Correlation between hysteroscopic features and specific microbial species in women with chronic endometritis.

Objective and rationale: Chronic endometritis (CE) has recently been associated with unexplained infertility and recurrent miscarriages. The current gold standard for CE detection is histopathological examination. However, office hysteroscopy and endometrial cultures are also significant, due to the possible link between CE and various microorganisms. Bacterial colonization of the endometrium has been associated with reduced success rates of in vitro fertilisation embryo transfer. Few studies have tried to correlate CE hysteroscopy findings with pathogenic microorganisms. This prospective cohort study sought to establish whether hysteroscopic diagnostic lesions correlate with specific microbial species. Methods: The study encompassed women undergoing diagnostic tests for a range of subfertility health issues. 189 women completed the standard office diagnostic hysteroscopy (DH). 181 had also endometrial samples taken for microbial culture investigation. Correlation analysis (χ2 and Fisher's exact test) between hysteroscopic findings suggestive of CE and endometrial cultures was carried out. Logistic regression models were also fitted to measure whether a positive endometrial culture could affect CE conditions. Results: A significant association of E. coli was observed between the hysteroscopically characterized CE + group with focal hyperplasia, when compared to the non-CE group. Logistic regression analysis revealed that women positive for E. coli were 4.423 times more likely to have focal endometrial hyperplasia. No other significant correlations were identified between DH and positive endometrial cultures. Conclusions: The presence of E. coli in the endometrium was significantly correlated with focal hyperplasia findings from hysteroscopy, emphasizing the importance of microbial cultures in the diagnosis and targeted treatment of CE in women with subfertility.

Performance characteristics of the boson rapid SARS-cov-2 antigen test card vs RT-PCR: Cross-reactivity and emerging variants.

Background: SARS-CoV-2 virus has undergone several mutations on its genome, since the onset of the pandemic. Multiple variants of concern (VOC) have emerged including Alpha, Beta, Gamma, and Delta with the more recent one being the Omicron (B.1.1.529). Specific rapid antigen tests (RADs) have been used for the detection of SARS-CoV-2. However, since the emergence of new VOCs, the performance characteristics of these RADs needs to be re-evaluated. Objectives: The main purposes of this clinical study were to determine the diagnostic sensitivity and specificity of the BOSON Rapid Antigen Test compared to the gold standard real time RT-PCR and to determine the ability of the RAD to accurately depict different VOC. Additionally, the cross reactivity to other viruses and pathogen, as well as, the possible interference of non Covid-19 hospitalized patients for various causes, were investigated. Results: A total of 623 individuals (symptomatic) were tested. The sensitivity, specificity and accuracy of the BOSON RAD was 95.27%, 100% and 98.45% (n = 448), meeting the WHO recommended standards. Additionally, the Delta (83.33%, Ct < 34) and Omicron (100%, Ct < 26) VOC were determined with high sensitivity. Also, there was no interference from hospitalized, non-Covid 19 patients, and no cross-reactivity was detected. Conclusions: The study showed that this RAD could rapidly identify individuals with SARS-CoV-2, including those with the new dominant Omicron VOC, with no cross reactivity from other pathogens.

Detection of Chlamydia trachomatis inside spermatozoa using flow cytometry: Effects of antibiotic treatment (before and after) on sperm count parameters.

There is increasing evidence that Chlamydia trachomatis (CT) infection can directly affect male fertility. However, only few have investigated the effects of CT on semen parameters, and mostly with inconclusive results. The main aims of this study were to identify CT inside spermatozoa, and the possible pre and post antibiotic treatment effects on the overall semen parameters. We developed a flow cytometric method for the detection of CT inside spermatozoa (SPI™). Briefly, sperm cells were fixed, membrane permeabilized and DNA was loosened using DNAse. Sperm cells were incubated with a primary monoclonal antibody against CT and with a secondary fluorescent antibody (vs primary), and analysed using a flow cytometer. Of 2415 infertile individuals, 48.61% were found positive for CT. 170 CT+ samples were included in the CT antibiotic treatment study. 78.82% (134/170) of the CT+ showed a significant reduction in the percentage of the iCT infected spermatozoa after the antibiotic treatment; 59.70% (80/134) decreased to non-detectable levels. Spermcount data were also recorded. Spermatozoa morphology (normal and teratozoospermia index, TZI) and motility (fast progressive and non-progressive spermatozoa) were statistically significant altered in CT+ pre-treatment vs control group. CT antibiotic treatment showed statistically significant effects on normal spermatozoa morphology, mid-piece and tail defects, and TZI. The study demonstrated that semen flow cytometric analysis of semen could be a valuable tool for faster and accurate identification of individuals with asymptomatic CT infection. It also identified a positive effect of antibiotic therapy on semen parameters, that could help males with infertility.

Evaluation of the Boson rapid Ag test vs RT-PCR for use as a self-testing platform.

The gold standard test available for detecting COVID-19 patients is Real Time RT-PCR. However, this method is expensive, needing special equipment and skilled laboratory staff. Recently, less expensive antigen tests have become available, that could easily and rapidly identify new COVID-19 cases. Our objective was to evaluate the Boson Rapid Antigen Test Card versus the RT-rtPCR, using samples taken both by laymen (self-testing) and professionals. The sensitivity, specificity and accuracy rates were, 98.18%, 100.00%, and 99.28%, respectively. The positive and negative predictive values were 100.00% and 98.82%, respectively. The detection rate for asymptomatic patients was 90.48%, and detection rate for Ct values ≥30 was 91.67%. Our results indicate a high coincidence rate between the Boson and the referencing RT-rtPCR method, meeting the performance standards recommended by the WHO. Therefore, this test could facilitate a fast self-testing screening method, for the detection of infected individuals.

Large-scale screening for factor V Leiden (G1691A), prothrombin (G20210A), and MTHFR (C677T) mutations in Greek population.

Background and aims: To provide a fair estimate of the prevalence of factor V Leiden (FVL) (G1691A), prothrombin (G20210A), and MTHFR (C677T) mutations in the Greek population. Methods: We genotyped a representative sample of 974 apparently healthy Greek adults by the method of real-time PCR and we calculated the allele frequencies of factor V Leiden (FVL) (G1691A), prothrombin (G20210A), and MTHFR (C677T) mutations. In addition, we determined the frequency of co-occurrence of FVL (1691A) and prothrombin (20210A), FVL (1691A) and MTHFR (677T), prothrombin (20210A) and MTHFR (677T) mutations. Results: Τhe career frequencies of FVL (1691A), prothrombin (20210A), and MTHFR (677T) alleles were 7.5%, 4.5%, and 49.3% while the allele frequencies were 4%, 2.25%, and 39.5%, respectively. The coexistence of the allele frequencies combinations of two, FVL (1691A) and Prothrombin (20210A), FVL (1691A) and MTHFR (677T), prothrombin (20210A) and MTHFR (677T) was found in 1 (0.9%), 29 (3.5%), and 22 (3%) samples, respectively. Triple heterozygous carriers were not found. Conclusion: Allele frequencies of the two (FVL and MTHFR) mutations are higher compared with published data. The large sample size of our study enhances the validity of our results and suggests a biological affinity of Greek population with Southern Italian populations.

Improvement of Sperm Quality in Hyperviscous Semen following DNase I Treatment.

Semen hyperviscosity impairs sperm motility and can lead to male infertility. This prospective study aimed at assessing the ability of exogenous DNase in improving sperm quality, taking into consideration that DNase has been found in the seminal plasma of several species and that neutrophils release chromatin in order to trap bacteria. A total of seventy-seven semen samples with high seminal viscosity (HSV) as the study group and sixty-two semen samples with normal seminal viscosity (NSV) as the control group were compared in this analysis. These semen samples were divided into three groups of receiving treatment (a) with DNase I at 37°C for 15 min, (b) by density gradient centrifugation, and (c) with a combination of the above two methods. Following a fifteen-minute treatment of hyperviscous semen, the motility of spermatozoa in 83% of semen samples increased to a statistically significant degree. On the contrary, DNase treatment of semen with normal viscosity had no such effects. The above treatment was also accompanied by a significant increase in the percentage of normal spermatozoa, resulting in a major decrease of the teratozoospermia index. Comparison between semen samples that underwent density gradient centrifugation following DNase I treatment, to those collected after density gradient treatment alone, showed that in the first case the results were more spectacular. The evaluation of each preparation in terms of yield (% total progressively motile sperm count after treatment in relation to the initial total sperm count) revealed that the combined approach resulted in 29.8% vs. 18.5% with density treatment alone (p=0.0121). DNase I treatment results in an improvement of sperm motility and morphology and could be beneficial to men with hyperviscous semen in assisted reproduction protocols.

Intracellular ESAT-6: A new biomarker for Mycobacterium tuberculosis infection.

BACKGROUND: Early secreted antigenic target 6 (ESAT-6) is a virulent factor of Mycobacterium tuberculosis (MTB). The identification of intracellular (i/c) ESAT-6 in host cells would be a direct marker of MTB infection. We developed a method to detect i/cESAT-6 by flow cytometry. The aim of this study is to investigate the expression of i/cESAT-6 in the host cells of individuals with MTB infection. METHODS: The expression of i/cESAT-6 was examined in the blood of 58 active TB patients, in 10 naïve to TB infection controls, in 17 patients who completed anti-TB treatment, and in 56 close contacts with an index TB case. Additionally, it was examined in the sputum of 12 active TB patients. RESULTS: The i/cESAT-6 was positively detected in the blood of 52 out of 58 (90%) active TB patients. All naïve to TB infection controls were negative. Three out of 17 (18%) patients at the end of anti-TB treatment were positive. Twenty-six out of 56 (46%) close contacts tested positive. The i/cESAT-6 was detected in all culture positive TB sputum specimens. CONCLUSIONS: The i/cESAT-6 is a promising biomarker of MTB infection that could be used in the evaluation of active TB patients and in the diagnosis of latent TB infection. Further studies are needed to validate its potential diagnostic role. © 2014 International Clinical Cytometry Society.

Molecular investigation of menstrual tissue for the presence of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis collected by women with a history of infertility.

AIM: At present, routine laboratory investigation of the infectious agents implicated in female genital infections is mainly based on culture/direct fluorescence antibody (DFA) (immunofluorescence antibody test) results of cervicovaginal secretions. In this study the use of the menstrual tissue is introduced for the molecular detection of pathogens which are implicated in female infertility. MATERIAL AND METHODS: Cervicovaginal secretions and menstrual tissue samples of 87 women (mean age 34.07 ± 5.17) experiencing infertility problems were screened for Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis presence using polymerase chain reaction (PCR, light cycler-PCR). Cervicovaginal secretions were also tested by the culture/DFA technique. The results were compared using the binomial test. RESULTS: In the overall study group, the prevalence of C. trachomatis was 25.3%, 18.3%, and 13.8%, the prevalence of U. urealyticum was 18.3%, 16.09% and 12.6% and the prevalence of M. hominis was 13.7%, 19.5% and 8.0% in the menstrual tissue, cervicovaginal secretions using PCR and cervicovaginal secretions culture/DFA, respectively. A statistically significant difference was revealed between the two methods for all three microbes and between menstrual tissue and cervicovaginal secretions PCR for chlamydia. CONCLUSIONS: The use of menstrual tissue along with the PCR method seems to be an effective and thus novel alternative for the investigation of the infectious agents lying in the genital tract. One of the main advantages of this technique compared to cervicovaginal secretions is that it is non-invasive and the sample can be collected at home, thus allowing the early detection and treatment of a condition that can otherwise lead to serious consequences, such as tubal obstruction, pelvic inflammatory disease, ectopic pregnancy, spontaneous abortions and unexplained infertility.

The effect of valacyclovir treatment on natural killer cells of infertile women.

PROBLEM: The aim of this study was to investigate the effect of valacyclovir treatment on natural killer (NK) cell concentration in the peripheral blood of infertile women. METHOD OF STUDY: Peripheral blood NK cell concentration of 104 non-pregnant women with a history of infertility was determined by flow cytometry. The controls were 14 fertile non-pregnant women. A cohort of 42 out of 104 women--whose NK cell levels were 175/microL or higher--was prospectively studied for the presence of HSV-1, 2, VZV, cytomegalovirus, HHV-6, HHV-7 and HHV-8 DNA in the peripheral blood and was orally administered valacyclovir (open label study). RESULTS: Herpes virus DNA was detected in 64.3% of the 42 women examined. Prior to valacyclovir treatment mean NK cell concentration in herpes-negative group was statistically higher from control group but lower from herpes positive group (P = 0.0007, ANOVA). Following valacyclovir treatment the mean NK cell concentration was statistically decreased in all studied women (P = 0.000453), in herpes-negative (P = 0.01622) and in herpes positive group (P = 0.0056). Sufficient decrease was observed in 31 (73.8%) of 42 women who received the drug. CONCLUSIONS: Valacyclovir treatment is associated with a decrease of NK cell levels in most of the women with a history of infertility.

Fraction of the peripheral blood concentration of CD56+/CD16-/CD3- cells in total natural killer cells as an indication of fertility and infertility.

OBJECTIVE: To determine whether the peripheral blood concentration of CD56(+)/CD16(-)/CD3(-) cells, the main natural killer (NK) subpopulation that colonizes the endometrium in the middle and late secretory phase, can be related to fertility or infertility status. DESIGN: A case control study. SETTING: Immunopathology department of an infertility laboratory. PATIENTS: A total 99 women were selected (group I: consecutive spontaneous aborters, n = 25; group II: sporadic spontaneous aborters, n = 30; group III: infertile, n = 33; group IV: controls, n = 11). INTERVENTION: Immunophenotyping of women grouped according to their fertility status. MAIN OUTCOME MEASURES: Peripheral blood lymphocytes were examined by two- and three-color flow cytometry. RESULTS: A statistically significant association between endometrial-type peripheral blood (PB) NK cell concentrations and fertility status (groups I and IV vs. groups II and III) was documented. The %(/TOTAL PB)(CD56+CD16-CD3-) NK cells was significantly higher [1] in fertile (groups I and IV) than in sporadic aborters/infertile (groups II and III) women, [2] in group I when compared with groups II and III, and [3] in group IV when compared with groups II and III. CONCLUSIONS: This study indicates that diminished numbers of the %(/TOTAL PB)(CD56+CD16-CD3-) NK cells are related to sporadic aborters and infertile women. Thus, the fraction could be used as an indicator of subsequent successful implantation and maintenance of gestation.