The present case-control study recruited 50 mothers aged less than 35 years at the time of conception of a child with DS that was confirmed by cytogenetic analysis and a control group of 50 healthy mothers with matched age who had no history of a child affected by DS or any other genetic disease. All were selected from the Genetic Clinic, Department of Human Genetics, Medical Research Institute, Egypt, between January 2015 and May 2016. Residency in urban and rural areas for studied cases is determined according to the data of Central Agency for Public Mobilization and Statistics (CAPMAS, 2015)  and The Egypt Demographic Health Survey (EDHS) .
Mothers aged more than 35 years or had a history of exposure to irradiation, chemotherapy, smoking, or oral contraceptive pills were excluded from the study.
The study was approved by the ethics committee, and a written informed consent was obtained from 50 DS mothers and 50 control mothers before participation in the study, according to the Declaration of Helsinki.
Detailed history and pedigree analysis were assessed. A comprehensive questionnaire was completed and included full data regarding the age, consanguinity, education, occupation, and income of the mother and her husband, place of living, reproductive history of abortion or stillbirth, and the presence of a previous child with DS or any other chromosome anomaly in the family or among other relatives.
Updated Fahmy scale  was used to assess the socio-economic status of the included mothers. High socioeconomic status was indicated at 70% or more of the total score while low socioeconomic status is less than 40%.
Peripheral venous blood samples were taken from all mothers in the study. Under complete aseptic conditions and with sterile disposable syringes, 4 ml of blood were collected in heparinized vacutainer tubes for peripheral blood lymphocyte culture. Two tubes for each mother were prepared, and blood lymphocyte cultures were set up within 24 h of sampling according to the conventional method .
Whole blood cultures were established by placing 0.5 ml of blood with an RPMI medium supplemented with 20% fetal calf serum and 1.5% phytohaemagglutinin.
Cultures were incubated at 37 °C for 48 h in one tube for the detection of chromosome breaks and 72 h in the other tube for the diagnosis of any numerical or structural chromosome anomalies.
The harvesting process was started by the addition of colchicine (0.1 mg/ml) for the last 2 h of incubation to arrest the cells at metaphases. Cells were incubated with hypotonic KCI (0.075 M) at 37 °C for 10 min and fixed in four changes of cold 3:1 methanol/acetic acid.
Slides were prepared by the heat drying technique and were stained with aqueous Giemsa solution for the tubes incubated for 48 h. Trypsin pretreatment was used before Giemsa staining for tubes incubated for 72 h.
For chromosome numerical and structural abnormalities, 30 metaphases were counted; 5 metaphases were analyzed and 2 were photographed for each sample prepared after the 72 h culture procedure.
For the samples prepared after the 48 h incubation, a total of 100 metaphase cells per sample were scored at random and analyzed for chromosome and chromatid aberrations according to the International System for Human Cytogenetic Nomenclature (ISCN) 2016 .
Achromatic areas less than a chromatid width (gaps) were excluded in the calculation of chromosomal breakage frequencies. Achromatic areas taking a chromatid width were scored as a single chromatid break, and chromatid breaks involving both chromatid widths were considered as chromosomal breaks and were scored as two breaks each .
Data obtained were analyzed using IBM SPSS software package version 20.0 (Armonk, NY: IBM Corp.), where a statistically significant difference between DS mothers and control mothers is set when P < 0.05. Qualitative data were described using number and percent and the association between studied factors (consanguinity, residency, social status, and chromosome breaks), and the risk of having a child with DS was calculated by chi-square test, odds ratio (OR), and 95% confidence interval (CI). Further analysis of the results was calculated in a 4 × 4 table to assess the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPP), and accuracy .