Assessment and analysis of outcome data
The primary outcome measures are: (i) Survival (ii) Development to the blastocyst stage (iii) Blastocyst quality (iv) MtDNA carryover.
Survival of manipulated zygotes: An experienced operator would be expected to achieve >95% fusion of both karyoplasts to the cytoplast and ~ 90% survival
Blastocyst formation and quality: We strongly recommend using autologous transfers to develop skills in the ePNT procedures. Autologous transfers are performed by removing and replacing the pronuclei in the same zygote and control for the technical procedures. Development to the blastocyst stage should not differ between unmanipulated controls and technical controls. The timing of blastocyst formation (day 5 or day 6) should be taken into account. For morphological assessment, we use a grading system that divides blastocysts into six classes (A to F), according to the degree of expansion, the size and degree of compaction of the ICM, and the TE morphology. Please see below on analysis of blastocyst morphology.
MtDNA carryover: MtDNA carryover should be measured in multiple samples of cells from ePNT blastocysts. This should be measured using a sensitive molecular technique, optimized for measuring low levels of heteroplasmy.
The aim is to carryover the smallest possible amount of mtDNA during the ePNT procedure. The amount of mtDNA carryover is influenced by the size of the karyoplast which can be minimized by shearing off excess cytoplasm prior to fusion, as described above. MtDNA carryover can also be increased by leakage of cytoplasm from the cytoplast. In our experience this problem is exacerbated in zygotes originating from vitrified oocytes. We find that, if using zygote pairs originating from fresh and vitrified oocytes, mtDNA is lowest when the cytoplast originates from a fresh oocyte and the karyoplast from a vitrified oocyte. Under these conditions, we aim for a maximum level of 2% mtDNA carryover and find that the procedures described above result in >40% of samples with undetectable levels.
Analysis of blastocyst quality: Given the limited number of oocytes available for research, a multiple-grade scoring system inevitably leads to very low numbers of blastocysts in some grades, which in turn, reduces the power of statistical analysis. We therefore recommend focusing the analysis on the proportion of blastocysts whose morphological characteristics are consistent with a high probability of implantation (grades A+B). We compare grades A+B with all other grades combined, using Fisher’s exact test. This approach provides a statistically robust and clinically relevant measure.
Comparing multiple experimental groups: Given the exploratory nature of this work and the limited number of human oocytes available to optimize the ePNT procedure, we chose not to apply corrections to multiple comparisons. Multiple test corrections reduce the risk of false positives (detecting an effect where none really exists) at the cost of increasing the false negative rate (not detecting an effect where one really exists). Because promising leads can be tested with follow-up experiments, we consider it more important to minimize the false negative rather than the false positive rate. By contrast, a high false negative rate entails the risk of prematurely eliminating potentially effective treatments. For example, in the case of mtDNA carryover, effects that might increase carryover could be missed. However, within each analysis, it is important to restrict the number of comparisons those of clinical relevance.