A infertilidade afeta aproximadamente 15% dos casais, sendo atualmente reconhecido o envolvimento de fatores masculinos em metade dos casos. Alterações nas análises seminais são detectadas na maioria dos homens inférteis e a mais frequente é a baixa concentração de espermatozoides no ejaculado, conhecida como oligozoospermia. Vários estudos mostram uma forte relação entre fatores genéticos e a infertilidade, incluindo alterações cromossômicas e microdeleções do cromossomo Y, porém as causas da oligozoospermia ainda permanecem obscuras. O desenvolvimento de novas tecnologias de investigação vem possibilitando a detecção de alterações a nível genômico, como mutações e variações no número de cópias (CNVs). O presente trabalho teve por objetivo a caracterização genômica de homens com oligozoospermia sem causa definida, visando estabelecer correlação entre alterações no número de cópias e perdas de heterozigosidade (LOHs) e o fenótipo de infertilidade. Foram selecionados 18 pacientes após rigorosa avaliação clínica e investigação do histórico reprodutivo, sendo excluídos pacientes portadores de alterações cromossômicas e portadores de microdeleções do cromossomo Y. Seis homens comprovadamente férteis foram selecionados para o grupo controle. A investigação genômica de ambos os grupos, amostral e controle, foi realizada pela técnica de hibridação genômica comparativa em microarranjos (aCGH) utilizando a plataforma de resolução 180K (Agilent®,US), analisada pelo software Nexus 8.0. Foram detectadas alterações possivelmente patogênicas no cromossomo Y, no cromossomo X e em autossomos. Um ganho na região de AZFc envolvendo apenas os genes DAZ1 e DAZ4 foi detectado em nove pacientes e em quatro controles, sendo classificado como alteração benigna. Porém, alterações na região de AZFc possivelmente relacionadas ao fenótipo de oligozoospermia foram detectadas em três pacientes e incluíram extensas duplicações e deleções envolvendo, entre outros genes, as quatro cópias do gene DAZ. Após comparação de regiões selecionadas com a literatura e com diferentes bancos de dados genéticos, sugerimos que os genes PLEC, SPATC1, COL1A1, MOV10L1, SYCE3 e ODF3B possam estar associados a alterações na produção espermática. Adicionalmente, entre os doze miRNAs presentes em regiões de LOH possivelmente relacionadas ao fenótipo de infertilidade, dez têm como alvo genes com funções relacionadas à espermatogênese e reprodução humana. Estudos adicionais a nível de expressão e sequenciamento gênico são necessários para confirmar a correlação entre o genótipo e o fenótipo de oligozoospermia. / Infertility affects about 15% of the couples, and it is currently recognized, that male factors are involved in about 50% of cases. Changes in seminal parameters are detected in most infertile men and the most common alteration, known as oligozoospermia, is a low concentration of sperm in the ejaculate. Several studies show a strong relationship between genetic factors and infertility, including chromosomal abnormalities and microdeletions of Y chromosome, however, the causes of oligozoospermia remain unclear. The development of new research technologies has allowed the detection of changes at genomic levels, such as mutations and copy number variations (CNVs). This study aimed to perform a genomic characterization of patients with idiopathic oligozoospermia to determine whether there is a correlation between changes of copy number and losses of heterozygosity (LOHs) in relation to the phenotype of infertility. Eighteen patients were selected for the cases after rigorous clinical examination and investigation of their reproductive history. Patients with chromosomal abnormalities or microdeletions of the Y chromosome were excluded. Six proven fertile men comprised the control group. Genomic investigation of both groups was performed by microarray comparative genomic hybridization (aCGH) using 4X180K platform (Agilent, US) analysed by Nexus 8.0 software. Potential pathogenic changes were detected on Y chromosome, as well as on the X and autosome chromosomes. A gain in AZFc region involving only DAZ1 and DAZ4 genes was detected in nine patients and four controls, and was considered as benign. However, changes in AZFc region, that could be related to the oligozoospermia phenotype were detected in three patients. These changes included extensive duplications and deletions involving the four copies of the DAZ gene together with copy number changes affecting other genes. After comparing the selected regions with the literature and with different databases, we suggest that changes such as LOH affecting PLEC, SPATC1, COL1A1, MOV10L1, SYCE3 and ODF3B genes may influence sperm production. Our analysis indicates that, ten out of the twelve miRNAs present in LOH regions could be involved in the infertility phenotype and could have target genes with functions related to spermatogenesis and human reproduction. Additional studies involving gene sequencing and expression analysis are needed to confirm the the correlation between the genotype and oligozoospermia phenotype.

A infertilidade afeta aproximadamente 15% dos casais, sendo atualmente reconhecido o envolvimento de fatores masculinos em metade dos casos. Alterações nas análises seminais são detectadas na maioria dos homens inférteis e a mais frequente é a baixa concentração de espermatozoides no ejaculado, conhecida como oligozoospermia. Vários estudos mostram uma forte relação entre fatores genéticos e a infertilidade, incluindo alterações cromossômicas e microdeleções do cromossomo Y, porém as causas da oligozoospermia ainda permanecem obscuras. O desenvolvimento de novas tecnologias de investigação vem possibilitando a detecção de alterações a nível genômico, como mutações e variações no número de cópias (CNVs). O presente trabalho teve por objetivo a caracterização genômica de homens com oligozoospermia sem causa definida, visando estabelecer correlação entre alterações no número de cópias e perdas de heterozigosidade (LOHs) e o fenótipo de infertilidade. Foram selecionados 18 pacientes após rigorosa avaliação clínica e investigação do histórico reprodutivo, sendo excluídos pacientes portadores de alterações cromossômicas e portadores de microdeleções do cromossomo Y. Seis homens comprovadamente férteis foram selecionados para o grupo controle. A investigação genômica de ambos os grupos, amostral e controle, foi realizada pela técnica de hibridação genômica comparativa em microarranjos (aCGH) utilizando a plataforma de resolução 180K (Agilent®,US), analisada pelo software Nexus 8.0. Foram detectadas alterações possivelmente patogênicas no cromossomo Y, no cromossomo X e em autossomos. Um ganho na região de AZFc envolvendo apenas os genes DAZ1 e DAZ4 foi detectado em nove pacientes e em quatro controles, sendo classificado como alteração benigna. Porém, alterações na região de AZFc possivelmente relacionadas ao fenótipo de oligozoospermia foram detectadas em três pacientes e incluíram extensas duplicações e deleções envolvendo, entre outros genes, as quatro cópias do gene DAZ. Após comparação de regiões selecionadas com a literatura e com diferentes bancos de dados genéticos, sugerimos que os genes PLEC, SPATC1, COL1A1, MOV10L1, SYCE3 e ODF3B possam estar associados a alterações na produção espermática. Adicionalmente, entre os doze miRNAs presentes em regiões de LOH possivelmente relacionadas ao fenótipo de infertilidade, dez têm como alvo genes com funções relacionadas à espermatogênese e reprodução humana. Estudos adicionais a nível de expressão e sequenciamento gênico são necessários para confirmar a correlação entre o genótipo e o fenótipo de oligozoospermia. / Infertility affects about 15% of the couples, and it is currently recognized, that male factors are involved in about 50% of cases. Changes in seminal parameters are detected in most infertile men and the most common alteration, known as oligozoospermia, is a low concentration of sperm in the ejaculate. Several studies show a strong relationship between genetic factors and infertility, including chromosomal abnormalities and microdeletions of Y chromosome, however, the causes of oligozoospermia remain unclear. The development of new research technologies has allowed the detection of changes at genomic levels, such as mutations and copy number variations (CNVs). This study aimed to perform a genomic characterization of patients with idiopathic oligozoospermia to determine whether there is a correlation between changes of copy number and losses of heterozygosity (LOHs) in relation to the phenotype of infertility. Eighteen patients were selected for the cases after rigorous clinical examination and investigation of their reproductive history. Patients with chromosomal abnormalities or microdeletions of the Y chromosome were excluded. Six proven fertile men comprised the control group. Genomic investigation of both groups was performed by microarray comparative genomic hybridization (aCGH) using 4X180K platform (Agilent, US) analysed by Nexus 8.0 software. Potential pathogenic changes were detected on Y chromosome, as well as on the X and autosome chromosomes. A gain in AZFc region involving only DAZ1 and DAZ4 genes was detected in nine patients and four controls, and was considered as benign. However, changes in AZFc region, that could be related to the oligozoospermia phenotype were detected in three patients. These changes included extensive duplications and deletions involving the four copies of the DAZ gene together with copy number changes affecting other genes. After comparing the selected regions with the literature and with different databases, we suggest that changes such as LOH affecting PLEC, SPATC1, COL1A1, MOV10L1, SYCE3 and ODF3B genes may influence sperm production. Our analysis indicates that, ten out of the twelve miRNAs present in LOH regions could be involved in the infertility phenotype and could have target genes with functions related to spermatogenesis and human reproduction. Additional studies involving gene sequencing and expression analysis are needed to confirm the the correlation between the genotype and oligozoospermia phenotype.

L’infertilité est définie par l’OMS comme l’incapacité à concevoir un enfant dans un couple, après au moins douze mois de rapports sexuels réguliers sans protection. Elle concerne 15 % des couples qui désirent avoir un enfant, un facteur masculin est retrouvé dans 50 % des cas. Dans le cadre de cette thèse, les principaux objectifs étaient de déterminer des causes génétiques d’infertilité masculine chez des patients atteints d'oligozoospermie sévère (OS) ou d'asthénozoospermie en lien avec des anomalies morphologiques des flagelles, afin de mieux comprendre la spermatogénèse, d’améliorer la prise en charge des couple infertiles et d'évaluer les risques de transmission à leur descendance. Nous avons abordé ces problématiques par le séquençage d’exome entier d'hommes ayant un de ces phénotypes au sein de deux familles consanguines. L'ensemble des résultats de cette thèse apportent des éléments de réponses sur de nouvelles causes génétiques d’infertilité masculine liée à une anomalie quantitative ou qualitative de la spermatogénèse. De plus, nos résultats concernant EXD1 nous interrogent sur la possibilité de transmission de mutations génétiques ou modifications de marques épigénétiques à la descendance en Assistance Médicale à la Procréation pour certains patients avec une oligozoospermie. / Infertility is defined by the WHO as the inability of a couple to conceive a child after twelve months of unprotected regular sexual intercourse. It concerns 15% of couples who wish to have a child, and a male factor is found in 50% of cases. In this thesis, the main objectives were to determine genetic causes of male infertility in patients with severe oligozoospermia (SO) or asthenozoospermia related to morphological abnormalities of the flagella, in order to better understand spermatogenesis, improve the care of infertile couples and inform risk evaluation for their offspring. We addressed these objectives by sequencing the whole exome of men with one of these phenotypes within two consanguineous families.The results presented in this thesis reveal new genetic causes of male infertility related to a quantitative or a qualitative abnormality of spermatogenesis. Moreover our findings concerning EXD1 raise questions about the risk that, for some patients with oligozoospermia, medically assisted reproduction could transmit de novo genetic or epigenetic modifications to future generations.

>Magister Scientiae - MSc / Male factor disorders affect more than 30% of infertile couples. Thus, it has become important to perform a andrological consultation and a basic semen evaluation in all male partners of couples consulting for infertility. The advent and development of assisted reproductive technologies has not only improved clinical results but also enhanced our basic understanding of the physiology of sperm and sperm preparation methods. Assisted reproduction has become among the more successful therapeutic modalities for a wide variety of sperm function disorders e.g. artificial insemination and in vitro fertilization (IVF) (Acosta et al. 1989). It is clear from recent experience that patients with male infertility showing oligozoospermia, asthenozoospermia, teratozoospermia (sometimes in combination), male immunological factor (antisperm antibodies) or ejaculatory problems as well as congenital abnormalities, can be successfully treated with IVF and embryo transfer. Prerequisite pre-fertilization changes by sperm, termed "capacitation" (Austin 1952) provides sperm with the capacity to fertilize eggs. These processes are generally regarded as encompassing all pre-fertilization changes occurring in sperm up 'to, but not including, loss of the acrosome (Bedford 1970). The endpoints of capacitation are often described as the acrosomal loss as well as changes in the motion characteristics. Capacitation alters the pattern of motility exhibited by freely swimming sperm, changing from a fairly rigid flagellar beat pattern to one of extreme flexure, often associated with increased thrust (Johnson et al. 1981) , which is referred to as hyperactivated motility (Yanagimachi 1981). without the transition to hyperactivated motility, sperm are unable to penetrate the zona pellucida (Fraser 1981), and possibly unable to fertilize eggs. Hyperactivation per se is marked by increased curvature in swimming trajectories and/or increased lateral displacement of the sperm head along their path (Burkman 1984). However, the physiological role of this change in motility is not clearly understood because almost all relevant data have been obtained under in vitro conditions. The association of specific seminal characteristics (sperm concentration, percentage motile cells and percentage normal sperm morphology) with the success rate of assisted particular has been The analysis of 1984; the reproductive techniques and IVF in under great scrutiny (Mahadevan and Trounson relationships between conventional semen parameters and fertilization rates in vitro has shown that sperm motility, concentration and morphology must be considered in estimating opportunities for successful intervention, as in the case of IVF and gamete intrafallopian transfer (GIFT) (Oehninger and Hodgen 1991). A reduction in the percentage of progressive motility alone does not seem to have a significant impact on IVF results unless it is below a threshold value of 10%. The semen sample should have an acceptable sperm concentration and morphology and/or that at least 1.5 X 106 motile spermatozoa can be recovered after swim-up separation (Acosta et al. 1989).

Male infertility may be due to oligozoospermia, asthenozoospermia and teratozoospermia. Intracytoplasmic sperm injection is used to address male infertility. However, the percentage of viable embryos obtained by this technique is very low. Pronucleus formation has been identified as one of the key events in fertilisation and gamete decondensation is vital for this process to take place. Decondensation can be initiated by chemicals such as DTT that reduce the disulphide groups between the protamine proteins that keep the DNA of the gamete condensed. An increase in decondensation should translate into a higher fertilization rate and a higher yield of embryos. The research from this thesis has compared the decondensation ability via DTT in human spermatozoa and bovine spermatozoa, to study pronucleus formation in bovine zygotes and bovine embryo formation in the presence of DTT; and lastly the cytotoxic effect of DTT using somatic cells in culture has been investigated. In this study 12 semen samples for either fertile or subfertile subjects were collected, isolated and exposed to 25 mM DTT for 0, 5, 7, and 10 minutes, washed and the morphological changes associated with decondensation was evaluated by phase contrast microscopy. After 5 and 7 minutes 11 of the 12 samples underwent decondensation while after 10 minutes several samples showed a lower rate of decondensation and this was associated with and unusual hypercondensed state, CMA3 staining revealed all spermatozoa samples evaluated were mature. However, after treatment with DTT for 5, 7 and 10 minutes an increase in fluorescence was observed indicating increased protamine thiol group reduction and subsequently increased CMA3 accessibility. For some samples reduced fluorescence was observed possible due to the supercoiling of the DNA. DTT successfully induces decondensation of human spermatozoa, however does this lead to the formation of viable embryos? Due to ethical issues associated with working with human embryos all further studies were done using bovine embryos. Spermatozoa used were derived from Friesian bulls and the samples were pooled to prevent sample bias and interindividual variation. Spermatozoa were exposed to 25 mM of DTT at 5, 7, and 10 minutes as used for human spermatozoa. No decondensation was observed using the same conditions as for human spermatozoa, therefore the ‘swim up’ medium containing heparin and regularly used in IVF procedures for bovines was used, and this resulted in successful decondensation of bovine spermatozoa after 30 minutes. The effects of DTT on pronucleus formation and embryo development were evaluated in three bovine specimens. In the first group, DTT had no significant effect on the parameters measured, namely the number of oocytes that were in metaphase II, with one pronucleus, with two pronuclei, with degeneration of the nucleus and polyspermia. In the second group the percentage cleavage and embryo formation was determined on Day 1 (group 2) and 7 (group 3) respectively and statistical differences were obtained between the control and the DTT group. DTT had no significant effect on all the early parameters measured however later in development DTT had a significant adverse effect on cleavage and eventual embryo development. <p)Cleavage and embryo formation is a process of multiple mitotic divisions resulting in an increase in the number of cells that become smaller with each cell division, while somatic cells also undergo mitotic division although the cell size remains constant. Therefore the L929 cell line, a standardized system used to test toxicity, can be used to investigate the toxic effects of DTT on a dividing cell population. In this study L929 cells were expose to 25mM DTT for 30 minutes, and lysosomal membrane integrity, cell viability and number was determined immediately following exposure and after 48 hours growth. In another experiment the L929 cell line was exposed to all concentrations used in this and other studies for 5, 10 and 20 minutes. At all concentrations and exposure times DTT was found to be cytotoxic to the L929 cell line. How exactly DTT mediates this toxic effect is unknown, however due to its high solubility DTT can cross the cell membranes. The tertiary structure of proteins, enzymes and DNA is vulnerable to the reducing effects of DTT. In conclusion, although DTT induces decondensation in human and bovine spermatozoa, in the bovine model it does not lead to viable embryo formation and this has been confirmed in cell culture where DTT at all concentrations used was found to be cytotoxic. / Dissertation (MSc (Anatomy))--University of Pretoria, 2006. / Anatomy / unrestricted