Distribution Of Follicles Research Articles

Effect of Jinfeng Pill on ovarian function of Tripterygium wilfordii polyglycoside tablets induced poor ovarian response rats

Objective: This study aimed to assess the effects of the Jinfeng pill on ovarian volume and endocrine function in rats with poor ovarian response and to elucidate the gene expression changes that contribute to the Jinfeng pill’s role in improving ovarian function. Methods: Thirty 8-week-old specific pathogen-free (SPF) female rats were randomly assigned to five groups: control, model, estradiol, Jinfeng pill, and Jinfeng pill + estradiol, with six rats in each group. All groups, except the control were administered Tripterygium glycoside tablets. After establishing the poor ovarian response model, the three intervention groups (estradiol, Jinfeng pill, and Jinfeng pill + estradiol) received their respective drug interventions. Hematoxylin-eosin staining was employed to assess follicle distribution, and immunohistochemistry was used to evaluate the expression of vascular endothelial growth factor and endostatin in rat ovaries. Hormone levels were measured using ELISA. Results: The uterine and total ovarian masses in the three intervention groups were higher than those in the model group; however, the differences were not statistically significant. The ovaries of the Jinfeng pill and Jinfeng pill + estradiol groups contained more follicles at various developmental stages. The three intervention groups showed significantly increased vascular endothelial growth factor expression and decreased endostatin expression compared with the model group, with the Jinfeng pill + estradiol group exhibiting the most pronounced differences. Anti-Müllerian hormone levels were slightly elevated in the Jinfeng pill group compared to the model group. However, only the Jinfeng pill + estradiol group showed a statistically significant decrease in follicle-stimulating hormone levels compared to the model group (P = 0.01). Conclusions: Jinfeng pill modulates ovarian protein expression, increases ovarian volume, and improves blood circulation. Additionally, it may elevate hormone levels and enhance ovarian response function in rats with poor ovarian function.

Ovarial Torsion in Young Children. Ultrasound Diagnostic

Background. Early adnexal torsion (AT) is extremely rare. It usually involves a torsion of the ovary together with the tube, which cannot be differentiated by transabdominal ultrasound, so the terms "adnexal torsion" and "ovarian torsion" for children are echographically identical.Objective. To study the features of echographic representation of ovarian torsion in infant girls.Materials and Methods. During 2015–2024, adnexal torsion was echographically diagnosed in 6 girls of the first year of life. All patients underwent transabdominal ultrasound in B-mode and color Doppler scanning. All children were operated, and the diagnosis was verified.Results. The frequency of adnexal torsion in children under 1 year of age was 13.3% of all patients (45) with this pathology. The complaints and clinical picture were nonspecific. In all cases, suspicion of adnexal torsion was expressed only when ultrasound was performed. There was an increase in the volume of the affected ovary by 6.91 ± 0.68 times compared to the contralateral ovary and increased echogenicity of the ovarian parenchyma, against which numerous anechogenic follicles were traced. Vascular pattern in the parenchyma of the affected ovary was detected in 2/6 cases (33.33%), uterine appendages were irreversibly changed. Hyperstimulated ovaries were observed in 4 out of 6 cases. The distribution of follicles in the affected ovary was chaotic, there was no "necklace symptom", there was no marked rounding of the ovarian shape, and the amount of stromal component in the tilted ovary was relatively small. In 5/6 (83.3%) girls, no pelvic cavity effusion was noted.ConclusionThere are no specific anamnestic and clinical data suggestive of adnexal torsion in infants, which determines the importance of ultrasound as virtually the only method of emergency diagnosis of this pathology.The peculiarity of ultrasound in the infant should be the maximum expansion of the area of examination with attempts to visualize the pelvic organs regardless of the degree of bladder filling.The basis of echographic diagnosis of adnexal torsion in infants are structural changes of the appendage in B-mode: increase in linear size of 1.5–2 times, volume of 4–10 times and increased echogenicity of the stroma. Rounding of the shape of the affected ovary is insignificant.A feature of twisted appendage in infants is a large number of follicles in the affected ovary (2/3 of observations). The "necklace" symptom is atypical.The presence of abdominal effusion in adnexal torsion in infants is not characteristic.Preservation of the vascular pattern in the ovary does not exclude adnexal torsion, is infrequent (in 1/3 of cases) and does not guarantee preservation of ovarian viability.

7395 Developmental Programming: Adverse Effects Of Prenatal Exposure To A Real-Life Environmental Chemical Mixture Via Biosolids On Ovarian Folliculogenesis In Adult Sheep

Abstract Disclosure: Y. Zhou: None. K.M. Halloran: None. M. Bellingham: None. N.P. Evans: None. R.G. Lea: None. K.D. Sinclair: None. V. Padmanabhan: None. The ovarian capacity to provide fertilizable oocytes, regulate folliculogenesis as well as activate and progress primordial follicles into preovulatory follicles are key contributing factors to female fertility and reproductive aging. In sheep, as in humans, while genetics play a role in establishing the ovarian reserve during fetal development, developmental insults such as inappropriate exposure to native steroids or environmental chemicals (ECs) can adversely impact the establishment of the ovarian reserve and, additionally can affect folliculogenesis. Considering humans are exposed to multiple ECs simultaneously which could have additive, synergistic, or antagonistic effects, real-life EC exposure models are needed to assess risks posed by EC exposure. Offspring from sheep grazed on biosolids-treated pasture offers one such real-life exposure model. Studies with day 140 fetuses found maternal exposure to a real-life EC mixture via biosolids increased the proportion of unhealthy transitory follicles (Lea et al., Sci Rep. 2016;6:22279). The present study tested if biosolids exposure during fetal life would impact folliculogenesis, follicle activation and follicle atresia during their adult life. Ovaries were derived from adult EasyCare ewes (age 31.4 ± 0.5 months) whose mothers were grazed on biosolids-treated (BTP, n=10) or inorganic fertilizer-treated (Control, n=10) pastures from preconception through to birth. Ovarian morphometry was undertaken to assess the distribution and health status of follicles. Follicular atresia was evaluated via the expression of Caspase-3, an apoptosis marker. Data analysis utilized Student's t-test for normally distributed variables and Mann-Whitney U tests for non-parametric comparisons. There were no differences in the absolute counts or the percentages of follicle classes at all stages of folliculogenesis, the density of primordial follicles in cortical tissue, or the follicular activation rate. An increase in the percentage of unhealthy activated follicles (post-primordial) was noted in the BTP group (p = 0.014), predominantly in the transitory stage (p = 0.021). Consistent with this, a higher density of Caspase-3 positive primary follicles was observed in the BTP group (p = 0.033). These findings indicate the adverse impacts of fetal exposure to an EC mixture via maternal biosolid exposure on the survival of early-stage activated follicles in adult sheep. The lack of difference in ovarian reserve between the control and BTP groups suggests compensatory mechanisms may be in place to prevent premature depletion of the ovarian reserve. Whether EC mixture exposure via biosolids also has an adverse effect on the quality of the surviving activated follicles remains to be determined. Funding source: NIH R01 ES030374. Presentation: 6/3/2024

A New Species of Creptotrematina (Trematoda: Allocreadiidae) from the Red Minor Tetra, Hyphessobrycon eques (Steindachner, 1882) (Characidae) from Brazil, with Comments on the Genetic Divergence of C. Aguirrepequenoi Jiménez-Guzmán, 1973 across a Wide Geographical Range in Middle America.

Allocreadiids are relatively small digeneans that appear to be restricted to freshwater systems distributed across the world. Allocreadiids are highly diverse in the Americas, particularly in the Neotropical biogeographical region. Their taxonomic history has been rather controversial, with several taxonomic reassessments and the description of new genera and species. We sampled Creptotrematina specimens from a characid collected in the Pardo River, Paranapanema River basin in Brazil, and specimens of C. aguirrepequenoi, from Astyanax spp. in several localities between northern Mexico and Costa Rica. The specimens were studied through integrative approaches using morphological and molecular analyses of the 28S rDNA gene and two different regions of the COI mtDNA gene. We describe a new species of Creptotrematina which is differentiated from other congeners by the overall body size, but in particular by the size and position of the cirrus-sac, distribution of the vitelline follicles, and extension of uterine loops in the posterior end of body. Phylogenetic analyses of the 28S rDNA and COI mtDNA genes placed the new species in a monophyletic clade together with all other sequenced species of Creptotrematina, and as a sister species of C. batalhensis. Genetic divergences between the new species and other Creptotrematina spp. varied from 1.1 to 1.2% for the 28S rDNA and 12.4-14.3% for the COI mtDNA. Phylogenetic analysis based on COI mtDNA showed the isolates of C. aguirrepequenoi grouped in four monophyletic clades representing populations geographically separated along a wide geographical range spanning between northern Mexico and Costa Rica, with an estimated genetic divergence between 3.9% and 8.9%. Our findings based on integrative analyses recognize Creptotrematina saltograndensis n. sp. from a characid collected in the Pardo River, Paranapanema River basin in Brazil and provide validation of the wide geographical distribution of C. aguirrepequenoi across Middle-America parasitizing Astyanax spp.; the genetic divergence of the species through the analysis of two regions of COI mtDNA points towards considering it represent a species complex, although we refrain at the moment on describing a new species, awaiting for further verification using other molecular markers, and obtaining fresh material for a more detailed taxonomic analyses. This study increases the known diversity of allocreadiids and contributes to the understanding of evolutionary relationships, host-parasite relationships, and biogeographic history of the group.

P-368 In vitro culture with rutin and VEGF-A before xenotransplantation protects ovarian tissue from ischemia and oxidative damage but not from growing follicle depletion

Abstract Study question Can pre-treatment with antioxidant and vascular endothelial factor (VEGF-A) help to maintain healthy tissue structure and follicle pool in xenotransplanted human ovarian tissue? Summary answer Rutin and VEGF-A help to prevent fibrosis and oxidative stress damage in grafted ovarian tissue but cannot preserve growing follicles. What is known already Nowadays, ovarian tissue (OT) cryopreservation and autotransplantation become established fertility preservation methods in numerous countries. However, even if hundreds of children has been born thanks to this procedure, most crucial process during which 70-80% of the follicles are lost in the graft is the transplantation itself. As the graft restores vascularisation and regains its function the tissue undergoes ischemia and oxidative stress. There have been attempts made to improve this critical postoperative period by administration of different growth factors, hormones, antoxidants or mechanical tissue damage. Study design, size, duration Pieces of frozen-tharwed OT (2,5 × 2,5 mm) from 10 women were transplanted into immunodeficient mice for 4 weeks. Before xenotransplantation OT fragments were in vitro cultured for 36h in culture media supplemented with 1 μg/ml rutin and 50 ng/ml VEGF-A (n = 10 OT, pretreated grafted) or without (n = 10 OT, non-pretreated grafted). Frozen-thawed OTs were used as non-grafted controls for ischemia-reperfusion injury, apoptosis and follicle loss evaluation. Participants/materials, setting, methods OTs were received from cancer patients (n = 10) undergoing cryopreservation at National Cancer Institute. Xenotransplantation was performed at Life Sciences Centre, Vilnius University. Retrieved grafts were devided into one small piece for immunochistochemical evaluation of α-SMA and apoptosis by TUNEL, and one bigger piece for qPCR analysis of genes related to tissue integrity (PRDX1, SOD1, FN1, COL6A1, CASP3, ANGPT2, mouse CD31) and follicle development (FOXO3, FSHR, GDF9), house keeping gene being GAPDH/Gapdh. Main results and the role of chance After 4 weeks of grafting, multiple genes and protein expressions were evaluated. Vascularisation evaluation: compared to non-grafted group, in grafted OTs we observed significantly more vessels (α-SMA staining, p < 0.0001 and ANGPT2 expression, p < 0.05), as well 1,5-fold higher relative expression of mouse CD31 in pretreated grafted group vs non-pretreated grafted group. Qxidative stress and apoptosis evaluation: expression of SOD2, PRDX1 and CASP3 genes was lower in both grafted groups compared to non-grafted control, significant alterations between xenotransplanted groups were found. Structural gene evaluation: in non-grafted control we could find higher expression of COL6A1 and FN1 genes compared to grafted groups and statistical difference of COL6A1 expression in between grafted groups (p < 0.05). Follicle pool evaluation: primordial follicle activation was measured by FOXO3 gene expression change, though we did not find significant differences between any of three groups, p = 0.06 was observed between grafted OT groups. Maturing follicles were evaluated by expression changes in GDF9 and FSHR genes. We observed significant decrese in expression of these genes in grafted groups (p < 0.05). Results from TUNEL are awaited, thus are the quantification of apoptotic cells, which will provide valuable insights for correlation with vascularisation and oxidative stress. Limitations, reasons for caution This study was performed only in a long-term xenotransplantation setting, we cannot know if rutin and VEGF-A had a significant influence during the first days of OT transplantation. Due to the small size of OT grafted, counting and distribution of follicles was not performed, Wider implications of the findings This study shows that pretreatment of OTs with rutin and VEGF-A could be potentially used in human setting due to protection of ischemia and oxidative stress, especially if the survival of growing follicles is not required, as it is in cases of prepubertal girls. Trial registration number not applicable

P-320 Ovarian tissue attached to stripped endometriomas: transforming the collateral damage to a potential source for oocyte preservation?

Abstract Study question To determine (antral) follicle density in the ovarian cortex tissue attached to stripped endometriomas and patient- and surgical characteristics affecting this density. Summary answer Ovarian tissue attached to stripped endometriomas offers a non-invasive source for antral follicles. Research with fresh tissue is necessary to further asses the quality. What is known already Ovarian endometriosis is associated with diminished ovarian reserve due to the disease itself as well as damage caused by excisional surgery, which may result in subfertility. When resecting endometriomas with the stripping technique, in the majority of cases, a thin line of adjacent ovarian cortex is attached to the endometrioma. Data regarding density and maturation of the follicles in this tissue are conflicting in previous publications. This study aims to quantify and characterise follicles and in particular antral follicles in ovarian tissue surrounding resected endometriomas as these may serve as a potential oocyte source for in vitro maturation (IVM). Study design, size, duration A retrospective cohort study was conducted at the Leiden University Medical Centre and the Endometriosis in Balance endometriosis expertise centre in Haaglanden Medical Centre (The Netherlands). Pathology slides of patients (≤50 years) that underwent uni- or bilateral endometrioma resection between 2011 and 2021 were analysed for the presence of follicles. Participants/materials, setting, methods Histological slides of previously removed endometriomas were assessed according to maturation and follicular density. Gougeon’s original follicle classification was modified by adding additional intermediate stages. Immunofluorescent staining of antral follicles in a subset of endometriomas was also performed. Main results and the role of chance In 90 out of 96 included endometriomas (93.7%), ovarian tissue attached to the cyst wall was observed. 1944 follicles at different maturation stages were identified (3 follicles/mm3). Follicle density was negatively associated with age (p < 0.001). Antral follicles (<7mm diameter) were present in the ovarian tissue attached to 35 endometriomas (36.5%) derived from younger patients compared to endometriomas where none were detected (30 versus 35 years, p = 0.003). Antral follicle density was 1 follicles/mm3. Based on immunofluorescence, healthy antral follicles were identified in 2 out of 4 examined endometriomas. Limitations, reasons for caution Tissue analysis was constrained by number of available pathology slides per patient, not fully representing the follicle distribution in the removed ovarian tissue. Immunofluorescence was performed on paraffin embedded tissue derived from a small number of endometriomas. Future research with fresh tissue is necessary to further assess viability. Wider implications of the findings Ovarian tissue attached to stripped endometriomas holds potential as a non-invasive source for antral follicles. In theory, application of IVM could be an interesting alternative fertility preservation option in young patients with endometriomas who undergo cystectomy in order to transform the surgical collateral damage to a potential oocyte source. Trial registration number not applicable

Uncovering further diversity of Ochoterenatrema Caballero, 1943 (Digenea: Lecithodendriidae) in South American bats

Ochoterenatrema Caballero, 1943 is a genus of lecithodendriid digeneans that prior to this study included 8 species parasitic in bats in the Western Hemisphere. Species of Ochoterenatrema possess a unique morphological feature in form of the pseudogonotyl on the sinistral side of the ventral sucker. In this study, we describe 2 new species of Ochoterenatrema from bats in Ecuador. The new species are readily differentiated from their congeners by a combination of morphological characters, including the distribution of vitelline follicles, length of oesophagus, sucker ratio and the body shape, among other features. We have generated partial nuclear 28S rDNA and mitochondrial cox1 gene DNA sequences from both new species. The newly obtained sequences were used to differentiate among species and study the phylogenetic interrelationships among Ochoterenatrema spp. The internal topology of the clade was weakly supported, although the cox1 tree was much better resolved than the 28S tree. Comparison of sequences revealed 0–1.2% interspecific divergence in 28S and 3.3–20.5% interspecific divergence in cox1 among Ochoterenatrema spp. The new findings demonstrate that bats in South America likely harbor multiple additional undescribed species of Ochoterenatrema. More extensive sampling from broader geographic and host ranges, especially in North America, should allow for a better understanding of the evolution of host associations and morphological traits of this lineage of lecithodendriid digeneans.

Determination of anti-Müllerian hormone levels in blood and urine in fertile cats

In recent years, blood anti-Müllerian hormone (AMH) levels have been investigated in female animals to diagnose many conditions, such as the presence of ovarian tissue, follicle reserve, and granulosa cell tumors. Since blood collection is an invasive method, diagnosis with a non-invasive method is important in terms of practicality and animal welfare. This study aimed to investigate the presence of AMH in cat urine and determine whether a correlation exists between blood and urine AMH levels. In addition, it was aimed at revealing whether there was a change in blood and urine AMH levels according to ovarian follicle distribution. Twenty-seven healthy, fertile female cats in the follicular phase were included. Following blood and urine sample collection, a routine ovariohysterectomy was performed. Histological analysis of the removed ovarian tissue was used to determine ovarian follicle types. While both AMH and estrogen levels were determined in blood samples, only AMH levels were investigated in urine samples. Blood AMH levels averaged 10.61 ± 0.75 ng/mL (range: 5–16 ng/mL), while urine AMH levels averaged 5.67 ± 0.91 ng/mL (range: 0.2–13 ng/mL). While urinary AMH level was <1 ng/mL in 7 cats, urinary AMH was >1 ng/mL in all remaining cats. While the study demonstrated AMH excretion in urine, no correlation was found between blood and urine AMH values. However, a significant positive correlation was observed between blood AMH levels and serum estrogen levels (P < 0.001). These findings suggest that urinary AMH may be a product of proteolytic degradation, potentially leading to inaccurate estimations of ovarian activity based solely on urine AMH levels.

Two new species of Scyphophyllidium (Cestoda: Phyllobothriidea) from Chaenogaleus macrostoma (Bleeker) (Elasmobranchii: Carcharhiniformes) from the Persian Gulf, Iran.

Two new species of Scyphophyllidium Woodland, 1927 are described from the Hooktooth shark, Chaenogaleus macrostoma (Bleeker), from the Persian Gulf, Iran. Scyphophyllidium hormuziense n. sp. is assigned to morphological category 2 of its genus because it possesses bothridial marginal loculi and an apical sucker on each flat bothridium, and lacks bothridial facial loculi. Within category 2, it is distinguished from its congeners, except for S. janineae (Ruhnke, Healy and Shapero, 2006), by possessing, rather than lacking, a prominent cephalic peduncle. It differs from S. janineae in the distribution of the vitelline follicles. Scyphophyllidium iraniense n. sp. is assigned to morphological category 5 because its bothridia are essentially flat and lack proximal apertures, marginal loculi, facial loci and semi-circular muscle bands, and bears weakly serrate gladiate spinitriches on its distal bothridial surfaces. Within its morphological category, the presence of a long cephalic peduncle distinguishes S. iraniense n. sp. from its congeners lacking this structure or possessing a short cephalic peduncle. It further differs from S. arnoldi (Ruhnke and Thompson, 2006) and S. typicum (Subhapradha, 1955) in total length, from S. paulum (Linton, 1897) in lacking a conspicuous band of muscles along the locular periphery of the bothridia, and from S. kirstenae (Ruhnke, Healy and Shapero, 2006) in the distribution of the vitelline follicles. This study brings the number of the valid Scyphophyllidium species of the Persian Gulf to five.

Effect of Oligopin Administration on Ovarian Morphology in Women with Polycystic Ovarian Syndrome (PCOS)

Background. The effects of oligopin as an antioxidant on polycystic ovarian morphology (PCOM) have not yet been examined. Therefore, the objective of this study was to evaluate the oligopin supplementation on PCOM among patients with polycystic ovarian syndrome (PCOS). Methods. This randomized, placebo‐controlled trial was carried out at Shariati Hospital, Arash Hospital, and Yas Hospital, Tehran, Iran, to determine the effect of oligopin (50 mg/d) or placebo in PCOS patients. The ultrasonographic ovarian morphology was assessed in women aged 18–40 years, before and after 3 months of intervention. Results. Among 45 randomized participants, 32 participants, of whom 17 were in the oligopin group and 15 were in the placebo group completed the trial. There was only one adverse event in the oligopin group. The mean (standard deviation) age of the patients was 30.47 (6.30) years and the median (interquartile range) BMI was 27.50 (23.42–33.55). Three months of oligopin therapy significantly decreased ovarian stromal area (p = 0.01) and stromal/total area (p = 0.003). However, no significant differences were observed in the ovarian volume, ovarian area, 2–9 mm antral follicle counts, or peripheral follicle distribution pattern at 3 months. Conclusion. Among participants with PCOS, the use of oligopin (50 mg) daily, as compared with a placebo, resulted in improvement of the stromal area and stromal/total area at the end of the 3 months of treatment. Further studies are, however, needed to evaluate the longer‐term efficacy and safety. This trial is registered with IRCT20140406017139N3.

#313 : The Correlation Between Ultrasound Morphological Parameters and Phenotypes of Polycystic Ovary Syndrome in Indonesian Women

Background and Aims: Polycystic ovary syndrome (PCOS) is a complex endocrine disorder that affects women of reproductive age. Ultrasound morphological parameters of ovarian morphology, ovarian volume, follicle number, endometrial plate thickness, and doppler analysis are necessary for the diagnosis of PCOS. The aim of this study is to evaluate the correlation between ultrasound morphological parameters and classic and nonclassic phenotypes of PCOS in Indonesian Women. Method: A single-center, observational analytic study from May 2022 until October 2022 was performed upon 50 women with PCOS at Halim Fertility Center (HFC), Indonesia. Fifty PCOS women were classified into two groups: 20 PCOS women with classic phenotype and 30 PCOS women with non-classic phenotype. Data on patient’s age, body mass index (BMI), LH level, FSH level, LH: FSH ratio, Testosterone, Free testosterone index, SHBG, HOMA-IR, Ultrasound parameters (antral follicle count, follicle size, ovarian volume, stromal volume, Stromal/total area (S/A) ratio, Pulsatility index (PI), Peak systolic velocity (PS) and follicle distribution) were assessed and compared between the two groups. Results: The antral follicle count was higher in classic phenotype of PCOS than in nonclassic phenotype and there was a significant difference between the groups (p&lt;0.05). The stromal volume and PI were higher in non-classic phenotype PCOS than in classic phenotype and there was significantly difference between the groups (p&lt;0.05). Most of the follicle distribution in the classic phenotype of PCOS was central and the follicle distribution in the non-classic phenotype was peripheral and there was significantly difference between the groups (p&lt;0.05). The number of antral follicles, stromal volume, PI, and follicle distribution were positively correlated with the PCOS phenotypes (r=0.734; r=0.594; r=0.577; r=0.196), respectively. Conclusion: Classic phenotype of PCOS in Indonesian women has high antral follicle count and central follicle distribution and nonclassic phenotype has high stromal volume, PI and peripheral follicle distribution.

A multifunctional Wnt regulator underlies the evolution of rodent stripe patterns.

Animal pigment patterns are excellent models to elucidate mechanisms of biological organization. Although theoretical simulations, such as Turing reaction-diffusion systems, recapitulate many animal patterns, they are insufficient to account for those showing a high degree of spatial organization and reproducibility. Here, we study the coat of the African striped mouse (Rhabdomys pumilio) to uncover how periodic stripes form. Combining transcriptomics, mathematical modelling and mouse transgenics, we show that the Wnt modulator Sfrp2 regulates the distribution of hair follicles and establishes an embryonic prepattern that foreshadows pigment stripes. Moreover, by developing in vivo gene editing in striped mice, we find that Sfrp2 knockout is sufficient to alter the stripe pattern. Strikingly, mutants exhibited changes in pigmentation, revealing that Sfrp2 also regulates hair colour. Lastly, through evolutionary analyses, we find that striped mice have evolved lineage-specific changes in regulatory elements surrounding Sfrp2, many of which may be implicated in modulating the expression of this gene. Altogether, our results show that a single factor controls coat pattern formation by acting both as an orienting signalling mechanism and a modulator of pigmentation. More broadly, our work provides insights into how spatial patterns are established in developing embryos and the mechanisms by which phenotypic novelty originates.

Disrupting Hedgehog signaling in melanocytes by SUFU knockout leads to ocular melanocytosis and anterior segment malformation.

Hedgehog (Hh) signaling is well known for its crucial role during development, but its specific role in individual cell lineages is less well characterized. Here, we disrupted Hh signaling specifically in melanocytes by using Cre-mediated cell-type-specific knockout of the Hh regulator suppressor of fused (Sufu). Interestingly, corresponding mice were fully pigmented and showed no developmental alterations in melanocyte numbers or distribution in skin and hair follicles. However, there were ectopic melanoblasts visible in the anterior chamber of the eye that eventually displayed severe malformation. Choroidal melanocytes remained unaltered. Surprisingly, the abnormal accumulation of anterior uveal melanoblasts was not the result of increased cell proliferation but of increased migration to ectopic locations such as the cornea. In melanoblasts in vitro, Sufu knockdown replicated the increase in cell migration without affecting proliferation and was mediated by an increased level of phosphorylated-ERK brought about by a reduction in the levels of the repressor form of GLI3. These results highlight the developmental divergence of distinct melanocyte subpopulations and may shed light on the pathogenesis of human ocular melanocytosis.

O-272 Oral administration of antioxidants can support follicle survival in xenografted human ovarian tissue

Abstract Study question Can oral administration of antioxidants improve follicle survival in human ovarian tissue through reduction of oxidative stress in a xenotransplantation model? Summary answer Daily oral administration of antioxidants for 7 days post-transplantation had a positive effect on follicle survival in grafted human ovarian tissue. What is known already Today cryopreservation and autotransplantation of ovarian tissue is an established fertility preservation method in many centres. Hundreds of live births have been achieved but the efficacy of this procedure is hampered by a critical loss of follicles following transplantation. The ovarian tissue is subjected to hypoxia and ischemia postoperative which leads to oxidative damage in the tissue. Postoperative supplementation with antioxidants has been shown to decrease oxidative stress in transplanted ovarian tissue and increase follicle survival, however, these results were mainly obtained through intraperitoneal injections to host animals. Oral administration of antioxidants could be a better alternative for clinical application. Study design, size, duration Pieces of frozen-thawed ovarian cortex tissue (total of 56) from 12 women were transplanted to immunodeficient mice in a short- and long-term xenograft study, for 7 and 28 days respectively. In each study the mice were administered daily doses of hazelnut cream (control group) for 7 days post-transplantation or hazelnut cream containing either 150 mg/kg N-acetylcysteine (NAC) or FERTILIX (containing Vitamin E, D and C, Mixed Tocopherols and Tocotrienols, Lycopene, and Coenzyme Q10). Participants/materials, setting, methods Cryopreserved ovarian tissue was donated by 12 women undergoing ovarian tissue cryopreservation at Copenhagen University Hospital. Grafts were retrieved after 7 and 28 days of xenografting and histologically processed for follicle counts, immunohistochemistry, and qPCR analysis. The number of surviving follicles and ovarian graft volume were evaluated histologically after 7 and 28 days xenografting. Gene expression analysis of antioxidant defence markers (Sod1, Hmox and Catalase) and angiogenic factor Vegfa were performed after 7 days xenografting. Main results and the role of chance After 7 days xenografting, the follicle density was similar for all groups (control group: 33.03±14.85 (mean±SEM) follicles/mm3; NAC-group: 26.34±10.54 follicles/mm3, FERTILIX-group: 35.12±10.88 follicles/mm3). However, the NAC- and FERTILIX-group had a 3- and 3.7-fold increase in follicle density respectively compared to the control group. However, after 28 days of xenografting there was a higher density of surviving follicles in both antioxidant groups, compared to control (control group: 9.51±2.9 follicles/mm3; NAC-group: 14.9±6.02 follicles/mm3, FERTILIX-group: 14.43±5.83 follicles/mm3), which resulted in a 3.2-fold increase in follicle density in the NAC-group and 2.4-fold increase in the FERTILIX-group compared to the control group. The findings were not statistically significantly different in either the 7 days or 28 days study (p = 0.7072; p = 0.5691). The relative gene expression of the antioxidant defence genes Hmox and Catalase was overall like the control for all treatment groups after 7 days xenografting, but for Sod1 the expression was significantly lower for the NAC- and FERTILIX-group compared to the control group (p = 0.0407; p = 0.0255). For Vegfa the expression was similar overall and not significantly different from the control group (p &amp;gt; 0.05). Limitations, reasons for caution The distribution of follicles in human ovarian cortex is highly heterogeneous and can vary tremendously between cortex pieces from the same woman and between patients. Statistically significant results were not obtained for the follicle data in the current study; thus, the conclusions should be interpreted with caution. Wider implications of the findings Oral administration of antioxidants had a positive effect on follicle survival in xenotransplanted human ovarian tissue. These findings provide a step toward potential clinical administration of antioxidants like NAC or FERTILIX postoperative of ovarian tissue transplantation, which could improve follicle outcomes in a feasible way for the patient. Trial registration number Not applicable

Serum Anti-Müllerian Hormone Is an Effective Indicator of Antral Follicle Counts but Not Primordial Follicle Counts.

Serum anti-Müllerian hormone (AMH) is a biomarker for predicting antral follicle counts but there is no clear consensus on whether AMH is indicative of primordial follicle counts in humans. Mice were used as a model species in this study to obtain accurate follicle counts across the reproductive phase of life. Serum AMH was measured in 62 female C57Bl6/J mice aged 25 to 401 days. Primordial and primary follicles were determined by stereological counts and all secondary and antral follicles were counted in serial histological sections. Serum AMH was most strongly correlated with small- and medium-sized antral follicles. Immunohistochemistry and stepwise multiple regression confirmed that these follicle development stages are the key determinants of serum AMH, with little contribution from other stages. Primordial follicles were not found to have strong correlations with serum AMH or antral follicle counts, particularly in younger females, but the strength of the association appeared to increase with age. This result is likely attributed to high interindividual variation in primordial follicle activation and preantral follicle survival rates. Recent large studies in human populations have shown similar results but the primary limitation of these studies was that primordial follicle counts were determined from ovarian cortical biopsies, where regional variation in follicle distribution may affect the quality of the data. In the present study, whole ovaries were surveyed, eliminating this limitation. The findings indicate that primordial follicle counts are not closely related with either serum AMH or antral follicle counts in females in the early phase of the reproductive phase of life.

Inhibition of phosphodiesterase PDE8B reduces activation of primordial follicles in mouse ovaries.

In the ovaries, cyclic adenosine 3',5'-monophosphate (cAMP) is a second messenger supporting the generation of steroids. Phosphodiesterases (PDEs) are regulators of intracellular cAMP, and therefore, potential regulators of ovarian function. Interestingly, the family of PDE genes are differentially expressed in human oocytes and granulosa cells from primordial and primary follicles, suggesting diverse roles. In this study, we addressed the functions of PDE3B and PDE8B in primordial follicle regulation using inhibitors of PDE3B and PDE8B in murine ovary primary in vitro cultures. Inhibition of PDE8B in ovarian cultures prevented primordial follicle activation, while inhibition of PDE3B had no effect on follicle distribution in the ovary, under the tested conditions. As cAMP levels may increase steroid levels, we assessed the protein levels of the steroidogenic acute regulatory protein (StAR) and aromatase enzymes, and found that inhibition of PDE3B reduced StAR protein levels, whereas inhibition of PDE8 did not alter StAR expression in our murine ovary culture system conditions. Our results showed that ketotifen-induced inhibition of PDE8B can decrease primordial follicle activation, whereas we observed no effect of follicle distribution, when PDE3B was inhibited. Expression of the StaR enzyme was not altered when PDE8B was inhibited, which might reflect not sufficient inhibition by ketotifen to induce StAR alterations, or redundant mechanisms.

Spatial distribution of preantral follicles in ovarian parenchyma of bovine species.

The goal of this study was to determine the distribution of preantral follicles in bovine ovaries. Follicular distribution in the ovaries (n = 12) was evaluated in the region of the greater curvature of the ovary (GCO) and the region close to the ovarian pedicle (OP) of Bos taurus indicus heifers of the Nelore breed. Two fragments were obtained from each region of the ovary (GCO and OP). The mean weight of the ovaries was 4.04 ± 0.32 g. The mean antral follicle count (AFC) was 54.58 ± 3.55 follicles (minimum and maximum variation of 30 and 71 follicles, respectively). In total, 1123 follicles were visualized in the region of the GCO; 949 (84.5%) of them were primordial follicles and 174 (15.5%) were developing follicles. The region close to the OP contained 1454 follicles, of which 1266 (87%) were primordial follicles and 44 (12.9%) were developing follicles. The OP region showed a higher proportion of intact follicles in the primordial (P < 0.0001) and primary (P = 0.042) stages compared with the GCO region. The proportion of secondary follicles was similar in the OP and GCO regions. The ovaries of two bovine females (16%; 2/12) contained multi-oocytes follicles, which were characterized as primary follicles. Therefore, the distribution of preantral follicles in the bovine ovary was heterogeneous, with the region close to the OP containing a greater number of preantral follicles compared with the GCO region (P < 0.05).

Spatio-temporal remodelling of the composition and architecture of the human ovarian cortical extracellular matrix during in vitro culture.

How does in vitro culture alter the human ovarian cortical extracellular matrix (ECM) network structure? The ECM composition and architecture vary in the different layers of the ovarian cortex and are remodelled during in vitro culture. The ovarian ECM is the scaffold within which follicles and stromal cells are organized. Its composition and structural properties constantly evolve to accommodate follicle development and expansion. Tissue preparation for culture of primordial follicles within the native ECM involves mechanical loosening; this induces undefined modifications in the ECM network and alters cell-cell contact, leading to spontaneous follicle activation. Fresh ovarian cortical biopsies were obtained from six women aged 28-38 years (mean ± SD: 32.7 ± 4.1 years) at elective caesarean section. Biopsies were cut into fragments of ∼4 × 1 × 1 mm and cultured for 0, 2, 4, or 6 days (D). Primordial follicle activation, stromal cell density, and ECM-related protein (collagen, elastin, fibronectin, laminin) positive area in the entire cortex were quantified at each time point using histological and immunohistological analysis. Collagen and elastin content, collagen fibre characteristics, and follicle distribution within the tissue were further quantified within each layer of the human ovarian cortex, namely the outer cortex, the mid-cortex, and the cortex-medulla junction regions. Primordial follicle activation occurred concomitantly with a loosening of the ovarian cortex during culture, characterized by an early decrease in stromal cell density from 3.6 ± 0.2 × 106 at day 0 (D0) to 2.8 ± 0.1 × 106 cells/mm3 at D2 (P = 0.033) and a dynamic remodelling of the ECM. Notably, collagen content gradually fell from 55.5 ± 1.7% positive area at D0 to 42.3 ± 1.1% at D6 (P = 0.001), while elastin increased from 1.1 ± 0.2% at D0 to 1.9 ± 0.1% at D6 (P = 0.001). Fibronectin and laminin content remained stable. Moreover, collagen and elastin distribution were uneven throughout the cortex and during culture. Analysis at the sub-region level showed that collagen deposition was maximal in the outer cortex and the lowest in the mid-cortex (69.4 ± 1.2% versus 53.8 ± 0.8% positive area, respectively, P < 0.0001), and cortical collagen staining overall decreased from D0 to D2 (65.2 ± 2.4% versus 60.6 ± 1.8%, P = 0.033) then stabilized. Elastin showed the converse distribution, being most concentrated at the cortex-medulla junction (3.7 ± 0.6% versus 0.9 ± 0.2% in the outer cortex, P < 0.0001), and cortical elastin peaked at D6 compared to D0 (3.1 ± 0.5% versus 1.3 ± 0.2%, P < 0.0001). This was corroborated by a specific signature of the collagen fibre type across the cortex, indicating a distinct phenotype of the ovarian cortical ECM depending on region and culture period that might be responsible for the spatio-temporal and developmental pattern of follicular distribution observed within the cortex. N/A. Ovarian cortical biopsies were obtained from women undergoing caesarean sections. As such, the data obtained may not accurately reflect the ECM distribution and structure of non-pregnant women. Clarifying the composition and architecture signature of the human ovarian cortical ECM provides a foundation for further exploration of ovarian microenvironments. It is also critical for understanding the ECM-follicle interactions regulating follicle quiescence and awakening, leading to improvements in both in vitro activation and in vitro growth techniques. Medical Research Council grant MR/R003246/1 and Wellcome Trust Collaborative Award in Science: 215625/Z/19/Z. The authors have no conflicts to declare. N/A.

Toxic Effects of Cadmium on the Female Reproductive Organs a Review

Abstract Cadmium (Cd) is a common environmental pollutant present in soil and associated with many modern industrial processes. Cadmium may adversely influence the health of experimental animals and humans and exert significant effects on the reproductive tract morphology and physiology. During embryonic development, cadmium suppresses the normal growth and development of the ovaries, and in adults it disrupts the morphology and function of the ovaries and uterus. The exposure to cadmium has adverse effects on the oocyte meiotic maturation affecting the structure of ovarian tissue. The distribution of follicles and corpus luteum in the ovarian tissues has been shown to be disrupted, affecting the normal growth and development of the follicles. In the ovarian cortex, the number of follicles at different stages of maturation decreased, and the number of atretic follicles increased. In the medulla, oedema and ovarian haemorrhage and necrosis appears at higher doses. Granulosa cells exposed to cadmium exhibited morphological alterations. Oocyte development was inhibited and the amount of oocyte apoptosis was higher. Cadmium exposure also caused changes in the structure of the ovarian blood vessels with reduction in the vascular area. Cadmium effects included increased uterine weight, hyperplasia and hypertrophy of the endometrial lining. Exposure to cadmium had specific effects on gonadal steroidogenesis by suppressing steroid biosynthesis of the ovarian granulosa cells and luteal cells. Progesterone, follicle stimulating hormone, and luteinizing hormone decreased significantly after CdCl2 administration. Cadmium can suppress the female’s ovulation process and cause temporary infertility.

Bone marrow-derived vasculogenesis leads to scarless regeneration in deep wounds with periosteal defects

Deep skin wounds with periosteal defects, frequently caused by traffic accidents or radical dissection, are refractory. Transplant surgery is frequently performed, but patients are subjected to stress for long operation periods, the sacrifice of donor regions, or several complications, such as flap necrosis or intractable ulcers. Even if the defects are covered, a scar composed of fibrous tissue remains in the body, which can cause itching, dysesthesia, or repeated ulcers because of the lack of distribution of peripheral nerves or hair follicles. Thus, treatments with the aim of regenerating lost tissue for deep wounds with periosteal defects are needed. Here, we show that the use of gelatin sponges (GS), which have been used as haemostatic materials in clinical practice, allowed the regeneration of heterogeneous tissues, including periosteum, skin, and skin appendages, when used as scaffolds in deep wounds with periosteal defects in rats. Bone marrow transplantation in rats revealed the mechanism by which the microenvironment provided by GS enabled bone marrow-derived cells (BMDCs) to form a vascular niche, followed by regeneration of the periosteum, skin, or skin appendages such as hair follicles by local cells. Our findings demonstrated that vascular niche formation provided by BMDCs is crucial for heterogeneous tissue regeneration.