Chromosomal meiotic segregation, embryonic developmental kinetics and DNA (hydroxy)methylation analysis consolidate the safety of human oocyte vitrification.

Oocyte vitrification has been introduced into clinical settings without extensive pre-clinical safety testing. In this study, we analysed major safety aspects of human oocyte vitrification in a high security closed system: (i) chromosomal meiotic segregation, (ii) embryonic developmental kinetics and (iii) DNA (hydroxy)methylation status. Fresh and vitrified sibling oocytes from young donors after intracytoplasmic sperm injection (ICSI) were compared in three different assays. Firstly, the chromosomal constitution of the fertilized zygotes was deduced from array comparative genomic hybridization results obtained from both polar bodies biopsied at Day 1. Secondly, embryo development up to Day 3 was analysed by time-lapse imaging. Ten specific time points, six morphokinetic time intervals and the average cell number on Day 3 were recorded. Thirdly, global DNA methylation and hydroxymethylation patterns were analysed by immunostaining on Day 3 embryos. The nuclear fluorescence intensity was measured by Volocity imaging software. Comprehensive chromosomal screening of the polar bodies demonstrated that at least half of the zygotes obtained after ICSI of fresh and vitrified oocytes were euploid. Time-lapse analysis showed that there was no significant difference in cleavage timings, the predictive morphokinetic time intervals nor the average cell number between embryos developed from fresh and vitrified oocytes. Finally, global DNA (hydroxy)methylation patterns were not significantly different between Day 3 embryos obtained from fresh and from vitrified oocytes. Our data further consolidate the safety of the oocyte vitrification technique. Nevertheless, additional testing in young and older sub-fertile/infertile patients and sound follow-up studies of children born after oocyte cryopreservation remain mandatory.

Tendon and fascial structure contributions to knee muscle excursions and knee joint displacement.

BACKGROUND: Semitendinosus and gracilis muscles whose tendons are used in surgical reconstruction of the anterior cruciate ligament maintain their contractile ability, and a limited decrease of hamstring muscles force is observed postoperatively despite important changes. The goal was to quantify the influence of the myofascial structures on excursions and moment arms of knee muscles to attempt explaining the above-mentioned post-surgical observations. METHODS: Hamstring harvesting procedures were performed by a senior orthopaedic surgeon on seven lower limbs from fresh-frozen specimens. Femoro-tibial kinematics and tendons excursion were simultaneously recorded at each steps of the surgery. FINDINGS: No significant difference was demonstrated for excursions and moment arms after tenotomies and gracilis tendon harvesting (P≥0.05). The first significant semitendinosus excursion (P<1.17×10(-4)) and moment arm (P<6.88×10(-5)) decrease was observed after semitendinosus tendon harvesting (46% of the initial excursion). INTERPRETATION: Gracilis and semitendinosus myofascial pathway is crucial for force transmission towards the knee joint.

Randomized sibling-oocyte study using recombinant human hyaluronidase versus bovine-derived Sigma hyaluronidase in ICSI patients.

BACKGROUND: The enzyme hyaluronidase from bovine origin is commonly used for oocyte-cumulus cell removal in ICSI. A recombinant human hyaluronidase (rHuPH20) has been introduced as a quality-controlled and safe alternative. METHODS: In order to validate its effectiveness, a non-inferiority trial was started on sibling cumulus-oocyte complexes (135 ICSI patients). Oocyte denudation involved enzyme incubation under Pasteur pipetting, followed by further mechanical stripping. Primary end-points were oocyte intactness after ICSI and fertilization rate. Secondary end-points were embryo development and positive hCG. RESULTS: Oocyte intactness after ICSI was 89.6% and 92.9% with rHuPH20 and bovine hyaluronidase, respectively [absolute difference -3.3% (-7.4 to 10.7)]. The fertilization rate was 73.9% after rHuPH20 and 77.1% after bovine hyaluronidase treatment [absolute difference -3.2% (-8.3 to 1.8)]. Embryo development was similar in both treatment groups up till Day 5. Positive hCGs were equally distributed over mixed transfers 21/45 (46.7%) and transfers of only embryos from rHuPH20 treatment 17/35 (48.6%) or transfers of only embryos from bovine hyaluronidase treatment 22/48 (45.8%). CONCLUSIONS: Our results indicate that rHuPH20 is not inferior to bovine hyaluronidase for oocyte denudation, with regard to oocyte survival and fertilization. rHuPH20 treatment of human oocytes is compatible with good embryo development, with positive hCG results and with live birth.

A comparison between different imaging strategies for diffusion measurements with the centric phase-encoded turboFLASH sequence.

The study compared the results of three centrally reordered phase-encoded turboFLASH sequences for diffusion-weighted imaging (DWI). The sequences were conventional turboFLASH, turboFLASH with subtraction of T1-related effects, and turboFLASH with correction for T1-related effects during the imaging period only. The relative merits were studied with respect to image quality and accuracy by computer simulation and by experimental validation on phantoms and on in vivo rat brain. A T1-related underestimation of the diffusion coefficient ranging from -30% (T1 approximately 200 ms) to -5% (T1 approximately 1 s) was found to exist for the conventional sequence. Image artifacts, caused by longitudinal relaxation during the imaging period, are reflected in calculated diffusion maps. When the correction sequence is used, the artifacts and the systematic errors are reduced but longitudinal relaxation during the delay between preparation and imaging periods remains large enough to induce significant errors (-15% for T1 approximately 200 ms to -3% for T1 approximately 1 s). The subtraction sequence eliminates the influence of T1 effects on the calibrations, but leads to identical artifacts for all diffusion-weighted images.