Cardiac autonomic modulation and anti-TPO antibodies in Subclinical Hypothyroidism – Does any correlation exit?

Background Heart rate variability (HRV) reects the balance of activities of sympathetic and parasympathetic components of the autonomic nervous system. Anti-thyroid antibodies have long been associated with thyroid dysfunction and inuence thyroid prole testing, the most common being anti-Thyroid Peroxidase (TPO) and anti-Thyroglobulin antibodies. Subclinical hypothyroidism (SCHypo) is characterized by elevated TSH with normal thyroid hormones.We hypothesized that autonomic function may be deranged in anti-TPO positive sub-clinical hypothyroid cases even before the onset of overt hypothyroidism. Objectives To investigate the association between anti-TPO antibodies (anti-TPOAb) positive SCHypo and sympathovagal imbalance (SVI). and SVI in SCHypo patients.

Introduction SCHYPO is de ned as a clinical entity in which the serum-free thyroxine (fT4) level remains within the reference range with an increased serum thyroid stimulating hormone (TSH) level (Cooper DS et al., 2012). Autoimmune thyroid disease (AITD) includes hyperthyroid Grave's disease, hypothyroid autoimmune thyroiditis, and subtle sub clinical thyroid dysfunctions (Yoo & Chung, 2016). Thyroid autoimmunity is characterized by easily detectable production of thyroid autoantibodies, to thyroglobulin (TG) and thyroid peroxidase (TPO) (Macdonald, Parsy-Kowalska, & Chapman, 2017). The sensitivity and speci city of anti-TPO is higher than that of anti-TG for identifying autoimmune thyroid disease (Spencer, 2000). TPO-Abs are the circulating hallmark of autoimmune thyroid disease and are present in majority of cases. (Roberts & Ladenson, 2004). SCHypo has found to be associated in various studies with an increased number of cardiovascular risk factors and altered autonomic activity (Christine, 2017; Selmer, Hoshi 2019) The thyroid gland and the Autonomic Nervous System (ANS) are closely linked by their effects on the cardiovascular system (Reeves, Fisher, Newman, & Granger, 2016;Mavai 2018). The ANS controls heart by complex mechanism of interactions between its two branches, which produces uctuations in heartbeat intervals. The greater the beat-to-beat interval, the better the cardiovascular system functions, to adapt and respond to internal and external stimuli (Billman, 2011&Maor et al., 2013. Heart rate variability (HRV) is an easy, non-invasive, sensitive and widely applied method for cardiac autonomic assessment. Lowered HRV is associated with increased risk of mortality, and HRV has been proposed as a marker for disease (Task Force, 1996).
Autonomic disturbances are common in patients with overt thyroid diseases, even in subclinical conditions (de Miranda et al., 2018; Mavai 2018) and it has been suggested that clinical impairments in SCHYPO may precede cardiac dysfunctions (Hoshi 2019(Hoshi , 2020. In light of the above, this study was planned to assess the cardiac autonomic functions by HRV parameters in anti-TPOAb positive and anti-TPOAb negative subclinical hypothyroid subjects and to assess the association between presence of anti-TPOAb in SCHypo and sympathovagal imbalance (SVI).

Materials And Methods
The study was commenced after obtaining formal approval from Institutional Ethics Committee.
Informed written consent was obtained from all the subjects included in the study. Age and BMI matched subclinical hypothyroid patients and healthy controls, 20-50 years of age, never previously treated for any endocrine disease and ready to participate were included in the study. Participants were strati ed into anti-TPOAb positive (n= 35), anti-TPOAb negative (n= 17) and control (n=20) groups. SCHYPO was de ned as patients with a TSH level of 4.5-10mU/ml and normal Individuals with history or complaints of any cardiac, hepatic or renal dysfunction, HIV / Immunode ciency disorders, neurological disease, any other systemic disease that may affect autonomic activity i.e. diabetes, hypertension and those taking any drug which affects autonomic activity were excluded. Serum levels of fT 3 , fT 4 , TSH, anti-TPOAb levels were measured by using the chemiluminescent immunoassay method (IMMULITE 2000 Systems Analyzer). The serum fT 3 , fT 4 , TSH levels for theeuthyroid state was between 1.8-4.2 pg/mL, 0.89-1.76 ng/dL and 0.4-4.0 μIU/mL.The level of anti-TPOAb was <35 IU/mL, respectively.The presence of antithyroid antibodies was determined by levels of serum antibodies to TPO. TPO antibodies were considered positiveabove these values.
Based on the above mentioned criteria, the following recruitment /screening plan was charted: see gure.
Based on the nding of the screening, the patients were grouped as Anti-TPOAb positive Subclinical Hypothyroid (35), Anti-TPOAb negative Subclinical Hypothyroid (17) age and BMI matched 25 healthy subjects (asymptomatic with normal clinical examination and biochemical tests and not on any medication) randomly selected from general population. Con rmation of clinical diagnosis was done by biochemical evaluation of FT 3 , FT 4 , TSH, anti-TPO Antibody.
Heart Rate Variability assessment was done by recording 5 minutes ECG by RMS ECG (DECG 1/ 63041/ ADBXB). The analogue signals were converted to digital signals by National Instrument Software NI- Experimental Protocol: 1. For HRV, the participants underwent 5 min of rest in supine position. Recording was done in quiet and comfortable room, temperature was maintained at 24-28°C. Subject was instructed to close the eyes and to avoid talking, moving hands, legs and body, coughing during test, sleeping.
2. The subjects were instructed to avoid food at least two hours before the procedure, abstain from coffee, nicotine or alcohol 24 hours prior to testing and to wear loose and comfortable clothing.
They were asked to report at 10.00 am. Their age, height, and body weight were recorded. Body Mass Index (BMI) was calculated by weight (kg) divided by the square of height (meter) (Quetelet's Index).
3. All standard limb leads were applied and the lead with upright R wave was selected for recording.
The ECG signals were continuously ampli ed, digitized and stored in the computer for o ine analysis. Processing including R wave and RR interval detection was done by the software.
Abnormal beats and areas of artifact were automatically identi ed and excluded from the recording.

Statistical analysis
Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA, USA). Descriptive characteristics are presented as means and standarddeviations (mean± SD).
Kolmogorov-Smirnov test was done to assess the normal distribution of variables. For parametric data, the level of signi cance among the groups was tested by One-way ANOVA followed by post-hoc Bonferroni test and for non-parametric data; Kruskal-Wallis One way ANOVA with post hoc Dunn's multiple comparison test was used. Spearman correlation tests were done to study associations between various variables. For all the analyses statistical signi cance was de ned at the level of p <0.05.

Results
The study population consisted of 52 SCHypo patients and 20 controls. Based on the presence of thy roid autoantibody (anti-TPOAb) status, subjects were divided into two groups: anti-TPOAb positive group (n=35) and anti-TPOAb negative group (n=17).    P<0.05 were considered statistically signi cant. TSH: Thyroid Stimulating Hormone; LF low frequency power(ms 2 ), HF high frequency power(ms2), LFnu normalized unit low frequency power, HFnu normalized high frequency power, LF/HF ratio of LF to HF, TP total power, SDNN standard deviation of normal to normal interval, RMSSD the square root of the mean of squares of the differences between adjacent NN intervals, pNN50 the proportion derived by dividing NN50 by the total number of NN intervals,SD1, SD2 and SD1/ SD2 ratio (Poincare index),, TINN triangular interpolation of NN interval.

Discussion
The study compared the autonomic activity in TPO Ab-positive' and -negative groups. The anti-TPO antibody-positive group demonstrated greater alterations in autonomic modulation, primarily characterized by diminished parasympathetic activity. Literature search could not retrieve any study on anti-TPOAb status of SCHypo patients and HRV. However research has shown signi cantl higher sympathetic and lower parasympathetic modulation at baseline conditions in SCHypo (Hoshi 2019).
No signi cant difference was observed in TSH levels between anti-TPOAb positive and anti-TPOAb negative patients, however there was a signi cant difference in the TSH levels between the control and SCHypo groups. Contrary to this nding, Brown J (2016) evidenced signi cantly higher serum TSH in antibody-positive than in antibody-negative individuals (Brown et al., 2016).
In the current study, signi cant increase in the LF-HF ratio in the anti-TPOAb positive SCHYPO patients was recorded in comparison to the control group, however no signi cant difference was observed between control and anti-TPOAb negative patients. The LH-HF ratio is a sensitive marker of SVI (Malliani A, 2005 &Task Force, 1996), hence the increase in LF-HF ratio represents considerable SVI in anti-TPOAb positive SCHYPO. In the Poincaré indices (SD1 and SD2) of HRV, width of SD1 re ects the parasympathetic modulation and the SD2 length re ects the sympathetic activity (Hsu et al., 2012). In our study, SD1 value was profoundly decreased (p<0.0001) and SD2 was increased (p<0.05) in antibody positive SCHYPO group than controls, which can be used as a sensitive indicator of sympathovagal changes. Akin to HF-LF ratio, the SD1-SD2 ratio also re ects the sympathovagal balance. The signi cant decrease in SD1/SD2 in Anti TPO-Ab positive group further con rms SVI in TPO-Ab positive SCHYPO.
In anti-TPOAb positive SCHypo patients, a signi cant decrease in HFnu indicates decreased parasympathetic activity in these patients, The time-domain indices of HRV -SDNN, RMSSD and pNN50 were signi cantly decreased in anti-TPOAb positive SCHypo patients than controls , further con rming the decreased vagal tone in these patients. The reduction in parasympathetic activity in TPO-positive group compared to healthy individuals can be further veri ed by the decrease in the SD1 indices. Hence, anti-TPO positive SCHypo group showed lower parasympathetic modulation than control group. A decrease vagal activity is related with an increased risk for all-cause morbidity and mortality and for the development of numerous other risk factors (Thayer & Lane, 2007).
In this study, no signi cant differences in cardiac vagal activity was observed in the two SCHypo groups, however TPOAb-positive group showed lower values in magnitude for parasympathetic indices in comparison to TPOAb-negative group. This points towards sympathovagal imbalance (SVI) in anti-TPOAb positive SCHypo group. Thus, from the present study, it may be presumed that SVI in anti-TPO positive SCHYPO could be due to diminished vagal activity. In another study, o reduced time domain indices and lower HF values in frequency domain analysis in SCHypo patients was founs in comparison to controls (Hoshi 2019).
In our study, anti-TPOAb were found to correlate positively with TSH and HRV indices. To the best of our knowledge, no study has assessed the correlation of anti-TPOAb with TSH levels and HRV indexes (linear as well as nonlinear) in SCHypo patients. It has been proved that Anti-TPOAb negative autoimmune thyroiditis has a milder course while the presence of anti-TPOAb is associated with an increased risk of overt hy pothyroidism (Radetti et al., 2012). Furthermore, presence of antibodies is linked with increased risk of atherosclerotic disease and myocardial infarction. Hence, anti-TPOAb positive SCHypo patients are more prone to cardiac risk than anti-TPOAb negative SCHypo patients, this has been proved further by this study. Low parasympathetic activity has been also proposed as an important risk factor for cardiovascular disease and mortality (Mavai et al., 2018) .
The limitations of the present study are that we could not assess the cardiac autonomic reactivity by Conventional Autonomic Function Tests (CAFT). Also, we have not studied the cardiac dysfunctions by echocardiography and their possible correlation with anti-TPOAb levels in subclinical hypothyroid subjects. Future studies should be planned to con rm our ndings in large sample size.