G 1 plus culture medium

Fertilization of oocytes was carried out in vitro with capacitated sperm (1 × 10⁶ cells/mL) and evaluated by the presence of a second polar body. The progression of cell division was monitored daily until it reached the 36-cell stage. Successfully fertilized oocytes were individually maintained in 25 μL of G-1 PLUS culture medium (Vitrolife Sweden AB, Sweden). The embryos were cultured in a ASTEC incubator (EC6S-MD, USA, PA) at 37 °C with 5% O₂/6% CO₂ until being transferred to women on day 5.
Two embryos with type I, II, or III quality on the third day of embryonic development were transferred to the uterine cavity during the implantation window using the Soft Cook technique and Flexible Pass intrauterine transfer cannula guided by an abdominal ultrasound apparatus equipped with a real-time, 5-MHz sector electronic array endovaginal probe (Philips Epiq CVx; MO, USA).
Fourteen days after embryo transfer, ultrasound was employed to examine the possibility of successful implantation, evidenced by the development of the embryonic sac. Based on the results, the patients were assigned to one of two groups: (1) implanted embryos (n = 25, pregnant patients) and (2) non-implanted embryos (n = 25, non-pregnant patients).

Cauda epididymal spermatozoa were assessed for their ability to undergo capacitation-associated tyrosine phosphorylation, a calcium ionophore (A23187) induced acrosome reaction and bind zona pellucidae as previously described^{53 (link),54 (link)}. Alternatively, 2 × 10⁵ capacitated spermatozoa were inseminated into a droplet of oocytes recovered from superovulated female C57BL/6 mice^{55 (link)}. The gametes were co-incubated for 4 h at 37 °C prior to the oocytes being assessed for fertilization (i.e. extrusion of second polar body and/or pronucleus formation). Zygotes were cultured in HTF medium overnight and transferred into G1 PLUS culture medium (Vitrolife, Stockholm, Sweden) on the morning of day 2 followed by an additional media change into G2 PLUS medium (Vitrolife) on Day 4^{55 (link)}. The percentage of fertilized oocytes as well as embryos that had reached blastocyst stage by the morning of day 5 was calculated.