Human MII Oocytes Research Articles

Visualization and Examination of the Meiotic Spindle in Human Oocytes with Polscope

The effect of detectability/location of meiotic spindle to fertilization of human MII oocytes and outcome of clinical IVF/ICSI-ET cycles was studied. The location of spindle relative to polar body positioned to 6 o'clock was detected with polscope prior to ICSI in oocytes. Data of 83 IVF/ICSI-ET cycle, including pregnancy results, and a total of 1033 oocytes examined were analyzed retrospectively. Polar body position does not always accurately predict spindle position. Similar fertilization was observed in the polscope detected and in the control group. The main number of oocytes and spindle positive oocytes collected was higher in the pregnant than in the non-pregnant group. Higher fertilization was found in that group where 2/3-d of the oocytes had detected spindle. The main number of oocytes and spindle positive oocytes obtained decreased with increasing age of patients. The presence of birefringent spindle predicts higher fertilization and development and greater probability of pregnancy.

The survival of human failed to fertilize MII-stage oocytes cryopreserved by microinjection of trehalose

Oocyte cryopreservation offers women an alternative approach to preserve fertility. We report here the stepwise optimization of a pre-clinical protocol wherein the feasibility of reproducibly cryopreserving human oocytes using failed to fertilized (FTF) MII stage oocytes cryopreserved with the addition of intracellular sugar in clinical practice is demonstrated. Four randomized comparison experiments were performed in a stepwise fashion whereby the experimental condition assessed in one study became the control condition for the following comparison study. Each condition in the experiments contained six replicates. Human FTF MII oocytes obtained from IVF patients under informed consent and IRB approval were utilized in these experiments. Oocytes were injected utilizing micromanipulation techniques with trehalose at an intracellular concentration of approximately 0.1 M. The non- injection control had no intracellular trehalose introduced. Oocytes were cryopreserved in straws in a choline chloride based freezing medium containing 0.4% BSA + 0.5M Trehalose + 0.5M DMSO (or PROH). Straws were seeded at -6 °C, cooled at 1°C/min to -60°C, then 5°C/min to -135°C, followed by plunging to liquid nitrogen. Oocytes were thawed and stepwise diluted in sugar solutions. Optimization of several original reagents and processes was undertaken. Experiments combining reagent changes of BSA to plasmanate, galactose to sucrose and DMEM/F12 to HTF were performed. Process optimization experiments carried out included the evaluation of the Cryo Bio System safety straw, a 30°C water thaw and an alternate sucrose concentration for post thaw dilution. Oocytes were defined as having survived based on the morphological criteria of having an intact zona pellucida, intact oolemma, and a refractive cytoplasm after 24 hour of culture. The results from 48 replicates over two months period are summarized in the Table. Microinjection of intracellular trehalose achieved greater than 60% survival (n=42, 116/182 (63.7%)) through serial optimization experiments. The injection of trehalose into FTF MII stage human oocytes prior to cryopreservation offers consistent and reproducible survival rates. Clinical studies are needed to demonstrate fertilization, embryo development, implantation, clinical pregnancies and live births from oocytes cryopreserved using the method of microinjection of trehalose.

Imaging living, human MII oocytes with the polscope reveals a high proportion of abnormal meiotic spindles.

Objective: Abnormal meiotic spindles are occasionally present in human oocytes and their rate increases significantly with maternal age (Battaglia et al., Human Reprod 11:2217–22). As spindles in living human oocytes can be imaged with a non-invasive, digital and orientation-independent Polscope (Wang et al., Fertil Steril 2001;75:348–53), such a technique may be useful to predict oocyte’s quality based on spindle morphology. In the present study we examined the morphology of spindles in living human oocytes imaged with Polscope and compared the results among the women of various ages.

Reconstruction of Human MII Oocytes by Partial Cytoplasmic Substitution

Reconstruction of Human MII Oocytes by Partial Cytoplasmic Substitution

P-130 Reconstruction of human MII oocytes by partial cytoplasmic substitution

P-130 Reconstruction of human MII oocytes by partial cytoplasmic substitution