Following semen collection and sperm processing (described in the “Male Factor” section), sperm are incubated in media for 3 to 4 hours to promote sperm capacitation and the acrosome reaction. Before fertilization, retrieved oocytes are cultured in media. Conventional IVF involves insemination concentrations of 100,000 to 800,000 motile sperm/mL per oocyte with each oocyte in a small droplet of media under oil (9,360,361). For every three cycles done for severe male factor, the use of ICSI prevents one case of fertilization failure when compared to conventional IVF (9). The indications for and the procedure and risks of ICSI are discussed under “Male Factor.”
Embryo Development Initial embryo development is typically assessed 15 to 20 hours after insemination or ICSI, when fertilization is characterized by the presence of two pronuclei and the extrusion of the second polar body (9,361,362). Embryos are examined again for cleavage after 24 to 30 hours of culture (9). The first embryo cleavage occurs approximately 21 hours after fertilization, and subsequent divisions occur every 12 to 15 hours up to the eight-cell stage on the 3rd day of embryo development (363). Compaction to form the 16-cell morula occurs on the 4th day of embryo development, and differentiation of the inner cell mass and trophectoderm to form a blastocyst (containing a ﬂuid-filled area called a blastocele) is completed by the 5th or 6th day (364,365).
Culture Environment Sequential media systems are preferred during embryo culture to adjust for each stage of embryo development. Prior to compaction, the embryo is under genetic control of the oocyte, it uses a pyruvate-based metabolism, it requires at least a few amino acids, and it prefers a relatively oxygenated environment (though much lower than atmospheric oxygen) similar to that found in the fallopian tube. Following compaction, amino acid needs increase (stable dipeptide glutamine instead of glutamine will avoid toxic ammonium buildup), the embryonic genome is activated, and metabolism requires both glucose and a very low oxygen environment similar as that found in the uterus (363,366). Supplementation of culture media with hyaluron and albumin is beneficial in postcompaction media preparations (363).
Extended Culture to Blastocyst Although precompaction human embryos can survive when placed in the uterus, the uterine cavity is a nonphysiological location for them, and there is greater uterine pulsatility during this period that may cause the embryos to be expelled. Therefore, the blastocyst stage represents a more physiologic time for embryo transfer. Since nearly 60% of morphologically normal cleavage embryos but only 30% of blastocysts are chromosomally abnormal, extended culture allows for better selection of embryos with improved quality (364,367).
Blastocyst versus Cleavage Transfer Outcomes Comparisons involving equal numbers of transferred embryos demonstrate that blastocyst transfer is associated with lower implantation failure, a higher pregnancy rate, and a 7% higher live birth rate than cleavage stage transfer. This is of particular interest in programs that offer elective single embryo transfer (364,367,368). Given that blastocyst formation rates range from only 28% to 60%, disadvantages of extended culture include the possibility that no embryos will survive to transfer (8.9% vs. 2.8% for cleavage transfer) and a reduced opportunity for embryo cryopreservation. Monozygotic twinning rates may be higher with blastocyst culture, although this has not been a consistent finding (364,369).