P-061 In situ microfluidics of fluidic walls: a novel deviceless and cost-effective approach for sperm selection in the same ICSI-dish
Authors: Ruiz - Jorro M , Bataller-Sánchez J J , Barberá-Alberola A , Gómez-Giménez B , Moliner-Aguilar V , Calatayud-Lliso C , Ferrer-Buitrago M 
Published: June 2023

Abstract

Study question
Is our novel deviceless method based on in-situ microfluidics a valuable strategy to select suitable sperm for ICSI?

Summary answer
The novel protocol allows selecting sperm for ICSI within 15 minutes, in the same ICSI dish and disregarding fungible supplies, centrifugation and washing steps.

What is known already
Microfluidics is an innovative operation in ART which integrates sperm guidance biomimicry during the in vitro sperm selection process. In contrast to other sperm preparation methods such as Wash-Swim-Up and Density- Gradients-Centrifugation, microfluidics disregards washing and centrifugation steps along the procedure. In addition, microfluidics are time-efficient methods which reduce risks associated with handling, gamete mix-up and ROS production. However, microfluidics devices are costly and likely to fail to select motile sperm in dispermic samples. With the aim of outlining the application of microfluidics in ART, we conceived a lab-on-a-chip approach for sperm selection by in-situ handmade microfluidics of fluidic walls.

Study design, size, duration
The microfluidics circuit conforms to Laplace and hydrostatic pressure laws and integrates the sperm guidance mechanisms of rheotaxis and boundary-following behavior. System’s efficacy is based on the volume differences, the distances between microdoplets, and their arrangement. It divides into Section 1 (dispersion and seminal plasma removal) and Section 2 (sperm sorting in response to positive rheotaxis). The system was adjusted to recover at least 20 selected progressive sperm for ICSI in less than 15 minutes.

Participants/materials, setting, methods
We prepared the system on a conventional IVF polystyrene round dish (15 mm diameter) using MOPS buffer, PVP and mineral oil only. We verified the flow-driven dynamics by registering the horizontal displacement (direction) and flow velocity (µm/s) using eosin-Y and methylene blue dyes. We confirmed the removal of the seminal plasma in a PSA assay. The proof-of-concept analysis was performed using fresh semen samples from 150 patients (Caucasian; mean age: 33,53±8,19).

Main results and the role of chance
Our design includes three main points to allow sperm separation: 1 (initial), 2 (rebalancing), 3 (final) with a total distance of 1,5 cm between 1 and 3. Our system assures a continuous microfluidic flow, with two opposed directions (from point 1 to point 2 and from point 3 to point 2). PSA assay showed a significant decrease in PSA levels &gt;200000 ng/ml in the fresh sample to undeterminable levels (&lt;0,0091 ng/ml) at point 3. To assess the utility to select a suitable number of sperm for ICSI we used sperm samples with the following characteristics: volume 3,6 (1,0-7,6) ml, concentration 68 (0,29-150) ×106/ml, progressive motility 24 (2-41) % and morphology 6 (1-15) % normal. The minimum and maximum times for sperm recovery at point 3 were recorded at 5 and 35 minutes, respectively. Normozoospermic samples required lower recovery times compared to dispermic (p &lt; 0,001), although according to the median, samples with at least 1 × 106/ml of motile sperm need less than ten minutes recovery time. Sperm parameters: concentration, motility and morphology were inversely correlated with the recovery time (p &lt; 0,0001), being morphology the parameter with the lesser influence on the outcome.

Limitations, reasons for caution
The present protocol depends on a free-hand preparation. Although the use of templates and the training reduces inter and intra-operator variability, a ready-to-use surface would benefit the operability. Non-inferiority studies are required to compare our method with the currently used ones (Wash-Swim-Up or Density-Gradients-Centrifugation) before clinical implementation.

Wider implications of the findings
Our deviceless methodology streamlines sperm selection for ICSI and reduces risks along the procedure. This novel approach represents a paradigm shift in sperm preparation, since it dispenses with centrifugation, washing and plasticware. The present protocol can be implemented to the clinic in the near-term, particularly due to its simplicity.

Trial registration number
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