Slow cooling of human oocytes: ultrastructural injuries and apoptotic status
Received 7 November 2007; received in revised form 11 January 2008; accepted 22 January 2008. published online 25 March 2008.
Objective
To identify the damages caused by slow cooling human metaphase II (MII) oocytes comparing the ultrastructure, inner mitochondrial membrane potential (ΔΨm), and apoptotic status of fresh and cryopreserved oocytes.
Design
Experimental study.
Setting
University biology research unit and private IVF unit.
Patient(s)
Fresh and cryopreserved supernumerary MII oocytes donated from women undergoing IVF cycles.
Main Outcome Measure(s)
Ultrastructure was assessed by transmission electron microscopy (TEM), mitochondrial function by means of the fluorescent ΔΨm reporter JC-1, and apoptotic status through fluorescent labeling with the pan-caspase inhibitor fluorescein isothiocyanate conjugate (FITC)-VAD FMK, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
Result(s)
Compared to fresh oocytes, frozen/thawed (F/T) oocytes showed reduced cortical granule densities (F/T 3.35 ± 1.94/10 μm vs. fresh 10.30 ± 3.9/10 μm), swelling of smooth endoplasmic reticulum (F/T 0.084 ± 0.03 μm2 vs. fresh 0.040 ± 0.02 μm2), decreased electron density of the mitochondrial matrix and damage to the mitochondrial membranes, low ΔΨm of pericortical mitochondria, but no signs of apoptosis.
Conclusion(s)
Slow cooling is associated with cortical granule exocytosis, swelling of smooth endoplasmic reticulum vesicles, and mitochondrial damage, but does not induce early or late apoptotic events. The observed injuries might be responsible for the reduced developmental competence of cryopreserved oocytes.
aDepartment of Structural and Functional Biology, University of Naples “Federico II”, Monte S Angelo, Naples, Italy
bMediterranean Center of Assisted Reproduction, Mediterranean Clinic, Naples, Italy
cDepartment of Biological Sciences, University of Naples “Federico II”, Monte S Angelo, Naples, Italy
Reprint requests: Roberto Gualtieri, Ph.D., Dipartimento di Biologia Strutturale e Funzionale, Università di Napoli “Federico II,” Complesso Universitario di Monte S Angelo, Via Cinthia, 80126 Napoli, Italy (FAX: 39-81679233).
R.G. has nothing to disclose. M.I. has nothing to disclose. V.M. has nothing to disclose. M.P. has nothing to disclose. S.I. has nothing to disclose. R.T. has nothing to disclose.
Supported by the Italian Ministry of University and Research, PRIN 2005 grant 2005073071_004 to Roberto Gualtieri “Influence of Cryopreservation on Qualitative Parameters of In Vivo Matured Human Oocytes.”
ABSTRACT