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Determination of biological characteristics of embryos fertilized in vitro by assaying for bioactive lipids in culture media

a technology of biological characteristics and culture media, which is applied in the direction of biochemistry apparatus and processes, instruments, enzymology, etc., can solve the problems of multiple pregnancies, emotional and economic problems in family planning, and many couples are not able to conceive in the traditional fashion

Inactive Publication Date: 2003-07-10
PARROTT JEFF A
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0009] Specifically, the invention contemplates measuring the presence or concentration of bioactive lipids, comparing these concentrations to a normal, pre-existing, or established values for the specific embryo or media, and correlating these differences to the underlying biochemical status of the embryo, including the probability that the embryo will accede through viability to normal development, or will result in a multiple-birth pregnancy. Also, The ability to analyze the underlying biochemical status of the embryo provides the ability to analyze the chemical composition of the media and the ability to alter the chemical composition of the media through supplementation with chemical or biochemical agents to affect the viability of the embryo or the probability of a multiple-birth pregnancy. By analyzing one or more lipids, a multi-component lipid profile can be constructed for the culture media such that the lipid profile of the culture media can be correlated to viability, or, where appropriate, the composition of the culture media may be changed according to the concentration of one lipid or the multi-lipid profile. Moreover, an individual embryo, or group of embryos, can be monitored over time to determine how the lipid profile and the culture media is altered through natural development or through chemical or biochemical intervention to titrate the composition of the culture media to reach a desired level indicative of viability or the avoidance of multiple pregnancies. Therefore, the invention enables both the selection of healthy embryos and the deselection of unhealthy embryos to increase the probability of success of in vitro fertilization procedures. The ability to transfer a single healthy embryo with an increased probability of success, instead of several embryos with unknown probabilities of success, will result in fewer undesired multiple birth events.
[0012] As discussed above, the ability to identify an embryo that is likely to implant properly in the uterine cavity and to develop normally to term is of great importance to the in vitro fertilization process. By eliminating the necessity to transfer multiple embryos for implantation, the overall success rate of the in vitro fertilization process may be enhanced, avoiding undesired multiple pregnancies. The present invention provides a method for determining the biological characteristics of embryos, including implantability and likelihood of normal development, by analyzing sample media from IVF embryo cultures at the very early stages of embryo development in order to determine which embryos are likely to develop successfully after transfer into a patient.
[0028] To determine viability of an embryo in question, a media specimen is collected from the IVF embryo culture, and then analyzed to determine the level of one or more bioactive lipids, precursors, or metabolites present in the specimen. Preferably, the level of the analyte is compared to a control media sample which has not been incubated with an embryo. Utilizing media controls can assist in correcting for natural variations in media compositions which may occur in culture media which are not wholly defined. By comparing the types of bioactive lipids and their concentrations to the standard levels, a determination is able to be made as to the likelihood of whether the embryo will implant and develop successfully to term. In the embodiments of the present invention, the level of a single bioactive lipid may be detected, or two or more bioactive lipids may be detected.
[0034] In normal IVF culture practices, the culture media is changed approximately two or three days after fertilization. The supernatant is decanted and fresh medium is added. In a preferred embodiment, the decanted media is used advantageously for the IVF culture specimen. The culture media, however, may be collected anytime during IVF. Preferably, the media specimen is collected when the embryo is between one hour to twenty days old, more preferably between one day and ten days old, and more preferably between two and five days old. For embodiments where the change of the detected bioactive lipid is assayed, two or more samples may be collected over a time interval within any of the above periods. Preferably, samples are collected over 1-5 days, more preferably over 1-3 days, and most preferably over 1 day or over 2 days. A small volume of media specimen (10-50 .mu.l) may be carefully extracted without disrupting development of the embryo.
[0045] In a preferred embodiment, microtiter plates may be used for small volumes of samples and reagents. An ELISA reader may also be used to monitor and help automate the assay, and the reduced processing times may in turn reduce variability between results. Also, micro-scale automated assay equipment, such as the Immuno I system available from Bayer, the Access system available from Beckman Coulter, or the Dimension R.times.L HM system available from Dade Behring may be used.

Problems solved by technology

However, even with treatment, many couples are not able to conceive in the traditional fashion.
Although a single pregnancy is desired, multiple pregnancies often result.
Multiple pregnancies pose health risks for the mother during pregnancy, and can cause emotional and economic problems in family planning.
The alternative of terminating one of the pregnancies poses a difficult ethical and emotional dilemma for many couples, and successful delivery of the remaining fetus cannot be ensured.
However, hormonal signaling in the early stages of development is complex, and a multitude of pathways and signaling mechanisms are involved in embryogenesis.
PAF alone has not proven a sufficient indicator of embryo viability, and the role of small hormone molecules both in indicating and maintaining embryo health is not well understood.
There is no current adequate molecular marker, or combination of markers, which indicates proper embryonic development at the very early stages of life.

Method used

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  • Determination of biological characteristics of embryos fertilized in vitro by assaying for bioactive lipids in culture media
  • Determination of biological characteristics of embryos fertilized in vitro by assaying for bioactive lipids in culture media
  • Determination of biological characteristics of embryos fertilized in vitro by assaying for bioactive lipids in culture media

Examples

Experimental program
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Effect test

example 1

Assay of Media Specimen from In vitro Fertilization Culture to Measure Total Lysophospholipid (LPX) and Total Glycerophosphatidyl Compounds (GPX) by a Redox-Coupled Enzymatic Reaction

[0050] IVF embryo conditioned culture media specimens were obtained from freshly cultured human embryos. Embryos were cultured in a media blend, containing RPMI media (from Irvine Scientific, Santa Ana, Calif.) as a base, with supplemental factors added. A control sample of the media blend was also obtained for comparison. Embryos were cultured in 0.1 ml media under a drop of oil to prevent evaporation. Embryos were maintained at 37.degree. C. in a humidified incubator. Media samples were collected by aspiration from individually cultured human IVF embryos at 72 hours of development. Additional media was added to the embryo culture if in vitro development were to be continued. The aspirated media specimen was either processed immediately or stored at -20.degree. C. or below until processing.

[0051] Reage...

example 2

Assay of Media Specimen from In vitro Fertilization Culture to Measure Lysophosphatidic Acid (LPA) and Glycerol-3-phosphate (G3P) By a Redox-Coupled Enzymatic Reaction

[0059] IVF embryo conditioned culture media were obtained from freshly cultured human embryos as described in Example #1.

[0060] Reagents

[0061] Lysophospholipase (LYPL) was purchased from Asahi Chemical (Tokyo, Japan). Glycerol-3-phosphate oxidase (GPO) was purchased from Toyobo (Osaka, Japan). 4-aminoantipyrine (AAP) was purchased from Sigma Chemical Co., St. Louis, Mo. Glycerol-3-phosphate dehydrogenase (GDH), peroxidase (POD) and beta-Nicotinamide-adenine dinucleotide (NADH) were purchased from Roche (Boehringer Mannheim, Indianapolis, Ill.). 3,5-Dichloro-2-hydroxybenzenesulfonic acid (HDCBS) was purchased from Biosynth (Naperville, Ill.). Amplex Red reagent (10-acetyl-3,7-dihydroxyp-henoxazine) was purchased from Molecular Probes, Inc. (Eugene, Oreg.). All lipid or glycerophosphatidyl standards were purchased from A...

example 3

Liquid Chromatography--Mass Spectrometry Analysis of Bioactive Lipids in Embryo Culture Media

[0068] IVF embryo conditioned culture media are obtained from freshly cultured human as described in Example #1.

[0069] Reagents

[0070] Methanol, chloroform and hydrochloric acid, are purchased from Fisher Scientific. All lipid or glycerophosphatidyl standards are purchased from Avanti Polar Lipids, Alabaster, Ala. or Sigma Chemical Co. LC-MS-MS triple quadrapole instrument is a Quattro Ultima (MicroMass, Beverly, Mass.). C18 columns are purchased from Keystone Scientific (Bellefonte, Pa.).

[0071] Sample Preparation

[0072] IVF media samples are diluted in methanol in preparation for analysis on the LC-MS. Samples are diluted 1:10 by combining 10 .mu.l of each sample with 90 .mu.l methanol. Samples are then centrifuged to remove precipitated protein and placed into maximum recovery autosampler vials. Alternatively, samples are extracted using methanol:chloroform (2:1), dried under nitrogen gas, a...

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Abstract

The present invention provides methods for determining various biological characteristics of in vitro fertilized embryos, including overall embryo health, implantability, and increased likelihood of developing successfully to term. More specifically, the present invention concerns analyzing media specimens of in vitro fertilization cultures for levels of bioactive lipids in order to determine these characteristics.

Description

RELATED INFORMATION[0001] This application is a continuation of U.S. Ser. No. 09 / 837,308 filed on Apr. 17, 2001, now U.S. Pat. No. 6,489,135. The priority of these applications are expressly claimed, and the disclosure is hereby incorporated by reference in their entirety.[0002] The present invention enables the determination of various biological characteristics of in vitro fertilized embryos, including overall embryo health, implantability, viability, and likelihood of successful development to term. More specifically, the present invention concerns analyzing media specimens of in vitro fertilization cultures for levels of bioactive lipids in order to determine the optional characteristics for media formulation to increase embryo viability and reduce the frequency of multiple births.[0003] As many as 30% of couples attempting to conceive suffer from infertility. Due in part to the increasing age of pregnancy, infertility is on the rise. For men, infertility is usually caused by lo...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12Q1/26C12Q1/28C12Q1/32C12Q1/44
CPCC12Q1/26C12Q1/28C12Q1/32Y10S435/975G01N2405/00G01N2405/06G01N2405/08C12Q1/44
Inventor PARROTT, JEFF A.
Owner PARROTT JEFF A
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