An apparatus for spermatozoa selection and an apparatus for spermatozoa guidance

Abstract

The present invention relates to microfluidic devices, in particular to an apparatus for spermatozoa selection and an apparatus for spermatozoa guidance. Provided are an apparatus for spermatozoa selection, the apparatus comprising an inlet chamber, an outlet chamber located radially inward from the inlet chamber, channels connecting the inlet and the outlet chambers, wherein the channels comprise blind branches having a radius of curvature ranging from 50 μm to 75 μm; and an apparatus for spermatozoa guidance, the apparatus comprising an inlet and an outlet openings, as well as blind branches having a radius of curvature ranging from 50 μm to 75 μm. The technical effect of the provided invention is in reducing the backflow of spermatozoa.

Description

[0001] AN APPARATUS FOR SPERMATOZOA SELECTION AND AN APPARATUS FOR

[0002] SPERMATOZOA GUIDANCE

[0003] TECHNICAL FIELD

[0004] The present invention relates to microfluidic devices, in particular to an apparatus for spermatozoa selection and an apparatus for spermatozoa guidance.

[0005] BACKGROUND OF THE INVENTION

[0006] Nowadays, with the general trend of increasing compactness of devices of all kinds and the desire for efficient and accurate devices, microfluidic devices are gaining importance. Microfluidic devices find a wide range of practical applications in the analysis and study of liquid samples in biology, pharmacology, biomedicine and medicine In a number of biological and medical applications, such as in vitro fertilization (IVF), artificial insemination and research in the field of reproductive medicine, the selection of high-quality spermatozoa with high motility, normal morphology and low degree of DNA fragmentation is one of the important problems. To solve these problems, microfluidic devices are used to sort and select spermatozoa according to characteristics such as motility, degree of DNA fragmentation and morphology. Typically, microfluidic devices comprise a microfluidic chip comprising microchannels, reservoirs, membranes, and other structures to allow fluid flow as well as fluid control. These devices may also comprise micropumps to provide fluid movement, valves to control fluid flows, and detectors to provide monitoring and the ability to analyze fluid contents.

[0007] It should be noted that microchannels play an important role in the microfluidic devices. The microchannels are used for mixing, separating, dosed dispensing fluids. The dimensions of microchannels are typically in the micrometer range, which allows an efficient guided control of fluid flows at the microscopic level. In the implementation of microchannels, an important task is to select such characteristics and parameters as a geometric shape, depth, length and width. Said characteristics and parameters affect operational efficiency and operational speed of the device / apparatus. The microchannels can have a different geometric shape, for example rectangular, circular, zigzag, spiral, etc. The geometry of the microchannels is selected based on specific objectives and requirements.

[0008] CA 2834007 Al (19.05.2015) discloses a microfluidic device for separating motile sperm. The known device comprises an inlet reservoir for receiving a biological fluid sample containing spermatozoa, an outlet reservoir for collecting spermatozoa, an array of microchannels arranged radially between the inlet and outlet reservoirs to provide fluid communication and to direct motile sperm from the inlet reservoir to the outlet reservoir. According to the known solution, the microchannels have a rectangular shape. Spermatozoa which entered the microchannels having rectangular shape can move unimpeded both in the forward direction, i.e., from the inlet reservoir to the outlet reservoir, and in the backflow direction, i.e., from the outlet reservoir to the inlet reservoir. Such unimpeded bidirectional movement of spermatozoa decreases concentration of motile sperm in the outlet reservoir, which adversely affects the efficiency and rate of selection of spermatozoa of the desired quality. To eliminate the above-mentioned disadvantage, in the known solution, the outlet reservoir can comprise a plurality of anti-return members preventing the motile sperm from returning back into the microchannels after they exit from these microchannels. However, the presence of such members in the outlet reservoir does not eliminate the backflow movement towards the inlet reservoir for the spermatozoa deployed in the microchannel or for the spermatozoa which entered again the microchannel.

[0009] The closest analog of the present invention is a solution disclosed in the article by P. Denissenko et al. “Human spermatozoa migration in microchannels reveals boundary-following navigation”, PNAS (2012). The article reveals the principle of spermatozoa selection using a ratchet- shaped channel, as well as the principle of spermatozoa movement in such channels. In particular, according to the article, spermatozoa travel along the intersection of the channel walls (channel comers). If the channel has curves, the spermatozoa leave the corner and continue moving forward until they collide with the opposite wall of the channel. The fact that spermatozoa depart from corners during their movement allows the use of channels having ratchet-type walls to ensure unidirectional spermatozoa movement. At the same time, it should be noted that, despite the known influence of the ratchet shape for the microchannel on the trajectory of spermatozoa movement, the influence of the ratchet- shape microchannel parameters on the efficiency of spermatozoa selection was not studied in the above article, which does not allow provision of an apparatus for spermatozoa selection having optimal configuration for rapid selection of spermatozoa of the desired quality.

[0010] Thus, in view of the disadvantages of the known solutions, there is a technical problem consisting in the absence of means and solutions that would provide a high efficiency of spermatozoa selection and minimize the time period for spermatozoa selection.

[0011] SUMMARY

[0012] Said technical problem is solved by the provided apparatus for spermatozoa guidance and the corresponding apparatus for spermatozoa selection. According to a first aspect of the invention, the provided apparatus for spermatozoa guidance comprises a channel for spermatozoa guidance and an inlet opening and an outlet opening, wherein the channel comprises blind branches arranged in a skewed manner in the direction of the inlet opening and terminating in rounded portions thereof on the side of the inlet opening, the rounded portions having a radius of curvature. In accordance with the most preferred embodiment of the invention, the radius of curvature takes values from 50 pm to 75 pm.

[0013] The technical effect is in reducing the backflow of spermatozoa, which allows for arranging a predominantly unidirectional movement of spermatozoa towards the outlet chamber, and also causes an increased concentration of motile sperm in the outlet chamber. Thus, the desired high concentration of motile sperm in the outlet chamber provides a high efficiency of spermatozoa selection within a minimum time period. In the context of the present invention, the high efficiency of spermatozoa selection and / or operational efficiency of the apparatus or of the microchannel is to be understood as providing a high concentration of motile sperm in the outlet chamber.

[0014] This technical effect is achieved, in particular, due to the fact that the channel comprises blind branches arranged in a skewed manner in the direction of the inlet opening and terminating in rounded portions thereof on the side of the inlet opening, the rounded portions having a radius of curvature. In particular, by providing a radius of curvature in the range of 50 to 75 pm, it is possible to redirect the spermatozoa deployed or entered again the channel towards the outlet chamber. Also, due to the fact that the most motile sperm enter the outlet chamber, and their concentration is high, a further technical effect is an improved quality of the sampled material as such. Spermatozoa selected with regard of their motility have high motility, good morphology and low DNA fragmentation.

[0015] In accordance with one embodiment of the invention, the radius of curvature takes values from 30 pm to 100 pm.

[0016] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 30 pm to 40 pm.

[0017] In accordance with yet another embodiment of the invention, the radius of curvature is equal to 30 pm.

[0018] In accordance with yet another embodiment of the invention, the radius of curvature is equal to 100 pm.

[0019] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 50 pm to 55 pm.

[0020] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 55 m to 60 pm. In accordance with yet another embodiment of the invention, the radius of curvature takes values from 60 m to 65 pm.

[0021] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 65 pm to 70 pm.

[0022] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 70 pm to 75 pm.

[0023] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 75 pm to 100 pm.

[0024] According to one of the embodiments of the invention, the blind branches are arranged asymmetrically relative to the axis of the corresponding channel.

[0025] According to the most preferred embodiment of the invention, the depth of the channel is 25 pm to 45 pm.

[0026] According to one embodiment of the invention, the depth of the channel is 25 pm to 30 pm.

[0027] According to another embodiment of the invention, the depth of the channel is 30 pm to 45 pm.

[0028] According to yet another embodiment of the invention, the width of the channel varies from the maximum value to the minimum value in the direction from the inlet opening to the outlet opening.

[0029] According to the most preferred embodiment of the invention, the minimum width of the channel is 20 pm to 80 pm.

[0030] According to another embodiment of the invention, the minimum width of the channel is 20 pm to 50 pm.

[0031] According to yet another embodiment of the invention, the minimum width of the channel is 50 pm to 80 pm.

[0032] According to a second aspect of the invention, the provided apparatus for spermatozoa selection comprises an inlet chamber having a non-enclosed circumferential shape and having at opposite ends thereof an inlet opening for introducing a seminal fluid sample into the inlet chamber and an outlet opening for withdrawing the seminal fluid sample from the inlet chamber, an outlet chamber located radially inward from the inlet chamber, channels connecting the inlet chamber and the outlet chamber and extending radially therebetween. The channels comprise blind branches arranged in a skewed manner in the direction of the inlet chamber and terminating in rounded portions thereof on the side of the inlet chamber, the rounded portions having a radius of curvature. In accordance with the most preferred embodiment of the invention, the radius of curvature takes values from 50 pm to 75 pm.

[0033] In accordance with one embodiment of the invention, the radius of curvature takes values from 30 pm to 100 pm.

[0034] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 30 pm to 40 pm.

[0035] In accordance with yet another embodiment of the invention, the radius of curvature is equal to 30 pm.

[0036] In accordance with yet another embodiment of the invention, the radius of curvature is equal to 100 pm.

[0037] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 50 pm to 55 pm.

[0038] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 55 pm to 60 pm.

[0039] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 60 pm to 65 pm.

[0040] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 65 pm to 70 pm.

[0041] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 70 pm to 75 pm.

[0042] In accordance with yet another embodiment of the invention, the radius of curvature takes values from 75 pm to 100 pm.

[0043] According to one of the embodiments of the invention, the blind branches are arranged asymmetrically relative to the axis of the corresponding channel.

[0044] According to yet another embodiment of the invention, the depth of the channels is 25 pm to 45 pm.

[0045] According to one embodiment of the invention, the depth of the channels is 25 pm to 30 pm.

[0046] According to another embodiment of the invention, the depth of the channels is 30 pm to 45 pm.

[0047] According to yet another embodiment of the invention, the width of the channels varies from the maximum value to the minimum value in the direction from the inlet chamber to the outlet chamber.

[0048] According to the most preferred embodiment of the invention, the minimum width of the channels is 20 pm to 80 pm. According to another embodiment of the invention, the minimum width of the channels is 20 pm to 50 m.

[0049] According to yet another embodiment of the invention, the minimum width of the channels is 50 pm to 80 pm.

[0050] According to yet another embodiment of the invention, the minimum distance between the channels is 10 pm to 60 pm.

[0051] According to yet another embodiment of the invention, a bottom portion of the apparatus for spermatozoa selection is fabricated from glass or biocompatible plastic.

[0052] According to yet another embodiment of the invention, a top portion of the apparatus for spermatozoa selection is fabricated from biocompatible plastic.

[0053] LIST OF DESIGNATIONS IN THE DRAWINGS

[0054] 1 - Apparatus for spermatozoa selection

[0055] 2 - Inlet chamber

[0056] 3 - Outlet chamber

[0057] 4 - Microchannel

[0058] 5 - Blind branches

[0059] 6 - Top portion

[0060] 7 - Bottom portion

[0061] 8 - Trajectory of deployed spermatozoa movement along the blind branches

[0062] 9 - Trajectory of spermatozoa movement in the forward direction

[0063] H - Minimum width

[0064] R - Radius of curvature

[0065] 1 - Length of the channel

[0066] BRIEF DESCRIPTION OF THE FIGURES

[0067] The essence of the invention is explained in more detail with reference to the accompanying drawings:

[0068] FIG. 1 depicts a general view of an apparatus for spermatozoa selection according to one embodiment;

[0069] FIG. 2 is a cross-sectional view of the apparatus for spermatozoa selection depicted in FIG. 1;

[0070] FIG. 3 depicts a top view of a microchannel in accordance with one of the embodiments; FIG. 4 depicts a top view of a microchannel in accordance with another embodiment;

[0071] FIG. 5 illustrates the mutual arrangement of the microchannels according to FIGS. 3- 4;FIG. 6 illustrates another variation of the mutual arrangement of the microchannels according to FIGS. 3-4;

[0072] FIG. 7 illustrates yet another variation of the mutual arrangement of the microchannels according to FIGS. 3-4; FIG. 8 shows a dual apparatus for spermatozoa selection;

[0073] FIG. 9 shows a trajectory of the spermatozoa movement in the microchannel;

[0074] FIG. 10 depicts a top view and cross-sectional views of the apparatus for spermatozoa selection according to embodiments of the present invention;

[0075] FIG. 11 depicts a general view of the apparatus for spermatozoa selection and a trajectory of spermatozoa movement in the microchannel according to one of the embodiments.

[0076] DETAILED DESCRIPTION OF THE EMBODIMENTS OF THE INVENTION

[0077] The embodiments of the present invention and various features thereof are explained in more detail with reference to non-limiting examples, which are described and / or depicted in the drawings and presented in detail in the following description. Within the scope of the present invention, the "channel angle" is to be understood as the junction between the side wall and the upper or lower wall. It should be noted that, according to a preferred embodiment of the present invention, the channel has vertical walls and the cross-section of the channel is rectangular at any location. However, within the scope of the present invention, the walls are understood to include all surfaces of the channel. It should be noted that optionally the channel may have other suitable shapes, in particular a cross-sectional shape other than rectangular.

[0078] In accordance with various embodiments of the present invention, a microchannel 4 is designed for guiding spermatozoa from an inlet chamber 2 of an apparatus 1 for spermatozoa selection to an outlet chamber 3 of the apparatus 1 for spermatozoa selection. According to the present invention, any suitable biocompatible material can be used to make the microchannel. In a preferred embodiment, the microchannel 4 is fabricated from a biocompatible plastic. For example, such biocompatible plastic may be one of the following: Poly(methyl methacrylate) (PMMA), polyurethane (PU), high-density and low-density polyethylene (PE), polyimide (PI), silicone rubber. It should be noted that said list of biocompatible plastics is not limiting, and the biocompatible plastic may be any suitable biocompatible plastic. In other embodiments, the microchannel 4 may be fabricated from Poly dimethylsiloxane (PDMS). In accordance with various embodiments of the present invention, the walls of the microchannel 4 on the interior side may have a hydrophilic coating to allow the microchannel 4 to be quickly filled with cell medium prior to use. Examples of such coatings may include polyethylene glycol, silanes, and polymer-based hydrophilic coatings. If necessary, the microchannel 4 may be fabricated from an optically transparent material to enable observation of the selection process by means of a microscope. In accordance with one of the non-limiting embodiments of the present invention, quartz or glass may be used as the optically transparent material.

[0079] In accordance with embodiments of the present invention, the microchannel 4 as presented in FIGS. 3-4 comprises an inlet opening (not shown), an outlet opening (not shown), and blind branches 5 arranged asymmetrically or symmetrically about the OX axis of the microchannel 4. The blind branches 5 are arranged in a skewed manner in the direction of the inlet opening and include rounded portions on the side of the inlet opening, the rounded portions having a radius of curvature R. According to the present invention, the presence of the blind branches 5 having rounded portions makes it possible to reduce the backflow of spermatozoa. This is due to the fact that when the spermatozoa make a turn to the side opposite to the direction of movement to the outlet chamber 3, when the deployed spermatozoa move along the branch 5 by virtue of the rounded portion, they will pass to the opposite wall of the microchannel 4 and then move towards the outlet chamber 3. If any spermatozoa leaving the outlet chamber 3 enter accidentally the microchannel 4, they will be redirected in a similar manner towards the outlet chamber 3. By reducing the backflow of the spermatozoa, the concentration of motile sperm in the outlet chamber 3 can be increased. As an example, in FIG. 9, the dashed lines depict a trajectory 8 of the deployed spermatozoa movement along the blind branches 5, and the solid line depicts a trajectory 9 of the spermatozoa movement toward the outlet chamber 3, which corresponds to the forward direction of movement.

[0080] It should be noted that the radius R of curvature is an important parameter characterizing the rounded portion and enabling the spermatozoa passage to the opposite wall of the microchannel 4.

[0081] According to various embodiments of the present invention, the radius R of curvature lies in the range of 30 pm to 100 pm.

[0082] In accordance with one embodiment of the present invention, the radius R of curvature is equal to 30 pm.

[0083] In accordance with another embodiment of the present invention, the radius R of curvature is equal to 100 pm.

[0084] In accordance with yet another embodiment of the present invention, the radius R of curvature lies in the range of 30 pm to 40 pm.

[0085] In accordance with yet another embodiment of the present invention, the radius R of curvature lies in the range of 75 pm to 100 pm. According to preferred embodiments of the present invention, the radius R of curvature lies in the range of 50 pm to 55 pm, or 55 pm to 60 pm, or 60 pm to 65 pm, or 65 pm to 70 pm, or 70 pm to 75 pm.

[0086] According to the most preferred embodiment of the present invention, the radius R of curvature lies in the range of 50 pm to 75 pm.

[0087] According to the present invention, the minimum value of the radius R of curvature at which spermatozoa can be trapped into the blind branches 5 is equal to 30 pm. If the radius R of curvature takes values less than 30 pm, the spermatozoa cannot enter the branches of the microchannel 4 due to their limited maneuverability. In particular, if the value R of curvature is less than 30 pm, the spermatozoa cannot be accommodated in the blind branch 5, so that the spermatozoa cannot make a turn as such.

[0088] The values R of curvature which are more than 100 pm result in an extended time period to make a turn; this is because of the increased path that the spermatozoa need to travel to make the turn in the correct direction. Also, with such values R of curvature, the microchannels 4 occupy a larger space, which results in fewer channels being able to be accommodated in the apparatus 1 for spermatozoa selection than, for example, at values R less than 100 pm. This, in consequence, results in a reduced efficiency of spermatozoa selection.

[0089] The values R of curvature ranging from 30 pm to 40 pm efficiently enable the spermatozoa to be accommodated in the blind branch 5; however, their ability to make a turn is impaired.

[0090] The values R of curvature ranging from 75 pm to 100 pm efficiently enable the spermatozoa to make a turn; however, the microchannel 4 occupies more space in the apparatus 1 for spermatozoa selection as compared to the aforementioned smaller values R of curvature, thus resulting in a lower concentration of spermatozoa in the output chamber 3 after 30 minutes of operation of the apparatus 1 for spermatozoa selection.

[0091] The values R of curvature ranging from 50 pm to75 pm most efficiently enable redirection of the spermatozoa trapped into the blind branch 5. In this case, the microchannels 4 remain compact to accommodate a sufficient number thereof in the apparatus 1 for sample selection.

[0092] The values R of curvature ranging from 50 pm to 55 pm, or from 55 pm to 60 pm, or from 60 pm to 65 pm, or from 65 pm to 70 pm, or from 70 pm to 75 pm also efficiently enable redirection of the spermatozoa trapped into the blind branch 5 while maintaining the compactness of the microchannels 4.

[0093] According to one of the embodiments of the present invention (FIG. 3), blind branches 5 are arranged asymmetrically about the OX axis of the microchannel 4. According to another embodiment of the present invention (FIG. 4), blind branches 5 are arranged symmetrically about the OX axis of the microchannel 4.

[0094] Another important parameter of the microchannel 4 is depth. The depth of the microchannels is of great importance in the context of guided controlling the fluid flow and controlling same in microfluidic devices. The fluid flow in the microfluidic devices can be caused by the difference in fluid levels in the microchannels due to large values of the depth of the microchannel. The high fluid flow causes immotile or poorly motile sperm to enter the microchannels together with motile sperm, resulting in a lower concentration of the motile sperm in the output chamber of the microfluidic device and consequently a lower quality of the selected spermatozoa and a lower efficiency of spermatozoa selection. In this regard, the microchannels 4 with different depths were studied within the scope of the present invention, thus obtaining, based on the studies performed, optimal values for the depth of the microchannel 4 in the context of the fluid flow based. In particular, the microchannels 4 with depth values of 18 pm, 25 pm, 30 pm, 45 pm, and 100 pm were the subject of the study. In this case, all parameters of the microchannels 4, except for the depths, were the same. The results of the study showed the following. In the case when the value of the depth of the microchannel 4 was equal to 18 pm there was a significant decrease in the concentration of spermatozoa in the outlet chamber 3. When the value of the depth of the microchannel 4 was 100 pm, a significant fluid flow was observed, which led to the entry of poorly motile sperm into the outlet chamber 3, resulting in a deterioration of the quality of the selected material. The most optimal depths of the microchannel 4 in terms of fluid flow were observed at values of the depth equal to 25 pm, 30 pm, and 45 pm. Based on the above-mentioned studies, the most preferable values of the depth of the microchannel 4 were selected, which lie in the range from 25 to 45 pm. At the boundaries of the above range, however, the following was observed. At values of depth less than 25 pm, a decreased concentration of spermatozoa was observed in the outlet chamber 3, and at values of depth more than 45 pm, a fluid flow from the inlet chamber 2 to the outlet chamber 3 occured, thereby causing entry of the poorly motile sperm into the outlet chamber 3. According to a preferred embodiment of the present invention, the value of the depth of the microchannel 4 lies in the range of 25 pm to 30 pm or in the range of 30 pm to 45 pm.

[0095] In accordance with various embodiments of the present invention, the effective operation of the microchannel 4 requires that the width of the microchannel 4 decreases in the direction from the inlet opening to the outlet opening, in particular this width being maximum at the inlet opening and minimum at the outlet opening. This configuration of the microchannel 4 creates conditions for selecting spermatozoa based on their motility. Motile sperm, due to their activity, more easily overcome the narrowing of the microchannel 4 and reach the outlet opening, while immotile or poorly motile sperm remain closer to the inlet opening. In accordance with the most preferred embodiment of the present invention, the minimum width H of the microchannel 4 lies in the range of 20 pm to 80 pm.

[0096] In accordance with a preferred embodiment of the present invention, the minimum width H of the microchannel 4 lies in the range of 20 pm to 50 pm or in the range of 50 pm to 80 pm.

[0097] In accordance with one embodiment of the present invention, the minimum width H of the microchannel 4 takes value equal to 20 pm.

[0098] In accordance with yet another embodiment of the present invention, the minimum width H of the microchannel 4 takes value equal to 80 pm.

[0099] The value of the minimum width H of the microchannel 4 affects the backflow of spermatozoa. In particular, by decreasing the value of the minimum width of the microchannel 4, the deployed spermatozoa pass to the opposite wall of the microchannel 4 at an acute angle, thereby reducing the backflow of spermatozoa. The values of the minimum width H more than 80 pm result in an increase in the size of the microchannel 4, which affects the number of microchannels 4 that can be accommodated in the apparatus 1 for spermatozoa selection and therefore entails a decreased operational efficiency of the apparatus 1 for spermatozoa selection.

[0100] Reducing the minimum width H to values less than 20 pm will result in a significant reduction in the number of cells that can simultaneously move in the forward direction. This is due to the fact that the diameter of the spermatozoon head is 5 pm. Consequently, only four spermatozoa will be able to pass through the narrow portions of the microchannel 4. Accordingly, the operational efficiency of the apparatus 1 for spermatozoa selection will be low.

[0101] According to a preferred embodiment of the present invention, the length 1 of the microchannel takes values from 5 mm to 10 mm.

[0102] In accordance with the most preferred embodiment, the length 1 of the microchannel is equal to 7.5 mm. Said values of the length 1 of the microchannel 4 are selected based on the following.

[0103] It is known from the prior art (see patent CA 2834007 Al, 19.05.2015) that concentration of spermatozoa, their viability and morphology depend linearly on the length of the microchannels. According to this document, the DNA fragmentation index (DFI) is minimal at the length of the microchannels equal to 7.5 mm, at the length of the microchannels less than 5 mm there is a high percentage of cells with abnormal morphology according to Kruger and high DNA fragmentation index (more than 4%), at the length of the microchannels more than 10 mm there is a low concentration of spermatozoa in the outlet chamber 30 min after the beginning of selection, the concentration being insufficient for IVF. Also, the length of the microchannels with values more than 10 mm results in an increased size of the apparatus, causing an increased cost of the apparatus.

[0104] Thus, it can be concluded that a shorter length of the microchannel yields a higher concentration of spermatozoa in the outlet chamber and a shorter time period required for spermatozoa selection, however their quality is lower; since a decreased length of the microchannel causes the poorly motile sperm to enter the outlet chamber, as well as causes an increase in abnormal shapes and an increase in the DNA fragmentation index of the selected spermatozoa.

[0105] The most preferred value of the length 1 of the microchannel 4, which is equal to 7.5 mm, is chosen on the basis that at such a length 1 the DNA fragmentation index is minimized, and furter this length 1 of the microchannel 4 enables designing the apparatus 1 for spermatozoa selection with the desired dimensions of 40x40 mm, according to the most preferred embodiment of the apparatus 1 for spermatozoa selection.

[0106] In accordance with one of the embodiments of the present invention, there is provided an apparatus 1 for spermatozoa selection comprising the above-described microchannels 4. In this regard, the description of the configuration, operating principles of the microchannels 4 comprised in the apparatus 1 for spermatozoa selection, as discussed hereinabove and hereinafter, also relates to the apparatus 1 for spermatozoa selection comprising the microchannels 4

[0107] Also, according to the present invention, optionally, a parallel selection of spermatozoa can be provided by means of a dual apparatus 1 for spermatozoa selection comprising two independently operating apparatuses 1 for spermatozoa selection (FIG. 8).

[0108] According to an embodiment of the present invention, the apparatus 1 for spermatozoa selection comprises from 40 to 100 microchannels 4.

[0109] According to another embodiment of the present invention, the apparatus 1 for spermatozoa selection comprises from 75 to 100 microchannels 4.

[0110] According to yet another embodiment of the present invention, the apparatus 1 for spermatozoa selection comprises from 40 to 50 microchannels 4.

[0111] According to the most preferred embodiment of the present invention, the apparatus 1 for spermatozoa selection comprises from 50 to 65 microchannels 4. According to various embodiments of the present invention, the apparatus 1 for spermatozoa selection comprises an inlet chamber 2, an outlet chamber 3, and microchannels 4 connecting the inlet and outlet chambers 2, 3.

[0112] According to various embodiments of the present invention, the inlet chamber 2 has a non-enclosed circumferential shape and has at opposite ends thereof an inlet opening for introducing a seminal fluid sample into the inlet chamber 2 and an outlet opening for withdrawing the seminal fluid sample from the inlet chamber 2. The opening for introducing the seminal fluid sample and the opening for withdrawing the seminal fluid sample are interchangeable, that is, the flow of the seminal fluid sample between the openings is designed in either direction.

[0113] According to embodiments of the present invention, optionally, the aforementioned inlet opening for introducing the seminal fluid sample and the outlet opening for withdrawing the seminal fluid sample may be sealed with a transparent film to prevent fluid flow between the inlet and outlet chambers 2, 3.

[0114] According to various embodiments of the present invention, the outlet chamber 3 is located radially inward from the inlet chamber 2.

[0115] According to embodiments of the present invention, the microchannels 4 extend radially between the inlet chamber 2 and the outlet chamber 3, wherein the microchannels 4 comprise blind branches 5 arranged in a skewed manner in the direction of the inlet chamber 2 and terminating in rounded portions thereof on the side of the inlet chamber 2, the rounded portions having a radius R of curvature.

[0116] According other embodiments of the present invention, the apparatus 1 for spermatozoa selection comprises a top portion 6 comprising the inlet chamber 2, the outlet chamber 3, and the microchannels 4 connecting the inlet and outlet chambers 2, 3. The apparatus 1 for spermatozoa selection further comprises a bottom portion 7. The top portion 6 can be fabricated from biocompatible plastic. Examples of such plastic can include Poly(methyl methacrylate) (PMMA), polyurethane (PU), high-density and low-density polyethylene (PE), polyimide (PI), silicone rubber. The bottom portion 7 is a flat plate fabricated from glass or biocompatible plastic. In particular, the bottom portion may be fabricated from a biocompatible plastic selected from: Poly(methyl methacrylate) (PMMA), polyurethane (PU), high-density and low-density polyethylene (PE), polyimide (PI), silicone rubber. The above list of biocompatible plastics is provided by way of example only, and the top portion 6 and the bottom portion 7 may be fabricated from any suitable biocompatible plastic, if desired.

[0117] In accordance with one of the embodiments of the present invention, the top portion 6 and the bottom portion 7 are hermetically sealed together in any suitable manner. In particular, such hermetically sealing together is known in the prior art and may include, for example, adhesive bonding.

[0118] In accordance with various embodiments of the present invention, the volume of the inlet chamber 2 is equal to 1 mL. Selection of such volume of the inlet chamber 2 is caused by standard volume of seminal fluid of patients, which is 3 mL. It should be noted, however, that attempts to reduce the volume of the inlet chamber 2 lead to a decreased concentration of motile sperm in the outlet chamber 3 after 30 minutes after the beginning of the selection of spermatozoa and, as a result, to a decreased operational efficiency of the apparatus 1 for spermatozoa selection. It should also be noted that the increase in the volume of the inlet chamber 2 entails increased dimensions of the apparatus 1 for spermatozoa selection, which leads to an increased cost of the apparatus 1 for spermatozoa selection.

[0119] In accordance with various embodiments of the present invention, the volume of the output chamber 3 lies in the range from 0.1 to 0.3 mL. This volume of the outlet chamber 3 is selected based on a standard IVF protocol that determines the required number and concentration of spermatozoa for subsequent studies.

[0120] According to various embodiments of the present invention, the apparatus 1 for spermatozoa selection may be used in conjunction with an automatic dosing dispenser to introduce or withdraw the test material or cell medium into the inlet chamber 2. According to various embodiments of the present invention, the automatic dosing dispenser may be any known suitable dosing dispenser. It should be noted that one way of increasing the operational efficiency and operational speed of the apparatus 1 for spermatozoa selection, in addition to those described above, is to increase the number of the microchannels 4 that can be accommodated in the apparatus 1 for spermatozoa selection. In this regard, when manufacturing the microchannels 4, it is advantageously to select the parameters of the microchannel 4 and the manufacturing technology in such a way that, while maintaining the required characteristics of the microchannels 4, allow accommodating the maximum possible number of the microchannels 4 in the apparatus 1 for spermatozoa selection.

[0121] In particular, one of the parameters affecting the number of microchannels 4 that can be accommodated in the apparatus 1 for spermatozoa selection is the radius R of curvature. Reducing the radius R of curvature in the microchannels 4 allows accommodating a larger number of the microchannels 4 in the apparatus 1 for spermatozoa selection, which leads to an increased operational efficiency of the apparatus 1 for spermatozoa selection due to the increased concentration of spermatozoa found in the outlet chamber 3 of the apparatus 1 for spermatozoa selection, as well as leads to an increased operational speed due to the reduced time period required for spermatozoa selection. At the same time, when selecting the radius R of curvature which ensures the most efficient operation of the apparatus 1 for spermatozoa selection, it is necessary to be guided not only by the number of the microchannels 4 which can be accommodated in the apparatus 1 for spermatozoa selection, but also to take into account the ability of the spermatozoa to make a turn, which, in particular, depends on the radius R of curvature. All of the examples given below are valid for an apparatus 1 for spermatozoa selection having dimensions of 40x40 mm. In various embodiments of the present invention, the radius R of curvature lies in the range of 30 pm to 100 pm, thereby allowing for the accommodation of 40 to 100 microchannels.

[0122] In accordance with yet another embodiment of the present invention, the radius R of curvature lies in the range of 30 pm to 40 pm, allowing for the accommodation of 75 to 100 microchannels 4.

[0123] In accordance with yet another embodiment of the present invention, the radius R of curvature lies in the range of 75 pm to 100 pm, allowing for the accommodation of 40 to 50 microchannels 4.

[0124] According to preferred embodiments of the present invention, the radius R of curvature lies in the range of 50 pm to 55 pm, or 55 pm to 60 pm, or 60 pm to 65 pm, or 65 pm to 70 pm, or 70 pm to 75 pm, allowing for the accommodation of 50 to 65 microchannels 4.

[0125] In accordance with the most preferred embodiment of the present invention, the radius R of curvature lies in the range of 50 pm to 75 pm, allowing for the accommodation of 50 to 65 microchannels 4.

[0126] When the value of the radius R of curvature is more than 100 pm, less than 40 microchannels 4 can be accommodated in the apparatus 1 for spermatozoa selection, and when the value of the radius R of curvature is less than 30, more than 100 microchannels can be accommodated in the apparatus 1 for spermatozoa selection. It should be noted that although at the values of the radius R of curvature less than 30 pm, the largest number of the microchannels 4 can be accommodated in the apparatus 1 for spermatozoa selection, this embodiment is not optimal for causing spermatozoa to make a turn; since at the values of the radius R of curvature less than 30 pm, the spermatozoa cannot make a turn as such.

[0127] In this regard, the most preferred embodiment of the present invention, which allows for accommodating the maximum number of the microchannels 4 in the apparatus 1 for spermatozoa selection while causing the spermatozoa to make the most efficient turn, is an embodiment, wherein the radius R of curvature lies in the range of 50 pm to 75 pm. In this case, the number of the microchannels 4 that can be accommodated in the apparatus 1 for spermatozoa selection will lie in the range of 50 to 65.

[0128] In addition to the most preferred embodiment of the present invention, in further optimal embodiments which provide accommodation of the maximum number of the microchannels 4 in the apparatus 1 for spermatozoa selection while causing the spermatozoa to make the most efficient turn, the radius R of curvature lies in the range of 50 pm to 55 pm, or 55 pm to 60 pm, or 60 pm to 65 pm, or 65 pm to 70 pm, or 70 pm to 75 pm.

[0129] Another parameter affecting the number of the microchannels 4 that can be accommodated in the apparatus 1 for spermatozoa selection is the minimum distance between the microchannels 4. FIGS. 5-7 show, by way of example, possible variants of the mutual arrangement of the two microchannels 4 according to FIGS. 3-4. In one of the embodiments of the present invention, the distance between the microchannels is from 10 to 60 pm, which allows at least 30 microchannels 4 to be accommodated in the apparatus 1 for spermatozoa selection. According to this embodiment, the apparatus 1 for spermatozoa selection is fabricated from Polydimethylsiloxane (PDMS) using soft lithography techniques. According to these techniques, the master for fabricating the apparatus 1 is produced by photolithography in a SU-8 photoresist on a silicon substrate.

[0130] FIG. 5 shows a preferred variant of the mutual arrangement of the microchannels 4 in the apparatus 1 for spermatozoa selection. This mutual arrangement is the most optimal arrangement of the microchannels 4 in terms of manufacturing labor intensity, as well as ensuring a minimum distance between the microchannels due to the shape of the microchannels 4. However, in view of the fact that minimum distance between the microchannels is provided, as a consequence, a larger number of the microchannels 4 can be accommodated in the apparatus 1 for spermatozoa selection. As shown in FIG. 5, the beginnings of two adjacent microchannels 4: the upper microchannel 4 and the lower microchannel 4 coincide, which simplifies the manufacturing process of the microchannels 4 since it is not necessary to take into account the displacement of the microchannels 4 relative to each other.

[0131] FIG. 6 shows another variant of the mutual arrangement of the microchannels 4 in the apparatus 1 for spermatozoa selection. The mutual arrangement of the microchannels 4 shown in FIG. 6 allows accommodating the same number of the microchannels 4 as in the embodiment shown in FIG. 5. However, as can be seen from FIG. 6, the beginnings of adjacent microchannels 4 are offset relative to each other, which complicates the manufacturing process of the microchannels 4 because, first, the offset relative to the previous microchannel 4 must be taken into account when forming each subsequent microchannel 4, and second, the number of blind branches in the adjacent microchannels may differ.

[0132] FIG. 7 shows another variant of the mutual arrangement of the microchannels 4 in the apparatus 1 for spermatozoa selection. This mutual arrangement of the microchannels 4 allows fewer microchannels 4 to be accommodated in the apparatus 1 for spermatozoa selection compared to the above-described mutual arrangements.

[0133] The apparatus 1 for spermatozoa selection operates as follows. Before beginning the selection, the inlet opening for introducing the seminal fluid sample into the inlet chamber 2 and the outlet opening for withdrawing the seminal fluid sample from the inlet chamber 2 are sealed with a transparent film to prevent fluid flow between the inlet and outlet chambers 2, 3. Next, a cell medium is introduced through the inlet opening designed for introducing the seminal fluid sample into the inlet chamber 2. Then, the cell medium is pumped out of the inlet chamber 2 through the outlet opening for withdrawing the seminal fluid sample by means of an automatic dispenser, and instead of the cell medium, a seminal fluid sample is loaded into the inlet chamber 2. The process of spermatozoa collection begins after loading the seminal fluid sample into the inlet chamber 2 and is carried out for 30 minutes under incubation conditions of the apparatus 1 for spermatozoa selection at a temperature of 37°C. After loading the seminal fluid sample into the inlet chamber 2, the motile sperm begin to move randomly within the inlet chamber 2. Further, the motile sperm enter the microchannels 4 connecting the inlet and outlet chambers 2, 3 and extending radially therebetween. In the microchannels 4, the spermatozoa begin to move predominately in the corners of the microchannels 4 in the direction from the inlet chamber 2 to the outlet chamber 3. If for some reason a spermatozoon made a turn and began to move in the opposite direction, the spermatozoon will be redirected to the outlet chamber 3 due to the presence of branches 5. The spermatozoa which had left the outlet chamber 3 and entered accidentally the microchannel 4 will be redirected in a similar manner. The present invention is not limited to the specific embodiments disclosed in the description for illustrative purposes and encompasses all possible modifications and alternatives falling within the scope of the present invention as defined by the claims.

Claims

CLAIMS1. An apparatus for spermatozoa selection, the apparatus comprising: an inlet chamber having a non-enclosed circumferential shape and having at opposite ends thereof an inlet opening for introducing a seminal fluid sample into the inlet chamber and an outlet opening for withdrawing the seminal fluid sample from the inlet chamber, an outlet chamber located radially inward from the inlet chamber, channels connecting the inlet chamber and the outlet chamber and extending radially therebetween, wherein the channels comprise blind branches arranged in a skewed manner in the direction of the inlet chamber and terminating in rounded portions thereof on the side of the inlet chamber, the rounded portions having a radius of curvature ranging from 50 pm to 75 pm.

2. The apparatus of claim 1, wherein any cross-section of each channel, including blind branches, is rectangular.

3. The apparatus of claim 1 or 2, wherein the blind branches are arranged asymmetrically relative to the axis of the corresponding channel.

4. The apparatus of any one of claims 1 to 3, wherein the depth of the channels is 25 pm to 45 pm.

5. The apparatus of any one of claims 1 to 4, wherein the width of the channels decreases from the maximum value at the inlet chamber to the minimum value at the outlet chamber.

6. The apparatus of claim 5, wherein the minimum width of the channels is 20 pm to 80 pm.

7. The apparatus of any one of claims 1 to 6, wherein the minimum distance between the channels is 10 pm to 60 pm.

8. The apparatus of any one of claims 1 to 7, wherein a bottom portion of the apparatus is fabricated from glass or biocompatible plastic.

9. The apparatus of any one of claims 1 to 8, wherein a top portion of the apparatus is fabricated from biocompatible plastic.

10. An apparatus for spermatozoa guidance, the apparatus comprising walls defining a channel for spermatozoa guidance and an inlet opening and an outlet opening, wherein the channel comprises blind branches arranged in a skewed manner in the direction of the inlet opening and terminating in rounded portions thereof on the side of the inlet opening, the rounded portions having a radius of curvature ranging from 50 pm to 75 pm.

11. The apparatus of claim 10, wherein any cross-section of the channel, including the cross-section of the blind branches, is rectangular.

12. The apparatus of claim 10 or 11, wherein the blind branches are arranged asymmetrically relative to the axis of the channel.

13. The apparatus of any one of claims 10 to 12, wherein the depth of the channel is 25 pm to 45 pm.

14. The apparatus of any one of claims 10 to 13, wherein the width of the channel decreases from the maximum value at the inlet opening to the minimum value at the outlet opening.

15. The apparatus of claim 14, wherein the minimum width of the channel is 20 pm to 80 pm.

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