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 (OmpA) 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 (data not shown). The presence of C. trachomatis DNA was detected with Momp primer using a nested PCR amplification method (outer primers followed by inner primer amplification) [15]. 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 [16]. Over half of the patients, i.e., 51.1 % (92/180), were diagnosed with genital C. trachomatis infection, suggesting a high incidence of sexually transmitted chlamydial infection among the female patients recruited (Table 2). 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 (data not shown).

Table 2

Chlamydial infection in patients with different symptoms. Numbers (percentages) of patients with or without C. trachomatis infection. n=180. The P values for all variables were measured with Fisher’s exact test. n.s.: non-significant

Parameters	All patients (n=180)	Non-infected (n=88)	C. trachomatis -infected (n=92)	P value	Odd Ratio (95 % CI)
Fertility
Fertile	112	76 (67.9 %)	36 (32.1 %)
Infertile	68	12 (17.6 %)	56 (82.4 %)	<0.0001***	9.852 (4.70 to 20.63)
- 1° or 2° infertility	43	4 (9.3 %)	39 (90.7 %)	< 0.0001***	20.580 (6.83 to 62.02)
- Miscarriage	25	8 (32.0 %)	17 (68.0 %)	0.0013**	4.486 (1.77 to 11.36)
Reproductive tract
Normal lining	143	79 (55.2 %)	64 (44.8 %)
Inflammation	37	9 (24.3 %)	28 (75.7 %)	0.00088***	3.84 (1.69 to 8.72)
- Mucopurulent Cervicitis	25	9 (36.0 %)	16 (64.0 %)	0.0876 n.s.	2.167 (0.90 to 5.23)
- Endometriosis	12	0 (0.0 %)	12 (100.0 %)	0.0001***	30.43 (1.77 to 524.2)
Menstrual Cycle
Regular	130	70 (53.8 %)	60 (46.2 %)
Irregular	50	18 (36.0 %)	32 (64.0 %)	0.0452*	2.074 (1.05 to 4.06)
Hormonal Disorder
Undiagnosed	170	87 (51.2 %)	83 (48.8 %)
PCOS	10	1 (10.0 %)	9 (90.0 %)	0.0184*	9.434 (1.17 to 76.14)

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*P<0.05 **P<0.01 ***P<0.001

Overall, C. trachomatis infection showed an association with infertility (OR:9.85, 95 % CI:4.70–20.63, P<0.0001^***) (Table 2). Among patients who were fertile, only 32.1 % (36/112) were infected by C. trachomatis, whereas among infertile patients, 82.4 % (56/68) had C. trachomatis infection. Notably, a high C. trachomatis prevalence of 90.7 % (39/43,) was detected among patients who were diagnosed with primary (1°) or secondary (2°) infertility (OR:20.580, 95 % CI:6.83–62.02, P<0.0001^***). Meanwhile, 68 % (17/25) of patients who experienced miscarriage were infected by C. trachomatis (OR:4.486, 95 % CI:1.77–11.36, P=0.0013^**).

The inflammation (cervicitis with mucopurulent discharge or endometriosis) of patients’ reproductive tract was recorded by clinicians during examination. C. trachomatis infection showed a close connection with the inflammation in the reproductive system (OR:3.84, 95 % CI:1.69–8.72, P=0.0008^***) (Table 2). Our data showed that approximately 64 % (16/25) of patients who were diagnosed with mucopurulent cervicitis (OR:2.167, 95 % CI:0.90–5.23, P=0.087) and 100 % (12/12) of patients with endometriosis (OR:30.43, 95 % CI:1.77–524.2, P<0.0001^***) had C. trachomatis infection.

In addition, we showed that C. trachomatis infection exhibited significant association with irregular menstrual cycles (OR:2.07, 95 % CI:1.05–4.06, P=0.045^*) as well as PCOS (OR:9.43, 95 % CI:1.17–76.14, P=0.018^*), 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 (which target Pgp1 and Pgp8 respectively) using quantitative real-time PCR analysis. Of the 92 C. trachomatis-infected patients, we detected that 93.5 % (86/92) were caused by plasmid (+) C. trachomatis whereas only 6.5 % (6/92) were caused by plasmid (−) C. trachomatis (Table 1).

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 (OR:0.76, 95 % CI:0.13–4.40, P=0.562), inflammation (OR:5.26, 95 % CI:0.28–98.68, P=0.155), irregular menses (OR:2.81, 95 % CI:0.31–25.24, P=0.315) and PCOS (OR:1.59, 95 % CI:0.08–30.6, P=0.529) (Table 3). Among the patients with infertility, 92.8 % (52/56) cases were diagnosed with plasmid (+) C. trachomatis, compared to only 7.14 % (4/56) cases with plasmid (−) C. trachomatis. Similarly, the majority of the patients who had inflammation in the reproductive tract (96.4 %, 27/28) and PCOS (100 %, 9/9) were caused by plasmid (+) C. trachomatis. Among the 6 patients infected by plasmid (−) C. trachomatis, 2 were fertile while 4 had infertility. However, 5 among the 6 showed no sign of inflammation and had regular menstrual cycles.

Table 3

C. trachomatis plasmid in patients with different symptoms. Numbers (percentages) of patients infected with plasmid (−) or plasmid (+) C. trachomatis variants. n=92. The P values for all variables were measured with Fisher’s exact test. n.s.: non-significant

Parameters	C. trachomatis-infected patients (n=92)	Plasmid (−) C. trachomatis (n=6)	Plasmid (+) C. trachomatis (n=86)	P value	Odd Ratio (95 % CI)
Fertility
Fertile	36	2 (5.55 %)	34 (94.4 %)
Infertile	56	4 (7.14 %)	52 (92.8 %)	0.5629 n.s.	0.764 (0.13 to 4.40)
- 1° or 2° infertility	39	3 (7.6 %)	36 (92.3 %)	0.5295 n.s.	0.755 (0.14 to 3.95)
- Miscarriage	17	1 (5.8 %)	16 (88.2 %)	0.6939 n.s.	0.875 (0.09 to 8.01)
Reproductive tract
Normal lining	64	5 (7.8 %)	59 (92.1 %)
Inflammation	28	1 (3.5 %)	27 (96.4 %)	0.4045 n.s.	2.288 (0.25 to 20.55)
- Mucopurulent Cervicitis	16	0 (0.0 %)	16 (100 %)	0.1550 n.s.	5.269 (0.28 to 98.68)
- Endometriosis	12	1 (8.3 %)	11 (91.6 %)	0.5786 n.s.	0.733 (0.078 to 6.88)
Menstrual Cycle
Regular	60	5 (8.3 %)	55 (91.6 %)
Irregular	32	1 (3.1 %)	31 (96.8 %)	0.3153 n.s.	2.818 (0.31 to 25.24)
Hormonal Disorder
Undiagnosed	83	6 (72.2 %)	77 (92.7 %)
PCOS	9	0 (0 %)	9 (100 %)	0.5293 n.s.	1.59 (0.083 to 30.6)

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DNA extraction

Endocervical swabs were collected by using a Floqswab (Copan, Brescia, Italy) and transferred to a laboratory in a UTM-RT universal transport medium tube (Copan). The samples were vortex mixed and the cells were centrifuged at 400× g for 10 min. The cells recovered were lysed and separated with phenol chloroform. The DNA was precipitated using 1:10 volume of 3 M sodium acetate and isopropanol at −20 °C overnight incubation. The samples were washed and eluted with TE buffer.

PCR amplification

Diagnosis of C. trachomatis was performed as described [15]. Nested PCR was performed to amplify the genes that encode for Major Outer Membrane Protein (MOMP) and plasmid using outer and inner primers (Table 4). PCR samples were run in a Veriti Thermal Cycler (Applied Biosystems, Essex, UK) at the following conditions: 95 °C for 3 min, 30 cycles of 95 °C for 30 s, 50 °C for 1 min, and 72 °C for 1 min, followed by a final extension at 72 °C for 7 min using HelixAmp Taq DNA polymerase (Nanohelix, Daejeon, South Korea). The first round PCR was amplified using 50 ng of DNA isolated from endocervical swabs with outer primer pairs. Then, 1 μl of first round PCR product was used as the template in the second round PCR and amplified with inner primer pairs. The amplicons were run on gel electrophoresis using 1.5 % agarose gel and viewed under a UV transilluminator. Human β-globin gene was used as a positive control to confirm successful DNA retrieval from patient swabs. PCR amplification for human β-globin primer was run separately in PCR master mix containing a total volume 50 μl as follows: 1 μl template DNA, 200 mM of each of dNTP, 25 pmol of each primer, 4 mM Tune Up solution, 5 μl of PCR buffer and 2.5 units of HelixAmp Taq DNA polymerase recombinant (Nanohelix, Daejeon, South Korea). PCR samples were run at the following conditions: 95 °C for 3 min, 30 cycles amplification consisted of 95 °C for 30 s, 50 °C for 1 min, and extension at 72 °C for 1 min, followed by final extension at 72 °C for 7 min.

Quantitative real-time PCR amplification

A quantitative real-time PCR assay was used to confirm the diagnosis and to determine the plasmid copy numbers in patient samples. DNA samples isolated from the vaginal swabs of C. trachomatis-infected patients were prepared. Mastermix containing 1 μl template DNA, 1× SsoAdvanced Universal SYBR Green Supermix (Biorad, Hercules, CA), 10 pmol primers were run using a Mx3000 Stratagene thermacycler (Agilent Technologies, Santa Clara, CA). Primers used were inner primers for Momp, inner primer for plasmid Pgp8, and plasmid Pgp1 primers. Plasmid copy number for each patient was calculated as below:

Sequencing

Amplification of an approximately 871 bp fragment of OmpA was performed by nested PCR. The first PCR step was carried out with outer primer pair (Table 1) using 10 μl of DNA extracted from swabs. Amplification was performed in a final reaction volume of 50 μl containing 0.3 μM of each primer, 0.2 mM of dNTPs, 3 μl of TuneUp solution (Nanohelix, Korea) and 5U of HelixAmp™Taq DNA polymerase (Nanohelix, Korea). The first amplification conditions consisted of initial polymerase activation at 94 °C for 2 min; 40 cycles of 94 °C for 45 s, 60 °C for 45 s and 72 °C for 90 s and a final elongation step at 72 °C for 5 min. In the second round PCR, 3 μl of product from the first PCR step was amplified using inner primer pairs (Table 1). Nested PCR conditions consisted of 95 °C for 5 min; 40 cycles of 94 °C for 1 min, 60 °C for 1 min and 72 °C for 2 min and a final elongation step at 72 °C for 10 min. The amplified products were visualized by electrophoresis on 1.5 % agarose gel stained with GelRed solution (Biotium).

The 871 bp OmpA fragments obtained were purified using a QIAquick Gel Extraction kit (Qiagen) and processed using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA). The reaction mixtures were loaded onto a 3730xL DNA Analyzers (Applied Biosystems). The primers used for sequencing were inner primer pairs. Nucleotide sequence data were assembled by Bioedit and residues corresponding to flanking primers were excluded from analysis. Sequences were submitted to the standard nucleotide BLAST search engine at the National Center for Biotechnology Information (blast.ncbi.nlm.nih.gov/Blast.cgi) to determine the genotype.

The authors would like to thank the volunteers and nurses at the University of Malaya Medical Center Obstetrics and Gynecology clinics for their assistance in sample collection. This study was supported by grants from Malaysia Ministry of Higher Education (HIR-MOHE E000013-20001) to WFW and Fundamental Research Grant Scheme (FRGS) FP027-2010B to NSS.