Hypothalamic-Pituitary-Adrenal Axis Responses in Women with Endometriosis-Related Chronic Pelvic Pain

Introduction

Endometriosis is commonly associated with pain including dysmenorrhea, non-menstrual pain, and dyspareunia [1]. Women currently diagnosed with endometriosis have similar rates of sensitization and myofascial dysfunction as women with pelvic pain without documented endometriosis, but those with any endometriosis history are most likely to have sensitization [2]. In considering the comorbidities of dysmenorrhea in women with and without endometriosis, additional symptoms are common and do not correlate with the presence or absence of lesions, supporting the role of central sensitization in the pain of dysmenorrhea [3]. Women with endometriosis-associated pain are more likely to suffer from anxiety and depression [4, 5], sleep disturbances [6], chronic fatigue syndrome, and fibromyalgia [7] compared with healthy individuals. Depression and anxiety can persist even after endometriosis lesions and pain are treated and quality of life improves, illustrating a complex relation among depression, anxiety, and pain in women with endometriosis [8].

The hypothalamic-pituitary-adrenal (HPA) axis plays a central role in the response to stress whereby axis activation results in release of the glucocorticoid cortisol by the adrenal cortex. In healthy individuals, the cortisol release undergoes a diurnal variation with a morning peak and nadir at midnight [9]. Cortisol is an essential aspect of the neuroendocrine response to pain or other stressful stimuli. Initially, an increase in cortisol secretion is an important part of the “fight or flight” reaction and serves as an adaptive physiologic response towards any stressor enabling the body to maintain homeostasis.

Exposure to chronic stress, such as pain, can result in glucocorticoid dysregulation. Disruption of HPA response is shown in fibromyalgia, low back pain, irritable bowel syndrome, rheumatoid arthritis, chronic headaches, anxiety, and depression [10–14]. Endometriosis-associated and chronic pelvic pain symptoms without endometriosis diagnosis may be associated with changes in neuroendocrine response. Dysmenorrhea alone, without an endometriosis diagnosis, is associated with activation of brain regions on functional MRI and hypocortisolism, which is negatively correlated with the duration of dysmenorrhea [15]. Women with endometriosis and pain have low salivary cortisol with dampening of diurnal variation [16]. However, high hair cortisol suggesting prolonged chronic stress is reported in women with endometriosis and pain compared with healthy volunteers [17].

Race also influences the HPA axis response. Blacks have similar baseline adrenocorticotropic-releasing hormone (ACTH) levels, but higher ACTH levels after corticotropin-releasing hormone (CRH) testing or exercise without a corresponding rise in cortisol, compared with non-blacks [18, 19].

Endometriosis, as an estrogen-dependent, chronic inflammatory condition associated with pain, may alter the stress response differently compared with those with pain but no endometriosis or healthy volunteers. We hypothesized that women with biopsy-proven endometriosis would be more likely to have a blunted response to CRH testing, compared with those with pelvic pain alone and healthy volunteers. We considered that HPA response may be altered by pain severity and duration, independent of endometriosis diagnosis, the presence of other symptoms, such as depression, anxiety, and fatigue as evidenced by sleep disturbances and tiredness, and demographic factors, including race, body mass index (BMI), and age [20–22].

Materials and Methods

Women aged 18 and 50 years with a chronic pelvic pain history suggestive of endometriosis and healthy volunteers from a prospective cohort study conducted at the National Institutes of Health Clinical Center and approved by the NICHD IRB (NCT00073801) from April 2004 to 2011 were included in this analysis [2]. All participants provided written informed consent. Women with pain symptoms reported having at least a 3-month history that included dysmenorrhea, dyspareunia, and non-menstrual pain. Controls were healthy women with no pelvic pain history or symptoms suggestive of endometriosis, and who had a normal pelvic examination and normal-appearing ovaries on ultrasound. Participants had regular menstrual cycles and were not pregnant. No subjects had used hormonal treatment or had surgery within the past 3 months.

Upon study entry, endometriosis, gynecologic, and reproductive health history were assessed, and participants underwent a physical examination including a pelvic examination and laboratory assessment (Fig. 1). Patients were excluded if their pain symptoms were attributable to other causes like pelvic infection, gastrointestinal disorders, untreated psychological disorders, thyroid disease, fibromyalgia, chronic fatigue syndrome, or autoimmune diseases. Those with abnormal renal or liver function more than twice the normal range, actively attempting pregnancy, lactating, or human immunodeficiency virus (HIV) positive were excluded.

After a month of study, participants with pelvic pain underwent laparoscopic surgery during which all lesions suspicious of endometriosis were excised and assessed by histologic evaluation to confirm endometriosis. At surgery, other potential causes of pelvic pain were diagnosed and treated. Participants were then categorized into three groups: those with pelvic pain and biopsy-proven endometriosis at study laparoscopy, those with pain alone but no endometriosis, and healthy volunteers.

Patient race was categorized as black if they self-reported (non-Hispanic) black race (n = 10), and non-black for all others (i.e., non-Hispanic white (n = 39), Asian (n = 4), and Hispanic (n = 1)). No one reported herself as Hispanic black.

Each woman’s cycle phase was confirmed by menstrual calendar, twice weekly estradiol and progesterone levels assessment, and LH kit use (OvuQUICK, Quidel, San Diego, CA). In the menstrual cycle follicular phase before surgery, an ovine corticotropin-releasing hormone (oCRH) test was performed, beginning at 7:30 a.m. following an overnight fast. After intravenous catheter placement, ACTH and cortisol levels were obtained at 5 and 0 min before stimulation. After oCRH was injected at 1 mcg/kg, ACTH and cortisol levels were measured at 15, 30, and 45 min. Specimens placed on ice were transported to the Clinical Center’s Department of Laboratory Medicine for assay by chemiluminescence immunoassay (Siemens Immulite 2500 analyzer). For plasma ACTH, intra-assay variation ranged from 1.6 to 3.4% and inter-assay variation ranged from 3.1 to 4.2%. Cortisol assay limit of quantitation was 0.5 μg/dL; intra-assay variation ranged from 4.6 to 8.4% and inter-assay variation from 6.4 to 13.5%.

Patients were assessed for the presence and severity of menstrual, non-menstrual, and coital pain using an 11-point visual analogue scale (VAS) (0 indicating no pain to 10 indicating worst pain imaginable); duration of chronic pain in years was obtained. Participants completed the Duke Health Profile and Endometriosis Health Profile-30 to assess the impact of pain on quality of life and other psychosocial variables [20, 23]. The Duke Health Profile (DUKE), a 17-item general health quality of life questionnaire, derived six health (physical, mental, social, general, perceived health, and self-esteem) and four self-reported functional measures (anxiety, depression, pain, and disability). The Endometriosis Health Profile-30 (EHP-30) questionnaire was used to measure coital pain and fatigue measures.

Study groups were defined in two ways: (1) women with biopsy-proven endometriosis diagnosed at study laparoscopy versus women with pelvic pain alone and no evidence of endometriosis (pain only) versus healthy volunteers without chronic pelvic pain or endometriosis; and (2) pain groups consisting of those reporting pelvic pain, either with biopsy-proven endometriosis or pain alone versus healthy volunteers. Adrenocorticotropic-releasing hormone and cortisol values representing baseline (0 min), delta (change between peak (highest value at 15, 30, or 45 min) and baseline), and area under the curve (AUC) using the linear trapezoidal rule were calculated over times 0, 15, 30, and 45 min. Data distributions were assessed, and log transformation was used as necessary to achieve normality. Data were additionally assessed for confounding and interactions with demographic covariates such as race, age, and BMI due to their potential effects on HPA response and were stratified and/or adjusted as necessary [21]. Presence and severity of menstrual, non-menstrual, and coital pain were analyzed among groups and in relation to ACTH and cortisol responses. Anxiety and depression, fatigue (being unable to sleep properly or needing to lie down because of the pain as indicated on EHP-30), and problems with sexual intercourse (avoiding sexual intercourse because of pain and problems with sexual intercourse score as calculated by EHP-30) were also assessed among groups and in relation to HPA response.

Continuous data for groups (study groups, pain groups, or groups by race) were analyzed by t tests or analysis of covariance (ANCOVA), with post hoc pairwise comparisons utilizing the Bonferroni correction for multiple comparisons. Fisher’s exact tests compared categorical data. Pearson’s correlation coefficients measured the relation between continuous independent variables (e.g., age) and HPA response outcomes. Univariable and multivariable regression modeling assessed the relation between relevant covariates (e.g., study group, pain severity, depression) and HPA response, and adjusted for study group or age, as applicable. Data are reported as mean ± standard deviation. A p value less than 0.05 was considered statistically significant; corrected p values are reported where applicable. All data were analyzed using SAS v 9.4 (SAS Institute, Inc., Cary, NC).

Results

Study groups consisted of women with chronic pelvic pain and biopsy-proven endometriosis (n = 22), pelvic pain alone with no evidence of endometriosis (n = 12), and healthy volunteers (n = 20), whose characteristics are listed in Table 1. Women were predominantly non-black (n = 44, 81.5%; of whom 39 were non-Hispanic white) with few black women (n = 10, 18.5%). As intended by the study design, both presence and severity of menstrual, non-menstrual, and coital pain were statistically significantly different among study groups but did not differ between the pain groups (Table 1) or racial groups. There were no statistically significant differences in any ACTH and cortisol responses among the three study groups (Fig. 2), nor were they different when the combined pain groups were compared with healthy volunteers. In considering the whole cohort, depression, anxiety, and fatigue measures (being unable to sleep properly because of pain, needing to lie down or go to bed because of pain) were worse for the pain groups than healthy volunteers (Table 1). None of these or other pain factors (presence and severity of menstrual, non-menstrual or coital pain) was related to ACTH and cortisol outcomes. However, outcome differences were observed between racial groups such that ACTH delta (129.9 ± 130.7 vs. 52.5 ± 66.0 pg/mL; p = 0.003), ACTH AUC (4813 ± 4707 vs. 2290 ± 2900 min*pg/mL; p = 0.013), and cortisol delta (26.3 ± 21.5 vs. 13.2 ± 9.7 μg/mL; p = 0.005) were statistically significantly higher in black versus non-black patients (Fig. 3).

An analysis by race was conducted because of these observed differences in HPA measures. Race was a consistent effect modifier; thus, analyses were stratified (Supplementary Figures 1 and 2). Black women had a higher BMI than non-black women (28.1 ± 4.3 vs. 23.9 ± 3.9, p = 0.004), but BMI was not associated with HPA response. Among predominately white, non-black women, those with pain only were older than those with endometriosis (39.1 ± 7.1 vs. 31.3 ± 7.5, p = 0.015), and age appeared to be related to HPA measures, particularly ACTH and cortisol deltas (r_p = 0.40, p = 0.007 and r_p = 0.37, p = 0.013, respectively). When stratified by race and adjusted for group and age, greater menstrual pain severity in these mostly white (non-black) women was associated with blunted cortisol and ACTH deltas (p = 0.023 and p = 0.015, respectively) and greater non-menstrual pain severity with a blunted cortisol delta (p = 0.017). Among the relatively small group of black women (n = 10), the presence of non-menstrual pain was associated with a higher baseline ACTH level (p = 0.015), and blunted ACTH delta and AUC (p < 0.001 and p = 0.003, respectively). HPA measures were not considered by race among those women who had intercourse due to the small sample size.

Discussion

We examined HPA axis response to early morning follicular phase CRH stimulation in women with biopsy-proven endometriosis-associated pelvic pain and women with pain alone and symptoms suggesting endometriosis, compared with healthy volunteers. A difference in HPA axis response was observed by race, in accordance with the literature [18, 19]. An analysis restricted to non-black subjects who were predominately white (89%) and combining all pain subjects found that a blunted response to dynamic testing was associated with greater menstrual and non-menstrual pain severity and was similar to the dampened circadian rhythm observed by others [16]. Black subjects with pain, although fewer in number, also seemed to experience a somewhat blunted ACTH, but not cortisol response, within their relatively higher magnitude of response. These findings support the hypothesis that there is an association between chronic pelvic pain severity and HPA axis dysfunction that is influenced by other variables including race.

The HPA axis response to CRH stimulation of those with pain and endometriosis did not differ from each other, or from healthy volunteers. Depression, anxiety, and sleep disturbance were associated with pelvic pain but not HPA axis response. Blunting of the HPA axis response in our study may have been masked by the response in (an albeit small number of) black women who had higher baseline and greater stimulated HPA axis measures independent of pain, an observation reported in healthy volunteers [18, 19]. However, lower mean cortisol reported in those with longer duration of dysmenorrhea [15] and with incapacitating pain [24] is consistent with our finding a blunted response associated with pain severity in the non-black subjects. This pattern of neuroendocrine dysregulation also is consistent with our findings regarding myofascial dysfunction and sensitization in this cohort [2].

Our observed association between blunted HPA axis response and pain severity in women with pain suggesting endometriosis, regardless of biopsy-confirmed diagnosis, may be explained as a response to chronic stress, similar to that observed in chronic fatigue syndrome and seasonal affective disorder [25]. Chronic conditions, commonly occurring in women with endometriosis and pain, including irritable bowel syndrome, fibromyalgia, depression, and facial pain are associated with hypocortisolism and blunting after stimulation [26–29]. Over time, the stress of chronic severe pain and the occurrence of other pain comorbidities, rather than the presence of endometriosis, may alter the HPA axis response. The mechanism by which the HPA axis may be suppressed is poorly understood but may represent axis “burnout” [30]. Chronic stress results in a continuous, prolonged hyper- or hypo- axis activation with blunting of cortisol circadian rhythm and response to CRH [21, 25, 31].

Importantly, hypocortisolism might contribute to the immune system disinhibition that, in turn, may contribute to the proinflammatory state reported in endometriosis [32, 33]. In animal models, exposure to chronic stress increases the size of endometriosis lesions and stress mitigation lessens lesion growth [34, 35]; treatment with a corticotrophin releasing hormone receptor-1 inhibitor reduces the size and number of endometriosis lesions [36]. In women with endometriosis and pain, several weeks of physical therapy and psychological intervention restore the diurnal variation in cortisol levels by raising the suppressed basal morning level [37].

Our findings also begin to inform us about the relationship among HPA axis dysfunction, pain, and race. While limited by sample size, the observed overall racial differences in HPA axis responsiveness are similar to those of Yanovski et al. in healthy black subjects [38] and others [39, 40]. They attributed the absence of increased cortisol levels to high levels of fragmented, but not necessarily bioactive, circulating ACTH, which hampered formation of the adrenocortical melanocortin type 2 receptor-hormone complex responsible for cortisol secretion [18, 19, 38].

Different types of testing may account for variability in results. Some studies measure salivary [16, 41], others serum or plasma cortisol [15], and others measured hair cortisol [17]. Shift work and adherence to instructions may affect salivary cortisol levels [42]. Sample collection for hair cortisol requires a standardized approach (hair length and location related to the hair follicle) [17]. HPA axis response to dynamic testing can only be assessed by timed measurement of serum. The presumed axis hyperactivation in endometriosis observed with hair cortisol measurement merits validation [17].

Other HPA axis studies in pelvic pain and endometriosis differ significantly in design, study population, and menstrual cycle timing that may, in turn, influence laboratory findings. Some performed dynamic testing in late afternoon, rather than morning [12]; others evaluated circadian rhythm changes [16] or measured basal cortisol only [15, 41], sometimes averaging levels across multiple days [15]. While silent when testing occurred in relation to surgery, few studied surgically diagnosed endometriosis and pain [16]. Most studied pelvic pain or dysmenorrhea without endometriosis diagnosis [12, 15, 41]. Studies generally did not consider race, even those with 10% black participants [16]. Despite reports of menstrual cycle variability in cortisol testing [43], participant oral contraceptive use prevented studying these effects [16, 41]; nonetheless, some described testing women in the luteal phase [16]. The diurnal variation of cortisol, differences between dynamic and static testing, type of specimens tested, and the potential impact of pain chronicity and characteristics, endometriosis diagnosis, contraceptive use, and menstrual cycle timing all merit consideration in future studies. Importantly, our study provides justification for sufficiently powered studies to consider the differing relationships between HPA axis response and pain variables by race.

Our study has several strengths. First, subjects were not using reproductive hormones, were assessed prior to surgical treatment, and were all studied in the mid-follicular phase of regular menstrual cycles. Pain and other symptoms of those with or without endometriosis were similar. Cortisol response testing with set intervals was conducted under investigator supervision in the early morning. Variables that could impact cortisol values, like time of day, menstrual cycle phase, or coexisting diseases, were eliminated. Ours may be the first study to consider the physiologic differences of HPA axis response by race in a pelvic pain cohort.

There were limitations. Measurement during a 45-min time period was better than one morning level, but longer CRH testing periods may provide additional information. Recruitment focused on women with pain symptoms suggesting endometriosis, making the chronic pain groups similar and limited the number with pain but no endometriosis. We were unable to recruit women with pain-free endometriosis or healthy volunteers undergoing laparoscopic surgery. Sample size estimations did not plan for analyses by race as we only included a population-based proportion of black women. As age and duration of pain are inextricably linked, adjusting for age likely serves as a proxy for adjusting for pain duration. Even though coital pain is common in women with endometriosis [44], few women were sexually active, precluding testing associations with coital pain. Fatigue symptoms were measured using single-item responses.

Our observation that race was associated with HPA axis responsiveness limited our ability to assess an association between chronic pelvic pain with (or without) endometriosis and HPA response in a racially diverse cohort. However, the findings suggest that HPA axis hypofunction may be related to pain severity in primarily white, non-black women, independent of endometriosis lesions. Whether the local peritoneal neurosensory, inflammatory environment in endometriosis, or the potential efficacy of hormonal treatment are related to variations in HPA axis responsiveness are areas of future research. Importantly, future studies could explore the influence of race on HPA function in patients with chronic pelvic pain. Better understanding of these processes may enhance targeted and individualized therapeutic approaches to endometriosis-related pain treatment.

Supplementary Material

Electronic supplementary material The online version of this article (https://doi.org/10.1007/s43032-020-00201-x) contains supplementary material, which is available to authorized users.