**2. Virus and endometriosis**

Pathogenic DNA virus infection has been associated with the aetiology of several human diseases. The double-stranded (ds) DNA viruses include herpesviruses, polyomaviruses, papillomaviruses, hepadnaviruses (e.g. hepatitis B virus (HBV)), adenoviruses and poxviruses. With the exception of poxviruses, these viruses often establish persistent or latent infections and can be reactivated in both healthy and immunosuppressed persons.

Only a few studies have addressed the possible involvement of a pathogenic virus in the aetiology of endometriosis (Oppelt et al., 2010; Vestergaard et al., 2010). The presence of specific human herpes viruses, human polyomaviruses, and human papillomaviruses was analysed. Also, the presence of human endogenous retroviruses have been investigated in endometriosis lesions (Hu et al., 2006; Oppelt et al., 2009), and these studies will be discussed in the following section.

#### **2.1 Herpes viruses**

250 Endometriosis - Basic Concepts and Current Research Trends

MMPs are important for the control of extracellular matrix turnover, and they are believed to influence the implantation of adhered endometriotic cells in endometriosis (Kyama et al., 2003). The level of MMPs is in up-regulated and tissue inhibitors of matrix metalloproteinases (TIMPs) down-regulated in the peritoneal fluid from women with endometriosis compared with controls (Bondza et al., 2009). MMPs are upregulated in response to the inflammatory cytokines TNFα and IL-1 and are expressed during the proliferative and menstrual phases but suppressed by progesterone in the secretory phase. In the absence of a normal progesterone response of the endometriosis lesions, sensitivity to TGFβ may be altered, and this may result in a failure to down-regulate MMPs, thus

Following implantation, the growth and maintenance of endometriosis lesions is influenced by a number of factors (figure 1). First of all, the implanted tissue is in need of vascularization and an increase in the concentration of several angiogenic factors has been reported. IL-6 and IL-8, which as stated above are up-regulated in endometriosis, both promote angiogenesis (Bondza et al., 2009). IL-1β, which is also elevated in the peritoneal fluid of endometriosis patients, induces IL-6, the vascular endothelial growth factor (VEGF), and an angiogenic phenotype in endometriotic but not endometrial stromal cells. Women with moderate and severe endometriosis have elevated levels of both IL-6 and VEGF in the peritoneal fluid compared with healthy controls (Kyama et al., 2003), underlining that angiogenesis is a feature of disease exacerbation. Furthermore, high proliferative activity of endometriosis lesions is associated with increased levels of VEGF and its receptor as well as

A positive feedback of activated immune response contributes further to the maintenance of endometriosis. The reduced NK cell activity has been suggested to result in less effective killing of autologous dendritic cells (DCs) loaded with endometrial self-antigens. This would facilitate self-antigen presentation to autoreactive T cells and the subsequent production of autoantibodies (Kyama et al., 2003), pointing towards a pathogenic contribution by an autoimmune disease mechanism. Indeed, the frequency of autoantibodies towards endometrial antigens is elevated in both serum and peritoneal fluid of women with endometriosis compared with healthy women (Osuga et al., 2011). Activation of Th2 immune response in endometriosis and elevated B cell levels has been reported both systemically and locally in the endometriosis lesions, supposedly causing sustained autoantibody production and continuous infiltration of immune cells, thus

maintaining the immunological contribution to the pathogenesis of endometriosis.

Pathogenic DNA virus infection has been associated with the aetiology of several human diseases. The double-stranded (ds) DNA viruses include herpesviruses, polyomaviruses, papillomaviruses, hepadnaviruses (e.g. hepatitis B virus (HBV)), adenoviruses and poxviruses. With the exception of poxviruses, these viruses often establish persistent or latent infections and can be reactivated in both healthy and immunosuppressed persons.

contributing to the implantation failure.

**1.1.4 Growth and maintenance of endometriosis** 

increased microvessel density (Bondza et al., 2009).

**2. Virus and endometriosis** 

The *Herpesviridae* are a large, diverse family of double-stranded, enveloped viruses. Eight human herpesviruses (HHVs) have been identified: Epstein-Barr virus (EBV, of the subfamily *Gammaherpesvirinae*), cytomegalovirus (CMV or HHV5 of the subfamily *Betaherpesvirinae*), herpes simplex virus type 1 and 2 (HSV-1 and HSV-2 of the subfamily *Alphaherpesvirinae*), varicella zoster (VZV), HHV6, HHV7, and the Karposi's sarcoma herpes virus (KHSV or HHV8). The seroprevalence of each of these herpes viruses varies according to a number of demographic factors (Prober, 2011). For EBV, CMV, and HSV-1 the prevalences range from 50 to 75%, whereas the prevalence for HSV-2 is about 25%. For VZV, HHV6 and HHV7 the seroprevalence is almost 100%, whereas the prevalence for HHV8 is less than 10%. The *Herpesviridae* family are found in many human tissue types, including the fallopian tubes (salpingitis) and the endometrium (see Vestergaard et al., 2010). These viruses are commonly distributed as asymptomatic infectious agents but are also all associated with diseases, such as genital sores or a variety of malignancies. EBV can cause infectious mononucleosis and has been associated with lymphomas of B, T and NK cell origin, but also cancers of epithelial origin like nasopharyngeal carcinomas and gastric adenocarcinomas (Dolcetti & Masucci, 2003). EBV maintains latency in B cells. CMV can cause pneumonitis and delayed neurological complications like sensory neural hearing deficits and learning disabilities (Brown & Abernathy, 1998). CMV maintains latency in monocytes and macrophages. HSV-1 and HSV-2 can cause encephalitis and genital ulcerative disease (Wilson et al., 2009). The HSVs maintain latency in neuronal cells. VSV cause chickenpox (varicella) as primary infection following which it becomes latent in neuronal cells (Kennedy & Cohrs, 2010). VZV can be reactivated and thus cause herpes zoster (shingles) and extremely painful vesicular eruption. HHV6 cause roseola during the primary infection (Prober, 2011). HHV6 becomes latent in salivary glands, the brain and in mononuclear cells or macrophages. Most reactived HHV6 infections are asymptomatic.

The presence of most of the herpes viruses in endometriosis has been analysed., Endometriosis samples from 32 patients were analysed for the presence of EBV, CMV, HSV-1 and HSV-2 by multiplex PCRs (Vestergaard et al., 2010). None of the clinical samples were positive for any of these four herpes viruses. In another study, 66 endometriosis samples from 56 patients were tested for the presence of EBV, CMV, HSV-1, HSV-2, VZV, and HHV6 by a PCR based analysis (Oppelt et al., 2010). Also this study failed to detect the presence of any of these six herpes viruses. Apart from HHV7 and HHV8, which has not yet been analysed, the six herpes viruses EBV, CMV, HSV-1, HSV-2, VZV, and HHV6 do not seem to be involved in the pathology of endometriosis.

Virus Infection and Type I Interferon in Endometriosis 253

estimated that 75% to 80% of sexually active individuals are infected with HPV during their lifetime, with the highest rates in women younger than 25 years of age. However, as the mean age of the women enrolled in this particular study were between 32 and 36 years of age, most HPV infections would have been cleared by the immune system, correlating well with the fact that none of these women had genital HPV infection in their cervical smears at the time of analysis. However, it is possible that transient HPV infections could cause the initiation of malignant-like processes by a "hit-and-run" mechanism and in this way contribute to the initiation of endometriosis. This theory is yet to be investigated further.

Retroviruses are RNA viruses, which integrate into the genome of the host cell in the form of a DNA copy, which is denominated the provirus. Endogenous retroviruses refer to proviruses integrated into germ line cells, which are transmitted from one generation to the next. Approximately 5% of the human genome consists of complete and partial sequences from human endogenous retroviruses (HERVs) (Muir et al., 2004). The significance and consequences of the presence of HERVs in the human genome have been the subject of intensive investigation. HERVs have been implicated in autoimmune diseases and neoplasia as well as in placental function and protection from exogenous retroviral infection. Considering the placental function, it is now believed that syncytin, a retroviral envelope protein encoded by the endogenous retrovirus HERV-W, is involved in the fusion of the cytotrophoblast cells to form the syncytial layer of the placenta, and the envelope protein encoded by ERV3 has been associated with cytotrophoblast differentiation. Other endogenous retroviruses like HERV-E also seem to be involved in placental function

Expression of HERVs in endometriotic tissues has been detected, indicating that endogenous retrovirus expression might be involved in endometriosis (Hu et al., 2006; Oppelt et al., 2009). Endometriosis samples from 14 women were analysed for the presence of HERV-E HERV-W, HERV-I/T, and HERV-H mRNA by PCR analysis (Hu et al., 2006). It was found that HERV-E was expressed at higher levels in the endometriosis samples than in normal endometrium control samples. In another study, 15 endometriosis samples showed low levels of RNA encoding the HERV-W encoded envelope protein syncytin, as analysed by reverse transcriptase PCR (Oppelt et al., 2009). It was also found in this study that the endometriosis samples did not show an elevated expression of the HERV-W encoded envelope protein syncytin. Interestingly though, samples from the endometrium of women with endometriosis showed an increased expression of syncytin RNA compared with endometrial samples from controls, suggesting that this protein might be involved in the pathology of endometriosis. At this point, the involvement of endogenous virus expression in the pathology of endometriosis is not clarified. Further studies are required, analysing larger groups of endometriosis samples and including tissue samples from the

We will now focus on the putative implication of the important immune modulating cytokine family type I interferons in endometriosis, as a line of circumstantial evidence

**2.4 Endogenous retroviruses** 

(reviewed in Muir et al., 2004).

endometrium of women with and without endometriosis.

**3. Type I interferon and endometriosis** 

#### **2.2 Polyomaviruses**

The well-described human polyomaviruses of the *Polyomaviridae* family, JC (JCV), BK (BKV) and simian virus 40 (SV40) are widely distributed in the general population (Moens & Johannessen, 2008). Thus antibodies against JCV and BKV have been found in more than 75% of the human adult population, and up to 15% of healthy humans are seropositive for SV40. These three polyomaviruses have been found in many different tissues (see Vestergaard et al., 2010). BKV has been found in the kidney tubule epithelium, urethral epithelium, the uterine cervix, and in the spleen. JCV has been found in tongue squamous cell epithelium, urethral epithelium, and in the spleen, whereas SV40 has been found in the liver and the mesothelium. Furthermore, these three polyomaviruses induce tumours in animal models and are able to transform cultured human cells (Moens & Johannessen, 2008). In 2007, two new human polyomaviruses, WU polyomavirus (WUV) and KI polyomavirus (KIV) were discovered in respiratory tract secretions and have subsequently been detected in faeces, blood, and lymphoid tissue (see Vestergaard et al., 2010). The full spectrum of their tissue tropism and their role in disease has yet to be elucidated. More recently, yet another human member of the *Polyomaviridae* family, the Merkel cell polyomavirus (MCV) was described in apparent association with Merkel cell carcinoma, an aggressive form of skin cancer (Moens & Johannessen, 2008). When analysed, none of these polyomaviruses were detected either in the endometrium or in endometriosis, suggesting an infrequent presence of polyomaviruses in the endometrium (Vestergaard et al., 2010).

#### **2.3 Human papillomaviruses**

Closely related to the polyomavirus family, the papillomavirus family is a very significant example of the large impact of a broad spectrum of pathogenic DNA viruses, which have emerged during the last two decades. More than 118 papillomaviruses have been fully described, and new virus types are constantly emerging (zur Hausen, 2009). The prevalence of human papillomavirus (HPV, of the family *Papillomaviridae*) in cervical carcinomas is 99.7%. Specific high-risk HPV types have been shown to cause the vast majority of cervical cancers as well as a substantial proportion of other anogenital and head and neck cancers as well as certain cutaneous cancers. HPV has not yet been detected in intraperitoneal tissues, but HPV has been detected in blood, including on the surface of peripheral blood mononuclear cells, suggesting a potential alternative route of transmission (see Vestergaard et al., 2010).

The prevalence of HPV in endometriosis has been analysed (Oppelt et al., 2010; Vestergaard et al., 2010). In one study, no HPV DNA was found in the endometriosis samples from 32 patients (Vestergaard et al., 2010). In another study, certain high-risk HPV types was found in endometriosis lesions (Oppelt et al., 2010). However, due to previous cervical HPV infections in the analysed patients, it was concluded that the detected HPV in the endometriosis samples possibly originated from these associated malignant transformations and might not have any association with the endometriosis *per se*.

In the endometrium, a HPV prevalence of less than 10% was found in samples from both women with endometriosis as well as controls, which is remarkably low compared with the well-known high frequencies of infection with these viruses (Vestergaard et al., 2010). It is estimated that 75% to 80% of sexually active individuals are infected with HPV during their lifetime, with the highest rates in women younger than 25 years of age. However, as the mean age of the women enrolled in this particular study were between 32 and 36 years of age, most HPV infections would have been cleared by the immune system, correlating well with the fact that none of these women had genital HPV infection in their cervical smears at the time of analysis. However, it is possible that transient HPV infections could cause the initiation of malignant-like processes by a "hit-and-run" mechanism and in this way contribute to the initiation of endometriosis. This theory is yet to be investigated further.
