38 Pregnancy

Lothar Thomas

38.1 Laboratory findings in normal pregnancy

Physiological changes in the maternal organism are an important prerequisite for a normal pregnancy and undisturbed development of the embryo and fetus. The adaptive processes, which affect nearly the entire maternal organism and lead to significant changes in the physiology, are mostly controlled by the embryo and the growing fetus /1/.

The placenta is a unique organ, arising de novo directly to the development and growth of the fetus. Placenta weighs normally 400–500 g and tends to be smaller in preeclampsia and pregnancy induced hypertension. Since substrates needed for embryonal and fetal metabolism come from the blood of the mother and catabolic products are passed back into the circulation of the mother the placenta gives a clear impression about gestational pathological changes during pregnancy.

Pregnancy produces profound changes in /1/:

• Hormone levels
• Metabolism of carbohydrates, lipids and proteins
• Blood volume.

These changes cause significant increase or decline in biochemical markers that increase during pregnancy.

38.1.1 Hormones

The physiological adjustments to pregnancy are mainly regulated by hormones produced by the placental trophoblast; however, as an incomplete endocrine organ, the placenta partly relies on maternal and fetal hormone precursors for the synthesis of steroids (feto-maternal placental unit). Pregnancy also causes significant changes to hormones of the maternal pituitary-adrenocortical axis, pituitary-somatotroph axis, and the pituitary-gonadal axis.

Refer to:

• Tab. 38.1-1 – Physiologic hormonal changes during pregnancy
• Fig. 38.1-1 – Synthesis of estrogens by the feto-maternal placental unit
• Fig. 38.1-2 – Placental synthesis of progesterone.

Adequate levels of thyroid hormones are of importance for normal reproductive function. Refer to Section 38.1.8 – Thyroid function during pregnancy.

38.1.2 Blood volume and hematopoiesis

About half of the physiological weight gain during pregnancy is due to maternal tissue growth and fluid retention. By the end of pregnancy, fluid retention amounts to approximately 4–6 liters. One important stimulus of the increased fluid and sodium retention is primary arterial vasodilatation, which has the following consequences /2/:

• Decrease in systolic and diastolic blood pressure
• Increase in cardiac output
• Non osmotic stimulation of the thirst mechanism and increased secretion of vasopressin
• Stimulation of the renin-angiotensin-aldosterone system (RAAS).

The RAAS is stimulated early in pregnancy in association with a decrease in vascular resistance despite an increase in blood volume. As a result, there is a progressive increase in the plasma concentrations of renin and aldosterone. There is a positive correlation between the increase in renin, aldosterone and progesterone during pregnancy. However, there is no hypokalemia, and serum sodium levels remain normal despite increased aldosterone. Refer to Chapter 31 – Mineralocorticoid hypertonus.

The above mechanisms lead to the following changes /3/:

• Increase in the maternal blood volume from 6 weeks’ gestation, by up to 1.6 L at 33 weeks
• Approximately 60–80% increase in the renal blood flow from mid-pregnancy
• Increase in the glomerular filtration rate from week 4 throughout the entire pregnancy and persistent 50% elevation until after delivery.

Despite increased erythropoietin synthesis, red blood cell count hemoglobin concentration and hematocrit decrease.

Hematopoiesis

Plasma volume increases progressively throughout normal pregnancy, however the distribution of red and white blood cells change /52/:

• Because the expansion of plasma volume is greater than the red blood cell mass, there is a decline in hemoglobin and hematocrit level.
• Increased spleen size and plasma volume contribute to mild thrombocytopenia
• Immune defense changes to support fetal development which increases white blood cells.

Refer to Tab. 38-1-9 – Trimester-specific reference intervals for hematology parameters.

Hemoglobin (Hb)

Anemia is most common disorder that increases maternal morbidity and is most common in underdeveloped countries. There are three causes of anemia:

• Decreased red cell production as in iron, vitamin B12 and folate deficiency
• Destruction of red cells because of hemoglobinopathies
• Loss of red blood cells in hemorrhage.

In normal pregnancy, the body’s total red blood cell mass may increase by 20–30% depending on the iron and vitamin status. Although the total red blood cell count and thus the capacity for transporting O₂ increases, the disproportionate increase in plasma volume results in a decrease in Hb and hematocrit and the development of physiological anemia in pregnancy. The WHO recommends that the Hb concentration should not fall below 110 g/L at any time during pregnancy.

The synthesis of maternal and fetal erythrocytes requires an extra 4 mg of iron per day during pregnancy, 2.5 mg during early pregnancy and 6.6 mg during the last trimester, in addition to the physiological iron demand /4/. Although dietary iron absorption increases during pregnancy, the daily iron requirement cannot be met even with an optimal diet. To ensure sufficient synthesis of Hb and iron containing enzymes, the body has to draw on iron stores. Since in many women of childbearing age these stores are as low as 0.2–0.5 g, and pregnant women often fail to properly follow the recommendations for oral iron supplementation, postpartum anemia has an incidence of 20–40%, depending on the population examined /5/.

Thrombocyte (platelet) count

Platelet count decreases by 11.9% between 20th week of gestation and delivery, and 8% of pregnant women have a platelet count below 130 × 10⁹/L.

Refer to Tab. 15.11-9 – Thrombocytopenia in pregnancy.

Leukocyte (WBC) count

The number of polymorphonuclear granulocytes increases during pregnancy /6/.

Refer to Tab. 38.1-2 – Maternal leukocyte count in pregnancy.

38.1.3 Kidney function

Compared to non-pregnant levels plasma volume, renal plasma flow, and glomerular filtration rate significantly increase during pregnancy. Renal plasma flow, measured by clearance (as result of renal vasodilatation), increases by 60–80%, the glomerular filtration rate (GFR, measured by inulin clearance), increases by 50% from the end of the first trimester. Maternal renal function tests show no significant difference between the reference intervals established with healthy non-pregnant women /34/.

Elevated levels are associated with increased risks of developing various pregnancy complications and adverse perinatal outcomes. Notably elevated third trimester renal function tests show strong risks of preterm birth and fetal growth retardation. The serum levels of uric acid and cystatin C are higher in the third trimester compared to the first trimester in healthy pregnant women. No significant difference exist in urea and creatinine between the first and the third trimester.

Creatinine

The concentration of serum creatinine decreases by 10% during the first trimester and by 30% during the last trimester. Mean creatinine levels prior to conception and in the following trimesters are shown in Tab. 38.1-3 – Maternal serum creatinine in pregnancy. A serum creatinine level above 0.85 mg/dL (75 μmol/L) is considered an indicator of early renal dysfunction /7/ and a level above 1.2 mg/dL (106 μmol/L) is highly suggestive of preeclampsia /8/.

The equations for estimating GFR from serum creatinine as well as creatinine clearance cannot be applied in pregnancy.

The upper reference limits for creatinine, cystatin C and neutrophil gelatinase-associated lipocalin change during pregnancy /25/.

Refer to Tab. 38.1-5 – Upper reference limits in pregnancy.

Urea

The urea concentration is low in pregnancy.

Uric acid

The uric acid concentration in serum increases progressively during pregnancy /8/. This is thought to be due to a reduction in tubular secretion and increased oxidative stress in the pregnant woman. A disproportionate increase in uric acid is seen in pregnant women with hypertension and preeclampsia.

Tab. 38.1-4 – Serum uric acid in pregnancy compares the uric acid levels of non-hypertensive and hypertensive pregnant women. The determination of serum uric acid is a screening test for suspected preeclampsia. Using a cutoff of ≥ 5.5 mg/dL (327 μmol/L) in pregnant women with newly onset hypertension, the diagnostic sensitivity for preeclampsia is 69%, with a specificity of 51% /9/.

Proteinuria

Albumin excretion increases slightly but progressively until term. Total protein excretion can be up to 300 mg/24 h.

Outcomes in pregnancy with CKD

The risk of pregnancy complications (odds ratios) among patients with CKD versus those without CKD were as follows /10/:

• Preeclampsia 10.36 (95% CI 6.28–17.09)
• Premature delivery 5.72 (95% CI 3.26–10.03)
• Small for gestational age/low birth weight 4.85 (95% CI 3.03–7.76)
• Cesarean section 2.67 (95% CI 2.01–3.54)
• Failure of pregnancy 1.80 (95% CI 1.03–3.13)

Renal function tests in twin pregnancy

In twin pregnancy renal function tests did not change to a greater extent in comparison to a single pregnancy. Refer to Tab. 38.1-8 – Reference intervals (95%) in the first and third trimester of singleton and twin pregnancy.

38.1.4 Liver function

The liver undergoes physiological changes during pregnancy, since increased synthetic and excretory functions have to be performed. However, the liver has sufficient reserve capacity to deal with this, and as a result there are no pathological laboratory findings.

Enzymes

Aminotransferases, GGT and cholinesterase are within the reference interval. Therefore, any elevation of these enzymes during pregnancy is indicative of cellular damage and needs to be investigated /11/. One exception is alkaline phosphatase (ALP), whose activity increases progressively from week 20 of gestation, reaching levels 2–4-fold the original level by the end of pregnancy. ALP is produced by the placenta.

38.1.5 Hemostasis

During pregnancy, the hemostatic balance shifts towards a hyper coagulable state to prevent complications associated with blood loss during delivery. For further information refer to Section 16.4 – Hemostasis in pregnancy. D-dimer levels may vary significantly in the pregnant women at term regardless of the presence or absence of complications /12/.

38.1.6 Glucose metabolism

During the first trimester of pregnancy, maternal insulin sensitivity is increased. The growing embryo releases increased insulin, which is most marked during the last trimester, because the hormones estradiol, progesterone, hCG and hPL cause mild insulin resistance in the tissues.

Pregnant women have lower fasting glucose levels than non pregnant women, since the fetus requires 30–50 g of glucose per day during the last trimester. Pregnant women are prone to ketosis as they feel hungry faster and their metabolism draws on alternative sources of energy, in particular fat, since glucose is reserved for the fetus. After an overnight fast, plasma ketone bodies and free fatty acids rise while plasma glucose is 63–75 mg/dL (3.5–4.2 mmol/L) i.e., about 15 mg/dL (0.8 mmol/L) lower than prior to conception /13/.

Food intake leads to a quick shift to an anabolic state, with increased levels of glucose, triglycerides and insulin, as well as suppression of glucagon. As a result /13/:

• More glucose is made available to the fetus
• The mother is provided with energy from lipids in the form of triglycerides
• There is a low tendency to activate gluconeogenesis, glycogenolysis and ketogenesis.

These processes lead to higher increases in blood glucose after meals and a more rapid decline before meals. Therefore, postprandial glucose levels, with 130–140 mg/dL (7.2–7.8 mmol/L), are slightly higher and pre prandial levels, with 63–75 mg/dL (3.5–4.2 mmol/L), are slightly lower than outside of pregnancy. However, the mean daily blood glucose level of 90–100 mg/dL (5.0–5.6 mmol/L) is the same in pregnant and non pregnant women.

Mild glucosuria in pregnancy is likely due to a reduced renal threshold.

Based on the recommendations of the American Diabetes Association’s Standards of Medical Care /14/, in healthy pregnancy fasting and pre prandial glucose levels should be ≤ 95 mg/dL (5.3 mmol/L) in capillary whole blood and ≤ 92 mg/dL (5,1 mmol/L) in plasma, and HbA_1c should be below 6.0% (Section 3.1.6 – Gestational diabetes mellitus (GDM)). All pregnant women should undergo a 75 g oGTT (Section 3.5. – Oral glucose tolerance test). In diabetic pregnancies therapeutic metabolic control should be monitored by measuring pre- and postprandial blood glucose levels (Tab. 3.1-9 – Glycemic goals in the treatment of diabetes mellitus).

Gestational diabetes causes birth of infants who are large for gestational age. In a study /32/ women at 24 to 32 weeks’ gestation were evaluated for gestational diabetes with the use of higher and lower criteria for diagnosis using a 75 g oral glucose tolerance test (oGTT) for gestational diabetes at 24 to 32 weeks’ gestation.

Gestational diabetes mellitus was diagnosed in early pregnancy:

• in the lower glycemia-criteria group: fasting plasma glucose ≥ 92 mg/dL (5.1 mmol/l) or 1 h level ≥ 180 mg/dL (10.0 mmol/L) or 2 h level ≥ 153 mg/dL (8.5 mmol/L)
• in the higher glycemia criteria group: fasting plasma glucose ≥ 99 mg/dL (5.5 mmol/l) or 2 h level ≥ 162 mg/dL (9.0 mmol/L).

In pregestational and gestational diabetes mellitus the risks of pregnancy complications were increased.

Pregnancy complications in women with pregestational and gestational diabetes mellitus were the following /33/:

• Gestational diabetes: associations are higher than in normal pregnancies. The disorders are relative elevated ratio for premature birth, large for gestational age, cesarean section, and transfer of the newborn to a special hospital.
• Pregestational diabetes: the associations were even stronger than in women with gestational diabetes and the risk of pregnancy complications increased over the study period.

38.1.6.1 Gestational diabetes mellitus diagnosed early in pregnancy

Immediate treatment of gestational diabetes before 20 weeks gestation leads to a lower incidence of adverse neonatal outcomes than no immediate treatment. Gestational diabetes mellitus is a common pregnancy complication e.g., preeclampsia, obstetrical interventions, large-for-gestational-age neonates, birth trauma, and neonatal hypoglycemia. Hyperglycemia shows accelerated fetal growth by 24 to 28 weeks gestation and has greater perinatal mortality than women who receive a diagnosis of gestational diabetes later in pregnancy. In a study /37/ immediate treatment of gestational diabetes before 20 weeks' gestation led to a modest lower incidence of a composite of adverse neonatal outcomes than no immediate treatment; no material differences were observed for pregnancy-related hypertension or neonatal lean body mass.

38.1.7 Protein metabolism

The total protein concentration decreases during pregnancy. At 28 weeks’ gestation it is 10–15 g/L lower than prior to conception. There is dysproteinemia, with a decrease in the albumin and γ-globulin fractions and a simultaneous increase in the α₁-, α₂- and β-globulin fractions. The albumin/globulin ratio falls from 1.4 : 1 outside of pregnancy to 1 : 1 during the third trimester. The dysproteinemia is caused by /15/:

• The increase in plasma volume
• The hormonally controlled, increased synthesis of acute-phase proteins and lipoproteins localized in the α- and β-globulin fractions, as well as the reduced synthesis of albumin and IgG.

Albumin

The albumin concentration decreases progressively with advancing gestation, reaching levels as low as 32–35 g/L at term, which is more than 20% lower than outside of pregnancy. This is due to the increased plasma volume, since the absolute intravascular albumin mass is increased by 20%. The urinary albumin excretion increases slightly but steadily up to 200 mg/24 h with advancing gestation.

Immunoglobulins

Significant reduction in IgG, IgA, and IgM.

Acute-phase proteins

Increase in α₁-antitrypsin, ceruloplasmin, C4, thyroxine-binding globulin (TBG), and corticosteroid-binding globulin (CBG).

38.1.8 Thyroid function

The placenta is impermeable to maternal TSH. During the first and second trimesters of pregnancy thyroid hormone is provided to the fetus mainly from transplacental thyroid hormones (e.g., mainly T4) because fetal thyroid follicular epithelial cells cannot yet synthesize thyroid hormones during the first 12 weeks of pregnancy.

Starting at the last trimester, the fetus produces thyroid hormones due to continued development of the hypothalamus and pituitary, from which TRH and TSH, respectively, are produced. From this time on the fetus competes with the maternal thyroid gland for any available iodide. As the fetus reaches term, TSH, T4 and T3 are all increased. TSH peaks at 30 minutes of life in the normal newborn in response to cold exposure, and steadily decreases over the next 24 hours. The concentration may remain elevated over the first week of life.

In healthy pregnant women the production of thyroxine (T4) and triiodothyronine (T3) and the iodine requirement increase by approximately 50%. The volume of the thyroid gland increases by 10% in iodine replete countries but by 20–40% in areas of iron deficiency /16/.

By week 7 following conception the concentration of thyroxine binding globulin increases, reaching a peak at week 16 of gestation and remain high until delivery.

Thyroid hormone seems to be important in placentation and regulation of early pregnancy which might explain the association between hypothyroidism and gestational hypertensive disease and pre tem birth. Thyroid hormone promotes growth via its effects on protein synthesis and plays an important role in differentiation, development and tissue maturation. Thyroid hormone is essential for brain proliferation prior to gestational week 20. Therefore, brain development depends on maternal serum thyroid hormone concentration.

Normal reference range for serum TSH in each trimester of pregnancy

In the first trimester measurement of maternal TSH results in a reduction in serum TSH level because hCG directly stimulates the TSH receptor, resulting in a TSH concentration below the non-pregnant lower limit of 0.4 mU/L.

According to the Guidelines of the American Thyroid Association for the Diagnosis and Management of Thyroid Disease during Pregnancy and Postpartum /16/ a downward ship of the TSH reference range occurs during pregnancy with a reduction in both the lower (decreased by about 0.1–0.2 mU/L) and the upper limit of maternal TSH (decreased by about 0.5–1.0 mU/L) relative to the typical nonpregnant reference range. The largest decrease is observed during the first trimester, because of elevated levels of serum hCG directly stimulating the TSH receptor and thereby increasing thyroid hormone production. Thereafter, serum TSH and its reference range gradually rise in the second and third trimesters, but nonetheless remain lower than in non-pregnant women. In pregnancies (i.e., mostly twins) and hCG levels > 200,000 IU/L TSH is suppressed (≤ 0.2 mU/L) in 67% of women /16/.

According to the 2014 European Thyroid Association Guidelines for the Management of Subclinical Hypothyroidism in Pregnancy and Children /17/ the following upper limits for TSH and recommendations are proposed if specific laboratory reference ranges are not available:

• First trimester 2.5 mU/L
• Second trimester 3.0 mU/L
• Third trimester 3.5 mU/L
• TSH should be measured at the beginning of pregnancy if screening is performed. IF TSH is elevated, FT4 and anti-thyroid peroxidase antibodies (anti-TPOAb) should be determined. This will unable sublinical or overt hypothroidism to be diagnosed.

Maternal thyroid diseases and adverse pregnancy outcomes

Thyroid diseases affect up to 4% of all pregnancies with primary hypothyroidism being the most prevalent disease. In a study /18/ of a large contemporary cohort of racially and ethnically diverse US women the primary hypothyroidism was associated with an increased odds of preeclampsia, pre term birth, gestational diabetes, induction, cesarean section, and intensive care unit admission.

A meta-analysis /19/ between maternal subclinical hypothyroidism and growth, development, and childhood intelligence showed an increased risk of adverse neonatal outcome, including delayed intellectual and motor development, low birth weight, fetal distress and fetal growth restriction.

Pregnant women with hyperthyroidism have an increased odds of hypertension, pre term birth and intensive care unit admission /18/. Maternal hyperthyroidism is associated with fetal growth restriction, tachycardia, and even fetal hypothyroidism, because thyroid hormones, TSH receptor antibodies, and antithyroid drugs cross the placenta.

38.1.9 Obesity

A body mass index of ≥ 30 is used to define obesity in non-pregnant persons. However this definition does not account for individual differences in frame size and lean body mass and does not take into consideration the fat distribution pattern, because visceral obesity posing greater metabolic risks than subcutaneous obesity /26/. In the United States with a life birth in 2020 only 40% entered pregnancy with a normal-range body mass index, where as 26.7% were overweight and 29.5% were obese /27/. Obesity is associated with a range of pregnancy-related perturbations e.g., disorders of the hypothalamic-pituitary-ovarian axis which may lead to menstrual dysfunction and other maternal complications that can affect endometrial implantation. Main risks of adverse outcomes of pregnancy with obesity are gestational diabetes, preeclapsia, macrosomia, failure of labor to progress, and postpartum infection. Refer also to Tab. 38.1-6 – Adverse outcomes in pregnancies associated with obesity.

Obesity before pregnancy is disproportionally prevalent among young women. In preconception care women should be informed by their treating doctor about the risks of obesity (e.g., subfertility, increased risk of miscarriage, preeclampsia, and gestational diabetes) and the benefits of weight loss before pregnancy.

38.1.10 Hypertension

Hypertension complicates pregnancy and contributes greatly to maternal and fetal morbidity and mortality. Gestational hypertension is used to describe any form of new-onset pregnancy-related hypertension. There are five types of hypertension: pregnancy induced, preeclampsia, eclampsia, preeclampsia superimposed on chronic hypertension, and chronic hypertension. Diagnosis of gestational hypertension is made in pregnant women whose blood pressure reaches > 140/90 mmHg for the first time during pregnancy. Gestational hypertension is also called transient hypertension because the blood pressure returns to normal by 3 months postpartum. Hypertensive disorders complicating pregnancy are common and along with hemorrhage and infection contribute greatly to maternal and fetal morbidity and mortality. Studies estimate that 2.6 to 7.6% of maternal deaths are associated with hypertension. As severity of hypertension increases the placental weight decreases. Placenta weighs normally 450–500 g. In cases of pregnancy induced hypertension and in preeclampsia mean placental weight is 450 to 413 g /31/.

38.1.11 Preeclampsia

Preeclampsia is the most common hypertensive disorder of pregnancy characterized:

• by sudden onset of high blood pressure usually after 20 weeks of gestation
• by high concentration of protein in urine
• in women with a history of hypertension during pregnancy have an increased risk of future hypertension, stroke, and cardiovascular disease
• in offsprings from pregnancies complicated by hypertensive disorders. The offsprings are small for gestational age and have an increased risk of cardiovascular disease in adulthood.

Preeclampsia is the most common hypertensive disorder of pregnancy. The disorder e.g., chronic hypertension, gestational hypertension, chronic hypertension with superimposed preeclampsia are primary causes of offspring morbidity. Findings reveal that most of the women with prior preeclampsia are not aware of hypertensive disorders that can occur during pregnancy before their diagnosis /36/.

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38.2 Pregnancy monitoring

Important aspects in prenatal testing are the diagnosis of

• Pregnancy associated disease and illness unrelated to pregnancy
• Embryonic and fetal disorders.

Illnesses unrelated to pregnancy are described in Ref. /1/.

38.2.1 Prenatal testing

Prenatal testing serves to monitor pregnancy and to identify high risk pregnancies. The aim is to detect fetal defects and possible gynecological complications at an early stage in order to keep perinatal morbidity and mortality to a minimum. Early identification of high risk pregnancies and deliveries allows suitable measures to be taken for the mother and child, as well as more intense and focused intrauterine monitoring and proper planning of delivery management and neonatal care. Fetal and maternal complications can be effectively detected and monitored by a combination of family history, medical examinations, ultrasound, laboratory and, as the case may be, genetic testing. Prenatal testing is regulated by law in many countries and its success depends on participation.

The most common fetal prenatal care tasks that can be influenced are:

• Premature birth (approximately 9%)
• Intrauterine retardation of fetal growth (approximately 5%)
• Fetal infectious disease (approximately 3%)
• Congenital childhood anomaly (1–2%).

Prenatal testing comprises the determination of /2/:

• hCG in serum or urine. The test is used for the early detection of pregnancy, for monitoring the viability of the embryo, and for diagnosing an ectopic (extrauterine) pregnancy.
• Antiphospholipid antibodies in the serum of women with a history of recurrent pregnancy loss. Antiphospholipid antibodies and high risk pregnancy are a common association. Refer to Section 16.22 – Antiphospholipid syndrome.
• Antibodies to pathogens in serum or direct detection of pathogens in maternal urine ad the genital tract smear. The aim is to prevent maternal and fetal infections and any complications they may cause.
• Hemoglobin in EDTA blood, as well as erythrocytic antigens and anti-erythrocytic antibodies in whole blood/serum to prevent fetal and maternal anemia
• α₁-fetoprotein in serum to screen for fetal neural tube defect
• PAPP-A and β-hCG in serum in combination with a nuchal translucency scan to screen for aneuploidy (e.g., Down syndrome) or molecular genetic analysis of cell-free fetal DNA in the maternal blood
• Blood glucose tolerance, using the glucose tolerance test to screen for diabetes mellitus of the pregnant
• Clinical chemistry and hematological tests in blood and urine to detect preeclampsia
• Surfactant in amniotic fluid to assess fetal lung maturity
• Karyotyping of fetal cells to identify aneuploidies
• Molecular genetic tests to identify monogenic diseases.

The main causes of perinatal mortality are pre term births (75%) and term births with fetal retardation. Premature births (prior to 38 weeks’ gestation) account for about 5% of all live births in Europe and North America. Pregnant women aged 35 years and older have a greater risk of obstetric complications and of giving birth to growth retarded and/or premature infants. From age 40, the risk of perinatal mortality is significantly increased.

The number of early embryonic deaths is significant. Roughly 50% of all conceptuses are lost within the first 1–4 weeks of pregnancy as a result of abnormal development. The incidence of spontaneous abortion at 6–8 weeks of gestation is about 18%, decreasing to 3% by 16 weeks’ gestation. Only about 30% of embryos survive to birth as normal fetuses; in addition, there are about 2% of infants with abnormalities /3/.

Refer to:

• Tab. 38.2-1 – Laboratory tests for the diagnosis and monitoring of pregnancy
• Tab. 38.2-2 – Laboratory tests for suspected high risk pregnancy or high risk delivery
• Tab. 38.2-3 – Laboratory tests for suspected embryonic and fetal infection
• Tab. 38.2-4 – Neonatal screening and laboratory findings of important aneuploidies.

38.2.2 Prenatal diagnosis of genetic disorders

A genetic disorder is a disease caused by an abnormality in an individual’s DNA sequence. It is usually a congenital condition. The disorder can range from a chromosomal abnormality to a monogenic disorder.

The risk of major structural birth defects among live births in the United States is approximately 3%. The defects are due to inherited or nouveau genetic rearrangements and mutations as well as with maternal factors (advanced age, diseases, exposure to teratogenic factors). Approximately 1 in 2,000 prenatal cases analyzed with conventional karyotyping has a nouveau, apparently balanced reciprocal translocation that carries a 6.1% risk of congenital malformation /4/. The genetic disorders include multiple congenital anomalies, unexplained developmental delays, intellectual disability, or autism spectrum disorders. Many of these genetic diseases do not manifest until childhood or later.

Monogenic disorders are due to changes or mutations in the DNA sequence of a single gene. There are more than 6,000 known monogenic disorders; they can be inherited and occur with an incidence of about 1 per 200 newborns. In most cases, a single nucleotide polymorphism (SNP, pronounced ”snip”) is present (Fig. 38.2-1 – Single nucleotide polymorphism). Most snips consist of the replacement of cytosine by thymidine. Cytosine is often methylated to methyl cytosine, followed by spontaneous deamination of 5-methyl cytosine to thymine. Depending on the replacement of bases, the information of the codon is changed and can lead to altered protein function with a defect.

For information on the inheritance pattern (autosomal dominant, autosomal recessive, X-linked) refer to Chapter 39 – Genetic diseases. The following are examples of monogenic disorders: cystic fibrosis, sickle cell anemia, Marfan syndrome, hemochromatosis, Huntington’s disease.

While monogenic disorders follow the simple Mendelian inheritance pattern, this is not the case with polygenic disorders, which tend to run in families.

38.2.2.1 Ultrasound examination

The human blueprint is complete in 10–12 weeks of pregnancy (embryonic stage). In the following phase (fetal stage), the state of health of the fetus can be determined. This is done by ultrasound examination, offered routinely between 18 and 20 weeks of gestation and allows detection of major fetal abnormalities /5/. Ultrasound unremarkable findings indicate healthy fetuses with high probability. Approximately 90% of infants with congenital anomalies are born to women without predisposing risk factors. An abnormal ultrasound finding necessitates counseling and discussion of a diagnostic procedure that can be used to assess the possibility that the abnormality has a genetic basis. About a quarter of the genetically diseased fetuses show no morphological abnormalities on ultrasound.

38.2.2.2 Invasive prenatal testing

Invasive methods are:

• Chorionic villus sampling (CVS): In CVS the amniotic sac is not touched, there is no possibility to retroamnial liquid penetration. CVS is carried out from the 10th week of pregnancy. Small cell associations of placental tissue are obtained. Enough amniocytes containing spontaneous mitoses are available, so that kariotyping can be carried out immediately.
• Amniocentesis (AC): In AC the amniotic sac is punctured, there is possibility of retroamnial liquid penetration. Occasionally amniocytes are obtained, but which still have to be increased through cultivation. This takes about 1 week before cultivation can begin.

With both methods the risk of miscarriage is about 1% /44/.

Traditional chromosome analysis using G-band technique (karyotyping) is the gold standard for diagnosis of cytogenetic fetal abnormalities. False positive rates are 5%.

38.2.2.2.1 Karyotyping

Karyotyping is performed following amniocentesis at 16–18 weeks of pregnancy. Fetal cells in the amniotic fluid are cultured in vitro by stimulating cell division through phytohemagglutinin. After cultivation for 2 weeks, cell division is blocked at metaphase of mitosis by adding colchicine, which ensures a high accumulation of chromosomal metaphase figures. The cell suspension is then placed on a slide in a hypotonic solution, thus fixing and spreading the chromosomes. Next, the chromosomes are stained with orcein (standard staining) or with Giemsa stain after pretreatment with trypsin (G bands), and evaluated microscopically. In routine diagnostics, faster and more sensitive karyotyping procedures are used.

Karyotyping identifies:

• Chromosomal abnormalities as aneuploidy (gain or loss of entire chromosome) by generating a karyogram
• Balanced rearrangements, and large unbalanced structural rearrangements with pathogenic copy number changes of greater than 5–10 MB.

Numerical abnormalities

The abnormal chromosomal composition can be 2n + 1, as in trisomy 21, or 2n – 1, as in monosomy X (Turner syndrome). The most common numerical chromosomal abnormalities are:

• Down syndrome 47, trisomy 21; 1 : 600; ♂ + ♀
• Klinefelter syndrome 47, XXY; 1 : 800; ♂
• YY syndrome 47, XYY; 1 : 900; ♂
• Triple X 47, XXX; 1 : 1,000; ♀
• Turner syndrome 45, XO; 1 : 2500; ♀

The tests are performed using amnion cell culture or chorionic villi samples.

Structural abnormalities

Structural abnormalities involve the loss of chromosomal material due to breakage or loss of function due to faulty fusion of chromosome fragments.

The following abnormalities are possible:

• Deletion: a piece of DNA has broken off from a chromosome, with the loss of genes
• Duplication: a segment of the chromosome is duplicated; affected individuals possess three copies of the genes of this chromosome segment
• Translocation: a chromosome or segment of a chromosome attaches to another chromosome or chromosome segment
• Inversion: a segment that has broken away from the chromosome is inverted from end to end, and reinserted into the chromosome
• Insertion: a chromosome segment that has broken away is inserted into a break of another chromosome.

Translocation and insertion lead to rearrangements. These can be balanced or unbalanced. Pathogenic copy number variants below 3 to 10 mega bases cannot be detected by karyotyping.

38.2.2.2.2 Microarrays

Microarrays (DNA biochips), also known as chromosomal microarrays or molecular karyotyping, are more sensitive than conventional karyotyping. This method detects small genomic deletions and duplications (called copy number variants). Copy number variants result in a variation from the expected number of copies of a segment of DNA (i.e., the number in a normal genome). Copy number variants can be either benign or pathogenic, depending on their location and genetic content /6/. They are identified with the use of chromosomal micro arrays, in which the test sample of DNA from the patient is compared directly or indirectly with a normal genome.

A DNA micro array is a collection of microscopic spots (picomoles of DNA) of a specific DNA sequence, known as probes or oligos. They are attached to a solid phase and can hybridize with a complementary DNA of the specimen, also referred to as the target. A large number of genes or multiple regions of the genome can be analyzed simultaneously.

Chromosomal micro array (CMA) detects 12–14% of cases in the spectrum of multiple congenital anomalies, unexplained developmental delay, intellectual disability and autism spectrum disorders compared to only 4% with karyotyping. The American College of Medical Genetics therefore recommends the use of CMA as the first-tier diagnostic test for the above mentioned chromosomal abnormalities /7/: overall, CMA provides additional relevant information in 1.7% of pregnancies with standard indications for prenatal diagnosis (such as advanced maternal age and positive aneuploidy screening result) and in 6% of cases with an anomaly on ultrasonography /7/. In the analysis of samples from stillbirths, CMA yields results in 24% more cases than does karyotyping /8/.

Limitations of CMA include its inability to detect balanced rearrangements, balanced trans locations, balanced inversions, and fetal triploidy /7/. Although these do not cause miscarriage or stillbirth, they are inherited. This is important to know in respect that a future newborn may be unbalanced. CMA also detects defects which are irrelevant at the time of testing and do not become evident until adulthood or not at all.

38.2.2.2.3 Specimens for invasive testing

Some laboratory tests for prenatal diagnosis are performed on maternal blood. Genetic tests are performed on fetal specimens or maternal blood.

Fetal specimens for prenatal diagnosis are obtained through the following procedures /9/:

• Amniocentesis from 15 weeks’ gestation to obtain amniotic fluid for chromosome analysis and biochemical tests
• Chorionic villus sampling between days 70 and 97 after the last menstruation for chromosome analysis and biochemical tests
• Fine-needle biopsy of fetal organs and body cavities from 15 weeks’ gestation
• Cordocentesis and cardiocentesis from 20 weeks’ gestation to obtain fetal blood samples
• Fetoscopy to obtain fetal blood and organ samples (skin, liver) from 18 weeks’ gestation
• Isolation of fetal cells from the maternal blood and testing of these cells with molecular biological methods /10/.

The risk of fetal infection from invasive procedures is about 1% and that of loss of pregnancy about 0.5% /9/.

38.2.2.4 Noninvasive prenatal testing

Cell-free DNA (cfDNA) tests are increasingly being offered to women in the first trimester of pregnancy at a high risk of trisomy 21. The principle of NIPT is the targeted increase of proportions of the human genom using next generation sequenching (NGS). The signal frequency is then compared with the signal pattern to be expected from a healthy person. Values of the expected limit values are rated as conspicuous. NIPT are screening tests and provide predictions based on mathematical statistical calculations The diagnostic sensitivity of NIPT for trisomy 21 is higher than 99%.

Procedure and physiology: DNA is extracted and amplified from the blood of pregnant women carrying a fetus. NIPT sequences cell-free DNA which contains a mixture of small maternal DNA fragments as well as placental DNA fragments that serve as a fetal surrogate /11/. Fetal DNA fragments are released from the placenta into the maternal circulation as cytotrophoblast and syncytiotrophoblast cells undergo physiologic cycles of fusion and apoptosis. The relative proportion of cell-free fetal DNA increases with gestational age and can reliably be detected by 9–10 weeks of gestation onward, since this is when the fetal fraction in the maternal circulation reaches the minimum amount needed for an informative test result /12/.

Two basic sequencing approaches are used to analyze cell free DNA:

• Targeted method: single nucleotide polymorphisms (SNPs) on the chromosomes of interest are amplified and sequenced. Ratios between heterozygous SNP alleles are compared with those of other targeted chromosomes. In aneuploidy of a targeted chromosome skewing of the ratio is measured /12, 13/.
• Whole genome sequenching method: cell free DNA molecules of mother and fetus are randomly sampled, sequenced, and mapped to specific chromosomes /12, 14/. The numbers of DNA molecules belonging to different chromosomes are then counted. For pregnancies involving a fetus with trisomy 21, the proportion of cell free DNA molecules derived from chromosome 21 is higher than that of samples of pregnancies with euploid fetuses /12/.

According to a systematic review and meta-analysis the diagnostic sensitivities and specificities of aneuploidy in high risk women were /15/:

• 97% and 99.7%, respectively for trisomy 21
• 93% and 99.7%, respectively for trisomy 18
• 95% and 99.9%, respectively for trisomy 13.

The false positive rate of cell-free DNA determination is < 0.2% compared to invasive testing which is considered the gold standard  /12/.

Analysis of cell free DNA is also used to test for fetal single gene disorders in couples who are at high risk, because of their personal or family history /12/.

Clinical practice standards recommend confirmation of a positive cell-free DNA screening results with determination of the karyotype or micro array assay.

Indications for the determination of cell-free DNA are shown in Tab. 38.2-5 – Indications for the determination of cell-free DNA and biologic causes of false results in Tab. 38.2-6 – Biologic causes of false positive and false negative results of cell-free DNA.

For the determination of cell-free fetal DNA in maternal plasma, blood collection tubes which stabilize nucleated blood cells for up to 7 days at room temperature should be used /42/. This is because, after blood collection, the concentration of cell-free DNA in the sample increases due to apoptosis, necrosis, and lysis of maternal nucleated cells /42/.

Centrifugation is important

To ensure that nucleated cells are not damaged, the following procedure is recommended /43/:

• Centrifugation at 1,600 × g for 15 min. at room temperature
• Remove plasma and centrifuge again at 2,500 × g for 10 min.
• Separate plasma from pellet and keep it frozen at ≤ –70 °C until the time of analysis.

An ISUOG (International Standard Obstet Gynecol) statement on the impact of cell free DNA (cfDNA) testing on screening policies and prenatal ultrasound practice is published in Ref. /46/.

38.2.2.5 Newborn screening

Many coutries carry out a screening program for inborn error of metabolism (IEM). The incidence of IEM can be up to 1 in 500 to 4,000. Minimal sample preparation and long term stability of analytes is guaranteed utilizing dried blood spot analysis. A study /47/ evaluated the analytical performance of 16-plex iSeq sequencing using 87-IEM-gene AmpliSeq panel (amplicon-based next generation sequencing panel) for second-tier test in newborn screening for IEM. There were 85 out of 87 genes with acceptable coverage. The turnaround time from DNA extraction to completion of procedures was 2.5 days.

38.2.2.6 Trisomy 21

Trisomy 21 (Down syndrome) is the most common chromosomal anomaly in humans and ocurs because of the extracopy of chromosome 21 which contains approximately 225 genes. Genetic mosaicism and translocations that develop in later states of embryogenesis are rare and associated with minor clinical signs of the Down syndrome. Individuals with trisomy 21 reach the age of 60. However in middle and old age these people have signs and symptoms that do not correspond to the normal population. Refer to Tab. 38.2-7 – Signs and symptoms of Down syndrome.

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38.3 Human chorionic gonadotropin (hCG)

Following conception and implantation of the fertilized ovum, hCG is produced by the trophoblast towards the end of the second week of conception. Approximately 10,000 trophoblastic cells are required to produce a measurable increase in hCG in serum. hCG stimulates progesterone synthesis in the corpus luteum from the time of implantation until the trophoblast takes over at around 8 weeks of pregnancy. From 12 weeks onward, hCG production begins to decline, because the trophoblast is inhibited by the fetal adrenal cortex.

In pregnancy hCG immunoreactivity consists of a number of hCG variants. Refer to Fig. 38.3-1 – Structures of hCG and related molecules.

The following molecules are of diagnostic importance in pregnancy:

• Intact hCG (β-hCG)
• hCGβ, the free β-subunit of the hCG molecule.

The antibodies of the commercially available β-hCG assays determine intact hCG and hCGβ

Intact hCG and hCGβ are clearly detectable in serum and urine. During pregnancy, the free β-subunit accounts for less than 1% of total hCG in serum and for 9–40% in urine /1/. Free β-subunit secretion is increased in hCG-synthesizing trophoblastic tumors and germ cell tumors. Most commercial hCG assays are designed to measure intact hCG and the free β-subunit, as well as other hCG variants, but with a different sensitivity of detection. These commercially available assays are named β-HCG test.

38.3.1 Indication

The determination of hCG in serum or urine in pregnancy is indicated:

• To detect early pregnancy
• To diagnose ectopic pregnancy
• In patients with gestational trophoblastic disease
• As a screening marker for aneuploidy in triple- or quadruple testing.

38.3.2 Method of determination

The determination of hCG in serum and urine is used in the diagnosis and monitoring of pregnancy and trophoblastic tumors. In these indications, hCG and hCG variants are present in different molar concentrations not only in serum, but also in urine. It is important for the determination in serum and urine that the assay used measures intact hCG and the free β-subunit well. All methods use an antibody directed against the β-subunit of the hCG molecule to differentiate between hCG and LH, since both molecules have identical α-subunits. For this reason, the term β-hCG refers to all tests that measure intact hCG and its free β-subunit. The terms hCG test and β-hCG test are therefore used synonymously in the following. Commercial assays use a large variety of antibodies directed against epitopes of the β-subunit. Different manufacturers’ assays thus measure intact hCG and a range of different hCG variants /2/. The principle of the quantitative determination of hCG in serum is explained in Section 28.16 – Human chorionic gonadotropin.

Qualitative tests in urine

Tests based on the competitive, immunometric and ELISA principles are used. In the ELISA assay, tubes, membrane filters or beads are coated with a monoclonal capture antibody which is directed against the α-subunit of the hCG molecule. If hCG and LH are contained in the sample to be analyzed, both hormones are bound to the tube wall or membrane filter. A second peroxidase or alkaline phosphatase conjugated antibody, which is directed against epitopes of the β-subunit, is used as a tracer and forms a sandwich. The addition of a chromogenic substrate solution leads to a color reaction. The practical sensitivity is approximately 10–20 IU/L.

38.3.3 Specimen

• Urine: 1 mL
• Serum: 1 mL

38.3.4 Reference interval

Refer to Tab. 38.3-1 – Reference intervals for serum hCG.

38.3.5 Clinical significance

In natural fertilization, implantation of the zygote occurs 5.5–6 days after conception, and on the 7th day hCG is synthesized by the trophoblast of the blastocyst and can be measured in serum.

38.3.5.1 Early diagnosis of pregnancy

Using a cutoff of 5 to 10 IU/L in serum, pregnancy is detected as early as at the beginning of the 4th week of pregnancy (i. e, on day 23–24 of the menstrual cycle if ovulation occurred on day 14 of the cycle). This threshold reduces false positive pregnancy findings, since peri- and post menopausal women with excessive pituitary hCG production may show levels up to 9 IU/L. The behavior of hCG levels during pregnancy is shown in Tab. 38.3-2 – Serum hCG levels during pregnancy.

The hCG concentration in urine is only half as high as in serum, and there is significant intraindividual variability due to the variation in fluid intake. Therefore, a urine test may detect early pregnancy several days later than a serum test.

38.3.5.2 hCG in reproductive medicine

To assess successful implantation and embryonic development after in vitro fertilization, the course of hCG levels is of importance. Measured on days 14, 16, 20 and 27 following ovulation induction, the serum concentrations of hCG are 4–125 IU/L, 20–294 IU/L, 97–2560 IU/L and 1860–16,200 IU/L, respectively. The doubling time of hCG is 1.4 days. hCG levels are significantly higher after artificial insemination than after in vitro fertilization /5/. It must be noted that, following gonadotropin induced ovarian stimulation, pregnancy cannot be detected until 14 days after ovulation. High gonadotropin levels cross react with the measurement of hCG, causing a false-positive pregnancy test result.

38.3.5.3 Determination of gestational age

The gestational age can be determined in the first 7 weeks of pregnancy by determining hCG in at least two serum samples 2–7 days apart, since the doubling time during this period of pregnancy is 2.5 days. The hCG levels (semi-logarithmically) are plotted against measuring days (linear). The gestational age can be determined only if the slope of the hCG level in the concentration time plot is normal.

When hCG levels (1st IRP) were related to the findings of transvaginal ultrasonography and the day of pregnancy /6/:

• A gestational sac of 1–3 mm was detected at days 30–34 of gestation, hCG levels ranged from 467 to 935 IU/L
• The yolk sac was detected at days 34–38, hCG levels ranged from 1,120 to 7,280 IU/L
• Fetal heart motion was visible at days 39–43, and hCG levels ranged from 5,280 to 22,950 IU/L.

38.3.5.4 Multiple pregnancy

Serum hCG levels increase more rapidly in multiple than singleton pregnancies, the doubling time is shorter; 10-week levels are higher than those in singleton pregnancies.

38.3.5.5 Early abortion

Problems during the first trimester of pregnancy will, at a certain point, become evident during serum hCG monitoring when levels are too low or they increase or decrease too slowly. The slope of hCG rise does not run parallel to the normal curve in the concentration-time plot. If monitoring shows a rise parallel to the normal curve but a doubling time with a significant time delay, and a dating error can be excluded, then an ectopic pregnancy or an abnormal intrauterine pregnancy must be suspected. Normal hCG levels, especially if tested only once, do not eliminate the possibility of late abortion and an ectopic pregnancy. Following complete curettage, hCG levels decrease with a half life of one day /7/.

38.3.5.6 Ectopic pregnancy

More than half of all women who present with abdominal pain or bleeding after about 7 weeks of amenorrhea have serum hCG levels < 2,000 IU/L. In these cases it is difficult to determine whether an empty uterus indicates early pregnancy or ectopic pregnancy. The incidence of ectopic pregnancy is approximately 10 per 1,000 births. Ectopic pregnancy accounts for 80% of first trimester maternal deaths. Approximately 98% of ectopic pregnancies occur in the Fallopian tubes. Important diagnostic tests include the combination of vaginal ultrasound and the determination of hCG in serum.

Tab. 38.3-3 – Criteria of a viable pregnancy in first trimester pregnancy of unknown location shows recommended diagnostic and management guidelines for ectopic pregnancy /8/. One useful indicator of ectopic pregnancy in the absence of a detectable gestational sac is the increase in hCG levels over a period of 48 h. Under stable conditions, the serum hCG concentration should double every 2.3 days. If this is not the case, an ectopic pregnancy is likely. The positive predictive value of a normal rise in hCG which excludes ectopic pregnancy is 94.7% /9/. If a corresponding rise does not occur, the diagnostic sensitivity for ectopic pregnancy is only 37%, with a specificity of 65%. Early ectopic pregnancy can also be diagnosed by determining hCG in serum (S) and Douglas pouch puncture fluid (DP). In an intact pregnancy, the S/DP > ratio is 1.3, in an ectopic pregnancy it is < 0.7 /10/.

38.3.5.7 hCG levels during pregnancy

Due to the large inter individual variation of serum hCG levels, it is not possible to quote an exact reference interval at any point of the pregnancy. Therefore, the determination of the individual percentage increase in serum hCG within defined time intervals (e.g. 48 h) provides positive indication of an intact pregnancy as absolute values. hCG levels reach a peak and plateau at 8–12 weeks of pregnancy before declining progressively in the 2nd and 3rd trimesters. In twin pregnancies, hCG levels are comparably higher especially during the 2nd and 3rd trimesters, although a diagnosis based on hormone findings is not reliable. After delivery, hCG serum levels decline with a half life of 24–36 h and are no longer detectable after 11–17 days.

Refer to Fig. 38.3-2 – hCG and hCG variants during pregnancy.

38.3.5.8 Gestational trophoblastic disease

Gestational trophoblastic disease (GTD) is a term used for various diseases of the trophoblast which, according to the WHO classification, comprise hydatidiform moles, non molar tumors, and benign trophoblastic disorders /11/. A finding of no embryo on ultrasound along with elevated serum hCG (500,000 – 1 million IU/L) is indicative of GTD, in particular hydatidiform mole. It must, however, be noted that in GTD there is little intact hCG, but predominantly hyperglycosylated hCG, nicked hCG, the free β-subunit in an intact or nicked form, or the β-core fragment. It is therefore essential that the laboratory uses assays or a combination of assays that can detect these forms /12/.

Refer to Tab. 38.3.4 – hCG and hCG variants in pregnancy and trophoblastic disease.

Hydatidiform moles

Hydatidiform moles, also known as molar pregnancies, are placentas with abnormal chorionic villi; a distinction is made between complete, partial, and invasive moles. Complete moles have one uniform villous population, partial moles have two distinct ones. Invasive moles are the most common form of persistent GTD and difficult to differentiate from choriocarcinoma.

Laboratory findings: although moles produce hCG, the serum hCG level of partial moles is within the reference range or only slightly elevated.

Non molar tumors

Among these, choriocarcinoma is differentiated from placental site trophoblastic tumor and epithelioid trophoblastic tumors.

• Choriocarcinoma is a highly malignant germ cell tumor which develops in pregnancy. It arises from the cells of the cytotrophoblast and syncytiotrophoblast and does not need to be differentiated from pregnancy related choriocarcinoma, which typically develops outside the uterus, usually together with other germ cell tumors. About 50% of choriocarcinomas are preceded by a molar pregnancy, 25% by spontaneous abortion, 22.5% by a normal pregnancy, and 2.5% by an ectopic pregnancy. Choriocarcinoma has the highest metastatic potential among all malignant tumors and usually metastasizes in the lungs, liver, and brain. The patient dies from pulmonary insufficiency or hemorrhage. The tumor often consists only of blood with a thin outer layer of tumor cells. Choriocarcinoma produces hCG.
• Placental site trophoblastic tumor is a malignant tumor which arises from the intermediate trophoblast. It is an extra villous cell type with important functions in placentation. Placental site trophoblastic tumor does not produce hCG, but expresses large amounts of hPL.
• Epithelioid trophoblastic tumor is a rare tumor which usually develops in the form of intramural masses in the myometrium or cervix in normal pregnancy. The tumor cells are of epithelial character and do not produce hCG.

Benign trophoblastic disorders

This group of disorders includes exaggerated placental site and placental micro nodules. Exaggerated placental site is characterized by an increased number of implantation-site intermediate trophoblastic cells that infiltrate the myometrium. The placental nodules are involuted and hyalinized parts of the trophoblast that have invaded the uterus and cervix.

Laboratory findings: the amounts of hCG secreted in these benign disorders are not significant enough to lead to elevated levels in serum.

Persistent low hCG values in women with past histories

Cases of low persistent elevation of hCG with serum levels up to 200 IU/L, occasionally up to 500 IU/L, and past histories of hydatidiform mole or gestational trophoblastic disease/gestational trophoblastic neoplasm ranging from 4 months to 12 years are registered /12/. These cases should be seen as a premalignant condition and monitored monthly by measuring hCG, since levels in this range are associated with invasive growth and will sooner or later increase suddenly /12/.

If hCG levels increase in the absence of such history and pregnancy can be excluded, hCG should be measured in order to exclude a trophoblastic disease. In invasive trophoblastic diseases, hyperglycosylated hCG accounts for ≥ 30% of total hCG.

If, in the case of hCG elevations not caused by invasive disease, false positive results due to heterophile antibodies are excluded, persistent elevations are usually due to pituitary hCG, which can sometimes reach levels of 20–40 IU/L in women and men and shows no variation in its concentration /13/.

For para neoplastic hCG secretion refer to:

• Section 28.16 – Human chorionic gonadotropin
• Tab. 29-7 – Para neoplastic hormone secretion.

38.3.6 Comments and problems

Standardization of hCG

See Section 28.16.6 – Comments and problems.

Method of determination

Urine assay

Urine contains intact hCG, hyperglycosylated hCG, nicked hCG, the free β-subunit and the β-core fragment (Fig. 38.3-1 – Structure of hCG and related molecules). About half of all commercial screening tests for the diagnosis of pregnancy do not detect hyperglycosylated hCG. Unlike serum tests, which usually detect hyperglycosylated hCG and therefore show a positive result as early as 2 weeks after conception, urine tests will still be negative /14/. Many assays also do not detect the β-core fragment which occurs only in urine and can account for about 50% of total hCG from week 4 and for an even higher proportion later in the pregnancy.

Serum assay

Most commercial assays detect intact hCG and the free β-subunit of hCG. For the diagnosis of suspected trophoblastic tumors, in which hyperglycosylated hCG is the dominant form, as well as for the monitoring and treatment follow-up of such tumors, specific immunoassays should also be used. Only 4 out of 11 tested immunoassays detected hyperglycosylated hCG to the same extent as intact hCG; all other immunoassays showed a variation by a factor of 0.5 to 1.7 /12/.

Interference factors

Heterophilic antibodies

The USA hCG Reference Service Experience /1/ found that false positive serum hCG levels are usually caused by the presence of heterophilic antibodies. Treatment of such sera with blocking antibodies produced by Scantibodies® leads to hCG levels within the reference interval. The urine finding in these cases is hCG negative, since heterophilic antibodies do not occur in urine. Women with false positive hCG results often also have false positive results in immunoassays for the determination of CEA, CA 19-9, or troponin. To test for heterophilic antibodies, serial serum dilutions have to be made. Low titer heterophilic antibodies then decline below a threshold where they no longer interfere.

Serum positive, urine negative: this finding can have the following causes:

• Heterophilic antibodies; the most common cause are human anti-mouse antibodies (HAMA) in serum. Two-side immunoassays reduce this problem. Many manufacturers also add animal proteins to the reagent to block the heterophilic antibodies. In many immunoassays, the urinary level of hCG is only half as high as the serum level.
• Significant dilution of the urine by diuresis. This may be the case after an ultrasonographic examination, which requires a full bladder.
• During the last trimester, the hCG production may be too low to be detected in urine, so that a serum test, which is more sensitive, may have to be used.
• Due to the short incubation time (3–5 min.), the detection limit of the qualitative hCG tests depends considerably on the temperature of the immunochemical reagents and of the urine. Unless these are at room temperature before being used for testing, the result may be false negative.

Interference of hCG in immunoassays

Due to the cross reactivity of hCG and LH in immunoassays, LH can be overestimated. For example, in a commercial assay /15/, the presence of 1,350 IU/L of hCG mimicked an LH concentration of 2.1 IU/L, an hCG concentration of 55,992 IU/L mimicked an LH concentration of 19.4 IU/L, and 143,828 IU/L an LH concentration of 25.6 IU/L.

Stability

• 2–3 days in urine, if 10 mL are stabilized with 0.1 mL of sodium azide (0.15 mol/L)
• Intact hCG in serum at 21 °C or 4 °C, recovery after 6 days of 94 ± 3.1% and 94 ± 8.3%, respectively /16/.
• Maternal serum free β-hCG following whole sample transit for trimester Down's syndrome is stable for at least 72 h of venepuncture /18/.

38.3.7 Pathophysiology

HCG belongs to the glycoprotein family of hormones whose members share a common α-subunit and vary in their β-subunits. The β-subunits determine the specificity for each individual hormone for its target receptor. Both subunits of hCG are held together by polar bonds (Fig. 38.3-1 – Structure of hCG and related molecules). Only 65% of the of 36 kDa protein consists of amino acids, the remainder are carbohydrates. Although the carbohydrate content is of great importance in relation to the biological activities, they are not particularly antigenic as compared to the protein itself /1/. For further pathophysiologic details Refer to Section 28.16 – Human chorionic gonadotropin.

The β-core fragment (not shown in Fig. 38.3-2) is the terminal degradation product of hCG. It is detectable only in urine and accounts for about 50% of the molar concentration of total hCG at 4–8 weeks of gestation. At 36–40 weeks its molar concentration is about 3 times higher than that of non-nicked hCG. β-core fragment is markedly elevated in pregnancies with Down syndrome and, in relation to creatinine clearance, is subject to a marked diurnal variation of 1.39–2.10 of the multiple of median (MoM) /17/.

References

1. O’Connor JF, Birken S, Lustbader JW, Krichevsky A, Chen J, Canfield RE. Recent advances in the chemistry and immunochemistry of human chorionic gonadotropin: impact on clinical measurements. Endocrine Reviews 1994; 15: 650–83.

2. Cole LA. Immunoassay of human chorionic gonadotropin, its free subunits, and metabolites. Clin Chem 1997; 43: 2233–43.

3. Alfthan H, Haglund C, Dabek J, Stenman UH. Concentrations of hCG, βhCG, and cβhCG in serum and urine of nonpregnant women and men. Clin Chem 1992; 38: 1981–7.

4. Stenman UH, Bidart JM, Birken S, Mann K, Nisula B, O’Connor J. Standardization of protein immunoprocedures. Choriongonadotropin (CG). Scand J Clin Lab Invest 1993; 216, Suppl: 42–78.

5. Ertzeid G, Tanbo T, Dale PO, Storeng R, Morkrid L, Abyholm T. Human chorionic gonadotropin levels in successful implantations after assisted reproduction techniques. Gynecol Endocrinol 2000; 14: 258–63.

6. Cacciatore B, Tiitinen A, Stenman UH, Ylöstalo P. Normal early pregnancy: serum hCG levels and vaginal ultrasonography findings. Brit J Obstetr Gynaecol 1990; 97: 899–903.

7. Taubert HD, Dericks-Tan JSE. Human-Chorion-Gonadotropin in der normalen und gestörten Schwangerschaft. Gynäkol Prax 1985; 9: 475–92.

8. Doubilet PM, Benson CB, Bourne T, Blaivas M. Diagnostic criteria for nonviable pregnancy early in the first trimester. N Engl J Med 2013; 369: 1443–51.

9. Dimitry ES, Atalla RK. Modern lines of management of ectopic pregnancy. BJCP 1996; 50: 376–80.

10. Hinney B, Osmers R, Tobler-Sommer M, Wilke G, Wuttke W, Kuhn W. Diagnose der frühen Extrauteringravidität durch hCG-Bestimmung aus Serum und Douglaspunktat. Geburtsh u Frauenheilk 1991; 51: 637–42.

11. Bentley RC. Pathology of gestational trophoblastic disease. Clin Obstet Gynecol 2003; 46: 513–22.

12. Cole LA. HCG tests in the management of gestational trophoblastic diseases. Clin Obstet Gynecol 2003; 46: 523–40.

13. Braunstein GD. False-positive serum human chorionic gonadotropin results: causes, characteristics, and recognition. Am J Obstet Gynecol 2002; 187: 217–24.

14. Butler SA, Khanlian SA, Cole LA. Detection of early pregnancy forms of human chorionic gonadotropin by home pregnancy test devices. Clin Chem 2001; 47: 2131–6.

15. Vivekanandan S, Andrew CE. Cross-reaction of human chorionic gonadotrophin in Immulite 2000 luteinizing hormone assay. Ann Clin Biochem 2000; 39: 318–9.

16. Kardana A, Cole LA. The stability of hCG and free β in serum samples. Prenat Diagn 1997; 17: 141–7.

17. Rotmensch S, Celentano C, Ellinger N, Sasan O, Lehman D, Golan A, et al. Diurnal variation of human chorionic gonadotropin β-core fragment concentrations in urine during second trimester pregnancy. Clin Chem 2001; 47: 1715–7.

18. Ballantyne A, Rashid L, Pattenden R. Stability of maternal serum free β-hCG following whole blood sample transit: First trimester Down’s syndrome screening in Scotland. Ann Clin Biochem 2022; 59 (1): 87–91.

38.4 Alpha-fetoprotein (AFP)

AFP belongs to the group of oncofetal proteins and is synthesized in the fetal liver and yolk sac. Small amounts of amniotic fluid AFP also are produced by other parts of the fetal gastrointestinal tract. AFP is excreted by the fetal kidneys into amniotic fluid and is transferred into the maternal circulation through diffusion across the placenta and fetal membranes. AFP is clinically significant in prenatal diagnosis and as a tumor marker. The significance of AFP as a tumor marker is described in Section 28.7 – Alpha-fetoprotein.

38.4.1 Indication

• Prenatal diagnosis of neural tube and abdominal wall defects in the second trimester
• Early detection of perinatal complications such as fetal anal atresia and other gastrointestinal obstructions
• Parameter in the quadruple test in Down syndrome screening.

38.4.2 Method of determination

Immunoassays based on competitive, immunometric or ELISA methods.

38.4.3 Specimen

• Serum: blood sampling should occur at 16–21 weeks of pregnancy: 1 mL
• Amniotic fluid: for chromosome analysis as part of amniocentesis or when two serum AFP levels are pathological: 1 mL

38.4.4 Reference interval

38.4.5 Clinical significance

AFP is an important screening test for the prenatal diagnosis of anencephaly, neural tube defects and abdominal wall defects. Determination of amniotic fluid acetylcholinesterase activity is a confirmatory test in cases with elevated AFP values.

38.4.5.1 Spina bifida

Spina bifida is a developmental malformation of the spinal cord /12/. Failure of the neural tube to fuse during the third week of gestation leads to an open neural-tube defect at the cranial level (anencephaly), the spinal level (myelomeningocele, spina bifida) or both, simply cranioarachischisis. Anencephaly and cranioarachischisis are lethal during gestation or soon after birth. A spina bifida is an open defect that has no dura, bone, muscle, or skin coverage. There is susceptibility to infection and cerebrospinal fluid leakage. Incomplete formation of the lower cord causes dysfunction of leg, bladder, and the bowel.

Studies indicate that neural-tube defects result in most cases from acquired or heritable disorders of the folate metabolism and are largely preventable with dietary folate supplementation. Spina bifida can be diagnosed by means of

• ultrasonography during the second trimester of pregnancy
• indirectly using laboratory investigations e.g., alpha fetoprotein.

Surgical approaches directed at ameliorating neurologic complications, together with multispeciality care, have improved survival and quality of life for children with spina bifida.

38.4.5.2 Spina bifida screening by means of alpha-fetoprotein

To screen for open neural tube defects, AFP should be measured in maternal serum at 16–20 weeks’ gestation. Multiples of medians (MoM) of healthy pregnancies are used as cutoffs for the relevant pregnancy. They are generally 2–3 multiples of the median of healthy pregnancies and are defined by the laboratories and gynecologists. At levels greater than 2.5 MoM, the diagnostic sensitivity for detecting fetuses with spina bifida aperta is 70%, with a specificity of 97%. The low incidence results in a positive predictive value of 3%, meaning that only three out of 100 AFP concentrations above the cutoff are due to spina bifida aperta.

Refer to Fig. 38.4-1 – AFP in serum and amniotic fluid as a function of gestational age/week.

If maternal serum AFP is elevated, two-thirds of false positive AFP results can be eliminated by repeating the serum test and by performing ultrasonography /1/. If the serum level remains elevated, the risk of the fetus having a neural tube defect is 5–10%.

If serum AFP levels are elevated on two occasions and the ultrasonography shows no abnormalities, then amniotic fluid AFP levels, and possibly acetyl cholinesterase (ACHE), need to be measured.

The results of a British collaborative study /3/ showed that if the amniotic AFP level is greater than 3 × median the likelihood of a singleton pregnancy with neural tube defect is 24 : 1. Refer to Fig. 38.4-1 – AFP in serum and amniotic fluid as a function of gestational age/week.

A German study on AFP /4/ evaluated the following results of serum AFP screening at 16–20 weeks of pregnancy: 96% of anencephalic fetuses and 71% of open neural tube defects were detected, but no neural tube defects covered by intact skin (myelomeningocele). Not counting the anencephalic fetuses, only 37% of all neural tube defects were detected by serum AFP screening.

Refer to Fig. 38.4-2 – Approach to the diagnosis of neural tube defects.

In only 10% of pregnancies with elevated serum AFP neural tube defects are diagnosed using special tests.

Possible causes of raised AFP levels in the absence of a neural tube defect are:

• Incorrect estimation of the gestational age (approximately 30%)
• Multiple pregnancy (approximately 20%); decreased and resorbed twin embryos pose a special problem as they are difficult to detect on ultrasonography and can cause a positive result in the amniotic fluid ACHE test
• Newborns who will have a birth weight of less than 2,500 g due to intrauterine malnourishment (approximately 10% of causes)
• Other causes (30%) (e.g., diabetes mellitus, preeclampsia, oligohydramnios) processes during which fetal blood passes into the maternal circulation, such as amniocentesis, chorionic villus sampling, trauma, attempted abortion, dying embryo.

An amniotic fluid AFP level within a MoM of 3.0 with elevated serum AFP excludes an open neural tube defect.

Elevated amniotic fluid AFP levels are due to the abnormalities listed in Tab. 38.4-1 – Causes of elevated AFP levels and pathologic ACHE findings. False positive AFP results are mainly due to contamination of the amniotic fluid sample with fetal blood and can be excluded by a normal acetylcholinesterase activity in the amniotic fluid /4/.

The results of large studies on serum AFP screening and on the follow-up tests shown in Fig. 38.4-2 – Approach to the diagnosis of neural tube defects suggest that about 20% of neural tube defects remain undetected by AFP screening and that the rate of abortion of healthy fetuses due to this diagnosis is approximately 1 : 10,000 /5/.

38.4.5.3 Acetylcholinesterase in prenatal diagnosis of fetal malformations

Acetycholinesterase (ACHE) in the amniotic fluid is determined enzymatically following separation by polyacrylamide gel electrophoresis or with immunoassays using the monoclonal antibody 4F19. The methods are equivalent. A review /6/, which summarizes all studies, states a diagnostic sensitivity of 95–99% with a specificity of 99% for spina bifida aperta. Falsely pathological ACHE findings were seen in two fetuses without abnormalities /4/.

Polyacrylamide gel electrophoresis /7/ separates amniotic fluid proteins towards the anode under alkaline conditions and visualizes the enzyme activity in the gel using acetylthiocholine as a substrate. In spina bifida or anencephaly pregnancies two activity bands are seen, of which the anode-side ACHE band, which is inhibitable by dibromide, is pathological. In pregnancies without a neural tube defect, no ACHE band is found near the anode. Tab. 38.4-1 – Causes of elevated AFP levels and pathologic ACHE findings demonstrates the high diagnostic value of ACHE electrophoresis.

The immunological determination of ACHE /8/ uses the monoclonal antibody 4F19 in an enzyme immunoassay.

38.4.5.4 Serum AFP and abnormal pregnancy

In comparison to normal concentrations decreased AFP levels after 10 weeks’ gestation maybe an indicator of an irreversibly damaged pregnancy, even if hCG is not clearly reduced.

The same interpretation applies to elevated AFP levels after 14 weeks’ gestation, unless the pregnant woman is carrying twins /8/.

In the last trimester, decreased AFP levels are seen in cases with hemorrhage, intrauterine growth retardation, and placental insufficiency, which underlies these disorders. Pregnancies with unspecifically elevated serum AFP are generally more likely to end in miscarriage, and newborns have a low birth weight.

Significantly lower maternal serum AFP levels than normal are seen in pregnancies with fetal anal atresia /9/.

38.4.6 Comments and problems

Time of testing

Amniocentesis for AFP measurement should be performed between 15 and 24 weeks of gestation, since levels can be normal before and after this time even in the presence of neural tube defects /10/.

Contamination of amniotic fluid by fetal blood

Fetal blood falsely elevates AFP levels, which rise with advancing gestational age. The contamination with fetal blood occurs during amniocentesis. At 13–15 weeks’ gestation, approximately 7% of samples are contaminated. Following puncture at 22–24 weeks, approximately 16% are contaminated /1/.

38.4.7 Pathophysiology

AFP is initially synthesized in the fetal yolk sac and, as the embryo develops, in the fetal liver. From there it reaches the blood, the cerebrospinal fluid and bile. It is released with the urine into the amniotic fluid; small amounts are contributed by the meconium. The AFP concentration in fetal plasma and CSF is 100–1,000 times higher than in amniotic fluid where, in turn, it is 100–1,000 times higher than in maternal serum.

AFP is transferred from the amniotic fluid into the maternal circulation by trans amniotic diffusion /5/. Maternal serum AFP continues to increase between 10 and 32 weeks of pregnancy before declining to the level of 24 weeks by term. The half life of AFP is 3 days. In amniotic fluid, AFP declines continuously between 16 and 22 weeks of pregnancy /11/.

From the 9th week of gestation the covering of the dura begins; it is usually completed by the 12th gestational week. Open neural tube defects clinically manifest as spina bifida aperta and anencephaly. Large amounts of AFP leak into the amniotic fluid with the cerebrospinal fluid; this also leads to elevated levels in the maternal blood. Closed spina bifida, however, does not cause an increase in AFP levels.

Elevated AFP in the amniotic fluid and maternal serum can also be caused by other severe fetal abnormalities in which large serous areas are exposed such as omphalocele, but also congenital nephrosis and atresia of the gastrointestinal tract.

References

1. Cuckle HS, Wald NJ, Lindenbaum RH. Maternal serum-alpha-fetoprotein measurement: a screening test for Down syndrome. Lancet 1984; 926–9.

2. Fuhrmann W, Weitzel HK. Maternal serum alpha-fetoprotein screening for neural tube defects. Report of a combined study in Germany and a short overview on screening in populations with low birth prevalence on neural tube defects. Human Genetics 1985; 69: 47–61.

3. Second report of the U.K. collaborative study on alpha-fetoprotein in relation to neural tube defects. Lancet 1979; 651–61.

4. Mühlhaus K, Weitzel HK, Schneider J. Pränatale Diagnostik von Neuralrohr- und Bauchwanddefekten im II. Trimenon. Geburtsh u Frauenheilk 1985; 45: 98–100.

5. Weitzel H. Alpha-Fetoprotein in der Geburtshilfe. Gynäkologe 1983; 16: 148–54.

6. Rasmussen-Loft AG. Determination of amnion fluid acetylcholinesterase activity in the antenatal diagnosis of fetal malformations: the first ten years. Clin Biochem 1990; 28: 893–911.

7. Read AP, Fennel SJ, Donnai D, Harris R. Amniotic fluid acetylcholinesterase: a retrospective and prospective study of the qualitative method. Brit J Obstet Gynecol 1982; 89: 111–6.

8. Rasmussen AG, Arends J, Larsen SO. Evaluation and quality control of a monoclonal antibody based enzyme immunoassay of acetylcholinesterase in amniotic fluid. Scand J Clin Lab Invest 1989; 49: 503–12.

9. van Rijn M, Christaens GCML, Hagenaars AM, Visser GHA. Maternal serum alpha-fetoprotein in fetal anal atresia and other gastro-intestinal obstructions. Prenat Diagn 1998; 18: 914–28.

10. NCCLS Document I/LA 17-P, Vol 13 No 13. Assessing the quality of systems for alpha-fetoprotein (AFP) assays used in prenatal screening and diagnosis of neural tube defects; proposed guideline. Villanova; NCCLS, 1993.

11. Canick JA, Kellner LH, Bombard AD. Prenatal screening for open neural tube defects. Clin Lab Med 2003; 23: 385–94.

12. Iskandar BJ, Finnell RH. Spina bifida. N Engl J Med 2022; 387 (5): 444–50.

38.5 Amniotic fluid bilirubin

The measurement of bilirubin in amniotic fluid in the last trimester is performed to estimate fetal hemoglobin (Hb) levels in pregnancies with suspected hemolytic disease. In prenatal diagnosis, the fetal Hb level is frequently measured directly in fetal blood obtained through cordocentesis, eliminating the need to determine bilirubin in amniotic fluid.

38.5.1 Indication

Prediction and assessment of the severity of hemolytic disease of the fetus and newborn.

38.5.2 Method of determination

Measurement of the relative absorption at 450 nm

Principle: the amniotic fluid sample is centrifuged at about 2,000 × g for 10 min. and the absorbance of the supernatant is measured with a recording spectrophotometer at 350–550 nm. Using semi log paper, the absorbance is plotted on the ordinate on a logarithmic scale versus the wavelength on the abscissa on a linear scale. On the absorbance plot a straight line (baseline) is drawn from the linear area of the curve at 350–375 nm to the linear area at 525–550 nm. If bilirubin is present in the amniotic fluid, a peak occurs at 450 nm. Then a line perpendicular to the x-axis from the absorbance peak at 450 nm to the baseline is constructed. The difference between the peak and the baseline represents the Δ A₄₅₀. The severity of the hemolysis as indicated by Δ A₄₅₀ is dependent on the gestational week and can be interpreted by correlation of the Δ A₄₅₀ value to the zones in the Liley diagram /1/.

38.5.3 Specimen

Amniotic fluid: 5 mL

Specimen is obtained by transabdominal puncture following ultrasonographic localization of the fetus and placenta.

38.5.4 Reference interval

Refer to Fig. 38.5-1 – Liley’s three zone chart.

38.5.5 Clinical significance

Hemolytic disease of the fetus and newborn (HDFN) is caused by an incompatibility between the blood groups of the mother and child. In most Western countries, the incidence of HDFN due to Rh allo immunization is 1–2 per 1,000 live births /2/. Approximately 1–2% of infants affected by HDFN have an antigen that belongs to neither the Rh(D) nor the AB0 blood group system. In Rh(D)-incompatible pregnancies in which anti-D prophylaxis was administered postpartum, the risk of sensitization for future pregnancies was only 1.6%.

If HDFN is present, it is important that fetal anemia is diagnosed early. Mild to moderate anemia is usually tolerated well by the fetus, but if the Hb level is less than 70 g/L, the fetus will develop hydrops. In HDFN, the bone marrow cannot compensate for the hemolysis, and hematopoiesis shifts to the liver, spleen, kidneys, intestinal mucosa, and adrenal glands.

38.5.5.1 Rhesus incompatibility and hemolytic disease

In the case of the constellation of a Rh(D)-negative mother and a Rh(D)-positive father, an antibody test must be performed early in the pregnancy, followed by a second test no later than at 16–20 weeks’ gestation /3, 4/. If the mother tests positive for Rh(D) antibodies in the indirect Coombs test, quantitative antibody testing should be performed every four weeks until 28 weeks’ gestation and then every two weeks until term.

Fetal anemia can be assessed by determining the following:

• Hb level in blood obtained by cordocentesis
• Bilirubin in amniotic fluid obtained through amniocentesis. This test is recommended from 17 weeks, when the maternal antibody titer is ≥ 1 : 16 or the anti-Rh(D) titer rises temporarily by two or more levels. An anti-Rh(D) titer of < 1 : 16 is usually not predictive of fetal erythroblastosis, with a titer of 1 : 32 the probability is 10%, with 1 : 64 it is about 25%, and with 1 : 128 about 50%.

The determination of bilirubin in amniotic fluid allows an assessment of the severity of the hemolytic process in the fetus. The extent of hemolysis correlates with the maternal antibody titer and is estimated based on the Liley diagram, expressed as ΔA 450 related to the gestational age in weeks. The probability of correct prediction of the degree of fetal risk based on the Liley diagram is high with an absorbance value in zone I or III and lower with an absorbance value in zone II. In this case serial sampling is required, which can increase the accuracy to 95%. A ΔA 450 in zone I corresponds to a fetal Hb concentration greater than 140 g/L, a value in zone II to a concentration of 140–100 g/L, and a value in zone III to a concentration of less than 100 g/L. In the latter case, an exchange transfusion may be required.

The main fetal risks associated with hemolysis are anemia and hypoxia, while the newborn is at risk for hyperbilirubinemia and kernicterus. A modified version of the Liley chart suggests a division into four zones /5/.

38.5.5.2 AB0 incompatibility and hemolytic disease

If the mother has blood group 0, AB0 incompatibility should be considered and a quantitative test for isoantibodies to blood group A and B should be performed at 36–37 weeks. Anti-A or anti-B titers greater than 1 : 1024 can be an indication to perform amniocentesis and to measure bilirubin in amniotic fluid /4/. AB0 HDFN is usually mild. Premature infants are less likely to develop the disease than full-term infants because the A and B antigens on the fetal erythrocytes are not fully developed. Erythrocytes with fully developed A and/or B antigens are produced only in the final weeks of prenatal development and only few newborns have antigens that are developed sufficiently for immune hemolysis to occur /4/. Hydrops fetalis associated with AB0 incompatibility occurs very rarely.

38.5.6 Comments and problems

Transabdominal puncture

Injury to the placenta (bloody amniotic fluid) can lead to fetal Rh(D)-positive erythrocytes entering the maternal circulation and thus to increased synthesis of anti-Rh(D) antibodies (booster effect). Since these added erythrocytes interfere with the spectrophotometry of amniotic fluid due to the hemoglobin released, immediate centrifugation is required.

Interference in spectrophotometry

Bilirubin shows maximum absorbance at 450 nm. Other high-absorbance contaminants such as meconium and oxyhemoglobin can falsely increase the ΔA 450 ; methemoglobin absorbs at a wavelength of 410 nm.

Misinterpretations

Misinterpretations are possible in the case of rapidly developing hydramnios prior to puncture or as a result of the aspiration of maternal or fetal urine or fetal ascites.

Stability

Bilirubin has a half life in amniotic fluid of approximately 15 min. under strong sunlight and of about 10 h under normal laboratory light. Storage and transport of amniotic fluid in lightproof containers, samples can be mailed uncooled provided they arrive within 24 h.

38.5.7 Pathophysiology

Maternal IgG antibodies directed against fetal blood group antigens cross the placenta, bind to the fetal erythrocytes and cause hemolysis through complement activation. Due to low glucuronyl transferase activity in the fetal liver, bilirubin produced during the breakdown of hemoglobin is only partly glucuronidated. Due to its good solubility in fat, non-glucuronidated bilirubin permeates the fetal surface layers and enters the amniotic fluid. Bilirubin levels in amniotic fluid are rarely above 1 mg/dL (17 μmol/L), even in highly pathological cases. The empirically determined degrees of risk of the Liley diagram correlate with the newborn’s postpartum hemoglobin levels.

References

1. Liley AW. Liquor amnii analysis in the management of the pregnancy complicated by rhesus sensitization. Am J Obstet Gynec 1961; 82: 1359–70.

2. Rubin LP, Hansen K. Testing for hematologic disorders and complications. Clin Lab Med 2003; 23: 317–43.

3. Golin YG, Copel JA. Management of the Rh-sensitized mother. In: Bifano EM, Ehrenkranz RA, eds. Perinatal hematology. Clinics in Perinatology 1995; 22: 545–59.

4. v Muralt G, Sidiropoulos D. Praktisches Vorgehen bei der Diagnose, Therapie und Prophylaxe des Morbus haemolyticus neonatorum. Ärztl Lab 1981; 27: 17–27.

5. Queenan JT, Thomas P, Tomasi BS, Serdar H, Ural MD, King JC. Deviation in amniotic fluid optical density at a wavelength of 450 nm in Rh-immunized pregnancies from 14 to 40 weeks’ gestation: a proposal for clinical management. Am J Obstet Gynecol 1993; 168: 1370–6.

38.6 Assessment of fetal lung maturity

The free transition of gas exchange from the placenta to the lungs of the newborn depends on a mature regulatory respiratory center, a complication free alteration of the pulmonary circulation, and mature, functional lungs /1, 2/. However, sufficient respiration is only made possible by the anti-atelectasis factor, or pulmonary surfactant.

38.6.1 Indication

Aid to decision making on an impeding desirable pre term delivery. If fetal lung maturity is still insufficient, it can be accelerated by the administration of glucocorticoids.

38.6.2 Method of determination

Lecithin/sphingomyelin ratio (L/S ratio)

Principle: the phospholipids are extracted from the centrifuged amniotic fluid sample with methanol/chloroform and fractionated by acetone precipitation. The precipitated phospholipids are separated by thin-layer chromatography and the fractions are visualized by staining with dyes or by charring. Following densitometric analysis, the lecithin/sphingomyelin ratio is calculated. The L/S ratio is the gold standard of lung maturity testing /3, 4/.

FLM-II assay

Principle: in the FLM II assay, the synthetic fluorescent dye N-[αN-palmitoyl-ε-N-(4-nitrobenzo-2oxa-1,3-diazole)-L-lysine]-2-aminoethanol-N-(trimethylamino ethanol) phosphate, which binds to albumin and surfactant, is added to the filtered amniotic fluid sample. The dye-albumin complex displays high polarization, whereas the surfactant complex shows low polarization. The measured total polarization therefore depends on the distribution of the dye between the surfactant and albumin and thus is used for the calculation of the surfactant/albumin ratio /5, 6/.

Immunological rapid slide-test for phosphatidylglycerol (Amniostat-FLM)

Principle: an aliquot of the centrifuged amniotic fluid sample is mixed with an alcoholic suspension of lecithin and cholesterol. The liposomes thus formed incorporate phosphatidylglycerol if it is present in the sample. 10 μL of liposome suspension is placed on a slide and mixed with 25 μL of antiserum which contains specific antibodies to phosphatidylglycerol. Subsequently, agglutination of the liposomes ensues; the extent of agglutination is roughly proportional to the phosphatidylglycerol content of the amniotic fluid sample /7/.

Lamellar body count

Principle: the number of lamellar bodies, which are roughly of the same size as platelets, is determined in the non centrifuged amniotic fluid sample in the platelet channel of a hematology analyzer /8/.

38.6.3 Specimen

Amniotic fluid, obtained preferably through amniocentesis: 10 mL

38.6.4 Reference interval

Refer to Tab. 38.6-1 – Reference intervals for surfactant.

38.6.5 Clinical significance

Lung maturity is usually reached at the 36th gestational week and is indicated by a characteristic increase in lecithin and phosphatidylglycerol in amniotic fluid.

38.6.5.1 Neonatal respiratory distress syndrome

Neonatal respiratory distress syndrome is one of the main causes of neonatal mortality. There are prenatal factors which either delay or accelerate lung maturation, leading to limited interpretability of lung maturity tests.

Lung maturation can be delayed as a result of maternal diabetes mellitus, non-hypertensive kidney disease, Rh(D) immunization, and medication with phenobarbital.

Factors accelerating lung maturation include fetal stress, (e.g., in the case of placental insufficiency) premature amniorrhexis, amnion infection, or tocolysis.

The result of a lung maturity test is an important adjunct tool for the obstetrician, as far as decisions are concerned on how to proceed in high risk pregnancies. Once lung maturity is reached, a possibly premature delivery can be timed to minimize the risk of respiratory distress syndrome. If the test indicates that the lung is not fully matured, glucocorticoids can be administered to accelerate maturation and lung maturity testing can be repeated (e.g., after 1 week).

All tests have a diagnostic sensitivity of 80–100% with a specificity of 50–70%.

Refer to Tab. 38.6-2 – Significance of lung maturity tests.

Incidence of respiratory distress syndrome

In Germany, 5% of pregnant women give birth after 34–36 weeks of gestation, 20% after 37–38 weeks, and 28% by caesarean section. The incidence of respiratory distress syndrome after delivery depends on the gestational age. The odds ratio of respiratory distress syndrome is 3.9 for infants delivered at 37 weeks and 1.9 for those delivered at 39 weeks of gestation /9/. An other study found that each additional week in utero before a cesarean delivery reduces the odds ratio of respiratory distress syndrome by 0.69.

Caesarean delivery increases the risk of respiratory distress syndrome, because for every 8 newborns delivered by caesarean section, one more than expected with vaginal delivery is hospitalized /10/.

Data from the US Centers for Disease Control /11/ show that children born at 34 weeks’ gestation had a significantly higher risk of the following than those born at 37–40 weeks’ gestation: respiratory distress syndrome (3.9% versus 0.2%), antibiotic treatment (10.8% versus 1%), and neonatal seizures (0.09% versus 0.03%).

38.6.5.2 Respiratory distress syndrome in very low birth weight infants

To ensure the survival of premature infants with a birth weight of less than 1 kg, considerable medical treatment is required, including prenatal administration of corticosteroids and postnatal administration of surfactant. All these premature infants will have respiratory distress syndrome, unless they were treated with corticosteroids immediately after birth. Prenatal inflammation accelerates the production of surfactant as well as lung maturation. This is not the case when chorioamnionitis was present. The synthesis of surfactant in response to corticosteriods would also be insufficient then /12/.

38.6.5.3 Twin gestation

Premature birth and premature amniorrhexis are common in twin pregnancies. Fetal lung maturation generally occurs earlier in twin than in singleton pregnancies (32 weeks versus 36 weeks of gestation). If premature amniorrhexis (i.e., rupture of membranes) occurs, it must be investigated whether the lungs of both fetuses have matured. The fetuses should show concordance of lung maturity at 28–29 and 36–37 weeks’ gestation, but not in the weeks in between. In diamniotic twin pregnancies, the L/S ratio should be determined in each amniotic sac at 30–35 weeks /13/.

38.6.5.4 Polyhydramnios, oligohydramnios

Both conditions lead to a change in the lecithin concentration of only 5–10% and therefore can be neglected in the evaluation /2/.

38.6.6 Comments and problems

Sample

Samples collected from the vaginal fluid after premature rupture of membranes often contain blood, bacteria, and vaginal secretion. The results are of limited value and can be of any use only if the sample was collected after the escape of amniotic fluid and cooled immediately at 4 °C.

Interference from blood or meconium

All methods, with the exception of immunological rapid tests, are affected, to a greater or lesser degree, by the presence of blood or meconium.

Centrifugation of the sample

If centrifugation is required, it should be performed for 2 min. at 400 × g. This sufficiently eliminates erythrocytes and clots of vaginal secretion, but also reduces the number of lamellar bodies by approximately 8%. The surfactant content is drastically reduced by higher speeds and longer times of centrifugation /14/.

FLM-II assay

Bilirubin in amniotic fluid does not interfere with the assay /17/. Samples are stable for 16 h when stored at room temperature and for 24 h when stored at 4 °C. Blood contamination up to 0.03 × 10¹² erythrocytes/L does not interfere with the assay /18/.

Lamellar body count

Because lamellar bodies settle to the bottom of the collection container if the sample is left to sit undisturbed, the sample should be mixed gently for about 2 min. before starting the analysis. To prevent foaming, it must not be mixed with a vortexer. Samples containing mucus and meconium are unacceptable for FLM-II analysis.

Stability

Stable for up to 2 days at 4 °C for determination of the L/S ratio, for up to 10 days for lamellar body count, and for up to 24 hours for FLM II analysis.

38.6.7 Pathophysiology

By 26 weeks’ gestation, the lungs are sufficiently developed to allow extrauterine gas exchange. Functionally, however, sufficient respiration is only made possible by the anti-atelectasis factor, or pulmonary surfactant. This substance, which can be found at the interface between the alveolar airspace and the alveolar wall, prevents the alveoli from collapsing during exhalation by reducing the surface tension at the interface between the liquid phase and the alveolar wall. This also prevents the exudation of serum and lymph into the alveolar space and thus the formation of the classic hyaline membranes /1, 2/.

If there is insufficient surfactant, ventilation and perfusion are difficult or impossible due to the formation of hyaline membranes. This results in respiratory distress syndrome in the neonate.

Pulmonary surfactant is a mixture of 90% phospholipids (phosphatidylcholine, phosphatidylglycerol) and 10% proteins (surfactant proteins A, B and C). Surfactant forms a monolayer over the surface epithelium of the alveoli at the air-liquid interface and reduces the surface tension. Surfactant is stored as a lipid-protein complex in the form of lamellar bodies in type II pneumocytes, which make up approximately 5% of the surface epithelium. Once secreted into the alveolar space, the lamellar bodies become hydrated in the surface water of the alveolar wall and form microtubular lattice-like structures which support the surfactant monolayer.

Surfactant production begins at 25–30 weeks of gestation, and surfactant or lamellar bodies are clearly detectable from 28–32 weeks. Surfactant synthesis increases by a factor of more than 10 with advancing gestation. However, prior to 34 weeks’ gestation, the amount and composition of surfactant are often insufficient to assure spontaneous respiration with adequate oxygenation.

Fetal breathing movements expel surfactant and lamellar bodies into the amniotic fluid where they can be measured. Thus, by measuring the phospholipids or lamellar bodies at 32–36 weeks’ gestation, the development of the fetal lung can be monitored and the risk of respiratory distress syndrome can be assessed.

From 37 weeks of gestation, the risk of respiratory distress syndrome is negligible, so that laboratory testing for fetal lung maturity is required only in exceptional cases.

References

1. Griese M. Pulmonary surfactant in health and human lung diseases: state of the art. Eur Resp J 1999; 13: 1455–76.

2. Torday JS, Rehan VK. Testing for fetal lung maturation: a biochemical window to the developing fetus. Clin Lab Med 2003; 23: 361–83.

3. Kulovich MV, Hallmann MB, Gluck L. The lung profile I. Normal pregnancy. Am J Obstet Gynecol 1979; 135: 57–63.

4. Kulovich MV, Gluck L. The lung profile II. Complicated pregnancy. Am J Obstet Gynecol 1979; 135: 64–70.

5. Russell JC, Cooper CM, Ketchum CH, Torday JS, Richardson DK, Holt JA, et al. Multicenter evaluation of TDx test for assessing fetal lung maturity. Clin Chem 1989; 35: 1005–10.

6. Herbert WNP, Chapman JF, Schnoor MM. Role of the TDx FLM assay in fetal lung maturity. Am J Obstet Gynecol 1993; 168: 808–12.

7. Weinbaum PJ, Richardson D, Schwartz JS, Gabbe SG. Amniostat FLM: a new technique for detection of phosphatidylglycerol in amniotic fluid. Am J Perinatol 1985; 2: 88–92.

8. Lu J, Gronowski AM, Eby C. Lamellar body counts performed on automated hematology analyzers to assess fetal lung maturity. Labmedicine 2008; 39: 419–23.

9. Hansen AK, Wisborg K, Uldbjerg N, Henriksen TB. Risk of respiratory morbidity in term infants delivered by elective caesarean section: cohort study. BMJ 2008; 336: 85–7.

10. Heinzmann A, Brugger M, Engels C, Prömpeler H, Superti-Furga A, Strauch K, et al. Risk factors of neonatal respiratory distress following vaginal delivery and caesarean section in the German population. Acta Paedriatrica 2009; 98: 25–30.

11. Been JV, Rours IG, Kornelisse RF, Jonkers F, de Krijger RR, Zimmermann LJ. Chorioamnionitis alters the response to surfactant in preterm infants. J Pediatr 2010; 156: 10–5.

12. Cheng YW, Kaimal AJ, Bruckner TA, Halloran DR, Caughey AB. Perinatal morbidity associated with late preterm deliveries compared with deliveries between 37 and 40 weeks of gestation. BJOG 2011; 118: 1446–54.

13. Mackenzie MW. Predicting concordance of biochemical lung maturity in the preterm twin gestation. J Matern Fetal Neonatal Med 2002; 12: 50–8.

14. Neerhof MG, Dohnal JC, Ashwood ER, et al. Lamellar body counts: a consensus protocol. Obstet Gynecol 2001; 97: 318–20

15. Moxness M, Lawson G, O’Brien J, Burritt M. Assessment of fetal lung maturity by the Abbott FLM II assay and three other methods. Clin Chem 1995; 41: S95.

16. Dilena BA, Ku F, Doyle I, Whiting MJ. Six alternative methods to the lecithin/sphingomyelin ratio in amniotic fluid for assessing fetal lung maturity. Ann Clin Biochem 1997; 34: 106–8.

17. Cariappa R, Parvin CA, Gronowski AM. Bilirubin in the amniotic fluid does not interfere with the Abbott TDx FLM II assay. Clin Chem 2003; 49: 986–7.

18. Grenache DG, Parvin CA, Gronowski AM. Preanalytical factors that influence the Abbott TDx FLM II assay. Clin Chem 2003; 49: 935–9.

38.7 Fetomaternal hemorrhage

The amount of fetal red cells in maternal blood is an indicator of the amounts of fetoplacental transfusion /1, 2/.

38.7.1 Indication

According to Ref. /3/:

• Detection and quantification of fetomaternal hemorrhage (e.g., after invasive obstetric procedures)
• Evaluation of the amount of fetal Rh(D)-positive erythrocytes in the circulation of an Rh(D)-negative mother
• Diagnosis of situations in the fetus or newborn, such as stillbirth, anemia, reduced activity
• Differentiation of maternal and fetal blood in vaginal hemorrhages during pregnancy.

38.7.2 Method of determination

Fetomaternal hemorrhage (FMH) is diagnosed by detecting fetal erythrocytes in the maternal blood.

38.7.2.1 Acid elution test

Principle: the Kleihauer-Betke test is based on a visual microscopic counting of fetal erythrocytes on a maternal blood film. In acid conditions, fetal hemoglobin (HbF) and adult hemoglobin (HbA) have differences in solubility properties. HbF resists to acid solution and fetal red blood cells are stained bright pink using an acidic solution consisting of FeCl₃ and hematoxylin. Hb is eluted from adult erythrocytes that appear as ghosts and are stained using erythrosin. The fetal cells are counted in relation to 1,000 erythrocytes (shadow erythrocytes and HbF cells) by means of a grid micrometer. The proportion of fetal blood (in mL) that has reached the maternal circulation can be calculated as follows /4/:

Fetal blood (mL) = HbF cells /total red cells × 5,000

The maternal blood volume is 5,000 mL

38.7.2.2 Flow cytometry (FC)

Flow cytometric strategies determine quantitatively:

• Fetal red cells (HbF cells; α2γ2) in the maternal blood
• Heterocellular red cells of the mother containing HbA and HbF (F cells; α2γ2 and α2β2)
• Adult red cells (HbA, α2β2)
• Rh(D)-positive fetal red cells in Rh(D)-negative maternal blood.

Flow cytometry using anti-HbF antibodies

Red cells containing HbF are labeled with fluorescein isothiocyanate (FITC)-conjugated anti-HbF antibodies.

Various strategies are used:

1. Intracellular labeling of HbF with FITC-labeled monoclonal anti-HbF antibodies. Prior to incubation with the antibody, the erythrocytes are fixed with glutaraldehyde and treated with permeabilization solution. Nucleated cells are labeled with propidium iodide. HbF cells show high fluorescence, F cells intermediate, and HbA cells no fluorescence /3/.

2. Combined use of FITC labeled anti-HbF antibodies and red cell anti-carbonic anhydrase antibodies. Carbonic anhydrase (CA) does not form in the erythrocytes until after birth, and only adult erythrocytes are labeled. A dual color test is used.

The following three cell populations are differentiated in maternal blood /5/:

• Fetal erythrocytes (HbF cells): HbF ⁺ / CA ^–
• F-cells: HbF ⁺ / CA ⁺
• Adult erythrocytes: HbF^– / CA ⁺

3. Combined use of antibodies to Rh(D) and HbF for the simultaneous analysis of FMH and the fetal Rh(D) phenotype /6/.

Calculation of fetomaternal hemorrhage

Assuming that the maternal red cell mass is 1,800 mL and the relative fetal red cell mass is 22% larger than that of adults, the mass (packed cell mass of fetal erythrocytes) of FMH is calculated as follows /16/:

• % positive events × 1,800/100 × 122/100
• FMH (mL packed cells) = % fetal cells × 22

38.7.3 Specimen

• 2 to 3 smears of maternal blood
• EDTA blood: 2 mL
• 2 to 3 smears of vaginal blood
• Bloody amniotic fluid, centrifuge prior to smear preparation, prepare smears from sediment.

38.7.4 Reference interval

Refer to Tab. 38.7-1 – HbF cells and HbF-containing red cells in blood.

38.7.5 Clinical significance

Fetomaternal hemorrhage (FMH) normally occurs in small volumes during pregnancy and increases during parturition. If there is a significant difference in the erythrocyte antigenicity between the mother and child, this can result in allosensitization of the maternal immune system, which can lead to disease and loss of the current and future pregnancies. The count of fetal erythrocytes in the maternal blood is important for determining the extent of FMH and for the treatment of fetomaternal Rh(D) incompatibility. FMH is more likely at parturition and often after invasive obstetric procedures during pregnancy.

The detection of fetal red cells in the maternal blood can provide answers to the following questions:

• Has a FMH occurred following a maternal trauma or after invasive obstetric procedures during pregnancy?
• Is a FMH the cause of an unexpected elevation of α₁-fetoprotein in maternal blood?
• Is a FMH the cause of an anemia in the fetus or newborn? Fetomaternal macro transfusions often cause no symptoms and can pose a significant risk to the fetus due to anemia.
• Is the dose of Rh antiserum administered postpartum to a Rh-negative mother with a Rh(D)-positive newborn sufficient?

38.7.5.1 Diagnosis of fetomaternal hemorrhage

FMH is determined with flow cytometry assays using:

• Anti-HbF or anti-Rh(D) antibodies
• A combination of anti-Rh(D) antibodies and anti-HbF antibodies, allowing simultaneous analysis of FMH and fetal Rh(D) phenotype
• A combination assay that detects cells containing fetal cells (HbF), F cells and adult red cells containing carbonic anhydrase.

Flow cytometry shows clear advantages in comparison to Kleihauer and Betke test, because it allows the differentiation of adult F cells from fetal HbF cells. Using the Kleihauer and Betke test, strongly stained F cells are erroneously counted as fetal erythrocytes, leading to false positive FMH results, in particular in pregnant women with a proportion of F cells greater than 5% /10/.

With the application of dual color flow cytometry (anti-HbF and anti-carbonic anhydrase antibodies) for the analysis of FMH during delivery it was demonstrated that small amounts of fetal cells (≥ 0.02%, corresponding to 1 mL of fetal blood) into the maternal circulation occurs in the majority of women during pregnancy and parturition. Significant FMH (≥ 5 mL) was detected in 11%, and severe FMH (≥ 30 mL) in 0.8% /11/. A different study for FMH detection /12/, in which HbF was labeled intracellularly with FITC labeled monoclonal anti-HbF antibodies and then measured with flow cytometry, found that 31% of pregnant women had FMH ≥ 1 mL, 5.5% had FMH ≥ 5 mL, and 1.7% had FMH ≥ 30 mL.

In neonatal anemia, FMH must be suspected if Hb is < 100 g/L and Hct < 0.30. In a study /13/, 24 of 219,853 live births had evidence of FMH with initial Hb levels of 14–102 g/L. All mothers in whom neonatal Hb level was < 30 g/L reported absent fetal movement as did two-thirds of mothers when the initial Hb was < 70 g/L. Outcomes were poorer in those with the lowest initial Hb. The adverse outcomes of death, intraventricular hemorrhage, periventricular leukomalacia, bronchopulmonary dysplasia or hypoxic-ischemic encephalopathy occurred in 71%, including all with an initial Hb < 50 g/L and all born at ≤ 35 weeks of gestation.

38.7.5.2 Management of Rh(D)-negative pregnant women

The risk of many women is of sensitization to incompatible fetal blood group antigens, because pregnant women are susceptible to making allo antibodies /10/.

In Central Europe, 18% of pregnant women are Rh(D) negative, and two-thirds of them will give birth to a Rh(D)-positive child, so that in 12% of pregnancies there is a rhesus constellation of a Rh(D)-negative mother and a Rh(D)-positive child and thus a risk of immunization /14/. The risk of immunization increases with the extent of fetal erythrocytes entering the maternal circulation. When FMH of less than 0.1 mL occurs, the risk of sensitization demonstrated in the sixth month after birth is 3%, if this blood volume is greater than 0.1 mL, the possibility of sensitization increases to 14% over the same time /15/. Due to the postpartum anti-D prophylaxis, 300 μg of anti-Rh(D) immunoglobulin, this risk is present in only 1.6% of this Rh(D) constellation and 0.3% of all pregnancies. It is further reduced by the anti-Rh(D) prophylaxis recommended as an additional preventive measure for Rh(D)-negative pregnant women at 28–30 weeks’ gestation.

It is important that anti-Rh(D) Ig is administered within 72 hours of delivery, an abortion or miscarriage (20–25 μg of anti-Rh(D) per mL of Rh(D)-positive fetal erythrocytes). In these situations, 300 μg of anti-Rh(D) Ig is generally administered in Germany and in the USA, and 100 μg in Great Britain. Anti-Rh(D) prophylaxis should also be given to Rh(D)-negative mothers whose newborns have the D^weak antigen.

The efficacy of treatment with anti-Rh(D) Ig can be assessed based on:

• The decline of HbF cells in the maternal blood. Determination shortly after delivery, on the 3rd day as well as the 7th day after parturition. Elimination can take up to 1 week even if anti-Rh(D) is given, and up to 80 days if no anti-Rh(D) is administered.
• Testing for excess levels of anti-Rh(D) antibodies in the maternal blood (previously negative indirect Coombs test becomes positive).

In 2–6% of cases of Rh(D) FMH, sensitization occurs even during the first pregnancy and an ill child is born.

38.7.5.3 Amniocentesis

If bloody amniotic fluid was aspirated during amniocentesis, it is recommended to determine HbF cells in the amniotic fluid to check whether the needle has passed the placenta and, if necessary, anti-Rh(D) prophylaxis is administered.

38.7.6 Comments and problems

The FMH volume estimated with the Kleihauer and Betke test is larger than that quantified using flow cytometry /16/. Depending on the flow cytometry method, a positive threshold of 4.3, 10 or 12.5 fetal erythrocytes per 10,000 maternal erythrocytes is used, depending on the study. The coefficient of variation is up to 20%, the inter laboratory variation in surveys up to 30%.

38.7.7 Pathophysiology

Fetal red cells contain exclusively fetal hemoglobin (HbF) until week 30 when synthesis of adult hemoglobin (HbA) is switched on. The fetal hemoglobin is a tetramer comprising two alpha chains and two gamma chains. Adult hemoglobin (HbA) is a tetramer comprising two alpha chains and two beta chains. At term the proportion of HbF in cord blood is about 50–70%. In adult nearly all erythrocytes contain HbA, a percentage lower than 5% contains HbF. These cells are known as F cells. In some hemoglobinopathies F cells are increased because of hereditary persistence (see Section 15.7 – Hemo­globino­pathies) /1/.

Fetal blood in the maternal circulation due to fetomaternal hemorrhage (FMH) following damage to the chorionic villi increases the number of fetal red cells (HbF cells) in the maternal circulation. In volume terms, only 0.1 mL of fetal blood or less is believed to penetrate the maternal circulation during pregnancy and in only 0.2% of births does more than 30 mL of fetal blood pass into the maternal circulation /2/. The life span of fetal erythrocytes in the maternal circulation is 80 days. The amounts of fetoplacental blood volume for different gestational ages (in weeks) are shown in Tab. 38.7-2 – Feto-placental blood volume.

For further information refer to Ref. /17/.

References

1. Chambers E, Davies L, Evans S, Birchall J, Kumpel B. Comparison of haemoglobin F detection by the acid elution test, flow cytometry and high-performance liquid chromatography in maternal blood samples analysed for fetomaternal haemorrhage. Transfusion Medicine 2012; 22: 199–204.

2. Meleti D, De oliveira LG, Junior EA, Ceatano ACR, Boute T, Nardozza LMM, Moron AF. Evaluation of passage of fetal erythrocytes into maternal circulation after invasive obstetric procedures. J Obstet Gynecol Res 2013; doi: 10.1111/jog.12073.

3. Pastoret C, Le Priol J, Fest T, Roussel M. Evaluation of FMH QuikQuant for the detection and quantification of fetomaternal hemorrhage. Cytometry Part B 2013; 84B: 37–43.

4. Betke K, Kleihauer E. Fetaler und bleibender Blutfarbstoff in Erythrozyten und Erythroblasten von menschlichen Feten und Neugeborenen. Blut 1958; 4: 241–9.

5. Porra V, Bernaud J, Gueret P, Bricca P, Rigal D, Follea G, et al. Identification and quantification of fetal red blood cells in maternal blood by dual-color flow cytometric method: evaluation of the Fetal Cell Count kit. Transfusion 2007; 47: 1281–9.

6. Radel DJ, Penz CS, Dietz AB, Gastineau DA. A combined flow cytometry-based method for fetomaternal hemorrhage and maternal D. Transfusion 2008; 48: 1886–91.

7. Ryan K, Bain BJ, Worthington D, et al. Significant haemoglobinopathies: guidelines for screening and diagnosis. Br J Haematol 2010; 149: 35–49.

8. Garner C, Tatu T, Rettie JE, Littlewood T, Darley J, Cervino S, et al. Genetic influences on F cells and other hematologic variables: a twin heritability study. Blood 2000; 95: 342–6.

9. Sebring ES, Polesky HF. Fetomaternal hemorrhage: incidence, risk factors, time of occurrence, and clinical effects. Transfusion 1990; 30: 344–57.

10. Kumpel BM, MacDonald AP, Bishop DR, Yates AF, Lee E. Quantitation of fetomaternal haemorrhage and F cells in unusual maternal blood samples by flow cytometry using anti-D and anti-HbF. Transfusion Medicine 2013; 23: 175–86.

11. Merz WM, Patzwaldt F, Fimmers R, Stoffel-Wagner B, Gembruch U. Dual-colour flow cytometry for the analysis of fetomaternal haemorrhage during delivery. J Clin Pathol 2012; 65: 186–7.

12. Chen JC, Davis BH, Wood B, Warzynski MJ. Multicenter clinical experience with flow cytometric method for fetomaternal hemorrhage detection. Clin Cytometry 2002; 50: 285–90.

13. Christensen RD, Lambert DK, Baer VL, Richards DS, Bennett ST, Ilstrup SH, Henry E. Severe neonatal anemia from fetomaternal hemorrhage: report from a multihospital health-care system. J Perinatol 2013; 33: 429–34.

14. von Muralt GV, Sidiropoulos D. Praktisches Vorgehen bei der Diagnose, Therapie und Prophylaxe des Morbus haemolyticus neonatorum. Ärztl Lab 1981; 27: 17–27.

15. Zipursky A, Israel SL. The pathogenesis and prevention of Rh immunization. Can Med Assoc 1967; 97: 1245–57.

16. Kumpel BM. Analysis of factors affecting quantification of fetomaternal hemorrhage by flow cytometry. Transfusion 2000; 40: 1376–83.

17. Kumpel BM, Manoussaka MS. Placental immunology and maternal alloimmune responses. Vox sanguis 2012; 102: 2–12.

38.8 Fetal aneuploidies: triple-test, quadruple test

Prenatal screening for fetal aneuploidies (differences in the number of chromosomes from euploidy or presence of chromosomal fragments) is a fundamental part of routine screening in many countries. Prenatal aneuploidy screening is a standard program for the diagnosis of:

• Down syndrome (trisomy 21), which accounts for about half of all chromosomal abnormalities
• Edwards syndrome (trisomy 18)
• Patau syndrome (trisomy 13)

Aneuploidies are not inherited, but are caused by nondisjunction of a chromosome in meiosis.

Noninvasive prenatal testing that combines maternal serum screening plus ultrasound to rule out fetal aneuploidies is carried out as

• Triple test in the first trimester
• Second trimester quadruple test.

The analysis of cell-free fetal DNA in maternal blood is a new screening test and results of ultrasound investigations are not included.

Refer to Section 38.2.2.3 – Noninvasive prenatal aneuploidy testing.

In cases with positive results all noninvasive methods have to be confirmed with the use of invasive methods (e.g., karyotyping of amniotic fluid).

38.8.1 Indication

• Advanced maternal age
• Suspected abnormality from previous tests
• Sonographic evidence of aneuploidy
• Parent or child with aneuploidy.

38.8.2 Method of determination

The combined first-trimester test is performed, which involves a nuchal translucency scan as well as laboratory tests /1/. The first-trimester test is not a diagnostic test, but merely a risk assessment using an algorithm. The test identifies about 90% of fetuses with trisomy 21 with a false positive rate of about 5%. This means that there is a high number of unaffected pregnancies and invasive test procedures or the determination of fetal cell-free DNA in the maternal plasma must be performed for verification.

38.8.2.1 Nuchal translucency scan

The measurement of fetal nuchal translucency thickness provides effective and early screening for chromosomal aneuploidy. In the fetus, fluid builds up in the tissue space within the nape of the neck up to 14 weeks of gestation. The nuchal thickness can be detected by ultrasonography as nuchal translucency /1, 2/. The more fluid that has accumulated, the greater the probability of a fetal abnormality being present. In aneuploidies, the increased nuchal translucency is thought to be caused by alterations of the extracellular matrix due to increased synthesis of collagen IV. The scan is of diagnostic value only if performed between weeks 11 to 13 weeks + 6 days /1/, because before 14 weeks’ gestation the lymphatic system and renal function are not sufficiently developed to adequately train the nuchal fluid collection /3/.

A nuchal translucency > 95th percentile for the gestational age is associated with an increased risk of aneuploidy and other diseases. In the FASTER study, the detection rate of nuchal translucency screening for fetal aneuploidy at 11 weeks was 70% with a false-positive rate of 5% /4/. However, increased nuchal translucency can also be a marker of other abnormalities. For example, the detection rate for cardiac defects is 52% with a false-positive rate of 5% /5/.

38.8.2.2 Laboratory biomarkers

The following biomarkers are used /4/:

• Free β-subunit (hCGβ) or total hCG (β-hCG)
• Pregnancy-associated plasma protein A (PAPP-A)
• α-fetoprotein (AFP)
• Inhibin A
• Unconjugated estriol.

Refer to Tab. 38.8-1 – Biomarkers in antenatal screening for aneuploidies.

38.8.2.3 Combined first-trimester test

The test is performed at 11–13 weeks and includes the results of nuchal translucency, the PAPP-A and hCGβ blood test results with maternal age. The probability of aneuploidy is calculated by combining the results. The following must be considered when calculating the results /1/:

• Gestational age; it is verified during the nuchal translucency scan by measuring the crown-rump length
• The discrimination of hCGβ increases with gestational age and is highest at 13 weeks
• PAPP-A is most discriminatory at 7–10 weeks of gestation and then becomes less discriminatory. When used together, PAPP-A and hCGβ complement each other and provide high diagnostic sensitivity from 11 to 13 weeks of gestation.
• In Trisomy 13 and 18, first-trimester PAPP-A and hCGβ levels are low, and in trisomy 21 hCGβ levels are elevated in comparison to a normal fetus.

38.8.2.4 Second-trimester quadruple test

Blood for this test is taken between 15 and 20 weeks of pregnancy. The test includes the serum levels of AFP, hCGβ, unconjugated estriol and inhibin A in combination with the maternal age. The behavior of the laboratory parameters between 15 and 22 weeks’ gestation is shown in Tab. 38.8-2 – Change of biomarkers for aneuploidy screening. Based on these values, the laboratories determine the gestational age-related risk for trisomy 21.

38.8.2.5 Integrated test

First-trimester nuchal translucency and PAPP-A, and second trimester AFP, hCGβ, unconjugated estriol and inhibin A combined with maternal age.

Tab. 38.8-3 – Test dependent detection rates for trisomy 21 provides an overview of the detection rates and reliability of the different diagnostic tests for trisomy 21.

38.8.3 Comments and problems

Specimen

Since PAPP-A is underestimated in EDTA and heparinized plasma, serum is used for the determination. It is important that all biomarkers are performed from the same blood sample. Therefore, serum is the specimen of choice /6/.

Very low concentration of free estriol

Very low estriol levels in the quadruple test are indicative of Smith-Lemli-Opitz syndrome, which has a prevalence of 1 : 10,000 or 1 : 20,000 live births and is caused by a mutation in the gene encoding 7-sterol reductase. This results in low cholesterol synthesis. Since steroids are synthesized from cholesterol, free estriol is reduced /9/.

References

1. Nicholaides KH. First-trimester screening for chromosomal abnormalities. Semin Perinatol 2005; 29: 190–4.

2. Bethune M. Literature review and suggested protocol for managing ultrasound soft markers for Down syndrome: thickened nuchal fold, echogenic bowel, shortened femur, shortened humerus, pyelectasis and absent or hypoplastic nasal bone. Australasian Radiology 2007; 51: 218–25.

3. von Kaisenberg CS, Krenn V, Ludwig M, Nicolaides KH, Brand-Saberi B. Morphological classification of nuchal skin in human fetuses with trisomy 21, 18, 13 at 12–18 weeks and in a trisomy 16 mouse. Anat Embryol (Berl) 1998; 197: 105–24.

4. Wald NJ, Bestwick JP, Huttly WJ. Improvements in antenatal screening for Down’s syndrome. J Med Screen 2013; 20: 7–14.

5. Wald NJ, Morris JK, Walker K, Simpson JM. Prenatal screening for serious congenital heart defects using nuchal translucency: a metaanalysis. Prenat Diagn 2008; 28: 1094–1104.

6. Spencer K. The influence of different sample collection types on the levels of markers used for Down’s syndrome screening as measured by the Kryptor immunoassay system. Ann Clin Biochem 2003; 40: 166–8.

7. Bindra R, Heath V, Liao A, Spencer K, Nicolaides KH. One-stop clinic for assessment of risk trisomy 21 at 11–14 weeks: a prospective study of 15,030 pregnancies. Ultrasound Obstet Gynecol 2002; 20: 219–25.

8. Wald NJ, Hackshaw AK. Combining ultrasound and biochemistry in the first-trimester screening for Down’s syndrome. Prenat Diagn 1998; 18: 511–23.

9. Suresh S, Rajan T, Tsutsumi R. New targets for old hormones: inhibins clinical role revisited. Endocrine J 2011; 58: 223–35.

10. Canick JA, Saller Jr, DN, Lambert-Messerlian GM. Prenatal screening for Down syndrome: current and future methods. Clin Lab Med 2003; 23: 395–411.

11. Wenstrom KD. First-trimester Down syndrome screening: component analytes and timing for optimal performance. Semin Perinatol 2005; 29: 195–202.

38.9 Preeclampsia, HELLP syndrome

Preeclampsia

The American College of Obstetrics and Gynecologists (ACOG) /1/ defines preeclampsia as a new onset of hypertension after 20 weeks' of gestation, and proteinuria, or new onset hypertension with evidence of end-organ damage. Preeclampsia is a placentally induced hypertensive disorder of pregnancy and multiorgan disease that can affect the maternal liver, hematopoietic, vascular, renal, neurologic, and respiratory systems with mild to severe impairments. Preeclampsia can also affect the fetus of new onset of growth restriction, placental abruption, or reduced amniotic fluid volume as isolated findings or in combination. Unpredictable disease evolution is a hallmark of preeclampsia such as progression from mild hypertension and proteinuria to HELLP syndrome (hemolysis, elevated liver enzymes, low platelet count), disseminated intravascular coagulation and posterior reversible encephalopathy. Preeclampsia is the leading cause of maternal and neonatal morbidity and mortality and affects about 8% of pregnancies worldwide. Approximately 30% of pregnancies develop some signs and symptoms of preeclampsia and about 20% of such patients are eventually diagnosed with preeclampsia /2/. The clinical diagnostic criteria for preeclampsia are presented in Tab. 38.9-1 – Criteria of preeclampsia (PE) severe PE and HELLP syndrome. Major risk factors for preeclampsia are: prior preeclampsia, chronic hypertension, pregestational diabetes mellitus, multiple gestation, and antiphospholipid syndrome.

Eclampsia

Eclampsia is the new onset of seizures before, during, or after labor, which is not attributable to other causes, in a women with preeclampsia. Seizures in a pregnant women who had not been previously diagnosed with preeclampsia must be accompanied by two of the following within 24 hours of presentation to be considered eclamptic: hypertension, proteinuria, thrombocytopenia, or increased aspartate aminotransferase /2/.

HELLP syndrome

The HELLP syndrome is a severe complication of preeclampsia and includes the laboratory constellation of hemolysis (H), elevated liver enzymes (EL) and low platelet (LP) count /3/.

38.9.1 Clinical findings in preeclampsia

Preeclampsia is one of the most common and most severe complications of pregnancy. It affects 3–8% of all pregnancies worldwide and accounts for 18% of maternal and up to 40% of fetal deaths as well as up to 15% of pre-term births.

Preeclampsia develops in three stages /4/:

• The first stage is characterized by an altered formation of the placenta. During placentation, a defective invasion of the extra villous trophoblast cells into the muscle layers of the spiral arteries occurs. This results in a reduced utero-placental blood flow that can result in fetal intrauterine growth restriction. Oxidative stress further aggravates vascular function in the placenta and this, in turn, gives rise to a further reduction of the blood flow, resulting in inflammation and apoptosis of placental cells.
• The second stage is characterized by the clinical manifestations of hypertension and proteinuria and appears from the 20 weeks of gestation onwards.
• As the disease progresses, angiospasms in the brain and brain edema may cause severe epileptic seizures (eclampsia).

According to the International Society for the Study of Hypertension in Pregnancy (ISSHP), preeclampsia is defined /5/:

• As nouveau hypertension occurring after 20 weeks of pregnancy together with proteinuria
• Hypertension is defined as a systolic blood pressure ≥ 140 mmHg and a diastolic blood pressure ≥ 90 mmHg, measured on two occasions at least 4 h apart.
• Proteinuria is defined as excretion ≥ 300 mg/24 h.

Based on the time of onset of clinical manifestations, preeclampsia is divided into early-onset preeclampsia (below 34 weeks’ gestation) and late-onset preeclampsia (≥ 34 weeks’ gestation). Based on the severity of manifestation, preeclampsia is distinguished from severe preeclampsia before 34 weeks’ gestation, and HELLP syndrome /6/. The incidence of severe preeclampsia ranges from 0.6–1.2% of pregnancies in Western countries. Preeclampsia below 37 weeks’ and severe preeclampsia below 34 weeks’ gestation complicates 0.6–1.5% and 0.3% of pregnancies, respectively /6/.

Severe features of preeclampsia include a systolic blood pressure of at least 160 mmHg or a diastolic pressure of at least 110 mmHg, platelet count less than 100 × 10⁹/L, aminotransferase levels two times the upper reference interval value, a doubling of serum creatinine level or a level greater 1.1 mg/dL (97 μmol/L), severe persistent right upper quadrant pain, pulmonary edema, or new-onset cerebral or visual disturbances. Preeclampsia can worsen or initially present after delivery /6, 7/.

Preeclampsia and renal dysfunction

The renal damage in preeclampsia is due to distinct glomerular lesions known as glomerular endotheliosis, defined by an enlarged glomerular volume with swelling of endothelial cells and occlusion of capillary lumens /8/. The podocytes are relatively normal despite the presence of proteinuria. Increased levels of soluble vascular endothelial growth factor (VEGF) type 1 is believed to initiate the onset of glomerular endotheliosis which may contribute to the increased proteinuria and decreased glomerular filtration rate observed in preeclampsia.

Preeclampsia and hypertension

In a normal pregnancy, cardiac output is increased with expanded circulatory blood volume and a decrease in peripheral vascular resistance /7, 8/. During normal gestation diastolic blood pressure is considerably reduced and the expansion of the vascular bed prevents placental hypo perfusion. In preeclampsia, the plasma volume is markedly reduced despite the presence of edemas. This results in decreased maternal systemic perfusion, which can potentially lead to damage to the maternal organs and those of the fetus.

The renin concentration acts as a volume sensor, and low renin levels are associated with volume expansion. This, however, does not mean that preeclampsia is associated with volume dependent hypertension. One theory on the pathology of hypertension suggests that preeclampsia patients have autoantibodies which bind to and activate angiotensin type I receptor, thereby causing vasoconstriction.

Refer to Tab. 38.9-1 – Clinical criteria of preeclampsia (PE), severe PE, and HELLP syndrome.

38.9.1.1 Screening for preeclampsia

Refer to Section 38.10 – PIGF and sFlt-1.

38.9.2 Clinical findings in HELLP syndrome

There is considerable variability in the terminology, cause, incidence and diagnostic criteria of HELLP syndrome /3/. Some investigators consider it an early form of severe preeclampsia, others a variant of preeclampsia. Thus, the incidence of HELLP syndrome also varies significantly. In one study /9/, the syndrome was observed in 30% of patients with postpartum eclampsia and in 28% of patients with preeclampsia prior to delivery.

Patients with HELLP syndrome are significantly older than those with severe preeclampsia. HELLP syndrome is more common among whites than blacks and occurs more frequently in multipara. The clinical symptoms are nonspecific and similar to those associated with a common cold. Many women give a history of malaise for several days before presentation. Often the pregnancy is still far away from the due date. One severe complication is acute fatty liver of pregnancy. These pregnants do not have hypertension or proteinuria.

Other complications which are less common than HELLP syndrome but have a similar clinical presentation include thrombotic thrombocytopenic purpura and hemolytic uremic syndrome.

38.9.2.1 Laboratory findings in HELLP syndrome

The diagnostic criteria used for HELLP syndrome are variable. The triad of HELLP syndome is hemolysis, caused by microangiopathic hemolytic anemia, disturbed liver function and low platelet count /3/.

Findings of microangiopathic hemolysis

Abnormal peripheral blood smear containing schistocytes, echinocytes, burr cells, elevated indirect bilirubin, low serum haptoglobin level, activity of LD increased.

Liver function

No consensus exists regarding pathology of disturbed liver function. Liver enzymes ALT and/or AST are elevated.

Low platelet count

There is no consensus regarding the pathology of thrombocytopenia.

References

1. Gestational hypertension and preeclampsia: ACOG Practice Bulletin number 222. Obstet Gynecol 2020; 135: e237–e60.

2. Docheva N, Arenas G, Nieman KM, Lopes-Perdigao J, Yeo KT, Rana S. Angiogenic biomarkers for risk stratification in women with preeclampsia. Clin Chem 2022; 68 (6): 771–81.

3. Barton JR, Sibai BM. Diagnosis and management of hemolysis, elevated liver enzymes, and low platelets syndrome. Clin Perinatol 2004; 31: 807–33.

4. Anderson UD, Olsson MG, Kristensen KH, Akerström B, Hansson SR. Review: Biochemical markers to predict preeclampsia. Placenta 2012; 26: S42–S47.

5. Brown MA, Lindheimer MD, de Swiet M, van Assche A, Moutquin JM. The statement from the International Society for the Study of Hypertension in Pregnancy (ISSHP). Hypertens Pregnancy 2001; 20: IX–XVI.

6. Publications Committee, Society for Maternal-Fetal Medicine, with the assistance of Baha M Sibai. Evaluation and management of severe preeclampsia before 34 weeks gestation. Am J Obstet Gynecol 2011; September: 191–8.

7. Leeman L, Dresang LT, Fontaine P. Hypertensive disorders of pregnancy. Am Fam Physician 2016; 93: 212–7

8. Magee LA, Nicolaides KH, von Dadelszen.P. Preeclampsia N Engl J Med 2022; 386 (19): 1817–32.

9. Sibai BM, Ramadan MK, Usta I, Salama M, Mercer BM, Friedman SA. Maternal morbidity and mortality in 442 pregnancies with hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome). Am J Obstet Gynecol 1993; 169: 1000–6.

10. Martin JN JL, Blake PG, Perry KG, Mc Caul JF, Hess LW, Martin RW. The natural history of HELLP syndrome: patterns of disease progression and regression. Am J Obstet Gynecol 1991; 164: 1500–9.

38.10 PlGF and sFlt-1

Placental production of soluble fms-like tyrosine kinase receptor-1 (sFlt-1), an antagonist of vascular endothelial growth factor (VEGF) and placental growth factor (PlGF) contribute to the pathogenesis of preeclampsia /1/. Increased serum levels of sFlt-1 and decreased levels of PlGF, thereby resulting in an increased sFlt-1/PlGf ratio can be detected in the second half of pregnancy diagnosed to have placenta-related disorders i.e., preeclampsia, intrauterine growth retardation or stillbirth /2/.

38.10.1 Indication

Risk for developing preeclampsia:

• Women with signs and symptoms of preeclampsia
• Asymptomatic women with increased risk of developing preeclampsia e.g., ethnicity, age, number of births, and a history of preeclampsia in a previous pregnancy.

Women with systemic disease and conditions that predispose to preeclampsia, including diabetes mellitus, essential hypertension, renal disease, antiphospholipid syndrome, autoimmune disease, obesity.

38.10.2 Method of determination

Sandwich immunoassays /3/ with streptavidin-biotin technology and electrochemiluminescence measurement.

38.10.3 Specimen

Serum: 1 mL

38.10.4 Reference interval

sFLt-1/PlGF ratio below 38 rules out preeclampsia. irrespective of gestational age, for at last 1 week /4, 5/.

38.10.5 Clinical significance

Clinical diagnosis of preeclampsia is commonly based on hypertension and proteinuria. However these signs are common in many other disorders. Furthermore the clinical course of preeclampsia ranges from severe and rapidly progressing early onset preeclampsia to late-onset preeclampsia at term /2/. The diagnosis based on blood pressure and proteinuria has a positive predictive value from only about 30% for predicting preeclampsia related adverse outcomes /6/.

The sFlt-1/PlGF ratio is a screening tool, especially for identifying all women developing preeclampsia from the mid trimester onwards and requiring delivery within the subsequent 4 weeks /2/. Estimation of the sFlt-1/PlGF ratio allows identification of women with high risk for imminent delivery and adverse maternal and neonatal outcome /2, 7/. The time-dependent slope of the sFlt-1/PIGF ratio between different measurements is predictive for pregnancy outcome and the risk of developing preeclampsia, and repeated measurements have been suggested /8/. High values are closely related to the need to deliver immediately.

A working group of the International Society of Ultrasound in Obstetrics and Gynecology /2/ has developed a consensus statement on the clinical use of the sFlt-1/PIGF ratio and the consequential management in pregnant women with suspected preeclampsia or at high risk of developing preeclampsia.

38.10.5.1 sFlt-1/PIGF ratio

The ratio cutoffs of the sFlt-1/PIGF ratio are shown in Tab. 38.10-1 – Consensus statements for the implementation of the sFlt-1/PlGF ratio for prediction and diagnosis of preeclampsia in singleton pregnancy.

38.10.6 Comments and problems

The described cutoff levels of sFlt-1/PIGF ratio are performed using the Elecsys® assay.

38.10.7 Pathophysiology

The angiogenesis factors placental growth factor (PlGF) and soluble fms-like tyrosine kinase-1 (sFlt-1), a soluble receptor of vascular endothelial growth factor (VEGFR), are important diagnostic biomarkers for differentiating normal pregnancy from preeclampsia.

SFlt-1 is a truncated splice variant of the membrane-bound receptor Flt-1. It consists of an extracellular binding domain, but lacks the intracellular signaling domain. In the circulation, sFlt-1 binds VEGFR and PlGF and neutralizes their effect /1/.

The placenta expresses VEGFR-1 mRNA and produces VEGFR-1 and, via an alternative splicing process, sFlt-1. While VEGFR-1 remains in the membrane of the trophoblast cell, sFlt-1 is released into the maternal circulation and acts as an antagonist of VEGF and PlGF. In normal pregnancy, PlGF rises steadily in the first two trimesters before declining towards the end of the pregnancy. SFlt-1 levels remains stable from early to mid-pregnancy before increasing steadily until birth /9/.

Changed serum concentrations of PlGF and sFlt-1 precede preeclampsia. The concentration of sFlt-1 increases about 5 weeks before the onset of preeclampsia while that of PIGF decreases from 13–16 weeks’ gestation onward.

The altered levels of angiogenesis factors are thought to be caused by placental dysfunction. In normal placentation, a network of branching vessels of fetal origin forms in the chorionic villi. The vascular bed is restructured so that the blood flow resistance decreases and the blood flow and thus oxygen supply increase.

Defective placentation leads to placental ischemia, hypoxia, angiogenic imbalance, and an anti-angiogenic state. The latter, stimulated by hypoxia, is maintained by the production of pro-angiogenic proteins of the endothelial cells, such as VEGF and its receptor Flt-1. The gene transcription for the production of VEGF and Flt-1 is regulated by hypoxia-inducible factor (HIF). Although the synthesis of VEGF and PlGF in the placenta is up regulated in hypoxia, their concentration in the circulation is low, effectively leading to an anti-angiogenic state. One reason for the reduced concentrations of VEGF and PlGF is thought to be the increase in the concentration of sFlt-1 in preeclampsia. By binding to VEGF and PlGF, sFlt-1 neutralizes their effect. As a result, for example, the modulating effect of VEGF on vascular function is canceled, and there is no vascular muscle relaxation in the myometrium of the uterus to counteract the hypoxemic state. In preeclampsia, the resistance of the uterine arteries is inadequately high, and the spiral arteries are not adequately remodeled to meet the needs of the growing fetus /1/.

Inflammation plays a role in the development of preeclampsia, but is not an etiological determinant /10/. The pathophysiologic pathway from normal pregnancy to preeclampsia is shown in Fig. 38.10-1 – Progression from normal pregnancy to preeclampsia.

Short interfering RNAs (siRNAs) selectively silence the sFLT1mRNA primarily responsible for placental overexpression of sFLT1 reduce levels of full-length FTL1mRNA. siRNAs can be a path toward a new treatment paradigm for patients with pre term preeclampsia /11/.

References

1. Maynard SE, Min JY, Merchan J, Lim KH, Li J, Mondal S, et al. Excess placental soluble fms-like tyrosine kinase 1 (sFLt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J Clin Invest 2003; 111: 649–58.

2. Stepan H, Herraiz I, Schlembach D, Verlohren S, Brennecke S, Chantraine F, et al. Implementation of the sFlt-1/PlGf ratio for prediction and diagnosis of pre-eclampsia in singleton pregnancy: implications for clinical practice. Untrasound in Obstetrics&Gynecology 2015; 45: 241–6.

3. Schneider E, Gleixner A, Hänel R, et al. Technical performance of the first fully automated assays for soluble fms-like tyrosine kinase 1 and human placental growth factor. Z Geburtshilfe Neonatol 2009; 213: 69.

4. Schittecate J, Russcher H, Anckaert E, Mees M, Leeser B, Tirelli AS, et al. Multicenter evaluation of thr first automated Elecsys sFlt-1 and PlGF assays in normal pregnancies and preeclampsia. Clin Biochem 2010; 43: 768–70.

5. Ohkuchi A, Hirashima C, Suzuki H, Takahashi K, Yoshida M, Matsubara S, et al. Evaluation of a new automated immunoassay for plasma sFlt-1 and PlGF levels in women mit preeclampsia. Hypertens Res 2010, 33: 422–7.

6. Zhang J, Klebanoff MA, Roberts JM. Prediction of adverse outcomes by common definitions of hypertension in pregnancy. Obstet Gynecol 2001; 97: 261–7.

7. Zeissler H, Llurba E, Chantraine F, Vatish M, Staff AC, Sennström O, et al. Predictive value of the sFlt:PlGF ratio in women with suspected preeclampsia. N Engl J Med 2016; 374: 13–22.

8. Schoofs K, Grittner U, Engels T, Pape J, Denk P,Henrich W, Verlohren S. The importance of repeated measurements of the sFlt-1/PlGF ratio for the prediction of preeclampsia and intrauterine growth restriction. J Perinat Med 2014; 42: 61–8.

9. Young BC, Karumanchi SA. Toward a better diagnosis of preeclampsia. Clin Chem 2016; 62 (7): 913–5.

10. Ramma W, Ahmed A. Is inflammation the cause of pre-eclampsia? Biochem Soc Trans 2011; 39: 1619–27.

11. Turanov AA, Lo A, Hassler MR, Makris A, Ashar-Patel A, Alterman JF, et al. RNAi modulation of placental sFLt-1 for the treatment of preeclampsia. Nature Biotechnology 2018; 36 (12): 1164–13.

38.11 Preexisting diabetes in pregnancy

Lothar Thomas

Approximately 0.4% of pregnancies occur in women with preexisting diabetes mellitus. About half of these pregnancies occur in women with type 1 diabetes, and half in women with type 2 diabetes. The classification of women with newly diagnosed hyperglycemia in pregnancy is difficult. The diagnosis of type 2 diabetes should be confirmed after pregnancy /1/. The International Association of the Diabetes in Pregnancy Study Group defined overt diabetes in pregnancy as requiring /2/:

• fasting glucose ≥ 7.0 mmol/L (127 mg/dL) or
• random glucose ≥ 11.1 mmol/L (200 mg/dl) or
• HbA_1c ≥ 48 mmol/L or
• 2 h result in oral glucose tolerance test (75 oGGT) ≥ 7.8 mmol/L (140 mg/dL)

Hyperglycemia, accelerated fetal growth by 24 to 28 weeks’ gestation, greater perinatal mortality.

38.11.1Glycemic control before pregnancy or below 20 weeks’ of gestation

Glycemic control before conception and below 20 weeks' gestation reduces the risks for mother and child because women with pre-existing diabetes in pregnancy are more likely to suffer adverse pregnancy outcomes /1/.

Gestational diabetes mellitus is associated with increased risks of the pregnant women and the fetus /3/.

Risks of pregnant women

Preeclampsia, obstetrical intervention, large-for-gestational-age, neonates, birth trauma, shoulder dystocia, and greater perinatal mortality.

A linear relationship exists between fasting glucose levels in early pregnancy and adverse pregnancy outcomes.

Risks of fetuses

Accelerated fetal growth, birth weight ≥ 4500 g, stillbirth or neonatal death.

In a study /4/ a 2-hour 75 g oGTT was performed before 20 weeks’ gestation fulfilling WHO diagnostic criteria. A fasting glucose level of ≥ 92 mg/dL (5.1 mmol/L), a 1-hour glucose level of ≥ 180 mg/dL (10.0 mmol/L) or a 2-hour glucose level of ≥ 153 mg/dL (8.5 mmol/L) before 20 weeks’ gestation were eligible for diabetes mellitus /4/. The conclusions of the authors was: Immediate treatment of gestational diabetes before 20 weeks’ gestation led to a modestly lower incidence of a composite of adverse neonatal outcomes than no immediate treatment.

Only 40% of pregnancies with type 2 diabetes, 15% with type 1 diabetes and < 50% are on folic acid prior to conception. Many women present with no pre-pregnancy planing for their first antenatal appointment from 8 to 12 weeks, when the period of fetal organogenesis is early complete /1/.

38.11.2 Glycemic control in pregnancy

Around pregnancy weeks 12 to 20 physiologically, a period of enhanced insulin sensitivity with hypoglycemia and reduced insulin requirements starts especially in type 1 diabetes. Around week 20 insulin resistance develops which results in increases in insulin treatment. In patients without sufficient insulin supplementation uncontrolled hyperglycemia and ketoacidosis will develop. In ketoacidosis 20 to 40% of offspring will not survive /3/. A women with diabetes in pregnancy is advised to keep:

• fasting blood glucose below 5.3 mmol/l (95 mg/dL)
• postprandial glucose below 7,8 mmol/L (140 mg/dL)
• 1 h and 2 h after a meal glucose ≥ 6.4 mmol/L (115 mg/dL)

Women with type 1 diabetes should be on insulin prior to pregnancy, women with type 2 diabetes are stable on metformin alone. Some women with type 2 diabetes need large doses of insulin during pregnancy, because placental hormones contribute worsening insulin resistance.

HbA_1c is generally believed to reflect glycemic control over the preceding 2 to 3 months however, it is not be reliable in pregnant women. The reasons are /1/:

• Red cell life span is variable in pregnancy
• HbA_1c is too slowly to assess the benefits of changing treatment.

38.11.3 Continuous glucose monitoring systems in diabetic pregnancies

Most glucose monitoring systems provide better accuracy than standard glucometers.

References

1. Meek CL. Monitoring motherhood: Monitoring and optimizing glycemia in women with pre-existing diabetes in pregnancy. Ann Clin Biochem 2022; 59 (1): 37–45.

2. International Association of Diabetes and Pregnancy Study Groups: Recommendations on the diagnosis and classification of hyperglycemia in pregnancy. Diabetes Care 2010; 33 (7): e97.

3. Diabetes in pregnancy: management of diabetes and its complications from preconception to the postnatal period. National Institute of Clinical Excellence (NICE) guideline NG3, London: NICE 2015.

4. Simmons D, Immanuel J, Hague WH, Teede H, Nolan CJ, Peek MJ, et al. Treatment of gestational diabetes mellitus diagnosed early in pregnancy. N Engl J Med 2023; 388 (23): 2132–44.