Volume 49, Issue 1

Introduction Metabolomics is a rapidly growing field that aims to understand the complex metabolic pathways involved in health and disease. Liquid chromatography mass spectrometry (LC-MS) based untargeted metabolomics has emerged as a powerful tool for investigating the metabolic changes associated with various diseases, and for identifying potential biomarkers for early disease detection and treatment monitoring. This review provides a comprehensive overview of LC-MS based untargeted metabolomics and its clinical applications. Content The advantages and challenges of untargeted metabolomics are discussed, encompassing sample preparation, data processing, sample storage, acquisition mode selection, column strategy, and annotation. The latest advancements in LC-MS technology and data analysis techniques are reviewed, which have facilitated the more accurate and sensitive detection of metabolites in biological samples. The clinical applications of untargeted metabolomics are emphasized, including its utilization in disease diagnosis, treatment monitoring, and personalized medicine. Examples are provided of how biomarkers for various diseases, such as cancer, coronary heart disease, and infectious disease, have been identified through the application of untargeted metabolomics. Summary and outlook Overall, LC-MS based untargeted metabolomics is a powerful tool for investigating the complex metabolic changes associated with various diseases, and has the potential to transform clinical diagnosis, treatment, and personalized medicine.

Societal habits’ continuation is expected to result in severe consequences for climate change, causing significant environmental damage and humanitarian crises. Sustainability, defined as meeting present needs without compromising future generations, balances environment, equity, and economy. Türkiye, a middle-income developing country, has committed to achieving net-zero emissions by 2053 under the United Nations’ framework. The construction sector is increasingly adopting eco-friendly practices, emphasizing green buildings and structures. Several green hospital certification systems, including BREEAM, LEED, and Australian Green Star, are now in use, with around 20 certified “green hospitals” in Türkiye. The “Zero Waste Project” initiated in Türkiye aims to reduce waste generation and resource usage efficiently. Recent efforts have focused on sustainability in high-carbon footprint medical laboratories, however, an international standard has not been established yet. Clinical chemistry and laboratory medicine federations have established working groups on the subject. Universities and nonprofits worldwide offer green laboratory certificate programs covering energy conservation, green chemistry, waste management, and water conservation. Laboratories’ sustainability efforts encompass inventory management, green purchasing, test request reduction, greenhouse gas management, efficient building design, transportation choices, carbon footprint calculations, and education. The guides published in Türkiye are “Health Institutions Wastewater/Liquid Waste Management Handbook” and “Guide for Laboratory and Dialysis Wastes”. Türkiye’s Ministry of Health introduced the “Rational Test Request Procedure” to enhance diagnostic accuracy and cost-effectiveness by reducing unnecessary tests. Collective efforts are essential to raise awareness and implement precautions, particularly in high-carbon footprint medical laboratories, addressing climate change and sustainability challenges in the healthcare sector.

This paper provides information on the significance of environmental sustainability and the potential benefits of implementing green laboratory processes. Sustainability is a broad concept that includes goals such as conservation of natural resources, energy efficiency, waste management, and reducing environmental footprints. Green laboratories strive to minimise environmental footprints by embedding sustainability principles into their everyday practices. This involves improvements in energy consumption, water usage, chemical waste reduction, and recycling, ultimately, as well as lower waste disposal costs. Implementing green laboratory approaches provides many benefits, such as a smaller environmental footprint, increased energy efficiency, water conservation, and cost savings. Moreover, a rational test ordering process can effectively contribute to reducing environmental impacts in medical laboratories. Rational test ordering provides cost-effectiveness and reduces the carbon footprint. Establishing a green and sustainable laboratory culture in medical laboratories requires a permanent organisation in where all personnel participate. Training of the hospital management teams, clinicians, and laboratory professionals is a vital point. Methodology and quality indicators should be defined by international and national laboratory associations within the framework of a green and sustainable laboratory plan, and data should be collected. In conclusion, adopting green laboratory practices reduces environmental impacts, saves costs, and accelerates innovation processes. Setting quality targets and rational test ordering practices are also essential for sustainability. Applying sustainability principles in laboratory workplaces is an important step towards protecting the environment and leaving a more liveable world for future generations.

Objectives The aim of this study was to compare the analytical performance of the KIMS (kinetic interaction of microparticles in solution) immunochemical method with a validated in-house and a commercial LC-MS/MS method. Methods The urine samples of the 100 subjects were included in the present study. The urine samples were analysed with Roche DAT immunochemical method based on KIMS method. In-house LC-MS/MS method was validated for 58 parameters according to the CLSI C62-A recommendations with the following parameters: matrix effect, lower limit of quantification (LLOQ), linearity, intra-day and inter-day precision and accuracy. Eureka Lab Division Drugs of Abuse kit was used as the commercial LC-MS/MS method. Results The immunochemical method had a satisfactory performance with specificity, sensitivity and accuracy values above 80 % and met the DRUID recommendation except benzodiazepines. The sensitivity and specificity of the immunochemical method were between 97–100 % and 84–100 %, respectively (except for benzodiazepines). The bias obtained for THC-COOH, morphine and codeine parameters were −17.5, 24.6 and 43.6 between two LC-MS/MS methods. The commercial method had a tendency to have a negative bias except for cannabinoids. Conclusions The analytical performance of the KIMS-based urine immunochemical method was found to be satisfactory for the intended use, except for benzodiazepines. The validated urine in-house LC-MS/MS method was found to be a good alternative for confirmation of substance abuse.

Objectives Peroxisome proliferator-activated receptor gamma (PPAR-γ) modifies many cellular processes that contribute to atherosclerosis. The increased concentrations of osteoprotegerin (OPG) are related with coronary artery disease, calcification in vascular tissue, advanced atherosclerosis, and diabetic complications has been informed. The aim of our study was to define the relation among PPAR-γ Pro12Ala and, OPG and PPAR-γ in Peripheral Vascular Disease (PVD) and hypertension (HT). Also, it was aim to investigate the relationship between flow-mediated dilatation (FMD) in HT and ankle brachial index (ABI) in PVD in terms of endothelial dysfunction (ED). Methods Fifty-four patients with HT, 47 with PVD, and 52 healthy for the controls were included. Blood samples were used for analyzing PPAR-γ and OPG by Enzyme-Linked Immunosorbent Assay (ELISA), and biochemical assays. The PPAR-γ Pro12Ala was examined using TaqMan with PrimerProbMix. p value less than 0.05 was accepted as the limit of significance. Results The PPAR-γ was significantly decreased in both HT and PVD (p<0.001). The serum concentrations of OPG were higher in HT (p<0.001) and increased in diabetic ones (p<0.05). CG genotype of PPAR-γ Pro12Ala was more frequent in HT patients (p<0.001). In the HT patients, increased OPG and decreased PPAR-γ were found in CC (p<0.001). In the PVD patients, PPAR-γ levels decreased in carrying with CC (p<0.05). Conclusions It may be significant that increased OPG, as a marker of endothelial dysfunction, is found in HT. Moreover, decreased PPAR-γ in those who have to carry CC may be protective in both HT and PVD.

Objectives Plaque-induced gingivitis is one of the most widely distributed periodontal disorder during childhood. The control of the pathogenic potential of the plaque is associated with oral hygiene status at individual, group, and population levels. We hypothesize that regular application of complex oral hygiene-prophylaxis could beneficially modulate salivary amino acids profile in children with different stage of plaque-induced gingivitis. Therefore, we aimed to study the salivary amino acids’ profile in relation to certain clinical indicators and environmental variables for plaque-induced gingivitis in children. Methods Fifty children (29 girls, 21 boys; mean age 8.18 ± 2.32 years) without anamnestic data for common diseases, no medication, and no data for allergy were selected. Plaque and gingival indexes were determined for assessment oral hygiene and plaque accumulation. Unstimulated whole saliva was collected, centrifuged and supernatants stored at −80 °C. Amino acid analysis was performed by liquid chromatography using analytical grade AccQ·Tag-Ultra-derivatization kit. Results Gingivitis was indicated in most of the examined children over 6 years. More than half (63.6 %) of them revealed moderate stage of the disease and a tendency to satisfactory good oral hygiene and degree of gingival inflammation. Salivary glycine, proline, arginine, serine, lysine, aspartate, glutamate, threonine, methionine, and isoleucine were higher in gingivitis children, while cysteine, tyrosine and phenylalanine decrease. In gingivitis children without regular oral hygiene-prophylaxis, some structural amino acids like glycine and proline were increased, while amino acids with protective antioxidant potential like cysteine were diminished. Conclusions Plaque-induced gingivitis is associated with increased salivary levels of certain amino acids. These may serve as distinguishing markers among children with gingivitis.

Objectives This study aims to investigate how the fibrosis index based on four factors (FIB-4) is altered in preeclampsia and whether the FIB-4 score differs with respect to the severity of preeclampsia and the presence of fetal and maternal adverse outcomes. Methods One hundred and forty-two patients with mild preeclampsia (34.6 %), one hundred and ninety patients who have preeclampsia with severe features (46.2 %), and 79 healthy pregnant controls (19.2 %) were included in the study. Fetal adverse outcomes occurred in 40.1 %, and maternal adverse outcomes only appeared as neurological symptoms in 20.5 % of the preeclampsia patients. Results Healthy controls had significantly lower FIB-4 scores than women with mild preeclampsia, and women with mild preeclampsia had significantly lower FIB-4 scores than women who had preeclampsia with severe features (respectively 0.58±0.29 vs. 0.68±0.44 vs. 1.93±4.92, p=0.003). The FIB-4 scores of preeclampsia patients with neurologic symptoms and preeclampsia patients with fetal adverse events were found to be similar to preeclampsia patients who did not have these problems. In ROC curve analysis, FIB-4 scores ≥0.758 indicated the presence of neurologic symptoms in preeclampsia patients, with a sensitivity of 0.66 and a specificity of 0.66 (p=0.004). Conclusions To the best of our knowledge, this is the first study to establish the FIB-4 scores of preeclampsia patients and determine if FIB-4 scores change with respect to maternal and fetal adverse outcomes. Our findings suggest that FIB-4 might be used to predict pregnancies destined to be complicated with preeclampsia and preeclampsia patients who are more likely to experience maternal and fetal adverse outcomes.

Objectives Hepcidin plays an important role in regulating iron metabolism. Elevated levels of hepcidin in renal failure contribute to the development of anemia. We aimed to evaluate the association between hepcidin and inflammation in hemodialysis patients and how dialysis affects hepcidin levels. Methods Hepcidin clearance with hemodialysis was investigated by measuring hepcidin concentrations by enzyme-linked immunosorbent assay (ELISA) method before and after hemodialysis of 40 patients in a single dialysis session. Hemogram parameters and ferritin, iron, total iron binding capacity (TIBC), and C-reactive protein (CRP) were measured and evaluated their relations with predialysis hepcidin levels. Results Hepcidin levels decreased significantly with dialysis treatment (p=0.009). Median hepcidin concentration before dialysis was measured as 330 ng/mL (83–459) and post-dialysis median hepcidin concentration was 250 ng/mL (94–384). There was a significant correlation between predialysis hepcidin levels and ferritin (r=0.858, p<0.001), TIBC (r=−0.451, p=0.004), and MCV (r=0.384, p=0.016). It was found that increases in ferritin levels in time were positively correlated with hepcidin before dialysis. Conclusions We think that understanding the removal of the hepcidin by dialysis, which causes a decrease in the amount of iron available in the anemia, is important in managing future therapy.

Objectives Although MOTS-C has been reported to have a role in diabetes mellitus, no human studies have evaluated the serum level of MOTS-C in GDM. It was aimed to investigate serum levels of MOTS-C in patients with gestational diabetes mellitus (GDM). Methods Comparisons were made of 44 pregnant patients diagnosed with GDM and a control group of 44 healthy pregnant women in respect of serum MOTS-C, insulin, and glucose levels, and serum lipid profile. Results A significantly higher level of fasting serum glucose and significantly lower serum levels of MOTS-C and high density lipoprotein were determined in the GDM group compared to the control group (p<0.05 for all). A cut-off value of 173.5 ng/mL for serum MOTS-C level had sensitivity of 81.8 % and specificity of 61.4 % for GDM diagnosis (p<0.001). A significant correlation was determined between the serum MOTS-C and serum glucose levels (r=−0.239, p=0.025). Conclusions For the first time in literature, the results of this study showed that patients with GDM had a decreased serum level of MOTS-C and that increasing serum MOTS-C levels were associated with a decrease in serum glucose levels, thereby supporting the view that mitochondrial dysfunction plays a role in GDM pathogenesis. Therefore, MOTS-C could be a promising diagnostic biomarker for GDM cases.

Objectives To determine the ideal timing of labor for term pregnancies using oxidative stress determiners. Methods A total of 150 low-risk pregnant women were divided into three groups according to gestational week periods: early term, full term, and late-term. Groups were matched in terms of age and body mass index. Thiol/disulfide homeostasis parameters were obtained from maternal blood samples at the beginning of the active phase and from cord blood immediately after delivery. Results Maternal and fetal native and total thiol, which indicates antioxidant status, were found to be significantly higher in full terms (maternal 397.5 ± 78.8, 435.9 ± 84.5, and fetal 472.5 ± 78.4, 513.7 ± 89.2 respectively; for all parameters p<0.05). The maternal oxidant balance was not significantly different between the groups; 1st-minute APGAR scores were significantly correlated with maternal antioxidant levels (p=0.042; r=0.199). Conclusions The highest level of antioxidants in thiol/disulfide homeostasis in full-term pregnancies can be explained by the mechanism that increases the antioxidant level of the mother, which works best in the period between 39 and 41 weeks of pregnancy.

Objectives Glioblastoma is a fast-growing and aggressive brain tumor. Despite the current treatment methods, such as chemical and surgical operations, the prognosis is still poor. Therefore, combined therapeutic strategies are proposed to maximize therapeutic efficacy and reduce toxicity. Thymoquinone has been shown to have neuroprotective effects in addition to its anti-cancer effects on different types of cancer. 5-Fluorouracil, on the other hand, is a cytotoxic chemotherapy agent used to treat cancer. As a synergistic combinational approach, this study aimed to examine the antiproliferative effects and production of reactive oxygen species in a glioblastoma cell line. Methods We have tested thymoquinone and 5-fluorouracil alone and in their combination to observe cellular growth with MTT assay. The combinational effects of the agents were determined by the CompuSYN software program. Cell proliferation was assayed with crystal violet assay. Reactive oxygen species production was analyzed by 2′,7′-dichlorodihydrofluorescein diacetate in glioblastoma cells. Results Thymoquinone and 5-fluorouracil inhibited cell growth of glioblastoma cells with half maximal inhibitory concentrations (IC 50 ) of 45.93 and 14.02 µM for 48 h, respectively. At synergistic combinational concentrations, the crystal violet assay demonstrated that there is a positive correlation between combination index values and cell proliferation. Also, an increment in the production of reactive oxygen species was observed upon combinational treatments. Conclusions Our results indicate that the combinational strategy of these two agents reduced cell viability and proliferation in glioblastoma cells and showed strong synergistic anticancer efficiency.

Objectives Type 1 diabetes mellitus is a metabolic disease characterized by dysbiosis. Modulation of the gut microbiota by oligosaccharides and aerobic training are proposed mechanisms that ameliorate the disease through affecting host-microbiota interactions. Methods Seventy-two male Wistar rats were randomly divided into 8 groups – 5 with streptozotocin-induced diabetes and 3 healthy controls. The effect of two oligosaccharides – xylo- and galactooligosaccharides, and of aerobic training on the blood glucose concentration, growth and diversity of the gut microbiota was evaluated in the current study. Results The galactooligosaccharides positively affected the glycemic status of the experimental animals as the diabetic and healthy rats had lower blood glucose concentration after 6 weeks of treatment (diabetic rats: week 4 vs. week 8, p=0.047; healthy rats: week 2,4,6,10 vs. week 8, p=0.001, p=0.000, p=0.025 and p=0.001, respectively). A positive effect of the galactooligosaccharides on body weight was observed when administered to diabetic rats in comparison to the diabetic control (p=0.020). Similar results were observed for the aerobically trained diabetic rats (p=0.004). The identification of bacterial species showed preserved microbiota diversity and indicated Bifidobacterium indicum , Lactobacillus feritoshensis and E. coli as the most abundant species among the analyzed genera. Conclusions Prebiotic treatment beneficially affected the hyperglycemia and growth of type 1 diabetic rats. The most significant effect of the aerobic training was the improvement of the morphological parameters. Oligosaccharide administration and exercise did not affect the diversity of the bacterial species.

Objectives We aimed to explore the association of obesity and hypertension and further their association with AdipoQ gene polymorphism in North Indian postmenopausal women of Punjab. Methods A total of 523 postmenopausal women (PMW) were enrolled (PMW with CVD=265 and PMW without CVD=258). Anthropometric measurements such as weight, height, hip circumference (HC), waist circumference (WC), waist hip ratio (WHR) and body mass index (BMI) for all the subjects were recorded in accordance to WHO 2000 protocol. For hypertension, guidelines of the Joint National Committee (JNC-VII) of high blood pressure were considered. Genotyping of AdipoQ (G>T) gene polymorphism was done by RFLP-PCR analysis. Results The comparison of the frequency distribution of alleles and genotypes of AdipoQ (+276G>T) gene polymorphism showed a significant distribution (p<0.05) among subjects with and without CVD. The risk for CVD was high (∼9 fold) among carriers of +276T allele towards CVD predisposition. Obese women with CVD under the recessive model conferred ∼8 fold high risk (p=0.001) and +276T allele and TT genotype of non-obese women with CVD for BMI <25 also conferred ∼9 fold high risk. Hypertension also acted as a strong risk factor related to CVD (p=0.0001). Under the recessive model, hypertensive PMW with CVD conferred 7–9 fold higher risk however, normotensive women with CVD also conferred 9∼10-fold risk towards CVD predisposition. Conclusions The T allele carriers of AdipoQ gene is strongly associated with risk factors such as obesity and hypertension pertaining to cardiovascular disease. Early detection of these risk factors may serve as a CVD preventative intervention.

Objectives A comprehensive understanding of the role of PON enzymatic activities may play an important role in the etiology and prevention of many cancers. PON1 is known as a potent antioxidant that scavenges free radicals in the human body. The enzymatic activities of paraoxonase (PON1) and mitogen-activated protein kinase (MAPK) signalling pathways in colorectal cancer are being investigated to determine whether they hold promise for novel diagnostic or therapeutic applications in colorectal cancer. Methods HT-29 colon cancer cell lines and CCD-18Co colon cell lines were used. B-Raf, p-B-Raf, ERK, and p-ERK proteins involved in MAPK signalling pathways and serum levels of PON1 were detected and analyzed by the Western blotting method. Results The levels and activity of PON1 enzyme were significantly decreased in HT-29 cells compared to CCD-18Co cells (p=0.0173 and p=0.0281, respectively). The levels of p-B-Raf and p-ERK, which activates the MAPK signalling pathway, were significantly increased in HT-29 cells (p=0.0037 and p=0.0074, respectively). Conclusions A positive correlation was found between PON1 level and PON1 enzyme activity (p=0.008). A negative correlation was found between PON1 level and p-B-Raf/p-ERK protein levels, which play a role in the MAPK signalling pathway (p=0.016 and p=0.036, respectively). A negative correlation was also found between PON1 enzyme activity and p-B-Raf/p-ERK protein levels (p=0.060 and p=0.037, respectively). It is suggested that the increase of proteins involved in the MAPK signaling pathway in cancer cells is caused by the decreasing serum levels of PON1 and enzymatic activity of PON1.

Objectives Resveratrol (RSV) is a stilbenoid compound that shows anticancer activity in many cancer cells. Exosomes might affect carcinogenesis and the development of colorectal cancer by affecting communication between tumor cells in the tumor microenvironment via their cargo content miRNA. The aim of this study is to determine the effect of RSV on the expression of Dicer, Ago2, eIf2α, CD-9, CD-63, and exosomal miRNA levels in COLO320 and COLO741 colon cancer cell lines. Methods The MTT method was used for cell growth and cytotoxicity in both COLO320 and COLO741 cell lines. Dicer, Ago2, eIF2α, CD-9, and CD-63 antibodies were used for the immunocytochemical evaluation. Total miRNA analysis was performed using a miRCURY Exosome Isolation Kit. Results As a result of immunocytochemical staining, increased CD-63 immunoreactivity was observed in RSV-treated COLO320 cells vs. RSV-treated COLO-741 cells. Dicer immunoreactivity increased after the RSV treatment in COLO320 cells. Higher eIF2α immunoreactivity was observed in RSV-treated COLO741 cells compared to both COLO741 control cells and RSV-treated COLO320 cells. Non-significant decreases were observed in miRNA concentration in RSV-treated COLO320 and COLO741 cells compared to control group cells. Conclusions RSV could increase miRNA biogenesis in COLO320 cancer cells and decrease it in COLO741 cancer cells.

Objectives Fibromyalgia syndrome (FMS) is a chronic pain syndrome characterized by widespread body pain over a long period, the cause of which is not yet clearly known. FMS patients usually have high pain sensitivity. We aimed to investigate whether rs4148855 and rs2288646 polymorphisms of acid-sensing ion channel 3 ( ASIC3 ), one of the factors contributing to pain, cause a predisposition to FMS in the Turkish population. Methods ASIC3 gene rs4148855 and rs2288646 polymorphisms in DNA samples obtained from blood samples of 175 patients with FMS and 176 healthy individuals were analyzed by real-time polymerase chain reaction (RT-PCR) using a hydrolysis probe. Statistical data were obtained by chi-square (χ 2 ) test and logistic regression analysis. Results No significant association was found between ASIC3 gene rs4148855 and rs2288646 polymorphisms and the Turkish population’s FMS group and control group (p>0.05). Conclusions As a result, no significant association was found between the genotype and allele distributions of ASIC3 polymorphism (rs4148855 and rs2288646) in patients with FMS compared to controls in the Turkish population. Further studies are needed to elucidate the relationship between ion channels and FMS to elucidate the mechanisms of FMS.

Objectives Adenomyosis is a benign uterine disease that occurs with the invasion of the endometrial gland and stoma into the myometrium. The etiology and molecular pathology of adenomyosis are not yet fully understood. Tissue samples of patients diagnosed with adenomyosis and healthy endometrial tissues were investigated for the lipogenesis and cholesterol synthesis pathways. It was aimed to determine the difference between adenomyosis and healthy endometrial tissues in terms of lipid metabolism and to investigate the mechanism of adenomyosis in this context. Methods Formalin-fixed paraffin-embedded archival tissues were used in the current retrospective study. A total of 76 patient samples and 3 groups were used. Group 1: adenomyotic tissue (n=28), Group 2: eutopic endometrial tissue (n=30), and Control Group (n=18). In these groups, Sterol regulatory element binding protein 1 ( SREBP1 ) molecule, fatty acid synthase ( FASN ), acetyl-CoA carboxylase ( ACACA ), ATP-citrate lyase ( ACLY ), HMG-CoA reductase ( HMGCR ), and HMG-CoA synthase ( HMGCS ) markers were evaluated by using RT-PCR method. Results Statistically significant differences (p<0.05) were found between the groups regarding expression levels of HMGCR , HMGCS , ACLY , ACACA , and SREBP1 . HMGCR , HMGCS , ACLY , and SREBP1 gene expression levels between Group 1 and Group 2 and HMGCS , ACACA , ACLY , and SREBP1 gene expression levels between Group 1 and Control Group were determined as statistically different. A significant difference was detected only in HMGCR gene expression levels between Group 2 and the Control Group. Conclusions These results show that genes involved in lipid metabolism may be associated with the molecular pathogenesis of adenomyosis.

Objectives To assess the frequency of multiple thrombophilia-associated mutations and polymorphisms in a selected population of high-risk pregnancies. Methods Thrombophilia screening was performed for 1,500 pregnant women with prior pregnancy complications or thrombotic events. Nine thrombophilia-associated mutations or polymorphisms were screened: factor V Leiden, factor V H1299R, prothrombin G20210A, MTHFR C677T, MTHFR A1298C, factor XIII V34L, PAI-1 4G/5G polymorphisms, EPCR G4600A, EPCR C4678G. Results Out of the 1,500 patients, 1,291 fulfilled the criteria for data interpretation. All patients had low-risk thrombophilia-associated genetic variants. Only 1.24 % of cases presented high-risk abnormalities (homozygous factor V Leiden/prothrombin G20210A, or both mutations in heterozygous form). Heterozygous factor V Leiden occurred in 10.38 % of cases, while only 5.81 % carried heterozygous prothrombin G20210A mutation. The frequency of prothrombin G20210A mutation was higher (10.37 %) in the subgroup associating factor V Leiden, than in the subgroup lacking it (5.36 %). Low-risk genetic variants occurred with a higher frequency: 23.78 % factor V H1299R, 57.32 % MTHFR C677T, 55.54 % MTHFR A1298C, 44.07 % factor XIII V34L, 73.20 % PAI-1 4G/5G polymorphisms, 69.64 % EPCR G4600A, and 69.63 % EPCR C4678G. Conclusions All patients had at least one prothrombotic genetic mutation or variant. Our data highlight the need for thrombophilia screening, including low-risk genetic variants, in a high-risk population of pregnant women with a history of pregnancy complications or thrombotic events.