Design of the Study
We conducted a transversal study at the National Institute of Perinatology (Neuroscience Department, Mexico City) together with the Department of Gynecology and Obstetrics (GO) at the General Hospital of Mexico (HGM, Dr. Eduardo Liceaga, Mexico City) from October 2014 to December 2016. Approval from the Institution Ethical Committee was obtained before the beginning of the study (HGM, D1/14/112/04/072, 2014–2016). Written informed consent was obtained from all women recruited before the study began. Pregnant women were admitted at the Prenatal Control Outpatient Unit of the GO Department during the second and third trimester of gestation. Clinical, sociodemographic, obstetric, and psychiatric characteristics of the recruited participants were recorded into a database. At entry, participants in the study required first, second, and third-trimester lab tests (blood count, biochemical testing, urinalysis, thyroid function), 2D fetal ultrasound, and Doppler Monitoring, including lab test for sex hormones, cortisol, and DHEA-S (data not shown). All patients were inhabitants of Mexico City and surrounding areas.
Patients recruited to the study attended the Prenatal Control Outpatient Unit. Women between 16- and 30-years-old, exhibiting a pregnancy between 20 and 39 gwks, were invited to participate in the study. In their first visit, a complete clinical evaluation was carried out, including anthropometric evaluation. Participants were asked to complete the self-reported questionnaires used to measure anxiety and depressive symptoms; in the same interview, an evaluation of socio-demographic variables was done, including marital status, education level, and working status. Patients exhibiting a cutoff score > 15 in the Hamilton Anxiety rating scale (HARS) were considered as having severe anxiety. Patients with a cutoff score as < 5 were included in the control group. We excluded patients who were receiving psychotropic medication, patients with illicit substance use, previous psychiatric diagnosis, obstetric pathologies (diabetes, hypertension, preeclampsia), infections, and medical diseases (past and present neurological, metabolic, cardiovascular, degenerative, and rheumatic disorders). Upon completion of the questionnaires, participants displaying high-rating scores for moderate to severe anxiety and/or depressive symptoms were remitted to a psychiatric outpatient service for mood-disorder management. After psychiatry clinical interview and completion of the questionnaires, patients were remitted to the GO Department for blood extraction and analysis of their serum cytokine profile.
We assessed a total of 298 pregnant women: patients, exhibiting low to high levels of depressive symptoms and comorbid anxiety (n = 141), pregnant women exhibiting anxiety symptoms without depression (n = 100) mostly during the 3rd trimester of pregnancy (28–40 gwks), and healthy pregnant women displaying no affective disorders (n = 57). All participants were screened for depressive symptoms and anxiety, serum cytokines and sociodemographic parameters. Some of the participants were excluded due to incomplete questionnaires, absence or incomplete laboratory tests, or inconsistencies in the evaluation of both depressive and anxiety symptoms. Our present work included three groups of participants a) patients exhibiting severe anxiety without depression (SA, n = 72); b) patients exhibiting severe anxiety and comorbid severe depression (SA + SD, n = 67); and c) healthy pregnant women as controls (CTRL, n = 40).
Criteria and evaluation instruments
Criteria considered for patient recruitment included the presence of severe anxiety according to the 14 item-Hamilton Anxiety Rating Scale (HARS) , which assesses the severity of anxiety symptoms with a cutoff score > 15 for severe anxiety. Depressive symptoms were evaluated using the 17-Item Hamilton Depression Rating Scale (HDRS), with a cutoff score > 19 for major depressive disorder [35, 36]; and evaluated by psychiatric clinical interview following the Diagnostic and Statistical Manual of Mental Disorders (DSM-5; American Psychiatric Association, 2015). HDRS and HARS were applied to healthy women to confirm the absence of depressive and anxiety symptoms.
Both instruments have been validated in the local language [36, 37] and have been demonstrated to be reliable, specific, and sensitive . Rating scores obtained from each instrument were checked by the Psychiatric Department, and final evaluations were recorded in a clinical database. Patients exhibiting an HDRS score > 19 and HARS score > 17 were referred to the psychiatry department for further evaluation and treatment.
Blood sampling was carried out during daylight under aseptic conditions, and 5.0 mL of venous blood was collected into sterile 13 × 100/Vacutainer BD Hemogard Tubes, containing Clot Activator/Polymer Gel for serum separation (Becton & Dickinson 367,977, USA). Tubes were allowed to clot for 1 h at 4 °C before serum separation. Fresh serum was collected by centrifuging samples at 1600 × G for 10 min, aliquoted into 1.5-mL Eppendorf tubes. and stored at − 70 °C until further use.
Quantification of serum cytokine profile
Serum cytokines (IFN-γ, TNF-α, IL-6, IL-2, IL-5, IL-13, IL-4, IL-10, IL-9, IL-17A, IL-17F, IL-21 and IL-22) were determined by an immunoassay based on multiplex bead array using the LEGENDplex™ Human Th Cytokine Panel (Cat. 740,001, BioLegend, USA) following the manufacturer’s protocol. Briefly, serum samples (50 μL) were incubated with specific IL-antibody coated beads in a mix buffer at room temperature (RT) for 1.5 h. An antigen-antibody reaction was carried out at RT for 1.5 h in the presence of specific fluorochrome per cytokine. After washes, samples were analyzed in a FACS Aria III flow cytometer (BD, USA). The concentration of analytes was calculated using the LEGENDplexTM Data Analysis Software v 7.0 (Biolegend, CA, USA). The limit of detection (LD) values detected for Th1 cytokines (IL-6, TNF-α, IL-2, IFN-γ) were 12.63 pg/ml, 78.13 pg/ml, and 9.88 pg/ml, respectively.
The LD values of Th17-related (IL-17A, IL-21, and IL-22) cytokines were 78.13 pg/ml, whereas the LD value of IL-17F was 14.1 pg/ml respectively; the LD values detected for Th2 cytokines (IL-4, IL-5, IL-13, IL-10, IL-9) were 78.13 pg/ml, 9.25 pg/ml, 11.68 pg/ml, 88.97 pg/ml and 6.77 pg/ml respectively.
Cytokine concentrations were presented as mean ± SEM. Demographic parameters were expressed as mean ± SD. Bivariate analyses, as well as parametric and non-parametric t-tests, were conducted to assess the associations among immune mediators, demographic parameters, and psychological distress scores. The non-parametric t-test analysis with Welch’s correction was used to detect significant differences between the demographic measures and groups, as well as Th-related cytokines among the tested groups. Partial correlation analysis was used to detect significant differences between serum cytokines and demographic parameters, after controlling specific parameters in the studied groups. A post hoc Tukey test analysis was performed to estimate the differences between cytokines and demographic parameters among groups. Also, a general linear model of analysis of variance was constructed using the group as a dependent variable, interleukin concentrations as independent variables, and HDRS/HARS scores and GWKS were introduced as covariables. Statistical analyses were performed using GraphPad Prism 7 (GraphPad Softwares Inc. USA) and SPSS software v.24.0 (Armonk, NY: IBM Corp). For all the statistical analysis, the p-value < 0.05 was considered significant.