Single-cell roadmap of human gonadal development.

Gonadal development is a complex process that involves sex determination followed by divergent maturation into either testes or ovaries1. Historically, limited tissue accessibility, a lack of reliable in vitro models and critical differences between humans and mice have hampered our knowledge of human gonadogenesis, despite its importance in gonadal conditions and infertility. Here, we generated a comprehensive map of first- and second-trimester human gonads using a combination of single-cell and spatial transcriptomics, chromatin accessibility assays and fluorescent microscopy. We extracted human-specific regulatory programmes that control the development of germline and somatic cell lineages by profiling equivalent developmental stages in mice. In both species, we define the somatic cell states present at the time of sex specification, including the bipotent early supporting population that, in males, upregulates the testis-determining factor SRY and sPAX8s, a gonadal lineage located at the gonadal-mesonephric interface. In females, we resolve the cellular and molecular events that give rise to the first and second waves of granulosa cells that compartmentalize the developing ovary to modulate germ cell differentiation. In males, we identify human SIGLEC15+ and TREM2+ fetal testicular macrophages, which signal to somatic cells outside and inside the developing testis cords, respectively. This study provides a comprehensive spatiotemporal map of human and mouse gonadal differentiation, which can guide in vitro gonadogenesis.

Mapping Human Reproduction with Single-Cell Genomics.

The trillions of cells in the human body develop as a result of the fusion of two extremely specialized cells: an oocyte and a sperm. This process is essential for the continuation of our species, as it ensures that parental genetic information is mixed and passed on from generation to generation. In addition to producing oocytes, the female reproductive system must provide the environment for the appropriate development of the fetus until birth. New genomic and computational tools offer unique opportunities to study the tight spatiotemporal regulatory mechanisms that are required for the cycle of human reproduction. This review explores how single-cell technologies have been used to build cellular atlases of the human reproductive system across the life span and how these maps have proven useful to better understand reproductive pathologies and dissect the heterogeneity of in vitro model systems.

An integrated single-cell reference atlas of the human endometrium.

The complex and dynamic cellular composition of the human endometrium remains poorly understood. Previous endometrial single-cell atlases profiled few donors and lacked consensus in defining cell types. We introduce the Human Endometrial Cell Atlas (HECA), a high-resolution single-cell reference atlas (313,527 cells) combining published and new endometrial single-cell transcriptomics datasets of 63 women with and without endometriosis. HECA assigns consensus and identifies previously unreported cell types, mapped in situ using spatial transcriptomics and validated using a new independent single-nuclei dataset (312,246 nuclei, 63 donors). In the functionalis, we identify intricate stromal-epithelial cell coordination via transforming growth factor beta (TGFß) signaling. In the basalis, we define signaling between fibroblasts and an epithelial population expressing progenitor markers. Integration of HECA with large-scale endometriosis genome-wide association study data pinpoints decidualized stromal cells and macrophages as most likely dysregulated in endometriosis. The HECA is a valuable resource for studying endometrial physiology and disorders, and for guiding microphysiological in vitro systems development.

In vitro exposure to benzo[a]pyrene damages the developing mouse ovary.

Females are born with a finite number of oocytes, collectively termed the ovarian reserve, established within the developing fetal ovary. Consequently, maternal exposure to reproductive toxicants can have harmful effects on the future fertility of her unborn female fetus. The chemical benzo[a]pyrene (B[a]P) is a prominent component of cigarette smoke. Despite it being a known ovotoxicant, around 8% of women in Europe smoke during pregnancy. The purpose of this research was to examine the effect of B[a]P on the developing ovary, using the mouse as a model and with experiments carried out in vitro. B[a]P-exposure to the fetal ovary prior to follicle formation reduced the number of germ cells and subsequently, the number of healthy primordial follicles, by up to 76%; however, while proliferation of germ cells was not affected, the germ cells contained higher levels of DNA double-strand breaks. Exposure to B[a]P also affected the proportion of oocytes progressing through prophase I of meiosis. B[a]P exposure to neonatal mouse ovaries, after follicle formation, resulted in an 85% reduction in the number of healthy follicles, with a corresponding increase in apoptotic cell death and reduction in somatic cell proliferation. Although there was a trend towards a higher level of oxidative stress in B[a]P-exposed ovaries, this was not statistically significant; likewise, the antioxidant melatonin failed to protect against the B[a]P-induced ovarian damage. Together, the results here demonstrate that B[a]P-exposure damages the developing ovary, both before and shortly after follicle formation, an effect that could lead to a subsequent decrease in fertility.

Early pregnancy maternal progesterone administration alters pituitary and testis function and steroid profile in male fetuses.

Maternal exposure to increased steroid hormones, including estrogens, androgens or glucocorticoids during pregnancy results in chronic conditions in offspring that manifest in adulthood. Little is known about effects of progesterone administration in early pregnancy on fetal development. We hypothesised that maternal early pregnancy progesterone supplementation would increase fetal progesterone, affect progesterone target tissues in the developing fetal reproductive system and be metabolised to other bioactive steroids in the fetus. We investigated the effects of progesterone treatment during early pregnancy on maternal and fetal plasma progesterone concentrations, transcript abundance in the fetal pituitary and testes and circulating steroids, at day 75 gestation, using a clinically realistic ovine model. Endogenous progesterone concentrations were lower in male than female fetuses. Maternal progesterone administration increased male, but not female, fetal progesterone concentrations, also increasing circulating 11-dehydrocorticosterone in male fetuses. Maternal progesterone administration altered fetal pituitary and testicular function in ovine male fetuses. This suggests that there may be fetal sex specific effects of the use of progesterone in early pregnancy, and highlights that progesterone supplementation should be used only when there is clear evidence of efficacy and for as limited time as necessary.

Label-free multiphoton microscopy reveals relevant tissue changes induced by alginate hydrogel implantation in rat spinal cord injury.

The development of therapies promoting recovery after spinal cord injury is a challenge. Alginate hydrogels offer the possibility to develop biocompatible implants with mechanical properties tailored to the nervous tissue, which could provide a permissive environment for tissue repair. Here, the effects of non-functionalized soft calcium alginate hydrogel were investigated in a rat model of thoracic spinal cord hemisection and compared to lesioned untreated controls. Open field locomotion tests were employed to evaluate functional recovery. Tissue analysis was performed with label-free multiphoton microscopy using a multimodal approach that combines coherent anti-Stokes Raman scattering to visualize axonal structures, two-photon fluorescence to visualize inflammation, second harmonic generation to visualize collagenous scarring. Treated animals recovered hindlimb function significantly better than controls. Multiphoton microscopy revealed that the implant influenced the injury-induced tissue response, leading to decreased inflammation, reduced scarring with different morphology and increased presence of axons. Demyelination of contralateral white matter near the lesion was prevented. Reduced chronic inflammation and increased amount of axons in the lesion correlated with improved hindlimb functions, being thus relevant for locomotion recovery. In conclusion, non-functionalized hydrogel improved functional outcome after spinal cord injury in rats. Furthermore, label-free multiphoton microscopy qualified as suitable technique for regeneration studies.