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Table of Contents
ORIGINAL ARTICLE
Year : 2020  |  Volume : 63  |  Issue : 4  |  Page : 163-170

Low-frequency electroacupuncture at acupoints guanyuan (CV4) and zhongji (CV3) lengthen ejaculatory latency and improves sexual behavior in male rats


1 Institute of Traditional Medicine, School of Medicine, National Yang-Ming University; Department of Urology, Taipei City Hospital Ren-Ai Branch, Taipei, Taiwan
2 Department of Urology, Taipei Medical University Hospital; School of Dental Technology, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan
3 Institute of Traditional Medicine, School of Medicine, National Yang-Ming University, Taipei; Department of Chemical Engineering, National United University, Miaoli, Taiwan
4 Department of Physical Therapy, Hungkuang University, Taichung, Taiwan

Date of Submission04-May-2020
Date of Acceptance21-Jul-2020
Date of Web Publication28-Aug-2020

Correspondence Address:
Dr. Kuei-Ying Yeh
Department of Physical Therapy, Hungkuang University, No. 1018, Sec. 6, Taiwan Boulevard, Shalu, Taichung 43302
Taiwan
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Source of Support: This study was supported by research grants obtained from the Ministry of Science and Technology, Taiwan (MOST 106-2410-H-241-002; MOST 107-2410-H-241-004), and the Department of Health, Taipei City Government, Taiwan (No. 10701-62-056)., Conflict of Interest: None


DOI: 10.4103/CJP.CJP_34_20

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  Abstract 

Acupuncture is a key component of Traditional Chinese Medicine in which needles are inserted into specific areas in the body to stimulate certain physiological reactions of the body. Clinical research shows that acupuncture is beneficial in treating a variety of illnesses, such as erectile dysfunction. Therefore, we investigated the effect of electroacupuncture (EA) stimulation of both low- and high frequencies at Guanyuan (CV4) and Zhongji (CV3) acupoints on male copulatory behavior in sexually experienced male rats. The animals were randomly divided into four groups: control, sham EA, EA, and only acupuncture. The administered low and high EA were 2 Hz and 80 Hz, respectively, for which the respective intensities were 1.5 mA (30 min/day for 5 days) and 1.5 mA (30 min once). The only acupuncture-administered group received acupunctures at CV4 and CV3 without electrical stimulation. Following the acupuncture treatments, copulatory behavior test was conducted. Eventually, animals were sacrificed and blood samples were collected for testing the serum hormonal profile including luteinizing hormone (LH), testosterone (T), and serotonin (5-HT). Results showed that low-frequency EA-treated rats exhibited increasing intromissions and ejaculation latency compared to control, sham EA, and acupuncture groups, while high-frequency EA-treated rats displayed lower intromissions and ejaculation frequency when compared with those in controls. Furthermore, serum levels of 5-HT and LH in low-frequency EA-treated rats were higher than all the other groups. These results indicate that compared to high-frequency EA, the low-frequency EA might be efficacious in the treatment of premature ejaculation, thereby improving sexual behavior.

Keywords: Ejaculation latency, electroacupuncture, luteinizing hormone, serotonin, sexual behavior


How to cite this article:
Huang AC, Liu MC, Tsai TH, Chang YH, Wu JM, Yeh KY. Low-frequency electroacupuncture at acupoints guanyuan (CV4) and zhongji (CV3) lengthen ejaculatory latency and improves sexual behavior in male rats. Chin J Physiol 2020;63:163-70

How to cite this URL:
Huang AC, Liu MC, Tsai TH, Chang YH, Wu JM, Yeh KY. Low-frequency electroacupuncture at acupoints guanyuan (CV4) and zhongji (CV3) lengthen ejaculatory latency and improves sexual behavior in male rats. Chin J Physiol [serial online] 2020 [cited 2020 Oct 21];63:163-70. Available from: https://www.cjphysiology.org/text.asp?2020/63/4/163/293584


  Introduction Top


Manual acupuncture is a key component of Traditional Chinese Medicine in which needles are inserted into the skin and underlying tissues at specific acupoints. Clinical research shows that acupuncture is a beneficial treatment for erectile dysfunction (ED).[1],[2] Notably, a few animal studies have demonstrated that after scrotal heat-treatment, the electroacupuncture (EA) might increase spermatogenesis in male rats[3] and restore sexual behavior in morphine withdrawal rats.[4]

In men, ED and premature ejaculation (PE) are considered as the two main types of sexual dysfunction that can lead to a reduced quality of life. ED affects up to 52% of men between the ages of 40 and 70 years,[5] while PE is the most common sexual problem affecting 20%–30% of all men. Several studies have reported the beneficial therapeutic effects of acupuncture on these sexual function disorders.[6],[7],[8] Additionally, previous reports indicate a high success rate of acupuncture therapy (>60%) in the treatment of ED, suggesting that acupuncture could be considered as an alternative adjuvant therapy for nonorganic ED.[1] However, these studies were conducted in humans, and factors such as psychological effects and questionnaires bias may influence the objectivity of reported clinical results.

Guanyuan (CV4), Zhongji (CV3), Sanyinjiao (SP6), and Zigong (Ex-CA1) are the four key acupuncture points that are commonly used for the treatment of infertility.[9] CV4 is an important acupoint located on the ventral midline of lower abdomen and exerts a large impact on urogenital and gynecological systems such as uterine dysfunction.[10] The CV4 is an acupoint associated with impotence and can therefore be used effectively for the treatment of male sexual dysfunction. In males, acupuncture at CV4 has been reported to exert therapeutic effects on PE.[2],[11] Moreover, it has had a great impact on the regulation of infertility[3] and sexual activity.[2],[4]

Morphine administration decreases sexual activity in rats and EA administered 30 min once daily for 7 days may facilitate the recovery of sexual behavior in morphine withdrawal male rats.[4] Furthermore, low-frequency electrical stimulation at CV4 every 3 days for 10 sessions significantly enhanced spermatogenesis in scrotal-heat-treated rats.[3] However, these EA studies were performed using only low-frequency stimulations and over several weeks. To date, the effects of acute and high-frequency electrical stimulation on male sexual behavior remain unknown. Additionally, most of the acupuncture studies on male sexual function are focused on infertility and ED, and to the best of our knowledge, no studies have investigated the effect of EA on male sexual performance.

It is further known that hormonal levels such as luteinizing hormone (LH), testosterone (T), and serotonin (5-HT) play a key role in the regulation of sexual behavior.[12],[13],[14] T is a primary male sex hormone and is essential in facilitation of male copulatory behavior. Studies have demonstrated that its deficiency after castration eliminates penile erection, and abolishes sexual behavior, and these effects could be reversed by T replacement.[15],[16]

Based on above-mentioned evidence, we aimed to determine the effects of low- and high-frequency EA stimulation at acupoint CV4 on male copulatory behavior in sexually experienced male rats through assessing their serum hormonal profile. It has been suggested that low- and high-frequency stimulations represent the frequencies ranging from 2 to 5 Hz and 20–200 Hz, respectively.[17],[18] Therefore, we used 2 Hz and 80 Hz for low- and high-frequency stimulations, respectively. The acupoint CV4 was chosen due to its role in both urological and reproductive system disorders, whereas CV3 was chosen as the matching acupoint.


  Materials and Methods Top


Animals

Long-Evans male (8-week-old) and female rats (8-week-old) were purchased from the Animal Center of National Science Council, Taipei, Taiwan. The animals were housed under a 12-h light/dark cycle (lights off at 12:00 p. m.), at temperature 22°C ± 1°C and humidity 55% ± 10%. Food and water were available adlibitum. The experimental protocols were approved by the Institutional Animal Care and Use Committee, HungKuang University (HK-10614), and all of the experimental procedures were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Female Long-Evans rats aged 8 weeks were ovariectomized (OVX) and immediately implanted with a 5 mm Silastic capsule filled with 17 β-estradiol (Sigma) under an intraperitoneal injection of tiletamine-zolazepam (20 mg/kg BW: Zoletil 50; Virbac, France) anesthesia. Approximately 1 week following surgery, each OVX female was subcutaneously injected with 500 μg progesterone (Sigma) at 4 h before behavioral testing. Female rats who displayed good sexual receptivity (proceptive behaviors and lordosis) were used.

Copulatory behavior

Copulation screening of test male rats

The copulation screening test was performed during the dark phase of the cycle (2 h after the lights-off) and under a dim red light. Each male rat was placed in a circular Plexiglas testing chamber (45 cm diameter) and a receptive female rat was introduced 3 min later. The male rats were tested thrice at intervals of 5–6 days, and those that had not ejaculated twice in three testing sessions were not used in subsequent sexual behavioral study.

Copulatory behavior testing

All behavioral tests were conducted between 14:00 h and 17:00 h under a dim red light. For acclimatization to the test environment, each male rat was placed in a circular Plexiglas testing chamber 3 min before the introduction of a sexually receptive female. Thereafter, male rat was allowed to copulate for 30 min. The copulatory behaviors were recorded by a term of observers. The following behavioral parameters were recorded: mount frequency (MF, number of mounts without penile insertion), intromission frequency (IF, number of mounts with penile insertion), ejaculation frequency (EF, number of ejaculations), mount latency (ML, latency from the introduction of the female to the first mount), intromission latency (IL, latency from the introduction of the female to the first intromission), and ejaculation latency (EL, latency from the first intromission to ejaculation). When a male rat achieved ejaculation, it developed violent trembling throughout its body accompanied with deep penetration. At the same time, they held the females tightly with their forelimbs for a few seconds. After ejaculation, male rats entered a post-ejaculatory refractory period that lasts 5–7 min.[19] Male rat who failed to ejaculate within 30-min observational period was excluded from the experiment.

Electroacupuncture stimulation

At low frequency: Twenty-four male rats were used in the experiment. The rats were divided into 4 groups: control, sham EA, EA, and acupuncture (n = 6 each). The acupoints CV4 and CV3 are located at points 3/5 and 4/5 down the ventral midline connecting the umbilicus to the pubic tubercle, respectively.[20] Two steel needles (0.3 mm in diameter) were inserted into CV4 and CV3 up to a depth of 5 mm each. Constant current square wave electrical stimulation produced by an EA apparatus (Model-05B; Ching-Ming Medical Device Co., Taipei, Taiwan) was administered via EA needles. The double pole method of needle acupuncture was used, as the impulses could pass from one needle to other. The EA group received EA stimulations at acupoints CV4 and CV3, whereas sham EA group received stimulations at non-EA points near areas of true acupuncture points (5 mm to the left and lateral to the CV4 and CV3). The stimulation parameters were 2 Hz frequency and 1.5 mA intensity. EA was performed by inserting the needles for 30 min daily for five consecutive days under anesthesia with intraperitoneal injection of Zoletil 50. The acupuncture group received treatment at acupoints CV4 and CV3 for 30 min daily for 5 days without electrical stimulation. Sexual behavior was measured before (pre-treatment) and after 5 days of treatment. The experimental design and procedure is shown in [Figure 1]a.
Figure 1: Experimental design and procedure. (a) Sexual behavior was tested prior to and after 5 days consecutive treatment of low-frequency EA (2 Hz, 1.5 mA, 30 min each). (b) Sexual behavior was tested pre- and post-treatment with high-frequency EA (80 Hz, 1.5 mA, 30 min once) at day 0, 2 and 6. (c) Schematic diagram showing the location of acupoints (CV4 and CV3) in the abdomen of the rat. EA: electroacupuncture, CV4: Guanyuan, CV3: Zhongji.

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At high frequency: Twelve male rats were used in the experiment. The rats were divided into two groups: control and EA (n = 6 each). The EA group received EA stimulations at acupoints CV4 and CV3 for 30 min once (frequency, 80 Hz; intensity, 1.5 mA). The control group received anesthesia. Copulatory behavior was measured before and 24 h after the EA stimulation. The EA rats underwent an additional sexual behavior test at day 6 after EA treatment [Figure 1]b. The experimental setting of at acupoints is illustrated in [Figure 1]c.

Serum assays in low frequency-treated animals

After the behavior test, animals were anesthetized by intraperitoneal administration of tiletamine–zolazepam (20 mg/kg BW: Zoletil 50; Virbac, France). Serum samples were obtained from the jugular vein to measure the levels of LH (Elabscience Biotechnology Inc.), T (Aviva Systems Biology), and 5-HT (Abnova) by ELISA kits according to the manufacturers' instructions. Eventually, death of the animals was ensured through exsanguination and absence of heartbeat under Zoletil anesthesia.

Statistical analysis

Quantitative data were analyzed for statistical significance using Statistica 12.0 (StatSoft Inc., Tulsa, OK, USA). One-way analysis of variance (ANOVA) with repeated measures (time as the repeated factor) was used to compare the copulatory behavior between the animal groups. A paired t-test was used to analyze sexual performance at different time points within the same group. One-way ANOVA was used to analyze serum levels of LH, T, and 5-HT. Post hoc comparisons using Fisher's least significant difference test were conducted to establish the significance between mean values. P < 0.05 was considered as statistically significant. All quantitative data were presented as mean ± standard error of the mean.


  Results Top


Low frequency

Sexual behavior

As shown in [Figure 2], the significant differences in EL (F3,20= 5.82, P < 0.01) and IF (F3, 20= 4.38, P < 0.05) were noted between compared groups. Post hoc analysis showed that EL was significantly longer in EA group when comparing to other three groups [P < 0.001; [Figure 2]c], indicating enhanced time from intromission to ejaculation. Furthermore, the EA group displayed significantly increased IF than the other groups [P < 0.01; [Figure 2]e], suggesting increased number of intromissions. No significant differences were observed in ML, IL, MF, and EF among the four groups.
Figure 2: Comparative effect of pre- and post-low-frequency EA treatment on sexual behavior of male rats. Mount latency (a), intromission latency (b), ejaculation latency (c), mount frequency (d), intromission frequency (e), and ejaculation frequency (f) in the male rats. The data are presented as mean ± standard error of the mean. **P < 0.01; ***P < 0.001, significant differences from EA group. Control: no treatment. Sham EA: Electroacupuncture at Sham EA acupoints with low-frequency electrical stimulation. EA: Electroacupuncture at Guanyuan (CV4) and Zhongji (CV3) with low-frequency electrical stimulation. Acupuncture: Acupuncture at Guanyuan (CV4) and Zhongji (CV3) without electrical stimulation. Pre-T: pretreatment.

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Serum levels of luteinizing hormone, T, and 5-HT

As shown in [Figure 3]a, ANOVA detected a significant difference in LH levels among the experimental groups (F3, 17= 12.76, P < 0.001). Post hoc analysis showed that rats in EA group had a higher LH levels compared to others, whereas no difference in T levels between the groups was observed (F3, 17= 1.98, P = 0.015) [Figure 3]b. Further, ANOVA detected a significant difference in 5-HT levels (F3, 17= 10.88, P < 0.001) [Figure 3]c. Post hoc analysis showed that rats in EA group had a higher 5-HT levels than the others.
Figure 3: Influence of electroacupuncture administration on levels of (a) luteinizing hormone (LH), (b) testosterone (T), and (c) 5-HT. The data are presented as mean ± standard error of the mean. *, ** and *** indicate P < 0.05, P < 0.01 and P < 0.001, respectively.

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High frequency

Sexual behavior

During high-frequency treatment, compared to control group, a remarkable decrease in EF was seen in male rats after EA treatment at day 2. The ANOVA revealed a significant difference in the treatment efficacy (F1, 10= 5.00, P < 0.05) and treatment time (day) and EF relation (F1, 10= 10.00, P < 0.05) between EA and control group. Post hoc analysis showed that rats receiving high-frequency EA treatment displayed a significantly lower EF than the control group (P < 0.001) and pre-treatment status [Figure 4]b. Surprisingly, we found that 50% of rats in EA group did not display ejaculatory behavior during the beginning 30 min of copulatory experiment. While determining IF, no significant statistical difference was noted at day 2 [Figure 4]a. Additionally, no significant differences in ML, IL, MF, and IF were observed between the controls and the EA group (data not shown).
Figure 4: Effect of high-frequency electroacupuncture treatment on intromission frequency (a) and ejaculation frequency (b) in the male rats. The data are presented as mean ± standard error of the mean for pre-treatment and Day 2. On ejaculation frequency (b), a represents the significant difference (***P < 0.001) between control group and EA group at Day 2, while b indicates the significant difference (*P < 0.05) between Pre-T and Day 2 of EA treated group. EA: Electroacupuncture at Guanyuan (CV4) and Zhongji (CV3) with high-frequency (80 Hz) electrical stimulation. Pre-T: pre-treatment.

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Further, comparing to the pre-treatment EA group, no significant changes were found in IF both at day 2 and 6 [Figure 5]a. Whereas, a significant difference in EF was noted on both day 2 and 6 and (df = 5, t = 3.87, P < 0.05). Surprisingly, on day 6, EF dramatically enhanced compared to day 2 (df = 5, t = -3.50, P < 0.05) and revert back almost equal to pre-T level. This suggests that high-frequency EA may temporarily impair the ejaculatory behavior, which might have reversed after 6 days of rest.
Figure 5: Effect of high-frequency EA treatment on sexual behavior of male rats. Intromission frequency (a) and ejaculation frequency (EF) (b). The data are presented as mean ± standard error of the mean for pre-treatment and 1 day after EA treatment (day 2). *P < 0.05. (a) Represent the EF at day 2 compared to Pre-T, whereas (b) represents EF at day 6 compared to day 2. EA: Electroacupuncture at Guanyuan (CV4) and Zhongji (CV3) with high-frequency (80 Hz) electrical stimulation, Pre-T: pre-treatment.

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  Discussion Top


This is the first animal study to demonstrate that low-frequency EA facilitates sexual function and extends ejaculatory latency in males. The advantage of animal research is that the results are objective. In the present study, low-frequency EA stimulation at CV4 and CV3 significantly improved copulatory performance in sexually experienced male rats. This was evidenced by increased EL and IF, whereas no changes were observed in EF, MF, ML, or IL [Figure 2]. On the other hand, high-frequency EA stimulation at CV4 and CV3 significantly inhibited copulatory performance in the male rats by decreasing EF; however, no any effects on IF, ML, or IL was exhibited.

MF, IF, and EF are considered as common indicators of vigor, libido, and potency.[21] On the other hand, prolonged EL is an indicative of enhanced sexual performance.[22] The significant increase in EL after low-frequency EA treatment also suggested that prolonged the duration of coitus, which indicates increasing sexual motivation and copulatory performance in the male rats was enhanced.[23] Recent studies also documented that herbal extracts of Allium tuberosum[24] and Moringa oleifera[25] have aphrodisiac action with similar rats' behavior findings.[26] In our study, we found that high-frequency EA stimulation resulted in decreased EF, which was restored on day 6 signifying that high-frequency EA stimulation of CV4 and CV3 exerted a temporary inhibitory effect on EF. However, the reduction in EF caused by high-frequency EA stimulation is temporarily and reversible.

ED or impotence is a sexual dysfunction characterized by the inability to develop or maintain an adequate erection during sexual activity. ED reportedly affects up to 40% of men between the ages of 40 and 70 and is more likely to develop with age. Unlike ED, which is more prevalent in older men, PE is considered as one of the most common male sexual disorders that occurs in men younger than 40 years of age. ED encompasses several ejaculation-associated disorders, including PE. A clinical study had reported that acupuncture can improve the quality of erection and sexual activity in ED.[2] Furthermore, the acupuncture at points CV3 has been shown to have a positive effect on intravaginal ejaculation latency time (IELT).[27] Collectively, these results suggest that acupuncture may be proved beneficial for the treatment of male sexual dysfunction. In the present study, low-frequency EA at points CV4 and CV3 significantly increased EL [Figure 2]c and IF [Figure 2]e in rats. Sildenafil (Viagra) was well known to be used for improvement of male sexual function and has been reported significantly increasing IF, EF, and EL in male rats.[26] Fouche et al. recently also reported that Monsoniaangustifolia significantly enhanced male sexual performance in rats, as revealed by an increase in the IF, EF, and EL.[26] Our results are similar to this study,[26] suggesting that low-frequency stimulation at these two acupoints may be used to improve sexual activity. An increase in the number of intromissions frequency indicates the efficiency of erection[28] and the prolonged EL signifies low-frequency EA may treat PE. This is the first animal study to show that low-frequency EA facilitates sexual function and extends ejaculatory latency in males.

5-HT plays an important role in regulation of EL.[29] Selective serotonin-reuptake inhibitors (SSRIs) are the first choice of treatment in PE. Clinical studies have shown that daily oral administration of the SSRI type drug, paroxetine, significantly increases IELT.[30] From a neurochemical point of view, dopaminergic activity in the brain influences sexual behavior, arousal, and motivation. Dopamine agonists facilitate sexual behavior and 5-HT which regulates erection or ejaculation in rodents. For example, stimulation of 5-HT2C may facilitate erections, but inhibit ejaculations.[31] Additionally, rats treated with clomipramine (5-HT reuptake inhibitor) significantly prolonged the EL.[32] It has been reported that acupuncture at CV4 and ST36 can increase serotonin levels in humans[33] and also in chronic, unpredictable, and mild stresses-exposed rats.[34] Our results show that low-frequency EA treated-group had higher serum 5-HT levels than other groups [Figure 3]c, suggesting that low-frequency EA prolonged EL in male rats, which may be attributed to elevated serum 5-HT levels after stimulation at CV3 and CV4.

It is well known that the hormones belonging to hypothalamic–pituitary–gonadal axis exerts facilitative effects on male reproductive behavior. It has also been reported that levels of T and LH decrease and increase, respectively, within hours following castration. The T replacement restores T levels and suppresses LH levels in serum, leading to gradual restoration of sexual behavior.[35] A research reported that serum T levels, but not LH levels, were significantly reduced in the hemicastrated male rats, which revealed poor sexual activity when compared to controls.[36] Our results showed that low-frequency EA treated-group had greater serum LH levels (but not T levels) than other groups. These results are in concord with the findings of Luo et al., who reported that Lyciumbarbarum polysaccharides (LBP) treatment significantly increased serum LH levels in heat exposed male rats. However, LBP had no effect on T levels of male rats exposed to heat.[36] These above-mentioned studies combined with ours suggest that low-frequency EA treatment may adjust sexual hormones secretion and then improve male sexual behavior.

Another finding in this study includes that high-frequency stimulation impaired male sexual performance in rats [Figure 5]. It is well known that low and high-frequency stimulation may activate different neural mechanisms. For example, analgesia can be induced at 4 Hz, but not at 200 Hz.[37] The injection of a selective endogenous opioid receptor agonist into the cerebral ventricle of mice[38] or the spinal subarachnoid space of rats,[39] reduced low-frequency EA (2 Hz) stimulation-induced analgesia; however, it revealed no effect on high-frequency (100 Hz) EA-induced analgesia. Furthermore, morphine-induced conditioned place preference can be suppressed successfully by EA at 2 Hz, but not at 100 Hz.[40] These findings suggest that different stimulation parameters acting on the acupuncture points have varying effects on brain function by activating different neural pathways.[41] In addition to strength of our study, a few limitations also exist, which includes the lack of sudden drop in EF during high-frequency EA treatment. Moreover, the detailed mechanism about therapeutic effect of low frequency is still needed to be investigated.


  Conclusion Top


Our results demonstrate that low-frequency EA (2 Hz) at CV4 and CV3 facilitates sexual function and extends EL in male rats, which was evidenced by prolonged EL, possibly through increasing LH and 5-HT levels. High-frequency EA stimulation at these acupoints impairs ejaculatory behavior in sexually experienced rats, which could be restored over time, implying that high-frequency EA stimulation may prove beneficial for treatment of hypersexuality, whereas low-frequency EA at these two points suggests it maybe be applied to improve sexual function and delay the time of ejaculation [Figure 6].
Figure 6: Schematic remarks. Summary of results demonstrating the effect of low and high EA at acupoints Guanyuan (CV4) and Zhongji (CV3) on sexual behavior.

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Financial support and sponsorship

This study was supported by research grants obtained from the Ministry of Science and Technology, Taiwan (MOST 106-2410-H-241-002; MOST 107-2410-H-241-004), and the Department of Health, Taipei City Government, Taiwan (No. 10701-62-056).

Conflicts of interest

There are no conflicts of interest.



 
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