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

PURPOSE: 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: 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.

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

PURPOSE: 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.

Membrane lipid changes in mouse blastocysts induced by ovarian stimulation, IVF and oocyte vitrification.

RESEARCH QUESTION: Is the membrane lipid profile of mice blastocysts affected by ovarian stimulation, IVF and oocyte vitrification? Could supplementation of vitrification media with L-carnitine and fatty acids prevent membrane phospholipid changes in blastocysts from vitrified oocytes? DESIGN: Experimental study comparing the lipid profile of murine blastocysts produced from natural mating, superovulated cycles or after IVF submitted or not to vitrification. For in-vitro experiments, 562 oocytes from superovulated females were randomly divided into four groups: fresh oocytes fertilized in vitro and vitrified groups: Irvine Scientific (IRV); Tvitri-4 (T4) or T4 supplemented with L-carnitine and fatty acids (T4-LC/FA). Fresh or vitrified-warmed oocytes were inseminated and cultured for 96 h or 120 h. The lipid profile of nine of the best quality blastocysts from each experimental group was assessed by multiple reaction monitoring profiling method. Significantly different lipids or transitions between groups were found using univariate statistics (P < 0.05; fold change = 1.5) and multivariate statistical methods. RESULTS: A total of 125 lipids in blastocysts were profiled. Statistical analysis revealed several classes of phospholipids affected in the blastocysts by ovarian stimulation, IVF, oocyte vitrification, or all. L-carnitine and fatty acid supplements prevented, to a certain extent, changes in phospholipid and sphingolipid contents in the blastocysts. CONCLUSION: Ovarian stimulation alone, or in association with IVF, promoted changes in phospholipid profile and abundance of blastocysts. A short exposure time to the lipid-based solutions during oocyte vitrification was sufficient to induce changes in the lipid profile that were sustained until the blastocyst stage.

Equilibration solution composition and extended exposure to equilibration phase affect embryo development and lipid profile of mouse oocytes.

RESEARCH QUESTION: Can exposure time to equilibration solutions during oocyte vitrification affect the lipid profile of oocytes and embryonic development? Could vitrification media supplemented with oleic, linoleic acids and L-carnitine effectively minimize damage induced by vitrification on embryo development and oocyte membrane lipid profile? DESIGN: Experimental study including 936 oocytes from C57BL/6J mice, randomly divided into fresh IVF (control) and equilibration solution groups. Oocytes were exposed to equilibration solution from Irvine Scientific, Tvitri-4 or Tvitri-4 supplemented with L-carnitine and fatty acids for 7 or 10 min, vitrified-warmed, and submitted to IVF. The lipid profile of oocytes immediately after equilibration solution exposure was also asessed using the same equilibration times and solution compositions. RESULTS: Longer equilibration time resulted in lower oocyte survival and blastocyst rates, and reduced relative abundance of structural lipids, i.e. phosphatidylcholines and sphingomyelins, varying according to equilibration solution composition. It also induced membrane disruptions resembling bubbles in the oocyte surface predominantly in equilibration solution from Irvine Scientific, rarely in Tvitri-4 and absent in Tvitri-4 supplemented with L-carnitine and fatty acids. To reveal the metabolic pathways associated with the equilibration phase of vitrification, lipid pathway analysis was conducted; both P-values and pathway impact values showed that the linoleic acid metabolism (P = 0.00223; impact =1) and alpha-Linolenic acid metabolism (P = 0.00084; impact = 0.33) were the most pathway perturbed, followed by glycerophospholipid metabolism (P = 0.0167; impact = 0.25) CONCLUSION: A longer equilibration phase pre-vitrification can influence embryo development and induce changes in oocyte lipid composition related to membrane integrity. The results suggest internalization of oleic and linoleic acids added to equilibration solution by the oocyte, which, to some extent, contributed to membrane phospholipids preservation, regardless of the equilibration times assessed.

Impact of superovulation and in vitro fertilization on LINE-1 copy number and telomere length in C57BL/6 J mice blastocysts.

OBJECTIVE: Millions of babies have been conceived by IVF, yet debate about its safety to offspring continues. We hypothesized that superovulation and in vitro fertilization (IVF) promote genomic changes, including altered telomere length (TL) and activation of the retrotransposon LINE-1 (L1), and tested this hypothesis in a mouse model. MATERIAL AND METHODS: Experimental study analyzing TL and L1 copy number in C57BL/6 J mouse blastocysts in vivo produced from natural mating cycles (N), in vivo produced following superovulation (S), or in vitro produced following superovulation (IVF). We also examined the effects of prolonged culture on TL and L1 copy number in the IVF group comparing blastocysts cultured 96 h versus blastocysts cultured 120 h. TL and L1 copy number were measured by Real Time PCR. RESULTS: TL in S (n = 77; Mean: 1.50 ± 1.15; p = 0.0007) and IVF (n = 82; Mean: 1.72 ± 1.44; p < 0.0001) exceeded that in N (n = 16; Mean: 0.61 ± 0.27). TL of blastocysts cultured 120 h (n = 15, Mean: 2.14 ± 1.05) was significantly longer than that of embryos cultured for 96 h (n = 67, Mean: 1.63 ± 1.50; p = 0.0414). L1 copy number of blastocysts cultured for 120 h (n = 15, Mean: 1.71 ± 1.49) exceeded that of embryos cultured for 96 h (n = 67, Mean: 0.95 ± 1.03; p = 0.0162). CONCLUSIONS: Intriguingly ovarian stimulation, alone or followed by IVF, produced embryos with significantly longer telomeres compared to in vivo, natural cycle-produced embryos. The significance of this enriched telomere endowment for the health and longevity of offspring born from IVF merit future studies.

The Impact of Oocyte Vitrification on Offspring: a Systematic Review.

Oocyte vitrification is a widespread and well-established assisted reproduction technique that has enabled some patient groups to obtain clinical results equivalent to those using fresh oocytes. However, as the number of babies born from vitrified oocytes has increased, so has the discussion regarding the method's safety for the offspring. Cryogenic oocyte damage caused by chemical, mechanical, and thermal stress has raised concern. In this systematic review, we asked the question of whether oocyte vitrification impacts offspring health. From 2007 to 2021, 13 studies were included in the analysis. All studies were observational and presented neonatal outcomes. A total of 4,159 babies were analyzed. Data from these studies were used to assess the following outcomes: multiple pregnancies, cesarean section, gestational age at delivery, the number of live births, birth weight, Apgar scores, congenital anomalies, and baby health. The most extended follow-ups evaluated children until 1, 2, and 6 years of age. According to the evidence appraised in this systematic review, vitrification seems to be a safe method for oocyte cryopreservation and child health, at least in the short term. Nevertheless, there is an urgent need for additional long-term data results from big databases and also for randomized controlled trials to improve the levels of evidence.

Control of LINE-1 Expression Maintains Genome Integrity in Germline and Early Embryo Development.

Maintenance of genome integrity in the germline and in preimplantation embryos is crucial for mammalian development. Epigenetic remodeling during primordial germ cell (PGC) and preimplantation embryo development may contribute to genomic instability in these cells, since DNA methylation is an important mechanism to silence retrotransposons. Long interspersed elements 1 (LINE-1 or L1) are the most common autonomous retrotransposons in mammals, corresponding to approximately 17% of the human genome. Retrotransposition events are more frequent in germ cells and in early stages of embryo development compared with somatic cells. It has been shown that L1 activation and expression occurs in germline and is essential for preimplantation development. In this review, we focus on the role of L1 retrotransposon in mouse and human germline and early embryo development and discuss the possible relationship between L1 expression and genomic instability during these stages. Although several studies have addressed L1 expression at different stages of development, the developmental consequences of this expression remain poorly understood. Future research is still needed to highlight the relationship between L1 retrotransposition events and genomic instability during germline and early embryo development.

Zidovudine inhibits telomere elongation, increases the transposable element LINE-1 copy number and compromises mouse embryo development.

PURPOSE: Millions of pregnant, HIV-infected women take reverse transcriptase inhibitors, such as zidovudine (azidothymidine or AZT), during pregnancy. Reverse transcription plays important roles in early development, including regulation of telomere length (TL) and activity of transposable elements (TE). So we evaluated the effects of AZT on embryo development, TL, and copy number of an active TE, Long Interspersed Nuclear Element 1 (LINE-1), during early development in a murine model. DESIGN: Experimental study. METHODS: In vivo fertilized mouse zygotes from B6C3F1/B6D2F1 mice were cultured for 48 h in KSOM with no AZT (n = 45), AZT 1 µM (n = 46) or AZT 10 µM (n = 48). TL was measured by single-cell quantitative PCR (SC-pqPCR) and LINE-1 copy number by qPCR. The percentage of morulas at 48 h, TL and LINE-1 copy number were compared among groups. RESULTS: Exposure to AZT 1 µM or 10 µM significantly impairs early embryo development. TL elongates from oocyte to control embryos. TL in AZT 1 µM embryos is shorter than in control embryos. LINE-1 copy number is significantly lower in oocytes than control embryos. AZT 1 µM increases LINE-1 copy number compared to oocytes controls, and AZT 10 µM embryos. CONCLUSION: AZT at concentrations approaching those used to prevent perinatal HIV transmission compromises mouse embryo development, prevents telomere elongation and increases LINE-1 copy number after 48 h treatment. The impact of these effects on the trajectory of aging of children exposed to AZT early during development deserves further investigation.

The Importance of the Study of the Embryonic Metabolome in Assisted Human Reproduction.

Since the advent of IVF in 1978, many novel techniques have arisen in Assisted Human Reproduction (AR). Every year thousands of people seek treatment, with success rates ranging from 35% to 40%. The way of assessing an embryo for intrauterine transfer is still carried out by means of morphological parameters, a traditional methodology that brings little information about embryo physiology. Analyses of embryo metabolic activity seem to be excellent predictors of embryo viability and implantation potential. This methodology is still new and experimental, presenting some degree of operational difficulty, for which reason it is not yet performed on a routine basis at major Assisted Reproduction centers. This paper intends to discuss the importance of studying the embryonic metabolome for the selection and transfer of human embryos.