Assay of nitric oxide metabolites in the culture media of human preimplantation embryos
The present invention relates, e.g., to a method for predicting the embryonic progression of a preimplantation embryo, comprising measuring in the insemination medium (IM) in which an oocyte was fertilized and subsequently incubated in vitro, thereby generating a pre-implantation embryo, at 18-24 hrs post fertilization, the levels of nitric oxide metabolites (NOx) in the insemination medium (IM), wherein a level of NOx of at least about 1.3 μM indicates that the preimplantation embryo is likely to progress to the blastocyst stage by culture day (CD) 5.
1. A method for predicting embryonic progression of a preimplantation embryo, comprising the steps of:
generating a preimplantation embryo by fertilizing an oocyte and subsequently incubating the fertilized oocyte in vitro in an insemination medium, thereby generating the preimplantation embryo;
measuring levels of nitric oxide metabolites in the insemination medium (IM) in which the oocyte was fertilized and subsequently incubated in vitro at 18-30 hours post fertilization; and
determining that the preimplantation embryo will progress to a blastocyst stage by culture day 5 based on whether the levels of nitric oxide metabolites in the insemination medium are at least about 1.3 uM.
2. The method of claim 1 , wherein the levels of nitric oxide metabolites are measured about 18 hours post fertilization.
3. The method of claim 2 , wherein the preimplantation embryo has been generated by intracytoplasmic sperm injection (ICSI).
4. The method of claim 2 , wherein the preimplantation embryo has been generated by introducing a deoxyribonucleic acid sequence of interest into the oocyte, a preliminary step for generating a transgenic animal.
5. The method of claim 2 , wherein the nitric oxide metabolites are measured with a Griess reaction.
6. The method of claim 2 , wherein the preimplantation embryo which has progressed to the blastocyst stage by culture day 5, is predicted to implant successfully in a female subject and to progress to a pregnancy.
7. The method of claim 2 , wherein the preimplantation embryo is human.
8. The method of claim 2 , which is automated.
9. The method of claim 1 , wherein the preimplantation embryo has been generated by intracytoplasmic sperm injection.
10. The method of claim 1 , wherein the preimplantation embryo has been generated by introducing a deoxyribonucleic acid sequence of interest into the oocyte, a preliminary step for generating a transgenic animal.
11. The method of claim 1 , wherein the nitric oxide metabolites are measured with a Griess reaction.
12. The method of claim 1 , wherein the preimplantation embryo which has progressed to the blastocyst stage by culture day 5 is predicted to implant successfully in a female subject and to progress to a pregnancy.
13. The method of claim 1 , wherein the preimplantation embryo is human.
14. The method of claim 1 , which is automated.
15. The method of claim 1 , further comprising a step of measuring a number of blastomeres at culture day 3, wherein a blastomere number of at least 6 indicates that the embryo will progress to the blastocyst stage by culture day 5.
16. The method of claim 1 , wherein the preimplantation embryo is implanted into a uterus of a female mammal on culture day 3 when said embryo has been predicted to progress to the blastocyst stage by culture day 5.
17. A kit for practicing the method of claim 1 , comprising one or more dipsticks that can measure nitric oxide metabolites levels and instructions enabling a user to practice the method.