There is no well documented correlation between the histological extent of adenomyosis and the severity of dysmenorrhea, the amount of blood loss or infertility in women [41]. Since rodents do not menstruate, the mouse model does not yield itself to the study of adenomyosis-related menorrhagia or dysmenorrhea. However, the induction of adenomyosis and its progression in mice is associated with increased sensitivity to noxious thermal stimuli [24, 38]. This is most likely due to central sensitization. It is possible that, similar to endometriosis [42], adenomyosis may be associated with features of neuropathic pain. As such, patients with adenomyosis may exhibit an array of sensory phenotypes including sensory gain [43]. In endometriosis such sensory gains [44] may be related to molecular changes in the central nervous system particularly in the dorsal root ganglia [45]. Since the severity of adenomyosis-associated pain in the mouse cannot be measured directly, sensitivity to noxious thermal stimuli has been used as a surrogate marker. For example, the hotplate test is performed by placing the mouse on a metal plate that is gradually heated to preset levels. The response latency is measured as the time from placement on the plate to the point when the mouse licks its hind paws. The hotplate test measures response thresholds to high intensity stimulus and is thus an “acute pain test” [46], but the response is mediated by spinal-brain stem-spinal reflexes [47]. Therefore, the test may reflect changes at the supraspinal level resulting from adenomyosis-induced pain, which supports its use as a surrogate measure of pain in adenomyosis. It also has the advantage of being simple to perform and of low cost. The tail-flick test is performed by directing a focused high-intensity light to generate a thermal stimulus 1–2 cm distal to the end of the tail. The time from start of stimulation to tail withdrawal determines tail-flick latency. The vaginal distention test was introduced as a non-thermal pain behavior measure [48]. The test is performed using a small latex balloon (10 mm long and 1.5 mm wide when not inflated) tied to a thin catheter. Immediately prior to the testing session, the un-inflated balloon is lubricated by K-Y jelly and inserted into the mid-vaginal canal. The balloon is gradually inflated in situ using a computer-controlled pump. The mouse is trained to respond to the noxious stimulus by interrupting the electric circuit which stops the pump. The test requires sophisticated equipments that are not widely available. Similar to dysmenorrhea, vaginal distention induces a form of visceral pain. Still, the relevance of these tests to women presenting with pelvic pain or dysmenorrhea is debatable. The mechanism of visceral pain is less well understood than somatic pain [49].

Caution should be exercised when using vaginal distention or response to noxious thermal stimuli tests to evaluate adenomyosis-associated pain behavior in mice. As in the case in neuropathic pain, sensory gain may well be a feature that is only observed in a subset of patients [43, 50]. Vaginal distention test, hotplate test, and other tests that measure the response of mice to noxious stimuli actually evaluate the severity of evoked pain, i.e. the hypersensitivity of withdrawal reflexes to sensory stimuli. This contrasts with the primary efficacy measure(s) used in clinical trials of adenomyosis where the focus is on dysmenorrhea or chronic pelvic pain which are forms of spontaneous pain. These types of pain have never been evaluated in animal models of adenomyosis or endometriosis, presumably due to the enormous difficulties this entails.

Outside of the field of adenomyosis, various attempts have been made to measure spontaneous pain in rodents. For example, ultrasound vocalization at 22–28 Hz was reported to be associated with chronic pain in rats [51], but the value of this test has been disputed [52, 53]. Two recent studies described the use of facial grimace scale which is used to assess pain in infants, for evaluating pain severity in mice and rats [54, 55]. This model seems promising, but it remains to be determined if it can be applied to the assessment of adenomyosis-related pain in mouse, assuming that adenomyosis in mouse does indeed induce pain.

Abnormal uterine contractility may contribute to adenomyosis-associated dysmenorrhea and pain. Intensified and somewhat deranged uterine contractility have recently been documented and found to correlate with reduced response latency to noxious thermal stimulus in mice with induced adenomyosis [38]. Uterine contractility could also be a functional measurement in the sense that it may be used in efficacy evaluation. There is a need to develop neural biomarkers and objective correlates of adenomyosis-associated pain as these may provide more reproducible measures.