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Association between high risk papillomavirus DNA and nitric oxide release in the human uterine cervix
Gynecologic Oncology 114 (2009) 323–326
Contents lists available at ScienceDirect
Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y g y n o
Association between high risk papillomavirus DNA and nitric oxide release in the human uterine cervix Paivi Rahkola, Tomi S. Mikkola, Olavi Ylikorkala, Mervi Vaisanen-Tommiska ⁎ Department of Obstetrics and Gynecology, Helsinki University Central Hospital, P.O. Box 610, FIN-00029 HUS, Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 9 March 2009 Available online 29 May 2009 Keywords: Human papillomavirus Nitric oxide Cytology Histology
a b s t r a c t Objective. Local cervical factors may determine the outcome of human papillomavirus (HPV) infection. Nitric oxide (NO) may be one such factor, since it is produced by uterine cervical cells and it takes part in both immunological and carcinogenic reactions. We studied the association between the presence of cervical high risk (hr) HPV DNA and NO in the cervical canal in women. Methods. High risk HPV DNA status was assessed from 328 women by using a specific DNA test and the release of cervical NO was assessed as nitrate/nitrite in cervical fluid. Cervical NO was then compared between women showing different status of hr HPV DNA and different cytological and histological findings. Results. High risk HPV DNA was present in 175/328 (53%) women. The cervical NO release in women with hr HPV DNA was 90% higher compared to hr HPV DNA negative women (p b 0.001) (median 45.2 μmol/L; 95% CI 35.2–53.1 vs. 23.8 μmol/L; 95% CI 21.0–26.1). This elevation was not affected by parity, use of oral contraception, intrauterine devices, or signs of bacterial vaginosis or candida infection. Cytologically healthy epithelium and epithelium with mild cytological or histological changes showed elevated NO release if hr HPV DNA was present. Conclusions. The presence of hr HPV DNA is associated with an increased release of NO in the human uterine cervix. The clinical significance of this phenomenon remains open. © 2009 Elsevier Inc. All rights reserved.
Introduction Up to 70% of all women experience genital human papillomavirus (HPV) infection during their lifespan [1]. This infection heals spontaneously through immunological defense in the great majority of cases [2,3], but in some women HPV persists and this may lead to cervical cancer [1], which rarely arises without a preceding HPV infection [4]. However, out of 40 HPVs capable of causing genital HPV infection, only 15–18 types (such as HPV 16 and 18) are pre-cancerous [1,4,5]. Therefore, HPV DNA tests have been made available for screening these high risk (hr) viruses [6–8]. It is unknown why hr HPV infection is cancerous in some women whereas in others it is eradicated. Individual differences in systemic or local immunological defense may be one explanation. One possible factor involved could be nitric oxide (NO), a free radical gas produced through three nitric oxide synthases (NOSs) (neuronal, endothelial and inducible) [9], which are all found in the human uterine cervix [10]. Nitric oxide interacts with HPV in experimental settings [11–13] and modifies immunological and cancerous responses in cell and animal models [14–16]. The response to cancer is biphasic: NO may promote or suppress carcinogenesis depending on the microenvironment, and concentration and duration of NO release [15,16]. In our
⁎ Corresponding author. Fax: +358 9 2412192. E-mail address: mervi.vaisanen-tommiska@hus.fi (M. Vaisanen-Tommiska). 0090-8258/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2009.05.003
earlier study we found that women with cervical cytological changes (likely related to HPV) show increased cervical NO release [17]. In the present study, we compared the release of NO in the human uterine cervix between women infected with hr HPV and in those who were free of such an infection. Methods Study subjects With the permission of the local Ethics Committee, and with written informed consent from the study subjects, we investigated 328 non-pregnant women between 18–69 years of age. The women participated in a cervical smear screening program (N = 91) or visited the outpatient department of our hospital in connection with a history of abnormal cervical cytology (N = 237). Women with any visible vaginal bleeding (N = 16) had been excluded. Samples Cervical fluid samples were collected from each woman as described before [18]. Briefly, a Dacron swab kept in the cervical canal for precisely 20 s was flushed in 1.5 mL of physiological saline for 2 min. The samples were assayed for nitrate/nitrite metabolites (NOx) spectrophotometrically using the Griess reaction [18]. The detection limit of the assay for NOx was 0.8 μmol/L and the intra- and interassay
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P. Rahkola et al. / Gynecologic Oncology 114 (2009) 323–326
Table 1 Clinical characteristics of the study population (mean ± SD, range, or %). Clinical characteristics
Table 3 Levels of cervical fluid nitric oxide metabolites (NOx) (μmol/L, median, 95% confidence interval) in human papillomavirus (HPV) -infected (+) and non-infected (−) women with cytologically normal cervical epithelium, low grade atypia, or with high grade atypia.
All
HPV+
HPV−
(N = 328)
(N = 175)
(N = 153)
31.3 ± 9.6 18–58
39.7 ± 12.0 19–69
20 (43%) 27 (57%)
23 (42%) 32 (58%)
0.9
68 (39%) 107 (61%)
92 (60%) 61 (40%)
b 0.001
76 (43%) 5 (3%)
36 (24%)
b 0.001
23 (13%) 6 (3%) 8 (5%) 6 (3%) 1 (0.5%)
21 (14%) 4 (3%) 21 (14%) 11 (7%) 6 (4%)
0.9
Age
35.2 ± 11.4 18–69 Phase of spontaneous menstrual cycle Follicular 43 (42%) Luteal 59 (58%) Parity Parous 160 (49%) Nulliparous 168 (51%) Use of oral contraceptives Combined 112 (34%) Progestogen only 5 (2%) IUD Levonorgestrel-releasing 44 (13%) Copper 10 (3%) Postmenopausal 29 (9%) Use of hormone therapy 17 (5%) Use of vaginal hormone therapy 7 (2%)
p b 0.001
0.004 0.04
Cytology
HPV DNA+
HPV DNA−
p
Normal epithelium Low grade atypia High grade atypia
40.0 (15.6–61.2) (N = 38) 56.0⁎ (48.3–62.9) (N = 97) 28.8⁎ (17.5–40.1) (N = 24)
21.5 (17.8–26.6) (N = 59) 26.2 (17.6–42.4) (N = 39)
0.02 0.002
Low grade atypia: atypical squamous cells with undetermined significance + low grade squamous intraepithelial lesion. High grade atypia: atypical squamous cells with undetermined significance, cannot exclude high grade squamous intraepithelial lesion + high grade squamous intraepithelial lesion. ⁎ p = 0.002.
14.0 for Windows, Chicago, IL. Probability values of b0.05 were considered statistically significant.
Results coefficients of variation were 1.6% and 2.4% respectively. In order to reduce the impact of interassay variation, the samples were assayed in only seven batches. The presence of hr HPV DNA in the cervical fluid was studied by means of Hybrid Capture 2 (HC2; Digene Corporation, Gaithesburg, MD) fluorescence immunoassay (N = 91) or by means of the AMPLICOR HPV test (Roche Molecular Systems, Inc., Brachburg, NJ) (N = 237), involving the PCR technique, according to the instructions of the manufacturers. These tests were carried out immediately after collection of the NOx sample. Both tests detect DNAs of the same hr HPV types (types 16,18,31,33,35,39,45,51,52,56,58,59,68). After NOx and hr HPV DNA sampling a cervical smear was taken from 257 (78%) women. One hundred and eighty-five (56%) women underwent conventional colposcopy and representative cervical biopsy samples were collected. Both cytological and histological samples were analyzed by experienced pathologists.
Statistical methods The NOx data, presented as medians and 95% confidence intervals, were evaluated by means of the non-parametric Mann–Whitney U test or Kruskal–Wallis test. Undetectable NOx levels were regarded as being equivalent to the detection limit. Categorical data were analyzed by means of Chi-square test. The tests were carried out by using SPSS
Table 2 Levels of cervical fluid nitric oxide metabolites (NOx) (μmol/L) in women with (+) and without (−) human high risk papillomavirus (HPV) DNA. HPV+ (N = 175)
HPV− (N = 153)
p
45.2 (35.2–53.1)
23.8 (21.0–26.1)
b 0.001
61 23.8 (19.0–26.6) 9224.1 (20.0–30.1)
0.001 b 0.001
NOx Parity Nulliparous
107 45.2 (33.1–55.0) Parous 6844.1 (33.0–61.8) Use of oral contraceptives Yes 78 38.8 (31.7–51.0) No 97 46.6 (33.8–61.8) Use of an intrauterine device Yes 27 57.0 (30.7–70.7) No 148 45.0 (35.2–50.9) Median and 95% confidence interval (CI).
34 25.4 (18.9–39.8) 119 23.3 (20.0–26.1) 24 30.2 (22.3–33.0) 129 22.4 (20.0–25.3)
0.028
Of the 328 women hr HPV DNA was detected in 175. These women with detectable hr HPV DNA were younger, more often nulliparous and users of oral contraceptives (OC) and they were less often postmenopausal and users of hormone therapy. The phases of the menstrual cycle and the use of IUDs were equally distributed between the groups (Table 1). The presence of cervical fluid NOx did not differ between the women with (94.5%) and the women without (89.5%) hr HPV DNA (p = 0.15). The NOx level in women showing the presence of hr HPV DNA by means of the HC2 test (33.6 μmol/L; CI 95% 36.5–56.5) did not differ significantly from that in women tested by means of the AMPLICOR method (47.5 μmol/L; CI 95% 14.5–57.3) (p = 0.2). The level of cervical fluid NOx was 90% higher (p b 0.001) in women showing the presence of hr HPV DNA compared to women without hr HPV DNA (Table 2). This elevation was not affected by parity, or use of OCs, or IUDs (Table 2). The cytological findings were classified as normal epithelium (N = 122), low grade atypia (N = 117) including ASC-US (atypical squamous cells with undetermined significance) (N = 79) and LSIL (low grade squamous intraepithelial lesion) (N = 38), and high grade atypia (N = 18) including ASC-H (atypical squamous cells with undetermined significance, cannot exclude high grade squamous intraepithelial lesion) (n = 10) and HSIL (high grade squamous intraepithelial lesion) (N = 8) [19]. The presence of hr HPV DNA in women with cytologically normal cervical epithelium or with low grade atypia was associated with elevated levels of NOx. However, when cytological change was more severe, the level of NOx was lower (Table 3). Thirty-nine women showed clue cells in cervical smears, which were taken as a sign of bacterial vaginosis. Bacterial vaginosis did not affect the level of cervical NOx (Table 4).Twenty-five women showed the presence of candida infection on their cervical smears. Of these women 16 were infected with hr HPV DNA and nine women were free of hr HPV DNA. The level of cervical fluid NOx did not differ between
Table 4 Levels of cervical fluid nitric oxide metabolites (NOx) (μmol/L) in women with (+) and without (−) human high risk papillomavirus (HPV) DNA and bacterial vaginosis (BV).
b 0.001
0.008 b 0.001
BV+ BV−
HPV DNA+
HPV DNA−
p
36.2⁎ (24.3–62.9) (N = 20) 48.7⁎ (36.5–56.6) (N = 139)
27.3⁎⁎ (10.1–54.6) (N = 19) 22.8⁎⁎ (18.3–26.2) (N = 79)
0.2 b0.001
Median, 95% confidence interval (CI). ⁎ p = 0.76. ⁎⁎ p = 0.37.
P. Rahkola et al. / Gynecologic Oncology 114 (2009) 323–326 Table 5 Levels of cervical fluid nitric oxide metabolites (NOx) (μmol/L, median, 95% confidence interval) in human papillomavirus (HPV) -infected (+) and non-infected (−) women (N = 185) grouped according to the cervical histology. Histology
HPV DNA+
HPV DNA−
p
Normal
9.1 (bdetect–88.0) (N = 8) 46.2⁎ (37.7–55.0) (N = 91) 33.0⁎⁎ (25.9–60.9) (N = 33)
27.9 (2.0–70.6) (N = 14) 24.8 (17.6–41.3) (N = 37) 104.6 (N = 2)
0.245
Cervical intraepithelial neoplasia grade 1 Cervical intraepithelial neoplasia grade ≥ 2
0.004
⁎ p = 0.01 vs. normal. ⁎⁎ p = 0.03 vs. normal.
women with or without candida infection alone (p = 0.3) or concomitantly with (p = 0.84) or without (p = 0.27) hr HPV DNA (data not shown). The histological findings were classified as normal (N = 22), CIN1 (cervical intraepithelial neoplasia grade 1) including HPV-related changes and low grade dysplasia (N = 128), and ≥ CIN2 (cervical intraepithelial neoplasia grade 2) including moderate dysplasia (N = 32), high grade dysplasia (N = 2), and adenocarcinoma in situ (N = 1) [20]. High risk HPV DNA in association with a histological CIN1 change was associated with elevated NOx levels, whereas the sole presence of hr HPV DNA without histological changes was accompanied by low levels of NOx (Table 5).
Discussion The biological factors that ultimately determine the outcome of cervical HPV infection remain unknown [4]. Nitric oxide could have a major role, since all cells present in the cervical canal (squamous or glandular epithelial, stromal, or haematopoietic cells) are capable of producing it [12,13,21] and NO affects both immunological and carcinogenic processes. Thus, we wanted to find out if cervical NO release was affected by the presence of hr HPV DNA in cervical cells. We measured cervical NO release as the level of nitrate/nitrite, as in many previous studies [18,21]. Although two HPV DNA tests were employed in our study, they detect DNAs of the same papillomaviruses with similar sensitivity [7,8], and no difference in NOx level was seen among women found to be positive according to these two tests. These tests do not detect low risk HPV strains and thus, it is possible that women infected with low risk HPV were classified as noninfected in our study. However, as low risk HPVs are not a major risk factor for cervical cancer we believe that this issue should not affect our results. A majority of the women (53%) were found to have hr HPV DNA in our study population, which was probably a result of the fact that many had a history of abnormal cervical cytology. It is well known that the risk of HPV infection is higher among younger women [2] and thus, our finding on younger age in hr HPV-infected women was expected. Nevertheless, the age was not an independent factor in the release for cervical fluid NOx. The individual cervical NO release data showed great variation in both the women with and without hr HPV DNA. However, the release of NO was 90% higher in women with hr HPV DNA. Elevated levels of NOx were independent of parity, or use of OCs or IUDs. Evidence from both cell and tissue experiments implies that HPV may induce the synthesis of NO, mainly through iNOS activity [11–13]. Human papillomavirus stimulates the synthesis of NO in cultured and cervical keratinocytes [11–13], and also in cervical stromal cells [12,13]. Alternatively, cervical NO can be derived from various kinds of inflammatory cells [13,22] which accumulate in the cervical canal following HPV infection [13,23–25]. Papillomavirus may trigger both epithelial and inflammatory cells to release several cytokines
325
[11,26,27], which may indirectly regulate the release of NO in host cells [16,28]. The clinical significance of increased NO release in HPV-infected women remains open. It is possible that a rise in NO accompanies any cervical infection. However, our data do not support this, since the presence of clue cells – a marker of bacterial vaginosis – or signs of candida infection were not accompanied by elevated NO release. The results of many studies imply that NO is induced by viral infections [29,30] and this may enforce cell-mediated immunological defense against such infections [29,30]. If this is the case in the human uterine cervix during HPV infection, increased NO release should contribute to the eradication of HPVs and possibly protect against cancerous changes. This hypothesis is supported by the finding that NO release has an antitumor activity [16] and that it has been found to be highest – both in our and in another study [13] – in women with low grade cervical changes, which most often heal spontaneously [31]. It is possible that HPV infection may take several months to stimulate the maximal release of NO [3], but unfortunately the time of HPV infection could not be determined in our study subjects. This idea is supported by the fact that there was a low level of NOx in a few women with hr HPV DNA and normal histology who were most likely in such an early phase of the infection that NO stimulation had not yet started. An alternative explanation could be that NO, or its active intermediates, may cause additional DNA changes in cervical cells [12] and thus promote cervical carcinogenesis. It is known that the effect of NO on the tumor growth is dependent on concentration; a high concentration arrests and a low concentration promotes the tumor growth [16]. This is supported by our finding that severe cell changes were associated with reduced release of NO. However, we need to recognize that the decrease in NO quantity in high grade dysplasia may be a consequence of selection bias of those women with poor immunological response to the low grade lesion. In conclusion, the presence of hr HPV DNA was associated with increased release of NO in the human uterine cervix. This may be biologically significant in cases of HPV infection, but further epidemiological and experimental studies must be conducted to determine the specificity and significance of this phenomenon. Conflict of interest statement The authors declare that there are no conflicts of interest.
Submission declaration We disclose prior presentation of the study abstract: EUROGIN 2008 Joining forces against cervical cancer prevention, Nice, France 14.11.2008. Acknowledgments This study was supported by the Clinical Research Fund of Helsinki University Central Hospital, the Emil Aaltonen Foundation, the Research Foundation of Instrumentarium Corporation, the Research Foundation of Orion Corporation, the Finnish Cultural Foundation, the Finnish-Norwegian Medical Research Foundation, and the Clinical Graduate School of Pediatrics and Obstetrics and Gynecology. References [1] Bosch FX, de Sanjose S. Chapter 1: Human papillomavirus and cervical cancer — burden and assessment of causality. J Natl Cancer Inst Monogr 2003;31:3–13. [2] Giuliano AR, Harris R, Sedjo RL, Baldwin S, Roe D, Papenfuss MR, et al. Incidence, prevalence, and clearance of type-specific human papillomavirus infections: the young women's health study. J Infect Dis 2002;186:462–9. [3] Stanley M. Immune responses to human papillomavirus. Vaccine 2006;24(Suppl 1):S16–22. [4] Bosch FX, Lorincz A, Munoz N, Meijer CJ, Shah KV. The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 2002;55:244–65.
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[5] Munoz N, Bosch FX, de Sanjose S, Herrero L, Castellsague X, Shah KV, et al. Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 2003;348:518–27. [6] Howard M, Sellors J, Kaczorowski J. Optimizing the hybrid capture II human papillomavirus test to detect cervical intraepithelial neoplasia. Obstet Gynecol 2002;100:972–80. [7] Sandri MT, Lentati P, Benini E, DellÒrto P, Zorzino L, Carozzi FM, et al. Comparison of the Digene HC2 assay and the Roche AMPLICOR human papillomavirus (HPV) test for detection of high-risk HPV genotypes in cervical samples. J Clin Microbiol 2006;44:2141–6. [8] Stevens MP, Garland SM, Rudland E, Tan J, Quinn MA, Tabrizi SN. Comparison of the Digene hybrid capture 2 assay and Roche AMPLICOR and LINEAR ARRAY human papillomavirus (HPV) tests in detecting high-risk HPV genotypes in specimens from women with previous abnormal pap smear results. J Clin Microbiol 2007;45: 2130–7. [9] Peranzoni E, Marigo I, Dolcetti L, Ugel S, Sonda N, Taschin E, et al. Role of arginine metabolism in immunity and immunopathology. Immunobiology 2007;212: 795–812. [10] Tornblom SA, Maul H, Klimaviciute A, Garfield RE, Bystrom B, Malmstrom A, et al. mRNA expression and localization of bNOS, eNOS and iNOS in human cervix at preterm and term labour. Reprod Biol Endocrinol 2005;3:33. [11] De Andrea M, Mondini M, Azzimonti B, Dellòste V, Germano S, Gaudino G, et al. Alpha- and betapapillomavirus E6/E7 genes differentially modulate pro-inflammatory gene expression. Virus Res 2007;124:220–5. [12] Hiraku Y, Tabata T, Ma N, Murata M, Ding X, Kawanishi S. Nitrative and oxidative DNA damage in cervical intraepithelial neoplasia associated with human papilloma virus infection. Cancer Sci 2007;98:964–72. [13] Mazibrada J, Ritta M, Mondini M, De Andrea M, Azzimonti B, Borgogna C, et al. Interaction between inflammation and angiogenesis during different stages of cervical carcinogenesis. Gynecol Oncol 2008;108:112–20. [14] Bogdan C, Rollinghoff M, Diefenbach A. Reactive oxygen and reactive nitrogen intermediates in innate and specific immunity. Curr Opin Immunol 2000;12: 64–76. [15] Fukumura D, Kashiwagi S, Jain RK. The role of nitric oxide in tumour progression. Nat Rev Cancer 2006;6:521–34. [16] Ridnour LA, Thomas DD, Donzelli S, Espey MG, Roberts DD, Wink DA, et al. The biphasic nature of nitric oxide responses in tumor biology. Antioxid Redox Signal 2006;8:1329–37.
[17] Rahkola P, Mikkola TS, Nieminen P, Ylikorkala O, Vaisanen-Tommiska M. Abnormal cervical cytology is associated with increased cervical nitric oxide release in the uterine cervix. Acta Obstet Gynecol Scand 2009;88:417–21. [18] Vaisanen-Tommiska M, Nuutila M, Aittomaki K, Hiilesmaa V, Ylikorkala O. Nitric oxide metabolites in cervical fluid during pregnancy: further evidence for the role of cervical nitric oxide in cervical ripening. Am J Obstet Gynecol 2003;188:779–85. [19] Smith JH. Bethesda 2001. Cytopathology 2002;13:4–10. [20] Benedet JL, Odicino F, Maisonneuve P, Beller U, Creasman WT, Heintz AP, et al. Carcinoma of the cervix uteri. Int J Gynaecol Obstet 2003;83(Suppl 1):41–78. [21] Vaisanen-Tommiska M, Mikkola TS, Ylikorkala O. Increased release of cervical nitric oxide in spontaneous abortion before clinical symptoms: a possible mechanism for preabortal cervical ripening. J Clin Endocrinol Metab 2004;89:5622–6. [22] Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. The chemokine system in diverse forms of macrophage activation and polarization. Trends Immunol 2004;25:677–86. [23] Woo YL, Sterling J, Damay I, Coleman N, Crawford R, van der Burg SH, et al. Characterising the local immune responses in cervical intraepithelial neoplasia: a cross-sectional and longitudinal analysis. BJOG 2008;115:1616–22. [24] Hammes LS, Tekmal RR, Naud P, Edelweiss MI, Kirma N, Valente PT, et al. Macrophages, inflammation and risk of cervical intraepithelial neoplasia (CIN) progression — clinicopathological correlation. Gynecol Oncol 2007;105:157–65. [25] Tay SK, Jenkins D, Maddox P, Hogg N, Singer A. Tissue macrophage response in human papillomavirus infection and cervical intraepithelial neoplasia. BJOG 1987;94:1094–7. [26] Fernandes AP, Goncalves MA, Duarte G, Cunha FQ, Simoes RT, Donadi EA. HPV16, HPV18, and HIV infection may influence cervical cytokine intralesional levels. Virology 2005;334:294–8. [27] Scott M, Nakagawa M, Moscicki AB. Cell-mediated immune response to human papillomavirus infection. Clin Diagn Lab Immunol 2001;8:209–20. [28] Karpuzoglu E, Ahmed SA. Estrogen regulation of nitric oxide and inducible nitric oxide synthase (iNOS) in immune cells: implications for immunity, autoimmune diseases, and apoptosis. Nitric Oxide 2006;15:177–86. [29] Akaike T, Maeda H. Nitric oxide and virus infection. Immunology 2000;101:300–8. [30] Zaki MH, Akuta T, Akaike T. Nitric oxide-induced nitrative stress involved in microbial pathogenesis. J Pharmacol Sci 2005;98:117–29. [31] Wright Jr TC, Cox JT, Massad LS, Carlson J, Twiggs LB, Wilkinson EJ, et al. 2001 consensus guidelines for the management of women with cervical intraepithelial neoplasia. J Low Genit Tract Dis 2003;7:154–67.
Contents lists available at ScienceDirect
Gynecologic Oncology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / y g y n o
Association between high risk papillomavirus DNA and nitric oxide release in the human uterine cervix Paivi Rahkola, Tomi S. Mikkola, Olavi Ylikorkala, Mervi Vaisanen-Tommiska ⁎ Department of Obstetrics and Gynecology, Helsinki University Central Hospital, P.O. Box 610, FIN-00029 HUS, Helsinki, Finland
a r t i c l e
i n f o
Article history: Received 9 March 2009 Available online 29 May 2009 Keywords: Human papillomavirus Nitric oxide Cytology Histology
a b s t r a c t Objective. Local cervical factors may determine the outcome of human papillomavirus (HPV) infection. Nitric oxide (NO) may be one such factor, since it is produced by uterine cervical cells and it takes part in both immunological and carcinogenic reactions. We studied the association between the presence of cervical high risk (hr) HPV DNA and NO in the cervical canal in women. Methods. High risk HPV DNA status was assessed from 328 women by using a specific DNA test and the release of cervical NO was assessed as nitrate/nitrite in cervical fluid. Cervical NO was then compared between women showing different status of hr HPV DNA and different cytological and histological findings. Results. High risk HPV DNA was present in 175/328 (53%) women. The cervical NO release in women with hr HPV DNA was 90% higher compared to hr HPV DNA negative women (p b 0.001) (median 45.2 μmol/L; 95% CI 35.2–53.1 vs. 23.8 μmol/L; 95% CI 21.0–26.1). This elevation was not affected by parity, use of oral contraception, intrauterine devices, or signs of bacterial vaginosis or candida infection. Cytologically healthy epithelium and epithelium with mild cytological or histological changes showed elevated NO release if hr HPV DNA was present. Conclusions. The presence of hr HPV DNA is associated with an increased release of NO in the human uterine cervix. The clinical significance of this phenomenon remains open. © 2009 Elsevier Inc. All rights reserved.
Introduction Up to 70% of all women experience genital human papillomavirus (HPV) infection during their lifespan [1]. This infection heals spontaneously through immunological defense in the great majority of cases [2,3], but in some women HPV persists and this may lead to cervical cancer [1], which rarely arises without a preceding HPV infection [4]. However, out of 40 HPVs capable of causing genital HPV infection, only 15–18 types (such as HPV 16 and 18) are pre-cancerous [1,4,5]. Therefore, HPV DNA tests have been made available for screening these high risk (hr) viruses [6–8]. It is unknown why hr HPV infection is cancerous in some women whereas in others it is eradicated. Individual differences in systemic or local immunological defense may be one explanation. One possible factor involved could be nitric oxide (NO), a free radical gas produced through three nitric oxide synthases (NOSs) (neuronal, endothelial and inducible) [9], which are all found in the human uterine cervix [10]. Nitric oxide interacts with HPV in experimental settings [11–13] and modifies immunological and cancerous responses in cell and animal models [14–16]. The response to cancer is biphasic: NO may promote or suppress carcinogenesis depending on the microenvironment, and concentration and duration of NO release [15,16]. In our
⁎ Corresponding author. Fax: +358 9 2412192. E-mail address: mervi.vaisanen-tommiska@hus.fi (M. Vaisanen-Tommiska). 0090-8258/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ygyno.2009.05.003
earlier study we found that women with cervical cytological changes (likely related to HPV) show increased cervical NO release [17]. In the present study, we compared the release of NO in the human uterine cervix between women infected with hr HPV and in those who were free of such an infection. Methods Study subjects With the permission of the local Ethics Committee, and with written informed consent from the study subjects, we investigated 328 non-pregnant women between 18–69 years of age. The women participated in a cervical smear screening program (N = 91) or visited the outpatient department of our hospital in connection with a history of abnormal cervical cytology (N = 237). Women with any visible vaginal bleeding (N = 16) had been excluded. Samples Cervical fluid samples were collected from each woman as described before [18]. Briefly, a Dacron swab kept in the cervical canal for precisely 20 s was flushed in 1.5 mL of physiological saline for 2 min. The samples were assayed for nitrate/nitrite metabolites (NOx) spectrophotometrically using the Griess reaction [18]. The detection limit of the assay for NOx was 0.8 μmol/L and the intra- and interassay
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P. Rahkola et al. / Gynecologic Oncology 114 (2009) 323–326
Table 1 Clinical characteristics of the study population (mean ± SD, range, or %). Clinical characteristics
Table 3 Levels of cervical fluid nitric oxide metabolites (NOx) (μmol/L, median, 95% confidence interval) in human papillomavirus (HPV) -infected (+) and non-infected (−) women with cytologically normal cervical epithelium, low grade atypia, or with high grade atypia.
All
HPV+
HPV−
(N = 328)
(N = 175)
(N = 153)
31.3 ± 9.6 18–58
39.7 ± 12.0 19–69
20 (43%) 27 (57%)
23 (42%) 32 (58%)
0.9
68 (39%) 107 (61%)
92 (60%) 61 (40%)
b 0.001
76 (43%) 5 (3%)
36 (24%)
b 0.001
23 (13%) 6 (3%) 8 (5%) 6 (3%) 1 (0.5%)
21 (14%) 4 (3%) 21 (14%) 11 (7%) 6 (4%)
0.9
Age
35.2 ± 11.4 18–69 Phase of spontaneous menstrual cycle Follicular 43 (42%) Luteal 59 (58%) Parity Parous 160 (49%) Nulliparous 168 (51%) Use of oral contraceptives Combined 112 (34%) Progestogen only 5 (2%) IUD Levonorgestrel-releasing 44 (13%) Copper 10 (3%) Postmenopausal 29 (9%) Use of hormone therapy 17 (5%) Use of vaginal hormone therapy 7 (2%)
p b 0.001
0.004 0.04
Cytology
HPV DNA+
HPV DNA−
p
Normal epithelium Low grade atypia High grade atypia
40.0 (15.6–61.2) (N = 38) 56.0⁎ (48.3–62.9) (N = 97) 28.8⁎ (17.5–40.1) (N = 24)
21.5 (17.8–26.6) (N = 59) 26.2 (17.6–42.4) (N = 39)
0.02 0.002
Low grade atypia: atypical squamous cells with undetermined significance + low grade squamous intraepithelial lesion. High grade atypia: atypical squamous cells with undetermined significance, cannot exclude high grade squamous intraepithelial lesion + high grade squamous intraepithelial lesion. ⁎ p = 0.002.
14.0 for Windows, Chicago, IL. Probability values of b0.05 were considered statistically significant.
Results coefficients of variation were 1.6% and 2.4% respectively. In order to reduce the impact of interassay variation, the samples were assayed in only seven batches. The presence of hr HPV DNA in the cervical fluid was studied by means of Hybrid Capture 2 (HC2; Digene Corporation, Gaithesburg, MD) fluorescence immunoassay (N = 91) or by means of the AMPLICOR HPV test (Roche Molecular Systems, Inc., Brachburg, NJ) (N = 237), involving the PCR technique, according to the instructions of the manufacturers. These tests were carried out immediately after collection of the NOx sample. Both tests detect DNAs of the same hr HPV types (types 16,18,31,33,35,39,45,51,52,56,58,59,68). After NOx and hr HPV DNA sampling a cervical smear was taken from 257 (78%) women. One hundred and eighty-five (56%) women underwent conventional colposcopy and representative cervical biopsy samples were collected. Both cytological and histological samples were analyzed by experienced pathologists.
Statistical methods The NOx data, presented as medians and 95% confidence intervals, were evaluated by means of the non-parametric Mann–Whitney U test or Kruskal–Wallis test. Undetectable NOx levels were regarded as being equivalent to the detection limit. Categorical data were analyzed by means of Chi-square test. The tests were carried out by using SPSS
Table 2 Levels of cervical fluid nitric oxide metabolites (NOx) (μmol/L) in women with (+) and without (−) human high risk papillomavirus (HPV) DNA. HPV+ (N = 175)
HPV− (N = 153)
p
45.2 (35.2–53.1)
23.8 (21.0–26.1)
b 0.001
61 23.8 (19.0–26.6) 9224.1 (20.0–30.1)
0.001 b 0.001
NOx Parity Nulliparous
107 45.2 (33.1–55.0) Parous 6844.1 (33.0–61.8) Use of oral contraceptives Yes 78 38.8 (31.7–51.0) No 97 46.6 (33.8–61.8) Use of an intrauterine device Yes 27 57.0 (30.7–70.7) No 148 45.0 (35.2–50.9) Median and 95% confidence interval (CI).
34 25.4 (18.9–39.8) 119 23.3 (20.0–26.1) 24 30.2 (22.3–33.0) 129 22.4 (20.0–25.3)
0.028
Of the 328 women hr HPV DNA was detected in 175. These women with detectable hr HPV DNA were younger, more often nulliparous and users of oral contraceptives (OC) and they were less often postmenopausal and users of hormone therapy. The phases of the menstrual cycle and the use of IUDs were equally distributed between the groups (Table 1). The presence of cervical fluid NOx did not differ between the women with (94.5%) and the women without (89.5%) hr HPV DNA (p = 0.15). The NOx level in women showing the presence of hr HPV DNA by means of the HC2 test (33.6 μmol/L; CI 95% 36.5–56.5) did not differ significantly from that in women tested by means of the AMPLICOR method (47.5 μmol/L; CI 95% 14.5–57.3) (p = 0.2). The level of cervical fluid NOx was 90% higher (p b 0.001) in women showing the presence of hr HPV DNA compared to women without hr HPV DNA (Table 2). This elevation was not affected by parity, or use of OCs, or IUDs (Table 2). The cytological findings were classified as normal epithelium (N = 122), low grade atypia (N = 117) including ASC-US (atypical squamous cells with undetermined significance) (N = 79) and LSIL (low grade squamous intraepithelial lesion) (N = 38), and high grade atypia (N = 18) including ASC-H (atypical squamous cells with undetermined significance, cannot exclude high grade squamous intraepithelial lesion) (n = 10) and HSIL (high grade squamous intraepithelial lesion) (N = 8) [19]. The presence of hr HPV DNA in women with cytologically normal cervical epithelium or with low grade atypia was associated with elevated levels of NOx. However, when cytological change was more severe, the level of NOx was lower (Table 3). Thirty-nine women showed clue cells in cervical smears, which were taken as a sign of bacterial vaginosis. Bacterial vaginosis did not affect the level of cervical NOx (Table 4).Twenty-five women showed the presence of candida infection on their cervical smears. Of these women 16 were infected with hr HPV DNA and nine women were free of hr HPV DNA. The level of cervical fluid NOx did not differ between
Table 4 Levels of cervical fluid nitric oxide metabolites (NOx) (μmol/L) in women with (+) and without (−) human high risk papillomavirus (HPV) DNA and bacterial vaginosis (BV).
b 0.001
0.008 b 0.001
BV+ BV−
HPV DNA+
HPV DNA−
p
36.2⁎ (24.3–62.9) (N = 20) 48.7⁎ (36.5–56.6) (N = 139)
27.3⁎⁎ (10.1–54.6) (N = 19) 22.8⁎⁎ (18.3–26.2) (N = 79)
0.2 b0.001
Median, 95% confidence interval (CI). ⁎ p = 0.76. ⁎⁎ p = 0.37.
P. Rahkola et al. / Gynecologic Oncology 114 (2009) 323–326 Table 5 Levels of cervical fluid nitric oxide metabolites (NOx) (μmol/L, median, 95% confidence interval) in human papillomavirus (HPV) -infected (+) and non-infected (−) women (N = 185) grouped according to the cervical histology. Histology
HPV DNA+
HPV DNA−
p
Normal
9.1 (bdetect–88.0) (N = 8) 46.2⁎ (37.7–55.0) (N = 91) 33.0⁎⁎ (25.9–60.9) (N = 33)
27.9 (2.0–70.6) (N = 14) 24.8 (17.6–41.3) (N = 37) 104.6 (N = 2)
0.245
Cervical intraepithelial neoplasia grade 1 Cervical intraepithelial neoplasia grade ≥ 2
0.004
⁎ p = 0.01 vs. normal. ⁎⁎ p = 0.03 vs. normal.
women with or without candida infection alone (p = 0.3) or concomitantly with (p = 0.84) or without (p = 0.27) hr HPV DNA (data not shown). The histological findings were classified as normal (N = 22), CIN1 (cervical intraepithelial neoplasia grade 1) including HPV-related changes and low grade dysplasia (N = 128), and ≥ CIN2 (cervical intraepithelial neoplasia grade 2) including moderate dysplasia (N = 32), high grade dysplasia (N = 2), and adenocarcinoma in situ (N = 1) [20]. High risk HPV DNA in association with a histological CIN1 change was associated with elevated NOx levels, whereas the sole presence of hr HPV DNA without histological changes was accompanied by low levels of NOx (Table 5).
Discussion The biological factors that ultimately determine the outcome of cervical HPV infection remain unknown [4]. Nitric oxide could have a major role, since all cells present in the cervical canal (squamous or glandular epithelial, stromal, or haematopoietic cells) are capable of producing it [12,13,21] and NO affects both immunological and carcinogenic processes. Thus, we wanted to find out if cervical NO release was affected by the presence of hr HPV DNA in cervical cells. We measured cervical NO release as the level of nitrate/nitrite, as in many previous studies [18,21]. Although two HPV DNA tests were employed in our study, they detect DNAs of the same papillomaviruses with similar sensitivity [7,8], and no difference in NOx level was seen among women found to be positive according to these two tests. These tests do not detect low risk HPV strains and thus, it is possible that women infected with low risk HPV were classified as noninfected in our study. However, as low risk HPVs are not a major risk factor for cervical cancer we believe that this issue should not affect our results. A majority of the women (53%) were found to have hr HPV DNA in our study population, which was probably a result of the fact that many had a history of abnormal cervical cytology. It is well known that the risk of HPV infection is higher among younger women [2] and thus, our finding on younger age in hr HPV-infected women was expected. Nevertheless, the age was not an independent factor in the release for cervical fluid NOx. The individual cervical NO release data showed great variation in both the women with and without hr HPV DNA. However, the release of NO was 90% higher in women with hr HPV DNA. Elevated levels of NOx were independent of parity, or use of OCs or IUDs. Evidence from both cell and tissue experiments implies that HPV may induce the synthesis of NO, mainly through iNOS activity [11–13]. Human papillomavirus stimulates the synthesis of NO in cultured and cervical keratinocytes [11–13], and also in cervical stromal cells [12,13]. Alternatively, cervical NO can be derived from various kinds of inflammatory cells [13,22] which accumulate in the cervical canal following HPV infection [13,23–25]. Papillomavirus may trigger both epithelial and inflammatory cells to release several cytokines
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[11,26,27], which may indirectly regulate the release of NO in host cells [16,28]. The clinical significance of increased NO release in HPV-infected women remains open. It is possible that a rise in NO accompanies any cervical infection. However, our data do not support this, since the presence of clue cells – a marker of bacterial vaginosis – or signs of candida infection were not accompanied by elevated NO release. The results of many studies imply that NO is induced by viral infections [29,30] and this may enforce cell-mediated immunological defense against such infections [29,30]. If this is the case in the human uterine cervix during HPV infection, increased NO release should contribute to the eradication of HPVs and possibly protect against cancerous changes. This hypothesis is supported by the finding that NO release has an antitumor activity [16] and that it has been found to be highest – both in our and in another study [13] – in women with low grade cervical changes, which most often heal spontaneously [31]. It is possible that HPV infection may take several months to stimulate the maximal release of NO [3], but unfortunately the time of HPV infection could not be determined in our study subjects. This idea is supported by the fact that there was a low level of NOx in a few women with hr HPV DNA and normal histology who were most likely in such an early phase of the infection that NO stimulation had not yet started. An alternative explanation could be that NO, or its active intermediates, may cause additional DNA changes in cervical cells [12] and thus promote cervical carcinogenesis. It is known that the effect of NO on the tumor growth is dependent on concentration; a high concentration arrests and a low concentration promotes the tumor growth [16]. This is supported by our finding that severe cell changes were associated with reduced release of NO. However, we need to recognize that the decrease in NO quantity in high grade dysplasia may be a consequence of selection bias of those women with poor immunological response to the low grade lesion. In conclusion, the presence of hr HPV DNA was associated with increased release of NO in the human uterine cervix. This may be biologically significant in cases of HPV infection, but further epidemiological and experimental studies must be conducted to determine the specificity and significance of this phenomenon. Conflict of interest statement The authors declare that there are no conflicts of interest.
Submission declaration We disclose prior presentation of the study abstract: EUROGIN 2008 Joining forces against cervical cancer prevention, Nice, France 14.11.2008. Acknowledgments This study was supported by the Clinical Research Fund of Helsinki University Central Hospital, the Emil Aaltonen Foundation, the Research Foundation of Instrumentarium Corporation, the Research Foundation of Orion Corporation, the Finnish Cultural Foundation, the Finnish-Norwegian Medical Research Foundation, and the Clinical Graduate School of Pediatrics and Obstetrics and Gynecology. References [1] Bosch FX, de Sanjose S. Chapter 1: Human papillomavirus and cervical cancer — burden and assessment of causality. J Natl Cancer Inst Monogr 2003;31:3–13. [2] Giuliano AR, Harris R, Sedjo RL, Baldwin S, Roe D, Papenfuss MR, et al. Incidence, prevalence, and clearance of type-specific human papillomavirus infections: the young women's health study. J Infect Dis 2002;186:462–9. [3] Stanley M. Immune responses to human papillomavirus. Vaccine 2006;24(Suppl 1):S16–22. [4] Bosch FX, Lorincz A, Munoz N, Meijer CJ, Shah KV. The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 2002;55:244–65.
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