The aims of the present study were to confirm that regulation of the PA and environment via TGF-β regulation of sperm by Percoll-separated in porcine uterine epithelial cells. And, it was performed to identify the cytokines (TGF-β1, 2 and 3, TGF-β receptor1 and 2; interleukin, IL-6, IL-8) and PA-related genes (urokinase-PA, uPA; tissue- PA, tPA; PA inhibitor, PAI; uPA-receptor, uPAR) by spermatozoa. The experiment used porcine uterus epithelial cells (pUECs) and uterine tissue epithelial cells, Boar sperm were separated by discontinuous Percoll density gradient (45/90%), and tissues were co-incubated with spermatozoa, followed by real-time PCR. PA activity was measured of sperm by discontinuous Percoll density gradient (45/90%) for 24 hours. To measure viability and acrosome damage of sperm double stained propidium iodide (PI) and SYBR- 14 or FITC-PNA were used. In results, binding ratio of Percoll-separated sperm was found no differences, but sperms isolated from 90% Percoll layer reduced PA activity (p < 0.05). when co-cultured sperm selected Percoll in porcine uterus tissues epithelial cells, 90% layer sperm increased TGF-β R1, contrastively tPA and PAI-1 in comparison with control (p < 0.05). 45% sperm was decreased the expression of uPA (p < 0.05). TGF-β decreased PA activity in the supernatant collected from pUECs (p < 0.05). Especially, The group including uPA, PAI-1 were induce sperm intact, while it was reduced in sperm damage when compared to control (p < 0.05). Also, there was no significant difference group of tPA and tPA+I in the dead sperm and acrosome damage compared to control. The expression of tPA and PAI showed a common response. Percoll-separated spermatozoa in 90% layer reduced tPA and IL-related gene mRNA expression. Thus, Percoll-sparated sperm in 90% layer show that it can suppress inflammation through increased expression of TGF-β and downregulation of PA and IL in epithelial cells compared to 45% layer Percoll.

Most of the commercial devices for vitrification are directly immersed into the warming solution (WS) for increasing of warming rate. However, the previous modified cut standard straw (MCS) which has reported is difficult to immerse into the WS. The aim of this study was to investigate whether the long cut straw (LCS) could be useful as a stable tool for vitrified-warmed human blastocysts. A total of 138 vitrified-warmed cycles were performed between November 2013 and November 2014 (exclusion criteria: women ≥38 years old, poor responder, surgical retrieval sperm, and severe male factor). The artificial shrinkage was conducted using 29-gauge needles. Ethylene glycol and dimethyl sulfoxide (7.5% and 15% (v/v)) were used as cryoprotectants. Freezing and warming were conducted using the LCS tool. The cap of LCS was removed using the forceps in the liquid nitrogen (LN2) and then directly immersed into the first WS for 1 min at 37℃ (1 M sucrose). Only re-expanded blastocysts were transferred after it was cultured in sequential media for 18-20 h. A total of 294 blastocysts were warmed, and all were recovered (100%). Two hundred eighty-five embryos were survived (96.9%). The vitrifiedwarmed blastocysts of all patients were transferred without any cancellation. We were able to achieve a reasonable implantation (24.2%), following by clinical pregnancy (36.2%), which then continued to ongoing pregnancy (36.2%), and live birth (31.2%). Using LCS is achieved the acceptable rates of survival, pregnancy and live birth. Therefore, the LCS could be considered as a stable and simple tool for human embryo vitrificaton.