Effect of Systemic and Auricular Acupuncture with a 2/100 Hz Frequency and Nogier Frequency in Fibromyalgia: a Randomized Clinical Trial, Pilot Study

Rosa Maria Moreira; Rhaynara Coelho Rosário; Érika Almeida Boggiss; Rosana Aparecida de Lima; Paula Aparecida Silva; Karol Priscila da Silva; Caroline Lima de Farias; Vanessa de Queiroz dos Santos; Josie Resende Torres da Silva; Rodrigo Polaquini Simões; Andréia Maria Silva Vilela Terra; Adriana Teresa Silva Santos

doi:10.51507/j.jams.2023.16.4.139

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Effect of Systemic and Auricular Acupuncture with a 2/100 Hz Frequency and Nogier Frequency in Fibromyalgia: a Randomized Clinical Trial, Pilot Study

Rosa Maria Moreira¹, Rhaynara Coelho Rosário², Érika Almeida Boggiss¹, Rosana Aparecida de Lima¹, Paula Aparecida Silva², Karol Priscila da Silva¹, Caroline Lima de Farias¹, Vanessa de Queiroz dos Santos¹, Josie Resende Torres da Silva³, Rodrigo Polaquini Simões², Andréia Maria Silva Vilela Terra², Adriana Teresa Silva Santos^2,*

¹Pos-Graduation Program in Rehabilitation Sciences, Human Performance Research Laboratory, Institute of Motor Sciences, Federal University of Alfenas, Santa Clara Campus, Alfenas-MG, Brazil
²Human Performance Research Laboratory, Institute of Motor Sciences, Federal University of Alfenas, Santa Clara Campus, Alfenas-MG, Brazil
³Institute of Motor Sciences, Federal University of Alfenas, Santa Clara Campus, Alfenas-MG, Brazil

Correspondence to:

Adriana Teresa Silva Santos
Human Performance Research Laboratory, Institute of Motor Sciences, Federal University of Alfenas, Santa Clara Campus, Alfenas-MG, Brazil
E-mail adriana.santos@unifal-mg.edu.br

Received: February 22, 2023; Revised: April 16, 2023; Accepted: July 18, 2023

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

J Acupunct Meridian Stud 2023; 16(4): 139-151

Published August 31, 2023 https://doi.org/10.51507/j.jams.2023.16.4.139

Abstract

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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
FUNDING
AUTHORS’ CONTRIBUTIONS
CONFLICT OF INTEREST
References

Background: Fibromyalgia is a syndrome of chronic, generalized muscular pain, accompanied by sleep disturbances, fatigue and cardic autonomic dysfunction that will affect the quality of life. There is currently no gold standard treatment. There are limitations of studies with electroacupuncture in auricular acupuncture.
Objectives: We evaluate the effects of systemic electroacupuncture (EA) with frequencies of 2/100 Hz associated of auricular acupuncture with a Nogier frequency (2.28, 4.56 and 9.12 Hz) for pain intensity, heart rate variability (HRV), and quality of life in fibromyalgia.
Methods: Randomized clinical trial, a pilot study. Eighteen volunteers were randomized into a control group (CG, n = 9) and an experimental group (EG, n = 9). Six systemic EA sessions systemic and auricular were applied in the EG for 20 min, twice a week, for six weeks consecutive. The Numerical Pain Assessment Scale (NPRS), 2010 diagnostic criteria of the American College of Rheumatology (FDC 2010), Fibromyalgia Impact Questionnaire (FIQ) and analysis of HRV were the instruments used. The independent t-test compared to the groups was applied.
Results: There was no statistically significant difference for the primary outcome for NPRS (p > 0.05). In the secondary outcome there was a significant difference in the total score and in some FIQ domains (p = 0.008) and some variables such as pain (p = 0.02) and anxiety (p = 0.006). There was no significant difference for the FDC 2010 and HRV variables (p > 0.05).
Conclusion: 2/100 Hz systemic EA associated with the Nogier frequency positively influenced some quality of life variables; however, pain intensity, diagnostic criteria, and HRV variables did not change.

Keywords

Fibromyalgia, Electroacupuncture, Acupuncture, Rehabilitation

INTRODUCTION

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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
FUNDING
AUTHORS’ CONTRIBUTIONS
CONFLICT OF INTEREST
References

Chronic, non-inflammatory pain syndrome known as fibromyalgia mostly affects the musculoskeletal system [1]. The patient also experiences a number of additional symptoms, including fatigue, sleep issues, paresthesia, headaches, anxiety, and irritable bowel syndrome, among others [2]. Thus, patients with fibromyalgia have a high degree of physical disability, professional, emotional, family, and social impairment, negatively affecting their quality of life [2,3]. Fibromyalgia’s etiology and pathogenesis are still poorly understood [3]. The central and autonomic nerve systems, neurotransmitters, hormones, the immunological system, environmental stressors, psychiatric factors, and others appear to be involved, according to certain hypotheses that are trying to be clarified. The primary mechanism thought to be at play is central sensitization, which is characterized by an augmented response to stimuli mediated by central nerve systems signaling [4]. Numerous multisystemic symptoms may be explained in part by the malfunction of this population’s autonomic nervous system [5], which will have a detrimental effect on their quality of life [6].

Because fibromyalgia has a number of symptoms and comorbidities, the diagnosis of this disorder are yet unknown. The American College of Rheumatology (FDC) in 2010 created new criteria that included the remaining symptoms and excluded palpation of tender points [1,7,8]. Its prevalence is 2% worldwide. In Brazil, 40.8% of women between 35 and 44 years of age have fibromyalgia, with a 3:1 ratio when compared to men [8].

Due to its high prevalence and multicausal etiopathogenesis, a multidisciplinary treatment is needed to improve fibromyalgia symptoms. The approach includes the association of pharmacological and non-pharmacological therapies; however, many patients may experience side effects due to prolonged use of medication and end up looking for non-pharmacological therapies [9]. Among the non-pharmacological therapies we can mention acupuncture [10]. According to the World Health Organization, acupuncture is defined as the insertion of needles in humans or animals for therapeutic purposes [10]. The advantages of acupuncture, when compared to anti-inflammatory and anti-depressant drugs, include its low rate of side effects when performed by an experienced professional [11].

Scientific evidence indicates that acupuncture promotes analgesic effects by activating peripheral and central pain control systems through the release of endogenous opioids [12,13]. Responses were also evidenced in the modulation of cardiac autonomic function [14]. These responses are dependent on the frequency variation used in the electroacupuncture device [14]. The 2 Hz frequency increases vagal activity while 50 Hz increases the sympathetic activity [15]. The change in HRV in patients with fibromyalgia is related to anxiety and pain, indicating sympathetic hyperactivity and vagal imbalance [16]. There is a growing number of studies that emphasize the attempt to understand the neurophysiological and neurochemical effects of acupuncture, but most studies are limited to researching systemic acupuncture and little research is done on auricular acupuncture and its effects [17]. Therefore, the aim of this study is to evaluate the effects of systemic electroacupuncture (EA) with frequencies of 2/100 Hz associated of auricular acupuncture with a Nogier frequency for pain intensity, HRV, and quality of life in fibromyalgia.

MATERIALS AND METHODS

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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
FUNDING
AUTHORS’ CONTRIBUTIONS
CONFLICT OF INTEREST
References

1. Trial design

The study was characterized as a randomized and parallel clinical trial, pilot study.

2. Study local and ethical aspects

This study occurred at the Federal University of Alfenas (UNIFAL - MG), the work occurred in the second half of 2019 until the end of 2020, and the recruitment took place from January 6, 2020, to September 30, 2020.

The work followed the norms of good practice in clinical studies involving human beings (Resolution 466/12 of the National Health Council). The Research Ethics Committee of the Federal University of Alfenas (CAAE number 24769719.0.0000.5142) approved the study. The present study is registered in the Brazilian Registry of Clinical Trials – REBEC (registration number: RBR–833mm2). All volunteers were properly informed about the study and signed an Informed Consent Form.

3. Sample study

The study consisted of adult women recruited from the Family Health Units in the city of Alfenas - MG and from the Physiotherapy Clinic of the Federal University of Alfenas (UNIFAL). After passing the eligibility criteria, the procedures were performed at the Physiotherapy Clinic at UNIFAL.

1) Inclusion criteria

Through screening, a tracking form was applied to characterize the volunteers who fit the inclusion criteria of the present study.

The following inclusion criteria were established: women aged over 40 to 78 years and clinical diagnosed with fibromyalgia according to the 2010 criteria of the FDC [7], having agreed to participate in the research and signed an informed consent form.

2) Exclusion criteria

Exclusion criteria included volunteers with needle phobia, hemorrhagic diathesis, or cardiac arrhythmia; those who were pregnant or lactating; who received physical therapy treatment, massage, or acupuncture in the last two weeks before the intervention; and those who received an anesthetic, analgesic, muscle relaxant, or anti-inflammatory drugs, or had inflammation in the last two days before the intervention, present central injury, peripheral nerve injury, open wound at the application site, patients with seizures and patients with coagulation deficit.

4. Evaluation instruments and procedures

The methodology of this study was based on the standards established by CONSORT [18] and STRICTA [19].

1) Evaluation instrument for the primary outcome

(1) Numerical Pain Rating Scale (NPRS)

The NPRS was used to measure pain intensity [20]. It was used because it is a simple, straight-line instrument with numbers from 0 to 10, with 0 representing “no pain” and 10 representing maximum pain. It proved to be a reliable instrument [21,22].

The scale was conducted at the initial assessment, after both groups’ sixth session, and at the beginning of each EG session.

2) Evaluation instrument for the secondary outcome

(1) Fibromyalgia Diagnostic Criteria – FDC 2010

The FDC 2010 was used to assess fibromyalgia. It addresses two indices: the Generalized Pain Index (GPI) and the Symptom Severity Scale (SSS). In the GPI, 19 regions are indicated in which the patient feels pain, resulting in a score between 0 and 19. In the SSS, the severity of symptoms of fatigue, non-restorative sleep, and cognitive issues are graded from 0 to 3. In this way, the SSS varies between 0 and 12. The fibromyalgia diagnosis is suggested when the GPI is greater than or equal to 7 and associated with an SSS that is greater than or equal to 5, or when the GPI is between 3 and 6 with an SSS greater than or equal to 9. In addition, for the fibromyalgia classification, the patient must have the painful condition for at least 3 months and not present any other disease that could justify the pain. There is an 88.1% accuracy in diagnosing fibromyalgia [7,23].

The questionnaire was applied at the initial assessment and after both groups’ sixth session.

(2) Fibromyalgia impact questionnaire - FIQ

The FIQ questionnaire was used to assess quality of life in this article [24]. It was comprised of 19 questions that were related to functional capacity, professional status, psychological disorders, and physical symptoms, and organized into 10 items. The higher the score, the greater the impact the fibromyalgia had on the patient’s quality of life. The Brazilian version of the FIQ was tested and proved to be an instrument that presents reliable and valid results for the Brazilian population [25].

The questionnaire was conducted at the initial assessment and after both groups’ sixth session.

(3) R-R interval (R-Ri) recording and heart rate variability analysis (HRV)

① Data collection

The subjects were instructed to abstain from caffeinated and alcoholic beverages, not to perform exercise on the day before data collection, to have a light meal at least 2 h prior to the tests and not to speak unnecessarily during the assessments to avoid interfering with the acquisition of signals. In addition, subjects were instructed to not perform activities requiring moderate-to-heavy physical exertion the day before the application of the protocols [26].

For data collection, the subject remained in the supine position, at rest and in silence, for 15 minutes before recording. The collection was carried out in the morning for a period of 5 minutes. The HR and R-Ri were recorded offline and continuously using a heart rate monitor telemetry system (Polar^Ⓡ RS800CX Heart Rate Monitor –Kempele –Finland) for further analysis of HRV. After capturing the data, the watch is connected to the computer by a USB cable, through which the data is synchronized to the Polar Flow Sync software (version 4), where it is stored. The validity and reliability of which have already been confirmed in the literature for this type of analysis [27].

② HRV processing and analysis

Signal processing was performed using Kubios HRV Standard software (MATLAB, version 3.5.0, Kuopio, Finland). For data analysis, sections containing the capture of 5 minutes of consecutive R-R intervals were selected. All artifacts were reviewed by visual inspection on the computer display. Only segments with > 90% pure sinus beats were included in final analyses and a very low-intensity filter was used, when necessary, to remove noise or ectopic beats. The section selected for HRV analysis was the portion considered most stable containing 256 R-Ri [26].

HRV was analyzed using linear statistical measures in the time and frequency domain and non-linear statistical measures. The mean of HR, mean of R-Ri, the root mean square of the successive differences (RMSSD), the number of pairs of successive R-Ri that differ by more than 50 ms (NN50), the proportion of NN50 divided by the total number of R-Ri (PNN50), the standard deviation of the mean of all normal R-Ri (SDNN), and the total number of R-Ri divided by the height of the histogram of all R-Ri (RRtri) and width of the R-Ri triangle distribution (TINN) were computed as time domain measures, with RMSSD, PNN50 and NN50 representing the parasympathetic modulation and the other indices representing the global HRV. For the frequency domain analysis, the power spectral components were reported at low (LF; 0.04 to 0.15 Hz) and high (HF; 0.15 to 0.4 Hz) frequencies (obtained using the fast Fourier transform in ms² and normalized units - n.u) and the LF/HF ratio. Signals in the LF band have been predominately related to high sympathetic modulation and low parasympathetic modulation, and signals in the HF have been attributed only to parasympathetic activity; at the same time, LH/HF represents the sympathetic-vagal balance [28].

Non-linear HRV analysis was performed to obtain standard deviation perpendicular the line of identity (SD1), plot standard deviation along the line of identity (SD2) (representing the parasympathetic and sympathetic modulation respectively) and the SD2/SD1 ratio (representing the sympathetic-vagal balance). In addition, non-linear statistical measures were calculated by detrended fluctuation analysis (DFA) as short-term fractal scaling exponent (DFAα1), and long-term (DFAα2) in the RR series data [29,30]. The complexity of the distribution of the R-Ri series was obtained through the sample entropy (SampEn) and approximate entropy (ApEn). Higher values of these indices represent greater complexity of the series and, therefore, better cardiac autonomic modulation [31].

5. Randomization

A researcher who did not participate in the assessment and intervention process randomized the volunteers with allocation concealment.

The randomization of volunteers followed the table of random numbers generated through the electronic Research Randomizer mechanism (www.randomizer.org). On this site, two columns (A and B) were created; column A represented the CG and column B represented the EG. The site then performed the random sequence within these two columns.

The researcher responsible for the intervention verified which group each volunteer fit into, and allocated them in equal numbers into two groups: CG and EG, both with n = 9. The EG received EA treatment and the CG received an informative lecture on daily care and managing pathology after a 30-minute assessment.

6. Intervention protocols

In the application of the intervention, there was no blinding of the researcher who evaluated and applied the intervention, there was only blinding for the analysis of statistical data.

1) Control group (CG)

The control group received no intervention, only an informative lecture of the thirty minutes, after the lasted assessment on daily care and management of the pathology.

2) Experimental group (EG)

The treatment protocol for the EG consisted of twice a week for six consecutive weeks for 20 minutes each session. Based on the results of surveys, the frequency and number of consultations were determined [32-37]. Asepsis of the ear and systemic points was performed with 70% alcohol. Needles were disposable and individual. The type of acupuncture used was systemic to Traditional Chinese Medicine and auricular to French. The treatment points were:

- Systemic: stomach 36 (ST36) [35] located 3 cun below the patella and 0.5 cun lateral to the tibia and bladder 60 (BL60) [36]. It is located at the apex of the lateral malleolus, at the midpoint between the malleolus and the Achilles tendon. Electrical stimulus power of 10 mA was applied bilaterally, with mixed stimulus of 2 and 100 Hz, with active time 3 seconds and deactivated time 3 seconds, and pulse width 200 µs, non-polarized pulse form, for 20 minutes. The stimulus occurred via the new model EL 608 (NKL^®, Brusque, SC, Brazil) device. Needle insertion was 3 mm deep, using a stainless-steel needle that was 25 × 30 mm in size and 0.2 mm in diameter. We followed regulatory norms (NR) 32 safety and hygiene standards.

- Auricular acupuncture: Shenmen, kidney, sympathetic, liver, lung [32], spleen-pancreas and ACTH [33]. The application in the acupoint was unilateral, with electrical current power of 10 mA, intermittent stimulus, pulse width 50 µs, and frequency [33] A (2.28 Hz), B (4.56 Hz), and C (9.12 Hz) with time 10 seconds at each point. Needle insertion was 3 mm deep, the stainless-steel needle was 0.18 × 8 mm in size, and 0.2 mm in diameter. There were six appointments, twice a week. To be more precise in locating the points, the EL30 Finder (NKL®,Brusque, SC, Brazil) ear point locator was used. Location of points is displayed in Table 1.

Table 1 . Location of points and frequencies used

Nogier frequency [33]	French ear points - unilateral application
C – 9.12 Hz - motor frequency - muscle cell stimulation.	1- Shenmen: superior portion of the apex of the triangular fossa.
	2- Kidney: upper shell in a juice below the beginning of the lower cross.
	3- Sympathetic: end of inferior root of antihelix.
B – 4.56 Hz - coherence frequency of multicellularity.	4- Liver: superior concha above the root of the helix of the right ear.
	5- Spleen- pancreas: superior concha above the root of the helix of the left ear.
	6- Lung: lower shell.
A – 2.28 Hz – cell frequency – used whenever there is cellular distress.	7- ACTH: intertragic notch.

Mixed frequency 2 e 100 Hz	Systemic points – bilateral application
	1 - ST36: located in the tibialis anterior muscle laterally to the tibial margin.
	2 - BL60: located near the lateral malleolus.

ACTH = adrenocorticotropic hormone; ST36 = stomach 36; B60 = bladder 60.

Source: NADA Protocol [32].

The professional responsible for the application has 15 years of experience, and is a specialist in the area. To perform EA, the volunteers were instructed to remain in the supine position on the stretcher (Table 1).

7. Statistical analysis

Statistical analysis was performed blindly. Data were tabulated in the Excel 2010 platform and transferred into the Statistical Package for Social Sciences (SPSS version 20.0) program. For sample calculation, the GPower software (3.0) was used and α = 0.05 and β = 0.80 were adopted.

The Shapiro Wilk test verified the normality of data distribution, and later, the independent t-test compared the groups of all variables analyzed in the study: ACR10 (GPI and SSS variables), FIQ, NPRS, and HRV. The groups were analyzed through variation (Δ) within each group, where the post-value – pre-value = delta value. Sample characterization data were normally distributed and presented as mean and standard deviation. Statistical significance was established at p < 0.05.

RESULTS

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Abstract
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
CONCLUSIONS
FUNDING
AUTHORS’ CONTRIBUTIONS
CONFLICT OF INTEREST
References

One hundred fifteen volunteers participated in the eligible sample. After applying the tracking form, 75 volunteers were excluded for not meeting the inclusion criteria (n = 52), giving up participating (n = 2), or for other reasons (n = 21). In the total study, 18 volunteers participated and were randomized into CG (n = 9) and EG (n = 9) (Fig. 1). Patients did not experience adverse reactions after an intervention.

Figure 1.Flowchart adapted from CONSORT (2010).

Table 2 shows the anthropometric data of the study volunteers: age, height, body mass, body mass index and diagnosis time. The variables are homogeneous, with no statistical difference between the groups (p > 0.05) (Table 2).

Table 2 . The anthropometric variables of the patients in control and experimental groups

Variables	Control group (n = 9) mean (SD) (95% CI)	Experimental group (n = 9) mean (SD) (95% CI)	p-value
Age (year)	57.00 (9.28) (49.26 to 64.74)	58.00 (9.89) (50.39 to 65.60)	0.76^a
Height (cm)	1.57 (0.09) (1.50 to 1.65)	1.57 (0.05) (1.57 to 1.61)	0.89^a
Body mass (kg)	71.56 (13.35) (60.39 to 82.72)	69.88 (8.70) (63.19 to 76.58)	0.80^a
Body mass index (kg/cm²)	29.02 (6.97) (23.19 to 34.86)	28.27 (3.54) (25.54 to 30.99)	0.81^a
Diagnostic time (months)	126.00 (82.89) (56.70 to 195.29)	66.00 (73.85) (9.23 to 122.76)	0.09^b

^aANOVA one-way test; ^bMann-whitney test. SD = standard deviation; CI = confidence interval.

Table 3 shows the results of the Numerical Pain Rating Scale (NPRS) and Fibromyalgia Diagnostic Criteria (FDC) 2010. These analyzed variables showed no statistical difference after the intervention (p > 0.05) (Table 3).

Table 3 . The effect of systemic and auricular acupunture on pain intensity and symptoms of fibromyalgia

		Control group (n = 9)			Experimental group (n = 9)
		Pre-intervention mean (SD) (95% CI)	Post-intervention at 6 sessions mean (SD) (95% CI)	Δ	Pre-intervention mean (SD) (95% CI)	Post-intervention at 6 sessions mean (SD) (95% CI)	Δ	p-value for Δ^*
NPRS (point) -		6.87 (2.47) (4.80 to 8.94)	6.75 (1.48) (5.50 to 7.99)	–0.14 (2.67) (–2.61 to 2.32)	6.66 (2.69) (4.59 to 8.73)	4.88 (3.14) (2.47 to 7.30)	–1.77 (2.90) (–1.77 to 2.90)	0.17
FDC 2010 (point)	GPI	11.37 (4.47) (7.63 to 15.11)	11.37 (4.59) (7.53 to 15.21)	0.14 (4.98) (–4.46 to 4.74)	11.22 (5.14) (10.26 to 18.17)	10.44 (6.22) (5.65 to 15.23)	–3.77 (3.86) (–6.74 to –0.80)	0.06
	SSS	8.37 (3.06) (5.81 to 10.93)	8.62 (2.50) (6.53 to 10.71)	0.28 (3.72) (–3.16 to –3.73)	8.22 (4.02) (5.12 to 11.31)	7.88 (3.68) (5.05 to 10.72)	–0.33 (2.44) (–2.21 to 1.54)	0.74

*Independent t-test. Δ = delta calculation (post-intervention - pre-intervention); CI = confidence interval; SD = standard deviation; NPRS = numerical pain rating scale; FDC = fibromyalgia diagnostic criteria; GPI = generalized pain index; SSS = symptoms severity scale.

Table 4 shows the results of the variables analyzed in the FIQ. The t test showed that the mean of the intervention group showed a significant increase in FIQ values (total score) in relation to the control group (t₁₅ = 3.08; p = 0.008; effect size = 1.39 and power = 0.86). The t test showed that the mean of the intervention group showed a significant reduction in pain domain values in relation to the control group (t₁₆ = 2.55; p = 0.002; effect size = 1.24 and power = 0.81). The t test showed that the mean of the intervention group showed a significant reduction in anxiety domain values in relation to the control group (t₁₅ = 3.20; p = 0.006; effect size = 1.65 and power = 0.94). All differences found are true. Zero is outside the mean confidence interval. All differences have high effect size and power above 0.80 (Table 4).

Table 4 . The effect of systemic and auricular acupunture on symptoms of fibromyalgia by FIQ

FIQ	Control group (n = 9)			Experimental group (n = 9)				Difference mean (95% CI)	Effect size, power
Domain	Pre-intervention mean (SD) (95% CI)	Post-intervention at 6 sessions mean (SD) (95% CI)	Δ	Pre-intervention mean (SD) (95% CI)	Post-intervention at 6 sessions mean (SD) (95% CI)	Δ	p-value for Δ^*	Difference mean (95% CI)	Effect size, power
Physical dysfunction	1.95 (1.10) (1.03 to 2.87)	1.63 (1.53) (0.33 to 2.90)	–0.23 (2.06) (–2.15 to 1.67)	3.70 (2.40) (1.85 to 5.54)	2.81 (1.71) (1.49 to 4.13)	–0.88 (1.30) (–1.88 to 0.11)	0.49
It felt good	5.72 (4.95) (1.57 to 9.86)	5.00 (4.18) (1.50 to 8.50)	–1.22 (4.55) (–5.43 to 2.98)	6.83 (4.15) (3.63 to 10.02)	4.29 (2.86) (2.09 to 6.48)	–2.54 (2.45) (–4.42 to –0.65)	0.30
Missed work	0.53 (1.51) (–0.73 to 1.80)	0.89 (1.69) (–0.52 to 2.31)	0.40 (2.70) (–2.09 to 2.90)	6.03 (4.89) (2.27 to 9.79)	3.17 (2.84) (0.99 to 5.36)	–2.86 (4.29) (–6.15 to 0.43)	0.08
Work done	5.50 (3.07) (2.93 to 8.06)	4.62 (3.96) (1.31 to 7.93)	–1.00 (5.13) (–5.74 to 3.74)	6.77 (3.59) (4.01 to 9.54)	4.00 (4.21) (0.76 to 7.23)	–2.77 (3.27) (–5.29 to –0.26)	0.34
Pain	6.62 (1.84) (5.08 to 8.16)	7.50 (2.56) (5.35 to 9.64)	1.00 (2.16) (–0.99 to 2.99)	7.88 (2.97) (5.60 to 10.17)	5.77 (3.03) (3.44 to 8.10)	–2.11 (2.80) (–4.26 to 0.04)	0.02^**	2.88 (0.48 to 5.29)	1.24, 0.81
Fatigue	7.00 (2.61) (4.81 to 9.18)	7.75 (1.83) (6.21 to 9.28)	0.57 (1.98) (–1.26 to 2.41)	6.66 (3.84) (3.71 to 9.61	4.88 (3.78) (1.97 to 7.80)	–1.77 (3.56) (–4.51 to 0.96)	0.07
Tiredness	7.12 (2.74) (4.82 to 9.42)	7.62 (2.44) (5.58 to 9.66)	0.28 (2.81) (–2.31 to 2.88)	6.22 (3.45) (3.56 to 8.87)	4.33 (3.53) (1.61 to 7.05)	–1.88 (2.36) (–3.70 to –0.06)	0.07
Rigidity	5.62 (3.96) (2.31 to 8.93)	5.75 (3.84) (2.53 to 8.96)	0.00 (2.76) (–2.56 to –0.11)	6.66 (4.12) (3.49 to 9.83)	4.88 (3.75) (2.00 to 7.77)	–1.77 (2.81) (–3.94 to 0.38)	0.17
Anxiety	6.12 (2.53) (4.00 to 8.24)	7.87 (1.55) (6.57 to 9.17)	2.00 (2.16) (0.002 to 3.99)	6.33 (4.21) (3.09 to 9.57)	4.55 (3.24) (2.06 to 7.04)	–1.77 (2.38) (–3.61 to 0.05)	0.006^**	3.52 (1.18 to 5.87)	1.65, 0.94
Depression	4.87 (3.52) (1.92 to 7.82)	6.37 (1.92) (4.76 to 7.98)	1.00 (2.44) (–1.26 to 3.26)	5.88 (4.96) (2.07 to 9.70)	4.77 (3.34) (2.20 to 7.34)	–1.11 (3.44) (–3.75 to 1.53)	0.10
Total score	51.08 (11.18) (41.73 to 60.43)	56.31 (14.27) (44.38 to 68.24)	4.27 (17.38) (–11.79 to 20.35)	63.01 (31.73) (38.61 to 87.41)	43.50 (24.03) (25.02 to 61.97)	19.51 (16.68) (–32.33 to –6.68)	0.008^**	24.73 (7.63 to 41.84)	1.39, 0.86

Data are presented with mean (SD) (95% CI). *Independent t-test; **p < 0.05. Δ = delta calculation (post-intervention - pre-intervention); CI = confidence interval; FIQ = fibromyalgia impact questionnaire; SD = standard deviation.

Table 5 shows the results of the variable HRV. These analyzed variables showed no statistical difference after the intervention (p > 0.05) (Table 5).

Table 5 . The effect of systemic and auricular acupuncture on HRV variables

	Control group (n = 9)			Experimental group (n = 9)
	Pre-intervention mean (SD) (95% CI)	Pos-intervention at 6 sessions mean (SD) (95% CI)	Δ	Pre-intervention mean (SD) (95% CI)	Pos-intervention at 6 sessions mean (SD) (95% CI)	Δ	p-value Δ
R-R (ms)	932.85 (127.83) (814.62 to 1052.08)	916.00 (59.36) (861.09 to 970.90)	–16.85 (79.03) (–89.95 to 56.23)	845.00 (134.76) (720.36 to 969.63)	880.14 (125.52) (764.05 to 996.23)	42.00 (52.87) (1.35 to 82.64)	0.09
HR (bpm)	65.57 (9.32) (56.94 To 74.19)	65.85 (4.09) (62.06 to 69.64)	0.28 (5.90) (–5.17 to 5.74)	72.42 (11.81) (61.50 to 83.35)	69.42 (9.99) (60.18 to 78.67)	–3.33 (5.22) (–7.34 to 0.67)	0.21
SDNN (ms)	21.17 (4.79) (16.73 to 25.60)	25.01 (11.20) (14.65 to 35.37)	3.84 (10.12) (–5.52 to 13.20)	21.64 (6.73) (15.41 to 27.86)	29.70 (17.78) (13.25 to 46.15)	22.23 (32.53) (–2.77 to 47.24)	0.36
RMSSD (ms)	20.05 (5.15) (15.28 to 24.82)	24.42 (13.02) (12.37 to 36.47)	4.37 (14.03) (–8.61 to 17.35)	49.45 (75.75) (–20.60 to 119.51)	30.42 (22.22) (9.87 to 50.98)	–1.35 (80.59) (–63.30 to 60.59)	0.56
NN50 (beats)	6.00 (3.21) (3.02 to 8.97)	10.57 (11.63) (–0.18 to 21.32)	4.57 (11.48) (–6.05 to 15.19)	9.57 (12.12) (–1.63 to 20.78)	18.42 (28.77) (–8.18 to 45.04)	12.77 (27.71) (–8.52 to 34.07)	0.52
pNN50 (%)	1.91 (0.93) 1.04 to 2.77	3.27 (3.65) –0.11 to 6.65	1.36 (3.60) –1.97 to 4.69	2.72 (3.43) –0.45 to 5.89	5.60 (8.88) –2.61 to 13.82	6.93 (10.84) –1.40 to 15.26)	0.31
RR triangular index	5.47 (1.37) (4.19 to 6.74)	5.67 (1.42) (4.35 to 6.99)	0.20 (1.33) (–1.03 to 1.44)	6.37 (1.69) (4.80 to 7.94)	7.19 (2.72) (4.67 to 9.71)	1.59 (2.68) (–0.46 to 3.65)	0.23
TINN (ms)	124.42 (30.67) (96.05 to 152.79)	154.14 (117.36) (45.59 to 262.68)	29.71 (102.92) (–65.47 to 124.90)	107.85 (34.89) (75.58 to 140.13)	185.57 (142.01) (54.23 to 316.90)	152.77 (203.55) (–3.68 to 309.24)	0.36
LF (ms2)	231.71 (147.50) (95.29 to 368.13)	273.00 (242.32) (48.88 to 497.11)	41.28 (133.03) (–81.74 to 164.32)	221.00 (175.26) (58.90 to 383.09)	329.71 (353.04) (3.20 to 656.22)	559.34 (1421.21) (–533.09 to 1651.78)	0.39
HF (ms2)	145.00 (69.23) (80.96 to 209.03)	312.71 (395.51) (–53.08 to 678.50)	167.71 (441.89) (–213.22 to 548.65)	183.28 (98.93) (91.78 to 274.78)	298.42 (363.24) (–37.51 to 634.37)	124.57 (334.62) (–132.64 to 281.78)	0.91
LF/HF	1.80 (1.12) (0.76 to 2.84)	1.62 (1.17) (0.54 to 2.71)	–0.01 (1.25) (–1.16 to 1.14)	1.16 (0.67) (0.53 to 1.78)	1.87 (1.07) (0.88 to 2.87)	1.54 (2.98) (–0.74 to 3.83)	0.18
LF (n.u)	59.81 (12.09) (48.63 to 71.00)	53.92 (23.48) (32.20 to 75.63)	–5.89 (24.41) (–28.47 to 16.68)	49.17 (17.87) (32.64 to 65.69)	58.52 (22.23) (37.96 to 79.09)	9.49 (14.67) (–1.78 to 20.77)	0.13
HF (n.u)	44.27 (14.86) (30.52 to 58.02)	46.04 (23.47) (24.33 To 67.75)	1.76 (28.91) (–24.98 to 28.51)	50.75 (17.87) (34.22 to 67.28)	41.43 (22.04) (21.05 to 61.82)	–9.39 (14.48) (–20.53 to 1.74)	0.32
SD1	14.18 (3.64) (10.81 to 17.55)	17.30 (9.21) (8.77 to 25.82)	3.11 (9.92) (–6.06 to 12.29)	14.75 (2.83) (12.13 to 17.38)	21.55 (15.75) (6.98 to 36.13)	14.96 (22.54) (–2.36 to 32.29)	0.31
SD2	26.18 (6.65) (20.03 to 32.33)	30.14 (12.67) (18.41 to 41.86)	3.95 (9.86) (–5.16 to 13.08)	26.60 (9.66) (17.66 to 35.53)	35.78 (20.41) (16.90 to 54.66)	27.32 (40.55) (–3.85 to 58.49)	0.39
SD2/SD1	2.00 (0.57) (1.46 to 2.53)	1.71 (0.48) (1.26 to 2.16)	–0.08 (0.78) (–0.81 to 0.63)	1.77 (0.47) (1.34 to 2.21)	1.83 (0.57) (1.29 to 2.36)	0.21 (0.43) (–0.12 to 0.55)	0.56
Approximate entropy (ApEn)	1.00 (0.01) (0.99 to 1.01)	0.98 (0.04) (0.93 to 1.02)	0.006 (0.12) (–0.10 to 0.11)	1.13 (0.05) (1.08 to 1.19)	1.10 (0.04) (1.06 to 1.14)	–0.09 (0.41) (–0.41 to 0.22)	0.36
Sample entropy (SampEn)	1.57 (0.53) (1.07 to 2.06)	1.69 (0.52) (1.21 to 2.17)	0.057 (0.44) (–0.35 to 0.47)	1.82 (0.32) (1.52 to 2.13)	1.69 (0.46) (1.25 to 2.12)	(–0.29 (0.68) (–0.82 to 0.23)	0.49
Short-term fluctuations α1	0.98 (0.15) (0.84 to 1.13)	0.88 (0.17) (0.72 to 1.04)	–0.08 (0.24) (–0.30 to 0.14)	0.98 (0.28) (0.72 to 1.25)	1.01 (0.27) (0.75 to 1.26)	0.01 (0.41) (–0.30 to 0.33)	0.71
Long-term fluctuations α2	0.40 (0.14) (0.26 to 0.53)	0.34 (0.08) (0.27 to 0.42)	–0.05 (0.13) (–0.17 to 0.06)	0.41 (0.07) (0.34 to 0.49)	0.44 (0.10) (0.34 to 0.54)	0.02 (0.20) (–0.13 to 0.18)	0.42

DISCUSSION

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DISCUSSION
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FUNDING
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References

The positive results associated with EA and the effects of the respective frequencies, 2 and 100 Hz, are well-known in the literature [37], but little is known about the effects of Nogier frequencies (2.28, 4.56 and 9.12 Hz), specifically for women with fibromyalgia. The aim of the study was to associate new techniques with those already known in the literature, but with different stimuli, and to observe their effects in the different domains that fibromyalgia directly affects. The main findings of the present study were that EA, with systemic frequencies of 2/100 Hz and associated with the Nogier frequency (2.28, 4.56 and 9.12 Hz) in auricular acupuncture, generated clinically relevant effects in reducing the impact of fibromyalgia, particularly in the domains of pain and anxiety. Regarding the NPRS variables, FDC 2010 and HRV, they remained unchanged.

Numerous experimental studies have shown that acupuncture, particularly low-frequency EA (2 to 4 Hz), causes the release of opioids in various regions of the hypothalamus, midbrain, and spinal cord that are related to information processing and ultimately influence sympathetic neural activity [38,39]. Thus, by releasing endorphins, endomorphins, or enkephalins, which act as neuromodulators that likely reduce the function of excitatory neurotransmitters, acupuncture appears able to inhibit sympathetic outflow and clinical events associated with increased sympathetic activity [40,41]. Other neurotransmitters that may be associated with the influence of acupuncture on sympathetic neural activity and are important in cardiovascular regulation include gamma-aminobutyric acid (GABA), serotonin or 5-hydroxydopamine (5-HT), acetylcholine, and nociceptin (also known as FQ orphanin) [40,42].

Electroacupuncture has been associated with different physiological analgesic mechanisms, with low frequency EA (at 2 to 4 Hz) inducing the release of enkephalin and activation of the µ receptor, and high frequency EA (50-100 Hz) inducing the release of dynorphin and activation of κ receptors [40]. High frequency EA (100 Hz) can influence the cardiovascular system through another opioid neurotransmitter/neuromodulator called dynorphin, which has been shown to have a potent analgesic effect [41,43].

An abundance of transient type 1 vanilloid potential (TRPV1) has been identified in the anatomical layers of ST36, indicating a possible pathway linking ion channel activation to neurological signaling and resulting in therapeutic benefits, such as analgesia [44]. Regarding the protocol used, studies that used the ST36 point [45] believe that it may have anti-inflammatory effects through the modulation of immunity. Deluze et al. [11] obtained positive results treating fibromyalgia using the ST36 point by using a rectangular, biphasic current of 10 volts and frequency 1-99 Hz (continuous frequency); 250 ms interval and 10 mA intensity. Vieira et al. [46] concluded that neuroactive mediators released during acupuncture-induced tissue injury may contribute to analgesia.

According to Nogier [33] in French auriculotherapy the A frequency (2.28 Hz) stimulates the cellular working mechanism. This frequency is used to treat inflammatory and degenerative pathologies. Frequency B (4.56 Hz) stimulates multicellular functions, such as cohesion, coherence, and cell recognition. This frequency is used to treat disorders related to nutrition, the digestive system, and immunity (allergies and autoimmune pathologies). Frequency C (9.12 Hz) stimulates muscle contraction and is used for spasms.

This study’s treatment protocol brought, as an innovation, the stimulation with mixed frequencies of 2 Hz/100 Hz and Nogier techniques not yet described in the literature, thus making it impossible to make further comparisons of the results found; however, Pirnia et al. [32] used the NADA protocol to treat a fibromyalgia patient, and concluded that there was no improvement in pain reduction. Dorsher [47] compared the effects of needle acupuncture with laser acupuncture, determining that laser stimulation showed superior results in relation to pain relief. However, many studies report the effectiveness of EA. A systematic review by Deare et al. [48] of the effectiveness of acupuncture in treating fibromyalgia concluded that EA is better than manual acupuncture for reducing pain and stiffness and improving general well-being, sleep, and fatigue. Lin et al. [49] concluded that EA alleviates myofascial pain and improves fibromyalgia symptoms, and Díaz-Toral et al. [16] showed that the sympathetic effect that EA caused in patients with fibromyalgia may be related to positive changes in quality of life. This is in accordance with Martin et al. [50] findings, where he defended the theory that the improvement of pain is one of the factors that results in the improvement of the quality of life.

The use of HRV analysis has been increasingly used to better understand the behavior of the ANS in various diseases, as seen in the study by Marães [51]. Changes in HRV standards provide a sensitive and early indicator of health impairments. High HRV is a sign of good adaptation, characterizing a healthy individual with efficient autonomic mechanisms. Conversely, low HRV is often an indicator of abnormal and insufficient adaptation of the ANS, which may indicate the presence of physiological malfunction in the individual [52]. We believe that the maintenance of the results obtained in the HRV could be due to some factors such as: intervention time, sample size, some drugs that can affect autonomic modulation (Beta blockers), stress level and sleep quality.

Possible limitations of this study include the non-blinding of researchers and volunteers, absence of a placebo and follow-up group, and considering the sample size. These limitations may influence the ability to explore the findings and draw conclusions. Despite the guidelines taken with the HRV, maybe not everyone has followed them correctly. Because fibromyalgia has several symptoms, other variables could have been checked, such as sleep quality and fatigue, which could influence heart rate variability responses and quality of life.

CONCLUSIONS

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MATERIALS AND METHODS
RESULTS
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CONCLUSIONS
FUNDING
AUTHORS’ CONTRIBUTIONS
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References

2/100 Hz systemic electroacupuncture associated with the Nogier frequency positively influenced some quality of life variables; however, pain intensity, diagnostic criteria, and HRV variables did not change. Due to the observed responses, it is feasible to perform a randomized clinical trial study; however, for better proof of data, there is a need to increase the sample size.

FUNDING

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This work was funded by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brazil (CAPES) - Código Financeiro 001, through the Post-Graduation Program in Rehabilitation Science of the Universidade Federal de Alfenas - UNIFAL-MG.

AUTHORS’ CONTRIBUTIONS

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Conceptualization: Andréia Maria Silva Vilela Terra, Adriana Teresa Silva Santos, Rodrigo Polaquini Simões; Methodology: Érika Almeida Boggiss, Rhaynara Coelho Rosário, Rosana Aparecida de Lima, Paula Aparecida Silva; Validation: Adriana Teresa Silva Santos and Josie Resende Torres da Silva; Formal analysis: Andréia Maria Silva Vilela Terra, Rodrigo Polaquini Simões; Investigation: Rosa Maria Moreira, Karol Priscila da Silva, Caroline Lima de Farias, Vanessa de Queiroz dos Santos; Resources: Érika Almeida Boggiss, Rosana Aparecida de Lima; Data curation: Paula Aparecida Silva and Josie Resende Torres da Silva; Writing-original draft preparation: Érika Almeida Boggiss, Andréia Maria Silva Vilela Terra and Josie Resende Torres da Silva; Writing - review and editing: Adriana Teresa Silva Santos, Rodrigo Polaquini Simões; Visualization: Andréia Maria Silva Vilela Terra; Supervision: Adriana Teresa Silva Santos; Funding acquisition: Andréia Maria Silva Vilela Terra; All authors have read and agreed to the published version of the manuscript.

CONFLICT OF INTEREST

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The authors declare no conflict of interest.

References

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References

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