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Obstetrics & Gynecology 1999;93:124-129
© 1999 by The American College of Obstetricians and Gynecologists
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ORIGINAL RESEARCH

Telomerase and Proliferative Activity in Placenta From Women With and Without Fetal Growth Restriction

TOSHIHIKO IZUTSU, MD, TOMOHIKO KUDO, MD, TAMOTSU SATO, MD, IWAO NISHIYA, MD, KAZUMA OHYASHIKI, MD and KANICHI NAKAGAWARA, PhD

From the Department of Obstetrics and Gynecology, Iwate Medical University, Morioka; the First Department of Internal Medicine, Tokyo Medical College, Tokyo; and Nihon Gene Research Laboratories Inc., Sendai, Japan.

Address reprint requests to: Toshihiko Izutsu, MD Department of Obstetrics and Gynecology Iwate Medical University Morioka, Iwate, 020 Japan


   Abstract
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Abstract
Materials and Methods
 Results
 Discussion
 References
 
Objective: To analyze telomerase and proliferative activity in placenta from women with and without fetal growth restriction (FGR).

Methods: Telomerase activity was analyzed in 30 first-trimester chorionic villi specimens (group A) and in 28 second- and third-trimester placenta specimens (group B) from women without FGR. Telomerase activity also was analyzed in 11 placenta specimens from women with asymmetric FGR (group C). The proliferative activity of these 69 specimens was assessed by immunohistochemical staining, using the MIB-1 monoclonal antibody.

Results: Telomerase activity was detected in 28 (93.3%) of 30 chorionic villi specimens and in 18 (64.3%) of 28 placenta specimens without FGR. In contrast, no telomerase activity was exhibited in the placenta specimens from any of the 11 women with asymmetric FGR by telomeric repeat amplification protocol assay. Telomerase activity also was detected by in situ telomeric repeat amplification protocol assay in trophoblastic cells from women without FGR but not in trophoblastic cells from women with asymmetric FGR. Thus, telomerase activity was detected significantly more often in groups A and B than in group C (P < .01). The rate of proliferative activity, evident as positive MIB-1 staining in trophoblastic cells, in groups A and B (28.1 ± 1.7% and 7.0 ± 2.9%, respectively) was significantly higher than that in group C (1.9 ± 0.6%; P < .01).

Conclusion: Telomerase and proliferative activity were minimal in placenta from women with asymmetrical FGR, suggesting placental senescence with asymmetrical FGR.

Human somatic cells have a finite proliferative capacity both in vitro and in vivo and enter a viable growth–arrested state called senescence. Recent studies have implicated telomeres and telomerase in the regulation of the life span of cells. Telomeres are the distal ends of human chromosomes and are composed of tandem repeats of the sequence TTAGGG.1 Possible functions of telomeres include stabilization of chromosome ends and prevention of their degradation, end-to-end fusion, rearrangement, and chromosome loss.2 Telomerase, the specialized reverse transcriptase enzyme that elongates telomeres and synthesizes telomeric DNA, is repressed in most human somatic cells. This results in telomere shortening with each cell division, leading to a process thought to contribute to senescence. Recent research indicates that activation of telomerase is important for cells to proliferate indefinitely and that all human cancer cells require activation of this enzyme to maintain telomeric DNA, to overcome cellular senescence, and to attain immortality.3 A highly sensitive polymerase chain reaction (PCR)-based telomeric repeat amplification protocol assay has been established recently.4 With the use of this method, strong telomerase activity in human cancer cells4–14 and weak telomerase activity in some normal somatic cells such as hematopoeitic cells, epidermal keratinocytes, and cervical epithelial cells have been detected.15–20 In situ telomeric repeat amplification protocol assay studies have lent support to these results.21 In a previous study,22 we identified telomerase activity in chorionic villi and placenta specimens from women without fetal growth restriction (FGR). However, telomerase activity was not observed in placenta from women with asymmetric FGR. Furthermore, telomerase as well as proliferative activity decreases gradually from the proliferative phase to the secretory or atrophic phase in the endometrium.20

In the present study, we analyzed telomerase and proliferative activity in chorionic villi and placenta specimens from women without FGR and in placenta specimens from women with asymmetric FGR.


   Materials and Methods
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Abstract
 Materials and Methods
Results
 Discussion
 References
 
HeLa 293 cells (cervical cancer cell line) were used as positive controls and culture medium was used as the negative control. Peripheral lymphocytes were adopted for human somatic cells.

Sixty-nine specimens were obtained from pregnant women between 5 and 42 weeks’ gestation. Thirty first-trimester chorionic villi specimens were obtained from women between 5 and 14 weeks’ gestation (group A). Twenty-eight second- and third-trimester placenta specimens were obtained from women without FGR between 23 and 42 weeks’ gestation (group B). The remaining 11 specimens were placenta tissue from women with asymmetric FGR between 26 and 39 weeks’ gestation (group C). Thirty first-trimester chorionic villi specimens were obtained from women who underwent pregnancy termination (group A). Twenty-eight second- or third-trimester placenta specimens were obtained from women who underwent preterm or full-term deliveries (group B). The remaining 11 specimens were placenta tissue from women with asymmetric FGR treated with cesarean delivery for fetal distress (group C). The presumed etiology of the 11 cases of asymmetric FGR was toxemia of pregnancy. The definition of asymmetric FGR is estimated weight below the 10th percentile of Lubchenco’s standards by ultrasound.23

All specimens were obtained within 30 minutes after delivery, frozen, and stored at -80C until use. Informed consent was obtained from all patients.

Samples of each specimen also were fixed in neutral 10% formalin and then embedded in paraffin. All 69 specimens were used in this study.

Telomerase activity was assayed using the modified protocol developed by Kim et al.4 Each frozen sample (50–100 mg of tissue) was washed in 1 mL of ice-cold buffer, consisting of 10 mM HEPES-KOH (pH 7.5), 1.5 mM MgCl2, 10 mM KCl, and 1 mM dithiothrenitol; and then homogenized in 200 µL of ice-cold lysis buffer consisting of 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, 10 mM Tris-HCl (pH 7.5), 1 mM MgCl2, 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetra-acetic acid, 10% glycerol, 5 mM ß-mercaptanol, and 0.1 mM phenylmethylsulfonyl fluoride. After incubation on ice for 30 minutes, the lysate was centrifuged at 100,000 x g for 30 minutes at 4C, and the supernatant was frozen rapidly and stored at -80C. An aliquot of extract containing 5 µg of protein was used for each telomeric repeat amplification protocol assay. Assay tubes were prepared by sequestering 0.1 µg of digoxigenin-labeling CX primer (5'-CCCTTACCCTTACCCTTACCCTTA-3') under a wax barrier (Boehringer, Mannheim, Germany). Each extract was assayed in 40 µL of reaction mixture, which contained 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.005% Tween 20, 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetra-acetic acid, 50 µM dNTP, 0.1 µg of TS primer (5'-AATCCGTCGAGCA-GAG TT-3'), 0.5 µM T4 gene 32 protein (Boehringer), and 2 µof Taq DNA polymerase (Boehringer). After 30 minutes of incubation at room temperature, for telomerase-mediated extension of the 5'-AATCCGTCGAG-CAGAG TT-3'primer, the elongated reaction mixture was amplified by PCR through 30 cycles of 95C for 30 seconds, 50C for 30 seconds, and 72C for 90 seconds. The PCR product was electrophoresed through a 15% polyacrylamide gel in 0.5 x Tris-borate at 150 volts for 1.5 hours. To estimate telomerase activity in tissue samples, we compared the intensity of the 6–base pair DNA ladder with that of the positive (HeLa 293) and negative (culture medium) controls.

Cells were washed in cold phosphate-buffered saline, cytospun (4000 rpm for 3 minutes,) spread onto non-fluorescent silane-coated glass slides, and quickly air dried. Reaction mix (final volume 25 µL) containing 20 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, 63 mM KCl, 0.05% Tween 20, 1 mM ethylene glycol bis(2-aminoethyl ether)-N,N,N',N'-tetra-acetic acid, 50 µM deoxynucleoside triphosphates, 10% glycerol, 1 µg T4 gene 32 protein, bovine serum albumin 0.1 mg/mL, 2 µof Taq DNA polymerase, and 10 pmol of fluorescein isothiocyanate-labeled (5'–end labeled using Fluore Prime; Pharmacia Biotech, Uppsala, Sweden) TS forward-primer (5'-AATCCGTCGAGCAGAG TT-3') was placed on each slide and the slide was incubated for 30 minutes at 22C in a dark box. After 5'-AATCCGTCGAGCAGAG TT-3'extension, 25 µL of the same solution, but with 10 pmol of fluorescein isothiocyanate-labeled (5–end labeled) CX reverse-primer (5'-CCCTTACCCTTACCCT-TACCCTTA-3'), was added, coverslips were sealed, and the slide was heated to 90C for 1.5 minutes, to inactivate telomerase. Amplification then proceeded with use of a Hybrid Omni Slide System thermocycler (National Labnet Co., Woodbridge, NJ) under the following PCR conditions: 30 cycles of 94C for 30 seconds, 50C for 30 seconds, and 72C for 1.5 minutes. Slides were washed in tap water and then sealed with a coverslip using fluorescent mounting medium solution (DAKO Corp., Glostrup, Denmark). Cells were observed with a fluorescence microscope fitted with a B-filter (Olympus, Tokyo, Japan).

The MIB-1 monoclonal antibody, which was raised against recombinant parts of the Ki-67 antigen, was used to detect proliferating cells in formalin-fixed paraffin sections.24 The tissue sections were deparaffinized and immersed for 20 minutes in 0.3% hydrogen peroxidase to block endogenous peroxidase activity. After being washed with phosphate-buffered saline, the tissue sections were treated with 10% normal mouse serum for 30 minutes to inhibit nonspecific binding. The sections then were incubated in MIB-1 monoclonal antibody (Immunotech, Marseilles, France) at a 200-fold dilution at 4C. After being rinsed in phosphate-buffered saline, the tissue sections were incubated for 30 minutes with biotin-labeled antimouse immunoglobulin G and then treated with avidin-biotin complex at room temperature. Cells with binding MIB-1 were visualized using a 3,3'-diaminobenzidine substrate kit (Nichirei, Tokyo, Japan). More than 1000 trophoblastic cells in chorionic villi and placenta were selected randomly for estimation of proliferative activity, which was determined as the mean ratio of MIB-1 positive to MIB-1 negative cells and expressed as the percentage of MIB-1 positive cells. Data are expressed as mean ± standard deviation. Significant differences were assessed using the {chi}2 test and the Kruskal-Wallis test. Statistical significance was established at P < .05.


   Results
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Abstract
 Materials and Methods
 Results
Discussion
 References
 
Telomerase-positive specimens produced a characteristic 6–base pair DNA ladder as seen in lane 1, which contained DNA from the telomerase-positive cancer sample (HeLa 293). Telomerase activity was identified in chorionic villi specimens taken from women at 6 weeks’ (lane 2), 7 weeks’ (lane 3), 7 weeks’ (lane 4), 7 weeks’ (lane 5), and 9 weeks’ (lane 6) gestation. A short 6–base pair DNA ladder was observed in specimen extracts from peripheral lymphocytes in lane 7. The 6–base pair DNA ladder was not evident in the telomerase-negative control (culture medium), as shown in lane 8 (Figure 1Go). Telomerase activity was detected in 28 (93.3%) of 30 chorionic villi specimens (Table 1Go). Telomerase activity was identified in samples of placenta, obtained from women without FGR. Lanes 2, 3, 4, 5, 6, and 7 represent samples of placenta from women at 25, 38, 39, 39, 39, and 42 weeks’ gestation, respectively (Figure 2Go). Telomerase activity was detected in 18 (64.3%) of the 28 placenta samples from women without FGR (Table 1Go). Telomerase activity was not detected in any of the 11 women with asymmetric FGR (Table 1Go). Lanes 2, 3, 4, 5, 6, and 7 represent samples of placenta from women at 28, 28, 30, 30, 34, and 39 weeks’ gestation with asymmetric FGR, respectively (Figure 3Go). Thus, telomerase activity was detected significantly more often in chorionic villi and placenta specimens from women without FGR than in specimens from women with FGR, using the {chi}2 test (P < .01).



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  		Figure 1. Telomerase activity in chorionic villi, positive control (HeLA 293), peripheral lymphocytes, and negative control (culture medium).

 

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  		Table 1. Telomerase Activity and Proliferative Activity in Chorionic Villi and Placenta From Women With Asymmetric FGR and Without FGR
 


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  		Figure 2. Telomerase activity in a placenta specimen from a woman without fetal growth restriction.

 


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  		Figure 3. Telomerase activity in a placenta specimen from a woman with fetal growth restriction.

 
The original telomeric repeat amplification protocol assay showed that normal resting lymphocytes display only weak telomerase activity, whereas cancer cells (HeLA 293) contain detectable levels of telomerase activity (Figure 1Go). Similar results were obtained with the in situ telomeric repeat amplification protocol assay. Normal resting lymphocytes had a weak fluorescence signal (Figure 4Go), whereas cancer cells showed intense fluorescence (Figure 5Go). Such an intense signal also was observed in trophoblastic cells obtained from chorionic villi specimens from women at 6 weeks’ gestation (Figure 6AGo) and trophoblastic cells obtained from placenta from women at 32 weeks’ (Figure 6BGo) and 39 weeks’ (Figure 6CGo) gestation. However, no signal was detected in trophoblastic cells from placenta from women with asymmetric FGR. These findings confirmed those of the original telomeric repeat amplification protocol assay.



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  		Figure 4. In situ telomeric repeat amplification protocol assay of peripheral lymphocytes (x500 original magnification).

 


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  		Figure 5. In situ telomeric repeat amplification protocol assay of cancer cell line (HeLa 293) (x500 original magnification).

 


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  		Figure 6. In situ telomeric repeat amplification protocol assay of trophoblastic cells from women at 6 weeks’ gestation (A), 32 weeks’ gestation (B), and 39 weeks’ gestation (C) (x500 original magnification).

 
The proliferative activity of the 30 chorionic villi specimens (28.1 ± 1.7%) and the 28 placenta specimens (7.0 ± 2.9%) from women without FGR was significantly higher than that of the 11 placenta specimens from women with asymmetric FGR (1.9 ± 0.6%), using the Kruskal-Wallis test (P < .01). Representative results of proliferative activity in chorionic villi and placenta are shown in Figure 7A, B, and CGo.



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  		Figure 7. A) Chorionic villi from a woman with normal pregnancy, exhibiting high MIB-1 activity. Nuclei of MIB-1 positive cells were stained with brown. MIB-1 labeling index was 28.1%. B) Placenta from a woman without fetal growth restriction (FGR), exhibiting low MIB-1 activity. MIB-1 labeling index was 8.7%. C) Placenta from a woman with FGR, exhibiting low MIB-1 activity. MIB-1 labeling index was 1.2% (x500 original magnification).

 

   Discussion
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Abstract
 Materials and Methods
 Results
 Discussion
References
 
The placenta is an organ that develops into a large complex structure, engages in intricate biologic processes, and becomes senescent in a 9-month period. The chorionic villi are primary components of the placenta. During pregnancy, the placenta serves as a means for interchange of many substances between the fetal and maternal bloodstreams, thereby maintaining fetal homeostasis. The placenta also performs many activities that are, in part, comparable to those of the gastrointestinal, respiratory, circulatory, and renal systems of adults. These functions of the placenta involve not only regulation of the activities of the reproductive organs throughout the period in which the offspring is dependent on the mother, but also maintenance of other organs, such as the adrenals and mammary glands, that are not concerned directly with the initiation, continuation, and termination of pregnancy.

As the placenta grows and ages, certain histologic changes suggest an increase in the efficiency of transport to meet the metabolic requirements of the growing fetus. In FGR, the growth of the fetus and placenta is restricted. A large variety of pathologic conditions have been found to correlate with FGR, such as a small placenta, persisting placental immaturity, a high degree of placental infarction, fetoplacental vasculopathy, villous hypermaturity, and terminal villus deficiency,25 as well as a reduction in cytotrophoblastic proliferation and increased incidence of pyknotic nuclei and syncytial knotting.26 Syncytial knot is a group of syncytiotrophoblastic specializations characterized by accumulation and aggregation of nuclei that were caused by a limited amount of true syncytiotrophoblastic outgrowth. The small nuclei in the syncytial knot show a more condensed chromatin and sometimes are severely pyknotic.27 The surrounding cytoplasm usually exhibits degenerative changes. These findings are a typical feature of senescence and are similar in the trophoblasts of normal-term placenta.27–29 Telomerase activity is expected to be a powerful tool for cancer diagnosis and chemotherapy because of its high incidence in various cancer tissues, weak expression in some peripheral blood cell types and some hematopoietic stem cells,15,16 and negative expression in most human somatic cell types. These findings were confirmed by the study of in situ telomeric repeat amplification protocol assay.21

In the present study, we detected telomerase activity in chorionic villi and placenta specimens from women without FGR but not in placenta specimens from women with asymmetric FGR by telomeric repeat amplification protocol and in situ telomeric repeat amplification protocol assay. We also detected significantly higher proliferative activity in chorionic villi and placenta specimens from women without FGR than in those from women with asymmetric FGR. These findings suggest that telomerase and proliferative activity play a role in placental senescence in women with and without FGR.


   Footnotes
 
PII S0029-7844(98)00383-4

Received March 30, 1998. Received in revised form June 22, 1998. Accepted July 17, 1998.


   References
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Abstract
 Materials and Methods
 Results
 Discussion
 References
 
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24. Cattoretti G, Becker MH, Key G, Duchrow M, Schuter C, Galle J, et al. Monoclonal antibodies against recombinant parts of the Ki-67 antigen (MIB-1 and MIB-3) detect proliferating cells in microwave-processed formalin-fixed paraffin sections. J Pathol 1992;168:357–62.[Medline]

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