The aims of this study were to examine, in a prospective, controlled way, the effect of the sperm deposition site in the oocyte and the mode of oolemma breakage in intracytoplasmic sperm injection (ICSI) on fertilization and embryo development rates. In the first trial (100 cycles in total), the spermatozoa were deposited further from the meiotic spindle (polar body at the 12 o'clock position) in half of the oocytes (n = 649), while in the other half (n = 605) the spermatozoa were deposited nearer to the meiotic spindle (polar body at the 6 o'clock position). In the second trial (6860 oocytes in 624 cycles), five different modes of membrane breakage (the reaction of the oolemma to the penetrating injection needle) at the moment of injection were noted: oolemma breakage, type A pricking once, no suction (n = 1401); type B, pricking once, small suction (n = 2761); type C, pricking once, long suction (n = 2310); type D, pricking twice or more, no or small amount of suction (n = 259); and type E, pricking twice or more, long suction (n = 129). No differences were observed between the 12 and 6 o'clock positions in the survival rate (90 and 90% respectively) and in the normal fertilization rates (78 and 77% respectively). Significantly more transfer quality embryos (< or = 50% fragmentation) were obtained in the 6 o'clock position group (83%) than in the 12 o'clock position group (79%). In the second trial, significantly lower survival rates were noted after membrane breakage type A (82%) than after breakages of types B, C, D and E (93, 92, 88 and 88% respectively). There were no significant differences present in the normal fertilization rates (70, 72, 70, 71 and 73% for types A-E respectively), but significantly more freeze quality embryos (< or = 20% fragmentation) were obtained after injection B (65%) than after injection types A, C, D and E (59, 61, 55 and 51% respectively). In conclusion, the site of sperm deposition in the oocyte does not influence the normal fertilization rate but does affect the embryo development rate. Furthermore, the mode of membrane breakage does not influence the normal fertilization rate but does affect oocyte survival and embryo development rates.

Oocyte vitrification has been introduced into clinical settings without extensive pre-clinical safety testing. In this study, we analysed major safetyaspects of human oocyte vitrification in a high security closed system: (i) chromosomal meiotic segregation, (ii) embryonic developmental kinetics and (iii) DNA (hydroxy)methylation status. Fresh and vitrified sibling oocytes from young donors after intracytoplasmic sperm injection (ICSI) were compared in three different assays. Firstly, the chromosomal constitution of the fertilized zygotes was deduced from array comparative genomic hybridization results obtained from both polar bodies biopsied at Day 1. Secondly, embryo development up to Day 3 was analysed by time-lapse imaging. Ten specific time points, six morphokinetic time intervals and the average cell number on Day 3 were recorded. Thirdly, global DNA methylation and hydroxymethylation patterns were analysed by immunostaining on Day 3 embryos. The nuclear fluorescence intensity was measured by Volocity imaging software. Comprehensive chromosomal screening of the polar bodies demonstrated that at least half of the zygotes obtained after ICSI of fresh and vitrified oocytes were euploid. Time-lapse analysis showed that there was no significant difference in cleavage timings, the predictive morphokinetic time intervals nor the average cell number between embryos developed from fresh and vitrified oocytes. Finally, global DNA (hydroxy)methylation patterns were not significantly different between Day 3 embryos obtained from fresh and from vitrified oocytes. Our data further consolidate the safety of the oocyte vitrification technique. Nevertheless, additional testing in young and older sub-fertile/infertile patients and sound follow-up studies of children born after oocyte cryopreservation remain mandatory.

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Prior to intracytoplasmic sperm injection (ICSI), seminal fluid is currently washed out from the ejaculated semen and further sperm selection is carried out by a discontinuous Percoll gradient. Possible deleterious effects from the sperm-separating substance Percoll on sperm function or embryo cleavage after in-vitro fertilization (IVF) have, to our knowledge, not yet been reported and the use of Percoll has been widely accepted in IVF. In this study, we examined whether the omission of the Percoll step in the sperm preparation has any influence on the outcome of the ICSI procedure. Two methods of sperm preparation for ICSI were compared in a controlled study on sibling oocytes: washing the semen sample once, followed by a Percoll gradient centrifugation versus washing the sperm sample twice without a Percoll gradient centrifugation. The mean fertilization rates were similar for the two sperm preparation methods: 78.2 +/- 21.4 and 75.0 +/- 24.1% respectively of the intact oocytes displaying two pronuclei. Cleavage rates did not differ statistically between the two groups. Whereas in both groups similar percentages of excellent, good and poor quality embryos were obtained, the percentage of fair quality embryos was significantly higher in the group without Percoll (16.3 +/- 20.1 versus 9.1 +/- 15.7%). However, no statistical differences were observed in either the percentage of transferable embryos or in the percentage of embryos actually transferred or frozen in the two groups. In conclusion, spermatozoa from ejaculates that are washed out from the seminal fluid without any further selection can be used for ICSI without any adverse effect on fertilization and embryo cleavage.

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