Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/48—Reproductive organs
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P15/00—Drugs for genital or sexual disorders; Contraceptives
  • A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/02—Coculture with; Conditioned medium produced by embryonic cells

Definitions

• the present invention relates to a composition which promotes pregnancy in human fertility treatment by blastocyst transfer, a method for production of the composition, as well as a method for promoting pregnancy in a human who undergoes blastocyst transfer.
• in vitro fertilization is a technique by which a patient is administered a fertility drug to induce generation of ova, which then are collected out of the body and mixed with sperm cells in a test tube (insemination) to have them got fertilized, and the fertilized eggs then are cultured and, on day 2 or 3 of culture in general, embryos at 4- or 8 cell stage are transferred into the uterine cavity in general, with a catheter.
• administration of luteinizing hormone is usually conducted in order to condition the uterine endometrium.
• Interleukin-1 for example, is a primary factor which modulates cross talks between the embryo and the uterine endometrium of the maternal organism, and a complete IL-1 system is found in a human embryo at all the stages of its development (see Non-patent Document 1).
• HCG human chorionic gonadotropin
• HCG human chorionic gonadotropin
• Non-patent Documents 3-9 several embryonic factors that modulate endometrial receptivity are detectable in the supernatant of embryo cultures. It also is known that, in vivo, an embryo developing in the oviduct induces differentiation of the uterine endometrium (see Non-patent Document 10). All these facts, taken together, indicate that the cross talks are brisk at the early stages of embryonic development, between the embryo and the uterine endometrium via the factors produced by the embryo.
• Non-patent Document 10 it has been shown that not only a preimplantation embryo in the uterine cavity, but also even an early embryo still remaining in the oviduct has the ability to modulate certain molecules in the uterine endometrium to place its implantation under its own control (see Non-patent Document 10).
• blastocyst transfer a new technique of in vitro fertilization, has been proposed and practiced clinically as a means of improving the success rate of implantation in human fertility treatment (see Non-patent Documents 11-13).
• this technique embryos produced by in vitro fertilization as described above are cultured for 5 to 6 days to let them develop into blastocysts and then injected into the uterine cavity.
• the objective of the present invention is to provide a means to increase the success rate of achieving pregnancy.
• the present inventors assumed that the lack of cross talks between the uterine endometrium and the embryos at the stage of development from earlier embryos to blastocysts is a factor causing unsuccessful blastocyst transfer. This lack of cross talks could cause insufficient modulation of the endometrial receptivity to embryos. Thus, the present inventors pursued investigations to increase the success rate of implantation and pregnancy in human blastocyst transfer, focusing on the modulation of the endometrial receptivity by embryos.
• blastocyst transfer after injecting in advance culture supernatant of human embryos into the uterine cavity of a recipient, the present inventors found that implantation and pregnancy was achieved with higher success rate than achieved by conventional blastocyst transfer.
• the technique of blastocyst transfer there has been no report known to us as to increasing the success rate of implantation and pregnancy by injecting culture supernatant of embryos in advance into the uterus.
• the present invention was completed based on the finding and further studies thereupon.
• An agent for promoting pregnancy in blastocyst transfer comprising the supernatant of culture obtained by culturing a human embryo in a medium until the embryo develops into a blastocyst.
• the agent for promoting pregnancy according to one of 1 to 3 above comprising a portion of the culture supernatant allotted to at least 0.3 embryo.
• a method for production of an agent for promoting pregnancy in blastocyst transfer comprising the steps of:
• a method for promoting pregnancy in a human comprising the following steps of:
• the pregnancy promoting agent according the present invention When injected into the uterine cavity prior to the transfer of a blastocyst into the uterus of the human recipient of an embryo, the pregnancy promoting agent according the present invention as described above remarkably increases the success rate of implantation of a transplanted blastocyst and pregnancy, compared with conventional blastocyst transfer.
• the present inventors assume that the marked improvement in the success rate of implantation and pregnancy in blastocyst transfer according to the present invention is due to the establishment of an optimal circumstance for transferred blastocyst, by stimulating the uterine endometrium with administered supernatant containing embryo-derived factors which, having been released from the embryo, modulate the endometrium receptivity.
• the pregnancy promoting agent of the present invention is a composition comprising the supernatant of a culture obtained by culturing one or more human embryos in a medium until they develop into blastocysts (e.g., culture supernatant itself).
• the supernatant of a culture to be collected is preferably that in which at least 2-day, more preferably 3-day culture was performed before collection.
• a medium to be employed may be any of conventional media conventionally used in blastocyst transfer.
• a serum-free medium is preferred considering that the risk of contamination with prion or other infectious agents should be eliminated.
• BlastAssist System media 1 and 2 examples of such serum-free media at present include BlastAssist System media 1 and 2, in particular BlastAssist System medium 2 (MediCult, Jyllinge, Denmark) which can be used suitably in the culture to be continued for some three days until blastocysts are formed.
• the above-mentioned culture of human embryos is conducted preferably in an amount to the medium which corresponds to 10-100 ⁇ L of the medium per human embryo, and more preferably in 10-60 ⁇ L of the medium per human embryo.
• the agent of the present invention comprises the culture supernatant in an amount corresponding to a portion allotted to at least 0.3 embryo out of the total amount of the culture in which blastocysts have developed. Namely, in the case where only one embryo is cultured in 50 ⁇ L of the medium until it develops into a blastocyst, it is preferred that 15 ⁇ L of the supernatant (i.e., 50 ⁇ L ⁇ 0.3) collected from the medium is contained in the composition according to the present invention.
• a unit amount of the composition should contain a portion of the culture medium allotted to at least 0.3 embryo.
• the collected culture supernatant may be used directly, or frozen-stored and thawed before use. It is also allowed to add one or more pharmaceutically inert diluents (e.g., sterile purified water, or aqueous solution containing human plasma albumin, glucose, sodium chloride and the like, which are compounds contained in BlastAssist System medium 2 as described in the section of Examples) to increase the volume by dilution into a volume which is easier to handle, e.g., 0.2 mL or 0.5 mL.
• pharmaceutically inert diluents e.g., sterile purified water, or aqueous solution containing human plasma albumin, glucose, sodium chloride and the like, which are compounds contained in BlastAssist System medium 2 as described in the section of Examples
• the pregnancy promoting agent according to the present invention in injected into the uterine cavity of a recipient prior to the transfer of blastocysts into her uterus in a blastocyst transfer procedure.
• the recipient of the blastocysts is in usual cases the very donor of the ova from which the embryos have developed, the recipient also may be a different individual. Therefore, the recipient of the pregnancy promoting agent according to the present invention may also be either the donor of the ova from which the embryos have developed, or a different individual.
• the pregnancy promoting agent according to the present invention is injected into the uterine cavity of a patient preferably 1-5 days before, more preferably 2-4 days before the transfer of blastocysts is carried out. Injection may be once (e.g., once, 3 days before) or repeated multiple times every other day or even everyday.
• the present invention also provides a method for promoting pregnancy in a human comprising: culturing a human embryo in a medium until the human embryo develops into a blastocyst, injecting a composition comprising the supernatant of the culture (e.g., the culture supernatant itself) into the uterine cavity of a human recipient who is to undergo blastocyst transfer, and then transferring the blastocyst into the uterine cavity of the recipient.
• the injection of the composition is done preferably 1-5 days, more preferably 2-4 days, and particularly preferably 3 days before the transfer of the blastocyst.
• the pregnancy promoting agent according to the present invention When injected into the uterine cavity prior to the transfer of a blastocyst into the uterus of a recipient, the pregnancy promoting agent according to the present invention remarkably increases the success rate of implantation and pregnancy in blastocyst transfer.
• HRT hormone replacement therapy
• the criteria of patients to be included in the study were that they were at least 32 years of age, had a history of one or more unsuccessful blastocyst transfer or two-step (consecutive double) embryo transfer, and that they had at least four early embryos on day 2 in oocytes retrieval cycles.
• the patients were treated with a long protocol. Briefly, the patients started to receive 600 ⁇ g of a gonadotropin-releasing hormone (GnRH) agonist on day 7 of the hyperthermic phase in the cycle of treatment, and then, on day 3 of menstruation and thereafter, received daily stimulation of ovaries with follicle-stimulating hormone (FSH preparation or HMG preparation) until the size of the second leading follicle reached 18 mm in diameter. Ovulation was triggered when the second leading follicle became greater than 18 mm in diameter. Ova were retrieved transvaginally under utrasonographic guidance 36 hours after intramuscular injection of 500 units of human chorionic gonadotropin (hCG). The follicles were measured by ultrasonic scanning (Mitsubishi RDF173H).
• GnRH gonadotropin-releasing hormone
• FSH preparation or HMG preparation follicle-stimulating hormone
• the ova thus retrieved were fertilized by insemination or intracytoplasmic sperm injection.
• the fertilized eggs were cultured in a 50- ⁇ L droplet of BlastAssist System medium 1 [containing synthetic serum replacement (SSR), human plasma albumin, glucose, sodium pyruvate, lactate, potassium sulfate, magnesium sulfate, sodium chloride, sodium hydrogen phosphate, non-essential amino acids, L-glutamine, taurine, sodium bicarbonate, HEPS, streptomycin 50 mg/L, penicillin 50,000 IU/L, and phenol red: MediCult, Jyllinge, Denmark] and early embryos were obtained on day 2.
• SSR synthetic serum replacement
• BlastAssist System medium 2 [containing synthetic serum replacement (SSR), human plasma albumin, glucose, sodium pyruvate, lactate, potassium sulfate, magnesium sulfate, sodium chloride, sodium hydrogen phosphate, essential amino acids, non-essential amino acids, L-glutamine, taurine, sodium bicarbonate, streptomycin 50 mg/L, penicillin 50,000 IU/L, and phenol red: MediCult, Jyllinge, Denmark], i.e., in 12.5-50 ⁇ L of the medium per embryo, under a covering layer of mineral oil (Oil Embryo Culture, Irvine Scientific Santa Ana Calif.
• SSR synthetic serum replacement
• the culture plates employed here were FALCON353002 Tissue Culture Dishes (Becton Dickinson, Franklin Lakes USA.
• the culture of the embryos were carried out in an incubator (TE-HER PRODUCT O 2 .CO 2 incubator CP O2-1800, Hirasawa, Tokyo, Japan) set at 5% CO 2 , 5% O 2 , 90% N 2 37° C., and 100% humidity.
• the fresh early embryos and a part of the blastocysts obtained by the culture were directly used for transplantation in the previous treatment cycle, and the other blastocysts, which were not used for transplantation in the cycle, were frozen-stored.
• the supernatant of the embryo culture in BlastAssist System medium 2 was frozen-stored at ⁇ 20° C.
• Estraderm M an estradiol preparation
• Two sheets of Estraderm M were applied every other day while gradually increasing the number applied in such a manner as three sheets on day 10, four sheets on day 12, and 6 sheets on day 14, and thereafter three sheets from day 16 to 30, which is the day when pregnancy was tested.
• administration of a progesterone vaginal suppository (400 mg/day) as a progesterone preparation was started together with chlorpromazine acetate 12 mg/day (p.o.).
• HRT hormone replacement therapy
• BT group blastocyst transfer group
• the basic characteristics of the patient of SEET and BT groups are shown in Table 1.
• the present invention is useful as a new type of pregnancy promoting agent which remarkably improves the success rate of pregnancy and implantation in blastocyst transfer.

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• 2007
  • 2007-07-31 WO PCT/JP2007/064961 patent/WO2008016039A1/fr active Application Filing
  • 2007-07-31 EP EP07791646.8A patent/EP2052726B1/fr not_active Not-in-force
  • 2007-07-31 JP JP2008527756A patent/JP5139294B2/ja not_active Expired - Fee Related
  • 2007-07-31 US US12/376,167 patent/US20100003339A1/en not_active Abandoned
  • 2007-07-31 CN CNA2007800289310A patent/CN101500585A/zh active Pending
• 2012
  • 2012-09-25 US US13/626,271 patent/US8759288B2/en not_active Expired - Fee Related

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