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.

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.

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.

The developmental potential of mature oocytes derived from rescue in vitro maturation.

OBJECTIVE: To examine the developmental competence of immature oocytes in stimulated cycles, that matured after rescue in vitro maturation (IVM) compared with their sibling in vivo matured oocytes. DESIGN: Retrospective cohort study. SETTING: IVF clinic. PATIENTS: A total of 182 patients underwent 200 controlled ovarian stimulation cycles with intracytoplasmic sperm injection cycles in which immature oocytes were retrieved and at least one mature oocyte was obtained through rescue IVM. INTERVENTION: In vitro culture of immature germinal vesicle (GV) and metaphase I (MI) oocytes, retrieved in stimulated cycles. MAIN OUTCOME MEASURES: Fertilization rate, cleavage rate, blastulation rate, ploidy of embryos evaluated using preimplantation genetic testing for aneuploidy, morphokinetic parameters and pregnancy outcomes. RESULTS: In total, 2,288 oocytes were retrieved from 200 cycles. After denudation, 1,056 of the oocytes (46% ± 16%) were classified as metaphase II (MII). A total of 333/375 (89%) of MI oocytes and 292/540 (54%) of GV oocytes matured overnight and underwent intracytoplasmic sperm injection. The fertilization rates of matured oocytes from MI rescue IVM (R-MI) and from GV rescue IVM (R-GV) were comparable with those of their sibling MII oocytes (71% vs. 66%; 66% vs. 63%, respectively). Early cleavage rates (80% ± 35% vs. 92% ± 20%; 80% ± 42% vs. 95% ± 28%, respectively) and blastulation rates (32 ± 40% vs. 62 ± 33%; 24 ± 37% vs. 60 ± 35%, respectively) were significantly decreased in rescue IVM matured oocytes (R-oocytes)-derived zygotes, but the blastocyst (BL) euploidy rate and "good quality" BL rate were comparable with those of MII sibling-derived embryos. In addition, rescue IVM embryos showed significantly higher levels of multinucleation at the 2- and 4-cell stages, as well as higher rates of zygote direct cleavage from one to 3 to 4 cells. Overall, 21 transfers of rescue IVM embryos resulted in 3 healthy live births. CONCLUSIONS: For patients with a low maturation rate and/or low numbers of mature oocytes at retrieval, rescue IVM may contribute more competent oocytes and additional viable BLs for transfer from the same stimulation cycle, maximizing the chances for pregnancy and live birth.

A new formula for estimating gestational age by crown-rump length.

BACKGROUND: The accurate estimation of gestational age by ultrasound is crucial in prenatal care for the monitoring of fetal growth and development. As changes in maternal childbearing age, body habitus, and ultrasound technology occur, previously published formulas may not be accurate for today's population. OBJECTIVE: This study aimed to develop new formulas for calculating the gestational age based on a first-trimester ultrasound scan and to compare the new formulas with preexisting formulas. STUDY DESIGN: This study was a single-center, retrospective observational study that included pregnancies conceived using in vitro fertilization. The pregnancies had known dates of embryo transfer and multiple standard ultrasound examinations in the first trimester of pregnancy. Pregnancies ending with a miscarriage or termination in the first trimester of pregnancy were excluded. A polynomial regression analysis was performed to determine the optimal model that represented the relationship between gestational age and crown-rump length. The models were evaluated using systematic error, random error, absolute difference of the calculated gestational age and actual gestational age, and proportion of estimation within 0 and 2 days of the known gestational age. The optimal model was chosen and compared with preexisting formulas. RESULTS: Overall, 1436 ultrasound results were included in the analysis. The analysis produced 3 models: linear, cubic, and quadratic models with correlation coefficients of 0.968, 0.989, and 0.991, respectively. The cubic formula was superior to the linear and quadratic formulas concerning systematic error, random error, absolute difference, and proportion of estimation within 0 and 2 days. The new formula had a lower systematic error, random error, and mean absolute difference (0.06%, 2.43%, and 0.97 days, respectively) and the highest proportion of estimation within 0 and 2 days (37.4% and 93.5%, respectively) than previously published formulas. CONCLUSION: The formula proposed in this study followed a cubic model and seemed to be able to more accurately estimate gestational age in the first trimester of pregnancy based on crown-rump length compared with previously published formulas.

First trimester human umbilical cord perivascular cells (HUCPVC) modulate the kynurenine pathway and glutamate neurotransmission in an LPS-induced mouse model of neuroinflammation.

BACKGROUND: The Kynurenine Pathway (KP) of tryptophan degradation and glutamate toxicity is implicated in several neurological disorders, including depression. The therapeutic potential of mesenchymal stromal cells (MSC), owing to their well documented phagocytosis-driven mechanism of immunomodulation and neuroprotection, has been tested in many neurological disorders. However, their potential to influence KP and the glutamatergic system has not yet been investigated. Hence, this study sought to investigate the effect of HUCPVC, a rich and potent source of MSC, on Lipopolysaccharide (LPS)-activated KP metabolites, KP enzymes, and key components of glutamate neurotransmission. METHODS: The immunomodulatory effect of peripherally administered HUCPVC on the expression profile of kynurenine pathway metabolites and enzymes was assessed in the plasma and brain of mice treated with LPS using LCMS and QPCR. An assessment of the glutamatergic system, including selected receptors, transporters and related proteins was also conducted by QPCR, immunohistochemistry and Western blot. RESULTS: HUCPVC were found to modulate LPS-induced activation of KP enzymes and metabolites in the brain associated with neurotoxicity. Moreover, the reduced expression of the glutamatergic components due to LPS was also found to be significantly improved by HUCPVC. CONCLUSIONS: The immunomodulatory properties of HUCPVC appear to confer neuroprotection, at least in part, through their ability to modulate the KP in the brain. This KP modulation enhances neuroprotective regulators and downregulates neurotoxic consequences, including glutamate neurotoxicity, which is associated with neuroinflammation and depressive behavior.

Surrogates' experience during the COVID-19 pandemic: mental health, social support, and relationship with intended parents.

PURPOSE: To examine surrogates' mental health, social support, and relationship with intended parents (IPs) during the COVID-19 pandemic from March 2020 to February 2022. METHODS: Data were collected between April 29, 2022 and July 31, 2022, at an academic IVF center in Canada using an 85-item online anonymous cross-sectional survey that included three standardized scales measuring mental health (PHQ-4), loneliness, and social support. Eligible surrogates actively involved in surrogacy during the study period received email invitations. RESULTS: The response rate was 50.3% (338/672); 320 submitted surveys were analyzed. Two-thirds (65%) of respondents experienced mental health concerns during the pandemic and were significantly less comfortable about seeking mental health support than those without concerns. Nonetheless, 64% were highly satisfied with their surrogacy experience; 80% received a high level of support from their IPs, and 90% reported a good relationship with them. The final hierarchical regression model identified five significant predictors, explaining 39.4% of the variance in PHQ-4 scores: a prior mental health history, COVID-19 impact on personal life, surrogacy satisfaction, loneliness, and social support. CONCLUSIONS: COVID-19 created an unprecedented challenge to surrogacy care, increasing surrogates' risk of experiencing mental health symptoms. Our data show that IP support and the surrogate-IP relationship were fundamentals to surrogacy satisfaction. The findings are relevant to fertility and mental health practitioners in identifying surrogates who are more susceptible to mental health challenges. Fertility clinics should ensure adequate psychological screening of surrogate candidates and proactively offer mental health support services.

Granulosa cell-derived miR-379-5p regulates macrophage polarization in polycystic ovarian syndrome.

Polycystic ovarian syndrome (PCOS) is associated with hyperandrogenemia and ovarian antral follicle growth arrest. We have previously demonstrated that androgen-induced exosomal release of miR-379-5p (miR379) from preantral follicle granulosa cells increases the proliferation of target cells via phosphoinositide-dependent kinase 1 (PDK1) upregulation. Androgen also increases inflammatory M1 macrophage abundance, but reduces anti-inflammatory M2 polarization in rat antral and preovulatory follicles. However, the role of small extracellular vesicles (sEVs; also known as exosomes) secretion in determining the cellular content and function of miRNAs in exosome-receiving cells is largely unknown. Our objectives were to determine: 1) the regulatory role of granulosa cells (GC)-derived exosomal miR379 on macrophage polarization and ovarian inflammation; 2) whether miR379-induced M1 polarization regulates GC proliferation; and 3) if this regulated process is follicular stage-specific. Compared with non-PCOS subjects, PCOS subjects had a higher M1/M2 ratio, supporting the concept that PCOS is an inflammatory condition. Ovarian overexpression of miR379 increased the number of M1 macrophages and the M1/M2 ratio in preantral follicles specifically. Transfection of macrophages with a miR379 mimic reduced the cellular content of PDK1 and induced M0→M1 polarization; whereas its inhibitor polarized M0→M2. Conditioned media from macrophages transfected with miR379 mimic and follicular fluid from PCOS subjects had higher galectin-3 content, a pro-inflammatory cytokine which specifically suppresses human antral follicle GC proliferation. These results indicate that miR379 inhibits M2 macrophage polarization, a condition which suppresses GC proliferation in a follicle stage-dependent manner, as exhibited in PCOS.

Androgen-induced exosomal miR-379-5p release determines granulosa cell fate: cellular mechanism involved in polycystic ovaries.

Polycystic ovarian syndrome (PCOS) is a complex multi-factorial syndrome associated with androgen excess and anovulatory infertility. In the current study, we investigated the role of dihydrotestosterone-induced exosomal miR-379-5p release in determining the destiny of the developing follicles. Our hypothesis was that androgen regulates granulosa cell miR-379-5p content by facilitating its exosomal release in a follicular-stage dependent manner, a process which determines granulosa cell fate. Compared to human non-PCOS subjects, individuals with PCOS exhibit higher follicular fluid free testosterone levels, lower exosomal miR-379-5p content and granulosa cell proliferation. Androgenized rats exhibited lower granulosa cell miR-379-5p but higher phosphoinositide-dependent kinase-1 (PDK1; a miR-379-5p target) content and proliferation. Androgen reduced granulosa cell miR-379-5p content by increasing its exosomal release in preantral follicles, but not in antral follicles in vitro. Studies with an exosomal release inhibitor confirmed that androgen-induced exosomal miR-379-5p release decreased granulosa cell miR-379-5p content and proliferation. Ovarian overexpression of miR-379-5p suppressed granulosa cell proliferation, and basal and androgen-induced preantral follicle growth in vivo. These findings suggest that increased exosomal miR-379-5p release in granulosa cells is a proliferative response to androgenic stimulation specific for the preantral stage of follicle development and that dysregulation of this response at the antral stage is associated with follicular growth arrest, as observed in human PCOS.

Time course and mechanistic analysis of human umbilical cord perivascular cell mitigation of lipopolysaccharide-induced systemic and neurological inflammation.

BACKGROUND AIMS: Because of their potent immunomodulatory and anti-inflammatory properties, mesenchymal stromal cells are a major focus in the field of stem cell therapy. However, the precise mechanisms underlying this are not entirely understood. Human umbilical cord perivascular cells (HUCPVCs) are a promising cell therapy candidate. This study was designed to evaluate the time course and mechanisms by which HUCPVCs mitigate lipopolysaccharide (LPS)-induced systemic and neurological inflammation in immunocompetent mice. To explore the underlying mechanisms, the authors investigated the biodistribution and fate of HUCPVCs. METHODS: Male C57BL/6 mice were randomly allocated to four groups: control, LPS, HUCPVCs or LPS + HUCPVCs. Quantitative polymerase chain reaction, enzyme-linked immunosorbent assay and cytokine arrays were used to assess changes in pro-inflammatory mediators systemically and in the brain. Depressive-like behavioral changes were evaluated via a forced swim test. Quantum dot (qDot) labeling and immunohistochemistry were used to assess the biodistribution and fate of HUCPVCs and interactions with recipient innate immune cells. RESULTS: A single intravenously delivered dose of HUCPVCs significantly reduced the systemic inflammation induced by LPS within the first 24 h after administration. HUCPVC treatment abrogated the upregulated expression of pro-inflammatory genes in the hippocampus and cortex and attenuated depressive-like behavior induced by LPS treatment. Biodistribution analysis revealed that HUCPVC-derived qDots rapidly accumulated in the lungs and demonstrated limited in vivo persistence. Furthermore, qDot signals were associated with major recipient innate immune cells and promoted a shift in macrophages toward a regulatory phenotype in the lungs. CONCLUSIONS: Overall, this study demonstrates that HUCPVCs can successfully reduce systemic and neurological inflammation induced by LPS within the first 24 h after administration. Biodistribution and fate analyses suggest a critical role for the innate immune system in the HUCPVC-based immunomodulation mechanism.

Personalization of IVF-ICSI workflow based on patient characteristics improves IVF laboratory outcomes and embryo ploidy by PGT-A.

BACKGROUND: Intracytoplasmic sperm injection (ICSI) has become a common method of fertilization in assisted reproduction worldwide. However, there are still gaps in knowledge of the ideal IVF-ICSI workflow including the optimal duration of time between induction of final oocyte maturation, oocyte denudation and ICSI. The aim of this study was to examine outcomes following different workflow protocols in IVF-ICSI procedures in blastocysts that have undergone undisturbed incubation and preimplantation genetic testing for aneuploidy (PGT-A) prior to transfer. METHODS: Retrospective secondary analysis of 113 patients (179 IVF cycles, 713 embryos), all of whom have gone through IVF-ICSI and PGT-A using undisturbed culture. Predictive test variables were the length of time from: trigger to OPU, OPU to denudation, and denudation to ICSI. Outcome metrics assessed were: maturation, fertilization, blastulation and euploid rates. Generalized Estimated Equations Linear Model was used to examine the relationship between key elements of a given cycle and continuous outcomes and LOESS curves were used to determine the effect over time. RESULTS: In a paired multi-regression analysis, where each patient served as its own control, delaying OPU in patients with unexplained infertility improved both maturation and blastulation rates (b = 29.7, p < 0.0001 and b = 9.1, p = 0.06, respectively). Longer incubation with cumulus cells (CCs) significantly correlated with improved ploidy rates among patients under 37, as well as among patients with unexplained infertility (r = 0.22 and 0.29, respectively), which was also evident in a multiple regression analysis (b = 6.73, p < 0.05), and in a paired analysis (b = 6.0, p < 0.05). Conversely, among patients with a leading infertility diagnosis of male factor, longer incubation of the denuded oocyte prior to ICSI resulted in a significantly higher euploid rate (b = 15.658, p < 0.0001). CONCLUSIONS: In this study we have demonstrated that different IVF-ICSI workflows affect patients differently, depending on their primary infertility diagnosis. Thus, ideally, the IVF-ICSI workflow should be tailored to the individual patient based on the primary infertility diagnosis. This study contributes to our understanding surrounding the impact of IVF laboratory procedures and highlights the importance of not only tracking "classic" IVF outcomes (maturation, fertilization, blastulation rates), but highlights the importance that these procedures have on the ploidy of the embryo.

Cannabis significantly alters DNA methylation of the human ovarian follicle in a concentration-dependent manner.

Cannabis is increasingly consumed by women of childbearing age, and the reproductive and epigenetic effects are unknown. The purpose of this study was to evaluate the potential epigenetic implications of cannabis use on the female ovarian follicle. Whole-genome methylation was assessed in granulosa cells from 14 matched case-control patients. Exposure status was determined by liquid chromatography-mass spectrometry (LC-MS/MS) measurements of five cannabis-derived phytocannabinoids in follicular fluid. DNA methylation was measured using the Illumina TruSeq Methyl Capture EPIC kit. Differential methylation, pathway analysis and correlation analysis were performed. We identified 3679 differentially methylated sites, with two-thirds affecting coding genes. A hotspot region on chromosome 9 was associated with two genomic features, a zinc-finger protein (ZFP37) and a long non-coding RNA (FAM225B). There were 2214 differentially methylated genomic features, 19 of which have been previously implicated in cannabis-related epigenetic modifications in other organ systems. Pathway analysis revealed enrichment in G protein-coupled receptor signaling, cellular transport, immune response and proliferation. Applying strict criteria, we identified 71 differentially methylated regions, none of which were previously annotated in this context. Finally, correlation analysis revealed 16 unique genomic features affected by cannabis use in a concentration-dependent manner. Of these, the histone methyltransferases SMYD3 and ZFP37 were hypomethylated, possibly implicating histone modifications as well. Herein, we provide the first DNA methylation profile of human granulosa cells exposed to cannabis. With cannabis increasingly legalized worldwide, further investigation into the heritability and functional consequences of these effects is critical for clinical consultation and for legalization guidelines.

Human platelet lysates stimulate in vitro proliferation of human endometrial cells from patients with a history of recurrent implantation failure.

OBJECTIVE: To optimize and compare the isolation of platelet-rich plasma (PRP) and its cryopreserved derivative, platelet lysate (PL), to a commercial human platelet lysate (HPL) product PLUS and investigate their proliferative stimulation on primary human endometrial cells in vitro. DESIGN: Basic research. SETTING: Academic fertility center. PATIENT(S): Three healthy blood donors and eight patients with a history of recurrent implantation failure. INTERVENTIONS(S): Stimulated proliferation of isolated primary endometrial epithelial cells and endometrial stromal cells in vitro with autologous and nonautologous HPL (PLUS; Compass Biomedical). MAIN OUTCOME MEASURE(S): Platelet-derived growth factor BB homodimer protein content in isolated PRP/PL and commercial HPL and endometrial epithelial cell and endometrial stromal cell proliferation after 24- or 48-hour stimulation with PL (measured by metabolic activity and Ki67 expression). RESULT(S): To optimize and compare the isolation of autologous PRP/PL, three double-centrifugation protocols were assessed by flow cytometry for platelet yield (CD45-CD41+CD61+) and platelet-derived growth factor BB homodimer protein content by enzyme-linked immunosorbent assay. Cryopreserved PL, especially isolated by our fastest protocol, contained higher protein concentrations and, thus, was optimal for experimental flexibility compared with fresh PRP. The autologous and commercial PLs displayed comparable immune and growth factor content and stimulation of cell proliferation in vitro. CONCLUSION(S): Our results provide the groundwork for the isolation and use of HPL to stimulate endometrial growth. Furthermore, commercial PL consistently stimulated cell proliferation and may allow standardization of clinical treatment for recurrent implantation failure.

Plasma Gelsolin Confers Chemoresistance in Ovarian Cancer by Resetting the Relative Abundance and Function of Macrophage Subtypes.

Ovarian cancer (OVCA) is the most lethal gynaecological cancer with a 5-year survival rate less than 50%. Despite new therapeutic strategies, such as immune checkpoint blockers (ICBs), tumor recurrence and drug resistance remain key obstacles in achieving long-term therapeutic success. Therefore, there is an urgent need to understand the cellular mechanisms of immune dysregulation in chemoresistant OVCA in order to harness the host's immune system to improve survival. The over-expression of plasma gelsolin (pGSN) mRNA is associated with a poorer prognosis in OVCA patients; however, its immuno-modulatory role has not been elucidated. In this study, for the first time, we report pGSN as an inhibitor of M1 macrophage anti-tumor functions in OVCA chemoresistance. Increased epithelial pGSN expression was associated with the loss of chemoresponsiveness and poor survival. While patients with increased M1 macrophage infiltration exhibited better survival due to nitric-oxide-induced ROS accumulation in OVCA cells, cohorts with poor survival had a higher infiltration of M2 macrophages. Interestingly, increased epithelial pGSN expression was significantly associated with the reduced survival benefits of infiltrated M1 macrophages, through apoptosis via increased caspase-3 activation and reduced production of iNOS and TNFα. Additionally, epithelial pGSN expression was an independent prognostic marker in predicting progression-free survival. These findings support our hypothesis that pGSN is a modulator of inflammation and confers chemoresistance in OVCA, in part by resetting the relative abundance and function of macrophage subtypes in the ovarian tumor microenvironment. Our findings raise the possibility that pGSN may be a potential therapeutic target for immune-mediated chemoresistance in OVCA.

Let the data do the talking: the need to consider mosaicism during embryo selection.

Chromosomal mosaicism, the coexistence of cells with different chromosomal content, has been documented in human embryos for 3 decades. Early versions of preimplantation genetic testing for aneuploidy (PGT-A) did not measure mosaicism, either because typically only a single cell was assessed or because the technique could not accurately identify it. Although this led to a straightforward diagnosis (an embryo was considered either normal or abnormal), it simply avoided the issue and, in hindsight, may have led to numerous misdiagnoses with negative clinical consequences. Modern PGT-A evaluates a multicellular biopsy specimen with techniques capable of recognizing intermediate copy number signals for chromosomes or subchromosomal regions. We are, therefore, inevitably confronted with the issue of mosaicism and the challenge of managing embryos producing such results in the clinic. Here we discuss recent data showing that not only mosaicism in general, but specific features of mosaicism detected with PGT-A, are associated with variable clinical outcomes. The conclusion is evident: mosaicism should be considered for more informed and improved embryo selection in the clinic.

Cannabis alters epigenetic integrity and endocannabinoid signalling in the human follicular niche.

STUDY QUESTION: Do phytocannabinoids (PCs) affect follicular endocannabinoid signalling and the epigenome in the surrounding granulosa cells (GCs)? SUMMARY ANSWER: Exposure to PCs increases the expression of endocannabinoid receptors and reduces DNA methylation enzyme expression and global DNA methylation in naïve GCs. WHAT IS KNOWN ALREADY: Cannabis plant derivatives, known as PCs, are used for medicinal and recreational purposes. The main PC, tetrahydrocannabinol (THC), is the third most commonly used substance by women of childbearing age, hence knowledge of the effect it has on reproduction is of utmost importance. THC exerts its effects via receptors of the endocannabinoid system (ECS) and can interfere with folliculogenesis, oocyte development and ovulation. Endocannabinoids have been measured in follicular fluid (FF) obtained during oocyte retrieval and are implicated in controlling folliculogenesis. It has been established that in the placenta, PCs disrupt endocannabinoid homeostasis via impairment of the synthetic and degrading enzymes, leading to a net increase of endocannabinoid levels. Finally, previous studies have shown that THC alters methylation and histone modifications in sperm, brain and blood cells. STUDY DESIGN, SIZE, DURATION: This study included an in vivo cohort assessment of cannabis exposure and its effects on the follicle and in vitro assays conducted to validate the in vivo findings and to explore possible mechanisms of action. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 318 FF samples, from 261 patients undergoing IVF treatment at a private fertility clinic who consented for biobanking biological waste material between January 2018 and July 2019, were included in this study. Concentrations of PCs and endocannabinoids were assessed in FF by liquid chromatography-mass spectrometry (LC-MS/MS). Exposure to PCs was determined based on these measured levels. Levels of both endocannabinoid receptors (CB1R, CB2R) and the de novo DNA methylating enzyme, DNMT3b, in GCs were assessed by flow cytometry both in vitro and in vivo and global DNA methylation was assessed in vitro by ELISA. In vivo effects were assessed by comparing samples positive for at least one PC, with samples negative for all measured PCs. In vitro effects were determined in naive GCs, obtained concurrently with FF samples that had tested negative for all PCs. These GCs were treated with different combinations of the main three PCs. MAIN RESULTS AND THE ROLE OF CHANCE: Overall, 17 patients (6.4%) were positive for cannabis consumption. Furthermore, the prevalence of cannabis positivity in the FF increased from 4% of the tested samples that were collected prior to national legalisation in October 2018 to 12% of those collected following legalisation. Of note, 59% of patients who tested positive for PCs (10 of 17) reported previous or ongoing exposure to cannabis upon their initial intake. Endocannabinoid levels were not affected by the presence of PCs. CB2R was more prevalent than CB1R in GCs and its expression increased following acute and chronic in vitro exposure to PCs. The expression of DNMT3b and global methylation decreased following exposure, suggesting that cannabis may affect the epigenome in the follicular niche. The acute changes were sustained throughout chronic treatment. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Our study is limited by lack of details regarding mode, frequency and timing of PC consumption. Moreover, we were not able to adequately assess the effect of PCs on immediate or long-term clinical outcomes, due to the small sample size and the lack of follow up. Future, large-scale studies should focus on assess the clinical implications of cannabis exposure, validate our findings, and determine to what extent cannabis affects the epigenome ovarian follicle and the developing oocyte. WIDER IMPLICATIONS OF THE FINDINGS: To our knowledge, this is the first study measuring PCs in FF by LC-MS/MS. We show that consuming cannabis alters the ECS in the developing follicle, and directly affects DNMT expression and global DNA methylation levels. Cannabis legalisation and use is increasing worldwide, therefore further understanding its role in female fertility and folliculogenesis is critical. STUDY FUNDING/COMPETING INTEREST(S): All funding was provided by CReATe Fertility Centre through the reinvestment of clinical earnings. The authors declare no competing interests.