Thyroid hormones act as mitogenic and pro survival factors in rat ovarian follicles.

PURPOSE: Thyroid disorders are clinically associated with impaired fertility in women, and these abnormalities can be improved by restoring the euthyroid state. The exact mechanisms of thyroid effect on female fertility are not well known; however, it is conceivable that thyroid hormones (THs) might act on ovarian physiology via receptors in granulosa cells. This work is aimed at evaluating the effects of THs on non-tumoral granulosa cells and follicles. METHODS: Freshly isolated rat ovarian follicles and granulosa cells were exposed to T3 or T4 (THs). Cell growth and viability were evaluated by cell counting and the MTT assay, respectively, follicle growth was evaluated by volume measurements. Apoptosis was evaluated by the TUNEL assay and active Caspase 3 staining. rGROV cells were exposed to T3, and apoptosis was induced by serum deprivation. Bcl2, Bcl-2-associated X protein (BAX), Akt and pAkt expression were evaluated by western blot. RESULTS: T3 induced a 40% increase in follicle volume (after 7 days). This increase was presumably due to the observed decrease (33%) in the apoptotic rate of the granulosa cell population. Both T3 and T4 caused a dose-dependent increase in rat granulosa cell number and viability. In addition, THs decreased the cell apoptotic rate in a dose-dependent manner. In both conditions, T3 appeared to be more efficient. In rGROV cells, 100 nM T3 induced cell growth and, in the absence of growth factors, reduced cell apoptosis by 40%, downregulating Caspase 3 and BAX. This effect was associated with an increase in pAkt levels. The involvement of the PI3 K pathway was confirmed by the ability of the PI3 K specific inhibitor (LY-294,002) to abolish T3 pro-survival action. CONCLUSIONS: THs influence cell survival of ovarian granulosa cells. This effect likely contributes to the TH-induced follicle volume increase.

Hyperhomocysteinemia in acute iatrogenic hypothyroidism: the relevance of thyroid autoimmunity.

PURPOSE: Hyperhomocysteinemia is a known cardiovascular risk factor and a key player in the inflammatory activation of autoimmune diseases. Hashimoto's thyroiditis (HT) is the leading cause of hypothyroidism which, in itself, has been associated with a significant raise of homocysteine (Hcy) levels and increased cardiovascular risk. Our aim was to assess the impact of HT on Hcy levels in patients with acute hypothyroidism. METHODS: We prospectively enrolled 121 patients (mean age: 46 years, F/M = 102/19) with acute post-surgical hypothyroidism. Based on the presence of anti-thyroid antibodies and the histological description of an inflammatory infiltrate, 26 and 95 patients were classified as HT and non-HT, respectively. Several parameters including thyroid-stimulating hormone (TSH), levels of serum free T3 and free T4, weight, glucose levels, total cholesterol, creatinine, vitamin B12, ferritin and erythrocyte sedimentation rate were obtained from all patients and correlated with Hcy levels. RESULTS: Median Hcy level in the whole cohort was 16.8 µmol/L (normal values: < 12 µmol/l). Among all parameters analysed, only Hcy levels were significantly different between HT and non-HT patients (median Hcy = 19.7 vs 16.2 µmol/L, respectively; p = 0.018, Mann-Whitney U test). Analysis of covariance showed the presence of HT to be the strongest predictor of Hcy levels (coefficient = 0.25534, p = 0.001). Serum TSH was not significantly associated with Hcy levels (p = 0.943). CONCLUSION: In patients with iatrogenic hypothyroidism, those with HT have significantly higher Hcy levels than those without HT. The increase of Hcy levels appears to be mainly determined by the HT-related immune-inflammatory condition.

Early detection of biochemically occult autonomous thyroid nodules.

OBJECTIVE: Autonomously functioning thyroid areas may be associated with subclinical or overt hyperthyroidism, but may exist even in the presence of normal TSH. This study was aimed at comparing the rate of autonomously functioning areas and their cardiac sequelae in patients with nodular goitre studied with the usual and a novel approach. DESIGN AND METHODS: In total 490 adult outpatients with thyroid nodular goitre, living in a mild iodine-deficient area, were selected in our referral centre for thyroid diseases from 2009 to 2014 on the basis of a suspicion of thyroid functional autonomy. They were divided in three groups according to a non-conventional approach (excessive response to thyroxine treatment: group 1) or conventional approach (low/normal TSH with clinical suspicion or low TSH: groups 2 and 3). All patients of the study with the suspicion of thyroid functional autonomy underwent thyroid scan with radioactive iodine (I131) uptake (RAIU). RESULTS: The percentage of confirmed thyroid functional autonomy was 319/490, being significantly higher in group 3 than in groups 1 and 2 (81.5 vs 64.7 vs 52.6%; chi-square P < 0.0001). However, the diagnosis with non-conventional approach was made at a significant earlier age (P < 0.0001). Cardiac arrhythmias as well as atrial fibrillation were similarly detected by conventional and non-conventional approaches (chi-square test: P = 0.2537; P = 0.8425). CONCLUSIONS: The hyper-responsiveness to thyroxine treatment should induce the suspicion of thyroid functional autonomy at an early stage, allowing to detect autonomous functioning areas in apparently euthyroid patients.

Individually-tailored thyroxine requirement in the same patients before and after thyroidectomy: a longitudinal study.

OBJECTIVE: Thyroxine (T4) requirement after total thyroidectomy for differentiated thyroid carcinoma (DTC) is a debated issue. As most of the studies in the area have been retrospective and/or performed with heterogeneous therapeutic approaches, we designed our study to determine T4 requirement in the same patients and treatment settings, before and after total thyroidectomy. DESIGN, PATIENTS AND METHODS: This was a longitudinal study including 23 goitrous patients treated with T4 in an individually tailored fashion. All patients exhibited a stable TSH (median TSH = 0.28 mU/l) at a stable T4 dose for at least 1 year before surgery (median T4 dose = 1.50 µg/kg per day). The patients underwent total thyroidectomy based on cancer suspicion or compressive symptoms. Eventually diagnosed as having DTC (pT1b-pT2N0) and following surgical and radiometabolic treatment, they were treated with the same pre-surgical doses of T4. RESULTS: Three months after surgery,using the same pre-surgical dose, median TSH increased up to 5.38 mU/l (P<0.0001) and so the T4 dose had to be increased (median T4 dose = 1.95 µg/kg per day; +30%; P < 0.0001). Once divided by patients' age, we observed that, after thyroidectomy and maintaining the same pre-surgical dose, serum TSH significantly increased both in younger and in older patients (median TSH = 4.57 and 6.11 mU/l respectively). Serum TSH was restored to the pre-surgical level by increasing the dose up to 1.95 and 1.77 µg/kg per day (+25 and +21%) respectively. CONCLUSIONS: Following the same treatment regimen, a thyroidectomized patient requires one-third higher therapeutic T4 dose than before surgery. Despite this increase, the dose of T4 needed in our patients remains significantly lower than that previously described in athyreotic patients.

Increased interleukin-4-positive lymphocytes in patients with Hashimoto's thyroiditis and concurrent non-endocrine autoimmune disorders.

A prevalent T helper type 1 (Th1) subset of lymphocytes has been described in Hashimoto's thyroiditis (HT), but whether a similar polarization may characterize HT when associated with non-endocrine autoimmune disorders (NEAD) is not known. The aim of the present study was to analyse the intracellular Th1 and Th2 distinctive cytokines in patients with isolated HT or associated with non-endocrine autoimmune disorders. Intracellular cytokine expression was assessed in peripheral blood lymphocytes (PBL) of 68 out-patients (females = 55; males = 13; median age = 6 years) with HT : 33 had isolated HT and 35 had a concurrent NEAD. The percentage of interferon (IFN)-γ and interleukin (IL)-2 Th1- and IL-4 Th2-positive cells was measured by flow cytometric analysis. We found an increased percentage of IL-2-positive cells in all patients, without differences between patients with isolated HT or associated with NEAD. IFN-γ(+) cells were also increased in both groups, but the median percentage of those with isolated HT was lower than in patients with HT+NEAD (19·0 versus 29·9%; P = 0·0082). An increased number of IL-4-positive cells was observed in three of 33 (9·1%) patients with isolated HT and in 25 of 35 patients with NEAD [71%; P < 0·0001; relative risk (RR) = 3·18]. The median values of IL-4(+) cells (HT = 5·0% versus HT + NEAD = 16·8%) confirmed this large difference (P < 0·0001). A clear-cut increase of IL-4(+) lymphocytes characterizes patients with autoimmune thyroiditis who have associated non-endocrine autoimmune disorders. These findings may represent an initial tool to detect patients with autoimmune thyroiditis in which additional non-endocrine autoimmune disorders may be awaited.

The presence of non-segmental vitiligo modifies intracellular cytokine subsets in patients with chronic lymphocytic thyroiditis.

Chronic lymphocytic thyroiditis and vitiligo often occur in association and seem to be characterized by a prevalent Th1-driven autoimmune process. The aim of this study is to analyze selected intracellular Τh1 and Th2 cytokines in patients with Hashimoto?s thyroiditis when associated with non-segmental vitiligo. We analyzed intracellular interleukin-2, interferon-gamma (Τh1) and interleukin-4 (Th2), in peripheral blood lymphocytes of 23 patients with isolated Hashimoto?s thyroiditis (group A) and of 11 patients with Hashimoto?s thyroiditis associated with non-segmental vitiligo (group B). Peripheral blood lymphocytes were stimulated and incubated with specific monoclonal antibodies. Intracellular cytokines were assayed by flow cytometric analysis. Interleukin-2 and interferon-gamma positive cells were increased in almost all patients but the median values were similar in patients with isolated Hashimoto?s thyroiditis and in those with concurrent vitiligo. In contrast, the number of patients with increased interleukin-4 positive cells was higher in patients with thyroiditis and vitiligo (9/11) than in those with isolated thyroiditis (2/23; p<0.0001). The median values of IL-4 positive cells in the two groups confirmed this difference (A: 5.8 percent, vs B: 20.6 percent; p=0.0011). Increased interleukin-4 positive lymphocytes characterize Hashimoto?s thyroiditis when associated with non-segmental vitiligo, suggesting a modified balance from highly prevalent Th1 to mixed Th1/Th2 subset.