SPERM SEPARATION DEVICES AND USES THEROF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63/422,803, filed on November 4, 2022, entitled “Sperm Separation Devices and Uses Thereof,” the contents of which is incorporated by reference herein in its entirety.

TECHNICAL FIELD

[0002] The subject matter disclosed herein is generally directed to devices for sperm separation and uses thereof

BACKGROUND

[0003] Rheotaxis in mammalian sperm has been proposed as one the long-range guiding mechanism for the journey of sperm toward the egg. Just recently, it was proved that higher rheotaxis ability is associated with higher fertilization rate and lower DNA integrity. Yet many of the microfluidic devices for sperm separation based on rheotaxis are not suitable for performing in vitro fertilization (IVF) since the retrieval efficiency (RE) of the output sample is usually less than two percent. As such there exists a need for compositions, techniques, and/or devices for rehotaxis based separation of sperm.

[0004] Citation or identification of any document in this application is not an admission that such a document is available as prior art to the present invention.

SUMMARY

[0005] In some aspects, the techniques described herein relate to a microfluidic device configured for rheotaxis-based sperm separation including: one or more channels, each channel including: an inlet at a first end of the channel, an outlet at a second end of the channel, a plurality of rows, each row including a plurality of structures forming a plurality of strictures, wherein each row extends across a width of the channel, wherein no structure contacts another structure so as to form a gap between each structure to generate the plurality of strictures, a plurality of straight guide veins extending across the width of the channel substantially perpendicular to a longest axis of the channel, wherein each straight guide veins is positioned equidistance between two different rows; a plurality of curved guide veins extending across the width of the channel, wherein a curved guide vein is placed on each side of each row with the curve extending away from the row, and a set of supports including a plurality of supports placed at a distance from each other, and wherein the microfluidic device is configured such that a fluid introduced at the inlet generates a fluid flow through the channel from the inlet to the outlet

[0006] In some embodiments, the techniques described herein relate to a microfluidic device further comprising a set of supports comprising a plurality of supports placed at a distance from each other, wherein a first set of supports is positioned between side walls of the channel and between the inlet and a curved vein on an inlet side of a first row and the inlet.

[0007] In some embodiments, the techniques described herein relate to a microfluidic device, the plurality of strictures generates regions of variable shear in the fluid flow.

[0008] In some embodiments, the techniques described herein relate to a microfluidic device, wherein one or more or all of the plurality of rows extend across the width of the channel substantially perpendicular to the longest axis of the channel.

[0009] In some embodiments, the techniques described herein relate to a microfluidic device, wherein the plurality of structures are prisms. In some embodiments, the techniques described herein relate to a microfluidic device, wherein the plurality structures are pyramids. [0010] In some embodiments, the techniques described herein relate to a microfluidic device, wherein each of the structures within the same row are substantially the same size, substantially the same shape, or both. In some embodiments, the techniques described herein relate to a microfluidic device, wherein all of the structures within two or more rows are substantially the same size, substantially the same shape, or both. In some embodiments, the techniques described herein relate to a microfluidic device, wherein all of the structures in all of the rows are substantially the same size, substantially the same shape, or both.

[0011] In some embodiments, the techniques described herein relate to a microfluidic device, wherein at least two or more structures within the same row are different. In some embodiments, the techniques described herein relate to a microfluidic device, wherein at least two or more structures within two different rows are different.

[0012] In some embodiments, the techniques described herein relate to the microfluidic device of the present disclosure, wherein the plurality of rows includes 2-100 rows.

[0013] In some embodiments, the techniques described herein relate to a microfluidic device, wherein each row is the same. In some embodiments, the techniques described herein relate to a microfluidic device, wherein at least two or more rows are different. In some embodiments, the techniques described herein relate to a microfluidic device, wherein all rows are different.

[0014] In some embodiments, the techniques described herein relate to a microfluidic device wherein the plurality of structures includes 10-1000 structures. In some embodiments, the techniques described herein relate to a microfluidic device, wherein the plurality of structures includes 40-50 structures. In some embodiments, the techniques described herein relate to a microfluidic device, wherein the plurality of structures includes 42 structures.

[0015] In some embodiments, the techniques described herein relate to a microfluidic device, wherein the set of supports includes 2-100 supports. In some embodiments, the techniques described herein relate to a microfluidic device, wherein the set of supports includes 3-6 supports. In some embodiments, the techniques described herein relate to a microfluidic device, wherein the set of supports includes 4 supports.

[0016] In some embodiments, the techniques described herein relate to a microfluidic device, wherein the microfluidic device includes 1-1000 or more channels.

[0017] In some embodiments, the techniques described herein relate to a microfluidic device, wherein the distance between each row is about 0.1 to 10 cm. In some embodiments, the techniques described herein relate to a microfluidic device, wherein the distance between each row is about 0.9 cm to about 1.2 cm. In some embodiments, the techniques described herein relate to a microfluidic device, wherein the distance between each row is about 1.1 cm. [0018] In some embodiments, the techniques described herein relate to a microfluidic device, wherein each channel is tapered at the inlet end, is tapered at the outlet end, or both. In some embodiments, the techniques described herein relate to a microfluidic device, wherein the tapers at the inlet end, the outlet end, or both form approximately 90° angles with the inlet, the outlet, or both being at the apex of each taper.

[0019] In some embodiments, the techniques described herein relate to a microfluidic device, wherein the device includes one or more suitable materials. In some embodiments, the techniques described herein relate to a microfluidic device, wherein the one or more suitable materials are biocompatible. In some embodiments, the techniques described herein relate to a microfluidic device, wherein the one or more suitable materials are each independently selected from a glass, a polymer, a ceramic, a metal, an alloy, or any combination thereof. In some embodiments, the techniques described herein relate to a microfluidic device, wherein the polymer is polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA), polytetrafluoroethylene (PTFE), or any combination thereof.

[0020] In some aspects, the techniques described herein relate to a method of using a microfluidic device of the present disclosure that includes injecting a fluid comprising sperm into an inlet of a microfluidic device and injecting a first suitable media via the inlet to induce a fluid flow within the device thereby removing incompetent sperm.

[0021] In some embodiments, the techniques described herein relate to a method, wherein the fluid is semen.

[0022] In some embodiments, the techniques described herein relate to a method, further including filling the microfluidic device with a second suitable media by injecting the second suitable media in the inlet prior to injecting the fluid including sperm into the inlet.

[0023] In some embodiments, the techniques described herein relate to a method, further including collecting competent sperm from the device after injecting the first suitable media.

[0024] In some embodiments, the techniques described herein relate to a method, wherein the second suitable media is injected at a flow rate of 1000 to 5000 pL/h. In some embodiments, the techniques described herein relate to a method, wherein the second suitable media is injected at a flow rate of about 3000 pL/h. In some embodiments, the techniques described herein relate to a method, wherein the first suitable media is injected at a flow rate of about 100 to 2000 pL/h. In some embodiments, the techniques described herein relate to a method, wherein the first suitable media is injected at a flow rate of about 350 pL/h.

[0025] In some embodiments, the techniques described herein relate to a method, wherein the fluid including sperm is injected at a flow rate of about 2000 to 5000 pL/h. In some embodiments, the techniques described herein relate to a method, wherein the fluid including sperm is injected at a flow rate of about 3000 pL/h.

[0026] In some embodiments, the techniques described herein relate to a method, wherein the fluid including sperm is injected at a flow rate that minimizes dilution of the fluid.

[0027] In some embodiments, the techniques described herein relate to a method, further including performing in vitro fertilization (IVF) using the collected competent sperm. In some embodiments, the techniques described herein relate to a method, wherein the IVF is chamber based IVF.

[0028] In some embodiments, the techniques described herein relate to a method of using a microfluidic device of the present disclosure, wherein one or more steps are automated. [0029] In some aspects, embodiments described herein relate to a composition containing a population of sperm enriched for competent sperm, wherein the population of sperm is enriched for competent sperm by performing a method of the present disclosure that uses the microfluidic device of the present disclosure.

[0030] In some aspects, embodiments described herein relate to a composition that includes a population of sperm enriched for competent sperm wherein the population of enriched for competent sperm have (i) a DNA integrity of at least 20% greater as compared to a population not enriched for competent sperm; (ii) has a higher rate of blastocyst formation, embryo formation, or both as compared to a population not enriched for competent sperm; (iii) has a lower DNA fragmentation index as compared to a population not enriched for competent sperm; or (iv) any combination of (i)-(iii). In some embodiments, the population of sperm enriched for competent sperm is prepared by performing a method of the present disclosure that uses the microfluidic device of the present disclosure.

[0031] These and other aspects, objects, features, and advantages of the example embodiments will become apparent to those having ordinary skill in the art upon consideration of the following detailed description of example embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

[0032] An understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention may be utilized, and the accompanying drawings of which:

[0033] FIG 1A-1D show (FIG. 1A) perspective view of the device with the inset showing the dimensions of the loading veins and prisms. (FIG. IB) The distance between the rows is 1.1 cm and the inlet and outlets are connected with 90° fans. The inlet area has 4 supports of 200 pm diameter. (FIG. 1C) The dimensions of the prisms. (FIG. ID) After semen is loaded, the medium (red, as represented in greyscale, Ci) is injected from the inlet at various flow rates and the simulation results show how the medium washes the semen (blue, as represented in greyscale).

[0034] FIG. 2A-2E show (FIG. 2A) ([) over time decreases faster for higher flow rates. The rate of semen discharge decreases since the fluid velocity near the walls is low. (FIG. 2B) The time required for the washing steps is estimated numerically at various flow rates. The washing can stop when is 3, 5, 10 or 15 %. (FIG. 2C) as the semen is washed from the chip, the motile sperm with high DNA integrity and higher velocity would swim against the flow and are guided by the strictures through the rheotaxis mechanism to the inlet area and the lesser motile spermatozoa and debris would discharge from the outlet. (FIG. 2D) The shear rate contours in isometric projection (i), on the xy plane at z = 18 pm (ii) and xz -plane at the middle of the stricture (iii). One of the prisms is cut to assist visualization. (FIG. 2E) Average shear rate versus flow rate in the device.

[0035] FIG. 3A-3C show accumulation of spermatozoa at the stricture at various shear rates. (FIG. 3A) As the shear rate increases the signal (S [a.u.]) increases and then decreases at higher shear rates, peaking at shear rate 9.2 s’ ¹ . (FIG. 3B) The rheotactic sperm population at the stricture moves downstream because of the increase in the drag forces; the maximum of the mean signal S shifts to higher x values (distance from contraction point between prisms). (FIG. 3C) X values start at zero at the smallest distance between prisms. Minus x values indicate the region in front of the stricture. (FIG. 3D) Shows the moving average of the signal. The signal before and after stricture is close to zero and peaks at 0-200 pm and it reduces for shear rates greater than 10 s’ ¹ .

[0036] FIG. 4A-4F show computer assisted sperm analysis (CASA) parameters and DNA fragmentation index (DFI) of the separated spermatozoa in comparison to raw semen and centrifugation sorting. (FIG. 4A) VAP. (FIG. 4B) Motility percentage. (FIG. 4C) Amplitude of lateral head displacement (ALH). (FIG. 4D) Concentration of spermatozoa. (FIG. 4E) Beat cross frequency (BCF). and (FIG. 4F) DFI. * p < 0.05, ** p < 0.01 and n > 3. Shaded areas show the 95% confidence intervals. The statistical significance of each group is represented by connecting letter on top of each group. Groups with no common letters are significantly different. Paired t-test was used to compare means.

[0037] FIG. 5A-5C show conventional IVF process using various shear rates and centrifugation-based sperm separation as control. (FIG. 5A) Spermatozoa sorted using the microfluidic device and the centrifugation-based semen sorted are evaluated using NucleoCounter device for their concentration or C. VNC=600/C microliter of the selected/sorted sperm and 60 - VNC microliter of media are mixed to produce samples of normalized concentration of 10 M mL’ ¹ . (FIG. 5B) Concentration of sorted semen; as the shear rate increases the concentration of separated spermatozoa decreases. (FIG. 5C) Proportion of sperm with membrane damage is reduces for rheotaxis-based separation in comparison to centrifugation, * p < 0.05, ** p < 0.01, *** p < 0.001.

[0038] FIG. 6A-6D show (FIG. 6A) The blastocysts for various groups. The red arrow shows hatched embryos. The scale bar is 200 pm. (FIG. 6B) Cleavage rate is approximately 80% for all the four groups; no statistical significance (n.s.). The fraction at each group shows the total number of cleaved embryos over total inseminated COCs. (FIG. 6C) As the shear rate increases, blastocyst rate increases while there is no significant difference between control and 3 and 5 s' ¹ groups. At y = 7 s’ ¹ , blastocyst rate increases to 37 % in comparison to 30 % of the control. The ratio shows the total number of blastocysts over total number of COCs. (FIG. 6D) Ratio of blastocysts over cleavage. * p < 0.05, ** p < 0.01.

[0039] FIG. 7 shows the procedure of the device operation. First, the empty chip is filled with media (I, and II). The capillary effect of the veins help load the device without air bubbles. Once the device is fully loaded, media is flushed and replaced by semen (III and IV). At last, after the semen is loaded, a syringe pump is used to wash the sperm with media injected from the inlet and the waste is collected in an outlet tube (V). For collecting the selected sperm that remained in the device at step VI, a pipette is used to aspirate the sample from the inlet.

[0040] FIG. 8 shows the velocity profile at the stricture.

[0041] FIG. 9 shows the velocity profile at the strictures for all the 3 rows of the device using computational fluid dynamics. Except for the strictures at the boundaries all the remaining strictures have the same velocity profile which results in the same shear rate. This, presumably, on its own results in the same accumulation of spermatozoa at the strictures.

[0042] FIG. 10 shows a graph demonstrating the filtering effect of the shear rate on the sperm VAP. VAP distribution of raw sample and samples of various shear rates of 3 s’ ¹ , 7 s’ ¹ and 11 s’ ¹ . The lines on the top of the curves indicate the mean of the VAP distributions which are color-coded based on the legend. The vertical axis is mirrored with respect to 0 to avoid overlapping curves.

[0043] FIG. 11A-11E show Minimal model of accumulation of microswimmers behind the barrier in the gate area (scope) of the prism (stricture).

[0044] FIG. 12 shows a graph demonstrating the variation of X chromosome vs shear rate. The shaded area shows the 95% confidence interval. The results are shown for two patients and the F-test revealed that there is no statistical significance for sex bias however the results are from limited observation and more data is required to evidently conclude the effect of shear rate on sperm paternal content.

[0045] FIG. 13 shows a graph demonstrating the retrieval efficiency of the sorted sperm. * p < 0.05, ** p < 0.01.

[0046] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.

DETAILED DESCRIPTION OF THE EXAMPLE EMBODIMENTS

[0047] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

[0048] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

[0049] All publications and patents cited in this specification are cited to disclose and describe the methods and/or materials in connection with which the publications are cited. All such publications and patents are herein incorporated by references as if each individual publication or patent were specifically and individually indicated to be incorporated by reference. Such incorporation by reference is expressly limited to the methods and/or materials described in the cited publications and patents and does not extend to any lexicographical definitions from the cited publications and patents. Any lexicographical definition in the publications and patents cited that is not also expressly repeated in the instant application should not be treated as such and should not be read as defining any terms appearing in the accompanying claims. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure. Further, the dates of publication provided could be different from the actual publication dates that may need to be independently confirmed. [0050] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0051] Where a range is expressed, a further aspect includes from the one particular value and/or to the other particular value. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. For example, where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, e.g., the phrase “x to y” includes the range from ‘x’ to ‘y’ as well as the range greater than ‘x’ and less than ‘y’. The range can also be expressed as an upper limit, e.g., ‘about x, y, z, or less’ and should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of Tess than x’, less than y’, and Tess than z’ . Likewise, the phrase ‘about x, y, z, or greater’ should be interpreted to include the specific ranges of ‘about x’, ‘about y’, and ‘about z’ as well as the ranges of ‘greater than x’, greater than y’, and ‘greater than z’. In addition, the phrase “about ‘x’ to ‘y’”, where ‘x’ and ‘y’ are numerical values, includes “about ‘x’ to about ‘y’”.

[0052] It should be noted that ratios, concentrations, amounts, and other numerical data can be expressed herein in a range format. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as “about” that particular value in addition to the value itself. For example, if the value “10” is disclosed, then “about 10” is also disclosed. Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms a further aspect. For example, if the value “about 10” is disclosed, then “10” is also disclosed.

[0053] It is to be understood that such a range format is used for convenience and brevity, and thus, should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. To illustrate, a numerical range of “about 0.1% to 5%” should be interpreted to include not only the explicitly recited values of about 0.1% to about 5%, but also include individual values (e.g., about 1%, about 2%, about 3%, and about 4%) and the subranges (e.g., about 0.5% to about 1.1%; about 5% to about 2.4%; about 0.5% to about 3.2%, and about 0.5% to about 4.4%, and other possible sub-ranges) within the indicated range.

General Definitions

[0054] As used herein, the singular forms “a”, “an”, and “the” include both singular and plural referents unless the context clearly dictates otherwise.

[0055] As used herein, "about," "approximately," “substantially,” and the like, when used in connection with a measurable variable such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value including those within experimental error (which can be determined by e.g. given data set, art accepted standard, and/or with e.g. a given confidence interval (e.g. 90%, 95%, or more confidence interval from the mean), such as variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. As used herein, the terms “about,” “approximate,” “at or about,” and “substantially” can mean that the amount or value in question can be the exact value or a value that provides equivalent results or effects as recited in the claims or taught herein. That is, it is understood that amounts, sizes, formulations, parameters, and other quantities and characteristics are not and need not be exact, but may be approximate and/or larger or smaller, as desired, reflecting tolerances, conversion factors, rounding off, measurement error and the like, and other factors known to those of skill in the art such that equivalent results or effects are obtained. In some circumstances, the value that provides equivalent results or effects cannot be reasonably determined. In general, an amount, size, formulation, parameter or other quantity or characteristic is “about,” “approximate,” or “at or about” whether or not expressly stated to be such. It is understood that where “about,” “approximate,” or “at or about” is used before a quantitative value, the parameter also includes the specific quantitative value itself, unless specifically stated otherwise.

[0056] The term “optional” or “optionally” means that the subsequent described event, circumstance or substituent may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0057] The recitation of numerical ranges by endpoints includes all numbers and fractions subsumed within the respective ranges, as well as the recited endpoints.

[0058] As used herein, a “biological sample” refers to a sample obtained from, made by, secreted by, excreted by, or otherwise containing part of or from a biologic entity. A biologic sample can contain whole cells and/or live cells and/or cell debris, and/or cell products, and/or virus particles. The biological sample can contain (or be derived from) a “bodily fluid”. The biological sample can be obtained from an environment (e.g., water source, soil, air, and the like). Such samples are also referred to herein as environmental samples. As used herein “bodily fluid” refers to any non-solid excretion, secretion, or other fluid present in an organism and includes, without limitation unless otherwise specified or is apparent from the description herein, amniotic fluid, aqueous humor, vitreous humor, bile, blood or component thereof (e.g. plasma, serum, etc.), breast milk, cerebrospinal fluid, cerumen (earwax), chyle, chyme, endolymph, perilymph, exudates, feces, female ejaculate, gastric acid, gastric juice, lymph, mucus (including nasal drainage and phlegm), pericardial fluid, peritoneal fluid, pleural fluid, pus, rheum, saliva, sebum (skin oil), semen, sputum, synovial fluid, sweat, tears, urine, vaginal secretion, vomit and mixtures of one or more thereof. Biological samples include cell cultures, bodily fluids, cell cultures from bodily fluids. Bodily fluids may be obtained from an organism, for example by puncture, or other collecting or sampling procedures.

[0059] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, murines, simians, humans, farm animals, sport animals, and pets. Tissues, cells and their progeny of a biological entity obtained in vivo or cultured in vitro are also encompassed.

[0060] The term “biocompatible”, as used herein, refers to a substance or object that performs its desired function when introduced into an organism without inducing significant inflammatory response, immunogenicity, or cytotoxicity to native cells, tissues, or organs, or to cells, tissues, or organs introduced with the substance or object. For example, a biocompatible product is a product that performs its desired function when introduced into an organism without inducing significant inflammatory response, immunogenicity, or cytotoxicity to native cells, tissues, or organs. Biocompatibility, as used herein, can be quantified using the following in vivo biocompatibility assay. A material or product is considered biocompatible if it produces, in a test of biocompatibility related to immune system reaction, less than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% of the reaction, in the same test of biocompatibility, produced by a material or product the same as the test material or product except for a lack of the surface modification on the test material or product. Examples of useful biocompatibility tests include measuring and assessing cytotoxicity in cell culture, inflammatory response after implantation (such as by fluorescence detection of cathepsin activity), and immune system cells recruited to implant (for example, macrophages and neutrophils).

[0061] Various embodiments are described hereinafter. It should be noted that the specific embodiments are not intended as an exhaustive description or as a limitation to the broader aspects discussed herein. One aspect described in conjunction with a particular embodiment is not necessarily limited to that embodiment and can be practiced with any other embodiment s). Reference throughout this specification to “one embodiment”, “an embodiment,” “an example embodiment,” means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases “in one embodiment,” “in an embodiment,” or “an example embodiment” in various places throughout this specification are not necessarily all referring to the same embodiment, but may. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to a person skilled in the art from this disclosure, in one or more embodiments. Furthermore, while some embodiments described herein include some but not other features included in other embodiments, combinations of features of different embodiments are meant to be within the scope of the invention. For example, in the appended claims, any of the claimed embodiments can be used in any combination.

[0062] All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference. OVERVIEW

[0063] Rheotaxis in mammalian sperm has been proposed as one the long-range guiding mechanism for the journey of sperm toward the egg. Just recently, it was proved that higher rheotaxis ability is associated with higher fertilization rate and lower DNA integrity. Yet many of the microfluidic devices for sperm separation based on rheotaxis are not suitable for performing in vitro fertilization (IVF) since the retrieval efficiency (RE) of the output sample is usually less than 2 %.

[0064] That said, embodiments disclosed herein can provide devices and techniques for rheotaxis-based separation of sperm. The devices and techniques described and demonstrated herein build upon Applicant’s prior finding that sperm with a greater rheotaxis capacity resulted in greater pregnancy outcomes (see e.g., M. Yaghoobi, M. Azizi, A. Mokhtare, F. Javi, A. Abbaspourrad, Rheotaxis quality index: a new parameter that reveals male mammalian in vivo fertility and low sperm DNA fragmentation. Lab on a Chip 22, 1486-1497 (2022)). Described in exemplary embodiments herein are microfluidic devices that generate various shear rates in a fluid flowing through the device. Without being bound by theory, as the shear rate in the fluid flow increases, sperm with a sufficient rheotaxis ability (i.e., competent sperm) will be able maintain their position within the device. In contrast, sperm with insufficient rheotaxis ability (i.e., incompetent sperm) will not be able to reorient and maintain their position within the device and will be flushed from the device with the fluid flow. This results in a population of sperm enriched for competent sperm that can be used for in vitro fertilization. [0065] Applicant observed that the device and method improved the retriever efficiency to more than 40%, which is at least over 20 times greater than conventional rheotaxis-based separation methods. Further, Applicant observed that DNA integrity of the samples separated via an embodiment of the device described herein with higher rheotaxis ability were higher than the raw sample at least by 20 %. At the highest rheotaxis capability as demontrated in the Working Examples herein, the DNA fragmentation index (DFI) was only 3 %; significantly lower than the raw sample DFI of 39 %, which evidences the viability of the device and techniques described herein.

[0066] Other compositions, compounds, devices, configurations, methods, features, and advantages of the present disclosure will be or become apparent to one having ordinary skill in the art upon examination of the following drawings, detailed description, and examples. It is intended that all such additional compositions, compounds, methods, features, and advantages be included within this description, and be within the scope of the present disclosure.

RHEOTAXIS-BASED SEPARATION DEVICES

[0067] Described in certain example embodiments herein are microfluidic devices configured for rheotaxis-based sperm separation. In some embodiments and as generally shown in FIG. 1A-1BC, the microfluidic device 1000 contains one or more channels 1010 (individually 1010a, 1010b, 1010c, etc.), each channel 1010 having an inlet 1020 at a first end of the channel 1010, an outlet 1030 at a second end of the channel 1010, a plurality of rows 1040, each row (1040a, 1040b, 1040c, etc.) containing a plurality of structures 1050 forming a plurality of strictures 1060, wherein each row 1040 extends across the width of the channel 1010 , wherein no structure 1050 contacts another structure 1050 so as to form a gap 1070 between each structure 1050 to generate the strictures 1060; a plurality of straight guide veins 1080 (individually 1080a, 1080b, 1080c, etc.) extending across the width of the channel 1010 and being substantially perpendicular to a longest axis of the channel 1010, wherein each straight guide vein 1080 is positioned equidistance between two different rows of the plurality of rows 1040; a plurality of curved guide veins 1090 extending across the width of the channel 1010, wherein a curved guide vein 1090 is placed on each side of each row of the plurality of rows 1040 with the curve of the curved guide vein 1090 extending away from the row 1040 (i.e., the concave side of the curved guide facing the row), and wherein the microfluidic device 1000 is configured such that a fluid introduced at the inlet 1020 generates a fluid flow through the channel from the inlet 1020 to the outlet 1030. In some embodiments, each channel 1010 further comprises a set of supports 1120 containing a plurality of supports 1100 placed at a distance from each other, wherein the first set of supports 1120 is positioned between side walls 1110 of the channel 1010 and between the inlet 1020 and a curved vein 1090 on an inlet side of a first row 1040 and the inlet 1020.

[0068] The channels, when viewed from above, can have any suitable two-dimensional shape. In some embodiments, the channels 1010 are hexagonal, rectangular, triangular, or oval. In some embodiments, the side walls of the channels 1010 are substantially linear. In some embodiments, the side walls of the channels 1010 are curved or have one or more curved regions. In some embodiments, the channel 1010 is tapered at the inlet end, the outlet end, or both ends of the channel 1010. In some embodiments, the inlet 1020, the outlet 1030, or both are placed at the apex of the taper, when present.

[0069] In some embodiments, one or more or all of the plurality of rows 1040 extend across the width of the channel 1010 such that it is substantially perpendicular to the direction of a fluid flow through the channel 1010 during operation. In some embodiments, one or more or all of the plurality of rows 1040 extend across the width of the channel 1010 such that they are not substantially perpendicular to the direction of fluid flow. In such embodiments, one or more of the plurality of rows 1040 can be so positioned in the channel 1010 such that they extend diagonally across the width of the channel 1010, with one end of the row.

[0070] In certain example embodiments, the microfluidic device 1000 generates regions of variable shear in the strictures 1060. In certain example embodiments, the device generates a region of high shear in the strictures 1060. In this context, “high shear” refers to a shear rate of at least 11 s ^-1 .

[0071] In certain example embodiments, one or more or all of the structures 1050 are prisms. The prisms can be triangular prisms. Exemplary prisms include, without limitation, pyramids, rectangular prisms, cuboid prisms, pentagonal prisms, hexagonal prisms, heptagonal prisms, and trapezoidal prisms. Other suitable prisms will be appreciated by those of ordinary skill in the art in view of the disclosure herein.

[0072] In certain example embodiments, the plurality of structures 1050 has about 10-1000 structures, or any value or range of values therein. In some embodiments the plurality of structures 1050 has about 10, to/or 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120,

121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139,

140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158,

159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,

178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196,

197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215,

216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,

235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, , 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272,, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291,, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310,, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329,, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348,, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367,, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386,, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405,, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424,, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443,, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462,, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481,, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500,, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519,, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538,, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557,, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576,, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595,, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614,, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633,, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652,, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671,, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690,, 692, 693, 694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709,, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724, 725, 726, 727, 728,, 730, 731, 732, 733, 734, 735, 736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747,, 749, 750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766,, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785,, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804,, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823,, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842,, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880,

881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899,

900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918,

919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937,

938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956,

957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975,

976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994,

995, 996, 997, 998, 999, 1000 structures. In some embodiments, the plurality of structures 1050 has about 40-50 structures. In some embodiments, the plurality of structures 1050 has a plurality of strictures 1060 formed by a gap 1070 between each of the structures in the plurality of structures 1050. In certain example embodiments, each of structures of the plurality of structures 1050 within the same row are substantially the same. In certain example embodiments, all of the structures of the plurality of structures 1050 within two or more rows are substantially the same. In some embodiments, all of the structures in the plurality of structures 1050 in all of the rows are substantially the same. In certain example embodiments, at least two or more structures within the same row are different. In certain example embodiments, at least two or more structures within two different rows are different.

[0073] In certain example embodiments, the plurality of rows 1040 has 2 to 10 or more rows. In some embodiments, the plurality of rows 1040 has 2, 3, 4, 5, 6, 7, 8, 9, 10 or more rows. In certain example embodiments, each row is the same. In certain example embodiments, at least two or more rows are different. In some embodiments, all the rows are different. Any difference between the row (e.g., number of structures, shape of structures, etc.) constitutes a difference. In some embodiments, the set of supports has 3-6 supports. In some embodiments, the set of supports has four supports. In certain example embodiments, the microfluidic device has 1-1000 or more channels, or any value or range of values therein. In certain example embodiments, the distance between each row is about 0.1 to 20 cm, or any value or range of value therein. In some embodiments, the rows of the plurality of rows 1040 are positioned in the channel 1010 such that they are about equidistant from each other. In some embodiments, the rows of the plurality of rows 1040 are positioned in the channel 1010 such that they are different distances from each other. In some embodiments, the rows of the plurality of rows 1040 are positioned in the channel 1010 such that some rows are the same distance from each other and some rows are different distances from each other. In some embodiments, the distance between any two rows is independently about 0.9cm to about 1.2cm, such as about 0.9 cm to/or 1 cm, 1.1 cm, 1.2 cm. In some embodiments, the distance between each row is about 0.9 cm to about 1.2 cm. In some embodiments, the distance between each row is about 1.1 cm.

[0074] In certain example embodiments, each channel 1010 is tapered at the inlet lend and the outlet end. In some embodiments, the tapers at each of the inlet 1020 end and outlet 1030 end form approximately 90° angles with the inlet 1020 or the outlet 1030 being at the point of each taper.

[0075] In some embodiments, one or more dimensions of each channel 1010 (e.g., a length, a width, a height, a diameter, and the like) can independently range from about 1-1,000 pm, nm, /zm, cm, or mm. In some embodiments, one or more dimensions of each channel 1010 are independently about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,

320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500,

510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690,

700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880,

890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 to/or about 1000 pm, nm, /zm, cm, or mm. In some embodiments, the largest dimension of each channel 1010 independently ranges from 1-1,000 pm, nm, /zm, cm, or mm. In some embodiments, the largest dimension of each channel 1010 is independently about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,

290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,

480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660,

670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850,

860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 to/or about 1000 pm, nm, /zm, cm, or mm. In some embodiments, the smallest dimension of each channel 1010 independently ranges from 1-1,000 pm, nm, /zm, cm, or mm. In some embodiments, the smallest dimension of the channel 1010 is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260,

270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450,

460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640,

650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 to/or about 1000 pm, nm, zm, cm, or mm.

[0076] In some embodiments, each channel 1010 has a volume. The volume of each channel 1010 independently ranges from about 1-1,000 pm ³ , nm ³ , rm ³ , cm ³ , or mm ³ . In some embodiments, each channel volume is independently about 1, to/or 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420,

430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610,

620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800,

810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990 to/or about 1000 pm ³ , nm ³ , rm ³ , cm ³ , or mm ³ .

[0077] In certain example embodiments, the microfluidic device 1000 is composed of one or more suitable materials. In some embodiments, the one or more suitable materials are biocompatible materials. Exemplary biocompatible materials include, but are not limited to glass, polymers (e.g., polydimethylsiloxane (PDMS), and others), ceramics, metals, and any combination thereof.

[0078] As used herein, ’’polymer” refers to molecules made up of monomers repeat units linked together. “Polymers” are understood to include, but are not limited to, homopolymers, copolymers, such as for example, block, graft, random and alternating copolymers, terpolymers, etc. and blends and modifications thereof. “A polymer” can be a three- dimensional network (e.g., the repeat units are linked together left and right, front and back, up and down), a two-dimensional network (e.g., the repeat units are linked together left, right, up, and down in a sheet form), or a one-dimensional network (e.g., the repeat units are linked left and right to form a chain). “Polymers” can be composed, natural monomers or synthetic monomers and combinations thereof. The polymers can be biologic (e.g., the monomers are biologically important (e.g., an amino acid), natural, or synthetic. As used interchangeably herein, “polymer blend” and “polymer mixture” refers to a macroscopically homogenous mixture of two or more different species of polymers. Unlike a copolymer, where the monomeric polymers are covalently linked, the constituents of a “polymer blend” and “polymer mixture” are separable by physical means and does not require covalent bonds to be broken. A “polymer blend” can have two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) different polymer constituents. [0079] Exemplary synthetic polymers include, without limitation, poly(hydroxy acids) such as poly(lactic acid), poly(glycolic acid), and poly(lactic acid-co-glycolic acid), poly(lactide), poly(glycolide), poly(lactide-co-glycolide), polyanhydrides, polyorthoesters, polyamides, polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as polyethylene glycol), polyalkylene oxides such as poly(ethylene oxide), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols, polyvinyl ethers, polyvinyl esters, polyvinyl halides such as poly(vinyl chloride), polyvinylpyrrolidone, polysiloxanes, poly(vinyl alcohols), poly(vinyl acetate), polystyrene, polyurethanes and co-polymers thereof, derivatized celluloses such as alkyl cellulose, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, methyl cellulose, ethyl cellulose, hydroxypropyl cellulose, hydroxy-propyl methyl cellulose, hydroxybutyl methyl cellulose, cellulose acetate, cellulose propionate, cellulose acetate butyrate, cellulose acetate phthalate, carboxylethyl cellulose, cellulose triacetate, and cellulose sulphate sodium salt (jointly referred to herein as “synthetic celluloses”), polymers of acrylic acid, methacrylic acid or copolymers or derivatives thereof including esters, poly(methyl methacrylate), poly(ethyl methacrylate), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly (isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate) (jointly referred to herein as “polyacrylic acids”), poly(butyric acid), poly(valeric acid), and poly(lactide-co-caprolactone), copolymers and blends thereof. As used herein, “derivatives” include polymers having substitutions, additions of chemical groups, for example, alkyl, alkylene, hydroxylations, oxidations, and other modifications routinely made by those skilled in the art. In some embodiments, the polymer is composed of polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA), Polytetrafluoroethylene (PTFE)), or any combination thereof.

[0080] As used herein, “glass” refers to any type of glass including, but not limited to silicate glasses (e.g., soda-lime glass, borosilicate glass, lead glass, aluminosilicate glass, glassceramics, and fiber glass), silica-free glasses (e.g., amorphous metals and polymers), and molecular liquids and molten salts. Glasses can contain additives that can modify e.g., the optical properties (e.g., transparency, color, refractivity etc.), conductive properties or other properties of the glass. [0081] As used herein, “metal” refers to Li, Be, Na, Mg, Al, K, Ca, Sc, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Ga, Rb, Sr, Y, Zr, Nb, Mo, Tc, Ru, Rh, Pd, Ag, Cd, In, Sn, Cs, Ba, La, Ce, Pr, Nd, Pm, Sm, Eu, Gd, Tb, Dy, Ho, Er, Rm, Yb, Lu, Hf, W, Re, Os, Ir, Pt, Au, Hg, Tl, Pb, Bi, Po, Ra, Ac, Th, Pa, U, Np, Am, Cm, Bk, Cf, Es, Fm, Md, No, Lr, Rf, Db, Sg, Bh, Hs, Mt, Ds, Rg, Cn, Nh, Fl, Me, Lv, and combinations thereof. As used herein, “metalloid” refers to B, Si, Ge, As, Sb, Te, At, and combinations thereof. As used herein, “non-metal” refers to He, H, C, N, O, F, Ne, P, S, Cl, Ar, Se, Br, Kr, I, Xe, Rn, and combinations thereof.

[0082] In some embodiments, all or one or more parts of the channels 1010 are hydrophilic. In some embodiments, all or one or more parts of the channels 1010 are hydrophobic. In some embodiments, all or one or more parts of the channels 1010 are superhydrophobic. It will be appreciated that the channels 1010 thus can contain one or more parts that are hydrophilic, one or more parts that are hydrophobic, one or more parts that are superhydrophobic, or any combination thereof. In some embodiments, one or more surfaces of a channel 1010 has a pattern. In some embodiments, patterns on a surface of a channel 1010 or other region of the microfluidic device 1000 can be formed by specific placement of hydrophobic and/or hydrophilic materials. In some embodiments, such patterns can, without limitation, can form features of the channel 1010 and/or microfluidic device 1000 and/or form conduits to provide sample, reactants, features, and the like to or in one or more regions of the channel 1010. As used herein, “hydrophilic”, refers to molecules which have a greater affinity for, and thus solubility in, water as compared to organic solvents. The hydrophilicity of a compound can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent, such as octanol, ethyl acetate, methylene chloride, or methyl tert-butyl ether. If after equilibration a greater concentration of the compound is present in the water than in the organic solvent, then the molecule is considered hydrophilic. As used herein, “hydrophobic”, refers to molecules which have a greater affinity for, or solubility in an organic solvent as compared to water. The hydrophobicity of a compound can be quantified by measuring its partition coefficient between water (or a buffered aqueous solution) and a water-immiscible organic solvent, such as octanol, ethyl acetate, methylene chloride, or methyl tert-butyl ether. If after equilibration a greater concentration of the compound is present in the organic solvent than in the water, then the molecule is considered hydrophobic. In some embodiments, hydrophobic and hydrophilic regions can be formed by particular materials that are hydrophobic or hydrophilic or can be formed by changing the texture of a surface (e.g., by etching, scoring, etc.) such that the contact angle or other interaction of water or liquid with the surface is changed such that that region such that it is hydrophobic or hydrophilic.

[0083] In some embodiments, the microfluidic device 1000 or component thereof contains a hydrophobic material. Suitable hydrophobic materials include, but are not limited to: acrylics (e.g., acrylic, acrylonitrile, acrylamide, and maleic anhydride polymers), polyamides and polyimides, carbonates (e.g., Bisphenol A -based carbonates), polydienes, polyesters, poly ethers, polyfluorocarbons, polyolefins (e.g., polyethylene, polypropylene, and copolymers thereof), polystyrenes and copolymers thereof, polyvinyl acetals, polyvinyl chlorides and polyvinylidene chlorides, poly vinyl ethers and polyvinyl ketones, polyvinylpyridines and polyvinypyrrolidones, Aculon’s Transition Metal Complex coting, SLIPS coating material (Adaptive Surface Technologies), and any combination thereof.

[0084] In some embodiments, the microfluidic device 1000 or component thereof contains a superhydrophobic material. Suitable superhydrophobic materials include, but are not limited to manganese oxide polystyrene, zinc oxide polystyrene, precipitated calcium carbonate, carbon nanotubes, silica nano-coatings, fluorinated silanes, and flurophopolymer coatings. See e.g., Meng et al. 2008, The Journal of Physical Chemistry C. 112 (30): 11454-11458; Hu et al. 2009. Colloids and Surfaces A: Physicochemical and Engineering Aspects. 351 (1-3): 65-70; Lin et al., Colloids and Surfaces A: Physicochemical and Engineering Aspects. 421 : 51-62; Das et al., RSC Advances. 4 (98): 54989-54997. doi: 10.1039/C4RA10171E; Torun et al., 2018. Macromolecules. 51 (23): 10011-10020; Warsinger et al. 2015., Colloids and Surfaces A: Physicochemical and Engineering Aspects. 421 : 51-62; Servi et al. 2017., Journal of Membrane Science. Elsevier BV. 523: 470-479

[0085] In some embodiments, the microfluidic device 1000 or component thereof contains a hydrophilic material. Hydrophilic materials include, but are not limited to, hydrophilic polymers such as poly(N-vinyl lactams), poly(vinylpyrrolidone), polyethylene oxide), polypropylene oxide), polyacrylamides, cellulosics, methyl cellulose, polyanhydrides, polyacrylic acids, polyvinyl alcohols, polyvinyl ethers, alkylphenol ethoxylates, complex polyol monoesters, polyoxyethylene esters of oleic acid, polyoxyethylene sorbitan esters of oleic acid, and sorbitan esters of fatty acids; inorganic hydrophilic materials such as inorganic oxide, gold, zeolite, and diamond-like carbon; and surfactants such as Triton X-100, Tween, Sodium dodecyl sulfate (SDS), ammonium lauryl sulfate, alkyl sulfate salts, sodium lauryl ether sulfate (SLES), alkyl benzene sulfonate, soaps, fatty acid salts, cetyl trimethylammonium bromide (CTAB) a.k.a. hexadecyl trimethyl ammonium bromide, alkyltrimethylammonium salts, cetylpyridinium chloride (CPC), polyethoxylated tallow amine (POEA), benzalkonium chloride (BAC), benzethonium chloride (BZT), dodecyl betaine, dodecyl dimethylamine oxide, cocamidopropyl betaine, coco ampho glycinate alkyl polyethylene oxide), copolymers of poly(ethylene oxide) and polypropylene oxide) (commercially called Poloxamers or Poloxamines), alkyl polyglucosides, fatty alcohols, cocamide MEA, cocamide DEA, cocamide TEA, Adhesives Research (AR) tape 90128, AR tape 90469, AR tape 90368, AR tape 90119, AR tape 92276, and AR tape 90741 (Adhesives Research, Inc., Glen Rock, Pa.). Examples of hydrophilic film include, but are not limited to, Vistex® and Visguard® films from (Film Specialties Inc., Hillsborough, N.J.), and Lexan HPFAF (GE Plastics, Pittsfield, Mass.). Other hydrophilic surfaces are available from Surmodics, Inc. (Eden Prairie, Minn.), Biocoat Inc. (Horsham, Pa.), Advanced Surface Technology (Billerica, Mass.), and Hydromer, Inc. (Branchburg, N.J.) and any combination thereof. Surfactants can be mixed with reaction polymers such as polyurethanes and epoxies to serve as a hydrophilic coating.

[0086] In some embodiments, the microfluidic device 1000 or component thereof contains a conductive and/or magnetic material. Conductive materials that can be included in the microfluidic device 1000 or component thereof include, without limitation, metals, electrolytes, superconductors, semiconductors and some nonmetallic conductors such as graphite and conductive polymers. Magnetic materials that can be included in the microfluidic device 1000 or component thereof include, without limitation, any magnetic material including those that are ferromagnetic, paramagnetic and diamagnetic. In some embodiments, the magnetic material can include those that are electromagnetic (i.e., those materials that become magnetic or become a more powerful magnet when an electric current is applied to them). Exemplary magnetic materials include, but are not limited to, iron, nickel, cobalt, steel, rare earth metals (e.g., gadolinium, samarium, and neodymium), and combinations thereof.

[0087] In some embodiments, the microfluidic device 1000 or component thereof contains an electric insulator material. Exemplary electric insulator materials include, but are not limited to, rubber, glass, oil, air, diamond, dry wood, dry cotton, plastic, fiberglass, porcelain, ceramics and quartz.

[0088] In some embodiments, the microfluidic device 1000 contains or is otherwise operatively, electrically, fluidically, coupled or in communication with, in addition to the channel(s) 1010 previously discussed, one or more channels, microchannels, reservoirs, chambers, pumps, computing devices, imaging devices, injectors, vacuums, electrical circuits, sensors, heating elements, cooling elements, optical devices, lights, tubes or tubing, syringes, and/or the like.

METHODS OF SPERM SEPARATION

[0089] The microfluidic device 1000 of the present disclosure can be used to separate competent sperm from incompetent sperm and/or generate a population of sperm enriched for competent sperm.

[0090] Described in certain example embodiments herein are methods of rheotaxis-based sperm separation including injecting a fluid containing sperm into an inlet 1020 of a microfluidic device 1000 of the present disclosure, wherein the fluid is optionally semen and injecting a first suitable media via the inlet 1020 to induce a fluid flow within the device, such as through the channel(s) 1010, thereby removing incompetent sperm. In some embodiments, the method further includes filling the microfluidic device 1000 of the present disclosure with a second suitable media by injecting the second suitable media in the inlet 1020 prior to injecting the fluid containing sperm into the inlet 1020. Without being bound by theory, sperm remaining in the channel(s) 1010 are enriched for competent sperm. In some embodiments the fluid containing sperm consists of undiluted semen. In some embodiments, the fluid containing sperm contains undiluted semen. In some embodiments, the fluid containing sperm contains diluted semen. In some embodiments, the concentration of sperm in the fluid contains sperm is about or is at least 50 M/mL. In some embodiments, the concentration of sperm is about or is at least 50 M/mL, to/or 50.5 M/mL, 51 M/mL, 51.5 M/mL, 52 M/mL, 52.5 M/mL, 53 M/mL,

53.5 M/mL, 54 M/mL, 54.5 M/mL, 55 M/mL, 55.5 M/mL, 56 M/mL, 56.5 M/mL, 57 M/mL,

57.5 M/mL, 58 M/mL, 58.5 M/mL, 59 M/mL, 59.5 M/mL, 60 M/mL, 60.5 M/mL, 61 M/mL,

61.5 M/mL, 62 M/mL, 62.5 M/mL, 63 M/mL, 63.5 M/mL, 64 M/mL, 64.5 M/mL, 65 M/mL,

65.5 M/mL, 66 M/mL, 66.5 M/mL, 67 M/mL, 67.5 M/mL, 68 M/mL, 68.5 M/mL, 69 M/mL,

69.5 M/mL, 70 M/mL, 70.5 M/mL, 71 M/mL, 71.5 M/mL, 72 M/mL, 72.5 M/mL, 73 M/mL,

73.5 M/mL, 74 M/mL, 74.5 M/mL, 75 M/mL, 75.5 M/mL, 76 M/mL, 76.5 M/mL, 77 M/mL,

77.5 M/mL, 78 M/mL, 78.5 M/mL, 79 M/mL, 79.5 M/mL, 80 M/mL, 80.5 M/mL, 81 M/mL,

81.5 M/mL, 82 M/mL, 82.5 M/mL, 83 M/mL, 83.5 M/mL, 84 M/mL, 84.5 M/mL, 85 M/mL,

85.5 M/mL, 86 M/mL, 86.5 M/mL, 87 M/mL, 87.5 M/mL, 88 M/mL, 88.5 M/mL, 89 M/mL, 89.5 M/mL, 90 M/mL, 90.5 M/mL, 91 M/mL, 91.5 M/mL, 92 M/mL, 92.5 M/mL, 93 M/mL,

93.5 M/mL, 94 M/mL, 94.5 M/mL, 95 M/mL, 95.5 M/mL, 96 M/mL, 96.5 M/mL, 97 M/mL,

97.5 M/mL, 98 M/mL, 98.5 M/mL, 99 M/mL, 99.5 M/mL, 100 M/mL.

[0091] In certain example embodiments, the method further includes collecting competent sperm from the microfluidic device 1000 after injecting the first suitable media. In some embodiments, the collected competent sperm are included in a composition. In some embodiments the collected competent sperm are diluted in the composition. In some embodiments, the composition contains a semen extender. Thus, also described herein are compositions a population of sperm enriched for competent sperm, wherein the population of sperm is enriched for competent sperm by performing the method described herein. In some embodiments, the composition further contains a suitable semen extender.

[0092] In some embodiment, a composition contains a population of sperm enriched for competent sperm wherein the population enriched for competent sperm (i) have a DNA integrity of at least 20% greater as compared to a population not enriched for competent sperm or other suitable control; (ii) has a higher rate of blastocyst formation, embryo formation, or both as compared to a population not enriched for competent sperm or other suitable control; (iii) has a lower DNA fragmentation index as compared to a population not enriched for competent sperm or other suitable control; or (iv) any combination of (i)-(iii), such as (i) and (ii), (i) and (iii), or (ii) and (iii). In some embodiments, the population of sperm enriched for competent sperm is prepared by performing a method of separating sperm and/or a device of the present disclosure. Without being bound by theory, enrichment of competent sperm so as to have (i) have a DNA integrity of at least 20% greater as compared to a population not enriched for competent sperm or other suitable control; (ii) has a higher rate of blastocyst formation, embryo formation, or both as compared to a population not enriched for competent sperm or other suitable control; (iii) has a lower DNA fragmentation index as compared to a population not enriched for competent sperm or other suitable control; or (iv) any combination of (i)-(iii) can be achieved by separation and concentration of competent sperm by the microfluidic devices and/or methods of the present disclosure.

[0093] In some embodiments, the DNA integrity of the population or sperm enriched for competent sperm is 20% to 1,000% or more greater as compared to a population of sperm not enriched for competent sperm. In some embodiments, the DNA integrity of the population or sperm enriched for competent sperm is 20%, to/or 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%,

44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%,

60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,

76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,

92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%,

300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%,

430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%,

560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%,

690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%,

820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%,

950%, 960%, 970%, 980%, 990%, 1000% or more greater as compared to a population of sperm not enriched for competent sperm or other suitable control. Methods and techniques of determining DNA integrity in sperm are generally known in the art.

[0094] In some embodiments, the rate of embryo formation as compared to a population not enriched for competent sperm is about 0.1%, to/or 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, 11%,

11.1%, 11.2%, 11.3%, 11.4%, 11.5%, 11.6%, 11.7%, 11.8%, 11.9%, 12%, 12.1%, 12.2%,

12.3%, 12.4%, 12.5%, 12.6%, 12.7%, 12.8%, 12.9%, 13%, 13.1%, 13.2%, 13.3%, 13.4%,

13.5%, 13.6%, 13.7%, 13.8%, 13.9%, 14%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%,

14.7%, 14.8%, 14.9%, 15%, 15.1%, 15.2%, 15.3%, 15.4%, 15.5%, 15.6%, 15.7%, 15.8%,

15.9%, 16%, 16.1%, 16.2%, 16.3%, 16.4%, 16.5%, 16.6%, 16.7%, 16.8%, 16.9%, 17%, 17.1%,

17.2%, 17.3%, 17.4%, 17.5%, 17.6%, 17.7%, 17.8%, 17.9%, 18%, 18.1%, 18.2%, 18.3%,

18.4%, 18.5%, 18.6%, 18.7%, 18.8%, 18.9%, 19%, 19.1%, 19.2%, 19.3%, 19.4%, 19.5%,

19.6%, 19.7%, 19.8%, 19.9%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,

31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,

63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,

79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%,

190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%,

320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%,

450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%,

580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%,

710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%,

840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%,

970%, 980%, 990%, 1000% or more higher as compared to a population of sperm not enriched for competent sperm or other suitable control.

[0095] In some embodiments, the rate of blastocyst formation as compared to a population not enriched for competent sperm is about 0.1%, to/or 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4%, 4.1%, 4.2%, 4.3%, 4.4%, 4.5%, 4.6%, 4.7%, 4.8%, 4.9%, 5%, 5.1%, 5.2%, 5.3%, 5.4%, 5.5%, 5.6%, 5.7%, 5.8%, 5.9%, 6%, 6.1%, 6.2%, 6.3%, 6.4%, 6.5%, 6.6%, 6.7%, 6.8%, 6.9%, 7%, 7.1%, 7.2%, 7.3%, 7.4%, 7.5%, 7.6%, 7.7%, 7.8%, 7.9%, 8%, 8.1%, 8.2%, 8.3%, 8.4%, 8.5%, 8.6%, 8.7%, 8.8%, 8.9%, 9%, 9.1%, 9.2%, 9.3%, 9.4%, 9.5%, 9.6%, 9.7%, 9.8%, 9.9%, 10%, 10.1%, 10.2%, 10.3%, 10.4%, 10.5%, 10.6%, 10.7%, 10.8%, 10.9%, 11%,

11.1%, 11.2%, 11.3%, 11.4%, 11.5%, 11.6%, 11.7%, 11.8%, 11.9%, 12%, 12.1%, 12.2%,

12.3%, 12.4%, 12.5%, 12.6%, 12.7%, 12.8%, 12.9%, 13%, 13.1%, 13.2%, 13.3%, 13.4%,

13.5%, 13.6%, 13.7%, 13.8%, 13.9%, 14%, 14.1%, 14.2%, 14.3%, 14.4%, 14.5%, 14.6%,

14.7%, 14.8%, 14.9%, 15%, 15.1%, 15.2%, 15.3%, 15.4%, 15.5%, 15.6%, 15.7%, 15.8%,

15.9%, 16%, 16.1%, 16.2%, 16.3%, 16.4%, 16.5%, 16.6%, 16.7%, 16.8%, 16.9%, 17%, 17.1%,

17.2%, 17.3%, 17.4%, 17.5%, 17.6%, 17.7%, 17.8%, 17.9%, 18%, 18.1%, 18.2%, 18.3%,

18.4%, 18.5%, 18.6%, 18.7%, 18.8%, 18.9%, 19%, 19.1%, 19.2%, 19.3%, 19.4%, 19.5%,

19.6%, 19.7%, 19.8%, 19.9%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%,

31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%,

47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,

63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%,

320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%,

450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%,

580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%,

710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%,

840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%,

970%, 980%, 990%, 1000% or more higher as compared to a population of sperm not enriched for competent sperm or other suitable control.

[0096] Methods and techniques for determining the rate of embryo formation and blastocyst rate are generally known in the art. The rate of embryo formation is defined herein by cleavage rate and blastocyst rate presented in the figure 6 of the current manuscript. Cleavage rate is defined herein as the ratio between the number of oocytes that cleaved after fertilization and the number of oocytes inseminated and blastocyst rate defined herein as the ratio between the number of oocytes that developed to the blastocyst stage and the number of oocytes inseminated. As discussed elsewhere herein and without being bound by theory, the microfluidic devices of the present disclosure can improve blastocyst rate, thus leading to increased rate of embryo formation.

[0097] In some embodiments, the DNA fragmentation index of the population of sperm enriched for competent sperm is about as compared to a population not enriched for competent sperm is about 1 to/or 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11,

11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 20.5, 21,

21.5, 22, 22.5, 23, 23.5, 24, 24.5, 25, 25.5, 26, 26.5, 27, 27.5, 28, 28.5, 29, 29.5, 30, 30.5, 31,

31.5, 32, 32.5, 33, 33.5, 34, 34.5, 35, 35.5, 36, 36.5, 37, 37.5, 38, 38.5, 39, 39.5, 40, 40.5, 41,

41.5, 42, 42.5, 43, 43.5, 44, 44.5, 45, 45.5, 46, 46.5, 47, 47.5, 48, 48.5, 49, 49.5, 50, 50.5, 51,

51.5, 52, 52.5, 53, 53.5, 54, 54.5, 55, 55.5, 56, 56.5, 57, 57.5, 58, 58.5, 59, 59.5, 60, 60.5, 61,

61.5, 62, 62.5, 63, 63.5, 64, 64.5, 65, 65.5, 66, 66.5, 67, 67.5, 68, 68.5, 69, 69.5, 70, 70.5, 71,

71.5, 72, 72.5, 73, 73.5, 74, 74.5, 75, 75.5, 76, 76.5, 77, 77.5, 78, 78.5, 79, 79.5, 80, 80.5, 81,

81.5, 82, 82.5, 83, 83.5, 84, 84.5, 85, 85.5, 86, 86.5, 87, 87.5, 88, 88.5, 89, 89.5, 90, 90.5, 91,

91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5, 100 fold lower as compared to a population of sperm not enriched for competent sperm or other suitable control. Methods and techniques for determining the DNA fragmentation index in sperm are generally known in the art.

[0098] In certain example embodiments, the second suitable media is injected at a flow rate of 1,000 to 5,000 pL/h, or any value or range of values therein. In some embodiments, the second suitable media is injected at a flow rate of about 1000 pL/h to/or 1010 pL/h, 1020 pL/h,

1030 pL/h, 1040 pL/h, 1050 pL/h, 1060 pL/h, 1070 pL/h, 1080 pL/h, 1090 pL/h, 1100 pL/h, 1110 pL/h, 1120 pL/h, 1130 pL/h, 1140 pL/h, 1150 pL/h, 1160 pL/h, 1170 pL/h, 1180 pL/h, 1190 pL/h, 1200 pL/h, 1210 pL/h, 1220 pL/h, 1230 pL/h, 1240 pL/h, 1250 pL/h, 1260 pL/h, 1270 pL/h, 1280 pL/h, 1290 pL/h, 1300 pL/h, 1310 pL/h, 1320 pL/h, 1330 pL/h, 1340 pL/h, 1350 pL/h, 1360 pL/h, 1370 pL/h, 1380 pL/h, 1390 pL/h, 1400 pL/h, 1410 pL/h, 1420 pL/h, 1430 pL/h, 1440 pL/h, 1450 pL/h, 1460 pL/h, 1470 pL/h, 1480 pL/h, 1490 pL/h, 1500 pL/h, 1510 pL/h, 1520 pL/h, 1530 pL/h, 1540 pL/h, 1550 pL/h, 1560 pL/h, 1570 pL/h, 1580 pL/h, 1590 pL/h, 1600 pL/h, 1610 pL/h, 1620 pL/h, 1630 pL/h, 1640 pL/h, 1650 pL/h, 1660 pL/h, 1670 pL/h, 1680 pL/h, 1690 pL/h, 1700 pL/h, 1710 pL/h, 1720 pL/h, 1730 pL/h, 1740 pL/h, 1750 pL/h, 1760 pL/h, 1770 pL/h, 1780 pL/h, 1790 pL/h, 1800 pL/h, 1810 pL/h, 1820 pL/h, 1830 pL/h, 1840 pL/h, 1850 pL/h, 1860 pL/h, 1870 pL/h, 1880 pL/h, 1890 pL/h, 1900 pL/h, 1910 pL/h, 1920 pL/h, 1930 pL/h, 1940 pL/h, 1950 pL/h, 1960 pL/h, 1970 pL/h, 1980 pL/h, 1990 pL/h, 2000 pL/h, 2010 pL/h, 2020 pL/h, 2030 pL/h, 2040 pL/h, 2050 pL/h, 2060 pL/h, 2070 pL/h, 2080 pL/h, 2090 pL/h, 2100 pL/h, 2110 pL/h, 2120 pL/h, 2130 pL/h, 2140 pL/h, 2150 pL/h, 2160 pL/h, 2170 pL/h, 2180 pL/h, 2190 pL/h, 2200 pL/h, 2210 pL/h, 2220 pL/h, 2230 pL/h, 2240 pL/h, 2250 pL/h, 2260 pL/h, 2270 pL/h, 2280 pL/h, 2290 pL/h, 2300 pL/h, 2310 pL/h, 2320 pL/h, 2330 pL/h, 2340 pL/h, 2350 pL/h, 2360 pL/h, 2370 pL/h, 2380 pL/h, 2390 pL/h, 2400 pL/h, 2410 pL/h, 2420 pL/h, 2430 pL/h, 2440 pL/h, 2450 pL/h, 2460 pL/h, 2470 pL/h, 2480 pL/h, 2490 pL/h, 2500 pL/h, 2510 pL/h, 2520 pL/h, 2530 pL/h, 2540 pL/h, 2550 pL/h, 2560 pL/h, 2570 pL/h, 2580 pL/h, 2590 pL/h, 2600 pL/h, 2610 pL/h, 2620 pL/h, 2630 pL/h, 2640 pL/h, 2650 pL/h, 2660 pL/h, 2670 pL/h, 2680 pL/h, 2690 pL/h, 2700 pL/h, 2710 pL/h, 2720 pL/h, 2730 pL/h, 2740 pL/h, 2750 pL/h, 2760 pL/h, 2770 pL/h, 2780 pL/h, 2790 pL/h, 2800 pL/h, 2810 pL/h, 2820 pL/h, 2830 pL/h, 2840 pL/h, 2850 pL/h, 2860 pL/h, 2870 pL/h, 2880 pL/h, 2890 pL/h, 2900 pL/h, 2910 pL/h, 2920 pL/h, 2930 pL/h, 2940 pL/h, 2950 pL/h, 2960 pL/h, 2970 pL/h, 2980 pL/h, 2990 pL/h, 3000 pL/h, 3010 pL/h, 3020 pL/h, 3030 pL/h, 3040 pL/h, 3050 pL/h, 3060 pL/h, 3070 pL/h, 3080 pL/h, 3090 pL/h, 3100 pL/h, 3110 pL/h, 3120 pL/h, 3130 pL/h, 3140 pL/h, 3150 pL/h, 3160 pL/h, 3170 pL/h, 3180 pL/h, 3190 pL/h, 3200 pL/h, 3210 pL/h, 3220 pL/h, 3230 pL/h, 3240 pL/h, 3250 pL/h, 3260 pL/h,

3270 pL/h, 3280 pL/h, 3290 pL/h, 3300 pL/h, 3310 pL/h, 3320 pL/h, 3330 pL/h, 3340 pL/h,

3350 pL/h, 3360 pL/h, 3370 pL/h, 3380 pL/h, 3390 pL/h, 3400 pL/h, 3410 pL/h, 3420 pL/h,

3430 pL/h, 3440 pL/h, 3450 pL/h, 3460 pL/h, 3470 pL/h, 3480 pL/h, 3490 pL/h, 3500 pL/h,

3510 pL/h, 3520 pL/h, 3530 pL/h, 3540 pL/h, 3550 pL/h, 3560 pL/h, 3570 pL/h, 3580 pL/h,

3590 pL/h, 3600 pL/h, 3610 pL/h, 3620 pL/h, 3630 pL/h, 3640 pL/h, 3650 pL/h, 3660 pL/h,

3670 pL/h, 3680 pL/h, 3690 pL/h, 3700 pL/h, 3710 pL/h, 3720 pL/h, 3730 pL/h, 3740 pL/h,

3750 pL/h, 3760 pL/h, 3770 pL/h, 3780 pL/h, 3790 pL/h, 3800 pL/h, 3810 pL/h, 3820 pL/h,

3830 pL/h, 3840 pL/h, 3850 pL/h, 3860 pL/h, 3870 pL/h, 3880 pL/h, 3890 pL/h, 3900 pL/h,

3910 pL/h, 3920 pL/h, 3930 pL/h, 3940 pL/h, 3950 pL/h, 3960 pL/h, 3970 pL/h, 3980 pL/h,

3990 pL/h, 4000 pL/h, 4010 pL/h, 4020 pL/h, 4030 pL/h, 4040 pL/h, 4050 pL/h, 4060 pL/h,

4070 pL/h, 4080 pL/h, 4090 pL/h, 4100 pL/h, 4110 pL/h, 4120 pL/h, 4130 pL/h, 4140 pL/h,

4150 pL/h, 4160 pL/h, 4170 pL/h, 4180 pL/h, 4190 pL/h, 4200 pL/h, 4210 pL/h, 4220 pL/h,

4230 pL/h, 4240 pL/h, 4250 pL/h, 4260 pL/h, 4270 pL/h, 4280 pL/h, 4290 pL/h, 4300 pL/h,

4310 pL/h, 4320 pL/h, 4330 pL/h, 4340 pL/h, 4350 pL/h, 4360 pL/h, 4370 pL/h, 4380 pL/h,

4390 pL/h, 4400 pL/h, 4410 pL/h, 4420 pL/h, 4430 pL/h, 4440 pL/h, 4450 pL/h, 4460 pL/h,

4470 pL/h, 4480 pL/h, 4490 pL/h, 4500 pL/h, 4510 pL/h, 4520 pL/h, 4530 pL/h, 4540 pL/h,

4550 pL/h, 4560 pL/h, 4570 pL/h, 4580 pL/h, 4590 pL/h, 4600 pL/h, 4610 pL/h, 4620 pL/h,

4630 pL/h, 4640 pL/h, 4650 pL/h, 4660 pL/h, 4670 pL/h, 4680 pL/h, 4690 pL/h, 4700 pL/h,

4710 pL/h, 4720 pL/h, 4730 pL/h, 4740 pL/h, 4750 pL/h, 4760 pL/h, 4770 pL/h, 4780 pL/h,

4790 pL/h, 4800 pL/h, 4810 pL/h, 4820 pL/h, 4830 pL/h, 4840 pL/h, 4850 pL/h, 4860 pL/h,

4870 pL/h, 4880 pL/h, 4890 pL/h, 4900 pL/h, 4910 pL/h, 4920 pL/h, 4930 pL/h, 4940 pL/h,

4950 pL/h, 4960 pL/h, 4970 pL/h, 4980 pL/h, 4990 pL/h, 5000 pL/h. In some embodiments, the second suitable media is injected at a flow rate of about 3,000 pL/h. Without being bond by theory, by filling the device with a suitable media prior to the introduction of a fluid containing sperm, the introduction of air bubbles is reduced or minimized as compared to directly injecting a fluid containing sperm to a dry channel 1010.

[0099] In certain example embodiments, the fluid containing sperm is injected at a flow rate of about 2000 to 5000 pL/h, or any value or range of values therein. In some embodiments, the fluid containing sperm is injected at a flow rate of about 2000 pL/h to/or 2010 pL/h, 2020 pL/h, 2030 pL/h, 2040 pL/h, 2050 pL/h, 2060 pL/h, 2070 pL/h, 2080 pL/h, 2090 pL/h, 2100 pL/h, 2110 pL/h, 2120 pL/h, 2130 pL/h, 2140 pL/h, 2150 pL/h, 2160 pL/h, 2170 pL/h, 2180 pL/h, 2190 pL/h, 2200 pL/h, 2210 pL/h, 2220 pL/h, 2230 pL/h, 2240 pL/h, 2250 pL/h, 2260 pL/h, 2270 pL/h, 2280 pL/h, 2290 pL/h, 2300 pL/h, 2310 pL/h, 2320 pL/h, 2330 pL/h, 2340 pL/h, 2350 pL/h, 2360 pL/h, 2370 pL/h, 2380 pL/h, 2390 pL/h, 2400 pL/h, 2410 pL/h, 2420 pL/h, 2430 pL/h, 2440 pL/h, 2450 pL/h, 2460 pL/h, 2470 pL/h, 2480 pL/h, 2490 pL/h, 2500 pL/h, 2510 pL/h, 2520 pL/h, 2530 pL/h, 2540 pL/h, 2550 pL/h, 2560 pL/h, 2570 pL/h, 2580 pL/h, 2590 pL/h, 2600 pL/h, 2610 pL/h, 2620 pL/h, 2630 pL/h, 2640 pL/h, 2650 pL/h, 2660 pL/h, 2670 pL/h, 2680 pL/h, 2690 pL/h, 2700 pL/h, 2710 pL/h, 2720 pL/h, 2730 pL/h, 2740 pL/h, 2750 pL/h, 2760 pL/h, 2770 pL/h, 2780 pL/h, 2790 pL/h, 2800 pL/h, 2810 pL/h, 2820 pL/h, 2830 pL/h, 2840 pL/h, 2850 pL/h, 2860 pL/h, 2870 pL/h, 2880 pL/h, 2890 pL/h, 2900 pL/h, 2910 pL/h, 2920 pL/h, 2930 pL/h, 2940 pL/h, 2950 pL/h, 2960 pL/h, 2970 pL/h, 2980 pL/h, 2990 pL/h, 3000 pL/h, 3010 pL/h, 3020 pL/h, 3030 pL/h, 3040 pL/h, 3050 pL/h, 3060 pL/h, 3070 pL/h, 3080 pL/h, 3090 pL/h, 3100 pL/h, 3110 pL/h, 3120 pL/h, 3130 pL/h, 3140 pL/h, 3150 pL/h, 3160 pL/h, 3170 pL/h, 3180 pL/h, 3190 pL/h, 3200 pL/h, 3210 pL/h, 3220 pL/h, 3230 pL/h, 3240 pL/h, 3250 pL/h, 3260 pL/h, 3270 pL/h, 3280 pL/h, 3290 pL/h, 3300 pL/h, 3310 pL/h, 3320 pL/h, 3330 pL/h, 3340 pL/h, 3350 pL/h, 3360 pL/h, 3370 pL/h, 3380 pL/h, 3390 pL/h, 3400 pL/h, 3410 pL/h, 3420 pL/h, 3430 pL/h, 3440 pL/h, 3450 pL/h, 3460 pL/h, 3470 pL/h, 3480 pL/h, 3490 pL/h, 3500 pL/h, 3510 pL/h, 3520 pL/h, 3530 pL/h, 3540 pL/h, 3550 pL/h, 3560 pL/h, 3570 pL/h, 3580 pL/h, 3590 pL/h, 3600 pL/h, 3610 pL/h, 3620 pL/h, 3630 pL/h, 3640 pL/h, 3650 pL/h, 3660 pL/h, 3670 pL/h, 3680 pL/h, 3690 pL/h, 3700 pL/h, 3710 pL/h, 3720 pL/h, 3730 pL/h, 3740 pL/h, 3750 pL/h, 3760 pL/h, 3770 pL/h, 3780 pL/h, 3790 pL/h, 3800 pL/h, 3810 pL/h, 3820 pL/h, 3830 pL/h, 3840 pL/h, 3850 pL/h, 3860 pL/h, 3870 pL/h, 3880 pL/h, 3890 pL/h, 3900 pL/h, 3910 pL/h, 3920 pL/h, 3930 pL/h, 3940 pL/h, 3950 pL/h, 3960 pL/h, 3970 pL/h, 3980 pL/h, 3990 pL/h, 4000 pL/h, 4010 pL/h, 4020 pL/h, 4030 pL/h, 4040 pL/h, 4050 pL/h, 4060 pL/h, 4070 pL/h, 4080 pL/h, 4090 pL/h, 4100 pL/h, 4110 pL/h, 4120 pL/h, 4130 pL/h, 4140 pL/h, 4150 pL/h, 4160 pL/h, 4170 pL/h, 4180 pL/h, 4190 pL/h, 4200 pL/h, 4210 pL/h, 4220 pL/h, 4230 pL/h, 4240 pL/h, 4250 pL/h, 4260 pL/h, 4270 pL/h, 4280 pL/h, 4290 pL/h, 4300 pL/h, 4310 pL/h, 4320 pL/h, 4330 pL/h, 4340 pL/h, 4350 pL/h, 4360 pL/h, 4370 pL/h, 4380 pL/h, 4390 pL/h, 4400 pL/h, 4410 pL/h, 4420 pL/h, 4430 pL/h, 4440 pL/h, 4450 pL/h, 4460 pL/h, 4470 pL/h, 4480 pL/h, 4490 pL/h, 4500 pL/h, 4510 pL/h, 4520 pL/h, 4530 pL/h, 4540 pL/h, 4550 pL/h, 4560 pL/h, 4570 pL/h, 4580 pL/h, 4590 pL/h, 4600 pL/h, 4610 pL/h, 4620 pL/h, 4630 pL/h, 4640 pL/h, 4650 pL/h, 4660 pL/h, 4670 pL/h, 4680 pL/h, 4690 pL/h, 4700 pL/h, 4710 pL/h, 4720 pL/h, 4730 pL/h, 4740 pL/h, 4750 pL/h, 4760 pL/h, 4770 pL/h, 4780 pL/h, 4790 pL/h, 4800 pL/h, 4810 pL/h, 4820 pL/h, 4830 pL/h, 4840 pL/h, 4850 pL/h, 4860 pL/h, 4870 pL/h, 4880 pL/h, 4890 pL/h, 4900 pL/h, 4910 pL/h, 4920 pL/h, 4930 pL/h, 4940 pL/h, 4950 pL/h, 4960 pL/h, 4970 pL/h, 4980 pL/h, 4990 pL/h, 5000 pL/h. In some embodiments, the fluid containing sperm is injected at a flow rate of about 3000 pL/h. In certain example embodiments, the fluid containing sperm is injected at a flow rate that minimizes dilution of the fluid.

[0100] In certain example embodiments, the first suitable media is injected at a flow rate of about 100 to 2,000 pL/h or any value or range of values therein. In some embodiments, the first suitable media is injected at a flow rate of about 100 pL/h to/or 110 pL/h, 120 pL/h, 130 pL/h, 140 pL/h, 150 pL/h, 160 pL/h, 170 pL/h, 180 pL/h, 190 pL/h, 200 pL/h, 210 pL/h, 220 pL/h, 230 pL/h, 240 pL/h, 250 pL/h, 260 pL/h, 270 pL/h, 280 pL/h, 290 pL/h, 300 pL/h, 310 pL/h, 320 pL/h, 330 pL/h, 340 pL/h, 350 pL/h, 360 pL/h, 370 pL/h, 380 pL/h, 390 pL/h, 400 pL/h, 410 pL/h, 420 pL/h, 430 pL/h, 440 pL/h, 450 pL/h, 460 pL/h, 470 pL/h, 480 pL/h, 490 pL/h, 500 pL/h, 510 pL/h, 520 pL/h, 530 pL/h, 540 pL/h, 550 pL/h, 560 pL/h, 570 pL/h, 580 pL/h, 590 pL/h, 600 pL/h, 610 pL/h, 620 pL/h, 630 pL/h, 640 pL/h, 650 pL/h, 660 pL/h, 670 pL/h, 680 pL/h, 690 pL/h, 700 pL/h, 710 pL/h, 720 pL/h, 730 pL/h, 740 pL/h, 750 pL/h, 760 pL/h, 770 pL/h, 780 pL/h, 790 pL/h, 800 pL/h, 810 pL/h, 820 pL/h, 830 pL/h, 840 pL/h, 850 pL/h, 860 pL/h, 870 pL/h, 880 pL/h, 890 pL/h, 900 pL/h, 910 pL/h, 920 pL/h, 930 pL/h, 940 pL/h, 950 pL/h, 960 pL/h, 970 pL/h, 980 pL/h, 990 pL/h, 1000 pL/h, 1010 pL/h, 1020 pL/h, 1030 pL/h, 1040 pL/h, 1050 pL/h, 1060 pL/h, 1070 pL/h, 1080 pL/h, 1090 pL/h, 1100 pL/h,

1110 pL/h, 1120 pL/h, 1130 pL/h, 1140 pL/h, 1150 pL/h, 1160 pL/h, 1170 pL/h, 1180 pL/h,

1190 pL/h, 1200 pL/h, 1210 pL/h, 1220 pL/h, 1230 pL/h, 1240 pL/h, 1250 pL/h, 1260 pL/h,

1270 pL/h, 1280 pL/h, 1290 pL/h, 1300 pL/h, 1310 pL/h, 1320 pL/h, 1330 pL/h, 1340 pL/h,

1350 pL/h, 1360 pL/h, 1370 pL/h, 1380 pL/h, 1390 pL/h, 1400 pL/h, 1410 pL/h, 1420 pL/h,

1430 pL/h, 1440 pL/h, 1450 pL/h, 1460 pL/h, 1470 pL/h, 1480 pL/h, 1490 pL/h, 1500 pL/h,

1510 pL/h, 1520 pL/h, 1530 pL/h, 1540 pL/h, 1550 pL/h, 1560 pL/h, 1570 pL/h, 1580 pL/h,

1590 pL/h, 1600 pL/h, 1610 pL/h, 1620 pL/h, 1630 pL/h, 1640 pL/h, 1650 pL/h, 1660 pL/h,

1670 pL/h, 1680 pL/h, 1690 pL/h, 1700 pL/h, 1710 pL/h, 1720 pL/h, 1730 pL/h, 1740 pL/h,

1750 pL/h, 1760 pL/h, 1770 pL/h, 1780 pL/h, 1790 pL/h, 1800 pL/h, 1810 pL/h, 1820 pL/h,

1830 pL/h, 1840 pL/h, 1850 pL/h, 1860 pL/h, 1870 pL/h, 1880 pL/h, 1890 pL/h, 1900 pL/h,

1910 pL/h, 1920 pL/h, 1930 pL/h, 1940 pL/h, 1950 pL/h, 1960 pL/h, 1970 pL/h, 1980 pL/h, 1990 pL/h, 2000 pL/h.In some embodiments, the first suitable media is injected at a flow rate of about 350 pL/h.

[0101] In certain example embodiments, the method further includes performing in vitro fertilization (IVF) utilizing competent sperm separated by a microfluidic device of the present disclosure. In some embodiments, the IVF is chamber based IVF. Methods of IFV in which the sperm or sperm containing composition prepared using a microfluidic device of the present disclosure are generally known in the art.

[0102] In certain example embodiments, one or more steps are automated.

EXAMPLES

[0103] Now having described the embodiments of the present disclosure, in general, the following Examples describe some additional embodiments of the present disclosure. While embodiments of the present disclosure are described in connection with the following examples and the corresponding text and figures, there is no intent to limit embodiments of the present disclosure to this description. On the contrary, the intent is to cover all alternatives, modifications, and equivalents included within the spirit and scope of embodiments of the present disclosure. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to perform the methods and use the probes disclosed and claimed herein. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in °C, and pressure is at or near atmospheric. Standard temperature and pressure are defined as 20 °C and 1 atmosphere.

Example 1

Introduction

[0104] Since its development assisted reproductive technologies, like in vitro fertilization (IVF), have allowed millions of human couples to conceive ¹ and increased the breeding efficiency of other mammalian species such as cattle. ² Early studies of IVF were more focused on the oocyte to improve the outcomes, but it was quickly found that the chance of fertilization in rabbits and mice increased dramatically when oocytes were exposed to in vivo capacitated sperm. ³ Sperm sorting was found to have an important role on fertilization efficiency and has since become a crucial part of efforts to improve the IVF process. ^{4 5}

[0105] Traditionally, sorting is done by washing semen through several rounds of centrifugation. ⁶ However, centrifugation techniques have been reported to cause damage to sperm DNA. ⁷ They do not select sperm similar to how they are naturally selected in FRT. ⁸ Recently researchers started investigating microfluidic methods to sort spermatozoa and began introducing the resulting devices into clinical settings. ⁹ These early clinical trials showed that sperm separation via microfluidics imposes less harmful effects on the sperm membrane integrity, mitochondrial activity, and morphology as well as reducing DFI. ⁵ ’ ^{6 9-11}

[0106] Much of the data about how spermatozoa find the oocyte in vivo, and the role of the FRT in the spermatozoa’s journey, are controversial. ^{12 13} For instance, Miki and Clapham ¹² argue that the fluid flow in FRT after coitus is sufficient for long-range sperm guidance. On the other hand, Hino and Yanagimachi ¹³ discuss the effect of active peristaltic contraction on the hydrodynamics of FRT contradicts any guiding mechanism for sperm finding the oocyte. Despite a lack of direct experimental evidence, many agree that the hydrodynamics, topology and chemical composition of the FRT place barriers in the spermatozoa’s path. ^{4 14} Spermatozoa are equipped with multiple features to overcome these barriers, and the FRT and the sperm have co-evolved for ideal selection conditions. ¹⁵ Microfluidic devices have made it easier to study and select for sperm features by implementing gentle flows, ¹⁶ ’ ¹⁷ filter-like components, ^{18 19} and the possibility of automation. ²⁰ These devices also include investigations of the four navigational mechanisms of sperm cells; thigmotaxis (swimming along boundaries), thermotaxis (swimming against the direction of a temperature gradient), chemotaxis (swimming against chemical gradients) and rheotaxis (swimming against the flow). ⁴ Chemotaxis and thermotaxis are short range mechanisms that guide sperm toward the egg in the oviduct and are only active in approximately 10% of the sperm population in mammalian species. ^21-23 Although sperm chemotaxis in mammals remains controversial, ^{24 25} recent strides toward sorting spermatozoa based on chemotaxis and thermotaxis has led to higher quality spermatozoa in humans. ²⁶

[0107] Cells are attracted to the walls when swimming in confined spaces. ²⁷ This hydrodynamic feature suggests that a significant amount of sperm motion takes place along the microenvironment walls within the FRT. ²⁸ This feature is proposed to lead spermatozoa to the fertilization site through the narrow crevices of the FRT ²⁹ and could be used for selection of highly motile spermatozoa. ^{30 31} Aligned with this, studies on sperm sorting based on boundaryfollowing characteristics showed improvement both in human and bovine spermatozoa in their motility parameters and DNA integrity. ³² ’ ³³ Although the sorting time is reduced using these methods, the fertilization ability of the sperm samples was not tested.

[0108] The mucosal fluid of the FRT, other post-copulation secretions, and ciliary motion, generate a robust flow through the narrow lumen of the mammalian oviduct from ovaries to the uterus. ¹² This fluid flow may guide or select sperm cells via rheotaxis. ^{12 34} Several microfluidic platforms have been designed to select spermatozoa based on rheotaxis using either a corral-based system or a platform with contraction and expansion channels to induce rheotaxis. ^{35 36} Other platforms contain a collection chamber and a loading reservoir connected to each other via a rheotaxis channel to obtain rheotactically capable spermatozoa. ^{10 37-39} These studies have explored sperm quality improvement via rheotaxis at one shear rate; the effect of modulating the shear rate has been recently reported, ⁴⁰ but its implication on sperm selection and embryonic development is still not well understood. Also the sperm yield in all the rheotaxis based sperm separation platforms as compared to the input sample, still needs improvement.

[0109] Previously, Applicant introduced the rheotaxis quality index (RHEOLEX) as a potential biomarker for fertility screening: the higher the number of spermatozoa with higher rheotaxis capacity, the higher the pregnancy outcomes. ⁴¹ Applicant has now designed a microfluidic channel to separate motile spermatozoa based on their rheotaxis capability and then how spermatozoa selected at different flow rates impacts early embryonic development. Applicant confirmed that the kinematic features of the separated spermatozoa (speed, beating amplitude and frequency) were tuned by the intensity of the flow rate. At higher flow rates only spermatozoa with high speed were selected at the cost of the total number of sperm. The DNA integrity of the selected spermatozoa was evaluated. Applicant selected ~2 million bovine spermatozoa and used these spermatozoa to perform chamber-based IVF. Applicant then compared the development of embryos resulting from spermatozoa selected at various speeds with each other and with sperm sorted by centrifugation. Under optimum conditions, selecting spermatozoa at high flow rates resulted in 23 % improvement in blastocyst rate when compared to centrifugation-based sperm sorting. Results and discussion

1 -Design layout and operation

[0110] To ensure that spermatozoa are guided upstream for rheotaxis-based sperm separation, the device was designed to have regions of high and low shear rates. Applicant designed strictures, using triangular prisms in a microfluidic channel: a network of 3 rows of 42 parallel prisms (FIG. 1A-1C). These strictures were inspired by the constrictions present in the uterotubal junction (UTJ) in the FRT of many mammalian species. ⁴² Curved veins around the triangular prisms and straight veins between each group of curved veins and prisms, were used to avoid trapping air bubbles while loading the device (FIG. 1A). ⁴³ The main channel is 180 pm deep, while the cross section of the veins run the width of the device, they are only 40 pm deep and 80 pm wide such that they create a bump for the media and sperm to flow over and keep air bubbles from forming (FIG. 1A and IB). The total device capacity is 80 pL of semen. Applicant used 3 rows of strictures to increase the chance of spermatozoa being oriented upstream reducing sperm loss due to a reorientation lag. But increasing the number of rows more than that would reduce the capacity of the device.

[OHl] The device is operated by loading the semen sample through the outlet and then washing the semen with media from the inlet, which sweeps the semen toward the outlet. Debris and nonmotile spermatozoa are washed away with the media and sperm that are capable of rheotaxis remain in the device after washing. Spermatozoa have multiple opportunities to pass through the strictures and be guided upstream, provided they have enough strength to swim against the flow (FIG. 7).

[0112] To estimate the required time for the washing step, a computational fluid dynamics simulation for a 2-dimensional layout of our device was done by ignoring the guiding veins. The contours of the relative concentration of media in the chip at 210 s intervals for a flow rate of 300 pL h' ¹ indicates that the media (red contours) washes the middle of the channel much faster than the regions near the walls (FIG. ID). C represents semen, which enters the device at a concentration of Ci. C can vary between 0 (media) and Ci, therefore the ratio of C/Ci varies between 0 and 1. Applicant attributed this to a lower velocity of the fluid near the boundaries. Thus, nonmotile sperm and debris near the side walls will take longer to be washed.

[0113] Our simulation indicates that the volume fraction ( >) of semen remaining in the chip decreases over time and with increasing flow rate (FIG. 2A). The time needed so that <[) = 3, 5, 10 and 15% versus various flow rates is then calculated (FIG. 2B). The diagram of time required for washing flattens for higher flow rates and it exponentially increases for smaller flow rates. Applicant chose the times for j> = 10% to ensure that theoretically 90% of debris and nonmotile spermatozoa would be discharged. Otherwise, to reach 95% removal (</> = 5%), the washing time nearly doubles that of (f = 10%. Increasing the washing time beyond this does not help with cleaning the sample and it over-exposes the spermatozoa to additional shear which might have harmful effects.

[0114] While washing, the motile spermatozoa trajectories are affected by the presence of the prisms. Spermatozoa starting either from the right-side within the stricture, or outside of the stricture, swim to the left side of the stricture against the flow (FIG. 2C). The shear rate contours on the xy-plane near the top wall and the xz-plane in the middle of the stricture show that right at the point of the strictures the shear rate is zero. However, near the top or the bottom walls (z = 18 pm, shown in FIG. 2D), and 20 pm before and after the strictures, the shear rate is high enough (3 s' ¹ ) to cause the spermatozoa to reorient upstream. ^{35 44} The reason that the shear rate at the strictures is zero is that the velocity profile is flat in the middle plane at the strictures due to symmetry (FIG. 8). Therefore, if the spermatozoa are moving near the left side of the prism and enter the stricture, they are dragged downstream, but once they reach the space between two prisms, they can reorient upstream.

[0115] The average shear rate (y) at the stricture, the red line at z = 18 pm (FIG. 2D), linearly changes as a function of the flow rate (FIG. 2E) so that shear rate (s' ¹ ) = 0.02 (h pL' ¹ s' ¹ ) x flow rate (pL h' ¹ ). Our results are expressed in terms of change in shear rate, as opposed to flow rate, as shear rate allows for cross comparison of results if, for example, the depth of the device is increased to accommodate a higher volume of semen. Expressing the results in terms of flow rate would introduce inconsistencies in the resulting rheotaxis information since flow rate varies by volume.

Accumulation of Sperm at the Strictures

[0116] Human and bovine spermatozoa undergo rheotaxis between shear rates of approximately 3 to 10 s' ¹ . Applicant confirmed this range by quantifying the number of human spermatozoa that accumulate at the strictures within the shear rate range of 2 to 46 s' ¹ (FIG. 3A). Applicant focused the microscope at one of the strictures and monitored the human sperm rheotaxis at various shear rates. To quantify the sperm accumulation in the strictures, Applicant has used the algorithm Applicant developed for RHEOLEX. ⁴² This algorithm calculates the changes in pixel intensity for consecutive frames and obtains the average intensity for 200 images to generate signal contours (FIG. 3A). The signal is then averaged vertically for each shear rate along the x-axis after eliminating the prisms from the image (FIG. 3B).

[0117] At low shear rates (2 s' ¹ ) Applicant found no accumulation since the shear rate is not sufficient to induce rheotaxis (Fig. 3D). This is consistent with the reported minimum shear rate for rheotaxis of bovine spermatozoa, 3 s' ¹ . ³⁵ Applicant found that for human spermatozoa, the RHEOLEX signal reaches a maximum at 9.2 s' ¹ and decreases as the shear rate increases. The maximum signal intensity occurs at a distance from stricture (x) indicating that higher shear rates drag the spermatozoa downstream. The signal decreases to 0.3 at x/L = 0.6, where x is the distance from the contraction point between two prisms and L is the total length of the prism from contraction point to full expansion point. Although these images were taken from only two strictures in the middle row of the device, the velocity profile and shear rate in all of the strictures is the same except for along the boundaries (FIG. 9).

[0118] Using a one-dimensional convective transport of active particles Applicant confirmed the accumulation of spermatozoa at the stricture. Applicant found that the accumulation of spermatozoa at the strictures can be simulated numerically only by tracking the direction of the spermatozoa’s motion and their location in a similar stricture geometry (FIG. 11A-11E). Previously, bacterial accumulation at similar contraction-expansion geometries have been studied using another one-dimensional approach. ⁴⁵

[0119] Spermatozoa accumulate at the strictures under medium shear rates in the range of 3 - 11 s' ¹ . At shear rates greater than 11 s' ¹ , the increased fluid velocity prevents spermatozoa from undergoing rheotaxis. While faster, stronger spermatozoa may stay in place at higher shear rates, the mechanical shear of the fluid at higher flow rates could cause damage. Therefore, sperm motility parameters determined by computer assisted sperm analysis (CASA), and other semen parameters such as DFI, and membrane integrity were used to evaluate selected sperm quality. See e.g., FIG. 4A-4F. For human spermatozoa, semen parameters were evaluated including sperm concentration, motility, normal morphology, and sperm chromatin fragmentation (SCF).

Characterization of Separated Human Sperm

[0120] Human semen analysis was carried out manually on a raw sample. Semen parameters were also accessed on selected spermatozoa isolated at various shear rates as well as conventional density gradient centrifugation (DGC)-processed spermatozoa. To further characterize the performance of our device, Applicant calculated the sperm retrieval efficiency (RE).

[0121] A comparison was made between the same semen parameters among raw sample, DGC and rheotaxis selection (Table 1). Morphology and motility increased, whereas concentration and SCF decreased, indicating that significantly superior spermatozoa resulted from rheotaxis-based selection but at the cost of concentration.

Table 1. Comparison of the selected sperm quality with that of raw sample and DGC.

*DGC vs selected spermatozoa (overall), paired t-test

[0122] In subanalysis, Table 2 shows the sperm parameters with respect to shear rate. The concentration decreased with increasing the shear rate while motility increased and then decreased. RE was 42% at the maximum that occurred at a shear rate 5 s' ¹ which is far more than the current rheotaxis-based sperm separation methods and minimum SCF was achieved at the same shear rate. Very similar to bovine spermatozoa, there is an optimum shear rate but since human sperm swims slower than bovine sperm, the optimum shear rate is also lower. Morphology did not show any changes with various shear rates. (Table 3 has additional details about data shown in Table 2.)

Table 2. Clinical quality parameters of rheotaxis-based human sperm selected at various shear rates. *ANOVA, comparison between shear rate 5 s' ¹ and 3 s' ¹

[0123] In an additional analysis, Applicant compared the proportion of X- and Y-bearing spermatozoa in relation to varying shear rates. Sperm cells possess a slightly different mass, depending on their gonosomal component. It is well-documented that the Y chromosome is smaller ⁴⁶ and, as such, would have a lower mass than the X; therefore, Applicant expected Y- bearing spermatozoa to possess a higher velocity and agility to perform rheotaxis. As the selection shear rate increased from 3 to 9 s’ ¹ , Applicant observed a gradual skew towards a greater proportion of Y-bearing spermatozoa (2-3%) (Table 4). These findings suggest that implementation of considerably higher shear rates may further skew a sperm population towards those carrying a Y chromosome. Although linear regression of the Y chromosome percentage and F-test showed no statistical significance (p-value = 0.1282 > 0.05, FIG. 12), this data is from limited samples and observations with a marginal difference. Applicant believes that more experiments are needed to confirm sex bias of the swimming velocity of Y- bearing spermatozoa.

Table 4. Fluorescent in situ hybridization (FISH) results in human sperm separation for two patients.

Sample ID Patient X % Y %

Raw Semen 1 52 48

3 s’ ¹ 1 52 48

5 s’ ¹ 1 51 49

7 s’ ¹ 1 52 48

9 s’ ¹ 1 51 49

Raw Semen 2 51 49

3 s’ ¹ 2 51 49

5 s’ ¹ 2 49 51

7 s’ ¹ 2 49 51

9 s’ ¹ 2 48 52

Characterization of the separated bovine spermatozoa

[0124] Sperm quality varies with separation conditions. Applicant used CASA and DFI parameters to evaluate quality. Applicant evaluated three types of bovine samples: raw semen; spermatozoa sorted via centrifugation (IVF control group); and spermatozoa selected in our microfluidic platform. Applicant considered the results of the control and raw as categorical variables, but since the shear rate is a continuous variable a regression model was fitted to the data to show the trend. The minimum shear rate for rheotaxis behavior is greater than 3 s’ ¹ , therefore, Applicant chose the optimum sorting conditions for bovine spermatozoa to be within the shear rate range of 3 to 11 s’ ¹ .

[0125] In CASA the head centroid is tracked and based on the head trajectory an averaged path is calculated. The velocity of the spermatozoa moving along this averaged path is called averaged-path velocity (VAP). The faster the speed of the spermatozoa the higher the VAP would be. VAP shifted to higher velocities as the shear rate increased but reached a maximum value. Applicant confirmed this trend in bovine spermatozoa by CASA for more than 150 sperm cells randomly tracked from among the hundreds of thousands of spermatozoa selected using our microfluidic device (FIG. 10). However, the control group showed a wide range of VAP distributions and had no significant differences with VAP at any of our shear rates or that of raw sample.

[0126] The total motility percentage of the samples increased as the shear rate increased up to 7 s’ ¹ , then decreased at higher shear rates as the concentration of sorted samples drops below 5% of the initial sample (FIG. 4B and 4D). With the concentration of the sorted sample and the motility percentage from CASA. RE was roughly 40% for y = 3 s’ ¹ and 5 s’ ¹ , but RE decreased to 28% for 7 s’ ¹ (FIG. 13). As expected from our calculations, the overall RE decreased as the washing flow rate increased. Total sperm count also significantly decreases by increasing shear rate from 1.72 million at shear rate 3 s’ ¹ to 0.31 million at shear rate 5 s’ ¹

(Table 5)

[0127] The deviation of the sperm head centroid from its averaged path is called amplitude of lateral head displacement (ALH); at higher amplitudes of beating, ALH is higher. In our experiments ALH decreased as the shear rate increased up to 7 s’ ¹ and increased thereafter. There is not a significant difference between ALH of the control and raw samples.

[0128] And finally, the frequency by which the head trajectory crosses the averaged path determines beat cross frequency (BCF). In theory, BCF and ALH are inverse to each other with respect to shear rate and our results follow this pattern (FIG. 4C, 4E). Further, the BCF of the control significantly increased over that of the raw sample.

[0129] VAP, motility, ALH and BCF are only characteristics of sperm movement. After the fusion of a spermatozoon and an oocyte, the zygote checks the genome by its correction mechanism. If spermatozoa carry a break in its DNA, embryonic development comes to a halt until the break is repaired which delays the growth. DNA breaks are quantified by DFI. For all sorted samples, DFI is lower than raw semen, with a minimum DFI found for samples selected at y = 7 s’ ¹ which is equivalent to the maximum and minimums of ALH, BCF, and motility.

[0130] In general, all of the quality assessments point to higher quality for both the control

(centrifuged) and the spermatozoa selected based on rheotaxis over raw sample. Also, in selected spermatozoa, ALH, BCF and motility exhibit optimum values at y = 7 s' ¹ . (Table 6 has the fitted lines for Fig. 4A-4F.)

Bovine IVF procedure and embryonic development

[0131] After sperm sorting, conventional IVF was performed using spermatozoa sorted via centrifugation (control) and spermatozoa separated by our microfluidic platform at shear rates of 3 s' ¹ , 5 s' ¹ and 7 s' ¹ . As the shear rate increased, fewer spermatozoa are able to swim against the flow, therefore, the concentration of the selected spermatozoa decreased significantly as the shear rate increased (FIG. 5B) For the control, the method is adjusted so that the concentration of sorted semen is closer to the raw sample.

[0132] The concentration of samples was measured using a NucleoCounter cell counter. The concentration of insemination dose was normalized by dilution of the sperm samples with warm media to 10 M mL' ¹ . A volume of VNC=600/C pL of selected/sorted sample, where C is the concentration of spermatozoa determined using the NucleoCounter, was added to 60 - VNC pL of media to produce 60 pL of samples with concentration of 10 M mL' ¹ . Normalized concentration samples were prepared for each group and 50 pL of the normalized sample was added to the chamber containing mature cumulus oocyte complexes (COCs) (FIG. 5A). Normalization is necessary because Applicant wanted to see only the effect of shear rate on the cleavage and blastocyst rates.

[0133] Applicant assessed the quality of the device separated spermatozoa by measuring the plasma membrane integrity. An intact plasma membrane will be able to keep non- membrane permeable stains, such as propidium iodide, out of the cell; whereas, in spermatozoa with damaged plasma membrane the stain will permeate the cell and stain the DNA in the nucleus. Some of the spermatozoa in raw semen may already have damage to their membrane reducing their success in the following IVF; thus, finding a method that effectively removes the damaged cells is important to successful IVF outcomes. Applicant found that for our device, the membrane damage was slightly decreased by about 4 % in comparison to the control (Fig. 5C). There was no significant difference between shear rates and membrane damage, leaving out the effect of mechanical shear on the integrity of the plasma membrane.

[0134] The embryos produced through IVF for spermatozoa separated at y = 3 s' ¹ and the control were smaller than those of spermatozoa separated at higher shear rates. At higher shear rates the embryos often included hatched or larger size embryos (FIG. 6A). There is no significant difference between the cleavage rate in control and any of the microfluidic-based sorting (FIG. 6B) The blastocyst rate, however, showed a significant difference between the control and spermatozoa separated at y = 7 s’ ¹ ; as the shear rate increases, the blastocyst rate also increases (FIG. 6C and 6D) This trend seems to reach a plateau as no significant difference between the blastocyst rate of 5 s’ ¹ and 7 s’ ¹ is observed, while there is a significant difference between shear rates of 3 s’ ¹ and 5 s’ ¹ and also between 3 s’ ¹ and 7 s’ ¹ .

Discussion

[0135] Previously Applicant developed a method of characterizing mammalian sperm rheotaxis and concluded that spermatozoa from bulls with higher fertilization rates show higher rheotaxis ability. ⁴¹ Here, Applicant postulated that sorting spermatozoa with higher rheotaxis capability could result in better fertilization outcomes in IVF cycles. To test this, Applicant designed and characterized a high throughput microfluidic platform to separate spermatozoa with various rheotaxis abilities within a network of parallel strictures. The semen was loaded into the chip followed by media at different flow rates. The media swept away both debris and low motility spermatozoa. The spermatozoa that were capable of rheotaxis remained in the chip. By tuning the flow rate, thus tuning the shear rate in the device, rheotactically competent spermatozoa were separated. The spermatozoa, selected based on various shear rates, were then used in IVF to assess their fertilization ability.

[0136] Previous researchers have shown that a minimum shear rate is required for rheotaxis in both human and bovine sperm. ⁴⁴ In our microfluidic platform we found that at shear rates below 3 s’ ¹ , there was no accumulation of spermatozoa at the stricture. As the shear rate increases, rheotaxis is induced and spermatozoa are oriented upstream once they are in the shear zone of the stricture (the open triangle space between the prisms). If the free-swimming velocity of the spermatozoa is higher than the fluid velocity at the stricture, the spermatozoa surpass the fluid drag force and moves upstream (FIG. 3B and 3D). This will cause faster sperm locomotion to be redirected toward the inlet area and lead to their accumulation over time (Movie SI of Yaghoobi et al., Faster sperm selected by rheotaxis leads to superior early embryonic development in vitro, In review., which is incorporated by reference as if expressed in its entirety herein). This redirection does not occur without fluid flow; defying the ratchet effect ²⁷ due to prisms’ shape in guiding the spermatozoa to the inlet.

[0137] At areas farther from the stricture, x > L or x < 0 (FIG. 3C), the velocity and the shear rate decrease and the spermatozoa follow their free-swimming motion again. Sperm accumulation occurs at x = 0; the highest signal intensity is observed at vicinity of x = 0 ⁺ at shear rates between 3 s' ¹ to 11 s' ¹ (FIG. 3A). However, as the shear rate increases, the drag force on the spermatozoa increases and the maximum peak of the signal sweeps downstream to the point that the signal intensity barely spikes. But the near zero intensity of sperm signals at x < 0 for shear rates of higher than 11 s' ¹ is an indication that spermatozoa cannot pass the barrier under these conditions. So, it is best to perform separation at shear rates 3 to 11 s' ¹ .

[0138] The CASA parameters and the DFI of the separated bovine spermatozoa showed the best sperm quality at y = 7 s' ¹ . That is, sperm speed becomes flat at y = 7 s' ¹ , DFI reached a minimum of 3% at this shear rate and ALH and BCF showed their minimum and maximum respectively. In comparison, human spermatozoa swims at lower speeds and our analysis of human sperm SCF showed that optimum quality occurs at y = 5 s' ¹ . Although the concentration of sorted spermatozoa declines with higher shear rate and VAP increases, the motility percentage reaches a maximum at y = 7 s' ¹ . The increase in motility is due to the washing effect, but the decrease in motility for shear rate more than 7 s' ¹ is attributed to the dilution effect. ⁴⁷ Applicant observed that as the seminal fluid content becomes diluted in the media, the spermatozoa’s affinity for the CASA chamber walls increased; this resulted in sperm head tethering to the walls and many motile spermatozoa were counted as nonmotile. However, those spermatozoa that did not stick to the walls, had higher VAP. Single VAP distributions versus shear rate (FIG. 10) had a bimodal distribution: one peak about 50 pm s' ¹ and the other at 135 pm s' ¹ . This distribution resembled a combination of raw sample’s VAP and that of selected sample. As the shear rate increases the intensity of the first peak weakens and that of the second peak becomes stronger. This is due to the spermatozoa that linger near the side walls that are not swept away as effectively as the spermatozoa in the middle of the channel at lower

Y (FIG. ID)

[0139] At y = 7 s’ ¹ , the ALH is at a minimum and the BCF is at its maximum. Nagata et al. showed that there are two types of sperm movement pattern in their rheotaxis sorted spermatozoa; transitional sinuous (TS) where sperm head sways laterally while moving forward, and progressive non-sinuous (PN) where sperm head stays on average trajectory while moving forward. ¹⁰ TS features larger ALH and lower BCF and VAP compared to the PN type. Our data indicate that as the shear rate increases from 3 to 7 s’ ¹ sorted spermatozoa shifts from TS to PN (FIG. 4C and 4E). The lateral movement exhibited by TS motion would allow the head of the spermatozoa to be exposed to the higher fluid velocity in the center of the stricture and be swept downstream, whereas spermatozoa exhibiting PN would remain closer to the wall and experience lower flow.

[0140] Nagata et al. also reported higher incidence of artificial insemination (Al)-related pregnancies in the case of TS type movement rather than PN. ¹⁰ This seems to be in contradiction to our IVF results which indicate as the shear rate increased, associated with PN type movement, the blastocyst rate increased significantly. However, since Nagata et al. performed Al, the FRT may play an important role in sperm selection whereas in our experiments, spermatozoa meet the oocytes directly. Also, In Al, the PN spermatozoa might undergo untimely hyperactivation before reaching the oocyte (transition to TS) and lose the chance of successful fertilization. However, in IVF, the activation of oocytes and the spermatozoa exposure to the capacitation media is controlled. Also, none of our separation experiments took more than two hours; less than required time for sperm capacitation. ⁴⁸ [0141] Up to the Y = 7 s’ ¹ the DFI decreased, however beyond 7s’ ¹ , there was a slight increase in DFI (although not statistically significant). The lack of damage to the plasma membrane means that the reduced DFI at higher shear rates is likely due to apoptosis in nonmotile sperm cells present in the selected sample (FIG. 5C). The trend of no change in membrane damage versus shear rate rules out the effect of shear damage causing the increase in DFI for shear rates more than 7 s’ ¹ . Therefore, the increase in DFI could be explained by a high level of reactive oxygen species (ROS) available in the sperm cells with higher velocities. ⁹ Thus, the spermatozoa separated at very high shear rates could have higher DFI. [0142] The fertilization rates of various groups showed no change except for 3 s' ¹ and 7 s’ ¹ which is merely due to the very high motility of the 7 s’ ¹ group despite the lower DFI of the spermatozoa at 7 s’ ¹ . As long as the motility of spermatozoa is not impaired, fertilization rates have been shown to be independent of high DFI because the paternal genome does not participate in the early stages of embryo development. ⁴⁹ Blastocyst formation, however, is affected by DNA fragmented spermatozoa because at this stage the paternal genome is involved. Applicant attributes the higher blastocyst rate of group 7 s’ ¹ with respect to the others to the lower DFI in the selected sample and not merely the motility percentage because the insemination dose was maximized at 10,000 spermatozoa per COC. Further, the motility of the group 5 s’ ¹ is lower than the group 7 s’ ¹ and yet the blastocyst rate does not vary significantly. Applicant also observed higher incidence of hatching and larger embryos within the group 7 s’ ¹ indicating a higher developmental rate. This higher rate could, in part, be attributed to oocyte’s DFI correction mechanism in group 5 s’ ¹ which delays the development rate and also optimizes the level of ROS in the fertilizing sperm which possibly regulates the metabolism of resulting embryos. Further experiments must be planned to characterize this observation and distinguish the underlying mechanisms.

[0143] Based on the results presented here, Applicant can conclude that our platform is a very efficient and suitable method of sperm separation in bovine and human based on rheotaxis since it resulted in less DNA damage and higher speed. Applicant e found the optimum of the performance of the device to be around shear rate of 7 s’ ¹ for bovine and 5 s’ ¹ for human spermatozoa. Further, by performing IVF cycles for nearly 2400 oocytes Applicant confirmed that as Applicant selected for higher rheotaxis ability in selected spermatozoa, the fertilization increases accordingly.

[0144] Since the concentration of sorted semen can be low for low motility samples, this method might not be optimal in the cases of male infertility due to low sperm counts or low motility, however, it can be useful in conventional IVF of non-male factors. Further, the idea of parallel strictures can be extended to higher capacity devices to accommodate low concentration samples to select the most competitive spermatozoa for the ICSI process.

Materials and Methods

Device fabrication

[0145] Standard photolithography was used for fabrication of the mold. ⁵⁰ The mold consisted of two layers. For the first layer SU-8 2100 was poured on silicon wafers (University Wafer) and spun at 3500 rpm for 30 s and baked for 5 and 35 min over 65 °C and 95 °C, respectively. Applicant exposed the basked masks to 365 nm UV light through the laser-printed patterns mask for 30 s and subsequently baked on 95 °C for 15 min to create the 140 pm layer. The second layer was fabricated using SU-8 2025 by spinning at 3000 rpm for 30 s and exposure time of 20 s to add a 40 pm layer with corresponding patterns. The two layers were submerged in SU8-Developer for 30 min before hard-baking.

Device loading and sperm collection

[0146] The device should be loaded from the outlet with media (BO-Semen Prep) with the flow rate of 3,000 pL h' ¹ for 70 pL and then the flow rate reduced to 350 pL h' ¹ for the area with the supports to reduce air entrapment. Overall, it takes 3 min to load each device with media. This loading media contained 0.2 % bovine serum albumin (BSA) to avoid tethering the sperm head to the glass and PDMS walls. After this 120 pL of semen was injected from the inlet via a pipette to replace the media and fill the device with semen. This step should be done quickly in order to avoid semen dilution (Fig. 7). After the washing step, the remaining sample was collected by setting the pipette on 80 pL and aspirating the sample from the inlet.

Numerical simulation of washing step

[0147] COMSOL multiphysics software 5.4a was used to solve the coupled fluid velocity and pressure as well as transport of diluted species utilizing the finite element method.

[0148] where C is the local concentration of semen in the chip, u is the fluid velocity vector, and p is the pressure, p = 1 mPa and D = 10' ⁹ m ² s' ¹ are viscosity and diffusion coefficient, respectively. For the calculation of the volume fraction of semen in the chip ( >) the following formula was used. b = H - ^J chi.p area C dA ( ^v 4) ⁷

[0149] where dA is the element of the surface in the integral and H is the depth of the device. Human sperm morphology and motility

[0150] Semen samples were incubated in 37 °C for 15 min to allow liquefaction. Semen analysis was performed manually in a Makler ® counting chamber according to WHO manual.51 Sperm concentration, motility, and morphology were evaluated on raw, DGC- processed (according to WHO manual), 51 and rheotactically selected spermatozoa.

Computer assisted sperm analysis

[0151] Motility, concentration, progressive motility, VSL, VCL, VAP, STL, LIN, ALH, BCF parameters were measured using the CASA system, Hamilton Thom, ltd. A minimum of 150 spermatozoa were measured and for the samples of very low concentrations 100 spermatozoa were measured.

Sperm DNA and chromatin integrity

[0152] Acridine orange (AO) test was used for assessment of bovine sperm DNA integrity as described elsewhere ⁴¹ and TUNEL assay was used to evaluate SCF following the previous report. ¹¹

Sperm membrane integrity

[0153] Total sperm concentration in the sample was determined by NucleoCounter SP-100 (ChemoMetic) by stripping cell membranes with a detergent S-100 which allows the propidium iodide which is a non-membrane permeable DNA stain to stain the nucleus of spermatozoa. The number of sperm nuclei are counted, and the concentration determined by multiplying the number of sperm nuclei by the volume and dilution factor to determine the proportion of membrane damaged sperm, a second sample was prepared with the sperm sample diluted in media with no detergent. The difference between the total sperm concentration and the concentration of cells with membrane damage was the concentration of cells with the intact plasma membrane.

Sperm samples

[0154] Bovine sperm samples were purchased from Genex corporation (Ithaca, NY, USA) from a single fertile bull. Human semen from 7 men were collected by masturbation following a 2 to 5 d period of abstinence. Patients gave informed written consent to participate (IRB 0712009553). Only specimens with normal semen parameters (based on WHO guidelines ⁵¹ ) were used for the experiments. Measurement of RE

[0155] Knowing the average motility of the raw sample, and concentration and motility of the sorted samples, RE is calculated using the following formula: 100 (5

[0156] Here M denotes motility percentage and C refers to the total concentration. The subscript sorted and Raw refer to the type of the samples. With this information, the RE of various groups are calculated for 3 replicates.

Theoretical modeling of sperm accumulation

[0157] Applicant have simplified our model to one dimensional motion of noninteracting spermatozoa using the Langevin equation:

[0158] in which Xi, 0i and Vsi are sperm position, direction of motion, and intrinsic velocity, respectively and subscript i indicates i-th sperm. Vf is the velocity of the fluid which is a function of x which denotes the x location. For the effect of rheotaxis on the directional change of the sperm motion Applicant used the following equation: ⁵²

[0159] In the first term on the right-hand side of the equation, A and y in the above equation are a constant and the shear rate respectively. If shear rate is between 11 s' ¹ and 3 s' ¹ this turning dynamics term is applied, otherwise it is ignored. ³⁵ In the second term, x is Gaussian noise with unit variance and mean zero which makes half of the spermatozoa right-turning and the other half left turning ⁵³ and D _e is the rotational diffusion coefficient taken as 0.01 rad ² s' ¹ . ⁵² = and W is the width of the channel. From the continuity equation, Vf can be calculated from the flow rate in the channel; meaning Vf = in which Q is the flow rate.

Oocytes collection and IVM

[0160] Cow ovaries were collected from a local slaughterhouse. The ovaries were washed several times in a sterile saline. COCs were aspirated from follicles (2-8 mm in diameter) using an 18-gauge needle attached to an aspiration unit aspirating at a flow rate of 22.5-25 mL H2O min' ¹ . COCs with dark homogenous cytoplasm and at least 2 intact layers of cumulus cells were selected and matured in IVM media (BO-IV IVF Bioscience, 61002) as 50 COCs per each well containing 700 pL of the media covered with mineral oil and were incubated at 38.5 °C for 22 h in a humidified atmosphere of 5 v/v % CO2 in air.

IVF o f bovine

[0161] Sperm preparation'. Frozen semen (from fertility-proven bulls) was thawed by immersing the straw in warm water (37 °C) for 20 s. For the control group, Spermatozoa were washed by centrifugation (350g for 5 min) in BO-Semen Prep (IVF Bioscience, 61004) as media. After removing the supernatant, the pellet was diluted with 1 mL of BO-Semen prep and centrifuged again for 5 min at 350g.

[0162] The microfluidic-based spermatozoa is sorted at shear rates of 3 s’ ¹ , 5 s’ ¹ and 7 s’ ¹ . The device was loaded with media. The media was replaced with 100 pL of the raw sample and then a syringe pump was used to generate 150, 250 and 350 pL h’ ¹ flow rates of media to wash the semen inside the chip for 35, 25 and 20 min respectively. These experiments were run in parallel. For the flow rates of 450 and 550 pL h’ ¹ which Applicant used for sperm characterization experiments, the washing time was set at 18 and 17 min, respectively. Then the wasted semen from the outlet was discarded, and the tube from the syringe pump was detached from the inlet port, and the sorted sample was aspirated from the inlet port using a 200 pL pipette.

[0163] The inseminating dose for fertilization of each group was calculated using the Nucleo-counter then volume was adjusted to be 50 pL for each group via formulation demonstrated in FIG. 5A. Fifty (50) matured COCs were washed twice in 100 pL BO-IVF (IVF Bioscience, 61003) then transferred to a well so that the final content of the well is 450 pL of BO-IVF (IVF Bioscience, 61003) containing 50 COCs. Then the previously adjusted inseminating dose was added to the wells so that the total volume in each well is 500 pL overlaid with mineral oil. Fertilization was carried out for 18 h at 38.5 °C in a humidified atmosphere of 5 v/v % CO2 in air.

[0164] After fertilization, cumulus cells were removed by vortexing at maximum speed for 30 seconds to denude the zygotes. Presumptive zygotes were transferred to a 5-well plate containing 500 pL BO-IVC (IVF Bioscience, 61001), overlaid with mineral oil as 50 embryos per well. Embryos were then cultured in a humidified atmosphere of 5 % O2, 5 % CO ₂ , and 90 % N ₂ at 38.5 °C for 7 d.

[0165] Assessment of cleavage and blastocyst rate'. Cleavage rate was assessed on day 2 of fertilization and blastocyst rate was assessed on day 7. Fluorescent in situ hybridization (FISH) analysis

[0166] In preparation for FISH, slides were fixed in Camoy’s fixative (3: 1 methanol :acetic acid) at room temperature (25 °C) for 15 min, then placed on a slide moat at 37 °C overnight. Sperm decondensation was achieved by immersing the slides in 10 mmol/L dithiothreitol (DTT; Sigma Chemical Co., St. Louis, MO, USA) in 100 mmol L' ¹ tris(hydroxymethyl) aminomethane (Trizma HC1; Sigma Chemical Co.). Slides were then washed for 1 min in 2x standard saline citrate (SSC; Vysis, Downers Grove, IL, USA), followed by hybridization with fluorescent probes. Sperm nuclei were counterstained by administering 7 pL of 4’,6-diamino- 2-phenylindole (DAPI; Abbott Molecular, Des Plaines, IL, USA) to each slide, which were then cover-slipped and assessed on a fluorescent microscope (Olympus BX61; New York/New Jersey Scientific, NJ, USA) at l,000x. A minimum of 1,000 cells per slide were assessed to determine the ratio of X:Y spermatozoa (Applied Imaging, Cyto Vision v3.93.2).

Statistical analysis

[0167] JMP 16.0 software was used to perform the statistical analysis for bovine sperm characterization. For continuous variables analysis of variance was employed with either linear model or polynomial regressions and 5% was chosen for statistical significance as the result of F-test. For the categorical variables t-test was used with 5% as the significance level. For human sperm experimentations, paired t-test was performed to compare DGC and rheotactically selected spermatozoa with 5% as significance level. ANOVA test was performed to compare semen parameters among samples selected by each shear rate with significance at 5%. To further check the power with the significance level of 0.05 in our data Applicant measured the common standard deviation as 5 %, considering 11 replicates the 5% increase in the blastocyst rate at y = 7 s' ¹ in comparison to the control group the sample size is valid with the power of 70 % using T statistics.

References for Example 1

[0168] 1. S. Franklin, Reprod. Biomed. Online, 2013, 27, 747-755.

[0169] 2. P. J. Hansen, Theriogenology, 2006, 65, 119-125.

[0170] 3. B. D. Bavister, Reproduction, 2002, 124, 181-196.

[0171] 4 . E. T. Leung, C.-L. Lee, X. Tian, K. K. Lam, R. H. Li, E. H. Ng, W. S. Yeung and P. C. Chiu, Nat. Rev. Urol., 2021, 1-21.

[0172] 5. R. Nosrati, P. J. Graham, B. Zhang, J. Riordon, A. Lagunov, T. G. Hannam, C.

Escobedo, K. Jarvi and D. Sinton, Nat. Rev. Urol., 2017, 14, 707. [0173] 6. M. Muratori, N. Tarozzi, F. Carpentiero, S. Danti, F. M. Perrone, M. Cambi, A.

Casini, C. Azzari, L. Boni and M. Maggi, Sci. Rep., 2019, 9, 7492.

[0174] 7. J. G. Alvarez, J. L. Lasso, L. Blasco, R. C. Nunez, S. Heyner, P. P. Caballero and B. T. Storey, Hum. Reprod., 1993, 8, 1087-1092.

[0175] 8. D. Sakkas, M. Ramalingam, N. Garrido and C. L. Barratt, Hum. Reprod. Update,

2015, 21, 711-726.

[0176] 9. W. Asghar, V. Velasco, J. L. Kingsley, M. S. Shoukat, H. Shafiee, R. M. Anchan,

G. L. Mutter, E. Tiizel and U. Demirci, Adv. Healthc. Mater., 2014, 3, 1671-1679.

[0177] 10. M. P. B. Nagata, K. Endo, K. Ogata, K. Yamanaka, J. Egashira, N. Katafuchi,

T. Yamanouchi, H. Matsuda, Y. Goto and M. Sakatani, Proc. Natl. Acad. Sci., 2018, 115, E3087-E3096.

[0178] 11. A. Parrella, D. Keating, S. Cheung, P. Xie, J. D. Stewart, Z. Rosenwaks and G.

D. Palermo, J. Assist. Reprod. Genet., 2019, 36, 2057-2066.

[0179] 12 . K. Miki and D. E. Clapham, Curr. Biol., 2013, 23, 443-452.

[0180] 13. T. Hino and R. Yanagimachi, Biol. Reprod., 2019, 101, 40-49.

[0181] 14. S. S. Suarez, Exp. Meeh., 2010, 50, 1267-1274.

[0182] 15 . C.-K. Tung and S. S. Suarez, Cells, 2021, 10, 1297.

[0183] 16. C. Tung, L. Hu, A. G. Fiore, F. Ardon, D. G. Hickman, R. O. Gilbert, S. S.

Suarez and M. Wu, Proc. Natl. Acad. Sci., 2015, 112, 5431-5436.

[0184] 17. D. Seo, Y. Agca, Z. C. Feng and J. K. Critser, Microfluid. Nanofluidics, 2007,

3, 561-570.

[0185] 18. T. Chinnasamy, J. L. Kingsley, F. Inci, P. J. Turek, M. P. Rosen, B. Behr, E.

Tuzel and U. Demirci, Adv. Sci., 2018, 5, 1700531.

[0186] 19 . S. S. Suarez and M. Wu, MHR Basic Sci. Reprod. Med., 2017, 23, 227-234.

[0187] 20. A. Mokhtare, B. Davaji, P. Xie, M. Yaghoobi, Z. Rosenwaks, A. Lal, G.

Palermo and A. Abbaspourrad, Lab. Chip, 2022, 22, 777-792.

[0188] 21. Y. Yan, B. Zhang, Q. Fu, J. Wu and R. Liu, Lab. Chip, 2021, 21, 310-318.

[0189] 22. L. Xie, R. Ma, C. Han, K. Su, Q. Zhang, T. Qiu, L. Wang, G. Huang, J. Qiao and J. Wang, Clin. Chem., 2010, 56, 1270-1278.

[0190] 23. S. Koyama, D. Amarie, H. A. Soini, M. V. Novotny and S. C. Jacobson, Anal.

Chem., 2006, 78, 3354-3359.

[0191] 24. H. Chang, B. J. Kim, Y. S. Kim, S. S. Suarez and M. Wu, PloS One. [0192] 25. M. Eisenbach and L. C. Giojalas, Nat. Rev. Mol. Cell Biol., 2006, 7, 276-285.

[0193] 26. M. R. Doostabadi, E. Mangoli, L. D. Marvast, F. Dehghanpour, B. Maleki, H.

Torkashvand and A. R. Talebi, Andrologia, 2022, el4623.

[0194] 27. V. Kantsler, J. Dunkel, M. Polin and R. E. Goldstein, Proc. Natl. Acad. Sci.,

2013, 110, 1187-1192.

[0195] 28. A. P. Berke, L. Turner, H. C. Berg and E. Lauga, Phys. Rev. Lett., 2008, 101,

038102.

[0196] 29. P. Denissenko, V. Kantsler, D. J. Smith and J. Kirkman-Brown, Proc. Natl.

Acad. Sci., 2012, 109, 8007-8010.

[0197] 30. A. Bukatin, P. Denissenko and V. Kantsler, Sci. Rep., 2020, 10, 1-8.

[0198] 31. F. Yazdan Parast, M. K. O’Bryan and R. Nosrati, Adv. Mater. TechnoL, 2022,

2101291.

[0199] 32. R. Nosrati, M. Vollmer, L. Earner, M. C. San Gabriel, K. Zeidan, A. Zini and

D. Sinton, Lab. Chip, 2014, 14, 1142-1150.

[0200] 33. M. Yaghoobi, M. Azizi, A. Mokhtare and A. Abbaspourrad, Lab. Chip, 2021,

21, 2791-2804.

[0201] 34. V. Kantsler, J. Dunkel, M. Blayney and R. E. Goldstein, Elife, 2014, 3, e02403.

[0202] 35. M. Zaferani, S. H. Cheong and A. Abbaspourrad, Proc. Natl. Acad. Sci., 2018,

115, 8272-8277.

[0203] 36. M. Zaferani, G. D. Palermo and A. Abbaspourrad, Sci. Adv., 2019, 5, eaav2111.

[0204] 37. J. Romero- Aguirregomezcorta, R. Laguna-Barraza, R. Fernandez-Gonzalez, M.

Stiavnicka, F. Ward, J. Cloherty, D. McAuliffe, P. B. Larsen, A. M. Grabrucker and A. Gutierrez- Adan, Reproduction, 2021, 161, 343-352.

[0205] 38. S. Sharma, M. A. Kabir and W. Asghar, Analyst, 2022, 147, 1589-1597.

[0206] 39. I. R. Sarbandi, A. Lesani, M. Moghimi Zand and R. Nosrati, Sci. Rep., 2021,

11, 18327.

[0207] 40. S. Zeaei, M. Z. Targhi, I. Halvaei and R. Nosrati, Lab. Chip, 2023, 23, 2241-

2248.

[0208] 41. M. Yaghoobi, M. Azizi, A. Mokhtare, F. Javi and A. Abbaspourrad, Lab. Chip,

2022, 22, 1486-1497.

[0209] 42. S. S. Suarez and A. A. Pacey, Hum. Reprod. Update, 2006, 12, 23-37. [0210] 43. A. Olanrewaju, M. Beaugrand, M. Yafia and D. Juncker, Lab. Chip, 2018, 18,

2323-2347.

[0211] 44. C. Tung, F. Ardon, A. Roy, D. L. Koch, S. S. Suarez and M. Wu, Phys. Rev.

Let., 2015, 114, 108102.

[0212] 45. E. Altshuler, G. Mino, C. Perez-Penichet, L. Del Rio, A. Lindner, A. Roussel et and E. Clement, Soft Mater, 2013, 9, 1864-1870.

[0213] 46. K. Cui and C. D. Matthews, Nature, 1993, 366, 117-118.

[0214] 47. R. A. P. Harrison, H. M. Dott and G. C. Foster, J. Exp. Zool., 1982, 222, 81-88.

[0215] 48. J. J. Parrish, J. L. Susko-Parrish and N. L. First, Biol. Reprod., 1989, 41, 683-

699.

[0216] 49. A. N. Fatehi, M. M. Bevers, E. Schoevers, B. A. J. Roelen, B. Colenbrander and

B. M. Gadella, J. Androl., 2006, 27, 176-188.

[0217] 50. Y. Xia and G. M. Whitesides, Annu. Rev. Mater. Sci., 1998, 28, 153-184.

[0218] 51. W. H. Organization, WHO laboratory manual for the examination and processing of human semen, World Health Organization, 2021.

[0219] 52. A. Guidobaldi, Y. Jeyaram, I. Berdakin, V. V. Moshchalkov, C. A. Condat, V.

I. Marconi, L. Giojalas and A. V. Silhanek, Phys. Rev. E, 2014, 89, 032720.

[0220] 53. A. Bukatin, I. Kukhtevich, N. Stoop, J. Dunkel and V. Kantsler, Proc. Natl.

Acad. Sci., 2015, 112, 15904-15909.

[0221]

[0222]

***

[0223] Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.

[0224] Further attributes, features, and embodiments of the present invention can be understood by reference to the following numbered aspects of the disclosed invention. Reference to disclosure in any of the preceding aspects is applicable to any preceding numbered aspect and to any combination of any number of preceding aspects, as recognized by appropriate antecedent disclosure in any combination of preceding aspects that can be made. The following numbered aspects are provided:

1. A microfluidic device configured for rheotaxis-based sperm separation comprising: one or more channels, each channel comprising: an inlet at a first end of the channel, an outlet at a second end of the channel, a plurality of rows, each row comprising a plurality of structures forming a plurality of strictures, wherein each row extends across a width of the channel, wherein no structure contacts another structure so as to form a gap between each structure to generate the plurality of strictures, a plurality of straight guide veins extending across the width of the channel substantially perpendicular to a longest axis of the channel, wherein each straight guide veins is positioned equidistance between two different rows; a plurality of curved guide veins extending across the width of the channel, wherein a curved guide vein is placed on each side of each row with the curve extending away from the row, and wherein the microfluidic device is configured such that a fluid introduced at the inlet generates a fluid flow through the channel from the inlet to the outlet.

2. The microfluidic device of aspect 1, further comprising a set of supports comprising a plurality of supports placed at a distance from each other, wherein a first set of supports is positioned between side walls of the channel and between the inlet and a curved vein on an inlet side of a first row and the inlet.

3. The microfluidic device of any one of aspects 1-2, wherein the plurality of strictures generates regions of variable shear in the fluid flow. 4. The microfluidic device of any one of aspects 1-3, wherein one or more or all of the plurality of rows extend across the width of the channel substantially perpendicular to the longest axis of the channel.

5. The microfluidic device of any one of aspects 1-4, wherein the plurality of structures are prisms.

6. The microfluidic device of aspect 5, wherein the plurality structures are pyramids.

7. The microfluidic device of any one of aspects 1-6, wherein each of the structures within the same row are substantially the same size, substantially the same shape, or both.

8. The microfluidic device of any one of aspects 1-7, wherein all of the structures within two or more rows are substantially the same size, substantially the same shape, or both.

9. The microfluidic device of any one of aspects 1-8, wherein all of the structures in all of the rows are substantially the same size, substantially the same shape, or both.

10. The microfluidic device of any one of aspects 1-6, wherein at least two or more structures within the same row are different.

11. The microfluidic device of any one of aspects 1-6 or 10, wherein at least two or more structures within two different rows are different.

12. The microfluidic device of any one of aspects 1-11, wherein the plurality of rows comprises 2-100 rows.

13. The microfluidic device of any one of aspects 1-12, wherein each row is the same.

14. The microfluidic device of any one of aspects 1-12, wherein at least two or more rows are different.

15. The microfluidic device of any one of aspects 1-12 and 14, wherein all rows are different.

16. The microfluidic device of any one of aspects 1-15, wherein the plurality of structures comprises 10-1000 structures.

17. The microfluidic device of aspect 16, wherein the plurality of structures comprises 40- 50 structures.

18. The microfluidic device of aspect 16, wherein the plurality of structures comprises 42 structures.

19. The microfluidic device of any one of aspects 1-18, wherein the set of supports comprises 2-100 supports. 20. The microfluidic device of aspect 19, wherein the set of supports comprises 3-6 supports.

21. The microfluidic device of aspect 19, wherein the set of supports comprises 4 supports.

22. The microfluidic device of any one of aspects 1-21, wherein the microfluidic device comprises 1-1000 or more channels.

23. The microfluidic device of any one of aspects 1-22, wherein the distance between each row is about 0.1 to 10 cm.

24. The microfluidic device of aspect 23, wherein the distance between each row is about 0.9 cm to about 1.2 cm.

25. The microfluidic device of aspect 23, wherein the distance between each row is about 1.1 cm.

26. The microfluidic device of any one of aspects 1-25, wherein each channel is tapered at the inlet end, is tapered at the outlet end, or both.

27. The microfluidic device of aspect 26, wherein the tapers at the inlet end, the outlet end, or both form approximately 90° angles with the inlet, the outlet, or both being at the apex of each taper.

28. The microfluidic device of any one of aspects 1-27, wherein the device comprises one or more suitable materials.

29. The microfluidic device of aspect 28, wherein the one or more suitable materials are biocompatible.

30. The microfluidic device of any one of aspects 28-29, wherein the one or more suitable materials are each independently selected from a glass, a polymer, a ceramic, a metal, an alloy, or any combination thereof.

31. The microfluidic device of aspect 30, wherein the polymer is polydimethylsiloxane (PDMS), poly(methyl methacrylate) (PMMA), polytetrafluoroethylene (PTFE), or any combination thereof.

32. A method of rheotaxis-based sperm separation comprising: injecting a fluid comprising sperm into an inlet of a microfluidic device of any one of aspects 1-31; and injecting a first suitable media via the inlet to induce a fluid flow within the device thereby removing incompetent sperm.

33. The method of aspect 32, wherein the fluid is semen. 34. The method of any one of aspects 32-33, further comprising filling the microfluidic device of any one of aspects 1-31 with a second suitable media by injecting the second suitable media in the inlet prior to injecting the fluid comprising sperm into the inlet.

35. The method of any one of aspects 32-34, further comprising collecting competent sperm from the device after injecting the first suitable media.

36. The method of any one of aspects 32-35, wherein the second suitable media is injected at a flow rate of 1000 to 5000 pL/h.

37. The method of aspect 36, wherein the second suitable media is injected at a flow rate of about 3000 pL/h.

38. The method of any one of aspects 32-37, wherein the first suitable media is injected at a flow rate of about 100 to 2000 pL/h.

39. The method of aspect 38, wherein the first suitable media is injected at a flow rate of about 350 pL/h.

40. The method of any one of aspects 32-39, wherein the fluid comprising sperm is injected at a flow rate of about 2000 to 5000 pL/h.

41. The method of aspect 39, wherein the fluid comprising sperm is injected at a flow rate of about 3000 pL/h.

42. The method of any one of aspects 32-41, wherein the fluid comprising sperm is injected at a flow rate that minimizes dilution of the fluid.

43. The method of any one of aspects 35-42, further comprising performing in vitro fertilization (IVF) using the collected competent sperm.

44. The method of aspect 43, wherein the IVF is chamber based IVF.

45. The method of any one of aspects 32-44, wherein one or more steps are automated.

46. A composition comprising: a population of sperm enriched for competent sperm, wherein the population of sperm is enriched for competent sperm by performing the method as in any one of aspects 35-45.

47. A composition comprising: a population of sperm enriched for competent sperm wherein the population of enriched for competent sperm have

(i) a DNA integrity of at least 20% greater as compared to a population not enriched for competent sperm (ii) has a higher rate of blastocyst formation, embryo formation, or both as compared to a population not enriched for competent sperm;

(iii) has a lower DNA fragmentation index as compared to a population not enriched for competent sperm; or any combination of (i)-(iii).

48. The composition of aspect 47, wherein the population of sperm enriched for competent sperm is prepared by performing a method as in any one of aspects 32-45.