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Delving into the Complexities of Endometrial Receptivity with Simple Western

"Our lab came across ProteinSimple’s Simple Western platforms in 2014 and was surprised by the sensitivity and specificity of the technology, as well as its minimal usage of consumables"

- Nageswara Rao Boggavarapu, PhD Assistant Professor, Women’s and Children’s Health, Karolinska Institute, Stockholm, Sweden

Nageswara Rao Boggavarapu

PUSHING THE BOUNDARIES OF EMBRYO IMPLANTATION RESEARCH

Though embryo implantation research has rapidly evolved in recent years, due to advances in both proteomics and genomics technology, there is still a significant lack of information surrounding the process of implantation. With failure to establish a receptive endometrium being one of the leading causes of female infertility, there is a push to identify molecules involved in the regulation of
endometrial receptivity.

Being primarily involved in translational research, Nageswara Rao Boggavarapu works at the world-renowned Karolinska Institute as an Assistant Professor in the Women’s and Children’s Health department. The Karolinska Institute is a medical university involved in basic and clinical research, and medical teaching, as well as awarding the Nobel Prize in Medicine and Physiology each year.

By researching endometrial receptivity and the mechanism of human embryo implantation in 3D in vitro cell culture models, Nageswara hopes his research can contribute to improved treatments for managing infertility and endometriosis in the future. He is currently investigating the synchrony between the receptive endometrium and embryo implantation, identifying the mechanisms involved in endometriosis and other gynaecological disorders.

FRUSTRATIONS WITH WESTERN BLOTTING

A major bottleneck in Nageswara’s clinical research is the availability of human samples from volunteers and patients. In the past, the limited availability of clinical material has led to his team not being able to run as many protein assays, as traditional Western blots require large sample volumes and the few human samples they have can only go so far.

Nageswara also had the typical frustrations associated with Western blotting: “Running a traditional Western blot is laborious, time demanding, consumes lot of chemicals and plastics, as well as needing numerous pieces of equipment,”
as well as experiencing issues with sensitivity and specificity. However, one of his biggest concerns was that it was always a subjective analysis.

This got Nageswara looking for a newer technology; one where he would be able to run completely automated Western blots with minimal human interference. He was searching for a technique that had the high sensitivity levels and quantitation associated with real-time PCR, but forprotein analysis. That’s where Wes comes in.

SENSITIVITY WITHOUT COMPROMISING SPECIFICITY

Nageswara’s lab first came across the Simple Western platforms in 2014. From the offset, he was blown away by the high specificity and sensitivity of the technology, as well as the limited usage of consumables.

The high-quality data obtained from Simple Western assays, using Wes, enabled Nageswara’s lab to show that specific molecules are affected in endometrial receptivity, influencing the human embryo implantation process in in vitro cell culture models. These molecules, such as Ectonucleotide Pyrophosphatase/ Phosphodiesterase 3 (ENPP3), have potential roles as biomarkers in assessing the status of endometrium before embryo transfer in vitro fertilization (IVF). The group confirmed the presence of ENPP3 in endometrium and uterine fluid using Simple Western assays (FIGURE 1).

Simple Western assay analysis showing expression of ENPP3 in endometrium and uterine fluid

Figure 1.  FIGURE 1. Simple Western assay analysis showing expression of ENPP3 in endometrium and uterine fluid. ENPP3 in receptive phase endometrial tissue lysates (A,B) and uterine fluid (C,D) showed good expression levels of ENPP3 in control samples (lanes 2–7). Antiprogestin treatment (A,C): (lanes 8–13) showed no detectable levels of ENPP3, both in endometrial tissue and uterine fluid, confirming the regulation of ENPP3 protein by progesterone. (B,D) show semiquantitative analysis of immunodetectable ENPP3, expressed in log2 AUC (area under the curve). Lane 1: protein molecular weight marker.

THE ONE-STOP SHOP FOR WESTERN BLOT

Overall, Nageswara has found Simple Western assays to be exactly as the name suggests: simple. This has led him and his colleagues to use the platform in multiple projects, optimizing many assays with different antibodies, all while using minimal sample material. Due to his success with Wes, Nageswara went a step further and upgraded to Jess. The additional capabilities, which include fluorescence detection and RePlex, have since enabled him to get more rich protein characterization data from just one sample.

The ease of ordering has also been a particular highlight for him. With each kit containing all the necessary components to carry out Simple Western assays, gone are the days of having to visit multiple websites to order reagents and plastics for Western blots!

The speed at which fully quantitated Western data can be produced with Wes and Jess, has enabled Nageswara to shorten the time between conducting assays and confidently publishing data. With two more projects incorporating Simple Western assays currently in progress, Nageswara is excited to further contribute to advances in embryo implantation and endometrial receptivity research. From his findings and future studies, less invasive methods of screening endometrial receptivity in women seeking IVF could be developed using uterine fluid samples. This is alongside possible new treatments for managing infertility, utilizing inhibitors of ENPP3 or other molecules associated with endometrial receptivity regulation. With this in mind, the future is looking bright for improved treatments of endometriosis, infertility and other gynaecological disorders.

SELECTED PUBLICATION

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

NR Boggavarapu, S Lalitkumar, V Joshua,S Kasvandik, A Salumets, PG Lalitkumar, K
Gemzell-Danielsson, Sci Rep., 2016 Sep 26;6:33811.

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