MINISTRY OF EDUCATION AND TRAINING CAN THO UNIVERSITY SUMMARY OF DOCTORAL DISSERTATION Specialization: Biotechnology Code: 42 02 01 STUDY ON SOME KINDS OF Y-CHROMOSOMAL ABNORMALITIES IN MEN SEEKING MEDICAL CARE FOR THEIR FERTILITY AT CAN THO OBSTETRICS AND GYNECOLOGY HOSPITAL Can Tho, 2018 THE STUDY WAS COMPLETED AT CAN THO UNIVERSITY The dissertation was defended at the university examination committee At.………………………………………., Cantho University At …………………………………………………………… Reviewer 1: Reviewer 2: Reviewer 3: The dissertation is available in Libraries: Central Library of Can Tho University National Library of Vietnam LIST OF PUBLICATION RELATED TO THE THESIS Cao Thi Tai Nguyen, Nguyen Trung Kien, Vu Thi Nhuan Nguyen Chung Vieng, Trinh Thi Bich Lien (2017), “Detection of AZFc microdeletions on Y chromosome in male infertility by QF-PCR assay”, Vietnam Medical Journal No 2/2017, pp 245249 Cao Thi Tai Nguyen, Trinh Thi Bich Lien, Nguyen Trung Kien, Vu Thi Nhuan, Nguyen Phan Vinh, Nguyen Thi Bich Ngoc, Trinh Minh Thiet, Phan Thi Nhan (2017), “Hormone FSH, LH and testosteronee of severe oligozoospermia and azoospermia male”, Journal of Practical Medicine No 5(1043)/2017, pp 178-181 Cao Thi Tai Nguyen, Nguyen Trung Kien, Vu Thi Nhuan, Nguyen Chung Vieng, Trinh Minh Thiet, Nguyen Phan Vinh, Nguyen Thi Bich Ngoc, Cao Luong Binh, Trinh Thi Bich Lien (2017), “An infertility SRY-negative 46,XX male detected by quantitative fluorescent polymerase chain reaction”, Journal of clinical case reports 7:1013, doi: 10.4172/21657920.10001013 Cao Thi Tai Nguyen, Nguyen Trung Kien, Trinh Thi Bich Lien, Vu Thi Nhuan, Nguyen Chung Vieng, Nguyen Dac Khoa, Nguyen Thi Bich Ngoc, Trinh Minh Thiet, Cao Luong Binh, Nguyen Phan Vinh, Nguyen Van Khuon (2018), “Using QF-PCR assay in detection of common genetic causes in infertile males”, Journal of Biotechnology No 16(2), pp 241-252 Chapter INTRODUCTION 1.1 Background Genetic factors are highlighted as the most frequent, contributed to 4-38%, of male infertility causes Up to date, Klinefelter syndrome and AZF deletions are common genetic causes in infertily men In Vietnam, these causes accounted for 23.1% in infertile males with sperm concentration ≤ million/mL Moreover, the proportion of Vietnamese infertile men with AZF deletions was range 5-12.8% in previous studies There are kinds of AZF deletions as AZFa, AZFb and AZFc Men with AZFc deletion can have a baby with his wife/partner due to reproductive technology (a successful rate was 70%), and others are very difficult for his wife/partner to have been pregnanced European Academy of Andrology and European Molecular Genetics Quality Network (EAA/EMQN) recommend that men with sperm concentration < million/mL should perform tests for genetic abnormalities before using assisted reproduction (Krausz et al., 2014) QF-PCR assay has many advantages such as fast feedback time, low cost, high accuracy; so many authors propose to use this technique in diagnosis of genetic causes in infertile men That is the reason behind the study of “Study on some kinds of Y-chromosomal abnormalities in men seeking medical care for their fertility at Can Tho Obstetrics and Gynecology hospital” 1.2 Objectives (1) To set up and to assure the procedure of QF-PCR assay using for detecting some kinds of Y-chromosomal abnormalities in men with sperm concentration ≤ million/mL (2) To determine the percentage of some kinds of Y-chromosomal abnormalities in men with sperm concentration ≤ million/mL by the setted up and assured procedure of QF-PCR assay (3) To describe characteristics of semenogram, endrocrines and some factors related to Y-chromosomal abnormalities in men with sperm concentration ≤ million/mL 1.3 Scientific significance of the dissertation 1.3.1 Scientific and educational values - Providing scientific data on the percentage of some kinds of Ychrosomal abnormalities, characteristics of semenogram, endrocrines and some factors related to Y-chromosomal abnormalities in men with sperm concentration ≤ million/mL - Making the premise for the following studies 1.3.2 Practical values This research has a great significance in supporting the diagnosis, counseling and treatment of infertility effectively and inexpensively in patients at Can Tho Obstetrics and Gynecology Hospital in particular and in the Mekong Delta in general 1.4 New contributions of the dissertation - The study was setted up and optimized a successful procedure of QF-PCR assay with 14 genetic markers (AMEL, SRY, sY1191, sY1192, TAF9B, DAZ, CDY, sY84, sY86, sY127, sY134, sY254, sY255 and sY1291) for detecting Y-chromosomal abnormalities in men seeking medical care for their fertility - The study of Rozen et al (2012) was firstly indicated that there were kinds of partial AZFc deletion in Vietnamese: gr/gr and b2/b3 Besides two of these deletions, de novo mutations were found in our research as sY1191-sY1192 deletion, sY1291 deletion, DAZ – CDY1 deletion and CDY1 deletion - The thesis was noted that the length polymorphism of sY1291 fragment, ranging from 507 bp – 527 bp Chapter OVERVIEW MATERIALS 2.1 General of infertility and spermatogenesis 2.1.1 Definition of infertility and male infertility About 80-85% of couples achieve pregnancy after year living together; but about 15-20% of them have trouble in fertility, also known as infertility (Ayensu-Coker et al., 2007; Jungwirth et al., 2012) Infertility is the inability of a sexually active, noncontracepting couple to achieve pregnancy in one year (WHO, 2000) Male infertility is the status of couples does not achieve pregnancy after year of unprotected intercourse and without using contraceptive method caused by men (Jungwirth et al., 2012) 2.1.2 General of spermatogenesis The spermatogonium take part in spermatogenesis and undergo stages as mitotic division, meiosis and differentiation to create mature sperm (Oliveira and Alves, 2015) A fertile man produces more than 40 million spermatozoa per day (Cheng and Mruk, 2013) Three main hormones regulate in spermatogenesis are FSH (FSH Follicle-stimulating hormon), LH (LH - Luteinizing hormon) and testosterone (Verhoeven et al., 2010; Rato et al., 2012) Besides, this process is regulated by some other factors such as temperature, reactive oxygen species (ROS) and antioxidant defence at testis (Oliveira and Alves, 2015) Testing for FSH, LH, and testosterone concentrations are important and necessary to figure out causes and treatments for male infertility caused by hormone (Hotaling and Walsh, 2009) 2.2 Semenogram and some risk factors of male infertility 2.2.1 Semenogram Up to now, most testing labs evaluate semenogram based on WHO guidelines (2010) 2.2.2 Some risk factors of male infertility Some risk factors relate to male infertility have been identified such as age, body mass index (BMI), family history of infertility, some of life habits (smoking, drinking, low physical activity), toxic enviroment, professions exposure to high temperatures, tight underwear and steam bath (Cavallini and Beretta, 2015) 2.3 Y chromosome and some kinds of Y-chromosomal abnormalities in male infertility 2.3.1 Y chromosome Y chromosome is approximately 60 Mb in size and includes two segments, pseudoautosomal regions (5%) and non-combining region (also known as male-specific region of Y chromosome (MSY)) (Jangravi et al., 2013) MSY comprises 95% of Y chromosome’s length and has regions They are euchromatin and heterochromatin (Fig.1) Fig 2.1 Structure of Y chromosome (Kandeel et al., 2007) 2.3.2 Some genetic markers on Y chromosome The MSY database contains many genetic markers For example, there are 1.287 Y-specific sequence tagged sites (STSs) on Y chromosome, in which 992 STSs are single-copy and 285 STSs are multiple-copy (Lange et al., 2008) Until now, most of studies have been used genetic markers (sY84, sY86, sY127, sY134, sY254 and sY255) to detect AZFa, AZFb and AZFc deletions (Krausz et al., 2014) Moreover, Rozen et al (2012) used sY1189, sY1191, sY1192 and sY1291 markers to detect partial AZFc deletions Based on these evidents, the study was used sY84, sY86, sY127, sY134, sY254, sY255, sY1191, sY1192 and sY1291 markesrs In addition, our study was used AMEL, TAF9B, DAZ, CDY, SRY markers to detect some kinds of Y-chromosomal abnormalities In conclusion, the thesis were used 14 genetic markers All primer sequences of genetic markers were tested on the NCBI (Version GRCh38.p7) to detect the location of the PCR fragments Figure 2.2 was presented the amplified PCR fragments of 13 genetic markers on Y chromosome, excluded TAF9B marker (amplifying PCR fragments on chromosome and X chromosome) Fig 2.2 PCR fragments were amplified by the genetic markers on Y chromosome 2.3.3 Y chromosome abnormalities in male infertility The genetic causes accounted for 30% includes single gene disorders, cell genetic abnormalities and AZF mutations on Y chromosome (Aston and Conrad, 2013) 2.4 Some assays using to detect genetic causes in male infertility Currently, not only cell genetic engineering but also many molecular assays have been used to detect abnormalities on Y chromosome Fluorescence in situ hybridization (FISH), Multiplex-PCR, Realtime PCR, Multiplex ligation dependent probe amplification (MLPA) and Quantitative flourescence (QF-PCR) are molecular assays 2.5 Scientific situation of genetic causes in men 2.5.1 In the world Many studies have shown that Klinefelter syndrome and AZF deletions are two of the most common genetic causes in infertility male with sperm concentration < million/mL (Cavkaytar et al., 2012; Choi et al., 2013; Zhang et al., 2013b) The percentage of AZF deletions were 438% (Mafra et al., 2011; Cavkaytar et al., 2012; Fu et al., 2012; Choi et al., 2013; Ambulkar et al., 2013; Nasasse et al., 2015) 2.5.2 In Vietnam The ratio of AZF deletions accounted for 5-12.8%; in which AZFc deletion was the most prevalent (Nguyen Minh Ha, 2011; Nguyen Thi Viet Ha, 2012; Nguyen Duc Nhu, 2015) Chapter STUDY METHODS 3.1 The main workups in research Fig.3.1 The primary steps in research 3.2 Study subjects Men seeking medical care for their fertility had sperm concentration ≤ million/mL at Infertility Department – Can Tho Obstetrics and Gynecology Hospital, according to WHO guidelines (2010) Exclusion criteria: men who did not get semen by masturbation and consent to participation in the study Additionally, men with ligation and vasectomy, taking sample at home and dropping sperm were excluded 3.3 Sample size - To apply the sample size estimation for a proportion n  Z12 / p.(1  p) d2 Where: n: sample size Z1-/2 = 1,96 with confidence interval is 95% p: the percentage of men seeking medical care for their fertility having sperm concentration ≤ million/mL had AZF deletions on Y chromosome Basing on the study of Nguyen Minh Ha (2011), the p value was 12.8% (p = 0.128) d: desired precision, d = 0,037 n = 313 patients Conclusion, number of sample size was 313 men seeking medical care for their fertility having sperm concentration ≤ million/mL In fact, 322 subjects were studied 3.4 A sampling method The patients were chosen conveniently without probability 3.5 Time and places 3.5.1 Study time From November 2014 to March 2017 3.5.2 Study places - Getting, testing and analysing semen samples were processed at Infertility Department – Can Tho Obstetrics and Gynecology Hospital - Testing for FSH, LH and testosterone levels; extracting DNA, doing QF-PCR assay and sequencing were performed at Genetics Analysis Department – Can Tho Obstetrics and Gynecology Hospital - Sending one blood sample and DNA samples to test the QFPCR results were done by the ISOLABO lab at 101/26 Nguyen Chi Thanh street, Area, District, Ho Chi Minh city 3.6 Equipments, instruments and chemicals - The research was used the chemicals and equipments to test for semens, endocrines, genetics (QF-PCR assay) and sequences - 14 primers (reverse primers) and fluorescent primers (forward primers) of genetic markers were shown in Table 3.1A The QF-PCR result of men with complete AZFc deletion (also known as b2/b4 deletion) was shown the absent peak of sY254, sY255, sY1191, sY1192 and sY1291 fragments; DAZ/DAZL ratio of 0:2, CDY2/CDY1 ratio of 2:0 and the other peaks were similar to the QF-PCR result of a man without Y-chromosomal abnormality The QF-PCR result of men with complete AZFbc deletion was shown the absent peak of sY127, sY134, sY254, sY255, sY1191, sY1192 CDY2/CDY1 and sY1291 fragments; DAZ/DAZL ratio of 0:2 and the other peaks were similar to the QF-PCR result of a man without Ychromosomal abnormality The QF-PCR result of men with DAZ duplication was shown DAZ/DAZL ratio of 6:2 or 8:2; the other peaks were similar to the QF-PCR result of a man without Y-chromosomal abnormality The QF-PCR result of men with not only Klinefelter syndrome but also gr/gr deletion was shown AMELX/AMELY ratio of 2:1; TAF9B3/TAF9BX ratio of 2:2 and the other peaks were similar to the QFPCR result of a man with gr/gr deletion The QF-PCR result of men with not only Klinefelter syndrome but also gr/gr deletion was shown AMELX/AMELY ratio of 2:1; TAF9B3/TAF9BX ratio of 2:2 and the other peaks were similar to the QFPCR result of a man with DAZ duplication The QF-PCR result of men with not only Klinefelter syndrome but also gr/gr deletion was shown AMELX/AMELY ratio of 2:1; TAF9B3/TAF9BX ratio of 2:2 and the other peaks were similar to the QFPCR result of a man with DAZ deletion 3.3.3 To describe characteristics of semenogram, endocrines and some factors related to Y-chromosomal abnormalities in men with sperm concentration ≤ million/mL - To counsel and instruct for patients the way of collecting semen - To evaluate of seminal parameters according to WHO guidelines (2010) - To interview the patients based on the questionnaire form - To examine clinic: BMI, genital abnormalities - To test endocrine of the pituitary-testicular axis - To analyze the results 13 Chapter RESULTS AND DISCUSSIONS 4.1 To set up and to test the procedure of QF-PCR assay using for detecting some kinds of Y-chromosomal abnormalities in men with sperm concentration ≤ million/mL 4.1.1 To set up the procedure of QF-PCR assay 4.1.1.1 To examine the conditions of QF-PCR assay Experimentally, the thesis had been optimized the annealing temperature in QF-PCR assay and was divided into sets Set consisted of genetic markers (sY254, sY255, sY1191, sY1192) and set had genetic markers (AMEL, CDY, DAZ, TAF9B, SRY) with annealing temperature of 560C Set consisted of genetic markers (sY84, sY86, sY127, sY1291, sY134) with annealing temperature of 580C Besides annealing temperature, final concentration and total volume of QF-PCR assay were optimized as pmol and 25 μl, respectively In addition, multiplex PCR 5X mastermix was also optimized in QF-PCR assay with a final concentration of 1X The cycling conditions of set and set were the following: 940C - minutes, {[940C - 30 seconds, 560C - minute, 680C - minute 30 seconds], 30 cycles}, 680C - 10 minutes, 40C - ∞ The cycling condition of QF-PCR in set had done similarly to set and set 2, except that annealing temperature was 580C Compared with the other studies, the differences in conditions, annealing temperature, and thermal cycle of the procedure of QF-PCR assay were probably understood due to the different types of chemicals and experimental conditions 4.1.1.2 To investigate the conditions of capillary electropherosis of fluorescent PCR fragments The thesis was used the procedure of the following capillary electrophoresis: mix sets of fluorescent PCR fragments into one tube Each tube contained μL Hidiformamide and standard marker + 0.5μL fluorescent PCR fragments which were diluted 100 times and placed in ABI 3500 Genetic Analyzer according to the Devyser or Elucigen programs The results were analyzed by Genemarker software V2.6.3 4.1.1.3 Results of QF-PCR With the optimized QF-PCR procedure, the results were shown by peaks of the fluorescent PCR fragments which were amplified by genetic 14 markers One primer in each genetic marker was labeled with FAM or HEX or NED fluorescent dye, which allowed the determination of the length of the different STR and STS alleles and for quantification of the relative AMELX/AMELY, TAF9BX/TAF9B3, DAZ/DAZL and CDY2/CDY1 ratios The primers of genetic markers was labeled with FAM fluorescent dye included: AMEL, sY255, TAF9B (T3), sY127, DAZ, sY254 and SRY Fluorescent PCR fragments of this group were demonstrated by blue peaks (Fig 4.1) Fig 4.1 Electrophoretogram of the QF-PCR analysis amplified by markers was labeled with FAM fluorescent dye With the optimal procedure, the normal results in a male DNA samples are presented by an AMELX/AMELY ratio of 1, due to the presence of one X and one Y chromosome, DAZ/DAZL ratio of 2, due to the presence of DAZ genes in the AZFc region of Y chromosome and two DAZL genes, one on each chromosome The normal TAF9BX/TAF9B3 ratio is 0.5 due to the presence of two copies of the gene on the chromosomes and one copy on the chromosome X in males The four STR markers on chromosome X, as well as the one in the AZFb region on chromosome Y generate one PCR fragment due to the presence of one allele of each of the investigated markers The non-polymorphic markers on Y chromosome: SRY, sY127 and sY254 gave PCR fragments of 465 bp, 192 bp and 380 bp in males without AZF rearrangements (Fig 4.1) The primers of genetic markers was labeled with VIC fluorescent dye included: CDY, sY86, sY1191 and sY1291 PCR fragments of this group were demonstrated by green peaks (Fig 4.2) 15 Fig 4.2 Electrophoretogram of the QF-PCR analysis amplified by markers was labeled with VIC fluorescent dye The normal results in a male DNA samples are presented by CDY2/CDY1 ratio of 1, due to the presence of CDY2 genes in the AZFb and CDY1 genes in the AZFc region of Y chromosome The nonplymorphic markers on Y chromosome: sY86 and sY1191 gave PCR fragments of 316 bp and 385 bp in males without AZF rearrangements (Fig 4.2) SY1291 fragments were found to be polymorphic in size: 507bp, 512 bp, 523 bp and 527 bp in patients (Fig 4.3) This meant there was a length polymorphism of sY1291 fragments This finding was in concordance with the results described by Lin et al (2006) and Evguenia (2016) To confirm the length polymorphism of sY 1291 fragments, our research was used sequencing assay and was presented more detail in 4.1.2 Fig 4.3 The length polymorphism of sY1291 fragments The primers of genetic markers was labeled with NED fluorescent dye included: sY1192, sY134 and sY84 PCR fragments of this group were demonstrated by black peaks (Fig 4.4) 16 Fig 4.4 Electrophoretogram of the QF-PCR analysis amplified by markers was labeled with NED fluorescent dye The non-plymorphic markers on Y chromosome: sY1192, sY134 and sY84 gave PCR fragments of 255 bp, 302 bp and 328 bp in males without AZF rearrangements (Fig 4.4) Moreover, there were a lot of extra peaks in Figures 4.1, 4.2 and 4.4 These extra peaks may be explained due to non-specific fluorescent PCR fragments This was caused by some primers of genetic markers can amplify the fragments that had > 600 bp in length, so the sizes of PCR fragments was shown to be shorter products (NCBI, GRCh38 p7) 4.1.2 Testing for the procedure of QF-PCR assay 4.1.2.1 Comparison of the research results to data on NCBI (GRCh38.p7) and some studies in the world The QF-PCR results were indicated that the sizes of the fluorescent PCR fragments were divided into three groups Group was amplified by sY84, sY254, sY1191 and sY1192 had fluorescent PCR fragments as equal as reference PCR fragments Group was amplified by SRY, sY86, sY127, sY255, CDY, AMEL, TAF9B and DAZ had the size of fluorescent PCR fragments less 2-5 bp than the reference PCR fragments Group had a longer fluorescent PCR fragment than the reference PCR was amplified by sY134 Moreover, our study was indicated that sY1291 has a fluorescent PCR fragments which were shorter than 4-20 nucleotides (507 bp, 512 bp, 523 bp) or equal to a reference PCR fragment (527 bp) Comparing with the study of Plaseska et al (2011), the results were consistent in size of fluorescent PCR fragments in groups and In group 2, the authors noted that the size of SRY fluorescence PCR fragment was 243 bp, shorter than nucleotides compared to the expected gene size of 248 bp The difference was due to the use of other primitive sequences The results were consistent with studies by Plaseska et al (2011) and Papoulidis et al (2012) is 103 bp and 109 bp Another study by Fodor et al (2007) and Majumder et al (2015) also noted the fluorescence range of PCR fragments of 104 bp and 110 bp 17 Thus, the fluorescence PCR fragments’ size may be 2-3 bp shorter than the reference PCR fragments’ size due to different QF-PCR reaction conditions This is totally suitable with the recommendation offering in Devyser kit for detecting AZF deletions Thus, the peak positions of fluorescent PCR fragments may be equal, shorter or longer than the reference PCR fragments The results of the thesis were shown that most of PCR fragments were 2-5 bp shorter or longer than the reference PCR fragments; except fluorescent PCR fragments amplified by sY1291 They were presented the length polymorphism in length 4.1.2.2 Comparison of the research results to the positive controls The study was carried out the positive controls (the ADN samples with AZFc deletion and Klinefelter syndrome) In our study, QF-PCR results were similar to the results of positive controls Thus, the optimized the procedure of QF-PCR assay was given similar results to other assays 4.1.2.3 Sending a blood sample and DNA samples of study subjects for confirming the QF-PCR results The confirming results of a blood sample also had shown that patient had an extra X chromosome (totally, 47 chromosomes) by culturing of peripheral blood lymphocyte, similar to the QF-PCR result In addition, the thesis was randomly selected three samples of DNA (one healthy DNA sample, one DNA sample having complete AZFbc deletion and one DNA sample having completed AZFc deletion) for submitting to the ISOLABO laboratory By using Devyser kit, they also gave the same results with our QF-PCR results 4.1.2.4 Sequencing to determine the length polymorphism of fluorescent PCR fragments amplified by sY1291 marker Trying 10 times to sequence PCR fragments amplified by sY1291 marker in one month, the results were shown the identities between samples DNA sequences and reference DNA sequences were 400 bp (77% of identities) (Figs 4.5A, B) 18 Fig 4.5A Identities between reference DNA sequence (REF) and sample DNA sequence (SA100) with a 527-bp-long PCR fragment Fig 4.5B Identities between reference DNA sequence (REF) and sample DNA sequence (SA207) with a 507-bp-long PCR fragment The percentage of correlation of nucleotide sequence was 77% because the trace data became mixed after a long mononucleotide T were recorded on both forward and reverse DNA fragments Mononucleotide runs are well-known hot spots for frameshift mutations, with DNA polymerase slippage typically resulting in loss or gain of one or a few nucleotides (Jinks-Robertson, 2002) Thus, the sequence results were seen the length polymorphism of sY1291 fragments The lengths of the fluorescent PCR fragments were 19 507 bp, 512 bp, 523 bp and 527 bp It was due to the length of mononucleotide T repeats Based on above evidences, the 14-plex QF-PCR assay was used for detecting some kinds of Y-chromosomal abnormalities in patients had high accuracy and reliability 4.2 To determine the percentage of some kinds of Y-chromosomal abnormalities in men with sperm concentration ≤ million/mL by the developed and tested procedure of QF-PCR assay0939668365 The study results of Y-chromosomal abnormalities in men with sperm concentration ≤ million/mL were 35.1% (Fig 4.6) were much higher than the other researches (4-24%) Fig 4.6 The percentage of Y-chromosomal abnormalities in patients There were three kinds of Y-chromosomal abnormalities They were AZF deletions (83.18%), DAZ duplication (13.27%) and Klinefelter syndrome with Y-chromosomal structural abnormalities 3.55% It was not similar to our data, most researches were found Klinefelter syndrome and AZF deletions The differences were probably the study methods and the number of genetic markers Four kinds of AZF deletions were found in our study Most of the AZF deletions were partial AZFc deletion (80.85%), complete AZFc deletion (9.57%), complete AZFbc deletion (6.38%) and partial AZFb deletion (3.2%) It was similar to published data in many studies (range 45-98%) (Choi et al., 2013; Zhang et al., 2013b; Fadlalla et al., 2014; Gallego et al., 2014; Bichile et al., 2016; Zhu et al., 2016; Asadi et al., 2017) Comparison with other studies also shown the same result with the most prevalent of AZFc deletion (Nguyen Minh Ha, 2011, Nguyen Thi 20 Viet Ha, 2012, Nguyen Duc Nhu, 2015) Data were published AZFc and AZFb deletion accounted for 11.4% and 1.43%, respectively in 70 men seeking medical care for their fertility at Tu Du hospital in 2011-2012 (Nguyen Minh Ha, 2011) Similarly, the other study was conducted by Nguyen Thi Viet Ha (2012) was also showed that AZFc deletion was the highest rate (4.4%), AZFa as 2.4%, AZFb and AZFabc as 0.4% equally In 2015, Nguyen Duc Nhu's study was shown that AZFc and AZFcd deletion accounted for 26.53% (13/49 cases), AZFbcd as 8/49 cases (16.33%), AZFd as 3/49 cases (6.12%) and AZFb as 2/49 cases (4.08%) In partial AZFc deletions, there were seven kinds of deletion: gr/gr, b2/b3, two DAZ genes, one CDY1 gene, two DAZ genes - one CDY1 gene, sY1191-s11119 and sY1291 SY1291, sY1191-1192, two DAZ genes, two DAZ genes - one CDY1 gene were the de novo mutations Our results were differed from many studies in the world because most studies rely solely on the absence of PCR fragments amplified by the primers of sY1291, sY1191, sY1192 markers to determine the gr/gr, b2/b3 and b1/b3 deletion Acccording to literature, it was suggested that gr/gr, b2/b3 and b1/b3 deletion were refered to two DAZ deletion and one CDY deletion In all types of partial AZFc deletion, the most prevalent deletion was gr/gr (31.6%), DAZ deletions (21.1%), sY1191-sY1192 deletion (14.5%), sY1291 deletion (10.5%), DAZ – CDY1 deletions (9.2%), b2/b3 deletion (7.9%) and CDY1 deletion (5.2%) It was similar to published data of Rozen et al., 2012; Sen et al., 2015; Olesen et al., 2017) After AZF deletions, DAZ duplication are the second most frequent Y-chromosomal abnormality The normal QF-PCR results in healthy men were presented by a DAZ/DAZL ratio of 4:2 However, our data had been shown peak ratio of DAZ/DAZL as 6:2 (Fig 4.7) or 8:2 (Fig 4.8) in 15/113 study subjects (13.27%) Fig 4.7 DAZ/DAZL ratio of 6:2 at 210 bp and 214 bp in sample A236 21 Fig 4.8 DAZ/DAZL ratio of 8:2 at 210 bp and 214 bp in sample A174 Many studies have been published of DAZ duplication such as Lu et al (2013); Saito et al (2015); Vaszko et al (2016) and Alimardanian et al (2016) In addition, some patients had two X chromosomes were presented on the QF-PCR results with AMELX/AMELY ratio of 1: and TAF9B3/TAF9BX ratio of 2: The QF-PCR results of AMELX/AMELY ratio and TAF9B3/TAF9BX ratio in men with one and two X chromosomes were shown on Fig 4.9A and 4.9B Fig 4.9A A patient with X chromosome was shown AMELX/AMELY and TAF9B3/TAF9BX ratio of 1:1 and 2:1, respectively in sample A95 Fig 4.9B A patient with X chromosomes was shown AMELX/AMELY and TAF9B3/TAF9BX ratio of 2:1 and 2:2, respectively in sample A97 Our data were seen cases not only had X chromosomes but also had Y-chromosomal abnormalities Y-chromosomal abnormalities in these cases were case of partial AZFc (gr/gr), cases of DAZ duplication and case of deletion of two DAZ The results were consistent with the previous studies conducting by Mitra et al (2006), Hadjkacem-Loukil et al (2009), Ceylan et al (2010) and Li et al (2015) 22 4.3 To describe characteristics of semenogram, endocrines and some factors related to Y-chromosomal abnormalities in men with sperm concentration ≤ million/mL 4.3.1 General characteristics and some risk factors referring to Ychromosomal abnormalities 4.3.1.1 Age characteristics and its relationship to Y-chromosomal abnormalities Mean age of the subjects was 31.62±5.53 The result was also similar to the published data in many researches The 30-34-year-old group accounted for the highest ratio (31.1%) There was no significant difference in ages with Y-chromosomal abnormalities in patients 4.3.1.2 Occupation characteristics and its relationship to Ychromosomal abnormalities Most of the infertile men in our study were farmers (30.1%), differed from the published data in other studies (civil servants were the most prevalent) Men who contacted to chemicals or pesticides had a 1.64fold increase in risk of Y-chromosomal abnormalities compared to noncontact groups (p > 0.05) 4.3.1.3 Characteristics of primary and secondary infertility We observed that the incidence of primary infertility was relatively high compared with previous studies It was 91.3% while this ratio was 61.5% (Nguyen Quoc Tuan and Nguyen Ngoc Han, 2011), 70.91% (Vo Xuan Dao, 2014) and 69.5% (Masoumi, 2015) The difference probably was explained due to our subjects had lower sperm concentration than the others 4.3.1.4 Characteristics of the duration of infertility The mean duration of infertility of the subjects was 3.74±3.25 years, which was similar to the studies done by Vu Minh Ngoc (2009) and Pham Chi Kong et al (2011) Most of the infertile men in our study had duration of infertility was 1-2 years (48.1%), coincided with the results of Radwan et al (2016) and Nguyen Xuan Bai (2010) 4.3.1.5 Characteristics of the habit and time of using cell phones There were 90.7% patients put a cell phone in their pockets during the day and took a cell phone on the bed when sleeping at night accounted for 83.9% Study subjects had a mean time of cell phone usage was 8.76±4.28 years Most of the infertile men in our study had time of cell phone usage 23 was 6-10 years (44.7%) These findings are in accordance with Radwan et al (2016) 4.3.1.6 Characteristics of the mumps history In this study, 42 out of 322 cases (13.0%) had a history of mumps This result was consistent with Radwan et al (2016) 4.3.1.7 Characteristics of BMI and the relationship of Ychromosomal abnormalities Normal weight men were the highest rate (52.8%) and the lowest rate was 3.1% of underweight men Similarly, Wang et al (2017) also had shown the same results This study did not find any relationship between BMI and Y-chromosomal abnormalities 4.3.1.8 Characteristics of testicular volume The research shown that the average testicular volume of the subjects was 9.972.22mL, lower than the study of Nguyen Duc Nhu (2015) (18.29±5.48mL) The study subjects had the largest proportion of 6-10mL testicular volume (67.4%) It was different from Nguyen Duc Nhu (2015) This was probably due to the different sample sizes and the study place 4.3.1.9 Characteristics of scrotum and the association between varicocele and Y-chromosomal abnormalities The frequency of scrotum abnormalities was 10.8% in patientsm was similar to Nguyen Duc Nhu (2015) Most of the infertile men in our study had both the different left and right testis size and varicocele were 37.1% The results of this study were suitable with Pham Chi Kong et al (2011), Olesen et al (2016) and Suganya et al (2016) The present study was shown that there was not any association between varicocele and the Y- chromosomal abnormalites 4.3.2 Characteristics of semenogram 4.3.2.1 Characteristics of semen a Seminal pH The mean pH was 7.51±0.46, which was similar to the results of Nguyen Xuan Bai (2010), Tran Thi Thu Trang et al (2014) and Tran Duc Long (2016) b Seminal volume In total, 78.9% of the 322 cases had the normal seminal volume (≥ 1.5 mL) The figure were similar to the results of Tran Thi Hoa et al (2008), Khan et al (2012), Nguyen Duc Nhu (2015) The mean seminal 24 volume was 2.85±1.85 mL, which was similar to the results of Nguyen Xuan Bai (2010); Le Hoang Anh et al (2012) and Ugwa (2015) 4.3.2.2 Characteristics of sperm parameters a Sperm concentration In totally, 71.1% of patients had severe oligozoospermia, leaving 28.9% diagnosed as azoospermia in the spermiogram The rate of azoospermia males was much lower than others researches This difference may be explained due to men with fertile troubles coming to private clinics for their treatment Moreover, azoospermia males went to Ho Chi Minh hospitals for examination and treatment Besides that, the average sperm concentration was 2.52±1.75 million/mL This result was higher than Song et al., (2010), but lower than Milardi et al (2012) These differences were probably due to the samples size and the study population b The other sperm parameters Total sperm count, progressive motility and non-progressive motility in the spermiogram were almost abnormal Particularly, there was only a case having normal total sperm count (0.4%) and cases having progressive motility 4.3.3 Characteristics of endocrine in pituitary-gonadal axis and association between Y-chromosomal abnormalities with hormonal levels 4.3.3.1 Characteristics of FSH level and association between Ychromosomal abnormalities and this hormonal level The group with normal FSH level was the highest (68.0%), which was similar to the study by Hoang Thi Thu Huong et al (2008) In addition, the results shown that the average FSH level was 10.55±10.64 mIU/mL, lower than the research of Evangelini et al (2016) Men with Y-chromosomal abnormalities had 1.43 times higher of risk of abnormality in FSH level than non-abnormal men However, it was not statistically significant with p> 0.05 Compared with Choi et al (2013), it was presented a similar result 4.3.3.2 Characteristics of LH level and the association between Ychromosomal abnormalities and this hormonal level The result shown that patients with normal LH level was the highest (92.9%), was similar to Hoang Thi Thu Huong et al (2008) In addition, our research shown that the mean LH level was 8.59±6.97 mIU/mL, consistented with studies by Evangelini et al (2016) 25 Men with Y-chromosomal abnormalities had 1.2 fold higher of risk of LH level However, it was not statistically significance (p > 0.05) 4.3.3.3 Characteristics of testosterone level and the association between Y-chromosomal abnormalities and this hormonal level Normal testosterone level accounted for 83.5%, similar to Hoang Thi Thu Huong et al (2008); but higher than Nguyen Xuan Bai (2010) and Jimoh et al (2012) This might be due to different sample sizes The average testosterone level was 4.05±1.88 ng/mL Men with Ychromosomal abnormalities had 1.39 folder times the risk of abnormal testosterone level The difference was not statistically significance (p> 0.05) 4.3.3.4 Characteristics of the results of endocrinal classification There were 41.3% (133/322 cases) had endocrinal disorders, which were similar to the result of Emokpa et al (2007) The research recorded that the normal endocrinal males accounted for 58.69% It was similar to Ishikawa et al (2004) In 133 cases of endocrine disruptors, primary germ cell damage was the most prevalent (44.3%) The result was similar to Pham Chi Kong (2012) Chapter CONCLUSIONS AND RECOMMENDATIONS 5.1 Conclusions In conclusion, we have developed and optimized the reliable 14plex QF-PCR method The percentage of men seeking medical care for their fertility had Y-chromosomal abnormalities was 35.1% (113/322 cases) AZF deletions, DAZ duplication and the Klinefelter syndrome with Y-chromosomal abnormalities were found Partial AZFc deletion was the most popular, accounting for 80.9% (76/94 cases) Our data found out four kinds of de novo partial AZFc deletions as: sY1191-sY1192 deletion, sY1291 deletion, two DAZ – one CDY1 deletion and one CDY1 deletion Characteristics of semenogram weren shown that the mean pH and semen volume were 7.51±0.46 and 2.85±1.85 mL, respectively Azoospermic men were 28.9% Moreover, mean sperm concentration was 2.52±1.75 million/mL 26 The normal FSH, LH and testosterone levels accounted for 68%, 92.85% and 83.54%, respectively Besides, the percentage of patients had endocrine disruptors was 41.3% (133/322 cases), in which primary germ cell damage the most common, accounted for 44.3% (59/133 cases) There was not significantly different between Y-chromosomal abnormalities and some general characteristics, FSH, LH and testosteron levels 5.2 Recommendations Some kinds of Y-chromosomal abnormalities in patients can be detected by the developed and optimized 14-plex QF-PCR However, some genetic markers should be added to the above QF-PCR assay for detecting the numerous of sex chromosomes To identify gr/gr deletion, we should base on not only sY1291 marker but also DAZ and CDY markers The endocrine disruptors was high prevalent among males being examed infertility Therefore, doctors should advice the patients to check whether their fertile troubles are caused by endrocine abnormalities or not Basing on the exact reason, it is easier for doctors to treat for them Sequencing of DNA fragments having single nucleotide repeat need be optimized to get the better results 27 ... Genetic markers sY84-F sY84-R sY86-F sY86-R sY127-F sY127-R sY134-F sY134-R sY254-F sY254-R sY255-F sY255-R sY1191-F sY1191-R sY1192-F sY1192-R sY1291-F sY1291-R SRY-F SRY-R CDY-F CDY-R AMEL-F AMEL-R... used sY1189, sY1191, sY1192 and sY1291 markers to detect partial AZFc deletions Based on these evidents, the study was used sY84, sY86, sY127, sY134, sY254, sY255, sY1191, sY1192 and sY1291 markesrs... study was setted up and optimized a successful procedure of QF-PCR assay with 14 genetic markers (AMEL, SRY, sY1191, sY1192, TAF9B, DAZ, CDY, sY84, sY86, sY127, sY134, sY254, sY255 and sY1291)