Driving Human Granulosa-Luteal Cells Recovered From In Vitro Fertilization Cycles Toward the Follicular Phase Phenotype.

Culture systems are available for human granulosa cells (GCs) that perpetuate luteinization. The present study examines the plating density effects and long-term serum-free culture on the in vitro dynamics differentiation of luteinizing human GCs. Cells were cultured in serum-free α-minimum essential medium (α-MEM) or serum-based tissue culture medium (TCM). The time course of GCs morphology and secretion of estradiol (E2), progesterone (P4), and relaxin were analyzed after 48, 96, and 144 hours of culture. Other functional markers as follicle-stimulating hormone/luteinizing hormone receptors and steroidogenic enzymes were investigated at the end of culture. The morphology of an α-MEM cell rather than a TCM cell resembles more closely that seen in vivo. Compared to TCM cultures, α-MEM cells secreted 93.7% and 87.2% more E2 and approximately 7% and 17% of the amount of P4 when cultured at densities of 2 × 10(4) or 4 × 10(4) cells/well, respectively. Relaxin secretion was significantly reduced in α-MEM cultures. α-MEM cells were estrogenic and expressed the CYP19 gene. Levels of CYP17 increased about 8-fold in α-MEM cells above the levels found in TCM cells. Our results reveal new insights into human GCs differentiation in vitro and demonstrate the critical importance of the culture system and cell-plating density on the establishment of estrogenic or progestogenic GC phenotypes.

Cultura de células da granulosa humanas com fenótipo de fase folicular: influência do sistema quimicamente definido na morfologia, ultraestrutura, secreção de esteroides e relaxina / Culture of human granulosa cells with follicular phase phenotype:influence of chemically defined system on morphology, ultrastructure,secretion of steroids and relaxin

Está bem descrito na literatura o padrão de cultivo de células da granulosa (CG) humanas que perpetua a luteinização, simulando a fase lútea do ciclo. Nesse sistema, há redução na secreção de estradiol (E2) e aumento na síntese de progesterona (P4) e relaxina (RLN). Objetivamos padronizar um sistema de cultura livre de soro, com o intuito de reverter o processo de luteinização de CG obtidas em ciclos de fertilização in vitro (FIV), pré-luteinizadas pela gonadotrofina coriônica humana (hCG), para aplicação na maturação in vitro de folículos ovarianos pré-antrais. Foi feito estudo experimental com GC obtidas de 10 mulheres em tratamento de reprodução assistida. As CG foram cultivadas em α-MEM contendo IGF-I, ITS, androstenediona, PVP-40 (meio quimicamente definido) ou TCM-199 contendo FSH/soro. Após 48, 96 e 144 horas, foram avaliados: morfologia das culturas, produção de E2, P4 (Quimioluminescência/Immulite), RLN (Elisa) e ultraestrutura (Microscopia Eletrônica). Os dados foram analisados por Anova e regressão linear com efeitos mistos (SAS versão 9.0). Células cultivadas em α-MEM apresentam alta capacidade estrogênica e padrão de produção hormonal característico da fase folicular, mantendo características morfológicas/ultraestruturais semelhantes a células in vivo. No sistema de cultura padronizado, as CG não completam in vitro o processo de luteinização deflagrado pela hCG, assumindo fenótipo de fase folicular.

It is well described in the literature the granulosa cells (GC) culture pattern that perpetuates human luteinizing simulating the luteal phase of the cycle. In this system, there is a reduction in the secretion of estradiol (E2) and increased synthesis of progesterone (P4) and relaxin (RLN). We aim to standardize a serum-free culture system, in order to reverse the luteinization process of GC obtained in IVF cycles, pre-luteinized by hCG, for use in in vitro maturation of preantral ovarian follicles. An experimental study was conducted with GC obtained from10 women undergoing treatment for assisted reproduction. The GC were cultured in α-MEM containing IGF-I, STI, androstenedione, PVP-40 (chemically defined medium) or TCM-199 containing FSH/serum. After 48, 96 and 144 h were analyzed: culture morphology, concentrations of E2, P4 (Chemioluminescence/Immulite), and RLN (Elisa), and ultrastructure (ElectronMicroscopy). Data were analyzed by Anova and linear mixed-effects regression (SAS version9.0). Cells cultured in α-MEM present estrogenic capacity and pattern of hormone production characteristic of the follicular phase, maintaining morphological/ultrastructural features similar that in vivo cell. In standard culture system, the CG not completes in vitro luteinization process triggered by hCG, assuming follicular phase phenotype.

Produção de oócitos em mamíferos adultos: o camundongo como modelo / Oocyte production in adult mammals: mice as the model

Objetivo: identificar produção de oócitos em mamíferos adultos com o uso de camundongo como modelo experimental. Método: empregamos a técnica de imuno-histoquímica em cortes de ovários de camundongos Balb-c (45 dias de idade) com o uso de anticorpo específico para marcação de células germinativas. Como controles positivos da reação, usamos cortes de testículos de camundongos. Resultados: as células germinativas (espermatogônias, espermatócitos e espermátides) dos controles positivos sofreram marcação, enquanto células não pertencentes a essa linhagem (células de Leydig e de Sertoli) mostraram negatividade de reação; nos cortes ovarianos observou-se marcação de oócitos de folículos em diferentes estágios de maturação, mas houve também marcação de células não englobadas pela estrutura folicular. Conclusões: os achados sugerem que durante a puberdade ovários de camundongos fêmeas contêm células da linhagem germinativa em estágios anteriores à formação folicular, o que corrobora estudos anteriores; o trabalho é pioneiro no Brasil e progredirá para a completa caracterização de células com potencial oogênico em outras espécies de mamíferos. Resultados positivos poderão alterar o entendimento da biologia reprodutiva e abrir novas portas para o tratamento de infertilidade.

Objective: to identify oocyte production in adult mammals using the mouse as the experimental model. Method: we used the immunohistochemistry technique on ovary sections of Balb-c mice (45 days old), with antibody that labels germline cells specifically. We used sections of mice’s testes as positive reaction controls.Results: in testes samples, germ cells (spermatogonia, spermatocytes and spermatids) were stained, while cells not belonging to germ lineage (Leydig and Sertoli cells) showed negativereaction; in ovarian samples, oocytes from follicles in different stages of maturation werestained, but the reaction was also positive for cells not enclosed by the follicular structure. Conclusions: the findings suggest that, during puberty, female mice ovaries contain germline cells in earlier stages before follicular formation, as was found in previous studies. Thework, pioneering in Brazil, must progress to a complete characterization of these cells (with oogenesis potential) in mice and in other mammal species. Positive results may change the understanding of the reproductive biology and open new possibilities for infertility treatment.

Cryopreservation and fertility: current and prospective possibilities for female cancer patients.

With the evolution of the treatment of malignant neoplasms, the survival rates of patients undergoing chemo- or radiotherapy are increasing. The continuous development of techniques of assisted human reproduction has led to important strategies in an attempt to maintain reproductive function in patients subjected to treatment of neoplastic diseases, among them cryopreservation of embryos, gametes, and ovarian cortical tissue. The freezing of ovarian tissue is currently being proposed with the primary purpose of preserving ovarian function in these patients. Currently, the major challenge of groups working with preservation of fertility is the use of cryopreserved ovarian tissue after disease remission. The main alternatives presented today are the implantation of hetero- or orthotopic tissue and isolation of immature follicles from ovarian tissue followed by in vitro maturation and assisted reproduction procedures.

Cryopreservation time does not decrease follicular viability in ovarian tissue frozen for fertility preservation.

OBJECTIVE: To determine the effect of storage duration on cryopreserved ovarian tissue using fresh and frozen-thawed samples. METHODS: Seventeen fertile patients underwent an ovarian biopsy during elective laparoscopic tubal ligation. The tissue sample was divided into three parts: one part was processed fresh (FG), and two were slowly frozen, cryopreserved for 30 (G30) or 180 days (G180), thawed and analyzed. Follicular density, follicular viability, and steroidogenic capacity were assessed. RESULTS: We observed no differences between the groups in follicular density, which was assessed in hematoxylin and eosin-stained tissue sections. A heterogeneous follicular distribution was observed in the parenchyma, with a mean density of 361.3±255.4, 454.9±676.3, and 296.8±269.0 follicles/mm(3) for FG, G30 and G180, respectively (p = 0.46). Follicular viability was greater in FG (93.4%) when compared with the cryopreserved tissues (70.8% for G30 (p<0.001) and 78.4% for G180 (p<0.001)), with no difference in viability between the frozen samples (p>0.05). The steroidogenic capacity of the tissue was not significantly reduced following cryopreservation. CONCLUSION: The slow freezing procedures used for ovarian cryopreservation are capable of preserving follicular viability and maintaining the steroidogenic capacity of the tissue despite a roughly 30% decrease in follicular viability. Furthermore, short-term storage of ovarian tissue does not appear to compromise follicle integrity.

Influência da interação entre o oócito e as células da granulosa nos resultados dos procedimentos de reprodução assistida / The influence of interaction between oocyte and granulosa cells on the results of procedures in assisted reproduction

A interação oócito-células da granulosa in vivo e sua influência na qualidade oocitária e embrionária tem sido alvo de inúmeros estudos, mas muitas questões ainda necessitam ser esclarecidas. O objetivo deste trabalho foi revisar a importância dessa comunicação, estabelecendo uma relação com a questão da maturação in vitro de oócitos imaturos humanos aplicando esses conhecimentos para definir possíveis marcadores moleculares que poderiam melhorar a seleção de oócitos e, consequentemente, selecionar embriões de boa qualidade para posterior transferência e sucesso de gravidez de pacientes submetidas ao tratamento da infertilidade conjugal. As células da granulosa têm um importante papel na maturação oocitária in vitro e os benefícios da presença dessas células durante essa etapa podem ser atribuídos à formação de um microambiente favorável (bioquímico e metabólico) ao redor do oócito. Foram identificados nesta revisão vários marcadores em potencial nas células do cumulus de oócitos competentes, incluindo vários genes que poderiam ser usados como preditores da competência oocitária, o que pode contribuir para a formulação de critérios mais objetivos e confiáveis para a seleção de oócitos e embriões, e consequente aprimoramento e otimização das técnicas em reprodução humana assistida que são aplicadas nos procedimentos clínicos atuais de fertilização in vitro.

The interaction of oocyte-granulosa cells in vivo and in vitro and its influence on oocyte and embryo quality has been the subject of numerous studies, but many issues still need to be clarified. The objective of this study was to promote a review about the importance of this communication establishing a connection with the issue of in vitro maturation of immature human oocytes by applying this knowledge to define potential molecular markers that could improve the selection of oocytes and consequently select good quality embryos for later transfer and success of pregnancy in patients undergoing treatment of infertility. The granulosa cells also have an important role in oocyte maturation in vitro and the venefits from the presence of these cells during this process can be atributed to the formation of a favorable micro-environment (biochemical and metabolic) around the oocyte. In this review, we identified several potential markers in the cumulus cells of competent oocytes, including several genes that could be used as predictors of oocyte competence, which contributes for more objective and reliable criteria for the selection of oocytes and embryos, thus improving and optimizing techniques in assisted human reproduction that are applied in current clinical in vitro fertilization.

Cryopreservation time does not decrease follicular viability in ovarian tissue frozen for fertility preservation

OBJECTIVE: To determine the effect of storage duration on cryopreserved ovarian tissue using fresh and frozenthawed samples. METHODS: Seventeen fertile patients underwent an ovarian biopsy during elective laparoscopic tubal ligation. The tissue sample was divided into three parts: one part was processed fresh (FG), and two were slowly frozen, cryopreserved for 30 (G30) or 180 days (G180), thawed and analyzed. Follicular density, follicular viability, and steroidogenic capacity were assessed. RESULTS: We observed no differences between the groups in follicular density, which was assessed in hematoxylin and eosin-stained tissue sections. A heterogeneous follicular distribution was observed in the parenchyma, with a mean density of 361.3±255.4, 454.9±676.3, and 296.8±269.0 follicles/mm3 for FG, G30 and G180, respectively (p = 0.46). Follicular viability was greater in FG (93.4 percent) when compared with the cryopreserved tissues (70.8 percent for G30 (p<0.001) and 78.4 percent for G180 (p<0.001)), with no difference in viability between the frozen samples (p>0.05). The steroidogenic capacity of the tissue was not significantly reduced following cryopreservation. CONCLUSION: The slow freezing procedures used for ovarian cryopreservation are capable of preserving follicular viability and maintaining the steroidogenic capacity of the tissue despite a roughly 30 percent decrease in follicular viability. Furthermore, short-term storage of ovarian tissue does not appear to compromise follicle integrity.