Immature Human Oocytes Research Articles

P-117 Extracellular vesicles from fallopian tubal fluid benefit human embryo development in vitro

Abstract Study question Do extracellular vesicles (EVs) from Fallopian tubes benefit human embryo development, and how do they affect preimplantation embryo development? Summary answer The Fallopian tubal EVs can be internalized by human embryos, and they contain miR-21-5p that downregulated P53 in blastocysts. What is known already EVs in the Fallopian tubes are identified as key mediators in embryo-oviduct interactions that contribute to a successful pregnancy in vivo. We previously showed Fallopian tubal EVs can benefit mouse embryo development in vitro. However, if a similar phenomenon can be observed in human embryos and what is the key component in Fallopian tubal EVs remains elusive. Study design, size, duration The EVs were isolated from the luminal fluid of human Fallopian tubes (n = 23). Immature human oocytes were collected and in vitro matured and fertilized, and 2-pronuclei embryos were included in this study and randomly divided (n = 87). We cocultured these zygotes with Fallopian tubal EVs until the blastocyst stage. Further, two-cell mouse embryos were used to explore the expression levels of miRNAs in Fallopian tubal EVs. The effects of miR-21-5p on mouse embryos were analyzed. Participants/materials, setting, methods EVs were isolated using ultracentrifugation. The uptake of labeled EVs by human embryos was analyzed using confocal microscopy. The fragmentation of the day-3 embryos and the blastocyst rate were compared between the control and the EV-treatment group. Then the stage-specific expression of miRNAs was compared in mouse embryos that were either developed in vivo or cultured in vitro. miR-21-5p were supplemented to the culture medium and the p53-related apoptosis in embryos was evaluated. Main results and the role of chance EVs were measured less than 200 nm in diameter with a typical bi-layer membrane structure and expressed protein markers Heat shock protein 70 (HSP70) and CD9. Most of the fluorescence-labeled EVs were attached to the zona pellucida within 2 h of co-culture. However, human embryos internalized EV within 4 h, with fluorescence signals observed in embryonic cells. The fragmentation rate was decreased and the blastocyst rate was increased by EVs. miR-21-5p, which was highly enriched in EVs, was found to be expressed at a significantly lower level in in-vitro-cultured mouse embryos compared to the in-vivo-developed embryos, through the two-cell stage to the eight-cell stage. miR-21-5p agomir supplementation to the culture medium was found to increase the mouse blastocyst formation rate, decrease apoptosis, and downregulate the P53 expression level in mouse blastocysts. Limitations, reasons for caution Though we observed that Fallopian tubal EVs could be internalized by human embryos and improve embryo development morphologically, the subsequent mechanism exploration was conducted using mouse embryos. Whether the stage-specific expression level of miR-21-5p and the effects of miR-21-5p are the same in human embryos remains to be determined. Wider implications of the findings This study showed miRNAs in Fallopian tubal EVs can act as embryonic survival signals, which opens the insight into further explorations to demonstrate the molecular mechanisms during early embryo development, as well as holds the potential to optimize the culture medium used in ART in the future. Trial registration number 2022YFC2702503

P-558 Characterization of the morphology of the outer-kinetochore expandable module, aka fibrous corona, and its role in ensuring proper chromosome segregation in human and bovine oocytes

Abstract Study question How does the Fibrous Corona (FC) behave in human and bovine oocyte meiosis and what is its function in chromosome segregation during meiosis I? Summary answer In human and bovine oocytes, FC shows extreme plasticity expanding beyond the centromeric region of the chromosomes. Pharmacological inhibition of FC’s expansion induces chromosome mis-segregation. What is known already Embryonic aneuploidy of meiotic origin is one of the major causes of implantation failure and early pregnancy loss. Erroneous microtubule (MT)-kinetochore attachments in human meiosis are the main cause of meiotic chromosome segregation errors. In somatic cells, the FC, a multiprotein complex, assembles on kinetochores and expands around the centromere during prometaphase to facilitate MT interactions and stimulate correct MT-kinetochore attachment. We have shown that in bovine oocytes FC expand beyond the centrosomes and is a potent MTs nucleator (10.1016/j.cub.2023.01.010). However, it is still unknown how FC behaves and what are its functions in MT-kinetochore interactions in human oocytes. Study design, size, duration FC morphology was studied in immature human oocytes discarded during ICSI procedures and donated (informed consent) by the patients for research (Ethical permit TCBio.21-754) during the years 2021-2024. Because of ethical concerns and reduced availability of human oocytes, functional studies on FC were performed on bovine oocytes collected from slaughterhouse ovaries. Participants/materials, setting, methods 180 immature human oocytes were matured in vitro and either fixed at different meiosis I stages or treated 2h with colcemid, a MT depolymerizing drug, to induce maximum FC expansion and fixed. The oocytes were then immuno-stained for chromatin(DAPI), FC(ZW10), MT(alpha tubulin) and kinetochores(CREST) and imaged with super-resolution confocal microscopy. Bovine immature oocytes were matured in vitro with or without lonafarnib (inhibitor of FC expansion). After maturation chromosome spreads were performed to evaluate oocyte ploidy. Main results and the role of chance In human oocytes, after nuclear envelope breakdown, before microtubule appearance when the chromosomes appear compacted, FCs are visible on the top of the kinetochores. During MT nucleation, FCs form thick mesh in the center of the compacted chromosomes. During prometaphase I, FCs expand beyond the centromeric region of the chromosomes forming a cup around the sister kinetochores of the paired homologous chromosomes. When the chromosomes starts to align at the metaphase plate, FCs start to be stripped from the kinetochores and colocalize to the MTs. At metaphase I and II, only kinetochore localization is visible. When oocytes are treated with colcemid, FCs extend to partially cover chromosome arms. We observed a wide variability in FCs expansion between and within patients. Inhibition of FC expansion in bovine oocytes increase the incidence of oocyte aneuploidy (90% in lonafarnib treated vs 37% in control oocytes). Altogether, our data reveal that in human oocytes FCs expand more compared to human somatic cells. Considering that inhibition of FC expansion induces chromosome segregation errors, we propose that the large FC size in human oocytes may play a role in achieving correct chromosome-MT attachments within their extremely large cytoplasm thus ensuring proper chromosome segregation. Limitations, reasons for caution The limited availability of human oocytes requires the use of an animal model for in depth mechanistic studies. Although bovine oocytes have similar aneuploidy rate and spindle assembly mechanisms as human oocytes, they show morphological differences in FC expansion which could underlie intrinsic functional discrepancies between the two organisms. Wider implications of the findings A better understanding of the chromosome segregation mechanisms in human meiosis will help understanding what causes the high incidence of aneuploidies in human oocytes and possibly contribute to the development of interventions or of oocyte selection procedures to reduce the incidence of oocyte aneuploidy during in vitro maturation. Trial registration number Not applicable

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

RESEARCH QUESTIONCan immature oocytes vitrified and warmed using a short protocol survive and resume meiosis? DESIGNThis study examined modifications of oocyte vitrification and warming protocols that shorten the time of exposure to vitrification and warming solutions. A total of 561 germinal vesicles (GV), and 218 metaphase I (MI) oocytes that were immature at oocyte retrieval were vitrified at room temperature in 2 minutes. Warming was done at 37°C in 2 minutes. Resumption of meiotic activity was evaluated after 24 and 48 hours of culture. Two different commercially available vitrification and warming kits were used for comparison. RESULTSNinety five percent of GV oocytes survived with no difference observed between kits. The survival of MI oocytes was on average 95.4% and not significantly different between the two kits. Of the 533 GV that survived, 491 converted to MI (92.1%). After culture for 48 hours, 54.4% converted to MII oocytes. In addition, of the 208 MI that survived the warming, 84.1% converted to MII oocytes after culture for 24 hours. CONCLUSIONSVitrification and warming of oocytes at different nuclear maturation stages can be done with a 2-minute exposure time to hypertonic and a 2-minute exposure time to hypotonic solutions. With this approach, the oocytes are less exposed to room temperature during dehydration and rehydration. Warming in 0.5M sucrose helps to maintain and support the potential to resume nuclear meiotic activity of oocytes and conversion from GV to MI and to MII oocytes.

P-266 Study on the mechanisms of ovol1/2 during the morula-to-blastocyst transition

Abstract Study question Screen the key gene groups that determine the development of blastocyst. Explore the effect of ovol1/2 gene on early embryonic development. Summary answer We founded that there were insufficient activation of the Major ZGA genes in the AE group,and focused on candidate gene ovol1/2. What is known already At present, the clinical success rate of assisted reproductive technology (ART) is less than 50%. A large number of embryos cultured in vitro can not develop to blastocyst, and there are cleavage or morula stage arrest. The developmental arrest of preimplantation embryos is an important reason for treatment failure of infertile patients. However, the cause of early embryonic development arrest is not clear, and there is also a lack of effective methods to improve embryo quality. Study design, size, duration In this study, fertilized embryos were obtained through ICSI and a single cell from eight-cell embryo was biopsied through micromanipulation for single-cell RNA sequencing (scRNA-seq). Subsequently, we tracked the developmental potential of remaining cells and divided them into high-quality blastocyst(Bla) group and high-quality eight-cell failed blastocyst formation(arrest embryo, AE) group. In addition,we employed a RNAi approach to study the function of candidate gene ovol1/2, by injecting ovol1/2-targeting small interfering RNAs (siovol1/2) into wild-type (WT) zygotes. Participants/materials, setting, methods The study collected 21 human blastocysts and 5 arrest embryos. Human immature oocytes from ICSI treatments were clinically discarded and donated by women after signing informed consent by donors. C57B6 background mouse strains were used in this study. Mice were maintained under specific pathogen free conditions in a controlled environment of 20–22 °C,with a 12/12 h light/dark cycle, 50–70% humidity, and food and water provided. Main results and the role of chance The results showed that the gene expression of embryos in AE group was significantly different from that of Bla group: 79.5% of differential expression genes were down-regulated, including 48.3% of the Major ZGA gene. Then we focused on the candidate gene ovol1/2. When ovol1/2 were knocked down alone, there was no significant difference in the development rate of embryos from 2-cell to blastocyst between knockdown group and normal control group. When ovol1/2 were knocked down at the same time, there was no significant difference in the development rate of embryos among three groups before the morula stage. But in the morula developed to the blastocyst stage, there were significant differences among three groups of embryos: about 73% of embryos in the normal control group formed blastocyst; about 39% of the embryos in the low-dose knockdown group; the high-dose knockdown group embryos were all arrested in the morula stage. Further investigation revealed that the number of inner cell mass(ICM) cells in low-dose knockdown group did not change significantly, while the number of trophoblastic ectoderm(TE) cells significantly decreased. In addition, the TE functions of low-dose knockdown group such as blastocyst cavity size, paved area, hatching and adhesion rate were damaged. Limitations, reasons for caution Further research of the mechanisms is needed. Wider implications of the findings This is significant to understand molecular mechanisms of early embryonic development, and will provide important theoretical basis for clinical diagnosis and intervention of embryonic development arrest. Our study provides clues for the causes of abnormal blastocyst formation and new ideas for possible clinical improvements in the success rate of ART. Trial registration number not applicable

O-040 Clinical competence of oocytes with developmental abnormalities

Abstract For decades we have looked for marker of embryo competence. And while we have come a long way in terms of identifying markers related to embryonic competence, a lot of research is still focused on finding this simple measurable indicator predicting a positive outcome of the individual treatment. Many potential indicators have been evaluated. This includes the cumulus oocyte complex, zona pellucida, perivitelline space, polar bodies, vacuoles, refractile bodies, SER clusters, cytoplasmic granularity, and many others. For some of these there are data suggesting potential prediction of negative outcome (for review see Bartolacci et al. 2022). Embryo autonomy may result in cleavage and development despite the lack of competence to establish a pregnancy. And pregnancies may occur without the competence to go all the way to a live birth. In a study by Lundin et al. we looked into chromosomal constitution after rescuing immature (developmentally abnormal) human oocytes. And while 15% of the oocytes developed into an embryo only very few appeared chromosomally normal after cytogenetic analysis. The study concluded that the majority of hCG exposed oocytes that have not reached the MII stage at time of pick-up are defect and consequently that GV or MI oocytes should not be used in ART programs. As reproductive specialists our main task is to assist our patients in achieving their dream of having a child. We are supposed to use all our combined expertise, knowhow, craftmanship, routine and experience to optimize the chance for the patients to have a baby. However, in our ambition to help our patients we sometimes lose touch of the underlying biology of human reproduction. Oocytes from a stimulated ovary are not like those we buy in the supermarket of equal quality. The depressing fact is that only 5.0 % of the oocytes in a first cycle is biologically competent and in around 2/3 of all cycles, none of the oocytes have the potential to result in the birth of a child (Lemmen et al 2016). We also have very limited ability to determine competence of individual sperm cells – the strongest prognosticator being if they look and move like a “normal” sperm sample. Are we getting a bit desperate when we start to look for normality in the abnormal as suggested in the title of this talk? Have we reached a point were patient expectations overrides our biological training and education resulting in low willingness to accept a negative outcome dictated by our biology? And is this reinforced by patient perceptions like “we did not pay a lot of money for the oocytes not being fertilized/developed/implanted”. Embryo competence evaluation is based on markers of what have happened rather than indicators of what will happen. At the end of the day, we as health care professionals as well as our patients needs to accept the fact that there is no such thing as “CLINICAL COMPETENCE OF OOCYTES WITH DEVELOPMENTAL ABNORMALITIES”. And what will be the consequence of a marker indicating a lower implantation potential in an otherwise ok embryo? It might affect the ranking of the embryos from a particular cycle, but we would be willing transfer it anyway if needed. Embryo evaluation don’t increase pregnancy chance per se. But it may decrease time to pregnancy by improved selection between the embryos available. Maybe it is time to re-focus our efforts and move from a binary normality approach identifying “developmental abnormalities” toward a more biological oriented approach mapping “developmental variation” in embryos and their impact on pregnancy potential and thus recognizing the multi-factorial nature of embryo maturation and ongoing developing competence.

Vitrification with Dimethyl Sulfoxide Induces Transcriptomic Alteration of Gene and Transposable Element Expression in Immature Human Oocytes.

Despite substantial advancements in the field of cryobiology, oocyte and embryo cryopreservation still compromise developmental competence. Furthermore, dimethyl sulfoxide (DMSO), one of the most commonly used cryoprotectants, has been found to exert potent effects on the epigenetic landscape of cultured human cells, as well as mouse oocytes and embryos. Little is known about its impact on human oocytes. Additionally, few studies investigate the effects of DMSO on transposable elements (TE), the control of which is essential for the maintenance of genomic instability. The objective of this study was to investigate the impact of vitrification with DMSO-containing cryoprotectant on the transcriptome, including on TEs, of human oocytes. Twenty-four oocytes at the GV stage were donated by four healthy women undergoing elective oocyte cryopreservation. Oocytes were paired such that half from each patient were vitrified with DMSO-containing cryoprotectant (Vitrified Cohort), while the other half were snap frozen in phosphate buffer, unexposed to DMSO (Non-Vitrified Cohort). All oocytes underwent RNA sequencing via a method with high fidelity for single cell analysis, and which allows for the analysis of TE expression through Switching Mechanism at the 5'-end of the RNA Transcript sequencing 2 (SMARTseq2), followed by functional enrichment analysis. Of the 27,837 genes identified by SMARTseq2, 7331 (26.3%) were differentially expressed (p < 0.05). There was a significant dysregulation of genes involved in chromatin and histone modification. Mitochondrial function, as well as the Wnt, insulin, mTOR, HIPPO, and MAPK signaling pathways were also altered. The expression of TEs was positively correlated with the expression of PIWIL2, DNMT3A, and DNMT3B, and negatively correlated with age. These findings suggest that the current standard process of oocyte vitrification, involving DMSO-containing cryoprotectant, induces significant transcriptome changes, including those involving TEs.

Detection of the effect of microvibrational stimulation on human discarded immature oocytes by single-cell transcriptome sequencing technology.

This study aimed to investigate the changes in oocytes at the transcriptome level after applying continuous microvibrational mechanical stimulation to human immature oocytes during in vitro maturation. The discarded germinal-vesicle stage (GV) oocytes with no fertilization value after oocytes retrieval in assisted reproduction cycles were collected. Part of them was stimulated with vibration (n = 6) at 10 Hz for 24 h after obtaining informed consent; the other was cultured in static condition (n = 6). Single-cell transcriptome sequencing was used to detect the differences in oocyte transcriptome compared with the static culture group. The applied 10-Hz continuous microvibrational stimulation altered the expression of 352 genes compared with the static culture. Gene Ontology (GO) analysis suggested that the altered genes were mainly enriched with 31 biological processes. The mechanical stimulation upregulated 155 of these genes and downregulated 197 genes. Among them, the genes related to mechanical signaling, such as protein localization to intercellular adhesion (DSP and DLG-5) and cytoskeleton (DSP, FGD6, DNAJC7, KRT16, KLHL1, HSPB1, MAP2K6), were detected. DLG-5, which was related to protein localization to intercellular adhesion, was selected for immunofluorescence experiments based on the transcriptome sequencing results. The protein expression of DLG-5 in the microvibration-stimulated oocytes was higher than that in the static culture oocytes. Mechanical stimulation affects the transcriptome during oocyte maturation, causing the express changes in intercellular adhesion and cytoskeleton-related genes. We speculate that the mechanical signal may be transmitted to the cell through DLG-5 protein and cytoskeleton-related protein to regulate cellular activities.

IVM of human immature oocytes for infertility treatment and fertility preservation.

Thousands of healthy babies are born from in vitro maturation (IVM) procedures, but the rate of efficiency differs with the source of immature oocytes obtained. Recently, there are different IVM protocols proposed for infertility treatment and fertility preservation. Based on the literature, the clinical application for IVM of immature oocytes was summarized. Immature oocytes may be retrieved from women after priming with or without the use of follicular stimulation hormone (FSH), human chorionic gonadotrophin (hCG) or a combination of both FSH and hCG. Successful pregnancy rates with IVM technology seem to be correlated with the number of immature oocytes obtained. With the source and culture course of immature oocytes, there are various IVM protocols. IVM of immature oocytes is profoundly affected by the culture conditions, but no breakthrough has been made by improving the IVM medium itself. Thus, the clinical application of IVM technology continues to evolve. IVM technology is a useful technique for infertile women and fertility preservation. Mild stimulation IVF combined with IVM of immature oocytes is a viable alternative to the conventional stimulation IVF cycle treatment as it may prove to be an optimal first-line treatment approach.

Impact of Ovarian Factor Mediums on the Apoptotic Gene Expression and Embryo Quality Derived From Vitrified Immature Human Oocytes.

Condition mediums have a potential role in oocyte development. In this study, we evaluated the effects of different mediums on the developmental potential of vitrified immature human oocyte after IVM and parthenogenesis by ionomycin. Immature oocytes were collected from 184 women after vitrification/thawing and maturation, in three types of IVM mediums separately. Finally, 151 IVM MΙΙ oocytes were obtained and randomly divided into six groups and underwent the following intervention. Fresh and vitrified-thawing MΙΙ oocytes were activated after IVM in three conditioned mediums by ionomycin. Mediums included 1) Minimum Essential Medium Alpha (α-MEM) (as control medium), 2) α-MEM supplemented with supernatants of Mesenchyme bone marrow (B.M), 3) α-MEM with ovarian growth factors (O.F). Then, scoring of parthenote embryos was undertaken in accordance with pertinent morphological properties. Moreover, the expression of Bax and Bcl2 were determined in the parthenote embryos. Percentage of the degenerated oocyte, 2-4 cells, 4-8 cells, and 16 cells, was different in the experimental groups. Also, cytoplasmic maturation and blastocyst formation rates were significantly different (p < 0.05) between the control and the other mediums. The highest mRNA expression levels of Bcl2 and Bax genes in parthenotes were observed in the fIVM O.F and vIVM α-MEM mediums, respectively. vIVM, α-MEM and fIVM O.F showed the lowest expression of Bcl2 and Bax genes, respectively. Our findings indicate that the O.F. medium had more potent effects on oocyte growth and cytoplasmic maturation up to the blastocyst stage with the highest expression level of the BCL2 gene and the lowest relative amount of the BAX gene in this medium. The results of the present study have been verified only for parthenogenetically activated embryos, and any positive effect of the environment on the egg/embryo fertilized with sperm requires more extensive studies.

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.

Effect of ovarian growth factors on ultra-structural maturation in frozen human immature oocytes after in vitro maturation: a comparative study

BackgroundIn artificial reproductive technique (ART), nearly 20% of human oocytes are immature in the germinal vesicle (GV) phase. Consequently, the best method for reserving them is cryopreserving GV oocytes, and in vitro maturation (IVM) is recommended. The aim of this study was to determine the ultrastructure characteristics of fresh and vitrified immature human oocytes after in vitro maturation in conditioned mediums.MethodsThis study was a comparative laboratory study carried out in 2018 at Afzalipur Infertility Center in Kerman. 170 fresh and 198 vitrified GV oocytes were cultured within three IVM mediums; α-mem as control medium, α-mem supplemented with human bone marrow mesenchymal stem cells (BM-MSCs) and α-mem supplemented with ovarian growth factors (O.F). After 48 h, the maturation rate and morphological feature of IVM oocytes [132 fresh IVM (fIVM) and 134 vitrified IVM (vIVM)] were evaluated. For the ultrastructure study, 10 IVM oocytes from each medium were compared with 10 fresh in vivo oocytes cancelled from ART.ResultsThe survival rate of vitrified GV oocyte after thawing was 88.88%. The oocyte maturation rate was reduced in vIVM compared to the fIVM group (76.33% vs. 77.95%); the highest oocyte maturation rate in the O.F fIVM and lowest in α-mem vIVM (82.35% vs. 71.42%). The lowest number of cortical granules was observed in α-mem vIVM, but the greatest presence of M-SER aggregates was in O.F fIVM. In vIVM oocytes, the oolemma contained irregular little microvillus organization.ConclusionsThe O.F mediums have shown the highest maturation which defends the oocyte ultra-structural conservation.

Effects of microvibration stimulation on developmental potential of discarded germinal vesicle oocytes of human.

This research aims to study the effects of continuous microvibration stimulation on the parthenogenetic development of human germinal vesicle oocytes. Ninety-five discarded germinal vesicle oocytes from intracytoplasmic sperm injection treatment (ICSI) cycles performed at Amcare Women's & Children's Hospital between January and December 2021 were used for conventional static culture as well as 10Hz microvibration culture. We investigated the differences between the two groups in terms of oocyte maturation rate, parthenogenetic activation rate, and parthenogenetic blastocyst formation rate. The static culture and 10Hz microvibration culture of 95 oocytes showed that the parthenogenetic blastocyst formation rate in the microvibration culture group was significantly higher than those in the traditional static culture group. A continuous microvibration stimulation can significantly improve the parthenogenetic developmental potential of human immature oocytes.

Oocyte diameter predicts the maturation rate of human immature oocytes collected ex vivo

PurposeTo study the impact of oocyte diameter and cumulus cell mass on the potential for final maturation of immature human oocytes in vitro.MethodsImmature oocytes (n = 1563) from 75 women undergoing fertility preservation by ovarian tissue cryopreservation (14–41 years) were collected. After preparation of the ovarian cortex for freezing, immature oocytes were collected from the surplus medulla. After collection, IVM was performed according to standard published methods. The mass of cumulus cell surrounding the immature oocyte was grouped according to size. After IVM, each oocyte was photographed, measured, and the diameter was calculated as a mean of two perpendicular measurements.ResultsThe diameter of the oocytes ranged from 60 to 171 µm with a mean of 115 µm (SD:12.1) and an interquartile range from 107 to 124 µm. The oocyte diameter was positively associated with a higher incidence of MII (p < 0.001). MII oocytes had a significantly larger mean diameter than MI, GV, and degenerated oocytes. The size of the cumulus cell mass was significantly associated with the MII stage (p < 0.001) and larger oocyte diameter (p < 0.001). The results further confirm that the diameter of the fully grown oocyte is reached relatively early in human follicular development and that the factors governing oocyte maturation in vitro are connected to the surrounding cell mass and the oocyte.ConclusionThe diameter of the oocyte is a highly determining factor in the nuclear maturation of the human oocyte during in vitro maturation, and the size of the cumulus cell mass is closely positively associated with a larger diameter.

P-257 Nanosphere-based assessment of zona pellucida permeability in humans and mammalian models

Abstract Study question What is the maximum particle size that can traverse through human zona pellucida (ZP) and that of other mammals? Summary answer Human oocytes ZP is impermeable to 40-100 nm nanospheres. ZP of other mammals is impermeable to 100 nm particles, and permeability changes along preimplantation development. What is known already ZP is a porous structure that protects oocytes and early embryos, being permeable to nourishing and signaling substances while blocking the entrance of immune cells and viruses into the perivitelline space. Size-dependent ZP permeability is poorly known and may differ between species, as in humans and rabbits ZP it is composed by 4 proteins (ZP1-4), while bovine and murine ZPs contain only 3 proteins (ZP2-4 in bovine, ZP1-3 in mice). Understanding permeability is crucial to determine embryo susceptibility to viral infections or large biological particles such as exosomes, and to assess possible damages inferred by artificial reproductive techniques. Study design, size, duration The objective has been to determine ZP permeability in mice, bovine, rabbit (wild-type or lacking ZP4) or human oocytes and/or embryos based on particle size. The assay was performed in 3 replicates of 3 samples per stage: mouse samples (oocytes, zygotes, 2-cell embryos and blastocysts) collected in vivo, bovine samples (oocytes, zygotes, 4-8 cell embryos and blastocysts) produced in vitro, rabbit samples (zygotes) collected in vivo and immature human oocytes discarded for clinical use. Participants/materials, setting, methods Permeability test consisted of incubating oocytes and embryos in 0.005 % solutions of carboxylate-modified polystyrene nanospheres (20, 40 and 100 nm) loaded with a fluorophore emitting at a 505/515 nm for 30 min. Following incubation and washing, samples were observed under a structured illumination fluorescence microscope to determine if particles are present inside ZP (i.e., ZP is permeable to that size of particle) or fluorescence cannot be detected (i.e., particles unable to traverse ZP). Main results and the role of chance ZPs of all species and stages blocked efficiently the pass of 100 nm-sized nanospheres. 40 mm nanospheres were blocked by the ZP of oocytes and zygotes in all species with the exception of rabbit zygotes lacking ZP4. ZP permeability changed during early development, as ZP from murine and bovine cleaved embryos and blastocyst were permeable to 40 nm-sized particles. Finally, with the exception or WT rabbit zygotes, ZP from all oocytes and embryos analyzed were permeable to 20 nm nanospheres. As a fluorescence control, murine and bovine oocytes without ZP were exposed to nanospheres of all sizes, which were clearly visualized attached to the oolema or into the ooplasm. Limitations, reasons for caution Experiments on cleavage and blastocysts stages were performed on animal models, so whether human ZP permeability changes following fertilization remains unknown. Wider implications of the findings Mammalian ZP acts as a selective barrier during early embryo development. Understanding particle size-dependent permeability is crucial to determine embryo susceptibility to viral infection or to signaling mediated by large particles such as extracellular vesicles and exosomes and to assess the impact of oocyte manipulation techniques. Trial registration number Not applicable

Recombinant anti-M\xfcllerian hormone in the maturation medium improves the in vitro maturation of human immature (GV) oocytes after controlled ovarian hormonal stimulation

BackgroundIn vitro maturation (IVM) of oocytes is a laboratory method that allows the maturation of immature (GV) oocytes retrieved from patients enrolled in the in vitro fertilization (IVF) programme. However, this method is still sparsely researched and used in clinical practice, leading to suboptimal clinical results. Anti-Müllerian hormone (AMH) is an important hormone with known effects on human ovaries, especially on follicles (follicular cells) during folliculogenesis. In contrast, the effect of AMH on the human oocyte itself is unknown. Therefore, we wanted to determine whether human oocytes express AMH receptor 2 (AMHR2) for this hormone. Recombinant AMH was added to the IVM medium to determine whether it affected oocyte maturation.MethodsIn total, 247 human oocytes (171 immature and 76 mature) were collected from patients enrolled in the intracytoplasmic sperm injection (ICSI) programme who were aged 20 to 43 years and underwent a short antagonist protocol of ovarian stimulation. The expression of AMHR2 protein and AMHR2 gene was analysed in immature and mature oocytes. Additionally, maturation of GV oocytes was performed in vitro in different maturation media with or without added AMH to evaluate the effect of AMH on the oocyte maturation rate.ResultsImmunocytochemistry and confocal microscopy revealed that AMHR2 protein is expressed in both immature and mature human oocytes. AMHR2 was expressed in a spotted pattern throughout the whole oocyte. The IVM procedure revealed that AMH in maturation medium improved GV oocyte maturation in vitro, as all oocytes were successfully matured in maturation medium containing recombinant AMH only. Furthermore, antagonism between AMH and follicle-stimulating hormone (FSH) during the maturation process was observed, with fewer oocytes maturing when both AMH and FSH were added to the maturation medium. Finally, AMHR2 gene expression was found in immature and in vitro matured oocytes but absent in mature oocytes.ConclusionsThe positive AMHR2 protein and AMHR2 gene expression in human oocytes shows that AMH could directly act on human oocytes. This was further functionally confirmed by the IVM procedure. These findings suggest the potential clinical application of recombinant AMH to improve IVM of human oocytes in the future.

A fertilin-derived peptide improves in vitro maturation and ploidy of human oocytes

To analyze the effect of a cyclic fertilin-derived peptide (cFEE) on invitro maturation of human oocytes. Randomized study. Fertility center in an academic hospital. Not applicable. Human immature germinal vesicle-stage oocytes (n = 1,629) donated for research according to French bioethics laws were randomly allocated to groups treated with 1 or 100 μM of cFEE or to a control group. They were incubated at 37 °C in 6% CO2 and 5% O2, and their maturation was assessed using time-lapse microscopy over 24 hours. Invitro maturated metaphase II oocytes were analyzed for chromosomal content using microarray comparative genomic hybridization, and their transcriptomes were analyzed using Affymetrix Clariom D microarrays. The percentage of oocytes undergoing maturation invitro was observed. Aneuploidy and euploidy were assessed for all chromosomes, and differential gene expression was analyzed in oocytes treated with cFEE compared with the control to obtain insights into its mechanism of action. cFEE significantly increased the percentage of oocytes that matured invitro and improved euploidy in meiosis II oocytes by the up-regulation of FMN1 and FLNA genes, both of which encode proteins involved in spindle structure. cFEE improves human oocyte maturation invitro and reduces aneuploidy. It may prove useful for treating oocytes before fertilization in assisted reproductive technology and for invitro maturation in fertility preservation programs to improve oocyte quality and the chances for infertile couples to conceive.

P–239 In vitro maturation of human oocytes: a systematic review and data analysis

Abstract Study question Based on published studies, how effective is in vitro maturation (IVM) in different patient groups, and how does the maturation rate correlate with culture conditions? Summary answer Clinical IVM is most effective when patients receive only hCG trigger prior to oocyte collection. Multiple additional parameters influencing the outcome were identified. What is known already Despite being used for more than fifty years, the overall efficacy of human IVM has not yet been determined, and results are often conflicting. Indeed, IVM is still perceived skeptically by many embryologists and doctors and not widely used in clinical practice. This review aims to collect all available data in the literature regarding the efficacy of IVM analyzing characteristics of patients, treatment, or laboratory conditions that may influence the MII-rate (MR). Study design, size, duration: A systematic search was performed in the PubMed database following Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. The search was limited to studies in the English language published before October 2020 using the following keyword: “oocyte in vitro maturation». Participants/materials, setting, methods Inclusion criteria for studies were: reporting data obtained on immature human oocytes, which transitioned to the MII stage after IVM. The requirement was that the numbers of cultured and matured oocytes were reported. If available, additional data were collected including patients’ characteristics (for example PCOS), hormonal stimulation prior to the procedure (administration of some FSH or hCG trigger or both), oocyte freezing before or after IVM, type of culture medium and supplements, etc. Main results and the role of chance A total of 350 publications were selected from 6866 search results, 436 abstracts, and 422 full read articles. Selected studies cover 21153 patients and 157420 immature oocytes cultured. It has been demonstrated that oocytes collected in vivo from adult, non-PCOS patients, who received only hCG trigger prior to the procedure had a statistically higher MII rate (66%) than oocytes from patients who received no gonadotropins or some FSH, or a combination of some FSH and hCG trigger (59%, 60% and 58% respectively). The same was valid for PCOS patients: MR in the trigger only cohort (66%) was significantly different from other cohorts. MR for in vivo collected oocytes (61%) from adult non-PCOS patients was significantly different from ex vivo collected oocytes (33%). MI stage oocytes at the moment of collection matured with a statistically higher rate (N = 4322, 73%), than GV oocytes (N = 3328, 54%). When in vitro matured oocytes were vitrified, their average survival rate was 81% (data from 50 studies on 1701 oocytes). Additionally, immature oocytes survived vitrification with a 75% rate (data from 30 studies on 4457 oocytes). Overall, ICSI fertilization rate for IVM oocytes was 69% (N = 59914). A total of 747 babies born from IVM were reported. Limitations, reasons for caution Among selected publications only 2 were randomized controlled trials and therefore the main challenge of this review is striking differences in setups among included studies. However, despite not being a meta-analysis, this study calculated MR for the most frequent treatment modalities and additional individual factors, which might influence MR. Wider implications of the findings: This review provides data regarding IVM efficiency in different cohorts of patients, performed under different culture conditions. Additional laboratory parameters influencing MR have been identified. Based on this new data, target groups benefiting the most were identified, and prognosis regarding the success of their treatment with IVM might be estimated. Trial registration number n/a

P–528 rDNA methylation of human oocytes of women undergoing intracytoplasmic sperm injection (ICSI) increases with maternal age

Abstract Study question Is there a correlation between the age of women undergoing ICSI and methylation pattern of rDNA core promoter and upstream control element in immature human oocytes? Summary answer Methylation levels of the upstream control element and the rDNA core promoter in immature human oocytes increase with age of women undergoing ICSI. What is known already Methylation of ribosomal DNA (rDNA) in germ cells regulates temporary and spatially highly coordinated nucleolar activity, cellular metabolism, and thus developmental potential of the early embryo. Alterations of methylation pattern may therefore cause dysregulation of genes and signal cascades resulting in limited fertility. It has been shown that the methylation of sperm rDNA increases with the donor’s age. The positive correlation between sperm rDNA methylation and age has been conserved among mammals during evolution including humans and mice. In contrast to sperm, little is known about the methylome of human oocytes and its role in human reproduction. Study design, size, duration Consecutive women undergoing ICSI because of male subfertility were included. Patients with endometriosis, polycystic ovary syndrome, ovarian, uterine or breast cancer, as well as patients with an anti-Mullerian hormone level &amp;lt;1ng/ml were excluded. Immature oocytes (germinal vesicle; GV) collected during oocyte pick-up at the Fertility Centre Dortmund between 2018 and 2020 were examined. Participants/materials, setting, methods Cumulus-free GV oocytes which were not usable for ICSI were rinsed in phosphate buffer and stored at –20 °C until further investigation. Multiplex-PCR followed by singleplex-PCRs were carried out on the rDNA core promoter and upstream control element. Methylation levels were quantified by bisulphite pyrosequencing. Two oppositely imprinted genes (hPEG3 and hGTL2) were used as controls to ensure correct amplification and bisulphite conversion. Spearman’s-rank-order-correlation and Mann-Whitney-U-Test were used for statistical analysis. Main results and the role of chance For each GV oocyte, nine different Cytosine-phosphate-Guanine dinucleotides (CpGs) were quantified by bisulphite pyrosequencing for the rDNA core promoter and 26 different CpGs for the upstream control element (UCE). 120 human single oocytes from 60 women were analyzed. Connected statistical analysis was used if one patient had more than one oocyte. The age of the included women ranged from 26 to 40 years (mean±SD 33.5±3.2). Only oocytes which showed a correct methylation pattern for at least one imprinting control gene (hPEG3 and hGTL2) were considered for analysis. Mean methylation level ranged from 2–31% (mean±SD 8.7±5.5) of the analyzed CpGs for the rDNA core promoter and from 3–36% (mean±SD 11.4±7.1) CpGs for UCE. Spearman’s correlation analysis revealed that the methylation levels of the human oocyte rDNA core promoter and rDNA UCE significantly increased with the age of the donor (p &amp;lt; 0.05). Correlation coefficient for rDNA core promoter was r = 0.22 and for upstream control element r = 0.21. It is also interesting to note that different oocytes from the same donors can display enormous methylation variation. Regarding clinical parameters, no correlation was observed between the methylation pattern of the rDNA core promoter or UCE and the body mass index or smoking status, respectively. Limitations, reasons for caution Limitations of this study include difficulties in extrapolating the findings to the general population, because no data of women not undergoing ICSI are available. Only GV-oocytes were analyzed. Additional research is needed to clarify the effect of different methylation pattern with increasing female age and its role in human reproduction. Wider implications of the findings: We propose that the increase of rDNA methylation in male and female germ cells with advanced age directly or indirectly influences the regulation of nucleolar activity, cellular metabolism, and thus the developmental potential of the early embryo. This age-dependent epigenetic effect may result in decreased human fertility. Trial registration number NCT03565107

O-231 In vitro maturation of human immature (GV) oocytes after controlled ovarian hormonal stimulation with recombinant AMH in the maturation medium

Abstract Study question Does the addition of recombinant AMH to the in vitro maturation (IVM) medium improve the maturation of GV oocytes after controlled ovarian hormonal stimulation? Summary answer Our results show that the addition of recombinant AMH to the in vitro maturation medium improves the maturation rate of GV oocytes. What is known already Anti-Müllerian hormone (AMH) is an important hormone involved in the process of sex differentiation during embryonic development. At the transition to the 21. century, more and more researchers have studied the role of AMH in ovarian function, especially its impact on folliculogenesis. AMH is becoming one of the main biomarkers of ovarian reserve and ovarian-specific disease, however, little is known about its effect on human oocyte maturation. Therefore, we matured immature GV (germinal vesicle) oocytes in IVM medium with recombinant AMH to assess its effect compared to the conventional IVM procedure with FSH and hCG. Study design, size, duration In this two-year prospective study, we compared the maturation rate of four groups of immature (GV) oocytes matured in maturation medium with added i) AMH (n = 15), ii) AMH+FSH+hCG (n = 44), iii) FSH+hCG (conventional; n = 22), and iv) hormone-free maturation medium (control; n = 15). Each oocyte was matured in vitro for a maximum of 28 hours and monitored by time-lapse microscopy to assess the time of GV breakdown (MI) and extrusion of the polar body (MII). Participants/materials, setting, methods Ninety-six GV oocytes of 46 patients (aged &amp;lt; 38 years, involved in the ICSI programme) after short antagonist protocol of controlled ovarian hormonal stimulation were included after written informed consent. IVM of oocytes was performed in the MediCult IVM System (LAG and IVM medium, Cooper Surgical, Denmark) with added hormones, and in a CO2 incubator equipped with the PrimoVision time-lapse microscope (Vitrolife, Sweden). Main results and the role of chance IVM medium with added recombinant AMH gave the best result with all (100 %) oocytes matured in vitro. In conventional IVM medium with FSH and hCG, the oocyte maturation rate was poorer, with 68 % of oocytes matured in vitro. An even lower oocyte maturation rate (34 %) was observed in IVM medium with AMH, FSH and HCG, which might be explained by the antagonistic action of these hormones. In a group of control oocytes, 25 % of oocytes matured in vitro. The mean time to GV breakdown (MI stage) was 3.7 hours and to polar body release (MII stage) 20,5 hours. The time to MI stage was quite comparable in all groups of oocytes (3.5, 3.8 and 3.7 hours). There was a tendency for the polar body to be released later if AMH was added to the maturation medium (21.5 and 20.2 vs. 19.9 hours) but differences were not statistically significant, as revealed by Student’s t-test. In the control group of oocytes, these times were prolonged (4.2 and 22.2 hours) due to slow spontaneous maturation. These preliminary results demonstrate that AMH could directly affect the oocyte maturation in vitro. Limitations, reasons for caution The limitation is the relatively small number of oocytes included; GV oocytes accounted for less than 10 % of all oocytes in the in vitro fertilisation (ICSI) programme. Moreover, the proportion of GV oocytes spontaneously matured to MI stage before the start of the experiment and were therefore not included. Wider implications of the findings Based on our data, we believe that AMH directly affects human oocyte maturation in vitro. Despite the common knowledge that AMH regulates the recruitment of growing ovarian follicles, it appears that the addition of AMH to the maturation medium can improve the human oocyte maturation in vitro. Trial registration number 0120-546/2018/6

Developmental potential of immature human oocytes aspirated after controlled ovarian stimulation.

Intracytoplasmic sperm injection (ICSI) for initially immature oocytes that mature in vitro is controversial and practice varies widely. While it may increase the number of usable embryos, it may also be time-intensive and potentially low-yield. This study sought to elucidate which patients may benefit from ICSI of initially immature oocytes that matured in vitro. A retrospective study comparing fertilization, cleavage, blastulation, and embryo usage rates between sibling initially immature and mature oocytes that underwent ICSI between 2015 and 2019 was performed. Outcomes of initially immature oocytes were stratified by initial maturation stage, timing of progression to metaphase II (MII) in vitro, percentage of mature oocytes in the cycle, and female age. Ten thousand eight hundred seventeen oocytes from 889 cycles were included. Of 3137 (29.0%) initially immature oocytes, 418 (13.3%) reached MII later on the day of retrieval (day 0) and 1493 (47.6%) on day 1. Overall, embryos originating from initially immature oocytes had lower cleavage and blastulation rates compared to those from initially mature oocytes (P<0.05, all groups). However, embryos from oocytes that matured later on day 0 comprised a unique subset that had clinically similar cleavage (75% vs 80%, RR 0.93, P=0.047) and blastulation rates (41% vs 50%, RR 0.81, P=0.024) compared to initially mature oocytes. Women with low percentages of mature oocytes in the cycle overall and women ≥40 in cleavage cyclesderived the highest relative benefit from theuse of immature oocytes. ICSI of immature oocytes, particularly those that mature later on the day of retrieval, may improve numbers of usable embryos. This study supports routine reassessment of immature oocytes for progression to MII and ICSI on day 0. An additional reassessment on day 1 may also be of use in older women or those with low percentage of mature oocytes.