Development Of Preantral Follicles Research Articles

Periovulatory PM2.5 constituent exposures and human clinically recognized early pregnancy loss: Susceptible exposure time windows and high–risk constituents

Evidence for effects of PM2.5 chemical constituent exposures during the periovulatory period on pregnancy complications was limited. We explored the associations of maternal PM2.5 and constituent exposures from the 12th week before to 4th week after ovulation with human clinically recognized early pregnancy loss (CREPL). From July 2017 to January 2024, 828 CREPL and 828 normal early pregnancy (NEP) participants were recruited in Tianjin, China. Daily residential exposures to PM2.5 and five main constituents of all participants were estimated using data of the Tracking Air Pollution in China platform. Nonlinear and linear associations between weekly pollutant exposures and CREPL were estimated using conditional logistic regression models combined with distributed lag nonlinear and distributed lag models, respectively. The risk of CREPL increased with per 10 μg/m³ increment in PM2.5 and per 1 μg/m³ increment in sulfate, nitrate, and ammonium exposures during specific weeks from the 5th week before to 2nd week after ovulation, with the largest OR (95% CI) of CREPL associated with PM2.5, sulfate, nitrate, and ammonium being 1.73 (1.07, 2.78), 1.71 (1.18, 2.46), 1.80 (1.12, 2.90), and 1.61 (1.01, 2.56), respectively. CREPL was positively associated with the 10th to 90th percentiles exposure to organic matter during the 2nd and 3rd week after ovulation. In analyses for constituent residuals, the five constituents were all independently related to CREPL, with organic matter being the highest risk constituent, and nitrate and ammonium affecting at the initial stage of preantral follicle development. In conclusion, periovulatory PM2.5 and constituent exposures were associated with increased risk of CREPL. Women planning a pregnancy are advised to take exposure precautions starting from the follicular development period.

Growth factor-loaded ovarian extracellular matrix hydrogels promote in vivo ovarian niche regeneration and enhance fertility in premature ovarian insufficiency preclinical models

Premature ovarian insufficiency (POI) means menopause before 40 years of age affecting about 1 % of women. Approaches based on cell therapy and the paracrine effects of stem cells or bioproducts such as platelet-rich plasma have been proposed, but concerns remain about undesired systemic effects, as well as the need to optimize delivery methods through bioengineering methods. This study explores the efficacy of decellularized bovine ovarian cortex extracellular matrix (OvaECM) hydrogels alone and as a growth factor (GF) carrier (OvaECM+GF) in a chemotherapy-induced POI murine model. In vitro assays showed a gradual release of GF from the OvaECM sustained for two weeks. Chemotherapy drastically reduced follicle numbers, but OvaECM+GF treatment restored pre-antral follicle development. Moreover, this treatment notably regenerated the ovarian microenvironment by increasing cell proliferation and microvessel density while reducing chemotherapy-induced apoptosis and fibrosis. Whole-ovary RNA sequencing and gene set enrichment analysis revealed an upregulation of regeneration-related genes and a downregulation of apoptotic pathways. The OvaECM+GF treatment also yielded significantly better outcomes following ovarian stimulation and in vitro fertilization. After two consecutive crossbreeding cycles, OvaECM+GF-treated mice showed normal reproductive function. This research showcases the biocompatibility and efficacy of OvaECM to reverse POI in mice, setting a foundation to explore innovative bioengineering-based POI therapies. Statement of significancePremature ovarian insufficiency (POI) affects about 1 % of women worldwide, causing early menopause before 40 years old. Current treatments alleviate symptoms but do not restore ovarian function. This study explores an innovative approach using ovarian cortex extracellular matrix hydrogels to deliver growth factors into the murine ovarian niche and reverse POI. In vitro release kinetic assays demonstrated a gradual and sustained release of growth factors. In a POI-induced mouse model, intraovarian injections of the hydrogel encapsulating growth factors restored pre-antral follicle development, increased cell proliferation, reduced apoptosis and fibrosis, and improved ovarian response and in vitro fertilization outcomes. Long-term benefits included larger litter sizes. This innovative technique shows promise in regenerating the ovarian environment and improving reproductive outcomes.

Bioengineered 3D ovarian model for long-term multiple development of preantral follicle: bridging the gap for poly(ε-caprolactone) (PCL)-based scaffold reproductive applications

BackgroundAssisted Reproductive Technologies (ARTs) have been validated in human and animal to solve reproductive problems such as infertility, aging, genetic selection/amplification and diseases. The persistent gap in ART biomedical applications lies in recapitulating the early stage of ovarian folliculogenesis, thus providing protocols to drive the large reserve of immature follicles towards the gonadotropin-dependent phase. Tissue engineering is becoming a concrete solution to potentially recapitulate ovarian structure, mostly relying on the use of autologous early follicles on natural or synthetic scaffolds. Based on these premises, the present study has been designed to validate the use of the ovarian bioinspired patterned electrospun fibrous scaffolds fabricated with poly(ε-caprolactone) (PCL) for multiple preantral (PA) follicle development.MethodsPA follicles isolated from lamb ovaries were cultured on PCL scaffold adopting a validated single-follicle protocol (Ctrl) or simulating a multiple-follicle condition by reproducing an artificial ovary engrafted with 5 or 10 PA (AO5PA and AO10PA). The incubations were protracted for 14 and 18 days before assessing scaffold-based microenvironment suitability to assist in vitro folliculogenesis (ivF) and oogenesis at morphological and functional level.ResultsThe ivF outcomes demonstrated that PCL-scaffolds generate an appropriate biomimetic ovarian microenvironment supporting the transition of multiple PA follicles towards early antral (EA) stage by supporting follicle growth and steroidogenic activation. PCL-multiple bioengineering ivF (AO10PA) performed in long term generated, in addition, the greatest percentage of highly specialized gametes by enhancing meiotic competence, large chromatin remodeling and parthenogenetic developmental competence.ConclusionsThe study showcased the proof of concept for a next-generation ART use of PCL-patterned scaffold aimed to generate transplantable artificial ovary engrafted with autologous early-stage follicles or to advance ivF technologies holding a 3D bioinspired matrix promoting a physiological long-term multiple PA follicle protocol.

Resveratrol attenuates doxorubicin-induced toxicity during in vitro culture of mouse-isolated preantral follicles.

The aims of this study were to evaluate the doxorubicin concentration that induces toxic effects on in vitro culture of isolated mouse secondary follicles and to investigate whether resveratrol can inhibit or reduce this toxicity. Secondary follicles were isolated and cultured for 12 days in control medium (α-MEM+) or in α-MEM+ supplemented with doxorubicin (0.1 µg/ml) or different concentrations of resveratrol (0.5, 2, or 5 µM) associated with doxorubicin (0.1 µg/ml) (experiment 1). For experiment 2, follicles were cultured in α-MEM+ alone or supplemented with doxorubicin (0.3 µg/ml) or different concentrations of resveratrol (5 or 10 µM) associated or not with doxorubicin (0.3 µg/ml) (experiment 2). The endpoints analyzed were morphology (survival), antrum formation, follicular diameter, mitochondrial activity, glutathione (GSH) levels and DNA fragmentation. In the first experiment, doxorubicin (0.1 µg/ml) maintained survival and antrum formation similar to the control, while 5 µM resveratrol showed increased parameters, maintained mitochondrial activity and increased GSH levels compared to the control. In the second experiment, doxorubicin (0.3 µg/ml) reduced survival, antrum formation and follicular diameter compared to the control. Resveratrol at a concentration of 10 µM attenuated the damage caused by doxorubicin by improving follicular survival and did not present DNA fragmentation. In conclusion, supplementation of the in vitro culture medium with 0.3 µg/ml doxorubicin reduced the survival and impaired the development of mouse-isolated preantral follicles. Resveratrol at 10 µM reduced doxorubicin-induced follicular atresia, without DNA fragmentation in the follicles.

Vitamin C Synergistically Enhances Protective Effects of Vitamin E Against Preantral Follicle Degeneration of Ovine Vitrified/Warmed Ovarian Tissue.

This study aimed to evaluate whether the addition of vitamins E and C as two conventional antioxidants improves the cryotolerance of preantral follicles enclosed in ovine ovarian tissue slices. For this purpose, ovarian slices were obtained from abattoired juvenile lambs and randomly distributed to the following groups: fresh, toxicity, vitrified (control), and three treatment groups in two experiments. Vitamin E, vitamin C, or vitamin E + C was added to the vitrification media alone in the first experiment and added to all vitrification, warming, and culture media in the second experiment. Finally, the treated tissues were cultured in vitro for 12 hours. The histological analysis showed that single or combined use of vitamins E and C increases intact preantral follicles in comparison to the control in two experiments (p < 0.05), and simultaneous use of vitamins E and C had a synergistic effect on increasing the percentage of normal preantral follicles in experiment 2 (p < 0.05). Due to the better results in Experiment 2, stromal cell density, antioxidant activity, and molecular evaluation were followed only in this experiment. The vitamin E + C group had higher stromal cell density compared with control group (p < 0.05). Vitamin E strengthened antioxidant capacity compared with the control and vitamin C groups (p < 0.05). This effect was exacerbated when used in combination with vitamin C (p < 0.05). The expression of all evaluated genes (BMP4, BMP15, GDF9, and KITLG) was significantly increased in ovarian tissue treated with vitamin E + C compared with the control group (p < 0.05). This increase was also observed in BMP4, GDF9, and KITLG genes compared with the vitamin C group (p < 0.05). In conclusion, this study revealed the positive effects of vitamins E and C on preantral follicle viability and to some extent a synergistic action of vitamin C on the protective effects of vitamin E against preantral follicle degeneration and increasing antioxidant capacity and development of preantral follicles after ovine ovarian tissue vitrification.

In Vitro Development of Mouse Preantral Follicle with Using Amniotic Membrane Extract-Loaded Hydrogels.

An artificial ovary based on the alginate (ALG) hydrogel has been widely implemented to preserve prepubertal female fertility. However, this platform is not fully capable of successful an ovary microenvironment simulation for follicle development, holding great potential for its improvement. Therefore, this experimental study aimed to evaluate the effect of an amniotic membrane extract (AME) -loaded hydrogel on the mouse preantral follicles in vitro development. In order to have better follicle development, first, the impact of different concentrations of follicle-stimulating hormone (FSH) was evaluated on the mouse preantral follicles encapsulated in ALG. Later, the appropriate dose was adjusted for the follicles encapsulated in the ALG-AME hydrogel. Results demonstrated that 100 mIU/ml FSH showed a significant follicle survival rate compared with 10 mIU/ml FSH (P=0.005). According to MTT assay finding, the rate of weight loss, and rheology evaluations, ALG containing 1 mg/ml AME was identified as an optimal sample of follicle culture instead of other AME concentrations. Follicle diameter significantly increased in the ALG-AME 1 hydrogel compared with the ALG control group without AME (P=0.027). The storage modulus of ALG-AME 1 was 773 Pa and retained the follicle morphology for 13 days. No statistically substantial difference was seen in survival, antrum cavity formation, and competent oocyte in terms of the normal chromosomal arrangement and meiotic spindle rate in comparison with the control group. It can be concluded that ALG-AME 1 could not significantly impact the mouse preantral follicle.

Improvement of Mouse Preantral Follicle Survival and Development following Co-Culture with Ovarian Parenchyma Cell Suspension.

The parallel and continued improvements in both infertility treatment and the management of malignancy cases have brought to the forefront the potential for fertility preservation. Using ovarian follicular resources can effectively improve reproductive capacity and prevent infertility. The primary aim of this research was to try to generate an appropriate in vivo environment for the growth of the mouse follicles. Hence, the possible effects of the ovarian parenchyma cell suspension were explored on the growth and maturation of preantral follicles in vitro. In this experimental study, ovarian parenchymal cells were mechanically dissociated from preantral follicles of 12-14 days-old NMRI mice and then divided into 5 experimental groups (G1: Control, G2: Fresh follicle with fresh parenchyma cell suspension, G3: Vitrified-warmed follicle with fresh parenchyma cell suspension, G4: Fresh follicle with frozen-thawed parenchyma cell suspension, and G5: Vitrified-warmed follicle with frozenthawed parenchyma cell suspension). The diameter of the follicles and immature oocytes, viability, antrum formation, resumption of meiosis, in vitro fertilization (IVF), and Gdf9, Bmp6, and Bmp15 gene expression were examined on different periods. The diameter of the follicles and the oocytes on days 4 and 8, as well as the survival rate of the follicles up to day 12, were significantly higher in G2 and G4 compared to the Ctrl group (G1: 73.66%, G2:87.99%, G3: 82.70%, G4: 94.37%, and G5: 78.59%). Expression of growth marker genes for G3, and G5 groups was significantly higher than other groups, which indicated the protective effects of parenchyma cell suspension on follicles damaged by vitrification solutions. The growth, survival, and maturation of preantral follicles could be enhanced by co-culturing them with ovarian parenchyma cells. Further studies are needed to optimize the conditions for a successful parenchyma cell suspension-induced in vitro maturation (IVM) to occur in infertility clinics.

Evaluation of the morphology and development of preantral ovarian follicles in mice submitted to a chronic diet of dietary supplementation with Pereskia aculeata Miller leaves.

This study aimed to investigate the effect of including mouse feed with different concentrations (5, 10, or 20%) of Pereskia aculeata Miller (PAM) leaves on the morphology and development of preantral ovarian follicles and ovarian stromal cell density. The oral toxicity was performed using repeated dose toxicity assays subdivided into experiments of 30 days and 90 days of treatment. After the experiments, the ovaries of each animal were collected and submitted to classical histology. At 30 and 90 days, there was an equivalent percentage of normal, primordial, and developing follicles (P > 0.05) between PAM treatments compared to the control. Regarding the different stages of follicular development, after 90 days, there was a higher percentage (P < 0.05) of developing follicles only in the control group compared to day 30. The PAM 5% treatment was the only one that affected the cell density in the stroma after 90 days of treatment. Thus, we observed that supplementing the diet with P. aculeata did not pose any risk concerning animal consumption; specifically, there were no toxic reproductive effects observed from adding Pereskia aculeata Miller to the mouse diet.

Kidney-tonifying formula facilitates the development and maturation of mouse preantral follicle in vitro.

Kidney-tonifying formulas are frequently used in clinical practices to enhance follicular development and maturation. This research explored the impacts of the Bushen Tiaojing formula (BSTJF) on the development of mouse preantral follicles in vitro and its relationship with granulosa cells and gonadotropins. Preantral follicles were extracted from mice and cultured with or without serum from rats that were previously treated with or without BSTJF. During cultivation, the follicles were monitored for morphological changes and developmental maturation. Exhausted medium was collected every other day for the measurement of progesterone and estradiol (E2) levels by ELISA. Granulosa cells in in-vitro medium were collected on days 8, 10, and 12 and analyzed for determining the expressions of apoptosis-associated genes (Bax, Bcl-2, and Caspase-3). Propagation and apoptosis rates of collected granulosa cells were measured by CCK-8 assay and flow cytometry. Compared with control follicles, follicles cultured with serum from BSTJF-treated rats had a higher survival rate, larger follicle diameter, higher Bcl-2 expression, and lower Bax and Caspase-3 expressions (all P ≤ 0.05). In addition, their granulosa cells presented substantially elevated proliferation (P ≤ 0.05) and a lower rate of apoptosis (P ≤ 0.05) compared with granulosa cells from control follicles. The level of E2 in the culture media of all groups increased slowly in the first 6 days. Subsequently, after formation of the antrum, the levels of E2 and progesterone were enhanced in the medium of follicles cultured with serum from BSTJF-treated rats compared with those in the media of control follicles (all P ≤ 0.05). Serum from BSTJF-treated rats facilitated the in vitro development and maturation of mouse follicles by increasing the expression of anti-apoptotic gene Bcl-2, reducing the expressions of pro-apoptotic genes Bax and Caspase-3 as well as the apoptosis of granulosa cells, promoting the proliferation of granulosa cells and increasing the secretion of E2 and progesterone in the cells.

Expression of luteinizing hormone receptor during development of bovine fetal ovary.

Steroids and gonadotrophins are essential for the regulation of late stages of preantral development and antral follicular development. Although the luteinizing hormone receptor (LHCGR) has been detected in the preantral follicles of rats, rabbits, and pigs, its expression, in bovine fetal ovary, has not been demonstrated. Based on this, we aimed to investigate the expression of the LHCGR and LHCGR mRNA binding protein (LRBP), as well as, to quantify bta-miR-222 (a regulatory microRNA of the LHCGR gene) during the development of bovine fetal ovary. In summary, LHCGR expression was observed in the preantral follicle in bovine fetal ovary, from oogonias to primordial, primary and secondary stages, and the mRNA abundance was lower on day 150 than day 60. However, the mRNA abundance of LRBP followed the opposite pattern. Similar to LRBP, the abundance of bta-miR-222 was higher on day 150 than day 60 or 90 of gestation. The LHCGR protein was detected in oogonia, primordial, primary, and secondary follicles. Moreover, both oocytes and granulosa cells showed positive immunostaining for LHCGR. In conclusion, we suggest the involvement of LHCGR/LRBP/bta-mir222 with mechanisms related to the development of preantral follicles in cattle.

Kisspeptin stimulates sheep ovarian follicular development in vitro through homologous receptors.

The present study was conducted to elucidate (1) the influence of kisspeptin (KP) on the in vitro development of preantral follicles (PFs) and (2) evolution of KP receptor gene (KISS1R) expression during ovarian follicular development in sheep. Kisspeptin was supplemented (0-100 µg/ml) in the culture medium of PFs for 6 days. The cumulus-oocyte complexes (COCs) from cultured PFs were subsequently matured to metaphase II (MII) for an additional 24 h. The proportions of PFs exhibiting growth, antrum formation, average increase in diameter, and maturation of oocytes to MII stage were the indicators of follicular development in vitro. The expression of the kisspeptin receptor gene at each development stages of in vivo developed (preantral, early antral, antral, large antral and COCs from Graafian follicles) and in vitro cultured PFs supplemented with KP was assessed using a real-time polymerase chain reaction. The best development in all the parameters under study was elicited with 10 µg/ml of KP. Supplementation of KP (10 µg/ml) in a medium containing other growth factors (insulin-like growth factor-I) and hormones (growth hormone, thyroxine, follicle-stimulating hormone) resulted in better PF development. The KISS1R gene was expressed in follicular cells and oocytes at all the development stages of both in vivo developed and in vitro cultured follicles. Higher KISS1R gene expression was supported by culture medium containing KP along with other hormones and growth factors. Accordingly, it is suggested that one of the mechanisms through which KP and other growth factors and hormones influence the ovarian follicular development in mammals is through the upregulation of expression of the KP receptor gene.

Optimisation of hormonal treatment to improve follicular development in one-day-old mice ovaries cultured under in vitro condition.

Base medium containing knock-out serum replacement (KSR) has been found to support formation and maintenance of follicles in one-day-old mice ovaries, but has not been shown to properly support activation and growth of primordial follicles. The present study was conducted to tailor the hormonal content of base medium containing KSR to enhance development of primordial follicles in neonatal ovaries. One-day-old mice ovaries were initially cultured with base medium for four days, and then, different hormonal treatments were added to the culture media and the culture was proceeded for four additional days until day eight. Ovaries were collected for histological and molecular assessments on days four and eight. In experiment I, the main and interactive effects of FSH and testosterone were investigated and FSH promoted activation of primordial follicles and development of primary and preantral follicles, and upregulated genes of phosphoinositide 3-kinase (Pi3k ), KIT ligand (Kitl ), growth differentiation factor 9 (Gdf9 ) and follicle stimulating hormone receptor (Fshr ) (P Bmp15 ), Connexin-43 (Cx43 ) and luteinising hormone and choriogonadotropin receptor (Lhcgr ) (P P Lhcgr (P P >0.05). Supplementation of culture medium containing KSR with gonadotropins, particularly hMG, could improve follicular growth and expression of factors regulating follicular development. This study was a step forward in formulating an optimal medium for development of follicles in cultured one-day-old mice ovaries.

Control of the growth and development of murine preantral follicles in a biomimetic ovary using a decellularized porcine scaffold

This study aimed to derive mature oocytes from murine preantral follicles cultured in a biomimetic ovary with a porcine scaffold using decellularization technology. We evaluated the DNA content and the presence of cell and extracellular matrix (ECM) components, including collagen, elastin, and glycosaminoglycans (GAGs), in decellularized (decell) porcine ovaries. The DNA content inthe decell ovarian tissues was approximately 94 % less than that in native tissues (66 ± 9.8 ng/mg vs. 1139 ± 269 ng/mg). Furthermore, the ECM component integrity was maintained in the decell ovarian tissue. The soluble collagen concentration of native ovarian tissue (native) was 195.34 ± 15.13 μg/mg (dry wt.), which was less than 878.6 ± 8.24 μg/mg for the decell ovarian tissue due to the loss of cellular mass. Hydrogels derived from decell porcine ovaries were prepared to develop an in vitro biomimetic ovary with appropriate ECM concentration (2−6 mg/mL). Scanning electron microscope (SEM) imagining revealed that the complex fiber network and porous structure were maintained in all groups treated with varying ECM concentration (2–6 mg/mL). Furthermore, rheometer analysis indicated that mechanical strength increased with ECM concentration in a dose-dependently. The preantral follicles cultured with 4 mg/mL ECM showed high rates of antral follicle (66 %) and mature oocyte (metaphase II) development (47 %). The preantral follicles cultured in a biomimetic ovary with a decell porcine scaffold showed a higher rate of antral follicle and mature oocytes than those cultured in other biomaterials such as collagen and Matrigel. In mature oocytes derived from antral follicles, meiotic spindles and nuclei were stained using a tubulin antibody and Hoechst, respectively. Two-cell embryos were developed from MII oocytes following parthenogenetic activation. Preantral follicles were cultured in a biomimetic ovary derived from the ECM of a decell porcine ovary, and embryos were generated from MII oocytes. This biomimetic ovary could contribute to restoring fertility in infertile women with reduced ovarian function, benefit mating efforts for endangered species, and maintain animals with valuable genetic traits.

A self-gelling hydrogel based on thiolated hyaluronic acid for three-dimensional culture of ovine preantral follicles

Three-dimensional (3D) ovarian follicle culture offers a promising option for fertility preservation in patients who cannot receive ovarian tissue transplantation. Our research evaluated the potential of a hydrogel composed of thiolated hyaluronic acid (HA-SH) for ovine preantral follicle development compared to routinely used alginate hydrogel (ALG). Synthesized via a carbodiimide reaction, HA-SH facilitated a self-crosslinking hydrogel through disulfide bond formation. Ovine preantral follicles (200-300 μm) retrieved through mechanical and enzymatic methods were encapsulated individually in either ALG or HA-SH hydrogels. Although both hydrogels adequately supported follicle survival, 3D integrity, and antrum formation over a 17-day in vitro culture, follicle growth was significantly higher within the HA-SH hydrogel. Gene expression analysis underscored that some folliculogenesis-related genes (ZP3, BMP7, and GJA1) and a steroidogenic gene (CYP19A1) demonstrated higher expression levels in HA-SH encapsulated follicles versus ALG. Collectively, our findings advocate for HA-SH hydrogel as a potent biomaterial for in vitro follicle cultures, attributing its efficacy to facile gelation, bio-responsiveness, and superior support for follicle growth.

Successful 3D culture and transplantation of mouse isolated preantral follicles in hydrogel of bioengineered Wharton's jelly.

Due to Human Wharton's Jelly (HWJ) could be applied in tissue engineering as a bio scaffold, the present study was conducted to investigate the effects of HWJ hydrogel on in vitro culture and auto-transplantation of mouse ovarian follicles. HWJ was isolated from umbilical cord and decellularized with SDS/Tris/EDTA. DNA, Collagen and Glycosaminoglycans (GAGs) were measured. Decellularized Wharton's Jelly (DWJ) was dissolved to make Wharton's Jelly Hydrogel (WJH), and composited with Alginate (ALG) (1.5%) in equal ratio (WJH+ALG). Then, mouse preantral follicles were isolated and encapsulated in 10μL droplets of WJH and randomly considered for both 14 days culture and auto-transplantation. Collagen, GAGs and DNA evaluations showed majority of WJ cells have been removed and MTT approved no toxicity. Degradation rate and rheological analysis represented optimal hydrogel compatibility. The data from in vitro culture revealed significant antral formation in WJH+ALG (P≤0.05). In transplantation, follicles failed to survive in ALG; however, survived in WJH+ALG to antral stage (P<0.05). VEGF and CD34 had greater expression in WJH+ALG than ALG (P< 0.05). Wharton's jelly hydrogel and Alginate compound is interesting composite for successful development of mouse preantral follicles in both 3D in vitro culture and transplantation.

Kisspeptin promotes follicular development through its effects on modulation of P450 aromatase expression and steroidogenesis in sheep ovarian follicles.

The present study was conducted to ascertain whether the role of kisspeptin in promoting in vitro development of preantral follicles was through the regulation of P450 aromatase gene expression and steroidogenesis in sheep. Accordingly, the cumulus cells and oocytes were collected from different development stages of preantral follicles grown in vivo and cultured in vitro in TCM199B (Group I), TCM199B + KP (10 μg/mL) (Group II) and Standard medium + KP (10 μg/mL). To measure the steroid (Estradiol-17β; E2 and Progesterone; P4 ) synthesis through ELISA, spent culture medium was collected separately from the same in vitro groups. E2 synthesis in the spent medium collected from all the three groups showed an increasing trend from PFs' exposed to respective culture media for 3 min to 2-day culture stage but decreased thereafter till 6-day culture stage. This is followed by a sharp increase in E2 concentration in the spent medium collected after in vitro maturation. However, P4 synthesis in group III followed increased pattern as the development progressed from PFs' exposed to culture medium for 3 min to in vitro maturation stage. The steroid production was observed at all stages of in vitro development in altered supplemented conditions. The steroid synthesis in the spent medium was highest in the 6 day cultured PFs' in Standard medium + KP matured in vitro for 24 h. Therefore, supplementation of kisspeptin along with other growth factors promoted steroid production in cultured preantral follicles far better than in other media.

Comparison of mouse ovarian follicular development and gene expression in the presence of ovarian tissue extract and sodium selenite: An experimental study.

Ovarian tissue extract (OTE) and sodium selenite (SS) enhance the growth and maturation of preantral follicles in a dose-dependent manner. The present study was designed to bring more information regarding the mechanism of OTE and SS on the mRNA expression of follicle-stimulating hormone receptors (FSHR) and the proliferation cell nuclear antigens (PCNA) of in vitro matured isolated follicles. The tissue extract was prepared from adult ovaries. The preantral follicles (n = 266) were isolated from 12-16-day-old mice and cultured in the control, experimental I (10 ng/ml SS), and experimental II (OTE) groups for 12 days. The follicular diameter, survival, and maturation rates, also, the production of 17-β-estradiol and progesterone, and the follicular expression of PCNA and FSH receptor genes were analyzed. The survival rate of follicles in the SS-treated group (84.58%) was significantly higher than that OTE (75.63%; p = 0.023) and control (69.38%; p = 0.032) groups. The mean diameter of culture follicles in experimental group I (403.8 μm) and experimental group II (383.97 μm) increased significantly in comparison with the control group (342.05 μm; p = 0.032). The developmental rate of follicles, percentages of antrum formation, released metaphase II oocytes (p = 0.027; p = 0.019 respectively), production of hormones and the expression of 2 studied genes were significantly increased in both experimental groups in compare with control group (p = 0.021; p = 0.023 respectively). The OTE and SS have a positive effect on development of mouse preantral follicles via over-expression of FSHR and PCNA genes.

Review: Roles of follicle-stimulating hormone in preantral folliculogenesis of domestic animals: what can we learn from model species and where do we go from here?

The pituitary gonadotropin FSH is a glycoprotein critical for the development of ovarian follicles. Upon binding to its G protein-coupled membrane receptor located on the granulosa cells of ovarian follicles, FSH elicits a cascade of downstream intracellular responses to promote follicle growth, maturation and steroidogenic activity, leading to the acquisition of meiotic and developmental competence of the enclosed oocyte. The essential role of FSH for proper antral follicle development and fertility is indisputable; over the decades, increasing evidence has also pointed toward survival and growth-promoting effects elicited by FSH in earlier-stage preantral follicles, deeming these follicles FSH-responsive as opposed to the FSH-dependent antral follicles. Transgenic mouse models lacking GnRH1, Fshβ or Fshr clearly demonstrate this difference by showing that, morphologically, preantral follicles develop to the secondary stage without FSH signaling; however, exogenous expression or administration of FSH to hormone-deficient mice promotes preantral follicle development, with more pronounced effects seen in earlier stages (i.e., primary follicles). In hypophysectomized sheep, FSH administration also promotes the growth of primary-stage preantral follicles. However, in vivo studies in this area are more challenging to perform in domestic animals compared to rodents, and therefore most of the research to date has been done in vitro. Here, we present the existing evidence for a role of FSH in regulating the growth and survival of preantral follicles from data generated in rodents and domestic animals. We provide an overview of the process of folliculogenesis, FSH synthesis and cellular signaling, and the response to FSH by preantral follicles in vivo and in vitro, as well as interactions between FSH and other molecules to regulate preantral folliculogenesis. The widespread use of FSH in ovarian stimulation programs for assisted reproduction creates a real need for a better understanding of the effects of FSH beyond stimulation of antral follicle growth, and more research in this area could lead to the development of more effective fertility programs. In addition to its importance as an agricultural species, the cow provides a desirable model for humans regarding ovarian stimulation due to similar timing of folliculogenesis and follicle size, as well as similar ovarian architecture. The refinement of minimally invasive methods to allow the study of preantral folliculogenesis in live animals will be critical to understand the short- and long-term effects of FSH in ovarian folliculogenesis.

Mind the mechanical strength: tailoring a 3D matrix to encapsulate isolated human preantral follicles.

Would a hydrogel with similar mechanical properties to the human ovarian cortex support preantral follicle development? Yes, our tailored PEGylated fibrin hydrogel was shown to significantly improve follicle growth in vitro. One of the main challenges in developing an engineered ovary is to provide a 3D matrix that supports the follicle architecture and the interaction between granulosa cells and the oocyte as they are essential for folliculogenesis. Thanks to its biocompatibility and bioactivity, fibrin has been employed to fabricate a 3D matrix to encapsulate ovarian follicles. However, follicles lose their physical support within a few days owing to rapid fibrin degradation. Therefore, different strategies, including physical and chemical modifications, have been developed to enhance the stability of fibrin. By developing a matrix made of a synthetic (polyethylene glycol: PEG) and natural polymer (fibrin), we aimed to overcome fibrin degradation by the chemical reaction of PEGylation and tailor a PEGylated fibrin hydrogel formulation with mechanical strength similar to the ovarian cortex in women of reproductive age. To this end, response surface methodology was employed to obtain a tailored formulation of PEGylated fibrin. This hydrogel was then tested to encapsulate and support isolated human preantral follicles in vitro. A PEGylated fibrin formulation was tailored using mathematical modeling software to mimic the mechanical properties of human ovarian tissue at reproductive age. Human preantral follicles were isolated from 11 patients of reproductive age and encapsulated in the tailored hydrogels, which were cultured in vitro for 4 or 7 days. Follicle survival and diameter were assessed on Days 1 and 7. Furthermore, the follicles were subjected to confocal microscopy to evaluate their growth (Ki67 staining) on Day 7 and analyze cell-cell communication (connexin 43 and transzonal projection staining) on Day 4. In this study, mathematical modeling was applied to achieve the biomechanically tailored PEGylated fibrin formulation by targeting the specific goal of 3178 ± 245 Pascal, Young's modulus of ovarian cortical tissue in reproductive-age women. Our results demonstrated that the PEGylated fibrin hydrogel consisting of 39.06 mg/ml of PEGylated fibrinogen and 50.36 IU/ml of thrombin was the optimum condition with the desirability of 97.5%. This tailored hydrogel yielded a high follicle survival rate (83%) after 7 days of in vitro culture and supported its development up to the secondary stage. Follicle growth was confirmed by the presence of Ki67-positive granulosa cells on Day 7. Additionally, connexin 43 and Phalloidin staining indicated the retention of connections between granulosa cells and the oocyte. N/A. In this study, our tailored hydrogel was only tested in vitro, which is not the same as the physiological environment. It is crucial to conduct a study assessing the follicles following their encapsulation in the tailored hydrogel and transplantation, which will be the next step of our investigation. The findings from this study introduced a suitable biomaterial similar to the ovarian cortex in reproductive-age women in terms of biomechanical properties for encapsulating human preantral follicles. This biomaterial allowed the radial growth of follicles and preserved their viability. Furthermore, PEGylation improved the stability of fibrin and the physical support of follicles. This study was supported by grants from the Fondation Louvain (PhD scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and PhD scholarship awarded to A.D. as part of a legacy from Mrs Ilse Schirmer). The authors declare no competing interests.

Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue.

This study evaluated the effects of different concentrations (10, 20, or 40 μM) of eugenol (EUG 10, EUG 20, or EUG 40), ascorbic acid (50 μg/mL; AA) or anethole (300 μg/mL; ANE 300) on the in-vitro survival and development of goat preantral follicles and oxidative stress in the cultured ovarian tissue. Ovarian fragments from five goats were cultured for 1 or 7 days in Alpha Minimum Essential Medium (α-MEM+) supplemented or not with AA, ANE 300, EUG 10, EUG 20 or EUG 40. On day 7 of culture, when compared to MEM, the addition of EUG 40 had increased the rate of follicular development, as observed by a decrease in the proportion of primordial follicles alongside with an increase in the rate of normally developing follicles. Furthermore, EUG 40 significantly increased both follicular and oocyte diameters. Subsequently, ovarian fragments from three goats were cultured for 1 or 7 days in α-MEM+ supplemented or not with AA, ANE 300 or EUG 40. All tested antioxidants, except ANE 300, were able to significantly decrease the levels of reactive oxygen species in the ovarian tissue, but EUG 40 could most efficiently neutralize free radicals. All ovarian tissues cultured in the presence of antioxidants, especially EUG 40, presented a significant decrease in H3K4me3 labeling, indicating a silencing of genes that play a role in the inhibition of follicular activation and apoptosis induction. When compared to cultured control tissues, both EUG 40 and ANE 300 significantly increased the intensity of calreticulin labeling in growing follicles. The mRNA relative expression of ERP29 and KDM3A was significantly increased when the culture medium was supplemented with EUG 40, indicating a response to ER stress experienced during culture. In conclusion, EUG 40 improved in-vitro follicle survival, activation and development and decreased ROS production, ER stress and histone lysine methylation in goat ovarian tissue.