Effects of group culture on the development of discarded human embryos and the construction of human embryonic stem cell lines

Oocytes with two pronucleus were individually cultured in 20 μl droplets of G1 medium (Vitrolife, Sweden) at 37 °C, 6 % CO2 for 48 h. According to the standard of Peter [17], embryos were examined for cell stage, fragmentation and multinucleation on day 3. We collected the one pronuclear (1PN), tri-pronucleus (3PN) and the cleavage-arrested embryos with two pronucleus (2PN). The group culture principles are: A, embryos in the same droplet were from the same patients; B, embryos in the same droplet were from the same pronuclear. All embryos were grouped by the number (single, two, three and four) and the pronuclear, then cultured in 30 μl droplets of G2 medium (Vitrolife, Sweden) at 37 °C, 6 % CO2.

On the Day 5, blastocyst formation was determined. According to scoring system of Gardner and Schoolcraft [18], blastocysts were scored from 1 to 6: 1, early blastocyst, the blastocoel being less than half the volume of the embryo; 2, blastocyst, the blastocoel being half or greater than half of the volume of the embryo; 3, full blastocyst, the blastocoel completely fill the embryos; 4, expanded blastocyst, the blastocoel volume is larger than that of the early embryo and the zona is thinner than before; 5, hatching blastocyst, the trophectoderm has started to herniate through the zona; 6, hatched Blastocyst, the blastocyst has completely escaped from the zona. The development of the inner cell mass (ICM) was graded as follows: A, tightly packed, many cells; B, loosely grouped, several cells; C, very few cells. The trophectoderm was assessed as follows: A, many cells forming a cohesive epithelium; B, few cells forming a loose epithelium; C, very few large cells. Those that did not reached the blastocyst stage on Day 5 were cultured one more day till Day 6. In this study, the blastocysts with grade 3BB or over were regarded as high-quality blastocysts [19].

The dishes were covered with Mitomycin C-inactivated mouse embryonic fibroblasts [20] in a density of 3.5*10⁵/cm² for future use. Trophectoderm cells and ICM were isolated with a syringe needle under a dissecting microscope. Trophectoderm cells around the ICM were removed as carefully as possible, avoiding damage to the ICM. The ICM was seeded in the feeder layer. After 48 h, the adherence and growth was observed and half of culture medium was changed. Cell passage was performed during 3–7 days in primary clone. Then each cell passage was performed every 4–7 days. These clones were incubated in H-DMEM (Gibco, USA) containing 20%serum replacement (Gibco,USA), penicillin/streptomycin(Gibco, USA),basic fibroblast growth factor (bFGF;Invitrogen,USA),insulin transferrin selenium(ITS;Gibco,USA),non-essential amino acids(Hyclone,USA),β-mercapto- eth anol(Sigma,USA),L-glutamine(Sigma,USA)at 37 °C,5 % CO2. Passage was perfor -med with the mechanical method. The pyknotic and solid clones were selected, and then the needle of 1 ml-syringed was used to mechanically cut these clones. The passage ratio was 1:3–1:5 [21].

Statistical analysis was performed with SPSS statistical package (SPSS version 13.0, Chicago, IL). Analysis of covariance (ANCOVA) was performed to compare the means between the patients in different groups. Data are expressed as percentage. Chi-square test was used in comparison between blastulation percentages in different group culture groups. All data are reported as the mean with their associated standard deviations and all tests were two-tailed. Test standard was set at a = 0.05, and P < 0.05 was considered significant.

In IVF/ICSI-ET cycles, there frequently are some abnormal zygotes, which developed into the embryos with 0PN, 1PN, or multi pronucleus (3PN, mainly 3PN) and the cleavage-arrested embryos with 2PN. Due to the sample size; we didn’t discuss the embryos with 0PN at this time. To our knowledge, many factors affect the oocyte quality and embryo quality, such as age and BMI [28, 29]. In this study, we excluded the influencing factors from the patients. Feng and coworkers [30] found that more than 50 % of zygotes with 1PN contained Y chromosome, and developed into the embryos with 1PN after cell cleavage in IVF cycles. Then, the cytogenetics indicated these embryos usually possessed diploid karyotype suggesting they had normal fertilization. The data from our study suggests that the embryos with 1PN have better developmental potential to form blastocysts and high-quality blastocysts. Therefore, the embryos with 1PN have the higher rates to have the normal chromosomes and can be used for establishment of hESCs.

At present, some researchers try to use the decidual cells, fibroblasts, granular cells and embryos were co-cultured in order to improve embryo blastocyst formation rate, but heterologous material that they bring can not meet the clinical grade human embryonic stem cell lines established requirements. Recently group culture of embryos has been beneficially applied in mice [31, 32], sheep [33], cow [34, 35, 36] and human [37, 38]. It has been reported that group culture may promote embryo development vie secretion of embryotrophic factors [39, 40], and embryos cultured in group developed better than those cultured individually [41, 42, 43]. Also, group culture of good-quality embryos and poor-quality embryos can improve blastocyst formation rate [44]. The study in the Salahuddin S ‘group suggests that specific factors are secreted from the embryos to influence embryo growth in an autocrine or paracrine manner. However, some researchers argue that embryos in group may deplete the media of substrates, and some other factors derived from poor quality embryos may have negative influences on the surrounding embryo development. It has been reported that the accumulation of toxic substances such as ammonia and oxygen-derived free radicals in the culture medium [45, 46, 47] may lead to the late developmental anomalies. Whether the modification of the group culture environment is beneficial or detrimental is not clear. The hypothesis generated in this study is that in addition to the positive effects, human embryo development in group culture may also be harmfully affected by negative factors that derived from poor quality of embryos.

At present, most hESCs come from the remnant embryos after assisted reproductive treatment cycles [48, 49]. In the ARTs, some couples have successfully got children; it is not necessary to store their frozen embryos. Therefore, many scholars believe that under the couples’ consent, the remaining frozen embryos may be used for scientific research [50]. At present, the use of clinical discarded fresh or frozen embryos can solve the problems including the lack of embryos and ethic disputes in the establishment of human embryonic stem cell bank. In our Reproductive Center, about 4,000 IVF cycles are performed every year; it provides an abundant embryo resource for the establishment of human embryonic stem cell bank. In this study, a total of 4hESCs have been established, and are all consistent with the current identification criteria of hESCs. When we use discarded fresh embryos to establish hESCs lines, the embryos group culture maybe increase the efficiency of hESCs establishment. This study has important significance for establishing hESCs lines with discarded embryos.