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During the course of our research, we sought to analyze the pathophysiological mechanisms incriminated in ovarian aging and endometriosis, by studying the possible impact of these conditions on the oocyte micro-environment. We have identified specific signatures for these pathologies in the follicular fluid, andin some cases, potential biomarkers. In cases of infertility secondary to isolated endometriosis, we did not find any alteration of the metabolic profile of the follicular fluid. The absence of an alteration in the oocyte micro-environment in women with endometriosis suggests that there is no impact of the disease on the oocyte quality. However, in cases of severe endometriosis, cytokine analysis showed that, in some patients, there is a different expression of MCP1 in the follicular fluid. We also found a modification of the oocyte micro-environment in severe endometriosis, associated with high levels of MCP1. In cases of low ovarian reserve, we found decreased levels of unsaturated plasmalogens and an increase in the methyl-transferase activity, suggesting oxidative stress in the follicular fluid. Moreover, the analysis of the cytokines profile showed a significant decrease in PDGF-BB concentrations in patients with low ovarian reserve. The alteration of the metabolic pathway simplicating PDGF-BB could negatively impact folliculogenesis and the antioxidant defense mechanisms, and could therefore be linked to the infertility seen in patients with low ovarian reserve. Finally, our future research will look to identify eventual biomarkers and to improve our understanding of the pathophysiologic mechanisms involved in recurrent pregnancy losses and recurrent implantation failures following in vitro fertilization treatment.; Nous avons, au cours de nos travaux, tenté d’approcher les mécanismes physiopathologiques liés au vieillissement ovarien et à l’endométriose en étudiant l’impact que ces pathologies pouvaient avoir sur le micro environnement ovocytaire. Nous avons, déterminé dans le fluide folliculaire des signatures spécifiques de la pathologie et, dans certains cas, d’éventuels biomarqueurs. En cas d’infertilité lié à une endométriose isolée, nous n’avons pas retrouvé d’altération du profil métabolique dans le fluide folliculaire. Nos résultats suggèrent qu’il n’existerait pas d’anomalies de l’environnement ovocytaire susceptible d’être lié à une altération de la qualité ovocytaire en cas d’endométriose isolée. En cas d’endométriose sévère, nos données sur l’analyse des cytokines suggèrent que l’endométriose entraine une expression différente de MCP1 dans le fluide folliculaire, en fonction des patientes. Nous constations aussi une modification du micro environnement ovocytaire en cas d’endométriose sévère associé à des niveaux de MCP-1élevé. En cas de réserve ovarienne diminuée, une diminution des plasmalogènes insaturés et une augmentation de l’activité méthyl transférase évoquent un contexte de stress oxydant au sein du fluide folliculaire. L’analyse des profils cytokiniques du FF a permis de constater une diminution significative des concentrations de PDGF-BB en cas de réserve ovarienne diminuée. Par conséquent, une infertilité liée à une réserve ovarienne diminuée pourrait être associée à une altération de voies métaboliques impliquant le PDGF-BB et pouvant être à l’origine de perturbation de la folliculogenèse ou des défenses antioxydantes. Enfin, nos futurs travaux s’intéresseront à la découverte éventuelle de biomarqueurs et à l’amélioration de la compréhension des mécanismes intervenant chez des femmes atteintes de fausses couches à répétition ou ayant vécu des échecs d’implantation répétés lors d’une prise en charge en FIV.

During the course of our research, we sought to analyze the pathophysiological mechanisms incriminated in ovarian aging and endometriosis, by studying the possible impact of these conditions on the oocyte micro-environment. We have identified specific signatures for these pathologies in the follicular fluid, andin some cases, potential biomarkers. In cases of infertility secondary to isolated endometriosis, we did not find any alteration of the metabolic profile of the follicular fluid. The absence of an alteration in the oocyte micro-environment in women with endometriosis suggests that there is no impact of the disease on the oocyte quality. However, in cases of severe endometriosis, cytokine analysis showed that, in some patients, there is a different expression of MCP1 in the follicular fluid. We also found a modification of the oocyte micro-environment in severe endometriosis, associated with high levels of MCP1. In cases of low ovarian reserve, we found decreased levels of unsaturated plasmalogens and an increase in the methyl-transferase activity, suggesting oxidative stress in the follicular fluid. Moreover, the analysis of the cytokines profile showed a significant decrease in PDGF-BB concentrations in patients with low ovarian reserve. The alteration of the metabolic pathway simplicating PDGF-BB could negatively impact folliculogenesis and the antioxidant defense mechanisms, and could therefore be linked to the infertility seen in patients with low ovarian reserve. Finally, our future research will look to identify eventual biomarkers and to improve our understanding of the pathophysiologic mechanisms involved in recurrent pregnancy losses and recurrent implantation failures following in vitro fertilization treatment., Nous avons, au cours de nos travaux, tenté d’approcher les mécanismes physiopathologiques liés au vieillissement ovarien et à l’endométriose en étudiant l’impact que ces pathologies pouvaient avoir sur le micro environnement ovocytaire. Nous avons, déterminé dans le fluide folliculaire des signatures spécifiques de la pathologie et, dans certains cas, d’éventuels biomarqueurs. En cas d’infertilité lié à une endométriose isolée, nous n’avons pas retrouvé d’altération du profil métabolique dans le fluide folliculaire. Nos résultats suggèrent qu’il n’existerait pas d’anomalies de l’environnement ovocytaire susceptible d’être lié à une altération de la qualité ovocytaire en cas d’endométriose isolée. En cas d’endométriose sévère, nos données sur l’analyse des cytokines suggèrent que l’endométriose entraine une expression différente de MCP1 dans le fluide folliculaire, en fonction des patientes. Nous constations aussi une modification du micro environnement ovocytaire en cas d’endométriose sévère associé à des niveaux de MCP-1élevé. En cas de réserve ovarienne diminuée, une diminution des plasmalogènes insaturés et une augmentation de l’activité méthyl transférase évoquent un contexte de stress oxydant au sein du fluide folliculaire. L’analyse des profils cytokiniques du FF a permis de constater une diminution significative des concentrations de PDGF-BB en cas de réserve ovarienne diminuée. Par conséquent, une infertilité liée à une réserve ovarienne diminuée pourrait être associée à une altération de voies métaboliques impliquant le PDGF-BB et pouvant être à l’origine de perturbation de la folliculogenèse ou des défenses antioxydantes. Enfin, nos futurs travaux s’intéresseront à la découverte éventuelle de biomarqueurs et à l’amélioration de la compréhension des mécanismes intervenant chez des femmes atteintes de fausses couches à répétition ou ayant vécu des échecs d’implantation répétés lors d’une prise en charge en FIV.

The quantitative and qualitative analysis of nanoparticles in human biological samples is a daunting scientific endeavour as there are no technical procedures capable to cope with the complexity of biological matrices. The central hypothesis of this work is that the increased presence of metal and metal oxide nanoparticles in pulmonary lavage and reproductive fluids may be associated with idiopathic interstitial lung disease and declining female and male fertility, respectively. The developed extraction procedures in the context of this thesis allowed the measuring of the the hydrodynamic size distribution of gold nanoparticles by DLS and their quantitation my means of ICP-OES. Due to the achieved extraction yields and the successful segregation of biological noise, extracted gold nanoparticles could be representatively observed at the nanoscale. Specifically, BSE could detect the dense gold particle cores at low magnification, and TEM and FESEM could be used to resolve the particles’ Feret diameter and morphology, respectively. Finally, the hard biomolecular corona of extracted gold nanoparticle from pulmonary lavages could be probed with AFM, AES, and XPS to acquire information on its morphology, elemental composition, and chemistry, respectively. The application of developed methodologies on patients did not return analytical data that could positively and significantly associate an elevated particle content with increased odds of idiopathic diseases or with low sperm count.; L'analyse des nanoparticules dans les échantillons cliniques est une entreprise scientifique redoutable puisqu'il n'existe pas de procédures capables de faire face à la complexité des matrices biologiques. L'hypothèse centrale de ce travail est que la présence accrue de nanoparticules d'oxyde de métal et de métal dans le lavage pulmonaire et les fluides reproducteurs peut être associée à une maladie pulmonaire interstitielle idiopathique et à une baisse de la fertilité féminine et masculine, respectivement. Les procédures d'extraction développées dans le cadre de cette thèse ont permis de mesurer la distribution de la taille hydrodynamique des nanoparticules d'or par DLS et leur quantification par ICP-OES. En raison des rendements d'extraction obtenus et de la ségrégation réussie du bruit biologique, les nanoparticules d'or extraites pourraient être observées de manière représentative à l'échelle nanométrique. Plus précisément, l'ESB pourrait détecter les noyaux denses de particules d'or à faible grossissement, et MET et MEB pourraient être utilisés pour résoudre le diamètre et la morphologie des feret des particules, respectivement. Enfin, la corona biomoleculaire dure de la nanoparticule d'or extraite à partir de lavages pulmonaires pourrait être sondée avec AFM, AES et XPS pour acquérir des informations sur sa morphologie, composition élémentaire et chimie, respectivement. L'application de méthodologies développées sur les patients n'a pas renvoyé de données analytiques qui pourraient associer de manière positive et significative une teneur élevée en particules avec une probabilité accrue de maladies pulmonaire interstitielle idiopathiques ou avec oligospermie.

Only 5% of oocytes lead to life birth after IVF. Currently, it is difficult to evaluate oocyte quality on the sole basis of morphology criteria. Embryo morphology is considered as a good marker of potential implantation of embryo after ET, however it is not enough. Therefore, we need to develop non invasive assessment of oocyte quality. Research performed on follicular fluid may represent a new tool of increasing interest. The presence of G-CSF in follicular fluid could predict ongoing pregnancy. Cytokines, IL-7 and IL-17 could be also a predictor of success.

Follicular fluid is a complex extracellular fluid, semi-viscous and yellow in colour, which accumulates in the antrum of ovarian follicles during their growing phase. Follicular fluid provides the microenvironment within which the cumulus-oocyte complex matures and granulosa cells differentiate. Scientists agree that follicular fluid derives mainly from plasma via the vascular compartment in the follicle wall. However, it also contains factors produced locally by the follicle cells, the production of which varies during different reproductive states. The aim of this paper is to review current knowledge on the formation, composition and roles of follicular fluid.

To evaluate the importance of follicular flushing on semi natural cycle IVF.We have compared prospectively the reproductive potential of oocytes obtained from follicular fluid (LF, N = 79) to those obtained from follicular flushing (R, N = 47) in 146 oocyte pick ups.The group LF and R were similar with regard to fertilization rate (79.7 versus 88.1%, respectively), percentage of superior grade embryos (28.8 versus 37.8%) and implantation rate (24.1 versus 44.1%).The practice of follicular flushing on semi natural IVF cycle improves the pregnancy rate. The oocytes obtained by follicular flushing had the same reproductive potential than those obtained on follicular fluid.

Modern gonadotrophin-releasing hormone (GnRH) antagonists such as cetrorelix and ganirelix reliably prevent premature LH surge in controlled ovarian hyperstimulation for assisted reproductive technologies. Nevertheless, GnRH antagonists do not allow homogenization of the follicle cohort, as observed with GnRH agonists. Administration of estrogens, progestogens or contraceptive pill in previous late luteal phase, may improve the size homogeneity of antral follicles. The coordination of follicular development may optimize ovarian response to r-FSH/GnRH antagonist protocols.

During their travel through the feminine genital tract, spermatozoa undergo a continuous, reversible and non specific maturation called "capacitation". Capacitation allows the ability to bind to the zona pellucida and to start acrosome reaction after induction. Capacitation is associated with biochemical modifications of the membrane and the cytoplasma. To fertilize oocytes, spermatozoa must first become capacitated, change their motility to an hyperactivated pattern, without undergoing a premature, spontaneous acrosome reaction. In vitro, follicular fluid, progesterone, pentoxifylline and adenosine analogues have been shown to improve capacitation without inducing acrosome reaction in case of male infertility.

It has been demonstrated that the preovulatory follicular fluid creates a micro-environment which is of importance for normal sperm function. In this paper, we attempt to evaluate the role of human follicular fluid (HFF) on the fertilizing capacity of spermatozoa.

The acrosome reaction that occurs after sperm capacitation, is an exocytotic event induced by a Ca++ influx. It plays an essential role during fertilization, by making spermatozoa able of penetrating the zona and capable of fusing with the egg plasma membrane. Zona pellucida is the natural inducer of the acrosome reaction. Binding of the sperm receptor to ZP3, a zona glycoprotein acting as ligand, triggers the molecular events leading to acrosomal exocytosis. G-proteins may be involved in the signal-transduction pathway during the acrosome reaction. Other known inducers of the human acrosome reaction include follicular fluid, progesterone and the calcium ionophore A23187. Progesterone acts through a receptor on the sperm plasma membrane, while the ionophore promotes non-physiological sperm Ca++ uptake. Several cytochemical procedures have been proposed for evaluating the acrosome reaction: the acrosomal status can be observed after staining or after labeling with lectins or antibodies. These methods both attempt to evaluate sperm viability and to distinguish degenerative acrosomal loss.

The effect of dietary energy balance (EB) on growth of ovarian follicles was tested. Cows (n = 9) were fed a high energy diet (HE diet; positive EB; n = 4) or switched to a low energy diet (LE diet; negative EB; n = 5) during the preovulatory period. Non-esterified fatty acids (NEFA) were greater in cows fed the LE diet. Concentrations of luteinizing hormone (LH) were similar in HE and LE cows. However, the growth of preovulatory follicles in cows fed the LE diet was 50% that of cows fed the HE diet. Insulin-like growth factor-I (IGF-I) in plasma was less in LE-fed cows compared with HE-fed cows, and plasma IGF-I was positively correlated to estrogen: progesterone ratio in follicular fluid of dominant follicles. In summary, slower follicular growth in cows fed an LE diet occurred despite normal plasma LH and coincided with reduced IGF-I and elevated NEFA in plasma.

The aim of this work is to study and compare lipid composition of peritoneal and ovulatory follicular fluid of women. The studied patients are involved in a FIV protocol. We have studied 53 peritoneal fluids and 90 follicular fluids from 33 patients. On each sample the following parameters are estimated: cholesterol, triglycerides, phospholipids, non esterified fatty acids (A.G.N.E.), HDL and LDL cholesterol, HDL and LDL phospholipids. Lipid composition of follicular and peritoneal fluids differ from these of blood plasma. The two mediums studies show no differences with regard to their lipid composition. In both cholesterol is only found as HDL cholesterol. Cholesterol arising from spermatozoa membranes may be implicated in capacitation phenomenon. HDL Cholesterol, the only form found in the studied mediums, may act as a cholesterol acceptor. Theses two points partially explain the fact that follicular and peritoneal fluids are good mediums for capacitation and survival of spermatozoa.

Plasmatic estradiol (E2), progesterone (P) and LH were measured during the follicular phase of 343 cycles induced for in vitro fertilization (IVF) using a LHRH agonist in a "long protocol" (Group I) and 76 cycles in a "short protocol" (Group II). Moreover measurements in the plasma and follicular fluid (FF) of E2, P, LH, Delta-4-androstenedione (A), Testosterone (T) and prolactin (PRL) were performed on the day of oocyte retrieval (DO) in 46 women of the group I (111 FF) and 27 of the group II (67 FF). In the group I, plasma LH always remains below 3 mUl/ml, whatever the type of agonist (Buserelin or DTRP6-LHRH) and the type of stimulation (HMG or FSH) are used. On the other hand in the group II, mean plasma LH and P levels from D-5 to D-2 and those of FF LH, T and A on DO are significantly higher than in the group I. These changes are associated with a significant decrease of retrieved oocytes (5.8 versus 7.8 p less than 0.0001), pregnancy rate (15% versus 30%, p less than 0.01) and ongoing pregnancy rate (10% versus 22%, per oocyte retrieval, p less than 0.01). They suggest that the pituitary desensitization could be unsatisfactory with the short protocol use of agonist.

The ultrastructure of rat granulosa cells from preovulatory follicles has been investigated after incubation in control fetal calf serum (FCS) medium with or without LH (1 micron/ml), and in total or steroid-free pig follicular fluid (FF) medium. While the cells had a similar ultrastructure after 1 day of incubation in these media, significant differences were detectable after 3 days. In FCS supplemented with LH, autolysis and phagocytotic activity remained elevated, and cells did not develop any of the typical ultrastructural features of steroid-secreting cells. In contrast, total FF induced complete differentiation of mitochondrial cristae from the lamelliform to the villiform type. Steroid-free FF induced the same array of effects as total FF, except that mitochondrial differentiation was not complete. These observations together with those of previous studies, in which progesterone secretion was measured in cells incubated in the same media, suggested that the development of the capacity to secrete progesterone in rat luteal cells could be directly related only to mitochondrial differentiation.

Changes in sexual behavior and progesterone, oestrone, oestradiol, testosterone and androstenedione concentrations have been studied in the follicular fluid of 25 pseudopregnant rabbits on Days 6, 9, 12, 15 and 18. The sexual behavior score seemed to rise at the end of pseudopregnancy and follicular steroid levels were higher on Days 6 and 18 of pseudopregnancy than on Days 9, 12 or 15. The generalized distance D2 of Mahalanobis showed the existence of four periods during pseudopregnancy: Day 6, Days 9 and 12 which were similar, Day 15 and Day 18. These stages were found to differ by a compound of the sexual behavior score and the concentrations of androgens and oestradiol in follicular fluid. There was no relationship between sexual behavior and follicular steroid concentrations. Nevertheless, undetectable levels of oestradiol in the follicular fluid were only observed in deeply dioestrus females.