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.

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.

The follicular fluid adipocytokine milieu could serve as a prediction tool for fertility treatment outcomes.

RESEARCH QUESTION: Can the adipocytokine milieu of the follicular niche improve the ability to predict treatment outcomes in infertile patients? DESIGN: Follicular fluid samples from overweight patients were analysed and compared with samples from matched normal-weight patients. Concentrations of adiponectin, chemerin, C-reactive protein, interleukin-6 (IL-6), IL-10, IL-18, insulin, leptin, prolactin, resistin, tumour necrosis factor alpha (TNF-α) and bone morphogenetic protein-15 (BMP-15) were assessed by multiple magnetic bead immunoassay (MMBI) and enzyme-linked immunosorbent assay and correlated with fertility treatment outcomes. RESULTS: Analysis of samples from 22 overweight and 22 normal-weight patients demonstrated that TNF-α can predict oocyte maturation rate. When stratified by body mass index (BMI), IL-10 emerges as a better predictor of oocyte maturation in normal-weight patients. Prolactin was a negative predictor for fertilization rate in the full cohort, and this prediction power was lost upon stratification. No adipocytokines were predictive of blastulation rate, and only age remained predictive. BMP-15 was a strong predictor of high-quality blastulation in the full cohort, more so in the normal-weight population. CONCLUSIONS: The adipocytokine milieu of the follicular fluid provides a snapshot of the growing oocyte's environment and can help predict fertility treatment outcomes, fine-tuning understanding of the dysregulation caused by increasing BMI. Inflammatory cytokines can predict oocyte maturation; prolactin, oocyte competence; and BMP-15, high-quality blastulation. Further analysis of these findings with a larger sample size and assessing individual oocytes will help shed more light on the clinical significance of these findings.

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.

Transcriptomics of cumulus cells - a window into oocyte maturation in humans.

BACKGROUND: Cumulus cells (CC) encapsulate growing oocytes and support their growth and development. Transcriptomic signatures of CC have the potential to serve as valuable non-invasive biomarkers for oocyte competency and potential. The present sibling cumulus-oocyte-complex (COC) cohort study aimed at defining functional variations between oocytes of different maturity exposed to the same stimulation conditions, by assessing the transcriptomic signatures of their corresponding CC. CC were collected from 18 patients with both germinal vesicle and metaphase II oocytes from the same cycle to keep the biological variability between samples to a minimum. RNA sequencing, differential expression, pathway analysis, and leading-edge were performed to highlight functional differences between CC encapsulating oocytes of different maturity. RESULTS: Transcriptomic signatures representing CC encapsulating oocytes of different maturity clustered separately on principal component analysis with 1818 genes differentially expressed. CCs encapsulating mature oocytes were more transcriptionally synchronized when compared with CCs encapsulating immature oocytes. Moreover, the transcriptional activity was lower, albeit not absent, in CC encapsulating mature oocytes, with 2407 fewer transcripts detected than in CC encapsulating immature (germinal vesicle - GV) oocytes. Hallmark pathways and ovarian processes that were affected by oocyte maturity included cell cycle regulation, steroid metabolism, apoptosis, extracellular matrix remodeling, and inflammation. CONCLUSIONS: Herein we review our findings and discuss how they align with previous literature addressing transcriptomic signatures of oocyte maturation. Our findings support the available literature and enhance it with several genes and pathways, which have not been previously implicated in promoting human oocyte maturation. This study lays the ground for future functional studies that can enhance our understanding of human oocyte maturation.

Does body mass index impact assisted reproductive technology treatment outcomes in gestational carriers.

BACKGROUND: The purpose of this study was to assess whether increased body mass index (BMI) negatively affects assisted reproductive technology (ART) outcomes among gestational carriers. METHODS: A retrospective matched case-control cohort, including all gestational carrier (GC) cycles performed at CReATe Fertility Centre (Toronto, ON, Canada) between 2003 and 2016. SETTING: A Canadian fertility clinic, with a large surrogacy program. PATIENTS: All gestational carriers that had undergone a cycle completed to a transfer at our clinic, and had BMI and outcome data available, were matched by BMI to infertile patients treated at our clinic during the same years provided they had undergone a cycle completed to a transfer, and had outcomes data available. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Clinical pregnancies rates, miscarriage rates and live birth rates. RESULTS: BMI was not a reliable prediction factor of any of the measured outcomes. Importantly, the gestational carrier population had better outcomes and a significantly lower overall incidence of maternal, fetal and neonatal complications when compared with infertile patients, treated at our clinic during the same years. CONCLUSION: BMI is not a reliable predictor of outcomes among gestational carriers.

Triggering method in assisted reproduction alters the cumulus cell transcriptome.

RESEARCH QUESTION: How does the choice of triggering final oocyte maturation affect the cumulus cell transcriptome? DESIGN: Sixty patients undergoing gonadotrophin-releasing hormone antagonist (GnRH-ant) IVF cycles were recruited for this nested case-control study. Patients were stratified into three subgroups based on their ovarian reserve (high, normal and low). Triggering final oocyte maturation was accomplished by either single trigger (with human chorionic gonadotrophin [HCG] only or gonadotrophin-releasing hormone agonist [GnRH-ag] only) or dual trigger combining HCG and GnRH-ag. The choice of trigger was at the discretion of the treating physician. Within each group patients receiving a dual trigger were matched by demographic and pre-stimulation parameters with patients receiving a single trigger. The matching was performed to minimize the biological variability within each subgroup. Thirty patients were included in the final analysis. Cumulus cells were stripped away from the retrieved oocytes. Cumulus cells from three sibling oocytes were pooled, the RNA extracted and libraries prepared. Next-generation sequencing was performed on all samples. RESULTS: Dual triggering supports key ovarian pathways of oocyte maturation and extracellular matrix remodelling, while attenuating vasculo-endothelial growth and providing antioxidant protection to the growing follicles. CONCLUSIONS: This is the first study to delineate key transcriptomic changes under dual triggering of final oocyte maturation, across different patient populations. The findings underline the need for larger-scale studies validating transcriptomic effects of methods for triggering final oocyte maturation. Furthermore, there is a need for large-scale clinical randomized controlled studies to relate the findings of this study with clinical outcomes.

Towards Improving Embryo Prioritization: Parallel Next Generation Sequencing of DNA and RNA from a Single Trophectoderm Biopsy.

Improved embryo prioritization is crucial in optimizing the results in assisted reproduction, especially in light of increasing utilization of elective single embryo transfers. Embryo prioritization is currently based on morphological criteria and in some cases incorporates preimplantation genetic testing for aneuploidy (PGT-A). Recent technological advances have enabled parallel genomic and transcriptomic assessment of a single cell. Adding transcriptomic analysis to PGT-A holds promise for better understanding early embryonic development and implantation, and for enhancing available embryo prioritization tools. Our aim was to develop a platform for parallel genomic and transcriptomic sequencing of a single trophectoderm (TE) biopsy, that could later be correlated with clinical outcomes. Twenty-five embryos donated for research were utilized; eight for initial development and optimization of our method, and seventeen to demonstrate clinical safety and reproducibility of this method. Our method achieved 100% concordance for ploidy status with that achieved by the classic PGT-A. All sequencing data exceeded quality control metrics. Transcriptomic sequencing data was sufficient for performing differential expression (DE) analysis. All biopsies expressed specific TE markers, further validating the accuracy of our method. Using PCA, samples clustered in euploid and aneuploid aggregates, highlighting the importance of controlling for ploidy in every transcriptomic assessment.

Survey on Barriers to Adoption of Laparoscopic Surgery.

OBJECTIVE: To identify challenges that impede wider adoption of laparoscopy in gynecologic surgery and assessing whether the current training programs are addressing these challenges adequately. METHODS: A survey was designed to examine barriers to adoption of laparoscopy for practicing gynecologists. The survey was piloted on gynecologic surgeons and was further refined following their feedback. Finally, the survey was deployed to 4273 gynecologists across the United States via e-mail using the national database of the American Medical Association. Respondents were grouped into two categories based on how often they report referral of patients for laparoscopy. Demographics, training, and practice characteristics were compared using Fisher exact tests for categorical variables and t tests for continuous variables. Participants rated factors that were thought to limit laparoscopy use on a 5-point Likert scale; median values of these scores were compared with Wilcoxon rank sum tests. RESULTS: We received 210 responses (29% of people who opened the e-mail and 93% of those who opened the survey). Physicians who perform their own laparoscopies were on average younger and tended to be more subspecialized. Some of the most highly rated limiting factors included lack of adequate surgical volume, reluctance of managing unexpected surgical scenarios, difficulty with video-eye-hand coordination, altered depth perception, and laparoscopic suturing. CONCLUSION: This survey identified barriers to adoption of laparoscopic surgical techniques beyond what has previously been identified. Based on these findings, novel simulation and continuing medical education curricula can be created to address the primary barriers in order to increase laparoscopic approach to surgery among gynecologists.

Vaginal cuff dehiscence: risk factors and associated morbidities.

BACKGROUND AND OBJECTIVES: To evaluate whether the route and surgical technique by which hysterectomy is performed influence the incidence of vaginal cuff dehiscence. METHODS: We performed a retrospective analysis of total hysterectomy cases performed at Brigham and Woman's Hospital or Faulkner Hospital during 2009 through 2011. RESULTS: During the study period, 2382 total hysterectomies were performed; 23 of these (0.96%) were diagnosed with cuff dehiscence, and 4 women had recurrent dehiscence. Both laparoscopic (odds ratio, 23.4; P = .007) and robotic (odds ratio, 73; P = .0006) hysterectomies were associated with increased odds of cuff dehiscence in a multivariate regression analysis. The type of energy used during colpotomy, mode of closure (hand sewn, laparoscopic suturing, or suturing assisted by a device), and suture material did not differ significantly between groups; however, continuous suturing of the cuff was a protective factor (odds ratio, 0.24; P = .03). Women with dehiscence had more extensive procedures, as well as an increased incidence of additional major postoperative complications (17.4% vs 3%, P = .004). CONCLUSION: The rate of cuff dehiscence in our cohort correlates with the current literature. This study suggests that the risk of dehiscence is influenced mainly by the scope and complexity of the surgical procedure. It seems that different colpotomy techniques do not influence the rate of cuff dehiscence; however, continuous suturing of the cuff may be superior to interrupted suturing.

Open power morcellation versus contained power morcellation within an insufflated isolation bag: comparison of perioperative outcomes.

STUDY OBJECTIVE: To compare perioperative outcomes, particularly operative time, between uncontained and in-bag power morcellation of uterine tissue at the time of laparoscopic surgery. DESIGN: Canadian Task Force classification II-3. SETTING: Academic tertiary care hospitals. PATIENTS: Women undergoing laparoscopic hysterectomy or myomectomy who required morcellation of uterine tissue for specimen extraction. INTERVENTIONS: Outcomes among patients who had in-bag power morcellation were compared with outcomes among patients who had traditional power morcellation. The technique for in-bag morcellation entails placing the specimen into a large containment bag within the abdomen, insufflating the bag within the peritoneal cavity, and then using a power morcellator to remove the specimen from inside the bag. MEASUREMENTS AND MAIN RESULTS: The cohort consisted of 85 consecutive patients who underwent surgery with morcellation of uterine tissue. Prospective data collected from 36 patients who underwent in-bag morcellation were compared with retrospective data collected from the immediately preceding 49 patients who had uncontained power morcellation. Baseline demographics were comparable between the 2 groups although women who underwent in-bag morcellation were on average older than the open morcellation group (mean age in years [standard deviation], 49.19 [1.12] vs 44.06 [8.93]; p = .01). The mean operating room time was longer in the in-bag morcellation group (mean time in minutes [standard deviation], 119.0 [55.91] vs 93.13 [44.90]; p = .02). The estimated blood loss, specimen weight, hospital length of stay, and perioperative complication rate did not vary between the 2 groups. Operative times did not vary significantly by surgeon. There were no cases of malignancy or isolation bag disruption. CONCLUSIONS: In-bag power morcellation, a tissue extraction technique developed to reduce the risk of tissue dissemination, results in perioperative outcomes comparable with the traditional laparoscopic approach. In this cohort, the mean operative time was prolonged by 26 minutes with in-bag morcellation but may potentially be reduced with further refinement of the technique.

Design and validation of a novel assessment tool for laparoscopic suturing of the vaginal cuff during hysterectomy.

OBJECTIVE: The aim of this study was to design and validate a new assessment tool for laparoscopic vaginal cuff suturing in a box trainer. DESIGN: A primary grading tool was constructed including a novel checklist component and a previously validated global rating scale. A four-step validation process was then undertaken. Face validity was determined by a survey of a panel of experts in the field of minimally invasive gynecologic surgery. Content validity was assessed via analysis of consistency and variability of the experts' ratings, and items were removed or rephrased according to the experts' comments. PARTICIPANTS: Overall, five novices and five expert laparoscopic surgeons were videotaped performing suture closure of a latex vaginal cuff model in a box trainer. The videotapes were reviewed by two raters. Discriminate validity, along with interrater and intrarater reliabilities, was assessed by analysis of the video ratings. SETTING: The Simulation, Training, Research, and Technology Utilization System center at Brigham and Women's Hospital, a tertiary medical center in Boston, MA. RESULTS: The final assessment tool is presented. CONCLUSION: We have validated an assessment tool for vaginal cuff suturing in a box trainer.

MAUDE: analysis of robotic-assisted gynecologic surgery.

STUDY OBJECTIVE: To evaluate the adverse events encountered during robotic gynecologic surgery, as reported to the FDA MAUDE database from January 2006 to December 2012. DESIGN: Database search (Canadian Task Force classification III). INTERVENTION: A search of the FDA MAUDE database was performed by brand name "da Vinci" and manufacturer "Intuitive Surgical." Reports reflecting gynecologic procedures either by description or procedure name were included. A record of reports was kept to ensure that no duplicates were added. The date and type of event (operator-related error, technical system failure, or surgical injuries attributed to use of the robot) and the clinical outcome were recorded. MEASUREMENTS AND MAIN RESULTS: Twenty-six percent of reported events (n = 73) resulted in injury, and 8.5% (n = 24) resulted in death. Of note, although adnexal procedures were performed in <3% of the cohort, they accounted for 20% of the fatalities. Twenty-one percent of injuries were attributed to operator-related error, and 14% to technical system failure; 65% were not directly related to use of the robot. Fifteen deaths were reported during planned hysterectomy. Four of those were due to injury to a major blood vessel (iliac artery in 3, and aorta in 1), although a detailed description of how the injury occurred was absent from the event description. CONCLUSION: It is important to continue to evaluate the occurrence of injuries during robot-assisted surgery in an effort to identify unique challenges associated with this advanced technology.