DNA spatial distribution in unfertilized human oocytes by confocal laser scanning microscope.

Abstract

The aim of this study is to investigate the DNA spatial distribution in unfertilized human oocytes by confocal laser scanning microscope (CLSM). One hundred and forty unfertilized oocytes after conventional in vitro fertilization (IVF) and one hundred and three after intracytoplasmic sperm injection (ICSI) were studied. The chromatin was stained with chromomycin A3 (CA3) initially and at a second step with propidium iodide, allowing the observation of the deprotaminated sperm heads in the ooplasm. CLSM offered the opportunity for studying the three-dimensional position of the spermatozoon in the oocyte and the description of the status of the maternal and paternal chromatin. For each of the IVF and ICSI group of unfertilized oocytes, subgroups were performed according to the status of the maternal and paternal chromatin. The maternal chromosomes were either aligned at metaphase II or disarrayed at metaphase II, and the sperm head was either condensed or swollen/decondensed. Few oocytes were activated and formed a pronucleus. The spatial approaching between paternal and maternal chromatin was calculated for all subgroups of unfertilized oocytes and the distances were compared among them. There was no statistical difference in the distance between the spermatozoon and the maternal chromatin among subgroups of the IVF group, while statistical differences occurred among subgroups of the ICSI group. The spermatozoa in the IVF group approached maternal chromatin more than those in the ICSI group. Movement of the spermatozoon into the oocyte and chromatin remodeling for the formation of pronucleus seem to occur independently and are possibly directed by different mechanisms.