Microfluidic device and method for separation of high-quality motile sperm based on hydrodynamic and motility-dependent properties

Abstract

The invention relates to a microfluidic device, method, and use for the continuous, label-free isolation of motile, morphologically normal spermatozoa from semen samples This invention offers a gentle, reliable, and efficient platform for sperm selection, serving as an alternative to centrifugation-based techniques in ART laboratories. By maintaining the structural and genetic integrity of sperm, it improves fertilization success, embryo quality, and pregnancy outcomes. The platform also reduces variability between technicians and limits exposure to reactive oxygen species (ROS), ensuring consistent clinical performance. Beyond its application in fertility treatments, the system can be used as a diagnostic tool to assess sperm motility disorders, susceptibility to oxidative stress, and DNA fragmentation dynamics under different environmental conditions or drug exposures.

Description

[0001] Microfluidic Device and Method for Separation of High-Quality Motile Sperm Based on Hydrodynamic and Motility-Dependent Properties

[0002] Field of the Invention

[0003] The present invention relates to the fields of reproductive biotechnology, assisted reproduction, and microfluidic engineering. More specifically, it concerns a microfluidic platform designed to isolate high-quality, motile, and genetically intact spermatozoa from semen samples.

[0004] Background of the Invention

[0005] This system provides a non-invasive, efficient, and physiologically safe alternative to conventional sperm preparation techniques such as swim-up and density gradient centrifugation, both of which may induce oxidative stress, DNA fragmentation, and loss of functional sperm (1).

[0006] Nowadays, fertility is decreasing among both humans and animals; therefore, understanding the underlying causes and developing effective interventions is one of the most pressing issues in modern reproductive science (2, 3).

[0007] Male infertility affects nearly 50% of infertile couples worldwide, and a significant proportion of cases are attributed to poor sperm quality, characterized by low motility, abnormal morphology, or high levels of DNA damage (4, 5). Current clinical sperm preparation methods are insufficient for consistently selecting sperm with optimal fertilizing potential, especially in cases of oligoasthenoteratozoospermia (OAT)(6, 7). There is thus a critical unmet need for a standardized, low-shear, label- free microfluidic system capable of isolating sperm with superior functional and genetic integrity for use in assisted reproductive technologies (ART) such as IVF and ICSI(8).

[0008] Traditional sperm selection methods rely on physical principles such as sedimentation or density gradients (9). While density gradient centrifugation (DGC) improves sperm purity, the process subjects cells to high centrifugal forces (>300 x g) and exposure to reactive oxygen species (ROS), which compromise DNA integrity (10). Similarly, the swim-up technique, though simple, suffers from low yield and variable reproducibility, depending on operator skill and semen viscosity (11).

[0009] Recent developments in microfluidic technology have demonstrated promise for label-free sperm sorting (12). Designs utilizing laminar flow, boundary-following, or chemotaxis-based principles have been reported. However, these systems are hindered by several issues, including low throughput and a tendency to clog because of their narrow channels (<50 pm). They also cannot simultaneously assess sperm based on both motility and morphology, lack physiologically relevant flow dynamics that mimic the female reproductive tract, and are not integrated with downstream collection modules needed for clinical application.

[0010] The present invention overcomes these deficiencies by integrating inertial focusing and motility-dependent sorting within a continuous-flow spiral microchannel. The design enables parallel selection of sperm based on both hydrodynamic focusing (size and shape) and self-propelled motility, achieving high-purity recovery of functional spermatozoa with intact DNA and low ROS levels.

[0011] Summary of the Invention

[0012] The present invention provides a microfluidic device, method and use for isolating motile, morphologically normal spermatozoa from semen samples in a continuous and label-free manner.

[0013] Description

[0014] The invention comprises a microfluidic device, method, and use for the continuous, label-free isolation of motile, morphologically normal spermatozoa from semen samples.

[0015] 1. Device Structure

[0016] The device comprises:

[0017] A spiral microchannel fabricated in polydimethylsiloxane (PDMS) or polymethyl methacrylate (PM MA) with: Channel height: 100 ± 10 m

[0018] Channel width: 200-400 pm

[0019] Length: 30-50 turns with an increasing radius (R = 1-10 mm)

[0020] Inlet ports:

[0021] (i) Sample inlet for semen (center stream)

[0022] (ii) Sheath flow inlet containing sperm washing medium (e.g., HTF or HEPES- buffered medium)

[0023] Outlet ports:

[0024] (i) Central outlet for high-quality, motile sperm (target fraction)

[0025] (ii) Lateral outlet for immotile or morphologically abnormal sperm

[0026] (iii) Waste outlet for debris and leukocytes

[0027] A temperature-controlled stage maintaining 37°C ± 0.5°C during operation.

[0028] The internal curvature of the spiral induces Dean vortices, secondary flows that differentially focus sperm cells according to their hydrodynamic size and shape. Concurrently, the motility-dependent migration allows active sperm to swim toward the central streamline, where shear stress and oxygen levels are optimal.

[0029] 2. Method of Operation

[0030] Sample Preparation

[0031] Liquefied semen is diluted 1 :1 with sperm washing medium (HTF + 0.3% BSA) to achieve a concentration of 5-10 x 106sperm / mL.

[0032] Flow Control

[0033] Sample inlet flow rate (Qs): 1-5 pL / min

[0034] Sheath flow rate (Qsh): 4-20 pL / min Total Reynolds number (Re): 0.1-1.5, ensuring laminar flow.

[0035] Hydrodynamic Focusing

[0036] The combined flow forms a stable laminar regime in which Dean forces (FD) and inertial lift forces (FL) guide sperm to distinct equilibrium positions based on morphology and swimming behavior.

[0037] Motility- Based Selection

[0038] Active spermatozoa overcome local streamlines and migrate toward the inner channel wall, while immotile and abnormal sperm follow the outer vortex path. The system thus passively enriches for progressively motile sperm without mechanical or chemical intervention.

[0039] Collection and Validation

[0040] The central outlet collects the motile sperm fraction in sterile tubes pre-warmed to 37°C.

[0041] Output fractions are analyzed by:

[0042] Computer-assisted sperm analysis (CASA) for motility and velocity

[0043] Morphology assessment via Papanicolaou or Diff-Quik staining

[0044] DNA fragmentation using the sperm chromatin dispersion (SCD) test

[0045] ROS quantification using DCFDA fluorescence.

[0046] 3. Key Technical Advantages

[0047] Physiological Safety: Shear stress within the channel is <0.3 Pa, preventing mechanical damage.

[0048] High Purity: >90% motile and morphologically normal sperm recovered in the target fraction. Low DNA Fragmentation: Reduction of DNA fragmentation index (DFI) by 40- 60% compared to initial semen sample.

[0049] High Yield: Recovery efficiency up to 80% for normozoospermic samples and 60% for OAT samples.

[0050] Automation and Scalability: Compatible with parallelization for processing 1 mL semen within 10-15 minutes.

[0051] Novelty

[0052] Dual-Mode Selection: First system combining inertial microfluidics and selfpropulsion behavior to sort sperm based on both morphology and motility.

[0053] Continuous Flow Operation: Eliminates centrifugation and manual handling.

[0054] Clinically Compatible Format: Designed for integration with ICSI and IVF workflows; the outlet is directly connectable to micromanipulation dishes.

[0055] Shear- Protective Geometry: Channel curvature optimized (Dean number IQ- 20) for gentle focusing.

[0056] Real-Time Monitoring: Optical window allows live imaging and automated Al- based tracking of sperm paths.

[0057] Protection Scope

[0058] Device Composition:

[0059] Spiral microchannel with channel height 100 ± 10 pm and width 200-400 pm, fabricated in PDMS bonded to glass.

[0060] At least three inlets and three outlets configured to generate laminar, inertial focusing flow.

[0061] Flow regime maintaining Re < 2.0 and wall shear stress <0.3 Pa.

[0062] Operational Method:

[0063] (a) Introducing semen into the sample inlet at 1-5 pL / min; (b) Introducing sheath medium at 4-20 pL / min;

[0064] (c) Allowing sperm to undergo inertial focusing and motility-driven migration;

[0065] (d) Collecting the central outlet fraction as the high-quality sperm population.

[0066] Performance Characteristics:

[0067] Recovered fraction with >85% progressive motility, <10% abnormal morphology, and DFI reduction >40%.

[0068] Throughput >105sperm / min with continuous operation.

[0069] Use Claims:

[0070] (a) For clinical sperm preparation prior to ICSI or IVF;

[0071] (b) For preclinical toxicology studies evaluating environmental or pharmaceutical effects on sperm motility;

[0072] (c) For cryopreservation of high-quality sperm fractions with superior post-thaw motility.

[0073] Clinical Impact

[0074] This invention provides a gentle, reproducible, and efficient sperm selection platform that replaces centrifugation-based methods in ART laboratories. By preserving sperm structural and genetic integrity, it enhances fertilization rates, embryo quality, and pregnancy outcomes. It also minimizes technician variability and ROS exposure, enabling consistent clinical results. Beyond fertility treatment, the platform can serve as a diagnostic tool for evaluating sperm motility disorders, oxidative stress susceptibility, and DNA fragmentation dynamics under various environmental or drug exposures. Brief Description of Drawings

[0075] [Fig 1]: Schematic representation of the microfluidic system used for sperm separation.

[0076] References:

[0077] 1. Hernandez-Silva G, Lopez-Torres AS, Maldonado-Rosas I, Mata-Martinez E, Larrea F, Torres-Flores V, et al. Effects of semen processing on sperm function: differences between swim-up and density gradient centrifugation. The world journal of men's health. 2020;39(4):740.

[0078] 2. Salomon Yunga-Ayavaca E, Ximena Quinche-Morocho R, Javier Angulo- Cubillan F. Fertilidad mejorada en ganado lechero con baja condition corporal al usar gonadotrofina corionica equina en insemination artificial a tiempo fijo. Revista Cientifica de la Facultade de Veterinaria. 2023;33(1).

[0079] 3. Skakkebaek NE, Lindahl-Jacobsen R, Levine H, Andersson A-M, Jorgensen N, Main KM, et al. Environmental factors in declining human fertility. Nature Reviews Endocrinology. 2022; 18(3): 139-57.

[0080] 4. Eisenberg ML, Esteves SC, Lamb DJ, Hotaling JM, Giwercman A, Hwang K, et al. Male infertility. Nature Reviews Disease Primers. 2023;9(1):49.

[0081] 5. Gorati A, Mantravadi K, Gedela D, Suresh O. P-013 Is Microfluidic Sperm Sorting an efficient and safe, advanced sperm processing intervention in assisted reproduction cycles? Human Reproduction. 2025;40(Supplement_1):deaf097. 322.

[0082] 6. Cariati F, Orsi MG, Bagnulo F, Del Mondo D, Vigilante L, De Rosa M, et al. Advanced sperm selection techniques for assisted reproduction. Journal of personalized medicine. 2024;14(7):726.

[0083] 7. Zhang X, Chao S, Ye N, Ouyang D. Emerging trends in sperm selection: enhancing success rates in assisted reproduction. Reproductive Biology and Endocrinology. 2024;22(1):67.

[0084] 8. Bouloorchi Tabalvandani M, Saeidpour Z, Habibi Z, Javadizadeh S, Firoozabadi SA, Badieirostami M. Microfluidics as an emerging paradigm for assisted reproductive technology: A sperm separation perspective. Biomedical Microdevices. 2024;26(2):23. 9. Vaughan DA, Sakkas D. Sperm selection methods in the 21st century. Biology of reproduction. 2019; 101 (6): 1076-82.

[0085] 10. Ali AH, Ajina T, Ali MB, Mehdi M. Efficacy of density gradient centrifugation technique (DGC) in enhancing sperm cell DNA quality for assisted reproductive technique. Middle East Fertility Society Journal. 2022;27(1):22.

[0086] 11. Vasilescu SA, Ding L, Parast FY, Nosrati R, Warkiani ME. Sperm quality metrics were improved by a biomimetic microfluidic selection platform compared to swim-up methods. Microsystems & Nanoengineering. 2023;9(1):37.

[0087] 12. Traini G, Ragosta M, Tamburrino L, Vignozzi L, Baldi E, Marchiani S. P-028 Optimizing sperm selection: A comparison of the Microfluidic device and Swim-up technique in samples with compromised semen quality. Human Reproduction. 2025;40(Supplement_1):deaf097. 337.

Claims

Claims1.- A microfluidic device for separation of motile spermatozoa, comprising:• A spiral microchannel of height 100 ± 10 pm and width 200-400 pm• At least one sample inlet, one sheath inlet, and three outlet ports• Configured for laminar flow (Re < 2) producing Dean vortices that separate sperm based on motility and hydrodynamic size.2.- The method of claim 1 , wherein the sperm sample is introduced at 1-5 pL / min and the sheath flow at 4-20 pL / min, generating selective enrichment of motile sperm at the inner outlet.3.- The system of claim 1 , wherein the collected sperm fraction exhibits:Progressive motility >85%;Abnormal morphology <10%;DNA fragmentation index reduced by >40% relative to the input.4.- The use of the device in assisted reproduction techniques (IVF, I CSI) for selecting sperm with optimal fertilizing potential.5.- The use of the device as a diagnostic and research platform for assessing sperm function and screening gonadotoxic agents.

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