**4.2 Upper genital tract infections**

The most frequent method of female upper genital tract infection is by ascension of members of the lower genital tract endogenous microflora, which may first cause disruption to the normal balance such as that seen in cases of bacterial vaginosis or vaginal candidiasis (Population Council, 2003). Following medical intervention, iatrogenic infections may result from the direct inoculation of microorganisms from the lower genital tract into the upper genital tract. Iatrogenic procedures associated with tubal ectopic pregnancy are tubal surgery and trans-vaginal oocyte retrieval for *in vitro* fertilisation (IVF).

Seminal fluid is also reportedly a mechanism of microbial transfer to the female upper genital tract. Furthermore, some microorganisms have the propensity to attach to the surface of spermatozoa, whilst others are obligate intracellular parasites within the spermatozoa *C*. *trachomatis*, *N*. *gonorrhoeae*, *Mycoplasma* spp., *Ureaplasma* spp., and *E*. *coli* have all been shown to adhere to the surface of spermatozoa or form intracellular inclusions within the spermatozoa (Friberg *et al.,* 1987; Hickey *et al.,* 2009; James-Holmquest *et al.,* 1974; Murthy *et al.,* 2009; Sanchez *et al.,* 1989; Wolner-Hanssen & Mardh 1984). Further, female partners of infected men with spermatozoa in their ejaculate had a significantly higher incidence of upper genital tract infection compared to infected men who have been vasectomised (Toth *et al.,* 1984). Interestingly it was recently reported that a significantly higher incidence of sperm-immobilizing antibodies (6.4%) was found in sera collected from 273 infertile women with a past *C. trachomatis* infection compared to that found in women without a past chlamydial infection (1.5%) (Hirano & Hoshino 2010). Thus, it may be that the production of sperm-immobilizing antibodies in infertile women is the result of a past *C*. *trachomatis* infection in these women and this may contribute to their infertility.

#### **4.3 Infections of the female upper genital tract (the endometrium, Fallopian tubes and ovaries)**

It is becoming increasingly accepted that the female upper genital tract is not a sterile site, but likely in fact to be asymptomatically colonised or infected with microorganisms (Horne et al 2008; Wira et al 2005). Endometritis, a persistent inflammation of the endometrial lining, has been reported in up to 19% of women (Farooki 1967). Endometritis is frequently asymptomatic, but similarly to other gynaecological infections, endometritis has been shown to reduce conception rates (Feghali et al 2003; Taylor & Frydman 1996). Excessive

Tubal Damage, Infertility and Tubal Ectopic Pregnancy:

*Chlamydia trachomatis* and Other Microbial Aetiologies 19

addition to causing symptomatic PID, *C. trachomatis* is also associated with subclinical upper genital tract disease in women (Horne *et al.,* 2008). The potentially serious sequelae of cervical infection with *C. trachomatis* can include infertility, ectopic pregnancy, pelvic pain and recurrent PID and these have recently been reviewed (Batteiger *et al.,* 2010; Darville and Hiltke, 2010; Haggerty *et al.,* 2010;). The extent to which disease sequelae eventuate following cervical infection with *C. trachomatis* is probably also significantly linked to natural processes that occur in the reproductive tract and include coitus-related phenomena and cyclical hormonal conditions. A novel paradigm that includes consideration of these and other aspects of reproductive biology particularly when using animal models to investigate potential vaccines for chlamydial genital tract infections in women has recently been proposed (Lyons *et al.,* 2009a). Continued investigations into the mechanisms of *Chlamydia*-induced tissue damage are required to further develop our understanding of the pathogenesis of genital tract disease caused by this organism, and to direct research into

effective ways to control *C. trachomatis* infection, including vaccine development.

In women it has previously been reported that a single chlamydial infection of the genital tract does not result in tubal scarring (Paavonen and Eggert-Kruse, 1999); however, prolonged exposure to *Chlamydia* due to a chronic persistent infection or frequent reinfection has been associated with (1) an autoimmune response to Chlamydial heat shock protein (which shares homology with human heat shock protein) and (2) the chronic inflammation associated with tubal factor infertility (Brunham and Peeling, 1994; Mardh, 2004; Ness *et al.,* 2008). The severity of the inflammatory response to chlamydial infection is enhanced during re-infection, causing inflammation, tissue damage and scarring. Recent studies by Hvid *et al.,* (2007) have concluded that damage to the Fallopian tubes is disproportional to the number of *C*. *trachomatis* infected cells, suggesting that Fallopian tube cell lysis does not occur as a direct result of infection. In their study, they instead

Based on the results of epidemiological studies, prospective data from studies of infertile women and on results from animal models, *C*. *trachomatis* infection of the female reproductive tract is known to be causally associated with tubal infertility. In the murine model, a primary chlamydial infection is sufficient to induce tubal damage and infertility (Swenson *et al.,* 1983) with Toll-like receptor 2 being identified as essential for oviduct pathology in this model (Darville *et al.,* 2003; Phillips *et al.,* 1984;). Derbigney and colleagues (2007) reported that *C*. *muridarum* infection of murine oviduct epithelial cell lines induced a beta-interferon response and implicated Toll-like receptor 3 as the source of this interferon. In female guinea pigs, long-term tissue damage was also caused following the host response to a primary chlamydial infection (Rank and Sanders, 1992), with chlamydial salpingitis also reported in female guinea pigs receiving oral contraceptives (Barron *et al.,* 1988). By contrast, in macaque monkeys a single upper genital tract infection with *Chlamydia* is usually selflimiting with tubal scarring only resulting from repeated episodes of salpingitis (Patton *et al.,* 1987; VanVoorhis *et al.,* 1997). It has been shown that *C. trachomatis* infection in monkeys induced delayed hypersensitivity, which is proposed to be the pathogenic mechanism of

There have been reports of serologic evidence of past chlamydial infections (Ness and Brooks-Nelson, 1999; Patton *et al.,* 1994b; Robertson *et al.,* 1987) in women with tubal infertility, and it has been reported that interleukin-1 (IL-1) initiates Fallopian tube

**4.4.1 Chlamydia, tubal pathology and tubal factor infertility** 

demonstrated that IL-1 had a toxic effect on ciliated Fallopian tube cells.

tubal damage in this species (Patton *et al.,* 1994).

inflammation in the endometrium at the time of implantation may be a cause of infertility. Endometritis represents an early stage in the continuum from lower genital tract infection through to salpingitis, the most serious form of female genital tract infection with respect to fertility.

Endometritis is a polymicrobial infection caused by the ascension of endogenous microorganisms or sexually transmitted infections. Endometritis is frequently reported in association with an altered lower genital tract microflora, such as that seen in women with bacteria vaginosis (Hillier et al., 1992; (Jacobsson et al 2002) or PID (Centres for Disease Control and Prevention, 2002). Alterations of the lower genital tract microbial milieu are not the only cause of endometiritis as this infection has been reported in women with 'normal' levels of lower genital tract microorganisms (Lucisano et al 1992). Microorganisms can also be introduced into the endometrium iatrogenically, during gynaecological investigations and treatment (Kiviat et al 1990).

PID results from ascension of microorganisms from the vagina to the upper genital tract (Holmes, 1984) causing post-infectious inflammation with potentially long-term sequelae including tubal infertility, ectopic pregnancy and pelvic pain (Cherpes *et al.,* 2006). Reportedly, up to 20% of women will be rendered infertile following a single diagnosis of PID (Westrom *et al.,* 1992) increasing to 50% following multiple episodes (Westrom *et al.,* 1980). In addition, women with a history of PID were twice as likely to have experienced an ectopic pregnancy when compared to women without any history of upper genital tract infection (Miller *et al.,* 1999). Similar to bacterial vaginosis, PID is also frequently polymicrobial. Opportunistic pathogens comprising anaerobic and facultative aerobic bacteria from the normal microflora of the lower genital tract, or species implicated in genital tract infections cause up to 50 % of PID, not the sexually transmitted bacteria *C*. *trachomatis* or *N*. *gonorrhoeae* (Soper, 2010).

Salpingitis is an infection in the Fallopian tube(s). A polymicrobial microflora has been reported for Fallopian tube tissue from women with salpingitis (Eschenbach *et al.,* 1975; Soper, 1994). Tubo-ovarian abscesses represent an extension of salpingitis and reportedly occur in up to 16% of women diagnosed with salpingitis.

Tubo-ovarian abscesses are usually complications of PID and represent inflammation of both the Fallopian tubes and the ovaries (Landers & Sweet 1983). The microbial aetiology of tubo-ovarian abscesses is predominantly polymicrobial (Landers & Sweet 1983; Wiesenfeld & Sweet 1993). Microbial invasion of the Fallopian tube(s) initiates an inflammatory response which results in oedema, increased pressure and restricted blood supply to the affected Fallopian tube(s) causing abscess formation and survival of the pathogens in a 'protected' environment (Osborne, 1986).

Numerous sexually transmitted and non-sexually transmitted pathogens have been isolated from infected upper genital tract tissues. Bacterial vaginosis associated bacteria have been detected independently of *C*. *trachomatis* and/or *N*. *gonorrhoeae* suggesting that investigations regarding causes of upper genital tract infection, but more specifically in the context of tubal ectopic pregnancy, and tubal damage, should focus on a diverse range of microorganisms.

#### **4.4** *Chlamydia trachomatis*

*C*. *trachomatis* is an important pathogen in the aetiology of acute PID and has been isolated from the upper genital tracts of approximately one quarter of patients with this disease. In

inflammation in the endometrium at the time of implantation may be a cause of infertility. Endometritis represents an early stage in the continuum from lower genital tract infection through to salpingitis, the most serious form of female genital tract infection with respect to

Endometritis is a polymicrobial infection caused by the ascension of endogenous microorganisms or sexually transmitted infections. Endometritis is frequently reported in association with an altered lower genital tract microflora, such as that seen in women with bacteria vaginosis (Hillier et al., 1992; (Jacobsson et al 2002) or PID (Centres for Disease Control and Prevention, 2002). Alterations of the lower genital tract microbial milieu are not the only cause of endometiritis as this infection has been reported in women with 'normal' levels of lower genital tract microorganisms (Lucisano et al 1992). Microorganisms can also be introduced into the endometrium iatrogenically, during gynaecological investigations

PID results from ascension of microorganisms from the vagina to the upper genital tract (Holmes, 1984) causing post-infectious inflammation with potentially long-term sequelae including tubal infertility, ectopic pregnancy and pelvic pain (Cherpes *et al.,* 2006). Reportedly, up to 20% of women will be rendered infertile following a single diagnosis of PID (Westrom *et al.,* 1992) increasing to 50% following multiple episodes (Westrom *et al.,* 1980). In addition, women with a history of PID were twice as likely to have experienced an ectopic pregnancy when compared to women without any history of upper genital tract infection (Miller *et al.,* 1999). Similar to bacterial vaginosis, PID is also frequently polymicrobial. Opportunistic pathogens comprising anaerobic and facultative aerobic bacteria from the normal microflora of the lower genital tract, or species implicated in genital tract infections cause up to 50 % of PID, not the sexually transmitted bacteria *C*.

Salpingitis is an infection in the Fallopian tube(s). A polymicrobial microflora has been reported for Fallopian tube tissue from women with salpingitis (Eschenbach *et al.,* 1975; Soper, 1994). Tubo-ovarian abscesses represent an extension of salpingitis and reportedly

Tubo-ovarian abscesses are usually complications of PID and represent inflammation of both the Fallopian tubes and the ovaries (Landers & Sweet 1983). The microbial aetiology of tubo-ovarian abscesses is predominantly polymicrobial (Landers & Sweet 1983; Wiesenfeld & Sweet 1993). Microbial invasion of the Fallopian tube(s) initiates an inflammatory response which results in oedema, increased pressure and restricted blood supply to the affected Fallopian tube(s) causing abscess formation and survival of the pathogens in a

Numerous sexually transmitted and non-sexually transmitted pathogens have been isolated from infected upper genital tract tissues. Bacterial vaginosis associated bacteria have been detected independently of *C*. *trachomatis* and/or *N*. *gonorrhoeae* suggesting that investigations regarding causes of upper genital tract infection, but more specifically in the context of tubal ectopic pregnancy, and tubal damage, should focus on a diverse range of

*C*. *trachomatis* is an important pathogen in the aetiology of acute PID and has been isolated from the upper genital tracts of approximately one quarter of patients with this disease. In

fertility.

and treatment (Kiviat et al 1990).

*trachomatis* or *N*. *gonorrhoeae* (Soper, 2010).

'protected' environment (Osborne, 1986).

microorganisms.

**4.4** *Chlamydia trachomatis*

occur in up to 16% of women diagnosed with salpingitis.

addition to causing symptomatic PID, *C. trachomatis* is also associated with subclinical upper genital tract disease in women (Horne *et al.,* 2008). The potentially serious sequelae of cervical infection with *C. trachomatis* can include infertility, ectopic pregnancy, pelvic pain and recurrent PID and these have recently been reviewed (Batteiger *et al.,* 2010; Darville and Hiltke, 2010; Haggerty *et al.,* 2010;). The extent to which disease sequelae eventuate following cervical infection with *C. trachomatis* is probably also significantly linked to natural processes that occur in the reproductive tract and include coitus-related phenomena and cyclical hormonal conditions. A novel paradigm that includes consideration of these and other aspects of reproductive biology particularly when using animal models to investigate potential vaccines for chlamydial genital tract infections in women has recently been proposed (Lyons *et al.,* 2009a). Continued investigations into the mechanisms of *Chlamydia*-induced tissue damage are required to further develop our understanding of the pathogenesis of genital tract disease caused by this organism, and to direct research into effective ways to control *C. trachomatis* infection, including vaccine development.

#### **4.4.1 Chlamydia, tubal pathology and tubal factor infertility**

In women it has previously been reported that a single chlamydial infection of the genital tract does not result in tubal scarring (Paavonen and Eggert-Kruse, 1999); however, prolonged exposure to *Chlamydia* due to a chronic persistent infection or frequent reinfection has been associated with (1) an autoimmune response to Chlamydial heat shock protein (which shares homology with human heat shock protein) and (2) the chronic inflammation associated with tubal factor infertility (Brunham and Peeling, 1994; Mardh, 2004; Ness *et al.,* 2008). The severity of the inflammatory response to chlamydial infection is enhanced during re-infection, causing inflammation, tissue damage and scarring. Recent studies by Hvid *et al.,* (2007) have concluded that damage to the Fallopian tubes is disproportional to the number of *C*. *trachomatis* infected cells, suggesting that Fallopian tube cell lysis does not occur as a direct result of infection. In their study, they instead demonstrated that IL-1 had a toxic effect on ciliated Fallopian tube cells.

Based on the results of epidemiological studies, prospective data from studies of infertile women and on results from animal models, *C*. *trachomatis* infection of the female reproductive tract is known to be causally associated with tubal infertility. In the murine model, a primary chlamydial infection is sufficient to induce tubal damage and infertility (Swenson *et al.,* 1983) with Toll-like receptor 2 being identified as essential for oviduct pathology in this model (Darville *et al.,* 2003; Phillips *et al.,* 1984;). Derbigney and colleagues (2007) reported that *C*. *muridarum* infection of murine oviduct epithelial cell lines induced a beta-interferon response and implicated Toll-like receptor 3 as the source of this interferon. In female guinea pigs, long-term tissue damage was also caused following the host response to a primary chlamydial infection (Rank and Sanders, 1992), with chlamydial salpingitis also reported in female guinea pigs receiving oral contraceptives (Barron *et al.,* 1988). By contrast, in macaque monkeys a single upper genital tract infection with *Chlamydia* is usually selflimiting with tubal scarring only resulting from repeated episodes of salpingitis (Patton *et al.,* 1987; VanVoorhis *et al.,* 1997). It has been shown that *C. trachomatis* infection in monkeys induced delayed hypersensitivity, which is proposed to be the pathogenic mechanism of tubal damage in this species (Patton *et al.,* 1994).

There have been reports of serologic evidence of past chlamydial infections (Ness and Brooks-Nelson, 1999; Patton *et al.,* 1994b; Robertson *et al.,* 1987) in women with tubal infertility, and it has been reported that interleukin-1 (IL-1) initiates Fallopian tube

Tubal Damage, Infertility and Tubal Ectopic Pregnancy:

pathogen and hence fertility outcomes in these women.

chronic pain, recurrent episodes of PID and ectopic pregnancy.

**4.4.3 Chlamydia screening** 

*Chlamydia trachomatis* and Other Microbial Aetiologies 21

74.1% of women without embryo (s) and in 69.5% of women with tubal occlusion (Jakus *et al.,* 2008). In the PID evaluation and clinical health (PEACH) study 443 women with clinical signs of mild to moderate PID were followed for 84 months and assessed for long-term sequelae of chlamydial infections of the genital tract including PID recurrence and time to pregnancy (Ness *et al.,* 2008). It was found that IgG antibody responses to CHSP60 and elementary bodies (the extracellular form) of *C. trachomatis* serovar D were independently associated with reduced pregnancy rates and increased rates of recurrent PID (Ness *et al.,* 2008). In another study of 72 female patients a significant seropositivity to CHSP60 antibodies was detected in patients with secondary infertility from an infertile cohort clinically characterised primary and/or secondary infertility. This indicated that specific antibodies to CHSP60 may aid in the early prognosis of immunopathologic sequelae following genital tract infections with *C. trachomatis* (Dutta *et al.,* 2008). The 10kDa chlamydial heat shock protein 10 (CHSP10) has also been identified as a target of cellmediated responses in human chlamydial infections. Women with tubal infertility have been shown to recognise CHSP10 more frequently than those women with current active chlamydial infections. Co-expression of both CHSP60 and CHSP10 were subsequently detected at higher levels in the infertile women compared to the fertile women (Jha *et al.,* 2009). CHSP10 and CHSP60 stimulation was reported to increase the cytokine responses of IFN-γ and IL-10 in *Chlamydia*-positive infertile women (Srivastava *et al.,* 2008). Srivastava *et al.,.* (2008) suggested that this could significantly affect the release of these cytokines from the cervical mononuclear cells, thus affecting the mucosal immune function against this

Linhares and Witkin (2010) recently reviewed the immunopathogenic consequences of CHSP60 expression in the female genital tract and they reported that scar formation and tubal occlusion resulted from the induction of pro-inflammatory immune responses following the release of CHSP60 from a *C. trachomatis* infection. They further reported that the production of CHSP60 cross-reacting antibodies and cell-mediated immunity to the human HSP60 was detrimental to subsequent pregnancy outcome in women infected in the

Screening for, and treatment of, chlamydial infection is aimed at reducing chlamydial transmission and preventing PID and the long-term sequelae of PID including infertility,

For the host to successfully clear infections of the female genital tract caused by *C. trachomatis* an adequate immune response is required following recognition of the pathogen by pattern recognition receptors (PRRs) of the Toll-like receptor (TLR) and nucleotide binding oligomerization domain (NOD) families. If functioning correctly, the host immune response clears the infection but in some females the infection is not cleared and this allows for a persistent infection to manifest in these hosts. Since most infections with *Chlamydia*  remain asymptomatic it is difficult to ascertain the risk of potential disease sequelae associated with previous chlamydial infections. There are several methods used to assess the risk of chlamydial infections in women that may lead to tubal factor sub-fertility and these have been reviewed (den Hartog et al., 2006). In particular it has been found that testing for anti-chlamydial IgG antibody in serum can indicate a previous infection but cannot predict a persistent infection. It has been noted that screening for serological markers of persistence

upper reproductive tract with this microbial pathogen (Linhares and Witkin 2010).

destruction following a *C. trachomatis* infection (Hvid *et al.,* 2007). In a retrospective study of 84 infertile women with tubal occlusion, the sera collected from 28% of these women were positive for chlamydial anti-IgG antibody, compared to only 11% positivity to the chlamydial anti-IgG antibody in sera collected from 253 infertile controls (Merki-Feld *et al.,* 2007). A study of 114 women with laparoscopically-verified tubal factor infertility (of which 96 cases showed evidence of past infection with *Chlamydia*) was undertaken to further elucidate the mechanisms of tubal damage in women with *Chlamydia*-associated infertility (Ohman *et al.,* 2009). The functional polymorphisms in selected cytokine genes [including IL-10, interferon gamma (IFN-γ), tumour necrosis factor alpha (TNF-α] revealed an increase in severe tubal damage in women with infertility caused by *Chlamydia* when certain IL-10 and TNF-α alleles were present (Ohman *et al.,* 2009). In terms of cytokine secretions in *Chlamydia*-positive infertile women, it has been reported that *Chlamydia*-stimulated cervical cells secreted significantly higher levels of IL-1ß, IL-6, IL-8 and IL-10. This indicated that the cytokine secretion profile of cervical cells may produce vital information to indicate the outcome (i.e. fertile or infertile) of a chlamydial infection of the female genital tract (Agrawal *et al.,* 2009). Others have reported that IL-1β, IL-4, IL-5 and IL-6 as well as IL-10 levels were found to be higher in *Chlamydia* membrane protein (Inc protein)-stimulated cervical cells of *C. trachomatis*-positive infertile women compared to fertile women infected with *Chlamydia* (Gupta *et al.,* 2009). More recently a unique link between elevated levels of anti-Chlamydial caseinolytic protease P (ClpP) and tubal factor infertility was identified in 21 tubal factor infertility patients (Rodgers *et al.,* 2010).

Host genetic factors are known to modulate the immune defence mechanisms to a *Chlamydia*  infection thus determining the occurrence of *Chlamydia*-induced tubal factor infertility. A study by Morre and colleagues (2002) reported that almost 45% of women infected with genital chlamydial infections cleared the infection after one year with no interventional treatments. However, some authors have claimed that this study by Morre *et al.,.* (2002) was methodologically flawed (Risser and Risser, 2007; Simms and Horner, 2008). An increased risk of tubal pathology (as a result of aberrant immune responses) has been reported in 227 sub-fertile women following a *C. trachomatis* infection and carrying two or more singlenucleotide polymorphisms (SNPs) in genes (toll-like receptor (TLR)-9, TLR-4, CD14, and caspase recruitment domain protein 15 (CARD15)/nucleotide-binding oligomerization domain containing 2(NOD2) that encode pattern recognition receptors (PRRs) involved in sensing bacterial components (den Hartog *et al.,* 2006). In a more recent report that investigated 214 infertile women, 42 of whom had tubal pathology, it was found that polymorphisms in the major histocompatibility complex class I chain related A gene (specifically allele 008) correlated with *C. trachomatis* anti-IgG antibodies in infertilewomen (Mei *et al.,* 2009).

#### **4.4.2 Chlamydia and PID**

PID is caused by infection of the female genital tract with microorganisms including *C*. *trachomatis* (Bakken and Ghaderi, 2009) and testing for serum antibody to the chlamydial 60kDa Heat shock protein(i.e**.**CHSP60 antibody) is an accurate means for predicting *Chlamydia*-associated tubal factor infertility (Claman *et al.,* 1997)**.** A prospective study into serologic parameters of tubal disease reported that antibodies to CHSP60 were also predictive for lower spontaneous conception and pregnancy outcome after a first episode of ectopic pregnancy (Sziller *et al.,* 2008). A retrospective study of follicular fluid from 253 IVF patients for IgG antibodies to CHSP60 reported that antibodies to CHSP60 were found in

destruction following a *C. trachomatis* infection (Hvid *et al.,* 2007). In a retrospective study of 84 infertile women with tubal occlusion, the sera collected from 28% of these women were positive for chlamydial anti-IgG antibody, compared to only 11% positivity to the chlamydial anti-IgG antibody in sera collected from 253 infertile controls (Merki-Feld *et al.,* 2007). A study of 114 women with laparoscopically-verified tubal factor infertility (of which 96 cases showed evidence of past infection with *Chlamydia*) was undertaken to further elucidate the mechanisms of tubal damage in women with *Chlamydia*-associated infertility (Ohman *et al.,* 2009). The functional polymorphisms in selected cytokine genes [including IL-10, interferon gamma (IFN-γ), tumour necrosis factor alpha (TNF-α] revealed an increase in severe tubal damage in women with infertility caused by *Chlamydia* when certain IL-10 and TNF-α alleles were present (Ohman *et al.,* 2009). In terms of cytokine secretions in *Chlamydia*-positive infertile women, it has been reported that *Chlamydia*-stimulated cervical cells secreted significantly higher levels of IL-1ß, IL-6, IL-8 and IL-10. This indicated that the cytokine secretion profile of cervical cells may produce vital information to indicate the outcome (i.e. fertile or infertile) of a chlamydial infection of the female genital tract (Agrawal *et al.,* 2009). Others have reported that IL-1β, IL-4, IL-5 and IL-6 as well as IL-10 levels were found to be higher in *Chlamydia* membrane protein (Inc protein)-stimulated cervical cells of *C. trachomatis*-positive infertile women compared to fertile women infected with *Chlamydia* (Gupta *et al.,* 2009). More recently a unique link between elevated levels of anti-Chlamydial caseinolytic protease P (ClpP) and tubal factor infertility was identified in 21 tubal factor

Host genetic factors are known to modulate the immune defence mechanisms to a *Chlamydia*  infection thus determining the occurrence of *Chlamydia*-induced tubal factor infertility. A study by Morre and colleagues (2002) reported that almost 45% of women infected with genital chlamydial infections cleared the infection after one year with no interventional treatments. However, some authors have claimed that this study by Morre *et al.,.* (2002) was methodologically flawed (Risser and Risser, 2007; Simms and Horner, 2008). An increased risk of tubal pathology (as a result of aberrant immune responses) has been reported in 227 sub-fertile women following a *C. trachomatis* infection and carrying two or more singlenucleotide polymorphisms (SNPs) in genes (toll-like receptor (TLR)-9, TLR-4, CD14, and caspase recruitment domain protein 15 (CARD15)/nucleotide-binding oligomerization domain containing 2(NOD2) that encode pattern recognition receptors (PRRs) involved in sensing bacterial components (den Hartog *et al.,* 2006). In a more recent report that investigated 214 infertile women, 42 of whom had tubal pathology, it was found that polymorphisms in the major histocompatibility complex class I chain related A gene (specifically allele 008) correlated with *C. trachomatis* anti-IgG antibodies in infertilewomen

PID is caused by infection of the female genital tract with microorganisms including *C*. *trachomatis* (Bakken and Ghaderi, 2009) and testing for serum antibody to the chlamydial 60kDa Heat shock protein(i.e**.**CHSP60 antibody) is an accurate means for predicting *Chlamydia*-associated tubal factor infertility (Claman *et al.,* 1997)**.** A prospective study into serologic parameters of tubal disease reported that antibodies to CHSP60 were also predictive for lower spontaneous conception and pregnancy outcome after a first episode of ectopic pregnancy (Sziller *et al.,* 2008). A retrospective study of follicular fluid from 253 IVF patients for IgG antibodies to CHSP60 reported that antibodies to CHSP60 were found in

infertility patients (Rodgers *et al.,* 2010).

(Mei *et al.,* 2009).

**4.4.2 Chlamydia and PID** 

74.1% of women without embryo (s) and in 69.5% of women with tubal occlusion (Jakus *et al.,* 2008). In the PID evaluation and clinical health (PEACH) study 443 women with clinical signs of mild to moderate PID were followed for 84 months and assessed for long-term sequelae of chlamydial infections of the genital tract including PID recurrence and time to pregnancy (Ness *et al.,* 2008). It was found that IgG antibody responses to CHSP60 and elementary bodies (the extracellular form) of *C. trachomatis* serovar D were independently associated with reduced pregnancy rates and increased rates of recurrent PID (Ness *et al.,* 2008). In another study of 72 female patients a significant seropositivity to CHSP60 antibodies was detected in patients with secondary infertility from an infertile cohort clinically characterised primary and/or secondary infertility. This indicated that specific antibodies to CHSP60 may aid in the early prognosis of immunopathologic sequelae following genital tract infections with *C. trachomatis* (Dutta *et al.,* 2008). The 10kDa chlamydial heat shock protein 10 (CHSP10) has also been identified as a target of cellmediated responses in human chlamydial infections. Women with tubal infertility have been shown to recognise CHSP10 more frequently than those women with current active chlamydial infections. Co-expression of both CHSP60 and CHSP10 were subsequently detected at higher levels in the infertile women compared to the fertile women (Jha *et al.,* 2009). CHSP10 and CHSP60 stimulation was reported to increase the cytokine responses of IFN-γ and IL-10 in *Chlamydia*-positive infertile women (Srivastava *et al.,* 2008). Srivastava *et al.,.* (2008) suggested that this could significantly affect the release of these cytokines from the cervical mononuclear cells, thus affecting the mucosal immune function against this pathogen and hence fertility outcomes in these women.

Linhares and Witkin (2010) recently reviewed the immunopathogenic consequences of CHSP60 expression in the female genital tract and they reported that scar formation and tubal occlusion resulted from the induction of pro-inflammatory immune responses following the release of CHSP60 from a *C. trachomatis* infection. They further reported that the production of CHSP60 cross-reacting antibodies and cell-mediated immunity to the human HSP60 was detrimental to subsequent pregnancy outcome in women infected in the upper reproductive tract with this microbial pathogen (Linhares and Witkin 2010).

#### **4.4.3 Chlamydia screening**

Screening for, and treatment of, chlamydial infection is aimed at reducing chlamydial transmission and preventing PID and the long-term sequelae of PID including infertility, chronic pain, recurrent episodes of PID and ectopic pregnancy.

For the host to successfully clear infections of the female genital tract caused by *C. trachomatis* an adequate immune response is required following recognition of the pathogen by pattern recognition receptors (PRRs) of the Toll-like receptor (TLR) and nucleotide binding oligomerization domain (NOD) families. If functioning correctly, the host immune response clears the infection but in some females the infection is not cleared and this allows for a persistent infection to manifest in these hosts. Since most infections with *Chlamydia*  remain asymptomatic it is difficult to ascertain the risk of potential disease sequelae associated with previous chlamydial infections. There are several methods used to assess the risk of chlamydial infections in women that may lead to tubal factor sub-fertility and these have been reviewed (den Hartog et al., 2006). In particular it has been found that testing for anti-chlamydial IgG antibody in serum can indicate a previous infection but cannot predict a persistent infection. It has been noted that screening for serological markers of persistence

Tubal Damage, Infertility and Tubal Ectopic Pregnancy:

health in the community (Kalwij et al., 2010).

**4.4.4 Chlamydia and vaccines** 

(Oakeshott et al., 2010).

*Chlamydia trachomatis* and Other Microbial Aetiologies 23

pregnancy EP, infertility diagnoses, IVF treatment and births in women. Results showed that no differences were found between the intervention group and the control groups of women for PID, ectopic pregnancy, infertility, IVF treatment and births. It was concluded that a populationbased offer to be tested for urogenital *C. trachomatis* infection using non-invasive samples and DNA amplification testing did not reduce the long-term risk of reproductive complications such as PID and ectopic pregnancies in asymptomatic women (Anderson *et al.,* 2011). This finding agrees with the conclusions made by authors of an earlier review of 12 databases who reported (from the one study that satisfied inclusion criteria for their review) the absence of valid evidence on the risk of tubal factor infertility following an infection of the genital tract with *C. trachomatis* (Wallace *et al.,* 2008) and is also in agreement with the finding of Oakeshott and colleagues

Of note from the Oakeshott study was that a screening intervention at 12 months would not have prevented the 10 reported cases of chlamydia-positive PID among women who were *Chlamydia*-negative at baseline and serves to highlight the ongoing transmission of *Chlamydia* as the elemental problem. The results of the POPI trial suggested that current levels of chlamydial screening are unlikely to have much impact on the overall incidence of PID (Low & Hocking 2010). A recent modelling study based on a comprehensive literature survey on the epidemiology of chlamydial infection and risk-estimates of its late complications has concluded that the risk of developing tubal infertility after a *Chlamydia*  lower genital tract is low (at around 4.6%), and these authors stated that high quality RCTs investigating the transition from cervicitis to tubal infertility are needed (Land et al., 2010). It has also been reported in a prospective study evaluating the sensitivity of multiple-site swab testing (cervix, urethra, vagina and Fallopian tubes) in 2,020 fertility patients over 12 months that multiple site sampling does not increase the detection rate of *C. trachomatis*  among infertile women and in fact that routine DNA testing for *C. trachomatis* should be confined to cervical sampling (Dietrich et al., 2010). A review from the National *Chlamydia* screening programme in London has highlighted the need for *Chlamydia* testing to be offered routinely to young people (under 25 years) as part of an overall approach to sexual

It has been noted recently that no studies have yet published results of the effects of greater than one round of screening or indeed screening for repeat *Chlamydia* infections on reproductive sequelae in women following asymptomatic *C. trachomatis* genital infection (Gottlieb *et al.,* 2010). However two new trials the *Chlamydia* Screening Implementation (CSI) Project (van den Broek *et al.,* 2010) and the Australian *Chlamydia* Control Effectiveness Pilot (ACCEPt) trial (Hocking *et al.,* 2008) are currently underway investigating multiple screening rounds and using *Chlamydia* prevalence (and not PID) as the end point. These trials should provide more conclusive information regarding the effectiveness of chlamydial

Fertility in women is overwhelmingly affected by unresolved or untreated infection of the female reproductive tract with *C. trachomatis*. Since greater than 70% of chlamydial genital infections in women are asymptomatic and sequelae of infection manifest as diseases resulting from severe pathological consequences such as tubal occlusion, a vaccine is likely to be imperative to control infections caused by this sexually transmitted mucosal pathogen. Many animal models of infection-induced immunity including murine and guinea pig

screening to control morbidity associated with genital chlamydial infection.

(including C-reactive protein) seems useful for identifying infected women at highest risk of tubal pathology. It has been proposed that three screening strategies would be useful for identifying tubal factor sub-fertility in women infected in the genital tract with *C. trachomatis*: (1) *C. trachomatis* IgG antibody testing, (2) high sensitivity CRP testing and (3) hysterosalpingography (den Hartog, 2008). A recent mathematical modelling study has analysed previously published data on the persistence of asymptomatic *C. trachomatis*  infection in women, and has estimated the mean duration of the asymptomatic period to be longer (433 days) than previously anticipated. These authors conclude that their study shows that a longer duration of the asymptomatic period results in a more pronounced impact of a screening programme (Althaus *et al.,* 2010).

The incidence of PID in untreated women infected with *C*. *trachomatis* has been reviewed and widely discussed in the literature in terms of (1) its cost-effectiveness as a screening program and (2) as a predictor of tubal damage in infertile patients (Aghaizu *et al.,* 2008; Althaus et al., 2010; Bakken & Ghaderi 2009; den Hartog *et al.,* 2008; Dietrich *et al.,* 2010; Kalwij *et al.,* 2010; Land *et al.,* 2010; Low *et al.,* 2009; Low & Hocking 2010; Oakeshott *et al.,* 2010; Risser & Risser 2007; Simms & Horner 2008). In a comprehensive study that evaluated all available original research and assessed the incidence of PID following *C. trachomatis* infection, it was concluded that no study could adequately answer the question and that many studies either had inaccuracies, validition problems or only indirect evidence to support their reported incidences (Risser and Risser, 2007). A similar review of the literature was undertaken by Simms and Horner (2008) who stated that a reasonable estimate of PID incidence in untreated women after *C. trachomatis* infection was likely to be in the range of 10-20%. A Norwegian registry-linkage study of 24,947 women who were tested for *C. trachomatis* infection reported a correlation between diagnosed *Chlamydia* infection and subsequent PID. The incidence rate of PID in this study was found to be higher in women with prior *C. trachomatis* infection than among women with negative *C. trachomatis* tests although the rates were notably low in both groups (Bakken & Ghaderi 2009). It has therefore been suggested that the benefits of current *Chlamydia* screening programmes may have been overestimated (Low *et al.,* 2006).

A comprehensive review of seven electronic databases covering 17 years of register-based reports (until 2007) and opportunistic screening programmes for *Chlamydia* found that there was no evidence to support the most commonly recommended approach of opportunistic *Chlamydia* screening in a general population younger than 25 years. Furthermore, it was proposed by these authors that an effective approach when assessing biological outcomes of chlamydial infection currently reuqires multiple rounds of screening in randomized control trials (RCT) (Low *et al.,* 2009). A recent RCT (the POPI-prevention of pelvic infection-trial) was undertaken to determine whether a single screening test and treating a subset of 2529 women for chlamydial infection can in fact reduce the incidence of PID over a 12-month period (Oakeshott et al., 2010). The baseline prevalence of *Chlamydia* was 5.4% in the screened population of 2529 sexually active female students (mean age 20.9 years) and 5.9% in (deferred screening) controls with the incidences of PID found to be 1.3% and 1.9% respectively in these cohorts. It was reported that after 12 months, most episodes of PID occurred in women who tested negative for *Chlamydia* at baseline (9.5%) when compared to the intervention group (1.6%) and these authors concluded that the effectiveness of a single *Chlamydia* test in preventing PID over 12 months may also have been overestimated (Oakeshott et al., 2010).

A Danish randomised trial was conducted with 9-year follow-up testing of 4000 asymptomatic women for the presence of urogenital *Chlamydia trachomatis*. Data were collected on PID, ectopic

(including C-reactive protein) seems useful for identifying infected women at highest risk of tubal pathology. It has been proposed that three screening strategies would be useful for identifying tubal factor sub-fertility in women infected in the genital tract with *C. trachomatis*: (1) *C. trachomatis* IgG antibody testing, (2) high sensitivity CRP testing and (3) hysterosalpingography (den Hartog, 2008). A recent mathematical modelling study has analysed previously published data on the persistence of asymptomatic *C. trachomatis*  infection in women, and has estimated the mean duration of the asymptomatic period to be longer (433 days) than previously anticipated. These authors conclude that their study shows that a longer duration of the asymptomatic period results in a more pronounced

The incidence of PID in untreated women infected with *C*. *trachomatis* has been reviewed and widely discussed in the literature in terms of (1) its cost-effectiveness as a screening program and (2) as a predictor of tubal damage in infertile patients (Aghaizu *et al.,* 2008; Althaus et al., 2010; Bakken & Ghaderi 2009; den Hartog *et al.,* 2008; Dietrich *et al.,* 2010; Kalwij *et al.,* 2010; Land *et al.,* 2010; Low *et al.,* 2009; Low & Hocking 2010; Oakeshott *et al.,* 2010; Risser & Risser 2007; Simms & Horner 2008). In a comprehensive study that evaluated all available original research and assessed the incidence of PID following *C. trachomatis* infection, it was concluded that no study could adequately answer the question and that many studies either had inaccuracies, validition problems or only indirect evidence to support their reported incidences (Risser and Risser, 2007). A similar review of the literature was undertaken by Simms and Horner (2008) who stated that a reasonable estimate of PID incidence in untreated women after *C. trachomatis* infection was likely to be in the range of 10-20%. A Norwegian registry-linkage study of 24,947 women who were tested for *C. trachomatis* infection reported a correlation between diagnosed *Chlamydia* infection and subsequent PID. The incidence rate of PID in this study was found to be higher in women with prior *C. trachomatis* infection than among women with negative *C. trachomatis* tests although the rates were notably low in both groups (Bakken & Ghaderi 2009). It has therefore been suggested that the benefits of current

*Chlamydia* screening programmes may have been overestimated (Low *et al.,* 2006).

A comprehensive review of seven electronic databases covering 17 years of register-based reports (until 2007) and opportunistic screening programmes for *Chlamydia* found that there was no evidence to support the most commonly recommended approach of opportunistic *Chlamydia* screening in a general population younger than 25 years. Furthermore, it was proposed by these authors that an effective approach when assessing biological outcomes of chlamydial infection currently reuqires multiple rounds of screening in randomized control trials (RCT) (Low *et al.,* 2009). A recent RCT (the POPI-prevention of pelvic infection-trial) was undertaken to determine whether a single screening test and treating a subset of 2529 women for chlamydial infection can in fact reduce the incidence of PID over a 12-month period (Oakeshott et al., 2010). The baseline prevalence of *Chlamydia* was 5.4% in the screened population of 2529 sexually active female students (mean age 20.9 years) and 5.9% in (deferred screening) controls with the incidences of PID found to be 1.3% and 1.9% respectively in these cohorts. It was reported that after 12 months, most episodes of PID occurred in women who tested negative for *Chlamydia* at baseline (9.5%) when compared to the intervention group (1.6%) and these authors concluded that the effectiveness of a single *Chlamydia* test in preventing PID over 12 months may also have been overestimated (Oakeshott et al., 2010). A Danish randomised trial was conducted with 9-year follow-up testing of 4000 asymptomatic women for the presence of urogenital *Chlamydia trachomatis*. Data were collected on PID, ectopic

impact of a screening programme (Althaus *et al.,* 2010).

pregnancy EP, infertility diagnoses, IVF treatment and births in women. Results showed that no differences were found between the intervention group and the control groups of women for PID, ectopic pregnancy, infertility, IVF treatment and births. It was concluded that a populationbased offer to be tested for urogenital *C. trachomatis* infection using non-invasive samples and DNA amplification testing did not reduce the long-term risk of reproductive complications such as PID and ectopic pregnancies in asymptomatic women (Anderson *et al.,* 2011). This finding agrees with the conclusions made by authors of an earlier review of 12 databases who reported (from the one study that satisfied inclusion criteria for their review) the absence of valid evidence on the risk of tubal factor infertility following an infection of the genital tract with *C. trachomatis* (Wallace *et al.,* 2008) and is also in agreement with the finding of Oakeshott and colleagues (Oakeshott et al., 2010).

Of note from the Oakeshott study was that a screening intervention at 12 months would not have prevented the 10 reported cases of chlamydia-positive PID among women who were *Chlamydia*-negative at baseline and serves to highlight the ongoing transmission of *Chlamydia* as the elemental problem. The results of the POPI trial suggested that current levels of chlamydial screening are unlikely to have much impact on the overall incidence of PID (Low & Hocking 2010). A recent modelling study based on a comprehensive literature survey on the epidemiology of chlamydial infection and risk-estimates of its late complications has concluded that the risk of developing tubal infertility after a *Chlamydia*  lower genital tract is low (at around 4.6%), and these authors stated that high quality RCTs investigating the transition from cervicitis to tubal infertility are needed (Land et al., 2010). It has also been reported in a prospective study evaluating the sensitivity of multiple-site swab testing (cervix, urethra, vagina and Fallopian tubes) in 2,020 fertility patients over 12 months that multiple site sampling does not increase the detection rate of *C. trachomatis*  among infertile women and in fact that routine DNA testing for *C. trachomatis* should be confined to cervical sampling (Dietrich et al., 2010). A review from the National *Chlamydia* screening programme in London has highlighted the need for *Chlamydia* testing to be offered routinely to young people (under 25 years) as part of an overall approach to sexual health in the community (Kalwij et al., 2010).

It has been noted recently that no studies have yet published results of the effects of greater than one round of screening or indeed screening for repeat *Chlamydia* infections on reproductive sequelae in women following asymptomatic *C. trachomatis* genital infection (Gottlieb *et al.,* 2010). However two new trials the *Chlamydia* Screening Implementation (CSI) Project (van den Broek *et al.,* 2010) and the Australian *Chlamydia* Control Effectiveness Pilot (ACCEPt) trial (Hocking *et al.,* 2008) are currently underway investigating multiple screening rounds and using *Chlamydia* prevalence (and not PID) as the end point. These trials should provide more conclusive information regarding the effectiveness of chlamydial screening to control morbidity associated with genital chlamydial infection.

#### **4.4.4 Chlamydia and vaccines**

Fertility in women is overwhelmingly affected by unresolved or untreated infection of the female reproductive tract with *C. trachomatis*. Since greater than 70% of chlamydial genital infections in women are asymptomatic and sequelae of infection manifest as diseases resulting from severe pathological consequences such as tubal occlusion, a vaccine is likely to be imperative to control infections caused by this sexually transmitted mucosal pathogen. Many animal models of infection-induced immunity including murine and guinea pig

Tubal Damage, Infertility and Tubal Ectopic Pregnancy:

their ability to induce protection (Cruz-Fisher *et al.,* 2011).

*gonorrhoeae* upper genital tract infection (Kamwendo *et al.,* 1996).

**4.5** *Neisseria gonorrhoeae*

**4.6 Mycoplasma species**

*Chlamydia trachomatis* and Other Microbial Aetiologies 25

third candidate antigen showing great promise particularly for prevention of infertility resulting from repeated infections with *C. trachomatis* is the recombinant chlamydial protease-like activity factor (rCPAF) (Murthy et al., 2011).This antigen has been reviewed as a potential vaccine candidate (Murthy *et al.,* 2009) and has successfully induced a combination of neutralising antibodies and cell-mediated responses against genital chlamydial challenge in a murine model of genital chlamydial infection (Li *et al.,* 2010). More recent studies have reported that mice vaccinated intranasally with rCPAF and the adjuvant CpG were significantly protected against infertility as seen by a reduction in hydrosaplinx in rCPAF+CpG vaccinated mice following a primary genital challenge with *C. muridarum* (Murthy *et al.,,* 2011). This latest finding augurs well for the inclusion of this

candidate antigen in a vaccine for use in humans to protect against female infertility.

Recently, a proteomics approach has been used to identify potential vaccine candidates for chlamydial infections. In one study three strains of mice, BALB/c, C3H/HeN and C57BL/6, were inoculated with live and inactivated *C. muridarum* by different routes of immunization. Using a protein microarray, serum samples collected from the mice after immunization were tested for the presence of antibodies against specific chlamydial antigens. This has identified a panel of seven *C. muridarum* dominant antigens (TC0052, TC0189, TC0582, TC0660, TC0726, TC0816 and, TC0828) (Molina, 2010). In a second study by the same group antigen identification was done by constructing a protein chip array by expressing the open reading frames (ORFs) from *C.muridarum* genomic and plasmid DNA and testing it with serum samples from *C.muridarum* immunized mice. This second approach has resulted in the identification of several new immunogens, including 75 hypothetical proteins thus identifying a new group of immunodominant chlamydial proteins that can be tested for

*N. gonorrhoeae* has been implicated in tubal infections. Both the bacteria themselves or components of the bacterial cell wall, lipopolysaccharide or peptidoglycan reportedly cause cessation of the ciliary activity (Mardh 1979) however, infection of the Fallopian tubes does not always result in ultrastructural damage to the mucosal surface (Woods & McGee 1986). Gonococci only invade the non-ciliated cells of the Fallopian tube mucosa, whereby the neighbouring ciliated cells become sloughy and detached (McGee 1981). *Neisseria* spp. infection of the Fallopian tubes results in a dose-dependent response to bacterial cells. Low numbers of bacterial cells induce secretion of TNF-α and subsequently apoptosis of infected cells, however, when bacterial cell numbers increase, the apoptosis appears to be inhibited, favouring bacterial survival (Dean and Powers, 2001). Studies have suggested that ectopic pregnancy is now more likely to be associated with non-gonococcal rather than *N*.

*M. hominis* reportedly causes ciliostasis and swelling of Fallopian tube cilia (Mardh & Westrom 1970). *Mycoplasma* spp. have been isolated from the female upper genital tract and Fallopian tubes (Cohen 2005; Heinonen & Miettinen 1994; Stagey *et al* 1992). Serological testing of women has confirmed an association between mycoplasmas and cases of PID (Moller *et al.,* 1985)and mycoplasma, PID and ectopic pregnancy (Jurstrand *et al.,* 2007). A prospective study of 212 infertile couples was undertaken to investigate the presence of

models continue to be essential in providing knowledge of the infection processes and immune responses to a variety species found within the Chlamydiaceae. These have recently been reviewed by several groups (Cochrane *et al.,* 2010; Farris & Morrison 2011; Hafner *et al.,* 2008; Hafner 2007; Hafner & McNeilly 2008; Lyons et al., 2006; Miyairi *et al.,* 2010; Rank & Whittum-Hudson 2010). These animal models have proved invaluable in providing knowledge of many novel candidate antigens for a vaccine (Barker *et al.,* 2008; McNeilly *et al.,* 2007; Murthy AK *et al.,* 2011; Murthy et al., 2009) as well as novel delivery vehicles (Xu *et al.,* 2011) and delivery routes such as oral and transcutaneous immunization for protection of genital infections (Hickey *et al.,* 2010; Hickey et al., 2009) and have investigated a myriad of potential adjuvants (reviewed in Cochrane *et al.,* 2010; Farris and Morrison, 2011; Hafner *et al.,* 2008) and immune responses elicited following animal immunization trials (Cunningham *et al.,* 2011; McNeilly et al., 2007; Patton *et al.,* 1983). For example it has recently been reported that a *Vibrio cholerae* ghost (VCG) multisubunit chlamydial vaccine delivered to mice by the intramuscular route stimulated immune memory in these animals (Eko *et al.,* 2011). Protection correlates that have been assessed in these models to determine vaccine efficiency have included reduced shedding of viable chlamydial infectious bodies, reduced duration of infection and reduced tissue pathologies such as hydrosalpinx; a vaccine that can achieve one and/or any of these outcomes will greatly aid in diminishing the pathological sequelae of chlamydial genital infections.

Results of recent studies using animal models have revealed many promising novel candidate antigens for eliciting protection against chlamydial genital tract infections in humans. The fusion protein CTH1 is composed of chlamydial proteins from two highly conserved (>97% homology) immune-dominant antigens CT443 (*omc*B) and CT521 (r116) that are targets both for cell-mediated and for humoral immunity and thus can be expected to allow for cross-protection amongst the various chlamydial serotypes (Olsen AW *et al.,* 2010). In addition, CT 521 has also been found to be a strong and frequent target for T cells during a natural *C. trachomatis* infection in humans (Olsen *et al.,* 2006). In 2009 a study investigating 55 chlamydial ORFs covering all putative type III secretion components and control molecules were expressed as fusion proteins. This study measured the reactivity of these fusion proteins with antibodies from sera collected from patient infected with *C. trachomatis* in the urogenital tract (24 antisera) (Wang *et al.,* 2009). It was reported that immunization of mice with the translocated actin recruiting phosphoprotein (Tarp) induced Th1-dominant immunity that significantly reduced the shedding of live bacteria from the lower genital tract and attenuated inflammatory pathologies in the Fallopian tube tissues (Wang *et al.,* 2009). Using the C3H/HeN murine model the subunit vaccine CtH1 delivered subcutaneously with a Th1-inducing adjuvant (CAF01) induced a protective CD4+T cell response and high levels of CTH1-specific antibodies in both the sera and genital tracts of immunised mice however it failed to provide a CD4 independent protective response needed for complete protection (Olsen et al., 2010). A second promising vaccine candidate that has induced CD4+Th1 cells both in *Chlamydia*-infected mice and in humans diagnosed with chlamydial genital tract infections is CT043 a highly conserved hypothetical protein that could potentially provide cross-serotype protection. DNA priming/protein boost immunization with this protein the bacterial load was also significantly reduced in the murine lung infection model (Meoni *et al.,* 2009). The fact that CTD43 has been shown to reduce (1) chlamydial infectivity in the murine model and (2) to prime a CD4+Th1 response in over 60% of patients infected with genital serovars of *C. trachomatis*, means that this antigen could be a promising vaccine candidate for chlamydial genital tract infections. A

models continue to be essential in providing knowledge of the infection processes and immune responses to a variety species found within the Chlamydiaceae. These have recently been reviewed by several groups (Cochrane *et al.,* 2010; Farris & Morrison 2011; Hafner *et al.,* 2008; Hafner 2007; Hafner & McNeilly 2008; Lyons et al., 2006; Miyairi *et al.,* 2010; Rank & Whittum-Hudson 2010). These animal models have proved invaluable in providing knowledge of many novel candidate antigens for a vaccine (Barker *et al.,* 2008; McNeilly *et al.,* 2007; Murthy AK *et al.,* 2011; Murthy et al., 2009) as well as novel delivery vehicles (Xu *et al.,* 2011) and delivery routes such as oral and transcutaneous immunization for protection of genital infections (Hickey *et al.,* 2010; Hickey et al., 2009) and have investigated a myriad of potential adjuvants (reviewed in Cochrane *et al.,* 2010; Farris and Morrison, 2011; Hafner *et al.,* 2008) and immune responses elicited following animal immunization trials (Cunningham *et al.,* 2011; McNeilly et al., 2007; Patton *et al.,* 1983). For example it has recently been reported that a *Vibrio cholerae* ghost (VCG) multisubunit chlamydial vaccine delivered to mice by the intramuscular route stimulated immune memory in these animals (Eko *et al.,* 2011). Protection correlates that have been assessed in these models to determine vaccine efficiency have included reduced shedding of viable chlamydial infectious bodies, reduced duration of infection and reduced tissue pathologies such as hydrosalpinx; a vaccine that can achieve one and/or any of these outcomes will

greatly aid in diminishing the pathological sequelae of chlamydial genital infections.

Results of recent studies using animal models have revealed many promising novel candidate antigens for eliciting protection against chlamydial genital tract infections in humans. The fusion protein CTH1 is composed of chlamydial proteins from two highly conserved (>97% homology) immune-dominant antigens CT443 (*omc*B) and CT521 (r116) that are targets both for cell-mediated and for humoral immunity and thus can be expected to allow for cross-protection amongst the various chlamydial serotypes (Olsen AW *et al.,* 2010). In addition, CT 521 has also been found to be a strong and frequent target for T cells during a natural *C. trachomatis* infection in humans (Olsen *et al.,* 2006). In 2009 a study investigating 55 chlamydial ORFs covering all putative type III secretion components and control molecules were expressed as fusion proteins. This study measured the reactivity of these fusion proteins with antibodies from sera collected from patient infected with *C. trachomatis* in the urogenital tract (24 antisera) (Wang *et al.,* 2009). It was reported that immunization of mice with the translocated actin recruiting phosphoprotein (Tarp) induced Th1-dominant immunity that significantly reduced the shedding of live bacteria from the lower genital tract and attenuated inflammatory pathologies in the Fallopian tube tissues (Wang *et al.,* 2009). Using the C3H/HeN murine model the subunit vaccine CtH1 delivered subcutaneously with a Th1-inducing adjuvant (CAF01) induced a protective CD4+T cell response and high levels of CTH1-specific antibodies in both the sera and genital tracts of immunised mice however it failed to provide a CD4 independent protective response needed for complete protection (Olsen et al., 2010). A second promising vaccine candidate that has induced CD4+Th1 cells both in *Chlamydia*-infected mice and in humans diagnosed with chlamydial genital tract infections is CT043 a highly conserved hypothetical protein that could potentially provide cross-serotype protection. DNA priming/protein boost immunization with this protein the bacterial load was also significantly reduced in the murine lung infection model (Meoni *et al.,* 2009). The fact that CTD43 has been shown to reduce (1) chlamydial infectivity in the murine model and (2) to prime a CD4+Th1 response in over 60% of patients infected with genital serovars of *C. trachomatis*, means that this antigen could be a promising vaccine candidate for chlamydial genital tract infections. A third candidate antigen showing great promise particularly for prevention of infertility resulting from repeated infections with *C. trachomatis* is the recombinant chlamydial protease-like activity factor (rCPAF) (Murthy et al., 2011).This antigen has been reviewed as a potential vaccine candidate (Murthy *et al.,* 2009) and has successfully induced a combination of neutralising antibodies and cell-mediated responses against genital chlamydial challenge in a murine model of genital chlamydial infection (Li *et al.,* 2010). More recent studies have reported that mice vaccinated intranasally with rCPAF and the adjuvant CpG were significantly protected against infertility as seen by a reduction in hydrosaplinx in rCPAF+CpG vaccinated mice following a primary genital challenge with *C. muridarum* (Murthy *et al.,,* 2011). This latest finding augurs well for the inclusion of this candidate antigen in a vaccine for use in humans to protect against female infertility. Recently, a proteomics approach has been used to identify potential vaccine candidates for chlamydial infections. In one study three strains of mice, BALB/c, C3H/HeN and C57BL/6, were inoculated with live and inactivated *C. muridarum* by different routes of immunization. Using a protein microarray, serum samples collected from the mice after immunization were tested for the presence of antibodies against specific chlamydial antigens. This has identified a panel of seven *C. muridarum* dominant antigens (TC0052, TC0189, TC0582, TC0660, TC0726, TC0816 and, TC0828) (Molina, 2010). In a second study by the same group antigen identification was done by constructing a protein chip array by expressing the open reading frames (ORFs) from *C.muridarum* genomic and plasmid DNA and testing it with serum samples from *C.muridarum* immunized mice. This second approach has resulted in the identification of several new immunogens, including 75 hypothetical proteins thus identifying a new group of immunodominant chlamydial proteins that can be tested for
