A sound approach for ova denudation.

Abstract

Objective: To introduce an innovative non-contact method for denudation process of cumulus-oocyte complexes (COCs) for intracytoplasmic sperm injection (ICSI).

Design: We designed and fabricated novel acousto-hydrodynamic tweezers (AHT) to perform contactless denudation and tested them in mouse model. Cumulus removal efficiency, preimplantation development and live birth were assessed and compared to conventional manual pipetting denudation.

Subjects: Fourteen female B6D2F1/J mice (∼4 weeks of age), 9 male B6D2F1/J mice (6-12 weeks of age), and 28 CD-1 female mice (∼6 weeks of age) were used for experiment INTERVENTION AND METHODS: We designed a contactless platform for oocyte denudation based on the principle of focalized acoustic waves. We first investigate the acoustic intensity and thermal variability by measuring the surface displacement and temperature with a thermal camera to ensure a safe operation. COCs were denuded by conventional manual pipetting (MP) with 40 IU/ml hyaluronidase serving as control, or by AHTs with reduced amount of hyaluronidase (15 IU/ml). Piezo-ICSI was performed on both experimental and control groups. A triplicate of denudation and insemination experiments were performed. All embryos were monitored in time-lapse incubator. Embryo developmental rates were compared by chi-square test. Embryo morphokinetic timing as a continuous variable was compared by one-way ANOVA. Embryo transfers were performed on some blastocysts.

Main outcome measures: The procedural time for each denudation method was measured and compared. Fertilization, embryo development and morphokinetics, pregnancy and live birth rate were compared between control and experimental cohorts.

Results: Facile non-contact denudation is achieved without any damage to oocyte. Acoustic induced fluidic shear is the main contributor to COC denudation. The average denudation time per oocyte is reduced by 46% (15 seconds per oocyte for control versus 8 seconds per oocyte for AHT) while using a lower concentration of hyaluronidase. Piezo-ICSI on oocytes processed by MP and AHTs resulted in comparable survival (86.1% vs 85.3%, P=0.80), fertilization (96.7% vs 94.1%, P=0.09) and blastocyst rates (88.0% vs 81.3%, P=0.06). Embryo morphokinetics for both experimental and control cohorts were comparable, showing no impact of sound waves on the embryo development. Eventual delivery rates were also comparable between the MP and AHT cohort (51.3% vs 55.4%).

Conclusion: Acousto-hydrodynamic tweezers (AHTs) are used for contactless removal of the cumulus cells from the COCs prior to ICSI in an expedited, safe, and reliable manner. Embryo development outcomes confirm their safety and validate their potential for a comprehensive ICSI-on-chip device.