Label-Free Proteomics Uncovers Energy Metabolism and Focal Adhesion Regulations Responsive for Endometrium Receptivity

The menstrual cycle
of the female uterus leads to periodic changes
of the endometrium. These changes are important for developing the
endometrial receptivity and for achieving competency of embryo implantation.
However, the molecular events underlying the endometrial receptivity
process remain poorly understood. Here we applied an LC–MS-based
label-free quantitative proteomic approach to compare the endometrial
tissues in the midsecretory (receptive) phase with the endometrial
tissues in the proliferative phase from age-matched woman (n = 6/group). The proteomes of endometrial tissues were
extracted using an SDS-based detergent, digested by the filter-aided
sample preparation procedures, and subsequently analyzed by nano-LC–MS/MS
(Orbitrap XL) with a 4 h gradient. Reliable protein expression profiles
were reproducibly obtained from the endometrial tissues in the receptive
and proliferative phases. A total of 2138 protein groups were quantified
under highly stringent criteria with a false discovery rate of <1%
for peptide and protein groups. Among these proteins, 317 proteins
had differences in expression that were statistically significant
between the receptive and proliferative phases. Direct protein–protein
interaction network analyses of these significantly changed proteins
showed that the up-regulation of creatine kinase B-type (CKB) in the
receptive phase may be related to endometrium receptivity. The interaction
network also showed that proteins related to cell–cell adhesion
were down-regulated. Moreover, the results from KEGG pathway analyses
are consistent with the protein–protein interaction results.
The proteins, including alpha-actinin (ACTN), extracellular matrix
proteins, integrin alpha-V, and so on, that are involved in the focal
adhesion pathway were down-regulated in the receptive phase compared
with the proliferative phase, which may facilitate the implantation
of the fertilized ovum. Selected proteins were validated by Western
blot analysis and indirect immunofluorescence, including the up-regulation
of CKB and down-regulation ACTN in the receptive phase. In summary,
our proteomic analysis study shows potential for predicting the endometrial
remodeling from the proliferative to the receptivity phase in women,
and these results also reveal the key biological mechanisms (such
as energy metabolism and focal adhesion) underlying human endometrial
receptivity.