FOLLICULAR FLUID METHOD

Abstract

Follicular fluid is used in in vitro fertilization. The use includes introducing follicular fluid in the uterus in one or more administrations during a time period of between six days before embryo transfer and the transfer day.

Claims

1-13. (canceled)

14. A method of in vitro fertilization with fresh follicular fluid, said method comprising withdrawing fresh follicular fluid from a plurality of follicles, introducing 0.1 cm.sup.3 to 5 cm.sup.3 of fresh follicular fluid in a uterus in one or more administrations during a time period between four days and six days before embryo transfer.

15. The method of in vitro fertilization with fresh follicular fluid according to claim 14, comprising withdrawing fluid at −80° C. or in liquid nitrogen.”

16. The method of in vitro fertilization with fresh follicular fluid according to claim 14, wherein said method comprises improving the blastocyst implantation success rate.

17. The method of in vitro fertilization with fresh follicular fluid according to claim 15, wherein said use is in improving the blastocyst implantation success rate.

18. The method of using follicular fluid according to claim 14, wherein said follicular fluid is autologous follicular fluid.

19. The method of in vitro fertilization with fresh follicular fluid according to claim 14, comprising collecting said fresh follicular fluid from mature follicles.

20. The method of in vitro fertilization with fresh follicular fluid according to claim 14, further comprising introducing one or more pharmacological agents selected from the group consisting of progesterone, nitric oxide donors, aromatase inhibitors, ascorbic acid, glucocorticoids, insulin or insulin-sensitizing drugs, relaxin, HCG, thyroxine, melatonin, phytotherapeutic extracts, verbascum, and one or more pharmaceutically acceptable carriers.

21. The method of vitro fertilization with fresh follicular fluid according to claim 20, comprising administering said one or more agents by introduction into the uterus and simultaneously introducing the follicular fluid into the uterus, or are administered in one or more administrations within a time period comprised between fourteen days before introduction of the follicular fluid and up to 3 months after the introduction of the follicular fluid, by an administration route selected from, subcutaneously, intraperitoneally, intramuscularly, sublingually, orally.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a block diagram representing the innovative method described herein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0018] Said follicular fluid is aspirated when the oocytes that will be subjected to vitro fertilization are aspirated, or it is aspirated at a different time. Typically, it is aspirated along with the oocytes. The oocytes are then isolated by aspiration from the follicular fluid, after which said follicular fluid is introduced into the uterus or stored for subsequent use. To this end, said follicular fluid is stored for the introduction into the uterus at a later time. In both cases, follicular fluid is introduced into the uterus according to the definition given for the purposes of the present invention.

[0019] For the purposes of the present invention, the follicular fluid is preferably collected from the follicles in phase with the complete maturation of the oocyte, i.e. in the form in which they are found at the time of the fine needle aspiration.

[0020] Said follicular fluid is introduced into the uterus using the procedures typically used for embryonic transfer. The day of introduction of said follicular fluid into the uterus is particularly critical for the success of the treatment. In a preferred embodiment, said follicular fluid is introduced into the uterus immediately after the collection and processing thereof, where processing involves the isolation of the oocytes contained therein. Preferably, 5 days after the introduction of the follicular fluid into the uterus, embryo transfer is carried out. In another embodiment, embryo transfer is carried out after four days, or after three days, or after 1 day of introduction of follicular fluid into the uterus. In an alternative embodiment, said follicular fluid is introduced into the uterus at the same time as the embryo transfer.

[0021] The author of the present invention has surprisingly demonstrated that follicular fluid, introduced into the uterus, has an advantageous role in promoting the successful outcome of the embryo implantation. This effect is observed where said follicular fluid has all the characteristics of the freshly explanted follicular fluid. In a preferred embodiment, said follicular fluid is introduced into the uterus in a volume comprised between 0.1 and 5 cm.sup.3, more preferably between 1.5 and 3 cm.sup.3, even more preferably in a volume of about 2 cm.sup.3.

[0022] Surprisingly, the authors of the present invention have shown that follicular fluid plays a crucial role in determining the temporal window of the endometrial implantation, where the presence of fresh follicular fluid, i.e. having the characteristics of the freshly aspirated follicular fluid, makes the maturation of the endometrium reach the optimum maturation stage for embryo implantation. This effect is obtained even more advantageously when the introduction of fresh follicular fluid into the uterus precedes the introduction of the fertilized embryo into the uterus by one, two, three, four, five, six days. The maximum effect is achieved by introducing fresh follicular fluid into the uterus from 2 to 4 days before the introduction of the fertilized embryo (embryo transfer, ET), even more preferably three days before.

[0023] For the purpose of the present invention, it is essential that said follicular fluid is properly stored follicular fluid, because after a few hours of aspiration, for example if maintained at room temperature, said follicular fluid undergoes a degradation of some of its fundamental components, a degradation which causes the follicular fluid to lose its surprising capacity shown herein to bring the endometrium to maturation. In a preferred embodiment, said fresh follicular fluid is not diluted with other culture media.

[0024] Follicular fluid is claimed herein for use in the treatment of infertility, characterized in that said follicular fluid is introduced into the uterus in one or more administrations during a time period comprised between six days before embryo transfer and the transfer day itself.

[0025] In a preferred embodiment, said use is in the embryo transfer process.

[0026] In a preferred embodiment, said use is in improving the implantation success rate in a human by transfer of blastocysts, where follicular fluid is introduced into the uterine cavity of a human patient undergoing embryo transfer in one or more administrations during a time period comprised between six days before the embryo transfer and the day of the transfer itself.

[0027] In a further aspect, a pharmaceutical composition is described, comprising fresh follicular fluid for therapeutic use in the IVF-ICSI treatment and other extra-uterine fertilization procedures, in a volume comprised between 0.1 and 5 cm.sup.3, more preferably between 1.5 and 3 cm.sup.3, even more preferably about 2 cm.sup.3. Said composition is introduced into the uterus one, two, three, four, five or six days before the embryo transfer.

[0028] A method for producing a composition for use in improving the implantation success rate by transfer of blastocysts is also claimed, said method comprising the steps of collecting follicular fluid from the same subject in which said blastocyst transfer will be carried out, optionally storing it at −80° C. or in liquid nitrogen, optionally admixing it with other active components. Said follicular fluid is preferably collected from follicles of at least 16 mm, or at least 20 mm.

[0029] A further object of the present invention is the follicular fluid for use in vitro fertilization, where said use comprises introducing follicular fluid in the uterus in one or more administrations during a time period comprised between six days before embryo transfer and the transfer day itself.

[0030] In a preferred embodiment, said follicular fluid is collected from mature follicles, preferably of at least 16 mm.

[0031] Even more preferably, said follicular fluid is autologous follicular fluid.

[0032] A further aspect of the present invention is a method for collecting and processing follicular fluid, comprising: [0033] collecting, preferably transvaginally, ovarian follicles from the ovary, by employing ultrasound techniques; [0034] puncturing the ovarian follicles and collecting the oocyte-containing follicular fluid; [0035] isolating the oocytes through aspiration; [0036] introducing the follicular fluid into the uterus or storing it.

[0037] A further aspect of the present invention is the combined intrauterine administration of a composition comprising the follicular fluid, or fractions thereof, together with one or more pharmacological agents, for example selected from progesterone, nitric oxide donors, aromatase inhibitors, ascorbic acid, glucocorticoids, insulin or insulin-sensitizing drugs, relaxin, HCG, thyroxine, melatonin, phytotherapeutic extracts, verbascum. The selection of said one or more pharmacological agents is carried out on the basis of specific patient's needs, as is well known to the man skilled in the art.

[0038] Alternatively, said introduction of the follicular fluid into the uterus is accompanied by the administration of one or more pharmacological agents that favor the uterine implantation. Said one or more pharmacological agents, selected from the group comprising, for example, progesterone, nitric oxide donors, aromatase inhibitors, ascorbic acid, glucocorticoids, insulin or insulin-sensitizing drugs, relaxin, HCG, thyroxine, melatonin, phytotherapeutic extracts, verbascum, is administered by an administration route selected, for example, from subcutaneously, intraperitoneally, intramuscularly, sublingually, orally. Said one or more agents are administered in one or more administrations within a time period comprised between fourteen days before the introduction of the follicular fluid into the uterus and up to 3 months after the introduction of the follicular fluid, or up to one month later, or up to fifteen days later, or up to five days later.

[0039] A further object of the present invention is a pharmaceutical composition comprising: follicular fluid according to the definition given in the present invention, one or more pharmacological agents selected from the group comprising, for example, progesterone, nitric oxide donors, aromatase inhibitors, ascorbic acid, glucocorticoids, insulin or insulin-sensitizing drugs, relaxin, HCG, thyroxine, melatonin, phytotherapeutic extracts, verbascum, one or more pharmaceutically acceptable carriers.

[0040] Said composition finds application for use in in vitro fertilization, where said use comprises introducing said composition in the uterus in one or more administrations during a time period comprised between six days before embryo transfer and the transfer day itself.

[0041] In a further aspect, a method for IVF-ICSI and other extra-uterine fertilization procedures is described herein, wherein said method comprises administering fresh autologous follicular fluid to a woman in one or more administrations, wherein said follicular fluid is introduced into the uterus in a volume of between 1 and 5 cm.sup.3, more preferably between 1.5 and 3 cm.sup.3, in a time period comprised between 6 days before the embryo transfer and the day of the transfer itself. Said method has surprisingly been shown to improve the implantation success rate.

[0042] Said method, described herein for humans, is advantageously applied also in other mammalian species.