Search

Study question Could collection temperature and spindle transfer (ST) potentially improve development of embryos derived from in vitro matured (IVM) ovarian tissue oocytes (OTO) of transgender men? Summary answer Spindle transfer, but not collection temperature, significantly improved embryo development of OTO-IVM oocytes from transgender men. What is known already For transgender men, the fertility preservation strategy of ovarian stimulation may interfere with the desired masculine characteristics and enhance gender dysphoria. Alternatively, ovarian tissue oocytes collected ex vivo could serve as potential gametes, not requiring ovarian stimulation. Oocytes can be collected during gender affirming surgery, matured, and vitrified. Ovarian tissue oocyte in vitro maturation (OTO-IVM) has successfully been used for cancer patients, as live births have been reported. OTO-IVM in transgender men demonstrated sufficient maturation rates and survival following vitrification. Nevertheless, a decreased fertilization potential of these oocytes and severely compromised embryonic development have been observed. Study design, size, duration Patients between 18-24 years were recruited for this study from November 2020 to September 2021. Ovaries from 14 transgender men were collected in either cold (4oC, OTO-Cold) or warm (37oC, OTO-Warm) collection medium, to verify the best collection method. Following ovarian manipulation, cumulus oocyte complexes (COCs) were harvested from spent medium and underwent maturation for 48hrs. ST was performed to overcome inferior fertilization and embryonic development. Participants/materials, setting, methods Injected IVM oocytes underwent calcium imaging or were monitored for embryonic developmental potential. In vitro matured GV (germinal vesicle), MI (metaphase I) and in vivo matured oocytes with clusters of smooth endoplasmic reticulum (SERa) served as controls and cytoplasmic recipients for ST. OTO-IVM or control oocytes were used as spindle donors (ST-OTO or Control-ST respectively). Genetic analysis was performed to detect chromosomal abnormalities in embryos from all groups. Main results and the role of chance In total, we collected 252 OTO-Cold and 230 OTO-Warm oocytes, showing similar maturation rates (53%). For calcium imaging, 39 control, 33 OTO-cold and 31 OTO-warm oocytes were analysed, determining the product of amplitude per frequency, in arbitrary units (AU). The average value for control oocytes was 2.30AU, significantly higher than OTO-Cold (1.47AU, p=0.046) and OTO-Warm oocytes (1.03AU, p=0.036). Calcium release was similar between OTO-Cold and OTO-Warm oocytes. Following ICSI, 19/47 OTO-Cold and 24/48 OTO-Warm oocytes normally fertilized, significantly lower than the control group (42/52) (p Limitations, reasons for caution A major limitation of our study is the lack of ovaries from cis women. Control oocytes used in this study originate from infertility patients that underwent ovarian stimulation. High abnormality rate in ST-OTO embryos might be concerning for the safety of ST, but the number of embryos analysed is limited. Wider implications of the findings Our data indicate that OTO-IVM oocytes from transgender men display poor cytoplasmic quality, demonstrated by embryonic arrest and calcium imaging. ST was able to overcome poor embryo development, and it could be of interest to use freshly donated oocytes as cytoplasmic recipients for this. Trial registration number Not applicable

Study question Can cryopreserved-thawed human ovarian cortex be cultured in vitro to produce growing follicles as efficiently as from fresh samples? Summary answer In contrast to fresh samples, cryopreserved-thawed ovarian cortex did not develop morphologically normal growing follicles, even when adding follicular growth stimulators. What is known already A multistep culture system has been shown to produce mature oocytes from immature follicles present in the human cortex, without the addition of follicular growth stimulators. However, these promising results were achieved using fresh human ovarian cortex tissue donated from cisgender women, which is not available for the purpose of fertility preservation. It remains unclear whether this multistep culture system can be used to mature follicles from cryopreserved-thawed human ovarian cortex. In addition, the applicability of the culture system to mature follicles in vitro has not been investigated using ovarian tissue from transmasculine people. Study design, size, duration Fresh and cryopreserved ovarian cortex tissue isolated from the same transmasculine people were cultured during 8 days (first step of the culture system) with or without the addition of a PI3K/Akt promotor, Sphingosine-1-Phosphate (S1P). The ovarian cortex fragments were collected a day 0 and day 8 for downstream morphological and quantitative analysis of the follicular population. This study also included data of in vitro culture of cryopreserved samples from oncological cisgender women. Participants/materials, setting, methods Ovarian cortex tissue was collected from six testosterone user transmasculine people (25,8 ± 4,9 years) and three oncological cisgender women (24,0 ± 6,0 years). Fresh cortex was either directly cultured or previously cryopreserved. All ovarian fragments were cultured in the same conditions for 8 days. After the culture period, follicular population was quantified on histological sections (haematoxylin-eosin) and compared to samples of day 0. Follicular cell types, cell proliferation and apoptosis were assessed by immunofluorescence. Main results and the role of chance Fresh ovarian cortex tissue isolated from transmasculine people showed morphologically normal primary follicles (PFs) and secondary follicles (SFs) after 8 days of culture and no antral follicles were observed. Moreover, the addition of the follicular growth stimulator, S1P, to the culture medium significantly increased the number of growing follicles (PFs, P Limitations, reasons for caution Limitations of this study are: small sample size and lack of fresh cortex tissue from oncological cisgender women. Due to the high variability of follicular density among patients, the use of a larger number of samples is recommended. Wider implications of the findings Optimization of culture systems to grow and mature follicles in the ovarian cortex of cryopreserved samples (both from cis and trans persons) is urgently needed. Current culture protocols can be applied to fresh samples, but further characterization is needed to consider this as an alternative in fertility preservation treatment. Trial registration number Not applicable

Study question What is the effect of CRISPR/Cas9-mediated KO of trophectoderm (TE) markers GATA2/3 on embryo development and lineage commitment in both mouse and human preimplantation embryos? Summary answer CRISPR/Cas9-mediated KO of GATA2/3 points to interspecies differences in TE regulation and potential cross-talk between trophectoderm and inner cell mass (ICM) in mouse preimplantation embryos. What is known already GATA3 and its isoform GATA2 are major TE markers regulating the first lineage segregation, operating downstream of the HIPPO-pathway. Although recent evidence suggests that the HIPPO-pathway is conserved across mouse and human, it is unknown whether GATA2/3 share similar interspecies function during preimplantation development. GATA3 RNA depletion experiments in mouse embryos revealed a compensatory upregulation of GATA2, that potentially masked the observed phenotype. Upon double KO (DKO), the phenotype appeared more severe, as embryos were unable to cavitate. However, the precise effect of the (D)KO on embryo development was not investigated thoroughly, and should be expanded towards human preimplantation embryos. Study design, size, duration Mouse embryonic stem cells (mESCs), mouse zygotes and donated human spare oocytes were targeted. CRISPR/Cas9 ribonucleoprotein complexes, either targeting Gata3/GATA3, Gata2 or both, were delivered via nucleofection, electroporation or co-injected with sperm, in mESCs, mouse zygotes or human oocytes, respectively. Appropriate non-targeted control groups were included. Morphological analysis, immunofluorescence and next-generation sequencing were applied to check for gene editing efficiency and the impact of KO on embryonic development. Participants/materials, setting, methods The targeted embryos and controls were cultured for 4.5 (mouse) or 6.5 days (human) in vitro. They were stained for different developmental markers, including TEAD4 and CDX2 (TE), OCT4 and SOX2 (early ICM), NANOG (epibast, EPI) and SOX17 (hypoblast, PrE). Immunostaining was used to determine cell number, TE/ICM fraction, marker localization and fluorescence intensity. Embryos were subjected to genetic analysis to determine on-target efficiency, while in silico predicted off-target sites were evaluated in targeted mESCs. Main results and the role of chance GATA3 KO mouse embryos exhibited morula arrest (94%; n = 16). All GATA3-edited mouse embryos exhibited a reduction of CDX2-positive cells. From the 12 full KO embryos, four showed a decreased number of NANOG-positive blastomeres. No effect was observed for TEAD4 and OCT4. Complete KO morulas were devoid of SOX2 expression. GATA2 KO mouse embryos could still form blastocysts (19% morula arrest, n = 21 embryos), even when harboring 100% frameshift mutations. KO did not noticeably influence cell number nor the expression of GATA3 or NANOG. GATA2/3 DKO mouse embryos could still form blastocysts (38% morula arrest, n = 21), showing a milder phenotype compared to GATA3 KO embryos. In the presumed DKO blastocysts, the whole ICM is NANOG-positive and increased in cell number. SOX2 expression was still retained in the ICM, but presumed polar TE was also SOX2-positive. Three out of four GATA3 KO human embryos harboring 100% frameshift mutations, were surprisingly able to form blastocysts. In one embryo, no morphological TE could be formed, while the others showed one or two CDX2-positive cells in TE. No effect was observed for TEAD4 and OCT4. In addition, all KO embryos displayed an increase in ICM/TE fraction. Some outer cells in KO blastocysts were NANOG-positive. Limitations, reasons for caution CRISPR/Cas9 is limited by the occurrence of mosaicism (more than one genotype present in an embryo) and potential off-target editing, which we will assess at in silico predicted off-target sites via NGS in mESCs. The observations of the study will be consolidated by increasing the sample size, especially in human. Wider implications of the findings Gene editing studies enable us to unravel the molecular interactions that are required for human preimplantation development. Obtaining novel insights into the molecular networks of the GATA transcription factor family could significantly improve our understanding of several pregnancy-related complications related to trophectoderm specification, such as early miscarriage or preeclampsia. Trial registration number NA

Contains fulltext : 51414.pdf (Publisher’s version ) (Closed access) The synovial sarcoma-associated protein SS18 (also known as SYT or SSXT) is thought to act as a transcriptional co-activator. This activity appears to be mediated through the SWI/SNF proteins BRG1 and INI1 and the histone acetyl transferase p300. Here, we report that disruption of the mouse Ss18 gene results in a recessive embryonic lethal phenotype, due to placental failure caused by impairment of placental vascularization and/or chorio-allantoic fusion. This phenotype resembles the p300 knockout phenotype, but is distinct from the Brg1 and Ini1 knockout phenotypes. Through expression profiling of knockout embryos, we observed altered expression of genes known to affect placental development, including the peroxisome proliferator-activated receptor-binding protein (Pparbp). Since Pparbp null mutant embryos display a similar, lethal phenotype with placental failure, we suggest that the functional and phenotypic co-linearities between Ss18 and p300 may also include the transcriptional co-activator Pparbp. Additional interbreeding of Ss18 and Ss18l1 (Crest) mutant mice indicates that these two functionally and structurally related genes may act synergistically during critical stages of embryonic development.