Reduced vagal tone in women with adenomyosis

Abstract Adenomyosis is an estrogen-dependent disease featuring chronic inflammation. This study was undertaken to investigate whether vagal tone is reduced in patients with adenomyosis compared with healthy women. We recruited 75 patients with adenomyosis, as diagnosed by a combination of ultrasound and gynecological examination, and 75 healthy women without adenomyosis, endometriosis, or other uterine disorders per ultrasound examination. All recruited subjects received an electrocardiogram evaluation, and their heart rate variability was assessed. In addition, lesional stiffness for patients with adenomyosis and myometrial stiffness for healthy controls were measured by ultrasound elastography. Severity of dysmenorrhea and the amount of menstrual blood loss were also evaluated. Patients with adenomyosis exhibited statistically significant sympatho-vagal imbalance, featuring domination of the sympathetic branch of the autonomic nervous system over the parasympathetic branch, as evidenced by reduced vagal tone and increased sympathetic activity. In addition, lesional stiffness, a proxy for the extent of lesional fibrosis, was found to be negatively associated with vagal tone. Patients with adenomyosis have reduced vagal tone. In addition, reduced vagal tone is likely attributable to increased lesional stiffness, a proxy for the extent of lesional fibrosis, which correlated with the severity of dysmenorrhea and the amount of menstrual blood loss. This raises the prospect of employing vagus nerve stimulation as a possible therapeutics approach. Future human studies are needed to determine whether vagus nerve stimulation can have any therapeutic effects. Lay summary Adenomyosis is a condition in which the inner lining of the uterus is found in the muscular wall of the uterus (called the myometrium). It is a common gynecological disease affecting mostly women of reproductive age. We found that patients with adenomyosis exhibited significant imbalance between the ‘flight-or-fight’ system and the ‘rest-and-digest’ system (sympatho-vagal imbalance), featuring reduced vagal tone (dampened calm-down system) and increased sympathetic activity (more anxiety and restlessness). In addition, we found that the stiffness of adenomyotic lesions, which can be viewed as a proxy for the ‘age’ of the lesions, was negatively associated with vagal tone. This reduced vagal tone may suggest that perhaps some vagal stimulating procedures, which are safe and non-invasive, can be used to boost vagal tone to achieve therapeutic purposes.

Adenomyosis is a complex gynecological condition defined as the presence of endometrial tissues in the myometrium and is responsible for dysmenorrhea, heavy menstrual bleeding (HMB), and subfertility (Vannuccini et al. 2017, Gordts et al. 2018). Traditionally viewed as an enigmatic disease, its pathogenesis and pathophysiology are poorly understood, due, in no small part, to the fact that it is under-researched (Guo 2020). Consequently, its management is a challenge, and the armamentarium for its treatment is quite limited, with surgery and hormonal drugs being the treatment of choice and hysterectomy being the ultimate solution (Vannuccini et al. 2018, Szubert et al. 2021, Rathinam et al. 2022). Epidemiological studies have shown that a history of depression is the only factor independently associated with adenomyosis (Taran et al. 2010), suggesting increased sympathetic tone in women with adenomyosis. This is due to the fact that adenomyotic lesions overexpress adrenergic receptor β2 (ADRB2) (Xu et al. 2021), which can be activated by catecholamines resulting from activation of stress/depression-induced hypothalamic-pituitary-adrenal and sympatho-adreno-medullary axes. In mice with induced endometriosis, ADRB2 can be activated by surgery (Long et al. 2016a ), chronic stress (Guo et al. 2017, Long et al. 2016b ), or early-life adverse events such as maternal separation (Long et al. 2020), which, in turn, can accelerate lesional progression. As wounds undergoing repeated tissue injury and repair (Guo 2018), adenomyotic lesions exhibit increased vascularization (Harmsen et al. 2022), inflammation (Carrarelli et al. 2017, Bourdon et al. 2021), and innervation (Berkley et al. 2004, Tokushige et al. 2006), which are known to be involved in all phases of tissue repair (Shaw & Martin 2009, Laverdet et al. 2015). In particular, adenomyosis is characterized by increased lesional infiltration of immune cells and increased production of proinflammatory cytokines and chemokines (Bourdon et al. 2021, Guo 2022), featuring activation of NF-κB (Nie et al. 2009, Li et al. 2013) and an imbalance of Th17-treg cells (Gui et al. 2014). While anti-inflammation may seem to be a promising therapeutic approach as in endometriosis (D’Hooghe et al. 2006), it should be noted that physiological endometrial repair also requires adequate inflammation (King et al. 2010, Critchley & Maybin 2011). Thus, we face a situation that is uncannily similar to the precariously narrow corridor of controlling estrogen levels to the desired ‘treatment window’ (Barbieri 1992). In the last two decades, the body-brain axis has emerged as a principal conductor of organismal physiology and pathophysiology. In particular, the concept of the cholinergic anti-inflammatory pathway (CAIP) has become more popular (Tracey 2002). The CAIP concept posits that a complex and intricate crosstalk between the nervous and immune systems exists via a complex set of cytokines, neurotransmitters, and hormones, which serve as counter-regulatory mechanisms capable of dampening inflammation and restoration of homeostasis (Steinman 2004, Sternberg 2006). A recent study reports that pro-inflammatory and anti-inflammatory cytokines communicate with distinct populations of vagal neurons to inform the body-brain circuit of an emerging inflammatory response, and the brain tightly modulates the course of the peripheral immune response (Jin et al. 2024). Within the CAIP, the vagus nerve (VN) plays a pivotal role since its activation, followed by activation of the α7 nicotinic acetylcholine receptor (α7nAChR), potently reduces inflammation in peripheral tissues (He et al. 2015, Chavan et al. 2017). With the pioneering discovery by Tracey and collaborators showing the significance of electrical VN stimulation (VNS) as a therapeutic strategy to attenuate inflammation (Borovikova et al. 2000) and the demonstration of the practical utility of transcutaneous auricular VNS (taVNS) in treating seizures (Ventureyra 2000), many clinical trials have demonstrated the safety and efficacy of taVNS in treating a plethora of disorders, such as anxiety, insomnia, and depression (Wang et al. 2022). This raises the possible prospect of transcending the narrow therapeutic anti-inflammatory corridor to treat adenomyosis by employing taVNS. We have previously reported reduced vagal tone in women with endometriosis and that taVNS can effectively treat endometriosis in mice (Hao et al. 2021). We have also reported that α7nAChR is suppressed in endometriotic lesions, and its activation by pharmacological means can impede the progression of endometriotic lesions (Hao et al. 2022). Just as many molecular aberrations are shared by both endometriotic and adenomyotic lesions (Guo 2020), adenomyotic lesions are also found to have reduced α7nAChR expression (Xu et al. 2021), raising the prospect that women with adenomyosis may also have reduced vagal tone as in endometriosis, and, if true, adenomyosis might be managed by taVNS. This study was conducted to test the hypothesis that women with adenomyosis have reduced vagal tone.

Ethics statement This study was approved by the Ethics Review Committee of Shuguang Hospital, Shanghai University of Traditional Chinese Medicine (approval number: 2021-976-51-01) in accordance with the ethical principles spelled out in the Declaration of Helsinki and its subsequent amendments. Informed consent was obtained from all participants, and all associated methods were performed in accordance with the request of the Ethics Committee. Patients and evaluation This was a cohort study that compared vagal tone between women with adenomyosis and those without. We recruited 75 age-comparable premenopausal patients from June 1st, 2021, to March 31st, 2022, who were diagnosed with adenomyosis by transvaginal ultrasound (TVUS), symptomology, and gynecological examination. The inclusion criteria were as follows: i) consent, in writing, to participate in the study; ii) aged between 20 and 50 years and premenopausal; iii) complaint of dysmenorrhea, HMB, or infertility; and iv) TVUS-diagnosis of adenomyosis, but without ovarian endometriomas or uterine fibroids. The exclusion criteria were: i) taking hormonal, anti-platelet drugs, oral contraceptives, or other medications to alleviate symptoms in the last 3 months; ii) having anemia, hypertension, cardiovascular disease, cancer, or thyroid disease, or complaint of chronic insomnia, neurological, or other diseases that may affect heart rate variability (HRV); iii) co-morbidity of either ovarian endometrioma, deep endometriosis, uterine fibroids, endometrial polyps, or endometritis; iv) being pregnant or lactating; and v) being a smoker. The diagnosis of adenomyosis was made by an experienced sonographer with 16 years of experience (FW) based on features spelled out in Ferraz et al. (2017), which, in turn, are based on the Morphological Uterus Sonographic Assessment (MUSA) criteria (Van den Bosch et al. 2015). The typical ultrasound signs of adenomyosis mainly included: i) enlarged uterus with asymmetric anterior and/or posterior walls and thickening of the posterior wall in most cases; ii) diffuse or localized thickening of the uterine muscle layer with coarse echoes, accompanied by a fence-like acoustic shadow in the back; iii) small cysts with hypoechogenicity or island-like hyperechogenicity between the layers of the uterine muscle, and linear or bud-like hyperechogenicity under the endometrium; iv) penetrating blood flow signals within the lesion; and v) abnormal morphology of the junctional zone (JZ), with thickening, irregularity, interruption, non-display, or difficulty in differentiation. Ovarian endometrioma and deep endometriosis were assessed based on the consensus of the International Deep Endometriosis Analysis (IDEA) group (Guerriero et al. 2016). While superficial or peritoneal lesions pose a challenge for ultrasound detection (Condous et al. 2024), they were excluded through a comprehensive assessment of medical history, relevant surgical history, gynecological examinations (such as speculum and palpation), digital rectal examination, and other imaging examinations (such as pelvic MRI). Once the patient agreed to participate, the participant was instructed to undergo transvaginal or transabdominal ultrasonography (TVUS/TAUS) and elastosonography (TVESG/TAESG) evaluation in the first week after menstruation. For controls, we recruited, after informed consent, 75 healthy and cycling women who visited the Health Check-Up Clinic in Shuguang Hospital and who happened to be in the first week after their menstruation during the same time period as those with adenomyosis. None of them complained of dysmenorrhea, and, as such, the 10 cm visual analog scale (VAS) for quantification of their dysmenorrhea was all 0. Aside from the exclusion criteria listed above, the recruited subjects had no previous gynecological disorders and symptoms, or any evidence of endometriosis or adenomyosis per sonographic examination. Once they consented to participate in the study, they received TAUS/TVESG (or, for a few unmarried women, TAUS/TAESG), and their reproductive history and the amount of menstrual blood loss (MBL) were queried and recorded. For patients with adenomyosis, their medical records, including clinical symptoms, were carefully reviewed, and their data retrieved. For both groups, demographic information on age, gravidity, parity, length of menstrual cycles, date of the last menstruation, VAS on dysmenorrhea, and the amount of MBL by the Pictorial Blood Loss Assessment Chart (PBAC) (light, if no more than one sanitary pad was used in each period; heavy, if more than three pads were used; otherwise moderate) as reported previously (Wyatt et al. 2001, Dasharathy et al. 2012), were collected. For the recruited adenomyosis patients and healthy controls, we evaluated and compared their HRV indices, controlling for possible confounding factors such as age, parity, uterine size, and body mass index (BMI). For patients with adenomyosis, we also evaluated the relationship, if any, between various HRV indices and age, uterine size, BMI, and lesional stiffness. Evaluation of the sonographic and elastographic images For all recruited subjects, conventional TVUS/TAUS (B-mode) and TVESG/TAESG were performed on a Canon Aplio i900 with a PVT-781VTE (11C3) transvaginal probe (3.6–10.5 mHz) or PVI-475BX (i8CX1) transabdominal probe (1.8–6.2 mHz) (all from Canon Medical Systems Corporation, Japan) to measure the stiffness (in kilopascal or kPa) of adenomyotic lesions, or, for controls, the myometrium. This ultrasound system has the capacity of providing real-time shear-wave or strain elastography that detects tissue stiffness while compressing the surface with the transducer. The transabdominal probe was used when the subject’s uterus was too large to acquire a satisfactory image and/or if she had not had sexual activity yet. In this study, we only used shear-wave measurement. The uterine size was measured by TVUS/TAUS. For patients with adenomyosis, we set a region of interest (ROI) in the typical lesional area of the uterus. For controls, the ROI was in the center myometrium of the posterior uterine wall. Irrespective of tissues of interest, the stiffness at the ROI of the uterus was measured with a transvaginal or transabdominal probe while the subject was instructed to hold her breath. In all cases, the tissue stiffness for an ROI was measured thrice, and the mean value was used. To avoid possible bias, the sonographer, while knowing the group identity of the subjects, was blinded to the purpose and procedures of this study. To maintain consistency, a single sonographer (FW) performed ultrasound scanning for all recruited subjects. Evaluation of HRV To evaluate possible autonomic imbalances in women with adenomyosis, we measured HRV, which has been used extensively for this purpose (Galinier et al. 2000). Right after the ultrasound examination, all recruited subjects underwent, between 8:00 and 10:00 in the morning, a 5 min long electrocardiogram (ECG) recording using a micro-ECG recorder (HeaLink-R211B, Healink Ltd, China) following standard recommendations. The ECG recording was performed in a quiet, bright, and temperature-controlled examination room with an ambient temperature of approximately 21–22°C. All subjects were put in a supine position for approximately 3 min to normalize their spontaneous breathing frequency. To avoid bias, all recruited subjects were instructed to refrain from coffee or tea before coming to the hospital for evaluation. The recorded ECG time series data were extracted by ECG Viewer software (HeaLink Ltd) and then Kubios HRV software (Kuopio, Finland) was used for HRV data analysis. The evaluation of HRV was performed in both time and frequency domains, separating vagal and sympathetic activities through characterization by several parameters, such as the standard deviation of normal-to-normal intervals (SDNN, expressed in ms), root mean square of successive differences (RMSSD, expressed in ms), percentage of differences between adjacent normal-to-normal intervals (or inter-beat intervals) >50 ms (pNN50 (%)), normalized high-frequency (HF) power (HF, expressed in ms2), normalized low-frequency (LF) power (LF, expressed in ms2), and frequency power ratio LF/HF. Of note, RMSSD, pNN50, and HF all reflect vagal tone, and RMSSD is highly sensitive to the fluctuation of high frequency of HRV and is an index of vagal control on the heart (Task Force of the European Society of Cardiology the North American Society of Pacing Electrophysiology 1996) and, as such, is often preferred to pNN50 (Laborde et al. 2017). In contrast, LF reflects a mix of sympathetic and vagal influences and shows an influence of both sympathetic and parasympathetic branches, and the LF/HF ratio has also been considered as indicative of sympathetic-to-parasympathetic autonomic balance despite its shortcomings (Laborde et al. 2017, Shaffer & Ginsberg 2017). Sample size calculation Based on our previous study, one major outcome measure was RMSSD, which had a difference of 7.3 between patients with endometriosis and control women (Hao et al. 2021). Based on the average standard deviation of 12.7 ≈ 13 (Hao et al. 2021), we would need a sample size of 69 in each group in order to have 95% power to detect the difference while keeping the type I error at 5%. We chose a sample size of 75 in each group to allow for possible drop-out. Statistical analysis The comparison of continuous variables between two groups was evaluated by Wilcoxon’s test. Fisher’s exact test was used for contingency table data. Pearson’s correlation coefficient was used when evaluating correlations between two continuous variables. Spearman’s rank correlation coefficient was used when one variable was ordinal. Multiple linear regression was used to evaluate possible associations of various HRV indices with age, parity, BMI, uterine size, and group identity (adenomyosis vs control). Multiple linear regression was also used to identify factors associated with HRV indices within patients with adenomyosis, incorporating age, parity, BMI, uterine size, lesional stiffness, VAS scores on dysmenorrhea, and the amount of MBL as covariates. P values of less than 0.05 were considered statistically significant. There were no missing data. All computations were made with R 4.4.0 (29) (www.r-project.org).

Characteristics of the recruited subjects The characteristics of the recruited patients with adenomyosis and healthy women are listed in Table 1. We can see that both groups were comparable in age, parity, and BMI, but patients with adenomyosis had significantly larger uterine size, greater amount of MBL, and more severe dysmenorrhea (Table 1). Characteristics of recruited subjects in this study. | Variable | Healthy subjects | Patients with adenomyosis | P-value | |---|---|---|---| | n | 75 | 75 | | | Age (years) | 0.21 | || | Mean ± SD | 36.5 ± 6.5 | 38.0 ± 7.3 | | | Median (range) | 36 (25–49) | 38 (24–50) | | | Parity | 0.60 | || | 0 | 16 (21.3%) | 11 (14.7%) | | | 1 | 46 (61.3%) | 49 (65.3%) | | | ≥2 | 13 (17.3%) | 15 (20.0%) | | | Body mass index | 0.16 | || | Mean ± SD | 22.1 ± 2.5 | 21.5 ± 1.4 | | | Median (range) | 21.9 (17.5–27.8) | 21.3 (19.2–24.9) | | | Uterine size (cm3) | 2.1 × 10−12 | || | Mean ± SD | 53.4 ± 25.6 | 109.5 ± 80.0 | | | Median (range) | 46.1 (18.6–142.5) | 85.5 (36.6–462.6) | | | Amount of menstrual blood loss | 2.6 × 10−8 | || | Light | 22 (29.3%) | 8 (10.7%) | | | Moderate | 44 (58.7%) | 25 (33.3%) | | | Heavy | 9 (12.0%) | 42 (56.0%) | | | Visual analog scale | <2.2 × 10−16 | || | Mean ± SD | 0.0 ± 2.5 | 5.1 ± 1.5 | | | Median (range) | 0 (0–0) | 5 (2–9) | | | Uterine stiffness (kPa) | 2.9 × 10−9 | || | Mean ± SD | 52.1 ± 27.9 | 76.9 ± 24.3 | | | Median (range) | 50.6 (12.4–157.5) | 75.1 (24.8–155.2) | We found that, overall, both the amount of MBL and the severity of dysmenorrhea correlated positively with the uterine size (Spearman’s r = 0.47, P = 1.6 × 10−9, and Spearman’s r = 0.50, P = 8.3 × 10−11; Fig. 1A and B). The lesional stiffness in patients with adenomyosis was significantly higher than the myometrial stiffness in healthy controls (P = 2.9 × 10−9). Overall, the tissue stiffness correlated positively with the uterine size (r = 0.32, P = 5.6 × 10−5; Fig. 1C), and it correlated positively with both the amount of MBL (Spearman’s r = 0.34, P = 2.5 × 10−5; Fig. 1D) and the severity of dysmenorrhea (Spearman’s r = 0.59, P = 1.5 × 10−15; Fig. 1E). Within patients with adenomyosis, the lesional stiffness correlated positively with the dysmenorrhea severity (Spearman’s r = 0.66, P = 9.9 × 10−11; Fig. 1F), but only marginally with the amount of MBL (Spearman’s r = 0.20, P = 0.078). The sympatho-vagal imbalance in women with adenomyosis We next investigated the state of sympatho-vagal tone through evaluation of HRV in patients with adenomyosis in comparison with their age-matched healthy controls. We found that, while patients with adenomyosis and controls had comparable SDNN (P = 0.52; Fig. 2A), patients with adenomyosis had significantly reduced RMSSD, pNN50 (%), and HF compared with healthy women (all three P-values ≤0.0007; Fig. 2B, C, D). Multiple linear regression incorporating age, parity, BMI, uterine size, and adenomyosis status confirmed these findings (all three P-values <0.0008). While women with adenomyosis appeared to have higher LF than controls, the difference did not reach statistical significance (P = 0.32; Fig. 2E). In contrast, the LF/HF ratio was significantly elevated in adenomyosis patients compared with controls (P = 0.0033 by Wilcoxon’s test, or P = 0.004 by linear regression; Fig. 2F). RMSSD correlated closely with HF (r = 0.18, P = 0.032), as expected (Kleiger et al. 2005). In addition, HF correlated negatively with the LF/HF ratio (r = −0.55, P = 3.2 × 10−13). Moreover, pNN50 correlated positively with both RMSSD (r = 0.34, P = 2.5 × 10−5) and HF (r = 0.31, P = 0.00014), as previously reported (Shaffer et al. 2014). In view of the above, our data indicate that patients with adenomyosis had reduced vagal activity and increased sympathetic activity, indicating that the sympatho-vagal balance was disrupted with reduced vagal tone, similar to endometriosis (Hao et al. 2021). Relationship between HRV parameters and phenotypes Among all HRV-derived parameters, we found that RMSSD and pNN50 were both negatively correlated with the amount of MBL (both Spearman’s r ≤ −0.21, and both P-values ≤0.0085; Fig. 3A and B). HF was found to be negatively, but not statistically significantly, associated with the amount of MBL (Spearman’s r = −0.15, P = 0.15). In addition, RMSSD, pNN50, and HF were all found to be negatively correlated (all Spearman’s r ≤ −0.25 and both P-values ≤0.0022; Fig. 3C, D, E) while the LF/HF ratio was positively correlated with the severity of dysmenorrhea (Spearman’s r = 0.24, P = 0.0027; Fig. 3F). In addition, both RMSSD and pNN50 were found to be negatively correlated with the uterine size (both r’s ≤ −0.21, and both P-values ≤0.0086; Fig. 3G and H). Within patients with adenomyosis, RMSSD correlated negatively with the lesional stiffness (P=-0.42, P=0.00017; Supplementary Fig. S1 (see section on Supplementary materials given at the end of the article)). Within women with adenomyosis, none of these HRV parameters was found to be associated with either the amount of MBL, the severity of dysmenorrhea, or other variables (all P’s > 0.16). Multiple linear regression incorporating age, parity, BMI, uterine size, lesional stiffness, VAS scores on dysmenorrhea, and the amount of MBL identified that lesional stiffness was the only variable that is negatively associated with RMSSD (estimated β =−0.0076, P = 0.0017, 95% confidence interval = (−0.0113, −0.0038), R 2 = 0.18). No single variable was found to be associated with other parameters. Since RMSSD provides a major assessment of vagal tone (Laborde et al. 2017), our data indicate that the intrinsic lesional phenotype, i.e. its stiffness, is likely responsible for the reduced vagal activity.

In this study, we have shown that, compared with healthy women, patients with adenomyosis display a significant sympatho-vagal imbalance, featuring the domination of the sympathetic branch of the autonomic nervous system over the parasympathetic branch, as evidenced by reduced vagal tone but increased sympathetic activity. Our results are consistent with our previous report of reduced α7nAChR but increased ADRB2 expression in adenomyotic lesions (Xu et al. 2021), which is suggestive of suppression of vagal activity as well as elevated sympathetic activity. Our results are also consistent with the seemingly elevated systemic inflammation in patients with adenomyosis, manifesting as increased serum levels of proinflammatory cytokines TNFα and IL-8 (Li et al. 2024). Indeed, it has been documented that a loss of vagal tone shifts the immune response to a proinflammatory state (Williams et al. 2019). Adenomyosis has been reported to be associated with a higher risk for anxiety and depression (Alcalde et al. 2021, Li et al. 2022). Conversely, depression may also increase the risk of developing adenomyosis (Chen et al. 2024). Thus, it is likely that adenomyosis and depression can be mutually promotional. Alleviating depression through boosting vagal tone may have potential in alleviating symptoms or as a potential adjuvant management for patients with adenomyosis. Depression has been shown to increase the production of proinflammatory cytokines (Ford & Erlinger 2004, Panagiotakos et al. 2004), elevate systemic prolactin levels (Kumar et al. 2025), and reduce α7nAChR expression (Picciotto et al. 2015) – all of which have been reported to be involved in adenomyosis (Xu et al. 2021, Wang et al. 2025). Inflammation can also facilitate the development of depression (Franklin et al. 2018, Leonard 2018). Thus, by boosting vagal tone, activating the CAIP, and improving adenomyosis-related depression, it is likely that we could restore parasympathetic balance and provide anti-inflammatory and anti-nociceptive benefits, effectively taming inflammation, desmotility, and visceral hypersensitivity (Yin 2025). Indeed, VNS (Calvillo et al. 2011, Kong et al. 2011) and α7nAChR agonists (Borovikova et al. 2000, Wang et al. 2003, de Jonge et al. 2005) can effectively suppress inflammation. In particular, taVNS is non-invasive, inexpensive, simple, and seemingly without any serious side effects, and has been used successfully in treating depression (Rong et al. 2016, Kong et al. 2018). This is particularly appealing since all existing hormonal drugs for treating adenomyosis are non-curative but merely alleviate symptoms, mainly by stopping menstruation (Brosens 1997). It is reported that, through the activation of α7nAChR, cholinergic agonists suppress the activation of NF-κB, a major transcription factor that regulates inflammation and angiogenesis in ectopic endometrium (Guo 2007), and hence inhibit the production of proinflammatory cytokines, such as TNFα (Wang et al. 2003, Parrish et al. 2008). TNFα can activate macrophages to release HMGB1 (Valdes-Ferrer et al. 2013), which has been reported to be elevated in adenomyosis (Liu & Cheng 2023). Given the chronic inflammatory nature of adenomyosis (Orazov et al. 2017) and in light of the report that α7nAChR is an essential regulator of inflammation (Wang et al. 2003), activation of α7nAChR through taVNS offers an attractive therapeutic option for adenomyosis. Of course, whether this would work for adenomyosis requires further investigation. In addition to inflammation, patients with adenomyosis are reported to have an increased risk of depression (Alcalde et al. 2021, Li et al. 2022). Depression is known to activate the sympathetic nervous system and the HPA/sympathetic-adrenal-medulla (SAM) axes (Kupfer et al. 2012), thus increasing nerve fiber density in or around lesions (Cheng et al. 2018), as seen in adenomyosis (da Cunha Vieira et al. 2024), as well as the secretion of epinephrine and norepinephrine. The released catecholamines activate ADRB2 in lesions, as indeed found in adenomyosis (Xu et al. 2021). Activation of the HPA/SAM axes resulting from stress or anxiety induces the ADRB2/CREB/PKA signaling pathway but suppresses the expression of DRD2, facilitating the progression of ectopic endometrium (Guo et al. 2017, Long et al. 2016b ), and very likely also in adenomyosis (Xu et al. 2021). Consistent with this notion, aged female DRD2 knockout mice developed adenomyosis in response to prolonged prolactin exposure (Kelly et al. 1997), while a vaginal ring containing bromocriptine, a DRD2 agonist, is reported to be effective in treating adenomyosis (Andersson et al. 2019). However, another clinical trial (NCT03749109) on endometriosis/adenomyosis employing a vaginal ring containing quinagolide, also a DRD2 agonist, did not find any significant difference between the placebo and the quinagolide-containing vaginal ring groups (ClinicalTrials.gov, accessed on August 12, 2024). At this moment, it is unclear what causes the discrepancy, but increased lesional fibrosis is likely the culprit (Guo 2025). Our study has several strengths. First, with a combined sample size of 150, this study appeared to be adequately powered. Second, we evaluated lesional stiffness for all patients with adenomyosis and found that RMSSD is negatively associated with stiffness. Since lesional stiffness is a surrogate measure for lesional ‘age’ (Shen et al. 2016, Zhang et al. 2016) and correlates with the severity of dysmenorrhea and the amount of MBL (Fig. 1 and (Liu et al. 2018, Mao et al. 2023)), it could be viewed as an intrinsic lesional feature. Our study also has several limitations. First, we did not classify adenomyosis due, in no small part, to the lack of a consensus classification system (Gordts et al. 2018, Munro 2020). However, accumulating evidence indicates that all lesions of ectopic endometrium are similar in the sense that they seem to progress through EMT, FMT, smooth muscle metaplasia, and fibrogenesis (Guo 2018), and lesional stiffness is a proxy for lesional ‘age’ (Shen et al. 2016, Zhang et al. 2016). Second, while we queried subjects about their amount of MBL, we did not convert the data into a more quantitative form, which would have been more informative. Future studies are warranted to validate our findings with more precise quantification of the amount of MBL, as well as adenomyosis classification. Finally, since the diagnosis of adenomyosis, endometrioma, or deep endometriosis was based on ultrasound without laparoscopy, women with superficial peritoneal endometriosis may not be detected due to the limitation of ultrasound imaging (Condous et al. 2024), hence there is a risk of misclassification. This is a technical challenge facing all clinical research of adenomyosis based on ultrasound diagnosis. However, since both cases and controls used similar ultrasound diagnostic workup, both groups arguably face similar risk. Using reliable non-invasive diagnostic biomarkers in future studies may resolve this challenge. To sum, this study found that patients with adenomyosis have autonomic imbalances, featuring reduced vagal tone. In addition, we also found that reduced vagal tone is likely attributable, at least in part, to increased lesional stiffness, a proxy for the extent of lesional fibrosis, which correlated with the severity of dysmenorrhea and the amount of MBL. However, caution should be exercised since lesional stiffness accounts for a modest amount of vagal tone variation, suggesting that there are other, yet unmeasured, contributors. In any event, our findings raise the prospect of employing VNS as a possible non-medicinal option to alleviate symptoms. Future human studies are needed to see whether taVNS can have any therapeutic effects or serve as an adjunctive management. Supplementary materials This is linked to the online version of the paper at https://doi.org/10.1530/RAF-25-0039. Declaration of interest S-WG is an Associate Editor of Reproduction & Fertility and was not involved in the review or editorial process for this paper, on which he is listed as an author. S-WG is a Board member of the Asian Society of Endometriosis and Adenomyosis, and a member of the Scientific Advisory Board of the Endometriosis Foundation of America (EndoFound). He is also a member of the Scientific Advisory Board of Heranova LifeSciences, FimmCyte AG, E3A Healthcare, and Maipl Therapeutics, and has provided consultancy advice to these companies, but these activities had no bearing on this work. He received a travel grant from Ziwig. All other authors state that they have no competing interest. No external factors influenced the fairness or objectivity of this study throughout the entire research, including study design, data collection, analysis, and reporting. Funding S-WG was supported by grant 82071623 from the National Natural Science Foundation of China, and WZ by grant 82004398 from the National Natural Science Foundation of China. The funders had no role in study design, data collection and analysis, preparation of the manuscript, or the decision to submit the manuscript for publication. Author contribution statement S-WG conceptualized the study. WWZ, TTZ, FW, and S-WG curated the data. S-WG was responsible for formal analysis. S-WG and WWZ were responsible for funding acquisition. WWZ, TTZ, FW, and S-WG investigated the study. S-WG, WWZ, TTZ, and FW provided methodology. TTZ administered the project. TTZ provided resources (software: not applicable). TTZ and S-WG supervised the study. WWZ, TTZ, FW, and S-WG validated the study. S-WG visualized the study. S-WG wrote the original draft. WWZ, TTZ, FW, and S-WG contributed to writing review and editing. Data availability The datasets used and analyzed in this study are available from the corresponding author upon reasonable and written request. Ethics approval and consent to participate The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of Shuguang Hospital, Shanghai University of Traditional Chinese Medicine (2021-976-51-01). Informed consent was obtained from all subjects involved in the study.

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