Ultrastructure of human mature oocytes after slow cooling cryopreservation using different sucrose concentrations

Abstract

Background: We studied the ultrastructural characteristics of human mature oocytes frozen/thawed (F/T) using different concentrations of sucrose. Fresh human mature oocytes were used as controls.

Methods: The oocytes (n = 48) were fixed in 1.5% glutaraldehyde at sampling (n = 16) or after freeze/thawing performed using a slow cooling method with propane-1,2-diol 1.5 mol/l and sucrose at either 0.1 mol/l (n = 16) or 0.3 mol/l (n = 16) in the freezing solution. The oocytes were then processed for electron microscopy observations.

Results: Fresh and F/T oocytes belonging to both study groups were regularly rounded in sections, with a homogeneous cytoplasm and an intact zona pellucida (ZP). Organelles (mainly mitochondria-smooth endoplasmic reticulum aggregates and mitochondria-vesicle complexes) were abundant and uniformly dispersed in the ooplasm. The amount and density of cortical granules appeared to be abnormally reduced in some F/T samples, independently of the sucrose concentration in the freezing solution: this feature was frequently associated with an increased density of the inner ZP, possibly related to the occurrence of zona 'hardening'. Furthermore, slight to moderate microvacuolization was revealed in the ooplasm of some F/T oocytes, particularly in those treated with sucrose 0.3 mol/l.

Conclusions: Freeze/thawing procedures are associated with ultrastructural alterations in specific oocyte microdomains, presumably linked to the reduced developmental potential of mature cryopreserved oocytes. Further work is needed to determine whether or not a high concentration of sucrose plays a role, at least in part, in producing the above alterations.