Compartmentalized gene expression profiling of receptive endometrium reveals progesterone regulated ENPP3 is differentially expressed and secreted in glycosylated form

The complexity of endometrial receptivity at the molecular level needs to be explored in detail to improve the management of infertility. Here, differential expression of transcriptomes in receptive endometrial glands and stroma revealed Ectonucleotide Pyrophosphatase/Phosphodiesterase 3 (ENPP3) as a progesterone regulated factor and confirmed by various methods, both at mRNA and protein level. The involvement of ENPP3 in embryo attachment was tested in an in vitro model for human embryo implantation. Interestingly, there was high expression of ENPP3 mRNA in stroma but not protein. Presence of N-glycosylated ENPP3 in receptive phase uterine fluid in women confirms its regulation by progesterone and makes it possible to use in a non-invasive test of endometrial receptivity.


Methods-Supplementary material Laser capture microdissection (LCMD)
Frozen endometrial tissues were sectioned at 10 µm using a cryotome (Reichert Jung Cryocut 1800, Leica) and fixed onto membrane slides (Membrane slide NF 1.0 PEN, Carl Zeiss Microimaging GmbH, Germany) with 70% ethanol at -20°C for 30 minutes. Prior to use, the membrane slides were made hydrophilic by UV irradiation for 30 minutes. The tissue sections were stained using Histogene ® staining solution (Applied Biosystems, Life Technologies, Carlsbad, CA) and serially dehydrated and stored at -80°C till they were used for laser dissection.
Epithelial glands and stromal cells were dissected out using LCM using a PALM Laser-Microbeam system and PALM Robosoftware (Carl Zeiss Microimaging GmbH, Germany) with a pulsed 355 nm diode laser to cut and catapult the sample onto a cup of 0.5ml MicroTube 500 (Carl Zeiss Microimaging GmbH, Germany) containing 35 µL of extraction buffer (Arcturus ® Picopure ® Frozen RNA Isolation kit, Applied Biosystems, Life Technologies, Carlsbad, CA). Approximately 200 cells were dissected from each section, briefly centrifuged, and stored at -80°C until further use. All the above-mentioned steps were performed in a strictly RNAse-free environment to maintain RNA integrity for microarray hybridizations. A separate set of dissections was carried out for real-time PCR analysis.

Microarray data analysis
The initial analysis of the data was performed with Affymetrix Expression Console™ software. The summarization algorithm used was Iterative Probe Logarithmic Intensity Error Estimation (IterPLIER). The advantage of IterPlier is that it performs PLIER estimation and iteratively discards the non-correlating probe sets. Background correction was done using GC-Composition-based background correction (PM-GCBG). Normalization was performed by the quantile sketch method and the data was log2-transformed after extraction from the console software. Probesets that were not annotated in the database were discarded; only annotated genes were considered for further analysis.
The data was tested for any interfering outliers using R software, and the data was filtered to remove background and noise before any statistical analysis was performed. Gene probes with median expression intensities less than 6 in either the control or treatment group were removed. Fold change was calculated for each gene probe and only those genes that were up or downregulated by two times in all pairs were used for further statistical analysis. Differentially-regulated significant genes were selected from a paired T-Test with Welch approximation, and P values based on T-distribution and fold change for each significant gene were calculated by a significance analysis of microarrays method (SAM) in Multiexperiment Viewer (MeV, part of TM4 Microarray software suite). The significant gene list obtained from this analysis was further analyzed for functional and canonical pathways by using Ingenuity Pathway Analysis (IPA) (Ingenuity System, www.ingenuity.com) software. IPA is a web-based application that performs analysis, integration, and interpretation of the given data using its own comprehensive, manually-curated content of the ingenuity knowledge base. This knowledge base includes information from various sources like Entrez gene, RefSeq, OMIM, GWAS, and Gene Ontology.

Real-time PCR
Real-time PCR was performed using a StepOne Plus instrument (Applied Biosystems). TaqMan Universal PCR Master Mix (Applied Biosystems) was used in a microamp fast optical 96 well reaction plate with a relative quantification method.
18S was used as a reference gene and samples were run in triplicate. Real-time PCR was run according to the manufacturer's instructions with an equal quantity of cDNA in all the samples. Real-time PCR data was analyzed using StepOne Plus software (Applied Biosystems).