Vitrification Procedure Research Articles

P-749 Maternal and obstetric outcomes following the transfer of embryos warmed with fatty acid supplemented solutions

Abstract Study question Does fatty acid (FA) supplementation into warming solutions influence maternal and obstetric outcomes after single vitrified-warmed cleavage stage embryo transfer (SVCT)? Summary answer FA-supplemented warming solutions improve the live birth rate after SVCTs without exerting any adverse effects on maternal and obstetric outcomes. What is known already Vitrification procedures decrease the intracellular lipid content and impair developmental competence. Adding fatty acids (FAs) to the warming solution has been shown to recover the lipid content of the cytoplasm and improve developmental competence and clinical pregnancy by stimulating the beta-oxidation pathway in warmed embryos. Recently, the FA-supplemented warming solutions have been commercially released. However, influence of the FA-supplemented supplementation on live birth rate after embryo transfers and perinatal outcomes remains unknown. Study design, size, duration The clinical records of 701 treatment cycles in 701 women who underwent SVCTs were retrospectively analysed. Vitrified embryos were warmed using solutions (from April 2022 to June 2022, control group, n = 340) or FA-supplemented solutions (from July 2022 to September 2022, FA group, n = 361). All pregnant women were invited to respond to the questionnaire at 9 weeks of gestation and after delivery. Maternal and obstetric outcomes were obtained from a self-reported questionnaire completed by the patients. Participants/materials, setting, methods The embryos in the FA group were warmed with the FA-supplemented solutions (VT526, Kitazato Corporation) while the embryos in the control group were warmed with the non-supplemented solutions (VT506, Kitazato Corporation). SVCTs were performed on day 2 after ovulation in natural cycles. The live birth rate, incidence of pregnancy complications, and neonatal outcomes were compared between the control and FA groups. Main results and the role of chance The live birth rate was higher in the FA group than in the control group (P = 0.0399). The miscarriage rates were comparable between the groups. Multivariate logistic regression analysis also demonstrated a higher probability of live births in the FA group than in the control group (adjusted odds ratio, 1.46; P = 0.0404). Patients were stratified by median maternal age for further analysis (37 years). This subgroup analysis showed that the live birth rate was significantly higher in the FA group than in the control group in women younger than 37 years of age. However, the live birth rate was comparable between the two groups for women aged 37 years or older. The incidence and types of pregnancy complications were comparable between the control and FA groups. Furthermore, caesarean section rates were similar between the groups. The average gestational age and incidence of preterm delivery were comparable between the groups. Birth length and weight, incidence of low birth weight, incidence of small for gestational age and large for gestational age, and infant sex were comparable between the groups. The incidence of birth defects was similar between the groups. Multivariate logistic regression analysis revealed a similar trend. Limitations, reasons for caution The single-centre retrospective study limits the strength of the study, necessitating further multicentre studies to ascertain the generalizability of these findings to other clinics with different protocols and/or patient demographics. Furthermore, the number of perinatal cycles was low. Wider implications of the findings The FA-supplemented warming solutions improved the live birth rates after SVCTs, particularly in young patients. Furthermore, the incidence of pregnancy complications and infant health was not affected by the FA-supplemented solutions. Therefore, FA-supplemented solutions can be considered safe and effective at improving clinical outcomes and reducing the patient burden. Trial registration number not applicable

Three live births after human embryo vitrification with the use of aluminum oxide as an intermediate cooling agent: a case report

To study the possibility of increasing the cooling rates of the vitrification procedure in a closed system with the use of aluminum oxide as an intermediate coolant. Case report. Six patients undergoing procedures for assisted reproduction. Comparative studies of cryopreservation of donor embryos with aluminum oxide as an intermediate cooling agent (experimental group) and without it (control group) have been performed. After thawing, the embryo morphology and its potential to develop to the blastocyst stage have been assessed. The methodology was then applied to clinical practice. Twenty embryos of 6 patients have been vitrified on day 4 after fertilization with the use of aluminum oxide as an intermediate coolant. Fourteen of them have been thawed. All have displayed normal morphology and 10 have formed blastocysts after 24 hours of culture. Four of the patients received embryo transfer with 2 embryos and the other 2 with single embryos. After preliminary comparative studies of embryos frozen with aluminum oxide and a control group, the results showed no statistically significant difference between their quality and potential to reach to blastocyst stage. That gave us ground to apply the methodology in clinical practice. After the embryo transfer, 3 clinical pregnancies with successful live births have been obtained. Our experience shows that preimplantation embryos can be cryopreserved aseptically, in closed systems, with the help of aluminum oxide as an intermediate coolant.

Comparative effects of a calcium chelator (BAPTA-AM) and melatonin on cryopreservation-induced oxidative stress and damage in ovarian tissue

Oncology treatments cause infertility, and ovarian tissue cryopreservation and transplantation (OTCT) is the only option for fertility preservation in prepubertal girls with cancer. However, OTCT is associated with massive follicle loss. Here, we aimed to determine the effect of supplementation of slow freezing and vitrification media with BAPTA-AM and melatonin alone and in combination on ovarian tissue viability, reactive oxygen species (ROS) levels, total antioxidant capacity (TAC), and follicular morphology and viability. Our results indicated that BAPTA-AM and melatonin can significantly improve ovarian tissue viability and the TAC/ROS ratio and reduce ROS generation in frozen-thawed ovarian tissues in slow freezing and vitrification procedures. BAPTA-AM was also found to be less effective on TAC compared to melatonin in vitrified ovarian tissue. While supplementation of slow freezing and vitrification media with BAPTA-AM and/or melatonin could increase the percentage of morphologically intact follicles in cryopreserved ovarian tissues, the differences were not significant. In conclusion, supplementation of cryopreservation media with BAPTA-AM or melatonin improved the outcome of ovarian tissue cryopreservation in both vitrification and slow freezing methods. Our data provide some insight into the importance of modulating redox balance and intracellular Ca2+ levels during ovarian tissue cryopreservation to optimize the current cryopreservation methods.

#334 : Tackling Complex Infertility Patient: A Case Report

Background: Infertility is a common problem presented in women with endometriosis especially as it is often associated with poor quality of life. Therefore, surgical Intervention has been considered as the first-line treatment of choice. On the other hand, surgery itself has detrimental effects on ovarian reserve and is associated with ovarian failure. Case Report: We present a case of a 39-year-old woman para 1 with multiple myoma uteri, bilateral endometriosis cyst, left hydrosalpinx, deep infiltrating endometriosis (DIE), and adhesion (Figure 1). She comes with a history of heavy abnormal uterine bleeding, lower abdominal pain, and unable to conceive 11 years after their first child despite adequate unprotected intercourse. The patient was anemic (Hemoglobin 7.30 g/dL) with low Anti Mullerian Hormone (AMH; 0.26 mg/ml). We decided to perform laparoscopic uterine myoma with multiple bilateral endometrial cysts, left salpingectomy, and adhesiolysis (Figure 2). Patient then underwent an ultra-long protocol with three cycles of leuprolide acetate 3.75 mg. Ovum pick-up was performed and we retrieved five M-II oocytes and from embryo culture, two embryos with good and moderate quality were collected for vitrification procedure. Prior frozen embryo transfer, extra acetylsalicylic acid 80 mg once a day and estradiol valerate 4 mg thrice a day were given for 18 days. The patient soon became pregnant after a few weeks of embryo transfer with βHCG of 185 mIU/m. Conclusion: Individualized treatment plans based on the patient’s symptoms and respective reproductive goals are important points to be discussed in endometriosis-associated infertility patients. Surgical intervention before IVF/ICSI is deemed as necessary to preserve ovarian reserve and to restore normal anatomy to increase the success rate of embryo implantation.

#208 : The Adverse Effects of Vitrification on Embryo Development and Metabolic Health in Mice Offspring

Background and Aims: Embryo vitrification is widely applied as a routine procedure of assisted reproductive technology. Cohort studies showed frozen embryo transfer (FET) was associated with an elevated risk of adverse maternal and neonatal outcomes compared with fresh embryo transfer. This study aimed to explore the effects of blastocyst vitrification on embryo development and long-term metabolic health in mice. Method: Mitochondrial function, apoptotic levels and cell numbers were examined in blastocysts with/without vitrification. Implantation rates, fetal growth and placentation were analysed after fresh and frozen blastocyst transfer. Energy consumptions were measured by indirect calorimetry. Metabolic tests were performed in adult mice offspring, including glucose tolerance, insulin tolerance and pyruvate tolerance. Serum lipid profiles were examined, and fetal and adult hepatic transcriptome status were detected by RNA-sequencing and qRT-PCR. Results: Mitochondrial dysfunction increased apoptotic levels and decreased cell numbers were observed in vitrified blastocysts. The implantation and live pup rates on embryonic day18.5 were not significantly different between the fresh and frozen groups. However, fetal weights and placental efficiencies such as the ratio of labyrinth layer/ junctional zone and microvascular density were significantly reduced after FET. Offspring aged 36 weeks in the FET group had decreased whole energy consumption, impaired glucose and pyruvate tolerance, insulin resistance, higher serum triglyceride, low-density lipoprotein and total cholesterol levels compared with the fresh group. However, these metabolic effects were not observed in mice offspring aged 14 weeks. The FET group also had altered hepatic gene expression and signaling pathways related to metabolic regulation, apoptotic cell clearance, and regulation of response to reactive oxygen species etc. Conclusion: The data indicated that blastocyst vitrification has short-term and long-term adverse effects in the mouse model. Further studies are needed to optimize vitrification procedure.

#67 : Fatty Acid Supplementation into Warming Solutions Improves Outgrowth Competence Human Vitrified Blastocysts

Background and Aims: The vitrification procedure decreased intracellular lipid content and subsequent developmental competence. Furthermore, a recent study reported that fatty acid (FA) supplementation into warming solutions increased intracellular lipid content and improved developmental competence by stimulating the beta-oxidation pathway in mice and bovines. However, in humans, the effects of FA addition to warming solutions on the viability of vitrified blastocysts remain unclear. In this study, we examined the implantation competence of blastocysts warmed by using the FA-supplemented solutions by comparing the in vitro outgrowth. Method: A total of 216 discarded human vitrified blastocysts donated for research by consenting couples were randomly allocated, to be warmed in solutions either with (FA, n = 98) or without FA (control, n = 118). Vitrified blastocysts were cultured for 120 min after warming. The blastocysts were plated on fibronectin-coated dishes and cultured for 96 h to assess the blastocyst competency for adhesion and outgrowth. Results: The embryo characteristics were comparable between the control and FA groups. The adhesion rate at 96 h after the outgrowth culture in the FA group (74.5%) was comparable with that in the control group (74.6%). The 1.3-fold increase in the outgrowth area was observed in the FA group, compared to the control group ([Formula: see text] = 0.0371). Furthermore, the sub-group analysis demonstrated that a more beneficial effect of FA supplementation on blastocyst outgrowth was observed when the trophectoderm was evaluated as grade A ([Formula: see text] = 0.0065). Conclusion: The FA supplementation into warming solutions increased the outgrowth competence of vitrified human blastocysts. These results suggest that the pregnancy outcomes, including the implantation rate, after frozen blastocyst transfers would be improved by using the FA-supplemented solutions.

Cryopreservation of rat embryos at all developmental stages by small-volume vitrification procedure and rapid warming in cryotubes

Intracellular ice formation during cryopreservation is lethal to the cell, including during warming. Here, we examined the effect of sample volume and warming rate on the cryopreservation success of 1-cell rat embryos based on successful development into blastocysts in vitro and to term in vivo following embryo transfer. Embryos were equilibrated in 5% propylene glycol solution for 10 min, held for 40 s at 0 °C in cryopreservation solution (5%PG + PEPeS), and cooled by immersion in liquid nitrogen. When 1-cell embryos were cryopreserved in a volume of 30–100 μL at a cooling rate of 5830–7160 °C/min and warmed at 35,480–49,400 °C/min by adding 1 mL of 0.3 M sucrose solution at 50 °C, 17.3–28.8% developed into blastocysts, compared with 57.0% of untreated embryos. However, when 1-cell embryos were cryopreserved in a smaller volume of 15 μl at 7950 °C/min and warmed at 68,850 °C/min, 58.8 ± 10.6% developed into blastocysts and 50.0 ± 7.4% developed to term, comparable to that of non-treated embryos (57.0 ± 5.4% and 51.4 ± 3.1%, respectively). Cryopreserved embryos at other developmental stages also showed high in vitro culture potential similar to that of the control. Using a conventional cryotube and a small-volume vitrification procedure with rapid warming, we achieved high levels of subsequent rat embryonic development at all developmental stages.

Robotic denudation of zygotes

The vitrification technology is used in the embryo freezing process. To ensure intracellular water removal and avoid ice crystal formation, the cumulus cells surrounding zygotes need to be removed. As operators have varying skill and long-time fatigue, it is challenging to completely remove the cumulus cells surrounding zygotes and reduce zygote loss inside the micropipette. The objective of this work is to develop a robotic zygote denudation system for the vitrification procedure. The robotic system enables accurate segmentation of cumulus cells for estimating the mass of cumulus zygote complexes (CZCs) inside the micropipette via the Res-Unet neural network. A mathematical model was built to describe the dynamic motion of CZCs inside the micropipette, and a model-based optimal controller was developed to aspirate and deposit CZCs inside the micropipette. To denude zygote completely, the Res-Attention neural network was used to detect cumulus cells for predicting the quality of zygote denudation. In the mouse zygotes experiments, the yield rate is 98.2% ± 1.6% and the denudation efficacy is 96.9% ± 0.2% for the robotic system. Compared with manual denudation, the survival rate and development rate of embryos cultured from zygotes denuded by the robotic system were both higher in subsequent vitrification procedure.

Post-thaw viability of mouse preantral follicles after cryopreservation with cryotube freezing and OPS vitrification procedures.

In the field of reproductive science, there is an increased interest in the application of ovarian preantral follicles. Since the ovary contains a great amount of preantral follicles (PAF), the cryopreservation and in vitro culture of such follicles support the fertility preservation of domestic animals with high genetic value, endangered or zoo animals, and women before anticancer therapy. To date, no standard freezing or vitrification protocol is available in human or animals. The aim of the present study was to examine the viability of preantral follicles cryopreserved using freezing or vitrification protocols: cryotube freezing or OPS vitrification.

Effect of α-tocopherol in the vitrification medium on the viability, lipid peroxidation, expression of key developmental, apoptotic and stress-related genes in ovine secondary follicles.

The present study aimed to evaluate the effect of α-tocopherol on viability, lipid peroxidation and the expression of apoptosis, stress and development-related genes in the vitrified sheep secondary follicles. Ovarian secondary follicles (200-300 μm) were isolated and distributed separately to the vitrification treatment and supplemented with 5 mM, 10 mM, 20 mM and 30 mM of α-tocopherol (while the control fresh group was without vitrification and supplementation of α-tocopherol). After a week, the follicles were thawed and evaluated for follicular viability by trypan blue dye exclusion method, lipid peroxidation and gene expression studies. The results showed that the vitrification with 10 and 20 mM of α-tocopherol positively affected (p < .05) the viability of vitrified follicles in comparison with vitrified ones without α-tocopherol but the higher concentration of α-tocopherol, i.e., 30 mM negatively affected the viability (p < .05) in comparison with the 10 and 20 mM of α-tocopherol groups. The malondialdehyde (MDA) levels were significantly (p < .05) higher in the vitrified without α-tocopherol group in comparison to the vitrified with 20 mM of α-tocopherol group. The expression of apoptotic-related gene, BCL2L1 was significantly higher in 10 mM α-tocopherol group compared to the control fresh and CASPASE 3, 9 expressions were significantly higher in the vitrified group when compared to the vitrified with 10 mM α-tocopherol group. Expressions of BAX, BAD, BAK, BMP-15 and GDF-9 showed no significant difference among the groups. The mRNA expression of SOD1 was significantly higher in the vitrified without α-tocopherol group when compared to other groups. We conclude that the supplementation of 10 and 20 mM α-tocopherol in vitrification solution was the efficient vitrification procedure for the vitrification of ovine secondary follicles.

Revealing nanoscale structure and interfaces of polymer and protein condensates via cryo-electron microscopy.

Liquid-liquid phase separation of macromolecules is a ubiquitous process studied across biological and material sciences. In one instance, phase separation of proteins can be driven by low complexity intrinsically disordered regions that permit multivalent interactions between polypeptides. In synthetic systems, a complexation of oppositely charged polyelectrolytes or the immiscibility of two blocks can drive phase separation of polyelectrolytes and block copolymers respectively. The result of these processes are condensates that display liquid-like characteristics. The nature of these condensates has mainly been investigated through methods based on optical microscopy, such as assessing condensate morphology, fusion dynamics, and diffusion of molecules. Importantly, current methods used to study condensates cannot probe the nanoscale structure of the material. Here, we developed a method that applies cryo-electron microscopy (EM) to investigate the internal structure of condensates in vitro at the nanoscale. By bypassing the drying of material for conventional EM, we preserve the internal organization of molecules. We tested a range of material preparation protocols, including varying the temperature, concentration, and buffer conditions of the solution, along with vitrification procedures. We are able to produce both micron-sized spherical droplets and thin films of condensed material that are amenable to cryo-EM imaging. We applied this method to study condensates composed of intrinsically disordered proteins, polyelectrolytes and block copolymers. Our results reveal the internal organization of the different materials at the nanoscale. Furthermore, we relate the material properties of condensates composed of intrinsically disordered proteins to their nanoscale structure. We anticipate the use of cryo-EM beyond single particle analysis to understand phase separation of intrinsically disordered proteins and other macromolecules.

Systematic review and meta-analysis on patented and non-patented vitrification processes to ovarian tissue reported between 2000 and 2021.

Due to the great interest in ovarian cryopreservation and, consequently conservation and restoration of female fertility in the last decades, different vitrification procedures (vitrification devices or solutions) have been developed, patented, and used both for academic research purposes and for clinical use. Therefore, the present study aimed to provide a systematic review and meta-analysis of data obtained from the application of different patented and non-patented vitrification devices and solutions in different countries. For this purpose, relevant observational studies published between the years 2000 to 2021 were selected to verify the efficiency of ovarian vitrification processes on parameters such as morphology, viability, and apoptosis in preantral ovarian follicles after transplantation or in vitro culture. Our research revealed that, although several countries were considered in the study, the United States and Japan were the countries that registered the most processes, and 22 and 16 vitrification devices and solutions out of a total of 51, respectively were patented. Sixty-two non-patented processes were also considered in the study in all countries. We also observed that transplantation and in vitro ovarian culture were the techniques predominantly used to evaluate the efficiency of the devices and vitrification solutions, respectively. In conclusion, this review showed that patented or non-patented protocols available in the literature are able to successfully preserve preantral follicles present in ovarian tissue. Despite the satisfactory results reported so far, adjustments in ovarian vitrification protocols in order to minimize cryoinjuries to the follicles remain one of the goals of cryopreservation and preservation of the female reproductive function. We found that vitrification alters the morphology and viability, and offers risks leading in some cases to follicular apoptosis. However, adjustments to current protocols to develop an optimal procedure can minimize damage by not compromising follicular development after vitrification/warming.

Potential Development of Vitrified Immature Human Oocytes: Influence of the Culture Medium and the Timing of Vitrification

How does the in vitro maturation (IVM) medium and the vitrification procedure affect the survival of germinal vesicle (GV) oocytes obtained from stimulated cycles and their development to the blastocyst stage? In total, 1085 GV human oocytes were obtained after women underwent a cycle of controlled ovarian stimulation, and these oocytes were subjected to IVM before or after their vitrification. IVM was carried out in two commercial culture media not specifically designed for maturation. MII oocytes were then activated and embryo development until day 6 was evaluated. According to the results, a higher percentage of oocytes reach the MII stage if they are vitrified before they undergo IVM. Nevertheless, the medium used and the sample size determine whether these differences become significant or not. Similar survival rates and development to blastocysts were observed in all the conditions studied.

Epididymal Spermatozoa Show Higher Cryoresistance to Vitrification Process Than Ejaculated Spermatozoa in Dogs.

The aim of this study was to investigate differences in the sperm response to a vitrification-warming process between ejaculated and epididymal dog spermatozoa, and to evaluate the efficacy of an animal protein-free extender for vitrification of both types of sperm cells. Vitrified-warmed spermatozoa from the epididymis showed greater (p < 0.001) progressive motility and total motility values than ejaculated spermatozoa, regardless of the diluent. The vitrification procedure returned better results for viability and intact acrosome when human tubal fluid (HTF®) was used (25.10 ± 7.90 and 56.50 ± 6.7, respectively) compared with Tris-Citric acid-Glucose (TCG) (15.20 ± 4.70 and 43.70 ± 7.9, respectively) in ejaculated samples. Similarly, higher total motility (34.5 ± 4.5) was observed in HTF postwarmed samples compared with TCG-treated samples (19.52 ± 5.1). The interaction source (epididymis, ejaculated) × extender had a significant effect (p < 0.001) on the values of total motile spermatozoa after warming. HTF-based extender improved (p < 0.001) total motility values in epididymal samples, but not in ejaculated samples. In conclusion, epididymal spermatozoa show higher cryoresistance to the vitrification process than ejaculated spermatozoa in dogs. The use of HTF is adequate for both ejaculated and epididymal canine sperm vitrification.

Quality assurance (QA) for monitoring the performance of assisted reproductive technology (ART) staff using artificial intelligence (AI).

Deep learning neural networks have been used to predict the developmental fate and implantation potential of embryos with high accuracy. Such networks have been used as an assistive quality assurance (QA) tool to identify perturbations in the embryo culture environment which may impact clinical outcomes. The present study aimed to evaluate the utility of an AI-QA tool to consistently monitor ART staff performance (MD and embryologist) in embryo transfer (ET), embryo vitrification (EV), embryo warming (EW), and trophectoderm biopsy (TBx). Pregnancy outcomes from groups of 20 consecutive elective single day 5 blastocyst transfers were evaluated for the following procedures: MD performed ET (N = 160 transfers), embryologist performed ET (N = 160 transfers), embryologist performed EV (N = 160 vitrification procedures), embryologist performed EW (N = 160 warming procedures), and embryologist performed TBx (N = 120 biopsies). AI-generated implantation probabilities for the same embryo cohorts were estimated, as were mean AI-predicted and actual implantation rates for each provider and compared using Wilcoxon singed-rank test. Actual implantation rates following ET performed by one MD provider: "H" was significantly lower than AI-predicted (20% vs. 61%, p = 0.001). Similar results were observed for one embryologist, "H" (30% vs. 60%, p = 0.011). Embryos thawed by embryologist "H" had lower implantation rates compared to AI prediction (25% vs. 60%, p = 0.004). There were no significant differences between actual and AI-predicted implantation rates for EV, TBx, or for the rest of the clinical staff performing ET or EW. AI-based QA tools could provide accurate, reproducible, and efficient staff performance monitoring in an ART practice.

Vitrification of the ovarian tissue in sturgeons

The aim of this study was to test whether vitrification of sterlet Acipenser ruthenus and Russian sturgeon Acipenser gueldenstaedtii ovarian tissue through needle-immersed vitrification (NIV) is an efficient strategy for the preservation of oogonia (OOG) in order to supplement the current conservation efforts for these endangered fish species. Histological analyses of the gonads displayed that the ovaries of both species were immature and contained predominantly OOG and primary oocytes. The germline origin of these cells was verified by localization of the vasa protein through immunocytochemistry. NIV protocol was optimized by testing different equilibration (ES) and vitrification solutions (VS) containing various concentrations of dimethyl sulfoxide (Me2SO), propylene glycol (PG) or methanol (MeOH). In sterlet, the highest average viability (55.7 ± 11.5%) was obtained by using a combination of 1.5 M PG and 1.5 M Me2SO in the ES, and 1.5 M MeOH and 5.5 M Me2SO in the VS. In Russian sturgeon, the highest average viability (49.4 ± 17.1%) was obtained by using a combination of 1.5 M MeOH and 1.5 M Me2SO in the ES, and 3 M PG and 3 M Me2SO in the VS. To test whether vitrified/warmed OOG are functional, we have conducted an intra-specific transplantation assay to verify whether transplanted sterlet OOG will colonize the gonads of recipient fish. Fluorescently labelled cells were detected within recipient gonads at 2 and 3 months post-fertilization (mpf). Colonization rates of vitrified/warmed OOG (70% at 2 mpf and 61% at 3 mpf) were similar to those of fresh OOG (80% at 2 mpf and 70% at 3 mpf). This study has demonstrated that vitrification of ovarian tissue is an effective method for the preservation of OOG, and that the vitrified/warmed cells are functional and are able to colonize recipient gonads after transplantation similarly to the fresh cells. Since the vitrification procedure displayed in this study is simple and does not require complex and expensive laboratory equipment, it can be readily applied in field conditions, and therefore it can be invaluable for the conservation efforts of the critically endangered sturgeon species. However, care needs to be taken that despite the research conducted so far, donor-derived progeny was not yet obtained in sturgeons.

Apoptosis and glucocorticoid-related genes mRNA expression is modulated by coenzyme Q10 supplementation during in vitro maturation and vitrification of bovine oocytes and cumulus cells

Oocyte in vitro maturation (IVM) and vitrification procedures lead to detrimental effects on the overall oocyte quality. The addition of antioxidants during IVM, such as the coenzyme Q10 (Q10), has been demonstrated to positively impact on the cumulus-oocyte complexes due to its role in protection from oxidative damage and modulating gene transcription. Furthermore, glucocorticoids (GC) regulate gene transcription, energy metabolism and apoptosis during the early steps of reproduction. In this sense, most GC actions are mediated by the glucocorticoid receptor (NR3C1), a transcription factor. However, the specific roles of GC in ovarian physiology and oocyte maturation are still unknown. In this regard, a better knowledge on the expression of GC-related and apoptosis-related genes during IVM and cryopreservation procedures could potentially benefit the refinement of assisted reproductive techniques in the bovine species. The present study aims to explore the expression of NR3C1 mRNA in fresh and vitrified bovine oocytes and cumulus cells in response to Q10 (50 μM), and the effect of cortisol addition (0.25 μM, 0.5 μM) on the expression of NR3C1. We also studied the mRNA expression of NR3C1-related genes belonging to the GC regulation pathway, such as hydroxysteroid dehydrogenases (HSD11B1; HSD11B2), immunophilins (FKBP4; FKBP5), signal transducers and activators of transcription (STAT3; STAT5A), the mineralocorticoid receptor (NR3C2), and to the apoptosis pathway, such as the anti- (BCL2) and pro-apoptotic (BAX) mRNA transcripts in oocytes and cumulus cells 1) after IVM, and 2) after vitrification, both in presence or absence of Q10 supplementation during IVM. Our results show that there is an increase in the NR3C1 receptor expression after vitrification of oocytes, but not after exogenous cortisol supplementation during IVM. In addition, Q10 reduces the mRNA expression of HSD11B1 and FKBP5 in oocytes at levels of immature oocytes (HSD11B1 mRNA expression also in cumulus cells), and the BAX:BCL2 ratio mRNA expression. After vitrification in the presence of Q10, HSD11B2 mRNA expression increases in cumulus cells, while HSD11B1 and BAX:BCL2 mRNA expression decreases significantly both in oocytes and cumulus cells. In conclusion, our results show for the first time the effect of IVM, vitrification and Q10 supplementation on the mRNA relative expression of GC-related and apoptosis genes, and the effect of vitrification in the protein expression of NR3C1.

Developing Graphene Grids for Cryoelectron Microscopy.

Cryogenic electron microscopy (cryo-EM) single particle analysis has become one of the major techniques used to study high-resolution 3D structures of biological macromolecules. Specimens are generally prepared in a thin layer of vitrified ice using a holey carbon grid. However, the sample quality using this type of grid is not always ideal for high-resolution imaging even when the specimens in the test tube behave ideally. Various problems occur during a vitrification procedure, including poor/nonuniform distribution of particles, preferred orientation of particles, specimen denaturation/degradation, high background from thick ice, and beam-induced motion, which have become important bottlenecks in high-resolution structural studies using cryo-EM in many projects. In recent years, grids with support films made of graphene and its derivatives have been developed to efficiently solve these problems. Here, the various advantages of graphene grids over conventional holey carbon film grids, functionalization of graphene support films, production methods of graphene grids, and origins of pristine graphene contamination are reviewed and discussed.

P-267 A continuous culture medium with a lower concentration of lactate has a pronounced effect on the percentage of usable blastocysts on day 5

Abstract Study question Does Continuous Single Culture Medium NX, an embryo culture medium containing 1mM lactate, support increased blastocyst development over high lactate Vitrolife G1/G2 Series sequential culture? Summary answer There is a statistically significant increase in day 5 usable blastocysts in low lactate culture medium compared to the one with high lactate medium. What is known already Studies have shown that day 5 is the most desirable day to obtain blastocysts that are of an expansion, grade and quality to be utilized for transfer and/or vitrification procedures as those result in the highest success of clinical pregnancy,as compared to day 6/ 7 blastocysts, that do not meet criteria. Moreover, recent studies have indicated that there is an increase in chromosomal correctness of embryos cultured in a 1mM lactate environment as opposed to the higher 6-10mM lactate concentrations that have historically been believed necessary for successful blastocyst culture and resulting pregnancy. Study design, size, duration A prospective split sibling oocytes study was performed on 50 ICSI and IMSI cycles from October 2020 through April 2021. Oocytes were divided into the low lactate medium and high lactate gradient medium immediately following ICSI/IMSI and thereafter cultured in those medium until the final day of blastocyst culture. All patient ages were included in the sample population. Participants/materials, setting, methods This study was carried out in a private clinic. All patient stimulation protocol information and diagnosis’ were recorded; however, there was no restriction on participation. The endpoint was to analyse the resulting usable blastocyst rates on day 5 and day 6 in both arms of the study, using a denominator of normal 2PN fertilization. If a blastocyst was transferred or cryopreserved on day 5 or day 6, it was determined to be usable. Main results and the role of chance The resulting data was stratified not only by day 5 and day 6 usable blastocyst rates but also by patient age. It illustrates a statistically significant improvement in day 5 usable blastocysts for patients &amp;lt;35 in CSCM-NXC vs G1/G2 at 56% and 42%, respectively, a 14% increase (p &amp;lt; .05). The overall day 5 usable blastocyst rate was also statistically significant in CSCM-NXC (47%) as compared to G1/G2 (36%), (p &amp;lt; .05) with all ages considered. Additionally, on day 5, there was a higher percentage of usable blastocysts demonstrated in low lactate vs high in patients aged 35-37 (65% vs 42%, respectively) and 41-42 (41% vs 15%). Statistical significance was reversed in patients &amp;lt;35 on day 6, with G1/G2 having 24% usable blasts and CSCM-NXC 10% (p &amp;lt; .05). Interestingly, though not significant, G1/G2 had an increase in usable blastocyst percentage on day 5 in patients &amp;gt;42 (20% vs 11%), but overall, CSCM-NXC saw an increase in that same age group by 16%. Limitations, reasons for caution Though statistical significance was found in this study, a greater number will help to bolster the statistical power of the observations. Additionally, more studies are needed in order to ascertain if low lactate has an effect on the development prior to ICSI and resulting culture. Wider implications of the findings The mechanism of action that leads to the successful embryo development in low lactate embryo culture medium is vastly unknown, so further studies are required in order to understand the complexities and the impact of the observations provided. Trial registration number not applicable.

P-444 Outcomes of oocyte vitrification in trans masculine persons

Abstract Study question What are the outcomes of oocyte vitrification treatment(OVT) in trans masculine persons (TMPs) prior to, and after testosteroneuse? And how do these patients reflect on their treatment? Summary answer TMPs show a normal response to controlled ovarian hyperstimulation(COH). Even though the OVT was considered burdensome, most patients were satisfied with their treatment and outcome. What is known already The desire to parent genetic offspring is a relevant topic in the lives of many TMPs. To preserve their fertility prior to gender affirming hormone treatment or –surgeries, fertility preservation should be discussed by healthcare providers. However, the procedure may lead to an increase in dysphoric distress in TMPs. Only few studies have been published describing the outcomes of TMPs undergoing OVT. Their data suggest a comparable oocyte retrieval number in TMPs compared to cis gender women as well as similar fertilization and pregnancy rates. Outcomes of OVT in TMPs and their experiences undergoing said treatment remain under-explored. Study design, size, duration This single center, retrospective cohort study was performed by the Center of Expertise on Gender and the fertility clinic at the Amsterdam UMC, location VUmc, Amsterdam, the Netherlands. Between January 2017 and June 2021, all TMPs who had undergone OVT where approached for participation and 24/30 TMPs were included in our cohort. Participants/materials, setting, methods Demographic characteristics and data on OVT were retrieved from the medical records. Oocyte vitrification at our center is performed in persons between the age of 16 and 39 using a long agonist COH protocol. TMPs who had initiated testosterone were advised 3 months cessation prior to stimulation. Evaluation of the oocyte vitrification procedure was collected via an online questionnaire comprising of 10 multiple choice and open ended questions. Main results and the role of chance The median age and BMI of participants was 21.1 years (IQR 19.4 – 24.1) and 22.1 kg/m2 (IQR 20.8 – 25.4), respectively. Seven persons were using testosterone, two persons were using puberty suppression (GnRH analogues) and seven persons were using other hormonal cycle regulation prior to their OVT. The median anteral follicle count on cycle day three was 28(IQR 24.5 – 35.0). After a median of 12 FSH stimulation days (IQR 10 – 13), a trigger was administered. The mean peak serum estradiol(E2) was 11062 pmol/L (SD 5385). A median of 20 oocytes (IQR 16 – 26) were found and a median of 17 oocytes (IQR 14 – 22) were frozen. Six participants (25%) developed an ovarian hyperstimulation syndrome(OHSS). Four of which were classified as severe. There were no significant differences between the prior testosterone users and non-testosterone users. The median time between OPU and taking the questionnaire was 19.2 months(IQR 3.2 – 27.5). The response rate was 100%. Almost half of participants(46%) were most anxious for the internal examination prior to starting OVT. Interestingly, afterwards only 13% described the internal examination as the most strenuous part of the treatment. Hormone injections were considered the most strenuous part of OVT (29%). Limitations, reasons for caution A limitation of this study is the small sample size, especially when comparing prior testosterone users to non-testosterone users. Another limitation is that the effect of previous testosterone on the chance of live birth rate remains unknown since no participants have chosen to pursue conception yet. Wider implications of the findings This is the first study describing OVT outcomes in the TMPs in the Netherlands. This study shows no difference between prior testosterone use or not. The survey results identify the most burdensome aspect of the procedure for TMPs and improve transgender specific fertility strategies. Trial registration number not applicable