Adriana Bos-Mikich

The Effects of Sample Size on the Outcome of Ovarian Tissue Cryopreservation

Cryopreservation of ovarian tissue is known to affect follicular survival. Several variables may be responsible for this. Little attention has focused on the effect of the size of the fragment to be cryopreserved. This study was conducted to assess the effect of the size of the tissue on follicular histology after freezing with 1,2-propanediol. Histological evaluations were performed of control and cryopreserved tissue. Fragments were cut 10 x 3 x 2 mm(3) (2 mm group) or 10 x 3 x 4 mm(3) (4 mm group). Percentages of normal follicles in control fragments cut into 2 and 4 mm slices were 56% and 34%, respectively. The relative risks to obtain normal follicles in the 2 mm and the 4 mm fragments after cryopreservation were 0.63 and 0.47, respectively. Freezing reduced follicle survival to a significantly greater extent in the larger tissue fragments. There is an increased risk of damage to primary and primordial follicles, when the tissue slices are cut with all dimensions larger than 2 mm.

Parthenogenesis and Human Assisted Reproduction.

Parthenogenetic activation of human oocytes obtained from infertility treatments has gained new interest in recent years as an alternative approach to create embryos with no reproductive purpose for research in areas such as assisted reproduction technologies itself, somatic cell, and nuclear transfer experiments and for derivation of clinical grade pluripotent embryonic stem cells for regenerative medicine. Different activating methods have been tested on human and nonhuman oocytes, with varying degrees of success in terms of parthenote generation rates, embryo development stem cell derivation rates. Success in achieving a standardized artificial activation methodology for human oocytes and the subsequent potential therapeutic gain obtained from these embryos depends mainly on the availability of gametes donated from infertility treatments. This review will focus on the creation of parthenotes from clinically unusable oocytes for derivation and establishment of human parthenogenetic stem cell lines and their potential applications in regenerative medicine.

A study on the vitrification of stage III zebrafish (Danio rerio) ovarian follicles.

Attempts to cryopreserve fish embryos have been conducted over the past three decades, nevertheless successful cryopreservation protocol for long-term storage still remains elusive. Fish oocytes offer some advantages when compared to embryos, which may help in improving the chances of cryopreservation. In the present study, a series of cryo-solutions were designed and tested for their vitrifying ability using different devices (0.25ml plastic straw, vitrification block and fibreplug™). Toxicity of vitrification solutions was evaluated by assessing follicle membrane integrity with trypan blue staining. In addition, the effect of vitrification protocol on stage III zebrafish ovarian follicles was investigated by measuring the cytoplasmic ATP content and the mitochondrial distribution and activity using JC-1 probe and confocal microscopy. After vitrification, follicles showed membrane integrity of 59.9±18.4% when fibreplug and V16 (1.5M methanol+4.5M propylene glycol) solution were employed. When vitrified in V2 (1.5M methanol+5.5M Me2SO) the membrane integrity decreased to 42.0±21.0%. It was observed that follicles located in the middle of the fragments were more protected from injuries and some of them showed good morphological appearance 2h post-warming. Mitochondria integrity of granulosa cells layer was clearly damaged by the vitrification protocol and ATP level in the follicles declined significantly after warming. Vitrification of zebrafish follicles in ovarian tissue fragments and its effect at sub-cellular level is reported here for the first time. Information gained from this study will help in guiding development of optimal protocol for cryopreservation of fish oocytes.

Viability of zebrafish (Danio rerio) ovarian follicles after vitrification in a metal container.

Cryopreservation of ovarian tissue has been studied for female germline preservation of farm animals and endangered mammalian species. However, there are relatively few reports on cryopreservation of fish ovarian tissue and especially using vitrification approach. Previous studies of our group has shown that the use of a metal container for the cryopreservation of bovine ovarian fragments results in good primordial and primary follicle morphological integrity after vitrification. The aim of this study was to assess the viability and in vitro development of zebrafish follicles after vitrification of fragmented or whole ovaries using the same metal container. In Experiment 1, we tested the follicular viability of five developmental stages following vitrification in four vitrification solutions using fluorescein diacetate and propidium iodide fluorescent probes. These results showed that the highest viability rates were obtained with immature follicles (Stage I) and VS1 (1.5 M methanol + 4.5 M propylene glycol). In Experiment 2, we used VS1 to vitrify different types of ovarian tissue (fragments or whole ovaries) in two different carriers (plastic cryotube or metal container). In this experiment, Stage I follicle survival was assessed following vitrification by vital staining after 24 h in vitro culture. Follicular morphology was analyzed by light microscopy after vitrification. Data showed that the immature follicles morphology was well preserved after cryopreservation. Follicular survival rate was higher (P < 0.05) in vitrified fragments, when compared to whole ovaries. There were no significant differences in follicular survival and growth when the two vitrification devices were compared.

Substrates and supplements for hESCs: a critical review

BackgroundDifferent laboratories around the world have succeeded in establishing human embryonic stem cell (hESC) lines. However, culture conditions vary considerably among the protocols used and the vast majority of the lines at some stage of their creation have been in contact with an animal derived component. One of the main problems to be overcome for the generation of a clinical-grade hESC line is the choice of a substrate and medium that allows derivation and culture, where animal derived components are kept to a minimum or completely excluded.Materials and methodsThe following review describes past and more recent achievements in the creation and culturing of hESC. It describes protocols, giving special attention to the matrices and supplements used for derivation, mantainance and cryostorage, considering whether they included defined, undefined and/or animal-derived components in their formulations.ConclusionThis information shall be useful for the creation and choice of new substrates and supplements for future research in the field of hESC for therapeutic purposes.

Derivation and culture of putative parthenogenetic embryonic stem cells in new gelatin substrates modified with galactomannan

Human embryonic stem cells (ESC) lines to be used for cell therapies must be created and maintained under strict conditions, excluding the use of undefined supplements. Two key steps in the creation of a new embryonic stem cell line are adherence to the substrate and derivation towards the formation of a primary colony. The bovine parthenote embryo model was used to test different matrices of gelatin nanofibers and gelatin/galactomannan films to be used for ESC derivation and culturing. Gelatin/galactomannan films were made in two concentrations of galactomannan, 0.1 and 0.3%, in an aqueous solution of gelatin and tested for gel cytotoxicity using cumulus cells (CCs). CCs showed normal cell morphology, with no sign of lysis or degeneration in any of the matrices tested. Inner cell masses of parthenote blastocysts (n=116) were placed onto the gel matrices for culture. There were three or four repeats for each matrix. Our results showed a good rate of inner cell mass (ICM) adherence on the gelatin/galactomannan films (41%–44%) and one derivative of the gel nanofiber (17% adherence to the substrate). These results encouraged us to try new gelatin formulations to increase the rates of derivation and cell proliferation under defined culture conditions to comply with good manufacturing practice directives for the potential therapeutic use of ESCs.

Vídeos agregam valor ao trabalho do professor de ciências da saúde

O trabalho apresenta exemplos de videos produzidos para o ensino medico. Justifica o uso de videos nesta area para apoiar a visualizacao de fenomenos cuja observacao in loco depende de situacoes nao reprodutiveis - como cirurgias ou dissecacoes - ou exigem alta capacidade de abstracao dos alunos, no caso de fenomenos microscopicos, quimicos, ou moleculares. Defende que os videos nao induzem a passividade dos alunos,, se forem produzidos de forma criativa e coerente, explorando os recursos da linguagem de video associados ao potencial da tecnologia digital. Os exemplos tambem apresentam recursos de producao e edicao, salientando-se a intencao didatica ou pedagogica que gerou a escolha por um ou outro recurso. Sugere que videos educacionais contribuem para a aprendizagem dos alunos e agregam valor ao trabalho do professor, porque apoiam a representacao e a exposicao do conhecimento e podem constituir objeto de estudo, pesquisa e profissionalizacao de equipes de producao, em todas as instituicoes que pretendem atualizar sua base tecnologica, segundo necessidades impostas pelo Sec. XXI.

Ovarian tissue vitrification: the use of a novel metal closed system for clinical grade cryopreservation

Objective: To investigate whether a metal chamber is an appropriate system for vitrification of ovarian tissue under clinical grade. Methods: Experimental study, control versus treatment. Bovine ovarian cortices cut in 1x1x1 mm fragments were vitrified using ethylene glycol and dimethyl sulfoxide inside steel cryovials, whose bases were in touch with Liquid Nitrogen (LN2). Screw caps closed the cryovials before plunging into LN2. Primordial (n=356) and primary (n=327) follicles and the stroma were analyzed after histological preparation using light microscopy. Results: High rate of primordial (93%) and primary (80%) follicles presented normal morphology in the rewarmed fragments. There was not a significant difference between controls and primordial follicles morphology (P=0.1519). Significant difference was observed for the primary follicles (P=0.0097). Important to point out that stromal cells and collagen fibers presented a remarkable integrity, without major alterations in the cryopreserved tissues. Conclusions: The steel cryovial seems to be a safe means of vitrification under clinical grade conditions, with very fast cooling rates and no direct contact of the biological material with the liquid nitrogen (LN2). Ovarian reserve represented by primordial and primary follicles and stroma are very well preserved in this vitrification system.

Dichorionic twins and monochorionic triplets after the transfer of two blastocysts

PurposeTo describe a unique case of MZ dichorionic twins and MZ monochorionic triplets in a quintuplet gestation after intracytoplasmatic sperm injection (ICSI) and blastocyst transfer.MethodsCase report. A 24-year-old woman underwent ICSI and received two blastocysts transferred. A quintuplet gestation was established .Transvaginal ultrasonography was performed sequentially during early pregnancy.ResultsThree intrauterine gestational sacs were revealed at about 5th week. At the 7th week, five gestational sacs presenting heart beats were detected and a quintuplet pregnancy consisting of two monozygotic (MZ) dichorionic twins and three MZ monochorionic triplets was determined. At the 10th week, a single gestational sac with heart beats was detected. The prenatal course was uneventful. A healthy baby was born at 36th week.ConclusionFew other reports have described the occurrence of a quintuplet gestation after the transfer of two blastocysts generated by ICSI. Our case is unique in that the two blastocysts underwent two different splitting processes, which occurred possibly at a similar time giving rise to MZ dichorionic twins and MZ monochorionic triplets.

Viabilidade e fertilização in vitro de oócitos bovinos após vitrificação

PURPOSE: to verify vitrification techniques using 6 M DMSO to cryopreserve in vitro matured bovine oocytes, and to assess the effects of the time of exposure to vitrification solutions (VS). METHODS: dilutions of VS were prepared from the stock VS (VS 100%) consisting of 6 M DMSO to give 25 and 65% DMSO solutions. Bovine oocytes were in vitro matured for 18-22 h. Matured oocytes were placed first into 25% VS, at room temperature for 5 min, then transferred to 65% VS, before being pipetted into the 100% VS in plastic straws. Three experimental groups were formed: in the first group, time of pipetting through 65% VS and loading the straw took up to 60 s, in the second group it did not exceed 30 s. For thawing, straws were held in air for 10 s and then in a water bath for 10 s. The contents of each straw were expelled in sucrose solution and held for 5 min. In the third experimental group, oocytes went through all VS, but were not vitrified. All retrieved oocytes were inseminated. For control, fresh, in vitro matured oocytes were inseminated. RESULTS: after vitrification, 69.1 and 59.8% of the oocytes were retrieved from the 30 s and 60 s groups, respectively, and 93 and 89% of these oocytes appeared morphologically normal 24 h after insemination, respectively. In the group of oocytes exposed without vitrification, 75.6% were retrieved and 84.7% were morphologically viable, 24 h after insemination. No fertilization was observed in the experimental groups. Among controls, 65.4% were fertilized. CONCLUSIONS: the vitrification technique using 6 M DMSO is not a feasible approach to cryopreserve in vitro matured bovine oocytes. Decreasing the time of exposure to VS did not overcome deleterious effects of the procedure on the fertilizability of oocytes. Improvements in the technique are needed to protect the zona pellucida and oolemma.