Spindle configuration of in vitro matured bovine oocytes vitrified and warmed in media supplemented with a biopolymer produced by an Antarctic bacterium

Biological molecules isolated from organisms that live under subfreezing conditions could be used to protect oocytes from cryoinjuries suffered during cryopreservation. Bacterial exopolysaccharides (EPS) constitute a common class of molecules that interact with ice in nature either by triggering ice nucleation or by inhibition of ice nucleation and growth. The aim of this work was to evaluate the spindle configuration of in vitro matured bovine oocytes vitrified/warmed in media supplemented with exopolysaccharide (M1) produced by Pseudomonas sp ID1 (Carrión et al., Carbohydr Polym 117:1028. 2015). After 22 h of in vitro maturation, a total of 546 oocytes form prepubertal (3 replicates) and 405 oocytes from adult cows (4 replicates) were vitrified/warmed in media supplemented with various concentrations of EPS M1 (0, 0.001, 0.01, 0.1 and 1 mg/ml). After warming, oocytes were allowed to recover for 2 additional hours in IVM medium. Fresh, non-vitrified oocytes were used as a control. At 24 h of IVM, oocytes from all treatments were fixed and immunostained with the Alexa-fluor 488 antibody and DAPI. Microtubule and chromosome distribution was analyzed by immunocytochemistry under a fluorescent microscope. ANOVA was performed to analyze differences in meiotic spindle configuration (P < 0.05). When cow oocytes were vitrified, similar percentages of normal spindle configuration were observed when compared to fresh control oocytes, except for the 0.1 mg/ml EPS M1 group that showed significantly lower rates compared to the fresh control group. Significantly higher rates of prepubertal oocytes exhibiting a normal spindle configuration were recorded in the non-vitrified group compared to all vitrified/warmed groups, regardless of the EPS M1 supplementation. However, the addition of EPS M1 to the vitrification/warming media decreased the ratio of decondensation or absence of chromosomes and microtubules in prepubertal oocytes. Although percentages of normal spindle configuration after vitrification were lower for prepubertal than for cow oocytes, no significant differences were observed when oocytes were vitrified with 0.001, 0.1 and 1 mg/ml EPS M1. In conclusion, supplementation with EPS M1 concentrations during vitrification and warming did not induce adverse changes in the spindle of bovine oocytes, regardless of the concentration used. Although a more severe damage on spindle configuration could be observed after vitrification of prepubertal oocytes, EPS supplementation during vitrification and warming seems to have a greater benefit during vitrification of prepubertal than adult bovine oocytes. Further experiments are required to investigate if in vitro-matured oocytes vitrified/warmed in presence EPS M1 can improve their development competence after being vitrified/warmed. This study was supported by the Spanish Ministry of Science and Innovation (Project AGL2016-79802-P and grant CTQ2014-59632-R)

Impact of Early Intrapatient Variability of Tacrolimus Concentrations on the Risk of Graft-Versus-Host Disease after Allogeneic Stem Cell Transplantation Using High-Dose Post-Transplant Cyclophosphamide

Tacrolimus (Tac) is a pivotal immunosuppressant agent used to prevent graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloHSCT). Tac is characterized by a narrow therapeutic window and a high inter-patient and intra-patient pharmacokinetic variability (IPV). Although high IPV of Tac concentrations has been associated with adverse post-transplant outcomes following solid organ transplantation, the effects of Tac IPV on alloHSCT recipients have not been determined. Tac IPV was therefore retrospectively evaluated in 128 alloHSCT recipients receiving high-dose post-transplant cyclophosphamide (PTCy) and the effects of Tac IPV on the occurrence of acute GVHD (aGVHD) were analyzed. Tac IPV was calculated from pre-dose concentrations (C0) measured during the first month after Tac initiation. The cumulative rates of grades II-IV and grades III-IV aGVHD at day +100 were 22.7% and 7%, respectively. Higher Tac IPV was associated with a greater risk of developing GVHD, with patients having IPV &gt; 50th percentile having significantly higher rates of grades II-IV (34.9% vs. 10.8%; hazard ratio [HR] 3.858, p &lt; 0.001) and grades III-IV (12.7% vs. 1.5%; HR 9.69, p = 0.033) aGVHD than patients having IPV ? 50th percentile. Similarly, patients with IPV &gt; 75th percentile had higher rates of grades II-IV (41.9% vs. 16.5%; HR 3.30, p &lt; 0.001) and grades III-IV (16.1% vs. 4.1%; HR 4.99, p = 0.012) aGVHD than patients with IPV ? 75th percentile. Multivariate analyses showed that high Tac IPV (&gt;50th percentile) was an independent risk factor for grades II-IV (HR 2.99, p = 0.018) and grades III-IV (HR 9.12, p = 0.047) aGVHD. Determination of Tac IPV soon after alloHSCT could be useful in identifying patients at greater risk of aGVHD

Impact of Early Intrapatient Variability of Tacrolimus Concentrations on the Risk of Graft-Versus-Host Disease after Allogeneic Stem Cell Transplantation Using High-Dose Post-Transplant Cyclophosphamide

Tacrolimus (Tac) is a pivotal immunosuppressant agent used to prevent graft-versus-host disease (GVHD) after allogeneic stem cell transplantation (alloHSCT). Tac is characterized by a narrow therapeutic window and a high inter-patient and intra-patient pharmacokinetic variability (IPV). Although high IPV of Tac concentrations has been associated with adverse post-transplant outcomes following solid organ transplantation, the effects of Tac IPV on alloHSCT recipients have not been determined. Tac IPV was therefore retrospectively evaluated in 128 alloHSCT recipients receiving high-dose post-transplant cyclophosphamide (PTCy) and the effects of Tac IPV on the occurrence of acute GVHD (aGVHD) were analyzed. Tac IPV was calculated from pre-dose concentrations (C0) measured during the first month after Tac initiation. The cumulative rates of grades II-IV and grades III-IV aGVHD at day +100 were 22.7% and 7%, respectively. Higher Tac IPV was associated with a greater risk of developing GVHD, with patients having IPV &gt; 50th percentile having significantly higher rates of grades II-IV (34.9% vs. 10.8%; hazard ratio [HR] 3.858, p &lt; 0.001) and grades III-IV (12.7% vs. 1.5%; HR 9.69, p = 0.033) aGVHD than patients having IPV &le; 50th percentile. Similarly, patients with IPV &gt; 75th percentile had higher rates of grades II-IV (41.9% vs. 16.5%; HR 3.30, p &lt; 0.001) and grades III-IV (16.1% vs. 4.1%; HR 4.99, p = 0.012) aGVHD than patients with IPV &le; 75th percentile. Multivariate analyses showed that high Tac IPV (&gt;50th percentile) was an independent risk factor for grades II-IV (HR 2.99, p = 0.018) and grades III-IV (HR 9.12, p = 0.047) aGVHD. Determination of Tac IPV soon after alloHSCT could be useful in identifying patients at greater risk of aGVHD

Cholesterol added prior to vitrification on the cryotolerance of immature and in vitro matured bovine oocytes

This study examines whether incorporating cholesterol-loaded methyl-β-cyclodextrin (CLC) in the bovine oocyte plasma membrane improves oocyte tolerance to vitrification. In vitro matured oocytes were incubated with 2 mg/ml BODIPY-labeled CLC for different time intervals in FCS or PVA supplemented medium or exposed to different CLC concentrations to examine the subcellular localization of cholesterol by confocal microscopy live-cell imaging. Subsequently, the effects of optimized CLC concentrations and incubation times prior to vitrification on early embryo development were assessed. Then, we evaluated the effects of pretreatment with 2 mg/ml CLC for 30 min before the vitrification of immature (GV) and in vitro matured (MII) oocytes on developmental competence and gene expression. Our results indicate a high plasma membrane labeling intensity after 30 min of incubation with 2 mg/ml CLC for 30 min, regardless of the holding medium used. When oocytes were incubated with 1 mg/ml, 2 mg/ml and 3 mg/ml of CLC, intense labeling was observed at the plasma membrane after 40, 30 and 20 min, respectively. CLC pre-treatment before the vitrification of bovine oocytes did not affect subsequent cleavage and embryo development rates irrespective of CLC concentrations, incubation times or meiotic stage. However, pretreatment seems to improve the quality of embryos derived from vitrified oocytes, mainly when oocytes were vitrified at the GV stage