A cross-sectional study included 115 Egyptian women, with ages ranged between 25 and 60 years; mean age 41.62 + 10.70 years. They were recruited and randomly chosen, from all employees and workers; of all categories; of the “National Research Centre”, Egypt. A written informed consent was obtained from all participants after being informed about the purpose of the study. This research paper was derived from a cross-sectional survey of a project funded by National Research Centre (NRC) Egypt, 2019–2022 entitled ‘‘Gut Microbiota in Obesity and Metabolic syndrome among obese women: Interactions of the Microbiome, Epigenetic, Nutrition and Probiotic Intervention.” (12th Research Plan of the NRC), which was approved from “Ethics Committee of NRC” (Registration Number is19/236) . All methods were performed in accordance with the relevant guidelines and regulations.
For each participated woman, blood pressure, anthropometric measurements, 24 hour dietary recall, laboratory investigations and microbiota analysis were done.
Blood pressure was measured using the standardized mercury sphygmomanometer with a suitable cuff size. It was measured on the left arm while the participated women were sitting relaxed for 5 minutes. Two readings were obtained and the average was recorded. Systolic blood pressure (SBP); determined by the onset of the “tapping” korotkoff sounds (K1), while the fifth korotkoff sound (K5), or the disappearance of korotkoff sounds, as the definition of diastolic blood pressure (DBP) were recorded.
- Anthropometric measurements
Body weight, height, neck, hip and waist circumferences were measured, following the recommendations of the “International Biological Program” . Body weight (Wt) was determined to the nearest 0.01 kg using a Seca Scale Balance, with the woman wearing minimal clothes and with no shoes. Body height (Ht) was measured to the nearest 0.1 cm using a Holtain portable anthropometer. Circumferences was measured using non-stretchable plastic tape; approximated to the nearest 0.1 cm. Neck Circumference was measured at a point mid-way between the collarbone and chin in the middle of the neck while Standing or sitting with a straight back. Waist circumference (WC) was measured at the midpoint between the lower curvature of the last fixed rib and the superior curvature of the iliac crest, with the woman in an upright standing position and their arms alongside the body, feet together, and abdomen relaxed. Hip circumference was measured at the maximum extension of the buttocks measuring the largest diameter above the symphisis pubis overlapping the apex of the buttocks. Waist/ hip ratio [WC/ Hip C in cm] and Body mass index (BMI) [BMI: weight (in kilograms) divided by height (in meters squared)] were calculated. The participated women were all chosen as obese; as their BMI ≥30 Kg/m2.The participant women were classified according to their BMI into 2 groups: 30 women with normal BMI (BMI=18-<25 Kg/m2) and 82 obese women (BMI ≥30Kg/m2).
Information from each participant about her usual pattern of food intake was obtained. Data was collected by means of dietary interview consisting of 24 h recall that repeated for 3 days, and a food frequency questionnaire. Analysis of food items was done using World Food Dietary Assessment System, (WFDAS), USA, University of California .
- Blood sampling and laboratory investigations
In the morning, venous blood samples (after 12-hour fasting) were drawn from the participated women, using venipuncture. Biochemical parameters were performed on fasting sera that were stored at -70 C° until used for assessment of liver enzymes: Aspartate amino-transferase (AST) and Alanine amino-transferase (ALT), leptin, Short Chain Fatty Acids (SCFA), C-reactive protein (CRP), fasting blood glucose (FBG), insulin, and lipid profile. All were done in the laboratory of “Medical Excellence Research Center MERC” which is a part of “NRC”, Egypt.
Serum concentrations of AST and ALT were determined using the automated clinical chemistry analyzer Olympus AU 400 analyzer (https://www.mybiosource.com).
The assay of human Leptin in serum was performed by ELIZA method, using kits of BioLegend, Inc., (San Diego – USA), according to the method of Considineet al. .
Human Short Chain Fatty Acids (SCFA) were assessed in serum using Enzyme Linked Immuno-sorbent Assay (ELISA) kits; Catalog Number: MBS7269061 according to the method described by den Bestenet al .
Fasting blood glucose (FBG) level was measured using the automated clinical chemistry analyzer Olympus AU 400 analyzer. Serum insulin was assessed using Enzyme Immunoassay Test Kit Catalog No. E29-072(Immunospec Corporation).
The assay of the serum CRP was performed by Enzyme Linked Immuno-sorbent Assay (ELISA) kits, Cat No.: RAP002 ,(https://www.mybiosource.com.)
Estimation of lipid profile: Serum levels of total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL-C) were measured by standardized enzymatic procedures; using kits supplied by Roche Diagnostics (Mannheim, Germany) on the Olympus AU 400 automated clinical chemistry analyzer. Low density lipoprotein cholesterol (LDL-C) was calculated according to formula of Friedewald et al.  as follows: LDL – C = Total cholesterol – Triglycerides/5 + HDL-C.
Clinically, a patient is considered to have MetS when three or more of the following five conditions exist, which are (i) waist circumference ≥88 cm in women, (ii) blood pressure ≥135/85 mmHg, (iii) triglycerides ≥150 mg/dl, (iv) HDL-C <50 mg/dl in women, and (v) fasting glucose ≥100 mg/ dl .
The proportion of Lactobacillus and Bifidobacteria; and Firmicutes/Bacteroidetes ratio strains were assessed in the stool of all participants by using the real time PCR (Polymerase Chain Reaction). Specimen collection and preparation: Stool was collected by defecation in a plain sterilized container allowed to be frozen. Specimen Storage and Preparation: stool was frozen on at -20 °. The primers and probes were used to detect Bifidobacterium spp. and Lactobacillus spp; and Firmicutes spp. and Bacteroidetes spp., where based on 16S rRNA gene sequences retrieved from the National Center for Biotechnology Information databases by means of the Entrez program .
Reagents provided by kits: DNA extraction Kit. Assay procedure: DNA extraction: The QIAamp DNA Stool Minikit (Qiagen) was used to extract DNA from one gram of fresh or frozen stool sample according to the manufacturer's instructions. Bacterial quantification by real-time PCR was done.
Data were analyzed using the Statistical Package for Social Sciences (SPSS/Windows Version 18, SPSS Inc., Chicago, IL, USA). Normality of data was tested using the Kolmogorov-Smirnov test. The data were normally distributed. So, the parametric tests were used.
The participated women were classified into: 33 with normal BMI (18- <25 Kg/m2) and 82 obese with BMI >30 Kg/m2. They obese women were classified according to the presence of MetS markers into two groups: 59 obese without MetS (have no or less than 2 markers of MetS), and 23 obese with MetS (have 3 or more markers of MetS).
The parametric data were expressed as mean + SE. The various parametric variables of the two groups were analyzed and compared using independent t test. Pearson's correlation test was used to assess the relations between Firmicutes/ Bacteroid ratio and the clinical and metabolic parameters, and between gut microbiota and daily intake of total fat, carbohydrate and fiber among the three groups.P < 0.05 was regarded as statistically significant for all tests.