An atlas of small non-coding RNAs in human preimplantation development.

Understanding the molecular circuitries that govern early embryogenesis is important, yet our knowledge of these in human preimplantation development remains limited. Small non-coding RNAs (sncRNAs) can regulate gene expression and thus impact blastocyst formation, however, the expression of specific biotypes and their dynamics during preimplantation development remains unknown. Here we identify the abundance of and kinetics of piRNA, rRNA, snoRNA, tRNA, and miRNA from embryonic day (E)3-7 and isolate specific miRNAs and snoRNAs of particular importance in blastocyst formation and pluripotency. These sncRNAs correspond to specific genomic hotspots: an enrichment of the chromosome 19 miRNA cluster (C19MC) in the trophectoderm (TE), and the chromosome 14 miRNA cluster (C14MC) and MEG8-related snoRNAs in the inner cell mass (ICM), which may serve as 'master regulators' of potency and lineage. Additionally, we observe a developmental transition with 21 isomiRs and in tRNA fragment (tRF) codon usage and identify two novel miRNAs. Our analysis provides a comprehensive measure of sncRNA biotypes and their corresponding dynamics throughout human preimplantation development, providing an extensive resource. Better understanding the sncRNA regulatory programmes in human embryogenesis will inform strategies to improve embryo development and outcomes of assisted reproductive technologies. We anticipate broad usage of our data as a resource for studies aimed at understanding embryogenesis, optimising stem cell-based models, assisted reproductive technology, and stem cell biology.

Non-invasively predicting euploidy in human blastocysts via quantitative 3D morphology measurement: a retrospective cohort study.

BACKGROUND: Blastocyst morphology has been demonstrated to be associated with ploidy status. Existing artificial intelligence models use manual grading or 2D images as the input for euploidy prediction, which suffer from subjectivity from observers and information loss due to incomplete features from 2D images. Here we aim to predict euploidy in human blastocysts using quantitative morphological parameters obtained by 3D morphology measurement. METHODS: Multi-view images of 226 blastocysts on Day 6 were captured by manually rotating blastocysts during the preparation stage of trophectoderm biopsy. Quantitative morphological parameters were obtained by 3D morphology measurement. Six machine learning models were trained using 3D morphological parameters as the input and PGT-A results as the ground truth outcome. Model performance, including sensitivity, specificity, precision, accuracy and AUC, was evaluated on an additional test dataset. Model interpretation was conducted on the best-performing model. RESULTS: All the 3D morphological parameters were significantly different between euploid and non-euploid blastocysts. Multivariate analysis revealed that three of the five parameters including trophectoderm cell number, trophectoderm cell size variance and inner cell mass area maintained statistical significance (P < 0.001, aOR = 1.054, 95% CI 1.034-1.073; P = 0.003, aOR = 0.994, 95% CI 0.991-0.998; P = 0.010, aOR = 1.003, 95% CI 1.001-1.006). The accuracy of euploidy prediction by the six machine learning models ranged from 80 to 95.6%, and the AUCs ranged from 0.881 to 0.984. Particularly, the decision tree model achieved the highest accuracy of 95.6% (95% CI 84.9-99.5%) with the AUC of 0.978 (95% CI 0.882-0.999), and the extreme gradient boosting model achieved the highest AUC of 0.984 (95% CI 0.892-1.000) with the accuracy of 93.3% (95% CI 81.7-98.6%). No significant difference was found between different age groups using either decision tree or extreme gradient boosting to predict euploid blastocysts. The quantitative criteria extracted from the decision tree imply that euploid blastocysts have a higher number of trophectoderm cells, larger inner cell mass area, and smaller trophectoderm cell size variance compared to non-euploid blastocysts. CONCLUSIONS: Using quantitative morphological parameters obtained by 3D morphology measurement, the decision tree-based machine learning model achieved an accuracy of 95.6% and AUC of 0.978 for predicting euploidy in Day 6 human blastocysts. TRIAL REGISTRATION: N/A.

Factors associated with decision outcomes along the planned oocyte cryopreservation pathway in a cohort of women who sought consultation before and during the COVID-19 pandemic.

PURPOSE: To identify factors associated with three decision outcomes along the planned oocyte cryopreservation (POC) pathway: fertility assessment completion, POC uptake, and multiple POC cycles uptake. METHODS: A single-site retrospective cohort study of 425 patients who sought POC consultation from 2018-2022 before and during the COVID-19 pandemic. RESULTS: The mean age of patients at consultation was 35.2 ± 4.0. 73% (n = 310) of the new consultations occurred since the COVID-19 pandemic. 335 patients completed fertility assessment following consultation. Of the 335 patients (78.8%) completing fertility assessment, 139 (32.7%) underwent at least one cycle. The odds of completing fertility assessment were 3.65 times greater for patients being seen pre-pandemic than for those being seen during the pandemic (p < 0.001). Not having a committed partner (OR 2.61, p < 0.001) and not having a prior pregnancy history (OR 1.99, p = 0.03) positively predicted POC uptake. Each additional number of cryopreserved MII oocytes in the first cycle reduced the odds of undergoing more cycles by 0.74 times (p < 0.001). Of the 196 patients with no POC uptake despite having a fertility consultation, 10 decided to do nothing, 16 decided to get pregnant with or without a partner, and the other 170 had not yet indicated their decision. CONCLUSION: Our centre experienced a surge of patients seeking POC consultation since the COVID-19 outbreak. Approximately one-third of these patients progressed to the POC stage. Our findings validate the complexity of POC decision-making and the value of providing ongoing decision support to patients along the POC pathway.

Extracellular vesicles and their content in the context of polycystic ovarian syndrome and endometriosis: a review.

Extracellular vesicles (EVs), particles enriched in bioactive molecules like proteins, nucleic acids, and lipids, are crucial mediators of intercellular communication and play key roles in various physiological and pathological processes. EVs have been shown to be involved in ovarian follicular function and to be altered in two prevalent gynecological disorders; polycystic ovarian syndrome (PCOS) and endometriosis.Ovarian follicles are complex microenvironments where folliculogenesis takes place with well-orchestrated interactions between granulosa cells, oocytes, and their surrounding stromal cells. Recent research unveiled the presence of EVs, including exosomes and microvesicles, in the follicular fluid (FFEVs), which constitutes part of the developing oocyte's microenvironment. In the context of PCOS, a multifaceted endocrine, reproductive, and metabolic disorder, studies have explored the dysregulation of these FFEVs and their cargo. Nine PCOS studies were included in this review and two miRNAs were commonly reported in two different studies, miR-379 and miR-200, both known to play a role in female reproduction. Studies have also demonstrated the potential use of EVs as diagnostic tools and treatment options.Endometriosis, another prevalent gynecological disorder characterized by ectopic growth of endometrial-like tissue, has also been linked to aberrant EV signaling. EVs in the peritoneal fluid of women with endometriosis carry molecules that modulate the immune response and promote the establishment and maintenance of endometriosis lesions. EVs derived from endometriosis lesions, serum and peritoneal fluid obtained from patients with endometriosis showed no commonly reported biomolecules between the eleven reviewed studies. Importantly, circulating EVs have been shown to be potential biomarkers, also reflecting the severity of the pathology.Understanding the interplay of EVs within human ovarian follicles may provide valuable insights into the pathophysiology of both PCOS and endometriosis. Targeting EV-mediated communication may open avenues for novel diagnostic and therapeutic approaches for these common gynecological disorders. More research is essential to unravel the mechanisms underlying EV involvement in folliculogenesis and its dysregulation in PCOS and endometriosis, ultimately leading to more effective and personalized interventions.

Prenatal cell-free DNA screening for chromosomal aneuploidies after euploid embryo transfer shows high concordance with preimplantation genetic testing for aneuploidy results and low positive predictive values.

OBJECTIVE: To evaluate the positive predictive value (PPV) of prenatal cell-free DNA (cfDNA) screening for chromosomal aneuploidies in pregnancies achieved either after single euploid transfer in in vitro fertilization or Preimplantation Genetic Testing for Aneuploidy (PGT-A) cycles or transfer of single untested embryo, and to assess the concordance of prenatal-cfDNA-screening and PGT-A results. DESIGN: Single center retrospective cohort study. SETTING: Fertility clinic. PATIENT(S): A total of 2,973 prenatal-cfDNA-screening results for the most common trisomies (T13, T18, T21, X, and Y) and microdeletions (1p36, 4p16.3, 5p15.2, 15q11.2, and 22q11.2) from singleton pregnancies allocated into two groups: PGT-A group (n = 1,204) pregnant after single euploid transfer and non-PGT-A group (n = 1769) pregnant after transfer of single untested embryo, between 2016 and 2023. INTERVENTION(S): Not applicable. MAIN OUTCOME MEASURE(S): The primary outcome measure was the accuracy of prenatal-cfDNA screening. Positive and negative prenatal-cfDNA-screening results and subsequent prenatal or postnatal diagnostic testing were used to classify each positive prenatal-cfDNA-screening result as a true or a false positive. Secondary endpoints were to evaluate the concordance of PGT-A and prenatal-cfDNA-screening results and to assess the differences in the fetal fraction of cfDNA used for prenatal-cfDNA-screening reports between the study groups. RESULT(S): Prenatal-cfDNA screening was performed at a mean 11.3 ± 1.8 weeks gestational age, and yielded results in 99.9% of the patients (0.1% cancellation rate). There was no difference in the fetal fraction between PGT-A tested and not tested pregnancies (9.5% ± 4% vs. 10.3% ± 4%). 13 positive prenatal-cfDNA-screening results (two T21, two X0, four XXX, one XYY, one indeterminate sex, two 22q11 del/dup, and one 15q11.2del) were received for PGT-A group. Only one (22q11 dup) was confirmed with amniocentesis and fetal autopsy, giving a PPV for an abnormal prenatal-cfDNA screening of 7.7%, the rest had results concordant with PGT-A. Sex chromosomes were 100% concordant between prenatal-cfDNA-screening and PGT-A results, giving a 100% PPV for PGT-A for sex chromosomes and 100% negative predictive value for aneuploidies. Positive prenatal-cfDNA-screening results were received for 27 pregnancies from untested embryos (1.5%), follow-up testing was electively performed for 21, and 8 had confirmed the prenatal-cfDNA-screening result, giving a PPV for the non-PGT-A group of 38%. CONCLUSION(S): This study demonstrates that patients undergoing in vitro fertilization/PGT-A and single euploid embryo transfer can reliably do prenatal-cfDNA screening during their first trimester. Fetal fraction in singleton pregnancies after PGT-A tested embryos is not different from pregnancies with untested embryos. Positive predictive value for an abnormal prenatal-cfDNA-screening result after euploid embryo transfer was reassuringly low (7.7%). PGT-A reliably selects against aneuploidy with 100% concordance with fetal sex.

CP-Net: Instance-aware part segmentation network for biological cell parsing.

Instance segmentation of biological cells is important in medical image analysis for identifying and segmenting individual cells, and quantitative measurement of subcellular structures requires further cell-level subcellular part segmentation. Subcellular structure measurements are critical for cell phenotyping and quality analysis. For these purposes, instance-aware part segmentation network is first introduced to distinguish individual cells and segment subcellular structures for each detected cell. This approach is demonstrated on human sperm cells since the World Health Organization has established quantitative standards for sperm quality assessment. Specifically, a novel Cell Parsing Net (CP-Net) is proposed for accurate instance-level cell parsing. An attention-based feature fusion module is designed to alleviate contour misalignments for cells with an irregular shape by using instance masks as spatial cues instead of as strict constraints to differentiate various instances. A coarse-to-fine segmentation module is developed to effectively segment tiny subcellular structures within a cell through hierarchical segmentation from whole to part instead of directly segmenting each cell part. Moreover, a sperm parsing dataset is built including 320 annotated sperm images with five semantic subcellular part labels. Extensive experiments on the collected dataset demonstrate that the proposed CP-Net outperforms state-of-the-art instance-aware part segmentation networks.

piRNA expression patterns in high vs. low fertility bovine sperm.

PIWI-interacting RNAs (piRNAs) are 24-32 nucleotide RNA sequences primarily expressed in germ cells and developing embryos that suppress transposable element expression to protect genomic integrity during epigenetic reprogramming events. We characterized the expression of piRNA sequences and their encoding clusters in sperm samples from an idiopathic fertility model of Holstein bulls with high and low Sire Conception Rates. The piRNA populations were determined to be mostly similar between fertility conditions when investigated by principal component and differential expression analysis, suggesting that a high degree of conservation in the piRNA system is likely necessary for the production of viable sperm. Both fertility conditions demonstrated evidence of 'ping-pong' activity - a secondary biogenesis pathway associated with active transposable element targeting and suppression. Most sperm-borne piRNAs were between 29-30 nucleotides in length and originated from 226 clusters across the genome, with the exception of chromosome 20. Mapping analysis revealed abundant targeting of several transposable element families, suggesting a suppressive function of sperm piRNAs consistent with their established roles. Expression of genes targeted by sperm-borne piRNAs is significantly reduced throughout early embryogenesis compared to the mRNA population. Limited transposable element expression is known to be essential for spermatogenesis, thus epigenetic regulation of this pathway is likely to influence sperm quality and fertilizing capacity.

A comprehensive analysis of spermatozoal RNA elements in idiopathic infertile males undergoing fertility treatment.

Current approaches to diagnosing male infertility inadequately assess the complexity of the male gamete. Beyond the paternal haploid genome, spermatozoa also deliver coding and non-coding RNAs to the oocyte. While sperm-borne RNAs have demonstrated potential involvement in embryo development, the underlying mechanisms remain unclear. In this study, 47 sperm samples from normozoospermic males undergoing fertility treatment using donor oocytes were sequenced and analyzed to evaluate associations between sperm RNA elements (exon-sized sequences) and blastocyst progression. A total of 366 RNA elements (REs) were significantly associated with blastocyst rate (padj < 0.05), some of which were linked to genes related to critical developmental processes, including mitotic spindle formation and both ectoderm and mesoderm specification. Of note, 27 RE-associated RNAs are predicted targets of our previously reported list of developmentally significant miRNAs. Inverse RE-miRNA expression patterns were consistent with miRNA-mediated down-regulation. This study provides a comprehensive set of REs which differ by the patient's ability to produce blastocysts. This knowledge can be leveraged to improve clinical screening of male infertility and ultimately reduce time to pregnancy.

Testing the generalizability and effectiveness of deep learning models among clinics: sperm detection as a pilot study.

BACKGROUND: Deep learning has been increasingly investigated for assisting clinical in vitro fertilization (IVF). The first technical step in many tasks is to visually detect and locate sperm, oocytes, and embryos in images. For clinical deployment of such deep learning models, different clinics use different image acquisition hardware and different sample preprocessing protocols, raising the concern over whether the reported accuracy of a deep learning model by one clinic could be reproduced in another clinic. Here we aim to investigate the effect of each imaging factor on the generalizability of object detection models, using sperm analysis as a pilot example. METHODS: Ablation studies were performed using state-of-the-art models for detecting human sperm to quantitatively assess how model precision (false-positive detection) and recall (missed detection) were affected by imaging magnification, imaging mode, and sample preprocessing protocols. The results led to the hypothesis that the richness of image acquisition conditions in a training dataset deterministically affects model generalizability. The hypothesis was tested by first enriching the training dataset with a wide range of imaging conditions, then validated through internal blind tests on new samples and external multi-center clinical validations. RESULTS: Ablation experiments revealed that removing subsets of data from the training dataset significantly reduced model precision. Removing raw sample images from the training dataset caused the largest drop in model precision, whereas removing 20x images caused the largest drop in model recall. by incorporating different imaging and sample preprocessing conditions into a rich training dataset, the model achieved an intraclass correlation coefficient (ICC) of 0.97 (95% CI: 0.94-0.99) for precision, and an ICC of 0.97 (95% CI: 0.93-0.99) for recall. Multi-center clinical validation showed no significant differences in model precision or recall across different clinics and applications. CONCLUSIONS: The results validated the hypothesis that the richness of data in the training dataset is a key factor impacting model generalizability. These findings highlight the importance of diversity in a training dataset for model evaluation and suggest that future deep learning models in andrology and reproductive medicine should incorporate comprehensive feature sets for enhanced generalizability across clinics.

Feasibility of intranasal human milk as stem cell therapy in preterm infants with intraventricular hemorrhage.

OBJECTIVE: Intraventricular hemorrhage (IVH) is a common cause of preterm brain injury. Fresh parent's own milk (POM) contains pluripotent stem cells (SCs) that produce neuronal cells in-vitro. The permeable neonatal blood brain barrier potentially allows SC delivery. We performed the first prospective trial (clinicaltrials.gov NCT04225286) of feasibility of intranasal POM (IPOM) in preterm infants with IVH and described SC content of POM samples. STUDY DESIGN: 37 Infants (mean gestation 27.7 ± 2.6 weeks, birthweight 1030 ± 320 g) with IVH (35.1% grade IV) were recruited from two tertiary Toronto NICUs. IPOM was given ideally twice daily until 28 days of age. Tolerance and adverse reactions were collected and 162 administering providers surveyed. RESULTS: There were no major adverse reactions. Provider surveys suggested acceptability, although potential provider and subject stress requires further study. Milk cell analysis suggests wide variability between parents. CONCLUSIONS: This phase 1 study demonstrated IPOM was tolerated and feasible in preterm infants.

Parenthood desire, perceived parenthood stigma, and barriers to achieving parenthood in childless sexual minority men.

PURPOSE: To examine the parenthood desire, perceived parenthood stigma, and barriers to achieving parenthood among sexual minority men (SMM) in Canada, and to investigate factors influencing their fertility and assisted reproductive knowledge. METHODS: Data were collected from March to mid-June 2023 using a 78-item anonymous online survey. Childless cisgender SMM (age 18+) living in Canada were recruited from the LGBTQIA+ community outside the fertility care networks. Chi-square, t-tests, ANOVA, reliability tests, Spearman's correlation, and hierarchical regression model were used for analysis. RESULTS: Over 160 people clicked the survey hyperlink during the study period and 112 completed surveys were analyzed. The mean age of participants was 33.2±8.5 (range: 19.7-60.0). Having a child by any means was "quite"/"very" important to 35.7% (n=40), yet 56.0% (n=61) thought it was "unlikely" to achieve parenthood. Financial readiness (n=90, 85.7%) and relationship stability (n=86, 81.9%) were the two most "important" parenthood considerations. Participants who were non-white (p=0.017), under age 30 (p=0.008), and had no siblings (p=0.024) had significantly higher means of parenthood desire compared to others. The final hierarchical regression model explained 43% of the variance in the knowledge scores (R2adj =0.353), predicted by the levels of (i) education (ß=0.37, p<0.001), (ii) family acceptance of sexual orientation (ß=0.39, p=0.004), and (iii) parenthood desire (ß=0.27, p=0.002). CONCLUSIONS: With an increasing number of SMM desiring children, it is pivotal to advance family-building equality through improving their fertility and assisted reproductive knowledge, removing disparities in accessing adoption and assisted reproductive services, and decreasing social stigma against SMM having children.

Single-Cell mRNA-sncRNA Co-sequencing of Preimplantation Embryos.

The development of single-cell multiomics has provided the ability to systematically investigate cellular diversity and heterogeneity in different biological systems via comprehensive delineations of individual cellular states. Single-cell RNA sequencing in particular has served as a powerful tool to the study of the molecular circuitries underlying preimplantation embryonic development in both the mouse and human. Here we describe a method to elucidate the cellular dynamics of the embryo further by performing both single-cell RNA sequencing (Smart-Seq2) and single-cell small non-coding RNA sequencing (Small-Seq) on the same individual embryonic cell.

Aberrant landscapes of maternal meiotic crossovers contribute to aneuploidies in human embryos.

Meiotic recombination is crucial for human genetic diversity and chromosome segregation accuracy. Understanding its variation across individuals and the processes by which it goes awry are long-standing goals in human genetics. Current approaches for inferring recombination landscapes rely either on population genetic patterns of linkage disequilibrium (LD)-capturing a time-averaged view-or on direct detection of crossovers in gametes or multigeneration pedigrees, which limits data set scale and availability. Here, we introduce an approach for inferring sex-specific recombination landscapes using data from preimplantation genetic testing for aneuploidy (PGT-A). This method relies on low-coverage (<0.05×) whole-genome sequencing of in vitro fertilized (IVF) embryo biopsies. To overcome the data sparsity, our method exploits its inherent relatedness structure, knowledge of haplotypes from external population reference panels, and the frequent occurrence of monosomies in embryos, whereby the remaining chromosome is phased by default. Extensive simulations show our method's high accuracy, even at coverages as low as 0.02×. Applying this method to PGT-A data from 18,967 embryos, we mapped 70,660 recombination events with ∼150 kbp resolution, replicating established sex-specific recombination patterns. We observed a reduced total length of the female genetic map in trisomies compared with disomies, as well as chromosome-specific alterations in crossover distributions. Based on haplotype configurations in pericentromeric regions, our data indicate chromosome-specific propensities for different mechanisms of meiotic error. Our results provide a comprehensive view of the role of aberrant meiotic recombination in the origins of human aneuploidies and offer a versatile tool for mapping crossovers in low-coverage sequencing data from multiple siblings.

A qualitative analysis of gestational surrogates' healthcare experiences during the COVID-19 pandemic.

OBJECTIVE: No empirical data are available on the healthcare experiences of surrogates during the COVID-19 pandemic. This study aimed to examine the impact of pandemic-control measures on surrogates' fertility, pregnancy and birthing experiences. METHODS: Sampling frame included eligible surrogates who were actively involved in a surrogacy process at an academic IVF centre during the pandemic (03/2020 to 02/2022). Data were collected between 29/04/2022 and 31/07/2022 using an anonymous 85-item online survey that included twelve open-ended questions. Free-text comments were analysed by thematic analysis. FINDINGS: The response rate was 50.7% (338/667). Of the 320 completed surveys used for analysis, 609 comments were collected from 206 respondents. Twelve main themes and thirty-six sub-themes grouped under 'vaccination', 'fertility treatment', 'pregnancy care', and 'surrogacy birth' were identified. Three in five surrogates found the control measures highly or moderately affected their surrogacy experiences. Themes involving loneliness and isolation frequently emerged when essential surrogacy support was restricted by the visitor protocols implemented at healthcare facilities. DISCUSSION: Our findings show that restricting or limiting intended parents' in-person involvement increased surrogates' feelings of isolation and made the overall surrogacy experience less rewarding and fulfilling. Furthermore, the childbirth experiences of surrogates were mostly negative, suggesting that hospitals were ill-equipped to manage all births, including surrogacy births, during the pandemic. IMPLICATIONS FOR PRACTICE: Our findings highlight the needs to rethink how surrogacy care and maternity services could be strengthened to better serve the needs of surrogates during times of public health crises, such as COVID-19, while still allowing for risk mitigation and maximising patient safety.

Chromosomal, gestational, and neonatal outcomes of embryos classified as a mosaic by preimplantation genetic testing for aneuploidy.

OBJECTIVE: To understand the clinical risks associated with the transfer of embryos classified as a mosaic using preimplantation genetic testing for aneuploidy. DESIGN: Analysis of data collected between 2017 and 2023. SETTING: Multicenter. PATIENTS: Patients of infertility treatment. INTERVENTION: Comparison of pregnancies resulting from embryos classified as euploid or mosaic using the 20%-80% interval in chromosomal intermediate copy numbers to define a mosaic result. MAIN OUTCOME MEASURES: Rates of spontaneous abortion, birth weight, length of gestation, incidence of birth defects, and chromosomal status during gestation. RESULTS: Implanted euploid embryos had a significantly lower risk of spontaneous abortion compared with mosaic embryos (8.9% [n = 8,672; 95% confidence interval {CI95} 8.3, 9.5] vs. 22.2% [n = 914; CI95 19.6, 25.0]). Embryos with mosaicism affecting whole chromosomes (not segmental) had the highest risk of spontaneous abortion (27.6% [n = 395; CI95 23.2, 32.3]). Infants born from euploid, mosaic, and whole chromosome mosaic embryos had average birth weights and lengths of gestation that were not statistically different (3,118 g and 267 days [n = 488; CI95 3,067, 3,169, and 266, 268], 3052 g and 265 days [n = 488; CI95 2,993, 3,112, and 264,267], 3,159 g and 268 days [n = 194; CI95 3,070, 3,249, and 266,270], respectively). Out of 488 infants from mosaic embryo transfers (ETs), one had overt gross abnormalities as defined by the Centers for Disease Control and Prevention. Most prenatal tests performed on pregnancies from mosaic ETs had normal results, and only three pregnancies produced prenatal test results reflecting the mosaicism detected at the embryonic stage (3 out of 250, 1.2%; CI95 0.25, 3.5). CONCLUSION: Although embryos classified as mosaic experience higher rates of miscarriage than euploid embryos (with a particularly high frequency shortly after implantation), infants born of mosaic ETs are similar to infants of euploid ETs. Prenatal testing indicates that mosaicism resolves during most pregnancies, although this process is not perfectly efficient. In a small percentage of cases, the mosaicism persists through gestation. These findings can serve as risk-benefit considerations for mosaic ETs in the fertility clinic.

Combination human umbilical cord perivascular and endothelial colony forming cell therapy for ischemic cardiac injury.

Cell-based therapeutics are promising interventions to repair ischemic cardiac tissue. However, no single cell type has yet been found to be both specialized and versatile enough to heal the heart. The synergistic effects of two regenerative cell types including endothelial colony forming cells (ECFC) and first-trimester human umbilical cord perivascular cells (FTM HUCPVC) with endothelial cell and pericyte properties respectively, on angiogenic and regenerative properties were tested in a rat model of myocardial infarction (MI), in vitro tube formation and Matrigel plug assay. The combination of FTM HUCPVCs and ECFCs synergistically reduced fibrosis and cardiomyocyte apoptosis, while promoting favorable cardiac remodeling and contractility. These effects were in part mediated by ANGPT2, PDGF-ß, and VEGF-C. PDGF-ß signaling-dependent synergistic effects on angiogenesis were also observed in vitro and in vivo. FTM HUCPVCs and ECFCs represent a cell combination therapy for promoting and sustaining vascularization following ischemic cardiac injury.

Neuropeptide Y directly reduced apoptosis of granulosa cells, and the expression of NPY and its receptors in PCOS subjects.

BACKGROUND: Most women with anovulatory infertility show polycystic ovarian syndrome (PCOS), and androgen excess is known as a key factor involved in pathogenicity of PCOS. However, the mechanism of follicular developmental arrest in PCOS is not completely understood. The reproductive function of Neuropeptide Y (NPY) in the ovary during folliculogenesis was previously reported; NPY function in apoptosis and proliferation of granulosa cells (GCs) is follicular-stage dependent. The objective of this study was to investigate the role of NPY in ovarian follicular development and the pathogenesis of PCOS. METHODS: To simulate the PCOS phenotype using a rat model, 21-day old Sprague Dawley rats were implanted with dihydrotestosterone (DHT) capsule (83 µg/day) and euthanized after 28 days. mRNA and protein content of NPY and its receptors were assessed in GCs from DHT treated rats using RT-qPCR and Western blot, respectively. Proliferation and apoptosis of GCs was assessed using Ki67- and TUNEL assays. Finally, NPY levels were measured in human follicular fluid (FF) from matched PCOS and non-PCOS patients using ELISA. RESULTS: GCs from DHT treated rats (PCOS-GCs) contained significantly less NPY protein and Npy mRNA by 0.16- and 0.56-fold, respectively, and more NPY receptor type 2 and 5 protein by 2.21- and 3.17-fold, respectively, when compared to sham control. Addition of recombinant NPY to PCOS-GCs culture did not alter Ki67-positive but significantly decreased TUNEL-positive cells by 0.65-fold, but not to baseline levels. There was no significant difference in NPY levels in FF between PCOS and non-PCOS subjects. CONCLUSIONS: These results indicate that DHT modulates expression of NPY and its receptors, NPY decreases DHT-induced GCs apoptosis. That alterations in NPY's function might be involved in follicular developmental failure of PCOS.

Obesity and PCOS radically alters the snRNA composition of follicular fluid extracellular vesicles.

Introduction: The ovarian follicle consists of the oocyte, somatic cells, and follicular fluid (FF). Proper signalling between these compartments is required for optimal folliculogenesis. The association between polycystic ovarian syndrome (PCOS) and extracellular vesicular small non-coding RNAs (snRNAs) signatures in follicular fluid (FF) and how this relates to adiposity is unknown. The purpose of this study was to determine whether FF extracellular vesicle (FFEV)-derived snRNAs are differentially expressed (DE) between PCOS and non-PCOS subjects; and if these differences are vesicle-specific and/or adiposity-dependent. Methods: FF and granulosa cells (GC) were collected from 35 patients matched by demographic and stimulation parameters. FFEVs were isolated and snRNA libraries were constructed, sequenced, and analyzed. Results: miRNAs were the most abundant biotype present, with specific enrichment in exosomes (EX), whereas in GCs long non-coding RNAs were the most abundant biotype. In obese PCOS vs. lean PCOS, pathway analysis revealed target genes involved in cell survival and apoptosis, leukocyte differentiation and migration, JAK/STAT, and MAPK signalling. In obese PCOS FFEVs were selectively enriched (FFEVs vs. GCs) for miRNAs targeting p53 signalling, cell survival and apoptosis, FOXO, Hippo, TNF, and MAPK signalling. Discussion: We provide comprehensive profiling of snRNAs in FFEVs and GCs of PCOS and non-PCOS patients, highlighting the effect of adiposity on these findings. We hypothesize that the selective packaging and release of miRNAs specifically targeting anti-apoptotic genes into the FF may be an attempt by the follicle to reduce the apoptotic pressure of the GCs and stave off premature apoptosis of the follicle observed in PCOS.

Non-identified and directed embryo donation: a questionnaire study on donor and recipient perspectives.

With the growing challenge of abandoned surplus embryos in the ART arena, and the limited traction of embryo donation as a viable embryo disposition choice, it is important to better understand barriers to wider adoption of this opportunity. We aim to learn about perspectives and experience of participants in directed and non-identified embryo donation programmes. This was a longitudinal cohort survey study, of all participants in an embryo donation programme in a single university affiliated clinic between 2016 and 2020. Clinical data were extracted from counselling reports. Based on these data, non-identified online questionnaires were constructed and refined via Delphi procedure for face and content validity. Sixty-five online questionnaires were emailed between March-April 2021. Descriptive statistics, cross-tabulation, Fisher's exact test and t-test were used for analyses. Source of patient awareness, factors influencing the decision-making process, patient perspective and satisfaction were explored. The response rate was 67.2%. Most participants in the non-identified programme learned of it through their treating physicians, whereas most participants in the directed programme learned of it online. The main driver to donate across both cohorts was wanting to give others the opportunity to experience the joy of parenthood. Overall, 45% described moderate to marked difficulty in decision making related to donating their embryos, and this did not differ between cohorts. Non-identified donors reported feeling highly attached to the donated embryos more often than directed donors. Level of satisfaction was higher in the directed donation programme. Participants were more satisfied following directed than non-identified donation, and some even consider their counterparts as extended family. Our findings should be validated in various settings, and on larger samples.