Cryopreservation and Preparation of Thawed Spermatozoa from Rhesus Macaques (Macaca mulatta) for In Vitro Fertilization.

Advances in assisted reproductive technologies in rhesus macaques have allowed the development of valuable models of human disease, particularly when combined with recent techniques for gene editing. While the ability to perform in vitro fertilization (IVF) in rhesus macaques is well established, this procedure has not yet been optimized. Specifically, damage to the sperm caused by cryopreservation (cryodamage) may lead to unsuccessful artificial insemination and low fertilization and blastocyst formation rates in vitro. To address this, we systematically assessed 2 cryopreservation methods and 4 recovery methods in the following 3 interdependent experiments: 1) comparing sperm survival after vitrification or slow-freezing; 2) comparing simple wash (SW), density gradient centrifugation (DGC), swim-up (SU), and glass wool filtration (GWF) for removal of cryoprotectants and isolation of motile sperm after thawing; and 3) evaluating the efficacy for IVF of the 2 best methods of isolating thawed sperm. We found that after vitrification, only 1.2 ± 0.3% of thawed sperm were motile, whereas after slow-freezing, 42 ± 5% of thawed sperm were motile. SW was significantly better than all other isolation methods for the recovery of total sperm and for the recovery of sperm with an intact plasma membrane. The isolation methods had no significant differences in the recovery of motile sperm or sperm with progressive motility. However, IVF of ova with sperm recovered by DGC resulted in 5% more embryos and 25% more blastocysts than did IVF with sperm recovered by SW. Although additional studies are required to optimize sperm cryopreservation in rhesus macaques, our study showed that slow-freezing, coupled with DGC, provided the highest efficacy in providing functional sperm for in vitro use.

Hepatitis C disease severity in living versus deceased donor liver transplant recipients: an extended observation study.

UNLABELLED: Donor factors influence hepatitis C virus (HCV) disease severity in liver transplant (LT) recipients. Living donors, because they are typically young and have short cold ischemic times, may be advantageous for HCV-infected patients. Among HCV-infected patients in the Adult-to-Adult Living Donor Liver Transplantation Cohort Study (A2ALL) surviving >90 days and followed for a median 4.7 years, advanced fibrosis (Ishak stage ≥3) and graft loss were determined. The 5-year cumulative risk of advanced fibrosis was 44% and 37% in living donor LT (LDLT) and deceased donor LT (DDLT) patients (P = 0.16), respectively. Aspartate aminotransferase (AST) activity at LT (hazard ratio [HR] = 1.38 for doubling of AST, P = 0.005) and biliary strictures (HR = 2.68, P = 0.0001) were associated with advanced fibrosis, but LDLT was not (HR = 1.11, 95% confidence interval [CI] 0.73-1.69, P = 0.63). The 5-year unadjusted patient and graft survival probabilities were 79% and 78% in LDLT, and 77% and 75% in DDLT (P = 0.43 and 0.32), with 27% and 20% of LDLT and DDLT graft losses due to HCV (P = 0.45). Biliary strictures (HR = 2.25, P = 0.0006), creatinine at LT (HR = 1.74 for doubling of creatinine, P = 0.0004), and AST at LT (HR = 1.36 for doubling of AST, P = 0.004) were associated with graft loss, but LDLT was not (HR = 0.76, 95% CI: 0.49-1.18, P = 0.23). CONCLUSION: Donor type does not affect the probability of advanced fibrosis or patient and graft survival in HCV-infected recipients. Thus, while LDLT offers the advantage of shorter wait times, there is no apparent benefit for HCV disease progression. Biliary strictures have a negative effect on HCV fibrosis severity and graft survival, and a high AST at LT may be an important predictor of fibrosis risk post-LT.

A randomized controlled trial of pretransplant antiviral therapy to prevent recurrence of hepatitis C after liver transplantation.

UNLABELLED: Hepatitis C virus (HCV) infection recurs in liver recipients who are viremic at transplantation. We conducted a randomized, controlled trial to test the efficacy and safety of pretransplant pegylated interferon alpha-2b plus ribavirin (Peg-IFN-α2b/RBV) for prevention of post-transplant HCV recurrence. Enrollees had HCV and were listed for liver transplantation, with either potential living donors or Model for End-Stage Liver Disease upgrade for hepatocellular carcinoma. Patients with HCV genotypes (G) 1/4/6 (n = 44/2/1) were randomized 2:1 to treatment (n = 31) or untreated control (n = 16); HCV G2/3 (n=32) were assigned to treatment. Overall, 59 were treated and 20 were not. Peg-IFN-α2b, starting at 0.75 µg/kg/week, and RBV, starting at 600 mg/day, were escalated as tolerated. Patients assigned to treatment versus control had similar baseline characteristics. Combined virologic response (CVR) included pretransplant sustained virologic response and post-transplant virologic response (pTVR), defined as undetectable HCV RNA 12 weeks after end of treatment or transplant, respectively. In intent-to-treat analyses, 12 (19%) assigned to treatment and 1 (6%) assigned to control achieved CVR (P = 0.29); per-protocol values were 13 (22%) and 0 (0%) (P = 0.03). Among treated G1/4/6 patients, 23 of 30 received transplant, of whom 22% had pTVR; among treated G2/3 patients 21 of 29 received transplant, of whom 29% had pTVR. pTVR was 0%, 18%, and 50% in patients treated for <8, 8-16, and >16 weeks, respectively (P = 0.01). Serious adverse events (SAEs) occurred with similar frequency in treated versus untreated patients (68% versus 55%; P = 0.30), but the number of SAEs per patient was higher in the treated group (2.7 versus 1.3; P = 0.003). CONCLUSION: Pretransplant treatment with Peg-IFN-α2b/RBV prevents post-transplant recurrence of HCV in selected patients. Efficacy is higher with >16 weeks of treatment, but treatment is associated with increased risk of potentially serious complications.