Hum. Reprod. Advance Access originally published online on December 11, 2006
Human Reproduction 2007 22(4):1123-1133; doi:10.1093/humrep/del463
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Ultrastructure of human mature oocytes after slow cooling cryopreservation using different sucrose concentrations
1 Department of Anatomy, University La Sapienza, Rome, Italy 2 Department of Experimental Medicine, Centre of Electron Microscopy 3 Department of Biomedical Sciences and Technologies, University of L'Aquila, L'Aquila, Italy 4 Tecnobios Procreazione, Bologna, Italy 5 IVF Centre, Vita-Salute University, H.S. Raffaele, Milan, Italy 6 CNESPS, National Health Institute, Rome, Italy 7 University of Bologna, Bologna, Italy
8 To whom correspondence should be addressed at: Laboratory for Electron Microscopy "Pietro M. Motta", Department of Anatomy, University of Rome La Sapienza, Via Alfonso Borelli 50, Rome 00161, Italy. Tel: + 39 06 4991 80 72; Fax: + 39 06 4991 80 81; E-mail: stefania.nottola{at}uniroma1.it
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.
Key words: cryopreservation/human/oocyte/slow cooling/ultrastructure
Submitted on June 16, 2006; resubmitted on October 20, 2006; accepted on November 2, 2006.
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