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.

Vitrification of pig embryos dysregulates the microRNA transcriptome profile.

This study examined how the vitrification of pig blastocysts using either the superfine open pulled straw (SOPS) or Cryotop method affects the expression profile of embryonic microRNA (miRNA) transcriptomes, as well as its relation to changes in the expression of target genes (TGs). Surgically collected pig blastocysts were vitrified using either the SOPS method (n = 60; 4-6 embryos/device) or the Cryotop system (n = 60; 20 embryos/device). Embryos were cultured in vitro for 24 h after warming. Fresh blastocysts (n = 60) cultured for 24 h served as controls. After in vitro culture, five pools of eight viable blastocysts from each group were prepared for miRNA expression analysis based on a microarray approach. Then, biological interpretation of miRNAs profiles and integrative analysis of miRNA and mRNA transcriptome data were performed. Survival after 24 h of in vitro culture was similar (>96 %) for both the vitrification systems and the control group (100 %). Compared with the controls, the SOPS-vitrified blastocysts had 94 (one upregulated and 93 downregulated) differentially expressed (DE) miRNAs, and the Cryotop-vitrified blastocysts had 174 DE miRNAs (one upregulated and 173 downregulated). One DE miRNA (miR-503) in the SOPS group and three DE miRNAs (miR-7139-3p, miR-214 and miR-885-3p) in the Cryotop group were annotated for Sus scrofa. The integrative analysis showed that 27 and 61 DE TGs were regulated by the DE miRNAs in blastocysts vitrified with the SOPS and Cryotop systems, respectively. The TGs enriched one pathway (the TGF-ß signaling pathway) for the SOPS system and four pathways (HIF-1, Notch, ascorbate and aldarate metabolism and glycosphingolipid biosynthesis-ganglio series) for the Cryotop system. In summary, vitrification via the SOPS and Cryotop systems dysregulates miRNAs, with slight differences between methods. The altered miRNAs identified in this study were related mainly to cell proliferation, apoptosis, and the response to cell stress. Further studies are needed to clarify the consequences of dysregulation of miRNAs involved in the TGF-ß (SOPS-vitrified blastocyst) and Notch (Cryotop-vitrified blastocyst) signaling pathways, particularly if they can affect embryonic development.

Experimental observation of cavity-free ice-free isochoric vitrification via combined pressure measurements and photon counting x-ray computed tomography.

Isochoric (constant-volume or volumetrically confined) vitrification has shown potential as an alternative cryopreservation-by-vitrification technique, but the complex processes at play within the chamber are yet poorly characterized, and recent investigations have prompted significant debate around whether a truly isochoric vitrification process (in which the liquid remains completely confined by solid boundaries) is indeed feasible. Based on a recent thermomechanical simulation of a high-concentration Me2SO solution, Solanki and Rabin (Cryobiology, 2023, 111, 9-15.) argue that isochoric vitrification is not feasible, because differential thermal contraction of the solution and container will necessarily drive generation of a cavity, corrupting the rigid confinement of the liquid. Here, we provide direct experimental evidence to the contrary, demonstrating cavity-free isochoric vitrification of a ∼3.5 M vitrification solution by combined isochoric pressure measurement (IPM) and photon-counting x-ray computed tomography (PC-CT). We hypothesize that the absence of a cavity is due to the minimal thermal contraction of the solution, which we support with additional volumetric analysis of the PC-CT reconstructions. In total, this study provides experimental evidence both demonstrating the feasibility of isochoric vitrification and highlighting the potential of designing vitrification solutions that exhibit minimal thermal contraction.

A study of the stabilization and solidification of heavy metals in co-vitrification of medical waste incineration ash and coal fly ash.

Medical waste incineration ash (MWIA) has significant concentrations of heavy metals, dioxins, and chlorine that, if handled incorrectly, might cause permanent damage to the environment and humans. The low content of calcium (Ca), silicon (Si), and aluminum (Al) is a brand-new challenge for the melting technique of MWIA. This work added coal fly ash (CFA) to explore the effect of melting on the detoxication treatment of MWIA. It was found that the produced vitrification product has a high vitreous content (98.61%) and a low potential ecological risk, with an initial ash solidification rate of 67.38%. By quantitatively assessing the morphological distribution features of heavy metals in ashes before melting and molten products, the stabilization and solidification rules of heavy metals during the melting process were investigated. This work ascertained the feasibility of co-vitrification of MWIA and CFA. In addition, the high-temperature melting and vitrification accelerated the detoxification of MWIA and the solidification of heavy metals.

NDT-C11 as a viable novel detergent for single particle cryo-EM.

Single particle cryo electron microscopy (cryo-EM) is now the major method for the determination of integral membrane protein structure. For the success of a given project the type of membrane mimetic used for extraction from the native cell membrane, purification to homogeneity and finally cryo-grid vitrification is crucial. Although small molecule amphiphiles - detergents - are the most widely used membrane mimetic, specific tailoring of detergent structure for single particle cryo-EM is rare and the demand for effective detergents not satisfied. Here, we compare the popular detergent lauryl maltose-neopentyl glycol (LMNG) with the novel detergent neopentyl glycol-derived triglucoside-C11 (NDT-C11) in its behavior as free detergent and when bound to two types of multisubunit membrane protein complexes - cyanobacterial photosystem I (PSI) and mammalian F-ATP synthase. We conclude that NDT-C11 has high potential to become a very useful detergent for single particle cryo-EM of integral membrane proteins.

New Approach to the Cryopreservation of GV Oocytes and Cumulus Cells through the Lens of Preserving the Intercellular Gap Junctions Based on the Bovine Model.

Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.

Vitrification of human blastocysts for couples undergoing assisted reproduction: an updated review.

Over the past 40 years there has been a worldwide critical change in the field of assisted reproduction technology (ART), leading to the increased application of single blastocyst transfer, which is extremely important to avoid the risks of multiple pregnancy and associated complications for both mother and babies. Indeed, advancements in ART over the last few decades have been obtained thanks to several improvements, including ovarian stimulation, embryo culture conditions and, of course, progress in cryopreservation methods, especially with the application of vitrification. The ability to cryopreserve human embryos has improved significantly with vitrification compared to the initially adopted slow-freezing procedures. Since the introduction of vitrification, it has become the gold standard method to effectively cryopreserve human blastocysts. However, some new protocols are now being explored, such as the short warming procedure and even shorter exposure to the equilibration solution before vitrification, which seem to provide optimal results. Therefore, the main aim of the current narrative review, will be to illustrate the benefit of vitrification as an effective method to cryopreserve the human blastocyst and to illustrate new protocols and variations which in future may increase the performance of vitrification protocols.

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 were 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-thaw oocyte viability and development in many species, improving outcomes and allowing other deleterious effects of the cryopreservation cycle to be independently studied.

Vitamin C Synergistically Enhances Protective Effects of Vitamin E Against Preantral Follicle Degeneration of Ovine Vitrified/Warmed Ovarian Tissue.

This study aimed to evaluate whether the addition of vitamins E and C as two conventional antioxidants improves the cryotolerance of preantral follicles enclosed in ovine ovarian tissue slices. For this purpose, ovarian slices were obtained from abattoired juvenile lambs and randomly distributed to the following groups: fresh, toxicity, vitrified (control), and three treatment groups in two experiments. Vitamin E, vitamin C, or vitamin E + C was added to the vitrification media alone in the first experiment and added to all vitrification, warming, and culture media in the second experiment. Finally, the treated tissues were cultured in vitro for 12 hours. The histological analysis showed that single or combined use of vitamins E and C increases intact preantral follicles in comparison to the control in two experiments (p < 0.05), and simultaneous use of vitamins E and C had a synergistic effect on increasing the percentage of normal preantral follicles in experiment 2 (p < 0.05). Due to the better results in Experiment 2, stromal cell density, antioxidant activity, and molecular evaluation were followed only in this experiment. The vitamin E + C group had higher stromal cell density compared with control group (p < 0.05). Vitamin E strengthened antioxidant capacity compared with the control and vitamin C groups (p < 0.05). This effect was exacerbated when used in combination with vitamin C (p < 0.05). The expression of all evaluated genes (BMP4, BMP15, GDF9, and KITLG) was significantly increased in ovarian tissue treated with vitamin E + C compared with the control group (p < 0.05). This increase was also observed in BMP4, GDF9, and KITLG genes compared with the vitamin C group (p < 0.05). In conclusion, this study revealed the positive effects of vitamins E and C on preantral follicle viability and to some extent a synergistic action of vitamin C on the protective effects of vitamin E against preantral follicle degeneration and increasing antioxidant capacity and development of preantral follicles after ovine ovarian tissue vitrification.

Use of membrane transport models to design cryopreservation procedures for oocytes.

Oocyte cryopreservation is increasingly being used in reproductive technologies for conservation and breeding purposes. Further development of oocyte cryopreservation techniques requires interdisciplinary insights in the underlying principles of cryopreservation. This review aims to serve this purpose by: (1) highlighting that preservation strategies can be rationally designed, (2) presenting mechanistic insights in volume and osmotic stress responses associated with CPA loading strategies and cooling, and (3) giving a comprehensive listing of oocyte specific biophysical membrane characteristics and commonly used permeation model equations. It is shown how transport models can be used to simulate the behavior of oocytes during cryopreservation processing steps, i.e., during loading of cryoprotective agents (CPAs), cooling with freezing as well as vitrification, warming and CPA unloading. More specifically, using defined cellular and membrane characteristics, the responses of oocytes during CPA (un)loading were simulated in terms of temperature- and CPA type-and-concentration-dependent changes in cell volume and intracellular solute concentration. In addition, in order to determine the optimal cooling rate for slow programmable cooling cryopreservation, the freezing-induced cell volume response was simulated at various cooling rates to estimate rates with tolerable limits. For vitrification, special emphasis was on prediction of the timing of reaching osmotic tolerance limits during CPA exposure, and the need to use step-wise CPA addition/removal protocols. In conclusion, we present simulations and schematic illustrations that explain the timing of events during slow cooling cryopreservation as well as vitrification, important for rationally designing protocols taking into account how different CPA types, concentrations and temperatures affect the oocyte.

The walnut-derived peptide TW-7 improves mouse parthenogenetic embryo development of vitrified MII oocytes potentially by promoting histone lactylation.

BACKGROUND: Previous studies have shown that the vitrification of metaphase II (MII) oocytes significantly represses their developmental potential. Abnormally increased oxidative stress is the probable factor; however, the underlying mechanism remains unclear. The walnut-derived peptide TW-7 was initially isolated and purified from walnut protein hydrolysate. Accumulating evidences implied that TW-7 was a powerful antioxidant, while its prospective application in oocyte cryopreservation has not been reported. RESULT: Here, we found that parthenogenetic activation (PA) zygotes derived from vitrified MII oocytes showed elevated ROS level and delayed progression of pronucleus formation. Addition of 25 µmol/L TW-7 in warming, recovery, PA, and embryo culture medium could alleviate oxidative stress in PA zygotes from vitrified mouse MII oocytes, furtherly increase proteins related to histone lactylation such as LDHA, LDHB, and EP300 and finally improve histone lactylation in PA zygotes. The elevated histone lactylation facilitated the expression of minor zygotic genome activation (ZGA) genes and preimplantation embryo development. CONCLUSIONS: Our findings revealed the mechanism of oxidative stress inducing repressed development of PA embryos from vitrified mouse MII oocytes and found a potent and easy-obtained short peptide that could significantly rescue the decreased developmental potential of vitrified oocytes, which would potentially contribute to reproductive medicine, animal protection, and breeding.

The Application of Mean Number of DNA Breakpoints in Sperm Cryopreservation.

Growing concerns over declining male semen quality and rising infertility have shifted attention to male fertility. Sperm cryopreservation emerges as a crucial tool in preserving male fertility, especially for patients who need proactive preservation, such as cancer patients before undergoing radiation or chemotherapy. Although cryopreservation does not directly address infertility, effective preservation can support future fertility. However, the process may compromise sperm DNA integrity. Despite their impairment, damaged sperm often retain vitality and may still have the potential to fertilize an egg. Nonetheless, if damaged sperm fertilize an egg, excessive DNA damage could impede embryo implantation and development, despite the egg's repair capabilities. Consequently, precise detection of sperm DNA damage is crucial and urgent. To better address the issue of sperm DNA damage detection, we have introduced a novel fluorescence biosensor technology known as the TDT/SD Probe. This technology utilizes terminal deoxynucleotidyl transferase (TdT) and strand displacement probes to accurately detect the number of sperm DNA breakage points during the cryopreservation process. Experimental results reveal that the number of sperm DNA breakpoints significantly increases after both sperm vitrification (8.17×105) and conventional slow freezing (10.80×105), compared to the DNA breakpoints of fresh semen samples (5.19×105). However, sperm vitrification has the least impact on sperm breakage points. This research provides innovative means for further optimizing sperm preservation techniques by offering a novel DNA damage detection method, enabling more precise assessment of sperm DNA damage during the freezing process.

Vitrification of medaka whole testis with a trehalose-containing solution and production of medaka individuals derived from the vitrified cells.

The cryopreservation of teleost eggs and embryos remains challenging, and there are no previous reports that demonstrate successful cryopreservation in medaka (Oryzias latipes). We have reported egg and sperm production, followed by the generation of donor-derived offspring by transplanting vitrified whole testes-derived testicular cells into surrogate fish. The vitrification solutions contained ethylene glycol, sucrose, and ficoll. In this study, we replaced sucrose with trehalose in the vitrification solution and medaka whole testis was vitrified with the solution. The post-vitrification survival (72.8±3.5%) was markedly improved compared with that achieved using the sucrose-containing solution (44.7±4.2%). Moreover, we demonstrated the production of eggs, sperm, and donor-derived offspring from testicular cells transplanted into surrogate recipients. The phenotype of donor-derived offspring was identical to that of transplanted testicular cells. These findings suggest that trehalose is effective for the vitrification of medaka whole testis and can be considered an effective and reliable method for the long-term preservation of their genetic resources.

Micropropagation and Shoot Tip Cryopreservation of 'Sunny Gold' Freesia.

Cryopreservation is a promising method for the long-term preservation of plant germplasm, especially for vegetatively propagated species like freesias. In this study, we investigate streamlining the cryopreservation process for 'Sunny Gold' Freesia, starting from effective in vitro initiation and proliferation using various plant growth regulator combinations. We also assess the impact of subculture on regrowth rates after cryopreservation. The shoot tips were successfully initiated in vitro after sterilization. The shoots were multiplied an average of three times in media containing N6-benzyladenine and kinetin. The regrowth rates of non-cryopreserved shoot tips excised from different subculture cycles did not differ significantly, with rates of 44% observed for plants from more than five subcultures and 47% for those from three subcultures. However, only the shoot tips excised from cultures subjected to three subculture cycles were able to recover after cryopreservation, with a regrowth rate of 31%. Our findings lay the groundwork for the development of an efficient cryopreservation protocol for freesias in the future.

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.

Developing and validating a prediction model of live birth following single vitrified-warmed blastocyst transfer.

RESEARCH QUESTION: Can the developed clinical prediction model offer an accurate estimate of the likelihood of live birth, involving blastocyst morphology and vitrification day after single vitrified-warmed blastocyst transfer (SVBT), and therefore assist clinicians and patients? STUDY DESIGN: Retrospective cohort study conducted at a Spanish university-based reproductive medicine unit (2017-2021) including consecutive vitrified-warmed blastocysts from IVF cycles. A multivariable logistic regression incorporated key live birth predictors: vitrification day, embryo score, embryo ploidy status and clinically relevant variables, i.e. maternal age. RESULTS: The training set involved 1653 SVBT cycles carried out between 2017 and 2020; 592 SVBT cycles from 2021 constituted the external validation dataset. The model revealed that female age and embryo characteristics, including overall quality and blastulation day, is linked to live birth rate in SVBT cycles. Stratification by vitrification day and quality (from day-5A to day-6 C blastocysts) applied to genetically tested and untested embryos. The model's area under the curve was 0.66 (95% CI 0.64 to 0.69) during development and 0.65 (95% CI 0.61 to 0.70) in validation, denoting moderate discrimination. Calibration plots showed strong agreement between predicted and observed probabilities. CONCLUSION: By incorporating essential predictors such as vitrification day, embryo morphology grade, age and preimplantation genetic testing for aneuploidy usage, this predictive model offers valuable guidance to clinicians and patients, enabling accurate forecasts of live birth rates for any given vitrified blastocyst within SVBT cycles. Additionally, it serves as a potentially indispensable laboratory tool, aiding in selecting the most promising blastocysts for optimal outcomes.

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.

Effect of post-vitrification cryopreservation duration on singleton birth-weight in frozen-thawed blastocysts transfer cycles.

Introduction: This study aimed to explore the effect of cryopreservation duration after blastocyst vitrification on the singleton birth-weight of newborns to assess the safety of long-term preservation of frozen-thawed blastocyst transfer (FBT) cycles. Methods: This was a retrospective observational study conducted at the Gynecological Endocrinology and Assisted Reproduction Center of the Peking Union Medical College Hospital. Patients who gave birth to singletons between January 2006 and December 2021 after undergoing FBT cycles were included. Five groups were formed according to the duration of cryopreservation of embryos at FBT: Group I included 274 patients with a storage time < 3 months. Group II included 607 patients with a storage time of 3-6 months. Group III included 322 patients with a storage time of 6-12 months. Group IV included 190 patients with a storage time of 12-24 months. Group V included 118 patients with a storage time of > 24 months. Neonatal outcomes were compared among the groups. Multivariate linear regression analysis was performed to evaluate birth-weights and other birth-related outcomes. Results: A total of 1,511 patients were included in the analysis. The longest cryopreservation period was 12 years. The birth-weights of neonates in the five groups were 3344.1 ± 529.3, 3326.1 ± 565.7, 3260.3 ± 584.1, 3349.9 ± 582.7, and 3296.7 ± 491.9 g, respectively (P > 0.05). The incidences of preterm birth, very preterm birth, low birth-weight, and very low birth-weight were similar in all groups (P > 0.05). The large-for-gestational-age and small-for-gestational-age rates did not differ significantly among the groups (P > 0.05). After adjusting for confounding factors that may affect neonatal outcomes, a trend for an increased risk of low birth-weight with prolonged cryopreservation was observed. However, cryopreservation duration and neonatal birth-weight were not significantly correlated (P > 0.05). Conclusion: The duration of cryopreservation after blastocyst vitrification with an open device for more than 2 years had no significant effect on the birth-weight of FBT singletons; however, attention should be paid to a possible increase in the risk of low birth-weight.