Protective Effect of Litchi chinensis Peel Extract-Prepared Nanoparticles on Rabbits Experimentally Infected with Eimeria stiedae.

The present study used Litchi chinensis peel extract to synthesize silver nanoparticles (AgNPs). This technique is eco-friendly and can be performed in a single step; thus, it has attracted great attention for NPs biosynthesis. Herein, we biosynthesized AgNPs with L. chinensis peel extract and examined their anticoccidial activity in rabbit hepatic coccidiosis induced by E. stiedae infection. Thirty-five rabbits were allocated into seven groups: a healthy group (G1), an infected control group (G2), four groups infected before treatment with 10 mg/kg L. chinensis peel extract-biosynthesized AgNPs (G3, G5) or 50 mg/kg amprolium (G4, G6), and rabbits infected after two weeks of pretreatment with 10 mg/kg L. chinensis eel extract-biosynthesized AgNPs (G7). In this study, both pre-and post-treatment with AgNPs produced a substantial reduction in fecal oocyst output, liver enzyme levels, and histopathological hepatic lesions relative to the infected group. In conclusion, L. chinensis peel extract-prepared AgNPs should be considered harmless and efficient in the cure of hepatic coccidiosis in rabbits.

Ameliorative effect of L-carnitine on lambda-cyhalothrin-induced anatomical and reproductive aberrations in albino mice.

The objective of this research was to look at how the pesticide lambda-cyhalothrin (LCT) affected the liver, kidney, testis, and ovary of albino mice; and on morphological and skeletal features of the newborn of treated females. The study also aimed to test the ameliorative effects of L-carnitine (LC) against (LCT). Five sets of mice were created, Group 1 acted as the control, while Group 2 received a high dose of LCT, Group 3 received a high dose of LCT + LC, Group 4 received a low dose of LCT that was a residue of in khat (Qat), and Group 5 received a low dose of LCT + LC. The findings revealed that the treated groups' body weights were reduced significantly, whereas the absolute and relative weights of the liver in all groups were statistically decreased insignificantly. There were histopathological changes in the tissues in groups 2 and 4. While the tissues of the ovary and testis showed recovery in groups 3 and 5. When compared to the control group, the values of the seminiferous tubules parameters were statistically significant in the 3 and 5 groups. The newborn had a high dose of pesticides and showed some malformations in the skeleton. However, in group 3 the skeletal malformation was minimized and in-group 5 the skeleton malformations had completely disappeared. It could be concluded that LCT is highly harmful to mouse tissues and caused neonatal malformations, whereas LC has a marked protective effect against LCT.

Association SOD2 and PON1 Gene Polymorphisms with Polycystic Ovary Syndrome in Saudi Women.

Polycystic ovary syndrome (PCOS) is a considered one of the most common female disorders associated with reproductive, metabolic, and psychological problems. The etiology of PCOS is still not yet disclosed; however, evidence for a genetic basis has been reported. In this study, we investigate the associations between superoxide dismutase 2 (SOD2) (rs4880) and paraoxonase 1 (PON1) (rs705379) polymorphisms in PCOS in Saudi women. The study included 99 females with PCOS and 98 healthy women as a control. Single nucleotide polymorphisms (SNPs) of promoter regions were determined using TaqMan genotyping assays. Regarding the polymorphism at SOD2 (rs4880), the CC, CT, and TT genotypes were present at rates of 32, 61, and 7% in PCOS patients, and 47, 43, and 10% in controls, respectively. The frequency of the CT genotype in PCOS patients (0.61) was significantly higher than in controls (0.43) (OR = 2.05, CI: 1.16-3.61; p = 0.015). The wild homozygous genotype (CC) with the phenotype alanine appears to confer protection against the disease compared to molecules sharing at least one valine (genotypes, CT + TT). Regarding the polymorphism at PON1 (rs705379), the rates of CC, CT, and TT genotypes were 34, 50, and 16% in PCOS patients and 33, 63, and 2% in controls, respectively. The rate of the TT genotype in PCOS patients was significantly higher than that in controls (p = 0.0058). SOD2 and PON1 polymorphisms may be genetic factors that affect the occurrence of PCOS in Saudi females.

Identification of Theileria spp. in sheep and goats from Jeddah, Saudi Arabia, using molecular techniques.

BACKGROUND: Thileriosis is a tick -born disease caused by hemoprotozoan parasites which has global veterinary and economic implications. METHODS: Blood samples were collected from 216 sheep and 83 goats from Jeddah, Saudi Arabia, were analyzed to determine whether the animals were infected with Theileria spp. parasites. The parasites were detected using a polymerase chain reaction (PCR) targeting the gene of 18S rRNA followed by sequencing. RESULTS: According to obtained findings, Theileria spp. were detected in sheep (57.8%, 48/83) and goats (51.9%, 112/216). Phylogenetic analysis to sequence data showed that T. ovis identified in this study were found to be closely connected to an isolate from Turkey, with 84.4-99.8% pairwise identity and 52.35-99.79% coverage.

Structural and Functional Characterization of Covalently Modified Proteins Formed By a Glycating Agent, Glyoxal.

Glycation, the main consequence of hyperglycemia, is one of the major perpetrators of diabetes and several other conditions, including coronary and neurodegenerative complications. Such a hyperglycemic condition is represented by a large increase in levels of various glycation end products including glyoxal, methylglyoxal, and carboxymethyl-lysine among others. These glycation end products are known to play a crucial role in diabetic complications due to their ability to covalently modify important proteins and enzymes, specifically at lysine residues (a process termed as glycation), making them non-functional. Previous studies have largely paid attention on characterization and identification of these reactive glycating agents. Structural and functional consequences of proteins affected by glycation have not yet been critically investigated. We have made a systematic investigation on the early conformational changes and functional alterations brought about by a glycating agent, glyoxal, on different proteins. We found that the early event in glycation includes an increase in hydrodynamic diameter, followed by minor structural alterations sufficient to impair enzyme activity. The study indicates the importance of glyoxal-induced early structural alteration of proteins toward the pathophysiology of hyperglycemia/diabetes and associated conditions.

Ameliorative effects of colostrum against DMBA hepatotoxicity in rats.

Colostrum, the sole diet for newborns, is an emerging nutraceutical. To date, the chemopreventive effect of Bovine Colostrum against liver injury induced by the potent carcinogen, 7,12-dimethyl-Benz[a]anthracene (DMBA) is unexplored. Humans are daily exposed to DMBA which is a highly lipophilic environmental organic pollutant. The study aimed to investigate the hepatoprotective role of Bovine Colostrum against DMBA-induced hepatotoxicity using a rat model. Fifty male rats were divided into five groups; GI (control), GII (olive oil, vehicle for DMBA), GIII (DMBA), GIV (DMBA + Bovine Colostrum), GV (Bovine Colostrum). After 12 weeks, body weight changes and mortality were calculated. Histological and ultrastructural examinations of liver tissue were performed. Expressions of p53, TGFß2, TNF-α, S6K2, and c20orf20 were assessed by RT-PCR. Post-treatment with Bovine Colostrum increased both the body weight and the survival rate of rats treated with DMBA. In addition, remarkable protection against the pathological effect of DMBA was noted. Ultrastructurally, Bovine Colostrum ameliorated/prevented most of the toxic effects of DMBA on hepatocytes, including irregularities of nuclear envelope, clumping, and margination of heterochromatin aggregates, segregated nucleoli, and mitochondrial pleomorphism. Bovine Colostrum administration down-regulated p53, C20orf20, and S6K2 mRNA levels, and up-regulated TNF-α and TGFß2. In conclusion, Bovine Colostrum have a protective effect against DMBA-induced toxicity on the liver of albino rats. Consequently, Bovine Colostrum may prevent polycyclic aromatic hydrocarbons-induced hepatotoxicity and may be useful in promoting human health if supplemented in the diet.

Effect of mesenchymal stem cells on cytochrome-c release and inflammation in colon cancer induced by 1,2-dimethylhydrazine in Wistar albino rats.

Colon cancer is one of the most common causes of deaths by cancer worldwide. Stem cells have immunosuppressive properties that promote tumor targeting and circumvent obstacles currently in gene therapy. Bone marrow stem cells are believed to have anticancer potential. The transplantation of mesenchymal stem cells (MSCs), a type of bone marrow stem cells, has been considered a potential therapy for patients with solid tumors due to their capability to enhance the immune response; MSC transplantation has received renewed interest in recent years. The present study aimed to evaluate the antiapoptotic effects of the MSCs on 1,2-dimethylhydrazine (DMH)-induced inflammation in the rat model of colorectal cancer. The rats were randomly allocated into four groups: control, treated with MSCs, induced by DMH, and induced by DMH and treated with MSCs. The MSCs were intra-rectally injected, and DMH was subcutaneously injected at 20 mg/kg body weight once a week for 15 weeks. The administration of MSCs into rats starting from day 0 of the DMH injection was found to enhance the histopathological picture. The MSC treatment resulted in fewer inflammatory cells than in the DMH group. Therefore, our findings suggest that BMCs have antitumor effects by modulating the cellular redox status and down-regulating the pro-inflammatory genes. Thus, BMCs may provide therapeutic value for colon cancer treatment.

Chlorogenic acid abates male reproductive dysfunction in arsenic-exposed mice via attenuation of testicular oxido-inflammatory stress and apoptotic responses.

Arsenic, a major environmental pollutant of global concern, is well-known for its reproductive toxicity. In this study, the protective potential of chlorogenic acid (CGA), a caffeoylquinic acid isomer abundantly found in many plants, was investigated against sodium arsenite (NaAsO2)-induced testicular dysfunctions. Adult male Swiss mice were either administered NaAsO2 alone at 5 mg kg-1 or co-treated with CGA at 100 mg kg-1 or 200 mg kg-1 body weight for 4 weeks. Results showed that NaAsO2-treated mice exhibited marked declines in testes weight, sperm count, and viability accompanied by decreases in sexual hormonal levels. Moreover, NaAsO2 toxicity evoked exhaustion of antioxidant markers (SOD, CAT, GPx, GR, and GSH), down-regulation of Nrf2 (nuclear factor erythroid 2-related factor 2) gene expression level, and elevations in malondialdehyde. Further, elevations in inflammatory cytokines (IL-1, TNF-α, and IL-6) together with the up-regulation of pro-apoptotic biomarkers (Bax and caspase- 3) and down-regulation of anti-apoptotic Bcl-2 were observed in NaAsO2 intoxication. Immunohistochemical analysis of testis sections of NaAsO2-treated mice showed high caspase-3 expression. These findings were well supported with testicular histopathological examination. However, pretreatment of mice with CGA resulted in noteworthy improvements in testicular damage induced by arsenic in a dose-dependent manner possibly mediated by the Nrf2 signaling pathway. Conclusively, CGA counteracted arsenic-induced testicular injury through its antioxidant, anti-inflammatory, and anti-apoptotic properties. Therefore, CGA could serve as a favorable intervention in the alleviation of arsenic-induced reproductive toxicity.

A preliminary study of cytokine gene polymorphism effects on Saudi patients with colorectal cancer.

OBJECTIVE:  To determine the possible associations of polymorphisms in interleukin (IL)-8 (rs4073 T/A), IL-10 (rs1800896 A/G), IL-22 (rs1179251 C/G and rs2227485 C/T), IL-27 (rs17855750 T/G), and transforming growth factor beta 1 (TGFß1) (rs1800469 C/T) with colorectal cancer (CRC) susceptibility in Saudi patients. METHODS: The case-control study was carried out between July 2019 and January 2020 in King Khaled University Hospital, Riyadh, Saudi Arabia. A total of 70 patients with CRC and 70 healthy controls were included  in  the  study.  Single nucleotide polymorphisms of promoter regions were determined using TaqMan genotyping assays. RESULTS:  A statistically significant reduction in CRC risk was identified for carriers of the IL-10 (rs1800896 A/G) AG genotype, IL-22 (rs1179251 C/G) G allele, IL-27 (rs17855750 T/G) G allele and TGFß1 (rs1800469 C/T) CT and TT genotype. While IL-10 (rs1800896 A/G) AA genotype and TGFß1 (rs1800469 C/T) CC genotype were significantly associated with increased susceptibility to CRC. No significant associations were identified between the cytokine polymorphisms of IL-8 (rs4073 T/A) and IL-22 (rs2227485 C/T), and CRC risk. Conclusion: Our findings indicate a significant impact of IL-10 (rs1800896 A/G), IL-22 (rs1179251 C/G), IL-27 (rs17855750 T/G) and TGF-ß1 (rs1800469 C/T) polymorphisms on risk of CRC; while the IL-8 (rs4073 T/A) and IL-22 (rs2227485 C/T) and polymorphisms were not associated with CRC risk.

Dipteran Carboxymethyl Chitosan as an Inexhaustible Derivative with a Potential Antiproliferative Activity in Hepatocellular Carcinoma Cells.

Traditional folk therapies indicate that insects have diverse medicinal potentials. However, the therapeutic application of insect chitosan and its derivatives has not been explored. To investigate the application of chitosan and its derivatives, the carboxymethyl derivative of chitosan (CM-Ch) was extracted from two dipteran larvae species, Chrysomya albiceps and Sarcophaga aegyptiaca. The degree of deacetylation (DD) and CM-Ch functional groups were validated using Fourier-transform infrared (FTIR) spectroscopy analysis and proton nuclear magnetic resonance spectroscopy (1H NMR), respectively. The molecular weight was estimated using MALDI-TOF MS analysis. The effect of CM-Ch on the morphology and proliferation of human liver HepG2 cancer cells was assessed. IC50 of CM-Ch induced significant growth-inhibitory effects in HepG2 cells. CM-Ch treatment altered the morphology of HepG2 in a dose-dependent manner and induced apoptosis in a caspase-dependent manner. CM-Ch treatment showed no signs of toxicity, and no alterations in liver and kidney biochemical markers were observed in albino rats. A CM-Ch derivative from commercial crustacean chitosan was used to assess the efficacy of the insect-derived CM-Ch. The data presented here introduce insect CM-Ch as a promising, inexhaustible, safe derivative of chitosan with antitumor potential in liver cancer. This is the first report highlighting the anticancer activity of insect CM-Ch in hepatocellular carcinoma cells.

Luteolin protects against testicular injury induced by lead acetate by activating the Nrf2/HO-1 pathway.

Lead (Pb) is a toxic heavy metal that is harmful to humans, especially male reproductive organs. Luteolin (LUT) is a naturally occurring flavonoid with numerous biological activities. Our aim was to investigate the possible reproprotective effect of LUT against testicular deficits induced by Pb intoxication. In the present study, 28 rats were distributed into 4 groups: control, LUT (50 mg/kg), lead acetate (PbAc, 20 mg/kg), and LUT + PbAc groups, in which rats were pre-treated with LUT 3 hr before PbAc injection. All animals were treated for 7 days. Oxidative stress, inflammatory and apoptotic markers along with histopathological changes have been examined using spectrophotometric, ELISA, real-time PCR, and histopathological methods. PbAc injection elevated Pb concentration in testicular tissue and decreased levels of sex hormones. PbAc intoxication exacerbated lipoperoxidation and nitric oxide formation, depleted superoxide dismutase, and catalase activities along with glutathione and its originated enzymes (glutathione peroxidase and glutathione reductase). At the molecular level, PbAc deactivated nuclear factor erythroid 2-related factor 2 and heme oxygenase-1 in the testicular tissue. In addition, PbAc toxicity induced inflammatory and apoptotic cascades in testicular tissue as evidenced by the increased tumor necrosis factor-alpha, interleukin-1 beta, inducible nitric oxide synthase, Bax, and caspase 3, while Bcl-2 was declined. Histopathological examination of testicular tissue also revealed that PbAc caused degeneration alterations in spermatogenic cells, the spermatogenic epithelial cells were disconnected from the basement membrane, and the seminiferous tubules were vacuolated. Remarkably, pre-treatment with LUT minimized significantly the testicular damage induced by PbAc. Therefore, we conclude that LUT may have a beneficial effect against PbAc-induced testicular injury through preventing oxidative challenge, inflammation, and finally apoptosis.

Association of IL-1ß, IL-6, TNF-α, and TGFß1 Gene Polymorphisms with Recurrent Spontaneous Abortion in Polycystic Ovary Syndrome.

Recurrent spontaneous abortion (RSA) is a common pregnancy-associated complication of polycystic ovary syndrome (PCOS) which is an endocrine malfunction disease. Patients with PCOS may have several underlying contributing and interrelated factors, which have been reported in women with RSA. The incidence rate between PCOS and RSA remains uncertain. The aim of this study is to determine the possible association of IL-1ß-511C/T, IL-6-174G/C, TNF-α-1031T/C, and TGFß1-509T/C with RSA patients with or without PCOS. A total of 140 RSA patients, 70 of which were PCOS patients, and 140 healthy females with no history of RSA or PCOS were included in this study. PCR amplification, genotyping, and sequence analysis were employed to investigate the presence of the polymorphisms. The genotypic and allelic frequencies were calculated separately for each subject. Out of the four studied polymorphisms, the IL-1ß-511C/T genotype in RSA without PCOS patients (12.7%) was significantly different compared with that in control subjects (p = 0.047). For IL-6-174C/G, there was a tendency towards more CC carriers among RSA with PCOS patients (10%) than in controls (3%). The GG genotype in RSA women with PCOS (60%) was significantly different compared with that in control subjects (p = 0.033), and the GC genotype in RSA with PCOS patients (30%) showed a marginal significant difference compared with that in control subjects (p = 0.050). Significant difference was identified in the allelic frequencies in RSA patients with PCOS compared to controls (p = 0.025). IL-6-174G/C and TNF-α-1031T/C polymorphisms are significantly associated with RSA patients in Saudi patients with PCOS, while the IL-1ß-511C/T polymorphism is significantly associated with RSA patients without PCOS.

The Beneficial Effect of Eco-Friendly Green Nanoparticles Using Garcinia mangostana Peel Extract against Pathogenicity of Listeria monocytogenes in Female BALB/c Mice.

Listeria monocytogenes is a psychrophilic bacterium, which causes widespread zoonosis in the natural environment, and mainly affects goat, sheep, and cattle herds. Recently, we predicted that it can be transmitted through food. It causes listeriosis, a severe infectious disease, which occurs with food contaminated with the pathogenic bacterium. Anti-inflammatory factors are important to treat the dangers of chronic inflammation associated with chronic diseases. Natural foodstuffs have made and are continuing to make vital contributions to the search for new antilisterial agents. The use of natural products in association with silver nanoparticles has drawn attention because of its easy, nonpathogenic, eco-friendly, and economical protocol. Hence, we aimed to biosynthesize silver nanoparticles (Ag-NPs) using Garcinia mangostana peel extract, which was found to be a good source for the synthesis of silver nanoparticles, their formation being confirmed by color change and stability in solution, and investigated the antilisterial activity of these nanoparticles in a murine model of L. monocytogenes infection. A total of 28 mice were divided into four groups-healthy control, infected, infected mice treated with green Ag-NPs biosynthesized with G. mangostana (5 mg/mL), and infected mice pretreated with Ag-NPs. From our results, oral treatment with Ag-NPs biosynthesized with G. mangostana peel extract resulted in a significant reduction in malondialdehyde (MDA), enhanced antioxidant enzyme activities, and increased the levels of the antiapoptotic protein, compared with the untreated mice. These results indicate that G. mangostana may provide therapeutic value against L. monocytogenes-induced oxidative stress and histopathological alterations, and that these effects may be related to antiapoptotic and antioxidant activities.

Antagonistic Efficacy of Luteolin against Lead Acetate Exposure-Associated with Hepatotoxicity is Mediated via Antioxidant, Anti-Inflammatory, and Anti-Apoptotic Activities.

The abundant use of lead (Pb; toxic heavy metal) worldwide has increased occupational and ecosystem exposure, with subsequent negative health effects. The flavonoid luteolin (LUT) found in many natural foodstuffs possesses antioxidant and anti-inflammatory properties. Herein, we hypothesized that LUT could mitigate liver damage induced by exposure to lead acetate (PbAc). Male Wistar rats were allocated to four groups: control group received normal saline, LUT-treated group (50 mg/kg, oral, daily), PbAc-treated group (20 mg/kg, i.p., daily), and LUT+PbAc-treated group (received the aforementioned doses via the respective routes of administration); the rats were treated for 7 days. The results revealed that PbAc exposure significantly increased hepatic Pb residue and serum activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT), and total bilirubin value. Oxidative reactions were observed in the liver tissue following PbAc intoxication, characterized by the depletion and downregulation of antioxidant proteins (glutathione, glutathione reductase, glutathione peroxidase, superoxide dismutase, catalase, nuclear factor erythroid 2-related factor 2, and heme oxygenase-1), and an increase in oxidants (malondialdehyde and nitric oxide). Additionally, PbAc increased the release and expression of the pro-inflammatory cytokines (tumor necrosis factor alpha and interleukin-1 beta), inducible nitric oxide synthase, and nuclear factor kappa B. Moreover, PbAc enhanced hepatocyte loss by increasing the expression of pro-apoptotic proteins (Bax and caspase-3) and downregulating the anti-apoptotic protein (Bcl-2). The changes in the aforementioned parameters were further confirmed by noticeable histopathological lesions. LUT supplementation significantly reversed all of the tested parameters in comparison with the PbAc-exposed group. In conclusion, our findings describe the potential mechanisms involved in the alleviation of PbAc-induced liver injury by luteolin via its potent anti-inflammatory, antioxidant, and anti-apoptotic properties.

Synergistic Anti-Breast-Cancer Effects of Combined Treatment With Oleuropein and Doxorubicin In Vivo.

CONTEXT: Breast cancer is a leading cause of cancer fatalities among women worldwide. Of the more than 80% of patients who receive adjuvant chemotherapy, approximately 40% relapse. The majority of these patients die of disseminated metastatic disease, which emphasizes the need for new therapeutic strategies. OBJECTIVE: The study intended to investigate the anticancer effects of oleuropein (OL) and doxorubicin (DOX) individually and in combination on breast tumor xenografts and also to evaluate the molecular pathways involved. DESIGN: The research team designed in vivo (animal) and in vitro (cell culture) studies. SETTING: The study was performed in the College of Science of King Saud University in the University Center for Women Students (Riyadh, Saudi Arabia). ANIMALS: The study involved 40 female, nude mice (BALB/c OlaHsd-foxn1). INTERVENTION: The mice were injected subcutaneously with MDA-MB-231 human breast cancer cells. After the growth of tumors, the animals were randomly divided into 4 groups to receive intraperitoneal injections: (1) group 1 (control group)-dimethyl sulfoxide, (2) group 2 (intervention group)-50 mg/kg of OL, (3) group 3 (intervention group)-2.5 mg/kg of DOX, and (4) group 4 (intervention group)-1.5 mg/kg of DOX, immediately followed by 50 mg/kg of OL. The OL was extracted from Manzanillo olive trees (Olea europaea) grown in Tabouk, Saudi Arabia. OUTCOME MEASURES: The measures included the isolation and primary culture of the tumor xenografts, apoptosis analysis by annexin V, cellular lysate preparation, and immunoblotting. RESULTS: The volume of the tumor increased aggressively, reaching 173 mm3 in the control animals in a time-dependent manner. On the other hand, a sharp drop, to 48.7 mm3, in the volume of the tumor was observed with the 2 drugs combined, a more than 3-fold decrease. The effect was mediated through the induction of apoptosis via the mitochondrial pathway. The combined treatment downregulated the antiapoptosis and proproliferation protein, nuclear factor-kappa Β, and its main oncogenic target cyclin D1. Furthermore, it inhibited the expression of BCL-2 and survivin. This inhibition could explain the cooperative suppression of the proliferation of breast tumor xenografts and the induction of apoptosis by the combined effect of the compounds used. CONCLUSIONS: The key findings clearly indicate the synergistic efficacy of DOX with natural and nontoxic OL against breast tumor xenografts.

The relationship between cytokine gene polymorphism and unexplained recurrent spontaneous abortion in Saudi females.

OBJECTIVE: To investigate the relationships between unexplained recurrent spontaneous abortion (RSA) and single nucleotide polymorphisms tumor necrosis factor-alpha (TNF-alpha) (-238 G/A, -308 G/A), interleukin (IL)-6 (-634 G/C) and IL-10 (-592 C/A) in the promoter region of 3 different interleukin (TNF-alpha, IL-6, and IL-10) genes. METHODS: The study group comprised 65 women (mean age: 34.1+/-6.2; range: 15-45 years) with unexplained RSA, consecutively referred to the Recurrent Abortion Clinic, King Khaled University Hospital, Riyadh, Kingdom of Saudi Arabia from January 2010 to January 2011. The control group consisted of 65 females with at least 2 successful pregnancies and no history of abortion. Blood samples were drawn and deoxyribonucleic acid (DNA) was extracted using Puregene DNA purification kit. Utilizing polymerase chain reaction, the promoter region was amplified and sequenced on an Applied Biosystems Integrated sequencer to study the polymorphic sites of interest. All polymorphisms were identified in the case and control samples. RESULTS: A significant association was identified only between the -308 G/A polymorphism in the TNF-alpha gene promoter and the occurrence of unexplained RSA, and there was no significant association with other positions. CONCLUSION: The TNF-alpha gene polymorphism at position -308 could be a genetic predisposing factor for unexplained RSA.