Vitrification of human immature oocytes before and after in vitro maturation: a review

Abstract

The use of immature oocytes subjected to in vitro maturation (IVM) opens interesting perspectives for fertility preservation where ovarian reserves are damaged by pathologies or therapies, as in PCO/PCOS and cancer patients. Human oocyte cryopreservation may offer some advantages compared to embryo freezing, such as fertility preservation in women at risk of losing fertility due to oncological treatment or chronic disease, egg donation and postponing childbirth. It also eliminates religious and/or other ethical, legal, and moral concerns of embryo freezing. In addition, a successful oocyte cryopreservation program could eliminate the need for donor and recipient menstrual cycle synchronization. Recent advances in vitrification technology have markedly improved the oocyte survival rate after warming, with fertilization and implantation rates comparable with those of fresh oocytes. Healthy live births can be achieved from the combination of IVM and vitrification, even if vitrification of in vivo matured oocytes is still more effective. Recently, attention is given to highlight whether vitrification procedures are more successful when performed before or after IVM, on immature GV-stage oocytes, or on in vitro matured MII-stage oocytes. In this review, we emphasize that, even if there are no differences in survival rates between oocytes vitrified prior to or post-IVM, reduced maturation rates of immature oocytes vitrified prior to IVM can be, at least in part, explained by underlying ultrastructural and biomolecular alterations.

Keywords: In vitro maturation; Oocyte; Transmission electron microscopy; Ultrastructure; Vitrification.

Fig. 1

Fresh control (a) and vitrified-warmed (b, c) human oocytes. LM shows the general morphology and organelle microtopography. No explicit differences in shape, dimensions, and organelle distribution were found between fresh and vitrified oocytes. Apparent variations of ZP thickness is an effect of the section plane (not equatorial). Vacuoles were detected only sporadically in both fresh (a) and vitrified-warmed (b, c) oocytes. O oocyte, Va vacuoles, ZP zona pellucida, M-SER mitochondria-smooth endoplasmic reticulum aggregates, PB polar body (modified from: [69] Bianchi et al., 2014 (a, c) and [67] Nottola et al., 2009 (b))

Fig. 2

Ultrastructure of human MII stage oocytes obtained from IVM of vitrified-warmed GV-stage oocytes. Mitochondria are generally rounded and provided with few peripheral or transverse cristae. Note the presence of complexes between mitochondria and vesicles of smooth endoplasmic reticulum (a, b). Dumbbell-shaped, possibly dividing mitochondria can be occasionally found in the ooplasm (asterisk in d). Extensive vacuolization is showed in c. Cortical granules are sparse in the ooplam (b) and form a discontinuous layer beneath the oolemma in vitrified-warmed oocytes (a, d). Microvilli are seen on the oolemma of vitrified-warmed oocytes (b, c). ZP zona pellucida, m mitochondria, mv microvilli, MV mitochondria-vesicle complexes, Ly lysosome, V vacuoles, CG cortical granules (modified from: [52] Shahedi et al., 2013)

Fig. 3

Fresh (a) and vitrified-warmed MII oocytes (b, c). TEM micrograph showing the presence of a continuous layer of cortical granules under the oolemmal membrane (a), differently to vitrified-warmed oocytes, irrespective of the open (b) or closed (c) cryodevices used. Note the increased compaction of the inner aspect of the ZP in b in comparison with the looser texture in a, c. CG cortical granules, ZP zona pellucida, PVS perivitelline space, mv microvilli, V vacuole, SER smooth endoplasmic reticulum (modified from: [67] Nottola et al., 2009 (a, b); [69] Bianchi et al., 2014 (c))

Fig. 4

Vitrified-warmed MII stage oocytes. Ultrastructure of multivesicular bodies (a) and lysosomes, sometimes associated with vacuoles (b). MVB multivesicular body, Ly lysosome, m mitochondria, MV mithocondria-vacuoles complexes, V vacuoles (modified from: [67] Nottola et al., 2009 (a) and [69] Bianchi et al., 2014 (b))

Fig. 5

Voluminous aggregates between mitochondria and elements of smooth endoplasmic reticulum are seen in fresh (a) and vitrified-warmed MII stage oocytes (b). Immature M-SER aggregates can be also found after vitrification (c). M-SER mitochondria-smooth endoplasmic reticulum aggregates, V vacuole (modified from: [72] Coticchio et al., 2010 (a); [67] Nottola et al., 2009 (b, c))