3. First Week of Development: Ovulation to Implantation

ECTOPIC TUBAL PREGNANCY (ETP)

1. An ETP occurs when the blastocyst implants within the uterine tube due to delayed transport. The ampulla of uterine tube is the most common site of an ETP. The rectouterine pouch (pouch of Douglas) is a common site for an ectopic abdominal pregnancy. 2. An ETP is frequently predisposed by chronic salpingitis, endometriosis, and post-operative adhesions. 3. An ETP is most commonly seen in women with endometriosis or pelvic inflammatory disease. 4. An ETP leads to uterine tube rupture and hemorrhage if surgical intervention (i.e., salpingectomy) is not performed. 5. An ETP must be differentially diagnosed from appendicitis, an aborting intrauterine pregnancy, or a bleeding corpus luteum of a normal intrauterine pregnancy. 6. Clinical signs of an ETP include: abnormal uterine bleeding, unilateral pelvic pain, increased levels of human chorionic gonadotropin (hCG) (but lower than originally expected with uterine implantation pregnancy), and a massive first-trimester bleed.

As soon as the spermatozoon has entered the oocyte, the egg responds in three ways:

1. Cortical and zona reactions. As a result of the release of cortical oocyte granules, which contain lysosomal enzymes, (1) the oocyte membrane becomes impenetrable to other spermatozoa, and (2) the zona pellucida alters its structure and composition to prevent sperm binding and penetration. These reactions prevent polyspermy (penetration of more than one spermatozoon into the oocyte). 2. Resumption of the second meiotic division. The oocyte finishes its second meiotic división immediately after entry of the spermatozoon. One of the daughter cells, which receives hardly any cytoplasm, is known as the second polar body; the other daughter cell is the definitive oocyte. Its chromosomes (22 plus X) arrange themselves in a vesicular nucleus known as the female pronucleus (Figs. 3.6 and 3.7). 3. Metabolic activation of the egg. The activating factor is probably carried by the spermatozoon. Activation encompasses the initial cellular and molecular events associated with early embryogenesis. The spermatozoon, meanwhile, moves forward until it lies ciose to the female pronucleus. Its nucleus becomes swollen and forms the male pronucleus (Fig. 3.6); the tail detaches and degenerates. Morphologically, the male and female pronuclei are indistinguishable, and eventually, they come into close contact and lose their nuclear envelopes (Fig. 3.7A). During growth of male and female pronuclei (both haploid), each pronucleus must rep lícate its DNA. If it does not, each cell of the two-cell zygote has only half of the normal amount of DNA. Immediately after DNA synthesis, chromosomes organize on the spindle in preparation for a normal mitotic division. The 23 maternal and 23 paternal (double) chromosomes split longitudinally at the centromere, and sister chromatids move to opposite poles, providing each cell of the zygote with the normal diploid number of chromosomes and DNA (Fig. 3.6D,E). As sister chromatids move to opposite poles, a deep furrow appears on the surface of the cell, gradually dividing the cytoplasm into two parts (Figs. 3.6F and 3.75).

The wall of the uterus consists of three layers

1. Endometrium or mucosa lining the inside wall 2. Myometrium, a thick layer of smooth muscle 3. Perimetrium, the peritoneal covering lining the outside wall (Fig. 3.11)

A male "pill"

A male "pill" has been developed and tested in clinical triais. It contains a synthetic androgen th at prevents both LH and FSH secretion and either stops sperm production [70% to 90% of men] or reduces it to a level of infertility.

Blastocyst Formation

A. Occurs when fluid secreted within the morula forms the blastocyst cavity. B. The inner cell mass, which becomes the embryo, is called the embryoblast. The embryoblast cells are pluripotent. Pluripotency refers to a stem cell that can differentiate into ectoderm, mesoderm, and endoderm. C. The outer cell mass, which becomes part of the placenta, is called the trophoblast.

Implantation

A. The zona pellucida must degenerate for implantation to occur. B. The blastocyst implants within the posterior superior wall of the uterus. C. The blastocyst implants within the functional layer of the endometrium during the secretory phase of the menstrual cycle. D. The trophoblast differentiates into cytotrophoblast and syncytiotrophoblast.

BLASTOCYST FORMATION

About the time the morula enters the uterine cavity, fluid begins to penetrate through the zona pellucida into the intercellular spaces of the inner cell mass. Gradually, the intercellular spaces become confluent, and finally, a single cavity, the biastocele, forms (Fig. 3.10A,B). At this time, the embryo is a biastocyst. Cells of the inner cell mass, now called the embryoblast, are at one pole, and those of the outer cell mass, or trophoblast, flatten and form the epithelial wall of the blastocyst (Fig. 3.10A,B). The zona pellucida has disappeared, allowing implantation to begin. In the human, trophoblastic cells over the embryoblast pole begin to penetrate between the epithelial cells of the uterine mucosa on about the sixth day (Fig. 3.10C). New studies suggest that L-selectin on trophoblast cells and its carbohydrate receptors on the uterine epithelium mediate initial attachment of the blastocyst to the uterus.

Adult Stem Cells

Adult tissues contain stem cells that may also prove valuable in treating diseases. These cells are restricted in their ability to form different cell types and, therefore, are multipotent, not pluripotent, although scientists are finding methods to circumvent this disadvantage. Adult stem cells isolated from rat brains have been used to cure Parkinson disease in rats, suggesting that the approach has promise. Disadvantages of the approach include the slow rates of cell division characteristic of these cells and their scarcity, which makes them difficult to isolate in sufficient numbers for experiments.

Corpus Luteum

After ovulation, granulosa cells remaining in the wall of the ruptured follicle, together with cells from the theca interna, are vascularized by surrounding vessels. Under the influence of LH, these cells develop a yellowish pigment and change into lutein cells, which form the corpus luteum and secrete estrogens and progesterone (Fig. 3.3C). Progesterone, together with some estrogen, causes the uterine mucosa to enter the progestational or secretory stage in preparation for implantation of the embryo.

ovarian cycle

At puberty, the female begins to undergo regular monthly cycles. These sexual cycles are controlled by the hypothalamus. Gonadotropin-releasing hormone (GnRH), produced by the hypothalamus, acts on cells of the anterior lobe (adenohypophysis) of the pituitary gland, which in turn secrete gonadotropins. These hormones, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), stimulate and control cyclic changes in the ovary. At the beginning of each ovarian cycle, 15 to 20 primary-stage (preantral) follicles are stimulated to grow under the influence of FSH. (The hormone is not necessary to promote development of primordial follicles to the primary follicle stage, but without it, these primary follicles die and become atretic.) Thus, FSH rescues 15 to 20 of these cells from a pool of continuously forming primary follicles (Figs. 3.1 and 3.2). Under normal conditions, only one of these follicles reaches full maturity, and only one oocyte is discharged; the others de- generate and become atretic. In the next cycle, another group of primary follicles is recruited, and again, only one follicle reaches maturity. Consequently, most follicles degenerate with out ever reaching full maturity. When a follicle becomes atretic, the oocyte and surrounding follicular cells degenerate and are replaced by connective tissue, forming a corpus atreticum. FSH also stimulates maturation of follicular (granulosa) cells surrounding the oocyte. In turn, proliferation of these cells is mediated by growth differentiation factor 9, a member of the transforming growth factor-𝛽 (TGF-𝛽) family. In cooperation, theca interna and granulosa cells produce estrogens: Theca interna cells produce androstenedione and testosterone, and granular cells convert these hormones to estrone and 17 𝛽-estradiol.

At the time of implantation,

At the time of implantation, the mucosa of the uterus is in the secretory phase (Fig. 3.12), during which time uterine glands and arteries become coiled and the tissue becomes suc- culent. As a result, three distinct layers can be recognized in the endometrium: a superficial compact layer, an intermedíate spongy layer, and a thin basal layer (Fig. 3.12). Normally, the human blastocyst implants in the endometrium along the anterior or posterior wall of the body of the uterus, where it becomes embedded between the openings of the glands (Fig. 3.12). If the oocyte is not fertilized, venules and sinu soidal spaces gradually become packed with blood cells, and an extensive diapedesis of blood into the tissue is seen. When the menstrual phase begins, blood escapes from superficial arteries, and small pieces of stroma and glands break away. During the following 3 or 4 days, the compact and spongy layers are expelled from the uterus, and the basal layer is the only part of the endometrium that is retained (Fig. 3.13). This layer, which is supplied by its own arteries, the basal arteries, fiinctions as the regenerative layer in the rebuilding of glands and arteries in the proliferative phase (Fig. 3.13).

Barrier methods of contraception

Barrier methods of contraception include the male condom, made of latex and often containing chemical spermicides, which fits over the penis, and the female condom, made of polyurethane, which lines the vagina. Other barriers placed in the vagina include the diaphragm, the cervical cap, and the contraceptive sponge.

Capacitatíon

Capacitatíon is a period of conditioning in the female reproductive tract that in the human lasts approximately 7 hours. Thus, speeding to the ampulla is not an advantage because capacitatíon has not yet occurred and such sperm are not capable of fertllizing the egg. Much of this conditioning during capacitation occurs in the uterine tube and involves epithelial interactions between the sperm and the mucosal surface of the tube. During this time, a glycoprotein coat and seminal plasma proteins are removed from the plasma membrane that overlies the acrosomal region of the spermatozoa.

Clomiphene citrate (Clomid)

Clomiphene citrate (Clomid) is a drug used to increase FSH concentrations to stim ulate ovulation. The drug is given early in the menstrual cycle to cause ovulation in women who do not ovulate or who ovulate irregulariy. AIso, it is given to stimulate egg development for use in in vitro fertilization procedures

Clinical Correlatas Ovulation

During ovulation, some women feel a slight pain, called mitteischmerz (German for " middie pain"), because it normally occurs near the middie of the menstrual cycle. Ovulation is aiso generally accompanied by a rise in basal temperature, which can be monitored to aid couples in becoming pregnant or preventing pregnancy. Some women fail to ovulate because of a low concentration of gonadotropins. In these cases, administration of an agent to stimulate gonadotrop in release, and hence ovulation, can be employed. Although such drugs are effective, they often produce multiple ovulations, so that the likelihood of multiple pregnancies is 10 times higher in these women than in the general population.

Embryonic stem cells (ES cells)

Embryonic stem cells (ES cells) are derived from the inner cell mass of the embryo. Because these cells are pluripotent and can form virtually any cell or tissue type, they have the potential for curing a variety of diseases, including diabetes, Alzheimer and Parkinson diseases, anemias, spinal cord injuries, and many others. Using animal model research with stem cells has been encouraging. For example, mouse ES cells in culture have been induced to form insulin-secreting cells, muscle and nerve stem cells, and glial cells. In whole animals, ES cells have been used to alleviate the symptoms of Parkinson disease and to improve motor ability in rats with spinal cord injuries. ES cells may be obtained from embryos after IVF, a process called reproductive cloning. This approach has the disadvantage that the cells may cause immune rejection because they would not be genetically identical to their hosts. The cells could be modified to circumvent this probiem, however. Another issue with this approach is based on ethical considerations, as the cells are derived from viable embryos. As the fieid of stem cell research progresses, scientific advances will provide more genetically compatible cells, and the approaches will be less controversial. Most recently, techniques have been devised to take nuclei from adult cells (e.g., skin) and introduce them into enucleated oocytes. This approach is called therapeutic cloning or somatic nuclear transfer. Oocytes are stim ulated to differentiate into blastocysts, and ES cells are harvested. Because the cells are derived from the host, they are compatible genetically, and because fertilization is not involved, the technique is less controversial.

Emergency contraceptive pills (ECPs)

Emergency contraceptive pills (ECPs) are used as birth control measures that may prevent pregnancy if taken 120 hours after sexual intercourse. These pills may be administered as high doses of progestin alone or in combination with estrogen [Plan B). Other types of ECPs [mifepristone [RU-486] and ulipristal acetate [Ella]] act as antihormonal agents. AIso, mifepristone is effective as an abortifacient if taken after the time of implantation.

FERTILIZATION

Fertilization, the process by which male and female gametes fuse, occurs in the ampullary region of the uterine tube. This is the widest part of the tube and is ciose to the ovary (Fig. 3.4). Spermatozoa may remain viable in the female reproductive tract for several days. Only 1% of sperm deposited in the vagina enter the cervix, where they may sundve for many hours. Movement of sperm from the cervix to the uterine tube occurs by muscular contractions of the uterus and uterine tube and very little by their own propulsion. The trip from cervix to oviduct can occur as rapidly as 30 minutes or as slow as 6 days. After reaching the isthmus, sperm become less motile and cease their migration. At ovulation, sperm again become motile, perhaps because of chemoattractants produced by cumulus cells surrounding the egg, and swim to the ampulla, where fertilization usually occurs. Spermatozoa are not able to fertilize the oocyte immediately upon arrival in the female genital tract but must undergo (1) capacitation and (2) the acrosome reaction to acquire this capability. Only capacitated sperm can pass through the corona cells and undergo the acrosome reaction.

UTERUSAT TIME OF IMPLANTATION

From puberty (11 to 13 years) until menopause (45 to 50 years), the endometrium undergoes changes in a cycle of approximately 28 days under hormonal control by the ovarles. During this menstrual cycle, the uterine endo metrium passes through three stages: 1. Follicular or proliferative phase 2. Secretory or progestational phase 3. Menstrual phase (Figs. 3.12 and 3.13) The proliferative phase begins at the end of the menstrual phase, is under the influence of estrogen, and parallels growth of the ovarían follicles. The secretory phase begins approximately 2 to 3 days after ovulation in response to progesterone produced by the corpus luteum. If fertilization does not occur, shedding of the endometrium (compact and spongy layers) marks the beginning of the menstrual phase. If fertilization does occur, the endometrium assists in implantation and contributes to formation of the placenta. Later in gestation, the placenta assumes the role of hormone production, and the corpus luteum degenerates.

Hormonal methods of contraception

Hormonal methods are another commonly used form of contraception. These approaches provide the female hormones estrogen and/ or progestin. These hormones produce their effects by inhibiting ovulation (by preventing the release of FSH and LH from the pituitary gland), changing the lining of the uterus, and thickening cervical mucus, making it difficult for sperm to enter the uterus. Hormonal contraception can be provided through birth control pills, a skin patch, vaginal ring, injection, or implant. There are two types of contraceptive pills: The first is a combination of estrogen and the progesterone analogue progestin; the second is composed of progestin alone. Both pills are effective, but one may suit some women better than others for various health-related issues.

Corpus Albicans

If fertilization does not occur, the corpus luteum reaches maximum development approximately 9 days after ovulation. It can easily be recognized as a yellowish projection on the surface of the ovary. Subsequently, the corpus luteum shrinks because of degeneration of lutein cells (luteolysis) and forms a mass of fibrotic scar tissue, the Corpus albicans. Simultaneously, progesterone production decreases, precipitating menstrual bleeding. If the oocyte is fertilized, degeneration of the corpus luteum is prevented by human chorionic gonadotropin, a hormone secreted by the syncytiotrophoblast of the developing embryo. The corpus luteum continúes to grow and forms the corpus luteum of pregnancy (corpus luteum graviditatis). By the end of the third month, this structure may be one third to one half of the total size of the ovary. Yellowish luteal cells continue to secrete progesterone until the end of the fourth month; thereafter, they regress slowly as secretion of progesterone by the trophoblastic component of the placenta becomes adequate for maintenance of pregnancy. Removal of the corpus luteum of pregnancy before the fourth month usually leads to abortion.

In humans, cleavage is

In humans, cleavage is holoblastic, which means the cells divide completely through their cytoplasm. Cleavage is asymmetrical, which means the daughter cells are unequal in size (i.e., one cell gets more cytoplasm than the other) at least during the first few cell divisions. Cleavage is asynchronous, which means only one cell will divide at a time; generally the largest daughter cell will divide next at least during the first few cell divisions

Ovulation

In the days immediately preceding ovulation, under the influence of FSH and LH, the vesicular follicle grows rapidly to a diameter of 25 mm to become a mature vesicular (graafian) follicle. Coincident with final development of the vesicular follicle, there is an abrupt increase in LH that causes the primary oocyte to complete meiosis I and the follicle to enter the preovulatory mature vesicular stage. Meiosis II is also initiated, but the oocyte is arrested in metaphase approximately 3 hours before ovulation. In the meantime, the surface of the ovary begins to bulge locally, and at the apex, an avascular spot, the stigma, appears. The high concentration of LH increases collagenase activity, resulting in digestion of collagen fibers surrounding the follicle. Prostaglandin levels also increase in response to the LH surge and cause local muscular contractions in the ovarian wall. Those contractions extrude the oocyte, which together with its surrounding granulosa cells from the region of the cumulus oophorus breaks free (ovulation) and floats out of the ovary (Fig. 3.3). Some of the cumulus oophorus cells then rearrange themselves around the zona pellucida to form the corona radiata (Figs. 3.25 to 3.6).

In vitro fertilization [IVF]

In vitro fertilization [IVF] of human ova and embryo transfer is the standard procedure used by laboratories through out the world. Follicle grow th in the ovary is stimulated by administration of gonadotropins. Oocytes are recovered by laparoscopy from ovarían follicles with an aspirator just before ovulation when the oocyte is In the late stages of the first meiotic division. The egg is placed In a simple culture medium, and sperm are added immediately. Alternatively, a single sperm can be injected into an egg's cytoplasm to produce fertilization.

Infertility

Infertility is a probiem fo r 15% to 30% of couples. Male infertility may be a result of insuffident numbers of sperm and /or poor motility. Normally, the ejaculate has a volum e of 2 to 6 mL, with as many as 100 million sperm per milliliter. Men with 20 million sperm per milliliter or 50 million sperm per total ejaculate are usually fertile. Infertility in a woman may be due to a number of causes, including occluded uterine tubes (most commonly caused by pelvic inflammatory disease), hostile cervical mucus, immunity to spermatozoa, absence of ovulation, and others.

Phase 1: Penetratíon of the Corona Radiata

Of the 200 to 300 million spermatozoa normally depositad in the female genital tract, only 300 to 500 reach the site of fertilization. Only one of these fertilizes the egg. It is thought that the others aid the fertilizing sperm in penetrating the barriers protecting the female gamete. Capacitated sperm pass freely through corona cells (Fig. 3.5).

CLEAVAGE

Once the zygote has reached the two-cell stage, it undergoes a series of mitotic divisions, increasing the numbers of cells. These cells, which become smaller with each cleavage division, are known as blastomeres (Fig. 3.8). Until the eight-cell stage, they form a loosely arranged clump (Fig. 3.9A). After the third cleavage, however, blastomeres maximize their contact with each other, forming a compact ball of cells held together by tight junctions (Fig. 3.95). This process, compaction, segregates inner cells, which communicate extensively by gap junctions, from outer cells. Approximately 3 days after fertilization, cells of the compacted embryo divide again to form a 16-cell morula (mulberry). Inner cells of the morula constitute the inner cell mass, and surrounding cells compose the outer cell mass. The inner cell mass gives rise to tissues of the embryo proper, and the outer cell mass forms the trophobiast, which later contributes to the placenta.

assisted reproductive technology (ART)

One percent to 2% of all pregnancies in the United States occur using assisted reproductive technology (ART). Offspring from these conceptions show increases in prematurity (<37 weeks' gestatlon), low birth weight (< 2,500 g], very low birth weight (<1,500g], and some types of birth defects. Most of these adverse outcomes are caused by increased rates of multiple births (twins, triplets, etc.), which occur more commonly among ART pregnancies. Recent studies in dícate, however, that even among singleton births after ART, there are increases in preterm birth and malformed infants. Some of the approaches used for ART include the following.

L-selectin

Selectins are carbohydrate-binding proteins involved in interactions between leuocytes and endothelial cells that allow leukocyte "capture" from flowing blood. A similar mechanism is now proposed for "capture" of the blastocyst from the uterine cavity by the uterine epithelium. Following capture by selectins, further attachment and invasion by the trophoblast involve integrins, expressed by the trophoblast and the extracellular matrix molecules laminin and fibronectin. Integrin receptors for laminin promote attachment, whereas those for fibronectin stimulate migration. These molecules also interact along signal transduction pathways to regulate trophoblast differentiation, so that implantation is the result of mutual trophoblastic and endometrial action. Henee, by the end of the first week of development, the human zygote has passed through the morula and blastocyst stages and has begun implantation in the uterine mucosa.

Severe male infertility

Severe male infertility, In which the ejaculate contains very few live sperm (oligozoospermia) or even no live sperm (azoospermia), can be overcome using ICSI. With this technique, a single sperm, which may be obtained from any point in the male reproductive tract, is injected into the cytoplasm of the egg to cause fertilization. This approach offers couples an alternative to using donor sperm for IVF. The technique carries an increased risk for fetuses to have Y chromosome deletions and appears to be associated with a higher incidence of birth defects than standard IVF techniques.

Oocyte Transport

Shortly before ovulation, fimbriae of the uterine tube sweep over the surface of the ovary, and the tube itself begins to contract rhythmically. It is thought that the oocyte, surrounded by some granulosa cells (Figs. 33 B and 3.4), is carried into the tube by these sweeping movements of the fimbriae and by motion of cilia on the epithelial lining. Once in the tube, cumulus cells withdraw their cytoplasmic processes from the zona pellucida and lose contact with the oocyte. Once the oocyte is in the uterine tube, it is propelled by peristaltic muscular contractions of the tube and by cilia in the tubal mucosa with the rate of transport regulated by the endocrine status during and after ovulation. In humans, the fertilized oocyte reaches the uterine lumen in approximately 3 to 4 days.

Sterilization

Sterilization is another form of birth control. The method for men is a vasectomy, which prevents the release of sperm by blocking the ductus deferens, the tube that transports sperm from the testes to the penis. The sterilization method for women is tubal sterilization in which the uterine tubes are blocked or ligated. These procedures for both men and women can be reversed in some cases.

Acrosome reaction

The acrosome reaction, which occurs after binding to the zona pellucida, is induced by zona proteins. This reaction culminates in the release of enzymes needed to penetrate the zona pellucida, including acrosin- and trypsin-like substances (Fig. 3.5).

Abnormal Zygotes

The exact number of abnormal zygotes formed is unknown because they are usually lost within 2 to 3 weeks of fertilization, before the woman realizes she is pregnant, and there fore are not detected. Estimates are that as many as 50% of pregnancies end in spontaneous abortion and that half of these losses are a result of chromosomal abnormalities. These abortions are a natural means of screening embryos for defects, reducing the incidence of congenital malform ations. Without this phenomenon, approximately 12% instead of 2% to 3% of infants would have birth defects. With the use of a combination of IVF and polymerase chain reaction, molecular screening of embryos for genetic defects is being conducted. Single blastom eres from early- stage embryos can be removed, and their DNA can be amplified for analysis. As the Human Genome Project provides more sequencing In formation, and as specific genes are linked to various syndromes, such procedures will become more commonplace.

Phase 3: Fusion of the Oocyte and Sperm Cell Membranes

The initial adhesion of sperm to the oocyte is mediated in part by the interaction of integrins on the oocyte and their ligands, disintegrins, on sperm. After adhesion, the plasma membranes of the sperm and egg fuse (Fig. 3.5). Because the plasma membrane covering the acrosomal head cap disappears during the acrosome reaction, actual fusion is accomplished between the oocyte membrane and the membrane that covers the posterior region of the sperm head (Fig. 3.5). In the human, both the head and the tail of the sper- matozoon enter the cytoplasm of the oocyte, but the plasma membrane is left behind on the oocyte surface.

intrauterine device [lUD]

The intrauterine device [lUD] is a small T-shaped unit and there are two types: hormonal and copper. The hormonal device releases progestin that causes thickening of cervical mucus to prevent sperm from entering the uterus. AIso, it may make sperm less active and both eggs and sperm less viable. The copper type releases copper into the uterus that prevents fertilization or inhibits attachm ent of the fertilized egg to the uterine wall. It also helps prevent sperm from entering the uterine tubes.

The process of cleavage eventually forms a___________, consisting of cells called ___________.

The process of cleavage eventually forms a blastula, consisting of cells called blastomeres.

Phase 2: Penetratíon of the Zona Pellucida

The zona is a glycoprotein shell surrounding the egg that facilitates and maintains sperm binding and induces the acrosome reaction. Both binding and the acrosome reaction are mediated by the ligand ZP3, a zona protein. Release of acrosomal enzymes (acrosin) allows sperm to penetrate the zona, thereby coming in contact with the plasma membrane of the oocyte (Fig. 3.5). Permeability of the zona pellucida changes when the head of the sperm comes in contact with the oocyte surface. This contact results in release of lysosomal enzymes from cortical granules lining the plasma membrane of the oocyte. In turn, these enzymes alter properties of the zona pellucida (zona reaction) to prevent sperm penetration and inactivate species-specific receptor sites for spermatozoa on the zona surface. Other spermatozoa have been found embedded in the zona pellucida, but only one seems to be able to penetrate the oocyte (Fig. 3.6).

intracytoplasmic sperm injection (ICSI)

This technique Is called intracytoplasmic sperm injection (ICSI) and can be used to circumvent male in fertility (see following discussion]. With either approach, fertilized eggs are monitored to the eight-cell stage and then placed in the uterus to develop to term. The success rate of IVF depends upon maternal age. Approxim ately 30% of couples will conceive after one attempt if the woman is younger than 35 years of age. The rate drops to 25% for women aged 35 to 37, 17% for those aged 38 to 40, and to < 5 % for those over 40. In addition to these relatively low success rates, the technique Is aiso associated w ith a higher rate of birth defects. To increase chances of a successful pregnancy, four or five ova are collected, fertilized, and placed in the uterus. This approach som etim es leads to m últiple births. The frequency of multiple births depends on maternal age (with a higher incidence in younger women) and the number of embryos transferred. For a 20- to 29-year-old woman and three embryos transferred, the risk is 46% . Multiple births are a disadvantage because they have high rates of morbidity and mortailty.

Steps in monozygotic (identical) twinning

a. A secondary oocyte arrested in metaphase of meiosis II is fertilized by one sperm. The nuclear contents and centriole pair of the sperm enter the oocyte cytoplasm. b. The secondary oocyte completes meiosis II, forming the second polar body. The female and male pronuclei form. c. The female and male pronuclei fuse and the centriole pair provides the cytoplasmic machinery for cleavage cell divisions to occur. A zygote is formed. d. Cleavage divisions produce a cluster of blastomeres called a morula surrounded by a zona pellucida. The molecular mechanisms that establish twin embryo- genesis are active in the morula and are responsible for the latter "splitting" of the inner cell mass. In other words, twinning causes the "splitting," not vice versa. The twinning morula can travel two different routes leading to either monochorionic or dichorionic twins. e. If "splitting" occurs AFTER the differentiation of the trophoblast, then monochorionic twins will form. f. If "splitting" occurs BEFORE the differentiation of the trophoblast, then dicho- rionic twins will form.

Steps in dizygotic (fraternal) twinning

a. A secondary oocyte arrested in metaphase of meiosis II is fertilized by two sperm. The nuclear contents and centriole pair of both sperm enter the oocyte cytoplasm. b. The secondary oocyte completes meiosis II, but does not form a secondary polar body. Instead, the DNA that would have been sequestered in second polar body forms another female pronucleus. There are now two separate cellular entities within the zona pellucida each containing a female and male pronucleus. c. The female and male pronuclei fuse and the centriole pair provides the cyto- plasmic machinery for cleavage cell divisions to occur. Two zygotes are formed with two different genotypes. d. Cleavage divisions produce a cluster of blastomeres called a morula surrounded by a zona pellucida. The morula is a chimera consisting of an assortment of cells with two different genotypes. The molecular mechanisms that establish twin embryogenesis are active in the chimeric morula and are responsible for the latter "splitting" of the inner cell mass. In other words, twinning causes the "splitting," not vice versa. The twinning chimeric morula can travel two differ- ent routes leading to either monochorionic or dichorionic twins. e. If "splitting" occurs AFTER the differentiation of the trophoblast, then mono- chorionic twins will form. f. If "splitting" occurs BEFORE the differentiation of the trophoblast, then dicho- rionic twins will form.

Conjoined (Siamese) twinning

a. Occurs exactly like monozygotic twins except that there is incomplete "splitting" of the inner cell mass. The exact molecular mechanisms involved are not clear. b. All conjoined twins (except parasitic twins) are symmetrical (i.e., like parts are fused to like parts). c. The most common types of conjoined twins are: (1) thoraco-omphalopagus (fusion from upper chest to lower chest), (2) thoracopagus (fusion from upper chest to lower abdomen), (3) omphalopagus (fusion at lower chest), craniopagus (fusion of skulls), and parasitic twins (asymmetrically conjoined; one twin is small and dependent on the larger twin). d. Conjoined twins are monoamniotic (i.e., one amnion) and monochorionic (i.e., one chorion).

A cluster of blastomeres (16-32 blastomeres) forms a

morula

At midcycle, there is an LH surge that:

■ Flevates concentrations of maturation- promoting factor, causing oocytes to complete meiosis I and initiate meiosis II ■ Stimulates production of progesterone by follicular stromal cells (luteinization) ■ Causes follicular rupture and ovulation

The phases of fertilization include the following:

■ Phase 1, penetration of the corona radiata ■ Phase 2, penetration of the zona pellucida ■ Phase 3, fusion of the oocyte and sperm cell membranes

The main results of fertilization are as follows:

■ Restoratíon of the diploid number of chromosomes, half from the father and half from the mother. Henee, the zygote contains a new combination of chromosomes diíferent from both parents. ■ Determinatíon of the sex of the new individual. An X-carrying sperm produces a female (XX) embryo, and a Y-carrying sperm produces a male (XY) embryo. Therefore, the chromosomal sex of the embryo is determined at fertilization. ■ Initiation of cleavage. Without fertilization, the oocyte usually degenerates 24 hours after ovulation.

As a result of this estrogen production:

■ The uterine endometrium enters the follicular or proliferative phase. ■ Thinning of the cervical mucus occurs to allow passage of sperm. ■ The anterior lobe of the pituitary gland is stimulated to secrete LH.