Cervical carcinoma continues to be a major public health challenge globally, ranking as the fourth most common cancer among women worldwide [1]. In India, the burden is disproportionately high, accounting for nearly one-fourth of the global cervical cancer deaths. Despite being a preventable disease with a long latent phase, late-stage presentation remains common due to a lack of awareness and organized screening programs [2]. The persistent infection with high-risk Human Papillomavirus (HPV) is established as the necessary cause for cervical cancer, driving the progression from mild dysplasia to invasive carcinoma [3]. The Papanicolaou (Pap) smear, introduced by George Papanicolaou in the 1940s, is widely regarded as the most successful cancer screening test in history. It is a simple, cost-effective, and non-invasive method to detect cytomorphological changes in cervical cells before they progress to invasive cancer [4]. The effectiveness of Pap smear screening in reducing cervical cancer incidence and mortality has been well-documented in developed countries. However, in low-and-middle-income countries (LMICs) like India, screening coverage remains suboptimal, often limited to opportunistic screening in tertiary care settings rather than population-based programs [5]. The interpretation of Pap smears has been standardized globally by The Bethesda System (TBS) for Reporting Cervical Cytology. TBS provides a uniform terminology that facilitates clear communication between the pathologist and the clinician, allowing for consistent management guidelines [6]. The most recent update, TBS 2019, refined definitions for non-neoplastic findings and epithelial cell abnormalities, improving the reproducibility of reporting. While national data on cervical cancer exists, regional variations in cytomorphological patterns are significant due to differences in socioeconomic status, hygiene practices, and sexual behavior [7]. Nashik, a rapidly growing urban center in Maharashtra with a significant rural periphery, serves as a hub for diverse patient demographics. There is a paucity of recent data regarding the specific cytological profile of women in this region. Understanding the local spectrum of cervical pathology—ranging from common infections to high-grade lesions—is essential for tailoring health education and screening initiatives. Significance and Research Gap: Most existing literature from Maharashtra focuses on retrospective data or dates back several years. There is a need for current prospective data reflecting the post-pandemic healthcare landscape, where screening behaviors may have shifted. Furthermore, the correlation between specific vaginal infections (like Bacterial Vaginosis) and inflammatory changes in this specific demographic requires documentation to guide syndromic management [8].

Study Design and Setting: This retrospective observational study was conducted at the Department of Pathology in collaboration with the Department of Obstetrics and Gynecology at a Tertiary Care Hospital in Nashik, Maharashtra. The study duration was six months, extending from January 1, 2025, to June 30, 2025. Sample Size: A total of 450 women were enrolled in the study. The sample size was calculated based on the prevalence of epithelial cell abnormalities in previous Indian studies (approximately 5%), with a 95% confidence interval and a 3% margin of error. Inclusion and Exclusion Criteria: Inclusion Criteria: 1. All pap 2. Sexually active women presenting to the gynecology outpatient department (OPD) with complaints such as vaginal discharge, post-coital bleeding, intermenstrual bleeding, or lower abdominal pain. 3. Asymptomatic women undergoing voluntary opportunistic screening. Exclusion Criteria: 1. Women who were menstruating at the time of examination. 2. Pregnant women (to avoid confounding cytological changes). 3. Women who had undergone a total hysterectomy. 4. Patients with a previously confirmed diagnosis of invasive cervical cancer or those currently undergoing chemotherapy/radiotherapy. Data Collection and Procedure A detailed clinical history was recorded, including age, parity, menstrual history, contraceptive use, and presenting complaints. The procedure was performed with the patient in the lithotomy position. A sterile Cusco’s bivalve speculum was inserted to visualize the cervix. The transformation zone was identified. A conventional Pap smear was taken using an Ayre’s wooden spatula (rotated 360 degrees) to collect ectocervical cells and a cytobrush for endocervical cells. The material was immediately smeared onto two clean glass slides and fixed wet in 95% ethyl alcohol for a minimum of 20 minutes to prevent air-drying artifacts. Staining and Interpretation The fixed slides were stained using the rapid Papanicolaou staining technique (utilizing Harris Hematoxylin, OG-6, and EA-36). The slides were mounted with DPX and examined under a light microscope. Reporting was done according to The Bethesda System (TBS) 2019 categories: 1. Specimen Adequacy: Satisfactory or Unsatisfactory. 2. Negative for Intraepithelial Lesion or Malignancy (NILM): This included normal findings, organisms (Trichomonas, Candida, Bacterial Vaginosis, Actinomycosis), and other non-neoplastic findings (reactive cellular changes associated with inflammation, atrophy, or IUD). 3. Epithelial Cell Abnormalities (ECA): • Squamous: Atypical Squamous Cells of Undetermined Significance (ASC-US), Atypical Squamous Cells–cannot exclude HSIL (ASC-H), Low-grade Squamous Intraepithelial Lesion (LSIL), High-grade Squamous Intraepithelial Lesion (HSIL), and Squamous Cell Carcinoma (SCC). • Glandular: Atypical Glandular Cells (AGC), Adenocarcinoma. Statistical Analysis: Data were entered into Microsoft Excel and analyzed using IBM SPSS Statistics for Windows, Version 26.0. Categorical variables were expressed as frequencies and percentages. Continuous variables (e.g., age) were expressed as mean ± standard deviation (SD). The Chi-square test was used to determine the association between age groups and the incidence of epithelial cell abnormalities. A p-value of <0.05 was considered statistically significant.

Demographic Profile A total of 450 women were included in the study. The age of the patients ranged from 21 to 65 years, with a mean age of 38.4 ± 11.2 years. The maximum number of patients fell into the reproductive age group of 31–40 years (38.2%). The most common presenting complaint was white discharge per vaginum (leukorrhea), reported in 215 (47.7%) patients, followed by lower abdominal pain in 95 (21.1%) patients. 80 (17.7%) women were asymptomatic and screened routinely. Specimen Adequacy Out of the 450 smears evaluated, 438 (97.3%) were reported as "Satisfactory for evaluation." 12 smears (2.7%) were "Unsatisfactory" due to obscuring inflammation, blood, or scant cellularity, and these patients were recalled for a repeat smear (though only the initial smear is included in this analysis). Cytomorphological Patterns The distribution of cytological findings is detailed in Table 1. The vast majority of satisfactory smears were Negative for Intraepithelial Lesion or Malignancy (NILM). Table 1: Distribution of Cytomorphological Patterns (N=438 Satisfactory Smears) Bethesda Category Findings Number (n) Percentage (%) NILM Total NILM 418 95.4% Normal / Within Normal Limits 65 14.8% Inflammatory smear (Non-specific) 272 62.1% Atrophic vaginitis 35 8.0% Specific Infections (see Table 2) 46 10.5% ECA Total Epithelial Cell Abnormalities 20 4.6% ASC-US 6 1.4% LSIL 9 2.1% HSIL 4 0.9% AGUS 1 0.2% Total 438 100% Note: NILM = Negative for Intraepithelial Lesion or Malignancy; ECA = Epithelial Cell Abnormalities. Infections and Inflammatory Changes Inflammation was the predominant finding. Among the NILM category, specific infectious agents were identified in a subset of cases. Candida species was the most common specific pathogen detected. Table 2: Spectrum of Specific Infectious Agents Detected (n=46) Organism Number (n) Percentage of Total Smears (N=438) Candida species 21 4.8% Bacterial Vaginosis (Shift in flora) 16 3.7% Trichomonas vaginalis 9 1.8% Total Specific Infections 46 10.5% Age Correlation with Abnormalities The relationship between patient age and the presence of Epithelial Cell Abnormalities (ECA) was analyzed. While inflammation was common across all age groups, premalignant and malignant lesions were significantly more frequent in older women. Table 3: Association Between Age and Epithelial Cell Abnormalities Age Group (Years) Total Cases (n) NILM n (%) ECA (ASC-US/LSIL/HSIL/AGUS) n (%) 21 – 30 110 108 (98.2%) 2 (1.8%) 31 – 40 172 168 (97.7%) 4 (2.3%) 41 – 50 105 98 (93.3%) 7 (6.7%) > 50 51 44 (86.3%) 7 (13.7%) Total 438 418 20 Chi-square statistic = 14.82; p-value = 0.002 (Significant). The data indicates a statistically significant increase in the rate of epithelial abnormalities as age increases, peaking in the post-menopausal age group (>50 years), where 13.7% of smears showed abnormalities ranging from ASC-US to AGUS. The single case of AGUS was found in a 58-year-old female which was confirmed as endometrial carcinoma on histopathology.

This prospective study conducted at a tertiary care center in Nashik analyzed 450 Pap smears to document the cytomorphological spectrum in the region. The study strictly adhered to The Bethesda System 2014 for reporting. Adequacy and NILM: In our study, the unsatisfactory rate was 2.7%, which is well within the acceptable range (<10%) recommended by quality assurance guidelines [9]. The majority of the smears (95.4%) were Negative for Intraepithelial Lesion or Malignancy (NILM). This is consistent with findings by Mulay et al., who reported a NILM rate of 95% in a similar Indian setting [10]. Within the NILM category, inflammatory smears (nonspecific) constituted the largest subgroup (62.1%). High prevalence of inflammation is a recurrent theme in Indian studies, often attributed to poor genital hygiene, low socioeconomic status, and lack of access to barrier contraceptives [11]. Persistent inflammation can be cytologically challenging as reactive cellular changes may mimic dysplasia, necessitating careful evaluation by the pathologist. Infectious Spectrum: Specific infectious agents were identified in 10.5% of cases. Candida species was the most common organism (4.8%), followed by Bacterial Vaginosis (3.7%) and Trichomonas vaginalis (1.8%). This pattern differs slightly from some western studies where Bacterial Vaginosis often predominates but aligns with several Indian studies such as those by Gupta et al., which highlight Candidiasis as a leading cause of leukorrhea in Indian women [12]. Epithelial Cell Abnormalities (ECA): The prevalence of ECA in our study was 4.6%. This figure is comparable to the range of 1.3% to 5.8% reported in various domestic studies [14, 15]. Specifically, our breakdown showed 1.4% ASC-US, 2.1% LSIL, 0.9% HSIL, and 0.2% AGUS. The rate of LSIL (2.1%) was higher than HSIL (0.9%), which is the expected epidemiological pyramid of cervical lesions. However, our ECA rate is slightly higher than that reported by Kothari et al. (3.5%) in a rural setting [16], possibly because our tertiary center in Nashik receives referral cases with higher symptom severity. The detection of one case of AGUS (0.2%) highlights the critical nature of opportunistic screening. Although the percentage seems low, in a population of millions, this translates to a significant disease burden. Age and Malignancy: Our statistical analysis revealed a significant association (p=0.002) between advancing age and the presence of epithelial abnormalities. The detection rate rose from 1.8% in the 21-30 age group to 13.7% in women over 50. This corroborates the natural history of cervical cancer, where HPV infection usually occurs in younger women and clears spontaneously, while persistent infection leads to precancerous lesions and cancer in the fourth and fifth decades of life [17]. This finding underscores the importance of targeting perimenopausal and postmenopausal women for screening, a group that often neglects reproductive health after childbearing years. The present study was conducted at Department

This six-month prospective study from Nashik provides valuable insight into the cervical cytological status of the local population in 2025. The results demonstrate that while non-specific inflammation and infections constitute the bulk of the disease burden, a significant percentage (4.6%) of women harbor potentially pre-malignant or malignant lesions. The study identifies Candida and Bacterial Vaginosis as common distinct etiologies for vaginal discharge, warranting specific treatment. More importantly, the strong correlation between advancing age and high-grade lesions reinforces the need for sustained screening efforts focusing on women above 40 years of age. The Pap smear remains an indispensable, cost-effective tool in a resource-limited setting like India. Regular screening, accurate reporting using the Bethesda system, and appropriate follow-up of abnormal smears are the cornerstones of reducing cervical cancer mortality. Healthcare policy in Nashik should focus on converting opportunistic screening into organized population-based programs to capture asymptomatic carriers of high-grade lesions.

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