Infertile patients cannot afford to wait for treatment while their eggs get older.
Dr. Sherman Silber, Infertility Center of St. Louis, is offering free video consultations for patients who need to plan now for their treatment while stay-at-home orders are in place. He is talking to and evaluating patients in their home via to comply with social distancing measures.
Dr. Silber is discovering that patients actually prefer this method of telemedicine consultation over the conventional office visit. Patients have conveyed that “it is so much more convenient and less stressful” to have a free telemedicine personal consultation than to take a day off from work to travel to the doctor’s office and sit with other nervous patients in the waiting room.
The COVID-19 pandemic is thus changing much of the way we will do things in the future, and for the better. "Our patients are surprisingly much happier with this approach. Of course, at some point we need to perform hands on treatment. But with this new manner of seeing patients, we can come to the right diagnosis and treatment plan for most patients more efficiently, quickly, and painlessly, with no loss of personal one-on-one communication.” This is a very welcome new era of telemedicine that has been forced on us by the current difficult times.
Getting pregnant is not an easy task, but understanding the essential physiology of the process is the best place to start. In this chapter I will describe the arduous journey that sperm must make through the female genitals to reach the egg, as well as the simultaneous adventure of the egg during which it matures to become genetically ready for fertilization, erupts from the ovary, and gets grabbed by the fallopian tube, fertilized, and then hustled along into the womb at exactly the right moment to implant. Failure of the sperm or egg to make an important connection anywhere along this complicated itinerary will prevent pregnancy from occurring.
A Brief Review of Female Anatomy
The vagina is an elastic canal, about four to five inches long. At the end of this canal, in the deepest recess of the vagina, is a structure called the cervix, which is the entrance to the womb, or uterus. The uterus is a hard, muscular, pear-shaped structure with a narrow, triangular cavity inside, so small that it would barely hold a teaspoonful of fluid. Yet this is where the fertilized egg must implant itself and grow during the next nine months into a full-size baby. The uterus has a remarkable capacity to expand to allow room for the developing baby, pushing aside and squashing all the other organs of the mother’s abdomen.When the baby is ready to be born, the muscles of the uterus contract during labor to squeeze the baby out into the world.
Far back in the corners of the uterus, on each side, are microscopic canals through which the sperm must squeeze in order to reach the fallopian tube, where it may encounter an unfertilized egg. Once the egg has been fertilized, it will pass through the canal in the opposite direction to reach the uterus. These microscopic canals leading from the uterus into the fallopian tubes are only about one-seventieth of an inch in diameter (the size of a pinpoint). The fallopian tubes are four inches long and hang freely in the abdomen. They widen at the ends into large, flowerlike openings called fimbriae.
The ovaries are the organs that make the female’s eggs and sex hormones. They lie outside of the uterus and fallopian tubes.When an egg is extruded every month from the surface of one of the ovaries (ovulation), it is released freely into the abdominal cavity rather than directly into the tube. The fimbria then comes to life like an octopus tentacle and actively grasps the egg, pulling it into the fallopian tube. The tube swallows the egg, nourishes it before and during fertilization for three days, and then transports it into the uterus.
While the male produces billions of sperm every week, the female matures only one of her existing eggs for ovulation each month. The ovaries mature and release about four hundred such eggs during the course of a woman’s lifetime. Generally, the most fertile eggs are released earlier in life, and of her limited supply of four hundred thousand, about one thousand eggs will die inexorably every month. Thus with advancing years, though a woman may still be able to get pregnant, she is much less fertile than she was in her youth.
How Do the Egg and the Sperm Reach the Fallopian Tube?
The journey of the egg, or ovum, through the fallopian tube and finally into the uterus after fertilization is extraordinarily hazardous. The woman’s tube is not simply a passive channel through which the egg is transferred; many events must work in precise synchrony for successful pregnancy to occur.
There are, on the surface of the fimbria, microscopic hairs called cilia, which constantly beat in the direction of the uterus at a fantastically rapid speed and create a kind of conveyor-belt effect for moving the egg into the tube and toward the uterus. The cilia work this magic by digging into the sticky gel, called the cumulus oophorus, that surrounds the egg, and they transport this whole sticky, gooey mass. The egg itself is invisible to the naked eye, but the gel that envelops it is easily visible. If this sticky substance were not present, and the egg were placed bare upon the surface of the fimbria, the beating of the cilia would never move the egg along. The cilia are only able to dig in and transport the egg with this sticky, gooey material encasing it.
The process of grasping the egg and moving it into the interior of the tube requires only about fifteen to twenty seconds. Once the egg is safely within the tube, it is transported quickly toward the narrower region of the tube, the ampullary-isthmic junction, located two-thirds of the way toward the uterus. Here, the egg must wait for a successful sperm coming from the opposite direction to fight its way into the egg’s tough outer membrane, the zona pellucida, score a direct hit, and thereby establish pregnancy.While the egg is held in this location by the tight resistance of the narrow region of the tube, the much tinier sperm nonetheless must struggle through this area of resistance to arrive from the opposite direction. Once it is fertilized, the egg must be nourished for several days in the ampulla of the tube before it can be allowed to pass into the uterus. If it is transferred into the uterus too soon, it will not be ready to implant, and it will die. If the transfer of the egg into the uterus is delayed too long, a tubal, or ectopic, pregnancy will occur (the fertilized egg will implant in the tube rather than the womb). Once the egg has been allowed to develop in the tube for three or more days, the isthmus suddenly opens up and the early embryo passes quickly into the uterus. Because the journey of the egg from the ovary to the site of fertilization, its nourishment in the tube, and the precise synchrony of the continuation of its journey into the womb are so intricate, problems with this egg and embryo transport process are frequently responsible for female infertility.
If the egg is not penetrated by sperm soon after ovulation, it becomes overripe and dies. After the egg is released from the ovary, it is only capable of fertilization for about twelve, or possibly at most twenty-four, hours. The likelihood of intercourse taking place during such a specific interval in any month is rather slight. So nature must provide some mechanism for providing a continuous flow of healthy sperm to the site of fertilization. That way, if intercourse is perhaps one or two days off schedule, some sperm can still arrive at the site of fertilization at the right time. For this reason, complicated barriers to sperm transport are necessary.
The success of IVF demonstrates that if eggs can be recovered at precisely the right time, they can be fertilized in the laboratory with only a small number of sperm. Then the complicated barrier mechanisms provided by nature to allow a slow, continuing flow of a small number of sperm at any moment is not necessary and the large numbers of sperm normally required for fertilization through intercourse are not needed.
Ejaculation into the Vagina
Most of the spermatozoa in the ejaculate are contained in the very first portion of fluid that squirts out of the penis and enters the vagina. The remaining squirts usually contain very little sperm. Thus, at the first moment of ejaculation the female’s cervix (the opening leading into her uterus) is bathed by a high concentration of sperm. Within just a few minutes after ejaculation, sperm begin to invade a very thick fluid (called cervical mucus) that is pouring out of the cervix. The sperm must be able to invade the cervix via the cervical mucus by virtue of their own swimming ability. Nothing about the sexual act will help those sperm get into the cervix. They simply have to swim into the mucus on their own, and this requires a great deal of coordinated, cooperative activity on their part.
Ejaculation is a challenging moment for the sperm, as the vagina presents a very harsh, acidic environment, which would normally immobilize them quickly. The alkalinity of the semen (the fluid that contains the sperm), as well as the alkalinity of the cervical mucus, allows the sperm to survive in this difficult vaginal milieu.Any acidity at all quickly kills sperm.
Yet even the semen is a potentially dangerous milieu for the sperm; any sperm that remain in the semen for more than two hours are likely to deteriorate. In order to survive long enough to get to the egg and fertilize it, the sperm must gain rapid access to the cervical mucus. Any sperm that have not penetrated the cervical mucus within a half hour after orgasm will not be able to do so later on, because by then they will have lost their ability to swim into the more friendly environment of the cervix.
Spermatozoa can be seen invading the cervical mucus within seconds after ejaculation, but most will not make it. Of some 200 million sperm deposited into the vagina near the cervix in a typical ejaculation, only 100,000 ever get into the womb. Thus, over 99.9 percent of the sperm never have a chance of getting beyond the vagina.
Once the sperm enter the canal of the cervix, they are capable of fertilizing the egg for as long as forty-eight to seventy-two hours, though they may actually live for up to six days. Remember, since the egg is only fertilizable for about twelve hours after ovulation, it is important to have a continuing flow of sperm across the tube so that whenever the egg appears, there will be sperm available. In this sense, the canal of the cervix can be looked upon as a receptacle through which platoons of spermatozoa migrate and in which some are detained in order to ensure a continuous supply of smaller numbers, over a prolonged period of time, to the deeper recesses of the female where fertilization takes place. Of course, these delaying mechanisms can do more harm than good in infertile couples if events do not allow the invasion of sperm to be mounted successfully.
To understand how this invasion of sperm gets launched effectively, we must first understand the remarkable liquid that covers the opening of the womb-the cervical mucus. The cervical mucus presents a very effective barrier to bacteria and thus protects the womb against infection. It is a selective filter, which favors normally active sperm and excludes other objects (including poor-quality sperm) from access. But it doesn’t even permit access to normal sperm except during a specific period at midcycle when ovulation is imminent and fertilization is possible. Cervical mucus resembles a thick, clear liquid that can be poured from one container into another. However, in a technical sense, it is not a liquid. As it is being poured, it can actually be cut with scissors; therefore, although it seems to behave as a thick liquid, it also has the characteristics of a very pliable, transparent plastic.
Cervical mucus is absent or very scanty during most of the monthly cycle, gradually becoming more abundant around the middle of the cycle, under the influence of increasing estrogen levels,when ovulation is about to occur. Just prior to ovulation it becomes almost optically clear, although it is translucent at other times. At the moment when fertilization is possible, near the time of ovulation, the mucus can be stretched out into a very thin strand; at other times in the cycle it is more sticky, and if stretched it will break. All of these changes in the cervical mucus, which occur around the time prior to ovulation, are designed to help sperm gain access to the uterus. The more liquidlike character, the greater transparency, and the greater stretchability (called Spinnbarkheit) are all characteristics that favor the successful invasion of an army of sperm. When the mucus is sticky and thick, not as abundant, and translucent rather than transparent, it is difficult if not impossible for any sperm to gain access.
Microscopically, the cervical mucus consists of a dense mesh that, during most of the monthly cycle, represents a solid barrier to invasion. Just prior to ovulation, under the effect of the female hormone estrogen, mucus production rises tenfold, and the water content of the mucus increases. The otherwise impenetrable mesh opens up and allows a successful invasion of sperm.When semen first reaches the cervical mucus after ejaculation, a clear barrier line can be seen separating the two different fluids. Semen does not “mix”with cervical mucus. Soon, however, phalanges of sperm begin to penetrate the mucus, forming branching structures that invade it.
Observing the sperm’s penetration of the cervical mucus under the microscope is an exciting event. Sperm at first seem to bounce against the cervical mucus without any evidence that they will ever be able to gain access. Their movements while in the ejaculate are haphazard and not specifically aimed toward the mucus. However, within a matter of minutes, one or two spermatozoa begin to make an indentation in the line separating the cervical mucus from the ejaculate. Once one sperm has been able to initiate the penetration of the mucus, other sperm then quickly follow at that same point of entry. Sperm then continue to invade the cervical mucus at that point much like a single-file line of army ants. Only one or two spermatozoa at a time can pass through this entrance.
The sperm swim in a straightforward direction along parallel rows of the invisible microscopic molecular structure of the mucus. Once this invasion of the cervical mucus has been established, sperm can reach the fallopian tubes in about thirty minutes.
Pregnancy would not be likely if all the sperm got into the fallopian tubes at one time, because they would soon pass on into the abdominal cavity, and not be available to fertilize the egg except during a very brief, lucky interval. If they were not lucky enough to pass through the fallopian tube at exactly the moment of ovulation (or within twelve hours of ovulation), they would be long gone by the time the egg arrived. Thus, nature had to invent some mechanism for allowing a continuous entry to the site of fertilization by a smaller number of sperm. To accomplish this, the cervix and the cervical mucus act as a reservoir from which spermatozoa are slowly released into the uterus and up to the fallopian tubes over a period of several days.
Capacitation of Sperm
During the course of their odyssey toward the site of fertilization, the sperm undergo capacitation, a process that was not fully understood before the advent of IVF. It used to be thought that unless sperm resided for a certain period of time outside the male reproductive tract and in the specific fluids of the female reproductive tract, they would not be capable of fertilization, even though in every other respect they looked normal. It was thought that this process of capacitation could occur only in the fluids of the female reproductive tract while the sperm migrated toward the egg. However, in vitro fertilization has demonstrated that capacitation of sperm (once considered one of the greatest problems in successfully achieving test-tube babies) can occur in relatively simple, nonspecific fluids available in any laboratory.
All that is necessary to start the capacitation process going is to remove the sperm from the semen by “washing” it. Removing the sperm from semen and placing them in any laboratory “culture media” fluid results in a dramatic tripling of their swimming velocity, so that even though they are mere human sperm, they begin to swim more like the sperm of horses or bulls. Thus, sperm seem to have a natural tendency toward developing capacitation for fertilization on their own and simply require a period of several hours outside the semen. In nature this happens when they leave the semen and enter the cervical mucus. In the IVF laboratory it happens when sperm are separated from the semen by virtually any washing technique.
Before egg and sperm can ever meet up in the fallopian tube, the egg must be matured and extruded from the ovary in a process called ovulation. Since many women who seem unable to have children owe their problems to a disturbance in ovulation, and since part of the IVF procedure involves stimulating the ovaries to prepare many eggs for fertilization, we should understand how the repeatable, monthly series of changes leading to ovulation occurs naturally in the ovary. Later we will unravel the hormonal events of the menstrual cycle, which regulate the clocklike orderliness of ovulation.
All of the hormonal events taking place during the month between menstrual periods are directed at preparing the egg to be genetically ready for fertilization, and preparing the uterus (womb) and the cervix for the moment of ovulation, so that the sperm and the egg have the best opportunity for joining up to form an embryo, which can then implant in a properly prepared uterus and result in successful pregnancy.
Formation of the Follicle
Each month, from the time of sexual maturity on, about one thousand undeveloped eggs, or oocytes, leave their prolonged resting phase and start to mature. This initiation of development is a continuous process, in marked contrast to ovulation, which occurs only once a month. Once the egg starts to develop, it proceeds inexorably and no longer has the choice of returning to being quiescent. It either wins the race to ovulate or must degenerate and die.
The most striking feature of the egg’s development is the growth of its surrounding fluid-filled compartment, called the follicle. The growth of this follicle is stimulated by the hormone FSH (follicle-stimulating hormone), which is produced by the pituitary gland in the early phase of the monthly cycle. The time required for the egg to develop the proper follicle necessary for ovulation is about fourteen days.Although the FSH stimulates all of the developing eggs during the month to form follicles, one of the eggs always gets a head start over the others, and once it obtains that lead it never relinquishes it. The other eggs developing that month then degenerate. However, if large doses of FSH were to be given to a woman at the beginning of the cycle, far in excess of what her pituitary would normally secrete, she would develop many follicles instead of just one.
The mature follicle is a spherical, bubblelike structure that bulges up from the surface of the ovary, and contains the egg. The egg (which is only 1/200 of an inch in diameter) is surrounded and protected by a mass of sticky, gelatinous fluid called the cumulus oophorus and hangs on a stalk attached to the inside of the follicle wall. The rest of the fluid in the follicle is clear yellow, and the follicle itself is fairly large (four- fifths of an inch in diameter). Occasionally two follicles successfully reach maturity and are both ovulated. In that circumstance the woman may have fraternal, or nonidentical, twins. Indeed, the drugs used to stimulate ovulation in women who would not otherwise ovulate usually cause the development of more than one follicle. Therefore, multiple births are common in women who require medical treatment to help them ovulate.
Two or three days prior to midcycle, when the follicle has reached its maximum size (usually two centimeters, or four-fifths of an inch), it produces an enormous amount of the hormone estrogen. This increased level of estrogen before ovulation stimulates the cervix to make more (and clearer) cervical mucus in order to allow sperm invasion. This dramatic increase in estrogen production by the follicle then stimulates the pituitary gland to release another hormone, different from FSH, called LH (luteinizing hormone). The sudden release of LH is what triggers ovulation (see fig. 1.6). The increase in estrogen indicates to the pituitary that the follicle is ripe, and this beautifully times the release of the LH hormone. Ovulation then occurs normally thirtyeight to forty-eight hours after the beginning of this LH surge.
Release of the Egg
Under the influence of the midcycle LH surge, the wall of the follicle weakens and deteriorates, and a specific site on its surface ruptures. The contents of the bulging follicle are then extruded from the surface of the ovary through this ruptured area. Observed under a microscope, ovulation appears similar to the eruption of a volcano.Occasionally women actually feel several hours of discomfort in their lower abdomen during ovulation; this discomfort is called Mittelschmerz. In women who require hormone treatment to stimulate ovulation, so many follicles may grow so large that when ovulation occurs it causes strong cramps, and a woman may even become sick enough to require several days of rest in the hospital. However, this sort of complication is not very likely with modern dosage monitoring. It is mentioned only to underscore what a dramatic intra-abdominal event ovulation is.
Production of Progesterone
The ruptured, empty follicle then undergoes another dramatic change, called luteinization. Luteinization is the process by which the follicle becomes able to make progesterone in addition to estrogen. Prior to ovulation, the follicle could produce only estrogen; after ovulation it can produce the other female hormone, progesterone, as well. Because it is impossible for the follicle to make progesterone before ovulation, the production of progesterone implies that ovulation has occurred. In the past, the presence of progesterone used to be the basis for all clinical methods of evaluating ovulation. The production of progesterone by the transformed follicle after ovulation is necessary for the successful implantation of the embryo in the womb during the second two weeks of the cycle.
The cystlike structure that forms monthly from the ruptured follicle is called the corpus luteum. This is Latin for “yellow body” and simply signifies that the follicle turns yellow as it changes its identity.As soon as the ruptured follicle begins to produce progesterone, the cervical mucus (which had become maximally receptive to sperm invasion just prior to ovulation) is suddenly caused to become sticky and totally impermeable to the invasion of sperm. In addition, progesterone causes the entrance of the cervix to close dramatically, even though just prior to ovulation it had been gaping in readiness for the entry of sperm. In the first half of the cycle, before ovulation, estrogen stimulates the buildup of a thick, hard layer of tissue called the endometrium to line the uterus, but this lining does not become receptive to the fertilized egg until after ovulation, when the secretion of progesterone causes it to soften. If the uterine lining is not softened by progesterone after ovulation (i.e., transformed from proliferative to secretory), implantation of the embryo cannot occur.
The corpus luteum manufactures this progesterone over a very limited time. If no pregnancy develops, the corpus luteum ceases to produce progesterone by ten to fourteen days after ovulation, and subsequently disappears.With this cessation of progesterone production by the ovary, the soft lining that was built up in the womb to prepare for the nourishment of the fertilized egg is shed and the woman menstruates. The decrease in progesterone (and estrogen) levels during menstruation then stimulates a renewed increase in FSH. A new follicle then develops, estrogen production resumes, and the cycle begins again.
A Review of the Hormones That Control Ovulation and the Menstrual Cycle
The reproductive cycle that animals go through is called the estrous cycle. Only humans and the apes have menstrual cycles. In a menstrual cycle the buildup of the lining of the womb is so lush, and the drop in hormone level supporting that lining so abrupt, that at the end of the cycle the lining actually sheds and the woman bleeds for four to five days in what is commonly known as her period. In all other animals, however, this shedding does not occur, and the thick lining of the womb merely returns to the thinned-out condition, marking the beginning of the next cycle. Furthermore, when animals are about to ovulate in their estrous cycle, they go into heat, or “estrus,”and know it is time to copulate. Since most woman are unaware of when they ovulate, they must try to understand the events of their menstrual cycle more fully, because unlike other animals,we do not automatically copulate at the right time. We will arbitrarily call the first day of the menstrual cycle “day one,” which is the day that bleeding commences. Bleeding usually ceases by day four or five and in most cases resumes after day twenty-eight of the cycle. Although the first day of menstruation represents a shedding of the lining of the uterus (womb) from the previous month’s cycle, it is actually the beginning (day one) of the next cycle.
On the first day of menstruation the pituitary hormone FSH is already stimulating development of a follicle that will take precedence over all other follicles that month. Interestingly, FSH,which in females causes the follicle to develop, is the exact same hormone that in males helps to stimulate sperm production. Estrogen from the developing ovarian follicle then inhibits further pituitary production of FSH. This is a “negative feedback”mechanism whereby the very estrogen that FSH causes to be produced by the ovary inhibits the pituitary from making more FSH.
By day twelve to fourteen of the menstrual cycle, the follicle appears on the surface of the ovary as a fluid-filled bubble ready to burst. In the meantime, the estrogen that has been produced by the follicle during this first half of the cycle has stimulated the uterus to prepare a thick “proliferative” lining. This thick, proliferative uterine lining is not ready to receive the egg until it is “softened” by progesterone in the second half of the cycle.
The final effect of estrogen (in high quantities at midcycle) is to trigger the release of a different pituitary hormone, LH. This enormous surge of LH from the pituitary is what causes the follicle to burst and then ovulate. But LH does more than simply cause ovulation (release of the egg from the ovary). LH triggers the chromosomes of the egg to separate and thereby prepares the egg genetically for fertilization.
How a Primitive Region of the Brain Called the Hypothalamus Controls the Menstrual Cycle
The entire cycle of follicle development, ovulation, and menstruation depends upon the precisely timed release of FSH and LH from the pituitary gland. In the male, FSH and LH production is constant, and therefore, sperm and hormone production are constant. In the female, there is a delicately synchronized increase in FSH at the beginning of the cycle to promote follicle growth, an LH surge at midcycle to promote ovulation, and then a gradual drop in pituitary hormones that causes a drop in estrogen and progesterone production by the ovary, resulting in menstruation.
We know that the release of FSH and LH from the pituitary is controlled by a hormone called GnRH (gonadotropin-releasing hormone), which originates in a primitive region of the brain called the hypothalamus.
The hypothalamus sits right at the base of the brain and above the pituitary gland, and causes the pituitary to release FSH and LH by sending the hormone GnRH directly to it. It used to be thought that the brains of males and females were different in this regard (and indeed they are in most other animals).We now know that this area of the brain in humans functions identically in the male and female, and that it is the ovary that directs the cyclical production of FSH and LH in the female pituitary.
By releasing GnRH, the hypothalamus is simply permissive in allowing the pituitary to stimulate the ovary in the female and the testicle in the male. The brain secretes small pulses (lasting only a minute or so) of the hormone GnRH about every ninety minutes in both men and women. It is the periodic, never-ending release of GnRH from the brain that causes the pituitary gland to start secreting FSH and LH, bringing on puberty, including menstruation in girls.
In men with deficient sperm or testosterone production, the FSH and LH levels are higher because the pituitary is overworking in an effort to compensate. The same phenomenon occurs in women. We know that when the ovary runs out of eggs, and women can no longer produce estrogen (thereby going into menopause), the FSH and LH levels from the woman’s pituitary go sky-high in an effort to stimulate what little ovarian reserve may still exist.
To understand estrogen’s role in controlling the pituitary hormones, we must look at what happens at midcycle in the woman. The surge in estrogen at midcycle causes the pituitary to suddenly release a high amount of LH (along with some extra FSH), and this stimulates ovulation. The cyclic pattern of hormone production in the female, which is quite different from the constant pattern of hormone production in the male, is not caused by any difference in the female brain’s release of GnRH. If the hypothalamus of any human being were destroyed (male or female), there would be no further GnRH secretion, the pituitary would cease to make FSH and LH, and the ovaries or testicles would shrivel up and completely stop functioning.
Clinical Importance of GnRH Release from the Brain for IVF
Why is the fascinating relationship of a primitive region of the brain to the pituitary, the ovaries, and the testicles so important? It bears very heavily on how we can obtain the best-quality eggs from the female for IVF.When the ovaries are stimulated to make more eggs by administering FSH (a necessary step in the in vitro fertilization process), the tremendous increase in estrogen production over a normal level can cause an early increase in LH secretion. This may result in premature ovulation with complete loss of the eggs or, at best, may hurt the subsequent pregnancy rate resulting from those eggs. In order to prevent this premature LH increase, we need to have a better understanding of GnRH, the hormone from the brain that allows the pituitary to release FSH and LH.
If GnRH were released constantly rather than at pulsatile intervals of ninety minutes, a peculiar reverse phenomenon would take place. The pituitary, rather than being stimulated to release FSH and LH, would become completely paralyzed after two to five days and would no longer secrete any FSH or LH until the constant release of GnRH was stopped and regular pulsatile ninety-minute secretion was resumed. Thus, we can completely turn off the pituitary whenever we want to by simply giving a constant rather than intermittent dose of GnRH. It’s as though the pituitary needs a ninety-minute rest before each new GnRH stimulus in order to function properly. If the pituitary doesn’t get this ninetyminute rest, it behaves just as though there were no GnRH at all. This process is called down regulation.
GnRH is chemically a very simple hormone called a polypeptide, which can be easily synthesized by drug companies.When a small modification is made in the structure of the GnRH, we have what is known as a GnRH agonist,which, if injected just one time a day, stays around in the bloodstream at a constant level rather than being immediately destroyed within minutes, as the brain’s normal GnRH would be. Thus, giving an injection of GnRH agonist once a day creates the same effect as infusing a constant level of GnRH all day long and giving the pituitary no rest.When you give the pituitary no rest, at first it pours out a lot of FSH and LH, but then several days later, the depleted pituitary can no longer release LH or FSH.
There are several GnRH agonists on the market, Lupron (leuprolide) being popular in the United States, and Suprefact (buserelin) being a popular one in Europe. Using Lupron along with a stimulation cycle completely turns off the pituitary and prevents a premature LH surge that would interfere with the proper development of the large number of eggs necessary for IVF.
A different variation of GnRH analogue is the GnRH antagonist, e.g., Cetrotide or Antagon. Instead of depleting the pituitary of FSH and LH, as Lupron does, GnRH antagonists work by directly and immediately blocking the pituitary’s release of FSH and LH by preventing GnRH from having any stimulating effect on the pituitary.
How Do Hormones Genetically Prepare the Egg for Fertilization?
Incredible genetic cellular changes take place in a woman’s developing eggs each month, beginning with the elevation of FSH at the start of menstruation. Very complex events are taking place in the egg during this monthly development and growth of the follicle. Furthermore, the release of LH stimulated by the estrogen surge at midcycle does much more than just cause ovulation. It finalizes the critical genetic preparation of the egg, without which fertilization would be impossible.
Thus far, only a superficial description of what happens during a menstrual cycle has been given: (1) follicular growth and estrogen production in the first half, (2) ovulation at midcycle, hopefully with fertilization, and (3) preparation of the uterine lining for embryo implantation in the second half of the cycle, stimulated by the production of progesterone from the corpus luteum (newly formed from the ovulated follicle). But these events are only the outward signs of an intricate genetic preparation for fertilization.
Reduction Division (Meiosis) of the Egg’s Chromosomes
Every cell in the body has forty-six chromosomes consisting of twenty-three pairs, which carry all of our genes. However, the sperm and the egg at the moment of fertilization must each have only twentythree single chromosomes, not forty-six, so that when the sperm and the egg unite, the fertilized egg has the normal number of chromosomes.
Like every other cell in the body, sperm precursors in the testicle have forty-six chromosomes. But in the process of sperm production, the chromosomes are reduced to half the normal number by a process called meiosis. So when sperm leave the testicle, they have only twentythree chromosomes. The eggs also have forty-six chromosomes until the very moment the sperm penetrates an egg and initiates fertilization. Fertilization cannot possibly occur unless the egg’s forty-six chromosomes can be reduced to twenty-three. The moment the sperm penetrates the egg, half of the egg’s chromosomes must be extruded. Then two half sets of chromosomes, one from the male, and one from the female, merge into a new individual with the normal number of fortysix chromosomes. Without the hormonal stimulation of FSH causing follicle development, followed by the release of LH at midcycle, the eggs would not be genetically prepared for this complex event of meiosis to occur.
The miracle of this separation of chromosomes is the most complicated event in the whole reproductive process; it determines the genetic makeup of the child and results in the genetic variability of the offspring.
Development of the Egg During Growth of the Follicle
At the time of a woman’s birth, all of her eggs are fixed in the beginning phase of the first meiotic division. The remaining stages of the meiotic division will not begin until years later, when her egg has finally matured in a developing follicle and the LH surge at midcycle causes the egg to resume meiosis. This resumption of meiosis, triggered by LH, would not occur without the prior preparation by FSH (meiotic competence) during the first two weeks of the cycle.
At the beginning of the cycle, from day one of menstruation, increased FSH production from the pituitary stimulates rapid growth in the egg. The egg will grow during this early follicular phase from a tiny 30 microns to its normal mature size of 140 microns (from 1/1,000 of an inch to approximately 1/200 of an inch in diameter). At this time, the very tough outer membrane, the zona pellucida, forms around the enlarging egg. Next, the follicle expands to form a fluid-filled cavity around the egg. The tiny forming follicle is visible on ultrasound at this point.
When the follicle forms, many compact layers of granulosa cells begin to surround the now enlarged egg, and the outer sheath of these granulosa cells produces the hormone estrogen. Even a brief deprivation of estrogen to the maturing egg during this stage will result in the egg’s immediate death. If FSH stimulation were to suddenly cease or be reduced dramatically, estrogen production by the granulosa cells would decline and the egg would die.
The egg remains embedded on one side of the follicle in a mound called the cumulus oophorus. The cells around the egg remain compact until the egg is ready for fertilization.When LH triggers the important genetic events that will allow fertilization after ovulation, these cells spread out in a radial pattern, giving a sunburstlike appearance referred to as corona radiata. If this widely dispersed appearance of cumulus cells surrounding the zona pellucida of the egg is present, physicians performing in vitro fertilization know that the egg is adequately mature for fertilization to occur. It is the most easily observable sign that the egg has gone through enough FSH stimulation to be ready for the genetic events of meiosis, which will ultimately lead to the possibility of fertilization.
It is quite astounding that there is little difference in the maximum diameter of the egg of almost any species, even though the size of the follicle containing the egg is generally related to the size of the animal. Thus, eggs of a whale could easily pass through the oviduct of the smallest mammal, like a rat, even though the whale’s follicle containing that small egg could easily be as large as a whole rabbit. The increasing size of the follicle has nothing to do with any increase in the size of the egg but is merely an indication that the egg is being properly prepared for what it has to do when it receives the surge of LH at midcycle.
Resumption of Meiosis After the LH Surge
LH begins the resumption of meiosis, but the penetration of the egg by a sperm is what causes the completion of that process. After the LH surge, the first meiotic division occurs, but this division does not reduce the number of chromosomes. This is an equal division in which fortysix chromosomes are still left within the egg nucleus. Actually, it is more complex than this, and I will explain it in detail in chapter 12. The “first polar body” is a small, divided nucleus that is pinched off from the main body of the egg prior to ovulation, about thirty hours after the LH surge. The extrusion of the first polar body from the egg shows that the first meiotic division has occurred under the influence of LH, meaning that the egg is now prepared to undergo the all-important second meiotic division.Many college biology students get confused by these two stages of meiosis. In the first division, all the chromosomes partly divide but do not split completely. In the second division, they actually complete the split. The egg is thus prepared during meiosis for the entrance of a sperm.
Penetration of the Egg by a Sperm
For a sperm to enter and fertilize the egg, it must dig its way through several layers of protective shields surrounding the egg. These outer walls safeguarding the inner confines of the egg represent an impressive barrier to sperm penetration, and a sperm cannot dig its way through these membranes without the aid of chemicals released from its warhead, the acrosome. The acrosome surrounds the front portion of the sperm and acts much like a battering ram. Chemicals released by the acrosome first dissolve the jellylike cumulus oophorus, enabling the sperm to pass through it and reach the tough zona pellucida. This very tough membrane, like the shell of a chicken egg, represents perhaps the most formidable obstacle to sperm. To penetrate this barrier, the sperm cannot just haphazardly liberate chemicals, or the egg might be damaged. The attacking chemicals must remain closely bound to the surface of the sperm and thereby cut an extraordinarily narrow slit into the membrane.
In order for the sperm to make its way through the sturdy zona pellucida, a process called the acrosome reaction is necessary. The acrosome is attached around the front two-thirds of the sperm head, where it is positioned much like an arrowhead. Its contents are tightly contained because premature leakage of acrosin (the dissolving chemical) would make it impossible for the sperm head to drill its way through the zona pellucida when it finally makes contact. Contact with the zona pellucida stimulates the acrosome to undergo its reaction, during which holes form in the inner and outer acrosomal membranes and acrosin is released, helping the sperm break through the zona pellucida.
Once a lucky sperm makes contact with the zona pellucida (which is purely a random event), it takes a minimum of fifteen minutes before penetration can begin. Some sperm can be seen struggling for as long as an hour before they make their initial penetration. If penetration hasn’t occurred within an hour, however, something is wrong and the egg probably won’t be fertilized. Sperm enter the zona pellucida at an angle almost exactly perpendicular to the surface of the egg and appear to develop a channel within the zona as they move forward. Despite the important “drilling” effect achieved by the release of acrosin from the outer acrosomal membrane of the sperm head, it is very clear that without the vigorous, hyperactive beating of the sperm tail providing strong mechanical propulsive force, the sperm still would not be able to get in.
Once penetration of the zona has begun, it requires an average of twenty minutes for the sperm to get completely through; once the sperm has broken through, it plunges directly into the egg membrane itself in less than a second. At that moment, the sperm tail immediately becomes paralyzed. Otherwise the thrashing of the sperm within the egg itself would kill the egg. Very soon after the sperm head becomes embedded in the egg, its tightly packed DNA begins to decondense (spread out a little), and the genetic material of the male becomes the male pronucleus.
Completion of Meiosis and Union of the Male and Female Genes
Once the first sperm has successfully invaded the zona pellucida of the egg, a remarkable event takes place. The membrane that surrounds the egg within the zona fuses with the membrane of the sperm, and the sperm and the egg become one. The egg literally swallows the sperm as these two microscopic entities initiate the development of a new human being. Also at this moment the outer zona pellucida becomes transformed into a rigid barrier so impenetrable that other sperm, despite all the chemicals in their acrosomes, cannot possibly enter. Many sperm can be seen attempting to enter the egg in competition with the one that made it first, but their efforts are in vain. Once the egg has been successfully penetrated by a single sperm it shuts its walls so tightly that none of the followers can get through. This protects the fertilized egg from the entrance of extra chromosomes (called polyploidy), which would cause a genetically impossible fetus, and a miscarriage.
Penetration of the egg membrane by the sperm head also sets in motion the second meiotic division of the egg with the release of the second polar body. It is this second meiotic division that reduces the number of the egg’s chromosomes to half so that sperm and egg genes can unite. When the sperm head enters the egg, its chromosomes are tightly and densely packed. After fertilization, the sperm head, with its twenty-three chromosomes, expands (decondenses) into what is called the male pronucleus. At the same time, the female nucleus (which is sitting on the opposite side of the egg) is triggered to undergo its second meiotic division shortly after sperm penetration and become the female pronucleus. This second meiotic division causes extrusion of half the egg’s chromosomes to the second polar body, leaving the female pronucleus with twenty-three, just like the sperm. Within eleven to eighteen hours the male and female pronuclei sitting on opposite sides of the egg appear extremely prominent and get ready to converge.
This is truly an amazing event. The two pronuclei (each with twentythree chromosomes) slowly and majestically move toward the center of the egg and join into one nucleus, which now has forty-six chromosomes and represents an entirely new human being. This merging of the male and female pronuclei is called syngamy. After syngamy, the fertilized egg is ready to divide. Division of the fertilized egg is called cleavage.
Early Development of the Fertilized Egg
Over the next three days the fertilized egg first divides (cleaves) into two, then four, then eight cells. The first cleavage into two cells occurs sometime before thirty-eight hours after penetration by the sperm. The second cleavage (four cells) begins sometime between thirty-eight and forty-six hours after fertilization. The third cleavage (eight cells) begins between fifty-one and sixty-two hours after fertilization. If any one of those cells were to be removed, the remaining ones would still continue to develop into a normal baby. That is, each cell is still totipotent, and the remaining cells could develop into a completely normal human being. Each one of these early cells formed by the first three or four divisions of the fertilized egg is called a blastomere.
Finally, by the fourth day, the embryo has 64 to 160 cells and is called a morula. These cells have now “compacted” and are no longer totipotent. It’s at this stage that the embryo is passed from the fallopian tube into the uterus. By the fifth or sixth day after fertilization, there are so many cells still packed into the same hard, tough zona pellucida that individual cells can no longer be recognized. At this stage the embryo is called a blastocyst. On the sixth or seventh day after fertilization this blastocyst thins out a spot in the otherwise hardened shell of the zona pellucida and actually “hatches,” just like a chicken hatches from its shell in an incubator. The blastocyst pushes its way out of this thinned-out crack in the zona pellucida and prepares for implantation (by the seventh day) into the wall of the uterus, or womb. Up until now the zona has protected the embryo. But as a blastocyst, the embryo is now ready for its most treacherous moment when it has to attach to the endometrial lining of the womb.When the blastocyst attaches successfully to the endometrium, that initiates pregnancy.
If a pregnancy has been achieved, seven days after fertilization, the embryo begins to secrete the hormone HCG (human chorionic gonadotropin), and this HCG stimulates the ovary to continue to produce progesterone and estrogen, which are necessary for the maintenance of the lining of the womb. Without continued production of progesterone, the pregnancy could not survive. The embryo begins to make HCG when the pregnancy is first established in the uterus, about seven days after ovulation. After three months the fetus, or rather the fetal placenta, actually makes its own progesterone, and the ovaries are no longer needed for production of hormones. After nine months, the baby is ready to be pushed out of the uterus by the mother during labor.
The presence of HCG only signifies that the embryo has implanted and is the basis for almost all of the routine pregnancy tests. Blood tests for pregnancy really just check for the presence of HCG. If it is present, then the pregnancy test is positive. Pregnancy can even be diagnosed with a simple urine test that the woman can perform herself within fourteen days of egg fertilization. However, the laboratory blood test is more reliable. If it is positive, i.e., there is more than twenty-five units of HCG, it should be repeated two days later to see if the HCG level has increased. Normally the HCG doubles every two days for the first month of pregnancy and reaches astronomic levels. If the HCG does not increase, a miscarriage is very likely, and the pregnancy is referred to as a chemical pregnancy.
If the HCG level goes up as it should, then an ultrasound five weeks after fertilization (defined as a seven-week gestational-age pregnancy) should show a normal fetal heartbeat. If there is no fetal heartbeat by seven weeks’ gestation, the pregnancy is not viable and miscarriage will follow. Thus, a positive pregnancy test alone does not ensure that the pregnancy is viable. For that you must have an ultrasound exam.
When you have a positive pregnancy test (which just means an elevated HCG level), the chances are 85 percent that you will have a favorable ultrasound at seven weeks and deliver a healthy baby. But miscarriage occurs commonly in early pregnancy despite an elevated HCG level. Because of the biological clock, miscarriage is more common in older women than in younger women.