Processing and Use of Fluids of the Reproductive Tract for Improving the IN VITRO Production of Embryos of Mammals

Abstract

Processing and use of fluids from the reproductive tract (biofluids) to improve the in vitro production of mammalian embryos comprising the following steps: a) fractionation and processing of biofluids through a sorting, purification, lyophilization and subsequent storage; b) a method of sperm capacitation in a culture medium supplemented with biofluids; c) in vitro fertilization in a medium enriched with biofluids and d) subsequent in vitro culture with development of the obtained embryos to any stage of preimplantational development in culture media supplemented with biofluids.

Claims

1. Method to increase the biological quality and survival of mammalian embryos produced in vitro by incorporating biofluids from specific phases of the reproductive cycle to culture media characterized in that it comprises the following steps: i) Fractionation and processing of the oviductal and uterine fluids of each of the phases of the reproductive cycle by a selection process, double centrifugation with subsequent lyophilization, optionally with pasteurization and cooling for its use in assisted reproduction techniques or biotechnology in general. ii) Processing of spermatozoa in a culture medium supplemented with biofluids obtained in i) wherein the selection of sperm for in vitro fertilization is based on their physiological ability to swim freely through this culture medium containing water, inorganic salts, energetic compounds, antibiotics and fractionated and treated oviductal fluid obtained from the late follicular phase of the cycle. iii) In vitro fertilization using sperm obtained in ii) in a culture medium supplemented with oviductal fluid. iv) Culture of embryos obtained in iii) to any stage of preimplantational development in media supplemented with oviductal fluid in a first phase (variable from 24 to 72 hours) and uterine fluid in a second phase (variable from 48 to morula or blastocyst stage).

2. The method according to claim 1, characterized in that the concentration of inorganic salts in the culture medium is between 90 and 130 mM, the concentration of energy substrates is between 120 and 160 mM and the concentration of fractionated and treated oviductal fluid is between 0.1 and 5%.

3. The method according to claim 1, wherein the concentration of fractionated and treated oviductal fluid, from late follicular or early luteal phase the reproductive cycle, in the IVF medium is between 0.1 and 5%.

4. The method according to claim 1, characterized in that step iv) is carried out by culturing embryos in the first 24-72 hours after fertilization in culture medium supplemented with fractionated and treated oviductal fluid, preferably obtained during the early luteal phase of the cycle at a concentration between 0.1 and 5%.

5. The method according to claim 1, characterized in that step iv) is carried out by culturing embryos to the blastocyst or morula stage in culture medium supplemented with fractionated and treated uterine fluid, preferably obtained during the luteal phase of the cycle at a concentration between 0.1 and 5%.

6. Method according to claim 1, wherein the biofluids are obtained from mammalian species.

7. The method according to claim 1, characterized in that the gametes, zygotes or embryos are obtained from mammalian species.

8. The method according to claim 1 wherein the biofluids used are from the same or different species.

9. The method according to claim 1 wherein the biofluids used may be of the same subject or a different subject (donor).

10. The use of fractionated and treated biofluids in the development of culture media, supplements or drugs to improve sperm functionality, in vitro fertilization, embryo culture, cryopreservation of gametes or embryos or embryo transfer to a recipient female according to claim 1.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention contemplates a method for increasing the quality of mammalian embryos obtained in vitro by a system that includes the addition of natural biological fluids (OF and/or UF) from specific phases of the reproductive cycle in the techniques of sperm capacitation, IVF and EC.

[0010] The authors of the present invention have developed on the one hand, a system for sperm capacitation that does not require centrifugation, where protein additive is OF and/or FU, which involves great advantages in reducing the stress that sperm cells are suffering during the process of separation of seminal plasma, and thus helps to select those sperm with better quality and DNA integrity. It has been managed to combine both strategies formulating a new culture medium for selecting sperm by Swim-up technique that offers better results than conventional procedures. On the other hand, it has carried out a new process for obtaining purified and lyophilized samples (dried) of both biofluids without losing their beneficial biological activity making thus possible to store dry samples that can be shipped at room temperature, what is an advantage against the conventional method used so far (liquid samples). All together increases the chances that this integrated system to obtain in vitro embryos can be implemented and developed in laboratories worldwide.

[0011] Currently, both the number and quality of mammalian embryos obtained through in vitro techniques are lower than that obtained in vivo. In the in vivo procedure, gametes contact with biofluid in isolation before fertilization and embryo development occur. So that, a complete protocol for obtaining high quality embryos should include this contact of gametes, in an individual way, with the biofluids; and replacing sperm selection systems such as Percoll (which involves centrifugation) by the swim-up technique where sperm swim freely.

[0012] The use of natural fluids that gametes and embryos specifically find in the reproductive tract along its development can increase the quality of embryos obtained in vitro. Thus, this invention proposes the introduction of these natural fluids of the reproductive tract, such as the OF and UF in the ARTs to improve the quality of embryos obtained in vitro. The invention describes a method for obtaining in vitro embryos by adding biofluids to the culture media in each of the stages of the process. The biofluids are obtained from genital tracts of mammalian females at specific phases of the reproductive cycle and are previously subjected to a process of purification and treatment for its preservation and transportation.

[0013] The invention comprises the following steps:

a) a method of classifying the OF and UF according to the stage of the reproductive cycle in which they are obtained, followed by double fractionation by centrifugation and subsequent processing by pasteurization and lyophilization.
b) a method of sperm capacitation without centrifugations in a specific culture medium supplemented with OF and/or UF.
c) IVF in a culture medium enriched with OF and/or UF.
d) in vitro culture and development of embryos obtained to any stage of development in media supplemented with OF and/or UF.

[0014] To carry out the fractionation method by double centrifugation and subsequent processing of biofluids by lyophilization, the OF and UF are obtained from animals slaughtered in the slaughterhouse (pig, swine, cattle, sheep, goat, rabbit and equine). Reproductive tracts are classified in the different phases of the estrous cycle: early follicular (F1), late follicular (F2), early luteal (L1) and late luteal (L2) according to the appearance and ovarian morphology (Carrasco et al 2008b, Reproduction 135: 19-27). In the human species biofluids are obtained from patients undergoing tubal ligation, hysterectomies or salpingectomy for health reasons and donors.

[0015] The OF is obtained by introducing the tip of an automatic pipette into the oviductal ampulla and exerting a manual pressure thus sucking all the oviductal content. For fractionation, the content is centrifuged (1-10 minutes, 4000-10000 g at 4° C.) and the cell pellet and mucosal phase is discarded. The supernatant of OF is recentrifuged under the same conditions being the aqueous phase at the top and the mucosal phase at the bottom of the tube. Only the aqueous phase of OF is drawn. Meanwhile, the UF is obtained by aspiration introducing the tip of an automatic pipette into the uterine horn and exerting an upward manual pressure. Such content is fractionated by a double centrifugation under the same conditions as the OF.

[0016] Once fractionated, biofluids are lyophilised at −60-−50° C. during 20-24 h at a pressure of 0.040-0.010 mbars. Once the process is finished, the biofluids are kept refrigerated until use. The biofluids obtained from the different species and phases of the cycle are stored in a biobank and can be used both in inter- or intraspecific manner as well as in autologous or heterologous way. Before being used as additives in a culture medium, the biofluids are resuspended with purified water to its original volume. One of the advantages of this new fractionation method is that biofluids can be transported at room temperature, retain their biological properties upon rehydration and can be pasteurized at 72-80° C. for 10-20 seconds before use, increasing the necessary sanitary warranties of the product.

[0017] The tests of the biological activity of the product have been performed to evaluate the ability of biofluids to harden the oocyte's zona pellucida (ZP) (Coy et al 2008b, Reproduction 135: 19-27). For that, mature oocytes were denuded and subjected to ZP digestion with a protease. The results show that biofluids subjected to fractionation and conservation process described in this patent retain their biological activity once they are resuspended and pasteurized.

[0018] For the development of sperm capacitation method without centrifugations and using media supplemented with biofluids, the present invention includes the formulation of a new culture medium for sperm selection that we call Swim-up-biofluids. To do this we rely on the media described in the literature (Alvarez et al. 1993, Hum Reprod 8: 1087-1092; Garcia-López et al. 1996, J Chromatogr B Biomed Appl 680: 137-143) but making the following changes on the composition: [0019] Adjusting the salt concentration: within this group we include NaHCO.sub.3, NaCl, KCl, MgSO.sub.4, K.sub.2HPO.sub.4, and CaCl.sub.2. They are responsible for maintaining the osmolarity and pH of the medium so that its concentration was adjusted to that described in the OF. The final osmolarity of the medium was kept in the physiological range of 280-320 mOsm. The concentration of inorganic salts was between 90 and 130 mM. [0020] Adjusting pH: it was adjusted to 7.2-7.8 using HEPES as buffering agent to prevent pH oscillations in the medium during the time that sperm are in contact with it. [0021] Adjusting the concentration of energy substrates: glucose, sodium pyruvate, sodium lactate and sucrose are within this group. Concentrations were adjusted to achieve optimal viscosity and density which allow the mobility of sperm through the medium. The concentration of energy substrates was between 120 and 160 mM. [0022] Protein: in the new medium designed, Swim-up-biofluids, the protein source consisted of addition of 0.1-5% of phase F2-OF preferably fractionated and treated as described in step a). This treatment was compared with BSA (bovine serum albumin), the protein source commonly used in the culture media for sperm capacitation.

[0023] The capacitation method Swim-up-biofluids was performed by mixing 0.5-1.5 ml of semen with 0.5-1.5 ml of culture medium Swim-up-biofluids and allowing sperm to swim to the top of the tube for a time of 10-50 min at a temperature of 37-38° C. After this time, the top 0.5-1 ml was collected containing the sperm to be used for IVF. The new method of sperm capacitation was compared with the traditional method of centrifugation (Percoll; Matas et al 2003, Reproduction 125: 133-114). Thorough their research, the inventors have shown that when sperm are capacitated in the Swim-up-biofluids system using a culture medium supplemented with OF the monospermy rate is significantly improved and therefore the number of zygotes capable of developing up to embryo stage.

[0024] In the embodiment of IVF method including addition of biofluids to fertilization medium, the porcine oocytes are matured in vitro for 42-44 h according to protocols described (Coy et al. 2008b, Reproduction 135: 19-27). After that, cumulus-oocyte complexes are mechanically denuded and 50-55 oocytes are transferred to each well containing 500 ul of TALP medium for fertilization (control group) (Matas et al. 2003, Reproduction 125: 133-141) or TALP supplemented with 0.1-5% OF preferably from F2 or L1 phase, fractionated and treated as described in step a) (biofluids group). The sperm used for IVF were obtained and processed by Swim-up-biofluids system described in step b). Optionally, before being inseminated the denuded oocytes may be incubated in OF, preferably from F2 or L1 phase, for 30-60 minutes. Gametes were co-cultured for 18 hours and after this time the presumptive zygotes were fixed and stained to evaluate the results of fertilization. With this method monospermy rates are improved after fertilization.

[0025] In developing the EC in culture media supplemented with biofluids, the cumulus-oocyte complexes are matured according to the protocols described (Coy et al. 2008b, Reproduction 135: 19-27). Two methods of EC were compared, the control method and the biofluids method using fluids fractionated and treated as described in step a). For the biofluids group, sperm used for IVF were capacitated by Swim-up-biofluids method described in step b) and the oocytes were inseminated in culture medium supplemented with 0.1-5% OF, as described in step c). The obtained zygotes were transferred to embryo culture medium NCSU-23 (Petters and Wells 1993, J Reprod Fertility Suppl 48: 61-73) supplemented the first two to three days with 0.1-5% of OF, preferably from L1 or L2 phase. After the first 48 hours, cleavage was evaluated and then the divided embryos were transferred to NCSU-23 medium supplemented with 0.1-5% UF, preferably from L1 or L2 phase, up to the blastocyst stage. In the control group the biofluids were not used in any phase of the method, sperm were capacitated with Swim-up-BSA method, the oocytes were inseminated in TALP medium and zygotes were transferred to NCSU-23 for embryo culture. After culture period, the embryo quality in both groups was assessed attending to morphology (Bó and Mapletoft 2013, Anim Reprod 10: 344-348) and mean number of cells per blastocyst. Results show that embryos produced in vitro with the biofluids method divide faster than the control and are of better quality (evaluated as number of cells per blastocyst and ability to hatch).

Embodiment of the Invention

[0026] Method Used to Obtain In Vitro Derived-Embryos by Fertilization and Embryo Culture Using Biofluids from Different Stages of Reproductive Cycle and Checking the Improved Embryo Quality

a.—Obtaining, Fractionation and Test of the Activity of the Biofluids

[0027] In farm animals, OF and UF are obtained from genital tracts of animals slaughtered in a slaughterhouse for meat consumption. In the human species they can be obtained from donors, patients undergoing tubal ligation or women undergoing salpingectomy or hysterectomies for health reasons. The biofluids are centrifuged (4000-10000 g for 1-10 minutes at 4° C.) and the aqueous phase recentrifuged under the same conditions. This liquid is lyophilized and pasteurized being then ready for its use as an additive to culture media after resuspension with purified water. Treatment efficacy was estimated by the ability of OF to induce hardening of the oocyte's zona pellucida. For this, in vitro matured oocytes are mechanically denuded and incubated for 30-60 minutes in OF at 38.5° C. After this time the eggs are incubated with a 0.5% protease solution and the time it takes to digest the ZP is recorded. As shown in Table 1 the biofluids treated by our method maintained their biological activity with respect to untreated biofluids (control):

TABLE-US-00001 TABLE 1 Digestion time of the zona pellucida (ZP) of in vitro matured oocytes after being incubated 30-60 minutes in OF from phase F2, fractionated, lyophilized and pasteurized. . Tratamiento del fluido oviductal N tdZP (seconds) Control (non fractionated) 14 3750.0 ± 665.2a Fractionated 14 7995.0 ± 70.8b  Fractionated and lyophilized 14 9432.8 ± 534.2b Fractionated, lyophilized and pasteurized 10 8874.0 ± 445.6b Data are expressed as mean ± SEM. Different letter indicate significant differences (P < 0.001) N is the number of oocytes employed
b.—Sperm Capacitation without Centrifugations in a New Culture Medium Enriched with Biofluids

[0028] The new culture medium was formulated by adjusting the osmolarity, pH and energy substrates and replacing bovine serum albumin (BSA) by biofluids. The capacitation was performed with ejaculated sperm from proven fertility males (12-24 months old). For the capacitation with Swim-up-biofluids method, semen was deposited on Swim-up-biofluids medium for 15-30 minutes at 37-38° C. After this time the sperm cells were collected from the top and sperm concentration was adjusted to 25.000 sperm/ml using with TALP culture medium (Matas et al. 2003, Reproduction 125: 133-114). This new method of sperm capacitation was compared with the traditional method of centrifugation (Percoll) (Matas et al. 2003, Reproduction 125: 133-114). The quality of the obtained sperm was assessed by their ability to fertilize porcine oocytes previously matured in vitro for 42-44 hours according to standard protocols (Coy et al. 2008b, Reproduction 135: 19-27). Oocytes were cocultured for 18 hours with spermatozoa capacitated in the different methods. After this time the presumptive zygotes were fixed and stained to evaluate the fertilization results (Coy et al. 2008b, 135 Reproduction: 19-27). The results showed that sperm capacitated with the Swim-up-biofluids system with medium supplemented with OF significantly improve monospermy by reducing the number of sperm binding to the zona pellucida of the oocyte (SPZ/ZP) and number of sperm penetrating into the oocyte (SPZ/OO), thereby obtaining higher performance (YIE) of the technique than with the conventional centrifugations system as shown in table 2.

TABLE-US-00002 TABLE 2 IVF results after capacitation of sperm in a traditional method with centrifugations (Percoll) or a method without centrifugations with a new culture medium in which it has been used as a protein source albumin (Swim-up-BSA) or the biofluids (Swim-up-biofluids). PEN (percentage of penetrated oocytes), MONO (percentage of monospermy), SPZ/OO (average number of spermatozoa per penetrated oocyte), SPZ/ZP (average number of sperm bound to the zona pellucida) and YIE (yield, percentage of viable zygotes from the total penetrated). The data are expressed as mean ± SEM. Different letters indicate significant differences (P < 0.05). Capacitation method N PEN (%) MONO (%) SPZ/OO SPZ/ZP YIE (%) Percoll 105 84.3 ± 3.6a 17.4 ± 4.1a 8.4 ± 0.7a 17.3 ± 2.3a  14.6 ± 0.1a Swim-up-BSA 180 69.6 ± 3.5b  42.7 ± 4.6ab 2.1 ± 0.1b 7.2 ± 0.5b 29.7 ± 0.2b Swim-up- 183 71.1 ± 3.4b 49.6 ± 4.5b 2.7 ± 0.1b 8.6 ± 0.5b 35.2 ± 0.2c biofluids N is the number of inseminated oocytes
c.—Obtaining of Embryos by IVF in Media Supplemented with Biofluids

[0029] Once the sperm are capacitated with the swim-up-biofluids system, IVF is performed in a culture medium (TALP) with or without biofluids. Oocytes are matured under standard protocols described above and transferred to fertilization medium. The results of this invention have shown that when the oocytes are inseminated in a culture medium supplemented with OF penetration rates are increased and high levels of monospermy remain (Table 3). This makes the performance (YIE) of the IVF with the biofluids method is significantly higher yielding a greater number of putative embryos.

TABLE-US-00003 TABLE 3 IVF results after supplementing the fertilization medium with biofluids. PEN (percentage of penetrated oocytes), MONO (percentage of monospermy), SPZ/OO (average number of spermatozoa per penetrated egg), SPZ/ZP (average number of sperm bound to the zona pellucida) and YIE (yield, percentage of viable zygotes from the total penetrated). Data are expressed as mean ± SEM. Different letters indicate significant differences at P < 0.05. Capacitation methods N PEN (%) MONO (%) SPZ/OO SPZ/ZP YIE (%) Control 32 43.7 ± 0.1a 78.6 ± 0.1 1.2 ± 0.1 13.4 ± 2.1 34.4 ± 0.1a Biofluids 33 66.6 ± 0.1b 72.7 ± 0.1 1.3 ± 0.1 19.3 ± 3.2 48.5 ± 0.1b N is the number of inseminated oocytes
d.—Embryo Culture with Biofluids and Assessment of the Quality of Obtained Embryos

[0030] After the fertilization period, zygotes were transferred to culture medium (NCSU-23) supplemented or not with OF from early luteal phase for 48 hours where they were cultured until 2-4 cells stage. After this time the embryos were transferred to NCSU-23 medium supplemented or not with UF from early luteal phase where they were culture up to the blastocyst stage. To check the effects of biofluids system on embryo quality parameters such as cleavage, number of blastocysts at each stage of development (from early blastocyst to hatched blastocyst), average number of cells per blastocyst and its functionality, evaluated as their ability to hatch (expand and contract rhythmically to get out of the zona pellucida), were assessed. It was found that embryos obtained by biofluids system had significantly better quality than the embryos that had not been in contact with these natural fluids, as evidenced by the increased number of cells per blastocyst (Table 4) and the highest percentage of blastocysts that initiate and complete the process of hatching (Table 5).

TABLE-US-00004 TABLE 4 Results of embryo culture with biofluids method. Blastocyst Cleavage formation YIE Cells/ Group N (%) (%)* (%) blastocyst Control 903 47.5 ± 1.6a 41.4 ± 2.4 19.6 ± 1.3 49.9 ± 3.7a Biofluids 961 42.1 ± 1.6b 44.5 ± 2.5 18.7 ± 1.2 81.8 ± 7.2b Data are expressed as mean ± SEM. Different letters indicate significant differences at P < 0.05. YIE (method performance, blastocyst rate with respect to embryos divided). *With respect to embryos divided. N is the number of employed zygotes

TABLE-US-00005 TABLE 5 Developemenatl stage of blastocysts (Blasto) obtained in vitro with biofluids method. Data are expressed as mean ± SEM. Different letters indicate significant differences at P < 0.05. Early Hatching Hatched Group N blasto Blasto Expand blasto blasto blasto Control 903 31.7 ± 6.1a 28.3 ± 5.9 40.0 ± 6.4 0a 0a Biofluido 961 12.8 ± 5.4b 30.8 ± 7.5 35.9 ± 7.8 15.4 ± 5.9b 5.1 ± 3.6b N is the number of employed zygotes

[0031] When it is compared the average number of cells from in vitro-derived blastocysts with in vivo-derived blastocyst, it is observed that the number of cells of the embryos obtained with biofluids method is similar to in vivo embryos, while the control method have about half cells (Table 6). This result shows that quality of embryos obtained with the biofluids method is similar to that obtained naturally, at least regarding this quality parameter.

TABLE-US-00006 TABLE 6 Average number of cells per blastocyst obtained in vivo and in vitro with the control and biofluids methods. Method Cells/blastocyst Control 49.9 ± 3.6a Biofluids 81.8 ± 7.2b In vivo 87.0 ± 7.2b Data are expressed as mean ± SEM. Different letters indicate significant differences with P < 0.001.