Occurrence of Mycoplasma genitalium in fertile and infertile women

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1 Occurrence of Mycoplasma genitalium in fertile and infertile women Joanna Grzesko, M.D., Ph.D., a Marek Elias, M.D., Ph.D., a Beata Mączynska, M.Sc., Ph.D., b Urszula Kasprzykowska, M.Sc., b Magdalena Tłaczała, M.D., Ph.D., a and Marian Goluda, M.D., Ph.D. a a Gynecology Clinic, 2nd Department of Gynecology and Obstetrics, and b Department and Institute of Microbiology of the Wroclaw Medical University, Wroc1aw, Poland Objective: To determine the frequency of occurrence of Mycoplasma genitalium in the reproductive organs of infertile women in comparison with a control group of healthy, fertile women. Design: Prospective study. Setting: Gynecology Clinic at the 2nd Department of Gynecology and Obstetrics of the Wroclaw Medical University, Poland. Patient(s): The study included 51 patients with primary infertility (24 women with idiopathic infertility) and 23 women with proven fertility. Intervention(s): Cervical smear and smear from the peritoneal cavity, performed during laparoscopy. Main Outcome Measure(s): Presence of the genetic material of M. genitalium in the collected material analyzed using polymerase chain reaction (PCR). Result(s): M. genitalium was found in the cervical canal of 19.6% of all infertile patients and in 4.4% of fertile patients. In addition, the pathogen was discovered in the cervical canal of 29% patients with unexplained (idiopathic) infertility, which in comparison with the fertile group was a statistically significant difference. In the abdominal cavity, M. genitalium was found in 5.8% of patients from the infertile group (in 8.4% patients with idiopathic infertility), whereas it was not detected in the material obtained from the studied fertile patients. Conclusion(s): The results obtained may suggest that M. genitalium is a species having an impact on impaired fertility in women. (Fertil Steril Ò 29;91: Ó29 by American Society for Reproductive Medicine.) Key Words: Mycoplasma genitalium, idiopathic infertility, cervical canal, PCR In the past 2 years, infertility has become a growing social and economic problem, especially in highly developed countries. It is estimated that 8% 12% couples in the world may experience problems conceiving at some point of their reproductive life (1). The World Health Organization (WHO) estimates that 6 8 million couples across the world experience unwanted infertility and has declared it a social disease (2). There exist many defined and diagnosable causes of infertility of partners but idiopathic infertility (infertility of unexplained origin) still stirs much controversy. The term is used to define infertile couples whose real cause of infertility cannot be discovered using standard diagnostic tests. To diagnose idiopathic infertility of partners, initially the hormonal profile and patency of fallopian tubes in the woman must be confirmed as well as sperm quality and undisturbed sexual Received February 4, 28; revised March 24, 28; accepted March 25, 28; published online June 19, 28. Joanna Grzesko and Marek Elias participate in clinical research sponsored by Organon Company. B.M. has nothing to disclose. U.K. has nothing to disclose. M.T. has nothing to disclose. M.G. has nothing to disclose. Supported by the Polish Committee of Scientific Studies (MNiSW Grant No 2 P5E 8229) and Wroc1aw Medical University (GR 632/25). Reprint requests: Joanna Grzesko, Ph.D., M.D., Gynecology Clinic, 2nd Department of Gynecology and Obstetrics, Wroclaw Medical University, Ul. Dyrekcyjna 5/7, 5-528, Wroc1aw, Poland (FAX: ; joannagrzesko@gmail.com). functions in both partners (3). Literature on the subject suggests that this group includes couples whose infertility is really unexplained, couples who have not been fully diagnosed, and in couples where a diagnostic mistake was made. Thus, clinicians should not treat this type of infertility as a diagnosis but as a diagnostic failure (4). Among insufficiently diagnosed patients can be women with oligosymptomatic infections of the urinary and genital system whose etiological factor has not been determined because of the lack of sufficient standards for diagnosing infertility. Bacteria from the family Mycoplasmataceae are the microorganisms that by causing chronic, oligosymptomatic genital infections may have a negative impact on fertility. They are the smallest and simplest prokaryotic organisms capable of self-replication. Their characteristic feature is the lack of cell wall, which makes them significantly different from other bacteria and is the reason of high cell plasticity (5). The family Mycoplasmataceae, belonging to the order Mycoplasmatales, class Mollicutes, includes two genera: Mycoplasma and Ureaplasma. In the genus Mycoplasma the existence of 118 species has been confirmed, and 7 species in the genus Ureaplasma. In this large group, 2 species of Mycoplasma and 2 species of Ureaplasma have been isolated from humans (6 8) Fertility and Sterility â Vol. 91, No. 6, June /9/$36. Copyright ª29 American Society for Reproductive Medicine, Published by Elsevier Inc. doi:1.116/j.fertnstert

2 Mycoplasma genitalium is the species first described in 1981 and most frequently associated with nongonococcal urethritis in men and women, especially in persistent and recurring urethritis (9 12), with simple or mucopurulent cervicitis (11, 13, 14), as well as with endometritis, pelvic inflammatory disease, and salpingitis (11, 15, 16). In organotypic cultures it has been discovered that M. genitalium is capable of adhering to human fallopian tube epithelial cells (17). Clausen et al. (18) also point to an observed higher ratio of fallopian tube lesions and the presence of antibodies to M. genitalium in the studied group of infertile women. In vitro tests of sperm infected with these mycoplasma revealed detrimental changes in sperm structure thickening around the neck, curving and looping of the tails, which caused a significant deterioration of motility, especially with regard to the correct progressive motility. On the other hand, sperms to which individual mycoplasma cells adhered in the area of the neck or at the beginning of the tail displayed correct motility, therefore they could transmit the infection during sexual contacts (19). However, because mycoplasmas are organisms causing infections whose progress is slow and often has very few symptoms, some patients and their doctors do not realize their significant medical history (2). It may be one of the reasons for the lack of association of M. genitalium infections with infertility in women and for the lack of performance of the relevant diagnostic tests on the treated partners. The purpose of the present study was to determine the frequency of occurrence of M. genitalium in the genitals of women with infertility of various etiologies, including women with idiopathic infertility, in comparison with fertile women. An attempt has also been made to study the correlation between the presence of this species in the reproductive tract and impaired fertility. MATERIALS AND METHODS Patients The study includes patients hospitalized at the Gynecology Clinic at the 2nd Department of Gynecology and Obstetrics of the Wroclaw Medical University from All the patients were admitted to the hospital for the performance of a planned laparoscopy. The study group was composed of 51 women with primary infertility for whom diagnostic laparoscopy had been planned and performed. In the course of the diagnostic and therapeutic process, the patients were divided into groups depending on the possible cause of their infertility: group A 24 women in whom, both before and after the laparoscopy, the cause of infertility remained unexplained (idiopathic infertility); group B 12 women in whom, during the laparoscopy, tubal obstruction was diagnosed; group C 7 women in whom the laparoscopy did not reveal the cause of infertility but the analysis of the sperm of the partner showed irregularities that were suspected of hindering or even preventing conception; and group D 8 women whose infertility was caused by ovulation disorders caused by polycystic ovary syndrome (PCOS) and by endometriosis of the pelvis. All of the patients in the study group were aged 24 4 years. The control group included 23 women with proven fertility (history of giving birth to at least one child). Treatment laparoscopy was performed on all the patients in this group and the indication for laparoscopy was the presence of noninflammatory, benign ovarian tumors and uterine myomas. The patients were aged years. A detailed gynecological examination and a general physical examination was performed on all women, which made it possible to exclude women with symptoms of reproductive organ infection. Material Sampling Cervical swabs were collected from all the patients using a sterile platinum loop under operating room conditions, after induction of general anesthesia. Cervical swabs were transported in 2 ml of 2SP medium (21) (.2 M sucrose in.2 M phosphate buffer, ph 7.2) containing gentamicin (5 mg/ ml) and nystatin (25 mg/ml) and frozen at 2 C for polymerase chain reaction (PCR) testing. Then, after introducing trocars into the peritoneal cavity, liquid was aspirated from the pouch of Douglas and 1 ml of the liquid was placed in a sterile, empty Eppendorf tube and was immediately frozen to 2 C. Sample Preparation for Polymerase Chain Reaction DNA isolation from frozen samples was carried with QIAamp DNA kit (Qiagen, Hilden, Germany) in accordance with the instructions of the suppliers. DNA isolates were dissolved in TE buffer (1 mm Tris-HCl, ph 8.; 1 mm ethylenediaminetetraacetic acid [EDTA]). The amount of DNA in the solution was quantified by measuring the optical density at 26 nm and appropriate DNA dilution for PCR was calculated. Polymerase Chain Reaction Assay All clinical samples were tested by PCR assay for the presence of M. genitalium. We used PCR for the detection of M. genitalium using primers that had been validated by other workers (22). The following specific primers were used to target the 14-kDa adhesion protein gene: MgPa-1 (5 -AGT TGA TGA AAC CTT AAC CCC TTG G-3 ) and MgPa-2 (5 -CCG TTG AGG GGT TTT CCATTT TTG C-3 ). The primers amplified the 281-bp adhesin gene fragment. The PCR assay was performed with Taq PCR Core Kit, a universal PCR master kit by Qiagen in a MJ Research PTC-2. After an initial 4 minutes at 95 C, there were 35 cycles of 95 C for 1 minute, 65 C for 1 minute, and 72 C for 1 minute, followed by a period of further extension of 6 minutes at 72 C and cooling to 4 C. As a positive control of Fertility and Sterility â 2377

3 the PCR reaction we used ATCC M. genitalium The PCR product was detected by looking for a band of the appropriate molecular weight in 1.2% agarose gel stained with ethidium bromide. FIGURE 1 Frequency of occurrence of Mycoplasma genitalium in the group of infertile patients and in control patients. Statistical Analysis The statistical analysis was carried out using the EPIINFO package of statistical software, version 3.2 (dated ) The hypothesis that the means for individual groups are equal was verified using the analysis of variance (ANOVA) method, and for groups with heterogeneous variance or with a small number of cases, using the nonparametric Wilcoxon test (variance homogeneity was tested using the Bartlett test). Analysis of the distribution of discrete parameters was made using the c 2 test with the Yates correction or, when the value expected in the cell was smaller than 5, using Fisher s test. P%.5 was considered statistically significant. RESULTS In total, material collected from 74 patients in two groups was analyzed the study group and the control group. In the infertile patient group, the presence of M. genitalium in the cervical canal was confirmed in 1 of 51 studied cases (19.6%), whereas in the control group only in 1 of 23 patients (4.4%). This was not statistically significant (P¼.156F). In the samples collected during laparoscopy from the abdominal cavity, genetic material of the microorganism was found in three patients from the study (infertile) group (5.88%) and in none from the control group. No statistical significance was observed (P¼.548F; Fig. 1). The analysis of the distribution of positive results in individual subgroups of infertile patients showed that the microorganism was isolated only from patients with idiopathic infertility (group A) and from patients whose infertility was caused by ovulation disorders (group D). What is curious, no M. genitalium was isolated from patients with infertility caused by tubal obstruction (group B) or by insufficient quality of the partner s sperm (group C). However, this may be a result of a relatively small number of patients in individual subgroups. The different potential causes of infertility caused problems in obtaining homogeneous study groups with a suitable number of patients in each group. Therefore, during the final stage of analyzing the groups covered by the study, the two most uniform groups were selected in which the frequency of occurrence of M. genitalium was compared. The two groups included 24 women with idiopathic infertility and 23 fertile patients from the control group. The presence of M. genitalium in the cervical canal was found in 7 (29.2%) of 24 women with idiopathic infertility, whereas in the control group a positive result was obtained Cervical canal Infertile patients only in 1 (4.4%) of 23 patients. The dependency observed was statistically significant (P¼.479F). In the fluid collected from the pouch of Douglas during laparoscopy, the presence of the microorganism was discovered in two infertile patients (8.4%) and in none from the control group, which did not, however, constitute a statistically significant difference (P¼.489F; Fig. 2). The higher frequency of detecting M. genitalium in the cervical canal of women in the study group in comparison with the control group may indicate the link of the microorganism with infertility. What seems particularly important is that, after the performance of a panel of mandatory tests, the reason of infertility remained unknown. DISCUSSION Infertility is a complex medical problem whose causes may be diverse. Taking into account the etiological factor 5.88 Rectouterine pouch Control group Grzesko. Mycoplasma genitalium and infertility. Fertil Steril 29. FIGURE 2 Frequency of occurrence of Mycoplasma genitalium in patients with idiopathic infertility and in the control group of fertile women Cervical canal 4.4 Patients with idiopatic infertility 8.4 Rectouterine pouch Control group Grzesko. Mycoplasma genitalium and infertility. Fertil Steril Grzesko et al. Mycoplasma genitalium and infertility Vol. 91, No. 6, June 29

4 responsible for the condition, infertility can be divided into functional infertility connected with ovulation disorders, infertility caused by anatomical abnormalities of reproductive organs, infertility connected with cervical canal pathologies, and infertility occurring in the course of endometriosis and as genital inflammatory complications. Idiopathic infertility, or infertility caused by unidentified reasons, is a separate problem. The role of an oligosymptomatic infection with genital mycoplasmas, including with M. genitalium, has been confirmed in the case of infertility caused by fallopian tube disorders. One of the examples is the study by Clausen et al. (18) in which antibodies against MgPa, the main adhesive protein of M. genitalium, were detected in 22% women with tubal infertility, whereas in the control group of women with unobstructed fallopian tubes these antibodies were found only in 6% of patients. This result allowed a conclusion that the studied species is an independent risk factor in the development of an inflammatory process damaging the fallopian tubes and causing infertility. Taylor-Robinson et al. (23) report a frequent (2% of cases) occurrence of these species in the cervical canal of patients visiting their doctors because of genital inflammations in comparison with healthy women without infection symptoms. This confirms the hypothesis that contrary to Ureaplasma urealyticum, M. genitalium does not colonize the reproductive organs and its presence is always connected with a pathology, although infection symptoms are very mild, unnoticeable both for patients and for their doctors (11, 24 26). This proposition was confirmed in other studies conducted simultaneously by the investigators of the present study. In the group of women treated at the Gynecology Clinic of the 2nd Department of Gynecology and Obstetrics of the Wroclaw Medical University for various genital infections, M. genitalium was found in nearly one-half of the patients (the ratio of positive PCR results reached 43.3% in the study group and 4.4% in the control group) (doctoral dissertation by J. Grzesko published in November 26). Thus, M. genitalium should always be treated as a pathogen, easily transmissible through sexual intercourse. It has been found that it occurs in more than 5% of sex partners of the infected women (13), which confirms the need to treat all patients with confirmed presence of this species as well as their partners. Interesting observations were made after comparing the groups of women with undetermined infertility with the control group of healthy women. It was demonstrated that M. genitalium is present significantly more frequently in the cervical canal of infertile women. Is it then the case that patients with idiopathic infertility and their partners suffer from undiagnosed, oligosymptomatic infection with M. genitalium leading to fertility problems? Tests to identify M. genitalium are not among the routine tests and can currently be performed only at specialized health care facilities. Because the problem of pathogenicity of M. genitalium and its impact on human fertility has been studied for only a relatively short time, literature on the subject is scarcer than in the case of M. hominis and U. urealyticum pathogens, which were discovered earlier and have been studied better. For a long time M. genitalium was classified as a microorganism of so-called undetermined pathogenicity. At present, advances in research and diagnostic methods, as in the case of many other pathogens previously regarded as nonpathogenic, are able to verify the existence of this pathogen. Despite of all of these conclusions, diagnosing of infections with M. genitalium is still not among the standard tests performed on infertile couples. The main reason is because the role of the bacteria in infertility is still being discussed and given many other factors having an impact on female fertility, it is impossible to provide conclusive evidence of their link with the condition. Another aspect are problems connected with the detection of M. genitalium. These microorganisms cannot be cultured on substrates typically used for mycoplasmas and the lack of commercial biochemical or serologic tests makes PCR the only diagnostic method available (22, 27). The results of tests performed for the purposes of this article seem sufficiently convincing to postulate introducing routine identification of M. genitalium in diagnostic and therapeutic procedures performed with respect to women treated for infertility. 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