Animal experiment:

Rats[2]Sprague-Dawley female rats (N=22) are used. Non-pregnant rats are divided into four groups: 1) control, 2) hypothyroidism, 3) hypothyroidism treated with low doses of L-Thyroxine (20 μg/kg/day) and 4) with high doses of L-Thyroxine (100 μg/kg/day). Control rats (group 1) are fed with standard diet, while the intervention rats are fed with iodine-free diet for 12 weeks to induce hypothyroidism (groups 2-4) which is continued for four more weeks to allow screening of hypothyroid status and L-Thyroxine-treatment. Food and water (iodine-free diet) are available ad libitum. The hypothyroid group treated with low (group 3) or high doses of L-Thyroxine (group 4) are injected intraperitoneally every 24 h with respectively 20 μg/kg/day and 100 μg/kg/day. Blood samples are collected for thyroid function screening at week 12 and 16 following the initiation of either the control or iodine-free diet. Hysterectomy is performed under general anesthesia (isoflurane 2%) at the end of the treatment and the two uterine horns are placed in physiological Krebs' solution until isometric tension measurements within no more than 1 h.

L-Thyroxine sodium salt pentahydrate (Levothyroxine; T4) is a synthetic hormone in the treatment of hypothyroidism. DIO enzymes convert biologically active thyroid hormone (Triiodothyronine,T3) from L-Thyroxine (T4). Thyroid Hormone Receptor

Deiodinases (DIOs), which catalyse the conversion of thyroxine (pro-hormone) to the active thyroid hormone, are associated with thyroid stimulating hormone (TSH) levels. DIO1 and DIO2 catalyze activation of thyroid hormone secretion in contrast to DIO3 playing role inactivation of the secretion. Activities of DIO1 and DIO2 play pivotal role in the negative feedback regulation of pituitary TSH secretion[1]. L-Thyroxine (T4) and Triiodothyronine (T3) hormones are known to modulate the expression of ionic channels, pumps and regulatory contractile proteins. Moreover, thyroid hormones have been shown to influence calcium homeostasis and flux responsible for excitation and contractility, with L-Thyroxine and Triiodothyronine modulating its pharmacological control and secretion. In rats fed 12 weeks with the iodine-free diet, a significant decrease in the levels of both Triiodothyronine and L-Thyroxine is observed when compared to the control group fed with standard diet (p<0.001). In the group treated with low doses of L-Thyroxine, an increase in L-Thyroxine levels is observed (p=0.02) while Triiodothyronine levels remain virtually similar to the control group (p=0.19). Rats treated with high doses of L-Thyroxine display a significant increase in both Triiodothyronine and L-Thyroxine circulating concentrations compared to the non-treated hypothyroid group (p<0.001 and p=0.004, respectively) and a significant increase in L-Thyroxine levels when compared to the control values (p=0.03)[2].

[1]. Arici M, et al. Association between genetic polymorphism and levothyroxine bioavailability in hypothyroid patients. Endocr J. 2018 Jan 11. [2]. Corriveau S, et al. Levothyroxine treatment generates an abnormal uterine contractility patterns in an in vitro animalmodel. J Clin Transl Endocrinol. 2015 Sep 9;2(4):144-149.