Abstract
Perinatal and childhood adverse outcomes associated with assisted reproductive technology (ART) has been reported, but it remains unknown whether the initial leukocyte telomere length (LTL), which is an indicator of age-related phenotypes in later life, is affected. Here, we estimated the LTLs of 1,137 individuals from 365 families, including 202 children conceived by ART and 205 children conceived spontaneously from two centers of the China National Birth Cohort, using whole-genome sequencing (WGS) data. One-year-old children conceived by ART had shorter LTLs than those conceived spontaneously (beta, −0.36; P = 1.29 × 10–3) after adjusting for plurality, sex and other potential confounding factors. In particular, blastocyst-stage embryo transfer was associated with shorter LTL (beta, −0.54, P = 2.69 × 10–3) in children conceived by ART. The association was validated in 586 children conceived by ART from five centers using different LTL quantification methods (that is, WGS or qPCR). Blastocyst-stage embryo transfer resulted in shorter telomere lengths in mice at postnatal day 1 (P = 2.10 × 10–4) and mice at 6 months (P = 0.042). In vitro culturing of mice embryos did not result in shorter telomere lengths in the late cleavage stage, but it did suppress telomerase activity in the early blastocyst stage. Our findings demonstrate the need to evaluate the long-term consequences of ART, particularly for aging-related phenotypes, in children conceived by ART.
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Data availability
WGS-based telomere length data, including the number of telomeric reads, the number of reads with GC content between 48–52%, and individual bam files of related aligned sequence, have been deposited to the ‘Download’ section of a publicly accessible website (http://omics.njmu.edu.cn:8012/search). An account can be easily registered with an academic email address and OMICS have provided a comprehensive ‘Help’ page that includes a step-by-step tutorial and details on how to use the website. Public datasets from other works50 can be accessed in the NCBI Gene Expression Omnibus (GEO) repository under accession numbers GSE136714 (mouse embryo scNOME-seq data) and GSE136715 (mouse embryo scRNA-seq data).
Code availability
The custom scripts used for WGS processing, telomere length estimation and single-cell multiomics data analysis can be found at https://github.com/NMUEPI/Telomere_Length.
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Acknowledgements
We thank all of the members of the collaborative group of the CNBC. We also thank all study participants for their support. This study was funded by grants from the China National Key Research & Development (R&D) Plan (2021YFC2700600 (to Z.H.), 2018YFC1004200 (to Z.H.), 2016YFC1000200 (to H.S.)).
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Z.H. and H.S. initiated, conceived, and supervised the study. C.W. performed bioinformatics and statistical analysis, and drafted the manuscript with J.Zhou, N.Q., W.D., and C.C. Y.G. and J.Zang conducted experiments with Y.J. and Y.H. X.Ling, H.Li, L.H., Bei Xu, B.Z., T.J., J.Du, J.Dai, F.D., C.L., X.G., R.H., J.L., Y.L., Y. X., G.J., H.M. and H.S. participated in the design and organization of the CNBC cohort study; H.Lv, X.H., K.Z., Bo Xu, X.Liu, S.T., and YQ.J. assisted in sample collection and clinical information organization. The manuscript was revised by all authors.
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Nature Medicine thanks Andres Salumets, Daniel Brison and the other, anonymous, reviewer(s) for their contribution to the peer review of this study. Primary Handling Editor: Ming Yang, in collaboration with the Nature Medicine team.
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Extended data
Extended Data Fig. 1 The correlation between LTL and age.
a,b The correlation between maternal LTL and maternal age (a), and paternal LTL and paternal age (b). Pearson’s correlation test was performed to calculate Pearson’s r and two-sided P-value. Abbreviations: LTL, leukocyte telomere length.
Extended Data Fig. 2 Comparison of LTL estimated by WGS and by qPCR assay.
qPCR assay was used to re-detect LTL in 70 singleton children from the discovery cohort with sufficient qualified DNA samples. Y-axis represents Z-standardized telomere length estimated by Telseq based on WGS data; X-axis represents Z-standardized telomere length estimated by qPCR assay. Pearson’s correlation test was performed to calculate the Pearson’s r and two-sided P-value. Abbreviations: LTL, leukocyte telomere length; WGS, whole-genome sequencing.
Extended Data Fig. 3 Forest plot of multivariable regression analysis including ET cycles and ET stages in children conceived by ART.
Linear mixed models were used for calculating beta coefficient and associated two-sided P-value. Data are estimates of beta (illustrated by the square symbol at center for error bars) and range of two-sided 95% confidence interval (CI) of the beta. Bold indicates statistically significant (P-value<0.05). Discovery: Children LTL ~ Parental conceived age + Parental LTL + ET stages + ET cycles + gestational age + plurality + sex. Validation I: Children LTL ~ Parental conceived age + ET stages + ET cycles + gestational age + plurality + sex. Validation II: Children LTL ~ Parental conceived age + ET stages + ET cycles + gestational age + plurality + sex. *Children number was counted. Abbreviations: LTL, leukocyte telomere length; ET, embryo transfer.
Extended Data Fig. 4 Forest plot for the stratified analysis based on the ET cycles and ET stages.
Data are estimates of the beta (illustrated by the diamond symbol or circle at center for error bars) and the range of two-sided 95% confidence interval (CI) of the beta. Cochran’s Q test was used to test for heterogeneity across subgroups (Het P-value). Linear mixed models were used for calculating beta coefficient and associated two-sided P-value. a, Adjusting for parental LTL, gestational age, plurality, and sex in children born from ART-conceived pregnancies in discovery cohort. b,c, Adjusting for gestational age, plurality, and sex in children conceived by ART in the validation I and II cohorts. * Children number was counted. Abbreviations: LTL, leukocyte telomere length; ET, embryo transfer.
Extended Data Fig. 5 Boxplot of telomere length in children born from spontaneous pregnancies, children born from ART-conceived pregnancies following cleavage-stage transfer and children born from ART-conceived pregnancies following cleavage-stage transfer.
The box plot displays the first and third quartiles (top and bottom of the boxes), the median (band inside the boxes), and the lowest and highest point within 1.5 times the interquartile range of the lower and higher quartile (whiskers). A linear mixed model was used for calculating beta coefficient and associated two-sided P-value. Abbreviations: ART, assisted reproductive technology.
Extended Data Fig. 6 The association between age and telomere length estimated by WGS in 1,185 healthy controls from Nanjing Lung Cancer Cohort aged 40–69.
The telomere length was estimated by WGS data. Telomere length showed significantly inverse association with age. After adjusting sex, telomere tended to lose 0.04 standardized units yearly. A generalized linear model was used for calculating beta coefficient and associated two-sided P-value. Abbreviations: LTL, leukocyte telomere length; WGS, whole-genome sequencing.
Extended Data Fig. 7 Years of aging between 40 and 69 years equivalent to LTL shortening caused by blastocyst-stage embryo transfer.
In the discovery and the validation I cohorts that estimated LTL by WGS, equivalent years were calculated as a quotient of the beta in Fig. 1 (blastocyst vs. cleavage, n = 120 following blastocyst-stage transfer and n = 82 following cleavage-stage transfer in discovery cohort, n = 107 following blastocyst-stage transfer and n = 73 following cleavage-stage transfer in validation I cohort) and the beta in Extended Data Fig. 6. In the validation II cohort that estimated LTL by qPCR, equivalent years were calculated as a quotient of the beta in Fig. 1 (blastocyst vs. cleavage, n = 180 following blastocyst-stage transfer and n = 226 following cleavage-stage transfer) and the beta published previously with UK Biobank26. Data are estimates of beta (illustrated by the square symbol at center for error bars) and range of two-sided 95% confidence interval (CI) of the beta. Fieller’s method was used to calculate the 95% CI and two-sided P-value. Abbreviations: LTL, leukocyte telomere length.
Extended Data Fig. 8 Telomere length of P1 mice born through different ET stages in different tissues.
The box plot displays the first and third quartiles (top and bottom of the boxes), the median (band inside the boxes), and the lowest and highest point within 1.5 times the interquartile range of the lower and higher quartile (whiskers). The Student’s t-test was performed to calculate the two-sided P-value. Abbreviations: P1, postnatal day 1; ET, embryo transfer.
Extended Data Fig. 9 The methylation level or upstream important transcription factors of gene Tert.
To explore the mechanisms underlying the telomere shortening, we included single-cell multi-omics sequencing (transcriptome and epigenome) data of mouse early embryos with different developmental stages. The box plot displays the first and third quartiles (top and bottom of the boxes), the median (band inside the boxes), and the lowest and highest point within 1.5 times the interquartile range of the lower and higher quartile (whiskers). a, The expression level of Tert increased at the early blastocyst stage (8-cell stage vs. 32-cell stage, 28 vs. 53, two-sided Wilcoxon rank sum test, P = 1.71 × 10−2). b, We observed a decreasing methylation level of Tert from the late cleavage stage to the early blastocyst stage (8-cell stage vs. 32-cell stage, 28 vs. 53, two-sided Wilcoxon rank sum test, P = 4.79 × 10−12). c, Embryos with low methylation level (average methylation level < 0.5, n = 58) of Tert showed significantly increased Tert expression (two-sided Wilcoxon rank sum test, P = 4.60 × 10−2) (d) SCENIC was used to identify the regulators of Tert expression and potential regulators of Tert expression (that is, Hif1a and Srebf1) were identified.
Extended Data Fig. 10 Association between ET stages and LTL in children detected by WGS and qPCR, respectively.
Beta and 95% confidence interval (CI) were generated from multivariate models adjusting for gestational age, sex, and embryo transfer cycles. Data are estimates of the beta based on WGS data (illustrated by the red circle) or qPCR (illustrated by the orange diamond symbol at center for error bars) and the range of two-sided 95% CI of the beta. A generalized linear model was used for calculating beta coefficient and associated two-sided P-value. *Children number was counted. Abbreviations: LTL, leukocyte telomere length; ET, embryo transfer.
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Wang, C., Gu, Y., Zhou, J. et al. Leukocyte telomere length in children born following blastocyst-stage embryo transfer. Nat Med 28, 2646–2653 (2022). https://doi.org/10.1038/s41591-022-02108-3
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DOI: https://doi.org/10.1038/s41591-022-02108-3
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