Vertical Temperature Gradient Apparatus and Method for Progressive Sperm Sorting

Abstract

To provide an apparatus and a method for sorting motile sperms from crude semen.SOLUTION: The enrichment of a population of spermatozoa with capacitated spermatozoa can be achieved by providing a 1-to-4-degree vertical temperature difference in combination with the swim-up method. An apparatus for processing sperm comprises a bottom chamber, a collection port, an injection port, a porous layer, and temperature control unit means.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention generally relates to an apparatus and method for sperm collection, and more particularly, to an apparatus and method for obtaining a sperm population rich in healthy and DNA-abnormal-free sperm cells at a high yield.

Background Art

[0002] Infertility is a major global health problem, estimated to affect 8-12% of couples of reproductive age (Agarwal et al., Lancet 2021; 10271: 319-333). Assisted reproductive technologies (ART) such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) are widely used in infertility treatment.

[0003] Selecting highly motile and morphologically normal sperm is an obvious key to the success of in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). The performance of current standard ART procedures such as swim-up technique and density gradient separation is not entirely satisfactory due to the generation of ROS and the increased level of oxidative DNA damage in sperm cells (Punjabi et al., J Assist Reprod Genet. 2019; 36: 1413-21).

[0004] Horizontal microfluidic chips enable the use of a small amount to separate sperm from non-viable or non-motile sperm. Only sperm with high motility were able to reach the constricted region of the channel against the flow field and be transported to the outlet. Such devices can be found, for example, in Patent Publication US10532357, CN102242055B, and US10450545. However, this process is often a time-consuming operation and may have significant volume limitations.

[0005] The device disclosed in U.S. Patent Application Publication No. 10,422,737 incorporates a filter that moves sperm relative to the filter and allows gravity to reach the outlet. However, in patients with low sperm concentration and / or low motility, it is necessary to overcome the low yield and low-quality separation results after sorting. In particular, the number of male sperm has decreased and has been deteriorating for some time in terms of swimming ability.

[0006] Thermotaxis of sperm is one of the mechanisms for selecting sperm that have acquired fertilizing ability to fertilize oocytes. Thermotaxis depends on the temperature gradient established within the fallopian tube. Due to this gradient, mammalian sperm that have acquired fertilizing ability can swim away from the uterotubal junction towards the warmer temperature where the oocyte awaits. In recent publications, sperm selection by thermotaxis in mice and humans has been shown to have higher DNA integrity and increased not only the live birth rate of intracytoplasmic sperm injection (ICSI) but also the production of blastocysts (Pérez-Cerezales et al., Scientific Reports 2018, 8:2902). In addition, mild heat treatment of azoospermic men has increased the number of motile sperm and the pregnancy rate (Kuschuk et al., J Assist Reprod Genet 2008, 25:235-238).

[0007] Current methods of sperm selection by thermotaxis have been operated on Petri dishes or microfluidic chips with a horizontal thermal gradient provided by a laboratory hot plate or resistive heater (Pérez-Cerezales et al., Scientific Reports 2018; 8:2902). Li et al. disclosed a horizontal temperature gradient system for sperm isolation (CN108504563A). However, even using the above methods, the throughput required to meet IVF criteria could not be obtained. Furthermore, in a horizontal temperature gradient system, in order to establish an effective temperature difference, it is necessary to separate the high-temperature reservoir and the low-temperature reservoir by a certain distance, resulting in spatial constraints in the system. Moreover, to date, there is no device based on thermotaxis to assist in the selection of motile sperm. SUMMARY OF THE INVENTION

Problems to be Solved by the Invention

[0008] Accordingly, in order to increase the quantity and quality of sperm selection, minimize the poor selection performed by an operator, and standardize sperm selection, the present invention provides a vertical temperature gradient sperm sorting apparatus for sperm isolation.

Means for Solving the Problems

[0009] In one aspect, the present invention relates to an apparatus for processing sperm, comprising a vertically arranged upper chamber and a lower chamber, a collection port connected to the upper chamber, an injection port connected to the lower chamber, a porous layer disposed at an interface between the upper chamber and the lower chamber to allow communication of particles between the upper chamber and the lower chamber and retard heat exchange therebetween, and temperature control unit means for maintaining the temperature of the upper chamber higher than the temperature of the lower chamber. The upper chamber has an inclined ceiling, the lower end of the inclined ceiling is close to the collection port, and the upper end of the inclined ceiling is away from the collection port.

[0010] In some embodiments, the temperature control unit comprises a heat source for heating the upper chamber.

[0011] In some embodiments, the porous layer is a biocompatible semipermeable membrane.

[0012] In some preferred embodiments, the biocompatible semipermeable membrane is a polycarbonate or polyvinyl alcohol track etching membrane.

[0013] In some embodiments, the pore size of the porous layer is 8 to 20 μm.

[0015] In some preferred embodiments, the inclined ceiling is provided with a strip vent at the upper end for discharging air bubbles.

[0016] In some embodiments, the inclined ceiling comprises a plurality of through holes for ventilating the upper chamber.

[0017] In another aspect, the present invention is a method for processing sperm using the apparatus, the method comprising providing the apparatus, introducing a population of unselected sperm through the injection port into the Lower chamber introducing into the upper chamber through the collection port, Biological introducing a compatibility buffer into the upper chamber through the collection port, Lower chamber maintaining the temperature of the upper chamber higher than the temperature of the

[0018] In some embodiments, the temperature control unit comprises a heat source in thermal contact with the upper chamber.

[0019] In some embodiments, the temperature of the lower chamber is Upward maintained at 1 to 4 °C. High maintained.

[0020] In some preferred embodiments, the temperatures of the upper chamber and the lower chamber are maintained between 35 to 38 °C and 30 to 36 °C, respectively.

[0021] In some embodiments, the period is 15 to 30 minutes.

[0022] In some embodiments, Biological the compatibility buffer is a sperm washing medium (bicarbonate or HEPES buffer).

Brief Description of the Drawings

[0023]

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DETAILED DESCRIPTION OF THE INVENTION

[0024] All features disclosed in this specification can be combined in any combination. Alternative features that serve the same, equivalent, or similar purpose can replace each feature disclosed in this specification. Thus, unless explicitly stated otherwise, each feature disclosed is merely an example of a general series of equivalent or similar features.

[0025] Device Manufacture

[0026] As shown in FIG. 1, the apparatus provided herein generally comprises a lower chamber (10) and an upper chamber (20) for semen sample deposits, and collection of sorted sperm, respectively. The lower chamber (10) has an injection port (11) for sample injection, while the upper chamber has a collection port (21) for loading buffer and collecting sorted sperm. A temperature control unit (400) is utilized to maintain the temperature of the upper chamber (20) higher than that of the lower chamber (10). A porous layer (30) having a typical pore size of 8-20 μm was placed between the two main chambers that can help maintain a temperature gradient and move motile sperm from the lower chamber (10) to the upper chamber (20).

[0027] Typically, the temperature difference between the upper chamber (20) and the lower chamber (10) is maintained at 1-4 °C. For example, the temperatures of the upper chamber (20) and the lower chamber (10) are maintained at 35-38 °C and 30-36 °C, respectively. During operation, motile sperm (50) gradually accumulates in the upper chamber (20), while non-motile sperm (60) remains in the lower chamber (10). Examples of the temperature control unit (400) can be a heating pad (410), a temperature gradient incubator (420), or simply a preheated metal sheet.

[0028] A heat source such as a heating pad (410) or a preheated metal sheet warms the internal fluid by contacting the outer wall of the upper chamber. The heating pad (410) is set to maintain the temperature. The preheated metal sheet is preheated to the temperature before operation. Preferably, the temperature is slightly higher than the desired temperature of the fluid in the upper chamber.

[0029] The thermal gradient incubator (420) is designed to provide heat from one end of the heat source (421). The other side is freely cooled by providing a plurality of holes (422) in the temperature gradient incubator.

[0030] The porous layer (30) separating the upper chamber and the lower chamber collects sperm against the gravity (50) of the upper chamber (20), not only separates the immotile sperm (60) in the lower chamber (10), but also delays the thermal equilibrium between the upper chamber and the lower chamber. Typically, the porous layer (30) is a biocompatible membrane that is either hydrophobic or hydrophilic. In some embodiments, the biocompatible membrane is a polycarbonate track etching membrane or a polyvinyl alcohol (PVA) membrane.

[0031] Handling of human sperm and sperm sorting

[0032] Human semen samples were obtained from donors after 3 days of sexual abstinence. Informed consent was obtained from each donor. After obtaining the semen samples from the hospital, the liquefied semen samples were analyzed by LensHooke X1pro (bonraybio). Subsequently, the semen samples were divided into two fractions to sort sperm in the presence and absence of temperature gradient conditions. Sperm were separated from the raw semen according to the present invention. Briefly, a 1.5 - 2 mL semen sample was injected into the bottom of the device, and the upper part of the device was filled with 1 - 1.5 mL of sperm washing medium (mHTF medium). The recovered sperm were incubated for 15 - 30 minutes in the presence or absence of a heating pad and then recovered. 40 microliters of the recovered sample was placed on a LensHooke CS0 chip (bonraybio) and analyzed with a LensHooke X1pro (bonraybio).

[0033] Immunofluorescence staining

[0034] To compare the quality of sperm before and after sorting, approximately 2×10 6 sperm were centrifuged at 400 g for 7 minutes and resuspended in PBS containing 4% paraformaldehyde. After fixation, the samples were centrifuged, washed twice with PBS, and then resuspended in PBS at 200 μL. Then, the fixed cells were aliquoted, smeared on glass microscope slides, and dried. The air-dried slides can be stored at -80 °C or directly subjected to immunofluorescence staining. The evaluation of sperm DNA fragmentation (sDF) in sperm was evaluated using the TUNEL method.

[0035] Example 1: Establishment of a vertical temperature gradient

[0036] In this example and the following examples, the temperature control unit (400) is in thermal contact with the outer wall of the upper chamber (20) and includes a heating source (410) that generates a temperature gradient between the upper chamber and the lower chamber. The porous layer (30) is a polycarbonate track etching membrane filter. As shown in Figure 2, a vertical temperature gradient system was set up, and the temperature of the mild heat source was detected by an infrared thermal imager (MT-4606, Proskit). Inserting the porous PCTE membrane not only enables motile sperm to pass from the lower chamber to the upper chamber but also has the direct advantage of increasing the temperature gradient as shown in Figure 4. As a result, the average temperature difference between the upper and lower chambers with and without the membrane was measured in the range of 3.80 - 4.97 °C and 0.73 - 1.10 °C, respectively, within 30 minutes. The temperature gradient was effective throughout the measurement range. The results suggest that placing the porous layer (30) between the sorting devices can maintain a temperature gradient of at least 3 °C. Compared with the conventional horizontal temperature gradient system for sperm separation, the temperature gradient can be achieved in a space of only 4 mm (4 mm in the upper chamber and 4 mm in the lower chamber), and only a simple heating source is required.

[0037] Example 2 Sperm motility, concentration, and separation efficiency

[0038] The improvement of sperm sorting using the vertical temperature gradient system was evaluated in parallel with sperm sorting without using the vertical temperature gradient system. When sperm were collected from the upper chamber (20), the concentration, motility, and sperm parameters were analyzed by CASA (LensHooke X1 pro bonraybio). As shown in Figure 5, sperm sorting using the vertical temperature gradient showed an increase in the total sperm count compared to sperm sorting at a uniform temperature, showing an increase of about 1.86 - 6 times (Figure 5A).

[0039] In FIG. 5B, the recovery rate of progressive sperm (PR sperm) increased by about 2 to 4 times in sperm sorting with a vertical temperature gradient compared to the control (sorting at a uniform temperature). Importantly, these results also demonstrated that the vertical thermal gradient system provided a higher percentage of PR sperm (96 ± 4.86%) compared to the uniform temperature system (89.27 ± 12.71%) and the unsorted samples (54 ± 16.84%).

[0040] Example 3 Sperm Velocity Analysis

[0041] The sperm and migration paths of the selected sperm were traced by computer-aided sperm analysis (LensHooke X1 pro bonraybio) as shown in FIGS. 6(A - C). The thermotactic selected sperm showed a series of significantly higher velocity parameters than the raw sperm samples. The values of VAP, VSL, and VCL tended to increase in the vertical temperature gradient system, suggesting that sperm responding to the temperature gradient have better velocity parameters.

[0042] Example 4 Quality of Thermotactic Sperm

[0043] The level of DNA fragmentation after sorting with the vertical sorting system by the TUNEL method was examined to evaluate the genetic quality of the sperm selected by thermotaxis. As shown in FIG. 7, the TUNEL index in sperm after selection by the vertical temperature gradient system decreased to almost 0% compared to the unselected samples.

[0044] The upper chamber with multiple through-holes reduced the number of immotile cells after sorting.

[0045] Bubbles were frequently observed when injecting the buffer into the upper chamber (20). It was found that tilting the ceiling of the upper chamber (20) and providing a strip vent (22) at the upper end of the tilt helps to improve the situation. Bubbles are generated on the ceiling during buffer injection, move along the tilt, and finally escape through the strip vent hole (22). On the other hand, by further arranging a plurality of through holes (23) in the ceiling, the negative pressure generated in the upper chamber (20) when collecting the sperm selected from the collection port (21) can be alleviated. FIGS. 8A and 8B illustrate one embodiment for achieving the above object.

[0046] As shown in FIG. 9, the performance of the device was compared at a uniform temperature (37° C.) regardless of the presence or absence of the through holes (23) in sperm selection. The sorting device with the through holes (23) reduced the number of immotile cells after sorting. The results indicate that an increase in the number of through holes (23) on the ceiling is an effective method for reducing the number of immotile sperm when performing sperm sorting.

[0047] The present invention mimics the sperm movement mechanism in the female reproductive tract and can be used as a tool in ART settings for selecting the best sperm as human sperm after the acquisition of fertilizing ability responds positively to changes in the temperature gradient.

[0048] From the above description, those skilled in the art can easily identify the essential features of the present invention and make various changes and modifications to the present invention without departing from its spirit and scope, so as to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

Claims

1. An upper chamber and a lower chamber arranged vertically, A collection port connected to the upper chamber, An injection port connected to the lower chamber, A porous layer arranged at the interface between the upper chamber and the lower chamber, enabling communication of sperm between them and delaying thermal equilibrium between them, Temperature control unit means for maintaining the temperature of the upper chamber higher than the temperature of the lower chamber, An apparatus for processing sperm, wherein the upper chamber has an inclined ceiling, the lower end of the inclined ceiling is close to the collection port, and the upper end of the inclined ceiling is away from the collection port.

2. The apparatus for processing sperm according to claim 1, wherein the temperature control unit comprises a heat source for heating the upper chamber.

3. The apparatus for processing sperm according to claim 1, wherein the porous layer is a biocompatible semipermeable membrane.

4. The apparatus for processing sperm according to claim 3, wherein the biocompatible semipermeable membrane is a polycarbonate or polyvinyl alcohol track-etched membrane.

5. The apparatus for processing sperm according to claim 1, wherein the pore size of the porous layer is 8 - 20 μm.

6. The apparatus for processing sperm according to claim 1, wherein the inclined ceiling is provided with a strip vent at the upper end for discharging bubbles.

7. The apparatus for processing sperm according to claim 1, wherein the inclined ceiling includes a plurality of through holes for ventilating the upper chamber.

8. a) Providing an apparatus for processing sperm according to claim 1, b) Injecting a population of unsorted sperm into the lower chamber through the injection port, c) Injecting a biocompatible buffer solution from the sampling port into the upper chamber, d) Maintaining the temperature of the upper chamber higher than the temperature of the lower chamber, e) Incubating for a certain period, f) Collecting a population of sorted sperm from the upper chamber through the collection port, A method for processing sperm using the apparatus for processing sperm according to claim 1, comprising the steps of.

9. The method for processing sperm according to claim 8, wherein the temperature control unit includes a heat source in thermal contact with the upper chamber.

10. The method for processing sperm according to claim 8, wherein the temperature of the upper chamber is maintained 1 - 4 °C higher than the temperature of the lower chamber.

11. The method for treating sperm according to claim 10, wherein the temperatures of the upper chamber and the lower chamber are maintained at 35 to 38 °C and 30 to 36 °C, respectively.

12. The method for treating sperm according to claim 8, wherein the period is 15 to 30 minutes.

13. The method for treating sperm according to claim 8, wherein the biocompatible buffer is a bicarbonate or HEPES buffered sperm washing medium.

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