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The Journal of Maternal-Fetal and Neonatal Medicine, 2013; 26(4): 347–350© 2013 Informa UK, Ltd.ISSN 1476-7058 print/ISSN 1476-4954 onlineDOI: 10.3109/14767058.2012.733770

Objective: In this study, we assessed Bcl-2 and Bax gene expres-sion patterns in human placental samples from intrauterine growth restriction (IUGR) pregnancies using normal pregnancy as control. Methods: We compared Bcl-2 and Bax gene expres-sion in placental samples from all IUGR pregnancies treated in our clinic between 1 January 2010–1 January 2011 vs. 140 normal pregnancy samples from the same study period. We also assessed clinical parameters such as maternal age, gesta-tional weight gain, gestational body mass index (BMI) change, and maternal birth weight. Results: In IUGR, the Bcl-2 gene was underexpressed compared to normal pregnancy. There was no difference in the Bax gene activity in the two groups. The degree of growth restriction within the IUGR group did not correlate with Bcl-2 or Bax gene activity. Conclusions: Our study revealed that it is the reduced inhibitory activity of the Bcl-2 gene rather than an enhanced stimulatory activity of the Bax gene in the background of the increased apoptosis observed in IUGR. IUGR appears to be more common with maternal age around 20 years and above 35 years. Gestational weight gain and gestational BMI change also predict the risk for IUGR.

Keywords: Placenta, apoptosis, intrauterine growth restriction, Bcl-2 gene, Bax gene

Abbreviations: AC: abdominal circumference; Bcl-2: B cell lymphoma 2; BH: Bcl-2 homologous domain; BMI: body mass index; BPD: biparietal diameter; FL: femur length; IUGR: intrauterine growth restriction; OFD: occipitofrontal diameter

IntroductionIntrauterine growth restriction (IUGR) is defined as fetal birth-weight at or below the tenth percentile for sex and gestational age [1]. (It must be remarked, that 5 and 3 percentile as a border-line of IUGR is also used in obstetrics.) IUGR may result from placental dysfunction, fetal malformation, intrauterine infection, or maternal factors. Although the most common etiology for IUGR is thought to be placental dysfunction, its molecular level pathology remains largely unknown [2,3].

During intrauterine growth, maternal glucose is the primary source of energy for the fetus. Fetal glucose production is minimal so most of fetal energy needs are met through transpla-cental glucose transport [4,5]. It is logical to assume that func-tional impairment of the placenta may often lead to disordered

fetal energy metabolism, especially if glucose transport is affected. Fetal hypoglycemia is in fact common in IUGR, with impairment of transplacental glucose transport as the main etiology [6].

Programmed cell death (apoptosis) plays a fundamental role in the physiological development of human placenta. This role is especially prominent in the final stages of pregnancy where apop-tosis is involved in physiological placental aging [1,3]. This is well described by the literal meaning of apoptosis in ancient Greek: a falling leaf [7].

Several genes are involved in the regulation of apoptosis. Among these, some are stimulatory (proapoptotic) while others are inhibitory (antiapoptotic). In this complex regulatory system, the B cell lymphoma 2 (Bcl-2) protein family plays a prominent role [8–11]. Bcl-2 genes may be either pro- or antiapoptotic. Each of these genes may possess one or several of the homologous domains BH1, BH2, BH3, or BH4 (BH = Bcl-2 Homologous domain.) These homologous domains appear to determine the interaction between the members of the gene family [12–14]. The antiapoptotic genes include Bcl-2, Bcl-xL (Bcl-extra long), A1, Bcl-w, and Boo. Each of these genes may have any of the homologous domains (BH1-4.). The proapoptotic genes including Bax, Bak, and Box may only have the BH3 homologous domain. In the antiapoptotic group, Bcl-2 is most important, whereas in the proapoptotic group the Bax gene is most prominent. Simply put, the initiation of programmed cell death is determined by the ratio of activitiy of the latter two genes [8,15,16]. Several studies suggest that apoptosis becomes more common toward the later stages of gestation. This is associated with increased activity of the Bcl-2 gene family [17–19]. For normal pregnancy and term delivery, physiological aging of the placenta is just as fundamentally important as other functional processes during placental development. Abnormal apoptotic activity of trophoblasts leads to several disease states, including IUGR, preterm delivery, postmature birth, and preeclampsia [8,20–22]. Recent research underlines the importance of inadequate or disrupted oxygenization (hypoxia-reoxygenization, (HR)) in the development of abnormal apoptosis. It is assumed that hypoxia will lead to the development of oxidative stress with production of free oxygen radicals that, in turn, will result in an imbalance of pro- vs. antiapoptotic gene activity. This imbalance in gene activity regulating apoptosis will result in abnormal apoptotic processes [23,24].

In this study, we assessed the expression patterns of the proapoptotic gene Bax, as well as the antiapoptotic gene Bcl-2,

The regulation of apoptosis in intrauterine growth restriction: a study of Bcl-2 and Bax gene expression in human placenta

Balázs Börzsönyi1, Csaba Demendi1, János Rigó Jr2, Imre Szentpéteri3, Attila Rab4 & József Gábor Joó2

1Semmelweis University, Second Department of Gynecology and Obstetrics, Budapest, Hungary, 2Semmelweis University, First Department of Gynecology and Obstetrics, Budapest, Hungary, 3Praxis für Gynäkologie und Geburtshilfe und allgemeine Medizin, Wehingen, Baden-Württemberg, Germany, and 4Hospital Telki, Telki, Hungary

Correspondence: Dr. Joó József Gábor, Semmelweis University, 1st Department of Obstetrics and Gynecology, Baross utca 27., Budapest, 1088 Hungary. E-mail: joogabor@hotmail.com

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Apoptotic gene expression in the background of IUGR

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in order to determine their possible role in the abnormal apop-tosis observed in IUGR. We hypothesized that altered expression of these regulatory genes (i.e. a decreased expression of Bcl-2 or an increased expression of Bax or both) may be involved in the enhanced apoptotic activity known to occur in IUGR. Our second aim was to clarify whether a correlation exists between the severity of IUGR and Bax or Bcl-2 gene activity. Finally, we aimed to establish whether maternal age, gestational weight gain, gestational body mass index (BMI) change, and maternal birth weight are predictors for the risk of IUGR.

Materials and methodsEthic statement

The research was ethically approved by the research ethics committee of the Hungarian Health Scientific Council (number: 6549-0/2010-1018EKU). Patients were recruited in the Semmelweis University, Budapest.

Patient population

We obtained placental samples for genetic studies from all patients treated for IUGR in our clinic at the Second Department of Gynecology and Obstetrics, Semmelweiss University, Budapest, in the study period between 1 January 2010–1 January 2011, as well as 140 placental samples from cases of normal pregnancy during the same time period. We also gathered clinical data from all patients including maternal age, gestational weight gain, gestational BMI change, and maternal birth weight. The diagnosis of IUGR was based on the standard definition as birthweight < 10 percentile for fetal sex and gestational age. (As we aimed to evaluate the genetic aspects of IUGR according severity, it seemed to be necessary to define IUGR as birthweight under 10 percen-tile.) The IUGR group was subdivided into two subgroups by the degree of growth restriction as below: less severe growth restric-tion defined as birth weight of 5–10 percentile vs. more severe growth restriction (0–5 percentile). Abdominal circumference (AC), biparietal diameter (BPD), occipitofrontal diameter (OFD), and femur length (FL) were also considered when establishing the clinical diagnosis of IUGR [17]. In the sonographic diagnosis Doppler velocitometry of the umbilical artery was also applied.

Only those placentas were included in the study where IUGR was likely to be due to placental dysfunction after the exclusion of intrauterine infections, chromosomal abnormalities, devel-opmental disorders, maternal malnutrition, multiple pregnancy, and structural abnormalities in the placenta [18].

Delivery was either vaginal or by cesarean section based on the clinical situation. In the final analysis, no distinction was made with respect to the type of delivery.

Placental tissue samples were taken from the neighborhood of the source of the umbilical cord right after the delivery in the delivery room (or in the operating theater) in a uniform manner with approximate dimensions of 8 cm3, which were then kept at –70°C for genetic expression testing.

Maternal demographics and relevant clinical data during preg-nancy or the perinatal period were collected including maternal and paternal age, obstetric history, genetic history, general medical

history, maternal birthweight, gestational age, fetal gender, weight gain, and BMI increase during pregnancy, pregnancy-related pathology including disorders of carbohydrate metabolism, neonatal weight and Apgar score. Consent was obtained in each case from the mother (signatures on file).

Primer design

Primers were designed using Primer Express Software (Applied Biosystems, Foster City, CA, USA) selecting for a primer annealing temperature of 55°C and amplicon length of < 100 bp. Primer sequences are detailed in Table I.

DNA standard analysis

To determine the quantification range, sensitivity, and viability just caused by the PCR assay, the DNA standard curve was amplified on real-time PCR cycler (realplex; Eppendorf, Hamburg, Germany).

RNA standard analysis

A comparable setup was chosen RNA standards to show the influ-ence of reverse transcription. Reverse transcription and subse-quent quantitative-PCR were done with RNA standards to look for technical variations induced by sampling and amplification process starting at RNA level.

RNA isolation and cDNA synthesis

Whole placental RNA content was isolated with Quick RNA microprep kit (Zymo Research, Irvine, CA, USA). RNA concen-tration was determined using NanoDrop spectophotometer (NanoDrop, Wilmington, DE, USA).

Complementary DNA (cDNA) synthesis out of RNA standard were performed following the next method: an amount of 1 μl of reaction mix was pipetted on six spots of each RNA standard dilution. One spot was used as negative reverse transcription control, without adding reverse transcriptase. The 1 μl-reaction mix was immediately covered by a 5 μl sealing solution to avoid evaporation.

Reverse transcription was performed in 20 μl target volume using 5 μg whole RNA, 75 pmol random hexamer primer, 10 mM dNTP (Invitrogen, Carlsbad, CA, USA), 20 U M-MuLV Reverse Transcriptase enzyme (MBI Fermentas, Hanover, MD, USA) and 1×-es buffer (MBI Fermentas). The reaction mix was incubated for 2 h at 42°C. Subsequently, the enzyme was inactivated at 70°C for 15 min.

Quantification by real-time PCR assay

All PCR experiments from cDNA were done with realplex (Eppendorf). Samples were measure in transparent, fully skirted, 96-well plates (Eppendorf), which were heat-sealed, using highly transparent films and the 4s2 heat-sealer (4titude, Berlin, Germany) to prevent from any evaporation during PCR.

The reverse transcriptase reaction solution was diluted three-fold with nuclease-free water. For the real-time PCR assay, 1 μl diluted cDNA (~15 ng RNA-equivalent) and 1× SYBR Green Master Mix (Applied Biosystems) were used. Real-time PCR was performed in 20 μl target volume using 1 μl cDNA, 1 pmol, gene-specific Forward and Reverse primer and 1× SYBR Green PCR

Table I. Primers and sequences in real-time PCR studies. Gene name and code Forward primer Reverse primerBcl-2 (NM_000633) 5ʹ-ATGTGTGTGGAGAGCGTCAACC-3ʹ 5ʹ-TGAGCAGAGTCTTCAGAGACAGCC-3ʹBax (NM_004324) 5ʹ-CCTTTTCTACTTTGCCAGCAAAC -3ʹ 5ʹ-GAGGCCGTCCCAACCAC -3ʹβ-Actin (M10277) 5ʹ-GGCACCCAGCACAATGAAG-3 5ʹ-GCCGATCCACACGGAGTACT-3ʹ

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Master mix. All real-time PCR were performed using the MX3000 Real-time PCR (Stratagen, Santa Clara, CA, USA) system with the following settings: 40 cycles at 95°C, denaturing process for 15 s, annealing at 60 °C, chain elongation and detection for 60 s. For each gene, relative expression was normalized using the human β-actin gene as standard.

Statistical analysis

For gene expression studies of Bcl-2 and Bax genes two-sample t-test was used with 95% confidence interval. Determination of degree of freedom was performed using the Welch–Satterthwaite correction. Values of gene expression testing were interpreted in the following manner: (1) overexpression = Ln value >1, p < 0.05; (2) underexpression = Ln value < -1, p < 0.05; (3) normal expression = Ln value < 1, >–1, p < 0.05. GraphPad Prism 3.0 (GraphPad Software Inc., La Jolla, CA, USA) software was used in all statistical analytic procedures.

Demographics and clinical data were analyzed with SPSS soft-ware. Logistic regression was used for dichotomous outcomes with multiple independent variables. For continuous outcomes, analysis of variance (ANOVA) and linear regression were used as appropriate. p value of < 0.05 was accepted for statistical significance.

ResultsClinical data

In the IUGR group (n = 101) the male to female ratio was 0.58, whereas in the control group (n = 140) it proved to be 1.09. In the IUGR group, median maternal age was 30.82 ± 4.34, not statistically different from the normal pregnancy group (31.45 ± 3.12, p > 0.05). IUGR was more common in age groups 17–24 and 35–44 when compared to age groups 25–31 and 32–34. There was a significant difference between the two groups in terms of gestational weight gain and gestational increase of BMI. In the IUGR group, mean gestational weight gain was 10.9 kg, whereas in the control group it was 14.8 kg. Similarly, in the IUGR group BMI increased by 4.1 compared to an increase of 5.3 in the control group. As far as maternal birthweight, those mothers who delivered babies with birth-weight between 0–5 percentile themselves had been born with a median birthweight of 2830 g, a birthweight significantly lower than that of mothers who delivered babies with birthweight between 5–10 percentile. Median maternal birthweight in the latter group of mothers was 3120 g.

Gene expression studies-bax gene expression

The Bax gene was elected for the assessment of proapoptotic activity. We obtained a total of 101 placental samples for the IUGR group vs. 140 control samples (Table II). There was no significant difference between the groups in Bax gene expression. Within the IUGR group there was no difference in the Bax gene activity between the two subgroups representing the more severe (0–5 percentile) or the less severe (5–10 percentile) degree of growth restriction (Table III).

Gene expression studies-Bcl-2 gene expression

In the IUGR group, the Bcl-2 gene was significantly underex-pressed compared to the control group (–2.17-fold underexpres-sion; p < 0.04) (Table II). Within the IUGR group there was no difference in Bcl-2 gene expression between the subgroups repre-senting the severity of growth restriction (Table III).

DiscussionThe genes affecting apoptosis can be detected during the entire gestational period [15,16,25]. There is controversy in the litera-ture as to the dynamics of Bcl-2 gene expression in normal fetal development during the final stages of gestation. Some studies [26,27] report an overexpression of Bcl-2 whereas there are even more reports suggesting that Bcl-2 is actually underexpressed in this time period [15,18,19,28,29]. In the case of Bax, most studies reveal increased gene activity toward the end of pregnancy [16,19]. This latter phenomenon fits with the observation of increased apoptotic activity of the trophoblasts in the time period immediately preceding delivery when this process is closely linked with physiological aging in the placenta. This represents a critical period necessitating intense gestational care with close follow up of both placental function and fetal activity.

In IUGR pregnancies some researchers report reduced Bcl-2 activity [29]; others [9] found that the imbalance of apoptotic regulatory genes in IUGR is explained by an overexpression of the Bax gene whereas Bcl-2 gene expression remains unchanged [9,29]. Our findings agree with those of Barrio, underlining the importance of a reduced inhibition of apoptosis with underex-pression of Bcl-2. Our study had a relatively large patient popu-lation compared to previous reports and adds strong support to Barrio’s findings. At the same time, we could not confirm the findings of Agata [9], or Heazell [10]. This would argue against the presence of an enhanced stimulatory effect by the Bax gene in IUGR.

Table II. Gene expression of the Bax and Bcl-2 genes in IUGR vs. controls (normal pregnancy). Gene name ΔCtnormal ± SE(I) ΔCtIUGR ± SE(III) α value ± SE (α)(III) Ln 2α LCL UCL p Gene expression

Bax 3.18 ± 0.82 4.04 ± 0.67 –0.86 ± 0.5 –0.59 0.43 –1.21 0.04 No changeBcl-2 3.18 ± 0.82 6.32 ± 0.86 –3.14 ± 0.81 –2.17 0.85 –3.79 0.04 Underexpression(I): ΔCtnormal = Ctexamined gene – Ctβ-actin; nIUGR = 101; nnormal = 140.(II): ΔCtIUGR = Ctexamined gene – Ctβ-actin.(III): α = ΔCtnormal – ΔCtIUGR.IUGR, intrauterine growth restriction; LCL, lower confidence limit; UCL, upper confidence limit.

Table III. Expression patterns of the Bax and Bcl-2 genes: 0–5 percentile birthweight vs. 5–10 percentile birthweight. Gene name ΔCtI ± SE ΔCtII ± SE α value ± SE (α) Ln 2α p Gene expression

Bax 4.32 ± 0.46 3.76 ± 0.3 0.56 ± 0.28 0.38 0.03 No changeBcl-2 6.92 ± 0.58 5.7 ± 0.64 1.22 ± 0.53 0.84 0.04 No change(I): 5–10 percentile IUGR placental sample; ntotal = 101 (nA = 60, nB = 41). (II): 0–5 percentile IUGR placental sample; ΔCtI = Ctexamined gene – Ctβ-actin; ΔCtII = Ctexamined gene – Ctβ-actin; α = ΔCtI – ΔCtII.IUGR, intrauterine growth restriction.

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De Falco suggested that the Bcl-2 and the Bax gene activity be assessed simultaneously, arguing that it is the ratio of the activities rather than the independent effect of either genes that determines the final outcome [16]. We propose that this argument is even more relevant in disease states like IUGR, when the regulation of apoptosis likely undergoes a complex change. In this context, our findings show that the ratio of activities did indeed change. However, this change was effected not by an increased stimula-tory influence, but rather a reduced anitapoptotic effect.

We found that the gene expression patterns of Bcl-2 and Bax did not correlate with the severity of growth restriction. We propose that the degree of growth restriction is not primarily determined by the activity of apoptotic genes, but rather by other presumably genetic factors.

While in the control group our findings approached the expected male to female ratio, in the IUGR group female fetal gender was significantly more common. Another clinical predictor of IUGR risk was maternal age. Although there was no difference between the two groups in terms of median maternal age, mothers within the age groups 17–24 years or 35–44 years had significantly more risk for IUGR than other age groups. This suggests that from the obstetric point of view, toward the extremes of maternal age, gestational fetal development may not be as optimal as with a maternal age closer to the median [30].

Both gestational weight gain and gestational BMI change were significantly reduced in IUGR pregnancies. Specifically, a less than average (12.5–13 kg) weight gain seemed to predict IUGR.

Maternal birth weight was associated with the degree of IUGR. Those mothers who gave birth to an infant with 0–5 percentile birth weight had themselves been born with a significantly lower birth weight than mothers giving birth to infants with 5–10 percentile birth weight.

In summary, Bcl-2 gene expression in human placental samples from IUGR pregnancies was found to be significantly reduced compared to normal pregnancy. There was no differ-ence in the activity of the Bax gene between the two groups. We conclude that the increased apoptotic activity observed in IUGR is primarily induced by a reduced inhibition of apoptosis by the Bcl-2 gene and not by increased stimulatory effect by Bax. The degree of IUGR in IUGR does not correlate with either Bcl-2 or Bax gene activity. The risk for IUGR is predicted by several epide-miological and clinical factors, including a maternal age close to 20 or above 35 years, reduced gestational weight or BMI gain and maternal birth weight.

Declaration of Interest: The authors report no declarations of interest.

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