Uterine cervical cancer is one of the leading causes of morbidity and mortality amongst the gynecological cancers worldwide, especially in underdeveloped countries. In Pakistan, it ranks the 3rd largest cancer killer of women after breast and oral cavity. It is the 2nd most common cancer among Pakistani women aged between 15-44 years of age. According to Information Centre on HPV and Cancer (ICO), in Pakistan, approximately 5,233 women are diagnosed with cervical cancer every year and approximately 2,876 die of this lethal disease. Several risk factors have been associated with pathogenesis of cervical cancer. Genital infection with high risk types of human papillomavirus (HPV) especially 16 and 18, is the most important risk factor in pathogenesis of cervical cancer. Among women with HPV infection, risk factors for cervical cancer include high parity (>7), long-term use of oral contraceptives (>5 years), active and passive smoking, exposure to ionizing radiation, and co-infection with Human Immunodeficiency Virus (HIV). Other risk factors for cervical cancer include history of genital warts, lack of circumcision and non-use of condoms in the male sexual partner, thus emphasizing the venereal nature of disease and pointing to HR HPV as an important agent. Multiple number of female sexual partners, number of female prostitutes as sexual partners, and detection of HPV DNA in husbands/male sexual partners are significant markers of exposure to HPV to a woman during her sexual relationship. HPV can also spread from one person to another during skin-to-skin contact. HPV infection precedes the development of cervical cancer and that persistent infection with HPV is a necessary factor for the development and progression of pre-cancerous cervical lesions to high grade cancer. Cervical cancer starts in the cells of mucosal lining of the cervix. Transitional zone of cervix is the most common site of cervical cancer and it is most susceptible to the tumorigenicity of HR HPV. The major histopathologic types of cervical cancers are squamous cell carcinoma and adenocarcinoma which constitute about 85% and 10-12% of all cases of cervical cancer, respectively. The squamous cell carcinoma arises in the squamocolumnar junction between the ectocervical squamous epithelium and the endocervical columnar epithelium and is preceded by a long phase of cervical intraepithelial neoplasia (CIN1, CIN2 and CIN3). The adenocarcinoma originates from glandular precursor lesions of the endocervical mucosa. Cancer of uterine cervix develops as a result of multiple genetic alterations that cause malignant transformation of cervical cells. Inactivation of tumor suppressor gene is a key event in cervical carcinogenesis. P53 is one of the most important tumor suppressor genes in the genome, and its mutations are found in approximately 50% of human cancers. Infection with human papillomavirus (HPV) is a critical event for the development of uterine cervical cancer. The pathogenesis of cervical cancer is thought to occur through a multistep process involving HPV infection in more than 95% of the cases. The viral proteins E6/E7 of HPV functionally interfere with cell cycle control by inactivating tumor suppressor gene p53 and the retinoblastoma protein. The complex binding between the p53 protein, and the E6 protein from the human papilloma virus may result in the disturbance of the growth-inhibitory effect of wildtype p53 which results in uncontrolled cell proliferation and malignant transformation. Present study was aimed to find out the frequency of mutation of p53 gene in Pakistani women which might be important in the origin or progression of cervical carcinoma. Secondly to find out the frequency of human papillomavirus infection in cervical cancer patients and correlate its association with p53 gene mutation in Pakistani population. Another aim was to study ultrastructural changes in cervical cancer cells under transmission electron microscope. For this study, 83 samples of cervical cancer were collected. All patients were females with age range from 19-70 years. Formalin fixed paraffin embedded tissue blocks were used. Out of 83 cervical cancer specimens, seventy four (89.16%) were of squamous cell carcinoma type and 9 (10.84%) were of adenocarcinoma type. DNA isolation was done by using Qiagen spin column method followed by PCR amplification. SSCP (single stranded conformation polymorphism) was done to detect mutant samples. Exons 4, 5, 6, 7 and 8 of p53 gene were studied for mutation. Mutations were confirmed by DNA sequencing. PCR of all samples was done to find out the E6 and E7 oncoproteins of human papillomavirus type 16 and 18. It was found that, 63 (75.9%) were HPV16 positive and only 2 cases (2.4%) were HPV18 positive. Among eighteen cases (21.68%), HPV16/18 were not identified. Two positive cases for HPV18 were of adenocarcinoma type and out of sixty three, HPV16 positive cases, seven were adenocarcinoma and 55 were of squamous cell variety. The frequency of mutation of p53 gene was calculated by using SPSS 21.0 software. It was found that in 59% of cervical cancer cases, p53 gene was mutated. One of the noteworthy observations in present study was that out of mutated samples, exon 4 of p53 gene was mutated in 40.8% of the cases. Exon 5, 6, and 7 were mutated in 16.32% cases each and exon 8 was mutated in only 10% of cases. Seven patients (14.28%) with p53 gene mutation were of age more than 60 years. Age of fourteen patients with p53 gene mutation (28.57%) ranged between 46 to 60 years of age. Twenty eight (57.14%) patients with p53 gene mutation were below 45 years of age. When further explored, it was found that among patients having adenocarcinoma 7 (77.8%) were of age above 45 years while among squamous cell carcinoma (SCC), 45(60.8%) had age below 45 years. This difference was significant with p-value (0.026). Association of cancer type with age, HPV 16/18 status, and p53 gene mutation was noted. It was found that the cases with HPV16/18 infection along with p53 mutation were significantly higher among age group younger than 45 years (p value 0.038), most common in patients with histological grade-II cervical cancer and mostly in squamous cell carcinoma type. It was also noted that out of a total 49 cases with p53 gene mutation, 44 (89.8%) were of squamous cell carcinoma type and only 5 (10.2%) were of adenocarcinoma variety with p value 0.05 which was statistically significant. For ultrastructural analysis of cervical cancer cells, fresh tissues were used for transmission electron microscopy (TEM). Ultrastructure of cervical squamous cell carcinoma cells showed that the nuclear/ cytoplasmic ratio was highly disturbed. Many of the nuclei showed marked irregularity of contour due to deep clefts and infoldings of the nuclear membrane. There was increased density of chromatin in nuclei either in form of clumps adherent to inner side of nuclear membrane or scattered throughout the nuclei. In most of the cervical cancer cells, nucleoli were multiple and hypertrophied and in some cells, meshbasket appearance of nucleoli can be appreciated. In cytoplasm of cells from squamous cell carcinoma of cervix, tonofilaments were noted in perinuclear area. Increased number of cytoplasmic vacuoles were also seen in cervical neoplastic cells. Cervical cancer is the only cancer that is almost completely preventable by safe, simple and inexpensive methods. Cervical cancer screening methods include periodic pap smears, visual inspection of cervix with acetic acid and Lugol’s iodine, and HPV genotyping. Immunizations against human papillomavirus (HPV) types 16 and 18 have the potential to prevent significant number of cervical cancer cases. Recognition of important precursor lesions may play an important role in cervical cancer prevention. It has been reported that p53 gene mutation and HPV 16/18 infection present in precancerous conditions of cervix. Future of cancer prevention depends on early detection and eradication of latent precancerous stage. The greater knowledge of the molecular etiology of cancer will contribute to disease screening strategies, diagnosis, staging of the disease and therapy." xml:lang="en_US