Survival Of Preantral Follicles Research Articles

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.

Melatonin acts through different mechanisms to control oxidative stress and primordial follicle activation and survival during in vitro culture of bovine ovarian tissue

This study aims to evaluate the effects of melatonin and its mechanisms of action on preantral follicle activation and survival, stromal cell density and collagen distribution in extracellular matrix (ECM). The involvement of melatonin receptors and mTORC1 pathway in these procedures were also investigated. To this end, ovarian fragments were cultured for six days in α‐MEM+ alone or supplemented with 1000 pM melatonin, 1000 pM melatonin with 1000 pM luzindole (inhibitor of melatonin receptors), or 1000 pM melatonin with 0.16 µg/ml rapamycin (mTORC1 inhibitor). At the end of culture period, tissues were processed for classical histology, and the follicles were classified as normal or degenerated, as well as in primordial or growing follicles. The ovarian stromal cell density and ECM collagen distribution were also evaluated. Samples of ovarian tissues were also destined to measure the levels of thiol and mRNA for CAT, SOD, GPX1 and PRDX1, as well as the activity of antioxidant enzymes CAT, SOD, and GPX1. The results demonstrated that ovarian tissues cultured with melatonin, melatonin with luzindole or melatonin with rapamycin had significantly higher percentage of morphologically normal follicles than those cultured in control medium (α‐MEM+). However, the presence of either luzindole or rapamycin, did not block the positive effects of melatonin on follicle survival (P > 0.05). Although the presence of melatonin in culture medium reduced the percentage of primordial follicles and increased the percentage of development follicles, these positive effects of melatonin were blocked by either luzindole or rapamycin (P < 0.05). Melatonin, melatonin with luzindole or melatonin with rapamycin did not influence the number of ovarian stromal cells. In contrast, melatonin significantly increased the percentages of collagen in ovarian tissues, but the positive effects of melatonin were blocked by either luzindole or rapamycin. Tissues cultured with melatonin and rapamycin had higher levels of mRNA for CAT and lower GPx activity when compared to those cultured in control medium. In conclusion, melatonin promotes primordial follicle activation, increases collagen fiber in ECM of in vitro cultured bovine ovarian tissue through its membrane-coupled receptors and mTORC1. Oppositely, melatonin increase follicles survival by acting through other pathways, since it can pass through cell membranes and directly regulate oxidative stress.

Influence of phenolic flavonols (Kaempferol, Querectin and Myricetin) on the survival and growth of ovine preantral follicles and granulosa cells cultured in vitro

Study was carried out to examine the influence of plant bioactive compounds [Kaempferol (KAE), Querectin (QUE) and Myricetin (MYR)] on the survival and growth parameters of cultured ovine preantral follicles (PFs) granulosa cells (GCs) and expression of some key developmental genes. Ovine PFs were isolated from slaughterhouse derived ovaries and KAE, QUE and MYR were supplemented to the standard culture medium of GCs and PFs at concentrations of 0, 5, 10, 25, 50 and 100 μM and cultured for 5 and 7 days respectively. PFs morphological and functional parameters [follicle and enclosed oocyte growth rate, viability of follicles, antrum formation rate, oocyte maturation rate, estradiol concentration, reactive oxygen species (ROS) production] and GC growth parameters (metabolic activity, viability rate, cell number increment, ROS production) were measured after culture. Significantly higher PF growth, viability rate and estradiol concentration was observed at 10 μM, 25 μM and 10 μM concentration of KAE, MYR and QUE respectively compared to the control. ROS production was significantly decreased in the PF culture media treated with 10 μM KAE or MYR 25 μM or 10 μM QUE compared to those observed in the control group. Likewise, metabolic activity of GCs, viability rate and cell number increment cultured with KAE, MYR and QUE was significantly higher at 10, 25 and 10 μM concentrations respectively compared to those observed in control group. ROS production was significantly lower in the GC cultured with KAE, MYR and QUE at 10, 25 and 10 μM concentrations respectively compared to the control. Based on the results of the growth parameters, gene expression of PFs and GCs were studied by qPCR at selected concentrations (KAE, MYR and QUE at 10, 25 and 10 μM concentrations respectively) in the cultured PFs and GCs. Gene expression of GDF9, FGF2, CYP19A1 was significantly higher and Bax, Bcl2 expression was significantly lower in the PFs and GCs cultured with the KAE or QUE at 10 μM concentration. KAE, MYR and QUE have dose dependant responses on PFs and GCs morphological and functional parameters; however, KAE is more potent amongst the three in augmenting the ovarian functions.

Mouse primary follicles experience slow growth rates after activation and progressive increases that influence the duration of the primary follicle phase†.

There are conflicting estimates of the duration of mouse primary follicle development. An accurate determination is needed for studies examining preantral follicle survival and mathematical modeling of folliculogenesis. Primary follicle granulosa cell proliferation rates are low and variable, which may explain the variation in duration estimates. In the present study, female C57Bl6/J mice were exposed to bromodeoxyuridine for 48hours, to label the proliferating granulosa cells in a large proportion of primary follicles. The bromodeoxyuridine-containing water was then withdrawn and replaced with drug-free water and the mice were euthanized at 0, 1, 3, 6, 10, or 13days post-bromodeoxyuridine withdrawal. Granulosa cells were bromodeoxyuridine labeled in 48% of primary follicles at day 0, but this decreased to 5% over the 13-day period, as the labeled primary follicles progressed to the secondary follicle stage. Curve-fitting estimated that the last of the bromodeoxyuridine-labeled primary follicles would progress to the secondary stage by 13.7days. Mathematical models that assumed constant rates of primary follicle proliferation were fitted to the data, but the observed pattern of bromodeoxyuridine-labeled primary follicle disappearance could not be replicated. The level of immunoreactivity for bromodeoxyuridine and proliferating-cell nuclear antigen in primary follicles revealed follicles with no granulosa cell proliferation during the 48-h bromodeoxyuridine-exposure period had resumed proliferation 1 or 3days later. Therefore, primary follicle granulosa cells proliferate after follicle activation, but proliferation rates gradually increase as the follicle develops. Prior estimates of primary follicle duration are inaccurate due to the assumption that follicles develop at a constant rate.

Reducing 3D Hydrogel Stiffness, Addition of Oestradiol in a Physiological Concentration and Increasing FSH Concentration Improve In Vitro Growth of Murine Preantral Follicles.

This study aimed to optimise culture conditions for murine preantral follicles to improve their growth and survival. Preantral follicles (diameter 100-130 µm) were isolated from prepubertal NMRI mice and individually cultured within alginate beads for 12 days. Three conditions were evaluated: (1) follicle re-encapsulation on day 6 of culture-reducing alginate concentration (0.5% to 0.25% w/v), (2) the presence of oestradiol (E2), and (3) increased follicle-stimulating hormone (FSH) concentration in the culture medium (from 10 to 100 mIU/mL FSH). Follicle morphology and growth, as well as anti-Müllerian hormone (AMH) production, were evaluated. From day 8, re-embedded follicles had a larger average diameter compared to follicles without alginate re-encapsulation (0.5% and 0.25% groups, p < 0.05). Oestradiol (1 µM) had a significantly positive effect on the mean follicular diameter and antrum formation (p < 0.001). Moreover, follicles cultured with 100 mIU/mL FSH showed faster growth (p < 0.05) and significantly higher antrum formation (p < 0.05) compared to the low FSH group. Nevertheless, AMH production was not affected by any of the culture conditions. In conclusion, the growth and survival of mouse preantral follicles during a 12-day period were improved by altering the alginate concentration midways during culture and adding E2 and FSH to the culture medium.

P-393 The mitochondrial targeted antioxidant Mitoquinol increases survival of isolated human preantral follicles in vitro

Abstract Study question Does Mitoquinol have a protective effect on survival and mitochondrial oxidative stress in cultured human preantral follicles? Summary answer Mitoquinol significantly increased survival of human preantral follicles cultured for 8 days and significantly decreased expression of genes related to the mitochondrial oxidative stress response. What is known already Culturing human ovarian follicles is a promising new source of mature oocytes for fertility preservation, as well as a key model system to investigate fundamental biology. Nevertheless, only a handful of studies have yielded mature human oocytes from preantral stage follicles and optimizing culture systems is challenging due to the scarcity of human material. Mitoquinol is an antioxidant that contains a form of Coenzyme Q10 called ubiquinone that has been modified to selectively target and protect mitochondria from oxidative damage. In human oocytes, the presence of Mitoquinol in the IVM media increased nuclear maturation and protected against chromosomal misalignment. Study design, size, duration Human preantral follicles (n = 213; mean diameter: 71 µm; range: 26-189 µm) were isolated from ovarian medulla tissue. Preantral follicles were mechanically and enzymatically isolated, encapsulated in 0.5% alginate and cultured for 8 days in three experimental groups: Control (n = 68); 50nM Mitoquinol (n = 71), 250nM Mitoquinol (n = 74). The primary endpoints were follicular growth and survival. Secondary endpoints included follicular gene expression and hormone analysis of the spent culture media. Participants/materials, setting, methods Ovarian medulla tissue was donated by 5 patients (aged 30-37 years) undergoing unilateral oophorectomy for ovarian tissue cryopreservation. Follicular growth and survival were assessed every second day during culture by microscopy. For follicles bigger than 95 µm, AMH and Estradiol concentrations were measured by ELISA in the spent media on day 8. Finally surviving follicles were snap-frozen and gene expression was analysed by qPCR; mitochondrial oxidative stress genes: HSP-60, TFAM and apoptosis genes: BAX/BCL-2. Main results and the role of chance After 8 days in culture, the follicular survival rate in the 50nM Mitoquinol group (68%; n = 51/74) was significantly higher than the control group (49%; n = 32/68; p = 0.0344) and the 250nM Mitoquinol (54%; n = 39/71; p = 0.0443). The protective effect of 50nM Mitoquinol was more pronounced in the subset of small follicles (Day0 &amp;lt; 70 µm, n = 135) in which survival rate of the 50nM Mitoquinol group (65%; n = 31/47) was significantly higher than the control (41%; n = 18/43; p = 0.005) and the 250nM group (44%; n = 20/4; p = 0.019). The average growth of surviving follicles was similar in the three experimental groups (Control: 40.7 ± 17.4 µm; Mitoquinol 50nM: 36.9 ± 19 μm; Mitoquinol 250nM: 36.4 ±16.5 μm). A significant positive association between the diameter of the follicle and the concentration of AMH and Estradiol was observed (AMH: p = 0.0215, R = 0.43 and Estradiol: p = 0.0413, R = 0.38) but no differences were found between the experimental groups. The relative expression of genes involved in mitochondrial oxidative stress response were significantly lower in the 50nM Mitoquinol group compared to the 250mM Mitoquinol group (HSP-60 p = 0.018; TFAM p = 0.013) and similar to the control group (HSP-60 p = 0.056; TFAM p = 0.071). Moreover, the gene expression of the ratio BAX/BCL2 was similar in the three groups. Limitations, reasons for caution A larger sample size is required to confirm the statistical analysis and to extrapolate the results to primordial, primary, and secondary stage follicles. Furthermore, long-term culture studies are required to determine the impact of Mitoquinol on antral follicle development and oocyte maturation Wider implications of the findings Our findings show that Mitoquinol can be used as an antioxidant in the culture of human preantral follicles since it increased follicle survival and reduced gene expression of genes related to mitochondrial oxidative stress response. Trial registration number Not appliclable

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.

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.

The protective effects of astaxanthin on pre-antral follicle degeneration in ovine vitrified/warmed ovarian tissue

This study assesses the protective effects of astaxanthin (AST) against vitrification/warming-induced cryoinjuries of ovarian tissue slices in sheep. Cortical slices of slaughterhouse acquired-ovine ovaries were randomly distributed in different groups: fresh, toxicity, and five vitrification groups including vitrification in presence of 0 (control group), 1, 10 and 100 μM astaxanthin or 100 μM vitamin E. After vitrification/warming and 24 h culturing, the samples were subjected to histological studies, antioxidant evaluation by TAC and TBAR assays, and assessment of relative expression of BMP4, BMP15, GDF9 and KITLG genes related to folliculogenesis and follicular growth regulation. According to the results, vitrification reduced the percentage of morphologically intact follicles compared to the fresh and toxicity groups (p < 0.05). In vitrification groups, vitamin E and all three concentrations of AST increased the percentage of intact pre-antral follicles and antioxidant activity relative to the vitrified control (p < 0.05). This enhancement significantly occurred in 10 μM AST group more than vitamin E (p < 0.05). Also, 10 μM concentration of AST enhanced the expression of all the examined genes compared to the control (p < 0.05), while the expression of BMP4, BMP15 and KITLG was higher in the AST than vitamin E (p < 0.05). The latter could increase only the expression of GDF9 compared to the control group (p = 0.011). In conclusion, AST is a highly effective antioxidant for maintaining the survival of pre-antral follicles, retaining cell density, increasing total antioxidant capacity, and increasing the expression of some genes related to follicular development after short-term culture of vitrified/warmed ovarian tissue slices.

Follicle-Stimulating Hormone Glycosylation Variants Distinctly Modulate Pre-antral Follicle Growth and Survival.

Follicle-stimulating hormone (FSH) is a key endocrine regulator of ovarian function. FSH is secreted as 2 macroglycosylation variants: partially glycosylated FSH (FSH21/18) and fully glycosylated FSH (FSH24). FSH21/18 is more potent than FSH24 at binding to and activating the FSH receptor (R). The ratio of FSH21/18:FSH24 has been shown to change with age, with FSH21/18 predominant at reproductive prime, and FSH24 predominant during perimenopause/menopause. How these FSH glycosylation variants modulate ovarian follicle functions remains largely unknown. The aim of this study was to investigate the effect of FSH glycosylation variants of pre-antral follicle function. Pre-antral follicles were isolated from 3- to 5-week-old C57BL/6 mice and treated ±10 ng/mL FSH21/18, FSH24, a ratio of 80:20 FSH21/18:FSH24 (to mimic reproductive prime), 50:50 FSH21/18:FSH24 (perimenopause), or 20:80 FSH21/18:FSH24 (menopause) for up to 96 hours. FSH21/18 and 80:20 FSH21/18:FSH24 increased follicle growth, in comparison with control, contrasting with FSH24 and 20:80 FSH21/18:FSH24. Survival rates were decreased in follicles treated with FSH24 or 20:80 FSH21/18:FSH24, with follicles undergoing basement membrane rupture and oocyte extrusion, increased Caspase3 gene and protein expression, and decreased markers of cell proliferation in FSH24 or 20:80 FSH21/18:FSH24-treated follicles. Moreover, this correlated with differential regulation of key genes modulating follicular functions. Pharmacological inhibitors of key FSH signal pathways suggests FSH21/18 and FSH24 initiate different FSHR signal pathway activation, which may determine their differential effects on follicle growth and survival. These data suggest that the nature of FSH glycosylation modulates the follicular cellular environment to regulate follicle growth and survival and may underpin the increasing ovarian resistance to FSH observed during aging.

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.

Impact of vitamin D3 supplementation on the in vitro growth of mouse preantral follicles

ObjectiveWe investigated the impact of vitamin D3 (VD3) supplementation during mouse preantral follicle culture in vitro and the mRNA expression of 25-hydroxylase (CYP2R1), 1-alpha-hydroxylase (CYP27B1), and vitamin D receptor (VDR) in mouse ovarian follicles at different stages.MethodsPreantral follicles were retrieved from 39 BDF1 mice (7–8 weeks old) and then cultured in vitro for 12 days under VD3 supplementation (0, 25, and 50 pg/mL). Follicular development and the final oocyte acquisition were assessed. Preantral follicles were retrieved from 15 other BDF1 mice (7–8 weeks old) and cultured without VD3 supplementation. Three stages of mouse ovarian follicles were obtained (preantral, antral, and ruptured follicles). Total RNA was extracted from the pooled cells (from 20 follicles at each stage), and then reverse transcriptase-polymerase chain reaction was performed to identify mRNA for CYP2R1, CYP27B1, and VDR.ResultsThe survival of preantral follicles, rates of antrum formation and ruptured follicles (per initiated follicle) and the number of total or mature oocytes were all comparable among the three groups. Both CYP2R1 and CYP27B1 were expressed in antral and ruptured follicles, but not in preantral follicles. VDR was expressed in all three follicular stages.ConclusionVD3 supplementation in vitro (25 or 50 pg/mL) did not enhance mouse follicular development or final oocyte acquisition. Follicular stage-specific expression of CYP2R1, CYP27B1, and VDR was observed.

Differential follicle stimulating hormone glycosylation modulates pre-antral follicle growth and survival rates

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

AlPc/ZnPc-based oncological photodynamic therapy for a selective eradication of leukemic cells from ovarian tissue

Due to the risk of reintroducing malignant cells, autotransplantation of cryopreserved ovarian tissue is not allowed in leukemia patients. In order to restore fertility in these patients, ex vivo purging of ovarian fragments could be proposed as a strategy to eradicate malignant cells before grafting. Photodynamic therapy (PDT), as a clinical-approved modality, is a minimally invasive and selective therapeutic for eliminating malignant cells. The present work aims therefore to evaluate the phototoxicity of two photosensitizers (aluminum phthalocyanine (AlPc) and zinc phthalocyanine (ZnPc)) on leukemia cells. To this end, two lines of malignant cells (K562 and HL-60) and isolated ovarian stromal cells (control) were treated by PDT using a diode laser with various energy densities. Cell viability after the treatment, the amount of generated reactive oxygen species, dark toxicity of the photosensitizers, and single-cell morphology were studied. Our results demonstrated that using irradiation with the energy density of 10 J/cm2, 1 µM AlPc could significantly reduce the viability of K562 (4.73 ± 0.14%) and HL-60 (2.74 ± 0.31%). Similarly, the viability of these cells was reduced (K562 cells: 3.84 ± 0.81%; HL-60 cells: 6.82 ± 3.21%) with 1 µM ZnPc and an energy density of 50 J/cm2. On the other hand, these PDT protocols had no significant effect on stromal cells. These findings indicate that our approach can be a promising strategy for the safe restoration of fertility in leukemia patients. However, further studies are necessary to assess its efficiency in ovarian fragments containing malignant cells to determine their eradication rate and the effect of our treatment on the survival of stromal cells and preantral follicles.

5-Fluorouracil disrupts ovarian preantral follicles in young C57BL6J mice.

5-Fluorouracil (5-FU), an anti-cancer drug, has been used for hepatoblastoma (HB) chemotherapy in children, who mayhave impaired ovarian follicle pool reserve with lasting effectsto reproduction. Therefore, this study aimed to investigate 5-FU effects on survival, growth, and morphology of ovarian preantral follicles from C57BL6J young mice. Experiments were carried-out both in vivo and in vitro. Mice were treated with 5-FU injection (450mg/kg i.p) or saline and sacrificed 3days after to obtain ovaries for histology and molecular biology. Ovaries for in vitro studies were obtained from unchallenged mice and cultured under basic culture medium (BCM) or BCM plus 5-FU (9.2, 46.1, 92.2mM). Preantral follicles were classified according to developmental stages, and as normal or degenerated. To assess cell viability, caspase-3 immunostaining was performed. Transcriptional levels for apoptosis (Bax, Bcl2, p53, Bax/Bcl2) and Wnt pathway genes (Wnt2 and Wnt4) were also analyzed. Ultrastructural analyses were carried-out on non-cultured ovaries. In addition, β-catenin immunofluorescence was assessed in mouse ovaries. The percentage of all-types normal follicles was significantly lower after 5-FU challenge. A total loss of secondary normal follicles was found in the 5-FU group. The highest 5-FU concentrations reduced the percentage of cultured normal primordial follicles. Large vacuoles were seen in granulosa cells and ooplasm of preantral follicles by electron microscopy. A significantly higher gene expression for Bax and Bax/Bcl2 ratio was seen after 5-FU treatment. A marked reduction in β-catenin immunolabeling was seen in 5-FU-challenged preantral follicles. In the in vitro experiments, apoptotic and Wnt gene transcriptions were significantly altered. Altogether, our findings suggest that 5-FU can deleteriously affect the ovarian follicle reserve by reducing preantral follicles survival.

Can frozen-thawed human ovary withstand refreezing-rethawing in the form of cortical strips?

The aim of this study was to elucidate whether ovarian tissue is able to withstand a double freezing-thawing procedure. Human ovarian cortical biopsies from 4 thawed whole ovaries were divided into 4 experimental subgroups: (a) frozen-thawed non-grafted group, (b) frozen-thawed xenografted group, (c) refrozen-rethawed non-grafted group, and (d) refrozen-rethawed xenografted group. Xenografting was performed using 8 severe combined immunodeficient mice for a total duration of 21days. The following analyses were conducted: classic hematoxylin and eosin staining, Ki67 immunolabeling, transmission electron microscopy, Masson's green trichrome, and double CD34 immunostaining. Morphologically normal preantral follicles were detected in all groups. We observed a dramatic decline of more than 65% in early preantral follicle survival rates after grafting of both frozen-thawed (p< 0.0001) and refrozen-rethawed (p< 0.0001) ovarian tissue. However, mean follicle densities remained comparable between the frozen-thawed and refrozen-rethawed non-grafted groups, as well as both grafted groups. Equivalent proportions of proliferating early preantral follicles were identified in frozen-thawed and refrozen-rethawed samples, whether the tissue was grafted or not. Furthermore, we did not observe any significant difference in atretic follicle rates between any of the four groups, and the ultrastructural quality of follicles appeared unaffected by the refreezing procedure. Similar proportions of fibrosis were noted in the frozen-thawed and refrozen-rethawed groups, irrespective of grafting. Finally, no significant differences were witnessed in terms of vascularization. We were able to demonstrate, for the first time, that refrozen-rethawed ovarian tissue has the same functional characteristics as frozen-thawed ovarian tissue.

Antral follicular count and its relationship with ovarian volume, preantral follicle population and survival, oocyte meiotic progression and ultrastructure of in vitro matured bovine cumulus-oocyte complexes.

This study aimed to evaluate the relationship between antral follicular count (AFC) and ovarian volume (OV), preantral follicular population and survival, meiotic progression and ultrastructure of cumulus-oocyte complexes (COCs) after in vitro maturation. In experiment 1, the relationship between AFC and preantral follicle population and survival was evaluated by classical histology. In experiment 2, the relationship among AFC, OV, ability of oocytes to resume meiosis and ultrastructure of in vitro matured bovine COCs was studied. A positive correlation (P < 0.05) between AFC and the numbers of healthy primordial, degenerate and total follicles was observed, as well as with healthy secondary follicles and total follicles. The numbers of grades I and II oocytes in ovaries of high AFC class were higher compared with those with intermediate or lower AFC. After in vitro maturation, COCs from ovaries of high AFC had a higher percentage of oocytes in metaphase II compared with those of intermediate and low AFC (P < 0.0001). Ovaries of intermediate AFC had a higher percentage of oocytes in metaphase II compared with ovaries with low AFC (P < 0.0001). The proportion of oocytes in metaphase I, telophase I and anaphase I in COCs from ovaries of intermediate AFC (26.04%) was higher (P < 0.05) compared with that seen in COCs of ovaries with high (8.55%) and low (14.15%) AFC. No differences in the ultrastructure of oocytes were seen. In conclusion, after in vitro maturation, cow ovaries with high AFC have higher numbers of oocytes that reach in metaphase II (MII), but they also have higher numbers of degenerated primordial and primary follicles.

The effect of bioidentical nanostructured progesterone in the in vitro culture of preantral follicles and oocyte maturation.

This study evaluated the effects of bioidentical nanostructured progesterone alone or in association with human chorionic gonadotropin (hCG) on the in vitro survival and development of preantral follicles (experiment 1) and oocyte maturation (experiment 2). Bioidentical hormones have a molecular structure identical with that of endogenous hormones; nanostructured substances refer to those reduced to a nanoscale. In experiment 1, fragments of goat ovarian tissue were cultured for 7 days in α-MEM+ alone or supplemented with nanoprogesterone (MEM+ + P4) or P4 and hCG (MEM+ + P4 + hCG). In experiment 2, two mediums of oocyte in vitro maturation (IVM) were compared. Medium 1 consisted of TCM 199+ + LH, and medium 2 consisted of TCM 199+ with nanoprogesterone and hCG. The MEM+ + P4 + hCG treatment showed the lowest percentage of follicular survival after 7 days of culture. MEM+ + P4 and MEM+ + P4 + hCG treatments showed higher percentage of follicular activation than MEM+. In experiment 2, there were no differences between mediums 1 and 2 for all endpoints evaluated. In conclusion, the addition of nanoprogesterone is advisable for in vitro culture of preantral follicles and oocyte maturation. However, the association of nanoprogesterone with hCG causes the cellular death of initial follicles but shows efficacy in IVM.

Effect of different extracts and fractions of Senecio biafrae (Oliv. &Hiern) J. Moore on in vivo and in vitro parameters of folliculogenesis in experimental animals.

Senecio biafrae is a medicinal plant widely used in traditional medicine to cure female infertility. Some effects have been pharmacologically demonstrated on immature female rats but in vivo and in vitro investigations are still necessary for determining its mechanism of action. The aim of the present study was to evaluate the estrogenic and FSH-like effects of the plant extracts and fractions on some fertility parameters in immature female rats and on in vitro survival and growth of swine preantral follicles. 21-23 days old female Wistar rats orally received extracts and fractions of S. biafrae at 0, 8 and 64mg/kg doses over 20 days. The LH, FSH, estradiol and progesterone serum levels were evaluated as well as the ovarian cholesterol, uterus and ovaries masses and proteins. The numbers of follicles at different developmental stages were recorded in ovarian cortexes after histology. Slices of swine ovarian cortexes were cultured along 1 or 7 days in alpha-minimum essential medium (α-MEM) and fixed for morphological analysis of preantral follicles. The fresh control, cultured control (CIV control) and different Senecio biafrae-treated ovarian fragments were analyzed for preantral follicles development. Treatments that showed the best follicle growth in culture were submitted to AgNOR test. The aqueous and MeOH/CH2Cl2 extracts as well as the ethyl acetate and hexane fractions of S. biafrae were submitted to the HPLC for analysis of polyphenolic secondary metabolites. Ovarian and uterine proteins were significantly high (p<0.01) in animals treated with the two dosages of ethyl acetate and n-butanol fractions. The same result was recorded with uterine proteins in animals treated with the hexane fraction. The FSH level significantly dropped with all ethanolic extract doses and with the 64mg/kg dosage of the methanol/methylene chloride (MeOH/CH2Cl2) extract while LH was reduced (p<0.01) in almost all the treated groups. Estradiol level was significantly increased (p<0.001) in the three groups receiving the extracts, but reduced (p<0.001) in the three groups receiving the fractions of the plant. The progesterone level increased with almost all the treated groups. Primary and secondary follicles augmented (p<0.01) in MeOH/CH2Cl2 extract and n-butanol fraction while tertiary follicles increased with the same extract and the ethyl acetate fraction (p<0.05). Treatments with aqueous and ethanolic extracts as well as ethyl acetate fraction led to a significant increase (p<0.05) in the number of morphologically normal follicles after 7 days of culture as compared to the CIV control. The number of AgNOR dots per follicle was significantly low (p<0.05) in all cultured groups as compared to the fresh control, except the ethyl acetate 2.8ng/ml dosage. The same observation was done with AgNOR dots per cell in the 2.8ng/ml dosage aqueous extract-treated fragments. The phenolic compounds mainly encountered in the plant, independently of the extract or fraction are apigenin, eugenol and rutin. Extracts and fractions of S. biafrae have an important FSH-like effect which induces follicular survival and growth.

The subtle balance of insulin and thyroxine on survival and development of in vitro cultured caprine preantral follicles enclosed in ovarian tissue

Numerous studies have reported the importance of thyroid hormones on the development of later preantral and antral follicles, but their interactions with other hormones and effects in regulating early preantral follicle growth remain unclear. Here we investigated the in vitro effects of thyroxine combined with insulin on caprine preantral follicle survival and development. Sliced ovarian tissues were cultured for 1 or 7 days using 10 ng/mL (low) or 10 μg/mL (high) insulin in the presence of thyroxine at 0, 0.5, 1 or 2 μg/mL. Post-culture, we evaluated the follicular survival and development, assessed the expression of apoptotic-related genes (Bcl2/Bax) and receptors of insulin and thyroid hormones, and quantified the estradiol and reactive oxygen species (ROS) production levels. Follicular survival in low-insulin culture conditions was enhanced by the presence of 0.5 μg/mL thyroxine (P < 0.05) as compared to the thyroxine-free medium but remained similar to non-cultured control in the presence of 2 μg/mL (P > 0.05). Significantly higher ROS production was measured from Day 1 to Day 7 in low-insulin culture media containing 0.5 or 2 μg/mL thyroxine (P < 0.05). When compared to high insulin level, the presence of thyroxine in low insulin culture conditions yielded higher stromal cell density (P < 0.05), increased estradiol production on Day 1, and higher Bcl2/Bax ratio on Day 7. Cultures with high levels of both insulin and thyroxine led to follicles and oocytes with larger diameters (P < 0.05). The RNA transcript levels of insulin and thyroid receptors were reduced in the presence of high insulin cultures when compared to controls (non-cultured). In conclusion, the combination of low concentrations of insulin and thyroxine better maintained follicle survival, while high levels ensured better follicular development.