Intertwin discordance of aldosterone levels in amniotic fluid with placental anastomoses in monochorionic twins: Insight into the pathophysiology of twin‐to‐twin transfusion syndrome

Abstract

Introduction

Our objective was to investigate the association between the presence of placental anastomoses and intertwin differences in renin‐angiotensin‐aldosterone activation in monochorionic twins using amniotic fluid aldosterone (AF‐ALD) levels. In addition, this study also examined the association between AF‐ALD and the ALD levels in the umbilical cord blood (UCB‐ALD) in monochorionic twins.

Material and Methods

This prospective study included monochorionic diamniotic (MD) twin pregnancies that were not complicated by twin‐to‐twin transfusion syndrome (TTTS) at delivery. Amniotic fluid and umbilical cord vein blood samples were collected from each twin at delivery, and the ALD levels were measured subsequently. The MD twins were divided into two groups: those with placental anastomoses and those without anastomoses owing to fetoscopic laser surgery. The differences in the AF‐ALD levels between the larger and smaller twins were analyzed.

Results

The AF‐ALD levels showed a strong and significant positive correlation with UCB‐ALD levels in 131 MD twins (r = 0.804, p < 0.001). Intertwin differences were examined in 41 and 28 pairs of MD twins with and without placental anastomoses, respectively. The AF‐ALD levels in the smaller twins were significantly higher than those in the larger twins among the pairs of MD twins with placental anastomoses (p = 0.003); however, no statistically significant intertwin differences were observed among the twins without placental anastomoses (p > 0.05).

Conclusions

The AF‐ALD levels reflect the UCB‐ALD levels in MD twins. The presence of placental anastomoses led to intertwin discordance in the ALD levels in MD twins even uncomplicated with TTTS. It was considered that monochorionic twins have this clinical background, and it leads to the development of TTTS.

An association of placental anastomoses with the intertwin discordance in the amniotic fluid aldosterone levels in monochorionic twins was found. The findings provide insights into the pathophysiology of RAAS upregulation and the development of twin‐to‐twin transfusion syndrome.

Abbreviations

AF

amniotic fluid

ALD

aldosterone

FLS

fetoscopic laser surgery

GA

gestational age

MD twins

monochorionic diamniotic twins

RAAS

renin‐angiotensin‐aldosterone system

TAPS

twin anemia‐polycythemia sequence

TP

total protein

TTTS

twin‐to‐twin transfusion syndrome

UCB

umbilical cord blood

Key message.

This study showed the association of placental anastomoses with intertwin discordance in the amniotic fluid aldosterone levels in monochorionic twins. The findings of this study provide insights into the pathophysiology of RAAS upregulation and the development of twin‐to‐twin transfusion syndrome.

1. INTRODUCTION

Twin‐to‐twin transfusion syndrome (TTTS), a severe complication occurring in monochorionic twin pregnancies, is associated with an elevated risk of perinatal morbidity and mortality. TTTS has been reported in 10%‐15% of monochorionic twins. ¹ The pathophysiological changes underlying the development of TTTS remain to be elucidated; however, the upregulation of the renin‐angiotensin‐aldosterone system (RAAS) is considered a key mechanism. ² Previous studies have reported discordant RAAS activation between the donor and recipient twins via placental anastomoses in TTTS. ³ , ⁴ , ⁵ However, RAAS discrepancies between twins before the development of TTTS have not been investigated.

Aldosterone (ALD) is an essential component of RAAS that maintains the homeostasis of liquid volume and electrolyte metabolism. ALD is produced by the activation of angiotensin II in the zona glomerulosa of the adrenal cortex. ⁶ , ⁷ The trend of the ALD levels in monochorionic twins and normal fetuses remains unclear. It was hypothesized that the amniotic fluid ALD (AF‐ALD) levels could be used to assess the status of RAAS activation in monochorionic twins because amniotic fluid samples are easier to obtain than blood samples, especially in the prenatal period. However, there are no reports about the fetal ALD levels in both blood samples and amniotic fluid samples.

Therefore, this study aimed to investigate the association between the presence of placental anastomoses and intertwin differences in the AF‐ALD levels in monochorionic twins. In addition, this study also examined the association between the AF‐ALD levels and ALD levels in the umbilical cord blood (UCB‐ALD) in monochorionic twins.

2. MATERIAL AND METHODS

This single‐center, prospective study was conducted at the Toho University Omori Medical Center (Tokyo, Japan). All consecutive monochorionic diamniotic (MD) twin pregnancies delivered between March 2016 and September 2021 with amniotic fluid samples available were included in this study. The study protocol was approved by the Institutional Ethics Committee of the Toho University Omori Medical Center (27‐158, M16103, and M19016). Written informed consent was obtained from all patients.

Chorionicity was confirmed in all pregnancies, and the gestational age (GA) was calculated based on the crown‐rump length determined via ultrasonography during the first trimester. The clinical records, including the obstetric examination results, complications, and ultrasonographic findings, of all patients were reviewed. The exclusion criterion was the presence of major fetal structural or chromosomal abnormalities.

The correlation between the AF‐ALD and UCB‐ALD levels was examined initially in all MD twins. The differences in the AF‐ALD levels between the larger and smaller twins among the pairs of MD twins with amniotic fluid samples available for both twins were analyzed subsequently. The AF‐ALD levels were compared between the larger and smaller twins as the larger and smaller twins became the recipient and donor twins, respectively, in most cases of TTTS. The pairs of MD twins were divided into two groups, MD twin pairs with and without placental anastomoses, to investigate the association between the presence of placental anastomoses and the intertwin discordance in the AF‐ALD levels. The inter‐twin differences were investigated in each group. The presence of placental anastomoses was determined by examining the delivered placentas. The pairs of MD twins with confirmed placental anastomoses who had not undergone fetal laser surgery (FLS) were grouped into the MD twin pairs with placental anastomoses group. MD twins without placental anastomoses were defined as pairs of MD twins with photocoagulated placental anastomoses who had undergone FLS in the second trimester. FLS is indicated for cases with TTTS or selective fetal growth restriction type II/III together with oligohydramnios (maximum vertical pocket ≤2 cm) in the smaller twin. ⁸ FLS was performed according to a previously described procedure followed at our institution. ⁹ , ¹⁰ , ¹¹ Cases were complicated by TTTS, twin anemia‐polycythemia sequence (TAPS), recurrence of TTTS, and severe neonatal asphyxia. TTTS, selective fetal growth restriction, and TAPS diagnosed according to standard criteria ¹² at the time of delivery were excluded from both groups at the time of delivery. A color dye injection test was used to confirm the presence of placental anastomoses in the delivered placenta in both groups.

2.1. Sample collection and measurement of ALD levels

Amniotic fluid samples and umbilical vein blood samples were collected from each twin at delivery. Amniotic fluid samples were obtained only from twins with intact membranes at the time of delivery and the sample collection. The AF‐ALD, amniotic fluid total protein (afTP), and UCB‐ALD levels were measured. Each sample (3 mL) was centrifuged at 3000g for 10 min at 4°C, aliquoted, and stored at −30°C until further analysis. The ALD levels were measured via radioimmunoassay (RIA) with a gamma counter (WALLAC 1460SRL) using the SPAC‐S ALD kit (FUJIREBIO Inc., Tokyo, Japan). The lowest detection level was 10.1 pg/mL. The afTP levels in each sample were also examined to assess the heterogeneity in the dilution of amniotic fluid. The afTP levels were measured using the Biuret method with a BioMajesty analyzer (JOEL, Tokyo, Japan). Both inter‐ and intra‐assay coefficient variations were <1.0%.

2.2. Statistical analyses

The normality of the data was assessed using the Shapiro‐Wilk test. Medians and nonparametric tests were used for the analyses as the variables were not normally distributed. Spearman's correlation coefficient was used to analyze the relationship between the AF‐ALD and UCB‐ALD levels. The Wilcoxon signed‐rank test was performed to examine the intertwin differences in the AF‐ALD levels between the larger and smaller twins. The Mann‐Whitney U test was performed to compare independent data between the two groups. Categorical data were compared using Pearson's chi‐square or Fisher's exact test, as appropriate. Two‐sided p‐values of <0.05 were considered statistically significant in all tests. SPSS software (version 20.0; IBM Corp., Armonk, NY, USA) was used to perform all statistical analyses.

3. RESULTS

In total, 191 MD twin pregnancies and 352 live births were recorded during the study period. Among these 352 twins, amniotic fluid samples of 183 from 110 pregnancies were available. The correlation between the AF‐ALD levels and each parameter was analyzed first in 183 twins from 110 pregnancies. In 110 MD pregnancies, the median maternal age was 33 years (interquartile range; 31‐36 years), the median GA at delivery was 35.6 weeks (interquartile range; 32.3‐37.1 weeks), and 77 samples of 183 samples (42.1%) were from MD twins after FLS. Eleven twins of 183 twins were delivered vaginally, and others were by C‐section. There were no cases either with iatrogenic or spontaneous septostomy. The AF‐ALD levels showed the strongest significant positive correlation with the UCB‐ALD levels among the parameters (r = 0.804, p < 0.001) (Table 1, Figure 1). Thus, the AF‐ALD levels rather than the AF‐ALD/afTP levels may be more useful for evaluating the association with the UCB‐ALD levels. Subsequent analyses were conducted using the AF‐ALD levels.

TABLE 1.

Correlation of amniotic fluid aldosterone levels with each parameter.

	UCB‐ALD	GA at delivery	Discordant rate of BW among twins
AF‐ALD	r = 0.804, p < 0.001 n = 131	r = 0.457, p < 0.001 n = 183	r = –0.216, p = 0.004 n = 175
AF‐ALD/TP	r = 0.350, p < 0.001 n = 131	r = 0.412, p < 0.001 n = 182	p = 0.153 n = 174

Note: “r” stands for Spearman's correlation coefficient.

Abbreviations: AF, amniotic fluid; ALD, aldosterone; BW, birthweight; n, number of cases; TP, total protein; UCB, umbilical cord blood.

FIGURE 1.

Association between the aldosterone (ALD) levels in the amniotic fluid and umbilical blood.

The intertwin differences (differences between the smaller and larger twins) in the AF‐ALD levels were investigated. Among the 191 MD twin pregnancies, 72 MD twin pregnancies with amniotic fluid samples from both twins available were identified. Amniotic fluid samples were not collected in 82 pregnancies of 191 pregnancies because of non‐reassuring fetal status, unstable maternal conditions, or rupture of membranes at the time of delivery and sample collection, and other 37 pregnancies were excluded because the fetal demise of one twin occurred during pregnancy or amniotic fluid samples of either of the twins were not collected. Three cases were excluded owing to TAPS (one case), severe neonatal asphyxia (one case), and TAPS after FLS (one case). Thus, 69 MD twin pregnancies were analyzed (41 MD twins with placental anastomoses and 28 MD twins without placental anastomoses) (Figure 2). Table 2 presents the clinical characteristics of the two groups. Vaginal delivery was four pregnancies in MD twins with placental anastomoses, and none in MD twins without placental anastomoses. The others were by C‐sections. There were no cases either with iatrogenic or spontaneous septostomy. Significant longer GA at delivery (p = 0.011) and lower discordance rate of birthweight (p = 0.004) were observed in MD twins with placental anastomoses. The AF‐ALD of the smaller twins was significantly higher than those of the larger twins among the pairs of MD twins with placental anastomoses (p = 0.003) (Figure 3A). However, no statistically significant differences in the AF‐ALD levels were observed among the pairs of MD twins without placental anastomoses (p > 0.005) (Figure 3B).

FIGURE 2.

The study population of analysis of intertwin aldosterone level difference. FLP, fetoscopic laser photocoagulation; MD, monochorionic diamniotic; TAPS, twin anemia‐polycythemia sequence.

TABLE 2.

Clinical characteristics of the two groups.

	MD twin pairs with placental anastomoses N = 41	MD twin pairs without placental anastomoses N = 28	p values
Maternal age, years	32 (30‐39)	34 (32‐35)	0.477
Pregnancies in ART, n	8 (19.5%)	4 (14.3%)	0.749
Parity, n	0 (0‐1)	0 (0‐1)	0.135
GA at delivery, weeks	36.9 (33.6‐37.3)	34.5 (32.9‐35.8)	0.011
Discordant rate of BW, %	13.1 (4.8‐19.2)	19.3 (9.5‐40.4)	0.004
GA at FLS, weeks	–	20.6 (17.3‐22.9)	–
AF‐ALD levels in larger twins, pg/mL	137 (97‐281)	162 (74‐251)	0.831
AF‐ALD levels in smaller twins, pg/mL	195 (103‐305)	195 (105‐244)	0.980
UCB‐ALD levels in larger twins, pg/mL	521 (415‐1190)	413 (383‐604)	0.119
UCB‐ALD levels in smaller twins, pg/mL	734 (497‐1110)	530 (436‐628)	0.031
AF‐TP levels in larger twins, g/dL	0.2 (0.2‐0.3)	0.3 (0.2‐0.4)	0.015
AF‐TP levels in smaller twins, g/dL	0.2 (0.2‐0.3)	0.3 (0.2‐0.4)	0.051
Difference in AF‐ALD levels between the twins, pg/mL	13 (0‐36)	3 (−11‐36)	0.347
Difference in UCB‐ALD levels between the twins, pg/mL	55 (−1‐123) N = 31	67 (45‐152) N = 12	0.565
Difference in AF‐TP levels between the twins, g/dL	0 (0‐0)	0 (−0.1‐0.1)	0.925

Note: Data are provided as median (interquartile range) or number (percentage), as appropriate. Discordant rate of birthweight was calculated as [(A – B)/A] × 100, where A is the birthweight of the larger twin, and B is that of the smaller twin. Difference between AF‐ALD, UCB‐ALD, and AF‐TP was calculated as each level of the smaller twin and that of the larger twin.

Abbreviations: AF, amniotic fluid; ALD, aldosterone; ART, artificial reproductive technology; FLS, fetoscopic laser surgery; BW, birthweight; GA, gestational age; MD twins, monochorionic diamniotic twins; TP, total protein; UCB, umbilical cord blood.

FIGURE 3.

(A) Intertwin differences in the amniotic fluid aldosterone (ALD) levels between the larger and smaller twins in monochorionic twins with placental anastomoses; (B) Intertwin differences in the amniotic fluid aldosterone levels between the larger and smaller twins in monochorionic twins without placental anastomoses.*p < 0.05, ns, not significant.

4. DISCUSSION

This is the first study to report the association of placental anastomoses with intertwin discordance in the ALD levels in MD twins and a strong positive correlation between the AF‐ALD and UCB‐ALD levels. These findings provide insights into the pathophysiology of RAAS upregulation and the development of TTTS.

The AF‐ALD levels reflected the UCB‐ALD levels in MD twins. No previous study has reported the presence of a correlation between the AF‐ALD and UCB‐ALD levels. An association between the AF‐ALD level and fetal blood has been reported in fetal sheep, ¹³ which is consistent with the findings of the present study. Although the heterogeneity of biomarkers in diluted amniotic fluid is controversial, the findings of the present study show that the AF‐ALD levels have a stronger correlation with the UCB‐ALD levels than with the AF‐ALD/afTP levels. The reason for this remains unclear, but we considered that amniotic fluid total protein levels could be influenced by other factors and afTP levels do not necessarily or appropriately reflect amniotic fluid dilution in fetuses. And so, the ratio of AF‐ALD/afTP levels might show a weaker correlation with UCB‐ALD levels. Thus, the AF‐ALD levels can be considered a potent biomarker in MD twins. In addition, the AF‐ALD level showed a positive correlation with GA and a weak negative correlation with the discordant rate of birthweight. Previous studies have reported an association between the AF‐ALD levels and GA. ¹³ , ¹⁴ However, the AF‐ALD level in MD twins was the strongest predictor of the UCB‐ALD level in the present study. Thus, the AF‐ALD levels reflect the UCB‐ALD levels, and the fetal blood ALD levels in MD twins can be considered to be elevated in cases with elevated AF‐ALD levels.

The presence of placental anastomoses could result in intertwin differences in the ALD levels between the smaller and larger twins. The donor twin is smaller, and the recipient twin is larger in the majority of MD twins with TTTS. The discrepancy in blood volume or body weight results in the upregulation of the RAAS in the donor twins, which paradoxically activates RAAS in the recipient twins via placental anastomoses during the development of TTTS. ³ , ⁴ The kidneys of the recipient twins show downregulation of RAAS. ³ , ⁴ In this study, the smaller twin in the MD twins with placental anastomoses tended to show higher ALD levels than the larger twin with statistical significance and we hypothesized that monochorionic twins have clinical background that there is different RAAS upregulation status between twins, and it leads to development of TTTS. In another past report, the plasma renin levels in the umbilical cord blood were activated in MD twins with birthweight discordance in a previous study, even among those without TTTS. ¹⁵ In the present study, significant differences could not be confirmed between the ALD levels of the smaller and larger MD twins without placental anastomoses; however, they showed that greater body weight discrepancies were observed than that in MD twins with placental anastomoses. In contrast, ALD difference was observed in MD twins with placental anastomoses, even among those without TTTS. It was hypothesized that the presence of weight discrepancy alone does not result in the upregulation of RAAS in one twin, but the blood flow, which includes the RAAS components, via placental anastomoses may cause the upregulation of RAAS in one twin. However, we cannot deny that laser surgery or experienced TTTS or selective fetal growth restriction type II/III in the second trimester influences present results. Further study is needed to confirm this finding.

This study revealed a strong association between the AF‐ALD and ALD levels in the blood of monochorionic twins before comparing the AF‐ALD levels between twins. Furthermore, this study used pairs of MD twins who had undergone FLS and did not have placental anastomoses as a control group, unlike previous studies, which used normal singleton fetuses or dichorionic twins as controls. It was considered that using MD twins with and without placental anastomoses was ideal.

A limitation of this study is that the ALD levels in the maternal blood were not examined. The ALD levels in the maternal blood influenced the ALD levels in the fetal blood in an animal model. ¹³ However, the ALD levels in the maternal blood did not influence the results as this study compared the intertwin ALD levels in the same pregnant women. Furthermore, the major components of RAAS, such as renin and angiotensin, were not evaluated in this study. These levels are difficult to measure owing to their instability. Another limitation of this study was that many cases were excluded from analyses because of non‐reassuring fetal status, unstable maternal conditions, rupture of membranes, or the fetal demise of one twin. In this study, 42% of samples are from MD twins after FLS. This proportion is high because our hospital is a referred fetal therapy center and some patients after fetoscopic laser surgery give birth in our hospital. In this study, we used the Wilcoxon signed‐rank test to determine whether the ALD levels differ between larger and smaller twins in the same placenta and mother and to exclude the effect of GA and other external factors. However, ideally, the values of difference between twins should be examined in the groups that have the same characteristics using a Mann‐Whitney U test.

5. CONCLUSION

The presence of placental anastomoses causes intertwin discordance in RAAS activation in MD twins, even among those without TTTS. In addition, the AF‐ALD levels can be used for the management of MD twins and TTTS in clinical settings.

AUTHOR CONTRIBUTIONS

Mayumi Takano: Data acquisition, sample collection, data analysis, and manuscript preparation. Mayu Tachihara, Mio Kamiya, Hikari Kotaki, Makiko Shimabukuro, and Sumito Nagasaki: Data analysis. Masahiko Nakata: Supervision.

FUNDING INFORMATION

This study was partially supported by JSPS KAKENHI (grant numbers 19 K09788 and 23 K15823).

CONFLICT OF INTEREST STATEMENT

None.

ETHICS STATEMENT

The study protocol of this study was approved by the Institutional Ethics Committee of the Toho University Omori Medical Center. Reference numbers were 27‐158 (approval date: October 10, 2015), M16103 (approval date: September 21, 2016), and M19016 (approval date: August 9, 2019), respectively. This study was registered with the Japanese Clinical Trial Registry “UMIN‐CTR” (http://www.umin.ac.jp/ctr/index‐j.htm; trial ID numbers UMIN000024486 and 000037702). The initial participant was enrolled on March 14, 2016. Written informed consent was obtained from all patients.

Takano M, Tachihara M, Kamiya M, et al. Intertwin discordance of aldosterone levels in amniotic fluid with placental anastomoses in monochorionic twins: Insight into the pathophysiology of twin‐to‐twin transfusion syndrome. Acta Obstet Gynecol Scand. 2024;103:1558‐1563. doi: 10.1111/aogs.14859