Ch03

The chain of events we take for granted for pregnancy to establish itself is such a long one that I'm reminded of Tolstoy's opening to his novel, Anna Karenin, when he was writing about what we take for granted in a successful marriage:

All happy families are alike, but an unhappy family is unhappy after its own fashion.

… which we might paraphrase as …

All successful pregnancies are alike, but an unsuccessful pregnancy is unsuccessful in its own particular way.

Just as many things need to go right, and to more or less keep going right, for a couple to stay in love and for their marriage to last, so it needs only one of the chain of many building steps to go awry for a pregnancy to fail, and to miscarry.

How do we come to grips with this chain? In parallel with the biological account I will run the metaphor of an international joint venture for a construction project.

An Overview of the Pregnancy Project

For an insight into the key biological events that lead to successful pregnancy, let's look at the project as if it were a joint construction venture developing in a foreign country. There are benefits to the foreign, host country (playing the role of the mother), but there is also some cost.

For the biology behind it you might want to revise WebPage 3 in preparation. As we go along the glossary will help too.

Here are the key steps to the initiation of "Project Pregnancy":

Step 1. The deal is done and the embryo forms

Fertilization has occurred ... by a genetically sound sperm, of an egg that is both genetically normal and, in its cytoplasm, metabolically capable and resourceful.

We have the raw ingredients for the joint venture between Man Inc. and Woman Inc. Both have brought to the table a bundled a set of instruction packs that will contribute to a balanced blueprint. The woman is supplying the resources to get started, the egg is packed with nutrients, proteins, last minute instructions to get through the first few days, as well as an enormous supply of mitochondria to power things along.

Step 2. Prior to implantation: the plan unfolds

Cleavage follows. The fertilized egg begins replicating, or dividing. By the third day the new genome has become indispensable, each of the 8 cells is referring to it. By the fourth day, the 16 or so cells are cooperating to form the first tissue, as the morula undergoes compaction. Next the cells on the periphery differentiate slightly: these are the placental stem cells, or trophectoderm. By the fifth day, off to one side, on the inside of the blastocyst, is the inner cell mass, destined to become the fetus. We started with a single cell: we now have more than 100 cells.

The project is underway, controlled by a hopefully good partnership of smart maternal and paternal owners. Their wisdom and codified instructions comprise the DNA blueprint that is being copied off for each cell, residing as a genome in each embryonic cell's nucleus. Their joint contributions to the developing idea are already being put to the test.

Step 3. Implantation: entry into foreign territory

The hatching blastocyst sheds its protective zona to expose itself to the intimacy of new surroundings, sending signals to an endometrium made responsive by progesterone from the host's corpus luteum.

The new project is hatched, admitted through the host's country's borders, and revealed to the host's regulators. The regulators happen to be related to the maternal partner to the project (a fact that might or might not prove helpful to the joint venture's survival).

For the duration of the project, the host will only support the project while there is an ample supply of progesterone to keep everybody happy and cooperative. Where the progesterone comes from is a secondary consideration. It will be supplied by the host for only another week without much cajoling from the pregnancy; even then the host will provide it only for another few weeks, after which time it must all be generated by the project itself.

The embryo's surrounding trophectoderm starts budding off trophoblast, which extends out into what should be a receptive endometrium, dissolving its carbohydrate, protein and nucleic acid-rich glands for nourishment, while the tissue between the glands, the endometrial stroma, is turned into decidua.

To pacify critical elements of the host's blood coagulation system and the immune system, and to keep up the progesterone supply, the trophoblast is churning out human chorionic gonadotropin (hCG) to drive the gradually more reluctant corpus luteum. To ensure that a bland, uniform, but persistent front is presented to the decidua and its gathering white blood cells, the trophoblast that will surround the growing gestation consists only of a single, extraordinarily stretched, single cell, the syncytiotrophoblast ... crawling, growing ... extending like an insatiable ameba ... fitting over the whole pregnancy like a glove.

Outside this, individual cells of the extravillous trophoblast (the other executive arm of the underlying cytotrophoblast) venture out deeply into the decidua and beyond, preparing not just the neighbourhood but the host immune system itself.

For more on the remarkable properties of the trophoblast, see the box, The 'ABCDEFG' of tissue groups ... .

The host's gathering militia comprises both a tolerant peace corps (resident, or uterine NK cells to facilitate the project) and more aggressive itinerant peace keepers (peripheral or circulating NK cells) as well as a few troopers (T cells) who are ready to call for reinforcements and to start shooting if things go wrong or if there's too much damage, either within the project or around it. As the infrastructure services begin developing, if the construction is too chaotic or if the policing immune system is provoked into a bad attitude, project pregnancy will be finished before it starts.

Communication helps the project take hold, but very many services need to be established simultaneously and quickly for the sensitive, rapidly developing project to go ahead in this potentially prickly environment. There's more than just hCG and progesterone involved. For details of the communication skills needed, see the box, ... and shaking hands with HLA-G.

Meanwhile the roving members of the consular staff, the cells of the extravillous trophoblast, are venturing deeply into host territory, concealing their foreignness while presenting a friendly face to the head honchos in the immune control centers, encouraging them to tolerate the project.

Step 4. Avoiding rejection

The surprise is, you might think, that in most cases the fetus is successful in keeping the mother's immune system so tolerant.

Isn't the fetus and the developing placenta a foreigner, getting half of its genes from a stranger (the father) or, in the case of gestational surrogacy, all of its genes from strangers? Why isn't it rejected, like a poorly matched kidney or liver or heart transplant?

It seems the answer lies with the trophoblast on one side and, on the other, the NK cells -- white blood cells that in evolutionary time stretch back more than 500 million years, before the evolution of immune systems that have memory. This was a time when the planet's oceans were inhabited only by invertebrates, animals accustomed to close proximity with each other, living communally as corals, sponges, molluscs and sea squirts. For more, see the box, Rejecting rejection.

If, for any reason, the development of the embryo is faltering, the trophoblast will also slow down, losing its capacity to sustain the rapid rise in hCG needed to maintain progesterone production from the corpus luteum. Bleeding will start and all the outside will know of the pregnancy is the transient circulation of hCG ... in other words a transiently positive pregnancy test, a biochemical pregnancy, or what we call a subclinical miscarriage.

The trophoblast at first does not need much instruction for its own function and growth, out on the periphery of the project. Its job description might be critical but it's not too complicated. But it does need plenty of support from the center. And it needs a friendly local militia. Without enough credibility at this early point, or with a too-prickly constabulary, the aggressive NK cells will take over from the facilitatory NK cells and will stick in the daggers, puncturing the trophoblast with molecules called perforins.

The trophoblast is only ever one big cell, stretched very thin, being fed by underlying cytotrophoblastic stem cells. Stick it with perforins at this early stage and it will dissolve away, with nothing left as a legacy but some still-circulating, but dwindling, hCG.

Step 5A. Placental construction: laying the project's foundations

Around the growing embryo, spaces appear within the expanding syncytiotrophoblast called lacunae, which as the trophoblast continues eating into the endometrium will coalesce and then briefly fill with maternal blood a day or two before the missed period (occasionally manifesting to the outside world as implantation bleeding).

For the footings of the nascent placenta between the lacunae, trunks of early chorionic villi form. These villi grow like trees, the trunks growing thicker as they bud ever finer branches into the lacunar, or "intervillous" spaces. At every stage, the lacunae (the developing intervillous space) is lined by the omnipresent cell that is the trophoblast. Down the track, about 12 weeks after the last menstrual period (LMP), these spaces will accept a regular flow of maternal blood. But for the first three months they are rather dry.

Having come to peaceful terms with the host officials, it's hard now to see who is contributing what to the scaffolding and support systems, so intermingled are the construction molecules. But one thing is for sure: none of the extravillous cells belonging to the project should wear conspicuous clothes. To display your unique tissue grouping molecules (such as HLA-A or HLA-B) is to attract instant reaction from the immune police. Discretion is the better part of valor.

Meanwhile the all important exchange system of the developing placenta, the linking spaces between the budding chorionic villi (the growing intervillous space) remains lined entirely by the living, breathing, growing syncytiotrophoblast, stretching over every last branch.

All the glucose, the oxygen, the nutrients the project needs pass through the syncytiotrophoblast. And all the carbon dioxide and excrement the embryo or developing fetus is ridding itself of pass through it in the opposite direction.

Step 5B. Meanwhile embryonic development proceeds: complicated contruction in the inner sanctum

Deep within the gestation, the purpose of the project, developing the fetus, should be going progressing full steam. By the time of the missed period, an embryo that will be successful is forming its third definitive layer of tissue. What was the inner cell mass had become first a disk of two layers, the ectoderm (or epiblast) on top, and the endoderm (or hypoblast) underneath. Then, in a move that creates a head-end and a tail-end to the embryo, an intervening tissue pushes between the two layers, the mesoderm -- in a process called gastrulation.

Reading off the blueprint, the embryonic genome (copies of which are available in every cell), already 1600 separate genes had been functioning before the blastocyst had started to implant. Since then new instructions have been called on by the hour, as the embryo progresses through the remarkable changes that will give it functioning tissues and organ systems, as well as a human form.

Before long, as many as 30,000 instruction sets will be being followed. A majority of them comprise instructions for molecular signalling systems.

Above the embryonic disk the ectoderm is extending to form a bubble, a membrane called the amnion, enclosing the at-first-tiny amniotic cavity.

Below the disc, and more prominent, is the chorionic cavity, as mesoderm envelops the hollow insides of the blastocyst's trophectoderm/cytotrophoblastic cavity with a new tissue called chorion. By a week after the missed period this growing cavity becomes the first visible sign of pregnancy on transvaginal ultrasound: the gestational sac.

If all continues to go well, the yolk sac forms inside the chorionic cavity, connected to the growing but still tiny embryo, and an early circulation system is developed, which will help carry nutrients from, and waste products to, the peripheral interface with the mother at the trophoblast surface.

Blood vessels push into the trophoblastic columns as these columns bud into branching trophoblastic villi. Soon, the earliest blood cells are formed inside them. Unlike later mature red blood cells, these primitive hemoglobin-containing red cells have a nucleus.

And the most prominent organ visible on transvaginal ultrasound after the yolk sac will be the developing heart, beating at around 120-160 beats per minute, and visible by 5½ weeks from the LMP ... if everything is going to plan.

Consider the chromosomes as bound volumes of the blueprint.

In the blueprint that every cell of the pregnancy is carrying, we need exactly two copies of each instruction, or gene -- one from each parent's genome. If the instructions are identical, then it's an unambiguous, homozygous instruction; if not, it's heterozygous, and the result is less predictable (though usually the correct instruction wins out). If there's only one instruction, it might not be heard. If there are three instructions, the action will be overstated.

But if both instructing genes for a particular step are faulty, and that step is a critical part of the plan that cannot be worked around, the project comes to a halt.

Sometimes this hitch can affect genes on the host side ... a failure of a maternal system within the decidua that prevents the host uterus from ever accepting an ongoing pregnancy.

Step 6. A placental circulation

Meanwhile, at the periphery, the syncytiotrophoblast, the thin envelope of the placenta that everywhere separates the mother from the gestation, is growing apace. By the time the baby is ready to be born, this single syncytiotrophoblast cell will have a surface area of 12 square meters.

It grows because it is continuously fed by trophoblastic stem cells, the cytotrophoblast, fusing into it. About 15% of the bulk of the fusing cytotrophoblast is for growth, as the placenta enlarges and extends and the villi keep branching. The other 85% is for turnover of the cell's internal workings, or organelles. As bits of the syncytiotrophoblast wear out or get tired, they are pinched off and enter the mother's circulation, reaching the mother's lungs, where they stick and are broken down and absorbed. If this dynamic is disturbed, and the syncytiotrophoblast gets fatigued, miscarriage will not be far behind.

The syncytiotrophoblast has many functions. A crucial early one is to manufacture hCG in rapidly increasing amounts to push a more and more resistant corpus luteum into continuing the production of enough progesterone to reach the uterus. From 6 to 8 weeks from the LMP, the syncytiotrophoblast takes over this responsibility. It feeds on cholesterol from the mother and turns it into progesterone, ensuring that there is plenty of it where it's needed: at the interface with the host decidua.

Without continuous encouragement from the center, represented both by the supply of fusing cytotrophoblast cells and various molecular messages from fetal tissues, the trophoblast loses its confidence in greasing the local immune system's border officials with progesterone, and starts to pack it in.

The construction project, while it was still small, would just have dissolved away from the hostility expressed by the NK cells and T cells.

But once it's too big to be so ignominiously ignored, an irritated host militia needs bigger guns as well as a way of expelling the project physically.

Many, many developments (or developmental lack, or delay) can initiate the breakdown in communication that can sooner or later cause the host defence force to turn nasty. When it does so it does so by turning on some bleeding and coagulation around the perimeter, followed by a call on the myometrium to contract and to expel the doomed project. Only uncommonly does it send in much of the way of sacrificial T cell troops from the immune battalion.

The extravillous trophoblast, like the syncytiotrophoblast, also has some special functions. These covert operators are sent out into the decidua from those areas of trophoblast not in direct contact with the intervillous space. Sometimes called "interstitial trophoblast" or "intermediate trophoblast", these cells seek out the maternal host's arteries that will be feeding the placenta. They enter the muscular walls of these arteries and subvert them, causing them to weaken and later to dilate. They enter the interior of the vessels, clogging them, before eventually forming a new lining that thus replaces the mother's cells with the fetus's cells.

All of this activity, remodelling the maternal host's arterial supply to the placenta, means that jus a trickle of maternal blood gets to the feto-maternal interface in the intervillous space for the first three months of pregnancy. If everything is developing normally, a transvaginal ultrasound will demonstrate an absence of flow through the placenta in a healthy preganancy. (Remember,you read it here. It's the reason you can biopsy the placenta during chorionic villus sampling, or CVS, without causing bleeding.)

On the other hand a pregnancy that is not doing well will show maternal blood pulsing into the intervillous space. It can be seen on ultrasound if "Doppler" is used. Miscarriage can follow within days.

If this "conversion" of the maternal arteries by the extravillous trophoblast is incomplete yet miscarriage is averted, preeclampsia (pregnancy-associated high blood pressure) is likely to occur later in the gestation.

Step 7. Serious growth and development

By the end of the first three months of pregnancy construction of the supply system is complete. The maternal arteries supplying the placenta have become large volume capacitance vessels, and it is time to gradually open the faucets to the intervillous space, irrigating the placental bed with maternal blood.

The embryo is now a fetus. It has a face, it has arms and legs, it has fingers and toes, it has a brain and a spinal cord, it has testes or ovaries. All its critical organ systems are close to being finalised, having formed in the lowest oxygen conditions imaginable -- conditions that have protected the millions of copies of the project blueprint from replication errors, or mutations.

From now on, mistakes become a little less serious. Cells with errors can be replaced by cells that do not.

As blood circulates through the placental bed, the chorionic villi, still immature, slowly refine their architecture for greater efficiency, but it will the 23rd week before fully mature "terminal" villi form. It becomes more and more important that the rather slowly flowing blood does not clot: to do so would cut off the oxygen supply and prove fatal. To encourage the circulation, the tips of the villi produce a special anticoagulant, annexin V.

Regarding the mother's white blood cells, delicate diplomacy continues, but the ground rules of engagement are now well and truly established. Occasional roving white blood cells from each side of the divide will travel deep into the other's territory. Most will be left behind long after the fetus and the host mother part company (called microchimerism), to show up many years later as an occasional Y-chromosome-bearing cell line in the mother, or as an X-bearing cell line in a boy.

What was once just a small blister, the amniotic cavity is growing faster than the chorionic cavity that surrounds it. Gradually the fluid between the chorionic membrane and the amniotic membrane (together "the membranes") disappears, and the outside of the amnion fuses with the inside of the chorion. From 14 or 15 weeks from the LMP the fusion is complete: the fetus floats in its own amniotic cavity: amniocentesis becomes possible for sampling the fetus's cells.

Step 8. Surviving threats

Threats to project pregnancy remain. Whereas the design features of the project and its host infrastructure are in place, the rapid growth taking place can strain the available resources.

If there is physical constraint, friction will occur at the boundary.

If there is logistic constraint, the fetus's growth will be restricted, especially in the last three months.

Occasionally a boundary gives way (see cervical incompetence on WebPage 8) and, to both sides' dismay, the project falls apart.

Faulty aspects of the fetal blueprint can still be operative. An extra volume of instruction set #21 (trisomy 21), for example (or for #13 and #18), may have permitted confident but abnormal development, escaping all internal checking procedures. The host mother could demand a formal examination of the blueprint by karyotype: a sample of chorionic villi (CVS) or a specimen of fluid and cells from the amniotic cavity (amniocentesis).

The ever present threat to the project by the host's immune guard (its white blood cells, or T cells) remains. Project pregnancy must keep its own house in order or face the consequences.

Generally the immune system is not out to cause trouble but if it sniffs it then it will vigorously respond to it.

If there is trouble -- if for example the fetus is invaded by infective organisms, or if shearing forces at the growth points provoke inflammation -- the mother will move quickly to safe guard her own future. (Pregnancy evolved millions of years before there were doctors and nurses with antibiotics.)

The immune system is called on and will be at work with a vengeance to protect the host. Collateral damage will be severe and separation of the fetus from the mother is the usual result.

More often, by now, the host environment is tolerant and helpful, ensuring a steadily increasing stream of supplies as the fetus grows, the host provides a constraining but yielding neighborhood, with the room to grow without excessive friction or suffocation, and providing adequate waste removal, until the fetus is mature enough to separate from the host into an independent life in a wider world.

Step 9. Birth

In the ideal pregnancy, the fetus signals to the host that he or she is ready to be born. It does this by having its trophoblast convert fetal hormones to a special estrogen, estriol. The mother obliges by switching off her receptors to progesterone. This starts a cascade of labour and birth, as she activates the powers to expel project pregnancy ... powers that, until now, she has kept in reserve.

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