Soluble Fas and Fas-ligand levels in mid-trimester amniotic fluid and their associations with severe small for gestational age fetuses: a prospective observational study

Journal of Reproductive Immunology 98 (2013) 39–44

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Soluble Fas and Fas-ligand levels in mid-trimester amniotic fluid and their associations with severe small for gestational age fetuses: a prospective observational study N. Vrachnis a,∗ , I. Dalainas a , D. Papoutsis b , E. Samoli c , D. Rizos a , Z. Iliodromiti a , C. Siristatidis d , P. Tsikouras e , G. Creatsas a , D. Botsis a a b c d e

Second Department of Obstetrics and Gynecology, University of Athens Medical School, Aretaieio Hospital, Athens, Greece Department of Obstetrics and Gynaecology, Royal Shrewsbury Hospital, West Midlands Deanery, United Kingdom Department of Hygiene, Epidemiology and Medical Statistics, Medical School, University of Athens, Athens, Greece Third Department of Obstetrics and Gynecology, University of Athens, Attiko Hospital, Athens, Greece Department of Obstetrics and Gynecology, University Hospital of Alexandroupolis, Democritus University of Thrace, Greece

a r t i c l e

i n f o

Article history: Received 13 January 2013 Received in revised form 12 February 2013 Accepted 19 February 2013 Keywords: Amniotic fluid Apoptosis sFas Fas-ligand Small for gestational age (SGA)

a b s t r a c t We aimed to determine the second-trimester amniotic fluid (AF) levels of soluble Fas (sFas) and Fas-ligand (FasL) and investigate their association with fetal growth. Therefore, sFas and FasL levels were measured by enzyme immunoassay in the AF of 21 small for gestational age (SGA), 13 large for gestational age (LGA), and 44 appropriate for gestational age (AGA) fetuses of pregnant women who underwent amniocentesis at between 15 and 22 weeks gestation. Our study results showed that sFas and FasL levels were detectable in AF. sFAS median (25th–75th centile) levels were 3.8 (2.8–4.6) ng/ml in SGA, 3.6 (3.1–4.5) ng/ml in AGA, and 4.0 (3.1–4.4) ng/ml in LGA. FasL median (25th–75th centile) levels were 26.0 (20.3–32.7) pg/ml in SGA, 22.7 (18.4–28.5) pg/ml in AGA, and 21.5 (15.8–30.9) pg/ml in LGA. The differences were not statistically significant. Nevertheless, statistically significant differentiation of FasL levels existed when SGA fetuses in the extremes of distribution (≤5th, ≤2.5th centile) were considered. This is the first study presenting sFas and FasL concentrations in early second-trimester amniotic fluid in AGA, SGA, and LGA fetuses. We found indications that severe and very severe SGA fetuses (≤5th and ≤2.5th centile) have high levels of FasL in the amniotic fluid. This finding probably reflects the increased rate of apoptosis that is assumed to exist in cases of extreme growth restriction. © 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Amniotic fluid provides a dynamic environment for the fetus; it is formed of fetal urine and lung excretions, and it contains molecules and factors that facilitate fetal growth (Malamitsi-Puchner et al., 2005, 2006). Small for gestational age (SGA) generally refers to a fetal weight below

∗ Corresponding author at: University of Athens Medical School, 124B Vas. Sofias, Postal Code 11526, Athens, Greece. Tel.: +30 2107789211. E-mail address: [email protected] (N. Vrachnis). 0165-0378/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.jri.2013.02.003

the 10th centile for gestational age (Vrachnis et al., 2006) and fetuses are considered large for gestational age (LGA) if their weight is greater than the 90th centile for gestational age (Alexander et al., 1996). If their weight is between the 10th and 90th centile for gestational age then the fetuses are considered appropriate for gestational age (AGA). Small for gestational age fetuses are considered to be at a higher risk of perinatal and later life complications (Botsis et al., 2006; Vrachnis et al., 2010). The underlying mechanism(s) that lead(s) to an SGA fetus remains undetermined, while reliable measures for prevention have not as yet been established. This raises the urgent need for the

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identification of biomarkers that will help to better understand the mechanisms leading to this condition so as to develop effective strategies for early detection, prevention and treatment of the condition (McMillen and Robinson, 2005; Wu et al., 2006). Fas (CD95) is a 45-kDa cell surface receptor of the tumor necrosis factor (TNF)/nerve growth factor family (Nagata and Goldstein, 1995). It is expressed as a type I membrane protein in many tissues and cells such as the heart, lung, liver, kidney, and ovary (Watanabe-Fukunaga et al., 1992). Fas-ligand (FasL) is also a member of the TNF family and is a type II membrane protein predominantly expressed in activated cytotoxic T-lymphocytes, natural killer cells, and neutrophils (Iwama et al., 2000). The FasL expressed on the surface of these cells binds to the Fas receptor оf the target cells, which results in downstream activation of a cascade of intracellular proteolytic enzymes, eventually leading to apoptosis (Nagata, 1994; Nagata and Goldstein, 1995). The soluble form of FasL, whose soluble molecule consists of the extracellular region of FasL, is cleaved from membrane FasL by matrix metalloproteinase (Kayagaki et al., 1995) and then binds to Fas receptor on target cells to induce apoptosis (Tanaka et al., 1995). Although the relationship between the membrane and soluble forms of Fas/FasL (sFas/sFasL) is still under investigation, it has been suggested that sFas has a suppressive effect on Fas/FasL-mediated apoptosis (Nagata and Goldstein, 1995), whereas sFasL may have both an augmentative and a diminutive effect (Iwama et al., 2000). Since the Fas/FasL signaling system may promote apoptosis, which is an essential physiological process for the normal development of an embryo (Kaponis et al., 2008), we sought to examine the levels of sFas, with its suppressive effect on apoptosis, and FasL, which promotes apoptosis in the amniotic fluid of second-trimester fetuses. We also explored the association between sFas and FasL concentrations in SGA/LGA fetuses, taking into account that amniotic fluid composition seems to be similar to fetal plasma during the first half of pregnancy (Underwood and Gilbert, 2005). It might therefore be concluded that sFas and FasL amniotic fluid concentrations reflect the sFas/FasL concentrations in the fetal plasma of normal (AGA) and abnormal (SGA/LGA) fetuses. Our study is the first to our knowledge to determine any detectable concentrations of sFas and FasL in early second-trimester amniotic fluid samples and to investigate any potential differences among the SGA, AGA, and LGA fetuses.

2. Materials and methods The study group consisted of 300 pregnant women who underwent an equal number of amniocenteses early in the second trimester of their pregnancy. Routine mid-trimester amniocentesis was carried out between 15 and 22 gestational weeks, for various indications, which included advanced maternal age, abnormal nuchal translucency screening, past history of genetic disorder and identification of fetal abnormality on the second-trimester ultrasound screening. Twin pregnancies or pregnancies with major congenital anomalies were excluded. The study

was approved by the Ethics Committee of our teaching hospital and patients’ informed consent was obtained. All patients were Caucasians. A questionnaire including maternal age, weight, height, parity, cigarette smoking, medical and obstetrical–gynecological history was completed before each procedure. Gestational age was calculated by the first day of the last menstrual period and confirmed by the crown rump length of the embryos, as determined in the first trimester ultrasound examination. The duration of pregnancy, mode of delivery, neonatal birth weight/gender, and the outcome were also recorded. The amniotic fluid samples were collected in pyrogenfree tubes and immediately centrifuged, and were kept frozen at −80◦ until the determination of sFas and FasL levels. In order to allocate the centile of each fetus at delivery, a gestation-related optimal weight (GROW) computer generated program was used (Gardosi and Francis, 2009). Fetuses below the 10th customized centile were characterized as SGA and those above the 90th customized centile as LGA. Our study sample consisted of 21 SGA fetuses and 13 LGA fetuses, which were matched for gestational age, sex, maternal height, and weight and compared with 44 AGA fetuses that comprised the control group. We additionally compared the concentrations of sFas and FasL in AGA fetuses with those in fetuses with more extreme somatometric characteristics, namely severe SGA/LGA fetuses as defined by the ≤5th and ≥95th centiles respectively, and very severe SGA/LGA fetuses as defined by the ≤2.5th and ≥97.5th centiles respectively. The determination of sFas and FasL was performed using the commercial enzyme immunoassay (ELISA) kits Quantikine Human sFas immunoassay and Quantikine Human Fas Ligand/TNFSF6 immunoassay respectively (R&D Systems Inc., Minneapolis, MN, USA). The minimum detectable concentration for sFas was 20 pg/mL and for FasL it was 2.7 pg/mL. The precision of the sFas kit, as estimated with %CV, ranged from 2.9% to 6.7% and for the FasL it ranged from 4.1% to 8.8%. Mainly because of the small sample sizes within each group, the distribution of the measured variables and the mothers’ characteristics deviate from the normality assumption. We used the Kruskal–Wallis test for comparison of the concentrations of substances among the three groups. Mean and standard deviations for quantitative variables are presented, as well as the number and percentages for qualitative variables. We also applied logistic regression to investigate the risk of SGA vs AGA and LGA vs AGA associated with sFas and FasL concentrations. To control for possible confounding factors, we adjusted the models for maternal age (continuously, in years), Body Mass Index (BMI) (categorically <25, 25–29, 30+ kg/m2 ), duration of gestation (continuously, in weeks), gender of offspring (boy vs girl), smoking during pregnancy (yes vs no, for the association between SGA and AGA), and parity (parous vs nulliparous). Significance was set at a p level of <0.05. 3. Results Table 1 presents the descriptive characteristics of mothers and fetuses. There were no statistically significant

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Table 1 Demographic data of participating mothers and fetuses (AGA, SGA or LGA). Mean and standard deviations for quantitative variables, number, and percentages for qualitative variables. AGA fetuses (n = 44) Maternal age (years) Maternal BMI (kg/m2 ) Parity 0 1+ Duration of pregnancy (weeks) Natural birth Yes No Birth weight (g) Gender of offspring Male Female Maternal smoking Yes No a

SGA fetuses [≤10th centile] (n = 21)

LGA fetuses [≥90th centile] (n = 13)

35.7 (3.1) 23.2 (3.8)

34.4 (3.7) 26.2 (6.6)

32.5 (5.4) 22.8 (3.4)

12 (30.0) 28 (70.0) 38.9 (0.8)

6 (31.6) 13 (68.4) 37.9 (2.5)

3 (25.0) 9 (75.0) 38.2 (1.2)

20 (45.5) 24 (54.5) 3339.8 (216.9)

12 (57.1) 9 (42.9) 2509.8 (531.5)

9 (69.2) 4 (30.8) 3798.5 (368.4)

21 (50.0) 21 (50.0)

12 (63.2) 7 (36.8)

3 (25.0) 9 (75.0)

1 (2.3) 42 (97.7)

4 (20.0) 16 (80.0)

0 (0.0) 12 (100.0)

p-Valuesa 0.075 0.356 0.923

0.116 0.284

≤0.001 0.116

0.020

Overall comparison of groups using the Kruskal–Wallis test for quantitative variables and Chi-squared test for qualitative variables.

differences with regard to maternal age, maternal BMI, parity, duration of gestation, mode of delivery or gender of offspring among the three groups. Nevertheless, there was a statistically significant difference in size status related to maternal smoking habits (p = 0.020), since none of the mothers with LGA fetuses were smokers. Spearman correlation coefficient between sFas and FasL in the amniotic fluid for the whole sample was r = 0.40 (p = 0.001) and for the AGA fetuses it was r = 0.43 (p = 0.004). It was positive, but non-statistically significant for SGA and LGA fetuses. The correlation was positive and statistically significant among SGA (r = 0.55, p = 0.010), while it was positive, but not statistically significant among LGA fetuses (r = 0.42, p = 0.150). Table 2 presents the comparison of the distribution of sFas levels by fetal size. Amniotic fluid sFas levels were not statistically significantly different by fetal size either when we considered SGA fetal sizes as usually defined (0th–10th centile definitions) or when we took into account severe sizes compared with AGA (p = 0.901 among the three groups; p = 0.534 between AGA and very severe SGA). However, sFas concentrations were increased, though not significantly, in women who had severe and very severe SGA fetuses (p = 0.878 and p = 0.534 respectively) for the comparison of the three groups. Fig. 1 presents the box-plot of sFas levels by fetal size.

Table 3 presents the comparison of the distribution of FasL levels by fetal size. Amniotic fluid FasL levels were statistically significantly different among the three groups, namely AGA and severe (at the 10% level of significance) or very severe (at the 5% level) SGA/LGA fetuses. These associations were driven by the significantly higher levels in SGA compared with AGA fetuses (p = 0.034 and p = 0.008 correspondingly for the comparisons between AGA and severe or very severe SGA). Fig. 2 presents the box-plot of FasL levels by fetal size. After adjustment for confounding factors the probability of an SGA or LGA fetus was not dependent on sFas

Table 2 Distribution of measured sFas (ng/ml) by group. Median levels (25th–75th centile). 25th–75th centile

a

Fetus

n

Median

AGA SGA ≤10th centile ≤5th centile ≤2.5th centile LGA ≥90th centile ≥95th centile ≥97.5th centile

44

3.6

3.1–4.5

p-Values

21 12 7

3.8 4.1 4.0

2.8–4.6 2.9–5.1 3.2–5.4

0.901 0.878 0.534

13 8 1

4.0 3.7 3.0

3.1–4.4 3.1–4.4

0.901 0.878 0.534

a Comparisons of sFas and FasL levels respectively, among AGA, SGA, and LGA fetuses, were performed using the Kruskal–Wallis test.

Fig. 1. Box-plot of sFas by fetal size. The symbol “o” indicates extreme values.

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Table 3 Distribution of measured Fas-ligand (pg/ml) by group. Median levels (25th–75th centile). 25th–75th centile

a

Fetus

n

Median

p-Values

AGA SGA ≤10th centile ≤5th centile ≤2.5th centile LGA ≥90th centile ≥95th centile ≥97.5th centile

44

22.7

18.4–28.5

21 12 7

26.0 27.0 33.4

20.3–32.7 23.0–35.1 26.0–76.2

0.307 0.097 0.026

13 8 1

21.5 23.2 25.0

15.8–30.9 16.0–32.3

0.307 0.097 0.026

a Comparisons of sFas and FasL levels respectively, among AGA, SGA, and LGA fetuses were performed using the Kruskal–Wallis test.

and FasL concentrations. However, the sample size was too small to adequately perform this type of analysis. 4. Discussion We calculated the levels of sFas and FasL in the amniotic fluid of fetuses between 15 and 22 weeks of gestation and we found that sFas concentrations were similar among SGA, LGA, and AGA fetuses. However, FasL was significantly increased at the extremes of distribution (≤5th and ≤2.5th centile) for SGA fetuses in comparison to AGA fetuses. Since in our study severe (≤5th centile) and very severe (≤2.5th centile) SGA fetuses had significantly increased FasL concentrations, this may reflect an accelerated condition of Fas/FasL-mediated apoptosis in cases of severe growth restriction. Apoptosis is a physiological procedure crucial for normal fetal development, serving the purpose of tissue homeostasis, removal of unwanted cells (Wyllie et al.,

Fig. 2. Box-plot of Fas-ligand by fetal size. The symbol “o” indicates extreme values.

1980), and regulation of immune responses (Ashkenazi and Dixit, 1998). In women undergoing in vitro fertilization (IVF), moderate levels of apoptosis were shown to be required for the healthy development of the preimplantation embryo, while increased apoptosis leads to embryo fragmentation (Hardy et al., 2003; MalamitsiPuchner et al., 2004). Apoptotic changes have also been documented in the cells of blastocysts on day 6 of embryonic development (Hardy, 1999). This apoptosis facilitates the removal of defective cells and also exerts a homeostatic role in regulating cell numbers (Hardy et al., 2003; Malamitsi-Puchner et al., 2004). During implantation of the zygote, there is an increase in apoptosis among glandular and stromal cells that allows the trophoblastic invasion to the endometrium, and this event has been related to increased expression of FasL by the endometrial cells (Kayisli et al., 2003; Harada et al., 2004). During pregnancy, the Fas/FasL system additionally plays a substantial role in placental development as it regulates trophoblast invasion and spiral artery remodeling (Ashton et al., 2005). In cases of increased trophoblast apoptosis, abnormal placentation has been demonstrated, resulting in uteroplacental insufficiency and growth restriction (Smith et al., 1997; Ishihara et al., 2002). Apoptosis also seems vital during transition from intrauterine to extrauterine life. Two reports found increased levels of the soluble form of FasL (sFasL) in the umbilical cord of healthy term fetuses measured at the time of delivery via either cesarean section or vaginal delivery, showing an acceleration of Fas/FasL-mediated apoptosis, probably due to placental degradation and in preparation for extrauterine adaptation (Iwama et al., 2000; Briana et al., 2010). Postpartum, sFasL serum levels are decreased compared with those at the time of delivery in both normal and IUGR neonates (MalamitsiPuchner et al., 2001; Briana et al., 2010). In adult life, the Fas/FasL system still maintains its significance as one of the main pathways triggering apoptosis in pathological conditions (Musial and Zwolinska, 2011); of note, the consequences of dysregulated Fas/FasL-mediated apoptosis have been closely linked with self-reactivity, malignant transformation, and immune dysfunction (Randhawa et al., 2010). A similar increase in Fas/FasL-mediated apoptosis has also been reported in pathological conditions. In early human pregnancy (first trimester), there are data suggesting that missed miscarriage is accompanied by increased Fas/FasL protein expression in fetal membranes and, therefore, aberration of the Fas-mediated apoptosis may lead to pregnancy loss (Kaponis et al., 2008). It has been reported that FasL is expressed by fetal membranes throughout the entire period of gestation (Runic et al., 1996) and that Fas signaling plays a role in the remodeling of fetal membrane architecture in parturition (Runic´ et al., 1998). In this latter study, in pregnancies complicated by infection, ischemia, preeclampsia, diabetes, and the premature rupture of membranes, a high apoptotic index was demonstrated in amnion epithelial cells, further suggesting that high levels of apoptosis may reflect a pathological condition before term. Other studies report elevated levels of FasL in preeclampsia, HELLP syndrome as well as IUGR (Kuntz

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et al., 2001; Hu et al., 2005; Prusac et al., 2011). In our study, the SGA fetuses lived in pathological centiles. In the present study, severe and very severe SGA fetuses demonstrated significantly higher levels of FasL in the amniotic fluid, thus indicating an increased state of apoptosis. The reason for this outcome is not yet known and only assumptions can be made. It is reported that apoptosis can either result from activation of an endogenous program or it may be induced by various stimuli (Hardy, 1999). In the first case, the increased FasL levels may reflect the increased apoptosis of fetal membrane cells and structures which, for reasons inherent to the fetus, lead to failure to achieve normal fetal growth. As to the second hypothesis, it is known that Fas/FasL-mediated apoptosis is involved in regulating immune tolerance at the feto-maternal interface (Straszewski-Chavez et al., 2005; Kauma et al., 1999). Possibly the increased FasL expression in SGA fetuses (severe, very severe) reflects the increased apoptosis in the fetus as a result of impaired maternal–fetal tolerance due to undetermined stimuli. There are thus far no literature data to confirm either of these two assumptions and further research is needed into these findings. 5. Conclusion An investigation was undertaken of the levels of sFas and FasL that are present in second-trimester amniotic fluid. The study did not demonstrate statistically significant differences in sFas concentrations among SGA fetuses, LGA fetuses, and AGA controls. However, in SGA fetuses below the 5th and 2.5th customized centile for birth weight we found higher levels of FasL compared with those of AGA and LGA fetuses. To our knowledge, this is the first study to investigate sFas and FasL levels in second-trimester amniotic fluid in relation to the size of fetuses. Further studies potentially validating our results will lead to greater understanding of these conditions and their implications, thus providing new insights for research concerning preventive and therapeutic interventions earlier in pregnancy. Conflict of interests We declare that we have no conflict of interest. Contributions Vrachnis, conceived the study design, performed the ultrasound investigations, collected samples, interpreted the data, drafted the manuscript, revised the manuscript. Dalainas, collected the samples, interpreted the data, edited the manuscript. Papoutsis, drafted the manuscript, edited the manuscript. Samoli, performed the data analysis, drafted the manuscript. Rizos, performed the determination of concentrations of sFas and FasL, revised the manuscript. Iliodromiti, Siristatidis, Tsikouras, and Creatsas, revised the manuscript. Botsis, performed the ultrasound investigations, collected samples, made critical revisions.

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