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Embryo and Endometrial Synchrony in Implantation Failure

  • Jason M. Franasiak
  • Richard T. Scott
Chapter

Abstract

Normal implantation requires synchronous timing between the endometrium and the embryo. A loss of this synchrony—termed dyssynchrony—occurs when the endometrium is not optimally receptive at the time the embryo is ready to implant. This issue related to timing may lead to implantation failure even when the endometrial is capable of being receptive and the embryo was capable of implantation and progressing through pregnancy to delivery. While the traditional view has represented dyssynchrony as pathology attributed singularly to the embryo or the endometrium, it is clear that both entities can have robust reproductive potential in isolation, and the issue lies in the combination of both of these entities at the appropriate time. The timing of the stimulus for secretory transformation may vary from cycle to cycle, and embryonic development can also vary with age. Thus, their respective contribution to dyssynchrony is not always reproducible from cycle to cycle—thus it may not be screened for in advance. All patients undergoing superovulation during IVF are at risk for embryonic-endometrial dyssynchrony based on timing when a critical level of progesterone is attained and the timing of embryonic blastulation.

Keywords

Blastocyst Endometrium Implantation Synchrony Timing 

Introduction

Normal embryo implantation requires synchronized interactions between the endometrium and the embryo. The concept of synchrony entails both of these components: the endometrium must be optimally receptive and it must be in that state at the same time that the embryo is ready to implant in order to attain optimal clinical outcomes. A loss of this synchrony—also termed dyssynchrony in the literature—occurs either when the endometrium is not optimally receptive or when the embryo is not developed to the point of optimal implantation capacity. When either of these scenarios occurs, dyssynchrony can cause implantation failure. Of great importance, this failure occurs in spite of the fact that the endometrium, given optimal timing, is capable of receiving an embryo and the embryo, given optimal developmental timing, is capable of implantation and progression to delivery of a healthy child. That is, dyssynchrony can cause implantation failure in the absence of true pathology—rather, this is a mishandling of physiology.

Much of the foundation of fundamental physiology of embryo implantation has been expertly reviewed elsewhere in this book—this chapter builds upon these concepts. Traditionally, dyssynchrony has been classified as true pathology. Faced with a poor outcome—failed implantation—it seems intuitive to attribute that failure to either an abnormality in the embryo or an impaired endometrium. Over the last 35 years, embryologist and endometrial physiologists have sought to isolate one factor or the other and identify the specific pathophysiologic changes resulting in failed implantation. In medicine and in science, we seek to employ Occam’s razor, or lex parsimoniae—the law of parsimony, whenever possible. Indeed, many clinical disorders can ultimately be attributed to a single underlying pathologic abnormality with enough thought. However, while investigating implantation failures, one cannot anticipate that all failures will be attributed to a singular pathologic abnormality. Furthermore, while it is often presumed that implantation failure is due to some identifiable pathology, one must also consider the physiology of the circumstances—perhaps the failure is due to a misunderstanding of the physiology at work during embryo and endometrial development during assisted reproduction.

Pathology or Physiology

The scientists investigating failed implantation could largely be divided into two groups—the embryologists and the endometrial physiologists. The embryologists have traditionally focused on the morphologic and temporal aspects of embryo development. The retrospective review of large clinical experiences allowed investigators to determine criteria for optimal embryo morphology as well as temporal milestones for both early cleavage events and for the timing of blastulation, which commonly ranged from day 5–6 and rarely even day 7 of development [1, 2]. Meanwhile, the endometrial physiologists have focused on abnormal endometrial development by evaluating endometrial sonography, histologic milestones, specific cytokines, and markers of inflammation and, most recently, evaluating more comprehensively the endometrial transcriptome [3, 4].

We have learned a great deal about the specific pathologic abnormalities which may impair implantation. However, a large question remains: Is it possible for a completely normal embryo and a completely normal endometrium which are dyssynchronous leading to failed implantation? Might a circumstance exist when you have a normal endometrium and a normal embryo and these independently regulated entities, which must be temporally coordinated, are not in synch? The answer is yes, and this physiologic change cannot be ignored when attempting to optimize outcomes or when evaluating patients who have implantation failure.

This concept is clearly demonstrated by considering the difference between natural conceptions and those during cycles following controlled ovarian stimulation during assisted reproduction. During natural cycles and conception, embryonic development and the window of endometrial receptivity are controlled by the orderly development of the follicle under the regulation of the hypothalamic-pituitary-gonadal axis. A meaningful rise in progesterone occurs shortly after ovulation (Fig. 2.1a). This timing results in the oocyte being exposed to the spermatozoa at approximately the same time that secretory transformation begins in the endometrium. If both are normal, then development will be synchronous and implantation will be optimized.
Fig. 2.1

Embryo and endometrial synchrony involves both the endometrium, whose window is determined by the progesterone stimulus, and the embryo, whose widow is relative to blastulation. During natural conception, a rise in progesterone follows the LH surge leading to the opening of the endometrial window of receptivity which overlaps with the window of embryonic blastulation and implantation (a). In the case of IVF, natural coordination can be lost. The rise in progesterone following ovulation trigger is faster and more robust, and the progesterone stimulus shifts the endometrial window of receptivity by 16–24 h. Additionally, blastulation may be delayed, particularly in older, low responders. These two factors, either alone or together, result in physiologic dyssynchrony which cannot be predicted prior to cycle start and may not necessarily be reproduced from cycle to cycle (b). Used with permission [5]

In the case of controlled ovarian stimulation during an IVF cycle, this natural coordination is often lost. Due to stimulation parameters, the rise in progesterone occurs earlier, and thus the hormonal signals which control the onset of secretory transformation occur earlier shifting the window of implantation. The result is that the endometrium is prepared for the embryo implantation event prior to the embryo reaching developmental maturity for optimal implantation—endometrial dyssynchrony (Fig. 2.1b).

The important difference between this physiologic phenomenon which leads to dyssynchrony and suboptimal implantation conditions and an underlying pathology is that the timing of the stimulus for secretory transformation varies from cycle to cycle. This results in the lack of reproducibility from cycle to cycle and dictates that this physiologic dyssynchrony is not something that can be screened for in advance. The practitioner and embryologist must coordinate in real time to optimize synchrony.

This concept of physiologic changes leading to dyssynchrony stands in contrast to true pathologic changes in the endometrium. The pathologic alterations in the rate of secretory transformation which has been hypothesized alter the timing of the window of receptivity, such as those studied with the endometrial receptivity array (ERA) test [6] among others, which seeks to characterize reproducible changes in the transcriptome which results in repeatedly altered windows of implantation—this effect is pathology in the cascade of events after the progesterone stimulus. While this is clearly important, receptivity pathology impacts only a relatively small percentage of the population. As noted, this alteration ought to be reproducible from cycle to cycle. In contrast, it could be hypothesized that all patients undergoing superovulation during IVF are at risk for embryonic-endometrial dyssynchrony based on timing when a critical level of progesterone is achieved as we will discuss further.

The Endometrium

The focus of the physiologic window of implantation in the uterus is progesterone. Progesterone represents the stimulus which, once a critical threshold is achieved, causes a well-timed and orderly secretory transformation. One can think of this threshold like a trigger which activates the natural timer with the window of receptivity opening several days later and then subsequently closing. Traditionally, there has been much emphasis placed on hormonal support. While this is required, the window of receptivity is more dependent on the timing of the onset of the stimulus than mid-luteal progesterone levels.

Traditionally, the window of endometrial receptivity had been thought of as being quite wide and forgiving, with implantations occurring in a 3–5-day window [7]. However, it is important to note that this concept was founded on data procured from the transfer of day 2 embryos from ovum donation cycles between days 16 and 24 of the cycle. Initially pregnancies were reported on days 17–19 with subsequent pregnancies reported from days 16–20. Subsequent studies have refined this window from what was possible to what is optimal—a very important distinction when discussing implantation failure with patients. The optimal window is in reality smaller than originally proposed with the highest rates occurring during a 2-day window [8]. In this study investigators utilized an ovum donation model with variation in the start of progesterone to control the window of implantation. Embryo transfers were performed following 2–6 days of progesterone. Pregnancies were achieved corresponding to days 17–20 with optimal days being 18–19. Rates began to fall by half on the late margin of the window. Indeed, delayed implantation on the far edge of the endometrial window may result in poor outcomes associated with abnormal placentation, reinforcing the difference between what is possible and optimal [9] (Fig. 2.2).
Fig. 2.2

The timing of implantation in naturally occurring pregnancies with increasing proportion of pregnancy loss with later implantation. The day of ovulation was defined as day 0. Modified from [9]

Given the apparent importance of the initial progesterone stimulus, it stands to reason that the natural question to follow would be: what is known about varied levels of progesterone and how these varied levels affect the secretory transformation which in turn leads to the optimal window of receptivity? Usadi et al., utilizing a controlled experimental design in which they varied progesterone dosing in healthy volunteers after controlled estrogen priming, showed that even very low levels of progesterone were able to cause differential expression of key genes known to be associated with the onset of secretory transformation leading to endometrial receptivity [10, 11]. These data and others, from the same investigators, suggest that even low serum levels of progesterone, perhaps level as low as 2.5 ng/mL, may initiate secretory transformation and ultimately control the window of time during which a reproductively competent embryo has the opportunity to implant.

In addition to the experimental and molecular evidence of a shift seen in response to the progesterone stimulus, there are several clinical studies in assisted reproduction showing that a premature rise in progesterone, and thus secretory transformation shift, causes an increase in failed implantations. Silverberg et al. measured serum progesterone on the day of ovulation trigger and noted that two breakpoints, 0.4 ng/mL and 0.9 ng/mL, were predictors of clinical pregnancy [3]. More recently, Bosch et al. evaluated serum progesterone levels on the day of hCG administration and found that patients with levels greater than 1.5 ng/mL had significantly lower ongoing pregnancy rates [4] (Fig. 2.3). Other investigators have shown similar detriment when there is premature progesterone elevation at levels of 1.5 ng/mL and 2 ng/mL [12].
Fig. 2.3

Elevated serum progesterone levels on the day of hCG administration are associated with reduced ongoing pregnancy rates. In particular, serum progesterone levels of 1.5 ng/mL were associated with lower ongoing pregnancy rates following IVF/ICSI cycles. Used with permission from [4]

It is important to interpret these data with caution. They do not necessarily mean that the endometrial secretory transformation begins at a progesterone level of 1.5 ng/mL. It is better to suggest that patients with that level of progesterone prior to the administration of the ovulation trigger are at increased risk of early-onset secretory transformation which would shift the window of implantation. It is important to note that those embryos which blastulate more slowly would then be at an even greater risk for being dyssynchronous with the endometrium—something we will discuss below.

As was mentioned before, it is important to note that in stimulated IVF cycles as compared to natural cycles, the progesterone rise is more rapid and robust following the ovulation stimulus. This is the result of the varied pharmacokinetics of hCG versus the natural LH surge. This can result in as much as a 16–24 h shift in the onset of the critical level of progesterone during stimulated cycles and would create a situation in which the endometrial window of receptivity is physiologically shifted in IVF. This window is of course all the more shifted if there is a premature rise in the progesterone prior to the administration of the ovulation trigger. This shift cannot be assessed prior to the cycle in question and is not reproducible from cycle to cycle—this must be actively managed in the current treatment cycle. Furthermore, this focuses on only one half of the puzzle—the embryo’s timing is also important.

The Embryo

Given that the time at which the embryo is ready to implant is the other half of the puzzle, it is important to look at what is known about variability in embryonic maturation. At the current time, the timing of blastulation is the best surrogate marker available in vitro. This physiology changes over time. It has been shown that blastulation rates differ given the woman’s age. Shapiro et al. showed that patients under age 30 had much higher blastulation rates prior to day 6 than did patients 31–34 and 35–40 [13]. Forman et al. have shown that patients age 35 and above have a significantly higher proportion of embryos which have failed to blastulate by day 5 when compared to those patients under age 35 [14] (Fig. 2.4).
Fig. 2.4

Embryos in extended culture were assessed on day 5 utilizing Gardner’s criteria for blastocyst grading. Those which were morula or B1 on the morning of day 5 were considered slowly blastulating. Patient over age 35 were at much higher risk for slowly blastulating embryos (p < 0.0001)

As for the clinical outcomes for these late blastulating embryos, similar to the shift in window seen with premature rises in progesterone in relation to the endometrium, the shift in the embryonic window confers a greater risk of implantation failure. Implantation rates of embryos which blastulate on day 6 versus day 5 were decreased by 15–18% [1, 2]. On first glance, one might suspect that this is due to some intrinsic deficit in the embryos. However, insightful studies have shown that cryopreservation of the late blastulating embryos and subsequent transfer in a synchronous programmed cycle allows for restoration of reproductive capacity [1, 14, 15]. This suggests that the decreased outcomes are due, in large part, to dyssynchrony and not to intrinsic deficits in embryonic reproductive competence.

Interestingly, these data also demonstrate why the impact of dyssynchrony may be greater in older women and contribute in part to the poorer outcomes in this population. The fact that the embryos from younger women complete blastulation earlier as compared to embryos in older patients may allow them to fall within the window of optimal endometrial receptivity even when the overall window is shifted 16–24 h earlier.

Management of Embryo and Endometrial Synchrony

Management of synchrony as it relates to these physiologic shifts requires monitoring and intervention in the current treatment cycle—the clinician is not able to anticipate it prior to initiation. Intuitively, it behooves the clinician to prevent dyssynchrony when possible. This may include changes in patient management during follicular stimulation, monitoring late follicular progesterone levels to determine if they exceed an “at-risk” threshold value, and observation of the timing of blastulation. Given the widespread availability of high-quality vitrification, it is possible to vitrify blastocysts and transfer them subsequently when embryo and endometrial synchrony may be assured.

Active management of the endometrium side of physiologic synchrony involves both a prevention and surveillance component. In order to prevent premature progesterone stimuli, it is necessary to keep progesterone levels low, below that stimulus level. Werner et al. have shown that the addition of an LH (or low-dose hCG) component to the ovarian stimulation regimen may help to prevent premature rises in progesterone [16]. Indeed, an LH-to-FSH ratio of 0.3–0.6 decreased the incidence of premature progesterone rise in all responders, both high and low (Fig. 2.5).
Fig. 2.5

The optimal ratio of exogenous LH-to-FSH to prevent a premature increase in progesterone according to response group (low, normal, and high). A ratio of 0.3–0.6 decreases the incidence or premature rise in all response groups. Used with permission [16]

The second component to active management of the endometrial window is prevention of an embryo transfer in the event of a premature progesterone rise. This has traditionally been assessed based upon serum progesterone drawn on the day of ovulation trigger administration. Of note, an absolute level which would trigger a decision to cryopreserve embryos in a given cycle is not uniform. This level will be dependent upon the progesterone assay utilized by the laboratory in a given program and based on the clinical experiences which result at various cutoff values. Of note, it is important to determine if patients are on any medications which may interact with your progesterone assay and, as such, would alter clinical management. For example, DHEA has been shown to alter results of the progesterone assay to the extent that clinical decision would change [17] (Fig. 2.6).
Fig. 2.6

Measurement of the manufacturer’s DHEA-S controls showed a linear increase in the progesterone detected, ranging from 0.5 ng/mL without DHEA-S (control) in the blank control to as high as 2.0 ng/mL in the high control where DHEA-S was 722 μg/mL (High). This linear increase in progesterone was seen on all platforms despite the complete absence of progesterone in the sample being analyzed. Mean and SE bars shown. Used with permission [17]

Active management of the embryonic window at this point includes only a prevention arm as there is not a way to proactively affect blastulation rates. Prevention would involve extended culture of the embryos with an assessment of the embryos, commonly on day 5, to determine if they have begun to blastulate. There has been great focus on time-lapse imaging and prediction of blastulation. As present, the day of blastulation can be predicted to some extent by time-lapse monitoring but not as accurately as required for management of synchrony based upon the rate of cleavage [15]. If the embryo begins blastulating on day 5, it can be assumed that, when transferred to an endometrium that did not receive a premature progesterone stimulus, a synchronous transfer will occur. If the embryos have not yet blastulated, it might be an indication for cryopreservation and subsequent transfer in a synchronous cycle in order to preserve reproductive competence capabilities [1].

It is paramount once again to note that both these factors, the embryo and the endometrium, must be accounted for in this active management paradigm.

Summary

When discussing physiologic embryo and endometrial dyssynchrony, we focus on an embryo, when analyzed in isolation is reproductively competent, and an endometrium, when analyzed in isolation is capable of being receptive to an embryo. It is when the two are assessed together that dyssynchrony occurs, either due to premature progesterone stimulus on the endometrium or late blastulation of the embryo or both.

There are limitations which exist in implementation of this paradigm. From the embryonic component, more data and detailed assessment of the timing of blastulation are needed. In terms of the endometrial window, it has been refined from a broad period of approximately 5 days to a more optimal time frame of approximately 2–3 days. However, more data is needed to define and refine the outer limits of and the most optimal time within this window of endometrial receptivity.

While all this additional physiologic data on the embryo and endometrium may be of great scientific interest, it is possible that another solution may preclude its necessity on the clinical side. Indeed, cryopreservation of the embryo after blastulation, whether on day 5 or 6, followed by a synthetic programmed cycle with known progesterone start may ensure a much more precise alignment of these two windows and may be the paradigm in the future.

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© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Sidney Kimmel Medical CollegeThomas Jefferson University PhiladelphiaPhiladelphiaUSA
  2. 2.IVI-RMA of New JerseyBasking RidgeUSA

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