O-124 Contact-free oocyte denudation in a chip-scale ultrasonic microfluidic device
Authors: Mokhtare A , Xie P , Davaji B , Abbaspourrad A , Rosenwaks Z , Palermo G 
Published: August 2021

Abstract

Study question
To design and test an automated microfluidic device to revolutionize the cumulus-oocyte-complex (COC) denudation procedure for intracytoplasmic sperm injection (ICSI) using murine oocytes.

Summary answer
Oocyte exposure to temperature variation, mechanical stress, and prolonged chemical treatment during denudation was mitigated using our microfluidic device based on surface acoustic waves (SAWs).

What is known already
COC denudation is a prerequisite for many ART procedures such as ICSI. However, this procedure is based on manual pipetting (MP), which lacks standardization and requires experienced embryologists to perform. Inadequate MP may damage oocytes through prolonged enzymatic treatment or high fluidic stresses and may jeopardize gamete competence. The use of microfluidic devices based on porous membranes or microchannels has been adopted by many laboratories for sperm selection. Of these, microchannel devices may also be adapted for denudation with minimal mechanical stress in a controlled microenvironment. However, oocyte manipulation and extraction have proven difficult to achieve.

Study design, size, duration
We developed a novel ultrasonic microfluidic device based on a microwell design manufactured with Polydimethylsiloxane (PDMS). The SAWs were generated by 4 interdigitated transducers (IDTs) arranged in an orthogonally symmetric pattern. A non-toxic dosage of ultrasonic waves, similar to those used in gynecology and obstetrics, was applied. COCs were denuded by induced acoustic streaming and acoustic radiation force. Denudation rate, embryo development, and pregnancy outcomes were assessed and compared to control oocytes denuded by MP.

Participants/materials, setting, methods
For each run, up to 10 individual COCs from super-ovulated B6D2F1 mice were loaded into the microwell alongside diluted hyaluronidase (20 IU/ml) and denuded by 80 or 200 MHz SAWs. Denuded oocytes were fertilized by piezo-actuated ICSI using spermatozoa from the same strain. Pre-implantation embryo development was assessed in a time-lapse incubator for up to 96 h. High-quality blastocysts were transferred to 2.5-dpc pseudo-pregnant CD-1 surrogates. Pregnancy and offspring health were observed.

Main results and the role of chance
Using alternating frequency sweep in a pulse-repetition mode, we swirled the fluid inside the microwell consistently and tumbled COCs inside the microwell to expose them to acoustic steaming-induced drag forces and acoustic radiation force. Using a high-speed camera and particle-tracking technique, we observed that the drag force generated by the SAWs fulfilled the denudation mechanism. Additionally, due to the small attenuation coefficient in water, thermal absorption heating remains minuscule, preventing any thermal-induced damage.
Our device significantly reduced the time and labor of the denudation process. It also yielded proper denudation quality without oocyte loss. To ensure that SAWs do not damage oocytes, 40 oocytes denuded by 80 MHz SAWs, 25 oocytes denuded by 200 MHz SAWs, and 30 oocytes denuded by MP were inseminated by piezo-actuated ICSI. The 80-MHz, 200-MHz, and MP groups yielded comparable post-ICSI survival (82.5% vs. 84.0% vs. 83.3%, respectively), fertilization (80.0% vs. 80.0% vs. 83.3%,respectively), and blastulation rates (72.5% vs. 82.0% vs. 66.7%, respectively). Embryo morphokinetics were also not impacted. After transferring all blastocysts into recipient mice, 8 live births were achieved from the 80-MHz group, while 5 were achieved from the 200-MHz group.

Limitations, reasons for caution
Although PDMS is a popular material due to its high optical transparency and biocompatibility, adverse effects due to gas permeability and small-molecule adsorption cannot be excluded. Large-scale mouse embryo assays should be performed to assess the teratogenicity of PDMS. Operation parameters must be optimized for human COCs in clinical application.

Wider implications of the findings
Adopting widely used ultrasound techniques with emerging SAW technology is a major step toward advancing and standardizing oocyte denudation—a laborious yet delicate procedure. We predict it will be further integrated with AI and miniaturized robotics, modules specialized in gamete assessment, ICSI, and embryo evaluation in the near future.

Trial registration number
‘not applicable’