Primary amenorrhoea - cytogenetic study in 40 Indian women.

BACKGROUND: Primary amenorrhoea (PA) refers to an ailment when adolescent girls do not attain menarche naturally. It is one of the most common gynaecological disorders specified. Chromosomal abnormalities play a pivotal role in PA. Cytogenetic analysis is an indispensable diagnostic tool to determine the abnormality of the chromosome. In an emerging country like India, cytogenetic analysis is at a nascent stage. There are very few studies on Cytogenetics present in eastern India, including West Bengal. In rural and suburban areas PA sufferers often experience late diagnosis and struggle to access suitable curative management. The aim of the study is to evaluate the various types of chromosomal abnormalities in patients suffering from PA for accurate, better management of the same and further counselling. METHODS: A total of 40 PA cases were referred by obstetricians and gynaecologists to the Department of Genetics of Nirnayan Health Care, Kolkata. To screen the chromosomal abnormalities, human leukocyte culture was accomplished with their peripheral venous blood followed by G-banding and then karyotyping was executed according to ISCN-2020. RESULT: Out of 40 patients, 29 were normal among which 46,XX was found in 70% cases (n = 28) and 46,XX,9qh + in 2.5% (n = 1). The remaining 11 showed different types of abnormalities. 45,X was found in 10% (n = 4), 46,X,i(X)(q10) in 2.5% (n = 1), 46,X,del(X)(p11.2) in 2.5% (n = 1), 46,X,del(X)(p22.1) in 2.5% (n = 1), 46,X,del(X)(q24) in 2.5% (n = 1), 46,XY in 2.5% (n = 1), mos 45,X[22]/46,Xi(X)(q.10)[8] in 2.5% (n = 1) and mos 45,X[16]/46,XY[14] (2.5%) in 2.5% (n = 1). CONCLUSION: This study indicates the importance of chromosomal study which must be included in early diagnosis of PA. Karyotyping at the appropriate phase of life will not only help in the judicial management of this disorder but will also give young girls a better lifestyle.

Primary amenorrhoea is a common gynecological disorder reported in adolescent girls, often linked to chromosomal abnormalities. In Eastern India, including West Bengal, where cytogenetic analysis is still in its nascent stage, late diagnosis and limited access to curative management are prevalent issues. A study conducted from January 2021 to May 2023 at Nirnayan Healthcare, Kolkata aimed to evaluate chromosomal abnormalities in 40 PA cases. Out of these, 28 exhibited normal karyotypes (46,XX); one patient was reported with 46,XX,9qh + which is considered a normal karyotype, while the remaining 11 revealed diverse abnormalities, including 45,X; sex reversal & several structural variations. The study underscores the significance of cytogenetic analysis in the early diagnosis of Primary Amenorrhoea. Early karyotyping not only facilitates judicious management but also ensures a better lifestyle for affected girls.

Heterochromatin extension: a possible cytogenetic fate of primary amenorrhea along with normal karyotype.

Primary amenorrhoea (PA) is considered to be one of the challenging and taxing problems for the gynaecologist. Previous studies suggested that different numerical and structural chromosome abnormalities are associated with this. Heterochromatin polymorphisms are considered to be normal variant but considering the recent research on crucial cellular effects of heterochromatin, we have aimed to find out the prevalence of heteromorphism along with other standard chromosomal abnormalities. This was an observational study which was conducted in Diamond Harbour Govt. Medical College and Hospital, West Bengal during March 2019-February 2021. Clinical features of 178 patients were noted and peripheral venous blood was taken following informed consent. This comprehensive study reveals that there are 10.11% of the females among 178 females having a heterochromatin extension which is significantly high. We hence suggest that heteromorphism may be associated with ovarian dysfunction leading to amenorrhoea as the region of heterochromatin acts as a key part in chromosome structure, histone modification and gene regulation. Analysis at the molecular level may be needed to unveil any relationship between heteromorphism and PA. Impact StatementWhat is already known on this subject? Primary amenorrhoea (PA) is a menstrual abnormality found in females with the prevalence of 1-3%. It may be associated with different types of numerical and structural chromosomal anomalies. Among them Turner's syndrome (pure and in variant form) is the commonest chromosomal aberration associated with PA. Some patients with PA are found to have a normal karyotype with heterochromatin extension on the large arm (q) of either chromosome 9 or chromosome 16. Chromosomal polymorphism with increase in heterochromatin region consists of highly repetitive sequences of satellite DNA, which normally does not encode any protein and thus considered to be a normal variant.What do the results of this study add? This comprehensive study reveals that there are 10.11% of the females among 178 females having a heterochromatin extension which is significantly high. PA and certain association of phenotypical stigmata like short stature in these patients with heterochromatin extension can be explained on the basis of histone modification and gene regulation by heterochromatin.What are the implications of these findings for clinical practice and/or further research? We will be able to know about involved transcription factors those are responsible for the histone modification directly linked to the heterochromatin extension by further molecular study. That will definitely help to find out the reason for PA as well as implementation of gene therapy in these cases.

Changes in Different Cytokines (IL-2, TNF-α, and IFN-γ) Profile in Acquired Aplastic Anemia Patients: A Study From Eastern India.

Although aplastic anemia has been extensively investigated, little is known about their circulating cytokine pattern. The present study was done to evaluate the severity of the disease with the 3 major anti-hematopoietic cytokines interleukin-2 (IL-2), tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ). This study is ethically cleared. A total of 102 bone marrow plasma and peripheral blood plasma paired samples were collected from the confirmed acquired aplastic anemia (AAA) patients and 10 control cases after taking written consent and analyzed by the quantitative enzyme-linked immunosorbent assay. The Mann-Whitney U test was used for statistical analysis. Considerably increased levels of IL-2, TNF-α, and IFN-γ were found in the peripheral blood plasma and bone marrow plasma of AAA patients as compared with controls, that is, 45.76±20.61 versus 1.99±1.25, P<0.00001; 26.51±15.62 versus 11.7±3.67, P=0.00188; 17.04±11.64 versus 5.27±1.92, P=0.00034 and 70.54± 37.57 versus 3.12±1.82, P<0.00001; 251.82±243.80 versus 15.66±6.35, P<0.00001; 39.35±22.58 versus 11.12±2.41, P=0.00012, respectively. The IL-2, TNF-α, and IFN-γ levels were observed to be extraordinarily elevated in AAA, but were very low in the control cases. The results confirm that IL-2, TNF-α, and IFN-γ may have an imperative association with the disaster in the bone marrow compartment of AAA patients. The levels and ranges of the observed cytokines can also be predicted by the severity basis of this study.

Study of frequency and types of chromosomal abnormalities in phenotypically female patients with amenorrhea in Eastern Indian population.

AIM: This comprehensive review article aims to comprehend the frequency and prevalence of chromosomal abnormalities in both primary amenorrhea (PA) and secondary amenorrhea (SA) cases and correlating it with their phenotypes, clinical features and hormonal profiles. METHODS: Research publications on prevalence of chromosomal abnormalities in both PA and SA cases worldwide and its etiology, clinical features, hormonal profiles; their correlation with chromosomal profiles were searched for on the internet, including general search engines and respective scientific sites. Only published, relevant and authentic data conducted on phenotypically female patients were considered. Another aspect of amenorrhea occurs due to several clinical conditions apart from cytogenetic viewpoint were not considered or discussed in detail. RESULTS: As literature study suggests; considering various etiology of amenorrhea counting anatomic defect of the hypothalamus or genetic defect, and various acquired causes of chromosomal anomalies contribute to be one of the major etiologies of both PA and SA; ranging from 15.9% to 63.3% in case of PA and from 3.9% to 44.4% in case of SA. In spite of the presence of any other factors responsible condition, the genetic factors need to be emphasized, which might include single gene disorders or chromosomal disorders. Individuals with chromosomal anomalies reported a wide range of abnormalities in phenotypes, as well as in other clinical features and hormonal profiles. CONCLUSION: This comprehensive review is the first structured review article that encompasses the cytogenetic profile of the amenorrhea cases and correlating it with their phenotypes, clinical features and hormonal profiles from Eastern India.

Cytogenetic analysis of patients with primary amenorrhea in Eastern India.

Amenorrhea is the absence of menstruation in women of reproductive age. Previous reports suggest that chromosomal abnormality is the second most common cause of amenorrhoea. Early referral for cytogenetic evaluation is recommended for the identification of underlying chromosomal aberrations in amenorrhoea patients. This was an observational study which was conducted in Vivekananda Institute of Medical sciences, Kolkata, during January 2013-December 2015. This study aims to estimate the frequency and types of chromosomal abnormalities in primary amenorrhoea (PA) patients in Eastern India and correlate their hormonal profile with chromosomal reports. Clinical features of 150 patients were recorded with clinical expertise. Peripheral venous blood was taken following informed consent, followed by karyotyping for chromosomal analysis. Results revealed 76.1% of PA with normal female karyotype (46, XX) and 23.9% with different abnormal karyotypes. Among the abnormal karyotype constituents, 50% numerical abnormalities, most frequent being Turner syndrome, pure (n = 12, 8%) and mosaic (n = 5, 3.3%). Three cases (2%) showed male (XY) karyotype. The other cases showed X structural abnormalities. This study emphasises the need for cytogenetic analysis as integral part of the diagnostic protocol in case of PA for precise identification of chromosomal abnormalities and for appropriate management and counselling of these patients.

Is There Any Role of Arsenic Toxicity in HPV Related Oral Squamous Cell Carcinoma?

Arsenic is a potent human carcinogen affecting the rate of cancer deaths worldwide. In India, West Bengal is the worst affected state by arsenic. To our best knowledge, this is the first study relating arsenic toxicity with oral carcinoma, along with HPV infection, the latter being well established in western countries. To find out a possible correlation between arsenic toxicity and oral carcinoma in the population of West Bengal, in or without any association with human papilloma virus infection. Ethical clearance of this study was obtained from the institutional committee. One hundred and four malignant and 103 premalignant cases were selected for this study along with 200 healthy age and sex-matched individuals selected as control (100 each for malignant and premalignant) (2013-2017). On proper consent, their buccal swab and hair samples were assessed for the presence of HPV DNA by DNA extraction, followed by PCR and arsenic estimation by flow injection hydride generation atomic absorption spectrometry respectively. A very highly significant correlation has been observed between arsenic toxicity, HPV infection and the occurrence of oral carcinoma (p value = 2.18e-06; p value = 0.00100 respectively). A correlation has also been observed between these two factors simultaneously, contributing to this malignancy (phi coefficient = 0.2194839). A statistically significant correlation observed between this metal toxicity and viral infection in the occurrence of oral carcinoma in this population indicates a possible symbiotic role between these two factors in this malignancy.

Comparative study of bone marrow and blood plasma levels of IL-2 in aplastic anaemia and their relationship with disease severity.

OBJECTIVES: Interleukin-2 (alias: IL-2, TCGF, Lymphokine), a type of interleukin, is also a potent signalling molecule in the signalling cascade of the immune-mediated activation of T Lymphocytes leading to the destruction of haematopoietic stem cell (HSC) which is the basis of acquired aplastic anaemia (AAA). The objective was to study the association of IL-2 in the bone marrow plasma (BMP) and peripheral blood plasma (PBP) in AAA patients. METHODS: A total of 52 BMP and PBP-paired samples (both from the same patients) was collected from the confirmed AAA patients and 10 healthy individuals. The level of IL-2 was measured by the quantitative enzyme-linked immunosorbent assay (ELISA). The Mann-Whitney U test was used for statistical analysis. RESULTS: Significantly increased level of IL-2 was observed in the BMP than PBP of AAA patients. The level of IL-2 in PBP and BMP was found to be very low in the control cases. Considerably increased levels of IL-2 were found in the PBP and BMP of AAA patients as compared to controls (48.54 ± 21.89 vs. 1.99 ± 1.25 p-value < 0.00001) and (75.33 ± 41.9 vs. 3.12 ± 1.82; p-value < 0.00001) respectively. Among these patients, the IL-2 levels were higher in patients with Very Severe Aplastic Anaemia (VSAA) and Severe Aplastic Anaemia (SAA) than those with Non-severe Aplastic Anaemia (NSAA) in the PBP (65.6 ± 23.61 vs. 31.72 ± 7.64; p-value 0.00338) and (45.37 ± 16.25 vs. 31.72 ± 7.64; p-value 0.01468) respectively. Again the IL-2 levels were higher in patients with VSAA and SAA than those with NSAA in the BMP (115.01 ± 38.91 vs. 38.32 ± 19.49; p-value < 0.00001) and (66.44 ± 23.34 vs. 38.32 ± 19.49; p-value 0.0006). The IL-2 level was higher in VSAA than SAA in PBP (65.6 ± 23.61vs. 45.37 ± 16.25; p-value 0.0114) and BMP (115.01 ± 38.91 vs. 66.44 ± 23.34; p-value 0.00044). CONCLUSION: This study emphasized on the bone marrow and blood plasma levels of IL-2 in aplastic anaemia and their relationship with disease severity. The results indicate towards the fact that IL-2 may have an important association with the marrow failure of AAA patients and thus can help in disease development. Further study is necessary for better understanding.

Cytogenetic and micronuclei study of human papillomavirus-related oral squamous cell carcinoma.

BACKGROUND: The association of human papilloma viral infection in oral squamous cell carcinoma is well studied in the Western countries, but its correlation with DNA damage in the form of micronuclei (MN) formation, ceased apoptosis or presence of chromosomal abnormalities has not yet been studied. AIM: The aim of this study is to find any possible correlation between human papillomavirus (HPV) infection and cytogenetic damage in the oral malignant and premalignant population of West Bengal. SETTINGS AND DESIGN: A total of 104 malignant and 103 premalignant cases were selected along with 200 controls. METHODS: The buccal smear samples were Pap stained for the detection of MN, apoptosis frequency and koilocytes. The buccal swab samples were processed for DNA extraction followed by polymerase chain reaction for the detection of HPV DNA. The peripheral venous blood samples were processed for the detection of any chromosomal abnormality, by the method of human leukocyte culture followed by Giemsa staining. STATISTICAL ANALYSIS USED: Correlation analysis using GraphPad Prism software was used in this study. RESULTS: About 34.6%, 42.3% and 6.73% of malignant and 6.79%, 3.88% and 20.38% of premalignant cases showed the presence of HPV DNA, koilocytes and apoptosis, respectively. The difference between the MN frequencies of premalignant and malignant oral lesions with the control group is significant with respect to various risk factors (P < 0.05). One percentage of malignant cases showed the presence of chromosomal break. CONCLUSION: A considerable percentage of malignant cases showing the presence of koilocytes and viral DNA may indicate the effect of HPV infection leading to the malignancy, which can be correlated with the MN and apoptosis frequency.

Incidence of Fanconi anaemia in phenotypically normal aplastic anaemia patients in West Bengal.

OBJECTIVES: Fanconi anaemia (FA) is a rare inherited bone marrow failure and autosomal recessive blood disorder. FA patients have a higher risk of cancer, including acute myeloid leukaemia and squamous cell carcinoma. Maximum, but not all, affected individuals have one or more somatic abnormalities, including skin, skeletal, genitourinary, gastrointestinal, cardiac and neurological anomalies, etc. Positive stress cytogenetics has immense implications for the treatment and management of FA. The aim of our study was to find out the incidence of FA in the population of phenotypically normal aplastic anaemia (AA) patients in West Bengal. METHODS: Ethical clearances were obtained from the corresponding institutional committees. A total of 117 AA cases was selected. Stress cytogenetics was performed from peripheral venous blood (PVB) samples of 63 AA patients (age ≤ 50 years) and 63 age- and sex-matched healthy individual (control) using Mitomycin C (MMC). RESULTS: Out of 63 AA patients, 6 (9.25%) cases showed positive stress cytogenetics suggestive of FA, which is statistically significant (p-value - 0.000532), analysed by chi-square test. DISCUSSION: A considerable percentage of patients showing sensitivity towards MMC, even if they are phenotypically normal and did not have any distinguishable features which are generally found in FA. CONCLUSION: This observation may indicate that stress cytogenetics analysis of phenotypically normal AA patients (≤50 years) is essential for the improvement of the treatment procedure.

Cytogenetic study is not essential in patients with aplastic anemia.

Depending on contemporary treatment approach of aggressive immunosuppression, Aplastic Anemia (AA) is caused by immunological destruction of otherwise normal hematopoietic stem cells. The aim was to summarize the cytogenetic abnormalities in AA patients and the frequency of Fanconi Anemia (FA) in morphologically normal AA patients in eastern India. Ethical clearances were obtained from both institutions involved in this study. Out of 72800 patients attending the outpatient department, 520 pancytopenia patients were screened for AA after Bone marrow (BM) aspiration and biopsy. Samples were collected from 117 cases in 3 phases. 51 peripheral venous blood (PVB) samples in the first phase, 19 BM & PVB paired samples in the second phase and 47 BM samples in third phase were collected followed by leukocyte and/or BM stem cell culture. Next GTG banding and karyotyping were performed. PVB was collected from 63 (< 50 years) AA patients and stress cytogenetics was done to diagnose FA. In the first phase of the study, out of 51 PVB samples, 1 (1.96%) showed a unique chromosomal abnormality, i.e. 45,XY,rob(14:21)(p10:q10)[20]. In the second phase of study, among 19 BM & PVB paired samples, 1 (5.26%) showed abnormal karyotype i.e. 45,X,-Y[3]/46,XY[47]. In the third phase of the study, 47 BM samples showed normal karyotype. Only 6 (9.52%) cases were found positive for stress cytogenetics. A negligible percentage showing cytogenetic abnormality in such a considerable number of AA cases indicates that routine cytogenetic analysis of AA patient is not essential. A significant percentage was positive for stress cytogenetics; suggestive for FA, even the patients were morphologically normal.

Study of arsenic exposure in oral/oropharyngeal carcinoma in West Bengal.

OBJECTIVES: To study any possible correlation between arsenic toxicity and the development of oral carcinoma in West Bengal population. MATERIAL AND METHODS: Ethical clearance for this study was obtained from the Vivekananda Institute of Medical Sciences. Out of 30 785 patients attending our hospital from November 2012 to July 2015, 107 cases and 50 control individuals were selected. The hair and buccal smear samples were obtained upon their consent for the purpose of the analysis of arsenic count and cytogenetic damage, respectively. RESULTS: Ninety-six percent of cases came from the highly arsenic affected districts and 81.3% showed their arsenic count above the safe limit (0.8 µg/g) whereas 96% of the controls' arsenic count was within the safe limit. The study showed a significant difference of the micronuclei and apoptosis frequency between the cases and controls. CONCLUSIONS: The difference of micronuclei and apoptosis frequency between cases and controls was significant. The maximum number of cases came from highly arsenic affected areas and a higher percentage of cases showed elevated arsenic count, as compared to controls, which may indicate a possible link between arsenic toxicity and this disease. However, a larger sample size is required for a proper correlation. Int J Occup Med Environ Health 2017;30(2):271-279.

Black tea (Camellia sinensis) as a chemopreventive agent in oral precancerous lesions.

Oral carcinoma is the most common malignancy found in adult Indian men and the third most common in adult Indian women. About half of all cases are found to be associated with precancerous lesions, chiefly leukoplakia. We wanted to explore the possible benefits of black tea (Camellia sinensis) administered to patients with oral leukoplakia. Eighty-two subjects with oral leukoplakia underwent micronuclei and chromosomal assays on exfoliated oral mucosal epithelium, after which they received black tea in a fixed regimen. The micronuclei assay was repeated at 6 months, and the chromosomal study at 1 year. After the first year, the first 15 patients entered onto this study showed a significant decrease in the micronuclei frequency and chromosomal aberrations, which correlated with the clinical improvement. Several in vitro and animal studies have suggested the efficacy of tea in the chemoprevention of cancer. To the best of our knowledge, this is the first report on the effect of black tea in oral leukoplakia.

Use of black tea in modulating clastogenic effects of arsenic in mice in vivo.

The concentration of arsenic in drinking water has far exceeded the permissible limit of 0.001 mg/L and has reached epidemic proportions, with a maximum of 3.7 mg/L in several districts of West Bengal and in the adjoining Bangladesh. Because inorganic arsenic is a documented human carcinogen, arsenic in drinking water may cause 200,000-270,000 deaths per year from cancer in India alone. Tea has a protective effect against the clastogenicity of arsenic. We investigated whether tea extracts could protect against the damage caused by arsenic in vivo. Our experiments were carried out with black tea in mice with the end points of incidence of chromosomal aberrations and damaged cells. Analysis of variance of chromosomal aberrations showed a significant difference in the toxic effects of arsenic, which were reduced by tea infusion. The frequency of chromosomal aberrations was close to the corresponding effects of tea alone. Continued dietary administration of black tea infusion protects against the chromosome-damaging effects of sodium arsenite at a statistically significant level. The degree of protection increases with duration of tea consumption, which may be attributed to the antioxidant and scavenging properties of tea infusion.

Increase in DNA damage in lymphocytes and micronucleus frequency in buccal cells in silica-exposed workers.

The alkaline single cell gel electrophoresis (comet assay) was applied to study the genotoxic properties of silica in human peripheral blood lymphocytes (PBL). The study was designed to evaluate the DNA damage of lymphocytes and the end points like micronuclei from buccal smears in a group of 45 workers, occupationally exposed to silica, from small mines and stone quarries. The results were compared to 20 sex and age matched normal individuals. There was a statistically significant difference in the damage levels between the exposed group and the control groups. The types of damages (type I -type 1V) were used to measure the DNA damage. The numbers of micronuclei were higher in the silica-exposed population. The present study suggests that the silica exposure can induce lymphocyte DNA damage and produces significant variation of micronuclei in buccal smear.

Association of genotoxic effects of arsenic with haematological malignancy in West Bengal.

Arsenic is a naturally occurring metalloid that has been associated with increased incidence of human cancers in certain highly exposed populations. This present work aimed to study whether arsenic plays any role in the increased incidence of blood cancer among residents of West Bengal, India, which is one of the worst arsenic-affected areas of the world. Our study group included both blood cancer patients and age-, sex-matched healthy controls. We studied the arsenic concentration in the hair and nail samples of our study group. We found significantly higher arsenic concentration in the biological tissues of patients, which also correlated with their greater incidence of chromosomal aberrations. Thus, arsenic may act as a predisposing factor for blood cancer.

Incidence of anemia and effect of nutritional supplementation on women in rural and tribal populations of eastern and northeastern India.

Screening of women of child bearing age among the tribal and rural population of different north eastern and eastern states of India was performed. More than 50% of women in some areas were found to have anemia. As part of the study nutritional supplementation was given to a proportion of the anemic population, comparing the effects of Spirulina, a compound containing vitamins and micronutrients and an iron-folic acid-B12 supplement. Follow-up of the cases was done over a period of one year and the impact of supplementation and a comparison of the two compounds made.

Anemia and hemoglobinopathies in tribal population of Eastern and North-eastern India.

It is estimated that out of approximately 31.4 million people living in North-eastern India, about 8.1 million are tribal people of the hills and plains. Among four of the seven north-eastern states, tribal people are in majority. Arunachal Pradesh is made of approximately 24 major tribal groups, which constitute about 70% of the total population, Tripura 29% and in Assam constitutes 11%. A total of 1726 cases were randomly selected in this study, out of which 1263 cases were from North-east India, namely from Arunachal Pradesh, Assam, Tripura and the rest were from West Bengal. Hematological parameters were estimated and agarose gel electrophoresis for identification of the Hb variants was performed. DNA was isolated, amplified and analysed by PCR-ARMS technology. The incidence of anemia among the tribal people of Assam was 59.82%, in Arunachal Pradesh 53.77% and Tripura 57.45%. The presence of hemoglobinopathies and thalassemia account for anemia in a sizeable population of the north-eastern states in certain tribes and urgent health resources are needed to deal with this. HbE appears to be the commonest hemoglobin among the different tribes of north-east.