On the 18th day after fertilization a flat strip of thickened cells appears along the back of the human embryo. The next day its edges begin to lift. They come toward each other, meet in the middle and leave one opening at each end. Around day 26 the front opening closes. Around day 28 the back one closes.
The whole movement fits into ten days. In the photographs of the corresponding stages the scale bar is 0.5 or 1 millimeter. Under it the tissue has already gone the way from a flat surface to a tube, out of which the brain and the spinal cord will develop.
This calendar creates an unusual problem. When the tube is closing, many people do not yet know that a pregnancy has begun. So the folic acid has to be there before the pregnancy test turns positive.
Ten days under a one-millimeter scale bar
At the start the cells lie like an elongated plate. Then they change their shape and their places. The plate lengthens, narrows and sinks in the middle; its raised edges form two folds. Around day 23 they have already fused in the central part. A tube has formed, but it is still open toward the future head and toward the future end of the spine.
Only now do the names help. The flat tissue is the neural plate. The raised edges are the neural folds. The hollow structure after they fuse is the neural tube. The last two openings close one after the other: first the one toward the head, then the one toward the lower end of the body.
The fusion also changes the neighboring surface. The cells on both sides meet above the new tube and cover it; out of this outer tissue the skin will form later. In this way one flat area splits into two structures with different fates: a closed tube below and an unbroken surface above it. The photographs catch not a finished organ but the rearranging itself.
The ten-day window is short only on the calendar. At the level of the cells, division, movement and change of shape run through it at the same time.
When the closing does not finish, neural tube defects arise, among them spina bifida and anencephaly. The observation raises two separate questions. The first is whether a nutrient can change the probability that this happens. The second is harder: when does it have to be there, so that it meets the tissue at the right stage?
A trial with four groups and a start before conception
In the early 1980s the evidence for the role of folic acid still came from smaller observations and trials. The Medical Research Council organized a trial that turned the question into four clearly distinguishable comparisons.
It enrolled 1,817 women, each of whom had already had a pregnancy affected by a neural tube defect. Their shared history is exactly what set what the study was testing: recurrence. The participants were assigned at random to four groups. One got folic acid, the second got seven other vitamins, the third got both, and the fourth got neither. The intake began before the next pregnancy.
This arrangement does more work than an ordinary comparison between one tablet and a placebo. The four groups form two crossing lines: with or without folic acid, and with or without the mixture of other vitamins. If the result changes along the first line but not along the second, the researchers can separate the contribution of the folic acid from the effect of the mixture.
For 1,195 completed pregnancies the outcome is known. In the groups with folic acid there were 6 cases of a neural tube defect, and in the two groups without it there were 21. The calculated relative risk is 0.28, or a 72% protective effect against recurrence. Across the four groups the result follows the intake of folic acid.
The numbers give an answer for women with a previous affected pregnancy. For the first occurrence another trial is needed, with another group of people and another design.
Six cases move the question to the first occurrence
In Hungary, Andrew Czeizel and István Dudás followed women who were planning a pregnancy and had no previous neural tube defect in their history. One group got a multivitamin containing 0.8 mg of folic acid. The control group got trace elements without vitamins. The intake began at least a month before conception and continued until the second missed period or longer.
The outcome is known for 2,104 pregnancies in the multivitamin group and 2,052 in the control group. In the first there was no case of a neural tube defect. In the second there were six.
So the Hungarian trial shows the prevention of the first occurrence with a multivitamin containing 0.8 mg of folic acid, compared with trace elements. Both trials began before conception, because the biology of the tissue runs ahead of the positive test.
How folate meets the clock of the dividing cell
The result of the trials is visible in the number of cases. To understand why the moment matters so much, we have to go down to the material that every new cell copies.
Before a cell divides into two, it makes a second copy of its DNA. That takes new molecules of the four building letters of the genetic code. The building of the purine nucleotides, which carry the letters A and G, and the production of thymidylate for the letter T use small carbon fragments carried by different forms of folate.
Chemists call these fragments one-carbon units, because each one carries a single carbon atom. The folate molecule takes them on, holds them in a particular chemical state and hands them over in the right reaction. In the building of the purine ring, such fragments supply two of its atoms. In the synthesis of thymidylate, another folate form hands a carbon fragment to a precursor molecule. After the handover the carrier comes back through a series of reactions and enters circulation again. A cell uses this cycle many times over while it copies all of its DNA.
In that ten-day window many cells copy their DNA. The folate forms carry the carbon parts needed for the new nucleotides. So the calendar of the neural tube and the chemistry of folate meet at the same early moment.
From the four groups in the trial to the everyday grain
Preconception intake covers the early window in a person who is planning a pregnancy and starts in time. To cover the conceptions that come before any planned intake, folic acid has to be present in everyday food already before the missed period or the test.
In the United States the answer became part of food production. The rule, fully in force from 1998, requires 140 micrograms of folic acid to be added to certain enriched grain products per 100 grams of product. The unit here describes a manufacturing standard, not a single moment of intake. In this way folic acid is present in widely eaten food before anyone has worked out the exact month of conception.
The change also shows in the data for the whole population. An analysis of 19 American surveillance programs compares the prevalence of anencephaly and spina bifida before and after mandatory fortification. Across all programs the overall drop is 28%. In the systems that also record the cases detected before birth, the drop is 35%. The researchers calculate about 1,326 cases prevented per year.
In the photograph from the Carnegie stage the 0.5 or 1 millimeter scale bar stands next to tissue that is closing day by day. Next to it the pregnancy test may still be lying unopened. That is exactly why folic acid has to be there already, while the neural tube is closing.
Sources
- Eric R. Detrait et al., Human Neural Tube Defects: Developmental Biology, Epidemiology, and Genetics, Neurotoxicology and Teratology, 2005.
- MRC Vitamin Study Research Group, Prevention of neural tube defects: results of the Medical Research Council Vitamin Study, The Lancet, 1991.
- Andrew E. Czeizel and István Dudás, Prevention of the First Occurrence of Neural-Tube Defects by Periconceptional Vitamin Supplementation, The New England Journal of Medicine, 1992.
- Anna E. Beaudin and Patrick J. Stover, Insights into Metabolic Mechanisms Underlying Folate-Responsive Neural Tube Defects: A Minireview, Birth Defects Research Part A, 2009.
- Jennifer Williams et al., Updated Estimates of Neural Tube Defects Prevented by Mandatory Folic Acid Fortification — United States, 1995–2011, Morbidity and Mortality Weekly Report, 2015.
