Pronuclear examination and embryo biopsy: Assessment and recording of the pronuclei
were performed using an inverted microscope 18-20 hours post ICSI.
ApoE3-Leiden mice were designed by Van den Maagdenberg and co-authors using microinjection of cosmid carrying the desired mutated DNA fragment into male pronuclei
of fertilized mouse eggs taken from super-ovulated females (32).
Sperm and oocyte treatments to improve the formation of male and female pronuclei
and subsequent development following intracytoplasmic sperm injection into bovine oocytes.
Fertilization was confirmed by the presence of two pronuclei
and two polar bodies on day 1.
The IVF technique involves removing the pronuclei
- the nucleus of the sperm and the egg during the process of fertilization - from an embryo containing the mother's unhealthy mitochondria.
Oocytes were examined under an optical microscope and classified as either (a) non fertilized: the presence of female and the absence of male chromatin; (b) normal fertilized: the presence of female and male chromatin in the cytoplasm, a decondensed sperm head, pronuclei
or cleavage; (c) abnormal fertilized: asynchronous oocytes (marked alteration in the formation of pronuclei
, as undecondensed sperm head or telophase II) and > 2 pronuclei
(oocytes in which more than 2 pronuclei
were observed in the cytoplasm); or (d) degenerate oocytes.
Following fertilisation, mitochondria cluster around the 2 pronuclei
[136, 138], plausibly, to meet increased energy demands and ensure an even distribution between dividing cells.
In contrast, PNT involves the removal of the female and male pronuclei
from the donor oocyte following fertilisation and before pronuclei
fusion has occurred.
Correct fertilization was confirmed 16-20 hours afterward when confirming presence of two pronuclei
. After the ICSI, the oocytes were immediately transferred into a Primo Vision micro well group culture dish (Vitolife, Goteburg, Sweden) with a 60 [micro]L droplet with preequilibrated sequential medium.
The fertilization rate was calculated as the number of oocytes with two pronuclei
divided by the number of injected oocytes when ICSI was performed and by all intact oocytes retrieved when IVF was performed.
"Although life is a continuous process, fertilization (which, incidentally, is not a 'moment') is a critical landmark because, under ordinary circumstances, a new genetically distinct human organism is formed when the chromosomes of the male and female pronuclei
blend in the oocyte." (Ronan O'Rahilly and Fabiola Muller, Human Embryology and Teratology, 3rd ed., New York: Wiley-Liss, 2001, p.8.)
The fixed and stained structures were placed between slide and cover slip and exposed to UV light for visualization and counting of male and female pronuclei
as well as first and second polar bodies.
Fertilization was recorded after 12-16 hours if two pronuclei
Options include the transfer of pronuclei
from the parents into a mitochondrial donor zygote,  or alternatively metaphase II spindle transfer into a mitochondrial donor oocyte.
Fertilization rate (FR) was calculated as percentage transformation of micro injected oocytes into two pronuclei
. Categorical variable of FR defined on the basis of 50% FR grouped females; Group I with FR [?]50% and Group II with FR >50%.