Comparison of 2, 5, and 20 % O2 on the development of post-thaw human embryos

  • Yu Yang
  • Yanwen Xu
  • Chenhui Ding
  • Rabea Youcef khoudja
  • Mingmei Lin
  • Awoniyi O. Awonuga
  • Jing Dai
  • Elizabeth E. Puscheck
  • Daniel A. Rappolee
  • Canquan Zhou
Embryo Biology



The objective of this study is to investigate the effect of 2, 5, and 20 % O2 on post-thaw day 3 human embryo culture until blastocyst stage.


One hundred fifty-five day 3 human embryos were used. One hundred twenty out of 155 embryos were recovered after thawing. Surviving embryos were distributed into 2, 5, or 20 % O2 groups and cultured for 2.5 days. At the end of culture, blastocyst formation was assessed, and then, embryos were collected for RT-qPCR or immunofluorescence analysis.


Using visible blastocoel to define blastocyst formation, 58.7 % (27/46) of surviving day 3 embryos formed blastocyst at 2 % O2, 63.6 % (28/44) at 5 % O2, and 66.7 % (20/30) at 20 % O2. The difference in blastocyst formation rates was not significant. Average blastocyst cell number was 119.44 ± 11.64 at 2 % O2, 142.55 ± 22.47 at 5 % O2, and 97.29 ± 14.87 at 20 % O2. Average apoptotic rate was 4.7 % ± 0.4 % for blastocyst formed at 2 % O2, 3.5 % ± 0.7 % at 5 % O2, and 5.8 % ± 1.1 % at 20 % O2. Apoptosis rate was significantly lower for blastocysts formed at 5 % O2 (p < 0.05). Compared with gene expression levels at 5 % O2, which were arbitrarily set as “1,” 20 % O2 is associated with significantly higher expression of BAX (2.14 ± 0.47), G6PD (2.92 ± 1.06), MnSOD (2.87 ± 0.88), and HSP70.1 (8.68 ± 4.19). For all genes tested, no significant differences were found between 2 and 5 % O2.


The result suggests that development of cryopreserved human embryos from day 3 to blastocyst stage benefits from culture at 5 % O2.


Human embryo Oxygen Stress PCR Apoptosis 



Thanks to Yuliang Liu (BS) and Yanhong Zeng (BS) for the technical support, Baomin Lu (BS) for the preparation of culture medium, Dr Tao Li for the advice on experimental design, and performance, and to Drs Husam Abu-Soud, Maik Huttemann, Mark Kibschull, and Michael Diamond for the analysis and comments on the manuscript as well as Rappolee lab members Drs Quanwen Li and Mohammed Abdulhasan.

Compliance with ethical standards

Written informed consent was obtained from all participants before the usage of their embryos. The institutional review board (IRB) of the First Affiliated Hospital of Sun Yat-Sen University approved the study.

Conflict of interest

The authors declare that they have no conflict of interest.


This research was supported by grants from National Basic Research Program of China (No. 2012CB947604), Science and Information Technology of Guangzhou (No. 201300000097), DAR and EEP from the Office of the Vice President for Research at Wayne State University, and from the funding of the Mary Iacobelli Endowed Chair.


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Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Yu Yang
    • 1
    • 2
    • 3
  • Yanwen Xu
    • 1
    • 4
  • Chenhui Ding
    • 1
    • 4
  • Rabea Youcef khoudja
    • 1
    • 4
  • Mingmei Lin
    • 1
    • 4
    • 5
  • Awoniyi O. Awonuga
    • 2
  • Jing Dai
    • 2
  • Elizabeth E. Puscheck
    • 2
  • Daniel A. Rappolee
    • 2
    • 3
  • Canquan Zhou
    • 1
    • 4
  1. 1.Center for Reproductive MedicineFirst Affiliated Hospital of Sun Yat-sen UniversityGuangzhouChina
  2. 2.Ob/GynWayne State University Medical SchoolDetroitUSA
  3. 3.Reproductive Sciences/PhysiologyWayne State University Medical SchoolDetroitUSA
  4. 4.Key Laboratory of Reproductive Medicine of Guangdong ProvinceGuangzhouChina
  5. 5.Department of Obstetrics and GynecologyPeking University Third HospitalBeijingChina

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