Implantation

The attachment and implantation of an embryo into a mother’s uterus is the critical event that defines an embryo’s fate. Implantation is not an efficient process, and is successful only about 10–15% of the time.¹ As a hallmark of development, implantation marks the very beginning of life, and proceeds from the tentative first attachment reaction (apposition) to subsequent aggressive embryonic intrusion and invasion into the maternal decidua. The earliest events of implantation have been difficult to study because the very act of examination disrupts the nature of the process. Animal models have shown a remarkable dissimilarity in implantation strategies, even when relatively closely related species are compared. However, with the development of modern molecular biology techniques and with the use of the donor egg model from assisted reproductive technologies (ART), we are now gaining increasing knowledge about implantation, especially at the biochemical level. We now know that synchronized development of both endometrium and embryo is a prerequisite for implantation to succeed, and that disruption of this developmental synchrony can result in infertility and/or pregnancy loss.

The events of early pregnancy are tightly coordinated by means of endocrine, paracrine, juxtacrine, and autocrine cellular communication pathways. Adhesion molecules and ligands promote cell-cell attachment and invasion, while proteases digest extracellular matrix (ECM) ahead of the invading embryo. The invading cells of the embryo, called trophoblasts, invade in a fashion not unlike cancer cells,² however, because of the extensive signaling mechanisms that exist between maternal and embryonic cells, uncontrolled spread and immunologic rejection are avoided. In this chapter we briefly describe a variety of animal models of implantation, then outline the classic studies that form the foundation of our current understanding of human implantation. Finally, we detail the known biochemical markers of uterine receptivity and describe their regulation. Our aim is to maintain this information within a context of clinical applicability for current treatment of infertility and recurrent pregnancy loss and to explore potential future directions of implantation-related research.

Historically, animal models have been the primary means for investigating implantation and they provide insight into various aspects of the process.^{3, 4, 5} Murid rodents can be induced to exhibit delayed implantation as a result of changes in hormonally controlled uterine development. This has been exploited as a model to study embryo-endometrial communication.^{6, 7} Implantation in mice occurs on the fourth day after coitus coincident with a rise in serum estrogen levels. Many of the proteins involved in human implantation were first identified in the mouse.⁸ The mouse is also an excellent model for the study of apoptosis during implantation because epithelial cells undergo programmed cell death after embryo apposition to allow penetration of the trophoblasts into the uterine basal lamina.⁹

Guinea pigs exhibit a different type of implantation, with similarities to the human. Here the syncytiotrophoblasts put forth appendages that extend through the zona pellucida to achieve apposition. Once contact is made, these appendages invade between uterine epithelial cells into the basal lamina and underlying stroma, thus making the guinea pig a useful model for the study of epithelial penetration.¹⁰

The rabbit has served as an outstanding model to study implantation. As induced ovulators (ovulating approximately 10 hours after mating), implantation in rabbits can be timed reliably.¹¹ Rabbit blastocysts are exceptionally large and form knob-like attachment sites with the uterine epithelium (Fig. 1).¹² Such specialized areas of attachment allow for the study of the cell-cell adhesion molecules and their regulation.

There have also been an increasing number of in vitro models to study implantation using purified endometrial epithelium and cytotrophoblast, but these have had limitations in their use.^{13, 14, 15} Such models may provide the most intimate look at the earliest moments of embryo-endometrial interaction (Fig. 2). Through the collective use of these various models, much has been learned about the basic principles of implantation. We discuss next some of the classic studies that established the timing of implantation as well as the stages of implantation in humans.

Our current understanding of human implantation has evolved from the pioneering work of Hertig and colleagues 50 years ago, to the present day receptor-mediated model with a more defined estimate of the endometrial window of implantation as described recently by Yoshinaga and Wilcox, respectively.^{16, 17, 18} Based on work from the infertility and ART clinics, we now know that embryo development is also critical before the overall window of receptivity is achieved.¹⁹ The sequence of events from the release of an ovum to the point of established pregnancy has been divided into discreet stages of implantation. As shown in Figure 3, these stages consist of preimplantation events for both the embryo and the endometrium, followed by apposition and attachment, and finally penetration.

Preimplantation events of the embryo

The earliest aspects of implantation in the human encompass the sequence of events that lead from fertilization to blastocyst attachment and invasion. After ovulation, the nascent oocyte is transported through the Fallopian tube, where fertilization occurs, thus defining stage I of implantation. Stage II is marked by the subsequent initiation of cell division in the embryo. At stage III, the ball of embryonic cells, now called a morula, enters the uterine cavity where further divisions result in formation of the blastocyst. It is estimated that only 20% of human embryos ever reach this point of development but for those that do, activation of the genome begins that is essential for successful implantation.^{20, 21} These initial events occur within a narrow time frame, as the embryo enters the uterine cavity at 72–96 hours after fertilization.^{20, 22}

Once in the uterine cavity, the embryo remains free for roughly 3 days before attaching to the endometrium.²⁰ During this time the embryo begins its communication with the mother even prior to attachment to the endometrium. Maternal secretions likely nurture the growing embryo.^{23, 24, 25} Human chorionic gonadotropin (hCG), produced by the embryo even before hatching from the zona pellucida (ZP), is the pregnancy recognition signal to the mother.^{26, 27} The ZP is important in prevention of polyspermy as well as providing an appropriate environment for early embryonic development; ultimately the embryo must hatch from the ZP prior to apposition, exposing surface receptors and ligands that are important for attachment. The process of hatching becomes less efficient with advancing maternal age and it is thought that zona hardening can entrap an embryo within the ZP.^{28, 29, 30} Several methods of assisted hatching techniques have been suggested to alleviate this problem in in vitro fertilization (IVF). Such methods to weaken the ZP allow for zona shedding that has improved implantation rates, in particular for older women or for prior implantation failures.

This progression of preimplantation events in the embryo is essential for implantation, and occurs within a narrow time frame during the midsecretory phase. As addressed in later sections, the endometrium is now thought to become permissive to implantation during cycle days 20–24 of a normalized 28-day cycle, the so-called window of receptivity.¹⁸ Navot and colleagues³¹ demonstrated that pregnancies can be achieved by embryo transfers (ET) in donor egg cycles on cycle days 15–20, with the vast majority of success between days 16 and 19 (Fig. 4).³¹ Actual implantation takes place between embryonic days 5 and 6, so the transfer of 2–3-day-old embryos prior to day 20 yields peak implantation rates.³¹ This concept was further expanded by this group by studying the timing the first hCG detection in donor egg recipients.¹⁹ They found the average detection of pregnancy on embryonic day 7 with 2-day-old embryos transferred on day 15 having hCG detection on day 20 and 2-day-old embryos transferred on day 19 having hCG detection on days 24.¹⁹ No detection was found when embryos were transferred after day 19. These data correlate well with a window of receptivity between cycle days 20–24 and agree with the studies by Wilcox and colleagues¹⁸ who also studied the timing of implantation. Such donor-recipient models are able to establish the triphasic pattern of early hCG, rise which has clinical applicability for many gynecologic problems such as ectopic pregnancies, abnormal intrauterine pregnancies, and gestational trophoblastic disease (Fig. 5).¹⁹

Preimplantation events of the uterus

STRUCTURAL CHANGES OF THE ENDOMETRIUM

During the first half of the menstrual cycle, called the proliferative phase, the endometrium thickens in response to rising estradiol levels (Fig. 6). As assessed by transvaginal ultrasound, the endometrium grows rapidly during the first 14 days of the cycle. It is thought that an endometrial thickness less than 6 mm is unlikely to support a pregnancy.^{32, 33} Once ovulation takes place and the granulosa transforms into the corpus luteum (CL), the endometrium is converted to a secretory structure under the influence of rising progesterone. This conversion follows an orderly progression as demonstrated by Noyes and coworkers³⁴ in their now classic paper on endometrial dating (Fig. 7). Initially, the endometrial glandular epithelium begins to synthesize and secrete glycogen and glycoproteins, and by midcycle, stromal edema develops, accompanied by proliferation of the spiral arterioles. Ultrastructurally, scanning electron microscopy of the midluteal luminal epithelium shows the development of small protrusions called pinopods, the appearance of which coincides well with the suspected time of maximal receptivity (Fig. 8). Such surface modifications are thought to play a functional role in embryo apposition and attachment.^{35, 36, 37}

Another progesterone-related uterine change is the frequency of uterine contractions. Animal studies have shown decreased implantation rates with increasing uterine contraction.^{38, 39} Work by Fanchin and coworkers⁴⁰ using digitized 5-minute ultrasound scans showed that contractility is inversely related to progesterone levels in humans as well. They also showed decreased clinical pregnancy rates with increasing uterine contractions. Lesny and coworkers⁴¹ also studied this phenomenon doing mock ET using 30 μL of an echogenic fluid medium. They noted that with easy transfers, uterine contractions remained at a stable baseline and the medium did not migrate significantly. However, difficult transfers were associated with strong, random contractions and the fluid medium migrated from the fundal region in six of seven patients (in two the medium migrated to the Fallopian tubes).⁴¹ This is still a relatively new concept but may lead to future therapeutic considerations in ART.

BIOCHEMICAL CHANGES OF THE ENDOMETRIUM

Aside from what is known about the structural changes that the endometrium undergoes during the secretory phase, there is also a growing body of information regarding the biochemical changes of the endometrium. Discoveries regarding the ECM, cell adhesion molecules, cytokines and growth factors, and the enzymes that degrade the ECM have shed light on embryo-endometrial interactions that occur during attachment and invasion.

Extracellular matrix

The ECM is formed from secreted proteins and glycoproteins, and forms the ground substance outside the cells in all tissues. The ECM appears to play an important role in the cell-cell interactions.⁴² Analysis of these substances in the endometrium throughout the menstrual cycle has shown constitutive expression of collagen types III and V, and fibronectin.⁴² Cycle-dependent variations in expression also exist; type VI collagen is produced throughout the proliferative phase, but declines during the secretory phase. There may also be differences in the expression of these various ECM components in endometrium of fertile and infertile women.⁴³ The luminal surface is an effective barrier to implantation during much of the reproductive cycle.³⁹ Early on, it was noticed that there is thinning of the carbohydrate-rich domain of the luminal uterine surface, known as the glycocalyx, and a decreased negative charge at the luminal epithelium at the midluteal phase.^{44, 45} Variations in the ECM that make up these phenomena may hinder embryo-epithelial contact necessary for apposition. One mucin component in particular, MUC-1, may serve as an antiadhesive molecule and limit apposition.⁴⁶ In rodents, MUC-1 disappears at the time of implantation and persistent expression results in implantation failure. Studies in human endometrium have been less convincing because MUC-1 persists throughout the luteal phase in women. Nevertheless, there may still be a role of MUC-1 during human implantation.⁴⁷

Matrix metalloproteinases and their inhibitors

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases that enzymatically digest certain ECM proteins and therefore play an important role in tissue remodeling processes.^{48, 49} There are at least three groups of these enzymes differentiated by their substrates, as well as a group of membrane-type matrix metalloproteinases (MT-MMPs) (Table 1). Endometrial expression of MMPs is highly regulated to facilitate successful implantation or timely degradation and remodeling of the endometrial lining during menstruation, in the absence of a successful pregnancy.^{50, 51}

Table 1. Matrix metalloproteinases

MMP, matrix metalloproteinases.

MMPs are produced as inactive precursors that require activation by other peptidases, denaturants, or heat.⁵² This cascade of activation is often initiated by the MT-MMPs and is tightly controlled by a variety of tissue inhibitors of metalloproteinases (TIMPs).⁵³ The ECM constituents can actually regulate cellular activity through a receptor-mediated process, thus, production and breakdown of the ECM influences a tissue’s characteristics through a dynamic reciprocity and is vital to invasion and maternal recognition of a pregnancy.^{50, 54} There are also other interactions between the MMPs and the embryo-endometrial complex that are likely involved with implantation. Cell adhesion receptors known as integrins appear to have a role in MMP expression and function. Engagement of a placental integrin with fibronectin induces MMP expression and the integrin αvβ3 directly binds to and activates MMP-2.^{55, 56} MMP-2 is one of the most active MMPs in the distal invasive cell column of human trophoblasts during implantation and allows these cells to invade in the direction of their migration.⁵⁷ MMP-9 is also essential for both rodent and human placental invasion.⁵⁸ Decidual cells strongly express the MMP inhibitor TIMP-2, presumably as part of the maternal mechanism to block uncontrolled invasion of the placenta.⁵⁹

The delicate interplay between these factors must be precisely coordinated for successful but controlled invasion of the trophoblast. Plasminogen, its activators, and its inhibitors appear to participate in this process.⁶⁰ Plasmin, a protease derived from plasminogen, activates certain MMPs and has a role in degradation of the ECM. Regulation of plasminogen to plasmin conversion is stimulated by urokinase-type plasminogen activator (uPA) and inhibited by alpha₂-macroglobulin (α2MG). Human endometrium, embryos, and trophoblasts express both uPA and its receptor, and mutations in these have been associated with implantation failure.^{61, 62, 63, 64, 65} Activity of plasminogen activator (PA) is tightly regulated by specific inhibitors of PAs (PAI-1 and PAI-2), which bind and inactivate PAs.⁶⁶ Plasmin activates transforming growth factor β (TGF-β), which in turn activates TIMP and PAI production, thus inhibiting further plasmin activation (Fig. 9). Polycystic ovary syndrome (PCOS) is associated with elevated PAI-1 activity, which may contribute to the higher than expected rate of pregnancy loss via this pathway.^{67, 68}

Integrin cell adhesion molecules

Integrins, one member of a larger family of cell adhesion molecules, have been well studied at the levels of the embryo, trophoblast, and endometrial epithelium and stroma throughout the menstrual cycle and into pregnancy (Fig. 10).^{69, 70, 71, 72, 73, 74, 75, 76} While many integrins are constitutively expressed in human endometrium, the expression of certain integrins coincides well with the period of peak uterine receptivity.^{71, 72} The timing of receptivity was first described in the classic studies by Hertig and colleagues.¹⁶ Over a 15-year time period, they examined 210 hysterectomy specimens, ranging from the day of ovulation to 17 days postovulation. They observed that in uteri obtained prior to cycle day 20, all embryos were free-floating in the tubes or uterus, while in uteri obtained after day 21 the embryos were attached to the uterine lining. Many subsequent studies have helped establish that peak uterine receptivity is between cycle days 20 and 24 of a normalized 28-day cycle.^{18, 31}

Three integrins (α1β1, α4β1, and αvβ3) have been shown to be coexpressed during the window of receptivity in humans, present together from cycle day 20–24 (Fig. 11). Of these three, the luminal epithelium expresses only αvβ3 at the apical pole, suggesting a potential role in embryo attachment.⁷⁷ This integrin recognizes the three amino acid sequence arg-gly-asp (RDG), which has been implicated in trophoblast attachment and outgrowth in several studies.^{78, 79} Interestingly, we have shown a loss of midluteal αvβ3 expression in certain women with unexplained infertility as well as in women with conditions known to have diminished implantation rates, including PCOS, endometriosis, and those with tubal disease and hydrosalpinges, further implicating the critical role of this integrin during implantation.^{74, 75, 76, 80} Animal studies have also demonstrated that blockade of this integrin will prevent or reduce implantation. This could prove to be useful for future diagnostic and therapeutic considerations for the infertile couple.

Other markers of uterine receptivity

A growing number of proteins involved in endometrial-embryo interaction both during and after implantation have been identified (Fig. 12).⁸¹ As mentioned earlier, mucins like MUC-1 have been suggested as markers of receptivity. Glycodelin (placental protein 14 [PP14]) is a glycoprotein produced by the glandular epithelium and has been associated with inadequate hormonal cycles.⁸² There are also other adhesion molecules that undergo upregulation during the time of uterine receptivity, such as trophinin, cadherin-11,^{83, 84} and the hyaluronate receptor, CD44.⁸⁵ Many cytokines and growth factors, such as leukemia-inhibitory factor (LIF), insulin-like growth factor (IGF)-II, heparin-binding epidermal growth factor (HB-EGF), and interleukin (IL)-1β also appear to be expressed during peak uterine receptivity.^{86, 87, 88, 89}

One of the most recently defined markers is a secreted glycoprotein called osteopontin (OPN). This RGD-containing protein has binding sites to two major receptors that are present on both the material and embryonic epithelium, αvβ3 and CD44. Osteopontin is a 70-kd glycosylated phosphoprotein secreted by the glandular epithelium and is expressed during the midsecretory phase, localized to the luminal endometrial epithelium.⁹⁰ Regulation of OPN is tied to progesterone.^{90, 91, 92} The apical localization of OPN suggests a role as a sandwich ligand that serves as a bridge binding surface receptors on the endometrial and embryonal surfaces (Fig. 13).

Apposition

Despite this wide array of structural and biochemical changes by the preimplantation embryo and endometrium, it has been difficult to ascribe specific function to many of them. Apposition, defined as stage IV of implantation, is still an elusive process to observe in humans, and there remains much uncertainty about its mechanism and exact timing. It appears many of the factors described above have critical roles in bringing the endometrium and embryo together. Both express a variety of adhesion molecules and ligands during the expected time of implantation. Current theories suggest that endometrial surface projections, known as pinopods, form a privileged site of receptor expression, raising the endometrial apical surface above anti-adhesive molecules such as MUC-1. Once contact is made between the embryo and the maternal surface, polar trophoblast displaces the endometrial cells by sending ectoplasmic protrusions between them and disrupting their desmosomes.^{93, 94} This invasiveness appears dependent on the formation of the syncytiotrophoblast, occurring less than 24 hours after embryo adhesion.⁹⁵

In the vasculature, leukocytes bind to endothelial cells through initial interaction with carbohydrate-binding proteins called selectins. This first step initiates further cellular interactions by integrins and such as are addressed above.⁹⁶ It has now been demonstrated that human endometrium up-regulates L-selectin during the window of receptivity and is also expressed on human trophoblasts.⁹⁷ This marker has shown decreased expression in controlled ovarian hyperstimulation and may provide another marker of endometrial function and receptivity.⁹⁸

Penetration and invasion

Apposition is followed quickly by epithelial penetration and invasion (stage V). Again, this is a process that is not clearly understood but must be tightly orchestrated by a multitude of components previously mentioned, namely the ECM, cell adhesion molecules, MMPs and their inhibitors, and a variety of growth factors and their receptors. The importance of the ECM on cell surface proteolysis has been studied and suggests that on invasion, cell migration and the acquisition of an invasive phenotype may be stimulated by exposed ECM and digested fragments of the endometrial or trophoblast ECM mediated in large part by activation of specific MMPs.^{99, 100} Rather than a destructive process, invasion of the placental cells appears to involve breaking the intercellular connections and selective apoptosis. The intruding trophoblast appears to adhere to the lateral surfaces of the luminal epithelium with formation of junctional complexes and pushes these cells aside as the mass of the embryo migrates into the underlying decidua.

With time, trophoblast invasion reaches the maternal circulation. Access to the maternal vasculature becomes a priority for the growing embryo, which requires increasing quantities of nutrients and oxygen and better management of cellular waste for its survival. This stage of implantation is marked by rapid expansion of both cytotrophoblast and syncytial trophoblast (Fig. 14).¹⁰¹ At stage Va of invasion, the maternal vasculature remains intact, but becomes surrounded by the expanding syncytium. With further growth, the syncytium and cytotrophoblast invade the maternal vasculature, and the cytotrophoblast is incorporated into the wall of maternal vessels. As detailed later, this ability to mimic endothelial cell characteristics is critical to this invasion, thus establishing a blood supply and a presence within the maternal tissues that will remain intact for the remainder of the pregnancy.

As shown in Figure 14C and 14D, stage Vb of invasion is characterized by expansion of the syncytium and cytotrophoblast and establishment of lacunae as a result of vasculature invasion. By stage Vc, the embryo is fully below the luminal surface and the circumference of the embryo is surrounded by a layer of cytotrophoblast that will rapidly bud to form villi. Development of the placenta and stage V ends by day 11–12 after ovulation with the development of primary villi.

As discussed, the implantation process is tightly regulated to allow proper attachment and penetration of the embryo while preventing uncontrolled invasion. Ovarian steroids provide the overriding endocrinologic control throughout the menstrual cycle while cytokines and growth factors work at the cellular level as paracrine and autocrine mediators of steroid action. As outlined below, highly coordinated events, involving receptors, cell adhesion molecules, and ECM components coupled with the enzymes and their inhibitors that degrade ECM, regulate implantation.

Steroid hormones and uterine receptivity

Ultimately, implantation of the nascent embryo is dependent on ovarian production of the sex steroids produced by the developing follicle and by the subsequent corpus luteum.¹⁰² Estradiol (E₂) derived from the developing follicle initiates endometrial growth and cell division increasing epithelial, stromal, and vascular components of the endometrium. Through hypertrophy and hyperplasia the endometrium grows in response to estrogen. After ovulation, progesterone, which is an essential hormone for successful pregnancy, transforms the endometrium into a secretory structure capable of supporting early pregnancy.¹⁰³

Steroid hormone receptors are expressed by the growing endometrium and dictate the action of the steroid hormones. Estrogen receptors-α and -β (ERα and ERβ), progesterone receptors A and B (PR-A and PR-B), androgen receptors (AR), and glucocorticoid receptors (GR) are all expressed in human endometrium.^{104, 105, 106} It is primarily ERα and the PRs that play the major role in endometrial development.^{107, 108} The receptors for estrogen and progesterone are regulated by these hormones in the endometrium. Estrogen upregulates both ERα and the PR with increased concentration of both during the proliferative phase in both epithelial and stromal cells.^{109, 110} Both ERα and PR are downregulated by progesterone and decline during in the secretory phase. Interestingly, ERα is decreased at the time of implantation in every mammal studied to date and may be a critical event for the establishment of uterine receptivity.¹¹¹ Although PR has been reported to also be reduced at the time of implantation,¹¹² Mote and colleagues¹⁰⁵ recently suggested that it is specifically epithelial PR-A that declines at the time of implantation. Thus, the relative increased activity of ERβ and PR-B by the midsecretory phase may have a role in regulating key implantation-specific genes.^{104, 105, 113} A diminished role for ERα is consistent with the previous demonstration by DeZiegler and coworkers¹¹⁰ that estrogen is unnecessary for the development of the secretory endometrium. In fact, failure to downregulate ERα may actually be detrimental to implantation, because it has been shown that estrogen inhibits the expression of several endometrial factors, including the integrin β3 subunit and OPN.¹¹⁴

The expression of certain proteins involved in the establishment of uterine receptivity may be regulated through indirect (paracrine) pathways. While an endocrine pathway represents a direct effect of hormones such as progesterone on the estrogen-primed endometrial epithelium, paracrine pathways also can be demonstrated, and represent an indirect effect of progesterone acting through endometrial stroma (Fig. 15). Cunha and colleagues^{115, 116, 117} demonstrated the importance of endometrial stroma in directing epithelial growth and differentiation using ER and PR knockout mice. In the human endometrium, we have demonstrated that both endocrine and paracrine pathways are functional.¹¹⁸ We have also defined defects in each of these two pathways that may contribute to infertility. Type I defects, as we have called them,⁷² are a result of deficient ovarian estrogen and/or progesterone and are associated with significant histologic delay as defined by Noyes and colleagues³⁴ (see Fig. 7). In this case progesterone-induced genes such as calcitonin or osteopontin are delayed in their expression. Type II defects represent a separate effect resulting from inappropriate paracrine activity. While endometrial histology appears unaffected, differentiation of the epithelium fails to occur perhaps as a result of an alteration in the action of stromal factors on epithelial cells. An example includes the loss of the αvβ3 integrin seen in some women with infertility and endometriosis.⁷⁵ These pathways can be independently disrupted but are functionally related. The topic of luteal phase defect (LPD), which is a common cause of both infertility and recurrent pregnancy loss, is the result of these related pathways, and will be expanded in detail in a later section.¹¹⁹

Cytokines and growth factors

Cytokines and growth factors are peptides that recognize and bind specific cell membrane receptors to signal intracellular events. Many cytokines have been recognized as important during implantation in preimplantation embryos and uteri.¹²⁰ These factors are important to an increasing list of processes including cellular growth, differentiation, and immunomodulation. While the list of biologic signals in the endometrium continues to grow, several factors have been established as essential for normal implantation.

COLONY STIMULATING FACTOR-1

Colony stimulating factor-1 (CSF-1), initially described as a hematopoietic growth factor, is a 50–70-kDa glycoprotein expressed by fibroblasts, monocytes, macrophages, and endothelial cells.¹²¹ The role of CSF-1 in fertility was first described in a mouse model in which mice deficient in CSF-1 were infertile as a result of implantation failure.¹²² Subsequent human studies have shown that both CSF-1 and its receptor (the c-fms proto-oncogene) are upregulated in the late secretory phase and in early pregnancy; other studies connect CSF-1 to trophoblastic growth and differentiation.^{123, 124} Thus, the temporal expression of CSF-1 and its receptor, its effect on trophoblasts, and data from CSF-1 null mice suggest an important role for CSF-1 in implantation.

LEUKEMIA-INHIBITORY FACTOR

LIF was originally shown to induce differentiation of a myeloid leukemia cell line.¹²⁵ Its association with fertility was established when it was discovered that peak uterine expression of LIF occurred at the time of implantation in mice, and in the delayed implantation model, injection of estradiol correlated with an increase in uterine LIF expression.¹²⁶ Interestingly, while LIF-null mutant male mice are fertile, female mice lacking LIF fail to implant, although their embryos transferred into wild-type mice will result in pregnancy, demonstrating the importance of LIF in the implantation process.⁸⁶

Human studies have also shown peak uterine expression during the window of implantation that may be decreased in some women with infertility.¹²⁷ LIF is produced in the luminal epithelium and causes increased trophoblastic production of fetal fibronectin and other proteins, thus implicating it as a direct maternal modulator of trophoblastic differentiation.^{127, 128}

INTERLEUKIN-1 FAMILY

IL-1 represents a family of peptides composed of IL-1α (159 amino acids), IL-1β (153 amino acids), IL-1 receptor antagonist (IL-1ra), and two IL-1 receptors (IL-1R, type I and II).¹²⁹ IL-1α increases fetal fibronectin production and immunoreactivity of MMP-9 by purified first trimester cytotrophoblasts in vitro.¹³⁰ IL-1β has been identified in oocytes, embryos, and endometrial stroma, and, along with IL-1ra and IL-1R type I, has been localized to macrophages in the villus trophoblast and maternal-trophoblast interface.^{131, 132, 133} Both IL-1Rs are found on the luminal epithelium and this could be a pathway through which an embryo can actually facilitate its own implantation. De los Santos and coworkers¹³⁴ demonstrated that not all embryos express these factors and suggested that embryonic competency relates to the presence or absence of these specific cytokine signals.

Further understanding of the role of IL-1 in implantation has come from the IL-1 receptor antagonist. This peptide is found in large amounts during the follicular phase of the menstrual cycle, but diminishes near the time of implantation.¹³⁵ Implantation failure occurs in mice injected with exogenous IL-1ra, thus implicating a vital role of IL-1 for successful implantation.¹³⁶ Interestingly, IL-1R type I null mutant mice display normal implantation, even in the presence of exogenous IL-1ra.¹³⁷ This may be caused by developmental compensation that overcomes the lack of IL-1 signaling in the null-mutant mice. Alternatively such studies may be demonstrating that IL-1 does not have an essential role during implantation. A precise role of this cytokine during implantation awaits further investigation.

GROWTH FACTORS

Epidermal growth factor and EGF-like molecules

EGF is a 6-kDa peptide that is a potent stimulator of proliferation in a variety of cell types.¹³⁸ The EGF family of growth factors has expanded to include EGF itself, TGF-α, HB-EGF, amphiregulin, and betacellulen. These factors act through binding to the ErbB receptors (ErbB 1 through 4), each specific for one of the aforementioned ligands.¹³⁹

The importance of EGF and EGF-like molecules in implantation has been well-demonstrated in the mouse model. It appears that these molecules stimulate embryo development, in that both EGF and TGF-α improve ZP hatching as well as stimulate trophoblast outgrowth in vitro.^{140, 141} In mice, uterine EGF expression is regulated by estrogen and may be part of the link between the estrogen rise and blastocyst activation/implantation in the delayed implantation model.¹⁴² This is further supported by the observation that exogenous EGF can trigger implantation in a similar fashion to exogenous estrogen in this model.¹⁴³ Inappropriate uterine TGF-α expression in transgenic mice also results in aberrant implantation.¹⁴⁴

The role of EGF and the EGF-like molecules are less clearly understood during human or primate implantation. Multiple studies show conflicting results regarding timing and location of expression of many of these factors.^{145, 146, 147} HB-EGF has been shown to be expressed during the window of implantation in multiple species, including human.¹⁴⁸ HB-EGF is regulated differentially in the endometrial epithelium and stroma by estrogen and progesterone, respectively.¹⁴⁹ Recent data show that HB-EGF is upregulated in endometrial stroma in the midluteal phase and that this stimulates epithelial expression of various important biomarkers of receptivity such as the αvβ3 integrin, HOXA-10, and LIF (Fig. 16).¹¹⁸

Insulin-like growth factors

The IGF family of growth factors includes two distinct hormones (IGF-I and IGF-II), two IGF receptors, and six insulin-like growth factor binding proteins (IGFBPs). IGF-I and IGF-II are mitogenic growth factors that are structurally similar to insulin and they are present in human endometrium throughout the menstrual cycle, promoting endometrial cellular mitosis and differentiation.¹⁵⁰ Both hormones are present in the endometrial stroma, but the receptors are found in both the stroma and epithelium.¹⁵¹ IGF-I is abundant during the proliferative phase and may promote proliferation; conversely, IGF-II is expressed more during the secretory phase.

The IGFBPs bind to and modulate the activity of IGF-I and IGF-II.¹⁵² All six are present in human endometrium with IGFBP-1 (also known as PP12 or α1-PEG) being a major secretory protein of the stroma.¹⁵³ IGFBP-1 is regulated by progesterone and it acts via two mechanisms.¹⁵⁴ First, as IGF-I and IGF-II inhibit decidualization, the presence of IGFBP-1 probably counteracts the effect of trophoblast IGF-II. Second, IGFBP-1 contains the integrin-binding domain arg-gly-asp (RGD) and is recognized specifically by the α5β1 integrin present on cytotrophoblast cells and endometrial stroma and decidua.¹⁵⁵ These interactions may act to limit invasion of the cytotrophoblast, although much is still unknown about these mechanisms.

Transforming growth factor β

The TGF-β superfamily is a group of related secretory proteins that regulate a variety of cellular processes. This group contains the four TGF-β isoforms (TGF-β1 through TGF-β4), inhibin, activin, Mullerian-inhibiting substance, VEGF-1, endometrial bleeding-associated factor (ebaf or LEFTY), the bone morphogenic proteins (BMPs), and the growth and differentiation factors (GDFs).¹²⁰ In humans, TGF-β mRNA is distributed throughout the endometrium with lowest levels during in the proliferative phase and highest found in maternal decidua. There is evidence that TGF-β affects embryonic development, as mouse embryos exposed to TGF-β in vitro show a greater differentiation compared to those that were not exposed.¹⁴⁰ Cultured human cytotrophoblasts secrete fetal fibronectin in the presence of TGF-β, likely promoting cellular adhesion. TGF-β may inhibit the production MMPs, thus preventing the normal menstrual breakdown of the endometrium.¹⁵⁶ Mice with null-mutations for TGF-β have not been shown to have implantation failure, so the importance of TGF-β may be indirect.¹⁵⁷

Other growth factors

Platelet-derived growth factors (PDGFs) are 30-kDa proteins that mediate intercellular signaling via tyrosine kinase receptors.¹²⁰ PDGFs are composed of two subunits (A and B) and can result in one of three dimers (AA, AB, BB). The PDGFs stimulate proliferation and may have a role in embryonic development. The PDGF-α receptor null mutant mouse (the Patch mouse) has normal fertility and placentation, however, raising the question of whether PDGF is essential for implantation.¹⁵⁸

Fibroblast growth factor (FGF) is an angiogenic growth factor that is highly expressed throughout the human menstrual cycle. FGF has been identified in four forms and the basic one (bFGF) increases endometrial stromal production of proteolytic enzymes and tissue plasminogen activator (t-PA), possibly aiding in trophoblast outgrowth and invasion.¹⁵⁹ FGF-3 and FGF-4 and their receptors have been found in mouse embryos suggesting a role in preimplantation embryo development.¹⁶⁰

Endometrial-embryonic dialogues

As evidenced by the information reviewed thus far, implantation is the result of an exceedingly complex series of cellular events (see Fig. 12). While much of the uterine preparation for implantation occurs without direct embryonic involvement, increasing evidence suggests that endometrium and the embryo may be targets for paracrine or juxtacrine signals that facilitate attachment and invasion. Cross-talk between the embryo and endometrium undoubtedly occurs. Data now exist that support a role for the endometrium in limiting placental invasion. Embryonic signals are also recognized as important for rescue of the CL that influences the reproductive tract directly through the action of progesterone. This early signal from the embryo may be essential for the recognition of pregnancy in primates and illustrates one example of this type of cross-talk.

As mentioned earlier, ECM may also play a role in embryo-endometrial signaling. Elaboration of the alternatively spliced form of fibronectin (fetal fibronectin) may form an adhesive interface between embryo and endometrium.¹⁶¹ Laminin produced by the blastocyst stimulates MMP-2 expression in both embryo and endometrium and the resulting proteolytic fragments of laminin have been shown to stimulate cell migration as an early inducer of the invasive phenotype.¹⁶² MMP production also promotes HB-EGF release from the endometrium and continues to promote embryo development.^{163, 164} Embryo quality is improved when it is cocultured with cells from the reproductive tract, likely as a result of these various interactions, and has led to improved outcomes in ART.^{165, 166}

We compared the invasiveness of the embryo during implantation to the metastic spread of cancer cells.² Complex cellular dialogues promote embryonic development and invasion, but also may at the same time prevent uncontrolled trophoblast invasiveness. One mechanism of regulation of embryonic invasiveness involves the TIMPs and inhibitors of plasmin. IGFBP-1 may also limit invasiveness through an interaction with the α5β1 integrin.¹⁵⁵ We are just beginning to appreciate the enormous complexity of these interactions. A thorough understanding of these influential factors will likely lead to development of novel therapeutic means to influence the reproductive process. In the next section we explore some clinical examples of implantation failure and suggest strategies for managing these common problems.

It is apparent from the data presented thus far that successful implantation depends on the synchronous development of both the endometrium and embryo. Loss of this synchrony results in the clinical manifestation of pregnancy loss and/or infertility in both animals and humans.^{167, 168} Luteal phase deficiency (LPD) is a major cause of infertility and recurrent pregnancy loss that results from either inadequate progesterone production or response.¹¹⁹ LPD has been difficult to study, however, because of a lack of consensus on its definition, and the estimated prevalence of the disorder ranges widely, from 4% to 60% of infertility patients. The true estimate likely will fall between 5% and 10% of truly infertile couples.¹¹⁹

Clinical features of luteal phase deficiency

The most stringent clinical diagnosis of LPD is based on a timed endometrial biopsy 7–9 days post-luteinizing hormone (LH) surge demonstrating a delay of 2 days in the dating criteria of Noyes and colleagues³⁴ for histologic progression of endometrium. Other means of demonstrating LPD are basal body temperature (BBT) testing demonstrating less than 11 days of temperature elevation, low luteal phase serum progesterone levels (by either single or multiple assessments), or lack of production of a variety of markers of receptivity.^{27, 169, 170} Inadequate progesterone effect (from lack of either production or response) results in a histologic lag that delays the onset of uterine receptivity. The precise reason why this might result in reproductive failure is unclear, but may be a result of loss of embryo viability as it awaits a receptive endometrium. More likely, the fault is because of a failure of timely rescue of the CL. Inappropriate or untimely expression of many of the previously mentioned factors, such as PR, MUC-1, glycodelin (PP14), ebaf, MMPs, or the αvβ3 integrin and its ligand OPN, potentially could be used to identify hormonally inadequate cycles.

LPD is best viewed not as a single distinct entity, but rather as a final common result of various predisposing factors or causes. The causes of LPD are summarized in Table 2. A key point is that the functional capacity of the CL depends on the relative health of the preovulatory ovarian follicle from which it is derived. Therefore, as can be seen from Table 2, many factors that disrupt the hypothalamic-pituitary-ovarian axis, and thus result in fewer developed granulosa cells from which to form the CL, potentially lead to LPD.¹⁷¹

Table 2. Causes of luteal phase deficiency

NSAIDs, nonsteroidal anti-inflammatory drugs; GnRH, gonadotropin-releasing hormone; PIF, prolactin inhibitory factor; TRH, thyrotropin-releasing hormone; CRH, corticotropic-releasing hormone; FSH, follicle stimulating hormone; CL, corpus luteum.

It is progesterone that causes the proliferative endometrium to undergo the characteristic secretory transformation and synthesis and secretion of factors essential for embryo implantation.¹⁷² Progesterone also influences the endometrial stroma, leading to the eventual conversion to the decidua, a highly specialized compartment that produces ECM, cytokines, and growth factors thought to be essential for implantation, but also to limit trophoblast invasion. It is noted that progesterone downregulates its receptor (PR) in the epithelium, but PR persists in the stroma.^{109, 113} This is a key event in development of the endometrium, marking a shift from epithelial to stromal responsiveness. Delay in PR (or ERα) downregulation may alter patterns of gene activation and thus render the endometrium unreceptive to embryo implantation.

As mentioned previously, alterations in hormonal stimulation or response may result in endometrial defects. The first, the classically defined LPD, is referred to as a type I luteal deficiency and is characterized by a delay in the histologic progression of the luteal phase endometrium.¹¹⁹ This is a result of insufficient progesterone action on the endometrium. LPD is commonly found in women with unexplained infertility and recurrent pregnancy loss, but it is also commonly seen in these groups of women even when serum progesterone concentrations are not clearly low.^{173, 174} Balasch and Vanrell¹⁷⁵ found that endometrial maturation was delayed by 2 or more days in 13.5% of infertile women and 32.5% of patients with a history of recurrent pregnancy loss, even though 86% overall had what they considered normal hormonal profiles. This demonstrates that LPD may result from factors influencing ovarian steroid production as well as the endometrium itself by means of its progesterone responsiveness. An important study by Wilcox and colleagues¹⁸ suggests that any delay in implantation will result in poor implantation efficiency. Women who had delayed implantation in this study were noted to have increasingly poor reproductive outcome and were reported to lack key proteins, such as αvβ3 integrins, that are normally present (Fig. 17).¹⁸

The utility of using histology to define LPD has, however, been seriously challenged by recent studies. First was a study in which the investigators attempted to refine the histologic criteria for dating first established by Noyes in 1950.¹⁷⁶  They systematically evaluated 130 biopsies timed to specific days following detected LH surges, all done independently by three gynecologic pathologists, using 32 pre-defined histologic features to accurately predict the histologic date. The endometrial dating criteria were shown to be much less temporally distinct and discriminating than originally described with considerable intersubject, intrasubject, and interobserver variability. Neither traditional dating criteria nor any combination of the best performing histologic features could reliably distinguish any specific cycle day or narrow interval of days. Subsequent to this, the Reproductive Medicine Network published its findings from its multicenter endometrial biopsy study comparing fertile to infertile women with regards to endometrial dating and likelihood of having out-of-phase endometrium.¹⁷⁷ They sampled 619 women and found that out-of-phase biopsies poorly discriminated fertile from infertile women, actually observing more out-of-phase biopsies in the fertile group (49%) than in the infertile group (43%). These findings combine to shed serious doubt in the clinical utility of endometrial biopsy in the assessment of infertile couples and in the role of endometrial histologic dating as a tool for research on uterine receptivity.

Evidence may exist for a novel type of endometrial receptivity defect that is unrelated to endometrial histology. This concept was first developed from the study of women with endometriosis, and later in women with unexplained infertility,⁷⁴ hydrosalpinges,⁷⁶ and PCOS.⁸⁰ We found that a subset of patients with endometriosis exhibited aberrant midluteal phase integrin expression despite normal histologic dating. Aberrant expression of various biomarkers has been described in such patients and may be caused by alterations in the signaling at the time of implantation. Such defects could be caused by the inhibitory effects of estrogen on epithelial expression of certain genes such as the αvβ3 integrin, interfering with the stimulatory effects of EGF or HB-EGF.¹¹⁸ Similar observations of integrin expression deficits were also made in patients with unexplained infertility⁷⁴ and we recently extended these findings to include the effect of hydrosalpinges on endometrial function.⁷⁶ Accumulating data support a detrimental effect on hydrosalpinx fluid on implantation in human reproduction, as an endometrial cavity bathed in hydrosalpinx fluid at the time of implantation decreases pregnancy rates in IVF.^{178, 179} Salpingectomy results in return of normal integrin expression in most women and yields improved pregnancy rates.⁷⁶ Similar to endometriosis, inflammatory cytokines in the tubal fluid may alter normal endometrial function and account for this dysfunctional endometrium. This concept of two distinct types of uterine receptivity defects was previously depicted in Figure 15.

Treatment of luteal phase deficiency

Although early observational studies suggested improved cycle fecundity with treatment of LPD, subsequent trials have not been as convincing.^{169, 180} Because the causes of LPD are varied, directed treatment of an identified causative factor would be merited. As for an otherwise idiopathic LPD, several therapies have been used, none of which has been shown decidedly to be superior, and thus the choice of therapy is generally one of individual preference.¹¹⁹ It was noted in a study by Daly and coworkers¹⁸¹ that in a group of 29 women with idiopathic LPD (biopsy proven) who were randomly assigned to receive intravaginal progesterone, clomiphene citrate, or human menotropins, all groups achieved some pregnancies, but only in women whose treatment normalized their histologic dating.

EXOGENOUS PROGESTERONE

Because LPD results from inadequate progesterone activity on the endometrium, giving supplemental progesterone makes intuitive sense. Progesterone can be given by vaginal suppository, intramuscular injection, or micronized oral administration.¹¹⁹ Several observational studies have shown improved fecundity with progesterone administration; however, data from randomized clinical trials (RCT) are scarce.^{108, 175, 182, 183} Two studies randomly assigning women with LPD to either progesterone or clomiphene citrate showed no difference between the groups.^{182, 183} Progesterone administration in ART cycles results in a thicker endometrium and higher pregnancy rates.¹⁸⁴ Because the granulosa is significantly disturbed by the retrieval process and is less adequately lutenized, it makes physiologic sense that progesterone supplementation would be beneficial. In general, however, the decision to give progesterone supplementation must consider the limited demonstrated benefit versus the theoretical risk of hyperandrogenizing a female infant through supraphysiologic uterine progesterone levels, although no congenital abnormalities have been directly attributable to progestin therapy.¹⁸⁵

CLOMIPHENE CITRATE

Clomiphene citrate is the most frequently used drug for ovulation induction. Because LPD often results from a poor follicular phase, clomiphene citrate is also often used for LPD. When given to ovulatory women with LPD, the intent of clomiphene citrate treatment is to enhance follicular development and, in turn, the functional capacity of the resultant CL.^{108, 186} Paradoxically, Cook and colleagues¹⁸⁷ reported that ovulatory women frequently develop LPD after receiving clomiphene citrate, an observation that suggests clomiphene citrate treatment may be detrimental in normally cycling women. Other reports show efficacy of clomiphene citrate on LPD, but only in women with profound degrees of histologic delay (5 days or more out of phase) or in women with demonstrated short luteal phase.^{188, 189} When given to women with unexplained infertility, clomiphene citrate reduces the incidence of histologic delay.¹⁹⁰

HUMAN CHORIONIC GONADOTROPIN

Exogenous hCG stimulates the CL directly and represents an alternative to progesterone supplementation.¹¹⁹ Supplemental hCG is most appropriate when specific defects in preovulatory or postovulatory LH secretion can be identified or in the management of recurrent pregnancy loss when delayed implantation is suspected.^{184, 191} Recommendations for dosing vary widely, with some suggesting 5000–10,000 international units (IU) at the time of ovulation followed by 5000 IU 5–7 days later; alternatively 10,000 IU can be given in divided doses every 3 days after ovulation. Unfortunately, there are few studies evaluating the effectiveness of hCG.

• Implantation is a complex process involving ovarian steroids, a multitude of paracrine and autocrine factors, and interactions between multiple cell types.
  
• Timing of implantation is important and involves synchronous activities in the embryo, endometrium, and ovary.
  
• The mechanism of implantation is not precisely known but likely involves cell attachment receptors as well as specific ligands.
  
• Problems with implantation and the development of uterine receptivity may be common and involve disruptions in the signaling pathways involved in embryonic or endometrial receptivity.
• Histological dating has very limited utility in clinically distinguishing fertile from infertile women and as a research tool for the future
  
• New studies are needed to better understand receptivity defects and to devise better methods to treat infertility and for purposes of contraception.