Determination of Autophagy in Human Cervicovaginal Smears by Cytological and Immunocytochemical Methods.

Background: Autophagy is a catabolic process whereby organelles and long-lived proteins are recycled through lysosomes to maintain cellular homeostasis. This process is being widely studied using culture techniques and animal models; however, cervicovaginal smears have not been used to detect autophagy. Aims: Our study aims to detect and evaluate autophagy in normal, malignant, infectious, and atypical cells in cervicovaginal smears by using cytological and immunocytochemical methods. Materials and Methods: Papanicolaou-stained 200 cervicovaginal smears were examined and 55 of 200 (27.5%) smears containing negative for intraepithelial lesion or malignancy (NILM) with identifiable infections and/or reactive/reparative changes (INF); briefly, NILM-INF (n = 31, 56.4%), atypical (n = 4, 7.3%), and malignant cells (n = 20, 36.3%) were evaluated as a study group. One hundred forty-five of 200 (72.5%) normal smears were accepted as the NILM without any identifiable infections (control group). The autophagy marker protein Microtubule-associated protein 1 light chain 3 A (MAP1LC3A) was used for immunocytochemical examination. Results: The staining intensity of the MAP1LC3A protein and autophagy positivity were lower in the malignant cells; however, they were higher in the NILM-INF and atypical cells. A statistically significant correlation between the malignant and normal cells was obtained for the autophagy positivity (P = 0.012). In view of the staining intensity of MAP1LC3A protein by the H-score method, a significant correlation was found between the NILM-INF and the normal cells (P = 0.015). Conclusions: Autophagy was detected in various cervicovaginal smears for the first time in this study. Our findings indicate that an autophagy process is essential in infectious cells as well as in the transformation of atypical cells into malignant cells in carcinogenesis.

Bacterial vaginosis in association with spontaneous abortion and recurrent pregnancy losses.

CONTEXT: Bacterial vaginosis (BV) is related to the increased risk of miscarriage, preterm labor, and postpartum endometritis. AIMS: The aim of this study was to evaluate the association between BV and the history of spontaneous abortion and recurrent pregnancy losses. We also examined periods of gestation, including the first and second trimester miscarriages. MATERIALS AND METHODS: The study population consisted of 200 fertile women. Sixty one (30.5%) of 200 women had the history of a spontaneous abortion in the last six months (N = 30) and at least three recurrent pregnancy losses (N = 31). BV was diagnosed either by using Papanicolaou staining, Gram staining, or by culturing with BV-associated bacteria, Gardnerella vaginalis. RESULTS: The presence of BV was statistically associated with the history of a spontaneous abortion in the last 6 months (P < 0.05), whereas there was no significant relationship between BV and recurrent pregnancy losses (P > 0.05). These women were also evaluated in view of periods of gestation. Forty-seven (77%) of 61 women had first trimester miscarriage (≤12 weeks) and 14 (23%) of 61 women had second trimester miscarriage (>12 weeks). There was a statistically significant relationship between BV and second trimester miscarriage (P < 0.05). Positive BV findings were not associated with discharge, itching, and pain (P > 0.05). CONCLUSION: BV may contribute to spontaneous abortion and second trimester miscarriage.

The relationship between beta-catenin and apoptosis: A cytological and immunocytochemical examination.

Disruption of the adhesive role of beta-catenin by caspases has been reported; however, the relationship between the Wnt/beta-catenin signaling pathway and apoptosis remains unclear. Therefore, we aimed to evaluate squamous epithelial cells in cervicovaginal smears by using cytological and immunocytochemical methods to observe changes in the presence and localization of beta-catenin during apoptosis, death receptor-, and mitochondria-mediated apoptosis. We investigated 224 cervicovaginal smears using the Papanicolaou method. Anti-beta-catenin and anti-cleaved caspase 3, 8, and 9 antibodies were used for immunocytochemical staining. Apoptotic cells were negative for beta-catenin. This showed that the Wnt/beta-catenin signaling pathway was inactive in apoptotic cells. However, beta-catenin showed intense positivity in the membrane, cytoplasm, and nucleus of non-apoptotic epithelial cells around these apoptotic cells. Therefore, the Wnt/beta-catenin signaling pathway was active in non-apoptotic epithelial cells, and this activity in non-apoptotic cells may have been induced by apoptotic cells. A highly significant association between the presence of death receptor-mediated apoptosis and the activity of the Wnt/beta-catenin signaling pathway was also found (P<0.001). In conclusion, the Wnt/beta-catenin signaling pathway was found to be inactive in apoptotic cells, but apoptotic cells may induce the Wnt/beta-catenin signaling pathway in non-apoptotic cells to compensate for a decrease in epithelial cells because of apoptosis in order to maintain epithelial tissue integrity.

Evaluation of the relationship between fungal infection, neutrophil leukocytes and macrophages in cervicovaginal smears: Light microscopic examination.

BACKGROUND: Right after opportunistic fungi become pathogenic, they face immune system cells including macrophages and neutrophil leukocytes. Although the relationship between fungi and immune cells are being widely studied by using animal models and culture techniques, cervicovaginal smears have not been used to evaluate this interaction yet. AIM: The aim of this study was to investigate the interactions between fungal infection, macrophages and neutrophil leukocytes in cervicovaginal smear. MATERIALS AND METHODS: Papanicolaou-stained cervicovaginal smears from 2307 women, aged between 18 and 73 years, were examined by light microscopy. Periodic acid-Schiff stain was also used to confirm the presence of fungal cell walls. RESULTS: Fungal infections were detected in 239 of 2307 patients (10.4%), and these cases were taken as the study group. Cases without any infectious agents (n = 1800, 78%) were considered as the control group. When the study and control groups were statistically compared in view of macrophages and neutrophil leukocytes, a significant relationship between presence of fungal infection, macrophages and neutrophil leukocytes was detected (P < 0.05). Furthermore, macrophages and neutrophil leukocytes were found to work against the fungal infection together (P < 0.05). Additionally, when the relationship between the existence of yeast or filamentous forms and these immune cells were evaluated, a significant correlation was not found (P > 0.05). CONCLUSIONS: Our findings indicate that macrophages and neutrophils may play a determining role in host defense against fungal infection together, but neither yeast nor filamentous forms affect the presence of neutrophil leukocytes and macrophages. As a result of this, both yeast and filamentous forms may have pathogenic effects.

Interactions of actinomyces -like organisms with host cells: light microscopic examination.

OBJECTIVE: To determine the interactions of Actinomyces-like organisms (ALOs) with host cells, including vaginal epithelial cells, polymorphonuclear leukocytes (PMNLs) and erythrocytes, using Pap smear microscopy and based on their light microscopic appearances. STUDY DESIGN: Cervicovaginal samples obtained from 200 patients were examined by both Pap smear microscopy and anaerobic culturing. Since the results obtained by these methods were not concordant for diagnosis of genital Actinomyces, the results of Pap smear microscopy were used as a reference, and the smears with ALOs were carefully screened with regard to interactions of ALOs with host cells. RESULTS: ALOs were detected as attached to vaginal epithelial cells, PMNLs and erythrocytes via their filament-like structures. At some attachment sites, the epithelial cell membrane and filaments of ALOs were almost fused with each other. A group of PMNLs surrounded the ALOs. However, ALOs were observed to form colonies to evade phagocytosis by PMNLs. At the connection points between erythrocytes and ALOs, the findings of interest were the changes in the shapes of the erythrocytes and filament-like structures of the ALOs on the erythrocyte membrane. CONCLUSIONS: The adhesiveness of ALOs can be observed in routine Papanicolaou-stained cervicovaginal smears at light microscopic level.

Comparison of PCR, culturing and Pap smear microscopy for accurate diagnosis of genital Actinomyces.

Members of the genus Actinomyces, Gram-positive, non-spore-forming anaerobic bacteria, are normal inhabitants of the mucosal surfaces of the oral, gastrointestinal and genital tracts. Identification of these bacteria using conventional methods is generally difficult because of their complex transport and growth requirements and their fastidious and slow-growing nature. However, in recent years, the advancement of molecular techniques has provided much improved identification and differentiation of closely related Actinomyces species. The aim of the present study was to evaluate the efficacy of the PCR technique in the diagnosis of genital Actinomyces in comparison with culturing and Papanicolaou (Pap) smear microscopy. Multiple sampling was conducted from 200 women using smear microscopy, culturing and PCR. Cyto-brushes were smeared on glass slides and stained using the routine Pap technique. Culturing was performed from a sterile swab, and Actinomyces were determined using the BBL Crystal ANR ID kit. PCR was performed from a second swab, and the Actinomyces type was determined using type-specific primers designed in our laboratory. Only one vaginal fluid sample (0.5%) revealed Actinomyces-like organisms on Pap smear examination. Actinomyces were detected in nine samples (4.5%) using the BBL Crystal ANR ID kit. Using PCR, eight samples (4%) were found positive for Actinomyces. No specimens that gave positive results by Pap smear microscopy and culturing could be confirmed by PCR. Pap smear microscopy and culturing were both found to have zero sensitivity for Actinomyces. PCR appears to be a sensitive and reliable diagnostic method for the detection of Actinomyces, which are difficult to cultivate from genital samples. PCR can be used for diagnostic confirmation in cases diagnosed by conventional methods, to prevent false-positive results.

The presence of eosinophil leucocytes in cervicovaginal smears with Actinomyces-like organisms: Light microscopic examination.

BACKGROUND: Actinomyces species are part of mucosal surfaces of oral cavity, gastrointestinal and genital tracts. When these mucosal surfaces disrupt, Actinomyces become pathogen and cause infection. Eosinophil leucocytes participate in host defense against helminthic infestation and they generally play a role in asthma and allergy. However, the role of eosinophil leucocytes in host defense against bacteria is conflicting. AIM: To determine whether there is a relationship between Actinomyces-like organisms (ALOs) and eosinophil leucocytes at light microscopic level. MATERIALS AND METHODS: Cervicovaginal samples obtained from 200 patients were examined by both Pap smear microscopy and anaerobic culturing. Since the results obtained by these methods were not concordant for diagnosis of genital Actinomyces, 6 of 200 patients (3%) diagnosed with ALOs by Pap smear microscopy became the study group. Patients without any infectious agents (n=134) were the control group. Statistical analyses were conducted with χ(2) test using SPSS program. RESULTS: The study and control groups were compared statistically in view of the presence of eosinophil leucocytes and it was found that there was a significant correlation between the presence of ALOs and eosinophil leucocytes (P<0.05). Abundant polymorphonuclear leucocytes (PMNLs) and macrophages were also detected in the study group. CONCLUSION: This study implies that eosinophil leucocytes might have a role in host defense against Actinomyces in addition to PMNLs and macrophages.

[Investigation of Chlamydia trachomatis positivity in women with and without gynecologic complaints by cytologic and direct immunofluorescence methods]. / Jinekolojik yakinmalari olan ve olmayan kadinlarda Chlamydia trachomatis pozitifliginin sitolojik ve direk immonofloresans yöntemleriyle arastirilmasi.

The asymptomatic nature of the majority of Chlamydia trachomatis infections leads to persistent infections and serious complications as well as continuous transmission of bacteria in the populations. The aim of this study was to investigate the presence of C. trachomatis in non-pregnant women with and without gynecologic signs and symptoms, and to detect the rate of asymptomatic carriage. Cervical specimens collected from 200 nonpregnant women (age range: 20-81 yrs; mean age: 40.2+/-10.4 yrs) who were admitted to Gynecology and Obstetrics Clinics of Hacettepe University Hospital were included to the study. Of them 68 had clinical complaints such as vaginal discharge, itching/irritation, inflammation and inguinal pain, while 132 had not any clinical complaints. All the samples were examined by direct immunofluorescence (DFA) method (Fluorotect Chlamydia, Omega Diagnostics, UK) with fluorescein isothiocyanate labeled monoclonal antibodies against C. trachomatis serotype specific major outer membrane proteins, and the samples were simultaneously screened cytologically by Papanicolaou staining method. As a result, C. trachomatis antigen positivity was found in 49 (24.5%) of the samples by DFA method, and chlamydial inclusion bodies were detected in 19 (9.5%) of women by cytologic method. Twelve (24.5%) of the 49 DFA positive samples, and 7 (4.6%) of the 151 DFA negative samples yielded positive results cytologically. The observed proportion of overall agreement (P) was estimated as 78% between the results of methods. C. trachomatis antigen positivity was detected in 16.2% (11/68) and 28.8% (38/132) of women with and without clinical symptoms, respectively. There was no statistically significant difference between C. trachomatis positivity and neither the presence of clinical signs and symptoms nor the characteristics of the signs and symptoms (p>0.05). In conclusion, the high asymptomatic carriage rate detected in our study population indicated that, for the prevention of bacterial transmission in the populations, the women who were admitted to gynecology and obstetrics clinics should be screened for C. trachomatis positivity even if they had no clinical complaints. The use of DFA method together with the widely used, practical and economical cytologic examination method, would increase the sensitivity and specificity of C. trachomatis diagnosis.

The association between copper containing IUCD and bacterial vaginosis.

This study was designed to investigate whether there is an association between bacterial vaginosis (BV) and the use of intrauterine contraceptive device (IUCD). Six hundred Papanicolaou stained cervicovaginal smears were analyzed cytologically. 56 of 600 patients (9.3%) were detected as having IUCD. Four of 56 (7,1%) and 10 of 544 (1.8%) were positive for BV [Bv (+)]. The duration of the use of IUCD was higher than 4 years in 3 of 4 patients who were BV (+). This study showed a significant correlation between the use of IUCD and the presence of BV statistically (p < 0.05). Our findings also suggest that time limited BV infection is associated with long term use of this device. It can be suggested that a periodic microscopic examination and use of IUCD (less than 5 years) are convenient to prevent this kind of anaerobic infection.

Cytologic findings in pap smears with Actinomyces-like organisms.

OBJECTIVE: To detect whether there is a relationship between the presence ofActinomyces-like organisms (ALOs) and cytologic findings. STUDY DESIGN: Papanicolaou-stained smears from 2290 women were examined cytologically. Nineteen (0.83%) of the 2290 were diagnosed with ALOs and became the study group. Patients without infectious agents (n=1792) were the control group. Statistical analyses were conducted with the chi2 test using the SPSS program (Chicago, Illinois, U.S.A.). RESULTS: The study and control groups were compared statistically. There was a significant correlation between the presence of ALOs and other cytologic findings, such as Trichomonas vaginalis, cocci, pseudoeosinophilia, endocervical cells, superficial cells, lactobacilli and polymorphonuclear leukocytes (p < 0.05), but there was no statistically significant difference between the presence of ALOs and metaplastic cells, parabasal cells or nuclear changes (p > 0. 05). CONCLUSION: ALOs in cervicovaginal smears might provide a milieu for growing some infectious agents, such as Trichomonas vaginalis and cocci. Lactobacilli were less plentiful in these cases. Vaginal discharge and abdominal pain were also important clinicalfindings for the detection ofALOs. Another finding was long-term usage of intrauterine contraceptive devices, which can cause the overgrowth of ALOs in vaginal mucosa.

Review of cytologic criteria of bacterial vaginosis: examination of 2,841 Papanicolaou-stained vaginal smears.

To review the cytologic criteria used in the diagnosis of bacterial vaginosis cases [Bv (+)], 2,841 Papanicolaou-stained vaginal smears were examined cytologically. Clue cells and other cytologic findings were observed in 94 of 2,841 (3.30%) vaginal smears. To detect Bv(+) cases, 94 vaginal smears were screened again. Clue cells, profuse free cocci among cornified type epithelial cells, absence of PMNLs, and vaginal Lactobacilli were detected in 43 of 94 (45.74%). The observation of free cocci which resembled a nebulous appearance was detected first in this study. These four cytologic criteria strongly suggested Bv(+) cases (n = 43). Although clue cells were seen in 51 of 94 (54.25%) vaginal smears, due to the presence of abundant PMNLs, metaplastic cells, vaginal Lactobacilli, and some infectious agents, mainly Trichomonas vaginalis and yeasts, they were considered bacterial vaginosis negative [Bv(-)]. It was postulated that 51 vaginal smears might be aerobic vaginitis. In conclusion, if vaginal smears contain profuse PMNLs, some infectious agents, and metaplastic cells, and even clue cells have been observed they are not accepted as Bv(+) cases.

[Bacterial vaginitis: general overview]. / Bakteriyel vajinit: genel bir bakis.

Bacteria are the most frequently detected agents in women, clinically complaining of vaginal discharge. The studies have shown that the vaginal microflora of women with bacterial vaginitis have altered from Lactobacillus spp. to various anaerobic bacteria. Gardnerella vaginalis is found in vaginal flora of women with bacterial vaginitis as well as in healthy women, while anaerobic bacteria such as Mobiluncus and Prevotella are the causative agents for bacterial vaginosis. For the laboratory diagnosis of bacterial vaginitis, direct microscopy is one of the most commonly used methods, and for this purpose cervicovaginal smears are examined by staining Papanicolaou and Gram stains. Because of the demonstration of bacterial vaginitis in association with the obstetric diseases such as preterm labor and postpartum endometritis, is a risk factor, its importance has increased recently. In this review article, the microorganisms that cause bacterial vaginitis, their biological characteristics, and the diagnostic laboratory methods of infection, have been discussed.