The patients included in this study were those who underwent CGM and autonomic function testing at the same time or within three months at the Division of Endocrinology and Metabolism of Samsung Medical Center, Seoul, Republic of Korea from March 2009 to July 2019.
Patients with type 1 or gestational diabetes mellitus (n = 108); cancer undergoing treatment (n = 5); history of thyroid dysfunction (n = 17); history of myocardial infarction, revascularization, or stroke (n = 39); severe liver disease defined by a Child–Pugh score of greater than seven points (n = 5); estimated glomerular filtration rate (GFR) of less than 30 mL/min/1.73 m2 derived from the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula; or lack of relevant clinical data such as HbA1c (n = 2) were excluded (Fig. 1). Finally, a total of 284 patients were included in this study.
The protocol of this study was approved by the institutional review board of Samsung Medical Center (no. 2020-03-111-001).
Clinical variables and biochemical measurements
Anthropometric measurements such as the height and weight of study participants were collected and the body mass index (kg/m2) was calculated as a result. Systolic and diastolic blood pressure values were recorded as the mean of two blood pressure values measured with the patient remaining in a stable sitting position for five minutes or longer. At the time of CGM, blood samples were collected for biochemical measurements including HbA1c, total cholesterol, high- and low-density lipoprotein cholesterol, triglycerides, C-peptide, and creatinine after a 12-hour overnight fast.
Data on medical history and the use of antihypertensive medications such as angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, and beta-blockers were collected. In addition, lipid-lowering agents and antidiabetic agents in use at the time of CGM implementation were reviewed. Hypertension was defined as having a blood pressure of 140/90 mmHg or higher or by the current use of antihypertensive medications.
The CGM was conducted in a blind manner by using the COLD™ system (Medtronic MiniMed, North Ridge, CA, USA) for three consecutive days and the collected data were retrospectively reviewed. Study participants calibrated the CGM by measuring capillary blood glucose levels at least twice a day from the first day of CGM implementation. In accordance with the international consensus recommendations for CGM metrics, TIR was defined as the percentage of time spent in the target range of 70 to 180 mg/dL for the blood glucose level . CGM metrics of hyperglycemia [time above range (TAR) > 250 mg/dL and > 180 mg/dL] hypoglycemia [time below range (TBR) < 70 mg/dL and < 54 mg/dL], and glycemic variability [standard deviation (SD), coefficient of variation (CV)] were also calculated.
Assessment of cardiovascular autonomic neuropathy
CAN was evaluated as the result of five cardiovascular autonomic function tests proposed by Ewing et al.  Patients were asked to avoid vigorous physical activity, smoking, and drinking for 24 hours before the test and to stop consuming coffee and food for three hours before the test. Drugs such as antihistamines, antidepressants, beta-blockers, acetaminophen, and diuretics, which may affect the test results, were stopped 12 hours before the test.
Assessments of the heart rate response to deep breathing (exhalation:inhalation ratio), to standing (30:15 ratio), and to the Valsalva maneuver (Valsalva ratio) were performed to evaluate parasympathetic functions. Two other tests performed to evaluate sympathetic functions included blood pressure responses to standing and to a sustained handgrip, respectively. The heart rate response was assessed automatically from electrocardiography recordings using the DICAN evaluation system (Medicore Co., Ltd., Seoul, Korea).
According to the age-specific reference, each of the three parasympathetic function tests was scored as zero points for normal or one point for abnormal results, while each of the two sympathetic function tests was scored as zero points for a normal result, 0.5 points for a borderline result, or one point for an abnormal result .
CAN was defined as an abnormal result in two or more parasympathetic tests and the severity of CAN was evaluated as the sum of the partial scores of the five cardiovascular autonomic function tests and categorized as either zero to 1.5 points, two to 2.5 points, and three to five points.
Continuous variables were expressed as mean (SD) values and categorical variables were expressed as ratio or percentages. In the comparison of the baseline characteristics according to the presence or absence of CAN, the Student’s t-test or the Mann–Whitney U test was used to discern the mean of continuous variables, while the chi-square test was performed for the categorical variables.
Multiple logistic regression analyses were performed to assess the odds ratio (OR) and 95% confidence interval (CI) for the association with the presence of CAN with a 10% increase in the TIR of 70 to 180 mg/dL, TAR (> 180 mg/dl, > 250 mg/dL), and TBR (< 54 mg/dL, < 70 mg/dL), after adjusting for confounding variables such as age, sex, duration of diabetes, and other known risk factors of CAN.
We also conducted subgroup analyses to evaluate the interaction between subgroups regarding age (< 65 vs. ≥ 65), duration of diabetes (≤ 10 years vs. > 10 years), glucose status (HbA1c 7.5% vs. > 7.5%), and the use of insulin or medications that can cause hypoglycemia. In addition, multivariate ordered logistic regression analysis was used to evaluate the association between the severity of CAN and CGM parameters and a trend test across ordered groups was used to evaluate the trend of TIR of 70 to 180 mg/dL according to the total CAN score.
Statistical analysis was executed using the Statistical Package for the Social Sciences version 25.0 (IBM Corporation, Armonk, NY, USA) and Stata version 16.0 (StataCorp LLC, College Station, TX, USA), and a p-value of less than 0.05 was considered to be statistically significant in two-tailed tests.