Isolated HELLP syndrome without hypertension or proteinuria exhibiting increases in serum levels of both PlGF and sFlt-1

Abstract

Aim: To compare serum levels of placental growth factor (PlGF) and soluble fms-like tyrosine kinase 1 (sFlt-1) and the serum sFlt-1/PlGF ratio in a case of isolated HELLP syndrome and cases of HELLP syndrome accompanied by either gestational hypertension (GH) or preeclampsia.

Methods: We measured serum levels of sFlt-1 and PlGF using Elecsys sFlt-1 and Elecsys PlGF assays in a patient with isolated HELLP syndrome (case patient) and 14 women with HELLP syndrome accompanied by either GH or preeclampsia (controls). The one-sample t-test was used to compare data between the case patient and controls.

Results: The serum log₁₀ PlGF level was markedly higher in the case patient than in controls (3.356 vs. mean [−SD–+SD]: 1.817 [1.547–2.087], P<0.001). The serum log₁₀ sFlt-1 level was significantly lower in the case patient than in controls (3.972 vs. 4.190 [3.899–4.481], P=0.015). The serum log₁₀ (sFlt-1/PlGF) was markedly lower in the case patient than in controls (0.616 vs. 2.373 [1.988–2.758], P<0.001).

Conclusion: Increases in serum levels of both PlGF and sFlt-1 may be characteristic of isolated HELLP syndrome.

Introduction

HELLP syndrome occurs in pregnancy and early postpartum and is characterized by hemolysis with a microangiopathic blood smear, elevated liver enzymes, and a low platelet count. The pathogenesis of HELLP syndrome remains unclear. Women with HELLP syndrome exhibit lower serum levels of placental growth factor (PlGF), higher serum levels of soluble fms-like tyrosine kinase 1 (sFlt-1), and higher serum sFlt-1/PlGF ratios compared to women with normal pregnancy.^1,2) These changes in angiogenesis-related factors among women with HELLP syndrome are almost identical to those observed in women with preeclampsia.^1,3) Since almost 80%–85% of patients with HELLP have antecedent hypertension or proteinuria,^4,5,6) changes in angiogenesis-related factors may be mainly related to the pathogenesis of preeclampsia. However, to the best of our knowledge, serum levels of angiogenesis-related factors in women with isolated HELLP syndrome, which is accompanied by neither hypertension nor proteinuria, have been reported by only one group.⁷⁾ We encountered a case of isolated HELLP syndrome, and measured the serum levels of PlGF and sFlt-1 and the sFlt-1/PlGF ratio at the onset of isolated HELLP syndrome. Our aim was to compare serum PlGF and sFlt-1levels and the sFlt-1/PlGF ratio between the case patient and women with HELLP syndrome accompanied by either gestational hypertension (GH) or preeclampsia.

Materials and methods

Patients and procedures

In April 2012–March 2023, a prospective cohort study was conducted to measure serum PlGF and sFlt-1 levels and the sFlt-1/PlGF ratio in women with GH, gestational proteinuria, preeclampsia, and preeclampsia-related diseases, such as HELLP syndrome or acute fatty liver of pregnancy, with a fund for contract research from Roche Diagnostics K.K. (Tokyo, Japan). The study was approved by the Ethics Committee of Jichi Medical University (Study Approval Number: I-12-78) and was carried out in accordance with the Declaration of Helsinki. All women gave written informed consent.

In 2020, we encountered a patient with HELLP syndrome presenting with neither GH nor preeclampsia. We previously reported the distribution of serum PlGF and sFlt-1 levels and sFlt-1/PlGF ratios in 14 women with HELLP syndrome,²⁾ as well as gestational-age-specific reference ranges of serum PlGF and sFlt-1 and sFlt-1/PlGF ratio in healthy pregnant women.³⁾ All of the 14 cases of HELLP syndrome were complicated with either GH or preeclampsia (four with GH [hypertension alone], and the remaining 10 with preeclampsia [both hypertension and proteinuria]). The present study used the aforementioned reference ranges of serum PlGF and sFlt-1 and sFlt-1/PlGF ratio, as well as raw data from the 14 cases of HELLP syndrome accompanied by GH or preeclampsia.

Measurement of sFlt-1, PlGF, and the sFlt-1/PlGF ratio

Serum levels of sFlt-1 and PlGF in the present case were measured using Elecsys sFlt-1 (Roche Diagnostics, Penzberg, Germany) and Elecsys PlGF (Roche Diagnostics) immunoassays in the laboratory of Roche Diagnostics Japan.

Definitions of HELLP syndrome, GH, and preeclampsia

HELLP syndrome was diagnosed when an elevated aspartate transaminase (AST) level, a low platelet count (normal reference range: ≥150,000/ml), and an increased lactate dehydrogenase (LDH) level were observed simultaneously.⁸⁾ In our laboratory, the normal reference range of AST is 11–30 U/l, and the normal reference range of LDH is 109–216 U/l. Preeclampsia and GH were diagnosed according to the definitions of preeclampsia, super-imposed preeclampsia, and GH in the “revised JSSHP classification of hypertensive disorders of pregnancy (HDP) in 2018,”⁹⁾ as described in detail elsewhere.¹⁰⁾ HDP was defined as either GH, preeclampsia, or super-imposed preeclampsia.

Statistics

Continuous data are presented as mean±SD, and categorical data are presented as frequency counts (%). For serum sFlt-1 and PlGF levels and the sFlt-1/PlGF ratio, statistical testing was conducted after logarithmic transformation according to our previous reports.^2,3) The one-sample t-test was used to compare data from the case patient and 14 women with HELLP syndrome accompanied by either GH or preeclampsia (controls). IBM SPSS Statistics (version 25 for Windows) was used for the analysis. P values less than 0.05 were considered statistically significant.

Results

Maternal and neonatal characteristics of women with HELLP syndrome

Maternal and neonatal characteristics of the 14 control patients have previously been reported.²⁾ Briefly, 10 of the 14 patients developed HELLP syndrome before delivery, and the remaining four developed HELLP syndrome in the puerperal period. All were singleton pregnancies. HELLP syndrome occurred at 34.0±3.7 weeks of gestation in the 10 patients with onset in the pregnancy period. Four patients experienced neither nausea nor epigastric pains. A total of 36% of HELLP syndrome patients delivered light-for-date infants.

Case report

Table 1 shows the maternal and neonatal characteristics of the present case. A 29-year-old primiparous woman was referred to our hospital due to suspicion of monochorionic monoamniotic twins at 8⁺⁰ weeks of gestation. She was diagnosed with Basedow disease at age 22 years but attained euthyroid status without any medication at age 23 years. In the current pregnancy, thyroid hormones were all within normal ranges (TSH: 1,78 μU/ml; F-T3: 2.17 pg/ml; F-T4: 1.24 ng/dl), and TSH receptor antibody was negative. Systolic and diastolic blood pressures at 8⁺⁰ weeks were 87 and 55 mmHg, respectively. There was no proteinuria. She was finally diagnosed with monochorionic diamniotic twins at 9⁺³ weeks. At 27⁺⁴ weeks, she was admitted to hospital due to suspicion of twin-to-twin transfusion syndrome (amniotic pocket: 32 mm for the smaller twin and 87 mm for the larger twin). Because she showed regular uterine contractions every 5 minutes at 27⁺⁶ weeks, intravascular infusion of ritodrine hydrochloride was started. Thereafter, intravascular infusion of magnesium sulfate was used in addition to the ritodrine infusion at 29⁺¹ weeks, as uterine contractions were not adequately controlled by maximum doses of ritodrine hydrochloride (200 μg/minute). Moreover, since she showed an AST level of 229 U/l at 29⁺⁴ weeks, we suspected that an adverse effect of ritodrine hydrochloride might have led to acute deterioration of liver function. Thus, the administration of ritodrine hydrochloride was discontinued, and intravascular infusion of isoxsuprine hydrochloride was started. At 28⁺⁰ weeks, she was diagnosed with gestational diabetes mellitus based on the 75-g oral glucose tolerance test (102–160–192 mg/dl at 0–60–120 min, respectively).

Table 1. Maternal and neonatal characteristics of the present case

	Isolated HELLP syndrome (n=1)
Sampling period
Gestational weeks at sampling (wk)	34+0
Information on HELLP syndrome
GH (hypertension alone)	(−)
PE (both hypertension and proteinuria)	(−)
HELLP onset† (wk)	34+0
Maternal profiles
Age (yr)	30
Nulliparous	Yes
Past history of PE/GH	No
Pre-pregnancy BMI (kg/m2)	19.1
Chronic hypertension	No
Cesarean section	Yes
SBP (mmHg) maximum	124
DBP (mmHg) maximum	88
AST (IU/l) maximum	124
ALT (IU/l) maximum	88
LDH (IU/l) maximum	470
Platelet count (×104/μl) at nadir	6.8
AT activity (%) at nadir	64.5
Neonatal profiles
Gestational age at delivery (wk)	34+0
Birthweight (g)	1,710/1,796
Sex	Male/Male
Light-for-date	Yes/No

Abbreviations: HELLP, hemolysis, elevated liver enzymes, and low platelet count; wk, weeks of gestation; yr, years old; PE, preeclampsia; GH, gestational hypertension; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; WBC, white blood cells; CRP, C-reactive protein; AST, aspartate aminotransferase; ALT, alanine aminotransferase; LDH, lactate dehydrogenase; AT, antithrombin.

AST and LDH levels and platelet count at 27⁺⁴ weeks were 28 U/l, 166 U/l, and 12.5×10⁴/μl, respectively. The AST level peaked at 29⁺⁴ weeks and then gradually decreased to 31 U/l at 32⁺⁰ weeks due to the cessation of ritodrine hydrochloride; however, it gradually increased again to 102 U/l at 34⁺⁰ weeks (the day of cesarean section). The platelet count was 16.9×10⁴/μl at 11⁺³ weeks and gradually decreased to 12.5×10⁴/μl at 27⁺⁴ weeks, 9.7×10⁴/μl at 33⁺⁰ weeks, and finally, 8.5×10⁴/μl at 34⁺⁰ weeks, possibly due to gestational thrombocytopenia often observed in twin pregnancies. The LDH level (normal reference range: 109–216 U/l) increased to 308 U/l at 29⁺⁴ weeks when AST increased due to the adverse effect of ritodrine hydrochloride, and then decreased to 218 U/l at 32⁺⁰ weeks along with a decrease in AST. However, the LDH level gradually increased again to 336 U/l at 34⁺⁰ weeks.

At 34⁺⁰ weeks, preterm premature rupture of the fetal membranes occurred. The serum level of creatinine increased from 0.52 mg/dl at 27⁺⁴ weeks to 0.95 mg/dl at 34⁺⁰ weeks. AST and LDH levels and platelet count at 34⁺⁰ weeks were 102 U/l, 336 U/l, and 8.5×10⁴/μl, respectively, suggesting the development of HELLP syndrome, although the patient did not show any symptoms related to this syndrome. Because of the deterioration of both renal and liver functions, we decided to perform an emergency cesarean section. The first infant was a male weighing 1,710 g, with Apgar scores at 1 and 5 minutes of 8 and 9, respectively, and the second infant was a male weighing 1,796 g, with Apgar scores at 1 and 5 minutes of 8 and 9, respectively, born at 34⁺⁰ weeks at 11:21. Only the first infant was light-for-date. Two placentas were fused, weighing 850 g. The pathological findings were almost normal (no chorioamnionitis, no infarctions).

AST and LDH levels and platelet count were 124 U/l, 445 U/l, and 7.5×10⁴/μl, respectively, 2.5 hours after the operation; 70 U/l, 470 U/l, and 6.8×10⁴/μl, respectively, one day after the operation; 43 U/l, 440 U/l, and 8.1×10⁴/μl, respectively, two days after the operation; 38 U/l, 334 U/l, and 13.3×10⁴/μl, respectively, four days after the operation; and 24 U/l, 171 U/l, and 33.5×10⁴/μl, respectively, 17 days after the operation. The peak AST level was noted at 2.5 hours after the operation, the peak LDH level was noted one day after the operation, and the nadir of the platelet count was noted one day after the operation. Given the prompt recovery of AST and LDH levels and platelet count, we finally judged that this patient had developed HELLP syndrome. However, the patient presented with no hypertension or proteinuria during the pregnancy and puerperal period, and she did not experience either nausea or epigastric pain. Systolic/diastolic blood pressures were 104/71 mmHg, and proteinuria was negative, 35 days after the operation.

Comparison of serum PlGF and sFlt-1 levels and the sFlt-1/PlGF ratio between the case patient and controls using the one sample t-test

In the case patient, both the serum PlGF level (raw value: 2,270 pg/ml; log₁₀ PlGF: 3.356) and the serum sFlt-1 level (raw value: 9,397 pg/ml; log₁₀ sFlt-1: 3.973) were elevated beyond the 95th percentile of the respective gestational-age-specific reference ranges. Moreover, the serum sFlt-1/PlGF ratio (raw value: 4.1; log₁₀ (sFlt-1/PlGF): 0.617) was almost at the 50th percentile of the gestational-age-specific reference range of the sFlt-1/PlGF ratio (Figures 1A–C).

Figure 1. Serum PlGF and sFlt-1 levels and the sFlt-1/PlGF ratio in a case of isolated HELLP syndrome and 14 cases of HELLP syndrome accompanied by either GH or preeclampsia (controls). A. An open square indicates the case, and closed circles indicate controls. Bold curves are the 5th and 95th percentiles of the gestational-age-specific reference range of serum PlGF, and the dotted curve is the mean. B. An open square indicates the case, and closed circles indicate controls. Bold curves are the 5th and 95th percentiles of the gestational-age-specific reference range of serum sFlt-1, and the dotted curve is the mean. C. An open square indicates the case, and closed circles indicate controls. Bold curves are the 5th and 95th percentiles of the gestational-age-specific reference range of serum sFlt-1/PlGF ratio, and the dotted curve is the mean. Abbreviations: PlGF, placental growth factor; sFlt-1, soluble fms-like tyrosine kinase 1; GH, gestational hypertension.

The serum log₁₀ PlGF level was markedly higher in the case patient than in controls (3.356 vs. mean [−SD–+SD]: 1.817 [1.547–2.087], P<0.001) (Figure 1A). The serum log₁₀ sFlt-1 level was significantly lower in the case patient than in controls (3.972 vs. mean [−SD–+SD]: 4.190 [3.899–4.481], P=0.015) (Figure 1B). The serum log₁₀ (sFlt-1/PlGF) was markedly lower in the case patient than in controls (0.616 vs. mean [−SD–+SD]: 2.373 [1.988–2.758], P<0.001) (Figure 1C).

Discussion

We encountered a case of isolated HELLP syndrome with elevated serum levels of both PlGF and sFlt-1 above the 95th percentile of the respective gestational-age-specific reference ranges. Interestingly, the sFlt-1/PlGF ratio was almost at the 50th percentile of the reference range, although HELLP syndrome occurred at 34 weeks of gestation. We compared serum PlGF and sFlt-1 levels and the sFlt-1/PlGF ratio between the case patient and controls (i.e., women with HELLP syndrome accompanied by either GH or preeclampsia) using the one-sample t-test. Notably, serum PlGF and sFlt-1 levels and the sFlt-1/PlGF ratio significantly differed between the case patient and controls, with a marked increase in PlGF level and a marked decrease in sFlt-1/PlGF ratio.

Increases in serum levels of both PlGF and sFlt-1 may be characteristic of isolated HELLP syndrome. Previously, we reported lower serum PlGF levels, higher serum sFlt-1 levels, and higher sFlt-1/PlGF ratios in 14 women with HELLP syndrome compared with normal pregnant women.²⁾ We confirmed that all 14 women with HELLP syndrome presented with either GH or preeclampsia. Engels et al.¹⁾ reported increases in both sFlt-1 levels and the sFlt-1/PlGF ratio, and a decrease in PlGF levels, in six women with HELLP syndrome. While these findings suggest that women with HELLP syndrome show a similar tendency to that observed in women with severe preeclampsia, the authors did not clarify whether the six women with HELLP syndrome were complicated with preeclampsia. Muñoz-Hernández et al.¹¹⁾ also reported increases in both sFlt-1 levels and the sFlt-1/PlGF ratio, and a decrease in PlGF levels, in 16 women with HELLP syndrome, again without clarifying whether the 16 women with HELLP syndrome were complicated with preeclampsia. Gullai et al.¹²⁾ reported that the plasma level of PlGF, which was measured using the Alere Triage PlGF assay, was below the 5th percentile in 95% of women with both HELLP syndrome and preeclampsia. Trottmann et al.⁷⁾ compared the levels of angiogenesis-related factors in 195 women with preeclampsia, 42 women with both HELLP syndrome and preeclampsia, and seven women with isolated HELLP syndrome using Elecsys sFlt-1 and Elecsys PlGF assays; serum sFlt-1 levels (median [range]) in the three groups were 9,791 pg/ml (1,122–42,222), 11,578 pg/ml (1,348–30,781), and 7,244 pg/ml (1,648–25,519), respectively; serum PlGF levels (median [range]) in the three groups were 66 pg/ml (10–716), 36 pg/ml (9–232), and 56 pg/ml (15–644), respectively; and serum sFlt-1/PlGF ratios (median [range]) in the three groups were 108 (5–662), 287 (51–948), and 49 (3–405), respectively. Although sFlt-1/PlGF ratios in the seven women with isolated HELLP syndrome were significantly lower compared with those of women with preeclampsia or HELLP syndrome/preeclampsia, most cases of isolated HELLP syndrome showed similar deviations in angiogenesis-related factors as women with either preeclampsia alone or HELLP syndrome with preeclampsia. Taken together, increases in sFlt-1 levels and the sFlt-1/PlGF ratio, and a decrease in PlGF levels, may reflect characteristic changes in angiogenesis-related factors in almost all women with HELLP syndrome accompanied by either GH or preeclampsia, as well as some women with isolated HELLP syndrome. In the present case, however, we observed marked increases in the levels of both angiogenesis-related factors: serum levels of both PlGF and sFlt-1 were increased beyond the 95th percentile of the respective gestational-age-specific reference ranges. In addition, the sFlt-1/PlGF ratio was almost at the 50th percentile of the reference range. Moreover, the serum PlGF and sFlt-1levels and sFlt-1/PlGF ratio of the case patient significantly differed from those of controls, showing a marked increase in PlGF level and marked decrease in sFlt-1/PlGF ratio. These findings suggest that increases in serum levels of both PlGF and sFlt-1 may be characteristic of isolated HELLP syndrome.

The present case of isolated HELLP syndrome presenting with neither hypertension nor proteinuria may shed light on the pathogenesis of a microangiopathic blood smear, elevated liver enzymes, and a low platelet count in HELLP syndrome, as well as the development of hypertension and proteinuria in preeclampsia. The slight increase in sFlt-1 levels and marked increase in PlGF levels in isolated HELLP syndrome suggest that a decrease in PlGF levels may mainly be associated with the development of hypertension or proteinuria, while an increase in sFlt-1 may mainly be associated with the development of a microangiopathic blood smear, elevated liver enzymes, and a low platelet count. These associations should be validated by accumulation of data from more cases.

The present case was not a typical case of HELLP syndrome: AST, LDH, and platelet count data gradually deteriorated before the operation, contrary to common cases of HELLP syndrome where abrupt changes in these markers are observed, accompanied with symptoms of either nausea or epigastric pain. However, given that AST, LDH, and platelet count rapidly recovered after delivery, we judged that this case was not inconsistent with the current standard criteria for diagnosing HELLP syndrome.⁸⁾

The reference ranges of PlGF and sFlt-1 and sFlt-1/PlGF ratio in twin pregnancies may slightly differ from those in singleton pregnancies.¹³⁾ In one study, serum sFlt-1 levels and sFlt-1/PlGF ratios were slightly higher in twins than in singletons throughout the course of pregnancy; meanwhile, PlGF levels were slightly higher until 26 weeks of gestation, and slightly lower after 26 weeks of gestation, in twins than in singletons.¹⁴⁾ Another study that compared median PlGF and sFlt-1 levels and sFlt-1/PlGF ratio in twins and singletons reported that the sFlt-1/PlGF ratio in twins rapidly increased after 29 weeks of gestation compared with that in singletons; sFlt-1 levels were almost twice as high in twins as in singletons during 15 weeks of gestation and delivery; and serum PlGF levels were almost the same until 36 weeks of gestation but rapidly increased after 36 weeks of gestation in twins.¹⁵⁾ Since definitive reference ranges of PlGF, sFlt-1, and sFlt-1/PlGF ratio have yet to be established, it may be possible to judge the levels of angiogenesis-related factors for women with twin pregnancies using the gestational-age-specific reference ranges in singletons.

Acknowledgments

This study was supported by Roche Diagnostics K.K. (Tokyo, Japan), which also provided the immunoassays used in this study. Roche Diagnostics K.K. had no involvement in the study design, collection, analysis, or interpretation of data, or in the writing of the report.

Conflict of interest

None.

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