METHOD FOR SEPARATION OF SPERM WITH UNDAMAGED INTACT HEADS FROM SPERM WITH DAMAGED HEADS AND SOMATIC CELLS

Abstract

A method of separation of sperm cells with undamaged intact heads from sperm cells with damaged heads and/or somatic cells and/or cell debris in a sperm sample obtained from a human, comprising the steps of: providing an anti-CD46 antibody or CD46-binding ligand bound to a carrier and/or to a tag; contacting the sperm sample with the anti-CD46 antibody or CD46 -binding ligand bound to the carrier and/or to the tag in order to bind CD46 presenting cells and/or cell debris from the sperm sample to the anti-CD46 antibody or CD46-binding ligand; removing the CD46 presenting cells bound via the anti-CD46 antibody or CD46 -binding ligand to the carrier and/or to the tag to obtain a sperm sample free of CD46 presenting cells, wherein CD46 presenting cells include sperm cells with damaged heads, somatic cells and cell debris, is disclosed.

Claims

1. A method of separation of sperm cells with undamaged intact heads from sperm cells with damaged heads and/or somatic cells and/or cell debris in a sperm sample obtained from a human, comprising the steps of: providing an anti-CD46 antibody or a CD46-binding ligand bound to a carrier and/or to a tag; contacting the sperm sample with the anti-CD46 antibody or the CD46-binding ligand bound to the carrier and/or to the tag in order to bind CD46 presenting cells and/or cell debris from the sperm sample to the anti-CD46 antibody or the CD46-binding ligand; removing the CD46 presenting cells bound via the anti-CD46 antibody or CD46-binding ligand to the carrier and/or to the tag to obtain a sperm sample free of CD46 presenting cells, wherein CD46 presenting cells include sperm cells with damaged heads, somatic cells and cell debris.

2. The method according to claim 1, wherein the sperm sample free of CD46 presenting cells is a sperm containing substantially only sperm cells with undamaged intact heads.

3. The method according to claim 1, wherein the starting sperm sample is an ejaculate or a pre-treated ejaculate, wherein the pre-treatment preferably includes removal of seminal fluid and/or tissues and/or cell debris from the ejaculate.

4. The method according to claim 1, which contains the following steps: providing at least one immunization peptide for anti-CD46 antibody production or a peptide sequence of a CD46-binding ligand; preparing an anti-CD46 antibody or a CD46-binding ligand; binding the anti-CD46 antibody or the CD46-binding ligand to a carrier and/or to a tag; contacting the anti-CD46 antibody or the CD46-binding ligand bound to the carrier and/or to the tag with a sperm sample in order to bind CD46 presenting cells, including sperm cells with damaged heads and/or somatic cells and/or cell debris; separating the sperm cells with damaged heads and/or somatic cells and/or cell debris bound to the carrier and/or to the tag via the anti-CD46 antibody or the CD46-binding ligand from the sperm sample, thereby obtaining two cell fractions: 1) sperm cells with undamaged intact heads and 2) sperm cells with damaged heads and/or somatic cells and/or cell debris.

5. The method according to claim 1, wherein the carrier is selected from the group comprising beads, magnetic beads, polymeric substrates, cellulosic substrates, metallic substrates, magnetic or magnetizable carriers.

6. The method according to claim 1, wherein the carrier is a vessel, such as a well, well plate, beaker, capillary or test tube, wherein the anti-CD46 antibody or CD46-binding ligand is coated or immobilized on the inner surface of the vessel; or wherein the carrier is coated or immobilized on the inner surface of a vessel, such as a well, well plate, beaker, capillary or test tube.

7. The method according to claim 1, wherein the removal of the CD46 presenting cells bound via anti-CD46 antibody or CD46-binding ligand to the carrier and/or to the tag is carried out by centrifugation, by sedimentation, by flow separation methods such as FACS, capillary flow separation, separation on chips, or by use of magnetic force such as MACS or magnetic separation.

8. A method of in vitro separation of sperm cells with undamaged intact heads from sperm cells with damaged heads and/or somatic cells and/or cell debris in a sperm sample obtained from a human, comprising the step of employing an anti-CD46 antibody or CD46-binding ligand.

Description

BRIEF DESCRIPTION OF FIGURES

[0042] FIG. 1: Schematic model of a sperm: (A) sperm with an undamaged intact head and localization of CD46 inside the intact head, which is not accessible for the antibody nor CD46-binding ligand. (B, C) sperm with the damaged head, wherein CD46 can be detected by a species specific antibody or CD46-binding ligand. (B) a partial membrane damage and partial acrosome damage. (C) an excessive head damage with a loss of acrosome.

[0043] FIG. 2: CD46 detection of damaged sperm, somatic cells and cell debris using fluorescent microscope. Top left panel: anti-CD46 antibody labels sperm cells with damaged heads (arrows) and somatic cells (*) and cell debris (**). Sperm cells with intact undamaged heads (+) are not labeled by anti-CD46 antibody. Top right panel: all present sperm, somatic cells and debris labeled by Hoechst. Bottom left panel: Sperm cells with intact undamaged heads are not labeled by anti-CD46 antibody (+). Bottom right panel: Sperm with intact undamaged heads labeled by Hoechst.

[0044] FIG. 3: CD46 detection of damaged sperm, somatic cells and cell debris using Flow Cytometer. Left panel: population of cells after gating (marked by polygon). Middle panel: visualization of selected singlets. Right panel: Sperm with intact undamaged heads are not labeled by anti-CD46 antibody (−). Sperm with damaged heads are labeled with anti-CD46 antibody (+).

[0045] FIG. 4: Schematic model of sperm magnetic selection example: Ejaculate sample incubation with an antibody against CD46 conjugated to a magnetic carrier. Damaged sperm and non-spermatic cells bound to the carrier via the antibody are attracted to the magnet. Intact sperm are simply transferred by a pipette and ready for further utilization.

[0046] FIG. 5: Sperm sample quantitative evaluation before and after selection using anti-CD46 antibody examined for acrosome integrity and damage. Sperm sample before selection contains 75% sperm with damaged acrosomes and 25% sperm with intact acrosome. Intact sperm samples after magnetic selection using CD46 marker contains 18% sperm with acrosome damage and 82% of sperm with intact undamaged heads. N(samples)=10, error bars represent standard error of the mean (SEM).

[0047] FIG. 6: Sperm sample quantitative evaluation before and after selection using anti-CD46 antibody examined for total and progressive motility by Computer Assisted Sperm Analysis (CASA). Sperm sample before selection contains 32% sperm displaying total motility, 30% of sperm with progressive motility, which in total represent 62% of motile sperm. Intact sperm samples after selection using CD46 marker contains 48% sperm detected with total motility and 44% of sperm with progressive motility, which in total represent 92% of motile sperm. N(samples)=10, error bars represents standard error of the mean (SEM).

[0048] FIG. 7: Sperm sample quantitative evaluation after selection using anti-CD46 antibody examined for DNA integrity by TUNEL assay in fraction with damaged sperm and fraction with intact sperm. Damaged sperm sample contains 73% sperm positive for DNA fragmentation and 27% of sperm negative for DNA fragmentation. Intact sperm samples after selection using CD46 marker contains 13% sperm positive for DNA fragmentation and 87% sperm negative DNA fragmentation showing no detectable DNA damaged. N(samples)=10, error bars represents standard error of the mean (SEM).

EXAMPLES OF CARRYING OUT THE INVENTION

Example 1: Differentiation of Sperm with Undamaged Intact Heads from Sperm with Damaged Heads, Somatic Cells and Cell Debris

[0049] Sperm cells in fresh human ejaculate were labeled for nucleus by Hoechst, and for the head integrity by an anti-CD46 primary antibody (clone M177, sc-52647, Santa Cruz Biotechnology, Inc.) visualized by a secondary fluorescent antibody (anti-mouse Alexa Fluor 488, Molecular Probes, Prague, Czech Republic). The Hoechst labeling showed all the cells present in the sample, while the labeling by anti-CD46 antibody showed only sperm cells with damaged head.

[0050] FIG. 2 shows an image obtained from the sample—Top left panel: anti-CD46 antibody labels sperm cells with damaged heads (arrows) and somatic cells (*) and cell debris (**). Top right panel: all present sperm, somatic cells and debris labeled by Hoechst. Bottom left panel: Sperm cells with intact undamaged heads are not labeled by anti-CD46 antibody (+). Bottom right panel: Sperm with intact undamaged heads labeled by Hoechst. The position of the sperm cell with undamaged intact head has been marked by a cross in the top and bottom left panels, for better understanding of the images by the reader.

Example 2: Preparation of Monoclonal Anti-Human CD46 Antibody

[0051] A monoclonal anti-human CD46 antibody was prepared as follows:

[0052] The monoclonal anti-human CD46 antibody was prepared after immunization of mice (BALB/c strain) with 50 μg of the human CD46 peptide (TFSEVEVFEYLDAVTYS) conjugated with KLH (KLH=keyhole limpet hemocyanin) in 50 μl of phosphate buffer and 50 μl of Freund's adjuvant, subcutaneously in two doses over 10 days. Reimmunization has taken place with 200 μg KLH-peptide in 200 μl intraperitoneally for another 10 days, followed by fusion of spleen cells with myeloma cells after 3 days.

Example 3: Magnetic Particle Synthesis

[0053] Maghemite nanoparticles size 150 nm were prepared by iron nitrate (Fe(NO.sub.3).sub.3.9H.sub.2O) reduction in the presence of the reducing agent, sodium borohydride. .sub.8 g of Fe(NO.sub.3).sub.3.9H.sub.2O was dissolved in 500 ml of water under continuous heating (100° C.) and stirring (600 rpm). While stirring, 1 g of NaBH.sub.4 was added, previously dissolved in 50 ml of 3.5% NH.sub.3. Then the obtained solution was heated at boiling temperature (130-150° C.) for 2.5 h under 900 rpm continuous stirring on magnetic stirrer. After cooling, the product was separated by external magnetic field and washed several times with dH.sub.2O. The prepared magnetic particles were used as core for further surface modification.

Example 4: Synthesis of Magnetic Particles having Average Size 35 nm

[0054] 25 g of FeCl.sub.3.6H.sub.2O and 17 g of FeSO.sub.4.7H.sub.2O was taken in a 500 ml flask. Then 200 mL of 0.01 M HCl solutions was added and the solution mixed gently with magnetic stirrer (1000 rpm). After 10 min the whole mixture was heated on hot plate at 85° C. for 30 min and 60 ml ammonia solutions were added to the mixture and Oleic acid (4.6 mL) added immediately. The reaction was carried out at 85° C. temperature for 2 hrs. The flask was taken out from the magnetic hot plate for cooling and precipitates were washed with 500 mL of deionized water under magnetic stirring (1000 rpm) for 2 hrs. Synthetized magnetic nanoparticles were separated by magnetic decantation.

Example 5: Surface Modification of Magnetic Particles

[0055] For surface modification of magnetic particles, polyethylene glycol (PEG) with molecular weight 6000, polyvinylpyrrolidone (PVP) with molecular weight 10,000 and dextran were used. PEG, PVP and dextran were dissolved (25% weight by volume) in PBS and added to magnetic particles at 1 ml/1 mg concentration. The mixture was placed in rotator for 24 hr rotation cycle at 37° C. temperature under continuous rotation and shaking. After rotation cycle the particles coated with modifying agent were separated by external magnetic field, and washed several times with dH2O, and kept in fridge for further

Example 6: Antibody Coating

[0056] CD46 antibody was diluted in PBS in 20 μg/mL concentration for antibody coating on magnetic particles. The coated particles were dispersed in antibody solution 1 mg/1 mL concentration. The solution was further placed in 37° C. with continuous rotation and shaking overnight. The final antibody coated particles were separated by external magnetic field and washed three times with dH2O, dispersed in PBS and kept in 4° C. for further use.

Example 7: Synthesis of Quantum Dots (QD)

[0057] Aqueous CdTe QDs were prepared as follows: 0.05 mL of 100 mM CdCl.sub.2 was placed in a conical flask first, then 5.25 mL of water, 0.25 mL of 40 mM mercaptopropionic acid (MPA), 0.25 mL of 4 mM Na.sub.2TeO.sub.3, 0.6 mg of NaBH.sub.4, and 34.2 mL of 85% N.sub.2H.sub.4 3H.sub.2O were added one after the other. The mixture was stored at room temperature to maintain the growth of QDs. After 2 h storage, the QDs with red emission were obtained. By altering the ratio of reagents, QDs with different emission colors were prepared. Meanwhile, the precipitates of QDs are gained by adding 2-propanol to QD solution and centrifugation.

Example 8: Synthesis of Quantum Dots (QD)

[0058] Cadmium (Cd) precursor was prepared by mixing water, ethanol, and oleic acid together in volume ratio of 15: 35: 12, then 0.001 mol cadmium acetate and 0.7 g sodium hydroxide were added in 48 mL of the mixed solvent. The reaction temperature was 80° C. Selenium (Se) precursor was prepared by mixing the 0.005 mol selenium powder, 0.01 mol sodium sulfite, and 0.01 mol sodium hydroxide, in 50 mL deionized water under continuous magnetic stirring. The mixture was heated to 130° C. then 0.01 mol sodium hydroxide was added, and the reaction system was kept under stirring and heating for 3 hours. After adequate reaction time the reactants were cooled down to room temperature. The CdSe QDs were synthesized by adding 10 mL Se precursor solution to the 48 mL Cd precursor solution, under magnetic stirring. The reactions were carried out at 55° C., 80° C., and 95° C. for 3 min, 8 min, 12 min, 15 min, and 20 min. The samples were centrifuged with ethanol and dispersed in hexane.

[0059] QD Capping: The capping was done with mercaptopropionic acid and triethylamine at 40° C. while magnetic stirring. A CD46-binding ligand or peptide is added for further use.

Example 9: General Procedure for Separation of Sperm with Undamaged Intact Heads from Sperm with Damaged Heads from Human Ejaculate

[0060] Material: Cell sample suspension (human ejaculate), primary anti-human antibody anti-CD46 (if not specified otherwise, sc-52647) conjugated to a carrier.

[0061] Method: After the liquefaction of ejaculate without using any separation method, the ejaculate is incubated for 30 minutes with the anti-CD46 antibody conjugated to the carrier at room temperature, in a physiological buffer (PBS, phosphate buffer saline). Standard procedures and materials were used for binding the antibody to the carrier. The incubation of the anti-CD46 antibody-carrier complex with the ejaculate was followed by separation of the carrier with the conjugated anti-CD46 antibody to which the CD46 presenting cells are bound. The remaining unbound sperm fraction with undamaged intact heads was transferred to a new container for further use in assisted reproduction such as intrauterine insemination (IUI), in vitro fertilization (WF) or intracytoplasmic sperm injection (ICSI), storage purpose, medicine, or any other technique or research.

Example 10: Separation of Sperm with Undamaged Intact Heads from Sperm with Damaged Heads from Human Ejaculate

[0062] The anti-human CD46 antibody is bound to magnetic beads as a carrier to form an anti-human CD46 antibody-carrier complex. The procedure of Example 9 was then followed. All the CD46 presenting sperm with damaged heads and somatic cells, mainly white blood cells, and cell debris are bound to the anti-human CD46 antibody-carrier complex and further separated by magnetic field, using a magnetic stand. The unbound sperm with undamaged intact heads remains in the suspension.

Example 11: Separation of Sperm with Undamaged Intact Heads from Sperm with Damaged Heads for Storage Purpose

[0063] The anti-human CD46 antibody is bound to polyacrylamide beads as a carrier to form an anti-human CD46 antibody-carrier complex. The procedure of Example 9 was then followed. All the CD46 presenting sperm with damaged heads and all somatic cells, mainly white blood cells, and cell debris are bound to the carrier via the anti-CD46 antibody, and further removed from the ejaculate by centrifugation. The unbound sperm with undamaged intact heads remain in the suspension and they are ready to be used for short or long-term storage such as cryopreservation or any kind of sample preservation.

Example 12: General Procedure for Separation of Sperm with Undamaged Intact Heads from Sperm with Damaged Heads from Cryopreserved Human Ejaculate

[0064] Material: Cryopreserved sample (human ejaculate), primary anti-human antibody anti-CD46 (if not specified otherwise, sc-52647) conjugated to a carrier or conjugated with a tag.

[0065] Method: After a sample thawing, the sample is incubated for 30 minutes with the anti-CD46 antibody conjugated to the carrier or conjugated with the tag, at room temperature, in a physiological buffer. Standard procedures and materials were used for binding the antibody to the carrier or to the tag. The incubation of the anti-CD46 antibody-carrier complex or anti-CD46 antibody-tag with the ejaculate was followed by separation of the carrier or tag with the conjugated anti-CD46 antibody to which the CD46 presenting cells are bound. The remaining unbound sperm fraction with undamaged intact heads is transferred to a new container for further use in assisted reproduction (artificial insemination) such as intrauterine insemination (IUI), in vitro fertilization (WF) or intracytoplasmic sperm injection (ICSI), storage purpose, medicine, or any other technique or research.

Example 13: Separation of Sperm with Undamaged Intact Heads from Sperm with Damaged Heads from Cryopreserved Sperm Sample

[0066] The procedure of Example 12 was followed, wherein the carrier are magnetic beads. All the CD46 presenting sperm with damaged heads and all somatic cells, mainly white blood cells, and cell debris are bound to the anti-human CD46 antibody-carrier complex and further separated by magnetic field. The unbound sperm with undamaged intact heads remain in the suspension.

Example 14: Sperm Motility Analysis (FIG. 6)

[0067] Material: Sperm sample after thawing (before selection) or after selection.

[0068] Method: Motility was measured with a Computer Assisted Sperm Analysis (CASA) system (ISAS, Proiser, Valencia, Spain). Total motility and progressive motility were evaluated based on manufacturer's setting (thresholds were VAP 10 um.s.sup.−and STR 80% for motile and progressively motile sperm, respectively). A 2 μL drop of each semen sample was placed in a 37° C. prewarmed Leja counting chamber (IMV Technologies, France). Six fields per sample were evaluated at 100× magnification negative phase-contrast objective. Constant temperature during analysis was ensured by a heating plate prewarmed to 37° C. as a part of microscope. The evaluation was based on the analysis of 60 consecutive digitized images, which were taken at a time lapse of 1 s with a camera at a frequency of 60 fps. At least 100 trajectories were analysed per field.

Example 15: Sperm Acrosomal Damage Detection by CD46 Antibody with Fluorescent Tag Using Fluorescent Microscopy (FIG. 2)

[0069] Material: Sperm sample after thawing (before selection) or after selection.

[0070] Method: Sperm samples were washed twice (300 g, 5 min) in PBS. Pellet was diluted in 100 μL of PBS buffer and 10 μL of mouse monoclonal anti-CD46 antibody (clone M177, ThermoFisher Scientific, USA) conjugated with fluorescence tag (FITC). After 30 minutes incubation in room temperature, spermatozoa were washed in PBS. 9 μL of sperm suspension were transferred onto slide and gently mixed with 3 μL of Vectashield/DAPI (Vector Laboratories Ltd., Peterborough, UK) and mounted under a coverslip. Images were captured in magnification 600× by camera as part of epifluorescent microscope Nikon Eclipse E600 (Tokyo, Japan) and analysed by software NIS Elements (Laboratory Imaging, Inc., Prague, Czech Republic).

Example 16: Sperm Acrosomal Damage Examined by CD46 Antibody Using Flow Cytometry (FIG. 3)

[0071] Material: Sperm sample after thawing or native liquified ejaculate.

[0072] Method: Sperm samples were washed twice (300 g, 5 min) in PBS. Sperm concentration was adjusted by PBS to 5−10×10.sup.6/mL. 10 μL of mouse monoclonal anti-CD46 antibody (clone M177, ThermoFisher Scientific, USA) conjugated with fluorescence tag (TRITC) was added into 100 μL aliquot. After 30 minutes incubation in room temperature, spermatozoa were washed twice in PBS. Sperm pellet was resuspended in 500 μL of PBS and samples were analysed by BD LSR Fortessa™ SORP (BD Biosciences, San Jose, Calif., USA). Based on fluorescence signal sperm was divided to CD46+ (Acrosome damaged) and CD46− (Acrosome intact).

[0073] Example 17: Sperm DNA Integrity Analysis (FIG. 7)

[0074] Material: Sperm sample after thawing sorted for damaged (CD46 positive) and intact (CD46 negative) sperm fraction.

[0075] Method: For sperm DNA integrity analysis (TUNEL) Apo-direct Kit (Phoenix Flow System, USA) was used. Spermatozoa were washed two times 250 ×gfor 5 min. Afterwards, 1 mL of Intracellular Perm Wash Buffer (Biolegend, San Diego, USA) was added to the pellet and suspension was incubated for 30 min in 4° C., centrifuged and washed two times. Sperm pellet was fixed in 1 mL of ice-cold 70% ethanol, vortexed and incubated for 30 min in 4° C. After fixation samples were centrifuged 300 ×g, 10 min, vortexed and washed two times (300 ×g, 10 minutes) in Wash buffer. Thereafter, a reaction mix was added for 60 min incubation in 37° C. Samples were washed twice with Rinse Buffer (300 ×g5 min) and 0.5 mL of Propidium iodide/RNAse A free was added for 30 min and samples were proceeded for analysis. Samples were analysed by flow cytometer BD LSR Fortessa™ SORP, BD Biosciences, San Jose, Calif., USA. For each sample at least 15000 events were recorded.

INDUSTRIAL APPLICABILITY

[0076] The present invention is a technology for selection and separation of CD46 presenting components from cells not presenting CD46 in cell samples. The cells not presenting CD46 are sperm with undamaged intact heads. The technology may thus serve for separation of sperm with undamaged intact heads from sperm with damaged heads, as well as from somatic cells and cell debris in a human sperm sample. This technology is designed to separate sperm with undamaged intact heads to be further used in assisted reproduction, human medicine and research. The invention results in an increased quality of the sperm cell sample, as well as allows separation of somatic cells and/or debris from sperm-containing samples. Such purified samples result in an increased fertilization or medical or research success. The invention also results in an increased quality of the sperm sample for long- or short-term storage under any condition and media such as cryopreservation. This invention also saves large amount of cryoprotectives and plastic by storage of the pre-selected quality sample.