Proteomic analysis of uterine washings

Summary

Using state of the art proteomics techniques and multiplex analyses we are identifying the proteins in the uterine cavity throughout the menstrual cycle and in women with infertility and other endometrial disorders. Validation is by Western blot and immunohistochemistry establishes the cellular sources. Extension of the work will determine functions for uterine fluid proteins on embryo development and uterine receptivity for implantation.

Description

Embryo transfer studies since the 1950s, in a range of species, have clearly demonstrated that the endometrium and the embryo must be in synchrony of development for implantation (a key step in establishing pregnancy) of the embryo into the uterine lining (the endometrium) is to be achieved.

In a woman's menstrual cycle, of about 28 days, implantation can only occur during a period of about 4 days in the mid-secretory phase. The changes in the endometrium that open this ‘window of implantation' are slowly being unraveled.

At this time, there is no way of testing whether or not a woman's endometrium will become receptive in any cycle: this is a key issue that must be addressed if we are to improve success rates of IVF or assist women with endometrial-based infertility.

Many proteins are secreted from the endometrium into the uterine cavity. One of the features of the receptive endometrium is that the epithelial cells lining the cavity become highly secretory.

We proposed that by flushing the uterine cavity with saline, identifying the proteins in this flushing fluid, and comparing these at different times of the cycle, in fertile and infertile women we would find a cohort of proteins whose analysis would provide the basis of a test for uterine receptivity and targets for strategies to improve fertility in some infertile women.

Uterine fluid contains an abundance of serum proteins. We have devised a method to remove these before further analysis, allowing detection of less abundant and biologically relevant proteins.

Using a combination of 2-dimensional differential in gel electorphoresis, liquid chromatography, Maldi-TOF mass spectrometry and multiplex analyses along with validation studies, we have identified a large cohort of proteins, previously unknown to be components of uterine fluid, and have shown that some of these differ between the stage of the cycle and in infertile women.

We are now defining the cellular source of these proteins, their potential functions and importance at the endometrial-embryo interface and their potential as markers of the receptive phase.

Funding

National Health and Medical Research Council
Monash IVF

Outcomes

We have identified a number of proteins previously unknown to be components of uterine fluid

Some of the identified proteins are different between the phases of the menstrual cycle and in women with infertility
Most of the proteins are products of the endometrial epithelium

Selected Publications

Hannan N, Stephens A, Rainczuk A, Hincks C, Salamonsen LA, Rombauts LR. Proteomic analysis of uterine washings reveals an abundance of proteins differentially present in proliferative and secretory phases (in preparation, submission July 2009)

Lois A. Salamonsen, Guiying Nie, Natalie Hannan, Evdokia Dimitriadis (2009) Preparing fertile soil: the importance of uterine receptivity. (The Society for Reproductive Biology Founder's Lecture) 2009. Reproduction Fertility and Development (in press)

Chen J I-C, Hannan NJ, Mak Y, Nicholls PK, Zhang J, Rainczuk, A Stanton PG, Robertson DM, Salamonsen LA, Stephens AN (2009). Proteomic characterization of mid-proliferative and mid-secretory human endometrium. Journal of Proteome Research [E-pub 17th Feb]

Hannan N, Stoikos C, Stephens A, Salamonsen L (2009) Depletion of high-abundance serum proteins from human uterine lavages enhances detection of lower abundant proteins. Journal of Proteome Research. 8(2):1099-1103.

Salamonsen LA (2007) The Menstrual and Estrous Cycles. In "The Endometrium: molecular, cellular and clinical perspectives", 2nd edition. Eds J Aplin, A Fazleabas, R Glasser and L Giudice. Informa UK Ltd, London, p.25-45. (ISBN 9780415385831).