Functional magnetic resonance imaging (magnetization transfer) and stereological analysis of human placentae in normal pregnancy and in pre-eclampsia and intrauterine growth restriction

Magnetic resonance imaging provides a non-invasive method for investigating functional changes in the human placenta in vivo. In this study, we combine a magnetic resonance imaging technique called magnetization transfer with established stereological methods in order to analyse and compare placentae from normal (16-36 weeks of gestation) and complicated (pre-eclampsia, intrauterine growth restriction) pregnancies. Magnetization transfer provided an in vivo measure of the ratio of bound protons:total protons and stereological analysis of histological sections was used to estimate a residual:total volume ratio (the ratio of non-vascular volume to total placental volume). Statistical comparisons were drawn using tests for related samples (longitudinal data) or one-way analysis of variance (cross-sectional data). We found no significant differences in magnetization transfer between gestational age groups or between uncomplicated pregnancies and pregnancies complicated by pre-eclampsia or intrauterine growth restriction. In comparable groups of different subjects, stereological analyses also failed to demonstrate significant differences in residual:total volume ratios. We conclude that [a] the ratio of non-vascular volume:total placental volume does not alter between 16 and 36 weeks of normal gestation, and [b] this integrated response is also conserved in pre-eclampsia and intrauterine growth restriction.     

A longitudinal study of angiogenic (placental growth factor) and anti-angiogenic (soluble endoglin and soluble vascular endothelial growth factor receptor-1) factors in normal pregnancy and patients destined to develop preeclampsia and deliver a small for gestational age neonate.

INTRODUCTION: Accumulating evidence suggests that an imbalance between pro-angiogenic (i.e., vascular endothelial growth factor (VEGF) and placental growth factor (PlGF)) and anti-angiogenic factors (i.e., soluble VEGF receptor-1 (sVEGFR-1, also referred to as sFlt1)) is involved in the pathophysiology of preeclampsia (PE). Endoglin is a protein that regulates the pro-angiogenic effects of transforming growth factor beta, and its soluble form has recently been implicated in the pathophysiology of PE. The objective of this study was to determine if changes in maternal plasma concentration of these angiogenic and anti-angiogenic factors differ prior to development of disease among patients with normal pregnancies and those destined to develop PE (preterm and term) or to deliver a small for gestational age (SGA) neonate. METHODS: This longitudinal nested case-control study included 144 singleton pregnancies in the following groups: (1) patients with uncomplicated pregnancies who delivered appropriate for gestational age (AGA) neonates (n = 46); (2) patients who delivered an SGA neonate but did not develop PE (n = 56); and (3) patients who developed PE (n = 42). Longitudinal samples were collected at each prenatal visit, scheduled at 4-week intervals from the first or early second trimester until delivery. Plasma concentrations of soluble endoglin (s-Eng), sVEGFR-1, and PlGF were determined by specific and sensitive ELISA. RESULTS: (1) Patients destined to deliver an SGA neonate had higher plasma concentrations of s-Eng throughout gestation than those with normal pregnancies; (2) patients destined to develop preterm PE and term PE had significantly higher concentrations of s-Eng than those with normal pregnancies at 23 and 30 weeks, respectively (for preterm PE: p < 0.036 and for term PE: p = 0.002); (3) patients destined to develop PE (term or preterm) and those who delivered an SGA neonate had lower plasma concentrations of PlGF than those with a normal pregnancy throughout gestation, and the maternal plasma concentration of this analyte became detectable later among patients with pregnancy complications, compared to normal pregnant women; (4) there were no significant differences in the plasma concentrations of sVEGFR-1 between patients destined to deliver an SGA neonate and those with normal pregnancies; (5) patients destined to develop preterm and term PE had a significantly higher plasma concentration of sVEGFR-1 at 26 and 29 weeks of gestation than controls (p = 0.009 and p = 0.0199, respectively); and (6) there was no significant difference in the increment of sVEGFR-1 between control patients and those who delivered an SGA neonate (p = 0.147 at 25 weeks and p = 0.8285 at 40 weeks). CONCLUSIONS: (1) Changes in the maternal plasma concentration of s-Eng, sVEGFR-1, and PlGF precede the clinical presentation of PE, but only changes in s-Eng and PlGF precede the delivery of an SGA neonate; and (2) differences in the profile of angiogenic and anti-angiogenic response to intrauterine insults may determine whether a patient will deliver an SGA neonate, develop PE, or both.     

Raised maternal serum alpha-fetoprotein in the absence of fetal abnormality--placental findings. A quantitative morphometric study

Ten placentae from pregnancies proceeding to term from mothers who on routine screening at 16-18 weeks gestation were found to have raised serum AFP but no increase in amniotic fluid AFP and no fetal abnormality, were studied using morphometric techniques. The results were compared with 20 placentae from normal term pregnancies where the maternal serum AFP level was not elevated. The mean total placental volume, volume of parenchyma and villous surface area were increased in the placentae associated with a raised maternal serum AFP. More of these placentae were infarcted and the fetal-placental weight ratio was significantly lower. The hypothesis that elevation of maternal serum AFP level is related to the increase in placental size is addressed.     

Placental CLIC3 is increased in fetal growth restriction and pre-eclampsia affected human pregnancies

Chloride intracellular channel (CLIC) proteins constitute a subgroup of the glutathione-S-transferase (GSTs) superfamily. In humans, the CLIC family of proteins consists of six members, designated CLIC 1-6, which have a conserved C-terminal 240 residue module and one major transmembrane domain. CLIC proteins regulate fundamental cellular processes including regulation of chloride ion concentration, stabilization of cell membrane potential, trans-epithelial transport, regulation of cell volume and stimulation of apoptotic processes in response to cellular stress. Previously, we described the expression profile of a member of the CLIC family of proteins, CLIC3, in human placentae and fetal membranes. In the current study, we determined CLIC3 expression in placentae from pregnancies complicated with either fetal growth restriction (FGR, n=19), pre-eclampsia (PE, n=16) or both FGR and PE combined (n=12) compared to gestation-matched controls (n=13) using real-time PCR and a CLIC3 specific immunoassay. Significantly increased CLIC3 mRNA and protein were detected in placental extracts from pregnancies with FGR, PE and PE with FGR compared to controls. Our results suggest that increased expression of CLIC3 may play a role in abnormal placental function associated with the human pregnancy disorders FGR and PE.     

Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational age infants

An examination of the maternal vascular response to placentation shows that physiological changes in the placental bed normally extend from the decidua into the inner myometrium. In pre-eclampsia and in a proportion of pregnancies with small-for-gestational age infants (SGA) the physiological changes are restricted to the decidual segments alone. In addition, complete absence of physiological changes throughout the entire length of some spiral arteries is seen in pre-eclampsia and SGA. This new observation is confirmed in a study of basal plates of placentas from abnormal pregnancies. Intraluminal endovascular trophoblast may be seen in the placental bed spiral arteries in the third trimester in pre-eclampsia and SGA, a feature not seen beyond the second trimester in normal pregnancy. These findings point to a defect in the normal interaction between migratory trophoblast and maternal uterine tissues in pre-eclampsia and in SGA.     

Inadequate maternal vascular response to placentation in pregnancies complicated by pre-eclampsia and by small-for-gestational age infants

Summary. An examination of the maternal vascular response to placentation shows that physiological changes in the placental bed normally extend from the decidua into the inner myometrium. In pre-eclampsia and in a proportion of pregnancies with small-for-gestational age infants (SGA) the physiological changes are restricted to the decidual segments alone. In addition, complete absence of physiological changes throughout the entire length of some spiral arteries is seen in pre-eclampsia and SGA. This new observation is confirmed in a study of basal plates of placentas from abnormal pregnancies. Intraluminal endovascular trophoblast may be seen in the placental bed spiral arteries in the third trimester in pre-eclampsia and SGA, a feature not seen beyond the second trimester in normal pregnancy. These findings point to a defect in the normal interaction between migratory trophoblast and maternal uterine tissues in pre-eclampsia and in SGA.     

2D-ultrasound and endocrinologic evaluation of placentation in early pregnancy and its relationship to fetal birthweight in normal pregnancies and pre-eclampsia

Objectives

To study the relationships between 2D ultrasound measurements of placentation and maternal serum (MS) levels of PAPP-A, inhibin A and fβhCG in early pregnancy and subsequent fetal growth in pregnancies with a normal and abnormal outcome.

Study design

Prospective population-based cohort study of 301 pregnancies with a normal outcome, 18 with a pregnancy complicated by pre-term delivery (PTD) and 14 with subsequent pre-eclampsia (PE).

Main outcome measures

Basal placental surface area, placental thickness, ellipsivity and volume; MS PAPP-A and fβhCG at 11–13 + 6 weeks, MS inhibin A at 15–22 weeks and birthweight centile at delivery.

Results

In the normal group, the basal surface area showed a significantly (P < 0.001) positive correlation with placental thickness and placental ellipsivity. With the exception of placental ellipsivity, all other placental ultrasound parameters were significantly related with birthweight centile. Inhibin A showed a significant (P < 0.005) correlation with birthweight centiles. The basal plate surface area and MS PAPP-A were significantly (P < 0.01 and P < 0.001, respectively) lower and MS inhibin A significantly (P < 0.01) higher in PE than in controls. No changes were found in pregnancies complicated by PTD.

Conclusion

The basal plate surface area at 11–14 weeks reflects indirectly normal and abnormal placentation and development of the definitive placenta. Combined with MS PAPP-A and/or inhibin A levels this parameter could be useful in identifying from the end of the first trimester, pregnancies subsequently complicated with PE.     

Pre-eclampsia: associated with increased syncytial apoptosis when the infant is small-for-gestational-age

The present investigation was undertaken to study the association between placental apoptosis and pre-eclampsia, discriminating between pre-eclamptic pregnancies with appropriate-, and small-for-gestational-age (SGA), infants. Twenty pregnancies with pre-eclampsia and SGA (birth weight at or below -2 standard deviations) infants were selected in a retrospective study. Subsequently, corresponding numbers of gestational age-matched pre-eclampsia cases with appropriate-gestational-age (AGA) (birth weight at or above the 50% centile) infants and AGA controls without pre-eclampsia were selected. Formalin-fixed placental tissue was obtained from all groups. Apoptosis was assessed by a monoclonal antibody (M30), detecting a neoepitope of cytokeratin that is generated early in the apoptotic cascade. M30-positive cells were counted in villous and decidual/ basal plate tissue fields, and results were given as numbers of M30-positive cells per field. The study was performed blinded. Increased apoptosis was found in the syncytiotrophoblast layer in pre-eclampsia with SGA infants (0.14 apototic incidents per field of villous tissue, with 0.04-0.23 as the corresponding 25-75% inter quartile range (IQR) (P=0.05)). Syncytial apoptosis in the syncytial layer in the pre-eclampsia group with AGA infants was lower (0.09, IQR 0.03-0.15) and corresponded to the level detected among controls (0.06, IQR 0.03-0.17). Apoptosis in other placental cellular compartments did not differ between groups. The increased syncytial apoptosis found in placentas from pregnancies with SGA infants may either be due to specific mechanisms associated with pre-eclampsia complicated with growth restriction, or may simply reflect the presence of syncytiotrophoblast layer damage, regardless of underlying pathological condition.     

Weights of placentae from small-for-gestational age infants revisited

The objective of the study was to investigate the association between placental weight and birthweight in appropriate (AGA) and small for gestational age (SGA) infants. Placental weight, birthweight and their ratio in chromosomally normal singleton pregnancies with SGA (n=1569) and AGA (n=15 047) infants were compared, and their determinants were studied by logistic regression. SGA infants had 24 per cent smaller placentae than AGA infants when gestational age was used as a covariate. Placental actual weight was also lower in SGA infants than in AGA infants of the same birthweight (P< 0.001). SGA infants had smaller placentae than the controls, suggesting that fetal growth depends on the actual weight of the placenta. Future studies should evaluate whether growth restriction could be reversed by therapeutic approaches increasing placental weight.     

Placental superoxide is increased in pre-eclampsia

One of the current hypotheses on the pathophysiology of pre-eclampsia (PE) states that the placenta secretes one or more cytotoxic factors resulting in maternal endothelial dysfunction. Among the candidate factors are the products of increased oxidative stress. Although there is circumstantial evidence of such an increase, direct evidence is still lacking. Electron paramagnetic spin trap resonance (EPR), the most direct method to detect free radicals in tissues, was used to measure superoxide levels in placentae from normal pregnancies (n=13) and pregnancies complicated by PE (n=10). The superoxide level was significantly increased in the placental tissue of pre-eclamptic women. Moreover, upon inhibition of Cu-Zn superoxide dismutase (SOD) activity the relative increase of the superoxide levels was significantly smaller in the placentae from the PE patients, implying decreased basal Cu-Zn SOD activity. These findings lend direct support to the hypothesis that oxidative stress in placental tissue is increased in PE.     

First trimester uterine Doppler and three-dimensional ultrasound placental volume calculation in predicting pre-eclampsia

OBJECTIVE: To compare the efficacy of uterine artery Doppler velocimetry and three-dimensional ultrasound placental volume calculation alone or in combination in predicting at 11-14 weeks of gestation those pregnancies who will develop pre-eclampsia. STUDY DESIGN: This was a prospective study of 348 nulliparous women scheduled for a routine prenatal ultrasound examination at 11-14 weeks. Color and pulsed wave Doppler was used to obtain uterine artery flow velocity waveforms transabdominally and the mean pulsatility index (PI) of the uterine arteries was calculated. The placental volume was measured by three-dimensional ultrasound using the virtual organ computer-aided analysis. Outcome variables considered were pre-eclampsia and pre-eclampsia requiring delivery <32 weeks. RESULTS: Pre-eclampsia developed in 4.1% of the patients studied and in 1.7% a delivery before 32 weeks was required. Placental volume resulted significantly lower in pregnancies who will develop pre-eclampsia (t=4.636, p<0.003) and this was particularly evident in those pregnancies delivering <32 weeks (t=9.704, p<0.0002). No relationship was found between placental volume and mean uterine artery PI (r=-0.08, p=0.327). Uterine artery PI and placental volume showed similar sensitivities in predicting pre-eclampsia (50% vs. 56%) and pre-eclampsia with delivery <32 weeks (66.7% vs. 66.7%). The combination of uterine artery PI and placental volume gave better results when compared to the single use of one of these parameters (pre-eclampsia sensitivity 68.7%, pre-eclampsia requiring delivery <32 weeks 83.3%). CONCLUSIONS: The combination of abnormal uterine artery Doppler and low placental volume at 11-14 weeks achieves better results than does either test alone in the prediction of pre-eclampsia.     

Morphometric placental villous and vascular abnormalities in early- and late-onset pre-eclampsia with and without fetal growth restriction

OBJECTIVE: To evaluate placental morphology in pregnancies complicated by early- and late-onset pre-eclampsia (PET) with and without fetal growth restriction (FGR) using stereological techniques. DESIGN: A total of 69 pregnant women were studied. Twenty women had pregnancies complicated by PET, 17 by FGR and 16 by both PET and FUR; the remaining 16 were from gestational-age-matched controls. Each group was further classified into early onset (<34 weeks) and late onsets (>34 weeks) based on gestational ages. SETTING: NPIMR at Northwick Park and St Marks Hospital. POPULATION: placentae from pregnant women. METHODS: Formalin-fixed, wax-embedded sections stained with anti-CD34 antibodies and counterstained with haematoxylin. MAIN OUTCOME MEASURES: Volumes, surface areas, lengths, diameters and shape factors of the villous tissues and fetal vasculature in the intermediate and terminal villi of all the groups studied. RESULTS: Terminal villi volume and surface area were compromised in early-onset PET cases, late-onset PET had no impact on peripheral villi or vasculature features. The morphology of the vascular and villous subcomponents in the intermediate and terminal villi was significantly influenced by late-onset FGR, whereas early-onset FGR caused a reduction in placental weight. Length estimates were not influenced by PET, FGR or age of onset. Intermediate arteriole shape factor was significantly reduced in late-onset FGR. CONCLUSIONS: Isolated early-onset PET was associated with abnormal placental morphology, but placentas from late-onset PET were morphologically similar to placentas from gestational-age-matched controls, confirming the existence of two subsets of this condition and supporting the hypothesis that late-onset PET is a maternal disorder and not a placental disease.     

Placental morphogenesis in pregnancies with Down's syndrome might provide a clue to pre-eclampsia

Insufficient perfusion of placenta in pre-eclampsia is commonly associated with oxidative stress leading to increased superoxide formation and reduced invasion of uterine spiral arteries by differentiated migratory cytotrophoblasts. The superoxide dismutase (SOD) level, responsible for eliminating toxic superoxides, drops significantly in pre-eclampsia. On the contrary, the SOD synthesis increases dramatically, compared to that of normal placenta, in pregnancies with trisomy 21 (T21) fetus. However, despite a low level of placental hypoplasia, the overall perfusion of T21 placentae is comparable to that of normal pregnancy. In the light of recent reports on alternative modes of SOD function and factors regulating pathways of cytotrophoblast differentiation, here we have attempted to reconcile the two seemingly disparate pregnancy conditions and suggest that trisomy 21 pregnancies might provide new insight into our understanding of placental morphogenesis in pre-eclampsia.     

Relative abundance of placental pro-atrial natriuretic factor mRNA in normal pregnancy and pre-eclampsia

Atrial natriuretic factor (ANF), produced by cytotrophoblast cells of the human placenta, may be involved in the regulation of uteroplacental blood flow. Pre-eclampsia is associated with maternal hypertension and reduced uteroplacental perfusion. The relationship between pre-eclampsia and placental production of ANF is not known. This study measured pro-ANF mRNA levels by Northern blot analysis in placentae delivered by caesarean section at preterm and term gestations from women with normotensive and pre-eclamptic pregnancies and found no significant difference between pre-eclampsia and normal pregnancy at either gestation. This result suggests that placental production of ANF is not altered at the pretranslational level during pre-eclampsia.     

An immunohistochemical study of type I insulin-like growth factor receptors in the placentae of pregnancies with appropriately grown or growth restricted fetuses.

Insulin-like growth factors (IGFs) and their receptors in the fetus are essential for growth and postnatal survival but the role of maternal IGFs is less well understood. Animal and in vitro evidence suggests that maternal IGF-I may have important effects on placental function. Recent work in humans suggests that although there is no relationship between maternal serum IGF-I and normal fetal growth, levels are low in pregnancies complicated by fetal growth restriction due to placental dysfunction. A prospective and observational study was undertaken of the distribution and concentration of placental type I IGF receptors (IGF-IR) in women with small for gestational age (n=26) or appropriately grown (n=14) fetuses. Women were scanned biweekly from 24 weeks to delivery and cases (birthweight <5th centile) were assigned to two groups: 'fetal growth restriction' (FGR; umbilical artery pulsatility index [UAPI] > +2 s.d.; n=16) and 'normal small for gestational age' (SGA; normal UAPI, growth velocity and amniotic fluid; n=10). Immunohistochemistry of the IGF-IR was performed on formol saline-fixed placental biopsies obtained at delivery. In control pregnancies IGF-IR were present in villous endothelium and stroma, trophoblast and decidua and their distribution and density were unchanged in both SGA and FGR pregnancies. We hypothesize that a therapeutic elevation of maternal IGF-I in FGR pregnancy might lead to enhanced placental function and so fetal growth. Our findings of normal localization and density of placental IGF-IR in FGR encourage us to extend our work to look at the effects of maternal IGF-I on the transport of glucose and amino acids.     

Maternal vascular lesions in placentae of small-for-gestational-age infants

Placentae from 140 term pregnancies were studied. Seventy-four were from uncomplicated pregnancies in which the neonates were within the normal weight range for their gestational age. The remaining 66 placentae were from pregnancies whose infants were small for gestational age (SGA). In eight cases of this latter group a curettage of placental bed was performed during caesarean section. Maternal arterial vessels with absence of trophoblastic migration were observed in basal decidua and basal plate, and acute atherosis in parietal decidua, basal decidua and basal plate, all of them in cases of the SGA group. Furthermore, chronic vasculitis-like lesions were observed in the parietal decidua of three cases from the SGA group and in two others of the control group. No vascular lesions were found in cases of the control group when an infant's birthweight was above the 25th percentile of the normal ponderal range. Atheromatous-like lesions have been described in placental bed arteries in pre-eclampsia, systemic lupus erythematosus and SGA infants: this type of vasculopathy has also been described in rejection of renal transplants. Moreover, lesions similar to those found in chronic vasculitis were also described in the latter pathological entity. It is suggested that these vasculopathies may represent different steps of the same lesion. On the other hand, they may also be the expression of a maternal immunological attack on placental tissues causing a deficit of placentation, low birthweight being the consequence of this deficit.     

Novel placental ultrasound assessment: Potential role in pre-gestational diabetic pregnancy

Objectives

Management of women with pre-gestational diabetes continues to be challenging for clinicians. This study aims to determine if 3D power Doppler (3DPD) analysis of placental volume and flow, and calculation of placental calcification using a novel software method, differ between pregnancies with type 1 or type 2 diabetes and normal controls, and if there is a relationship between these ultrasound placental parameters and clinical measures in diabetics.

Methods

This was a prospective cohort study of 50 women with diabetes and 250 controls (12–40 weeks gestation). 3DPD ultrasound was used to evaluate placental volume, vascularisation index (VI), flow index (FI) and vascularisation-flow index (VFI). Placental calcification was calculated by computer analysis. Results in diabetics were compared with control values, and correlated with early pregnancy HbA1c, Doppler results and placental histology.

Results

Placental calcification and volume increased with advancing gestation in pre-gestational diabetic placentae. Volume was also found to be significantly higher than in normal placentae. VI and VFI were significantly lower in diabetic pregnancies between 35 and 40 weeks gestation. A strong relationship was seen between a larger placental volume and both increasing umbilical artery pulsatility index and decreasing middle cerebral artery pulsatility index. FI was significantly lower in cases which had a booking HbA1c level ≥6.5%. Ultrasound assessed placental calcification was reduced with a histology finding of delayed villous maturation. No other correlation with placental histology was found.

Conclusions

This study shows a potential role for 3D placental evaluation, and computer analysis of calcification, in monitoring pre-gestational diabetic pregnancies.     

Measurement of the mass of syncytiotrophoblast in a range of human placentae using an image analysing computer

Placentae from normal term pregnancies, from "normal" preterm pregnancies and from pregnancies complicated by pre-eclampsia have been analysed by computerized histometry. An immunoperoxidase technique was used to stain specifically human placental lactogen in the syncytiotrophoblast. The percentage and mass of syncytiotrophoblast were measured using an image analysing computer. The percentage of syncytiotrophoblast was found to be remarkably constant throughout gestation. Strong evidence was found that a linear relationship exists between syncytiotrophoblast mass and gestation period and that on average pre-eclampsia placentae have a lower percentage and a reduced mass of syncytiotrophoblast.     

Maternal cardiac function and uterine artery Doppler at 11-14 weeks in the prediction of pre-eclampsia in nulliparous women

Objective  To assess maternal cardiac function in nulliparous women in the first trimester of pregnancy and evaluate its potential role for predicting pre-eclampsia and small for gestational age (SGA).
Design  Prospective, observational, cross-sectional study.
Setting  Maternity unit of a teaching hospital.
Population  Nulliparous women with singleton pregnancies presenting consecutively for routine antenatal care ( n = 534).
Methods  Two-dimensional and M-mode echocardiography and uterine artery Dopplers were carried out at 11-14 weeks.
Main outcome measures  Cardiac output (CO), stroke volume (SV), mean arterial pressure (MAP), total vascular resistance and uterine artery pulsatility index (UAPI) were compared in four outcome groups according to the development of pre-eclampsia and/or SGA.
Results  Compared with the normal outcome group ( n = 457), in those with pre-eclampsia but not SGA ( n = 8), CO and MAP were increased; in the group with pre-eclampsia and SGA ( n = 19) MAP, TRP and UAPI were increased and in the group with SGA but no pre-eclampsia ( n = 50) total peripheral resistance and UAPI were increased. Independent predictors of pre-eclampsia were MAP, SV and UAPI and of SGA SV and UAPI.
Conclusions  Alterations in maternal cardiac function and UAPI are observed in the first trimester of pregnancy in nulliparous women that subsequently develop pre-eclampsia and/or SGA.