This is a hospital-based prospective study carried out at Neuropsychiatry Department, Tanta University Hospitals throughout a 12-month period, started from June 2018. The study protocol was approved by the ethical committee in Tanta University, Egypt, under the code number (3226/04/18) on April 2018. Participation was voluntary and all contributors received detailed information about the aims of this research work and an informed written consent was obtained prior to the commencement of the study.
Forty female patients with RRMS aged from 20 to 45 years old fulfilling McDonald criteria of MS 2017 , without a history of receiving disease-modifying therapy in addition to 10 age-matched healthy females were included in the study who served as a control group.
Patients with other autoimmune disorders, primary or secondary progressive MS, breastfeeding patients, and patients receiving medications that affect the hormonal status (antipsychotics, antiemetic, or antidepressants) were excluded from the study.
All patients were subjected to the following: history taking (age, disease duration, number of relapse and full past, and current medical history) general, neurological examination, and assessment by Expanded Disability Status Scale (EDSS) . A relapse was defined as an increase of two points in one or more Kurtzke Functional Systems, or an increase of one point in two or more KFS (except changes in bowel or bladder function or cognition), in the absence of fever, lasting for at least 24 h, preceded by at least 30 days of clinical stability or improvement. Remission considered on return to base line score .
Magnetic resonance image (MRI) of the brain and spinal cord were carried out for all patients using Magnetom Sempra 1.5 Tesla, Siemens, Germany, to detect site, size, and number of MS plaques and to exclude other structural lesions. The MRI examination included axial T1-weighted sequences as well as T2-weighted dual fast spin echo sequence and a FLAIR sequence. Axial and coronal T1-weighted sequences were acquired 6 min after intravenous injection of 0.1 mmol/kg gadolinium chelate (Omniscan(R)).
Serum levels of PRL and TNFα were measured for all patients (during the relapse phase before steroid pulse therapy administration, then during the remission phase) and control subjects included in this study. Serum PRL was estimated mainly during the follicular phase of the menstrual cycle [15, 16]. This was performed at Unit of Clinical Chemistry, Clinical Pathology Department, Tanta University. Peripheral blood samples were collected without anticoagulant. All of the samples were immediately centrifuged at 3000 rpm for 15 min, and the sera aliquots were stored in − 20 °C for TNFα and PRL estimation. Serum PRL level was measured by immunofluorescence techniques on AIA 1800, Tosoh Bioscience, Tokyo, Japan. TNFα level in the serum was estimated by ELISA double antibody sandwich technique using BT LAB, Shanghai Crystal Day Biotech CO., LTD. Shanghai, China, Cat.no: E0082Hu. The assay was performed following the manufacturer’s instructions and the developed color was measured at 450 nm on a Tecan Spectra II microplate reader (Switzerland). The results were calculated from the standard curve. The sensitivity was 1.52 pg/ml, the intra-assay CV was < 10%, and the inter-assay CV was < 12%.
The collected data were organized, tabulated, and statistically analyzed by SPSS version 19, 2011 created by IBM, Illinois, Chicago, USA. Parametric numerical data were expressed as mean and standard deviation and compared among the groups using ANOVA test and Tukey post hoc test. Non-parametric data were expressed as median and range and compared among the studied groups using the Kruskal-Wallis test. The performance characteristics of serum PRL and TNFα were assessed by AUROC analysis and their optimal cutoff was determined via Youden index. Serum PRL and TNFα were correlated with the studied parameter using Pearson correlation. For all statistical tests done, the threshold of significance was fixed at 5% level (P value), i.e., when < 0.05 significant results are indicated.