Key words endometrial receptivity array (ERA) - microRNA - window of implantation - implantation
failure - assisted reproduction
Schlüsselwörter endometriale Rezeptivität - Implantationsfenster (WOI) - assistierte Reproduktionstechniken
(ART) - micro-Ribonucleinsäure (RNA)
Introduction
Successful implantation of the embryo in the maternal endometrium is the result of
a perfect synchrony between a viable blastocyst, the receptive endometrium, and appropriate
communication between them [1 ]. The most investigated element in the implantation triad is the embryo, which seeks
to adhere to the endometrial epithelium and invade the decidualized stroma, initiating
trophoblast invasion and placentation. Indeed, the understanding of human pre-implantation
development is critical (for review see [2 ]), as are the soluble ligands produced and received by their receptors to mediate
this fundamental process (for review see [3 ]). However, research to develop an understanding of the endometrial component of
implantation has been largely neglected.
The maternal endometrium is receptive to an embryo only during the specific period
of time in the menstrual cycle known as the window of implantation (WOI). Classically,
this period is considered as occurring 8 to 10 days after ovulation and lasting 2
or 3 days, during which time a functional and transient ovarian steroid-dependent
status is acquired to enable the blastocyst to implant. This classical definition
was established on the grounds of a relevant clinical study [4 ] but without basic research supporting it. In this important contribution, published
by Wilcox et al. in 1999 [4 ], the day of ovulation was defined on the basis of changes in urinary excretion of
the estradiol metabolite estrone 3-glucuronide and the progesterone metabolite pregnanediol
3-glucuronide, which were measured by radioimmunoassay. The authors developed an algorithm
to identify the day of ovulation based on the ratio of these urinary hormone metabolites,
and claimed the test was similar to measurement of the luteinizing hormone (LH) peak
[4 ]. However, 26 years later, the method proposed by these authors to identify ovulation
has not been clinically adopted. Further, we now recognize limitations of the use
of LH measurements in urine or even in blood to predict ovulation [5 ]. Nevertheless, the clinical community has since assumed that the endometrium in
all patients becomes receptive during the indicated time frame (8 to 10 days after
ovulation), regardless of individual characteristics or hormonal treatments received
(i.e., natural cycles or controlled ovarian stimulation).
Human Endometrial Receptivity
Human Endometrial Receptivity
To date no single molecular or histological biomarker has been identified to objectively
and reliably diagnose endometrial receptivity. In the absence of such a diagnosis,
the endometrium has been supported by progesterone or human chorionic gonadotropin
(hCG) as the only “endometrial treatment” in patients undergoing assisted reproductive
techniques (ART). Accordingly, embryo transfer (ET) has been guided only by the quality
and developmental stage of the embryo and the thickness of the endometrial layer.
However, we have demonstrated that in 25 % of cases repeated implantation failure
is attributable to endometrial origin [6 ], which is consistent with the clinical relevance of endometrial receptivity in successful
pregnancy [7 ].
In 1950 Noyes et al. histologically defined the endometrial dating criteria for evaluating
the endometrium [8 ]. However, multiple randomized [9 ], [10 ] and prospective studies [11 ], [12 ], [13 ], [14 ], [15 ], [16 ], [17 ] questioned the accuracy and reproducibility of the Noyes method to diagnose endometrial
receptivity or fertility status.
Subsequent research has focused on discovering biochemical markers to assess endometrial
status. Although myriad molecular mediators, including growth factors, cytokines,
chemokines, lipids, and adhesion molecules, have been identified in the endometrium
[1 ], [7 ], so far, none of these molecules has been established as an endometrial biomarker
in clinical practice [18 ].
Developments in molecular biology techniques, along with global transcriptomic analyses,
have enabled the investigation of the genomics of human endometrial development [19 ]. Transcriptomic analyses identify actively expressed genes at the mRNA level at
any given time [20 ]. Human endometrial transcriptomic analyses reveal that differential gene expression
patterns exist during different phases of the menstrual cycle [21 ], [22 ], including during the receptive phase [19 ], [23 ]. Further, differential transcriptomic profiles have been uncovered in patients with
repetitive implantation failure [24 ], [25 ], [26 ] as well as endometrial pathologies such as endometriosis or endometrial cancer [27 ], [28 ], and gene expression patterns have been defined during controlled ovarian stimulation
(COS) and hormonal replacement therapy (HRT) cycles [29 ], [30 ]. These efforts enabled the discovery of the unique genomic signature of endometrial
receptivity that became the basis of the endometrial receptivity array (ERA) [31 ]. This assay diagnoses the molecular status of the receptive endometrium according
to its transcriptomic signature, regardless of its histological appearance [31 ].
Endometrial Receptivity Array
Endometrial Receptivity Array
The ERA is a novel diagnostic method clinically available worldwide that classifies
the endometrium as receptive, pre-receptive, or post-receptive [6 ]. The test requires a small biopsy of endometrial tissue taken during scheduled treatment
at either 7 days after the luteinizing hormone peak (LH + 7) in a natural cycle, or
at the end of 5 days of progesterone administration after estrogen priming in a hormonal
replacement therapy cycle (P + 5). RNA extracted from the tissue is applied to a microarray
to determine the transcriptomic profile of 238 genes. This transcriptomic profile,
when coupled to a computational predictor, objectively identifies whether this endometrium
is receptive, pre-receptive or post-receptive by clustering analysis against sample
training sets [6 ], [31 ]. The 238 genes analyzed by ERA were chosen according to the expression data of 14
previous papers by our group searching for the transcriptomic signature of endometrial
receptivity in natural cycles, COS, HRT and even in patients with intrauterine device
(IUD) (for review see [19 ]). Although these genes were selected by t-test with an absolute fold change > 3
and a false discovery rate < 0.05, the clinical validation was done with a training
set in real patients [31 ]. Importantly, the result obtained by ERA is independent of the histological appearance
of the endometrium, and has been demonstrated to be more accurate than histological
dating [32 ] and completely reproducible even with up to 40 months between samples [32 ]. This finding is consistent with the idea that the receptivity status remains the
same within an individual woman throughout her lifetime, but that different hormonal
treatments and states such as pregnancy may change the endometrium since it is a hormonally
regulated organ.
Analysis of over 6000 ERA results, performed by our group, indicates that, in approximately
30 % of patients, the endometrial biopsy is classified as non-receptive. In these
instances, the predictor describes whether the tissue is pre-receptive (85.0 %) or
post-receptive (12.6 %). Based on these findings, the algorithm then recommends the
timing of progesterone treatment for the individual patient to find her personalized
WOI, thereby obtaining an optimal chance of successful implantation through personalized
embryo transfer (pET) ([Fig. 1 ]).
Fig. 1 Clinical algorithm for personalized embryo transfer (pET), including the percentage
probability (unpublished data provided by C. Simon).
Personalized Embryo Transfer
Personalized Embryo Transfer
The clinical application of the ERA has been studied in a prospective, interventional,
multicenter, clinical trial in 85 patients with recurrent implantation failure (RIF)
versus 25 controls undergoing IVF for the first time [6 ]. The endometrial biopsy was classified as receptive in 74.1 % of patients with RIF;
when embryo transfer was performed according to the timing indicated by ERA diagnosis,
patients achieved a 33.9 % implantation rate and a 51.7 % pregnancy rate. However,
displacement of the WOI was observed in one out of four patients with RIF as diagnosed
by ERA [6 ]. In these 26.3 % of patients, when embryo transfer was performed according to ERA-diagnosed
timing of the WOI, pregnancy and implantation rates rose to the level of normally
receptive controls, in this initial study 7 patients underwent pET.
A clinical case of a successful personalized embryo transfer in a patient having experienced
four IVF and three oocyte donation failures has been reported [34 ]. This patient was diagnosed with a displacement of the WOI using the ERA. Therefore,
personalized embryo transfer of two blastocysts was performed after 7 days of progesterone
(P + 7) in an HRT cycle, resulting in a successful twin pregnancy after 7 previous
repeated implantation failures. Similarly, in a pilot study of 17 patients undergoing
oocyte donation who had experienced failed implantations with routine embryo transfer,
the implantation rate was increased from 12.9 to 34.5 % and the pregnancy rate from
23.5 to 52.9 % when pET was performed following ERA diagnosis [33 ]. All 17 patients were initially diagnosed with a displaced WOI, whereby the endometrium
biopsy was classified pre-receptive in 16 patients and post-receptive in one patient
[33 ].
The value of the diagnosis of endometrial receptivity during the routine infertility
work-up of patients undergoing assisted reproductive technology is currently being
explored in an international, multicenter, prospective, randomized, interventional
and controlled study – “The ERA as a diagnostic guide for personalized embryo transfer”
– comparing fresh embryo transfer versus elective delayed embryo transfer or pET (Clinical
trails.gov, Identifier: NCT01954758).
MicroRNAs: New Molecules Advancing Our Reproductive Knowledge
MicroRNAs: New Molecules Advancing Our Reproductive Knowledge
Despite the wealth of information uncovered in recent years, technologies continue
evolving to discover all transcripts across the transcriptome. In 2014, Hu et al.
reported the first global gene expression profile of the human endometrium using next-generation,
high-throughput RNA sequencing (RNA-seq) [34 ]. This RNA-seq-based transcriptome comparison of pre-receptive and receptive human
endometrium revealed a total of 2372 differentially expressed genes, including metallothionein
family members, HAP1, ZCCHC12, MRAP2, OVGP1, regulatory factors (GLI2, CDC25A, TLR9,
MT1G and SLC5A1), and transcription factors (AP2 and SP1) that have not previously
been linked to endometrial receptivity. In addition, the discovery of microRNAs (miRNAs)
as potential post-transcriptional regulators of gene expression [35 ] represents a breakthrough in biology during the last ten years and has become an
extremely active research field [36 ], [37 ], [38 ], [39 ].
MiRNAs are small, non-coding RNA sequences of 18 to 25 nucleotides that regulate gene
expression post-transcriptionally [40 ]. These molecules do not encode proteins; instead, miRNAs target mRNAs through complementary
base pairing to the 3′-untranslated region for degradation or repression, thereby
functioning as gene silencers [41 ]. Based on the degree of sequence homology, one miRNA can potentially target a broad
range of genes and one gene can be regulated by several miRNAs [40 ]. Initially, long precursors (pri-miRNAs) are transcribed and processed to shorter
precursors (pre-miRNAs) in the nucleus [37 ]; these precursors are exported into the cytoplasm and incorporated into the RNA-induced
silencing complex (RISC) to bind an mRNA target [42 ], as shown in [Fig. 2 ].
Fig. 2 Diagram showing the process of miRNA synthesis and the potential role of miRNAs in
the embryo-maternal dialogue.
As has been found for mRNAs, miRNAs are differentially expressed in the endometrium
across the menstrual cycle [43 ]. Further, the endometrial epithelium releases miRNAs that are secreted into the
endometrial fluid [43 ]. Profiling of miRNA and mRNA transcripts in human endometrium suggests that the
hormonal regulation of miRNAs leads to a suppression of cell proliferation by down-regulating
the expression of some cell cycle genes in the endometrial epithelium during the secretory
phase [44 ]. By isolating endometrial epithelial cells from endometrial biopsies of 14 fertile
women in the late-proliferative and mid-secretory phases, Kuokkanen et al. identified
miRNA-29B, miRNA-29C, miRNA-30B, miRNA-30D, miRNA-31, miRNA-193A-3P, miRNA-203, miRNA-204,
miRNA-200C, miRNA-210, miRNA-582-5P, and miRNA-345 as significantly increased in the
secretory endometrium. Indeed, a subset of miRNAs, namely hsa-miR-30b and hsa-miR-30d,
are significantly upregulated, whereas hsa-miR-494 and hsa-miR-923 are downregulated,
in receptive endometrium (LH + 7) versus pre-receptive endometrium (LH + 2) in fertile
women [45 ]. These findings support the previously reported upregulation of hsa-miR-30b and
hsa-miR-30-d during the acquisition of endometrial receptivity [46 ]. The involvement of miRNAs in failed embryo implantation has been suggested in patients
with recurrent implantation failure [47 ]. The existence of 13 differentially expressed miRNAs (miRNA-145, miRNA-23b, miRNA-99a,
miRNA-27b, miRNA-652, miRNA-139-5p, miRNA-195, miRNA-342-3p, miRNA-150, miRNA-374b,
miRNA-32, miRNA628-5b, miRNA-874) has been described in patients with recurrent implantation
failure; these may regulate the expression of up to 3800 genes [47 ].
Recently, our group has demonstrated that hsa-miR-30d is secreted by the human endometrial
epithelium into the endometrial fluid either free or in exosome-associated form, and
can be incorporated into the pre-implantation embryo to potentially modify its transcriptome
[43 ]. The internalization of this miRNA results in an indirect overexpression of genes
encoding for certain molecules involved in embryonic adhesion, such as ITGB3, ITGA7 , and CDH5
[43 ]. Furthermore, it has been suggested that miRNAs can be secreted by the human embryo
[48 ]; hsa-miR-191, hsa-mi-372, and hsa-miR-645 are differentially expressed according
to the fertilization method, chromosomal status, and pregnancy outcome. Together,
these findings reinforce the concept of maternal-embryonic cross-talk that uses many
different languages, with miRNAs as one of them.
Conclusion
The receptivity status of the endometrium can now be diagnosed reliably by the ERA
test, an objective molecular tool based on the transcriptomic signature of human endometrial
receptivity, to identify the WOI. The ERA can guide and improve our clinical practice
by introducing and enabling a personalized diagnosis of the WOI and, accordingly,
a personalized embryo transfer. In the near future, the challenge will be to identify
biomarkers of endometrial receptivity that could be assessed by non-invasive methods.
MiRNAs may be interesting candidate molecules to consider, particularly with the potential
role of maternal endometrial miRNAs as transcriptomic modifiers of the preimplantation
embryo.
