BACKGROUND
chlamydia trachomatis is a common bacterial sexually transmitted disease  worldwide.
chlamydial urogenital infection has increased at a fast rate, with more than  <dig>  million new cases diagnosed each year  <cit> .
in malaysia, a high prevalence of chlamydial antibody was detected among urban citizens  <cit> , particularly young females and sex workers  <cit> .
long term asymptomatic persistency of the pathogen among 50–70 % of individuals results in wide spread of the disease  <cit> , and leads to delayed treatment, which in turn causes prostatitis and epididymitis in males or infertility in females  <cit> .
in females, the pathogen can ascend the reproductive tract to the endometrial epithelium and fallopian tubes, leading to pelvic inflammatory disease  in approximately 20–40 % of the infected patients; among which 11 % develop tubal factor infertility while 9 % show ectopic pregnancy  <cit> .
c. trachomatis-mediated inflammation in the genital tract can result in cervicitis or endometriosis, causing lesions, abnormal mucopurulent discharge and thickening of the uterus inner layer  <cit> .
inflammation can also contribute to hormonal disorders, i.e., polycystic ovarian syndrome  and irregular menses  <cit> .
additionally, newborn infants can be infected at birth when delivered through an infected birth canal, causing severe conjunctivitis or pneumonia  <cit> .fig.
<dig> plasmid copy numbers among c. trachomatis-infected patients with different clinical parameters.
dot plot graphs show the ratio of plasmid:momp as determined by quantitative real-time pcr assay.
each dot represents data from a single patient in each group .
the p values were measured with unpaired student’s t-test.
data was considered significant when *p <  <dig> .
n.s.
: non-significant
in recent years, several research groups suggest that the c. trachomatis without plasmid demonstrates a weaker degree of virulence.
the plasmid-deficient strains show impaired ability to infect the female mouse genital tract  <cit> .
c. trachomatis infection damages the reproductive tract by activating tlr <dig> and initiates a th1-dependent inflammatory response.
in a study using plasmid-cured c. muridarum mutant infection in a murine model, tlr2-dependent cytokine was not detected and no sign of oviduct damage can be observed, suggesting that the plasmid-cured mutant has lost virulence  <cit> .
also, intra-vaginal immunization of the plasmid-deficient strains provide protection in the murine model of genitourinary tract infection  <cit> ; as well as in trachoma infected macaques  <cit>  due to the failure to trigger a toll-like receptor 2-dependent immune response  <cit> .
these findings have led to research on use of plasmid-deficient strains as a potential human vaccine.
in fact, an intravaginal infection of attenuated plasmidless c. trachomatis l <dig> has been shown to provide protection to the host because it is nonpathogenic and raises systemic antibody in the c3h/hej mouse model  <cit> .
despite compelling evidence in animal models, there is no equivalent finding from human clinical study confirming the significance of plasmid in c. trachomatis infection.
a recent study in human ocular c. trachomatis infection reveals that no direct association exists between plasmid copy numbers and disease severity  <cit> .
therefore, further work should be done to establish the plausible connection.
in this study, we investigated the prevalence of plasmid-bearing  or plasmid-free  c. trachomatis infection and the plasmid copy numbers among obstetrics and gynecology patients in kuala lumpur, the capital city of malaysia.
by associating the data with the patients’ clinical presentations, we evaluated the prevalence and risk of plasmid  and plasmid  variants in affecting gynecological disorders.
RESULTS
demographics of the study population
the patient cohort from this study comprised  <dig> female patients of child-bearing age  who visited the obstetrics and gynecology clinic at the university of malaya medical centerfrom  <dig> to  <dig> .
a total number of  <dig> patients were married,  <dig> were single or had divorced, and the remaining  <dig> patients did not reveal their marital status.
most of the patients were of malay ethnicity , followed by indians , chinese , and others , reflecting the demographics of the multiethnic malaysian population.
a majority of the patients recruited to the study presented with gynecological complications or subfertility issues, and had a higher risk of std infection.
the main reason for patients’ visit to the clinic was due to infertility .
other reasons included abdominal pain, bleeding, menorrhagia, irregular menstrual cycles and others.
two patients had cystectomy whereas one had a history of resolved pid.table  <dig> patient demographics.
numbers  of patients with or without c. trachomatis infection; and patients infected with plasmid  or plasmid  variants.
n =  <dig>  the p values for all variables were measured with fisher’s exact test.
for age, a t-test  was used.
n.s.
: non-significant
c. trachomatis infection
c. trachomatis
c. trachomatis
a
a
c. trachomatis infection is associated with various gynecological disorders
genital c. trachomatis infection in each patient was first diagnosed by detection of the c. trachomatis momp  gene.
total dna was first isolated from the patients’ endocervical swabs and pcr amplified using a human β-globin control primer to ensure the successful extraction of dna from the endocervical swabs .
the presence of c. trachomatis dna was detected with momp primer using a nested pcr amplification method   <cit> .
the results were further confirmed using a quantitative real-time pcr method with inner primer alone.
momp is expressed on the cell surface with a porin activity that is often used for diagnosis of c. trachomatis  <cit> .
over half of the patients, i.e.,  <dig>  % , were diagnosed with genital c. trachomatis infection, suggesting a high incidence of sexually transmitted chlamydial infection among the female patients recruited .
no significant difference was detected among patients by age, marital status or ethnic background.
we then sequenced the momp pcr product and performed a nucleotide blast to assess the diversity of c. trachomatis strains in the local population.
interestingly, our data revealed that all samples examined showed a d serotype .table  <dig> chlamydial infection in patients with different symptoms.
numbers  of patients with or without c. trachomatis infection.
n =  <dig>  the p values for all variables were measured with fisher’s exact test.
n.s.
: non-significant
c. trachomatis
*p< <dig>  **p< <dig>  ***p< <dig>
overall, c. trachomatis infection showed an association with infertility  .
among patients who were fertile, only  <dig>  %  were infected by c. trachomatis, whereas among infertile patients,  <dig>  %  had c. trachomatis infection.
notably, a high c. trachomatis prevalence of  <dig>  %  was detected among patients who were diagnosed with primary  or secondary  infertility .
meanwhile, 68 %  of patients who experienced miscarriage were infected by c. trachomatis .
the inflammation  of patients’ reproductive tract was recorded by clinicians during examination.
c. trachomatis infection showed a close connection with the inflammation in the reproductive system  .
our data showed that approximately 64 %  of patients who were diagnosed with mucopurulent cervicitis  and 100 %  of patients with endometriosis  had c. trachomatis infection.
in addition, we showed that c. trachomatis infection exhibited significant association with irregular menstrual cycles  as well as pcos , when compared to the uninfected controls.
higher incidence of plasmid-bearing c. trachomatis infection among patients with infertility, inflammation and pcos
next, we investigated if the patients enrolled were infected by plasmid  or  c. trachomatis variants.
total dna extracted from the vaginal swabs of the c. trachomatis-infected patients were amplified with two sets of cryptic plasmid primers  using quantitative real-time pcr analysis.
of the  <dig> c. trachomatis-infected patients, we detected that  <dig>  %  were caused by plasmid  c. trachomatis whereas only  <dig>  %  were caused by plasmid  c. trachomatis .
when comparing the clinical parameters of the patients, we noted no significant correlation between the plasmid and the tendency for the patients to exhibit infertility , inflammation , irregular menses  and pcos  .
among the patients with infertility,  <dig>  %  cases were diagnosed with plasmid  c. trachomatis, compared to only  <dig>  %  cases with plasmid  c. trachomatis.
similarly, the majority of the patients who had inflammation in the reproductive tract  and pcos  were caused by plasmid  c. trachomatis.
among the  <dig> patients infected by plasmid  c. trachomatis,  <dig> were fertile while  <dig> had infertility.
however,  <dig> among the  <dig> showed no sign of inflammation and had regular menstrual cycles.table  <dig>
c. trachomatis plasmid in patients with different symptoms.
numbers  of patients infected with plasmid  or plasmid  c. trachomatis variants.
n =  <dig>  the p values for all variables were measured with fisher’s exact test.
n.s.
: non-significant
higher relative plasmid copy numbers associated with pcos
to examine if the abundance of plasmid in c. trachomatis is associated with the presence of the clinical symptoms, we quantified the plasmid copy numbers in each patient .
the average ct value from two cryptic plasmid primers  for each patient was obtained.
relative plasmid copy numbers per bacterium  for all  <dig> c. trachomatis-infected patients were calculated.
in general, we noticed no direct connection between fertility, the reproductive tract lining and menstrual cycle, consistent with the previous studies in human ocular infection and in vitro tissue tropism  <cit> .
notably, pcos patients showed a higher plasmid copy number , relative to the non-pcos patients.
although endometriosis patients also demonstrated a comparatively higher plasmid copy number, , no statistical significance was noted.
discussion
in this study, we reported a high prevalence  of c. trachomatis infection among female adults of child bearing age, with subfertility or gynecological problems who visited obstetrics and gynecology clinics in malaysia.
this indicates that there is a pressing need for wider population screening to increase awareness and prevent the spread of the disease among the community.
most of the patients who demonstrated symptoms were diagnosed with genital c. trachomatis infection, including infertility , reproductive system lesions , irregular menses  and pcos , suggesting the c. trachomatis is a leading factor for female reproductive system disorders.
although a high rate of genital c. trachomatis prevalance was detected in our study, the patients recruited were suspected to be at risk of the bacterial infection based on the clinical examination.
the rates of c. trachomatis infection vary in different studies depending on the group of patients recruited and the study region.
for example, a study using  <dig> infertile female patients showed a 40 % infection rate by c. trachomatis  <cit>  whereas other studies reported only a 8 %  <cit>  or 15 % c. trachomatis infection rate.
among patients with tubal infertility, the prevalence of c. trachomatis was reported to be  <dig>  % among  <dig> patients  <cit> .
consistent to the previous study, the presence of plasmid  c. trachomatis was high .
however, the prevalence using real-time pcr amplification depends highly on the assay sensitivity.
for data validation, a glycogen-positive test for bacterial isolates can be used to confirm the presence of plasmid in the bacteria  <cit> .
various research groups have established that the plasmid  c. trachomatis demonstrated a higher virulence in animal models of ocular or genital infections .
consistent with these findings, our results showed that high percentages of those who had infertility , inflammation in the reproductive tract , irregular menses  and pcos  were diagnosed with plasmid  but not plasmid  variants of c. trachomatis.
although these observations provide support for previous work which suggests the usage of plasmidless c. trachomatis as a potential human vaccine  <cit> , caution must be taken as some of the patients who showed symptoms were infected by the plasmid  strains.
also note that the genetic diversity of human populations may contribute to results contradicting experiments performed using inbred animals.
therefore, additional surveys which involve a larger human cohort should be conducted to ensure the over-all safety of the plasmidless strains among individuals from diverse backgrounds.
plasmid  or  c. trachomatis strains demonstrated no difference with regards to growth kinetics, plaquing efficiency and size but showed a defect in glycogen granule accumulation and intrainclusion movement  <cit> .
a recent study showed that c. trachomatis is capable of inducing alteration to global host histone modifications and double strand break repair, thus generating an environment favorable for malignant transformation  <cit> .
in fact, c. trachomatis infection poses risk for infected individuals to develop cervix intraepithelial neoplasia  <cit> , in a similar way to other tumor-inducing pathogens.
extra-chromosomal plasmid dna may play a role in integrating with the host genome which leads to this pathological damage.
the conserved  <dig>  kb cryptic plasmid is a small, non-conjugative and non-integrative extrachromosomal dna that contains genes encoding  <dig> proteins  <cit> .
the plasmid pgp <dig> encodes for immunogenic trimers which trigger specific antibody production in infected individuals  <cit> ; and is secreted into the cytosol of infected cells during chlamydial infection  <cit> .
meanwhile, pgp <dig> encodes for a protein which comprises a putative helix-loop-helix domain which functions as transcriptional regulator for virulence-associated genes  <cit> .
therefore, expression of plasmid genes may be crucial in leading to clinical symptoms.
to support this notion, a quantitative protein analysis could be carried out in the future to measure the amount of plasmid-derived proteins in the patient sample.
CONCLUSIONS
in conclusion, we suggest that plasmids maybe a potential risk factor for reproductive system disorders including infertility, inflammation in the reproductive tract, irregular menses and pcos in genital chlamydial infection.
however, continued work is required to substantiate plasmids as a virulence factor in future studies with a larger human cohort.

 chlamydia trachomatis is a common bacterial sexually transmitted disease worldwide. chlamydial urogenital infection has increased at a fast rate, with more than million new cases diagnosed each year. in malaysia, a high prevalence of chlamydial antibody was detected among urban citizens, particularly young females and sex workers. long term asymptomatic persistency of the pathogen among 50–70 % of individuals results in wide spread of the disease, and leads to delayed treatment, which in turn causes prostatitis and epididymitis in males or infertility in females. in females, the pathogen can ascend the reproductive tract to the endometrial epithelium and fallopian tubes, leading to pelvic inflammatory disease in approximately 20–40 % of the infected patients; among which 11 % develop tubal factor infertility while 9 % show ectopic pregnancy. c. trachomatis-mediated inflammation in the genital tract can result in cervicitis or endometriosis, causing lesions, abnormal mucopurulent discharge and thickening of the uterus inner layer. inflammation can also contribute to hormonal disorders, i.e., polycystic ovarian syndrome and irregular menses. additionally, newborn infants can be infected at birth when delivered through an infected birth canal, causing severe conjunctivitis or pneumonia.fig. plasmid copy numbers among c. trachomatis-infected patients with different clinical parameters. dot plot graphs show the ratio of plasmid:momp as determined by quantitative real-time pcr assay. each dot represents data from a single patient in each group. the p values were measured with unpaired student’s t-test. data was considered significant when *p < . n.s.: non-significant in recent years, several research groups suggest that the c. trachomatis without plasmid demonstrates a weaker degree of virulence. the plasmid-deficient strains show impaired ability to infect the female mouse genital tract. c. trachomatis infection damages the reproductive tract by activating tlr and initiates a th1-dependent inflammatory response. in a study using plasmid-cured c. muridarum mutant infection in a murine model, tlr2-dependent cytokine was not detected and no sign of oviduct damage can be observed, suggesting that the plasmid-cured mutant has lost virulence. also, intra-vaginal immunization of the plasmid-deficient strains provide protection in the murine model of genitourinary tract infection; as well as in trachoma infected macaques due to the failure to trigger a toll-like receptor 2-dependent immune response. these findings have led to research on use of plasmid-deficient strains as a potential human vaccine. in fact, an intravaginal infection of attenuated plasmidless c. trachomatis l has been shown to provide protection to the host because it is nonpathogenic and raises systemic antibody in the c3h/hej mouse model. despite compelling evidence in animal models, there is no equivalent finding from human clinical study confirming the significance of plasmid in c. trachomatis infection. a recent study in human ocular c. trachomatis infection reveals that no direct association exists between plasmid copy numbers and disease severity. therefore, further work should be done to establish the plausible connection. in this study, we investigated the prevalence of plasmid-bearing or plasmid-free c. trachomatis infection and the plasmid copy numbers among obstetrics and gynecology patients in kuala lumpur, the capital city of malaysia. by associating the data with the patients’ clinical presentations, we evaluated the prevalence and risk of plasmid and plasmid variants in affecting gynecological disorders.  demographics of the study population the patient cohort from this study comprised female patients of child-bearing age who visited the obstetrics and gynecology clinic at the university of malaya medical centerfrom to. a total number of patients were married, were single or had divorced, and the remaining patients did not reveal their marital status. most of the patients were of malay ethnicity, followed by indians, chinese, and others, reflecting the demographics of the multiethnic malaysian population. a majority of the patients recruited to the study presented with gynecological complications or subfertility issues, and had a higher risk of std infection. the main reason for patients’ visit to the clinic was due to infertility. other reasons included abdominal pain, bleeding, menorrhagia, irregular menstrual cycles and others. two patients had cystectomy whereas one had a history of resolved pid.table patient demographics. numbers of patients with or without c. trachomatis infection; and patients infected with plasmid or plasmid variants. n =  the p values for all variables were measured with fisher’s exact test. for age, a t-test was used. n.s.: non-significant c. trachomatis infection c. trachomatis c. trachomatis a a c. trachomatis infection is associated with various gynecological disorders genital c. trachomatis infection in each patient was first diagnosed by detection of the c. trachomatis momp gene. total dna was first isolated from the patients’ endocervical swabs and pcr amplified using a human β-globin control primer to ensure the successful extraction of dna from the endocervical swabs. the presence of c. trachomatis dna was detected with momp primer using a nested pcr amplification method. the results were further confirmed using a quantitative real-time pcr method with inner primer alone. momp is expressed on the cell surface with a porin activity that is often used for diagnosis of c. trachomatis. over half of the patients, i.e.,  %, were diagnosed with genital c. trachomatis infection, suggesting a high incidence of sexually transmitted chlamydial infection among the female patients recruited. no significant difference was detected among patients by age, marital status or ethnic background. we then sequenced the momp pcr product and performed a nucleotide blast to assess the diversity of c. trachomatis strains in the local population. interestingly, our data revealed that all samples examined showed a d serotype.table chlamydial infection in patients with different symptoms. numbers of patients with or without c. trachomatis infection. n =  the p values for all variables were measured with fisher’s exact test. n.s.: non-significant c. trachomatis *p< **p< ***p< overall, c. trachomatis infection showed an association with infertility. among patients who were fertile, only  % were infected by c. trachomatis, whereas among infertile patients,  % had c. trachomatis infection. notably, a high c. trachomatis prevalence of  % was detected among patients who were diagnosed with primary or secondary infertility. meanwhile, 68 % of patients who experienced miscarriage were infected by c. trachomatis. the inflammation of patients’ reproductive tract was recorded by clinicians during examination. c. trachomatis infection showed a close connection with the inflammation in the reproductive system. our data showed that approximately 64 % of patients who were diagnosed with mucopurulent cervicitis and 100 % of patients with endometriosis had c. trachomatis infection. in addition, we showed that c. trachomatis infection exhibited significant association with irregular menstrual cycles as well as pcos, when compared to the uninfected controls. higher incidence of plasmid-bearing c. trachomatis infection among patients with infertility, inflammation and pcos next, we investigated if the patients enrolled were infected by plasmid or c. trachomatis variants. total dna extracted from the vaginal swabs of the c. trachomatis-infected patients were amplified with two sets of cryptic plasmid primers using quantitative real-time pcr analysis. of the c. trachomatis-infected patients, we detected that  % were caused by plasmid c. trachomatis whereas only  % were caused by plasmid c. trachomatis. when comparing the clinical parameters of the patients, we noted no significant correlation between the plasmid and the tendency for the patients to exhibit infertility, inflammation, irregular menses and pcos. among the patients with infertility,  % cases were diagnosed with plasmid c. trachomatis, compared to only  % cases with plasmid c. trachomatis. similarly, the majority of the patients who had inflammation in the reproductive tract and pcos were caused by plasmid c. trachomatis. among the patients infected by plasmid c. trachomatis, were fertile while had infertility. however, among the showed no sign of inflammation and had regular menstrual cycles.table c. trachomatis plasmid in patients with different symptoms. numbers of patients infected with plasmid or plasmid c. trachomatis variants. n =  the p values for all variables were measured with fisher’s exact test. n.s.: non-significant higher relative plasmid copy numbers associated with pcos to examine if the abundance of plasmid in c. trachomatis is associated with the presence of the clinical symptoms, we quantified the plasmid copy numbers in each patient. the average ct value from two cryptic plasmid primers for each patient was obtained. relative plasmid copy numbers per bacterium for all c. trachomatis-infected patients were calculated. in general, we noticed no direct connection between fertility, the reproductive tract lining and menstrual cycle, consistent with the previous studies in human ocular infection and in vitro tissue tropism. notably, pcos patients showed a higher plasmid copy number, relative to the non-pcos patients. although endometriosis patients also demonstrated a comparatively higher plasmid copy number,, no statistical significance was noted. discussion in this study, we reported a high prevalence of c. trachomatis infection among female adults of child bearing age, with subfertility or gynecological problems who visited obstetrics and gynecology clinics in malaysia. this indicates that there is a pressing need for wider population screening to increase awareness and prevent the spread of the disease among the community. most of the patients who demonstrated symptoms were diagnosed with genital c. trachomatis infection, including infertility, reproductive system lesions, irregular menses and pcos, suggesting the c. trachomatis is a leading factor for female reproductive system disorders. although a high rate of genital c. trachomatis prevalance was detected in our study, the patients recruited were suspected to be at risk of the bacterial infection based on the clinical examination. the rates of c. trachomatis infection vary in different studies depending on the group of patients recruited and the study region. for example, a study using infertile female patients showed a 40 % infection rate by c. trachomatis whereas other studies reported only a 8 % or 15 % c. trachomatis infection rate. among patients with tubal infertility, the prevalence of c. trachomatis was reported to be  % among patients. consistent to the previous study, the presence of plasmid c. trachomatis was high. however, the prevalence using real-time pcr amplification depends highly on the assay sensitivity. for data validation, a glycogen-positive test for bacterial isolates can be used to confirm the presence of plasmid in the bacteria. various research groups have established that the plasmid c. trachomatis demonstrated a higher virulence in animal models of ocular or genital infections. consistent with these findings, our results showed that high percentages of those who had infertility, inflammation in the reproductive tract, irregular menses and pcos were diagnosed with plasmid but not plasmid variants of c. trachomatis. although these observations provide support for previous work which suggests the usage of plasmidless c. trachomatis as a potential human vaccine, caution must be taken as some of the patients who showed symptoms were infected by the plasmid strains. also note that the genetic diversity of human populations may contribute to results contradicting experiments performed using inbred animals. therefore, additional surveys which involve a larger human cohort should be conducted to ensure the over-all safety of the plasmidless strains among individuals from diverse backgrounds. plasmid or c. trachomatis strains demonstrated no difference with regards to growth kinetics, plaquing efficiency and size but showed a defect in glycogen granule accumulation and intrainclusion movement. a recent study showed that c. trachomatis is capable of inducing alteration to global host histone modifications and double strand break repair, thus generating an environment favorable for malignant transformation. in fact, c. trachomatis infection poses risk for infected individuals to develop cervix intraepithelial neoplasia, in a similar way to other tumor-inducing pathogens. extra-chromosomal plasmid dna may play a role in integrating with the host genome which leads to this pathological damage. the conserved  kb cryptic plasmid is a small, non-conjugative and non-integrative extrachromosomal dna that contains genes encoding proteins. the plasmid pgp encodes for immunogenic trimers which trigger specific antibody production in infected individuals; and is secreted into the cytosol of infected cells during chlamydial infection. meanwhile, pgp encodes for a protein which comprises a putative helix-loop-helix domain which functions as transcriptional regulator for virulence-associated genes. therefore, expression of plasmid genes may be crucial in leading to clinical symptoms. to support this notion, a quantitative protein analysis could be carried out in the future to measure the amount of plasmid-derived proteins in the patient sample.  in conclusion, we suggest that plasmids maybe a potential risk factor for reproductive system disorders including infertility, inflammation in the reproductive tract, irregular menses and pcos in genital chlamydial infection. however, continued work is required to substantiate plasmids as a virulence factor in future studies with a larger human cohort. 
however, no statistical significance was detected among patients with plasmid versus plasmid c. trachomatis infection. the  kb cryptic plasmid of chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. our data showed that genital c. trachomatis infection was associated with infertility issues, inflammation in the reproductive tract, irregular menstrual cycles and polycystic ovarian syndrome. our findings show a high incidence of c. trachomatis infection among women with infertility or gynecological problems in malaysia. a total of female patients of child bearing age with gynecological complications and subfertility issues, who visited obstetrics and gynecology clinics in kuala lumpur, malaysia were recruited for the study. interestingly, plasmid c. trachomatis was detected in all patients with pcos, and the plasmid copy numbers were significantly higher among pcos patients, relative to non-pcos patients. the aim of this study was to investigate the prevalence and potential involvement of the c. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome among genital c. trachomatis–infected patients. of the chlamydia-infected patients,  % were infected with plasmid-bearing c. trachomatis while the remaining  % were caused by the plasmid-free variant. however, due to the low number of plasmid c. trachomatis cases, a significant role of the plasmid in causing virulence in human requires further investigation of a larger cohort. prevalence of genital chlamydial infection among these patients was alarmingly high at  %. chlamydia trachomatisreproductive system disordersinfertilityplasmidhigh impact research granthir-mohe e000013-20001wong won fen fundamental research grant scheme fp027-2010bsabet negar shafiei issue-copyright-statement© the author 2016
 the  kb cryptic plasmid of chlamydia trachomatis has been shown to be a virulence factor in animal models, but its significance in humans still remains unknown. the aim of this study was to investigate the prevalence and potential involvement of the c. trachomatis cryptic plasmid in causing various clinical manifestations; including infertility, reproductive tract disintegrity, menstrual disorder, and polycystic ovarian syndrome among genital c. trachomatis–infected patients.  a total of female patients of child bearing age with gynecological complications and subfertility issues, who visited obstetrics and gynecology clinics in kuala lumpur, malaysia were recruited for the study. prevalence of genital chlamydial infection among these patients was alarmingly high at  %. of the chlamydia-infected patients,  % were infected with plasmid-bearing c. trachomatis while the remaining  % were caused by the plasmid-free variant. our data showed that genital c. trachomatis infection was associated with infertility issues, inflammation in the reproductive tract, irregular menstrual cycles and polycystic ovarian syndrome. however, no statistical significance was detected among patients with plasmid versus plasmid c. trachomatis infection. interestingly, plasmid c. trachomatis was detected in all patients with pcos, and the plasmid copy numbers were significantly higher among pcos patients, relative to non-pcos patients.  our findings show a high incidence of c. trachomatis infection among women with infertility or gynecological problems in malaysia. however, due to the low number of plasmid c. trachomatis cases, a significant role of the plasmid in causing virulence in human requires further investigation of a larger cohort. keywords chlamydia trachomatisreproductive system disordersinfertilityplasmidhigh impact research granthir-mohe e000013-20001wong won fen fundamental research grant scheme fp027-2010bsabet negar shafiei issue-copyright-statement© the author 2016 