Swim-up as a strategy for isolation of spermatozoa without viral incorporation in men with chronic hepatitis B: A pilot study.

Hepatitis B virus (HBV) incorporates into spermatozoa which raises safety concerns about paternofetal transmission performing intracytoplasmatic sperm injection (ICSI) in men with chronic hepatitis B (cHB). HBV reduces sperm cell motility, assuming spermatozoa with highest motility are least HBV-incorporated. This study investigates an ICSI preparation technique (swim-up) to isolate most motile spermatozoa in order to select HBV-free spermatozoa. Semen and blood samples were collected from four patients with cHB. Spermatozoa were incubated in trajectories of gamete medium to create non-motile, motile/non-progressive and motile/progressive fractions. After DNA-extraction, HBV DNA loads were determined in every fraction. Participants (mean age 31) were HBsAg+(4/4), anti-HBc+(4/4) and HBV DNA+(2/4). They were treated (3/4) with entecavir(1/4) or tenofovir (2/4) and had no adverse sperm parameters(3/4). CRP-gene was detected in 95/96 sample fractions, proving successful DNA-extraction. HBV DNA was detected in none of the sample fractions, except for the motile, non-progressive fraction of one patient (HBeAg+, HBV DNA+). Since no HBV DNA was detected in progressive fractions, this study suggests swim-up a successful strategy to select HBV-free spermatozoa. Since all but one fraction was HBV DNA-negative, this study also suggests that patients with well-controlled disease have no HBV-contaminated sample fractions. This study encourages evaluation of guidelines restricting reproductive possibilities in men with cHB.

Stickler syndrome caused by COL2A1 mutations: genotype-phenotype correlation in a series of 100 patients.

Stickler syndrome is an autosomal dominant connective tissue disorder caused by mutations in different collagen genes. The aim of our study was to define more precisely the phenotype and genotype of Stickler syndrome type 1 by investigating a large series of patients with a heterozygous mutation in COL2A1. In 188 probands with the clinical diagnosis of Stickler syndrome, the COL2A1 gene was analyzed by either a mutation scanning technique or bidirectional fluorescent DNA sequencing. The effect of splice site alterations was investigated by analyzing mRNA. Multiplex ligation-dependent amplification analysis was used for the detection of intragenic deletions. We identified 77 different COL2A1 mutations in 100 affected individuals. Analysis of the splice site mutations showed unusual RNA isoforms, most of which contained a premature stop codon. Vitreous anomalies and retinal detachments were found more frequently in patients with a COL2A1 mutation compared with the mutation-negative group (P<0.01). Overall, 20 of 23 sporadic patients with a COL2A1 mutation had either a cleft palate or retinal detachment with vitreous anomalies. The presence of vitreous anomalies, retinal tears or detachments, cleft palate and a positive family history were shown to be good indicators for a COL2A1 defect. In conclusion, we confirm that Stickler syndrome type 1 is predominantly caused by loss-of-function mutations in the COL2A1 gene as >90% of the mutations were predicted to result in nonsense-mediated decay. On the basis of binary regression analysis, we developed a scoring system that may be useful when evaluating patients with Stickler syndrome.