuted blocks with a block size of eight, these men were assigned to one of four groups. Group 1 (n = 25) received no treatment. Group 2 (n = 25) were treated with doxycycline 100 mg orally twice per day for 7 days and then 100 mg once per day for 21 days. Group 3 (n = 25) were told to ejaculate frequently (at least every 3rd day) for 1 month but received no antibiotic treatment. Group 4 (n = 27) were treated with the same antibiotic regimen of group 2 but also were told to ejaculate frequently during the treatment phase. All men were told to keep an ejaculation calendar during the treatment phase. Females whose husbands randomized to the antibiotic treatment groups were treated concomitantly with 10 days of doxycycline to prevent a rebound effect. The men were followed at monthly intervals for 3 months with repeat semen smears stained with the Bryan-Leishman stain. Repeat monthly prostatic massages were not done because of difficulty with patient compliance. Leukocytes were counted per 100 sperm and leukocyte concentrations were calculated from sperm concentrations. Resolution of the leukocytospermia was achieved if the calculated leukocyte concentration was <10 6 /ml. The slides of borderline results (leukocyte concentrations between 500,000 and 1,000,000) were recounted and calculated three times. All three of the results needed to be <10 6 for resolution. Comparison between the various groups at each monthly time interval was analyzed using x 2 analysis. RESULTS Of the 755 men screened for leukocytospermia in this study, 87 met both criteria for diagnosis of leukocytospermia and prostatitis used in the study on the initial exam. Thirty six others met one criterion, either ~6 WBCs/100 sperm on semen smear or ~20 WBCs/high power field and/or WBC clumping on expressed prostatic secretion but did not meet both criteria. These 36 men had both semen smear and expressed prostatic secretion repeated and those meeting both criteria (15/36) were randomized to treatment groups. A total of 102 or 13.5% of the 755 men screened entered the study. The treatment groups did not differ significantly with respect to age, sperm concentration, motility, or percent normal sperm morphology. Average age of males was 38.1 years with average sperm concentration, 67.4 X 10 6 /ml; motility, 52%; and normal morphology, 50.7%. Thirty-two percent of the couples had the diagnosis of unexplained infertility, 28% had mild male factor infertility (defined as one abnormality in their semen analysis below World Vol. 62, No.3, September 1994 Branigan and Muller Treatment of leukocytospermia 581

3 Table 1 Effectiveness of Treatment at 1 Month No. of Resolution of Treatment group subjects leukocytospermia p 1 No treatment 25 1 of 25 (4)* 2 Antibiotics alone of 25 (40)t < Frequent ejaculation alone 25 8 of 25 (32)t < Antibiotic and frequent ejaculation of 25 (68)t < * Values in parentheses are percentages. t Significant differences when compared by x 2 analysis with no treatment group. Health Organization standards, (1) but not severe in nature), 17% had mild ovulation problems corrected by clomiphene citrate, and the rest had an assortment of diagnoses. None of the men initially were seen specifically for any genitourinary complaints, but 4 7% had postvoid dribbling, 21% had nocturia, and 18% had pain with ejaculation or urination on review of systems. Together, 68% of men reported at least one of these symptoms. The frequency of ejaculation of the men before starting the study was 6 ± 2.1 SEM/mo (range, 1 to 9 times/roo) and none of the men ejaculated on average more than every 3 days. The men in the frequent ejaculation groups (groups 3 and 4) increased their frequency from 6 times/roo to 15 times/roo during the 1-month treatment phase as reported on their ejaculation calendar. At 1 month after treatment, 4% (1/25) men in group 1 (no treatment), 40% (10/25) in group 2 (antibiotic alone), 32% (8/25) in group 3 (frequent ejaculation alone), and 68% (17/25) in group 4 (antibiotic treatment with frequent ejaculation) had their leukocytospermia resolve. All of the treatment groups (groups 2, 3, and 4) showed significant improvement in resolution of leukocytospermia by x 2 analysis compared with the no treatment group (group 1). These results are summarized in Table 1. The effectiveness of treatment was sustained at 2 and 3 months follow-up only in group 4 (antibiotic with frequent ejaculation). At 2 months, 56% (13/ 23) of men in group 4 continued to have resolution of their leukocytospermia compared with 5% (2/24) in group 1 (no treatment), 17% (4/23) in group 2 (antibiotic alone), and 13% (3/22) in group 3 (frequent ejaculation alone). Similar results were seen at 3 months follow-up. Group 4 had 53% (12/23) resolution of leukocytospermia compared with 5% (2/24) in group 1, 17% (4/23) in group 2, and 9% (2/23) in group 3. Group 4 (antibiotic treatment with frequent ejaculation) had significantly improved resolution of leukocytospermia (P < 0.01) compared with all other groups by x 2 analysis; these results are summarized in Table 2. Eleven pregnancies occurred in the various treatment groups during the 3 months of the study. Ten occurred in the treatment groups and one in the untreated group. All of the pregnancies occurred in the men who responded to treatment. The single pregnancy in the untreated group occurred by one of the men who spontaneously resolved his leukocytospermia. The number of pregnancies was too small to make statistically meaningful comparisons between groups. One pregnancy (1/25) occurred in the no treatment group, two (2/25) in the antibiotic group, three (2/ 25) in the frequent ejaculation group, and four (4/ 27) in the antibiotic treatment with frequent ejaculation group. DISCUSSION In this study a rigorous definition of leukocytospermia was used to identify asymptomatic infertile men with leukocytospermia, so that the effectiveness of various treatments and recurrence rates after treatment could be evaluated. At 1 month follow-up all the treatment groups showed significant improvement in resolution of leukocytospermia compared with the no treatment group. However, this resolution was only sustained at 2 and 3 month follow-up in the group that received antibiotic treatment and frequently ejaculated. All of the pregnancies in the study occurred by men who had resolution of their leukocytospermia. Many studies have shown that leukocytes can have significant adverse effects on sperm function. The presence of leukocytes is the best predictor of Table 2 Effectiveness of Treatment at 2 and 3 Months Treatment group No. of subjects 1 No treatment 24 2 Antibiotics alone 23 3 Frequent ejaculation alone 22 4 Antibiotic and frequent ejaculation 23 Resolution of leukocytospermia 2 months 3 months 2/24 (5)* 2/24 (5) 4/23 (17) 4/23 (17) 3/22 (13) 2/22 (9) 13/23 (56) 12/23 (55)t * Values in parentheses are percentages. t Significantly different (P = 0.007) when compared by x 2 analysis with groups 1, 2, and Branigan and Muller Treatment of leukocytospermia

4 I an abnormal SPA in men with normal semen analysis. (15) The addition of the supernatant of WBCs or peripheral blood leukocytes to semen of fertile donors results in decreased fertilizing ability in the SPA (5). Leukocytospermia reportedly adversely affects sperm motility, total motile sperm, and sperm velocity (4). Increased number of WBCs in semen generally is regarded as a clinical sign of genital tract infection. Comhaire et al. (16) have shown that leukocytospermia is significantly more common whenever >10 4 bacteria/ml (either pathogenic or nonpathogenic bacteria) are present in the ejaculate. However, the relationship between leukocytes and genital tract infection is by no means clear-cut. Berger et al. (15) found no correlation between bacteria in semen and SPA results or between leukocytes and bacteria. Electron microscopy studies of seminal leukocytes show leukocytes contain bacteria 30% of the time and 60% contain sperm fragments (17). This study used leukocytospermia as a marker to identify those men most likely to benefit from treatment and to measure the success of various treatment options and the recurrence rates. Antibiotic treatment of men with leukocytospermia implies an infectious etiology. However, leukocytospermia has a heterogeneous etiology, including inflammation and autoimmunity as well as infection (9). For this reason antibiotics and frequent ejaculation were used alone and in combination in this study. Frequent ejaculation was used to help clear the prostatic gland and seminal vesicles of their stored secretions. This may allow antibiotics to work more effectively and decrease accessory gland stagnation, which may increase white blood cells through noninfectious inflammation. Doxycycline was chosen because of its broad spectrum, penetration into accessory glands, and efficacy in improving SPAs (11). The diagnosis of leukocytospermia is difficult because of similarities in size and morphology between leukocytes and immature germ cells in semen. Monoclonal antibodies directed against leukocyte antigens provide the gold standard of leukocyte identification. However, immunocytochemical methods are costly and time consuming to use on a routine basis and are used primarily in research settings. The Bryan-Leishman stain (14) used in this study is performed on all routine semen analyses at the University of Washington and provides a clinically useful method of identifying total leukocytes per 100 sperm. We have found <5% variation in our laboratory using this staining and counting method when compared with monoclonal antibody HLe-1 (panleukocyte)-stained slides. The expressed prostatic secretion provides further evidence of increased leukocytes in the male accessory glands. These two methods were used in combination in this study to identify more accurately men with leukocytospermia, to follow them clinically, and to demonstrate the effectiveness of therapy on resolution of leukocytospermia. In conclusion, leukocytospermia and prostatitis were a frequent finding in the men without any initial genitourinary complaints screened in this study. Of the 755 men in this study, 102 (13.5%) who were screened met the rigorous definition of leukocytospermia and 70 (68%) had at least one symptom of prostatitis on review of symptoms. Leukocytospermia can be treated effectively in most but not all men with a low recurrence rate. The role of leukocytes in the male reproductive tract warrants further study. REFERENCES 1. World Health Organization. WHO laboratory manual for the examination of human and semen-cervical mucus interaction. 2nd ed. Cambridge: The Press Syndicate of the University of Cambridge Wolff H, Anderson DJ. Immunohistologic characterization and quantitation of leukocyte subpopulations in human semen. Fertil Steril1988; 49: Barratt CLR, Kessopoulou LA, Tomlinson MJ, Cooke ID. The functional significance of leucocytes in human reproduction. Reprod Med Rev 1991;1: Wolff H, Politch JA, Martinez A, Haimovici F, Hill JA, Anderson DJ. Leukocytospermia is associated with poor semen quality. Fertil Steril 1990;53: Maruyama DK, Hale RW, Rogers BJ. Effects of white blood cells on the in vitro penetration of zona-free hamster eggs by human spermatozoa. J Androl1985;6: HillJA, Cohen J, Anderson DJ. The effects oflymphokines and monokines on human sperm fertilizing ability in the zona free hamster egg penetration test. Am J Obstet Gynecol1989;160: Vander Verr HH, Jeyendran RS, Perez-Pelaez M, Al-Hasami S, Diedrich K, Krebs D. Leucospermia and the fertilizing capacity of spermatozoa. Eur J Obstet Gynecol Reprod Biol 1987;4: Cohen J, Edwards R, Fehilly C, Fishel S, Hewitt J, Purdy J, et al. In vitro fertilization: a treatment for male infertility. Fertil Steril 1985;43: Barratt CLR, Bolton AE, Cooke ID. Functional significance of white blood cells in the male and female reproductive tract. Hum Reprod 1990;5: Vol. 62, No.3, September 1994 Branigan and Muller Treatment of leukocytospermia 583

5 10. Hillier SL, Rabe LK, Muller CH, Zarutskie PW, Keyan FB, Stenchever MA. Relationship of bacteriologic characteristics to semen indices in men attending an infertility clinic. Obstet Gynecol 1990;75: Berger RE, Smith WD, Critchlow CW, Stenchever MA, Moore DE, Spadoni LR, et a!. Improvement in the sperm penetration (hamster ova) assay (SPA) results after doxycycline treatment of infertile men. J Androl1983;4: Harrison RF, Blades M, Delouvois J, Hulley R. Doxycycline treatment and human infertility. Lancet 1975;1: Mathews CD, Clapp KH, Tansing JA, Coy LW. T-mycoplasma genital infection, the effect of doxycycline therapy on human unexplained infertility. Fertil Steril 1978;30: Couture M, Ulstein M, Leonard JM, Paulsen CA. Improved staining method for differentiated immature germ cells from white blood cells in human seminal fluid. Andrologia 1976;8: Berger RE, Karp LE, Williamson RA, Koehler J, Moore DE, Holmes KK. The relationship ofpyospermia and seminal fluid bacteriology to sperm function as reflected in the sperm penetration assay. Fertil Steril 1982;37: Comhaire F, Verschraegen G, Vermeulen L. Diagnosis of accessory gland infection and the possible role in male infertility. In: Edited by G. Frajese, EFE Hafez, C. Conti, A Fabbrini pg Oligozoospermia: recent progress in andrology. New York: Raven Press, Hughes L, Ryder TA, McKenzie ML, Pryse-Davies J, Stendronska J, Hendry WF. The use of transmission electron microscopy to study non-spermatozoa cells in semen. In: Edited by G. Frajese, EFE Hafez, C. Conti, A Fabbrini pg Oligozoospermia: recent progress in andrology. New York: Raven Press, Branigan and Muller Treatment of leukocytospermia