Clinical Efficacy of Acupuncture on the Liver Meridian in Women With Diminished Ovarian Reserve

Study Overview
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Brief Summary
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Diminished ovarian reserve (DOR) refers to a decrease in the number and quality of oocytes in the ovary, which results in impaired ovarian function and decreased fertility. Meanwhile, levels of the anti-Müllerian hormone (AMH), antral follicle count (AFC), and Follicle-Stimulating Hormone (FSH) also decrease in patients with DOR. In general, there is a decline in fertility and premature menopause. Some patients will have low menstrual volume, oligomenorrhea or even amenorrhea, abnormal uterine bleeding, ovulation disorders, infertility and perimenopausal performance before the age of 40, and eventually develop into premature ovarian failure. In recent years, with the changes of social culture, living environment, work pressure and other factors, the incidence of this disease has increased year by year, which has a great impact on women's fertility, mental health, quality of life, family relations and other aspects. As a green and safe complementary and alternative therapy, acupuncture has been proved to be effective. According to the statistics, 904 (33.54%) of the 2695 syndromes indicated by acupoints of the liver meridian recorded in 93 ancient medical books are reproductive disorders, ranking first in the diseases indicated by the liver meridian and the 14 meridians. The body surface course of the liver meridian is closely related to the genitals, and there is a close relationship between the liver meridian and the genitals in physiology and pathology. According to the theory of the relationship between meridians and zangfu organs, the study aims to verify the efficacy and safety of acupuncture of the liver meridian and provide high-level research evidence for meridian syndrome differentiation of reproductive system diseases via treatment from the liver.

Detailed Description
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This is an exploratory multicenter randomized controlled trial involving four clinical research centers, including the First Affiliated Hospital of Hunan University of Traditional Chinese Medicine, Hubei Hospital of Traditional Chinese Medicine, Shaanxi Hospital of Traditional Chinese Medicine, and Guizhou Hospital of Traditional Chinese Medicine. Each clinical research center strictly screened cases according to the diagnostic criteria, inclusion criteria and exclusion criteria. After confirmation of enrollment, random numbers were obtained according to the application for random coding process until the total number of observation was completed. According to the sample size, a total of 198 patients with DOR need to be enrolled. The 29-week study period consists of a 4-week screening period, 1-week baseline period, 12-week treatment period, 12-week follow-up period, and assessments at baseline. He patients were randomly divided into three groups: the liver meridian, stomach meridian, and non-acupoint acupuncture groups. Due to the particularity of acupuncture research, it is impossible to blind acupuncture operators. Blinded evaluation will be adopted in the trial, and the third party who does not know the grouping will evaluate the efficacy. Blind statistical analysis was used in the data summary stage, and the researchers, operators and statisticians were separated. In the experimental group (liver meridian group), EA was applied to Taichong, Li gou, Ququan and Jimai. In the control group 1 (gastric meridian group), EA was applied to Chongyang, Fenglong, Zusanli and Biguan. In the control group 2 (the non-meridian non-acupoint group), 4 non-meridian non-acupoints were selected from the lateral thigh, the lateral calf and the lateral calcaneus, respectively (see the treatment methods of the non-meridian non-acupoint group for details) and stimulated with EA. The main evaluation index will be Anti-Müllerian hormone(AMH), The secondary evaluation index include serum sex hormones (bFSH, bE2, bLH, bFSH/bLH), antral follicle count(AFC),, menstrual status, the modified Kupperman Index(KI), perimenopausal quality of life score (MENQOL) ,Self-Rating Anxiety Scale( SAS ), self-rating depression scale(SDS), Menstrual conditions. The patients were evaluated at different time points during the baseline period, treatment period and follow-up period, and the incidence of adverse events will be recorded. Double data entry will be used in this trial to ensure the accuracy of data entry. A three-level quality control system (self-inspection by each center, supervision by project management team, and third-party audit) was implemented to ensure that the implementation, recording and reporting of clinical trials were in accordance with the trial protocol, standard operating procedures, and relevant laws and regulations. The statistical analysis plan was developed by the statistical analysis principal and the principal investigators in accordance with the protocol and was finalized before the database was locked. The tables for the statistical analysis were developed. Statistical analysis will be calculated by SPSS25.0 statistical analysis software, P<0.05 (i.e., =0.05) will be considered statistically significant, in order to clarify the efficacy and safety of acupuncture on DOR.

Official Title
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Clinical Study of Liver Meridian in the Treatment of Male Impotence and Female Premature Ovarian Failure

Conditions
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Ovarian Reserve, Acupuncture Therapy, Meridian

Intervention / Treatment
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* Other: electropuncture


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: - The inclusion criteria are as follows: patients with the detection indicators within 3 months before enrollment met the diagnostic criteria of DOR. patients aged between 18 and 40 years old. patients who have received no other treatment in the 2 months before enrollment. (4) patients who voluntarily participate in this study and can actively cooperate with the study process. (5) patients who have signed an informed consent form. Patients who meet all five criteria will be eligible for this study. Exclusion Criteria: (1) Patients with polycystic ovarian syndrome, hyperprolactinemia, pituitary or hypothalamic amenorrhea, thyroid dysfunction, congenital malformations, and organic lesions in the reproductive organs. (2) Patients with a history of local ovarian surgery, such as oophorectomy and ovarian tumor surgery. (3) Patients with a history of chemotherapy or pelvic radiotherapy and a history of therapy with immunosuppressive agents. (4) Patients with comorbidities in the cardiovascular system, liver, kidney, hematopoietic system, nervous and mental system, diabetes, hypertension, and other diseases. (5) Patients being administered estrogen, progesterone, or dehydroepiandrosterone (DHEA) within 2 months before enrollment. (6) Patients who cannot tolerate blood or ultrasound testing. (vii) Patients who are also participating in other clinical trials. (7) Patients who refuse to sign the informed consent form. Patients who meet any of the above criteria will be excluded.

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 40 Years

Sexes Eligible for Study
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Female

Accepts Healthy Volunteers
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No

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Treatment
Allocation: Randomized
Intervention Model: Parallel Assignment
Interventional Model Description: The subjects were randomly assigned to the liver meridian group, the stomach meridian group and the non-acupoint acupuncture groups group at a ratio of 1:1:1.
Masking: Triple


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: liver meridian group<br>Four acupoints; LR3 Taichong, LR5 Ligou, LR8 Ququan, and LR12 Jiman. Based on the national standard of the People's Republic of China in 2006 (GB/T 12346-2006). The patient will be in a supine position. Taichong will be punctured at a 25-mm depth using a 0.35×25-mm filiform needle, Ligou will be punctured at a 15-20-mm depth with a 0.35×25-mm filiform needle, Ququan at a 20-25-mm depth with a 0.30×40-mm filiform needle, and an acute pulse of 15-20 mm. Each acupoint will be subject to a small amount of uniform lifting and twisting to the degree of local acid distension. An auxiliary needle (0.16×13 mm) will be inserted approximately 5 mm into the needle, 2 mm proximal to the heart of each acupoint along the meridian. The negative electrode will be connected to the main point, and the positive electrode will be connected to the auxiliary needle, and connect the electroacupuncture instrument. The needles will be retained for 30 min respectively, once every other day, 36 treatments. | Other: electropuncture<br>* Electroacupuncture (EA) is a method of treating diseases by inserting a needle (usually a filiform needle) into the skin or tissue of a patient at a certain Angle, and then passing through the needle (sensing) micro-current waves of human bioelectricity to stimulate specific parts of the human body (acupoints). Disposable sterile acupuncture filiform needle (0.16mm×13mm, 0.30mm×25mm, 0.30mm× 40mm), Guizhou Andi Medical Equipment Co, LTD, Guizhou Food and Drug Supervision and Equipment Production Xu 20170011. Electroacupuncture apparatus: SDZ-Ⅱ electroacupuncture apparatus of Huatuo brand, Suzhou Medical Supplies Factory Co., LTD., Registration No. : 20172270675.<br>* Other names: acupuncture;|
| Active Comparator: stomach meridian group<br>Four acupoints have been selected for the gastric meridian group, including ST42 Chongyang, ST40 Fenglong, ST36 Zusanli, and ST31 Biguan. The acupoint locations are based on the national standard of the People's Republic of China in 2006 (GB/T 12346-2006). The patient will be supine during the procedure, and the needles will be routinely sterilized. Straight needling will be performed in the hip region using a 0.30×50-mm-filiform needle, a 0.30×50-mm filiform needle in Zusanli, and a 0.30×40-mm filiform needle in Fenglong. A 0.35×25-mm filiform needle will be used to stimulate Chongyang for 10-15 mm, and each acupoint will be gently and evenly lifted, inserted, and twisted to local acid distension. Electroacupuncture will be applied in the same manner as for the liver meridian group. The needles will be retained for 30 min respectively, once every other day, then thrice weekly for a total of 36 treatments in the three groups. | Other: electropuncture<br>* Electroacupuncture (EA) is a method of treating diseases by inserting a needle (usually a filiform needle) into the skin or tissue of a patient at a certain Angle, and then passing through the needle (sensing) micro-current waves of human bioelectricity to stimulate specific parts of the human body (acupoints). Disposable sterile acupuncture filiform needle (0.16mm×13mm, 0.30mm×25mm, 0.30mm× 40mm), Guizhou Andi Medical Equipment Co, LTD, Guizhou Food and Drug Supervision and Equipment Production Xu 20170011. Electroacupuncture apparatus: SDZ-Ⅱ electroacupuncture apparatus of Huatuo brand, Suzhou Medical Supplies Factory Co., LTD., Registration No. : 20172270675.<br>* Other names: acupuncture;|
| Placebo Comparator: non-acupoint acupuncture group<br>Four points: (1) On the lateral thigh, between the vastus lateralis and biceps femoris, the midpoint of the popliteal stria, and the highest point of the greater trochanter. (2) On the lateral side of the calf, beside the level of Zusanli, and at the lateral edge of the tibia. (3) On the fibular side of the calf, the midpoint of the stomach meridian, and the bile meridian, 3 cm above the tip of the lateral malleolus and in front of the hanging bell. (4) On the lateral side of the calcaneus, and the servant enters the midpoint of the line connecting the posterior edge of the calcaneus at the same level. The 0.30×25-mm filiform needles are inserted straight for 3-5 mm at the points, the needles can stand without Deqi. The auxiliary needle will be inserted and electroacupuncture will be connected (same as in the other group). The internal wire of the electroacupuncture instrument will be interrupted, no current passed through. The treatment time is the same as the other two groups. | Other: electropuncture<br>* Electroacupuncture (EA) is a method of treating diseases by inserting a needle (usually a filiform needle) into the skin or tissue of a patient at a certain Angle, and then passing through the needle (sensing) micro-current waves of human bioelectricity to stimulate specific parts of the human body (acupoints). Disposable sterile acupuncture filiform needle (0.16mm×13mm, 0.30mm×25mm, 0.30mm× 40mm), Guizhou Andi Medical Equipment Co, LTD, Guizhou Food and Drug Supervision and Equipment Production Xu 20170011. Electroacupuncture apparatus: SDZ-Ⅱ electroacupuncture apparatus of Huatuo brand, Suzhou Medical Supplies Factory Co., LTD., Registration No. : 20172270675.<br>* Other names: acupuncture;|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| The value of anti-mullerian hormone(AMH) | This index is used to measure ovarian hormone secretion function and assess ovarian reserve function. Instrument:Beckman Coulter Access automatic immune analyzer. Method: Venous blood will be collected on an empty stomach at 3-5mL in the morning on the 2nd to 4th day of menstruation, centrifuged at 5000r/min for 3min with a high-speed centrifuge, and the upper serum is taken and stored in a -80℃ refrigerator for test. Unit: ng/mL. | 3 checkpoints. Baseline period(1 week before enrollment), enrollment 12 weeks (Change from Baseline at 12weeks) and enrollment 24 weeks(Changes at 12 weeks of treatment and 12 weeks of follow-up). | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| The number of antral follicle count (AFC) | The number of antral follicles was detected by three-dimensional B-ultrasound to evaluate ovarian reserve function. The smaller the number, the more severe the symptoms. | 3 checkpoints. Baseline period(1 week before enrollment), enrollment 12 weeks (Change from Baseline at 12weeks) and enrollment 24 weeks(Changes at 12 weeks of treatment and 12 weeks of follow-up). | 
| Sex hormone: follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2） | Use Chemiluminescence immunoassay to detected the ovarian hormone secretion. On the 2nd to 4th day of menstruation, 5 ml of venous blood will be collected from subjects in the morning under the condition of fasting for 8 h and put into the test tube without anticoagulant. The collected samples will be centrifuged to separate the upper serum and stored in the refrigerator at -70℃ for examination. Unit: FSH(mLU/mL), LH(mLU/mL), E2( pg/mL). | 3 checkpoints. Baseline period(1 week before enrollment), enrollment 12 weeks (Change from Baseline at 12weeks) and enrollment 24 weeks(Changes at 12 weeks of treatment and 12 weeks of follow-up). | 
| The total score of the modified Kupperman scale | To evaluate the severity of perimenopausal syndrome symptoms.Each item has a different basic and severity score, the severity score is calculated according to 0-3 points, and the symptom score is calculated as follows:Symptom score=basic score×severity score.The sum of each symptom score is the total score; the higher the total score, the more severe the disease. Patients with a normal score have <6 points. This with mild, moderate, and severe disease have 6-15, 16-30, and >30 points, respectively. | 3 checkpoints. Baseline period(1 week before enrollment), enrollment 12 weeks (Change from Baseline at 12weeks) and enrollment 24 weeks(Changes at 12 weeks of treatment and 12 weeks of follow-up). | 
| The total score of the perimenopausal quality of life score | 29 items, divided into Vasomotor symptoms, psychosocial function, physical function, sexual life, each item scored 1-7 points. Dimension score = Total score of item questions in each dimension ➗ Number of questions Total score = Sum of four dimensions scores | 3 checkpoints. Baseline period(1 week before enrollment), enrollment 12 weeks (Change from Baseline at 12weeks) and enrollment 24 weeks(Changes at 12 weeks of treatment and 12 weeks of follow-up). | 
| The total score of the self-rating anxiety Scale | Reflect anxiety level. 20 items, each item 1-4 points, total score and standard score (total score ×1.25), the higher the score, the more severe symptoms. | 3 checkpoints. Baseline period(1 week before enrollment), enrollment 12 weeks (Change from Baseline at 12weeks) and enrollment 24 weeks(Changes at 12 weeks of treatment and 12 weeks of follow-up). | 
| The total score of the self-rating Depression Scale | Reflect the degree of depression. 20 items, each item 1-4 points, total score and standard score (total score ×1.25), the higher the score, the more severe symptoms. | 3 checkpoints. Baseline period(1 week before enrollment), enrollment 12 weeks (Change from Baseline at 12weeks) and enrollment 24 weeks(Changes at 12 weeks of treatment and 12 weeks of follow-up). | 
| Menstrual assessment | Menstruation was assessed throughout the study period (a total of 7 times). The evaluation consists of 6 items, each item is scored (0 to 4 points), and the total score is calculated for each time. The lower the total score is, the better the menstrual condition is. | Enrollment 1、4、8、12、16、20、24 weeks. |