Vitrification versus Slow Freezing of Human Oocytes: Effects on Ultrastructure and Developmental Potential

Objective:This study compared spindles,cytoskeleton,and developmental potential between vitrified and slow-frozen oocytes using PolScope and electron microscopy.Methods:Oocytes were randomly divided into control,slow freeze-thaw,and vitrification freeze-thaw groups(0,1,and 3 h).PolScope was used to observe spindles,angle of spindles to the first polar bodies,surface areas of oocytes,and lining and outer ret of zona pellucida.Surfaces and ultrastructure of oocytes were observed by scanning electron microscopy and transmission electron microscopy.These measures were used to characterize the impacts of two freezing methods on the developmental capacity of human oocytes.Results:The visible frequency of spindle formation was 92.4%,56.4%,11.2%,24.8%,and 61.1%in control group,slow freeze-thaw group,and the three vitrification freeze-thaw groups(0,1,and 3 h),respectively.Compared to oocytes in the slow freeze group,the angle of the spindle to the first polar body in oocytes in the 3-h vitrification freeze-thaw group was less(37.3°vs.68°,P=0.023).No significant differences were observed between the surface area of oocytes,or lining and outer ret of oocyte zona pellucida between freeze-thaw in these same two groups.Microvilli appeared normal.However,protrusions on oocyte surfaces were increased,and microvilli were laid down on the membrane surface in the 3-h vitrification freeze-thaw group in comparison to the slow-freeze group.Similar comparisons showed better recovery of perivitelline space and mitochondria between the 3-h vitrified and slow-frozen groups.Bipronuclear(2PN)fertilization rate observed in the slow-freeze group(65.7%)was lower than the rate seen in controls(79.2%,P=0.041).No significant differences were observed in 2PN fertilization,cleavage,and blastocyst formation rates between the 3-h vitrification freeze-thaw and control groups.Conclusions:Results suggest that vitrification freeze-thaw for oocyte cryopreservation was a better choice than slow freeze-thaw.

First Babies from Cryopreserved Oocytes in Hungary

Objective To evaluate the value of oocyte cryopreservation （CP） in our clinical ICSI program. Methods Freezing procedure with medium containing 1.5 mol/L propanediol （PrOH） ＋ 0. 3 mol/L succrose and traditional slow-freezingprotocol were used. Thawed oocytes were fertilized with ICSI （4-6 h after thawing）, and fertilization was assessed 12-16 h later. Laser assisted hatching was performed on all transferred embryos and embryo transfer was carried out 48-72 h after ICSI. Results Eighty-five eggs were thawed and survival rate of 75.3% （64/85） was obtained. Sixty-four oocytes were inseminated with ICSI, 47fertilized （47/64; 73.4%） and a cleavage rate of 85% （40/47） was obtained. Embryo transfers were performed in 18 patients, and 4 （19%） resulted in clinical pregnancies. One of the pregnancies encountered first trimester abortion. Implantation rate were 17.2% （5/29） per embryo and 5.8% （5/85） per egg thawed. In all cases, chorion biopsy was performed resulting 46 XY kariotype. Conclusion Our results provide further evidence of that although egg freezing cannot currently claim to be a routine procedure in human IVF,, there will certainly be a place for oocyte CP in reproductive medicine in the future.

Improving native human sperm freezing protection by using a modified vitrification method

Slow freezing is the most commonly used technique for the cryopreservation of spermatozoa in clinical practice.However,it has been shown to have a negative impact on sperm function and structure.Vitrification as a successful alternative method has been proved to have better protective effects on human embryos,but vitrification of spermatozoa is still subject to low recovery rates.In this study,a modified vitrification method for native spermatozoa was developed.A total of 28 semen samples were included;each sample was divided into three equal parts and assigned to fresh,slow freezing,and vitrification groups.Sperm vitality,motility,morphology,DNA integrity,and acrosome reaction were assessed for each of the groups.The results showed that vitrification achieves better results for several sperm protection parameters than slow freezing;vitrification achieves a higher recovery rate(P<0.05),motility(P<0.05),morphology(P<0.05),and curve line velocity(P<0.05)than slow freezing.Furthermore,DNA fragmentation was decreased(P<0.05)and better acrosome protection(P<0.05)was exhibited in the spermatozoa after vitrification.Principal component analysis of all sperm parameters revealed that the vitrification cluster was closer to the fresh cluster,indicating that spermatozoa are better preserved through vitrification.In conclusion,while both slow freezing and vitrification have negative effects on sperm function and structure,the vitrification protocol described here had a relatively better recovery rate(65.8%)and showed improved preservation of several sperm quality parameters compared with slow freezing.