Serine threonine kinase receptor associated protein regulates early follicle development in the mouse ovary.

The molecular mechanisms involved in regulating the development of small, gonadotrophin-independent follicles are poorly understood; however, many studies have highlighted an essential role for TGFB ligands. Canonical TGFB signalling is dependent upon intracellular SMAD proteins that regulate transcription. STRAP has been identified in other tissues as an inhibitor of the TGFB-SMAD signalling pathway. Therefore, in this study we aimed to determine the expression and role of STRAP in the context of early follicle development. Using qPCR, Strap, Smad3 and Smad7 revealed similar expression profiles in immature ovaries from mice aged 4-16 days containing different populations of early growing follicles. STRAP and SMAD2/3 proteins co-localised in granulosa cells of small follicles using immunofluorescence. Using an established culture model, neonatal mouse ovary fragments with a high density of small non-growing follicles were used to examine the effects of Strap knockdown using siRNA and STRAP protein inhibition by immuno-neutralisation. Both interventions caused a reduction in the proportion of small, non-growing follicles and an increase in the proportion and size of growing follicles in comparison to untreated controls, suggesting inhibition of STRAP facilitates follicle activation. Recombinant STRAP protein had no effect on small, non-growing follicles, but increased the mean oocyte size of growing follicles in the neonatal ovary model and also promoted the growth of isolated preantral follicles in vitro Overall findings indicate STRAP is expressed in the mouse ovary and is capable of regulating development of small follicles in a stage-dependent manner

SMAD3 directly regulates cell cycle genes to maintain arrest in granulosa cells of mouse primordial follicles

Primordial follicles, consisting of granulosa cell (GC)-enveloped oocytes are maintained in a state of developmental arrest until activated to grow. The mechanism that operates to maintain this arrested state in GCs is currently unknown. Here, we show the TGFβ-activated transcription factor SMAD3 is expressed in primordial GC nuclei alongside the cell cycle proteins, cyclin D2 (CCND2) and P27. Using neonatal C57/Bl6 mouse ovaries densely populated with primordial follicles, CCND2 protein co-localised and was detected in complex with P27 by immunofluorescence and co-immunoprecipitation, respectively. In the same tissue, SMAD3 co-precipitated with DNA sequences upstream of Ccnd2 and Myc transcription start sites implicating both as direct SMAD3 targets. In older ovaries follicle growth was associated with nuclear exclusion of SMAD3 and reduced P27 and CCND2 in GCs, alongside elevated Myc expression. Brief (2 H) exposure of neonatal ovaries to TGFβ1 (10 ng/ml) in vitro led to immediate dissociation of SMAD3 from the Ccnd2 and Myc promoters. This coincided with elevated Myc and phospho-S6, an indicator of mTOR signalling, followed by a small increase in mean primordial GC number after 48 H. These findings highlight a concentration-dependent role for TGFβ signalling in the maintenance and activation of primordial follicles, through SMAD-dependent and independent signalling pathways, respectively

Imaging seminiferous tubules – a 9.4T MRI mouse model

Fertility problems affect many couples. Research into male fertility commonly uses mouse models due to their availability and similar spermatogenesis to humans. A common target is the seminiferous tubules, the site of spermatozoa production, typically studied using biopsies and histological analysis. High-field Magnetic Resonance (MR) may offer a non-invasive alternative to investigate testicular function in infertility models. Here, we assess seminiferous tubules structure in sacrificed mice to determine the usefulness of MR compared to histology. Twelve mice (11 aged 35-57 days, one >9 months) were sacrificed and MR imaged at 9.4T with a Rapid Acquisition with Relaxation Enhancement sequence. Testes were scanned in situ for all mice, and excised in a subset of mice (n=4). A second subset of mice (n=4) had their testes selected for histological analysis. Seminiferous tubule diameter was measured manually from MRI and histology images. Custom image analysis scripts were created for the automated segmentation of seminiferous tubules and calculation of tissue volumes. All ex vivo and in situ images of testes exhibited clear outlines of seminiferous tubules. Ratio of total testis volume to volume of seminiferous tubules did not differ significantly between ex vivo and in situ measurements, and were similar in mature and younger mice. Both total testis volume and seminiferous tubule volume were larger in the mature animal. While histological slices trended towards larger average seminiferous tubules diameter than MRI images, we found no significant differences between MRI and histological measurements. High-field MRI can be used in a mouse model to assess testicular structure in situ. All volumetric measurements compared favourably with histological data. In situ scans also clearly showed identifiable extra-testicular tissues, such as epididymis and prostate tissues. The potential to image tissues associated with sperm maturation as well as spermatogenesis emphasises how MR could be a useful technique in mouse models of fertility, however further work is required to optimize tissue segmentation and validate this method for use in longitudinal studies. This type of measurement could be extended to human fertility studies in the future

XXVI Congreso Nacional y II Congreso Internacional de SEDEM

Organizan: Sociedad Española de Educación Médica y Facultad de Medicina y Enfermería, Universidad del País Vasco (UPV/EHU)Comunicaciones aceptadas en el XXVI Congreso de la Sociedad Española de Educación Médica, celebrado en Bilbao del 28 al 30 de noviembre de 2024

Nicotine exposure is associated with targeted impairments in primordial follicle phenotype in cultured neonatal mouse ovaries

The ovarian reserve consists of a limited supply of primordial follicles (PFs), each containing an oocyte surrounded by a layer of granulosa cells (GCs). PFs are relatively quiescent and must remain viable for a long period, thereby making them susceptible to environmental and lifestyle influences. Given the widespread prevalence of e-cigarette use, this study aimed to investigate the effects of nicotine and its metabolite cotinine in a mouse model and to elucidate the mechanisms by which nicotine influences the ovarian reserve. Neonatal ovaries were cultured for 7-days in nicotine or cotinine reflective of concentrations in plasma of e-cigarette users. From histological evaluation, nicotine or cotinine had no impact on the number of PFs or early growing follicles; however, the medium (15 ng/ml) and high (45 ng/ml) concentrations of nicotine (but not cotinine) caused a small reduction in oocyte and GC size within PFs relative to controls (0 ng/ml; both P<0.01). These morphological effects were not associated with changes in immunofluorescent markers of apoptosis (active caspase-3) or proliferation (Pcna), but were associated with increased gH2AX in PF oocytes, indicative of DNA damage and repair. RNA-sequencing of cultured ovaries exposed to nicotine (45 ng/ml) relative to control (0 ng/ml), revealed a suite of differentially expressed candidates, as well as numerous gene ontology biological processes associated with increased DNA damage, metabolism, respiration and immune function, alongside suppression of meiosis, cell adhesion, differentiation and morphogenesis. Findings from this study indicate that direct nicotine exposure has a limited effect on the quantity of PFs, but importantly highlights a range of processes that could impinge on the quality of the ovarian reserve

Imaging seminiferous tubules – a 9.4T MRI mouse model

Fertility problems affect many couples. Research into male fertility commonly uses mouse models due to their availability and similar spermatogenesis to humans. A common target is the seminiferous tubules, the site of spermatozoa production, typically studied using biopsies and histological analysis. High-field Magnetic Resonance (MR) may offer a non-invasive alternative to investigate testicular function in infertility models. Here, we assess seminiferous tubules structure in sacrificed mice to determine the usefulness of MR compared to histology. Twelve mice (11 aged 35-57 days, one &gt;9 months) were sacrificed and MR imaged at 9.4T with a Rapid Acquisition with Relaxation Enhancement sequence. Testes were scanned in situ for all mice, and excised in a subset of mice (n=4). A second subset of mice (n=4) had their testes selected for histological analysis. Seminiferous tubule diameter was measured manually from MRI and histology images. Custom image analysis scripts were created for the automated segmentation of seminiferous tubules and calculation of tissue volumes. All ex vivo and in situ images of testes exhibited clear outlines of seminiferous tubules. Ratio of total testis volume to volume of seminiferous tubules did not differ significantly between ex vivo and in situ measurements, and were similar in mature and younger mice. Both total testis volume and seminiferous tubule volume were larger in the mature animal. While histological slices trended towards larger average seminiferous tubules diameter than MRI images, we found no significant differences between MRI and histological measurements. High-field MRI can be used in a mouse model to assess testicular structure in situ. All volumetric measurements compared favourably with histological data. In situ scans also clearly showed identifiable extra-testicular tissues, such as epididymis and prostate tissues. The potential to image tissues associated with sperm maturation as well as spermatogenesis emphasises how MR could be a useful technique in mouse models of fertility, however further work is required to optimize tissue segmentation and validate this method for use in longitudinal studies. This type of measurement could be extended to human fertility studies in the future

Autologous cell therapy with CD133+ bone marrow-derived stem cells for Asherman Syndrome: a phase 1/2 trial

Abstract Autologous CD133+ bone marrow-derived stem cell (BMDSC) therapy has been designated as an Orphan Drug by the EMA and FDA for the treatment of Asherman Syndrome (AS). This phase 1/2, non-randomized, open-label, single-arm trial assessed the safety and efficacy of this novel therapy in 20 infertile women with moderate to severe AS, unresponsive to prior hysteroscopic treatments. Primary endpoints were safety and tolerability over 15 months follow-up, including during pregnancy and after live birth. The therapy was well tolerated with a mean dosage of 125.41 × 106 cells, with no treatment-related serious adverse events and only reversible events such as arm pain, headache, and nausea. In pregnant patients, minor obstetric complications were reflux-related cough (n = 1), gestational diabetes (n = 2), cervical shortening requiring pessary placement (n = 2), and postpartum placenta accreta (n = 1). No preterm labor occurred, and all six newborns remained free of significant adverse events. Our findings suggest that autologous CD133 + BMDSC therapy is a safe and effective treatment for AS. Clinical trial registration (Eudra CT): 2016-003975-2

Chronic coronary syndromes without standard modifiable cardiovascular risk factors and outcomes: the CLARIFY registry

Background and Aims: It has been reported that patients without standard modifiable cardiovascular (CV) risk factors (SMuRFs—diabetes, dyslipidaemia, hypertension, and smoking) presenting with first myocardial infarction (MI), especially women, have a higher in-hospital mortality than patients with risk factors, and possibly a lower long-term risk provided they survive the post-infarct period. This study aims to explore the long-term outcomes of SMuRF-less patients with stable coronary artery disease (CAD). Methods: CLARIFY is an observational cohort of 32 703 outpatients with stable CAD enrolled between 2009 and 2010 in 45 countries. The baseline characteristics and clinical outcomes of patients with and without SMuRFs were compared. The primary outcome was a composite of 5-year CV death or non-fatal MI. Secondary outcomes were 5-year all-cause mortality and major adverse cardiovascular events (MACE—CV death, non-fatal MI, or non-fatal stroke). Results: Among 22 132 patients with complete risk factor and outcome information, 977 (4.4%) were SMuRF-less. Age, sex, and time since CAD diagnosis were similar across groups. SMuRF-less patients had a lower 5-year rate of CV death or non-fatal MI (5.43% [95% CI 4.08–7.19] vs. 7.68% [95% CI 7.30–8.08], P = 0.012), all-cause mortality, and MACE. Similar results were found after adjustments. Clinical event rates increased steadily with the number of SMuRFs. The benefit of SMuRF-less status was particularly pronounced in women. Conclusions: SMuRF-less patients with stable CAD have a substantial but significantly lower 5-year rate of CV death or non-fatal MI than patients with risk factors. The risk of CV outcomes increases steadily with the number of risk factors