Assignment 2: Review Of A Paper

Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis.

K.O'Donoghue, M.Choolani, J. de la Fuente, S. Kumar, C. Campagnoli, P.R. Bennett, I.A.G. Roberts and N.M. Fisk.

Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis (2003) K. O'Donoghue, M. Choolani, J. Chan, J. de la Fuente, S. Kumar, C. Campagnoli, P.R. Bennett, I.A.G. Roberts and N.M. Fisk.Molecular Human Reproduction, Vol. 9, No. 8, 497-502


Summary:

My paper is based off the recent identification of mesenchymal stem cells (MSC) in first trimester fetal blood. The authors of my paper state that strategies for genetic prenatal diagnosis on fetal cells in the maternal circulation have been limited by lack of a cell type present only in fetal blood. By discovering MSC in first trimester fetal blood it offers the prospect of targeting MSC for non-invasive prenatal diagnosis. The authors of the paper developed protocols for fetal MSC enrichment from maternal blood and determined sensitivity and specificity in mixing experiments of male fetal MSC added to female blood. They also used the optimal protocol to isolate fetal MSC from maternal blood in the first trimester using blood taken after surgical termination of pregnancy as a model of increased feto-maternal haemorrhage. Their experiments showed that they could isolate fetal MSC from maternal blood, however, it is rare in post-termination blood which suggest they are unlikely to have a role in non-invasive prenatal diagnosis.

The aim of this study was to investigate fetal MSC as potential targets for non-invasive prenatal diagnosis in first trimester maternal blood. They established a protocol for fetal MSC enrichment from maternal blood and determined sensitivity and specificity in mixing experiments with adult nucleated cells. They then applied this method to the isolation of fetal MSC from maternal blood after first trimester termination of pregnancy, which is a biological model of increased feto-maternal haemorrhage.

According to the paper, invasive procedures limit the uptake of prenatal diagnosis for chromosomal and monogenic disorders, because of their associated risk of fetal loss. Throughout the last 20 years there has been much interest in the development of non-invasivie techniques. Of these, isolation of fetal cells from the maternal circulation early in preganacy could replace existing methods such as serum and nuchal screening, since it should allow exact genetic diagnosis without risk to the fetus.

In non-invasive prenatal diagnosis, fetal cells must be distinguished from maternal cells, usually by enriching them to an acceptable level of purity before identifying them as uniquely of fetal orgin. Many approaches have been designed to recover fetal cells from maternal blood, but all are problematic: enrichment procedures result in significant loss of rare fetal cells, and most known fetal cells types are also found in adult blood, rendering identification difficult. The recent identification of mesenchymal stem cells (MSC) in first trimester fetal blood offers the prospect of targeting fetal MSC for non-invasive prenatal diagnosis, since MSC are not known to circulate in healthy adults.

Since between 11-13 weeks is recognized as the optimal time for prenatal diagnosis, as most of the risk of spontaneous miscarriage has passed and late first trimester diagnosis is still possible, the researchers used this as the optimal time for taking fetal cells. Unlike other candidates for non-invasive prenatal diagnosis, fetal MSC have a characteristic morphology and immunophenotype and are readily expandable in vitro and like their counterparts in adult bone marrow they have the capacity to differentiate into a number of mesenchymal lineages.

In their study to establish optimal cell purification and culture systems for detection of fetal MSC in maternal blood, cultured male fetal MSC were mixed with 20-40 ml adult female whole blood, to achieve dilutions of 1 in 10^5 to 1 in 10^8 nucleated cells. Numbers of MSC > 100 were obtained by serial dilutions from a larger known concentration, and, for greater accuracy, cell number of MSC were counted.

Since feto-maternal haemorrhage after first trimester termination of pregnancy results in an 80-fold increase in fetal cell numbers in maternal blood, and is a useful biological model with which to evalute fetal cell enrichement and expansion strategies under development. The researchers applied their enrichment protocol to the isolation of MSC from first trimester post-termination maternal blood. Although it was possible to isolate fetal MSC in maternal blood, fetal cells were identified in only one of 20 patients tested. This suggested to the researchers that fetal MSC are likely to circulate at a very low frequency in maternal blood, since their enrichment protocol in model mixtures was sensitive enough to detect one MSC among 2.5x10^7 adult nucleated cells or one cell in 3.3 ml whole blood. The researchers came up with possible reasons for the low frequency of fetal MSC detected in maternal blood, thinking that fetal MSC may only be present in the cirulation in extremely low numbers at or below the level of sensitivity of the culture assay they used. Among others within the paper.

The authors concluded that they have isolated fetal MSC from maternal blood for the first time, but acknowledge that the rarity of fetal MSC circulating in maternal blood appears to preclude clinical application in non-invasive prenatal testing. Besides this, the discovery of a unique fetal stem cell cirulating in maternal blood with the potential to persist in tissues year after pregnancy provides information about feto-maternal trafficking in early pregnancy and emphasizes increasing awareness that cellular trafficking may have far-reaching biological consequences.

Critique:

Though I found the information within this paper extremely interesting, I found that it lacked necessary information which would of made the paper clearer. I understood that the point of the paper was to look into the identification of mesenchymal stem cells and their use in non-invasive diagnosis. However, the exact reasoning behind the enrichment protocol and how this was useful in identifying and determing fetal MSC was not clear. It was obviously important to the researchers in determining the amount of fetal MSC that was present and identifying them as fetal MSC but it was unclear why it was so important. I found the diagrams to be interesting, since they gave a visual of the cells and tissues which we are discussing, however, it would of been more helpful if the authors of the paper had included some more figures and diagrams of the different trials that they did and the results they obtained. Over all it was an extremely interesting and illuminating paper, giving me more idea of the types of study which are being done with fetal cells, however, the topic is still much discussed and studied and therefore is obviously not fully represented as it may be in the future.

Savannah Isaacs

Assignment #1: Fetal Tissue: Description, Structure, Function & Pathology

The human fetus is a complicated and interesting tissue. The development from a single cell into a newborn infant with many cells, tissues and organs is an interesting and complex series of steps, starting with conception, continuing until birth and even during postnatal development. The ability for our bodies to develop such a small but important concept of life is what interests me most about human fetal tissues. In the following few blogs I will discuss the growth and development of fetal tissues, showing the structure and function of these tissues as they become a tiny human being. From conception until the fetus has developed, the different tissues change and grow, taking over the important functions of our body, such as breathing. I've also included pictures and given information to show this development and their functions. Enjoy!!

Conception:

Figure 1: Journey of the ovum to the uterus (http://www.getceusnow.com/portal/file/ca_clip_image004.gif)

About once every 28 days, in the middle of a woman's menstrual cycle, an ovum bursts from one of her ovaries two walnut-sized organs deep inside her abdomen (Figure to the Right). Surrounded by thousands of nurse cells to feed and protect it along its path, the ovum is drawn into one of two fallopian tubes - long, thin structures that lead to the hollow, soft-lined uterus. While the ovum travels, the spot on the ovary from which it was released, now called the corpus luteum, secretes hormones that prepare the lining of the uterus to receive a fertilized ovum. If pregnancy does not occur, the corpus luteum shrinks, and the uterine lining is discarded 2 weeks later with menstruation (Berk, 2003).

The male produces sperm in vast numbers - an average of 300 million a day. In the final process of maturation, each sperm develops a tail that permits it to swim long distances, upstream in the female reproductive tract and into the fallopian tube, where fertilization usually takes place. The journey is difficult, and many sperm die. Only 300 to 500 reach the ovum, if one happens to be present. Sperm live for up to 6 days and can lie in wait for the ovum, which survives for only 1 day after being released into the fallopian tube. However, most conception result from intercourse during a 3-day period - on the day of or during the 2 days preceding ovulation (Wilcox, Weinberg & Baird, 1995).

With conception, the story of prenatal development begins to unfold. The vast changes that take place during the 38 weeks of pregnancy are usually divided into three periods: the zygote, the embroyo and the fetus.



The Period Of The Zygote:

Figure 2: Zygote (http://encarta.msn.com/media_461533430_761563311_-1_1/Zygote.html)

The period of the zygote lasts about 2 weeks, from fertilization until the tiny mass of cells drifts down and out of the fallopian tube and attaches itself to the wall of the uterus. The zygote's first cell duplication is long and drawn out; it is not complete until about 30 hours after conception. Gradually, new cells are added at a faster rate. By the fourth day, 60 to 70 cells exist that form a hollow, fluid-filled ball called a blastocyst. The cells on the inside, called the embryonic disk, will become the new organism; the outer ring of cells, termed the trophoblast, will provide protective covering and nourishment (Berk, 2003)
.

Implantation:

Figure 3: Implantation of the blastocyst into the uterine lining.

Sometime between the seventh and ninth day, implantation occurs: the blastocyst burrows deep into the uterine lining. Surrounded by the woman's nourishing blood, it starts to grow in earnest. At first, the trophoblast (protective outer layer) multiplies fastest. It forms a membrane, called the amnion, that encloses the developing organism in amniotic fluid. The amnion helps keep the temperature of the prenatal world constant and provides a cushion against any jolts caused by the woman's movement. A yolk sac also appears. It produces blood cells until the developing liver, spleen, and bone marrow are mature enough to take over this function (Moore & Persaud, 1998).

The Placenta and Umbilical Cord:

Figure 4: Placenta & Umbilical Cord

(http://www.bpk.cz/img/schema.jpg)

By the end of the second week, cells of the trophoblast form another protective membrane - the chorion, which surrounds the amnion. From the chorion, tiny hairlike villi, or blood vessels, emerge. As these villi burrow into the uterine wall, a special organ called the placenta starts to develop. By bringing the embryo's and mother's blood close together, the placenta will permit food and oxygen to reach the organism and waste products to be carried away. A membrane forms that allows these substances to be exchanged but pervents the mother's and embryo's blood from mixing directly (Berk, 2003).

The placenta is connected to the developing organism by the umbilical cord. In the period of the zygote, it appears as a primitive body stalk, but during the course of the pregnancy, it grows to a lenght of 30 to 90 centimetres (1 to 3 feet). The umbilical cord contains one large vein that delivers blood loaded with nutrients and two arteries that remove waste products. The force of the blood flowing through the cord keeps it firm so it seldom tangles while the embryo, like a space-walking astronaut, floats freely in its fluid-filled chamber (Moore & Persaud, 1998).

By the end of the period of the zygote, the developing organism has found food and shelter in the uterus. Already, it is a complex being. These dramatic beginnings take place before most mothers know they are pregnant (Berk, 2003).

The Period Of The Embryo:

The period of the embryo lasts from implantation through the eighth week of pregnancy. During these brief 6 weeks, the most rapid prenatal changes take place. Because the groundwork for all body structures and internal organs is laid down, the embryo is especially vulnerable to interference with healthy development. But a short time span of embryonic growth helps limit opportunities for serious harm (Berk, 2003).

Figure 1: 6 week old developing embryo.

Last Half Of The First Month:

In the first week of this period, the embryonic disk forms three layers of cells: (1) the ectoderm, which will become the nervous system and skin; (2) the mesoderm, from which will develop the muscles, skeleton, circulatory system, and other internal organs; and (3) the endoderm, which will become the digestive system, lungs, urinary tract, and glands. These three layers give rise to all parts of the body (Berk, 2003).

Figure 2: Cell Differentiation (http://en.wikipedia.org/wiki/Image:Cell_differentiation.gif)

At first, the nervous system develops fastest. The ectoderm folds to form a neural tube, or primitive spinal cord. At 3 1/2 weeks, the top swells to form a brain. Production of neurons (nerve cells that store and transmit information) begins deep inside the neural tube. Once formed, neurons travel along tiny threads to their permanent locations, where they will form the major parts of the brain (Nelson & Bosquet, 2000).

While the nervous system is developing, the heart begins to pump blood, and muslces, backbone, ribs, and digestive tract appear. At the end of the first month, the curled embryo, only 0.6 centimetres (one-quarter inch) long, consists of millions of organized groups of cells with specific functions (Berk, 2003).

The Second Month:

In the second month, growth continues rapidly. The eyes, ears, nose, jaw and neck form. Tiny buds become arms, legs, fingers, and toes. Internal organs are more distinct: the intestines grow, the heart develops separate chambers, and the liver and spleen take over production of blood cells so the yolk sac is no longer needed. Changing body proportions cause the embryo's posture to become more upright. Now 2.5 centimetres (1 inch) long and 4 grams (one-seventh of an ounce) in weight, the embryo can sense its world. It responds to touch, particularly in the mouth area and on the soles of the feet. And it can move, although its tiny flutters are still too light to be felt by the mother (Nilsson & Hamberger, 1990).

The Period Of The Fetus:

Lasting from the ninth week until the end of pregnancy, the period of the fetus is the "growth and finishing" phase. During this longest prenatal period, the organism increases rapidly in size, especially from the ninth to the twentieth week (Moore & Persaud, 1998).

Figure 1: Fetal Development from 8 weeks til 40 weeks.

(http://www.lpch.org/photos/greystone/ei_0128.jpg)

The Third Month:


In the third month, the organs, muscles, and nervous system start to become organized and connected. The brain signals, and in response, the fetus kicks, bends its arms, forms a fist, curls its toes, opens its mouth, and even sucks its thumb. The tiny lungs begin to expand and contract in an early rehearsal of breathing movements. By the twelfth week, the external genitals are well formed, and the sex of the fetus can be detected with ultrasound. Other finishing touches appear, such as fingernails, toenails, tooth buds, and eyelids that open and close. The hearbeat is now stronger and can be heard through a stethoscope (Berk, 2003).

Prenatal development is often divided into trimesters, or three equal time periods. At the end of the third month, the first trimester is complete.


The Second Trimester:

By the middle of the second trimester, between 17 and 20 weeks, the new being has grown large enough that the mother can feel its movements. If we could look inside the uterus at this time, we would find the fetus completely covered with a white, cheeselike substance called the vernix. It protects the skin from chapping during the long months spent in the amniotic fluid. A white, downy hair covering called lanugo also appears over the entire body, helping the vernix stick to the skin (Berk, 2003).

At the end of the second trimester, many organs are quite well developed. And a major milestone is reached in brain development, in that most neurons are in place; few will be produced after this time. However, glial cells, which support and feed the neurons, continue to increase at a rapid rate throughout the remaining months of pregnancy, as well as after birth (Nowakowski, 1987).

Brain growth means new behavioural capacities. The 20-week-old fetus can be stimulated as well as irritated by sounds. And if a doctor has reason to look inside the uterus with fetoscopy, fetuses try to shield their eyes from the light with their hands, indicating that sight has begun to emerge (Nilsson & Hamberger, 1990). Still, a fetus born at this time cannot survive. Its lungs are too immature, and the brain cannot yet control breathing and body temperature (Berk, 2003).


The Third Trimester:

During the final trimester, a fetus born early has a chance for survival. The point at which the baby can first survive is called the age of viability. It occurs sometime between 22 and 26 weeks (Moore & Persaud, 1998). If born between the seventh and eighth months, a baby would still have trouble breathing , and oxygen assistance would be necessary. Although the respiratory centre of the brain is now mature, tiny air sacs in the lungs are not yet ready to inflate and exchange carbon dioxide for oxygen (Berk, 2003).

The brain continues to make great strides during the last 3 months. The cerebral cortex, the seat of human intelligence, enlarges. As neurological organization improves, the fetus spends more time awake. At 20 weeks, heart rate variability reveals no periods of alertness. But by 28 weeks, fetuses are awake about 11 percent of the time, a figure that rises to 16 percent just before birth (DiPietro et al, 1996).

Figure 2: Image of a third trimester baby

(http://distractible.org/wp-content/uploads/2007/09/28-week-36364-7.jpg)

The third trimester brings greater responsiveness to external stimulation. Around 24 weeks, fetuses can first feel pain, so after this time painkillers should be used in any surgical procedures (Royal College of Obstetricians and Gynecologists, 1997). By 25 weeks, fetuses react to nearby sounds with body movements and in the last weeks of pregnancy, they learn to prefer the tone and rhythm of their mother's voice (Berk, 2003).

During the final 3 months, the fetus gains more than 2300 grams (5 pounds) and grows 18 centimetres (7 inches). As it fills the uterus, it gradually moves less often. In addition, brain development, which permits the organism to inhibit behaviour, may contribute to a decline in physical activity (DiPietro et al, 1996).

In the eighth month, a layer of fat is added under the skin to assist with temperature regulation. The fetus also receives antibodies from the mother's blood to protect against illnesses, since the newborn's own immune system will not work well until several months after birth. In the last weeks, most fetuses assume an upside-down position, partly because of the shape of the uterus and because of gravity: the head is heavier than the feet. Growth slows, and birth is about to take place (Berk, 2003).

Prenatal Environmental Influences:

In this blog I will discuss the many environmental factors which influence the growth and development of fetal tissues. Though we only require one pathology which may influence our tissue, i've gone into detail of all the important ones which influence fetal tissue because it is extremely interesting to see the different things which could of affected our own individual growth. Feel free to read as much as you choose, though everything is extremely interesting.

Teratogens:

The term teratogen refers to any environmental agent that causes damage during the prenatal period. It come from the Greek word teras, meaning "malformation" or "monstrosity." This label was selected because scientists first learned about harmful prenatal influences from babies who had been profoundly damaged (Berk, 2003).

(1) Prescription and Nonprescription Drugs:

In the early 1960s, the world learned a tragic lesson about drugs and prenatal development. At that time, a sedative called thalidomide was prescribed in Canada, Europe, and South America to help with symptoms associated with morning sickness. When taken by mothers between the fourth and sixth week after conception, thalidomide produced gross deformities of the embryo's developing arms and legs, less frequently, damage to the ears, heart, kidneys, and gentials. Also children exposed to thalidomide grew older, many scored below average in intelligence (Berk, 2003).

Another medication, a synthetic hormone called diethylstilbestrol (DES), was widely prescribed between 1941 and 1971 to prevent miscarriages. As daughters of these mothers reached adolescence and young adulthood, they showed unusally high rates of cancer of the vagina and malformations of the uterus (Berk, 2003).

Any drug taken by the mother that has a molecule small enough to penetrate the placental barrier can enter the embryonic or fetal bloodstream. Despite the lesson of thalidomide, many pregnant women continue to take over-the-counter drugs, such as aspirin, without consulting their doctors which may lead to low birth weight of the baby (Berk, 2003).

(2) Illegal Drugs:

The use of highly addictive mood-altering drugs, such as cocaine and heroin has become widespread. Babies born to users of cocaine, heroin, or methadone are at risk for a wide variety of problems, including prematurity, low birth weight, physical defects, breathing difficulties, and death around the time of birth. In addition, these infants arrive drug addicted. They are often feverish and irritable and have trouble sleeping (Berk, 2003).

Throughout the first year, heroin and methadone exposed infants are less attentive to the environment, and their motor development is slow. After infancy, some children get better, whereas others remain jittery and inattentive. The kind of parenting these youngsters receive may explain why problems last for some but not for others (Cosden, Peerson, & Elliott, 1997).

Growing evidence on cocaine suggests that many prenatally exposed babies have lasting difficulties. Cocaine constricts the blood vessels, causing oxygen delivered to the developing organism to fall dramatically for 15 minutes following a high dose. It also alters the production and functioning of neurons and the chemical balance in the fetus's brain. These effects may contribute to a specific set of cocaine-linked physical defects, including eye, bone, genital, urinary tract, kidney, and heart deformities, as well as brain hemorrhages and seizures (Berk, 2003).

(3) Tobacco:

The most well-known effect of smoking during pregnancy is low birth weight. But the likelihood of other serious consequences, such as miscarriage, prematurity, impaired heart rate and breathing during sleep, infant death, and cancer later in childhool, is also increased. The more cigarettes a mother smokes, the greater the chances that her baby will be affected. If a pregnant woman stops smoking at any time, even during the last trimester, she reduces the likelihood that her infant will be born underweight and suffer from future problems (Berk, 2003).

(4) Alcohol:

Fetal Alcohol Syndrome (FAS) is the name for a constellation of symptoms that include mental retardation, overactivity, and impairments in motor coordination, attention, memory, language, planning, and problem solving. Accompanying physical symptoms include slow physical growth and a particular pattern of facial abnormalities: widely spaced eyes; short eyelid openings; a small, upturned nose; a thin upper lip; and a small head, indicating that the brain has not developed fully. Other defects may include the eyes, ears, nose, throat, heart, genitals, urinary tract, or immune system (Berk, 2003).

In all babies with FAS, the mother drank heavily through most or all of her pregnancy. Sometimes children display only osme of the physical abnormalities associated with FAS. In these cases, the child is said to suffer from fetal alcohol effects (FAE). Usually, their mothers drank alcohol in smaller quantities or less often. The particular defects of FAE children vary with timing and length of alcohol exposure during pregnancy (Berk, 2003).

(5) Radiation:

When mothers are exposed to radiation during pregnancy, harm can come to the embryo or fetus. Defects due to radiation were tragically apparent to the children born to pregnant Japanese women who survived the atomic bombing of Hiroshima and Nagasaki during World War 2. Similar abnormalities surfaced in the 9 months following the 1986 Chernobyl, Ukraine, nuclear power plant accident. After each disaster, the incidence of miscarriage, small head size (indicating an undeveloped brain), physical deformities, and slow physical growth rose dramatically (Berk, 2003).

(6) Environmental Pollution:

An astounding number of potentially dangerous chemicals are released into the environment in industrialized nations. Over 100 000 are in common use, and many new ones are introduced each year.

Mercury: is an established teratogen. In the 1950s, an industrial plant released waste containing high levels of mercury into a bay providing food and water for the town of Minimata, Japan. Many children born at the time were mentally retarded and showed other serious symptoms, including abnormal speech, difficulty in chewing and swallowing, and uncoordinated movements. Autopsies of those who died revealed widespread brain damage (Dietrich, 1999).

Lead: is another teratogen. It is present in old paint and in certain industrial materials. High levels of lead exposure are consistently related to prematurity, low birth weight, brain damage, and a wide variety of physical defects (Berk, 2003).

Polychlorinated biphenyls (PCBs): were used to insulate electrical equipment for many years, until research showed that they found their way into waterways and entered the food supply. Prenatal exposure to very high levels of PCBs in rice oil resulted in low birth weight, discoloured skin, deformities of the gums and nails, brain-wave abnormalities, and delayed cognitive development (Berk, 2003).

(7) Maternal Disease:

Five percent of women catch an infectious disease while pregnant. Most of the illnesses, such as the common cold, seem to have no impact on the embryo or fetus. However, a few diseases can cause extensive damage.

Viruses: Rubella ~ is a well known teratogen. In the mid-1960s, a worldwide epidemic of rubella led to the birth of many thousands of babies with serious defects. The greatest damage occurs when rubella strikes during the embryonic period. More than 50 percent of infants whose mothers become ill during that time show heart defects; eye cataracts; deafness; gential, urinary, and intestinal abnormalities; and mental retardation. Infection during the fetal period is less harmful, but low birth weight, hearing loss, and bone defects may still occur (Berk, 2003).
The Human Immunodeficiency Virus (HIV) ~ which leads to acquired immune deficiency syndrome (AIDS), a disease that destroys the immune system, has infected increasing numbers of women over the past decade. When AIDS victims become pregnant, about 20 to 30 percent of the time they pass the deadly virus to the developing organism. AIDS progresses rapidly in infants. By 6 months, weight loss, diarrhea, and repeated respiratory illnesses are common. The virus also causes brain damage. Most prenatal AIDS babies survive only 5 to 8 months after the appearance of symptoms (Berk, 2003).

Bacterial and Parasitic Diseases: Among the most common is toxoplasmosis, cuased by a parasite found in many animals. Pregnant women may become infected from eating raw or undercooked meat or from contact with the feces of infected cats. About 40 precent of women who have the disease transmit it to the developing organism. If it strikes during the first trimester, it is likely to cause eye and brain damage. Later infection is linked to mild visual and cognitive impairments (Berk, 2003).

Reference Page

Berk, L. 2003. Child Development. Can Ed. Pearson Education Canada Inc., Toronto, Ontario.

Cosden, M., Peerson, S., & Elliott, K. 1997. Effects of prenatal drug exposure on birth outcomes and early child developmental. Journal of Drug Issues, 27, pp 525-539.

Dietrich, K. N. 1999. Environmental toxicants and child development. In H. Tager-Flusberg (Ed.), Neuro-developmental disorders. Boston: MIT Press. Pp 469-490.

DiPietro, J. A., Hodgson, D. M., Costigan, K.A., & Hilton, S. C. 1996. Fetal neurobehavioral development. Child Development, 67, pp 2553-2567.

Moore, K. L., & Persaud, T. V. N. 1998. Before we are born. 5th ed. Philadelphia: Saunders.

Nelson, C. A., & Bosquet, M. 2000. Neurobiology of fetal and infant development: Implications for infant mental health. In C. H. Aeanah, Jr. (Ed.), Handbook of infant mental health. New York: Guilford.

Nilsson, L., & Hamberger, L. 1990. A Child is born. New York: Delacorte.

Nowakowski, R.S. 1987. Basic concepts of CNS development. Child Development, 58, 568-595.

Royal College of Obstetricians and Gynecologists. 1997. Report of thepanel to review fetal pain. London: Author.

Wilcox, A. J., Weinberg, C. R., & Baird, D. D. 1995. Timing of sexual intercourse in relation to ovulation: Effects on the probability of conception, survival of the pregnancy, and sex of the baby. New England Journal of Medicine, 333, 1517-1519.