Abstract
BackgroundHuman mature oocytes are very susceptible to cryodamage. Several reports demonstrated that vitrification might preserve oocyte better than slow freezing. However, this is still controversial. Thus, larger clinical, biological and experimental trials to confirm this concept are necessary. The aim of the study was to evaluate and compare fine morphological features in human mature oocytes cryopreserved with either slow freezing or vitrification.MethodsWe used 47 supernumerary human mature (metaphase II) oocytes donated by consenting patients, aged 27-32 years, enrolled in an IVF program. Thirtyfive oocytes were cryopreserved using slow freezing with 1.5 M propanediol +0.2 M sucrose concentration (20 oocytes) or a closed vitrification system (CryoTip Irvine Scientific CA) (15 oocytes). Twelve fresh oocytes were used as controls. All samples were prepared for light and transmission electron microscopy evaluation.ResultsControl, slow frozen/thawed and vitrified/warmed oocytes (CO, SFO and VO, respectively) were rounded, 90–100 μm in diameter, with normal ooplasm showing uniform distribution of organelles. Mitochondria-smooth endoplasmic reticulum (M-SER) aggregates and small mitochondria-vesicle (MV) complexes were the most numerous structures found in all CO, SFO and VO cultured for 3–4 hours. M-SER aggregates decreased, and large MV complexes increased in those SFO and VO maintained in culture for a prolonged period of time (8–9 hours). A slight to moderate vacuolization was present in the cytoplasm of SFO. Only a slight vacuolization was present in VO, whereas vacuoles were almost completely absent in CO. Amount and density of cortical granules (CG) appeared abnormally reduced in SFO and VO, irrespective of the protocol applied.ConclusionsEven though, both slow freezing and vitrification ensured a good overall preservation of the oocyte, we found that: 1) prolonged culture activates an intracellular membrane “recycling” that causes the abnormal transformation of the membranes of the small MV complexes and of SER into larger rounded vesicles; 2) vacuolization appears as a recurrent form of cell damage during slow freezing and, at a lesser extent, during vitrification using a closed device; 3) premature CG exocytosis was present in both SFO and VO and may cause zona pellucida hardening.
Highlights
IntroductionSeveral reports demonstrated that vitrification might preserve oocyte better than slow freezing
Human mature oocytes are very susceptible to cryodamage
The 1st polar body (PB) was detected by light microscopy (LM) in the perivitelline space (PVS) (Figure 1a)
Summary
Several reports demonstrated that vitrification might preserve oocyte better than slow freezing. The aim of the study was to evaluate and compare fine morphological features in human mature oocytes cryopreserved with either slow freezing or vitrification. Mature MII oocytes, commonly used for cryopreservation, are among the largest cells in the human body and contain the delicate meiotic spindle. As their cytoplasm contains a high proportion of water in comparison to other cells, damage due to ice crystal formation was a mandatory problem to overcome to achieve viability after thawing [5,6]. Cryopreservation of mature oocytes may cause hardening of the zona pellucida (ZP), impairing fertilization [8]; intracytoplasmic sperm injection (ICSI) may be applied for overcoming this problem [9]
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