L’hormone anti-Müllerienne (AMH), une glycopréotéine exclusivement produite par les cellules de la granulosa (CG) des follicules ovariens de la femme, est un marqueur unique de du statut folliculaire ovarien. Contrairement à l’inhibine B, l’estradiol et la FSH, l’AMH est produite par un large éventail de follicules allant des follicules primaires aux follicules à petit antrum. Cependant, les mécanismes précis régulant la production d’AMH par les CG restent mal connus. Nous avons montré que la sélection folliculaire précoce au cours de la phase de transition lutéo-folliculaire, un phénomène fréquemment retrouvé chez les femmes ayant un vieillissement ovarien, caractérisé par la présence d’au moins un follicule surdéveloppé au cours de la phase folliculaire précoce, n’altérait pas la puissance de la relation entre le compte folliculaire antral et les concentrations sériques d’AMH. En revanche, cette situation perturbait significativement celle entre le nombre de follicules antraux et les taux sériques de FSH, d’inhibine B et d’estradiol. Nous avons par la suite mis en évidence, en utilisant un nouvel outil, nommé Follicular Output RaTe (FORT), que le pourcentage de follicules qui répondent effectivement à la FSH exogène en atteignant la maturation pré-ovulatoire, était négativement et indépendamment lié aux taux sériques d’AMH, ce qui va dans le sens de l’hypothèse d’un effet inhibiteur de l’AMH sur la sensibilité des follicules à la FSH. Ensuite, nous avons regardé si la production d’AMH par ovaire et par follicule était altérée chez les femmes n’ayant plus qu’un seul ovaire suite à une ovariecomie unilatérale. En effet, tout indique que chez ces femmes, des réarrangements majeurs de la folliculogenèse sont mis en place pour maintenir une fonction ovarienne malgré la perte brutale d’une partie du pool folliculaire. Ainsi, par une analyse extensive et comparative de la folliculogenèse utilisant des marqueurs hormono-folliculiares, nous n’avons pu mettre aucune modification significative chez les femmes avec un ovaire unique, comparativement aux contrôles. A l’aide du modèle précédemment utilisé, nous avons constaté une augmentation de la sensibilité des follicules antraux à la FSH exogène, évaluée par le FORT, chez des femmes avec un seul ovaire, comparativement aux femmes avec 2 ovaires. Ces résultats supportent l’hypothèse d’une augmentation de la sensibilité folliculaire à la FSH, qui pourrait faire partie des possibles mécanismes compensatoires en jeu dans le maintien d’une folliculogenèse efficace chez les femmes ayant eu une ovariectomie unilatérale.Finalement, à l’aide de 2 approches complémentaires, in vitro and in vivo, nous avons montré que la FSH et l’AMPc stimulaient la transcription de l’AMH, et que la LH avait un effet additif. Nous avons montré que les gonadotrophines et l’AMPc agissaient à travers la protéine kinase A et la P38 MAP Kinase, impliquant notamment les facteurs de transcription GATA binding factor-4 et le steroidogenic factor-1. Par ailleurs, nous avons également mis en évidence que l’expression d’AMH pouvait être régulée de manière différentielle par l’estradiol, en fonction du type de récepteur aux estrogènes exprimés par les CG. Ainsi, la chute d’expression de l’AMH au sein des CG des follicules matures, qui expriment essentiellement ERβ, est probablement liée à un effet de l’estradiol. En résumé, ces travaux de thèse ont permis d’apporter de nouvelles données sur la régulation de la croissance folliculaire et sur la production d’AMH chez la femme. / Anti-Müllerian hormone (AMH), a glycoprotein that is exclusively produced by the granulosa cells (GC) of ovarian follicles in the adult female, is a unique biomarker of ovarian follicular status. In contrast with inhibin B, estradiol and FSH, AMH is produced in a wide range of follicles that goes from the primary to the small antral stages of folliculogenesis. However, the precise mechanisms that drive AMH expression by GC remain poorly understood.We showed that untimely and/or accelerated antral follicle growth during the luteal–follicular transition, a phenomenon that is frequent in ovarian-aged women and that is characterized by the presence of at least one overdeveloped antral follicle during the first days of the follicular phase does not alter the strength of the relationship between antral follicle count and serum AMH levels but does affect the relationship between serum FSH, inhibin B and estradiol levels and the number of antral follicles. The heftiness of AMH in relation to advanced antral follicle growth provides a further explanation for the reported stronger association between serum AMH levels and antral follicle counts as compared with the other hormonal markers of the ovarian fertility status. We subsequently demonstrated, using an innovative tool, the Follicular Output RaTe (FORT), that the percentage of follicles that effectively respond to exogenous FSH by reaching pre-ovulatory maturation is negatively and independently related to serum AMH levels, which is in keeping with the hypothesis that AMH inhibits follicle sensitivity to FSH. Given this hypothesis, we wondered if per-ovary and per-follicle AMH production could be altered in patients having a single ovary as a result of unilateral oophorectomy. Indeed, all indicate that major rearrangements of folliculogenesis occur to preserve and maintain ovarian function despite the abrupt halving of follicular stockpile in these patients. We performed an extensive and comparative evaluation of the folliculogenesis using homono-follicular markers failing to show major changes in unilaterally oophorectomized when compared with control women. Using the same model, we demonstrated an increased antral follicle responsiveness to exogenous FSH, as assessed by FORT, in normo-ovulating unilaterally oophorectomized women undergoing controlled ovarian hyperstimulation. These results support the hypothesis that increased FSH sensitivity ranks among the possible compensating mechanisms at stake in the maintenance of successful folliculogenesis after unilateral oophorectomy.Finally, using complementary approaches, in vitro and in vivo, we showed that FSH and cAMP enhance AMH transcription, and LH has an additive effect. Gonadotropins and cAMP act through protein kinase A and p38 MAPK signaling pathways and involve the GATA binding factor-4 and steroidogenic factor-1 transcription factors, among others. The expression profile of AMH and the dynamics of serum AMH after gonadotropin stimulation have been interpreted as a down-regulating effect of FSH upon AMH production by GC. We also demonstrated that AMH expression can be differentially regulated by estradiol depending on the estradiol receptors by GC. Therefore the decrease in AMH expression by GC of mature follicles, which mainly express ERβ, is likely due to the effect of estradiol.In short, this Ph.D. work offers new insight into the regulation of the follicular growth and AMH production in woman.

Alterações morfogênicas do aparelho cardiorespiratório de bovinos provenientes de fecundação in vitro e transferência nuclear são um dos principais fatores responsáveis pela alta incidência de mortalidade embrionária, fetal e pós-natal. Utilizamos técnicas empregadas em microscopia de luz para estudar o desenvolvimento do coração e pulmão nestes animais. Verificamos que em embriões provenientes de fecundação in vitro, aos 28 dias de gestação, aparece o tubo laringotraqueal e sua septação através da prega traqueoesofágica. Neste mesmo período os embriões apresentaram cavidade pericárdica, átrio dividido em direito e esquerdo, cone cardíaco, seio venoso, camada de miocárdio e epicárdio. O brônquio traqueal foi observado em embriões com idade gestacional de 36 dias a partir de um brotamento na porção lateral direita da traquéia, cranial a sua bifurcação. Aos 44 dias de gestação os brotos pulmonares dos embriões apresentaram brônquios principais originando brotamentos de brônquios segmentares. O mesênquima de sustentação em diferenciação continha vasos sangüíneos dispersos, diferentemente de embriões provenientes de TN, que com 68 dias de gestação apresentou pulmão em fase pseudoglandular contendo brotos de bronquíolos e poucos vasos sangüíneos nos cortes obtidos e analisados. Com 70 dias, o coração do feto apresentava ventrículo significativamente grande, pequeno átrio e pulmão não desenvolvido. A partir dos nossos resultados, concluímos que alterações genéticas, incompletas informações e comunicações celulares e modificação no metabolismo celular são os prováveis responsáveis pelas anomalias presentes nas técnicas de manipulação de embrião, causando um desenvolvimento mais lento e falho quando comparados com embriões in vivo, explicando o alto índice de perda gestacional / Morphogenetic changes of cardio-respiratory system of bovine from in vitro fertilization and nuclear transfer is a major factor responsible for high incidence of both periods embryonic, fetal and postnatal mortality. We used techniques of light microscopy to study the development of heart and lung in these animals. We found that in embryos derived from in vitro fertilization, at 28 days of gestation, appears the laryngotracheal tube and its fold through tracheoesophageal septation. In the same period the embryos showed pericardial cavity, atrium divided into left and right, cardiac cone, venous sinus, layer of myocardium and epicardium. In embryos with 36 days of gestational age was observed the tracheal bronchus from a bud in the right lateral portion of the trachea, cranial to its bifurcation. At 44 days of gestation, the lung buds of the embryos showed main bronchi giving rise to budding of segmental bronchi. The sustentation mesenchyme in differentiation contained scattered blood vessels, unlike embryos from TN, which with 68 days of gestation showed lung in pseudoglandular stage, containing bronchioles buds and few blood vessels in histological sections obtained and analyzed. With 70 days, the fetal heart ventricle had significantly large, small atrium and lungs not developed. From our results we conclude that genetic alterations, incomplete information in cellular communications and change in cellular metabolism are likely responsible for the anomalies in the techniques of embryo manipulation, causing a slower and flawed development when compared to embryos in vivo, explaining the high rate of pregnancy loss

In vitro fertilization (IVF) is used to help infertile couples achieve a live birth. Clinical studies have suggested that multiple, consecutive cycles of IVF can increase live birth rate significantly. Others have documented improved live birth rates from the use of new laboratory techniques for preimplantation genetic screening (PGS). This genetic screening technique seeks to determine the ploidy of the embryo prior to implantation into the woman. To date, no study has examined the cost-effectiveness of using IVF in conjunction with PGS compared to that of IVF alone for 3 consecutive cycles in achieving a live birth. This study compared the incremental cost-effectiveness ratios (ICER) from each intervention arm based on the clinical probabilities for each outcome and this study was grounded in the protection motivation theory. Costs were obtained from secondary sources, such as the literature and government databases. The model was constructed using a decision-analytical approach that allowed for z test statistical analysis of the outcomes, where the ICER is the dependent variable and the independent variables are the 2 interventions. The robustness of the model was tested through univariate and probabilistic sensitivity analysis and stratified by age groups. The results showed that PGS with IVF was cost-effective for women aged under 40 and women aged 40-42, but not for women over 42. Based on a willingness-to-pay threshold of $100,000, IVF with PGS was the most cost-effective strategy in all age groups. The positive social change implication of this study is such that understanding the costs associated with a new technology to achieve a live birth is significant for society to help guide clinical treatment of these patients.

The number of women diagnosed as infertile continues to grow every year. The psychological impact of the infertility experience has been said to create distress equivalent to that associated with life-threatening illnesses and has been linked with posttraumatic stress disorder (PTSD). Using shattered assumption and stress-buffering theory, this quantitative causal comparative study explored the potential functional relationship between infertility and PTSD. The majority of the (all-female) participants were 24- to 34-year-old college graduates. A 2 X 3 factorial between-subjects ANOVA examined and compared the cause and effect of the independent variables, fertility treatment and psychological intervention, on the dependent variable, PTSD. A multiple linear regression was conducted to understand PTSD symptomology scores. The results revealed that the type of infertility treatment does not impact PTSD symptomatology in medically diagnosed women. However, the main effect of psychological treatment was significant, as was infertility treatment by psychological treatment interaction. Additionally, the impact of fertility problems on the participant's physical health was the highest ranking predictor, which suggests that stress levels in women receiving infertility treatment are equivalent to those in women with cancer, AIDS, and heart disease, as suggested by other researchers. This study has implications for positive social change, in that it may promote better understanding of the psychological impact of infertility and decreased PTSD symptomatology for medically diagnosed infertile women. It opens the door for future research about the effectiveness of psychological intervention, and provides awareness of possible PTSD susceptibility.

The follicle and its enclosed oocyte share intimate and critical communication that regulates folliculogenesis and produces a mature oocyte. Protein and RNA accumulated in the oocyte during oogenesis control fertilization and direct embryonic development until the embryonic genome activates. Most knowledge of mammalian oocyte biology has been derived from eutherian species. Marsupials deserve more detailed studies because they have a distinct reproductive biology that offers a unique perspective from which to consider mammalian reproduction. The oocyte biology of the tammar wallaby, Macropus eugenii, is the focus of research in this thesis. Cold storage, a simple method for transporting ovarian tissue, was evaluated using histological techniques and follicle culture to assess the structure and function of tammar ovarian tissue. In vitro techniques were used to examine and compare: -folliculogenesis in prepubertal and adult animals, - fertilization of in vivo and in vitro matured oocytes, - and embryo development in follicular and tubal oocytes. / Tammar ovaries were placed in cold storage (PBS at 4?C) for 24 or 48 hours. Necrotic changes were minimal in ovarian follicles after cold storage and preantral follicles isolated from ovarian tissue after cold storage grew by similar amounts as non-stored follicles when cultured for 4 days in vitro. Although the general morphology and growth of follicles are unaffected after cold storage for up to 48 hours, the viability of the oocyte is of prime importance. The next important stages of this study were to develop in vitro techniques for follicle culture and for oocyte maturation and fertilization for future assessment of oocytes after cold storage. / A defined (serum-free) culture system was developed to grow isolated preantral follicles from prepubertal and adult tammars. FSH promoted follicle growth and antrum formation in follicles from prepubertal tammars. Although FSH promoted growth in follicles from adult tammars, other factors present in serum were required for antrum formation. Therefore, once the hypothalmo-pituitary-gonadal axis is mature, hormones and growth factors modify the mechanism of antrum formation. Only follicles that developed an antrum in the presence of serum had granulosa and theca layers that had appropriately differentiated. While FSH stimulates follicle growth in vitro, more complex conditions are required to promote granulosa and theca differentiation. / Intra-cytoplasmic sperm injection (ICSI) was successfully used to compare fertilization of in vivo and in vitro matured oocytes as well as the development of mature oocytes collected from the ovary (surrounded by zona pellucida) or from the oviduct (surrounded by zona pellucida and mucoid coat). In vitro matured oocytes proceeded though the early stages of fertilization (e.g. sperm nuclear decondensation, pronuclear formation), but not syngamy. After sperm injection, in vivo matured oocytes cleaved as far as the 8-cell stage. Oocytes do not lose their ability to fertilize after acquisition of the mucoid coat, since tubal oocytes cleaved as far as the 8-cell stage after sperm injection. Follicular oocytes develop as far as the 5-cell stage after sperm injection, but embryos had a large cleavage cavity that hindered cell-cell contact. While the mucoid coat is not required for cleavage, it is important for appropriate cell-cell interaction and normal early development of the embryo. / This, the most detailed in vitro study of marsupial oocyte biology, has shown that there are many similarities in the biology of marsupial and eutherian oocytes but that the unique biology of marsupials offers a significant perspective on mammalian reproduction. This work also lays the foundation for the effective use of assisted reproductive techniques for conservation of Australia’s unique mammalian fauna.

<p>The main aims were to investigate emotional reactions during in vitro fertilization (IVF) treatment among women and men, attitudes of IVF couples towards cryopreserved embryos and public attitudes towards various aspects of oocyte donation in Sweden. Assessments of emotional, physical and social reactions were made on a daily reaction scale and attitudes on the basis of study-specific questionnaires. Both women and men experienced the stages of oocyte retrieval and embryo transfer, as well as the luteal phase, as being particularly stressful and there was a similarity in the type of reaction pattern. Presence of supernumerary embryos for cryopreservation increased the women’s optimism, but did not seem to reduce their level of distress on the day of embryo transfer. One-third of IVF couples chose not to use their cryopreserved embryos. The main reasons among the respondents concerned family planning and too short a legally allowed time of embryo storage. There was strong support for oocyte donation among a subset of the Swedish population. Women were more positive than men towards oocyte donation and to disclosure of the origin to offspring. Factors that might increase the likelihood of women becoming oocyte donors were talking to women with experience of donating oocytes, proximity to the clinic, accessibility of counselling and having children of their own. These results indicate that different approaches to psychological care for women and men may not be warranted during IVF treatment. Contact should be maintained during the cryopreservation period in order to adress the couple’s questions and concerns about the embryos. To increase the donor pool, IVF clinics could provide information about donation to potential donors through the Internet and through experienced donors. The information to donors and recipient couples about different consequences of donation seems to be of great importance.</p>

The first goal of this dissertation is to investigate the status of the fetus in Swedish law. The second goal is to contribute to the debate concerning attitudes toward mankind, attitudes toward the fetus, and the value of human life as these are expressed in our everyday concepts of law. The tbird goal is to contribute to greater uniformity in the future in the laws which touch upon human beings in the early phases of life (the fertilized egg, the blastula and the fetus). The study is based on an analysis of printed official government documents and records, instructions, motions, bills, minutes of the Riksdag, replies to official queries from experts and affected parties, and laws and government studies made prior to the drafting of legislation, such as SOU and Ds. The areas of the law which are analyzed include (i) the Abortion Act, (ii) the Transplant Act, (iii) the Act concerning Measures for Purposes of Research or Treatment Involving Fertilized Human Ova (LBÄ), (iv) the Act on the Treatment of Alcoholics and Drug Abusers (LVM) in certain cases, (v) the Inheritance Code, (vi) the National Registration Act, and (vii) the Burial Legislation. There are divergent views of the fetus in the various laws and statutes. This is partly due to the dissimilar purposes of the different laws, and partly due to the fact that the fetus is seldom or never the actual subject of these laws. The laws have also come into being at different points in time. The result is that the fetus is not regarded in the same way legally and morally in the divergent laws and statutes. In the conclusion the acceptability of these discrepancies is discussed. Special attention is paid to the debate concerning the underlying values. the divergence among them, and the extent to which such differences are acceptable. Finally, a discussion follows about what might be done, which leads into an appeal for an unbiased study of the need for uniform legal protection for the fetus in Swedish law.

The main aims were to investigate emotional reactions during in vitro fertilization (IVF) treatment among women and men, attitudes of IVF couples towards cryopreserved embryos and public attitudes towards various aspects of oocyte donation in Sweden. Assessments of emotional, physical and social reactions were made on a daily reaction scale and attitudes on the basis of study-specific questionnaires. Both women and men experienced the stages of oocyte retrieval and embryo transfer, as well as the luteal phase, as being particularly stressful and there was a similarity in the type of reaction pattern. Presence of supernumerary embryos for cryopreservation increased the women’s optimism, but did not seem to reduce their level of distress on the day of embryo transfer. One-third of IVF couples chose not to use their cryopreserved embryos. The main reasons among the respondents concerned family planning and too short a legally allowed time of embryo storage. There was strong support for oocyte donation among a subset of the Swedish population. Women were more positive than men towards oocyte donation and to disclosure of the origin to offspring. Factors that might increase the likelihood of women becoming oocyte donors were talking to women with experience of donating oocytes, proximity to the clinic, accessibility of counselling and having children of their own. These results indicate that different approaches to psychological care for women and men may not be warranted during IVF treatment. Contact should be maintained during the cryopreservation period in order to adress the couple’s questions and concerns about the embryos. To increase the donor pool, IVF clinics could provide information about donation to potential donors through the Internet and through experienced donors. The information to donors and recipient couples about different consequences of donation seems to be of great importance.

El objetivo del presente trabajo consistió en valorar el efecto de las células epiteliales del oviducto porcino y del cumulus oophorus sobre distintos parámetros relacionados con la fecundación in vitro en la especie porcina. Las variables estudiadas fueron el porcentaje de penetración, el número medio de espermatozoides por ovocito, el porcentaje de formación de pronúcleo masculino, el porcentaje de monospermia, el porcentaje de cigotos divididos a las 48 horas postfecundación, el porcentaje de blastocistos, el número medio de núcleos por blastocisto, la resistencia de la zona pelúcida a la digestión con pronasa y la densidad y distribución de los gránulos corticales en los diferentes grupos experimentales. / The objective of this experimental work was to study the effect of porcine epithelial oviductal cells and cumulus cells on several parameters related to in vitro fertilization in pigs. Studied variables were rate of penetration, mean number of sperm cells per penetrated oocyte, rate of male pronuclear formation, cleavage rate at 48h postinsemination, blastocyst formation rate, mean number of cells per blastocyst, resistance of zona pellucida to pronase digestion and density and distribution of cortical granules in the different experimental groups.

Con el presente trabajo se ha pretendido investigar la influencia de diversos factores relacionados con el cocultivo de los gametos porcinos, sobre los resultados de la fecundación" in vitro" (AV), fundamentalmente con la intención de mejorar la eficacia actual del sistema de AV en cuanto a la consecución de embriones viables (fecundaciones monospérmicas). Para ello, se han utilizado 129 hembras porcinas prepúberes a las que se indujo la ovulación mediante un tratamiento con 1250 U.I. de PMSG y 750 U.I. de HCG. El tratameinto empleado resultó eficaz para los fines perseguidos en un 80'62% de las hembras y el número medio de ovocitos recogidos fue de 19'07 + 1'52 por animal, utilizándose un total de 1984 ovocitos.En relación a las condiciones del sistema de fecundación, el primer factor investigado ha sido el tiempo de cocultivo, entendido como tiempo de contacto entre los gametos. En las dos experiencias realizadas, utilizando tiempos de 4, 6 u 8 horas (experiencia la), o de 1, 2, 3 ó 4 horas (experiencia lb), los mejores resultados se obtuvieron tras 4 horas de cocultivo, ya que los porcentajes de penetración de mantuvieron altos con respecto al máximo alcanzado a las 8 horas (82,68 vs. 93,96%), mientras que los de monospermia no disminuyeron excesivamente con respecto a los obtenidos con tiempos de cocultivo menores, teniendo en cuenta que la concentración de espermatozoides empleada fue intencionadamente elevada (12 x 105 esp/ml). El segundo factor analizado fue la concentración espermática. Se utilizaron concentraciones de 3, 6 y 12 x 105 esp vivos/ml, deduciéndose de los resultados que la mayor efectividad en nuestro sistema correspondía a la concentración de 6 x lO 5 esp/ml, ya que los porcentajes de penetración fueron significativamente diferentes a los obtenidos con la concentración espermática más alta (71'62% vs. 76'83%), y los porcentajes de monospermia tampoco se diferenciaron de los obtenidos con la concentración espermática más baja (62'26 vs. 68'08%). El tercer factor estudiado ha sido la influencia de la presencia o ausencia en el medio de cocultivo del "cumulus" expandido que acompaña al ovocito en la ovulación. Por los resultados obtenidos, se puede pensar que la presencia estas células junto con la correspondiente matriz intrecelular de ácido hialúrico es altamente beneficiosa para la mejora del rendimiento de la FIV debido a que los porcentajes de penetración en los ovocitos denudados (53'69 fueron menores (p<0'0l) que los obtenidos en los ovocitos con "cumulus" (69'10%), mientras que los porcentajes de monospermia fueron superios (p < 0'01) en el segundo caso (39'45% vs. 60'97%). Por último, se ha investigado el efecto de la reducción del volumen medio de cocultivo más comúnmente utilizado (2 mI) a otro menor (0'4 n obteniéndose resultados equivalentes en ambos casos para los porcentajes penetración, pero mayores porcentajes de monospermia (p<0'05) con volumen de 0'4 mI (57'53% vs. 78'12%). Del conjunto de los resultados se deduce que los porcentajes de penetración y polispermia en la AV porcina son consecuencia de la influencia de diferentes factores, entre los que se encuentran el tiempo de cocultivo, concentración espermática, la presencia del "cumulus oophorus" y el volumen de medio de cocultivo utilizados. / In the present work, we have investigated the influence of differen factors, related to porcine gametes coculture, on the results of "in vitro" fertilization (IVF). We have try to improve the efficiency of the current system to get viable embryos (monospermic fertilizations). 129 prepuberal gilts have been used after the induction of ovulation by administration of 1250 I.U. of PMSG followed, 55 hours later, by 750 I.U. of HCG. The results showed that the best moment for the recovery of oocytes was 44 h after HCC administration. In the same way, the treatment followed was effective for the required objectives in 80.62% of the studied females and the medium number of recovered oocytes was 19.07 + 1.52 per animal, giving a total number 01 1984 oocytes used. In relation with the conditions of the fertilization system, the first investigated factor was the coculture time, understanding it as contact time between gametes. To study the effect of this factor, two experiences were realized; fot the first one, 4, 6 or 8 hours of coculture time were used (experience lb) The best results were obtained at 4 hours of coculture, because the percentage of penetration was maintained high (82.68%) and, at the same time, the percentage of monospermy increased (p<O.Ol), although the sperm concentration employed was deliberately high (12 x lOS spx/ml). The second investigated factor was the sperm concentration. The results showed that, among the used concentrations (3, 6 and 12 x lO 5 alive spz/ml), the maximum effectiveness in our system was obtained for the concntration of 6 x lOS spz/ml, since the percentage of penetration was not signficatively different of that obtained with the highest sperm concentration The third studied factor was the influence of the presence or absence of the expanded ."cumulus", which is shed with the oocytes at the ovulation, in the coculture medium. The presence of these cells joined with the intercellular matrix of hialuronic acid was highly beneficious for the improvement of the IVF, because of the percentage of penetration with the denuded oocytes (53.69%) was lower (p<0.01) than that obtained with the .cumulus" enclosed oocytes (69.16%), and the percentage of monospermy was higher (p<0.01) at the second case (39.45% vs. 60.97%). Finally, the percentage of penetration and monspermy was investigated using two different coculture medium volume, one the commonly used by other authors (2 ml) and another minor volume (0.4 mI). The results showed that the percentage of monospermy was higher (p<0.01) with the 0.4 mI volume (57.53 vs.78312%). We may deduce from the total results that the percentages of and monospermy in porcine IVF are due to the influence of different factors, some of them being the coculture time, the sperm concentrarían, the presence of "cumulus". and the coculture medium volume.