The placentas were collected as part of a study designed to identify factors that increase risk for structural and functional neurological disorders in extremely low gestational age newborns (ELGANs). With approval of the individual institutional review boards, women delivering before 28 weeks gestation at one of 14 participating institutions were asked to enrol in the study. 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%), last menstrual period (LMP) without fetal ultrasound (7%), and gestational age recorded in the log of the neonatal intensive care unit (1%).
The enrolment period covered years 2002–2004. A total of 1250 mothers of 1506 infants consented (an estimated 260 mothers were missed or declined to participate) but only 1411 placentas were submitted for pathological evaluation (totals refer to the number of umbilical cords; i.e., twins are counted as two placentas). The 947 placentas from singletons are described here.
Placentas were examined grossly by the available pathologist, often a resident, within 24 hours of delivery. Gross parameters recorded included trimmed weight, disc dimensions, the presence of an accessory lobe and membrane insertion (marginal, circummarginate, circumvallate). The cord was described with respect to insertion, twist, knot or pseudoknots and number of vessels. The disc parenchyma was examined and coded for focal lesions including infarcts, intervillous thrombus, retroplacental haematoma and vascular thrombosis. All other lesions were described in a free text section.
Histological examination of the placenta was performed following College of American Pathologists guidelines.6
Representative sections were taken from all abnormal areas, as well as routine sections of the umbilical cord and a membrane roll, and two full thickness sections from the centre and a paracentral zone of the placental disc.
After the creation of a manual with definitions and illustrations, and completion of consensus case exercises to minimise observer variability, a pathologist at each site examined the slides for the histological characteristics listed on the ELGAN study data form (available on request).
Infarcts and intervillous fibrin, fetal stem vessel thrombosis, and decidual haemorrhage and fibrin deposition consistent with abruption were coded as present or absent. So, too, was the presence of decidual vasculopathy, absence of villous oedema, chorioangiosis and chorioangioma. Vascular lesions were not quantified (i.e., number and size of infarcts). Chorionic villi were scored for subjective increase in syncytial knots.
Inflammation of the membranes was described in detail. At the chorionic plate of the disc, acute inflammation was assigned a stage from 0 to 3 (0, none; 1, neutrophils collecting in subchorionic space; 2, neutrophils into chorionic plate; 3, neutrophils up to amnionic epithelium). The severity of inflammation at the plate was assigned a grade from 1 to 3 (1, 1–9 neutrophils/20×; 2, 10–19 neutrophils/20×; 3, >20 neutrophils/20×). Inflammation of the free membranes (chorion/decidua) was graded from 0 to 4 (0, none; 1, single focus of 5–10 neutrophils; 2, several small foci or single focus of >10 neutrophils; 3, numerous large or confluent foci; 4, necrotising). Inflammation in the amnion was similarly but separately graded from 0 to 4.
The fetal inflammatory response was gauged by inflammation in the umbilical cord which was graded from 0 to 5 (0, none; 1, neutrophils within the inner third of one umbilical vessel; 2, neutrophils within the inner third of at least two umbilical vessels or through the wall of one vessel; 3, neutrophils in perivascular Wharton’s jelly; 4, inflammation extending deep into Wharton’s jelly; 5, ‘halo lesion’ ring of precipitate in Wharton’s jelly encircling each vessel). Neutrophilic and eosinophilic infiltration into fetal stem vessels in the chorionic plate was also noted as present or absent.
Other lesions specifically noted as present or absent included subamniotic haematoma, maternal floor infarction, maternal sickle cells, trophoblast inclusions, chronic villitis, intervillositis, acute villitis, fibromuscular sclerosis (muscular hypertrophy with lumen obliteration), obliterative endarteritis (encroachment of lumen by subendothelial expansion associated with trapped red blood cells), placenta accreta and infectious agents.
At most of the study institutions, the participating pathologist had a special interest in reproductive pathology. The pathologists helped in the development of the procedures manual and definitions as well as in the design of the data collection form. Placentas were processed as part of the daily workflow of the departments and then were reviewed by the ELGAN pathologist.
We did not conduct a central review of the histology. Each pathologist was provided with an illustrated manual of the characteristics and scoring systems as described. Fourteen sets of slides from 13 placentas were circulated among the pathologists for evaluation using the data form. Responses were summarised for the group and discussed by e-mail.
Once the study data were collected, we assessed observer variability by comparing among pathologists the rate of diagnosis for each data-form element. This approach assumes that the placentas at each institution are similar to the placentas at other institutions, and that much of the variability among institutions reflects the tendency of the pathologist at that institution to see each histological feature (). Since the study entry criteria were the same across institutions, this assumption seemed reasonable.
Percent of placentas with each histological characteristic according to each reader (column percents)
The pathologists were generally consistent in their use of diagnoses. The measures of inflammation were similar across institutions. Infarction and fetal vessel thrombosis were consistent with one outlier in each. Quantitation of syncytial knots, decidual haemorrhage and intervillous fibrin deposition are difficult to standardise7,8
and pathologists fall into two groups, conservative and more liberal.
The data were entered into a central database and analysed using Stata Release 9.2 (2007; StataCorp, USA) statistical software. The χ2 test for linear trend was used to evaluate trends with gestational age. Fisher’s exact test was used for pair-wise comparisons of the histological characteristics.