Optimization of In Vitro Mammalian Blastocyst Development: Assessment of Culture Conditions, Ovarian Stimulation and Experimental Micro-Manipulation

Factors currently at the forefront of human in vitro fertilization (IVF) that collectively influence treatment success in the form of blastocysts development were investigated during early mammalian embryology with concentration on infertile patients presenting with diminished ovarian reserve or preliminary ovarian failure. A novel experimental technique, Graft Transplant-Embryonic Stem Cells (GT-ESC) was introduced in the mouse model, as the first inclusive approach for embryo selection in IVF treatments resulting in successful graft integration of sibling cells, stage-dependent (day 4) blastocysts. E-Cadherin-catenin bonds play an integral role in trophectoderm cell viability and calcium removal, inducing disruption of cell-to-cell bonds at the blastocyst stage was detrimental to continued blastocyst development. One of the leading methods for embryo selection for uterine transfer in human IVF is application of pre-implantation genetic screening (PGS) methods such as next generation sequencing (NGS). Female patients <35 y do not benefit from this treatment when outcome is measured by presence of fetal heart beats at 10 weeks of gestation. Patients 35-37 y benefit from PGS with no significant difference of outcome based on form of PGS method utilized. Therefore, small nucleotide polymorphism array (snp-array) or targeted-NGS should be selected for this age range to lessen the financial burden of the patient. Embryos from women >40 y have a higher rate of mosaic cell lines which can be detected by NGS. Therefore NGS is most beneficial for women >40 y. Additionally, ovarian stimulation of the patient during human IVF can notably influence outcome. Anti-Müllerian hormone (AMH) is a more conducive indicator of blastocysts development per treatment compared to basal follicle stimulating hormone (FSH). Actionable variables included in a decision tree analysis determined a negative influence (0% success, n=11) of high dose gonadotropin use (>3325 IUs) in good prognosis patients (>12 mature follicles at trigger, AMH >3.15 ng/mL). A positive relationship exists (80% success, n=11) between poor responders (AMH <1.78 ng/mL, <12 mature follicles at trigger) and high dose gonadotropin use (>3025 IUs). Utilizing the decision tree during IVF treatment can be beneficial to treatment success. Moreover, a parallel relationship of the fundamental principles of culture medium pH, pCO2 and pO2 was found with respect to blastocyst development. Human infertility patients' gametes predisposed to primary stressors (i.e., age, genetics and etiology) are negatively impacted (~30% success, n=7) for cleavage stage (day 3) embryo development when primary culture medium has pCO2 <30mmHg given age >31 y and <14 oocytes retrieved. When day 3 embryo development is measured at >65% good quality embryos per treatment (based on SART grading criteria), blastocysts development success is highest when secondary culture medium pO2 is 69-88 mmHg (~90% success, n=12). Thus, IVF treatment outcome can be optimized with utilization of predictive model analyses in the form of decision trees providing greater success for the IVF laboratories, ultimately decreasing the emotional and financial burden to infertility patients.