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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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Human SFO and VO at MII stage. Presence of vacuoles. By TEM, numerous vacuoles (Va) are seen in the ooplasm of SFO (a,b), whereas only a few vacuoles are visible in VO (c). Vacuoles appear empty (a-c) or contain cell debris (a,b). Note some interruptions in the vacuole membrane (a-c). A close association between a vacuole and a lysosome (Ly) is seen in (c). A typical M-SER aggregate (b), isolated mitochondria (M) and MV complexes (c) are seen in the areas adjacent to vacuoles. Bar is: 1 μm (a-c).
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Fig3: Human SFO and VO at MII stage. Presence of vacuoles. By TEM, numerous vacuoles (Va) are seen in the ooplasm of SFO (a,b), whereas only a few vacuoles are visible in VO (c). Vacuoles appear empty (a-c) or contain cell debris (a,b). Note some interruptions in the vacuole membrane (a-c). A close association between a vacuole and a lysosome (Ly) is seen in (c). A typical M-SER aggregate (b), isolated mitochondria (M) and MV complexes (c) are seen in the areas adjacent to vacuoles. Bar is: 1 μm (a-c).

Mentions: Using TEM, the most numerous organelles found in all the CO, SFO and VO cultured for 3–4 hours, consisted of aggregates of anastomosing tubuli of smooth endoplasmic reticulum (SER) surrounded by mitochondria (M-SER aggregates). The diameter of the tubular network of the M-SER aggregates varied from 1 to 5 μm (Figure 2a and b). Small vesicles, 0.3 – 0.5 μm in diameter, containing a slighlty electrondense material were associated with mitochondria forming the so-called mitochondria-vesicle (MV) complexes (Figure 2b). No overt qualitative differences in the fine structural morphology of M-SER aggregates and MV complexes came out from the comparison among CO, SFO and VO after 3–4 hours of culture. M-SER aggregates appeared instead partially replaced by numerous, large MV complexes, up to 2.5 μm in vesicle diameter, when SFO and VO extended culture for a prolonged period of time (8–9 hours) (Figure 2c and d). Mitochondria, either associated with membranes or isolated, revealed a normal fine structure in all the samples observed (CO, SFO and VO). They were rounded or oval in profile, with a diameter varying from 0.5 to 0.8 μm and a few peripheral arch-like or transverse cristae and contained a moderately electrondense matrix (Figure 2a, b and d; Figure 3c).Figure 2


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 SFO and VO at MII stage. Presence of vacuoles. By TEM, numerous vacuoles (Va) are seen in the ooplasm of SFO (a,b), whereas only a few vacuoles are visible in VO (c). Vacuoles appear empty (a-c) or contain cell debris (a,b). Note some interruptions in the vacuole membrane (a-c). A close association between a vacuole and a lysosome (Ly) is seen in (c). A typical M-SER aggregate (b), isolated mitochondria (M) and MV complexes (c) are seen in the areas adjacent to vacuoles. Bar is: 1 μm (a-c).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Human SFO and VO at MII stage. Presence of vacuoles. By TEM, numerous vacuoles (Va) are seen in the ooplasm of SFO (a,b), whereas only a few vacuoles are visible in VO (c). Vacuoles appear empty (a-c) or contain cell debris (a,b). Note some interruptions in the vacuole membrane (a-c). A close association between a vacuole and a lysosome (Ly) is seen in (c). A typical M-SER aggregate (b), isolated mitochondria (M) and MV complexes (c) are seen in the areas adjacent to vacuoles. Bar is: 1 μm (a-c).
Mentions: Using TEM, the most numerous organelles found in all the CO, SFO and VO cultured for 3–4 hours, consisted of aggregates of anastomosing tubuli of smooth endoplasmic reticulum (SER) surrounded by mitochondria (M-SER aggregates). The diameter of the tubular network of the M-SER aggregates varied from 1 to 5 μm (Figure 2a and b). Small vesicles, 0.3 – 0.5 μm in diameter, containing a slighlty electrondense material were associated with mitochondria forming the so-called mitochondria-vesicle (MV) complexes (Figure 2b). No overt qualitative differences in the fine structural morphology of M-SER aggregates and MV complexes came out from the comparison among CO, SFO and VO after 3–4 hours of culture. M-SER aggregates appeared instead partially replaced by numerous, large MV complexes, up to 2.5 μm in vesicle diameter, when SFO and VO extended culture for a prolonged period of time (8–9 hours) (Figure 2c and d). Mitochondria, either associated with membranes or isolated, revealed a normal fine structure in all the samples observed (CO, SFO and VO). They were rounded or oval in profile, with a diameter varying from 0.5 to 0.8 μm and a few peripheral arch-like or transverse cristae and contained a moderately electrondense matrix (Figure 2a, b and d; Figure 3c).Figure 2

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