Original Research

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Finding NEMO in preeclampsia Agata Sakowicz, PhD; Paulina Hejduk, PhD; Tadeusz Pietrucha, PhD; Magdalena Nowakowska, PhD; _ Elzbieta Płuciennik, PhD; Karolina Pospiech, PhD; Agnieszka Gach, MD, PhD; Magda Rybak-Krzyszkowska, MD, PhD; Bartosz Sakowicz, PhD; Marek Kaminski, PhD; Grzegorz Krasomski, MD, PhD; Lidia Biesiada, MD, PhD

BACKGROUND: The mechanism of preeclampsia and its way of inheritance are still a mystery. Biochemical and immunochemical studies reveal a substantial increase in tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6 concentrations in the blood of women with preeclampsia. The level of these factors is regulated by nuclear facxtorkappa B, whose activation in a classical pathway requires inhibitory kappa B kinase gamma (known as NEMO or IKBKG). Moreover, NEMO can schedule between cytoplasma and the nucleus. In the nucleus, IKBKG interacts with other proteins, and thus, it is implicated in the regulation of different gene expressions, which are related to cell cycle progression, proliferation, differentiation, and apoptosis. OBJECTIVE: This is the first study investigating the association between the level of NEMO gene expression and the presence of preeclampsia. We tested the hypothesis that the simultaneous increase in NEMO gene expression both in the mother and her fetus may be responsible for the preeclampsia development. Moreover, the relationships between clinical risk factors of preeclampsia and the levels of NEMO gene expression in blood, umbilical cord blood, and placentas were investigated. STUDY DESIGN: A total of 91 women (43 preeclamptic women and 48 controls) and their children were examined. Real-time reverse transcriptionepolymerase chain reaction was used to assess the amount total NEMO messenger ribonucleic acid (mRNA) content and the mRNA

P

reeclampsia (PE) is a disorder that appears only during pregnancy and affects up to 5e7% of low-risk nulliparous women. The higher rates of preeclampsia (15e20%) are found in some groups of women, including those with multifetal gestation, chronic hypertension, obesity, pregestational diabetes, or previous preeclampsia.1,2 The family nature of preeclampsia has been known since the 19th century. Studies report a higher occurrence of preeclampsia in daughters of women whose pregnancy was complicated by preeclampsia (28.5e38.1%) compared with the frequency of this disorder in

Cite this article as: Sakowicz A, Hejduk P, Pietrucha T, et al. Finding NEMO in preeclampsia. Am J Obstet Gynecol 2016;214:538.e1-7. 0002-9378/$36.00 ª 2016 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ajog.2015.11.002

level of each NEMO transcript from exons 1A, 1B, and 1C in maternal blood, umbilical cord blood, and placentas. Univariate analyses and correlation tests were performed to examine the association between NEMO gene expression and preeclampsia. RESULTS: Newborn weight and height, maternal platelet number, and gestational age (week of delivery) were lower in the group of women with preeclampsia than controls. NEMO gene expression level was found to be almost 7 times higher in the group of women with preeclampsia than healthy controls. The correlation analysis found that a simultaneous increase in the expression level of total NEMO mRNA in maternal blood and the mRNA for total NEMO (Rs ¼ 0.311, P < .05), transcripts 1A (Rs ¼ 0.463, P < .01), 1B (Rs ¼ 0.454, P < .01), and 1C (Rs ¼ 0.563, P < .001) in fetal blood was observed in preeclamptic pregnancies. In addition, the mRNA levels for total NEMO and transcripts 1A, 1B, and 1C were lower in placentas derived from pregnancies complicated by preeclampsia. CONCLUSION: Simultaneous increase of NEMO gene expression in maternal and fetal blood seems to be relevant for preeclampsia development. The results of our study also suggest that a decreased NEMO gene expression level in preeclamptic placentas may be the main reason for their intensified apoptosis. Key words: NEMO gene, preeclampsia, real-time reverse

transcriptionepolymerase chain reaction

their daughters-in-law (4.4e10.6%, the approximate rate of preeclampsia in the whole population).3 The most plausible genetic model postulates that maternal genes are strongly associated with the development of preeclampsia. But the expression of preeclampsia in 2 pregnancies of the same woman may differ. For this reason the fetal genotype is considered to be implicated in the development of this disorder.3,4 Moreover, current data show that male fetus is associated with an increased risk of preeclampsia.5 The pathogenesis of PE is not completely understood. The most probable pathomechanism is related to the reduction of placental flow caused by shallow (insufficient) endovascular throphoblast invasion into uterus spiral arteries in early pregnancy.6 Because this insufficient endovascular throphoblast invasion leads to prolonged hypoxia and poor perfusion in the placenta, it

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synthesizes and releases an increased amount of the antiangiogenic factor, soluble fms-like tyrosine kinase-1 (sFlt-1); the antiapoptotic factor, soluble form of Fas ligand; and the following cytokines: tumor necrosis factor (TNF) alpha, interleukin (IL)-6, IL-1, and nitric oxide synthetase inhibitors.7-9 Biochemical and immunochemical studies conducted among patients with preeclampsia revealed a substantial increase of TNF alpha, IL-1 beta, and IL-6 concentration in the blood of pregnant women.10,11 As the level of these factors is increased, a number of various cellular mechanisms dependent on factor nuclear factor kappa B (NFkB) are also stimulated.6,12 The development of human pregnancy is associated with a shift from Th1 type of lymphocytes toward a Th2 type immune response.13 The Th1 cells secrete TNF alpha, interferon gamma, and interleukins, whereas Th2 cells

ajog.org secrete IL-4 and IL-10. In pregnancies complicated by preeclampsia, cytokine profiles in peripheral blood are mainly Th1.14 The study conducted by Lederer et al15 pointed out that the activation and nuclear translocation of NFkB is necessary for the development of the Th1 response. Some observations reveal that higher levels of NFkB are found in mononuclear blood cells in cases of preeclampsia than in nonpregnant women or women experiencing an uncomplicated pregnancy. The NFkB level is also higher in the blood cells of women with preeclampsia than in women during parturition, in which NFkB is known to be one of the main factors responsible for the regulation of labour.16-18 The activation of NFkB in the classical pathway requires the presence of multicomponent protein kinase complex (IKK), consisting of catalytic subunits IKK alpha and IKK beta, and the regulatory scaffolding subunit IKK gamma (known as NEMO or IKBKG).19 Genetic studies demonstrate the essential role of NEMO in regulating NFkB activation on the classical pathway. The increase in NFkB activation leads to an increase in the levels of TNF alpha, IL-1, and IL-6. This may explain why women with preeclampsia demonstrate increased levels of TNF alpha, IL-1, and IL-6 in the blood and why in preeclampsia it is impossible to achieve an immunosuppression state characteristic for pregnancy.11,20 The disruption and inactivation of the NEMO one copy gene, located on sex chromosome Xq28, is associated with syndromes such as incontinentia pigmenti and X-linked immunodeficiency syndrome.21 A lack of NEMO in keratinocytes results in their spontaneous death and triggers the expression of proinflammatory mediators by adjacent wild-type cells.22 Moreover, the inactivation of NEMO in cardiomyocytes leads to the inactivation of antioxidant processes and the consequent accumulation of free oxygen radicals, spontaneously pathological tissue remodeling, cell death, fibrosis, and contractile dysfunction.19 Very similar pathomorphological observations have been noted in several studies conducted on placentas

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obtained from pregnancies complicated by preeclampsia.23,24 In this study we tested the hypothesis that the simultaneous increase in NEMO gene expression both in mother and her fetus may be responsible for the preeclampsia development. In addition, the relationships between clinical risk factors of preeclampsia and levels of NEMO gene expression in blood, umbilical cord blood, and placentas were investigated.

Materials and Methods Patient selection and data collection The present study included 91 women in single pregnancy (43 women with preeclampsia and 48 controls) and their children. Informed consent was obtained from each mother before delivery, and the study protocol was approved by the Medical University of Lodz Ethical Committee. A venous blood sample was taken from each woman about 1e2 hours before the beginning of the delivery. An umbilical cord blood sample and a fragment of the placenta were taken immediately after the delivery of the baby. Placental fragments of about 2 cm3 were trimmed approximately 5 cm from the site of the umbilical cord insertion into the placenta. The decidua and amnion were removed. Following the drainage of excess blood, the placental samples were immediately washed in sterile phosphate-buffered saline (pH 7.4) and set in RNAlater (Ambion Inc, Grand Island, NY) to protect the ribonucleic acid (RNA) from degradation. Inclusion criteria to the study group were as follows: preeclampsia, single pregnancy, no hypertension and diabetes mellitus before pregnancy, no gestational diabetes mellitus, no chromosomal aberration in the fetus, maternal body mass index (BMI) before pregnancy < 30 kg/m2, and no other chronic maternal disorders. Preeclampsia was diagnosed on the basis of the following symptoms: maternal blood pressure greater than 140/90 mm Hg (measured twice with an interval of at least 6 hours) with accompanied proteinuria (> 300 mg per 24 hours or at least 2þ during a single urine

Original Research

test), which developed after week 20 of gestation. Patients with early (< 34 weeks of pregnancy; n ¼ 19) and late ( 34 weeks of gestation; n ¼ 24) preeclampsia were both qualified to the study group. All women qualified to the study group delivered by cesarean delivery. Inclusion criteria to the control group were as follows: single pregnancy, no chronic diseases, maternal BMI before pregnancy < 30 kg/m2, no fetal chromosomal abnormalities, and no uterus activity that signalizes the delivery. Indications for cesarean delivery in the control group were as follows: transverse or breech position of the fetus, ophthalmological indications, orthopedic indications, or an increased risk of uterine rupture because of a previously performed cesarean delivery.

RNA isolation and complementary dexoyribonucleic acid (cDNA) synthesis Total RNA was isolated from blood samples and placentas using the Total RNA minikit (A&A Biotechnology, Gdynia, Poland), according to the manufacturer’s protocols for blood or tissue. The RNA quality and purity of the sample were determined spectrophotometrically (NanoDrop; Thermo Fisher Scientific, Grand Island, NY). Samples were qualified for further analysis when the optical density260/280 ratio was between 1.8 and 2.0. Reverse transcription of RNA into cDNA was conducted by the Maxima first-strand cDNA kit (Thermo Fisher Scientific) according to the manufacturer’s instruction. The obtained cDNA was stored at e20 C for further analysis.

Gene expression analysis The primers used for the analysis of the total NEMO transcript are as follows: TN forward, 5’-TACTGGGCGAAGAGT CTCC-3’, TN reverse, 5’-AGAATCTGG TTGCTCTGCC-3’. The specificity of the primers was determined with the Primer-Blast program. Transcripts 1A, 1B, and 1C failed when we analyzed them directly by real-time reverse transcriptionepolymerase chain reaction (PCR) because of the low-level gene expression, and for this reason we

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decided to assess their level expression using nested PCRs. Our observations were in consistency with others.25 The first step of the nested PCR was conducted with the following outer primers: 1A_out forward, 5’-GAACGC CCATCAAGCCC-3’, 1A_out reverse, 5’-GCTCTTGATTCTCCTCCAGGC-3’; 1B_out forward, 5’-GAAGCGTGGTAG GGAAGG-3’, 1B_out reverse, 5’-GCTC TTGATTCTCCTCCAGGC-3’, and 1C_ out forward, 5’-CTGTTCACCAAACT TGACTGCG-3’, 1C_out reverse, 5’-GC TCTTGATTCTCCTCCAGGC-3’. The real-time PCRs were conducted with the following inner primers: 1A_in forward, 5’-CACCCTTGCCCTGTTGG AT-3’, 1A_in reverse, 5’-GGAGACTCTT CGCCCAGTA-3’, 1B_in forward, 5’-CT GACGGACTCTGCTGACA-3’, 1B_in reverse, 5’-AGGAGACTCTTCGCCCAG TA-3’, and 1C_in forward, 5’-TAGCC CTTGCCCTGTTGGA-3’, and 1C_in reverse, 5’-GGAGACTCTTCGCCCAG TA-3’. The stability of the housekeeping gene candidates was analyzed using NormFinder software.26 The normalization of each run was calculated using a reference sample prepared from 80 pooled randomly selected samples of first-strand cDNA from the study (40 samples) and control (40 samples) groups in equal volumes. Normalized gene expression was calculated as described by Pfaffl.27

Statistical analysis All data were analyzed using Statistica software version 12 (StatSoft, Tulsa, OK). The normal distribution of the data was tested using the Shapiro-Wilk test. Clinical and personal characteristics between groups were compared using the Student t test for normally distributed data, and the c2 test for nominal variables. The Mann-Whitney U test was used for nonparametric data. In the nonparametric data comparison between 3 groups of women (early, late preeclampsia, and controls), the Kruskal-Wallis and Dunn’s post-hoc tests were used. The nonparametric Spearman rank correlation test was used to assess the correlation between the expression levels of studied transcripts and between the expression level of maternal total

NEMO mRNA and clinical parameters of women. Values of P < .05 were considered statistically significant.

Results Clinical details of the study are given in Table 1. Newborn weight and height, maternal platelet number, and gestational age (week of delivery) were lower in the group of women with preeclampsia than controls. The study group displayed significantly higher BMI values and primiparas and son numbers. The IKBKG gene expression level was found to be almost 7 times higher in the group of women with preeclampsia than healthy controls. In addition, the expression of NEMO gene transcripts (1A, 1B, and 1C) was also found to be significantly higher in the blood of women with preeclampsia (Table 2). Only the level of the NEMO 1A transcript was significantly higher in the umbilical blood of children born from pregnancy complicated by preeclampsia

than children born to healthy mothers (Table 2). Differences were also observed between placentas from normal and preeclamptic pregnancies with regard to the level of IKBKG gene expression. The expression of both the total NEMO transcript and the 1A, 1B, and 1C transcripts were significantly lower in placentas of women with preeclampsia in comparison with controls (Table 2). After the division of women with preeclampsia into groups according to the labor term before and after the 34th week of pregnancy, we found that the levels of total NEMO and 1A, 1B, and 1C transcripts in both subgroups were significantly higher than in the control group (P < .01 for all analyses). The results of all the studied NEMO transcripts in children do not differ significantly between the subgroups (late and early preeclampsia) and the control group. The most interesting results were noticed in the placental analysis. We

TABLE 1

Comparison of clinical data within the study population Preeclamptic group (n ¼ 43)

Clinical data a

Age of women, y 2a

BMI, kg/m

WBC, 10 /mL 3

a

RBC, 10 /mL 6

a

Controls (n ¼ 48)

P value

30.4  6.61

31.4  3.81

26  4.86

24  4.06

10.26  2.61

11.30  2.12

.051

4.16  0.37

4.12  0.49

.675

.387 < .01c

12.27  1.32

12.15  1.22

.692

a

35.9  3.48

35.7  2.97

.783

a

HB, g/dL

a

HCT, %

86  4.00

86.5  5.65

.636

MCHC, g/dLa

34.3  2.36

33.7  1.37

.180

PLT, 10 /mL

201.3  59.70

233.5  52.60

< .05c

36.3  3.13

38.5  1.03

< .001c

2563.9  926.97

3313.9  421.86

< .001c

49.5  5.27

54.2  3.21

< .001c

8.8  1.24

9.2  0.94

MCV, fL

3

a

Week of deliverya Baby weight, ga Baby height, cma Apgar score, 1 min

a

b

Primiparas, n, %

Miscarriage, n, %b b

Baby sex (son), n, %

.057 < .001c

32 (74%)

16 (33%)

12 (28%)

6 (12%)

.066

27 (63%)

20 (42%)

< .05c

HB, hemoglobin; HCT, hematocrit; MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; PLT, platelet count; RBC, red blood count; WBC, white blood count. The values are presented as mean  SD. For data analysis, a Student t test was used; b For data analysis, a c2 test was used; c Denotes statistical significance. Sakowicz et al. Finding NEMO in preeclampsia. Am J Obstet Gynecol 2016.

a

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TABLE 2

Comparison of NEMO gene expression level between study and control groups Preeclamptic gestation median (interquartile range)

Normal gestation median (interquartile range)

P valuea

Total NEMO

3.40 (1.12e11.00)

0.52 (0.13e2.00)

< .001b

1A

1.15 (0.42e3.44)

0.18 (0.10e0.39)

< .001b

1B

1.32 (0.47e3.03)

0.26 (0.07e0.46)

< .001b

1C

2.62 (0.03e11.24)

0.26 (0.02e0.80)

< .010b

Total NEMO

4.68 (1.19e15.30)

5.88 (1.20e14.60)

1A

1.93 (0.81e3.78)

0.71 (0.22e2.03)

1B

1.46 (0.89e3.49)

1.13 (0.38e2.29)

.248

1C

2.94 (0.27e13.44)

1.50 (0.10e4.63)

.153

Total NEMO

0.76 (0.27e1.87)

3.08 (1.28e9.99)

< .001b

1A

0.22 (0.08e0.61)

1.04 (0.22e2.96)

< .001b

1B

0.24 (0.09e0.42)

0.47 (0.21e2.17)

< .001b

1C

0.57 (0.04e1.16)

1.68 (0.53e8.32)

< .001b

Transcripts Mothers

Children .922 < .050b

Placentas

The values are presented as relative gene expression levels calculated by the Pfaffl method.27 P value was calculated using the Mann-Withney U test; b Denotes statistical significance. Sakowicz et al. Finding NEMO in preeclampsia. Am J Obstet Gynecol 2016. a

observed that both the total NEMO and all the transcript gene expressions differ according to the term of labor (Table 3). A correlation analysis was made between the level of maternal total NEMO mRNA and each of their NEMO transcripts and another between the level of maternal total NEMO mRNA and the levels of fetal and placental total NEMO mRNA and its 1A, 1B, and 1C transcripts. The correlations were observed between the levels of the maternal total NEMO mRNA and the fetal NEMO gene transcripts but only in the study group. More detailed information is given Table 4. The correlation was also analyzed between the expression level of each NEMO gene transcript and various maternal clinical factors. Although no correlation was found between the expression level of each NEMO gene transcript and maternal age or BMI, a statistically significant correlation was observed between the sex of delivered children (sons) and the expression of

mRNA for the transcripts 1A (Rs ¼ 0.326, P < .01) and 1B (Rs ¼ 0.229, P < .05) in the maternal group; 1A (Rs ¼ 0.233, P <.05), 1B (Rs ¼ 0.249, P <.05), and 1C (Rs ¼ 0.249, P < .05) in the children’s group; and 1A (Rs ¼ e0.209, P < .05) and 1C (Rs ¼ e0.248, P < .05) in the placental group.

Comment This is the first study to examine the relationship between the level of NEMO gene expression and the presence of preeclampsia. At the time of this writing, the existing literature points at the correlation between IL-1, IL-6, TNF alpha, soluble receptor of vascular endothelial growth factor (sFlt1), Fas ligand, and NFkB in the serum of preeclamptic women and hypertension during pregnancy.9,28,29 NEMO is an essential activator of NFkB on the canonical pathway.21 Activation of NFkB leads to its translocation into the nucleus, in which it acts as a transcription factor. It is known to regulate the expression of a number of

Original Research

genes including those coding for cytokines (TNF alpha, IL-1, IL-2, IL-6, and IL-12), chemokines, metalloproteinases, Fas ligand, sFlt1, vascular adhesion molecule 1, and intercellular adhesion molecule 1.21,30 Luppi et al16 reported that preeclampsia is related to the activation of circulating leukocytes via the NFkB signal transduction pathway. In their study, the NFkB level was found to be higher in preeclamptic blood cells than in healthy women during delivery. Furthermore, higher IL-6 and TNF alpha levels were found, these being the main factors that activate the canonical NEMO-dependent NFkB pathway. Although the present study does not examine the level of NFkB gene expression, it can indirectly confirm these observations because NEMO gene expression was found to be higher in women with preeclampsia than controls. The higher level of NEMO gene expression in women with preeclampsia blood was noticed after subdivision of the study group into 2 subgroups: early and late preeclampsia. The study assesses the level of total NEMO mRNA (the common mRNA level for 3 different transcripts of the IKBKG gene) and the levels of each NEMO gene transcript: 1A, 1B, and 1C identified in GeneBank as NM_001099856, NM_001099857, and NM_003639, respectively, which are derived from different exons. These different transcripts of IKBKG gene are the result of the existence of 2 promoters. The study conducted by Fusco et al31 noted that promoter A is distally located in the intron 2 of the G6PD gene (glucose-6-phosphatase dehydrogenase). The G6PD and NEMO genes are arranged head to head on the X chromosome. Promoter A drives the transcription from the 1D and 1A exons, whereas promoter B is located in the CpG islands and drives the transcription from the 1B and 1C exons.31 The transcript that drives from 1D exon is typical for hepatic cells, whereas the 1A, 1B, and 1C exon products are observed in various tissues of the human body.25 Our findings indicate that both promoter A and promoter B are more active

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TABLE 3

Comparison of placental NEMO gene expression level between subgroups of women with preeclampsia (early and late) and controls Transcripts

PE < 34th weeka

Controlsa

P valueb

PE  34th weeka

Controlsa

P valueb

Total NEMO

2.85 (0.59e4.17)

4.46 (1.30e14.90)

.136

0.76 (0.23e1.87)

4.46 (1.30e14.90)

< .001c

1A

1.33 (0.15e1.86)

1.04 (0.22e2.96)

.143

0.12 (0.02e0.54)

1.04 (0.22e2.96)

< .001c

1B

0.35 (0.25e0.74)

0.47 (0.21e2.17)

.175

0.22 (0.08e0.37)

0.47 (0.21e2.17)

< .001c

1C

2.38 (0.31e2.33)

1.68 (0.53e8.32)

.889

0.44 (0.01e1.07)

1.68 (0.53e8.32)

< .001c

The values are presented as relative gene expression levels calculated by the Pfaffl method. The results were statistically analyzed by the Kruskal-Wallis test (P < .05) followed by Dunn’s test. PE, preeclampsia. 27

a Results are presented as median and interquartile range; b P value was calculated by the Dunn’s post-hoc test; c Denotes statistical signifance. Sakowicz et al. Finding NEMO in preeclampsia. Am J Obstet Gynecol 2016.

in women with preeclampsia than controls. The higher level of NEMO gene expression may be related to the increase of its protein in the cells, which is known to induce NFkB activation. Moreover, the correlation analysis found that a simultaneous increase of NEMO gene expression in the mother and her fetus

(child) is observed in preeclamptic pregnancies, with no such correlation observed in the control group. It is highly probably that the simultaneous NEMO increase in mother and fetus is relevant for the development of preeclampsia. NEMO gene expression was also found to be statistically significantly

TABLE 4

Spearman rank correlation in the study and in the control groups between maternal total NEMO mRNA level and each of maternal transcripts as well as between maternal total NEMO mRNA level and mRNA of total NEMO, 1A, 1B, and 1C transcripts of their babies and placentas Study group

Transcripts

Control group Spearman correlation coefficienta

P value

Mothers

Transcripts

Spearman correlation coefficienta

P value

Mothers

1A

0.436

< .010b

1A

0.663

< .001b

1B

0.537

< .001b

1B

0.505

< .001b

1C

0.594

< .001b

1C

0.449

< .050b

0.311

< .050b

Total NEMO

0.230

.115

0.463

< .010

1A

0.420

.148

0.454

< .010

1B

0.205

.166

0.563

< .001

1C

0.082

.584

Total NEMO

e0.025

.876

Total NEMO

0.165

.262

1A

e0.037

.814

1A

0.104

.488

1B

0.219

.162

1B

0.036

.812

1C

0.319

1C

0.111

.459

Children Total NEMO 1A 1B 1C

Children b b b

Placentas

Placentas

< .050b

a

The Spearman rank correlation was conducted using the relative gene expression levels of studied transcripts calculated by the Pfaffl method27; b Denotes statistical significance. Sakowicz et al. Finding NEMO in preeclampsia. Am J Obstet Gynecol 2016.

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lower in placentas derived from preeclapmtic pregnancies with regard to the levels of mRNA for total NEMO and transcripts 1A, 1B, and 1C. NEMO is most widely recognized as playing a role in the regulation of the NFkB pathway. However, Legarda-Addison et al reported that NEMO plays a novel role in blocking cell death via a pathway independent of NFkB signaling by inhibiting the reaction of RIP1 (receptor interacting protein-1) with caspase-8.32 The low level of NEMO gene expression in preeclamptic placentas may lead to the decrease in NEMO protein in placental cells. If the level of NEMO is insufficient, RIP1 may interact with caspase-8, leading to the induction of placenta cell apoptosis, which is observed in preeclampsia.32-34 The results of our study with regard to total NEMO gene expression are contrary to the results of the GDS2080 microarray study conducted by Nishizawa et al35 (http://www.ncbi.nlm.nih. gov/geoprofiles/?term¼GDS2080þikbkg). They found no significant discrepancy in the level of NEMO gene expression between the studied preeclamptic subgroups (late, n ¼ 5, and early, n ¼ 5) and the control group (n ¼ 4). This contradiction to our study may be related to the different number of cases (n ¼ 91 vs n ¼ 14) and probably different criteria of patient inclusion to both studies. We did not find some essential information about participant/ patient selection criteria in study of Nishizawa et al35 such as multiple or single pregnancy, diabetes mellitus,

ajog.org hypertension before pregnancy, or chronic diseases and whether before the cesarian delivery, the uterus contracted, indicating the onset of the labor. In our study we noticed that the level of NEMO gene expression depends on the gestational age. The level of total NEMO and each of IKBKG transcripts were significantly lower in preeclamptic placentas obtained from pregnancies termined after the 34th week of gestation compared with the controls. We did not observe a significant discrepancy between NEMO gene expression levels in the controls and the subgroup of early preeclampsia. The results of our study may suggest that depletion of the NEMO gene expression level in placentas is typical for pregnancy. During preeclampsia the decrease in the IKBKG expression level may be more intensive or the top of the NEMO expression level from which depletion starts may be lower compared with controls. We suspect that the lower IKBKG gene expression level may be responsible for the decrease in NEMO protein in placental cells. Insufficient NEMO level in placentas negatively affects their cellular processes. Up until now some published studies have pointed out that in addition to the cytoplasmatic role of NEMO, it also plays a role in the nucleus. NEMO can schedule between cytoplasma and nucleus in which it is involved in the regulation of different gene expressions.21,36 Moreover, in the nucleus, NEMO can also influence the stabilization of various proteins.21 The study conducted by Kim et al37 reported that IKBKG can directly interact with transcription factor c-Myc in the nucleus, leading to its phosphorylation and stabilization. c-Myc plays a significant role in the proliferation and differentiation of placental cells, especially in the first trimester of pregnancy.38,39 Moreover, c-Myc is essential for vascular endothelial growth factor gene expression, which is decreased in placentas derived from pregnancies complicated by preeclampsia.40-42 Hence, an insufficient level of NEMO in placentas also may be implicated in incorrect implantation, which is

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regarded as the primary cause of preeclampsia. The present study demonstrates the association between NEMO gene expression and preeclampsia. Up until now, there has been a lack of research that pointed at the role of essential modulator of NFkB in the development of hypertension during pregnancy. Our findings indicate that the observed increase of IKBKG gene expression, both in the mother and the fetus, may be essential for the appearance of preeclampsia. In addition, NEMO gene expression was found to be statistically significantly lower in placentas derived from pregnancies complicated by preeclampsia than in controls. Further studies are needed to determine whether this lower level of NEMO gene expression is responsible for causing preeclampsia or is an effect of increased simultaneous IKBKG gene expression in the mother and the fetus. n Acknowledgment We thank all mothers who agreed to participate in this study.

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Author and article information From the Departments of Medical Biotechnology (Drs A. Sakowicz, Hejduk, and Pietrucha) and Molecular Cancerogenesis (Drs Nowakowska, Płuciennik, and Pospiech), Medical University of Lodz, and Departments of Genetic (Dr Gach) and Obstetrics and Gynecology (Drs Krasomski and Biesiada), Polish Mother’s Memorial HospitaleResearch Institute in Lodz, Department of Obstetrics and Perinatology (Dr Rybak-Krzyszkowska), University Hospital in Krakow, and Department of Microelectronics and Computer Science (Drs B. Sakowicz and Kaminski), Lodz University of Technology, Poland. Received July 17, 2015; revised Oct. 24, 2015; accepted Nov. 5, 2015. The views expressed herein are those of the authors, and The National Science Center had no involvement in the study design or in the collection, analysis, and interpretation of the data or in the writing of the report or in the decision to submit the article for publication. This work was supported by grant DEC-2013/11/D/ NZ5/01783 from the National Science Center (Poland). The authors report no conflict of interest. Corresponding author: Agata Sakowicz, PhD. agata. [email protected]