This invention relates generally to apparatus and methods for sperm collection, and more particularly to apparatus and methods for high yielding sperm populations that are enriched with health and DNA intact sperm cells.
Infertility is a major health problem worldwide and is estimated to affect 8-12% of couples in the reproductive age group (Agarwal et. al., Lancet 2021; 10271:319-333). Assisted reproductive technology (ART) such as in-vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) has been widely used for infertility treatment.
While selecting highly motile and morphologically normal sperm is the obvious key to in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) success. The performance of current standard ART procedures, such as swim-up technique and density gradient separation, is not entirely satisfactory due to ROS generation and increasing the level of oxidative DNA damage in sperm cells (Punjabi et al., J Assist Reprod Genet. 2019; 36:1413-21).
Horizontal microfluidic chips enable the use of small volumes for the separation of sperm into viable and motile sperm from non-viable or non-motile sperm. Only sperms with high motility could reach the contracted region of the channel against the flow field and be transported to the outlet. Such devices can be found, for example, in patent publications U.S. Ser. No. 10/532,357, CN 102242055B. and U.S. Ser. No. 10/450,545. However, the process is often a time-consuming task and can have severe volume restrictions.
The device disclosed in patent publication U.S. Pat. No. 10,422,737 incorporates a filter to cause sperm to move against the filter and gravity to reach the outlet. However, when patients with low sperm concentration and/or low motility, the separation result of the low yield and lower quality after sorting need to be overcome. Especially, men's sperm has been decreased in number and getting worse in swimming for some time now.
Sperm thermotaxis is one of the mechanisms for selecting capacitated spermatozoa to fertilize the oocyte. Thermotaxis is dependent on a temperature gradient established within the fallopian tube. Guided by this gradient, capacitated mammalian spermatozoa can swim away from the utero-tubal junction towards the warmer temperature where the oocyte awaits. In a recent publication, spermatozoa selection by thermotaxis in mice and human possess higher DNA integrity and increased blastocyst production as well as live birth rate of ICIS (Perez-Cerezales et al. Scientific Reports 2018, 8:2902.). In addition, a mild heat treatment of asthenozoospermic males increased the number of motile sperm and pregnancy rate (Küçük et al. J Assist Reprod Genet 2008, 25:235-238).
Current methods of sperm selection by thermotaxis were operating on a petri dish or microfluidic chip 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 spermatozoa isolation (CN 108504563A). However, using the methods described above did not provide the throughput needed to meet IVF criteria. Furthermore, in a horizontal temperature gradient system, to establish an effective temperature difference, the hot and cold reservoirs need to be separated by a distance, resulting in space constraints for the system. Besides, there's no thermotaxis-based device to assist the sorting of motile spermatozoa on the market so far.
Therefore, to increase the quantity and quality of spermatozoa selection and minimize a poor selection made by the operator and to standardize spermatozoa selection, the present invention provides a vertical temperature-gradient sperm sorting device for spermatozoa isolation.
In one aspect, the present invention relates to an apparatus for processing sperm, comprising: an upper chamber and a bottom chamber arranged vertically; a collection port connected to the upper chamber; an injection port connected to the bottom chamber; a porous layer that placed at the interface between the upper and bottom chamber to allow particles communication between them and delay heat exchange therebetween; and a temperature control unit means for maintaining the temperature of upper chamber higher than that of the bottom chamber.
In some embodiments, the temperature control unit comprises a heating source to heat the upper chamber.
In some embodiments, the porous layer is a biocompatible semipermeable membrane.
In some preferred embodiments, the biocompatible semipermeable membrane is a polycarbonate or polyvinyl alcohol track-etched membrane.
In some embodiments, the pore diameter of the porous layer is between 8 to 20 μm.
In some embodiments, the upper chamber comprises an inclined ceiling, the lower end of the inclined ceiling is close to the collection port and the higher end of the inclined ceiling is away from the collection port.
In some preferred embodiments, the inclined ceiling comprises a strip vent at the higher end to release air bubbles.
In some embodiments, the inclined ceiling comprises a plurality of through-holes to ventilate the upper chamber.
In another aspect, the present invention relates to a method for processing sperm using the said apparatus, comprising: providing the said apparatus; injecting a population of unsorted sperm into the bottom chamber through the injection port; injecting a compatible buffer into the upper chamber through the collection port; maintaining the temperature of the upper chamber higher than that of the bottom chamber; incubating a period; harvesting a population of sorted sperm from the upper chamber through the collection port.
In some embodiments, the temperature control unit comprises a heating source in thermal contact with the upper chamber.
In some embodiments, the temperature of upper chamber higher than that of the bottom is maintained between 1 to 4° C.
In some preferred embodiments, the temperatures of the upper and bottom chambers are maintained between 35 to 38° C. and 30 to 36° C., respectively.
In some embodiments, the period is between 15 to 30 minutes.
In some embodiments, the compatible buffer is a sperm washing medium (bicarbonate or HEPES buffered).
All the features disclosed in this specification may be combined in any combination. An alternative feature serving the same, equivalent, or similar purpose may replace each feature disclosed in this specification. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.
Device Fabrication
As shown in
Typically, the temperature difference between the upper chamber (20) and the bottom chamber (10) is maintained to be 1-4° C., for example, the temperatures of the upper chamber (20) and bottom chamber (10) were kept at 35-38° C. and 30-36° C., respectively. During the operation, the motile sperm (50) gradually accumulate in the upper chamber (20), while the immotile sperm (60) stay in the bottom chamber (10). Examples of the temperature control unit (400) can be a heating pad (410), a thermal gradient incubator (420), or simply a pre-warmed metal sheet.
A heating source such as a heating pad (410) or a pre-warmed metal sheet warms the fluid inside by contacting the outer walls of the upper chamber. The heating pad (410) is set to maintain a temperature. The pre-warmed metal sheet pre-warmed to a temperature before operation. Preferably, the said temperatures are slightly higher than the desired temperature of the fluid in the upper chamber.
A thermal gradient incubator (420) is designed to provide the heat from the one end of the heating source (421). The other side is either subjected to free cooling by providing a plurality of holes (422) of the thermal gradient incubator.
The porous layer (30) separating the upper and lower chambers not only concentrates the spermatozoa swum-up against gravity (50) in the upper chamber (20) and leaves immotile spermatozoa (60) in the bottom chamber (10), but also delays the thermal equilibrium of the upper and bottom chambers. Typically, the porous layer (30) is a biocompatible membrane, either hydrophobic or hydrophilic. In some embodiments, the biocompatible membrane is a polycarbonate track-etched membrane or a polyvinyl alcohol (PVA) membrane.
Human Sperm Handling and Sperm Sorting
Human semen samples were obtained from donors after 3 days of sexual abstinence. Informed consent was obtained from each donor. After obtaining semen samples from the hospital, liquefied-semen samples were then analyzed by LensHooke X1 pro (bonraybio). Subsequently, a semen sample was split into two fractions for sperm sorting in the presence and absence of temperature gradient condition. Spermatozoa were separated from crude semen by the present invention. Briefly, a semen sample of 1.5˜2 mL was injected into the bottom of the device; the upper part of the device was filled with 1˜1.5 mL of sperm washing medium (mHTF medium). Recovered sperms were collected after 15 to 30 minutes incubation in the presence or absence of heating pad. Forty microliters of recovery samples were placed on a LensHooke CS0 chip (bonraybio) and analyzed with LensHooke X1 pro (bonraybio).
Immunofluorescence Staining
To compare the quality of spermatozoa before and after sorting, approximately 2×106 spermatozoa were centrifuged at 400 g for 7 minutes and resuspended in PBS containing 4% of paraformaldehyde. After fixation, the samples were centrifuged and washed with PBS twice, and then resuspended in PBS at 200 μL. Fixed cells were then aliquoted and smeared onto a glass microscope slide and left to dry. The air-dried slides can be placed into storage at −80° C. or performed immunofluorescence staining directly. The assessment of sperm DNA fragmentation (sDF) in spermatozoa was evaluated by using with TUNEL assay.
In this and the following examples, the temperature control unit (400) comprises a heating source (410) in thermal contact with the outer wall of the upper chamber (20) to generate a temperature gradient between the upper and bottom chambers, and the porous layer (30) is a polycarbonate track-etched membrane filter. A vertical thermal gradient system was set up as described in
The improvement of sperm sorting with a vertical thermal gradient system was assessed in parallel to sperm sorting without a vertical thermal gradient system. Once the sperm was collected from the upper chamber (20), we analyzed the concentration, motility as well as sperm parameters by CASA (LensHooke X1 pro bonraybio). As shown in
In
The sperms and moving paths of the sorted sperm were tracked by the computer-aided sperm analysis (LensHooke X1 pro bonraybio), as shown in
To assess the genetic quality of spermatozoa selected by thermotaxis, we examined DNA fragmentation level after sorting with our vertical sorting system by TUNEL assay. As shown in
Bubbles were frequently observed when injecting buffer into the upper chamber (20). Sloping the ceiling of the upper chamber (20) and deploying a strip vent (22) at the higher end of the slope was found helpful to improve the situation. The bubbles arise during buffer injection contact the ceiling, move along the slope, and finally release through the strip vent (22). On the other hand, further disposing a plurality of through-holes (23) on the ceiling can relieve the negative pressure generated in the upper chamber (20) when collecting the sorted sperm through the collection port (21).
The performance of the devices with or without through-holes (23) in sperm sorting was compared at a uniform temperature (37° C.), as shown in
This invention imitates the avigation mechanism of spermatozoa in the female genital tract, it could be used as a tool in the ART setting to select the best spermatozoa as human spermatozoa after capacitation responds positively to a temperature gradient change.
From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.