Method for evaluating the fertilising capacity of a sperm sample or for recovering human sperm using zonae pellucidae isolated from bovine oocytes

Abstract

The present invention relates to an in vitro method for evaluating the fertilising capacity of sperm and / or for selecting capacitated, acrosome-free sperm that penetrate the zona pellucida (ZP), by means of the use of zonae pellucidae previously isolated from bovine oocytes obtained from ovaries in slaughterhouses, and heterologous in vitro fertilisation between sperm (preferably human sperm) and the zonae pellucidae isolated from bovine oocytes. Furthermore, the present invention relates to the use of the selected sperm as a medicament, particularly in assisted reproductive therapies.

Description

[0001] DESCRIPTION

[0002] Method for evaluating the fertilizing capacity of a sperm sample or for recovering human sperm using zona pellucida isolated from bovine oocytes

[0003] The present invention relates to an in vitro method for evaluating the fertilizing capacity of spermatozoa and / or for selecting capacitated, acrosome-free spermatozoa that penetrate the zona pellucida (ZP), by using zona pellucida isolated from bovine oocytes obtained from ovaries of slaughterhouses and in vitro heterologous fertilization between spermatozoa (preferably human spermatozoa) and the zona pellucida isolated from bovine oocytes.

[0004] BACKGROUND OF THE INVENTION

[0005] The effectiveness of assisted reproduction is relatively low, partly because in vitro there is very little gametic selection, unlike in vivo, where the female genital tract offers a great selection, such that of the millions of sperm in an ejaculate, only a few manage to reach the oviduct to fertilize the oocyte.

[0006] Furthermore, in most assisted reproductive technology (ART) clinics, the most commonly used treatment to fertilize oocytes is intracytoplasmic injection (ICSI), a much more invasive method than in vitro fertilization (IVF), which produces relatively low results, but is used preferentially, partly because there is no in vitro system to analyze the fertilizing capacity of sperm, nor to select the best quality sperm, and it is unknown whether the sperm sample contains sperm capable of penetrating the zona pellucida (ZP) and fertilizing the oocyte by IVF.

[0007] Male infertility is the inability of a man to induce pregnancy in a clinically fertile woman, affecting 50% of couples seeking assisted reproductive technologies. The condition is multifactorial and presents heterogeneous phenotypic characteristics. In the clinic, these men usually undergo conventional andrological diagnostic procedures, which rely on descriptive criteria to define their fertility based on the number of morphologically normal and motile sperm found in their ejaculate. These diagnoses have many limitations, and consequently, there is growing awareness of the importance of assessing sperm quality to more accurately predict a man's fertility status and decide which assisted reproduction technique to use.

[0008] That sperm are capacitated is important because it is known that only capacitated human sperm can respond to chemotaxis in response to the progesterone gradients that occur very close to the egg (V. G atica et al., 2013, Pico molar gradients of progesterone select functional human sperm even in subfertile samples. Mol Hum Reprod 19, 559-569) and only if they are capacitated can they respond to thermotaxis (S. Perez-Cerezales et al., 2018, Sperm selection by thermotaxis improves ICSI outcome in mice. Sci Rep 8, 2902), mechanisms that act in the female genital tract as sperm selection systems; and the ability to capacitate has been shown to predict sperm fertility (A. Keller, K. Kerns, Sperm capacitation as a predictor of boar fertility. Molecular Reproduction and Development 10. 1002 / mrd.23690 (2023)).

[0009] Sperm capacitation refers to the physiological changes that occur in sperm within the female reproductive tract, which allow them to acquire the ability to fertilize an egg. It is a complex process involving several biochemical and structural modifications in sperm, including changes in the sperm membrane, ion channel activity, and sperm motility, all of which are essential for traversing the zona pellucida. While sperm capacitation is an essential step in the fertilization process, it is not typically used as a direct predictor of fertility. Instead, fertility assessments typically focus on evaluating aspects such as sperm count, motility, morphology, and other factors that can affect sperm's ability to reach and fertilize an egg.

[0010] There is no current technology that accurately determines the fertilizing capacity of human sperm or that can select sperm capable of capacitating and penetrating the ZP. For a time, hamster oocytes free of the ZP were used as a method to analyze whether sperm could penetrate the oocyte, since it was shown that the ZP is a barrier to prevent crossing between species and does not allow the penetration of sperm from another species (Vieira A, Miller DJ. 2006. Gamete interaction: Is it species-specific? Mol Reprod Dev 73 1422-1429). Furthermore, it is known that the degree of fragmentation is related to the effectiveness of reproductive techniques and to the appearance of embryonic and fetal alterations (C. Alvarez Sedo et al., 2017, Effect of sperm DNA fragmentation on embryo development: clinical and biological aspects.JBRA Assist Reprod 21, 343-350)', and that male age affects DNA fragmentation and, as a consequence, embryonic development is reduced and fetal losses and abortions increase (E. Martinez et al., 2021, Sperm DNA fragmentation and male age: results of in vitro fertilization treatments. JBRA Assist Reprod 25, 533-539).

[0011] DESCRIPTION OF THE INVENTION

[0012] The present invention relates to a method for evaluating the fertilizing capacity of human sperm (determining whether a sperm sample has fertilizing capacity) and / or for selecting those sperm from the ejaculate that have the potential to capacitate (hyperactivate and undergo the acrosomal reaction) and penetrate the zona pellucida (ZP) (recovering from a sample the sperm that have penetrated the zona pellucida of an oocyte). The invention, through the use of isolated zona pellucidas from bovine oocytes obtained from the ovaries of slaughterhouses and carrying out an in vitro test of heterologous fertilization between sperm (preferably human sperm) and zona pellucidas of bovine oocytes, makes it possible to determine the fertilizing capacity of sperm by counting those that bind and those that hyperactivate and are capable of penetrating the free zona pellucidas of the cytoplasm.

[0013] Furthermore, in this way, it is possible to select and recover those sperm that have penetrated, such as those of the millions in the ejaculate that have the capacity to penetrate the ZP, to become hyperactive, and have also lost the acrosome and its enzymatic component, since these acrosomal enzymes can be toxic to the oocyte.

[0014] The methodology allows for the identification of assisted reproduction patients who could benefit from in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) technology, and also allows for the identification of patients whose sperm samples are unable to bind and / or penetrate the zona pellucida. Furthermore, it allows for the selection and recovery of sperm that have penetrated the ZP as optimal for use in reproductive techniques, primarily ICSI, because in addition to being capacitated sperm (having undergone hyperactivation and acrosomal reaction), they have less DNA fragmentation compared to sperm that have not crossed the zona.

[0015] Thus, a first aspect of the present invention relates to an in vitro method for determining whether a sperm sample has fertilizing capacity and / or for recovering from a sample sperm that have penetrated the zona pellucida of an oocyte, comprising: a) removing cytoplasmic material from oocytes previously isolated from bovine ovaries, obtaining the isolated zona pellucida free from the rest of the oocyte components; b) carrying out heterologous in vitro fertilization between the free zona pellucidas of oocytes of a) with a sample of sperm previously isolated from a mammal; c) determining the % of sperm in the sample that penetrate the zona pellucida; d) determining whether the sperm sample has fertilizing capacity based on the % penetrability obtained in c), wherein when at least 50% of the sperm present in the sample have penetrated the zona pellucida, it is determined that the sample has fertilizing capacity;and / or e) when it is determined in step d) that the sample has fertilizing capacity, the sperm that have penetrated the zona pellucida are recovered;

[0016] The term "oocytes previously isolated from bovine ovaries," as used herein, refers to female germ cells (female gametocytes) that participate in reproduction, i.e., they are the immature precursors of the ovum or egg cell. Oocytes are produced in the embryo's ovary during female gametogenesis. As described above, in the method of the invention, the oocytes are previously obtained from bovine ovaries in slaughterhouses, i.e., they are bovine oocytes that have never been in contact with oviductal fluid.

[0017] In many species, the zona pellucida undergoes modifications that make it impossible for sperm from another species to penetrate. These modifications occur in the oviduct, when the oocyte comes into contact with the oviductal fluid. However, the method of the invention uses oocytes obtained from slaughterhouse ovaries that have never been in contact with oviductal fluid and, therefore, lack the species-specific modifications. Thus, these oocytes have zones that can be penetrated by other species.

[0018] As described herein, oocytes are isolated from grade I or grade II cumulus oocyte complexes (COCs), which are characterized by increased compaction between the cumulus cells and oocytes with smaller lipid droplets and greater dispersion in the ooplasm,

[0019] Thus, in a preferred embodiment of the invention of the method of the invention, the oocytes previously isolated from bovine ovaries are isolated from cumulo oocyte complexes (COC) of grade I or grade II.

[0020] In step a) of the method of the invention, all cytoplasmic material is removed from the bovine oocytes, obtaining the isolated empty zona pellucida free of oocytes. The term "oocyte cytoplasmic material" refers to all components of the oocyte except the ZP, that is, the cytoplasm with all its organelles, mainly the plasma membrane, nucleus, mitochondria, and polar body.As those skilled in the art know, there are numerous methods and instruments in the state of the art for removing cytoplasmic material from oocytes. The zona pellucida can primarily be pierced with a microinjector with a piezoelectric system, or with a beveled microcapillary attached to a micromanipulator, or perforated with a laser system. Any of these methods makes it possible to create a small cavity sufficient for a microcapillary with a diameter of 6 to 10 micrometers to penetrate the gap generated in the ZP and suction out the remaining components of the oocyte.

[0021] In the method of the invention, a microinjector with a coupled piezoelectric system was used to eliminate the cytoplasm of the oocytes. This system is used to make a hole in the oocyte's ZP using a blunt injection needle using pulses from the piezoelectric injector. Once inside the pelvic space, and proceeding in the same way, more pulses are applied to the plasma membrane of the oocyte, and all the cytoplasmic content is aspirated (Figure 1), obtaining the emptied ZP. The zona pellucida can be sterilized with UV and frozen at -20°C for storage.

[0022] Thus, in another preferred embodiment of the method of the invention, in step a) the cytoplasmic material is removed from the oocyte by means of a microinjector with a piezoelectric system. Subsequently, once the empty pellucid zones (free from the oocyte) have been isolated, heterologous in vitro fertilization is carried out between said oocyte-free pellucid zones with a sample of sperm isolated from a mammal.

[0023] The term “heterologous in vitro fertilization,” as used herein, refers to heterologous in vitro fertilization in mammals, which refers to a process by which the gametes (eggs and sperm) used to fertilize an egg come from individuals of different species or varieties within the same species. The method of the invention uses ZPs from bovine oocytes and sperm from different mammalian species, preferably humans.

[0024] Heterologous fertilization is performed with a sperm sample isolated from a mammal. The term "sperm sample" refers to any semen sample collected from the ejaculate of any mammal. In another preferred embodiment of the method of the invention, the sperm sample comes from a mammal selected from: humans, mountain goats, horses, dolphins, guinea pigs, mice, and cats.

[0025] In another more preferred embodiment of the method of the invention, the sperm sample comes from a human being.

[0026] In another preferred embodiment of the method of the invention, the sperm concentration of the sample is between 1x10 4 1x10 8 sperm / mL, preferably between 1x10 5 1x10 7 sperm / mL, more preferably 1x10 6 sperm / mL.

[0027] Once heterologous fertilization has been carried out, in step c) the percentage of sperm in the sample that penetrate the zona pellucida is determined, where said percentage is calculated using the formula:

[0028] Number of spermatozoa that penetrate the ZP

[0029] _ X 100

[0030] Total number of sperm in the sample

[0031] Subsequently, in step d), it is determined whether the sperm sample has fertilizing capacity based on the % penetrability obtained in c), where when at least 50% of the sperm present in the sample have penetrated the zona pellucida, it is determined that the sample has fertilizing capacity; and / or, in step e), when it is determined in step d) that the sample has fertilizing capacity, the sperm that have penetrated the zona pellucida are recovered.

[0032] The term “fertilization capacity,” as used herein, refers to the ability of the sperm in the isolated sample to fertilize a physiologically normal and structurally intact oocyte, based on their ability to penetrate the emptied (oocyte-free) zona pellucida according to the method of the invention. To determine the fertilization capacity of a sperm sample, the number of sperm adhering to the ZP, the percentage of ZPs penetrated, and the average number of sperm passing through the ZP (referred to as % penetrability) are determined relative to or relative to the total sperm in the sample.Those samples with a penetrability percentage (sperm that penetrate the ZP / total sperm in the sample x 100) with at least 50% penetration are considered valid for use in in vitro fertilization, that is, when at least 50% of the sperm present in the sample have penetrated the zona pellucida, it is determined that the sample has fertilizing capacity.

[0033] Thus, in another preferred embodiment of the method of the invention, in step d) it is determined that the sperm sample has fertilizing capacity when the % of sperm that penetrate the zona pellucida is equal to or greater than 70%, 75%, 80%, 85%, 90% or 95% with respect to 100% of sperm present in the sample.

[0034] Sperm capable of penetrating the zona pellucida, i.e., sperm categorized as having a positive fertilizing capacity, have undergone sperm capacitation, hyperactivation and acrosomal reaction, and have an elongated and elliptical shape, are longer and narrower, have a larger area and smaller perimeter, characteristics of sperm from fertile males, and have less DNA fragmentation than the rest of the sperm that have not penetrated the ZP.

[0035] The term “recovering sperm that have penetrated the zona pellucida” as used herein refers to the recovery of sperm that have penetrated the ZP, for which the ZPs with the penetrated sperm are placed under the inverted microscope and with the help of a micromanipulator the ZP is penetrated and the penetrated sperm are recovered (by means of negative pressure), then these sperm are deposited in a drop of medium, allowing them to be used in assisted reproduction therapies, such as fertilization by ICSI.

[0036] Therefore, another aspect of the present invention relates to the sperm recovered after step e) of the method of the invention, hereinafter “the sperm of the invention”.

[0037] As understood by those skilled in the art, the sperm of the invention may be included in a composition, preferably a pharmaceutical composition.

[0038] Thus, another aspect of the present invention relates to a composition, hereinafter the “composition of the invention”, comprising the sperm of the invention.

[0039] In another preferred embodiment, the composition of the invention is a pharmaceutical composition. The term "pharmaceutical composition" refers to a product, solution, or composition comprising the sperm of the invention in any concentration, which has at least one application in improving the physical, physiological, or psychological well-being of a subject, implying an improvement in the general state of health or a reduction in the risk of disease. Said pharmaceutical composition may be a medicament. The term "medicament" has a narrower meaning than the meaning of "pharmaceutical composition" as defined herein, since a medicament necessarily implies a preventive or therapeutic effect.

[0040] In another preferred embodiment of the use of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier and / or excipient.

[0041] The term "pharmaceutically acceptable carrier" refers to those substances, or combinations of substances, known in the pharmaceutical industry, used in the preparation of pharmaceutical dosage forms that facilitate the incorporation of other compounds to allow for improved dosage and administration or to give consistency and shape to the composition. Examples of carriers include, but are not limited to, solids, liquids, solvents, or surfactants.

[0042] The term "pharmaceutically acceptable excipient" refers to a substance that helps to absorb any of the components of the composition of the invention, stabilizes said components, or assists in the preparation of the composition by giving it consistency or, if necessary, providing flavors that make them more pleasant. Therefore, the excipients could have the function of keeping the components together, such as, for example, starches, sugars, or celluloses, a sweetening function, a coloring function, the function of protecting the composition, such as, for example, isolating it from air and / or moisture, a filler function for a tablet, capsule, or any other form of formulation, such as, for example, dibasic calcium phosphate, a disintegration function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.Therefore, the term "excipient" is defined as any material included in dosage forms that is added to the active ingredients or their combinations to allow their preparation and stability, modify their organoleptic properties, or determine the physicochemical properties of the composition and its bioavailability. The "pharmaceutically acceptable" excipient must allow the activity of the compounds in the pharmaceutical composition, that is, to be compatible with said components. Examples of excipients are binders, fillers, disintegrants, lubricants, coating products, sweeteners, flavorings, and colorants. More specific, non-limiting examples of acceptable excipients are starches, sugars, xylitol, sorbitol, calcium phosphate, spheroid fats, talc, silica, or glycerin, among others.

[0043] As understood by those skilled in the art, the ZPs freed from the rest of the oocyte and sterilized by UV, the sperm of the invention, or the composition of the invention may be included in a kit. Thus, another aspect of the present invention relates to a kit comprising the ZPs freed from the rest of the oocyte, the sperm, or the composition of the invention.

[0044] As described above, the sperm selected by the method of the invention are fertile sperm capable of fertilizing a physiologically normal and structurally intact oocyte, and can therefore be used as a medicament, preferably for the treatment of infertility. As shown in Figure 5, when oocytes are fertilized with sperm that have penetrated the ZPs, many more blastocyst-stage embryos are produced than when sperm that have not penetrated are used.

[0045] Thus, another aspect of the present invention relates to the sperm or the composition of the invention for use as a medicine.

[0046] Furthermore, another aspect of the present invention relates to the sperm or composition of the invention for use in assisted reproduction therapies. The term "assisted reproduction therapies" as used in the present invention refers to treatments, procedures, and the set of techniques that replace the natural reproductive process in order to achieve and carry a pregnancy to term, giving birth to a live and healthy offspring. Examples of assisted reproduction therapies include, but are not limited to, artificial insemination (AI), in vitro fertilization (IVF), or intracytoplasmic sperm injection (ICSI).

[0047] In another preferred embodiment, the assisted reproduction therapy is in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).

[0048] DESCRIPTION OF THE FIGURES

[0049] Figure 1. Methodology used to empty the bovine zona prelucida of its cytoplasmic contents, the oocyte. The diagrams are shown on the left and a photograph corresponding to the manipulation shown in the diagram is shown on the right.

[0050] Figure 2. Bovine zona pellucida free of cytoplasm and other oocyte components, showing human spermatozoa penetrated and / or attached to the ZP after 3 hours of coincubation.

[0051] Figure 3. Bovine zona pellucida (ZP) free of other oocyte components, with penetrated spermatozoa after 3 h coincubation. (A) Bovine ZP with spermatozoa inside the ZP and outside the ZP stuck to the external part of the ZP. (B) Appearance of spermatozoa inside the ZP without acrosome (B) or outside the ZP with acrosome (C) (Staining with Hemacolor kit, Merck, Darmstadt, Germany). Figure 4. Percentages of fragmented DNA determined by neutral SCGE (mean ± SEM, n = 4 for human, 50-100 spermatozoa per determination). (P < 0.001, two-way ANOVA).

[0052] Figure 5. Development of mouse embryos produced by intracytoplasmic sperm injection (ICSI) using sperm that have penetrated the bovine cytoplasm-free ZP (penetrated) and sperm retrieved outside the ZP (unpenetrated). Morphological assessment 24 h (% reaching the 2-cell stage) and 5 days (% of embryos developing to blastocyst) after ICSI (mean ± SEM, n = 5 experiments, 140 ICSI-injected oocytes per sperm type). An asterisk (*) indicates significant differences (P < 0.01 ANOVA).

[0053] EXAMPLES

[0054] Materials and Methods

[0055] Recovery of bovine oocytes from slaughterhouse ovaries

[0056] The ovaries of slaughtered heifers or cows were collected from the slaughterhouse and transported to the laboratory in saline solution (0.9% NaCl (Sigma) supplemented with 0.1% gentamicin) at 35–37°C. Once in the laboratory, the ovaries were washed with water at 35–37°C and subsequently with saline solution at the same temperature to remove any blood. The ovaries were then kept in saline solution and in a water bath to maintain the temperature between 35–37°C. Follicular fluid was aspirated from ovarian follicles measuring between 2 mm and 8 mm using a 5 mL syringe and an 18 g needle. The aspirated follicular fluid was stored in a sterile 50 mL falcon tube, also located in the 37°C water bath. The aspirated follicular fluid was allowed to settle for 10 minutes.After the time was elapsed, the supernatant was removed, while the pellet in which the cumulus oocyte complexes (COC) sedimented was resuspended in a PBS solution. This suspension was poured into a 100 mm culture dish (Biofil), which was placed on a plate at 37°C to locate the COCs under a stereomicroscope. Only grades I and II COCs were selected, which are characterized by greater compaction between cumulus cells and oocytes with smaller lipid droplets and greater dispersion in the ooplasm (de Loos et al., 1989. Morphology of immature bovine oocytes. Gamete Res. 24:197-204).

[0057] In vitro maturation of COCs

[0058] Selected COCs were washed three times in maturation medium (TCM-199 supplemented with 10% FCS and 10 ng / mL EGF). Selected COCs were then plated in 4-well plates (Thermo Scientific) with 500 pL of maturation medium in each well. Each well contained groups of 50 COCs that were cultured for 22–24 h at 38.5°C, 5% CO2, and 100% humidity.

[0059] Sperm capacitation

[0060] As a control for homologous coincubation between zona pellucida (ZP) and bovine sperm, frozen semen from a previously tested bull (ASEA VA, Spain) was thawed in a 37°C water bath for 1 min. It was then centrifuged at 280g for 10 min in a gradient of 1 mL of an isotonic saline solution containing 40% silane-coated colloidal silica particles and 1 mL of the same solution at 80% to form the gradient. The sperm pellet was isolated and washed in 3 mL of sperm-specific isotonic saline solution (pH 7.25–7.35 at room temperature) by centrifugation at 280g for 5 min. Once centrifuged, the supernatant was removed and the pellet was resuspended in the 300 µL of isotonic saline. Sperm concentration was adjusted to achieve a final concentration of 1x10 6spermatozoa / mL for co-incubation. Male and female gametes were then co-incubated for 18–22 hours in 500 pL of fertilization medium (Tyrode's medium with 25 mM bicarbonate, 22 mM sodium lactate, 1 mM sodium pyruvate, and 6 mg / mL fatty acid-free BSA supplemented with 10 mg / mL hepahn's sodium salt) in a 4-well plate. Each well contained groups of 50 COCs that were cultured at 38.5°C, 5% CO2, and maximum humidity. Oocyte cytoplasm was then removed by micromanipulation to leave the ZPs free.

[0061] Emptying and sterilization of zona pellucida of bovine oocytes

[0062] After maturation of bovine oocytes obtained from slaughterhouse ovaries, cumulus cells were removed by treatment with hyaluronidase (Sigma H3506), and those oocytes that presented the first polar body and morphology characteristic of quality and correct maturation were selected. These oocytes were placed on a manipulation plate with M199 medium containing Hepes and 10% FCS (Fetal Calf Serum, Sigma F2442).

[0063] To eliminate the cytoplasm from the oocytes, a microinjector with a coupled piezo system (Piezo Impact Drive Unit PMM100, Prime Tech Ltd) was used. The oocyte was fixed using a holder needle, while the Piezo injection needle, similar to those used for murine ICSI, was used to make a hole in the oocyte's PZ using pulses from the piezoelectric motor (Figure 1A, 1B). Once inside the perivitelline space, and proceeding in the same way, more pulses were applied to the oocyte's plasma membrane, and all the cytoplasmic content was aspirated (Figure 1C, 1D). The injection needle was removed from the perivitelline space and the emptied PZ was set aside. Once all the oocytes had been processed, they were washed in PBS and frozen at -20°C in groups of 20 in the smallest possible volume (Fig. 1E).

[0064] Sterilization was performed by applying short-wave ultraviolet light (UVC) radiation (253.7nm) for 3 minutes.

[0065] Coincubation of zona pellucida with spermatozoa

[0066] For heterologous coincubation between bovine ZPs and human sperm, patient semen is thawed in a water bath at 37°C, volume, motility and concentration of the sample are assessed in a Neubauer chamber; an equal volume of IVF-G medium (Vitrolife) is added and centrifuged at 1800 rpm for 10 min, the supernatant is discarded and 200 mL of IVF-G is added to the pellet, kept at 38.5°C for 45 min to swim-up (the semen sample is placed in a test tube and then a suitable isotonic medium is added that allows the most vigorous sperm to move upwards) and subsequently the supernatant is collected, the concentration is calculated and a concentration of 1 million sperm per mL is added to the ZPs.

[0067] Penetrated zona pellucida staining

[0068] After co-cultivation of the ZPs with sperm, they are fixed by washing for 30 minutes in 4% paraffin aldehyde at 4°C and then stored in cold PBS at 4°C. Staining is performed with Hoechst 33342 dye for 15 minutes, then the samples are mounted and viewed under a fluorescence microscope. Evaluation of the morphometry of the head of the spermatozoa that penetrated the ZP

[0069] Sperm head morphometry, including length, width, perimeter, and head area, was assessed using Hemacolor® fast stain (Merck, Madrid, Spain) for both penetrated and non-penetrated groups after coincubation. 5 µL of the sample was spotted onto a microscope slide, smeared, and allowed to air dry at room temperature. Hemacolor staining was performed following the kit recommendations. Slides were immersed in a fixative solution for 3 minutes, then for 2 minutes each in a red color reagent containing Eosin Y and a blue color reagent containing Azure B. A total of 32 slides were analyzed, with 8 slides from each replicate (4 slides per experimental group). On each slide, 25 sperm with well-formed heads were measured using a Motic BA210 microscope equipped with a Moticam 3.0 MP CMOS digital camera.A photograph of the sperm was taken using a 100* magnification objective, and the head area was measured using Motic Images Advanced 3.2 software.

[0070] Based on the measurement data of the sperm head, calculations were made for the parameters related to the shape of the structure: ellipticity (length / width), roughness (4TT X area / perimeter A2), elongation [(length - width) / (length + width)] and regularity (TT X length x width / area). Elongation and ellipticity values ​​describe cell size in terms of length (a higher value indicates a longer cell) and width (a value above 1 indicates an elliptical cell, while a value equal to 1 indicates round cells), respectively. Regularity provides a more accurate description of the format within the ellipse variations, with a value equal to 1 indicating a perfect ellipse. Roughness characterizes amorphous cells, with a lower value indicating a rougher head surface. This parameter can be positively correlated with head length and serves as an indicator of the cell's susceptibility to damage or rupture.

[0071] Assessment of sperm DNA fragmentation

[0072] DNA fragmentation in sperm was assessed using single cell gel electrophoresis (SCGE). Microgels were prepared on sheets pre-coated with 1% agarose by depositing 85 pL of a sperm suspension (2 x 10 6 ) in 0.5% low melting point (LMP) agarose in PBS, and then covered with a 22 x 22 mm coverslip.

[0073] To solidify the LMP agarose, the slides were placed in a humid box at 4°C for 2 hours. After removing the coverslips, the slides were incubated at 37°C for 1 hour in a lysis solution (2 M NaCl, 55 mM EDTA-Na2, 8 mM Tris, 4% Triton X-100, 0.1% SDS, 1 mM DTT, and 0.5 mg / mL proteinase K, pH 8). The slides were then washed twice in an alkaline (0.3 M NaOH, 1 mM EDTA-Na2, pH 12) or neutral (90 mM Tris, 90 mM boric acid, and 2 mM EDTA, pH 8.5) electrophoresis solution and then placed in an electrophoresis cell filled with the same solution to cover the slides with approximately 1 cm of solution. Electrophoresis was carried out for 10 min at 25 V, and the slides were subsequently neutralized in a 0.4 M Tris-HCl solution at pH 7.5 for 10 min.The slides were then fixed in methanol for 3 minutes and air-dried before being stained with ethidium bromide for observation by fluorescence microscopy on a Nikon Optiphot-2 (Nikon, Japan). The comets were digitized using a Nikon 5100 digital camera (Nikon, Japan) attached to the microscope, always using the same settings in manual mode. The images obtained were analyzed using the free Komet casplab_1.2.3b2 software (CaspLab.com).

[0074] ICSI in mouse oocytes with sperm that have penetrated the ZP

[0075] Metaphase II oocytes were collected from the fallopian tubes of 6- to 8-week-old female B6D2 mice superovulated with 7.5 IU of eCG and with an equivalent dose of hCG 48 h later. Briefly, 14 h after hCG administration, fallopian tubes were removed from superovulated female mice and placed in a Petri dish containing culture medium at room temperature. After washing, the collected fallopian tubes were placed in fresh medium, and cumulus-oocyte complexes were released from the ampulla with the help of dissecting forceps. The cumulus-oocyte complexes (COCs) were then transferred to a dispersion drop.In ICSI experiments, cumulus cells were dispersed during a 3- to 5-minute incubation with 350 IU / ml hyaluronidase; after washing, oocytes were maintained in culture medium at 37°C in a 5% CO2 atmosphere until fertilization by ICSI following the standard protocol (Perez-Cerezales et al., Sperm selection by thermotaxis improves ICSI outcome in mice. Sci Rep 8, 2902 (2018)).

[0076] Results

[0077] Penetration capacity of the ZP of sperm from subfertile bulls

[0078] To analyze whether bulls with different fertility levels, but with normal spermiogram, have different capacity to penetrate ZPs of oocytes from which the cytoplasm with the nucleus was removed by micromanipulation, leaving only the ZP (Fig. 1), the ZPs were coincubated with sperm from four bulls, one with proven fertility and three with subfertility, but which maintained good criteria of motility and sperm quantity, similar to fertile bulls.

[0079] As shown in Figure 2, after 3 hours of coincubation, the ZPs were collected and the areas penetrated by oocyte were quantified. The first thing that was observed was that all penetrated sperm lacked acrosome, while 95% of the sperm that had not penetrated retained the acrosome (Figure 3). Therefore, it can be deduced that sperm lose the acrosome when penetrating the ZP, then only if they undergo capacitation (hyperactivation and undergo the acrosomal reaction) can they penetrate the ZP (Fig. 2). Table 1 shows the results of the four bulls, observing that subfertile bulls had fewer penetrated sperm per zone and a lower range of sperm per zone.

[0080] Table 1. Differences in penetrability of empty sperm ZPs from fertile and subfertile bulls with normal motility and vitality. Esp: sperm. a - b - cIndicates significant differences in each column (Anova, p<0.05). Preservation of the free zones pellucida of the oocyte

[0081] The inventors also analyzed whether the areas can be frozen at -20°C without cryoprotectant and subsequently used for penetrability testing. The results indicate that the ZPs isolated from the oocyte can be used up to 6 months after freezing. Furthermore, they demonstrated that they can be treated with ultraviolet radiation for 3 minutes to eliminate 99.9% of potential microorganisms. The areas can then be sterilized and frozen and subsequently distributed for use in other laboratories.

[0082] Penetration capacity of the ZP of bovine oocytes by spermatozoa of different mammal species

[0083] Although the zona pellucida (ZP) is considered a species-specific barrier in biology, the inventors have demonstrated that bovine ZPs can be penetrated by sperm from other mammalian species, including humans. Results in Table 2 indicate that human, ibex, horse, dolphin, guinea pig, mouse, and cat sperm are able to penetrate the ZP of bovine oocytes. Similar penetration results were observed among the different species analyzed, ranging from 35% in the case of dolphins to 65% in cats.

[0084] Table 2. Penetration test of free ZPs from bovine oocytes obtained in vitro by sperm from other mammalian species.

[0085] Effect of oviductal fluid on the heterologous penetration capacity of the oocyte and / or zona pellucida

[0086] To analyze whether the penetrability of bovine oocytes is lost once the oocyte comes into contact with oviductal fluid and the ZP glycoproteins are modified, the inventors analyzed whether a 30-minute exposure of oocytes to bovine oviductal fluid modified their penetration capacity by human sperm. As shown in Table 3, oocytes that had been in the presence of oviductal fluid lost their ability to be penetrated by human sperm.

[0087] Table 3. Effect of oviductal fluid on the ability of bovine oocytes obtained from the ovary to be fertilized by human sperm. a - b Indicates significant differences (Anova, p<0.05).

[0088] To analyze whether the effect was due to the presence of the oocyte or if it was exclusive to the ZP, the inventors have analyzed whether bovine ZPs isolated from oocytes that were in contact with bovine oviductal fluid for 30 min lost their ability to penetrate human sperm. As we see in Table 4, bovine ZPs that had been in contact with bovine oviductal fluid are not penetrated by human sperm, demonstrating that it is the oviductal fluid that produces the modifications in the ZPs that determine species specificity. The same phenomenon has been observed with ZPs isolated from mouse oocytes obtained directly from the ovary, which are penetrated by human or bovine sperm, but once in contact with oviductal fluid they lose this ability, indicating that the phenomenon is not exclusive to bovines but occurs in other mammalian species.

[0089] Table 4. Effect of oviductal fluid on the ability of the bovine ZP to be penetrated by human sperm Morphometry of the head of spermatozoa penetrated into the ZP

[0090] To analyze whether sperm that were able to penetrate the ZP had different morphometric characteristics than those that did not, the inventors analyzed the length, width, perimeter, and area of ​​200 sperm that had penetrated and compared them with another 200 that had not penetrated but had lost the acrosome, which occurs with all sperm that penetrate. In addition, four morphometric characteristics were analyzed: ellipticity, roughness, elongation, and regularity. The results are shown in Table 5. The results showed that sperm found within the ZP were distinguished by being longer and narrower, with a larger area and a smaller perimeter than sperm found outside the ZP. Furthermore, they were characterized by being more elliptical and elongated cells, with slightly greater roughness and less regularity. The only parameter that did not show differences between the two groups was perimeter.

[0091] Table 5. Mean values ​​and standard deviations of the morphometric parameters corresponding to the cells inside and outside the ZP. * Indicates the degree of significance.

[0092] Outside Inside

[0093] Length (L, pm) 8.85 ± 0.36 9.12 ± 0.31**

[0094] Width (W, pm) 4.20 ± 0.21 4.13 ± 0.20*

[0095] Perimeter (P, pm) 30.05 ± 2.23 29.66 ± 1.39

[0096] Area (A, pm) 2 ) 33.76 ± 1 .91 34.96 ± 2.09**

[0097] Ellipticity (L / W) 2.11 ± 0.13 2.21 ± 0.13**

[0098] Roughness (4TTA / P 2 ) 0.48 ± 0.07 0.50 ± 0.05**

[0099] Elongation [(LW) / (L+W)] 0.36 ± 0.03 0.38 ± 0.02**

[0100] Regularity (TTLW / 4A) 0.87 ± 0.04 0.85 ± 0.06 **

[0101] Sperm that penetrate the ZP have less DNA fragmentation than sperm that do not penetrate.

[0102] To assess the genetic quality of human sperm penetrating the bovine ZP, DNA fragmentation levels were examined using the single cell electrophoresis assay (SCGE or comet assay), using the neutral version.

[0103] By SCGE, human sperm that had penetrated the ZP showed significantly less DNA fragmentation than control sperm that had not penetrated (Figure 4), with a clear enrichment in the percentage of sperm with poorly fragmented DNA.

[0104] Sperm that penetrate the ZP produce more mouse blastocysts than sperm that do not penetrate.

[0105] To evaluate the quality of sperm penetrating the cytoplasm-free bovine ZP, heterologous coincubation between mouse sperm and cytoplasm-free bovine ZP was performed, and the penetrated sperm were recovered. Immediately after, mouse oocytes were fertilized by ICSI with these sperm recovered from inside and outside the bovine ZP, and as can be seen in Figure 5, both penetrated and non-penetrated sperm produced similar numbers of 2-cell stage embryos, but when we cultured these embryos in vitro, it was observed that penetrated sperm produced many more blastocyst stage embryos than non-penetrated sperm, indicating that the penetrated sperm population is of higher quality than non-penetrated sperm, which is reflected in higher blastocyst production.

Claims

CLAIMS 1. An in vitro method for determining whether a sperm sample has fertilizing capacity and / or for recovering from a sample sperm that have penetrated the zona pellucida of an oocyte, comprising: a) removing cytoplasmic material from oocytes previously isolated from bovine ovaries, obtaining the isolated zona pellucida free from the rest of the oocyte components; b) performing heterologous in vitro fertilization between the free zona pellucida of oocytes from a) with a sperm sample previously isolated from a mammal; c) determining the % of sperm in the sample that penetrate the zona pellucida; d) determining whether the sperm sample has fertilizing capacity based on the % penetrability obtained in c), wherein when at least 50% of the sperm present in the sample have penetrated the zona pellucida, the sample is determined to have fertilizing capacity;and / or e) when it is determined in step d) that the sample has fertilizing capacity, the sperm that have penetrated the zona pellucida are recovered; 2. The method according to claim 1, wherein the sperm sample comes from a mammal selected from: human, mountain goat, horse, dolphin, guinea pig, mouse and cat.

3. The method according to claim 2, wherein the mammal is the human being.

4. The method according to any one of claims 1 to 3, wherein the oocytes previously isolated from bovine ovaries are isolated from grade I or grade II cumulo oocyte complexes (COC).

5. The method according to any one of claims 1 to 4, wherein the sperm concentration of the sample is between 1x10 4 1x10 8 sperm / mL, preferably 1x10 6 sperm / mL.

6. The method according to any one of claims 1 to 5, wherein the material Cytoplasmic is removed from the oocyte in stage a) by means of a microinjector with a piezoelectric system.

7. The method according to any one of claims 1 to 6, wherein in step d) it is determined that the sperm sample has fertilizing capacity when the % of sperm that penetrate the zona pellucida is equal to or greater than 70%, 75%, 80%, 85%, 90% or 95% with respect to 100% of sperm present in the sample.

8. Sperm recovered after step e) of the method according to any one of claims 1 to 7.

9. Composition comprising the sperm according to claim 8.

10. Composition according to claim 9, wherein the composition is a pharmaceutical composition.

11. The composition according to claim 10 further comprising a pharmaceutically acceptable carrier and / or excipient.

12. Kit comprising the sperm according to claim 8 or the composition according to any one of claims 9 to 11.

13. Sperm according to claim 8 or the composition according to any of claims 9 to 11 for use as a medicament.

14. Sperm according to claim 8 or the composition according to any of claims 9 to 11 for use in assisted reproduction therapies.

15. Sperm according to claim 8 or the composition according to any of claims 9 to 11 for use according to claim 14, wherein the assisted reproduction therapy is in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI).

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