Fertilization, Pregnancy, and Lactation

Transport of Gametes and Fertilization

Cilia and smooth muscle transport the egg and sperm within the female genital tract

Following ovulation, the fimbriae of the fallopian tube sweep over the ovarian surface and pick up the oocyte—surrounded by its complement of granulosa cells, the cumulus oophorus, and corona radiata (see p. 1122 )—and deposit it in the fallopian tube. Shortly after ovulation, movements of the cilia and the smooth muscle of fallopian tube propel the oocyte-cumulus complex toward the uterus.

A man normally deposits 150 to 600 million sperm cells into the vagina at the time of ejaculation. Only 50 to 100 of these cells actually reach the ampullary portion of the fallopian tube, where fertilization normally occurs. However, the sperm get there very quickly, within ~5 minutes of ejaculation. The swimming motion of the sperm alone cannot account for such rapid transport. Forceful contractions of the uterus, cervix, and fallopian tubes propel the sperm into the upper reproductive tract during female orgasm. Prostaglandins in the seminal plasma may induce further contractile activity.

The “capacitation” of the spermatozoa that occurs in the female genital tract enhances the ability of the sperm cell to fertilize the ovum

Maturation of sperm continues while they are stored in the epididymis (see pp. 1102–1103 ). In most species, neither freshly ejaculated sperm cells nor sperm cells that are removed from the epididymis are capable of fertilizing the egg until these cells have undergone further maturation—capacitation—in the female reproductive tract or in the laboratory. Capacitation is a poorly understood physiological process by which spermatozoa acquire the ability to penetrate the zona pellucida of the ovum. The removal or modification of a protective protein coat from the sperm cell membrane appears to be an important molecular event in the process of capacitation. N56-0

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Role of a Bicarbonate-Activated Adenylyl Cyclase in Sperm Capacitation

It has long been recognized that the capacitation of mammalian sperm requires both and adenylyl-cyclase activity, which led to the discovery of a -stimulated soluble adenylyl cyclase (sAC). Recall that the adenylyl cyclase activated by heterotrimeric G proteins is a membrane-bound enzyme ( p. 53 ). In contrast, sAC is present in the cytosol.

Reference

Wuttke MS, Buck J, Levin LR: Bicarbonate-regulated soluble adenylyl cyclase. J Pancreas 2001; 2: pp. 154-158.

Sperm cells do not need to pass through the cervix and uterus to achieve capacitation. Successful pregnancy can occur with gamete intrafallopian transfer (GIFT), in which spermatozoa and oocytes are placed directly into the ampulla of the fallopian tube, and also with direct ultrasound-guided intraperitoneal insemination, in which the sperm are deposited in the peritoneal cavity, near the fimbria. Thus, capacitation of sperm in the reproductive tract is not strictly organ specific. As evidenced by the success of in vitro fertilization (IVF) and embryo transfer (ET) —discussed in Box 56-1 —capacitation is possible even if the sperm does not make contact with the female reproductive tract.

Box 56-1

In Vitro Fertilization and Embryo Transfer

I VF is a procedure in which an oocyte or oocytes are removed from a woman and then fertilized with sperm under laboratory conditions. Early development of the embryo also proceeds under laboratory conditions. Finally, the physician transfers one or more embryos to the uterine cavity, where the embryo will hopefully implant and develop.

Indications for IVF-ET

Indications for IVF-ET include disorders that impair the normal meeting of the sperm and the egg in the distal portion of the fallopian tube. In addition to ovulatory dysfunction, these disorders include tubal occlusion, tubal-peritoneal adhesions, endometriosis, and other disease processes of the female peritoneal cavity. In addition, IVF-ET is indicated in some cases of male-factor infertility (abnormalities in male reproductive function) or unexplained infertility.

Ovarian Stimulation

Because the success rates are <100% for each of the stages of IVF-ET, the physician needs several oocytes, all obtained in a single ovarian cycle. However, woman normally develops a single dominant follicle each cycle (see p. 1123 ). Thus, to obtain the multiple oocytes for IVF-ET, the physician must stimulate the development of multiple follicles in the woman by controlled ovarian hyperstimulation. Although this procedure qualitatively mimics the hormonal control of the normal cycle, the high dose of gonadotropins triggers the development of many follicles. The physician administers some combination of FSH and LH, or pure FSH, either intramuscularly or subcutaneously. Because exogenous gonadotropins stimulate the ovaries directly, GnRH analogs (see Box 55-2 ) are often used to downregulate the hypothalamic-pituitary axis during controlled ovarian stimulation. One usually administers these GnRH analogs before initiating gonadotropin therapy, primarily to prevent a premature LH surge and ovulation.

Cycle Monitoring

After administering the gonadotropins, the physician monitors the stimulated follicular growth in the ovaries with sonographic imaging. Size, number, and serial growth of ovarian follicles may be assessed daily or at other appropriate intervals. Serum estradiol levels provide an additional measure of follicular growth and function. When estradiol levels and follicular growth indicate—by established criteria—appropriate folliculogenesis, the physician simulates a natural LH surge by injecting hCG, which mimics the actions of LH (see p. 1111 ). However, in this case, the simulated LH surge completes the final maturation of multiple follicles and oocytes. Because ovulation usually occurs 36 to 39 hours following the beginning of the LH surge (see p. 1123 ), the physician plans oocyte retrieval in such a way that maximal follicular maturation can occur but the oocytes are still harvested prior to ovulation. Thus, retrievals are scheduled for 34 to 36 hours following the administration of hCG.

Oocyte Retrieval

The physician retrieves oocytes by aspirating them from individual follicles under sonographic guidance. With the patient under conscious or unconscious sedation, and after a local anesthetic is applied to the posterior vaginal wall, the physician inserts a probe equipped with a needle guide into the vagina. After inserting a 16- to 18-gauge needle through the vaginal wall, the physician aspirates the follicular fluid from each mature follicle and collects it in a test tube containing a small amount of culture medium. The eggs are identified in the follicular fluid, are separated from the fluid and other follicular cells, and are then washed and prepared for insemination. This procedure normally yields 8 to 15 oocytes.

Insemination

The sperm sample is subjected to numerous washes, followed by gradient centrifugation to separate the sperm cells from the other cells and from debris found in the ejaculate. Each egg is inseminated with 50,000 to 300,000 motile sperm cells in a drop of culture medium and is incubated overnight. Fertilization can usually be detected by the presence of two pronuclei in the egg cytoplasm after 16 to 20 hours. Fertilization rates generally range from 60% to 85%. Embryo development is allowed to continue in vitro for another 48 to 120 hours until embryos are transferred to the uterus.

Among couples whose male partner has very low numbers of motile sperm, high fertilization rates can be achieved using intracytoplasmic sperm injection (ICSI). Micromanipulation techniques are used to inject a sperm cell into the cytoplasm of each egg in vitro. Fertilization rates after ICSI are generally 60% to 70%, or approximately equivalent to conventional insemination in vitro.

Embryo Transfer

After culturing the cells for 48 to 120 hours, the physician transfers three or four embryos to the uterus at the four- to eight-cell stage (after 2 days) or fewer embryos at the blastocyst stage (after 5 days). Embryos are selected and are loaded into a thin, flexible catheter, which is inserted into the uterine cavity to the desired depth under ultrasonic guidance. The woman usually receives supplemental progesterone to support implantation and pregnancy. In certain cases, the embryos are transferred to the fallopian tube during laparoscopy. This procedure is referred to as tubal embryo transfer (TET). The rationale for this procedure is that the fallopian tube contributes to the early development of the embryo as it travels down the tube to the uterus.

Success Rates

Implantation rates usually range from 8% to 15% per embryo transferred. In the United States, the mean live birth rate per ET procedure is ~33%. Success rates in IVF-ET depend on numerous factors, including age as well as the type and severity of the disease causing infertility.

Fertilization begins as the sperm cell attaches to the zona pellucida and undergoes the acrosomal reaction, and it ends with the fusion of the male and female pronuclei

After ovulation, the egg in the fallopian tube is in a semidormant state. If it remains unfertilized, the ripe egg will remain quiescent for some time and eventually degenerate. When fertilization occurs, the sperm normally comes into contact with the oocyte in the ampullary portion of the tube, usually several hours after ovulation. Fertilization causes the egg to awaken (activation) and initiates a series of morphological and biochemical events that lead to cell division and differentiation. Fertilization occurs in eight steps:

Step 1: The sperm head weaves its way past the follicular cells and attaches to the zona pellucida that surrounds the oocyte ( Fig. 56-1 ). The zona pellucida is composed of three glycoproteins; ZP1 cross-links the filamentous ZP2 and ZP3 into a latticework. Receptors on the plasma membrane of the sperm cell bind to ZP3, thereby initiating a signal-transduction cascade.
Step 2: As a result of the sperm-ZP3 interaction, the sperm cell undergoes the acrosomal reaction, a prelude to the migration of the sperm cell through the mucus-like zona pellucida. The acrosome (see p. 1103 ) is a unique sperm organelle containing hydrolyzing enzymes that are necessary for the sperm to penetrate the zona pellucida. During the acrosomal reaction, an increase in intracellular free Ca ²⁺ concentration ([Ca ²⁺ ] _i ) triggers fusion of the outer acrosomal membrane with the sperm cell's plasma membrane, resulting in the exocytosis of most of the acrosomal contents.
Step 3: The spermatozoon penetrates through the zona pellucida. One mechanism of this penetration is the action of the acrosomal enzymes. N56-2 Protease inhibitors can block the penetration of spermatozoa through the zona pellucida. The sperm cell also penetrates the zona pellucida by mechanical action. The sperm head rapidly oscillates about a fulcrum that is situated in the neck region. This rapid, vigorous, rocking action occurs at a frequency of ~6 to 8 per second. The sperm penetrates the zona pellucida at an angle, which creates a tangential cleavage slit and leaves the sperm head lying sideways against the oocyte membrane.

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Acrosomal Enzymes

Among the many enzymes in the acrosome are acid hydrolases, the best characterized of which is proacrosin, the precursor to acrosin. Acrosin is a member of the serine protease superfamily; it is expressed only in spermatogenic cells. Two other enzymes released from the acrosome are neuraminidase and a special form of hyaluronidase. This particular hyaluronidase can be distinguished from the common lysosomal form of the enzyme, and it appears to be a spermatogenic cell–specific isozyme. Acrosin, hyaluronidase, and neuraminidase help the sperm penetrate the zona pellucida by hydrolyzing the sugar chains and the peptide chains of the glycoproteins of the zona pellucida.
Step 4: The cell membranes of the sperm and the oocyte fuse. Microvilli on the oocyte surface envelop the sperm cell, which probably binds to the oocyte membrane via specific proteins on the surfaces of the two cells. The posterior membrane of the acrosome—which remains part of the sperm cell after the acrosomal reaction—is the first portion of the sperm to fuse with the plasma membrane of the egg. The sperm cell per se does not enter the oocyte. Rather, the cytoplasmic portions of the head and tail enter the oocyte, leaving the sperm-cell plasma membrane behind, an action similar to a snake's crawling out of its skin.
Step 5: The oocyte undergoes the cortical reaction. As the spermatozoon penetrates the oocyte's plasma membrane, it initiates formation of inositol 1,4,5-trisphosphate (IP ₃ ); IP ₃ causes Ca ²⁺ release from internal stores (see p. 60 ), which leads to an increase in [Ca ²⁺ ] _i and [Ca ²⁺ ] _i waves. This rise in [Ca ²⁺ ] _i , in turn, triggers the oocyte's second meiotic division—discussed below (see step 6)—and the cortical reaction. In the cortical reaction, small electron-dense granules that lie just beneath the plasma membrane fuse with the oocyte's plasma membrane. Exocytosis of these granules releases enzymes that act on glycoproteins in the zona pellucida, causing the zona pellucida to harden. This hardening involves the release of polysaccharides that impede the progression of the runners-up (i.e., sperm cells still in the zona pellucida). The cortical reaction also leads to the destruction of ZP receptors, which prevents further binding of sperm cells to the zona pellucida. From a teleological perspective, the cortical granule reaction prevents polyspermy. N56-3 Polyspermic embryos are abnormal because they are polyploid. They do not develop beyond the early cleavage stages.

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Block to Polyspermy

Unlike the situation in some laboratory animals, in humans the block to polyspermy during fertilization does not involve receptors on the zona pellucida or on the cell membrane. It appears that the block to polyspermy in humans is due only to alterations of the inner aspect of the zona pellucida. In experiments on monospermic oocytes fertilized in vitro, other sperm cells added later can partially penetrate the zona pellucida but do not reach the inner half of the zona. When unfertilized oocytes, monospermic oocytes, and polyspermic oocytes are examined in vitro, similar numbers of sperm are found on and within the zona pellucida of each type of oocyte. Further evidence that the zona pellucida is the primary barrier to polyspermy in humans comes from experiments in which the zona pellucida is removed. In this case, the egg is usually penetrated by many sperm. In the in vitro fertilization of normal human oocytes, the rate of polyspermy is quite low, ~5% to 8%, even though 50,000 to 300,000 sperm are available to the oocyte. Thus, the zona pellucida block to polyspermy in humans is highly efficient.
Step 6: The oocyte completes its second meiotic division. The oocyte, which had been arrested in the prophase of its first meiotic division since fetal life (see p. 1073 ), completed its first meiotic division at the time of the surge of luteinizing hormone (LH), which occurred several hours before ovulation (see p. 1116 ). The result was the first polar body and a secondary oocyte with a haploid number of duplicated chromosomes (see Fig. 53-2 C ). Before fertilization, this secondary oocyte had begun a second meiotic division, which was arrested in metaphase. The rise in [Ca ²⁺ ] _i inside the oocyte—which the sperm cell triggers, as noted in step 5—causes not only the cortical reaction, but also the completion of the oocyte's second meiotic division. One result is the formation of the second polar body, which contains a haploid number of unduplicated maternal chromosomes. N56-4 The oocyte extrudes the chromosomes of the second polar body, together with a small amount of ooplasm, into a space immediately below the zona pellucida; the second polar body usually lies close to the first polar body. The nucleus of the oocyte also contains a haploid number of unduplicated chromosomes. As its chromosomes decondense, the nucleus of this mature ovum becomes the female pronucleus.

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Three Polar Bodies

Interestingly, although the small polar bodies are nonfunctional, they contain a full set of chromosomes and are responsive to cell cycle–regulatory mechanisms. Consequently, the first polar body also divides during the second meiotic division to produce a total of three polar bodies and a mature oocyte, each of which contain a haploid number of chromosomes. Thus, as with spermatogenesis, one diploid oogonia produces four haploid daughters; however, in oogenesis only one daughter becomes an functional gamete.
Step 7: The sperm nucleus decondenses and transforms into the male pronucleus, which, like the female pronucleus, contains a haploid number of unduplicated chromosomes (see Fig. 54-7 ). The cytoplasmic portion of the sperm's tail degenerates.
Step 8: The male and female pronuclei fuse, forming a new cell, the zygote. The mingling of chromosomes (syngamy) can be considered as the end of fertilization and the beginning of embryonic development. Thus, fertilization results in a conceptus that bears 46 chromosomes, 23 from the maternal gamete and 23 from the paternal gamete. Fertilization of the ovum by a sperm bearing an X chromosome produces a zygote with XX sex chromosomes; this develops into a female (see pp. 1073–1075 ). Fertilization with a Y-bearing sperm produces an XY zygote, which develops into a male. Therefore, chromosomal sex is established at fertilization.

Implantation of the Developing Embryo

As discussed on page 1129 , the ovum is fertilized in the ampullary portion of the fallopian tube several hours after ovulation ( Fig. 56-2 ), and the conceptus remains in the fallopian tube for ~72 hours, during which time it develops to the morula stage (i.e., a mulberry-shaped solid mass of 12 or more cells), receiving nourishment from fallopian-tube secretions. During these 3 days, smooth-muscle contractions of the isthmus prevent advancement of the conceptus into the uterus while the endometrium is preparing for implantation. The mechanisms by which the ovum is later propelled through the isthmus of the fallopian tube to the uterus probably include beating of the cilia of the tubal epithelium and contraction of the fallopian tube.

Figure 56-2, Transport of the conceptus to the uterus.

After the morula rapidly moves through the isthmus to the uterine cavity, it floats freely in the lumen of the uterus and transforms into a blastocyst (see Fig. 56-2 ). A blastocyst is a ball-like structure with a fluid-filled inner cavity. Surrounding this cavity is a thin layer of trophoectoderm cells that forms the trophoblast, which develops into a variety of supporting structures, including the amnion, the yolk sac, and the fetal portion of the placenta. On one side of the cavity, attached to the trophoblast, is an inner cell mass, which develops into the embryo proper. The conceptus floats freely in the uterine cavity for ~72 hours before it attaches to the endometrium. Thus, implantation of the human blastocyst normally occurs 6 to 7 days following ovulation. Numerous maturational events occur in the conceptus as it travels to the uterus. The embryo must be prepared to draw nutrients from the endometrium on arrival in the uterine cavity, and the endometrium must be prepared to sustain the implantation of the blastocyst. Because of the specific window in time during which implantation can occur, temporal relationships between embryonic and endometrial maturation assume extreme importance.

The presence of an embryo leads to decidualization of the endometrium

During the middle to late secretory phase of the normal endometrial cycle, the endometrium becomes more vascularized and thicker, and the endometrial glands become tortuous and engorged with secretions (see pp. 1125–1126 ). These changes, driven by progesterone from the corpus luteum, peak at ~7 days after ovulation. Additionally, beginning 9 to 10 days after ovulation, a process known as predecidualization (see pp. 1125–1126 ) begins near the spiral arteries. During predecidualization, stromal cells transform into rounded decidual cells, and these cells spread across the superficial layer of the endometrium to make it more compact (zona compacta) and separating it from the deeper, more spongy layer (zona spongiosa; see Fig. 55-11 ). If conception fails to occur, the secretory activity of the endometrial glands decreases, followed by regression of the glands 8 to 9 days after ovulation, which is ultimately followed by menstruation.

When pregnancy occurs, the predecidual changes in the endometrium are sustained and extended, which completes the process of decidualization. The decidua is the specialized endometrium of pregnancy. Its original name was membrana decidua, a term referring to the membranes of the endometrium that are shed following pregnancy, like the leaves of a deciduous tree. Because the degree of decidualization is considerably greater in conception cycles than in nonconception cycles, it is likely that the blastocyst itself promotes decidualization. Indeed, either the presence of the embryo or a traumatic stimulus that mimics the embryo's invasion of the endometrium induces changes in the endometrium. N56-5

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Onset of Decidualization

One of the earliest signs that the blastocyst has transmitted an embryonic signal to the endometrium is a marked increase in the permeability of endometrial capillaries. One can detect this permeability increase by injecting laboratory rats intravenously with a dye, such as Evans blue, which binds to albumin. Accumulation of blue dye in the area of the blastocyst is an index of increased capillary permeability to albumin. Increased endometrial capillary permeability precedes the decidual response and may be triggered by vasoactive substances released by the blastocyst just prior to implantation. Because inhibitors of histidine decarboxylase (which converts histidine to histamine) interrupt implantation, histamine is one candidate for the vasoactive substance.

The area underneath the implanting embryo becomes the decidua basalis ( Fig. 56-3 ). Other portions of the decidua that become prominent later in pregnancy are the decidua capsularis, which overlies the embryo, and the decidua parietalis, which covers the remainder of the uterine surface. The upper zona compacta layer and the middle zona spongiosa layer of the nonpregnant endometrium are still recognizable in the decidualized endometrium of pregnancy. The glandular epithelium within the zona spongiosa continues its secretory activity during the first trimester. Some of the glands take on a hypersecretory appearance in what has been referred to as the Arias-Stella phenomenon of early pregnancy—named after the pathologist Javier Arias-Stella. Although the decidualized endometrium is most prominent during the first trimester, prior to the establishment of the definitive placenta, elements of decidualization persist throughout gestation.

Figure 56-3, Three decidual zones during early embryonic development (about 13 to 14 days postfertilization). The figure shows a sagittal section through a pregnant uterus, with the anterior side to the right.

Uterine secretions nourish the preimplantation embryo, promote growth, and prepare it for implantation

Before the embryo implants in the endometrium and establishes an indirect lifeline between the mother's blood and its own, it must receive its nourishment from uterine secretions. Following conception, the endometrium is primarily controlled by progesterone, which initially comes from the corpus luteum (see p. 1124 ). The uterine glandular epithelium synthesizes and secretes several steroid-dependent proteins ( Table 56-1 ) that are thought to be important for the nourishment, growth, and implantation of the embryo. The endometrium secretes cholesterol, steroids, and various nutrients, including iron and fat-soluble vitamins. It also synthesizes matrix substances, adhesion molecules, and surface receptors for matrix proteins, all of which may be important for implantation.

TABLE 56-1

Endometrial Proteins, Glycoproteins, and Peptides Secreted by the Endometrial Glands during Pregnancy

Mucins
Prolactin
Insulin-like growth factor–binding protein 1 (IGFBP-1)
Placental protein 14 (PP14) or glycodelin
Pregnancy-associated endometrial α ₂ -globulin (α ₂ -PEG)
Endometrial protein 15 (EP15)
Fibronectin
Laminin
Entactin
Collagen IV
Heparin sulfate
Proteoglycan
Integrins
Albumin
β-lipoprotein
Relaxin
Acidic fibroblast growth factor
Basic fibroblast growth factor
Pregnancy-associated plasma protein A (PAPP-A)
Stress response protein 27 (SRP-27)
Cancer antigen 125 (CA-125)
β endorphin
Leu-enkephalin
Diamine oxidase
Plasminogen activator (PA)
Plasminogen activator inhibitor (PAI)
Renin
Progesterone-dependent carbonic anhydrases
Lactoferrin

Pinopodes appear as small, finger-like protrusions on endometrial cells between day 19 (about the time the embryo would arrive in the uterus) and day 21 (about the time of implantation) of the menstrual cycle; they persist for only 2 to 3 days. Pinopode formation appears to be progesterone dependent and is inhibited by estrogens. Pinopodes endocytose macromolecules and uterine fluid and absorb most of the fluid in the lumen of the uterus during the early stages of embryo implantation. By removing uterine luminal fluid, the pinopodes may allow the embryo and the uterine epithelium to approximate one another more closely. Because apposition and adhesion of the embryo to the uterus are the first events of implantation, the presence and action of pinopodes may determine the extent of the implantation window.

The blastocyst secretes substances that facilitate implantation

If the blastocyst is to survive, it must avoid rejection by the maternal cellular immune system. It does so by releasing immunosuppressive agents ( Table 56-2 ). The embryo also synthesizes and secretes macromolecules that promote implantation, the development of the placenta, and the maintenance of pregnancy.

TABLE 56-2

Substances Secreted by the Blastocyst

Immunoregulatory Agents

Platelet-activating factor (PAF)
Immunosuppressive factor
PGE ₂
Interleukins 1α, 6, and 8
Interferon-α
Leukemia inhibitory factor
Colony-stimulating factor
HLA-6
Fas ligand

Metalloproteases (facilitate invasion of the trophoblast into the endometrium)

Collagenases: digest collagen types I, II, III, VII, and X
Gelatinases: two forms, digest collagen type IV and gelatin
Stromelysins: digest fibronectin, laminin, and collagen types IV, V, and VII

Serine Proteases (facilitate invasion of trophoblast into the endometrium)

Other Factors or Actions

hCG: autocrine growth factor
Ovum factor
Early pregnancy factor (EPF)
Embryo-derived histamine-releasing factor
Plasminogen activator and its inhibitors
Insulin-like growth factor 2 (IGF-2): promotes trophoblast invasiveness
Estradiol
β ₁ integrin
Fibroblast growth factor (FGF)
Transforming growth factor-α (TGF-α)
Inhibins

Both short-range and long-range embryonic signals may be necessary for implantation, although the nature of some of these signals remains enigmatic. One short-range signal from the blastocyst may stimulate local changes in the endometrium at the time of its apposition to the endometrium. A long-range signal that is secreted by the early blastocyst is human chorionic gonadotropin (hCG), which is closely related to LH (see p. 1111 ) and sustains the corpus luteum in the face of rapidly falling levels of maternal LH.

hCG is one of the most important of the factors secreted by the trophoblast of the blastocyst, both before and after implantation. Besides rescuing the corpus luteum, hCG acts as an autocrine growth factor and promotes trophoblast growth and placental development. hCG levels are high in the area where the trophoblast faces the endometrium. hCG may have a role in the adhesion of the trophoblast to the endometrium and also has protease activity.

During implantation, the blastocyst apposes itself to the endometrium, adheres to epithelial cells, and finally invades the stroma

As noted on pages 1132–1133 , the conceptus lies unattached in the uterine cavity for ~72 hours. About halfway through this period (i.e., 5 to 6 days after ovulation), the morula transforms into the blastocyst ( Fig. 56-4 A ). Before the initiation of implantation, the zona pellucida that surrounds the blastocyst degenerates. This process, known as hatching of the embryo, occurs 6 to 7 days after ovulation. Lytic factors in the endometrial cavity appear to be essential for the dissolution of the zona pellucida. The blastocyst probably also participates in the process of zona lysis and hatching; when an un fertilized egg is placed in the uterus under the same conditions, its zona pellucida remains intact. A factor produced by the blastocyst may activate a lytic factor that is derived from a uterine precursor. Plasmin, produced from plasminogen, is a plausible candidate for this uterine factor, because plasmin exhibits a lytic effect on the zona pellucida in vitro, and inhibitors of plasmin block in vitro hatching of rat blastocysts. Implantation occurs in three stages: (1) apposition, (2) adhesion, and (3) invasion.

Apposition

The earliest contact between the blastocyst wall, the trophoectoderm, and the endometrial epithelium is a loose connection called apposition (see Fig. 56-4 B ). Apposition usually occurs in a crypt in the endometrium. From the standpoint of the blastocyst, it appears that apposition occurs at a site where the zona pellucida is ruptured or lysed and where it is possible for the cell membranes of the trophoblast to make direct contact with the cell membranes of the endometrium. N56-6 Although the preimplantation blastocyst is asymmetric, it seems that the entire trophoectoderm has the potential to interact with the endometrium, and the final correct orientation—with the inner cell mass pointing toward the endometrium—occurs by free rotation of the inner cell mass within the sphere of overlying trophoectoderm cells.

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Mechanisms of Apposition

A mutual reduction in the electrostatic repulsion between the blastocyst and the endometrial membranes occurs at the time of implantation. The loss of zona pellucida proteins during zona lysis and hatching, as well as changes in glycoproteins and their terminal sugars, may decrease the electrostatic repulsive forces between the conceptus and the endometrium. Inhibitors of protein glycosylation may diminish blastocyst attachment. Further evidence that changes in the zona pellucida are necessary to complete apposition is the fact that neither an unfertilized egg nor the preblastocyst-stage conceptus, each of which is still surrounded by a zona pellucida, shows evidence of adhesion. Changes in the integrity of the cytocalyx lining the surface of the uterine epithelium are also important for apposition.

Adhesion

The trophoblast appears to attach to the uterine epithelium via the microvilli of the trophoblast; ligand-receptor interactions are probably involved in adhesion (see Fig. 56-4 C ). The receptors for these ligand-receptor interactions are often members of the integrin family (see p. 17 ) and can be either on the blastocyst or on the endometrium. Integrins are bifunctional integral membrane proteins; on their intracellular side, they interact with the cytoskeleton, whereas on their extracellular side, they have receptors for matrix proteins such as collagen, laminin, fibronectin, and vitronectin. Therefore, ligand-receptor interactions have two possible orientations. For the first, the extracellular surface of the trophoblast has integrins for binding fibronectins, laminin, and collagen type IV. Thus, during implantation, the trophoblast binds to the laminin that is distributed around the stromal (decidual) cells of the endometrium. Fibronectin, a component of the basement membrane, probably guides the implanting embryo (see below) and is subsequently broken down by the trophoblast.

For the second orientation of matrix-integrin interactions, the extracellular surface of the glandular epithelium also expresses integrins on days 20 to 24 of the menstrual cycle, the implantation window (see p. 1126 ). The expression of receptors for fibronectin and vitronectin (i.e., integrins) may serve as markers of the endometrial capacity for implantation. Small peptides containing sequences that are homologous to specific sequences of fibronectin block blastocyst attachment and outgrowth on fibronectin.

In addition to the integrin-matrix interactions, another important class of ligand-receptor interactions appears to be between heparin proteoglycans or heparan sulfate proteoglycans (see p. 39 ), which are attached to the surface of the blastocyst, and surface receptors on the uterine epithelial cell. These endometrial proteoglycan receptors increase in number as the time of implantation approaches.

Any of the foregoing ligand-receptor interactions can lead to cytoskeletal changes. Thus, adhesion of the trophoblast via ligand-receptor interactions may dislodge the uterine epithelial cells from their basal lamina and thereby facilitate access of the trophoblast to the basal lamina for penetration.

Invasion

As the blastocyst attaches to the endometrial epithelium, the trophoblastic cells rapidly proliferate, and the trophoblast differentiates into two layers: an inner cytotrophoblast and an outer syncytiotrophoblast (see Fig. 56-4 D ). The syncytiotrophoblast arises from the fusion of cytotrophoblast cells to form a giant multinucleated cell that constitutes the principal component of the maternal-fetal interface. During implantation, long protrusions from the syncytiotrophoblast extend among the uterine epithelial cells. The protrusions dissociate these endometrial cells by secreting tumor necrosis factor-alpha (TNF-α), which interferes with the expression of cadherins (cell-adhesion molecules; see p. 17 ) and β catenin (an intracellular protein that helps anchor cadherins to the cytoskeleton). N56-7 The syncytiotrophoblast protrusions then penetrate the basement membrane of the uterine epithelial cells and ultimately reach the uterine stroma.

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Uterine E-Cadherin and β-Catenin at the Implantation Site

The blastocyst can only implant into a receptive endometrium. Conversely, the endometrium can become receptive only when it receives the appropriate signals from the nearby blastocyst, which result in the upregulation of certain genes such as those encoding E-cadherin and β-catenin.

In the endometrial epithelial cells of the peri-implantation uterus, E-cadherin (see pp. 17 and 44–45 )—a transmembrane protein that mediates cell-cell contacts—is expressed at high levels at the apical membrane at the prospective sites of implantation, and much less elsewhere. In addition, β-catenin (see pp. 44–45 and 50 )—a cytoskeletal anchor protein that attaches to the cytosolic domain of the cadherin—is also expressed at the apical membrane of these same cells. These changes are part of the adhesive phenotype of the endometrium during the implantation window.

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