Employing multidimensional proteomics analysis of amniotic fluid coupled with bioinformatic algorithms we were able to identify a novel subgroup of patients at risk for preterm birth that share common clinical, biochemical and proteomic features. These cases were distinct from those characterized by intra-amniotic inflammation or bleeding. The findings of the present study further support the notion that preterm birth is the result of several distinct pathogenetic pathways for which increased uterine contractility, cervical ripening and/or PPROM are simply common clinical features of the end product.
Proteomics encompasses protein separation and identification techniques along with the necessary bioinformatic tools that allow for biomarker discovery. Two current and rather opposing proteomic approaches have emerged. One relies on generating proteomic patterns from biological samples using high-throughput mass spectrometry approaches while minimizing the need to know the identity of the discriminatory biomarkers (proteomic pattern diagnostic approach) 
. The other focuses on identification of proteins by digesting them into peptides and sequencing them using tandem mass spectrometry and database searching (identification-centered proteomic approach) 
. In several prior studies, we and others have mined the amniotic fluid proteome for combinations of biomarkers that would separate pathogenetic pathways leading to preterm birth and could potentially be used as predictors of outcome 
In the present study we used a multidimensional proteomic approach that combined SELDI-based diagnostic pattern proteomics with identification-based techniques (2D-DIGE) to find and understand changes in amniotic fluid proteome associated with spontaneous preterm birth in the absence of inflammation and bleeding. While in all prior studies 
the relevant proteomic profiles were derived by comparing a “diseased set” versus a “normal set” as defined by other non-proteomic clinical, biochemical or microbiological criteria (differential proteomics), in the present study we used a reverse approach. Since none of the current tools available in clinical practice were of use to discriminate our patient pool despite significant differences in their pregnancy outcomes we sought to define a novel clinical instrument. After we eliminated the proteomic tracings characterized by diagnostic patterns indicative of the presence of inflammatory and bleeding, among the remaining cases we found a distinct cluster of patients characterized by presence of 5 SELDI peaks in the 10–12.5 kDa region (Q-profile
). By exploring the common clinical characteristics of this subgroup we determined that these women presented most often with preterm labor and intact membranes in the context of normal amniotic fluid clinical and microbiological results. However, the majority of cases in this subgroup delivered prematurely despite tocolytic and expectant management per standard protocols. To our knowledge the Q-profile
is the first proteomic pattern with the ability to identify women at risk for preterm birth in the absence of intra-amniotic inflammation and bleeding.
With few exceptions, the current stage of medical practice emphasizes disease classification based on shared clinical features which together are representative of a syndrome. Assignment of individual cases to these diagnostic categories frequently dictates treatment choices. However, while in clinical instances such as tumors or infectious etiologies the medical treatment is further conditioned based on strict histopathologic or microbiologic examinations, obstetrical practice lags seriously behind in this respect. Perhaps, our failure to recognize and prevent preterm birth rests with the inability to rapidly and accurately discern among the several distinct pathogenetic mechanisms leading to premature delivery. Personalized medicine, stands at the opposite end of this “one size fits all” approach and relies heavily on development of classification algorithms based on subclinical features and biomarkers 
. Nowhere is a “pathway specific targeted treatment approach” more needed than in preventing premature delivery and its consequences to the newborn baby. To further identify novel pathways leading to preterm birth which may allow for individualized targeted treatment we proceeded with a proteomic identification-centered approach using the classical differential proteomics paradigm by defining the “disease set” as the samples expressing the Q-profile
in the context of a preterm delivery. In the current study we have determined that the differentially expressed proteins which characterize the Q-profile
are primarily involved in protein metabolism, signal transduction and transport. The identities of the proteins matched in the database pointed to a metabolic rather than inflammatory abnormality since none of the protein identities had apparent inflammatory function.
APO A-I and APO A-IV are associated with high density lipoprotein (HDL) and chylomicrons and have important roles in cholesterol, trygliceride and phospholipid transport and metabolism 
. Most recently it has been proposed that APO A-IV is a satiety factor acting centrally in the brain. In fasted young animals there is a dramatic increase in liver mRNA synthesis and serum levels of APO A-I and A-IV in response to glucocorticoids and insulin increased by fasting stress 
. APO A-I and A-IV have been described previously in amniotic fluid, but to our knowledge there has been no study investigating their levels or phenotypic variants as it relates to the fetus and to preterm birth 
IGFBP-1 is the predominant insulin-like growth factor binding protein in amniotic fluid 
. It is produced by the decidua and fetal liver and it is thought to play an important role in fetal growth 
. Higher mid-pregnancy levels of IGFBP-1 have been associated with growth failure 
. Using an in vitro
model Popovici et al demonstrated that hypoxia up-regulates fetal hepatocyte IGFBP-1 mRNA steady-state levels and protein, which is the major IGFBP derived from the fetal hepatocytes 
. Another protein found differentially abundant in samples with the Q-profile
was lumican, a member of the small leucine-rich proteoglycan family (SLRP) with important roles in embryonic development, tissue repair, tumor growth, organization of collagens 
and maintenance of corneal transparency 
Bikunin (also known as the “complex-forming glycoprotein heterogeneous in charge”) is a Kunitz-type serin-protease inhibitor predominantly found in amniotic fluid and to a lesser degree in urine and serum where it circulates bound to the inter-alpha inhibitor proteins 
A number of studies have shown that many serine protease inhibitors have complex effects on cellular growth and differentiation unrelated to their anti-proteolytic function 
Bikunin in particular has the ability to modulate cell growth and to block cellular calcium uptake and is currently being evaluated as a novel anti-metastatic agent 
. Additionally, bikunin has a significant anti-inflammatory function by blocking systemic cytokine induction in response to endotoxin challenge in vivo 
Finally, bikunin inhibits entotoxin-induced preterm labor in mice, 
inhibits prostaglandin expression from amnion cells 
suppresses uterine contractions 
by reducing calcium influx in myometrial cells 
and suppresses premature cervical ripening 
which are all key pathogenetic processes necessary for preterm birth. The observed upregulation in bikunin levels may reflect an endogenous compensatory mechanism aimed to prolong pregnancy and downplay inflammatory processes. This may explain the longer duration to delivery in women with Q profile compared to the other groups exhibiting abnormal proteomic profiles, despite the similar gestational age at amniocentesis.
All proteins found differentially down-regulated in amniotic fluid expressing the Q-profile
(pigment epithelium-derived factor [PEDF] and alpha-1 antitrypsin) were members of serpin superfamily. The roles of these factors in amniotic fluid are not well understood. PEDF is known to be the most potent inhibitor of angiogenesis in the mammalian ocular compartment 
. It also has neurotrophic activity, both in the retina and in the central nervous system, and is highly up-regulated in young versus senescent fibroblasts 
. No prior study investigated the presence PEDF in amniotic fluid or defined “normal levels”. One study found higher levels of trypsin activity and lower alpha 1-antitrypsin concentration in amniotic fluid of women with PPROM 
but a subsequent study found no difference between amniotic fluid alpha-1 antitrypsin in premature birth with intact versus ruptured membranes or in those with intra-amniotic infection 
The present study demonstrates how diagnostic pattern proteomics can be used in conjunction with identification-centered proteomic techniques and bioinformatic tools to provide insight into a problem which has frustrated clinicians for decades. We acknowledge that at this time due to the limitation of the current technology we have not been able to identify the specific proteins responsible for the five SELDI peaks of the Q-profile. Furthermore as SELDI and 2D-DIGE technologies are not equivalent, the biomarker peaks comprising the Q profile may not be part of the differentially expressed identities. Yet the SELDI diagnostic pattern itself proved essential in isolating a subgroup of preterm birth cases with unique features at the level of their amniotic fluid proteome. This will perhaps prompt a future research direction and paradigm into the prevention of prematurity.