Uterine Fluid Proteins for Minimally Invasive Assessment of Endometrial Receptivity

Sergo Kasvandik; Merilin Saarma; Tanel Kaart; Ilmatar Rooda; Agne Velthut-Meikas; Aivar Ehrenberg; Kristina Gemzell; Parameswaran Grace Lalitkumar; Andres Salumets; Maire Peters


J Clin Endocrinol Metab. 2020;105(1) 

In This Article

Abstract and Introduction


Context: Clinically used endometrial (EM) receptivity assays are based on transcriptomic patterning of biopsies at midsecretory endometrium (MSE) to identify the possible displacement or disruption of window of implantation (WOI) in patients with recurrent implantation failure (RIF). However, biopsies are invasive and cannot be performed in the same cycle with in vitro fertilization embryo transfer, while uterine fluid (UF) analysis is considered minimally invasive and can immediately precede embryo transfer.

Objective: To determine whether UF proteome can be used for WOI monitoring and whether it would highlight the etiology of RIF.

Patients: Paired early secretory endometrial (ESE) and MSE UF samples from six fertile control women for discovery, and an additional 11 paired ESE/MSE samples from controls and 29 MSE samples from RIF patients for validation.

Results: Using discovery mass spectrometry (MS) proteomics we detected 3158 proteins from secretory phase UF of which 367 undergo significant (q < 0.05) proteomic changes while transitioning from ESE to MSE. Forty-five proteins were further validated with targeted MS, and 21 were found to display similar levels between control ESE and RIF MSE, indicating displacement of the WOI. A panel of PGR, NNMT, SLC26A2 and LCN2 demonstrated specificity and sensitivity of 91.7% for distinguishing MSE from ESE samples. The same panel distinguished control MSE samples from RIF MSE with a 91.7% specificity and 96.6% sensitivity.

Conclusion: UF proteins can be used for estimating uterine receptivity with minimal invasiveness. Women with RIF appear to have altered MSE UF profiles that may contribute to their low IVF success rate.


The uterine micromilieu and its main medium, the uterine fluid (UF), play an important role in reproductive success, influencing sperm movement through the uterus to fallopian tubes, embryo development, and implantation processes.[1] UF is a complex mixture of molecules secreted primarily by the endometrial (EM) glandular epithelial cells, but also by the immune cells and exosomes derived from EM cells.[2,3] Additionally, detached or nonadherent cells and passively diffused molecules may contribute to the final repertoire of molecules found in the UF. The lower molecular weight fraction has been described to consist of compounds such as amino acids, lactate, pyruvate, oxygen, glucose, antioxidants, ions, growth factors, hormones, and lipids (reviewed in[1,4]). Proteomics studies have established that UF contains at least 600–1500 different proteins,[5–7] depending on the sampling procedures and analysis methods used. The proteomic component of UF reflects not only protein expression patterns in EM tissue but may also contain components from other reproductive tract fluids, such as cervicovaginal fluid[8] and from the fallopian tubes or of peritoneal origin.[9]

Regardless of the origin of the compounds identified in UF, all of them provide a suitable buffer for the developing embryo in transit and facilitate its arrival to the correct intrauterine location for subsequent implantation.[10] During the menstrual cycle, successful implantation is considered to be possible in a short period of time, known as the window of implantation (WOI),[11] starting on cycle days 19 or 20 and lasting for about 4–5 days. Determination of the WOI has utmost importance in the in vitro fertilization (IVF) procedure to increase chances of successful commencement of pregnancy. Patients who suffer recurrent implantation failure (RIF) form one of the most complex groups of patients whose conception is cumbersome and can require large amounts of resources and time, while causing emotional stress to both infertile couples and clinicians. RIF is defined when at least three implantation failures with good-quality embryo transfers have occurred or when conception was not achieved after transfer of at least 10 good-quality embryos.[12] In some patients with RIF, WOI may be temporally displaced, leading to the asynchrony between the developing embryo and EM tissue that may result in implantation failure. Alternatively, endometrial RIF may also arise from a molecularly disrupted WOI without a temporal shift. Recent work has shown that mRNA signatures supporting both temporally displaced and molecularly disrupted WOI can be present in the same RIF patient,[12] indicating that the whole concept of RIF pathogenesis requires additional studies that will pave the way to new treatment options for this complex group of patients. Another factor that needs to be addressed is whether the widely used mRNA data in EM receptivity studies predictably translates into the proteome level, as significant discordance between mRNA and protein, especially in dynamic biologic contexts, has recently been emphasized.[13]

Currently, there are few approaches in clinical use that enable determination of EM receptivity by gene expression profiling of midsecretory endometrial (MSE) tissue.[14] However, tissue-based methods require an invasively obtained EM biopsy that excludes embryo transfer during the same IVF cycle. Aspiration of UF or uterine lavage is a less invasive approach, and even if dilution of UF contents occurs during the sample collection, it can be performed prior to embryo transfer in the same cycle, without any adverse effect on pregnancy rates.[15–18] As the molecular composition of the UF changes across the menstrual cycle,[19] the alterations may specifically reflect the receptivity status of the EM tissue, providing ways to develop minimally invasive receptivity tests for determination of the most appropriate day for embryo transfer in an IVF cycle.[20] New therapies are certainly welcome to improve treatments for RIF patients, which has not changed significantly in recent decades.

Despite published studies unveiling molecular changes in UF composition during the transition towards WOI,[8,21–25] there are no UF-based biomarker tests in clinical use. This can be attributed to insufficient use of rigorous statistical methods, lack of subsequent validation, and overall limited concordance between studies to derive reliable markers or marker panels.[26] Proteomics approaches used in discovery studies are also technically complex and time-consuming,[25] hindering their translation into clinical practice. Therefore, there is a need for more time-effective and simpler techniques enabling minimally invasive EM receptivity evaluation before the planned IVF embryo transfer.

Our study set out to find receptivity-related protein markers from the UF by using comprehensive state-of-the-art discovery proteomics, validating candidate proteins using targeted mass spectrometry (MS) in an independent cohort of women, and studying their contribution to the pathogenesis of RIF (Figure 1). Finally, we propose a conveniently small panel of proteins that show sufficiently high sensitivity and specificity for determining the EM receptivity status from UF, and which can be further pursued into translational studies.

Figure 1.

Overview of the experimental design for determining proteomic receptivity biomarkers from uterine fluid. Abbreviations: ESE, early secretory phase; MSE, midsecretory phase; UF, uterine fluid; FDR, false discovery rate; FC, fold change; LC/MS/MS, liquid chromatography tandem-mass spectrometry; MSE, midsecretory endometrium; RIF, recurrent implantation failure; UF, uterine fluid.