This was a cross-sectional study conducted on 40 Egyptian female patients with relapsing-remitting multiple sclerosis diagnosed according to the McDonald criteria 2010 with regular menstrual cycles, and all were in remission. They were recruited from the MS unit of the Neurology Department in the Kasr Al-Ainy Hospital, Cairo University, from January 2015 to November 2016. The age of patients ranged from 18 to 38 years with mean age of 30.22 ± 4.68.
Excluded patients presented with progressive multiple sclerosis, history of steroid intake, or immunosuppressive treatment in the past 2 months prior to involvement in the study or history of immunomodulatory treatment and conditions known to be associated with changed cytokine level as post-traumatic head injury, post-stroke epilepsy, Alzheimer’s disease, Parkinson’s disease, cerebral palsy, impaired cognition, malignant brain tumors, hypothyroidism, liver or renal disease, immune-mediated diseases, or inflammatory diseases and patients on histamine receptor H2 antagonists or anti-inflammatory drugs. We also excluded patients on hormonal contraceptive or with history of hormone replacement therapy and with polycystic ovaries, pituitary adenoma, or hypophysectomy.
Twenty healthy female (age-matched) subjects with regular menstrual cycles, with ages ranging from 25 to 38 years and mean age of 30.67 ± 3.71, served as a control group.
Informed consents were taken from all subjects, and the study was approved by the local ethics committee.
All subjects were subjected to the following: full history taking including the history of hormonal contraception or replacement therapy and history of previous treatments with immunosuppressive or immunomodulatory medications to verify exclusion criteria; complete general and neurological examinations; and evaluation of disability using the Expanded Disability Status Scale (EDSS).
Laboratory investigations were done to detect serum levels of anti-inflammatory cytokine (interleukin 10 (IL-10) and interleukin 4 (IL-4)), pro-inflammatory cytokine (tumor necrosis factor alpha (TNF-α)), and hormonal profile (estrogen and testosterone).
Peripheral blood samples were withdrawn from all subjects in fasting condition and without anticoagulant. Samples were withdrawn during the follicular phase (day 3 to day 9 of the menstrual cycle) and measured using a sandwich enzyme-linked immunosorbent assay (ELISA).
Regarding interleukin-10, interleukin 4, and tumor necrosis factor alpha assay (supplied by eBioscience, San Diego, USA), samples were coagulated at room temperature, then centrifuged at approximately 1000×g for 10 min and stored at − 20 °C. An anti-human (IL-10, IL-4, or TNF alpha) coating antibody was adsorbed onto microwells, human (IL-10, IL-4, or TNF alpha) in the sample or standard binds to antibodies adsorbed to the microwell. Following incubation, unbound biotin-conjugated anti-human (IL-10, IL-4, or TNF alpha) antibody was removed during the wash step. Streptavidin-horseradish peroxidase (HRP) was added and binds to the biotin-conjugated anti-human (IL-10, IL-4, or TNF alpha) antibody. Following incubation, unbound streptavidin-HRP was removed during the wash step and substrate solution reactive with HRP is added to the wells. A colored product was formed in proportion to the amount of human (IL-10, IL-4, or TNF alpha) present in the sample or standard. The reaction is terminated by the addition of acid, and absorbance is measured at 450 nm. A standard curve was prepared from seven human (IL-10, IL-4, or TNF alpha) standard dilutions and human (IL-10, IL-4, or TNF alpha) sample concentration determined.
Regarding estrogen and testosterone assay (supplied by ALPCO Diagnostics, New Hampshire, USA and Diagnostic Biochem Canada Inc., London, Ontario, Canada, respectively), samples were left to coagulate at room temperature then centrifuged and stored at − 10 °C. These assays were based on a standard sandwich enzyme-linked immunosorbent assay technology. Fifty microliters of each calibrator, control, and specimen sample was pipetted into correspondingly labeled wells in duplicate. One hundred microliters of the conjugate working solution was pipetted into each well and incubated on a plate shaker (200 rpm) for 1 h at room temperature. The wells were washed three times with 300 μl of diluted wash buffer per well, and the plate was tapped firmly against absorbent paper to ensure it is dry. One hundred and fifty microliters of tetramethylbenzidine substrate was pipetted into each well at timed intervals and incubated on a plate shaker for 10–15 min at room temperature, then, 50 μl of stopping solution was pipetted into each well at timed intervals, and finally, the plate on the microwell plate reader was read at 450 nm within 20 min after the addition of stopping solution.
The expected normal value for testosterone in females is 0.2–1 nanograms/milliliter (ng/ml)
The expected normal value for estrogen during the follicular phase is 15–169 picograms/milliliter (pg/ml)
The data will be summarized using mean and standard deviation (SD) for the quantitative data and frequency distribution for the qualitative data.
For quantitative data, a comparison between two groups was carried out using a nonparametric t test.
The Pearson correlation test is used to estimate the correlation between given random variables. The correlation coefficient indicates the strength and direction of a linear relationship between random variables. Multivariate linear regression analysis was done.
p values less than 0.05 were considered statistically significant. All statistical calculations were done using the computer program SPSS (Statistical Package for the Social Sciences; SPSS Inc., Chicago, IL, USA) version 17 for Microsoft Windows.