Screening Of Differentially Expressed Genes In Human Endometrium During The Implantation Window Establishment Of The Technical Plateform And Its Application

Embryo implantation is a critical step of the reproductive process in mamalians, which involves a complex sequence of biological events. Successful implantation requires a receptive endometrium, a normal embryo and a synchronized dialogue between mother and embryo. It has been established that the endometrium is receptive to the embryo only in a short period during the cycle. During this period (implantation window), the endometrium, under the influence of ovarian hormones, undergoes the defined changes (both acquisition and loss of certain molecules) to establish the receptivity. For better understanding the biological events and gaining an insight into their molecular regulation during the implantation window, we took the endometrial samples from volunteers to study the specific gene expression profile "by using suppression subtractive hybridization approach. Result:1. The samples were obtained from each patient at two different days of two menstrual cycles (dayl and day7 after ovulation) and validated by scanning electron microscopic observation, vaginal ultrasonography monitoring, routine histological examination and PR immunohistochemical staining. The samples were histologically in phase and dated as early-secretory phase and implantation window respectively. It was the solid base for subsequent molecular experiments.2. By means of SMART^TM PCR cDNA Synthesis and suppression subtractive hybridization, two subtractive libraries were constructed. One was named as window of implantation against early-secretory-phase endometrium cDNA library (HWE, forward library), and the other named as early-secretory-phase against window of implantation endometrium cDNA library (HEW, reverse library).3.911 clones were screened by dot-blotting with forward and reverse subtracted probes respectively, resulting 192 and 162 positive clones respectively. Partial clones were sequenced. At last, we got 94 satisfied ESTs (59 from forward library and 35 from reverse library)4. Sequence homology search in the GenBank by BLAST showed that, the distribution of ESTs of known genes, known ESTs and novel ESTs was 50.85%, 42.37% and 6.78%, respectively with specific cDNA of implantatin window, and 48.57%, 48.57% and 2.86%, respectively with specific cDNA of early secretory phase.5. These genes were assigned to several different groups by function: transcriptional factors, cytoskeleton proteins, protein synthesis, and signaling transduction, cell proliferation, differentiation and apoptosis, ECM/Cell adhesion and tissue remodeling. Most of the known genes function in transcription, cytoskeleton, and signal transduction.6. One of known genes, NID-1(82G7 from forward library), was examined by in situ hybridization. Gene expression of NID-1 was observed in endometrial stromal cells of implantation window but not in the early-secretory-phase endometrium. This result was coincident with other reports. It suggested that NID-1 may play important roles in embryogenesis, implantation and decidualization in human.7. The novel EST 82F4 was submitted to Genbank after RACE (rapid amplification of cDNA ends), and its accession number was EF063108.8. Northern blotting showed a significantly strong expression of the novel EST (82F4) in the endometrium of implantation window. In situ hybridization localized its expression in glandular epithelium and stroma cells.In general, a forward and a reverse subtractive cDNA libraries (HWE and HEW) were constructed. There were 192 and 162 positive clones identified in the libraries respectively. The differentially expressed genes were assigned to several different groups by function. Most of the known genes function in transcription, cytoskeleton, and signal transduction. Endometrium underwent great changes from ovulation to implantation to prepare for the embryo implantation. Many genes were involved in this process. Some genes were expressed or up-regulated and some were inhibited or down-regulated. These results increase our likelihood of finding new potential genes involved in the window of implantation.