[Oocyte activation with calcium ionophore A23187 and puromycin on human oocytes that fail to fertilize after intracytoplasmic sperm injection].
OBJECTIVE
To investigate the effect of assisted oocyte activation with calcium ionophore A23187 and puromycin on human oocytes that fail to fertilize after intracytoplasmic sperm injection (ICSI).


METHODS
All 113 discarded oocytes that showed no evidence of fertilization at 16 - 18 hours after in vitro maturation (IVM)-ICSI cycles and conventional ICSI were assigned to four groups according to the time after ICSI: IVM-ICSI 22-hour group (n = 33), IVM-ICSI 44-hour group (n = 18), ICSI 44-hour group (n = 37) and ICSI 68-hour group (n = 25). All unfertilized oocytes were exposed to calcium ionophore A23187 (5 micromol/L) for 5 minutes and subsequently incubated with puromycin (10 microg/ml) for 4 hours. After incubation, the oocytes were cultured in vitro for 3 - 5 days. The activation rate, proportion of oocytes that showed pronucleus formation and cleavage rate were calculated after activation. Sex chromosomal analysis was performed by dual color fluorescence in situ hybridization (FISH) on the embryos that displayed two pronuclei and a second polar body.


RESULTS
The combination of calcium ionophore A23187 with puromycin could activate the unfertilized oocytes 22 - 68 hours after ICSI. Best results were achieved in IVM-ICSI 22-hour group, which elicited 88% (29/33) of activation rate, 62% (18/29) of cleavage rate and 28% (5/18) of 4-cell embryos. One embryo in this group developed to the morular stage. The activation rate and developmental potential of the activated embryos in IVM-ICSI 44-hour group, ICSI 44-hour group and ICSI 68-hour group decreased. FISH analysis showed 4 embryos with XX and 9 embryos with XY in 16 embryos.


CONCLUSIONS
The combination of calcium ionophore A23187 with puromycin could effectively activate unfertilized oocytes 22 - 68 hours after ICSI. The cultured time of unfertilized oocytes after ICSI affects activation efficiency and developmental potential of the activated embryos. The activated zygotes that display two pronuclei and a second polar body can develop normally.
Lu
Qun
Q
Reproductive Medicine Center, Shandong Provincial Hospital, Shandong University, Jinan 250021, China.
Chen
Zi-jiang
ZJ
Gao
Xuan
X
Ma
Shui-ying
SY
Li
Mei
M
Hu
Jing-mei
JM
Li
Yuan
Y
D000001:Calcimycin / Q000494:pharmacology*; D002478:Cells, Cultured; D002970:Cleavage Stage, Ovum / Q000187:drug effects; D004622:Embryo, Mammalian / Q000166:cytology / Q000187:drug effects; D005260:Female; D005307:Fertilization in Vitro; D006801:Humans; D017404:In Situ Hybridization, Fluorescence; D007476:Ionophores / Q000494:pharmacology; D007621:Karyotyping; D008297:Male; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011691:Puromycin / Q000494:pharmacology*; D020554:Sperm Injections, Intracytoplasmic; D013997:Time Factors; D015053:Zygote / Q000166:cytology / Q000187:drug effects / Q000254:growth & development
D007476:Ionophores; D000001:Calcimycin; D011691:Puromycin