Ultrastructure of human mature oocytes after slow cooling cryopreservation with ethylene glycol

Abstract

The morphological characteristics of frozen-thawed human mature oocytes (n = 12) were studied by light and transmission electron microscopy following cryopreservation using a slow cooling protocol including increasing concentrations of ethylene glycol (0.5-1.5 mol/l) and sucrose 0.2 mol/l in the freezing solution. Fresh human mature oocytes (n = 12) were used as controls. Fresh and frozen-thawed oocytes appeared rounded in section, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Disorganization of mitochondria-smooth endoplasmic reticulum aggregates and a decreased complement of microvilli and cortical granules were frequently observable in frozen-thawed oocytes. Increased density of the inner zona pellucida, possibly related to the occurrence of zona 'hardening', was sometimes found associated with a reduced amount of cortical granules. In addition, delamination of the zona pellucida was evident in some frozen-thawed samples. Finally, numerous vacuoles and secondary lysosomes were detected in the ooplasm of most frozen-thawed oocytes. In conclusion, frozen-thawed oocytes treated with ethylene glycol may show a variety of ultrastructural alterations, possibly related, at least in part, to the use of this cryoprotectant. Thus, the ethylene glycol-based protocol of slow cooling herein described does not seem to offer significant advantages in terms of oocyte structural preservation.

Qualitative and morphometric analysis of the ultrastructure of human oocytes cryopreserved by two alternative slow cooling protocols

“…Similar findings were described by Gualtieri et al [24]. Consistent with this, we found that oocytes cryopreserved by a CRSC protocol including ethylene glycol (EG) as an intracellular CPA [23] or by vitrification [39] were also affected by a loss of CG. Here, we extend and confirm our previous experience and provide quantitative data suggesting that about two thirds of the original population of CG is lost, and presumably their contents are released into the perivitelline space, as a result of cryopreservation.…”

“…The one-step protocol seems to be associated to a higher clinical outcome [3], although this has not been confirmed through a prospective randomized control trial. Rather consistently, the presence of vacuoles was described by our group as well as by others in association with a variety of CRSC protocols [19,23,36]. It is tempting to hypothesize that vacuoles located peripherally may evolve from crypt-like invaginations and clusters of endocytic vesicles which form in the oocyte cortex following simple exposure to CPA, as shown by Schalkoff et al [41].…”

“…Conversely, other cryopreservation conditions may be in fact deleterious to mitochondria-SER aggregates. In particular, in oocytes cryopreserved by a CRSC protocol including EG as an intracellular CPA, we noticed pronounced disorganization of these aggregates [23]. This lead us to exclude this protocol as a candidate method applicable for clinical purposes.…”

“…With the exception of an analysis of Van Blerkom and Davies published in the mid-nineties [21], further evidence on the ultrastructure of frozen-thawed oocytes has been gained only recently. Ultrastructural alterations involving the cortical granules (CG), ZP, organelle associations, and ooplasm were reported in coincidence with the application of particular dehydration and rehydration conditions [19,[22][23][24] or possibly specific CPAs [23], suggesting that TEM can represent a first line investigative tool.…”

Vitamin E-analog Trolox prevents endoplasmic reticulum stress in frozen-thawed ovarian tissue of capuchin monkey (Sapajus apella)

“…Ethylene glycol is widely and successfully used for ovarian follicles vitrification because of its low toxicity (Amorim et al 2013;Sheikhi et al 2013;Ting et al 2013). However, when this cryoprotectant is employed during controlled-rate freezing, an increase in ooplasm vacuolization and multivesicular bodies but a decrease in volume of mitochondria/smooth ER aggregates are observed in human oocytes (Nottola et al 2008). Therefore, to minimize cryoprotectant damage, supplementation of the freezing medium with protecting compounds such as antioxidants that might counteract organelle damage will safeguard oocyte quality after freezing-thawing.…”