In vitro test method for early detection of endometriosis and/or uterine adenomyosis

Abstract

An in vitro test method for early detection of endometriosis and/or uterine adenomyosis in a female patient, comprising the following steps: a) providing menstrual blood of the patient to be tested, b) determining expression of the genes ESR2 and/or CXCL12 and/or CXCR4 in comparison with at least one control sample, wherein an increased expression of one or more of the genes indicates endometriosis and/or uterine adenomyosis.

Claims

1. In vitro test method for early detection of endometriosis and/or uterine adenomyosis in a female patient, comprising the following steps: a) providing menstrual blood of the patient to be tested, b) determining expression of the genes ESR2 and/or CXCL12 and/or CXCR4 in comparison with at least one control sample, wherein an increased expression of one or more of the genes indicates endometriosis and/or uterine adenomyosis.

2. The method according to claim 1, wherein the provided menstrual blood comes from a vaginal examination of the patient that was carried out on any of cycle days 1 to 7 or from a tampon used by the patient.

3. The method according to claim 2, wherein the menstrual blood was frozen at −25° C. or below and/or admixed with a stabilization reagent immediately after the examination or after the removal of the tampon from the body.

4. The method according to claim 1, wherein the patient is acutely suffering from dysmenorrhoea and/or has a previous history of dysmenorrhoea.

5. The method according to claim 4, wherein the dysmenorrhoea can be assigned to ICD 94-4 or ICD 94-5.

6. The method according to claim 4, wherein the dysmenorrhoea can be assigned to class II° or III° according to the classification system described in the present application.

7. The method according to claim 1, wherein the patient is of 18 to 35 years of age and/or is a nulligravida.

8. The method according to claim 1, wherein the patient has not undergone hormone therapy within 3 months before menstrual blood collection.

9. The method according to claim 1, wherein, for the sample to be tested and for the control sample(s), the expression of the genes ESR2 and/or CXCL12 and/or CXCR4 is determined in comparison with a constitutively expressed housekeeping gene, so that it is possible to normalize expression.

10. The method according to claim 1, wherein a simultaneous increase in the expression of the genes ESR2 and CXCL12, or a simultaneous increase in the expression of the genes ESR2 and CXCR4, or a simultaneous increase in the expression of the genes CXCL12 and CXCR4, or a simultaneous increase in the expression of the genes ESR2, CXCR4 and CXCL12 indicates endometriosis and/or uterine adenomyosis.

11. The method according to claim 1, wherein an increase in expression of relevance to making a diagnosis can be assumed when the expression is increased by at least 1.5-fold, compared to the at least one control sample, wherein, in the case of use of more than one control sample, the average values of the expression in the control samples are used for the purposes of comparison.

12. The method according to claim 1, wherein the at least one control sample comes from the samples of menstrual blood from one or more women who have no history of dysmenorrhoea and for whom there is no laparoscopically detectable endometriosis or uterine adenomyosis, wherein the women are nulligravidae and are of 18-35 years of age upon sample collection.

13. The method according to claim 1, wherein expression is determined on the basis of mRNA determination or on the basis of protein determination.

14. The method according to claim 13, wherein the mRNA determination is carried out by means of Northern blotting, by means of in situ hybridization, by means of an RNAse protection assay, by means of an RNA microarray or by means of PCR.

15. The method according to claim 14, wherein the mRNA is first transcribed into cDNA and subsequently quantitative real-time-PCR is carried out by means of Scorpion primers, Lux primers, lanthanide-labelled probes or FRET probes.

16. The method according to claim 13, wherein the protein determination is carried out by means of Western blotting, by means of a protein microarray or by means of an immunoassay.

17. In vitro test kit for use in the early detection of endometriosis and/or uterine adenomyosis in a female patient on the basis of quantitative real-time PCR, comprising: one or more oligonucleotide primer pairs homologous to transcribed regions of the genes ESR2 and/or CXCL12 and/or CXCR4; optionally an RNA stabilization reagent; optionally a set of reagents for extraction of mRNA from a blood sample; optionally a set of reagents for transcription of mRNA into cDNA; optionally at least one oligonucleotide primer pair homologous to the transcribed region of a housekeeping gene, especially c-Abl; and at least one FRET probe homologous to the target transcript(s).

18. In vitro test kit for use in the early detection of endometriosis and/or uterine adenomyosis in a female patient on the basis of an immunoassay, comprising: one or more antibodies which bind to the gene products of the genes ESR2 and/or CXCL12 and/or CXCR4; optionally a protein stabilization reagent; optionally a set of reagents for extraction of protein from a blood sample; and optionally at least one antibody which binds to the gene product of a housekeeping gene, especially c-Abl.

19. Oligonucleotide primer pairs homologous to the transcripts of the genes ESR2 and/or CXCL12 and/or CXCR4 for use in the early detection of endometriosis and/or uterine adenomyosis in patients.

20. Antibodies which bind to the gene products of ESR2 and/or CXCL12 and/or CXCR4 for use in the early detection of endometriosis and/or uterine adenomyosis in patients.

21. Primer pairs according to claim 19, wherein the patients have dysmenorrhoea.

22. Primer pairs according to claim 19, wherein the patients are nulligravidae and are of 18-35 years of age upon sample collection.

23. Antibodies according to claim 20, wherein the patients have dysmenorrhoea.

24. Antibodies according to claim 20, wherein the patients are nulligravidae and are of 18-35 years of age upon sample collection.

Description

FIGURES

[0054] FIG. 1 Primers and probe for the target gene CXCL12

[0055] FIG. 2 Sequences of the primers and probe for the target gene ESR2

[0056] FIG. 3 Bar chart showing the expression of the target genes CXCL12 and ESR2

EXEMPLARY EMBODIMENT 1—ANALYSIS OF SAMPLES BY MEANS OF QUANTITATIVE REAL-TIME PCR

[0057] Samples of menstrual blood were collected from three young nulligravidae (average age: 31 years) suffering from severe analgesic-dependent dysmenorrhoea during a vaginal examination on cycle day 2 (CD 2) in the practice of the principal investigator. All three women were assigned to group III° according to the above-described system for classification of dysmenorrhoea. For two of the three women, there was laparoscopic detection of fresh endometriosis. For the third woman, there were laparoscopic signs of an already long-standing, relatively old endometriosis and possibly adenomyosis.

[0058] The controls used were samples of menstrual blood from three young nulligravidae (average age: 31 years) who had no history of dysmenorrhoea and were gynaecologically healthy.

[0059] All patients including the control group were not allowed to have taken hormone therapy within 3 months before menstrual blood analysis or to have taken analgesics directly before menstrual blood collection (24 hours).

[0060] Menstrual blood containing menstrually desquamated basal endometrium was expelled from the uterus at about 1-2 uterine contractions per minute. In the analysis presented here, the menstrual blood was obtained by means of a gynaecological speculum examination (alternatively, it would also be possible without any problems for the blood to be received via a syringe with sterile irrigation cannula attached or via a groove-containing vaginal speculum). One to two millilitres of the menstrual blood collected was promptly transferred to a sterile commercially available Nunc tube. The samples were stored at −30° C. in the laboratory at the practice immediately after the examination.

[0061] The processing of the menstrual blood samples and the expression analyses were carried out as follows:

[0062] Total RNA was extracted from the menstrual blood samples by means of the Direct-zol Miniprep Kit (Zymo Research, Irvine, Calif., United States) according to the manufacturer's protocol.

[0063] Complementary DNA (cDNA) was then synthesized using the Super-Script™ Reverse Transcriptase (Invitrogen™, Carlsbad, Calif., United States) in combination with random hexamer primers according to details from the manufacturer.

[0064] Quantitative PCR (qPCR) was carried out with a 7500 Real Time PCR System (Applied Biosystems™, California, United States) using a TaqMan probe. The target genes were CXCL12 (gene ID: 6387, chromosome 10811.21) and ESR2 (gene ID: 2100, chromosome 14q23.2-q23.3). The quencher used was BBQ, and the reporter fluorescent dye used was 6FAM.

[0065] The sequences of the primers and probe for the target gene CXCL12 and the positions of the oligonucleotides on the gene can be seen in FIG. 1. Four primer combinations were used. Here, the mix CXCL-2,1 corresponds to the primer combination CXCL12F/CXCL12As, the mix CXCL-2,2 corresponds to the primer combination CXCL12F/CXCL12Lo, the mix CXCL-2,3 corresponds to the primer combination CXCL12Se/CXCL12As and the mix CXCL12-2,4 corresponds to the primer combination CXCL12Se/CXCL12Lo.

[0066] The sequences of the primers and probe for the target gene ESR2 and the positions of the oligonucleotides on the gene can be seen in FIG. 2. In the case of ESR2, two primer combinations were used. Here, the mix ESR-2,1 corresponds to the primer combination ESR2_Ex2 S/ESR2_Ex2/3 A, and the mix ESR-2,2 corresponds to the primer combination ESR2_Ex2 F/ESR2_Ex3 R.

[0067] All reactions were carried out in duplicate with a final volume of 30 microlitres using 5× Hot Start Taq Probe qPCR Mix (Axon Labortechnik, Kaiserslautern, Germany).

[0068] The following cycles were carried out:

TABLE-US-00001 Cycle No. Temperature Duration 1st 50.0°  2:00 min 2nd 95.0° 10:00 min 3rd-45th 95.0°  0:05 min 60.0°  0:32 min 72.0°  0:32 min

[0069] Relative quantification (RQ) of mRNA expression was calculated using the 2(−Delta Delta CT) method (Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) Method. Methods 2001; 25: 402-408) using a pool of normal samples as calibrator. The reference gene ABL1 (gene ID: 25, chromosome 9q34.12) was used for the normalization of the quantity of mRNA.

Result:

[0070] The relative expression of the genes CXCL 12 and ESR2 for the test group (T) and for the control group (C) is presented in the following table and in FIG. 1.

TABLE-US-00002 Control Test group (T) group (C) Factor (T)/(C) Mean of ratio for total 0.092057464 0.03465358 2.65650677 ESR Mean of ratio for ESR-2,1 0.044303902 0.03523657 1.2573273 Mean of ratio for ESR-2,2 0.163687806 0.03407058 4.80437323 Mean of ratio for total 1.77088021 0.30959724 5.71994823 CXCL Mean of ratio for CXCL- 1.128916467 0.25311336 4.46012206 2,1 Mean of ratio for CXCL- 1.022933426 0.15446975 6.62222489 2,2 Mean of ratio for CXCL- 3.309422411 0.52423829 6.31282081 2,3 Mean of ratio for CXCL- 1.622248534 0.30656758 5.291650716 2,4

[0071] All three patients suffering from severe dysmenorrhoea exhibited distinct overexpression of the CXCL12-ESR2 complex compared to the three control patients. This can be established by the significantly increased rise in the expression pattern in comparison with the housekeeping gene (Ct abl).

[0072] The distinct overexpression of essential constituents (CXCL12 and ESR2) of the morphogenetic complex indicates that an invasion and activation of mesenchymal stem cells occurs in the context of uterine wound healing after auto-traumatization. Detection of the overexpression of the gene products of the stated genes from the menstrual blood makes it possible to diagnose the disease endometriosis or adenomyosis at a very early stage.

[0073] Accordingly, using the in vitro method presented here, it is possible to develop a screening test for women, especially for young women suffering from severe dysmenorrhoea. In the event of a positive test result, it would be possible to take adequate preventive measures to prevent progression of uterine destruction in endometriosis or adenomyosis. It is also proposed that the screening test be used for quantification of the extent of endometriosis or adenomyosis, even in the case of endometriosis or adenomyosis that has already been diagnosed. Such quantification makes it possible to optimize the options for therapy, especially with a view to treating infertility. Specifically, it is known that both the spontaneous pregnancy rate and the pregnancy rate following assisted reproductive technology (ART) are associated with the severity of uterine adenomyosis in an inversely proportionally manner.

EXEMPLARY EMBODIMENT 2

[0074] The analysis of the expression of the genes ESR2 and CXCL12 that is presented in detail in exemplary embodiment 1 can be carried out in an analogous manner for the gene pair ESR2 and CXCR4 and the gene pair CXCL12 and CXCR4. It is also possible to use the expression of all three genes ESR2, CXCL12 and CXCR4 for the test method.

[0075] For the practical implementation of the in vitro test method for early detection of endometriosis and/or uterine adenomyosis in a female patient, it is not only the method described in exemplary embodiment 1 that is of particular significance, but also the analysis of the expression of the genes CXCL12 and CXCR4: patients suffering from severe dysmenorrhoea exhibited distinct overexpression of the CXCL12-CXCR4 complex compared to control patients.