Patent Description:
This invention belongs generally to the field of Assisted Reproduction in mammals, specifically to non therapeutical method to increase the biological quality and survival of mammalian embryos produced in vitro.

The procedure currently known for in vitro obtaining of an embryo involves making different assisted reproductive techniques (ART) that differ between laboratories and species, and comprises the following steps: <NUM>) obtaining and in vitro maturation (IVM) of female gametes (oocytes); <NUM>) the preparation and capacitation of male gametes (sperm); <NUM>) the process of IVF itself either by co-culture of gametes or assisted microinjection of a sperm into the oocyte, and <NUM>) the EC of the zygotes up to they reach the stage of desired development, usually the blastocyst stage.

A strategy to increase the efficiency of the ART has been to mimic in the laboratory the conditions that gametes-zygotes-embryos find in vivo where, after ovulation, the mature oocyte is captured by the oviduct (fallopian tubes) interacting with the natural medium, the oviductal fluid (OF). Meanwhile sperm "swim" from the vagina or cervix (depending on the species) to the site of fertilization (oviduct) contacting during its passage through the uterus with the uterine fluid (UF) and then, when they are already in near the oocyte, with the FO. Once both gametes are in the oviduct, fertilization and early embryonic development occur, being the OF the natural environment in which these reproductive events occur. Subsequently the embryo passes into the uterus, with the UF as the physiological medium where implantation and placentation will happen. Therefore, in order to develop and/or adapt new culture media employed in vitro, and to improve the current results offertilization and embryo development, many studies have examined the composition of these natural fluids ( <NPL>;<NPL> ). However, there have been no studies to reproduce in a combined and complete form the physiological environment designing systems a whole culture system (from IVF to EC) with culture media close to the composition of natural biological fluids (OF and UF). Even though, considering that it has been proven in humans that the addition to the culture medium of a "simple" oviductal protein increases more than <NUM>% of births ( <NPL> ).

Undoubtedly, the oviductal and uterine secretions provide better conditions for fertilization and embryonic development that conventional in vitro systems since the composition of these natural media varies along the different phases of the female reproductive cycle thus providing the oocyte, zygote and/or embryo the ideal conditions for their development at all times ( <NPL>; <NPL>). In general, the natural biofluids contain salts, electrolytes, amino acids, protein and energy compounds. Among the more than <NUM> oviductal proteins that have been identified (<NPL>), some are already commercially available and used as a supplement to the in vitro culture media (<NPL>; <NPL>). However, the complete composition and exact concentration of all components of biofluids is unknown, so the direct addition of the biofluids to the artificial culture media is the cheapest, fast and reliable strategy to supplement them with all the compounds that gametes, zygotes and / or embryos needed in each of their development stages.

In mammals, including humans, the OF and/or UF are obtained by different techniques and are often processed with a single centrifugation and subsequent freezing of the liquid fraction ( <NPL>;<NPL>;<NPL>;<NPL>;<NPL>). These biofluids improve fertilization rates and increase embryo quality (<NPL>; <NPL>; <NPL>; <NPL>). Despite these encouraging results, to date the biofluids have not been used as additives in human ARTs which opens a new field of research, especially considering that, in most cases, the beneficial effect on gametes and embryos is interspecific, i.e. biofluids from a mammalian species can be used for ART in different species ( <NPL>;<NPL> ). So far the processing, storage and transport of biofluids samples require a controlled temperature to maintain the cold chain, conditioning the implementation and extension of its use in the field of reproductive biology.

It has been described the beneficial effect of gametes contact with OF compounds before IVF (<NPL>;<NPL> ) and it has been found that sperm processed by Swim-up technique show an increased DNA integrity and a lower percentage of morphological abnormalities than those obtained by Percoll method ( <NPL>;<NPL> ). Although the process of sperm capacitation may seem a mere formality in the ART (it lasts only <NUM>-<NUM> minutes), the stress suffered by sperm is transmitted to offspring with metabolic and behavioural changes ( <NPL> ).

It has been found <NUM> documents related to the use of specific substances in IVF and/or CE protocols but none of them describe the combined use of OF and UF from specific phases of the reproductive cycle in the generation of embryos. In these patents it is not described a purification or fractionation method of biofluids neither its use in sperm capacitation.

Other documents are:
<NPL>. The document discloses bovine processed OF for the culture of oocytes, and further IVF.

The paper is related about the identification of the growth factor and cytokine profile of human uterine fluid from fertile women. The objective of the document is to determine the relevant endometrial secretions for embryo implantation.

This document describes the superoxide dismutase activity in the oviductal and uterine fluid of the bitch and the effects of the enzyme on viability, motility, and hyperactivation of canine sperm in vitro.

The document refers to the effects of oviductal fluid on sperm penetration and cortical granule exocytosis during fertilization of pig oocytes in vitro.

<NPL>",discloses centrifuged bovine oviductal fluid for culturing oocyte before IVF. The document refers to refers to the effects of oviductal fluid in porcine oocytes produced in vitro. The aim is to determine whether the exposure of oocytes to oviductal fluid prior to fertilization followed by a shorter sperm oocyte co-incubation, would also affect subsequent pre-implantation embryo development states.

The 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 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 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.

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.

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 non therapeutical 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.

The present invention contemplates an oviductal and uterine fluids for use in a non therapeutical method to increase the biological quality and survival of mammalian embryos produced in vitro by incorporating mammalian oviductal and uterine fluids from specific phases of the reproductive cycle to culture media, wherein the non therapeutical method comprises the following steps:.

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 (<NPL>). In the human species biofluids are obtained from patients undergoing tubal ligation, hysterectomies or salpingectomy for health reasons and donors.

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 (<NUM>-<NUM> minutes, <NUM>-<NUM> at <NUM>) 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.

Once fractionated, biofluids are lyophilised at -<NUM> - -<NUM> during <NUM>-<NUM> at a pressure of <NUM>-<NUM> 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 <NUM>-<NUM> for <NUM>-<NUM> seconds before use, increasing the necessary sanitary warranties of the product.

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) ( <NPL>). 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.

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 ( <NPL>;<NPL> ) but making the following changes on the composition:
Adjusting the salt concentration: within this group we include NaHCO3 , NaCl, KCI, MgSO4 , K2 HPO4 , and CaCl2. 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 <NUM>-320mOsm. The concentration of inorganic salts was between <NUM> and <NUM>.

Adjusting pH: it was adjusted to <NUM>-<NUM> using HEPES as buffering agent to prevent pH oscillations in the medium during the time that sperm are in contact with it.

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 <NUM> and <NUM>.

Protein: in the new medium designed, Swim-up-biofluids, the protein source consisted of addition of <NUM>-<NUM>% of phase F2-OF preferably fractionated and treated as described before.

This treatment was compared with BSA (bovine serum albumin), the protein source commonly used in the culture media for sperm capacitation.

The capacitation method Swim-up-biofluids was performed by mixing <NUM>-<NUM> of semen with <NUM>-<NUM> of culture medium Swim-up-biofluids and allowing sperm to swim to the top of the tube for a time of <NUM>-<NUM> at a temperature of <NUM>-<NUM>. After this time, the top <NUM>-<NUM> was collected containing the sperm to be used for IVF. The new method of sperm capacitation was compared with the traditional method of centrifugation (<NPL> ). 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.

In the non therapeutic method including addition of biofluids to fertilization medium, the porcine oocytes are matured in vitro for <NUM>-<NUM> according to protocols described ( <NPL>). After that, cumulus-oocyte complexes are mechanically denuded and <NUM>-<NUM> oocytes are transferred to each well containing 500ul of TALP medium for fertilization (control group) ( <NPL>)or TALP supplemented with <NUM>-<NUM>% OF preferably from F2 or L1 phase, fractionated and treated as described before (biofluids group). The sperm used for IVF were obtained and processed by Swim-up-biofluids system. Optionally, before being inseminated the denuded oocytes may be incubated in OF, preferably from F2 or L1 phase, for <NUM>-<NUM> minutes. Gametes were cocultured for <NUM> hours and after this time the presumptive zygotes were fixed and stained to evaluate the results of fertilization. With this non therapeutic method monospermy rates are improved after fertilization.

In developing the EC in culture media supplemented with biofluids, the cumulus-oocyte complexes are matured according to the protocols described ( <NPL>). Two methods of EC were compared, the control method and the biofluids method using fluids fractionated and treated as described before. For the biofluids group, sperm used for IVF were capacitated by Swim-up-biofluids method and the oocytes were inseminated in culture medium supplemented with <NUM>-<NUM>% OF. The obtained zygotes were transferred to embryo culture medium NCSU-<NUM> (<NPL>) supplemented the first two to three days with <NUM>-<NUM>% of OF, preferably from L1 or L2 phase. After the first <NUM> hours, cleavage was evaluated and then the divided embryos were transferred to NCSU-<NUM> medium supplemented with <NUM>-<NUM>% 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-<NUM> for embryo culture. After culture period, the embryo quality in both groups was assessed attending to morphology (B6 and <NPL>) 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).

Non therapeutic 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.

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 (<NUM>-<NUM> for <NUM>-<NUM> minutes at <NUM>) 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 <NUM>-<NUM> minutes in OF at <NUM>. After this time the eggs are incubated with a <NUM>% protease solution and the time it takes to digest the ZP is recorded. As shown in Table <NUM> the biofluids treated by our method maintained their biological activity with respect to untreated biofluids (control):.

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 (<NUM>-<NUM> months old). For the capacitation with Swim-up-biofluids method, semen was deposited on Swim-up-biofluids medium for <NUM>-<NUM> minutes at <NUM>-<NUM>. After this time the sperm cells were collected from the top and sperm concentration was adjusted to <NUM> sperm/ml using with TALP culture medium (<NPL>). This new method of sperm capacitation was compared with the traditional method of centrifugation (Percoll) <NPL>). The quality of the obtained sperm was assessed by their ability to fertilize porcine oocytes previously matured in vitro for <NUM>-<NUM> hours according to standard protocols (<NPL>). Oocytes were cocultured for <NUM> hours with spermatozoa capacitated in the different methods. After this time the presumptive zygotes were fixed and stained to evaluate the fertilization results (<NPL>). 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 <NUM>.

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 <NUM>). This makes the performance (YIE) of the IVF with the biofluids method is significantly higher yielding a greater number of putative embryos.

After the fertilization period, zygotes were transferred to culture medium (NCSU-<NUM>) supplemented or not with OF from early luteal phase for <NUM> hours where they were cultured until <NUM>-<NUM> cells stage. After this time the embryos were transferred to NCSU-<NUM> 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 <NUM>) and the highest percentage of blastocysts that initiate and complete the process of hatching (Table <NUM>).

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 <NUM>). This result shows that quality of embryos obtained with the biofluids method is similar to that obtained naturally, at least regarding this quality parameter.

Claim 1:
Non therapeutical method to increase the biological quality and survival of mammalian embryos produced in vitro by incorporating mammalian biofluids from specific phases of the reproductive cycle to culture media, characterized in that it comprises the following steps:
Culture of the mammalian embryos to any stage of preimplantational development in media supplemented with oviductal fluid in a first phase between <NUM> to <NUM> hours and uterine fluid in a second phase between <NUM> hours to morula or blastocyst stage,
wherein the embryos have been obtained by in vitro fertilization using sperm that has been processed in a culture medium supplemented with oviductal fluid,
wherein the selection of said sperm for in vitro fertilization is based on its physiological ability to swim freely through a culture medium containing water, inorganic salts, energetic compounds, antibiotics and fractionated and treated oviductal fluid obtained from the late follicular phase of the cycle and,
wherein the oviductal and uterine fluids of each of the phases of the reproductive cycle are fractionated and processed by double centrifugation with subsequent lyophilization, optionally with pasteurization and cooling for their use in assisted reproduction techniques or biotechnology in general.