Participants

TPO autoantibody titers in healthy prenatal participants (n = 414) in a study of postpartum thyroiditis were measured, and the subjects were characterized thereafter as TPO positive or TPO negative. The study was approved by the university institutional review board, and all participants gave informed consent. Recruitment took place in 2 large university obstetrics practices by trained recruiters from 2006-2009. Pregnant women were screened for eligibility by a research nurse and given information about the study at a prenatal visit. Participants then gave formal consent and were enrolled in a subsequent visit after having the opportunity to review all materials and ask questions. The number of participants who declined to participate was 44% of all who were eligible. No demographics are available for those who did not wish to participate. The reasons for declining varied, but by far the most common reasons were inability or unwillingness to be enrolled potentially in a 6-month postpartum phase for evaluation of postpartum thyroiditis or unwillingness to have venipunctures. Participants were between 16 and 25 gestational weeks of pregnancy (to ensure that TPO-positive women would be in the second trimester, when the risk of spontaneous abortions is decreased from first trimester). Exclusion criteria for the study included immune diseases, immune altering medications, in vitro fertilization, human immunodeficiency virus disease, illegal drug abuse, extreme thinness, and age <18 years or >45 years.

Measures

Participants completed a demographic questionnaire and the Profile of Mood States (POMS) at the time of blood draw. The POMS is a 65-item dysphoric moods instrument, with 7 subscales: depression-dejection, tension-anxiety, confusion-bewilderment, fatigue-inertia, anger-hostility, vigor-activity, and a total mood disturbance score. Respondents indicated their responses over the past week to items using 4-point rating scales with verbal anchors. The POMS-depression/dejection score (POMS-D) has been shown to be highly correlated with the Beck’s Depression Index–II (r = 0.81). The POMS-D is also reported to correlate with the modified Hopkins Symptom Distress Scales (range, 0.43–0.86), Taylor Manifest Anxiety Scale (range, 0.51–0.80), and the Minnesota Multiphasic Personality Index Depression scale (.65); (POMS Manual). The POMS-tension/anxiety score correlates with the Spielberger State Trait Anxiety scale score at 0.71. The POMS was chosen because it is an excellent screening instrument for a panel of dysphoric moods. The original aims of the study were not to evaluate clinical depression but rather to examine general negative moods at pregnancy and through the postpartum period.

Research nurses and participants were blind to TPO and T gondii status at the time of enrollment and screening. TPO antibody status was measured on all participants at the time of data collection. Aliquots of frozen plasma were analyzed for antibodies to T gondii with the use of previously published methods. A set of aliquots was shipped to the Medical University of Innsbruck, Austria (D.F.), for analysis of tryptophan, kynurenine, and neopterin. Cytokines were measured in batches in frozen plasma samples.

Procedures

Each participant completed a demographic questionnaire (the POMS) and provided a 15-mL heparinized blood sample that had been collected by venipuncture at her prenatal provider’s visit between 16 and 25 weeks of pregnancy. The blood was brought to the laboratory within 2 hours in a cold biohazard container and then centrifuged at 1500 g for 25 minutes at 4°C. The plasmas were aliquotted into Eppendorf tubes and stored at –80°C until TPO antibodies and cytokines were measured in batches. The TPO antibodies and cytokines were measured within a few weeks of collection. TPO antibodies were measured in plasma by an enzyme-linked immunosorbent assay (ALPCO, Salem, NH); a value of ≥20 IU/mL was considered TPO positive. Cytokines (IFN-γ, interleukin [IL]-10, TNF-α) were measured in undiluted plasma samples by Luminex technology (Luminex Corporation, Austin, TX), with the use of multiplex kits from Millipore (St. Louis, MO). Stored plasma samples (–80°C) from 2006-2009 were sent to the laboratory (R.H.Y.) for quantitation of IgG antibodies to whole Toxoplasma organisms (tachyzoites). Titers were measured with a commercially available assay (VIR-ELISA; Viro-Immun Labor-Diagnostika, Oberursel, Germany). For this assay, a result was defined as positive or negative based on comparison with the reactivity of specific antibody standards that were assayed along with the plasma samples. A ratio of the test optical density to the standard ≥1.1 is considered positive (10 IU). Serotyping of IgG antibodies to type-specific Toxoplasma peptides was performed in the following manner: peptides were diluted to 8 μg/mL in 0.1 mol/L carbonate buffer, pH 8.5; 50 μL of each peptide solution was loaded per well of a polystyrene microtiter plate and incubated overnight at 4°C. After unbound peptide was removed, wells were washed and blocked with 300 μL of Starting Block blocking buffer (Pierce Protein Research Products, Rockford, IL) for 1-2 minutes at room temperature. Sera were tested by the addition of 50 μL of diluted human plasma (1:100) to each well and incubation for 3 hours at 37°C. The microtiter plates were washed 4 times with phosphate-buffered saline solution that contained 0.1% Tween 20 solution and then reacted with horseradish peroxidase-coupled goat antibody against human IgG (Southern Biotech, Birmingham, AL) for 2 hours at 37°C. After being washed in phosphate-buffered saline solution with 0.1% Tween 20 solution, color was developed by the addition of 50 μL of H ₂ O ₂ with 2,2′-azinobis(3-ethylbenzthiazoline-sulfonic acid) reagent (Kirkegaard and Perry Laboratories, Inc, Gaithersburg, MD) for 25 minutes. Absorbance was measured with a microplate (Vmax; Molecular Devices Corporation, Sunnyvale, CA) colorimeter with a 405-nm filter.

Plasma samples were analyzed for tryptophan (micromoles per liter) and kynurenine (micromoles per liter) by high-performance liquid chromatography. The kynurenine-to-tryptophan ratio was calculated (expressed as micromoles of kynurenine per millimoles of tryptophan). Neopterin was measured by enzyme-linked immunosorbent assay (BRAHMS, Hennigsdorf, Germany) according to the manufacturer’s instructions, with a detection limit of 2 nmol/L.

Statistical analysis

Data were examined for normality and log transformed if necessary.

Pearson product-moment correlations were performed between T gondii IgG titers and POMS scores. The T gondii –positive group (n = 44) was compared with the negative group (n = 370) by t tests. The T gondii –positive group was evaluated separately, and correlational analyses were performed between depression and T gondii titers in this group. The effects of race, age, number of pregnancies, and smoking on this relationship were controlled in linear regression; we did not have complete data on income or marital status for the sample. Correlations were also performed on the kynurenine-to-tryptophan ratio and neopterin in the total sample and in the Toxoplasmosis -positive subgroup.

χ ² analysis was done to examine the relationship between Toxoplasmosis ( T gondii antibody status >10 IUs) and TPO status.

t tests and analyses of variance were used to compare the T gondii –positive and –negative groups and serotypes on tryptophan, kynurenine, kynurenine-to-tryptophan ratio, neopterin, and mood states.

Cytokine data (IL-10, TNF-α, and IFN-γ) were available on a subset of the population ( T gondii –positive, n = 38; T gondii –negative, n = 104). The levels of these cytokines in the 2 groups were compared by t tests.