Cushing Syndrome: A Review.

Importance: Cushing syndrome is defined as a prolonged increase in plasma cortisol levels that is not due to a physiological etiology. Although the most frequent cause of Cushing syndrome is exogenous steroid use, the estimated incidence of Cushing syndrome due to endogenous overproduction of cortisol ranges from 2 to 8 per million people annually. Cushing syndrome is associated with hyperglycemia, protein catabolism, immunosuppression, hypertension, weight gain, neurocognitive changes, and mood disorders. Observations: Cushing syndrome characteristically presents with skin changes such as facial plethora, easy bruising, and purple striae and with metabolic manifestations such as hyperglycemia, hypertension, and excess fat deposition in the face, back of the neck, and visceral organs. Cushing disease, in which corticotropin excess is produced by a benign pituitary tumor, occurs in approximately 60% to 70% of patients with Cushing syndrome due to endogenous cortisol production. Evaluation of patients with possible Cushing syndrome begins with ruling out exogenous steroid use. Screening for elevated cortisol is performed with a 24-hour urinary free cortisol test or late-night salivary cortisol test or by evaluating whether cortisol is suppressed the morning after an evening dexamethasone dose. Plasma corticotropin levels can help distinguish between adrenal causes of hypercortisolism (suppressed corticotropin) and corticotropin-dependent forms of hypercortisolism (midnormal to elevated corticotropin levels). Pituitary magnetic resonance imaging, bilateral inferior petrosal sinus sampling, and adrenal or whole-body imaging can help identify tumor sources of hypercortisolism. Management of Cushing syndrome begins with surgery to remove the source of excess endogenous cortisol production followed by medication that includes adrenal steroidogenesis inhibitors, pituitary-targeted drugs, or glucocorticoid receptor blockers. For patients not responsive to surgery and medication, radiation therapy and bilateral adrenalectomy may be appropriate. Conclusions and Relevance: The incidence of Cushing syndrome due to endogenous overproduction of cortisol is 2 to 8 people per million annually. First-line therapy for Cushing syndrome due to endogenous overproduction of cortisol is surgery to remove the causative tumor. Many patients will require additional treatment with medications, radiation, or bilateral adrenalectomy.

Pregnancy complicated with adrenal adenoma causing ACTH-independent Cushing's syndrome, accompanied by obstetric antiphospholipid syndrome and severe pre-eclampsia: case report and literature review.

This case report shares the management experience of a patient with pregnancy combined with adrenal adenoma causing ACTH-independent Cushing's syndrome (CS), accompanied by obstetric antiphospholipid syndrome (OAPS) and severe pre-eclampsia. The case was a 26-year-old that presented with typical clinical symptoms and signs of CS. The patient had a history of 4 spontaneous abortions in the last 4 years. The 24-hour urinary free cortisol was significantly increased, an abnormal cortisol circadian rhythm was demonstrated by a high late-night salivary cortisol, blood ACTH was suppressed (< 1ng/dL), anticardiolipin antibody was positive, and imaging examination showed an adrenal tumor. The patient underwent laparoscopic adrenal tumor resection under general anesthesia at 23 weeks of gestation. The tumor was pathologically confirmed to be an adrenocortical adenoma. The patient underwent a cesarean section at 39 weeks of gestation to give birth to a healthy baby girl with an Apgar score of 10. Pregnancy complicated by CS is clinically rare, easily masked by normal physiological changes of pregnancy, and is difficult to diagnose. The determination of 24-hour urinary free cortisol, the circadian rhythm of serum cortisol, ultrasound, and MRI can be helpful in the diagnosis of CS during pregnancy. Surgery is the first choice for the treatment of CS during pregnancy. As a subtype of antiphospholipid syndrome, patients with OAPS are prone to thrombotic events and recurrent miscarriages if not treated accordingly. To our knowledge no cases of CS with OAPS and severe pre-eclampsia have been reported. We summarize the experience of the treatment of this patient and review the literature to improve clinicians' awareness of this disease.

Eleven-Year Experience with Selective Adrenal Vein Sampling in Management of Primary Adrenal Hormonal Hypersecretion.

Introduction: Nearly half of the adult population in the United States has been diagnosed with hypertension. Adrenal hormonal hypersecretion is a leading cause of secondary hypertension. Adrenal vein sampling (AVS) may assist in differentiating between unilateral and bilateral adrenal hormonal hypersecretion to identify patients who are candidates for adrenalectomy. We reviewed the use of AVS at our institution along with associated outcomes after adrenalectomy. Materials and Methods: A retrospective chart review was conducted of patients with a diagnosis of primary hyperaldosteronism (PA) or adrenocorticotropic hormone-independent Cushing syndrome (AICS) and who underwent adrenalectomy between January 1, 2010, and December 1, 2021. Patient data of baseline characteristics, preoperative workup, including AVS, and postoperative outcomes were collected and analyzed. Results: Seventy-one patients were identified in the study period (48 PA and 23 AICS). Computed tomography scan identified unilateral adrenal nodules in 52 patients (29 left; and 23 right), bilateral nodules in 13 patients, and no nodules in 6 patients. AVS was performed in 45 patients with PA (93%) and 5 patients with AICS (21%). After surgery, the number of PA patients with hypokalemia or requiring potassium supplementation significantly decreased after adrenalectomy (before surgery: 33 [68.7%]; and after surgery: 5 [10.4%], P < .01). The number of medications required for hypertension in AICS patients also significantly decreased. No major adverse events were noted. Conclusions: Our long-term experience demonstrates the ongoing use of AVS during workup of patients with primary hyperaldosteronism and for select patients with adrenocorticotropic hormone-independent Cushing syndrome. However, a low level of discordance between imaging and AVS findings in PA patients suggests that there may be a subset of patients in whom preoperative AVS is not necessary.

Inflammatory biomarkers in the evaluation of pediatric endogenous Cushing syndrome.

OBJECTIVE: Inflammatory biomarkers, such as absolute neutrophil and lymphocyte counts, neutrophil-to-lymphocyte ratio (NLR), platelet (PLT)-to-lymphocyte ratio (PLR) and monocyte-to-lymphocyte ratio (MLR), are associated with the progression and development of several disorders. Although patients with Cushing syndrome (CS) have immunosuppression with altered leucocyte counts, the profile of the inflammatory biomarkers in these patients has not been extensively studied. DESIGN: We compared a panel of inflammatory biomarkers in patients with active endogenous CS (n of complete blood count (CBC) reports = 319) and eucortisolemic subjects of similar age, gender and BMI (n of CBC reports = 93). Patients were divided into two age groups (6-12 years at the time of CBC and >12 years at the time of CBC) based on age differences in normal reference ranges. RESULTS: Patients with CS had higher NLR vs controls (6-12 years: 2.47 (1.86, 3.32) vs 1.35 (1.11, 2.27), P < 0.0001; >12 years: 3.00 (2.23-4.17) vs 1.80 (1.23-2.31), P < 0.0001). Similarly, absolute neutrophil and lymphocyte counts, MLR and PLR differed between patients with CS and controls. The inflammatory biomarkers correlated with indices of cortisol secretion, such as midnight serum cortisol, 24-h urinary free cortisol and morning cortisol. On receiver operating characteristic analysis, NLR showed high area under the curve (AUC) (6-12 years: cutoff of 1.72 had AUC: 0.77, >12 years: cutoff of 2.35 had AUC: 0.81). CONCLUSIONS: We conclude that multiple inflammatory biomarkers differed between patients with CS and controls suggesting substantial effects of hypercortisolemia on the immune system.

Recent Advances in the Clinical Application of Adrenal Vein Sampling.

We reviewed clinical research investigating the applications of adrenal vein sampling (AVS). AVS could be applied not only to primary aldosteronism (PA) but also to other endocrine diseases, such as adrenocorticotropic hormone (ACTH) independent Cushing syndrome (AICS) and hyperandrogenemia (HA). However, the AVS protocol requires improvements to increase its success rate. Using the computed tomography image fusion, coaxial guidewire technique, and fast intraprocedural cortisol testing (CCF) technique could improve the success rate of catheterization in AVS for PA. ACTH loading could be considered in medical centers with a low selectivity of AVS for PA but is not essential in those with mature AVS technology. The continuous infusion method should be recommended for ACTH stimulation in AVS for PA to reduce adverse events. AVS has not been routinely recommended before management decisions in AICS, but several studies verified that AVS was useful in finding out the source of excess cortisol, especially for distinguishing unilateral from bilateral disease. However, it is necessary to reassess the results of AVS in AICS with the use of reference hormones to fully normalize cortisol levels. In addition, it is essential to determine the optimal model that combines AVS results and mass size to guide the selection of surgical plans, including identifying the dominant gland and presenting the option of staged adrenalectomy, to minimize the impact of bilateral resection. For HA, AVS combined with ovarian intravenous sampling to locate excess androgens could be considered when imaging results are equivocal.

Evaluation of procoagulant imbalance in Cushing's syndrome after short- and long-term remission of disease.

OBJECTIVE: Patients with Cushing's syndrome (CS) are at high risk of venous thromboembolism related to a hypercoagulability due to procoagulant imbalance. However, whether these alterations are reversible after disease remission is still unclear. The endogenous thrombin potential (ETP) measured with and without the addition of thrombomodulin provides a global representation of coagulation and previous data confirmed hypercoagulable profile in patients with active hypercortisolism. Aim of this study was to assess the short- and long-term modification of ETP in patients with CS after disease remission. DESIGN AND METHODS: Nineteen patients with CS for whom surgical remission was achieved, were prospectively evaluated for clinical characteristics, cortisol secretion profile and ETP at different time points: (i) before surgical intervention; (ii) after 6 months and (iii) 5 years from the time of persistent remission. Nineteen healthy subjects matched for age and gender were also evaluated as control group. RESULTS: Before surgery, patients showed higher ETP-ratio (with/without thrombomodulin) than controls (0.62 ± 0.09-vs-0.56 ± 0.09, p = 0.034). No significant correlation between ETP-ratio and cortisol secretion was found. 6 months after remission, ETP-ratio was still significantly increased compared to controls (0.64 ± 0.09-vs-0.56 ± 0.09, p = 0.01), but was similar to baseline (0.64 ± 0.09-vs-0.62 ± 0.09, p = 0.87). At 5 years, ETP-ratio showed a significant decrease (0.55 ± 0.14-vs-0.62 ± 0.09, p = 0.02) and was comparable to controls (0.55 ± 0.14-vs-0.56 ± 0.09, p = 0.7). CONCLUSIONS: Plasma hypercoagulability detected in patients with active hypercortisolism persists at short-term evaluation and seems to be completely reversible after long-term remission of disease. These data, as part of a whole evaluation of thrombotic risk, can contribute to make appropriate therapeutic choice in these patients.

Interpretation of Abnormal Dexamethasone Suppression Test is Enhanced With Use of Synchronous Free Cortisol Assessment.

CONTEXT: Interpretation of dexamethasone suppression test (DST) may be influenced by dexamethasone absorption and metabolism and by the altered cortisol binding. OBJECTIVE: We aimed to determine the normal ranges of free cortisol during DST in participants without adrenal disorders and to identify the population of patients where post-DST free cortisol measurements add value to the diagnostic workup. DESIGN AND SETTING: Cross-sectional study conducted in a tertiary medical center. PARTICIPANTS: Adult volunteers without adrenal disorders (n = 168; 47 women on oral contraceptive therapy [OCP], 66 women not on OCP, 55 men) and patients undergoing evaluation for hypercortisolism (n = 196; 16 women on OCP). MEASUREMENTS: Post-DST dexamethasone and free cortisol (mass spectrometry) and total cortisol (immunoassay). MAIN OUTCOME MEASURES: Reference range for post-DST free cortisol, diagnostic accuracy of post-DST total cortisol. RESULTS: Adequate dexamethasone concentrations (≥0.1 mcg/dL) were seen in 97.6% volunteers and 96.3% patients. Only 25.5% of women volunteers on OCP had abnormal post-DST total cortisol (>1.8 mcg/dL). In volunteers, the upper post-DST free cortisol range was 48 ng/dL in men and women not on OCP, and 79 ng/dL in women on OCP. When compared with post-DST free cortisol, diagnostic accuracy of post-DST total cortisol was 87.3% (95% CI, 81.7-91.7); all false-positive results occurred in patients with post-DST cortisol between 1.8 and 5 mcg/dL. OCP use was the only factor associated with false-positive results (21.1% vs 4.9%, P = 0.02). CONCLUSIONS: Post-DST free cortisol measurements are valuable in patients with optimal dexamethasone concentrations and post-DST total cortisol between 1.8 and 5 mcg/dL.

Targeted Mutational Analysis of Cortisol-Producing Adenomas.

CONTEXT: Somatic gene mutations have been identified in only about half of cortisol-producing adenomas (CPAs). Affected genes include PRKACA, GNAS, PRKAR1A, and CTNNB1. OBJECTIVE: This work aims to expand our understanding of the prevalence of somatic mutations in CPAs from patients with overt Cushing syndrome (OCS) and "subclinical" mild autonomous cortisol excess (MACE), with an immunohistochemistry (IHC)‒guided targeted amplicon sequencing approach using formalin-fixed paraffin-embedded (FFPE) tissue. METHODS: We analyzed FFPE adrenal tissue from 77 patients (n = 12 men, 65 women) with either OCS (n = 32) or MACE (n = 45). Using IHC for 17α-hydroxylase/17,20-lyase (CYP17A1) and 3ß-hydroxysteroid dehydrogenase (HSD3B2), we identified 78 CPAs (32 OCS CPAs and 46 MACE CPAs). Genomic DNA was isolated from the FFPE CPAs and subjected to targeted amplicon sequencing for identification of somatic mutations. RESULTS: Somatic mutations were identified in 71.8% (56/78) of the CPAs. While PRKACA was the most frequently mutated gene in OCS CPAs (14/32, 43.8%), somatic genetic aberrations in CTNNB1 occurred in 56.5% (26/46) of the MACE CPAs. Most GNAS mutations were observed in MACE CPAs (5/7, 71.4%). No mutations were observed in PRKAR1A. In addition to the known mutations, we identified one previously unreported mutation in PRKACA. Two patients with MACE harbored 2 adjacent tumors within the same adrenal gland - one patient had 2 CPAs, and the other patient had a CPA and an aldosterone-producing adenoma (identified by IHC for aldosterone synthase). CONCLUSION: A comprehensive FFPE IHC-guided gene-targeted sequencing approach identified somatic mutations in 71.8% of the CPAs. OCS CPAs demonstrated a distinct mutation profile compared to MACE CPAs.

Cushing syndrome and glucocorticoids: T-cell lymphopenia, apoptosis, and rescue by IL-21.

BACKGROUND: Pediatric endogenous Cushing syndrome (eCs) is mainly caused by pituitary corticotropin-producing adenomas, and most glucocorticoid-dependent effects progressively regress upon tumor removal. eCs reproduces long-term, high-dose glucocorticoid therapy, representing a clean, natural, and unbiased model in which to study glucocorticoid bona fide effects on immunity. OBJECTIVE: We performed extensive immunologic studies in otherwise healthy pediatric patients with eCs before and 6 to 13 months after tumor resection, as well as in in vitro glucocorticoid-treated control cells. METHODS: Flow cytometry, immunoblotting, enzyme-linked immunosorbent assay, real-time quantitative PCR, and RNA-Seq techniques were used to characterize patients' and in vitro glucocorticoid treated cells. RESULTS: Reduced thymic output, decreased naive T cells, diminished proliferation, and increased T-cell apoptosis were detected before surgery; all these defects eventually normalized after tumor removal in patients. In vitro studies also showed increased T-cell apoptosis, with correspondingly diminished NF-κB signaling and IL-21 levels. In this setting, IL-21 addition upregulated antiapoptotic BCL2 expression and rescued T-cell apoptosis in a PI3K pathway-dependent manner. Similar and reproducible findings were confirmed in eCs patient cells as well. CONCLUSIONS: We identified decreased thymic output and lymphocyte proliferation, together with increased apoptosis, as the underlying causes to T-cell lymphopenia in eCs patients. IL-21 was decreased in both natural and in vitro long-term, high-dose glucocorticoid environments, and in vitro addition of IL-21 counteracted the proapoptotic effects of glucocorticoid therapy. Thus, our results suggest that administration of IL-21 in patients receiving long-term, high-dose glucocorticoid therapy may contribute to ameliorate lymphopenia and the complications associated to it.

Identification of glucocorticoid-related molecular signature by whole blood methylome analysis.

OBJECTIVE: Cushing's syndrome represents a state of excessive glucocorticoids related to glucocorticoid treatments or to endogenous hypercortisolism. Cushing's syndrome is associated with high morbidity, with significant inter-individual variability. Likewise, adrenal insufficiency is a life-threatening condition of cortisol deprivation. Currently, hormone assays contribute to identify Cushing's syndrome or adrenal insufficiency. However, no biomarker directly quantifies the biological glucocorticoid action. The aim of this study was to identify such markers. DESIGN: We evaluated whole blood DNA methylome in 94 samples obtained from patients with different glucocorticoid states (Cushing's syndrome, eucortisolism, adrenal insufficiency). We used an independent cohort of 91 samples for validation. METHODS: Leukocyte DNA was obtained from whole blood samples. Methylome was determined using the Illumina methylation chip array (~850 000 CpG sites). Both unsupervised (principal component analysis) and supervised (Limma) methods were used to explore methylome profiles. A Lasso-penalized regression was used to select optimal discriminating features. RESULTS: Whole blood methylation profile was able to discriminate samples by their glucocorticoid status: glucocorticoid excess was associated with DNA hypomethylation, recovering within months after Cushing's syndrome correction. In Cushing's syndrome, an enrichment in hypomethylated CpG sites was observed in the region of FKBP5 gene locus. A methylation predictor of glucocorticoid excess was built on a training cohort and validated on two independent cohorts. Potential CpG sites associated with the risk for specific complications, such as glucocorticoid-related hypertension or osteoporosis, were identified, needing now to be confirmed on independent cohorts. CONCLUSIONS: Whole blood DNA methylome is dynamically impacted by glucocorticoids. This biomarker could contribute to better assessment of glucocorticoid action beyond hormone assays.

Circulating MicroRNA Expression, Vitamin D, and Hypercortisolism as Predictors of Osteoporosis in Elderly Postmenopausal Women.

BACKGROUND: MicroRNAs (miRNA) identified as critical molecular regulators for bone development, function, and modeling/remodeling process and could be predictable for osteoporotic fractures in postmenopausal elderly women. AIM: The potential diagnostic role of circulating miRNAs, miR-148a and miR-122-5p, in the pathogenesis of osteoporosis and its association with bone markers, hypercortisolism, and vitamin D deficiency were explored in postmenopausal elderly women with osteoporosis. METHODS: A total of 120 elderly women aged 50-80 years old were recruited in this study, of which only 100 eligible women with amenorrhea of at least 12 consecutive months or surgical menopause participated in this study. Based upon bone mineral density (BMD) measurements, the participants were classified according into two groups: normal (n = 45; T score of ≥-1.0) and osteoporosis (n = 55; T score: ≤-2.5). Circulating miRNAs, miR-148a and miR-122-5p, were estimated by real-time RT-PCR analysis. In addition, bone markers, hypercortisolism, and vitamin D deficiency were colorimetrically and ELISA immune assay estimated. The potential role of miR-148a, miR-122-5p, cortisol, and vitamin D in the diagnosis of osteoporosis was predicted using the analysis of the respective area under the receiver operating characteristic curve (AUC-ROC). RESULTS: The expressed level of miR-148a significantly increased and miR-122-5p significantly decreased in the serum of osteoporotic patients compared to healthy controls. In addition, a significant increase in the levels of cortisol, s-BAP, and CTx and significant decrease in the levels of T-BMD, the levels of OC, and s-Ca were also identified. All parameters significantly correlated with fracture risk parameters; BMD, and T score lumbar spine (L2-L4). Thus, the data showed AUC cut off values (miR-148a; 0.876, miR-122-5p; 0.761) were best evaluated for clinical diagnosis of patients with osteoporosis and that AUC cut off values of 0.748 for cortisol and 0.635 for vitamin D were the best cut off values, respectively, reported for the prediction of osteoporosis clinical diagnosis. CONCLUSION: In this study, expressed miRNAs miR-148a and miR-122-5p and changes in the levels of both cortisol and vitamin D status are significantly associated with bone loss or osteoporosis. Thus, circulation miRNAs alone or in combination with cortisol and vitamin D status might be considered predictable biomarkers in the diagnosis or the pathogenesis of osteoporosis in elderly postmenopausal women; however, more studies are recommended.

An Optimized Pathway for the Differential Diagnosis of ACTH-Dependent Cushing's Syndrome Based on Low-Dose Dexamethasone Suppression Test.

Context: Traditionally, low-dose dexamethasone suppression test (LDDST) was used to confirm the diagnosis of Cushing's syndrome (CS), and high-dose dexamethasone suppression test (HDDST) was used to differentiate Cushing's disease (CD) and ectopic adrenocorticotropin (ACTH) syndrome (EAS), but some studies suggested that HDDST might be replaced by LDDST. For the differential diagnosis of CS, dexamethasone suppression test was usually combined with other tests such as bilateral petrosal sinus sampling (BIPSS) and pituitary magnetic resonance imaging, but the optimal pathway to incorporate these tests is still controversial. Objectives: To develop an optimized pathway for the differential diagnosis of CD and EAS based on LDDST. Design and Setting: Single-center retrospective study (2011-2019). Patients: Two hundred sixty-nine CD and 29 EAS patients with pathological diagnosis who underwent consecutive low- and high-dose DST. Results: For the differential diagnosis of CD and EAS, the area under curve (AUC) of LDDST using urine free cortisol (0.881) was higher than that using serum cortisol (0.685) (p < 0.001) in head-to-head comparison among a subgroup of 108 CD and 10 EAS. The AUC of LDDST (0.883) was higher than that of HDDST (0.834) among all the included patients. With the cutoff of <26%, the sensitivity and specificity of LDDST were 39.4% and 100%. We designed a new pathway in which BIPSS was only reserved for those patients with unsuppressed LDDST and adenoma <6mm, yielding an overall sensitivity of 97.7% and specificity of 86.7%. Conclusion: LDDST had similar value to HDDST in differentiating CD and EAS using the specific cutoff point. The pathway that combined LDDST and BIPSS could differentiate CD and EAS accurately.

Case Report: A Challenging Localization of a Pulmonary Ectopic ACTH-Secreting Tumor in a Patient With Severe Cushing's Syndrome.

Background: Ectopic adrenocorticotropic syndrome (EAS) is a rare cause of endogenous ACTH-dependent Cushing's syndrome, usually associated with severe hypercortisolism as well as comorbidities. Tumor detection is still a challenge and often requires several imaging procedures. In this report, we describe a case of an ectopic ACTH secretion with a misleading localization of the responsible tumor due to a concomitant rectal carcinoma. Case presentation: A 49-year-old man was referred to our Endocrinology Unit due to suspicion of Cushing's syndrome. His medical history included metastatic rectal adenocarcinoma, diagnosed 5 years ago and treated with adjuvant chemotherapy, radiotherapy and surgical resection. During follow-up, a thoracic computed tomography scan revealed two pulmonary nodules located in the superior and middle lobes of the right lung with a diameter of 5 and 10 mm, respectively. However, these nodules remained radiologically stable thereafter and were not considered relevant. All biochemical tests were suggestive of EAS (basal ACTH levels: 88.2 ng/L, nv 0-46; basal cortisol levels: 44.2 µg/dl, nv 4.8-19.5; negative response to CRH test and high dose dexamethasone suppression test) and radiological localization of the ectopic ACTH-secreting tumor was scheduled. The CT scan revealed a dimensional increase of the right superior lung nodule (from 5 to 12 mm). [68Ga]-DOTA-TOC PET/CT scan was negative, while [18F]-FDG-PET/CT showed a tracer accumulation in the superior nodule. After a multidisciplinary consultation, the patient underwent thoracic surgery that started with two atypical wedge resections of nodules. Frozen section analyses showed a neuroendocrine tumor on the right middle lobe nodule and a metastatic colorectal adenocarcinoma on the superior lesion. Then, a right superior nodulectomy and a right middle lobectomy with mediastinal lymphadenectomy were performed. The final histopathological examination confirmed a typical carcinoid tumor, strongly positive for ACTH. A post-surgical follow-up showed a persistent remission of Cushing's syndrome. Conclusions: The present report describes a case of severe hypercortisolism due to EAS not detected by functional imaging methods, in which the localization of ACTH ectopic origin was puzzled by a concomitant metastatic rectal carcinoma. The multidisciplinary approach was crucial for the management of this rare disease.

Intratumoral steroid profiling of adrenal cortisol-producing adenomas by liquid chromatography- mass spectrometry.

Endogenous Cushing syndrome (CS) is an endocrine disorder marked by excess cortisol production rendering patients susceptible to visceral obesity, dyslipidemia, hypertension, osteoporosis and diabetes mellitus. Adrenal CS is characterized by autonomous production of cortisol from cortisol-producing adenomas (CPA) via adrenocorticotropic hormone-independent mechanisms. A limited number of studies have quantified the steroid profiles in sera from patients with CS. To understand the intratumoral steroid biosynthesis, we quantified 19 steroids by mass spectrometry in optimal cutting temperature compound (OCT)-embedded 24 CPA tissue from patients with overt CS (OCS, n = 10) and mild autonomous cortisol excess (MACE, n = 14). Where available, normal CPA-adjacent adrenal tissue (AdjN) was also collected and used for comparison (n = 8). Immunohistochemistry (IHC) for CYP17A1 and HSD3B2, two steroidogenic enzymes required for cortisol synthesis, was performed on OCT sections to confirm the presence of tumor tissue and guided subsequent steroid extraction from the tumor. LC-MS/MS was used to quantify steroids extracted from CPA and AdjN. Our data indicated that CPA demonstrated increased concentrations of cortisol, cortisone, 11-deoxycortisol, corticosterone, progesterone, 17OH-progesterone and 16OH-progesterone as compared to AdjN (p < 0.05). Compared to OCS, MACE patient CPA tissue displayed higher concentrations of corticosterone, 18OH-corticosterone, 21-deoxycortisol, progesterone, and 17OH-progesterone (p < 0.05). These findings also demonstrate that OCT-embedded tissue can be used to define intra-tissue steroid profiles, which will have application for steroid-producing and steroid-responsive tumors.

Dysfunction in Sertoli cells participates in glucocorticoid-induced impairment of spermatogenesis.

The effect of stress on male fertility is a widespread public health issue, but less is known about the related signaling pathway. To investigate this, we established a hypercortisolism mouse model by supplementing the drinking water with corticosterone for four weeks. In the hypercortisolism mice, the serum corticosterone was much higher than in the control, and serum testosterone was significantly decreased. Moreover, corticosterone treatment induced decrease of sperm counts and increase of teratozoospermia. Increased numbers of multinucleated giant cells and apoptotic germ cells as well as downregulated meiotic markers suggested that corticosterone induced impaired spermatogenesis. Further, upregulation of macrophage-specific marker antigen F4/80 as well as inflammation-related genes suggested that corticosterone induced inflammation in the testis. Lactate content was found to be decreased in the testis and Sertoli cells after corticosterone treatment, and lactate metabolism-related genes were downregulated. In vitro phagocytosis assays showed that the phagocytic activity in corticosterone-treated Sertoli cells was downregulated and accompanied by decreased mitochondrial membrane potential, while pyruvate dehydrogenase kinase-4 inhibitor supplementation restored this process. Taken together, our results demonstrated that dysfunctional phagocytosis capacity and lactate metabolism in Sertoli cells participates in corticosterone-induced impairment of spermatogenesis.

The usefulness of the combined high-dose dexamethasone suppression test and desmopressin stimulation test in establishing the source of ACTH secretion in ACTH-dependent Cushing's syndrome.

Bilateral inferior petrosal sinus sampling (BIPSS) is the current gold standard test for differentially diagnosing ACTH-dependent Cushing's syndrome (CS). However, BIPSS is an invasive procedure, and its availability is limited. We retrospectively analysed the 24-hour urinary free cortisol (UFC) level during the high-dose dexamethasone suppression test (HDDST) and plasma ACTH/cortisol levels after the desmopressin stimulation test (DDAVP test) in subjects with confirmed Cushing's disease (CD) (n = 92) and ectopic ACTH-dependent CS (EAS) (n = 16), and evaluated the positive predictive value (PPV) of the two combined-tests in the aetiological diagnosis of ACTH-dependent CS. The percent changes in UFC levels after the HDDST and in ACTH/cortisol levels after DDAVP administration relative to the corresponding basal levels and the area under the receiver operating characteristic (ROC) curve (AUC) were analysed. UFC suppression below 62.7% suggested a pituitary origin with a sensitivity (SE) of 80% (95% CI: 70-88) and a specificity (SP) of 80% (95% CI: 52-96). A threshold increase in the ACTH level after DDAVP stimulation of 44.6% identified CD with an SE of 91% (95% CI: 83-97) and an SP of 75% (95% CI: 48-93). The combination of both tests yielded an SE of 95.5% and PPV of 98.4% for CD, and significantly improved the efficiency of the differential diagnosis between CD and EAS. These dual non-invasive endocrine tests may substantially reduce the need for BIPSS in the etiological investigation of ACTH-dependent CS.

Plasma Cortisol and Risk of Atrial Fibrillation: A Mendelian Randomization Study.

CONTEXT: Atrial fibrillation (AF), cardiac arrhythmias, and related risk factors are common in patients with Cushing's syndrome, or clinical chronic hypercortisolism. While hypercortisolism may be associated with AF, this association has not yet been ascertained causally. OBJECTIVE: To determine whether plasma cortisol is causally associated with AF using a 2-sample Mendelian randomization (MR) design. METHODS: Three genetic variants in the SERPINA1/SERPINA6 locus and functionally associated with plasma cortisol were identified in the CORtisol NETwork consortium (12 597 participants). Summary-level genome-wide association study (GWAS) data for the associations between the cortisol-associated variants and AF were obtained from a GWAS meta-analysis of 6 studies (60 620 AF cases and 970 216 noncases) and the FinnGen consortium (17 325 AF cases and 97 214 noncases). The fixed-effects inverse-variance weighted approach accounting for genetic correlations between variants was used for analysis. Multivariable MR analyses were conducted to assess potential mediating effects of systolic blood pressure (SBP) and waist circumference (WC). Summary-level GWAS data for SBP and WC were obtained respectively from the International Consortium of Blood Pressure (757 601 participants) and the Genetic Investigation of ANthropometric Traits consortium (232 101 participants). RESULTS: One standard deviation increase in genetically predicted plasma cortisol was associated with greater risk of AF (odds ratio [OR] 1.20, 95% CI 1.06-1.35). The association attenuated when adjusting for genetically predicted SBP and WC (OR 0.99, 95% CI 0.72-1.38). CONCLUSION: Evidence derived from the MR study suggests a positive association between plasma cortisol and risk of AF, likely mediated through SBP and WC.