High blood glucose levels early in pregnancy deprive the embryo of oxygen

The babies of women with diabetes are two to five times more likely to develop birth defects than offspring of women without the disease.

A recent study in animals by scientists at Joslin Diabetes Center in Boston helps explain why. The research, appearing in the October issue of the American Journal of Physiology: Endocrinology and Metabolism, suggests that high blood glucose levels early in pregnancy deprive the embryo of oxygen, interfering with its development.

"Until recently, it was not understood how diabetic pregnancy could cause birth defects. My laboratory wanted to explore this research because the more we know about the effects of the mother's diabetes on the embryo, the more tools we have to identify therapies that may prevent birth defects in diabetic pregnancy," says the study's lead investigator, Mary R. Loeken, Ph.D., an investigator in Joslin's Section on Developmental and Stem Cell Biology and Assistant Professor of Medicine at Harvard Medical School.

Women with both type 1 and type 2 diabetes run a high risk of having babies with birth defects, especially of the heart and spinal cord. Because these organs form during the first few weeks of pregnancy, coinciding with the time that a woman may first learn she is pregnant, aggressive control of blood glucose levels just before and after conception is critical. "Women with diabetes should be consulting with their healthcare team to be sure they have good glycemic control before becoming pregnant," says Dr. Loeken. Maintaining blood glucose control continues to be important throughout the pregnancy, but it is particularly important during the first eight weeks, when an embryo's organs are forming.

In addition to recommending that women with diabetes have good control of their glucose levels before becoming pregnant, Dr. Loeken recommends that obese women who don't know if they have diabetes but who are planning to become pregnant be tested for diabetes. There have been several recent reports of increased birth defects in the pregnancies of obese women. "Many obese individuals have type 2 diabetes and do not know it, so it is a good idea to bring glucose levels to within the normal range before becoming pregnant, and to monitor women with pre-diabetes closely during pregnancy to make sure that they don't develop diabetes," Dr. Loeken says.

In the new study, Dr. Loeken and her colleagues examined embryos of pregnant mice injected with glucose (the sugar that is elevated in the blood during diabetes) to mimic diabetic pregnancy. The researchers knew that oxygen is needed by cells to break down glucose and produce energy, and that normally, when oxygen is consumed, more oxygen is delivered to tissues by increasing blood flow to those tissues. However, at the stage of embryonic development in which birth defects in women with diabetes frequently are believed to occur, the embryo does not yet have a heart or blood supply, and so the scientists theorized it might not be possible to replace oxygen as rapidly as it is consumed. This has the potential to cause hypoxic stress, or damage to cells caused by low oxygen (hypoxia).

Working in collaboration with Peter Smith, Ph.D., Director of the BioCurrents Research Center at the Marine Biological Laboratory at Woods Hole, Mass., Dr. Loeken found that the oxygen concentrations in embryos of mice injected with glucose were significantly lower than in control embryos. This demonstrated that breaking down higher amounts of glucose caused oxygen to be used up faster than it could be delivered.

The researchers then injected pregnant mice with glucose, or exposed them to varying levels of oxygen to see if raising and lowering oxygen delivery to the embryos had the same effect as raising and lowering glucose. The scientists' goal was to see whether oxygen deprivation is what mediates the effects of high glucose on the embryo in pregnant diabetic mice. Dr. Loeken's lab had previously found that inducing high blood glucose levels in pregnant mice suppressed Pax3 expression in embryos. Pax3 is a gene required for healthy formation of the brain and spinal cord.

In the new study, the researchers found that restricting oxygen delivery (by housing pregnant mice for one day in cages containing 12 percent oxygen – a concentration that did not cause any stress to the mothers, but which might significantly reduce the amount of oxygen delivered to the uterus – instead of 20 percent oxygen contained in room air at sea level) had the same effect as high glucose. In fact, embryos from pregnant mice with high blood glucose levels, or oxygen-restricted mice, had five-fold decreases in Pax3 expression and eight-fold increases in a severe type of birth defect called neural tube defects. Conversely, increasing the oxygen delivery to pregnant diabetic mice (by housing them in cages containing 30 percent oxygen) blocked the decrease in Pax3 expression and neural tube defects in their embryos.

Neural tube defects occur when parts of the brain, spinal cord, or their protective coverings fail to develop properly. For example, spina bifida – the most common neural tube defect in humans – results from the incomplete closure of the spinal cord. Neural tube defects and heart abnormalities are the most common birth defects affecting babies born to women with diabetes.

Previous research by Dr. Loeken and others has shown that in pregnant mice, high blood glucose levels boost an embryo's production of free radicals – products of metabolism that cause oxidative stress, and that oxidative stress leads to birth defects. These new experiments showed that glucose also caused hypoxic stress in embryos. However, what Dr. Loeken and her colleagues did not know was whether glucose caused two different disturbances, hypoxic stress and oxidative stress, each having separate effects on Pax3 expression, or whether the two disturbances were linked. To their surprise, when they investigated the production of free radicals, they found that embryos of oxygen-restricted mice showed the same three- to five-fold changes in markers of oxidative stress as did embryos of glucose-injected mice. Conversely, the markers of oxidative stress were suppressed in embryos of oxygen-supplemented diabetic mice. These results suggest that the lack of oxygen caused by increased glucose consumption triggers the production of free radicals, which then causes birth defects, Dr. Loeken explains.

The researchers also found that administration of high levels of antioxidants – which keep free radicals from wreaking cellular havoc – prevented the decrease in Pax3 expression and birth defects in embryos of diabetic mice. This lends weight to the theory that lack of oxygen and the accompanying increase in free radical production that occurs in mice with high blood glucose levels are what ultimately increase risk of birth defects. "We're trying to pinpoint all the steps that occur between oxygen deprivation and gene expression, and to identify the signals and molecules that regulate Pax3," Dr. Loeken says.