The thesis evaluated the safety and improvement of spermatogenesis of Y10 capsules in experimental animals; Effect of improving the fertility of capsules Y10 in patients with impaired sperm.
Trang 1INTRODUCTIONMale infertility is a disease that greatly affects the society WHO predicted it would be the 3rd common dangerous disease in 21st century after cancer and heart diseases. Infertility couples in the world accounts for 15% and the figure for Vietnam is 8%.
Causes of male infertility are enormous, but the most frequent is sperm decline. Modern medicine has gained achievements in infertility due to sperm decline treatment; however, it also presents side effects.
Traditional oriental medicine that has experienced thousands of years of experience has also had specific male infertility treatments and brought good results Many scientific studies have proven that traditional medicine treatments have good effects on the treatment of male infertility in general and sperm decline in particular
Cornu cervi parvum and Cordyceps militaris cultured in Vietnam are two valuable medicinal herbs, which are widely used in the treatment of infertility and male reproductive dysfunction, with good potential. However, there have been are no scientific studies on the effects of Cornu cervi parvum and cordyceps and their therapeutic effects on treating reproductive functions We carry out the research with the following objectives:
1 Assess the safety and effects on sperm production of Y10 on experimental animals
2. Assess the effects on the sperm production of Y10 on patients with sperm decline
PRACTICAL MEANINGS AND NEW CONTRIBUTIONS OF THE DISSERTATION
The reproductive health in Vietnam has changed significantly in recent years. The rate of infertile couples is increasing, especially infertility due to sperm decline in men. Modern medical treatments have shown a positive role and effectiveness in the treatment of sperm decline, but it can cause physiological trauma and side effects Thus, it is always necessary to continue the search for effective, economical and safe herbalbased medicines, it is also the research direction that scientists in the country and in the world are interested in The dissertation is systematic and experimental, and scientific work
The research and its application on the treatment of male infertility due
to sperm decline by traditional remedies has contributed to elucidate the
Trang 2STRUCTURE OF THE DISSERTATION
The dissertation consists of 126 pages: 02 pages of introduction, 39 pages of literature review, 22 pages of subjects and research methods,
34 pages of research results, 25 pages of discussion, 03 pages of conclusion and 01 page of recommendation The thesis has 141 references (42 in Vietnamese, 64 in English, and 35 in Chinese), 45 tables, 2 charts, 01 diagram, and 15 photos and appendices
Chapter 1. LITERATURE REVIEW
1.1 The view of modern medicine on sperm decline
1.1.1. The causes of sperm decline
Sperm decline is caused by factors that affect sperm growth and disrupt that process. There are 3 main types of causes, as followed:
* Reproductive hormonal disorders: Some reproductive hormones play a decisive role in sperm production in general and its single differentiation stage in particular. Diseases caused by reproductive hormonal disorders lead to a huge change in the sperm count and quality
* Genetic causes: testicular, microbiological and temperature factors; varicose veins of the spermatic cord damage, Xrays, radiation and chemicals, PH of semen, or medical drugs might cause sperm decline, etc
* Other causes: diet, immune, living and working environment, or stress
1.1.2. Directions of treatment of sperm decline according to modern medicine
* Using modern medicine: Antioxidants: Glutathion, Larginin, vitamin C; Hormones: FSH (humegon, puregon), LH (pregnyl), hMG or hCG; andriol testocaps, mesterolone, testosterone undecanoate; Corticoids are used for infertility due to antisperm antibodies; Antibiotics are indicated for urinary and genital infections. The common bacteria are Chlamydia trachomatis, E.coli, and U. Urealyticum
Using of traditional medicine: the trend of research using alternative therapies and traditional medicine to treat sperm decline is increasing
Surgery: surgery should be indicated for patients with impaired spermatozoa that affect the sex life, such as varicose veins, ectopic
Trang 3be indicated for patients with no sperms by connecting vas deferens.IUI: a method of putting a small volume of sperm that has been selected
by sperm washing technique into the uterus near the time of ovulation; IVF and embryo transfer into the uterus: a technique for in vitro fertilization ICSI: sperm injection directly into the ovule to create embryos
1.2. Traditional medicine theory on sperm decline
1.2.1. Traditional medicine theory on male genital and reproductive functions
1.2.3. Treatment of sperm decline according to Traditional Medicine
Traditional methods of treatment of sperm decline include traditional medicine, acupuncture, elecaccupuncture, macrobiotic medicine, or a combination of the methods for the most effective treatment
* Medication therapies
Deficiency of Kidney and Jing: Therapy: warming Kidney and strenthening yang. Formulas: wu zi yan zong wan in combination with gui wan Ingredients:Aconitum Camichaeli Debx.,Laurus cassia L,
Codonopsis pilosula , Astragalus propinquus, Rehmannia glutinosa Libosch, Dioscorea opposita, Cornaceae , Cortex Moutan,Cortex Paeoniae Suffuticosae, Alisma plantago aquatic L., Poria cocos Wolf.,Zingiber officinale, Glycyrrhiza uralensis, and Glycyrrhiza uralensis
Deficiency of Gate of life Fire: Therapies: warming kidneys,
strengtening Yang Nourishing kidneys to promote Jing production.
Formulas: jin gui shen qi wan in combination with bao yuan tang. Ingredients: Aconitum fortunei Hemsl., Cinnamomum cassia Nees &
Eberth, Codonopsis pilosula (Franch) Nannf., Astragalus propinquus., Rehmannia glutinosa (Gaertn.) Libosch., Dioscorea persimilis Prain et
Trang 4Burk., Cornus controversa., Paeonia suffruticosa Andr., Alisma plantago aquatica L, Poria cocos Wolf., Zingiber officinale, and Glycyrrhiza uralensis.
Deficiency of spleens and Jing: Therapies:Warming and tonifying
Hominis, Cornaceae, Codonopsis pilosula, Astragalus propinquus, Atractylodes macrocephala, Wolfiporia extensa, Dioscorea persimilis Prain et Burk, Glycyrrhiza uralensis, Angelica sinensis, Paeonia lactiflora Pall., Rehmannia glutinosa Libosch, Lycium barbarumL., Gynochthodes officinalis, and Epimedium.
Stagnation of liver qi: Therapies: Dispersing the stagned liver qi.
Activating Qi and blood circulation to promote the Jing production.
Formulas: Ingredients: xue fu zhu yu tang . Prunus persica Stokes,
Carthamus tinctorius, Paeonia liacliflora Pall, Ligusticum striatum, Angelica sinensis, Bupleurum chinesnis DC., Liquidambar formosana Hance, and Squama Manidis.
Wetness heat evil attacking the lower warmer: Therapies: Clearing heat. Eliminating dampness. Expelling toxic. Formulas: ong dan zuo gan tang in combination with liu wei di huang wan. Ingredients:
Gentiana scabra Bunge., Phellodendron amurense, Gardenia jasminoides ellis, Lonicera japonica Thunb., Forsythia suspensa, Semen Plantaginis, Alisma plantagoaquatica, Rehmannia glutinosa Libosch, Cornaceae, Dioscorea persimilis Prain et Burk., and Cortex Moutan Cortex Paeoniae Suffuticosae
* Alternative therapies
Common therapies are accupuncture and elecaccupuncture which are often used together to get highest effectiveness.
* Accupunture: Use the ponts of Ming men, Yao yuan guan (Du.3) ,
Guan yuan (Ren.4), Zhong ji (Ren.3), San jin jiao (Sp. 6), Zhi shi (UB.52), Thai yuan (Lu.9), Zu san li (St.36), etc
Trang 5Benefit liver and kidney meridians. Effects: Strengthen Yang. Nourishing Jing blood Strengthening bones Regulating meridian
points. It is used in cases of kidney Yang imbalance, deficiency of Jing blood, impotence, fatigue, dizziness, back ache, etc.
Cordyceps militaris: Characteristics: Sweet. Neutral. Benefit lung and kidney meridians. Effects: According to ‘ben cao gang mu shi yi’ the herb maintains lung Qi, nourishes kidneys and good for oldage.
In traditional medicine, Cornu cervi parvum và Cordyceps militaris are two precious herbs that tonify kidneys and nourish Jing. When they are combined, they would be beneficial for both kidney Yang and Yin, Jing and Qi. They, hence, have been used for longs in male infertility effectively. They are soaked in wine.
Chapter 2. SUBJECTS AND METHODS OF THE STUDY
2.1. Research material
Studied madication: Y10 made from Cornu cervi parvum purchased in Huong Son Ha Tinh and Cordyceps militaris cultured at Military Medical Academy, meeting its standards
2.2. Research subjects
Studied animals: Adult white mice, purebred Swiss lines, 2 sexes, weight 18 22g, total of 470 mice. Adult white rats, purebred Wistar lines, both sexes, weighing 160 180g, a total of 80 rats
Studied patients: 30 military men with sperm examined at the Embryo Technology Training and Research Center Military Medical Academy. They were consistent with disease selection and exclusion criteria according to traditional medicine and modern medicine, and volunteered
to the research
2.3. Location and time
The experimental research was conducted at the Department of Clinical Pharmacy Military Medical Academy, from May 2017 to October
2017. The clinical research was conducted at the Embryo Technology Training and Research Center Military Medical Academy, from October 2017 to December 2017
2.4. Research Methods
2.4.1. The study on the safety and effects of Y10 on the experimental animals.
Trang 6The acute toxicity was determined in accordance with the guidelines of the Ministry of Health and the OECD by oral route with increasing doses. LD50 was calculated by the LitchfieldWilcoxon method
The semichronic toxicity was assessed according to the regulations of the Ministry of Health of Vietnam and the guidance of OECD, WHO.
30 mice were divided into 3 groups, 10 each; the control group drank distilled water. Study group 1: Y10 224mg decoction / kg / day. Study group 2: Y10 672mg decoction / kg / day in 90 consecutive days. Their general condition, body weight, hematopoietic function, liver and kidney function, liver and kidney histopathology were observed and recorded before and after the treatment
* The study of toxicity on reproductive abiliity of Y10
The white mice of both sexes were randomly divided into 5 study groups, with 60 each including 20 males and 40 females. The control group consisted of 20 males and 40 females drinking distilled water
Group 1: 20 males and 40 females taking Y10 384mg decoction/kg/ day
Group 2: 20 males and 40 females taking Y10 1152mg decoction /
kg / day
Group 3: 20 males taking Y10 384mg decoction / kg / day and 40 females taking distilled water
Group 4: 20 males taking Y10 1152mg decoction / kg / day and 40 females taking distilled water
The treatment duration was 60 days, at a certain time everyday (8 am).After 60 days, a male was kept with two females in a separate cage (generation P) and the reproduction process was monitored
The conception and fetal development were monitored
* The mutagenic toxicity study on chromosomes of Y10 on white miceAccording to OECD guideline 475 (2002), 90 healthy white mice, divided into 3 groups, each group of 30 rats including 15 male rats and
Trang 7in the middle of cell division, when the chromosomes were most typical
2 hours after the colcemide injection, the chromosome samples from the bone marrow were made by Ford method and the chromosomes from the testes by Evan method. 50 chromosome patterns of each mouse were analyzed at the Diakinesismetaphase stage when the chromosomes were short, evenly sprung and were not overlapped
* Evaluation of the effects of sperm enhancement of Y10 on white rats that caused sperm decline with Natri valproate
The group of mice that were caused sperm decline by Natri valproate at the dose of 500mg / kg / day in 7 weeks. Mice in nonspermdeclinecaused groups were given distilled water at the same volume in 7 weeks.After 6 weeks, mice were killed to serve as samples for the evaluation of research indicators, including: testosteron, sperm density, sperm motility, sperm morphology, percentage of structural morphology sperm abnormalities, testicular histopathological changes, size of spermatogenesis diameter, genital organs (testes, epididymis, seminal vesicles, Cowper glands, glans, prostate gland, and levator ani muscles). The genital organs were determined out of every 100g the mice’s body weight
2.4.2. Clinical research
We conducted inclusive studies and open clinical trials, compared the differences in the indexes between before and after the treatment. The patients with sperm decline were examined according to modern and traditional medicine, they undertook all tests and if they were eligible, they would be selected for the study groups
* Criteria for selecting patients
Trang 8 Criteria for selecting patients according to modern medicine: Age: 16
to 56; Sperm decline according to WHO standards onseminogram (2010)
Criteria for selecting patients according to traditional medicine: men had ‘deficiency of kidney and Jing’
Criteria for excluding patients: The patients who did not agree to take part in the study, did not strictly followed guidances, or refused to continue the treatment
* Dosage: 4 tablets per day, twice, 2 hours after eating, in 2 months.
* Methods of clinical examination and clinical monitoring
The medical record for each patient is made according to a unified form based on the criteria of the World Association of Southern Studies, combined with v i ớ four methods for physical examination of traditional medicine, the patients were examined and their test results were
recorded
* Laboratory tests
Biochemical blood before and after the treatment: urea, creatinine, AST, ALT
Quantification of LH, FSH, and serum testosterones before the treatment; quantification of LH, FSH, and serum testosteron in selected patients after the treatment
Some clinical signs due to unwanted effects of the medication: rashes, digestive disorders (loose stool, constipation , etc), dizziness, etc.
Clinical symptoms due to renal impairment before and after the treatment
ALT, AST, urea, serum creatinine before and after the treatment
Serum testosteron, LH, FSH concentrations before and after the treatment
Semen before and after treatment (Table 2.3)
Trang 9* Data processing
The research data is processed by biomedical statistical method, using statistical software SPSS.17.0 The difference was statistically significant when p <0.05
Chapter 3. RESULTS
3.1. Research results on the acute and semichronic toxicity study
3.1.1 Acute toxicity results: Rats took up to 20.0g decoction / kg body
weight (equivalent to 25.0 g of powder in a capsule / kg body weight) which was the maximum dose that mice could take orally to evaluate the acute toxicity of the reagent but no mouse died, no abnormal symptoms appeared after 72 hours and during 7 days after the experiment
3.1.2. Result of the semichronic toxicity study
During the experiment, the rats in all 3 groups were normal, agile, eat well, had silky hair and firm feces Mice weight in all 3 groups increased. The frequency and amplitude of ECG did not change. The number of red blood cells, hemoglobin content, hematocrit concentration, leukocyte count, percentage of neutrophils and lymphocytes, number platelet count, enzyme activity of AST, ALT, total bilirubin urea concentration and serum creatinine concentration of white rats in all groups were not different (p> 0.05)
* Changes in histopathology: macroscope morphology images of liver
organs, spleens, and kidneys of rats in research groups 1 and 2 had even dark reddish brown color, smooth surface, no lumps or hemorrhages. When being pressed down, there were no differences compared to that
Trang 11Number of stillbirths / 1
Trang 12mother mouseControl
Trang 13Comments: The rate of chromosomal structural disorders in the
chromosomal samples of bone marrow cells in the mice taking the