In 39.2% of participants, cytological abnormalities had resolved: 29.6% in the common care group and 50% in the probiotic group. Figure 1 shows the PAP smear results at outcome in both groups.
The significance of ASCUS is, as the term describes, unclear. The policy in Belgium in this case is to repeat a PAP smear after 1 year. If ASCUS smears are considered as ‘no cure’, there is a tendency toward a higher chance for cure in the probiotic group [12/24 or 50.0 vs. 8/27 or 29.6%, odds ratio (OR) 2.37, P=0.137]. If, however, on the basis of the uncertainty of the diagnosis, ASCUS smears are excluded from analysis, the difference in the likelihood of resolution of cellular abnormalities between both groups is statistically significant (12/20 or 60 vs. 8/26 or 30.7%, OR 3.38, P=0.047) in favor of the probiotic group. The number of participants in this pilot is too small to perform a complete multivariate analysis; however, controlling the effect for several combinations of confounders did not substantially alter the results. More specifically, the significant effect of probiotics was still present in a model that contained the two factors in which the intervention group and the control group tended to differ: smoking and consumption of fruit. Taking into account the fact that a minority of participants was not randomized but allocated to one or another group (because they entered the study too late to be in the intervention group or because they had a strong preference for one or the other group), we checked whether there was a difference in outcome between randomized and nonrandomized participants within each group. No difference was observed.
At the beginning of the study, all women were positive for HPV. After 3 months, HPV had been cleared in 16% of the study participants; in 25.0% of probiotic takers; and in 7.7% of control participants (P=0.13). In 24.0% of participants, HPV DNA was no longer detectable after 6 months; this was the case in 19.2% of the control participants and in 29.2% of probiotic takers (P=0.41).
In agreement with existing evidence, smoking tended to lower the chance of clearing the infection and the cytological lesions (Kjaer et al., 1996; Guarisi et al., 2009; Maucort-Boulch et al., 2010). Similarly, genetic vulnerability (in the form of cervical cancer/abnormal cytology in the family) tends to be a risk for persistence of HPV-induced cellular abnormalities (Bosch et al., 2002; Wang et al., 2010). Genetic factors are perceived as the major risk factors by patients (Baay et al., 2004; Ackermann et al., 2005). Acquisition of HPV is well known to be age dependent, but once HPV has been acquired, age is not associated with clearance rates (Syrjänen et al., 2005). This finding is supported by our data. Tampon use seems to be a limited risk in our study – an association with acquisition or delayed clearance has been found, but inconsistently, in the literature (Rousseau et al., 2000; Richardson et al., 2005). Daily fruit consumption seems to be protective, which is consistent with several observations of a protective effect of the regular intake of fruit and fresh vegetables, which contain micronutrients that may help in preventing cellular DNA damage (Sedjo et al., 2002; Richardson et al., 2005). Vitamin intake was not beneficial in our study; on the contrary, vitamin takers did markedly worse in clearing their infection. We found no evidence of a protective effect of vitamin supplementation on existing lesions in the scientific literature. Possibly, vitamin intake may be a marker of a more general lack of well-being related to a suboptimal health status. In our study, we found no tendency toward a protective effect of condom use. Although condom use is probably more important in the prevention of HPV acquisition, some studies report a protective effect of consistent condom use even after HPV infection (Hogewoning et al., 2003).
Hormonal contraceptive (OAC) use tends to be protective in our sample. Although OAC has been proposed as a cofactor in cervical carcinogenesis (Murthy and Mathew, 2000; Castellsagué and Muñoz, 2003), most observational studies do not find an effect on the clearance of HPV or cytological abnormalities (Syrjänen et al., 2006). Some studies report a protective effect (faster clearance) with OAC, similar to our observation (Muñoz et al., 2002; Richardson et al., 2005). In general, the findings in our study are in accordance with existing evidence, which supports the representativeness of our sample.
Several limitations of this pilot study need to be pointed out. There was a substantial heterogeneity among study participants (irrespective of the groups they were allocated to), especially in terms of age and (often unknown) the duration of HPV infection before entering the study. Furthermore, the fact that some of the participants chose their group instead of being randomized may have led to some form of bias, although we registered most relevant health-associated attitudes in the study questionnaire, and no significant difference could be found between the groups. The choice for taking the probiotic in some individuals might fit in with a healthy lifestyle in general, as probiotic takers tended (although not significantly) to smoke less and to eat more fruit. However, after controlling for these two factors in multivariate analysis, the significant effect of the probiotic remained. Furthermore, within each group, there was no difference in outcome for randomized versus nonrandomized participants.
Although a recent study has shown a beneficial effect (higher complete remission rate) of probiotics when added to conventional therapy on cutaneous warts (Cassano et al., 2011), which are also induced by HPVs, to our knowledge, our study is the first to show an effect of probiotics on mucosal HPV and its consequences. In this study, we propose a model for examining a possible effect of a nutritional intervention on HPV infection and on LSIL abnormalities, which represent early-stage disease in the progression to cervical cancer. As LSIL often regresses spontaneously, it is not a sensitive predictor of cancer, but it has the advantage of being reasonably prevalent and to require a strict clinical follow-up, which makes research feasible. The short-term (6-month) follow-up period and the availability of well-defined biomarkers (cytological abnormalities, DNA-confirmed presence of HPV) are important advantages of this model, which should make it more feasible to prove or to negate an effect of probiotics or lifestyle interventions on this type of cancer.
Because of the small sample size and the methodological considerations mentioned above, this study cannot provide conclusive evidence on the effect of the probiotic on HPV infection and on cytological abnormalities, even if statistical significance is achieved. However, the surprisingly huge effect that was found appears to be promising and warrants further exploration. In the future, the study should be repeated with a randomized, placebo-controlled design and, if possible, inclusion should be limited to women with a first diagnosis of LSIL. Finally, it would be advisable to perform cytology in a single laboratory to account for interobserver variation in cytological diagnosis (Stoler and Schiffman, 2001).
In this study, we used cytological and viral endpoints to study a possible effect of probiotics on cervical abnormalities. The likelihood of clearance of LSIL abnormalities was twice as high in the group of probiotics users compared with nonusers. A larger and randomized study is warranted to confirm these encouraging results.
We would like to thank Dr Jean Renard for recruiting patients and all women who voluntarily participated in our study.
This study was initiated by V.V. and partly funded by university resources. We contacted Bart Degeest, Yakult Belgium, for additional funding and for the study product. Yakult Belgium made no contribution in study design, had no access to study data, and made no contribution toward the manuscript. In an earlier large study on Yakult and respiratory tract infections in the elderly, we found no beneficial effect of the probiotic. That study has been published recently in Van Puyenbroeck et al., 2012.
There are no conflicts of interest.
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