Blastocyst formation after intracytoplasmic sperm injection (ICSI) and culture in two sequential culture media preparations differing with respect to the method of protein supplementation

Abstract

Objective: Embryo development in vitro may be influenced by the protein in culture media. Our objectives were to learn about 1) early embryo development of sibling oocytes after ICSI and culture in two sequential media preparations differing with respect to the method of protein supplementation 2) pH buffering behavior of these two sequential culture media preparations during the course of embryo culture. Design: Prospective, randomized. Materials and Methods: All media were purchased from Irvine Scientific, Santa Ana, California. After ICSI, metaphase II sibling oocytes from 13 patients were randomly cultured in P1 media with 10% synthetic serum substitute (SSS) added just prior to equilibration (P1, n = 104) and complete P1 media with 10% SSS added at the time of manufacturing (CP1, n = 102). Fertilization and oocyte/zygote degeneration were checked 16–18 h post ICSI (hpi). Embryo cleavage was observed 44–48 hpi. On day 3 of culture, cleaved embryos were transferred from P1 to blastocyst media with 10 % SSS added just prior to equilibration (BM) and from CP1 to complete blastocyst media with 10% SSS added at the time of manufacturing (CBM). Blastocyst formation was determined on day 5 and 6 post ICSI. All cultures were carried out at 37°C in equilibrated 100 μl media droplets under oil, 5% CO2, 5% O2, and balanced N2 at pressure of 2.5 psi. The pH of culture media droplets under similar conditions was determined daily during the course of culture. Data were analyzed using Chi-square test and F test where appropriate. Results: The data is presented as mean (±SD) or otherwise mentioned. Age of oocyte source was 32.7±7.5 years. A total of 301 oocytes (23.2 ± 9.8 oocytes/retrieval) with nuclear maturation rate of 74.8% were recovered. Number of oocytes cultured in P1/BM and CP1/CBM were 8.0 ± 2.8 and 7.8 ± 2.7 differing non-significantly (p > 0.05). Fertilization (74% vs. 78.4%), multiple (≥3) pronucleation (4.8% vs. 4.9%), degeneration (3.8% vs. 5.8%) 16– 18 hpi and cleavage rates (98.7% vs. 95%) 44–48 hpi were not significantly different between P1 and CP1 (p > 0.05), respectively. Premature compaction (6–8 cells stage) was significantly higher in embryos cultured in CP1 (19.7%) than those in P1 (6.6%, p < 0.05). Percent good quality embryos (8 cell grade 1 on day 3) was higher in P1 (76.6%) as compared to CP1 (62.5%) although the difference was not significant (p > 0.05). Blastocyst formation rates from zygote (45.5% vs. 23.8%) as well as from good quality embryos (59.3% vs. 38.0%) were significantly higher in P1/BM than CP1/CBM group (p < 0.05). In 53.8% of patients, blastocysts transferred were selected from both the groups. Percentages of patients with blastocysts transferred exclusively from P1/BM or CP1/CBM were 38.5, and 7.7, respectively. After overnight equilibration, pH of P1, CP1, BM, and CBM were 7.30±0.12, 7.33±0.12, 7.34±0.08, and 7.39±0.11, respectively. By day 3 of culture, pH of P1, CP1, BM, and CBM dropped to 7.27±0.09, 7.20±0.06, 7.29±0.12, and 7.36±0.11, respectively. Conclusions: Under our laboratory conditions, 1) more good quality embryos were generated in P1, and culture in CP1 caused premature embryo compaction. 2) Both sequential culture media preparations supported blastocyst formation. However, P1/BM media were better in terms of producing transferable and cryopreservable blastocysts. 3) CP1 showed poorer buffering capacity than any other media studied. Critical attention given to culture conditions can optimize embryo development in vitro and improve IVF outcome.