A METHOD SUITABLE FOR USE IN DETERMINATION OF NON-APOPTOTIC

SPERMS

Technical Field

The present invention is related to a method enabling the selection of viable non-apoptotic sperms in semen samples which can be used in assisted reproduction techniques .

Prior Art

The microinjection method (ICSI-Intracytoplasmic sperm injection), which is a common method used in infertility treatments, sperm is microinjected into an oocyte by using a micromanipulator.

Sperm and oocyte quality are the two major factors that directly affect the embryo development and the success of the treatment. Since the number of oocytes is limited (average 10), it is not possible to select the oocytes, and all oocytes are used for microinjection, the large number of sperms (approximately 10 million) makes it possible to select sperms. However, the only criteria used is the morphological criteria are for choosing sperm selection, which generally does not reflect the genetic, metabolic and, functional status of the sperm. As in all other cells, the apoptosis mechanism in sperms is triggered by a problem in the internal dynamics of the cell. The cell destructs itself as a result of this mechanism which is also defined as ‘programmed cell death’. Since a big variety of reasons (genetic, metabolic, or functional) may activate the internal control mechanism in a cell, apoptosis indirectly indicates these problems.

The selection of non-apoptosic sperm will make it possible to select a healthy sperm.

DNA fragmentation and activation of caspase cascade , are the main indicators of apoptosis, which are used to diagnose apoptosis in sperms. However ut all the techniques used to detect apoptosis can only be applied to fixed or lysed dead sperm cell. The apoptosis rates obtained can only be used only to inform and counsel the patient instead of treating them.

A 30% DNA fragmentation rate is accepted as the cut-off value for normal fragmentation index, and the below rates are known to decrease fertilization rates, embryo development, embryo quality, implantation, pregnancy and live birth rates and increase abortus rates during in individuals. This is of great importance in an in vitro fertilization cycle in which the chance of success does not exceed 40% even in couples with the best cohort because the pregnancy failure after each IVF cycle the patient have no other chance than starting a new cycle, which is cost and time consuming, which increases the possibility of medical complications and which is physiologically, psychologically and socially challenging for couples. The high chance of success in patients with a low or no apoptosis rate provide evidence for the importance of this situation. However, approximately half of the patients undergoing infertility treatment are because of male factor, who are known to have higher apoptosis rates than normal population. It has been shown that the rate is increasing in the unexplained infertility group which is suggested to be one of the causes of the problem in in this population.

In the light of all this information, it is of great importance to select the healthy sperm cells to use during infertility treatments.

In the prior art, the process of separating apoptotic and non-apoptotic sperms can be performed by using the flow-cytometry method. However, the reasons such as both the technique and the consumables used in this method are very expensive, requires extra training and experience, and the non- sterility, it is available only in very few centers, , for research purposes only.

Considering the current situation, it is obvious that there is a need for a cost-effective, easily applicable method that can be used to separate non-apoptotic sperms in a sperm sample by in vivo labeling.

For this purpose, unlike known techniques, instead of a sperm selection method based solely on motility and morphology, it is aimed to develop a method that permits the selection of non- apoptotic sperm cells thus giving indirect information about the metabolic, genetic, and functional status of the sperm.

Description of the Figures Figure 1 shows the microinjection technique used in the selection of non-apoptotic sperm selected by the method according to the invention and microinjecting them with oocytes. The equivalents of the numbers in the figure are shown below.

1: Microinjection media

101: Polyvinylpyrrolidone (PVP) drops

102: Second washing drop

103: First washing drop

104: Drop-in which oocytes are placed (Microinjection drop)

105: Mixture of labeled bead and sperm sample

Detailed Description of the Invention

The present invention is directed to the use of a technique that allows labeling of apoptotic sperms in a sperm sample in in vitro conditions, thus allowing to select sperm cells with no apoptosis.

The method of the invention differs from known methods in that it enables analysis in living sperms, allows sperm cells with no apoptosis for microinjection (ICSI) method, sterile operation, and does not require extra equipment, and training.

In one aspect, the invention is related to a method to be used in determination of non- apoptotic (healthy) sperms in a sperm sample under in vitro conditions, said method comprises the steps of; a. Preparing latex beads coated with a protein with phosphatidylserine affinity or b. Preparing streptavidin polystyrene beads coated with a protein with a biotinylated

Phosphatidylserine affinity and c. Treating the beads obtained by the method described in steps a) or b) with a sperm sample and d. Observing the sperm sample with a light microscope or inverted microscope or phase- contrast microscope, and e. Defining sperms that do not have adhered beads as non-apoptotic (healthy) sperm.

Within the scope of the present invention, sperm with adhered beads are defined as apoptotic sperm.

In a preferred embodiment of the invention, the method to be used to determine non-apoptotic (healthy) sperms in the sperm sample under in vitro conditions comprises the steps of; a. Preparing latex beads coated with a protein with phosphatidylserine affinity b. Treating the beads obtained by the method described in step a) with a sperm sample c. Observing the sperm sample with a light microscope or inverted microscope or phase- contrast microscope d. Defining sperms that do not have adhered beads as non-apoptotic (healthy) sperm.

In a preferred embodiment of the invention, the method to be used to determine non-apoptotic (healthy) sperms in the sperm sample under in vitro conditions comprises the steps of; a. Preparing streptavidin polystyrene beads coated with a protein with a biotinylated Phosphatidylserine affinity b. Treating the beads obtained by the method described in step a) with a sperm sample c. Observing the sperm sample with a light microscope or inverted microscope or phase- contrast microscope d. Defining sperms that do not have adhered beads as non-apoptotic (healthy) sperm.

Studies show that streptavidin, biotin, and polystyrene do not harm sperm viability, which provides an important advantage in selecting a living and healthy sperm sample and obtaining a healthy embryo by using it in microinjection.

In a preferred embodiment of the invention, after the identification of apoptotic sperms with the method according to the invention, healthy sperms that do not adhere to beads are individually distinguished.

This differentiation process can be performed by aspiration or by selecting sperm that do not adhere to beads.

In a preferred embodiment of the invention, the healthy sperm selected by the method according to the invention is injected into the oocyte in the microinjection process.

In a preferred embodiment of the invention, the healthy sperm selected with the method according to the invention is tested for sperm toxicity.

In a preferred embodiment of the invention, the embryos obtained by injecting the healthy sperm selected with the method according to the invention into the oocyte in the microinjection process, an embryotoxicity test is applied.

In a preferred embodiment of the invention, a sterility test is applied to the healthy sperm selected by the method according to the invention and the embryo obtained by injecting this sperm into the oocyte.

In a preferred embodiment of the invention, step a) includes the steps of al) pretreating the latex beads and a2) coating the pretreated latex beads with a protein with an affinity for phosphatidylserine. In a preferred embodiment of the invention, al) the process of pretreatment of latex beads includes the steps of diluting the latex bead solution, preferably 40 mg/ml of IDC UltraClean Amidine Latex bead solution, in a suitable buffer, preferably 0.025M, pH 6 MES buffer (2-(N-morpholino)ethanesulfonic acid), centrifuging the resulting mixture, centrifuging by diluting the particles with buffer again, preferably with 0.025M, pH 6 MES buffer (2-(N-morpholino)ethanesulfonic acid) after discarding the supernatant, and after discarding the supernatant, suspending the particles with buffer, preferably 0.025M, pH 6 MES buffer (2-(N-morpholino)ethanesulfonic acid), preferably to form a 2% solution.

In a preferred embodiment of the invention, a2) the process of coating pre-treated latex beads with a protein with phosphatidylserine affinity includes the steps of; adding the solution containing EDAC (l-Ethyl-3-(3-dimethylaminopropyl carbodiimide) (e.g., 100 mg of EDAC in 2 ml of MES) at a rate of 50 mg/ml in a suitable buffer, preferably in 0.025M, pH 6 MES buffer (2-(N-morpholino)ethanesulfonic acid) to the latex bead solution obtained in step al), adding the protein with the affinity of the phosphatidylserine in a suitable buffer, preferably, 0.025M, pH 6 MES buffer (2-(N-morpholino)ethanesulfonic acid) to the resulting mixture, incubating the mixture at room temperature for 2-5 hours, then centrifuging the mixture to separate free protein with latex beads binding protein with phosphatidylserine affinity, diluting and centrifuging the particles with PBS (phosphate buffered saline/buffer, 0.1M, pH 7.2) after discarding the supernatant, optionally repeating this process 3-4 times, and suspending the precipitate part (pellet) in stock buffer (0.1 M PBS, 0.1% glycine, 0.1% sodium azide, pH 7.2) and bringing to a concentration of 1% after discarding the supernatant.

In a preferred embodiment of the invention, microscope examination in step d) is performed with a 10X or 20X, or 40X objective.

The term “protein with phosphatidylserine affinity" as used herein refers to proteins that are a lipid bilayer structure and tend to bind to phosphatidylserine found on the outer surface of apoptotic cells.

The proteins with phosphatidylserine affinity are selected from the group consisting of annexins, protein kinase C (PKC), synaptotagmin, phospholipase C (PLC), rhabfilin, DOC1, DOC2, MARCKS, Vinculin, Lactadherin, Gas-6, Raf-1, DGK, SKI, NO synthase, FGF. The Annexins mentioned here can be selected from a group consisting of 12 members identified in humans, preferably from a group consisting of Annexin type I, Annexin type II, Annexin type III, Annexin type IV, Annexin type V (or Annexin A5), Annexin type VI, Annexin giardin, Annexin type VII, Annexin type VIII, Annexin Type X, Annexin type XI, Annexin type XIII, Annexin type XIV, Annexin type XXXI.

In a preferred embodiment of the invention, Annexins, the protein with affinity for phosphatidylserine, particularly preferably Annexin V (Annexin A5) are used.

In one embodiment of the invention, proteins with phosphatidylserine affinity can be used as conjugated with a fluorescent or enzymatic tracer or biotin.

The terms "non-apoptotic sperm" and "healthy sperm" used within the scope of the invention are equivalent to each other and can be used interchangeably within the scope of this invention.

During the implementation of the method according to the invention, solutions to aid in carrying out the process, such as water or suitable buffer solutions that are readily accessible to the skilled person in the state of the art can be used. Another aspect of the invention is the use of the method according to the invention to quantify apoptotic sperms in a sperm sample.

Another aspect of the invention is the use of the method according to the invention to separate healthy sperms from apoptotic sperms in a sperm sample.

In a preferred embodiment of the invention, with the method according to the invention, healthy sperms can be selected under the microscope and separated from sperms determined to be apoptotic sperms.

In one embodiment of the invention, Aldehyde/Sulphate latex beads from super active latex beads will be used.

The latex bead diameters used in the method of the invention can be 4, 5, 6, 7, 8, or 9 pm. In a preferred embodiment of the invention, beads of 4 pm or 9 pm in diameter will be used. The crosslink density of said latex beads can be 0.02 or 0.1 or 0.3 or 0.5 or 1 or 3 or 5 or 9.

Streptavidin polystyrene beads coated with a biotinylated protein with phosphatidylserine affinity used in the method according to the invention can have a diameter of 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8 or 5.9 pm. In a preferred embodiment of the invention, the diameter of the streptavidin polystyrene beads coated with a protein with biotinylated phosphatidylserine affinity is 5 pm or 5.9 pm. The Crosslink density of said polystyrene beads can be 0.05 or 0.3 or 0.4 or 0.7 or 1 or 1.5 or 2 or 3 or 4 or 5 or 6 or 10 or 14 or 18 or 38 or 70 or 90 or 196.

The invention will now be described regarding the following examples, which are for exemplary purposes only and should not be construed as limiting the scope of the present invention in any way.

EXAMPLES:

Example 1: Production of Annexin V Coated Latex Beads

A) Pretreatment of Latex Beads First, 2.5 ml (40 mg/ml) IDC UltraClean Amidine Latex bead solution is diluted in 10 ml 0.025M, pH 6 MES buffer (2-(N-morpholino)ethanesulfonic acid). Afterward, the mixture is centrifuged at 4°C (3.000 g, 20 min) to precipitate the particles. The supernatant is discarded and 10 ml of MES buffer is added to the mixture again, centrifuged again and the supernatant is separated from the particles and discarded. By adding 5 ml of MES buffer onto the obtained latex beads and suspending the beads, Annexin V ready-to-be-coated Latex beads are obtained (20 mg/ml as a 2% solution).

B) Coating of Pre-treated Latex Beads with Annexin V, a Protein with an Affinity for

Phosphatidylserine

Annexin V protein is prepared as 1 mg/ml in MES buffer. 2 mg of latex beads are added to 100 mg of Annexin V protein solution. This amount is for a 1 pm bead. If latex beads of 3 mih or different size are used, the amount of Annexin V is calculated according to the formula below the required amount of antibody (protein) per 1 pm particle antibody (protein) mass particle diameter (pm)

According to the formula given above; for 2 mg 3 mih aldehyde/sulfate latex bead (5/3) 33.2 mg of Annexin V is used. The Latex/ Annexin V mixture is gently stirred at 4°C overnight. To separate protein-bound latex particles from free protein, centrifugation of 3.000 g at 4°C for 20 minutes is performed. For protein determination, the supernatant is separated and stored. The separated supernatant is used to quantify the protein that is not bound to the latex bead. The precipitate obtained after centrifugation is resuspended in 1 ml of PBS. The resulting suspension is centrifuged again to precipitate the beads. This washing step is repeated at least 3 times.

The final latex is resuspended in a storage buffer to give a solid concentration of 1%. It is stored at 4°C until it is used and is not frozen. Example 2: Determination of Apoptotic Sperm Cells Using Annexin V-bound Latex Beads and Its Use in Microinjection Process

The sperm sample will be suspended at lxlO ⁶ cells/ml in Puresperm buffer (Nidacon).

Latex beads coated with Annexin V will be combined with 20 pi of beads/100 mΐ of a sperm sample, the volume will be completed to 1.5 ml with Annexin V binding buffer (10 mM HEPES, 140 mM NaCl and 2.5 mM CaC12, pH 7.4). Since this buffer contains CaCl2, it provides an appropriate pH environment for Annexin V to bind to phosphatidyl serine and for sperm to survive. The sperm-latex bead mixture is incubated for half an hour at room temperature. 5 mΐ is taken of the mixture and a drop is put into the dish prepared for microinjection. With the binding of Annexin V to phosphatidylserine in the sperm membrane, sperms (with beads) and healthy sperms (without beads) that undergo apoptosis are separated under the light microscope.

For the microinjection process, an appropriate number of sperm is drawn into the injection pipette from healthy sperms that are not bonded with bead, that is, non-apoptotic. These sperms are transferred to 2 separate 5 mΐ droplets containing the appropriate medium and washed. The washed sperm sample is drawn back into a pipette and taken to a 5 mΐ polyvinylpyrrolidone (PVP) droplet and is immobilized and microinjected (Figure 1).

The latex bead/sperm sample ratio can be as given in this example, or it can be at different rates. The skilled person in the state of the art can obtain the optimum result with different trials.

Example 3: Streptavidin-Polystyrene Coated with Biotinylated Annexin V Protein Preparation of Beads

It is incubated at 4°C overnight with 50 mg of biotinylated Annexin V with 2 mg (200 mΐ 1%) of streptavidin-coated polystyrene particles. To separate the annexin-bound polystyrene particles from free protein, 3.000 g centrifugation is performed for 10 minutes at 4°C. The supernatant is separated and stored so that the amount of protein released without binding to the bead can be determined. The pellet obtained after centrifugation is resuspended in 1 ml sodium phosphate buffer 0.1 M, pH 5.5. The mixture particles are centrifuged again to allow them to settle. This washing step is repeated at least 3 times.

Finally, the polystyrene is dissolved in sodium phosphate buffer 0.1 M, pH 5.5, to give a solid concentration of 1%. It is stored at 4°C until it is used and is not frozen.

Example 4: Combining biotinylated Annexin V Coated Streptavidin-Polystyrene Beads with sperm sample and its use in microinjection process

The sperm sample is suspended at lxlO ⁶ cells/ml in Puresperm buffer (Nidacon).

Streptavidin polystyrene bead coated with 25 mΐ of Annexin V is combined with 100 mΐ (lOxlO ⁶ sperm/ml) sperm sample and the total volume is completed with Annexin V binding buffer to 1.5 ml.

The annexin V bead and sperm mixture are incubated for half an hour at room temperature.

5 mΐ is taken of the mixture and a drop is put into the dish prepared for microinjection (105).

With the binding of Annexin V to phosphatidylserine in the sperm membrane, sperms (with beads) and healthy sperms (without beads, in other words, non-apoptotic) that undergo apoptosis are separated under the light microscope.

For the microinjection process, an appropriate number of sperm that are not bonded with beads are drawn into the injection pipette from healthy sperms.

These sperms are transferred to 2 separate 5 mΐ droplets (102, 103) containing the appropriate medium and washed.

It will be pipetted again and taken to a 5 mΐ polyvinylpyrrolidone (PVP) droplet (101) and immobilized and microinjected (104).

The latex bead/sperm ratio will be optimized by trials at different rates (Figure 1).

Within these basic concepts, it is possible to develop a wide variety of applications for the method of the present invention, and the invention cannot be limited to the examples described herein, it is essentially as stated in the claims.

A person skilled in the art can introduce the innovation disclosed in the invention by using similar embodiments and/or apply this embodiment to other fields of similar purpose used in the related art. Therefore, it is obvious that such embodiments will lack the criteria of innovation and especially exceeding the state of the art.