Peter W Nathanielsz. American Scientist. Volume 84, Issue 6. Nov/Dec 1996.
In the fifth century B.C., the Greek philosopher, physician and scientist Hippocrates suggested that a baby decides the timing of its own birth: “When the child has grown big and the mother can no longer support him with food, he struggles and breaks forth into the world, free from all bonds.” Hippocrates believed that the signal that begins the process is a failure of the placenta to keep up with the increasing nutritional needs of the fetus.
More than 2,300 years later, in the 1930s, Sir Joseph Barcroft at Cambridge University in England was the first to attempt direct experiments with pregnant sheep to assess various functions of the fetus while the fetal lamb was still in the uterus. Sir Joseph developed techniques for placing small catheters in blood vessels in the fetal lamb and the pregnant ewe under anesthesia. With these catheters in place, he could take samples simultaneously from both the maternal and fetal blood and measure the amounts of oxygen and other molecules that these blood samples contained. As a result of his studies Barcroft proposed that as the fetus grows, fetal needs for oxygen gradually exceed the ability of the mother to provide enough oxygen through the placenta. He viewed the normal birth process as a race between the danger of the fetus dying while still in the uterus because of lack of oxygen, on the one hand, and on the other hand being born before the oxygen supply gave up.
The ideas of Hippocrates two millennia ago and those of Barcroft 50 years ago were similar in suggesting that fetal awareness of failing supplies of life-supporting substances acted as a trigger to stimulate the fetus to actively initiate the mechanisms that would lead to delivery. Hippocrates believed that the fetus can sense when nutrient supply is getting low. Barcroft considered that a lack of oxygen is the critical condition monitored by the fetus.
These views introduce the concept of the fetal brain as a decision-making computer, analyzing input and acting accordingly. However, the ideas put forward by both Hippocrates and Barcroft had three major deficiencies. First, neither of them had conclusive experimental evidence to prove that the fetus runs short of oxygen, nutrients or any other factor at the end of pregnancy. Hippocrates did no experimental studies, and Barcroft lacked the ability we now possess to study pregnant animals over many weeks in order to observe significant trends in hormonal secretion and production of paracrine regulators, and to evaluate myometrial contractility as pregnancy progresses. Second, neither put forward any ideas on the precise mechanism the fetus might use to sense impending danger. Finally, even if the fetus can sense a shortage of vital materials, neither of them could describe the specific nature of the mechanisms that the fetus could utilize to bring about its own birth. The writings of Hippocrates and Barcroft make no mention of the location of the clock, or clocks, that decide that pregnancy has gone on long enough and that determine that it is now the time to be born.
It took the observation of unusual disease conditions in pregnant women and in pregnant animals to point investigators in the right direction to solve the mystery of the fetal role in the initiation of birth. In 1933 Percy Malpas, an obstetrician, wrote a paper describing the prolongation of human pregnancy that occurs when a fetus is born with part of the brain missing or deformed, a condition called anencephaly. The anterior portions of the brain of the anencephalic fetus have failed to develop, and the baby is generally born dead. If the anencephalic baby survives birth, it can live only a few hours without the support derived from the mother through the placenta. Unless other complications are present, anencephalic babies are generally born well past the normal expected time of birth. This was one of the first clues that the critical signals that start the process of birth might have been missing because they were lodged in the part of the fetal brain that was itself absent in the anencephalic baby.
The next major clue came in the early 1960s when reports began to circulate in the western states of the U.S. regarding three related naturally occurring situations, two in cows and one in sheep, in which the duration of pregnancy was altered. In each case there were similar abnormalities in the fetal endocrine system. In both adult animals and humans, the endocrine system to a very large extent is controlled by the brain. The main pathway of neuroendocrine control is through the hypothalamus, which is connected by a special set of blood vessels, the pituitary portal system, to the pituitary gland. The pituitary portal blood system is a private communication pathway between the hypothalamus and the pituitary. Acting on instructions from the hypothalamus, the pituitary controls several other endocrine glands by secreting an array of hormones directly into the blood. The function of the adrenal cortex is regulated by the brain through the hypothalamus. The hypothalamus regulates the pituitary by secreting a hormone, corticotropin-releasing hormone (CRH). CRH stimulates the pituitary to secrete adrenocorticotropin (ACTH). ACTH is a hormone that regulates how much cortisol is secreted by the adrenal cortex. Cortisol from the adrenal regulates the function of several different tissues. Any of these steps can provide opportunities for control of the whole hypothalamo-pituitary-adrenal system taken together. They are also a potential cascade that permits rapid and pronounced amplification of the system.
A very dramatic clue to the fetal signals that initiate parturition came from the alpine meadows of Idaho. Pregnant sheep that had eaten a particular plant–the corn lily, Veratrum californicum–early in their pregnancy were carrying their lambs well past 200 days of pregnancy. This degree of prolongation of pregnancy was remarkable since sheep normally give birth after about 150 days of pregnancy.
The fetal lambs of ewes that had eaten Veratrum californicum were not born. They had to be removed surgically from the ewes by cesarean section. Some of the lambs had not been born even as late as 250 days of pregnancy. The fetuses that had not been delivered had hideous deformities, including just one central cyclopean eye. The base of the brain in affected fetuses was also deformed in the region of the hypothalamus and pituitary. Detailed chemical analysis proved that these deformities were caused by a specific toxin (2-deoxyjervine) in the Veratrum californicum. If embryogenesis was at a critical point of its development when the plant was eaten, the fetal brain developed abnormally. The developing neuraxis of the fetal sheep must be exposed to 2-deoxyjervine on the 14th day of ovine pregnancy in order to produce the changes that will lead to prolongation of pregnanacy.
All these observations of prolonged pregnancy have one underlying theme. They all suggest involvement of the fetal hypothalamus, pituitary and adrenal gland in the process of birth. But what the signal is, where in the fetus it originates and when it is given remained questions to be tackled. The time was now right for controlled experimental animal studies to determine the nature of the abnormality that was causing pregnancy to continue far longer than normal in these situations, namely: human anencephaly, the pituitary and adrenal abnormalities of cattle, and the deformities produced following the consumption of Veratrum californicum by pregnant ewes.
Critical Experiments
The clues described above strongly suggested, but did not prove, that the fetal pituitary is involved in determining how long pregnancy will last. A series of classical studies was performed by Professor, now Sir Graham, Mont Liggins. He reasoned that if he could surgically take away the fetal pituitary early in pregnancy, he could mimic the prolongation of pregnancy that occurs in the much more complex situation of the anencephalic human fetus and the cyclopean fetal lambs. He conducted a series of elegant, precisely controlled experimental studies.
When Liggins removed the pituitary gland from fetal lambs at around 115 days of pregnancy, pregnancy went on well past its normal length of 150 days. In separate studies he found that removal of both of the fetal adrenal glands also led to prolongation of pregnancy. If twin lambs were present, the pituitary-adrenal axis had to be surgically interrupted in both fetuses to prolong pregnancy. In the wake of Liggins’s experiments, many laboratories set out to understand the physiological consequences of increasing levels of fetal cortisol, the adrenal hormone that is released following the secretion of CRH and then ACTH. If premature maturation of the fetal adrenal is stimulated in the fetal lamb by the infusion of ACTH while the fetus is still in the uterus, the duration of the pregnancy is shortened. At 120 days of pregnancy Liggins infused a fetal lamb that was still in the uterus with ACTH to cause the fetal adrenal glands to grow prematurely. The lamb was born within about four days. When he skipped a step in the cascade process and infused fetal lambs with cortisol, the fetal lambs were born in about three days. Fetal lambs infused with saline as a control experiment did not deliver early; they remained in the uterus and were born at the correct time, around 150 days of pregnancy. These elegant studies firmly established that in the sheep the fetus is the originator of the signal to be born. The studies also clearly established the fetal pituitary and adrenal system as the major pathway for the signals that initiate the birth process.
The production of ACTH in the fetal lamb is controlled, as it is in the adult sheep, by CRH (and probably other hormones as well) from the fetal hypothalamus. CRH is secreted by specific neurons in the two hypothalamic paraventricular nuclei, one on either side of the fetal hypothalamus. When radiofrequency waves are used experimentally to destroy both of these nuclei in the fetal hypothalamus at 120 days of pregnancy, the pregnancy is prolonged and the normal fetal and maternal endocrine changes that take place at term are prevented.
There is now a firm body of experimental evidence to show that late in pregnancy, about 25 days before birth, the fetal lamb begins to increase the secretion of ACTH from the pituitary gland, stimulating the fetal adrenal cortex to grow and to secrete more cortisol. The fetal sheep is in large measure protected from rapid alterations in the maternal hypothalamo-pituitaryadrenal system since late in pregnancy fetal plasma cortisol concentrations are higher than those circulating in maternal blood, and cortisol does not cross the placenta from mother to fetus up this concentration gradient.
Taken together, the series of studies in pregnant sheep show that as a result of increased activity of the fetal brain, some time in the last third of pregnancy the fetus secretes more ACTH and cortisol, producing the changes that lead to birth.
Most of the experiments on which these findings are based were carried out on pregnant sheep because in outline there are many similarities between pregnancy in sheep and pregnancy in humans, as well as for the convenience that the sheep provides. Obviously there are differences between the sheep and human systems involved in parturition. We have some guide as to what these differences might be from work done on other primates such as monkeys and baboons, which are our near evolutionary relatives.
Two lines of evidence from studies in the monkey suggest that mechanisms similar to those so carefully and clearly shown in fetal sheep are also important in initiating normal birth at the end of normal pregnancy in nonhuman primates. In the last 10 percent of the time the sheep fetus spends in the uterus, the fetal adrenal begins to secrete more and more cortisol into the fetal bloodstream until at delivery the concentration of cortisol in fetal blood has risen to levels 20 times those that were present 15 days before birth. Cortisol stimulates the production of more estrogen and less progesterone by the placenta, changing the balance of factors regulating the level of contraction of the uterine muscle.
By different biochemical mechanisms the fetal monkey also stimulates processes that result in increased maternal plasma-estrogen concentrations. The fetal monkey adrenal predominantly secretes the steroid dihydroepiandrosterone sulfate (DHEAS). DHEAS is converted to estrogen in the placenta. If we draw a graph of the rise in cortisol in the blood of the fetal sheep in the last 15 percent of pregnancy and superimpose on the graph the rise in DHEAS in fetal monkey blood over the same portion of pregnancy, the lines are virtually identical. The strategy of the two species is slightly different, but the end result is the same. The fetal monkey instructs the fetal adrenal glands to produce precursors, or building blocks, from which the cells in the placenta can make estrogen. The fetal sheep, on the other hand, uses cortisol to produce enzymes in the placenta, which convert progesterone to estrogen. The overriding point of importance is that in both species it is the fetal brain, through the mechanism of the hypothalamo-hypophyseal-adrenal axis, that plays the fundamental role in deciding the length of pregnancy. In addition, as we shall see, the final link between the fetus and mother in both groups of animals appears to be increased production of estrogen.
Further confirmation of the role of the fetus in the initiation of parturition in primates comes from studies in which the fetus is removed from the uterus of the pregnant monkey at about 125 days of pregnancy but the placenta is left behind. When this is done, the placenta is retained in the uterus well past the normal time at which the fetal monkey would normally be born. The interpretation of this study is that, in the absence of the fetus, the normal signal to deliver does not happen.
So the fetus is not a passive participant in the birth process after all. He or she actually initiates the events that result in normal birth at the end of normal pregnancy. The fetus does this by manufacturing and secreting a cascade of hormonal messengers and local instructional molecules. The initiating process takes place gradually over several days.
Uterine Contractility
Extensive studies in pregnant sheep and many other species show that the uterine muscle is active through the majority of pregnancy. Throughout pregnancy periodic epochs of activity occur that last three minutes or longer and only give rise to a small increase in intrauterine pressure. These episodes of myometrial contractility, contractures, are very different from the well coordinated labor and delivery contractions which occur at the time of delivery. Contractures probably correspond to Braxton-Hicks activity described by many pregnant women. During contractures, the rise in intraamniotic pressure is very small compared with the increase that occurs during contractions. At the time of delivery it is necessary for contractures to change to contractions. This switch is brought about by the preparation of the muscle cell as a result of increased maternal plasmaestrogen concentrations.
The muscle layers of the uterus (the myometrium) are regulated by both inhibitory and stimulatory regulatory molecules. During pregnancy the balance is in favor of inhibition of uterine contraction so that the pregnancy will be maintained. At the end of pregnancy, so that birth may occur, the balance has to be switched to stimulation.
In sheep and other species, the hormone progesterone has an inhibitory effect on the myometrium. In contrast, estrogens are stimulatory to many processes that excite the myometrium. As the blood from the fetal lamb passes through the placenta, cortisol in the fetal blood stimulates the placenta to in crease the activity of the enzymes 17betahydroxylase and 17-20 lyase. These enzymes convert progesterone into estrogens. Thus, maternal plasma-progesterone concentration falls over the last three days of pregnancy, followed by a dramatic rise in maternal plasma estrogen. Progesterone is known to exert a quieting effect on the activity of uterine muscle. If progesterone production declines prior to labor in sheep pregnancy, it is helpful in producing the switch from less frequent lowgrade contractures to more frequent, regular, efficient contractions. However, in many species, including all primates studied to date, progesterone concentrations in maternal blood do not fall at the end of pregnancy. The steroid pathways in the placenta differ in primates, including monkeys and humans, when compared with sheep. The pregnant primate does not use placental progesterone as a precursor for estrogen production. Instead, the primate placenta utilizes androgen from the fetal adrenal as an obligate precursor for estrogen production.
For many years the absence of any evidence of progesterone withdrawal in human pregnancy has been seen as a major difficulty in trying to understand how the process of active labor is finally stimulated. It is accepted by virtually every investigator that progesterone keeps the uterus quiet during pregnancy. Two related questions can be posed. What is the major stimulator(s) of contractility at term? Are there any similarities between species?
Extensive studies suggest a key central role for estrogen as the major promoter of the underlying processes that bring about successful parturition. Maternal plasma estrogen increases markedly in late gestation in several species. We have recently demonstrated that infusion of the androgen androstenedione into pregnant rhesus monkeys eight-tenths of the way through gestation leads to a switch of contractures to contractions, dilatation of the cervix, rupture of the fetal membranes and normal delivery of live young.
Estrogen has been shown to stimulate several processes that eventually lead to contraction of the uterine muscle of the type that brings about labor and delivery. These changes include: increased maternal oxytocin production; increased numbers of oxytocin receptors on the muscle cells of the uterine wall; and increased prostaglandin production. Estrogens have also been shown to stimulate the formation of gap junctions between the individual uterine muscle cells. In the pregnant monkey study referred to above, the infusion of androgen did not alter the concentration of progesterone in the mother’s blood, yet normal delivery occurred.
The uterus is a muscular bag, similar in many ways to the heart. Both are beyond conscious control. There is nothing anyone can do to influence the operation of either the heart or the uterus as a conscious act of will: They are composed of involuntary muscles. Although there are, of course, differences, the fine structure of the individual muscle cells is similar in the heart, the digestive system and the uterus. On the other hand, uterine muscle cells are very different from the cells in muscles, such as those of the limbs that can be moved voluntarily as a conscious act of will. A pregnant woman cannot consciously control the contraction of her uterus, just as she cannot consciously control the beating of her heart or the activity of her stomach.
The lining of the uterus uses paracrine messages to talk to the muscle cells beneath the lining, while at the same time receiving paracrine messages from the fetal membranes that surround the fetus. Several of these endocrine and paracrine regulatory molecules alter the contractile properties of uterine muscle. As mentioned above, progesterone, a steroid hormone like many of the molecules involved in the process of birth, has inhibitory effects on uterine muscle contraction. Acting in opposition to progesterone, estrogens stimulate uterine muscle contractility. They do this, in part, by instructing the lining of the uterus (the decidua) to increase the production of prostaglandins, which are paracrine regulatory compounds. The prostaglandins diffuse from cells of the decidua to the muscle cells beneath it and stimulate them to contract. The level, type and pattern of uterine muscle contraction is determined by the balance of factors that tend to keep the uterine muscle relatively inactive and the factors that tend to stimulate activity. Maintenance of a normal pregnancy for its full duration requires the balance to be in favor of the inhibitory factors. At birth, the regulatory balance is tilted in favor of stimulation of the muscle of the uterus. At this time the muscle switches from the irregular, weak pattern of activity it has been undergoing throughout pregnancy to strong well-coordinated labor contractions.
Maternal switching of contractures to contractions demonstrates that the fetal signal has now been transduced into a change in uterine muscle activity that will lead to labor and delivery. Several agents are now recruited to stimulate contraction of the uterine muscle. These agents are called uterotonins. Oxytocin is the strongest uterotonin known. However, its role in promoting labor has been one of the major areas of contention in the study of the physiology of late pregnancy. This is an interesting story with important lessons for understanding the biology of the mother during late pregnancy. Recent studies in pregnant rhesus monkeys have elucidated the changes that take place in maternal oxytocin production. Although there are no marked changes in maternal plasmaoxytocin concentration during the daytime, there is a nocturnal rise in maternal plasma oxytocin that increases over the final 30 days or so of gestation. This increase in maternal plasma concentrations of oxytocin occurs at the time of the day when the switch from contractures to contractions occurs. Further evidence that oxytocin is critically involved in this switch is provided by studies showing that the switch can be reversed by administration of the specific oxytocin antagonist Atosiban. It is of interest that previous attempts to show increases in maternal plasma oxytocin prior to labor were probably doomed to failure because, in general, sampling of experimental animals and pregnant women did not take place around the hours of darkness! The demonstration that a rise in oxytocin can be measured if the appropriate set of samples are taken is another indication that when studying biological systems “the absence of evidence is not the evidence of absence.”
Oxytocin has also been shown to be produced locally within the pregnant human and rat uterus by both the endometrium and the chorio-decidua. Oxytocin produced in these locations could exert a direct paracrine stimulatory effect on the underlying myometrium. In addition, interesting differences in the responsiveness of the myometrium to oxytocin have been demonstrated at different times of the 24-hour day. In monkeys, sensitivity to oxytocin is highest in the early hours of darkness. In sheep there is a concurrent increase in the message for oxytocin-receptor synthesis and the numbers of oxytocin receptors in both the endometrium and the myometrium when premature labor is precipitated by the infusion of glucocorticoids into the fetus using the model first introduced by Liggins. Thus, both the increased levels of oxytocin in maternal blood and the heightened responsiveness to oxytocin are involved in explaining why the switch from contractures to contractions generally occurs around the time of onset of darkness.
In summary, it can be seen that the fetus determines the duration of pregnancy. When the fetus is adequately mature it activates an endocrine cascade that involves the hypothalamus pituitary and adrenal. By mechanisms that differ slightly in sheep and monkeys, the placenta is stimulated to produce more estrogen. Estrogen constitutes the link between the fetal mechanisms and the maternal effectors that bring about labor and delivery–oxytocin, prostaglandins, critical receptors on the cells that line the uterus, and the uterine muscle cells and gap junctions.
In studies in pregnant monkeys and baboons in which electrodes have been placed on the myometrium, we have shown that the switch from contractures to contractions recurs with an approximate 24-hour periodicity when pregnant monkeys are kept in a uniform low-level light environment for the final two months of pregnancy. What regulates the remarkable rhythmicity of the contractures-to-contractions switch? Why does it occur at nighttime? This periodicity in the absence of external clues demonstrates that the switch is under true circadian control. The maternal production of oxytocin is hence either directly under the control of the mother’s circadian clock in her hypothalamic suprachiasmatic nucleus or indirectly via the control of estrogen which itself fluctuates throughout the 24-hour day as a result of the interlocked circadian rhythms of the maternal and fetal adrenals.
One final piece of evidence that maternal estrogen is the prime coordinator of the switching process at the end of pregnancy comes from the observation that when androgens are infused into pregnant monkeys 20 days before delivery would normally take place, the switch from contractures to contractions occurs prematurely. This switch can be inhibited by simultaneously administering to the mother an enzyme inhibitor that prevents the conversion of the androgen being infused to estrogen. Each species has an appointed time of the day at which it is safest for the young to be delivered in relation to presence of predators or prevailing environmental conditions. Thus, the mother completes the job the fetus began and life refreshes itself with a new start.