**5. Ectopic pregnancy and chlamydia**

Infectious agents cause damage to the Fallopian tube mucosa either directly or because of the host inflammatory response aimed at clearing the infection. Alteration to the mucosa can result in poor transport of the embryo and subsequent implantation of the blastocyst outside the endometrial lining of the uterine cavity – an ectopic pregnancy. Tubal ectopic pregnancy is a result not only of impaired transport of the embryo causing the embryo to be maintained in the Fallopian tube but also a result from alterations in the tubal environment that allows early implantation to occur (reviewed in Shaw *et al.,* 2010). A recent review also summarises the results of investigations of the Fallopian tube with respect to the roles of of caspase 1, cannabinoid receptor and Dicer 1 knockout mice and how these contribute to tubal dysfunction and contribute to ectopic pregnancies (Shao 2010).

A recent review highlighted the many risk factors for ectopic pregnancy and these are summarised in Table 1.


Table 1. Risk Factors for ectopic preganacy (adapted from Kulp and Barnhart, 2008)

Tubal Damage, Infertility and Tubal Ectopic Pregnancy:

**6. Endometriosis and Fallopian tube function** 

secrete elevated levels of pro-inflammatory cytokines.

the genital tracts (Mol *et al.,* 2010).

Mol *et al.,* 2011).

ectopic pregnancy.

*Chlamydia trachomatis* and Other Microbial Aetiologies 29

due to a decrease in ectopic pregnancy in women over 35 years of age. The report also states that women born between 1985 and 1990 and less than 25 years of age are now at an increased risk of ectopic pregnancy and this rise has not been preceded by a peak incidence of PID (Mol *et al.,* 2010). These authors further conclude that the significant rise in ectopic pregnancies may in fact be related to an increase in positive tests for chlamydial infection of

The management of ectopic pregnancy has recently been addressed (Kulp & Barnhart 2008;

Women with endometriosis have an increased incidence of tubal ectopic pregnancy, suggesting that endometriosis results in impairment of tubal transport of gametes and embryos. Previous *in vitro* studies have revealed that peritoneal fluid collected from women with endometriosis caused a decrease in the Fallopian tube ciliary beat frequency (Lyons *et al.,* 2006), which supports the increased incidence of implantation of the embryo within the Fallopian tubes of these women. The pelvic inflammation associated with endometriosis can also cause adhesion and scar tissue formation within the Fallopian tubes creating a physical obstruction to embryo transport (Halis & Arici 2004). The pathogenesis of endometriosis involves changes both in cellular and in humoral immunity. Impaired natural killer cell activity results in inadequate removal of debris following retrograde menstruation. Elevated primary inflammatory mediators, which are characterised by increased numbers of macrophages, result in the production of secondary inflammatory mediators such as cytokines, chemokines and growth factors (Harada *et al.,* 2001) However, the secretion of primary inflammatory mediators can also be induced by microbial stimuli (Wira *et al.,* 2005). This highlights the relevance of a non-sterile endometrium in this aetiology. It may be that the retrograde menstruation of colonised or contaminated (by microorganisms) menstrual blood enhances the pathology of endometriosis by recruiting macrophages, which then

A small study investigating eutopic and ectopic endometrium, identified DNA with a 96% homology to the Gram-negative bacterium *Shigella* spp. in ectopic but not in eutopic endometrium. Therefore, an infection hypothesis was proposed for the pathogenesis of endometriosis (Kodati *et al.,* 2008). Recently, Khan *et al.,*. (Khan *et al.,* 2010) reported a significant increase in the number of colony forming units of *E*. *coli* recovered from the menstrual blood of women with endometriosis when compared to women without the disease. In their study, the bacterial endotoxin concentration was also higher both in the menstrual blood and in the peritoneal fluid samples from women with endometriosis. The relative level of *E*. *coli* within the peritoneal fluid of women with endometriosis was likely due to retrograde menstruation through the Fallopian tubes and into the pelvic cavity. The 'open' nature of the female genital tract makes it unlikely that secretions from the uterus, Fallopian tubes and peritoneal cavity remain compartmentalised. Transport of microorganisms within the upper genital tract may well be an area requiring further investigation in the pathogenesis of endometriosis and its increased association with tubal

Interestingly, *E*. *coli* has also been cultured from tubo-ovarian abscesses in women with ovarian endometriomas and pelvic endometriosis (Kavoussi *et al.,* 2006; Lin *et al.,* 2010).The fluid-filled ovarian endometrioma may provide an excellent growth medium for

Ectopic pregnancy accounts for up to 11% of all pregnancies and there is serological evidence that links ectopic pregnancies with *C. trachomatis* infection in women (Chow *et al.,* 1990; Swenson & Schachter 1984) It was noted in one study that 19/21 *C. trachomatis* seropositive women with ectopic pregnancies had antibodies to the chlamydial 57kDa antigen and it was suggested that perhaps immune responses to this antigen may be involved in the immunopathogenesis of ectopic pregnancy associated with *C. trachomatis*  infections (Brunham *et al.,* 1992). A recent review has reported that one third of ectopic pregnancies could be attributable to chlamydial infection (Bebear and de Barbeyrac, 2009).

The finding of chlamydial RNA in Fallopian tube biopsy samples collected from women with ectopic pregnancies suggested that viable, metabolically active bacteria were present in the Fallopian tubes of these women (Gerard *et al.,* 1998). Chlamydial DNA has also been detected in the Fallopian tube tissue collected from women at the time of ectopic pregnancy (Barlow *et al.,* 2001; Noguchi *et al.,* 2002). A report to the contrary regarding the detection of chlamydial DNA in fresh tissue from the Fallopian tubes of women with ectopic pregnancy has, however, also been published and suggested that persistent chlamydial infection of Fallopian tubes was rare in ectopic pregnancy (Bjartling *et al.,* 2007). A retrospective study looking at births and ectopic pregnancy in 20, 762 women in Norway has reported that the risk of ectopic pregnancy increased in a dose-dependent manner with the increasing number of prior chlamydial infections (Bakken *et al.,* 2007). In a comprehensive review by the same author it has been reported that relatively low risks of ectopic pregnancy are recorded after a positive *C. trachomatis* diagnosis with, in Sweden for example, a cumulative incidence before the age of 35 years of rates of 2.7% *C. trachomatis*-positive and 2% *C. trachomatis*-negative (Bakken 2008).

In a small study of 14 ectopic pregnancy patients who were serologically-positive for *C. trachomatis*, subjective immunohistochemistry techniques were used to show an increase in the expression of inducible nitric oxide synthase (iNOS) (which is related to inflammation and infection and which can generate nitric oxide) and activin A (a member of the transforming growth factor beta family that has been reported to increase inflammation and repair) in Fallopian tubes from these women (Refaat *et al.,* 2009). It was proposed by these authors that tubal activin A and nitric oxide (NO) could perhaps be involved in microbial– mediated damaging immune response within the Fallopian tubes of *Chlamydia*-infected women and that their pathological expression may lead to ectopic pregnancy development (Refaat *et al.,* 2009). Nitric oxide is similarly proposed as the damaging agent of cells in the uterine tubes of female mice in the murine model of chlamydial genital infection. It is proposed that nitric oxide expressed in macrophages in response to a *C. muridarum* infection in mice could perhaps be the cause of damage to oviduct interstitial cells of Cajal (ICC-OVI) that have been identified as oviduct pacemaker cells critical for egg transport along the Fallopian tubes (Dixon *et al.,* 2010). The expression of iNOS by human Fallopian tubes has recently been reported as being cyclical during different stages of the menstrual cycle and the intensity of expression of iNOS was found to be higher in the Fallopian tubes of 15 women bearing an ectopic pregnancy when compared with pseudo-pregnant women (Al-Azemi *et al.,* 2010). These results suggested that increased iNOS levels in response to a microbial infection could lead to an increased expression of nitric oxide which may in turn affect the contraction of muscles and/or the ciliary beat in Fallopian tubes, ultimately leading to retention of the embryo at this site.

A recent report from The Netherlands has presented the finding that a peak incidence of admissions for PID in 1983 preceded a peak incidence of ectopic pregnancy in 1988 mainly

Ectopic pregnancy accounts for up to 11% of all pregnancies and there is serological evidence that links ectopic pregnancies with *C. trachomatis* infection in women (Chow *et al.,* 1990; Swenson & Schachter 1984) It was noted in one study that 19/21 *C. trachomatis* seropositive women with ectopic pregnancies had antibodies to the chlamydial 57kDa antigen and it was suggested that perhaps immune responses to this antigen may be involved in the immunopathogenesis of ectopic pregnancy associated with *C. trachomatis*  infections (Brunham *et al.,* 1992). A recent review has reported that one third of ectopic pregnancies could be attributable to chlamydial infection (Bebear and de Barbeyrac, 2009). The finding of chlamydial RNA in Fallopian tube biopsy samples collected from women with ectopic pregnancies suggested that viable, metabolically active bacteria were present in the Fallopian tubes of these women (Gerard *et al.,* 1998). Chlamydial DNA has also been detected in the Fallopian tube tissue collected from women at the time of ectopic pregnancy (Barlow *et al.,* 2001; Noguchi *et al.,* 2002). A report to the contrary regarding the detection of chlamydial DNA in fresh tissue from the Fallopian tubes of women with ectopic pregnancy has, however, also been published and suggested that persistent chlamydial infection of Fallopian tubes was rare in ectopic pregnancy (Bjartling *et al.,* 2007). A retrospective study looking at births and ectopic pregnancy in 20, 762 women in Norway has reported that the risk of ectopic pregnancy increased in a dose-dependent manner with the increasing number of prior chlamydial infections (Bakken *et al.,* 2007). In a comprehensive review by the same author it has been reported that relatively low risks of ectopic pregnancy are recorded after a positive *C. trachomatis* diagnosis with, in Sweden for example, a cumulative incidence before the age of 35 years of rates of 2.7% *C. trachomatis*-positive and 2% *C.* 

In a small study of 14 ectopic pregnancy patients who were serologically-positive for *C. trachomatis*, subjective immunohistochemistry techniques were used to show an increase in the expression of inducible nitric oxide synthase (iNOS) (which is related to inflammation and infection and which can generate nitric oxide) and activin A (a member of the transforming growth factor beta family that has been reported to increase inflammation and repair) in Fallopian tubes from these women (Refaat *et al.,* 2009). It was proposed by these authors that tubal activin A and nitric oxide (NO) could perhaps be involved in microbial– mediated damaging immune response within the Fallopian tubes of *Chlamydia*-infected women and that their pathological expression may lead to ectopic pregnancy development (Refaat *et al.,* 2009). Nitric oxide is similarly proposed as the damaging agent of cells in the uterine tubes of female mice in the murine model of chlamydial genital infection. It is proposed that nitric oxide expressed in macrophages in response to a *C. muridarum* infection in mice could perhaps be the cause of damage to oviduct interstitial cells of Cajal (ICC-OVI) that have been identified as oviduct pacemaker cells critical for egg transport along the Fallopian tubes (Dixon *et al.,* 2010). The expression of iNOS by human Fallopian tubes has recently been reported as being cyclical during different stages of the menstrual cycle and the intensity of expression of iNOS was found to be higher in the Fallopian tubes of 15 women bearing an ectopic pregnancy when compared with pseudo-pregnant women (Al-Azemi *et al.,* 2010). These results suggested that increased iNOS levels in response to a microbial infection could lead to an increased expression of nitric oxide which may in turn affect the contraction of muscles and/or the ciliary beat in Fallopian tubes, ultimately

A recent report from The Netherlands has presented the finding that a peak incidence of admissions for PID in 1983 preceded a peak incidence of ectopic pregnancy in 1988 mainly

*trachomatis*-negative (Bakken 2008).

leading to retention of the embryo at this site.

due to a decrease in ectopic pregnancy in women over 35 years of age. The report also states that women born between 1985 and 1990 and less than 25 years of age are now at an increased risk of ectopic pregnancy and this rise has not been preceded by a peak incidence of PID (Mol *et al.,* 2010). These authors further conclude that the significant rise in ectopic pregnancies may in fact be related to an increase in positive tests for chlamydial infection of the genital tracts (Mol *et al.,* 2010).

The management of ectopic pregnancy has recently been addressed (Kulp & Barnhart 2008; Mol *et al.,* 2011).
