# Patent

## Title
Use of relaxin to treat placental syndromes

## Abstract

The subject application relates to methods for treating a placental syndrome, wherein relaxin is administered during the late secretory/luteal (LS) phase of the menstrual cycle in women who have a propensity for developing the placental syndrome. In certain embodiments, administration of relaxin continues beyond the LS phase and into pregnancy.

## Background
CROSS-REFERENCE TO A RELATED APPLICATION
This application claims the benefit of U.S. provisional application Ser. No. 61/858,282 filed Jul. 25, 2013, which is incorporated herein by reference in its entirety.


The subject invention was made with government support under Grant Nos. RO1 HD030325; RO1 HL067937; RO1 DK063321; PO1 HD030367; and PO1 HD065647 research projects supported by the National Institute of Health (NIH). The government has certain rights in the invention.


BACKGROUND OF INVENTION
Preeclampsia (also known as toxemia and referred to herein as PE) is a dangerous and volatile hypertensive disease that affects pregnant women, usually late in the second or third trimester, and postnatal women in the first six weeks after delivery. It is a leading cause of maternal, fetal and neonatal, morbidity and mortality. Further, both women and their children who survive PE are at greater risk for future adverse cardiovascular events.
The condition affects the kidneys, liver, brain, heart and placenta of the pregnant woman. PE occurs in approximately three to five percent of pregnancies and is only alleviated by ending the pregnancy, either by induction of labor or cesarean. PE most commonly occurs during a first pregnancy. The risk for preeclampsia is also known to be moderately increased for certain groups of pregnant women, including women who are over 35 years of age or under 18 years of age; women who are genetically predisposed to this condition; women who suffer from preexisting hypertension, diabetes, autoimmune diseases like lupus, various inherited thrombophilias like Factor V Leiden, or renal disease; obese women, and in women with multiple gestations (twins, triplets, and more). The single most significant risk for developing preeclampsia is having had preeclampsia in a previous pregnancy.
PE can develop either gradually or suddenly, and may remain mild throughout the pregnancy or become severe. PE is diagnosed by new onset protein in the urine (proteinuria) and high blood pressure. Common symptoms in addition to high blood pressure and proteinuria are elevated uric acid, vision problems such as blinking lights or blurry vision, persistent headaches, extreme swelling of hands and feet, fluid retention, pain in the upper right abdomen. If untreated, preeclampsia can damage the mother's liver or kidneys, cause pulmonary edema, deprive the fetus of oxygen, and cause eclampsia (seizures). A pregnant woman with signs of preeclampsia must be closely monitored by a physician. Moderate to severe preeclampsia is often treated in the hospital with bed rest, magnesium sulfate, and medication for high blood pressure. Unfortunately, delivery is still the only true “cure” for preeclampsia. In fact, when a woman has severe preeclampsia or is near term with mild to moderate preeclampsia, delivery is still the best remedy to date. Labor is then started with medication, unless a cesarean section is deemed necessary. Within the first few days following delivery, the mother's blood pressure usually returns to normal; however, with severe preeclampsia, it may take several weeks for blood pressure to return to normal.
The pathogenesis of PE has been investigated in the last decade, e.g., circulating factors emanating from the placenta have been identified in the blood that injure the endothelium, thereby producing maternal symptoms including hypertension and proteinuria. In contrast, PE etiology remains uncertain and infrequently addressed. One reason why so little is known about what causes PE is that the disease likely begins in early pregnancy, secondary in large measure to “shallow” placentation, i.e., deficient trophoblast (Tr) invasion of uterine spiral arteries, which starts in the 1st trimester. Normally, placental cells called Tr invade the endometrium, inner 1⁄3 of the myometrium and the uterine spiral arteries, ultimately remodeling the latter, which allows for large increases of blood flow, oxygen and nutrients to the developing placenta and fetus. In PE, Tr invasion is impaired and hence blood flow and oxygen delivery to the placenta and fetus is compromised, leading to release of factors from the placenta that circulate and injure the maternal endothelium, thereby producing disease manifestations. Thus, the Tr (“seed”) has been the focus of much investigative attention. In contrast, the uterine milieu (“soil”) has been less explored.
Endometrial maturation is a necessary precursor for healthy placentation. Stromal cells, uterine and glandular epithelial cells, as well as spiral arteries undergo distinct morphologic and functional changes, which begin before pregnancy in the late secretory/luteal (LS) phase of the menstrual cycle (“pre-decidualization”) continuing after conception (“decidualization”). Unfortunately, little data is available regarding whether certain elements in the LS endometrium (i.e., before conception) play a role in the later development of PE during pregnancy.
Because of the current lack of effective treatments for placental syndromes (i.e., PE and IUGR), there is a strong need to develop new therapeutic approaches for protecting both mother and child from the harmful effects of placental syndromes.
BRIEF SUMMARY
The present invention provides methods for treatment, diagnosis and/or prevention of placental syndromes including, but not limited to, PE, deficient Tr invasion of uterine spiral arteries, defective decidualization, compromised endometrial maturation (e.g., pre-decidualization) in the LS phase of the menstrual cycle and inadequate placentation. Advantageously, the present invention facilitates treating woman in a timely fashion to decrease the risk for developing placental syndromes, such as PE, following conception.
In one embodiment, the invention provides a method for treating and/or reducing the likelihood of development of a placental syndrome comprising the steps of: (a) determining whether a woman has experienced a placental syndrome during a previous pregnancy; (b) if the woman has experienced a placental syndrome during a previous pregnancy, determining the late secretory/luteal (LS) phase of the menstrual cycle; and (c) administering a therapeutically effective amount of relaxin to the woman during the LS phase of menstruation to treat and/or reduce the likelihood of development of the placental syndrome. Preferably, the placental syndrome is PE and/or IUGR.
In a related embodiment, the method further comprises administering relaxin from the LS phase of the menstrual cycle throughout a portion or entirety of the first trimester of pregnancy. In another embodiment, the method further comprises administering relaxin from the LS phase of the menstrual cycle throughout a portion or entirety of the second trimester of pregnancy. In yet another embodiment, the method further comprises administering relaxin from the LS phase of the menstrual cycle throughout a portion or entirety of the third trimester of pregnancy. In another embodiment, the method further comprises administering relaxin from the LS phase of the menstrual cycle throughout pregnancy and following delivery.
In another embodiment, the invention provides methods comprising the steps of: (a) determining whether the woman is at risk for developing a placental syndrome; (b) if the woman is at risk, determining the late secretory/luteal (LS) phase of the menstrual cycle; and (c) administering natural or exogenous relaxin to the woman during the LS phase of menstruation. In a related embodiment, step (a) comprises obtaining a biological sample from the woman (such as blood, urine, uterine tissue (e.g., endometrial biopsy) or secretions) and measuring in the sample biomarker(s) that reflect endometrial maturation (pre-decidualization) or a deficiency thereof.
In certain related embodiments, the step of determining whether the woman is at risk for developing a placental syndrome comprises diagnosing whether the woman has a genetic predisposition or propensity for impaired relaxin production and/or activity. For example, this determination may comprise determining the nucleotide sequence of at least a portion of a gene that is involved with deficient decidualization in PE.
The subject invention is directed to addressing PE disease etiology that starts in the first trimester with antecedents before conception. In certain embodiments, the method of the subject invention can be used to (1) correct deficient endometrial maturation (“(pre)decidualization”), thereby improving histiotrophic nutrition of the placenta and fetus before 10 weeks; (2) promote trophoblast invasion and physiological remodeling of uterine spiral arteries, and hence, fetoplacental blood flow after 10 weeks; and (3) directly stimulate survival and invasion of trophoblast, and thus, improve placentation and placental function; and augment maternal circulatory adaptations to pregnancy, which are deficient in women destined to develop severe preeclampsia or intrauterine growth restriction, particularly in the first trimester. The treatments described herein promote proper endometrial maturation prior to conception, which is beneficial in obviating PE development, as well as intrauterine growth restriction (IUGR) during pregnancy
Relaxin employed by the subject invention can be, for example, synthetic or recombinant relaxin, or a pharmaceutically effective relaxin agonist or mimetic. In one embodiment of the invention, relaxin is H1 human relaxin. In another embodiment, relaxin is H2 human relaxin. In yet another embodiment, relaxin is H3 human relaxin. In a further embodiment, relaxin is synthetic or recombinant human relaxin, or a pharmaceutically effective relaxin agonist or relaxin mimetic. Thus, a non-pregnant female at risk for preeclampsia can be treated with a pharmaceutical formulation of isolated, synthetic or recombinant human relaxin or relaxin agonist or mimetic during the LS phase of menstruation and potentially throughout pregnancy (vide supra).
In one embodiment, a human female is treated with synthetic human relaxin at least during the LS phase of menstruation, preferably through the end of the first trimester. In another embodiment, a human female is treated with recombinant human relaxin at least during the LS phase of menstruation, preferably through the end of the first trimester. In yet another embodiment, a human female is treated with a pharmaceutically effective relaxin agonist or mimetic during the LS phase of menstruation, preferably through the end of the first trimester. Relaxin or mimetics can be administered to the female through a number of different routes, including but not limited to, subcutaneously, intramuscularly, intravenously, sublingually, intranasally, orally, topically (e.g., by dermal patch) or via inhalation. One preferred route of administration is subcutaneous (SQ) administration



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a flow diagram illustrating factors involved in endometrial and placental maturation.
FIG. 2 is a slide highlighting the analysis of genes isolated from CVS tissues in spontaneously conceived pregnancies to determine dysregulated genes associated with PE.
FIG. 3 is a slide regarding the analysis of the dysregulated genes to determine whether hypoxia or oxidative stress regulated genes were upregulated.
FIG. 4 is a venn diagram highlighting the twenty-four (24) genes associated with both deficient decidualization and PE.
FIG. 5 is a slide regarding the identification of additional genes associated with both deficient decidualization and PE.
FIG. 6 illustrates steps involved in a prospective study to determine endometrial antecedents of preeclampsia.
FIG. 7 are graphs illustrating relaxin ability to stimulate human trophoblast invasion in vitro. The effect of recombinant human relaxin-2 (rhRLX) and the general matrix metalloproteinase inhibitor (MMP), GM6001, on FIG. 7A, HTR-8/SVNeo cell invasion after 24 h in the Matrigel Invasion Assay (see following FIG. 8). Concentration dependence of rhRLX (3, 30, and 300 ng/ml) compared to vehicle for rhRLX (VEH), and 10% fetal bovine serum as positive control. FIG. 7B demonstrates the inhibition of rhRLX stimulated invasion by GM6001 (25 mM). Data from 2 experiments, 2 or 3 replicate wells per treatment, and one 20× magnification field per well. Expressed as number of counted cells with values presented as mean±SEM. *p<0.05 and ***p<0.001 vs VEH in FIG. 7A; ***p<0.001 vs all other treatments in FIG. 7B by Newman-Keuls Multiple Comparison test.
FIG. 8 are images and graphs illustrating the analysis of Matrigel Invasion Assay by epifluorescent microscopy and ImageJ. Pictures were obtained by epifluorescent microscopy (FIG. 8A) and than loaded in ImageJ software***. Images were converted to grayscale 8-bit pictures (FIG. 8B) and than analyzed with a Nucleus Counter plugin***. Analysis by ImageJ of pictures (n=70) taken at 100× magnification were correlated (C) to manual counting.
FIG. 9 are images and graphs demonstrating relaxin's ability to attenuate trophoblast cell apoptosis in vitro. Illustrated are the effects of recombinant human relaxin-2 (rhRLX) on HTR-8/SVneo cell death following serum starvation and hypoxia-reoxygenation injury as measured by TUNEL staining, microscopy and counting on ImageJ software. FIG. 9A are images of total nuclei stained with Hoechst for normoxia cells (1), hypoxia non-treated cells (2) and hypoxia rhRLX-treated cells (3). Note that the number of rhRLX-treated adherent cells (3) is similar to control (1) demonstrating the anti-apoptotic effect of the hormone. FIG. 9B is a graph illustrating the average of three separate experiments. In FIG. 9C, each experiment (1-3) is shown. The different treatments were normoxia (n=3 dishes per experiment), hypoxia untreated (n=2-3), and hypoxia with 300 ng/mL rhRLX (n=2-3). FIG. 9D are images depicting total nuclei stained with Hoechst dye (1); apoptotic nuclei stained with TUNEL reagent (2); and (3) the merged images. ImageJ software and a ratio of TUNEL positive to total nuclei was calculated to determine percent apoptosis.
FIG. 10 are images and graphs demonstrating relaxin's ability to attenuate trophoblast cell death in vitro. FIG. 10A depicts the effect of recombinant human relaxin-2 (rhRLN) on HTR-8/SVneo cell death following serum starvation and hypoxia-reoxygenation injury as measured by PI staining and flow cytometry. FIG. 10B illustrates the concentration dependence of rhRLN (1, 3, 10, 30, 100 and 300 ng/mL) compared to vehicle (VEH) and 5% fetal bovine serum as a positive control. P<0.001 by ANOVA.* P<0.05, §P<0.1 vs VEH.
FIGS. 11A-11C are Venn Diagrams showing differentially expressed genes (DEG) down-regulated in chorionic villous samples obtained from women  ̃11 gestational weeks who developed preeclampsia 5-6 months later (PE-CVS; relative to CVS from women with normal pregnancy) and their overlap with DEG up-regulated in late secretory endometrium (LSE; relative to proliferative phase endometrium) and with endometrium with intermediate decidual (intDEC) changes from tubal ectopic pregnancies (EP; relative to EP endometrium without decidual changes). FIG. 11A is a Venn Diagram showing significant overlap (*P<0.0001 by Pearson's chi-square test) between DEG down-regulated in PE-CVS and DEG up-regulated in LSE (38 DEG, Table 10). FIG. 11B is a Venn Diagram showing significant overlap (*P<0.0001 by Pearson's chi-square test) between DEG down-regulated in PE-CVS and DEG up-regulated in endometrium with intDEC changes from EP, which lacks local trophoblast influence (32 DEG, Table 11). FIG. 11C is a Venn Diagram showing significant overlap (*P<0.0001 by Pearson's chi-square test) between DEG down-regulated in PE-CVS and DEG up-regulated in intDEC-EP endometrium and LSE (16 DEG, Table 12). There is also significant overlap between DEG up-regulated in intDEC-EP endometrium and LSE (382 DEG; *p<0.0001).
FIGS. 12A-12D are Venn Diagrams showing differentially expressed genes (DEG) down-regulated in chorionic villous samples obtained from women  ̃11 gestational weeks who developed preeclampsia 5-6 months later (PE-CVS; relative to CVS from women with normal pregnancy) and their overlap with DEG up-regulated in endometrium and with intermediate (intDEC) and confluent (confDEC) decidual changes from intrauterine pregnancy (IUP; relative to EP endometrium without decidual changes), but not with DEG up-regulated in cultured DEC stromal cells incubated with trophoblast conditioned médium (TrCM). FIG. 12A is a Venn Diagram showing significant overlap (*P<0.0001 by Pearson's chi-square test) between DEG down-regulated in PE-CVS and DEG up-regulated in intDEC endometrium (37 DEG, Table 13). FIG. 12B is a Venn Diagram showing significant overlap (*P<0.0001 by Pearson's chi-square test) between DEG down-regulated in PE-CVS and DEG up-regulated in confDEC endometrium (46 DEG, Table 14) both from IUP with local trophoblast present. FIG. 12C is a Venn Diagram showing significant overlap (*P<0.0001 by Pearson's chi-square test) between DEG down-regulated in PE-CVS and DEG up-regulated in intDEC-EP endometrium (local trophoblast absent; also see FIG. 11B), and in intDEC-IUP endometrium (local trophoblast present). The majority of these DEG, in turn, are overlapping (30 DEG, Table 15; *p<0.0001) suggesting minimal trophoblast contribution to the overlap. There is also significant overlap between DEG up-regulated in intDEC-EP and intDEC-IUP endometrium (689 genes, *p<0.0001). FIG. 12D is a Venn Diagram showing no significant association (n=4 DEG, p=0.5) between DEG down-regulated in PE-CVS and DEG up-regulated by exposure of DEC stromal cells in culture to trophoblast conditioned medium (TrCM). There is significant overlap with DEG down-regulated in PE-CVS and up-regulated in confDEC-IUP endometrium (46 DEG), as well as 69 DEG in common between DEG up-regulated in TrCM and confDEC-IUP endometrium (both *p<0.0001). For abbreviations, also see Legend to FIG. 11.
FIG. 13 is a Venn Diagram illustrating the confluence of overlapping differentially expressed genes (DEG) down-regulated in chorionic villous samples obtained from women  ̃11 gestational weeks who developed preeclampsia 5-6 months later (PE-CVS; relative to CVS from women with normal pregnancy) and DEG up-regulated in: LSE (relative to proliferative endometrium); endometrium with intDEC and confDEC changes from IUP; and intDEC changes from EP (all relative to EP endometrium without decidual changes). There are 20 DEG down-regulated in PE-CVS and uniquely up-regulated in LSE. There is significant overlap (*p<0.0001 by Pearson's chi-square test) between DEG down-regulated in PE-CVS and up-regulated in intDEC-EP and —IUP and confDEC-IUP endometrium, but not LSE (13 DEG); and in LSE, intDEC-EP and —IUP, and confDEC-IUP endometrium (16 DEG). See Table 16 for individual genes and FIG. 14 for average expression levels of these genes. For abbreviations, also see Legends in FIGS. 11 and 12.
FIGS. 14A-14D illustrate average expression levels (log base 2) of differentially expressed genes (DEG) in samples obtained from endometrium at different stages of endometrial maturation and from PE-CVS. FIG. 14A illustrates the average expression of 20 DEG down-regulated in chorionic villous samples obtained from women  ̃11 gestational weeks who developed preeclampsia 5-6 months later (PE-CVS; relative to CVS from normal pregnancy) and up-regulated in mid and late secretory endometrium (MSE and LSE, respectively; relative to proliferative phase endometrium, PrE). FIG. 14B illustrates the average expression of 13 DEG down-regulated in PE-CVS and up-regulated in intermediate (intDEC-IUP and -EP) and confluent decidualized (confDEC-IUP) endometrium, but not LSE. FIG. 14C illustrates the average expression for 16 DEG down-regulated in PE-CVS and upregulated in LSE, intDEC-IUP and -EP and confDEC-IUP endometrium (16 DEG). FIG. 14D is a heat map corresponding to FIG. 14C. The individual DEG in FIGS. 14A, 14B and 14C are listed in Table 16. nonDEC (non-decidualized endometrium from EP); for more abbreviations, see also Legends to previous figures. Significantly different (p<0.05) from: a, PrE; b, ESE; c, MSE; d, LSE; e, intDEC-EP; f, intDEC-IUP; g, nonDEC; h, PE-CVS.
FIG. 15 is a Venn Diagram illustrating the intersection of differentially expressed genes (DEG) down-regulated in chorionic villous samples obtained from women  ̃11 gestational weeks who developed preeclampsia 5-6 months later (PE-CVS; relative to CVS from women with normal pregnancy) and DEG up-regulated in: (A) late secretory endometrium (LSE, relative to proliferative endometrium)+intermediate DEC endometrium from ectopic pregnancy (intDEC-EP, relative to ectopic pregnancy endometrium without decidual changes) and decidual Natural Killer cells (dNK, relative to peripheral blood CD56dim NK or CD56bright NK cells); (B) endometrium with intermediate+confluent decidual changes from intrauterine pregnancy (intDEC and confDEC-IUP) and dNK.
FIGS. 16A-16C illustrate the intersection of differentially expressed genes (DEG) down-regulated in chorionic villous samples obtained from women  ̃11 gestational weeks who developed preeclampsia 5-6 months later (PE-CVS; relative to CVS from women with normal pregnancy) and in 112 DEG up-regulated in dNK, and in LSE+intDEC-EP endometrium (highlighted by dotted line). FIG. 16A illustrates the intersection of differentially expressed genes (DEG) down-regulated in chorionic villous samples obtained from women  ̃11 gestational weeks who developed preeclampsia 5-6 months later (PE-CVS; relative to CVS from women with normal pregnancy) and in 93 DEG up-regulated in dNK, and in intDEC- and confDEC-IUP (p<0.0001). Seventy-four of these 112 and 93 DEG are in common (p<0.0001, Table 17). In FIGS. 16A and 16B (yellow shading), 16 DEG up-regulated in dNK are down-regulated in PE-CVS (p<0.0001). The gene symbols of these 16 DEG are listed in panel of FIG. 16C.
FIG. 17 is an illustration of the five-stages of preeclampsia. Based on a systems biology approach, the findings of Example 2 described herein raised the possibility that impaired endometrial maturation and deficient decidual NK cell number and/or function in the secretory phase (pre-decidualization) and during early pregnancy (decidualization) precede the development of preeclampsia. As pre-decidualization, decidualization and associated decidual NK cell function are emerging as important players in the regulation of trophoblast invasion, and hence, spiral artery remodeling, perturbation of these biological processes may contribute to the etiology of preeclampsia.



BRIEF DESCRIPTION OF THE TABLES
Table 1. Genes differentially expressed in first trimester PE: Fold change data for CVS microarray.
Tables 2-6. Genes expressed in decidualization.
Table 7. DEG up-regulated in PE-CVS compared to NP-CVS.
Table 8. DEG down-regulated in PE-CVS compared to NP-CVS.
Table 9. Enriched Biological processes for DEG down-regulated in PE-CVS compared to NP-CVS (DAVID Bioinformatics Resources 6.7).
Table 10. Overlap of DEG down-regulated in PE-CVS and up-regulated in LSE.
Table 11. Overlap of DEG down-regulated in PE-CVS and up-regulated in intDEC-EP.
Table 12. Overlap of DEG down-regulated in PE-CVS and up-regulated in intDEC-EP & LSE.
Table 13. Overlap of DEG down-regulated in PE-CVS and up-regulated in intDEC-IUP.
Table 14. Overlap of DEG down-regulated in PE-CVS and up-regulated in confDEC-IUP.
Table 15. Overlap of DEG down-regulated in PE-CVS and up-regulated in intDEC-IUP and intDEC-EP.
Table 16. PE-CVS down-regulated genes linked to decidualization.
Table 17. Overlap of DEG up-regulated in dbNK and LSE plus intDEC-EP and intDEC-IUP plus confDEC-IUP (74 genes).
Table 18. Class prediction applying the k-Nearest Neighbors (kNN) algorithm for classification and the K-fold cross validation method as classifier.
DETAILED DISCLOSURE
The invention provides methods for treating and/or reducing the likelihood of development of a placental syndrome. In certain embodiments, the method for treating and/or reducing the likelihood of development of a placental syndrome comprises the steps of: (a) determining whether a woman has experienced a placental syndrome during a previous pregnancy; (b) if the woman has experienced a placental syndrome during a previous pregnancy, determining the late secretory/luteal (LS) phase of the menstrual cycle; and (c) administering a therapeutically effective amount of relaxin to the woman during the LS phase of menstruation to treat the placental syndrome.
In other embodiments, the methods for treating a placental syndrome comprise the steps of: (a) determining whether the woman is at risk for developing a placental syndrome; (b) if the woman is at risk, determining the late secretory/luteal (LS) phase of the menstrual cycle; and (c) administering natural or exogenous relaxin to the woman during the LS phase of menstruation. In a related embodiment, step (a) comprises obtaining a biological sample from the woman (such as blood, urine, uterine tissue (e.g., endometrial biopsy) or secretions) and measuring in the sample biomarker(s) that reflect endometrial maturation (pre-decidualization) or a deficiency thereof.
Abnormalities in (Pre)Decidualization and Disposition to PE
As described herein, numerous genes of putative decidual and NK cell origin being mostly downregulated in CVS from women who later developed PE (vide supra; Example 1 and Table 1) have been discovered. Some of the dysregulated genes identified as decidual in origin may also be expressed by Tr, e.g., EPAS1 or HIF-2α.
(pre)Decidualization begins in the LS phase. Abnormalities in decidual gene expression, as observed in the 1st trimester CVS of women who developed PE, may have commenced before conception. Thus, dysregulated gene expression in the LS endometrium appears to be an important factor in PE disease etiology.
According to the subject invention, supplemental hormonal support with relaxin, congener or mimetic during the LS phase, and in certain instances into early pregnancy, will improve endometrial maturation, thereby improving the uterine environment for Tr invasion and spiral artery remodeling, and thus treat and/or reduce the likelihood of development of placental syndromes, including PE.
Definitions
The terms “endogenous relaxin” or “natural relaxin” are used interchangeably herein and refer to the naturally occurring peptide hormone relaxin, which is well known in the art. Examples of endogenous relaxin include, but are not limited to, proteins associated with Relaxin 1 (RLN-1), Relaxin 2 (RLN-2), and Relaxin 3 (RLN-3) as well as with Relaxin/Insulin-Like Family Peptide Receptor (RXFP1) activity. In women, relaxin is produced by the corpus luteum of the ovary, the breast and, during pregnancy, also by the placenta, chorion, and decidua. Endogenous relaxin levels rise after ovulation as a result of its production by the corpus luteum and peak in the mid and late luteal phase of the menstrual cycle. If the cycle is nonconceptive, relaxin concentrations decline to undetectable. However, if the cycle is conceptive, relaxin concentrations rapidly increase and peak in the first trimester. Relaxin concentrations then begin a slow decline but remain elevated throughout gestation. The term relaxin (natural relaxin and endogenous relaxin) as used herein in reference to human subjects refers to H2 relaxin, unless otherwise specified.
The term “exogenous relaxin”, as used herein, means non-endogenous human relaxin, including intact full length human relaxin or a portion of the relaxin molecule that retains biological activity. The term “exogenous relaxin” encompasses human H1 preprorelaxin, prorelaxin, and relaxin; H2 preprorelaxin, prorelaxin, and relaxin; and H3 preprorelaxin, prorelaxin, and relaxin. The term “relaxin” further includes biologically active (also referred to herein as “pharmaceutically active”) relaxin from recombinant, synthetic or native sources as well as relaxin variants, such as amino acid sequence variants. As such, the term encompasses synthetic human relaxin and recombinant human relaxin, including synthetic H1, H2 and H3 human relaxin and recombinant H1, H2 and H3 human relaxin. The term further encompasses active agents with relaxin-like activity, such as relaxin agonists, relaxin mimetics and/or relaxin analogs and portions thereof that retain biological activity, including all agents that competitively displace bound relaxin from a relaxin receptor (e.g., LGR7 receptor, LGR8 receptor, GPCR135, GPCR142, etc.). Thus, a pharmaceutically effective relaxin agonist or mimetic is any agent with relaxin-like activity that is capable of binding to a relaxin receptor to elicit a relaxin-like response. In addition, the nucleic acid sequence of human relaxin as used herein does not necessarily have to be 100% identical to nucleic acid sequence of human relaxin (e.g., H1, H2 and/or H3) but may be at least about 40%, 50%, 60%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of human relaxin. Relaxin, as used herein, can be made by any method known to those skilled in the art. Examples of such methods are illustrated, for example, in U.S. Pat. No. 5,759,807 as well as in Bullesbach et al. (1991) The Journal of Biological Chemistry 266(17):10754-10761. Examples of relaxin molecules and analogs are illustrated, for example, in U.S. Pat. No. 5,166,191.
Naturally occurring biologically active relaxin may be derived from human, murine (i.e., rat or mouse), porcine, or other mammalian sources. Also encompassed is relaxin modified to increase in vivo half life, e.g., PEGylated relaxin (i.e., relaxin conjugated to a polyethylene glycol), modifications of amino acids in relaxin that are subject to cleavage by degrading enzymes, and the like. The term also encompasses relaxin comprising A and B chains having N- and/or C-terminal truncations. In general, in H2 relaxin, the A chain can be varied from A(1-24) to A(10-24) and the B chain from B(1-33) to B(10-22); and in H1 relaxin, the A chain can be varied from A(1-24) to A(10-24) and the B chain from B(1-32) to B(10-22). Also included within the scope of the term “relaxin” are other insertions, substitutions, or deletions of one or more amino acid residues, glycosylation variants, unglycosylated relaxin, organic and inorganic salts, covalently modified derivatives of relaxin, preprorelaxin, and prorelaxin. Also encompassed in the term is a relaxin analog having an amino acid sequence which differs from a wild-type (e.g., naturally-occurring) sequence, including, but not limited to, relaxin analogs disclosed in U.S. Pat. No. 5,811,395. Possible modifications to relaxin amino acid residues include the acetylation, formylation or similar protection of free amino groups, including the N-terminal, amidation of C-terminal groups, or the formation of esters of hydroxyl or carboxylic groups, e.g., modification of the tryptophan (Trp) residue at B2 by addition of a formyl group. The formyl group is a typical example of a readily-removable protecting group. Other possible modifications include replacement of one or more of the natural amino-acids in the B and/or A chains with a different amino acid (including the D-form of a natural amino-acid), including, but not limited to, replacement of the Met moiety at B24 with norleucine (Nle), valine (Val), alanine (Ala), glycine (Gly), serine (Ser), or homoserine (HomoSer). Other possible modifications include the deletion of a natural amino acid from the chain or the addition of one or more extra amino acids to the chain. Additional modifications include amino acid substitutions at the B/C and C/A junctions of prorelaxin, which modifications facilitate cleavage of the C chain from prorelaxin; and variant relaxin comprising a non-naturally occurring C peptide, e.g., as described in U.S. Pat. No. 5,759,807.
Also encompassed by the term “relaxin” are fusion polypeptides comprising relaxin and a heterologous polypeptide. A heterologous polypeptide (e.g., a non-relaxin polypeptide) fusion partner may be C-terminal or N-terminal to the relaxin portion of the fusion protein. Heterologous polypeptides include immunologically detectable polypeptides (e.g., “epitope tags”); polypeptides capable of generating a detectable signal (e.g., green fluorescent protein, enzymes such as alkaline phosphatase, and others known in the art); therapeutic polypeptides, including, but not limited to, cytokines, chemokines, and growth factors. All such variations or alterations in the structure of the relaxin molecule resulting in variants are included within the scope of this invention so long as the functional (biological) activity of the relaxin is maintained. Preferably, any modification of relaxin amino acid sequence or structure is one that does not increase its immunogenicity in the individual being treated with the relaxin variant. Those variants of relaxin having the described functional activity can be readily identified using in vitro and in vivo assays known in the art.
In some embodiments, the subject invention provides methods comprising administration of a relaxin agonist. In some methods, the relaxin agonist activates one or more relaxin-related G-protein coupled receptors (GPCR) examples of which include, but are not limited to, RXFP1, RXFP2, RXFP3, RXFP4, FSHR (LGR1), LHCGR (LGR2), TSHR (LGR3), LGR4, LGR5, LGR6LGR7 (RXFP1) and LGR8 (RXFP2). In some embodiments, the relaxin agonist comprises the amino acid sequence of Formula I of WO 2009/007848 of Compugen (herein incorporated by reference for the teaching of relaxin agonist sequences). Exemplary relaxin agonists are also disclosed in international application PCT/US2009/044251 of Corthera, which is hereby incorporated by reference for the teaching of relaxin agonist sequences of SEQ ID NOS:4-8. Contemplated relaxin agonists also include those disclosed by Xiao et al., “Identification and optimization of small-molecule agonists of the human relaxin hormone receptor RXFP1,” Nat Commun, 4:1953 (2013).
The present disclosure also encompasses homologues of Formula I polypeptides, such homologues can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 85%, at least 90%, at least 95% or more say 100% identical to the amino acid sequence of an exemplary relaxin agonist (e.g., SEQ ID NO:5 or SEQ ID NO:6 of PCT/US2009/044251 of Corthera), as can be determined using BlastP software of the National Center of Biotechnology Information (NCBI) using default parameters, optionally and preferably including the following: filtering on (this option filters repetitive or low-complexity sequences from the query using the Seg (protein) program), scoring matrix is BLOSUM62 for proteins, word size is 3, E value is 10, gap costs are 11, 1 (initialization and (initialization and extension). Optionally and preferably, nucleic acid sequence identity/homology is determined with BlastN software of the National Center of Biotechnology Information (NCBI) using default parameters, which preferably include using the DUST filter program, and also preferably include having an E value of 10, filtering low complexity sequences and a word size of 11. Finally the present disclosure also encompasses fragments of the above described polypeptides and polypeptides having mutations, such as deletions, insertions or substitutions of one or more amino acids, either naturally occurring or artificially induced, either randomly or in a targeted fashion.
The term “late secretory/luteal (LS) phase” of the menstrual cycle refers to the phase that commences several days following ovulation (where an egg is released from the egg follicle on the ovary). This will depend upon the woman's cycle; in general the LS phase can commence at around 4 to 8 days following ovulation, preferably at around 5 to 7 days following ovulation, and more preferably at about 6 days following ovulation. This phase terminates if the egg is fertilized by sperm and then implants in the endometrium, following which pregnancy begins, or if the egg is not fertilized or does not implant and the endometrium begins to break down. The term “pregnancy” refers to the nine months (40 weeks from the last menstrual period) of pregnancy which is traditionally divided into three trimesters, i.e., distinct periods of roughly three months in which different phases of fetal development take place. The first trimester is a time of basic cell differentiation. It is believed to end at the mother's first perception of fetal movement (quickening), which usually occurs around the end of the third month (or about 12 to about 14 weeks of gestational age). The second trimester is a period of rapid growth and maturation of body systems (about 15 to about 28 weeks of gestational age). A second-trimester fetus born prematurely may be viable, depending on the hospital care. The third trimester marks the final stage of fetal growth, in which systems are completed, fat accumulates under the fetus' skin, and the fetus moves into position for birth (about 29 to about 42 weeks of gestational age). This trimester ends with the birth itself.
The term “placental syndromes” includes pre-eclampsia, intra-uterine growth restriction, pre-term labor, pre-term birth, impaired decidualization, recurrent spontaneous abortions, and/or compromised endometrial maturation.
The term “about” when used in the context of a stated value, encompasses a range of up to 10% above or below the stated value (e.g., 90-110% of the stated value). For instance, an intravenous (IV) infusion rate of about 30 mcg/kg/day, encompasses IV infusion rates of 27 mcg/kg/day to 33 mcg/kg/day.
“Therapeutically effective” refers to the amount of pharmaceutically active relaxin that will result in a measurable desired medical or clinical benefit to a patient, as compared to the patient's baseline status or to the status of an untreated or placebo-treated (e.g., not treated with relaxin) subject.
The term “nucleic acid” can be understood to mean, according to the present invention, either a double-stranded DNA, a single-stranded DNA or products of transcription of the said DNAs (e.g., RNA molecules).
The term “preeclampsia” as used herein refers to a condition that occurs during pregnancy, diagnosed by the new onset of high blood pressure accompanied by the presence of proteins in the urine and may include edema (swelling). Preeclampsia, sometimes called toxemia of pregnancy, is related to a more serious disorder called “eclampsia”, which is preeclampsia together with seizure. These conditions usually develop during the second half of pregnancy (after 20 weeks), though they may develop shortly after birth (postpartum) or before 20 weeks of pregnancy.
The term “primer extension reaction” as used herein refers to any polymerization process mediated by the action of a nucleotide polymerase, e.g., a DNA polymerase, by extending a predetermined polynucleotide sequence that is at least partially complementary to a template sequence under appropriate conditions.
Probe set ID Nos: 207016_s_at; 231040_at; 236514_at; 242868_at; 1568736_s_at; 202363_at; 204041_at; 210164_at; 205291_at; 205495_s_at; 231798_at; 217143_s_at; 227238_at; 206785_s_at; 205992_s_at; 209763_at; 205445_at; 229839_at; 223786_at; 227816_at; 204741_at; 235019_at; 205493_s_at; 205067_at; 204580_at; 205870_at; 230748_at; 231818_x_at; 214702_at; 202768_at; 203592_s_at; 206067_s_at; 205302_at; 215388_s_at; 217767_at; 217552_x_at; 205654_at; 229902_at; 208084_at; 206638_at; 202917_s_at; 221286_s_at; 206859_s_at; 230848_s_at; 219759_at as set forth in Table 1 on the AFFYMETRIX GeneChip system (Affymetrix, Santa Clara, Calif.; HG-U133 Plus 2.0 GeneChips containing 53,613 probe sets), as used herein, refer to nucleic acid sequences found on the aforementioned AFFYMETRIX GeneChip system. The polynucleotide sequences are identified by database accession numbers (e.g., NM_006433.2, etc.) in Table 1 and each of the accession numbers are hereby incorporated by reference in their entireties.
“Standard control” or “control sample” as used herein refers to a sample suitable for use in a method of the present invention, e.g., in order for quantitatively determining the amount of a nucleic acid. Such a sample contains a known amount of the nucleic acid that closely reflects the average level of the nucleic acid in an average non-preeclamptic woman without symptoms of placental syndromes. In certain embodiments, a “standard control” may be derived from an average healthy non-pregnant woman.
“An increase and decrease in the amount of the nucleic acid or polypeptide species in the test sample as compared to the standard control” refers to a positive or negative change in amount from the standard control. An increase is preferably at least 2.00 fold, 2.25 fold, 2.50 fold, 2.75 fold, 3.00 fold, 3.25 fold, 3.5 fold, 3.75 fold, 4.00 fold, 4.25 fold, 4.50 fold, 4.75 fold, of 5.00 fold. Similarly, a decrease is at least 2.00 fold, 2.25 fold, 2.50 fold, 2.75 fold, 3.00 fold, 3.25 fold, 3.5 fold, 3.75 fold, 4.00 fold, 4.25 fold, 4.50 fold, 4.75 fold, of 5.00 fold. For example, an increase of 2+ or greater or −2 or below would be considered significant difference from control. These expression levels (+2 or −2) can also be referred to as “overexpressed”/“overexpression” or “underexpressed”/“underexpression”.
A “polynucleotide hybridization method” as used herein refers to a method for detecting the presence and/or quantity of a polynucleotide based on its ability to form Watson-Crick base-pairing, under appropriate hybridization conditions, with a polynucleotide probe of a known sequence. Examples of such hybridization methods include Southern blotting and Northern blotting.
“PCR primers” as used herein refer to oligonucleotides that can be used in a polymerase chain reaction (PCR) to amplify a nucleotide sequence originating from a nucleic acid (RNA transcript). Some aspects of the invention provide for primers that comprise the sequences of probe set ID Nos: 207016_s_at; 231040_at; 236514_at; 242868_at; 1568736_s_at; 202363_at; 204041_at; 210164_at; 205291_at; 205495_s_at; 231798_at; 217143_s_at; 227238_at; 206785_s_at; 205992_s_at; 209763_at; 205445_at; 229839_at; 223786_at; 227816_at; 204741_at; 235019_at; 205493_s_at; 205067_at; 204580_at; 205870_at; 230748_at; 213818_x_at; 214702_at; 202768_at; 203592_s_at; 206067_s_at; 205302_at; 215388_s_at; 217767_at; 217552_x_at; 205654_at; 229902_at; 208084_at; 206638_at; 202917_s_at; 221286_s_at; 206859_s_at; 230848_s_at; 219759_at as set forth in Table 1 on the AFFYMETRIX GeneChip system (Affymetrix, Santa Clara, Calif.; HG-U133 Plus 2.0 GeneChips containing 53,613 probe sets). Various combinations of the aforementioned primers can be included in a primer kit as set forth herein.
As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Additionally, the terms “comprising”, “consisting of” and “consisting essentially of” are defined according to their standard meaning. The terms may be substituted for one another throughout the instant application in order to attach the specific meaning associated with each term. The phrases “isolated” or “biologically pure” refer to material that is substantially or essentially free from components which normally accompany the material as it is found in its native state. Thus, isolated peptides or nucleic acids, in accordance with the invention, preferably do not contain materials normally associated with the peptides in their in situ environment.
Determining Previous Placental Syndrome
Various methods are available for determining whether a woman has experienced a placental syndrome during a previous pregnancy. Such methods include, but are not limited to, determining whether the woman was previously diagnosed with PE, IUGR, preterm labor/birth and/or abnormal endometrial maturation/intrauterine growth. According to one embodiment of the subject invention, the woman's past health experience, particularly in pregnancy, would be used to determine whether the woman has experienced a placental syndrome.
Determining Risk for Developing a Placental Syndrome
In certain embodiments, the methods described herein include the step of determining whether a woman is at risk for developing a placental syndrome. In one embodiment, determining the risk for developing a placental syndrome comprises the following steps: (a) obtaining a biological sample from a woman and measuring in the sample biomarker(s) that reflect endometrial maturation (pre-decidualization) or a deficiency thereof. According to the subject invention, the biological sample can be blood, urine, uterine tissue (e.g., endometrial biopsy) or secretions, and the like. The biomarkers include proteins of relevant genes described herein.
Immunoassays can be used to detect at least one secreted protein disclosed herein, the expression levels of said at least one secreted protein, and comparison of said at least one secreted protein to a control (standard control) sample. Protein expression (secretion) can be detected by any suitable method, such as gas chromatography-mass spectrometry. In some embodiments, proteins are detected by immunoassays.
In one embodiment, the step for determining the risk for developing a placental syndrome comprises the steps of: obtaining a biological sample from a woman; quantitatively determining the amount of endogenous relaxin in the biological sample; and comparing the amount of the quantified amount of endogenous relaxin to a standard control representing the amount of the endogenous relaxin in the corresponding sample from an average non-preeclamptic woman without symptoms of placental syndromes; wherein an increase and decrease in the amount of the endogenous relaxin in the biological sample as compared to the standard control indicates an increased risk of developing placental syndromes, such as preeclampsia. In a preferred embodiment, at least one of the probe sets for RLN-1, RLN-2, RLN-3 and RXFP1 is provided and the amount of one or more nucleic acid species in the biological sample obtained from the woman that hybridizes with any one or more probe set ID is quantified and compared against a control sample.
In another embodiment, the step for determining the risk for developing a placental syndrome comprises the steps of:
obtaining a biological sample from a woman;
quantitatively determining the amount of one or more nucleic acid species or corresponding proteins or activities in the biological sample obtained from the woman that hybridizes with any one or more probe set ID Nos: 207016_s_at; 231040_at; 236514_at; 242868_at; 1568736_s_at; 202363_at; 204041_at; 210164_at; 205291_at; 205495_s_at; 231798_at; 217143_s_at; 227238_at; 206785_s_at; 205992_s_at; 209763_at; 205445_at; 229839_at; 223786_at; 227816_at; 204741_at; 235019_at; 205493_s_at; 205067_at; 204580_at; 205870_at; 230748_at; 213818_x_at; 214702_at; 202768_at; 203592_s_at; 206067_s_at; 205302_at; 215388_s_at; 217767_at; 217552_x_at; 205654_at; 229902_at; 208084_at; 206638_at; 202917_s_at; 221286_s_at; 206859_s_at; 230848_s_at; 219759_at as set forth in Table 1 on the AFFYMETRIX GeneChip system (Affymetrix, Santa Clara, Calif.; HG-U133 Plus 2.0 GeneChips containing 53,613 probe sets); and
comparing the amount of the quantified nucleic acid species to a standard control representing the amount of the nucleic acid species in the corresponding sample from an average non-preeclamptic woman without symptoms of placental syndromes; wherein an increase and decrease in the amount of the nucleic acid species in the biological sample as compared to the standard control indicates an increased risk of developing placental syndromes, such as preeclampsia. The biological sample can be blood, washing from the reproductive tract, urine, saliva, amniotic fluid, or uterine tissue (e.g., endometrial biopsy or chorionic villus). One aspect of the invention provides for increased expression of nucleic acids that hybridize with 205827_at; 215141_at; 202917_s_at; 215733_x_at; 234601_x_at; and decreased expression of nucleic acids that hybridize with 227238_at; 239010_at; 214702_at; 1553319_at; 235592_at; 229839_at; 230748_at; 203789_s_at; 226482_s_at; 215388_s_at; 1562053_at; 219911_s_at; 209351_at; 1552858_at; 215108_x_at; 226403_at; 207607_at; 228293_at; 210251_s_at; 1561318_at; 241036_at; 219759_at; 203592_s_at; 205302_at; 1568554_x_at; 1554276_at; 242842_at; 242868_at; 206859_s_at; 204580_at; 221286_s_at; 206859_s_at; 230848_s_at; 219759_at; and 207509_s_at.
In some embodiments, the first step can comprise the use of a reverse transcriptase polymerase chain reaction (RT-PCR). In other embodiments, the first step comprises using a polynucleotide hybridization method, or using a primer extension reaction.
Various other embodiments provide a kit for diagnosing whether a woman will have a propensity for developing a placental syndrome in a non-pregnant woman. This kit comprises the following: (i) PCR primers for quantitatively determining the amount of one or more nucleic acid species in a biological sample obtained from the non-pregnant woman, wherein the nucleic acid species hybridize with probe set ID Nos: 207016_s_at; 231040_at; 236514_at; 242868_at; 1568736_s_at; 202363_at; 204041_at; 210164_at; 205291_at; 205495_s_at; 231798_at; 217143_s_at; 227238_at; 206785_s_at; 205992_s_at; 209763_at; 205445_at; 229839_at; 223786_at; 227816_at; 204741_at; 235019_at; 205493_s_at; 205067_at; 204580_at; 205870_at; 230748_at; 213818_x_at; 214702_at; 202768_at; 203592_s_at; 206067_s_at; 205302_at; 215388_s_at; 217767_at; 217552_x_at; 205654_at; 229902_at; 208084_at; 206638_at; 202917_s_at; 221286_s_at; 206859_s_at; 230848_s_at; 219759_at as set forth in Table 1 on the AFFYMETRIX GeneChip system (Affymetrix, Santa Clara, Calif.; HG-U133 Plus 2.0 GeneChips containing 53,613 probe sets) and (ii) a standard control representing the amount of the nucleic acid species in the corresponding sample from an average non-preeclamptic woman without symptoms of placental syndromes.
As discussed above, immunoassays can be used to detect at least one secreted protein disclosed herein, the expression levels of said at least one secreted protein, and comparison of said at least one secreted protein to a control (standard control) sample. Protein expression (secretion) can be detected by any suitable method, such as gas chromatography-mass spectrometry. In some embodiments, proteins are detected by immunoassays.
Antibody binding is detected by techniques known in the art (e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), “sandwich” immunoassays, immunoradiometric assays or Western blots. In some embodiments, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, the secondary antibody is labeled. Many methods are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. In aspects of this invention, an automated detection assay is utilized. Methods for the automation of immunoassays include those described in U.S. Pat. Nos. 5,885,530, 4,981,785, 6,159,750, and 5,358,691, each of which is herein incorporated by reference. In some embodiments, the analysis and presentation of results is also automated. In other embodiments, the immunoassay described in U.S. Pat. Nos. 5,599,677 and 5,672,480; each of which is herein incorporated by reference.
In various aspects of the invention, the woman being examined is examined prior to pregnancy.
Determining Late Secretory/Luteal (LS) Phase of the Menstrual Cycle
The luteinizing hormone (LH) is always present in urine and increases 24-48 hours prior to ovulation. The LH surge triggers ovulation, which is the release of an egg from one of a woman's ovaries. The LS phase commences following ovulation and terminates either with implantation of an inseminated egg into the endometrium or when the endometrium commences breaking down. In certain embodiments of the invention, determination of the LS phase of the menstrual cycle is performed using a kit that is able to detect the LH surge. Such kits are readily available and have been disclosed in U.S. Pat. Nos. 3,991,174; 4,208,187; 6,234,974; and 6,451,619, all of which are incorporated by reference in their entirety.
Relaxin Compositions and Formulations
Relaxin, relaxin agonists, relaxin mimetics and/or relaxin analogs can be formulated as pharmaceuticals to be used in the methods of the invention. Any composition or compound that can stimulate a biological response associated with the binding of biologically or pharmaceutically active relaxin (e.g., synthetic relaxin, recombinant relaxin) or a relaxin agonist (e.g., relaxin analog or relaxin-like modulator or relaxin mimetic) to relaxin receptors can be used as a pharmaceutical in the disclosure. General details on techniques for formulation and administration are well described in the scientific literature (see Remington's Pharmaceutical Sciences, Maack Publishing Co, Easton Pa.). Pharmaceutical formulations containing pharmaceutically active relaxin can be prepared according to any method known in the art for the manufacture of pharmaceuticals. The formulations containing pharmaceutically active relaxin or relaxin agonists used in the methods of the invention can be formulated for administration in any conventionally acceptable way including, but not limited to subcutaneously (SQ), intramuscularly, intravenously, sublingually, topically, orally and via inhalation. Illustrative examples are set forth below. In one preferred embodiment, relaxin is administered subcutaneously (SQ).
When the drugs are delivered subcutaneously (SQ), the formulations containing pharmaceutically active relaxin or a pharmaceutically effective relaxin agonist can be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oleaginous suspension. For example, relaxin can be diluted in sodium acetate at pH 5.0 where it is very soluble and stable. Patients can be treated with a relaxin composition via continues infusion as long as necessary. For example, relaxin infusion pumps deliver relaxin through a cannula to a needle that is applied subcutaneously and the pumps can be worn on a belt under the patient's clothes. Relaxin can also be administered via timely relaxin injections while the patient is being monitored for symptoms of preeclampsia. Doses can be adjusted on a patient by patient basis. Relaxin suspensions can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation can also be a sterile injectable solution or suspension in a nontoxic parenterally-acceptable diluent or solvent. Among the acceptable vehicles and solvents that can be employed are water and Ringer's solution, an isotonic sodium chloride. In addition, sterile fixed oils can conventionally be employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can likewise be used in the preparation of injectables.
Aqueous suspensions of the disclosure contain relaxin in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethylene oxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol (e.g., polyoxyethylene sorbitol mono-oleate), or a condensation product of ethylene oxide with a partial ester derived from fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension can also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose, aspartame or saccharin. Formulations can be adjusted for osmolarity.
Oil suspensions can be formulated by suspending relaxin in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspensions can contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents can be added to provide a palatable oral preparation. These formulations can be preserved by the addition of an antioxidant such as ascorbic acid.
Dispersible powders and granules of the disclosure suitable for preparation of an aqueous suspension by the addition of water can be formulated from relaxin in admixture with a dispersing, suspending and/or wetting agent, and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those disclosed above. Additional excipients, for example sweetening, flavoring and coloring agents, can also be present.
The pharmaceutical formulations of the disclosure can also be in the form of oil-in-water emulsions. The oily phase can be a vegetable oil, such as olive oil or arachis oil, a mineral oil, such as liquid paraffin, or a mixture of these. Suitable emulsifying agents include naturally-occurring gums, such as gum acacia and gum tragacanth, naturally occurring phosphatides, such as soybean lecithin, esters or partial esters derived from fatty acids and hexitol anhydrides, such as sorbitan mono-oleate, and condensation products of these partial esters with ethylene oxide, such as polyoxyethylene sorbitan mono-oleate. The emulsion can also contain sweetening and flavoring agents. Syrups and elixirs can be formulated with sweetening agents, such as glycerol, sorbitol or sucrose. Such formulations can also contain a demulcent, a preservative, a flavoring or a coloring agent.
Administration and Dosing Regimen of Relaxin Formulations
The formulations containing pharmaceutically active H2 relaxin or a pharmaceutically effective H2 relaxin chimera, agonist, or mimetic used in the methods of the disclosure can be administered in any conventionally acceptable way including, but not limited to, subcutaneously, intramuscularly, intravenously, sublingually, topically, orally and via inhalation. Administration will vary with the pharmacokinetics and other properties of the drugs and the patients' condition of health. General guidelines are presented below.
The methods of the disclosure reduce the likelihood of the development of placental syndromes, such as preeclampsia, following insemination. The amount of relaxin alone or in combination with another agent or drug that is adequate to accomplish this is considered the therapeutically effective dose. The dosage schedule and amounts effective for this use, i.e., the “dosing regimen,” will depend upon a variety of factors, including the general state of the patient's health, the patient's physical status, the type of pregnancy (e.g., single vs. multiple pregnancy) age, and the like.
In calculating the dosage regimen for a patient, the mode of administration is also taken into consideration. The dosage regimen must also take into consideration the pharmacokinetics, i.e., the rate of absorption, bioavailability, metabolism, clearance, and the like. Based on those principles, relaxin can be used to reduce or prevent development of preeclampsia in pregnant women. The subject invention also provides relaxin or a relaxin agonist or mimetic and, optionally, another drug for simultaneous, separate or sequential administration. For example, the disclosure provides relaxin and, optionally, a hypertensive medication for combined use in therapy if needed. In another example, the disclosure further provides relaxin and, optionally, MgSO4 for seizure prophylaxis in combined therapy.
The subject invention also provides the use of relaxin in the manufacture of a medicament for reducing or preventing the development of placental syndromes, such as preeclampsia or IUGR, in pregnant women. As such, the medicament is prepared for administration during the LS phase of the menstrual cycle. The subject invention further provides relaxin or a relaxin analog or mimetic for use in a method of reducing the likelihood of the development of placental syndromes, such as preeclampsia, wherein relaxin is prepared for administration to non-pregnant women during the LS phase of the menstrual cycle.
The state of the art allows the clinician to determine the dosage regimen of relaxin for each individual woman. As an illustrative example, the guidelines provided below for relaxin can be used as guidance to determine the dosage regimen, i.e., dose schedule and dosage levels, of formulations containing pharmaceutically active relaxin administered when practicing the methods of the invention. As a general guideline, it is expected that the daily dose of pharmaceutically active H1, H2 and/or H3 human relaxin (e.g., synthetic, recombinant, analog, agonist, mimetic, etc.) is typically in an amount in a range of about 0.1 to about 100 μg/kg of subject body weight per day. In one preferred embodiment, the dosage of relaxin is 0.1 to 30 μg/kg/day throughout the LS phase of the menstrual cycle. More preferably, the daily dose of pharmaceutically active H1, H2 and/or H3 human relaxin (e.g., synthetic, recombinant, analog, agonist, mimetic, etc.) is administered to a woman to result in serum concentrations of relaxin of about 0.1-10.0 ng/ml during the LS phase of the menstrual cycle. In another embodiment, these dosages result, for example, in serum concentrations of relaxin of about 0.1-3.0 ng/ml during the LS phase of the menstrual cycle.
In one preferred embodiment, pharmaceutically effective relaxin or an agonist thereof is administered at about 0.1 to 30 μg/kg/day throughout the LS phase of the menstrual cycle and through the 1st, 2nd or 3rd trimester of pregnancy. In another preferred embodiment, pharmaceutically effective relaxin or an agonist thereof is administered at about 0.1 to about 30 μg/kg/day throughout the LS phase of the menstrual cycle and through the 1st trimester of pregnancy. In another embodiment, the administration of relaxin is continued as to maintain a serum concentration of relaxin of from about 0.5 ng/mL to about 20 ng/ml, more preferably from about 0.5 to about 15 ng/ml, and most preferably from about 1 to about 10 ng/ml. Most preferably, the administration of relaxin is continued as to maintain a serum concentration of relaxin of 10 ng/ml or greater throughout pregnancy following administration during the LS phase of the menstrual cycle.
These relaxin concentrations can reduce the likelihood of the development of placental syndromes, including preeclampsia. In addition, these relaxin concentrations may treat and/or prevent symptoms in the mother that are associated with preeclampsia such as hypertension, high blood pressure, proteinuria, renal insufficiency and mortality Furthermore, these relaxin concentrations can reduce or prevent the likelihood of low birth weight in infants and associated risks as well as infant deaths. Depending on the subject, the relaxin administration is maintained for as specific period of time or for as long as needed to achieve the therapeutic efficacy described herein. For example, relaxin can be administered through continuous infusion through the LS phase. This can be achieved via an infusion pump or other means. Alternatively, relaxin can be administered through the LS phase and during the first and/or second trimester only if needed.
In certain embodiments, the method of the subject invention can be used to (1) correct deficient endometrial maturation (“(pre)decidualization”), thereby improving histiotrophic nutrition of the placenta and fetus before 10 weeks; (2) promote trophoblast invasion (see FIGS. 7 and 8) and physiological remodeling of uterine spiral arteries, and hence, fetoplacental blood flow after 10 weeks; and (3) directly stimulate survival and invasion of trophoblast (see FIGS. 9 and 10), and thus, improve placentation and placental function; and augment maternal circulatory adaptations to pregnancy, which are deficient in women destined to develop severe preeclampsia or intrauterine growth restriction, particularly in the first trimester. The treatments described herein promote proper endometrial maturation prior to conception, which is beneficial in obviating PE development, as well as intrauterine growth restriction (IUGR) during pregnancy.
EXAMPLES
Identification of Differentially Expressed Genes
Materials and Methods:  ̃160 surplus chorionic villus sampling (CVS) over a 4-5 year period were collected. CVS is typically performed in women of advanced maternal age at 10-12 weeks of gestation to check for fetal chromosomal abnormalities. CVS also contains maternal decidual tissue. Four of the  ̃160 women developed PE  ̃6 months later and they were matched to 8 women with normal pregnancy (NP) outcome. These CVS tissues were subjected to DNA microarray, and it was discovered that 168 genes differentially expressed by J5 and/or fold change (FC) analyses in CVS between women who developed PE and those who experienced NP outcome (see FIG. 2 and Application Publication No. 20110171650, which is incorporated by reference in its entirety, including all figures, tables and amino acid or nucleic acid sequences). There was no evidence for upregulation of hypoxia or oxidative stress regulated genes at this early stage of pregnancy as observed in 3rd trimester PE placentas, suggesting these changes to be later events in the disease (see FIG. 3). Rather, there was dysregulation of several genes related to (pre)decidualization indicating that endometrial maturation, which occurs before (pre-decidualization) and after conception (decidualization) was impaired.
These data were reanalyzed specifically in the context of (pre)decidualization, and it was found that as many as 45 of the 168 dysregulated genes in CVS of women who developed PE  ̃6 months later are associated with this biological process (See Tables 1-6). Because decidual genes and pathways do not exist in commercially available bioinformatics software, the “Decidual Gene Set” consisting of 658 genes from Duncan W C et al. (“Ectopic pregnancy as a model to identify endometrial genes and signaling pathways important in decidualization and regulated by local trophoblast,” PLOS One. 6(8): e23595 (2011)) was used (see FIG. 4). Twenty-four of the 168 dysregulated genes in CVS from women who developed PE overlap with this Decidual Gene Set. The probability of such extensive overlap occurring by chance is p<10−20 (1-sided binomial exact test). Moreover, 20 of the 24 genes changed in a direction consistent with deficient decidualization in PE (i.e., opposite in direction to that observed for the same genes in the Decidualization Gene Set, p<0.0008).
It was discovered that an additional 21 of the 168 dysregulated genes in the CVS study (not represented in the “Decidual Gene Set” of Duncan et al.) were also previously identified in the literature to be associated with (pre)decidualization bringing the total number to 45 genes (Table 1; FIG. 5). Once again, it is highly significant (p<0.00001) that expression of 38 (highlighted in bold and italics in Table 1) of these 45 genes changed in a direction opposite to that observed for the biological process of (pre)decidualization. Noteworthy is the marked downregulation of genes classically associated with the decidual response such as prolactin −7.86, glycodelin −15.64 and IGFBP1 −10.35 in the CVS of women who developed PE compared to those with NP outcome (suggesting relative impairment of (pre)decidualization in the former). Ingenuity Pathway Analysis further revealed “Lack of endometrial Natural Killer (NK) Cells” (p=0.00092) and deficient “Activation of NK Cells” (p=0.00096) consistent with the downregulation of genes shown in Table 1 that are associated with NK cells (but not necessarily exclusively so) including granulysin, granzyme B, IL2RB, IL-15, IKZF1, and KLRC2. Indeed, uterine (u)NK cells comprise a large percentage of leukocytes in the (pre)decidualized endometrium. Taken together, these results from early pregnancy placentas suggest “decidualopathy” as part of the etiology of PE.
By extension, the results suggest that the antecedents of PE may actually precede conception residing in the LS endometrium. That is, dysregulation of endometrial gene expression may begin in the LS phase compromising endometrial maturation before conception (pre-decidualization), which persists and perhaps worsens after conception (decidualization), thereby disposing to impaired Tr invasion, inadequate placentation and PE (see FIG. 1). As the nomenclature implies, pre-decidualization and decidualization are a biological continuum. Uterine spiral arteries undergo morphological changes associated with the decidual response. Decidualization of spiral arteries may be permissive or play a direct role in promoting Tr invasion during early pregnancy (see FIG. 1). On this basis, inadequate (pre)decidualization may preclude optimal Tr invasion and spiral artery remodeling, thereby restricting blood flow to the placenta and fetus in women who develop PE.
To further support the findings herein, reduced glycodelin expression persists in the decidua of delivered placentas from women with PE, and serum IGFBP1 is decreased in women before developing PE. Moreover, uterine Natural Killer (uNK) cells normally increase in number during the LS phase associated with pre-decidualization, rising even further during the 1st trimester. Besides immune modulation, these cells play key roles in Tr lineage decisions, directing Tr invasion and mediating angiogenesis in the decidua.
Underscoring the importance of uNK and Tr cell interaction, certain polymorphic combinations of killer-cell immunoglobulin-like receptors (KIRs) and Tr HLA-C molecules increase the risk of PE. Thus, inadequate uNK cell number or maturation in the (pre)decidua as suggested in CVS from women who developed PE may play a causal role in the disease. Impaired (pre)decidualization could deprive the maternal-fetal interface of critical immune cells including uNK cells, cytokines and growth factors crucial for optimal Tr invasion and spiral artery remodeling that, in turn, are important for NP outcome (see FIG. 1).
The incidence of PE in women with endometriosis-associated infertility has been investigated. Unexpectedly, it was found that this condition significantly lowered the risk of PE compared to a control group (case group—0.8% vs. control group—5.8%, P=0.002). This finding establishes a precedent in which a pathological condition affecting the endometrium before pregnancy bears a significant relationship with the development of PE. More recently, data has been reported supporting aberrant pre-decidualization as a potential mechanism for recurrent pregnancy loss, again underscoring an important role for optimal endometrial maturation in NP outcome.
Prospective Study to Characterize LS Endometrium in Women Who Experienced Severe PE During Pregnancy
Women who experienced severe PE will be enrolled before hospital discharge, using established diagnostic criteria of severe PE (n=8). Women matched for maternal age, race, ethnicity and parity with NP outcomes will likewise be recruited (n=8). After discontinuation of breastfeeding and resumption of normal menstrual cycles, the subject will be instructed to refrain from intercourse or use barrier contraception after a menstrual period. She will report to the clinic during the LS phase, day 10-12 post-LH surge, as documented by the Ovulation Predictor kit provided. Serum hCG will be analyzed; and if negative, endometrial biopsy (EnBx) will be obtained. Tissue will be processed for histology, flow cytometry, RNA, DNA and protein. Specimen quality and histology will be evaluated. Endometrial gene expression will be assessed by DNA microarray and selected genes validated using real time PCR. Endometrial proteins will be identified by iTRAQ and mass spectrometry. uNK cell number and maturation will be assessed by flow cytometry.
Post-partum patients in Labor and Delivery and on the Obstetrics Floor who experienced severe PE or NP and who matched for maternal age, race and ethnicity will be approached. If the patient is interested in the study, then Study Coordinator will contact the subject to administer informed consent explaining the study, and provide the subject with instructions on how to complete the study. The Study Coordinator will schedule the EnBx and supervise collection and processing of endometrial tissue, blood and urine (see FIG. 6).
By restricting enrollment to severe PE without preexisting medical complications, disease heterogeneity should decrease, thereby increasing the likelihood of finding significant differences between the PE and NP cohorts.
Endometrial biopsy: EnBx will be obtained using a Pipelle biopsy catheter. This causes discomfort to the subject for  ̃10 seconds, but is safe and complications are rare. Because the subjects will not be actively bleeding, the expectation is to obtain mostly tissue and not blood or clots. The usual sample size is 0.25-0.5 g, sufficient tissue to perform the analyses; however, if any given sample is inadequate, then gene and protein expression will be analyzed first.
Blood and urine collection: Spot urine and blood samples for future study of biomarkers (as informed by EnBx study results) will be obtained immediately before EnBx.
DNA microarray: RNA will be extracted from endometrial tissues using TRI Reagent, and RNA quantification and integrity determined by Nanodrop and Agilent Bioanalyzer, respectively. cDNA will be synthesized from total RNA, and the cDNA will be used as a template for in vitro transcription (IVT). The antisense RNA synthesized during the IVT reaction will be used to generate sense DNA. Sense strand DNA will be fragmented, biotin labeled, and hybridized with rotation at 45° C. for 16 hours to the Affymetrix GeneChip® Human Gene 1.0 ST array. The arrays are washed and stained with reagents supplied in the GeneChip® Hybridization Wash and Stain kit on an Affymetrix Fluidics Station 450, and scanned with a GeneChip® 7G Scanner. Bioinformatics will be accomplished using Database for Annotation, Visualization and Integrated Discovery; and Kyoto Encyclopedia of Genes and Genome. Pathway Studio will be used to identify, build and visualize pathways that differ between subject groups. Published “Decidual Gene Sets and Pathways” will also be queried.
iTRAQ and Mass Spectrometry: For quantitative proteomics, a gel-free approach will be used with iTRAQTM reagents from Applied Biosystems [38] and LC/MS/MS using the ThermoFinnigan LTQ-orbitrap that is available in the ICBR proteomics core. The experiment will be repeated three times in order to obtain data that can be evaluated statistically. To calculate a false discovery rate (FDR) for peptide-protein assignments, Proteomics System Performance Evaluation Pipeline (ProteomicS PEP, Applied Biosystems) in Protein PilotTM will be used to create a reversed human decoy database to search.
Flow cytometry: Endometrial NK cell subsets will be analyzed using a BD LSRFortessa cell analyzer. Based on the gene array data, both CD56bright and CD56dim are expected to be diminished in frequency with a greater loss likely seen in the important pCD56bright CD16dim population. The analysis of cell subsets in small samples is ideally performed using FACS approaches and has been performed previously to quantitate and clone CD1d-restricted iNKT cells from human placenta.
Study to Determine if Defective Endometrial Maturation May Lead to Development of Preeclampsia
The following describes a unique discovery based approach to study the etiology of PE. Surplus 1st trimester placental villi were rapidly dissected and snap frozen within 5 minutes of extraction from women undergoing chorionic villous sampling (CVS) for prenatal genetic screening. Genome-wide gene expression profiling was utilized to study the placental (CVS) transcriptome of women who developed PE 5-6 months later or experienced a normal pregnancy. Unexpectedly, several characteristic molecular markers for decidualization (DEC) of the maternal endometrium, namely prolactin, insulin-like growth factor-binding protein 1 and glycodelin, were strongly down-regulated in CVS from PE relative to NP women (−7.7, −10.4 and −15.6 fold change, respectively).
The decidualized tissue in these isolated villi from CVS at least partly derives from placental septae projecting upward from the basal to the chorionic plate, which contains an admixture of decidual and immune cells, and Tr. DEC is a process of endometrial maturation that begins in the secretory phase of the menstrual cycle (preDEC) continuing after conception. An important part of this biological process is the appearance of decidual Natural Killer (dNK) cells in the secretory endometrium eventually comprising 30-40% of cells in the stromal compartment or 70-80% of all leukocytes in early pregnancy (32, 33). The origin of dNK cells is unclear, but they may arise from peripheral blood (pb) CD56bright NK (34-36) or CD56dim NK cells. In essence, preDEC and DEC are a biological continuum in preparation of the “soil” for the “seed” (conceptus).
Optimal endometrial maturation is emerging as an important precursor of successful pregnancy outcome. The following study findings obtained from CVS in the 1st trimester of women remote from disease onset provide prospective evidence that one possible etiology of PE is deficient endometrial maturation.
Methods:
Publically available microarray datasets were analyzed in order to determine differentially expressed genes (DEG), which increase expression in late secretory endometrium (also known as pre-decidualization or endometrial maturation before implantation), during decidualization (endometrial maturation after implantation), the latter in the presence or absence of local trophoblast. In addition, DEG up-regulated in decidual relative to peripheral blood NK cells were analyzed using other microarray datasets. These up-regulated DEG were then compared to DEG down-regulated in chorionic villous samples (CVS) obtained at  ̃11 gestational weeks from 4 women who developed preeclampsia 5-6 months later matched to 8 women with normal pregnancy. This overall approach was chosen to support the provision that genes, which expression is increased during the process of normal endometrial maturation before and after implantation, will be decreased in the endometrium of women destined to develop preeclampsia.
Microarray datasets: Microarray dataset searches were performed in two public functional genomics data repositories: Gene Expression Omnibus (GEO) from the National Center for Biotechnology Information (NCBI) and the European Bioinformatics Institute from the European Molecular Biology Laboratory (EMBL-EBI). Both data repositories support MIAME-compliant data submissions.
One pre-condition for microarray dataset searches was the selection of microarray data in which RNA was hybridized to the Affymetrix Human Genome U133 Plus 2.0 Array (GPL570 for GEO; A-AFFY-44 for EMBL-EBI), the same platform used for the interrogation of CVS from PE and NP women (GSE12767). This pre-condition is necessary because it enables the direct comparison of the microarray data in the present work.
To search for preDEC data, the keywords entered were “endometrium” AND “menstrual cycle” AND GPL570 (or A-AFFY-44). Two datasets were selected from the GEO database: GSE4888 (Talbi S et al. (2006) Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women. Endocrinology 147:1097-1121) and GSE6364 (Burney R O et al. (2007) Gene expression analysis of endometrium reveals progesterone resistance and candidate susceptibility genes in women with endometriosis. Endocrinology 148:3814-3826). The dataset GSE4888 consisted of 27 samples obtained from women with normal ovulatory cycles. Twenty-one had histologic phenotypes of proliferative (PrE; n=4), early secretory (ESE; n=3), mid-secretory (MSE; n=8) or late secretory (LSE; n=6) endometrium, while 6 had ambiguous histological reading. The dataset GSE6364 consisted of 37 endometrial biopsies obtained from women without pathology (n=16) or diagnosed with some degree of endometriosis (n=21). Biopsy samples of the former were from PrE (n=5), ESE (n=3), and MSE (n=8). The 21 and 16 normal endometrial samples from GSE4888 and GSE6364, respectively, were pooled. The LSE phase was only represented by the 6 samples from GSE4888. To maintain equal number of replicates per stage of the endometrial cycle, 3 samples were randomly selected from each dataset for PrE, ESE and MSE. Thus, each of the 4 menstrual cycle phases was comprised of 6 endometrial samples (n=24 total).
To search for DEC data, the keywords employed were “endometrium” AND “decidualization” AND GPL570 (or A-AFFY-44). One dataset was selected from EMBL-EBI database: E-MTAB-680 (Duncan W C et al. (2011) Ectopic pregnancy as a model to identify endometrial genes and signaling pathways important in decidualization and regulated by local trophoblast. PLoS One 6:e23595). This dataset consisted of 24 endometrial samples collected at approximately 59 days of gestation. Of these, 13 were obtained from intrauterine pregnancies (IUP) and 11 from ectopic tubal pregnancies (EP). As reported by the authors, these samples presented different degrees of decidualization as assessed by morphology in H&E stained sections. The IUP samples were classified as confluent DEC (confDEC-IUP, n=7) or intermediate DEC (intDEC-IUP, n=6), while the EP samples were intermediate DEC (intDEC-EP, n=6) or without DEC changes (nonDEC, n=5). The presence or absence of trophoblast was determined by cytokeratin staining.
The keyword employed to evaluate the trophoblast influence on the decidualization process were “trophoblast” AND “decidualization” AND “endometrium” AND GPL570 (or A-AFFY-44). One dataset met the search criteria GSE5809 (GEO database) or E-GEOD-5809 (EMBL-EBI database) (Hess A P et al. (2007) Decidual stromal cell response to paracrine signals from the trophoblast: amplification of immune and angiogenic modulators. Biol Reprod 76:102-117). Human endometrial stromal cells were decidualized in culture or left untreated serving as a control. The DEC and nonDEC cells were then incubated with conditioned media from human trophoblast (TrCM) for 0 (n=3), 3 (n=6) and 12 (n=5) hours. Cytotrophoblasts were isolated from placentae obtained after elective pregnancy termination (6-22 gestational weeks), and they were cultured on Matrigel-coated matrix for 48 hours before harvesting of the conditioned media.
To approximate DEG up-regulated in NK cells during the preDEC or DEC process, a comparison between gene expression of decidual (d)NK or endometrial (e)NK cells and peripheral blood (pb)NK cells was conducted. To this end, Koopman et al. generously provided the microarray datasets performed on dNK cells (n=9) and pbNK cells (n=10) cells (Koopman L A et al. (2003) Human decidual natural killer cells are a unique NK cell subset with immunomodulatory potential. J Exp Med 198:1201-1212). In this study, decidual samples were collected from pregnant woman between 6 to 12 weeks of gestation after elective termination, and dNK cells were isolated by fluorescence-activated cell sorting. The same technique was employed to isolate pbNK cells from peripheral blood mononuclear cells of healthy donors (n=5 CD56bright pbNK and n=5 CD56dim pbNK). The isolated RNA was amplified, labeled and hybridized to the Affymetrix Human Genome U95 Version 2 Array (GPL8300). This platform is not the same as the others employed for the datasets described above. However, results from the analysis of this dataset are comparable, insofar as data imputation, normalization and transformation are the same.
Data Analysis:
Data input: Bioconductor software for the R software environment was employed for all the analyses. The gcRMA package was employed to import the raw data into R, perform background correction, as well as normalize and summarize the data. Then, rows of each data set were collapsed, in order to retain the microarray probe with the highest mean value (Max mean) from the group of the genes with the same official symbol. The function applied was the “collapseRows” from the WGCNA package (Langfelder P, Horvath S (2008) WGCNA: an R package for weighted correlation network analysis. BMC Bioinformatics 9:559). The purpose of row collapsing is to obtain unique identifiers for each gene in the working data set. Thus, from the original platform GPL570, containing 54675 probes, 21049 probes belonging to unique genes were retained for further analysis. For the platform GPL8300 employed in the NK cell dataset, 9127 probes related to unique genes were retained from 12625 probes.
Statistical analysis—LSE (or preDEC): data from biopsy samples in GSE4888 and GSE6364 (n=24) were analyzed using time as an ordinal variable. The Bayesian Estimation of Temporal Regulation (BETR) algorithm (Aryee M J et al. (2009) An improved empirical bayes approach to estimating differential gene expression in microarray time-course data: BETR (Bayesian Estimation of Temporal Regulation). BMC Bioinformatics 10:409) was used to identify the DEG at a False Discovery Rate (FDR) of <0.05. The first phase of the endometrial cycle (PrE) was considered as the baseline measurement and was compared to subsequent stages of the endometrial cycle, in order to correlate the differential expression among the various stages. This method, which is applied with the BETR package, provides the probabilities of differential expression for each gene in the data set. Genes with a probability higher than 99.99% were considered as differentially expressed genes (DEG).
Next, DEG selected by the BETR analysis were subjected to a supervised weighted gene co-expression network analysis employing the WGCNA package. The automatic method was employed for block-wise network construction and module detection. The co-expression similarity was raised to a soft thresholding power (β) of 12 to calculate adjacency. The adjacency for the signed network is defined as aij=|(1+cor(xi,xj))/2|β (Zhang B, Horvath S (2005) A general framework for weighted gene co-expression network analysis. Stat Appl Genet Mol Biol 4:Article17). The resulting modules for each network were related to the phase of the endometrial cycle in order to identify modules or clusters of co-expressed genes showing increasing expression pattern with progression through the endometrial cycle and peaking in the late secretory phase. Gene significance (GS) was defined as the correlation of i-th gene with a temporal pattern. Module membership (MM) was defined as the correlation of the i-th gene with respect to its corresponding module (the higher the MM the more connected is the i-th gene with the other genes of the corresponding modules). The correlation coefficient of MM and GS was measured for each module, plotting MM versus GS. Higher correlation between MM and GS indicates that genes that are highly associated in a temporal pattern are also the central elements of a given module (73). The module with the highest positive correlation between MM and GS was selected for further comparison with DEG down-regulated in PE- vs NP-CVS.
Statistical analysis—DEC: data from the intDEC-EP (n=6) or intDEC-IUP (n=6) and confDEC-IUP (n=7) endometrium in the E-MTAB-680 database were compared to nonDEC samples (n=5) from EP to determine DEG up-regulated during the biological process of DEC. The limma package was used for the statistical analysis, applying the empirical Bayes method proposed by Smyth (Smyth G K (2004) Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat Appl Genet Mol Biol 3:Article3). This method calculates a moderated t-statistic for differential expression of each gene by performing a linear model fit of the data. Then, an empirical Bayes step is applied to moderate the standard errors of the estimated log-fold changes in order to produce more stable estimates, especially when the number of replicates is small. A gene was considered to be significantly differentially expressed, if both of the following conditions were met: 1) the ratio of the normalized intensity of the intermediate or confluent DEC to normalized intensity of the nonDEC endometrial samples was higher than a 2-fold change; and 2) differences were considered statistically significant at P≦0.05.
Statistical analysis—Potential Influence of Trophoblast: data from cultured endometrial stromal cells in GSE5809 (n=14) were analyzed over time (0, 3 and 12 hours incubation with TrCM) and by two conditions (DEC and nonDEC cultured endometrial cells). The BETR algorithm was used to identify DEG between decidualized endometrial cells treated with TrCM and nonDEC endometrial stromal cells treated with TrCM at a FDR <0.05 as a function of TrCM incubation time (0, 3 and 12 hours). This method yields the probability of differential expression for each gene in the data set. Genes with a probability of 99.9% were considered as DEG. Co-expressed genes as determined by WGCNA (see above) increasing in expression by 12 hours of incubation with TrCM were selected for further comparison with DEG down-regulated in PE-vs NP-CVS.
Statistical analysis—Decidual NK Cells: data from dNK (n=9) were compared to CD56dim pbNK (n=5) or CD56bright pbNK (n=5) by the empirical Bayes method as described above. A gene was considered to be significantly differentially expressed if both of the following conditions were met: 1) the ratio of the normalized intensity of the dNK to normalized intensity of the pbNK samples was higher than a 2-fold change; and 2) differences were considered statistically significant at P≦0.05.
Statistical analysis—PE-CVS: data from PE-CVS (n=4) and NP-CVS (n=8) in the dataset GSE12767 were compared by the empirical Bayes method. The DEG were considered if both of the following conditions were met: 1) the ratio of normalized intensity in PE-CVS to normalized intensity in CVS samples from normal pregnancy exceeded a 1.5-fold change; and 2) differences were considered statistically significant at P≦0.05. To expand the number of genes, down-regulated DEG determined by J5 and FC analysis were also included from Founds and coworkers (see Table 2 and Table S1 in Founds S A et al. (2009) Altered global gene expression in first trimester placentas of women destined to develop preeclampsia. Placenta 30:15-24).
Class prediction: in order to evaluate the performance of the selected DEG in each dataset, class prediction applying the k-Nearest Neighbors (kNN) algorithm for classification and the K-fold cross validation method as classifier was performed. The methodology was performed with the RWeka package for R (Hornik K, Buchta C, Zeileis A (2009) Open-source machine learning: R meets Weka. Computational Statistics 24:225-232). Specifically, after gene selection by the corresponding statistical method, each sample was examined for that dataset to determine if it would be able to predict to which class it belongs according the Euclidean distance to its kNN. For this, the K-folds number was set to the n samples for each dataset, known as leave-one-out cross validation (LOOCV). The k number for KNN was set as ni−1, for ni being the number of samples in the class of interest. The corresponding K-fold and k numbers, and the number of correct classifications for each dataset are shown in Table 18 below:













TABLE 18







Dataset
LSE
intDEC-EP
intDEC-IUP
confDEC-
PE-CVS
dNK






IUP




Classes
PrE, ESE,
intDEC-EP,
intDEC-IUP,
confDEC-IUP,
PE-CVS,
dNK,



MSE, LSE
NonDEC
NonDEC
NonDEC
NP-CVS
pbNK



(n = 24)
(n = 11)
(n = 11)
(n = 13)
(n = 8)
(n = 17)


Class of
LSE
intDEC-EP
intDEC-IUP
intDEC-IUP
PE-CSV
dNK


interest
(ni = 6)
(ni = 6)
(ni = 6)
(ni = 7)
(ni = 4)
(ni = 9)


K-fold
24
11
11
13
12
17


kNN
 5
 5
 5
 6
 3
 9


Number of
24
11
11
13
12
13


correct
(100%)
(100%)
(100%)
(100%)
(100%)
(100%)


classifications









Data comparison: the DEG down-regulated in PE-CVS were compared to: (i) the cluster of co-expressed endometrial genes increasing expression by the late secretory phase of the menstrual cycle (preDEC); (ii) DEG up-regulated in intermediate DEC endometrium from IUP or EP with and without trophoblast, respectively, and confluent DEC from IUP; (iii) the cluster of co-expressed genes increasing expression in decidualized endometrial stromal cells in culture after 12 hours of incubation with TrCM; and (iv) DEG up-regulated in decidual relative to peripheral blood NK cells. Statistical comparisons were made by the test of independence (Pearson's chi-square test) to determine the relatedness between down-regulated DEG in PE-CVS and up-regulated DEG in LSE, intermediate DEC (IUP or EP) and confluent DEC endometrium, in decidualized stromal cells in culture treated with TrCM, and decidualized NK cells.
Systematic Literature Search
Systematic review of the literature was undertaken by electronic searches in Medline through PubMed without language or publication date restrictions. The goal was to identify all publications related to decidualization that also reported one or more of the DEG down-regulated in PE-CVS (n=195). To enable identification of all relevant publications, Human Genome Organisation (HUGO) approved gene symbols were searched, as well as previous symbols and synonyms as listed by HUGO. The electronic search strategy was based on the Medical Subject Heading (MeSH) for each gene name, and when applicable, combined with title/abstract searches with all gene symbol synonyms. Synonyms that were not specific for a gene and generated too many irrelevant abstracts were omitted from the search string. By the use of Boolean operators individual gene searches (n=195) were combined with a search strategy identifying titles/abstracts related to “decidua/decidualization” based on the MeSH “decidua” or a title/abstract search for “decidua*”.
Retrieved references reporting DEG(s) down-regulated in PE-CVS and “decidua/decidualization” in title/abstract were selected by two reviewers (EPU and KPC) who independently scrutinizing titles and abstracts. Full-text articles of any ambiguous references were selected by one reviewer (EPU) and further scrutinized by two reviewers (EPU and KPC) to determine whether there was a clear relationship between the DEG(s) down-regulated in PE-CVS with decidua/decidualization. As a reference, the PubMed identifier (PMID) of one of the most relevant publications for each gene related to decidualization was provided (Table 8). For all the DEG down-regulated in PE-CVS (n=195), the test of independence (Pearson's chi-square test) was applied to determine the relatedness between DEG identified by the system biology approach (n=67, FIG. 13), and genes identified by the literature search in Pubmed (n=31).
Differentially expressed genes (DEG) between PE and NP-CVS: chorionic villous samples (CVS) obtained at  ̃11 gestational weeks from 4 women who developed preeclampsia 5-6 months later matched to 8 women with normal pregnancy, as described in Example 1 herein. Women were diagnosed with PE according to published criteria, three delivered after 34 weeks of gestation, and all met criteria for severe disease. The 12 women did not have associated co-morbidities except those with PE tended to have higher BMI. A wide net was cast and included differentially expressed genes (DEG) determined by FC, t-test (p<0.05) and J5 for subsequent bioinformatics analysis. The results of the J5 analysis were taken from Example 1 herein, those from t-test were obtained by re-analyzing the original Affymetrix data GSE12767, and FC data stemmed from both the original (< or >2.0) and re-analysis (< or >1.5). There was a total of 396 DEG between PE-CVS and NP-CVS of which 201 were up-regulated and 195 down-regulated in PE-CVS (Tables 7-8).
Significant overlap between DEG in PE-CVS and DEG associated with decidualization: there was a significant overlap between DEG in PE- relative to NP-CVS and DEG in late secretory endometrium (LSE; relative to proliferative endometrium, PrE) (75 DEG); endometrium with intermediate decidual (intDEC) changes from tubal ectopic pregnancy (EP) (70 DEG); as well as endometrium from intDEC (71 DEG) and confluent (99 DEG) decidual (confDEC) changes from intrauterine pregnancy (IUP), all compared to EP endometrium without decidual changes (nonDEC) (p<0.0001 except LSE p<0.003). Twenty-four of these DEG were in common. Moreover, most of the overlapping DEG down- or up-regulated in PE-CVS changed in the opposite direction in the other data sets: 54/75, 52/70, 55/71 and 70/99, respectively, with 18 in common.
Analyses were then performed on DEG up-regulated during the biological processes of LSE, intDEC, and confDEC reasoning that if significant numbers of these DEG were down-regulated in PE-CVS, then the concept of deficient preDEC and DEC in women destined to develop PE would be supported. The top Enriched Biological Processes for these 195 DEG down-regulated in PE were defense response, inflammatory response, response to wounding and negative regulation of cell proliferation (all Benjamini corrected p<0.05; Table 9).
Significant overlap between DEG down-regulated in PE-CVS and DEG up-regulated in LSE and intDEC-EP endometrium: gene expression in normal endometrium from different phases of the menstrual cycle were analyzed (GSE4888 and GSE6364) using time as an ordinal variable to determine temporal changes in DEG relative to the proliferative phase of the menstrual cycle. These genes were clustered by supervised weighted gene co-expression analysis, in order to identify the cluster of co-expressed genes strongly increasing expression in the endometrium throughout the menstrual cycle and peaking in the LSE. There was a significant overlap of 38 genes between the LSE cluster of 1315 up-regulated DEG and the 195 down-regulated DEG in PE-CVS (p<0.0001 by Pearson's chi-square test; FIG. 11A, see also Table 10 for gene list).
Gene expression in endometrium from women with EP showing intDEC changes was compared to gene expression in nonDEC endometrium obtained from women with EP (E-MTAB-680, (Duncan W C et al. (2011) Ibid.). The up-regulated DEG in intDEC endometrium (873 DEG) was compared to the down-regulated DEG in PE-CVS. There was a significant overlap of 32 genes between DEG up-regulated in intDEC endometrium and down-regulated in PE-CVS (p<0.0001; FIG. 11B, Table 11). There was also a large and significant overlap of 382 DEG increasing in LSE endometrium with DEG up-regulated in intDEC endometrium from EP. Of these, 16 DEG significantly overlapped with the DEG down-regulated in PE-CVS (p<0.0001; FIG. 11C; Table 12).
DEG down-regulated in PE-CVS and up-regulated in int- and confDEC-IUP endometrium overlap significantly: gene expression in endometrium from women with IUP (both int- and confDEC) was first compared to gene expression in nonDEC endometrium obtained from women with EP (E-MTAB-680 (Duncan W C et al. (2011) Ibid.)). The up-regulated DEG in int- (1007 DEG) and conDEC (1581 DEG) endometrium were compared to the 195 down-regulated DEG in PE-CVS. Thirty-seven and 46 DEG up-regulated in int- and conDEC endometrium, respectively, overlapped with DEG down-regulated in PE-CVS (both p<0.0001; FIGS. 12A and 12B; Tables 13 and 14, respectively).
Comparison of DEG down-regulated in PE-CVS with DEG up-regulated in intDEC-IUP and -EP endometrium: because IUP endometrium was influenced by local trophoblast (Tr) but EP endometrium was not (as verified by immunohistochemistry for cytokeratin), the potential Tr contribution to the overlap of DEG down-regulated in PE-CVS and up-regulated in intDEC endometrium was estimated. There was large overlap of 689 DEG up-regulated in intDEC endometrium from EP and IUP, compared to nonDEC endometrium from EP (p<0.0001, FIG. C). As further illustrated in FIG. 12C, 30 of these 689 DEG overlapped significantly with DEG down-regulated in PE-CVS (p<0.0001, Table 15). The majority of overlapping DEG between those up-regulated in intDEC from IUP or EP and down-regulated in PE-CVS were the same genes (30 of 37 for intDEC-IUP and 30 of 32 for intDEC-EP).
Expression of DEG down-regulated in PE-CVS was not significantly associated with genes expressed in cultured DEC endometrial cells by trophoblast conditioned medium (TrCM): DEG down-regulated in PE-CVS were compared to genes induced by TrCM in cultured endometrial cells decidualized in vitro. TrCM was obtained from cytotrophoblasts isolated from placentae between 6 and 22 weeks of gestation after elective termination and culture on Matrigel-coated substrate for 48 hours (GSE5809, (56)). DEC endometrial cell gene expression was analyzed over time (0, 3 and 12 hours of incubation with TrCM) and between treated and untreated DEC endometrial cell cultures. Selected genes were clustered by supervised weighted gene co-expression analysis, in order to identify the cluster of co-expressed genes strongly increasing expression after 12 hours of TrCM incubation. As expected, there was significant overlap between the cluster of 304 DEG increasing expression in DEC endometrial cells incubated with TrCM and DEG up-regulated in confDEC-IUP endometrium influenced by local Tr (69 DEG, p<0.0001; FIG. 12D). However, there was no significant intersection of endometrial genes increasing in expression after treatment with TrCM and DEG down-regulated in PE-CVS with only 4 DEG in common (FIG. 12D, p=0.5).
Confluence of overlapping genes: the confluence of DEG down-regulated in PE-CVS and up-regulated in LSE, intDEC-IUP and -EP, as well as confDEC-IUP endometrium was investigated. As portrayed by the Venn Diagram in FIG. 13, there were 20 down-regulated DEG in PE-CVS, which were up-regulated in LSE but not in int- or confDEC; 13 DEG down-regulated in PE-CVS and up-regulated in int- and confDEC, but not in LSE (p<0.0001), and 16 DEG down-regulated in PE-CVS and up-regulated in LSE, int- and confDEC endometrium (p<0.0001). The individual DEGs are presented in Table 16 and their mean expression values are illustrated in FIGS. 14A-14D.
FIG. 14A depicts the log 2 mean expression values for the DEG down-regulated in PE-CVS (relative to NP-CVS) and up-regulated in LSE relative to proliferative endometrium (PrE; DEG). FIG. 14B depicts the log 2 mean expression values for the DEG down-regulated in PE-CVS (relative to NP-CVS) and up-regulated in int- and confDEC relative to nonDEC-EP (13 DEG). FIG. 14C depicts the log 2 mean expression values for the DEG down-regulated in PE-CVS (relative to NP-CVS) and up-regulated in LSE+int- and confDEC endometrium (16 DEG). The heat map shown in FIG. 14D corresponds with the bar graph in FIG. 14C.
Twenty DEG were identified as uniquely up-regulated in LSE and down-regulated in PE-CVS; therefore, their average expression did not further increase with DEC (FIG. 14A). Average gene expression of these 20 DEG was significantly less in PE-CVS than in MSE and LSE (p<0.05), and comparable to PrE or ESE endometrium. The 13 DEG down-regulated in PE-CVS and uniquely up-regulated in int- and confDEC endometrium, slightly increased in LSE, but mostly rose during decidualization (FIG. 14B). Average gene expression for these 13 DEG was markedly less in PE-CVS than in int- and confDEC endometrium (p<0.05). Finally, the 16 DEG down-regulated in PE-CVS and up-regulated in LSE, and int- and confDEC endometrium increased expression beginning in the MSE and progressively rose thereafter (FIG. 14C). In this case, average gene expression of the 16 DEG was also dramatically less in PE-CVS compared to int- and confDEC endometrium (p<0.05).
Comparison of DEG down-regulated in PE-CVS with DEG up-regulated in dNK Cells: in contrast to published gene expression of peripheral blood and endometrial (e)NK cells derived from different microarray platforms (Koopman L A et al. (2003) Ibid., and Kopcow H D et al. (2010) Human decidual NK cells from gravid uteri and NK cells from cycling endometrium are distinct NK cell subsets. Placenta 31:334-338), gene expression between dNK and CD56dim pbNK or CD56bright pbNK cells (Koopman L A et al. (2003) Ibid.) were compared because the same microarray platform was employed. As expected, there was a large confluence of 380 shared DEG up-regulated in dNK relative to CD56dim pbNK or CD56bright pbNK (p<0.000001). There was also high overlap (112 DEG) between DEG up-regulated in dNK relative to CD56dim pbNK or CD56bright pbNK, and DEG up-regulated in LSE (relative to PrE)+intDEC-EP (relative to nonDEC-EP) in the absence of local Tr influence (p<0.00001; FIG. 16A); and a high number of overlapping DEG (93 DEG) up-regulated in dNK relative to CD56dim pbNK or CD56bright pbNK, and intDEC-IUP+confDEC-IUP endometrium (relative to nonDEC-EP) in the presence of local Tr influence (p<0.00001; FIG. 16B). The majority of these 112 and 93 overlapping DEG were the same (74 DEG, p<0.00001, Table 17). Finally, 16 DEG up-regulated in dNK relative to CD56dim pbNK or CD56bright pbNK were down-regulated in PE-CVS (relative to NP-CVS; p<0.0001; FIGS. 16A and 16B).
Systematic Literature Search: because the biological process of “decidualization” is not available in public bioinformatic databases for pathway analysis, a systematic and comprehensive literature search was conducted of all 195 DEG down-regulated in PE-CVS. Thirty-one were previously associated with decidua/decidualization in the literature (FIG. 16, Table 16 and Table 8). The overlap of these 31 DEG identified by literature search was evaluated against the overlapping DEG determined by systems biology approach, i.e., those down-regulated in PE-CVS and up-regulated in: LSE (38 DEG; FIG. 11A), intDEC-EP endometrium (32 DEG; FIG. 11B), intDEC-IUP endometrium (37 DEG, FIG. 12A) and confDEC-IUP endometrium (46 DEG; FIG. 12B), all together 67 unique genes (see also FIG. 13). It was found that 18 of the 31 DEG identified in the literature were in common with 67 DEG identified by systems biology (p=0.001). The majority (15, p=0.03) was up-regulated in LSE or intDEC-EP in which (local) trophoblast influence is absent.
Findings
In conducting the analysis of the original microarray dataset from PE- and NP-CVS (GSE12767, Founds S A et al. (2009) Ibid.; Example 1 above), a wide net was cast to identify DEG in PE- relative to NP-CVS (396 total; Tables 6 and 7). An impressive number of these DEG (from 70 up to 99) significantly overlapped with DEG associated with the various stages of endometrial maturation before and after conception. Moreover, at least 70% of the overlapping DEG down- or up-regulated in PE-CVS were changed in the opposite direction in the other data sets dealing with normal endometrial maturation. These findings suggest impairment of preDEC and DEC in the women who developed PE.
Analysis was then performed on DEG down-regulated in PE-CVS that were up-regulated during the biological process of (pre)decidualization. Of the 396 total DEG, 195 were down-regulated in PE- relative to NP-CVS. A cluster of 1315 co-expressed genes was found to be up-regulated during the process of endometrial maturation in the menstrual cycle culminating with the LS phase (GSE4888 and GSE6364). There was significant overlap of 38 DEG between the PE-CVS and LSE datasets (FIG. 11A). Microarray analysis of DEC in early pregnancy (E-MTAB-680) revealed 873 up-regulated genes in intDEC endometrium from ectopic pregnancy relative to nonDEC-EP endometrium. There was significant overlap of 32 DEG between those up-regulated in intDEC-EP endometrium and down-regulated in PE-CVS (FIG. 11B). Finally, the confluence of all 3 datasets yielded 16 DEG in common (FIG. 11C).
Remarkably, 54 of the 195 DEG down-regulated in PE-CVS were up-regulated during LS or intDEC endometrium from EP. Taken together, these results bolster the notion that there is impairment of endometrial maturation in the LS phase and during early pregnancy in the women destined to develop PE. Included among the genes which expression failed to increase in decidua of CVS from the women who developed PE are those classically associated with the biological process of decidualization in the literature including IGFBP-1, PAEP or glycodelin, and PRL (Tables 10-12). The results also implicate a primary defect in preDEC and DEC rather than Tr, because Tr are lacking altogether in the LS phase and local Tr influence was absent in intDEC-EP endometrium. Another, albeit less plausible explanation is that after conception defective Tr reversed preDEC in the LS phase and inhibited DEC after conception in the women who developed PE.
Further inspection of the microarray analyses from decidualization in early pregnancy revealed >1000 genes each up-regulated in int- and confDEC endometrium from intrauterine pregnancy compared to nonDEC endometrium from EP. Thirty-seven and 46 of these up-regulated DEG, respectively, overlapped significantly with DEG down-regulated in PE-CVS (FIGS. 12A and 12B, and Tables 13 and 14). There was also a large confluence of 689 DEG up-regulated in intDEC-IUP and intDEC-EP endometrium (matched for the degree of DEC). Although this degree of overlap was substantial and highly significant, it was by no means a complete overlap most likely explained by the presence of local Tr influence in the intDEC-IUP. Of note, the vast majority of DEG up-regulated in intDEC-EP and down-regulated in PE-CVS (32 DEG), and those up-regulated in intDEC-IUP and down-regulated in PE-CVS (37 DEG) were themselves overlapping (30 DEG; FIG. 12C and Table 15). These observations suggest that there was little if any contribution of Tr gene expression per se to the overlap of DEG down-regulated in PE-CVS and up-regulated in intDEC-IUP and -EP, or of Tr influence on DEC stromal, epithelial or immune cells either through cell-to-cell contact or paracrine interactions, because the vast majority of DEG down-regulated in PE-CVS were up-regulated in intDEC regardless of the presence (IUP) or absence (EP) of local Tr influence. A caveat is that the comparison of these datasets does not take into account a potential role for (circulating) endocrine factors secreted by Tr, which may not be the same for EP and IUP.
To further scrutinize the potential contribution of Tr to the impaired DEC of PE-CVS, DEG down-regulated in PE-CVS was compared with DEG up-regulated in cultured decidualized endometrial stromal cells after incubation with trophoblast conditioned medium (TrCM; GSE5809). Despite the significant overlap between DEG up-regulated in DEC endometrial stromal cells exposed to TrCM and in confDEC-IUP endometrium (69 DEG; FIG. 12D), there were virtually no DEG in common between those up-regulated in DEC endometrial stromal cells treated with TrCM (304 DEG) and down-regulated in PE-CVS (195 DEG), the overlap being a non-significant 4 genes (FIG. 12D). This finding reinforces the idea that there was minimal Tr contribution to the overlap observed between DEG down-regulated in PE-CVS and DEG up-regulated in either int- or confDEC-IUP endometrium, which is consistent with the concept that there may have been a primary defect of endometrial maturation in the women destined to develop PE.
The confluence of DEG down-regulated in PE-CVS and up-regulated in LSE, intDEC-IUP and -EP, as well as confDEC-IUP endometrium was examined (i.e., intersection of all 4 data sets; FIG. 3 and Table 16). Mean expression of the 20 DEG down-regulated in PE-CVS and uniquely up-regulated in secretory relative to PrE was significantly increased in mid-secretory endometrium (MSE), maintained in LSE, but not further increased during DEC (i.e., after implantation; FIG. 14A). Of note, the mean expression for these 20 DEG was significantly lower in PE-CVS compared to MSE and LSE by  ̃5-fold, and comparable to PrE and early secretory endometrium. Taken together, this analysis suggests that impairment of endometrial maturation in the women destined to develop PE may actually have begun before pregnancy in the secretory phase.
Mean expression of the 13 DEG down-regulated in PE-CVS and up-regulated in intDEC-EP and -IUP and in confDEC-IUP, but not LSE is shown in FIG. 14B. Examination of the mean expression relative to PrE revealed a significant increase in intDEC-EP and -IUP with a further rise in confDEC-IUP endometrium (FIG. 14B). The mean expression level in PE-CVS was markedly reduced relative to int- and confDEC by  ̃15-fold. These results suggest that, in addition to a defect in preDEC as described above there was also impairment of DEC after implantation in the women who developed PE.
Finally, a core set of 16 DEG was down-regulated in PE-CVS and up-regulated in LSE, intDEC-EP and -IUP, and confDEC-IUP endometrium (FIG. 14C). The average level of expression of these 16 genes relative to PrE increased progressively beginning with MSE and peaking in confDEC-IUP endometrium. Mean gene expression for PE-CVS was considerably lower than MSE, LSE, int- and confDEC, the latter by  ̃50-fold. On balance, these data present a composite picture of FIGS. 14A and 14B underscoring the notion that both preDEC and DEC were compromised in the women who developed PE.
Because DEG known to be involved in dNK function emerged from the aforementioned analyses (e.g., IL-15, IL2RB, etc., see Table 16), the overlap of DEG up-regulated in isolated dNK (relative to CD56dim pbNK or CD56bright pbNK) cells and down-regulated in PE-CVS (FIG. 15) was explored. Despite lacking the corresponding eNK cell match (at least for LSE), it was discovered that a large confluence of 112 DEG up-regulated in isolated dNK and in LSE+intDEC-EP with local Tr absent (FIG. 15A). These common DEG may be expressed by dNK alone or co-expressed with other cell-types in the LSE including other immune, DEC stromal, epithelial and glandular cells. When up-regulated DEG in dNK were compared to those up-regulated in intDEC+confDEC-IUP with local Tr present, the overlapping genes (93 DEG, FIG. 15B) were mostly the same as in LSE+in DEC-EP (74 DEG, Table 17). Taken together, these results suggest minimal contribution of Tr gene expression per se, or Tr contact with or paracrine effects on decidual cells to the intersection of DEG. Finally, in addition to impaired DEC (vide supra), the women destined to develop PE were also likely to have deficient dNK cell number and/or function, because 16 DEG up-regulated in dNK were down-regulated in PE-CVS (FIGS. 15A and 15B).
In addition to the evidence provided by bioinformatics approaches linking the DEG down-regulated in PE-CVS to impaired endometrial maturation, a different tack was taken to marshal further evidence associating these down-regulated DEG to inadequate preDEC and DEC. Because preDEC and DEC biological pathways are not represented in public bioinformatic databases, a systematic and comprehensive literature search of all 195 DEG down-regulated in PE-CVS (FIG. 16 and Table 8) was conducted. It was discovered that 31 of the 195 DEG had been previously linked to decidua or decidualization in the literature. There was a significant overlap of 18 genes with the decidualization genes identified by bioinformatics approach. Of these, only 3 were exclusively up-regulated during DEC in intra-uterine pregnancies, the remaining 15 genes were also up-regulated during the process of preDEC or DEC in the absence of Tr (Table 16, FIG. 16 and Table 8). Thus, the systematic literature search further strengthens the argument that endometrial maturation was impaired in the women destined to develop PE.
In summary, substantial evidence is provided for deficient DEC in the LS phase and during early pregnancy in women destined to develop PE as illustrated in FIG. 17. This conclusion is based on a systems biology approach, which was employed to evaluate microarray analysis of 1st trimester placentas from women who developed late onset, severe PE or experienced a normal pregnancy in the context of other microarray studies in the public domain related to normal maturation of endometrium and of dNK cells. Remarkably, one-third of the DEG down-regulated in PE- relative to NP-CVS were up-regulated during the biological process of endometrial maturation, and a number of these were uniquely up-regulated in dNK cells. Moreover, the analysis performed above further suggests that inadequate endometrial maturation may be a primary event, because the majority of these DEG were up-regulated in LSE or DEC endometrium from ectopic pregnancy in the absence of local Tr influence. The latter conclusion is reinforced by the finding that an insignificant few of the DEG down-regulated in PE-CVS overlapped with DEG up-regulated in decidualized stromal cells in culture exposed to TrCM. The subject study provides evidence supporting a link between deficient endometrial maturation and the development of preeclampsia.
In accordance with the subject application, supplemental hormonal support with relaxin, congener or mimetic during the LS phase, and in certain instances into early pregnancy, will improve endometrial maturation, thereby improving the uterine environment for Tr invasion and spiral artery remodeling to facilitate normal placentation, and thus treat and/or reduce the likelihood of development of placental syndromes, including PE.
All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application.







TABLE 1







Gene Expression associated with Decidualization


(Fold Change Data for CVS Microarray: PE vs. NP)













Fold
Condition PE



Gene Symbol
Probe ID*
Change
Group
Target Description














ALDH1L2
207016_s_at
−7.8
Underexpressed
gb:AB015228.1






/DB_XREF=gi:3970845






/GEN=RALDH2 /FEA=FLmRNA






/CNT=10 /TID=Hs.95197.0 /TIER=FL






/STK=0 /UG=Hs.95197 /LL=8854






/DEF=Homo sapiens mRNA for






RALDH2-T, complete cds.






/PROD=RALDH2-T






/FL=gb:AB015228.1 gb:AB015227.1






gb:AB015226.1 gb:NM_003888.1


RORB
231040_at
−13.2
Underexpressed
gb:AW512988 /DB_XREF=gi:7151066






/DB_XREF=xt76b02.x1






/CLONE=IMAGE:2792331 /FEA=EST






/CNT=9 /TID=Hs.184780.0






/TIER=Stack /STK=9 /UG=Hs.184780






/UG_TITLE=ESTs


ACOT8
236514_at
2.1
Overexpressed
gb:AI885067 /DB_XREF=gi:5590231






/DB_XREF=wl89c05.x1






/CLONE=IMAGE:2432072 /FEA=EST






/CNT=7 /TID=Hs.6511.0






/TIER=ConsEnd /STK=6 /UG=Hs.6511






/UG_TITLE=ESTs


EPAS1
242868_at
−15.3
Underexpressed
gb:T70087 /DB_XREF=gi:681235






/DB_XREF=yc17g11.s1






/CLONE=IMAGE:80996 /FEA=EST






/CNT=7 /TID=Hs.307559.0






/TIER=ConsEnd /STK=1






/UG=Hs.307559 /UG_TITLE=ESTs


DLGAP1
1568736_s_at
−4.4
Underexpressed
gb:BC030096.1






/DB_XREF=gi:22535265






/TID=Hs2.371203.1 /CNT=5






/FEA=mRNA /TIER=ConsEnd /STK=2






/UG=Hs.371203 /UG_TITLE=Homo






sapiens, clone IMAGE:4795078,






mRNA /DEF=Homo sapiens, clone






IMAGE:4795078, mRNA. (All probes






match over all sequences)


SPOCK1
202363_at
−5.5
Underexpressed
gb:AF231124.1






/DB_XREF=gi:7248844






/FEA=FLmRNA /CNT=190






/TID=Hs.93029.0 /TIER=FL+Stack






/STK=68 /UG=Hs.93029 /LL=6695






/UG_GENE=SPOCK /DEF=Homo






sapiens testican-1 mRNA, complete






cds. /PROD=testican-1






/FL=gb:NM_004598.1 gb:AF231124.1


MAOB
204041_at
−2.7
Underexpressed
gb:NM_000898.1






/DB_XREF=gi:4505092 /GEN=MAOB






/FEA=FLmRNA /CNT=79






/TID=Hs.82163.0 /TIER=FL+Stack






/STK=39 /UG=Hs.82163 /LL=4129






/DEF=Homo sapiens monoamine






oxidase B (MAOB), nuclear gene






encoding mitochondrial protein,






mRNA. /PROD=monoamine oxidase B






/FL=gb:NM_000898.1 gb:M69177.1


GZMB
210164_at
−2.7
Underexpressed
gb:J03189.1 /DB_XREF=gi:338010






/FEA=FLmRNA /CNT=28






/TID=Hs.1051.1 /TIER=FL+Stack






/STK=11 /UG=Hs.1051 /LL=3002






/UG_GENE=GZMB






/UG_TITLE=granzyme B (granzyme 2,






cytotoxic T-lymphocyte-associated






serine esterase 1) /DEF=Human






proteolytic serine esterase-like protein






(SECT) gene, complete cds.






/FL=gb:M17016.1 gb:J03189.1






gb:NM_004131.2 gb:J04071.1


IL2RB
205291_at
−2.8
Underexpressed
gb:NM_000878.1






/DB_XREF=gi:4504664 /GEN=IL2RB






/FEA=FLmRNA /CNT=45






/TID=Hs.75596.0 /TIER=FL+Stack






/STK=26 /UG=Hs.75596 /LL=3560






/DEF=Homo sapiens interleukin 2






receptor, beta (IL2RB), mRNA.






/PROD=interleukin 2 receptor, beta






/FL=gb:NM_000878.1 gb:M26062.1


GNLY
205495_s_at
−23.5
Underexpressed
gb:NM_006433.2






/DB_XREF=gi:7108343 /GEN=GNLY






/FEA=FLmRNA /CNT=32






/TID=Hs.105806.1 /TIER=FL+Stack






/STK=18 /UG=Hs.105806 /LL=10578






/DEF=Homo sapiens granulysin






(GNLY), transcript variant NKG5,






mRNA. /PROD=granulysin, isoform






NKG5 /FL=gb:NM_006433.2


NOG
231798_at
−5.3
Underexpressed
gb:AL575177






/DB_XREF=gi:12936088






/DB_XREF=AL575177






/CLONE=CS0DI063YJ07 (3 prime)






/FEA=FLmRNA /CNT=7






/TID=Hs.248201.0 /TIER=ConsEnd






/STK=1 /UG=Hs.248201 /LL=9241






/UG_GENE=NOG






/UG_TITLE=noggin






/FL=gb:NM_005450.1


TRA@
217143_s_at
−4.9
217143_s_at
gb:X06557.1 /DB_XREF=gi:37003






/FEA=mRNA /CNT=2 /TID=Hs.2014.2






/TIER=ConsEnd /STK=0 /UG=Hs.2014






/LL=6964 /UG_GENE=TRD@






/UG_TITLE=T cell receptor delta locus






/DEF=Human mRNA for TCR-delta






chain.


MUC15
227238_at
−8
Underexpressed
gb:W93847 /DB_XREF=gi:1422970






/DB_XREF=zd97a07.s1






/CLONE=IMAGE:357396






/FEA=mRNA /CNT=89






/TID=Hs.24139.0 /TIER=Stack






/STK=21 /UG=Hs.24139






/UG_TITLE=Homo sapiens cDNA:






FLJ23137 fis, clone LNG08842


KLRC2
206785_s_at
−2.9
Underexpressed
gb:NM_002260.2






/DB_XREF=gi:7108353 /GEN=KLRC2






/FEA=FLmRNA /CNT=12






/TID=Hs.177605.0 /TIER=FL /STK=1






/UG=Hs.177605 /LL=3822






/DEF=Homo sapiens killer cell lectin-






like receptor subfamily C, member 2






(KLRC2), mRNA. /PROD=killer cell






lectin-like receptor subfamily






C,member 2 /FL=gb:NM_002260.2






gb:AF260134.1


IL15
205992_s_at
−2.8
Underexpressed
gb:NM_000585.1






/DB_XREF=gi:10835152 /GEN=IL15






/FEA=FLmRNA /CNT=33






/TID=Hs.168132.0 /TIER=FL /STK=0






/UG=Hs.168132 /LL=3600






/DEF=Homo sapiens interleukin 15






(IL15), mRNA. /PROD=interleukin 15






/FL=gb:NM_000585.1 gb:U14407.1


CHRDL1
209763_at
−7.3
Underexpressed
gb:AL049176 /DB_XREF=gi:4808226






/FEA=FLmRNA /CNT=84






/TID=Hs.82223.0 /TIER=Stack






/STK=46 /UG=Hs.82223 /LL=57803






/UG_GENE=LOC57803






/UG_TITLE=chordin-like






/DEF=Human DNA sequence from






clone 141H5 on chromosome Xq22.1-






23. Contains parts of a novel Chordin






LIKE protein with von Willebrand






factor type C domains. Contains ESTs,






STSs and GSSs /FL=gb:BC002909.1


PRL
205445_at
−7.9
Underexpressed
gb:NM_000948.1






/DB_XREF=gi:4506104 /GEN=PRL






/FEA=FLmRNA /CNT=352






/TID=Hs.1905.0 /TIER=FL+Stack






/STK=19 /UG=Hs.1905 /LL=5617






/DEF=Homo sapiens prolactin (PRL),






mRNA. /PROD=prolactin






/FL=gb:NM_000948.1


SCARA5
229839_at
−8.3
Underexpressed
gb:AI799784 /DB_XREF=gi:5365256






/DB_XREF=wc43b08.x1






/CLONE=IMAGE:2321367 /FEA=EST






/CNT=10 /TID=Hs.49696.0






/TIER=Stack /STK=9 /UG=Hs.49696






/UG_TITLE=ESTs


CHST6
223786_at
−2.2
Underexpressed
gb:AF280086.1






/DB_XREF=gi:12060803






/GEN=GST4beta /FEA=FLmRNA






/CNT=9 /TID=Hs.157439.1 /TIER=FL






/STK=0 /UG=Hs.157439 /LL=4166






/DEF=Homo sapiens N-






acetylglucosamine 6-O-sulfotransferase






GST-4beta mRNA, complete cds.






/PROD=N-acetylglucosamine 6-O-






sulfotransferaseGST-4beta






/FL=gb:AF280086.1


NTN1
227816_at
−3.6
Underexpressed
gb:BF591483 /DB_XREF=gi:11683807






/DB_XREF=nab98c06.x1






/CLONE=IMAGE:3275890 /FEA=EST






/CNT=23 /TID=Hs.96917.0






/TIER=Stack /STK=15 /UG=Hs.96917






/UG_TITLE=ESTs


BICD1
204741_at
2.2
Overexpressed
gb:NM_001714.1






/DB_XREF=gi:4502408 /GEN=BICD1






/FEA=FLmRNA /CNT=47






/TID=Hs.164975.0 /TIER=FL+Stack






/STK=10 /UG=Hs.164975 /LL=636






/DEF=Homo sapiens Bicaudal D






(Drosophila) homolog 1 (BICD1),






mRNA. /PROD=Bicaudal D






(Drosophila) homolog 1






/FL=gb:U90028.1 gb:NM_001714.1


ADCYAP1R1
221286_s_at
3.1
Overexpressed
gb:NM_016459.1






/DB_XREF=gi:7706002






/GEN=LOC51237 /FEA=FLmRNA






/CNT=10 /TID=Hs.122492.1






/TIER=FL /STK=1 /UG=Hs.122492






/LL=51237 /DEF=Homo sapiens






hypothetical protein (LOC51237),






mRNA. /PROD=hypothetical protein






/FL=gb:NM_016459.1


CPM
235019_at
−2.1
Underexpressed
gb:BE878495 /DB_XREF=gi:10327271






/DB_XREF=601492515F1






/CLONE=IMAGE:3894722 /FEA=EST






/CNT=32 /TID=Hs.267158.0






/TIER=ConsEnd /STK=0






/UG=Hs.267158 /UG_TITLE=ESTs


DPYSL4
205493_s_at
2
Overexpressed
gb:NM_006426.1






/DB_XREF=gi:11321616






/GEN=DPYSL4 /FEA=FLmRNA






/CNT=29 /TID=Hs.100058.0






/TIER=FL /STK=1 /UG=Hs.100058






/LL=10570 /DEF=Homo sapiens






dihydropyrimidinase-like 4 (DPYSL4),






mRNA. /PROD=dihydropyrimidinase-






like 4 /FL=gb:NM_006426.1






gb:AB006713.1


IL1B
205067_at
−2.1
Underexpressed
gb:NM_000576.1






/DB_XREF=gi:10835144 /GEN=IL1B






/FEA=FLmRNA /CNT=97






/TID=Hs.126256.0 /TIER=FL+Stack






/STK=15 /UG=Hs.126256 /LL=3553






/DEF=Homo sapiens interleukin 1, beta






(IL1B), mRNA. /PROD=interleukin 1,






beta /FL=gb:M15330.1 gb:M54933.1






gb:K02770.1 gb:NM_000576.1


MMP12
204580_at
−17.2
Underexpressed
gb:NM_002426.1






/DB_XREF=gi:4505206






/GEN=MMP12 /FEA=FLmRNA






/CNT=72 /TID=Hs.1695.0






/TIER=FL+Stack /STK=18






/UG=Hs.1695 /LL=4321 /DEF=Homo






sapiens matrix metalloproteinase 12






(macrophage elastase) (MMP12),






mRNA. /PROD=matrix






metalloproteinase 12 preproprotein






/FL=gb:L23808.1 gb:NM_002426.1


BDKRB2
205870_at
−2.7
Underexpressed
gb:NM_000623.1






/DB_XREF=gi:4557358






/GEN=BDKRB2 /FEA=FLmRNA






/CNT=34 /TID=Hs.250882.0






/TIER=FL+Stack /STK=17






/UG=Hs.250882 /LL=624 /DEF=Homo






sapiens bradykinin receptor B2






(BDKRB2), mRNA.






/PROD=bradykinin receptor B2






/FL=gb:M88714.1 gb:NM_000623.1


SLC16A6
230748_at
−8.4
Underexpressed
gb:AI873273 /DB_XREF=gi:5547322






/DB_XREF=wf41c12.x1






/CLONE=IMAGE:2358166 /FEA=EST






/CNT=20 /TID=Hs.42645.0






/TIER=Stack /STK=10 /UG=Hs.42645






/UG_TITLE=ESTs


COL5A1
213818_x_at
2.1
Overexpressed
gb:AI862325 /DB_XREF=gi:5526432






/DB_XREF=tw71h04.x1






/CLONE=IMAGE:2265175 /FEA=EST






/CNT=23 /TID=Hs.146428.3






/TIER=Stack /STK=19 /UG=Hs.146428






/LL=1289 /UG_GENE=COL5A1






/UG_TITLE=collagen, type V, alpha 1


FN1
214702_at
−8.2
Underexpressed
gb:AJ276395.1






/DB_XREF=gi:12053816 /GEN=FN






/FEA=mRNA /CNT=52






/TID=Hs.321592.0 /TIER=ConsEnd






/STK=4 /UG=Hs.321592 /DEF=Homo






sapiens mRNA for MSF-FN70 (FN






gene). /PROD=migration stimulation






factor FN70


PP14
206859_s_at
−15.6
Underexpressed
gb:NM_002571.1






/DB_XREF=gi:4505582 /GEN=PAEP






/FEA=FLmRNA /CNT=18






/TID=Hs.82269.0 /TIER=FL+Stack






/STK=13 /UG=Hs.82269 /LL=5047






/DEF=Homo sapiens progestagen-






associated endometrial protein






(placental protein 14, pregnancy-






associated endometrial alpha-2-






globulin, alpha uterine protein) (PAEP),






mRNA. /PROD=progestagen-associated






endometrial protein(placental protein






14, pregnancy-associated






endometrialalpha-2-globulin, alpha






uterine protein) /FL=gb:NM_002571.1






gb:J04129.1


FOSB
202768_at
2.3
Overexpressed
gb:NM_006732.1






/DB_XREF=gi:5803016 /GEN=FOSB






/FEA=FLmRNA /CNT=167






/TID=Hs.75678.0 /TIER=FL+Stack






/STK=67 /UG=Hs.75678 /LL=2354






/DEF=Homo sapiens FBJ murine






osteosarcoma viral oncogene homolog






B (FOSB), mRNA. /PROD=FBJ






murine osteosarcoma viral oncogene






homologB /FL=gb:NM_006732.1






gb:L49169.1


FSTL3
203592_s_at
−10.1
Underexpressed
gb:NM_005860.1






/DB_XREF=gi:5031700 /GEN=FSTL3






/FEA=FLmRNA /CNT=129






/TID=Hs.25348.0 /TIER=FL+Stack






/STK=33 /UG=Hs.25348 /LL=10272






/DEF=Homo sapiens follistatin-like 3






(secreted glycoprotein) (FSTL3),






mRNA. /PROD=follistatin-like 3






glycoprotein /FL=gb:U76702.1






gb:NM_005860.1


WT1
206067_s_at
−5.3
Underexpressed
gb:NM_024426.1






/DB_XREF=gi:13386509 /GEN=WT1






/FEA=FLmRNA /CNT=24






/TID=Hs.1145.1 /TIER=FL+Stack






/STK=11 /UG=Hs.1145 /LL=7490






/DEF=Homo sapiens Wilms tumor 1






(WT1), transcript variant D, mRNA.






/PROD=Wilms tumor 1 isoform D






/FL=gb:NM_024424.1






gb:NM_024426.1


IGFBP1
205302_at
−10.4
Underexpressed
gb:NM_000596.1






/DB_XREF=gi:4504614






/GEN=IGFBP1 /FEA=FLmRNA






/CNT=268 /TID=Hs.102122.0






/TIER=FL+Stack /STK=117






/UG=Hs.102122 /LL=3484






/DEF=Homo sapiens insulin-like






growth factor binding protein 1






(IGFBP1), mRNA. /PROD=insulin-like






growth factor binding protein 1






/FL=gb:NM_000596.1 gb:M31145.1






gb:M20841.1


CFH/CFHR1
215388_s_at
−8.5
Underexpressed
gb:X56210.1 /DB_XREF=gi:30132






/GEN=H 36-2 /FEA=mRNA /CNT=4






/TID=Hs.296941.0 /TIER=ConsEnd






/STK=0 /UG=Hs.296941 /LL=3079






/DEF=H.sapiens mRNA for






complement Factor H-related protein 1,






clone H 36-2. /PROD=FHR-1;






complement Factor H-related protein 1


C3
217767_at
−3.3
Underexpressed
gb:NM_000064.1






/DB_XREF=gi:4557384 /GEN=C3






/FEA=FLmRNA /CNT=473






/TID=Hs.284394.0 /TIER=FL+Stack






/STK=213 /UG=Hs.284394 /LL=718






/DEF=Homo sapiens complement






component 3 (C3), mRNA.






/PROD=complement component 3






precursor /FL=gb:K02765.1






gb:NM_000064.1


CR1
217552_x_at
2
Overexpressed
gb:AI432713 /DB_XREF=gi:4283899






/DB_XREF=th43e02.x1






/CLONE=IMAGE:2121050 /FEA=EST






/CNT=5 /TID=Hs.241053.0






/TIER=ConsEnd /STK=4






/UG=Hs.241053 /UG_TITLE=ESTs


C4BPA
205654_at
−2.7
Underexpressed
gb:NM_000715.1






/DB_XREF=gi:4502502 /GEN=C4BPA






/FEA=FLmRNA /CNT=53






/TID=Hs.1012.0 /TIER=FL+Stack






/STK=22 /UG=Hs.1012 /LL=722






/DEF=Homo sapiens complement






component 4-binding protein, alpha






(C4BPA), mRNA. /PROD=complement






component 4-binding protein, alpha






/FL=gb:NM_000715.1 gb:M31452.1


Flt-4
229902_at
−2.1
Underexpressed
gb:AW083785 /DB_XREF=gi:6038937






/DB_XREF=xc35b04.x1






/CLONE=IMAGE:2586223 /FEA=EST






/CNT=14 /TID=Hs.8941.0






/TIER=Stack /STK=11 /UG=Hs.8941






/UG_TITLE=ESTs


ITGB6
208084_at
−2.1
Underexpressed
gb:NM_000888.3






/DB_XREF=gi:9966771 /GEN=ITGB6






/FEA=FLmRNA /CNT=2






/TID=Hs.123125.0 /TIER=FL /STK=0






/UG=Hs.123125 /LL=3694






/DEF=Homo sapiens integrin, beta 6






(ITGB6), mRNA. /PROD=integrin, beta






6 /FL=gb:NM_000888.3 gb:M35198.3


HTR2B
206638_at
−2.5
Underexpressed
gb:NM_000867.1






/DB_XREF=gi:4504538 /GEN=HTR2B






/FEA=FLmRNA /CNT=13






/TID=Hs.2507.0 /TIER=FL+Stack






/STK=10 /UG=Hs.2507 /LL=3357






/DEF=Homo sapiens 5-






hydroxytryptamine (serotonin) receptor






2B (HTR2B), mRNA. /PROD=5-






hydroxytryptamine (serotonin) receptor






2B /FL=gb:NM_000867.1


S100A8
202917_s_at
13.2
Overexpressed
gb:NM_002964.2






/DB_XREF=gi:9845519






/GEN=S100A8 /FEA=FLmRNA






/CNT=257 /TID=Hs.100000.0






/TIER=FL+Stack /STK=93






/UG=Hs.100000 /LL=6279






/DEF=Homo sapiens S100 calcium-






binding protein A8 (calgranulin A)






(S100A8), mRNA. /PROD=S100






calcium-binding protein A8






/FL=gb:NM_002964.2


ERAP2
219759_at
−9.9
Underexpressed
gb:NM_022350.1






/DB_XREF=gi:11641260






/GEN=LOC64167 /FEA=FLmRNA






/CNT=18 /TID=Hs.280380.0






/TIER=FL /STK=0 /UG=Hs.280380






/LL=64167 /DEF=Homo sapiens






aminopeptidase (LOC64167), mRNA.






/PROD=aminopeptidase






/FL=gb:AF191545.1 gb:NM_022350.1





*Probe ID in Affymetrix HG-U133 Plus 2.0 Genechip













TABLE 2







Gene Expression associated with Decidualization


(Duncan et al., PLOS One. 6(8): e23595 (2011))










Gene Symbol
Fold Change













ALDH1L2
2



RORB
2.3



ACOT8
2.3



EPAS1
2.6



DLGAP1
2.6



SPOCK1
3



MAOB
3.3



GZMB
3.7



IL2RB
3.9



GNLY
5



NOG
5.6



TRA@
6.1



MUC15
6.4



KLRC2
6.8



IL15
8.9



CHRDL1
32



PRL
47



SCARA5
181



CHST6
−2.2



NTN1
−4.3



BICD1
−4.4



ADCYAP1R1
−5.2



CPM
−5.4



DPYSL4
−5.8



HTR2B
5.1



S100A8
8.9



ERAP2
4.1
















TABLE 3







Gene Expression associated with Decidualization*










Gene Symbol
Fold Change/Condition






ALDH1L2
↑ (overexpressed)



GNLY
↑ (overexpressed)



IL15
2.2



IL1B
4.3



MMP12
2.4



BDKRB2
2  



SLC16A6
4.5



COL5A1
−2.3 



PP14
↑ (overexpressed)



FSTL3
 ↑11 (overexpressed)



WT1
 ↑12 (overexpressed)



IGFBP1
5.9



CFH/CFHR1
6.2



C3
↑ (overexpressed)



CR1
↑ (overexpressed)



C4BPA
↑ (overexpressed)



Flt-4
↑ (overexpressed)



ITGB6
5.5





*As disclosed in Hess A P et al. “Decidual stromal cell response to paracrine signals from the trophoblast: amplification of immune and angiogenic modulators.” Biol. Reprod. 76: 102-17 (2007); Guidice L C. “Application of functional genomics to primate endometrium: insights into biological processes.” Repro. Biol. Endocrinol. 4(Suppl I): S4 doi: 10.1186/1477-7827-4-S1-S4; Savaris R F et al. “Endometrial gene expression in early pregnancy: lessons from human ectopic pregnancy.” Repro. Sci. 1797-816 (2008); Guidice L C et al. “Steroid and peptide regulation of insulin-like growth factor-binding proteins secreted by human endometrial stromal cells is dependent on stromal differentiation.” J. Clin. Endocrinol. Metab. 75: 1235-41 (1992); and Popovici R M et al. “Discovery of new inducible genes in in vitro decidualized human endometrial stromal cells using microarray technology.” Endocrinol. 141: 3510-13 (2000).













TABLE 4







Gene Expression associated with Decidualization


(Koopman L A et al. J Exp Med. 198: 1201-12 (2003))










Gene Symbol
Fold Change/Condition













GZMB
10



KLRC2
5



SLC16A6
No change



PP14
5



FOSB
3



MAX
−4
















TABLE 5







Gene Expression associated with Decidualization


(Irwin J C et al. Fertil Steril. 52: 761-8 (1989))










Gene Symbol
Fold Change/Condition






PRL
↑ (overexpressed)



FN1
↑ (overexpressed)
















TABLE 6







Gene Expression associated with Decidualization


(Tseng L et al. DNA and Cell Biol. 22: 633-40 (2003))










Gene Symbol
Fold Change/Condition






FN1
↑ (overexpressed)
















TABLE 7







DEG up-regulated in PE-CVS compared to NP-CVS










Approved





symbol
Approved name
HGNC ID
Location





ABL1
c-abl oncogene 1, non-receptor tyrosine kinase
HGNC: 76
9q34.1


ABLIM2
actin binding LIM protein family, member 2
HGNC: 19195
4p16.1


ACOT8
acyl-CoA thioesterase 8
HGNC: 15919
20q13.12


ACP5
acid phosphatase 5, tartrate resistant
HGNC: 124
19p13.2


ACSS1
acyl-CoA synthetase short-chain family member 1
HGNC: 16091
20p11.23-p11.21


ADCY4
adenylate cyclase 4
HGNC: 235
14q11.2


AHSG
alpha-2-HS-glycoprotein
HGNC: 349
3q27.3


ANKRD20A1
ankyrin repeat domain 20 family, member A1
HGNC: 23665
9p12


ANXA13
annexin A13
HGNC: 536
8q24.13


AOAH
acyloxyacyl hydrolase (neutrophil)
HGNC: 548
7p14-p12


AP1S2
adaptor-related protein complex 1, sigma 2 subunit
HGNC: 560
Xp22


AP3M2
adaptor-related protein complex 3, mu 2 subunit
HGNC: 570
8p11.2


ARFGEF2
ADP-ribosylation factor guanine nucleotide-exchange
HGNC: 15853
20q13.13



factor 2 (brefeldin A-inhibited)




ARL2
ADP-ribosylation factor-like 2
HGNC: 693
11q13


ATP7B
ATPase, Cu++ transporting, beta polypeptide
HGNC: 870
13q14.3


AUTS2
autism susceptibility candidate 2
HGNC: 14262
7q11.22


BCL2A1
BCL2-related protein A1
HGNC: 991
15q24.3


BICD1
bicaudal D homolog 1 (Drosophila)
HGNC: 1049
12p11.2-p11.1


BOLA2
bolA family member 2
HGNC: 29488
16p11.2


C11orf45
chromosome 11 open reading frame 45
HGNC: 28584
11q24.3


DHRS4-AS1
DHRS4 antisense RNA 1
HGNC: 23175
14q11.2


C2orf44
chromosome 2 open reading frame 44
HGNC: 26157
2p23.3


NOP14-AS1
NOP14 antisense RNA 1
HGNC: 20205
4p16.3


NDNF
neuron-derived neurotrophic factor
HGNC: 26256
4q27


CARD16
caspase recruitment domain family, member 16
HGNC: 33701
11q23


CASK
calcium/calmodulin-dependent serine protein kinase
HGNC: 1497
Xp11.4



(MAGUK family)




CCBL2
cysteine conjugate-beta lyase 2
HGNC: 33238
1p22.2


CCDC159
coiled-coil domain containing 159
HGNC: 26996
19p13.2


CCK
cholecystokinin
HGNC: 1569
3p22.1


CCL3
chemokine (C—C motif) ligand 3
HGNC: 10627
17q12


CD52
CD52 molecule
HGNC: 1804
1p36


CD58
CD58 molecule
HGNC: 1688
1p13


CD83
CD83 molecule
HGNC: 1703
6p23


CDH15
cadherin 15, type 1, M-cadherin (myotubule)
HGNC: 1754
16q24.3


CDH26
cadherin 26
HGNC: 15902
20q13.33


CDH3
cadherin 3, type 1, P-cadherin (placental)
HGNC: 1762
16q22.1


CDH6
cadherin 6, type 2, K-cadherin (fetal kidney)
HGNC: 1765
5p13.3


CDK16
cyclin-dependent kinase 16
HGNC: 8749
Xp11


CENPBD1
CENPB DNA-binding domains containing 1
HGNC: 28272
16q24.3


CHST15
carbohydrate (N-acetylgalactosamine 4-sulfate 6-O)
HGNC: 18137
10q26



sulfotransferase 15




COL5A1
collagen, type V, alpha 1
HGNC: 2209
9q34.2-q34.3


COL9A3
collagen, type IX, alpha 3
HGNC: 2219
20q13.3


CR1
complement component (3b/4b) receptor 1 (Knops blood group)
HGNC: 2334
1q32


CTAG2
cancer/testis antigen 2
HGNC: 2492
Xq28


CXCL9
chemokine (C—X—C motif) ligand 9
HGNC: 7098
4q21


DDX3Y
DEAD (Asp-Glu-Ala-Asp) box helicase 3, Y-linked
HGNC: 2699
Yq11


DGKD
diacylglycerol kinase, delta 130 kDa
HGNC: 2851
2q37


DHX30
DEAH (Asp-Glu-Ala-His) box helicase 30
HGNC: 16716
3p24.3-p22.1


DLC1
deleted in liver cancer 1
HGNC: 2897
8p22


DOK4
docking protein 4
HGNC: 19868
16q13


DPYSL3
dihydropyrimidinase-like 3
HGNC: 3015
5q32


DPYSL4
dihydropyrimidinase-like 4
HGNC: 3016
10q25.2-q26


EGR1
early growth response 1
HGNC: 3238
5q23-q31


ELOVL4
ELOVL fatty acid elongase 4
HGNC: 14415
6q14


EVC2
Ellis van Creveld syndrome 2
HGNC: 19747
4p16.2-p16.1


EXD2
exonuclease 3′-5′ domain containing 2
HGNC: 20217
14q24.1


EXOSC6
exosome component 6
HGNC: 19055
16q22.1


F8A1
coagulation factor VIII-associated 1
HGNC: 3547
Xq28


FAM132B
family with sequence similarity 132, member B
HGNC: 26727
2q37.3


FAM189A2
family with sequence similarity 189, member A2
HGNC: 24820
9q21.11


FAM57A
family with sequence similarity 57, member A
HGNC: 29646
17p13.3


FAT1
FAT atypical cadherin 1
HGNC: 3595
4q35.2


FIZ1
FLT3-interacting zinc finger 1
HGNC: 25917
19q13.42


FJX1
four jointed box 1 (Drosophila)
HGNC: 17166
11p13


FKBP1A
FK506 binding protein 1A, 12 kDa
HGNC: 3711
20p13


FLRT2
fibronectin leucine rich transmembrane protein 2
HGNC: 3761
14q24-q32


FOS
FBJ murine osteosarcoma viral oncogene homolog
HGNC: 3796
14q24.3


FOSB
FBJ murine osteosarcoma viral oncogene homolog B
HGNC: 3797
19q13.3


FPR3
formyl peptide receptor 3
HGNC: 3828
19q13.3-q13.4


FUT6
fucosyltransferase 6 (alpha (1,3) fucosyltransferase)
HGNC: 4017
19p13.3


FZD5
frizzled class receptor 5
HGNC: 4043
2q33.3


GBP5
guanylate binding protein 5
HGNC: 19895
1p22.2


GDNF
glial cell derived neurotrophic factor
HGNC: 4232
5p13.1-p12


GPR183
G protein-coupled receptor 183
HGNC: 3128
13q32.3


HBEGF
heparin-binding EGF-like growth factor
HGNC: 3059
5q23


HCFC1
host cell factor C1 (VP16-accessory protein)
HGNC: 4839
Xq28


HEXA
hexosaminidase A (alpha polypeptide)
HGNC: 4878
15q24.1


HINFP
histone H4 transcription factor
HGNC: 17850
11q23.3


HLA-DQA1
major histocompatibility complex, class II, DQ alpha 1
HGNC: 4942
6p21.3


HP
haptoglobin
HGNC: 5141
16q22.2


LGALSL
lectin, galactoside-binding-like
HGNC: 25012
2p14


IKZF1
IKAROS family zinc finger 1 (Ikaros)
HGNC: 13176
7p12.2


IL18BP
interleukin 18 binding protein
HGNC: 5987
11q13


IPCEF1
interaction protein for cytohesin exchange factors 1
HGNC: 21204
6q25.2


ISG20
interferon stimulated exonuclease gene 20 kDa
HGNC: 6130
15q26


ISL1
ISL LIM homeobox 1
HGNC: 6132
5q11.2


ITFG2
integrin alpha FG-GAP repeat containing 2
HGNC: 30879
12p13.33


ITGA9
integrin, alpha 9
HGNC: 6145
3p21.3


KIF22
kinesin family member 22
HGNC: 6391
16p11.2


KLHL6
kelch-like family member 6
HGNC: 18653
3q27.3


ERAP2
endoplasmic reticulum aminopeptidase 2
HGNC: 29499
5q15


LRP1
low density lipoprotein receptor-related protein 1
HGNC: 6692
12q13.3


PPP1R37
protein phosphatase 1, regulatory subunit 37
HGNC: 27607
19q13.32


LRRC8D
leucine rich repeat containing 8 family, member D
HGNC: 16992
1p22.2


LY96
lymphocyte antigen 96
HGNC: 17156
8q13.3


MAGEL2
MAGE-like 2
HGNC: 6814
15q11-q12


MAP1S
microtubule-associated protein 1S
HGNC: 15715
19p13.12


MAP2K7
mitogen-activated protein kinase kinase 7
HGNC: 6847
19p13.3-p13.2


MED21
mediator complex subunit 21
HGNC: 11473
12p12


MED22
mediator complex subunit 22
HGNC: 11477
9q34.1


MGA
MGA, MAX dimerization protein
HGNC: 14010
15q15


MGMT
O-6-methylguanine-DNA methyltransferase
HGNC: 7059
10q26


MIIP
migration and invasion inhibitory protein
HGNC: 25715
1p36.22


MPPED2
metallophosphoesterase domain containing 2
HGNC: 1180
11p13


MRVI1
murine retrovirus integration site 1 homolog
HGNC: 7237
11p15


MSR1
macrophage scavenger receptor 1
HGNC: 7376
8p22


MTRR
5-methyltetrahydrofolate-homocysteine
HGNC: 7473
5p15.31



methyltransferase reductase




MYL9
myosin, light chain 9, regulatory
HGNC: 15754
20q11.23


NAAA
N-acylethanolamine acid amidase
HGNC: 736
4q21.1


NAP1L3
nucleosome assembly protein 1-like 3
HGNC: 7639
Xq21.3-q22


NDN
necdin, melanoma antigen (MAGE) family member
HGNC: 7675
15q11-q12


NINJ2
ninjurin 2
HGNC: 7825
12p13


NKX2-5
NK2 homeobox 5
HGNC: 2488
5q34


NMNAT3
nicotinamide nucleotide adenylyltransferase 3
HGNC: 20989
3q23


NNAT
neuronatin
HGNC: 7860
20q11.2-q12


NOTCH4
notch 4
HGNC: 7884
6p21.3


NUB1
negative regulator of ubiquitin-like proteins 1
HGNC: 17623
7q36


OPRL1
opiate receptor-like 1
HGNC: 8155
20q13.33


ADCYAP1
adenylate cyclase activating polypeptide 1 (pituitary)
HGNC: 241
18p11


PDE4B
phosphodiesterase 4B, cAMP-specific
HGNC: 8781
1p31


PDE9A
phosphodiesterase 9A
HGNC: 8795
21q22.3


PDXP
pyridoxal (pyridoxine, vitamin B6) phosphatase
HGNC: 30259
22q12.3


PGA3
pepsinogen 3, group I (pepsinogen A)
HGNC: 8885
11q13


PLA2G4A
phospholipase A2, group IVA (cytosolic, calcium-
HGNC: 9035
1q25



dependent)




PLEKHG4B
pleckstrin homology domain containing, family G
HGNC: 29399
5p15.33



(with RhoGef domain) member 4B




POMZP3
POM121 and ZP3 fusion
HGNC: 9203
7q11.2


PPM1H
protein phosphatase, Mg2+/Mn2+ dependent, 1H
HGNC: 18583
12q14.1


PPM1M
protein phosphatase, Mg2+/Mn2+ dependent, 1M
HGNC: 26506
3p21.31


PPP1R13B
protein phosphatase 1, regulatory subunit 13B
HGNC: 14950
14q32.33


PPP1R9A
protein phosphatase 1, regulatory subunit 9A
HGNC: 14946
7q21.3


PTPRN
protein tyrosine phosphatase, receptor type, N
HGNC: 9676
2q35-q36.1


RAD52
RAD52 homolog (S. cerevisiae)
HGNC: 9824
12p13-p12.2


REST
RE1-silencing transcription factor
HGNC: 9966
4q12


RFTN1
raftlin, lipid raft linker 1
HGNC: 30278
3p24.3


RGPD1
RANBP2-like and GRIP domain containing 1
HGNC: 32414
2p11.2


RIN1
Ras and Rab interactor 1
HGNC: 18749
11q13.2


RPGR
retinitis pigmentosa GTPase regulator
HGNC: 10295
Xp11.4


RPL31
ribosomal protein L31
HGNC: 10334
2q11.2


RPS4Y1
ribosomal protein S4, Y-linked 1
HGNC: 10425
Yp11.3


S100A12
S100 calcium binding protein A12
HGNC: 10489
1q21


S100A8
S100 calcium binding protein A8
HGNC: 10498
1q12-q22


SACS
sacsin molecular chaperone
HGNC: 10519
13q11


SCARB2
scavenger receptor class B, member 2
HGNC: 1665
4q21.1


SCML2
sex comb on midleg-like 2 (Drosophila)
HGNC: 10581
Xp22


SEZ6L
seizure related 6 homolog (mouse)-like
HGNC: 10763
22q12.1


SH3BP1
SH3-domain binding protein 1
HGNC: 10824
22q13.1


SH3BP5L
SH3-binding domain protein 5-like
HGNC: 29360
1q44


SIRT5
sirtuin 5
HGNC: 14933
6p23


SLC13A5
solute carrier family 13 (sodium-dependent citrate
HGNC: 23089
17p13.1



transporter), member 5




SLC22A7
solute carrier family 22 (organic anion transporter),
HGNC: 10971
6p21.1



member 7




SLC25A29
solute carrier family 25 (mitochondrial
HGNC: 20116
14q32.2



carnitine/acylcarnitine carrier), member 29 




SNRNP25
small nuclear ribonucleoprotein 25 kDa (U11/U12)
HGNC: 14161
16p13.3


SNRPN
small nuclear ribonucleoprotein polypeptide N
HGNC: 11164
15q11.2


SNX16
sorting nexin 16
HGNC: 14980
8q21.13


SORD
sorbitol dehydrogenase
HGNC: 11184
15q15-q21.1


SPDEF
SAM pointed domain containing ETS transcription
HGNC: 17257
6p21.3



factor




SPRR2B
small proline-rich protein 2B
HGNC: 11262
1q21-q22


SPRY1
sprouty homolog 1, antagonist of FGF signaling
HGNC: 11269
4q



(Drosophila)




SRF
serum response factor (c-fos serum response element-
HGNC: 11291
6p



binding transcription factor)




STAG3
stromal antigen 3
HGNC: 11356
7q22


STXBP2
syntaxin binding protein 2
HGNC: 11445
19p13.3-p13.2


TBC1D7
TBC1 domain family, member 7
HGNC: 21066
6p23


TGIF2
TGFB-induced factor homeobox 2
HGNC: 15764
20q11.23


TMEM100
transmembrane protein 100
HGNC: 25607
17q23.1


TMEM106C
transmembrane protein 106C
HGNC: 28775
12q13.1


TMEM216
transmembrane protein 216
HGNC: 25018
11q13.1


TMEM229B
transmembrane protein 229B
HGNC: 20130
14q23.3-q24.1


TMSB15A
thymosin beta 15a
HGNC: 30744
Xq21.33-q22.3


TNFSF12
tumor necrosis factor (ligand) superfamily, member 12
HGNC: 11927
17p13.1


TPTE
transmembrane phosphatase with tensin homology
HGNC: 12023
21p11


TRIM3
tripartite motif containing 3
HGNC: 10064
11p15.5


TRIM55
tripartite motif containing 55
HGNC: 14215
8q13.1


TRMT2B
tRNA methyltransferase 2 homolog B (S. cerevisiae)
HGNC: 25748
Xq22.1


TRPV2
transient receptor potential cation channel, subfamily V, member 2
HGNC: 18082
17p11.2


TRRAP
transformation/transcription domain-associated protein
HGNC: 12347
7q21.2-q22.1


TSSC1
tumor suppressing subtransferable candidate 1
HGNC: 12383
2p25.3


TUBB1
tubulin, beta 1 class VI
HGNC: 16257
20q13.32


UGT2B7
UDP glucuronosyltransferase 2 family, polypeptide B7
HGNC: 12554
4q13


ULK3
unc-51 like kinase 3
HGNC: 19703
15q24.1


WNT10B
wingless-type MMTV integration site family, member 10B
HGNC: 12775
12q13


ZFP57
ZFP57 zinc finger protein
HGNC: 18791
6p22.1


ZKSCAN2
zinc finger with KRAB and SCAN domains 2
HGNC: 25677
16p12.1


ZMYM3
zinc finger, MYM-type 3
HGNC: 13054
Xq13.1


ZNF101
zinc finger protein 101
HGNC: 12881
19p13.11


ZNF383
zinc finger protein 383
HGNC: 18609
19q13.13


ZNF385A
zinc finger protein 385A
HGNC: 17521
12q13.13


ZNF469
zinc finger protein 469
HGNC: 23216
16q24


ZNF542P
zinc finger protein 542, pseudogene
HGNC: 25393
19q13.43


ZNF571
zinc finger protein 571
HGNC: 25000
19q13.12


ZNF581
zinc finger protein 581
HGNC: 25017
19q13.42


ZP3
zona pellucida glycoprotein 3 (sperm receptor)
HGNC: 13189
7q11.23


LOC100131366
. . .
. . .
. . .


LOC100132147
. . .
. . .
. . .


LOC100132999
. . .
. . .
. . .


LOC100271836
. . .
. . .
. . .


LOC100505956
. . .
. . .
. . .


LOC151146
. . .
. . .
. . .


LOC643529
. . .
. . .
. . .


LOC728377
. . .
. . .
. . .


MGC34034
. . .
. . .
. . .


PK155
. . .
. . .
. . .
















TABLE 8







DEG down-regulated in PE-CVS compared to NP-CVS











Approved



Literature


symbol
Approved name
HGNC ID
Location
DEC PMID














ACACA
acetyl-CoA carboxylase alpha
HGNC: 84
17q21



ACOT1 ///
acyl-CoA thioesterase 1 /// 2
HGNC: 33128 ///
14q24.3



ACOT2

HGNC: 18431




AGAP3
ArfGAP with GTPase domain, ankyrin repeat
HGNC: 16923
7q36.1




and PH domain 3





AIF1L
allograft inflammatory factor 1-like
HGNC: 28904
9q34.13-q34.3



AKR7A3
aldo-keto reductase family 7, member A3
HGNC: 390
1p36.13




(aflatoxin aldehyde reductase)





ALDH1A2
aldehyde dehydrogenase 1 family, member A2
HGNC: 15472
15q21.2



ALS2CL
ALS2 C-terminal like
HGNC: 20605
3p21.31



AOC1
amine oxidase, copper containing 1
HGNC: 80
7q36.1
20668027


APC
adenomatous polyposis coli
HGNC: 583
5q21-q22



AQP2
aquaporin 2 (collecting duct)
HGNC: 634
12q12-q13



ART1
ADP-ribosyltransferase 1
HGNC: 723
11p15



ASCL2
achaete-scute family bHLH transcription factor 2
HGNC: 739
11p15.5



AXIN1
axin 1
HGNC: 903
16p13.3



BAIAP2L1
BAI1-associated protein 2-like 1
HGNC: 21649
7q22.1



BDKRB2
bradykinin receptor B2
HGNC: 1030
14q32.1-q32.2



BEAN1
brain expressed, associated with NEDD4, 1
HGNC: 24160
16q21



BEX1
brain expressed, X-linked 1
HGNC: 1036
Xq22.1



BLNK
B-cell linker
HGNC: 14211
10q23.2-q23.33



BSG
basigin (Ok blood group)
HGNC: 1116
19p13.3
12141934


C12orf75
chromosome 12 open reading frame 75
HGNC: 35164
12q23.3



C3
complement component 3
HGNC: 1318
19p13.3-p13.2
8311932


C4BPA
complement component 4 binding protein, alpha
HGNC: 1325
1q32



C7orf71
chromosome 7 open reading frame 71
HGNC: 22364
7p15.2



CA12
carbonic anhydrase XII
HGNC: 1371
15q22



CA2
carbonic anhydrase II
HGNC: 1373
8q21.2
9692790


CC2D2B
coiled-coil and C2 domain containing 2B
HGNC: 31666
10q23.33



CCDC113
coiled-coil domain containing 113
HGNC: 25002
16q21



CCDC125
coiled-coil domain containing 125
HGNC: 28924
5q13.2



CFH ///
complement factor H /// complement factor
HGNC: 4883 ///
1q32



CFHR1
H-related 1
HGNC: 4888




CHERP
calcium homeostasis endoplasmic reticulum
HGNC: 16930
19p13.1




protein





CHRDL1
chordin-like 1
HGNC: 29861
Xq23



CHST2
carbohydrate (N-acetylglucosamine-6-O)
HGNC: 1970
3q24




sulfotransferase 2





CHST6
carbohydrate (N-acetylglucosamine 6-O)
HGNC: 6938
16q22




sulfotransferase 6





CLASP2
cytoplasmic linker associated protein 2
HGNC: 17078
3p24.3



CLCN7
chloride channel, voltage-sensitive 7
HGNC: 2025
16p13



CLDN6
claudin 6
HGNC: 2048
16p13.3



CMAHP
cytidine monophospho-N-acetylneuraminic
HGNC: 2098
6p23-p22




acid hydroxylase, pseudogene 





CMTM4
CKLF-like MARVEL transmembrane domain containing 4
HGNC: 19175
16q22.1-q22.3



COL27A1
collagen, type XXVII, alpha 1
HGNC: 22986
9q33.1



COTL1
coactosin-like F-actin binding protein 1
HGNC: 18304
16q24.1



CPM
carboxypeptidase M
HGNC: 2311
12q15



CPXM2
carboxypeptidase X (M14 family), member 2
HGNC: 26977
10q26



CRH
corticotropin releasing hormone
HGNC: 2355
8q13
159239


CRYBB1
crystallin, beta B1
HGNC: 2397
22q12.1



CUL1
cullin 1
HGNC: 2551
7q36.1



CYP4A11
cytochrome P450, family 4, subfamily A,
HGNC: 2642
1p33




polypeptide 11





CYTH2
cytohesin 2
HGNC: 9502
19q13.32



DEPDC7
DEP domain containing 7
HGNC: 29899
11p13



DHRS2
dehydrogenase/reductase (SDR family) member 2
HGNC: 18349
14q11.2



DLGAP1
discs, large (Drosophila) homolog-
HGNC: 2905
18p11.3




associated protein 1





DNAJC6
DnaJ (Hsp40) homolog, subfamily C, member 6
HGNC: 15469
1p31.3



DSC2
desmocollin 2
HGNC: 3036
18q12.1



DUXAP10
double homeobox A pseudogene 10
HGNC: 32189
14q11.2



EFCAB2
EF-hand calcium binding domain 2
HGNC: 28166
1q44



EGLN3
egl-9 family hypoxia-inducible factor 3
HGNC: 14661
14q12



ELL2
elongation factor, RNA polymerase II, 2
HGNC: 17064
5q15



EPAS1
endothelial PAS domain protein 1
HGNC: 3374
2p21-p16



ERAP2
endoplasmic reticulum aminopeptidase 2
HGNC: 29499
5q15
24331737


ERO1L
ERO1-like (S. cerevisiae)
HGNC: 13280
14q22.1



F11R
F11 receptor
HGNC: 14685
1q21.2-q21.3



F2R
coagulation factor II (thrombin) receptor
HGNC: 3537
5q13
12549865


FABP7
fatty acid binding protein 7, brain
HGNC: 3562
6q22-q23



FAM3B
family with sequence similarity 3, member B
HGNC: 1253
21q22.3



FHL2
four and a half LIM domains 2
HGNC: 3703
2q12.2



FKBP11
FK506 binding protein 11, 19 kDa
HGNC: 18624
12q13.12



FLT4
fms-related tyrosine kinase 4
HGNC: 3767
5q34-q35
11297624


FN1
fibronectin 1
HGNC: 3778
2q34
14611684


FSTL3
follistatin-like 3 (secreted glycoprotein)
HGNC: 3973
19p13
15130517


GATA1
GATA binding protein 1 (globin transcription factor 1)
HGNC: 4170
Xp11.23



GDA
guanine deaminase
HGNC: 4212
9q21.13



GNG4
guanine nucleotide binding protein (G protein), gamma 4
HGNC: 4407
1q42.3



GNG7
guanine nucleotide binding protein (G protein), gamma 7
HGNC: 4410
19p13.3



GNLY
granulysin
HGNC: 4414
2p12-q11
21623991


GOLGA8B
golgin A8 family, member B
HGNC: 31973
15q14



GPR158
G protein-coupled receptor 158
HGNC: 23689
10p12.31



GTPBP2
GTP binding protein 2
HGNC: 4670
6p21



GZMB
granzyme B (granzyme 2, cytotoxic T-
HGNC: 4709
14q11.2
16451356



lymphocyte-associated serine esterase 1)





HBE1
hemoglobin, epsilon 1
HGNC: 4830
11p15.5



HBZ
hemoglobin, zeta
HGNC: 4835
16p13.3



HCAR3
hydroxycarboxylic acid receptor 3
HGNC: 16824
12q24.31



HIVEP2
human immunodeficiency virus type I
HGNC: 4921
6q23-q24




enhancer binding protein 2





HOXB7
homeobox B7
HGNC: 5118
17q21.32



HPS3
Hermansky-Pudlak syndrome 3
HGNC: 15597
3q24



HTR2B
5-hydroxytryptamine (serotonin) receptor 2B,
HGNC: 5294
2q36.3-q37.1




G protein-coupled 





HYDIN
HYDIN, axonemal central pair apparatus protein
HGNC: 19368
16q22.2



IGFBP1
insulin-like growth factor binding protein 1
HGNC: 5469
7p13-p12
1385468


IGKC
immunoglobulin kappa constant
HGNC: 5716
2p11.2



IL15
interleukin 15
HGNC: 5977
4q31
10952908


IL1B
interleukin 1, beta
HGNC: 5992
2q14
16860880


IL1RL1
interleukin 1 receptor-like 1
HGNC: 5998
2q12
23300625


IL2RB
interleukin 2 receptor, beta
HGNC: 6009
22q13
21248224


INPP4B
inositol polyphosphate-4-phosphatase, type II, 105 kDa
HGNC: 6075
4q31.1



ITCH
itchy E3 ubiquitin protein ligase
HGNC: 13890
20q11.22



ITGB6
integrin, beta 6
HGNC: 6161
2q24.2



KCNH2
potassium voltage-gated channel, subfamily H
HGNC: 6251
7q36.1




(eag-related), member 2





KCNIP3
Kv channel interacting protein 3, calsenilin
HGNC: 15523
2q21.1



KCNQ1
potassium voltage-gated channel, KQT-like
HGNC: 6294
11p15.5




subfamily, member 1





KISS1R
KISS1 receptor
HGNC: 4510
19p13.3
24225150


KLRC2
killer cell lectin-like receptor subfamily C, member 2
HGNC: 6375
12p13
16488482


KRT14
keratin 14
HGNC: 6416
17q21.2



LAIR2
leukocyte-associated immunoglobulin-like receptor 2
HGNC: 6478
19q13.4



LAMA4
laminin, alpha 4
HGNC: 6484
6q21



LIAS
lipoic acid synthetase
HGNC: 16429
4p14



LIPH
lipase, member H
HGNC: 18483
3q27



LIPT1
lipoyltransferase 1
HGNC: 29569
2q11.2



LSS
lanosterol synthase (2,3-oxidosqualene-
HGNC: 6708
21q22.3




lanosterol cyclase)





LTBR
lymphotoxin beta receptor (TNFR
HGNC: 6718
12p13




superfamily, member 3)





MAGEB6
melanoma antigen family B, 6
HGNC: 23796
Xp22.12



MAOB
monoamine oxidase B
HGNC: 6834
Xp11.4-p11.3



MAP3K5
mitogen-activated protein kinase kinase kinase 5
HGNC: 6857
6q22.33



MLIP
muscular LMNA-interacting protein
HGNC: 21355
6p12.2-p12.1



MMD
monocyte to macrophage differentiation-associated
HGNC: 7153
17q



MMP12
matrix metallopeptidase 12 (macrophage
HGNC: 7158
11q22.3
20802175



elastase)





MUC15
mucin 15, cell surface associated
HGNC: 14956
11p14.3
17720698


MUC4
mucin 4, cell surface associated
HGNC: 7514
3q29



MVK
mevalonate kinase
HGNC: 7530
12q24



NDP
Norrie disease (pseudoglioma)
HGNC: 7678
Xp11.4-p11.3
16035034


NDUFV2
NADH dehydrogenase (ubiquinone)
HGNC: 7717
18p11.22




flavoprotein 2, 24 kDa





NOG
noggin
HGNC: 7866
17q22
11158592


NOTUM
notum pectinacetylesterase homolog (Drosophila)
HGNC: 27106
17q25.3



NTN1
netrin 1
HGNC: 8029
17p13-p12



NTN4
netrin 4
HGNC: 13658
12q22



NUDT13
nudix (nucleoside diphosphate linked moiety X)-
HGNC: 18827
10q22.3




type motif 13





OXGR1
oxoglutarate (alpha-ketoglutarate) receptor 1
HGNC: 4531
13q32.2



P4HA3
prolyl 4-hydroxylase, alpha polypeptide III
HGNC: 30135
11q13



PAEP
progestagen-associated endometrial protein
HGNC: 8573
9q34
3194393


PARP16
poly (ADP-ribose) polymerase family, member 16
HGNC: 26040
15q22.2



PAWR
PRKC, apoptosis, WT1, regulator
HGNC: 8614
12q21.2



PDE4C
phosphodiesterase 4C, cAMP-specific
HGNC: 8782
19p13.11
14715868


PITPNC1
phosphatidylinositol transfer protein,
HGNC: 21045
17q24.3




cytoplasmic 1





PLAC8
placenta-specific 8
HGNC: 19254
4q21.22



PLCXD2
phosphatidylinositol-specific phospholipase
HGNC: 26462
3q13.2




C, X domain containing 2





PPDPF
pancreatic progenitor cell differentiation and
HGNC: 16142
20q13.33




proliferation factor





PPP1R3C
protein phosphatase 1, regulatory subunit 3C
HGNC: 9293
10q23-q24



PRDM1
PR domain containing 1, with ZNF domain
HGNC: 9346
6q21



PRG2
proteoglycan 2, bone marrow (natural killer
HGNC: 9362
11q12




cell activator, eosinophil granule major basic






protein)





PRKAB2
protein kinase, AMP-activated, beta 2 non-
HGNC: 9379
1q21.2




catalytic subunit





PRL
prolactin
HGNC: 9445
6p22.3
10611264


PSG11
pregnancy specific beta-1-glycoprotein 11
HGNC: 9516
19q13.2



PTPRS
protein tyrosine phosphatase, receptor type, S
HGNC: 9681
19p13.3



PVR
poliovirus receptor
HGNC: 9705
19q13.2



RAB12
RAB12, member RAS oncogene family
HGNC: 31332
18p11.22



RBP4
retinol binding protein 4, plasma
HGNC: 9922
10q23.33



RHD
Rh blood group, D antigen
HGNC: 10009
1p36.11



RNF14
ring finger protein 14
HGNC: 10058
5q23.3-q31.1



RORB
RAR-related orphan receptor B
HGNC: 10259
9q22



RSRC1
arginine/serine-rich coiled-coil 1
HGNC: 24152
3q25.32



RUFY3
RUN and FYVE domain containing 3
HGNC: 30285
4q13.3



SART3
squamous cell carcinoma antigen recognized
HGNC: 16860
12q24.11




by T cells 3





SCARA5
scavenger receptor class A, member 5 (putative)
HGNC: 28701
8p21.1
21858178


SDHAP1
Succinate dehydrogenase complex, subunit A,
HGNC: 32455
3q29




flavoprotein pseudogene 1





SEC24D
SEC24 family member D
HGNC: 10706
4q26



SEMA3C
sema domain, immunoglobulin domain (Ig),
HGNC: 10725
7q21-q31




short basic domain, secreted, (semaphorin) 3C





SERPINA3
serpin peptidase inhibitor, clade A (alpha-1
HGNC: 16
14q32.1
8951488



antiproteinase, antitrypsin), member 3





SFI1
Sfi1 homolog, spindle assembly associated (yeast)
HGNC: 29064
22q12.2



SGSM1
small G protein signaling modulator 1
HGNC: 29410
22q11.23



SLC13A4
solute carrier family 13 (sodium/sulfate
HGNC: 15827
7q33




symporter), member 4





SLC16A6
solute carrier family 16, member 6
HGNC: 10927
17q24.2



SLC25A15
solute carrier family 25 (mitochondrial
HGNC: 10985
13q14




carrier; ornithine transporter) member 15





SLC26A7
solute carrier family 26 (anion exchanger), member 7
HGNC: 14467
8q23



SLC2A3
solute carrier family 2 (facilitated glucose
HGNC: 11007
12p13.3
12915684



transporter), member 3





SLC36A1
solute carrier family 36 (proton/amino acid
HGNC: 18761
5q33.1




symporter), member 1





SLC44A3
solute carrier family 44, member 3
HGNC: 28689
1p22.1



SLCO4A1
solute carrier organic anion transporter
HGNC: 10953
20q13.1




family, member 4A1





SNX25
sorting nexin 25
HGNC: 21883
4q35.1



SOWAHC
sosondowah ankyrin repeat domain family
HGNC: 26149
2q13




member C





SP140L
SP140 nuclear body protein-like
HGNC: 25105
2q37.1



SPG20
spastic paraplegia 20 (Troyer syndrome)
HGNC: 18514
13q13.1



SPOCK1
sparc/osteonectin, cwcv and kazal-like
HGNC: 11251
5q31.2




domains proteoglycan (testican) 1





SSTR1
somatostatin receptor 1
HGNC: 11330
14q13



ST3GAL6
ST3 beta-galactoside alpha-2,3-sialyltransferase 6
HGNC: 18080
3q12.2



ST6GALNAC4
ST6 (alpha-N-acetyl-neuraminyl-2,3-beta-
HGNC: 17846
9q34




galactosyl-1,3)-N-acetylgalactosaminide






alpha-2,6-sialyltransferase 4





SYCP2L
synaptonemal complex protein 2-like
HGNC: 21537
6p24.2



SYT1
synaptotagmin I
HGNC: 11509
12q21.2



TES
testis derived transcript (3 LIM domains)
HGNC: 14620
7q31.2



THBS4
thrombospondin 4
HGNC: 11788
5q13



THUMPD2
THUMP domain containing 2
HGNC: 14890
2p22.2



TIAM1
T-cell lymphoma invasion and metastasis 1
HGNC: 11805
21q22.1



TLN2
talin 2
HGNC: 15447
15q15-q21



TMC4
transmembrane channel-like 4
HGNC: 22998
19q13.42



TMEM62
transmembrane protein 62
HGNC: 26269
15q15.2
24767823


TOX3
TOX high mobility group box family member 3
HGNC: 11972
16q12.1



TPM1
tropomyosin 1 (alpha)
HGNC: 12010
15q22.1



TRA@
T cell receptor alpha locus
HGNC: 12027
14q11.2



TREML2
triggering receptor expressed on myeloid cells-like 2
HGNC: 21092
6p21.1



TSTD1
thiosulfate sulfurtransferase (rhodanese)-like
HGNC: 35410
1q23.3




domain containing 1





TTC18
tetratricopeptide repeat domain 18
HGNC: 30726
10q22.3



USP5
ubiquitin specific peptidase 5 (isopeptidase T)
HGNC: 12628
12p13



WT1
Wilms tumor 1
HGNC: 12796
11p13
11739471


XRCC4
X-ray repair complementing defective repair
HGNC: 12831
5q14.2




in Chinese hamster cells 4





ZFP62
ZFP62 zinc finger protein
HGNC: 23241
5q35.3



ZNF165
zinc finger protein 165
HGNC: 12953
6p21



LOC153546
. . .
. . .
. . .
. . .


LOC440157
. . .
. . .
. . .
. . .


FLJ13744
. . .
. . .
. . .
. . .
















TABLE 9







Enriched Biological processes for DEG down-regulated in PE-CVS compared to NP-CVS


(DAVID Bioinformatics Resources 6.7)












Enriched Biological


Fold




Process
#Genes
Genes
Enrichment
p value
Benjamini















GO: 0006952~defense
23
F11R, KLRC2, C3,
3.285228593
1.58E−06
0.002391642


response

IL1RL1, GNLY,







PRG2, CHST2,







C4BPA, BDKRB2,







IL15, COTL1,







TRA@, CFHR1,







ITGB6,







SERPINA3, CRH,







CFH, IL1B, ITCH,







GNG7, F2R, FN1,







BLNK





GO: 0006954~inflammatory
16
F11R, C3, CHST2,
4.324635365
4.13E−06
0.003132854


response

C4BPA, BDKRB2,







IL15, CFHR1,







ITGB6,







SERPINA3, CFH,







CRH, IL1B, ITCH,







F2R, FN1, BLNK





GO: 0009611~response to
19
F11R, NOG, C3,
3.149130115
3.04E−05
0.015286151


wounding

CHST2, C4BPA,







BDKRB2, IL15,







TPM1, CFHR1,







ITGB6,







SERPINA3, CRH,







CFH, IL1B,







IGFBP1, ITCH,







F2R, FN1, BLNK





GO: 0008285~negative
15
RBP4, NOG,
3.650034176
6.03E−05
0.02264638


regulation of cell

BDKRB2, IL15,





proliferation

PAWR, DHRS2,







ALDH1A2,







KISS1R, SSTR1,







IL1B, ITCH,







FABP7, CUL1,







F2R, APC





GO: 0042127~regulation of
21
XRCC4, RBP4,
2.343999076
5.61E−04
0.156679481


cell proliferation

NOG, FLT4,







EGLN3, BDKRB2,







IL15, PAWR,







NTN1, MMP12,







DHRS2,







ALDH1A2,







KISS1R, SSTR1,







IL1B, ITCH,







FABP7, PRL,







CUL1, F2R, APC





GO: 0002526~acute
7
CFHR1, C3, CFH,
6.27458256
8.49E−04
0.193443436


inflammatory response

SERPINA3, IL1B,







C4BPA, FN1





GO: 0046883~regulation of
6
RBP4, KISS1R,
7.985832349
8.89E−04
0.17553566


hormone secretion

GPR109B, CRH,







IL1B, KCNQ1





GO: 0048871~multicellular
6
RBP4, EPAS1,
6.200763942
0.002749326
0.407110431


organismal homeostasis

SERPINA3,







ACACA, IL1B,







F2R





GO: 0048584~positive
9
CFHR1, PVR, C3,
3.349988994
0.005404771
0.599354927


regulation of response to

CFH, CRH, IL1B,





stimulus

IL15, C4BPA,







AXIN1





GO: 0001894~tissue
5
RBP4, EPAS1,
6.9717584
0.005586825
0.573020053


homeostasis

SERPINA3,







ACACA, F2R





GO: 0050778~positive
7
CFHR1, PVR, C3,
4.240752351
0.006079294
0.569176788


regulation of immune

CFH, IL1B, IL15,





response

C4BPA





GO: 0060249~anatomical
6
RBP4, EPAS1,
4.972310708
0.007035029
0.590846406


structure homeostasis

SERPINA3,







ACACA, F2R,







APC





GO: 0010627~regulation of
9
MAP3K5, LTBR,
3.17508997
0.007392779
0.5798009


protein kinase cascade

IL1RL1, IL1B,







HTR2B, PRL,







F2R, AXIN1, APC





GO: 0046887~positive
4
RBP4, KISS1R,
9.76046176
0.007706954
0.568049167


regulation of hormone

GPR109B, CRH





secretion







GO: 0001655~urogenital
6
ALDH1A2, RBP4,
4.79149941
0.008197726
0.565508003


system development

NOG, CA2, WT1,







APC





GO: 0044057~regulation of
10
KISS1R, EPAS1,
2.84285294
0.008537529
0.556922869


system process

GPR109B, CRH,







IL1B, BDKRB2,







KCNH2, TPM1,







KCNQ1, F2R





GO: 0006956~complement
4
CFHR1, C3, CFH,
8.36611008
0.011796833
0.653664906


activation

C4BPA





GO: 0006959~humoral
5
CFHR1, C3, CFH,
5.559756699
0.012269978
0.647200259


immune response

C4BPA, BLNK





GO: 0002541~activation of
4
CFHR1, C3, CFH,
8.171549381
0.012578263
0.636500333


plasma proteins involved in

C4BPA





acute inflammatory







response







GO: 0051130~positive
7
KISS1R, C3,
3.397287795
0.016896621
0.725900409


regulation of cellular

TIAM1, IL1B,





component organization

TPM1, NTN1,







APC





GO: 0015669~gas transport
3
HBZ, CA2, HBE1
14.64069264
0.017267525
0.716325311


GO: 0046717~acid
3
KISS1R,
14.64069264
0.017267525
0.716325311


secretion

SLC26A7,







BDKRB2





GO: 0030097~hemopoiesis
8
XRCC4, DHRS2,
2.977767995
0.017831541
0.711279469




HOXB7, EPAS1,







HBZ, IL15,







BLNK, APC





GO: 0002684~positive
8
CFHR1, PVR, C3,
2.952744734
0.018585144
0.710319549


regulation of immune

CFH, IL1B, IL15,





system process

ITCH, C4BPA





GO: 0003013~circulatory
7
ERAP2, BDKRB2,
3.305962854
0.019095627
0.70485324


system process

KCNH2, HTR2B,







TPM1, KCNQ1,







AQP2





GO: 0008015~blood
7
ERAP2, BDKRB2,
3.305962854
0.019095627
0.70485324


circulation

KCNH2, HTR2B,







TPM1, KCNQ1,







AQP2





GO: 0070163~regulation of
2
GPR109B, IL1B
87.84415584
0.022492665
0.748989022


adiponectin secretion







GO: 0060341~regulation of
8
SYT1, RBP4,
2.833682447
0.022691482
0.738410497


cellular localization

KISS1R,







GPR109B, CRH,







IL1B, KCNQ1,







APC





GO: 0001822~kidney
5
ALDH1A2, NOG,
4.57521645
0.023438757
0.736669831


development

CA2, WT1, APC





GO: 0009894~regulation of
5
PPP1R3C,
4.57521645
0.023438757
0.736669831


catabolic process

GPR109B, IL1B,







ITCH, APC





GO: 0009725~response to
10
ALDH1A2, RBP4,
2.393573729
0.023681675
0.727519347


hormone stimulus

BSG, FHL2, IL1B,







CA2, IGFBP1,







GNG4, RNF14,







GNG7





GO: 0046942~carboxylic
6
SLC36A1,
3.585475749
0.025707542
0.744403169


acid transport

KISS1R,







SLC16A6,







SLC26A7,







BDKRB2,







SLC25A15





GO: 0015849~organic acid
6
SLC36A1,
3.561249561
0.026373782
0.741618489


transport

KISS1R,







SLC16A6,







SLC26A7,







BDKRB2,







SLC25A15





GO: 0051046~regulation of
7
SYT1, RBP4,
3.04410441
0.027285984
0.742205581


secretion

KISS1R,







GPR109B, CRH,







IL1B, KCNQ1





GO: 0007155~cell adhesion
15
PVR, F11R, TLN2,
1.882374768
0.027332218
0.73165635




CLDN6, CPXM2,







PTPRS, SPOCK1,







MUC4, LAMA4,







COL27A1, ITGB6,







DSC2, APC, FN1,







THBS4





GO: 0002697~regulation of
5
PVR, RBP4, C3,
4.348720586
0.02760069
0.72426926


immune effector process

IL1B, IL15





GO: 0022610~biological
15
PVR, F11R, TLN2,
1.879689497
0.027622076
0.713902032


adhesion

CLDN6, CPXM2,







PTPRS, SPOCK1,







MUC4, LAMA4,







COL27A1, ITGB6,







DSC2, APC, FN1,







THBS4





GO: 0048534~hemopoietic
8
XRCC4, DHRS2,
2.702897103
0.028401277
0.713626389


or lymphoid organ

HOXB7, EPAS1,





development

HBZ, IL15,







BLNK, APC





GO: 0010817~regulation of
6
ALDH1A2,
3.490496259
0.028432612
0.703907645


hormone levels

DHRS2, RBP4,







SLCO4A1,







FAM3B, CRH





GO: 0034754~cellular
4
ALDH1A2,
5.955535989
0.029086816
0.702351713


hormone metabolic process

DHRS2, RBP4,







CRH





GO: 0001890~placenta
4
BSG, EPAS1,
5.856277056
0.030370631
0.708537337


development

NDP, PRDM1





GO: 0006040~amino sugar
3
CHST6,
10.5412987
0.032161637
0.720077035


metabolic process

ST3GAL6, CHST2





GO: 0042445~hormone
5
ALDH1A2,
4.143592257
0.032177273
0.711200708


metabolic process

DHRS2, RBP4,







SLCO4A1, CRH





GO: 0042129~regulation of
4
IL1B, PAWR,
5.667364893
0.033026613
0.711845228


T cell proliferation

IL15, ITCH





GO: 0043122~regulation of
5
LTBR, IL1RL1,
4.104867096
0.033142832
0.704468375


I-kappaB kinase/NF-

IL1B, HTR2B,





kappaB cascade

F2R





GO: 0008219~cell death
15
LTBR, GPR109B,
1.832631902
0.033238101
0.697027136




MMD, EGLN3,







GZMB, PAWR,







NTN1, KCNIP3,







MAP3K5, TIAM1,







SPG20, IL1B,







CUL1, F2R,







AXIN1





GO: 0043009~chordate
9
ASCL2, XRCC4,
2.38851179
0.034502798
0.702447499


embryonic development

ALDH1A2, RBP4,







NOG, HOXB7,







EPAS1, PRDM1,







TPM1





GO: 0016265~death
15
LTBR, GPR109B,
1.819975605
0.034936457
0.698923988




MMD, EGLN3,







GZMB, PAWR,







NTN1, KCNIP3,







MAP3K5, TIAM1,







SPG20, IL1B,







CUL1, F2R,







AXIN1





GO: 0043408~regulation of
5
MAP3K5, IL1B,
4.029548433
0.035124446
0.692945523


MAPKKK cascade

F2R, AXIN1, APC





GO: 0009792~embryonic
9
ASCL2, XRCC4,
2.367058092
0.036095685
0.695217503


development ending in

ALDH1A2, RBP4,





birth or egg hatching

NOG, HOXB7,







EPAS1, PRDM1,







TPM1





GO: 0007565~female
5
BSG, CRH, IL1B,
3.992916175
0.036140548
0.688038826


pregnancy

PRL, PSG11





GO: 0045732~positive
3
IL1B, ITCH, APC
9.76046176
0.037082855
0.69007507


regulation of protein







catabolic process







GO: 0002520~immune
8
XRCC4, DHRS2,
2.546207416
0.037432747
0.686212032


system development

HOXB7, EPAS1,







HBZ, IL15,







BLNK, APC





GO: 0016477~cell
8
PVR, IL1B,
2.546207416
0.037432747
0.686212032


migration

SEMA3C,







CLASP2, NTN1,







THBS4, FN1, APC





GO: 0009968~negative
7
NOG, IL1RL1,
2.782394077
0.039681478
0.700599905


regulation of signal

FSTL3, IL1B,





transduction

PAWR, AXIN1,







APC





GO: 0006915~apoptosis
13
LTBR, GPR109B,
1.896966821
0.040454891
0.700702813




EGLN3, GZMB,







PAWR, NTN1,







KCNIP3,







MAP3K5, TIAM1,







IL1B, CUL1,







AXIN1, F2R





GO: 0009719~response to
10
ALDH1A2, RBP4,
2.168991502
0.04063477
0.695453292


endogenous stimulus

BSG, FHL2, IL1B,







CA2, IGFBP1,







GNG4, RNF14,







GNG7





GO: 0051050~positive
7
RBP4, KISS1R,
2.75743987
0.041164097
0.693469585


regulation of transport

C3, GPR109B,







CRH, IL1B, F2R





GO: 0010740~positive
6
LTBR, IL1B,
3.156077455
0.041169601
0.686857828


regulation of protein kinase

HTR2B, PRL,





cascade

F2R, AXIN1





GO: 0030334~regulation of
6
LAMA4, KISS1R,
3.118727426
0.042950909
0.6960231


cell migration

TPM1, NTN1,







F2R, APC





GO: 0012501~programmed
13
LTBR, GPR109B,
1.869024592
0.044440699
0.702228226


cell death

EGLN3, GZMB,







PAWR, NTN1,







KCNIP3,







MAP3K5, TIAM1,







IL1B, CUL1,







AXIN1, F2R





GO: 0007262~STAT
2
PRL, F2R
43.92207792
0.044482643
0.696292693


protein nuclear







translocation







GO: 0002824~positive
3
PVR, C3, IL1B
8.784415584
0.044962609
0.69407898


regulation of adaptive







immune response based on







somatic recombination of







immune receptors built







from immunoglobulin







superfamily domains







GO: 0008284~positive
10
XRCC4,
2.121839513
0.045617995
0.693355795


regulation of cell

ALDH1A2, NOG,





proliferation

FLT4, IL1B, IL15,







PRL, NTN1,







MMP12, F2R





GO: 0048568~embryonic
6
ALDH1A2, RBP4,
3.064331018
0.04575653
0.688483791


organ development

NOG, HOXB7,







EPAS1, PRDM1





GO: 0050678~regulation of
4
NOG, PRL,
4.948966526
0.046415088
0.687892226


epithelial cell proliferation

MMP12, APC





GO: 0002821~positive
3
PVR, C3, IL1B
8.50104734
0.047714819
0.692355325


regulation of adaptive







immune response
















TABLE 10







Overlap of DEG down-regulated in PE-CVS and up-regulated in LSE (FIG. 11A)










Approved





symbol
Approved name
HGNC ID
Location





BAIAP2L1
BAI1-associated protein 2-like 1
HGNC: 21649
7q22.1


BDKRB2
bradykinin receptor B2
HGNC: 1030
14q32.1-q32.2


BLNK
B-cell linker
HGNC: 14211
10q23.2-q23.33


C12orf75
chromosome 12 open reading frame 75
HGNC: 35164
12q23.3


C3
complement component 3
HGNC: 1318
19p13.3-p13.2


C4BPA
complement component 4 binding protein, alpha
HGNC: 1325
1q32


CA12
carbonic anhydrase XII
HGNC: 1371
15q22


CHST2
carbohydrate (N-acetylglucosamine-6-O)
HGNC: 1970
3q24



sulfotransferase 2




CMAHP
cytidine monophospho-N-acetylneuraminic acid
HGNC: 2098
6p23-p22



hydroxylase, pseudogene




DNAJC6
DnaJ (Hsp40) homolog, subfamily C, member 6
HGNC: 15469
1p31.3


DSC2
desmocollin 2
HGNC: 3036
18q12.1


ELL2
elongation factor, RNA polymerase II, 2
HGNC: 17064
5q15


EPAS1
endothelial PAS domain protein 1
HGNC: 3374
2p21-p16


ERO1L
ERO1-like (S. cerevisiae)
HGNC: 13280
14q22.1


GNG4
guanine nucleotide binding protein (G protein), gamma 4
HGNC: 4407
1q42.3


GNLY
granulysin
HGNC: 4414
2p12-q11


GTPBP2
GTP binding protein 2
HGNC: 4670
6p21


GZMB
granzyme B (granzyme 2, cytotoxic T-lymphocyte-
HGNC: 4709
14q11.2



associated serine esterase 1)




HPS3
Hermansky-Pudlak syndrome 3
HGNC: 15597
3q24


IGFBP1
insulin-like growth factor binding protein 1
HGNC: 5469
7p13-p12


IL15
interleukin 15
HGNC: 5977
4q31


IL1B
interleukin 1, beta
HGNC: 5992
2q14


IL2RB
interleukin 2 receptor, beta
HGNC: 6009
22q13


INPP4B
inositol polyphosphate-4-phosphatase, type II, 105 kDa
HGNC: 6075
4q31.1


ITGB6
integrin, beta 6
HGNC: 6161
2q24.2


LAMA4
laminin, alpha 4
HGNC: 6484
6q21


MAOB
monoamine oxidase B
HGNC: 6834
Xp11.4-p11.3


MAP3K5
mitogen-activated protein kinase kinase kinase 5
HGNC: 6857
6q22.33


PAEP
progestagen-associated endometrial protein
HGNC: 8573
9q34


PVR
poliovirus receptor
HGNC: 9705
19q13.2


RBP4
retinol binding protein 4, plasma
HGNC: 9922
10q23-q24


RUFY3
RUN and FYVE domain containing 3
HGNC: 30285
4q13.3


SCARA5
scavenger receptor class A, member 5 (putative)
HGNC: 28701
8p21.1


SLCO4A1
solute carrier organic anion transporter family, member
HGNC: 10953
20q13.1



4A1




SPOCK1
sparc/osteonectin, cwcv and kazal-like domains
HGNC: 11251
5q31.2



proteoglycan (testican) 1




TES
testis derived transcript (3 LIM domains)
HGNC: 14620
7q31.2


TIAM1
T-cell lymphoma invasion and metastasis 1
HGNC: 11805
21q22.1


ZNF165
zinc finger protein 165
HGNC: 12953
6p21
















TABLE 11







Overlap of DEG down-regulated in PE-CVS and up-regulated in intDEC-EP (FIG. 11B)










Approved





symbol
Approved name
HGNC ID
Location





AIF1L
allograft inflammatory factor 1-like
HGNC: 28904
9q34.13-q34.3


CHRDL1
chordin-like 1
HGNC: 29861
Xq23


CHST2
carbohydrate (N-acetylglucosamine-6-O)
HGNC: 1970
3q24



sulfotransferase 2




COTL1
coactosin-like 1 (Dictyostelium)
HGNC: 18304
16q24.1


DNAJC6
DnaJ (Hsp40) homolog, subfamily C, member 6
HGNC: 15469
1p31.3


ELL2
elongation factor, RNA polymerase II, 2
HGNC: 17064
5q15


EPAS1
endothelial PAS domain protein 1
HGNC: 3374
2p21-p16


F2R
coagulation factor II (thrombin) receptor
HGNC: 3537
5q13


GNG4
guanine nucleotide binding protein (G protein),
HGNC: 4407
1q42.3



gamma 4




GNLY
granulysin
HGNC: 4414
2p12-q11


GZMB
granzyme B (granzyme 2, cytotoxic T-lymphocyte-
HGNC: 4709
14q11.2



associated serine esterase 1)




HTR2B
5-hydroxytryptamine (serotonin) receptor 2B, G
HGNC: 5294
2q36.3-q37.1



protein-coupled




IGFBP1
insulin-like growth factor binding protein 1
HGNC: 5469
7p13-p12


IL15
interleukin 15
HGNC: 5977
4q31


IL1RL1
interleukin 1 receptor-like 1
HGNC: 5998
2q12


IL2RB
interleukin 2 receptor, beta
HGNC: 6009
22q13


LAMA4
laminin, alpha 4
HGNC: 6484
6q21


LIPH
lipase, member H
HGNC: 18483
3q27


MAOB
monoamine oxidase B
HGNC: 6834
Xp11.4-p11.3


MAP3K5
mitogen-activated protein kinase kinase kinase 5
HGNC: 6857
6q22.33


MUC15
mucin 15, cell surface associated
HGNC: 14956
11p14.3


NDP
Norrie disease (pseudoglioma)
HGNC: 7678
Xp11.4-p11.3


NOG
noggin
HGNC: 7866
17q22


P4HA3
prolyl 4-hydroxylase, alpha polypeptide III
HGNC: 30135
11q13


PPP1R3C
protein phosphatase 1, regulatory subunit 3C
HGNC: 9293
10q23-q24


PRL
prolactin
HGNC: 9445
6p22.3


RBP4
retinol binding protein 4, plasma
HGNC: 9922
10q23-q24


SCARA5
scavenger receptor class A, member 5 (putative)
HGNC: 28701
8p21.1


SLC25A15
solute carrier family 25 (mitochondrial carrier;
HGNC: 10985
13q14



ornithine transporter) member 15




SNX25
sorting nexin 25
HGNC: 21883
4q35.1


SPOCK1
sparc/osteonectin, cwcv and kazal-like domains
HGNC: 11251
5q31.2



proteoglycan (testican) 1




TLN2
talin 2
HGNC: 15447
15q15-q21
















TABLE 12







Overlap of DEG down-regulated in PE-CVS and up-regulated


in intDEC-EP & LSE (FIG. 11C)










Approved





symbol
Approved name
HGNC ID
Location





CHST2
carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2
HGNC: 1970
3q24


DNAJC6
DnaJ (Hsp40) homolog, subfamily C, member 6
HGNC: 15469
1p31.3


ELL2
elongation factor, RNA polymerase II, 2
HGNC: 17064
5q15


EPAS1
endothelial PAS domain protein 1
HGNC: 3374
2p21-p16


GNG4
guanine nucleotide binding protein (G protein), gamma 4
HGNC: 4407
1q42.3


GNLY
granulysin
HGNC: 4414
2p12-q11


GZMB
granzyme B (granzyme 2, cytotoxic T-lymphocyte-
HGNC: 4709
14q11.2



associated serine esterase 1)




IGFBP1
insulin-like growth factor binding protein 1
HGNC: 5469
7p13-p12


IL15
interleukin 15
HGNC: 5977
4q31


IL2RB
interleukin 2 receptor, beta
HGNC: 6009
22q13


LAMA4
laminin, alpha 4
HGNC: 6484
6q21


MAOB
monoamine oxidase B
HGNC: 6834
Xp11.4-p11.3


MAP3K5
mitogen-activated protein kinase kinase kinase 5
HGNC: 6857
6q22.33


RBP4
retinol binding protein 4, plasma
HGNC: 9922
10q23-q24


SCARA5
scavenger receptor class A, member 5 (putative)
HGNC: 28701
8p21.1


SPOCK1
sparc/osteonectin, cwcv and kazal-like domains
HGNC: 11251
5q31.2



proteoglycan (testican) 1
















TABLE 13







Overlap of DEG down-regulated in PE-CVS and up-regulated in intDEC-IUP (FIG. 12A)










Approved





symbol
Approved name
HGNC ID
Location





ACOT1 ///
acyl-CoA thioesterase 1 /// acyl-CoA thioesterase 2
HGNC: 33128 ///
14q24.3


ACOT2

HGNC: 18431



AIF1L
allograft inflammatory factor 1-like
HGNC: 28904
9q34.13-q34.3


CFH ///
complement factor H /// complement factor H-related 1
HGNC: 4883 ///
1q32


CFHR1

HGNC: 4888



CHRDL1
chordin-like 1
HGNC: 29861
Xq23


CHST2
carbohydrate (N-acetylglucosamine-6-O)
HGNC: 1970
3q24



sulfotransferase 2




COTL1
coactosin-like 1 (Dictyostelium)
HGNC: 18304
16q24.1


CPXM2
carboxypeptidase X (M14 family), member 2
HGNC: 26977
10q26


DNAJC6
DnaJ (Hsp40) homolog, subfamily C, member 6
HGNC: 15469
1p31.3


ELL2
elongation factor, RNA polymerase II, 2
HGNC: 17064
5q15


EPAS1
endothelial PAS domain protein 1
HGNC: 3374
2p21-p16


F2R
coagulation factor II (thrombin) receptor
HGNC: 3537
5q13


GNG4
guanine nucleotide binding protein (G protein), gamma 4
HGNC: 4407
1q42.3


GNLY
granulysin
HGNC: 4414
2p12-q11


GZMB
granzyme B (granzyme 2, cytotoxic T-lymphocyte-
HGNC: 4709
14q11.2



associated serine esterase 1)




HTR2B
5-hydroxytryptamine (serotonin) receptor 2B, G protein-
HGNC: 5294
2q36.3-q37.1



coupled




IGFBP1
insulin-like growth factor binding protein 1
HGNC: 5469
7p13-p12


IL15
interleukin 15
HGNC: 5977
4q31


IL1RL1
interleukin 1 receptor-like 1
HGNC: 5998
2q12


IL2RB
interleukin 2 receptor, beta
HGNC: 6009
22q13


LAMA4
laminin, alpha 4
HGNC: 6484
6q21


LIPH
lipase, member H
HGNC: 18483
3q27


LSS
lanosterol synthase (2,3-oxidosqualene-lanosterol
HGNC: 6708
21q22.3



cyclase)




MAOB
monoamine oxidase B
HGNC: 6834
Xp11.4-p11.3


MAP3K5
mitogen-activated protein kinase kinase kinase 5
HGNC: 6857
6q22.33


MUC15
mucin 15, cell surface associated
HGNC: 14956
11p14.3


NDP
Norrie disease (pseudoglioma)
HGNC: 7678
Xp11.4-p11.3


P4HA3
prolyl 4-hydroxylase, alpha polypeptide III
HGNC: 30135
11q13


PPP1R3C
protein phosphatase 1, regulatory subunit 3C
HGNC: 9293
10q23-q24


PRG2
proteoglycan 2, bone marrow (natural killer cell activator,
HGNC: 9362
11q12



eosinophil granule major basic protein)




PRKAB2
protein kinase, AMP-activated, beta 2 non-catalytic
HGNC: 9379
1q21.2



subunit




PRL
prolactin
HGNC: 9445
6p22.3


RBP4
retinol binding protein 4, plasma
HGNC: 9922
10q23-q24


SCARA5
scavenger receptor class A, member 5 (putative)
HGNC: 28701
8p21.1


SLC16A6
solute carrier family 16, member 6
HGNC: 10927
17q24.2


SLC25A15
solute carrier family 25 (mitochondrial carrier; ornithine
HGNC: 10985
13q14



transporter) member 15




SNX25
sorting nexin 25
HGNC: 21883
4q35.1


SPOCK1
sparc/osteonectin, cwcv and kazal-like domains
HGNC: 11251
5q31.2



proteoglycan (testican) 1
















TABLE 14







Overlap of DEG down-regulated in PE-CVS and up-regulated in confDEC-IUP (FIG. 12B)










Approved





symbol
Approved name
HGNC ID
Location





ACOT1 ///
acyl-CoA thioesterase 1 /// acyl-CoA thioesterase 2
HGNC: 33128 ///
14q24.3


ACOT2

HGNC: 18431



AIF1L
allograft inflammatory factor 1-like
HGNC: 28904
9q34.13-q34.3


CFH ///
complement factor H /// complement factor H-related 1
HGNC: 4883 ///
1q32


CFHR1

HGNC: 4888



CHRDL1
chordin-like 1
HGNC: 29861
Xq23


CHST2
carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2
HGNC: 1970
3q24


COTL1
coactosin-like 1 (Dictyostelium)
HGNC: 18304
16q24.1


CPXM2
carboxypeptidase X (M14 family), member 2
HGNC: 26977
10q26


DNAJC6
DnaJ (Hsp40) homolog, subfamily C, member 6
HGNC: 15469
1p31.3


EFCAB2
EF-hand calcium binding domain 2
HGNC: 28166
1q44


ELL2
elongation factor, RNA polymerase II, 2
HGNC: 17064
5q15


EPAS1
endothelial PAS domain protein 1
HGNC: 3374
2p21-p16


F2R
coagulation factor II (thrombin) receptor
HGNC: 3537
5q13


FSTL3
follistatin-like 3 (secreted glycoprotein)
HGNC: 3973
19p13


GNG4
guanine nucleotide binding protein (G protein), gamma 4
HGNC: 4407
1q42.3


GNLY
granulysin
HGNC: 4414
2p12-q11


GZMB
granzyme B (granzyme 2, cytotoxic T-lymphocyte-
HGNC: 4709
14q11.2



associated serine esterase 1)




HTR2B
5-hydroxytryptamine (serotonin) receptor 2B, G protein-
HGNC: 5294
2q36.3-q37.1



coupled




IGFBP1
insulin-like growth factor binding protein 1
HGNC: 5469
7p13-p12


IL15
interleukin 15
HGNC: 5977
4q31


IL1B
interleukin 1, beta
HGNC: 5992
2q14


IL1RL1
interleukin 1 receptor-like 1
HGNC: 5998
2q12


IL2RB
interleukin 2 receptor, beta
HGNC: 6009
22q13


LAMA4
laminin, alpha 4
HGNC: 6484
6q21


LIPH
lipase, member H
HGNC: 18483
3q27


LSS
lanosterol synthase (2,3-oxidosqualene-lanosterolcyclase)
HGNC: 6708
21q22.3


MAOB
monoamine oxidase B
HGNC: 6834
Xp11.4-p11.3


MAP3K5
mitogen-activated protein kinase kinase kinase 5
HGNC: 6857
6q22.33


MUC15
mucin 15, cell surface associated
HGNC: 14956
11p14.3


NDP
Norrie disease (pseudoglioma)
HGNC: 7678
Xp11.4-p11.3


NOG
noggin
HGNC: 7866
17q22


P4HA3
prolyl 4-hydroxylase, alpha polypeptide III
HGNC: 30135
11q13


PPP1R3C
protein phosphatase 1, regulatory subunit 3C
HGNC: 9293
10q23-q24


PRG2
proteoglycan 2, bone marrow (natural killer cell activator,
HGNC: 9362
11q12



eosinophil granule major basic protein)




PRKAB2
protein kinase, AMP-activated, beta 2 non-catalytic
HGNC: 9379
1q21.2



subunit




PRL
prolactin
HGNC: 9445
6p22.3


PVR
poliovirus receptor
HGNC: 9705
19q13.2


RBP4
retinol binding protein 4, plasma
HGNC: 9922
10q23-q24


RNF14
ring finger protein 14
HGNC: 10058
5q23.3-q31.1


RORB
RAR-related orphan receptor B
HGNC: 10259
9q22


SCARA5
scavenger receptor class A, member 5 (putative)
HGNC: 28701
8p21.1


SERPINA3
serpin peptidase inhibitor, clade A (alpha-1 antiproteinase,
HGNC: 16
14q32.1



antitrypsin), member 3




SLC16A6
solute carrier family 16, member 6
HGNC: 10927
17q24.2


SNX25
sorting nexin 25
HGNC: 21883
4q35.1


SPOCK1
sparc/osteonectin, cwcv and kazal-like domains
HGNC: 11251
5q31.2



proteoglycan (testican) 1




TLN2
talin 2
HGNC: 15447
15q15-q21


WT1
Wilms tumor 1
HGNC: 12796
11p13
















TABLE 15







Overlap of DEG down-regulated in PE-CVS and up-regulated in intDEC-IUP and


intDEC-EP (FIG. 12C)










Approved





symbol
Approved name
HGNC ID
Location





AIF1L
allograft inflammatory factor 1-like
HGNC: 28904
9q34.13-q34.3


CHRDL1
chordin-like 1
HGNC: 29861
Xq23


CHST2
carbohydrate (N-acetylglucosamine-6-O) sulfotransferase 2
HGNC: 1970
3q24


COTL1
coactosin-like 1 (Dictyostelium)
HGNC: 18304
16q24.1


DNAJC6
DnaJ (Hsp40) homolog, subfamily C, member 6
HGNC: 15469
1p31.3


ELL2
elongation factor, RNA polymerase II, 2
HGNC: 17064
5q15


EPAS1
endothelial PAS domain protein 1
HGNC: 3374
2p21-p16


F2R
coagulation factor II (thrombin) receptor
HGNC: 3537
5q13


GNG4
guanine nucleotide binding protein (G protein), gamma 4
HGNC: 4407
1q42.3


GNLY
granulysin
HGNC: 4414
2p12-q11


GZMB
granzyme B (granzyme 2, cytotoxic T-lymphocyte-
HGNC: 4709
14q11.2



associated serine esterase 1)




HTR2B
5-hydroxytryptamine (serotonin) receptor 2B, G protein-
HGNC: 5294
2q36.3-q37.1



coupled




IGFBP1
insulin-like growth factor binding protein 1
HGNC: 5469
7p13-p12


IL15
interleukin 15
HGNC: 5977
4q31


IL1RL1
interleukin 1 receptor-like 1
HGNC: 5998
2q12


IL2RB
interleukin 2 receptor, beta
HGNC: 6009
22q13


LAMA4
laminin, alpha 4
HGNC: 6484
6q21


LIPH
lipase, member H
HGNC: 18483
3q27


MAOB
monoamine oxidase B
HGNC: 6834
Xp11.4-p11.3


MAP3K5
mitogen-activated protein kinase kinase kinase 5
HGNC: 6857
6q22.33


MUC15
mucin 15, cell surface associated
HGNC: 14956
11p14.3


NDP
Norrie disease (pseudoglioma)
HGNC: 7678
Xp11.4-p11.3


P4HA3
prolyl 4-hydroxylase, alpha polypeptide III
HGNC: 30135
11q13


PPP1R3C
protein phosphatase 1, regulatory subunit 3C
HGNC: 9293
10q23-q24


PRL
prolactin
HGNC: 9445
6p22.3


RBP4
retinol binding protein 4, plasma
HGNC: 9922
10q23-q24


SCARA5
scavenger receptor class A, member 5 (putative)
HGNC: 28701
8p21.1


SLC25A15
solute carrier family 25 (mitochondrial carrier; ornithine
HGNC: 10985
13q14



transporter) member 15




SNX25
sorting nexin 25
HGNC: 21883
4q35.1


SPOCK1
sparc/osteonectin, cwcv and kazal-like domains
HGNC: 11251
5q31.2



proteoglycan (testican) 1
















TABLE 16







PE-CVS down-regulated genes linked to decidualization









DEG down-regulated in PE-CVS and up-



regulated in:















Approved




intDEC-
intDEC-
confDEC-
Pubmed


symbol
Approved name
HGNC ID
Location
LSE
EP
IUP
IUP
DEC





ACOT1 ///
acyl-CoA thioesterase 1 /// 2
HGNC: 33128
14q24.3


✓
✓



ACOT2

///










HGNC: 18431








AIF1L
allograft inflammatory factor 1-like
HGNC: 28904
9q34.13-

✓
✓
✓






q34.3







BAIAP2L1
BAI1-associated protein 2-like 1
HGNC: 21649
7q22.1
✓






BDKRB2
bradykinin receptor B2
HGNC: 1030
14q32.1-
✓









q32.2







BLNK
B-cell linker
HGNC: 14211
10q23.2-
✓









q23.33







C12orf75
chromosome 12 open reading frame 75
HGNC: 35164
12q23.3
✓






C3
complement component 3
HGNC: 1318
19p13.3-
✓



✓





p13.2







C4BPA
complement component 4 binding
HGNC: 1325
1q32
✓







protein, alpha









CA12
carbonic anhydrase XII
HGNC: 1371
15q22
✓






CFH ///
complement factor H /// complement
HGNC: 4883
1q32


✓
✓



CFHR1
factor H-related 1
///










HGNC: 4888








CHRDL1
chordin-like 1
HGNC: 29861
Xq23

✓
✓
✓



CHST2
carbohydrate (N-acetylglucosamine-6-
HGNC: 1970
3q24
✓
✓
✓
✓




O) sulfotransferase 2









CMAHP
cytidine monophospho-N-
HGNC: 2098
6p23-
✓







acetylneuraminic acid hydroxylase,

p22








pseudogene









COTL1
coactosin-like F-actin binding protein
HGNC: 18304
16q24.1

✓
✓
✓




1









CPXM2
carboxypeptidase X (M14 family),
HGNC: 26977
10q26


✓
✓




member 2









DNAJC6
DnaJ (Hsp40) homolog, subfamily C,
HGNC: 15469
1p31.3
✓
✓
✓
✓




member 6









DSC2
desmocollin 2
HGNC: 3036
18q12.1
✓






EFCAB2
EF-hand calcium binding domain 2
HGNC: 28166
1q44



✓



ELL2
elongation factor, RNA polymerase II, 2
HGNC: 17064
5q15
✓
✓
✓
✓



EPAS1
endothelial PAS domain protein 1
HGNC: 3374
2p21-
✓
✓
✓
✓






p16







ERO1L
ERO1-like (S. cerevisiae)
HGNC: 13280
14q22.1
✓






F2R
coagulation factor II (thrombin)
HGNC: 3537
5q13

✓
✓
✓
✓



receptor









FSTL3
follistatin-like 3 (secreted
HGNC: 3973
19p13



✓
✓



glycoprotein)









GNG4
guanine nucleotide binding protein (G
HGNC: 4407
1q42.3
✓
✓
✓
✓




protein), gamma 4









GNLY
granulysin
HGNC: 4414
2p12-
✓
✓
✓
✓
✓





q11







GTPBP2
GTP binding protein 2
HGNC: 4670
6p21
✓






GZMB
granzyme B (granzyme 2, cytotoxic T-
HGNC:4709
14q11.2
✓
✓
✓
✓
✓



lymphocyte-associated serine esterase










1)









HPS3
Hermansky-Pudlak syndrome 3
HGNC: 15597
3q24
✓






HTR2B
5-hydroxytryptamine (serotonin)
HGNC: 5294
2q36.3-

✓
✓
✓




receptor 2B, G protein-coupled

q37.1







IGFBP1
insulin-like growth factor binding
HGNC: 5469
7p13-
✓
✓
✓
✓
✓



protein 1

p12







IL15
interleukin 15
HGNC: 5977
4q31
✓
✓
✓
✓
✓


IL1B
interleukin 1, beta
HGNC: 5992
2q14
✓


✓
✓


IL1RL1
interleukin 1 receptor-like 1
HGNC: 5998
2q12

✓
✓
✓
✓


IL2RB
interleukin 2 receptor, beta
HGNC: 6009
22q13
✓
✓
✓
✓
✓


INPP4B
inositol polyphosphate-4-phosphatase,
HGNC: 6075
4q31.1
✓







type II, 105 kDa









ITGB6
integrin, beta 6
HGNC: 6161
2q24.2
✓






LAMA4
laminin, alpha 4
HGNC: 6484
6q21
✓
✓
✓
✓



LIPH
lipase, member H
HGNC: 18483
3q27

✓
✓
✓



LSS
lanosterol synthase (2,3-
HGNC: 6708
21q22.3


✓
✓




oxidosqualene-lanosterol cyclase)









MAOB
monoamine oxidase B
HGNC: 6834
Xp11.4-
✓
✓
✓
✓






p11.3







MAP3K5
mitogen-activated protein kinase
HGNC: 6857
6q22.33
✓
✓
✓
✓




kinase kinase 5









MUC15
mucin 15, cell surface associated
HGNC: 14956
11p14.3

✓
✓
✓
✓


NDP
Norrie disease (pseudoglioma)
HGNC: 7678
Xp11.4-

✓
✓
✓
✓





p11.3







NOG
noggin
HGNC: 7866
17q22

✓

✓
✓


P4HA3
prolyl 4-hydroxylase, alpha
HGNC: 30135
11q13

✓
✓
✓




polypeptide III









PAEP
progestagen-associated endometrial
HGNC: 8573
9q34
✓



✓



protein









PPP1R3C
protein phosphatase 1, regulatory
HGNC: 9293
10q23-

✓
✓
✓




subunit 3C

q24







PRG2
proteoglycan 2, bone marrow (natural
HGNC: 9362
11q12


✓
✓




killer cell activator, eosinophil granule










major basic protein)









PRKAB2
protein kinase, AMP-activated, beta 2
HGNC: 9379
1q21.2


✓
✓




non-catalytic subunit









PRL
prolactin
HGNC: 9445
6p22.3

✓
✓
✓
✓


PVR
poliovirus receptor
HGNC: 9705
19q13.2
✓


✓



RBP4
retinol binding protein 4, plasma
HGNC: 9922
10q23.33
✓
✓
✓
✓



RNF14
ring finger protein 14
HGNC: 10058
5q23.3-



✓






q31.1







RORB
RAR-related orphan receptor B
HGNC: 10259
9q22



✓



RUFY3
RUN and FYVE domain containing 3
HGNC: 30285
4q13.3
✓






SCARA5
scavenger receptor class A, member 5
HGNC: 28701
8p21.1
✓
✓
✓
✓
✓



(putative)









SERPINA3
serpin peptidase inhibitor, clade A
HGNC: 16
14q32.1



✓
✓



(alpha-1 antiproteinase, antitrypsin),










member 3









SLC16A6
solute carrier family 16, member 6
HGNC: 10927
17q24.2


✓
✓



SLC25A15
solute carrier family 25 (mitochondrial
HGNC: 10985
13q14

✓
✓





carrier; ornithine transporter) member










15









SLCO4A1
solute carrier organic anion transporter
HGNC: 10953
20q13.1
✓







family, member 4A1









SNX25
sorting nexin 25
HGNC: 21883
4q35.1

✓
✓
✓



SPOCK1
sparc/osteonectin, cwcv and kazal-like
HGNC: 11251
5q31.2
✓
✓
✓
✓




domains proteoglycan (testican) 1









TES
testis derived transcript (3 LIM
HGNC: 14620
7q31.2
✓







domains)









TIAM1
T-cell lymphoma invasion and
HGNC: 11805
21q22.1
✓







metastasis 1









TLN2
talin 2
HGNC: 15447
15q15-

✓

✓






q21







WT1
Wilms tumor 1
HGNC: 12796
11p13



✓
✓


ZNF165
zinc finger protein 165
HGNC: 12953
6p21
✓
















TABLE 17







Overlap of DEG up-regulated in dbNK and LSE plus intDEC-EP and intDEC-IUP plus


confDEC-IUP (74 genes)










Approved





symbol
Approved name
HGNC ID
Location





ADCY3
adenylate cyclase 3
HGNC: 234
2p23.3


ADM
adrenomedullin
HGNC: 259
11p15.4


APOBEC3G
apolipoprotein B mRNA editing enzyme, catalytic
HGNC: 17357
22q13.1-q13.2



polypeptide-like 3G




APOC2
apolipoprotein C-II
HGNC: 609
19q13.2


APOD
apolipoprotein D
HGNC: 612
3q29


ARHGEF6
Rac/Cdc42 guanine nucleotide exchange factor (GEF) 6
HGNC: 685
Xq26


C19orf10
chromosome 19 open reading frame 10
HGNC: 16948
19p13.3


C1R
complement component 1, r subcomponent
HGNC: 1246
12p13.31


CAMK1
calcium/calmodulin-dependent protein kinase I
HGNC: 1459
3p25.3


CAPG
capping protein (actin filament), gelsolin-like
HGNC: 1474
2p11.2


CCR1
chemokine (C—C motif) receptor 1
HGNC: 1602
3p21


CD38
CD38 molecule
HGNC: 1667
4p15.32


CD3E
CD3e molecule, epsilon (CD3-TCR complex)
HGNC: 1674
11q23


CD59
CD59 molecule, complement regulatory protein
HGNC: 1689
11p13


CD96
CD96 molecule
HGNC: 16892
3p13-q13.2


CDHR1
cadherin-related family member 1
HGNC: 14550
10q23.1


CLU
clusterin
HGNC: 2095
8p21-p12


CORO1A
coronin, actin binding protein, 1A
HGNC: 2252
16p11.2


CRYAB
crystallin, alpha B
HGNC: 2389
11q22.3-q23.1


CTSA
cathepsin A
HGNC: 9251
20q13.12


CTSL
cathepsin L
HGNC: 2537
9q21.33


DLEU1
deleted in lymphocytic leukemia 1 (non-protein coding)
HGNC: 13747
13q14.3


DOCK10
dedicator of cytokinesis 10
HGNC: 23479
2q36.3


DPYSL2
dihydropyrimidinase-like 2
HGNC: 3014
8p22-p21


EPAS1
endothelial PAS domain protein 1
HGNC: 3374
2p21-p16


FAM49A
family with sequence similarity 49, member A
HGNC: 25373
2p24.3


FASLG
Fas ligand (TNF superfamily, member 6)
HGNC: 11936
1q23


FCER1G
Fc fragment of IgE, high affinity I, receptor for; gamma
HGNC: 3611
1q23



polypeptide




FGR
feline Gardner-Rasheed sarcoma viral oncogene homolog
HGNC: 3697
1p36.2-p36.1


FKBP1A
FK506 binding protein 1A, 12 kDa
HGNC: 3711
20p13


GADD45A
growth arrest and DNA-damage-inducible, alpha
HGNC: 4095
1p31.2


GAS1
growth arrest-specific 1
HGNC: 4165
9q21.3-q22


GLUL
glutamate-ammonia ligase
HGNC: 4341
1q31


GNLY
granulysin
HGNC: 4414
2p12-q11


GPX3
glutathione peroxidase 3 (plasma)
HGNC: 4555
5q23


GZMA
granzyme A (granzyme 1, cytotoxic T-lymphocyte-
HGNC: 4708
5q11-q12



associated serine esterase 3)




HOPX
HOP homeobox
HGNC: 24961
4q12


IGFBP1
insulin-like growth factor binding protein 1
HGNC: 5469
7p13-p12


IGFBP2
insulin-like growth factor binding protein 2, 36 kDa
HGNC: 5471
2q33-q34


IL1B
interleukin 1, beta
HGNC: 5992
2q14


ITGA1
integrin, alpha 1
HGNC: 6134
5q11.1


ITGAD
integrin, alpha D
HGNC: 6146
16p13.1-p11


ITM2A
integral membrane protein 2A
HGNC: 6173
Xq13.3-q21.2


KIR3DL1
killer cell immunoglobulin-like receptor, three domains,
HGNC: 6338
19q13.4



long cytoplasmic tail, 1




KIR3DL2
killer cell immunoglobulin-like receptor, three domains,
HGNC: 6339
19q13.4



long cytoplasmic tail, 2




LCP2
lymphocyte cytosolic protein 2 (SH2 domain containing
HGNC: 6529
5q35.1



leukocyte protein of 76 kDa)




LILRP2
leukocyte immunoglobulin-like receptor pseudogene 2
HGNC: 15497
19q13.4


MDFIC
MyoD family inhibitor domain containing
HGNC: 28870
7q31.1-q31.2


MIR22HG
MIR22 host gene (non-protein coding)
HGNC: 28219
17p13.3


MTHFD2
methylenetetrahydrofolate dehydrogenase (NADP+
HGNC: 7434
2p13.1



dependent) 2, methenyltetrahydrofolate cyclohydrolase




MYL9
myosin, light chain 9, regulatory
HGNC: 15754
20q11.23


NCAM1
neural cell adhesion molecule 1
HGNC: 7656
11q23.2


NUCB2
nucleobindin 2
HGNC: 8044
11p15.1


NUPR1
nuclear protein, transcriptional regulator, 1
HGNC: 29990
16p11.2


OSTF1
osteoclast stimulating factor 1
HGNC: 8510
9q13-q21.2


PECAM1
platelet/endothelial cell adhesion molecule 1
HGNC: 8823
17q23.3


PLCG2
phospholipase C, gamma 2 (phosphatidylinositol-specific)
HGNC: 9066
16q24.1


PNP
purine nucleoside phosphorylase
HGNC: 7892
14q11.2


PSTPIP1
proline-serine-threonine phosphatase interacting protein 1
HGNC: 9580
15q24-q25.1


PTGIS
prostaglandin I2 (prostacyclin) synthase
HGNC: 9603
20q13


PTPN6
protein tyrosine phosphatase, non-receptor type 6
HGNC: 9658
12p13.31


RRAS2
related RAS viral (r-ras) oncogene homolog 2
HGNC: 17271
11p15.2


SEPT11
septin 11
HGNC: 25589
4q21.1


SERPING1
serpin peptidase inhibitor, clade G (C1 inhibitor), member 1
HGNC: 1228
11q12.1


SKAP2
src kinase associated phosphoprotein 2
HGNC: 15687
7p15.2


SLA
Src-like-adaptor
HGNC: 10902
8q24.22


SPINK2
serine peptidase inhibitor, Kazal type 2 (acrosin-trypsin
HGNC: 11245
4q12



inhibitor)




SPTSSA
serine palmitoyltransferase, small subunit A
HGNC: 20361
14q13.1


TGM2
transglutaminase 2
HGNC: 11778
20q12


TIMP3
TIMP metallopeptidase inhibitor 3
HGNC: 11822
22q12.3


TRAF3IP3
TRAF3 interacting protein 3
HGNC: 30766
1q32.3-q41


TRD
T cell receptor delta locus
HGNC: 12252
14q11.2


TRGC2
T cell receptor gamma constant 2
HGNC: 12276
7p14


TSPAN5
tetraspanin 5
HGNC: 17753
4q22.3

## Claims

1. A method for treating a placental syndrome in a woman comprising the steps of: (a) identifying a woman who has experienced a placental syndrome during a previous pregnancy; (b) determining the late secretory/luteal (LS) phase of the menstrual cycle of the woman from step (a); and (c) administering a therapeutically effective amount of relaxin to the woman from step (a) only during the LS phase of the menstrual cycle to treat the placental syndrome, wherein the LS phase of the menstrual cycle refers to the phase that commences several days following ovulation in the woman.

2. The method according to claim 1, wherein the relaxin is an RXFP-1 agonist or mimetic.

3. The method according to claim 1, wherein determination of the LS phase of the menstrual cycle is performed using a kit that is able to detect LH surge.

4. The method according to claim 1, wherein the therapeutically effective amount of relaxin is 0.1 to about 100 μg/kg of subject body weight per day.

5. The method according to claim 4, wherein the administration of the relaxin results in serum concentrations of relaxin of about 0.1-10.0 ng/ml during the LS phase of the menstrual cycle.

6. The method according to claim 4, wherein administration of the relaxin results in serum concentrations of relaxin of about 0.1-3.0 ng/ml during the LS phase of the menstrual cycle.

7. The method according to claim 1, further comprising the step of (d) determining whether the woman is pregnant and (e) if the woman is determined to be pregnant following step (d), administering a therapeutically effective amount of relaxin to the woman through the 1st trimester of pregnancy.

8. The method according to claim 7, wherein the relaxin is an RXFP-1 agonist or mimetic.

9. The method according to claim 1, wherein the placental syndrome is preeclampsia.

10. A method for treating a placental syndrome in a woman comprising the steps of: (a) determining that a woman has a propensity for developing a placental syndrome; (b) determining the late secretory/luteal (LS) phase of the menstrual cycle of the woman from step (a); and (c) administering a therapeutically effective amount of a relaxin to the woman of step (a) only during the LS phase of the menstrual cycle,
wherein the LS phase of the menstrual cycle refers to the phase that commences several days following ovulation in the woman.

11. The method according to claim 10, wherein the relaxin is an RXFP-1 agonist or mimetic.

12. The method according to claim 10, wherein determination of the LS phase of the menstrual cycle is performed using a kit that is able to detect LH surge.

13. The method according to claim 10, wherein the therapeutically effective amount of relaxin is 0.1 to about 100 μg/kg of subject body weight per day.

14. The method according to claim 13, wherein administration of the relaxin results in serum concentrations of relaxin of about 0.1-10.0 ng/ml during the LS phase of the menstrual cycle.

15. The method according to claim 13, wherein administration of the relaxin results in serum concentrations of relaxin of about 0.1-3.0 ng/ml during the LS phase of the menstrual cycle.

16. The method according to claim 10, further comprising the step of (d) determining whether the woman is pregnant and (e) if the woman is determined to be pregnant following step (d), administering a therapeutically effective amount of relaxin (or RXFP-1 agonist or mimetic) to the woman through the 1st trimester of pregnancy.

17. The method according to claim 10, wherein the placental syndrome is preeclampsia.

18. The method according to claim 17, wherein step (a) comprises:
obtaining a biological sample from the woman during the LS phase;
performing analysis of the biological sample to determine whether the following one or more genes are downregulated: ALDH1L2; RORB; EPAS1; DLGAP1; SPOCK1; MAOB; GZMB; IL2RB; GNLY; NOG; TRA@; MUC15; KLRC2; IL15; CHRDL1; PRL; SCARA5; CHST6; NTN1; CPM; IL1B; ITGB6; MMP12; BDKRB2; SLC16A6; FN1; PP14; FSTL3; WT1; IGFBP1; CFH/CFHR1; C3; C4BPA; Flt-4; HTR2B; and ERAP2; and
performing analysis of the biological sample to determine whether the following one or more genes are upregulated: ACOT8; BICD1; ADCYAP1R1; DPYSL4; COL5A1; FOSB; CR1; and S100A8.

19. The method according to claim 18, wherein step (a) further comprises the use of an immunoassay for (i) binding one or more polypeptide species in the biological sample to an antibody and quantitatively determining the amount of said one or more polypeptide species in said biological sample to determine downregulation or upregulation of the one or more genes, and (ii) comparing the amount of the polypeptide species from step (i) to a standard control representing the amount of the polypeptide species in the corresponding sample from an average non-preeclamptic woman, wherein an increase or a decrease in the amount of the polypeptide species from the standard control indicates upregulation or downregulation of the one or more genes.

20. The method according to claim 19, wherein said immunoassay is selected from the group consisting of: a radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), sandwich immunoassay, immunoradiometric assay and Western blot.

21. The method according to claim 18, wherein the biological sample can be blood, washing from the reproductive tract, urine, saliva, or endometrial biopsy.

22. The method according to claim 17, wherein step (a) comprises:
obtaining a biological sample from the woman during the LS phase;
quantitatively determining the amount of endogenous relaxin in the biological sample; and
comparing the amount of the quantified amount of endogenous relaxin to a standard control representing the amount of the endogenous relaxin in the corresponding sample from a woman without symptoms of placental syndromes; wherein an increase and decrease in the amount of the endogenous relaxin in the biological sample as compared to the standard control indicates an increased risk of developing preeclampsia.

23. The method according to claim 22, wherein the step of quantitatively determining the amount of endogenous relaxin in the biological sample comprises determining the amount of one or more nucleic acid species in the biological sample that hybridizes with any one or more probe set for RLN-1, RLN-2, RLN-3 and RXFP1.