Assess for the presence of antinuclear antibodies (ANA) via the ANA by IFA, Reflex to Titer and Pattern. 

Antinuclear antibodies are a group of antibodies that attack the nucleus of cells. They can attack various components of the nucleus, including DNA, histones, nucleolar proteins, and nuclear antigens. The specific pattern in staining can provide insight into the type of attack. They are most associated with systemic lupus erythematosus but can also be found in other autoimmune conditions.

ANA testing is reported as a titer (concentration). ANA titers are represented as a dilution factor in ratio to the highest dilution at which ANAs are still detectable. For example, 1:40 means the patient’s serum was diluted 40 times before ANAs were still detected. The higher the titer, the more autoantibodies are present.

ANA antibodies should not be present in pregnancy. They can affect placental development and lead to early pregnancy loss. ANA antibodies have been shown to be higher in pregnancy, even in normal pregnancies. 

ANA antibodies are not uncommon in women with unexplained recurrent miscarriages, suggesting a role for autoimmune disease in cases of unexplained pregnancy loss.

Titers of >1:160 are highly associated with unexplained recurrent pregnancy loss, while titers of <1:80 are not.

Some studies have found that antinuclear antibodies occur more often in patients with preeclampsia, intrauterine growth retardation, fetal death, and placental abruption. Other studies, however, have not confirmed these results; therefore, there is not a clear connection.

​Low titers are not associated with pregnancy complications.

The c-reactive protein (CRP), quantitative test measures the concentration of CRP in the blood.

​CRP is primarily produced by the liver in response to certain signals from the immune system, particularly pro-inflammatory cytokines. These cytokines are released by various cells, including macrophages and adipocytes, in response to tissue damage, infection, or inflammation.

CRP plays a role in the body’s immune response by binding to certain substances released by damaged or inflamed cells. It binds to certain substances released by damaged or inflamed cells, including phosphocholine residues on the surface of bacteria, damaged cell membranes, and nuclear material from dead cells. By binding to these substances, CRP helps to activate the complement system, enhance phagocytosis, and promote inflammation at the site of injury or infection. It is a key compound in the clearance of bacteria and dead cells.

There are two CRP tests: quantitative and high-sensitivity.

Quantitative CRP (qCRP): The qCRP test measures CRP levels in the blood within a broader range. This test is often used to assess moderate to high levels of inflammation, such as those seen in acute infections, autoimmune diseases, or inflammatory conditions like rheumatoid arthritis. It is less sensitive than the high-sensitivity CRP assay and may not detect low levels of CRP associated with lower-grade inflammation.

High-Sensitivity CRP (hs-CRP): The hs-CRP assay is more sensitive than quantitative CRP tests and can measure CRP levels within a narrower range. This test is specifically designed to detect low levels of CRP associated with chronic low-grade inflammation. High sensitivity CRP testing is used as a marker of cardiovascular risk and is often included as part of cardiovascular risk assessment panels. It is more accurate in detecting subtle changes in CRP levels, even in the absence of clinically apparent inflammation.

​Pregnancy is a state of low-grade to moderate-grade inflammation. In response, c-reactive protein levels are increased in pregnancy. A significant increase is seen from 24 weeks through the first week of postpartum.

​Some elevation is normal and protective in pregnancy.

Elevated CRP has been associated with increased risks of preeclampsia, gestational diabetes, intrauterine infections, and preterm birth.

Elevated CRP levels have been found in preeclamptic women, with a median value of 8.8 and a range of 0.3 to 25.5.
​
CRP >5.5 μg/mL increases the risk of spontaneous preterm labor.

​Not considered pathogenic

 The Homocysteine test measures the amount of homocysteine in the bloodstream.

Homocysteine is a metabolic intermediate produced by the demethylation of methionine. Homocysteine is commonly found in the blood in three forms: free, bound to albumin, and in a complex with other homocysteine thiols. Homocysteine is then further metabolized in the body through two main pathways: remethylation, which converts homocysteine back to methionine, and transsulfuration, which converts homocysteine to cysteine.

There are two pathways of remethylation and one pathway of transsulfuration.

The remethylation of homocysteine to methionine on the folate side involves a series of enzymatic reactions that require the presence of folate (specifically, 5-methyltetrahydrofolate) and the cofactors riboflavin (vitamin B2) and cobalamin (vitamin B12). Folate is first converted to 5,10-methylenetetrahydrofolate through a reaction catalyzed by the enzyme dihydrofolate reductase (DHFR). Then 5,10 methylenetetrahydrofolate is converted to 5-methyltetrahydrofolate (5-methyl-THF) by the enzyme methylenetetrahydrofolate reductase (MTHFR), which requires riboflavin. This step involves the transfer of a methyl group.

The methyl group carried by 5-methyl-THF is transferred to homocysteine in a reaction catalyzed by the enzyme methionine synthase (also known as cobalamin-dependent methionine synthase). This transfer is facilitated by vitamin B12, which serves as a cofactor for methionine synthase.

​The choline betaine pathway, also known as the alternative or non-folate remethylation pathway, is another mechanism for remethylating homocysteine. In the liver, choline is oxidized to betaine in a reaction catalyzed by the enzyme choline oxidase. Betaine donates a methyl group to homocysteine in a reaction catalyzed by the enzyme betaine-homocysteine methyltransferase (BHMT).

The transsulfuration pathway involves the conversion of homocysteine to glutathione.

Homocysteine levels decrease in normal pregnancies. This drop is attributed to both hemodilution as well as increased use during pregnancy. Homocysteine levels decrease during early pregnancy, reaching the lowest point during the second trimester, and steadily increase during the later third trimester reaching early pregnancy levels.

There is an association between elevated homocysteine levels and pregnancy complications such as early miscarriage, preeclampsia, IUGR, and placental abruption, as well as in certain cases of maternal and fetal mortality.

Preconception homocysteine levels >12.4 μ mol/L increase the prevalence of preterm labor by four times.

A 5 μ mol/L increased change in homocysteine concentration was associated with 63% increased odds of placental mediated complications.

Studies have found that higher homocysteine levels within six weeks postpartum are associated with postpartum depression, regardless of folate and vitamin B12 levels.

There is little research on hypohomocysteinemia and none related to pregnancy.

In one study, 41% of patients with idiopathic peripheral neuropathy were found to have low homocysteine levels.18

The methylmalonic acid (MMA) test measures the amount of MMA in a sample. This can be tested via serum/plasma or through a urine test in ratio to creatinine.

Methylmalonic acid is a derivative of malonic acid in which one of the methylene hydrogens is replaced by a methyl group. L-methylmalonyl-CoA is then isomerized to form succinyl-CoA. This reaction is catalyzed by the enzyme methylmalonyl-CoA mutase, which requires adenocobalamin. This reaction is reversible. Succinyl-CoA enters the Krebs cycle, where it is converted to succinate, releasing energy. A dysfunction in the isomerization step can cause an increase in methylmalonic acid. MMA is excreted through the urine. 

There is a gradual increase in methylmalonic acid levels throughout pregnancy, with a peak in the third trimester. There is a twofold increase in the need for vitamin B12 by the third trimester, with much of this being distributed to the fetus. Maternal vitamin B12 stores are depleted, thus causing a normal elevation in methylmalonic acid. In addition, there are changes in renal excretion of MMA.

Postpartum research on MMA levels is limited, but some studies suggest a sharp decrease in both serum and urinary methylmalonic acid immediately after delivery.

​Amniotic fluid and maternal urine MMA concentrations in the middle term of neural-tube defect (NTD)-affected pregnancies (22.1 +/- 12.6 nmol/mmol creatinine) were significantly higher than that of normal pregnancies (2.5 +/- 1.1 nmol/mmol creatinine).

One study from Turkey found that 36% of the women tested had urinary MMA levels >6 μmol/mmol crt. Of those, 50% had serum vitamin B12 levels <180 pg/mL.

​Not considered pathogenic

The cobalamin (vitamin B12) test measures the amount of vitamin B12 in a serum sample. Vitamin B12 is also known as cobalamin because, structurally, it contains cobalt surrounded by a corrin ring structure.

Vitamin B12 is released from food by the actions of stomach acid and proteolytic enzymes in the mouth and stomach. Transcobalamin I is a haptocorrin protein produced by the salivary glands and stomach mucosa that binds to B12 to help stabilize it and facilitate absorption. It is synthesized via the TCN1 gene. In the stomach, B12 binds to a protein called intrinsic factor, which is produced in conjunction with hydrochloric acid. The complex protects B12 from degradation by stomach acids and facilitates absorption in the small intestines.

The absorption rate of B12 varies based on the amount of intrinsic factor produced and the binding capacity of intrinsic factor. Once in the bloodstream, transcobalamin I-bound vitamin B12 is transferred to transcobalamin II, and the complex binds to transcobalamin, a transport protein that delivers the vitamin to various tissues. TCN2 codes for the synthesis of transcobalamin II.

Once in the cells, vitamin B12 is converted to additional forms that include hydroxocobalamin, ​methylcobalamin and adenosylcobalamin. 

Serum vitamin B12 levels drop drastically throughout pregnancy as cellular need increases and there is increased hemodilution. During normal pregnancies, serum B12 levels drop 30% by the third trimester, yet dietary needs have increased twofold.

In the first few days postpartum, there is a sharp rise in serum vitamin B12 levels, as the hemodilution of pregnancy resolves. Levels return to normal pre-pregnancy levels by a few weeks postpartum.

High maternal vitamin B12 (>600 pmol/L or 810 pg/mL) and folate have been associated with an increased risk for childhood autism.
​
​This is an interesting correlation with no known etiology or mechanism of action yet. Other studies are in process regarding genetic variations and analysis of other biomarkers, as well as the link to unmetabolized folates.

Vitamin B12 deficiency has been associated with cognitive alternations in fetal brains, causing cognitive impairment that includes poor memory, language, and motor skills as well as preterm birth, anemia, and insulin resistance.

The 25OH vitamin D (250H-D) is considered the most accurate test to assess vitamin D status.

When the skin is exposed to UVB radiation from sunlight, 7-dehydrocholesterol, a compound in the skin, absorbs the UVB rays. UVB radiation converts 7-dehydrocholesterol into previtamin D3. Previtamin D3 undergoes thermal isomerization (a change in structure induced by heat) in the skin, converting it into vitamin D3 (cholecalciferol). 

Dietary vitamin D, primarily in as vitamin D2 (ergocalciferol) or vitamin D3 (cholecalciferol).

Vitamin D3 is then transported to tissues for conversion to calcidiol (the active hormonal form of vitamin D). This is primarily done in the kidneys, but other tissues, such as the placenta. 

During pregnancy, serum calcitriol rises from the first trimester, doubling by the end of the third trimester. This increase is due to an actual increase in production and not a reduction in renal clearance. This rise is driven by maternal kidney and placental production, with most conversion from maternal sources.

​While serum calcitriol levels rise, serum 25OH-D levels decrease due to increased demands.

Maternal need for dietary vitamin D increases in the breastfeeding period. A study found that a maternal intake of 6,400 IU of vitamin D was equivalent to supplementing 400 IU in the infant. This means that the higher consumption of vitamin D delivered the required 400 IU of vitamin D needed in breast milk to supply infant needs.

Studies have shown no negative effects in pregnancy with serum levels <80 ng/mL. Greater than 100 ng/mL is associated with hyperkalemia risk.

Studies have found that vitamin D deficiency increases the risk of preeclampsia, gestational diabetes, low birth weight, and preterm birth. Maternal vitamin D deficiency in pregnancy is also associated with long-term health risks for babies.

A 2023 study from Sweden found that serum vitamin D levels ≤30.1 nmol/L were associated with an increased risk of preeclampsia.11 Vitamin D status prior to 20 weeks is associated with gestational diabetes risk, with >30 ng/mL being protective.
​
Studies have also found that a serum concentration of 40 ng/mL is needed to maintain calcitriol balance in pregnancy. To maintain these levels, a supplemental dosage of 4,000 IU per day was required.

The serum progesterone test measures the amount of the hormone progesterone in the blood.

​Progesterone synthesis is a complex biochemical process called steroidogenesis. It occurs in the adrenal glands, corpus luteum of the ovaries, and the placenta.

Nutrients shown to affect progesterone production via enzymatic action include vitamin D, vitamin A, vitamin K, riboflavin, and iron.

 During pregnancy, progesterone levels increase, starting at conception. 

Progesterone in early pregnancy helps prime the endometrial tissues for implantation. The initial production of progesterone is regulated by hCG and thyroid hormones and is secreted by the corpus luteal in the ovaries and the endometrial tissues of the uterus. After the 10th week, the placenta takes over progesterone production. 

Progesterone production steadily increases throughout pregnancy as the placenta increases size and health. Levels peak around 34 weeks and decrease toward the onset of labor.

Within hours after delivery, following the birth of the placenta, maternal progesterone levels decrease abruptly. These levels reach their lowest point 24 to 48 hours after delivery. This progesterone drop helps signal the initiation of lactation and uterine involution. Over the next several weeks and months, progesterone levels remain low. In breastfeeding women, the progesterone levels will remain low until the return of menstrual cycles.

​There is limited data on the consequences of high progesterone in pregnancy.

Studies show a relationship between progesterone deficiency in the first trimester and first-trimester miscarriage. A 2024 systematic–scoping review indicates the possible benefits of progesterone replacement therapy in maintaining a healthy pregnancy and fetal development. 

My Maternal Lab Analysis Course covers this and more. MEAC credits available.

The urinary iodine clearance (UIC) test measures the amount of iodine present in a urine sample. It can be done using a random sample, a 24-hour sample, or an iodine:creatinine ratio.

The Random UIC is the most common assessment for iodine deficiency used in research.
The 24-hour UIC is slightly more accurate than the Random UIC, but it is rarely assessed in research and can be variable based on consumption and methodological errors. There are no established pregnancy ranges.

The iodine:creatinine ratio is corrected for urinary output and kidney function, adding accuracy to the UIC spot test.

In cases of iodine deficiency, more iodine is reabsorbed, and less is excreted. When iodine levels are sufficient, more iodine is excreted through the urine.

Urinary iodine clearance increases during pregnancy, with the highest clearance seen during the first trimester.

​There is limited data on the consequences of low UIC in pregnancy. (See Serum Iodine Article)

Learn more about iodine testing, thyroid health and more!

The iodine, serum, or plasma test (also known as the serum iodine concentration, or SIC, test) analyzes the amount of iodine present in a blood sample. It represents the iodide pool. 

Iodine, serum, or plasma testing is less commonly used in research to assess iodine deficiency. The more common measure is urinary iodine clearance. Studies show that the iodine, serum, or plasma test accurately correlates with the UIC:creatinine ratio.

Iodide is absorbed through the stomach and small intestines, with between 70% and 90% of dietary iodide being absorbed. Iodine absorption is lower and may vary depending on other factors, as it is not naturally found in the diet as iodine.

Once absorbed, iodide enters the bloodstream and becomes part of the iodide pool. The iodide pool is the serum-accumulated iodide that the body can pull from. This can be recycled iodide from the deiodination of thyroxine as well as recent intake.

During pregnancy, there is a 50% increase in the dietary need for iodine. Much of the iodine is used to increase maternal thyroid hormone production. Another portion of iodine is transferred to the fetus for storage, starting around 10 to 12 weeks of gestation until the fetus begins producing its own thyroid hormones at around 16 to 20 weeks. Iodine is also essential for developing the fetal brain and nervous system.

Iodine absorption and clearance are increased during pregnancy.

Elevated serum iodine levels, particularly in the first trimester, may have a slight negative impact on motor and physical development and infant intellect, according to a 2024 study.

It has been suggested that excess iodine in the first trimester may be a cause of nausea as a means of preventing excess consumption. Pregnancy nausea is closely linked to iodine intake and thyroid function.

In the US, between 2011 and 2021, 38% of the population was classified as iodine deficient.
A 2021 sampling of pregnant women in Michigan found that 23% had inadequate iodine intake.

It is estimated that an average of 44% of Europeans are iodine deficient, and more than 50% of those in central and western Europe are deficient.

A 2011 study from Spain found that only 14.4% of pregnant women in the first trimester and 26.8% in the second trimester had adequate iodine levels.

In Southeast Asia and the Eastern Mediterranean, iodine deficiency is estimated to affect 36% to 43% of the general population.

Serum iodine levels are associated with urinary iodine clearance (UIC) and thyroid function in pregnancy. Low serum iodine is associated with an increased risk for iodine deficiency and hypothyroxinemia.

Visit the page below to learn more about my advanced maternal blood work analysis course - MEAC CEUs Available

The thyroid receptor antibodies (TRAb) test measures the levels of antibodies that target the thyroid-stimulating hormone (TSH) receptor, also known as the thyrotropin receptor, on the surface of thyroid cells.

This test detects two main types of thyroid receptor antibodies.

Thyroid-Stimulating Immunoglobulin (TSI): TSI antibodies mimic the actions of TSH by binding to and stimulating that TSH receptor. Continuous stimulation causes a decrease in TSH and an increase in T4 and T3 indices, generally with an increase in reverse T3.

Thyroid-Binding Inhibitory Immunoglobulins (TBIIs): TBIIs completely binds to TSH receptors but do not stimulate the thyroid. Instead, they lead to a decrease in the production of thyroid hormones. In rare cases, they have been associated with hyperstimulation, but this is typically in association with TSI.

TSI can be measured on its own. It is a separate and specified analyte. TRAb is typically used to rule out Graves’ disease or autoimmune hyperthyroidism.

It is not uncommon for all thyroid antibodies to increase in the first trimester if present preconception. Levels may then increase or decrease based on individual presentations. With changes in immune-system function and globulin production, in normal pregnancy, antibodies generally decrease slightly in the second and third trimesters.

​All Graves’ disease patients should be medically managed by endocrinologists. During pregnancy, TRAb crosses the placenta and may induce fetal hyperthyroidism. TRAb levels over three times the upper limit of normal are associated with hyperthyroidism in the fetus and newborn.

Check out this and more in my Maternal Blood Work Analysis Course.
MEACH Approved for Continuing Education Credits.