Reduced placental telomere length during pregnancies complicated by intrauterine growth restriction

Abstract

Objectives: Recent studies have shown that telomere length was significantly reduced in placentas collected at delivery from pregnancies complicated by intrauterine growth restriction secondary to placental insufficiency. Placental telomere length measurement during ongoing pregnancies complicated by intrauterine growth restriction has never been reported. This was the main objective of our study.

Methods: In our center, late chorionic villus samplings were performed between 18 and 37 weeks of amenorrhea in 24 subjects with severe intrauterine growth restriction (cases) and in 28 subjects with other indications for prenatal diagnosis (controls). Placental insufficiency was assessed by histo-pathological examination. Relative measurement of telomere length was carried out prospectively by quantitative Fluorescent In Situ Hybridization using fluorescent Peptide Nucleic Acid probes on interphase nuclei obtained from long-term cultured villi and with an automated epifluorescent microscope. A quantitative Polymerase Chain Reaction technique was performed to confirm the quantitative Fluorescent In Situ Hybridization results. The number of copies of gene loci encoding the RNA template (hTERC) and the catalytic subunit (hTERT) of the enzyme complex telomerase were also estimated in these placentas by Fluorescent In Situ Hybridization.

Results: Mean fluorescence intensity of telomere probes estimated by quantitative Fluorescent In Situ Hybridization was significantly less for cases compared to controls (p<0.001). This result indicated that mean telomere length was significantly reduced in placentas during pregnancies complicated by intrauterine growth restriction. Reduced telomere length was confirmed by the quantitative Polymerase Chain Reaction technique. No copy number variation of the hTERC and hTERT loci was noticed for cases, or for controls.

Conclusion: This study clearly demonstrates a reduction of placental telomere length in ongoing pregnancies (from 18 to 37 weeks of amenorrhea) complicated by severe intrauterine growth restriction secondary to placental insufficiency.

Figure 1. Placental telomere length estimated from late chorionic villus samplings by two quantitative techniques.

A. Quantitative Fluorescence In Situ Hybridization (Q-FISH). Telomere fluorescence intensity of Peptide Nucleic Acid probes (arbitrary units) as a function of pregnancy term of placental biopsy (expressed in weeks of amenorrhea (WA)) for the 28 controls (light grey circles) and the 24 cases with intrauterine growth restriction (IUGR) (dark grey circles). B. Quantitative Polymerase Chain Reaction (Q-PCR). T/S ratios (no unit) as a function of pregnancy term of placental biopsy (expressed in WA) for the 28 controls (light grey circles) and the 24 cases with IUGR (dark grey circles).

Figure 2. Comparison of placental telomere length between controls and cases with intrauterine growth restriction (IUGR), with two quantitative techniques.

A. Quantitative Fluorescence In Situ Hybridization (Q-FISH). Mean placental telomere fluorescence intensity of Peptide Nucleic Acid probes with the Q-FISH technique ± standard deviation (arbitrary units) for the 28 controls (light grey histogram) and the 24 cases with IUGR (dark grey histogram) (**: p<0.001, Student’s t-test). B. Quantitative Polymerase Chain Reaction (Q-PCR). Mean placental telomere T/S ratio with the Q-PCR technique ± standard deviation (no unit) for the 28 controls (light grey histogram) and the 24 cases with IUGR (dark grey histogram) (**: p<0.001, Student’s t-test).

Figure 3. Copy number evaluation by Fluorescence In Situ Hybridization (FISH) of the loci carrying hTERC and hTERT in late chorionic villus samplings.

A. Percentage of interphase nuclei displaying no gain or loss, relative losses, relative gains, and absolute gains of hTERC locus with the FISH technique ± standard deviation (%) for 12 controls (light grey histograms) and 12 cases with intrauterine growth restriction (IUGR) (dark grey histograms) randomly selected (p = non-significant (NS), Student’s t-test). B. Percentage of interphase nuclei displaying no gain or loss, relative losses, relative gains, and absolute gains of hTERT locus with the FISH technique ± standard deviation (%) for 12 controls (light grey histograms) and 12 cases with IUGR (dark grey histograms) randomly selected (p = NS, Student’s t-test).