Embryo apoptosis identification: Oocyte grade or cleavage stage?

Noraina Mohd Bakri; Siti Fatimah Ibrahim; Nurul Atikah Osman; Nurhaslina Hasan; Farah Hanan Fathihah Jaffar; Zulaiha Abdul Rahman; Khairul Osman

doi:10.1016/j.sjbs.2015.10.023

Embryo apoptosis identification: Oocyte grade or cleavage stage?

Saudi J Biol Sci. 2016 Jan;23(1):S50-5. doi: 10.1016/j.sjbs.2015.10.023. Epub 2015 Nov 10.

Authors

Noraina Mohd Bakri¹, Siti Fatimah Ibrahim¹, Nurul Atikah Osman¹, Nurhaslina Hasan², Farah Hanan Fathihah Jaffar¹, Zulaiha Abdul Rahman³, Khairul Osman⁴

Affiliations

¹ Physiology Department, Preclinical Building, Faculty of Medicine, Canselor Tuanku Muhriz Hospital, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaacob Latif, Bandar Tun Razak, 56000 Cheras, Wilayah Persekutuan, Kuala Lumpur, Malaysia.
² Faculty of Dentistry, Universiti Teknologi MARA, 40450 Shah Alam, Selangor, Malaysia.
³ Faculty of Dentistry, Universiti Sains Islam Malaysia, Level 15, Tower B, Persiaran MPAJ, Jalan Pandan Utama, 55100 Kuala Lumpur, Malaysia.
⁴ Department of Forensic Sciences, School of Diagnostic and Applied Health Sciences, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia.

Abstract

Apoptosis is a programed cell death that is vital for tissue homeostasis. However, embryo apoptosis had been known to be related to embryo fragmentation which should be avoided in in vitro fertilization (IVF). The purpose of this study was to evaluate the relationship of embryo apoptosis with the grade of immature oocytes and cleavage stage of in vitro produced (IVP) cattle embryos. This study consisted of 345 oocytes collected through ovary slicing. Immature oocytes were graded as A, B and C. This grading was based on cumulus cell thickness and compactness. All oocytes then underwent an in vitro maturation (IVM) procedure. An IVF was done 24 h after IVM culture. Prior to staining, stage of cleaved embryos was determined and classified as either 2, 4, 8 or >8-cell embryo stage. Apoptosis status of cleaved IVP embryos was determined by using annexin V-FITC staining technique at 48 and 72 h post insemination (hpi). Apoptosis status for each embryo was classified as either early or late. The result showed that there was no significant difference (p > 0.05) of apoptosis status among grade A, B and C embryos. All grades of oocytes showed embryo apoptosis where 1.5% late apoptosis for grade A, 4.5% and 10.4% of early and late apoptosis for grade B and grade C. Early apoptosis was not seen in grade A embryo. We also noted no significant difference (p > 0.05) of apoptosis status between 2, 4, 8 and >8-cell embryo stage. Early apoptosis was also not seen in >8-cell stage. Even though there were no differences in apoptosis expression between the three classes, the cleavage rate of grade A oocytes was significantly higher (p < 0.01) than grade B and grade C. In conclusion, the apoptosis expression in the embryo can occur regardless of the oocyte quality and the cleavage stage of the embryo produced.

Keywords: ART, assisted reproductive technologies; Apoptosis; BO, Brackett and Oliphant; BSA, bovine serum albumin; CC, cumulus cells; CO2, carbon dioxide; COC, cumulus–oocyte complex; CR1aa, Charles Rosenkran’s 1 amino acid; CaI, calcium ionophore; Cleavage stage; DNA, deoxyribonucleic acid; DO, denuded oocyte; EA, early apoptosis; FBS, fetal bovine serum; FITC, fluorescein isothiocyanate; FSH, follicle stimulating hormone; GSH, glutathione; IVC, in vitro culture; IVF, in vitro fertilization; IVM, in vitro maturation; IVP, in vitro produced; LA, late apoptosis; LH, luteinizing hormone; Oocyte grading; PBS, phosphate buffered saline; PI, propidium iodide; PS, phosphatidylserine; TUNEL, terminal deoxynucleotidyl transfer-mediated dUTP nick end-labeling.; hpi, hours post insemination.