The Placenta as a Research Biospecimen

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The Placenta as a Research Biospecimen Jane G. Ryan, Rene´e Krysko Davis, and Joan Rosen Bloch

Correspondence Jane G. Ryan, PhD, CNM, College of Nursing and Health Professions, 245 N. 15th St. MS 501, Philadelphia, PA 19102. [email protected]

ABSTRACT The placenta provides a unique opportunity to study the prenatal environment of the fetus to better understand subsequent infant and child health and illness. In this article we describe the role of the placenta as a research biospecimen, including placental morphology and cytokine biomarkers. Because of the role of the placenta in contemporary research, members of the perinatal health care team involved in birth have an important role in advancing science.

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Keywords placenta National Children’s Study cytokines biospecimens pregnancy maternal fetal exposures fetal development developmental biology biomarkers

Jane G. Ryan, PhD, CNM, is a clinical assistant professor in the College of Nursing and Health Professions, Drexel University, Philadelphia, PA. Rene´e Krysko Davis, MD, MPH, is a senior research associate in the Department of Community Health and Prevention, Drexel University School of Public Health, Philadelphia, PA. Joan Rosen Bloch, PhD, CRNP, is an associate professor in the College of Nursing and Health Professions and School of Public Health, Drexel University, Philadelphia, PA.

Accepted May 2012

he health of the placenta is crucial for the health of the developing fetus, and the health of the fetus is crucial for the health of the developing infant and child. Thus, understanding the placenta provides a critical link to identifying key physiological processes in subsequent infant and child health and illness. The study of the placenta includes clinical, sonographic, laboratory, and anatomic data. The placenta (Salafia et al., 2008) as well as other birth specimens including maternal serum, cord blood, meconium, and breast milk (Barr, Wang, & Needham, 2005) provide invaluable scientific and clinical information about the health of the mother and fetus during gestation. Because of its ability to store biochemical information about events during gestation, the placenta has been referred to as the diary of pregnancy (Aagaard-Tillery, Thornburg, Bernstein, & Washburn, 2011; Baergen, 2007). Examination of the placenta provides a unique window into the prenatal environment of the fetus and may predict the future life course of the child (Burton, Barker, Moffett, & Thornburg, 2011).

T

natal and infant health research. Therefore, we describe cytokine biomarkers identified in specimens retrieved during birth. Although we only present a synopsis, this article frames some of the key components that may be studied in large-scale studies requiring the collection of birth specimens such as the National Children’s Study (NCS) (Kent, Mancini, Pacholski, & Janisak, 2012).

The Placenta Overview The placenta is a very complex organ responsible for three major functions during pregnancy: metabolism, interfacing between the mother and fetus, and serving as an endocrine organ. The placenta, a unique repository or holding place of biochemical and genetic information, begins developing during the first 2 weeks of gestation. Early placental development occurs during the pre-embryonic period when the fertilized egg begins forming the placenta, the fetal membranes, and an interconnecting vascular space between the mother and fetus known as the intervillous network (Evain-Brion & Malassine, 2003; Maltepe, Bakardjiev, & Fisher, 2010).

The authors report no conflict of interest or relevant financial relationships.

In this article, we provide an overview of how the placenta is studied as a biospecimen by researchers seeking to understand the relationship between in-utero exposures and subsequent infant and child health outcomes. We provide background information and describe the morphologic examination of the placenta. The study of molecular biomarkers is an active area of peri-

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 C 2012 AWHONN, the Association of Women’s Health, Obstetric and Neonatal Nurses

The placental barrier separating the mother’s blood from that of the fetus is composed of one layer of multinucleated cells, the syncytiotrophoblasts. The two sides (maternal and fetal)

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Ryan, J. G., Davis, R. K., and Bloch, J. R.

of this single-layered barrier, the sycytium, are structurally and functionally different. On the maternal-facing side of the syncytium, a brush border membrane comes into direct contact with the mother’s blood. Conversely, on the fetal-facing side, a basal membrane containing microvillous protrusions faces the fetal blood but does not come into direct contact. Interface between maternal blood and fetal trophoblastic cells takes place at this single-cell layer barrier (Prouillac & Lecoeur, 2010). The placenta is able to adapt to its own growth needs and those of the fetus based on available maternal resources (Burton et al., 2011). The composition of the placenta is fluid, and changes throughout pregnancy. At one point of gestation, a placenta may be considerably different in size and molecular composition than at a different point in gestation (Burton et al.; Salafia et al., 2008). The weights of the placenta and the infant at birth are often used to determine the health of the intrauterine milieu. Specifically, low birth weight has been linked to increased lifetime risks of cardiovascular disease and diabetes (Barker, 2007; Landrigan et al., 2006). Accordingly, Salafia et al. (2008) suggested that the characteristics of the placenta might be used as indicators of the fetal experience. These researchers analyzed data collected between 1959 and 1965 on 24,061 singleton infants born between 34 and 42 weeks gestation. They measured six placental characteristics: (a) distance from the umbilical cord insertion to the margin of the placental disc, (b) size of the smallest placental disc diameter, (c) size of the largest placental disc diameter, (d) thickness of the placental disc, (e) length of the umbilical cord, and (f) weight of the placenta. They proposed that measurements of the placenta reflect placental development during gestation and may indicate placental function, including most importantly the ability of the placenta to carry fetal blood to and from the maternal villious interface. Furthermore, they suggested that without adequate placental disc surface area and disc thickness, the placenta may not be able to adequately provide the nutrition and waste removal necessary for the fetus to grow and flourish (BaptisteRoberts et al., 2008; Salafia et al., 2008). Salafia and colleagues suggested comprehensive and in-depth research of the placenta is needed to advance the current understanding of the intricate interactions between the fetus, mother, and placenta.

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Understanding the placenta provides a critical link to identifying key physiological processes in subsequent infant and child health and illness.

Importance of Placenta Research The study of the placenta is multifaceted and contributes significantly to understanding the in-utero experience of the developing fetus. Placenta research has been conducted internationally since at least the mid-1800s. The unusual interface between maternal-fetal circulation was reported in the Journal of the British Royal Society of Medicine in 1841 when Dalrymple published an article describing microscopic examination of placental tissue. The article included a series of three sketches depicting the vascular structure of the umbilical arteries and vein and the segments of the placenta (Dalrymple). Research of placenta morphology and function continues to this day. The International Federation of Placenta Associations is devoted to the scientific study of the placenta. The official scientific journal of this organization, Placenta, has been published since 1980. To conduct research on the placenta, scientists need access to tissue specimens. Clinicians, including nurses, are the most common bridge between scientists conducting biological research and patients whose placentas would otherwise be discarded. Maternal/child health clinicians, educators, and researchers need to understand their critically important roles in retrieving specified birth biospecimens, such as the placenta, according to specific study protocols. Researchers are currently studying the extent to which maternal exposure to environmental chemicals influences fetal development and childhood health via placental transfer. Environmental exposures that adversely affect the developing fetus include air pollution (Chalupka & Chalupka, 2010; Myren, Mose, Mathiesen, & Knudsen, 2007), metals such as lead and mercury (Chalupka & Chalupka; Dietrich et al., 2005), and pesticides (Dietrich et al.; Fenske, Bradman, Whyatt, Wolff, & Barr, 2005). Furthermore, a growing number of researchers are examining the adverse effects of maternal exposures to asbestos, pesticides, and leaded gasoline on long-term childhood illness (Landrigan et al., 2006; Swanson, Entringer, Buss, & Wadhwa, 2009; Wang, Needham, & Barr, 2005). Scientific examination of birth biospecimens is an established method to measure perinatal exposure (Evain-Brion & Malassine, 2003; Myren

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The Placenta as a Research Biospecimen

The placenta, described as a dual biomarker, is capable of recording maternal and fetal exposures.

et al., 2007; Vahakangas & Myllynen, 2006). The placenta, described as a dual biomarker (Iyengar & Rapp, 2001, p. 221), is capable of recording maternal and fetal exposures. Therefore, the placenta is scientifically invaluable, as it provides a window into the experiences of mother and fetus (Salafia et al., 2008).

Examination of the Placenta as a Birth Biospecimen Clinical and basic science research involves examination of multiple dimensions of the placenta, including morphology and various biomarkers such as cytokines and genetics. The macrolevel morphology presents a broad perspective, whereas cytokine biomarkers present a very narrowly focused topic for study. The area of science related to cytokines is emerging and involves assessing specified birth specimens with particular maternal and neonatal clinical outcomes.

Morphological Examination of the Placenta Consistent with its complexity, numerous morphological characteristics of an individual placenta can be examined at the macro- and microlevels. These characteristics differ among individual samples and are associated with various clinical outcomes. Table 1 provides a summary of important terminology used during morphological examination to describe potential macrolevel parameters of the placenta and terms associated with the umbilical cord. Microscopic morphologic examination of the placenta is beyond the scope of this article, and the reader is referred first to a maternal/fetal medicine textbook (Creasy, Resnik, Iams, Lockwood, & Moore, 2009) to frame the histology and cytology underpinning placental research involving microscopic examination methods.

Molecular Biomarkers: Placental Cytokines Biological substances within tissues are increasingly being used by researchers to study the effects of exposures to various environmental toxins on health (Lobdell & Mendola, 2005). These biological substances, biomarkers, may be cellular, molecular, or biochemical and can include genetic material, precancerous tissue, and serum mark-

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ers, such as lead levels. Biomarkers can also be the by-product of the interaction between living tissue and something foreign to the tissue, such as metabolites following exposure to environmental chemicals. Biological sites in which biomarkers can be located include human tissue, cells, and bodily fluids (Lobdell & Mendola). One class of molecular biomarkers are cytokines, small proteins that regulate and mediate molecular processes involved in immunity, inflammation, and blood cell formation (Lyon et al., 2010). Cytokines can be found in maternal, neonatal, and umbilical cord blood as well as in the placenta and amniotic fluid (Pickler et al., 2010). Research involving cytokines during pregnancy has revealed significant associations with various clinical outcomes, such as chorioamnionitis (Takahashi et al., 2010), preeclampsia (Bowen, Chamley, Keelan, & Mitchell, 2002), preterm birth (Bowen, Chamley, Keelan, et al.; Pickler et al.; Takahashi et al.), neonatal infection and neurological insult in the newborn (Goines et al., 2011; Pickler et al.). Table 2 provides a summary of selected cytokines that have been reported in birth biospecimens and their associations with various clinical outcomes. This table is not intended to be a comprehensive review of cytokines and pregnancy but instead an overview of some of the research being conducted on this topic. In Table 2, cytokines may be identified as pro-inflammatory or anti-inflammatory and then classified as either Th1-type or Th2-type. This classification refers to the subset of CD-4 T cells and immune response that produces the specified cytokine. The reader is referred to Sompayrac (2008) for more details. The production, distribution, and concentrations of many cytokines vary from site to site and with gestational age. For example, the concentration of Interlukin-8 (IL-8) increases throughout gestation and at labor in the amniotic fluid (Bowen, Chamley, Mitchell, & Keelan, 2002) but is inversely correlated with gestational age in umbilical cord blood (Lyon et al., 2010). In addition, maternal cytokines may cross the placenta and affect the fetus directly (e.g., IL-6), or they may affect the fetus indirectly via interactions with placental cells and alteration of the placental environment. A growing body of literature suggests proinflammatory cytokines may be predictive of adverse birth outcomes, such as preterm birth, neonatal infection, and neurologic insults in the newborn (Bowen, Chamley, Keelan, et al., 2002; Pickler et al., 2010). Notably, the proinflammatory cytokines IL-1β, IL-6, IL-8, and tumor necrosis factor (TNF-α)

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Table 1: Morphological Examination of the Placenta Term

Definition

Significance

Placental

Complete = all cotyledons present, no

Incomplete indicates probable retained tissue

completeness

Placental disc diameter

velamentous vessels, and vessels taper to the

that is associated with postpartum

edge of the placenta

hemorrhage and infection.

Diameters (large and small) of chorionic disc recorded in cm (normal is about 18–22 cm).

Diameters have been associated with newborn birth weight; small diameter has been associated with intrauterine growth restriction (IUGR).

Placental disc thickness

Placental thickness measured at the center of the chorionic disc; recorded in cm (normal is 2.0–2.5 cm).

Thin placenta associated with placental insufficiency, IUGR, and rarely placenta membranacea. Thick placenta associated with maternal diabetes mellitus, fetal hydops, and fetal infections. Placental thickness has been associated with newborn birth weight.

Placental weight

Measured in the delivery room and recorded in

A relatively light or heavy placenta often indicates

grams (normal is about 470 g). In a term

an abnormal pregnancy. Placental weight has

gestation, the infant usually weighs 7–8 times

been associated with gestational age and

the placental weight (ratio is decreased earlier in gestation).

newborn birth weight. Standard weight tables for gestational age are available.

Placental disc shape

Shapes include round-to-oval and a variety of

Shape may be altered by multiple lobes and/or

atypical shapes (bipartite, tripartite,

placenta accreta or placenta percreta all of

succenturiate, crescent, membranous, or

which indicate probable retained tissue and

“irregular”). In some research, binary variable

are associated with an increased risk of

“0” indicates round-to-oval shape and “1” indicates an atypical variant.

postpartum hemorrhage and infection. Placental shape has been associated with newborn birth weight.

Placental surfaces

Maternal surface = basal plate decidua Fetal surface = chorionic plate

Maternal surface abnormalities include pallor, placental infarcts, fibrin deposition, placental clots/bleeding, chorioanginoma, choriocarcinoma, and hydatidiform mole. Fetal surface abnormalities include pallor, thick ring of membranes (circumvallate placenta), thinner ring of membranes (circummarginate placenta), various types of nodules representing different clinical conditions, and amniotic bands. The majority of these surface abnormalities have been associated with various clinical outcomes.

Umbilical cord length

Measured in the delivery room and recorded in cm (normal is about 40–70 cm).

Both short and long cord lengths have been associated with poor fetal outcomes. Cord length has been associated with newborn birth weight.

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Table 1: Continued Term

Definition

Significance

Umbilical cord

Measured in the delivery room and recorded in

Narrow areas have been associated with torsion

thickness

cm; usually the diameter of the umbilical cord

and fetal death.

is uniform throughout its length (2.0–2.5 cm). Umbilical cord inflammation

May be assessed via grossly examining for edema or through histological examination.

Inflammation can be diffuse, focal, or segmental (necrotizing funisitis) with each type being associated with various maternal and fetal poor outcomes.

Umbilical cord insertion

Normally, the cord inserts near the center of the chorionic disc (central or eccentric insertion). Marginal insertion (7% of pregnancies) – the cord

Umbilical cord insertion has been associated with newborn birth weight. Marginal insertions are generally insignificant. Velamentous (membranous) insertion is

inserts at the edge of the placenta. Velamentous insertion (1%) – the cord does not

associated with increased risk of fetal

insert into the placenta, but into the external

hemorrhage, vascular compression, and

membranes instead.

thrombosis, and has been linked to various maternal characteristics as well as fetal malformations.

Umbilical cord structure

Cord knot/entanglement (fetus passes through a Number of vessels = normally two arteries and

Abnormal number of vessels, especially if only

one vein are present; vessels should be

one artery, is associated with a high risk of

counted at a level > 5 cm from the placental

numerous fetal anomalies affecting multiple organ systems.

end of the cord. Cord vessel thrombosis = thrombosis within one

Cord vessel thrombosis has been associated with fetal compromise.

of the cord vessels. Fetal membrane

Cord knot and/or entanglement may be associated with fetal compromise if it is tight.

loop of cord).

Normal membranes are thin, grey, and glistening.

appearance

Abnormalities may be seen in membrane color and/or odor. Thick, dull, discolored or malodorous membranes may indicate infection. Green discoloration may indicate meconium staining, past bleeding events, or infection.

Note. Adapted from Yetter (1998). Examination of the placenta. American Family Physician, 57(5), 1045–1054. Used with permission.

were found to be increased in umbilical cord blood of preterm infants diagnosed with white matter lesions shortly after birth, suggesting that infants who produce an immune response in utero may be at higher risk of neurologic insult (Pickler et al.). The study of cytokines in birth biospecimens has led to numerous associations with various clinical outcomes, and the study of the biological pathways between various cytokines and clinical outcomes is constantly evolving. However, limitations in these studies exist and care must be taken when interpreting results. Nevertheless, the study of cytokine biomarkers in birth biospecimens will likely play a key role in future research protocols.

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Retrieving Placental Specimens for Research: A Methodological Debate No single approach has been identified for the collection and study of birth biospecimens including the placenta, and study of this tissue is a matter of ongoing debate among scientists (Mayhew, 2008; Nelson & Blair, 2011; Yuen & Robinson, 2011). Nelson and Blair (2011) described the need for more clear articulation of procedures used to evaluate the placenta as well as descriptive nomenclature. Mayhew (2008) argued for the need for more precise description of sample design in placenta research, including explanations of the

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Table 2: Summary of Selected Cytokine Biomarkers in Birth Specimens and Associations with Maternal and Neonatal Outcomes Cytokines

Reported Sites of Production

Selected Associations with Maternal and Neonatal

(Bowen, Chamley, Mitchell, &

Health Outcomes

Keelan, 2002) in Gestational Tissues and Sites Sampled in Selected Research As a group:

Pro-Inflammatory

• Levels in AF normally increase towards term and at labor (Bowen,

Cytokines

Chamley, Keelan, et al., 2002) • Can be found in cervico-vaginal secretions in the 3rd trimester (especially in BV+ women or women who deliver preterm with associated intra-amniotic infections) (Bowen, Chamley, Keelan, et al., 2002) • Cervico-vaginal fluid levels increase with labor; peak at complete cervical dilation (Bowen, Chamley, Keelan, et al., 2002) • High levels increase risk for PTB (Bowen, Chamley, Keelan, et al., 2002; Lyon et al., 2010; Takahashi et al., 2010) • Placenta, AF, UCB, MS levels associated with neonatal infection and neurologic insult (Pickler et al., 2010) • Associated with CLD in PT infants (Takahashi et al., 2010) IL-1β

Production sites:

• Associated with histologic chorioamnionitis (Takahashi et al., 2010),

PL, D, FM

preeclampsia with and without IUGR (Bowen, Chamley, Keelan,

Other sites sampled:

et al., 2002), PTB (Bowen, Chamley, Keelan, et al.; Matoba et al.,

AF, UCB

2009), maternal obesity (Lyon et al., 2010), neonatal infectiona (Pickler et al., 2010), neurologic insult in the newborn (can cross the blood brain barrier) (Bowen, Chamley, Keelan, et al.; Pickler et al.; Takahashi et al.), and BPD in the newborn (Bowen, Chamley, Keelan, et al.) • IL-1β, IL-6, IL-8, and TNF-α were found to be increased in infants who produced an in-utero immune response (Pickler et al., 2010) • Levels in AF of Black women with PTB reported higher than in Whites (Lyon et al., 2010) • Does not cross the placenta (Aaltonen, Heikkinen, Hakala, Laine, & Alanen, 2005)

IL-6 (Th2 cytokine)

Production sites: PL, D, FM Other sites sampled: AF, UCB, MS

• AF level is a marker of intrauterine infection and rapid onset of delivery (Bowen, Chamley, Keelan et al., 2002) • Associated with preeclampsia (Bowen, Chamley, Keelan, et al., 2002), chorioamnionitis (Takahashi et al., 2010), PTBa (Bowen et al.; Lyon et al., 2010; Matoba et al., 2009), gestational diabetes (Lyon et al.), maternal obesity (Lyon et al.), IUGR (Takahashi et al.), neonatal RDS, BPD, and CLD (Bowen, Chamley, Keelan, et al.; Takahashi et al.), white matter damage and cerebral palsy (can cross blood brain barrier) (Bowen, Chamley, Keelan, et al.; Pickler et al., 2010; Takahashi et al.), and neonatal infection (Pickler et al.)

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Table 2: Continued Cytokines

Reported Sites of Production

Selected Associations with Maternal and Neonatal

(Bowen, Chamley, Mitchell, &

Health Outcomes

Keelan, 2002) in Gestational Tissues and Sites Sampled in Selected Research • Levels in AF of White women with PTB were higher than in Blacks (Lyon et al., 2010) • Elevated UCB IL-6 may be a marker of a systemic fetal inflammatory response (Bowen, Chamley, Keelan, et al., 2002) • IL-1β, IL-6, IL-8, and TNF-α were found to be increased in infants who produced an in-utero immune response (Pickler et al., 2010) • The profile of elevated MS concentrations of IL-2, IL-4, IL-6, GM-CSF, and MIP-1α was associated with developmental delays without autism (Goines et al., 2011) IL-12 (Th1 cytokine)

Production site:

• No association between PTB and UCB levels (Matoba et al., 2009)

Unknown

• UCB levels higher in infants without NRFS (Takahashi et al., 2010)

Other sites sampled:

• May be associated with infection and white matter damage in the

AF, UCB TNF-α/β (Th1 cytokine)

Production sites:

newborna (Pickler et al., 2010) • Increased TNF-α levels associated with chorioamnionitis (Takahashi

PL, D, FM

et al., 2010), preeclampsia with and without IUGR (Bowen,

Other sites sampled:

Chamley, Keelan, et al., 2002), maternal obesity (Lyon et al., 2010),

AF, UCB (TNF-α)

early pregnancy loss (Bowen, Chamley, Mitchell, et al., 2002), PTB (Bowen, Chamley, Keelan, et al.; Matoba et al., 2009; Takahashi et al., 2010), white matter damage in newborn (can cross blood brain barrier) and may be associated with neonatal infection and BPD (Bowen, Chamley, Keelan, et al.) • AF levels of Black women with PTB reported to be higher than in Whites (Lyon et al., 2010) • TNF-α levels have been implicated as a negative regulator of trophoblast growth in normal placentae and choriocarcinoma cells (Bowen, Chamley, Mitchell, et al., 2002) • TNF-α does not cross the placenta (Aaltonen et al., 2005) • IL-1β, IL-6, IL-8, and TNF-α were found to be increased in infants who produced an in-utero immune response (Pickler et al., 2010)

IFN-γ (Th1 cytokine)

Production sites:

• Associated with histologic chorioamnionitis (Takahashi et al., 2010),

PL, D, FM

early pregnancy loss (Bowen, Chamley, Mitchell, et al., 2002),

Other sites sampled:

some evidence of associations with PTBa (Lyon et al., 2010;

a

AF , UCB, MS

Matoba et al., 2009), preeclampsia and IUGR (Bowen, Chamley, Keelan, et al., 2002) may be associated with white matter damage in the newborn (Pickler et al., 2010) • Implicated as a negative regulator of trophoblast growth in normal placentae and choriocarcinoma cells (Bowen, Chamley, Mitchell, et al., 2002) • The profile of elevated MS levels at mid-gestation of IFN-γ, IL-4, and IL-5, was significantly associated with an increased risk of autism spectrum disorders (Goines et al., 2011)

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Table 2: Continued Cytokines

Reported Sites of Production

Selected Associations with Maternal and Neonatal

(Bowen, Chamley, Mitchell, &

Health Outcomes

Keelan, 2002) in Gestational Tissues and Sites Sampled in Selected Research MIF

Production sites: PL, D, FM Other sites sampled: AF, UCB, MS

• AF levels associated with preeclampsia with IUGR (Cardaropoli et al., 2012), chorioamnionitis, and PTB (these associations are not seen in MS samples) (Chaiworapongsa et al., 2005) • UCB levels not associated with PTB (Matoba et al., 2009) • No correlation between levels in AF and MS levels (Chaiworapongsa et al., 2005)

Lymphocyte-Derived Mediators IL-2 (Th1 cytokine)

Production sites:

• Reports of increased levels in cases of recurrent miscarriages

PL, D, FM

(Bowen, Chamley, Mitchell, et al., 2002), spontaneous abortions

Other sites sampled:

(Bowen, Chamley, Mitchell, et al.), preeclampsia (Bowen, Chamley,

AFa , UCBa , MS

Keelan, et al., 2002), PTB (Matoba et al., 2009), and IUGR (Makhseed et al., 2001) • The profile of elevated MS concentrations of IL-2, IL-4, IL-6, GM-CSF, and MIP-1α was associated with developmental delays without autism (Goines et al., 2011)

IL-5 (Th2 cytokine)

Production site:

• UCB levels higher in infants without NRFS (Takahashi et al., 2010)

D (mRNA detected)

• Increased UCB levels associated with PTB (Matoba et al., 2009)

Other sites sampled:

• The profile of elevated MS levels at mid-gestation of IFN-γ, IL-4,

AF, UCB, MS

and IL-5, was significantly associated with an increased risk of autism spectrum disorders (Goines et al., 2011)

Macrophage-Derived Mediators IL-15 (Th1 cytokine)

Production sites: PL, D, FM Other sites sampled:

• May be reduced in preeclampsia (Bowen, Chamley, Keelan, et al., 2002) • Increased in women with recurrent miscarriages (Toth et al., 2010)

AF, UCBa • Collectively, levels do not appear to be greatly influenced by labor

Anti-Inflammatory

(Bowen, Chamley, Keelan, et al., 2002; Lyon et al., 2010)

Cytokines IL-4 (Th2 cytokine)

Production sites:

• Important in maintaining a pregnancy (Piccinni et al., 2001)

PL, D, FM

• Has been associated with PTB (Bowen, Chamley, Keelan, et al.,

Other sites sampled: AFa , UCB, MS

2002; Matoba et al., 2009) and recurrent pregnancy loss (Bowen, Chamley, Mitchell, et al., 2002; Piccinni et al., 2001) • Does not cross the placenta in detectable amounts (Takahashi et al., 2010) • Elevated MS levels with several different combinations of other biomarkers associated with risk of autism spectrum disorders and developmental delays without autism (Goines et al., 2011)

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Table 2: Continued Cytokines

Reported Sites of Production

Selected Associations with Maternal and Neonatal

(Bowen, Chamley, Mitchell, &

Health Outcomes

Keelan, 2002) in Gestational Tissues and Sites Sampled in Selected Research IL-10 (Th2 cytokine)

Production sites:

• Important in maintaining a pregnancy (Piccinni et al., 2001)

PL, D, FM

• Associated with IUGR (Bowen, Chamley, Keelan, et al., 2002; Amu

Other sites sampled:

et al., 2006), preeclampsia (Bowen, Chamley, Keelan, et al.), PTBa

AF, UCB, MS

(Bowen, Chamley, Keelan, et al.; Lyon et al., 2010; Matoba et al., 2009), recurrent pregnancy loss (Bowen, Chamley, Mitchell, et al., 2002; Piccinni et al., 2001), chorioamnionitis (Takahashi et al., 2010), placental abruption (Takahashi et al.), gestational diabetes (Lyon et al.), and neonatal RDS (Takahashi et al.) may be associated with BPD in the newborn (Bowen, Chamley, Keelan, et al.) • Increased MS levels found in cases of autism spectrum disorder and developmental delay (borderline significance) (Goines et al., 2011)

IL-13 (Th2 cytokine)

Production site: PL Other sites sampled: AF, UCB, MS

• Limited data regarding its production (Bowen, Chamley, Mitchell, et al., 2002) • UCB levels have been associated with placental abruption (Takahashi et al., 2010) • Does not cross the placenta in detectable amounts (Takahashi et al., 2010)

Chemokines IL-8

Production sites:

• Associated with chorioamnionitis (Takahashi et al., 2010), NRFS

PL, D, FM

(Takahashi et al.), PTB (Bowen, Chamley, Keelan, et al., 2002;

Other sites sampled:

Matoba et al., 2009; Lyon et al., 2010; Takahashi et al., 2010),

AF, UCB

gestational diabetes (Lyon et al.), BPD, CLD, and patent ductus arteriosis in the newborn (Bowen, Chamley, Keelan, et al.; Takahashi et al.), and neurologic insult in the newborn (Bowen, Chamley, Keelan, et al.; Pickler et al., 2010) may be associated with preeclampsiaa (Bowen, Chamley, Keelan, et al.) and neonatal infectiona (Pickler et al.) • IL-1β, IL-6, IL-8, and TNF-α were found to be increased in infants who produced an in-utero immune response (Pickler et al., 2010)

RANTES

Production sites: PL, D, FM Other sites sampled: AF, UCB

MIP-1α/β

Production sitesa : D, FM Other sites sampled: AF, UCB, MS

• No association found between PTB and UCB levels (Matoba et al., 2009) • Elevated AF levels reported in PTB (Bowen, Chamley, Keelan, et al., 2002) • May be associated with PTBa (Bowen, Chamley, Keelan, et al., 2002; Matoba et al., 2009; Takahashi et al., 2010) • The profile of elevated MS concentrations of IL-2, IL-4, IL-6, GM-CSF, and MIP-1α was associated with developmental delays without autism (Goines et al., 2011)

MCP-1/2/3/4

Production sites:

• UCB levels of MCP-1 has been associated with maternal pregnancy

PL, D, FM

induced hypertension, chorioamnionitis, threatened PT labor, and

Other sites sampled:

neonatal RDS and CLD (Matoba et al., 2009; Takahashi et al., 2010)

AF, UCB (MCP-1)

842

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Ryan, J. G., Davis, R. K., and Bloch, J. R.

Table 2: Continued Cytokines

Reported Sites of Production

Selected Associations with Maternal and Neonatal

(Bowen, Chamley, Mitchell, &

Health Outcomes

Keelan, 2002) in Gestational Tissues and Sites Sampled in Selected Research TGF-β Superfamily

Production sites:

• Reviews did not consistently differentiate between subtypes

PL, D, FM

• Associated with IUGR (Amu et al., 2006) and BPD in the newborn

Other sites sampled:

(Bowen, Chamley, Keelan, et al., 2002) may be associated with

AF, UCB, MS

PTB (Lyon et al., 2010), preeclampsia (Bowen, Chamley, Keelan, et al.), and recurrent spontaneous abortion (Bowen, Chamley, Mitchell, et al., 2002) • No association between PTB and UCB levels (Matoba et al., 2009)

Note. AF = amniotic fluid; BPD = bronchopulmonary dysplasia; BV = bacterial vaginosis; CLD = chronic lung disease; D = decidua; FM = fetal membranes; IUGR = intrauterine growth restriction; MS = maternal serum; NRFS = non-reassuring fetal status; PL = placenta; PT = preterm; PTB = preterm birth; RDS = respiratory distress syndrome; UCB = umbilical cord blood. If not stated explicitly, the associations presented in this table may be positive or negative. The reader is directed to the listed references for more thorough discussions of the directionality and strength of these associations. a denotes conflicting reports.

area of the placenta from which the tissue sample was derived. This argument was based on the fact that significant differences occur within the same placenta depending upon the location of tissue sampling (i.e., proximal to or distant from the umbilical cord insertion). These within-placental differences include genetic expression (Yuen & Robinson) and enzymatic activity (Brameld, Hold, & Broughton Pipkin, 2011) among others. Furthermore, Barr et al. (2005) proposed that because environmental toxins are absorbed, metabolized, and excreted differently in various tissues of the body, careful consideration needs to be given to which tissue matrix is studied, the timing of the chemical exposure, and the conclusions that can be drawn from the study of the tissue.

Implications for Practice The Importance of the Research Protocol Depending on the nature of the research questions in each study, exact protocols for retrieving, storing, and transporting the placenta may vary. Clinical research may be focused on a broad range of maternal health factors (e.g., genetics, endocrinology, etc.). The health outcomes of infants and children may also be the research focus as in the NCS. Basic science research may be the focus with a wide range of topics. Regardless of the type of research, protocols must be approved by the Institutional Review Boards (IRBs) of the involved institutions. Although health care providers, specifically, the nurses and doctors present in the JOGNN 2012; Vol. 41, Issue 6

delivery room, are typically not responsible for obtaining IRB approval, staff from the research study team should be available to answer any questions regarding this process. In most cases, the research team is responsible for ensuring that the placenta is retrieved as per the IRB-approved protocol. Therefore, clear instruction of this protocol is critical for all who may be involved in collection. Nurses and other health care workers who are asked to assist in research studies should expect full orientation to the study protocol so they can completely understand their roles in participating in the research. When implementing study protocols aimed to collect birth specimens, the research team, including participating clinical staff, must follow prescribed methods for collecting, storing, and transporting specimens, and specifications will vary from study to study. Thus, if any questions or concerns arise regarding the exact collection procedures, roles, and responsibilities, a member of the study’s research team should be contacted without hesitation, and strong partnerships between clinical staff and researchers are critical. To date, the NCS has not publically released specific research protocols for collection, storage, and transport of birth biospecimens. It is likely that rather than establishing one protocol, multiple protocols will be developed to account for geographic variability between study sites and longitudinal changes associated with the length of the study.

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The Placenta as a Research Biospecimen

Cytokines can be found in maternal, neonatal, and umbilical cord blood, the placenta, and amniotic fluid.

An Integral Role for Nurses Those unaccustomed to the complexities of the interface between research and clinical practice might expect the collection of birth biospecimens and placentas to be seamless. However, as Kent et al. (2012) reported, the experiences of research nurses at the Children’s Hospital of PhiladelphiaStudy Center (CHOP-SC) demonstrated that success in specimen collection rested on the interpersonal skills and communication skills of the nurses. The research nurses educated all involved parties about the importance of the NCS research. Additionally, the CHOP-SC research team designed its protocol such that the research personnel were nurses (RNs). The research nurse was present at the hospital when the birth was taking place. Ultimately, the dedication and perseverance of the hospital staff nurses as well as their full collaboration with the research team were pivotal factors in the success of their efforts in retrieving birth biospecimens.

critical research specimen that offers researchers a unique view to study antenatal and fetal antecedents of subsequent infant, child, and adult health.

Acknowledgment Disclaimer: The views expressed in this article are the responsibility of the authors and do not represent the position of the National Children’s Study, the National Institutes of Health, or the U.S. Department of Health and Human Services.

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