Human reproduction entails a fundamental paradox: although it is critical to the survival of the species, the process is relatively inefficient. Maximal fecundity (the probability of conception during one menstrual cycle) is approximately 30 percent. Only 50 to 60 percent of all conceptions advance beyond 20 weeks of gestation. Of the pregnancies that are lost, 75 percent represent a failure of implantation and are therefore not clinically recognized as pregnancies. Failed implantation is also a major limiting factor in assisted reproduction. A better understanding of the molecular mechanisms responsible for implantation and placentation may improve clinicians’ ability to treat disorders related to these processes, including infertility and early pregnancy loss.
Early Embryonic Development
Very few specimens exist that document the first weeks of embryonic development in humans. In some cases, information about a particular stage of development comes from a single specimen. Other crucial events, such as the initial adhesion of the blastocyst to the uterine epithelium, have never been observed. Therefore, much of our understanding of early human development is inferred from studies in animals. Given that the cellular interactions culminating in implantation and placentation vary greatly even among primates, the relevance of this information is unclear. Nevertheless, certain important steps that have been identified in implantation and placentation in animals probably apply to humans. This review emphasizes those steps for which data already exist.
Fertilization occurs in the fallopian tube within 24 to 48 hours after ovulation. The initial stages of development, from fertilized ovum (zygote) to a mass of 12 to 16 cells (morula), occur as the embryo, encased in a nonadhesive protective coating known as the zona pellucida, passes through the fallopian tube. The morula enters the uterine cavity approximately two to three days after fertilization. The appearance of a fluid-filled inner cavity within the mass of cells marks the transition from morula to blastocyst and is accompanied by cellular differentiation: the surface cells become the trophoblast (and give rise to extraembryonic structures, including the placenta), and the inner cell mass gives rise to the embryo. Within 72 hours after entering the uterine cavity, the embryo hatches from the zona, thereby exposing its outer covering of syncytial (multinucleate) trophoblasts.
Implantation occurs approximately six or seven days after conception (fertilization). Insofar as it is analogous to the events that occur in several primate species, implantation in humans probably includes three stages. The initial adhesion of the blastocyst to the uterine wall, called apposition, is unstable. Microvilli on the apical surface of syncytiotrophoblasts interdigitate with microprotrusions from the apical surface of the uterine epithelium, known as pinopodes (Figure 1). Apposition, and consequently implantation, occurs most commonly in the upper posterior (fundal) wall of the uterus. The next stage, stable adhesion, is characterized by increased physical interaction between the blastocyst and the uterine epithelium. Shortly thereafter, invasion begins, and syncytiotrophoblasts penetrate the uterine epithelium. By then, the blastocyst is oriented with its embryonic pole toward the uterine epithelium.
The diagram shows a preimplantation-stage blastocyst (approximately six to seven days after conception) and the processes thought to be necessary for uterine receptivity and blastocyst apposition and adhesion. COX-2 denotes cyclooxygenase-2, EGF epidermal growth factor, and LIF leukemia inhibiting factor.
By the 10th day after conception, the blastocyst is completely embedded in the stromal tissue of the uterus, the uterine epithelium has regrown to cover the site of implantation, and mononuclear cytotrophoblasts stream out of the trophoblast layer. Eventually, cytotrophoblasts invade the entire endometrium and the inner third of the myometrium (a process termed interstitial invasion), as well as the uterine vasculature (endovascular invasion). The latter process, which establishes the uteroplacental circulation, places trophoblasts in direct contact with maternal blood.
Uterine Receptivity and Blastocyst Activation
Successful implantation is the end result of complex molecular interactions between the hormonally primed uterus and a mature blastocyst (Figure 1, Figure 2, and Figure 3). The failure to synchronize the component processes involved in these interactions results in a failure of implantation.
The diagram shows an invading blastocyst (about 9 to 10 days after conception) and the processes necessary for trophoblast invasion.
The diagram shows an implanted embryo (approximately 14 days after conception) and the processes necessary for the maintenance of an early pregnancy. VEGF denotes vascular endothelial growth factor, and hCG human chorionic gonadotropin.
Uterine receptivity is defined as the state during the period of endometrial maturation when the blastocyst can become implanted. Those involved in the development and use of assisted reproductive techniques for transferring embryos into the uterine cavity have identified days 20 to 24 of a regular 28-day menstrual cycle as the optimal period for implantation. The features of uterine receptivity include histologic changes (the endometrium becomes more vascular and edematous, the endometrial glands display enhanced secretory activity, and pinopodes develop on the luminal surface of the epithelium). Although these changes are useful predictors of the outcome of pregnancy, the molecular mechanisms underlying them are largely unknown.
Multiple signals synchronize the development of the blastocyst and the preparation of the uterus. Of the many aspects of the synchronization process, the role of steroid hormones is the best understood. Implantation requires a preovulatory increase in the secretion of estradiol-17β, which stimulates the proliferation and differentiation of uterine epithelial cells. The continued production of progesterone by the corpus luteum stimulates the proliferation and differentiation of stromal cells. Downstream effectors of steroid-hormone actions include peptide hormones, growth factors, and cytokines.
Several factors have been identified as potential markers of endometrial receptivity. The level of leukemia inhibiting factor in both the luminal and glandular epithelium of the uterus rises dramatically in the midsecretory phase of the menstrual cycle, and diminished secretion of this factor is associated with recurrent pregnancy loss. Other molecules that are probably involved in endometrial receptivity include adhesion molecules and proteins called mucins that have high sugar content and that cause an increase in the expression of oligosaccharide receptors on the surface of endometrial epithelial cells.
The blastocyst actively participates in the process of implantation. Mechanisms that enable the blastocyst to initiate implantation (a process termed activation) include catecholestrogens, a class of estrogen metabolites. Medium in which preimplantation-stage embryos have been cultured in vitro contains many bioactive substances, including leukemia inhibiting factor, transforming growth factor α, transforming growth factor β, platelet-derived growth factor, insulin-like growth factor II, colony-stimulating factor 1, interleukin-1, interleukin-6, prostaglandin E2, and platelet-activating factor. Evidence of signaling between the blastocyst and the uterus comes from studies in mice in which implantation has been delayed indefinitely by the manipulation of the hormones. During this delay, the expression of endometrial heparin-binding epidermal growth factor genes does not increase, even when the blastocyst is positioned next to the uterine lining. When estrogen is injected, the implantation process resumes, with the activation of the blastocyst and a rapid increase in the expression of endometrial heparin-binding epidermal growth factor genes at the site of apposition of the blastocyst.
Completing the loop, embryos at or near the implantation stage express epidermal-growth-factor receptors and heparan sulfate proteoglycans, both of which interact with epidermal growth factor–like ligands. The addition of heparin-binding epidermal growth factor to cultured embryos stimulates their proliferation and maturation. These findings are probably applicable to implantation in humans, because heparin-binding epidermal growth factor has similar effects on human embryos in vitro.
The interaction between an activated blastocyst and a receptive uterus is part of a complex process that leads to implantation and the early stages of placental development. Many of the regulatory mechanisms that have been identified govern multiple important transitions involved in this process. Thus, associating their functions with any single event draws an arbitrary distinction that does not exist in vivo. Leukemia inhibiting factor, for example, appears to be important for both decidualization and implantation. It is produced not only before implantation in response to estrogen in progesterone-primed uterine glands, but also at the time of implantation by stromal cells surrounding the active blastocyst
Implantation requires the biosynthesis of prostaglandin. Cyclooxygenase (COX), the rate-limiting enzyme in the conversion of arachidonic acid to prostaglandin H2, exists in two isoforms: constitutive (COX-1) and inducible (COX-2). In the endometrium, COX-1 production decreases in response to progesterone and estradiol-17β, and the endometrial content of COX-1 falls precipitously in the midluteal phase of the menstrual cycle in anticipation of implantation. In contrast, COX-2 production, which is not affected by steroid hormones, is restricted to the site of implantation and depends on the presence of a blastocyst that is ready to implant. Moreover, interleukin-1, detected in the medium in which the human embryos have been cultured, induces the expression of COX-2 genes in cultured endometrial stromal cells. Prostaglandin I2 produced by the action of COX-2 is a ligand for the nuclear receptor peroxisome-proliferator–activated receptor δ (PPARδ). This interaction is probably critical, given that fetal mice lacking a related receptor (PPARγ) die in the middle of the gestational period because of defective placentation.
Once implantation begins, a brief interval of stable adhesion is followed by a much longer period during which trophoblasts invade the uterus (Figure 2). As in other biologic systems in which stable adhesion is followed by invasion, such as the extravasation of leukocytes and tumor cells, changes in the production of adhesion molecules and proteinases are implicated. The invasion of cytotrophoblasts leads to a decrease in the expression of adhesion receptors characteristic of cytotrophoblast stem cells and an increase in the expression of adhesion receptors that are characteristic of vascular cells. Besides allowing cytotrophoblasts that line maternal vessels to masquerade as vascular cells, these receptors also improve the cells’ ability to invade the uterus.
Invading cytotrophoblasts also increase their production of proteinase. For example, they increase their production and activation of matrix metalloproteinase-9, which contributes to the invasiveness of cytotrophoblasts in vitro. The simultaneous increase in the production of tissue inhibitor of metalloproteinase-3 provides a mechanism for restricting matrix metalloproteinase–mediated invasion. Matrix metalloproteinases and tissue inhibitors of metalloproteinases in maternal decidua appear to have a similar role in regulating the invasion of trophoblasts. Other trophoblast proteinases that may be important in invasion include cathepsin B and L.
The molecular mechanisms that regulate the differentiation and invasion of trophoblasts are not well understood. The temporal and spatial expression of several growth factors and cytokines within the uterus (e.g., leukemia inhibiting factor, interleukin-1 and its receptors, insulin-like growth factors I and II and their binding proteins, colony-stimulating factor 1, and transforming growth factors α and β) suggests that they may have important functional roles. For example, interleukin-1 increases the production of matrix metalloproteinase-9 by cytotrophoblasts, and interleukin-1 concentrations in embryo culture medium correlate with reproductive success after in vitro fertilization. Decidual vascular endothelial growth factor probably promotes angiogenesis and localized vascular permeability, other key elements in implantation. Physiologic regulators may also be important. For example, oxygen tension promotes some aspects of trophoblast differentiation, including the production of integrin α1β1.
From the Divisions of Maternal–Fetal Medicine and Reproductive Endocrinology, Department of Obstetrics, Gynecology, and Reproductive Biology, Brigham and Women’s Hospital and Harvard Medical School, Boston (E.R.N., D.J.S.); and Department of Stomatology, University of California, San Francisco (S.J.F.).
1. Zinaman MJ, Clegg ED, Brown CC, O’Connor J, Selevan SG. Estimates of human fertility and pregnancy loss. Fertil Steril 1996;65:503-509.
2. Wilcox AJ, Weinberg CR, O’Connor JF, et al. Incidence of early loss of pregnancy. N Engl J Med 1988;319:189-194.
3. Spandorfer S, Rosenwaks Z. The impact of maternal age and ovarian age on implantation efficacy. In: Carson DD, ed. Embryo implantation: molecular, cellular and clinical aspects. New York: Springer-Verlag, 1999:12-9.
4. Enders AC, Lopata A. Implantation in the marmoset monkey: expansion of the early implantation site. Anat Rec 1999;256:279-299.
5. Hertig AT, Rock J, Adams EC, Menkin MC. Thirty-four fertilized human ova, good, bad and indifferent, recovered from 210 women of known fertility: a study of biologic wastage in early human pregnancy. Pediatrics 1959;23:202-211.
6. Pijnenborg R, Robertson WB, Brosens I, Dixon G. Trophoblast invasion and the establishment of haemochorial placentation in man and laboratory animals. Placenta 1981;2:71-91.
7. Early development of the human placenta. In: Benirschke K, Kaufmann P. Pathology of the human placenta. New York: Springer-Verlag, 1991:13-21.
8. Pijnenborg R, Bland JM, Robertson WB, Dixon G, Brosens I. The pattern of interstitial trophoblastic invasion of the myometrium in early human pregnancy. Placenta 1981;2:303-316.
9. Enders AC. Trophoblast-uterine interactions in the first days of implantation: models for the study of implantation events in the human. Semin Reprod Med 2000;18:255-263.
10. Psychoyos A. Uterine receptivity for nidation. Ann N Y Acad Sci 1986;476:36-42.
11. Bergh PA, Navot D. The impact of embryonic development and endometrial maturity on the timing of implantation. Fertil Steril 1992;58:537-542.
12. Cullinan EB, Abbondanzo SJ, Anderson PS, Pollard JW, Lessey BA, Stewart CL. Leukemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation. Proc Natl Acad Sci U S A 1996;93:3115-3120.
13. Chaouat G, Menu E, Delage G, et al. Immuno-endocrine interactions in early pregnancy. Hum Reprod 1995;10:55-59.
14. Lessey BA. Endometrial integrins and the establishment of uterine receptivity. Hum Reprod 1998;13:Suppl 3:247-261.
15. Lagow E, DeSouza MM, Carson DD. Mammalian reproductive tract mucins. Hum Reprod Update 1999;5:280-292.
16. Paria BC, Huet-Hudson YM, Dey SK. Blastocyst’s state of activity determines the “window” of implantation in the receptive mouse uterus. Proc Natl Acad Sci U S A 1993;90:10159-10162.
17. Paria BC, Das SK, Dey SK. Embryo implantation requires estrogen-directed uterine preparation and catecholestrogen-mediated embryonic activation. Adv Pharmacol 1998;42:840-843.
18. Stewart CL, Cullinan EB. Preimplantation development of the mammalian embryo and its regulation by growth factors. Dev Genet 1997;21:91-101.
19. Das SK, Wang XN, Paria BC, et al. Heparin-binding EGF-like growth factor gene is induced in the mouse uterus temporally by the blastocyst solely at the site of its apposition: a possible ligand for interaction with blastocyst EGF-receptor in implantation. Development 1994;120:1071-1083.
20. Das SK, Das N, Wang J, et al. Expression of betacellulin and epiregulin genes in the mouse uterus temporally by the blastocyst solely at the site of its apposition is coincident with the “window” of implantation. Dev Biol 1997;190:178-190.
Errol R. Norwitz, M.D., Ph.D., Danny J. Schust, M.D., and Susan J. Fisher, Ph.D.
The New England Journal of Medicine