Description:Frozen Cell Sperm Lift:--
SYSTEM AND METHOD; HOW ONLY HEALTHY SPERM IS LIFTING TO THE UPPER COMPARTMENT?
The device is made up of virgin plastic crystal. It has two compartments separated by a microporous membrane (pore size from 6 microns to 10 microns).
As the name indicates the lower compartment is on the lower side. There is a tunnel that connects the lower compartment with an inlet. The inlet portion of the tunnel is designed to make it fit with a syringe without any leakage.
The upper compartment is positioned just above the lower compartment. The length width and shape of the upper compartment are the same as with the lower compartment. But the upper compartment differs in depth. There is a small protrusion from the upper compartment, and that protrusion can be used to collect healthy sperm by a syringe or a tube. Healthy sperm can also be collected directly from the upper compartment.
The compartments are separated by a membrane that has numerous minute pores. The role of these minute pores is to allow the traveling of sperm from the lower compartment to the upper compartment. The travel of sperm is against the law of gravity is by the inherited property of human sperm by a process of POSITIVE RHEOTAXIS.
There is a lid (cover) that covers the upper compartment as well as the inlet and outlet. This lid has three small holes. This cover prevents falling minute particles or debris in the upper compartment. Which is possible during the procedure and while keeping the device inside an incubator. The purpose of holes is to maintain the pressure inside the lid. Or maintaining the inside and outside ambiance pressure. This lid also blocks the inlet with a silicon stopper to prevent the return flow of semen from the lower compartment.

BACKGROUND OF INVENTION
This invention is meant to be a system and method to separate healthy and robust sperm for Assisted Reproductive Technology (ART).
According to a Google search, 186 million individuals worldwide live with infertility. Infertility is not only a disease, it is also a social stigma. Ultimately resulting in psychological illness. Approximately 60% of infertile couples look for medical management for their infertility. Here it is worth mentioning that the male factor is responsible for up to 50% of infertility cases. Different modules of Assisted Reproductive Technology (ART) are used globally e.g. ICSI, IVF, IUI. Moreover, the world is witnessing an increase in male infertility day by day. The reason behind this seems to be environmental, psychological, and changing lifestyles. This has resulted in increasing demand for infertility treatment in infertility clinics and the demand appears to increase gradually. Separation of motile, healthy, robust, and morphologically normal as well as capable sperm to fertilize an ovum is an integral part of ICSI, IVF, and IUI. At present, the success rate of IVF/ICSI is near 50%. However, the final result will be compromised if the selected sperm is not suitable. Also, the amount of reactive oxygen species (ROS) production affects infertility management significantly.

At present, human sperm are being separated either by swim-up or swim-down method. Both methods use centrifugation of sperm. None of the presently available methods uses the mechanism of Positive Rheotaxis (Positive Rheotaxis is the movement of an organism against the flow). The present available methods of sperm separation (swim-up or swim-down) have disadvantages of centrifugation. Moreover, the presently available methods are time-consuming and need training and there is also variation in results. Further, centrifugation (350 G) itself results in the deterioration of sperm quality. ART is a technique that needs delicacy. Centrifuging the sperm at 350 G for more than one time has a direct effect on sperm quality and outcome. Last but not least is ROS generation. Lengthy procedure and centrifugation adversely affect the sperm by excessive ROS generation.
Sperm separation based on Rheotaxis needs less time to perform, no centrifugation, and no variation in result also very simple to perform. A semen sample of 1.5 mL volume can be processed by one device within a period of 30 to 45 minutes from the time of semen collection. The separated sperm can be collected in a volume between 0.5 mL to 1.5 mL as per requirement.
During the process of ICSI few best sperm are required. It is difficult to select the best one from a mixture of sperm, where all types of sperm are jumbled. Certainly, it will be preferred to select the best sperm among the best ones only.
Therefore, it will be preferable to separate the best-quality sperm before the initiation of a process like IVF, ICSI, or IUI. Above all the sperm separation method should not cause harm to sperm by any means.
SUMMARY OF INVENTION;
The present invention is a method and system to separate the most suitable sperm for ART procedures. By using this invention one can eliminate most of the factors which are responsible for the compromised or poor quality of sperm separation. The final aim of this device is to offer the best quality of sperm for ART management.
This invention includes compartments of squircle shape. Which has the benefits of a circle and a square both. Because of its square shape, it accommodates more fluid in comparison to a circle shape. On the other hand, its round corners help to not trap any liquid in the corners.
Additionally, the device has a lid. Which covers the upper compartment, inlet, and outlet portion of the device. The lid has three tiny holes that maintain the pressure of the upper compartment and facilitate the movement of active sperm from the lower compartment to the upper compartment by equalizing the pressure.
The upper compartment works as a collection compartment for best-quality sperm. The upper and lower compartments are separated by a membrane with a pore size of 5 microns to 10 microns (a microporous membrane of 10 microns has pores of different diameters ranging from 5 microns to 10 microns). And having a thickness of 10 microns. The seminal fluid, dead sperm, debris, leukocytes, any foreign bodies, and weak sperms are left in the lower compartment. Only healthy, actively motile robust sperm travel to the upper compartment.
This device does not use any chemical (silane-coated colloidal silica) or centrifugal force to separate sperm. The sperm uses its inherent nature of positive Rheotaxis and swimming-up ability to cross the membrane and collect in an upper compartment.
This device takes less time and gives the best output for all ART procedures. There is minimum human or mechanical interference.
The other benefits of this invention are mentioned in the following part of the description and diagrams.

A DETAILED DESCRIPTION OF THE INVENTION
This invention is based on the inherent property of Positive Rheotaxis and the swim-up capability of human sperm. Also, we are aware that good quality and viable sperm have a very good capability to swim in a favorable liquid medium.
This invention offers an effective way to separate good-quality sperm using the principle of positive Rheotaxis and the swim-up by using the swimming ability of human sperm. Moreover, this invention offers a method of sperm separation with minimal or no disturbance to sperm quality and function, even this invention eliminates the use of centrifugation. Therefore, there is good control over ROS generation, DNA fragmentation, and other parameters.
As shown in Picture A-1 and Picture A-2; the whole system is made up of three plates and one lid. Assembled to suit the effective method of sperm separation. All components including the lid are made up of virgin Plastic Crystal. Which is inert in nature and does not change color after sterilization by gamma radiation.
The invention has a base plate that makes the base and offers support in assembling the rest of the plate. The base plate or plate no:1 also makes the floor of the lower compartment. The system includes a lower compartment as well as an upper compartment. Plate - 2 is the second plate placed over plate: 1. The position of both plates is secured with pre-built grooves in plate no: 1 and holes for pillars in plate no: 2. Plate no: 1 and plate no: 2 make the lower compartment and a tunnel to transfer semen from the inlet to the lower compartment. There is a hole made at 90 degrees with the inlet end of the tunnel. Which (the hole) is a perfect fit with a two mL syringe. Plate: 3 is a smaller in dimension thicker (4 mm). Plate: 3 makes the upper compartment and protrusion/outlet. Plate: 3 has three pillars to securely assemble all three plates.
The partition between the lower compartment and the upper compartment is a membrane having a pore diameter of 5 microns to 10 microns. Track length 10 microns. Membrane thickness 10 microns. The membrane material may be Polycarbonate or Polyethersulfone. Both of them work but Polycarbonate gives better result.
The upper compartment is perfectly similar in shape to the lower compartment. Squircle shape is common for both the upper compartment and lower compartment. Squircle shape has the added benefit of not trapping liquid in corners and having a capacity for more liquid.
The upper compartment has a protrusion. Which is a syringe fit. The output can be collected by syringe, or transfer pipette of a catheter directly (for IUI).
The capacity of the lower compartment is 1.5 mL. Which is near normal to the collection amount (from 1 mL to 4 mL) of semen. In case the collection is less than 1.5 mL the volume can be increased by adding washing medium with collected semen to make it 1.5 mL. If the volume of collected semen is more than 1.5 mL, more than one device can be used.
The capacity of the upper compartment is 2.5 mL. In case we add 1 mL of washing medium in the upper compartment, the output will be 0.6 to 0.7 mL. Approx 0.2 mL of wash medium is traveling down in the lower compartment. This travel from the upper compartment to the lower compartment is gravity-dependent. This gravity-dependent movement of the washing medium is responsible for initiating positive Rheotaxis movement in the sperm. That brings the healthy and robust sperm into the upper compartment. And leaving the dead sperm, weak sperm, leucocytes, and debris in the lower compartment. The output may be decided from 0.6 mL to 1.5 ml according to the requirement.
ROS minimization; It’s a well-established factor that the main source of ROS in semen is Sperm and Leukocytes. The sperm, (Particularly dead and immature sperm) are responsible for generating ROS. The invention has been designed to work as fast as possible. After placing the liquified semen in the lower compartment only 20 minutes is required to go up the most motile and robust sperm to the upper compartment by the mechanism of Positive Rheotaxis. It is worth mentioning, ROS does not have any mechanism to go to the upper compartment. The second source is leukocytes. Here also the same mechanism works. With this invention, we separate the sperm from semen as quickly as possible. Therefore the sperm are being in touch with ROS for a minimum time only. Moreover, once the sperm reaches to upper compartment there is no ROS-generating factor.
Sperm Travel mechanism; The human sperm is made up of three parts; head, midpiece, and tail. The head of the human sperm is oval in shape and its size is 5 microns in length and 3.5 microns in width. The midpiece (Neck) is the part that connects the head with the tail its length is 8 microns. The tail of the flagellum is the longest part of sperm its length is approx 50 to 60 microns. The tail is part of the sperm that gives movement to the sperm through its screw-type rotating movement. If a microporous membrane with a pore size bigger than the sperm head, it will allow the sperm to travel across the membrane. The travel will be facilitated in case the pore is almost straight in nature. The healthy sperm with a fast progressive movement crosses the membrane for a period of 20 minutes. The healthy sperm choose to move up due to its Rheotaxis and swimming capability. Dead or damaged or immature sperm whose progressive movement is either nil or poor cannot cross the membrane. Hence dead, damaged, abnormal, or immature sperm stay in the lower compartment.
Thus, with this invention, there is no need for centrifugation, no need for any density gradient. Only actively motile and morphologically normal sperm will travel to the upper compartment with minimum ROS generation. Even if some ROS is being generated it will remain in the lower compartment. Lastly, this invention takes a minimum time (20 minutes only) to separate active sperm, therefore time-dependent ROS generation has less time.

Assembly of Frozen Cell Sperm Lift;
Material required:
1. Plastic crystal mould-shaped components.
2. Double-sided adhesive tape (pressure sensitive, polyester carrier, Methyl methacrylate adhesives, thickness with adhesive < 0.2 mm)
3. Microporous membranes
4. Ultrasonic cleaning bath for cleaning plastic components.
We designed the invention in four parts and assembled them to give the final shape of the invention. Named Plate: 1, Plate; 2 Plate:3 , and Lid. Plate:1 is the base and Lid is the top portion. Each part is made up of virgin plastic crystal. The virgin plastic crystal has the property that it is non-reacting at normal temperatures, is long-lasting, transparent, and does not change color after gamma radiation.
To assemble parts, pressure-sensitive, polyester carrier, Methyl methacrylate adhesives, (thickness without liner < 0.2 mm) is being used.
We tried with 5 types of membrane, Polyethersulfone membrane: 8 microns, 47 mm diameter. Polycarbonate Track Etch membrane: 8 microns, 10 microns, 12 microns, and 14 microns, each 47 mm diameter.
The base was made up of Plate: 1. It also forms the floor of the lower compartment. The thickness of the base plate is 2 mm, length – 70 mm, width – 40 mm. It has three circular grooves to hold the pillars of plate “C”.
The Plate:2 is 2.5 mm thick, length - 70 mm, and width - 40 mm. It has a squircle-shaped cut. Dimension on each side of the cut is 25 mm. Its corners are round to give the shape of a squircle. It has three holes to accommodate the pillars of Plat: 3. Each hole is of a different diameter, therefore each hole is specific for a particular pillar of Plate - 3. There is one syringe fit hole in Plate: 2, that makes an inlet. The inlet is connected to the lower compartment with a tunnel. Through the inlet and tunnel, the semen reaches the lower compartment.
The Plate: 3 is smaller in comparison to plate: 1 and Plate; 2. Its thickness is 4 mm. It also has a squircle-shaped cut. The cut is a mirror image of plate “B” cut. The dimensions of plate C are 40 mm in length and 40 mm in width (square in shape). The Plate: 3 has three pillars, that fit perfectly with the holes of Plate:2 and grooves of Plate: 1. Plate: 3 has a protrusion on the left-upper corner. The distal part of the protrusion is syringe fit. The protrusion also works as an outlet.
The Lid is a curved plate to cover the upper compartment, inlet, and protrusion. The dimensions of Lid are 48 mm in length and 40 mm in width. The Lid is a curved plate has three tiny holes. The purpose of making tiny holes is to maintain the pressure inside the cover. By maintaining the pressure the very nominal flow of liquid from the upper compartment to the lower compartment is facilitated like a normal environment. This flow is gravity-dependent. This gravity-dependent microflow initiates and maintains the positive rheotaxis movement of sperm, which is responsible for lifting or bringing up healthy sperm to the upper compartment. The other very important function of Lid is to keep the content of the upper compartment, inlet, and outlet safe from any contamination or foreign particles (which may fall while keeping the device in the incubator) in the upper compartment.
Microporous membrane; We tried with microporous membrane of 5 types, Polyethersulfone membrane: 8 microns, 47 mm diameter. Polycarbonate Track Etch membrane: 8 microns, 10 microns, 12 microns, and 14 microns, each 47 mm diameter. The shape of the membrane was cut to suit the size and shape of the upper and lower compartments.

Final assembly;
1. All the plates were cleaned with an ultrasonic bath to clean any dust or oily material used during the manufacturing process.
2. The lower surface was kept on a flat and alcohol-cleaned surface. The whole assembly is being conducted in laminar flow with full sterilization.
3. A mould cut double-sided tape was fixed with the Plat: 1. We took care that no part of double-sided tape should come in the lower compartment. Also, the tunnel from the inlet to the lower compartment was kept free from the double-sided tunnel to maintain a free flow of semen from the inlet to the lower compartment.
4. Plate: 2 was fixed over Plat: 2. Full care was taken to maintain the alignment for grooves in Plate: 1 and holes of Plate: 2.
5. A second mold-cut double-sided adhesive tape was used over plate “B”.
6. The pre-decided microporous membrane is fixed gently and loosely over double-sided tape. The membrane was kept loose to allow some flexibility in the space of the lower compartment. First, a loose membrane is helpful to prevent membrane tears. Second, we need some additional space for the flow of liquid from the upper compartment to the lower compartment. Third, there should be some volumetric change during the rheotaxis movement of sperm, loose membrane helps to accommodate this volumetric change.
7. Once again a mould-cut double-sided adhesive tape is applied over the membrane. The use of this membrane is to fix plate “B” and microporous membrane with plate “C”.
8. The microporous membrane separates the lower compartment from the upper compartment.
9. The plate “D” or lid makes the cover of the upper compartment, inlet, and outlet.
10. The whole invention is sterilized by Gamma Radiation with a dose of 25 gray.

How does it work?
The purpose of this invention is to separate the most actively motile, morphologically normal, and robust sperm for Assisted Reproductive Technology (ART). The invention works on the principle of positive Rheotaxis and also takes the support of sperm swimming capability.
The liquified semen is injected into the lower compartment in a quantity of 1.5 mL. In the upper compartment washing medium with HEPES, a buffer is spread gently in a volume of 1 ml or more according to the requirement. The cover is applied and the device with semen and washing medium is kept in an incubator at 37 degrees C for 20 minutes.
In these twenty minutes, some part of the washing medium very slowly travels down by the microporous membrane to the lower compartment. This movement of liquid facilitates the positive Rheotaxis mechanism of sperm. Rheotaxis is a property of many aquatic organisms, e.g. fish. Rheotaxis may be positive or negative. Positive rheotaxis indicates orientation and swimming against the current. Negative rheotaxis implies swimming downstream, which could allow an organism to use the current for faster motion.
It is worth mentioning that for Assisted Reproductive technology we need only sperm, the best in quality. The said positive Rheotaxis is responsible for lifting only the healthy, morphologically normal, and robust sperm to the upper compartment. By keeping the pores on a little higher side it is easy to lift the sperm to the upper compartment in minimum time. A little bit bigger pore is also required because sperm have a zig-zag movement rather than a straight-line movement.

How to use Frozen Cell Sperm Lift?
• Incubate the sample, sperm washing medium, and disposables at 35-37 degrees C for 10-20 minutes. Check the liquefaction of semen.
• Mix the semen sample properly. The semen sample must be completely liquefied.
• Take 1.5 mL of sample in a 2 mL syringe (if the volume is less than 1.5 mL add sperm washing medium to make 1.5 mL)
• Be careful not to get air-bubble in the syringe. Air bubbles reduce the functional area of the device and its performance.
• Remove the lid from the device.
• Pass 1.5 mL of sample in the lower compartment through inlet.
• Place 1 mL of sperm wash medium in the upper compartment with a pasture pipette or syringe and spread properly.
• Cover the device with lid. Incubate at 35-37 degrees C for 20 minutes.
• Take out the separated sperm from the outlet. The separated sperm can be collected by syringe, pasture pipette, or catheter directly.
• Sample is ready for procedure or study.

What solution do we offer with this invention?
At present or to date, for all Assisted Reproductive Technology sperm is being separated either by swim-up method or swim-down method (Density Gradient Centrifugation or DGC).
Drawbacks of the present method of sperm separation;
1. Both methods require a minimum of two times of centrifugation. Maybe more.
2. Both methods take enough time.
3. Both methods require training.
4. The chances of human error or man-to-man variation are very high in these methods.
5. There is enough ROS generation by centrifugation.
6. A considerable quantity of dead sperm, debris, and seminal plasma remains in the final output with the present methods. {WHO Laboratory Manual for the examination and processing of human semen Sixth Edition, 2021 - Swim-up and DGC also produce different levels of contamination with seminal components in the final sperm preparation. (page - 177)}
With our invention “Frozen Cell Sperm Lift”, we can effectively almost eliminate or reduce to a maximum extent above mentioned drawbacks. The positive aspects of this invention are:
1) No centrifugation is required.
2) Takes less time; 30 minutes to 40 minutes after semen collection.
3) Minimum training is required because processing is much less.
4) Minimum chance of human error.
5) Less generation of ROS; a) as there is no centrifugation, b) processing time is less, c) lastly but most importantly – even if there is some ROS generation it will not come to the upper compartment. Therefore, the output is free from ROS.
6) Dead sperm, debris, and seminal plasma do not reach to the upper compartment.

The drawback of this invention:
• Do not work well in viscous semen.
• Poor sample: Poor result.
• Do not work in case of Asthenozoospermia (a condition in which a person has reduced or nil sperm motility). Motility is required to lift the sperm from the lower compartment to the upper compartment.

DETAIL OF DRAWING
1. Picture A-1 is a simple one taken from an upward direction. This picture is with the properly placed lid. Picture A-2 is a diagrammatic view of invention.
2. Plate: 1, Plate: 2, Plate: 3, and Lid in color.
3. Picture B is a diagrammatic view movement of sperm through the device.
4. Bar Chart - one is a graph showing the concentration of sperm in the upper compartment after 10 minutes, 20 minutes, 30 minutes, and 40 minutes after placing the semen in lower compartment.
5. Bar chart – Two is a graphic illustration of the motility % recovered sperm of Frozen Cell Sperm Lift in comparison to the swim-up and density gradient centrifugation method.
6. Bar Chart - Three is a graphic illustration of the morphology of Frozen Cell Sperm Lift in comparison to the swim-up and density gradient centrifugation method and morphology of raw semen.
NOTE; Data for bar chart prepared after processing of six semen samples. And chart was prepared after taking their average.

Abbreviations -
• ART: Assisted reproductive technology.
• IVF: In Vitro fertilization
• ICSI: Intracytoplasmic sperm injection
• IUI: Intrauterine insemination
• ROS: Reactive oxygen species
• DNA: Deoxyribonucleic acid
• DGC: Density gradient centrifugation

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, Claims:1. The invention was assembled using two types of plastic; Acrylic and Plastic Crystal. Following gamma radiation the color of acrylic changed to yellow. Therefore, it was decided to continue with virgin-grade plastic crystal.
2. This invention is made up of four mould-shaped crystal plates. Three plates were assembled by using double-sided adhesive. While the fourth component was simply a lid with various functions. Two compartments were made by separating by using a microporous membrane of various size pores. There was an inlet and tunnel to push the semen into the lower compartment. In the upper compartment, a sperm-washing medium with H.S.A. and HEPES buffer is placed. The healthy sperm travel from the lower compartment to the upper compartment by the mechanism of positive rheotaxis. From the upper compartment, very active, motile, morphologically normal, and robust sperm is collected for assisted reproductive technology.
3. With this invention 1.5 mL of semen can be processed at a time. In case the volume is less it can be increased by adding a sperm-washing medium. The volume of semen normally ejaculated is 1 mL to 4 mL. Therefore, this invention can process at least a volume, which is normally ejaculated.
4. The output volume can be decided as per requirement. May be from 0.6 mL to 1.5 mL.
5. We used a microporous membrane to separate the most active sperm. We observed that a microporous membrane of 10 microns pore size gives the best result in a time frame of 20 minutes. A microporous membrane of 10 microns has pores from 6 microns to 10 microns. Bigger than the size of the human sperm head.
6. We observed that 20 minutes is enough to lift the most actively motile sperm in the upper compartment. Provided we used a microporous membrane of 10 microns.
7. We claim that the mechanism of Positive Rheotaxis is responsible for lifting the only healthy sperm from the lower compartment to the upper compartment. For positive rheotaxis motility of sperm is a necessary condition. Non-motile or dead sperm will stay in the lower compartment.
8. We claim a Protrusion of the upper compartment. This protrusion is used as outlet or collection point of separated sperm.
9. We claim that the base of the protrusion part is solid. The purpose is to prevent damage to the microporous membrane while collecting the output from outlet.
10. ROS; The main source of ROS generation is sperm and leucocytes. Particularly dead and immature sperm. With this invention, the positive point is that it takes minimum time and, therefore less time to generate ROS. Secondly, there is no centrifugation with this invention. A compact pellet is not formed. Keeping the dead sperm along with the live sperm in a close contact generates more ROS.
11. We claim that 20 minutes is enough to lift the most healthy and actively motile sperm. After 20 minutes it was observed that sperm count is reducing marginally. The reason may be the sperm of the upper compartment is traveling back to the lower compartment.
12. Morphology of sperm; For the morphological study of sperm we stained the raw sperm and sperm separated in the upper compartment by Polyethersulfones: 8 microns pore size and Polycarbonate membrane: 6 microns, 8 microns, 10 microns and 12 microns pore size. In almost all the cases the out have a very good morphology
13. Our claim regarding sperm recovery in the upper compartment; We observed the best output was with 10 micron pore size polycarbonate membrane. In a 12-micron pore size polycarbonate membrane, we observed less sperm concentration in the upper compartment. It seems, that with 12 microns pore size, there are both ways of sperm movement. With Polyethersulfone, sperms are being separated, but not in the required quantity. The reason may be the tortuous path of track of the Polyethersulfone membrane. On the other hand, the Polycarbonate membrane gives an almost straight path to travel sperm from the lower compartment to the upper compartment.
14. Claim of lid; This invention has a lid with three tiny holes, which are funnel shapes. The lid also has a silicon bar that closes the inlet. Thus not allowing the return of semen from the inlet. The tiny holes of the lid are funnel-shaped. The outer side of the hole is narrow. The lid in this invention has three advantages.
a. It protects the upper compartment, inlet, and outlet from contamination, from falling debris or dust particles, or even gushes of air e.g. breathing.
b. The tiny holes in the lid maintain the air pressure in the upper compartment the same as ambient. Pressure maintenance is a necessary pre-condition to lift up the sperm by the mechanism of positive rheotaxis.
c. While shifting the invention from a laminar work platform to an incubator there is a fair possibility that the device may tilt on one side and semen may come out from the inlet. The attached silicon bar prevents spillage and waste of semen.

15. Claim of sperm separation; This invention is based on the principle of positive rheotaxis. The 1.5 mL semen is pushed into the lower compartment through a syringe. The upper compartment is filled with a washing medium (with H.S.A. and HEPES buffer). Because of gravity, a very small amount of washing medium moves to the lower compartment. This movement of fluid (washing medium with H.S.A. and HEPES buffer) helps to travel up the sperm to the upper compartment by using the principle of positive rheotaxis. For positive rheotaxis swimming ability of sperm plays a crucial role. Only stronger sperm can cross the membrane in the given time. While weaker sperm, dead sperm, leucocytes, debris, and seminal fluid stay held up in the lower compartment. Thus, the outcome is actively motile, healthy, morphologically normal and robust sperm. Moreover, the final outcome is free from seminal plasma.
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