Impact of chemotherapy and age on human ovarian tissue

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Signaling pathways governing the in vitro follicular activation in mouse and human model: Impact of ovarian tissue processing and prior chemotherapy exposure

Advancements in oncology have improved the overall survival rate of cancer patients. However, chemotherapeutic regimens can be gonadotoxic and induce infertility. To ensure good quality of life after treatment, preservation of fertility is highly recommended. Among the available techniques, ovarian tissue cryopreservation (OTC) is an attractive approach that can be performed without postponing cancer treatment or after first-line chemotherapy. It is also the only available option for children. This technique consists of freezing the cortical tissue followed by grafting after remission in order to restore fertility. The major limitation is the risk of recurrence of the disease if cancer cells are present in the stored tissue. As an alternative, in vitro follicle growth until maturation with subsequent fertilization of the oocyte appears to be a promising option for all patients. However, this procedure is challenging and has poor efficacy in humans. To optimize this method, a better understanding of the mechanisms regulating primordial follicle activation (PFA) in vitro as well as the factors that modulate it is essential. This project aimed to investigate two signaling pathways, the PI3K/AKT/mTOR and Hippo signaling pathways, during spontaneous and chemotherapy induced-PFA in vitro using mouse model. The impact of a first-line chemotherapy exposure was further studied in ovarian tissue from adults and children exposed (or not) to chemotherapy before OTC. Finally, we assessed the impact of two inhibitors, everolimus (EVE) and verteporfin (VERT), to control these pathways in a short term culture system using mouse model. Mouse experiments demonstrated that the removal of post-natal day 3 mouse ovaries from their physiological environment impaired the Hippo signaling pathway, inducing spontaneous PFA. After tissue fragmentation, alteration of both signaling pathways occurred in mouse model. To evaluate the effect of chemotherapy on subsequent follicular activation, we first exposed ovarian tissue to 4-hydroperoxycyclophosphamide (4HC), a derivative of cyclophosphamide, in the animal model. In mice, we observed an induction of the PI3K/AKT/mTOR and, surprisingly, also a disruption of the Hippo signaling pathways after 4HC exposure. We confirmed an acute deleterious effect on DNA in ovarian tissues from adult and prepubertal patients previously treated with chemotherapy. However, the effect on the PI3K and Hippo pathways was less clear, as only triggering of mTOR signaling was observed in prepubertal tissue previously exposed to chemotherapy. Alterations in vascularization were also observed in prepubertal patients exposed to chemotherapy. However, ovarian tissue collection was usually performed several weeks after the administration of first-line chemotherapy. Taken together, these results suggest that the processing procedure for OTC and chemotherapy exposure may exacerbate spontaneous in vitro PFA through both the PI3K and Hippo pathways. To decrease the massive PFA occurring in vitro, we evaluated the potential benefit of using inhibitors of PI3K/AKT/mTOR and Hippo signaling pathways. In our mouse model, EVE (mTORC1 inhibitor) was able to prevent chemotherapy-induced PFA through a decrease in both the PI3K/AKT/mTOR and Hippo signaling effectors, suggesting crosstalk between the pathways. Using VERT, we aimed to avoid Hippo signaling disruption in vitro in mouse tissues. VERT transiently prevented the YAP activity induced by culturing and sectioning. However, the inhibitory activity of VERT was insufficient to control the disruption of Hippo signaling following chemotherapy exposure. Surprisingly, we noticed that VERT was able to prevent the induction of the mTOR pathway under 4HC exposure. These results suggest that tissue processing, in vitro culture, and chemotherapy exposure alter the signaling pathways regulating PFA. Experiments in mouse model highlight a potential two-way interaction between the PI3K and Hippo signaling pathways that controls ovarian reserve. Additionally, inhibition of PI3K/AKT/mTOR signaling using EVE may be a promising approach to regulating both signaling pathways in vitro and to reducing chemotherapy-induced signaling.Grâce aux progrès diagnostiques et thérapeutiques, la survie des patientes atteintes d’un cancer a grandement augmenté ces dernières décennies. Néanmoins, certains agents chimiothérapeutiques peuvent être gonadotoxiques et mener à une infertilité. Afin d’assurer une qualité de vie adéquate aux patients après rémission, il est grandement recommandé de préserver leur fertilité. Parmi les techniques disponibles en clinique, la cryopréservation de tissu ovarien est une procédure intéressante puisqu’elle peut être réalisée sans devoir postposer le traitement ou même après une première cure de chimiothérapie. De plus, elle est la seule option disponible pour les enfants. Elle consiste en la congélation du cortex ovarien en vue d’une greffe après rémission, pour autant que le tissu ne contienne pas de cellules néoplasiques. Afin de palier à cette limitation, la culture du tissu ovarien in vitro afin d’obtenir des ovocytes matures fertilisables est une alternative prometteuse actuellement en développement en recherche. Cependant, ce système de folliculogenèse in vitro montre de grandes limitations dû à une activation massive des follicules en culture. Afin d’améliorer cette technique, il est essentiel d’avoir une meilleure compréhension des mécanismes régulant l’activation des follicules in vitro. De plus, les impacts de la procédure de congélation du tissu et d’un éventuel traitement antérieur doivent être considérés. Les objectifs de ce projet sont d’étudier deux voies de signalisations, la voie PI3K/AKT/mTOR et Hippo, durant l’activation folliculaire spontanée et induite en modèle murin. L’impact du traitement chimiothérapeutique a quant à lui été étudié plus en détail en comparant le tissu de patientes adultes et prépubères qui ont eu de la chimiothérapie ou non avant la cryopréservation. Enfin, nous avons tenté de contrôler ces deux voies de signalisations in vitro en utilisant deux inhibiteurs, l’everolimus (EVE) et la verteporfine (VERT), en modèle animal. Nos analyses en modèle murin ont indiqué que l’extrusion de l’ovaire de son environnement physiologique suffit à altérer la voie Hippo, induisant l’activation spontanée des follicules en culture. Après fragmentation du tissu, une altération des deux voies de signalisation a été observée en modèle murin. Afin de mieux comprendre les mécanismes de gonadotoxicité de la chimiothérapie, nous avons analysé les impacts de l’exposition au 4HC, un dérivé du cyclophosphamide, en modèle animal. En souris, nos analyses ont montré une augmentation de dommages à l’ADN, une induction de la voie PI3K/AKT/mTOR et également une altération de la voie Hippo après exposition au 4HC. Cet effet délétère aigu de l’exposition à la chimiothérapie a été confirmé sur le tissu provenant de patientes adultes et prépubères. Par contre, l’effet sur les voies de signalisation semble moins clair. Seul une induction de la voie mTOR a été mise en évidence chez les patientes prépubères exposées à la chimiothérapie, ainsi qu’une atteinte de la vascularisation. Ceci pourrait être expliquer par le délai de plusieurs semaines généralement observé entre la chimiothérapie de première ligne et le prélèvement ovarien. Ces résultats suggèrent donc que le processing de cryopréservation et l’exposition à la chimiothérapie pourrait exacerber l’activation folliculaire spontanée. Afin de diminuer cette activation massive, nous avons évalué le potentiel contrôle des deux voies en utilisant des inhibiteurs. En modèle murin, EVE (inhibiteur de mTORC1) a pu prévenir l’induction chimiothérapeutique de l’activation folliculaire en diminuant les effecteurs des deux voies de signalisation, suggérant une interaction entre elles. Nous avons ensuite testé l’impact de la VERT pour rétablir la voie Hippo en modèle murin. VERT a montré un contrôle partiel de l’activité de YAP induite par la culture et le sectionnement. Néanmoins, ce contrôle s’est avéré insuffisant pour palier l’altération de la voie Hippo suite à l’exposition chimiothérapeutique. En outre, nous avons constaté que la VERT pouvait diminuer l’induction de la voie mTOR sous 4HC. En conclusion, nos résultats soulignent que le processing du tissu, la culture in vitro et le traitement chimiothérapeutique impactent sur les voies de signalisation régissant l’activation folliculaire. Nos différentes études en modèle murin ont suggéré une potentielle interaction bidirectionnelle entre la voie PI3K et Hippo afin de contrôler la réserve ovarienne. Cette étude fournit de nouvelles connaissances sur les voies de signalisation impactées au sein de l’ovaire qui permettront d’améliorer le système de culture de follicules humains. Enfin, nous mettons en évidence l’approche prometteuse de l’utilisation de l’EVE pour réguler à la fois la voie PI3K/AKT/mTOR et Hippo in vitro et diminuer l’impact de la chimiothérapie sur ces voies.Doctorat en Sciences biomédicales et pharmaceutiques (Médecine)info:eu-repo/semantics/nonPublishe

Evaluation de l'activation des follicules ovariens primordiaux en modèle murin -- Master Sciences biomédicale

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Ethnic Diversity Matters: Putting Implicit Associations between Weapons and Ethnicity in Context

Weapons are implicitly associated with Black Americans. We examined the extent to which this implicit stereotype fluctuates as a function of the ethnic diversity of contexts. Across 351 U.S. metropolitan areas, we tested whether three distinct indicators of ethnic diversity predicted implicit associations between the concept of “weapons” (vs. “harmless objects”) and Black Americans vs. White Americans. As predicted, implicit Black-weapon stereotypes were weaker in areas characterized by the presence of multiple ethnic groups (variety) and greater dispersion of ethnic groups at the neighborhood level (integration). Additionally, the negative association between integration and implicit stereotypes was strongest when minority representation was low compared to high. Considering multiple dimensions of ethnic diversity proved useful to document reliable relations between implicit associations and characteristics of local contexts

Implicit American Identity and Ethnic Diversity across Metropolitan Areas

Prior research documents that Asian Americans are implicitly seen as less American than European Americans (implicit American = White effect). The aim of the present research was to test whether this effect is weaker in more ethnically diverse metropolitan areas. Data from the 2010 U.S. Census were utilized to compute three indicators of context ethnic diversity: minority representation, variety, and integration. Implicit ethnic-American associations were assessed using data collected through Project Implicit. A total of 304 metropolitan areas were included in the analyses. The sample (N = 271,006) included 44.8% White and 31.7% Asian participants; it was composed mostly of relatively young adults (M = 26.54, SD = 11.16) and included more women (60.9%) than men. Respondents completed an Implicit Association Test measuring associations between the concepts “American” vs. “foreign” and two ethnic groups (“Asian American” vs. “European American”). Data were analyzed using multilevel modeling. The implicit American = White effect was less pronounced in metropolitan areas characterized by higher proportions of Asian Americans (minority representation). The presence of multiple ethnic groups (variety) was associated with a weaker implicit American = White effect only when minority representation was high. Greater dispersion of ethnic groups at the neighborhood level (integration) was not a source of reliable variation in implicit ethnic-American associations. These findings highlight the value of a multi-faceted perspective on context ethnic diversity. The extent to which the American identity is implicitly associated with Asian Americans and European Americans fluctuates as a function of socio-structural characteristics of local contexts

Implicit Weapon Associations and Ethnic Diversity across Metropolitan Areas

Weapons are implicitly associated with Black Americans. We examined the extent to which this implicit stereotype fluctuates as a function of the ethnic diversity of contexts. Across 351 U.S. metropolitan areas, we tested whether three distinct indicators of ethnic diversity predicted implicit associations between the concept of “weapons” (vs. “harmless objects”) and Black Americans vs. White Americans. As predicted, implicit Black-weapon stereotypes were weaker in areas characterized by the presence of multiple ethnic groups (variety) and greater dispersion of ethnic groups at the neighborhood level (integration). Additionally, the negative association between integration and implicit stereotypes was strongest when minority representation was low compared to high. Considering multiple dimensions of ethnic diversity proved useful to document reliable relations between implicit associations and characteristics of local contexts

Context Diversity Predicts the Extent to Which the American Identity Is Implicitly Associated with Asian Americans and European Americans

Prior research documents that Asian Americans are implicitly seen as less American than European Americans (implicit American = White effect). The aim of the present research was to test whether this effect is weaker in more ethnically diverse metropolitan areas. Data from the 2010 U.S. Census were utilized to compute three indicators of context ethnic diversity: minority representation, variety, and integration. Implicit ethnic-American associations were assessed using data collected through Project Implicit. A total of 304 metropolitan areas were included in the analyses. The sample (N = 271,006) included 44.8% White and 31.7% Asian participants; it was composed mostly of relatively young adults (M = 26.54, SD = 11.16) and included more women (60.9%) than men. Respondents completed an Implicit Association Test measuring associations between the concepts “American” vs. “foreign” and two ethnic groups (“Asian American” vs. “European American”). Data were analyzed using multilevel modeling. The implicit American = White effect was less pronounced in metropolitan areas characterized by higher proportions of Asian Americans (minority representation). The presence of multiple ethnic groups (variety) was associated with a weaker implicit American = White effect only when minority representation was high. Greater dispersion of ethnic groups at the neighborhood level (integration) was not a source of reliable variation in implicit ethnic-American associations. These findings highlight the value of a multi-faceted perspective on context ethnic diversity. The extent to which the American identity is implicitly associated with Asian Americans and European Americans fluctuates as a function of socio-structural characteristics of local contexts

Interaction between PI3K/AKT and Hippo pathways during in vitro follicular activation and response to fragmentation and chemotherapy exposure using a mouse immature ovary model

Understanding and control of the massive and accelerated follicular growth that occurs during in vitro culture of ovarian tissue is a crucial step toward the development of efficient culture systems that offer an attractive alternative to ovarian tissue transplantation for fertility restoration in cancer survivors. One outstanding question focuses on processes that occur prior to cryopreservation, such as tissue sectioning or chemotherapeutic treatment, might exacerbate this follicular activation. Although the PI3K/AKT/mTOR pathway is well known as a major trigger of physiological and chemotherapy-induced follicular activation, studies have shown that disruption of Hippo pathway due to ovarian fragmentation acts as an additional stimulator. This study aimed to characterize the possible interactions between these pathways using post-natal day 3 mouse ovaries cultured for 4 or 48 h. Morphology, gene transcription, and protein levels were assessed to investigate the impact of sectioning or chemotherapy exposure (4-hydroperoxycyclophosphamide [4HC], 3 and 20 μM). The effect of an mTORC1 inhibitor, Everolimus, alone or as a 4HC co-treatment to prevent follicle activation was evaluated. The results showed that organ removal from its physiological environment was as effective as sectioning for disruption of Hippo pathway and induction of follicle activation. Both PI3K/AKT/mTOR and Hippo pathways were involved in chemotherapy-induced follicular activation and responded to fragmentation. Surprisingly, Everolimus was able to prevent the activation of both pathways during chemotherapy exposure, suggesting cross-talk between them. This study underscores the major involvement of PI3K/AKT/mTOR and Hippo pathways in in vitro follicle activation and provides evidence that both can be regulated using mTORC1 inhibitor.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Deciphering PI3K/Akt/mTOR and Hippo signaling pathways interactions during in vitro spontaneous and chemotherapy-induced follicular activation in mice ovaries

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