TW202348794A - Vertical thermal gradient equipment and method for progressive sperm sorting - Google Patents

Abstract

This disclosure relates to an apparatus and method for sorting motile sperms from crude semen. The enrichment of a population of motile spermatozoa with low level DNA fragmentation can be achieved by providing a 1-to-4-degree vertical temperature difference in combination with the swim-up method.

Description

Device and method for sorting sperm using vertical temperature gradient

The present invention relates to a device and method for collecting sperm, and in particular, to a device and method for high-yield sperm cells rich in health and DNA integrity.

Infertility is a major medical problem worldwide, estimated to affect 8-12% of couples of reproductive age (Agarwal et. al., Lancet 2021 ; 10271:319-333). Assisted reproductive technologies such as in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) have been widely used to treat infertility.

For the success rate of in vitro fertilization and intracytoplasmic sperm injection, selecting sperm with high motility and normal morphology is obviously the key. However, the current conventional techniques used in assisted reproduction, such as swimming upstream and density gradient centrifugation, will produce reactive oxygen species (ROS) due to centrifugation or long operation time during the operation, exposing sperm to excessively high levels. Oxidative stress can cause damage to the DNA of sperm cells (Punjabi et al., J Assist Reprod Genet . 2019 ; 36:1413-21), increasing the chance of poor embryo development, lower implantation rate, and miscarriage rate (Borges et al., Fertil Steril . 2019 ; 112(3):483-90; Zhao et al., Fertil Steril . 2014 ; 102(4)998-1005).

In order to reduce the damage to sperm DNA caused by centrifugation and long processing time, many technologies for sperm sorting using microfluidic channels have recently been developed. Horizontal microfluidic chips can sort sperm with good motility in a small volume. Only high-motility sperm can pass through the flow field and reach the concentrated area of the channel, and then be transported to the outlet. Similar patented devices include US patents US 10532357, US 10450545, and mainland China patent CN 102242055B. However, such devices are often time-consuming to operate, and because the operating capacity is too small, the recovery quantity is insufficient and cannot be used for assisted reproductive treatments.

The device disclosed in US Patent No. 10,422,737 includes a filter membrane, so that the sperm must overcome the filter membrane and gravity before reaching the outlet. However, this patent needs improvement in that when the sperm density is too low and/or the motility is insufficient, the yield and sperm quality after sorting will also be poor, especially in patients with low sperm count and poor swimming ability. Suffer.

Sperm thermotaxis is a form of sperm guidance in mammals, in which sperm actively change their swimming direction in response to temperature gradients. Because the core temperature of a woman is higher during ovulation, a temperature difference is felt in the fallopian tubes. Sperm feel the temperature difference and swim to the high temperature place to reach the egg (Lottero et al., Translational Cancer Research 2017 , 6:S427-430). Recent studies have pointed out that mouse and human sperm selected using thermotaxis have more complete DNA, can produce more blastocysts, and have a higher live birth rate by intraplasmic injection (Pérez-Cerezales et al. Scientific Reports 2018 , 8:2902.). In addition, mild heat treatment of sperm from patients with asthenozoospermia can increase the number of active sperm and the pregnancy rate (Küçük et al. J Assist Reprod Genet 2008 , 25:235–238).

The current method of thermotaxis screening of sperm is operated on a petri dish or a microfluidic chip, and a horizontal temperature gradient is provided by a laboratory heating plate or a resistance heater (Pérez-Cerezales et. al., Scientific Reports 2018 ; 8:2902). Mainland China patent CN 108504563A also discloses a horizontal temperature gradient system for sperm cell sorting. However, using the above methods does not provide the throughput required to meet IVF standards. Furthermore, in order to establish an effective temperature difference in a horizontal temperature gradient system, the cold and hot tanks must be separated by a sufficient distance, which also creates space limitations. At the same time, there is currently no thermotropic device on the market to assist in sorting motile sperm.

Therefore, in order to increase the quality and quantity of selected sperm and standardize the sorting process, the present invention provides a device for vertical temperature gradient sorting of sperm to achieve the above objectives.

In one aspect, the present invention provides a sperm sorting device, which includes: 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; disposed in the upper chamber A porous layer at the interface between the chamber and the lower chamber allows particles to pass between them and delays the heat exchange therebetween; a temperature control unit is used to maintain the temperature of the upper chamber higher than that of the lower chamber.

In some embodiments of the present invention, the temperature control unit includes a heat source for heating the upper chamber.

In some embodiments of the present invention, the hole layer is a biocompatible semipermeable membrane.

In some preferred embodiments of the present invention, the biocompatible semipermeable membrane is a polycarbonate track-etched permeable membrane or a polyvinyl alcohol track-etched permeable membrane.

In some embodiments of the present invention, the pore diameter of the hole layer is between 8-20 microns.

In some embodiments of the present invention, the upper chamber includes an inclined roof with a lower end close to the collection port and a higher end away from the collection port.

In some preferred embodiments of the present invention, the inclined roof includes a long exhaust port at the higher end to discharge air bubbles.

In some embodiments of the present invention, the inclined top plate includes a plurality of through-holes to allow the upper chamber to circulate air.

Another aspect of the present invention relates to a method for sorting sperm using the device, including: providing the device; injecting an unsorted sperm group into the lower chamber through the injection port; injecting a compatible buffer solution through the collection port In the upper chamber; maintain the temperature of the upper chamber higher than that of the lower chamber; let it stand for a culture period; collect a sorted sperm group in the upper chamber through the collection port.

In some embodiments of the invention, the temperature control unit includes a heat source in thermal contact with the upper chamber.

In some embodiments of the present invention, the temperature of the upper chamber is maintained 1-4°C higher than that of the lower chamber.

In some preferred embodiments of the present invention, the temperatures of the upper chamber and the lower chamber are maintained at 35-38°C and 30-36°C respectively.

In some embodiments of the present invention, the incubation period ranges from 15 to 30 minutes.

In some embodiments of the present invention, the compatible buffer is a commercially available sperm washing solution containing bicarbonate or HEPES buffer.

All technical features disclosed in this specification can be combined in any way. Each technical feature disclosed in this specification may be replaced by alternatives serving the same, equivalent or similar purpose. Therefore, unless expressly stated otherwise, each technical feature disclosed is merely a series of generic examples of equivalent or similar features.

Device assembly

As shown in Figure 1, the present invention provides a device, which generally includes a lower chamber 10 for storing sperm samples and an upper chamber 20 for collecting sorted sperm. The lower chamber 10 has an injection port 11 for injecting samples, and the upper chamber has a collection port 21 for injecting buffer and collecting sorted sperm. The temperature control element 400 is used to maintain the temperature of the upper chamber 20 to be higher than that of the lower chamber 10 . A porous layer 30 is provided at the junction of the two chambers, with a pore diameter of 8-20 microns, for maintaining a temperature gradient and allowing motile sperm to swim from the lower chamber 10 to the upper chamber 20 .

Generally speaking, the temperature difference between the upper chamber 20 and the lower chamber 10 is maintained at 1-4°C. In a preferred embodiment, the temperature of the upper chamber 20 is maintained at 35-38 degrees, and the temperature of the lower chamber 10 is maintained at 30-36 degrees. During sorting, the motile sperm 50 will gradually gather into the upper chamber 20, while the non-motile sperm 60 stay in the lower chamber 10. Specific examples of the temperature control element 400 may be a heating plate 410, a temperature gradient incubator 420, or simply a preheated metal sheet.

A heat source such as a heating pad 410 or a preheated metal sheet heats the fluid within the chamber by contacting the upper chamber outer wall. The heating pad 410 can be set to maintain a certain temperature, while the preheating metal sheet is preheated to a certain temperature before operation. Preferably, the temperature will be slightly higher than the desired temperature of the fluid in the chamber.

The design of the ladder temperature incubator 420 is that the heat source 421 at one end provides heat energy, and the other side has a plurality of heat dissipation holes 422 for natural cooling, or the temperature sensing element is combined with a fan for temperature control, as shown in Figure 3.

The porous layer 30 that separates the upper and lower chambers not only collects sperm swimming against gravity in the upper chamber, allowing inactive sperm to remain in the lower chamber, but also delays the thermal equilibrium between the upper and lower chambers. Generally speaking, the hole layer 30 is a biocompatible permeable membrane, which can be hydrophobic or hydrophilic. In some embodiments, the biocompatible permeable membrane may be a polycarbonate track-etched (PCTE) permeable membrane or a polyvinyl alcohol (PVA) permeable membrane.

Human sperm processing and sorting

Human sperm samples were obtained from donors who had abstained from sex for three days and whose consent was obtained. After receiving the sperm samples from the hospital, the liquefied sperm samples were first analyzed with LensHooke X1 pro (bonraybio) instrument, and then the sperm samples were divided into two groups for sorting with and without temperature gradient environment. The process of sperm sorting in the present invention is briefly described as follows. 1.5-2 ml of semen sample is injected into the lower chamber, and 1-1.5 ml of sperm washing solution (mHTF medium) is injected into the upper chamber. The heating plate and the sperm are used according to the group. No, collect the sorted sperm after 15-30 minutes of incubation. 40 μl of the recovered sperm was placed on the LensHooke CS0 chip and analyzed again with the LensHooke X1 pro (bonraybio) instrument.

Immunofluorescence staining

In order to compare the quality difference before and after sperm sorting, a sperm quantity of approximately 2×10 ⁶ was centrifuged at 400 g for 7 minutes and resuspended in PBS containing 4% paraformaldehyde. After fixation, the samples were centrifuged and rinsed with PBS. This step was repeated twice and finally resuspended in 200 μl of PBS. The fixed cells were divided into several equal parts, and then smeared on the glass slides respectively and waited for air drying. Air-dried slides can be immediately processed for immunofluorescence staining or stored at -80°C. The degree of sperm DNA fragmentation (sDF) in spermatozoa was assessed using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.

Example 1: Establishing a vertical temperature gradient.

In this and the following examples, the temperature control unit 400 includes a heat source 410 in thermal contact with the outer wall of the upper chamber 20 to generate a temperature gradient between the upper and lower chambers. The porous layer 30 is a polycarbonate track-etched transparent membrane. As shown in Figure 2, a vertical temperature gradient system is established, using an Infrared thermal imager (MT-4606, Proskit) to detect the temperature released by a mild heat source and a probe-type thermometer to measure the upper and lower liquid temperatures (Center thermometer 300 series) . The porous polycarbonate track-etched permeable membrane not only allows motile sperm to pass from the lower chamber to the upper chamber, but also has a direct effect of strengthening the temperature gradient, as shown in Figure 4. The data shows that within 30 minutes, the average temperature difference between the upper and lower chambers was measured to be 3.80-4.97 ℃ with a permeable membrane, and 0.73~1.10 ℃ without a permeable membrane. This result shows that the temperature difference can be maintained at least 3°C by incorporating the porous layer 30 into the device. Compared with the previous technology of horizontal temperature gradient sorting of sperm, the present invention only requires 4 mm of space (4 mm in the upper chamber and 4 mm in the lower chamber) to achieve the temperature gradient and only requires a simple heat source.

Example 2 Sperm motility, concentration and sorting efficiency.

The improvement of the sperm sorting effect of vertical temperature gradient can be evaluated by comparing the groups without vertical temperature gradient. Use CASA (LensHooke X1 pro bonraybio) to analyze the concentration, motility and sperm-related parameters of the sperm collected in the upper chamber 20. As shown in Figure 5, sorting using a vertical temperature gradient increased the number of sperm approximately 1.86-6 times compared to sorting at a uniform temperature (Figure 5A).

As shown in Figure 5B, using a vertical temperature gradient to sort sperm increased the recovery rate of progressive sperm (PR sperm) by 2-4 times compared to the control (using uniform temperature sorting). More importantly, this result also demonstrates that using vertical temperature gradient sorting provides a higher ratio of forward motility sperm numbers (96±4.86%) compared to uniform temperature sorting (89.27±12.71%) and unsorted of sperm samples (54±16.84%).

Example 3 Sperm speed analysis.

As shown in Figures 6A-6C, a computer-assisted sperm analysis system (LensHooke X1 pro bonraybio) was used to track the movement path of sperm after sorting. Sperm sorted using a vertical temperature gradient had significantly higher velocity parameters than unsorted sperm. Sperm sorted under a vertical temperature gradient system have higher average path speed, linear speed and curve speed, indicating that sperm that respond to temperature gradients have better speed parameters.

Example 4: Quality of Thermotropic Sperm

The TUNEL test was used to examine the degree of DNA fragmentation of the thermotropic sperm sorted by the vertical temperature gradient system to evaluate their genetic quality. As shown in Figure 7, compared with unsorted samples, the sperm selected by the device of the present invention Its fragmented DNA index can be reduced to nearly 0%.

Embodiment 5: The upper chamber has multiple holes to reduce the number of inactive sperm sorted out

When buffer solution is injected into the upper chamber 20, bubbles are often observed. This situation can be improved if the top plate of the upper chamber 20 is tilted and a long exhaust port 22 is provided at the higher end of the top plate. The generated bubbles will rise, hit the top plate and move along the slope, and finally be discharged from the elongated exhaust port 22 . On the other hand, further providing a plurality of through holes 23 on the top plate can reduce the negative pressure generated by collecting the sorted sperm in the upper chamber 20 through the collection port 21. Figures 8A and 8B are one embodiment of achieving the above purpose.

Comparing the performance of the device with and without multiple through holes 23 in sorting sperm at a temperature of 37 degrees, as shown in Figure 9, if the sorting device has multiple holes 23, the number of inactive sperm can be reduced. The results show that adding a plurality of through holes 23 to the top plate is one of the ways to effectively reduce the collection of inactive sperm.

The invention imitates the navigation mechanism of sperm in the female reproductive tract and can be used as a tool for assisting reproductive technology. By using the positive response of capacitated sperm to temperature gradients, sperm with good mobility and DNA integrity are selected.

From the above description, those skilled in the art can easily determine the essential features of the present invention, and without departing from the spirit and scope of the present invention, can make various changes and modifications to the present invention to adapt to various uses and conditions. Accordingly, other embodiments are within the scope of the following claims.

10:Lower chamber 11:Injection port 20: Upper chamber 21:Collection port 22:Long exhaust port 23:Through hole 30: Porous layer 100:Device lower layer 200: Upper level of device 400:Temperature control unit 410:Heating plate 420:Ladder temperature incubator 421:Heat source 422:Heat dissipation hole 50: Motile sperm 60: Immobile sperm

Figure 1 is a schematic diagram of the device design and working principle of the present invention. The injection port 11 is used to inject sperm samples, the collection port 21 is used to inject buffer solution and collect sorted sperm, the upper chamber 20 and the lower chamber 10 are separated by a porous layer 30, and the temperature control unit 400 is used to generate vertical temperature gradient, so that the temperature of the upper chamber 20 is higher than that of the lower chamber 10 .

Figure 2 is another schematic diagram of the device design and working principle of the present invention. Injection port 11 is used to inject sperm samples, and collection port 21 is used to inject buffer and collect sorted sperm. The upper chamber 20 and the lower chamber 10 are separated by a porous layer 30 . In order to generate a vertical temperature gradient, the temperature control unit 400 can be a heat source 410, such as a heating device such as a gentle heating pad or a handheld lukewarm heater. The heat source 410 is in contact with the upper chamber 20 to heat the buffer solution therein.

Figure 3 is an example of a vertical temperature gradient device.

Figure 4 shows that in some embodiments, when the heat source 410 is placed on the top of the device, the impact of the arrangement of the porous layer 30 on the temperature difference between the upper and lower chambers. When there is a permeable membrane in the device, the temperature difference is about 4°C, and when there is no permeable membrane, the temperature difference is about 0.8°C.

Figure 5 shows the effect of the temperature gradient system on sperm sorting: (A) Sperm concentration after sorting (/ml); (B) Progressive motility (PR) recovery percentage (the number of motile sperm in the upper chamber divided by Number of motile sperm in untreated samples); (C) Proportion of forward motility sperm. (Each point in the figure represents the measurement result of an experimental subject).

Figure 6 shows various sperm quality parameters of unprocessed sperm samples and sperm sorted with or without temperature gradient: average path velocity (VAP), straight-line velocity (VAL), curve velocity (curvilinear velocity, VCL).

Figure 7 shows the proportion of sperm DNA fragmented during the sorting process. The TUNEL method was used to immunostain sperm, and the results showed that the use of the device of the present invention can effectively reduce the degree of sperm fragmentation.

Figure 8 is an embodiment of the present invention for sorting human sperm, and a schematic structural diagram of the device: (A) top view of the upper chamber; (B) cross-sectional view of the upper chamber; (C) top view of the lower chamber; (D) ) Cross-sectional view of the lower chamber; (E) Device assembly diagram.

Figure 9 is a preferred embodiment of the present invention for sorting human sperm. It shows that if the upper chamber is equipped with a plurality of through holes, it can effectively reduce the number of inactive sperm after sorting and slightly increase the proportion of forward moving sperm.

without

10:Lower chamber

11:Injection port

20: Upper chamber

21:Collection port

30: Porous layer

50: Motile sperm

60: Immobile sperm

410:Heating plate

Claims (14)

A sperm sorting device containing: An upper chamber and a lower chamber are arranged vertically; connected to a collection port of the upper chamber; connected to an injection port of the lower chamber; A porous layer is provided at the interface of the upper chamber and the lower chamber to allow particles to pass between them and to delay the heat exchange therebetween; A temperature control unit is used to maintain the temperature of the upper chamber to be higher than that of the lower chamber. The device of claim 1, wherein the temperature control unit includes a heat source for heating the upper chamber. The device of claim 1, wherein the hole layer is a biocompatible semipermeable membrane. The device of claim 3, wherein the biocompatible semipermeable membrane is a polycarbonate track-etched permeable membrane or a polyvinyl alcohol track-etched permeable membrane. membrane. The device as claimed in claim 1, wherein the hole diameter of the hole layer is between 8-20 microns. The device as claimed in claim 1, wherein the upper chamber includes an inclined roof with a lower end close to the collection port and a higher end away from the collection port. The device of claim 6, wherein the inclined roof includes a long exhaust port at a higher end to discharge air bubbles. The device of claim 1, wherein the inclined top plate includes a plurality of through holes to allow air to circulate between the upper chamber. A method of sorting sperm using the device described in claim 1, comprising: a) Provide the device mentioned in request 1; b) Inject an unsorted sperm group into the lower chamber through the injection port; c) Inject a compatible buffer into the upper chamber through the collection port; d) Maintain the temperature of the upper chamber higher than that of the lower chamber; e) Let stand for one culture period; f) Collect a sorted sperm group in the upper chamber through the collection port. The method of claim 9, wherein the temperature control unit includes a heat source in thermal contact with the upper chamber. The method according to claim 9, wherein the temperature of the upper chamber is maintained at 1-4°C higher than that of the lower chamber. The method of claim 11, wherein the temperatures of the upper chamber and the lower chamber are maintained at 35-38°C and 30-36°C respectively. The method as described in claim 9, wherein the incubation period is between 15-30 minutes. The method of claim 9, wherein the compatible buffer is a buffer containing bicarbonate or HEPES buffer.