Methods for increasing the embryo implantation rate in mammals

Abstract

The present invention relates to a method for increasing embryo implantation rate in mother's uterus in mammals by administering to the uterus of a mammal an effective amount of beta-galactoside-binding lectin or derivatives thereof, as well as to a product comprising said lectin.

Claims

1. A method for increasing embryo implantation rate in the uterus of mammals, characterized in that it comprises administering to the uterus of a mammal an effective amount of a galectin or an alkylated form thereof that binds beta-galactoside simultaneously with or up to 17 days after administration of semen, an oocyte, or an embryo.

2. A method according to claim 1, characterized in that an amount ranging from 0.0001 to 1.0 mg of an active form of a galectin or an alkylated form thereof is administered per kilogram of body weight of the mammal.

3. A method according to claim 2, characterized in that the active form of a galectin or an alkylated form thereof is in the form of a sterile, stable, endotoxin-free, isotonic carrier having a pH between 6.8 and 7.4.

4. A method according to claim 3, characterized in that the carrier is a buffered solution.

5. A method according to claim 4, characterized in that the buffered solution is selected from a saline phosphate buffered solution (PBS) or physiological serum.

6. A method according to claim 1, characterized in that the galectin or alkylated form thereof is selected from Galectin-1, Galectin-3, Galectin-9, Galectin-13, Galectin-15 or an alkylated thereof.

7. A method according to claim 1, characterized in that the galectin or alkylated form thereof is supplied to the uterus of the mammal mixed with semen, oocyte or embryo.

8. A method according to claim 7, characterized in that the semen, oocyte or embryo is provided in a maintenance medium.

9. A method according to claim 8, characterized in that the semen is fresh, chilled or frozen.

10. A method according to a claim 8, characterized in that the embryo is fresh, frozen or vitrified, and comes from embryo transfer (ET), in vitro fertilization (IVF), a clone or a transgenic embryo.

11. A method according to a claim 8, characterized in that the oocyte is fresh, chilled or vitrified.

12. A method according claim 7, characterized in that the galectin or alkylated form thereof, together with semen, oocyte or embryo, is administered through uterine or vaginal route.

13. A method according to claim 1, characterized in that the galectin or alkylated form thereof is supplied to the uterus of the mammal separately from but simultaneously with the administration of semen, oocyte or embryo.

14. A method according to claim 1, characterized in that the galectin or alkylated form thereof is supplied to the uterus of the mammal separately from and subsequently to the administration of semen, oocyte or embryo.

15. A method according to claim 14, characterized in that the galectin or alkylated form thereof is supplied to the uterus of the mammal together with and semen, oocyte or embryo in a subsequent manner, and the lapse of time between the supply of galectin or alkylated form thereof, and the supply of semen, oocyte or embryo extends up to 17 days.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) In the present invention, the implantation rate relates to the number of embryos that actually adhered to the endometrium of mammals after fertilization (union of the male and female gamete), whether or not generated by assisted reproduction technology.

(2) The objects of the present invention are achieved by supplying to the mother's uterus of a mammal, either by uterine or vaginal route, an effective amount of an active form of a beta-galactoside-binding lectin or derivative thereof in a conventional buffered solution, alone or mixed with sperm, oocyte or embryo in a maintenance medium.

(3) The buffered solution aims at maintaining, handling and transferring the semen, oocyte or embryo, and it is preferably a carrier composed of a saline buffered solution (Phosphate Buffered SalinePSB) or a sterile, stable, endotoxin-free, isotonic physiological serum with a pH between 6.8 and 7.4.

(4) The maintenance medium is a complex and serum-free medium to maintain embryos under atmospheric air for a variable time in accordance with the temperature. A maintenance medium is usually composed of an isotonic buffered solution, which contains essential amino acids, growth factors, enzymes, energy substrates, cell nutrients and antibiotics.

(5) An example of a maintenance medium is a dilution agent, i.e., a diluting liquid added to or mixed with semen to preserve the fertilization ability of the latter. Special dilution agents for freezing also have cryogenic properties, thus enabling transportation, freezing and thawing. Dilution agents consist of isotonic buffer, energetic substrates and cell nutrients, antibiotics, anti-oxidants and cryoprotectants.

(6) The objects of the present invention may also be achieved by supplying, in a simultaneous or subsequent manner, a beta-galactoside-binding lectin or derivative thereof, together with semen, oocyte or embryo to the uterus of a mammal (these steps will be further defined).

(7) Some of the advantages provided by the present invention are as follows: increased reproductive efficiency of livestock; qualitative and quantitative improvements on progeny; reduced length of time between birth and slaughter; reduced feeding costs; increased possibility of heterozygosis; improvements in handling and reduced technical, health and reproductive expenses; among other aspects later described herein.

(8) The present invention comprises the use of a beta-galactoside-binding lectin or derived thereof, preferably Galectin-1, Galectin-3, Galectin-9, Galectin-13 and Galectin-15, for increasing the embryo implantation rate in the uterus of mammals.

(9) The increase of the embryo implantation rate in the uterus of mammals by means of reproductive biotechnologies directly reflects the feasibility of using such procedures as a tool for genetic progress, considering that it improves the results and/or success of techniques to economically viable levels.

(10) Additionally, it is better to use genetic material from genetically superior individuals on reproductive biotechniques, since it maximizes the spread of these animals and provides an alternative to the shortage of clearly superior individuals. Reproductive biotechnologies also allow reduced generation intervals.

(11) In addition, one of today's global challenges is to conquer hunger in a World where cultivatable land areas are shrinking, and with an estimate 50% population growth over the next 25 years. Thus, an increase on embryo implantation rates in the mother's uterus of bovine, porcine, ovine, and other species means increasing food production as a whole, which includes meat production, the demand for which is estimated to double over the next 25 years, reaching more than 127 million tons per year.

(12) The impact of increased embryo implantation rates in the uterus of mammals in productive herds is as follows: Qualitative and quantitative improvements on progenyin beef cattle, both in weight and qualityincreased daily weight gain, improved quality of marbling, tenderness, flesh flavor, and also improved production of prime cuts, which adds more value to the end product. In dairy herd, it provides increased productivity and improved milk quality. It also provides a reduced length of time until slaughter, i.e., the increased daily weight gain deriving from genetic improvement reduces the length of time from birth to slaughter by up to 30%. Therefore, the early growth of the animals is significantly increased. Reduction in feeding costs: in spite of the increasing investments needed to achieve a superior genetic quality, there is a more than satisfactory consideration with the reduction on animal production costs, mainly because of the increase in precocity, which reduces the length of time until slaughter. In view of the above, production costs are reduced in at least 10%. Increased likelihood of heterozygosis: it becomes more viable to cross-breed cattle breeds that are not adapted to the tropical climate of Brazil, but which provide improvements in carcass, precocity and prolificacy of the herd. Improvements in handling and reduced technical, health and reproductive expenses: the use of reproductive biotechnologies provides better health conditions, since it prevents the spread of sexually transmitted diseases to cattle, reducing expenses on drugs to treat diseases, and thus reducing production costs.

(13) More particularly, the present invention is a result of tests on the transfer of in vivo produced embryos in female cows in order to prove an increase on embryo implantation rates in the uterus of mammals in the presence of Galectin-1.

EXAMPLES

(14) A single 100 g dose in-22 L of a sterile, active, alkylated, and endotoxin-free recombinant human Galectin-1 was administered to the embryo recipients through uterine route. It should be noted that the above dose is dramatically low when compared to the bovine body weight, and favors the biosecurity aspects of the treated females, which, unlike mice (usually used as experimental animals), have the body prepared for a single pregnancy at a time.

(15) In order to assess the effects of Galectin-1 as a regulator agent for fertility in mammals, we have used non-isogenic, confined animals, with normal handling conditions.

(16) Donor bovine females underwent superovulation and were inseminated on D0 (day zero) of fertilization. In parallel, the recipients underwent estrus synchronization protocols in order to ovulate on D0.

(17) On D7 (7th day), the donors entered the phase of collection and evaluation of embryos in morula and/or blastocyst stage. Poor (grade 3), good (grade 2) and excellent (grade 1) embryos were bottled in order to be transferred, on the same day, to the recipients, which were previously selected according to the development of the corpus luteum.

(18) In general, embryo classification is performed in view of morphological parameters, and encompasses three main stages identified according to their stage of development. At the best, the embryo should not have visible fragments, and the cells should have homogeneous size. It should be also noted that not all embryos with excellent morphology are internally healthy, because they are subject to conditions related with the ovule, the spermatozoon and the fertilization process itself.

(19) Embryos on a column of TQC maintenance medium and Galectin-1 diluted in sterile physiological serum on another column (the embryo does not come into contact with the Galectin solution at the time of bottling) were bottled in the same straw and transferred to the uterus of the recipients, synchronized with the donors, with the aid of an insemination pipette. All of the straw content is transferred at once and, in this embodiment, the embryos and Galectin-1 reach the uterus of the recipient in a separate and simultaneous manner.

(20) The purpose of the tests was to analyze the number of confirmed embryo implantations in the uterus of the mammal on 30 and 60 days after the date of embryo transfer in (i) recipients which received local treatment with Galectin-1 (ii) recipients of a transferred embryo without the presence of Galectin-1. To this end, recipients of a transferred embryo were divided into two distinct groups for each mating (donorsemen), as follows: The Control Group (embryos without the presence of Galectin-1), and the Treated Group (embryos with the presence of Galectin-1).

(21) Pregnancy diagnosis was performed both in the Treated Group and in the Control Group by ultrasound examination at two distinct moments: the first measurement was performed 30 days after embryo transfer (represented in the table below as P30), and the second evaluation of the embryo implantation rate in the uterus of a mammal was performed 60 days after embryo transfer (represented in the table below as P60).

(22) Methodology:

(23) The recipients were bovine females with good milk production and known reproductive ratings. The recipients were Holstein females with high milk production and known reproductive ratings.

(24) The procedure for selection of mating refers to the choice of the breeding animal (semen doses) for each donor. After the collection and evaluation of embryos, about half of the generated embryos were transferred to the uterus of recipients belonging to the CONTROL GROUP and the other half of the embryos was transferred to the uterus of recipients belonging to the TREATED GROUP. There was an effort to equally share the embryos according to the degree of quality shown between the CONTROL GROUP and the TREATED GROUP, in order to render the groups comparable.

(25) Methodology: CONTROL GROUP

(26) The embryo transfer procedure in the Control Group was performed in accordance with the traditional approach to such events, i.e., the embryo transfer occurred in the uterine horn ipsilateral to the corpus luteum (where ovulation occurred at the time of estrus) of the recipients. The straw used, consisting of three main columns, packed one column of TQC maintenance medium, followed by a column of air, followed by a column of TQC maintenance medium containing the embryo, followed by another column of air, followed by another column of TQC maintenance medium. The entire content of the straw was transferred at once.

(27) Methodology: TREATED GROUP

(28) The embryo transfer procedure of the Treated Group was performed in accordance with the traditional approach, i.e., the embryo transfer occurred in the uterine horn ipsilateral to the corpus luteum (where ovulation occurred at the time of estrus) of the recipients. The straw used, consisting of three main columns, packed one column of TQC maintenance medium, followed by a column of air, followed by a column of TQC maintenance medium containing the embryo, followed by another column of air, followed by another column of maintenance medium containing Galectin-1. All the straw content was transferred at once and, in this embodiment, the embryos and Galectin-1 reached the uterus of the recipient in a separate and simultaneous manner.

(29) Results:

(30) In the two illustrated tables below, the first column to the left (Repetition) indicates the repetitions of the transfers of embryos generated by each female bovine, corresponding to a total of 12 repetitions. Meanwhile, two other columns are subdivided to separate the results obtained in view of the Control Group and the Treated Group, followed by two other columns showing the relationship between the rates of the additional embryo implantation rate in the uterus of the recipients obtained with the use of Galectin-1 in the procedure.

(31) The column corresponding to the Quantity Control indicates the number of embryos generated from embryo transfers without the presence of Galectin-1. The subsequent column to the right (Control P30) is subdivided to show both the number of implantations 30 days after embryo transfer (Quantity 30) with regard to the respective percentage corresponding to the implantation rate during the same period (T30). The same reasoning is used for the subsequent column to the right (Control P60), subdivided in order to demonstrate both the number of implantations 60 days after embryo transfer (Quantity 60) and the respective percentage corresponding to the implantation rate during the same period (T60).

(32) The column corresponding to the Treated Quantity indicates the number of embryos generated from embryo transfers in the presence of Galectin-1. The subsequent column to the right (Treated P30) is subdivided to show both the number of implantations 30 days after embryo transfer (Quantity 30) with regard to the respective percentage corresponding to the implantation rate during the same period (T30). The same reasoning is used for the subsequent column to the right (Treated P60), subdivided in order to demonstrate both the number of implantations 60 days after the embryo transfer (Quantity 60) and the respective percentage corresponding to the implantation rate during the same period (T60).

(33) The column corresponding to implantation rate is subdivided to indicate the difference in the implantation rate obtained with the Control Group and the Treated Group P30 (30), as well as with the Control Group and the Treated Group P60 (60).

(34) Tables:

(35) TABLE-US-00001 TABLE 1 Control P30 Control P60 Treated P30 Control P60 Control Quantity Quantity Treated Quantity Quantity implantation rate Repetition Quantity 30 T30 60 T60 Quantity 30 T30 60 T60 30 60 #01 16 6 37.5% 4 25.0% 15 5 33.3% 5 33.3% 4.2% 8.3% #02 11 5 45.5% 4 36.4% 15 8 53.3% 8 53.3% 7.9% 17.0% #03 6 4 66.7% 4 66.7% 4 3 75.0% 2 50.0% 8.3% 16.7% #04 19 7 36.8% 6 31.6% 18 13 72.2% 9 50.0% 35.4% 18.4% #05 6 3 50.0% 3 50.0% 7 7 100.0% 7 100.0% 50.0% 50.0% #06 10 5 50.0% 5 50.0% 11 8 72.7% 4 36.4% 22.7% 13.6% #07 9 7 77.8% 5 55.6% 17 10 58.8% 9 52.9% 19.0% 2.6% #08 1 1 100.0% 2 2 100.0% 2 100.0% 100.0% #09 8 5 62.5% 5 62.5% 6 5 83.3% 4 66.7% 20.8% 4.2% #10 6 2 33.3% 1 16.7% 10 4 40.0% 4 40.0% 6.7% 23.3% #11 5 5 2 40.0% 1 20.0% 40.0% 20.0% #12 6 6 3 50.0% 3 50.0% 50.0% 50.0% TOTAL 103 45 43.7% 37 35.9% 116 70 60.3% 58 50.0% 16.7% 14.1% Results of the implantation rate of grade 1, 2 and 3 embryos from the Control Group (embryo transfer without the presence of Galectin-1) and the Treated Group (embryo transfer in the presence of Galectin-1) after 30 and 60 days, respectively.

(36) As seen in Table 1, the analysis of the total result shows that 30 days after the embryo transfer, the Treated Group showed higher rates of implantation in the mother's uterus, with 60.3% of implantation over 43.7% of the Control Group. The analysis of the final outcome 60 days after embryo transfer showed that the Treated Group rates had 50% implantation in the mother's uterus, while the Control Group had a 35.9%.

(37) In view of the results of Table 1, it is possible to conclude that the use of Galectin-1 increased the number of embryo implantations in the mother's uterus by 16.7 percentage points after 30 days and 14.1 percentage points after 60 days.

(38) TABLE-US-00002 TABLE 2 Control P30 Control P60 Treated P30 Control P60 Control Quantity Quantity Treated Quantity Quantity Pregnancy Rate Repetition Quantity 30 T30 60 T60 Quantity 30 T30 60 T60 30 60 #01 11 4 36.4% 3 27.3% 14 5 35.7% 5 35.7% 0.6% 8.4% #02 9 4 44.4% 3 33.3% 12 6 50.0% 6 50.0% 5.6% 16.7% #03 5 3 60.0% 3 60.0% 4 3 75.0% 2 50.0% 15.0% 10.0% #04 12 4 33.3% 4 33.3% 11 8 72.7% 6 54.5% 39.4% 21.2% #05 4 2 50.0% 2 50.0% 4 4 100.0% 4 100.0% 50.0% 50.0% #06 3 1 33.3% 1 33.3% 5 3 60.0% 2 40.0% 26.7% 6.7% #07 5 3 60.0% 2 40.0% 10 6 60.0% 5 50.0% 10.0% #08 1 1 100.0% 1 1 100.0% 1 100.0% 100.0% #09 3 3 100.0% 3 100.0% 1 1 100.0% 1 100.0% #10 4 1 25.0% 7 2 28.6% 2 28.6% 3.6% 28.6% #11 4 3 2 66.7% 1 33.3% 66.7% 33.3% #12 2 2 1 50.0% 1 50.0% 50.0% 50.0% TOTAL 63 26 41.3% 21 33.3% 74 42 56.8% 36 48.6% 15.5% 15.3% Results of the implantation rate considering only grade 2 embryos from the Control Group (embryo transfer without the presence of Galectin-1) and the Treated Group (embryo transfer in the presence of Galectin-1) after 30 and 60 days, respectively.

(39) The results listed in Table 2 showed that the Treated Group had a higher number of implantations in the mother's uterus with a 56.8% implantation rate over 41.3% of implantation achieved by the Control Group 30 days after embryo transfer of grade 2 embryos. The analysis of the final outcome 60 days after the transfer of grade 2 embryos showed that the Treated Group rates had a 48.6% implantation in the mother's uterus, while the Control Group had a 33.9% rate.

(40) In view of the results shown in Table 2, it is possible to conclude that Galectin-1 supplied to the uterus of a bovine recipient of the Treated Group increased the number of implantations by 15.5 percentage points after 30 days and by 15.3 percentage points after 60 days, enhancing the implantation rates of grade 2 embryos in the uterus of cows.

(41) Considering that, in this experiment, recombinant human Galectin-1 acts as an antigen in the mother's uterus of bovines, mainly because it comes from different species, studies are being made in order to prove (i) a greater interaction between Galectins and glycans from the same species, and, consequently, (ii) a better performance of Galectin-1 in the increase of the embryo implantation rate in the mother's uterus of mammals.

(42) The physiological mechanisms triggered by Galectin-1 that make it possible to increase the embryo implantation rate in the uterus of mammals are still being studied, since the pregnancies of the bovine recipients to which embryos had been transferred did not come into term yet.

(43) In bovines, the placenta has numerous units with different sizes and shapes which, in turn, consist in a swell of interdigitated fetal villosities with cryptform invaginations reunited in the uterus. For the maintenance of pregnancy, the embryo must participate by sending signs of its existence in the uterine environment through the production of IFNT. Thus, luteolysis remains inhibited and, consequently, P4 levels are kept high. Therefore, both IFNT and P4 stimulate the mRNA to increase the level of Galectins in the uterus. After the maternal environment recognizes and accepts the embryo, the trophoblast cells differentiate and unite to the cells of the uterine epithelium, thus coming into direct contact with maternal tissues.

(44) It is believed, therefore, that the increased embryo implantation rate in the mother's uterus of mammals is explained by the participation of Galectin-1 in the regulation of the mechanisms related to immune tolerance and/or in promoting the process of blastocyst elongation and embryo adhesion in the endometrium.

(45) The present invention further discloses that a beta-galactoside-binding lectin or derived thereof, preferably selected from Galectin-1, Galectin-3, Galectin-9, Galectin-13 or Galectin-15, or a derivative thereof, can be used to act as agent to regulate the fertility of semen, oocytes or embryos, thereby increasing the embryo implantation rate in the uterus of mammals.

(46) The amount of beta-galactoside-binding lectin or a derivative thereof supplied to the mother's uterus of a mammal may vary in accordance with the body weight of the species, and its concentration rate should preferably range from 0.0000001 to 1.0 mg per kilogram of body weight of the mammal.

(47) In one embodiment, the beta-galactoside-binding lectin or derived thereof is diluted in a buffered solution, preferably phosphate buffered saline (PBS) or physiological serum, and it is supplied to the uterus by means of a conventional straw mixed with semen, oocyte or embryo kept in a maintenance medium (capable of maintaining the survival of cells and the biological integrity during handling, transporting, freezing and thawing), so that the entire content of the straw is supplied to the uterus of the mammal at once.

(48) In a preferred variation, the beta-galactoside-binding lectin or derived thereof is packed in two steps, in a separate manner and subsequent to semen, oocyte or embryo, and, in this procedure, two filling straws are used. In the first application, the semen, oocyte or embryo is put into a maintenance medium and packed in a conventional straw, so that all content is supplied to the uterus of the mammal. In the second application, the beta-galactoside-binding lectin or derivative thereof diluted in a buffered solution is packed in a different straw and, subsequently, it is supplied to the uterus of the mammal. The lapse of time between the supply of beta-galactoside-binding lectin or a derivative thereof and the supply of sperm, oocyte or embryo will depend, essentially, on the mammalian species handled and the method of treatment employed and may extend up to 17 days in accordance with the treatment being employed.

(49) It is also disclosed that the beta-galactoside-binding lectin or a derivative thereof diluted in a buffered solution is packed on a column of a given conventional straw, interspersed between other columns containing sperm, oocyte or embryo in a maintenance medium, so that all the straw content is subsequently supplied to the uterus of the mammal.

(50) In all variations provided, the beta-galactoside-binding lectin or derivative thereof, together with semen, oocyte or embryo, is administered through uterine or vaginal route. The semen can be in fresh, chilled or frozen; the oocyte can be fresh, frozen or vitrified; and the embryo can be fresh, frozen or vitrified, and may also be from embryo transfer, in vitro fertilization, as well as a clone or a transgenic embryo.

(51) Moreover, it is known that in vitro embryo production systems allow the determination of the sex of the embryo after fertilization, and thus said systems are an important tool dairy cattle and beef cattle breeders. Nevertheless, in vitro produced embryos do not exhibit satisfactory survival rates in the uterus of mammals. Hence, the present invention provides a solution to regulate semen, oocyte or embryo fertility, thus increasing the embryo implantation rate in the uterus of mammals.

(52) On the other hand, one of the limitations for the commercial application of reproduction biotechnologies is the distance to be traveled between the location for collection of semen, oocyte or donor's embryo and the location where the recipient species is; as well as the distance separating the donor species from the recipient species. In this sense, cryopreservation (freezing process) and vitrification (ultra-fast freezing method) have become a common practice in animal production. However, although cryopreservation and vitrification techniques have commercial advantages, the spermatozoons may suffer changes in the membrane, early capability, DNA changes and oxidative stress during freezing, which compromises their fertility.

(53) In a preferred embodiment, the present invention discloses the use of a product comprising an effective amount of a beta-galactoside-binding lectin or derivatives thereof to enhance the embryo implantation rate in the uterus of mammals.

(54) As several examples of preferred embodiments had been disclosed, it should be understood that the scope of the present invention encompasses other possible embodiments and it is only limited by the content of the appended claims, including therein the possible equivalents.