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Telomere shortening in intra uterine growth restriction placentas
Early Human Development 90 (2014) 465–469
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Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Telomere shortening in intra uterine growth restriction placentas☆ Tal Biron-Shental a,b,⁎, Rivka Sukenik-Halevy a,b,c, Yudith Sharon c,d, Ido Laish b, Moshe D. Fejgin a,b,c, Aliza Amiel c,d a
Department of Obstetrics and Gynecology, Meir Medical Center, Kfar Saba, Israel Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Genetic Institute, Meir Medical Center, Kfar Saba, Israel d Faculty of Life Science, Bar Ilan University, Ramat Gan, Israel b c
a r t i c l e
i n f o
Article history: Received 15 April 2014 Received in revised form 29 May 2014 Accepted 4 June 2014 Available online xxxx Keywords: Placenta Trophoblasts Telomere hTERT Telomere capture IUGR Senescence
a b s t r a c t Introduction: Placentas from pregnancies complicated with IUGR (intrauterine growth restriction) express altered telomere homeostasis. In the current study, we examined mechanisms of telomere shortening in these placentas. Methods: Placental biopsies from 15 IUGR and 15 healthy control pregnancies were examined. The percentage of trophoblasts with fragmented nuclei: senescence-associated heterochromatin foci (SAHF), was calculated using DAPI staining. The amount of human telomerase reverse transcriptase (hTERT) mRNA was evaluated using RtPCR levels of telomere capture using FISH in those samples were estimated. Results: The percentage of trophoblasts with SAHF was higher in IUGR compared to control samples, (25 ± 13.4% vs. 1.6 ± 1.6%, P b 0.0001), hTERT mRNA was decreased (0.5 ± 0.2 vs. 0.9 ± 0.1, P b 0.0001) and telomere capture was increased (13.2 ± 9.7% vs.1.3 ± 2.5%, P b 0.001). Conclusions: We suggest that IUGR placentas express increased signs of senescence as part of the impaired telomere homeostasis. One factor that mediates telomere shortening in these placentas is decreased hTERT mRNA, leading to decreased protein expression and therefore, reduced telomere elongation. Telomere capture, which is a healing process, is increased in IUGR trophoblasts as a compensatory mechanism. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Intrauterine fetal growth restriction (IUGR), defined as fetal growth below the tenth percentile, is associated with increased short and long term morbidities [1–3]. IUGR is caused by genetic and environmental factors related to maternal, placental or fetal abnormalities [1–3]. The placenta, as a mediator of all communications between the mother and the fetus, has a key role in the pathways of fetal growth and development [2,3]. Therefore, placental research is essential for enhancing our understanding of IUGR secondary to placental insufficiency. Telomeres are nucleoprotein structures located at the termini of chromosomes that protect them from fusion and degradation. Telomeres are progressively shortened with each mitotic cycle and by environmental
Abbreviations: hTERT, human telomerase reverse transcriptase; SAHF, senescence associated heterochromatin foci; RtPCR, real time PCR; FISH, fluorescence in situ hybridization. ☆ Condensation: Telomere homeostasis is impaired in IUGR placentas by increased senescence, decreased hTERT-mRNA, leading to telomere shortening, and by increased telomere capture, as a compensatory mechanism. ⁎ Corresponding author at: Department of Obstetrics and Gynecology, Meir Medical Center, Tel Aviv University, Kfar Saba 44281, Israel. Tel.: +972 528362331. E-mail address: [email protected] (T. Biron-Shental).
http://dx.doi.org/10.1016/j.earlhumdev.2014.06.003 0378-3782/© 2014 Elsevier Ltd. All rights reserved.
factors [4,5]. Telomere biology was suggested as a mechanism connecting fetal intrauterine programming and subsequent health conditions [6]. Intrauterine exposure to stress such as hypoxia, which causes placental insufficiency leading to IUGR, might cause changes in the telomere system that accelerate telomere shortening, cellular dysfunction, aging, and disease susceptibility [6]. Telomere homeostasis is impaired in placentas from pregnancies complicated by IUGR secondary to placental insufficiency [7–9]. Telomeres are shorter in IUGR placentas than in placentas from healthy controls [7,8]. Moreover, telomeres tend to cause aggregates, reflecting dysfunction unrelated to their length [8,9]. Telomerase is an enzyme complex that elongates telomeres. hTERT (human telomerase reverse transcriptase) is the catalytic component of telomerase. Protein levels of hTERT are significantly lower in IUGR placentas [8,9]. One component of telomerase, which serves as the RNA template for the addition of telomeric repeats, is encoded by the telomerase RNA component gene (TERC). The TERC gene copy number is decreased in IUGR trophoblasts [10]. These data suggest that impaired telomere homeostasis might play a role in the pathophysiology of IUGR and might affect intrauterine programming related to the origin of some diseases that arise in adulthood [6,11–13]. However, the mechanisms of telomere shortening in these placentas are not fully understood.
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Senescence is an arrest of cellular proliferation and is involved in the biological process of tissue aging. It is driven by cell division, as well as oxidative stress, DNA damage and repair response, inflammation and telomere shortening [4,14]. Nuclei in senescent human cells are often granulated, containing senescence-associated heterochromatin foci (SAHF) [15]. The relationship between cellular senescence and telomere shortening is well established [14–18], but has not been described in IUGR placentas. When telomeres are shortened to a critical length, repair pathways are recruited. One repair pathway is telomere capture, through which a shortened telomere acquires a new telomeric sequence from a sister chromatid or from another chromosomal end [16–18]. In this study, we examined mechanisms of telomere shortening that have not been previously described in IUGR placentas. 2. Materials and methods 2.1. Study groups and sample collection The study was approved by the institutional ethics review board. We examined biopsies from 15 third-trimester placentas derived from pregnancies complicated by IUGR and 15 placentas matched for gestational age from uncomplicated pregnancies, as controls. All patients signed an informed consent. IUGR was defined as neonatal birth weight below the 10th percentile for gestational age, based on the standardized Israeli curves [19]. Only placental specimens from pregnancies complicated with IUGR attributed to placental insufficiency were included. Other reasons for IUGR, such as intrauterine viral infections, chromosomal abnormalities, congenital anomalies, poor pregnancy dating, or maternal smoking were ruled out. Exclusion criteria were multi-gestational pregnancies, patients with no prenatal care who lacked information regarding possible reasons for IUGR other than placental insufficiency or inaccurate dates. All newborns were evaluated by a neonatologist within 24 h of delivery. Major malformations or suspected genetic congenital syndrome that could be related to small fetus were ruled out. All placentas were biopsied immediately after delivery under sterile conditions. From the samples of intermediate trophoblasts, 1 cm2 was taken from the area midway between the cord insertion and the edge of the placenta. The placental biopsy area was based on hypoxic gene expression in different areas in the placenta, as described by Wyatt et al. [20]. Each specimen was divided into 5 pieces of 2 mm2, kept on ice during the entire processing procedure to prevent RNA degradation and frozen at −80 °C within 20 min of delivery. 2.2. Fluorescence in situ hybridization (FISH) assay Frozen placental tissue sections of 4 μm were prepared for fluorescence in situ hybridization (FISH) using the tissue pretreatment kit (Biomedicals, North America, Solon, OH, USA). Initially, the slides were fixed in a 1:3 acetic acid and ethanol solution for 15 min. The slides were then placed in fresh 100% ethanol for 5 min, twice, allowed to air dry and then placed on a plate warmed to 80 °C for 60 min. The slides were incubated for 25 min in a glass jar containing the pretreatment solution preheated to 45 °C (12 g of pretreatment powder in 40 cm3 of 2XSCC), rinsed twice in 2XSSC buffer at room temperature, then incubated in a glass jar containing a protein digesting enzyme preheated to 45 °C for 45 min (400 μl in 40 cm3 of 2XSCC), rinsed twice in 2XSCC buffer at room temperature, dehydrated for 1 min in 70%, 80%, and 100% ethanol at room temperature and left to air dry for 5 min. Afterwards, the slides were placed on 94 °C warming plate for 4 min. The telomere capture was performed using a CY3 labeled telomere capture-specific peptide nucleic acid probe (vial 2 in K 532 DAKO, Glostrup, Denmark) following manufacturer's instructions. The slides were stained with 4′-6-diamidino-w-phenylindole (DAPI) (1000 ng/Ml, VYS-32-804830, Vysis, Abbott Laboratories. Abbott Park, IL, USA) and finally overlaid with glass cover slips for observation. Each placental specimen was
placed under an AX70 Olympus Provis fluorescent microscope (Olympus, Tokyo, Japan) at ×100 magnification. For each specimen, 100–200 trophoblasts were counted. The nuclei identified were selected as belonging to cytotrophoblasts by their appearance and arrangement in the villi. The percentage of trophoblasts with fragmented nuclei (SAHF) in each specimen was evaluated. The slides were blindly scored twice (and a third time whenever there was a difference of more than 10% between the counts). Positive and negative controls were used to ensure reliability of the assay. 2.3. Telomere capture In the nuclei, the number of the specific loci of the specific chromosome (SNRPN or 13q 14.3: orange, the “normal disomic loci”) and its telomere (15qter or 13qter: green) were compared. The number of signals of the SNRPN or 13q 14.3 locus was compared to the number of signals of the sub-telomeric region of the specific chromosome (green signals). For example, the normal appearance is two orange and two green signals, while an abnormal appearance, which represents telomere capture is two orange compared with one, three, or more green signals (the rearranged-captured sub-telomeric regions), previously described by Amiel et al. [21]. Approximately 200 nuclei from each sample were analyzed. Only cells in which two orange signals (normal “disomic loci”) appeared were analyzed. Positive and negative controls were used to ensure the reliability of the assay. 2.4. RT-PCR and RT-quantitative PCR (RT-qPCR) RNA was purified from placental biopsies using a Perfect-Pure RNA kit (5 Prime, Inc., Gaithersburg, MD, USA) according to the manufacturer's instructions. Complementary DNA (cDNA) was prepared from 300 ng RNA using the TaqMan Gold RT-PCR kit with the random hexamer primers supplied (Applied Biosystems, Inc., Foster City, CA, USA). For quantitative PCR (qPCR), cDNA was prepared using TaqMan RT-PCR kit (Applied Biosystems, Foster City, CA, USA) in a 20 μl reaction mix that contained 300 ng total RNA, 1× RT buffer, 5.5 mM MgCl, deoxy-NTP mixture (500 μM of each dNTP), 2.5 μM random hexamers, 0.4 units μl−1 RNase inhibitor, and 1.25 units ml−1 Multi-Scribe reverse transcriptase at 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. PCR was performed using 3 μl RT product, produced with 0.3 μM of the primer pairs in duplicate, with SYBR Green PCR Master Mix (Applied Biosystems, Inc., Foster City, CA, USA) in a total reaction volume of 25 μl. Reactions were analyzed using Geneamp 7500 Fast Instrument System (Applied Biosystems, Inc., Foster City, CA, USA). Dissociation curves were ran on all reactions to ensure amplification of a single product with the appropriate melting temperature. Fold change was computed relative to control using the 2−ΔΔCT method [21]. Samples were normalized to GAPDH expression [22,23], determined in parallel reactions. 2.5. Statistical analysis A 2-tailed sample t test and the nonparametric Mann–Whitney U test were applied to evaluate the differences between the groups, with P b .05 considered statistically significant. SPSS software (SPSS, Inc, Chicago, IL) was used for statistical analysis. 3. Results The study and the control groups were comparable in terms of demographic and baseline characteristics. As expected, birth weights were significantly lower and characteristics of placental insufficiency were significantly more prominent in the IUGR group. Although the inclusion criteria were birth weight below the 10th percentile, and the actual birth weights of the IUGR patients in the study were below the 5th percentile. All the IUGR fetuses had clinical and histological characteristics
T. Biron-Shental et al. / Early Human Development 90 (2014) 465–469
of placental insufficiency, 60% had oligohydramnios, and 53.3% had an abnormal umbilical artery Doppler. Each IUGR fetus had oligohydramnios and/or abnormal umbilical artery Doppler (Table 1). To evaluate senescence in IUGR placentas, we looked at the percentage of trophoblasts with SAHF in placental samples from 15 IUGR pregnancies and 15 healthy controls. Trophoblast nuclei with and without fragmentations are shown in Fig. 1. We found that the IUGR placentas had a significantly higher percentage of trophoblasts with fragmented nuclei, SAHF (25 ± 13.4%) compared to (1.6 ± 1.6%) the control samples, P b 0.0001 (Fig. 2). To understand some of the mechanisms of telomere shortening in the senescent placentas, we sought to confirm the down-regulation of telomerase by real time PCR and found that hTERT was decreased in IUGR placentas (0.5 ± 0.2) compared to controls (0.96 ± 0.1) (P b 0.0001) (Fig. 3). To evaluate the compensatory response of the IUGR trophoblasts to telomere shortening, we estimated the presence of cells with telomere capture in the study and control samples. An example of nuclei with and without telomere capture is shown in Fig. 4. The percentage of trophoblasts with telomere capture in the IUGR samples was significantly higher than in the controls (13.3 ± 9.8% vs. 1.4 ± 2.5%; P b 0.001) (Fig. 5). About 50% of the samples were drawn from placentas delivered via cesarean section. There were no differences in the percentage of cesarean sections between the study and control groups. Moreover, no differences in telomere homeostasis were found when analyzing the results based on mode of delivery. 4. Comment
Fig. 1. DAPI stained nuclei from placental trophoblasts displaying senescence. (A) A nucleus with 3 fragments from a placenta from a pregnancy complicated by IUGR (intrauterine growth restriction). (B) A nucleus from a normal placental trophoblast.
along with shorter telomeres in those placentas [8,9]. Moreover, TERC which is the telomerase RNA component and considered the gene that encodes telomerase, was found significantly less often in IUGR trophoblasts. This observation supports the role of telomerase activity and telomere shortening in IUGR caused by placental insufficiency [10]. In order to expand and strengthen these observations, we evaluated hTERT mRNA and found that it is reduced in IUGR placentas, suggesting that the lower levels of hTERT protein in these placentas is the consequence of decreased mRNA leading to reduced protein production. The mechanisms of telomere maintenance include not only elongation by telomerase, but also an alternative lengthening by recombination, based on elongation or telomere capture. The telomere capture mechanism involves rearrangement and acquisition of a telomeric sequence from another chromosome in an attempt to stabilize the terminal chromosomal region. It can be detected by the use of chromosome specific sub-telomeric FISH probes that are characterized cytogenetically [21, 32–34]. In the current study, we found significantly increased telomere capture in IUGR trophoblasts compared to the control samples. These results may suggest a compensatory response of the IUGR placentas to the shortened telomeres in order to maintain telomere homeostasis [35]. Further studies are indicated to pursue these findings.
% of trophoblasts with SAHFs 45 40 35
*
Control IUGR
30 25
%
This study investigated some of the mechanisms of telomere shortening not previously described in IUGR placentas. The results expand previous knowledge regarding telomere homeostasis in IUGR placentas. There is evidence of senescence, expressed by nuclear granulations known as SAHF [15]. We found that trophoblasts from IUGR placentas expressed a significantly higher amount of nuclei with SAHF, supporting the hypothesis that senescence has an important role in IUGR due to placental insufficiency. Senescence is known as metabolic cell arrest resulting from stress, inhibitors of specific enzymatic activities and DNA damage [14,24–26]. IUGR is a consequence of sub-optimal intrauterine conditions and hypoxia leading to short- and long-term morbidities [2,11–13]. Senescence is known to reflect cellular aging and cause shortened telomeres. However, premature senescence can occur in the presence of oxidative stress, even without critically shortened telomeres. Hence, this leads to telomere dysfunction that can initiate a DNA damage response, causing senescence with a typical granulation appearance [14,16,27–30]. Telomerase is a ribonucleic protein complex that contains a reverse transcriptase domain that elongates chromosomes by adding TTAGGG sequences to the chromosomal ends [31]. It has been shown that the telomerase reverse transcriptase protein (hTERT) is decreased in placentas from pregnancies with IUGR secondary to placental insufficiency,
467
20 Table 1 Demographic and clinical characteristics.
15
Characteristics
IUGR
Control
P value
Maternal age (year ± SD) Maternal BMI (kg/m2 ± SD) Gestational week at delivery (week ± SD) Fetal birth-weight (gram ± SD) Oligohydramnion n (%) of patients Abnormal umbilical artery Doppler n (%) of patients Placental pathology of insufficiency n (%) of patients
31 ± 0.5 25.8 ± 2.7 37 ± 0.2 1930 ± 320 9 (60%) 8 (53.33%)
30 ± 1.1 26.9 ± 1.8 37.2 ± 0.1 3240 ± 180 0 0
NS NS NS P b 0.001 P b 0.001 P b 0.001
15 (100%)
0
P b 0.001
10 5 0
* P < 0.001 Fig. 2. The percentage of trophoblasts with senescence associated heterochromatin foci (SAHF) in IUGR and control placentas.
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TERT mRNA, RT-PCR
% of trophoblasts with Telomere Capture
1.2
25.00
Control
Control
1
IUGR
IUGR
15.00
%
Fold
0.6
10.00
0.4
0.2
* P < 0.001
20.00
*
0.8
0
*
5.00
1
Fig. 3. RT-PCR, hTERT (human telomerase reverse transcriptase) mRNA in IUGR and in control placentas.
To conclude, pregnancy complications, including IUGR have been shown to be related to oxidative stress, DNA and protein damage that can result in trophoblasts with shortened telomeres, which undergo cell cycle arrest and enter a state of senescence. This study expands on previous observations of short telomeres in IUGR placentas by demonstrating the presence of senescence-associated heterochromatin bodies, by exposing the involvement of hTERT mRNA and telomere capture mechanisms. The study was observational and cannot show causation between these findings and IUGR; larger studies are indicated to investigate this matter. There is evidence supporting cellular senescence as a contributor to later onset diseases of intrauterine origin. Therefore, understanding mechanisms of placental telomere homeostasis might improve our knowledge regarding IUGR and its short- and long-term implications.
Conflict of interest The authors report no conflict of interest.
Fig. 4. Samples of nuclei with and without telomere capture. The normal appearance is two orange and two green signals (A). The abnormal appearance, which represents telomere capture, is two orange compared to one, three, or more green signals (the rearranged-captured sub-telomeric regions) (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
0.00
* P < 0.001 Fig. 5. The percentage of trophoblasts with telomere capture in IUGR and in control placentas.
Acknowledgment We would like to thank Mrs. Faye Schreiber for English editing assistance.
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Contents lists available at ScienceDirect
Early Human Development journal homepage: www.elsevier.com/locate/earlhumdev
Telomere shortening in intra uterine growth restriction placentas☆ Tal Biron-Shental a,b,⁎, Rivka Sukenik-Halevy a,b,c, Yudith Sharon c,d, Ido Laish b, Moshe D. Fejgin a,b,c, Aliza Amiel c,d a
Department of Obstetrics and Gynecology, Meir Medical Center, Kfar Saba, Israel Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Genetic Institute, Meir Medical Center, Kfar Saba, Israel d Faculty of Life Science, Bar Ilan University, Ramat Gan, Israel b c
a r t i c l e
i n f o
Article history: Received 15 April 2014 Received in revised form 29 May 2014 Accepted 4 June 2014 Available online xxxx Keywords: Placenta Trophoblasts Telomere hTERT Telomere capture IUGR Senescence
a b s t r a c t Introduction: Placentas from pregnancies complicated with IUGR (intrauterine growth restriction) express altered telomere homeostasis. In the current study, we examined mechanisms of telomere shortening in these placentas. Methods: Placental biopsies from 15 IUGR and 15 healthy control pregnancies were examined. The percentage of trophoblasts with fragmented nuclei: senescence-associated heterochromatin foci (SAHF), was calculated using DAPI staining. The amount of human telomerase reverse transcriptase (hTERT) mRNA was evaluated using RtPCR levels of telomere capture using FISH in those samples were estimated. Results: The percentage of trophoblasts with SAHF was higher in IUGR compared to control samples, (25 ± 13.4% vs. 1.6 ± 1.6%, P b 0.0001), hTERT mRNA was decreased (0.5 ± 0.2 vs. 0.9 ± 0.1, P b 0.0001) and telomere capture was increased (13.2 ± 9.7% vs.1.3 ± 2.5%, P b 0.001). Conclusions: We suggest that IUGR placentas express increased signs of senescence as part of the impaired telomere homeostasis. One factor that mediates telomere shortening in these placentas is decreased hTERT mRNA, leading to decreased protein expression and therefore, reduced telomere elongation. Telomere capture, which is a healing process, is increased in IUGR trophoblasts as a compensatory mechanism. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Intrauterine fetal growth restriction (IUGR), defined as fetal growth below the tenth percentile, is associated with increased short and long term morbidities [1–3]. IUGR is caused by genetic and environmental factors related to maternal, placental or fetal abnormalities [1–3]. The placenta, as a mediator of all communications between the mother and the fetus, has a key role in the pathways of fetal growth and development [2,3]. Therefore, placental research is essential for enhancing our understanding of IUGR secondary to placental insufficiency. Telomeres are nucleoprotein structures located at the termini of chromosomes that protect them from fusion and degradation. Telomeres are progressively shortened with each mitotic cycle and by environmental
Abbreviations: hTERT, human telomerase reverse transcriptase; SAHF, senescence associated heterochromatin foci; RtPCR, real time PCR; FISH, fluorescence in situ hybridization. ☆ Condensation: Telomere homeostasis is impaired in IUGR placentas by increased senescence, decreased hTERT-mRNA, leading to telomere shortening, and by increased telomere capture, as a compensatory mechanism. ⁎ Corresponding author at: Department of Obstetrics and Gynecology, Meir Medical Center, Tel Aviv University, Kfar Saba 44281, Israel. Tel.: +972 528362331. E-mail address: [email protected] (T. Biron-Shental).
http://dx.doi.org/10.1016/j.earlhumdev.2014.06.003 0378-3782/© 2014 Elsevier Ltd. All rights reserved.
factors [4,5]. Telomere biology was suggested as a mechanism connecting fetal intrauterine programming and subsequent health conditions [6]. Intrauterine exposure to stress such as hypoxia, which causes placental insufficiency leading to IUGR, might cause changes in the telomere system that accelerate telomere shortening, cellular dysfunction, aging, and disease susceptibility [6]. Telomere homeostasis is impaired in placentas from pregnancies complicated by IUGR secondary to placental insufficiency [7–9]. Telomeres are shorter in IUGR placentas than in placentas from healthy controls [7,8]. Moreover, telomeres tend to cause aggregates, reflecting dysfunction unrelated to their length [8,9]. Telomerase is an enzyme complex that elongates telomeres. hTERT (human telomerase reverse transcriptase) is the catalytic component of telomerase. Protein levels of hTERT are significantly lower in IUGR placentas [8,9]. One component of telomerase, which serves as the RNA template for the addition of telomeric repeats, is encoded by the telomerase RNA component gene (TERC). The TERC gene copy number is decreased in IUGR trophoblasts [10]. These data suggest that impaired telomere homeostasis might play a role in the pathophysiology of IUGR and might affect intrauterine programming related to the origin of some diseases that arise in adulthood [6,11–13]. However, the mechanisms of telomere shortening in these placentas are not fully understood.
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Senescence is an arrest of cellular proliferation and is involved in the biological process of tissue aging. It is driven by cell division, as well as oxidative stress, DNA damage and repair response, inflammation and telomere shortening [4,14]. Nuclei in senescent human cells are often granulated, containing senescence-associated heterochromatin foci (SAHF) [15]. The relationship between cellular senescence and telomere shortening is well established [14–18], but has not been described in IUGR placentas. When telomeres are shortened to a critical length, repair pathways are recruited. One repair pathway is telomere capture, through which a shortened telomere acquires a new telomeric sequence from a sister chromatid or from another chromosomal end [16–18]. In this study, we examined mechanisms of telomere shortening that have not been previously described in IUGR placentas. 2. Materials and methods 2.1. Study groups and sample collection The study was approved by the institutional ethics review board. We examined biopsies from 15 third-trimester placentas derived from pregnancies complicated by IUGR and 15 placentas matched for gestational age from uncomplicated pregnancies, as controls. All patients signed an informed consent. IUGR was defined as neonatal birth weight below the 10th percentile for gestational age, based on the standardized Israeli curves [19]. Only placental specimens from pregnancies complicated with IUGR attributed to placental insufficiency were included. Other reasons for IUGR, such as intrauterine viral infections, chromosomal abnormalities, congenital anomalies, poor pregnancy dating, or maternal smoking were ruled out. Exclusion criteria were multi-gestational pregnancies, patients with no prenatal care who lacked information regarding possible reasons for IUGR other than placental insufficiency or inaccurate dates. All newborns were evaluated by a neonatologist within 24 h of delivery. Major malformations or suspected genetic congenital syndrome that could be related to small fetus were ruled out. All placentas were biopsied immediately after delivery under sterile conditions. From the samples of intermediate trophoblasts, 1 cm2 was taken from the area midway between the cord insertion and the edge of the placenta. The placental biopsy area was based on hypoxic gene expression in different areas in the placenta, as described by Wyatt et al. [20]. Each specimen was divided into 5 pieces of 2 mm2, kept on ice during the entire processing procedure to prevent RNA degradation and frozen at −80 °C within 20 min of delivery. 2.2. Fluorescence in situ hybridization (FISH) assay Frozen placental tissue sections of 4 μm were prepared for fluorescence in situ hybridization (FISH) using the tissue pretreatment kit (Biomedicals, North America, Solon, OH, USA). Initially, the slides were fixed in a 1:3 acetic acid and ethanol solution for 15 min. The slides were then placed in fresh 100% ethanol for 5 min, twice, allowed to air dry and then placed on a plate warmed to 80 °C for 60 min. The slides were incubated for 25 min in a glass jar containing the pretreatment solution preheated to 45 °C (12 g of pretreatment powder in 40 cm3 of 2XSCC), rinsed twice in 2XSSC buffer at room temperature, then incubated in a glass jar containing a protein digesting enzyme preheated to 45 °C for 45 min (400 μl in 40 cm3 of 2XSCC), rinsed twice in 2XSCC buffer at room temperature, dehydrated for 1 min in 70%, 80%, and 100% ethanol at room temperature and left to air dry for 5 min. Afterwards, the slides were placed on 94 °C warming plate for 4 min. The telomere capture was performed using a CY3 labeled telomere capture-specific peptide nucleic acid probe (vial 2 in K 532 DAKO, Glostrup, Denmark) following manufacturer's instructions. The slides were stained with 4′-6-diamidino-w-phenylindole (DAPI) (1000 ng/Ml, VYS-32-804830, Vysis, Abbott Laboratories. Abbott Park, IL, USA) and finally overlaid with glass cover slips for observation. Each placental specimen was
placed under an AX70 Olympus Provis fluorescent microscope (Olympus, Tokyo, Japan) at ×100 magnification. For each specimen, 100–200 trophoblasts were counted. The nuclei identified were selected as belonging to cytotrophoblasts by their appearance and arrangement in the villi. The percentage of trophoblasts with fragmented nuclei (SAHF) in each specimen was evaluated. The slides were blindly scored twice (and a third time whenever there was a difference of more than 10% between the counts). Positive and negative controls were used to ensure reliability of the assay. 2.3. Telomere capture In the nuclei, the number of the specific loci of the specific chromosome (SNRPN or 13q 14.3: orange, the “normal disomic loci”) and its telomere (15qter or 13qter: green) were compared. The number of signals of the SNRPN or 13q 14.3 locus was compared to the number of signals of the sub-telomeric region of the specific chromosome (green signals). For example, the normal appearance is two orange and two green signals, while an abnormal appearance, which represents telomere capture is two orange compared with one, three, or more green signals (the rearranged-captured sub-telomeric regions), previously described by Amiel et al. [21]. Approximately 200 nuclei from each sample were analyzed. Only cells in which two orange signals (normal “disomic loci”) appeared were analyzed. Positive and negative controls were used to ensure the reliability of the assay. 2.4. RT-PCR and RT-quantitative PCR (RT-qPCR) RNA was purified from placental biopsies using a Perfect-Pure RNA kit (5 Prime, Inc., Gaithersburg, MD, USA) according to the manufacturer's instructions. Complementary DNA (cDNA) was prepared from 300 ng RNA using the TaqMan Gold RT-PCR kit with the random hexamer primers supplied (Applied Biosystems, Inc., Foster City, CA, USA). For quantitative PCR (qPCR), cDNA was prepared using TaqMan RT-PCR kit (Applied Biosystems, Foster City, CA, USA) in a 20 μl reaction mix that contained 300 ng total RNA, 1× RT buffer, 5.5 mM MgCl, deoxy-NTP mixture (500 μM of each dNTP), 2.5 μM random hexamers, 0.4 units μl−1 RNase inhibitor, and 1.25 units ml−1 Multi-Scribe reverse transcriptase at 25 °C for 10 min, 48 °C for 30 min, and 95 °C for 5 min. PCR was performed using 3 μl RT product, produced with 0.3 μM of the primer pairs in duplicate, with SYBR Green PCR Master Mix (Applied Biosystems, Inc., Foster City, CA, USA) in a total reaction volume of 25 μl. Reactions were analyzed using Geneamp 7500 Fast Instrument System (Applied Biosystems, Inc., Foster City, CA, USA). Dissociation curves were ran on all reactions to ensure amplification of a single product with the appropriate melting temperature. Fold change was computed relative to control using the 2−ΔΔCT method [21]. Samples were normalized to GAPDH expression [22,23], determined in parallel reactions. 2.5. Statistical analysis A 2-tailed sample t test and the nonparametric Mann–Whitney U test were applied to evaluate the differences between the groups, with P b .05 considered statistically significant. SPSS software (SPSS, Inc, Chicago, IL) was used for statistical analysis. 3. Results The study and the control groups were comparable in terms of demographic and baseline characteristics. As expected, birth weights were significantly lower and characteristics of placental insufficiency were significantly more prominent in the IUGR group. Although the inclusion criteria were birth weight below the 10th percentile, and the actual birth weights of the IUGR patients in the study were below the 5th percentile. All the IUGR fetuses had clinical and histological characteristics
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of placental insufficiency, 60% had oligohydramnios, and 53.3% had an abnormal umbilical artery Doppler. Each IUGR fetus had oligohydramnios and/or abnormal umbilical artery Doppler (Table 1). To evaluate senescence in IUGR placentas, we looked at the percentage of trophoblasts with SAHF in placental samples from 15 IUGR pregnancies and 15 healthy controls. Trophoblast nuclei with and without fragmentations are shown in Fig. 1. We found that the IUGR placentas had a significantly higher percentage of trophoblasts with fragmented nuclei, SAHF (25 ± 13.4%) compared to (1.6 ± 1.6%) the control samples, P b 0.0001 (Fig. 2). To understand some of the mechanisms of telomere shortening in the senescent placentas, we sought to confirm the down-regulation of telomerase by real time PCR and found that hTERT was decreased in IUGR placentas (0.5 ± 0.2) compared to controls (0.96 ± 0.1) (P b 0.0001) (Fig. 3). To evaluate the compensatory response of the IUGR trophoblasts to telomere shortening, we estimated the presence of cells with telomere capture in the study and control samples. An example of nuclei with and without telomere capture is shown in Fig. 4. The percentage of trophoblasts with telomere capture in the IUGR samples was significantly higher than in the controls (13.3 ± 9.8% vs. 1.4 ± 2.5%; P b 0.001) (Fig. 5). About 50% of the samples were drawn from placentas delivered via cesarean section. There were no differences in the percentage of cesarean sections between the study and control groups. Moreover, no differences in telomere homeostasis were found when analyzing the results based on mode of delivery. 4. Comment
Fig. 1. DAPI stained nuclei from placental trophoblasts displaying senescence. (A) A nucleus with 3 fragments from a placenta from a pregnancy complicated by IUGR (intrauterine growth restriction). (B) A nucleus from a normal placental trophoblast.
along with shorter telomeres in those placentas [8,9]. Moreover, TERC which is the telomerase RNA component and considered the gene that encodes telomerase, was found significantly less often in IUGR trophoblasts. This observation supports the role of telomerase activity and telomere shortening in IUGR caused by placental insufficiency [10]. In order to expand and strengthen these observations, we evaluated hTERT mRNA and found that it is reduced in IUGR placentas, suggesting that the lower levels of hTERT protein in these placentas is the consequence of decreased mRNA leading to reduced protein production. The mechanisms of telomere maintenance include not only elongation by telomerase, but also an alternative lengthening by recombination, based on elongation or telomere capture. The telomere capture mechanism involves rearrangement and acquisition of a telomeric sequence from another chromosome in an attempt to stabilize the terminal chromosomal region. It can be detected by the use of chromosome specific sub-telomeric FISH probes that are characterized cytogenetically [21, 32–34]. In the current study, we found significantly increased telomere capture in IUGR trophoblasts compared to the control samples. These results may suggest a compensatory response of the IUGR placentas to the shortened telomeres in order to maintain telomere homeostasis [35]. Further studies are indicated to pursue these findings.
% of trophoblasts with SAHFs 45 40 35
*
Control IUGR
30 25
%
This study investigated some of the mechanisms of telomere shortening not previously described in IUGR placentas. The results expand previous knowledge regarding telomere homeostasis in IUGR placentas. There is evidence of senescence, expressed by nuclear granulations known as SAHF [15]. We found that trophoblasts from IUGR placentas expressed a significantly higher amount of nuclei with SAHF, supporting the hypothesis that senescence has an important role in IUGR due to placental insufficiency. Senescence is known as metabolic cell arrest resulting from stress, inhibitors of specific enzymatic activities and DNA damage [14,24–26]. IUGR is a consequence of sub-optimal intrauterine conditions and hypoxia leading to short- and long-term morbidities [2,11–13]. Senescence is known to reflect cellular aging and cause shortened telomeres. However, premature senescence can occur in the presence of oxidative stress, even without critically shortened telomeres. Hence, this leads to telomere dysfunction that can initiate a DNA damage response, causing senescence with a typical granulation appearance [14,16,27–30]. Telomerase is a ribonucleic protein complex that contains a reverse transcriptase domain that elongates chromosomes by adding TTAGGG sequences to the chromosomal ends [31]. It has been shown that the telomerase reverse transcriptase protein (hTERT) is decreased in placentas from pregnancies with IUGR secondary to placental insufficiency,
467
20 Table 1 Demographic and clinical characteristics.
15
Characteristics
IUGR
Control
P value
Maternal age (year ± SD) Maternal BMI (kg/m2 ± SD) Gestational week at delivery (week ± SD) Fetal birth-weight (gram ± SD) Oligohydramnion n (%) of patients Abnormal umbilical artery Doppler n (%) of patients Placental pathology of insufficiency n (%) of patients
31 ± 0.5 25.8 ± 2.7 37 ± 0.2 1930 ± 320 9 (60%) 8 (53.33%)
30 ± 1.1 26.9 ± 1.8 37.2 ± 0.1 3240 ± 180 0 0
NS NS NS P b 0.001 P b 0.001 P b 0.001
15 (100%)
0
P b 0.001
10 5 0
* P < 0.001 Fig. 2. The percentage of trophoblasts with senescence associated heterochromatin foci (SAHF) in IUGR and control placentas.
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TERT mRNA, RT-PCR
% of trophoblasts with Telomere Capture
1.2
25.00
Control
Control
1
IUGR
IUGR
15.00
%
Fold
0.6
10.00
0.4
0.2
* P < 0.001
20.00
*
0.8
0
*
5.00
1
Fig. 3. RT-PCR, hTERT (human telomerase reverse transcriptase) mRNA in IUGR and in control placentas.
To conclude, pregnancy complications, including IUGR have been shown to be related to oxidative stress, DNA and protein damage that can result in trophoblasts with shortened telomeres, which undergo cell cycle arrest and enter a state of senescence. This study expands on previous observations of short telomeres in IUGR placentas by demonstrating the presence of senescence-associated heterochromatin bodies, by exposing the involvement of hTERT mRNA and telomere capture mechanisms. The study was observational and cannot show causation between these findings and IUGR; larger studies are indicated to investigate this matter. There is evidence supporting cellular senescence as a contributor to later onset diseases of intrauterine origin. Therefore, understanding mechanisms of placental telomere homeostasis might improve our knowledge regarding IUGR and its short- and long-term implications.
Conflict of interest The authors report no conflict of interest.
Fig. 4. Samples of nuclei with and without telomere capture. The normal appearance is two orange and two green signals (A). The abnormal appearance, which represents telomere capture, is two orange compared to one, three, or more green signals (the rearranged-captured sub-telomeric regions) (B). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
0.00
* P < 0.001 Fig. 5. The percentage of trophoblasts with telomere capture in IUGR and in control placentas.
Acknowledgment We would like to thank Mrs. Faye Schreiber for English editing assistance.
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