Human papillomavirus type-specific persistence and recurrence after treatment for cervical dysplasia.

Human papillomavirus (HPV) infection is a necessary factor in the cervical cancer development. Also after treatment for cervical dysplasia, HPV can be present and promote the recurrence of cervical disease. In the present study, the aim was to perform a long-term follow-up on the ability of HPV testing with genotyping, as compared with cytology, to predict recurrence of high-grade cervical intraepithelial neoplasia and to evaluate the effectiveness of treatment with loop electrosurgical excision procedure (LEEP) conization. Cervical samples for HPV DNA testing and cytological analysis were obtained from 178 women with abnormal smears referred for treatment with LEEP conization. These women were scheduled for HPV DNA testing and Pap smears before and 3, 6, 12, 24, and 36 months after treatment. Three years after treatment 3.1% (N = 4) of women were still persistently HPV-positive with the same type as had been detected at treatment. Recurrent or residual cervical intraepithelial neoplasia II+ in histopathology was found among 9 (5.1%) women during follow-up. All of these women had type-specific HPV-persistence (sensitivity 100% [95% CI 63-100%] and specificity 94.7% [89.8-97.4%]), but only 7/9 had abnormal cytology (sensitivity 77.8% [40.2-96.1%] and specificity 94.7% [89.8-97.4%]). No recurrent or residual disease was found among women with any other patterns of HPV positivity (e.g., type change or fluctuating positivity) (sensitivity 0% [95% CI 0-37.1%] and specificity 80.5% [73.5-86.0%]). In conclusion, only type-specific HPV persistence predicted recurrent or residual disease, and HPV genotyping appears useful to improve the specificity when using HPV testing in post-treatment follow-up.

Galectin-1 influences trophoblast immune evasion and emerges as a predictive factor for the outcome of pregnancy.

Galectin-1 (gal-1) is expressed at the feto-maternal interface and plays a role in regulating the maternal immune response against placental alloantigens, contributing to pregnancy maintenance. Both decidua and placenta contribute to gal-1 expression and may be important for the maternal immune regulation. The expression of gal-1 within the placenta is considered relevant to cell-adhesion and invasion of trophoblasts, but the role of gal-1 in the immune evasion machinery exhibited by trophoblast cells remains to be elucidated. In this study, we analyzed gal-1 expression in preimplantation human embryos and first-trimester decidua-placenta specimens and serum gal-1 levels to investigate the physiological role played by this lectin during pregnancy. The effect on human leukocyte antigen G (HLA-G) expression in response to stimulation or silencing of gal-1 was also determined in the human invasive, proliferative extravillous cytotrophoblast 65 (HIPEC65) cell line. Compared with normal pregnant women, circulating gal-1 levels were significantly decreased in patients who subsequently suffered a miscarriage. Human embryos undergoing preimplantation development expressed gal-1 on the trophectoderm and inner cell mass. Furthermore, our in vitro experiments showed that exogenous gal-1 positively regulated the membrane-bound HLA-G isoforms (HLA-G1 and G2) in HIPEC65 cells, whereas endogenous gal-1 also induced expression of the soluble isoforms (HLA-G5 and -G6). Our results suggest that gal-1 plays a key role in pregnancy maternal immune regulation by modulating HLA-G expression on trophoblast cells. Circulating gal-1 levels could serve as a predictive factor for pregnancy success in early human gestation.

Contribution of TMC6 and TMC8 (EVER1 and EVER2) variants to cervical cancer susceptibility.

Cervical cancer (CxCa) is caused by persistent human papillomavirus (HPV) infection; genetic predisposition is also suspected to play a role. Our study is a targeted candidate gene follow-up based on: (i) strong clinical evidence demonstrating that mutations in the TMC6 and TMC8 (EVER1 and EVER2) genes associate with the HPV-associated disease epidermodysplasia verruciformis (EV) and (ii) recent epidemiological data suggesting a genetic susceptibility conferred by polymorphisms in such genes for skin and CxCa. Clarifying the association of the TMC6/8 genes with risk of CxCa will help in understanding why some HPV-infected women develop persistent infection, cervical lesions and eventually cancer while others do not. Twenty-two single nucleotide polymorphisms (SNPs) harboring the TMC6/8 genes were genotyped in 2,989 cases with cervical intraepithelial neoplasia grade III or invasive CxCa and 2,281 controls from the Swedish population. Association was evaluated in logistic regression models. Two SNPs displayed association with cervical disease: rs2290907 [odds ratio (OR)(GGvsAA) = 0.6, 95% confidence interval (95% CI): 0.3-0.9, p = 0.02)] and rs16970849 (OR(AGvsGG) = 0.8, 95% CI: 0.66-0.98, p = 0.03). The present data support the involvement of the TMC6/8 region in CxCa susceptibility but further analyses are needed to replicate our findings, fully characterize the region and understand the function of the genetic variants involved.

Dietary factors and in situ and invasive cervical cancer risk in the European prospective investigation into cancer and nutrition study.

Some dietary factors could be involved as cofactors in cervical carcinogenesis, but evidence is inconclusive. There are no data about the effect of fruits and vegetables intake (F&V) on cervical cancer from cohort studies. We examined the association between the intake of F&V and selected nutrients and the incidence of carcinoma in situ (CIS) and invasive squamous cervical cancer (ISC) in a prospective study of 299,649 women, participating in the European Prospective Investigation into Cancer and Nutrition study. Cox proportional hazard models were used to estimate adjusted hazard ratios (HRs) and 95% confidence intervals (95% CI). A calibration study was used to control measurement errors in the dietary questionnaire. After a mean of 9 years of follow-up, 253 ISC and 817 CIS cases were diagnosed. In the calibrated model, we observed a statistically significant inverse association of ISC with a daily increase in intake of 100 g of total fruits (HR 0.83; 95% CI 0.72-0.98) and a statistically nonsignificant inverse association with a daily increase in intake of 100 g of total vegetables (HR 0.85: 95% CI 0.65-1.10). Statistically nonsignificant inverse associations were also observed for leafy vegetables, root vegetables, garlic and onions, citrus fruits, vitamin C, vitamin E and retinol for ISC. No association was found regarding beta-carotene, vitamin D and folic acid for ISC. None of the dietary factors examined was associated with CIS. Our study suggests a possible protective role of fruit intake and other dietary factors on ISC that need to be confirmed on a larger number of ISC cases.

Human placenta expresses and secretes NKG2D ligands via exosomes that down-modulate the cognate receptor expression: evidence for immunosuppressive function.

During mammalian pregnancy maternal-fetal tolerance involves a number of immunosuppressive factors produced by placenta. Recently, placenta-derived exosomes have emerged as new immune regulators in the maternal immune tolerance. Exosomes are membrane nanovesicles with defined morphology, which are secreted from endosomal multivesicular bodies (MVB) upon fusion with the plasma membrane. Previously, we reported that the MHC class I chain-related (MIC) proteins A and B, human ligands of the activating NK cell receptor NKG2D, are expressed by placenta, sorted to MVB of syncytiotrophoblast and probably released via MIC-bearing exosomes. In this report, we show that the second family of human NKG2D ligands, the UL-16 binding proteins (ULBP), is also expressed by placenta. Importantly, this expression was not due to placental CMV infection. Immunoelectron microscopy disclosed that ULBP1-5 are produced and retained in MVB of the syncytiotrophoblast on microvesicles/exosomes. Using human placenta explant cultures and different assays, we demonstrate that exosomes bearing NKG2D ligands are released by human placenta. Isolated placental exosomes carried ULBP1-5 and MIC on their surface and induced down-regulation of the NKG2D receptor on NK, CD8(+), and gammadelta T cells, leading to reduction of their in vitro cytotoxicity without affecting the perforin-mediated lytic pathway. Release of placental NKG2D ligands via exosomes is an alternative mechanism for generation of bioactive soluble form of these ligands. These findings highlight a role for NKG2D ligand-bearing placental exosomes in the fetal immune escape and support the view of placenta as a unique immunosuppressive organ.

Association of HLA-DRB1, interleukin-6 and cyclin D1 polymorphisms with cervical cancer in the Swedish population--a candidate gene approach.

High-risk human papillomavirus (hrHPV) infection is the major risk factor for cervical cancer (CxCa). The role of genetic susceptibility in the disease has been suggested, but the existing data lack consistency. We conducted a nested case-control study on 973 CxCa cases and 1,763 matched controls, from two Swedish population-based cohorts to examine the association of common genetic variants with CxCa risk. Human leukocyte antigen (HLA) alleles and 24 other polymorphisms in 14 genes were selected on the basis of reported association or mechanistic plausibility with an HPV infection or cervical cancer development. Genotyping was conducted using multiplex PCR and Luminex technology. A significant association of CxCa with various polymorphisms was observed: rs1800797 in the IL-6 gene (odds ratio [OR] = 0.88, 95% confidence intervals [CI]: 0.79-0.99); rs1041981 in the LTA gene (OR = 0.87, 95% CI: 0.78-0.98), and rs9344 in the CCND1 gene (OR = 1.14, 95% CI: 1.02-1.27), for those individuals carrying the rare allele. Additionally, the alleles 0401 and 1501 of the HLA class II DRB1 locus were associated with an increased risk (OR = 1.23, 95% CI: 1.04-1.45 and OR = 1.29, 95% CI: 1.11-1.50, respectively), and allele 1301 was associated with decreased risk (OR = 0.59, 95% CI: 0.47-0.73). The effects of CCND1 and the HLA*DRB1 alleles were independent of the effect of smoking. We did not find any association of risk with polymorphisms in genes related to the innate immune system. In conclusion, our study provides evidence for genetic susceptibility to CxCa due to variations in genes involved in the immune system and in cell cycle.

Maternal Foxp3 expressing CD4+ CD25+ and CD4+ CD25- regulatory T-cell populations are enriched in human early normal pregnancy decidua: a phenotypic study of paired decidual and peripheral blood samples.

PROBLEM: Regulatory T cells (Treg cells), a small subset of CD4(+) T cells maintaining tolerance by immunosuppression, are proposed contributors to the survival of the fetal semiallograft. We investigated Treg cells in paired decidual and peripheral blood (PB) samples from healthy women in early pregnancy and PB samples from non-pregnant women. METHOD OF STUDY: Distribution, location, cytokine mRNA, and phenotype were assessed in CD4(+) CD25(+) Treg cells from paired samples using immunohistochemistry, immunofluorescence, flow cytometry, and real-time quantitative RT-PCR. RESULTS: The presence and in situ distribution of CD4(+) Foxp3(+) Treg cells in decidua are hereby demonstrated for the first time. Three Foxp3(+) cell populations, CD4(+) CD25(++) Foxp3(+), CD4(+) CD25(+) Foxp3(+), and CD4(+) CD25(-) Foxp3(+), were enriched locally in decidua. In contrast, no statistically significant difference in numbers of circulating Treg cells between pregnant and non-pregnant women was found. The Foxp3(+) cells expressed the surface molecules CD45RO, CTLA-4, CD103, Neuropilin-1, LAG-3, CD62L, and TGFß1 mRNA consistent with Treg phenotype. The population of CD4(+) CD25(-) Foxp3(+) cells, not described in human decidua before, was enriched 10-fold compared with PB in paired samples. Their cytokine expression was often similar to Th3 profile, and the Foxp3 mRNA expression level in CD4(+) CD25(-) cells was stable and comparable to that of CD4(+) CD25(+) Treg cells implying that the majority of CD4(+) CD25(-) Foxp3(+) cells might be naïve Treg cells. CONCLUSION: (i) There is a local enrichment of Treg cells in decidua (ii) The exclusive accumulation of decidual CD4(+) CD25(-) Foxp3(+) cells suggests an additional reservoir of Foxp3(+) naïve Treg cells that can be converted to 'classical' Treg cells in uterus.

Endogenous sex steroids and risk of cervical carcinoma: results from the EPIC study.

BACKGROUND: Epidemiologic data and animal models suggest that, despite the predominant role of human papillomavirus infection, sex steroid hormones are also involved in the etiology of invasive cervical carcinoma (ICC). METHODS: Ninety-nine ICC cases, 121 cervical intraepithelial neoplasia grade 3 (CIN3) cases and 2 control women matched with each case for center, age, menopausal status and blood collection-related variables, were identified in the European Prospective Investigation into Cancer and Nutrition (EPIC) study. Circulating levels of testosterone (T) and estradiol (E(2)); dehydroepiandrosterone sulfate (DHEAS); progesterone (premenopausal women); and sex hormone-binding globulin (SHBG) were measured using immunoassays. Levels of free (f) T and E(2) were calculated from absolute concentrations of T, E(2), and SHBG. Odds ratios (ORs) and 95% confidence intervals (CI) were computed using regularized conditional logistic regression. RESULTS: Among premenopausal women, associations with ICC were observed for fT (OR for highest vs. lowest tertile = 5.16, 95% CI, 1.50-20.1). SHBG level was associated with a significant downward trend in ICC risk. T, E(2), fE(2), and DHEAS showed nonsignificant positive association with ICC. Progesterone was uninfluential. Among postmenopausal women, associations with ICC were found for T (OR = 3.14; 95% CI, 1.21-9.37), whereas E(2) and fT showed nonsignificant positive association. SHBG level was unrelated to ICC risk in postmenopausal women. No associations between any hormone and CIN3 were detected in either pre- or postmenopausal women. CONCLUSIONS: Our findings suggest for the first time that T and possibly E(2) may be involved in the etiology of ICC. IMPACT: The responsiveness of cervical tumors to hormone modulators is worth exploring.

Urinary, climacteric and sexual symptoms 1 year after treatment of cervical cancer without brachytherapy.

OBJECTIVE: Prospective studies elucidating the impact of the treatment of cervical cancer on urinary and climacteric symptoms and sexual life are relatively rare. The aim of this study was to seek information about the occurrence of urinary, climacteric and sexual symptoms in women with cervical cancer before and 1 year after treatment without brachytherapy. METHODS: This prospective study evaluated 39 women treated for cervical cancer. Data were collected by two questionnaires (before and 1 year after treatment). In order to supplement the data from the questionnaires, some data were selected from the patient's medical records. RESULTS: The number of voluntary micturitions, urgency, urinary incontinence and climacteric symptoms had not increased 1 year after treatment. Vaginal dryness and dyspareunia had increased and sexual desire was reduced 1-year post-treatment. CONCLUSION: This study has shown that urinary and climacteric symptoms are not frequent 1 year after treatment of cervical cancer without brachytherapy. However, there is an increased occurrence of vaginal dryness and sexual disorders 1-year post-treatment, mainly in the form of dyspareunia and reduced sexual desire. Taken together these symptoms affect the women's quality of life and it is, therefore, crucial that the medical providers become more aware of and skilled to deal with these conditions before and after treatment.

An efficient optimized method for isolation of villous trophoblast cells from human early pregnancy placenta suitable for functional and molecular studies.

PROBLEM: The uniqueness of the human placenta cannot be replaced by animal models. In vitro studies are compulsory to elucidate the biology of human placenta and require isolation and purification of villous trophoblasts, which can be used in molecular and functional studies. Constant improvement in the isolation technique is required to obtain a high yield of pure trophoblast cells with high viability and well preserved morphology. METHOD OF STUDY: Optimized isolation procedure for human villous trophoblasts based on mild enzymatic treatment, Percoll gradient centrifugation and additional purification step involving positive or negative immunoselection on magnetic beads is described. RESULTS: A simple and effective isolation protocol gave a reasonably high yield of villous trophoblast cells with high purity and viability, and excellent morphology as assessed by flow cytometry and electron microscopy. CONCLUSION: This protocol provides an efficient, optimized method for isolation and enrichment of villous trophoblast cells, suitable for phenotypic, ultrastructural, molecular and functional analyses and for establishment of primary cultures.

'See and treat' regime by LEEP conisation is a safe and time saving procedure among women with cytological high-grade squamous intraepithelial lesion.

BACKGROUND: Assess the value of colposcopic evaluation preceding loop electrosurgical excision procedure (LEEP) conisation of cytological high grade squamous intraepithelial lesion (HSIL), and study risk factors for recurrence. METHODS: Consecutive follow-up among women undergoing LEEP conisation from January 2001 to December 2004. RESULTS: Some 528 LEEP conisations were performed because of suspected or verified cervical dysplasia. On classified samples, cytology, punch biopsy and histopathology of the cone specimen showed cervical intraepithelial neoplasia (CIN)2 or a higher degree in 48.5, 36.2 and 58.6%, respectively. Sensitivity for HSIL out of cytology and colposcopically directed punch biopsy was 74.4 and 73.3%, respectively. Likewise, among 286 women with all 3 samples, positive and negative predictive value for HSIL in Papanicolaou (Pap) smear and punch biopsy was 78.5, 73.2% and 60.3, 63.6%, respectively. Positive cone margins were found in 16.8%. Residual/recurrent disease, defined as any grade of dysplasia at cytological follow-up, was found among 9.4%. Significant risk for recurrent/residual disease was found among those with positive marginal status. Median time from colposcopy to conisation was 2 months. CONCLUSIONS: An immediate colposcopically-guided LEEP conisation after HSIL Pap smear may be a safe and time saving strategy. Positive cone margins are a risk factor for residual/recurrent disease.