The Bone Morphogenetic Protein (BMP) family reiteratively signals to direct disparate cellular fates throughout embryogenesis. In the developing dorsal spinal cord, multiple BMPs are required to specify sensory interneurons (INs). Previous studies suggested that the BMPs act as concentration-dependent morphogens to direct IN identity, analogous to the manner in which sonic hedgehog patterns the ventral spinal cord. However, it remains unresolved how multiple BMPs would cooperate to establish a unified morphogen gradient. Our studies support an alternative model: BMPs have signal-specific activities directing particular IN fates. Using chicken and mouse models, we show that the identity, not concentration, of the BMP ligand directs distinct dorsal identities. Individual BMPs promote progenitor patterning or neuronal differentiation by their activation of different type I BMP receptors and distinct modulations of the cell cycle. Together, this study shows that a ‘mix and match’ code of BMP signaling results in distinct classes of sensory INs.

STUDY QUESTION Does LH protect mouse oocytes and female fertility from alkylating chemotherapy? SUMMARY ANSWER LH treatment before and during chemotherapy prevents detrimental effects on follicles and reproductive lifespan. WHAT IS KNOWN ALREADY Chemotherapies can damage the ovary, resulting in premature ovarian failure and reduced fertility in cancer survivors. LH was recently suggested to protect prepubertal mouse follicles from chemotoxic effects of cisplatin treatment. STUDY DESIGN, SIZE, DURATION This experimental study investigated LH effects on primordial follicles exposed to chemotherapy. Seven-week-old CD-1 female mice were randomly allocated to four experimental groups: Control (n = 13), chemotherapy (ChT, n = 15), ChT+LH-1x (n = 15), and ChT+LH-5x (n = 8). To induce primary ovarian insufficiency (POI), animals in the ChT and ChT+LH groups were intraperitoneally injected with 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan, while control mice received vehicle. For LH treatment, the ChT+LH-1x and ChT+LH-5x animals received a 1 or 5 IU LH dose, respectively, before chemotherapy, then a second LH injection administered with chemotherapy 24 h later. Then, two animals/group were euthanized at 12 and 24 h to investigate the early ovarian response to LH, while remaining mice were housed for 30 days to evaluate short- and long-term reproductive outcomes. The effects of LH and chemotherapy on growing-stage follicles were analyzed in a parallel experiment. Seven-week-old NOD-SCID female mice were allocated to control (n = 5), ChT (n = 5), and ChT+LH-1x (n = 6) groups. Animals were treated as described above, but maintained for 7 days before reproductive assessment. PARTICIPANTS/MATERIALS, SETTING, METHODS In the first experiment, follicular damage (phosphorylated H2AX histone (γH2AX) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay), apoptotic biomarkers (western blot), and DNA repair pathways (western blot and RT-qPCR) were assessed in ovaries collected at 12 and 24 h to determine early ovarian responses to LH. Thirty days after treatments, remaining mice were stimulated (10 IU of pregnant mare serum gonadotropin (PMSG) and 10 IU of hCG) and mated to collect ovaries, oocytes, and embryos. Histological analysis was performed on ovarian samples to investigate follicular populations and stromal status, and meiotic spindle and chromosome alignment was measured in oocytes by confocal microscopy. Long-term effects were monitored by assessing pregnancy rate and litter size during six consecutive breeding attempts. In the second experiment, mice were stimulated and mated 7 days after treatments and ovaries, oocytes, and embryos were collected. Follicular numbers, follicular protection (DNA damage and apoptosis by H2AX staining and TUNEL assay, respectively), and ovarian stroma were assessed. Oocyte quality was determined by confocal analysis. MAIN RESULTS AND THE ROLE OF CHANCE LH treatment was sufficient to preserve ovarian reserve and follicular development, avoid atresia, and restore ovulation and meiotic spindle configuration in mature oocytes exposed at the primordial stage. LH improved the cumulative pregnancy rate and litter size in six consecutive breeding rounds, confirming the potential of LH treatment to preserve fertility. This protective effect appeared to be mediated by an enhanced early DNA repair response, via homologous recombination, and generation of anti-apoptotic signals in the ovary a few hours after injury with chemotherapy. This response ameliorated the chemotherapy-induced increase in DNA-damaged oocytes and apoptotic granulosa cells. LH treatment also protected growing follicles from chemotherapy. LH reversed the chemotherapy-induced depletion of primordial and primary follicular subpopulations, reduced oocyte DNA damage and granulosa cell apoptosis, restored mature oocyte cohort size, and improved meiotic spindle properties. LARGE SCALE DATA N/A. LIMITATIONS, REASONS FOR CAUTION This was a preliminary study performed with mouse ovarian samples. Therefore, preclinical research with human samples is required for validation. WIDER IMPLICATIONS OF THE FINDINGS The current study tested if LH could protect the adult mouse ovarian reserve and reproductive lifespan from alkylating chemotherapy. These findings highlight the therapeutic potential of LH as a complementary non-surgical strategy for preserving fertility in female cancer patients. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Regional Valencian Ministry of Education (PROMETEO/2018/137), the Spanish Ministry of Science and Innovation (CP19/00141), and the Spanish Ministry of Education, Culture and Sports (FPU16/05264). The authors declare no conflict of interest.

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Study question Could controlled ovarian stimulation (COS) protocols with letrozole supplementation induce changes in the molecular footprints of estrogen receptor-positive (ER+) breast cancer (BC) women? Summary answer Ovarian stimulation with letrozole and gonadotropins does not change the gene expression profile of estrogen receptor-positive (ER+) breast malignant tumours as compared to non-stimulated patients. What is known already Fertility preservation (FP) strategies in ER+ BC patients involve modifications of the classical COS protocols to minimise the impact of estrogen levels during ovarian stimulation. The most common approaches are using the aromatase inhibitor letrozole to block the conversion of testosterone into estradiol in the granulosa cells or the use of tamoxifen to block the ER in the breast tissue. Although retrospective studies suggest that these strategies will have no impact on patient’s survival and disease progression, little is known regarding the effect of the ovarian stimulation on the genomic fingerprints of the exposed cancerous tissue. Study design, size, duration Retrospective, non-randomized, comparative study. Gene expression profiles were compared in biopsies at diagnosis and during excision surgery in two groups of ER+ BC patients: Patients undergoing COS for FP before surgery (COS group, n = 10) and patients not undergoing any type of COS (Control group, n = 11). The former received letrozole (5mg/day) supplements during COS. The latter included 7 patients not doing FP and 4 undergoing ovarian cortex cryopreservation. Patients were recruited between 2009 and 2021. Participants/materials, setting, methods Single 5-µm sections of formalin-fixed, paraffin-embedded (FFPE) needle core biopsy (NCB) and breast surgery (BS) tumour samples were obtained prior to starting gonadotoxic therapies. Differential gene expression and gene set analysis (GSA) was performed in tumour samples before (NCB-sample) and after COS (BS-sample). Simultaneous, quantitative detection of 2549 genes associated with tumour biology was performed with the HTG EdgeSeq Oncology Biomarker Panel. Tumour cell proliferation was also assessed by Ki67 staining. Main results and the role of chance Patients were younger in the COS group (COS: 30.2±3.4 vs Control: 34.5±2.3; p = 0.004). None of the patients experienced any relapse during the observation period. The length of COS was 11.5±3.5 days and 10.6±6.8 MII oocytes were vitrified. The exploratory analysis using principal component analysis revealed no relationships between the two BC biopsy samples and gene expression levels in the experimental groups. The differential expression analysis just revealed 6 genes significantly over-expressed after ovarian stimulation (DUSP1, FOS, EGFR1, NR4A1, JUN and CYR61). From these, DUSP1, FOS, and NR4A1 were also significantly upregulated in the unstimulated group. The GSA showed the cytochrome P450 pathway was significantly enriched after COS. No pathways related to cell proliferation were differentially expressed between groups. However, in unstimulated patients, 6 KEGG pathways were upregulated, with the cytokine-cytokine receptor interaction and the Jak-STAT signalling pathway being the most enriched. Ki67 immunostaining showed no differences in cell proliferation after COS (22.8±7.2 vs 24.0±6.9, p = 0.723). The rate of cell proliferation also remained constant in unstimulated patients (22.9±8.4 vs 25.1±8.1, p = 0.560). Limitations, reasons for caution The main limitations of the present study are the retrospective design and the associated risk of bias, although the population is very homogeneous when it comes to clinical characteristics; and the fact that molecular footprints are only interim markers of survival/response to the treatment. Wider implications of the findings The present study provides molecular evidence supporting the safety of COS using letrozole for oocyte vitrification in young BC patients undergoing ovarian stimulation. This information is crucial to support patient guidance during discussions about fertility preservation. Trial registration number Not applicable