144ARE BLASTOCYST RATES A RELIABLE INDICATOR OF THE QUALITY OF AN IVP SYSTEM?

Abstract

Normally blastocyst rates are used to document the efficiency of an IVP system, because routine transfer of all embryos is not a realistic approach. Even though pregnancies are established, there will only be a weak correlation to a given IVP system because the embryos for transfer have been highly selected. The aim of this study was to analyze the in vitro development of bovine IVM/IVF oocytes after culture in SOFaa medium with or without the presence of bovine oviduct cells (BOEC) under 5% or 20% O2 in 5% CO2 and 38.5°C in order to select the optimal IVC system under the given circumstances. The study was based on six replicates and 2373 inseminated oocytes retrieved from abattoir ovaries, and the quality markers were Day 8 blastocyst rates (BL per inseminated oocytes), morphology, kinetics, and cell count. From the relative proportion of BL, XB, and H, an average developmental stage (kinetics) could be assigned. Ranking was based on BL rate, rates of A and B graded BL, and the average developmental stage. Established standard procedures were used for IVM (23h in DMEM with 5% serum and eCG/hCG), and IVF (23h in TALP with heparin), and the inseminated oocytes were randomly allocated into four IVC groups (5% O2, 5% O2/BOEC; 20% O2, and 20% O2/BOEC) to be cultured in groups of 25 in 0.1mL oil-covered droplets of SOFaa with 5% serum (Holm P et al. 1999 Theriogenology 52, 683–700). The morphology was graded as A: compact and distinct inner cell mass, regular morphology of trophoblast cells, development corresponding to the expected; B: smaller or less distinct inner cell mass, a few degenerated trophoblast cells or slight fragmentation, development corresponding to the expected; C: dispersed or no inner cell mass, degenerated trophoblast cells or much fragmentation, developmental arrest. For cell counts the zona and cytoplasm from the individual blastocysts were lysed in 0.01M HCL and 0.1% Tween 20, leaving the isolated nuclei to be fixed in 3:1 methanol:acetic acid on a slide (Viuff D et al. 2002 Biol. Reprod. 63, 1143–1148). The kinetics were assessed as hatched per total BL at Day 8 (Fisher’s exact test, P<0.01). The BL rates were significantly lower in the 20% O2 group (23% v. 31%, 32%, 33% in the other groups, respectively), while the hatching rate was significantly higher in the 5% O2 group (35% v. 12%, 10%, 18%). The frequency of A-quality blastocysts was significantly higher in the 5% O2 and 20% O2/BOEC groups (46%, 41%) than in the 20% O2 and 5% O2/BOEC groups (27%, 22%). The B-quality frequency did not differ between the four groups (41%, 40% v. 48%, 45%), whereas the C-quality inversely reflected the A-quality (13%, 19% v. 25%, 33%). There were no differences in the cell counts between the same quality grades in the four systems. An A-grade expanded BL had 134±50 cells (mean±SD), a B-grade 94±45; a hatched A-grade BL had 168±48 cells, a B-grade 143±54. This study shows that regardless of differences in average developmental stages (kinetics) and morphology, similar blastocyst rates can be obtained. Using these criteria our four IVC groups would be ranked as (1) 5% O2, (2) 20% O2/BOEC, (3) 5% O2/BOEC (4) 20% O2. In conclusion, when evaluating the suitability of an IVP system, morphology and kinetics should be considered as well as blastocyst rates.