SPERM SORTING APPARATUS AND SPERM SORTING METHOD

A sperm sorting apparatus and a sperm sorting method are provided. The sperm sorting apparatus includes a medium chamber, a waste chamber and a sorting channel communicated with and extending along a first direction in between. The medium chamber is configured to contain a medium solution. The waste chamber disposed aside the medium chamber is configured to contain a residual solution after sorting. The residual solution includes low motility sperms and/or dead sperms. The sorting channel is configured to be inserted with a sperm sample, such that sperms in the sperm sample are sorted in corresponding to a medium solution flow from the medium chamber. The sorting channel has first and second portions. The first portion is closer to the medium chamber than the second portion. A width of the first portion measured along a second direction is greater than a critical dimension, ranging from 200 μm to 400 μm.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the priority benefit of Taiwan application serial no. 110107226, filed on Mar. 2, 2021. The entirety of the above-mentioned patent application is hereby incorporated by reference herein and made a part of this specification.

BACKGROUND

Technical Field

The present invention relates to a sperm sorting apparatus and a sperm sorting method.

Description of Related Art

Infertility has become one of the common problems in modern society. Various artificial fertilization methods have been developed for addressing such problem. For instance, current artificial fertilization methods include intrauterine insemination (IUI), in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) and so forth. These methods require high motility sperms for each test. Hence, a method for sorting out high motility sperms is important in the field of artificial fertilization. Currently, finding a method for sorting out great amount of the high motility sperms from a sperm sample is quite challenging.

SUMMARY

In an aspect of the present invention, a sperm sorting apparatus is provided. The sperm sorting apparatus comprises: a medium chamber, configured to contain a medium solution; a waste chamber, disposed aside the medium chamber, and configured to contain a residual solution obtained after sorting, wherein the residual solution includes low motility sperms and/or dead sperms; and a sorting channel, extending between and communicated with the medium chamber and the waste chamber along a first direction, and configured to be inserted with a sperm sample, such that sperms in the sperm sample are sorted in corresponding to a medium solution flow entering the sorting channel from the medium chamber, wherein the sorting channel has a first portion and a second portion, the first portion is closer to the medium chamber than the second portion, a width of the first portion measured along a second direction is greater than a critical dimension, the second direction is intersected with the first direction, and the critical dimension ranges from 200 μm to 400 μm.

In some embodiments, sperm sorting apparatus further comprises parallel micro-channels, extending along the second direction and disposed between the medium chamber and the sorting channel.

In some embodiments, the first portions of the sorting channel converges toward the medium chamber, a width of at least a section of the first portion measured along the second direction is greater than the critical dimension.

In some embodiments, the sorting channel is communicated to outside of the sperm sorting apparatus through an input/output hole.

In some embodiments, the input/output hole is communicated with the first portion of the sorting channel.

In some embodiments, the second portion of the sorting channel converges toward the waste chamber.

In some embodiments, an end portion of the sorting channel communicated with the waste chamber is a narrow channel, and a width of the sorting channel measured along the second direction is at its minimum at the narrow channel.

In another aspect of the present invention, a sperm sorting method is provided. The sperm sorting method comprises: providing a sperm sorting apparatus, comprising a medium chamber, a waste chamber and a sorting channel extending between and communicated with the medium chamber and the waste chamber along a first direction, a width of at least a section of the sorting channel measured along a second direction is greater than a critical dimension, and the critical dimension ranges from 200 μm to 400 μm; inserting a medium solution to the medium chamber and the waste chamber, till liquid levels in the medium chamber, the sorting channel and the waste chamber are balanced; inserting a sperm sample into the sorting channel; additionally inserting a medium solution into the medium chamber, such that the medium solution in the medium chamber flows into the sorting channel to form a medium solution flow, wherein sperms in the sperm sample are sorted in corresponding to the medium solution flow; and extracting a sorted solution from the sorting channel.

In some embodiments, the sperm sorting method further comprises: rinsing the medium chamber, the sorting channel and the waste chamber with an additional medium solution before the step of inserting the medium solution to the medium chamber and the waste chamber.

In some embodiments, the sperm sorting method further comprises: intermittently repeating the step of additionally inserting a medium solution into the medium chamber before the step of extracting the sorted solution from the sorting channel.

The sperm sorting apparatus according to embodiments of the present invention utilizes both of a behavioral tendency that the high motility sperms swim against the medium solution flow and a behavioral tendency that the high motility sperms gather across the inner surface of the sorting channel, thus a great amount of high motility sperms can be extracted by using the sperm sorting apparatus. As a result, in addition to be applicable for the ICSI artificial fertilization method, the sperm sorting apparatus10may be further applicable for the WF artificial fertilization method. During a sperm sorting process, the high motility sperms at least mostly stay in the sorting channel since being inserted into the sorting channel, while the low motility and/or dead sperms are carried to the waste chamber along the medium solution flow. Subsequently, the sorted solution in the sorting channel may be extracted from the sorting channel. In other words, a moving distance of the high motility sperms is short, thus the sorting can be performed in a short time, and energy consumption of the high motility sperms can be low. Moreover, since the sperm sample is directly inserted into the sorting channel, substantially all of the sperms in the sperm sample can be ensured to be sorted, thus a great sorting efficiency can be promised.

DESCRIPTION OF THE EMBODIMENTS

FIG. 1Ais a three-dimensional perspective view schematically illustrating a sperm sorting apparatus10according to some embodiments of the present invention.FIG. 1Bis a three-dimensional view schematically illustrating the sperm sorting apparatus10in an assembled state, according to some embodiments of the present invention.

Referring toFIG. 1A, the sperm sorting apparatus10includes a medium chamber100, a waste chamber110and a sorting channel120extending between and communicated to the medium chamber100and the waste chamber110. The medium chamber100is configured to contain a medium solution. As will be further described, high motility sperms in a sperm sample inserted into the sorting channel120swim against the medium solution flowing into the sorting channel120from the medium chamber100, and stay in the sorting channel120, to be extracted. On the other hand, a residual solution obtained after sorting and including low motility and/or dead sperms flow into the waste chamber110along the sorting channel120, and can be collected. In this way, the sperm sample can be sorted by the sperm sorting apparatus10.

In some embodiments, the sperm sorting apparatus10is formed by attaching a top substrate SB1with a bottom substrate SB2. In these embodiments, the bottom substrate SB2may be a flat plate, and the top substrate SB1may have openings and a recess at its bottom surface. For instance, the openings of the top substrate SB1may include an opening P100for forming the medium chamber100after the top substrate SB1is attached with the bottom substrate SB2, and may include an opening P110for forming the waste chamber110after the top substrate SB1is attached with the bottom substrate SB2. In addition, the recess of the top substrate SB1may include a recess R120for forming the sorting channel120after the top substrate SB1is attached with the bottom substrate SB2. A portion of the bottom substrate SB2overlapped with the opening P100may define a bottom surface of the medium chamber100, and a sidewall of the opening P100may define a sidewall of the medium chamber100. A portion of the bottom substrate SB2overlapped with the opening P110may define a bottom surface of the waste chamber110, and a sidewall of the opening P110may define a sidewall of the waste chamber110. In addition, a portion of the bottom substrate SB2overlapped with the recess R120may define a bottom surface of the sorting channel120, and a surface of the recess R120(i.e., the recessed surface at the bottom surface of the top substrate SB1) may define a top surface and a sidewall of the sorting channel120.

The sperm sorting apparatus10may further include an input/output hole H120communicated with the sorting channel120. The sperm sample may be inserted into the sorting channel120from outside of the sperm sorting apparatus10through the input/output hole H120, and the high motility sperms from the sperm sample may be extracted from the sorting channel120through the input/output hole H110. In those embodiments where the sperm sorting apparatus10is formed by attaching the top substrate SB1with the bottom substrate SB2, the input/output hole H110penetrates through a portion of the top substrate SB1having the recess R120, so as to communicate with the sorting channel120defined by the recess R120of the top substrate SB1and the portion of the bottom substrate SB2overlapped with the recess R120.

Referring toFIG. 1B, after the top substrate SB1is attached with the bottom substrate SB2, the medium chamber100and the waste chamber110are defined. However, since the sorting channel120may be an inner space in the sperm sorting apparatus10, the sorting channel120may not be observed from the appearance of the sperm sorting apparatus10, except for the input/output hole H120communicated to the sorting channel120. In some embodiments, a thickness T1of the portion of the top substrate SB1having the recess R120is less than a thickness T2of the portions of the top substrate SB1having the openings P100, P110. The thickness T1may be slightly greater than a height D120of the sorting channel120as will be described with reference toFIG. 3. Further, in these embodiments, a ratio of the thickness T2with respect to the thickness T1may range from, for example, 4 to 5. For instance, the thickness T2may range from 4 mm to 20 mm, while the thickness T1may range from 1 mm to 4 mm. On the other hand, in some embodiments, the bottom substrate SB2has a consistent thickness T3. For instance, the thickness T3may range from 1 mm to 4 mm.

FIG. 2Ais a plan view schematically illustrating the bottom surface of the top substrate SB1as shown inFIG. 1A.FIG. 2Bis an enlarged three-dimensional view schematically illustrating micro-channels MC as shown inFIG. 1AandFIG. 2A.

Referring toFIG. 1AandFIG. 2A, the sorting channel120may have a first portion120aclose to the medium chamber100, and may have a second portion120bclose to the waste chamber110. The first portion120aof the sorting channel120may converge toward the medium chamber100, while the second portion120bof the sorting channel120may converge toward the waste chamber110. In other words, a width W120aof the first portion120amay gradually decrease toward the medium chamber100, and a width W120bof the second portion120bmay gradually decrease toward the waste chamber110. The high motility sperms have a behavioral tendency of swimming against flow (e.g., a medium solution flow), such that the high motility sperms may gather in a region where a flow velocity is relatively high. By having the portion of the sorting channel120close to the medium chamber100(i.e., the first portion120a) converges toward the medium chamber100, the medium solution may enter the sorting channel120with a relatively high flow velocity, and may gradually slow down while away from the medium chamber100. In this way, the high motility sperms may gather in the portion of the sorting channel120close to the medium chamber100(i.e., the first portion120a), whereas low motility and/or dead sperms may move toward the waste chamber110along the medium solution flow. In addition, by having the portion of the sorting channel120close to the waste chamber110(i.e., the second portion120b) converges toward the waste chamber110, the medium solution flow that carries the low motility and/or dead sperms and leaves for the waste chamber110from the sorting channel120may be accelerated. In some embodiments, an end portion of the second portion120bof the sorting channel120(i.e., an end of the sorting channel120directly communicated with the waste chamber110) is designed as a narrow channel NC, such that the medium solution flow that carries the low motility and/or dead sperms may enter the waste chamber110with a further accelerated flow velocity. In these embodiments, the narrow channel NC may be a necking portion of the sorting channel120, and the width W120bis significantly reduced to a minimum at the narrow channel NC. Moreover, in some embodiments, a convergence rate of the first portion120aof the sorting channel120is less than a convergence rate of the second portion120bof the sorting channel120. In other words, a reduction rate of the width W120amay be less than a reduction rate of the width W120b. By such design, the medium solution flow from the medium chamber100may be avoided from entering the sorting channel120with an excessively high flow velocity, which may result in carrying the high motility sperms to the portion of the sorting channel120close to the waste chamber110(i.e., the second portion120b). For instance, the width W120amay range from 20 mm to 30 mm. In addition, the width W120bmay range from 1 mm to 28 mm, and the width W120bmay be 1 mm at the narrow channel NC. On the other hand, a length L120aof the first portion120amay range from 17 mm to 20 mm, while a length L120bof the second portion120bmay range from 15 mm to 18 mm.

In some embodiments, the input/output hole H120is communicated with the first portion120aof the sorting channel120, where the high motility sperms swim against the medium solution flow and gather. Accordingly, more of the high motility sperms can be extracted from the input/output hole H120. However, the input/output hole H120should be laterally spaced apart from the medium chamber100by an appropriate distance, in order to prevent the sperms in the sperm sample inserted into the sorting channel120through the input/output hole H120from flowing back to the medium chamber100. For instance, the input/output hole H120may be close to a side of the first portion120athat is away from the medium chamber100.

In some embodiments, the sorting channel120further has a third portion120cbetween the first portion120aand the second portion120b. A width W120cof the third portion120cmay be substantially equal to a maximum value of the width W120aof the first portion120aand a maximum value of the width W120bof the second portion120b. For instance, the width W120cof the third portion120cof the sorting channel120may range from 28 mm to 30 mm. Further, a length L120cof the third portion120cof the sorting channel120may range from 9 mm to 12 mm.

Referring toFIG. 2AandFIG. 2B, in some embodiments, the first portion120aof the sorting channel120is communicated with the medium chamber100through parallel micro-channels MC. A width WMCof each micro-channel MC (shown inFIG. 2B) is significantly less than a width of the sorting channel120(e.g., the width W120aas described with reference toFIG. 2A), in order to prevent the high motility sperms gathering in the first portion120aof the sorting channel120from flowing back to the medium chamber100through the micro-channels MC. For instance, the width WMCof each micro-channel MC may range from 0.2 mm to 0.4 mm. In some embodiments, the micro-channels MC are defined by fin structures FN at the bottom surface of the top substrate SB1. The fin structures FN protrude from the recessed surface of the recess R120, and are arranged side-by-side between the medium chamber100and a side of the sorting channel120. Each of the fin structures FN may be located between adjacent micro-channels MC. In addition, after the top substrate SB1is attached with the bottom substrate SB2(as shown inFIG. 1B), the fin structures FN may contact the bottom substrate SB2, such that the medium chamber100may be communicated with the sorting channel120only through the micro-channels MC. Accordingly, a height of the fin structures FN may be substantially equal to a depth of the recess R120.

FIG. 3is a cross-sectional view schematically illustrating the sorting channel120in the sperm sorting apparatus10according to some embodiments of the present invention. It should be noted that, behavioral tendencies of the high motility sperms HM and low motility and/or dead sperms LM will be described with the first portion120aof the sorting channel120. However, such behavioral tendencies can be also observed in the second portion120band the third portion120cof the sorting channel120. In addition, an arrow AR indicates a flow direction of the medium solution flow.

Referring toFIG. 2AandFIG. 3, in addition to the behavior tendency of swimming against the medium solution flow, the high motility sperms further tend to gather at a top surface, a sidewall and a bottom surface of the sorting channel120in certain condition. Specifically, the high motility sperms HM in the sperm sample has such behavior tendency (i.e., gathering at the top surface, the sidewall and the bottom surface of the sorting channel120) while being in a segment of the sorting channel120having a width greater than a critical dimension. On the other hand, the low motility and/or dead sperms LM may be left in a central passage of the sorting channel120, and may be carried to the waste chamber110(as described with reference toFIG. 2A) by the medium solution flowing through such central passage (as indicated by the arrow AR). Consequently, more of the high motility sperms HM may gather in the sorting channel120, then extracted from the sorting channel120. In some embodiments, the width W120a(even its minimum value) of the first portion120aof the sorting channel120is controlled to be greater than the critical dimension, such that the high motility sperms HM tend to gather in the first portion120aof the sorting channel120. For instance, the critical dimension may range from about 200 μm to about 400 μm (e.g., substantially equal to about 400 μm), and the width W120amay range from 20 mm to 30 mm. Further, the width W120cof the third portion120cof the sorting channel120may also be greater than the critical dimension, such that a portion of the high motility sperms HM may gather at a top surface, a sidewall and a bottom surface of the third portion120c. Similarly, the width W120bat a certain section of the second portion120bof the sorting channel120may be greater than the critical dimension as well, such that a portion of the high motility sperms HM may gather in the second portion120b.

On the other hand, if a width of a segment of the sorting channel120through which the sperm sample flows is less than the critical dimension, the high motility sperms HM may otherwise gather at corners of such segment (e.g., a corner defined by a top surface and a sidewall of such segment and a corner defined by the sidewall and a bottom surface of such segment), and may not spread across the top surface, the sidewall and the bottom surface of such segment. Therefore, fewer high motility sperms HM may gather in such segment. If a width of the sorting channel120is mostly or completely less than the critical dimension, then an amount of the extracted high motility sperms HM may be significantly limited.

In some embodiments, a height Duo of the sorting channel120may be about 100 μm. Moreover, portions of the sorting channel120(including the first portion120a, the second portion120band the third portion120c) may have substantially identical height (i.e., the height D120). In other words, both of the top surface and the bottom surface of the sorting channel120may be substantially flat surfaces.

The sperm sorting apparatus10as described utilizes both of a behavioral tendency that the high motility sperms swim against the medium solution flow and a behavioral tendency that the high motility sperms gather across the inner surface of the sorting channel120, thus a great amount of high motility sperms can be extracted by using the sperm sorting apparatus10. As a result, in addition to be applicable for the ICSI artificial fertilization method, the sperm sorting apparatus10may be further applicable for the WF artificial fertilization method. For instance, by inserting a 100 μl sperm sample with sperm concentration about 12 M/ml into the sperm sorting apparatus10, about 186,000 high motility sperms can be extracted. Further, by such extraction, a content of high motility sperms in the sperm sample is significantly raised from about 43.1% to about 91.3%. As another example, by inserting a 100 μl sperm sample with sperm concentration about 20.2 M/ml into the sperm sorting apparatus10, about 156,000 high motility sperms can be extracted. In addition, by such extraction, a content of high motility sperms in the sperm sample is significantly raised from about 39.6% to about 92.3%.

FIG. 4is a flow diagram illustrating a sperm sorting method according to some embodiments of the present invention. Such sperm sorting method is performed by using the sperm sorting apparatus10as described above, and will be described with reference toFIG. 4andFIG. 2A.

Referring toFIG. 4andFIG. 2A, step S400is performed, and the sperm sorting apparatus10is rinsed by a few medium solution. In some embodiments, the medium solution is inserted into the medium chamber100, and flows to the waste chamber110through the micro-channels MC and the sorting channel120, to rinse the whole sperm sorting apparatus10. In addition, in some embodiments, the medium solution is Vitromed Sperm Wash.

At step S402, more medium solution is inserted into the medium chamber100and the waste chamber110. In some embodiments, the medium chamber100and the waste chamber110are respectively inserted with 500 μl medium solution in the current step. Liquid levels in the medium chamber100, the sorting channel120and the waste chamber110may become balanced after a period of time.

At step S404, a sperm sample is inserted into the sorting channel120. The sperm sample may be inserted into the sorting channel120through the input/output hole H120. In some embodiments, the sperm sample is a solution including sperm specimen and medium solution. In addition, in some embodiments, 100 μl of the sperm sample may be inserted into the sorting channel120.

At step S406, additional medium solution is inserted into the medium chamber100. Such additional medium solution may flow to the sorting channel120after the insertion, and form a medium solution flow in the sorting channel120. In corresponding to the medium solution flow, the high motility sperms in the sorting channel120may exhibit the behavioral tendencies of swimming against the medium solution flow and gathering across the inner surface of the sorting channel120. In some embodiments, the step of inserting additional medium solution into the medium chamber100is intermittently performed multiple times. For instance, 120 μl of medium solution is inserted into the medium chamber100for every 2 minutes in a total time of 10 minutes.

At step S408, a sorted solution is extracted from the sorting channel120. A pipette may be used for extracting the sorted solution from the sorting channel120through the input/output hole H120. The sorted solution includes the high motility sperms and the medium solution, and may include a few low motility and/or dead sperms. In some embodiments, the sorted solution of about 65 μl may be extracted from the sorting channel120.

Up to here, the sperm sorting process according to some embodiments of the present invention has been completed. As described above, the high motility sperms at least mostly stay in the sorting channel120since being inserted into the sorting channel120, while the low motility and/or dead sperms are carried to the waste chamber110along the medium solution flow. Subsequently, the sorted solution in the sorting channel120may be extracted from the sorting channel120. In other words, a moving distance of the high motility sperms is short, thus the sorting can be performed in a short time, and energy consumption of the high motility sperms can be low. Moreover, since the sperm sample is directly inserted into the sorting channel120, substantially all of the sperms in the sperm sample can be ensured to be sorted, thus a great sorting efficiency can be promised.