Effect of the flow-force relationships used in two parameter modelling of freezing and vitrification protocols for bovine embryos.

Cells may become damaged by strong volume changes and related intracellular changes during slow freezing or vitrification. These osmotic events can be modelled mathematically, using descriptions of transmembrane flow of solute and water. We compared different variants of an often used 2-parameter (2P) formalism in fitting of an empirical shrink-swell curve of a bovine embryo in 5 vol% glycerol, and in simulations of CPA loading and removal in a vitrification protocol. In its original form, the 2P model uses a flow-force relationship for the flux of CPA that is not analogous to that for water (asymmetrical), but in the other variants used, the flow-force relationships for water and CPA are analogous to each other (symmetrical). The effect of used model on estimated values for Lp and Ps in 5 vol% glycerol was small. Also the effect on shrinking and swelling in vitrification media was small, but the original 2P model predicted stronger swelling of embryos during one-step CPA removal. One variant that we compared simply assumes Raoult's law, i.e. M = m, even in very concentrated solutions We conclude that this simple model is easy and appropriate for simulating osmotic events of embryo's. But if a method for correcting for the deviation from Raoult's law is used, a symmetrical model seems more appropriate than the original (asymmetrical) 2P model.

The importance of social oocyte cryopreservation in supporting local municipalities: A prospective study.

BACKGROUND: With the trend toward late marriages and late childbearing, cryopreservation of oocytes for fertility preservation is attracting attention as a method to counteract the declining birthrate. OBJECTIVES: To examine the impact of social oocyte cryopreservation on local communities by assessing the significance of government assistance for cryofreezing and capturing the participants' subsequent feelings regarding this assistance. DESIGN: Descriptive study. METHODS: A prospective study was conducted on city-dwelling women <35 years old attending monthly seminars on oocyte retrieval/cryopreservation to whom the study concept was explained. Egg collection and storage management costs were free for 3 years after the project completed, and subsequent actual storage costs were borne by the individuals. After oocyte retrieval, we conducted a questionnaire on oocyte cryopreservation and administrative assistance. RESULTS: Of the 62 seminar participants, 2 became pregnant naturally without oocyte retrieval. Oocytes were retrieved in 34 women (average age: 32.8 years, number of oocytes obtained: 8.3), among whom 4 subsequently became pregnant and gave birth through natural pregnancy or artificial insemination, and 1 became pregnant and gave birth using frozen oocytes. In a follow-up questionnaire given to these 34 subjects, all responded that they were glad to have oocyte cryopreservation, but 23 subjects (67.6%) answered that they could not perform cryopreservation without financial assistance. Twenty-five participants (73.5%) wanted to try to conceive without using frozen oocytes as a post-cryopreservation plan. CONCLUSIONS: As a countermeasure against the declining birthrate, oocyte cryopreservation and associated workshops that can provide the information and education needed to conduct this task in a "planned" manner may be useful in providing women with additional reproductive options. Financial assistance will also be required to offer this service to the women who need it.

Women benefit when egg freezing is subsidized by local municipalitiesWhy was the study done? To prospectively examine the significance of egg freezing in a society in which the declining birthrate is an issue, particularly with regard to those who wish to undergo egg freezing and their trends when it is supported by the government. What did the researchers do? This project was conducted as a three-year endowed course by a local city government. Participants were women aged 20 to 34 who lived in the city and were recruited through the city's newsletter and website. They then attended a fertility workshop that was held once a month. Participants who wished to freeze their eggs were offered one free egg retrieval and three years of frozen storage. Participants were also asked to complete a questionnaire about their progress three years after the project ended. What did the researchers find? Sixty-two women participated in the three-year project, of whom 34 chose to freeze their eggs. Those who did not plan to conceive early, and two conceived naturally. Of those who froze their eggs, only one gave birth using the frozen eggs, and seven conceived naturally or through fertility treatments without using frozen eggs, two of whom had two pregnancies, resulting in 10 children being born. What do the findings mean? Three years after the project ended, the findings suggested that egg freezing itself may not have had a significant effect on pregnancy and childbirth but that holding workshops on fertility may have acted as an incentive for women to become pregnant and give birth.

Liquid Overlay-Induced Donor Plant Vigor and Initial Ammonium-Free Regrowth Medium Are Critical to the Cryopreservation of Scrophularia kakudensis.

Cryopreservation, storing biological material in liquid nitrogen (LN, -196 °C), offers a valuable option for the long-term conservation of non-orthodox seeds and vegetatively propagated species in the sector of agrobiodiversity and wild flora. Although the large-scale cryobanking of germplasm collections has been increasing worldwide, the wide application of cryopreservation protocols in wild flora is hampered by difficulties in vitro propagation and a lack of universal cryopreservation protocols, among others. This study established a systematic approach to developing an in vitro culture and droplet-vitrification cryopreservation procedure for shoot tips of Scrophularia kakudensis. The standard procedure includes a two-step preculture with 10% sucrose for 31 h and with 17.5% sucrose for 16 h, osmoprotection with loading solution C4-35% (17.5% glycerol + 17.5% sucrose, w/v) for 30 min, cryoprotection with A3-80% (33.3% glycerol + 13.3% dimethyl sulfoxide + 13.3% ethylene glycol + 20.1% sucrose, w/v) at 0 °C for 60 min, and cooling and rewarming using aluminum foil strips. After unloading, a three-step regrowth procedure starting with an ammonium-free medium with growth regulators was essential for developing normal plantlets from cryopreserved shoot tips. Liquid overlay on the gelled medium two weeks after inoculation resulted in vigorous growth during subcultures. Moreover, liquid overlay increased LN regeneration by up to 80%, i.e., 23% higher than no liquid overlay.

Oocytes Vitrification Using Automated Equipment Based on Microfluidic Chip.

Oocyte vitrification has a wide range of applications in assisted reproduction and fertility preservation. It requires precise cryoprotectant agents (CPAs) loading and removal sequences to alleviate osmotic shock, which requires manual manipulation by an embryologist. In this study, a microfluidic system was developed to facilitate the precise adjustment of the CPA concentration around the oocyte by linear loading and removal of CPA. In addition, the microfluidic-based automated vitrification (MAV) device combines CPA loading/removal process, with vitrification process, thereby achieving automated oocyte vitrification. Oocytes were vitrified by Cryotop/QC manual method and MAV method. The results showed that the survival, cleavage, and blastocyst rates of oocytes were 80.44, 54.17, and 32.95% for the MAV method, which were significantly higher than Cryotop manual method (73.35, 43.73, and 23.67%) (p < 0.05). In MAV, solution injection rate during CPA loading/removal process was designed as a 1-segment, 2-segment, and 4-segment function. Accordingly, three concave loading and convex removal protocols were adopted to vitrify oocytes. Oocytes vitrified using the 4-segment function group exhibited increased survival (86.18%), cleavage (63.29%), and blastocyst (45.58%) rates compared to those vitrified using the 1-segment and 2-segment groups. The oocytes vitrification with the highest concentration of CPA, denoted as VS1-TS1, exhibited the highest survival rate after rewarming (86.18%). In contrast, the VS3-TS3 group, characterized by a CPA concentration half that of VS1-TS1, exhibited lower survival (74.14%) and cleavage (59.31%) rates, but displayed the higher blastocyst rate (50.79%) following oocyte activation. Our study demonstrates potential of the MAV device for oocyte or embryo vitrification.

Unraveling the occurrence of hyperhydricity in oil palm somatic embryos during somatic embryogenesis process.

The propagation of oil palm through somatic embryogenesis is the most effective method of cloning this palm tree; however, in vitro cultivation can lead to abnormalities in plant tissue, such as hyperhydricity. The present study aimed to evaluate the difference in anatomical, morphological, and histochemical characteristics, and gene expression in normal (Nm) and hyperhydric (Hh) somatic embryos of oil palm. For this purpose, Nm and Hh somatic embryos were collected from the differentiation medium and were submitted to anatomical and histochemical analyses to assess the nucleus/cytoplasm ratio (toluidine blue), starch (Lugol), and proteins (XP), as well as ultrastructural analyses via transmission electron microscopy. Additionally, gene expression analyses were performed to gain a better understanding on the molecular aspect of hyperhydric abnormality. A higher quantity of differentiated Nm somatic embryos per explant was observed, with a germination rate close to zero in Hh somatic embryos. Additionally, a higher accumulation of proteins and starch was found in Nm somatic embryos when compared to Hh embryos. It was also noted that in Nm somatic embryos, protein reserves were primarily located in the proximal region (embryonic axis), whereas starch reserves were mainly accumulated in the distal region of the somatic embryos. Hh somatic embryos exhibit insignificant starch reserves, and a greater number of intercellular spaces were observed compared to Nm somatic embryos. However, some Hh somatic embryos displayed histochemical characteristics similar to Nm, which could explain the occurrence of reversions from the Hh state to the Nm state observed in this study. Regarding molecular analyses, the gene expression results obtained showed that out of the 19 genes analyzed, 17 were upregulated in hyperhydric embryos when compared to the control condition (normal somatic embryos). Genes involved in stress response, energy metabolism, defense, membrane transport, hormonal regulation, and development were positively regulated, especially those involved in ethylene synthesis and energetic metabolism. To the best of our knowledge, this is the first in-depth study addressing hyperhydricity in oil palm during somatic embryogenesis.

Good laboratory practice for PGT-M: Turkish Society of Reproductive Medicine guidelines.

This guideline was prepared by the Turkish Society of Reproductive Medicine to define the conditions and requirements for an outsourced preimplantation genetic testing (PGT) programme in line with the experience and needs of practitioners. This guideline is intended to be a reference document for assisted reproductive technology centres, genetic diagnosis centres, non-governmental organizations working on reproductive health, legal experts, consultants working on laboratory accreditation, academicians specializing in ethical issues, and policy makers. The Consortium aims to provide recommendations addressing the challenges of genetic testing, especially PGT for monogenic diseases (PGT-M) due to the high rate of consanguineous marriage in Turkey. For this purpose, this summary document specifically includes challenges and recommendations regarding PGT-M practice, and aims to identify and aid in prevention of errors leading to misdiagnosis. The recommendations can be modified to fit other locations.

Rutin enhances mitochondrial function and improves the developmental potential of vitrified ovine GV-stage oocyte.

Vitrification of oocyte has become an important component of assisted reproductive technology and has important implications for animal reproduction and the preservation of biodiversity. However, vitrification adversely affects mitochondrial function and oocyte developmental potential, mainly because of oxidative damage. Rutin is a highly effective antioxidant, but no information is available to the effect of rutin on the mitochondrial function and development in vitrified oocytes. Therefore, we studied the effects of rutin supplementation of vitrification solution on mitochondrial function and developmental competence of ovine germinal vesicle (GV) stage oocytes post vitrification. The results showed that supplementation of vitrification solution with 0.6 mM rutin significantly increased the cleavage rate (71.6 % vs. 59.3 %) and blastocyst rate (18.9 % vs. 6.8 %) compared to GV-stage oocytes in the vitrified group. Then, we analyzed the reactive oxygen species (ROS), glutathione (GSH), mitochondrial activity and membrane potential (ΔΨm), endoplasmic reticulum (ER) Ca2+, and annexin V (AV) of vitrified sheep GV-stage oocytes. Vitrified sheep oocytes exhibited increased levels of ROS and Ca2+, higher rate of AV-positive oocytes, and decreased mitochondrial activity, GSH and ΔΨm levels. However, rutin supplementation in vitrification solution decreased the levels of ROS, Ca2+ and AV-positive oocytes rate, and increased the GSH and ΔΨm levels in vitrified oocytes. Results revealed that rutin restored mitochondrial function, regulated Ca2+ homeostasis and decreased apoptosis potentially caused by mitophagy in oocytes. To understand the mechanism of rutin functions in vitrified GV-stage oocytes in sheep, we analyzed the transcriptome and found that rutin mediated oocytes development and mitochondrial function, mainly by affecting oxidative phosphorylation and the mitophagy pathways. In conclusion, supplementing with 0.6 mM rutin in vitrification solution significantly enhanced developmental potential through improving mitochondrial function and decreased apoptosis potentially caused by mitophagy after vitrification of ovine GV-stage oocytes.

The effect of resveratrol on the developmental competence of feline oocytes vitrified at the metaphase II stage.

The objective of this research was to assess the viability and developmental potential of feline oocytes following in vitro maturation (IVM), vitrification, and post-warming incubation with resveratrol. In the first experiment, warmed oocytes were incubated with 0.2 µM, 2 µM, or 20 µM resveratrol for 2 h. Oocytes treated with 0.2 µM resveratrol had the highest viability (68.89 %), as assessed by fluorescein diacetate and ethidium bromide staining, while higher concentrations were associated with diminished oocyte viability. In the second experiment, the warmed oocytes were inseminated following the 2-h incubation with the three concentrations of resveratrol. The presumptive zygotes were then maintained in culture and their development evaluated. The highest cleavage rate was observed when the oocytes had been incubated with 0.2 µM resveratrol (88.34 %), which was higher than for the control group (without resveratrol (75 %)). Moreover, this concentration of resveratrol also augmented the blastocyst formation rate. While the vitrification of oocytes often results in diminished developmental potential in the ensuing embryos, attributed to cryopreservation-induced injury, the utilization of low concentrations of resveratrol enhances the procedure's efficacy.

Comparison of CellRox green fluorescence upon thawing on in vitro Bos taurus and Bos indicus embryos cryopreserved by slow freezing or vitrification.

The aim of this study was to compare the levels of reactive oxygen species (ROS) in Bos taurus and Bos indicus in vitro embryos cryopreserved using either slow freezing or vitrification. Embryos were divided into four groups based on subspecies and freezing method: Bos indicus slow freezing (BiSF; n = 8), Bos indicus vitrification (BiVT; n = 10), Bos taurus slow freezing (BtSF; n = 9), and Bos taurus vitrification (BtVT; n = 6). After thawing, the embryos were incubated with CellRox Green and images were obtained using a confocal microscope. The fluorescence intensity of each cell was measured and expressed as arbitrary units of fluorescence (auf) and compared using a multiple regression and unpaired t-test with α = 0.05. Results showed that subspecies and the freezing method significantly affected auf (P < 0.001; R2 = 0.1213). Bos indicus embryos had higher auf than Bos taurus embryos, whether frozen by slow freezing (67.05 ± 23.18 vs 51.30 ± 16.84, P < 0.001) or vitrification (64.44 ± 23.32 vs 47.86 ± 17.53, P < 0.001). Slow freezing induced higher auf than vitrification in both Bos taurus (51.30 ± 16.84 vs 47.86 ± 17.53, P < 0.001) and Bos indicus (67.05 ± 23.18 vs 64.44 ± 23.32, P < 0.014). In conclusion, Bos taurus embryos had lower ROS levels when frozen using vitrification, while Bos indicus embryos had consistent ROS patterns regardless of the freezing method. However, Bos indicus embryos frozen by slow freezing tended to have a higher number of cells with elevated ROS levels.

Investigating cryopreservation techniques for maintaining morphology and in vitro viability of cartilage and skin from Spix's yellow-toothed cavies (Galea spixii Wagler, 1831) for conservation through biobanks.

Conservation of the genetic diversity through skin and cartilage biobanks represents an essential strategy for maintaining biodiversity. Biobanks for the wild species of the order Rodentia have been little studied. Considering that the cryopreservation technique has specific relationships with the tissue and species of interest, we propose investigating different techniques for preserving tissue integrity and cell viability after cartilage and skin culture from Spix's yellow-toothed cavies. Subsequently, two techniques [solid-surface vitrification (SSV) vs. slow freezing (SF)] were used for cartilage and skin cryopreservation. Tissues not subjected to cryopreservation were used as controls. All tissues were evaluated for morphology and proliferation by histological techniques. Moreover, fragments were cultured, and cells were evaluated for viability, proliferation, metabolism, and apoptosis. Regardless of the cryopreservation technique, no differences were observed for the thickness of the epidermis, dermis, skin, spinous and basal layers, fibroblasts, and proliferative activity regarding the number of nucleolar organizer regions (NOR). SSV ensured better maintenance of epidermal cells, normal chondrocytes, filled gaps, collagen fibers, proliferative activity by NOR area/cell, and reduced perinuclear halos and empty gaps compared to SF. SF ensured the conservation of corneum thickness compared to the control. Although both techniques promoted cell recovery after culture, cells from SF resulted in better subconfluence time and day with cell growth around fragments compared to SSV. In conclusion, both cryopreservation techniques resulted in viable cells after culture. However, SSV promoted better maintenance of tissue morphological integrity, and SF ensured the preservation of all cell quality parameters in Spix's yellow-toothed cavies.

Prospective solutions to ovarian reserve damage during the ovarian tissue cryopreservation and transplantation procedure.

Birth rates continue to decline as more women experience fertility issues. Assisted reproductive technologies are available for patients seeking fertility treatment, including cryopreservation techniques. Cryopreservation can be performed on gametes, embryos, or gonadal tissue and can be used for patients who desire to delay in vitro fertilization treatment. This review focuses on ovarian tissue cryopreservation, the freezing of ovarian cortex containing immature follicles. Ovarian tissue cryopreservation is the only available treatment for the restoration of ovarian function in patients who undergo gonadotoxic treatments, and its wide adoption has led to its recent designation as "no longer experimental" by the American Society for Reproductive Medicine. and subsequent transplantation can restore native endocrine function and can support the possibility of pregnancy and live birth for the patient. Importantly, there are multiple steps in the procedure that put the ovarian reserve at risk of damage. The graft is highly susceptible to ischemic reperfusion injury and mass primordial follicle growth activation, resulting in a "burnout", phenomenon. In this review, we summarize current efforts to combat the loss of primordial follicles in grafts through improvements in freeze and thaw protocols, transplantation techniques, and pharmacologic adjuvant treatments. We conducted a review of the literature, with emphasis on emergent research in the last 5 years. Regarding freeze and thaw protocols, we discuss the widely accepted slow freezing approach and newer vitrification protocols. Discussion of improved transplantation techniques includes consideration of the transplantation location of the ovarian tissue and the importance of graft sites in promoting neovascularization. Finally, we discuss pharmacologic treatments being studied to improve tissue performance postgraft. Of note, there is significant research into the efficacy of adjuvants used to reduce ischemic injury, improve neovascularization, and inhibit hyperactivation of primordial follicle growth activations. Although the "experimental" label has been removed from ovarian tissue cryopreservation and subsequent transplantation, there is a significant need for further research to better understand sources of ovarian reserve damage to improve outcomes. Future research directions are provided as we consider how to reach the most hopeful results for women globally.

Cryopreservation of human teeth using vitrification method with cryoprotectant cocktails and N-acetylcysteine for banking and clinical applications.

Preserving freshly-extracted healthy human teeth offers an optional resource for potential tooth transplantation and cell therapy. This study aimed to assess the impact of vitrification, utilizing a blend of cryoprotectant agents and N-acetylcysteine (NAC), on the cryopreservation of periodontal ligament tissues, and investigate the underlying mechanisms of NAC on the tooth cryopreservation. Periodontal ligament cells were isolated from freshly-extracted healthy human permanent teeth, and cell sheets of PDLCs were fabricated. The samples including cell sheets, freshly-extracted human and rat teeth were cryopreserved with or without NAC for three months. The viability, ROS level, gene expressions and microstructure of PDLCs within cell sheets were assessed. The expression of SOD-2, Caspase3, LC3A/B and Catalase were evaluated through western blotting. Histological assessments of cryopreserved cell sheets and teeth were conducted. PDLCs were isolated from cryopreserved teeth, and their immunophenotype and differentiation ability were evaluated. The data was analyzed using one-way analysis of variance. The vitrification method showed good performance in preserving the viability and differentiation potential of PDLCs. Cryopreservation supplemented with NAC improved the survival rate of PDLCs, enhanced osteogenic differentiation ability, upregulated the expression of SOD-2 and Catalase, and inhibited cell apoptosis. Additionally, mRNA sequencing analysis revealed a significant activation of the PI3K-AKT pathway following cryopreservation via vitrification. Adding a PI3K-AKT activator improved the survival rates of PDLCs post-cryopreservation. The vitrification strategy combining various CPAs and NAC proved to be feasible for tooth cryopreservation. Targeting the PI3K-AKT pathway may improve the efficacy of tooth cryopreservation.

Purification and Cryo-Electron Microscopy Analysis of Bacterial Appendages.

A number of extracellular helical protein polymers are crucial for supporting bacterial motility. The bacterial flagellum is a polymeric appendage used to support cellular motility. Historically, structural studies of flagellar and other filaments were limited to those present as or locked into straightened states. Here, we present a robust workflow that produces biologically relevant high-resolution cryo-electron microscopy (cryo-EM) structures of bacterial flagellar filaments. We highlight how a simple purification method, centered around several centrifugation steps, exploits the process of filament ejection in Caulobacter crescentus and results in isolated filaments amenable to transmission electron microscopy (TEM) studies. The quality of the sample is validated by SDS-PAGE and negative stain TEM analysis before a sample is vitrified for cryogenic electron microscopy (cryo-EM) data collection. We provide a detailed protocol for reconstructing either straight or curved flagellar filaments by cryo-EM helical reconstruction methods, followed by an overview of model building and validation. In our hands, this workflow resulted in several flagellar structures below 3 Å resolution, with one data set reaching a global resolution of 2.1 Å. The application of this workflow supports structure-function studies to better understand the molecular interactions that regulate filament architecture in biologically relevant states. Future work will not only examine interactions that regulate bacterial flagellar and other filament organization but also provide a foundation for developing new helical biopolymers for biotech applications. Key features • Rapid high-quality purification of bacterial flagella via simple bacterial culturing, centrifugation, and resuspension methods. • High-throughput cryo-EM data collection of filamentous objects. • Use of cryoSPARC implementations of helical reconstruction algorithms to generate high-resolution 3D structures of bacterial flagella or other helical polymers.

Effects of Cryoprotectant Concentration and Exposure Time during Vitrification of Immature Pre-Pubertal Lamb Cumulus-Oocyte Complexes on Nuclear and Cytoplasmic Maturation.

Oocyte vitrification allows for the storing of endangered breed female gametes. Cryoprotectant (CPA) concentration and exposure time should ensure cell protection with minimal toxicity. In the present study, a high concentration-rapid exposure (HC-RE) and a low concentration-slow exposure (LC-SE) vitrification protocol, using dimethyl sulfoxide (DMSO) and ethylene glycol (EG) as permeating CPAs, were evaluated on meiotic competence and bioenergetic-oxidative status of pre-pubertal lamb immature COCs after in vitro maturation (IVM). For each protocol, COCs vitrified through a traditional protocol and fresh ones were used as controls. Both protocols allowed COC morphology preservation after vitrification-warming (V-W) and cumulus expansion after IVM. The maturation rate (7% and 14%) was comparable to the vitrified control (13% and 21%) but not satisfactory compared to fresh ones (58% and 64%; p < 0.001). The rate of mature oocytes displaying a perinuclear/subcortical (P/S) mitochondrial distribution pattern, an index of cytoplasmic maturity, was comparable between vitrified and fresh oocytes. The LC-SE vitrification protocol did not affect quantitative bioenergetic-oxidative parameters compared to both controls whereas HC-RE protocol significantly reduced intracellular reactive oxygen species (ROS) levels, indicating cell viability loss. In conclusion, to improve pre-pubertal lamb immature COC vitrification, the combination of low CPA concentrations with prolonged exposure time could be more promising to investigate further.

Transcriptomic Signatures of the Foetal Liver and Late Prenatal Development in Vitrified Rabbit Embryos.

Assisted reproduction technologies (ARTs) are generally considered safe; however, emerging evidence highlights the need to evaluate potential risks in adulthood to improve safety further. ART procedures like rederivation of embryos by vitrification differ from natural conditions, causing significant disparities between in vitro and in vivo embryos, affecting foetal physiology and postnatal life. This study aims to investigate whether hepatic transcriptome and metabolome changes observed postnatally are already present in foetal livers at the end of gestation. This study compared fresh and vitrified rabbit embryos, finding differences between foetuses obtained by the transfer of fresh and vitrified embryos at 24 days of gestation. Rederived embryos had reduced foetal and liver weights and crown-rump length. However, the offspring of vitrified embryos tended to be born with higher weight, showing compensatory growth in the final week of gestation (59.2 vs. 49.8 g). RNA-Seq analysis revealed 43 differentially expressed genes (DEGs) in the foetal liver of vitrified embryos compared to the fresh group. Notably, downregulated genes included BRAT1, CYP4A7, CYP2B4, RPL23, RPL22L1, PPILAL1, A1BG, IFGGC1, LRRC57, DIPP2, UGT2B14, IRGM1, NUTF2, MPST, and PPP1R1B, while upregulated genes included ACOT8, ERICH3, UBXN2A, METTL9, ALDH3A2, DERPC-like, NR5A2-like, AP-1, COG8, INHBE, and PLA2G4C. Overall, a functional annotation of these DEGs indicated an involvement in lipid metabolism and the stress and inflammatory process or immune response. Thus, our results suggest that vitrification and embryo transfer manipulation induce an adaptive response that can be observed in the liver during the last week of gestation.

Ice formation and its elimination in cryopreservation of oocytes.

Damage from ice and potential toxicity of ice-inhibiting cryoprotective agents (CPAs) are key issues in assisted reproduction of humans, domestic and research animals, and endangered species using cryopreserved oocytes and embryos. The nature of ice formed in bovine oocytes (similar in size to oocytes of humans and most other mammals) after rapid cooling and during rapid warming was examined using synchrotron-based time-resolved x-ray diffraction. Using cooling rates, warming rates and CPA concentrations of current practice, oocytes show no ice after cooling but always develop large ice fractions-consistent with crystallization of most free water-during warming, so most ice-related damage must occur during warming. The detailed behavior of ice at warming depended on the nature of ice formed during cooling. Increasing cooling rates allows oocytes soaked as in current practice to remain essentially ice free during both cooling and warming. Much larger convective warming rates are demonstrated and will allow routine ice-free cryopreservation with smaller CPA concentrations. These results clarify the roles of cooling, warming, and CPA concentration in generating ice in oocytes and establish the structure and grain size of ice formed. Ice formation can be eliminated as a factor affecting post-warming oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.

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.

Effect of breast cancer prognostic factors on ovarian reserve and response in fertility preservation.

RESEARCH QUESTION: Do breast cancer prognostic factors influence ovarian reserve and response to ovarian stimulation in the context of fertility preservation? DESIGN: Observational, bicentric retrospective study of 352 women with breast cancer who underwent ovarian stimulation using a random start gonadotrophin releasing hormone antagonist protocol and vitrified oocytes between November 2015 and August 2022. Serum anti-Müllerian hormone (AMH) levels and antral follicle count (AFC) were measured. The number of oocytes recovered, maturation rate and follicular output rate (FORT) were analysed according to patients' characteristics and breast cancer prognostic factors. RESULTS: Median age was 34 years (31.1-37.1). Median AFC and serum AMH level were 17 (12-26) follicles and 2 (1.2-3.4) ng/ml, respectively. After ovarian stimulation, 10.5 (6.0-16.0) oocytes were recovered, with eight (4-13) being mature. Mean oocyte maturation rate was 79% (62-92). Antral follicle count (>12) significantly affected the risk of recovering fewer than eight mature oocytes (P < 0.0001, multivariate analysis). Follicular responsiveness to FSH, assessed by the follicular output rate (FORT index) and number of oocytes recovered, were 31% (21-50) and 10.5% (6.0-16.0), respectively. FORT index and ovarian stimulation outcomes were not influenced by breast cancer prognostic factors. CONCLUSION: Breast cancer prognostic factors do not influence ovarian reserve markers or response to ovarian stimulation in fertility preservation. Therefore, tumour grade, triple-negative status, HER2 overexpression and high Ki67 should not alter the fertility-preservation strategy when considering ovarian stimulation for oocyte vitrification.

Protective Effect of Bio-Scaffold Against Vitrification Damage in Mouse Ovarian Tissue.

Ovarian tissue cryopreservation is regarded as useful method for fertility preservation. This study aimed to preserve most of the follicular reserve from the destructive effects of cryoprotectant solutions and liquid nitrogen. For this purpose, 48 female NMRI mice (8 weeks old) were randomly divided into six groups: Fresh (not vitrified), Vitrification (not encapsulated), Alginate 1 (encapsulated in 1% alginate hydrogel before placing in vitrification solutions), Alginate 2 (encapsulated in 1% alginate hydrogel before placing in liquid nitrogen), Aloe vera 1 (encapsulated in Aloe vera pieces before placing in vitrification solutions), Aloe vera 2 (encapsulated in Aloe vera pieces before placing in liquid nitrogen). After vitrification and warming, the histological evaluation showed that the average number of intact primordial follicles decreased significantly in all groups compared to the Fresh group. (P < 0.05). Results of evaluating the expression of apoptosis-related genes showed that the ratio of Bax/Bcl2 and P53 significantly decreased in the Alginate 2 group compared with the vitrification group. The level of Kit gene (KIT proto-oncogeni receptor tyrosine kinase gene) expression was either the same or lower in the experimental groups than in the vitrification group, but there was no statistically significant difference. Levels of tissue nitric oxide (NO) and malondialdehyde (MDA) in Alginate groups 1 and 2 showed a significant decrease compared with the vitrification group (P < 0.05). To conclude, Encapsulation of ovaries in 1% alginate hydrogel before immersion in liquid nitrogen may reduce the damage caused by cryopreservation.