WO2019166634A1

WO2019166634A1 - Method of predicting abnormal pregnancy outcome

Info

Publication number: WO2019166634A1
Application number: PCT/EP2019/055156
Authority: WO
Inventors: Ariane GERMEYER; Volker DANIEL; Alexander Freis
Original assignee: Universität Heidelberg
Priority date: 2018-03-01
Filing date: 2019-03-01
Publication date: 2019-09-06

Abstract

The present invention relates to a method of predicting abnormal pregnancy outcome and a kit for carrying out the method. In particular, it relates to a method of predicting abnormal pregnancy outcome comprising measuring in a sample obtained from a patient, for example, a maternal blood or urine sample obtained early in the first trimester of gestation the amount of one or more markers and determining a ratio between said markers, wherein a change in the ratio is predictive for an abnormal pregnancy outcome.

Description

Method of predicting abnormal pregnancy outcome

The present invention relates to a method of predicting abnormal pregnancy outcome and a kit for carrying out the method. In particular, it relates to a method of predicting abnormal pregnancy outcome comprising measuring in a sample obtained from a patient, for example, a maternal blood or urine sample obtained early in the first trimester of gestation, the amount of one or more markers and determining a ratio between said markers, wherein a change in the ratio is predictive for an abnormal pregnancy outcome.

Background of the Invention

Spontaneous abortion is one of the most common complications in early pregnancy and affects 10-20% of all pregnancies. Symptoms include vaginal bleeding, and/or uterine cramping. Despite such symptoms in the first trimester, only 28% of these symptomatic patients experience a spontaneous abortion in their pregnancy. In clinical practice, ultrasound is performed to verify the embryos viability. However, as ultrasound cannot predict pregnancy outcome, different hormone assessments have been published in order to help predict pregnancy outcome, such as human chorionic gonadotropin (hCG), progesterone, kisspeptin, activin A, activin B, follistatin, CA-125, pregnancy associated plasma protein A (PAPP-A) or macrophage inhibitory cytokine- 1. However, the clinical significance for the predictive power of these markers remains moderate. Even the most frequently used marker hCG is not prognostic, as hCG serum levels can demonstrate a wide variability despite an expected normal pregnancy outcome.

In more than 80% of cases spontaneous abortions occur within the first 12 weeks of gestation and present an enormous distress for the patient and challenges the medical professionals, especially as pregnancy outcome is difficult to estimate when the patient presents with the first symptoms. Moreover, patients with a previous abortion present with a higher risk of severe complications in the following pregnancy, such as preeclampsia or preterm birth.

The causes of early pregnancy loss are various, including cytogenetic abnormalities, maternal comorbidities (eg. diabetes mellitus, lupus erythematosus), uterine malformations, smoking as well as inadequate placental development.

Activation of maternal immunological tolerance seems to be essential for early fetal development and implantation and various cytokines have been described in different stages of pregnancy. For example, lower levels of human interleukin 1 -receptor antagonist (hIL-lra) prior to embryo transfer in IVF ( In-Vitro fertilization) cycles were associated with lower pregnancy rates. Higher levels of human thrombopoietin (hTPO) and lower concentrations of human Granulocyte-Colony Stimulating Factor (hGCSF) in maternal serum were significantly associated with the risk of spontaneous abortion in women prior to the report of the event itself. However, concerning hTPO, this effect is not evident starting before approximately 8 weeks of gestation and hGCSF alone was only predictive when focusing on women with an upcoming spontaneous abortion within 14 days. Furthermore, chemokine ligand 3 (CCL-3), also known as human macrophage inflammatory protein 1- alpha (hMIP-la) is recognized as a local chemoattractant for natural killer cells, whose increased levels in decidual tissue are associated with recurrent pregnancy losses in decidual tissue within the first ten weeks of gestation. In addition, human transforming growth factor (hTGF) beta-l is described as an important factor in immunological reactions and immunological tolerance and shows a lower expression in peripheral blood mononuclear cell cultures of women who experience an abortion or biochemical pregnancy, measured in blood samples prior to the time of oocyte retrieval.

A shift between Thl- and Th2- guided immunological response also seems to play an important role in early pregnancy. The cytokine shift towards Th2-cytokines like hIL-6, as well as human tumor necrosis factor (hTNF)-alpha is considered important in early pregnancy in maternal blood. Various factors influencing this balance, e.g. CCL-2, also called human monocyte chemoattractant protein 1 (hMCP-l),) are considered to cause a shift towards the Th2-response of decidual leukocytes in uteri. Despite this essential Th2 response in the local environment in early pregnancy, elevated serum levels of hMCP-l were observed in women with recurrent spontaneous abortion (RSA) after spontaneous abortion occurred, whereas other studies did not see any significant alteration.

The assessment of ratios of biological factors for predicting progression of diseases or course of pregnancy is well known in the art. Recently, the predictive value of the ratio of soluble fms-like tyrosine kinase 1 (sFlt-l) to placental growth factor (P1GF) in women with suspected preeclampsia was demonstrated (Zeisler et al., N Engl J Med, 374;l, 2016). The authors of this study identified an sFlt-l: P1GF ratio cutoff of 38 as having predictive value.

Although both ultrasound and serial hCG values can, to some extent, identify women at risk of spontaneous abortion, no reliable predictive biomarkers have been found yet and there is a high unmet need to prospectively identify at-risk pregnancies. Most of the currently available studies evaluate symptomatic women; therefore, the present inventors are the first ones to develop a method for predicting abnormal pregnancy outcome even before patients become symptomatic in order to identify women who will experience a spontaneous abortion. The development of a predictive test for an abnormal pregnancy outcome, such as spontaneous abortion has several benefits: Patients with a negative outcome for a potential spontaneous abortion can be reassured. Especially in cases of patients with a RSA (recurrent spontaneous abortion) background, patients experience enormous psychological stress during early pregnancy. As a clinical consequence of negative prediction, the frequency of surveillance visits might be reduced thereby facilitating a normal daily routine of the patient. Furthermore, hormone treatment (i.e. progesterone) in relation to in vitro fertilization-based pregnancies might be adapted based on the outcome of such a predictive test. In cases of positive predictive outcome for a potential spontaneous abortion the patient‘s medical surveillance might be intensified or additional diagnostic and treatment options might be applied. In severe high-risk cases, the psychological support in preparation of the expected miscarriage might highly benefit the patient. Furthermore, unnecessary prolongation of the pregnancy by i.e. hormone treatment might be counter-indicated to shorten unnecessary psychological suffering of the patient or to prepare further in vitro fertilization attempts given the shortened biological window of elderly women. Such a predictive test might also contribute to find better methods for stratification of patients to optimize or identify new subsequent treatment options.

Summary of the Invention

In a first aspect, the present invention provides a method of predicting abnormal pregnancy outcome, comprising measuring, in a sample obtained from a patient, the level of:

(i) one or more cytokines and the level of human chorionic gonadotropin (hCG), and determining a ratio of the level(s) of one or more cytokines and the level of hCG, wherein the one or more cytokines are selected from the group consisting of human interleukin 1 receptor antagonist (hlF-lra), human tumor necrosis factor alpha (hTNF alpha), human macrophage inflammatory protein 1 alpha (hMIPla), human monocyte chemoattractant protein 1 (hMCPl), human thrombopoietin (hTPO), human interleukin 6 (hIF-6), human transforming growth factor beta 1 (hTGF-betal) and human granulocyte-colony stimulating factor (hG-CSF); and/or

(ii) human granulocyte-colony stimulating factor (hG-CSF) and the level of hlF-lra and determining a ratio of the level of hG-CSF and the level of hlF-lra; and/or

(iii) human macrophage inflammatory protein 1 alpha (hMIPla) and the level of hTGF- betal and determining a ratio of the level of hMIPla and the level of hTGF-betal, wherein: (a) an increased ratio for (i), an increased ratio for (iii), and/or a decreased ratio for (ii) in comparison to the respective ratio in a normal pregnancy; and/or

(b) a ratio for (i) hIL-lra/hCG [pg/mlU] of 3.0 or higher, hTNFalpha/hCG [pg/mlU] of 0.021 or higher, hMIPla/hCG [pg/mlU] of 0.20 or higher, hMCPl/hCG [pg/mlU] of 0.66 or higher, hTPO/hCG [pg/mlU] of 3.2 or higher, hTGF-betal/hCG [pg/mlU] of 91 or higher, hIL-6/hCG [pg/mlU] of 0.012 or higher, hG-CSF/hCG [pg/mlU] of 0.19 or higher; a ratio for (iii) hMIPla/hTGF-betal of 0.0041 or higher; or a ratio for (ii) hG-CSF/hIL-lra of 0.063 or lower

is predictive for an abnormal outcome during pregnancy.

In a second aspect, the present invention relates to a kit comprising one or more reagents that allow measuring the level of one or more cytokines selected from the group consisting of hCG, hIL-lra, hMCPl, hTPO, hIL-6, hTNF alpha, hTGF-betal, hMIPla and hG-CSF and optionally the level of hCG, and instructions for carrying out the method according to the first aspect of the invention.

List of Figures

In the following, the content of the figures comprised in this specification is described. In this context please also refer to the detailed description of the invention above and/or below.

Figure 1: Preferred combinations of cytokines. The preferred combinations of ratios of the first aspect of the invention are specified.

Detailed Descriptions of the Invention

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

Throughout this specification and the claims, which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being optional, preferred or advantageous may be combined with any other feature or features indicated as being optional, preferred or advantageous.

Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions etc.), whether supra or infra, is hereby incorporated by reference in its entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein are characterized as being“incorporated by reference” . In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

In the following, the elements of the present invention will be described. These elements are listed with specific embodiments; however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments, which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

Definitions

In the following, some definitions of terms frequently used in this specification are provided. These terms will, in each instance of its use, in the remainder of the specification have the respectively defined meaning and preferred meanings.

As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents, unless the content clearly dictates otherwise.

As used herein,“abnormal pregnancy outcome” is a nonviable pregnancy, including missed abortions, spontaneous abortion, miscarriage, abortus imminens, abortus completus, molar pregnancy, abortus incompletus etc. Preferably, an“abnormal pregnancy outcome is an abnormal early pregnancy outcome”.

As used herein,“gestation” is time of pregnancy. In other words, gestation is the period of pregnancy starting two weeks prior to the time of fertilization of the oocyte (ovum) up to birth.

As used herein,“pregnancy testing” is the first time of hCG testing in the blood.

Usually, the testing is performed in the 5^th week of gestation.

As used herein, the term“spontaneous abortion”,“pregnancy loss” and“miscarriage” are used interchangeably herein. Spontaneous abortion is the natural death of an embryo or fetus before it is able to survive independently. Usually, this means a spontaneous pregnancy loss within the first 24 weeks of gestation. A spontaneous abortion is considered to be an “early spontaneous abortion” if the pregnancy loss occurs within the first trimester, i.e. week 1 to 12 of gestation.

As used herein, a“sample” refers to any biological sample obtained from a patient. Preferably, a“sample” refers to blood or urine obtained from a patient.

As used herein, a“patient” is any human being. Preferably, a“patient” is a female human being after fertilization.

The terms“level” and“amount” are used interchangeably herein. Usually, the level of any of the cytokines of the present invention is specified in picogram [pg]/ milliliter [ml] and the level of human chorionic gonadotropin (hCG) is specified in milli-International Units [mlU]/ milliliter [ml] . Thus, a ratio as determined according to the present invention is either specified in [pg/mlU] or has no measuring unit, if both levels are determined in the same measuring unit. It is well known in the art, how to determine the levels of the respectively indicated marker.

As used herein the term“hCG” refers to human chorionic gonadotropin, which is a hormone produced by the placenta and the fetus after implantation. It is well known in the art, how to determine the“mlU/ml” level of hCG in a sample, preferably it can be determined by ELISA as described by Duan L et al., Arch Gynecol & obstet, 2010.

As used herein the term“hMCPl” refers to human monocyte chemoattractant protein 1, which is also known as chemokine (C-C motif) ligand 2 (CCL-2) and small inducible cytokine A2. It is well known in the art, how to determine the“pg/ml” level of hMCPl in a sample, preferably it can be determined by multiplex analysis as described by Penova- Veselinovic B et al., J Reprod Immunol., 2015. As used herein the term“hIL-lra” refers to human interleukin- 1 receptor antagonist, which is a protein that is encoded by the IL1RA gene. It is well known in the art, how to determine the“pg/ml” level of hIL-lra in a sample, preferably it can be determined by multiplex analysis as described by Villani S et al., J Neuroimmunol., 2017.

As used herein the term“hTPO” refers to human thrombopoietin, also known as megakaryocyte growth and development factor (MGDF), which is a glycoprotein hormone produced by the liver and kidney which regulates the production of platelets. It is well known in the art how to determine the“pg/ml” level of hTPO in a sample, preferably it can be determined as described by Westerink J et al., Lipids , 2014.

As used herein the term“hIL-6” refers to human interleukin 6, which is a protein encoded by the IL6 gene. It is well known in the art, how to determine the“pg/ml” level of hlL- 6 in a sample, preferably it can be determined as described by Westerink J et al., Lipids, 2014.

As used herein the term“hTNF alpha” refers to human tumor necrosis factor alpha, also known as cachexin or cachectin, which is a cytokine that is involved in systemic inflammation. It is well known in the art, how to determine the“pg/ml” level of hTNF alpha in a sample, preferably it can be determined as described by Westerink J et al., Lipids, 2014.

As used herein the term“hTGF-betal” refers to human transforming growth factor beta 1, which is encoded by the TGFB1 gene. It is well known in the art, how to determine the “pg/ml” level of hTGF-betal in a sample, preferably it can be determined as described by Flanders et al., Oncotarget, 2017.

As used herein the term“hMIPla” refers to human macrophage inflammatory protein a, which is also known as CCL-3. It is well known in the art, how to determine the“pg/ml” level of hMIPla in a sample, preferably it can be determined as described by Hang et al., Mol Vis, 2014.

As used herein the term“hG-CSF” refers to human granulocyte-colony stimulating factor, also known as colony-stimulating factor 3 (CSF 3). It is well known in the art, how to determine the“pg/ml” level of hG-CSF in a sample, preferably it can be determined as described by Hang et al., Mol Vis, 2014.

Embodiments

In the following different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous. In a first aspect, the present invention provides a method of predicting abnormal pregnancy outcome comprising measuring, in a sample obtained from a patient, the level of:

(i) one or more cytokines and the level of human chorionic gonadotropin (hCG), and determining a ratio of the level(s) of one or more cytokines and the level of hCG, wherein the one or more cytokines are selected from the group consisting of human interleukin 1 receptor antagonist (hIL-lra), human tumor necrosis factor alpha (hTNF alpha), human macrophage inflammatory protein 1 alpha (hMIPla), human monocyte chemoattractant protein 1 (hMCPl), human thrombopoietin (hTPO), human interleukin 6 (hIL-6), human transforming growth factor beta 1 (hTGF-betal) and human granulocyte-colony stimulating factor (hG-CSF); and/or

(ii) human granulocyte-colony stimulating factor (hG-CSF) and the level of hIL-lra and determining a ratio of the level of hG-CSF and the level of hIL-lra; and/or

(iii) human macrophage inflammatory protein 1 alpha (hMIPla) and the level of hTGF- betal and determining a ratio of the level of hMIPla and the level of hTGF-betal, wherein:

(a) an increased ratio for (i), an increased ratio for (iii), and/or a decreased ratio for (ii) in comparison to the respective ratio in a normal pregnancy; and/or

is predictive for an abnormal outcome during pregnancy.

In a preferred embodiment of the first aspect of the present invention, the ratio for hlL- lra/hCG [pg/mlU] of 3.0 or higher, preferably of 3.2 or higher, more preferably of 3.4 or higher, more preferably of 3.6 or higher, more preferably of 3.8 or higher, most preferably of 4.0 or higher, such as 4.4 or higher is predictive for an abnormal outcome during pregnancy. For example, a ratio for hIL-lra/hCG [pg/mlU] of about 7.83 ± 2.30 is predictive for an abnormal outcome during pregnancy.

In a preferred embodiment of the first aspect of the present invention, the ratio for hTNFalpha/hCG [pg/mlU] of 0.021 or higher, preferably of 0.025 or higher, preferably of 0.03 or higher, more preferably of 0.035 or higher, more preferably of 0.04 or higher, most preferably of 0.045 or higher, such as 0.05 or higher is predictive for an abnormal outcome during pregnancy. For example, a ratio for hTNFalpha/hCG [pg/mlU] of about 0.07 ± 0.02 is predictive for an abnormal outcome during pregnancy.

In a preferred embodiment of the first aspect of the present invention, the ratio for hMIPla/hCG [pg/mlU] of 0.2 or higher, preferably of 0.3 or higher, more preferably of 0.4 or higher, more preferably of 0.5 or higher, more preferably of 0.6 or higher, such as 0.70 or higher is predictive for an abnormal outcome during pregnancy. For example, a ratio for hMIPla/hCG [pg/mlU] of about 1.02 ± 0.38 is predictive for an abnormal outcome during pregnancy.

In a preferred embodiment of the first aspect of the present invention, the ratio for hMCPl/hCG [pg/mlU] of 0.66 or higher, preferably of 0.67 or higher, more preferably of 0.68 or higher, more preferably of 0.69 or higher, most preferably of 0.70 or higher, such as 0.71 or higher is predictive for an abnormal outcome during pregnancy. For example, a ratio for hMCPl/hCG [pg/mlU] of about 1.44 ± 0.40 is predictive for an abnormal outcome during pregnancy.

In a preferred embodiment of the first aspect of the present invention, the ratio for hTPO/hCG [pg/mlU] of 3.2 or higher, preferably of 3.4 or higher, more preferably of 3.6 or higher, more preferably of 3.8 or higher, more preferably of 4.0 or higher, most preferably of 4.5 or higher, such as 4.75 or higher is predictive for an abnormal outcome during pregnancy. For example, a ratio for hTPO/hCG [pg/mlU] of about 5.47 ± 1.31 is predictive for an abnormal outcome during pregnancy.

In a preferred embodiment of the first aspect of the present invention, the ratio for hTGF- betal/hCG [pg/mlU] of 91 or higher, preferably of 95 or higher, more preferably of 100 or higher, more preferably of 110 or higher, more preferably of 120 or higher, most preferably of 130 or higher, such as 140 or higher is predictive for an abnormal outcome during pregnancy. For example, a ratio for hTGF-betal/hCG [pg/mlU] of about 149.35 ± 33.89 is predictive for an abnormal outcome during pregnancy.

In a preferred embodiment of the first aspect of the present invention, the ratio for hlL- 6/hCG [pg/mlU] of 0.012 or higher, preferably of 0.015 or higher, such as 0.019 or higher is predictive for an abnormal outcome during pregnancy. For example, a ratio for hIL-6/hCG [pg/mlU] of about 0.02 ± 0.003 is predictive for an abnormal outcome during pregnancy.

In a preferred embodiment of the first aspect of the present invention, the ratio for hG- CSF/hCG [pg/mlU] of 0.19 or higher, preferably of 0.21 or higher, more preferably of 0.23 or higher, more preferably of 0.25 or higher, most preferably of 0.27 or higher, such as 0.29 or higher is predictive for an abnormal outcome during pregnancy. For example, a ratio for hG- CSF/hCG [pg/mlU] of about 0.296 ± 0.078 is predictive for an abnormal outcome during pregnancy.

In a preferred embodiment of the first aspect of the present invention, the ratio for hMIPla/hTGF-betal of 0.0041 or higher, preferably of 0.005 or higher, more preferably of 0.006 or higher, more preferably of 0.007 or higher, more preferably of 0.008 or higher, most preferably of 0.009 or higher, such as 0.011 or higher is predictive for an abnormal outcome during pregnancy. For example, a ratio for hMIPla/hTGF-betal of about 0.012 ± 0.005 is predictive for an abnormal outcome during pregnancy.

In a preferred embodiment of the first aspect of the present invention, the ratio for hG- CSF/hlL- lra of 0.063 or lower, preferably of 0.060 or lower, more preferably of 0.055 or lower, most preferably of 0.050 or lower, such as 0.045 or lower is predictive for an abnormal outcome during pregnancy. For example, a ratio for hG-CSF/hIL- lra of about 0.043 ± 0.005 is predictive for an abnormal outcome during pregnancy.

In a preferred embodiment of the first aspect of the present invention, the abnormal pregnancy outcome is an abnormal early pregnancy outcome.

In a preferred embodiment of the first aspect of the present invention, the level of two or more cytokines according to claim 1 and optionally the level of hCG is measured. Each combination of the ratios specified in claim 1 is within the scope of the present invention. Preferably, the cytokines comprise or consist of hIL-lra, hTNFalpha and hMIPla.

In a preferred embodiment of the first aspect of the present invention, the two or more cytokines are combined as specified in Figure 1.

In a preferred embodiment of the first aspect of the present invention, the ratios of all cytokines according to claim 1 and optionally the level of hCG are determined.

In a preferred embodiment of the first aspect of the present invention, the sample is obtained within 3 months of gestation. Preferably, the sample is obtained within 1 month of gestation.

In a preferred embodiment of the first aspect of the present invention, the sample is obtained at the time of pregnancy testing.

In a preferred embodiment of the first aspect of the present invention, the sample is blood or urine. Preferably, the sample is whole blood, plasma or serum.

In a preferred embodiment of the first aspect of the present invention, the abnormal pregnancy outcome is early spontaneous abortion or spontaneous abortion.

In a second aspect, the present invention relates to a kit comprising one or more reagents that allow measuring the level of one or more cytokines selected from the group consisting of hCG, hIL-lra, hMCPl, hTPO, hIL-6, hTNF alpha, hTGF-betal, hMIPla and hG-CSF and optionally the level of hCG, and instructions for carrying out the method according to the first aspect of the invention. The instructions can be provided in the form of a leaflet.

In a preferred embodiment of the second aspect of the present invention, the reagents allow measuring the level of one or more cytokines selected from the group consisting of hCG, hIL-lra, hTNF alpha and hMIPla.

Suitable reagents are known individually for each of the cytokines in the art. They comprise antibodies or other specific ligands, if the amount of the respective cytokine is measured on the protein level or suitable primers for, e.g. QT-PCR, if the amount of the respective cytokines is measured on the mRNA or cDNA level.

Examples

Methods:

The prospective pilot study was approved by Heidelberg University Ethical Committee (protocol S-243/2015) and the experimental testing complied with the principles specified in the Declaration of Helsinki. Blood samples were obtained at the time of pregnancy testing after informed consent from women who underwent ovarian hyperstimulation to perform in vitro fertilization by IVF or ICSI at the University Hospital in Heidelberg, Germany. Exclusion criteria were autoimmune diseases, essential hypertonia, diabetes mellitus or the intake of confounding medication (e.g. acetylsalicylic acid).

Dependent on the follow-up, patients with a positive pregnancy test were subsequently divided in two groups: (1) patients with ongoing pregnancy (n=22) and (2) patients with miscarriage (missed abortion, abortus incompletus or abortus completus, n=l7) in the first trimester of this pregnancy. Immunological and endocrine profiling of maternal plasma of women with and without miscarriage was performed for each group at the time of pregnancy, in order to predict pregnancy outcome.

Determination of cytokine levels

hIL-6, hTNF-a, (Luminex Performance Assay, Human High Sensitivity Cytokine Base Kit A; R&D systems, Wiesbaden, Germany), hIL-lra, hMCP-l, hMIP-l alpha, hG-CSF, hThrombopoietin /hTPO (Human Luminex Performance Assay Base Kit, Panel A; R&D systems, Wiesbaden, Germany), hTGF-beta 1 (Luminex Performance Assay 3-plex Kit; R&D systems, Wiesbaden, Germany) were determined in 39 samples using Multiplex and ELISA analysis. Assays were performed according to the instructions of the manufacturer. Measurements were performed as pg/ml and values given as mean +/- SEM.

Determination of hCG-levels

hCG-levels (mIE/ml, mean+/- SEM) were measured routinely in clinical practice at the time of pregnancy testing from central laboratory, University Hospital Heidelberg, Germany.

Example 1

There was no significant difference in age (33.64 ±6.49 years vs. 33.94+4.4 years), nor in body mass index in group 1 and 2 with 23.81 + 4.29 kg/m² in group 1 vs. 26.48+ 5.27 kg/m² in group 2 (table 1) or transfer day (table 2).

Table 1: Patient characteristics

Table 2: Embryo quality and day of embryo transfer

hCG-results and immunological profiles of patients with ongoing pregnancy (n=22, No. 1-22) and abortion (n=l7, No. 23-39) were analysed. Quantitative difference in hGCSF-, hMCP-l-, hTPO- or hIL-6-expression showed a trend towards a change in values, however did not yet reach statistical significance. hCG levels were significantly decreased in patients with abortion compared to those with ongoing pregnancy (151.75 ± 25.29 IU/l vs. 351.27 ± 111.02 IU/l, p<0.05). Nevertheless, the hCG values were within normal limits for early pregnancy and therefore not predictive for pregnancy outcome. Similarly, levels of hIL-lra (655.80±78.80 pg/ml vs. 398.69±32.73pg/ml, p<0.0l), hMIP-la (73.74±9.9l pg/ml vs. 34.20±8.25 pg/ml, p<0.0l) and hTNF-alpha (5.l l±0.40 pg/ml vs. 4.00±0.26 pg/ml, p<0.05) were significantly increased.

In order to get a potentially more stable predictor, we investigated if the relative expressions of immunological factors among themselves show a significant alteration. Here, we found that the ratio of hGCSF to IL-lra was 1.66-fold decreased (p<0.05) in patients suffering from miscarriage (0.043±0.005) compared to patients with ongoing pregnancy (0.072±0.009; p<0.05). In addition, the ratio of hMIPla to hTGF-beta was 3.45-times higher in patients with miscarriages (0.0l2±0.005) compared to patients without ongoing pregnancy (0.004±0.00l; p<0.05) (data are shown in table 3). The other relative expressions of the different cytokines analyzed did not show significant changes.

Table 3: Significant alterations of relative immunological profile. hGCSF, hIL-lra, hMIPla and hTGF-beta were assessed in pg/ml

Due to the observation, that patients with miscarriage showed lower levels of hCG and increased levels in immunological factors at the same time, we investigated if we get a higher predictive value, if we analyze the relative expression of immunological factors compared to hCG (table 4). Table 4: Alterations in immunological profile relative to hCG-levels. hMIPla, hIL-lra, hTNF alpha, hMCPl, hIL-6, hTPO and hTGF-betal were assessed in pg/ml, hCG in mlU/ml.

Comparing patients with ongoing pregnancy to patients experiencing a miscarriage, we could demonstrate significant alterations of the ratios hMIPla/hCG (0.l6±0.04pg/mIU vs. l.02±0.38 pg/mlU, p<0.05), hIL-lra/hCG (2.22±0.72 pg/mlU vs. 7.83±2.30 pg/mlU, p<0.05), hTNFalpha /hCG (0.02±0.0l pg/mlU vs. 0.07±0.02 pg/mlU, p<0.05), hMCPl /hCG (0.50±0.l6 pg/mlU vs. l.44±0.40 pg/mlU, p<0.05), hIL-6/hCG (0.007±0.002 pg/mlU vs 0.0l6±0.003 pg/mlU, p<0.05), hTPO/hCG (2.50±0.70 pg/mlU vs. 5.47±l.3l pg/mlU, p<0.05) and hTGF- beta 1 /hCG (68.04±22.37 pg/mlU vs. l49.35±33.89 pg/mlU, p<0.05).

Most of the studies trying to establish biomarkers to follow this approach include symptomatic women at a later stage of pregnancy. One of the most interesting aspects concerning the data of the present invention is the fact that we discovered in total 3 absolute and 9 relative alterations in immunological profile parameters of asymptomatic patients at the time of first pregnancy testing, that could present a very helpful tool in clinical practice to discover patients at risk even before first symptoms occur.

Altogether, the present inventors observed a significant alteration towards the immunological factor in relation to hCG levels in patients, who experience a miscarriage in their course of pregnancy. The strongest effects were seen for the ratio hMIPla/hCG (ratio 0.15) for patients with ongoing pregnancy compared to patients with abortion, hIL-lra (ratio

0.28) and hTNFalpha (ratio 0.29). Example 2

In a further example, hMCP-l (Human Luminex Performance Assay Base Kit, Panel A; R&D systems, Wiesbaden, Germany) and hTGF-beta 1 (Luminex Performance Assay 3-plex Kit; R&D systems, Wiesbaden, Germany) were determined in serum samples using ELISA analysis. Assays were performed according to the instructions of the manufacturer. Measurements were performed as pg/ml and values given as mean +/- SEM.

Table 5: Alterations in immunological profile relative to hCG-levels. hMCPl and hTGF-betal were assessed in pg/ml, hCG in mlU/ml

Claims

1. A method of predicting abnormal pregnancy outcome, comprising measuring in a sample obtained from a patient the level of:

(i) one or more cytokines and the level of human chorionic gonadotropin (hCG), and determining a ratio of the level(s) of the one or more cytokines and the level of hCG, wherein the one or more cytokines are selected from the group consisting of human interleukin 1 receptor antagonist (hIL-lra), human tumor necrosis factor alpha (hTNF alpha), human macrophage inflammatory protein 1 alpha (hMIPla), human monocyte chemoattractant protein 1 (hMCPl), human thrombopoietin (hTPO), human interleukin 6 (hIL-6), human transforming growth factor beta 1 (hTGF-betal) and human granulocyte-colony stimulating factor (hG-CSF); and/or

is predictive for an abnormal outcome during pregnancy.

2. The method according to claim 1, wherein the level of two or more cytokines and optionally the level of hCG according to claim 1 is measured.

3. The method according to claim 1 or 2, wherein the cytokines comprise or consist of hlL- lra, hTNFalpha and hMIPla.

4. The method according to any of the preceding claims, wherein the ratios of all cytokines and optionally the level of hCG according to claim 1 are determined.

5. The method according to any of the preceding claims, wherein the sample is obtained within 3 months of gestation.

6. The method according to claim 5, wherein the sample is obtained within 1 month of gestation.

7. The method according to any of the preceding claims, wherein the sample is obtained at the time of pregnancy testing.

8. The method according to any of the preceding claims, wherein the sample is blood or urine.

9. The method according to any of the preceding claims, wherein the abnormal pregnancy outcome is spontaneous abortion.

10. The method according to any of the preceding claims, wherein the abnormal pregnancy outcome is early spontaneous abortion.

11. A kit comprising :

one or more reagents that allow measuring the level of one or more cytokines selected from the group consisting of hCG, hIL-lra, hMCPl, hTPO, hIL-6, hTNF alpha, hTGF- betal, hMIPla and hG-CSF and optionally the level of hCG, and

instructions for carrying out the method of any one of claims 1 to 10.