Clinicopathologic Study of Placentas From Women With a Fontan Circulation

Abstract

Background: Pregnant women with a Fontan circulation have a high risk of obstetric complications, such as preterm delivery and small for gestational age (SGA), which may be affected by low blood flow to the placenta and hypoxia. This study investigated placental pathology in a Fontan circulation.

Methods and Results: Eighteen pregnancies in 11 women with a Fontan circulation were reviewed. Pregnancy outcomes showed 9 miscarriages and 9 live births, with 4 preterm deliveries. Five neonates were SGA (<5th percentile). Eight placentas from live births in 7 women were available for the study. Five placentas had low weight placenta for gestational age, and 7 grossly showed a chronic subchorionic hematoma. Histological examination revealed all placentas had some form of histological hypoxic lesions: maternal vascular malperfusion in 7, fetal vascular malperfusion in 1, and other hypoxia-related lesions in 8. Quantitative analyses, including immunohistochemistry (CD31, CD68, and hypoxia inducible factor-1α antibodies) and Masson’s trichrome staining, were also performed and compared with 5 control placentas. Capillary density and the area of fibrosis were significantly greater in placentas from women with a Fontan circulation than in control placentas.

Conclusions: Placentas in a Fontan circulation were characterized by a high frequency of low placental weight, chronic subchorionic hematoma, and constant histological hypoxic changes, which could reflect altered maternal cardiac conditions and lead to poor pregnancy outcomes.

The success of treatment for congenital heart disease (CHD) has increased the number of pregnancies complicated by CHD. CHD can interfere with the normal increases of 40–50% in plasma volume and 30–50% in cardiac output during pregnancy.¹ Studies have shown that the risk of obstetric complications in these pregnancies is high, with reported rates of miscarriage, preterm delivery, and small for gestational age (SGA) of 19.4%, 12.0%, and 25.0%, respectively.² The Fontan procedure is a palliative operation for patients with CHDs who lack biventricular circulation, and complications of the procedure include high central venous pressure, low cardiac output, and hypoxia. High rates of miscarriage (45.0–54.8%), preterm delivery (59.0–70.4%), and SGA babies (20.0–55.6%) have been reported among pregnant women who have a Fontan circulation.^3,4

The placenta is an organ that connects the mother and fetus and is essential for successful pregnancy and fetal health. Throughout pregnancy, the placenta has many roles, including in gas exchange, immunity, nourishment, and hormone production. Abnormal placental function is a cause of pregnancy complications such as miscarriage, preterm delivery, pre-eclampsia (PE), and SGA.

However, little is known regarding the effect of the maternal Fontan circulation on placental histology. Maternal perfusion pressure is crucial to placental diffusion and transfer. We hypothesized that in women with a Fontan circulation, the maternal cardiac condition would cause placental hypoxia, which, in turn, could be an important cause of poor obstetric outcomes.

The aim of this study was to investigate placental pathology from pregnancies in women with a Fontan circulation, with a particular focus on hypoxic changes.

Methods

Study Population

The study subjects were 11 women with a Fontan circulation who had 18 pregnancies registered at the National Cerebral and Cardiovascular Center (NCVC) in Suita, Japan, between January 2006 and September 2019. Eight placentas from 7 women and chorionic villi from 6 miscarriages in 4 women were also examined. The study was approved by the Institutional Research Ethics Board of the NCVC, Suita, Japan (M30-161).

Data Collection

Data were obtained from the medical records and samples stored in the NCVC Biobank. The demographic and clinical data of mothers and babies are presented in Table 1. Adverse cardiovascular events were defined as heart failure, arrhythmia requiring medication and/or any intervention, worsening of atrioventricular valve regurgitation (AVVR) requiring termination of pregnancy, thromboembolism, and cardiac arrest or death.

Table 1. Baseline Characteristics of 8 Deliveries in 7 Women With a Fontan Circulation

Delivery case no.	Age (years)	BMI (kg/m2)	CHD	Prior surgery (age [years])	Systemic ventricle	SVEF (%)	AVVR	NYHA FC	Peak V̇O2 (mL/kg/min)	SpO2 (%)	CVPB (mmHg)	CIB (L/min/m2)	Medication		Delivery		Indication for CS	BW (g)/ percentile20	SCH
													Before pregnancy	During pregnancy	Gestational age	Mode
1	27	18.8	Right iso SV	TCPC (16)	RV	68A	Moderate	II	NA	96	10	2.76	ASA DA	H (–22w)	30w3d	CS	pPROM	1,450/44.1	+
2–1	28	22.3	TA	APC (11) TCPC (23)	LV	72A	Mild	II	22.3	94	11	2.75	ASA DA AAD	ASA H (–20w) B DA	35w6d	CS	NRFS	1,760/2.9	+
2–2	34	21.6	TA	APC (11) TCPC (23)	LV	55B	Trivial	II	18.6	96	14	2.7	ASA ACEI/ARB DA AAD	ASA B DA	33w0d	CS	NRFS	1,451/3.1	−
3	29	19.9	MS Hypo LV	APC (5)	RV	72A	Moderate	I	25.6C	94	NA	NA	–	H (–28w)	36w4d	CS	Worsening TR	2,456/36.8	−
4	19	20.7	Right iso SV	TCPC (3)	RV	60A	Mild	I	NA	91	10	2.25	ASA	ASA	37w0d	VD	–	1,620/0.2	−
5	33	23.5	Left iso DILV	TCPC (15)	LV	57B	Trivial	I	27.4	96	16	2.19	Warfarin ASA ACEI/ARB DA	ASA H DA	37w0d	CS	Breech	2,312/23.9	−
6	28	20.8	TA	APC (8) TCPC (23)	LV	58B	Mild	II	16	95	8	2.51	ASA	ASA (–27w) H (27w–)	37w5d	CS	NRFS	1,726/0.1	−
7	28	23.9	DIRV	TCPC (7)	RV	44B	Mild	II	20.5	95	9	3.67	Warfarin ASA	ASA H	37w6d	CS	Delivery arrest	1,902/0.4	−

^AEstimated by echocardiography or cardiac magnetic resonance imaging. ^BMeasured by a catheterization study. ^CExamined 14 months after delivery. AAD, antiarrhythmic drugs; ACEI, angiotensin-converting enzyme inhibitor; APC, atriopulmonary connection; ARB, angiotensin II receptor blocker; ASA, acetylsalicylic acid; AVVR, atrioventricular valve regurgitation; B, β-blocker; BMI, body mass index; BW, birth weight; CHD, congenital heart disease; CI, cardiac index; CS, cesarean section; CVP, central venous pressure; d, days; DA, diuretic agent; DILV, double inlet left ventricle; DIRV, double inlet right ventricle; H, heparin; Hypo LV, hypoplastic left ventricle; Left iso, left isomerism; LV, left ventricle; MS, mitral stenosis; NA, not available; NRFS, non reassuring fetal status; NYHA FC, New York Heart Association functional class; pPROM, preterm premature rupture of membranes; Right iso, right isomerism; RV, right ventricle; SV, single ventricle; SVEF, systematic ventricular ejection function; TA, tricuspid atresia; TCPC, total cavopulmonary connection; TR, tricuspid regurgitation; VD, vaginal delivery; w, weeks.

Placental Pathologic Examinations

The placentas were weighed fresh after removal of umbilical cords and membranes. In the gross examination, we evaluated the degree of chronic subchorionic hematoma (SCH) and the presence of disorders of membrane development. Placental histologic examination was performed using a standard protocol. At least 4 samples were taken from each placenta: 1 at the cord insertion, 2 from the central tissue that appeared normal on gross examination (if the appearance was grossly abnormal, additional samples were obtained), and 1 from the umbilical cord and membrane roll. Specimens were inspected for evidence of placental hypoxia. Placental lesions were classified according to the criteria of the 2014 Amsterdam Placental Workshop Group⁵ and previous studies of placental hypoxia^6,7 as follows:

• Lesions associated with maternal vascular malperfusion (MVM): villous infarcts, increased syncytial knots, villous agglutination, increased intervillous fibrin deposition, distal villous hypoplasia, accelerated villous maturation, and decidual vasculopathy (acute atherosis and mural hypertrophy)

• Lesions associated with fetal vascular malperfusion (FVM): thrombosis of the chorionic plate and stem villous vessels and avascular villi

• Lesions associated with placental hypoxia other than MVM and FVM: villous stromal fibrosis, hypervascularity, chorangiosis, perivillous fibrin deposition, laminar necrosis and delayed villous maturation

Hypervascularity was defined as the presence of 6–9 capillaries in the terminal villi of a lesion, whereas chorangiosis was defined as ≥10 capillaries in >10 terminal villi in several areas of the placenta visualized using a ×10 objective.⁸ In cases of miscarriage, we also performed a histological analysis of the chorionic villus from the products of conception.

Quantifying Immunohistological Findings and Villous Fibrosis

Of the 8 placentas, 7 placentas from 7 women with a Fontan circulation had additional tissue sections available, provided by the NCVC Biobank, to quantify the immunohistological findings and villous fibrosis. These placentas were compared to 5 control placentas obtained from planned cesarean deliveries at term without maternal or fetal complications (all women were non-smokers). Control placentas, delivered by healthy women without any complications at NCVC, were also provided by NCVC Biobank.

Formalin-fixed and paraffin-embedded sections were stained by immunohistochemistry with anti-CD 31 antibody (M0823; DAKO) for endothelial cells and anti-CD 68 antibody (M0876; DAKO) for macrophages termed Hofbauer cells in the villi, and were examined using a Leica Bond-III autostainer (Leica Biosystems, Wetzlar, Germany) as described previously.⁹ Immunohistochemistry using anti-hypoxia inducible factor (HIF)-1α antibody (ab51608; Abcam) to show evidence of hypoxia was outsourced (Applied Medical Research). Briefly, paraffin-embedded sections were deparaffinized and rehydrated. Antigen retrieval was conducted with EDTA buffer (pH 9.0) in a water bath. After antigen retrieval at 98℃, the sections were treated with 3% H₂O₂ for 10 min and then incubated with anti-HIF-1α (diluted 1 : 100) at 4℃ overnight. The sections were then washed and incubated with diaminobenzidine solution. Masson’s trichrome stain was used to evaluate villous stromal fibrosis.

Five random ×10 objective field images per slide were taken, and the number of capillaries was counted and the total villous area was determined using ImageJ software (National Institutes of Health, Bethesda, MD, USA). At first, the images were converted to 8-bit size and binarized using the ImageJ thresholding algorithm. Then, the number of capillaries was counted using the ImageJ command of analyzing particles. The total villous area was measured using the ImageJ command of measuring the area after applying the erosion processes 3 times to binarized images. Capillary density was then calculated as the number of capillaries divided by the total villous area. Five random ×40 objective field images per slide were taken and the number of macrophages was counted by 2 observers (T.Y.-K., K.O.-O.). The observers scored HIF-1α staining as follows: 0, no staining; 1, mild staining; 2, moderate staining; 3, intense staining. The area of fibrosis was analyzed for 20 villi cut along the short axis of ×20 objective field images, and the percentage area of fibrosis was determined automatically using Aperio ImageScope (Leica Biosystems).

Statistical Analysis

Data with a normal (Gaussian) distribution are presented as the mean±SD and data that were not normally distributed are presented as the median with minimum–maximum. Student’s t-test was used to compare normally distributed continuous variables and the Mann-Whitney U test was used for comparisons of non-parametric variables. Pearson’s correlation coefficient was used to evaluate correlations between capillary density and the area of fibrosis. Statistical analyses were performed with GraphPad Prism 7. Two-sided P<0.05 was considered statistically significant.

Results

Of 18 pregnancies in 11 women in the present study, 9 pregnancies in 6 women ended before 22 weeks of gestation. From the 9 continuing pregnancies, 8 placentas were available for detailed clinical and histopathological description, and 7 placentas were available for quantification analysis of immunohistological findings and villous fibrosis. Of the 9 miscarriages, the chorionic villi of 6 miscarriages in 4 women for which tissue was available were evaluated histologically.

Maternal Cardiac Characteristics of the 8 Deliveries

Table 1 provides baseline characteristics of the 8 deliveries whose placentas were available for histological examination. Five of 8 women showed decreased systemic saturation (≤95%) levels at rest. Regarding systemic ventricular function, all but 1 woman had preserved ejection function (≥55%), but 3 of the 7 women in whom cardiac index (CI) was measured had a decreased CI, and 3 of the remaining 4 women had a CI just above the lower limit of the normal range (2.6–4.2 L/min/m²). Furthermore, 4 of 6 women had decreased peak V̇O₂. Because of the Fontan circulation, central venous pressure was elevated, with a median value of 10 mmHg. Women taking antiplatelet agents before pregnancy continued with this medication, except for 1 woman (Delivery case 1 in Table 1), who stopped taking the antiplatelet agent because of SCH identified on an ultrasound scan. Women taking warfarin before pregnancy changed from warfarin to prophylactic doses of unfractionated heparin during pregnancy. Women who were not taking warfarin before pregnancy were started on heparin only if there was an increased risk of thromboembolism (e.g., due to arrhythmia or hospitalization due to preterm delivery). Three women took a diuretic agent and 2 women took a β-blocker during pregnancy.

Cardiovascular Complications of the 8 Pregnancies With Placental Histology

Two women had cardiovascular events during pregnancy. One (Delivery case 2-1) experienced atrial tachycardia, which required defibrillation and medication at 14 weeks of gestation. Because this woman also had sick sinus syndrome, she experienced extreme bradycardia with antitachyarrhythmic medications and proceeded to pacemaker insertion at 20 weeks of gestation. Another woman (Delivery case 3) suffered worsening AVVR at 36 weeks of gestation, and underwent an emergency cesarean section. Cardiovascular events occurred after delivery in 4 women: atrial tachycardia 2 days after delivery (n=1; Delivery case 1); intraventricular thrombosis 8 days after delivery (n=1; Delivery case 2-2); and heart failure 4 weeks (n=1; Delivery case 5) and 6 weeks (n=1; Delivery case 7) after delivery. There was no significant factor predicting maternal cardiovascular complications.

Obstetrics and Neonatal Outcomes of the 8 Pregnancies With Placental Histology

In the 8 pregnancies with placental histology, the median gestational age at delivery was 36 weeks. There were 4 preterm deliveries, and 4 women delivered at 37 weeks. Two (25.0%) women had SCH (Delivery cases 1 and 2-1) and gestational diabetes (Delivery cases 2-1, 2-2), and 1 (12.5%) woman had hypertensive disorders of pregnancy (Delivery case 2-2). All neonates, including the 5 who were SGA (62.5%), survived to hospital discharge without complications. None of them had CHD. Of the 8 women, 5 were examined by cardiopulmonary exercise testing before pregnancy; the median interval between the examination and pregnancy was 15 months (minimum–maximum 5–39) and 1 case was examined 14 months after delivery. The peak V̇O₂ in the mothers of SGA infants was lower than that in mothers of appropriate for gestational age infants (19.35±2.69 vs. 26.6±1.27 mL/kg/min; P=0.026).

Gross and Histological Findings of the 8 Placentas

Five placentas had low placental weight for gestational age (<25th percentile) and had a low fetal/placental ratio (<25th percentile; Table 2). The presence of a low-weight placenta and SGA status was concordant in 6 of 8 neonates. Of the remaining 2 neonates, 1 baby with SGA had a normal-weight placenta and the other had a low-weight placenta but a normal birth weight. Of the 8 placentas, all but 1 showed chronic SCH (massive, n=1; multifocal, n=6); 4 were circummarginate and 1 was circumvallate. Histologically, all specimens showed evidence of placental hypoxia (MVM, n=7; FVM, n=1). None showed decidual vasculopathy. All 8 placentas showed hypervascularity, and 7 had chorangiosis and villous stromal fibrosis (Table 2; Figure 1).

Table 2. Placental Findings of 8 Deliveries in 7 Women With a Fontan Circulation

	Delivery case no.
	1	2–1	2–2	3	4	5	6	7
Circummarginate or circumvallate	Circumvallate	–	–	Circummarginate	–	Circummarginate	Circummarginate	Circummarginate
Chronic subchorionic hematoma	Focal	Focal	Focal	Massive	–	Focal	Focal	Focal
Placental weight (g)/percentile21	365/50–75th	465/50–75th	220/3–5th	500/50–75th	288/<3th	285/<3th	242/<3th	375/10–25th
Fetal-placental weight ratio/percentile21	3.98/<25th	3.84/<3th	6.59/>50th	4.91/<10th	5.62/<25th	8.11/>75th	7.13/>50th	5.07/<5th
Maternal vascular malperfusion
Villous infarcts	+	−	−	+	−	−	+	−
Increased syncytial knots	+	+	+	+	+	−	+	+
Villous agglutination	−	−	+	−	−	−	+	+
Increased intervillous fibrin deposition	−	+	−	+	+	−	−	+
Distal villous hypoplasia	−	−	−	−	−	−	+	−
Accelerated villous maturation	−	−	−	−	−	−	−	−
Decidual vasculopathy	−	−	−	−	−	−	−	−
Fetal vascular malperfusion
Fetal vessels thrombosis	−	−	−	−	−	−	+	−
Avascular villi	−	−	−	−	−	−	−	−
Other
Villous stromal fibrosis	+	+	+	+	+	+	−	+
Hypervascularity	+	+	+	+	+	+	+	+
Chorangiosis	+	+	+	+	+	+	+	−
Perivillous fibrin deposition	+	−	+	+	+	−	−	+
Laminar necrosis	−	−	+	−	+	−	−	−
Delayed villous maturation	−	+	−	−	−	−	−	−

Delivery cases 2–1, 2–2, 4, 6, and 7 were babies born small for gestational age.

Figure 1.

Representative images showing findings in placental specimens. (A) Control placenta (hematoxylin-eosin [H&E] staining). Scale bar, 200 µm. (B) Increased number of syncytial knots (H&E staining). Note the aggregation of syncytiotrophoblastic nuclei in tight clusters. Scale bar, 100 µm. (C) Villous stromal fibrosis (H&E staining). Extracellular matrix is increased in chorionic villi. Scale bar, 50 µm. (D) Perivillous fibrin deposition (H&E staining). Large deposits of fibrin are seen surrounding the villi. Scale bar, 200 µm. (E) Chorangiosis (H&E staining). More than 10 terminal villi with ≥10 capillaries are seen in several areas of the placenta. Scale bar, 100 µm. (F) Representative histological findings of placental specimens after Masson’s trichrome staining. Scale bar, 100 µm. (G–I) Representative images of immunohistochemical findings of placental specimens. (G) Endothelial cells are for CD31. Scale bar, 200 µm. (H) Macrophages stained for CD68. Scale bar, 50 µm. (I) Moderate hypoxia-inducible factor-1α expression in the nuclei of trophoblast cells. Scale bar, 20 µm.

Immunohistological Findings and Stromal Fibrosis

The quantitative findings of 7 of the 8 placentas in women with a Fontan circulation and 5 control placentas are presented in Table 3. Placentas of the study group had significantly greater capillary density (537 [minimum–maximum 471–639] vs. 424 [minimum–maximum 292–543] number/mm²; P=0.03) and area of fibrosis (44.3 [minimum–maximum 39.2–50.8] vs. 32.4 [minimum–maximum 29.1–43.8] %; P=0.01) than control placentas, and there was a significant correlation between capillary density and area of fibrosis (P=0.0007; Figure 2). Although there were no significant differences in HIF-1α staining scores in the villi between placentas of women with a Fontan circulation and control placentas (1.0 [minimum–maximum 0–2] vs. 0.0 [minimum–maximum 0–1] P=0.07), 6 of 7 placentas in women with a Fontan circulation expressed HIF-1α, with 3 have a score 2. The number of macrophages in the villous area was not significantly different between placentas from women with a Fontan circulation and control placentas (8.2 [minimum–maximum 3.4–12.6] vs. 5.8 [minimum–maximum 5.4–10.2] P=0.66).

Table 3. Immunohistological and Villous Fibrosis Data in 7 of 8 Placentas of the Study Group (Case 2–2 Not Available) and in 5 Placentas in the Control Group

	Delivery case no.							Control	Control	Control	Control	Control
	1	2–1	3	4	5	6	7
Capillary density (number/mm2)	537±40	581±121	527±108	639±76	476±67	563±44	471±61	444±27	424±49	543±64	292±73	378±45
Number of macrophages	3.4±2.0	6.6±4.1	9.2±1.9	8.2±3.1	12.6±5.6	8.2±3.7	7.4±2.9	5.8±1.4	9.2±3.1	5.6±3.6	5.4±3.9	10.2±3.8
HIF-1α score	2	2	1	1	1	2	0	1	0	0	1	0
Area of fibrosis (%)	41.9±4.9	41.0±5.8	39.2±7.2	50.8±9.2	45.9±6.9	44.3±10.8	46.1±7.1	36.9±8.8	32.4±6.3	43.8±8.2	29.1±5.7	30.5±3.9

Data are shown as mean±SD. HIF, hypoxia-inducible factor.

Figure 2.

Correlation between capillary density and area of fibrosis in placentas from women with a Fontan circulation (F) and control placentas (C).

Interaction Between Hemodynamic Features and Placental Pathology

Most placentas in women with a Fontan circulation showed both MVM and other hypoxic lesions, but 1 did not show MVM (Delivery case 5). This woman had the highest peak V̇O₂ and systemic saturation among the cases studied.

Interaction Between Placental Pathology and Obstetric Outcome

Both MVM lesions and other hypoxic lesions were seen in all 5 cases of SGA. Specifically, among the other hypoxic lesions, all cases showed hypervascularity, and 4 of 5 cases showed villous stromal fibrosis and chorangiosis.

Miscarriage

Nine pregnancies in 6 women resulted in miscarriage: 7 at very early gestation (including 1 twin pregnancy), and 1 each at 12 and 17 weeks of gestation. There were no therapeutic terminations. Of the 6 women, 3 miscarried twice, and 2 of these 3 did not have a live birth during the study period. The chorionic villi in 6 miscarriages were examined and the baseline characteristics are presented in Table 4. In 1 miscarriage at 17 weeks of gestation, SCH was present from 8 weeks of gestation, fetal weight was normal, and placental weight was large for gestational age. Histologically, villous stromal fibrosis, perivillous fibrin deposition and chronic SCH were observed. There was no evidence of infection. The histological findings in the chorionic villi in the other 5 miscarriages were similar to those seen in miscarriages in mothers with normal hearts.

Table 4. Baseline Characteristics of 4 Women With 6 Miscarriages

Case	Corresponding delivery case no.	Miscarriage week	Age (years)	BMI (kg/m2)	CHD	Prior surgery (age [years])	Systemic ventricle	SVEF (%)	AVVR	NYHA FC	Peak V̇O2 (mL/kg/min)	SpO2 (%)	CVPB (mmHg)	CIB (L/min/m2)	Medication before pregnancy
1	2–1, 2–2	7	30	21.4	TA	APC (11) TCPC (23)	LV	73A	Mild	II	18.6	94	13	2.75	ASA DA
2	6	5 8	25 26	20.8	TA	APC (8) TCPC (23)	LV	58B	Mild	II	21.7	95	8	3.25	–
3	–	9	31	18.8	Right iso CAVC	TCPC (1)	RV	62B	Mild	II	18.5	97	7	2.34	B AAD
4	–	17 8	28 29	24.3	TA	APC (3) TCPC (21)	LV	60A	Mild	I	18.1	95	13	2.63	ASA DA

^AEstimated by echocardiography or cardiac magnetic resonance imaging. ^BMeasured by a catheterization study. AAD, antiarrhythmic drugs; APC, atriopulmonary connection; ASA, acetylsalicylic acid; AVVR, atrioventricular valve regurgitation; B, β-blocker; BMI, body mass index; CAVC, common atrioventricular canal; CHD, congenital heart disease; CI, cardiac index; CVP, central venous pressure; DA, diuretic agent; LV, left ventricle; NYHA FC, New York Heart Association functional class; Right iso, right isomerism; RV, right ventricle; SVEF, systemic ventricular ejection function; TA, tricuspid atresia; TCPC, total cavopulmonary connection.

Discussion

In this study, we observed 2 important findings in placentas from mothers with a Fontan circulation that could be associated with poor pregnancy outcomes: (1) they frequently showed low weight for gestational age and chronic SCH; and (2) all placentas demonstrated some histological evidence of placental hypoxia, particularly MVM, hypervascularity/chorangiosis, and villous stromal fibrosis.

Placental weight is an indicator of placental growth and a surrogate for placental function.⁵ The high frequency of low placental weight in this study (n=5/8; 62.5%) suggests that women with a Fontan circulation could be more susceptible to impaired placental growth, which is presumably associated with underlying maternal hypoxia and cardiac dysfunction. Four of the 5 newborns with a low-weight placenta were SGA. The reported risk factors for SGA in mothers with CHD are maternal cyanosis,¹⁰ low cardiac output, and low peak V̇O₂.¹¹ The present study also identified low peak V̇O₂ as a good marker for SGA. Macroscopically, most placentas (n=7/8; 87.5%) had noticeable chronic SCH, consistent with a previous report,¹² indicating that even if it is not detected clinically, subclinical SCH could be common in placentas of women with a Fontan circulation. SCH may have had negative effects on placental function, and is possibly associated with aspirin and heparin use during pregnancy.

As we hypothesized, all placentas demonstrated some histological evidence of placental hypoxia, most commonly MVM (n=7/8; 87.5%). A previous study identified MVM in 8.4% of 856 healthy women and found a link between MVM lesions and higher rates of PE, SGA, and preterm delivery compared with the non-MVM group.¹³ Our results are in agreement with this observation, as shown by the high rate of SGA (n=5/8; 62.5%) and preterm delivery (n=4/8; 50.0%). The most common MVM lesion in PE is decidual vasculopathy.¹⁴ However, no PE or decidual vasculopathy was observed in the placentas of women with a Fontan circulation in the present study. Although both PE and a Fontan circulation are associated with placental hypoxia, the mechanisms are different: PE occurs due to the failure of remodeling of the spiral artery, whereas in a Fontan circulation the preplacental problems are more likely to manifest as MVM lesions other than decidual vasculopathy. In a previous study of 954 placentas of SGA with a healthy mother, the rates of any MVM, multiple MVM or any FVM were 84.8%, 46.0%, and 25.1%, respectively.¹⁵ Compared with these cases of SGA, placentas of women with a Fontan circulation were much more likely to have MVM (any, 87.5%; multiple, 87.5%) and less likely to have FVM (12.5%). This comparison also indicates that the high rates of MVM in placentas of women with a Fontan circulation are caused by maternal cardiac conditions.

Among hypoxic lesions other than MVM and FVM, the high occurrence of chorangiosis (n=7/8; 87.5%) in placentas of women with a Fontan circulation is intriguing. Chorangiosis is found in 6.6% of the general population,¹⁶ in 13.3% of the population at high altitude,¹⁷ and in 21.2% of smokers.¹⁸ When it occurs in such populations, it is associated with low-grade hypoxia and is considered an adaptive mechanism to increase gas exchange.¹⁹ Although it is reported that placentas with chorangiosis become large for gestational age,²¹ in the present study 4 of 7 (57.1%) placentas were low weight despite having chorangiosis. Furthermore, chorangiosis was present in all but 1 placenta, regardless of SGA status. These data suggest that, in many cases, the chorangiosis induced in the placentas of women with a Fontan circulation is not enough to compensate for the hypoxic state and impaired placental growth resulting from maternal cardiac conditions. Regarding the Delivery case 5, which showed a small placenta and normal weight baby, the chorangiosis could have compensated for the placental insufficiency because this case did not show MVM. As shown in this case, in addition to placental weight, placental quality is important for normal fetal growth. MVM and other hypoxic lesions, including chorangiosis, show placental insufficiency. Conversely, chorangiosis results from compensation for placental insufficiency, so whether the fetus appears small or not depends on both the degree of chorangiosis and the degree of other lesions.

The quantifying histological findings confirmed that the placentas of women with a Fontan circulation had a greater capillary density and area of stromal fibrosis than control placentas, and these findings are considered to be the result, in part, of the hypoxic state. There was a significant correlation between capillary density and the area of fibrosis. This may be one piece of evidence that indicates that chorangiosis is the result of a placenta’s response against the other lesions. Placental hypoxia in women with a Fontan circulation in this study is also supported by the immunohistochemical detection of HIF-1α expression in most of the placentas.

Therefore, the placentas of women with a Fontan circulation are considered highly exposed to hypoxia and low perfusion, which could influence placental growth and function. Because uteroplacental blood flow is perfusion dependent, improving cardiac output to the level required by pregnancy is important to improve pregnancy outcomes. However, there is no way to directly increase cardiac output. Bed rest may improve pregnancy prognosis by increasing the blood supply to the uterus. With regard to hypoxia, oxygen supply and correcting any iron deficiency anemia may improve obstetric outcomes.

This study have some limitations. First, the dataset was small. Second, we quantified only hypervascularity, villous stromal fibrosis, the number of macrophages, and HIF-1α expression. Third, because the pathophysiology of a Fontan circulation involves multiple organs, we cannot completely rule out the effects of factors other than hypoxia. However, we found no blood test abnormalities suggestive of undernutrition or abnormal liver function.

Conclusions

The placentas in women with a Fontan circulation were characterized by hypoxic changes, growth impairment, and chronic SCH, which could play important roles in poor pregnancy outcomes.

Acknowledgment

T.Y.-K., K.O.-O., and J.Y. designed the study. T.Y.-K., K.O.-O., and H.I.-U. analyzed pathological examination. T.Y.-K., K.O.-O., and CA.K. analyzed the clinical data. T.Y.-K. and K.O.-O. wrote the manuscript through a fruitful discussion with and supervision by CA.K., T.S., A.N., N.I., H.O., K.K., H.I., T.N., H.I.-U., and J.Y. The authors thank Philip Steer (Emeritus Professor, Imperial College London) for help editing the manuscript.

Sources of Funding

This study was supported by the Intramural Research Fund for Cardiovascular Diseases of the National Cerebral and Cardiovascular Center (30-6-17) and was performed using the National Cerebral and Cardiovascular Center Biobank resource (http://www.ncvc.go.jp/biobank/).

Disclosures

T.N. is a member of Circulation Journal’s Editorial Team. The remaining authors have no conflicts of interest to declare.

IRB Information

This study was approved by the Institutional Research Ethic Board of the National Cerebral and Cardiovascular Center, Suita, Japan (M30-161) on January 16, 2019.

Data Availability

The deidentified participant data will not be shared.

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