Regulation and expression of sFlt-1 in twin pregnancy - first step towards understanding placental physiology in multiple pregna

Background
Severe intrauterine growth restriction (IUGR) is one of the major complications of pregnancy and accounts for significant neonatal mortality and morbidity 1. Approximately 50% of women with severe growth restricted fetuses will develop preeclampsia 2. Severe growth restriction and preeclampsia often necessitate preterm delivery, further impacting upon neonatal morbidity. True growth restriction is present when the fetus fails to achieve its growth potential 3 and most cases are related to a defect in proper placental development 4. Histomorphological studies indicate that IUGR is associated with structural alterations of the placenta such as abnormally developed terminal capillary loops and sparse arrangement of functional terminal villi 5 which are responsible for increased feto-placental vascular resistance and consequently decreased blood flow and availability of nutrients and oxygen to the fetus.
The soluble VEGF receptor 1, also known as soluble fms-like tyrosine kinase-1 (sFlt-1), is a truncated form of the VEGFR-1 gene that is physiologically secreted by the human placenta 6. Circulating sFlt-1 binds VEGF and placental growth factor (PlGF) with high affinity thereby decreasing their availability for binding to the trans-membrane receptors VEGFR-1 and VEGFR-2 6. Recent studies have shown that both circulating and placental sFlt-1 expression levels are markedly increased in preeclamptic pregnancies 7-9. We have recently shown that sFlt-1 is regulated by oxygen via HIF-1, supporting a key role for low oxygen in regulating sFlt-1 expression in the human placenta 10. Doppler studies indicate that pregnancies complicated by IUGR exhibit decreased uterine blood flow and higher resistance indices. This is due both to failure in spiral artery remodeling found in IUGR pregnancies 11 and to abnormal development of the tertiary villous vasculature 12;13 which then limits the amount of oxygen available in the placenta.
Twin pregnancy is a unique condition in which two fetuses and placentae share the same maternal environment. It is known that growth restriction as well as preeclampsia are more common in twins though the cause for this increase is not known. Data regarding twin placentae development or physiology in the molecular level is very limited to absent.

Purpose
In the present study we examined the expression of sFlt-1 in placental samples from singletons with severe IUGR pregnancies, discordant twin samples in which one twin was severely growth restricted and twins with preeclampsia. The singleton and twin IUGR pregnancies were rigorously classified according to onset and severity as documented by Doppler analysis. Singleton IUGR placentae were also compared to normal control tissue and to placentae from small for gestational age pregnancies.

Methods
Patient population
IUGR was defined as birth weight < 5th centile accompanied by abnormal umbilical artery Doppler defined as absent or reverse end diastolic velocity (AREDV) and in most of cases with increased resistance to flow in uterine arteries defined as early diastolic notch or pulsatility index >1.45. Newborns with birth weight ≤ 10 centile but with normal umbilical and uterine artery Doppler were defined as small for gestational age. Controls were selected as age-matched healthy pregnancies with normally grown fetuses that did not have signs of placental dysfunction.
Discordant twins were selected as a separate group in which a normal twin and a growth-restricted twin developed in the same maternal and uterine environment. Discordant growth was defined as discordancy of more than 25% in birth weight in conjunction with IUGR in one fetus with AREDV and normal growth and Doppler study in the co-twin. Dichorionic and monochorionic twins were considered as two separate groups. Additionally, dichorionic twin pregnancies with signs of preeclampsia, but without IUGR, were included as a separate group and were compared to control twins without preeclampsia. Placental samples from all patients subgroups were collected immediately after delivery.
Measurement of sFlt-1 expression
Total RNA was isolated and qPCR for sFlt-1 was performed. Western blot analysis for sFlt-1 was performed using mouse monoclonal anti Flt-1 antibody.
Paraffin embedded sections were used for immunohistochemistry.

Results
sFlt-1 expression in placentae of singletons with severe IUGR
Real-time qPCR analysis showed a significant increase (> 2.5 fold) in sFlt-1 mRNA expression in severe singleton IUGR placentae compared to the SGA, preterm control (PTC) and term control (TC) placentae. No significant changes in sFlt-1 mRNA expression were found between SGA, PTC and TC placentae.
Western blot analysis demonstrated 2-fold increase in sFlt-1 protein levels in IUGR placentae compared to SGA, PTC and TC placentae, in agreement with increased mRNA expression. Strong positive immunoreactivity for sFlt-1, mostly localized to trophoblast layers, was observed in IUGR placentae. Negative or weak staining for sFlt-1 was found in PTC and SGA placentae, respectively. Low to absent stromal or perivascular staining was noted in both IUGR and control placentae.
sFlt-1 expression in IUGR discordant twin placentae
In dichorionic twins, sFlt-1 transcript level was significantly greater in the IUGR twin placenta compared to its normally grown co-twin placenta (IUGR twin vs. co-twin: 3.52±0.5 vs 1.08±0.13 arbitrary sFlt-1 mRNA levels, P<0.05) and placenta of normal control twins. In line with the dichorionic twins, sFlt-1 mRNA expression in monochorionic twins was also increased in the growth-restricted twin placenta compared to the co-twin placenta (IUGR twin vs. co-twin: 1.7±0.19 vs. 0.79±0.2 arbitrary sFlt-1 mRNA levels; P<0.05) and placentae of normal control twins without growth restriction. Interestingly, sFlt-1 transcript levels were greater in the dichorionic than the monochorionic growth restricted twin placentae (dichorionic vs. monochorionic IUGR: 3.52±0.5 vs. 1.7±0.19 arbitrary sFlt-1 mRNA levels).
Western blot and densitometric analysis of placental lysates from the discordant twins showed increased sFlt-1 protein content in the placentae from monochorionic and dichorionic IUGR twins when compared to their co-twins and control twins without growth restriction.
sFlt-1 expression in placentae from preeclamptic twins
Unlike measurements of circulating sFlt-1 that do not differentiate between the different sources of sFlt-1, sampling of each placenta separately enabled us to quantify the relative sFlt-1 expression in each placenta. Real-time qPCR analysis of preeclamptic twin placentae consistently showed increased sFlt-1 transcripts in one placenta relative to the other. Therefore, the preeclamptic and control twin pairs were divided into high expression (HE) and low expression (LE) subgroups according to the level of sFlt-1 RNA expression detected. We found significant greater sFlt-1 mRNA expression in HE preeclamptic twin placenta compared to the LE preeclamptic twin placenta and that of twin controls (HE twin vs. LE twin: 5.01±0.82 vs. 1.57±0.71 arbitrary sFlt-1 mRNA levels, p<0.05). The protein content of sFlt-1 in HE preeclamptic twin placenta was greater than that of the LE preeclamptic twin placenta and control twin placentae.
Conclusion / new knowledge
The characterization of specific placental sub-pathologies is of utmost importance when studying the basis of molecular events underlying pregnancy related disorders. In the present study we show that placental sFlt-1 expression is increased in placentae from severe early onset IUGR pregnancies with documented AREDV, but not in small for gestational age pregnancies with normal Doppler. Secondly, we demonstrate that placental sFlt-1 expression is increased in singleton IUGR pregnancies and in the growth restricted twin from discordant twins pregnancies compared to control twins. Additionally, we show that elevated sFlt-1 expression in preeclamptic twin pregnancies with no sign of growth discordancy originates in one, not both placentae.
We used discordant twin pregnancies as a natural model in which the IUGR and normal fetus share the same maternal environment. Hypothetically, the maternal environment should affect the two placentae and the fetuses in a similar manner. Our data demonstrate that in discordant twin pregnancies sFlt-1 expression is increased in the placental tissue from the growth-restricted twin confirming that fetal discordancy is associated with molecular placental differences 14. The increased sFlt-1 expression in IUGR placentae is likely due to reduced placental perfusion experienced by the growth restricted placenta resulting in low oxygenation. Thus, in discordant twins, in spite of the same maternal environment, the two placentae present different morphology which is accompanied by differences in sFlt-1 expression. Our findings indicate that sFlt-1 expression was higher in dichorionic growth-restricted twins compared to the monochorionic (3-fold increase in IUGR dichorionic vs. 1.7-fold increase in monochorionic). The cause for increased sFlt-1 levels in dichorionic placenta can be related to differences in the mechanism underlying the pathogenesis of the chorionicity in twin pregnancies. While the alteration found in dichorionic placenta are likely due to impaired placental development similar to that found in singletons IUGR, the placental pathology in monochorionic IUGR is further complicated by the unequal sharing of the placenta in which the IUGR twin has smaller area of the placenta and the presence of vascular anastomoses between the two twins 15.
Elevated placental sFlt-1 production, leading to increased sFlt-1 circulating levels in the feto-placental circulation 16, can bind locally produced VEGF and PlGF, impairing their binding to the VEGF receptors. This would result in an anti-angiogenic balance in IUGR placentae thereby altering the terminal differentiation of placental capillary loops. This may contribute to the abnormal angiogenesis found in IUGR in the intermediate and terminal IUGR villi.
While it is established that preeclampsia is more prevalent in twin pregnancies, it remains to be established whether both placentae contribute equally to the development of preeclampsia. Our finding of high sFlt-1 expression in one placenta, but not in the other, suggests that only one placenta can trigger preeclampsia in this subgroup of not discordant twin pregnancies. The notion that one placenta in twins can cause preeclampsia is known from cases in which selective fetocide reverses preeclampsia in discordant twins 17 or resolution of mirror syndrome after demise of the diseased fetus 18.

Transdisciplinary
Encouraged by our results we have started to examine the occurrence rate of preeclampsia in discordant twins, since we found imbalance in sFlt-1 expression between the two placentae and wondered what the clinical implications are. Our question was whether the rate of preeclampsia in singletons with IUGR is the same as discordant twins with IUGR in one twin. We performed a data base and chart review study of all deliveries in our institution during 2002-2007. Using singletons and twins growth curves with growth restriction defined below 10 centile we found that: a) the rate of preeclampsia in twins is about double the rate in singletons, as expected. b) preeclampsia is significantly more common in singletons with IUGR ( 19.1 %) compared to discordant twins (9.9%). Our conclusion is that the presence of a normal grown fetus and placenta in twin pregnancy may be protective for the development of preeclampsia or alternatively, the pathogenesis of IUGR in twins is different from singletons. Our centre is a tertiary care centre and thus the patient population does not necessarily reflect the general population. Population epidemiological data may be useful in better defining differences between singletons and twins pregnancies.

Question
1. What other interdisciplinary aspects can be examined?
2. What may be the pathogenesis behind the differences in twins placentae?

Reference List

1. Thornton JG, Hornbuckle J, Vail A, Spiegelhalter DJ, Levene M. Infant wellbeing at 2 years of age in the Growth Restriction Intervention Trial (GRIT): multicentred randomised controlled trial. Lancet 2004;364:513-20.
2. Karsdorp VH, van Vugt JM, van Geijn HP, Kostense PJ, Arduini D, Montenegro N et al. Clinical significance of absent or reversed end diastolic velocity waveforms in umbilical artery. Lancet 1994;344:1664-68.
3. Chaddha V, Viero S, Huppertz B, Kingdom J. Developmental biology of the placenta and the origins of placental insufficiency. Semin.Fetal Neonatal Med. 2004;9:357-69.
4. Regnault TR, Galan HL, Parker TA, Anthony RV. Placental development in normal and compromised pregnancies-- a review. Placenta 2002;23 Suppl A:S119-S129.
5. Krebs C, Macara LM, Leiser R, Bowman AW, Greer IA, Kingdom JC. Intrauterine growth restriction with absent end-diastolic flow velocity in the umbilical artery is associated with maldevelopment of the placental terminal villous tree. Am.J.Obstet.Gynecol. 1996;175:1534-42.
6. Clark DE, Smith SK, He Y, Day KA, Licence DR, Corps AN et al. A vascular endothelial growth factor antagonist is produced by the human placenta and released into the maternal circulation. Biol.Reprod. 1998;59:1540-48.
7. Zhou Y, McMaster M, Woo K, Janatpour M, Perry J, Karpanen T et al. Vascular endothelial growth factor ligands and receptors that regulate human cytotrophoblast survival are dysregulated in severe preeclampsia and hemolysis, elevated liver enzymes, and low platelets syndrome. Am.J.Pathol. 2002;160:1405-23.
8. Koga K, Osuga Y, Yoshino O, Hirota Y, Ruimeng X, Hirata T et al. Elevated serum soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) levels in women with preeclampsia. J.Clin.Endocrinol.Metab 2003;88:2348-51.
9. Levine RJ, Maynard SE, Qian C, Lim KH, England LJ, Yu KF et al. Circulating angiogenic factors and the risk of preeclampsia. N.Engl.J.Med. 2004;350:672-83.
10. Nevo O, Soleymanlou N, Wu Y, Xu J, Kingdom J, Many A et al. Increased expression of sFlt-1 in in vivo and in vitro models of human placental hypoxia is mediated by HIF-1. Am.J.Physiol Regul.Integr.Comp Physiol 2006;291:R1085-R1093.
11. Brosens I, Dixon HG, Robertson WB. Fetal growth retardation and the arteries of the placental bed. Br.J.Obstet.Gynaecol. 1977;84:656-63.
12. Kingdom JC, Burrell SJ, Kaufmann P. Pathology and clinical implications of abnormal umbilical artery Doppler waveforms. Ultrasound Obstet.Gynecol. 1997;9:271-86.
13. Todros T, Sciarrone A, Piccoli E, Guiot C, Kaufmann P, Kingdom J. Umbilical Doppler waveforms and placental villous angiogenesis in pregnancies complicated by fetal growth restriction. Obstet.Gynecol. 1999;93:499-503.
14. Roh CR, Budhraja V, Kim HS, Nelson DM, Sadovsky Y. Microarray-based identification of differentially expressed genes in hypoxic term human trophoblasts and in placental villi of pregnancies with growth restricted fetuses. Placenta 2005;26:319-28.
15. Victoria A, Mora G, Arias F. Perinatal outcome, placental pathology, and severity of discordance in monochorionic and dichorionic twins. Obstet.Gynecol. 2001;97:310-15.
16. Schlembach D, Wallner W, Sengenberger R, Stiegler E, Mortl M, Beckmann MW et al. Angiogenic growth factor levels in maternal and fetal blood: correlation with Doppler ultrasound parameters in pregnancies complicated by pre-eclampsia and intrauterine growth restriction. Ultrasound Obstet.Gynecol. 2007;29:407-13.
17. Heyborne KD, Porreco RP. Selective fetocide reverses preeclampsia in discordant twins. Am.J.Obstet.Gynecol. 2004;191:477-80.
18. Pirhonen JP, Hartgill TW. Spontaneous reversal of mirror syndrome in a twin pregnancy after a single fetal death. Eur.J.Obstet.Gynecol.Reprod.Biol. 2004;116:106-07.