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Telomere length: a marker for reproductive aging?

Pirtea, Paul; Keefe, David L; Ayoubi, Jean Marc; de Ziegler, Dominique
The improvements accomplished in assisted reproductive technology have emphasized more than ever the role played by chronological age, notably for predicting oocyte quality. Studies in cellular aging have directed research on telomere length measurements as possible markers of functional aging and, notably, female reproductive outcomes. Although further research is still needed, encouraging results are already available on the possibility that leucocyte telomere length may be a useful parameter for assessing reproductive potential in aging women.
PMID: 37914069
ISSN: 1556-5653
CID: 5620382

Telomere dynamics and reproduction

Robinson, LeRoy G; Kalmbach, Keri; Sumerfield, Olivia; Nomani, Wafa; Wang, Fang; Liu, Lin; Keefe, David L
The oocyte, a long-lived, postmitotic cell, is the locus of reproductive aging in women. Female germ cells replicate only during fetal life and age throughout reproductive life. Mechanisms of oocyte aging include the accumulation of oxidative damage, mitochondrial dysfunction, and disruption of proteins, including cohesion. Nobel Laureate Bob Edwards also discovered a "production line" during oogonial replication in the mouse, wherein the last oocytes to ovulate in the adult-derived from the last oogonia to exit mitotic replication in the fetus. On the basis of this, we proposed a two-hit "telomere theory of reproductive aging" to integrate the myriad features of oocyte aging. The first hit was that oocytes remaining in older women traversed more cell cycles during fetal oogenesis. The second hit was that oocytes accumulated more environmental and endogenous oxidative damage throughout the life of the woman. Telomeres (Ts) could mediate both of these aspects of oocyte aging. Telomeres provide a "mitotic clock," with T attrition an inevitable consequence of cell division because of the end replication problem. Telomere's guanine-rich sequence renders them especially sensitive to oxidative damage, even in postmitotic cells. Telomerase, the reverse transcriptase that restores Ts, is better at maintaining than elongating T. Moreover, telomerase remains inactive during much of oogenesis and early development. Oocytes are left with short Ts, on the brink of viability. In support of this theory, mice with induced T attrition and women with naturally occurring telomeropathy suffer diminished ovarian reserve, abnormal embryo development, and infertility. In contrast, sperm are produced throughout the life of the male by a telomerase-active progenitor, spermatogonia, resulting in the longest Ts in the body. In mice, cleavage-stage embryos elongate Ts via "alternative lengthening of telomeres," a recombination-based mechanism rarely encountered outside of telomerase-deficient cancers. Many questions about Ts and reproduction are raised by these findings: does the "normal" T attrition observed in human oocytes contribute to their extraordinarily high rate of meiotic nondisjunction? Does recombination-based T elongation render embryos susceptible to mitotic nondisjunction (and mosaicism)? Can some features of Ts serve as markers of oocyte quality?
PMID: 37993053
ISSN: 1556-5653
CID: 5608742

Should we use CRISPR gene editing in human embryos? [Editorial]

Kubikova, Nada; Keefe, David L; Wells, Dagan; Oktay, Kutluk H; Feinberg, Eve C
PMID: 37656090
ISSN: 1556-5653
CID: 5618102

A pilot study of LINE-1 copy number and telomere length with aging in human sperm

Berteli, Thalita S; Wang, Fang; Navarro, Paula A; Kohlrausch, Fabiana B; Keefe, David L
PURPOSE/OBJECTIVE:Unlike other cells in the body, in sperm, telomere length (TL) increases with age. TL can regulate nearby genes, and the subtelomeric region is rich in retrotransposons. We hypothesized that age-related telomere lengthening in sperm might suppress Long Interspersed Element 1 (LINE-1/L1), the only competent retrotransposon in humans. METHODS:We measured L1 copy number (L1-CN) and sperm telomere length (STL) from young and older men to evaluate the relationship between age, TL and L1-CN. We also evaluated L1-CN and TL in individual sperm to determine whether these variables influence sperm morphology. STL was assayed by Multiplex quantitative polymerase chain reaction method (mmqPCR) and L1-CN by Quantitative polymerase chain reaction (qPCR). RESULTS:We found that STL increased, and L1-CN decreased significantly with paternal age. STL in normal single sperm was significantly higher than in abnormal sperm. L1-CN did not differ between normal and abnormal sperm. Furthermore, morphologically normal sperm have longer telomeres than abnormal sperm. CONCLUSIONS:Elongation of telomeres in the male germline could repress retrotransposition, which tends to increase with cellular aging. More studies in larger cohorts across a wide age span are needed to confirm our conclusions and explore their biological and clinical significance.
PMID: 37382785
ISSN: 1573-7330
CID: 5540392

Transposon insertion profiling by sequencing (TIPseq) identifies novel LINE-1 insertions in human sperm

Berteli, Thalita S; Wang, Fang; McKerrow, Wilson; Navarro, Paula A; Fenyo, David; Boeke, Jef D; Kohlrausch, Fabiana B; Keefe, David L
PURPOSE/OBJECTIVE:Long interspersed nuclear element-1 (LINE-1 or L1) comprises 17% of the human genome. Retrotransposons may perturb gene integrity or alter gene expression by altering regulatory regions in the genome. The germline employs a number of mechanisms, including cytosine methylation, to repress retrotransposon transcription throughout most of life. Demethylation during germ cell and early embryo development de-represses retrotransposons. Intriguingly, de novo genetic variation appearing in sperm has been implicated in a number of disorders in offspring, including autism spectrum disorder, schizophrenia, and bipolar disorder. We hypothesize that human sperm exhibit de novo retrotransposition and employ a new sequencing method, single cell transposon insertion profiling by sequencing (scTIPseq) to map them in small amounts of human sperm. METHODS:Cross-sectional case-control study of sperm samples (n=10 men; ages 32-55 years old) from consenting men undergoing IVF at NYU Langone Fertility Center. scTIPseq identified novel LINE-1 insertions in individual sperm and TIPseqHunter, a custom bioinformatics pipeline, compared the architecture of sperm LINE-1 to known LINE-1 insertions from the European database of Human specific LINE-1 (L1Hs) retrotransposon insertions (euL1db). RESULTS:scTIPseq identified 17 novel insertions in sperm. New insertions were mainly intergenic or intronic. Only one sample did not exhibit new insertions. The location or number of novel insertions did not differ by paternal age. CONCLUSION/CONCLUSIONS:This study for the first time reports novel LINE-1 insertions in human sperm, demonstrating the feasibility of scTIPseq, and identifies new contributors to genetic diversity in the human germ line.
PMID: 37310664
ISSN: 1573-7330
CID: 5591902

Vitrification with Dimethyl Sulfoxide Induces Transcriptomic Alteration of Gene and Transposable Element Expression in Immature Human Oocytes

Wiltshire, Ashley; Schaal, Renata; Wang, Fang; Tsou, Tiffany; McKerrow, Wilson; Keefe, David
Despite substantial advancements in the field of cryobiology, oocyte and embryo cryopreservation still compromise developmental competence. Furthermore, dimethyl sulfoxide (DMSO), one of the most commonly used cryoprotectants, has been found to exert potent effects on the epigenetic landscape of cultured human cells, as well as mouse oocytes and embryos. Little is known about its impact on human oocytes. Additionally, few studies investigate the effects of DMSO on transposable elements (TE), the control of which is essential for the maintenance of genomic instability. The objective of this study was to investigate the impact of vitrification with DMSO-containing cryoprotectant on the transcriptome, including on TEs, of human oocytes. Twenty-four oocytes at the GV stage were donated by four healthy women undergoing elective oocyte cryopreservation. Oocytes were paired such that half from each patient were vitrified with DMSO-containing cryoprotectant (Vitrified Cohort), while the other half were snap frozen in phosphate buffer, unexposed to DMSO (Non-Vitrified Cohort). All oocytes underwent RNA sequencing via a method with high fidelity for single cell analysis, and which allows for the analysis of TE expression through Switching Mechanism at the 5'-end of the RNA Transcript sequencing 2 (SMARTseq2), followed by functional enrichment analysis. Of the 27,837 genes identified by SMARTseq2, 7331 (26.3%) were differentially expressed (p < 0.05). There was a significant dysregulation of genes involved in chromatin and histone modification. Mitochondrial function, as well as the Wnt, insulin, mTOR, HIPPO, and MAPK signaling pathways were also altered. The expression of TEs was positively correlated with the expression of PIWIL2, DNMT3A, and DNMT3B, and negatively correlated with age. These findings suggest that the current standard process of oocyte vitrification, involving DMSO-containing cryoprotectant, induces significant transcriptome changes, including those involving TEs.
PMID: 37372413
ISSN: 2073-4425
CID: 5538612

The Landscape of Telomere Length and Telomerase in Human Embryos at Blastocyst Stage

Wang, Fang; McCulloh, David H; Chan, Kasey; Wiltshire, Ashley; McCaffrey, Caroline; Grifo, James A; Keefe, David L
The telomere length of human blastocysts exceeds that of oocytes and telomerase activity increases after zygotic activation, peaking at the blastocyst stage. Yet, it is unknown whether aneuploid human embryos at the blastocyst stage exhibit a different profile of telomere length, telomerase gene expression, and telomerase activity compared to euploid embryos. In present study, 154 cryopreserved human blastocysts, donated by consenting patients, were thawed and assayed for telomere length, telomerase gene expression, and telomerase activity using real-time PCR (qPCR) and immunofluorescence (IF) staining. Aneuploid blastocysts showed longer telomeres, higher telomerase reverse transcriptase (TERT) mRNA expression, and lower telomerase activity compared to euploid blastocysts. The TERT protein was found in all tested embryos via IF staining with anti-hTERT antibody, regardless of ploidy status. Moreover, telomere length or telomerase gene expression did not differ in aneuploid blastocysts between chromosomal gain or loss. Our data demonstrate that telomerase is activated and telomeres are maintained in all human blastocyst stage embryos. The robust telomerase gene expression and telomere maintenance, even in aneuploid human blastocysts, may explain why extended in vitro culture alone is insufficient to cull out aneuploid embryos during in vitro fertilization.
PMID: 37372380
ISSN: 2073-4425
CID: 5538602

Telomere fusions as a signal of term placental aging? A pilot study

Kohlrausch, Fabiana B; Wang, Fang; Luo, Danxia; Mahn, Rebecca; Keefe, David L
The placenta plays an essential role at the beginning of life, nourishing and supporting the fetus, but its life span is limited. In late pregnancy, the placenta develops signs of aging, including inflammation and impaired function, which may complicate pregnancy. Placentas also show another sign of aging - cells with extra or missing chromosomes. Chromosomally abnormal cells could gather in the placenta if they get stranded there and/or if the cells do not separate normally. Chromosome separation goes wrong in aging cells when the DNA sequences, which protect the ends of the chromosomes, erode. When chromosomes lose their protective caps, they fuse which leads to abnormal numbers of chromosomes. In this pilot study, for the first time, we found fusions between the caps in a human placenta when it reaches full term. More studies are needed to decide whether this has an influence on how the placenta works and outcomes of pregnancy.
PMID: 36374285
ISSN: 2633-8386
CID: 5381622


Wiltshire, A M; Schaal, R F; WANG, F; Tsou, T; McKerrow, W; Keefe, D L
Objective: DMSO alters the epigenetic state of mouse oocytes and human cultured cells. The effect of vitrification with DMSO containing cryoprotectant on gene and TE expression in human oocytes is unknown.
Material(s) and Method(s): A prospective paired controlled cohort laboratory study was performed from February - June 2021. Twenty-four discarded oocytes in the germinal vesical (GV) stage were donated from four patients. All oocytes were paired such that half of the oocytes from each patient were vitrified with DMSO-containing cryoprotectant, while the other half were frozen, unexposed to DMSO. All oocytes underwent RNA sequencing via Switching Mechanism At the end of the 5'-end of the RNA Transcript sequencing 2 (SMARTseq2). Reads containing adapters, bases that could not be determined >10% and low quality reads were excluded. Gene mapping to the human reference genome, followed by gene quantification, were performed. Next, differential gene expression analysis between the two cohorts was performed. Then functional enrichment analyses of dysregulated gene sets were performed with Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Human Disease Ontology (DO). Raw data obtained from RNA sequencing was used to analyze TE transcripts using the BonaFide-TEseq method, which were mapped to specific TE loci using Software for Quantifying Interspersed Repeat Expression (SQuIRE), to identify differentially expressed TEs. Real time-qPCR validated results of selected genes and TEs.
Result(s): Of the 27,837 genes identified by SMARTseq2, 7,331 (26.3%) were differentially expressed (p<0.05). Specifically, 3,987 genes were upregulated and 3,344 genes were downregulated in oocytes exposed to DMSO. Genes involved in chromatin and histone modification, and mitochondrial function, as well as WNT, insulin, MTOR, HIPPO and MAPK signaling pathways were affected by DMSO. There was no significant over expression of human disease ontology terms within our data set. Expression of a number of TEs was also affected by exposure to DMSO, including Alu, endogenous retrovirus family members 1 and K (ERV1, ERVK) and long interspersed nuclear elements 1 (LINE-1). Notably, the effects of DMSO on TE expression were most pronounced in the oldest patient. The expression of TEs was negatively correlated with age, and positively correlated with the expression of PIWI-like protein 2 (PIWIL2), DNA Methyltransferase (DNMT) 3A and 3B.
Conclusion(s): Vitrification with DMSO exposure leads to significant changes in gene and TE expression in human GV oocytes. Future experiments should determine whether MII oocytes respond similarly. Impact Statement: Oocyte vitrification with DMSO containing cryoprotectant induces significant transcriptome changes, including those involving TEs, in human GV oocytes. Further studies are needed to evaluate the clinical significance of these findings. Support: This study was supported by the Stanley H Kaplan Fund.
ISSN: 1556-5653
CID: 5366962

Prenatal phthalate exposure and placental telomere length: Prenatal DEHP exposure and placental telomere length [Letter]

Hawks, Rebecca Mahn; Kahn, Linda G; Fang, Wang; Keefe, David; Mehta-Lee, Shilpi S; Brubaker, Sara; Trasande, Leonardo
PMID: 35853584
ISSN: 2589-9333
CID: 5278972