Discussion
This is the first study performed to characterize the vaginal microbiome in women with POI. The study revealed that, for women with POI using systemic HT, and by means of analysis by DNA extraction, the vaginal flora is predominantly Lactobacillus sp., which is the flora considered healthy and suitable for estrogenized women. There was a predominance of L. cripatusin the same proportion of L. iners , followed at a smaller proportion by L. gasseri and L. jensenii . It should also be considered that Ravel group IV bacteria were present in 15.2% of the cases, even though these women were adequately estrogenized by systemic HT ( 15-19).
Studies with evaluation of the vaginal flora, through different methods, reveal that to maintain a healthy vaginal ecosystem, it is necessary to have superior counting of Lactobacillus sp colonies in order to maintain adequate pH through the production of acids, especially lactic acid, to prevent pathogenic bacterial proliferation (20-22). A study in which the authors compared the vaginal flora (but not the microbiome) of women with POI using HT, with women of the same age with preserved gonadal function by means of bacterioscopy and culture for fungi, showed similar floras, with no difference between groups. Thus, it can be assumed that systemic HT was able to provide sufficient estrogenic levels to restore the vaginal flora, despite loss of ovarian function (23).
Considering that common evaluation methods of the vaginal ecosystem have no specificity for which species of lactobacilli are present and that different species of lactobacilli produces different types of acids and concentrations, the present study used up-to-date techniques (DNA study of the vaginal flora). The specific phylogenetic classification of women with POI may provide evidence of HT action on the vaginal flora restoration (24).
Molecular biology has become a great ally in the search for vaginal flora identification. Efforts to characterize vaginal microbial communities using culture methods undoubtedly led to significant improvements in understanding the role of microorganisms in vaginal health, but they were limited because of the biases inherent in culturing methods. It is now known that most environmental microorganisms associated with the host are not identified in the laboratory using traditional cultivation techniques (25).
Culture of microorganisms is fundamental for the understanding of their physiological and phenotypic characteristics, and it continues being a very useful tool in studies of microbial ecology. Promising developments in the cultivation of fastidious bacteria using state-of-the-art techniques are likely to enable the cultivation of many previously inaccessible microorganisms (26-29). However, studies aimed at evaluating fine-scale variation in host-associated microbial communities within and between individuals or exploring ecological relationships within those communities require methods that provide detailed information on microbial diversity and, at the same time, be scalable for processing samples and also cost-effective. In response to this need, independent culture methods have become, in recent years, the standard approach to characterize the diversity of microorganisms residing in the human body (30-34).
Ravel et al. found that the group I communities in healthy women of reproductive age, which occurred in 26.2% of the women that they have studied, were dominated by L. crispatus, while groups II (6.3%), III (34, 1%) and V (5.3%) were dominated by L. gasseri, L iners and L. jensenii, respectively. Curiously, communities dominated by Lactobacillus species other than L. crispatus have slightly higher pH, ranging from 4.4 (group III) to 5.0 (group II), indicating that these communities as a whole may not produce as much acid as group I (4). These results are in agreement with the findings in the present study, where there was also a predominance of groups I (L. crispatus ) and III (L. iners ), indicating a healthy vaginal flora and a percentage expected for estrogenized women. Thus, it is possible to infer that HT in women with POI may be able to maintain adequate vaginal flora.
Two important points will have to be clarified in the future. Intriguingly, we found 15.2% of bacteria associated with bacterial vaginosis in adequately estrogenized women. Second, there is no predominance of L. crispatius on L. iners. These two points may point out that the use of systemic estrogens is not sufficient to determine vaginal colonization with L crispatus, producers of lactic acid. Probably, other factors such as frequency of sexual intercourse, type of sexual practices, feeding, individual immune response, among others, may interfere with vaginal colonization.
Although this study is one of the first in literature to evaluate the vaginal flora of young women, with POI undergoing systemic hormone therapy, by using advanced and reliable technique such as the evaluation of vaginal microbiology through molecular biology with the 16S rRNA pyrosequencing, we understand that the fact of having been a descriptive study only limits our conclusions or future inferences, as is already known in the descriptive studies. The small sample size also makes us understand that the findings are limited to this group studied and that further studies are needed with larger cases where there is comparison with women using topical replacement therapy and/or with women at menacme with preserved ovarian function. On the other hand, we have to consider that the studies with POI, do not have large case-by-case studies due to the difficulties inherent in this nosological entity. Work with large case-by-case studies about POI often come from databases involving several institutions.