Telomere Homeostasis and Senescence Markers Are Differently Expressed in Placentas From Pregnancies With Early- Versus Late-Onset Preeclampsia

Abstract

Background:

Early-onset preeclampsia (EOPE; <34 weeks’ gestation) usually has more severe morbidity for the mother and fetus compared to late-onset preeclampsia (LOPE). Telomere homeostasis is disrupted in preeclampsia (PE) and senescence markers are increased. The pathophysiologic differences between early and LOPE are not fully unraveled yet.

Methods:

We studied placental biopsies from 7 pregnancies with EOPE, 6 pregnancies with LOPE, and 13 healthy gestational age-matched controls. Telomere length and aggregate formation were assessed using qualitative fluorescence in situ hybridization and electronic quantitative methods. Senescence markers were evaluated including senescence-associated heterochromatin foci, β-galactosidase (SAβ-Gal), and P16 staining, as was the expression of P16 complementary DNA (cDNA) using real-time quantitative polymerase chain reaction (RT-qPCR).

Results:

There were no differences in maternal age, gravidity, parity, body mass index, and mode of conception between the study and the control groups. The percentage of trophoblasts with short telomeres was higher in placental samples from EOPE (52.61% [12.27%]) versus LOPE (28.72% [10.14%]); both were higher compared to controls (7.53% [5.14%], P = .03). Aggregate formation was enhanced in EOPE (8.72% [2.49%]) compared to LOPE (4.54% [1.45%]); both were higher than in healthy controls (2.72% [1.08%], P = .03). Trophoblasts from EOPE versus LOPE were more likely to stain positive for SAβ-Gal and P16 compared to controls (P < .001). P16 cDNA expression assayed by RT-qPCR was 7.51 times higher in EOPE compared to controls and 5.86 times higher than in LOPE.

Conclusions:

Impaired telomere homeostasis and senescence markers are more prominent in EOPE versus LOPE. These findings may contribute to our understanding of the pathophysiology and explain their different clinical presentations and outcomes.

Keywords

early-onset preeclampsia late-onset preeclampsia telomeres senescence

Introduction

Preeclampsia (PE) is a major placental-related complication that affects 5% to 8% of pregnancies. It is characterized by increased blood pressure (BP) and new-onset proteinuria.¹ Delivery is indicated when symptoms pose a risk to the mother or the fetus. Premature delivery exposes the newborn to complications in addition to those related to PE.^1
–3 Despite optimal treatment, serious maternal–fetal morbidity and mortality still occur. Neonates face both direct (ie, growth restriction and placental abruption) and indirect sequelae from PE due to early delivery-induced prematurity. Maternal complications include eclamptic seizures or sequelae related to renal injury and high BP.^1
–3

Preeclampsia is a heterogeneous disease that can be categorized into early-onset preeclampsia (EOPE, diagnosed ≤34 weeks of gestation) and late-onset preeclampsia (LOPE, diagnosed >34 weeks of gestation).^1,4,5 It has been suggested that maternal characteristics and perinatal morbidity and mortality differ between these subgroups and that these differences may be related to different pathophysiological processes.^4

–8

Early-onset preeclampsia is associated with intrinsic placental factors. Inadequate spiral artery remodeling and poor placental perfusion lead to oxidative stress, which is the basis of the pathogenesis of the more severe early-onset phenotype. In contrast, LOPE shows relatively normal initial placentation.

Telomeres are nucleoprotein structures located at the end of chromosomes. They are important for chromosome stability and cell survival. Telomere length is dynamic. Telomeres shorten with each cell division or in response to other physiological and pathological environmental factors, such as stress or disease. Telomere elongation is controlled by the activity of the telomerase enzyme. When telomeres reach a critically short length, they can no longer be protected from degradation or from the cell’s own DNA damage response mechanisms, which trigger senescence. Dysfunctional telomeres tend to form aggregates, which consist of end-to-end fusion of telomeres.^9

–13

Cellular senescence is a mechanism that irreversibly arrests cell cycle progression of potentially harmful cells.^14
–16 As a result, it prevents tumorigenesis and limits tissue damage but also contributes to aging of tissues.^17
–19 Senescence can be induced by several triggers, including telomere shortening, DNA damage, expression of activated oncogenes, and other forms of cellular stress.^14,20

–23

Cellular senescence in normal placentas is triggered by the process of syncytiotrophoblast formation created by cytotrophoblast fusion.¹⁴ In the initial stages of pregnancy, trophoblasts have the unique capacity to proliferate rapidly and invade the uterine wall, which is a tumor-like characteristic.^14,24 As the pregnancy progresses, the placenta enters a state of senescence.^13,25

Telomere homeostasis is disrupted in PE and senescence markers are enhanced.^10,12,13 However, differences between EOPE and LOPE regarding these parameters have not been reported yet. This study assessed telomere homeostasis and cellular senescence in placentas from pregnancies with EOPE and LOPE.

Methods

Patients

Placental samples from pregnancies complicated with EOPE (diagnosed ≤34 weeks of gestation; 7 samples), LOPE (diagnosed >34 weeks of gestation; 6 samples), and from normal pregnancies matched for gestational age (13 samples) were collected. Controls for EOPE were cases of preterm delivery due to preterm premature rupture of membranes or spontaneous preterm labor. Pregnancies in which preterm delivery was the result of labor induction were not included in the control group, nor were cases of preterm delivery related to intrauterine infection, nonreassuring fetal monitoring, or other obstetrical causes.

To overcome potential differences in telomere length between different gestational weeks, we matched the gestational week at the time of the delivery between EOPE and the control group. Since preterm delivery is always for a reason and may influence telomere length by itself, we included in the preterm control group only pregnancies that were completely normal until uneventful asymptomatic ruptured membranes occurred. Preeclampsia was defined, based on American college of obestetrics and gynecology criteria, as new onset of BPs ≥140 mm Hg systolic or ≥90 mm Hg diastolic on 2 occasions at least 4 hours apart after 20 weeks of gestation in a woman with previously normal BP and proteinuria ≥300 mg per 24-hour urine collection.

Patients with multiple gestations, fetal malformations, and maternal chronic diseases such as hypertension, renal disease, or diabetes mellitus were excluded. The study and control groups consisted of Caucasian patients 18 to 45 years old. The study was approved by institutional ethics review board and all patients provided signed an informed consent form.

Placenta Sampling

Villous tissue samples from maternal aspect were taken midway between the cord insertion site and the placental margin. Samples were collected within 20 minutes of delivery and frozen at −80°C. After freezing, placental specimens were cut to the thickness of 12 μm and placed on slides. Hematoxylin and eosin staining was performed.

Quantitative Fluorescence In Situ Hybridization for Telomere Length

PNA FISH Kit/Cy3 kit (Dako, Glostrup, Denmark) was used. Slides were incubated for 2 minutes in Tris-buffered saline (TBS) followed by fixation in formamide 3.7% diluted 1:10 in phosphate-buffered saline (PBS) for 2 minutes. Slides were placed in TBS for 5 minutes twice, in pretreatment solution for 10 minutes, in TBS solution for 5 minutes twice, dehydrated in a graded ethanol series, and left to air dry. This was followed by addition of 10 μL of CY3-labeled telomere-specific peptide nucleic acid probe (vial 2 in K 532; Dako) and hybridization at 80°C for 5 minutes. Slides were then left at room temperature overnight in a dark environment. They were rinsed for 1 minute, in wash solution at 65°C for 5 minutes, and dehydrated in a graded ethanol series. Slides were counterstained in 4′,6-diamidino-2-phenylindole (DAPI)-II (Abbott) antifade solution and finally overlaid with glass coverslips for observation with a fluorescent automatic exposure, ×100 magnification on an AX70 Olympus Provis microscope (Olympus, Tokyo, Japan).

Nuclei from 300 to 400 trophoblasts were counted for each slide. Telomere length was quantified by signal intensity and by the number of signals. The cells were grouped into 4 categories: high (strong) or low (weak) fluorescence and number of telomere signals (dots per cell) <10 or ≥10. We then calculated the percentage of cells in each category for each slide. Short telomeres are consistent with fewer signals and lower intensity signals, while long telomeres with more signals and higher intensity. Telomere aggregates were demonstrated under fluorescent microscopy by clusters of telomeric signals.

The manual qualitative fluorescence in situ hybridization (qFISH) counts were also assessed by an additional (automatic) computer-assisted analysis of the digital microscope images. Signals representing the telomers were analyzed. In order to separate the telomeric data from the varying background, spatial frequency filtering was applied to the sample. Briefly, the microscope image is Fourier transformed, multiplied with a filter function to reduce low spatial frequencies, and then retransformed to the spatial domain. Since low spatial frequencies are filtered out, the picture is reconstructed with only its highly varying features, that is, data regarding the telomers (without background). Since a “fingerprint” of the telomere activity (ie, number of telomers and their corresponding length) is related to the number of dots and their signal intensity in the processed image, the computer-assisted analysis calculated the average of the processed image intensity, that is, the telomere activity.

4′,6-Diamidino-2-Phenylindole Staining for Senescence-Associated Heterochromatin Foci

The percentage of fragmented DAPI-stained nuclei was evaluated in placental samples.

Immunohistochemistry for P16

Slides of 12-μm frozen sections of placental tissue samples were blocked for nonspecific binding with 4% horse serum and 1% body surface area. Primary antibody recognizing p16 (Abcam, Cambridge, United Kingdom) was applied for an hour at room temperature. Staining was developed using DAB (Vector Laboratories, Burlingame, California) followed by hematoxylin counterstaining. Primary sections were visualized with an Olympus microscope, and images were analyzed using CellP Software (Diagnostic Instruments, Sterling Heights, Michigan).

Detecting Senescence-Associated β-Galactosidase Activity

Frozen sections of placental tissue were fixed with 0.5% glutaraldehyde in PBS for 15 minutes, washed with PBS supplemented with 1 mM MgCl₂, and stained overnight in PBS containing 1 mM MgCl₂, 1 mg/mL X-Gal, and 5 mM each of potassium ferricyanide and potassium ferrocyanide. Sections were counterstained with nuclear fast red (Sigma, St Louis, Missouri). Positive staining was analyzed using ImageJ software.

Real-Time Quantitative Polymerase Chain Reaction

For quantitative real-time quantitative polymerase chain reaction (RT-qPCR), total RNA was isolated using NucleoSpin kit (Macherey Nagel, Düren, Germany) and 1 µm was reverse transcribed using the RevertAid H Minus First-Strand cDNA Synthesis Kit (Fermentas, Glen Burnie, Maryland). The complementary DNA (cDNA) samples were amplified using Fast SYBR Green Master Mix in StepOnePlus Real-Time PCR System (both Applied Biosystems, Foster City, California). The relative expression of p16 was normalized using GAPDH expression levels. Primer sequences are available upon request.

Statistical Analysis

Data are presented as numbers and percentage for nominal variables and as mean and standard deviation for continuous parameters. Fisher exact or χ² tests were used to compare proportions. Continuous variables were compared using Mann-Whitney nonparametric analysis (more conservative). P < .05 was considered statistically significant. All analyses were done with SPSS version 23 software (IBM, Armonk, New York).

Results

Clinical data regarding the pregnancies from which samples were collected are presented in Table 1. There was no difference in maternal age, gravidity, parity, body mass index, and mode of conception between the study and control groups.

Table1.

Clinical Data.

Parameter	EOPE (n = 7)	Control (n = 7)	P Value	LOPE (n = 6)	Control (n = 6)	P Value	P Value EOPE vs LOPE
Maternal age (SD), years	32.8 (6.5)	29 (7.4)	.341	35.25 (5.5)	27.8 (3.6)	.65	0.553
BMI (at term) (SD)	32 (5.6)	31.5 (8.2)	.907	31 (0)	25.5 (1.2)	.145	0.981
Gravidity	2.1 (1.46)	2 (1.26)	.855	3 (2.7)	2.4 (1.7)	.694	0.5
Parity	0.71 (0.95)	0.5 (0.84)	.677	0.5 (0.57)	0.8 (1.3)	.685	0.695
Mode of conception
Spontaneous	57.1%	100%	.188	100%	100%	1	0.308
COH/IUI	14.3%	0
IVF	28.6%	0
GA at diagnosis	32.3 (2.7)	NA	NA	37 (1.1)	NA	NA	NA
% patients with severe symptoms	100%	NA	NA	0	NA	NA	NA
% patients with IUGR	14.3%	0	NA	0	0	NA	NA
GA at delivery (weeks [SD])	34.1 (2.19)	35.6 (0.53)	.12	37 (1.09)	39.3 (0.816)	.002	0.015
Mode of delivery
Cesarean	85.7%	14.3%	.029	0	0	1	0.005
Vaginal	14.3%	85.7%		100%	100%
Birth weight (g)	2068 (638)	2850 (328)	.021	3554 (630)	3376 (336)	.601	0.005
1-Minute Apgar score ≥7	85.7%	100%	.56	100%	100%	1	0.52
5-Minute Apgar score ≥7	100%	100%	.82	100%	100%	.26	0.67

Abbreviations: BMI, body mass index; COH, controlled ovarian hyperstimulation; EOPE, early-onset preeclampsia; GA, gestational age; IUI, intrauterine insemination; IUGR, intrauterine growth restriction; IVF, in vitro fertilization; LOPE, late-onset preeclampsia; NA, not applicable; SD, standard deviation.

The EOPE group had a more severe disease as expected by the study design. All 7 cases in the EOPE group had severe features. In the EOPE group, 6 of 7 cases were delivered by cesarean section. All cases in the control groups were delivered vaginally.

As expected by the nature of the study design, the EOPE group delivered at an earlier gestational age (34.1 [2.19] weeks) than the LOPE group (39.3 [0.816] weeks; P = .015). Birth weights were significantly lower in the EOPE group (2068 [638] g) compared to the LOPE group (3554 [630] g; P = .005), but both were within the normal range for gestational age, except for one case of intrauterine growth restriction in the EOPE group. There were no differences in neonatal Apgar scores between the groups.

Placental specimens taken from EOPE showed areas of distal separated villi with increased syncytial knots, intervillous fibrin deposition, decreased villous branching, areas of villous infarcts which represent areas of ischemic necrosis overlying occluded spiral arteries, and accelerated villous maturation. We did not find these changes in placentas from patients with LOPE or in placentas from healthy patients.

The percentage of trophoblasts with short telomeres in placentas from EOPE (52.6% [12.27%] standard deviation, SD) was higher compared to LOPE (9.26% [10.1%] SD; P = .005) and compared to the controls (P = .003, P = .015, respectively). The percentage of trophoblasts with long telomers from EOPE (8.3% [6.79%]) was lower compared to the control group (47.2% [5%]; P = .003) and compared to LOPE (34.1% [12.1%]; P = .001), as presented in Figure 1. More telomere aggregates were found in EOPE (8.72% [2.49%] SD) compared to controls (2.82% [1.14%] SD; P = .003) and compared to LOPE (2.5% [3.25%] SD; P = .007; Figure 1).

The results of the manual qFISH counts (shown in the graphs) and those obtained by the computer-assisted analysis showed the same trend. A computer-assisted analysis assigned a parameter related to the telomere signal intensity. This activity was lower in EOPE group (2.3 [0.2]) compared to the control group (3.3 [0.2]) and compared to LOPE (3 [0.2]; P < .05).

The placental trophoblasts in the study groups exhibited characteristics of senescent cells which were more prominent in EOPE as presented in Figure 2. Senescence-associated β-galactosidase (SA-β-Gal) activity was more prominent in the EOPE trophoblasts (55.36% [9.5%] SD) compared to LOPE (23.93% [6.64%] SD; P = .009). We also analyzed trophoblasts by immunohistochemistry for P16, an important molecular marker of senescence. The EOPE trophoblasts exhibited stronger specific staining for p16 compared to LOPE (56.95% [0.65%] SD vs 33.15% [5.56%] SD, respectively; P = .011).

The P16 cDNA expression assayed by quantitative RT-qPCR was 7.5 times higher in EOPE than in the control group and 5.86 times higher than in the LOPE group. The expression of P16 in LOPE was 1.28-fold higher than in the control (Figure 3).

Discussion

Preeclampsia has been studied for many years, and despite numerous studies, there are still unknown aspects of the pathophysiology of this disease. Abnormal placentation is known to be strongly associated with PE²⁶ and is expressed through abnormal placental vascular lesions. The placental component in different clinical manifestations of PE (term/preterm, appropriate for gestational age fetus/fetal growth restriction, PE with/without severe features) has been studied thoroughly in an effort to find a correlation between the placental lesion and the severity of the clinical expression.^26
–28 However, much of the pathophysiology related to these diverse clinical aspects is still unraveled.

Early-onset preeclampsia is considered a fetal disorder typically associated with placental dysfunction, while LOPE is considered a maternal disorder, due to an underlying constitutional disorder.^7,29

–32 Thus, the placenta seems like the most promising candidate for studying the specific pathophysiology of these 2 conditions.³³

Small and distal villi, increased syncytial knots, intervillous fibrin, and accelerated villous maturation are histologic changes seen in placentas from PE patients.^34
–36 We found these changes to be more pronounced in EOPE than in LOPE trophoblasts. Our results are in agreement with previous studies that showed abnormal placental morphology and vascular pathology are enhanced in EOPE compared to LOPE. Maternal vasculature shows major involvement in EOPE placentas.^26,37

This study focused on telomere homeostasis and senescence markers expressed in placentas from EOPE and LOPE. We show significant differences in these parameters between EOPE, LOPE, and controls. We found that placentas from EOPE compared to LOPE have enhanced pathological abnormalities, including shorter telomeres with increased aggregate formation.

Previous studies reported shorter telomeres and increased aggregate formation in placental samples from pregnancies complicated with PE.^9,12 We report that these parameters are not only different between PE and normal pregnancies but also that they are different between early EOPE and LOPE, with EOPE showing much more profound telomere shortening and aggregate formation in placentas from EOPE. This observation suggests that these parameters may be closely related to the pathogenesis of PE.^12,13

Increased senescence in PE trophoblasts was previously reported and is consistent with telomere shortening.^12,13,38,39 However, no study investigated senescence while differentiating PE into early and late diseases. We revealed in the current study increased cellular senescence in trophoblasts from pregnancies complicated with EOPE compared to LOPE and compared to controls.

We found in EOPE increased expression of P16 and SA-β-GAL, which are senescence-related markers in PE trophoblasts.^14,15,18,23 Following the same trend as with telomere shortening, the expression was stronger in EOPE than LOPE trophoblasts. P16 expression, measured by RT-qPCR, showed consistent results of increased expression in EOPE compared to LOPE and controls.

Senescence-associated heterochromatin foci (SAHF) are distinct chromatin structures enriched with heterochromatin proteins that prevent activation of genes that promote cell progression.^9

–13 We detected SAHF only in PE trophoblasts and not in controls. Our findings are in agreement with previous reports which suggest that increased SAHF are an indication of senescence in PE placentas.^9

–13 Interestingly, we could not detect statistically significant differences between EOPE and LOPE in SAHF expression. This can be explained by the fact that the evaluation of SAHF is qualitative and not sensitive enough as a marker of senescence.

The strengths of the study are based on its novelty in evaluating telomere homeostasis and senescence in EOPE versus LOPE. Also, the semiquantitative data obtained by qFISH was validated by a computer-assisted analysis, which eliminated observer bias. However, this is an observational study, with a relatively small number of samples, with no interventions on the tissues examined.

To conclude, there is controversy whether EOPE and LOPE represent 2 different entities or do they represent a phenotypical spectrum of a single condition. Early- and late-onset diseases are associated with different biochemical markers, genetic and environmental risk factors, prognosis, heritability, and clinicopathological features.^7,40
–42 Our findings suggest that telomere shortening in EOPE trophoblasts is linked to increased cellular senescence, which is much more prominent than in LOPE, supporting the hypothesis of 2 different pathogenic mechanisms for EOPE and LOPE. These data add to previous reports regarding the role of telomeres and senescence in the pathogenesis of trophoblastic dysfunction in PE. Further research is indicated to achieve better understanding of the pathophysiological mechanisms which contribute to these differences in telomere homeostasis and cellular senescence.

Footnotes

Acknowledgments

I gratefully acknowledge the support of Erez Gershnabel and Meytal Liberman, without which the present study could not have been completed.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

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