RESUMO
Vitrification is mainly used to cryopreserve female gametes. This technique allows maintaining cell viability, functionality, and developmental potential at low temperatures into liquid nitrogen at -196°C. For this, the addition of cryoprotectant agents, which are substances that provide cell protection during cooling and warming, is required. However, they have been reported to be toxic, reducing oocyte viability, maturation, fertilization, and embryo development, possibly by altering cell cytoskeleton structure and chromatin. Previous studies have evaluated the effects of vitrification in the germinal vesicle, metaphase II oocytes, zygotes, and blastocysts, but the knowledge of its impact on their further embryo development is limited. Other studies have evaluated the role of actin microfilaments and chromatin, based on the fertilization and embryo development rates obtained, but not the direct evaluation of these structures in embryos produced from vitrified immature oocytes. Therefore, this study was designed to evaluate how the vitrification of porcine immature oocytes affects early embryo development by the evaluation of actin microfilament distribution and chromatin integrity. Results demonstrate that the damage generated by the vitrification of immature oocytes affects viability, maturation, and the distribution of actin microfilaments and chromatin integrity, observed in early embryos. Therefore, it is suggested that vitrification could affect oocyte repair mechanisms in those structures, being one of the mechanisms that explain the low embryo development rates after vitrification.
RESUMO
The empty follicle syndrome (EFS) is defined as a failure to aspirate any oocyte (s) from the follicles after ovarian hyperstimulation in preparation for IVF/ICSI. It is a frustrating and vague syndrome; and a controversial one concerning its existence, causes and possible treatment. Recurrent EFS or the recovery of immature oocytes thereafter is a more challenging problem. Delayed injection after leaving the immature oocytes for in vitro-maturation (IVM) has been suggested to be a possible option if immature oocytes are retrieved. Here, we present a case of repeated retrieval of a few immature oocytes after a first incidence of EFS. IVM was tried twice for those immature oocytes. Unfortunately, in this case IVM was unsuccessful and the oocytes failed to mature in vitro. Assistance is required for future management of these unfortunate couples.
Assuntos
Técnicas de Maturação in Vitro de Oócitos , Recuperação de Oócitos , Oócitos , Doenças Ovarianas/fisiopatologia , Adulto , Feminino , Humanos , Oócitos/patologia , Oócitos/fisiologia , Injeções de Esperma IntracitoplásmicasRESUMO
BACKGROUND: Most studies carried out to evaluate recovery and development after porcine oocyte vitrification, reported better rates when cryopreserved in embryonic development stages or zygotes, but not in immature oocytes. For this reason, many studies are performed to improve immature oocyte vitrification protocols testing the use of different cryoprotectant concentrations, cooling devices, incubation times; but only a few of them have evaluated which fertilization procedure enhances blastocyst rates in vitrified oocytes. Therefore, this study was aimed to evaluate: 1) if the sperm selection with hyaluronic acid (HA) or polyvinylpyrrolidone (PVP) before injection could play a key role in increasing fertilization and blastocyst formation and 2) the embryo developmental ability and blastocyst production of porcine immature oocytes retrieved after vitrification-warming and co-cultured with granulosa cells during IVM, using different fertilization techniques: in vitro fertilization (IVF), intracytoplasmic sperm injection (ICSI) and conventional ICSI with hyaluronic acid (HA) sperm selection, known as physiological intracytoplasmic sperm injection (PICSI) and. RESULTS: Sperm selected with HA-PICSI displayed a higher percentage of live/acrosome reacted status compared to those in control and exposed to PVP. Higher dead/acrosome reacted rates were obtained after PVP exposure compared to control and HA. In oocytes, viability significantly decreased after IVM in vitrified oocytes. Besides, IVM rates were not different between control denuded oocytes cultured with granulosa cells (DO-GC) and vitrified oocytes. Regarding fertilization parameters, IVF showed higher percentages of total fertilization rate than those obtained by ICSI and PICSI. However, results demonstrate that PICSI fertilization increased the blastocysts formation rate in control DO-GC and vitrified oocytes compared to IVF and ICSI. CONCLUSIONS: To achieve high blastocyst formation rates from vitrified GV oocytes, it is recommended that sperm should be selected with HA instead of PVP before injection since high viability and acrosome reaction rates were obtained. Also, PICSI fertilization was the best method to produce higher blastocyst rates compared to the IVF and ICSI procedures.
RESUMO
O objetivo foi avaliar diferentes meios na presença e ausência de soro fetal bovino (SFB; 10%) durante o transporte por 24 h de ovários a 4°C sobre a recuperação e a qualidade oocitária bovina. Cinco experimentos foram realizados comparando: (E1) NaCl vs. NaCl+SFB vs. controle (não resfriado), (E2) PBS vs. PBS+SFB vs. controle, (E3) DMEM vs. DMEM+SFB vs. controle, (E4) DPBS vs. DPBS+SFB vs. controle e (E5) melhores resultados dos experimentos anteriores. Após a aspiração folicular, oócitos foram avaliados quanto à qualidade por critérios morfológicos e ensaio de azul cresil brilhante. Ainda, células dos cumulus de oócitos viáveis foram avaliadas quanto à viabilidade pelo azul de tripan. No E1, NaCl permitiu uma maior taxa de recuperação em relação ao NaCl+SFB (46,0% vs. 39,6%), enquanto que os demais parâmetros não foram alterados. Já no E2, o SFB em PBS influenciou positivamente a taxa de recuperação (41,2% vs. 32,8%) e a viabilidade celular (46,3% vs. 41,7%), quando comparado ao PBS. No E3, o SFB em DMEM teve uma influência negativa sobre a taxa de recuperação (39,9% vs. 40,7%) e avaliação morfológica (59,0% vs. 73,1%). Contudo, a adição de SFB ao DMEM se mostrou benéfica à viabilidade celular (42,0% vs. 34,5%). Em relação ao E4, a presença de SFB em DPBS influenciou positivamente a viabilidade celular (50,7% vs. 47,0%). Já no E5, comparando os melhores grupos [NaCl, PBS+SFB, DMEM, DPBS+SFB], a viabilidade celular mostrou uma maior porcentagem em DMEM (54,0%) e DPBS (54,5%), quando comparada a NaCl (48,3%) e PBS (50,1%). Em conclusão, a presença do SFB em meios com alta capacidade de tamponamento (PBS e DPBS) pode se mostrar benéfica. Contudo, DMEM e DPBS resultam num ambiente mais propício para o resfriamento de ovários bovinos.
The aim was to evaluate different media in the presence and absence of fetal bovine serum (FBS; 10%) during 24 h transport of ovaries at 4°C on bovine oocyte recovery and quality. Five experiments were performed comparing: (E1) NaCl vs. NaCl+FBS vs. control (not cold), (E2) PBS vs. PBS+FBS vs. control, (E3) DMEM vs. DMEM+FBS vs. control, (E4) DPBS vs. DPBS+FBS vs. control and (E5) better results from previous experiments. After follicular aspiration, oocytes were evaluated for quality by morphological criteria and brilliant cresyl blue assay. Also, cumulus cells of viable oocytes were evaluated for viability by trypan blue. In E1, NaCl allowed a higher rate of recovery compared to NaCl+FBS (46.0% vs. 39.6%), while the other parameters were not altered. Already in E2, FBS in PBS positively influenced recovery rate (41.2% vs. 32.8%) and cell viability (46.3% vs. 41.7%) when compared to PBS. In E3, FBS in DMEM had a negative influence on the recovery rate (39.9% vs. 40.7%) and morphological evaluation (59.0% vs. 73.1%). Nevertheless, the addition of SFB to DMEM was shown to be beneficial to cell viability (42.0% vs. 34.5%). Regarding E4, the presence of FBS in DPBS positively influenced cell viability (50.7% vs. 47.0%). Already in E5, comparing the best groups (NaCl, PBS+FBS, DMEM, DPBS+FBS), cell viability showed a higher percentage in DMEM (54.0%) and DPBS (54.5%) when compared to NaCl (48.3%) and PBS (50.1%). In conclusion, the presence of FBS in media with high buffering capacity (PBS and DPBS) may show to be beneficial. Nevertheless, DMEM and DPBS result in an environment more favorable to the cooling of bovine ovaries.
Assuntos
Animais , Bovinos , Oócitos , Recuperação de Oócitos/métodos , Recuperação de Oócitos/veterináriaRESUMO
O objetivo foi avaliar diferentes meios na presença e ausência de soro fetal bovino (SFB; 10%) durante o transporte por 24 h de ovários a 4°C sobre a recuperação e a qualidade oocitária bovina. Cinco experimentos foram realizados comparando: (E1) NaCl vs. NaCl+SFB vs. controle (não resfriado), (E2) PBS vs. PBS+SFB vs. controle, (E3) DMEM vs. DMEM+SFB vs. controle, (E4) DPBS vs. DPBS+SFB vs. controle e (E5) melhores resultados dos experimentos anteriores. Após a aspiração folicular, oócitos foram avaliados quanto à qualidade por critérios morfológicos e ensaio de azul cresil brilhante. Ainda, células dos cumulus de oócitos viáveis foram avaliadas quanto à viabilidade pelo azul de tripan. No E1, NaCl permitiu uma maior taxa de recuperação em relação ao NaCl+SFB (46,0% vs. 39,6%), enquanto que os demais parâmetros não foram alterados. Já no E2, o SFB em PBS influenciou positivamente a taxa de recuperação (41,2% vs. 32,8%) e a viabilidade celular (46,3% vs. 41,7%), quando comparado ao PBS. No E3, o SFB em DMEM teve uma influência negativa sobre a taxa de recuperação (39,9% vs. 40,7%) e avaliação morfológica (59,0% vs. 73,1%). Contudo, a adição de SFB ao DMEM se mostrou benéfica à viabilidade celular (42,0% vs. 34,5%). Em relação ao E4, a presença de SFB em DPBS influenciou positivamente a viabilidade celular (50,7% vs. 47,0%). Já no E5, comparando os melhores grupos [NaCl, PBS+SFB, DMEM, DPBS+SFB], a viabilidade celular mostrou uma maior porcentagem em DMEM (54,0%) e DPBS (54,5%), quando comparada a NaCl (48,3%) e PBS (50,1%). Em conclusão, a presença do SFB em meios com alta capacidade de tamponamento (PBS e DPBS) pode se mostrar benéfica. Contudo, DMEM e DPBS resultam num ambiente mais propício para o resfriamento de ovários bovinos.(AU)
The aim was to evaluate different media in the presence and absence of fetal bovine serum (FBS; 10%) during 24 h transport of ovaries at 4°C on bovine oocyte recovery and quality. Five experiments were performed comparing: (E1) NaCl vs. NaCl+FBS vs. control (not cold), (E2) PBS vs. PBS+FBS vs. control, (E3) DMEM vs. DMEM+FBS vs. control, (E4) DPBS vs. DPBS+FBS vs. control and (E5) better results from previous experiments. After follicular aspiration, oocytes were evaluated for quality by morphological criteria and brilliant cresyl blue assay. Also, cumulus cells of viable oocytes were evaluated for viability by trypan blue. In E1, NaCl allowed a higher rate of recovery compared to NaCl+FBS (46.0% vs. 39.6%), while the other parameters were not altered. Already in E2, FBS in PBS positively influenced recovery rate (41.2% vs. 32.8%) and cell viability (46.3% vs. 41.7%) when compared to PBS. In E3, FBS in DMEM had a negative influence on the recovery rate (39.9% vs. 40.7%) and morphological evaluation (59.0% vs. 73.1%). Nevertheless, the addition of SFB to DMEM was shown to be beneficial to cell viability (42.0% vs. 34.5%). Regarding E4, the presence of FBS in DPBS positively influenced cell viability (50.7% vs. 47.0%). Already in E5, comparing the best groups (NaCl, PBS+FBS, DMEM, DPBS+FBS), cell viability showed a higher percentage in DMEM (54.0%) and DPBS (54.5%) when compared to NaCl (48.3%) and PBS (50.1%). In conclusion, the presence of FBS in media with high buffering capacity (PBS and DPBS) may show to be beneficial. Nevertheless, DMEM and DPBS result in an environment more favorable to the cooling of bovine ovaries.(AU)