Characterization of vaginal Lactobacillus species as a predictor of fertility among Iranian women with unexplained recurrent miscarriage and fertile women without miscarriage history using machine learning modeling.

BACKGROUND: Lactobacillus spp. are the predominant bacteria of the vaginal tract, the alteration of which has been previously linked to miscarriage. Here, we investigated differences between selected vaginal Lactobacillus species of women with a history of recurrent miscarriages and fertile women without a history of miscarriage in Iran. METHODS AND RESULTS: Vaginal swabs were taken from 29 fertile and 24 infertile women and quantitative real-time PCR (qPCR) assay was used to determine a selection of vaginal Lactobacillus species in both groups. The logistic regression (LR) model, Naive Bayes (NB) model, support vector machine model (SVM), and neural network model (NN) were developed to predict disease outcome by selected variables. LR analysis was used to construct a nomogram indicating predictions of the risk of miscarriage. The most abundant species among the patients were L. rhamnosus, L. ruminis, and L. acidophilus, while L. gasseri, L. vaginalis, L. fermentum, and L. iners were more abundant in healthy subjects. The distribution of L. ruminis, L. iners, and L. rhamnosus was higher in patients, while L. acidophilus, L. gasseri, and L. fermentum were highly distributed among healthy subjects. Higher AUC in predicting the disease outcome was observed for L. gasseri, L. rhamnosus, L. fermentum, and L. plantarum. CONCLUSION: Our findings provide experimental evidence of vaginal Lactobacillus imbalance in infertile women and a suitable predictor for miscarriage based on the AUC algorithms. Further studies with larger sample size and using high-throughput technologies are needed to boost our understanding of the role of lactobacilli in miscarriage.

An intron variant in the FLT1 gene increases the risk of preeclampsia in Iranian women.

Background: Preeclampsia is a hypertensive disorder that affects pregnancy, mother, and fetus. Pathogenesis of preeclampsia could be associated with the angiogenesis pathways. The vascular endothelial growth factor (VEGF) family is one of the important factors for normal pregnancy and angiogenesis. Genetic variations in the gene family members may play a role in the etiology of preeclampsia. We investigated the possible association between VEGFA gene rs3025039, and VEGFR1 (FLT1) gene rs722503 polymorphisms and preeclampsia in a sample of Iranian patients. Methods: Genotyping was performed in 395 women, including, 204 pre-eclamptic pregnant women and 191 healthy normotensive pregnant women by using the PCR-RFLP method. Results: The rs722503 polymorphism was associated with preeclampsia under the dominant model (P = 0.04, OR = 1.53, 95% CI: 1.03-2.27). No significant difference was observed for the rs3025039 alleles and genotypes in the studied groups. Conclusions: Based on our study, rs722503 polymorphism in the FLT1 gene may play an important role in susceptibility to preeclampsia.

Electrical stimulation promotes the wound-healing properties of diabetic human skin fibroblasts.

This study evaluated the effect of low (20 and 40 mV/mm) intensities of electrical stimulation on the proliferation and migration of skin fibroblasts from diabetic donors. We also examined the effect of electrical stimulation on modulating the capacity of fibroblasts to contract collagen gel, express alpha-smooth muscle actin, and secrete proteolytic enzymes involved in regulating extracellular matrix synthesis and degradation. Our study shows that 20 and 40 mV/mm of stimulation increased the growth of fibroblasts extracted from diabetic patients but not from non-diabetic donors. Electrical stimulation increased the migration of diabetic fibroblasts, their capacity to contract collagen gel, and the expression of alpha-smooth muscle actin and promoted different proteolytic enzymes involved in accelerating wound healing. Overall results confirm the effectiveness of electrical stimulation in modulating the wound healing activities of fibroblasts extracted from diabetic skin donors. This study, therefore, suggests the possible use of electrical stimulation to promote diabetic foot ulcer healing by stimulating the wound healing properties of skin fibroblasts.

Engineering diabetic human skin equivalent for in vitro and in vivo applications.

The prevalence of diabetes is increasing worldwide. Diabetes contributes to 70% of all non-traumatic lower-limb amputations, which are directly caused by diabetic foot ulcers (DFU) that are difficult to heal. Non-healing diabetic ulcers represent one of modern society's most difficult medical challenges. One of the promising initiatives to treat DFU is the grafting of autologous skin or stimulating the skin cells at the edge of the wound to proliferate and close the wound. The present study was to engineer a diabetic human skin equivalent (DHSE) that contains fibroblasts and keratinocytes extracted from the skin collected from diabetic patients. The DHSE was used to investigate whether exposure to low-intensity electrical stimulation (ES) could promote diabetic cell activity. The ES was generated by a direct current (DC) electric field of 20 or 40 mV/mm. We demonstrated that the fibroblasts and keratinocytes could be extracted from older diabetics, cultured, and used to engineer DHSE. Interestingly, the exposure of DHSE to ES led to a structural improvement through tissue stratification, increased Ki-67 expression, and the deposition of basement membrane proteins (laminin and type IV collagen). The DHSE exposed to ES showed a high level of keratin 5 and 14 expressions in the basal and supra-basal layers. The keratinocyte proliferation was supported by an increased secretion of the keratinocyte growth factor (FGF-7). Exposure to ES decreased the activity of metalloproteinases (MMP) 2 and 9. In conclusion, we extracted keratinocytes and fibroblasts from the skin of diabetic-old donors. These cells were used to engineer skin equivalents and demonstrate that ES can promote diabetic wound healing. This DHSE can be a promising tool for various in vitro studies to understand the wound healing mechanisms under chronic inflammatory conditions such as diabetes. The DHSE could also be used as an autologous substrate to cover the DFU permanently.

Effect of Electrical Stimulation on Diabetic Human Skin Fibroblast Growth and the Secretion of Cytokines and Growth Factors Involved in Wound Healing.

Diabetic foot ulcers are indicative of an impaired wound healing process. This delay may be resolved through electrical stimulation (ES). The goal of the present study was to evaluate the effect of ES on diabetic fibroblast adhesion and growth, and the secretion of cytokines and growth factors. Diabetic human skin fibroblasts (DHSF) were exposed to various intensities of direct current ES (100, 80, 40 and 20 mV/mm). The effect of ES on fibroblast adhesion and growth was evaluated using Hoechst staining, MTT and trypan blue exclusion assays. The secretion of cytokine and growth factor was assessed by cytokine array and ELISA assay. The long-term effects of ES on DHSF shape and growth were determined by optical microscopy and cell count. We demonstrated that ES at 20 and 40 mV/mm promoted cell adhesion, viability and growth. ES also decreased the secretion of pro-inflammatory cytokines IL-6 and IL-8 yet promoted growth factor FGF7 secretion during 48 h post-ES. Finally, the beneficial effect of ES on fibroblast growth was maintained up to 5 days post-ES. Overall results suggest the possible use of low-intensity direct current ES to promote wound healing in diabetic patients.

Electrical stimulation promotes the proliferation of human keratinocytes, increases the production of keratin 5 and 14, and increases the phosphorylation of ERK1/2 and p38 MAP kinases.

Effective wound healing remains a significant clinical challenge in reducing patient morbidity and improving quality of life. Wound healing is a complex process involving the endogenous electrical field. The electrical field can contribute to wound healing by activating keratinocytes to promote reepithelialization. The objective of this study was to determine the effects of exogenous electrical stimulation (ES) on human keratinocyte viability and proliferation and on production of IL-6, IL-8, and keratins (K5 and K14) and to investigate the activated signalling pathways in keratinocytes exposed to ES. Keratinocytes were cultured under ES at different intensities for 6 or 24 hr. Cell proliferation, cytokines and growth factors, K5 and K14, as well as phosphorylated ERK1/2 and p38 MAP kinases, were evaluated. The results showed that the keratinocytes exposed to ES between 100 and 150 mV/mm for 6 or 24 hr showed a significantly increased proliferation rate. However, a 24 hr exposure to 200 mV/mm revealed no significant effect in cell growth. ES at 100 and 200 mV/mm for 6 hr increased the secretion of epidermal growth factor and vascular endothelial growth factor, and the production of K5 and K14. K14 was more sensitive than K5 to ES. However, ES down-regulated the secretions of IL-6 and IL-8. Finally, ES increased the phosphorylation of ERK1/2 and p38 MAP kinases. Overall results suggested that ES can be useful in supporting skin wound healing by activating keratinocytes.

RAR-related orphan receptor A: One gene with multiple functions related to migraine.

AIMS: RAR-related orphan receptor (RORA) involves in regulation of several biological processes including inflammation and circadian rhythm that probably are involved in migraine pathophysiology. In the current study, the association between RORA rs11639084 and rs4774388 variants and susceptibility to migraine were investigated in a sample of Iranian migraine patients for the first time. METHODS: In a case-control study including 400 participants, 200 migraineurs and 200 healthy controls, genotyping of RORA rs4774388 and rs11639084 polymorphisms was performed using tetra-primer amplification refractory mutation system-polymerase chain reaction (TP-ARMS-PCR). RESULTS: The distribution of rs4774388 C/T and T/T genotypes differed significantly between the studied groups. Moreover, an association was observed between rs4774388 and migraine under the recessive mode of inheritance (P = 0.002; OR = 1.89.; CI = 1.25-2.87). The distribution of rs11639084 alleles and genotypes was not significantly different between migraineurs and healthy controls. CONCLUSION: Current results suggest RORA, as a molecular link, may explain inflammation and circadian rhythm dysfunction in migraine. Further studies in different ethnicities are required to confirm the function of RORA in migraine development.

MED12 Exon 1 Mutational Screening in Iranian Patients with Uterine Leiomyoma.

BACKGROUND: Uterine leiomyoma, also called fibroid, is a benign tumor that arises due to monoclonal transformation of myometrium, the smooth muscle cell layer of the uterus. Fibroids cause several complications including infertility, miscarriage, bleeding, pain, and dysmenorrhea. Recent studies have revealed the role of mutations in MED12 gene exon 2 in various populations; however, the reported frequency of these mutations differs between reports. In addition, it is suggested that mutations in exon 1 may also play a role in leiomyoma. The aim of the present study was to screen for MED12 exon 1 mutations in leiomyoma tissue samples of Iranian patients. METHODS: We performed mutational analysis of exon 1 and the flanking intronic regions using multi-temperature single-strand conformational polymorphism (MSSCP) and sequencing analyses in 120 uterine leiomyoma samples. RESULTS: No mutations were detected in exon 1 of MED12 in our samples. CONCLUSION: According to the literature and the present results, mutations in the MED12 exon 1 are rare. However, we could not ignore the role of these mutations in developing leiomyoma.

LncRNA SRA1 may play a role in the uterine leiomyoma tumor growth regarding the MED12 mutation pattern.

BACKGROUND: Uterine leiomyomas (ULMs) are benign uterine tumors that are estrogen-dependent. Recent studies suggest that the abnormal expression of the steroid receptor RNA activator 1 (SRA1) long non-coding RNA (lncRNA) might participate in the mechanisms of tumorigenesis of some hormone-dependent tumors including breast cancer. SRA1 is known to enhance the transcriptional activity of steroid receptors and also promotes steroidogenesis. The level of steroid hormones, such as estrogen and the progesterone, and their receptors play an important role in the development and growth of leiomyoma. The aim of the present study was to determine the expression level of lncRNA SRA1 in ULM tissues considering the MED12 mutation pattern. METHODS: Mutation screening was performed for MED12 exons 1 and 2 and the intronic flanking regions using Sanger sequencing in 60 ULM tissues. Quantitative real-time polymerase chain reaction (qRT-PCRs) was performed in order to estimate the expression of lncRNA SRA1 in leiomyoma samples with and without MED12 gene mutations. The expression results were analyzed by using LinReg and REST software. RESULTS: Mutations were detected in exon 2 of the MED12 in 28 (46.67%) ULM samples; including, 21 (75%) missense mutations and 7 (25%) in-frame deletions. No mutation was detected in the MED12 exon 1. LncRNA SRA1 was over-expressed in ULM samples without MED12 mutation compared with ULM samples harboring MED12 mutation (Expression ratio=2.5, P-value=0.004). CONCLUSION: Present results suggest that lncRNA SRA1 may explain the phenotypic difference observed in the tumor size of ULM samples considering MED12 mutation pattern. Therefore, it serves as a good therapeutic target and provides new insight into understanding the disease molecular mechanism.

ACE gene rs4343 polymorphism elevates the risk of preeclampsia in pregnant women.

The multifactorial basis of preeclampsia (PE) implies that there are several genes and risk factors that are important in the development of the disease. Therefore, the exact etiology and pathogenesis of preeclampsia remains unclear. It is suggested that inappropriate regulation of the renin-angiotensin system (RAS) is a risk factor for hypertension during pregnancy. The angiotensin I-converting enzyme (ACE) serum level, a key component of the RAS, affects the blood pressure. It is hypothesized that the ACE gene polymorphisms contribute to preeclampsia development. In a case-control study containing 296 subjects (165 PE patients and 131 normotensive controls), we aimed to examine the association of the ACE gene I/D and rs4343 polymorphisms with preeclampsia in Iranian women. Genotyping for rs4343 and ACE I/D polymorphisms was performed by using TP-ARMS-PCR and conventional PCR, respectively. The rs4343 G allele frequency was higher in the case group (OR = 1.90, 95% CI, 1.37-2.65; P = 0.0001). Besides, a significant difference was detected for the genotype frequencies between the studied groups under dominant (OR = 3.94, 95% CI, 2.05-7.56; P < 0.0001) and recessive (OR = 2.21, 95% CI, 1.22-4.01; P = 0.009) inheritance models. For the I/D polymorphism, no significant differences were detected in the genotype and allele frequencies or any of the inheritance models between PE patients and controls. To verify the current results and validate the significance of the studied genetic variations, additional studies in diverse ethnic populations are required.

Lactobacilli Differentially Modulate mTOR and Wnt/ ß-Catenin Pathways in Different Cancer Cell Lines.

BACKGROUND: Lactobacilli are a group of beneficial bacteria whose anti cancer effects have been evaluated in different cancer cell lines as well as animal models and human subjects. Such anti cancer effects can be exerted via different mechanisms such as modulation of immune response as well as inhibition of pathogens colonization. In addition, lactobacilli have direct cytotoxic effects against cancer cells which may be exerted through modulation of expression cancer related pathways. OBJECTIVES: The aim of this study is to find the mechanism of anti cancer effects of two lactobacilli strains, Lactobacillus. crispatus (LC) and Lactobacillus. rhamnosus (LR). MATERIALS AND METHODS: We analyzed expression of some mTOR and Wnt/ ß-catenin pathways genes in three cancer cell lines (HeLa, MDA-MB-231 and HT-29) following treatment with LC and LR culture supernatants. RESULTS: Of note, the expression of CCND1 as a marker of cell proliferation, survival, and angiogenesis, has been decreased following LR treatment in all cell lines. In addition, the expression of SFRP2, an antagonist of Wnt pathway, has been increased in HT-29 following LR treatment and in HeLa cells following LR and LC treatments. Furthermore, we have demonstrated the downregulation of S6K1 expression, a marker of poor prognosis, following LR treatment in HT-29 and following LR and LC treatments in MDA-MB-231 cell line. CONCLUSIONS: Consequently, lactobacilli can modulate expression of mTOR and Wnt/ ß-catenin pathways genes in cancer cell lines in a strain specific as well as cell type specific manner.

Lactobacilli Modulate Hypoxia-Inducible Factor (HIF)-1 Regulatory Pathway in Triple Negative Breast Cancer Cell Line.

OBJECTIVE: Hypoxia-Inducible Factor (HIF)-1 plays an essential role in the body's response to low oxygen concentrations and regulates expression of several genes implicated in homeostasis, vascularization, anaerobic metabolism as well as immunological responses. Increased levels of HIF-1α are associated with increased proliferation and more aggressive breast tumor development. Lactobacilli have been shown to exert anti-cancer effects on several malignancies including breast cancer. However, the exact mechanism of such effect is not clear yet. The aim of this study was to analyze the expression of selected genes from HIF pathway in a triple negative breast cancer cell line (expressing no estrogen and progesterone receptors as well as HER-2/Neu), MDA-MB-231, following treatment with two lactobacilli culture supernatants. MATERIALS AND METHODS: In this experimental study, we analyzed the expression of HIF-1α, SLC2A1, VHL, HSP90, XBP1 and SHARP1 genes from HIF pathway in MDA-MB-231 cells, before and after treatment with Lactobacillus crispatus and Lactobacillus rhamnosus culture supernatants (LCS and LRS, respectively) by means of quantitative reverse-transcription polymerase chain reaction (qRT-PCR). RESULTS: Both LRS and LCS had cytotoxic effects on MDA-MB-231 cells, while the former type was more cytotoxic. LRS dramatically down-regulated expression levels of the HIF-1α, HSP90 and SLC2A1 in the MDA-MB-231 cells. LCS had similar effect on the expression of HSP90, to what was observed in the LRS treatment. The expression level of tumor suppressor genes VHL and SHARP1 were also decreased in LCS treated cells. CONCLUSION: Although both LCS and LRS had cytotoxic effects on the MDA-MB-231 cells, it is proposed that LRS could be more appropriate for pathway directed treatment modalities, as it did not decrease expression of tumor suppressor genes involved in HIF pathway. Down-regulation of HIF pathway mediated oncogenes by LRS suggests that the cytotoxic effects of this Lactobacillus may at least be partly caused by this mechanism. As previous studies have shown that inhibition of HIF-1α and HSP90 expressions have therapeutic impact on cancer treatment, the inhibitory effect of LRS on expression of these genes implies that this Lactobacillus can be used in treatment strategies.

Immunomodulatory effects of Lactobacillus strains: emphasis on their effects on cancer cells.

Lactobacilli are a group of normal microbiota whose immunomodulatory effects have been known for a long time. Recently, they have gained more attention for their direct and indirect effects on cancer cells. Several cell line experiments, animal model studies as well as clinical trials have indicated their inhibitory effects on cancer initiation and progression. Different lactobacilli strains could modulate innate and adoptive immune system. Such effects have been documented in modulation of function of T cells, dendritic cells and macrophages as well as cytokine production. In this review, the various immunomodulatory effects of lactobacilli on tumor cells as well as their direct cytotoxic effects on cancer cells are discussed.