Various medical conditions and treatments in young adults, adolescents, and children can impact future fertility, including increasing risk of premature gonadal failure. Preservation of fertility can decrease the overall risk of fertility in these patients, but its use remains low and is correlated with social determinants of health. This article is a summary of the components of comprehensive fertility preservation care as well as clinical recommendations for currently available methods of preservation. Components of comprehensive fertility care include timely initiation of counseling, rapid referrals, completion of treatment, post-treatment counseling, documentation, and assistance with financial navigation. Patient counseling should include all available options and all known and unknown risks to fertility associated with their medical condition(s) and treatment(s). Concrete documentation of consent, disposition of preserved tissue upon death, and storage arrangements should be kept by each party. Fertility preservation methods for female patients include mature oocyte cryopreservation and embryo cryopreservation, each with variations such as genetic testing, ovarian stimulation, or various starting protocols depending on the urgency of treatment. In addition, ovarian cryopreservation is now considered a safe and effective method, particularly for patients who cannot wait to begin treatment to undergo oocyte or embryo cryopreservation or ovarian stimulation. Safety considerations in female patients include medical complexity impacting the effectiveness and response to treatments, prevention of ovarian hyperstimulation syndrome (OHSS), consideration of ovarian suppression with GnRH analogs during chemotherapy treatment, and consideration of ovarian transposition in patients needing local pelvic radiation. In cases of gynecologic cancer, fertility-sparing surgical treatments should be considered. Emerging evidence has shown potential for the efficacy of newer treatments, including in vitro maturation, uterine fixation and transposition, and the application of existing strategies in adolescent patients and occasionally children. Methods of fertility preservation for male patients include ejaculated sperm cryopreservation, medically-assisted ejaculated semen collection, surgical sperm retrieval, and electroejaculation. Each method addresses specific fertility barriers, but all available options should be presented during patient counseling. Similar to female patients, clinics should have concrete policies and documentation surrounding the disposition of cryopreserved tissue after a patient’s death. All patients who are at risk of reproductive impairment from medical conditions or treatments should be made aware of all available options for fertility preservation and be provided with access to a multidisciplinary team to counsel with regarding preservation decisions. Each method of preservation has benefits and disadvantages, and each should be considered in the context of the situation of each individual patient. In minors, careful counseling should be undertaken with both the patients and their parents or guardians to achieve the best possible outcomes with informed consent from all involved. (Summarized from Practice Committee of the American Society for Reproductive Medicine. Fertility preservation in patients with medical indications: a committee opinion. Fertil Steril . 2026;125:247–259. doi: 10.1016/j.fertnstert.2025.12.001)

Successful cell division during meiosis is dependent on accurate chromosome segregation; the role of centromeres, kinetochores, and spindle microtubules is well characterized in mitosis, but the role of certain proteins surrounding them remains uncertain in meiosis. This is important to understand because the arrest of maturation of oocytes is a common origin of female infertility, but the mechanisms for this are largely unknown. This study was designed to assess the regulatory mechanism of the farnesylation of centromere protein F on its meiotic function as well as evaluate any association between mutations in centromere protein F and female oocyte maturation disorders. This study used oocyte microinjection, western blotting, co-immunoprecipitation, and immunofluorescence to characterize the role of centromere protein F. During mitosis, this protein assists in chromosome segregation, but it is currently unknown if its role remains the same during meiosis. This study used a mouse model to explore both genetic and pharmacologic methods of farnesylation, and to verify the mechanism of mutations in oocyte maturation. Microinjection of centromere protein F siRNA reduced maturation rates significantly, with many halting maturation at metaphase I ( P <0.05). Farnesylation likewise reduced the rate of oocyte maturation ( P <0.01), and additionally weakened the interationc between centromere protein F and Aurora kinase B, which was confirmed by co-immunoprecipitation ( P <0.01). Farnesylation also disrupted kinetochore localization, contributing to the arrest of oocyte maturation. Immunofluorescence analysis further showed that centromere protein F localized at kinetochores by metaphase I, causing arrested development. Screening human patients with infertility resulted in the identification of 4 individuals with rare heterozygous variants in the CENP-F gene that were associated with the arrest of oocyte maturation. When testing centromere protein F derived from patients who were identified to have a genetic mutation in this protein, microinjection of the patient-derived centromere protein F also caused significantly reduced maturation in mouse oocytes ( P <0.01). Two identified mutations reduced the fanesylation of centromere protein F as well as disrupted kinetochore localization and damaged the Aurora kinase B interaction. These results indicate that there is a direct association between centromere protein F mutations and infertility, specifically the arrest of oocyte maturation during meiosis. These findings contribute evidence to the controversy surrounding the influence of farnesylation on the localization of CENP- F, with some previous studies showing no effect of farneslyation and others showing a direct interaction between molecules that is dependent on farneslyation. This study emphasizes the need for further clinical research to validate the pathology of these mutations in diverse populations. Future studies should also attempt to ethically validate the results of these mouse models in human models and examine other possible mechanisms for arrested maturation of oocytes such as disruption of microtubule binding. (Summarized from Zhong O, Wang C, Zhang J, et al. Farnesylation-dependent kinetochore targeting of centromere protein F is essential for oocyte meiotic progression and female fertility. Am J Obstet Gynecol. 2026;234:116-140. doi: 10.1016/j.ajog.2025.08.031)

In women of reproductive age, the most common form of cancer is breast cancer; due to its being the most common, the impact of the malignancy and its treatment on fertility has been well-characterized. Individual risks to fertility are difficult to quantify; however, based on available evidence, clinical recommendations conclude that patients should be referred for fertility preservation if interested. Some studies suggest that the majority of pregnancies that occur after breast cancer treatment are spontaneous, but there is limited evidence surrounding fertility treatment and outcomes in this patient demographic. This study was designed to assess long-term outcomes of fertility preservation and treatment among women who had a live birth after being diagnosed with breast cancer. This was a multicenter prospective cohort study of women who were diagnosed with breast cancer before the age of 40 between the years 2006 and 2016, and was titled the Young Women’s Breast Cancer Study (YWS). Individuals who had been diagnosed with breast cancer in stages 0 to III and reported a subsequent live birth were recruited for a survey concerning the mode of conception and the use of fertility treatment. A total of 115 patients completed the survey, with patients excluded due to incomplete responses (1), diagnosis of breast cancer during pregnancy (18), using a surrogate (2), or adopting (1). Final analysis included 94 patients, and the median time to the survey after the first childbirth after breast cancer diagnosis was 5 years. The majority of patients were diagnosed with stage I or II hormone receptor-positive breast cancer; at the time of diagnosis, 51% of patients were nulligravida, and 15% were BRCA 1 or BRCA 2 carriers. The majority of patients also received chemotherapy and endocrine therapy (67% and 65%, respectively). Median time to first childbirth after cancer diagnosis was 4.9 years. A total of 35% of patients who completed the survey used fertility preservation after diagnosis and before treatment for breast cancer. Of these, 33% used frozen oocytes or embryos to conceive (12% of live births in this cohort). Most patients who conceived using fertility treatment were more than 35 years of age at cancer diagnosis, and 29% of patients who conceived spontaneously underwent fertility preservation as well. The most common reasons for fertility treatment were known infertility before cancer diagnosis, wanting to resume cancer treatment quickly, and infertility after cancer treatment. Of patients who underwent in vitro fertilization, 38% used oocytes or embryos obtained during fertility preservation, 45% used oocytes or embryos obtained after cancer treatment, and 17% used donor oocytes or embryos. A total of 69% of patients who used fertility treatment attempted spontaneous conception, and when compared with those who conceived spontaneously, those who used treatment were more likely to be nulligravida when diagnosed with cancer and less likely to be BRCA carriers ( P < 0.0001 and P = 0.029, respectively). This study was limited by a small sample size and a homogenous patient population, as well as the fact that pregnancy loss was not within the scope of this study. Future research should attempt to address these factors as well as assess other factors affecting the utilization of fertility preservation in patients with breast cancer who are of reproductive age. (Summarized from Sorouri K, Zheng U, Sella T, et al. Fertility preservation and fertility treatment use in young breast cancer survivors reporting a live birth. Fertil Steril. 2026; 125:166-168. doi: 10.1016/j.fertnstert.2025.09.031)

Over the past decade, the field of human embryo editing has witnessed remarkable advancements and triggered significant ethical debates. The groundbreaking tool, CRISPR/Cas9, has revolutionized the landscape of genetic engineering by enabling modifications at the genomic level in germ cells. Since the first case of human embryo gene editing in 2015, the field has rapidly progressed, presenting promising avenues for therapeutic interventions. However, it still grapples with safety concerns, including off-target effects, mosaicism, and the long-term impacts of genetic alterations, as well as ongoing ethical controversies. In this review, we will systematically overview the significant research in this field and provide insights into the potential applications of basic research in early embryonic development and the treatment of genetic diseases.

(Abstracted from J Assist Reprod Genet 2025;42:753–762) Oxidative phosphorylation (OXPHOS) occurs within the mitochondria to produce ATP and is driven by mitochondrial DNA (mtDNA). Mutations in mtDNA have been associated with rare genetic disorders as well as cellular dysfunction, with these mutations often attributable to limited capacity for DNA repair and proximity to reactive oxygen species generated during OXPHOS.

Fertility in women decreases with age, but the molecular basis for age-related, unexplained infertility remains elusive. Here, we reveal distinct transcriptome changes in oocytes and surrounding cumulus cells from women in their mid-thirties, as evidenced by notably increased transcription of ribosome genes. Additionally, meiosis genes and actin and cohesin components are downregulated in oocytes with age. Lysosomes and proteostasis are also disrupted in cumulus cells. Moreover, DNA hypomethylation and altered heterochromatin deposition at specific genomic loci are linked to increased transcription of ribosome genes. Rapamycin effectively reduces translation and promotes protein homeostasis in cumulus cells. Remarkably, short-term rapamycin allows patients who fail repeated in vitro fertilization cycles with embryo developmental arrest to achieve high-quality blastocysts that yield successful pregnancy and live birth. These data suggest a causal role for elevated transcription of ribosome genes in aging oocytes and cumulus cells and identify rapamycin as a promising treatment for age-related infertility. This study is registered at Chinese Clinical Trial Registry (ChiCTR2300069828).

Increased chromosomal instability impairs oocyte quality, contributing to female reproductive aging. The telomeric DNA damage response (DDR) is essential for genomic stability; however, how oocytes respond to telomeric damage remains elusive. Here, we observed that aged human germinal vesicle (GV) oocytes accumulated telomeric DNA damage. We next established a telomeric DNA damage model with CRISPR/Cas9 in mouse oocytes, which exhibited increased chromosome instability and impaired meiotic maturation. Furthermore, telomeric DNA damage in oocytes did not initiate telomere fusion but rather accelerated telomere movement and triggered break-induced telomere synthesis (BITS). Mechanistically, RPA32 and RAD51 were recruited to damaged telomeres, and contributed to BITS along with ATR and PARP1. However, telomeric DNA damage recruited few RNF8 in fully grown oocytes, possibly impeding the 53BP1 recruitment. Despite minimal changes in the overall activity of RAD51-promoted DNA repair in GV oocytes with maternal age, this DDR machinery was preferentially involved in non-telomeric regions in aged oocytes. Consequently, upon encountering telomeric DNA damage, aged oocytes might undergo insufficient telomeric DDR and BITS. Together, our study illustrates that telomeric DDR recruits key factors, such as RAD51, to activate BITS, and that insufficient telomeric DDR increases chromosomal instability in aged oocytes.

Induction of meiotic competence is a major goal of the controlled ovarian stimulation used in ART. Do factors intrinsic to the oocyte contribute to oocyte maturation? Deletions in mtDNA accumulate in long-lived post mitotic tissues and are found in human oocytes. If oogenesis cleanses the germline of deleterious deletions in mtDNA, meiotically competent oocytes should contain lower levels of mtDNA deletions vs. meiotically arrested oocytes. We tested this hypothesis using a novel PCR assay for a deletion ratio in human oocytes derived from IVF. A real-time PCR assay was developed to measure total mtDNA copy number (mtDNACN) and mtDNA harboring the 5 Kb “common deletion” to enable calculation of the mtDNA deletion ratio (mtDNADR) in 143 cultured oocytes. Kruskal-Wallis test was carried out to compare the total mtDNACN and the mtDNADR among oocytes which matured to metaphase II (MII) vs. oocytes arrested at GV or metaphase I (MI). 51.75

(Abstracted from J Clin Med 2024;13(15):4283 Approximately 10% to 20% of pregnancies end in miscarriage, or early pregnancy loss (EPL), with most losses occurring in the first trimester. An estimated half of EPLs are due to chromosomal abnormalities, including trisomies, monosomies, and sex chromosome abnormalities, with another 10% due to polyploidy, uniparental disomy, and copy-number variants.

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.

PurposeRetrotransposons play important roles during early development when they are transiently de-repressed during epigenetic reprogramming. Long interspersed element-1 (L1), the only autonomous retrotransposon in humans, comprises 17% of the human genome. We applied the Single Cell Transposon Insertion Profiling by Sequencing (scTIPseq) to characterize and map L1 insertions in human embryos.MethodsSixteen cryopreserved, genetically tested, human blastocysts, were accessed from consenting couples undergoing IVF at NYU Langone Fertility Center. Additionally, four trios (father, mother, and embryos) were also evaluated. scTIPseq was applied to map L1 insertions in all samples, using L1 locations reported in the 1000 Genomes as controls.ResultsTwenty-nine unknown and unique insertions were observed in the sixteen embryos. Most were intergenic; no insertions were located in exons or immediately upstream of genes. The location or number of unknown insertions did not differ between euploid and aneuploid embryos, suggesting they are not merely markers of aneuploidy. Rather, scTIPseq provides novel information about sub-chromosomal structural variation in human embryos. Trio analyses showed a parental origin of all L1 insertions in embryos.ConclusionSeveral studies have measured L1 expression at different stages of development in mice, but this study for the first time reports unknown insertions in human embryos that were inherited from one parent, confirming no de novo L1 insertions occurred in parental germline or during embryogenesis. Since one-third of euploid embryo transfers fail, future studies would be useful for understanding whether these sub-chromosomal genetic variants or de novo L1 insertions affect embryo developmental potential.

Background: Products of conception samples are often collected and analyzed to try to determine the cause of an early pregnancy loss. However, sample collection may not always be possible, and maternal cell contamination and culture failure can affect the analysis. Cell-free DNA-based analysis of a blood sample could be used as an alternative method in early pregnancy loss cases to detect if aneuploidies were present in the fetus. Methods: In this prospective study, blood samples from early pregnancy loss patients were analyzed for the presence of fetal aneuploidies using a modified version of a noninvasive prenatal testing assay for cell-free DNA analysis. Results from cell-free DNA analysis were compared against the gold standard, microarray analysis of products of conception samples. This study was registered with ClinicalTrials.gov, identifier: NCT04935138. Results: Of the 76 patient samples included in the final study cohort, 11 were excluded from performance calculations. The 65 patient samples included in the final analysis included 49 with an abnormal microarray result and 16 with a normal microarray result. Based on results from these 65 samples, the study found that genome-wide cell-free DNA analysis had a sensitivity of 73.5% with a specificity of 100% for the detection of fetal aneuploidies in early pregnancy loss cases. Conclusions: This prospective study provides further support for the utility of cell-free DNA analysis in detecting fetal aneuploidies in early pregnancy loss cases. This approach could allow for a noninvasive method of investigating the etiology of miscarriages to be made available clinically.

Scientific enquiry is typically accompanied by a kaleidoscope of differing opinions, with champions of competing hypotheses seeking the final data that will prove the validity of their preferred ideas and the folly of those holding conflicting notions. Debate is not only healthy, but essential for the vitality of science and for the rigor with which new concepts are evaluated. In no field is this truer than in reproductive science. Consider the many differences of opinion with respect to scientific theory and clinical practice that sometimes divide us from our dear colleagues. So numerous are they in our discipline, that the journal in which we are writing today has little difficulty dedicating a section to a different "Fertile Battle" every other month. In this context, perhaps it seems strange that the scientific community has found an unusual degree of unanimity with respect to the question of germline genome editing. This was evidenced by the near universal dismay, disapproval, and disbelief that met the 2018 announcement of twin girls born in China after the treatment of in vitro fertilization (IVF) embryos using the genome editing technology known as CRISPR. If the scientist who led the work initially failed to appreciate the distinction between fame and infamy, he probably fully comprehends it today. The news of the "CRISPR births" stunned the scientific community and sent out societal shockwaves that continue to reverberate today. Presenting a heretical viewpoint can be an entertaining philosophical exercise for scientists, but with a subject as important and controversial as germline genome editing it is important to be clear that we, the investigators of this article, do not dissent from majority view. We fully agree that the clinical application of genome editing technologies to IVF embryos is premature given that significant questions concerning safety and efficacy remain to be answered and ethical considerations have not been fully resolved. The statement above should bring this debate to a rather early conclusion, leaving us an embarrassing 1000 words short of fulfilling our obligation to the journal. Luckily, however, there is more to be said. Adding IVF to genetics unites 2 topics that have long been favored themes in certain sections of the media. Think of "three-parent-embryos," "designer babies," "savior siblings," and "slippery slopes." It would be flippant to dismiss the genuine potential for technologies to be abused, and the legitimate ethical concerns that some may have. Nonetheless, it is not difficult to present a positive view of the possibilities that genome editing methods may offer us in the future. We will attempt to outline just a few of these below. It is sometimes argued that genome editing is "a step too far," a radical intervention, which is unnecessary given that there are well-established alternatives for avoiding inherited disease transmission. It is true that prenatal diagnostics, using techniques such as amniocentesis and chorionic villus sampling to collect fetal DNA, are available for most common inherited conditions. Indeed, prenatal diagnosis has been used for some monogenic conditions since the late 1980s. However, the major limitation of this approach is that prevention of disease is only achieved by terminating the pregnancies that prenatal testing indicates to be affected. This strategy carries its own ethical questions and is also problematic from the perspective of some religious teachings. It is therefore unsurprising that prenatal diagnosis is unacceptable to some patients. Over 30 years ago, preimplantation genetic testing (PGT) was introduced as an alternative to prenatal testing, with the advantage that the vast majority of pregnancy terminations are avoided. This is achieved by sampling one or more cells from cultured preimplantation embryos, produced using IVF technology. The genetic material of the cell(s) is tested to establish the disease status of the embryo and only those found to be unaffected are considered for transfer to the uterus. Thus, any pregnancy established should be free of the familial disorder. Although many patients consider diagnosis at the preimplantation stage to be preferable to prenatal diagnosis, the reality is that PGT cannot help all patients. If the embryo is viewed as having an equal status to the fetus, which could be considered the case if following strict Catholic doctrine, then the destruction of several viable (but affected) embryos in a PGT cycle might not be deemed an improvement on the termination of a single affected pregnancy after prenatal testing. For this reason, some patients are unable to accept PGT. Another important limitation of PGT is that as many as 10% of cycles do not have any embryos that are both unaffected and suitable for transfer (1Gutiérrez-Mateo C. Sánchez-García J.F. Fischer J. Tormasi S. Cohen J. Munné S. et al.Preimplantation genetic diagnosis of single-gene disorders: experience with more than 200 cycles conducted by a reference laboratory in the United States.Fertil Steril. 2009; 92: 1544-1556Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar). Of course, it is not uncommon for an IVF cycle to yield only 1 or 2 competent embryos, and if PGT shows those embryos to be affected, then the cycle cannot produce an unaffected child. Thus, a patient may endure the substantial costs (financial, physical, and emotional) of an IVF cycle with PGT, yet still be denied the outcome they desire. Genome editing has been successfully applied to human embryos in a research context, allowing the precise targeting and disruption of individual genes (2Fogarty N.M.E. McCarthy A. Snijders K.E. Powell B.E. Kubikova N. Blakeley P. et al.Genome editing reveals a role for OCT4 in human embryogenesis.Nature. 2017; 550: 67-73Crossref PubMed Scopus (258) Google Scholar). Unquestionably, this sort of approach provides us with powerful tools for increasing our understanding of early development. However, in a debate such as this, the inevitable focus is on the question of whether it is desirable to use genome editing technology clinically, removing mutations responsible for serious inherited disorders. It could be argued that the successful application of genome editing would "rescue" affected conceptions. Instead of being discarded at the preimplantation stage after PGT, or terminated at fetal stages after prenatal testing, their inherited mutation would be removed, the DNA restored to the same "wild-type" sequence carried by the vast majority of the global human population. From a PGT perspective, this would result in all embryos becoming eligible for transfer, and no human conception would have to be discarded. This would seem to circumvent some of the ethical quandaries associated with PGT and prenatal testing. Most of us can probably agree that it is preferable to avoid being affected by a genetic disorder, at least one that significantly impacts the length or quality of life, but why complicate things by adding IVF into the equation? Why not simply treat affected individuals after they are born? Unfortunately, many the >10,000 monogenic disorders caused by inherited mutations disrupt developmental processes and would already have had an irreversible impact within days or weeks of conception. In such cases, removal of mutations would need to be undertaken at a very early stage. An additional consideration is that heritable disorders often exert their effects on the phenotype by altering the function of millions of cells, contributing to multiple tissues and organs. This leaves scientists with the problem of how to deliver the components required for genome editing into huge numbers of cells located at numerous sites, deep inside the body. Somatic gene therapy is limited: often too late, with too many cells to correct or reach in vivo, and consequently with limited capacity to prevent the broad spectrum of inherited diseases that afflict children and adults. Theoretically, the application of genome editing to IVF embryos has several important advantages. Removal of a mutation during the preimplantation phase is likely to be sufficiently early in development that the disease phenotype is entirely avoided. Indeed, if treatment was undertaken before the 4-cell stage (when the major wave of embryonic gene expression begins), it is likely that the affected gene would not be transcribed until after the wild-type sequence had been restored. Genome editing at the preimplantation stage also has the advantage that the number of cells requiring targeting is very small. For example, at the zygote stage, delivery of genome editing reagents can be virtually guaranteed by microinjection into the single cell. For later preimplantation stages, microinjection may become impractical, but until around day 3, all blastomeres have part of their surface in contact with the external environment. This gives a high likelihood that genome editing components could be successfully introduced using various methods of transfection. Naturally, if genome editing is successfully accomplished in all cells of a preimplantation embryo, the phenotype associated with the inherited mutation should not occur. At first consideration, this seems the perfect outcome. However, if all cells are edited, this will include those destined to give rise to the germ cells. So, any alterations to the genome could be passed to subsequent generations, potentially remaining in the human gene pool forever. For some, the possibility of a heritable change to the human genome brings a sense of unease, but for others, the idea of permanently removing a mutation that may have brought misery to a family for generations seems less problematic, and maybe even desirable. Interestingly, a positive view of germline genome editing is often held by those who are perhaps best placed to consider its appropriateness, namely those who have experienced the devastation wrought by inherited disease within their own families. In recent years, distinguished organizations have carefully weighed the merits of heritable genome editing and the concerns. The National Academy of Sciences and National Academy of Medicine acknowledged that such an approach has potential to alleviate the suffering caused by inherited diseases, stating "There are circumstances in which genome editing in germline cells or embryos might be the only or most acceptable option for prospective parents who wish to have a genetically related child while minimizing the risk of transmitting a serious disease or disability" (3National Academy of Sciences, National Academy of MedicineHuman genome editing: science, ethics and governance. The National Academies Press, Washington, DC2017Google Scholar). Similarly, in the United Kingdom, the well-respected Nuffield Council on Bioethics agreed that "the use of heritable genome editing to influence the characteristics of future generations could be ethically acceptable in some circumstances, if it is consistent with, the welfare of a person who may be born as a consequence of interventions and it does not increase disadvantage, discrimination, or division in society" (4Nuffield Council on BioethicsGenome editing and human reproduction: social and ethical issues.http://nuffieldbioethics.org/project/genome-editing-human-reproductionDate accessed: August 7, 2023Google Scholar). It is true that some of these caveats are not trivial, but nonetheless, these learned opinions represent a consistent view that there are circumstances in which genome editing (even that which affects the germline) can be considered beneficial and appropriate. There is evidence that the public shares this opinion; a survey conducted by the Royal Society in the United Kingdom reported that 83% of participants were supportive of germline genome editing to treat incurable disease, although many drew the line at editing for "enhancement" (e.g. 60% were opposed to heritable gene editing to improve intelligence) (https://royalsociety.org/topics-policy/projects/genetic-technologies/). If genome editing is ever to be applied to human embryos, safety concerns will have to be addressed. There is mounting evidence that the cells of early preimplantation embryos struggle to process the double-strand DNA breaks induced by CRISPR/Cas9 technology, leading to unresolved damage with potentially serious consequences for the embryo (5Alanis-Lobato G. Zohren J. McCarthy A. Fogarty N.M.E. Kubikova N. Hardman E. et al.Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos.Proc Natl Acad Sci USA. 2021; 118e2004832117Crossref PubMed Scopus (95) Google Scholar, 6Kubikova N. Esbert M. Titus S. Coudereau C. Savash M. Fagan J. et al.Deficiency of DNA double-strand break repair in human preimplantation embryos revealed by CRISPR-Cas9.Hum Reprod. 2023; 38: i46Crossref PubMed Google Scholar). Nonetheless, genome editing technologies continue to evolve and more nuanced methods, such as base editing and base editing, which can be considered "gentler" on the DNA, might assuage some fears over safety. Base editing, which avoids the creation of double-strand DNA breaks, has been applied to human embryos in vitro in a research context with promising results (7Zeng Y. Li J. Li G. Huang S. Yu W. Zhang Y. et al.Correction of the Marfan syndrome pathogenic FBN1 mutation by base editing in human cells and heterozygous embryos.Mol Ther. 2018; 26: 2631-2637Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). Even if genome editing is eventually adjudged to be safe, legitimate concerns may remain over equity of access, and the societal impact of genome editing. However, it is worth noting that from a health economics perspective, the permanent removal of a mutation from a family is likely to be far less costly than a lifetime of medical care (and potentially other forms of support), not just for one individual, but also for all their future descendants. This is food for thought for countries with national healthcare systems. As with many scientific advances, there is a risk that genome editing technologies could be abused—employed to modify non-disease characteristics or for the introduction of "enhancements." Although this possibility can never be entirely ruled out, is it a reason to deny families a method that could prevent the suffering, emotional trauma, and death that accompanies many inherited diseases? Perhaps the introduction of mitochondrial replacement therapies (MRTs) in the United Kingdom could be taken as a model for how genome editing technologies could be introduced in the future. MRTs were developed to prevent the transmission of incurable mitochondrial diseases from mother to child. They involve the transfer of the nuclear DNA from an oocyte or zygote, produced by a woman carrying a mitochondrial DNA mutation, into the cytoplasm of an enucleated donor egg. The defective mitochondria are left behind and the nuclear DNA is placed in a cell populated by functional mitochondria, thus avoiding the cause of the disorder (8Hyslop L.A. Blakeley P. Craven L. Richardson J. Fogarty N.M. Fragouli E. et al.Towards clinical application of pronuclear transfer to prevent mitochondrial DNA disease.Nature. 2016; 534: 383-386Crossref PubMed Scopus (232) Google Scholar, 9Costa-Borges N. Nikitos E. Späth K. Miguel-Escalada I. Ma H. Rink K. et al.First pilot study of maternal spindle transfer for the treatment of repeated in vitro fertilization failures in couples with idiopathic infertility.Fertil Steril. 2023; 119: 964-973Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar). This has some parallels with genome editing, because the mitochondrial DNA of the resulting embryo has a different DNA sequence to that of the original oocyte/zygote and this change can be inherited by future generations through the maternal line. Like genome editing, implementation of MRT raised profound questions of ethics and safety. In the United Kingdom, an extensive consultation process was launched, engaging experts, religious groups, patients, and the public to understand their opinions. New legislation was then debated in the UK Parliament, ultimately culminating in The Human Fertilisation and Embryology (Mitochondrial Donation) Regulations 2015. This created a framework for a rigorous licensing process, with independent oversight, ensuring that clinics meet strict scientific, ethical, and safety standards, that the process of obtaining patient consent is adequate, and guaranteeing a commitment to long-term follow-up studies. It is not inconceivable that a similar chain of events could lead to the clinical application of heritable genome editing. In time, perhaps sooner than many anticipate, genome editing methods will be developed that are safe and efficacious. Before that occurs, efforts must be undertaken to demystify the process. It is of vital importance that scientists do not abdicate the responsibility of educating the public, for if they do, the void may be filled by the writers of tabloid newspaper headlines, or others with agendas where the needs of the patient are not adequately considered. A comprehensive and transparent public consultation should be undertaken, and robust regulation introduced to ensure responsible governance and to earn the confidence of the public. Of course, it must be acknowledged that establishing universally acceptable guidelines may prove difficult as attitudes toward embryo manipulation vary substantially across cultures, nations, governments, and religions. However, this is a worthy challenge because the potential prize is great. If practical regulations can be created, the benefits of genome editing may at last be realized, transforming the fates of afflicted families for generations to come. With thousands of heritable mutations known to cause disease, coupled with steady advancements in assisted reproductive technology (ART) and genetic technologies, embryo selection after ART and preimplantation genetic testing for monogenic disease (PGT-M) has become the predominant option for couples at risk of transmitting genetic disorders to their children. Despite its success, embryo selection comes with several limitations, most notably the finite number of embryos found in an IVF cycle that are both developmentally competent and unaffected by the familial mutation, of which there are often none. Germline gene editing has been proposed as a strategy for targeted correction of mutations in the germline that could potentially replace embryo selection. The development of this novel technique has the potential to rescue the number of transferable embryos, thus possibly leading to better outcomes. So far, the great majority of such attempts have utilized CRISPR-Cas9 technology (shortened to CRISPR). Both technical and ethical considerations, however, render germline gene editing unacceptable for clinical application at the current time. CRISPR induces double-stranded DNA breaks (DSBs) that can be repaired by 1 of 2 mechanisms—an error-prone pathway, called non-homologous end joining (NHEJ), which is the most common response. Less frequently, homology directed repair (HDR) can be employed to correct the DSB, which uses the embryo's own repair machinery. The earliest stage of development, particularly the fertilization stage, is the most optimal for genome editing if it facilitates delivery of edits into all cells of the future organism. However, embryos also appear to be most vulnerable to DNA damage at this stage, presumably because of deficiency in DNA repair present before the activation of the embryonic genome. This observation alone makes a compelling argument against the clinical use of CRISPR-Cas9. However, when coupled with other technical and biological factors impeding the success of the technique in its current format, as well as the complicated ethics, one must wonder whether it would be safer to avoid gene editing in human embryos altogether. In fact, as far as we can tell, most of the evidence suggests that gene editing interventions in the germline have the potential to negatively impact embryonic development, and we think it should, therefore, be avoided in clinical form. Thus far, the utilization of CRISPR in the human germline has taken a mostly pre-clinical focus, aimed at investigating whether disease-causing mutations could be disrupted or repaired. Several studies now have established a proof-of-principle as well as technical and methodological framework for the application of gene editing technique in human embryos donated or created for research (2Fogarty N.M.E. McCarthy A. Snijders K.E. Powell B.E. Kubikova N. Blakeley P. et al.Genome editing reveals a role for OCT4 in human embryogenesis.Nature. 2017; 550: 67-73Crossref PubMed Scopus (258) Google Scholar, 10Ma H. Marti-Gutierrez N. Park S.W. Wu J. Lee Y. Suzuki K. et al.Correction of a pathogenic gene mutation in human embryos.Nature. 2017; 548: 413-419Crossref PubMed Scopus (1) Google Scholar, 11Kang X. He W. Huang Y. Yu Q. Chen Y. Gaoet X. et al.Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.J Assist Reprod Genet. 2016; 33: 581-588Crossref PubMed Scopus (219) Google Scholar, 12Liang D. Mikhalchenko A. Ma H. Marti Gutierrez N. Chen T. Lee Y. et al.Limitations of gene editing assessments in human preimplantation embryos.Nat Commun. 2023; 14: 1219Crossref PubMed Scopus (3) Google Scholar, 13Liang P. Xu Y. Zhang X. Ding C. Huang R. Zhang Z. et al.CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes.Protein Cell. 2015; 6: 363-372Crossref PubMed Scopus (827) Google Scholar, 14Tang L. Zeng Y. Du H. Gong M. Peng J. Zhang B. et al.CRISPR/Cas9-mediated gene editing in human zygotes using Cas9 protein.Mol Genet Genomics. 2017; 292: 525-533Crossref PubMed Scopus (158) Google Scholar). Initial reports from China showed low efficiency, as well as significant off-target consequences and mosaicism 456 with CRISPR. Later studies using refined methods showed improved editing frequency (>90%) in early human embryos (2Fogarty N.M.E. McCarthy A. Snijders K.E. Powell B.E. Kubikova N. Blakeley P. et al.Genome editing reveals a role for OCT4 in human embryogenesis.Nature. 2017; 550: 67-73Crossref PubMed Scopus (258) Google Scholar, 10Ma H. Marti-Gutierrez N. Park S.W. Wu J. Lee Y. Suzuki K. et al.Correction of a pathogenic gene mutation in human embryos.Nature. 2017; 548: 413-419Crossref PubMed Scopus (1) Google Scholar, 11Kang X. He W. Huang Y. Yu Q. Chen Y. Gaoet X. et al.Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.J Assist Reprod Genet. 2016; 33: 581-588Crossref PubMed Scopus (219) Google Scholar). It appears that editing efficacy increases when microinjection of the CRISPR components is performed at fertilization or early in development. Optimization of the half-life of CRISPR reagents can reduce mosaicism (10Ma H. Marti-Gutierrez N. Park S.W. Wu J. Lee Y. Suzuki K. et al.Correction of a pathogenic gene mutation in human embryos.Nature. 2017; 548: 413-419Crossref PubMed Scopus (1) Google Scholar). After generation of DSBs, HDR uses either the uncut homologous wild-type DNA sequence or alternatively a synthetic homologous template harboring the correct sequence for restoration of the damaged copy. When changing the timing of microinjection and HDR template availability, it also appears that human embryos prefer to use the endogenous template over the synthetic one, by a mechanism that is largely not understood. One could imagine this to be a protective strategy, inherent to embryonic cells, to prevent genotoxic damage. The downside for the prospect of clinical application of CRISPR is the fact that most embryos still resolve generated DSBs by mutagenic NHEJ rather than the HDR, which is required for correction of most mutations associated with human disease. NHEJ introduces additional mutations (small insertions and deletions known as indels) rather than correcting the existing ones. This predominant form of repair is, therefore, suboptimal for gene editing, except when the intent is to disrupt gene function entirely. Because HDR occurs infrequently (<10%) in cells of early human embryos, the success of editing is low. This is especially problematic for humans, because IVF provides only a limited number of embryos. The second concern precluding the clinical application of CRISPR in the human germline is the possibility that the technique causes DNA damage that is not readily repaired in early human embryos, which are already predisposed to genomic instability. Several papers report large deletions and structural abnormalities affecting chromosome segments after CRISPR (5Alanis-Lobato G. Zohren J. McCarthy A. Fogarty N.M.E. Kubikova N. Hardman E. et al.Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos.Proc Natl Acad Sci USA. 2021; 118e2004832117Crossref PubMed Scopus (95) Google Scholar, 12Liang D. Mikhalchenko A. Ma H. Marti Gutierrez N. Chen T. Lee Y. et al.Limitations of gene editing assessments in human preimplantation embryos.Nat Commun. 2023; 14: 1219Crossref PubMed Scopus (3) Google Scholar, 15Kosicki M. Tomberg K. Bradley A. Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements.Nat Biotechnol. 2018; 36: 765-771Crossref PubMed Scopus (53) Google Scholar, 16Adikusuma F. Piltz S. Corbett M.A. Turvey M. McColl S.R. Helbig K.J. et al.Large deletions induced by Cas9 cleavage.Nature. 2018; 560: E8-E9Crossref PubMed Scopus (199) Google Scholar, 17Cullot G. Boutin J. Toutain J. Prat F. Pennamen P. Rooryck C. et al.CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.Nat Commun. 2019; 10: 1136Crossref PubMed Scopus (219) Google Scholar). Segmental abnormalities are known to be detrimental to embryonic viability and can produce congenital abnormalities in offspring. In 2020, Alanis-Lobato et al. (5Alanis-Lobato G. Zohren J. McCarthy A. Fogarty N.M.E. Kubikova N. Hardman E. et al.Frequent loss of heterozygosity in CRISPR-Cas9-edited early human embryos.Proc Natl Acad Sci USA. 2021; 118e2004832117Crossref PubMed Scopus (95) Google Scholar) observed loss-of-heterozygosity in the edited cells that spanned regions beyond the on-target locus (4 kb to at least 20 kb outside the POU5F1 gene), as well as segmental gains and losses of chromosome 6 (the site of the POU5F1 gene) present in approximately 22% of the samples, significantly higher than observed in the control group. Recently, Liang et al. (12Liang D. Mikhalchenko A. Ma H. Marti Gutierrez N. Chen T. Lee Y. et al.Limitations of gene editing assessments in human preimplantation embryos.Nat Commun. 2023; 14: 1219Crossref PubMed Scopus (3) Google Scholar) reported that embryonic cells are often subject to loss of heterozygosity extending out of the target site, which would indicate the absence of repair in the broken strand. The study showed that, besides technical challenges of gene editing, assessments resulting from the minute amount of DNA processed, allelic dropouts, and amplification biases, DSBs can produce large deletions at the target site extending outside of the targeted MYBPC3 locus. Furthermore, some embryonic stem (ES) cells derived from the targeted embryos show copy-neutral loss of heterozygosity at the cleavage site, likely caused by interallelic gene conversion (12Liang D. Mikhalchenko A. Ma H. Marti Gutierrez N. Chen T. Lee Y. et al.Limitations of gene editing assessments in human preimplantation embryos.Nat Commun. 2023; 14: 1219Crossref PubMed Scopus (3) Google Scholar). This study, along with work of others, indicate that human embryos have a DNA damage repair deficiency, presumably because of the lack of gene expression before the activation of the embryonic genome at around the cleavage stage. Continued mitotic division, despite the presence of DSBs, suggests cellular mechanisms that usually preserve genetic integrity lack stringency, ultimately failing to ensure repair. The results provide a strong warning against the therapeutic use of CRISPR-Cas9 in human embryos, and underline the importance of basic research to evaluate the safety of genome editing techniques in the human germline. If gene editing was to replace embryo selection, it is essential that the intervention specifically target the locus where the mutation is found. However, when evaluating data from CRISPR screens and validations, it is apparent that most CRISPR constructs have a degree of off-target activity, especially at DNA sequences that share close homology to the targeted locus. Algorithms predicting off-target activity are becoming more accurate with time, but they mostly lack data from actual human embryonic cells, because of scarcity of the material, with ES cells commonly used as a proxy. However, it appears that ES cells exhibit significantly lower targeting efficiencies and they lack the diversity of mutational profiles resulting from NHEJ, which make them unsuitable for predicting off-targets effects (2Fogarty N.M.E. McCarthy A. Snijders K.E. Powell B.E. Kubikova N. Blakeley P. et al.Genome editing reveals a role for OCT4 in human embryogenesis.Nature. 2017; 550: 67-73Crossref PubMed Scopus (258) Google Scholar, 12Liang D. Mikhalchenko A. Ma H. Marti Gutierrez N. Chen T. Lee Y. et al.Limitations of gene editing assessments in human preimplantation embryos.Nat Commun. 2023; 14: 1219Crossref PubMed Scopus (3) Google Scholar). Also worth noting is the fact that these tools depend on in silico predictions. Confirmatory tests to exclude the possibility of transferring an embryo harboring additional mutations would be required for clinical application of CRISPR. Such a test would necessitate whole-genome sequencing of the embryo to examine all putative off-target consequences, a

Importance: Genetic testing of gamete donors is becoming increasingly comprehensive and now often includes expanded carrier screening. Some argue that testing has gone too far, whereas others propose that testing is not extensive enough. Thinking critically about how much genetic testing is appropriate for gamete donors is crucial for ensuring that market forces alone do not determine the level of testing that is performed.Objective: The goal of this paper is to highlight contradictions in the current approach toward genetic testing of gamete donors and to suggest that we either embrace the value of preventing the birth of children with hereditary diseases and do so in a logical and consistent manner or consider reducing our level of genetic testing for gamete donors. Evidence Review: The Food and Drug Administration requires screening for infectious diseases and the American Society for Reproductive Medicine recommends screening for a small number of common recessive conditions. However, private donor banks are increasingly performing karyotype testing and expanded carrier screening.Findings: There are 2 major inconsistencies in our current approach to genetic testing of gamete donors: (1) if genetic information is valued by gamete recipients, why should testing stop with recessive conditions, and not expand to dominant conditions or even polygenic risk scoring? (2) Why should gamete donors be asked to undergo testing that may or may not be reciprocated by gamete recipients? Addressing these inconsistencies requires us to consider the ultimate goal of testing gamete donors' genes. We argue that the present, default goal is empowerment of gamete recipients, whereas an alternative and more laudable mission is to avoid preventable, heritable disease in offspring. However, the latter brings its own ethical and practical challenges, including the issue of which diseases are worth preventing.Conclusion and Relevance: A more comprehensive and well-reasoned approach to genetic testing of gamete donors is needed. Otherwise, testing will continue to be haphazard and guided by the free market, rather than deeper societal values. (Fertil Sterile 2023;120:1042-7.(c) 2023 by American Society for Reproductive Medicine.) El resumen esta disponible en Espanol al final del articulo.

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?

Telomeres influence cancer, aging and development [1]. The gene encoding telomerase reverse transcriptase, which elongates telomeres, contains an estrogen response element, and estradiol (E2) elongates telomeres in postmenopausal women taking estrogen [2]. We also found increased leukocyte telomere length (LTL) in a woman with telomeropathy undergoing fertility preservation [3]. Telomere elongation increases cancer risk, and thus has implications for women with cancer. Here we report effects of ovarian hyperstimulation (OS) and programmed frozen embryo transfer (FET) on LTL. 93 consented subjects (NYU IRB #S16-00154) provided blood at baseline and peak E2 - 60 were doing OS for IVF or oocyte cryopreservation and 33 were doing FET. Leukocyte DNA was extracted, quantified, and real-time PCR measured LTL. LTL was compared between baseline and peak E2 samples, so each subject served as her own control. Paired data was statistically analyzed with GraphPad Prism 9. As expected, peak E2 in OS exceeded that in FET cycles (3,054±1,470 vs 482.3±663.5, P<0.0001, unpaired t test). Maternal age, baseline E2, and baseline LTLs did not differ between OS and FET. OS markedly reduced LTL (Paired t test, -0.032±0.078, P=0.002). Intriguingly, maternal age blunted the LTL reduction (r=-0.299, P=0.021) during OS. Body mass index (BMI) (r=-0.22, P=0.09) or days of OS (r=-0.086, P=0.515) had no effect. By contrast, FET produced a lesser, but significant increase in LTL (Paired t test, 0.025±0.067, P=0.042). Maternal age did not affect this response. This study, for the first time, reports LTL shortening in women undergoing OS. Since telomere elongation increases cancer risk, this finding provides reassurance to women undergoing fertility preservation. The subtle, but significant stimulatory effect of FET on LTL requires further study. The differing responses of LTL to OS reported here and that reported here [3] may be related the fact that telomeropathy lowers response to ovarian stimulation [3], so the LTL elongation in telomeropathy patients may parallel that observed following mild E2 elevation, e.g. during FET. E2 seems to have a biphasic effect on LTL, such that high E2, e.g. from robust OS, reduces LTL, while modest E2, e.g. from FET, elongates LTL. Future studies should elucidate the mechanisms underlying the relationship between LTL and reproductive interventions.

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.

Telomere length is related to longevity and cancer, and resets during early embryo development. A recent study reports shortened telomeres, equivalent to 10.8-13.4 years of aging, in children born following blastocyst transfer, compared to children born after cleavage-stage embryo transfer or spontaneous conception. Mice conceived following blastocyst culture also have decreased telomere length and telomerase activity compared to cleavage stage culture [1]. How culture to blastocyst shortens telomeres remains unclear. Expression of the gene encoding TERT, the regulatory subunit of telomerase, is regulated by oxygen (O2) tension [2], so we hypothesized that it is the decreased O2 employed during blastocyst culture that inhibits telomerase and shortens telomeres in preimplantation embryos. Mouse zygotes (B6C3F1 X B6D2F1) (Embryotech Laboratories) were thawed and cultured in KSOM Medium (Sigma) for 90 hours in 7% CO2 and 370C in incubators supplied with 5% or 20% O2. DNA and mRNA were simultaneously isolated from individual blastocysts after removal of zona pellucidae (ZP). Telomere length and gene expression were measured by qPCR and RT-qPCR, respectively. Telomerase activity of individual blastocysts was measured by Telomere Repeat Amplification Protocol (TRAP), and telomerase proteins were labeled and imaged by anti-TERT fluorescent immunostaining. Data (Mean±Std) were analyzed by unpaired t test with GraphPad Prism 9. Blastocyst rate under 5% O2 did not differ significantly to that under 20% O2 (94.8% vs. 91.2%, P=0.41, Fisher's exact test). Nor did O2 tension affect pluripotent gene Oct4 and Sox2 mRNA levels (0.96±0.24 vs. 0.99±0.29 and 0.72±0.32 vs. 0.81±0.39; P=0.66 and P=0.38; respectively). Surprisingly, blastocysts developed in 5% O2 had significantly shorter telomeres (0.78±0.18 vs. 0.89±0.12, P=0.02), decreased Tert mRNA level (1.40±1.05 vs. 2.47±1.28, P=0.003), decreased fluorescence intensity of anti-TERT staining (678.3±115.5 vs. 755.9±174.9, P<0.0001), and lower telomerase activity (0.12±0.05 vs. 0.15±0.06, P=0.04) compared to blastocysts developed in 20% O2. Our study demonstrates for the first time that decreased O2 concentration, in the range used for preimplantation embryo culture, decreases telomerase activity and shortens telomeres in blastocysts grown from zygote stage, despite similar blastocyst formation rates and pluripotent gene expression. The mechanisms underlying inhibition of telomerase and telomeres by low O2 tension in preimplantation embryos require further investigations.

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.

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.