In order for solid tumors to metastasize, tumor cells must acquire the ability to invade the surrounding tissue and intravasate into blood- or lymph vessels, survive in the circulation and then extravasate at a distant site to form a new tumor. Overexpression of the glycosaminoglycan hyaluronan, and its adhesion receptor CD44, correlate with breast cancer progression. This thesis focuses on the role of hyaluronan in tumor invasion and metastasis. In paper I, we demonstrated that upregulation of the hyaluronan synthesizing enzyme hyaluronan synthase 2 (HAS2) was crucial for transforming growth factor β (TGFβ)-induced epithelial-mesenchymal transition (EMT) in mammary epithelial cells. In paper II, we further demonstrated that silencing of HAS2 decreased the invasive behavior of bone-metastasizing breast cancer cells, via upregulation of tissue inhibitor for metalloproteinase 1 (TIMP1), and dephosphorylation of focal adhesion kinase (FAK). During tumorigenesis, stromal cells, such as fibroblasts, play important roles and several growth factors are synthesized, promoting crosstalk between different cell surface receptors. In paper III, we investigated the crosstalk between the hyaluronan receptor CD44 and the receptors for TGFβ and platelet-derived growth factor BB (PDGF-BB) in dermal fibroblasts. We found that the receptors for the three molecules form a ternary complex, and that PDGF-BB can activate the Smad pathway downstream of TGFβRI. Importantly, CD44 negatively modulated the signaling of both PDGF-BB and TGFβ. In paper IV, we studied the process by which breast cancer cells invade blood-vessels and the role of hyaluronan and CD44 in angiogenesis. Importantly, CD44, or the hyaluronan degrading enzyme hyaluronidase 2 (HYAL2), decreased the capacity of endothelial cells to form tubes in a 3D in vivo-like assay.  Collectively, our studies add to the understanding of the role of hyaluronan in tumor progression.

No description available.

Il est reconnu que le passage de la matrice extracellulaire du cumulus ainsi que de la zone pellucide par le spermatozoïde sont assurés, chez plusieurs espèces de mammifères, par la protéine spermatique PH-20. Cette protéine possède une activité hyaluronidase qui lui permet de cliver les liens d'acide hyaluronique contenus dans le cumulus et est responsable de la liaison du spermatozoïde ayant subi la réaction de l'acrosome à la zone pellucide. Une protéine spermatique bovine de 80 kDa (p80) possédant une homologie aVec PH-20 fut identifiée au sein de notre laboratoire. Puisque PH-20 n'est pas identifiée chez le bovin, les travaux présentés dans cette thèse étaient destinés à déterminer si la protéine p80 est la PH-20 bovine et si elle est impliquée dans l'interaction entre les gamètes au cours de la fécondation. Dans un premier temps, la caractérisation de la protéine fut effectuée à l'aide d'une approche PCR et immunologique. L'homologie entre la protéine spermatique bovine p80 et PH-20 fut établie à l'aide de ces résultats. L'analyse de la séquence déduite en acides aminés de p80 a permis de mettre en lumière la présence de caractéristiques uniques chez la protéine dont l'absence d'une ancre-GPI, la présence d'un domaine transmembranaire, de sites de phosphorylation et de N- et O-glycosylation. Deuxièmement, à l'aide du développement d'un anticorps dirigé contre l'extrémité C-terminale de p80 et d'un anticorps dirigé contre l'extrémité N -terminale, l'orientation de p80 dans les membranes spermatiques fut établie. Les résultats montrent que deux populations de p80 existent chez le spermatozoïde bovin. La première est internalisée dans la région antérieure de la tête des spermatozoïdes alors que la deuxième est localisée dans la région post -acrosomale de la tête et expose son domaine hyaluronidase à l'environnement extracellulaire. De plus, les analyses protéomiques et immunologiques démontrent que la population post-acrosomale est un isofo~e plus court de p80 et est d'origine épididymaire alors que la forme pleine longueur présente dans la région antérieure de la tête du spermatozoïde est d'origine testiculaire. La présence de sites d'O-glycosylation fut aussi confirmée expérimentalement. Afin de déterminer le rôle fonctionnel de p80, l'état de phosphorylation sur les résidus tyrosines de la protéine au cours de la capacitation et de la réaction de l'acrosome fut étudié. De même, l'implication de p80 dans la modulation de la concentration des niveaux de calcium intracellulaire spermatique en présence d'acide hyaluronique ainsi que son implication au cours de l'interaction entre le spermatozoïde et la zone pellucide furent déterminées. Les résultats démontrent que p80 n'est pas phosphorylée sur ses résidus tyrosines au cours de la capacitation et de la réaction acros·omique et n'est pas impliquée dans la modulation des concentrations de calcium intracellulaire. Toutefois, p80 est impliquée, via son extrémité C-terminale, dans la liaison du spermatozoïde à la zone pellucide puisque la présence de la protéine purifiée ainsi que l'anticorps C-terminal de p80 dans le milieu inhibe cette liaison. De plus, la glycosylation de la protéine n'est pas responsable de son interaction avec la zone pellucide puisque la présence de la forme déglycosylée de p80 dans le milieu d'incubation produit le même taux d'inhibition que sa forme native. Les résultats présentés dans cette thèse démontrent clairement que la protéine p80 est la PH-20 bovine, que deux populations sont présentes sur le spermatozoïde et qu'elle est responsable de l'activité hyaluronidase requise par le spermatozoïde pour franchir le cumulus ainsi que pour l'interaction entre ceux -ci et la zone pellucide entourant l'ovule au cours de la fécondation.

Les nanoparticules à base d’acide hyaluronique (AH) sont utilisées pour de nombreuses applications pharmaceutiques. Elles peuvent cibler les tumeurs par interaction avec le CD44,qui est un récepteur biologique surexprimé à la surface de certaines cellules cancéreuses. Dans ce projet nous explorons l’application potentielle de ces nanoparticules dans les domaines cosmétiques, car l’AH est aussi un ingrédient important pour l’hydratation et le renouvellement de la peau. Les copolymères à bloc à base de polypeptides et de polysaccharides ont été synthétisés par une combinaison de polymérisation par ouverture de cycle et de couplage par chimie « click ». Les nanoparticules ont été obtenues par l’auto assemblage de ces copolymères en utilisant un procédé de nano precipitation, dont la taille et la morphologie sont contrôlées par les paramètres expérimentaux. L’interaction entre les nanoparticules d’AH et le CD44 a été quantifiée par la résonance de plasmon de surface(RPS). En comparant avec l’AH libre en solution, les nanoparticules d’AH ont montré une interaction plus efficace avec le CD44, mettant ainsi en évidence un effet de multivalence des nanoparticules. Finalement, la dégradation enzymatique de ces nanoparticules d’AH a été évaluée avec deux types de hyaluronidases, HYAL1 et SPAM-1. La digestion des nanoparticules de l’AH a été significativement ralentie par rapport à l’AH libre. De manière très surprenante, ces nanoparticules de AH ont pu inhiber l’activité de l’enzyme HYAL1 et protéger l’AH libre dans la solution. Enfin, des ligands du récepteur TLR2 de type lipopeptide ont été synthétisés et leurs performances via TLR2 ont été évaluées par RPS. / Nanoparticles based on hyaluronic acid (HA) are widely used in pharmaceutics. They can target the tumor by the interaction with CD44, a biological receptor overexpressed in some cancer cells. In this project, we investigate the potential applications of these nanoparticles in cosmetics, since HA is also an important ingredient for the skin hydration and renewing. Block copolymers based on polypeptides and polysaccharides were synthesized using a combination of ring opening polymerization and “click chemistry”. The nanoparticles were formed by the self-assembly of these block copolymers using a nanoprecipitation process, and their size and morphology were controlled by the experimental conditions. The interaction between nanoparticles and CD44 were measured by surface plasmon resonance(SPR). Compared to free hyaluronic acid chains in solution, the HA-based nanoparticles could interact more efficiently with CD44, thus demonstrating a multivalent effect. The enzymatic degradation of these HA nanoparticles was then evaluated with twohyaluronidases: HYAL1 and SPAM-1. The digestion of the HA nanoparticles was significantly slower than that of free hyaluronic acid. Surprisingly, these HA nanoparticles could even inhibit the activity of the enzyme HYAL1 and protect free HA chains in the solution. Finally, lipopeptide-based ligands of the biological receptor TLR2 were also synthesized and their performances were evaluated by SPR.

No description available.

Hyaluronidase 2 (HYAL2) is a GPI-linked protein that is proposed to initiate the degradation of hyaluronan (HA), a major extracellular matrix component of many vertebrate tissues. Hyal2 knockout (KO) mice displayed craniofacial abnormalities and severe preweaning lethality. 54% of the surviving KOs developed a grossly dilated left or right atrium, requiring euthanasia, by 3 months of age. We hypothesize that the absence of HYAL2 leads to the accumulation of HA in organs/tissues where HA is normally abundant resulting in developmental defects and organs dysfunction. Molecular and histological analysis of HYAL2 KO hearts demonstrated extracellular accumulation of high molecular mass (HMM) HA in the heart valves, myocardium, serum and lungs which was associated with severe cardiopulmonary dysfunction. Further, structural and functional analyses of Hyal2 KO mouse hearts using high-frequency ultrasound revealed atrial dilation accompanied by diastolic dysfunction that was evident as early as 4 weeks of age, and progressed with age. Further, 50% of HYAL2 KO mice exhibited a triatrial heart (cor-triatriatum). Histological analyses revealed that the atrial dilation was the result of excess tissue, and did not correlate with the presence of cor triatrium. Hyal2 KO mice were found to have increased numbers of mesenchymal cells at early stages of development, presumably due to the presence of excess HA, that lead to cardiac dysfunction. Further examination of HYAL2 distribution in a broad range of mouse tissues, and accumulation of HA in its absence demonstrated that HYAL2 is mainly localized to endothelial cells and some specialized epithelial cells, and plays a major role in HMM-HA degradation. These studies demonstrated that HYAL2 is important for HA degradation and organ development. In the longer term, our findings will be valuable for understanding pathologies associated with the disruption of HA catabolism, and potentially in the identification of HYAL2-deficient patients. / May 2016

O Ácido Hialurônico, HA, é um dos principais componentes da matriz extracelular dos vertebrados. É um glicosaminoglicano hidrolisado pelas enzimas da família da Hialuronidase, envolvidas na regulação de importantes processos biológicos, como a permeabilidade vascular e a angiogênese. Conforme o interesse pelo desenvolvimento de uma rota de síntese para esta enzima, objetivamos a obtenção de um plasmídeo, contendo a sequência que codifica a variante 8 do gene Hyal-1. Para isto, planejou-se a inserção de dois sítios de restrição para a sub-clonagem, sítio dirigida, BamH-1 na região 5\' e Not-1 na 3\' na sequência do códon da Hyal-1. O inserto foi sub-clonado no plasmídeo, pET28-a, e transfectado para expressão em Escherichia coli Bl-21. A expressão foi induzida por IPTG no melhor tempo, de 4 horas, e a confirmação da expressão protéica foi realizada por Western blotting. Observou-se uma proteína de 45 kDa, confirmando a presença da Hyal-1. Realizou-se a expressão em 4 litros de cultura para obtenção de uma quantia suficiente para purificação em coluna de níquel-agarose. O rendimento neste experimento foi de 25µg por litro. A rota sintética sugerida neste estudo mostrou-se eficiente para obtenção da proteína Hyal-1 recombinante, justificando futuros investimentos na otimização deste processo / Hyaluronic Acid, HA is a major component of the extracellular matrix of vertebrates. It is a glycosaminoglycan hydrolyzed by enzymes of the hyaluronidase family, involved in the regulation of important biological processes such as angiogenesis and vascular permeability. As interest in the development of a synthesis route for this enzyme, we aim to obtain a plasmid containing the coding sequence of the gene variant 8 Hyal-1. To obtain the plasmid insert was planned and two restriction sites for sub-cloning site directed at the 5\' Bam H-1 and 3\' Not-1 in codon sequence of Hyal-1. The insert was sub-cloned into plasmid pET28-a, and transfected for expression in Escherichia coli Bl-21. The expression was induced by IPTG in the best time of 4 hours and confirmation of protein expression was performed by Western blotting. There was a 45 kDa protein, thus confirming the presence of Hyal-1. Purification was performed on nickel-agarose column to obtain a larger amount of the protein, approximately 25µg/L. The route suggested in this study was efficient attainment of Hyal-1 recombinant protein.

A hialuronidase (HAase) é um complexo enzimático que age sobre o tecido conjuntivo frouxo. Existem vários estudos sobre a aplicação da HAase na região perineal, com a finalidade de reduzir a ocorrência de episiotomia e lacerações espontâneas. Apesar dos resultados positivos, as limitações metodológicas destes estudos justificam a controvérsia sobre os benefícios de sua utilização no parto normal. O objetivo do presente estudo foi comparar a frequência de trauma perineal e o grau de laceração perineal espontânea no parto normal, com e sem uso da HAase injetável no períneo. Trata-se de um ensaio clínico aleatório, placebo-controlado e duplo-cego, realizado em 2008, no Centro de Parto Normal do Amparo Maternal, na cidade de São Paulo. A amostra foi de 156 parturientes, alocadas aleatoriamente nos grupos experimental (n=76) e controle (n = 80). Foram incluídas apenas mulheres com feto único e gestação a termo, sem partos vaginais anteriores, que tiveram parto normal na posição semissentada, sem anestesia por bloqueio intra ou extradural. No segundo período do parto, as mulheres do grupo experimental receberam injeção de HAase na região perineal posterior e as do grupo controle, injeção de placebo. Os frascos com a solução foram previamente mascarados e identificados por código numérico. A avaliação do desfecho perineal também foi realizada de forma mascarada por uma enfermeira-juíza. Para a análise inferencial dos dados o teste exato de Fisher foi realizado de forma monocaudal, sendo considerados estatisticamente significantes os valores de p < 0,025. Os resultados mostraram que a integridade perineal (ausência de laceração de qualquer grau na região posterior do períneo ou episiotomia) ocorreu em 34,2% das mulheres do grupo experimental e em 32,5% do grupo controle, sem diferença estatisticamente significante entre os grupos (p = 0,477). O trauma perineal grave (lacerações de 2º e 3º graus e episiotomia) ocorreu em 28,9% das mulheres do grupo experimental e em 38,8% do grupo controle, sem diferença estatisticamente significante (p = 0,131). A profundidade da laceração de 2º grau nos grupos experimental e controle, avaliada com a Peri-Rule®, foi de 1,9 e 2,3 cm, respectivamente. A episiotomia foi realizada em 11 parturientes e quatro mulheres do grupo controle tiveram laceração de 3º grau. Entre as mulheres de ambos os grupos, o trauma mais frequente na região perineal posterior foi a laceração de primeiro grau (56% e 42,6% nos grupos experimental e controle, respectivamente). As únicas alterações perineais observadas, entre 24 a 48 horas após o parto, foram edema ou equimose, presentes em 14,7% e 32,5% das mulheres dos grupos experimental e controle, respectivamente. A média do perímetro cefálico dos recém-nascidos foi maior entre as mulheres com episiotomia e laceração de 3º grau, com valores do IC de 95% não coincidente para os casos de integridade perineal e laceração de 1º e 2º graus, sugerindo que a medida do perímetro cefálico dos recém-nascidos de mães submetidas a episiotomia era maior que dos demais bebês. A quase totalidade dos recém-nascidos apresentou Apgar maior ou igual a sete nos primeiro e quinto minutos de vida (94,2% e 99,4%, respectivamente), indicando a boa vitalidade ao nascer. Todos os bebês permaneceram internados com a mãe no Alojamento Conjunto. Concluiu-se que a utilização da HAase injetável não aumentou a proporção de períneo íntegro nem reduziu a proporção de trauma perineal grave na amostra estudada / Hyaluronidase (Haas) is a complex enzyme that acts on the connective tissue. There are several studies on the implementation of Haas in the perineal area in order to reduce the incidence of episiotomy and spontaneous lacerations Despite positive results, methodological limitations of these studies justify the controversy about the benefits of its use in spontaneous delivery. The aim of this study was to compare the frequency of trauma and the degree of perineal lacerations in vaginal deliveries, with and without the use of injectable Haase in the posterior region of the perineum. This is a randomized clinical trial, placebo-controlled, double-blind study, conducted in 2008 in the Amparo Maternal Birth Center located in São Paulo city. The sample of 156 pregnant women were randomly allocated to experimental groups (n = 76) and control (n = 80). We included only women with singleton pregnancies, pregnancy to term without previous vaginal deliveries and who had spontaneous delivery in semi-sitting position without anesthesia by intra or extra-dural. In the second stage of labor, women in the experimental group received injections of Haas in the posterior perineal area and those of the control group received injections of placebo. The vials of solution were previously masked and identified by code number. The assessment of perineal outcome was also performed under masked by a nurse-judge. For the inferential analysis of data, we performed the Fisher exact test, so tailed and were considered statistically significant p values <0.025. The results showed that the perineal integrity (absence of any laceration in the posterior region of the perineum or episiotomy) occurred in 34.2% of women in the experimental group and 32.5% in the control group, no statistically significant difference between groups (p = 0.477). The severe perineal trauma (second and third degree lacerations and episiotomy) occurred in 28.9% of women in the experimental group and 38.8% in the control group, no statistically significant difference (p = 0.131). The depth of the perineal laceration of 2nd degree in experimental and control groups measured by the Peri-Rule ®, was 1.9 and 2.3 cm, respectively. Episiotomy was performed in 11 pregnant women and four women in the control group had 3rd degree lacerations. Among women in both groups, the most common trauma in the posterior region of the perineum was the first degree (56% and 42.6% in the experimental and control groups, respectively). The only perineal changes observed between 24 to 48 hours after delivery, were swelling or bruising, present in 14.7% and 32.5% of women in the experimental and control groups, respectively. The average head circumference of newborns was higher among women with episiotomy and 3rd degree tears, with CI of 95% not matched to the cases of integrity and perineal laceration at 1st and 2nd degrees, suggesting that the extent head circumference of newborns whose mothers undergo episiotomies was larger than the other babies. Almost all of the newborns had Apgar scores greater than or equal seven in the first and fifth minutes of life (94.2% and 99.4%, respectively), indicating good vitality at birth. All babies roomed-in with the mother during hospital stay. It was concluded that the use of injectable Haase did not increase the proportion of intact perineum and did not reduce the proportion of severe perineal trauma in our sample

As hialuronidases de peçonhas animais são capazes de clivar o hialuronan presente na matriz extracelular, facilitando a difusão das toxinas no tecido da vítima. Essas enzimas têm sido negligenciadas, devido à instabilidade enzimática e à baixa concentração na peçonha. Assim, a expressão heteróloga das hialuronidases permite a sua obtenção em quantidades que viabilizam seu estudo estrutural e funcional. Associado a isso, o transcriptoma propicia a identificação de novas toxinas e componentes de baixa proporção na peçonha. Portanto, o presente trabalho realizou o transcriptoma da glândula de peçonha do escorpião Tityus serrulatus e a clonagem e expressão heteróloga da hialuronidase. No transcriptoma foram obtidos 558 ESTs, dos quais 61,8% correspondem às toxinas, dentre elas neurotoxinas com ação em canais iônicos, metaloproteinas, hipotensinas, peptídeos antimicrobianos, dentre outros. Foram também identificadas novas toxinas como o neuropeptídeo e Ts16.1, descritos pela primeira vez para o gênero Tityus. Dentre os transcritos obtidos, foi identificado apenas um clone correspondente ao C-terminal incompleto de uma hialuronidase de T. serrulatus. Consequentemente, foi produzido o gene sintético contendo a sequência da TsHyal-1, obtida em bancos de dados, no vetor de expressão pPICZ?A para expressão heteróloga em P. pastoris. A rTsHyal-1 foi expressa em escala laboratorial em meio não suplementado (BMM) em pH 7,0 durante 96 h da indução com alimentação diária de metanol a 0,75%. A rTsHyal-1 foi produzida na sua forma solúvel e ativa (838,31 UTR/mg) e resultou em um rendimento proteico de 250 mg/L de material expresso. O secretoma do meio de expressão mostrou que além da rTsHyal-1, a P. pastoris também secreta proteínas nativas associadas à ligação do ATP, metabolismo de carboidrato e resposta ao estresse oxidativo. A rTsHyal-1 foi parcialmente purificada em troca catiônica fraca e apresentou atividade específica de 1.097,45 UTR/mg. A rTsHyal-1 apresenta massa molecular de 49,5 kDa e o tratamento com PNGase F seguido da análise por espectrometria de massas (MALDI-TOF) indicou que uma possível N-glicosilação de 4,5 kDa. Adicionalmente, o sequenciamento dos digestos trípticos da enzima realizado no MALDI-TOF e pelo Q-Exactive resultaram em 46,8% de cobertura da sequencia da proteína. A rTsHyal-1 apresenta especificidade pelo substrato hialuronan, seguido da condroitina C, A e B e apresentou atividade ótima em pH 6,0 e a 40°C. Adicionalmente, o ensaio do MTT indicou que a enzima recombinante não apresenta citotoxicidade in vitro. Os resultados obtidos determinaram as melhores condições para a expressão heteróloga da rTsHyal-1, que corresponde à primeira hialuronidase recombinante de escorpião expressa em P. pastoris com atividade enzimática preservada / The venom hyaluronidases of animals are able to cleave hyaluronan present in the extracellular matrix, acting as a spreading factor for the toxins into the tissues of the victim. These enzymes have been neglected due to their instability and low concentration in the venom. Thus, heterologous expression of hyaluronidases permits the obtainment of sufficient amount for structural and functional studies. Moreover, the transcriptome allows the identification of new toxins or components with low proportion in the venom. In this way, this study aimed the construction of the transcriptome of Tityus serrulatus venom gland and cloning/heterologous expression of hyaluronidase. In the transcriptome, 558 ESTs were identified and 61.8% corresponded to toxins, such as neurotoxins with action on ion channels, metalloproteins, hypotensins, antimicrobial peptides, among others. In addition, new toxins were identified, comprising one neuropeptide and Ts16.1, described for the first time to the genus Tityus. Among the obtained transcripts, one identified clone corresponded to an incomplete C-terminal of a hyaluronidase. Consequently, a synthetic gene was synthesized containing the sequence of TsHyal-1 (obtained from databases) in the pPICZ?A vector for heterologous expression in P. pastoris. The rTsHyal-1 was expressed at laboratorial scale in unsupplemented medium (BMM) at pH 7.0 for 96 h, after induction time, with daily feeding of 0.75% methanol. The rTsHyal-1 was produced in soluble and active form (838.31 UTR/mg) and resulted in a protein yield of 0,266 mg/mL in final expressed material. Besides, the secretome of the medium showed that P. pastoris also secretes native proteins bound with ATP, proteins related to carbohydrate metabolism and oxidative stress response. The rTsHyal-1 was partially purified in a weak cation exchange and presented specific activity of 1097.45 UTR/mg. The rTsHyal-1 has molecular mass of 49.5 kDa and the treatment with PNGase F and analysis by mass spectrometry (MALDI-TOF) indicated a potential N-glycosylation of 4.5 kDa. Additionally, the sequencing of tryptic digests performed in the MALDI-TOF and Q-Exactive resulted in 46.8% of protein sequence coverage. The rTsHyal-1 presents substrate specificity for hyaluronan followed by chondroitin C, A and B and showed an optimum activity at pH 6.0 and 40°C. Furthermore, the MTT assay indicated that the recombinant enzyme does not display in vitro cytotoxicity. These results validate the biotechnological process of the heterologous expression of rTsHyal-1. This is the first recombinant hyaluronidase from scorpions expressed in P. pastoris system with enzymatic activity preserved.

A hialuronidase (HAase) é um complexo enzimático que age sobre o tecido conjuntivo frouxo. Existem vários estudos sobre a aplicação da HAase na região perineal, com a finalidade de reduzir a ocorrência de episiotomia e lacerações espontâneas. Apesar dos resultados positivos, as limitações metodológicas destes estudos justificam a controvérsia sobre os benefícios de sua utilização no parto normal. O objetivo do presente estudo foi comparar a frequência de trauma perineal e o grau de laceração perineal espontânea no parto normal, com e sem uso da HAase injetável no períneo. Trata-se de um ensaio clínico aleatório, placebo-controlado e duplo-cego, realizado em 2008, no Centro de Parto Normal do Amparo Maternal, na cidade de São Paulo. A amostra foi de 156 parturientes, alocadas aleatoriamente nos grupos experimental (n=76) e controle (n = 80). Foram incluídas apenas mulheres com feto único e gestação a termo, sem partos vaginais anteriores, que tiveram parto normal na posição semissentada, sem anestesia por bloqueio intra ou extradural. No segundo período do parto, as mulheres do grupo experimental receberam injeção de HAase na região perineal posterior e as do grupo controle, injeção de placebo. Os frascos com a solução foram previamente mascarados e identificados por código numérico. A avaliação do desfecho perineal também foi realizada de forma mascarada por uma enfermeira-juíza. Para a análise inferencial dos dados o teste exato de Fisher foi realizado de forma monocaudal, sendo considerados estatisticamente significantes os valores de p < 0,025. Os resultados mostraram que a integridade perineal (ausência de laceração de qualquer grau na região posterior do períneo ou episiotomia) ocorreu em 34,2% das mulheres do grupo experimental e em 32,5% do grupo controle, sem diferença estatisticamente significante entre os grupos (p = 0,477). O trauma perineal grave (lacerações de 2º e 3º graus e episiotomia) ocorreu em 28,9% das mulheres do grupo experimental e em 38,8% do grupo controle, sem diferença estatisticamente significante (p = 0,131). A profundidade da laceração de 2º grau nos grupos experimental e controle, avaliada com a Peri-Rule®, foi de 1,9 e 2,3 cm, respectivamente. A episiotomia foi realizada em 11 parturientes e quatro mulheres do grupo controle tiveram laceração de 3º grau. Entre as mulheres de ambos os grupos, o trauma mais frequente na região perineal posterior foi a laceração de primeiro grau (56% e 42,6% nos grupos experimental e controle, respectivamente). As únicas alterações perineais observadas, entre 24 a 48 horas após o parto, foram edema ou equimose, presentes em 14,7% e 32,5% das mulheres dos grupos experimental e controle, respectivamente. A média do perímetro cefálico dos recém-nascidos foi maior entre as mulheres com episiotomia e laceração de 3º grau, com valores do IC de 95% não coincidente para os casos de integridade perineal e laceração de 1º e 2º graus, sugerindo que a medida do perímetro cefálico dos recém-nascidos de mães submetidas a episiotomia era maior que dos demais bebês. A quase totalidade dos recém-nascidos apresentou Apgar maior ou igual a sete nos primeiro e quinto minutos de vida (94,2% e 99,4%, respectivamente), indicando a boa vitalidade ao nascer. Todos os bebês permaneceram internados com a mãe no Alojamento Conjunto. Concluiu-se que a utilização da HAase injetável não aumentou a proporção de períneo íntegro nem reduziu a proporção de trauma perineal grave na amostra estudada / Hyaluronidase (Haas) is a complex enzyme that acts on the connective tissue. There are several studies on the implementation of Haas in the perineal area in order to reduce the incidence of episiotomy and spontaneous lacerations Despite positive results, methodological limitations of these studies justify the controversy about the benefits of its use in spontaneous delivery. The aim of this study was to compare the frequency of trauma and the degree of perineal lacerations in vaginal deliveries, with and without the use of injectable Haase in the posterior region of the perineum. This is a randomized clinical trial, placebo-controlled, double-blind study, conducted in 2008 in the Amparo Maternal Birth Center located in São Paulo city. The sample of 156 pregnant women were randomly allocated to experimental groups (n = 76) and control (n = 80). We included only women with singleton pregnancies, pregnancy to term without previous vaginal deliveries and who had spontaneous delivery in semi-sitting position without anesthesia by intra or extra-dural. In the second stage of labor, women in the experimental group received injections of Haas in the posterior perineal area and those of the control group received injections of placebo. The vials of solution were previously masked and identified by code number. The assessment of perineal outcome was also performed under masked by a nurse-judge. For the inferential analysis of data, we performed the Fisher exact test, so tailed and were considered statistically significant p values <0.025. The results showed that the perineal integrity (absence of any laceration in the posterior region of the perineum or episiotomy) occurred in 34.2% of women in the experimental group and 32.5% in the control group, no statistically significant difference between groups (p = 0.477). The severe perineal trauma (second and third degree lacerations and episiotomy) occurred in 28.9% of women in the experimental group and 38.8% in the control group, no statistically significant difference (p = 0.131). The depth of the perineal laceration of 2nd degree in experimental and control groups measured by the Peri-Rule ®, was 1.9 and 2.3 cm, respectively. Episiotomy was performed in 11 pregnant women and four women in the control group had 3rd degree lacerations. Among women in both groups, the most common trauma in the posterior region of the perineum was the first degree (56% and 42.6% in the experimental and control groups, respectively). The only perineal changes observed between 24 to 48 hours after delivery, were swelling or bruising, present in 14.7% and 32.5% of women in the experimental and control groups, respectively. The average head circumference of newborns was higher among women with episiotomy and 3rd degree tears, with CI of 95% not matched to the cases of integrity and perineal laceration at 1st and 2nd degrees, suggesting that the extent head circumference of newborns whose mothers undergo episiotomies was larger than the other babies. Almost all of the newborns had Apgar scores greater than or equal seven in the first and fifth minutes of life (94.2% and 99.4%, respectively), indicating good vitality at birth. All babies roomed-in with the mother during hospital stay. It was concluded that the use of injectable Haase did not increase the proportion of intact perineum and did not reduce the proportion of severe perineal trauma in our sample