We stratified IOE patients into those whose preoperative RMSSD values were below (Group 1a = 7) versus above (Group 1b = 9) a cut-off threshold of 31 ms . In group 1a, all HRV values significantly increased after surgery. In group 1b, time-domain parameters significantly decreased postoperatively. Conclusions: Our results suggest that in IOE, HRV may be either decreased in parasympathetic tone or increased globally in both sympathetic and parasympathetic tones. We found no evidence that insulo-opercular surgeries lead to major autonomic dysfunction when a good seizure outcome is reached. The increase in parasympathetic tone observed preoperatively may be of clinical concern, as it was positively correlated with the SUDEP-7 score.Background: We aimed to evaluate heart rate variability (HRV) changes in insulo-opercular epilepsy (IOE) and after insulo-opercular surgery. Methods: We analyzed 5-min resting HRV of IOE patients before and after surgery. Patients’ SUDEP-7 risk inventory scores were also calculated. Results were compared with age- and sex-matched patients with temporal lobe epilepsy (TLE) and healthy individuals. Results: There were no differences in HRV measurements between IOE, TLE, and healthy control groups (and within each IOE group and TLE group) in preoperative and postoperative periods. In IOE patients, the SUDEP-7 score was positively correlated with pNN50  ( The role of the insula in drug-resistant epilepsy has been increasingly recognized with the report of several cases of insular, insulo-opercular or temporo-insular epilepsy successfully treated by resection, radio-frequency thermocoagulation, or laser ablation ,3,4,5,6.Heart rate variability (HRV) is considered to be an index of autonomic activity that reflects brain–heart interactions and the balance between sympathetic and parasympathetic activity . HRV hasGiven that chronic autonomic dysfunction or cardiovascular dysfunction may be involved in the mechanisms of SUDEP ,10, seven = 7) or ≥25% (n = 1) of their insula resected compared to those whose insula was not (n = 4) or minimally/marginally (n = 9) resected. Because most patients had temporal or temporal plus epilepsy, the effect of insular epilepsy per se remains unknown. Furthermore, because nearly half of patients continued to have seizures post-operatively, it is unclear whether HRV changes were due to insular surgery or persistent seizures. Finally, there was no control group to help account for the physiological decrease in HRV that occurs with aging.While several studies have looked at the effect of acute ischemic damage to the insula on HRV ,44, the In this study, we assessed pre- and post-operative HRV in patients with insulo-opercular epilepsy (IOE). Considering the role of the insula in autonomic and cardiovascular control, we hypothesized that HRV could be altered by long-standing insulo-opercular seizures, as well as insulo-opercular resections. We also sought to look at the relationship between HRV changes and SUDEP risk factors.We retrospectively reviewed all patients with IOE investigated and treated at our institution between 2000 and 2021. An insulo-opercular focus was assumed based on the presence of an epileptogenic lesion, a clear cluster of dipoles on magnetoencephalography, or intracranial EEG-recorded seizures in that area. Patients were included if: (1) they had at least one pre-operative video-electroencephalography (VEEG) monitoring study with electrocardiographic (ECG) lead derivations available for analysis review; and (2) had undergone epilepsy surgery in which insulo-opercular areas  were part of the epileptogenic zone. Exclusion criteria were: (1) a history of cardiac disease  or any condition that could impair autonomic function ; and (2) use of antiarrhythmic agents .The study was performed in two parts. Since the primary purpose of our study was to determine HRV changes in IOE, in the first part of the study, we only included patients who became free or nearly free of disabling seizures for more than one year (Engel I or II) after epilepsy surgery limited to the insulo-opercular region . We excluded patients who had surgery outside the insulo-opercular area (in the same or in a previous surgery). For this first part of our study, an active control group and a healthy control group were established. The active control group consisted of 16 age- and sex-matched TLE patients who had a good seizure outcome after an anterior temporal lobectomy. Similar inclusion and exclusion criteria were used, except for the location of the seizure focus and surgery. Since for IOE and TLE patients, HRV comparisons were carried out twice, pre- and post-operatively, two separate healthy control groups were constituted, matched for age and sex with patients for both study periods.In the second part of the study, because we wished to see whether HRV changes were different in patients with temporo-insular epilepsy compared to patients with IOE patients, and also whether failed insular surgery had a negative impact on HRV, we included all patients whose epilepsy surgery consisted of the removal of the insulo-opercular region with or without the ipsilateral temporal lobe and with or without a good seizure outcome. For HRV comparisons in the preoperative period, each patient was matched for sex and age with one healthy individual. For HRV comparisons in the postoperative period, an age-correction method  were calculated from seven elements: (1) more than three tonic-clonic seizures in the last year; (2) one or more tonic-clonic seizures in the last year ; (3) one or more seizures of any type over the last 12 months ; (4) more than 50 seizures of any type per month over the last 12 months; (5) duration of epilepsy ≥ 30 years; (6) current use of three or more antiseizure medications; and (7) mental retardation, IQ < 70, or too impaired to test .For each patient, 12 five-minute segments were selected from VEEG recordings performed during their presurgical assessment. Three lead ECGs were recorded and digitized at a sampling frequency of 200 Hz. Periods were selected based on the following criteria, inspired by Myers et al. : (1) awaA one-hour continuous three-lead ECG awake recording at resting state in a lying position was carried out on all control subjects and epileptic patients in the post-operative periods. From that one-hour recording a 5-min segment was extracted for HRV analysis .Consecutive R-R intervals were measured using the Brain Vision Analyser 2.1 commercial software . R peaks were initially automatically detected from ECG data and then subsequently visually inspected to detect and manually correct artifacts, missed beats, or ectopic beats. Ectopic beats were removed from the recordings, and were replaced by an interpolated R-R interval . The KubOut of the time-domain parameters, RMSSD is considered to be a primary measure to assess the vagal tone. Both RMSSD and pNN50 are mainly influenced by the parasympathetic nervous system. Out of the frequency-domain parameters, the HF power represents parasympathetic influences on the heart rate, while the LF power reflects predominantly sympathetic activity .The statistical analysis was performed using the Statistical package for the social sciences (SPSS) version 25 .p < 0.05.Since data were not normally distributed and there was a small number of studied populations, non-parametric statistical tests were used. The median and interquartile ranges (IQR) were calculated for each HRV variable, and values were expressed as median (IQR). HRV values of all three groups  were compared using the Kruskal–Wallis test. The Mann–Whitney U test was performed to compare two independent samples. Within a group, a Wilcoxon signed-rank test was used to compare pre-operative and post-operative HRV values. Spearman’s rank correlation coefficients were calculated to evaluate the relationship between HRV values and the SUDEP-7 score. The results were considered significant at p-value < 0.01:-2, r = 0.9982For RMSSD: y = 75.78 − 1.618x + 0.011x-2, r = 0.9989For pNN50: y = 60 − 1.89x + 0.015x-2, r = 0.9964For LF: y = 310.114 − 6.6771x + 0.03857x-2, r = 0.9983,For HF: y = 271.214 − 7.4701x + 0.05407x With y = HRV value, x = age.In part 2 of the study, pre-operative HRV changes of epileptic patients were compared to healthy controls matched for age and sex. Since the interval between time of surgery and post-operative HRV measures were different between epileptic patients, an age correction method was used to adjust HRV values of each healthy control at the postoperative age of the matched IOE patients, as previous studies have shown that both time- and frequency HRV features are age-dependent. In 2012, Voss et al. calculated reference values for 5-min resting HRV indices from a population of 1906 healthy subjects aged 25 to 74 years , which wThe value determined for each parameter from this quadratic regression was considered as the “theoretical value.” The adjusted value of each parameter at the desired postoperative time point (age 2) was calculated by adding the theoretical value of each parameter at age 2 to the difference between the actual value and its theoretical value at age 1 :Adjusted value at age 2 = Theoretical value at age 2 + .This study was approved by our institutional ethics committee (Project 16.146). All participants signed written informed consent.Sixteen patients  were enrolled in the first part of the study, aged from 19 to 47 years (median (IQR) = 34.50 (9)) at the time of presurgical evaluation. Findings from the presurgical evaluation, presurgical revised SUDEP-7 score, and surgical data for all patients are summarized in The TLE group consisted of 11 females and 5 males, aged from 21–46 years (median (IQR) = 32.50 (12)) at the time of presurgical evaluation . Median Two control groups of 16 healthy subjects, matched for age and sex with the patients at the pre- and post-operative period, were established. Median age for pre-operative and postoperative control groups was 34 (10) and 37.5(16), respectively Table A2In the second part of the study, the group of interest was composed of twenty-seven patients  with IOE without or with temporal epilepsy. Preoperative age ranged from 19 to 54 (median age (IQR) = 35(10)). Postoperative age ranged from 19 to 56 (median (IQR) = 38 (15)). A summary of their demographic and clinical findings can be found in HRV parameters for each participant can be found in Regarding the laterality of the epileptic focus, there was no significant difference for IOE and TLE groups.There was also no difference between median pre- and post-operative HRV values within the IOE group as well as the TLE group.p = 0.008), and RMSSD . We then evaluated the effect of each factor of the SUDEP-7 inventory on HRV; we found that RMSSD and pNN50 values of patients who had at least one bilateral tonic-clonic seizure during the last year were significantly higher than those of patients without bilateral tonic-clonic seizure (p = 0.039 and 0.030 respectively). The difference between preoperative and postoperative HRV was not correlated with the presence of the bilateral tonic-clonic seizure.We plotted the revised SUDEP-7 score against each HRV parameter for IOE and TLE groups. In the IOE group, we found a positive relationship between the revised SUDEP-7 score and pNN50 , RMSSD, and HF values were significantly lower than those of the control group preoperatively . On the other hand, all HRV parameter values were significantly higher for group 1b (n = 9) compared to the control group . After surgery, all HRV values in group 1a significantly increased (p < 0.05) even though postoperative median values were not statistically different from those of matched controls. On the contrary, in group 1b, all HRV values decreased postoperatively, but only RMSSD and pNN50 met statistical significance. As for group 1a, all postoperative HRV values of group 1b were not statistically different from those of control subjects.Next, we looked at the behavior of RMSSD pre- and post-operatively for each individuals A. Two diUsing the same methodology for the TLE group, we noted that only two TLE patients (12.5%) had very high RMSSD values than their matched controls B. When ep = 0.014).Although, in the TLE group there was no relationship between the SUDEP-7 score and the HRV parameters, the median SUDEP-7 score of group 2a  was significantly higher than that of group 2b  (4 (2) vs. 1 (2); To assess HRV changes associated with insulo-opercular seizures on a larger set of patients, we included in this second part not only the 16 patients from part 1 with a definite IOE  but also four more with a poor outcome  with those of the control group both in the pre- and post-operative periods. Furthermore, within the former group, there was no significant difference between preoperative and postoperative HRV values.Once again, we used the threshold of 31 ms to divide the epileptic group into two subgroups, and similar observations were found as in part 1, i.e., patients with low pre-operative HRV values, which were significantly increased, whereas those with high pre-operative HRVs were significantly decreased post-operatively.More importantly, all post-operative HRV parameters were not significantly different from those of matched controls.We further analyzed pre- and post-operative HRV by taking into account the side of foci, the presence of an epileptogenic lesion, and whether a limited operculo-insular surgery or a larger temporo-insulo-opercular epilepsy surgery was performed, and found no statistical differences.Unlike when analysis was limited to the IOE group, when including all patients within the epileptogenic zone, which included the insulo-opercular region , no correlation was found between HRV parameters and the revised SUDEP-7 score.In this study, we sought to look at HRV changes in patients with IOE/IOE ‘plus’ before and after epilepsy surgery, and compare them with patients with TLE as well as healthy controls. To our knowledge, no other study has done this before.We did not find significant differences in median HRV values between patients with definite IOE versus TLE patients and healthy controls. The analysis for patients in which the insulo-opercular area was a part of their epileptogenic zone (definite or probable IOE and IOE ‘plus’ patients) showed similar findings. We did not find any differences in HRV related to the side of the focus either. This was somewhat unexpected, since the insula is known to be part of the central autonomic network ,50. WhilIn our study, median pre-operative HRV values did not significantly change after insulo-opercular surgery. At first glance, these results appear to contradict findings from ,46. In 2n = 7) or extensive  insular removal compared to temporal surgeries with no (n = 4) or marginal (n = 9) insular damage. Once again, because almost half of the patients had persistent seizures (12 ILAE outcome class 1–2/21 patients), one cannot exclude the possibility that HRV changes were related to ongoing seizures rather than surgical damage to the insula. Since they did not include a healthy control group, and the duration between pre- and postsurgical recordings was very variable (from 9 to 168 months), it was unclear whether pre- and post-operative HRV values were in the normal ranges. In any case, patients in their series were hardly comparable to those in our series. In the former, most patients had temporal lobe epilepsy and surgeries systematically included the temporal lobe, but only variable portions of the insula . In the latter, all had IOE with only seven whose resection extended to the temporal.In 2019, the same group looked at HRV changes in patients undergoing temporal epilepsy surgery with variable resection/damage to the insula . AuthorsIn an exploratory subanalysis, we observed a normalization of low or high preoperative HRV values in individuals operated in the insulo-opercular region . Thus, it may be that the removal of the insula has a positive impact on autonomic function in the long run, at least if such a resection leads to a good post-operative seizure outcome (as was the case in 23/27 of our patients). Potential explanations for such a recovery include (a) arrest of seizure activity in the epileptogenic insula and areas of propagation involved in the central autonomic network; (b) gradual compensation by the intact contralateral insula; (c) reduction or withdrawal of antiepileptic drug after successful epilepsy surgery; (d) reduction in co-morbid anxiety and depressive disorders, which may impact HRV as well; (e) improvement in quality of life and a favorable psychosocial outcome ,54,55.In our study, median pre-operative HRV values in the TLE group did not significantly differ from those with IOE, as well as healthy controls. Fewer than a dozen studies have previously looked at interictal HRV in TLE, generating conflicting results. Similar to us, Varon et al.  failed tIn our study, no significant changes were noted between median pre-operative and post-operative HRV parameters. This is in line with findings from Persson et al.  who founIn patients with definite IOE (first part of the study), we found a positive correlation between pNN50 and RMSSD with the SUDEP-7 score. This positive correlation between these parasympathetic parameters and the SUDEP-7 score may suggest that, in IOE, the increase in parasympathetic tone could be more worrisome than its decrease. This is partly supported by case reports of ictal bradycardia, atrioventricular block, or asystole in patients with insular epilepsy ,61,62. IWhen evaluating each factor of the SUDEP-7 inventory in the IOE group, we found that the influence of each factor on HRV parameters was different. While the presence of at least one bilateral tonic-clonic seizure was significantly associated with high values of two HRV parameters (pNN50 and RMSSD), the others did not affect any HRV parameters. These results were not replicated in the TLE group, possibly because of the low prevalence of bilateral tonic-clonic seizures in that group (4/16 versus 7/16~in the IOE group). This suggests that HRV may rather reflect the severity of the chronic cardiac consequence of generalized/bilateral tonic-clonic seizures, rather than the combination of many factors. In a case series of 25 drug-refractory epileptic patients, Novak et al.  reportedWe acknowledge that our study has several limitations, mainly related to its retrospective design: the need to resort to ECG recordings obtained during pre-operative VEEG sessions (rather than dedicated ECG recording sessions), variable time intervals between surgery and post-operative ECG acquisitions, and variable antiseizure medications between patients. We also recognize that the trend towards normalization observed in IOE subgroups could reflect regression to the median rather than a true effect from successful epilepsy surgery. However, this effect was only noted in the IOE group and not in the TLE group, which could support a real contribution from successful insulo-opercular epilepsy surgery. Even if the regression to the mean is genuinely responsible for these observed findings, the good news is that the insular resection does not appear to induce further HRV impairments in patients with IOE. We also recognize that in part 1 of our study, it would have probably been preferable to only include IOE patients with Engel 1 outcomes (leaving out the only patient with an Engel 2 outcome) for a more reliable comparison with TLE patients ; however, when this patient was excluded from the analysis, the results were unchanged (data not shown). We also acknowledge that our IOE and TLE groups, while matched for age and sex, were unmatched for lateralization of the epileptic focus. Finally, we are also aware of the implications of conducting an exploratory study with a modest number of patients in which several pairwise comparisons were conducted and for which an accurate correction to control for Type I errors could not be estimated.p-values could be interesting enough to be worth reporting to encourage further work. On the other side, one must contend with the fact that OIE is relatively rare. To reach adequate power would require a multicenter collaboration. A standard protocol among sites for ECG recording and HRV analyses, both pre- and post-operatively, would need to be set in place to ensure that autonomic activity is assessed at a resting state, during sleep stages, and during autonomic activation tests. Adding other biomarkers for sudden cardiac death, such as corrected QT dispersion and T-wave alternans in refractory epilepsy, may improve the ability to identify the patients at high risk. Such better-designed studies should obviously not be limited to IOE, but should also include other types of epilepsy.Nevertheless, we deemed that some of the significant observations found with low In refractory IOE, our preliminary findings suggest that HRV may be either lower in parasympathetic tone or higher in both parasympathetic and sympathetic tones. Higher parasympathetic tone was also positively correlated with the revised SUDEP-7 inventory score. More importantly, we found no evidence that insulo-opercular surgeries lead to major autonomic dysfunction when a good seizure outcome is reached. Hence, when one is convinced that the seizure focus lies in the insula and believes that a good outcome can be reasonably attained with surgery rather than medical therapy, epilepsy surgery in this region is justified. The same goes for refractory temporal lobe epilepsy, as temporal lobectomies leading to a good outcome do not negatively impact on HRV. Our findings may pave the way for a multicenter prospective study with a larger number of patients that could ultimately impact patient care in favor for earlier surgical intervention for eligible candidates and better long-term monitoring of cardiac markers of SUDEP risk.


The impact of adrenalectomy on morbidity in patients with mild hypercortisolism and non-functioning adrenocortical adenoma is unclear. The present study evaluated morbidity before and after adrenalectomy in patients with benign adrenocortical tumour with Cushing´s syndrome (CS), autonomous cortisol secretion (ACS) and non-functioning adrenocortical adenoma as assessed by national and quality registries.Patients registered in the Scandinavian Quality Register for Thyroid, Parathyroid and Adrenal Surgery (SQRTPA) 2009–2017 with CS, ACS or non-functioning adrenocortical adenoma, were included in this retrospective study and analysed with age- and sex-matched controls, 1:3. Morbidity associated with CS was assessed pre- and postoperatively by analysing data from the Swedish National Patient Register and the Swedish Prescribed Drug Register.Some 271 patients were included, CS (127), ACS (45) and non-functioning adrenocortical adenoma (99), with 813 matched controls. The frequency of hypertension was almost 50% in all tumour groups. Antihypertensive medication preoperatively was more frequent in all tumour groups compared with controls. No preoperative differences in medication were detected between patients with CS and ACS. A decrease in the use of hypertensive drugs was noticed annually for all patient groups after adrenalectomy.Hypertension is common in patients with benign adrenocortical tumours regardless of cortisol hypersecretion. The use of antihypertensive drugs in patients with CS, ACS and non-functioning adrenocortical adenoma was reduced after adrenalectomy. These findings highlight the need for a randomized controlled trial to investigate the impact of adrenalectomy on morbidity in patients with mild hypercortisolism.The online version contains supplementary material available at 10.1007/s00268-021-06214-0. Adrenal incidentalomas are frequently detected in routine imaging with a prevalence of 1–4 percent . The majIt is still unclear if patients with ACS benefit from surgical treatment, comprehensive patient series are absent , 14, 17.The aim of this study was to evaluate the impact of adrenalectomy on morbidity potentially related to cortisol excess, in patients with CS, ACS and non-functioning adrenocortical adenoma, as assessed by national and quality registries, with a special focus on cardiovascular disease and related risk factors.The Scandinavian Quality Register for Thyroid, Parathyroid and Adrenal Surgery (SQRTPA) was established in 2004 and is recognized by the Swedish National Board of Health and Welfare as the national register within the field . The SweThe National Board of Health and Welfare administrate national registers to facilitate analysis and development of Swedish healthcare . QualityPatients registered as undergoing adrenalectomy 2009–2017 in SQRTPA, with CS, ACS or non-functioning adrenocortical adenoma, were included in the study. Patients were identified by preoperative laboratory findings and histopathology . CS and ACS was diagnosed by a specialist in endocrinology and based on applicable criteria at the time of diagnosis with 1-mg-overnight dexamethasone-suppression (DST) for initial evaluation , 25. NonMorbidity in patients with CS, ACS and non-functioning adrenocortical adenoma were compared to controls before and after adrenalectomy.th, 2018. Drugs used for treatment of hypertension, diabetes mellitus, hyperlipidaemia, osteoporosis, infectious diseases and depressive disorders were identified through ATC-codes  through the Swedish Population register . The cont test or Wilcoxon´s Rank-Sum test for continuous variables. The pre- and postoperative incidence density rates of disease diagnoses are calculated as the sum of diagnoses (ICD-10) in each disease group relative to the amount of person time at risk from start of follow up until surgery and from surgery until end of follow up respectively. The confidence intervals for the incidence density rate ratios (IRR) were calculated under the assumption of a Poisson distribution. All analyses were conducted using Stata v16.1 . p values below 0.05 were considered statistically significant.Aggregated data are presented as frequencies, medians with interquartile range (IQR) or with mean and standard deviation (SD). Frequencies were assessed based on provided data, no data are excluded or imputed. Statistical hypothesis tests were performed using the Chi-squared test for categorical variables and either Student´s The ethics committee, Lund university approved the study (No. 2017/800).Some 271 patients undergoing adrenalectomy were included in the study  is shown in Table Thirteen patients died within a year from adrenalectomy . Median postoperative follow-up for drug consumption and ICD-diagnoses for the different tumour groups were: CS 3.0 (min–max 0–9.9) years, ACS 2.6 (0–9.6) years, and non-functioning adrenocortical adenoma 3.2 (0–9.9) years. Differences in drug consumption one year before and one year after adrenalectomy are presented in Table The annual change in medication after adrenalectomy is presented in Fig. Analysis of data from NPR in Sweden showed no large differences in morbidity among the patient groups. Incidence rate ratios increased after surgery, or the corresponding date in controls, for ischemic heart disease in all patient groups as well as in controls Fig. .Fig. 3MoCushing’s syndrome is associated with high morbidity if untreated , 28. ThiIn the present study there were no major differences in morbidity, as evaluated with ATC-codes, for patients with CS and ACS before adrenalectomy. Nor for patients with ACS compared with non-functioning adrenocortical adenoma other than osteoporosis. Patient in all three groups used more antihypertensive drugs as compared to their controls, both before and after adrenalectomy. Medication increased the first year after adrenalectomy for all patient groups followed by a decrease annually. Distinct reductions annually were also detected for patients with CS for diabetes, hyperlipidaemia, osteoporosis, antibiotics and antidepressant medication. The results should be interpreted with some caution because of large statistic variability in the patient population and also because of possible reductions in medication for osteoporosis due to absence of fractures. The annual decrease in medication for diabetes and hyperlipidaemia were less distinct for patients with ACS compared with patients with CS. However, there were no differences in medication for hypertension or osteoporosis. Medication for all studied diseases decreased slightly over time including that of the control group. Possible causes include non-adherence, competing risks and secular trends. Interestingly, the frequency of hypertension and the use of antihypertensive drugs were higher for patients with non-functioning adrenocortical adenoma compared with controls, although lower than for patients with CS and ACS. Non-functioning adrenocortical adenoma are considered to compose no health risk, in this study they had an increased risk for antihypertensive medication. One reason for this could be to detection bias, although subtle hormonal hypersecretion not detected by current biochemical methods cannot be ruled out. Indeed, some reports indicate disturbances in cortisol secretion in patients with non-functioning adrenocortical adenoma compared with controls . UnfortuHypertension is the most frequent treatable risk factor for cardiovascular disease with prevalence in European countries of 25–44 percent , 31. In Most studies agree that non-functioning adrenocortical adenoma and ACS rarely progress to CS , 7, 32. Despite many reports of increased prevalence of cardiovascular risk factors in patients with non-functioning adrenocortical adenoma, there is a lack of data about the effects of adrenalectomy . It may Evaluation of drug use indicated a higher risk for hypertension, hyperlipidaemia, diabetes, osteoporosis and depression in patients with ACS compared with healthy controls but improvements could not be verified postoperatively neither in drug consumption, nor hospital care as assessed by in-patient ICD-10-codes. Current evidence does not allow general treatment guidelines in patients with ACS, individual evaluation and care are recommended , 37. TheThe accuracy of drug-based morbidity derived from ATC-codes has been debated. ATC-codes used in this study are comparable with earlier evaluations . A numbeThe risk for hypertension for patients with CS, ACS and non-functioning adrenocortical adenoma were high in this register-study. After the initial postoperative year an annual decrease in medication with antihypertensive drugs was noted for these patients. Adrenalectomy failed to decrease morbidity in cardiovascular disease based on inpatient-diagnosis in a short perspective. A prospective, randomized trial is hence warranted to evaluate the long-term impact of adrenalectomy on morbidity in patients with ACS and in patients with non-functioning adrenocortical adenoma.Supplementary file1 (PDF 183 kb)Below is the link to the electronic supplementary material.


Exercise-induced atrial fibrillation has been described in the literature and is a well-known phenomenon. It has been mostly described in long-distance runners. We present a case of a 69-year-old white male who had recurring atrial fibrillation with rapid ventricular response while paddle boarding, but not during other activities such as tennis and cycling. This case highlights the rationale behind different activities provoking atrial fibrillation and the need for multidisciplinary management of this entity including consulting with an electrophysiologist for possible early ablation. Age is the dominant risk factor for new-onset atrial fibrillation (AF). In one cross-sectional study of almost 2 million subjects in a health maintenance organization in the United States, the overall prevalence of AF was 1 percent; 70 percent were at least 65 years old and about half were ≥ 75 years old [The patient’s first episode of AF occurred in 2005 after being hospitalized for idiopathic small bowel obstruction requiring abdominal exploration and subsequently complicated by deep venous thrombosis/pulmonary infarction. In 2014, the patient had an episode of transient global amnesia associated with paddle boarding. At that time, no evidence of cerebrovascular accident or ongoing AF was noted. In 2019, the patient developed AF during paddle boarding. His only other past medical history was mild hypercholesterolemia. There was no history of illicit or performance-enhancing drugs or thyroid abnormalities.The patient was a participant in organized sports during early childhood , and long-term thereafter . In addition to organized sports, he regularly exercised from middle school onwards, scoring 470/500 on the President’s fitness program in 9th grade, and running a 4:30 mile his senior year of high school. At the age of 28, he ran a 66-minute ten-mile marathon. From age thirty until now, he has done some kind of aerobic exercise 3-5 times weekly, first running, then Nordic track, then indoor cycling on a spinner bike standing up , and going up and downstairs with a 45 lb. backpack (30 minutes each session). His most recent aerobic sport was stand up paddle boarding, which started in 2015 and was done year-round  until October 2019, when it was stopped due to AF.Levels of aerobic conditioning performing different exercises prior to the appearance of AF are shown in Figure The onset of a predisposition toward post paddle boarding AF likely occurred between July and September of 2019 Figure .After treatment with metoprolol, normal sinus rhythm (NSR) returned and the patient was discharged on propafenone (225mg twice daily), after consultation with an Electrophysiology (EP) Cardiologist (author JG). The hospitalization was otherwise significant for a negative cardiac stress test, normal cardiac echo , and normal troponins and TSH. A subsequent sleep study was negative for obstructive sleep apnea.After discussion with the EP Cardiologist, paddle boarding was reattempted resulting in a return of AF with RVR, in spite of paddling easily and being on propafenone Figure . The patThe patient eventually underwent atrial catheter ablation on May 4th 2020, after which he developed AF again during paddle boarding in August 2020. He was then started on low dose metoprolol succinate (25 mg daily) and resumed paddle boarding with no recurrence of AF on more than 40 different 2-mile paddle boarding outings, while still allowing near-maximum age-predicted HR  is the most prevalent arrhythmia in the world . It has It is important to note that the patient never developed AF or symptoms suggesting AF with any of the other exercises mentioned above, and his I-Watch was checked after each exercise episode was completed. The consistency with which AF occurred only with paddle boarding led us to search the literature for other cases of AF provoked by paddle boarding. To our knowledge, this is the first case of post-exercise AF associated only with paddle boarding.Exercise-induced AF is an infrequent yet well-known entity, it has been described in the medical literature as a syndrome called paroxysmal AF in young and middle-aged athletes (acronym: PAFIYAMA) . PAFIYAMLong-term endurance exercise can cause anatomical changes in an athlete’s heart including myocardial fibrosis, especially in the atria, which can be a risk factor for AF. Other factors that might link long-term excessive exercise and AF include increased vagal and sympathetic tone, increased atrial size, inflammatory changes, and bradycardia. In roughly 20% of competitive athletes, the left atrium may be enlarged which may be a predictor for AF. It is important to note that the relationship between left atrial size and AF is more complex in athletes than in non-athletes .Guideline documents recommend that PAFIYAMA be managed in consultation with an electrophysiologist ,5-7. EarIt is worth speculating about why post paddle boarding AF occurred in this aging athlete. Based on HR alone, paddle boarding produced higher sustained HR (160s) than stand up cycling, going up and downstairs with a 45 lb. backpack, running, or playing competitive tennis, suggesting it was responsible for the greatest stress on the heart. This is corroborated using the estimated maximum HR calculation 220 - age equal 150 for a 70-year-old, so heart rates in the 160s are likely to be at the high end of heart stress, even with a prior history of 50 plus years of aerobic exercise. As stated above, both increased sympathetic and parasympathetic heart stimulation can trigger AF, and this is especially true for aging athletes ,11. AlthThe American Heart Association and American College of Cardiology both recommend the following tests be done for any athlete with AF: performance-enhancing/illicit drug use questioning, thyroid function testing, electrocardiography, and echocardiography (Class I recommendations) . It has In summary, a 69-year-old athlete developing AF only after paddle boarding. Two antiarrhythmic drugs alone  were unsuccessful. Atrial ablation and low-dose metoprolol prevented any further paddle board-associated episodes of AF. The fact that metoprolol was successful likely means that for this athlete, increased sympathetic tone was the dominant trigger. Further research needs to be done to know whether this approach has value for the treatment of paroxysmal AF in other athletes.


Improvements in generalized anxiety symptoms, worry, intolerance of uncertainty, and mood were observed at post-treatment, maintained at 3-months, and the intervention received high ratings of treatment satisfaction. This is the first study to examine an augmented CBGT for perinatal women with GAD during the pandemic and supports the inclusion of strategies that target intolerance of uncertainty as well as specific pandemic and perinatal worry content for effective outcomes.The perinatal period is considered a window of vulnerability given the increased risk of psychiatric difficulties during this time, such as mood and anxiety disorders (ADs). Pre-pandemic rates of ADs in perinatal women were one in five but have since increased with the onset of the COVID-19 pandemic (COVID). In addition, recent research suggests that the focus of worry has shifted during the pandemic, with perinatal women reporting significantly more COVID-specific worries. The objective of this study was to augment our current evidence-based Cognitive Behavioural Group Therapy (CBGT) for perinatal anxiety protocol by targeting intolerance of uncertainty and tailoring existing strategies to address COVID-related worry and impact. Pregnant ( Pregnancy and the postpartum, often referred to as the perinatal period, are associated with increased risk of experiencing psychiatric difficulties such as mood and anxiety disorders ,2. AnxieGeneralized Anxiety Disorder (GAD), characterized by excessive and difficult to control worry , is the Given that the prevalence of perinatal anxiety has increased substantially since the onset of COVID, and the focus of worry often reflects the current context  and circumstance  of life, our team qualitatively examined the worry content of 84 treatment seeking pregnant and postpartum women during the pandemic (between April 2020 to October 2020), the majority (94%) of which, had a diagnosis of GAD ,22,23.Intolerance of uncertainty, defined as a dispositional characteristic that results from a set of negative beliefs about uncertainty and its implications and involves the tendency to react negatively on an emotional, cognitive, and behavioral level to uncertain situations and events , is assoKnowing that perinatal women with GAD are uniquely impacted by the COVID-19 pandemic provides justification for augmenting current treatment strategies to meet their unique mental health needs during this time. To date, no evidence-based psychological treatments have been developed for perinatal women with GAD that specifically target identified COVID-related worry content and its related impact despite the tremendous negative burden of COVID on pregnant and postpartum women . As suchn = 19) and postpartum (n = 49) women recruited from regular clinic patient flow, participated in this study. Pre-treatment assessments took place between September 2020 and March 2021. Eligibility criteria were as follows: (1) 18–45 years old; (2) pregnant or within the first 12 months postpartum; (3) a principal diagnosis of GAD confirmed through use of the Mini International Neuropsychiatric Interview for DSM-5 . Of the 14 pregnant participants who completed group treatment, 10 transitioned to postpartum by the post-treatment assessment, and an additional 3 transitioned by the 3-month follow-up.In the present study, 68 .All pre- to post-treatment outcomes for pregnant and postpartum women are presented in p = 0.08). Pregnant women however, demonstrated clinically meaningful reductions in worry symptoms, as post-treatment PSWQ scores were below the clinical threshold for this measure. Pregnant women showed statistically significant and clinically meaningful reductions in depression (p = 0.001), and statistically significant reductions in intolerance of uncertainty symptoms (p = 0.010) from pre- to post-treatment. Postpartum women demonstrated statistically significant and clinically meaningful reductions in worry (p < 0.001) and depression (p < 0.001), as well as statistically significant reductions in intolerance of uncertainty symptoms (p < 0.001) from pre- to post-treatment. There was no significant medication use by worry, depression, or intolerance of uncertainty symptom interactions from pre- to post-treatment in pregnant or postpartum women (p > 0.05).With regard to mean clinical change in secondary symptom outcomes, there were no statically significant reductions in worry symptoms for pregnant women from pre- to post-treatment (p = 1.00), worry (PSWQ: p = 0.13), depression (EPDS: p = 0.27), or intolerance of uncertainty (IUS: p = 0.31) for pregnant women, suggesting that treatment gains were maintained at 3-months follow-up. Similarly, there were no significant differences in GAD symptoms (GAD-7: p = 0.52), worry (PSWQ: p = 0.14), depression (EPDS: p = 0.43), or intolerance of uncertainty (IUS: p = 0.54) for postpartum women from post-treatment to 3-months follow-up. There were no significant medication use by GAD symptom, worry, depression, or intolerance of uncertainty interactions from post-treatment to 3-months follow-up in pregnant or postpartum women .All symptom improvements made by pregnant and postpartum women during treatment were maintained from post-treatment to 3-months follow-up see . Specifin = 7 pregnant, 50%; n = 23 postpartum, 62.2%) or ‘good’ , reported that the treatment ‘helped’  or ‘helped a great deal’ , and reported that they would recommend the treatment to others. The majority of pregnant  and postpartum  women reported being satisfied with the program.Treatment satisfaction was assessed at post-treatment and is reported for 14 pregnant women and 37 postpartum women. The majority of participants rated the treatment as ‘excellent’  were no longer experiencing excessive anxiety related to COVID-19 by the end of treatment. Similarly, 93.2% of participants  reported that the treatment addressed their worry content.Qualitative data collected at post-treatment also suggested that 71.7% of participants  from pre- to post-treatment. Again, gains on all secondary outcomes were maintained at 3-months follow-up for pregnant and postpartum women and were not influenced by psychotropic medication use. Given that worry is a defining feature of GAD , and hasWith regard to depression symptomatology, depression is highly comorbid with GAD ,62,63. FReductions in, and maintenance of intolerance of uncertainty symptoms in pregnant and postpartum women is another critical finding from our study, as intolerance of uncertainty in pregnancy has been linked to higher risk of anxiety worsening postnatally . FurtherThe present study has several limitations. First, we did not use a comparison condition and our sample size was relatively small, which limits generalizability and interpretation of results. As such, future studies should consider evaluating augmented CBGT in a larger sample and against a waitlist-control or other active comparison conditions to further establish its effectiveness. In addition, given the small sample size and limited number of pregnant women in our sample by post-treatment and follow-up, we were unable to evaluate what effect gestational age or weeks postpartum had on treatment outcomes. Future studies should consider evaluating whether participating in treatment at various times throughout the perinatal period affects treatment effectiveness. Further, since we did not evaluate the effectiveness of our traditional 6-session CBGT protocol with perinatal women with GAD during the pandemic, we are not certain as to whether that protocol would have been less effective, as effective, or even more effective than our augmented CBGT protocol. In addition, although the present study evaluated maintenance of treatment gains over a three-month period, future studies would benefit from assessing the longer-term impact of the augmented CBGT on primary and secondary outcome measures. Given that the sociodemographic characteristics of our sample were relatively homogenous , it is plausible that our augmented CBGT may not be effective for perinatal women of diverse sociodemographic backgrounds. Future studies should evaluate the effectiveness of our augmented CBGT in heterogenous samples of perinatal women. Finally, participants were allowed to use psychotropic medications during this study thus making our sample ecologically valid. While we found no medication use by symptom improvement interaction in our analyses, a future medication-free study could also be very informative.To our knowledge, this was the first study to examine the effectiveness of an augmented CBGT for perinatal GAD that is tailored to address the unique mental health needs of pregnant and postpartum women during the COVID-19 pandemic. Our study provides empirical support for a brief, eight-session augmented CBGT protocol to address symptoms of GAD, worry, intolerance of uncertainty, and depression in pregnant and postpartum women irrespective of psychotropic medication use during the COVID-19 pandemic.


Studies of outpatients with mild or moderate COVID-19 are uncommon. We studied: 1) association of symptoms with reverse transcriptase polymerase chain reaction (RT-PCR) test results; and 2) association of initial RT-PCR cycle threshold (Ct) in relation to duration of RT-PCR positivity in outpatients with mild or moderate COVID-19.This was a cohort study of outpatients with confirmed COVID-19 and at least one symptom. Participants had repeat nasopharyngeal swabs and symptom checklists every 3–5 days until two consecutive RT-PCR tests were negative. RT-PCR tests were used to assess viral load. Antibody tests for COVID-19 were performed at 2 weeks, 4 weeks, and 8 weeks after symptom onset.Twenty-five patients  were enrolled, ranging in age from 19–58 (median age 28 years). All patients reported at least one symptom, with a median of six symptoms per patient. Symptoms persisted for 6–67 days (median duration 18 days). In all 25 patients, blood samples collected a median of 13 days after symptom onset were positive for SARS-CoV-2 antibodies in 15 (60%). After a median of 28 days following symptom onset, 23/23 patients with available samples tested positive for antibodies. The longest duration of positive RT-PCR test was 49 days from first positive PCR test . Initial Ct was significantly associated with longer duration  of RT-PCR positivity.In mildly or moderately ill COVID-19 outpatients, RT-PCT tests remained positive for as long as 49 days and test positivity and symptom duration correlated with initial viral load. COVID-19, caused by SARS-CoV-2, has resulted in severe illness and hospitalization in a relatively small proportion of patients. Most patients remain asymptomatic or develop symptoms that do not require hospitalization. However, studies of outpatients with mild or moderate COVID-19 are uncommon. There is limited information in the published literature about the time period of resolution of symptoms in outpatients and the association of symptoms with viral load, as estimated by the initial reverse transcription polymerase chain reaction (RT-PCR) test results.A meta-analysis of 77 studies on viral shedding in hospitalized SARS-CoV-2 patients summarized that RT-PCR tests in this relatively ill group of patients remained positive for up to 63 days . To our To address gaps related to viral load and association of symptoms with viral load in outpatients with mild-to-moderate disease, we conducted a cohort study with the following aims: 1) to determine the association of symptoms with RT-PCR test results; 2) to study the association of initial PCR cycle threshold (Ct) in relation to duration of RT-PCR positivity in outpatients with mild-to-moderate COVID-19 who did not require hospital admission.The study was IRB-approved by NorthShore University HealthSystem (NSUHS), Evanston, Illinois. Since the beginning of the pandemic in 2020, NSUHS has operated immediate care facilities that offered clinical evaluation and RT-PCR testing to outpatients, with results usually available within 24 hours. At the time of this study, patients reporting at least one symptom consistent with possible COVID-19 infection were eligible for RT-PCR testing. NSUHS protocols followed Centers for Disease Control (CDC) guidelines to identify symptoms consistent with COVID-19 infection, which included: fever, chills, cough, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, new loss of taste or smell, sore throat, congestion or runny nose, nausea, vomiting, or diarrhea [According to NSUHS protocols at the time of this study, all patients with a positive COVID-19 RT-PCR result received a phone call with the test result from a member of the infection control team or a healthcare provider in the immediate care division. During this call, patients were asked about their willingness to be contacted about COVID-19 research studies for which they might qualify. If they agreed to be contacted, a CITI-trained  study teWe excluded individuals who presented with severe symptoms or signs such as SpO2 < 94%, or who required emergency department evaluation or hospitalization at any point during their illness course, or who were high risk by age (age of 65 or older), or who were pregnant. Eligible patients were contacted, and the purpose of the study was explained. If interested in participation, informed consent was sent electronically and an initial study visit was scheduled. The informed consent was reviewed at the beginning of the initial study visit to answer questions, ensure understanding and obtain a hard copy signature.Study participants had nasopharyngeal swabs collected every 3–5 days until the participant had two consecutive negative RT-PCR tests. Samples were transported within four hours of collection, and RT-PCR testing was done using the Abbott m2000 or Abbott Alinity m EUA reagent kits, or an in-house version of the CDC assay  on a LC4We used the RT-PCR test results and cycle threshold (Ct) to estimate viral load. Ct is the number of cycles required to pass the threshold for positivity and is a semiquantitative measure that is inversely correlated with viral genomic load. While Ct counts can vary by instrument, a typical RT-PCR assay runs a maximum of 40 cycles to detect the presence of viral genetic material in a sample .A symptom survey  was completed at each visit (performed every 3–5 days) or until the patient had two consecutive negative symptom surveys  between December 30, 2020 and February 15, 2021. Statistical significance was determined at p<0.05.Following exclusion of 2 patients due to early dropout, we determined the association of initial Ct count with duration of illness. The longest duration of positive RT-PCR test was 49 days from first positive PCR test . Initial Ct was significantly associated with longer symptom duration  of RT-PCR positivity . We alsoNine patients reported dyspnea and therefore were considered to have “moderate” COVID-19 compared to 15 patients with complete data for follow-up and no dyspnea. Comparing patients without dyspnea to those with, there were no significant differences in first Ct value , in duration of positive PCR test , and in duration of symptoms . Additional results for means of these measurements are shown in All patients were asked to provide blood samples for serologic antibody testing during study visits in weeks 2, 4 and 8. During week 2, samples were collected a median of 13 days after the onset of symptoms (range 10–20 days). Of the samples collected during week 2 in all 25 patients, 15 (60%) were positive for SARS-CoV-2 antibodies.During week 4, blood samples were collected a median of 28 days after symptom onset (range 24–34 days). Of the 23 patient samples collected during study week 4, all 23 were positive for SARS-CoV-2 antibodies.During week 8, samples were collected a median of 54 days after the onset of symptoms (range 52–65 days) in 21 patients, and all 21 samples that were collected were positive for antibodies.All available data for all patients, including symptoms, Ct, and antibody results are contained in This study of symptomatic mild or moderate COVID-19 outpatients resulted in several findings. First, we found a correlation between initial viral load, as measured by Ct values, and the duration of positive RT-PCR test results. Second, we found that the number of symptoms that patients experienced was also correlated with duration of positive RT-PCR tests. Third, there was considerable variability in the Ct values of outpatients presenting to an immediate care clinic. This number  is rarely reported to patients or physicians. To our knowledge, this is the first study of patients with mild-to-moderate COVID-19 in which serial respiratory samples were collected until RT-PCR tests became negative. In these non-hospitalized patients, we found that positive RT-PCR tests persisted for as long as 49 days after initial positive test.There is a small number of previous reports in symptomatic patients with mild or moderate COVID-19. Zou, et al reported on 17 mildly symptomatic patients from China . SimilarYilmaz, et al.  did seriThis study has both strengths and limitations. One of the strengths is the focus on mildly or moderately ill patients, in contrast to the numerous reports in hospitalized patients. We found only 8 previous studies focused on symptomatic outpatients despite the prevalence of this form of COVID-19. Another strength of this study is the serial testing for RT-PCR and careful tracking of symptoms in all patients. The major weakness of the study is its small numbers, but our findings, taken along with those in other reports, forms a picture of viral load dynamics in mildly or moderately ill patients.Our findings indicate that viral load tests by Ct value can be used to estimate duration of “viral shedding” by RT-PCR tests and resolution of symptoms. Since positive RT-PCR tests can be found for up to 49 days, RT-PCR test results should not be used as a guide to return to work or other daily activities.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Among multiple other functional roles of tissue factor (TF) and other coagulation proteins in the development and targeting of malignant disease, some scientific groups are attempting to modify TF and target the molecule or truncated forms of the molecule to tumor vasculature to selectively induce local blood vessel thromboembolic occlusion resulting in tumor infarction. This review briefly describes the characteristics and development of some of these proteins and structures, including tTF-NGR, which as the first drug candidate from this class has entered clinical trials in cancer patients.Besides its central functional role in coagulation, TF has been described as being operational in the development of malignancies and is currently being studied as a possible therapeutic tool against cancer. One of the avenues being explored is retargeting TF or its truncated extracellular part (tTF) to the tumor vasculature to induce tumor vessel occlusion and tumor infarction. To this end, multiple structures on tumor vascular wall cells have been studied at which tTF has been aimed via antibodies, derivatives, or as bifunctional fusion protein through targeting peptides. Among these targets were vascular adhesion molecules, oncofetal variants of fibronectin, prostate-specific membrane antigens, vascular endothelial growth factor receptors and co-receptors, integrins, fibroblast activation proteins, NG2 proteoglycan, microthrombus-associated fibrin-fibronectin, and aminopeptidase N. Targeting was also attempted toward cellular membranes within an acidic milieu or toward necrotic tumor areas. tTF-NGR, targeting tTF primarily at aminopeptidase N on angiogenic endothelial cells, was the first drug candidate from this emerging class of coaguligands translated to clinical studies in cancer patients. Upon completion of a phase I study, tTF-NGR entered randomized studies in oncology to test the therapeutic impact of this novel therapeutic modality. Amonggulation ,4. As figulation . As throgulation , standarConnected with its role in coagulation and thromboembolism, but also far beyond, TF biology in cancer is complex ,9. The mSince many tumor entities have been described as being TF-positive in immunohistochemistry and the presence of higher amounts of TF in tumors correlates with worse prognosis ,10,17,18In an attempt opposite to what has been described so far, several groups including our laboratory have tried to target TF or procoagulatory parts of the molecule at the tumor vasculature to selectively induce tumor vessel occlusion with resulting tumor infarction, tumor shrinkage, or inhibition of further tumor growth and spread. In general, this field was kicked off by the ground-breaking hypothesis of J. Folkman in 1971  that tumP. Thorpe’s laboratory was the first to propose a strategy of targeting the extracellular part of TF to a marker specific to the tumor endothelium . At thatThorpe’s group subsequently followed with a study using VCAM-1 as a target . VCAM-1 The VCAM-1 targeting of TF, initially started in P. Thorpe’s laboratory , was stuThe antibody technology described above was modified by D. Neri’s group. On the basis of a broad laboratory program designing and testing immunocytokines to target a growing number of tumor vascular markers described , the autThese observations were then further followed by T.S. Edgington’s laboratory, who used PSMA as a target . PSMA isWithin a search for novel tumor vascular targets T.S. Edgington’s laboratory next concentrated on the vascular endothelial growth factor (VEGF) isoform containing a heparin-binding domain (HBDt) as a potential target . They haLater, a similar fusion, in which the peptide SP5.2 with a sequence of 16 amino acids selectively binding the VEGFR1, was N-terminally linked to tTF by X. Li’s group at the Xiamen University in Fujian, China. This construct was functionally studied in vitro for binding to human umbilical vein endothelial cells (HUVECs) and retaining procoagulatory activity. In vivo it showed tumor homing upon systemic application of a fluorescent derivative and inhibitory effects in a sarcoma mouse model .Neuropilin-1 (NRP-1) is a co-receptor for VEGF. Recently, the same university in Fujian, China, chemically coupled an anti-NRP-1 antibody into an anti-NRP-1-streptavidin:tTF-biotin “composite system” and obtained similar tumor vascular thrombosis and tumor growth inhibition effects . The autR. Pasqualini in E. Ruoshlathi’s group described organ targeting in vivo by phage display of peptide library techniques  and lateDespite the disappointing results with RGD/tTF, we kept our parallel project active, in which we generated a fusion protein consisting of tTF and an RGD motif, GRGDSP, positioned to the C-terminus of tTF . This fuIn contrast to the negative results of RGD/tTF, a model fusion protein in which the targeting peptide was bound to the N-terminus of tTF, the COOH-terminal positioning of the targeting RGD-moiety in our tTF-RGD was unexpectedly successful. We had theoretically deduced this conformation by replacement of the transmembrane peptide present in the identical C-terminus position of the naturally occurring TF, which renders the molecule procoagulative by forming the TF:FVIIa complex and activating FX on the cell membrane in the neighbourhood of PS-rich phospholipids. When considering the crystal structure of the TF:FVIIa complex , linkageIn 2000 Rippmann et al. reported on a fusion protein composed of a humanized scFv targeting FAP, abundantly present in tumor tissues and the extracellular domain of TF . This coD in the higher nanomolar range, and the in vivo therapeutic effects against human tumor xenografts were disappointing with a rather small therapeutic window.Within our search for better targets directing tTF via peptides linked to the C-terminus of the molecule into the tumor vasculature, we have also tested NG2 as a surface antigen on angiogenic pericytes . NG2 is 3) was described as binding to fibronectin-fibrin microthrombi inside tumors undergoing EMT, and thus was used for magnetic resonance imaging (MRI) detection of 4T1 breast cancer micrometastasis models [In vivo screening of phage-displayed peptide libraries for tumor homing in tumor-bearing MMTV-PyMT transgenic breast cancer mice by Ruoslahti’s group also identified a pentapeptide, CREKA . As CREKs models .Recently, G. Nie’s group in Beijing described a tTF-CREKA fusion protein as procoagulatory, accumulating in tumors in biodistribution studies, and inducing tumor-selective intravascular thrombosis. tTF-CREKA inhibited growth in model tumors of breast (4T1), hepatocellular (MHCC97H) and colon (LS174T) origin .In the series of antitumor tTF-constructs made and tested by G. Nie’s group, in some model tumors tTF-CREKA was more efficient than a fusion protein, tTF-pHLIP, a construct forming a helical structure inserting into cellular membranes at low pH . HoweverIt was again R. Pasqualini’s laboratory group who proposed the binding of small NGR (asparagine-glycine-arginine)-containing peptides to aminopeptidase N  for tumor vascular targeting . Later, In 1998, stimulated by P. Thorpe’s work , our labDuring these studies, his-tag-tTF-GNGRAHA (tTF-NGR) evolved as the lead compound due to most stable antitumor activity. The fusion protein is graphically depicted in tTF-NGR retained the procoagulatory activity of TF in a FX/FXa assay ,70,71 anWe have performed orienting dose-response studies in our early xenograft mouse experiments. To this end, we have repeatedly found single doses of 0.5 mg/kg body weight to be ineffective for tumor growth inhibition and 0.75 mg/kg as a dose with marginal activity. These early studies were done also in the HT1080 fibrosarcoma model and the results are among those given in In addition to the tumor growth inhibition in the xenograft models of human tumors, an antimetastatic effect was seen in a syngeneic B-16 melanoma model when targeted tTF was given close to the surgical removal of the primary tumor .As locoregional toxicity leading to necrosis of mouse tail tips after repeated intravenous injections of the concentrated tTF-NGR occurred, the molecule was polyethylene glycol conjugated (PEGylated) in a random  modificaInterestingly, therapeutic combination protocols using tTF-NGR in specific sequences with regional ultrasound , radiothWhereas TF or procoagulatory parts of the molecule retargeted to tumors with different molecular constructs and within multiple studies in the last 20 years have shown promising anti-tumor activity in various preclinical models, none of the molecules studied have been consequently developed into an investigational new product (IMP) for clinical application in cancer patients. Certainly, there is a medical need to improve the outcome of different advanced cancer entities by novel treatment modalities. Among other reasons for this hesitation are concerns about off-target procoagulatory effects of tTF not bound to the targets on tumor vasculature cells leading to systemic thromboembolism. To this end, it is important to note that soluble TF without the transmembrane domain was reported to have minor procoagulatory effects, but its activity is restored once TF is anchored into a phospholipid PS rich cellular membrane ,83. On ttTF-NGR primarily binds to the aminopeptidase CD13. In contrast to regular mature vascular tissue, CD13 is predominantly prevalent in juvenile and growing endothelial tissue and is therefore mainly found in the endothelial cells (EC) of tumor vasculature . AdditioE. coli fermenters and a 4-step purification process on HPLC as described in detail [After setting up a GMP process and laboratory using n detail , and obtn detail . In addin detail , and thetTF-NGR has recently been investigated in a first-in-class phase I study, which recruited a total of 17 patients suffering from a variety of different solid tumor entities with previously exhausted standard therapy ,88. The 2 body surface area (bsa) per day. The protocol allowed for intraindividual dose escalation and once dose-limiting toxicity (DLT) was observed, maximum tolerated dose (MTD) was established in verification cohorts of 6 patients. The primary objectives of the study were safety and tolerability including establishing DLT and MTD for the protocol applied. Secondary objectives were to assess for tumor blood flow reduction under therapy as proof of principle, tumor response, and to perform pharmacokinetic studies of tTF-NGR. MTD was reached at 3 mg/m2 tTF-NGR/day × 5, q day 22. DLT was observed as an isolated significant increase of high sensitivity (hs) Troponin T hs. This increase remained without clinical sequelae and was completely reversible upon discontinuation of treatment. Three thromboembolic events (grade 2), tTF-NGR-related besides other relevant risk factors, were observed within this group of 17 patients treated. The dose levels were 3, 4, and 5 mg/m2 bsa (1 event at each level) respectively, and although the numbers were small, a dose-related occurrence is possible. All 3 thromboembolic events were completely reversible upon anticoagulation and the ceasing of further infusions. A daily application of tTF-NGR for 5 consecutive days  with rest periods of 2 weeks between cycles was chosen, and the starting dose was 1 mg/mImportantly, blood flow was significantly and specifically reduced in the tumor tissue of all measurable lesions as shown by dynamic contrast-enhanced ultrasound (CEUS) and dynamic contrast-enhanced (DCE) magnetic resonance imaging (MRI), serving as evidence for the mechanism of action of tTF-NGR. An example of a CEUS imaging series in one patient is shown in The last two decades have seen multiple experimental projects incorporating TF into bifunctional proteins and retargeting the molecule to the cells of the tumor vasculature wall for induction of tumor infarction. With some structural exceptions ,51, the However, with the reproducibility of the efficacy and safety data in animal models and the urgent medical need to improve therapy for advanced cancer by new treatment modalities, our group decided to translate the drug candidate tTF-NGR into clinical testing in cancer patients.The primary target for tTF-NGR binding to tumor vascular cells is aminopeptidase N (APN), classified as CD13 . CD13 isTo shed some more light on the field, similar, but also methodologically separate studies have attempted to increase intratumoral coagulation without targeting a specific molecule within the neoplastic tissue via a bifunctional fusion protein. To this end, it is interesting that soluble TF when coadministered with low-dose lipopolysaccharides has been described as inducing coagulation on tumor endothelial cells , an effeIn addition, Seidi et al. have used the GNGRAHA peptide for targeting molecules than other TF, e.g., staphylococcus coagulase in tumor vessels, and observed tumor accumulation and infarction in a PC3 human prostate tumor model with tolerability at therapeutic doses .Meanwhile, as phase I data are available, tTF-NGR has entered randomized clinical trials in cancer patients, one of which will compare progression free survival of patients suffering from advanced soft tissue sarcomas treated in 2nd line with trabectedin versus trabectedin plus tTF-NGR (EudraCT-Nr: 2020-005858-21). We hope these randomized trials will give conclusive data on the clinical impact of the first drug candidate for this novel treatment modality. Only then can we answer the question of whether one of the approaches described can reveal an “upside of coagulation in cancer”  and can The last two decades have seen multiple experimental projects to incorporate TF into bifunctional proteins and retarget the molecule to tumor vasculature wall cells for inducting vascular occlusion and tumor infarction. This has been successfully exploited in different experimental models for cancer therapy. A first drug candidate, tTF-NGR, was translated into the clinic and has shown applicability in a phase I trial. It has now entered a randomized study in patients with metastasized sarcoma. However, due to potential side effects, further studies with tTF-NGR must be performed with caution and in a clinical setting with specific hemostaseology expertise available.W.E.B. and R.M.M. are inventors of patents on vascular targeting with tissue factor-constructs.


The maturation of the oocyte is influenced by cumulus cells (CCs) and associated with pregnancy rate, whereas the influencing factors have not been completely elucidated in the CCs. In this study, we identified new regulators of CCs for high-quality oocytes and successful pregnancies during assisted reproductive techniques. CCs were collected from cumulus–oocyte complexes (COCs) in young (≤33 years old) and old (≥40 years old) women undergoing intracytoplasmic sperm injection (ICSI) procedures. We screened for factors differentially expressed between young vs. old CCs and pregnancy vs. non-pregnancy using whole mRNA-seq-next-generation sequencing (NGS). We characterized the transcriptome of the CCs to identify factors critical for achieving pregnancy in IVF cycles. Women in the young and old pregnancy groups exhibited the up- and downregulation of multiple genes compared with the non-pregnancy groups, revealing the differential regulation of several specific genes involved in ovarian steroidogenesis in CCs. It was shown that the low-density lipoprotein (LDL) receptor to the steroidogenesis pathway was upregulated in CCs with higher maturity rates of oocytes in the pregnancy group. In conclusion, a higher pregnancy rate is related to the signaling pathway of steroidogenesis by the LDL receptor in infertile women undergoing IVF procedures. In assisted reproductive technology (ART), age is a major determinant of successful pregnancy outcomes ,2,3. CurCCs have different roles and sustain the activities of different factors required for the production of competent oocytes depending on the phase of folliculogenesis ,13. The Until now, several studies aimed at identifying the predictive biomarkers of oocyte and embryo competence have reported the RNA profiling of specific factors in human CCs using microarrays and next-generation sequencing (NGS) ,18. HoweHere, we focus on identifying factors that are differentially regulated between young and advanced age women and between those who have achieved successful pregnancies and those who have not . We aimed to use NGS-based, whole-transcriptome databases to detect new regulators of ovulation in high-quality oocytes that are likely to increase the rate of successful pregnancies in advanced age IVF patients.n = 8); (2) young non-pregnancy ; (3) old pregnancy ; and (4) old non-pregnancy . No personal information about study subjects was collected other than check-up age and category of infertility diagnosis. Patients participating in the study had been diagnosed with infertility due to male, tubal, or uterine factors. Women with infertility due to endocrine diseases, such as polycystic ovarian syndrome (PCOS) or women with genetic diseases were excluded. All patients underwent conventional controlled ovarian stimulation (COS) or minimal stimulation protocols with a Gonadotropin-releasing hormone (GnRH) antagonist protocol during the IVF procedure. When there was at least one or more 18-mm follicle, ovulation was triggered by recombinant human chorionic gonadotropin (hCG) injection and oocytes were retrieved 36 h later. After ovum pick-ups, we isolated CCs by denuding oocytes with 0.1% hyaluronidase  for ICSI. Isolated CCs were washed in PBS and stored at −80 °C until mRNA purification. Eight samples were collected for each group. All clinical data were collected using electronic medical records, including baseline characteristics , infertility factors, previous IVF cycles, etc.) and basal hormone levels. Parameters related to IVF cycles were also investigated, including number of retrieved oocytes, and maturation, fertilization, and clinical pregnancy rates. A high-quality embryo at the cleavage stage was defined as a 4-cell embryo on day 2 and a 7- or 8-cell embryo on day 3, containing <20% anucleate fragments, and exhibiting no apparent morphological abnormalities. As for blastocyst-stage embryos, a good-quality embryo was defined according to the Gardner and Schoolcraft criteria [This study was approved by the Institutional Research and Ethical Committees of CHA University , Republic of Korea. All participating researchers underwent training and received a certificate for biomedical research with human materials. All women gave written informed consent to provide material for this study. All procedures followed the rules for studies with human-origin materials established by the IRB. Patients were divided into four groups according to age (“young” ≤ 33 years old vs “old” ≥ 40 years old) and pregnancy: (1) young pregnancy . mRNA was quantified using a NanoDrop ND-1000 spectrophotometer . Purified mRNA was subjected to mRNA-seq using a MiSeq sequencer, , and sample sheets were prepared to provide run details. Bioinformatic analysis was performed on the whole-transcriptome raw data obtained from NGS.Whole-transcriptome data were analyzed using the human genome sequence as reference . From the initial NGS data, 27,685 genes were identified under the 4 sample conditions using the stringTie-e option. The data were then normalized to fragments per kilobase of exon per million fragments mapped (FPKMs) using Cufflinks. To filter potentially significant gene expression, genes with more than one FPKM value of 0 were excluded. To clarify the relationship between samples, unsupervised hierarchical clustering analysis and multidimensional scaling (MDS) analysis were performed on the expression data for 17,317 genes; we then sought to confirm the presence of outlier samples and similar expression patterns between biological replicates.p-value ≤ 0.05 were included as DEGs in the subsequent analysis . To display DEG expression patterns, hierarchical clustering analysis was performed using complete linkage and Euclidean distance as a measure of similarity. The top 10 Gene Ontology terms, in ascending order of p-values, are shown. Next, we analyzed the list of top-ranking up- and downregulated genes and plotted each up and down gene cluster as a function of age and pregnancy outcome. Next, we performed differential expression gene (DEG) analysis. For this purpose, log2(FPKM + 1) values were calculated and normalized by the quantile method. Transcripts with fold-change values >2 with p-values were adjusted by the Benjamini–Hochberg procedure. However, because all adjusted p-values were statistically insignificant, we used p < 0.05 instead of adjusted p < 0.05 to identify DEGs.Each group was investigated according to age and pregnancy competence. To identify key DEGs associated with pregnancy competence, we compared three pairs: (1) old pregnancy vs. old non-pregnancy; (2) young pregnancy vs. young non-pregnancy; and (3) pregnancy vs. non-pregnancy, regardless of age. In the first two comparisons, we used fold-change values (>2 or <0.5) to identify DEGs, as each group contained only one sample. In the final comparison, we analyzed the data using the t-test, and http://david.abcc.ncifcrf.gov/tools.jsp, v.6.8 web software). After GO analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed to identify ovary specific signaling pathways in the gene enrichment data of DEGs with p-value < 0.05. The threshold was set as modified Fisher’s Exact p-value (EASE score) ≤ 0.05.DEG data were uploaded to the online Database for Annotation, Visualization, and Integrated Discovery (DAVID) program . Functio® CycleScript RT PreMix  with poly-dT. qPCR was performed with AccuPower Taq PCR PreMix (Bioneer) in a spectrofluorometric thermal cycler . Based on the KEGG signal pathways, we selected up- and downregulated target genes involved in ovarian steroidogenesis as a function of age and pregnancy outcome. Real-time qPCR was performed using the SsoAdvanced Universal SYBR Green Supermix  with specific primers . All data are expressed as means ± standard error of the mean (SEM) of triplicate measurements. Statistical analyses were carried out using t-tests with significance level set at * n = 16) or old  and pregnancy or non-pregnancy. Age-related AMH was significantly reduced in the old group relative to the young group. However, the clinical outcomes did not significantly differ within each age group as a function of BMI, number of previous IVF cycles, or basal FSH level. All IVF patients were stimulated using GnRH antagonist protocols with no significant difference in the doses of gonadotropin administered. The oocyte properties such as the number of retrieved oocytes and the fertilization rate did not significantly differ among groups. However, the oocyte maturation rates were significantly different among groups. Based on the clinical outcomes, pregnancy was related to oocyte and embryo quality in both the young and old groups. The clinical pregnancy group had a significantly greater number of blastocyst formation rates and higher number of high-quality blastocysts than the non-pregnancy group. Hence, we performed whole-transcriptome analysis by NGS following n = 836)  C. Table Both young and old pregnancy groups showed the upregulation of genes in the category “cellular component” such as: zinc finger protein, NME1-NME2 readthrough, and the HECT and RLD domain containing E3 ubiquitin protein ligase 2 pseudogene. The old pregnancy group exhibited elevated expressions of “single-organism process” genes such as NGFR. Among “cellular component” related genes, the young and old pregnancy groups showed significantly high expression of “cell part” and “cell-related” gene processes. Among molecular function genes, both young and old pregnancy groups showed significantly high expression of cell–cell adhesion related genes. Therefore, we performed a key signal pathway analysis of the functional network of each gene in young and old pregnancy patients.In both the real and simulated data of the young vs. old and pregnancy vs. non-pregnancy groups, we assessed the impact of the normalization methods using the results of the DEG analysis A. We perNext, we performed real-time qPCR to validate the mRNA-seq data. The samples used for NGS were subjected to RT-PCR with primers specific for up- and downregulated genes. In this study, we found specific factors in CCs that were associated with successful pregnancy in advanced aged IVF women using whole-transcriptome NGS analysis. Except for embryo quality and blastocyst rates, the clinical criteria  did not differ significantly between the age and pregnancy groups. The pregnancy group had significantly higher blastocyst quality than the non-pregnancy group. Hierarchical clustering and multidimensional scaling analysis of whole-transcriptome NGS data revealed different up- and downregulated DEG clusters depending on young and age groups with archive pregnancy; upregulated genes included LDLR and StAR, and downregulated genes included ADCY and HSD17B, whose expression is associated with the ovarian steroidogenesis signaling pathway. In addition, based on the validated data, the whole-transcriptome NGS data showed a similar up- and downregulation of gene expression profiles between the young and old pregnancy groups.Based on the DEG data from whole-transcriptome NGS, the pregnancy group (both young and old) exhibited an upregulation of genes including ZNF117, ITGA2, and SFRP4, which are involved in the functional regulation of GCs, oogenesis, and follicular development in the ovary ,28,29,30DEG data were analyzed using the DAVID functional annotation clustering tool. DEG data used to visualize the KEGG pathway result revealed the up- and downregulation of gene signaling pathways. KEGG pathway mapping of gene set enrichment analysis data revealed that ovarian steroidogenesis was strongly associated with successful clinical pregnancy in patients of advanced age. The presence of LDL is required for maximal progesterone secretion by cultured human granulosa cells . The NGSThis study identified LDLR-linked steroidogenesis activity in cumulus cells as a potentially important factor for reducing infertility in women of advanced age. Infertility is a global health issue, and it is considered that about one in seven couples may have difficulty conceiving. In the clinical field, the number of women of advanced age continues to increase, and accordingly, the successful pregnancy rate through IVF procedure is declining. Globally, the average age at which people marry has increased, and the mean age at which women experience infertility has also dramatically increased over the last decade. Older infertile women have an unmet need to rid themselves of the age-related issues that prevent successful pregnancies and to experience a healthy birthing experience. This study opens the prospect of the development of a new ovarian stimulation protocol that overcomes the issues that prevent pregnancy at an advanced age.We identified a specific transcriptome profile in the CCs of young and old IVF women. Up-regulation of LDLR and StAR was associated with embryo quality and clinical pregnancy in infertile women. Those data could be applied to new COS protocols to activate LDLR and StAR in infertile women of advanced age, which may help to increase maturation ratios and pregnancy rates in the IVF cycles. Further studies of LDLR upregulation should be performed in infertile women who have not previously had a successful pregnancy in a large size prospective model.