Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients.

Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients.

Introduction: Intracytoplasmic sperm injection (ICSI) has been a successful assisted reproductive technique for men with severe male-factor infertility. However, ICSI requires the subjective selection of normal looking sperm, which does not preclude the transmission of genetically abnormal sperm. Correlation between abnormal sperm morphology and chromosomal abnormalities has been suggested but not been conclusive and less is known about the connection between sperm morphology and DNA integrity. Sperm morphology will be evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. To further focus this investigation on sperm morphology, men with infertility isolated to abnormal sperm morphology (isolated teratozoopsermia) are examined. Materials and Methods: Sperm from isolated teratozoopsermic men (n=10) were analysed by fluorescent in situ hybridization (FISH) and terminal dUTP nick-end labelling (TUNEL) assays to determine the level of aneuploidy and DNA fragmentation, respectively. These results were also compared to that of sperm from control men (n=9) of proven fertility and normal seminal parameters. Results: Sperm from teratozoospermic men, compared to control men, had higher rates of total chromosomal abnormality (5.90±3.74% vs. 2.35±0.87%, P=0.0128), total aneuploidy (4.90±2.82% vs. 1.99±0.65%, P=0.0087), and chromosome 13 disomy (0.77±0.50% vs. 0.20±0.14%, P=0.0046). In control samples, incidence of tapered heads associated with supernumerary chromosomal abnormalities (rs=0.9747, P=0.0167). In teratozoospermic samples, incidence of amorphous heads associated to chromosome 13 disomy and sex chromosome aneuploidy (rs=0.6391, P= 0.0466; rs=0.8049, P=0.0050, respectively). Tail abnormalities were associated with chromosomal abnormalities (bent tail-disomy 13: rs=0.7939, P=0.0061; 2-tailed-disomy 13: rs=0.8193, P=0.0037; 2-tailed-supernumerary chromosomal abnormalities: rs=0.7534, P=0.0119). Levels of DNA fragmented sperm were higher in teratozoospermic men than control men (60.28±21.40% vs. 32.40±17.20%, P=0.0121). DNA fragmentation in sperm positively correlated with the incidence of sperm with bent necks in control samples (rs=0.8571, P=0.0238) and round headed sperm in teratozoospermic samples (rs=0.6727, P=0.0390). Conclusions: Sperm of isolated teratozoospermic men have elevated rates of chromosomal abnormalities and DNA fragmentation compared to that of fertile controls. Specific abnormal sperm morphology can be correlated wiht chromosomal abnormalities and level of DNA fragmentation in sperm and this may prove useful in sperm selection for ICSI when applied to isolated teratozoospermic patients. / Medicine, Faculty of / Obstetrics and Gynaecology, Department of / Graduate

Teratospermia (ejaculation of ≥ 60% structurally abnormal spermatozoa) is prevalent among felids facing extinction risk, including the cheetah. This trait also occurs in certain domestic cat populations, providing a valuable research model. Multiple components of sperm function are disrupted in teratospermic cats, and even structurally normal spermatozoa from these ejaculates may be functionally compromised. Teratospermic ejaculates are highly sensitive to damage during cryopreservation, limiting the success of genome resource banking programs for species conservation. Although both teratospermia and cryopreservation are linked to disruptions in multiple energy-dependent sperm processes, the metabolism of these cells has not been investigated. This project explored how cellular metabolism of domestic cat and cheetah spermatozoa is influenced by species physiology, teratospermia, and sperm cryopreservation. The project scope was divided into four studies that collectively examined the two main energy-producing pathways in spermatozoa, i.e., glycolysis and oxidative phosphorylation. Each study compared three animal populations: normospermic cat, teratospermic cat, and cheetah. First, rates of glycolytic and oxidative substrate utilization were correlated to standard metrics of sperm function. Second, the influence of exogenous substrate availability and glycolytic enzyme activity was investigated. Third, mitochondrial activity and the role of oxidative metabolism were assessed. Lastly, sperm metabolic function was examined after cryopreservation and postthaw processing.enzyme activity was essential for sperm function, but, unexpectedly, the importance of this pathway appeared to be linked to glycerol rather than glucose metabolism. Sperm oxidative metabolism was severely compromised in the cheetah, and comparison with the teratospermic cat proved this defect to be species-specific. Spermatozoa from both species experienced metabolic damage during cryopreservation. Post-thaw processing recovered a metabolicallynormal sperm subpopulation in the cat, but cheetah spermatozoa remained functionally compromised. Collectively, these studies provided key insight into metabolism and cryosensitivity of felid spermatozoa and highlighted the importance of domestic animal models for wildlife research. Patterns of substrate utilization were similar in spermatozoa of the cat and cheetah, including an unexpected lack of glucose uptake. However, rates of sperm pyruvate uptake and lactate production were reduced in the teratospermic cat and cheetah compared to the normospermic cat. Lactate production predicted ejaculate quality in each study. Glycolytic

L'infertilité masculine concerne plus de 20 millions d'homme à travers le monde et représente un véritable enjeu de santé public. Bien que multifactorielle, l'infertilité masculine a une composante génétique importante qui jusqu'à présent n'a été que peu étudiée. L'objectif de mon travail a été d'initier et de poursuivre les investigations génétiques sur trois phénotypes de tératozoospermie: les spermatozoïdes macrocéphales, la globozoospermie et les anomalies morphologiques multiples des flagelles (AMMF).Pour le premier phénotype, nous avons étudié 87 patients, dont 83 cas-index, présentant un phénotype de macrozoospermie. Nous avons trouvé la mutation c.144delC dans le gène AURKC chez 82% des patients (68/83) confirmant qu'il s'agit de l'évènement génétique prépondérant pour ce phénotype. Une nouvelle mutation récurrente, p.Y248*, entrainant la dégradation totale du transcrit anormal par nonsense-mediated mRNA decay, a été retrouvée chez 10 patients non‐apparentés. L'identification de deux mutations ancestrales dans AURKC maintenues au cours de l'évolution malgré leur effet délétère sur la reproduction chez l'homme homozygote, ouvre la question d'un potentiel avantage sélectif procuré par l'haplo-insuffisance d'AURKC.Pour le second phénotype, nous avons analysé une cohorte de 34 patients globozoospermiques par séquençage et MLPA (multiplex ligation-dependent probe amplification). Au total, la délétion homozygote de DPY19L2 a été retrouvée chez 22 patients sur 30 cas non apparentés (73.3%) et 3 nouvelles mutations ponctuelles ont été identifiées. Ces résultats indiquent que l'analyse moléculaire de DPY19L2 des patients globozoospermiques ne devrait pas être limitée à la recherche de la délétion homozygote de DPY19L2. Dans un second temps, nous avons démontré que la délétion récurrente de DPY19L2 était médiée par le mécanisme de recombinaison homologue non allélique (NAHR) entre deux séquences répétées homologues (LCR) de 28kb situées de chaque côté du gène. La très grande majorité des points de cassure surviennent dans une région de 1,2 kb située dans la partie centrale des LCRs. Cette région minimale de recombinaison est elle‐même centrée sur une séquence consensus de 13 nucléotides reconnue par PRDM9, une protéine à doigts de zinc qui favorise la survenue des cassures doubles brins initiant les processus de recombinaisons. Les modèles théoriques prédisent que, lors de la méiose, le mécanisme NAHR génère de novo plus d'allèles recombinés délétés que dupliqués. Étonnamment, dans la population générale les allèles DPY19L2 dupliqués sont trois fois plus fréquents que les allèles délétés. Nous avons développé une PCR digitale sur le sperme afin de mesurer le taux de délétions et de duplications de novo à ce locus chez des témoins. Tel qu'il était prédit par le modèle de NAHR, nous avons identifié un taux de délétions supérieur à celui des duplications. Ce paradoxe peut s'expliquer par la sélection qui s'opère à l'encontre des hommes infertiles porteurs de la délétion homozygote et potentiellement des hommes porteurs d'une délétion hétérozygote.Enfin pour le troisième phénotype, nous avons réalisé l'analyse par cartographie par homozygotie de 20 patients infertiles, dont 18 cas-index, présentant des anomalies morphologiques du flagelle. Cinq mutations homozygotes ont été identifiées dans le gène DNAH1 parmi les 18 patients non-apparentés (28%). Ce gène code pour une chaine lourde des bras internes de dynéine exprimée dans le testicule. Des analyses d'immunofluorescence et de RT-PCR ont confirmé le caractère pathogène d'une de ces mutations situées sur un site donneur d'épissage. Les analyses par microscopie électronique ont révélé une désorganisation générale de l'axonème incluant une disparition des doublets centraux et des bras internes de dynéine suggérant que DNAH1 est une protéine clé dans la biogenèse du flagelle du spermatozoïde. / Male infertility affects more than 20 million men worldwide and represents a major health concern. Although multifactorial, male infertility has a strong genetic basis which has so far not been extensively studied. The objectives of my thesis were to initiate and conduct some genetic investigations on three specific phenotypes of teratozoospermia: macrozoospermia, globozoospermia and multiple morphological abnormalities of the flagella (MMAF).For the first phenotype, we studied 87 patients with macrozoospermia, including 83 index cases, and identified c.144delC, a pathogenic mutation in the AURKC gene in 82% of patients (68/83) confirming that this variant is the main cause of macrozoospermia. A new recurrent mutation, p.Y248*, leading to degradation of the mutant transcripts by non-sense mediated mRNA decay was identified in 10 unrelated patients. Patients with no identified AURKC mutation have a decreased rate of spermatozoa with a large head and multiple flagella. Identification of two ancestral mutations in AURKC maintained during evolution despite their negative effect on reproduction in homozygous men, raises the question of a potential selective advantage provided by the AURKC haploinsufficiency.For the second phenotype, we first analyzed 34 patients presenting with globozoospermia using MLPA (multiplex ligation-dependent probe amplification) and Sanger sequencing. In total, 22 of the 30 unrelated patients where homozygous for the DPY19L2 deletion (73.3%) and 3 novel point mutations were identified. These results suggest that the molecular investigation of the DPY19L2 gene in globozoospermic patients should not be limited to the detection of the DPY19L2 genomic deletion and open interesting perspectives for the identification of DPY19L2 partners during acrosome biogenesis. Subsequently, we demonstrated that the genomic deletion was mediated by Non-Allelic Homologous Recombination (NAHR) between two homologous 28-Kb Low Copy Repeats (LCRs) located on each side of the gene. The vast majority of genomic breakpoints fell within a 1.2-Kb region central to the 28-Kb LCR. A 13-mer consensus sequence is located in the centre of that 1.2-Kb region recognized by PRDM9, a multi-unit zinc finger binding protein that promotes the formation of double-strand breaks (DSBs) initiating the homologous recombination process. The accepted theoretical NAHR model predicts that during meiosis, NAHR produces more deleted than duplicated alleles. Surprisingly, array-CGH data show that, in the general population, DPY19L2 duplicated alleles are approximately three times as frequent as deleted alleles. In order to shed light on this paradox, we developed a sperm-based digital PCR to measure the de novo rates of deletions and duplications at this locus. As predicted by the NAHR model, we identified an excess of de novo deletions over duplications. These discording results may be explained by the purifying selection against sterile, homozygous deleted men. Heterozygous deleted men might also suffer a small fitness penalty.Lastly, for the third phenotype, homozygosity mapping was carried out on a cohort of 20 North African individuals, presenting with primary infertility resulting from impaired sperm motility caused by a mosaic of multiple morphological abnormalities of the flagella (MMAF). Five unrelated subjects out of 18 (28%) carried a homozygous variant in DNAH1, which encodes an inner dynein heavy chain and is expressed in testis. RT-PCR and immunostaining studies confirmed the pathogenic effect of one of these mutations located on a donor splice site. Electronic microscopy revealed a general axonemal disorganization including mislocalization of the microtubule doublets and loss of the inner dynein arms suggesting that DNAH1 plays a critical role in sperm flagellum biogenesis and assembly.

>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).