Introdução: A criopreservação de fragmentos de tecido ovariano previamente ao início da terapia oncológica e posterior reimplante do tecido ovariano tem sido sugerido como promissora alternativa para preservação de fertilidade. Neste sentido, um novo sistema de vitrificação denominado Ovarian Tissue Cryosystem (OTC), vem sendo desenvolvido visando aperfeiçoar a conservação do tecido congelado. Objetivo: Avaliar o impacto da criopreservação sobre a viabilidade do tecido ovariano murino vitrificado por três diferentes protocolos, dentre eles o sistema OTC. E avaliar o impacto do reimplante heterotópico do tecido ovariano murino fresco e descongelado pelos mesmos protocolos, sobre a sua viabilidade. Metodologia: Este é um estudo experimental onde foram utilizados tecido ovariano de camundongos C57BL/6. Três protocolos de vitrificação foram testados simultaneamente: protocolo murino (PrM); protocolo humano (PrH); protocolo OTC (PrOTC) em comparação ao grupo frescos (GF). As amostras foram analisadas imediatamente após o descongelamento (Etapa 1) ou após 15 ou 30 dias de reimplante retroauricular (Etapa 2). Para análise da viabilidade utilizou-se imunohistoquímica para proliferação celular (Ki-67), dano celular (NF-kB) e dano oxidativo (4-HNE e Nitrotirosina). Os resultados foram obtidos utilizando-se o teste Q-quadrado para verificar a distribuição entre os grupos e a imunomarcação (% de folículos marcados), sendo que o nível de significância adotado foi p<0,05. Resultados: Etapa 1: Os grupos PrOTC e PrM apresentaram melhor desempenho em relação à proliferação celular. Entretanto, estes mesmos protocolos apresentaram graus variados de dano oxidativo quando analisados pela imunomarcação de nitrotirosina, aonde o PrOTC teve maior marcação do que o GF, portanto maior dano, e o PrM teve maior marcação em relação ao HNE em comparação com os demais protocolos. Não houve nenhuma diferença na marcação do NF-kB entre os grupos descongelados. Etapa 2: O reimplante em si não pareceu comprometer significativamente o tecido, uma vez que as amostras GF e GF reimplantado com 15 e 30 dias tiveram desempenho semelhante em relação à todos os marcadores, com exceção do GF 30 dias que mostrou algum grau de dano oxidativo pela nitrotirosina (p<0,05). E ao se avaliar a somatória de efeitos entre congelamento e reimplante entre os diferentes protocolos apenas o PrH evidenciou dano tecidual imediatamente após o descongelamento e dano progressivo após o reimplante. Conclusão: Os PrM e PrOTC foram semelhantes ao GF na conservação da amostra durante o processo de criopreservação, sendo que o PrOTC causou algum grau de dano oxidativo. O reimplante retroauricular do tecido não impactou sobre a sua viabilidade do mesmo nem no GF e nem nos PrM e PrOTC. O PrH mostrou-se inadequado para a conservação de tecido ovariano murino. / Introduction: The Cryopreservation of fragments of ovarian tissue prior to initiation of oncotherapy and subsequent reimplantation of ovarian tissue has been suggested as promising alternative for fertility preservation. In this sense, a new system of vitrification called Ovarian Tissue Cryosystem (OTC), has been developed in order to improve the frozen ovarian tissue conservation. Objective: To compare the viability of murine ovarian tissue vitrified by three different vitrification protocols and to verify the efficiency of the Ovarian Tissue Cryosystem (OTC). Methods: This is an experimental study using ovarian tissue of C57BL/6 mice. Three vitrification protocols were tested simultaneously: Murine protocol (PrM); Human Protocol (PrH) and Protocol OTC (PrOTC); in comparison to Fresh Tissue (GF). Samples were analyzed immediately after thawing (Step 1) or after 15 or 30 days of retroauricular reimplantation (Step 2). For viability analysis immunohistochemistry for cell proliferation (Ki-67), cell damage (NF-kB) and oxidative damage (4-HNE and Nitrotyrosine) was used. The results were obtained using the Q-square test to analyze immunostaining counted as % of labeled follicles and the level of significance was set at p <0.05. Results: Step 1: The PrOTC and PrM presented the best performance in relation to cell proliferation. However, these same protocols presented varying degrees of oxidative damage when analyzed by the nitrotyrosine immunolabeling, where PrOTC had higher marking than GF, thus greater damage, and PrM had a greater marking in relation to HNE compared to the other protocols. There was no difference in the labeling of NF-kB between the thawed groups. Step 2: The reimplantation procedure did not appear to significantly impair the ovarian tissue quality once day 15 and 30 after fresh tissue engrafment of was similar for all markers except for nitrotyrosine after 30 days (p<0,05). And when assessing the sum of effects of freezing and reimplantation among the 3 different protocols only PrH showed tissue damage immediately after thawing and progressive damage after reimplantation. Conclusion: The PrM and PrOTC were similar to the GF in the preservation of the sample during the cryopreservation process, and PrOTC caused some degree of oxidative damage. Retroauricular reimplantation of the tissue did not impact on its viability in either the GF or PrM and PrOTC. PrH was found to be unsuitable for the cryopreservation of murine ovarian tissue.

Diethylstilbestrol (DES) is one of the synthetic estrogen available today for therapeutic use. It is also referred to as a carcinogenic agent, with a great number of side effects reported between maternal ingestion of DES during pregnancy, as a postovulatory agent to prevent implantation and possible occurrence of carcinoma in the progeny of women known to be aborterse. In the present study, experiments were done to determine the effects of DES on the estrogen titer of the maternal, fetal and neo-natal plasma ovarian homogenates of female Long-Evans rats. Adult and 30-day old rats were treated via stomach intubation with 35 mg/2cc/kg body wt of DES. The estrous cycle of the rats was monitored and vaginal smear cell counts were determined in 0.5 cc saline smear volume. Radioimmunoassay was used to determine the estradiol levels in the plasma and ovarian homogenates. Results showed no changes in the estrous cycle synchrony of the maternal rats, while the estrous cycle of the treated 30-dayold rats has synchronous. The vaginal smear cell count was significantly greater in the two groups of treated rats studied than in the controls. Comparing the mean estradrol value for the plasma and ovarian homogenates in the maternal rats, an obvious increase in the plasma E2 level was obtained as compared to small, decrease in the mean value of ovarian E2 level. There was no difference in the mean value of pooled ovarian homogenate E2 level of the control saline, control and DES-treated 19-1/2-day old rats. A significant decrease in the plasma E2 mean value of 30-day old treated rats was obtained when compared to both control groups. There was no significant increase in ovarian homogenate E2 level of both treated and the control groups. In light of these results, DES has a secondary effect, asynchronous cell proliferation in the vaginal epithelia leading to prolonged specific stages of the estrous cycle of the young adult female rats. Also, DES causes an increase in plasma E2 levels of treated maternal rats and a decrease in plasrna E2 levels of 30-dayold rats. Finally, DES has no effect on the estrogen level of the ovarian homogenates of the maternal, fetal and 30-clay-old young Long-Evans female rats.

Introdução: a alta prevalência do câncer e o aumento significativo da sobrevivência em longo prazo geraram interesse quanto à preservação da fertilidade em mulheres jovens expostas a quimioterapia e radioterapia. Neste sentido estudos de congelamento de tecido ovariano para posterior transplante, abriram uma nova perspectiva de aplicação no tratamento e prevenção da infertilidade feminina. Objetivos: comparar dois protocolos de congelamento de tecido ovariano, um lento não automatizado e um por vitrificação, com o intuito de avaliar a viabilidade dos tecidos para posterior transplante autólogo. Método: Foram utilizadas 30 camundongos fêmea CF1 com aproximadamente 8 semanas e pesando 29,29g±2,9. •Os ovários extraídos foram vitrificados ou congelados, mantidos em nitrogênio líquido por 30 dias e descongelados. Após o descongelamento, o ovário esquerdo foi destinado às análises histológicas e caracterização por imuno histoquímica para o marcador mouse vasa homologue (MVH) e o ovário direito foi utilizado para os testes de viabilidade celular com exclusão por azul de trypan. Resultados: Nas análises de Hematoxilina e Eosina (HE) foram contados folículos primordiais, primários, pré-antrais e antrais. Não houve diferença significativa na proporção de folículos primordiais, primários e pré-antrais após descongelamento entre os grupos testados. A contagem de folículos antrais foi significativamente maior no grupo de vitrificação (p = 0,004). No ensaio de imunohistoquímica para o marcador MVH, folículos MVH + e MVH- foram contados e comparados com o número total de folículos. O grupo congelamento lento apresentou maior número de células não marcadas (p = 0,012). Conclusão: Embora ambos os protocolos tenham apresentado resultados semelhantes na análise histológica das contagens foliculares, o protocolo de vitrificação foi significativamente melhor para preservar a população de células tronco ovarianas. / Introduction: The high prevalence of cancer and the significant increase in long-term survival have generated interest as the preservation of fertility in young women exposed to chemotherapy and radiotherapy. Experimental techniques have been tried in an attempt to reverse the ovarian failure induced by these treatments. In this regard studies of ovarian tissue freezing for subsequent transplantation disclose a new application perspective in the treatment and prevention of female infertility. Objective: two ovarian tissue freezing protocols were tested, a non-automated slow-freezing and by vitrification, in order to assess the viability of the tissues for subsequent autologous transplantation. Methods: as ovaries donors, were used 30 female CF1 mice approximately 8 weeks and weighing 29,29g±2,9. • The ovaries were vitrified or frozen, stored in liquid nitrogen for 30 days and thawed. After thawing, the left ovary was intended for histological and immunohistochemical characterization by histochemical marker for MVH and right ovary was used for the tests with cell viability by trypan blue exclusion. Results: In HE slides was counting primordial, primary, pre antral and antral follicles. No significant difference was found in the proportion of high-quality primordial, primary and pre antral follicles after thawing/warming in the slow-freezing and vitrification group, respectively. The antral follicle counting was significant higher in vitrification group (p=0,004). In immunohistochemistry assay for MVH Antibody , MVH+ and MVH- follicles were counted and compared with the total number of follicles and slow freeze group had a higher number of not marked cells (p=0,012). Conclusion: Although both protocols showed similar results in the histological analysis for follicular counts, the vitrification protocol was significantly better for preserve the ovarian stem cell population.

Introdução: a alta prevalência do câncer e o aumento significativo da sobrevivência em longo prazo geraram interesse quanto à preservação da fertilidade em mulheres jovens expostas a quimioterapia e radioterapia. Neste sentido estudos de congelamento de tecido ovariano para posterior transplante, abriram uma nova perspectiva de aplicação no tratamento e prevenção da infertilidade feminina. Objetivos: comparar dois protocolos de congelamento de tecido ovariano, um lento não automatizado e um por vitrificação, com o intuito de avaliar a viabilidade dos tecidos para posterior transplante autólogo. Método: Foram utilizadas 30 camundongos fêmea CF1 com aproximadamente 8 semanas e pesando 29,29g±2,9. •Os ovários extraídos foram vitrificados ou congelados, mantidos em nitrogênio líquido por 30 dias e descongelados. Após o descongelamento, o ovário esquerdo foi destinado às análises histológicas e caracterização por imuno histoquímica para o marcador mouse vasa homologue (MVH) e o ovário direito foi utilizado para os testes de viabilidade celular com exclusão por azul de trypan. Resultados: Nas análises de Hematoxilina e Eosina (HE) foram contados folículos primordiais, primários, pré-antrais e antrais. Não houve diferença significativa na proporção de folículos primordiais, primários e pré-antrais após descongelamento entre os grupos testados. A contagem de folículos antrais foi significativamente maior no grupo de vitrificação (p = 0,004). No ensaio de imunohistoquímica para o marcador MVH, folículos MVH + e MVH- foram contados e comparados com o número total de folículos. O grupo congelamento lento apresentou maior número de células não marcadas (p = 0,012). Conclusão: Embora ambos os protocolos tenham apresentado resultados semelhantes na análise histológica das contagens foliculares, o protocolo de vitrificação foi significativamente melhor para preservar a população de células tronco ovarianas. / Introduction: The high prevalence of cancer and the significant increase in long-term survival have generated interest as the preservation of fertility in young women exposed to chemotherapy and radiotherapy. Experimental techniques have been tried in an attempt to reverse the ovarian failure induced by these treatments. In this regard studies of ovarian tissue freezing for subsequent transplantation disclose a new application perspective in the treatment and prevention of female infertility. Objective: two ovarian tissue freezing protocols were tested, a non-automated slow-freezing and by vitrification, in order to assess the viability of the tissues for subsequent autologous transplantation. Methods: as ovaries donors, were used 30 female CF1 mice approximately 8 weeks and weighing 29,29g±2,9. • The ovaries were vitrified or frozen, stored in liquid nitrogen for 30 days and thawed. After thawing, the left ovary was intended for histological and immunohistochemical characterization by histochemical marker for MVH and right ovary was used for the tests with cell viability by trypan blue exclusion. Results: In HE slides was counting primordial, primary, pre antral and antral follicles. No significant difference was found in the proportion of high-quality primordial, primary and pre antral follicles after thawing/warming in the slow-freezing and vitrification group, respectively. The antral follicle counting was significant higher in vitrification group (p=0,004). In immunohistochemistry assay for MVH Antibody , MVH+ and MVH- follicles were counted and compared with the total number of follicles and slow freeze group had a higher number of not marked cells (p=0,012). Conclusion: Although both protocols showed similar results in the histological analysis for follicular counts, the vitrification protocol was significantly better for preserve the ovarian stem cell population.

Introdução: a alta prevalência do câncer e o aumento significativo da sobrevivência em longo prazo geraram interesse quanto à preservação da fertilidade em mulheres jovens expostas a quimioterapia e radioterapia. Neste sentido estudos de congelamento de tecido ovariano para posterior transplante, abriram uma nova perspectiva de aplicação no tratamento e prevenção da infertilidade feminina. Objetivos: comparar dois protocolos de congelamento de tecido ovariano, um lento não automatizado e um por vitrificação, com o intuito de avaliar a viabilidade dos tecidos para posterior transplante autólogo. Método: Foram utilizadas 30 camundongos fêmea CF1 com aproximadamente 8 semanas e pesando 29,29g±2,9. •Os ovários extraídos foram vitrificados ou congelados, mantidos em nitrogênio líquido por 30 dias e descongelados. Após o descongelamento, o ovário esquerdo foi destinado às análises histológicas e caracterização por imuno histoquímica para o marcador mouse vasa homologue (MVH) e o ovário direito foi utilizado para os testes de viabilidade celular com exclusão por azul de trypan. Resultados: Nas análises de Hematoxilina e Eosina (HE) foram contados folículos primordiais, primários, pré-antrais e antrais. Não houve diferença significativa na proporção de folículos primordiais, primários e pré-antrais após descongelamento entre os grupos testados. A contagem de folículos antrais foi significativamente maior no grupo de vitrificação (p = 0,004). No ensaio de imunohistoquímica para o marcador MVH, folículos MVH + e MVH- foram contados e comparados com o número total de folículos. O grupo congelamento lento apresentou maior número de células não marcadas (p = 0,012). Conclusão: Embora ambos os protocolos tenham apresentado resultados semelhantes na análise histológica das contagens foliculares, o protocolo de vitrificação foi significativamente melhor para preservar a população de células tronco ovarianas. / Introduction: The high prevalence of cancer and the significant increase in long-term survival have generated interest as the preservation of fertility in young women exposed to chemotherapy and radiotherapy. Experimental techniques have been tried in an attempt to reverse the ovarian failure induced by these treatments. In this regard studies of ovarian tissue freezing for subsequent transplantation disclose a new application perspective in the treatment and prevention of female infertility. Objective: two ovarian tissue freezing protocols were tested, a non-automated slow-freezing and by vitrification, in order to assess the viability of the tissues for subsequent autologous transplantation. Methods: as ovaries donors, were used 30 female CF1 mice approximately 8 weeks and weighing 29,29g±2,9. • The ovaries were vitrified or frozen, stored in liquid nitrogen for 30 days and thawed. After thawing, the left ovary was intended for histological and immunohistochemical characterization by histochemical marker for MVH and right ovary was used for the tests with cell viability by trypan blue exclusion. Results: In HE slides was counting primordial, primary, pre antral and antral follicles. No significant difference was found in the proportion of high-quality primordial, primary and pre antral follicles after thawing/warming in the slow-freezing and vitrification group, respectively. The antral follicle counting was significant higher in vitrification group (p=0,004). In immunohistochemistry assay for MVH Antibody , MVH+ and MVH- follicles were counted and compared with the total number of follicles and slow freeze group had a higher number of not marked cells (p=0,012). Conclusion: Although both protocols showed similar results in the histological analysis for follicular counts, the vitrification protocol was significantly better for preserve the ovarian stem cell population.

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.

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.

Thesis (MScMedSc (Obstetrics and Gynaecology))--Stellenbosch University, 2008. / BACKGROUND: The cryopreservation and transplantation of ovarian tissue have been shown to restore ovarian function temporarily and may also preserve the fertility of young female cancer patients until after their sterilizing cancer treatment. Since tissue samples are large and morphologically complex, the cryopreservation methodology is difficult to optimize and standardise. Ovarian tissue cryopreservation is therefore, still in its experimental stages and is not a routine option offered to cancer patients. OBJECTIVES: Our main aim was to initiate, develop and implement a practical ovarian tissue cryopreservation and re-transplantation protocol which would restore ovarian function, and possibly fertility, in young female cancer patients undergoing sterilizing cancer therapies in South Africa. The objective of this study was to improve the slow cryopreservation protocol for human ovarian tissue. The ultrastructural effects after cryopreservation with two well-known cryoprotectants, dimethyl sulfoxide (DMSO) and 1,2-propylene glycol (PROH), on early human follicles were investigated and compared to identify and the better cryoprotectant. MATERIALS AND METHODS: A single group experimental study design was used. The participants consisted cancer patients of the Gynaecological Oncology Unit of Tygerberg Hospital who entered on a basis of voluntary informed consent. Ovarian tissue was obtained by laparoscopic oophorectomy. After dissection of the ovary(ies), some fresh cortical tissue was sent for metastatic analysis and a few strips taken as fresh control. Remaining dissected ovarian cortical tissue sections of each patient were equally divided into the two cryoprotectant groups. Five resulting groups could be compared: i) fresh tissue (control group); tissue equilibrated in ii) DMSO; or iii) PROH and tissue equilibrated and cryopreserved in iv) DMSO or v) PROH. Five tissue samples per patient were therefore fixed for standard histological haematoxylin and eosin (HE) staining and transmission electron microscopy (TEM). Tissue samples showing early follicles on HE slides were sent for TEM processing. Ultrastructural studies on micrographs of primordial and primary follicles were assessed according to a scoring system which gave an indication of follicular health. Appropriate statistical tests were applied to analyse the mean scores where P≤0.05 was considered as statistically significant. RESULTS: No significant overall cryopreservation treatment effect was evident in any of the follicular ultrastructures evaluated. This result indicated that the cryopreservation protocol used did not induce significant damage to the cortex tissue compared to the fresh control group. Comparison of the effect of PROH and DMSO on the follicular ultrastructures showed that PROH tend to induce more extensive damage, especially after cryopreservation. Correlation studies showed significant positive relationships between the majority of the evaluated ultrastructures, especially between the oocyte and granulosa cell layer and basal membrane. The stromal cells and extracellular matrix did not correlate well with other structures. Correlations indicated that the granulosa cells, oocyte and basal lamina are affected similarly and that the damage in one of these structures may be representative of the damage in the other structures. CONCLUSION: The main aim of the study was achieved since results showed that no significant damage was induced by the cryopreservation protocols. Ovarian tissue cryopreserved in this study has shown to restore endocrine function temporarily after heterotopic autotransplantation in menopausal patients. From the electron microscopy evaluations, DMSO showed better cryopreservation results. The DMSO cryopreservation protocol was also more time efficient and has shown the most successful outcomes in the literature. The stromal tissue seemed to be affected differently by cryopreservation compared to the primordial follicle ultrastructures. Younger patients are needed for future studies since a larger initial follicular reserve may allow for larger follicle sample sizes.

La cryoconservation de tissu ovarien peut être proposée, avant traitements hautement gonadotoxiques, à des patientes afin de préserver leur fertilité. L'autogreffe de tissu ovarien est actuellement la seule méthode de réutilisation du tissu ovarien disponible, mais en cas de pathologie néoplasique, elle présente un risque de réintroduction d'éventuelles cellules malignes via le greffon. L'objectif de ce travail a été de développer une méthode pour détecter la maladie résiduelle (MRD) dans le tissu ovarien par cytométrie en flux multicouleurs (CMF) en cas de leucémie aiguë. Une technique de dissociation de cortex ovarien a été mise au point à partir de tissu ovarien de référence provenant de résections percoelioscopiques. Un modèle expérimental de détection de la MRD a été validé et consistait à ajouter des cellules de leucémie aiguë lymphoblastique (LAL) ou myéloïde (LAM) à une suspension de cellules ovariennes isolées de référence. La méthode a ensuite été utilisée pour rechercher la MRD dans le tissu ovarien cryoconservé de 11 patientes atteintes de leucémie aiguë (7 LAL et 4 LAM).Le modèle expérimental a permis de valider une sensibilité de 10"4 et une spécificité élevée tant pour les LAL que pour les LAM. Lorsqu'un marqueur moléculaire était disponible pour la recherche de la MRD, nous avons observé une bonne corrélation entre la CMF et la PCR quantitative. La détection par CMF de la MRD dans le tissu ovarien des patientes leucémiques était positive chez 3 des 11 patientes étudiées.Cette technique est essentielle pour évaluer le risque carcinologique avant de proposer la réutilisation du tissu ovarien cryoconservé par technique d'autogreffe. / Ovarian cryopreservation together with autograft of frozen/thawed ovarian tissue is a real option to preserve and restorefertility in cancer patients. However in cases of leukemia, there is a real concern regarding the presence of metastaticcells in the ovarian tissue, which could lead to the recurrence of the primary disease. The aim was to validate multicolorflow cytometry (MFC) as an original technique for minimal residual disease (MRD) detection in ovarian cortex fromacute leukemia patients.We developed an experimental model which consisted in adding serial dilutions of leukemic cells into isolated ovariancell suspensions obtained from healthy cortex. The modelization was validated for acute lymphoblastic leukemia (ALL)and acute myeloid leukemia (AML). Then the method was applied to MRD detection of leukemic cells in cryopreservedovarian cortex from 11 leukemia patients (7 ALL and 4 AML).This experimental model made it possible to obtain a high specificity and a robust sensitivity of 10~4 for MRD detectionby MFC for both types of acute leukemic cells. When a molecular marker was available, we observed a good correlationbetween CMF and quantitative PCR. Ovarian MRD was positive by MFC in one T-ALL and 2 AML patients.MRD detection in the ovarian cortex is essential to evaluate the risk of cancer reseeding before ovarian tissue autograft.

De nos jours, la cryoconservation est une technique très largement utilisée dans les protocoles d’assistance à la reproduction ou comme outil pour la sauvegarde des ressources génétiques. Toutefois, la chienne est un modèle animal complexe pour l’application des biotechnologies de la reproduction du fait de ses nombreuses singularités anatomiques et physiologiques. L’objectif de notre travail était d’étudier et de développer une méthode de cryoconservation des ressources génétiques chez la chienne par le biais de deux types de ressources : les embryons et le tissu ovarien. Après avoir mis au point une méthode de collecte d’embryons, nous nous sommes appliqués à la constitution d’un stock d’embryons cryoconservés en prévision d’un transfert embryonnaire. L’étude et le développement d’un protocole de cryoconservation du tissu ovarien ont été abordés après avoir adapté et validé nos méthodes d’analyses in vitro. L’utilisation de plans d’expériences factoriels fractionnaires a permis de mettre en évidence les facteurs les plus influents sur la qualité de la réserve folliculaire (nature du cryoprotecteur pénétrant, cinétique de congélation, étapes d’équilibration) et de proposer un protocole de cryoconservation. La combinaison du DMSO incorporé en un seul bain d’équilibration avec une vitesse de congélation de 0,3°C/min est apparue comme la combinaison la plus appropriée à la cryoconservation de tissu ovarien chez la chienne et a permis d’observer, après xénogreffe de tissu ovarien cryoconservé, une reprise de la croissance folliculaire et de l’activité hormonale du tissu greffé / Nowadays, cryopreservation is widely used in animal assisted reproduction or safeguarding of genetic resources. Nevertheless, the bitch is a complex animal model concerning the use of this biotechnology, due to numerous anatomical and physiological peculiarities. The aim of our research work was to investigate and develop a method of cryopreservation of genetic resources in the bitch by exploring two kinds of resources: embryos and ovarian tissue. After the setting up of a method for embryo collection, we have built up a stock of cryopreserved embryo for subsequent embryo transfer. After a preliminary validation of our in vitro assessment methods, the investigation and development of a cryopreservation protocol has been conducted. The use of fractional experimental design allowed us to highlight the main factors affecting the follicular pool quality (CPA nature, freezing rate and equilibration steps). The combination of DMSO incorporated in a unique equilibration bath with a freezing rate of 0.3°C/min appeared to be suitable for the cryopreservation of bitch ovarian tissue. Finally, Follicular growth and hormonal activity resumption have been observed after xenotransplantation of cryopreserved bitch ovarian tissue