A Real-world Clinical Study on the Treatment of Menopausal Syndrome With Liuwei Dihuang Pills
Conditions: Menopausal Syndrome
Sponsor: China Academy of Chinese Medical Sciences

A Real-world Clinical Study on the
Treatment of Menopausal Syndrome
With Liuwei Dihuang Pills
Study Protocol and Statistical
Analysis Plan
2025.01.23

Name of test drug
Liuwei Dihuang Pills
Title
A Real-world Clinical Study on the Treatment of Menopausal Syndrome
With Liuwei Dihuang Pills
Observational
purpose
To explore the clinical efficacy and application value of Liuwei Dihuang
Pills in treating menopause syndrome.
Research
technique
Multicenter, large sample, prospective cohort study
Sample capacity
1000 menopausal patients were observed. 800 cases in the exposed group
and 200 cases in the non-exposed group.
Study population
1. Age: 45-55 years old, Gender: female;
2. Syndrome of deficiency of kidney yin;
3. Patients with the climacteric syndrome.
Entry information
1. Treatment information: reasons for treatment, treatment departments,
etc.
2. General information: gender, age, height, weight, body mass index
(Body Mass Index, BMI), lifestyle (such as smoking, drinking, etc.), family
history, past history, allergy history, basic diseases and their treatment, etc.
3. Disease related data: western medicine diagnosis, Chinese medicine
diagnosis and syndrome differentiation classification, menstrual condition,
disease course, medication information (course of treatment, dosage and
medication), before and after the symptoms, signs, examination results
(such as Kupperman score scale, sex hormones, perimenopausal survival
quality scale (MENQOL), TCM syndrome scale, etc.), etc.
Observational
indicators
1.General information: gender, age, course of disease, past history, etc
2.Safety measures (required before and after treatment)
(1) General physical examination items: heart rate and blood pressure;
(2) Laboratory examination items: Blood, Urine, Stool Routine; Fasting
Blood Glucose (FPG), Fasting Insulin (FINS),Blood lipids[Total
Cholesterol (TC), Triglycerides (TG), High-density Lipoprotein
Cholesterol (HDL-C), Low-density Lipoprotein Cholesterol
(LDL-C)],Liver function [Alanine Aminotransferase(ALT), Aspartate
Aminotransferase(AST)], Renal function [Creatinine(Cr), Blood Urea
Nitrogen(BUN)], Thyroid hormone indicators[serum free

triiodothyronine (FT3), free thyroxine (FT4), thyroid stimulating
hormone (TSH)],Electrocardiogram; Gynecological B ultrasound, etc.;
(3) Record of adverse events and adverse reactions;
3. Efficacy Indices (Compulsory Pre-treatment and Post-treatment Items):
(1) Primary Efficacy Indices:
①Change in Menopausal Symptoms: Modified Kupperman Scale
Score;
②Serum Hormone Level Measurement: Serum Follicle-Stimulating
Hormone (FSH), Luteinizing Hormone (LH), Estradiol (E2);
(2) Secondary Efficacy Indices:
①Menopausal-Specific Quality of Life Questionnaire;
②Changes in TCM Syndrome Scores;
4.Exploratory Analysis
Metabolomics, Modified Proteomics, Micro-RNA, and Other Omics
Sample Testing and Analysis.
Therapeutic
regimen
In this study, it were divided into exposed and non-exposed
groups. Patients in the non-exposed group were conventional treatment,
and patients in the exposed group were conventional treatment. It is
required to use drugs rationally and dialectically according to the
instructions for at least 4 consecutive weeks. Without intervention in
the treatment plan, concomitant medication is allowed, and the use of
medication and concomitant medication shall be truthfully recorded,
including dose, course of treatment, frequency, etc. Type of combined
drugs and the corresponding daily dose, daily frequency and course of
treatment.During the treatment, both groups had a follow-up
assessment for 4 weeks.
Follow-up time
Patients were followed up and cohort analyzed before and 4 weeks
after medication, and follow-up for patients with long medication (> 4
weeks) was extended to 3 months. Data were collected by the
clinicians.Test patients for liver and kidney function,
electrocardiogram, sex hormones and fill in the Kupperman score scale,
perimenopausal quality of life scale (MENQOL) and other indicators.
The registration observation form is recorded as the original record into
the electronic data acquisition system (EDC) for data management. Set
up the data verification plan in the study and edit the verification in real

time.
Scheduling
Completed within 6 months of observation initiation.
1. Research background
1.1 Basis for project approval
Menopause syndrome is a series of symptoms of autonomic dysfunction due to a
decline in sex hormones before and after menopause. Often accompanied by
menstrual cycle disorders, hot flushes, night sweats, sleep disorders, mood disorders
and other neuropsychological symptoms [1]. The core reason lies in the gradual decline
of ovarian function, including natural menopause and artificial menopause. Due to the
irreversible low-level estrogen state, postmenopausal aging lasts for at least 30 to 40
years, and the postmenopausal state in women occupies the ⅓for even longer, during
which the incidence of complications also significantly increases
[2-4]. About 25
million women worldwide enter menopause every year, and 10 million women in
China enter menopause every year. The United Nations World Health Organization
estimates that by 2030, the population of menopausal women worldwide will reach
more than 1.2 billion, and the number of menopausal women in China will exceed
210 million, accounting for about 1/7 of the total population, which needs to attract
attention to public health [5].
The western medicine treatment of menopausal syndrome mainly focuses on
psychological therapy and hormone therapy, but hormone supplementation will often
lead to diseases of other organs, such as breast cancer, cervical cancer and other [6],
which has certain limitations. Treatment of traditional Chinese medicine menopausal
syndrome has thousands of years of history, menopausal syndrome in traditional
Chinese medicine called "before and after the disease", belongs to the "lily disease"
"dirty manic" category [7], the pathogenesis of the disease of the basic pathogenesis for
menopause before and after female kidney qi deficiency, decyl day, kidney imbalance
of Yin and Yang, is the key to the syndrome before and after the menopause. The
imbalance of Yin and Yang in the kidney often involves other viscera, mainly in the

heart, liver and spleen. If the kidney Yin is insufficient, can not help heart fire, the
heart fire is high; kidney Yin deficiency, kidney Yin, liver deficiency, liver flexibility,
liver Yang hyperactivity; kidney and spleen days, kidney Yang warm, kidney
deficiency Yang failure, fire is not warm soil, often lead to spleen kidney Yang
deficiency, water dampness, phlegm turbidity, blood, blood stasis, depression, and
complex pathological mechanisms and various clinical symptoms [8].
Liuwei Dihuang Pills were first recorded in the "Direct Formula of Children",
which is a classic prescription for tonifying kidney, and was listed as the first batch of
"Ancient Classic prescriptions" by the State Administration of Traditional Chinese
Medicine. The prescription is composed of six traditional Chinese medicines, namely
rehmannia, cornwood, peony skin, yam, poria cocos and diarrhea. It has the effect of
nourishing Yin and tonifying kidney. It is suitable for kidney Yin loss, dizziness,
tinnitus, acid and soft waist and knee, bone steaming and heat, night sweat and sperm
and thirst elimination. Compound powder, nourishing Yin and tonifying kidney,
tuckahoe removing dampness and invigorating the spleen, Chinese yam nourishing
the spleen and kidney, nourishing the liver and kidney, and reducing the heat of the
peony peel. It enhances immunity, reduces blood lipid, blood pressure, blood sugar,
anti-fatigue, aging, low temperature, and resistance to hypoxia to improve renal
function, plant nervous system function, and has a promoting effect on metabolism [9].
It can improve the estrogen level of menopausal women, promote the feedback
control of the hypothalamic-pituitary-ovarian axis, improve the endocrine function
and the internal environment of the body, and reduce the clinical symptoms [10].
Traditional Chinese medicine treatment of menopausal syndrome from syndrome
differentiation has distinct characteristics and advantages. Clinical observation and
research have proved that TCM treatment of menopausal syndrome has definite effect
and good safety.
Menopause syndrome in addition to menstrual disorders, changeable mood and
other main symptoms, some female patients will appear abnormal glucose and lipid
metabolism [11,12]. In the perimenopausal stage female estrogen level decreased or
fluctuation, resulting in female blood lipid level change [13], estradiol (E2) is an

important lipid metabolism substances, it can promote the decomposition of lipids,
excretion,
reduce
plasma
cholesterol,
β-lipoprotein
levels,
make
the
serum
phospholipid,
α-lipoprotein
content
increased[14].
In
addition,
during
the
perimenopausal
period,
endogenous
estrogen
was
decreased
significantly,
accompanied by weight gain, adipose tissue redistribution and metabolic levels.
Studies showed that visceral adipose tissue quality and central obesity were
significantly associated with insulin resistance, which led to an increased risk of
T2DM in perimenopausal women[15]. To this end, the present study analyzed the
clinical effect of the application and the effect of the level of glucose and lipid
metabolism. Previous studies have shown that inflammatory response can reflect the
degree of insulin resistance, and the heavier the inflammatory response, the more
severe the insulin resistance[16]. IL-6, TNF-α and CRP are the commonly used
inflammatory markers in clinical practice[17]. Immune function and endocrine
dysfunction has obvious correlation between them, and to influence each other,
menopausal women body abnormal hormone secretion, can lead to the cell activity is
affected by different degree, at the same time if the endocrine balance between
ovarian and pituitary disorders, can cause the immune system function, can cause the
occurrence of various clinical symptoms of menopausal syndrome. Therefore, the
immune function [18] can be observed in patients with menopausal syndrome.
Studies have shown that Liuwei Dihuang Pills combined with western medicine
can treat hyperthyroidism kidney Yin deficiency in children, and regulate the
corresponding serum free triiodithyronine (FT3), free thyroxine (FT4) and thyroid
stimulating hormone (TSH) levels [19]. Sex hormone and thyroid hormone are jointly
governed by the thalamic-pituitary axis. At the same time, thyroid hormone can
improve the secretion of sex hormone, and sex hormone and thyroid hormone
mutually influence[18]. Thyroid hormone plays an important auxiliary role in the
regulation of sex hormones. Therefore, the abnormal secretion of thyroid gland makes
the clinical symptoms of patients aggravate[20]. In addition, there are few clinical
studies on thyroid hormone and glucose and lipid metabolism in perimenopausal
women, so further research is needed.

The immune system is an indispensable defense system of the human body, and
it plays a very important role in menopause. Studies have shown that the immune
system of the human body is closely related to the neuroendocrine system. With the
arrival of menopause, the neuroendocrine system of women is chaotic and its disease
resistance gradually decreases. The ratio of CD4/CD8 in T lymphocyte subsets
reflects the immune function of the human body. And the ratio of T cell subsets in the
peripheral blood of healthy adults is between 1.5 and 2.0. With the decline of immune
function of menopausal women, the composition, efficacy and transmission pathway
of T cell subsets are change, mainly manifested as a drop of CD4 and CD8 T cells. As
a pleiotropic cytokine produced by a variety of cells, IL-6 mainly participates in the
inflammatory response and immune response of the human body through humoral
immunity. In the normal immune state, most of them are in the "dormant" state. With
the decline of the body's immune function, the serum content of IL-6 increases
significantly to enhance the ability of foreign foreign body antigen[21].
1.2 Introduction of the study drugs
Name: Liuwei Dihuang Pills.
Ingredients: cooked rehmannia, wine wood meat, peony skin, yam, poria cocos,
diarrhea.
Character: this product is brown to black brown honey Pills; sweet and sour
taste.
Indications: nourishing the Yin and tonifying the kidney. For kidney Yin loss,
dizziness and tinnitus, waist and knee acid and soft, bone steaming hot flashes, night
sweat spermatorrhea.
Usage and dosage: Use drugs according to the instructions.
Specification: No limit.
Storage: seal.
1.3 Overview of human experience
98 patients with menopausal syndrome were randomly divided into treatment
group (56 patients) and control group (42 patients). The treatment group took 240 mg
each day per day, and the control group took 0.625 mg per day. Both groups were

treated continuously for 3 months. According to the menopause Kupperman symptom
score scale, the treatment group hot flashes sweating score decreased significantly (P
<0.05), the efficacy was better than that of the control group (P <0.05), and insomnia,
emotional excitement and other symptoms also improved [22] to varying degrees. In
addition, 120 patients with menopausal syndrome were randomly selected. According
to the analysis of Kupperman score results, liuwei Dihuang Pills alone could
significantly improve the symptoms of insomnia, anxiety and depression in
menopausal patients (P<0.05), but the combined effect with combined estrogen
tablets was better [23]. In addition, previous studies have shown that Liuwei Dihuang
Pills and its formula combined with metformin have good efficacy in the treatment of
diabetes mellitus[24]. Similarly, some studies have shown that Liuwei Dihuang Pills
combined with low-dose estrogen progesterone replacement therapy has a certain
impact on blood lipid therapy in patients with menopausal syndrome[25].
1.4 Preliminary foundation
This study investigated the effect of Liuwei Dihuang Pills (LWDHP) on
menopause syndrome and clarified the underlying mechanism. In this study, using the
method of network pharmacology, the KEGG pathway analysis of menopausal
syndrome, screening the top 20 pathways, using inflammatory proteomics and
menopausal syndrome Menendelian randomization analysis, obtained
OR> 1
inflammatory protein, and query the genes regulating inflammatory protein through
the NCBI website, screening the target genes of the inflammatory pathway and the
inflammatory genes obtained in Mendelian analysis, to construct the corresponding
PPI network map. Finally, it is concluded that LWDHP provides a new targeted
treatment strategy for menopausal syndrome by regulating inflammatory-related
targets and alleviating menopausal syndrome.
1.5 Support topics
This study belongs to the national Key Research and Development Program of
Traditional Chinese Medicine modernization research &quot;The establishment of
clinical efficacy and safety evaluation of proprietary Chinese patent Medicine based
on Systems Biology&quot; (No.2022YFC3502004).

2. Research purpose
To evaluate the efficacy and safety of Liuwei Dihuang Pills in the treatment of
menopausal syndrome and its effect on glucose and lipid metabolism.
3. Study design
3.1 Study type
The study type is a multicenter, large sample, prospective cohort study.
3.2 Study subjects
Women aged 45–55 years with menopausal syndrome from multiple centers
nationwide were consecutively included in this study.
3.3 Diagnostic criteria

Climacteric syndrome:
According to the standard [26-28] for the diagnosis of menopausal syndrome
stipulated in Obstetrics and Gynecology (9th edition), Guiding Principles for Clinical
Research of New Drugs of Traditional Chinese Medicine and Guidelines for the
Diagnosis and Treatment of Integrated Chinese and Western Medicine for
Menopausal Syndrome (2023 edition), Those who meet the following standards, Can
be diagnosed with menopausal syndrome: ①Age 45-55 years old; ②Menstrual
disorders for> 3 months or amenorrhea for 3 to 12 months; ③Accompanied by hot
and wet sweating, menstrual disorders, irritability, irritability, anxiety, insomnia,
palpitations, chest tightness, headache, dizziness, pain in sexual intercourse, frequent
urination, back and limb pain symptoms (hot and wet sweating necessary, Have 4 or
more other symptoms); ④Sex hormone index: serum follicle tin (FSH)> 10 IU / L,
Or AMH <1.1µ g/L, It suggests that the ovarian reserve function is reduced; Sealing:
FSH> 40 IU / L, And estradiol (E2)<73.4pmol/L (20 pg/mL), The ovarian failure was
indicated.
Menopause stage criteria: (2011 reproductive aging stage + 10 system menstrual
cycle length as the standard).

1. The length of the menstrual cycle varies (i. e., menstrual disorder), and the 10
menstrual cycles have 2 or more changes in the adjacent menstrual cycle for 7 days,
that is, the early transition of menopause.
2. The menstrual cycle is 60 days, and FSH> 25 IU / L, or late menopausal
transition.
3. One year after menstruation, that is, early menopause
These three periods can be called menopause

TCM diagnosis of menopausal syndrome:
It meets the clinical diagnostic standard [27,29] of before and after menopause in
Gynecology of Traditional Chinese Medicine and Guiding Principles of Clinical
Research of New Chinese Medicine, and is kidney Yin deficiency. Main syndrome:
temporary hot flashes, accompanied by sweating; secondary syndrome: dizziness,
tinnitus, irritability, dry vagina, dry mouth and constipation; tongue pulse: red tongue,
dry moss, fine pulse count.
One or more of the above items can be diagnosed.
3.4 Inclusion criteria
(1) Age: 45-55 years old (including both ends), female;
(2) Meet the western medicine diagnostic criteria for menopausal syndrome;
(3) Meet the TCM syndrome differentiation standard of kidney and Yin deficiency
syndrome;
(4) Informed consent, and the voluntary signing of the informed consent form.
3.5 Exclusion criteria
(1) Those allergic to the ingredients and excipients;
(2) Patients with other endocrine diseases;
(3) The presence of the reproductive system organic lesions;
(4) Patients with bilateral oophorectomy, hysterectomy, and ovarian dysfunction;
(5) Patients with endometrial hyperplasia, unexplained vaginal bleeding;
(6) Patients with malignant tumors, hematopoietic system and immune system
diseases;
(7) Serious diseases of cardiovascular and cerebrovascular diseases, liver and kidney
organs (serum transaminase level is more than 1.5 times higher than the upper limit of
normal value, and serum creatinine is higher than the upper limit of normal value);

(8) Psychiatric illness and alcohol or drug dependence;
(9) with mental illness or communication disorder;
(10) According to the judgment of the investigator, other lesions that reduce the
possibility of enrollment or complicate the enrollment are not suitable to participate in
this clinical study;
3.6 Test discontinuation criteria
(1) With serious adverse events or serious adverse reactions;
(2) Request withdrawal from the investigator;
(3) Patients with serious complications or other serious diseases who occurred during
the study.
3.7 Discontinue the handling of the cases
Record the reason for the discontinuation, investigate the treatment history,
record the time of the last treatment and the evaluation index at the discontinuation.
Analyze the possible impact of discontinued cases on the study conclusions, complete
and complete the observation form.
3.8 Treatment protocol
In this study, it were divided into exposed and non-exposed groups. Patients in
the non-exposed group were conventional treatment, and patients in the exposed
group were conventional treatment + Liuwei Dihuang Pills. It is required to use drugs
rationally and dialectically according to the instructions for at least 4 consecutive
weeks. Without any intervention in the treatment plan, concomitant medication is
allowed, and the use of medication and concomitant medication shall be truthfully
recorded, including dose, course of treatment, frequency, etc. Type of combined drugs
and the corresponding daily dose, daily frequency and course of treatment. During the
treatment, both groups underwent a follow-up assessment for 4 weeks. Study
termination follow-up conditions include patient death, loss to follow-up, or reaching
the study endpoint.
3.9 General Information:
(1) Treatment information: reasons for treatment, treatment departments, etc.
(2) Basic information: gender, age, height, weight, occupation; family history, past
medical history, allergy history, menstrual history, basic diseases and their treatment,
etc.
(3)
Disease-related
data:
diagnostic
information,
including
western
medicine

diagnosis, TCM diagnosis and syndrome differentiation; onset and duration of onset,
diagnostic
examination
results,
description
and
symptoms
and
signs
before
medication, etc. Medication information, including course of treatment, dose,
combined medication (description of frequency of hormone combination, frequency
of nourishing Yin and kidney drugs, frequency of other combined drugs);
3.10 Outcome indicators:
Comparative analysis of clinical characteristics of patients before and after
medication, including:
（1）Efficacy indicators:
1) scale:

Western medicine scale: Kupperman score scale [33], perimenopausal quality of
life scale [34];

Chinese medicine scale: Chinese medicine syndrome score table.
2) Inspection indicators:

Sex hormones: serum folliculogectin (FSH), luteinizing hormone (LH), estradiol
(E2);

Glucose: fasting blood glucose (FPG), fasting insulin (FINS),Glycated Albumin
(GA);

Blood lipids: total cholesterol (TC), triglycerides (TG), high-density lipoprotein
cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C);

Thyroid hormone indicators: serum free triiodothyronine (FT3), free thyroxine
(FT4), thyroid stimulating hormone (TSH).
(2) Safety indicators:
①Safety indicators adverse events / reactions clinical manifestations (symptoms
and signs);
②Vital signs: blood pressure, respiration, heart rate and body temperature;
③Blood, urine routine, stool occult blood;
④Liver
and
kidney
function:
glutamate
aminotransferase,
glutamate
transaminase, creatinine, urea nitrogen;
And a ⑤12-lead ECG.

(3) Exploratory indicators:
Biosample collection, biomarker detection and analysis.Thirty blood samples
were collected from patients in each group before and after treatment for
pre-treatment
and post-treatment
testing.
Metabolomics,
modified
proteomics,
Micro-RNA, and other omics sample testing and analysis were performed to assess
treatment efficacy.
3.11 Case Screening and Sample Size
In this study, 1,000 patients were collected for the menopausal syndrome. There
were 800 patients in the exposed group and 200 patients in the non-exposed group
(including 100 patients with blood collection; 900 patients without blood collection).
3.12 Relevant information collected for each follow-up time point
Patients were followed up and cohort analyzed before and 4 weeks after
medication, and follow-up for patients with long medication (> 4 weeks) was
extended to 3 months. Data were collected by the clinicians. The liver and kidney
function, electrocardiogram, sex hormones, glucose and lipid metabolism were tested
and the Kupperman score scale, perimenopausal period quality of life scale and other
indicators were filled in. The registration observation form is recorded as the original
record into the electronic data acquisition system (EDC) for data management. Set up
the data verification plan in the study and edit the verification in real time.

Specific follow-up steps
1)
Visit 1 (V1) —— Screening Period / Enrollment
Before enrollment
(1) Sign a written informed consent form;
(2) Obof demographic data;
(3) Medical history, treatment history, surgery history, personal history, allergy
history, menstrual history;
(4) Measurement of vital signs;
(5) Complete the Kupperman score scale and perimenopausal quality of survival
scale (MENQOL);
(6) TCM syndrome differentiation;
(7) Fill in the TCM certificate score;
(8) Investigate the situation of menopause;

(9) Laboratory examination:

Routine examination: blood routine, urine routine, liver and kidney function;

Sex hormones: serum folliculogectin (FSH), luteinizing hormone (LH), estradiol
(E2);

Blood glucose: fasting blood glucose (FPG), fasting insulin (FINS),Glycated
Albumin (GA);

Blood lipids: total cholesterol (TC), triglycerides (TG), high-density lipoprotein
cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C);

Thyroid hormone indicators: serum free triiodothyronine (FT3), free thyroxine
(FT4), thyroid stimulating hormone (TSH).

RNA test: serum;
(10) A 12-lead ECG;
(11) Abdominal ultrasound, pelvic cavity (gynecological) ultrasound, breast
ultrasound, thyroid ultrasound;
(12) Check the inclusion / exclusion criteria;
(13) Issue the patient's electronic questionnaire (questionnaire star).
2)
Visit 2 (V2)——Treatment Period
The first 15 ± 2 days after enrollment.
(1) Fill in the symptom Kupperman score scale and perimenopausal quality of
life scale (MENQOL);
(2) Fill in the TCM certificate score form;
(3) Investigate the situation of menopausal syndrome;
(4) Record the concomitant medication;
(5) Record the adverse events;
(6) Recovery the subject's medication diary card, used drug packaging and
unused drug packaging
Drug, and record;
(7) Recovery and distribution of patient questionnaires;
(8) Issuance of medication and subject medication diary cards.
3)
Visit 3— medication completion / follow-up period
Day 30 of medication after enrollment.
(1) Measure the vital signs;
(2) Recovery the patient questionnaire;
(3) Complete the Kupperman score scale and perimenopausal quality of survival

scale (MENQOL);
(4) Fill in the TCM certificate score form;
(5) To investigate the situation of menopausal syndrome;
(6) Record the concomitant medication;
(7) Record the adverse events;
(8) Laboratory test of blood routine, urine routine, liver and kidney function;

Routine examination: blood routine, urine routine, liver and kidney function;

Sex hormones: serum folliculogectin (FSH), luteinizing hormone (LH), estradiol
(E2);

Glucose: fasting blood glucose (FPG), fasting insulin (FINS);

Blood lipids: total cholesterol (TC), triglycerides (TG), high-density lipoprotein
cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C);

Thyroid hormone indicators: serum free triiodothyronine (FT3), free thyroxine
(FT4), thyroid stimulating hormone (TSH);

RNA test: serum;
(9) Abdominal ultrasound, pelvic cavity (gynecological) ultrasound, breast
ultrasound, thyroid ultrasound;
(10) Fill in the study completion summary form.

Unscheduled visit / early exit visit
If the subject has an AE during the study or requires additional visit /
examinations, record in the unscheduled visit; if the subject withdraws from the trial
early, the investigator should conduct safety assessment and effectiveness assessment
whenever possible, record in the early exit visit and complete a summary of trial
completion.
4. Analysis method
4.1 Hierarchical analysis
(1) Different medication duration and daily dosage (high and low dose), presence
of age, menstruation, course of disease and whether the concomitant medication and
disease as stratification factors;
(2) Explore the correlation between hormone indicators of menopausal syndrome
and blood glucose and lipid indicators;
(3) After taking Liuwei Dihuang Pills, blood lipid index in patients with

abnormal glucose metabolism; blood glucose index in patients with menopausal
syndrome
with abnormal
dyslipidemia;
and
blood glucose
in
patients
with
menopausal syndrome with abnormal glucose and lipid metabolism.
4.2 Analysis of the influencing factors of disease recovery
According to the results of the stratified analysis and combined with the medical
opinions, the independent variables were determined, and the effect index was taken
as the dependent variable, and the Logistic stepwise regression model was established
to analyze the influencing factors of disease recovery.
4.3 Tendency matching analysis
Considering that the baseline imbalance between the indicated populations may
affect the analysis results, so the propensity matching method was adopted for
matching, and factors such as whether to use drugs were taken as dependent variables,
and clinical characteristics were used as independent variables to estimate the
tendency score.
6. Statistical methods
The general demographic characteristics of the study subjects at baseline were
first descriptive and group described. For continuous variables (such as age) with
normal distribution and mean standard deviation, the differences between groups were
compared by t-test or analysis of variance (analysisofvariance, ANOVA); without
normal distribution, the median (first quartile, third quartile) was used, and the
differences between groups were compared by Wilcoxon rank sum test or
Kruskal-Wallis rank sum test. Categorical variables were expressed using frequency
and percentage (n,%), and differences between groups were compared using the
Pearson x2 test. Linear regression model was used for continuity variables and
Mantel-Haenszel x2 test for multi-group variables.
7. Quality control
Standardized training. Information collection should be carried out when the
environment is quiet and clean, the air is fresh, natural light or artificial lighting, and
the patients are emotionally stable and relaxed. After signing the informed consent,

the basic information should be collected. Follow the principle of objectivity and
standard to collect data and fill in scales to ensure the authenticity and reliability of
information. Be familiar with the specific meanings of all scales and representative
items in this study, so as to reduce the impact of subjective factors on the trial. After
the collection, the information should be checked again to avoid missing filling and
misfilling. The physician with the title of chief physician should judge the information
again to ensure the data quality.
8. Ethics issues
This study adhered with the Declaration of Helsinki and protected the treatment
information of all subjects. This study is a specific study based on the database of the
registration study of menopausal syndrome cases, through the national clinical
research ethics approval.
9. Participants gave their informed consent
The investigator and the patient signed the informed consent form and gave
copies to the patient.
10. Medical treatment and protection of the subjects
In the study, patient privacy as well as personal information will be strictly
protected.
11. Definition and collection method of relevant indicator
variables
11.1according to the modified Kupperman scoring scale [33]
Scoring method: symptom degree * symptom index
Symptom index: hot flashes and sweating 4 points, abnormal sensation (cold, hot,
numbness, pain, etc.), insomnia, irritability, sex pain, urinary tract symptoms 2 points,
depression, dizziness, fatigue, joint muscles, limb pain, headache, etc. 1 points.
Degree of symptoms: asymptomatic 0 points, occasional symptoms 1 point,
symptoms last 2 points, affecting life 3 points. Evaluation of the severity of the
disease: mild total score was 15-20, moderate total score was 21-35, and severe total

score was greater than 35.
Modified Kupperman rating scale
Modified Kupperman rating scale
surname and personal name:
age:
contact number:
symptom
base
score
0
Points
1 Points
Two points
Three points
grade
Hot and sweaty
four
not
have
<3 Times /
day
3-9 times / day
Vacation 10
times /
sky
abnormal
sensation
two
not
have
With the
weather
close
Usually there is cold
Hot pain numbness
feel
Hot and cold
pain
lose
lose sleep
two
not
have
once in a
while
Often, Ann
Sleep pills are
effective
Affect the
work
life
mood swing
two
not
have
once in a
while
Often, no
Self-consciousness
Know, no
Can
self-control
Depression,
suspicion
heart
one
not
have
once in a
while
Often, can
automatic control
Lost life
confidence
circumgyration
one
not
have
once in a
while
Often, no
Influence life
Influence daily
life
Life is tired
one
not
have
once in a
while
On the fourth floor
sleepy
difficult
daily life
Restricted
Osteoarthralgia
one
not
have
once in a
while
Often, no
Influence function
dysfunction
headache
one
not
once in a
Often, can
Need to take

have
while
bear
medicine
cardiopalmus
one
not
have
once in a
while
Often, no
influence
Need treatment
formication
one
not
have
once in a
while
Often, can
bear
Need treatment
sexual life
two
not
have
Under the
normal sex
desire
fall
Sexual life is
difficult
difficult
sexual
anesthesia
Urinary infection
two
not
have
once in a
while
> 3 times / year, can
from
heal
> 3 times /
year, required
medicine

11.2 Perimenopausal Quality of survival scale (MENQOL) [34]
Perimenopausal Quality of Survival Scale (MENQOL)
Perimenopausal Quality of Survival Scale (MENQOL)
During the last month, did you have the symptoms in the following questionnaire? Please choose one of the levels, "0", means no impact at
all, "6" means extreme impact, the closer to 0, the less affected, the closer to 6, the more affected. Please select a best situation for you in
0-6, and outline "(".
symptom
The degree of symptoms
0 Points
1 Points
Two points
Three
points
4 Points
5 Points
Six points
1. Heat (burst of heat)

□
□
□
□
□
□
2. Night sweats (sweating after
falling asleep at night)
□
□
□
□
□
□
□
3. Sweat comes out during the day
□
□
□
□
□
□
□
Not satisfied with their own life
□
□
□
□
□
□
□
5. Feel anxious or nervous
□
□
□
□
□
□
□
Memory loss
□
□
□
□
□
□
□
Do things are not as handy as
before
□
□
□
□
□
□
□
Feel
depressed,
depressed
or
depressed
□
□
□
□
□
□
□
Lack of patience with others
□
□
□
□
□
□
□
Always want to stay there alone
□
□
□
□
□
□
□
11, gastrointestinal flatulence or
distension pain
□
□
□
□
□
□
□
12, muscle and joint pain
□
□
□
□
□
□
□
Feel tired or exhausted
□
□
□
□
□
□
□
Sleep problems
□
□
□
□
□
□
□
15. Cervical pain or headache
□
□
□
□
□
□
□
Physical strength decline
□
□
□
□
□
□
□

Poor appearance and spirit
□
□
□
□
□
□
□
Feel a lack of energy
□
□
□
□
□
□
□
Tired and dry
□
□
□
□
□
□
□
20, and the weight gain
□
□
□
□
□
□
□
Face hair increased
□
□
□
□
□
□
□
Appearance, skin quality changes
□
□
□
□
□
□
□
23. The joints feel swollen and
uncomfortable
□
□
□
□
□
□
□
Low back pain
□
□
□
□
□
□
□
Urinary frequency
□
□
□
□
□
□
□
26.
Urinated
control
when
laughing or coughing
□
□
□
□
□
□
□
Sexual desire changes
□
□
□
□
□
□
□
Dry vagina during sex
□
□
□
□
□
□
□
29. Avoidative sexual behavior
□
□
□
□
□
□
□

11.3 The scoring standard of TCM syndrome is formulated with reference to the
relevant contents in the Guiding Principles for Clinical Research of New Chinese
Medicine and the Standard for Diagnosis and Efficacy of TCM Certificate (the
fourth edition).
Traditional Chinese medicine syndrome score table
Symptoms /
Score
No (0)
Light (1)
Medium (2)
Heavy (3)
Heat sweat out
not have
Occasionally the
head is hot sweat
Hot flashes in
the back and
chest
Body and hot
flashes, sweat out
of the water
lose sleep
not have
4-5 Hours
2-3 Hours
pernoctation
Menstrual
disorders or
amenorrhea
not have
Accidental
often
repeatedly
dysphoria in
chestpalms-soles
not have
Accidental
often
repeatedly
soreness and
weakness of
waist and knees
not have
Sometimes it hurts
Change
position is
required
Persistent pain,
medication
Dry mouth and
dry throat
not have
light
Drinking
water to
relieve
Drink and don't
understand
dizzy
not have
Accidental
often
repeatedly
tinnitus
not have
Accidental
often
repeatedly
Panpulsive
palpitations
not have
mild
moderate
severe
Urine little color
yellow
not have
once in a while
often
repeatedly
Both eyes dry
not have
once in a while
often
repeatedly

The skin is dry
and itchy
not have
Accidental
often
repeatedly
The pussy is dry
not have
mild
moderate
severe
constipation
not have
once in a while
often
repeatedly

一、Blood samples were collected for the SOP
1 Blood collection package
1.1 Blood vessel collection
（1）2
common blood vessels
(hospital
equipped,
red
trunk
tube,
without
anticoagulant) Use 1 dose before medication and 1 dose after medication;
（2）14 EP tubes (issued by the general research group) 7 before and 7 after
medication;
1.2 4 disposable straws (equipped by the general research group);
1.3 Frozen storage box (equipped by the general research group);
10 translucent frozen storage boxes (frozen storage EP tube, 81 holes);
1.4 Blood sample Collection and FreeStorage Registration Card
1.5 marker for marking patient number on each wall (avoid label);
1.6 Low-temperature centrifuge (4℃, 4000rpm), refrigerated refrigerator (4℃), -80℃
ultra-cold refrigerator.
2 Blood sampling process
2.1 Preparation for patient blood collection
5ml of median elbow venous blood samples were collected at 2-4 days of
menstruation and fasting for over 8 hours; for rare menstruation and amenorrhea for>
2 months, urine pregnancy test was negative and color ultrasound examination: no
bilateral ovarian follicles> 10mm, endometrial thickness of <5mm, 5 mL of median
elbow venous blood samples were collected during the fasting state for over 8 hours;
5ml of median elbow venous blood samples were collected during fasting for over 8

hours.
2.2 Special personnel shall be responsible for preparing for blood collection
The department shall designate a special person to guide the subject to the
subject to the special blood collection department, and the special person shall be
responsible for blood collection. The blood collection person shall check whether the
blood collection consumables labels are consistent with the patient, and sign in the
column of "Blood Collection and Frozen Storage Registration Card", "Blood
Collection", "whether", "Blood collection time" and "Blood collection amount". If
there is any abnormality in blood sample collection, it should be explained in the
remarks column (such as blood collection delay, hemolysis, etc.).
2.3 Collect venous blood
(1) 4ml of each common blood collection vessel (special label with patient
number);
Note: gentle gently to avoid physical hemolysis; blood collection should be
sufficient. After the blood sample is collected, the designated person shall process and
preserve the blood sample in time.
3 Blood samples
3.1Ordinary blood vessels
Blood sampling was used with 1 branch before medication.
3.2 EP vessels
After blood collection, the designated specimens in 4℃refrigerator for 1 hour,
serum fully precipitated, low temperature (4℃) for 10 minutes (4000rpm), serum into
EP tube, packaging specifications: 200 micro liters / tube * 5 tubes, 0.5ml / tube * 2
tubes, a total of 14 tubes, paste a special label with patient number, before and after
medication.
3.3 RNA Blood Collection Tube
Attach a label with the patient’s ID. Collect 2.5 mL of blood, then invert the tube
gently several times to mix the additive with blood.
The above operation requires special personnel to cooperate with the blood

collector, so as not to expose the blood sample for too long at room temperature.
4 Blood samples frozen storage
All samples shall be stored in the corresponding frozen storage box after
completion and shall be stored in the-80℃refrigerator immediately. If it cannot be
placed in the-80℃refrigerator immediately, it should be temporarily stored in the 4℃
refrigerator for no more than 30 minutes, and then placed in the-80℃refrigerator
before and after medication.
5 Precautions
5.1 Do not shake or tilt the blood vessel after centrifugation.
5.2 Always tighten the EP pipe cap to prevent leakage.
5.3 Strictly prevent repeated freezing and thawing.
5.4 The distributor shall sign in the column of "distributor" and "Assembly" of Blood
sample Collection and Frozen Storage Card.
5.5 Frozen holder sign in the column of "Frozen holder", "Frozen holder", "Frozen
time", "refrigerator No." and "Frozen layer".
6 Transportation of the blood samples
All kinds of frozen samples are regularly transferred (every 3 months) to the cold
chain transportation company for transfer and recovery to the biological sample bank
of the Institute of Clinical Traditional Chinese Medicine of China Academy of
Chinese Medical Sciences, and the unified testing unit is the Medical Experimental
Center of China Academy of Chinese Medical Sciences. The cold chain transportation
company is Beijing Yingyu Logistics Co., LTD. Before transportation, the Institute of
Clinical Basic Medicine of Traditional Chinese Medicine of China Academy of
Chinese Medicine has a special person to communicate with the contact person of the
clinical hospital about the time and handover procedures.
6.1 Before transportation
The head of biological samples of the general research group shall regularly
coordinate with the project leader of the cold chain transportation company, the head
of biological samples of the sub-center and the head of the biological sample bank to
confirm the number of biological samples to be transferred, the number of incubators

required for transportation and the transportation time. Confirm the dry ice quantity
and the thermometer quality with the cold chain transportation project leader. Ensure
the sample safety during transportation.
6.2 After transportation
After the transportation, ask from the project leader of the cold chain
transportation company for the temperature record table;
The person in charge of the biological samples of the sub-center shall report the
biological samples of the sub-center regularly. Fill in the Blood sample Delivery
Form and Statistical Form and transport them with the box. And send the electronic
version to the research group email: wlxing@126.com.
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