Cellular atlas of the human ovary using morphologically guided spatial transcriptomics and single-cell sequencing.

The reproductive and endocrine functions of the ovary involve spatially defined interactions among specialized cell populations. Despite the ovary's importance in fertility and endocrine health, functional attributes of ovarian cells are largely uncharacterized. Here, we profiled >18,000 genes in 257 regions from the ovaries of two premenopausal donors to examine the functional units in the ovary. We also generated single-cell RNA sequencing data for 21,198 cells from three additional donors and identified four major cell types and four immune cell subtypes. Custom selection of sampling areas revealed distinct gene activities for oocytes, theca, and granulosa cells. These data contributed panels of oocyte-, theca-, and granulosa-specific genes, thus expanding the knowledge of molecular programs driving follicle development. Serial samples around oocytes and across the cortex and medulla uncovered previously unappreciated variation of hormone and extracellular matrix remodeling activities. This combined spatial and single-cell atlas serves as a resource for future studies of rare cells and pathological states in the ovary.

Fresh and cryopreserved ovarian tissue from deceased young donors yields viable follicles.

OBJECTIVE: Ovarian tissue cryopreservation is one of the crucial options for fertility preservation. Transplantation of cryopreserved ovarian tissue was proven to restore ovarian endocrine function in patients with premature ovarian insufficiency. Ovaries from deceased donors potentially serve as an excellent and readily available tissue for the translational and basic research. In this study, we used ovaries obtained from 5 deceased donors aged 18-26 years, to evaluate the number and quality of ovarian follicles isolated before and after cryopreservation. DESIGN: Preclinical. SETTING: Academic biomedical research laboratory. PATIENTS: De-identified deceased human donors. INTERVENTIONS: Slow-freeze cryopreservation and thawing. MAIN OUTCOME MEASURES: Follicle count, follicle density, follicle viability using immunohistochemical staining (TUNEL). RESULTS: The follicle density negatively correlated with age in both cryopreserved/thawed and fresh group. A total of 2803 follicles from fresh and 1608 follicles from cryopreserved tissues were classified and analyzed using Hematoxylin and eosin staining. There was no significant difference in the percent of morphologically normal follicles between two groups. TUNEL assay indicated no higher DNA damage in the follicles and the stroma cells after cryopreservation. Morphologically normal preantral follicles were enzymatically isolated from both fresh and cryopreserved tissue with 88.51 ± 5.93% (mean ± SD) of the isolated follicles confirmed viable using LIVE/DEAD evaluation. CONCLUSIONS: Our results indicate the ovarian tissue from deceased donors maintain high quality after long time extracorporeal circulation and transportation from the hospital to the laboratory. High survival rate of follicles at different developmental stages suggested tolerance to the cryopreservation process. Human ovarian tissues obtained from deceased donors is an ample source tissue and can be applied to promoting research and future clinical applications.

Ovarian tissue cryopreservation and novel bioengineering approaches for fertility preservation.

PURPOSE OF REVIEW: Breast cancer patients who cannot delay treatment or for whom hormone stimulation and egg retrieval are contraindicated require alternative methods of fertility preservation prior to gonadotoxic treatment. Ovarian tissue cryopreservation is an alternative approach that may offer patients the opportunity to preserve fertility and carry biologically-related children later in life. Various experimental approaches are being explored to obtain mature gametes from cryopreserved and thawed ovarian tissue for fertilization and implantation using biomimetic tissue culture in vitro. Here we review the most recent developments in ovarian tissue cryopreservation and exciting advances in bioengineering approaches to in vitro tissue and ovarian follicle culture. RECENT FINDINGS: Slow freezing is the most widely accepted method for ovarian tissue cryopreservation, but efforts have been made to modify vitrification for this application as well. Numerous approaches to in vitro tissue and follicle culture are in development, most prominently two-step culture systems for ovarian cortical tissue and encapsulation of ovarian follicles in biomimetic matrices for in vitro culture. SUMMARY: Refinements to slow freeze and vitrification protocols continue to address challenges associated with cryopreservation, such as ice crystal formation and damage to the stroma. Similarly, improvements to in vitro tissue and follicle culture show promise for utilizing patients' cryopreserved tissues to obtain mature gametes after disease treatment and remission. Development of an effective and reproducible culture system for human ovarian follicles will serve as a broad assisted reproductive technology for cancer survivors who cryopreserved tissue prior to treatment.

Follicle development as an orchestrated signaling network in a 3D organoid.

The ovarian follicle is the structural and functional unit of the ovary, composed of the female gamete (the oocyte) and supportive somatic cells. Follicles are not only the source of a female's germ cell supply, but also secrete important hormones necessary for proper endocrine function. Folliculogenesis, the growth and maturation of the follicular unit, is a complex process governed by both intrafollicular crosstalk and pituitary-secreted hormones. While the later stages of this process are gonadotropin-dependent, early folliculogenesis appears to be controlled by the ovarian microenvironment and intrafollicular paracrine and autocrine signaling. In vitro follicle culture remains challenging because of the limited knowledge of growth factors and other cytokines influencing early follicle growth. Here we discuss the current state of knowledge on paracrine and autocrine signaling influencing primary follicles as they develop into the antral stage. Given the importance of intrafollicular signaling and the ovarian microenvironment, we reviewed the current engineering approaches for in vitro follicle culture, including 3D systems using natural hydrogels such as alginate and synthetic hydrogels such as poly(ethylene glycol). Our discussion is focused on what drives the proliferation of granulosa cells, development of the thecal layer, and antrum formation-three processes integral to follicle growth up to the antral stage. Further research in this area may reveal the mechanisms behind these complex signaling relationships within the follicle, leading to more successful and physiologically-relevant in vitro culture methods that will translate well to clinical applications.

Efficacy of a Chronic Care-Based Intervention on Secondary Stroke Prevention Among Vulnerable Stroke Survivors: A Randomized Controlled Trial.

BACKGROUND: Disparities of care among stroke survivors are well documented. Effective interventions to improve recurrent stroke preventative care in vulnerable populations are lacking. METHODS AND RESULTS: In a randomized controlled trial, we tested the efficacy of components of a chronic care model-based intervention versus usual care among 404 subjects having an ischemic stroke or transient ischemic attack within 90 days of enrollment and receiving care within the Los Angeles public healthcare system. Subjects had baseline systolic blood pressure (SBP) ≥120 mm Hg. The intervention included a nurse practitioner/physician assistant care manager, group clinics, self-management support, report cards, decision support, and ongoing care coordination. Outcomes were collected at 3, 8, and 12 months, analyzed as intention-to-treat, and used repeated-measures mixed-effects models. Change in SBP was the primary outcome. Low-density lipoprotein reduction, antithrombotic medication use, smoking cessation, and physical activity were secondary outcomes. Average age was 57 years; 18% were of black race; 69% were of Hispanic ethnicity. Mean baseline SBP was 150 mm Hg in both arms. SBP decreased to 17 mm Hg in the intervention arm and 14 mm Hg in the usual care arm; the between-arm difference was not significant (-3.6 mm Hg; 95% confidence interval, -9.2 to 2.2). Among secondary outcomes, the only significant difference was that persons in the intervention arm were more likely to lower their low-density lipoprotein <100 md/dL (2.0 odds ratio; 95% confidence interval, 1.1-3.5). CONCLUSIONS: This intervention did not improve SBP control beyond that attained in usual care among vulnerable stroke survivors. A community-centered component could strengthen the intervention impact. CLINICAL TRIAL REGISTRATION: URL: https://clinicaltrials.gov. Unique identifier: NCT00861081.

Randomized, controlled trial of an intervention to enable stroke survivors throughout the Los Angeles County safety net to "stay with the guidelines".

BACKGROUND: Stroke is the leading cause of adult disability. Inpatient programs optimize secondary stroke prevention care at the time of hospital discharge, but such care may not be continued after hospital discharge. METHODS: To improve the delivery of secondary stroke preventive services after hospital discharge, we have designed a chronic care model-based program called SUSTAIN (Systemic Use of STroke Averting INterventions). This care intervention includes group clinics, self-management support, report cards, decision support through care guides and protocols, and coordination of ongoing care. The first specific aim is to test, in a randomized, controlled trial, whether SUSTAIN improves blood pressure control among an analytic sample of 268 patients with a recent stroke or transient ischemic attack discharged from 4 Los Angeles County public hospitals. Secondary outcomes consist of control of other stroke risk factors, lifestyle habits, medication adherence, patient perceptions of care quality, functional status, and quality of life. A second specific aim is to conduct a cost analysis of SUSTAIN from the perspective of the Los Angeles County Department of Health Services by using direct costs of the intervention, cost equivalents of associated utilization of county system resources, and cost equivalents of the observed and predicted averted vascular events. CONCLUSIONS: If SUSTAIN is effective, we will have the expertise and findings to advocate for its continued support at Los Angeles County hospitals and to disseminate the SUSTAIN program to other settings serving indigent, minority populations. Clinical Trial Registration- URL: http://www.clinicaltrials.gov. Unique identifier: NCT00861081.