Nghiên cứu sự biến đổi hình thái cấu trúc, siêu cấu trúc ống sinh tinh, tinh trùng sau uống khang bảo tử trên bệnh nhân vô tinh TT TIENG ANH

MINISTRY OF EDUCATION MINISTRY OF NATIONAL AND TRAINING DEFENSE VIETNAM MILITARY MEDICAL UNIVERSITY QUACH THI YEN STUDY ON THE CHANGES OF STRUCTURE AND MICROSTRUCTURE OF SEMINIFEROUS TUBULES AND SPERM AFTER TAKING KHANG BAO TU ON AZOOSPERMIA PATIENTS Major: Biomedical science Code: 72 01 01 MEDICAL DOCTORAL THESIS SUMMARY CÔNG TRÌNH ĐƯỢC HỒN THÀNH TẠI HỌC VIỆN QN Y HANOI, 2021 THE WORK WAS COMPLETED AT VIETNAM MILITARY MEDICAL UNIVERSITY Supervisors: Assoc Prof PhD Quan Hoang Lam PhD Trinh Quoc Thanh Opponent 1: Assoc Prof PhD Nguyen Khang Son Opponent 2: Assoc Prof PhD Ngo Van Tai Opponent 3: Assoc Prof PhD Trinh The Son The thesis will be defended before the university-level thesis Examination Council at: , date … The thesis can be found at: National Library Library of Vietnam Military Medical University QUESTION According to the World Health Organization, infertility is one of the main reproductive health problems The infertility rate accounts for about – 12% of the population depending on the region, in North America, the rate is even up to 15% In Vietnam, the rate of infertility is about 7.7% Among the causes of male infertility, infertility is the most serious cause, accounting for about 10 – 20% of all male infertility cases Infertility is divided into two groups: obstructive sterile and non – obstructive sterility The choice of treatment is often based on the cause of infertility In 1992 with the introduction of the technique of injecting sperm into the cytoplasm of the oocyte along with the technique of collecting sperm in the epididymis or testicles helped infertile couples have had their own children However, before the procedure, patients are often given medical treatment to stimulate spermatogenesis to increase their chances of collecting sperm Traditional medicine is used to treat infertility for a long time and gives positive results Khang bao tu capsule is registered as a trading name instead of Hoi xuan hoan capsules, including Radix Rehmanniae glutinosae praeparata, Fructus Corni, Rhizoma Dioscorea persimilis, Radix Glycyrrhizae, Cortex Cinnamomi, Radix Aconiti lateralis praeparata, Cortex Eucommiae, Fructus Lycii, Colla Cornus Cervi has the good effect of moderating the kidneys, beneficial blood Based on the initial research on the efficacy of rejuvenating capsules in sperm depleted patients, we conducted this project on infertile patients with two goals: Description of morphological and structural characteristics, sperm superstructure, spermatogenesis tube after drinking Khang bao tu on a sterile patient Evaluation of results, some factors related to sperm collection after drinking Khang bao tu in a patient with infertility The signification of the thesis:  This is the first research in the country on the morphological change of the structure of the – spermatogen tube and sperm after using traditional medicine in a sterile patient  Initially assess the spermatogenetic effects of traditional medicine in infertile patients  The rate of sperm obtained by TESE micro – TESE method after taking Khang bao tu is 46.34% In the study, some patients with small testicles 4mL still obtained sperm The structure of the thesis: The thesis consists of 147 pages (the references and appendices are not included), structured into chapters: Question (02 pages); Chapter 1: Literature review (33 pages); Chapter 2: Subject and research method (25 pages); Chapter 3: Research results (43 pages); Chapter 4: Discussion (40 pages); Conclusion (2 pages); Recommendations (1 page); The thesis limitations (1 page); List of related articles; references (117 documents, including 15 Vietnamese documents, 07 Chinese documents and 95 English documents) and appendices Chapter LITERATURE REVIEW 1.1 Characteristics of structure and microstructure of seminiferous tubule and sperm 1.1.1 Characteristics of structure and microstructure of seminiferous tubules 1.1.2 Characteristics of structure and microstructure of sperm 1.1.3 The process of spermatogenesis and its affecting factors 1.2 Azoospermia 1.2.1 Definition and classification Infertility is the absence of sperm in the semen, not retrograde ejaculation In the past, semen samples were compulsorily centrifuged at 3000 rpm for 15 minutes, under a magnification microscope, at two different semen analyses Currently, azoospermia can be classified into two categories: Obstructive Azoospermia – OA and Non – Obstructive Azoospermia – NOA 1.2.2 The causes of azoospermia 1.2.2.1 The causes of non – obstructive azoospermia 1.2.2.2 The causes of obstructive azoospermia 1.2.3 The structural morphological characteristics of seminiferous tubule in azoospermia patients According to the guidelines of the European Urological Association 2010 division of testicular histopathology into four groups: (1) Impaired spermatogenesis (HP); (2) Stop half – life (MA) spermatogenesis; (3) Sertoli cell – only syndrome (SCOS); (4) Hyaline seminiferous tubes 1.2.4 Medical treatment for male infertility and azoospermia The role of hormones as well as antioxidants 1.2.5 Some techniques to collect sperm in azoospermia patients Percutaneous epididymal sperm aspiration (PESA); Microsurgical epididymal sperm aspiration (MESA); Percutaneous Epididymal Sperm Aspiration (TESA); Testicular sperm extraction (TESE); Micro – surgery to collect sperm from the testicles (micro TESE) 1.3 Azoospermia and male infertility according to traditional medicine 1.3.1 Opinions of traditional medicine about mechanism of azoospermia 1.3.2 Pathogenesis of azoospermia according to traditional medicine The pathologenesis underlying of infertile male with azoospermia is considered to divide into patterns: (1) Deficient kidney essence; (2) Deficient Qi and blood, deficiently cold spleen and stomach; (3) Inharmonious diet, damp and heat located in lower san Qi; (4) Depression and anxiety, stagnant liver Qi; (5) Damp stagnancy and blood stasis; (6) External pathogenic factors 1.3.3 Treatment categories of azoospermia in traditional medicine (1) Cold located in vas deferen and seminal vesicle; (2) Deficient yin and excessice yang; (3) Insufficent Qi – blood; (4) Deficient spleen cum kidney; (5) Qi stagnancy and blood stasis; (6) Insufficient kidney essence; (7) Stagnant heat 1.3.4 Potential therapeutic mechanisms of traditional medicine for male infertility Regulation of the reproductive endocrine system; boosting the function of Sertoli cells and Leydig cells; modulating the proliferation and apoptosis of germ cells; preventing oxidative stress; decreasing the level of anti – sperm antibody; ameliorating the microcirculation of the testis; alleviating inflammation 1.3.5 Traditional medicine studies in azoospermia and male infertility 1.3.5.1 In the world A lot of researches on the effectiveness of traditional medicine in the treatment of male infertility The results have been shown to increase the quantity and quality of spermatozoa in oligospermia 1.3.5.2 In Vietnam Some authors have also shown the effect of traditional medicine on spermatogenesis in experiment and in patients with impaired spermatogenesis However, there are no studies on patients with azoospermia 1.4 The overview of Khang bao tu capsule 1.4.1 The origin and composition of Khang bao tu capsule Khang bao tu capsule consists of medicinal ingredients and the content is equivalent to Hoi xuan hoan capsule of Doan Minh Thuy and is manufactured based on the complete rejuvenation remedy Hoi xuan hoan with ingredients: Radix Rehmanniae glutinosae praeparata 24g, Fructus Corni 12g, Rhizoma Dioscorea persimilis 16g, Radix Glycyrrhizae 12g, Cortex Cinnamomi 12g, Radix Aconiti lateralis praeparata 12g, Cortex Eucommiae 16g, Fructus Lycii 16g, Colla Cornus Cervi 10g The rejuvenating remedy Hoi xuan hoan is original from the “Chinese Organic regulation of spices” 1.4.2 Studies on Hoi xuan hoan capsule have been performed Hoi xuan hoan capsules have been studied for acute toxicity, semi – chronic toxicity, chromosomal toxicity and reproductive toxicity It has also been studied experimentally causing testicular heat damage and in patients with impaired spermatogenesis resulting in increased sperm count and quality Chapter RESEARCH SUBJECTS AND METHODS 2.1 Research subjects, time and place 2.1.1 Criteria for selecting patients The patients selected were those who had no sperm in the semen defined in accordance with WHO 2010 standards, not retrograde ejaculation 2.1.2 Exclusion criteria Patients with retrograde ejaculation, with secondary hypogonadism; complete discontinuation of AZFa or AZFb, Chapelle syndrome; patients suffering from acute diseases, HIV, and sexually transmitted diseases; patients are on therapies or drugs that affect sperm production; the patient does not agree to cooperate or strictly abide by the treatment regimen 2.1.3 Place, time of study The research was conducted at the Military Clinical Embryo Institute – Military Medical University and the Department of Morphology Institute 69, Ho Chi Minh Mausoleum Security Command from May 2017 to May 2020 2.2 Reaserch materials Ingredients of 01 Khang bao tu capsule (Hoi xuan hoan) 500mg capsule: Radix Rehmanniae glutinosae praeparata 2.4g, Fructus Corni 1.2g, Rhizoma Dioscorea persimilis 1.6g, Radix Glycyrrhizae 1.2g, Cortex Cinnamomi 1.2g, Radix Aconiti lateralis praeparata 1.2g, Cortex Eucommiae 1.6g, Fructus Lycii 1.6g, Colla Cornus Cervi 1.0g and other excipients Standard of the product: + Medicinal herbs have met the standards of Vietnamese Pharmacopoeia standards IV + Dried medicinal herbs have met the standard of Vietnamese pharmacopoeia standards IV + Hard capsule 500mg, with the material, is a mixture of dried high powder from medicinal herbs Capsules have met the standards based on high standards of drugs in the Pharmacopoeia of Vietnam IV + Number VISA: 1497/2015/ATTP – XNCB + Manufatory address: Institute of Natural products chemistry, Building H1, 18 Hoang Quoc Viet, Nghia Do, Cau Giay, Hanoi, Vietnam 2.3 Research methods, sample size and ways of choosing samples Research method is a randomized controlled clinical trial Apply the sample size formula for the study of randomized controlled clinical trial, to test the difference between the two rates n= [ ⁄ √ ̅ ̅ √ ] p1: sperm collection rate in the group not treated with micro TESE method, according to one study estimated to be 28.1% → p1 = 0.281 p2: sperm collection rate (desired) in Khang bao tu using group by micro TESE method In order to ensure that the smallest sample size was statistically significant, we took the rate of sperm collection as 50% → p2 = 0.5 Instead of the formula, we got the minimum sample size for each study group of 49, round the sample size to 66 patients From qualified infertile patients, numbered by time to examine and randomized into three groups, each group had 66 patients: Group 1: select patients with ordinal numbers 1,4,7, were treated with Khang bao tu 500mg capsule with dose: 10 tablets per day in divided doses Group 2: choose patients with sequence numbers 2,5,8, are treated with a regimen of zinc and vitamin E Dosage is as follows: + Vitamin E 400UI: take capsules a day in divided doses + Subzex (zinc): capsules per day in divided doses Group 3: choose patients with sequence numbers 3,6,9, and not receive any treatment Patients in group and group received at least three months of treatment The patients were then given a PESA technique to find sperm; If sperm cannot be obtained, the patients would be given micro TESE 2.3.1 Techniques used in research Semen analysis (from WHO 2010); hormonal tests; measure testicular volume with a Prader gauge Sample techniques: PESA, micro TESE The techniques to assess sperm micro – characteristics obtained from the epididymis and testicles are according to WHO 2010 standards Techniques to identify and assess the structural injury of the spermatogenesis tube: HE staining, quantitative and semi – quantitative according to Johnsen 1970; measuring the tube diameter and fiber shell thickness according to McVicar C.M (2005) Method of making testicular tissue superstructure specimen for transmission electron microscopy and scanning electron microscope 1.3.2 Research criteria 2.3.2.1 Criteria about clinical characteristics of research subjects Age, BMI, infertility duration, infertility type, history, testicular volume 2.3.2.2 Variables on subclinical characteristics of study subjects: endocrine, AZF, histopathology 2.3.2.3 Characteristics of spermatozoa obtained from the crest and testicles 2.3.2.4 Results of micro TESE techniques Sperm collection rate, sperm microcrystalline characteristics Comparing morphological characteristics of the spermatogenesis tube among research groups on the following criteria: Johnsen score, tube diameter of spermatogenesis; the fibrous sheath thickness, the number of sperm epithelial cells 2.3.2.5 The association of some factors with micro – TESE sperm collection in a patient with non – obstructive spermatozoa Evaluation factors: age, BMI, time of infertility, preoperative testicular volume, endocrine, AZF, histopathology, spermatogenesis tube diameter, genital canal thickness, Johnsen score sperm epithelial cells 2.4 Data processing Data was processed by using spss 20.0 software 2.5 Ethical issues in research Rights and responsibilities of participating in the study were explained to patients All patient information is kept secretly 11 Median testicular volume in group OA was 10mL, NOA was 6mL There were differences in testicular volume in the two groups (p < 0.001) 3.1.3 Subclinical of characteristics of patients 3.1.3.1 Endocrine characteristics of study patients There was no difference in endocrine concentrations between the study groups in azoospermia patients as well as OA and NOA groups (p > 0.05) The comparison in the two groups of OA and NOA showed that: the mean FSH concentration was 3.8mIU/mL and 18.25mIU/mL, respectively; The corresponding LH was 3.93mIU/mL and 8.79mIU/mL; The corresponding testosterones were 4.43ng/mL and 3.73ng/mL There was a difference in the endocrine concentration of FSH, LH in the two groups OA and NOA (p < 0.001), but there was no difference in testosterone in the two groups (p > 0.05) 3.1.3.2 Characteristics AZF of research patients Normal AZF accounted for 88.38% and abnormal AZF accounted for 11.62%, of which 43.48% were combined with AZF; 39.13% AZFc; 13.04% AZFb and 4.35% AZFa AZF abnormalities were fairly evenly distributed in groups 3.2 Microscopic characteristics of sperm in azoosperm patients 3.2.1 Microscopic characteristics of sperm obtained from epidymis In group 1: the median sperm density was 10 million/mL, the lowest was 0.5 million/mL and the highest was 100 million/mL; sperm concentration obtained mainly from – less than 15 million/mL; immobilized sperm are predominantly; median percentage of living spermatozoa is 42% (from – 100%); the median rate of normal sperm morphology is 2% (from – 5%); 12 among the types of abnormalities, the first type of abnormality accounts for the highest rate of 37.47% There was no difference in sperm microbiological characteristics of spermatophytes in the study groups (p > 0.05) 3.2.2 Microscopic characteristics of sperm obtained from testicular 3.2.2.1 Characteristics of micro sperm obtained from testicular In group 1: median sperm density was million/mL (from 0.5 to 30 million/mL); the percentage of immobile sperm was mainly; median survival rate of spermatozoa was 31% (from 10 to 80%); normal morphological sperm accounted for 2.37%; of the abnormal spermatozoa, the coordinated abnormal form accounted for the highest proportion However, there was no difference in most of the sperm microbiological characteristics obtained from the testicles in the study groups (p > 0.05) but there was a difference in sperm density in group compared with that of group (p < 0.01) 3.2.2.2 Characteristics of supermicro sperm obtained from testicular In a total of 13 testicular tissue samples did superstructure, samples found sperm Most head abnormalities manifested were in the cell membrane of the head, wrinkled, or even discontinuous, the terminal sac had an abnormally distorted shape, the chromosome was heterogeneous, and there were areas of defects that showed equal to low electron density region The cytoplasmic neck was thick and the tail mitochondria lost their folds 3.3 Characteristics of structure and superstructure of seminiferous tubules in patients with non obstructive azoospermia 3.3.1 Characterisitics of structure of seminiferous tubeles 3.3.1.1 Characteristics of histopathology 13 The tissue lesions of testicular pathology in group NOA patients met four morphological forms: SCOS, MA, HP, and hyalin, respectively, of 60.98%; 19.51%; 12.20%, and 7.32% There was no difference in histopathology between the study groups (p > 0.05) 3.3.1.2 Qualitative degree of seminiferous tubules degeneration The degree of seminiferous tubules degeneration in NOA patients in the study had many morphologies: the degeneration was irregular, there were only Sertoli cells Sometimes there was still a picture of the spermatogenesis tube with full sperm cells, but the number was reduced and the cell order changes compared to normality 3.3.1.3 Semi quantitative degree of seminiferous tubules The median of the semi – quantitative point of NOA patients group was The difference was statistically significant between groups – 3; group – with p < 0.001, there is no difference between group – (p > 0.05) 3.3.1.4 Quantify degree of seminiferous tubules * The tube diameter of seminiferous tubules and the thickness of the fiber shell The median diameter of the seminiferous tubules and fiber shell thickness in group was 126.65µm; 9.90µm respectively There was no difference in the diameter of the seminiferous tubules and the fiber shell thickness in the study groups with p > 0.05 * Characteristics of the seminiferous tubules epithelium 14 Table 3.28 Characterisitcs of seminiferous tubules epithelium in the study groups with NOA Seminal Group (n = 820) Group (n = 880) Group (n = 940) epithelial cells Median Median Median ( ̅ ±SD) (min – max) (min – max) (min – max) 8.93 ± 8.34 10.87 ± 6.40 13.40 ± 8.46 (0 – 37) 11 (0 – 35) 13 (0 – 44) Sertoli cell Sertoli average: 11.17 ± 8.00; median (min – max): 10 (0 – 44); p12* < 0.001; p13* < 0.001; p23* < 0.001; p** < 0.001 spermatogonium 8.08 ± 13.24 7.56 ± 13.01 3.09 ± 5.72 (0 – 78) (0 – 48) (0 – 80) Spermatogonium average : 6.13 ± 11.30 Median (min – max): (0 – 80); p12* = 0.200; p13* < 0.001; p23* < 0.05; p** < 0.001 spermatocyte 4.75 ± 8.44 3.19 ± 7.18 2.26 ± 5.15 (0 – 54) (0 – 44) (0 – 25) Spermatocyte average: 3.35 ± 7.05; median (min – max): (0 – 54) p12* < 0.01; p13* < 0.001; p23* = 0.143; p** < 0.001 Sperm cell 2.60 ± 5.88 1.67 ± 4.05 0.59 ± 2.16 (0 – 51) (0 – 31) (0 – 20) Sperm cell average: 1.57 ± 4.31; median (min – max): (0 – 51) p12* = 0.915; p13* < 0.001; p23* < 0.001; p** < 0.001 Sperm 1.06 ± 4.10 0.50 ± 2.15 0.41 ± 2.96 (0 – 31) (0 – 22) (0 – 30) Sperm average: 0.64 ± 3.16; median (min – max): (0 – 31); p12* = 0.363; p13* < 0.001; p23* < 0.001; p** < 0.001 *Mann – Whitney test; ** Kruskal – wallis test 15 Sertoli: There was a difference in the number of cells between the study groups (p < 0.001) Regarding sperm cells: the number of group sperm cells tended to be higher than group and group When comparing groups – 2: there was the only difference in spermatocyte (p < 0.05); group – comparison: the difference was statistically significant for all sperm cells (p < 0.01 and p < 0.001); group – comparison: the difference was statistically significant for spermatogonium, sperm cells and sperm (p < 0.001), but there was no difference for spermatogonium (p > 0.05) 3.3.2 Characteristics of superstructure of seminiferous tubules 3.3.2.1 The diameter and thickness of the fibrous shell of the seminiferous tubules The majority of NOA patients in the groups had an increase in the thickness of the spermatogenesis tubular fibrous shell with an increase in the number and size of the collagen fiber bundles and the connective cells The diameter of the spermatogenesis tube was atrophy small and unevenly, sometimes there were a few normal – sized spermatic tubes and sperm epithelium was thick with many rows of cells 3.3.2.2 Seminiferous tubules epithelial cells The appearance of vacuoles of various sizes in both Sertoli and Sertoli cells were one of the most common images appearing in NOA patients Most Sertoli cells were poorly structured with sparse organelles, no phagocytosis, and no folds in the nuclear membrane 3.4 Results of sperm collection on azoospermia patients The relationship of some factors to sperm collection rate in a patient with non – obstructive azoospermia 3.4.1 Results of sperm collection on azoospermia The rate of sperm collection by the PESA method and/or micro TESE is 60.10% There was a difference in sperm collection rate between groups – and group – (p < 0.05), there was no difference between groups – (p > 0.05) The rate of sperm obtained by the PESA method accounted for 33.33%; the highest was group with 37.88% and the 16 lowest was group with 28.79% The rate of sperm collection by micro TESE method accounted for 40.15%, the highest rate in group was 46.34% However, there was no difference in the study groups (p > 0.05) 3.4.2 The relationship of some factors to sperm collection rate in a patient with non – obstructive azoospermia There was no relationship between age, BMI, and duration of infertility, and sperm collection rate in NOA patients There is a relationship between testicular volume, endocrine concentration, and sperm collection in NOA patients We found the cut – off point of the bilateral testicular volume was 5.5mL; The FSH was 13.41mIU/mL, the LH was 7.64mIU/mL and the testosterone was 3.02ng/mL with the sperm collection rate in the NOA patient There was no difference in normal, abnormal AZF with sperm collection rate in NOA patients (p > 0.05) 75% of AZFc found sperm There were histopathological differences in the two groups that found no sperm in NOA patients with p < 0.001 There was a relationship between normal spermatogenesis tube diameter, normal fibrous shell thickness, and Johnsen scored ≥ with sperm collection rate in NOA patients There was a difference in the number of sperm cells between the two groups of finding and not finding sperm with p < 0.001, but no difference between the two groups in the number of Sertoli cells (p > 0.05) CHAPTER DISCUSSION 4.1 Discussion on study subjects and esearch methods 4.1.1 Grouping of patients and research methods There was no relationship between age, BMI, and duration of infertility, and sperm collection rate in NOA patients There was a relationship between testicular volume, endocrine concentration, and sperm collection in NOA patients We found the cut – off point of the bilateral testicular volume was 5.5mL; The FSH was 17 13.41mIU/mL, the LH was 7.64mIU/mL, and the testosterone was 3.02ng/mL with the sperm collection rate in the NOA patient Out of 198 infertility patients were divided equally into three groups The duration of drug use for group and group was three months However, infertile patients often have severe testicular damage Therefore, to evaluate the effectiveness of the drug requires a longer time of taking the drug At the same time, to accurately evaluate the ability of the drug to stimulate spermatogenesis, the optimal design for the study is to compare before and after This was difficult and a limitation of the research The proportion of patients with OA accounted for 33.33% and NOA accounted for 66.67% This proves that in infertile patients, the rate of NOA patients was more, this is a worrying problem for the reproductive health of patients with infertility 4.1.2 Clinical characteristics of study patients 4.1.2.1 Age, BMI, infertility duration and type of infertility The median age in the study was 32 years old; the median BMI was 22.68; the median time of infertility was four years Primary infertility accounted for 93.43% and secondary to 6.57% There was no difference in age, BMI, infertility time, and infertility type between the two groups of patients with OA and NOA as well as three groups in azoospermia patients and OA and NOA groups (p > 0.05) 4.1.2.2 History related to infertility In the study, the highest proportion of patients with a history of mumps (37.37%) was also a common cause of atrophic testicular, chronic atrophy leading to infertility 4.1.2.3 Testicular volume The median volume of the right and left testicles were 8mL (from to 25mL), much lower than the average testicular volume of Vietnamese adult men is from 12 to 30mL There was a difference in testicular volume in the two groups of patients with OA and NOA (p < 0.001) but no difference in the groups of azoospermia patients and OA, NOA patients (p > 0.05) 18 4.1.3 Characteristics of subclinical 4.1.3.1 Endocrine characteristics The FSH concentration in the study was higher than the normal value (normal from – 10mIU/mL); The smallest FSH concentration in the study was 0.20mIU/mL and the highest was 68.56mIU/mL This proved that increased FSH in the blood was a marker of fertility problems, but normal FSH did not guarantee intact spermatogenesis When comparing the endocrine concentration in groups of azoospermia patients, OA and NOA patients, there was no difference with p > 0.05 However, there were differences in FSH, LH in the two groups OA and NOA (p < 0.001) 4.2 Characteristics of micro and super – micro of sperm in infertility patients 4.2.1 Characteristics of micro sperm collected from epididymis Characteristics of micro sperm from epididymis in the group using Khang bao tu as well as the other two groups were almost all lower than WHO standards (2010) This proved that the obstruction has some what affected the quality of the sperm as well as the spermatogenesis process inside the testicles We did not see any difference in the micro characteristics of sperm from epididymis in study groups (p > 0.05) Thus, whether the treatment was used or not, it did not affect the density, mobility, survival/death rate, and sperm morphology in the infertile patient However, for accurate assessment, it is necessary to have a longer time to use the drug 4.2.2 Characteristics of micro and super – micro of sperm obtained from testicles 4.2.2.1 Characteristics of micro sperm obtained from testicles The characteristics of micro sperm obtained from the testicles in groups were all lower than WHO 2010 standards Particularly, sperm density in group was better than in group (p < 0.01) Other micro characteristics did not differ between the three groups (p > 0.05) The above results proved that the process of spermatogenesis in the testicles 19 of NOA patients was severely damaged And it can be seen that the patients treated with Khang bao tu capsules had better sperm density than the untreated group However, to evaluate the effectiveness of the drug requires a longer dosing time and a larger sample size 4.2.2.2 Characteristics of super – micro sperm obtained from testicles Most sperm were head abnormally with a distorted, or even discontinuous, the head membrane with an abnormally distorted appearance; The condensed chromosome is heterogeneous, with defect regions represented by low electron density regions The cytoplasmic neck was thick and the tail mitochondria lost folds 4.3 Characteristics of structure and superstructure of seminiferous tubules in patients with NOA 4.3.1 Characteristics of structure of seminiferous tubules 4.3.1.1 Histopathology of the seminiferous tubules The results showed that the histopathological lesions of patients with NOA group met mainly SCOS with 60.98%; followed by MA with 19.51%, HP with 12.20%, and the lowest Hyalin with 7.32% There was no difference in histopathology in the study groups (p > 0.05) 4.3.1.2 Semi – quantitative level of seminiferous tubules degeneration NOA patients in all three groups had low Johnsen scores, with group and group tending to be higher than group The difference in Johnsen score between group – and group – was statistically significant with p < 0.001 This implied that the patients in group have the most severe damage to testicular tissue 4.3.1.3 Quantify the degree of seminiferous tubules degeneration * The of diameter of seminiferous tubules and the thickness of the fiber shell * Characteristics of sperm epithelial cells The number of sperm cells in groups and tended to be higher than in group (p < 0.001 and < 0.01) This can bring hope that when patients undergo longer-term treatment, they will increase the number of sperm cells more, thereby increasing their chances of collecting sperm 20 Although the number of sperm cells in group increased compared with group 2, the difference was only found in the spermatocyte (p < 0.01) 4.3.2 Characteristics of super – micro structure of seminiferous tubules 4.3.2.1 The diameter of the seminiferous tubules and the thickness of the fibrous shell In the majority of cases, the seminiferous tubules were irregular in size, many tubules shrink, the thickness of the fibrous tube layer increases That proves the process of spermatogenesis is greatly reduced The thickening of the fibroblast shell of seminiferous tubules was often accompanied by the appearance of many Mast cells in this layer because Mast cells also act to activate fibroblasts and stimulate collagen synthesis However, due to the small number of superstructure samples, the results of the study are not fully representative 4.3.2.2 Sperm cells In addition to normal structures, most were head abnormal with distorted, or even discontinuous, head membranes manifested in an abnormally distorted appearance; The condensed chromosome was heterogeneous, with defect regions represented by low electron density regions The cytoplasmic neck was thick, the tail mitochondria lost folds Most Sertoli cells had a structure of reduced functional activity expressing the nuclear membrane is not folded, the nucleus can be seen or not, the cytoplasm is not polar, the organelles are sparse, and the phagocytosis is absent However, due to the small number of superstructure samples, the superstructure of sperm epithelial cells is not fully reflected 4.4 Results of sperm collection on patients with azoospermia Relationship of several factors to sperm collection in patients with non – obstructive azoospermia 4.4.1 Results of sperm collection on patients with azoospermia The rate of sperm collection by PESA method and/or micro TESE in patients with infertility is 60.10% If counting patients with NOA alone, the rate of sperm collection by micro TESE is 40.15% Analyzing 21 each group individually, we found that: the results of sperm collection by PESA and/or micro TESE method in patients with group infertility was the highest (66.67%), there was a difference between group – and group – (p < 0.05), however, there was no difference between groups – (p > 0.05) For NOA patients, the highest rate of sperm collection by micro TESE in group was 46.34%, but the difference was not statistically significant (p > 0.05) * Explain the treatment mechanism and effectiveness of group and group According to research by Doan Minh Thuy (2010), the Khang bao tu capsule (Hoi xuan hoan) has been shown to stimulate the spermatogenesis process in experiments and in patients with reduced spermatogenesis Antioxidants can purify and eliminate the effects of ROS by inhibiting the formation and countervailing effects of ROS Many studies have shown that the use of antioxidants can have an impact on sperm quantity and quality Simultaneously with the proliferation of sperm cells in the sperm epithelium may partly explain the higher sperm collection rate in groups and than in group However, for NOA patients, studies are needed study with a longer drug use time, the larger sample size to evaluate the real effectiveness of the drug At the same time, optimal for the study is pre-post assessment This is a real challenge for research because repeated testicular biopsy increases the risk of secondary testicular damage 4.4.2 Relationship of several factors to sperm collection in patients with non – obstructive azoospermia The study found no correlation between age, BMI, and duration of infertility, and sperm collection rate in NOA patients We found a correlation between testicular volume, endocrine concentration, and sperm collection rate in NOA patients We found the cutoff point of the right and left testicular volume was 5.5mL; The FSH cutoff point was 13.41mIU/mL, the LH was 7.64mIU/mL and that of the testosterone was 3.02ng/mL But the cutoff points differ widely between 22 studies This proves that the prognostic value of sperm collection of testicle volume cutoff point, as well as endocrine concentration, was low Larger sample size and many factors used in combination to increase the validity of the study are needed There were differences in the types of histopathological lesions in the two groups with and without sperm (p < 0.001) When comparing the sperm collection rate in each type of histopathological injury, it showed that the rate of sperm found in the cases of HP, MA, SCOS, and Hyalin was 90.91%, respectively; 55.55%; 32.91%, and 13.33% Most studies conclude that there is no single factor can be used clinically to predict the success of micro TESE However, the synthesis of all three factors (endocrine, testicular volume, histopathology) can help clinicians predict the rate of sperm collection, especially histopathology CONCLUSION Through the study of 198 infertile patients and divided equally into groups, of which 66 patients were given Khang bao tu, we obtained the following results Characteristics of structure and superstructure of sperm, seminiferous tubules of azoospermia after taking Khang bao tu 1.1 Characteristics of structure and superstructure of sperm of azoospermia patients after taking Khang bao tu  For sperm collected from epididymis: median sperm density was 10 million/mL, minimum 0.5 million/mL and maximum 100 million/mL, of which 48.0% were affected density ≥ 15 million/mL; High incidence of malformed sperm, live sperm, and poor sperm motility  For sperm obtained from testicles: median sperm density was million/mL, minimum 0.5 million/mL and maximum 30 million/mL; There was a difference in sperm density in the Khang bao tu group compared with the untreated group (p < 0.01); only 10.53% of patients 23 had density ≥ 15 million/mL; a high proportion of malformed and immobile sperm; the low survival rate of spermatozoa  When viewed under an electronic microscope, it is common that the first abnormal sperm is characterized by a heterogeneous dense chromosome, thick cytoplasmic part, and tail mitochondria lose folds 1.2 Characteristics of structure and superstructure of seminiferous tubules of azoospermia patients after taking Khang bao tu  The epithelium of the seminiferous tubules was quite severely damaged with the manifestations of thickened fibrous, the size of the seminiferous tubules was atrophic The common spermatophore epithelium in forms was the Sertoli cell – only syndrome (60.98%), halving spermatogenesis (19.51%), impaired spermatogenesis (12.20%), and hyalinization tube (7.32%)  Khang bao tu increased the number of sperm cells compared to the untreated group (p < 0.001) and significantly increased the number of sperm cells compared with the zinc + vitamin E treatment group (p < 0.01) Patients treated with zinc + vitamin E had an increase in the number of spermatogonium, sperm cells, and sperm compared with the untreated group (p < 0.001 and p < 0.05) Results of sperm collection on azoospermia patients The relationship of some factors to sperm collection in a non – obstructive azoospermia patients  The results of sperm collection by one of two methods PESA/micro TESE in infertile patients after taking Khang bao tu was 66.67%; This rate was different than the group without treatment (p < 0.05)  46.34% of patients obtained sperm using the micro TESE method after taking Khang bao tu; however, there was no difference compared with the group of patients treated with zinc + vitamin E as well as the group without treatment (p > 0.05)  There was a relationship between testicular volume, FSH, LH, testosterone, and sperm collection rate in patients with non-obstructive 24 azoospermia: cut – off point of testicular volume was 5.5mL; FSH was 13.41mIU/mL; LH is 7.64mIU/mL; testosterone was 3.02ng/mL There was a relationship between histopathological characteristics, normal seminiferous tubules diameter, normal tubular fibrous shell thickness, Johnsen score, and the number of sperm epithelial cells and sperm collection rate in NOA patients RECOMMENDATIONS Khang bao tu capsules can be selected to treat infertile patients before the procedure to find sperm in the epididymis or testicles It is necessary to continue to study with a larger sample size and a longer duration of use to evaluate the effectiveness of Khang bao tu capsule Study of pregnancy rate and follow-up of babies born from sperm of infertile patients treated with Khang bao tu capsule Follow up, evaluate the function of the testicles in patients with non – obstructive azoospermia after performing the micro TESE technique SOME LIMITATIONS OF THE THESIS This is a randomized controlled clinical trial, so to accurately evaluate the effect of the drug, it is necessary to have a comparative study before and after However, this is a challenge for research Patients with infertility often have a severe cause of damage, prolonged infertility, so three months of medication may not be enough to evaluate the effect of the drug Due to the limited budget for making the superstructure specimen, the number of samples is small (13 samples), so it cannot represent the research results The rate of pregnancy after taking the drug has not been evaluated 25 LIST OF AUTHOR'S SUCCESSFUL RESEARCH WORKS RELATED TO THE THESIS Quach Thi Yen, Quan Hoang Lam, Trinh Quoc Thanh, Doan Minh Thuy, Vu Thi Hao (2020) The effects of „Hoi Xuan Hoan‟ capsules on sperm collection efficiency and morphological sperm structure obtained from micro in nonobstructive azoospermia patients Journal of Military Pharmaco-medicine Vol 45, N06, 2020 Vietnamese pp15-22, English pp104-111 Quach Thi Yen, Quan Hoang Lam, Trinh Quoc Thanh, Nguyen Huyen Trang, Vuong Mai Linh (2020) Characteristic structure of sperm obtained from epididymal in azoospermia patients due to obstructive Vietnam Medical Journal August N001, Vol 493/2020 pp160-163 ... studies on patients with azoospermia 5 1.4 The overview of Khang bao tu capsule 1.4.1 The origin and composition of Khang bao tu capsule Khang bao tu capsule consists of medicinal ingredients and... superstructure, spermatogenesis tube after drinking Khang bao tu on a sterile patient Evaluation of results, some factors related to sperm collection after drinking Khang bao tu in a patient with infertility... MEDICAL UNIVERSITY Supervisors: Assoc Prof PhD Quan Hoang Lam PhD Trinh Quoc Thanh Opponent 1: Assoc Prof PhD Nguyen Khang Son Opponent 2: Assoc Prof PhD Ngo Van Tai Opponent 3: Assoc Prof PhD Trinh