Bacteriological and Cytokine Profiles for Women Using Hormonal Contraceptive Drugs

Background: Hormonal contraceptives that contain estrogen and that contained only progesterone, while 30 women were healthy women never use contraception as a control. Samples taken from all women were high vaginal swab for Bacterial isolation and blood for measurement of serum interferon –gamma (IFN- γ ), interleukin-2 (IL-2) and interleukin-8 (IL-8) by enzyme linked immunosorbent assay (ELISA) technique. Results: Lactobacillus spp were the predominant among the control group (93.3%), while only (5%) in both contraceptive groups. Gram negative and Gram positive bacteria were isolated (68.39%, and 31.56%) respectively from women with contraceptive drugs. Immunological assay showed a significant decrease in serum concentration of both IFN- γ and IL- 8 in both contraceptive group compared to control group (p value less than 0.05). While, there is no significant difference in serum level of IL-2 among study groups. Conclusions: Both types of hormonal contraceptives lead to inhibition of vaginal Lactobacilli and decrease serum level of both IFN- ɣ and IL-8.


INTRODUCTION
Contraceptives or birth control, are devices or methods used to prevent pregnancy, provision and the use of birth control is also called family planning [1]. Birth control methods have been used since ancient times, but the effective and safe methods only became available in the 20 th century [2]. Contraceptive methods include behavioral methods, hormonal contraceptive, intrauterine devices (IUDs), barrier methods and sterilization, each methods usually used during or before sex while emergency birth control are effective for up to a limited days after sex [3]. The most effective methods of birth control is a sterilization, implantable contraceptives, IUDs and hormonal contraceptives which including vaginal rings, patches, oral pills and injectable contraceptives [4]. About 60% of married fertile women use contraceptives worldwide [5], in Iraq the prevalence of contraceptives among women is 53.7% [6].
Hormonal contraceptive alter the genital tract epithelial configuration [7], that have been suggested to be associated with shifts in the vaginal flora, artificial changes that occurring as a result of hormonal contraceptives usage may disrupt the normal vaginal flora [8]. Normal vaginal inhabitants which maintain vaginal pH between 3.5-4.5 [9]. Lactobacilli play an important role in the maintenance of vaginal health, and prevention of vaginal infection [10]. Lactobacilli produce a variety of bacteriocins (proteins/peptides) that inhibit the growth of bacterial vaginosis-associated bacteria and other pathogens such as N. gonorrhoea, Escherichia coli, and C. albicans [11].Vaginal infections primarily occur when normal vaginal flora is disrupted, since vagina is normally dominated by lactobacilli and its metabolites [12].
Estradiol and progesterone decrease the infiltration of macrophages, neutrophils and leukocytes to the upper and lower women genital tract and inhibits the expression of chemokine monocyte chemo-attractant protein-1 by human endometrial stromal cells [13]. Contraceptives that contain only progesterone also change the expression of cytokines that normally involve in T helper2 responses [14]. Usage of injectable (medroxy progesterone) contraceptives exert a number of effects on the T cell-mediated immune response including the inhibition of cytotoxic activity of T cells and blocking perforins expression in T cells [15]. A current study focused on evaluation of the effect of oral combined contraceptive pills (estrogen and progesterone) and injectable (medroxy progesterone) contraceptive on the vaginal bacteria and on the serum levels of cytokines, interferon-gamma, Interleukines-2 and 8.

Patients
The study involved 60 randomly selected women using hormonal contraceptives.
Forty of them used oral combined contraceptive pills that contained progesterone and estrogen, and twenty used injectable contraception that contained only progesterone. Thirty healthy women who never used contraception were involved as a control. Blood samples and high vaginal swab using vaginal speculum during late follicular phase of menstrual cycle were collected from all women. All samples were collected from family planning clinic in Babylon hospital for maternity and children. Women excluded from this study were pregnant, diabetics, those who were under steroid, anti-cancer and antibiotics treatment and those with signs and symptoms of genital tract infection. Bacterial isolation and identification was carried out according to Forbes et al. [16]. Immune parameters: Serum IFN-γ, IL-2 and IL-8 were estimated by ELISA technique according to the instruction company [Boster, China].

Ethical Approval
The necessary ethical approval from family planning clinic in Babylon hospital for maternity and children / Hilla -Iraq was obtained. Moreover, all subjects involved in this work were informed and the agreement required for doing the experiments and publication of this work was obtained from each one prior the collection of samples.

Statistical Analysis
The statistical analysis of this study was obtained by the SPSS and Excel programs, the statistical parameter that used in this study were include Z Test, Mean, Standard deviation, percentage and P value(<0.05) considered statistic significant.

Gram Stain Distribution of Bacterial Isolates
The types, number and percentage of Gram negative and Gram positive bacteria isolated from women of both groups are shown in Table 1.
The percentage of Gram negative isolates in oral combined contraceptive pills and injectable (medroxy progesterone) administered women was (68.39%), E-coli isolates had the highest percentage (28.07%) between them, followed by Enterobacter cloacae (19.29%) and Klebsiella pneumonia (14.03%), the lowest percentage was represented in both Pseudomonas aeruginosa and Neisseria gonorrhea, which recorded (3.50%) for each one. While, there is no bacterial isolate in control group. These findings are similar to those obtained by Gupta et al. [17] who found that there was an inverse association between vaginal lactobacilli and E. coli colonization. Furthermore, after controlling for intercourse, antimicrobial use and birth control method in the preceding week. The risk of E. coli colonization at any visit was 2-fold greater among women without Lactobacilli as compared with women with Lactobacilli. Other study done in Pakistan showed that E. coli was isolated from 24% of women with hormonal contraceptives [18]. The presence of K. pneumoniae in cases of contraceptive women can be attributed to absence or decrease in numbers of lactobacilli and subsequently their defense factors [19]. P. aeruginosa had also been isolated from both groups. It is potentially opportunistic bacteria within the vagina. Such microorganism may become increasingly prevalent upon minor alterations of the vaginal environment. Investigators had isolated this bacteria from cases of vaginitis and showed that this bacteria was prevalent among non-pregnant women using intra uterine device [20]. Only two isolate of N. gonorrhoeae had been isolated in this study from both groups. These organisms are gram negative intracellular diplococci organisms that are sexually-transmitted causing infection in the genital tract which may disseminate to other organs [21].
The percentage of Gram positive isolates in oral combined contraceptive pills and injectable progesterone administered women was (31.56%). Staphylococcus epidermidis isolates had the highest percentage (14.03%) followed by Staphylococcus aureus (10.52%) and Lactobacillus (5.26%). Streptococcus feacalis represents the lowest percentage (1.75%). While in control group the highest percentage was Lactobacillus spp (93.3%). Hiller et al. [22] were studied the vaginal flora of some women and they found that the organisms commonly isolated in women with normal smears were lactobacilli, coagulase negative Staphylococci, S. aureus, diphtheroids, Candida and group B Streptococci.
There is an inhibitory effect of Lactobacillus reuteri on some pathogenic bacteria that cause bacterial vaginosis, including Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus and Streptococcus agalactiae [25]. No bacterial isolates obtained from control group.

Distribution of Lactobacillus Isolates among Study Groups
There is a significant difference in the number and percentage of Lactobacillus spp isolates between contraceptive and control groups. These results reflect the effect of contraceptives on vaginal Lactobacilli, since Lactobacilli were highly prevalent in control group (93.3%) while highly decreased in contraceptive groups (5%). These results are similar to other results obtained by other researchers who studied the effects of variety of factors on vaginal micro flora. Some researchers show that vaginal flora can be disrupted by a variety of factors including sexual intercourse, antimicrobial use and douching [26]. Long-term use of depot medroxy progesterone acetate results in endothelial dysfunction which may result from hypoestrogenism and serum estradiol was significantly reduced in users of depot medroxy progesterone acetate compared with controls [27]. Other study showed that hypoestrogenism state associated with decreased H 2 O 2 -positive Lactobacillus colonization and slight thinning of the glycogen vaginal epithelial layer in users of depot medroxy progesterone acetate. Such change may compromise the vaginal barriers to infection [28]. Lactobacilli play important role in protection of the vagina from colonization by pathogens and this could be attributed to blocking the attachment of pathogens to the vaginal epithelium and production of substances that inhibit their multiplication [29].

Interferon gamma (IFNγ)
There is a significant difference between control and injectable contraceptive users (P value less than 0.05), also there is significant difference between control and oral combined contraceptive pills users (P value less than 0.05) Table 3.
This finding indicates that injectable and oral combined contraceptive pills uses may diminish the production of IFN-γ. These results are similar to Vicetti-Miguel et al. [30] who showed that contraceptive usage reduce the ability of cytotoxic T cell mainly CD 8 T cells for activation and secretion of cytokines especially IFN-γ. Gonadotropin releasing hormone and Hypothalamic-pituitary-gonadal activation is involved in thymus maturation and exerts a potent immune-stimulatory effect leading to activation of T helper cells and increased levels of serum interferon-gamma(IFN-γ) [31]. Oral combined contraceptive administration lead to suppression of Hypothalamic-pituitary-gonadal axis [32] which leads to reduce activation of T helper cells and decrease serum level of IFN-γ.
Many studies have shown that bacterial endotoxin and exotoxin in vaginal infection of women without contraceptive are the cause of hyper production of pro-inflammatory cytokines [33]. Loncar [34] found that serum level of IFN-γ was elevated in pelvic inflammatory disease and in vaginal bacterial infection of women without contraceptives. The results of this work exhibited a decreasing level in the concentration of IFN-γ in women with bacterial infection and using contraceptive drugs. This result may give an indicator for the inhibiting effects of these drugs on the production of TH1 cytokines which is supported by Koldehoff [35].

Interleukin -2 (IL-2)
There was non-significant increase in serum level of IL-2 in both oral and injectable contraceptive users groups compared to control group (p value less than 0.05) Table 4.
Antigens such as bacterial cells are internalized by endocytosis and processed by the antigen presenting cells then presented to immunocompetent lymphocytes to initiate the early steps of the immunological response. Processing by a macrophage for example results in attaching antigenic materials to the surface of the membrane in association with MHC II molecules on the surface of the cell. The antigenclass II MHC complex presented antigens to Thelper cells causing activation of these cells that begin to produce Interleukin 2 (IL-2) and to express a membrane receptor for IL-2. The secreted IL-2 autostimulates proliferation of the TH2 cells. Stimulated TH2 cells produce a variety of lymphokines including IL-2, IL-4, IL-6 and gamma Interferon which mediate various aspects of the immune response [36]. Other study showed that serum progesterone levels did not correlate with the activation of cytokine-secreting cells [37]. The effects of synthetic hormones on IL-2 level were studied and the results were conflicting: Oral combined contraceptive use did not affects IL-2 level [38]. Injectable contraceptive of Depot Medroxy progesterone acetate levels did not correlate with the production of IL-2 [37].

Interleukin -8 (IL -8)
Serum level of IL-8 has a significantly decrease in injectable contraceptive users group compared to control group (P value less than 0.05) also IL-8 was significanty decrease in oral combined contraceptive pill users group compared to control group (P value less than 0.05) Table 5.
IL-8 produces by monocytes, neutrophils, endothelial cells, epithelial cells and fibroblasts [39]. IL-8 exerts several effects on neutrophils [40]. These effects include chemo-attraction, increase expression of adhesion molecules, modulation of trans endothelial cell migration and respiratory burst [41]. Progestin inhibits the expression of IL-8 by human endometrium stromal cells which leads to virtual reduction of neutrophils after progesterone-dominated midluteal phase human endometrium [42].
Other study showed that medroxy-progesterone acetate and estradiol cause reduction in the secretion of IL-8 by human endometrium stromal cells monolayers [43]. Moreover, IL-8 production by endometrial stromal cells markedly decreases during progesterone-induced decidualization [44] also other study concluded that progestogens inhibit IL-8 and other chemokines [45].

CONCLUSIONS
Hormonal contraceptives lead to inhibition of vaginal lactobacilli and decrease serum level of both IFN-ɣ and IL-8 while IL-2 level not affected by hormonal contraceptives.

CONSENT
All authors declare that written informed consent was obtained from the patients for publication of this research article.