Profil clinique, biologique et traitement des drépanocytaires régulièrement suivis au Centre Hospitalier Universitaire de Libreville / Clinical, biological and treatment profile of sickle cell patients regularly followed at Libreville University Hospital Center

Introduction: La mise en place depuis Septembre 2016 au Centre Hospitalier Universitaire (CHUL) d'une consultation d'hématologie dédiée aux adultes drépanocytaires a été l'occasion de mener cette étude dont le but principal était d'établir les profils clinique et paraclinique de l'adulte drépanocytaire régulièrement suivi.Patients et méthodes : Il s'agissait d'une étude rétrospective. La population d'étude était constituée de patients drépanocytaires homozygotes de 18 ans et plus, qui avaient effectués au moins trois consultations d'hématologie sur une année. Les informations recueillies, après étude du dossier médical et entretien téléphonique avec le patient, concernaient les données socio-démographiques, l'histoire de la drépanocytose, les antécédents, les complications de la maladie, les examens biologiques et radiologiques et le traitement.Résultats : Au total 88 patients répondaient aux critères d'inclusion sur les 233 drépanocytaires vus durant la période de l'étude. L'âge moyen était de 30,4 ± 7,8 ans. L'interrogatoire révélait que la crise vaso-occlusive (CVO) était la principale complication aiguë et la lithiase vésiculaire (36,3%) la première complication chronique. L'hémoglobine moyenne était de 7,8 g/dl et pour 49,3% des patients elle se situait entre 7 et 9 g/dl. Les leucocytes étaient augmentés dans 65,7%. L'échographie cardiaque réalisée chez 35 patients retrouvait 11,1% d'hypertension artérielle pulmonaire et 22,8% d'hypertrophie ventriculaire gauche. L'intensité de la crise douloureuse motivait une consultation au service des urgences du CHU dans 81,6% des cas dont plus de 62% déploraient un retard dans l'exécution de cette prise en charge bien qu'ils en aient été satisfaits dans 54,9% des cas.Conclusion : Les drépanocytaires adultes régulièrement suivis au CHUL sont peu nombreux. La transition entre le suivi pédiatrique et adulte doit se faire avec une transmission des informations du dossier médical sur l'histoire de la drépanocytose.

Introduction: The establishment since September 2016 at the University Hospital Center (CHUL) of a hematology consultation dedicated to adults with sickle cell disease was an opportunity to conduct this study, the main purpose of which was to establish the clinical and paraclinical profiles of the adults with sickle cell disease regularly monitored. Patients and methods: This was a retrospective study. The study population consisted of homozygous sickle cell patients aged 18 and over, who had performed at least three hematology consultations over a year. The information collected, after studying the medical file and telephone interview with the patient, concerned socio-demographic data, history of sickle cell disease, history, complications of the disease, biological and radiological examinations and treatment. Results: A total of 88 patients met the inclusion criteria out of the 233 sickle cell patients seen during the study period. The mean age was 30.4 ± 7.8 years. The questioning revealed that vaso-occlusive crisis (VOC) was the main acute complication and cholelithiasis (36.3%) the first chronic complication. The average hemoglobin was 7.8 g/dl and for 49.3% of the patients it was between 7 and 9 g/dl. Leukocytes were increased in 65.7%. Cardiac ultrasound performed in 35 patients found 11.1% pulmonary arterial hypertension and 22.8% left ventricular hypertrophy. The intensity of the painful crisis motivated a consultation in the emergency department of the CHU in 81.6% of cases, of which more than 62% complained of a delay in the execution of this care although they were satisfied with it in 54 .9% of cases.Conclusion: Few adult sickle cell sufferers are regularly monitored at the CHUL. The transition between pediatric and adult follow-up must be made with a transmission of information from the medical file on the history of sickle cell disease

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.

BACKGROUND: 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. METHODS: 21-23 days old female Wistar rats orally received extracts and fractions of S. biafrae at 0, 8 and 64 mg/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. RESULTS: 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 64 mg/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 2.8 ng/ml dosage. The same observation was done with AgNOR dots per cell in the 2.8 ng/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. CONCLUSION: Extracts and fractions of S. biafrae have an important FSH-like effect which induces follicular survival and growth.

A qualitative study about quality of life in Brazilian families with children who have severe or profound intellectual disability.

BACKGROUND: This study investigated the psychocultural perspectives concerning family quality of life among Brazilian families with children who have severe or profound intellectual disability. METHODS: Individual in-depth semi-structured interviews conducted with 15 mothers, selected by convenience, were analysed using a categorical thematic analysis technique. The themes were examined to allow for an interpretative approach of the results. RESULTS: Mothers revealed that their children with disabilities had insufficient access to services and support related to health care, transportation and recreation. Family quality of life was negatively affected by financial restrictions and social interaction difficulties. Caring for a child with disabilities seemed to be centred on the mother and religious coping appeared as a common psychological adjustment strategy. CONCLUSIONS: Improving emotional and psychological cares, as well as social and practical measures comprising income support and access to appropriate health care, were inferred to be the mothers' priorities to improve their families' quality of life.

Anethole reduces oxidative stress and improves in vitro survival and activation of primordial follicles.

Primordial follicles, the main source of oocytes in the ovary, are essential for the maintenance of fertility throughout the reproductive lifespan. To the best of our knowledge, there are no reports describing the effect of anethole on this important ovarian follicle population. The aim of the study was to investigate the effect of different anethole concentrations on the in vitro culture of caprine preantral follicles enclosed in ovarian tissue. Randomized ovarian fragments were fixed immediately (non-cultured treatment) or distributed into five treatments: α-MEM+ (cultured control), α-MEM+ supplemented with ascorbic acid at 50 µg/mL (AA), and anethole at 30 (AN30), 300 (AN300), or 2000 µg/mL (AN2000), for 1 or 7 days. After 7 days of culture, a significantly higher percentage of morphologically normal follicles was observed when anethole at 2000 µg/mL was used. For both culture times, a greater percentage of growing follicles was observed with the AN30 treatment compared to AA and AN2000 treatments. Anethole at 30 and 2000 µg/mL concentrations at days 1 and 7 of culture resulted in significantly larger follicular diameter than in the cultured control treatment. Anethole at 30 µg/mL concentration at day 7 showed significantly greater oocyte diameter than the other treatments, except when compared to the AN2000 treatment. At day 7 of culture, levels of reactive oxygen species (ROS) were significantly lower in the AN30 treatment than the other treatments. In conclusion, supplementation of culture medium with anethole improves survival and early follicle development at different concentrations in the caprine species.

In vivo and in vitro strategies to support caprine preantral follicle development after ovarian tissue vitrification.

The aim of the present study was to compare fresh and vitrified goat ovarian tissue after autotransplantation and in vitro culture. Adult goats were completely ovariectomised and each ovarian pair was sliced and distributed among six different treatment groups: fresh control, fresh transplant, fresh culture, vitrified control, vitrified transplant and vitrified culture. Follicular morphology, development, growth, density, revascularisation and hormone production were evaluated in all groups. Three antral follicles (two in the fresh transplant and one in the vitrified transplant groups) were observed on the surface of the graft 90 days after transplantation. The percentage of morphologically normal follicles was similar in the fresh control, fresh transplant and vitrified transplant groups. The percentage of developing (transition, primary and secondary) follicles was higher after in vitro culture of fresh or vitrified tissue. Transplantation resulted in a lower follicle density. Serum oestradiol concentrations remained constant during the entire transplantation period. In contrast, progesterone production decreased significantly. Expression of CD31 mRNA was lower in fresh culture. In conclusion, restoration of goat ovarian function can be successfully achieved following transplantation of both fresh and vitrified goat ovarian tissue. However, transplantation induced higher follicle loss than in vitro culture.

Anethole reduces oxidative stress and improves in vitro survival and activation of primordial follicles

Primordial follicles, the main source of oocytes in the ovary, are essential for the maintenance of fertility throughout the reproductive lifespan. To the best of our knowledge, there are no reports describing the effect of anethole on this important ovarian follicle population. The aim of the study was to investigate the effect of different anethole concentrations on the in vitro culture of caprine preantral follicles enclosed in ovarian tissue. Randomized ovarian fragments were fixed immediately (non-cultured treatment) or distributed into five treatments: α-MEM+ (cultured control), α-MEM+ supplemented with ascorbic acid at 50 μg/mL (AA), and anethole at 30 (AN30), 300 (AN300), or 2000 µg/mL (AN2000), for 1 or 7 days. After 7 days of culture, a significantly higher percentage of morphologically normal follicles was observed when anethole at 2000 μg/mL was used. For both culture times, a greater percentage of growing follicles was observed with the AN30 treatment compared to AA and AN2000 treatments. Anethole at 30 and 2000 µg/mL concentrations at days 1 and 7 of culture resulted in significantly larger follicular diameter than in the cultured control treatment. Anethole at 30 µg/mL concentration at day 7 showed significantly greater oocyte diameter than the other treatments, except when compared to the AN2000 treatment. At day 7 of culture, levels of reactive oxygen species (ROS) were significantly lower in the AN30 treatment than the other treatments. In conclusion, supplementation of culture medium with anethole improves survival and early follicle development at different concentrations in the caprine species.

Role of EGF on in situ culture of equine preantral follicles and metabolomics profile.

The effects of epidermal growth factor (EGF) concentrations (0, 10, 50, and 100ng/ml) on in vitro culture (IVC) of equine preantral follicles were evaluated using histology, estradiol and reactive oxygen species (ROS) production and metabolomics. After IVC, the percentage of normal follicles was lower (P<0.05) for all treatments when compared to non-cultured control. EGF 50ng/ml treatment had more (P<0.05) normal follicles at Day 7 of culture when compared with EGF 0 and 100ng/ml. EGF 50ng/ml had more (P<0.05) developing follicles than the 0ng/ml and 10ng/ml EGF treatments. Follicular and oocyte diameters were greater (P<0.05) with EGF 50ng/ml than the other cultured treatments, but similar (P>0.05) to the non-cultured control. From Day 1 to Day 7 estradiol production increased (P<0.05) in all EGF treatments. EGF 50ng/ml was the only treatment that maintained ROS production through IVC. Metabolomics profiles of the spent media indicated that eleven ions from variable influence in the projection (VIP) scores were higher represented in the EGF 50ng/ml treatment. In conclusion, EGF 50ng/ml treatment maintained follicle survival and ROS production, and promoted activation of cultured equine preantral follicles enclosed in ovarian tissue.

Modulation of aquaporins 3 and 9 after exposure of ovine ovarian tissue to cryoprotectants followed by in vitro culture.

Our aim has been to evaluate the effect of cryoprotective agents (CPAs) on the exposure, vitrification (VIT), and in vitro culture (IVC) of ovarian tissue with regard to the expression and immunolocalization of aquaporins (AQPs) 3 and 9 in ovine preantral follicles. Tissues were treated as follows: Experiment I: (1) control (without exposure to CPAs), (2) e-EG (exposure to ethylene glycol), (3) er-EG (exposure to and removal of EG), (4) e-DMSO (exposure to dimethyl sulfoxide), (5) er-DMSO (exposure to and removal of DMSO), (6) e-EG+DMSO (exposure to EG+DMSO), (7) er-EG+DMSO (exposure to and removal of EG+DMSO); Experiment II: (1) control, (2) VIT, (3) IVC, (4) VIT-IVC. In Experiment I, following er-EG or er-DMSO, tissue showed the down-regulation (P < 0.05) of AQP3 mRNA. The mRNA transcript levels were reduced (P < 0.05) for AQP9 in tissue following er-EG+DMSO. Immunolocalization was positive for both proteins (AQP3 and AQP9) on ovine preantral follicles following all treatments, except in the e-EG+DMSO group. In Experiment II, the mRNA levels of AQP3 and AQP9 following VIT treatment were similar (P > 0.05) to that of the control group. Nevertheless, VIT-IVC treatment led to the down-regulation of mRNA of AQP3 and AQP9. Thus, AQP3 and AQP9 act in a mutually dependent way, maintaining the cell homeostasis that is essential for the ovary cryopreservation process. Furthermore, the changes in the expression profiles of mRNA and protein after culture are a strong indicator that in vitro conditions have to be strictly controlled to ensure follicle viability and functionality.

FSH supplementation to culture medium is beneficial for activation and survival of preantral follicles enclosed in equine ovarian tissue.

This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FSH treatment had a greater (P < 0.05) percentage of morphologically normal follicles when compared to the other groups, except the 10-ng/mL FSH treatment at Day 1 of culture. The percentage of developing follicles (transition, primary, and secondary), and follicular and oocyte diameters were higher (P < 0.05) in the 50-ng/mL FSH treatment compared to the other groups after Day 7 of culture. Furthermore, estradiol secretion and ROS production were maintained (P > 0.05) throughout the culture in the 50-ng/mL FSH treatment. In conclusion, the addition of 50 ng/mL of FSH promoted activation of primordial follicles to developing follicles, improved survival of preantral follicles, and maintained estradiol and ROS production of equine ovarian tissue after 7 days of culture.

Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and reduces reactive oxygen species production after culture.

This study investigated the effect of insulin concentration on the in vitro culture of equine preantral follicles enclosed in ovarian tissue. Ovarian tissue samples were immediately fixed (noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0 ng/mL, 10 ng/mL, or 10 µg/mL insulin. Ovarian tissues were processed and analyzed by classical histology. Culture medium samples were collected after 1 and 7 days of culture for steroid and reactive oxygen species (ROS) analyses. The percentage of morphologically normal follicles was greater (P < 0.001) in insulin-treated groups after 1 day of culture; likewise, more (P < 0.02) normal follicles were observed after 7 days of culture in medium supplemented with 10-ng/mL insulin. Furthermore, an increase (P < 0.01) in developing (transition, primary, and secondary) follicles between Days 1 and 7 of culture was observed only with the 10-ng/mL insulin treatment. ROS production after 1 or 7 days of culture was lower (P < 0.0001) in medium with 10-ng/mL insulin than the other treatments. Ovarian tissues containing preantral follicles were able to produce estradiol and progesterone after 1 and 7 days of culture; however, treatments did not differ in steroid production. In conclusion, the use of a physiological concentration (10 ng/mL) of insulin rather than the previously reported concentration (10 µg/mL) for in vitro culture of equine preantral follicles improved follicular survival and growth and lowered oxidative stress. Results from this study shed light on new perspectives for producing an appropriate medium to improve equine preantral follicle in vitro survival and growth.

Expression of angiotensin II receptors in the caprine ovary and improvement of follicular viability in vitro.

This study aimed to evaluate mRNA levels of angiotensin II (ANG II) receptors (AGTR1 and AGTR2) in caprine follicles and to investigate the influence of ANG II on the viability and in vitro growth of preantral follicles. Real-time polymerase chain reaction (PCR) was used to quantify AGTR1 and AGTR2 mRNA levels in the different follicular stages. For culture, caprine ovaries were collected, cut into 13 fragments and then either directly fixed for histological and ultrastructural analysis (fresh control) or placed in culture for 1 or 7 days in α-minumum essential medium plus (α-MEM+) with 0, 1, 5, 10, 50 or 100 ng/ml ANG II. Then, the fragments were destined to morphological, viability and ultrastructural analysis. The results showed that primordial follicles had higher levels of AGTR1 and AGTR2 mRNA than secondary follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA than their respective cumulus-oocyte complex (COCs). After 7 days of culture, ANG II (10 or 50 ng/ml) maintained the percentages of normal follicles compared with α-MEM+. Fluorescence and ultrastructural microscopy confirmed follicular integrity in ANG II (10 ng/ml). In conclusion, a high expression of AGTR1 and AGTR2 is observed in primordial follicles. Granulosa/theca cells from antral follicles had higher levels of AGTR1 mRNA. Finally, 10 ng/ml ANG II maintained the viability of caprine preantral follicles after in vitro culture.

Vascular endothelial growth factor-A(165) (VEGF-A(165)) stimulates the in vitro development and oocyte competence of goat preantral follicles.

The aim of this study was to evaluate the effect of vascular endothelial growth factor-A(165) (VEGF-A(165)) on the in vitro development of goat secondary preantral follicles. Preantral follicles (≥150 µm in diameter) were isolated from the ovaries of adult mixed-breed goats and individually cultured for 18 days in αMEM in the absence (control) or presence of VEGF-A(165) at concentrations of 10 ng/ml (VEGF10) and 100 ng/ml (VEGF100). Analyses of follicular survival, diameter, antrum formation and rate of daily growth were performed every 6 days. At the end of the culture period, morphologically normal oocytes (≥110 µm in diameter) were taken for in vitro maturation (IVM). The results demonstrated that all follicles presented oocytes and granulosa cells that were morphologically normal and after labeling with calcein-AM, high rates of oocyte viability were observed in all treatments. The follicular diameter and the growth rate achieved in the presence of VEGF10 were higher than those of the control. Both treatments with VEGF-A(165) showed higher rates of oocyte recovery for IVM when compared with the control. Moreover, only the addition of VEGF-A(165) permitted oocytes grown in vitro to reach metaphase II. Thus, the addition of VEGF-A(165) to the culture medium improves the development of goat preantral follicles cultured in vitro, allowing the production of mature oocytes.

Steady-state level of bone morphogenetic protein-15 in goat ovaries and its influence on in vitro development and survival of preantral follicles.

This study investigates steady-state level of bone morphogenetic protein-15 (BMP-15) mRNA in caprine follicles, and the effects of BMP-15 on in vitro development of preantral follicles. Ovarian fragments were cultured for one or seven days in Minimal Essential Medium (MEM(+)) with BMP-15 (0, 1, 10, 50, 100 or 200 ng/mL), and further analyzed by histology, transmission electron and fluorescent microscopy. BMP-15 mRNA in secondary follicles was higher than in primordial and primary follicles. After seven days, 10, 50 or 100 ng/mL of BMP-15 maintained the percentage of normal follicles similar to the control (non-cultured), and increased the oocyte and follicle diameters when compared to the control and MEM(+). BMP-15 at 100 ng/mL increased the secondary follicles and maintained their ultrastructural integrity. In conclusion, the BMP-15 mRNAs were detected in all follicular categories. BMP-15 (100 ng/mL) maintained the integrity and promoted the growth of caprine preantral follicles cultured for seven days.

Expression of follicle-stimulating hormone receptor (FSHR) in goat ovarian follicles and the impact of sequential culture medium on in vitro development of caprine preantral follicles.

This study evaluated the expression of FSH receptors (FSHR) in the different stages of goat follicle development and investigated whether the addition of increasing concentrations of FSH throughout the culture period influences the survival, growth and antral formation of in vitro-cultured caprine preantral follicles. The expression of FSHR was analysed before and after culturing follicles using real-time RT-PCR. For the culture, preantral follicles (≥150 µm) were isolated from ovarian fragments and cultured for 18 days in α-MEM+ alone or associated with recombinant FSH (rFSH: 100 or 1000 ng/ml), or in α-MEM+ supplemented with increasing concentrations of FSH throughout culture periods as follows: (a) sequential medium 1: FSH 100 ng/ml (from day 0 to 6), FSH 500 ng/ml (from day 6 to 12) and FSH 1000 ng/ml (from day 12 to 18); and (b) sequential medium 2: FSH 500 ng/ml (from day 0 to 9) and 1000 ng/ml (from day 9 to 18). Follicle development was evaluated on the basis of antral cavity formation, follicular and oocyte growth, and cumulus-oocyte complex health. The expression of FSHR in isolated caprine follicles increased from the preantral to antral phase. Regarding the culture, after 18 days, sequential medium 1 promoted follicular survival, antrum formation and a reduction in oocyte extrusion. Both sequential media promoted a higher rate of meiotic resumption compared with the other treatments. In conclusion, the addition of increased concentrations of FSH (sequential medium) has a significant impact on the in vitro development of caprine preantral follicles.

In vitro development of primordial follicles after long-term culture of goat ovarian tissue.

This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH+FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro.

Effect of the medium replacement interval on the viability, growth and in vitro maturation of isolated caprine and ovine pre-antral follicles.

The aim of the present study was to evaluate the effects of different medium replacement intervals on the viability, antral cavity formation, growth and in vitro maturation (IVM) of oocytes from caprine and ovine pre-antral follicles. Pre-antral ovarian follicles (≥ 150 µm) were isolated from the ovarian cortex of goats and sheep and were individually cultured for 24 days using two different medium replacement intervals [2 days (T(1) ) or 6 days (T(2) )]. Follicle development was evaluated on the basis of antral cavity formation, increases in follicular diameter and the presence of healthy cumulus oocyte complexes and fully grown oocytes. For caprine species, results showed a higher percentage (p<0.05) of viable follicles in T(1) than T(2) from day 6 until the end of the culture. In addition, when comparing both treatments after the same culture duration, the rate of antrum formation was significantly higher in T(1) than in T(2) from day 12 onwards. Yet, in ovines, when both treatments were compared on day 24 of the culture, there were more viable follicles in T(2) than in T(1) (p<0.05). In the caprine species, percentages of fully grown oocytes (≥ 110 µm) acceptable for IVM after 24 days of culture were significantly higher in normal follicles cultured in T(1) (30.0%) than in T(2) (6.7%; p<0.05). On the other hand, in ovines, at the end of the culture, the percentage of oocytes destined for IVM was higher in T(2) than in T(1) (23.5% vs 2.9%; p<0.05). In conclusion, under the same conditions, the frequency of medium replacement significantly affected the in vitro development of caprine and ovine pre-antral follicles. To improve the efficiency of the culture system, the medium must be replaced every 2 and 6 days for goat and sheep pre-antral follicles, respectively.

Dynamic medium produces caprine embryo from preantral follicles grown in vitro.

The aim of this study was to develop a dynamic culture medium containing FSH, LH and EGF to promote the in vitro development of oocytes obtained from goat preantral follicles to complete maturation and to improve the capacity of these oocytes for in vitro fertilization (IVF) and embryo production. For experiment I, preantral follicles were cultured for 18 days in medium supplemented with increasing concentrations of FSH (T1 - control) or in control medium added LH alone or in association with EGF: T2 (LH 50 ng/ml), T3 (LH 50 ng/ml + EGF 50 ng/ml), T4 (LH 50 ng/ml + EGF 100 ng/ml), T5 (LH 100 ng/ml), T6 (LH 100 ng/ml + EGF 50 ng/ml) and T7 (LH 100 ng/ml + EGF 100 ng/ml). For experiment II, preantral follicles were cultured only in the culture medium used in T7, and after 18 days, their oocytes underwent in vitro maturation (IVM) followed by IVF. At the end of the culture period, T3, T4 and T7 had a positive influence on the daily follicular growth rate. Oocytes grown in T4 and T7 had a meiosis resumption percentage significantly superior to the other treatments. Two embryos were obtained, in which preantral follicles in medium supplemented with 100 ng/ml LH and 100 ng/ml EGF (T7). In conclusion, our sequential culture system was able to promote the in vitro growth of preantral follicles, promoting their oocyte maturation and caprine embryo production from preantral follicles.

Vasoactive intestinal peptide improves the survival and development of caprine preantral follicles after in vitro tissue culture.

The aim of this study was to evaluate the effect of vasoactive intestinal peptide (VIP)on the survival, activation and growth of goat preantral follicles after in vitro culture. The ovarian cortex was divided into small pieces and one fragment was immediately fixed (control). The remaining fragments were cultured in vitro for 1 or 7 days at 39 degrees C and 5% CO(2), in supplemented minimum essential medium (MEM(+)) with or without different concentrations of VIP (1, 10, 50, 100 or 200 ng/ml). Noncultured (fresh control) and cultured ovarian fragments were processed for histological analysis and transmission electron microscopy. Follicles were classified as primordial or developing, and as normal or degenerated. Our findings indicate that when compared with control, addition of all concentrations of VIP except 200 ng/ml resulted in similar percentages of normal preantral follicles after 1 and 7 days of culture. Culture of ovarian cortex tissue for 1 and 7 days increased the percentage of follicular activation in all treatments when compared with control, except with 1 ng/ml of VIP after 1 day. However, no difference was observed between VIP-treated and MEM(+)-treated follicles. In addition, after 7 days of culture, the highest follicular and oocyte diameters were observed in follicles cultured with 10 ng/ml VIP relative to MEM(+) alone. Transmission electron microscopy showed ultrastructural integrity of follicles after 7 days of culture in 10 ng/ml VIP. In conclusion, this study demonstrates that VIP maintains follicular integrity and stimulates caprine preantral follicle growth.

Interaction between estradiol and follicle-stimulating hormone promotes in vitro survival and development of caprine preantral follicles.

The aim of this study was to investigate the effects of estradiol and follicle-stimulating hormone (FSH) on survival and growth of caprine preantral follicles. Pieces of ovarian tissue were cultured for 1 or 7 days in minimum essential medium (MEM) containing estradiol (1, 5, 10, 20 or 40 pg/ml), FSH (50 ng/ml), or a combination of the two hormones. Cultured and noncultured control ovarian tissues were processed for histological and ultrastructural studies. The results showed that after 7 days of culture, the treatments that yielded the highest percentage of normal follicles relative to MEM alone were those that combined FSH with estradiol at 1, 5 or 20 pg/ml. The addition of FSH to 1-day cultures containing 1 pg/ml estradiol or to 7-day cultures with 1 or 5 pg/ml estradiol increased the percentage of normal follicles compared to estradiol alone at the same concentrations. After 7 days of culture, all treatments generated higher percentages of developing follicles as compared to control and MEM alone. The addition of either FSH or 10 pg/ml of estradiol to the culture media or estradiol (1, 5, 10 or 20 pg/ml) and FSH in combination significantly increased follicular diameter as compared with MEM alone following 7 days of culture. Ultrastructural studies confirmed follicular integrity after 7 days of culture in the presence of 1 pg/ml estradiol plus FSH. In conclusion, this study demonstrated that the interaction between estradiol and FSH maintains ultrastructural integrity and stimulates activation and further growth of cultured caprine preantral follicles.

Expression of vascular endothelial growth factor (VEGF) receptor in goat ovaries and improvement of in vitro caprine preantral follicle survival and growth with VEGF.

The aim of the present study was to evaluate the effect of vascular endothelial growth factor (VEGF) on the survival and growth of goat preantral follicles after in vitro culture and to verify the expression of VEGF receptor (VEGFR)-2 in goat ovaries. Ovarian fragments were cultured for 1 or 7 days in minimal essential medium (MEM) with different concentrations of VEGF (1, 10, 50, 100 or 200 ng mL(-1)). Non-cultured (fresh control) and cultured tissues were processed for histological and ultrastructural studies. The results showed that 200 ng mL(-1) VEGF resulted in a similar percentage of normal preantral follicles after 1 and 7 days of culture compared with control. Compared with basic culture medium alone, an increase in follicular and oocyte diameters was observed in the presence of 10 ng mL(-1) VEGF after 7 days culture. Ultrastructural analysis confirmed follicular integrity after 7 days culture in the presence of 200 ng mL(-1) VEGF. Immunohistochemical studies demonstrated the expression of VEGFR-2 in oocytes and granulosa cells of all follicular stages, except in granulosa cells of primordial follicles. In conclusion, the present study has shown that VEGF maintains follicular ultrastructural integrity and promotes follicular growth. In addition, VEGFR-2 is expressed in oocytes of caprine ovarian follicles at all developmental stages and in granulosa cells of developing follicles.