In vivo evidence of significant placental growth factor release by normal pregnancy placentas

Placental growth factor (PlGF) is an angiogenic factor identified in the maternal circulation, and a key biomarker for the diagnosis and management of placental disorders. Furthermore, enhancing the PlGF pathway is regarded as a promising therapy for preeclampsia. The source of PlGF is still controversial with some believing it to be placental in origin while others refute this. To explore the source of PlGF, we undertook a prospective study enrolling normal pregnant women undergoing elective caesarean section. The level of PlGF was estimated in 17 paired serum samples from the uterine vein (ipsilateral or contralateral to the placental insertion) during caesarean section and from a peripheral vein on the same day and second day post-partum. PlGF levels were higher in the uterine than in the peripheral vein with a median difference of 52.2 (IQR 20.1–85.8) pg/mL p = 0.0006. The difference when the sampled uterine vein was ipsilateral to the placenta was 54.8 (IQR 37.1–88.4) pg/mL (n = 11) and 23.7 (IQR −11; 70.5) pg/mL (n = 6) when the sample was contralateral. Moreover, PlGF levels fell by 83% on day 1–2 post-partum. Our findings strongly support the primary source of PlGF to be placental. These findings will be of value in designing target therapies such as PlGF overexpression, to cure placental disorders during pregnancy.


Methods
The study was conducted at the John Radcliffe Hospital, Oxford, United Kingdom. We recruited women admitted for an elective cesarean section. All pregnancies were uncomplicated and indications for caesarean section were previous caesarean section or breech presentation. sFlt-1 levels in this cohort have previously been reported 23 .
All women included in the study had an ultrasound scan to assess placental location prior to the elective caesarean section. Patients with a midline placenta or with any signs of early labour were excluded.
Serum samples were collected from the antecubital fossa (peripheral vein) and the uterine vein (UV) during the caesarean section, prior to delivery of the fetus. Uterine veins were identified laterally to the uterus and exposed by gentle displacement of the uterus towards the midline. Patients had a second peripheral sample taken on day 1-2 postnatally.
Uterine vein samples were labelled as contralateral or ipsilateral depending on their location in relation to the placenta. In the majority of cases the surgeon collected one sample either from the ipsilateral or contralateral uterine vein, at their own description or depending on surgical accessibility. In three cases of extreme lateral placentas it was possible to take bilateral uterine vein samples. Extreme lateral placenta was defined as a placenta located mostly on one side of the uterus: lateral left: all or almost all the placenta is located to the left of the midline; lateral right: all or almost all the placenta is located to the right of the midline; Midline placenta was defined as a placenta with a similar percentage of tissue towards the left and the right of the midline.
Non-parametric analysis, Wilcoxon rank test, was used to assess differences between paired PlGF results and P < 0.05 was considered significant.
Samples were centrifuged within 3 hours of collection and frozen at −80C until analysis. PlGF was measured on a Roche e411 analyzer (Roche Diagnostics Limited, Burgess Hill, UK). Inter-assay percentage coefficient of variation was 3.0% at 106.1 pg/mL and 2.9% at 1068.5 pg/mL.
All patients provided informed written consent prior to the start of the study. The study was approved by the Central Oxfordshire Research Ethics Committee C (07/H0607/74) and was in accordance with the ethical standards of the institutional and/or national research committee and with the Helsinki Declaration and its amendments.

Discussion
PlGF is an angiogenic factor that belongs to the vascular endothelial growth factor (VEGF) family. It has an important role in normal placental function and can be detected in maternal circulation as early as the first trimester 24 . PlGF is tightly linked with the pathogenesis of preeclampsia and fetal growth restriction; thus understanding PlGF biodynamics will be crucial to making progress in therapeutics in these diseases. We demonstrate that the placenta is an important source of maternal circulating PlGF by showing a significant gradient between uterine and peripheral PlGF levels. This is most evident in uterine vein samples acquired from the same side as the placenta (ipsilateral). In support of this, bilateral samples collected from lateral placentas consistently showed higher PlGF levels in the ipsilateral uterine vein compared to the contralateral. Finally, when the placenta is removed (i.e. postnatal period) there is an 83% fall in PlGF levels in the peripheral circulation. It has been  www.nature.com/scientificreports www.nature.com/scientificreports/ suggested that the uterus itself could be a key source of PlGF, rather than the placenta. In vitro studies showing significant production and expression of PlGF by normal placentas 1,25 and our data from bilateral samples and postnatal samples (showing a significant drop of PlGF after the placenta is removed) suggests that this is unlikely. This study was performed on term patients and therefore the conclusions may not be applicable to other trimesters.
Most of our knowledge of placental biology is derived from human in vitro and ex vivo data, which is easier to access than in vivo samples, but remains a model. In vitro studies show that both VEGF and PlGF are produced by the placenta 25 . Intriguingly, in contrast with PlGF, in vivo data suggests that maternal circulating VEGF is taken up by the placenta and mostly produced by peripheral organs (shown by a lower concentration of VEGF in the uterine vein when compared to the radial artery) 10 . This not only demonstrates the value of in vivo data, but also has important implications. For example, when designing target-specific strategies, such as the recent use of RNAi to downregulate the production of sFlt-1 only in the placenta (the main source of sFlt-1) and not in other organs 26 .
In summary, these data strongly suggest that the placenta is the main source of maternal PlGF in normal pregnancy.