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Study question Do human sperm contain novel LINE–1 insertions and are they affected by paternal age? Summary answer Human sperm contain novel LINE–1 insertions. Their location or number are not affected by paternal age. What is known already LINE–1 comprises 17% of the human genome and some LINE–1s are the only autonomous retrotransposons in humans. Retrotransposons influence genomic instability and/or regulation if new retrotransposition events disrupt coding or regulatory regions in the host genome. Demethylation during germ cell development de-represses retrotransposons. Advanced paternal age is associated with genomic instability. Previously we showed that sperm LINE–1 copy number decreases with paternal age. We hypothesize that human sperm exhibit de novo retrotransposition and that sperm from older men contain increased novel LINE–1 insertions. Study design, size, duration Cross-sectional case–control study with semen samples collected between February to July 2020. Participants/materials, setting, methods Normospermic sperm samples (n = 10; 5 Main results and the role of chance TIPseq identified 17 novel insertions in sperm, 8 from older (≥ 45 years) and 9 in younger men ( Limitations, reasons for caution The small sample-size and use of normospermic specimens limit interpretation of paternal age effect on LINE–1. Besides, the novel insertions could be polymorphic sites that have low allele frequency and thus have not yet been described. Wider implications of the findings: This study for the first time reports novel LINE–1 insertions in human sperm, demonstrating that scTIPseq method is a feasible technique, and identifying new contributions to genetic diversity in the human germ line. Further studies are needed to evaluate the impact of these insertions on sperm function. Trial registration number Not applicable

Study question Do young women with idiopathic early ovarian ageing have changes in telomere length and epigenetic age indicating accelerated biological aging? Summary answer The telomere length and epigenetic age were comparable to those in young women with normal ovarian ageing. What is known already Increased risk of several health events usually considered to be age-related such as cardiovascular disease, osteoporosis, over-all morbidity and mortality have been associated with premature and early menopause when compared to the risk in women with normal menopausal age suggesting an accelerated general ageing process associated to early ovarian ageing. It is unclear whether the onset of this process may start before menopause. Study design, size, duration A prospective cohort study. Young women (≤ 37 years) having ART at two Danish Public fertility clinics during the period 2016 to 2018 were divided into two groups dependent on their ovarian reserve status: early ovarian ageing (EOA) (N = 55) and normal ovarian ageing (NOA)( N = 52). Number of oocytes harvested in first and subsequent cycles was used as a marker of ovarian reserve. Blood samples was drawn at time of oocyte retrieval to assess biological age. Participants/materials, setting, methods EOA was defined as ≥ 2 IVF cycles with ≤ 5 harvested oocytes despite sufficient stimulation with FSH and NOA as ≥ 8 oocytes harvested in minimum 1 cycle. Known causes influencing the ovarian reserve (endometriosis, ovarian surgery, etc.) was reason for exclusion. Relative telomere length (qPCR) and epigenetic age acceleration (DNA methylation levels) were measured in white blood cells as markers of accelerated biological ageing. Main results and the role of chance Relative telomere length was comparable with a mean of 0.46 (± sd 0.12) in the EOA group and 0.47 (0.14) in the normal ovarian ageing group (p = 0.64). The difference of predicted mean epigenetic age and mean chronological age (i.e. epigenetic age acceleration) was, insignificantly, 0.5 years older in the EOA group when compared to the NOA group( (–1.02 years (2.62) and –1.57 years (2.56), respectively, p = 0.27)), but this difference disappeared when adjusting for chronological age. Limitations, reasons for caution Discrete changes in epigenetic age acceleration may not have been captured as the study only had power to detect an age acceleration of ≥ 2 years. Wider implications of the findings: By analysis of biomarkers for ageing in whole blood, we did not find any indications of a premenopausal accelerated aging in young women with idiopathic EOA. Further investigations in a similar cohort of premenopausal women is needed to fully elucidate the potential relationship between premenopausal accelerated biological ageing and EOA. Trial registration number The study was approved by the Danish Data protection Agency (nr 1–16–02–320–14) and the Regional committee on health research ethics of Central Region Denmark (jr.no 1–10–72–142–14).

STUDY QUESTION Does resveratrol counteract age-associated infertility in a mouse model of reproductive aging? SUMMARY ANSWER Long-term-oral administration of resveratrol protects against the reduction of fertility with reproductive aging in mice. WHAT IS KNOWN ALREADY Loss of oocytes and follicles and reduced oocyte quality contribute to age-associated ovarian aging and infertility. Accumulation of free radicals with age leads to DNA mutations, protein damage, telomere shortening, apoptosis and accelerated ovarian aging. Increasing evidence shows that resveratrol, enriched in certain foods, for example red grapes and wine, has anti-tumor and anti-aging effects on somatic tissues by influencing various signaling pathways, including anti-oxidation, as well as activating Sirt1 and telomerase. We investigated the potential of resveratrol to stave off ovarian aging in the inbred C57/BL6 mouse model. STUDY DESIGN, SIZE, DURATION Young C57/BL6 females (aged 2-3 months) were fed with resveratrol added to drinking water at 30 mg/l (providing ∼7.0 mg/kg/day) for 6 or 12 months, and the fertility and ovarian functions were compared among mice treated with or without resveratrol, and young mice served as reproductive controls. Experiments were repeated three times, with an average of 25 females randomly allocated to each treatment group for each repeat. PARTICIPANTS/MATERIALS, SETTING, METHODS Reproductive performance of female mice was determined by litter size, ovarian follicles and oocyte quantity and quality, and compared with age-matched controls. The impact of resveratrol on telomeres and telomerase activity, and expression of genes associated with cell senescence also was evaluated. MAIN RESULTS AND THE ROLE OF CHANCE Young mice fed with resveratrol for 12 months retained the capacity to reproduce, while age-matched controls produced no pups. Consistently, mice fed with resveratrol for 12 months exhibited a larger follicle pool than controls (P < 0.05). Furthermore, telomerase activity, telomere length and age-related gene expression in ovaries of mice fed with resveratrol resembled those of young mice, but differed (P < 0.05) from those of age-matched old mice. Resveratrol improved (P < 0.05) the number and quality of oocytes, as evidenced by spindle morphology and chromosome alignment. Also, resveratrol affected embryo development in vitro in a dose-dependent manner. LIMITATIONS, REASONS FOR CAUTION The doses of resveratrol and the experimental conditions used by different research groups have varied considerably, and the dosage influences both the effectiveness and toxicity of resveratrol. Fine-tuning the dosage of resveratrol likely will optimize its anti-aging effects on ovarian function. WIDER IMPLICATIONS OF THE FINDINGS Our data provide a proof of principle of the fertility-sparing effect of resveratrol in female mice. Although depletion of the ovarian reserve of high-quality oocytes also contributes to increased infertility with reproductive aging in women, the data obtained using a mouse model may not extrapolate directly to human reproduction, and more extensive research is needed if any clinic trials are to be attempted. STUDY FUNDING/COMPETING INTEREST(S) This work was supported by MOST of China National Basic Research Program (grant number: 2010CB94500 and 2012CB911200). The authors have no competing interests to declare.

Background Spindles are formed from microtubules and are exquisitely sensitive to changes in temperature. An orientation-independent polarized light microscope, the Polscope, can be used to image spindles in living oocytes allowing analysis of spindle kinetics in the living state. This study examined the effects of cooling on spindle disassembly in living human oocytes and spindle recovery after rewarming. Methods Oocytes were imaged continuously with the Polscope during cooling and rewarming. The quantity of microtubules in the spindles was measured by its birefringence using the Polscope. Results Spindles had completely disassembled by 5 min after cooling and recovered by 20 min after rewarming to 37 degrees C if rewarming started soon after the oocyte's temperature dropped to room temperature. However, when oocytes were cooled and kept at 33, 28 or 25 degrees C for 10 min and then warmed, it was found that warming allowed 5/5, 2/5 and 0/5 oocytes of the spindles to recover respectively. Conclusions These results indicate that human meiotic spindles are exquisitely sensitive to alterations in temperature. The maintenance of temperature at 37 degrees C during in-vitro manipulation is important for spindle integrity and, therefore, is likely to be important for normal fertilization and subsequent embryo development.

The zona pellucida (zona) is a glycoprotein coat that envelopes the oocyte and embryo, binds sperm during fertilization and facilitates transfer of the embryo through the Fallopian tube. Before implantation can occur, the blastocyst must hatch from the zona. Several lines of evidence suggest that the zona is multilaminar. We hypothesized that the multilaminar structure of the zona filaments could be imaged non-destructively with the polarized light microscope. A recent modification of the polarized light microscope (pol-scope), which combines innovations in polarization optics with novel image processing software, allows measurement of birefringence at all points of the image. Hamster metaphase II oocytes were placed on glass coverslips which replaced the bottom of culture dishes, imaged under differential interference contrast (DIC) and pol-scope optics, then digitized and processed to measure birefringence magnitude and orientation. The pol-scope revealed the zona to be divided into outer and inner layers separated by a zone of low retardance. This finding is consistent with filaments in the outer layer oriented tangentially and in the inner layer oriented radially. The multilaminar structure of the mammalian zona suggested by differential lectin binding and by scanning electron microscopy could be imaged non-destructively with the pol-scope. Because the pol-scope provides a non-destructive method to identify macro-molecular organization of the zona, it may prove useful in developmental studies of hatching and to direct resection of the zona.