State Enterprise “L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health of Ukraine”, Kyiv, Ukraine
Abstract. In previous studies, high serum concentrations of manganese and nickel were established in women with polycystic ovary syndrome (PCOS). Increased level of exposure of the population of Ukraine to manganese and nickel has been demonstrated.
The objective of this study is studying the incidence of inflammatory processes and composition of vaginal bacterial flora in healthy women and in women with PCOS.
Materials and methods. The control group consisted of 57 women without reproductive system conditions and 64 women who had established PCOS according to the Rotterdam Consensus criteria. In both groups, the presence of sexually transmitted infections was excluded by PCR. The analysis of the incidence of inflammatory processes was carried out according to the microscopic characteristics of vaginal microbiocenosis. Vaginal microflora was examined by the culture method. Evaluation of the resulting validity was performed using Student’s t-test, Mann-Whitney U-test and χ2.
Results. Studies have shown that women with PCOS are much more likely to show signs of inflammatory processes in the vagina, as well as colonization of vagina with bacteria and fungi. Compared to control, lactobacilli are less common, while the diversity of bacterial flora is increasing, with a predominance of the representatives of intestinal flora (Enterococcus faecalis, Escherichia coli) and Candida albicans. The analysis of quantitative parameters showed a significant decrease in Lactobacillus spp. (Р=0,0003), increase in Escherichia coli (Р=0,0026), Enterococcus faecalis (Р=0,0003), and Enterococcus Durans (Р=0,0272). Meanwhile, the quantitative parameters of other representatives of the vaginal microflora virtually did not change compared with the control.
Conclusion. Inflammatory processes in PCOS may not be associated with sexually transmitted infections or body mass index. Increased exposure to manganese and nickel can affect vaginal microflora and its individual representatives.
Key words: polycystic ovary syndrome, inflammation, microflora, vagina, manganese, nickel.
Introduction. Our previous studies observed high concentrations of manganese and nickel in the serum of women with polycystic ovary syndrome (PCOS). [1]. Our further studies have shown that the population of Ukraine experiences an increased level of exposure to manganese and nickel [2,3]. Studying of possible sources of exposure requires a separate study. However, without a doubt, the drinking water consumed by the residents of Ukraine relates to the risk factors of exposure to manganese. It has been proved that in drinking water from underground sources, the maximum permissible manganese values are exceeded 4–28-fold [4], including in Kyiv [5].
Polycystic ovary syndrome (PCOS) is a multiaethiological disease. In recent years, the role of the inflammatory process in the pathogenesis of PCOS has been discussed. Obesity plays not the least role in the occurrence of the inflammatory process in PCOS [6]. The role of ecological factors in the pathogenesis of PCOS is virtually not reflected in the literature. Available literature data suggest the possible participation of trace elements in the occurrence of hormonal disorders and inflammatory process [7].
The objective of our study was studying the incidence of inflammatory processes and composition of bacterial flora in conditions of high population exposure to manganese and nickel in healthy women and women with PCOS in the absence of pathogenic flora, similar same body weight.
Materials and methods. The control group consisted of 57 women without reproductive system conditions and 64 women who had established PCOS according to the Rotterdam Consensus criteria (2003). Female citizens of Kyiv of child-bearing potential with normal body weight participated in the study. All women underwent testing of urogenital smears for the presence of genital pathogens (gonococci, chlamydia, trichomoniasis, herpes 1 and 2 types, cytomegalovirus, M.genitalium). Tests were performed using Real-time PCR, FEMOFLOR® SCREEN Real-Time PCR Detection Kit (DNA-Technology LLC).
Microbiological tests of vaginal discharges were performed by culture method using digest media of HiMedia Laboratories Pvt Ltd. Subsequently, pure cultures of microorganisms were isolated with the determination of sensitivity to antibiotics and bacteriophages. The characteristics of vaginal microbiocenosis were analysed according to Kira Ie. F. [8]. Body mass index (BMI) was calculated using the BMI formula (kg/m2) = weight: (height × weight).
Women were examined at Yurinmed clinics (Kyiv). Laboratory tests were performed by the laboratory “Klinika Markova” (Kyiv).
The determination of the rate of representativity of detected bacteria and analysis of quantitative parameters was carried out. Statistical processing was carried out using the STATISTICA software. Evaluation of the results validity was performed using Student’s t-test and Mann — Whitney U-test The association factor was determined using χ2.
Results. As Table 1 shows, 89.48 % of women in the control group had 1st and 2nd type smears. This suggests the absence of an inflammatory process.
Table 1. Microscopic characteristics of vagina in the control group and in women with polycystic ovary syndrome (PCOS).
The inflammatory type of smears in the control group was observed in 10.52 % of cases. Women with PCOS had more common smears of 3rd and 4th type (P = 0,001). Signs of the inflammatory process were in 84.38 % of cases. Smears of the 1st and 2nd types were detected in only 15.62 % of women.
BMI parameters in the control group were 21.50 + 1.51 kg/m2 and in the group of women with PCOS — 21.87 + 1.64 kg/m2, and they were virtually identical (P = 0.198).
Thus, we have shown that in women with PCOS, signs of the inflammatory process are more common. At the same time, we excluded the presence of pathogens in the body and factor of the increased body mass index.
Analysis of bacterial flora showed that Lactobacillus spp. is most common in women of the control group. (42.19 %) (Table 2).
Table 2. Structure of vaginal microflora in women of the control group and in PCOS as per culture test method.
In 14.06 % of the control group, Enterococcus faecalis is found, in 9.37 % — Candida albicans and 7.81 % — Staphylococcus epidermidis. Significantly less common are Enterococcus durans and Staphylococcus aureus — 4.69 %, Cl.pneumonis, Proteus mirabilis — 3.13 %, St.Haemolyticus, St.saprophitis 1.56 %. Seven cases (12.28 %) in the control group showed an association of two microorganisms, most common — associations of Enterococcus faecalis: with Cl.pneumonis and Eschеrichia coli (2 cases for each), and St. aureus (1 case). Another case showed an association between St. aureus and Candida albicans and between Lactobacillus spp. and Staphylococcus epidermidis.
In contrast to the control group, women with PCOS more commonly had representatives of conditionally pathogenic flora: Enterococcus faecalis (23.16 %), Eschеrichia coli (16.84 %) and Candida albicans (13.68 %). Lactobacilli were found in only 11.58 % of cases. The rate of detection of Staphylococcus epidermidis (9.47 %), Enterococcus durans (6.32 %), St. aureus (4.21 %), Cl.pneumonis (3.16 %) St. Haemolyticus (1.05 %) was virtually unchanged. Furthermore, women with PCOS had bacteria that were not detected in the control group: Proteus mirabilis, Candida tropicalis (2.11 %), Streptococcus Viridans, Candida glabrata, Enterobacter Aerogenus, Corynebacterium, Enterococcus faecium (1.05 %). In 27 (42.19 ) of cases, associations with 2 (25) and 3 bacteria (2) were found, that is 3.44-fold higher than in the control group.
The most common associations of bacteria were with Enterococcus faecalis (14), Eschеrichia coli (9), Candida spp. (9) Staphylococcus epidermidis (5), and Lactobacillus spp. (4).
Thus, conducted studies have shown that women with PCOS have colonization of the vagina. Change in vaginal bacterial flora is observed. In this case, lactobacilli are less common in comparison with control, whereas representatives of intestinal flora (Enterococcus faecalis, Eschèrichia coli) and fungal flora of Candida spp. prevails.
The next stage of the study was the determination of the quantitative parameters of the most common bacteria. As Table 1 shows, 3 in women with PCOS had changes in the quantitative parameters of representatives of the dominant flora. There is a decrease not only in the rate of detection but also a decrease of quantitative parameters of lactobacilli by 30.79 %.
Table 3. Comparison of quantitative parameters of microflora (Lg CFU) in the control group and in PCOS.
The decrease in the number of lactobacilli in PCOS was accompanied by a significant increase in the quantitative parameters of E. coli (by 61.39 %), E. faecalis (by 55.28 %) and E. Durans (30.95 %). The fungal flora of Candida spp. is also more common in women with PCOS, however quantitative parameters were virtually unchanged. Both groups had virtually identical parameters of St. epidermis.
Therefore, the analysis of quantitative parameters showed that in PCOS under conditions of increased exposure to manganese and nickel, there is an increase in the diversity and number of representatives of conditionally pathogenic flora on the background of a reduced number of lactobacilli.
Discussion and results. Our studies have shown that in women with PCOS, in the absence of sexually transmitted infections and normal BMI, the inflammatory process is present. The reasons for the occurrence of this process in PCOS remain unknown. Since the influence of environmental factors on the state of the human microflora has not been studied sufficiently, it is possible that the trace elements may be involved in violations of the body microbiota [7]. Therefore, it was important to find out the effect of some trace elements on vaginal microflora and their role in the development of PCOS. No publications are available in the literature devoted to the study of the effects of high manganese and nickel concentrations on vaginal biocoenosis. Therefore, we tried to assess the possible effects of manganese and nickel exposure to the vaginal biocoenosis.
Our study has shown that under conditions of increased exposure to manganese and nickel, change in vaginal microflora is observed in PCOS. They are accompanied by a decrease in the number of lactobacilli, an increase in the number of conditionally pathogenic flora, associations of 2 or 3 representatives of conditionally pathogenic flora are more common. However, according to our data, there is an increase in only certain types of bacteria, while others, such as Candida spp and Staphylococcus epidermidis, remain virtually unchanged. We believe that such changes can be caused by the peculiarities of trace elements metabolism by bacteria under conditions of increased exposure to manganese and nickel. The relationship between exposure to manganese and activation of infection is confirmed by recent studies. It has been shown that a diet rich in manganese causes activation of Staphylococcus aureus and the development of endocarditis [9].
According to the literature, trace elements can change the composition of body microflora. Available publications are mainly experimental or devoted to the study of the effects of man-made exposure and mainly relate to the gastrointestinal tract and respiratory system.
It is known that many bacteria use manganese in large quantities to provide their functioning. Such bacteria include lactobacilli. Apparently, the presence of manganese should improve the microbiota state of the body. However, manganese is required for other representatives, including conditionally pathogenic microflora. Moreover, the composition of body microflora may change due to changes in intermicrobial interaction under the influence of manganese [10]. We believe that it is precisely by these processes an increase in the diversity of bacterial flora in women with PCOS in the background of increased exposure to manganese and nickel can be explained.
However, it is known that high concentrations of manganese cause oxidative stress in the body. This is accompanied by dysfunction of the immune, endocrine and central nervous systems. But it turns out that the microbiota of the body can increase the manifestations of oxidative stress in the host. Bacteria can oxidase Mn2 + to toxic form — Mn3 +with multicopper oxidase. It was shown in the experiment that manganese-exposed female rats have increased multicopper oxidase level [7,10]. Therefore, at high levels of manganese exposure, manganese toxicity can be not only due to the direct effect of the trace element on the host but also due to the toxic forms of manganese that are formed by bacteria.
In addition, to change in biocoenosis, increased virulence of bacteria (such as Salmonella enterica, Mycobacterium tuberculosis, Staphylococcus aureus, Yersinia pestis and Streptococcus pneumoniae) and increased sensitivity to the host organism to them can be observed under the exposure to manganese [11]. It has been shown that under stress, enterobacteria significantly increase the consumption of manganese. It is necessary for bacteria to form antioxidant protection. It is believed that the formation of non-protein antioxidant manganese complexes is, on the one hand, an important factor in the virulence of certain bacteria, and, on the other hand, allows bacteria to survive unfavourable conditions that contribute to body colonization [7]. Currently, relatively little is known about bacterial protection systems, this issue needs further study.
Women with PCOS presented a decreased number of lactobacilli and an increased number of conditionally pathogenic microflora. It is quite logical to ask why, under conditions of increased exposure, the number of lactobacilli, which actively and in large quantities consume Mn decreases. It turns out that the consumption of Mn by bacteria occurs with the involvement of manganese transporters. Bacteria have a large number of transport systems that, under adverse conditions, help bacteria survive and adapt. Transport systems may differ in different types of bacteria. Majority of bacteria use MntH and MntABCD transporters. Sometimes bacteria use both types of transport systems. However, some of them, in particular, Lactobacillus spp, use MntP type transporters. With an excess of manganese, these transporters are blocked, but other transport systems may be activated [7]. We believe that it is precisely this that can explain the change in the vaginal biocoenosis under conditions of increased exposure to manganese. The diversity of enzyme systems can be one of the causes of dysbacteriosis under the development of adverse environmental factors. We do not exclude the possible involvement of other regulatory mechanisms as this issue needs further research.
In addition to manganese, high serum concentrations of nickel in women with PCOS were found. It seems that nickel is also able to play a role in the formation of biocoenosis. This is due to the genotoxic and mutagenic properties of nickel in bacteria. In addition, nickel reduces body resistance that also contributes to colonization [12].
Therefore, our studies have shown that under conditions of high exposure levels to manganese and nickel in the absence of sexually transmitted infections, normal body mass index, signs of inflammatory process are more common in women with PCOS. Increase in the diversity of bacterial flora, the incidence of detection and quantitative parameters of certain representatives of the conditionally pathogenic flora is detected. These processes are accompanied by a decrease in the number of lactobacilli. The reasons for such changes can be the result of manganese and nickel toxicity.
Conclusion
1. Increased concentrations of manganese and nickel in the body in women with PCOS can be the cause of dysbacteriosis of the vaginal microflora.
2. Decrease in the number of lactobacilli and decrease in the number of optionally-anaerobic microorganisms and fungi of Candida spp. may be a reason of chronic inflammatory processes in women with PCOS.
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Надійшла до редакції 31.05.2018 р.