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Fine morphological assessment of quality of human mature oocytes after slow freezing or vitrification with a closed device: a comparative analysis.

Bianchi V, Macchiarelli G, Borini A, Lappi M, Cecconi S, Miglietta S, Familiari G, Nottola SA - Reprod. Biol. Endocrinol. (2014)

Bottom Line: 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).Amount and density of cortical granules (CG) appeared abnormally reduced in SFO and VO, irrespective of the protocol applied.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University, Rome, Italy. stefania.nottola@uniroma1.it.

ABSTRACT

Background: Human 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.

Methods: We 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.

Results: Control, 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.

Conclusions: Even 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.

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Related in: MedlinePlus

Human CO, SFO and VO at MII stage. Main characteristics. By LM (a-c) and TEM (d-f) no overt difference in shape, dimensions and organelle distribution is seen among CO (a,d), SFO (b,e) and VO (c,f). Note the intact ZP (a-c) and the presence of microvilli (mv) on the oolemma (d-f). Numerous vacuoles (Va) are seen in SFO (b,e). O: oocyte; PB: 1st PB; arrow: MII spindle with chromosomes. Bar is: 45 μm (a-c); 5 μm (d-f).
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Fig1: Human CO, SFO and VO at MII stage. Main characteristics. By LM (a-c) and TEM (d-f) no overt difference in shape, dimensions and organelle distribution is seen among CO (a,d), SFO (b,e) and VO (c,f). Note the intact ZP (a-c) and the presence of microvilli (mv) on the oolemma (d-f). Numerous vacuoles (Va) are seen in SFO (b,e). O: oocyte; PB: 1st PB; arrow: MII spindle with chromosomes. Bar is: 45 μm (a-c); 5 μm (d-f).

Mentions: LM and TEM techniques allowed analyzing and comparing size, shape and organelle distribution in CO, SFO and VO. CO, SFO and VO were all generally rounded, 90–100 μm in diameter, with normal ooplasm showing uniform distribution of organelles. All CO, SFO and VO showed an intact ZP, separated by a narrow PVS from the oolemma, continuous and provided with microvilli (Figure 1a, b, c, d, e and f).


Fine morphological assessment of quality of human mature oocytes after slow freezing or vitrification with a closed device: a comparative analysis.

Bianchi V, Macchiarelli G, Borini A, Lappi M, Cecconi S, Miglietta S, Familiari G, Nottola SA - Reprod. Biol. Endocrinol. (2014)

Human CO, SFO and VO at MII stage. Main characteristics. By LM (a-c) and TEM (d-f) no overt difference in shape, dimensions and organelle distribution is seen among CO (a,d), SFO (b,e) and VO (c,f). Note the intact ZP (a-c) and the presence of microvilli (mv) on the oolemma (d-f). Numerous vacuoles (Va) are seen in SFO (b,e). O: oocyte; PB: 1st PB; arrow: MII spindle with chromosomes. Bar is: 45 μm (a-c); 5 μm (d-f).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4255960&req=5

Fig1: Human CO, SFO and VO at MII stage. Main characteristics. By LM (a-c) and TEM (d-f) no overt difference in shape, dimensions and organelle distribution is seen among CO (a,d), SFO (b,e) and VO (c,f). Note the intact ZP (a-c) and the presence of microvilli (mv) on the oolemma (d-f). Numerous vacuoles (Va) are seen in SFO (b,e). O: oocyte; PB: 1st PB; arrow: MII spindle with chromosomes. Bar is: 45 μm (a-c); 5 μm (d-f).
Mentions: LM and TEM techniques allowed analyzing and comparing size, shape and organelle distribution in CO, SFO and VO. CO, SFO and VO were all generally rounded, 90–100 μm in diameter, with normal ooplasm showing uniform distribution of organelles. All CO, SFO and VO showed an intact ZP, separated by a narrow PVS from the oolemma, continuous and provided with microvilli (Figure 1a, b, c, d, e and f).

Bottom Line: 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).Amount and density of cortical granules (CG) appeared abnormally reduced in SFO and VO, irrespective of the protocol applied.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, La Sapienza University, Rome, Italy. stefania.nottola@uniroma1.it.

ABSTRACT

Background: Human 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.

Methods: We 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.

Results: Control, 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.

Conclusions: Even 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.

Show MeSH
Related in: MedlinePlus