Female and male partners of 25 and 28 years old, respectively, comprised a heterosexual married couple from Saratov city. During the past six years, they had frequent sexual contacts to attempt pregnancy. Moreover, this couple had been engaged in receptive oral and genital, but not anal intercourse. Each individual had regular annual physical and laboratory examinations by a gynecologist or andrologist, an endocrinologist and a family physician. Both partners had neither somatic nor endocrinal abnormality nor symptoms, nor any clinically apparent genital manifestations of Chlamydia or other Sexually Transmitted Infections (STIs). Nevertheless, they demonstrated marked symptoms of bacterial conjunctivitis, such as unilateral hyperemia and mucopurulent discharge that were observed for the last 6 months. During the last three years, the patients underwent several unsuccessful attempts at IVF. The female partner disclaimed any sexual contacts outside legal marriage. However, the male partner reported frequent and irregular relationships with at least 3 to 5 female partners annually. Both partners denied any pernicious habits or addiction to alcohol, as well as animal or homosexual contacts. Both of them declared that they had never traveled to Sweden or other EU countries.

Clinical specimens were taken in October 2013, from both genital (urethra & cervix) and extra-genital (oropharynx, conjunctive, blood) sites of each individual one year after a single course of antibiotic therapy. Clinical samples from similar sites of both partners were also obtained in October, 2012 prior to initiation of antibiotic therapy according to recommendations of the European STD Guidelines, and CDC STDs Treatment Guidelines [16, 17]. All procedures performed in studies involving human participants were in accordance with the ethical standards of the 1964 Helsinki declaration and its later amendments or comparable ethical standards. The research protocol was approved by the Human Bioethics Committee of the Saratov Scientific and Research Veterinary Institute No. IRB00008288 (http://ohrp.cit.nih.gov/search/IrbDtl.aspx), and both participants provided written informed consent.

Semen analysis was performed on the male partner according to the procedures outlined by the World Health Organization (WHO) [18], twice in October 2012, and three times in August 2014 by means of an automated sperm analyzer SQA-V (MES, Austria-Israel).

Direct Immunofluorescent Test (DIFT) with a mixture of chlamydia monoclonal antibodies (MAbs) to synthetic serogroup-specific MOMP-associated epitopes of 17 С.trachomatis serovars A-L (NiarMedic Plus, Moscow, Russia) [19] was used to test for chlamydial Elementary Bodies (EBs) in individual clinical samples.

Total DNA was isolated from clinical samples including blood specimens using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany). Each DNA sample was tested by: (i) End-point PCR with conventional and fluorescent product detection using commercial kits developed by the Central Research Institute of Epidemiology, Moscow, Russia (AmpiSens-Eph and AmpiSens-FL, respectively) to reveal the plasmid DNA of the typical C. trachomatis strains (wtCT) in the clinical material [20]; (ii) PCR with two pairs of primers, such as orf2_F/orf2_R and orf8_F/orf8_R (Table 1), designed by us to recognize genes orf2 and orf8 of C. trachomatis plasmid (PCR-orf2 and PCR-orf8, respectively).

We used plasmid pBour of C. trachomatis strain E/Bour (GeneBank accession no. HE603212) to obtain sequences of both orf2 and orf8 genes. These primers amplified fragments of 150 bp and 145 bp for orf2 and orf8, respectively. The reaction was done in a 30 µL volume with 10 pmol of each primer, and 1 µL of DNA isolated from the clinical sample. The conditions of amplification were as follows: 94° C for 2 min, 15 cycles of 94° C for 30 sec, 61° C for 20 sec, and 72° C for 30 sec followed by 45 cycles of 94° C for 30 sec, 58° C for 20 sec, and 72° C for 45 sec with the final extension at 72° C for 10 min; (iii) PCR with the pair of primers developed by Catsburg et al. [21] to identify the presence of DNA of novel Swedish strains of C. trachomatis (nvCT) – PCR-nvCT, respectively. The forward primer swCT_serE_F was used in the original design. The reverse primer swCT_serE_R was modified by us in a single nucleotide to fit better the sequence of genovar E (Table 1). These primers amplified a product with a size of 474 bp and 97 bp in the presence of wtCT and nvCT templates, respectively. The DNA of genovar E of C. trachomatis strain E/Bour (#VR-348BD, ATCC) was used as a control for each experiment. The reaction was done in 30 µL with 10 pmol of each primer, and 1 µL of DNA isolated from the clinical sample. The conditions of amplification were the following: 94° C for 2 min, 35 cycles of 94° C for 30 sec, 57° C for 30 sec, and 72° C for 45 sec followed by the final extension at 72° C for 10 min. Then, the fragments of amplification were purified from gel by using the QIAquick Gel Extraction Kit (Qiagen), and sequenced commercially by the EVROGEN Company (Moscow, Russia).

The individual C. trachomatis-positive DNA samples were genotyped by sequencing of the variable domain 2 (VD2) region of ompA as previously described by Quint et al. [22]. This system of genotyping included three forward and four reverse primers that in a multiplex format could amplify the VD2 region of all known C. trachmatis genovariants generating amplicons of 157 to 160 bp. The fragments of amplification were extracted from gel and sequenced. To determine the genovar, the sequence reads from the forward and reverse primers were searched in the GenBank for homology by the BLAST program.

The ompA gene was amplified with the use of primers F11 and B11, which produced the 1156 bp fragment containing all four variable regions of the gene [23]. To sequence the amplicons, additional primers complementary to the internal part of the ompA gene were designed (MOMP-E_F1.seq; MOMP-E_F3.seq; MOMP-E_F5.seq; MOMP-E_R2.seq; MOMP-E_R4.seq; MOMP-E_R6.seq, Table 1, Fig. S1) allowing reading of the fragment sequence at least twice from the direct and reverse chains. The sequencing was done commercially by the EVROGEN Company. The consensus sequence of the ompA derived from the DNA of clinical sample was compared with the reference sequences of the strains of genovar E, such as E/Bour (HE601870), E/IU-TC0755ut (FJ261948), and E/H38 (AF265238), as well as strains of the genovar D, such as D/SotonD1 (HE601798), D/SotonD5 (HE601799) as reported by Harris et al. [13]. The nucleotide sequence alignment was done using the MultAlin (http://multalin.toulouse.inra.fr/multalin/multalin.html program).

All representative genovar sequences reported in this research were deposited in GenBank (accession numbers MF288583-MF288585).

Each DNA sample was also tested by real-time multiplex commercial kit developed by the Vector-Best (Novosibirsk, Russia) to reveal DNA of Ureaplasma spp., Mycoplasma hominis, Mycoplasma genitalium, Trichomonas vaginalis, Neisseria gonorrhoeae, Candida albicans, Gardnerella vaginalis, cytomegalovirus, herpes simplex virus (HSV) types 1/2 and human papillomavirus (HPV) types 16 and 18 [20].

C.trachomatis immunoglobulin G (IgG) antibody titres (CAT) of the patients were tested with an ELISA kit (“VECTOR-BEST” Company, Novosibirsk-117, Russia) according to the manufacturer’s instructions. Results were considered to be positive at ELISA titers ≥1:32.

The initial screening in October 2012 prior to the planned IVF procedure revealed the lack of С.trachomatis DNA in the genital specimens (cervix and urethra) of both partners using AmpliSens Chlamydia trachomatis-Eph and -FL kits (AmpiSens-Eph and AmpiSens-FL). Nevertheless, specific chlamydial antigens were identified in these clinical specimens by DIFT (data not shown).

Further we tested the samples for the presence of chlamydial DNA in PCR by using primers targeting different parts of the C. trachomatis cryptic plasmid (orf2 and orf8, PCR-orf2 and PCR-orf8), as well as primers swCT_serE_F/swCT_serE_R (Table 1) flanking the area of deletion identified in the orf1 of the Swedish strains (nvCT, PCR-nvCT, respectively). Although amplification with the primers recognizing both orf2 and orf8 provided negative results, specific DNA of the Swedish nvCT variant bearing a cryptic plasmid with a 377-bp deletion in the orf1 was detected in the samples from the genital sites tested. The exact position of the 377-bp specific deletion within the orf1 plasmid gene was confirmed by sequencing (data not shown). Additional examination of the female patient revealed scanty mucous vaginal discharge and moderate hyperemia in uterine and cervical mucosa in the absence of symptoms or any other clinical manifestations. After a single course of antibiotic therapy with doxycycline (Unidox Solutab®) 100 mg twice a day for seven days, and subsequent analysis of clinical specimens one year later (October 2013), her genital samples (urethra and cervix) were negative for wtCT DNA; however, the PCR analysis still revealed the presence of nvCT DNA in her cervix (Table 3). The urethral specimen of the male partner was also initially (October, 2012) positive for nvCT alone (Table 2). Later (October, 2013), in contrast to the results of the female patient testing, we detected chlamydial DNA with primers recognizing both wtCT and nvCT variants suggesting mixed infection. The presence of chlamydial EBs in clinical samples from genital sites of both partners was confirmed by DIFT (data not shown).

During the first screening in October 2012, both AmpiSens-Eph and AmpiSens-FL kits failed to detect the wtCT DNA in extra-genital specimens of both partners. PCR-orf2 did not detect the presence of Chlamydia. Nevertheless, PCR-orf8 revealed Chlamydia in specimens derived from two out of three extra genital sites (conjunctiva and oropharynx). Moreover, PCR-nvCT produced positive signals with all three of their extra-genital samples, namely conjunctiva, oropharynx and blood (Table 2). Testing the specimens obtained one year later showed that both male and female patients were positive for Chlamydia in all extra-genital sites. AmpiSens-Eph and AmpiSens-FL were less efficient, although both PCR-orf2 and PCR-orf8 produced positive reactions in most specimens. PCR-nvCT detected Chlamydia in all samples. Overall, both wtCT and nvCT were detected indicating a mixed infection in extra-genital sites of this couple. Chlamydial EBs were found by DIFT in all clinical samples.

The ompA genotype was identified as genovar E in all clinical samples from genital sites and in each of the three extra-genital sites tested (Tables 2 and 3). Only novel ‘Swedish’ variant (nvCT) of С.trachomatis genovar E was detected in the patients at the first screening (Table 2). One year later, specific chlamydial DNA of genovar D was also found in extra-genital sites of both partners, namely in conjunctiva and oropharynx of the female partner and in oropharynx of her husband, indicating a mixed infection in these extra-genital sites (Table 3). When we determined the nucleotide sequence of the ompA gene of these genovar D variants, we identified SNPs that are not present in the current version of the GenBank for this genotype. The SNP at position 971 was matched in the samples obtained from the oropharynx of both partners, suggesting the identity of the strains that infected both patients. This SNP displayed a C→A substitution (compared with D/SotonD5) that resulted in an alanine-to-aspartic acid residue change within the VD4 region of the MOMP. Moreover, two additional SNPs were found in the conjunctiva of the female patient, those at positions 991 and 1020 (Table 3). The SNP at position 991 was also located within the VD4 region, and displayed an A→G substitution resulting in a threonine-to-alanine amino acid change. The SNP at position 1020 was mapped at the second amino acid after the end of the VD4 region. The A→C substitution did not change the amino acid residue. A single SNP for the genovar E was identified at position 997 in the blood sample of the male patient. This SNP displayed a G→A substitution (compared with E/Bour) that resulted in an alanine-to-threonine amino acid change within the VD4 region of the MOMP. The ompA sequence found in this clinical specimen was identical with that existing in the strains E/IU-TC0755ut and E/H38 found in the GenBank. Importantly, since AmpiSens-Eph and AmpiSens-FL recognized the typical strains of С.trachomatis (wtCT), such as E/Bour, PCR-nvCT detected the ‘Swedish’ variant with the 377-bp deletion in the orf1 of the cryptic plasmid. The ompA sequence analysis revealed the strain with the SNP at position 997, and there were at least three different strains of the genovar E present in the clinical specimens of our two patients.

The female partner demonstrated a CAT IgG titre of 1:160 at both initial screen and on re-examination one year later. No CAT IgG was registered in the male partner during this study.

The characteristics of the sperm in samples obtained in 2014 in comparison with those taken in 2012: progressive were, 29.8% versus 45% (the lower reference limit is 32%); non-progressive, 19.1% versus 23.4%; immotile, 51.1% versus 21.6%; vitality 46% versus 68% (the lower reference limit is 58%); pH 7.9 versus 7.6 (normal range is 7.2 – 7.8). Other major sperm parameters, such as total fluid volume, and spermatozoa concentration, were 4.5 ml (the lower reference limit is ≥ 1,5 ml) and 76.5 × 10⁶/ml (the lower reference limit is 15 × 10⁶ spermatozoa per ml), respectively, were maintained at the normal level. Overall, the sperm quality of the male partner collected in 2014 had declined after two years of chlamydial infection.