Slow cooling of human oocytes: ultrastructural injuries and apoptotic status

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.

Key Words: Assisted reproduction, cryopreservation, human oocyte, ultrastructure, mitochondria, apoptosis, cryodamage