Longitudinal measurements of NO-mediated vasodilation and physical activity over 1 yr following endometriosis excision surgery: a passive experiment

For each study the patient participated in, the Institutional Review Board at the Pennsylvania State University approved all experimental procedures and protocols. A Food and Drug Administration Investigational Drug Number was obtained for all protocols (IND 78,954). Verbal and written informed consent was voluntarily obtained in accordance with the guidelines set forth by the Declaration of Helsinki. At the time of this submission, data from only one experiment visit included in this report is published ( Williams et al., 2025 ); data from the remaining experiment visits is part of ongoing research. Data were collected on one Caucasian cis-gendered female beginning one month following endometriosis excision surgery. The patient had previously reported to the Emergency Department for acute lower right quadrant pain and was referred to a surgeon for intussusception of the intestine. Upon surgical intervention, it was discovered that the patient was experiencing a complication of previously undiagnosed endometriosis. An endometriosis lesion was biopsied at this time for confirmation. The patient was then scheduled for endometriosis excision surgery to occur 16 August 2023 (t = 0 days). During this surgery, a complete excision of endometriosis was performed (by author AD, fellowship-trained complex gynecologic surgeon specializing in endometriosis excision); lesions were removed from the superficial peritoneal space, left fallopian tube, left ureter, and retrocervical space with evidence of posterior Cul-de-Sac obliteration, collectively giving this patient an American Association of Gynecological Laparoscopists classification score of 28 (Stage IV) and American Society for Reproductive Medicine classification score of 14 (Stage II). Following excision surgery, the patient participated in research studies over the course of the subsequent 13 months. At the time of the excision surgery, the patient was 25 years 9 months. The patient participated in investigations of the impact of endometriosis on cutaneous microvascular and brachial conduit artery endothelial function (described below) at t = 37 days, t = 247 days, and t = 402 days. The participant had an IUD inserted (Mirena) at t = 83 days and attended five sessions of pelvic floor therapy ~1-year post-op (t = 352, 366, 373, 380, 415 days), but otherwise had no significant medical intervention. To investigate microvascular endothelial function by quantifying NO-mediated dilation, we utilized the progressive acetylcholine (ACh) concentration intradermal microdialysis dose-response protocol ( Williams et al., 2023 ). Two microdialysis probes were placed in the dermal layer of the skin of the ventral forearm, and a minimum of 60 minutes was allowed for the resolution of the hyperemic response to probe placement as well as a wash-in period of 0.015 M N G -nitro-L-arginine methyl ester (L-NAME; “L-NAME site”) solution in one of the probes. The other probe during this time perfused lactated Ringer’s solution alone (“control site”). Following resolution of hyperemia, local heaters were placed on the skin over the semi-permeable membrane of the microdialysis probe and clamped to 33 °C, a thermoneutral skin temperature. A laser Doppler flowmetry probe was placed within the heater to continuously monitor red blood cell flux (RBC flux; perfusion units, PU) to record a baseline RBC flux over 10 minutes. Following these baseline measurements, ACh was perfused in progressively greater concentrations for 5-minute increments (10 −10 to 10 −1 M). At the end of each 5-minute increment, blood pressure was measured (Connex Spot Monitor, WelchAllyn, Skaneateles Falls, NY). The final ~2 minutes of RBC flux was divided by mean arterial pressure to calculate cutaneous vascular conductance (CVC). Following the final dose of ACh, sodium nitroprusside (28 mM; USP, Rockville, MD) was perfused while the local heaters were raised to 43 °C to induce maximal local vasodilation. RBC flux was allowed to plateau for ~5 min before the experiment was terminated. An average value was taken over this time period and used to normalize all blood flow data to this maximum value (CVC %max ). The dose-response to ACh at the control site as well as the L-NAME site were plotted, and the NO-mediated vasodilation is represented by the calculation: L - N A M E - s e n s i t i v e c o m p o n e n t = c o n t r o l s i t e A U C - L - N A M E s i t e A U C and is represented in absolute arbitrary units. After the microdialysis probes were placed in the left forearm, the participant was positioned in a fully supine position, the lights were dimmed, and she was allowed to rest quietly for 10 – 15 min. During this time, an experienced investigator imaged the brachial artery of the right arm proximal to the antecubital fossa with high-resolution ultrasonography (GE Healthcare LOGIQe Ultrasound NextGen, Wauwataosa, WI). This ultrasound was connected to a data collection laptop with automated edge-detection software (Quipu Cardiovascular Suite FMD Studio, Pisa, Italy) to continuously record brachial artery diameter and blood flow. Baseline was recorded for 1.5 – 2 min with minimum 1 min stable period used for analysis. At the end of baseline, a pneumatic cuff placed distal to the antecubital fossa was rapidly inflated to suprasystolic pressure (210 – 220 mmHg; Rapid Cuff Inflation System, D.E. Hokanson Inc., Bellevue, WA at visit 1; DS400 Aneroid Sphygmomanometer and SC12 ™ Straight Segmental Cuff, Hokanson Inc., Bellevue, WA at visit 2 and 3) and maintained for 5 min then rapidly deflated. The hyperemic response was recorded for an additional 4 min. Movement artifact was manually removed during data analysis. We asked the participant to retroactively complete short-form International Physical Activity Questionnaires (IPAQ) using physical activity data logged in the Fitness app (© Apple Inc) on the participant’s smartphone for the 4 weeks leading up to the experiment dates. The participant regularly engages in high intensity full body resistance exercise beginning December 2023 (t = 133) and has reliably tracked all exercise bouts during her time participating in these studies. We calculated the participant’s metabolic equivalent (MET) minutes per week according to the completed IPAQ surveys.

Figure 1 is a timeline of events for this passive experiment. Figure 2 shows the results of the patient’s microvascular data. At day 37, we calculated the L-NAME-sensitive component to microvascular dilation to be −33.9 AUC. This increased to 178.1 AU at day 247. At day 402, the L-NAME-sensitive component decreased to 3.0 AUC. Figure 3 shows the results of the patient’s macrovascular data. FMD percentages at each visit were −0.06%, 3.69%, and 0.43%, chronologically. IPAQ results for the week prior to each experiment are shown in Figure 4 . At day 37, the participant demonstrated 1746 MET minutes (vigorous = 0 MET-min; walking = 1386 MET-min). The week prior to day 247 included 3759 MET minutes (vigorous = 1680 MET-min; walking = 2079 MET-min). The week prior to the final visit at day 402 was 2019 MET minutes vigorous = 1920 MET-min; walking = 99 MET-min).

We fortuitously collected vascular endothelial function data on a single endometriosis patient at three distinct time points in the 13 months following endometriosis excision surgery. The key findings of this passive experiment are that, in this patient, microvascular and macrovascular endothelial function as indicated by NO-mediated vasodilation (or the L-NAME-sensitive component to ACh dose-response) is not initially improved following endometriosis excision but increases substantially over months of recovery only to return to near baseline months later. These alterations in endothelial function occurred in lock-step with weekly physical activity. Compared with healthy young women, our participant demonstrated low NO-mediated dilation in the microvasculature at the first and third study visits, but comparatively normal NO-mediated dilation at the second ( Content et al., 2024 ; Greaney et al., 2022 ). Her FMD values, despite appearing to similarly fluctuate, remained well below that which has been reported in healthy groups ( Eagan et al., 2021 ; Santoro et al., 2012 ). The improvement of endothelial function that occurs at the second experiment visit may be driven in its entirety by the increased physical activity – combined high intensity resistance training and frequent walking – the participant engaged in during the days leading to the experiment visits. Collectively, this suggests that the recovery of vascular endothelial function following endometriosis excision surgery, if it occurs at all, is neither linear nor a direct result of surgical excision. Brachial artery FMD is the most commonly utilized research methodology for interrogating NO-mediated dilation (Green et al., 2013) in the macrovasculature in humans, the results of which are considered one of the earliest indicators of atherosclerosis and overt CVD development ( Yeboah et al., 2009 ). We utilized the FMD technique in our investigations and found that this patient, not unlike many other endometriosis patients, demonstrates lower FMD values than groups of young healthy women ( Santoro et al., 2012 , Kinugasa et al., 2011). Moreover, despite the marked improvement in microvascular NO-mediated vasodilation, her FMD values did not meet previously reported healthy ranges ( Eagen et al., 2021 ; Santoro et al., 2012 ) over the course of the 13 months following surgical excision. We also examined agonist-dependent endothelial function in the cutaneous microcirculation. Endothelial dysfunction in the microcirculation may occur in conjunction with or perhaps even prior to macrovascular endothelial dysfunction in populations with known cardiovascular disease risk factors ( Dillon et al., 2021 ; Stanhewicz et al., 2022 ; Greaney et al., 2022 ). In women, microcirculatory dysfunction underlies the pathogenesis of several highly prevalent cardiovascular diseases included non-obstructive cardiac disease. This participant demonstrated no functional NO-mediated dilation in the cutaneous microvasculature one month following her first endometriosis excision surgery. Eight months post-surgery, she showed marked improvement that rivaled that of similarly aged healthy young women ( Content et al., 2024 , Greaney et al., 2022 ), but then regressed by the next point of measurement five months later. The trend of vascular endothelial function in this participant follows that of the IPAQ-calculated MET minutes of physical activity per week the participant engaged in during the days leading up to her laboratory experiment visits. The microvasculature may be more sensitive to alterations in systemic function than the macrovasculature and thus may more acutely reflect the impact of exercise on vascular endothelial function. Previous studies have suggested that endothelial dysfunction in endometriosis can be improved if the systemic inflammatory or oxidant stress can be pharmacologically countered ( Dillon et al., 2022 ), or surgically removed ( Santoro et al., 2014 ). Our recent finding that oral salsalate, a nuclear factor κ B knockdown drug, did not increase NO-mediated microvascular endothelial dilation ( Williams et al., 2025 ) suggests a more targeted anti-inflammatory approach is required to achieve this. Exercise, although pro-inflammatory during acute bouts promoting interleukin production, downregulates systemic inflammation over time through multiple pathways (Gleeson et al., 2011). We can therefore speculate that the mechanism instigating increased NO-mediated vasodilation in the microvasculature in this patient is related to the acute and specific anti-inflammatory environment induced by the distinct increase in MET minutes per week. This passive experiment affords us the opportunity to contextualize the seemingly linear improvement in conduit artery endothelial function in a cohort of women in the Lazio region of Italy two years following endometriosis excision surgery ( Santoro et al., 2012 , Santoro et al., 2014 ). Within the Lazio region, sedentary behavior as categorized by aerobic activity (walking or cycling) is similar to that of Pennsylvania, where our data were collected, with sedentary Laziali citizens constituting ~29% ( EpiCentro ) and sedentary Pennsylvanians constituting ~25% of the population ( Center for Disease Control and Prevention ). The impact of regular physical activity on endometriosis risk, severity, recurrence, and symptoms is currently debated and inconsistent in the global literature, owing largely to different prioritized outcomes, intervention types, and poor experimental designs ( Ricci et al., 2016 ; Shafrir et al., 2018 ; Bonocher et al., 2014 ; Tennfjord et al., 2021 ). The results of this case as well as our previous findings in women who have undergone endometriosis excision surgery and continued to demonstrate impaired macrovascular ( Dillon et al., 2022 ; Williams et al., 2025 ) and microvascular ( Williams et al., 2025 ) endothelial function suggests that a cardioprotective treatment or lifestyle change that Italian endometriosis patients undergo is not occurring in American endometriosis patients. This passive experiment allows us to posit that changes in resistance exercise may be a potential avenue of explanation given the dearth of other available data. Larger scale studies should evaluate the relation of lifestyle behavior changes including physical activity or exercise and diet on the longitudinal progression of vascular endothelial function in endometriosis patients following excision surgery. These data collected in a single endometriosis patient over the course of one-year post-excision surgery indicates that the surgical approach itself did not recover vascular endothelial function as determined by pharmacologic targeting of the microvasculature and FMD in the macrovasculature. However, the added benefit of resistance exercise in combination with regular walking accomplishes this in the microvasculature, and although the improvement is slight and does not quite reach ranges reported in healthy groups, an increase in FMD values in this same session allows us to speculate that several weeks of exercise intervention of such MET minutes per week as this participant demonstrated at her second experiment visit may recover FMD values. Clinicians should consider promoting resistance exercise or physical activities that involve resistance physical activity (e.g., mountain biking, volleyball, martial arts training, etc) in patients with endometriosis as a means of improving vascular health in this population. Endometriosis is a disease that is not isolated to its lesions and requires longitudinal intervention. Collectively, these data suggest that recovery of vascular function following endometriosis excision surgery may not occur linearly within the year of surgical intervention or perhaps at all in some patients. This case study demonstrates the need for further research into the mechanisms of vascular dysfunction in this population as well as a critical analysis of post-operative care, pharmacologic treatment, and lifestyle intervention. Further longitudinal research may even reveal an association of this continued dysfunction with endometriosis recurrences or the effect of exercise programming on sustained improvements of cardiovascular health in women with endometriosis.

Endometriosis has been identified as a female-specific independent risk factor for cardiovascular disease (CVD; Farland et al., 2022 ; Mu et al., 2017 ), the leading cause of death worldwide ( Tsao et al., 2022 ). Affecting approximately one in ten women ( Buck Louis et al., 2011 ; Giudice et al., 2004 ; Missmer et al., 2004 ), a diagnosis of endometriosis is associated with a ~50% increased risk of myocardial infarction and ~40% increased risk of stroke ( Farland et al., 2022 ). Much earlier in life, women with endometriosis demonstrated attenuated macrovascular ( Santoro et al., 2012 ), and microvascular ( Dillon et al., 2022 ) endothelial dysfunction, defined as attenuated nitric oxide (NO) bioavailability, compared with healthy age-matched women. Vascular endothelial dysfunction is a critical precursor to major adverse cardiovascular events as well as CVD ( Jung et al., 2013 ). In 2012, Santoro et al. demonstrated that surgically confirmed endometriosis is associated with attenuated endothelial function in the brachial artery as determined with the flow-mediated dilation (FMD) technique ( Santoro et al., 2012 ). Two years following the surgical excision of endometriosis, a large percentage of participants from this study returned for repeat measurement. The FMD values measured in the endometriosis group two years after surgical excision improved such that the difference between groups was abolished at this follow-up ( Santoro et al., 2014 ). Women who undergo surgical removal of pathologic endometrial tissue tend to experience distinct relief of symptoms ( Lewin et al., 2024 ). However, endometriosis recurrence is frequent (~22%), and this disease often necessitates follow-up surgeries ( Nirgianakis et al., 2020 , Schippert et al., 2020 ). The discrepancy between the linear recovery of macrovascular function and the high recurrence rate of endometriosis warrants further investigation. Our laboratory has been successful in identifying mechanisms of microvascular dysfunction across disease states ( Dillon et al., 2021 ; Dillon et al.,2022 ; Stanhewicz et al., 2022 ; Greaney et al., 2022 ). In recent years, we have directed our focus to endometriosis and have identified impaired microvascular endothelial function in this population ( Dillon et al., 2022 ). Since this time, we have pursued investigations into the mechanisms of this cardiovascular pathophysiology ( Williams et al., 2025 ), most of which are ongoing. Several participants in each of these studies have completed more than one study. However, one participant has participated in several studies across the course of a year beginning one month following her first surgical intervention. Concomitantly, this participant diligently recorded her regular resistance exercise routine. We were therefore fortuitously able to, in a passive experiment approach, collect data on her macro- and microvascular endothelial function over the course of ~13 months following surgical excision of endometriosis in tandem with her exercise habits. These data demonstrate the time course of the vascular impact of endometriosis surgical excision in an otherwise healthy, regularly exercising young woman.