The thyroid gland is an important endocrinological organ, and its secretory products, thyroxine (T4) and triiodothyronine (T3), are significantly involved in the regulation of many metabolic processes. The main thyroid hormone is T4, of which approximately 99% in the blood are bound to proteins and less than 1% is free (fT4) [1]. Only the unbound hormones can pass into cells, bind to the DNA and influence enzyme expression [2].

Measurement of thyroid hormones in blood serum is the first diagnostic tool used in cases of suspected thyroid disease [3]. Hypothyroidism is one of the most common endocrinologic diseases in the dog [3]. As in most species, measurements of T4 concentrations generate more reliable information than T3 concentrations [4,5]. Another indicator of thyroid function is the blood serum concentration of thyroid-stimulating hormone (TSH, also known as thyrotropin). TSH is produced by the pituitary gland, which in turn is stimulated by thyrotropin-releasing hormone (TRH) from the hypothalamus. Furthermore, to assess a patient with thyroid hormone concentrations outside of the physiological range, the clinical picture must be taken into account, as the thyroid can likewise be impaired by other diseases of the organism, also known as non-thyroidal illness (NTI). Medication or other factors, e.g. breed or age also influence thyroid function [[6], [7], [8], [9], [10]]. To avoid these effects, it can be helpful to determine only the free T4 concentrations, as they are less affected by an alteration of protein binding, which is often the cause for changes in thyroid hormone concentrations [5].

Hence, it has been recommended to begin an assessment of canine thyroid function with the measurement of baseline total (t) T4 concentrations (sum of all protein-bound and free T4) [3,11]. If tT4 is low and there is no evidence of NTI, practitioners should either measure TSH concentrations or perform a TSH-stimulation test [5,12,13]. As previously described, fT4 can aid the assessment if an NTI is thought to be likely but difficult to diagnose [3,11,13].

As thyroid hormones are critically important for almost all aspects of fetal development, their concentrations during pregnancy can be cause for concern [14,15]. In humans, it has been demonstrated that thyroid-binding proteins (TBG) substantially increase in concentrations during pregnancy [16]. Moreover, tT3 and tT4 concentrations are considerably higher during pregnancy than one year postpartum, while TSH remains relatively equal and fT4 is lower during pregnancy [17]. tT4 levels are similarly high throughout pregnancy, whereas tT3 increases with each trimester and fT4 decreases slightly [17]. These known differences have led to trimester-specific reference intervals, to ascertain that the thyroid is functioning physiologically and to avoid misdiagnoses in pregnant women [[18], [19], [20]].

In dogs, thyroid hormone concentrations during normal pregnancy have been the subject of few studies. The first study to show an effect of reproductive state on concentrations of T3 and T4 was published in 1984 [21]. Albeit based on a small number of animals, the study showed that tT4 concentrations were elevated in pregnant and non-pregnant diestrus bitches compared with bitches in anestrus, proestrus, female dogs in lactation and male dogs. In addition, tT3 was higher in diestrus bitches than in all other groups. No bitches in estrus were sampled in this study. Some recent studies also found that tT4 concentrations were significantly higher during estrus and diestrus than during proestrus and anestrus [14,22,23] with group sizes of 130, 33 and 17 bitches respectively, although one study reported tT4 concentrations within the normal range in a sample of 12 bitches [24]. A significant positive correlation between progesterone (P4) and tT4 has also been described during pregnancy or diestrus [22,23]. TSH was found to be higher during pregnancy [23].

Whether an alteration of thyroid hormone concentrations has direct effects on reproduction in dogs is controversial in the available literature. While it has often been stated that hypothyroidism can lead to weak estrual signs, short interestrous intervals and even infertility [[25], [26], [27], [28], [29], [30]], few studies are available confirming these statements. A case report from 1989 described these symptoms in one hypothyroid bitch, and their resolution after levothyroxine treatment [31]. Another case report from 1997 suggested a connection between hypothyroidism and galactorrhea in a nulliparous bitch [32]. One study published in 2009 evaluated 204 dogs of five different breeds and found that only 11.7% of dogs with disorders of fertility were borderline hypothyroid, while three “true hypothyroid” dogs showed no signs of reproductive disease [33]. The authors also found that thyroid hormone concentrations did not differ between the normal and the subfertile dogs. In two consecutive studies by Panciera et al., hypothyroidism was experimentally induced, and the reproductive effects examined [34,35]. While fertility was unaffected in short-term hypothyroid bitches and only slightly decreased in prolonged hypothyroid subjects, effects on the puppies were more pronounced with higher periparturient mortality, lower birth weight and lower vitality at birth. In conclusion, the described studies outline that a relationship between thyroid hormones and reproductive status seems to exist, but is far from being understood.

Over the past years the authors have observed several cases of suspicious thyroid hormone concentrations in pregnant bitches. This led to the consideration that a study on thyroid hormones during normal canine pregnancy will offer valuable insight and guide practitioners through diagnosing their patients. On the basis of well acknowledged publications [[36], [37], [38]], it was decided to include around 120 bitches in this study, to allow for statistical power while still aiming for an attainable goal. The parameters focused on were TSH, tT4, fT4 and Progesterone (P4).