Three-dimensional genome architecture persists in a 52,000-year-old woolly mammoth skin sample.

Analyses of ancient DNA typically involve sequencing the surviving short oligonucleotides and aligning to genome assemblies from related, modern species. Here, we report that skin from a female woolly mammoth (†Mammuthus primigenius) that died 52,000 years ago retained its ancient genome architecture. We use PaleoHi-C to map chromatin contacts and assemble its genome, yielding 28 chromosome-length scaffolds. Chromosome territories, compartments, loops, Barr bodies, and inactive X chromosome (Xi) superdomains persist. The active and inactive genome compartments in mammoth skin more closely resemble Asian elephant skin than other elephant tissues. Our analyses uncover new biology. Differences in compartmentalization reveal genes whose transcription was potentially altered in mammoths vs. elephants. Mammoth Xi has a tetradic architecture, not bipartite like human and mouse. We hypothesize that, shortly after this mammoth's death, the sample spontaneously freeze-dried in the Siberian cold, leading to a glass transition that preserved subfossils of ancient chromosomes at nanometer scale.

Unified understanding of the impact of semiflexibility, concentration, and molecular weight on macromolecular-scale ring diffusion.

Conformationally fluctuating, globally compact macromolecules such as polymeric rings, single-chain nanoparticles, microgels, and many-arm stars display complex dynamic behaviors due to their rich topological structure and intermolecular organization. Synthetic rings are hybrid objects with conformations that display both ideal random walk and compact globular features, which can serve as models of genomic DNA. To date, emphasis has been placed on the effect of ring molecular weight on their unusual behaviors. Here, we combine simulations and a microscopic force-level theory to build a unified understanding for how key aspects of ring dynamics depend on different tunable molecular properties including backbone rigidity, monomer concentration, degree of traditional entanglement, and molecular weight. Our large-scale molecular dynamics simulations of ring melts with very different backbone stiffnesses reveal unanticipated behaviors which agree well with our generalized theory. This includes a universal master curve for center-of-mass diffusion constants as a function of molecular weight scaled by a chemistry and thermodynamic state-dependent critical molecular weight that generalizes the concept of an entanglement cross-over for linear chains. The key physics is how backbone rigidity and monomer concentration induced changes of the entanglement length, interring packing, degree of interpenetration, and liquid compressibility slow down space-time dynamic-force correlations on macromolecular scales. A power law decay of the center-of-mass diffusion constant with inverse molecular weight squared is the first consequence, followed by an ultraslow activated hopping transport regime. Our results set the stage to address slow dynamics and kinetic arrest in different families of compact synthetic and biological polymeric systems.

The adverse effects of vitrification on mouse embryo development and metabolic phenotype in offspring.

Embryo vitrification is a standard procedure in assisted reproductive technology. Previous studies have shown that frozen embryo transfer is associated with an elevated risk of adverse maternal and neonatal outcomes. This study aimed to explore the effects of mouse blastocyst vitrification on the phenotype of vitrified-warmed blastocysts, their intrauterine and postnatal development, and the long-term metabolic health of the derived offspring. The vitrified-warmed blastocysts (IVF + VT group) exhibited reduced mitochondrial activity, increased apoptotic levels, and decreased cell numbers when compared to the fresh blastocysts (IVF group). Implantation rates, live pup rates, and crown-rump length at E18.5 were not different between the two groups. However, there was a significant decrease in fetal weight and fetal/placental weight ratio in the IVF + VT group. Furthermore, the offspring of the IVF + VT group at an age of 36 weeks had reduced whole energy consumption, impaired glucose and lipid metabolism when compared with the IVF group. Notably, RNA-seq results unveiled disturbed hepatic gene expression in the offspring from vitrified-warmed blastocysts. This study revealed the short-term negative impacts of vitrification on embryo and fetal development and the long-term influence on glucose and lipid metabolism that persist from the prenatal stage into adulthood in mice.

Tardigrades Use Intrinsically Disordered Proteins to Survive Desiccation.

Tardigrades are microscopic animals that survive a remarkable array of stresses, including desiccation. How tardigrades survive desiccation has remained a mystery for more than 250 years. Trehalose, a disaccharide essential for several organisms to survive drying, is detected at low levels or not at all in some tardigrade species, indicating that tardigrades possess potentially novel mechanisms for surviving desiccation. Here we show that tardigrade-specific intrinsically disordered proteins (TDPs) are essential for desiccation tolerance. TDP genes are constitutively expressed at high levels or induced during desiccation in multiple tardigrade species. TDPs are required for tardigrade desiccation tolerance, and these genes are sufficient to increase desiccation tolerance when expressed in heterologous systems. TDPs form non-crystalline amorphous solids (vitrify) upon desiccation, and this vitrified state mirrors their protective capabilities. Our study identifies TDPs as functional mediators of tardigrade desiccation tolerance, expanding our knowledge of the roles and diversity of disordered proteins involved in stress tolerance.

Cryopreservation of human kidney organoids.

Recent advances in stem cell research have led to the creation of organoids, miniature replicas of human organs, offering innovative avenues for studying diseases. Kidney organoids, with their ability to replicate complex renal structures, provide a novel platform for investigating kidney diseases and assessing drug efficacy, albeit hindered by labor-intensive generation and batch variations, highlighting the need for tailored cryopreservation methods to enable widespread utilization. Here, we evaluated cryopreservation strategies for kidney organoids by contrasting slow-freezing and vitrification methods. 118 kidney organoids were categorized into five conditions. Control organoids followed standard culture, while two slow-freezing groups used 10% DMSO (SF1) or commercial freezing media (SF2). Vitrification involved V1 (20% DMSO, 20% Ethylene Glycol with sucrose) and V2 (15% DMSO, 15% Ethylene Glycol). Assessment of viability, functionality, and structural integrity post-thawing revealed notable differences. Vitrification, particularly V1, exhibited superior viability (91% for V1, 26% for V2, 79% for SF1, and 83% for SF2 compared to 99.4% in controls). 3D imaging highlighted distinct nephron segments among groups, emphasizing V1's efficacy in preserving both podocytes and tubules in kidney organoids. Cisplatin-induced injury revealed a significant reduction in regenerative capacities in organoids cryopreserved by flow-freezing methods, while the V1 method did not show statistical significance compared to the unfrozen controls. This study underscores vitrification, especially with high concentrations of cryoprotectants, as an effective approach for maintaining kidney organoid viability and structure during cryopreservation, offering practical approaches for kidney organoid research.

A strategic approach for efficient cryo-EM grid optimization using design of experiments.

In recent years, cryo-electron microscopy (cryo-EM) has become a practical and effective method of determining structures at previously unattainable resolutions due to advances in detection, automation, and data processing. However, sample preparation remains a major bottleneck in the cryo-EM workflow. Even after the arduous process of biochemical sample optimization, it often takes several iterations of grid vitrification and screening to determine the optimal grid freezing parameters that yield suitable ice thickness and particle distribution for data collection. Since a high-quality sample is imperative for high-resolution structure determination, grid optimization is a vital step. For researchers who rely on cryo-EM facilities for grid screening, each iteration of this optimization process may delay research progress by a matter of months. Therefore, a more strategic and efficient approach should be taken to ensure that the grid optimization process can be completed in as few iterations as possible. Here, we present an implementation of Design of Experiments (DOE) to expedite and strategize the grid optimization process. A Fractional Factorial Design (FFD) guides the determination of a limited set of experimental conditions which can model the full parameter space of interest. Grids are frozen with these conditions and screened for particle distribution and ice thickness. Quantitative scores are assigned to each of these grid characteristics based on a qualitative rubric. Input conditions and response scores are used to generate a least-squares regression model of the parameter space in JMP, which is used to determine the conditions which should, in theory, yield optimal grids. Upon testing this approach on apoferritin and L-glutamate dehydrogenase on both the Vitrobot Mark IV and the Leica GP2 plunge freezers, the resulting grid conditions reliably yielded grids with high-quality ice and particle distribution that were suitable for collecting large overnight datasets on a Krios. We conclude that a DOE-based approach is a cost-effective and time-saving tool for cryo-EM grid preparation.

Testing an artificial intelligence algorithm to predict fetal heartbeat of vitrified-warmed blastocysts from a single image: predictive ability in different settings.

STUDY QUESTION: Could an artificial intelligence (AI) algorithm predict fetal heartbeat from images of vitrified-warmed embryos? SUMMARY ANSWER: Applying AI to vitrified-warmed blastocysts may help predict which ones will result in implantation failure early enough to thaw another. WHAT IS KNOWN ALREADY: The application of AI in the field of embryology has already proven effective in assessing the quality of fresh embryos. Therefore, it could also be useful to predict the outcome of frozen embryo transfers, some of which do not recover their pre-vitrification volume, collapse, or degenerate after warming without prior evidence. STUDY DESIGN, SIZE, DURATION: This retrospective cohort study included 1109 embryos from 792 patients. Of these, 568 were vitrified blastocysts cultured in time-lapse systems in the period between warming and transfer, from February 2022 to July 2023. The other 541 were fresh-transferred blastocysts serving as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Four types of time-lapse images were collected: last frame of development of 541 fresh-transferred blastocysts (FTi), last frame of 467 blastocysts to be vitrified (PVi), first frame post-warming of 568 vitrified embryos (PW1i), and last frame post-warming of 568 vitrified embryos (PW2i). After providing the images to the AI algorithm, the returned scores were compared with the conventional morphology and fetal heartbeat outcomes of the transferred embryos (n = 1098). The contribution of the AI score to fetal heartbeat was analyzed by multivariate logistic regression in different patient populations, and the predictive ability of the models was measured by calculating the area under the receiver-operating characteristic curve (ROC-AUC). MAIN RESULTS AND THE ROLE OF CHANCE: Fetal heartbeat rate was related to AI score from FTi (P < 0.001), PW1i (P < 0.05), and PW2i (P < 0.001) images. The contribution of AI score to fetal heartbeat was significant in the oocyte donation program for PW2i (odds ratio (OR)=1.13; 95% CI [1.04-1.23]; P < 0.01), and in cycles with autologous oocytes for PW1i (OR = 1.18; 95% CI [1.01-1.38]; P < 0.05) and PW2i (OR = 1.15; 95% CI [1.02-1.30]; P < 0.05), but was not significantly associated with fetal heartbeat in genetically analyzed embryos. AI scores from the four groups of images varied according to morphological category (P < 0.001). The PW2i score differed in collapsed, non-re-expanded, or non-viable embryos compared to normal/viable embryos (P < 0.001). The predictability of the AI score was optimal at a post-warming incubation time of 3.3-4 h (AUC = 0.673). LIMITATIONS, REASONS FOR CAUTION: The algorithm was designed to assess fresh embryos prior to vitrification, but not thawed ones, so this study should be considered an external trial. WIDER IMPLICATIONS OF THE FINDINGS: The application of predictive software in the management of frozen embryo transfers may be a useful tool for embryologists, reducing the cancellation rates of cycles in which the blastocyst does not recover from vitrification. Specifically, the algorithm tested in this research could be used to evaluate thawed embryos both in clinics with time-lapse systems and in those with conventional incubators only, as just a single photo is required. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Regional Ministry of Innovation, Universities, Science and Digital Society of the Valencian Community (CIACIF/2021/019) and by Instituto de Salud Carlos III (PI21/00283), and co-funded by European Union (ERDF, 'A way to make Europe'). M.M. received personal fees in the last 5 years as honoraria for lectures from Merck, Vitrolife, MSD, Ferring, AIVF, Theramex, Gedeon Richter, Genea Biomedx, and Life Whisperer. There are no other competing interests. TRIAL REGISTRATION NUMBER: N/A.

Cumulative pregnancy rates of two strategies: Day 3 fresh embryo transfer followed by Day 3 or Day 5/6 vitrification and embryo transfer: a randomized controlled trial.

STUDY QUESTION: Are cumulative pregnancy rates better if supernumerary embryos are vitrified on Day 5/6 instead of Day 3? SUMMARY ANSWER: The results do not show a significant difference in cumulative pregnancy rates between the Day 3 and Day 5/6 vitrification groups. WHAT IS KNOWN ALREADY: Pregnancy and live birth rates following IVF or ICSI treatment are higher after extended embryo culture and blastocyst transfer (Day 5/6) compared to cleavage-stage (Day 3) transfer. Cumulative pregnancy rates from one oocyte retrieval (OR) cycle show no significant difference after fresh and frozen embryo transfers, but only one study has used vitrification for the cryopreservation of supernumerary embryos while four studies have used a slow freezing protocol. STUDY DESIGN, SIZE, DURATION: Our prospective randomized controlled trial was performed in an academic centre between January 2018 and August 2020. Patients were randomized into vitrification Day 3 (n = 80) or Day 5/6 (n = 81) groups. The primary outcome was the cumulative ongoing pregnancy rate (cOPR), considering only the first pregnancy for each couple. The power calculation revealed that 75 patients were required in each group, when assuming a 50% cOPR with four embryo transfers in the vitrification Day 3 group vs two transfers in the vitrification Day 5/6 group. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients <38 years undergoing their first or second OR cycles were randomized at the start of the first cycle. Up to two cycles were included in the analysis. A fresh embryo transfer was performed on Day 3. Supernumerary embryos (with ≥6 cells, <25% fragmentation, and equal blastomeres) or blastocysts (with expansion grade ≥2 with inner cell mass and trophectoderm score A/B) were vitrified on Day 3 or Day 5/6, respectively, and then transferred at a later date. A time-to-event analysis was performed with the patient's first ongoing pregnancy as the event of interest and the number of embryo transfers as the time component. The statistical comparison was performed by a Cox proportional hazards model. Cumulative costs of vitrification on Day 3 vs Day 5/6 were explored and compared using Mann-Whitney U tests. MAIN RESULTS AND THE ROLE OF CHANCE: By December 2021, 233 transfers (96 fresh and 137 frozen) in 77 patients were performed in the vitrification Day 3 group and 201 transfers (88 fresh and 113 frozen) in 77 patients were performed in the vitrification Day 5/6 group. The time-to-event analysis did not show a difference between the two arms with regard to the patient's first ongoing pregnancy as the primary study outcome (hazard ratio [HR] 1.25, 95% CI 0.82; 1.92, P = 0.30). The cumulative ongoing pregnancy rate after eight transfers (from one or two ORs) was 57% in the vitrification Day 3 group vs 58% in the vitrification Day 5/6 group. The median number of embryo transfers until a pregnancy was achieved was five vs four, respectively, in the vitrification Day 3 group vs the Day 5/6 group. Similar results were found for the secondary study outcome, i.e. clinical pregnancy with foetal heart rate (HR 1.19, 95% CI 0.78; 1.80, P = 0.41). The cumulative clinical pregnancy rate (cCPR) after eight embryo transfers was 62% in the vitrification Day 3 group vs 59% in the vitrification Day 5/6 group. The median number of transfers until a pregnancy was achieved was four in both groups. The healthcare consumption pattern differed between the two groups and we observed higher costs for the vitrification Day 3 group compared to the vitrification Day 5/6 group, although these differences were not statistically significant. LIMITATIONS, REASONS FOR CAUTION: Although our power calculation revealed that only 75 patients were needed in each study group (ß = 0.87, α < 0.05), the numbers were low. Also, different numbers of single and double embryo transfers were performed between the two groups, which may have affected the results. The cost analysis was performed on a subset of the patients and is therefore exploratory. WIDER IMPLICATIONS OF THE FINDINGS: Our study shows no difference in the cumulative pregnancy rate nor costs after fresh and frozen embryo transfers of at most two sequential OR cycles between the Day 3 and Day 5/6 vitrification groups; however, obstetric and perinatal outcomes should be taken into account to determine the best strategy. STUDY FUNDING/COMPETING INTEREST(S): This study was funded as an investigator-sponsored study of S.D. by Merck nv/sa Belgium, an affiliate of Merck KGaA, Darmstadt, Germany, and by Gedeon Richter Benelux (PA18-0162). The authors declare no conflict of interest related to this study. TRIAL REGISTRATION NUMBER: NCT04196036. TRIAL REGISTRATION DATE: 15 January 2018. DATE OF FIRST PATIENT'S ENROLMENT: 15 January 2018.

Trophectoderm biopsy of blastocysts following IVF and embryo culture increases epigenetic dysregulation in a mouse model.

STUDY QUESTION: Does trophectoderm biopsy (TEBx) of blastocysts for preimplantation genetic testing in the clinic affect normal placental and embryo development and offspring metabolic outcomes in a mouse model? SUMMARY ANSWER: TEBx impacts placental and embryonic health during early development, with some alterations resolving and others worsening later in development and triggering metabolic changes in adult offspring. WHAT IS KNOWN ALREADY: Previous studies have not assessed the epigenetic and morphological impacts of TEBx either in human populations or in animal models. STUDY DESIGN, SIZE, DURATION: We employed a mouse model to identify the effects of TEBx during IVF. Three groups were assessed: naturally conceived (Naturals), IVF, and IVF + TEBx, at two developmental timepoints: embryonic day (E)12.5 (n = 40/Naturals, n = 36/IVF, and n = 36/IVF + TEBx) and E18.5 (n = 42/Naturals, n = 30/IVF, and n = 35/IVF + TEBx). Additionally, to mimic clinical practice, we assessed a fourth group: IVF + TEBx + Vitrification (Vit) at E12.5 (n = 29) that combines TEBx and vitrification. To assess the effect of TEBx in offspring health, we characterized a 12-week-old cohort (n = 24/Naturals, n = 25/IVF and n = 25/IVF + TEBx). PARTICIPANTS/MATERIALS, SETTING, METHODS: Our mouse model used CF-1 females as egg donors and SJL/B6 males as sperm donors. IVF, TEBx, and vitrification were performed using standardized methods. Placenta morphology was evaluated by hematoxylin-eosin staining, in situ hybridization using Tpbpa as a junctional zone marker and immunohistochemistry using CD34 fetal endothelial cell markers. For molecular analysis of placentas and embryos, DNA methylation was analyzed using pyrosequencing, luminometric methylation assay, and chip array technology. Expression patterns were ascertained by RNA sequencing. Triglycerides, total cholesterol, high-, low-, and very low-density lipoprotein, insulin, and glucose were determined in the 12-week-old cohort using commercially available kits. MAIN RESULTS AND THE ROLE OF CHANCE: We observed that at E12.5, IVF + TEBx had a worse outcome in terms of changes in DNA methylation and differential gene expression in placentas and whole embryos compared with IVF alone and compared with Naturals. These changes were reflected in alterations in placental morphology and blood vessel density. At E18.5, early molecular changes in fetuses were maintained or exacerbated. With respect to placentas, the molecular and morphological changes, although different compared to Naturals, were equivalent to the IVF group, except for changes in blood vessel density, which persisted. Of note is that most differences were sex specific. We conclude that TEBx has more detrimental effects in mid-gestation placental and embryonic tissues, with alterations in embryonic tissues persisting or worsening in later developmental stages compared to IVF alone, and the addition of vitrification after TEBx results in more pronounced and potentially detrimental epigenetic effects: these changes are significantly different compared to Naturals. Finally, we observed that 12-week IVF + TEBx offspring, regardless of sex, showed higher glucose, insulin, triglycerides, lower total cholesterol, and lower high-density lipoprotein compared to IVF and Naturals, with only males having higher body weight compared to IVF and Naturals. Our findings in a mouse model additionally support the need for more studies to assess the impact of new procedures in ART to ensure healthy pregnancies and offspring outcomes. LARGE SCALE DATA: Data reported in this work have been deposited in the NCBI Gene Expression Omnibus under accession number GSE225318. LIMITATIONS, REASONS FOR CAUTION: This study was performed using a mouse model that mimics many clinical IVF procedures and outcomes observed in humans, where studies on early embryos are not possible. WIDER IMPLICATIONS OF THE FINDINGS: This study highlights the importance of assaying new procedures used in ART to assess their impact on placenta and embryo development, and offspring metabolic outcomes. STUDY FUNDING/COMPETING INTEREST(S): This work was funded by a National Centers for Translational Research in Reproduction and Infertility grant P50 HD068157-06A1 (M.S.B., C.C., M.M.), Ruth L. Kirschstein National Service Award Individual Postdoctoral Fellowship F32 HD107914 (E.A.R.-C.) and F32 HD089623 (L.A.V.), and National Institutes of Health Training program in Cell and Molecular Biology T32 GM007229 (C.N.H.). No conflict of interest.

Is large for gestational age in singletons born after frozen embryo transfer associated with freezing technique or endometrial preparation protocol? A longitudinal national French study.

STUDY QUESTION: Is large for gestational age (LGA) observed in babies born after frozen embryo transfer (FET) associated with either the freezing technique or the endometrial preparation protocol? SUMMARY ANSWER: Artificial cycles are associated with a higher risk of LGA, with no difference in rate between the two freezing techniques (vitrification versus slow freezing) or embryo stage (cleaved embryo versus blastocyst). WHAT IS KNOWN ALREADY: Several studies have compared neonatal outcomes after fresh embryo transfer (ET) and FET and shown that FET is associated with improved neonatal outcomes, including reduced risks of preterm birth, low birthweight, and small for gestational age (SGA), when compared with fresh ET. However, these studies also revealed an increased risk of LGA after FET. The underlying pathophysiology of this increased risk remains unclear; parental infertility, laboratory procedures (including embryo culture conditions and freezing-thawing processes), and endometrial preparation treatments might be involved. STUDY DESIGN, SIZE, DURATION: A multicentre epidemiological data study was performed through a retrospective analysis of the standardized individual clinical records of the French national register of IVF from 2014 to 2018, including single deliveries resulting from fresh ET or FET that were prospectively collected in fertility centres. Complementary data were collected from the participating fertility centres and included the vitrification media and devices, and the endometrial preparation protocols. PARTICIPANTS/MATERIALS, SETTING, METHODS: Data were collected from 35 French ART centres, leading to the inclusion of a total of 72 789 fresh ET, 10 602 slow-freezing FET, and 39 062 vitrification FET. Main clinical outcomes were presented according to origin of the transferred embryos (fresh, slow frozen, or vitrified embryos) and endometrial preparations for FET (ovulatory or artificial cycles), comparing five different groups (fresh, slow freezing-ovulatory cycle, slow freezing-artificial cycle, vitrification-ovulatory cycle, and vitrification-artificial cycle). Foetal growth disorders were defined in live-born singletons according to gestational age and sex-specific weight percentile distribution: SGA and LGA if <10th and ≥90th percentiles, respectively. Analyses were performed using linear mixed models with the ART centres as random effect. MAIN RESULTS AND THE ROLE OF CHANCE: Transfers led to, respectively, 19 006, 1798, and 9195 deliveries corresponding to delivery rates per transfer of 26.1%, 17.0%, and 23.5% after fresh ET, slow-freezing FET, and vitrification FET, respectively. FET cycles were performed in either ovulatory cycles (n = 21 704) or artificial cycles (n = 34 237), leading to 5910 and 10 322 pregnancies, respectively, and corresponding to pregnancy rates per transfer of 31.6% and 33.3%. A significantly higher rate of spontaneous miscarriage was observed in artificial cycles when compared with ovulatory cycles (33.3% versus 21.4%, P < 0.001, in slow freezing groups and 31.6% versus 21.8%, P < 0.001 in vitrification groups). Consequently, a lower delivery rate per transfer was observed in artificial cycles compared with ovulatory cycles both in slow freezing and vitrification groups (15.5% versus 18.9%, P < 0.001 and 22.8% versus 24.9%, P < 0.001, respectively). Among a total of 26 585 live-born singletons, 16 413 babies were born from fresh ET, 1644 from slow-freezing FET, and 8528 from vitrification FET. Birthweight was significantly higher in the FET groups than in the fresh ET group, with no difference between the two freezing techniques. Likewise, LGA rates were higher and SGA rates were lower in the FET groups compared with the fresh ET group whatever the method used for embryo freezing. In a multivariable analysis, the risk of LGA following FET was significantly increased in artificial compared with ovulatory cycles. In contrast, the risk of LGA was not associated with either the freezing procedure (vitrification versus slow freezing) or the embryo stage (cleaved embryo versus blastocyst) at freezing. Regarding the vitrification method, the risk of LGA was not associated with either the vitrification medium used or the embryo stage. LIMITATIONS, REASONS FOR CAUTION: No data were available on maternal context, such as parity, BMI, infertility cause, or maternal comorbidities, in the French national database. In particular, we cannot exclude that the increased risk of LGA observed following FET with artificial cycles may, at least partially, be associated with a confounding effect of some maternal factors. No information about embryo culture and incubation conditions was available. Most of the vitrification techniques were performed using the same device and with two main vitrification media, limiting the validity of a comparison of risk for LGA according to the device or vitrification media used. WIDER IMPLICATIONS OF THE FINDINGS: Our results seem reassuring, since no potential foetal growth disorders following embryo vitrification in comparison with slow freezing were observed. Even if other factors are involved, the endometrial preparation treatment seems to have the greatest impact on LGA risk following FET. FET during ovulatory cycles could minimize the risk for foetal growth disorders. STUDY FUNDING/COMPETING INTEREST(S): This work has received funding from the French Biomedicine Agency (Grant number: 19AMP002). None of the authors has any conflict of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Vitrification preservation of good-quality blastocysts for more than 5 years reduces implantation and live birth rates.

STUDY QUESTION: Does vitrification cryopreservation of embryos for more than 5 years affect the pregnancy outcomes after frozen embryo transfer (FET)? SUMMARY ANSWER: Vitrification cryopreservation of good-quality blastocysts for more than 5 years is associated with a decrease in the implantation rate (IR) and live birth rate (LBR). WHAT IS KNOWN ALREADY: Previous studies have predominantly focused on embryos cryopreserved for relatively short durations (less than 5 years), yet the impact of extended cryopreservation duration on pregnancy outcomes remains a controversial issue. There is a relative scarcity of data regarding the efficacy and safety of storing embryos for 5 years or longer. STUDY DESIGN, SIZE, DURATION: This retrospective study involved 36 665 eligible vitrified-thawed embryo transfer cycles from 1 January 2016 to 31 December 2022, at a single fertility center in China. PARTICIPANTS/MATERIALS, SETTING, METHODS: Patients were divided into three groups according to embryo storage time: Group 1 consisted of 31 565 cycles, with storage time of 0-2 years; Group 2 consisted of 4458 cycles, with a storage time of 2-5 years; and Group 3 included 642 cycles, with storage time exceeding 5 years. The main outcome measures were IR and LBR. Secondary outcome variables included rates of biochemical pregnancy, multiple pregnancy, ectopic pregnancy, and miscarriage, as well as neonatal outcomes. Reproductive outcomes were analyzed as binary variables. Multivariate logistic regression analysis was used to explore the effect of preservation time on pregnancy outcomes after correcting for confounding factors. In addition, we also assessed neonatal outcomes, such as large for gestational age (LGA) and small for gestational age (SGA). MAIN RESULTS AND THE ROLE OF CHANCE: IRs in the three groups (0-2, 2-5, and >5 years) were 37.37%, 39.03%, and 35.78%, respectively (P = 0.017), and LBRs in the three groups were 37.29%, 39.09%, and 34.91%, respectively (P = 0.028). After adjustment for potential confounding factors, compared with the 0-2 years storage group, prolonged embryo vitrification preservation time (2-5 years or >5 years) did not affect secondary outcomes such as rates of biochemical pregnancy, multiple pregnancy, ectopic pregnancy, and miscarriage (P > 0.05). But cryopreservation of embryos for more than 5 years reduced the IR (adjusted odds ratio (aOR) 0.82, 95% CI 0.69-0.97, P = 0.020) and LBR (aOR 0.76, 95% CI 0.64-0.91, P = 0.002). Multivariate stratified analysis also showed that prolonging the cryopreservation time of blastocysts (>5 years) reduced the IR (aOR 0.78, 95% CI 0.62-0.98, P = 0.033) and LBR (aOR 0.68, 95% CI 0.53-0.87, P = 0.002). However, no effect on cleavage embryos was observed (P > 0.05). We further conducted stratified analyses based on the number and quality of frozen blastocysts transferred, and the results showed that the FET results after transfers of good-quality blastocysts in the >5 years storage group were negatively affected. However, the storage time of non-good-quality blastocysts was not significantly associated with pregnancy outcomes. Regarding the neonatal outcomes (of singletons), embryo vitrification preservation time had no effect on preterm birth rates, fetal birth weight, or neonatal sex ratios. However, as the storage time increased, rates of SGA (5.60%, 4.10%, and 1.18%) decreased, while rates of LGA (5.22%, 6.75%, and 9.47%) increased (P < 0.05). After adjusting for confounding factors, the increase in LGA and the decrease in SGA were significantly correlated with the duration of storage time. LIMITATIONS, REASONS FOR CAUTION: This was a retrospective study using data from a single fertility center, even though the data had been adjusted, our findings still need to be validated in further studies. WIDER IMPLICATIONS OF THE FINDINGS: With the full implementation of the two-child policy in China, there may be more patients whose embryos have been frozen for a longer time in the future. Patients should be aware that the IR and LBR of blastocysts are negatively affected when the cryopreservation time is longer than 5 years. Couples may therefore consider shortening the time until FET treatment. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Nature Science Foundation of China (No. 82101672), Science and Technology Projects in Guangzhou (No. 2024A03J0180), General Guidance Program for Western Medicine of Guangzhou Municipal Health Commission (No. 20231A011096), and the Medical Key Discipline of Guangzhou (2021-2023). None of the authors have any conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Impacts of double biopsy and double vitrification on the clinical outcomes following euploid blastocyst transfer: a systematic review and meta-analysis.

STUDY QUESTION: Compared to the 'single biopsy + single vitrification' approach, do 'double biopsy + double vitrification' or 'single biopsy + double vitrification' arrangements compromise subsequent clinical outcomes following euploidy blastocyst transfer? SUMMARY ANSWER: Both 'double biopsy + double vitrification' and 'single biopsy + double vitrification' led to reduced live birth/ongoing pregnancy rates and clinical pregnancy rates. WHAT IS KNOWN ALREADY?: It is not uncommon to receive inconclusive results following blastocyst biopsy and preimplantation genetic testing for aneuploidy (PGT-A). Often these blastocysts are warmed for re-test after a second biopsy, experiencing 'double biopsy + double vitrification'. Furthermore, to achieve better workflow, IVF laboratories may choose to routinely vitrify all blastocysts and schedule biopsy at a preferred timing, involving 'single biopsy + double vitrification'. However, in the current literature, there is a lack of systematic evaluation of both arrangements regarding their potential clinical risks in reference to the most common 'single biopsy + single vitrification' approach. STUDY DESIGN, SIZE, DURATION: A systematic review and meta-analysis were performed, with the protocol registered in PROSPERO (CRD42023469143). A search in PUBMED, EMBASE, and the Cochrane Library for relevant studies was carried out on 30 August 2023, using the keywords 'biopsy' and 'vitrification' and associated variations respectively. Only studies involving frozen transfers of PGT-A tested euploid blastocysts were included, with those involving PGT-M or PGT-SR excluded. PARTICIPANTS/MATERIALS, SETTING, METHODS: Study groups included blastocysts having undergone 'double biopsy + double vitrification' or 'single biopsy + double vitrification', with a 'single biopsy + single vitrification' group used as control. The primary outcome was clinical pregnancy, while secondary outcomes included live birth/ongoing pregnancy, miscarriage, and post-warming survival rates. Random effects meta-analysis was performed with risk ratios (RR) and 95% CIs were used to present outcome comparisons. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 607 records were identified through the initial search and nine studies (six full articles and three abstracts) were eventually included. Compared to 'single biopsy + single vitrification', 'double biopsy + double vitrification' was associated with reduced clinical pregnancy rates (six studies, n = 18 754; RR = 0.80, 95% CI = 0.71-0.89; I2 = 0%) and live birth/ongoing pregnancy rates (seven studies, n = 20 964; RR = 0.72, 95% CI = 0.63-0.82; I2 = 0%). However, no significant changes were seen in miscarriage rates (seven studies, n = 22 332; RR = 1.40, 95% CI = 0.92-2.11; I2 = 53%) and post-warming survival rates (three studies, n = 13 562; RR = 1.00, 95% CI = 0.99-1.01; I2 = 0%) following 'double biopsy + double vitrification'. Furthermore, 'single biopsy + double vitrification' was also linked with decreased clinical pregnancy rates (six studies, n = 13 284; RR = 0.84, 95% CI = 0.76-0.92; I2 = 39%) and live birth/ongoing pregnancy rates (seven studies, n = 16 800; RR = 0.79, 95% CI = 0.69-0.91; I2 = 70%), and increased miscarriage rates (five studies, n = 15 781; RR = 1.48, 95% CI = 1.31-1.67; I2 = 0%), but post-warming survival rates were not affected (three studies, n = 12 452; RR = 0.99, 95% CI = 0.97-1.01; I2 = 71%) by 'single biopsy + double vitrification'. LIMITATIONS, REASONS FOR CAUTION: All studies included in this meta-analysis were retrospective with varying levels of heterogeneity for different outcomes. Not all studies had accounted for potential confounding factors. Only one study reported neonatal outcomes. WIDER IMPLICATIONS OF THE FINDINGS: Our data indicated adverse impacts of 'double biopsy + double vitrification' and 'single biopsy + double vitrification' on clinical outcomes following euploid blastocyst transfers. Patients should be carefully consulted about the risks when offered such approaches. The biopsy process should be carried out as carefully and competently as possible to minimize an inconclusive diagnosis. STUDY FUNDING/COMPETING INTEREST(S): R.W. is supported by a National Health and Medical Research Council Emerging Leadership Investigator Grant (2009767). There is no other external funding to report. All authors report no conflict of interest. REGISTRATION NUMBER: CRD42023469143.

Electroporation-mediated genome editing in vitrified/warmed porcine zygotes obtained in vitro.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated 9 (Cas9) system is the most efficient and widely used technology for genome editing in all sorts of organisms, including livestock animals. Here, we examined the feasibility of CRISPR/Cas9-derived genome editing (GE) in vitrified porcine zygotes, where the flexible planning of experiments in time and space is expected. OCT4 and CD46 genes were targeted, and the Cas9/sgRNA ribonucleoprotein complexes (RNP) were electroporated into zygotes at 2 h after warming. Vitrification or GE alone did not significantly reduce the developmental rates to the blastocyst stage. However, vitrification followed by GE significantly reduced blastocyst development. Sequencing analysis of the resultant blastocysts revealed efficient GE for both OCT4 (nonvitrified: 91.0%, vitrified: 95.1%) and CD46 (nonvitrified: 94.5%, vitrified: 93.2%), with no significant difference among them. Immunocytochemical analysis showed that GE-blastocysts lacked detectable proteins. They were smaller in size, and the cell numbers were significantly reduced compared with the control (p < 0.01). Finally, we demonstrated that double GE efficiently occurs (100%) when the OCT4-RNP and CD46-RNP are simultaneously introduced into zygotes after vitrification/warming. This is the first demonstration that vitrified porcine zygotes can be used in GE as efficiently as nonvitrified ones.

Effect of melatonin on developmental competence, mitochondrial distribution, and intensity of fresh and vitrified/thawed in vitro matured buffalo oocytes.

BACKGROUND: In livestock breeding, oocyte cryopreservation is crucial for preserving and transferring superior genetic traits. This study was conducted to examine the additional effect of melatonin to maturation and vitrification media on the in vitro developmental capacity, mitochondrial distribution, and intensity of buffalo oocytes. The study involved obtaining ovaries from a slaughterhouse and conducting two phases. In the first phase, high-quality oocytes were incubated in a maturation medium with or without 10-9M melatonin for 22 h (at 38.5°C in 5% CO2). Matured oocytes were fertilized in vitro and cultured in SOF media for seven days. In the second phase, vitrified in vitro matured oocytes were stored in vitrified media (basic media (BM) containing a combination of cryoprotectants (20% Ethyl Glycol and 20% Dimethyl sulfoxide), with or without melatonin, and then stored in liquid nitrogen. Normal vitrified/thawed oocytes were fertilized in vitro and cultured as described. Finally, the matured oocytes from the fresh and vitrified/thawed groups, both with and without melatonin, were stained using DAPI and Mitotracker red to detect their viability (nuclear maturation), mitochondrial intensity, and distribution using a confocal microscope. The study found that adding 10-9M melatonin to the maturation media significantly increased maturation (85.47%), fertilization rate (84.21%)cleavage (89.58%), and transferable embryo (48.83%) rates compared to the group without melatonin (69.85%,79.88%, 75.55%, and 37.25% respectively). Besides that, the addition of melatonin to the vitrification media improved the recovery rate of normal oocytes (83.75%), as well as the cleavage (61.80%) and transferable embryo (27.00%) rates when compared to the vitrified TCM group (67.46%, 51.40%, and 17.00%, respectively). The diffuse mitochondrial distribution was higher in fresh with melatonin (TCM + Mel) (80%) and vitrified with melatonin (VS2 + Mel groups) (76.70%), Furthermore, within the same group, while the mitochondrial intensity was higher in the TCM + Mel group (1698.60) than other group. In conclusion, Melatonin supplementation improves the developmental competence and mitochondrial distribution in buffalo oocytes in both cases(in vitro maturation and vitrification).

Advancements in fertility preservation strategies for pediatric male cancer patients: a review of cryopreservation and transplantation of immature testicular tissue.

Recently, there has been increasing emphasis on the gonadotoxic effects of cancer therapy in prepubertal boys. As advances in oncology treatments continue to enhance survival rates for prepubertal boys, the need for preserving their functional testicular tissue for future reproduction becomes increasingly vital. Therefore, we explore cutting-edge strategies in fertility preservation, focusing on the cryopreservation and transplantation of immature testicular tissue as a promising avenue. The evolution of cryopreservation techniques, from controlled slow freezing to more recent advancements in vitrification, with an assessment of their strengths and limitations was exhibited. Detailed analysis of cryoprotectants, exposure times, and protocols underscores their impact on immature testicular tissue viability. In transplantation strategy, studies have revealed that the scrotal site may be the preferred location for immature testicular tissue grafting in both autotransplantation and xenotransplantation scenarios. Moreover, the use of biomaterial scaffolds during graft transplantation has shown promise in enhancing graft survival and stimulating spermatogenesis in immature testicular tissue over time. This comprehensive review provides a holistic approach to optimize the preservation strategy of human immature testicular tissue in the future.

Fast and furious: pregnancy outcome with one-step rehydration in the warming protocol for human blastocysts.

RESEARCH QUESTION: Do embryos warmed using a one-step rehydration protocol with a more efficient workflow result in comparable pregnancy rates to the standard multi-step rehydration protocol? DESIGN: A retrospective cohort study of 3439 frozen embryo transfers (FET). Clinical outcomes of 833 FETs using a one-step rehydration protocol were reviewed and compared with results from the control group (2606 FETs using standard multi-step rehydration protocol). Primary outcome was ongoing pregnancy rate. Secondary outcomes were survival, positive pregnancy, clinical pregnancy, implantation and miscarriage rates. RESULTS: Survival rates were identical between the two groups (99.5%). Clinical pregnancy rate was 63.0% in the one-step warming protocol, comparable to 59.9% in the multi-step rehydration protocol. A significant increase was observed in the ongoing pregnancy rate with 60.4% in the one-step rehydration versus 55.4% in the multi-step rehydration group (P = 0.011); implantation rate was 63.6% versus 57.0% (P = 0.0005). The miscarriage rate of 4.0% in the one-step rehydration protocol was significantly lower compared with 7.6% in the multi-step rehydration protocol (P = 0.0001). Comparable outcomes persisted even when the analysis was extended to embryos that had and had not undergone preimplantation genetic testing (PGT), as well as day of development of the blastocysts. When controlling for variables of age, PGT, blastocyst development day and embryo expansion, rapid warming significantly increased chances of an ongoing pregnancy (adjusted OR 1.264, 95% CI 1.076 to 1.484). CONCLUSION: A one-step rehydration protocol resulted in identical survival rates and improved ongoing pregnancy rates compared with the multi-step rehydration technique.

A compact, high-throughput semi-automated embryo vitrification system based on hydrogel.

RESEARCH QUESTION: What is the efficiency and efficacy of the novel Biorocks semi-automated vitrification system, which is based on a hydrogel? DESIGN: This comparative experimental laboratory study used mouse model and human day 6 blastocysts. Mouse oocytes and embryos were quality assessed post-vitrification. RESULTS: The Biorocks system successfully automated the solution exchanges during the vitrification process, achieving a significantly improved throughput of up to 36 embryos/oocytes per hour. Using hydrogel for cryoprotective agent delivery, 12 vessels could be processed simultaneously, fitting comfortably within an assisted reproductive technology (ART) workstation. In tests involving the cryopreservation of oocytes and embryos, the system yielded outcomes equivalent to the manual Cryotop method. For example, the survival rate for mouse oocytes was 98% with the Biorocks vitrification system (n = 46) and 95% for the manual Cryotop method (n = 39), of which 46% and 41%, respectively, progressed to blastocysts on day 5 after IVF. CC-grade day 6 human blastocysts processed with the Biorocks system (n = 39) were associated with a 92% 2 h re-expansion rate, equivalent to the 90% with Cryotop (n = 30). The cooling/warming rates achieved by the Biorocks system were 31,900°C/minute and 24,700°C/minute, respectively. Oocyte quality was comparable or better post-vitrification for Biorocks than Cryotop. CONCLUSIONS: The Biorocks semi-automated vitrification system offers enhanced throughput without compromising the survival and developmental potential of oocytes and embryos. This innovative system may help to increase the efficiency and standardization of vitrification in ART clinics. Further investigations are needed to confirm its efficacy in a broader clinical context.

Fast and furious: successful survival and resumption of meiosis in immature human oocytes vitrified and warmed using a short protocol.

RESEARCH QUESTION: Can immature oocytes vitrified and warmed using a short protocol survive and resume meiosis? DESIGN: This study examined modifications of oocyte vitrification and warming protocols that reduce the length of exposure to vitrification and warming solutions. In total, 561 germinal vesicles and 218 metaphase I oocytes that were immature at oocyte retrieval were vitrified at room temperature for 2 min. Warming was performed at 37°C for 2 min. Resumption of meiotic activity was evaluated after 24 and 48 h of culture. Two different commercially available vitrification and warming kits were used for comparison. RESULTS: Ninety-five percent of germinal vesicles survived, with no difference observed between the kits. The survival of metaphase I oocytes was, on average, 95.4% and did not differ significantly between the kits. Of the 533 germinal vesicles that survived, 491 converted to metaphase I oocytes (92.1%). After culture for 48 h, 54.4% converted to metaphase II oocytes. In addition, of the 208 metaphase I oocytes that survived warming, 84.1% converted to metaphase II oocytes after 24 h of culture. These maturation rates were similar to those of non-vitrified oocytes. CONCLUSIONS: Vitrification and warming of oocytes at different nuclear maturation stages can be performed with 2 min of exposure to hypertonic solution and 2 min of exposure to hypotonic solution, respectively. This approach reduces exposure of the oocytes to room temperature during dehydration and rehydration. Warming in 0.5M sucrose helps to maintain and support the potential of oocytes to resume nuclear meiotic activity, and conversion from germinal vesicles to metaphase I and metaphase II oocytes.

First report on successful delivery after retransplantation of vitrified, rapid warmed ovarian tissue in Europe.

RESEARCH QUESTION: Cryopreservation of ovarian tissue is one feasible option to preserve female fertility prior to cancer treatment. The slow freezing protocol represents the current standard approach, while vitrification has been suggested as a promising alternative. This paper reports the follow-up and first successful delivery after retransplantation of vitrified, rapid warmed ovarian tissue in Europe. DESIGN: After the patient received a diagnosis of breast cancer, ovarian tissue was removed laparoscopically and sent via overnight transportation to University Hospital Bonn for vitrification on site. The patient was treated with chemotherapy, leading to ovarian failure. After 2 years, retransplantation of the vitrified, rapid warmed tissue was conducted on site. RESULTS: Two months after grafting, the patient reported regular menstrual cycles. After 1 further month a clinical pregnancy occurred, which ended in a spontaneous abortion at the 8th week of pregnancy. Six months after grafting, another naturally conceived pregnancy was determined, resulting in the birth of a healthy boy 14 months after retransplantation of the ovarian tissue. CONCLUSIONS: Complementing the successful deliveries reported by the groups of Suzuki (Japan) and Silber (USA) regarding vitrified tissue, the current results confirm the high potential of this cryopreservation method in a clinical routine setting as an alternative approach to the widespread slow freezing method.

Clinical outcomes of vitrified-warmed autologous oocyte cycles with 15-year follow-up at a single UK centre: consistent and predictable results.

RESEARCH QUESTION: What were the clinical outcomes from 332 autologous vitrified- warmed oocyte cycles derived from 3182 elective autologous oocyte freeze cycles carried out between 2008 and 2022 in a single-centre series? DESIGN: In this retrospective observational study, outcomes in 299 patients returning to use their frozen oocytes between 2015 and 2023 were analysed. RESULTS: A total of 3328 elective oocyte vitrification cycles were performed in 2280 patients. The return rate to use oocytes was 14% (299/2171). Mean ages were 37.6 years at storage and 40 at warming. Ninety-three clinical pregnancies and 77 healthy live births were recorded. The live birth rate (LBR) was 24% (39/163) per fresh transfer and 17% (39/227) per embryo transferred. Stratified by age at freezing, the LBR per embryo transferred was 26% (12/47) in participants under 35 years, 20% (24/118) in those 35-39 years and 5% (3/62) in those 40+ years. Frozen embryo transfers (FET) achieved a 30% (24/80) LBR per embryo transfer and a 27% (24/90) LBR per embryo transferred. PGT-A for embryo selection doubled the LBR compared with FET from an untested embryo after one attempt (40% versus 21%). In patients aged over 40 years, the cumulative LBR reached 42% per patient in euploid FET. CONCLUSION: The proportion of patients who returned to use their stored oocytes and the clinical outcomes were consistent with other recent reports and challenges the prevalent critical narrative regarding elective oocyte freezing for fertility preservation. The results are now comparable to routine IVF. Not everyone who returns to use their oocytes will conceive, but for those choosing to preserve their fertility, oocyte freezing can provide reproducible and reassuring results.