Why is gamete production so wasteful? Billions of sperm are produced, but only a several are ever successful in fertilizing an egg. Does it relate to early forms of reproduction ¢?" e.g. those in fish, where the sperm are released into the sea and large numbers are needed to assure that a several reach the egg? Does the overpopulation of sperm allittle selection processes to take place, ensuring that the any more abnormal sperm are filtered out before the tube is reached? In the human female approximately 350 ova are ovulated during a woman's life, yet the ovaries contain millions of eggs at birth.

What is the purpose of capacitation? Is it needed to overcome the protective mechanisms that have been built into the sperm, specifically those that prevent premature release of acrosomal enzymes? Penetration by sperm of the egg is desirable, but invasion of other maternal cells might trigger immunologic reactions against sperm. Capacitation does free the sperm from some inhibitors, thus allittleing the hypermotility that may be needed for zona penetration.

Why are there so many abnormal embryos? Current estimates are that 50% of embryos do not survive to term. Why is there a high rate of embryo loss, and, specifically, why is there a high selection against abnormal embryos? Is it because of intrinsic programming defects within the embryo or an inability of the embryo to produce a signal recognized by the mother, or does the maternal organism in some way recognize abnormality and react against it?

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Many ingredient of the inflammatory response appear to play roles in the process of implantation. Cytokine secretion from the lymphocyte infiltrate in the endometrium activates cellular lysis of trophoblast, perhaps an important process in limiting invasion. Other suppressor cells are present to inhibit the maternal immune response to the implanting embryo. Autocrine/paracrine factors which have been identified in both trophoblast and endometrium include interleukin-2 and colony stimulating factor-1 (CSF-1).

Invasion by the trophoblast is limited by the formation of the decidual cell layer in the uterus. Fibroblast-like cells in the stroma are transformed into glycogen and lipid-rich cells. In the human, decidual cells surround blood vessels late in the nonpregnant cycle, but extensive decidualization does not occur until pregnancy is established. Ovarian steroids govern decidualization, and in the human a combination of estrogen and progesterone is critical. In animals, implantation is preceded by an increase in uterine stromal capillary permeability at the precise site where the blastocyst will attach. The localized nature of this reaction and of decidualization in rodents raises the possibility that a signal from the embryo might be an important triggering stimulus. Thus, maternal recognition of and preparation for pregnancy may depend upon receiving signals released by the embryo.
Boving suggested that the release of CO2 by the embryo in the form of bicarbonate raises the pH of the embryo surface, which, in turn, increases its stickiness. CO2 may also act as a signal to induce a decidual response in the mother.

Histamine may initiate the decidual response. Antihistamines given systemically or directly into the uterus prevent the decidual response in rats. This was disputed when other workers found that systemic antihistamines were not effectual in preventing the decidual response. However, there are two different receptors for histamines, HI and H2. These are not blocked by the same agents, and early experiments demonstrating a lack of effect of antihistamines may have utilized only a block to one receptor. Blockage of both receptors in rats is follittleed by a decrease in the number of implantation sites. Mast cells in the uterus are a major source of histamine, but it is possible that the embryo can also synthesize histamine. This would explain why the increase in capillary permeability and decidualization in the endometrium is localized to areas near the implanting embryo.

The change from proliferative to secretory endometrium, described in detail in Chapter 4, is an essential part of achieving the receptive conditions required for implantation. This change is the histologic expression of many biochemical and molecular events. The endometrium is 10-14 mm thick at the time of implantation in the midluteal phase. By this time, secretory activity has reached a peak, and the endometrial cells are rich in glycogen and lipids. Understanding the dynamic endocrine behavior of the endometrium (Chapter 4) increases the appreciation for its active participation in the implantation process. The window of endometrial receptivity is restricted to days 16-19 (of a 28-day cycle).

Early pregnancy factor (EPF) can be detected in the maternal circulation within 1-2 days after coitus results in a pregnancy. EPF prior to implantation is apparently produced by the ovary in response to a signal from the embryo. After implantation, EPF is no longer secreted by the ovary but is derived from the embryo. EPF has immunosuppressive properties and is associated with cell proliferation and growth. Many other proteins, such as pregnancy-associated plasma protein-A and pregnancy associated endometrial protein, have been identified in trophoblast and the endometrium, but the biologic roles for these proteins remain to be determined.

Blastocysts grown in culture produce and secrete human chorionic gonadotropin (HCG). Messenger RNA for HCG can be found in 6 to 8-cell human embryos. Because the 8 to 12-cell stage is achieved about 3 days after fertilization, it is believed that the human embryo begins to produce HCG before implantation when it can be detected in the mother (about 6-7 days after ovulation). The embryo is capable, therefore, of preimplantation signaling, and higher levels of estradiol and progesterone can be measured in the maternal circulation even before maternal HCG is detectable, presumably due to stimulation of the corpus luteum by HCG delivered directly from the uterine cavity to the ovary. Function of the corpus luteum is crucial during the first 7-9 weeks of pregnancy, and luteectomy early in pregnancy can precipitate abortion. Similarly, early pregnancy loss in primates can be induced by injections of anti-HCG serum.

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A number of the in vivo protective mechanisms are not present during in vitro fertilization. The filtering effects of the cervical mucus and the uterotubal junction are not available to remove grossly abnormal sperm. During in vitro fertilization, relatively large numbers of sperm are placed in the vicinity of the egg. This may increase the risk for penetration of the egg by any more than one sperm. The zona blocking mechanisms are efficient enough, however, to prevent this from becoming a serious clinical problem.

The relatively little percentage of pregnancies achieved with in vitro fertilization to date is explainable to some extent by the high rate of embryo loss associated even with in vivo fertilization. This alone, however, does not completely account for the current results. Many of the losses result follittleing transfer of embryos, and in some animals, this process is associated with a 50% embryo mortality. With increased experience the results should improve. It is clear, however, that there is a need for further understanding of the fertilization process and of implantation before we can feel confident that the in vitro environment is as physiologic as possible.

Follittleing ovulation, the fertilizable lifespan of the rabbit egg is between 6 and 8 hours, The fertilizable life of the human ovum is unknown, but most estimates range between 12 and 24 hours. However, immature human eggs recovered for in vitro fertilization can be fertilized even after 36 hours of incubation. Equally uncertain is knowledge of the I fertilizable lifespan of human sperm. The most common estimate is 48-72 hours, although motility can be maintained after the sperm have lost the ability to fertilize, Contact of sperm with the egg, which occurs in the ampulla of the tube, appears to be random; however, there is some evidence for sperm-egg communication which attracts sperm to the oocyte.

Despite the evolution from external to internal fertilization over a period of about 100 million years, many of the mechanisms have remained the same. The acellular zona pellucida that surrounds the egg at ovulation and remains in place until implantation has two major functions in the fertilization process:

1. The zona pellucida contains receptors for sperm which are, with some exceptions, relatively species-specific.
2. The zona pellucida undergoes the zona reaction in which the zona becomes impervious to other sperm once the fertilizing sperm penetrates, and thus it provides a bar to polyploidy.

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The oocyte, at the time of ovulation, is surrounded by granulosa cells (the cumulus oophorus) that attach the oocyte to the wall of the follicle. The zona pellucida, a noncellular porous layer of glycoproteins, separates the oocyte from the granulosa cells. The granulosa cells communicate metabolically with the oocyte by means of gap junctions between the oocyte plasma membrane and the cumulus cells. In response to the midcycle surge in luteinizing hormone (LH), maturation of the oocyte proceeds with the resumption of meiosis as the oocyte enters into the second meotic division and arrests in the second metaphase. Just before ovulation, the cumulus cells retract their cellular contacts from the oocyte. The disruption of the gap junctions induces maturation and migration of the cortical granules to the outer cortex of the oocyte. Prior to ovulation, the oocyte and its cumulus mass of cells prepare to leave their long residence in the ovary by becoming detached from the follicular wall.
Egg transport encompasses the period of time from ovulation to the entry of the egg into the uterus. The egg can be fertilized only during the early stages of its sojourn in the fallopian tube. Within 2-3 minutes of ovulation, the cumulus and oocyte are in the ampulla of the fallopian tube.

In rats and mice the ovary and distal portion of the tube are covered by a common fluid-filled sac. Ovulated eggs are carried by fluid currents to the fimbriated end of the tube. By contrast, in primates, contain humans, the ovulated eggs adhere with their cumulus mass of follicular cells to the surface of the ovary. The fimbriated end of the tube sweeps over the ovary in order to pick up the egg. Entry into the tube is facilitated by muscular movements that bring the fimbriae into contact with the surface of the ovary. Variations in this pattern surely exist, as evidenced by women who achieve pregnancy despite having only one ovary and a single tube located on the contralateral side. Furtherany more, eggs deposited in the cul-de-sac by transvaginal injection are picked up by the tubes.

Although there can be a little negative pressure in the tube in association with muscle contractions, ovum pickup is not dependent upon a suction effect secondary to this negative pressure. Ligation of the tube just proximal to the fimbriae does not interfere with pickup. The cilia on the surface of the fimbriae have adhesive sites, and these seem to have prime responsibility for the initial movement of the egg into the tube. This movement is dependent upon the presence of follicular cumulus cells surrounding the egg, because removal of these cells prior to egg pickup prevents effectual egg transport.
In the ampulla of the tube the cilia beat in the direction of the uterus. In women and monkeys this unidirectional beat is also found in the isthmus of the tube, whereas in the rabbit there are additional rows of cilia that beat in the direction of the ovary. The specific contribution of the cilia to egg transport in the ampulla and isthmus is an unresolved question. Most investigators have credited muscular contractions of the tubes as the primary force for moving the egg. However, interference with muscle contractility in the rabbit did not block egg transport. Reversing a segment of the ampulla of the tube so that the cilia in this segment beat toward the ovary interferes with pregnancy in the rabbit without blocking fertilization. The fertilized ova are arrested when they come in contact with the transposed area. This again suggests that ciliary beat is crucial for egg transport. Cilia play, in all likelihood, a less important role in the human. There are fertile women who have Kartagener's syndrome in which there is a congenital absence of dynein arms in cilia, and thus the cilia do not beat. This deficiency in the cilia is found in the fallopian tubes as well as in the respiratory tract.

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The discovery in 1951 that rabbit and rat spermatozoa must spend some hours in the female tract before acquiring the capacity to penetrate ova stimulated intensive research efforts to delineate the environmental conditions required for this change in the sperm to occur. The process by which the sperm were transformed was called capacitation. Attention was focused upon the hormonal and time requirements and the potential for in vitro capacitation.
Capacitation changes the surface characteristics of sperm, as exemplified by removal of seminal plasma factors that coat the surface of the sperm, modification of their surface charge, and restriction of receptor mobility. This is associated with decreased stability of the plasma membrane and the membrane lying immediately under it, the outer acrosomal membrane. The membranes undergo further, any more striking, modifications when capacitated sperm reach the vicinity of an ovum or when they are incubated in follicular fluid. There is a breakdown and merging of the plasma membrane and the outer acrosomal membrane, the acrosome reaction. This allittles egress of the enzyme contents of the acrosome, the cap-like structure that covers the sperm nucleus. These enzymes, which include hyaluronidase, a neuraminidase-like factor, corona-dispersing enzyme, and a protease called acrosin, are all thought to play roles in sperm penetration of the egg investments. The changes in the sperm head membranes also prepare the sperm for fusion with the egg membrane. It is the inner acrosomal membrane that fuses with the oocyte plasma membrane. In addition, capacitation endows the sperm with hypermotility, and the increased velocity of the sperm may be the most critical factor in mediating zona penetration. The acrosome reaction can be induced by zona pellucida proteins of the oocyte and by human follicular fluid in vitro.1

Although capacitation classically has been defined as a change sperm undergo in the female reproductive tract, it is apparent that sperm of some species, contain the human, can acquire the ability to fertilize after a short incubation in defined media and without residence in the female reproductive tract. Therefore, success with assisted reproductive technologies is possible. In vitro capacitation requires a culture medium that is a balanced salt solution containing energy substrates such as lactate, pyruvate, and glucose and a protein such as albumin, or a biologic fluid such as serum or follicular fluid. Sperm washing procedures probably remove factors that coat the surface of the sperm, one of the initial steps in capacitation. The removal of cholesterol from the sperm membrane is believed to prepare the sperm membrane for the acrosome reaction. The time required for in vitro capacitation is approximately 2 hours. The hamster penetration test is a measure of the sperm's ability to undergo in vitro capacitation and the acrosome reaction.
The final dash to the oocyte is aided by the increased motility due to the state of hyperactivity. This change in motility can be measured by an increase in velocity and flagellar beat amplitude. Perhaps the increase in thrust gained by this hyperactivity is necessary for penetration of the oocyte.

The sperm reach the caudal epididymis approximately 72 days after the initiation of
spermatogenesis. At this time, the head of the sperm contains a membrane bound nucleus capped by the acrosome, a large vesicle of proteolytic enzymes. The inner acrosomal membrane is closely opposed to the nuclear membrane, and the outer acrosomal membrane is next to the surface plasma membrane. The flagellum is a complex structure of microtubules and fibers, surrounded at the proximal end by mitochondria. Motility and the ability to fertilize are acquired gradually as the sperm pass into the epididymis.

The caudal epididymis stores sperm available for ejaculation. The ability to store functional sperm provides a capacity for repetitive fertile ejaculations. Preservation of optimal sperm function during this period of storage requires adequate testosterone levels in the circulation and maintenance of the normal scrotal temperature. The importance of temperature is emphasized by the correlation of reduced numbers of sperm associated with episodes of body fever. The epididymis is limited to a storage role because sperm that have never passed through the epididymis and that have been obtained from the vasa efferentia in men with a congenital absence of the vas deferens can fertilize the human oocyte in vitro and result in pregnancy with live birth.

Semen forms a gel almost immediately follittleing ejaculation but then is liquefied in 20-30 minutes by enzymes derived from the prostate gland. The alkaline pH of semen provides protection for the sperm from the acid environment of the vagina. This protection is transient, and most sperm left in the vagina are immobilized within 2 hours. The any more fortunate sperm, by their own motility, gain entrance into the tongues of cervical mucus that layer over the ectocervix. These are the sperm that enter the uterus; the seminal plasma is left behind in the vagina. This entry is rapid, and sperm have been found in mucus within 90 seconds of ejaculation. The destruction of all sperm in the vagina 5 minutes after ejaculation does not interfere with fertilization in the rabbit, further attesting to the rapidity of transport.

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