The pathogenesis of ovarian cancer, the most lethal of gynecologic malignancies with 16-51% 5-year survival globally, is to a great extent still unknown. Infections by microbes causing chronic inflammatory disease have achieved increasing interest as possible initiators/promoters of various cancers, and infectious etiologies have been established for some cancers. Helicobacter pylori colonization of the stomach may lead to gastric cancer, particular subtypes of human papillomavirus (HPV) initiate cervical cancer, and chronic inflammation induced by hepatitis B and C virus infections can cause liver cancer. Pelvic inflammatory disease (PID) has been associated with ovarian cancer but the mechanism connecting PID with ovarian cancer is as yet unknown.

Chlamydia trachomatis is the most common cause of PID and tubal factor infertility (TFI) in the developed world, and infertility is in itself a suggested risk factor for ovarian cancer. Due to these associations, C trachomatis has been investigated as a possible risk factor for ovarian cancer by studying C trachomatis serum or plasma antibodies with contradicting results. Ness et al found an association of C trachomatis IgG antibodies and ovarian cancer and a monotonic trend toward a higher risk for ovarian cancer associated with higher levels of chlamydial heat shock protein 60-1 IgG antibodies. However, these results were not supported by a later study, in which a reduced odds ratio for ovarian, fallopian tube, and primary peritoneal cancer was detected among women (<49 years) with the highest titers of C trachomatis IgG. Likewise Wong et al did not find any associations of chlamydial serum antibodies and ovarian cancer. Our recent data indicate a possible association of C trachomatis antibodies and ovarian cancer risk, particularly when prospective plasma samples are analyzed (unpublished data). There are, to this date, no reports on the presence or absence of C trachomatis in ovarian cancer tissues although it has been found in 36% of ovarian tissues of women with pelvic adhesions/TFI. C trachomatis has also been detected in ovarian tissues of women with ectopic pregnancies, women with TFI, and control women.

Mycoplasma genitalium has in the last years evolved as an important sexually transmitted infectious agent causing PID, possibly with negative effects on fertility. Recently M genitalium was also studied as a possible tumor initiator/promoter of the ovaries and an association of plasma IgG and ovarian borderline tumors was found (unpublished data). The presence of Mycoplasma DNA (polymerase chain reaction [PCR] targeting 15 different species) has been found in 59% of ovarian cancer tissues. Neisseria gonorrhoeae is another sexually transmitted bacterium known to cause PID and TFI and the infection remains asymptomatic in up to 80% of women. N gonorrhoeae is not previously studied in relation to ovarian cancer but a history of gonorrhea is linked to prostate cancer in several metaanalyses.

HPV is a well-established cause of cervical cancer and the high-risk types are present in >95% of cervical cancer biopsy specimens. Regarding HPV in ovarian tissues there are, today, a great number of reports focusing on a possible tumor-initiating/-promoting role of the virus. Some have detected HPV in ovarian carcinoma tissues whereas others have not. Serum HPV antibodies was not associated with ovarian cancer in 1 study. The polyomaviruses BK virus (BKV) and JC virus (JCV) have been found in several different human tumors (urinary tract, kidney, bone, pancreatic islet, and a wide variety of brain tissue and skin tumors, including Merkel cell carcinoma) but a causal link has not been established. In vitro BKV and JCV are known to cause transformation of human cells, and they can induce different types of tumors in several rodent species. The presence of these viruses in ovarian tumors is not previously explored.

To study a possible role of chronic infections in ovarian tumorigenesis we examined the presence of the microorganisms C trachomatis , M genitalium , N gonorrhoeae , HPV, and the polyomaviruses BKV and JCV in ovarian tissues of women with ovarian carcinomas, borderline tumors, and benign conditions.

Materials and Methods

A cross-sectional study was undertaken to investigate the presence of 6 different microorganisms in ovarian tissues of women going through surgery due to suspected ovarian pathology. The study was approved by the human ethics committee of the medical faculty at Umeå University.

Clinical samples

From 1994 through 2001, 430 women who underwent surgery at the Department of Obstetrics and Gynecology, the University Hospital of Northern Sweden (Umeå, Sweden), due to suspected ovarian pathology were included in the study after oral and written informed consent. The women were mainly from Västerbotten County in northern Sweden. Ovarian tissue from the primarily affected ovary was obtainable in 186 women, and from the contralateral ovary in 126 women ( Figure ). Tissues were snap-frozen and stored at –80°C. Information on histopathologic diagnosis, according to the World Health Organization classification was extracted from the medical records and reviewed by a specialist in gynecologic pathology at the Department of Clinical Pathology Laboratory of the University Hospital of Northern Sweden.

Study cohort originating from women undergoing surgery due to suspected ovarian pathology

Shaded boxes show number of women with ovarian tissue obtainable. A = Number of tissues from primarily affected ovaries. C = Number of tissues from contralateral ovaries. All women with tissue from contralateral ovary had tissue obtainable from primarily affected ovary as well.

Idahl. C trachomatis, M genitalium, N gonorrhoeae , HPV, and polyomavirus in ovarian tissues. Am J Obstet Gynecol 2010.

DNA and RNA extraction

Tissue, 25-30 mg for each extraction, was cut frozen in a clean area with a sterile knife. RNA was extracted with RNeasy Mini Kit (Qiagen, Hilden, Germany) and DNA was extracted with QIAamp Blood Mini Kit (Qiagen) according to manufacturer instructions. We used the human β-globin gene as positive extraction control. Normal human cells contain 2 copies of the β-globin gene, which are therefore excellent positive extraction controls.

C trachomatis and N gonorrhoeae transcription mediated amplification

A total of 50 μL of extracted RNA solution was added to an Aptima Combo 2 (Gen-Probe Inc, San Diego, CA) collection tube and analyzed using a nucleic acid amplification test assay for detection of C trachomatis and N gonorrhoeae . As positive controls ovarian tissue samples from 4 different women were spiked with 10; 100; 1000; 10,000; and 100,000 C trachomatis bacteria. RNA was similarly extracted and added to collection tubes and examined with the Aptima Combo 2 test. For further validation urine samples positive or negative for C trachomatis with the BD ProbeTec (Becton Dickinson Diagnostics, Sparks, MA) were also tested with the same RNA extraction method and Aptima Combo 2.

M genitalium PCR

The M genitalium real-time PCR reaction was carried out in a 20-μL LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) glass capillary. The 15-μL reaction mix contained FastStart DNA Master Hybridization Probe mix, containing FastStart Taq DNA polymerase, reaction buffer, deoxyribonucleotide triphosphate mix, 10 mmol/L of MgCl ₂ (Roche Diagnostics, Mannheim, Germany), 1.0 μmol/L each of the forward primer M genitalium adhesion protein (MgPa)-355F and the reverse primer MgPa-432R (Scandinavian Gene Synthesis, Köping, Sweden), and 0.1 μmol/L of MgPa-380 TaqmanMGB probe (Applied BioSystems, Warrington, UK). Additional MgCl ₂ was added to a final concentration of 5 mmol/L. A 5-μL template DNA was added to the mixture. Amplification was performed in a LightCycler PCR system under the following conditions: FastStart Taq DNA polymerase activation at 95°C for 10 minutes, followed by 50 cycles of denaturation at 95°C for 5 seconds, annealing at 60°C for 15 seconds, and extension at 72°C for 5 seconds. Included in each run was a positive control containing M genitalium and distilled water as a negative control.

HPV, polyomavirus, and β-globin PCR

For HPV, polyomavirus, and β-globin PCR analyses 5 μL of DNA were used as template in 50-μL PCR mix containing Taq DNA polymerase (Roche, Penzberg, Germany). All PCR mixes were prepared in a DNA-free area and the transferring of PCR template from the first PCR to the nested PCR mixture was done on a separate bench to avoid contamination (only for polyomavirus-specific PCR). To amplify the 268-base pair (bp) long fragment of the β-globin gene, primers PCO4/GH20 were used. The samples were analyzed regarding presence of HPV and polyomavirus (JCV and BKV) DNA. For HPV a 1-step PCR program was used to amplify a sequence corresponding to the late region L1 (6764-6902) in HPV 6 and the equivalent regions in other genital HPV. The primers for polyomaviruses were designed using the primer express program based on the National Center for Biotechnology Information for sequence information. JCV primers amplified a template (2657-2914) in Tag encoding gene, using a nested PCR system. For the first PCR amplification 2 outside primers JCV1/JCV2 were used, yielding a 257-bp long product and the second PCR was performed with 2 inner primers JCV3/JCV4 yielding a 167-bp long product. For BKV PCR, a sequence (1324-1515) in the VP1 gene acted as a template and first PCR amplification was performed with the outer primer pair BKV1/BKV2 yielding a 233-bp long product and second PCR was performed with the 2 inner primers BKV3/BKV4 resulting in an 85-bp long sequence. The PCR products were separated on a 2% agarose gel (Nusieve Agarose; Cambrex Bio Science, Rockland, ME) stained with ethidium bromide (VWR International, LLC, West Chester, PA) and visualized by means of ultraviolet irradiation.

HPV and polyomavirus controls

HPV DNA extracted from SiHa cells (American Type Culture Collection nr HTB-35™ LGC Promochem, Teddington, UK) was used as a positive HPV PCR control and polyoma DNA extracted from JCV- and BKV-infected SVG cells (JCV strain Mad-1, BKV strain ASV enteric genome cloned in pBR322, Lewis AM; Division of Viral Products, Food and Drug Administration, Bethesda, MD), donated by Rosina Girones, Department of Microbiology, University of Barcelona, Barcelona, Spain, was used as a positive polyomavirus-specific PCR control. Autoclaved water was used as a negative PCR control.