Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, Laboratory of Veterinary Physiology and Biochemistry, Gamete Research Centre, University of Antwerp, Universiteitsplein 1, Gebouw U, 2610, Wilrijk, Belgium.
J Assist Reprod Genet. 2018 Jul;35(7):1187-1199. doi: 10.1007/s10815-018-1208-3. Epub 2018 May 24.
Individual follicle cryopreservation techniques, without hydrogel support, are labor-intensive and a substantial proportion of isolated follicles are lost during handling and after warming. Therefore, the viability and morphology of isolated bovine (as a model for human) pre-antral follicles after vitrification and warming, when encapsulated in alginate beads, were investigated.
Bovine pre-antral follicles were mechanically isolated and divided into four different groups: (1) culture in 2% alginate beads (3D system) and vitrification in beads using mesh cups (3DVIT), (2) culture in 2% alginate beads (3DCUL), (3) culture in 96-well plates (2D system) and vitrification using High Security Vitrification straws® (2DVIT), (4) culture in a 2D system (2DCUL). The same vitrification and warming protocols were used for embedded (3DVIT) and non-embedded follicles (2DVIT).
No differences were observed in follicle viability between group 2DCUL and 3DCUL. Group 3DVIT showed the lowest viability (45.9%) according to calcein and neutral red staining among all groups. Group 2DVIT displayed the highest viability (87.5%) and largest percentage of follicles with a well-preserved morphology.
Our results show that, using a vitification protocol optimized for non-embedded follicles, 2D culture is more effective in vitrifying isolated follicles. However, embedding in alginate allow to handle follicles more efficiently, i.e., without excessive manipulation and thus less labor-intensive in combination with a reduced loss of follicles during the procedure. Based on the increased work efficiency, but lower viability and higher proportion of follicles showing impaired morphology, we consider it advantageous to optimize the protocol for the vitrification of embedded follicles to increase survival and maintain morphology after vitrification.
没有水凝胶支持的单个卵泡冷冻技术劳动强度大，在处理和解冻后，很大一部分分离的卵泡会丢失。因此，研究了在海藻酸钠珠粒中包埋后玻璃化和解冻时，分离的牛（作为人类模型）原始卵泡的活力和形态。
牛原始卵泡通过机械分离，分为四组：（1）在 2%海藻酸钠珠粒（3D 系统）中培养，并使用网杯进行珠粒玻璃化（3DVIT），（2）在 2%海藻酸钠珠粒（3DCUL）中培养，（3）在 96 孔板中培养（2D 系统），并使用高安全性玻璃化吸管（2DVIT）进行玻璃化，（4）在 2D 系统中培养（2DCUL）。对包埋（3DVIT）和非包埋卵泡（2DVIT）使用相同的玻璃化和解冻方案。
2DCUL 和 3DCUL 组的卵泡活力没有差异。根据钙黄绿素和中性红染色，3DVIT 组的活力最低（45.9%）。2DVIT 组的活力最高（87.5%），形态保存良好的卵泡比例最大。
我们的结果表明，使用针对非包埋卵泡优化的玻璃化方案，2D 培养在玻璃化分离卵泡方面更有效。然而，在海藻酸钠中包埋可以更有效地处理卵泡，即在操作过程中不需要过度处理，从而减少劳动力，并且在处理过程中卵泡的损失也更少。基于提高的工作效率，但活力降低且更多的卵泡显示出受损的形态，我们认为优化包埋卵泡的玻璃化方案以提高存活率和保持玻璃化后形态是有利的。
