This genital infection can result in adverse reproductive
outcomes such as infertility, premature delivery, ectopic
pregnancy, low birth weight, and miscarriage (
Enzyme linked immunosorbent assay (ELISA) and
polymerase chain reaction (PCR) are two common
methods for detection of CT. In 2003-2006, a group in
Poland evaluated the frequency of CT infection in women
suffering from spontaneous miscarriage by PCR and
ELISA IgG and IgA (
This study was a case-control study and samples were
collected starting in October 2013 through June 2014.
The sample size was calculated with regard to the reported
prevalence of CT (
All participants were married and had one sex partner. Local Ethical Committee approval and participants’ consent were obtained. A questionnaire containing demographic information, anti-biotherapy history, and previous adverse pregnancy outcome was completed by participants. The criteria for participant selection were no use of any chlamydia-related antibiotics during the last three months, no bleeding, and submitting a completed questionnaire. In order to exclude cases who most likely had genetic problems, questions regarding possible products of conception with congenital malformation and developmental delay, past karyotype tests, and a history of infertility and genetic disorders in family members were asked in the questionnaires.
Vaginal samples were collected using sterile cotton swabs and were conserved in phosphate buffer saline (PBS) at -70°C until tested. Blood samples were collected in 5-ml volumes and the sera were separated by centrifugation at 2500 rpm (1090×g). All the sera were aliquoted into several tubes to avoid excessive freeze-thaw cycles and were stored at -20°C prior to analysis.
We used boiling method to extract DNA from vaginal
samples, since it has been reported as a rapid and cost-
effective method with a high DNA efficiency (
The presence of human cells and the absence of inhibitory
elements in the extracted DNA were evaluated
by amplification of a 268-bp fragment of the
GH20: 5'-GAAGAGCCAAGGACAGGTAC-3' (
PCR was carried out on 2 µl of the extracted DNA samples in a 25 µl reaction volume consist of 20 pmol of each primer, 2 mM MgCl2, 0.3 mM dNTP and 1 U of Taq DNA polymerase. All PCR reagents were purchased from Cinna Gene Company (Tehran, Iran). The PCR protocol was as follows: an initial step 10 minutes at 95°C; 30 cycles of 1 minute at 94°C, 1 minute at 58°C, and 1 minute at 72°C; and a final step 8 minutes at 72°C.
KL2: 5'-AATCAATGCCCGGGATTGGT-3' (
PCR was performed on a final volume of 25 µl containing 5 µl DNA, 6 pmol of each primer (Genfanavaran, Iran), 3 mM MgCl2, 0.2 mM dNTP and 1 U of Taq DNA polymerase were used for each experiment. The PCR protocol was as follows: an initial step 2 minutes at 95°C; 30 cycles of 30 seconds at 95°C, 30 seconds at 58.3°C, and 30 seconds at 72°C; and a final step 5 minutes at 72°C.
Serum samples were tested by MOMP-based ELISA
kits (Euroimmun, Germany) to detect anti-CT IgA and
IgG antibodies. All steps were performed according to the
manufacturer’s instructions. The IgA kit used in this experiment
had 100% sensitivity and 97.4% specificity and
the IgG kit had 78.2% sensitivity and 97.1% specificity.
There was no cross reactivity with other
This was a case-control study and data analysis was carried out using GraphPad Prism version 6.07 for Windows. The Chi-square and Fishers exact tests were used for analysing diagnostic findings (PCR, IgA and IgG). Student’s t test was used to determine the mean and the standard deviations for comparing ages among participants with and without miscarriage. P<0.05 were considered statistically significant.
PCR and ELISA were performed on vaginal swabs and blood samples of 157 participants, respectively. Then the relationship between the number of previous miscarriages and the prevalence of CT infection was evaluated. The number of miscarriages was given under three categories (0, 1-2, and = 3 miscarriages).
Internal control PCR showed that all samples were free
of inhibitory elements (
Human beta globin polymerase chain reaction (PCR) as internal control.
Gel electrophoresis of amplified human
Chlamydia trachomatis (CT) plasmid polymerase chain reaction (PCR). Gel electrophoresis of amplified CT plasmid presenting 241 bp amplicons. The gel electrophoresis results on the left show the presence of CT infection in women in the miscarriage group. The gel electrophoresis results on the right present the absence of CT infection in the participants. L; 100 bp ladder, PC; Positive control, S1-S6; Samples, and NC; Negative control.
The number of C. trachomatis positive cases in the control and both miscarriage groups together
|Diagnostic tools||Miscarriage group n=97||Control group n=60||P value|
The results of the relationship between the number of previous miscarriages and prevalence of CT infection are shown in Table 2. According to the PCR data, none of the participants without a history of miscarriage were positive for CT infection. On the other hand, 5 out of 55 women with 1-2 miscarriages and 6 out of 42 women with three or more miscarriages were positive for CT as indicated by PCR. The difference between these three categories was statistically significant.
Results reported for C. trachomatis infection by three diagnostic tools in women regarding their history of previous miscarriages
|Number of miscarriages||Count||PCR+ (%)||IgG+ (%)||IgA+ (%)|
Three out of 38 women (7.9%) in =25-year age group, 4 out of 97 women (4.1%) in 26-35-year age group, and four out of 22 women (18%) in 36-45-year age group were positive for CT by PCR. Evaluation of association of mother’s age with CT infection revealed that there was a significantly higher correlation between 36-45-year age group and CT infection compared to other age groups, as indicated by PCR (P=0.042).
In the miscarriage groups, 4.1 and 2.1% of women were
positive for CT IgG and IgA antibodies, respectively.
However, in the control group, these ratios were 1.7 and
6.7% of the cases (
The relationship between the number of previous miscarriages
and the prevalence of anti-CT antibodies was
evaluated by ELISA IgA and IgG as well (
CT IgG antibodies were detected in 1 out of 60 women without miscarriage history, 3 out of 55 women with 1-2 miscarriages and 1 out of 42 women with three or more miscarriages. The difference among these three categories was not statistically significant.
According to our PCR results, there is a positive relationship
between miscarriage and underlying CT infection.
The association between molecular evidence of CT
infection and miscarriage has been reported by previous
Furthermore, in our study a significant molecular relationship
was shown between the 36-45-year age group and the
incidence of chlamydia positivity, which was in agreement
with other studies. In 2010 Jenab et al. (
Our ELISA results showed no significant relationship between
the number of previous miscarriages and CT infection,
which was in accordance with earlier serologic studies
on women suffering from recurrent spontaneous abortion
Despite the accuracy of the tests, the CT-positive samples
were surprisingly confirmed by only one of our three
diagnostic tools (PCR, ELISA IgG and ELISA IgA). For
example, all PCR-positive samples were IgG/IgA-negative
or IgG-positive samples were PCR/IgA-negative.
This contradiction may happen due to different reasons.
Positive serologic and negative molecular detection of
CT may be due to an old infection or resolution of CT
At present, these alternative explanations for the discrepancy
between molecular and serological results are not evident at
this point, as they are case-dependent. This reflects the unique
adaptive immunity in the genital tract compared to other mucosal
sites. There is an association between specific host immune
responses and susceptibility to or protection from CT infection
Taken together, to improve the precision and the efficiency of chlamydia detection in the current CT screening tests in clinical laboratories (usually ELISA), it is recommended that molecular tests, such as PCR, be performed as gold standard tests. Moreover, serological tests are helpful in evaluating disease conditions to differentiate ongoing from past damages caused by CT. In cases of past CT infections or upper genital infections not amenable to sampling, a serological test is an effective method to detect the infection and its importance has not faded. However, PCR is the test of choice to detect current CT infection, CT infection at the earliest days of transmission, and persistent CT infection in arrested-immunity cases. Thus, the inclusion of CT molecular and serological screening tests to other pregnancy and prenatal tests, which could allow for early detection and treatment of this infection, would decrease adverse reproductive outcomes.