Les chromosomes sexuels subissent pendant la spermatogenèse de multiples modifications qui entrainent d’importantes variations dans le niveau d’expression des gènes qu’ils portent. Notamment, ils sont inactivés au cours de la méiose et la majorité reste réprimé tout au long de la spermiogenèse. Cette étude met en évidence l’existence de transcrits alternatifs particuliers de gènes sur le chromosome X et Y, dont les profils d’expressions témoignent de leur rôle au cours de ces deux phases de la spermatogenèse. Sur le chromosome X nous avons isolé, chez l’homme et chez la souris, trois gènes ubiquitaires (Uba1x, Prdx4, Atp11c) réactivés dans les spermatides via un transcrit alternatif exprimé de façon majoritaire dans les testicules. Le gène Prdx4 code, pour deux isoformes de protéines différentes par leur domaine N-terminal. Nous avons mis au point des anticorps spécifiques de chaque isoforme et nous avons démontré que, chez la souris, le transcrit réactivé est traduit dans les spermatides et produit une protéine dans un compartiment cellulaire distinct de l’autre isofome ubiquitaire. Un total de cinq mutations, affectant ces transcrits exprimés dans les spermatides, ont été retrouvées dans les gènes UBA1X et PRDX4 chez des hommes infertiles. Sur le chromosome Y chez la souris, nous avons étudié les gènes Zfy1 et Zfy2, deux homologues testicules spécifiques codant pour des protéines à doigt de zinc avec un long domaine d’activation. Zfy2, mais pas Zfy1, promeut l’élimination apoptotique des spermatocytes contenant un chromosome X univalent. Nous avons identifié un transcrit alternatif du gène testicule spécifique Zfy1 exprimé dans les spermatocytes et les spermatides. La protéine putative issue de ce transcrit, possédant un domaine acidique réduit de moitié qui pourrait être à l’origine des différences fonctionnelles entre les gènes homologues Zfy1 et Zfy2 au cours de la méiose murine. Chez l’homme, l’orthologue de ces gènes ZFY est ubiquitaire et nous avons montré qu’il produisait un transcrit alternatif testicule spécifique, codant une protéine avec le même domaine acidique raccourci que Zfy1. Nos données indiquent que ce transcrit alternatif est prédominant dans les spermatocytes et les spermatides chez l’homme et chez la souris. Ces résultats apportent la première évidence d’une fonction du gène ZFY au cours de la spermatogenèse chez l’homme et de son implication possible dans la fertilité masculine. / Sex chromosomes undergo many modifications during spermatogenesis, leading to dramatic variations in the expression levels of their genes. In particular, they are inactivated during meiosis with most genes remain silent throughout spermiogenesis. Our study describes specific alternative transcripts produced by X and Y chromosome genes, whose expression indicates roles in early spermatocytes (meiosis) and in spermatids (spermiogenesis). On the X chromosome, we have shown that three widely transcribed genes, Uba1x, Prdx4, and Atp11c, are reactivated in mouse and human spermatids via an alternative transcript that is expressed mainly in the testis. The Prdx4 gene codes two isoforms of the peroxiredoxin 4 that differ in their N-terminal domain. We have raised antibodies specific for each PRDX4 isoform and demonstrate, in mouse, that the reactivated transcript is translated in spermatids, producing a protein in a distinct cellular compartment from the ubiquitous isoform. Altogether, five mutations, affecting the spermatid-reactivated transcripts uniquely, of UBA1x and PRDX4, have been found specifically in our group of infertile men. On the mouse Y chromosome, we have studied Zfy1 and Zfy2, nearly identical testis specific zinc finger genes with long acidic (activation) domains. Zfy2, but not Zfy1, promotes the apoptotic elimination of spermatocytes with an unpaired X chromosome. We have identified an alternatively spliced transcript of Zfy1 that lacks half the acidic domain, and could explain the functional difference between Zfy1 and Zfy2. In human, the ZFY gene is widely transcribed, but we show that ZFY produces a testis specific variant transcript, encoding the same short acidic domain as Zfy1. Our data indicate that the alternative transcripts predominate in spermatocytes and spermatids, in both human and mouse. This provides the first evidence that human ZFY may play a conserved role during spermatogenesis, and contribute to human male fertility.

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

Testicular germ cell tumors (TGCTs) are thought to arise during early embryogenesis due to the arrest of germ cell differentiation at primordial germ cells (PGCs) or gonocytes. Oxidative stress (OS) is implicated in cancer development as a factor leading to DNA damage. Reactive oxygen species (ROS) -induced instability occurs as a series of progressive steps. The cell has several defense mechanisms against the deleterious effect of ROS (e.g. antioxidants and DNA repair). When the defense mechanisms are exhausted by increasing OS, DNA damage leads to genomic instability with subsequent mutations that can be transmitted during cell division. On the other hand, male infertility is a representation of testicular dysgenesis syndrome, which carries a risk for TGCTs development. The mechanisms underlying both TGCTs and male infertility are thought to be overlapping to some extent. The central hypothesis of this work is that OS induces germ line genomic instability leading to testicular germ cell tumors. To test this hypothesis, mouse germ cell lines were established and subjected to different doses of OS in the form of H2O2. The mutation frequency was associated with the treatment dose 2 uM at days 3, 6, and 9 (p<0.001, p<0.001, and p<0.0003, respectively). The mBAT27 marker showed a mutation frequency fitting quadratic response surface regression. The mutation frequencies pointed to the possible role of OS leading to accumulation of DNA damage and initiating events that lead to TGCTs development that may occur early in life, possibly during the prenatal period. In addition, different panels of microsatellite markers from across the genome were analyzed to test for differential instability in both somatic cells and germline cells. Blood and semen samples from 18 infertile patients and 7 ethnically matched controls were used. Microsatellite markers were selected; 26 on the Y chromosome, 16 on the X chromosome, and 20 on different autosomes. Microsatellite instability was detected in markers located near genes responsible for testis development, spermatogenesis, cell differentiation, and proteins involved in mismatch repair mechanisms. This supports the hypothesis that testicular germ cell tumors may arise during early embryogenesis through acquiring multiple mutations that accumulate over time.

Thesis (MScMedSc)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Much has been documented about the detrimental effects of adverse lifestyle factor exposure on the body. Exposure to factors, such as cigarette smoke, have proved to not only be a burden on global health and economy, but have also led to growing concerns about effects on systemic functions such as reproduction. The aim of the present study was to determine the effects of in utero and in vitro nicotine exposure on spermatozoal function and the antioxidant enzyme activity and lipid peroxidation (LPO) status of the male reproductive system. A better understanding of this process is necessary to combat the respective burdens of smoking and male infertility and for the prospective development of treatment strategies. Two experimental models were employed: Wistar rats were exposed to nicotine in utero while human and rat spermatozoa were exposed to nicotine in vitro. In utero studies were achieved by selecting healthy pregnant rats and treating them with 1 mg/kg-bodyweight/day nicotine or 1 ml/kg-bodyweight/day 0.85% physiologic saline throughout gestation and lactation. Male rat pups were selected and sacrificed at each of the following age groups (n=6): 42 days, 84 days and 168 days old. The pups were only exposed to the treatment/saline via placental uptake or lactation. Biochemical analyses of the tissue comprised of measurement of LPO and antioxidant enzyme activity. Results indicated a significant association of maternal nicotine exposure to decreased levels of primary antioxidant enzymes in rat testes. Of particular note was the observation that the treatment group, of which each of the respective antioxidant enzyme levels were significantly less than the control group, was the oldest (d168) rat group. In vitro studies were achieved by collecting sperm samples from healthy human donors (n=12), healthy rats (n=6) and obese rats (n=6). Samples were washed and exposed to different concentrations of high levels of nicotine (Control, 0.1mM, 1mM, 5mM, 10mM) in vitro. Semen parameters such as motility, viability and acrosome reaction were monitored at different time points (30min, 60min, 120min, 180min). Results revealed increasing in vitro nicotine concentrations were associated with decreased viability and acrosomal status of human spermatozoa and decreased progressive motility and viability of rat spermatozoa. Obesity was also associated with decreases in progressive motility and viability of rat spermatozoa. These results indicate that the acute in vitro exposure of spermatozoa to high levels of nicotine could adversely affect semen quality and may be an additive factor to the impediment of male fertility. In utero results reveal maternal nicotine exposure adversely affects male fertility in later life and seems to elicit more detrimental effects on the reproductive system than that of direct nicotine exposure to spermatozoa. Obesity also inhibits parameters of male fertility and these effects are exacerbated by nicotine exposure. The authors believe these adverse effects on the reproductive system to be related to an increased activation of leukocytes, excess production in reactive oxygen species (ROS) and consequent onset of oxidative stress (OS). Nevertheless this study agrees with other studies that nicotine exposure may be an additive factor to the impediment of male fertility. / AFRIKAANSE OPSOMMING: Daar is reeds baie bekend oor die moontlik newe effekte vir die liggaam wat met ‘n ongesonde lewenstyl gepaard gaan. Menslike blootstelling aan sulke faktore, soos sigaret rook, is wêreldwyd ‘n las vir gesondheid en ekonomie en het gelei tot geweldige kommer onder navorsers oor die moontlike komplikasies vir liggaamlike funksies soos voortplanting. Die doel van die betrokke projek was om die effekte van in utero en in vivo nikotien blootstelling op die antioksiderende ensiem aktiwiteit en lipied peroksidasie status van reproduktiewe weefsel en die funksionele parameters van spermatozoa te bepaal. ‘n Beter begrip van hierdie proses is noodsaaklik om die las van rook en vetsug teen te werk en vir die moontlike ontwikkeling van behandelingsstrategieë. Twee eksperimentele modelle is ontwerp: Wistar rotte is in utero blootgestel aan nikotien terwyl mens- en rot- spermatosoë ook in vitro aan nikotien blootgestel is. Vir die in utero studie is gesonde dragtige rotte gedurende swangerskap en laktasie met 1 mg/kgliggaamsgewig/ dag nikotien of 1 ml/kg-liggaamsgewig/dag 0.85% fisiologiese soutoplossing behandel. Manlike welpies is gekies en geoffer op elk van die volgende ouderdomme (n=6): 42 dae, 84 dae en 168 dae. Die welpies is slegs aan nikotien blootgestel deur plasentale opname en laktasie. Biochemiese analise van die testikulêre weefsel het ‘n beduidende assosiasie getoon tussen maternale nikotien blootstelling en verminderde vlakke van die primêre antioksiderende ensieme. Die 168 dag oue groep het ‘n merkbare vermindering getoon tussen kontrole en nikotien weefsel vir elk van die antioksiderende ensieme. Vir die in vitro studie is sperm monsters verkry vanaf gesonde mans (n=12), gesonde rotte (n=6) en vet rotte (n=6). Monsters is gewas en in vitro blootgestel aan verskeie hoë vlakke van nikotien (kontrole, 0.1mM, 1mM, 5mM, 10mM). Seminale parameters soos motiliteit, lewensvatbaarheid en akrosoom status is by verskei tydpunte gemeet (30min, 60min, 120min, 180min). Dit blyk dat verhoging in in vitro nikotien konsentrasies verband hou met verlaagde lewensvatbaarheid en akrosoom status van menslike spermatosoë en verlaagde progressiewe motilteit en lewensvatbaarheid van rot spermatosoë. Vetsug is ook geassosieer met verlagings in progressiewe beweeglikheid en lewensvatbaarheid van rot spermatosoë. In utero resultate openbaar dat maternale nikotien blootstelling manlike vrugbaarheid nadelig beïnvloed in latere lewe en blyk dat dit meer van ‘n nadelige uitwerking op die voortplantingstelsel het as dié van direkte nikotien blootstelling aan spermatosoë. In vitro blootstelling van spermatosoë aan hoë vlakke van nikotien, het wel ook semen kwaliteit nadelig beïnvloed. Vetsug inhibeer ook manlike vrugbaarheids parameters en hierdie effek word vererger deur nikotien blootstelling. Die outeure glo dat hierdie nadelige uitwerking op die voortplantingstelsel verband hou met 'n verhoogde aktivering van leukosiete, oortollige produksie van reaktiewe suurstof spesies en die gevolglike aanvang van oksidatiewe stres bevorder. Hierdie studie stem wel ooreen met ander studies wat nikotien blootstelling bestempel as ‘n bydraende faktor tot die struikelblok van manlike onvrugbaarheid. / Harry Crossley Foundation (South Africa)

L’infertilité affecte jusqu’à 15-20% des couples en âge de se reproduire. C’est pourquoi, mieux comprendre les mécanismes à la base de la fécondation est essentiel pour l’identification de nouvelles causes d’infertilité et l’optimisation des techniques de reproduction assistée. La capacitation est une étape de la maturation des spermatozoïdes qui se déroule dans le tractus génital femelle. Elle est requise pour la fécondation d’un ovocyte. Notre laboratoire a démontré que des protéines du plasma séminal bovin, appelées protéines Binder of SPerm (BSP), se lient aux phospholipides portant des groupements choline à la surface de la membrane des spermatozoïdes lors de l’éjaculation et promeuvent la capacitation. Ces protéines exprimées par les vésicules séminales sont ubiquitaires chez les mammifères et ont été étudiées chez plusieurs espèces dont l’étalon, le porc, le bouc et le bélier. Récemment, l’expression de gènes homologues aux BSP a été découverte dans les épididymes d’humains (BSPH1) et de souris (Bsph1 et Bsph2). Notre hypothèse est que les BSP chez ces deux espèces sont ajoutées aux spermatozoïdes lors de la maturation épididymaire et ont des rôles dans les fonctions spermatiques, similaires à ceux des protéines BSP bovines. Les protéines BSP humaines et murines représentent une faible fraction des protéines totales du plasma séminal. Pour cette raison, afin d’étudier leurs caractéristiques biochimiques et fonctionnelles, des protéines recombinantes ont été produites. Les protéines recombinantes ont été exprimées dans des cellules Escherichia coli origami B(DE3)pLysS en utilisant un vecteur d’expression pET32a. Suivant la lyse cellulaire, les protéines ont été dénaturées avec de l’urée et purifiées par chromatographie d’affinité sur ions métalliques immobilisés. Une fois liées à la colonne, les protéines ont été repliées à l’aide d’un gradient d’urée décroissant avant d’être éluées. Cette méthode a mené à la production de trois protéines recombinantes (rec-BSPH1 humaine, rec-BSPH1 murine et rec-BSPH2 murine) pures et fonctionnelles. Des expériences de chromatographie d’affinité et de co-sédimentation nous ont permis de démontrer que les trois protéines peuvent se lier à des ligands connus des protéines BSP comme la gélatine et l’héparine en plus de pouvoir se lier aux spermatozoïdes. Nos études ont également révélées que les deux protéines rec-BSPH1 peuvent se lier aux liposomes de phosphatidylcholine (PC) et sont capable de promouvoir la capacitation des spermatozoïdes. À l’opposé, rec-BSPH2 ne peut ni se lier aux liposomes de PC, ni stimuler la capacitation. Finalement, les protéines recombinantes n’ont aucun effet sur la réaction acrosomique ou sur la motilité des spermatozoïdes. Chez les bovins, les protéines BSP induisent la capacitation grâce des interactions avec les lipoprotéines de haute densité (HDL) et les glycosaminoglycanes. Puisque le HDL est également un joueur important de la capacitation chez la souris, le rôle de la protéine native BSPH1 murine au niveau de la capacitation induite par le HDL a été étudié. Les résultats obtenus suggèrent que, in vivo, la protéine BSPH1 de souris serait impliquée dans la capacitation via une interaction directe avec le HDL. Comme les protéines BSPH1 humaines et murines sont orthologues, ces résultats pourraient aussi s’appliquer à la fertilité humaine. Les résultats présentés dans cette thèse pourraient mener à une meilleure compréhension de la fertilité masculine et aider à améliorer les techniques de reproduction assistée. Ils pourraient également mener au développement de nouveaux tests diagnostiques ou de contraceptifs masculins. / Infertility can affect as much as 15-20% of couples of reproductive age. Therefore, elucidating mechanisms occurring during fertilization is needed to resolve cases of infertility and optimize assisted reproductive technology procedures. Sperm capacitation is a maturation step that takes place in the female genital tract and is deemed to be essential for sperm to fertilize an oocyte. Our laboratory has demonstrated that proteins from bovine seminal plasma called Binder of SPerm (BSP) proteins bind to choline phospholipids on the sperm membrane upon ejaculation and promote capacitation. These proteins expressed in seminal vesicles are ubiquitous amongst mammals and have been studied in many species including stallion, boar, ram and goat. More recently, the expression of BSP-homologous genes has been discovered in the epididymis of humans (BSPH1) and in mice (Bsph1 and Bsph2). We hypothesized that the BSP homologs in these two species are added to sperm during epididymal maturation and play similar roles in sperm functions as bovine BSP proteins. BSP proteins in humans and mice constitute only a minute percentage of the seminal plasma proteins. Thus, to study their biochemical and functional characteristics recombinant proteins were produced. Recombinant proteins were expressed in Escherichia coli origami B(DE3)pLysS cells using a pET32a expression vector. Following cell lysis, proteins were denatured using urea and purified by immobilized metal ion affinity chromatography. Once bound to the resin, proteins were refolded using a decreasing urea gradient after which they were eluted. This method led to the production of three pure, functional recombinant proteins (human rec-BSPH1, mouse rec-BSPH1 and mouse rec-BSPH2). Using affinity chromatography and co-sedimentation experiments, we were able to demonstrate that all three recombinant proteins bind known ligands of BSP proteins including gelatin, heparin and have the ability to bind to sperm. Studies also revealed that both rec-BSPH1 proteins bind to phosphatidylcholine (PC) liposomes and promote sperm capacitation. However, rec-BSPH2 neither binds to PC liposomes nor stimulates capacitation. Recombinant proteins had no effect on acrosome reaction or sperm motility. In bovine, BSP proteins promote sperm capacitation through interactions with high-density lipoproteins (HDL) and glycosaminoglycans. Since in mice HDL is also a major factor implicated in capacitation, the role of the native murine BSPH1 protein in HDL-induced capacitation was investigated. Results obtained suggest that, in vivo, murine BSPH1 protein could act in capacitation via a direct interaction with HDL. As human and murine BSPH1 are orthologs, these results could possibly also apply to human fertility. The results presented in this thesis could lead to a better understanding of male fertility and help improve assisted reproduction technology procedures. They could also lead to the development of diagnostic tests as well male contraceptives.

Infertility is a widespread health problem, caused by the male factor in about half of all cases, and in about a half of the infertile men the cause is unknown. In a significant number of these men, genetic etiology is assumed. Current routine methods of laboratory diagnostics, which include karyotype examination, exclusion of mutations in the CFTR gene, and Y chromosome microdeletions, do not usually reveal the cause of infertility. That is why researchers' efforts aim at detecting mutations in other genes that are causing male infertility. In recent years, animal models have been used to identify many genes necessary for fertility. Based on these findings, 12 candidate genes have been selected (CAPZA3, CDC14B, CDC42, CNTROB, CSNK2A2, GOPC, HOOK1, HRB, OAZ3, ODF1, RIMBP3, SPATA16) that are essential for spermatogenesis. Mouse or rat mutants in these genes are primarily associated with oligoasthenoteratozoospermia, since they are involved in sperm morphogenesis. However, the phenotype spectrum may comprise also azoospermia. The purpose of the thesis was to determine the sequence of the afore mentioned genes in infertile men with impaired spermatogenesis and to reveal presence or absence of pathogenic mutations in these genes, using cDNA and genomic DNA from peripheral blood. The candidate genes were...

INTRODUÇÃO: Infertilidade atinge aproximadamente 15% dos casais em idade reprodutiva e afeta de maneira profunda a vida dessas pessoas. Dentre as causas identificáveis de infertilidade masculina, a varicocele é a mais frequente e acomete cerca de 40% dos homens inférteis ou subférteis. Um dos maiores desafios na abordagem cirúrgica da varicocele é a identificação de indivíduos que apresentarão maior benefício com o tratamento, uma vez que muitos pacientes não apresentam melhora da análise seminal. OBJETIVOS: Identificar um padrão histológico testicular como prognóstico da melhora da capacidade reprodutiva em pacientes submetidos à varicocelectomia microcirúrgica. METODOLOGIA: Estudo retrospectivo composto pela análise de 60 biópsias testiculares bilaterais de homens inférteis atendidos em clínica especializada de fertilidade masculina, entre os anos de 2006 e 2014. Como critérios de inclusão foram considerados homens com diagnóstico de varicocele clínica e subclínica entre 19 e 50 anos de idade com resultados de análise histopatológica testicular. Os sujeitos de pesquisa foram divididos em dois grupos. Grupo 1: homens com diagnóstico de varicocele subclínica (n = 20). Grupo 2: homens com diagnóstico de varicocele clínica (n =40). Foram excluídos do estudo homens com diagnóstico de criptorquidia, azoospermia obstrutiva e não-obstrutiva, usuários de drogas e anabolizantes, além de pacientes portadores de doenças sexualmente transmissíveis e de neoplasias no trato geniturinário. Os participantes foram submetidos ao exame físico urológico com a avaliação do volume testicular por meio de ultrassonografia da bolsa escrotal com Doppler-Colorido. O diagnóstico da varicocele foi realizado por meio da palpação cuidadosa do plexo pampiniforme com o paciente em posição ortostática. A manobra de Valsava foi utilizada para a classificação clínica do grau de varicocele. Para a determinação de um padrão histológico capaz de predizer a melhora da capacidade reprodutiva, foram criados valores de corte que associam os scores de Johnsen, os índices de Copenhagen e o volume testicular à melhora dos parâmetros seminais. RESULTADOS: No grupo 1, para a melhora da concentração espermática o score de Johnsen deve ser superior a 8,2 (lado esquerdo) e o volume testicular acima de 12,8 mL (lado direito). Adicionalmente, para a avaliação da motilidade total de espermatozoides os scores de Johnsen devem ser superiores a 8,2 (bilateral) e o dígito II de Copenhagen inferior a 2,5 em ambos os testículos. Todavia, para a motilidade progressiva de espermatozoides o score de Johnsen deve ultrapassar a 9,1 (bilateral) e na avaliação da morfologia espermática, este deve se apresentar acima de 7,9 e com volume testicular acima de 13,6 mL (lado direito). Quanto aos valores de corte obtidos no grupo 2, para a concentração de espermatozoides, os scores de Johnsen devem ser superiores a 5,5 com volume testicular acima de 11,5 mL em ambos os testículos. Finalmente, quanto à motilidade espermática total e progressiva, o dígito III do índice de Copenhagen deve ser inferior a 1,5 (lado direito). CONCLUSÃO: Valores prognósticos da melhora da capacidade reprodutiva obtidos por meio de biópsia testicular podem auxiliar com eficácia no prognóstico e na avaliação dos pacientes candidatos à correção microcirúrgica da varicocele / BACKGROUND: Infertility affects approximately 15% of couples in reproductive age and profoundly changes the lives of these people. Among the identifiable causes of male infertility, varicocele is the most common and affects about 40% of infertile or subfertile men. One of the challenges in the surgical approach is the identification of individuals who will present benefits with the treatment, since many patients do not show improvement of semen analysis. OBJECTIVE: To identify a testicular histological pattern as prognostic value of improved reproductive capacity in patients submitted to microsurgical correction of varicocele. METHODS: we retrospectively analyzed bilateral testicular biopsies of 60 men attending specialized clinic of male fertility between the years 2006 and 2014. As inclusion criteria were considered men diagnosed with clinical and subclinical varicoceles between 19 and 50 years old with results of testicular histopathology and seminal analysis. The patients were divided into two groups. Group 1: Men diagnosed with subclinical varicocele (n = 20). Group 2: men diagnosed with clinical varicocele (n = 40). Men diagnosed with cryptorchidism, obstructive and non-obstructive azoospermia, users of drugs and anabolic steroids were excluded of the study. All Participants were submitted to urological physical examination with the evaluation of testicular volume by ultrasonography of the scrotum with Color Doppler. The diagnosis of varicocele was performed by careful palpation of pampiniform plexus with the patient in standing position. The Valsalva maneuver was used to classify the grade of varicocele. The determination of a testicular histological pattern as prognostic value of the improved reproductive capacity was performed by the creation of cut-off values that associate Johnsen scores, Copenhagen indices and testicular volume to improvement in semen parameters. RESULTS: In Group 1, for improvement of sperm concentration, the Johnsen score must be greater than 8.2 (in the left testicle) and testicular volume must be greater than 12.8 mL (in the right testicle). Concerning evaluation of sperm total motility, the Johnsen score must be greater than 8.2 (bilateral) and digit II of Copenhagen indices must be less than 2.5 (bilateral). However, for sperm progressive motility, the Johnsen score must exceed 9.1 (bilateral) and evaluation of sperm morphology must be greater than 7.9 with right testicular volume greater than 13.6 mL. In Group 2, the cut-offs values for sperm concentration indicates that Johnsen scores must be greater than 5.5 with testicular volume greater than 11.5 mL in both testicles. Finally, regarding the sperm total and progressive motility, the digit III of Copenhagen indice must be less than 1.5 (in the right testicle). CONCLUSION: Prognostic values of improved reproductive capacity obtained from testicular biopsy can assist effectively in the prognosis and evaluation of patients candidates for microsurgical correction

Apesar da maioria dos estudos mostrarem que parâmetros seminais anormais, redução do volume testicular e elevação dos níveis de radicais livres de oxigênio (ROS) no sêmen estão associados à presença de varicocele em homens inférteis, a maioria dos homens com varicocele apresenta os parâmetros seminais normais e são capazes de estabelecer gravidez em suas esposas. Nesse sentido, a avaliação desses parâmetros ainda não foi adequadamente estudada em homens com fertilidade comprovada e que apresentam varicocele. Esse estudo teve como objetivo estudar o impacto da varicocele clínica em homens férteis avaliando o volume testicular, parâmetros tradicionais da análise seminal e níveis de radicais livres de oxigênio no sêmen. Esses parâmetros foram avaliados em 113 homens férteis sem varicocele, 43 homens férteis com varicocele e 38 pacientes inférteis com varicocele. A medida do volume testicular foi realizada utilizando-se orquidômetro de Prader. Os parâmetros seminais foram avaliados manualmente segundo os critérios da Organização Mundial da Saúde (OMS) e a morfologia avaliada conforme os critérios da OMS e estrito de Kruger. Os níveis de ROS foram mensurados em sêmen puro utilizando-se a quimioluminescência. Os pacientes inférteis com varicocele apresentaram diminuição dos parâmetros seminais, redução do volume testicular e elevação dos níveis de ROS no sêmen em comparação com os homens férteis, com ou sem varicocele. Os homens férteis com varicocele não apresentaram diferenças significativas em nenhum dos parâmetros avaliados quando comparados aos homens férteis sem varicocele. No entanto, os homens férteis com varicocele grau 3 apresentaram menor concentração espermática e níveis de ROS no sêmen mais altos quando comparados àqueles com grau 1 ou 2. Além disso, o grau de varicocele nos homens férteis apresentou correlação negativa com a concentração espermática e positiva com os níveis de ROS no sêmen. A dosagem dos ROS no sêmen poderá ajudar, principalmente, na avaliação dos homens com varicocele que apresentam parâmetros seminais normais, e quando o potencial de fertilidade ainda não foi testado. A dosagem de ROS no sêmen pode representar um marcador mais sensível e específico no diagnóstico precoce da disfunção testicular, principalmente, nos homens férteis com varicocele grau 3 / Although the majority of the studies showed that abnormal seminal parameters, reduced testicular volume and high levels of seminal reactive oxygen species (ROS) are frequently associated with varicocele in infertile population, the majority of the men with varicocele present normal seminal parameters and are able to establish a pregnancy. In view of that, the evaluation of these semen parameters in fertile men with varicocele has not yet been well documented. The objective of our study was to evaluate the impact of clinical varicocele in fertile men assessing testicular volume, routine seminal parameters and seminal ROS levels. These parameters were evaluated in 113 fertile men without varicocele, 43 fertile men with clinical varicocele and 38 infertile patients with varicocele. Testicular volume was measured with a Prader orchidometer. Seminal parameters were assessed according to World Healthy Organization (WHO) guidelines and morphology according to WHO as well as Krugers strict criteria. Measurement of seminal ROS in neat semen was performed using a chemiluminescence assay. Infertile men with varicocele presented lower semen parameters, reduced testicular volume and higher seminal ROS levels compared with fertile men with or without varicocele. Although fertile men with varicocele presented all parameters evaluated similar to fertile men without varicocele, fertile men with varicocele grade 3 presented lower sperm concentration and higher seminal ROS levels compared with fertile men with varicocele grade 1 or 2. In addition, varicocele grade infertile men presented a negative correlation with sperm concentration and a positive correlation with seminal ROS levels. Men with unknown fertility status presenting with palpable varicocele and normal seminal parameters, the presence of increased ROS levels may be indicative of an early recognition of those subjects who will experience a progressive decrease in fertility potential if left untreated, especially in those presenting with varicocele grade 3

Infertilidade conjugal é uma doença do sistema reprodutivo que acomete cerca de 20% dos casais e na qual o fator masculino responde por metade desses casos. A infertilidade masculina é um fenótipo complexo que abrange diferentes fatores. Os fatores genéticos envolvidos variam desde mutações pontuais, microdeleções no cromossomo Y, até alterações cromossômicas, como a Síndrome de Klinefelter. Mesmo após avaliação clínicolaboratorial detalhada, metade dos pacientes permanece sem a identificação de um fator causal, caracterizando a infertilidade idiopática. Nesse grupo, observamos com maior frequência os pacientes com falha espermatogênica primária, que clinicamente apresentam oligozoospermia grave ou azoospermia não obstrutiva (ANO) e, no qual, preponderam fatores genéticos ainda desconhecidos. Para auxiliar na compreensão de possíveis alterações genômicas, sejam as variantes de número de cópias (CNVs) ou as regiões de perda de heterozigosidade (LOHs), envolvidas com infertilidade masculina idiopática, 16 pacientes com ANO e 6 controles foram investigados pela técnica de hibridação genômica comparativa (aCGH) utilizando a plataforma 4x180 CGH+SNP Agilent® com análise dos dados pelo software Nexus 8.0. Não foram observadas diferenças significativas tanto no número, como no tamanho das alterações genômicas em ambos os grupos. Foram descritas 18 novas alterações genômicas com efeito sobre a produção espermática, distribuídas na forma de 12 ganhos, 3 perdas e 3 LOHs. Os ganhos mais significativos para o fenótipo azoospermia não obstrutiva foram descritos em 7q36.3, 17q21.33, Xq21.1 e Yp11.2. Nessas regiões, os genes com maior impacto sobre o fenótipo foram, respectivamente, SHH, COL1A1, COX7B e LINC00279. Ganhos envolvendo a sub-banda Yq11.223 e contendo cópias dos genes DAZ1 e DAZ4 foram considerados benignos. As três perdas detectadas em 2q31.1, 3p21.1-21.31 e 15q11.2, contendo, respectivamente, os genes DLX1, CACNA2D2 e representantes da família de receptores olfatórios foram consideradas relevantes. A análise das LOHs em fenótipos complexos é escassa e desafiadora. No presente trabalho, foram descritas 3 dessas alterações, localizadas em 1p31.1, 7q21.1 e 12q21.1-21.2 e compartilhadas por mais de um indivíduo infértil. A descrição dessas alterações genômicas contribui para a compreensão de mecanismos complexos e ainda pouco estudados, que resultam em azoospermia não obstrutiva decorrente da falha espermatogênica primária. / Infertility is a disease of the reproductive system that affects about 20% of all couples, with half of the cases being related to the male factor. Male infertility is a complex phenotype associated with an interaction of different factors. The genetic factors involved may range from point mutations, microdeletions on the Y chromosome to chromosomal changes such as Klinefelter syndrome. Even after detailed clinical-laboratory evaluation, the etiology may remain unknown in approximately half of the patients, and, in such cases, the infertility can be classified as idiopathic. This group of patients more frequently present with primary spermatogenic failure, with severe oligozoospermia or non-obstructive azoospermia (NOA). Nevertheless, the underlying genetic factors are still largely unknown. In order to better understand the potential genomic changes involved with idiopathic male infertility, sixteen patients with NOA and 6 controls were investigated in this study. Copy number variants (CNVs) and regions of loss of heterozygosity (LOHs) were assessed by array comparative genomic hybridization technique (aCGH), using the Agilent® 4x180 CGH + SNP platform. Data analyses was performed using Nexus 8.0 software. No significant differences between the groups were observed in relation to either the number or the size of the genomic changes. Eighteen new genomic alterations were described that were associated with sperm production (12 gains, 3 losses and 3 LOHs). The most important gains for the nonobstructive azoospermia phenotype were observed in 7q36.3, 17q21.33, Xq21.1 and Yp11.2. In these regions, the genes related to greatest impact on the phenotype were SHH, COL1A1, COX7B and LINC00279, respectively. Gains involving the Yq11.223 sub-band and containing copies of the DAZ1 and DAZ4 genes were considered benign. All 3 losses detected in 2q31.1, 3p21.1-21.31 and 15q11.2, containing, respectively, the DLX1, CACNA2D2 genes and representatives of the olfactory receptor family were considered relevant. Analysis of LOHs in complex phenotypes such as male infertility has been infrequently reported and is challenging. In the present study, three significants LOHs were found (1p31.1, 7q21.1 and 12q21.1-21.2) and were identified in more than one infertile individual. The description of these genomic alterations contributes to a better understanding of this complex and poorly explored mechanisms that results in non-obstructive azoospermia due to primary spermatogenic failure.

Este trabalhou avaliou o stress e a ansiedade em 30 casais, que se submeteram à reprodução assistida no momento anterior à coleta dos óvulos e/ou espermatozóides, tendo um tempo de infertilidade que variou de 1 a 17 anos. Para avaliação da ansiedade foi aplicado o STAI-STATE TRAIT ANXIETY INVENTORY (STAI) e o Stress foi avaliado através do SCOPE-STRESS. No STAI foi acrescentada uma escala visual analógica para medir também a intensidade da ansiedade. A Ficha de Identificação avaliou questões como: idade, sexo, nacionalidade, profissão, ocupação, religião, grau de instrução, renda mensal, estado civil, tempo de casado e questões como: tempo de infertilidade, filhos naturais ou adotivos, profissionais implicados no tratamento, a existência de tentativas anteriores e os momentos de maior tensão emocional nas tentativas anteriores e a atual. Dentre os 36 sujeitos que já haviam feito tentativas anteriores de Reprodução Assistida, um dos três momentos de maior tensão emocional foi o de aguardar a gravidez. Dentre os 60 sujeitos, ou seja, todos os que estão na tentativa atual, aguardar a gravidez foi também um dos três momentos que geraram maior tensão. O diagnóstico de infertilidade foi misto em 33,3% dos casais, apenas feminino em 20% e apenas masculino em 46,7% dos casais. As mulheres apresentaram grau de ansiedade significantemente maior que os homens quanto às escalas Stai-Trait freqüência e intensidade e semelhantes quanto às escalas Stai-State freqüência e intensidade. Não houve diferença significante entre os escores médios dos homens e mulheres quanto às medidas descritivas do Scope-Stress. / This work evaluated stress and anxiety in 30 couples submitted to assisted reproduction, with an infertility period that ranged from 1 to 17 years, the moment just before the oocyte retrieval and/or semen sample. For anxiety evaluation the Stai-State Trait Anxiety Inventory (STAI) was applied, and stress was evaluated using the Scope-Stress. While applying STAI, a visual analogic scale was added to measure the intensity of anxiety. The identification form included information such as: age, gender, nationality, profession, occupation, religion, school level, monthly income, marital status, married time and issues such as: infertility period, existence of natural or adoptive children, professionals involved in infertility treatment, existence of previous attempts and the moments of major emotional stress during the previous attempts and during the present one. Among the 36 subjects submitted to previous attempts of Assisted Reproduction, one of the three moments of major emotional stress was the attendance of pregnancy confirmation. Among the all 60 subjects submitted to the present attempt, attendance of pregnancy confirmation also was one of the three moments of major emotional stress. Infertility diagnosis was mixed in 33,3% of the couples, exclusively feminine in 20% and exclusively masculine in 46,7% of the couples. Women presented a significantly higher anxiety degree than men, regarding the STAI-TRAIT scales of frequency and intensity and similar regarding the STAI-STATE scales of frequency and intensity. There was no significant difference between the mean scores of men and women regarding descriptive measures of the SCOPE-STRESS.