Types of human papillomavirus observed in hospital-based population.

Background and Objectives: Human papillomavirus (HPV) is the causative agent of cervical cancer, a major cause of cancer mortality in Indian women. The current study was undertaken to add information to the existing data on HPV type distribution in Indians, in an attempt to document HPV types for future vaccination programme, if any. Materials and Methods: HPV infection was screened in 223 cervical cancer cases and 2408 healthy women without cancer and cervical intraepithelial neoplasia (control). HPV was typed using polymerase chain reaction, Southern hybridisation using specific probes and HPV GenoArray (Hybribio) test. Results: HPV DNA was found in 92.8% of cases and 7.3% of controls. Of the 383 HPV-infected women, 30.0% had single infection; 50.9% had multiple infections (two or more types) and 19.1% were infected with HPV types other than HPV-16, -18, -6 and -11. Besides HPV-16, HPV-51 and HPV-33 were also seen as single infection in cases. In cases, HPV-18 or its homologous HPV-45 was always present as co-infection with HPV-16 or with other high-risk type. Binary logistic regression (backward) analysis highlighted significant association of age, parity and socioeconomic status with HPV infection. The present study highlighted the presence of multiple HPV infection (186 of 207, 89.9%) along with HPV-16 in women with cervical cancer. In control, 27.3% were co-infected with other sexually transmitted infections, while Chlamydia trachomatis infection was seen in 13% of cases. Conclusions: The study highlighted the type of HPV infection seen among the hospital-based population. For better screening, HPV tests available in the market should include all the types seen in the population.

Cervical cancer in Indian women reveals contrasting association among common sub-family of HLA class I alleles.

We studied the relationship between human leukocyte antigen (HLA) class I alleles and cervical cancer among Indian women. Seventy-five cervical cancer cases were compared with 175 noncancer controls. Cervical biopsy tissue specimen from cancer cases and cervical swab specimen from controls were collected for HPV detection and typing. Blood was taken for HLA typing by PCR-SSOP method. The impact of HLA class I alleles on cervical cancer risk was evaluated using StatCalc program (Epi Info version 6.0.4. CDC Atlanta, GA, USA), and confirmed with Bonferroni correction. Results revealed HLA-B*37, HLA-B*58 were associated significantly with increased risk while HLA-B*40 with decreased risk for cervical cancer. At high-resolution analysis after Bonferroni correction, HLA-B*37:01 allele was associated with increased risk, whereas HLA-B*40:06 was with decreased risk for cervical cancer. HLA-B*37:01 and HLA-B*40:06 belong to the same superfamily of HLA-B44. In silico analysis revealed different binding affinities of HLA-B*37:01 and HLA-B*40:06 for the epitopes predicted for E6 and L1 proteins of HPV16. The higher binding affinity of epitopes to B*40:06, as revealed by docking studies, supports the hypothesis that this allele is able to present the antigenic peptides more efficiently than B*37:01 and thereby can protect the carriers from the risk of cervical cancer. Thus, there is a clear indication that HLA plays an important role in the development of cervical cancer in HPV-infected women. Identification of these factors in high-risk HPV-infected women may help in reducing the cervical cancer burden in India.

Variations in immunogenetics, human papillomavirus (HPV) infection & predisposition to cervical cancer in Indian women.

BACKGROUND & OBJECTIVES: Human papillomavirus (HPV) is the main causative agent for cervical cancer. Variability in host immunogenetic factors is important in determining the overall cellular immune response to the HPV infection. This study was carried out to confirm the association between human leukocyte antigen (HLA) class II alleles and cervical cancer in HPV infected women. METHODS: Both low and high resolution methods were used to genotype HLA class II (DRB1 and DQB1) alleles in 75 women with cervical cancer (cases) and 75 HPV positive women and 100 HPV negative women with healthy cervix (controls). odds ratio and 95% confidence interval were calculated. Co-occurring HLA alleles (haplotype) across cases and controls were also studied. RESULTS: Significant association was found for HLA-DRB1*03(*13:01) and - DQB1*02(*02:01) with increased risk for cervical cancer. Also, HLA-DRB1*13(*13:01); -DQB1*06 and -DQB1*03:02 were significantly associated with decreased risk for cervical cancer. Haplotype analysis highlighted the significant association of HLA- DRB1*07:01-DQB1*02:02 and HLA DRB1*10:01-DQB1*05:01 with cervical cancer, while HLA-DRB1*14:04-DQB1*05:03 and DRB1*15:01-DQB1*06:01 conferred decreased risk for cervical cancer. Multivariate analysis highlighted the association of specific alleles with cervical cancer after adjusting for confounding factor age. INTERPRETATION & CONCLUSIONS: There were possible associations of specific HLA class II alleles either with risk of developing cervical cancer, or with its protection. Our results confirmed the assessment of DRB1*13 as a protective marker in HPV infection outcome. our study also revealed protective association of homozygous haplotype DRB1*15- DQB1*06 with cervical cancer.

CTLA-4 gene polymorphism at position +49 A>G in exon 1: a risk factor for cervical cancer in Indian women.

Single nucleotide polymorphisms (SNPs) in the CTLA-4 gene exert differential effects on T-cell response to viral infection. We aimed to evaluate the association of two SNPs of the CTLA-4 gene with cervical cancer in Indian women. The two polymorphic loci, one in the promoter region -318 C>T, rs5742909 (100 cervical cancer cases and 101 controls) and the other in exon 1 +49 A>G, rs231775 (104 cervical cancer cases and 162 controls) were genotyped using polymerase chain reaction-restriction fragment length polymorphism methods. Haplotype block structure was determined using Haploview 4.2. The statistical analyses were performed using a commercially available statistical software package, whereas PyPop was used to calculate the haplotypic frequencies. In this case-control study, the A/A genotype frequency (30.76% vs. 17.6%, P = 0.01) as well as the allelic frequency for A (52.8% vs. 43.5%, P = 0.04) was significantly higher in cases compared to controls. No significant association was seen in the -318 C>T polymorphism. In forward stepwise binary logistic regression analysis considering age and parity as potential confounders, significant association was demonstrated between +49 A/A and cervical cancer. Most likely, this is the first study from India to highlight the significant association between the CTLA-4 gene +49 A/A SNP and cervical cancer, thus adding to the global knowledge of the association of this SNP with cervical cancer.

Methylation status of imprinted genes DLK1-GTL2, MEST (PEG1), ZAC (PLAGL1), and LINE-1 elements in spermatozoa of normozoospermic men, unlike H19 imprinting control regions, is not associated with idiopathic recurrent spontaneous miscarriages.

OBJECTIVE: To study methylation aberrations in spermatozoa at developmentally important imprinted regions to ascertain their role in early embryo loss in idiopathic recurrent spontaneous miscarriages (RSM). DESIGN: Case-control study. SETTING: Academic research setting at National Institute for Research in Reproductive Health, Parel, Mumbai. PATIENT(S): Male partners of couples with a history of RSM and male partners of couples with proven fertility (control group). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): DNA methylation levels at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) by Epityper Massarray and global methylation levels as measured by LINE-1 methylation and anti-5-methyl cytosine antibody in spermatozoa of 23 men in control group and 23 men in RSM group. RESULT(S): We did not observe any aberration in the total methylation levels in any of the imprinted genes or global methylation analyzed. CONCLUSION(S): Our results indicate that paternal methylation aberrations at imprinting control regions of DLK1-GTL2, MEST (PEG1), and ZAC (PLAGL1) and global methylation levels are not associated with idiopathic RSM and may not be good epigenetic markers (unlike the H-19 imprinting control region) for diagnosis of idiopathic RSM.

Methylation analysis of idiopathic recurrent spontaneous miscarriage cases reveals aberrant imprinting at H19 ICR in normozoospermic individuals.

OBJECTIVE: To study H19 ICR methylation levels in association with sperm parameters routinely analyzed in idiopathic recurrent spontaneous miscarriage cases. DESIGN: Case-control study. SETTING: Academic research setting. PATIENT(S): Male partners of couples with a history of idiopathic recurrent spontaneous miscarriage (RSM group) and male partners of couples with proven fertility (control group). INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Paternal age, sperm concentration, motility, chromatin compaction status, morphology, and H19 ICR methylation were assessed in control and idiopathic RSM group participants. RESULT(S): Paternal age and basic semen parameters analyzed did not show any significant difference between the two groups; however H19 ICR methylation levels were reduced significantly in the idiopathic RSM group compared with the control group. CONCLUSION(S): Significant reduction in the H19 ICR methylation without significant difference in the sperm parameters demonstrates aberrant imprinting to be associated with idiopathic RSM.

Current Chlamydia trachomatis Infection, A Major Cause of Infertility.

BACKGROUND: In India, the impact of current Chlamydia trachomatis (C. trachomatis) in reproductive health remains a neglected area of investigation. The present study evaluates if current Chlamydia infection is associated with any clinical complication that needs the attention of clinical investigators. METHODS: In this cross-sectional study, we enrolled 896 women attending the Gynecology Out Patient for the detection of C. trachomatis infection. Polymerase chain reaction was used to diagnose current C. trachomatis infection and ELISA for past infections. Bacterial vaginosis, Candida and Trichomonas were screened. The results of symptomatic and asymptomatic groups were compared. The data was analyzed using Epi Info version 6 and "Z" test. A probability value of p≤0.05 was considered as significant.. RESULTS: Statistical analysis revealed significant association between current C. trachomatis infection with infertility when comparing infected fertile (18.6% vs. 9.4%, odds ratio: 2.19, p<0.0005) and uninfected infertile women (45.6% vs. 27.3%, odds ratio: 2.24, p<0.0001). Average infection rate was 12.1%, highest in women with infertility (18.6%) or with ectopic pregnancy (25%). Significant proportions of infected women with infertility (p<0.01) or with recent pregnancy (p<0.001) were asymptomatic. Follow up of infected women who became negative after treatment [28 women from infertility group and 9 women with recurrent spontaneous abortion (RSA)] revealed live birth in 8 (21.6%) women within one year, 4 with infertility and 4 with RSA. CONCLUSION: Study findings suggest association between current C. trachomatis infection and infertility. Absence of signs and symptoms associated with this infection highlights its diagnosis in women with a history of infertility and RSA for their better management, as revealed by live births with one year of follow up.

Detection, characterization, and spontaneous differentiation in vitro of very small embryonic-like putative stem cells in adult mammalian ovary.

The present study was undertaken to detect, characterize, and study differentiation potential of stem cells in adult rabbit, sheep, monkey, and menopausal human ovarian surface epithelium (OSE). Two distinct populations of putative stem cells (PSCs) of variable size were detected in scraped OSE, one being smaller and other similar in size to the surrounding red blood cells in the scraped OSE. The smaller 1-3 µm very small embryonic-like PSCs were pluripotent in nature with nuclear Oct-4 and cell surface SSEA-4, whereas the bigger 4-7 µm cells with cytoplasmic localization of Oct-4 and minimal expression of SSEA-4 were possibly the tissue committed progenitor stem cells. Pluripotent gene transcripts of Oct-4, Oct-4A, Nanog, Sox-2, TERT, and Stat-3 in human and sheep OSE were detected by reverse transcriptase-polymerase chain reaction. The PSCs underwent spontaneous differentiation into oocyte-like structures, parthenote-like structures, embryoid body-like structures, cells with neuronal-like phenotype, and embryonic stem cell-like colonies, whereas the epithelial cells transformed into mesenchymal phenotype by epithelial-mesenchymal transition in 3 weeks of OSE culture. Germ cell markers like c-Kit, DAZL, GDF-9, VASA, and ZP4 were immuno-localized in oocyte-like structures. In conclusion, as opposed to the existing view of OSE being a bipotent source of oocytes and granulosa cells, mammalian ovaries harbor distinct very small embryonic-like PSCs and tissue committed progenitor stem cells population that have the potential to develop into oocyte-like structures in vitro, whereas mesenchymal fibroblasts appear to form supporting granulosa-like somatic cells. Research at the single-cell level, including complete gene expression profiling, is required to further confirm whether postnatal oogenesis is a conserved phenomenon in adult mammals.

Human Papillomavirus infection in asymptomatic population.

OBJECTIVES: The present study aimed to evaluate type specific Human Papillomavirus (HPV) strains in women with different clinical manifestations but with normal cervical cytology, attending a gynecology out patient clinic and HPV infection in males attending a private pathology laboratory for routine check up in Mumbai. METHODS: Cervical swab specimens from 470 women with normal cervical cytology as detected by Pap were used for detection and typing of HPV by PCR, southern blotting and sequencing. In 104 males, 30 ml of first void/random urine specimens were used for HPV screening. RESULTS: Thirty-eight women (8.1%) tested positive for HPV. HPV 16, 18, 6, 11 and mixed infection was observed in 26.3%, 10.5%, 36.8%, 5.2% and 15.8% of these infected women, respectively, while 36.8% had other HPV types, indicating high rate of high-risk HPV types 16/18. Among the 104 males, 12 (11.5%) had HPV infection, 50% (n=6) of them were below 30 years. Nine of them were married and three were unmarried. CONCLUSIONS: The present study revealed presence of high risk HPV infection in women with normal cervical cytology. This is the first report from the Western region of India on HPV infection in males using urine specimen.

Thrombophilia and unexplained pregnancy loss in Indian patients.

BACKGROUND: The role of acquired and congenital thrombophilias in the aetiology of unexplained pregnancy loss in the Indian population has not been studied in detail. We studied the association of acquired and inherited markers of thrombophilia in a large group of patients with unexplained pregnancy loss. METHODS: A total of 602 women with pregnancy loss were referred to us for evaluation of thrombophilia between April 2000 and June 2005. After investigations to rule out cytogenetic, hormonal, anatomical and microbiological causes, no cause was ascertained in 430 women for the pregnancy loss. Of these, 49 women, who had a history of only one pregnancy loss, were excluded. The remaining 381 women comprised the study group. These patients and 100 age-matched women who did not have any obstetric complication and had at least one normal healthy child (controls) underwent detailed investigations for the presence of thrombophilia markers. These included screening coagulations tests, tests for lupus anticoagulant (LA), IgG and IgM antibodies to anticardiolipin antibodies (ACA), beta2 glycoprotein 1 (beta2GP1) and annexin V. The genetic markers studied included protein C (PC), protein 5 (PS), antithrombin III (AT III), factor V Leiden (FVL), PT gene G20210A, MTHFR C677T, EPCR 23 bp insertion and PAI 4G/5G polymorphisms. RESULTS: Of the 381 women with pregnancy loss, 183 had 2 and 198 had > or = 3 pregnancy losses. Early pregnancy loss occurred in 136 patients, late pregnancy loss in 119, and both early and late pregnancy losses in 126. The strongest association was observed with ACA (OR 32.5, 95% CI: 8.6-21.8, p < 0.001) followed by annexin V (OR 17.1, 95% CI: 2.9-99.4, p < 0.001), LA (OR 8.2, 95% CI: 1.4-47.7, p = 0.01) and anti-beta2GP1 (OR 5.8, 95% CI: 1.6-22.1, p = 0.007). No association of antiphospholipid antibodies with the time of pregnancy loss was found except LA which was significantly associated with early pregnancy loss compared with late pregnancy loss (p < 0.05). The risk of pregnancy loss with PS deficiency (OR 17.8, 95% CI: 3.1-102.9, p < 0.001) was the highest observed for any heritable thrombophilia followed by PC deficiency (OR 5.8, 95% CI: 1-34, p = 0.06). There were no statistically significant differences in the frequency of any of the genetic thrombophilias studied between women with early and late pregnancy loss. A combination of > or = 2 genetic factors was observed in 41 (10.8%) while that of genetic and acquired risk factors were observed in 79 (20.7%) patients. No more than one risk factor was observed in any of the controls. In all, 176 (46.2%) patients had at least one acquired thrombophilia while 143 (37.5%) had at least one genetic thrombophilia marker. Overall, 288 patients (75.6%) had either an acquired, genetic or both markers of thrombophilia. CONCLUSION: Thrombophilia is an important factor in both early and late pregnancy losses. Women with unexplained pregnancy loss should be screened for the presence of thrombophilias.

Successful pregnancy outcome in women with bad obstetric history and recurrent fetal loss due to thrombophilia: effect of unfractionated heparin and low-molecular weight heparin.

Acquired and inherited thrombophilias are known to be associated with unfavorable pregnancy outcome including recurrent fetal loss. There are differences of opinion whether these patients need to be treated with aspirin, unfractionated heparin, low-molecular weight heparin, corticosteroids, or intravenous immunoglobulins. In all, 25 consecutive patients with a history of fetal loss and 7 patients who presented in early pregnancy with deep-vein thrombosis were treated, and their pregnancy outcome was noted. All the women were positive either for a solitary or for a combination of acquired and heritable thrombophilia markers. In all, 23 patients were treated with unfractionated heparin and 9 with low-molecular weight heparin. In all, 16 out of 23 patients (69.6%) treated with unfractionated heparin and 9 out of 9 (100%) treated with low-molecular weight heparin had successful pregnancy outcome. There was a complete resolution of thrombus in all the cases. None of the patients had any adverse reactions such as heparin-induced thrombocytopenia, thrombosis, or fracture. Both unfractionated heparin and low-molecular weight heparin were effective in cases of bad obstetric history and recurrent pregnancy loss due to thrombophilia. However, low-molecular weight heparin was found to be more effective than unfractionated heparin along with other advantages of not requiring laboratory monitoring and easy administration. None of the patients in either group had to interrupt the therapy for any adverse treatment-related complications.

A comprehensive screening analysis of antiphospholipid antibodies in Indian women with fetal loss.

OBJECTIVE: The present study was aimed at a comprehensive analysis of acquired thrombophilia in a large series of Indian women with fetal loss. STUDY DESIGN: Four hundred and thirty women (median age 26 years, range 18-39 years) with unexplained fetal loss (median number of abortions 3, range 1-13) were screened for the presence of antiphospholipid antibodies (APA), i.e. lupus anticoagulant (LA), IgG/M antibodies for cardiolipin (ACA), beta 2 glycoprotein 1 (beta2 GP1) and annexin V. We also studied 100 normal healthy women (median age 24 years, range 18-30 years) who had at least one healthy child and did not have any miscarriage or other obstetric complications. RESULTS: The prevalence of persistently positive LA was 8.1% and 1% in the patients and controls, respectively (OR 8.7; 95% CI, 1.4-51; P<0.05). The overall prevalence of IgG and/or IgM antibodies for cardiolipin, beta 2 GP1 and annexin V were as follows-ACA 27.9% (OR 18.9; 95% CI, 5-70; P<0.05), beta 2 GP1 12.2% (OR 6.8; 95% CI, 1.8-25; P<0.05) and annexin V 14.6% (OR 17; 95% CI, 2.9-98; P<0.05). The conventional LA and ACA tests were positive 23.2% of the cases as against 1% in the controls (OR 14.8; 95% CI, 3.9-55; P<0.05). The prevalence of LA, ACA, beta 2 GP1 and annexin V antibodies as independent risk factors were observed in 0.5%, 16.5%, 5.4% and 7.8% in the patients as against 1% each in the controls. The overall positivity for any one of the APA studied was 42.6% (OR 10.2; 95% CI, 4.5-23; P<0.05). CONCLUSION: The present study thus indicates the importance of APA in women experiencing fetal loss where all the conventional causes of miscarriages have been ruled out. It also suggests that conventional APA assays (LA and ACA) are effective in the detection of a majority of APA positive cases and by the addition of other cofactor-dependent (beta 2 GP1 and annexin V) APA assays, there is a considerable increase in the diagnostic efficiency in the detection of APA.

Deep venous thrombosis in the antenatal period in a large cohort of pregnancies from western India.

BACKGROUND: Deep venous thrombosis (DVT) is an important complication in the peripartal and postpartal period. METHODS: We followed up prospectively the prevalence of DVT in 34720 prenatal mothers between June 2002 and July 2006 attending the antenatal clinics of two major hospitals in Mumbai, India. Thirty two women (0.1%) presented for the first time with symptomatic DVT i.e. 17 in the first trimester, 6 in the second and 9 in the third trimester of pregnancy. Nine had history of fetal loss while in the remaining twenty three there was no history of fetal loss. RESULTS: The evaluation of both acquired and heritable thrombophilia showed a conglomeration of thrombophilia in this group when compared to 100 normal pregnant women who have given birth to at least one healthy baby with no history of fetal death, DVT or other obstetrical complications. The relative risks for all the antiphospholipid antibodies (APA) studied i.e lupus anticoagulant (LA), IgG/IgM antibodies for cardiolipin (ACA), beta2 glycoprotein 1 (beta2 GP 1) and annexin V were significantly higher in women with pregnancy associated DVT (RR 7.4 95% CI 4.3-11.3 P < 0.05). Among the genetic thrombophilia markers studied, Protein S (PS) deficiency was the strongest risk factor (RR 5.00 95% CI 3.02-5.00 P < 0.05) followed by factor V Leiden (FVL) mutation (RR 4.57 95% CI 2.23-4.57 P < 0.05) and PAI 4G/4G homozygosity (RR 3.24 95% CI 1.85-5.12 P < 0.05). Protein C (PC) and endothelial protein C receptor (EPCR) 23 bp insertion polymorphism was also increased in the patient group as compared to controls but the difference was not statistically significant. The MTHFR C677T, fibrinogen gene beta448 Arg/Lys polymorphisms were not significantly different from the normal controls, while antithrombin III (AT III) deficiency and PT G20210A polymorphism were absent in both controls and patients. Two or more risk factors were present in 22 out of 32 cases (68.75%). CONCLUSION: We conclude that the prevalence of DVT in India is more or less similar to other reports published and both acquired and heritable thrombophilia show strong association with DVT associated with pregnancy.

Successful embolization of an ovarian artery pseudoaneurysm complicating obstetric hysterectomy.

Transcatheter arterial embolization is becoming the therapy of choice for controlling obstetric hemorrhage, affording the ability to control persistent bleeding from pelvic vessels while avoiding the morbidity of surgical exploration. The clinicians are left with little choice if pelvic hemorrhage continues after hysterectomy and ligation of anterior division of both internal iliac arteries. We present one such case of intractable post-obstetric hysterectomy hemorrhage in which an ovarian artery pseudoaneurysm was diagnosed angiographically and successfully embolized, highlighting the role of transcatheter embolization.

The spectrum of bleeding disorders in women with menorrhagia: a report from Western India.

In order to evaluate the incidence of hereditary bleeding disorders in patients presenting with menorrhagia, where the usual gynecological and endocrinal causes of bleeding were ruled out by various local ultrasonography (USG) and relevant endocrine investigations, 120 women aged between 18 and 35 years presenting with menorrhagia without any discernable cause were studied using an open design, where the investigators knew that these patients had menorrhagia. These patients were investigated for inherited coagulation defects. Of the 120 women investigated, 19.16% (23 cases) had an inherited coagulation disorder to account for their menorrhagia. Although a majority (11.6%) are patients with von Willebrand's disease (VWD), other rare platelet disorders such as Glanzmann's thrombasthenia (3.3%), Bernard-Soulier syndrome (0.83%), coagulation factor deficiencies such as factor VIII (0.83%), factor X (0.83%), and factor XI (0.83%), and immune thrombocytopenia (0.83%) were also found. Taking a detailed history for bleeding from other sites however minor, paternal consanguinity as well as family history of bleeding tendencies appeared as a very strong predictor for such kinds of disease in patients with menorrhagia. Patients with menorrhagia without a discernable cause, therefore, need evaluation for the congenital coagulation disorders.