The current study was prospectively designed and performed at the Department of Microbiology, Aretaieion General Hospital, National and Kapodistrian University of Athens, from January 2012 to December 2017. A total of 125 Greek females of reproductive age (range, 22-45 years; median age, 32), undergoing vaginal/cervical fluid examination as prenatal screening were recruited into the present study. Patients were classified into two groups: 111 infertile females enrolled into an IVF program (group A) and 104 fertile females (having at least one child; Group B; considered to be the control). Epidemiologic data, symptoms and GTIs were compared between groups.

In all patients receiving IVF, a long ovulation induction protocol was applied, which included the daily nasal administration of gonadotropin releasing hormone (2 mg/ml; Synarela^®; Pfizer, Inc., New York, NY, USA) on the 21st day of the pre-treatment cycle, which included the administration of combined oral contraceptives (0.03 mg ethinyl estradiol and 3 mg drospirenone; each, Yasmin^®; Bayer Hellas AG, Athens, Greece) administered for 2 to 4 weeks. Following the downregulation of estradiol-2 by the aforementioned drugs (E2; <50 pg/ml) recombinant follicle stimulating hormone (FSH) was administered subcutaneously (beta-follitropin; Puregon^®; Organon International BV, OSS, The Netherlands) in a daily dose of 75-300 IU (depending on the patient's characteristics). When ≥3 follicles of 18 mm in diameter were detected via a trans-vaginal ultrasound, 10,000 IU of human chorionic gonadotropin (hCG) was administered subcutaneously (Pregnyl^®; Organon International BV) and oocyte pick-up was performed transvaginally via a thin needle 35-36 h later, under ultrasonography. Following oocyte removal, blood-free follicular fluid samples were centrifuged at 600 x g for 10 min, at 25˚C and supernatants were stored at -80˚C. The in vitro fertilization of oocytes was accomplished using the spermatozoa of the patient's respective husband and all embryo transfers were performed on day 3 with a Wallace catheter under ultrasound guidance.

Patients in group A were assessed for IVF success and for potential predictors of failure. Patients that successfully became pregnant (Group C; 32 patients) were compared with patients unable to achieve pregnancy (Group D; 79 patients) in regards to the type of pathogen isolated In patients scheduled for IVF, basic characteristics including E2 levels, the number of oocytes retrieved, the number of oocytes fertilized and the number of transferred embryos were recorded. Serum E2 levels (centrifuged at 600 x g for 10 min at 25˚C) were measured using the Abbott Architect i-1000SR auto-analyzer (Abbott Pharmaceutical Co., Ltd., Lake Bluff, IL, USA; sensitivity, 10 pg/ml; intra-assay and inter-assay coefficients of variability, 5.5 and 6.7%, respectively). Additionally, factors contributing to infertility including endometriosis, male factor (male infertility) or tubal factor (anatomical disorders) were reported.

In all patients, specimens of vaginal discharge were obtained from the posterior fornix for microscopy, amine testing, and gram staining at 25˚C for 5 min. Solution A (90% Crystal violet; 20 ml of 95% Ethanol) and Solution B (0.8 g Ammonium oxalate with 80 ml Distilled water) were mixed to obtain a crystal violet staining reagent. Samples were stored for 24 h at 25˚C and filtered through paper prior to use. At the completion of the Gram Stain, gram-negative bacteria stained pink/red and gram-positive bacteria stained blue/purple. Patients from which vaginal samples were not collected were excluded from the current study. Amine testing (‘whiff’ test) was performed by adding a drop of potassium hydroxide 10% to vaginal discharge samples. The test was considered positive if the typical fishy odor was produced. A gram-stained smear was examined via light microscopy (magnification, x1,000) and the presence of yeast and leukocytes was also determined. Specimens for yeast culture, were incubated at 37˚C for 24-48 h and placed in modified Stuart's medium and plated on Sabouraud dextrose agar (both from BioMerieux, Marcy l’Etoile, France). Vaginal samples were cultured for aerobic pathogens at 37˚C for 24-48 h and the automated system VITEK2 (BioMerieux) was used to identify the pathogens present.

BV was diagnosed via the Nugent scoring system (19). This method assesses the quantity of three different bacterial morphotypes: Large gram-positive rods (representing lactobacilli), small gram-variable rods (representing Gardnerella and Bacteroides/Prevotella species) and curved rods (representing the Mobiluncus species). On the basis of these results, samples were assigned a score from 0 to 10, with 1-3 being considered as normal, 4-6 being considered as intermediate bacterial count (where patients receive chemoprophylaxis treatment) and 7-10 being considered as BV. BV diagnosis was further verified using the Amsel criteria (20), which includes the presence of at least three of the following four findings: homogeneous vaginal discharge, vaginal pH >4.5, ‘fishy’ odour on addition of potassium hydroxide to vaginal fluid (positive ‘whiff test’) and the presence of clue cells in microscopy of wet preparation (20). When each test (Nugent scoring system with Amsel criteria) was positive for either the presence of fungus, trichomonas vaginalis or bacterial vaginosis, the diagnosis of BV was confirmed.

The presence of AV was determined using an ‘AV’ score (21). The score was calculated using high-power field light microscopy (magnification, x1,000) to assess the presence or absence of healthy lactobacilli, the number of leukocytes, the number of leukocytes with toxic granulation, the type of vaginal flora and the number of parabasal epithelial cells. An ‘AV’ score of <3 corresponds to no signs of AV, 3-4 to light AV, 5-6 to moderate AV and >6 to severe AV. Candidiasis was diagnosed in symptomatic patients when yeast culture in modified Stuart's medium and plated on Sabouraud dextrose agar was positive for any Candida species and no other pathogen was detected.

Ureaplasma urealyticum and Mycoplasma hominis were detected using the Mycoplasma IST2 kit (BioMerieux). The kit contained strips that provide information on the presence or absence of Ureaplasma urealyticum and Mycoplasma hominis, and also provides additional information on susceptibility to different antibiotics. Samples were vortexed rapidly at 3,500 rpm at 25˚C for 1 min and they were used (in their liquid form) to rehydrate the lyophilized growth medium provided in the kit. The Mycoplasma strips were then inoculated with 100 µl of the rehydrated growth medium overlaid with two drops of oil, then incubated at 37˚C and observed for colour changes. The results were interpreted following 24 and 48 h of incubation at 37˚C according to the manufacturer's protocol. Samples with titres ≥10⁴ CCU/ml were considered positive. Finally, Chlamydia trachomatis was detected using the COBAS AMPLICOR test kit (cat. no. 04341341 190; Roche Diagnostics, Indianapolis, IN, USA).

HPV was detected using the LINEAR ARRAY HPV Genotyping Test (Roche Diagnostics). This method detects 21 HPV genotypes of high risk (including 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 67, 68, 69, 70, 73 and 82) and 16 HPV genotypes of low risk (6, 11, 40, 42, 54, 55, 61, 62, 64, 71, 72, 81, 83, 84, IS39 and CP6108) in cervical cells from each group preserved in PreservCyt^® solution, (ThinPrep^® PreservCyt^® Solution; Hologic, Inc., Marlborough, MA, USA) according to the manufacturer's protocol. Additionally, anti-CMV, anti-Toxoplasma gondii and anti-Rubella IgG and IgM antibodies were measured in serum using a chemiluminescent microparticle immunoassay (Architect i-1000SR; Abbott Pharmaceutical Co., Ltd.). For the diagnosis of HIV-1/HIV-2 infection, the Architect HIV Ag/Ab Combo assay, a chemiluminescent microparticle immunoassay was utilized (Abbott Pharmaceutical Co., Ltd.). Additionally, serum Hepatitis B surface antigen, anti-HBV and anti-HCV titers were determined via an enzyme immunoassay using the ARCHITECT platform (Abbott Pharmaceutical Co., Ltd.) according to the manufacturer's protocol.

The incidence of demographics and symptoms were reported in all study groups (group A vs. group B) and subgroups of women receiving IVF (group C vs. group D). The type and number of symptoms as well as the type and number of pathogens were compared among all groups. To compare nominal data between two groups, the Chi-Square or Fisher test were utilized, while for all data expressed in numeric format, a Student's t-test was used. P<0.05 was considered to indicate a statistically significant difference. All P-values were calculated based on absolute values. Subgroup analysis was performed to determine potential predictors of fertility or IVF success. Furthermore to exclude any confounding factors and calculate the relative risk (RR) for the two groups, multivariate analysis using logistic regression was used. All statistical analyses were performed using IBM SPSS v.24 software (IBM Corp., Armonk, NY, USA).

Finally, the protocol for this research project was approved by a suitably constituted Ethics Committee of Aretaieion Hospital within which the work was undertaken and it conforms to the provisions of the Declaration of Helsinki (as revised in Tokyo 2004).

A total of 215 females undergoing prenatal cervical/vaginal fluid testing in Aretaieion hospital (within a period of 5 years) were included in the current study. These comprised 111 infertile females (Group A) scheduled for IVF and 104 fertile females (Group B). The mean age of patients did not differ significantly between the two groups, although a greater number of females from Group A were >30 years of age compared with Group B (P=0.0064). Additionally, fertile females had a mean number of 1.23 children. Group A had a higher education level compared with Group B (P=0.0001), as well as a higher smoking prevalence (P=0.007). However, no significant differences in symptoms were observed between groups. In Group A, only BV (P=0.0013) was significantly more prevalent. Furthermore, infertile females possessed >2 pathogens more frequently when compared with fertile females (P=0.006; Table I).

Of the 111 infertile females enrolled in the IVF program, 32 had a successful pregnancy (Group C) and the remaining 79 patients did not (Group D). The mean E2 levels for all patients undergoing IVF was 2234.38±1614.06 pg/ml, with E2 levels being higher in Group C compared with Group D (2853.75±1393.58 vs. 1983.49±1619.40 pg/ml; P=0.007). Furthermore, the number of fertilized oocytes (P=0.002) and the number of resulting embryos (P=0.004) was higher in Group C compared with group D. Male factor was more prevalent in Group C (P=0.0009) while tubal factor was more prevalent in Group D (P=0.028). Other factors, including endometriosis or ovulation disorder, did not significantly differ between the two groups. Of the pathogens examined in the current study, only Mycoplasma was detected more frequently in Group D (P=0.03; Table II).

The results of multivariate analysis revealed that a higher education [relative risk(RR)=2.31; 95% confidence interval (CI), 1.825-2.939; P<0.0001] was the strongest predictor associated with infertility. Other factors associated with infertility were as following: An age of >30 years, smoking, the presence of decreased lactobacillus species, the presence of >2 pathogens and the presence of BV (Table III). Furthermore, tubal factor (RR=1.32; 95% CI, 1.064-1.649; P=0.012), E2 levels <2,500 pg/ml (RR=1.78; 95% CI, 1.265-2.501; P=0.0009) and Mycoplasma infection (RR=1.36; 95% CI, 1.110-1.660; P=0.003) were determined to be the strongest predictors for IVF failure (Table III).