Population and sample collection

During the years 2002-2004, women delivering before 28 weeks’ gestation at 1 of 14 participating institutions in 11 cities in 5 states were asked to enroll in a study to identify factors that increase risk for structural and functional neurologic disorders in extremely low gestational age newborns (ELGANs). The enrollment and consent processes were approved by the individual institutional review boards. Mothers were approached for consent either on antenatal admission or shortly after delivery, depending on clinical circumstance and institutional preference. In total, 1249 mothers of 1506 infants consented. The details of the population are described elsewhere.

For this report, we limited the sample to the 798 children for whom we had day 1 blood protein measurements and whose placenta was evaluated histologically.

Demographic and clinical variables

After delivery, a trained research nurse interviewed each mother in her native language using a structured data collection form and following procedures defined in a manual. The mother’s report of her own characteristics and exposures, as well as the sequence of events leading to preterm delivery were taken as truth, even when her medical record provided discrepant information.

The clinical circumstances that led to each maternal admission and ultimately to each preterm delivery were operationally defined using both data from the maternal interview and data abstracted from the medical record. Each mother/infant pair was assigned to the category that described the primary reason for the preterm delivery. Preterm labor (PTL) was defined as progressive cervical dilation with regular contractions and intact membranes. The diagnosis of pPROM was defined as the presence of vaginal pooling with either documented nitrazine positive testing or ferning before regular uterine activity. PE was defined as new onset hypertension and proteinuria of sufficient severity to warrant delivery for either a maternal or fetal indication. For a diagnosis of CI, a woman had to present with cervical dilation of greater than 2 cm, in the absence of membrane rupture and detected or perceived uterine activity.

PA was defined as presentation with a significant amount of vaginal bleeding (either documented in the medical record or a postpartum hematocrit <24%) and a clinical diagnosis of placental abruption in the absence of cervical change.

Presentations under the category of FI/IUGR included severe intrauterine growth restriction based on antepartum ultrasound examination, nonreassuring fetal testing, oligohydramnious, and Doppler abnormalities of umbilical cord blood flow. We allowed each center to apply its local definitions of these conditions and record any of the above as the delivery indications if the delivering teams cited 1 or more of these abnormalities in the medical record as the justification for delivery.

The gestational age estimates were based on a hierarchy of the quality of available information. Most desirable were estimates based on the dates of embryo retrieval or intrauterine insemination or fetal ultrasound before the 14th week (62%). When these were not available, reliance was placed sequentially on a fetal ultrasound at 14 or more weeks (29%), LMP without fetal ultrasound (7%), and gestational age recorded in the log of the neonatal intensive care unit (1%). The birthweight Z-score is the number of standard deviations the infant’s birthweight is above or below the median weight of infants at the same gestational age in a standard dataset. Prepregnancy body mass index (BMI) was defined as weight in kilograms divided by the square of height in meters (kg/M ² ).

Blood spot collection

Drops of blood were collected on Schleicher and Schuell 903 filter paper (Whatman International Ltd, Florham Park, NJ) on the first postnatal day (range, 0–1 days). All blood was from the remainder after specimens were obtained for clinical indications.

Dried blood spots were stored at −70°C in sealed bags with desiccant until processed. Blood was available for 798 neonates.

Blood spot elution and protein analysis

For protein elution, the frozen dried blood spots (DBS) were punched using 12 mm disposable biopsy AcuPunch (Acuderm, Inc, Fort Lauderdale, FL). The punched paper specimen was submerged in 300 μL PBS-based buffer containing 0.1% Triton X100 (Sigma-Aldrich, St Louis, MO) and 0.03% Tween-20 (Fisher, Hampton, NH) vortexes for 30 seconds and incubated on a shaker for 1 hour at 4°C. The punched paper along with the buffer were then transferred over the filter of a SpinX tube (Corning Fisher), centrifuged at 2000× g , followed by collection of the filtered eluted blood. An additional wash of the punch was performed in 100 μL for a final elution volume of 400 μL. The eluted blood samples were aliquoted and stored frozen at −70°C in bar-coded air-tight microtubes (USA Scientific, Orlando, FL). The eluted blood samples were analyzed for cytokines and other protein markers of immunoinflammatory function in duplicate using the Meso Scale Discovery (MSD) multiplex platform and Sector Imager 2400 (MSD, Gaithersburg, MD), which has been previously validated by comparisons with traditional ELISA. MSD multiplex assays measuring up to 10 proteins simultaneously were optimized to allow detection of each biomarker within the linearity range of the DBS-eluted samples. The MSD Discovery Workbench Software was used to convert relative luminescent units into protein concentrations using interpolation from several log calibrator curves. Split quality control blood pools tested on each plate showed interassay variation of <10-20% for each individual marker. In each DBS-eluted sample, the total protein level was determined by BCA assay (Thermo Scientific, Rockford, IL) using a multilabel Victor 2 counter (Perkin Elmer, Boston, MA) and the expression levels of each of the analytes normalized to mg total protein.

The 25 analytes chosen for study were grouped in the following fashion:

Cytokines and their receptors

Interleukin (IL)-1β, IL-6, IL-6 receptor (R), tumor necrosis factor-alpha (TNFα), TNF-R1, TNF-R2.

Other markers of systemic inflammation

C-reactive protein (CRP), serum amyloid A (SAA), myeloperoxidase (MPO).

Chemokines

IL-8 (CXCL8), monocyte chemotactic protein-1 (MCP-1, CCL2), MCP-4 (CCL13), macrophage inflammatory protein (MIP)-1β (CCL4), regulated on activation normal T-cell expressed and presumably secreted (RANTES, CCL5), interferon-inducible T cell alpha-chemoattractant (I-TAC, CXCL11).

Adhesion molecules

Intercellular adhesion molecule-1 (ICAM-1, CD54), intercellular adhesion molecule-3 (ICAM-3, CD50), vascular cell adhesion molecule-1 (VCAM-1, CD106), E-selectin (ESEL, CD62E).

Metalloproteinases

Matrix metalloproteinase-1 (MMP-1), MMP-9.

Proteins with angiogenic properties

Vascular endothelial growth factor (VEGF), VEGF-R1 (FLT1), VEGF-R2 (KDR), insulin growth factor binding protein-1 (IGFBP-1).

Sixteen of the 25 proteins were detectable within the assay linearity range in all samples. For the other 9 proteins, the percent of samples with undetectable concentrations were MIP-1β (11%), IL-1β (11%), VEGF-R1 (6%), VEGF (5%), TNFα (3%), MCP-4 (0.4%), IL-6 (0.4%), I-TAC (0.2%), and IGF-BP1 (0.1%). The small percentage of undetectable values could not affect our analysis based on quartile distribution of protein concentrations.

Statistical analysis

Because the distributions of many of the proteins were not normal, we categorized children into quartiles of each protein concentration. Because concentrations of these proteins may be developmentally regulated, we defined each quartile of each protein within gestational age categories (23-24, 25-26, 27 weeks). We evaluated the generalized null hypothesis that the risk of a blood protein concentration in the highest quartile for gestational age was not associated with the pregnancy disorder that led to preterm delivery.

Our measure of association is the odds ratio (and 99% confidence interval) that children whose mother had each pregnancy disorder were more likely to have a protein concentration in the top quartile than children born to women who were delivered because of severe PE. We selected PE as the referent pregnancy disorder because our experience suggests it would be least likely to lead to a fetal inflammatory response. We selected a 99% confidence interval rather than the conventional 95% confidence interval because we wanted to modify our analyses for multiple comparisons (25 proteins and 6 pregnancy disorders), although not appreciably increasing the risk of a type 1 (false positive) error.

Demographic and clinical characteristics of the mothers and infants ( Table 1 )

Women delivered for PE and FI/IUGR tended to have higher BMI (≥30 kg/M ² ) than women with other complications. Women who presented with PTL, PE, or abruption were more often primigravid than women who presented with the other pregnancy disorders. The use of some form of conception assistance was uniform across the pregnancy disorders with the exception of an approximate halving of the frequency among those delivered for PE. Consistent with PE and FI/IUGR as “iatrogenic” causes of preterm delivery, the incidence of cesarean delivery was higher for these 2 indications than for the other disorders. The incidence of urinary tract infection (UTI) was highest among deliveries for PTL and PE, and the incidence of vaginitis was lowest among pregnancies delivered for PE and FI/IUGR. The incidence of multiple gestations tended to be higher among deliveries caused by PTL and FI/IUGR and prominently lowest among deliveries complicated by PE.

TABLE 1

Demographic and clinical characteristics of the 709 mothers and 861 infants classified by pregnancy complications (column percents)

	Indication for preterm delivery
Characteristic	PTL	pPROM	CI	PA	FI/IUGR	PE	Row N
Prepregnancy BMI, kg/M 2
<18.5	10	11	0	7	12	1	56
18.5, <30	74	65	84	77	58	64	477
≥30	17	24	16	16	31	35	146
Primigravid	42	30	28	48	27	46	271
Conception assistance	17	14	19	19	23	7	106
Cesarean delivery	50	55	74	71	88	96	444
During this pregnancy
Fever >38°	6	6	6	11	15	4	47
UTI	17	12	13	11	12	20	105
Vaginitis	16	15	22	12	8	9	99
Antibiotic	33	29	34	30	27	30	213
Multiple gestation	25	20	21	16	38	5	143
Antenatal corticosteroid
None	14	5	2	18	21	5	94
Partial	30	18	18	15	24	28	213
Complete	56	77	80	67	56	67	554
Gestational age at delivery, wk
23-24	24	20	33	27	3	9	182
25-26	44	47	49	48	50	45	395
27	32	34	18	25	47	45	284
Birthweight Z-score
<−2	1	3	0	5	12	25	44
≥−2, <−1	9	8	2	8	29	42	114
≥−1	90	89	98	87	59	34	703
Male sex	56	59	60	51	29	36	455
Maximum N mothers	302	160	34	83	26	101	709
Maximum N infants	390	189	45	97	34	106	861

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