Preliminary clinical experience with human blastocyst development in vitro without co-culture

Abstract

This preliminary analysis was designed to quantify blastocyst development of supernumerary embryos without the use of feeder cells, conditioned medium or whole serum. Embryos derived from in-vitro fertilization (IVF) that were not transferred or cryopreserved were included in this study. Ova were harvested for IVF after a standard ovarian stimulation with gonadotrophin-releasing hormone agonist/ human menopausal gonadotrophin (GnRHa/HMG) or follicle-stimulating hormone (FSH). Ova were collected and culture in 150 microliters droplets of P1 medium under mineral oil, in groups at 37 degrees C under 5% CO2, 5% O2, 90% N2 (group A) or under 5% CO2 in air (group B) environment. Embryo transfer was performed 72 h post-harvest. Viable embryos not transferred or cryopreserved were placed in blastocyst medium and cultured for an additional 48 h in 5% CO2 in air. Embryos that exhibited an expanded blastocoelic cavity and well-defined inner cell mass at 120 h were counted. Of 838 supernumerary embryos cultured, 448 (53.5%) reached the expanded blastocyst stage by 120 h of culture. Patients were given the option of cryopreservation at that time. The embryos were cryopreserved using a standard protocol with serial addition of glycerol. Embryos reaching the blastocyst stage after more than 120 h of culture were not included. There was no difference in the proportions of blastocyst development between group A, 217/410 (53.5%) and group B, 231/428 (54%). To date, 16 patients have each had up to three thawed blastocysts transferred, out of whom seven became pregnant. This report demonstrates that a simple system of sequential culture generated acceptable, viable blastocyst development (54%) with supernumerary embryos, without the use of feeder cells, conditioned medium or whole serum. Recognizing the differential metabolic requirements of early and late cleavage stage embryos has enabled the application of a glucose/phosphate-free simple culture medium (P1) for up to 72 h of culture and a complex, glucose-containing medium (blastocyst medium) for subsequent blastocyst development.

The impact of pronuclear orientation to select chromosomally normal embryos

“…Most of embryos from IVF patients (40-60%) are genetically abnormal (15), and as many as 40% of blastocysts exhibit some degree of chromosomal mosaicism in inner cell mass (16). The high rate of genetic dysfunction correlates with previously reported blastocyst conversion rates of 50% (17), as well as with our laboratory findings, that almost half embryos on day 3 seems will not survive until day 5. Since absolute correlations are seldom seen, embryo selection remains one of the most difficult endeavors in assisted reproduction.…”

“…With improved culture media systems, it is possible to extend culture until blastocyst and some groups have believed in down-selection through that (17,(42)(43)(44). This process enables to choose embryos with higher viability, although day 3 morphology seems not to be predictive for further blastocyst development (45).…”

Receiver operating characteristic (ROC) analysis of day 5 morphology grading and metabolomic Viability Score on predicting implantation outcome

“…The current study is the first report on utilization of NIR spectroscopy-based metabolomics for model development and blinded analysis to determine viability of day 5 embryos. Extended culture to blastocyst stage, made possible by the advent of more physiological culture media and improvements in the IVF laboratory, resulted in high implantation and pregnancy rates [33][34][35][36]. A meta-analysis by Blake et al [37] including 18 randomized controlled trials, reported that blastocyst stage embryo transfer is associated with increased pregnancy rates compared to embryo transfer at cleavage stage.…”

The effect on human sex ratio at birth by assisted reproductive technology (ART) procedures – an assessment of babies born following single embryo transfers, Australia and New Zealand, 2002–2006

“…The reduction in SSR after ICSI has often been attributed to male infertility, because ICSI is predominantly used to treat male infertility. 12 Both advances in the development of embryo culture media and evidence of higher pregnancy rates by blastocyst transfer (BT) [13][14][15][16] have provided fertility clinics with the opportunity to offer BT to patients for treatment. In 1994, Pergament et al 17 observed that the transfer of fast growth embryos resulted in more male births than female births.…”