METHOD FOR ANALYSING FOETAL NUCLEIC ACIDS

• US 20150031030A1
• Filed: 02/08/2013
• Published: 01/29/2015
• Est. Priority Date: 02/10/2012
• Status: Abandoned Application

• Current Assignee: IVF Zentren Prof. Zech - Bregenz GmbH
• Original Assignee: IVF Zentren Prof. Zech - Bregenz GmbH
• Inventors: Zech, Nicolas
• Application Number:
  US14/377,909
• Publication Number:
  US 20150031030A1
• Time in Patent Office: Days
• US Class Current: 435/6.11
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6879 for sex determination
• CPC Class Codes: C12Q 1/6881 for tissue or cell typing, e.g. human leukocyte antigen [HLA] probes
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2600/124 Animal traits, i.e. production traits, including athletic performance or the like
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers

Abstract

The invention relates to a method for analyzing fetal nucleic acids from the supernatant of the culture medium of fetal cell cultures of an in vitro fertilization, comprising the steps: a) removing from the culture vessel used for cultivating the fertilized egg cell the cell-free supernatant of the in vitro fertilization culture medium, which supernatant contains fetal nucleic acid, b) enriching the fetal nucleic acids contained in the supernatant, and c) carrying out an analysis of the nucleic acids present in the cell-free supernatant. The invention further relates to a method for analyzing fetal nucleic acids from the liquid surrounding the embryo within the zona pellucida, the blastocoel or the blastocyst cavity, comprising the steps: a) removing the fluid containing the cell-free fetal nucleic acid from the liquid surrounding the embryo within the zona pellucida, the blastocoel or the blastocyst cavity of the blastula or blastocyst, b) enriching the nucleic acid contained in the fluid surrounding the embryo within the zona pellucida, or the fetal nucleic acids contained in the blastocoel or the blastocyst cavity, and c) carrying out an analysis of the nucleic acids present in the cell-free supernatant.

Claims

1. A method for analyzing fetal nucleic acids from the supernatant of the culture medium of fetal cell cultures of in vitro fertilization comprising the steps:

a) extracting cell-free supernatant containing fetal nucleic acid from the in vitro fertilization culture medium from the culture vessel used for cultivating the fertilized egg cell, b) isolating the fetal nucleic acids contained in the supernatant and c) performing an analysis of the nucleic acids present in the cell-free supernatant.

2. The method as claimed in claim 1, wherein DNA and/or RNA is analyzed as fetal nucleic acid.

3. The method as claimed in claim 1, wherein the cell culture supernatant of mono and/or sequential culture media is analyzed.

4. The method as claimed in claim 1, wherein the cell-free supernatant containing the nucleic acid is analyzed on at least one of days 1 to 6 of the in vitro culture.

5. The method as claimed in claim 1, wherein the supernatant of the culture medium (3) of the culture vessel (1) is extracted from an area (4, 5) surrounding the fertilized egg cell (2) which has at least twice the diameter of the cultivated fertilized egg cell (2).

6. The method as claimed in claim 1, wherein at least 2 μ

l of the cell culture supernatant is analyzed.

7. The method as claimed in claim 1, wherein the egg cell membrane (zona pellucida) of the fertilized egg cell is opened mechanically, chemically or by laser 1 to 6 days prior to the extraction of the cell culture supernatant.

8. The method as claimed in claim 1, wherein the isolation of the fetal nucleic acids is performed by enrichment, in particular by a method selected from a group comprising centrifugation, filtration, binding to beads and amplification, in particular a polymer chain reaction.

9. The method as claimed in claim 1, wherein the analysis of the fetal nucleic acids comprises a method selected from a group comprising sequencing, amplification and in situ hybridization, in particular fluorescence in situ hybridization.

10. The method as claimed in claim 1, wherein diseases, in particular monogenetic diseases, blood groups, the sex, aneuploidies and HLA types of the embryo are detected.

11. The method for analyzing fetal nucleic acids from the fluid surrounding the embryo within the egg cell membrane (zona pellucida), from the blastocoel or from the blastocyst cavity comprising the steps:

a) extracting the cell-free fluid containing fetal nucleic acid from the fluid surrounding the embryo within the egg cell membrane (zona pellucida), the blastocoel or the blastocyst cavity of the blastula or blastocyst, b) enriching the fetal nucleic acids contained in that fluid surrounding the embryo within the egg cell membrane (zona pellucida), in the blastocoel or the blastocyst cavity and c) performing an analysis of the nucleic acids present in the cell-free supernatant.

12. The method as claimed in claim 11, wherein a quantity of fluid selected from a range with a lower limit of 0.004 pl, in particular 0.004 nl, and an upper limit of 0.1 nl is removed from the fluid surrounding the embryo within the egg cell membrane (zona pellucida), from the fluid contained in the blastocoel or blastocyst cavity of the blastula or blastocyst and transferred into a fluid volume, preferably consisting of or containing the culture medium, of at least 2 μ

l and then analyzed.

13. The method as claimed in claim 11, wherein the enrichment of the nucleic acid is performed by amplification, in particular a polymer chain reaction.

14. The method as claimed in claim 11, wherein the isolation of the fetal nucleic acids is performed by enrichment, in particular by a method selected from a group comprising centrifugation, filtration, binding to beads and amplification, in particular a polymerase chain reaction.

15. The method as claimed in claim 11, wherein the analysis of the fetal nucleic acids comprises a method selected from a group comprising sequencing, amplification and in situ hybridization, in particular fluorescence in situ hybridization.

16. The method as claimed in claim 11, wherein diseases, in particular monogenetic diseases, blood groups, the sex, aneuploidies and HLA types of the embryo are detected.