Chlamydial infection-a female and/ or male infertility factor?

Transcription

1 FERTILITY AND STERILITY Vol. 53, No.6, June 1990 Copyright The American Fertility Society Printed on acid-free paper in U.S.A. Chlamydial infection-a female and/ or male infertility factor? Waltraud Eggert-Kruse, M.D.*t Ingrid Gerhard, M.D.* Horst Naber, M.D.:j: WolfgangTilgen, M.D.:j: Benno Runnebaum, M.D.* University of Heidelberg, Heidelberg, Federal Republic of Germany After screening a large series (n = 491) of asymptomatic males of infertile partnerships for chlamydia! immunoglobulin (lg) G antibodies (Chlam AB), no significant influence of past chlamydia! infection was found with regard to semen analysis, postcoital testing, in vitro sperm-cervical mucus penetration tests with hormonally standardized cervical mucus, circulating antisperm antibodies (detected with three different methods), local lgg and lga antibodies (detected by means of the mixed antiglobulin reaction test) on the sperm surface, the sperm-cervical mucus contact test, and a microbial screening of semen samples for mycoplasmas and other potentially pathogenic micro-organisms. However, when the findings were correlated with infertility factors of patients' female partners and the subsequent pregnancy rate in a prospective study, a significant positive correlation of male Chlam AB with a tubal factor in their wives as cause of the couple's infertility was found. The results suggest that the main influence of Chlamydia trachomatis on male fertility is based on sexual transmission and negative influence on tubal function offemale partners, but not on reduced sperm functional capacity. Fertil Steril53:1037, 1990 Chlamydia trachomatis is the most frequently sexually transmitted micro-organism in industrial countries. 1 2 With the cervix uteri as a reservoir, C. trachomatis causes widespread genital tract infection in both men and women. For example, 20% to 50% ~f female nongonococcal salpingitis 2 and most cases of male nongonococcal urethritis and epididymitis3-5 is caused by these micro-organisms. Whereas there is strong evidence for a negative influence of C. trachomatis on tubal function with severe postinfectious tubal damage in case of ascending infection, 6--8 not much is known about the in- Received October 30, 1989; revised and accepted February 16, *Division of Gynecological Endocrinology, Women's Hospital Infertility Unit. t Reprint requests: Waltraud Eggert-Kruse, M.D., Division of Gynecological Endocrinology, Women's Hospital Unit, University of Heidelberg, Vo13stral3e 9, 6900 Heidelberg, Federal Republic of Germany. :j: Department of Dermatology. fluence of these micro-organisms on the fertility of the man. Although they are known to attach to spermatozoa, 9 it is unclear if chlamydiae affect sperm function. Sperm functional capacity is not sufficiently examined by the parameters of microscopical sperm analysis. 10 The in vitro sperm -cervical mucus penetration test (SCMPT) is a reliable test for sperm function which has been shown to be of prognostic value for subsequent fertility. 11 The aim of the present study was to assess the clinical significance of a past chlamydia! infection on the fertility of the male and female partner of involuntarily childless couples. Therefore, the results of chlamydia! antibody testing in both men and women were correlated with a variety of other infertility factors (tubal and ovarian function, parameters of sperm analysis, testing of circulating and local antisperm antibodies [ ASA], postcoital test [PCT], crossed in vitro SCMPT, and results of microbial examination of genital secretions) and Vol. 53, No.6, June 1990 Eggert-Kruse et al. Chlamydia[ infection and male fertility 1037

2 Table 1 Significance of Past Chlamydia! Infection in Female Patients for the Tubal Factor Both sides free One side impaired Both sides Both sides impaired closed Total Tubal factor No. Percent No. Percent Chlam AB negative a Chlam AB slightly pos b Chlam AB pos c Total a Chlamydia (Chlam) IgG antibody (AB) titer< 1:32. b Chlam lgg AB titer 1:32 to 1:128. No. Percent No. Percent No. Percent c Chlam IgG AB titer~ 1:256; P < when a+b tested versus c. analyzed for subsequent pregnancy rate in a prospective study. MATERIALS AND METHODS A total of 586 couples were randomly chosen from those presenting at the infertility unit of the University of Heidelberg. Enrolled in this study were only asymptomatic patients without clinical signs or symptoms of infection of the genital tract. Couples had been involuntarily childless for > 1 year with a median of 5 years and a maximum of 19 years. Primary infertility was found in 79%, secondary infertility in 21%. The age of the women ranged from 22 to 45 years (median 30 years) and of the men from 24 to 53 years (median 33 years). After medical history and careful clinical examination, blood was taken from both partners on the same day for detection of immunoglobulin (Ig) G antibodies to C. trachomatis in serum using the indirect fluorescent antibody test (Electro Nucleonics Incorporation, Viramed, Martinsried, West Germany). In female patients, the tubal factor was evaluated by hysterosalpingography (50.5%) and/or laparoscopy (38.1% ), and/ or perturbation (11.4%). Examination by two or three methods was done in 32.6% of patients; one method only was used in 67.4%. The results are shown in Table 1. The hormonal factor was carefully examined. The evaluation included tests for ovarian, pituitary, thyroid, and adrenal function in the early follicular phase, multiple determinations of progesterone and estradiol (E 2) in the luteal phase, and premenstrual endometrial biopsy. Semen was collected in hospital by masturbation into sterile glass jars after a sexual abstinence of 5 days and was examined directly after liquefaction. For sperm analysis, standard criteria of the World Health Organization were used. The results included the whole variability of andrological find- ings except azoospermia. The PCT was performed as described in detail previouslyp Briefly, the number of progressively motile spermatozoa in cervical mucus (CM) was counted 8 to 12 hours after intercourse and results were classified as good when ~2 motile sperm were found (mean of 20 high power fields [HPF]). The in vitro SCMPT was evaluated after hormonal standardization of the mucus quality by oral administration of ethinyl E 2 for ~7 days (80 ~g/d) before testing. 11 Additionally, CM (also hormonally standardized) and semen of fertile donors was used for a cross-matching penetrability test. Results were classified as adequate, when a cumulative SCMPT score, taking into account the penetration distance, sperm density, and the quality and duration of motility in CM, was ~6. Blood for detection of chlamydia! antibodies ( Chlam AB) in serum of both partners was usually taken on the day of SCMPT. A simultaneous microbial screening included cultures for mycoplasmas, for potential pathogenic aerobic and anaerobic bacteria, and additionally in females, for Herpes simplex virus and yeasts. The microbial methods used have been described in detail elsewhere.13 Cervical swabs for detection of C. trachomatis by means of McCoy cell culture were taken in 205 women. Additionally, a cervical swab was taken in these patients for the direct immunofluorescent test using monoclonal antibodies (Micro Trak; Syva, Palo Alto, CA). One hundred fortyfour randomly chosen women were screened by endometrial biopsies for endometrial infection with chlamydia by means of McCoy cell culture (three subcultures were performed in the majority of the samples).14 To evaluate immunological causes of infertility, in addition to the crossed SCMPT, serum samples of females and males were screened for circulating ASA with a standard tray agglutination test (TAT, n = 302 couples), a radioimmunoassay (RIA, n = 173 couples), and a commercially available en Eggert-Kruse et al. Chlamydial infection and male fertility Fertility and Sterility

3 zyme-linked immunoadsorbent assay (ELISA; Elias Inc., Freiburg, West Germany, n = 66 males and 83 females). Additionally, semen was evaluated for local ASA (IgG and IgA) using the mixed antiglobulin reaction (MAR) test according to Jager et al. 15 (n = 118 ejaculates). Briefly, IgG- and IgA-coated red blood cells were used to detect antibodies on the sperm surface. The percentage of spermatozoa covered with IgG and/or IgA ASA and involved in "mixed agglutinates" was determined. Furthermore, the sperm-cervical mucus contact (SCMC) 16 test was performed in 95 couples. All patients were treated in a standardized way, independent of serological findings, according to results of hormonal examinations, sperm analyses, PCT, and results of microbial cultures, but not with in vitro fertilization or gamete intrafallopian transfer. Pregnancy rate was determined 6 months after Chlam AB testing. Data were processed using the statistical analysis system, x 2 tests, Fisher's two-tailed exact tests, Spearman rank correlation, Wilcoxon rank sum tests, and Friedman tests. The level of significance was set at P < RESULTS Significance of Female Chlamydia! Infection for the Tubal Factor The results of tubal examinations in the 586 randomly chosen females of infertile couples presenting at the infertility clinic are shown in Table 1. Free patency of both fallopian tubes was found in 67.8% of women. A moderate or severe tubal factor was diagnosed in 32.2% of patients. Highly elevated titers of Chlam IgG AB in serum samples of female patients correlated significantly with a tubal factor as cause of infertility (P < , x 2 analysis). Incidence of Chlam AB in Int._rtile Couples Serum samples for evaluation of Chlam AB in both partners and complete data of infertility investigation (tubal, hormonal, and male factor) could be obtained in 491 of the 586 couples. These patients (86.6%) were taken for further analysis. The incidence of Chlam IgG AB is shown in Table 2. Low titers were found in 37% of women and 44% of men. Strong positive titers of Chlam IgG AB (~1:256) were found in 15% of male and 18% of the female serum samples. According to Punnonen et al., 8 this group of patients was classified as Table2 Incidence of Chiaro lgg AB in Serum Samples Female Male Titer No. Percent No. Percent ChlamlgGAB Negative :S1: :S1: :S1: : Total Chlam AB positive (pos) and was compared with the other infertile couples. A titer of 1:256 was used as a cutoff for statistical analysis, but all statistical evaluations were also performed for cutoffs at lower and higher Chlam IgG AB titers in serum. Past chlamydia! infection was detected significantly more frequently when a patient's partner was Chlam AB pos: 41.8% (38/91) of husbands of the Chlam AB pos women offered IgG titers of ~1/256 versus 9.3% (37/400) of the other male patients (P < 0.001, x 2 analysis). Chlamydia! IgG Antibodies and Microbial Screening Chlam AB pos and negative patients were compared with regard to colonization of genital secretions with potentially pathogenic micro-organisms and species of the physiological flora. No significant differences were found for the prevalence of any of the cultured micro-organisms in both groups of patients, e.g., mycoplasmas (M. hominis and/or Ureaplasma urealyticum) were cultured in 21.7% (10/46) in semen specimens of the Chlam AB pos men compared with 17.4% (37/213) in the other samples. Potentially pathogenic aerobic bacteria (as classified previously 13 ) were found in 57.9% (252/435) of ejaculates and in comparable frequency with regard to all species in the Chlam AB pos and negative patients; potentially pathogenic anaerobes 13 were cultured in 14% and in 19.5%, respectively. Species of the physiological flora were found in the majority of patients. The percentage of semen samples being completely negative in aerobic cultures was very low (8.1%, 35/435) and did not differ with regard to Chlam AB status. Negative results in all the different cultures were found in only 1.5% (7/471). Semen samples of the first 44 patients with Chlam AB titers~ 1:256 were examined by means of MicroTrak, but C. trachomatis was not detected in any of these cases. The female partners of the Chlam AB pos and Vol. 53, No.6, June 1990 Eggert-Kruse et al. Chlamydia[ infection and male fertility 1039

4 negative men as well did not differ with respect to colonization of cervical secretions with the above mentioned potentially pathogenic micro-organisms and with the finding of cervical Herpes simplex (4%, 17 /430) or vaginal/cervical yeasts (9.2%, 17 j 430). Furthermore, no correlation was found between microbial results and Chlam AB in female serum samples. C. trachomatis in cervical swabs was found in 1% (1/205) and in endometrial biopsies in 3.5% (5/144). We reported on this previously.14 None of the patients offered mucopurulent cervical or urethral discharge on clinical examination. More than 10 leucocytes (X1,000) in gram-stained cervical smears were found in 6.4% (31/487) of women, and> 10 round cells per HPF were counted in 4 7% of semen samples, but these findings did not correlate with Chlam AB titer in serum. Influence of Chlamydia! Infection on Semen Characteristics and ASA When results of sperm analysis were compared with regard to Chlam AB status, no significant differences were found; e.g., when sperm count was reduced to <20 X 10 6 /ml, 7.1% (4/56) of patients were Chlam AB pos compared with 16.1% (70/435) in the group with higher sperm counts. When motility was <20%, high Chlam AB titers were found in 11.4% (5/44) compared with 15.4% (69/447) in patients with >20% of sperm progressive motility. The results of semen analysis analyzed by means of Wilcoxon tests are shown in Table 3. No significant difference was found for any ofthe tested variables. Circulating ASA (TAT ~ 1:32) in males were found in 11.8% (36/306) and 11.6% (20/173, RIA ~ 20% binding capacity), respectively, but did not correlate with results of Chlam AB testing in serum samples. No differences were found either when results of ELISA testing for circulating ASA were ' compared. Antisperm IgG AB on the sperm surface (~60% of sperm MAR positive) were found in 6.8% and surface MAR IgA AB were detected in 5.1% (see Table 3). Both MAR tests correlated significantly with each other (r = , P < ; Spearman rank correlation) but not with testing of Chlam IgG ABs in male and female serum samples. Results of SCMC tests did not offer any significant differences, either, between Chlam pos and negative male and female patients. Influence of Chlamydia! Infection on Sperm Function Sperm functional capacity was evaluated with in vivo and in vitro CM penetration tests. Results of Table 3 Chlamydia! lgg AB and Semen Characteristics and ASA a Parameter of sperm analysis d Sperm volume (ml) ph Sperm count (X10 6 /ml) Progressive motility(%) Morphology(% normal) Viability (%) Fructose concentration (ng/ml) Round cells (n/hpf) ASA Circulating ASA TAT (~1/32) RIA (~20%) ELISA (~20%) Loca!ASA MAR lgg (~60%) MAR lga (~30%) ChlamAB negativeb 3.1 (0.6 to 9.5) 7.5 (6.7 to 8) 45 (1 to 249) 40 (1 to 80) 60 (10 to 75) 65 (10 to 75) 2.0 (0.2 to 4.9) 10 (0 to 30) 12.6 (32/254) 13.1 (19/145) 17.2 (10/58) 7.6 (8/105) 5.7 (6/105) Chlam AB pos' 2.8 (1 to 7.2) 7.5 (6.4 to 7. 7) 49.5 (8 to 186) 45 (1 to 70) 62 (47to69) 70 (50 to 80) 1.8 (0.2 to 4.8) 10 (Oto35) 8.3 (4/48) 3.6 (1/28) 12.5 (1/8) 0 (0/13) 0 (0/13) a All differences not significant. b <1:256; n = 416. '~1:256; n = 75. d Values are the medians with range in parentheses. Values are percent of patients positive with number of patients positive/total patients in parentheses. PCT were poor in 45.2% (211/467) and good in 54.8% (256/467). No negative influence was seen when male and/or female serum samples offered evidence of past chlamydia! infection. When only results of PCT obtained after hormonal standardization of the CM quality with oral estrogens were taken into consideration, again no significant correlation of PCT outcome and Chlam AB status in both men and women was seen (n = 393). The SCMPT was evaluated with CM and semen of the infertile couple and as a cross-matching penetrability test. The CM of both patients and donors was hormonally standardized. Sperm-cervical mucus penetration test results (performed with semen and CM of the infertile couples) were inadequate in 39.7% (192/484) and adequate in 60.3% (292/484). The incidence of males with high titers of Chlam AB was 12% in the inadequate SCMPT group and 18% in the adequate SCMPT group. As demonstrated in Table 4, past chlamydia! infection did not influence the SCMPT results in a negative way (x 2 analysis). Furthermore, males with high titers of Chlam AB were not found in higher frequency when results of SCMPT were very poor or negative. Adequate SCMPT was significantly more frequent when penetration testing was performed with donor's spermatozoa (85%, 406/475), showing that the male factor accounted to a significantly 1040 Eggert-Kruse et al. Chlamydia/ infection and male fertility Fertility and Sterility

5 Table 4 No Negative Influence of Past Chlamydia! Infection on Sperm-Mucus Interaction In Vivo (PCT) and In Vitro (SCMPT)" PCT SCMPT Poor Good Inadequate Adequate Woman Chlam AB pos b Man Chlam AB pos b Crossed SCMPT with donors'cm Woman Chlam AB pos b Man Chlam AB pos b Crossed SCMPT with donors' sperm Woman Chlam AB pos b Man Chlam AB pos b 16.1 (34/211) 14.2 (30/211) 19.5 (50/256) 15.6 (40/256) 19.3 (37/192) 11.5 (22/192) 16.5 (31/188) 12.2 (23/188) 21.7 (15/69) 18.8 (13/69) 18.2 (53/292) 17.8 (52/292)c 19.9 (59/297) 17.2 (51/297) 17.5 (71/406) 14.3 (58/406) " All differences not significant; values are percents with no. patients/total in parentheses. b,;;1:256. SCMPT with wives' CM. higher degree than the CM quality to deficient sperm-mucus interaction (P < , Friedman tests). However, the results of the crossed sperm CM penetration assay as well were not significantly impaired when serum samples offered evidence of past chlamydia! infection. Correlation of Past Chlamydia! Infection with Socioeconomic Factors and Subsequent Fertility The overall pregnancy rate 6 months after Chlam AB testing was 23% (113/490). One couple was lost for follow-up. The subsequent fertility was lower when the testing of the male partner offered a Chlam antibody titer of ~1:256 (13.5%, 10/74) than in case of lower or negative titer (24.7%, 103/ 416) (P < 0.05, x 2 analysis). In addition to the above mentioned andrological characteristics, results of medical history and clinical examination in patients with and without evidence of previous chlamydia! infection were compared but did not show significant differences. For example, varicocele was found in 17.9% in the Chlam AB pos and in 12.9% in the other patients. Positive Chlam AB in males were found in 16.0% in case of primary sterility and in 13.7% in case of secondary sterility. In both Chlam AB pos and negative groups, 30.2% of men reported on stress due to daily working conditions, and 17.1% and 23.3%, respectively, were stressed by the procedures of infertility investigation. The incidence of Chlam AB was slightly higher when patients came from other countries than West Germany (19.4% versus 14.8%). The majority of patients represented a population of middle and upper social class, but in patients with a lower social status, Chlam AB ~ 1: 256 were detected significantly more frequent (19% versus 12%) than in the other patients (P < 0.05). Patients were comparable for female and male age, duration of infertility, andrological medication, smoking and drinking habits, and hormonal medication of their wives. In the 491 female partners of patients, the tubal factor was good (both sides open, no adhesions) in 70.7% and impaired in 29.3%, and tubal patency correlated significantly with female Chlam AB status (P < 0.001). A tubal factor was also found significantly more frequently in couples with Chlam AB pos males (38.7%, 29/75) versus in other couples (27.6%, 115/416) when the patency of the female's fallopian tubes was analyzed with respect to the Chlam AB status of their husbands. Past chlamydia! infection of the males correlated significantly with tubal disease as cause of the couples infertility (P < 0.04, x 2 analysis). DISCUSSION Although it has been shown that chlamydiae as well as other bacteria can adhere to spermatozoa, 9 there is a lack of information about the influence of C. trachomatis on male fertility. Chlamydia trachomatis is the main cause of urethritis and epididymitis in males. 3-5 Changes of the testis parenchyma by unspecific epididymitis and subsequent inhibition of spermatogenesis have been de~ scribed. 17 Therefore, the possible significance of male chlamydia! infection was studied in a large series of males of randomly chosen infertile partnerships (n = 491). Past chlamydia! infection, reflected by IgG antibodies in serum, was correlated with a variety of tests to evaluate semen characteristics, sperm function, and immunological causes of male j I! Vol. 53, No.6, June 1990 Eggert-Kruse et al. Chlamydia[ infection and male fertility 1041

6 infertility as well as female infertility factors and subsequent fertility. The present study did not reveal significant differences between parameters of classical sperm analysis in males with and without past exposure to C. trachomatis, which is consistent with the report of others. 18 However, the parameters of semen recorded by microscopical clinical analysis do not totally reflect male fertility potential Additional information can be obtained by examination of sperm-mucus interaction as a parameter for sperm function. The PCT is a useful screening test during infertility investigation, but in the present investigation results did not correlate with past chlamydia! infection of the male as well as of the female. The in vitro SCMPT with a hormonally standardized approach has been shown to be superior to PCT in assessing sperm functional capacity with regard to fertility prognosis. 12 However, in the present study, no negative influence of past C. trachomatis infection of male patients on results of SCMPT was found. To test sperm functional capacity, the in vitro SCMPT was additionally performed as a cross-test with CM of fertile donors, but the incidence of Chlam AB pos men was not higher, even lower in the group of patients with inadequate SCMPT outcome. This confirms our previous results in a smaller study population. 19 It has been hypothesized that bacterial colonization might influence sperm-mucus interaction, but when the association of chlamydia! antibodies with a variety of potentially pathogenic micro-organisms colonizing the lower male and/ or female genital tract was examined, no significant correlation was found. On the other hand, bacterial infections might stimulate the immune system. 20 In particular, C. trachomatis is supposed to be a potent immune stimulator. The present investigation did not reveal any significant correlation between circulating ASA (determined with three different methods) and Chlam AB in male as well as female serum samples. However, circulating ASA may not properly reflect immunological causes of infertility. Local ASA on the sperm surface, in particular lga antibodies detected with the MAR test, 15 are supposed to be more important than serum ASA for sperm functional capacity. No significant correlation of Chlam AB with sperm surface lgg or lga AB was found. In the group of Chlam AB pos men, none of the patients offered MAR positive semen samples, indicating that couples' infertility was caused by other factors in this group. Chlamydia! infection can lead to a total or partial blockage of the ductus deferens causing azoospermia or severe oligozoospermia. 5 This was very rare in this study population. It can be hypothesized that these cases were detected earlier during the course of infertility investigation and were therefore not in the group of patients referred to the infertility center. On the other hand, males. with a motility < 20% or sperm counts of <20 X 10 6 /ml were not more frequent in the Chlam AB pos group. However, the subsequent fertility was lower when males offered strong positive Chlam AB titers in serum samples. Male fertility can only be analyzed properly when infertility factors of both members of the infertile partnership are taken into consideration. Chlam AB pos and negative males were comparable for age, duration of infertility, age and hormonal factor in women, and medical treatment of males and females. However, female partners of Chlam AB pos men offered a tubal factor as cause of infertility significantly more frequently than wives of the other patients. The tubal disease of patients' wives explains the lower fertility rate within 6 months after Chlam AB testing, but other effects of C. trachomatis on the male reproductive organs not detected with the above mentioned methods cannot be excluded. Sperm -adhering chlamydiae, as "bacterial hitchhikers" can easily gain entrance to the upper female genital tract after sexual transmission. There is a dose-response relationship between acute salpingitis and infertility, and the rate of infertility due to tubal dysfunction increases with the number and severity of episodes of salpingitis. 1 A tubal factor is an important cause for female sterility. For example, in the present investigation in an unselected study population of a large infertility unit, it was32.2%. Chlamydia! infection is asymptomatic to a high degree, e.g., only 14.3% of the Chlam AB pos male patients remembered genital tract infection. The studied population consisted of 491 asymptomatic, and apart from their infertility problem, healthy individuals. None of the patients offered cervical or urethral discharge on clinical examination. Screening for C. trachomatis in cervical swabs and endometrial biopsies offered a quite low incidence of positive results ( <1% and 3.5%, respectively), although care was taken to get enough cellular material, and problems of transportation could be avoided (same building). The laboratory proved to be effective in detecting chlamydiae in a high percentage in a sexually transmitted disease population. Other authors as well failed to detect 1042 Eggert-Kruse et al. Chlamydial infection and male fertility Fertility and Sterility

7 C. trachomatis in cell culture in an asymptomatic infertile population, maybe because of socioeconomic factors. 1 The monoclonal fluorescent antibody technique was found to be of high reliability 23 but did not offer more positive results. However, screening for C. trachomatis AB in serum samples demonstrated that a high percentage of presently asymptomatic infertile couples had experienced infection with these micro-organisms. A significant positive correlation between past chlamydia! infection of female patients and a tubal factor as cause of infertility was found, confirming the reports of other authors. 6-8 Furthermore, the data of the present investigation demonstrate that male seropositivity to C. trachomatis is also a significant risk factor for tubal disease in their female partners. The results underline the need for a treatment as soon as possible in case of C. trachomatis infection in both males and females, preferably as a partner therapy even if the infection is only suspected. Future efforts are necessary to define the clinical significance of Chlam lga and lgm testing. Maybe the burden of suffering from infertility many years later could have been eliminated if chlamydia! infection in both men and women had been diagnosed and successfully treated before sexual transmission and tubal damage. In summary, the present study did not reveal past chlamydia! infection to affect adversely semen characteristics and sperm-mucus interaction or to correlate with ASA. The results suggest that infections with C. trachomatis of both female and male members of infertile partnerships are a risk factor for tubal disease (via sexual transmission), but not for reduced sperm functional capacity. REFERENCES 1. 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