Abstract

Despite many advances in assisted reproductive technologies (ART), implantation rates are still low. The process of implantation requires a reciprocal interaction between blastocyst and endometrium, culminating in a small window of opportunity during which implantation can occur. This interaction involves the embryo, with its inherent molecular programme of cell growth and differentiation, and the temporal differentiation of endometrial cells to attain uterine receptivity. Implantation itself is governed by an array of endocrine, paracrine and autocrine modulators, of embryonic and maternal origin. Implantation failure is thought to occur as a consequence of impairment of embryo developmental potential and/or impairment of uterine receptivity and the embryo–uterine dialogue. Therefore a better comprehension of implantation, and the relative importance of the factors involved, is warranted. New techniques for monitoring changes in the endometrium and/or the embryo at the level of gene regulation and protein expression may lead to the identification of better markers for implantation. Moreover, the use of predictive sets of markers may prove to be more reliable than a single marker. Continuing refinements to ART protocols, such as optimizing ovarian stimulation regimens, the timing of human chorionic gonadotrophin injection, or the timing of embryo transfer, should help to increase implantation rates further.

Introduction

The techniques used in assisted reproductive technologies (ART) have advanced considerably since the first in vitro fertilization (IVF) birth in 1978. Tools are now available that enable the selection of high-quality embryos or assessment of endometrial status. Furthermore, ART protocols continue to evolve with the aim of achieving higher pregnancy rates, fewer multiple births and healthy babies from genetically affected progenitors. However, despite these advances, pregnancy rates are still relatively low and have not increased significantly in the last decade ( Nygren and Andersen, 2001 ; Andersen et al. , 2005 ). This suggests that implantation rates in stimulated cycles remain suboptimal. Other factors, which are yet to be identified, must play a role.

Historically, both endometrial receptivity and embryo quality have been judged using morphological assessments, and the search for predictors of implantation has focused primarily on the analysis of single markers. There is now a movement towards more sophisticated, high-throughput technologies, such as DNA chips and proteomic arrays, capable of rapidly monitoring small changes in the levels of thousands of different genes or proteins, respectively. This not only enables the sampling of many more potential molecular candidates, but also the identification of characteristic molecular profiles (e.g. gene expression clusters or cytokine ‘fingerprints’) rather than single biomarkers. This strategy may be particularly relevant in the field of implantation because numerous factors are involved, many of these have multiple functions, and there is potentially a large amount of redundancy.

The aim of this review is to outline the current understanding of implantation in humans and to describe and critique the tools currently available for the study of the human preimplantation embryo, the receptivity of the endometrium and the embryo–uterine dialogue. In addition, this review will identify key areas in implantation research and methodology where efforts need to be focused in the future.

Implantation

On the basis of studies in rhesus monkeys, it is thought that human implantation involves a number of different stages ( Enders et al. , 1986 ). Prior to implantation, the blastocyst shows evidence of polarity, assuming a particular orientation as it approaches the endometrium. Once the blastocyst is oriented correctly (apposition), the zona pellucida is shed. The blastocyst then comes into contact with the epithelial layer and adheres to the endometrial surface (adhesion). Finally, the blastocyst penetrates the epithelial layer and invades the stroma (invasion).

Successful implantation requires the appropriately timed arrival of a viable blastocyst into a receptive endometrium. The endometrium is remodelled throughout the menstrual cycle, and exhibits only a short period of receptivity, known as the ‘implantation window’. In humans, during a natural cycle, the embryo enters the uterine cavity ∼4 days after ovulation ( Croxatto et al. , 1978 ). The endometrium becomes receptive to blastocyst implantation 6–8 days after ovulation and remains receptive for ∼4 days (cycle days 20–24) ( Bergh and Navot, 1992 ). The importance of endometrial environment is highlighted by the observation that high-quality embryos transferred into women involved as embryo recipients in a surrogacy procedure have a higher probability of implanting than if they are transferred back into the donor women ( Check et al. , 1992 ; Stafford-Bell and Copeland, 2001 ). Poor embryo quality has also been identified as a major cause of implantation failure ( Urman et al. , 2005 ).

It is clear that to improve implantation rates in stimulated cycles, it is important to find ways to pinpoint the window of implantation, ensure that the best embryo is selected and synchronize embryo transfer with the time of optimal endometrial receptivity. Importantly, ways of evaluating and enhancing endometrial receptivity and embryo quality without disrupting the delicate process of implantation itself must be identified.

Implantation in humans is controlled by a complex and sophisticated interaction between embryo and endometrium, which begins at the early stages of oocyte maturation ( Emiliani et al. , 2005 ). This dialogue enables synchronous development of the oocyte and maturation of the endometrium, followed by embryo orientation, apposition, adhesion and endometrial invasion by the blastocyst ( Enders et al. , 1986 ). By understanding the activity and function of the hormones and factors involved in this dialogue, it may be possible to use them as predictors of endometrial receptivity or embryo quality to maximize implantation rates in hormonally stimulated ART cycles.

Epidemiology

The majority of spontaneous human conceptions fail to complete implantation and to achieve ongoing pregnancy. Evidence from sperm donation programmes have indicated that the maximal chance of achieving successful implantations under optimal conditions is ∼40% per cycle, and this rate declines with age ( Ferrara et al. , 2002 ; Achard et al. , 2005 ). The proportion of human conceptions that fail to implant remains uncertain, as data are limited. More is known about the fate of the embryo post-implantation. Using markers of early pregnancy, such as human chorionic gonadotrophin (hCG), it has been demonstrated that one-third of post-implantation early pregnancy losses occur during the pre-clinical stages of pregnancy in fertile women ( Wilcox et al. , 1999 ). The situation is even worse for recipients in ovum donation programmes or in patients undergoing IVF, who show rates of early pregnancy loss as high as 37 and 48%, respectively ( Simon et al. , 1999a ).

A high incidence of chromosomal abnormalities has been reported for human embryos ( Munné, 2001 ) and a significant proportion of pregnancy wastage is caused by numerical or structural chromosomal abnormalities ( Hassold et al. , 1980 ). The frequency of embryonic genetic abnormality increases with maternal age ( Hassold et al. , 1980 ) and is higher among infertile couples than in the general population ( Munné, 2001 ). Therefore, genetic abnormalities are thought to be a major factor contributing to implantation failure in ART. Patients undergoing ART procedures often hold unrealistically high expectations of achieving pregnancy ( Peddie et al. , 2005 ), and this may stem from a lack of awareness about the low implantation rates observed in natural cycles.

Morphology and cellular composition of the endometrium

The endometrium is a multilayered, dynamic organ overlaying the myometrium and comprises a functional layer and a basal layer. Each month, cells in the functional layer are separated from the basal layer during menstruation. The basal layer is attached to the myometrium and remains intact during menstruation, serving as a base for endometrial regeneration. The endometrium is composed of several different cell types, including luminal and glandular epithelial cells, stroma with stromal fibroblastic cells, immunocompetent cells and blood vessels. The numbers, activity, structure and function of these cells change throughout the menstrual cycle and change again during pregnancy.

In the early 1950s, Noyes and co-workers ( Noyes et al. , 1950 ; Noyes and Haman, 1953 ) examined the histological features of endometrial biopsies taken during 8000 spontaneous cycles in 300 women. By associating histological changes with natural changes in basal body temperature, they were able to link distinct histological patterns to particular time points during the menstrual cycle. The criteria for endometrial dating that resulted from this work have since remained the gold standard approach for evaluating endometrial responsiveness and detecting endometrial abnormalities.

Endometrial biopsy is known to disrupt normal anatomical layering. Biopsies may contain different parts of the endometrial layers, low uterine segment fragments and variable amounts of glands or stroma. Nevertheless, compared with other biopsy techniques, analysis of samples using the Noyes method generally allows evaluation of the cellular architecture. Other key advantages of the Noyes method are that it enables differential component analysis and both the morphology and function of the cells can be assessed (Table  1 ) ( Bourgain et al. , 1994 ).

Table 1:

Strengths and limitations of morphological and immunohistochemistry assessments of endometrial receptivity

Advantages Limitations 
Established technique Subjective interpretation (high intraobserver variability) 
Widely used and accepted Snapshot analysis 
Architecture preserved Sample bias 
Provides information about morphology and function Inter-cycle association poor 
Differential component analysis Disregards embryo interaction 
Advantages Limitations 
Established technique Subjective interpretation (high intraobserver variability) 
Widely used and accepted Snapshot analysis 
Architecture preserved Sample bias 
Provides information about morphology and function Inter-cycle association poor 
Differential component analysis Disregards embryo interaction 

As well as these benefits, a number of weaknesses in Noyes' approach have been identified. Biopsies can only provide a snapshot of the real situation in the endometrium, and sample bias is unavoidable because it is not applicable to take large numbers of samples. Histological interpretation is inherently subjective, both intra- and interobserver variability are high and intraobserver variability has been shown to be highest among infertile women during the implantation window (intraclass correlation coefficient = 0.65) ( Murray et al. , 2004 ; Myers et al. , 2004 ). Variability is also introduced because of the differences between women and the differences between cycles in the same woman ( Murray et al. , 2004 ). Furthermore, ovarian stimulation in artificial cycles may lead to differences in the timing of endometrial maturation compared with natural cycles ( Papanikolaou et al. , 2005 ).

The issue of timing based on endometrial dating is critical (Fig.  1 ). During the 2 days following ovulation, the morphological features of the endometrium do not change significantly. Therefore, an error of 2 days is introduced into endometrial dating for biopsies taken during this period. A similar situation is evident for biopsies taken during the mid-luteal phase, where there is lack of positive morphology criteria for a period of 4–5 days (stromal oedema is the only feature that changes significantly during this period). It is clear that more stringent criteria are needed to improve the precision of timing with endometrial dating.

Figure 1:

Graph illustrating the Noyes method of endometrial dating, which highlights the uncertainty in timing introduced during the post-ovulatory period, the mid-luteal phase and by measuring the LH surge. The distribution over time of many observed changes is too diffuse to allow precise endometrial dating, for example 2 days of the post-ovulatory period and 4–5 days in the mid-luteal phase

Figure 1:

Graph illustrating the Noyes method of endometrial dating, which highlights the uncertainty in timing introduced during the post-ovulatory period, the mid-luteal phase and by measuring the LH surge. The distribution over time of many observed changes is too diffuse to allow precise endometrial dating, for example 2 days of the post-ovulatory period and 4–5 days in the mid-luteal phase

Using the luteinizing hormone (LH) surge to predict ovulation is one approach that has been investigated, although there would still remain a degree of uncertainty in timing, as the LH surge occurs over a period of 30 h ( Acosta et al. , 2000 ). Electron microscopy allows the examination of endometrial ultrastructures present during the implantation window, such as pinopodes and nucleolar channels, which may prove useful markers of endometrial receptivity ( Bentin-Ley et al. , 1999 ; Isaac et al. , 2001 ). Methods that associate morphology and function (e.g. immunohistochemistry, molecular markers) may help to improve the precision of endometrial dating. Unfortunately, immunohistochemistry suffers from the same problems as morphological assessments. Furthermore, the most promising molecular candidates for markers of the implantation window have, so far, failed as predictors of endometrial status ( Acosta et al. , 2000 ). However, other markers may be more successful (e.g. mucin (MUC-1), integrins) ( Lessey et al. , 1996 ; DeLoia et al. , 1998 ). In the future, laser capture microdissection may be coupled with gene expression analysis, providing another useful tool that could be used to link endometrial morphology and function ( Yanaihara et al. , 2004 ).

Another key consideration when using endometrial dating is that it disregards the status of the embryo. Ensuring the endometrium is receptive is of little use if a poor-quality embryo is introduced. Therefore, to ensure optimal conditions for implantation, endometrial dating should not be used in isolation, but should be combined with other techniques that provide information about embryo quality.

Endocrinological aspects

Progesterone and estrogen are the dominant hormonal modulators of endometrial development. Ovarian estrogen and progesterone condition the uterus for implantation, and knowledge about the precise temporal action of these hormones within the menstrual cycle has allowed the development of hormone-based contraception. Both the epithelial and stromal compartments express progesterone and estrogen receptors, and the response depends on the levels of these receptors as well as on the concentration of the hormones themselves. The interactions of progesterone and estrogen with estrogen receptors (ER) during endometrial development are illustrated in Fig.  2 . In recent years, a better understanding has been gained in terms of the types of receptors involved (ERα, ERβ, PRA, PRB) and the dynamics of receptor expression (Fig.  3 ) (Cooke et al. , 1997; Mote et al. , 1999 ). It is apparent that the appropriate cyclical pattern of receptor expression is crucial for achieving endometrial receptivity and successful implantation ( Lessey, 2003 ; Ma et al. , 2003 ).

Figure 2:

The roles of progesterone and estrogen (E 2 ; E 3 , estriol) and estrogen receptors (ER) during endometrial development

Figure 2:

The roles of progesterone and estrogen (E 2 ; E 3 , estriol) and estrogen receptors (ER) during endometrial development

Figure 3:

Changes in the expression of progesterone receptors (PRA, PRB) in glandular epithelial cells and stromal cells during the different phases of the menstrual cycle. Adapted with permission from Mote et al. (1999)

Figure 3:

Changes in the expression of progesterone receptors (PRA, PRB) in glandular epithelial cells and stromal cells during the different phases of the menstrual cycle. Adapted with permission from Mote et al. (1999)

Although progesterone and estrogen are the key modulators of endometrial maturation, their roles in this process are complex and sophisticated ( Punyadeera et al. , 2003 ). Hormonal activity depends on not only the levels of progesterone, estrogen and their receptors, but also on the rates of progesterone and estrogen metabolism (e.g. up-regulation of enzymes that convert estradiol (E 2 ) to estrone or estrone sulphate or remove sulphate from E 2 and estrone) ( Punyadeera et al. , 2003 ). The activities of progesterone and estrogen are also influenced by the effects of co-activators and repressors ( Punyadeera et al. , 2003 ). Furthermore, both hormones regulate the expression of numerous endometrial proteins (paracrine signalling) (Cooke et al. , 1997).

In addition to progesterone and estrogen, a number of other endocrinological factors are known to mediate endometrial function ( Kodaman and Taylor, 2004 ). In rodents, prostaglandins (PGs) are thought to facilitate increased vascular permeability during implantation ( Kennedy, 1979 ), and enzymes involved in PG production (COX-1 and COX-2) shown cyclical changes in expression ( Chakraborty et al. , 1996 ; Das et al. , 1999 ). hCG is thought to have direct effects on the endometrium and also mediates cross-talk between the embryo and the endometrium, through chorionic gonadotrophin receptors present on epithelial cells ( Srisuparp et al. , 2003 ). The effects of androgens are often overlooked in the female reproductive cycle. However, androgen receptors are present on stromal and epithelial cells in the endometrium, and both androstenedione and testosterone induce changes in endometrial function that may be important during implantation ( Kodaman and Taylor, 2004 ).

Endometrial modulators of implantation

Endometrial factors are critical mediators of all phases of the implantation process (Fig.  4 ). Once the embryo reaches the uterus, the first cells it encounters are the epithelial cells of the endometrium. These cells secrete a range of factors into the uterine lumen, which may affect embryonic attachment as well as further development of the early placenta and embryo. However, the precise roles of individual factors as well as the molecular interactions involved have mostly not been elucidated for humans, and the current understanding of these processes stems primarily from research in rodents (reviewed in Dimitriadis et al. , 2005 ; Tranguch et al. , 2005 ; Wang and Dey, 2005 ).

Figure 4:

Factors regulated during the early stages of implantation. Adapted with permission from Dimitriadis et al. (2005)

Figure 4:

Factors regulated during the early stages of implantation. Adapted with permission from Dimitriadis et al. (2005)

In humans, one factor that has attracted particular interest is leukaemia-inhibitory factor (LIF), which is an interleukin (IL)-6 cytokine expressed in endometrial epithelial cells at the appropriate time for which receptors are present on preimplantation embryos. Studies in mice demonstrate that LIF plays a role in implantation and may also promote embryonic development. Observational studies in humans are suggestive of a possible role for LIF in humans ( Robb et al. , 2002 ; Dimitriadis et al. , 2005 ). Indeed, infertility in some women has been associated with the dysregulation of LIF, and also of IL-11, which is produced in the same manner in the endometrial glands during the receptive phase ( Dimitriadis et al. , 2006 ). However, the importance of LIF in implantation is still under debate, as promising results in animal models have failed to translate to humans ( Kimber, 2005 ).

Embryonic factors and the impact of ART

With the increasing trend towards single embryo transfer in ART ( Vilska et al. , 1999 ; Hamberger et al. , 2005 ), the selection of viable embryos is becoming more and more important. Morphological assessment is currently the standard tool for embryo selection in ART (Table  2 ) ( Borini et al. , 2004 ). Over the years, with an improving understanding of embryonic development and advances in in vitro culture techniques, the developmental stage at which an embryo can be transferred has become more advanced, and embryo selection criteria have evolved accordingly. However, no single method of embryo selection has emerged, with some groups selecting blastocyst stage embryos, and others still opting to select at the 2PN stage or cleavage stage ( De Neubourg et al. , 2002 ). Furthermore, it is recognized that morphological assessment of embryo quality is still highly subjective and, therefore, a number of alternative approaches are currently being explored, such as assessment of the embryo culture medium to detect nutrient uptake or metabolite secretion ( Sakkas and Gardner, 2005 ).

Table 2:

Strengths and limitations of morphological assessments of embryo viability

Advantages Limitations 
Established technique Subjective approach 
Widely used and accepted There is no consensus on the timing of selection (2PN versus cleavage stage versus blastocyst) 
Non-invasive Selection criteria cannot yet guarantee an embryo with developmental potential is transferred 
Advantages Limitations 
Established technique Subjective approach 
Widely used and accepted There is no consensus on the timing of selection (2PN versus cleavage stage versus blastocyst) 
Non-invasive Selection criteria cannot yet guarantee an embryo with developmental potential is transferred 

Preimplantation genetic diagnosis (PGD) was initially developed as a preconception test for couples carrying genetic disorders who were at risk of having a child affected by the disorder ( Thornhill et al. , 2005 ). However, more recently, the technique has been used extensively in the context of optimizing IVF outcomes in infertile patients who are not carriers of a heritable disease (Sermon et al. , 2007). Chromosomal analysis of human gametes and embryos has revealed that chromosome aberrations occur at high frequency in the early preimplantation embryo. Preimplantation genetic screening (PGS) enables the testing of gametes and embryos for numerical chromosomal aberrations commonly found in early pregnancy loss, with the aim of replacing only euploid embryos and increasing pregnancy rates after IVF in groups of women who have poor IVF success rates ( Munné, 2003 ; Verlinsky et al. , 2004 ; Caglar et al. , 2005 ; Kearns et al. , 2005 ). Genetic analysis can be performed on polar bodies extracted from the oocyte before fertilization (first polar body) and/or after fertilization (second polar body) ( Verlinsky et al. , 2001 ). At later stages, genetic testing can be performed on one or two blastomeres from the cleavage stage embryo (day 3), or on trophectoderm tissue of the blastocyst (day 5) ( Staessen et al. , 2004 ; McArthur et al. , 2005 ). Although data are emerging from clinical studies investigating the use of PGS in cleavage stage embryos and blastocysts ( Staessen et al. , 2004 ; McArthur et al. , 2005 ; Platteau et al. , 2005a , b ; Twisk et al. , 2006 ), it remains to be established whether advantages from genetic selection are counteracted by a detrimental effect of the biopsy procedure and the removal of embryonic cells, respectively. Furthermore, mosaicism (presence of both aneuploid and euploid cells in an embryo) is commonly found in cleavage-stage embryos, although the clinical relevance of this phenomenon remains unclear ( Bielanska et al. , 2005 ; Baart et al. , 2006 ).

Another key issue in ART protocols that is still under debate is the timing of embryo transfer. Whereas in an unselected patient population a clinical benefit of day-5 transfer (blastocyst transfer) with respect to live-birth rate and multiple-pregnancy rate has not been shown ( Blake et al. , 2005 ), in patients with a good prognosis (young patients, minimum number of good quality embryos on day 3) blastocyst transfer yields a significantly higher live-birth rate ( Papanikolaou et al. , 2006a , b ; The Practice Committee of the American Society for Reproductive Medicine and the Practice Committee of the Society for Assisted Reproductive Technology, 2006 ). Blastocyst transfer gives the option to select the morphologically best embryo, whereas it has also been indicated that good-quality blastocysts have a decreased incidence of aneuploidy (Fig.  5 ) ( Staessen et al. , 2004 ).

Figure 5:

Relationship between chromosomal abnormalities and developmental stage on ( A ) day 3 or ( B ) day 5 of embryonic development. Figure reproduced with permission from Staessen et al. (2004)

Figure 5:

Relationship between chromosomal abnormalities and developmental stage on ( A ) day 3 or ( B ) day 5 of embryonic development. Figure reproduced with permission from Staessen et al. (2004)

Failure of the blastocyst to release from the zona pellucida has been identified as a potential cause of implantation failure in assisted cycles, particularly in older women ( Seif et al. , 2006 ). A potential solution to this is artificial disruption of the zona pellucida or assisted hatching. A systematic review of studies investigating the effects of this technique on conception found that assisted hatching significantly improved pregnancy rates, but had no effect on live-birth rates or spontaneous abortion rates, and multiple-pregnancy rates were significantly increased ( Seif et al. , 2006 ). Unfortunately, there were insufficient data for this analysis to investigate the impact of assisted hatching on a number of other important outcomes, such as monozygotic twinning, embryo damage, congenital and chromosomal abnormalities and in vitro blastocyst development.

When optimizing ART procedures to mimic nature as closely as possible, it is important to remember that controlled ovarian stimulation itself interrupts natural physiological processes and is likely to alter key parameters such as the rate of embryonic development and the extent and timing of endometrial receptivity. Artificial stimulation affects the levels of progesterone and estrogen, the ratio between these two hormones and endometrial expression of their receptors ( Beckers et al. , 2000 ; Papanikolaou et al. , 2005 ). There is evidence that supraphysiological steroid levels impair the luteal phase, and this is true, even when stimulation is started in the late follicular phase ( Bourgain et al. , 1994 ; Ubaldi et al. , 1996 ; Macklon and Fauser, 2000a ; Kolibianakis et al. , 2003 ). Therefore, if the luteal phase is not supplemented, premature luteolysis can occur and pregnancy may not be achieved ( Beckers et al. , 2000 , 2003 ). In ART cycles, the aim is to produce multiple mature follicles, which leads to elevated levels of progesterone and estrogen compared with natural cycles, and this can induce changes in the endometrium ( Bourgain and Devroey, 2003 ) that can be detected using standard histological techniques ( Garcia et al. , 1984 ) and scanning electron microscopy ( Kolb et al. , 1997 ).

To overcome some of the effects potentially associated with hormonal stimulation, various modified stimulation protocols have been investigated. For example, milder stimulation regimens have been studied, in which gonadotrophins were administered at a lower dose, or later in the cycle, or that used gonadotrophin-releasing hormone antagonists for pituitary downregulation ( Macklon and Fauser, 2000b ; Hohmann et al. , 2001 , 2003 ). Early administration of hCG for final oocyte maturation (as soon as three follicles ≥17 mm are present) appears beneficial in terms of pregnancy rates, especially when day 3 embryo transfers are performed ( Kolibianakis et al. , 2005 ).

Animal models and human in vitro systems

The implantation process itself has never been observed directly in vivo in humans ( Lee and DeMayo, 2004 ). However, studies in animals, primarily rodents, sheep and primates, have provided clues about the hormonal and morphological changes that might occur in women prior to and during implantation ( Lee and DeMayo, 2004 ). Indeed, the three stages of endometrial development observed in animals (endometrial neutrality, receptivity and refractoriness) are also thought to occur in humans ( Rogers, 1995 ). It is recognized that different species show a wide variety of mechanisms by which implantation occurs ( Ringler and Strauss, 1990 ) and, therefore, different animals may be more suited as models for particular steps in the human implantation process. For example, pigs and sheep are potential candidates for the study of the early stages of implantation, as they have extended apposition and attachment phases ( Lee and DeMayo, 2004 ). Conversely, macaques and humans have similar mechanisms for trophoblast invasion and, therefore, macaques are a suitable model for studying the later phases of implantation ( Lee and DeMayo, 2004 ).

Although information about the physiology of implantation has been gained from a range of different animal models, the current understanding about this process on a molecular level results largely from studies in mice ( Lee and DeMayo, 2004 ). However, the mechanisms of implantation in mice and humans are quite distinct. During implantation in mice, the luminal epithelium forms an invagination that surrounds the trophoblast (eccentric mechanism) and is subsequently shed by apoptosis, whereas in humans, the trophoblast invades the stroma by penetrating the luminal epithelium (interstitial mechanism) ( Wimsatt, 1975 ).

Studies on LIF illustrate how promising findings in mice have translated to disappointing results in humans. Targeted mutagenesis studies in mice clearly established an essential role for LIF in mouse implantation, prompting intensive investigation into its role in humans. However, LIF expression varies widely in humans, and although putative LIF mutations have been identified ( Giess et al. , 1999 ; Kralickova et al. , 2006 ), their functional significance is unclear. Moreover, low LIF levels have been associated with increased success in IVF/embryo transfer programmes in some studies ( Ledee-Bataille et al. , 2002 ), whereas others have found no association ( Olivennes et al. , 2003 ). Collectively, these data question an essential role for LIF in human implantation and are cause for reflection as to the translatability of animal studies to human biology.

This issue of translatability has important implications for future research, as rodent models are best suited for testing the functional role of genes and proteins. Consequently, animal studies should be validated using alternative in vivo models, including primates, and in vitro systems that can reproduce critical stages of the implantation process with fidelity, prior to the initiation of large-scale clinical trials or development of methods to assess endometrial receptivity or improve implantation rates. To address this need, a number of in vitro models using human cell culture systems have been developed to study various aspects of embryo–endometrial interaction.

Bentin-Ley et al. (1994) constructed a complex 3D endometrial cell culture system containing stromal cells embedded in a collagen matrix and separated from an epithelial monolayer by basement membrane material (‘Matrigel’: Becton and Dickinson Biosciences, San Jose, CA, USA). Using this model, they demonstrated that human blastocysts attach preferentially to pinopode-presenting areas on the endometrial surface ( Bentin-Ley et al. , 1999 ). Another group cultured a complete endometrial biopsy of the upper functional layer of the endometrium onto collagen gel ( Landgren et al. , 1996 ). Although they were able to observe human blastocyst adhesion of the stromal layer in this ‘miniorgan’, there was evidence of tissue degeneration after 48 h.

Simon et al. have developed in vitro models to specifically study the apposition and adhesion phases of implantation ( Simon et al. , 1999b ; Mercader et al. , 2003 ). In the apposition model, embryos obtained after ovarian superovulation and insemination (IVF or intracytoplasmic sperm injection) were co-cultured with luteal phase endometrial epithelial cells. This model resulted in a clinical programme where embryos could be co-cultured with epithelial cells until blastocyst stage and transferred back to the mother ( Mercader et al. , 2003 ). For the adhesion model, a 3D culture was prepared, comprising epithelial and stromal cells cultured from endometrial biopsies. Blastocysts cultured on these endometrial epithelial cells attached to the epithelial surface and could be immunologically localized using anti-β-hCG staining ( Galan et al. , 2000 ; Meseguer et al. , 2001 ). These models have provided information about the embryonic regulation of endometrial epithelial molecules such as anti-adhesion molecules ( Meseguer et al. , 2001 ), cytoskeletal proteins ( Martin et al. , 2000 ), chemokines ( Dominguez et al. , 2003 ) and the leptin system ( Cervero et al. , 2004 ) during the apposition and adhesion phases of human implantation.

An in vitro model has also been developed to study the process of blastocyst invasion ( Carver et al. , 2003 ). Carver et al. (2003) were able to observe structural and hormonal changes occurring during blastocyst invasion using time-lapse photography, immunostaining and measurement of hCG levels for human hatched blastocysts co-cultured with human endometrial stromal cell monolayers.

Molecular approaches

Advances in biotechnology have lead to the development of new techniques that allow the examination of changes in the endometrium and embryo at the molecular level. DNA microarrays enable analysis of the simultaneous expression of thousands of genes in a single sample. Bioinformatic tools have been developed, which can quantify and link such molecular changes (Table  3 ). These genomic and proteomic techniques have been used to study changes occurring throughout the cycle, examine the impact of artificial stimulation and determine the patterns of gene expression in different cell types.

Table 3:

Strengths and limitations of measuring changes in protein or gene expression to assess endometrial receptivity and/or embryo viability

Advantages Limitations 
Objective approach Not accessible to many groups (expensive and high level of technical skill required to analyse and interpret results) 
Provides information about related groups of molecules (clusters) New technology, methodology needs to be standardized 
Large amounts of information generated in a small amount of time Poor reproducibility between experiments in different groups 
May be more representative of biological phases than morphological methods Correct sample preparation is essential and should be consistent 
Advantages Limitations 
Objective approach Not accessible to many groups (expensive and high level of technical skill required to analyse and interpret results) 
Provides information about related groups of molecules (clusters) New technology, methodology needs to be standardized 
Large amounts of information generated in a small amount of time Poor reproducibility between experiments in different groups 
May be more representative of biological phases than morphological methods Correct sample preparation is essential and should be consistent 

The expression of many endometrial genes has been shown to change over the course of the menstrual cycle ( Ponnampalam et al. , 2004 ; Talbi et al. , 2006 ). However, some of these expression patterns do not appear to associate with histopathological changes occurring in the endometrium ( Ponnampalam et al. , 2004 ). Possibly, gene expression may be a better marker of the biological phases and may be a more reliable predictor of endometrial receptivity than morphology.

To date, five studies have examined changes in endometrial gene expression during the receptive phase and all have reported genes that are strongly up- or down-regulated when the endometrium is receptive ( Carson et al. , 2002 ; Kao et al. , 2002 ; Borthwick et al. , 2003 ; Riesewijk et al. , 2003 ; Mirkin et al. , 2005 ). One striking observation is that only a single gene (osteopontin) was differentially expressed (up-regulated) in all five of these studies ( Carson et al. , 2002 ; Kao et al. , 2002 ; Borthwick et al. , 2003 ; Riesewijk et al. , 2003 ; Mirkin et al. , 2005 ). The divergent results from different studies have been attributed to differences in study design and the software/statistics used in the analysis of the data ( Riesewijk et al. , 2003 ). This finding highlights the need for standardization of methodology if meaningful conclusions are to be made from genomic and proteomic studies.

Microarray studies comparing natural and stimulated cycles indicate that controlled ovarian stimulation has a profound effect on endometrial gene expression during the window of implantation (7 days after the LH surge compared with 2 days; Fig.  6 ) ( Horcajadas et al. , 2005 ; Simon et al. , 2005 ). Over 200 genes were differentially expressed in stimulated cycles ( Horcajadas et al. , 2005 ), and the pattern of expression depended upon the type of down-regulation protocol used (agonist or antagonist) ( Simon et al. , 2005 ). Studies have also examined gene expression changes in human endometrial cells in vitro during decidualization ( Popovici et al. , 2000 ; Brar et al. , 2001 ; Tierney et al. , 2003 ), in response to progesterone ( Okada et al. , 2003 ), or in endometrial biopsies taken at different phases of the menstrual cycle ( Ponnampalam et al. , 2004 ; Punyadeera et al. , 2005 ; Talbi et al. , 2006 ).

Figure 6:

Principle component analyses (PCA) of endometrial gene expression showing clustering of samples from ( A ) 2 versus 7 days after the LH surge or ( B ) natural versus stimulated cycles. Adapted with permission from Riesewijk et al. (2003) and Horcajadas et al. (2005)

Figure 6:

Principle component analyses (PCA) of endometrial gene expression showing clustering of samples from ( A ) 2 versus 7 days after the LH surge or ( B ) natural versus stimulated cycles. Adapted with permission from Riesewijk et al. (2003) and Horcajadas et al. (2005)

Laser capture microdissection coupled with gene expression analysis enables accurate comparison of gene expression patterns between different cell types from the same tissue. To date, one study has used this technique to examine differences in normal human endometrial tissues from the secretory phase ( Yanaihara et al. , 2004 ). A total of 28 genes were found to be differentially expressed in epithelial and stromal cells, and a number of these genes have known immunological functions ( Yanaihara et al. , 2004 ).

As well as array technologies being used to study gene expression, methods are also being developed to study proteomic changes occurring during implantation. Endometrial secretion aspiration is one such approach and enables the measurement of protein changes in the uterine lumen during treatment cycles ( van der Gaast et al. , 2003 ). A key advantage of this approach is that the technique itself does not appear to have an adverse effect on implantation ( van der Gaast et al. , 2003 ). However, it is important to note that secretion aspirations may contain cellular contaminants, such as leukocytes, stromal cells or epithelial cells, which must be removed prior to analysis, or taken into consideration when interpreting the results.

As well as studying the molecular changes occurring in the endometrium, it is equally important to conduct molecular studies on oocytes and embryos, but unfortunately these have been few ( Neilson et al. , 2000 ; Stanton et al. , 2002 ; Dobson et al. , 2004 ; Katz-Jaffe et al. , 2006 ). Two of these studies have investigated differential gene or protein expression in human embryos ( Dobson et al. , 2004 ; Katz-Jaffe et al. , 2006 ). Dobson et al. (2004) characterized global changes in gene expression during the first 3 days of embryonic development and found that embryonic transcriptional programmes were already established within 3 days of fertilization. Katz-Jaffe et al. (2006) examined changes in the proteome of individual human blastocysts and observed characteristic expression profiles that associated with changes in morphology or embryo degeneration. Such studies could reveal molecular signatures that are consistent with high-quality gametes and embryos and, more importantly, identify candidate secreted molecules that could be assessed non-invasively for association with implantation success. However, when assessing oocytes and embryos, much smaller amounts of material can be obtained compared with endometrial samples. The fewer cells that are removed from the embryo (or blastocyst), the less disruption is likely to be caused. Therefore, highly accurate methods of amplification and detection are required.

Investigators at Serono International SA (Geneva, Switzerland) have begun to develop tools and a strategy to enable molecular-based embryo selection (Fig.  7 ). Using this approach, RNA amplification from a single blastomere provided sufficient amplified RNA for microarray analysis. The expression of over 8300 genes was detected in day 3 human embryos and enzyme-linked immunosorbent assay (ELISA) is in progress to confirm the expression of these genes. In parallel, detection of proteins produced by embryos in culture using antibody arrays or ELISA provided sufficient sensitivity to identify secreted proteins from a single embryo, with the potential to assess embryo quality at day 3 of culture. Finally, DNA fingerprinting of embryos from single blastomere biopsy and later of amniocytes and fetal cord cells would enable matching of genomic and proteomic profiles to the embryo that successfully develops. Both microsatellite DNA analysis (short tandem repeat) and single-nucleotide polymorphism (SNP) analysis have demonstrated early potential for use with a single cell.

Figure 7:

A molecular approach being developed to improve phenotypic selection of human embryos for transfer using genomic and proteomic tools. Some initial data from the ‘straight to proteomics’ and ‘genomic to proteomic’ approaches are also presented. RNA, ribonucleic acid; STR, short tandem repeat polymorphism

Figure 7:

A molecular approach being developed to improve phenotypic selection of human embryos for transfer using genomic and proteomic tools. Some initial data from the ‘straight to proteomics’ and ‘genomic to proteomic’ approaches are also presented. RNA, ribonucleic acid; STR, short tandem repeat polymorphism

Moving beyond genomics and proteomics, metabolic profiles as well as embryonic signalling molecule profiles can be targeted as predictors of developmental potential (‘metabolomics’). However, to date, most data on embryo metabolism stem from rodent research and need to be evaluated in humans.

The way forward

Animal studies and in vitro experiments have improved understanding about the hormonal and morphological changes that occur during implantation in natural cycles. In addition, numerous paracrine factors that mediate implantation processes have been identified, and a next step would be to associate this information with endocrinology and morphology. It is also clear that stimulated cycles behave differently to natural cycles, so establishing where these differences lie, in terms of both the endometrium and the embryo, is another important area of focus for future studies.

Current morphological markers of endometrial receptivity are poor predictors of pregnancy. Therefore, there is a need for non-disruptive in vivo methods to study endometrial receptivity and the implantation process itself, particularly in those women in whom pregnancy is achieved. Endometrial secretion aspiration may be one useful approach to this problem, providing aspiration does not affect implantation rates ( van der Gaast et al. , 2003 ).

In the past, the focus for improving embryo selection has been morphological criteria and the detection of chromosomal abnormalities. However, while chromosomal abnormalities may be responsible for a large proportion of implantation failures, they are not the cause for all of them. Therefore, trials that examine the causes of implantation failure, particularly in older women, in whom levels of chromosomal abnormality are high, would be useful. Ideally, single embryo transfer would be used, although the policy of single embryo transfer may not be ethical in this particular group of patients.

New molecular techniques are becoming available for measuring both embryonic and endometrial changes prior to and during implantation. However, these approaches are still in their infancy, and although they hold much promise, it is important that standardized ways of working are devised at an early stage. Ultimately, the aim is to use these tools to increase implantation in artificial cycles and consequently improve live-birth rates.

Appendix

Evian Current Reproductive Medicine Workshop Group

Veronica Alam (Serono International SA, Geneva, Switzerland), Paul Bischof (University of Geneva, Geneva, Switzerland), Claire Bourgain (University Hospital, Dutch-speaking Brussels Free University, Brussels, Belgium), Paul Devroey (Centre of Reproductive Medicine, Dutch-speaking Brussels Free University), Klaus Diedrich (University Hospital of Schleswig-Holstein, Lübeck, Germany), Aliza Eshkol (Serono International SA), Bart Fauser (University Medical Center, Utrecht, The Netherlands), Georg Griesinger (University Hospital of Schleswig-Holstein), Christoph Keck (Serono International SA), Nick Macklon (University Medical Centre, Utrecht, The Netherlands), Steve Palmer (Serono Reproductive Biology Institute, Rockland, MA, USA), Evangelos Papanikolaou (AZ-VUB, Centre for Reproductive Medicine, Brussels, Belgium), Lois Salamonsen (Prince Henry's Institute of Medical Research, Melbourne, Australia), Gamal Serour (Egyptian IVF-ET Center, Cairo, Egypt), Carlos Simon (Valencia University, Valencia, Spain), Catherine Staessen (University Hospital, Dutch-Speaking Brussels Free University) and Jerome F. Strauss III (Virginia Commonwealth University School of Medicine, Richmond, Virginia, USA).

The Evian Current Reproductive Medicine Workshop was held in February 2006. The workshop and the preparation of this manuscript were both sponsored by Serono International SA, Geneva, Switzerland.

References

Achard
V
Perrin
J
Saias-Magnan
J
Noizet
A
Grillo
JM
Paulmyer-Lacroix
O
Optimization of artificial inseminations with donor semen: a four-year experience
Gynecol Obstet Fertil
 , 
2005
, vol. 
33
 (pg. 
877
-
883
)
Acosta
AA
Elberger
L
Borghi
M
Calamera
JC
Chemes
H
Doncel
GF
Kliman
H
Lema
B
Lustig
L
Papier
S
Endometrial dating and determination of the window of implantation in healthy fertile women
Fertil Steril
 , 
2000
, vol. 
73
 (pg. 
788
-
798
)
Andersen
AN
Gianaroli
L
Felberbaum
R
de Mouzon
J
Nygren
KG
Assisted reproductive technology in Europe, 2001. Results generated from European registers by ESHRE
Hum Reprod
 , 
2005
, vol. 
20
 (pg. 
1158
-
1176
)
Baart
EB
Martini
E
van den Berg
I
Macklon
NS
Galjaard
RJ
Fauser
BC
Van Opstal
D
Preimplantation genetic screening reveals a high incidence of aneuploidy and mosaicism in embryos from young women undergoing IVF
Hum Reprod
 , 
2006
, vol. 
21
 (pg. 
223
-
233
)
Beckers
NG
Laven
JS
Eijkemans
MJ
Fauser
BC
Follicular and luteal phase characteristics following early cessation of gonadotrophin-releasing hormone agonist during ovarian stimulation for in-vitro fertilization
Hum Reprod
 , 
2000
, vol. 
15
 (pg. 
43
-
49
)
Beckers
NG
Macklon
NS
Eijkemans
MJ
Ludwig
M
Felberbaum
RE
Diedrich
K
Bustion
S
Loumaye
E
Fauser
BC
Nonsupplemented luteal phase characteristics after the administration of recombinant human chorionic gonadotropin, recombinant luteinizing hormone, or gonadotropin-releasing hormone (GnRH) agonist to induce final oocyte maturation in in vitro fertilization patients after ovarian stimulation with recombinant follicle-stimulating hormone and GnRH antagonist cotreatment
J Clin Endocrinol Metab
 , 
2003
, vol. 
88
 (pg. 
4186
-
4192
)
Bentin-Ley
U
Pedersen
B
Lindenberg
S
Larsen
JF
Hamberger
L
Horn
T
Isolation and culture of human endometrial cells in a three-dimensional culture system
J Reprod Fertil
 , 
1994
, vol. 
101
 (pg. 
327
-
332
)
Bentin-Ley
U
Sjogren
A
Nilsson
L
Hamberger
L
Larsen
JF
Horn
T
Presence of uterine pinopodes at the embryo-endometrial interface during human implantation in vitro
Hum Reprod
 , 
1999
, vol. 
14
 (pg. 
515
-
520
)
Bergh
PA
Navot
D
The impact of embryonic development and endometrial maturity on the timing of implantation
Fertil Steril
 , 
1992
, vol. 
58
 (pg. 
537
-
542
)
Bielanska
M
Jin
S
Bernier
M
Tan
SL
Ao
A
Diploid-aneuploid mosaicism in human embryos cultured to the blastocyst stage
Fertil Steril
 , 
2005
, vol. 
84
 (pg. 
336
-
342
)
Blake
D
Proctor
M
Johnson
N
Olive
D
Cleavage stage versus blastocyst stage embryo transfer in assisted conception
Cochrane Database Syst Rev
 , 
2005
 
CD002118
Borini
A
Lagalla
C
Sciajno
R
Distratis
V
Bonu
MA
Cattoli
M
Coticchio
G
Artificial reproductive technology achievements for optimizing embryo quality
Ann NY Acad Sci
 , 
2004
, vol. 
1034
 (pg. 
252
-
261
)
Borthwick
JM
Charnock-Jones
DS
Tom
BD
Hull
ML
Teirney
R
Phillips
SC
Smith
SK
Determination of the transcript profile of human endometrium
Mol Hum Reprod
 , 
2003
, vol. 
9
 (pg. 
19
-
33
)
Bourgain
C
Devroey
P
The endometrium in stimulated cycles for IVF
Hum Reprod Update
 , 
2003
, vol. 
9
 (pg. 
515
-
522
)
Bourgain
C
Smitz
J
Camus
M
Erard
P
Devroey
P
Van Steirteghem
AC
Kloppel
G
Human endometrial maturation is markedly improved after luteal supplementation of gonadotrophin-releasing hormone analogue/human menopausal gonadotrophin stimulated cycles
Hum Reprod
 , 
1994
, vol. 
9
 (pg. 
32
-
40
)
Brar
AK
Handwerger
S
Kessler
CA
Aronow
BJ
Gene induction and categorical reprogramming during in vitro human endometrial fibroblast decidualization
Physiol Genomics
 , 
2001
, vol. 
7
 (pg. 
135
-
148
)
Caglar
GS
Asimakopoulos
B
Nikolettos
N
Diedrich
K
Al-Hasani
S
Preimplantation genetic diagnosis for aneuploidy screening in repeated implantation failure
Reprod Biomed Online
 , 
2005
, vol. 
10
 (pg. 
381
-
388
)
Carson
DD
Lagow
E
Thathiah
A
Al-Shami
R
Farach-Carson
MC
Vernon
M
Yuan
L
Fritz
MA
Lessey
B
Changes in gene expression during the early to mid-luteal (receptive phase) transition in human endometrium detected by high-density microarray screening
Mol Hum Reprod
 , 
2002
, vol. 
8
 (pg. 
871
-
879
)
Carver
J
Martin
K
Spyropoulou
I
Barlow
D
Sargent
I
Mardon
H
An in-vitro model for stromal invasion during implantation of the human blastocyst
Hum Reprod
 , 
2003
, vol. 
18
 (pg. 
283
-
290
)
Cervero
A
Horcajadas
JA
MartIn
J
Pellicer
A
Simon
C
The leptin system during human endometrial receptivity and preimplantation development
J Clin Endocrinol Metab
 , 
2004
, vol. 
89
 (pg. 
2442
-
2451
)
Chakraborty
I
Das
SK
Wang
J
Dey
SK
Developmental expression of the cyclo-oxygenase-1 and cyclo-oxygenase-2 genes in the peri-implantation mouse uterus and their differential regulation by the blastocyst and ovarian steroids
J Mol Endocrinol
 , 
1996
, vol. 
16
 (pg. 
107
-
122
)
Check
JH
Nowroozi
K
Chase
J
Nazari
A
Braithwaite
C
Comparison of pregnancy rates following in vitro fertilization-embryo transfer between the donors and the recipients in a donor oocyte program
J Assist Reprod Genet
 , 
1992
, vol. 
9
 (pg. 
248
-
250
)
Cooke
PS
Buchanan
DL
Young
P
Setiawan
T
Brody
J
Korach
KS
Taylor
J
Lubahn
DB
Cunha
GR
Stromal estrogen receptors mediate mitogenic effects of estradiol on uterine epithelium
Proc Natl Acad Sci USA
 , vol. 
94
 (pg. 
6535
-
6540
)
Croxatto
HB
Ortiz
ME
Diaz
S
Hess
R
Balmaceda
J
Croxatto
HD
Studies on the duration of egg transport by the human oviduct. II. Ovum location at various intervals following luteinizing hormone peak
Am J Obstet Gynecol
 , 
1978
, vol. 
132
 (pg. 
629
-
634
)
Das
SK
Wang
J
Dey
SK
Mead
RA
Spatiotemporal expression of cyclooxygenase 1 and cyclooxygenase 2 during delayed implantation and the periimplantation period in the Western spotted skunk
Biol Reprod
 , 
1999
, vol. 
60
 (pg. 
893
-
899
)
De Neubourg
D
Mangelschots
K
Van Royen
E
Vercruyssen
M
Ryckaert
G
Valkenburg
M
Barudy-Vasquez
J
Gerris
J
Impact of patients' choice for single embryo transfer of a top quality embryo versus double embryo transfer in the first IVF/ICSI cycle
Hum Reprod
 , 
2002
, vol. 
17
 (pg. 
2621
-
2625
)
DeLoia
JA
Krasnow
JS
Brekosky
J
Babaknia
A
Julian
J
Carson
DD
Regional specialization of the cell membrane-associated, polymorphic mucin (MUC1) in human uterine epithelia
Hum Reprod
 , 
1998
, vol. 
13
 (pg. 
2902
-
2909
)
Dimitriadis
E
White
CA
Jones
RL
Salamonsen
LA
Cytokines, chemokines and growth factors in endometrium related to implantation
Hum Reprod Update
 , 
2005
, vol. 
11
 (pg. 
613
-
630
)
Dimitriadis
E
Stoikos
C
Stafford-Bell
M
Clark
I
Paiva
P
Kovacs
G
Salamonsen
LA
Interleukin-11, IL-11 receptoralpha and leukemia inhibitory factor are dysregulated in endometrium of infertile women with endometriosis during the implantation window
J Reprod Immunol
 , 
2006
, vol. 
69
 (pg. 
53
-
64
)
Dobson
AT
Raja
R
Abeyta
MJ
Taylor
T
Shen
S
Haqq
C
Pera
RA
The unique transcriptome through day 3 of human preimplantation development
Hum Mol Genet
 , 
2004
, vol. 
13
 (pg. 
1461
-
1470
)
Dominguez
F
Galan
A
Martin
JJ
Remohi
J
Pellicer
A
Simon
C
Hormonal and embryonic regulation of chemokine receptors CXCR1, CXCR4, CCR5 and CCR2B in the human endometrium and the human blastocyst
Mol Hum Reprod
 , 
2003
, vol. 
9
 (pg. 
189
-
198
)
Emiliani
S
Delbaere
A
Devreker
F
Englert
Y
Embryo-maternal interactive factors regulating the implantation process: implications in assisted reproductive
Reprod Biomed Online
 , 
2005
, vol. 
10
 (pg. 
527
-
540
)
Enders
AC
Schlafke
S
Hendrickx
AG
Differentiation of the embryonic disc, amnion, and yolk sac in the rhesus monkey
Am J Anat
 , 
1986
, vol. 
177
 (pg. 
161
-
185
)
Ferrara
I
Balet
R
Grudzinskas
JG
Intrauterine insemination with frozen donor sperm. Pregnancy outcome in relation to age and ovarian stimulation regime
Hum Reprod
 , 
2002
, vol. 
17
 (pg. 
2320
-
2324
)
Galan
A
O'Connor
JE
Valbuena
D
Herrer
R
Remohi
J
Pampfer
S
Pellicer
A
Simon
C
The human blastocyst regulates endometrial epithelial apoptosis in embryonic adhesion
Biol Reprod
 , 
2000
, vol. 
63
 (pg. 
430
-
439
)
Garcia
JE
Acosta
AA
Hsiu
JG
Jones
HW
Jr
Advanced endometrial maturation after ovulation induction with human menopausal gonadotropin/human chorionic gonadotropin for in vitro fertilization
Fertil Steril
 , 
1984
, vol. 
41
 (pg. 
31
-
35
)
Giess
R
Tanasescu
I
Steck
T
Sendtner
M
Leukaemia inhibitory factor gene mutations in infertile women
Mol Hum Reprod
 , 
1999
, vol. 
5
 (pg. 
581
-
586
)
Hamberger
L
Hardarson
T
Nygren
KG
Avoidance of multiple pregnancy by use of single embryo transfer
Minerva Ginecol
 , 
2005
, vol. 
57
 (pg. 
15
-
19
)
Hassold
T
Chen
N
Funkhouser
J
Jooss
T
Manuel
B
Matsuura
J
Matsuyama
A
Wilson
C
Yamane
JA
Jacobs
PA
A cytogenetic study of 1000 spontaneous abortions
Ann Hum Genet
 , 
1980
, vol. 
44
 (pg. 
151
-
178
)
Hohmann
FP
Laven
JS
de Jong
FH
Eijkemans
MJ
Fauser
BC
Low-dose exogenous FSH initiated during the early, mid or late follicular phase can induce multiple dominant follicle development
Hum Reprod
 , 
2001
, vol. 
16
 (pg. 
846
-
854
)
Hohmann
FP
Macklon
NS
Fauser
BC
A randomized comparison of two ovarian stimulation protocols with gonadotropin-releasing hormone (GnRH) antagonist cotreatment for in vitro fertilization commencing recombinant follicle-stimulating hormone on cycle day 2 or 5 with the standard long GnRH agonist protocol
J Clin Endocrinol Metab
 , 
2003
, vol. 
88
 (pg. 
166
-
173
)
Horcajadas
JA
Riesewijk
A
Polman
J
van Os
R
Pellicer
A
Mosselman
S
Simon
C
Effect of controlled ovarian hyperstimulation in IVF on endometrial gene expression profiles
Mol Hum Reprod
 , 
2005
, vol. 
11
 (pg. 
195
-
205
)
Isaac
C
Pollard
JW
Meier
UT
Intranuclear endoplasmic reticulum induced by Nopp140 mimics the nucleolar channel system of human endometrium
J Cell Sci
 , 
2001
, vol. 
114
 (pg. 
4253
-
4264
)
Kao
LC
Tulac
S
Lobo
S
Imani
B
Yang
JP
Germeyer
A
Osteen
K
Taylor
RN
Lessey
BA
Giudice
LC
Global gene profiling in human endometrium during the window of implantation
Endocrinology
 , 
2002
, vol. 
143
 (pg. 
2119
-
2138
)
Katz-Jaffe
MG
Gardner
DK
Schoolcraft
WB
Proteomic analysis of individual human embryos to identify novel biomarkers of development and viability
Fertil Steril
 , 
2006
, vol. 
85
 (pg. 
101
-
107
)
Kearns
WG
Pen
R
Graham
J
Han
T
Carter
J
Moyer
M
Richter
KS
Tucker
M
Hoegerman
SF
Widra
E
Preimplantation genetic diagnosis and screening
Semin Reprod Med
 , 
2005
, vol. 
23
 (pg. 
336
-
347
)
Kennedy
TG
Prostaglandins and increased endometrial vascular permeabiltiy resulting from the application of artificial stimulus to the uterus of the rat sensitized for the decidual cell reaction
Biol Reprod
 , 
1979
, vol. 
20
 (pg. 
560
-
566
)
Kimber
SJ
Leukaemia inhibitory factor in implantation and uterine biology
Reproduction
 , 
2005
, vol. 
130
 (pg. 
131
-
145
)
Kodaman
PH
Taylor
HS
Hormonal regulation of implantation
Obstet Gynecol Clin North Am
 , 
2004
, vol. 
31
 (pg. 
745
-
766
)
Kolb
BA
Najmabadi
S
Paulson
RJ
Ultrastructural characteristics of the luteal phase endometrium in patients undergoing controlled ovarian hyperstimulation
Fertil Steril
 , 
1997
, vol. 
67
 (pg. 
625
-
630
)
Kolibianakis
EM
Bourgain
C
Platteau
P
Albano
C
Van Steirteghem
AC
Devroey
P
Abnormal endometrial development occurs during the luteal phase of nonsupplemented donor cycles treated with recombinant follicle-stimulating hormone and gonadotropin-releasing hormone antagonists
Fertil Steril
 , 
2003
, vol. 
80
 (pg. 
464
-
466
)
Kolibianakis
EM
Bourgain
C
Papanikolaou
EG
Camus
M
Tournaye
H
Van Steirteghem
AC
Devroey
P
Prolongation of follicular phase by delaying hCG administration results in a higher incidence of endometrial advancement on the day of oocyte retrieval in GnRH antagonist cycles
Hum Reprod
 , 
2005
, vol. 
20
 (pg. 
2453
-
2456
)
Kralickova
M
Sima
R
Vanecek
T
Sima
P
Rokyta
Z
Ulcova-Gallova
Z
Sucha
R
Uher
P
Hes
O
Leukemia inhibitory factor gene mutations in the population of infertile women are not restricted to nulligravid patients
Eur J Obstet Gynecol Reprod Biol
 , 
2006
, vol. 
127
 (pg. 
231
-
235
)
Landgren
BM
Johannisson
E
Stavreus-Evers
A
Hamberger
L
Eriksson
H
A new method to study the process of implantation of a human blastocyst in vitro
Fertil Steril
 , 
1996
, vol. 
65
 (pg. 
1067
-
1070
)
Ledee-Bataille
N
Lapree-Delage
G
Taupin
JL
Dubanchet
S
Frydman
R
Chaouat
G
Concentration of leukaemia inhibitory factor (LIF) in uterine flushing fluid is highly predictive of embryo implantation
Hum Reprod
 , 
2002
, vol. 
17
 (pg. 
213
-
218
)
Lee
KY
DeMayo
FJ
Animal models of implantation
Reproduction
 , 
2004
, vol. 
128
 (pg. 
679
-
695
)
Lessey
BA
Two pathways of progesterone action in the human endometrium: implications for implantation and contraception
Steroids
 , 
2003
, vol. 
68
 (pg. 
809
-
815
)
Lessey
BA
Ilesanmi
AO
Lessey
MA
Riben
M
Harris
JE
Chwalisz
K
Luminal and glandular endometrial epithelium express integrins differentially throughout the menstrual cycle: implications for implantation, contraception, and infertility
Am J Reprod Immunol Microbiol
 , 
1996
, vol. 
35
 (pg. 
195
-
204
)
Ma
WG
Song
H
Das
SK
Paria
BC
Dey
SK
Estrogen is a critical determinant that specifies the duration of the window of uterine receptivity for implantation
Proc Natl Acad Sci USA
 , 
2003
, vol. 
100
 (pg. 
2963
-
2968
)
Macklon
NS
Fauser
BC
Impact of ovarian hyperstimulation on the luteal phase
J Reprod Fertil
 , 
2000a
, vol. 
55
 
Suppl
(pg. 
101
-
108
)
Macklon
NS
Fauser
BC
Regulation of follicle development and novel approaches to ovarian stimulation for IVF
Hum Reprod Update
 , 
2000b
, vol. 
6
 (pg. 
307
-
312
)
Martin
JC
Jasper
MJ
Valbuena
D
Meseguer
M
Remohi
J
Pellicer
A
Simon
C
Increased adhesiveness in cultured endometrial-derived cells is related to the absence of moesin expression
Biol Reprod
 , 
2000
, vol. 
63
 (pg. 
1370
-
1376
)
McArthur
SJ
Leigh
D
Marshall
JT
de Boer
KA
Jansen
RP
Pregnancies and live births after trophectoderm biopsy and preimplantation genetic testing of human blastocysts
Fertil Steril
 , 
2005
, vol. 
84
 (pg. 
1628
-
1636
)
Mercader
A
Garcia-Velasco
JA
Escudero
E
Remohi
J
Pellicer
A
Simon
C
Clinical experience and perinatal outcome of blastocyst transfer after coculture of human embryos with human endometrial epithelial cells: a 5-year follow-up study
Fertil Steril
 , 
2003
, vol. 
80
 (pg. 
1162
-
1168
)
Meseguer
M
Aplin
JD
Caballero-Campo
P
O'Connor
JE
Martin
JC
Remohi
J
Pellicer
A
Simon
C
Human endometrial mucin MUC1 is up-regulated by progesterone and down-regulated in vitro by the human blastocyst
Biol Reprod
 , 
2001
, vol. 
64
 (pg. 
590
-
601
)
Mirkin
S
Arslan
M
Churikov
D
Corica
A
Diaz
JI
Williams
S
Bocca
S
Oehninger
S
In search of candidate genes critically expressed in the human endometrium during the window of implantation
Hum Reprod
 , 
2005
, vol. 
20
 (pg. 
2104
-
2117
)
Mote
PA
Balleine
RL
McGowan
EM
Clarke
CL
Colocalization of progesterone receptors A and B by dual immunofluorescent histochemistry in human endometrium during the menstrual cycle
J Clin Endocrinol Metab
 , 
1999
, vol. 
84
 (pg. 
2963
-
2971
)
Munné
S
Preimplantation genetic diagnosis of structural abnormalities
Mol Cell Endocrinol
 , 
2001
, vol. 
183
 
Suppl 1
(pg. 
S55
-
S58
)
Munné
S
Preimplantation genetic diagnosis and human implantation—a review
Placenta
 , 
2003
, vol. 
24
 
Suppl B
(pg. 
S70
-
S76
)
Murray
MJ
Meyer
WR
Zaino
RJ
Lessey
BA
Novotny
DB
Ireland
K
Zeng
D
Fritz
MA
A critical analysis of the accuracy, reproducibility, and clinical utility of histologic endometrial dating in fertile women
Fertil Steril
 , 
2004
, vol. 
81
 (pg. 
1333
-
1343
)
Myers
ER
Silva
S
Barnhart
K
Groben
PA
Richardson
MS
Robboy
SJ
Leppert
P
Coutifaris
C
Interobserver and intraobserver variability in the histological dating of the endometrium in fertile and infertile women
Fertil Steril
 , 
2004
, vol. 
82
 (pg. 
1278
-
1282
)
Neilson
L
Andalibi
A
Kang
D
Coutifaris
C
Strauss
JF
III
Stanton
JA
Green
DP
Molecular phenotype of the human oocyte by PCR–SAGE
Genomics
 , 
2000
, vol. 
63
 (pg. 
13
-
24
)
Noyes
RW
Haman
JO
Accuracy of endometrial dating; correlation of endometrial dating with basal body temperature and menses
Fertil Steril
 , 
1953
, vol. 
4
 (pg. 
504
-
517
)
Noyes
RW
Hertig
AT
Rock
J
Dating the endometrial biopsy
Fertil Steril
 , 
1950
, vol. 
1
 (pg. 
3
-
25
)
Nygren
KG
Andersen
AN
Assisted reproductive technology in Europe, 1997. Results generated from European registers by ESHRE. European IVF-Monitoring Programme (EIM), for the European Society of Human Reproduction and Embryology (ESHRE)
Hum Reprod
 , 
2001
, vol. 
16
 (pg. 
384
-
391
)
Okada
H
Nakajima
T
Yoshimura
T
Yasuda
K
Kanzaki
H
Microarray analysis of genes controlled by progesterone in human endometrial stromal cells in vitro
Gynecol Endocrinol
 , 
2003
, vol. 
17
 (pg. 
271
-
280
)
Olivennes
F
Ledee-Bataille
N
Samama
M
Kadoch
J
Taupin
JL
Dubanchet
S
Chaouat
G
Frydman
R
Assessment of leukemia inhibitory factor levels by uterine flushing at the time of egg retrieval does not adversely affect pregnancy rates with in vitro fertilization
Fertil Steril
 , 
2003
, vol. 
79
 (pg. 
900
-
904
)
Papanikolaou
EG
Bourgain
C
Kolibianakis
E
Tournaye
H
Devroey
P
Steroid receptor expression in late follicular phase endometrium in GnRH antagonist IVF cycles is already altered, indicating initiation of early luteal phase transformation in the absence of secretory changes
Hum Reprod
 , 
2005
, vol. 
20
 (pg. 
1541
-
1547
)
Papanikolaou
EG
Camus
M
Fatemi
HM
Tournaye
H
Verheyen
G
Van Steirteghem
A
Devroey
P
Early pregnancy loss is significantly higher after day 3 single embryo transfer than after day 5 single blastocyst transfer in GnRH antagonist stimulated IVF cycles
Reprod Biomed Online
 , 
2006a
, vol. 
2
 (pg. 
60
-
65
)
Papanikolaou
EG
Camus
M
Kolibianakis
EM
Van Landuyt
L
Van Steirteghem
A
Devroey
P
In vitro fertilization with single blastocyst-stage versus single cleavage-stage embryos
New Engl J Med
 , 
2006b
, vol. 
354
 (pg. 
1139
-
1146
)
Peddie
VL
van Teijlingen
E
Bhattacharya
S
A qualitative study of women's decision-making at the end of IVF treatment
Hum Reprod
 , 
2005
, vol. 
20
 (pg. 
1944
-
1951
)
Platteau
P
Staessen
C
Michiels
A
Van Steirteghem
A
Liebaers
I
Devroey
P
Preimplantation genetic diagnosis for aneuploidy screening in patients with unexplained recurrent miscarriages
Fertil Steril
 , 
2005a
, vol. 
83
 (pg. 
393
-
397
)
Platteau
P
Staessen
C
Michiels
A
Van Steirteghem
A
Liebaers
I
Devroey
P
Preimplantation genetic diagnosis for aneuploidy screening in women older than 37 years
Fertil Steril
 , 
2005b
, vol. 
84
 (pg. 
319
-
324
)
Ponnampalam
AP
Weston
GC
Trajstman
AC
Susil
B
Rogers
PA
Molecular classification of human endometrial cycle stages by transcriptional profiling
Mol Hum Reprod
 , 
2004
, vol. 
10
 (pg. 
879
-
893
)
Popovici
RM
Kao
LC
Giudice
LC
Discovery of new inducible genes in in vitro decidualized human endometrial stromal cells using microarray technology
Endocrinology
 , 
2000
, vol. 
141
 (pg. 
3510
-
3513
)
Punyadeera
C
Verbost
P
Groothuis
P
Oestrogen and progestin responses in human endometrium
J Steroid Biochem Mol Biol
 , 
2003
, vol. 
84
 (pg. 
393
-
410
)
Punyadeera
C
Dassen
H
Klomp
J
Dunselman
G
Kamps
R
Dijcks
F
Ederveen
A
de Goeij
A
Groothuis
P
Oestrogen-modulated gene expression in the human endometrium
Cell Mol Life Sci
 , 
2005
, vol. 
62
 (pg. 
239
-
250
)
Riesewijk
A
Martin
J
van Os
R
Horcajadas
JA
Polman
J
Pellicer
A
Mosselman
S
Simon
C
Gene expression profiling of human endometrial receptivity on days LH + 2 versus LH + 7 by microarray technology
Mol Hum Reprod
 , 
2003
, vol. 
9
 (pg. 
253
-
264
)
Ringler
GE
Strauss
JF
III
Recent advances in understanding the process of implantation
Curr Opin Obstet Gynecol
 , 
1990
, vol. 
2
 (pg. 
703
-
708
)
Robb
L
Dimitriadis
E
Li
R
Salamonsen
LA
Leukemia inhibitory factor and interleukin-11: cytokines with key roles in implantation
J Reprod Immunol
 , 
2002
, vol. 
57
 (pg. 
129
-
141
)
Rogers
PA
Current studies on human implantation: a brief overview
Reprod Fertil Dev
 , 
1995
, vol. 
7
 (pg. 
1395
-
1399
)
Sakkas
D
Gardner
DK
Noninvasive methods to assess embryo quality
Curr Opin Obstet Gynecol
 , 
2005
, vol. 
17
 (pg. 
283
-
288
)
Seif
M
Edi-Osagie
E
Farquhar
C
Hooper
L
Blake
D
McGinlay
P
Assisted hatching on assisted conception (IVF & ICSI)
Cochrane Database Syst Rev
 , 
2006
 
CD001894
Sermon
KD
Michiels
A
Harton
G
Moutou
C
Repping
S
Scriven
PN
Sengupta
S
Traeger-Synodinos
J
Vesela
K
Viville
S
, et al.  . 
ESHRE PGD Consortium data collection VI: cycles from January to December 2003 with pregnancy follow-up to October 2004
Hum Reprod
 , vol. 
22
 (pg. 
323
-
336
)
Simon
C
Landeras
J
Zuzuarregui
JL
Martin
JC
Remohi
J
Pellicer
A
Early pregnancy losses in in vitro fertilization and oocyte donation
Fertil Steril
 , 
1999a
, vol. 
72
 (pg. 
1061
-
1065
)
Simon
C
Mercader
A
Garcia-Velasco
J
Nikas
G
Moreno
C
Remohi
J
Pellicer
A
Coculture of human embryos with autologous human endometrial epithelial cells in patients with implantation failure
J Clin Endocrinol Metab
 , 
1999b
, vol. 
84
 (pg. 
2638
-
2646
)
Simon
C
Oberye
J
Bellver
J
Vidal
C
Bosch
E
Horcajadas
JA
Murphy
C
Adams
S
Riesewijk
A
Mannaerts
B
, et al.  . 
Similar endometrial development in oocyte donors treated with either high- or standard-dose GnRH antagonist compared to treatment with a GnRH agonist or in natural cycles
Hum Reprod
 , 
2005
, vol. 
20
 (pg. 
3318
-
3327
)
Srisuparp
S
Strakova
Z
Brudney
A
Mukherjee
S
Reierstad
S
Hunzicker-Dunn
M
Fazleabas
AT
Signal transduction pathways activated by chorionic gonadotropin in the primate endometrial epithelial cells
Biol Reprod
 , 
2003
, vol. 
68
 (pg. 
457
-
464
)
Staessen
C
Platteau
P
Van Assche
E
Michiels
A
Tournaye
H
Camus
M
Devroey
P
Liebaers
I
Van Steirteghem
A
Comparison of blastocyst transfer with or without preimplantation genetic diagnosis for aneuploidy screening in couples with advanced maternal age: a prospective randomized controlled trial
Hum Reprod
 , 
2004
, vol. 
19
 (pg. 
2849
-
2858
)
Stafford-Bell
MA
Copeland
CM
Surrogacy in Australia: implantation rates have implications for embryo quality and uterine receptivity
Reprod Fertil Dev
 , 
2001
, vol. 
13
 (pg. 
99
-
104
)
Stanton
JL
Bascand
M
Fisher
L
Quinn
M
Macgregor
A
Green
DP
Gene expression profiling of human GV oocytes: an analysis of a profile obtained by Serial Analysis of Gene Expression (SAGE)
J Reprod Immunol
 , 
2002
, vol. 
53
 (pg. 
193
-
201
)
Talbi
S
Hamilton
AE
Vo
KC
Tulac
S
Overgaard
MT
Dosiou
C
Le Shay
N
Nezhat
CN
Kempson
R
Lessey
BA
, et al.  . 
Molecular phenotyping of human endometrium distinguishes menstrual cycle phases and underlying biological processes in normo-ovulatory women
Endocrinology
 , 
2006
, vol. 
147
 (pg. 
1097
-
1121
)
The Practice Committee of the American Society for Reproductive Medicine and the Practice Committee of the Society for Assisted Reproductive Technology
Blastocyst culture and transfer in clinical-assisted reproduction
Fertil Steril
 , 
2006
, vol. 
86
 
Suppl 5
(pg. 
S89
-
S92
)
Thornhill
AR
deDie-Smulders
CE
Geraedts
JP
Harper
JC
Harton
GL
Lavery
SA
Moutou
C
Robinson
MD
Schmutzler
AG
Scriven
PN
, et al.  . 
ESHRE PGD Consortium ‘Best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS)
Hum Reprod
 , 
2005
, vol. 
20
 (pg. 
35
-
48
)
Tierney
EP
Tulac
S
Huang
ST
Giudice
LC
Activation of the protein kinase A pathway in human endometrial stromal cells reveals sequential categorical gene regulation
Physiol Genomics
 , 
2003
, vol. 
16
 (pg. 
47
-
66
)
Tranguch
S
Daikoku
T
Guo
Y
Wang
H
Dey
SK
Molecular complexity in establishing uterine receptivity and implantation
Cell Mol Life Sci
 , 
2005
, vol. 
62
 (pg. 
1964
-
1973
)
Twisk
M
Mastenbroek
S
van Wely
M
Heineman
MJ
Van der Veen
F
Repping
S
Preimplantation genetic screening for abnormal number of chromosomes (aneuploidies) in in vitro fertilisation or intracytoplasmic sperm injection
Cochrane Database Syst Rev
 , 
2006
 
Art. No: CD005291.pub2. DOI: 10.1002/14651858.CD005291.pub2
Ubaldi
F
Camus
M
Smitz
J
Bennink
HC
Van Steirteghem
A
Devroey
P
Premature luteinization in in vitro fertilization cycles using gonadotropin-releasing hormone agonist (GnRH-a) and recombinant follicle-stimulating hormone (FSH) and GnRH-a and urinary FSH
Fertil Steril
 , 
1996
, vol. 
66
 (pg. 
275
-
280
)
Urman
B
Yakin
K
Balaban
B
Recurrent implantation failure in assisted reproduction: how to counsel and manage. A. General considerations and treatment options that may benefit the couple
Reprod Biomed Online
 , 
2005
, vol. 
11
 (pg. 
371
-
381
)
van der Gaast
MH
Beier-Hellwig
K
Fauser
BC
Beier
HM
Macklon
NS
Endometrial secretion aspiration prior to embryo transfer does not reduce implantation rates
Reprod Biomed Online
 , 
2003
, vol. 
7
 (pg. 
105
-
109
)
Verlinsky
Y
Cieslak
J
Ivakhnenko
V
Evsikov
S
Wolf
G
White
M
Lifchez
A
Kaplan
B
Moise
J
Valle
J
, et al.  . 
Chromosomal abnormalities in the first and second polar body
Mol Cell Endocrinol
 , 
2001
, vol. 
183
 
Suppl 1
(pg. 
S47
-
S49
)
Verlinsky
Y
Cohen
J
Munné
S
Gianaroli
L
Simpson
JL
Ferraretti
AP
Kuliev
A
Over a decade of experience with preimplantation genetic diagnosis: a multicenter report
Fertil Steril
 , 
2004
, vol. 
82
 (pg. 
292
-
294
)
Vilska
S
Tiitinen
A
Hyden-Granskog
C
Hovatta
O
Elective transfer of one embryo results in an acceptable pregnancy rate and eliminates the risk of multiple birth
Hum Reprod
 , 
1999
, vol. 
14
 (pg. 
2392
-
2395
)
Wang
H
Dey
SK
Lipid signaling in embryo implantation
Prostag Other Lipid Mediat
 , 
2005
, vol. 
77
 (pg. 
84
-
102
)
Wilcox
AJ
Baird
DD
Weinberg
CR
Time of implantation of the conceptus and loss of pregnancy
New Engl J Med
 , 
1999
, vol. 
340
 (pg. 
1796
-
1799
)
Wimsatt
WA
Some comparative aspects of implantation
Biol Reprod
 , 
1975
, vol. 
12
 (pg. 
1
-
40
)
Yanaihara
A
Otsuka
Y
Iwasaki
S
Koide
K
Aida
T
Okai
T
Comparison in gene expression of secretory human endometrium using laser microdissection
Reprod Biol Endocrinol
 , 
2004
, vol. 
2
 pg. 
66