Study population

The Extremely Low Gestational Age Newborns (ELGAN) study was designed to identify pregnancy and neonatal characteristics and exposures that increase the risk of neurologic disorders in ELGANs. During the years 2002-2004, women delivering between a gestation of 22 weeks 0 days and 27 weeks 6 days at 1 of 14 participating institutions in 11 cities in 5 states were asked to enroll in the study. The individual institutional review boards approved the enrollment and consent processes. Mothers were approached for consent either upon antenatal admission or shortly after delivery, depending on clinical circumstance and institutional preference.

Of 1506 infants enrolled in the ELGAN study, placentas were collected from 1411, ribonucleic acid (RNA) was prepared from 1370, and 1219 contained nondegraded RNA. These 1219 placentas constitute the sample for all the tables. The 3 samples (1506, 1411, and 1219) are comparable in their proportion of children delivered for maternal and fetal reasons and the distribution of gestational ages and birthweights.

Pregnancy variables

The clinical circumstances that led to 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 hospitalization: preterm labor was defined as progressive cervical dilation with regular contractions and intact membranes. The diagnosis of preterm premature rupture of fetal membranes (pPROM) was defined as the presence of vaginal pooling with either documented nitrazine positive testing or ferning prior to regular uterine activity. Preeclampsia 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 cervical insufficiency, 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. Placental abruption was defined as presentation with 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. Although painful uterine contractions were not required, most women given this diagnosis tended to present with vaginal hemorrhage often accompanied or very soon followed by labor.

In addition, placenta abruption, as defined, tended to be much more like the other spontaneous disorders of labor, prelabor rupture of membranes, and cervical insufficiency. Presentations under the category of fetal indication/intrauterine growth restriction included severe intrauterine growth restriction based on antepartum ultrasound examination, nonreassuring fetal testing, oligohydramnious, and Doppler abnormalities of umbilical cord blood flow.

Preterm labor, prelabor premature rupture of fetal membranes, cervical insufficiency, and placental abruption were then grouped into spontaneous deliveries because they were initiated without medical assistance. “Induced for maternal or fetal reasons” is applied to deliveries that were not initiated by the mother or fetus but by the physician to preserve the health of mother or fetus.

Placenta characteristics

Delivered placentas were placed in a sterile examination basin and transported to a sampling room. Eighty-two percent of the samples were obtained within 1 hour of delivery.

CRH mRNA quantitation by quantitative reverse transcription-polymerase chain reaction (RT-PCR)

Complementary DNA was prepared in 10 μL reactions using iScript cDNA synthesis (Bio-Rad Laboratories, Hercules, CA) and 0.5 μg of placental RNA. Subsequently, 2 μL of this reaction was amplified by qPCR using IQ SYBR Green Supermix (Bio-Rad Laboratories), a Bio-Rad iQ5 qPCR apparatus, and the following primers: CRH forward primer, AGA GAA AGC CCC CGG AGA, CRH reverse primer, ATG TTA GGG GCA CTC GCT TC, ACTB forward primer, CGC GAG AAG ATG ACC CAG AT, ACTB reverse primer, GTA CAT GGC TGG GGT GTT G.

The qPCR conditions consisted of denaturation (95.0°C for 3 minutes) and amplification (95.0°C for 10 seconds, 60.0°C for 40 seconds, repeated for 45 cycles). The standards were prepared by PCR amplification using these same primers and control placenta RNA (see the preceding text). ACTB results were used to confirm the quality of the mRNA and correct for total RNA amount. Of the 1370 samples, 151 contained degraded RNA (as judged by lack of ACTB and CRH signals by qRT-PCR), so that final analyses were performed on 1219 samples.

To normalize the results among the different experimental runs, all CRH standards across all 17 qPCR runs were averaged, and each of the 1219 experimental samples was evaluated against that average CRH standard curve. Similarly, all ACTB standards across all 17 qPCR runs were averaged and each of the 1219 experimental ACTB values was evaluated against that average ACTB standard curve. Each CRH value was then divided by its corresponding ACTB value to obtain the relative CRH mRNA concentrations, which are displayed in Figure 1 and Tables 1-7 .

Placental CRH mRNA (qRT-PCR), gestational ages 23-27 weeks

Abrupt, placental abruption (n = 125); CI, cervical incompetence (n = 74); CRH, corticotropin-releasing hormone; FI, fetal indication (n = 62); PE, preeclampsia (n = 163); pPROM, prelabor premature rupture of membranes (n = 253); PTL, preterm labor (n = 542); qRT-PCR, quantitative reverse transcription-polymerase chain reaction.

Trivedi. Fetal-placental inflammation, not adrenal activation, in extreme preterm delivery. Am J Obstet Gynecol 2012.

TABLE 1

Percent of infants by delivery indication (column) and characteristic (row)

Characteristic	Delivery indication						Row number
	PTL	pPROM	CI	Abrupt	FI	PE
Antenatal steroid course
Complete	55	73	78	62	66	67	771
Partial	32	21	19	16	18	30	320
None	13	6	3	22	16	3	126
Cesarean delivery
Yes	52	60	62	68	95	98	782
Sex
Male	57	53	59	52	48	44	652
Type of gestation
Singleton	59	70	58	74	44	94	812
Gestational age, wks
23-24	32	26	39	33	16	14	340
25-26	40	43	51	42	48	45	521
27	28	32	9	25	35	40	358
Birthweight, g
≤750	38	39	42	38	68	67	537
751-1000	42	39	42	47	19	26	469
>1000	20	22	16	14	13	7	213
Birthweight
<−2	1	5	0	4	32	31	92
Z-score a
≥−2, <−1	10	9	3	6	23	39	166
≥−1	89	86	97	90	45	30	961
Column number	542	253	74	125	62	163	1219

Columns contain column percents.

Abrupt, placental abruption; CI, cervical insufficiency; FI, fetal indication; PE, preeclampsia; pPROM, prelabor premature rupture of membranes; PTL, preterm labor.

Trivedi. Fetal-placental inflammation, not adrenal activation, in extreme preterm delivery. Am J Obstet Gynecol 2012.

a Yudkin standard.

TABLE 2

Percent ^a of placentas with CRH expression in highest quartile for gestational age and delivery indication

Gestational age, wks	Delivery indication						Row percentage
	PTL	pPROM	CI	Abrupt	FI	PE
23-24	6	14	17	17	30	70	15
25-26	15	9	13	23	37	72	24
27	22	24	29	26	55	79	35
Column percentage	14	15	16	22	42	74	25

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