Programmed Cell Death (Apoptosis) Differs in IVF Versus ICSI Blastocysts from Non-Human Primates

Abstract

Objective: To compare the cell numbers, allocation to the trophectoderm (TE) and inner cell mass (ICM) and proportions of cells undergoing programmed cell death between IVF (in vitro) and ICSI (intracytoplasmic sperm injection) fertilized blastocysts.Design: Mature oocytes from hyperstimulated rhesus monkeys were allocated for fertilization via IVF or ICSI.Materials and Methods: A terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling (TUNEL) assay kit was used to assay apoptosis. Blastocysts were fixed (2% formaldehyde; pH 7.4; 30 min), rinsed, and permeabilized with 0.1% Triton X-100—0.1% Na Citrate (4°C; 2 min). Hoechst 33342 was used to visualize total DNA. Images were obtained by confocal microscopy and 3-dimensional reconstructions of the blastocysts were generated. Reconstruction permitted determination of the total cell number by counting nuclei serially. Cell nuclei were allocated either to TE or ICM depending on their peripheral or interior location in 3-D images, respectively. Data analyzed by Statview Software.Results: ICSI blastocysts have an increased number of both ICM and TE cells over IVF blastocysts (31 ± 4 ICM and 200 ± 21 TE versus 15 ± 0.5 ICM and 120 ± 5.1 TE, respectively). However, the ratio of ICM to TE cells in IVF versus ICSI is not significantly different, IVF = 13% ± 1.3, ICSI = 15% ± 1.9. The apoptotic ICM dead cell index for IVF blastocysts is higher than for ICSI generated blastocysts (13.2 ± 3.3 versus 4.8% ± 2.5, p=.07). In both IVF and ICSI, apoptosis appears to be correlated with cell number. The lower the cell number of the blastocyst, the higher the total dead cell index.Conclusions: Even though morphologically indistinguishable, the viability of blastocysts derived from different fertilization protocols seems to be different. Higher proportions of ICM cells undergoing apoptosis in IVF blastocyst is surprising. The TUNEL assay can be used to reveal differences between embryos and the viability of ICM may depend on the type of manipulation required to generate embryos. Objective: To compare the cell numbers, allocation to the trophectoderm (TE) and inner cell mass (ICM) and proportions of cells undergoing programmed cell death between IVF (in vitro) and ICSI (intracytoplasmic sperm injection) fertilized blastocysts. Design: Mature oocytes from hyperstimulated rhesus monkeys were allocated for fertilization via IVF or ICSI. Materials and Methods: A terminal deoxynucleotidyl transferase-mediated dUPT nick-end labeling (TUNEL) assay kit was used to assay apoptosis. Blastocysts were fixed (2% formaldehyde; pH 7.4; 30 min), rinsed, and permeabilized with 0.1% Triton X-100—0.1% Na Citrate (4°C; 2 min). Hoechst 33342 was used to visualize total DNA. Images were obtained by confocal microscopy and 3-dimensional reconstructions of the blastocysts were generated. Reconstruction permitted determination of the total cell number by counting nuclei serially. Cell nuclei were allocated either to TE or ICM depending on their peripheral or interior location in 3-D images, respectively. Data analyzed by Statview Software. Results: ICSI blastocysts have an increased number of both ICM and TE cells over IVF blastocysts (31 ± 4 ICM and 200 ± 21 TE versus 15 ± 0.5 ICM and 120 ± 5.1 TE, respectively). However, the ratio of ICM to TE cells in IVF versus ICSI is not significantly different, IVF = 13% ± 1.3, ICSI = 15% ± 1.9. The apoptotic ICM dead cell index for IVF blastocysts is higher than for ICSI generated blastocysts (13.2 ± 3.3 versus 4.8% ± 2.5, p=.07). In both IVF and ICSI, apoptosis appears to be correlated with cell number. The lower the cell number of the blastocyst, the higher the total dead cell index. Conclusions: Even though morphologically indistinguishable, the viability of blastocysts derived from different fertilization protocols seems to be different. Higher proportions of ICM cells undergoing apoptosis in IVF blastocyst is surprising. The TUNEL assay can be used to reveal differences between embryos and the viability of ICM may depend on the type of manipulation required to generate embryos.