Journal of Assisted Reproduction and Genetics

, Volume 32, Issue 9, pp 1343–1358 | Cite as

Ultrastructural markers of quality are impaired in human metaphase II aged oocytes: a comparison between reproductive and in vitro aging

  • S. Bianchi
  • G. Macchiarelli
  • G. Micara
  • A. Linari
  • C. Boninsegna
  • C. Aragona
  • G. Rossi
  • S. Cecconi
  • S. A. Nottola
Gamete Biology



Childbearing delay contributes to the increase of subfertile couples that require assisted reproductive technology (ART). Subfertility relates with reproductive aging (RA). In vitro aging (IvA) (due to extended culture) may also impair oocyte competence. Aims of this study were to evaluate and compare the oocyte ultrastructure after RA and IvA.


Cumulus-oocyte complexes (COCs) (n = 68), with metaphase II oocyte and expanded cumulus, from consenting patients (<35 years old and ≥35 years old, n = 36), were selected by phase contrast microscopy and fixed at pick up, or after 24 h culture. COCs (n = 44) were studied by light and qualitative/morphometric transmission electron microscopy. Two-way ANOVA, with age and culture as grouping factors, was applied for statistical analysis (p < 0.05). Metaphase II cumulus-free oocytes (n = 24) were selected for confocal microscopy observations.


Significant decrease of mitochondria-smooth endoplasmic reticulum aggregates, increase of mitochondria-vesicle complexes size and amount, decrease of cortical granules and microvilli, and alterations of the spindle structure characterized both RA and IvA oocytes. These changes were significantly more evident in the RA oocytes submitted to IvA. RA oocytes also showed changes of the zona pellucida and occurrence of vacuoles after culture. Cumuli appeared re-compacted after culture, irrespective of the age of the patients.


These data demonstrated that aging is related to decay of oocyte ultrastructural quality, and that oocytes from elder women are more sensitive to prolonged culture (IvA) than the oocytes from younger women. These morphological results should be considered when applying ART in aged patients, rescue ICSI, or artificial oocyte activation.


Oocyte Aging Quality Assisted reproduction Human Ultrastructure 



Assisted reproductive technology




Cumulus-oocyte complexes

CC cells

Cumulus-corona cells


Cortical granules




Intracytoplasmic sperm injection


In vitro fertilization


In vitro aging


Light microscopy


Metaphase II


Mitochondria-smooth endoplasmic reticulum






1st polar body


Phase contrast microscopy


Perivitelline space


Reproductive aging




Smooth endoplasmic reticulum


Transmission electron microscopy


Ultrasound scan


Zona pellucida


Zona pellucida thickness



The authors wish to acknowledge Prof. Stefano Necozione of the Department of Life, Health and Environmental Sciences, University of L’Aquila, for providing statistical advice and Mr. Ezio Battaglione of the Laboratory for Electron Microscopy “Pietro M. Motta”, Department of Anatomy, Histology, Forensic Medicine and Orthopaedics, “Sapienza” University, Rome, Italy, for his technical assistance.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.


The present study was supported by grants from the Italian Ministry of Education, University and Research (Sapienza and L’Aquila university grants), years 2011–2013.

Conflicts of interest

The authors declare that they have no conflict of interest.


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Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • S. Bianchi
    • 1
  • G. Macchiarelli
    • 1
  • G. Micara
    • 2
  • A. Linari
    • 2
  • C. Boninsegna
    • 2
  • C. Aragona
    • 2
  • G. Rossi
    • 1
  • S. Cecconi
    • 1
  • S. A. Nottola
    • 3
  1. 1.Department of Life, Health and Environmental SciencesUniversity of L’AquilaL’AquilaItaly
  2. 2.Department of Gynaecology-Obstetrics & Urology“Sapienza” UniversityRomeItaly
  3. 3.Department of Anatomy, Histology, Forensic Medicine and Orthopaedics“Sapienza” UniversityRomeItaly

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