Follicles In Group Research Articles

Survival and growth of isolated pre-antral follicles from human ovarian medulla tissue during long-term 3D culture.

Can human pre-antral follicles isolated enzymatically from surplus medulla tissue survive and grow in vitro during long-term 3D culture? Secondary human follicles can develop to small antral follicles and remain hormonally active in an alginate-encapsulation culture system for more than 30 days. Ovarian tissue cryopreservation followed by transplantation is a promising fertility preservation approach for cancer patients. However, transplantation of cryopreserved tissue to patients may carry the risk of re-implanting malignant cells. Grafting of follicles enzymatically isolated from ovarian tissue or developing a method for follicular culture and maturation in vitro may provide fertility to such patients without the risk of reintroducing the malignancy. However, the growth of pre-antral follicles isolated by enzymatic digestion from medulla tissue during long-term culture has received only little attention. Two to ten human pre-antral follicles were encapsulated together within an alginate bead and cultured with or without ovarian interstitial tissue for either 7 days or >30 days. Follicles were cultured in either 20% oxygen or 5% oxygen or encapsulated in a lower concentration of alginate together with a lower concentration of FSH in high oxygen. A total of 395 pre-antral follicles from 16 cancer patients, aged 9-37 years, were co-cultured for either 7 days or >30 days. A proportion of follicle (64) were removed from culture on Day 7 and assessed for viability using confocal fluorescence microscopy following calcein-AM and ethidium homodimer-1 staining or histology. The remaining follicles (331) were continued in culture for >30 days then assessed for survival and growth. Anti-Müllerian hormone (AMH) and estradiol levels were quantified in the medium. An optimized protocol for isolation of intact healthy pre-antral follicles from ovarian medulla was developed. After 7 days of culture, secondary follicles had a significantly higher survival rates compared with primary and primordial follicles (70 versus <38%). Primordial and primary follicles did not develop into the antral follicle stage. In contrast, secondary follicles continued to develop in all culture conditions examined. Based on growth rate and morphology, four distinct cohorts of surviving follicles, 'fast growth', 'slow growth', 'no growth' and 'extruded oocyte' were identified. From Day 1 to Day 30, the mean diameter of follicles increased from 184 ± 35 to 661 ± 120 μm (significant from Day 18), 145 ± 19 to 318 ± 68 μm and 136 ± 15 to 162 ± 25 μm (mean ± SD) in the 'fast growth', 'slow growth' and 'no growth' patterns, respectively. The fast growth follicles also contained a larger diameter oocyte than other follicle groups. From the pre-antral follicle to antral stage, follicles became steroidogenically active and secretion of AMH and estradiol increased. No significant difference between the follicles cultured with or without ovarian interstitial tissue was observed. The number of surviving follicles at the end of study was low in each of the culture conditions therefore whether there is a benefit with any of the conditions is difficult to ascertain. Multiple pre-antral follicles were cultured within the same alginate bead which may affect the in vitro development of the secondary follicles. These findings show that pre-antral follicles, isolated enzymatically from surplus medulla tissue that is normally discarded, possess a developmental potential which may be used to devise safer fertility preservation methods for patients who are at high risk of malignant contamination of their ovarian tissue. The Child Cancer Foundation in Denmark (2012-26) and the EU interregional project ReproHigh are thanked for having funded this study; and the Key Program of Medical Science and Technology Innovation of Nanjing Military Area Command in China (14ZX06; 11Z010). They had no role in the study design, collection and analysis of data, data interpretation or in writing the report. The authors have no conflicts of interest to disclose.

Changes in intrafollicular concentrations of free IGF-1, activin A, inhibin A, VEGF, estradiol, and prolactin before ovulation in mares

Changes in intrafollicular growth factors and hormones were evaluated in vivo in postdeviation and impending ovulation follicles. Mares (n = 30) were randomly assigned to five experimental groups based on target diameters of 25, 30, 35, 40 mm, and impending signs of ovulation. Furthermore, data belonging to two or more proximal diameter groups that were not different were combined and regrouped for each factor separately. Follicular fluid-free insulin-like growth factor 1 was highest (P < 0.003) in 35-mm follicles, followed by the 40-mm and impending ovulation follicle group, and the 25- to 30-mm follicle group. However, concentrations of insulin-like growth factor binding protein 2 in follicular fluid did not differ (P > 0.05) among groups. Additionally, follicular fluid activin A tended (P < 0.06) to be higher in impending ovulation follicles when compared with the 25- to 40-mm follicle group. Concentrations of intrafollicular estradiol were higher (P < 0.0001) in 40-mm and impending ovulation follicles than in the other follicle groups. Follicular fluid concentrations of inhibin A and vascular endothelial growth factor were lower (P < 0.05) in the 40-mm and the impending ovulation follicle group when compared with the 25- to 35-mm follicle group. Systemic and intrafollicular prolactin levels were lower (P < 0.05) in the impending ovulation group when compared with the 25- to 40-mm follicle group. Prolactin concentrations were higher (P < 0.05) in the follicular fluid than in the plasma. The novel findings of this study, a decrease in intrafollicular-free insulin-like growth factor 1, inhibin A, vascular endothelial growth factor, and prolactin during the final stages of follicular growth, document for the first time the occurrence of dynamic changes among intrafollicular factors and hormones during the stages of follicle dominance and as ovulation approaches.

Association between three glycosidases activity [±-mannosidase (±-oe'), ²--acetyloglucosaminidase (NAGASE) and ²-galactosidase (²-GAL)] andin vitro fertilization of bovine oocytes collected from different-sized follicles

We studied the role of three glycosidases (α- mannosidase - α-ΜΑΝ, β-N-acetyloglucosaminidase - NAGASE and β-galactosidase - β-GAL) in follicular fluid (FF) and in fertilization medium (FM) of bovine oocytes. Oocytes were allocated into 3 groups according to the follicular size (controls - CF: 2-8 mm, small follicle group - SF: 2-5 mm, large follicle group - LF: >5-8 mm). Bovine embryos were produced in vitro either in groups (experiment 1, n = 2099 oocytes) or individually (experiment 2, n = 79 oocytes). In both experiments, the activity of all glycosidases in the FF of large follicles was significantly lower than in the FF of small follicles group. In the FM of LF-group oocytes, α- MAN and NAGASE were significantly higher compared to SF- and CF-group oocytes (experiment 1) and β-GAL was significantly higher in SF- compared to CF-group oocytes (experiment 2). Cleavage rate was similar among all groups in both experiments; however significantly higher blastocyst formation was noted in CF-group compared to LF- (days 7, 8, 9) and SF- (days 8, 9) groups (experiment 1). In follicular fluid of small follicle group, β-GAL was associated positively with degenerating oocytes’ number and negatively with blastocyst rate at days 7, 8 (P = 0.065) and 9 (experiment 1). In fertilization medium of control group, α-MAN related negatively to cleavage rate (P < 0.05) and β-GAL to blastocyst rate at day 8 (P = 0.089) or day 9 (P = 0.072) (experiment 1). During fertilization, in experiment 1 all oocytes consumed β-GAL and only control or small follicle oocytes consumed α-MAN; in experiment 1, only large follicle oocytes released NAGASE, whereas all oocytes released all three glycosidases in experiment 2. In conclusion, glycosidases affect the developmental competence of oocytes collected from different sized follicles during in vitro fertilization, performed either in groups or individually; their role in follicular fluid is different from that in fertilization medium.

Establishment of an in vitro culture model of single human hair follicle

To establish an in vitro culture model of single human hair follicle, and observe their morphological and histological changes. Human hair follicles were isolated from the volunteer patients. After dissecting follicles into single, follicles in growth phase were cultured in Williams E without any serum. This experiment included 3 groups: single follicle without sebaceous gland and other surrounding tissue (control group); single follicle with sebaceous gland and without the other surrounding tissue( experiment group A); single follicles with sebaceous gland and the other surrounding tissue (experiment group B). The survival rate, survival time, growth rate, multiplication capacity and apoptosis of cultured follicles and their morphological and histological changes were observed sequentially. The hair follicles in experiment groups showed a better viability and a higher growth rate than those in control group. And the follicles in group B could keep growing for more than 25 days, which was longer than those in group A. Moreover, the sebaceous gland and the other surrounding tissue in group B showed great induction effect on follicle-cell proliferation and anti-apoptosis. The in vitro culture model of signal human hair follicles (single follicles including epidermis, sebaceous gland and the other surrounding tissue) had optimized internal environment which is similar to in vivo internal environment.

Improved fertility in suckled beef cows ovulating large follicles or supplemented with long-acting progesterone after timed-AI

We aimed to evaluate the effects and the interaction of size of the preovulatory follicle (POF) and long-acting progesterone (P4) supplementation after timed-AI on CL function and pregnancy success in beef cows. In experiment 1, ovulations of beef cows were synchronized starting on Day −10, and cows were split to receive sodium cloprostenol (large follicle group; LF; n = 31) or nothing (small follicle group; SF; n = 35). Ovulations were induced on Day 0, and cows were inseminated. Ovulated cows were assigned to receive placebo (LF/control group, n = 14; and SF/control group, n = 9) or 150 mg of long-acting P4 on Day 4.5 (LF/P4 group, n = 13; and SF/P4 group, n = 12). Diameter of POF, blood flow in POF wall, ovulation rate, and size and vascularization of CL were greater (P < 0.05) in LF group. In experiments 2 (unknown cyclic status) and 4 (noncycling), ovulations were synchronized, and beef cows received placebo or 150 mg of long-acting P4 on Day 4 after timed-artificial insemination. In experiment 2, pregnancy/AI (P/AI) did not differ (P > 0.1) between P4-treated (53.2%; 209/393) and control cows (56.2%; 219/390), but P/AI was greater in cows with a CL < 0.9 cm2 on Day 4 that were P4-treated (57.9%, 22/38) versus placebo-treated (40.4%, 21/52; P < 0.05). In Experiment 4, P/AI was greater (P < 0.05) in P4-treated cows (55.6%, 105/189 vs. 46.0%, 86/187). In Experiment 3, cyclic–suckled beef cows were treated as described in Experiment 1 to generate animals with small (SF; n = 111) or large POF (LF; n = 109), and subdivided to receive placebo or P4 on Day 4. POF size, ovulation rate, CL area, and P/AI were greater (P < 0.007) in the LF group. Pregnancy/AI in ovulated cows were lower (P = 0.05) in the SF/control group (41.5%, 17/41) compared to LF/control group (62%, 31/50) and were similar for the SF/P4 group (55.6%, 25/45) and LF/P4 group (57%, 28/49) compared to others. In summary, smaller and less vascularized POF results in less functional CL and reduces ovulatory rate and P/AI in cyclic beef cows; the long-acting P4 injection on Day 4 after timed-artificial insemination may attenuate the negative effects of small POF/CL; and postovulatory P4 supplementation improved fertility in anestrous beef cattle.

125 CHANGES IN INTRAFOLLICULAR CONCENTRATIONS OF FREE INSULIN-LIKE GROWTH FACTOR-1, ACTIVIN-A, INHIBIN-A, VASCULAR ENDOTHELIAL GROWTH FACTOR, ESTRADIOL, AND PRL BEFORE OVULATION IN MARES

Intrafollicular factors and hormones during the process of follicle deviation have been studied extensively in mares. However, knowledge is still lacking concerning the dominance and preovulatory (postdeviation) phases of the oestrous cycle. Furthermore, information about intrafollicular growth factors and hormones during postdeviation growth is critical for understanding the ovulatory and post-ovulatory physiological mechanisms that have important in vivo and in vitro applications, such as oocyte maturation, ovulation, and embryo development and survival. Changes in intrafollicular growth factors and hormones were evaluated in vivo in postdeviation and impending ovulation follicles. Hormone and growth factor concentrations were determined by ELISA or RIA. Mares (n = 30) were randomly assigned to 5 experimental groups based on target diameters of 25, 30, 35, 40 mm, and impending signs of ovulation. Data were analysed by one-way ANOVA, paired-test, and Pearson correlation. Data belonging to 2 or more proximal diameter groups that were not different were combined and regrouped for each factor separately. When the data were regrouped (all results shown below), follicular fluid-free insulin-like growth factor-1 was higher (P &lt; 0.003) in 35-mm follicles than in the 25- to 30-mm and 40-mm impending ovulation follicle groups. The 25- to 30-mm follicle group had the lowest insulin-like growth factor-1 concentration. However, concentrations of insulin-like growth factor binding protein-2 in follicular fluid did not differ (P &gt; 0.05) among groups. Additionally, follicular fluid activin-A tended (P &lt; 0.06) to be higher in impending ovulation follicles when compared to the 25- to 40-mm follicle group. Concentrations of intrafollicular oestradiol were higher (P &lt; 0.0001) in 40-mm impending ovulation follicles than in the other follicle groups. Follicular fluid concentrations of inhibin-A and vascular endothelial growth factor were lower (P &lt; 0.02–0.002) in the 40-mm impending ovulation follicle group when compared to the 25- to 35-mm follicle group. Systemic and intrafollicular PRL were lower (P &lt; 0.05) in the impending ovulation group when compared to the 25- to 40-mm follicle group. PRL concentrations were higher (P &lt; 0.05) in the follicular fluid than in the plasma. The novel findings of this study, a decrease in intrafollicular free insulin-like growth factor-1, inhibin-A, vascular endothelial growth factor, and PRL during the final stages of follicular growth, document for the first time the occurrence of dynamic changes among intrafollicular factors and hormones during the stages of follicle dominance and as ovulation approaches. Overall, the elucidation of the role of follicular fluid factors and hormones and their interactions need to be studied further to allow a better understanding of the ovulatory process in mares. This knowledge will potentially assist in obtaining better outcomes during the use of assisted reproductive technologies and will also help to understand mechanisms of ovulatory dysfunctions in mares and perhaps in other species.

126 EFFECTIVE METHOD FOR IN VITRO CULTURE OF CRYOPRESERVED OVINE OVARIAN TISSUE

Today, ovarian tissue cryopreservation is used for preserving the reproductive function of women, as well as the genetic material of rare and endangered species or domestic animals breeds. In the last 20 years genetic diversity of farm animals breeds suffered considerable losses in Kazakhstan; therefore, genetic preservation of valuable local breeds is desirable. The aim of this study was to compare the effectiveness of different in vitro culture media on morphology of ovine ovarian tissue cryopreserved by a slow-freezing protocol with 1.5 M dimethyl sulfoxide (DMSO). Ovaries were collected from indigenous Chuyi breed and immediately transported to the laboratory at 30°C within 1 h. Ovaries were rinsed several times in PBS supplemented with antibiotics (75 mg L–1 of penicillin-G, 50 mg L–1 of streptomycin sulfate). In Hepes-buffered medium 199, halved and the medulla removed with curved iris. Using a scalpel, the cortex was cut into 5- × 3- × 1-mm strips. Ovarian strips were equilibrated sequentially in freezing medium containing 0.25, 0.75, and 1.5 M DMSO with 0.5 M sucrose (5 min each). Then, ovarian strips were frozen in plastic straws using a programmable freezer Planer Kryo-360 3,3 (Planer, UK) and cooled as follows: stabilised at 20°C for 5 min, cooled from 20°C to –70°C at 5°C min–1, seeded to the temperature –7°C, cooled again to –30°C at 0.3°C min–1, cooled to –150°C at 35°C min–1, and finally plunged into liquid nitrogen and stored for 10 days. The straws were thawed at room temperature for 1 min, and then immersed in a water bath at 37°C for 2 min, warmed at room temperature with Dulbecco’s PBS (DPBS), supplemented with 10% FCS and 0.75 M sucrose (15 min), then DPBS + 10% FCS (30 min), and finally placed in the culture media for 10 min. Fresh and frozen tissue pieces were randomly distributed into 12 groups for further culture: 1) TCM 199 + 10% FBS; 2) TCM 199 + 10% native ovine serum (NOS); 3) TCM-Hepes + 10% FBS; 4) TCM-Hepes + 10% NOS; 5) DMEM + 10% FBS; 6) DMEM + 10% NOS; with and without 7.5 mg mL–1 of FSH. After 7 days of culture, the effects of different culture media on ovarian tissue morphology was evaluated by light microscopy after hematoxylin and eosin staining of tissue sections. The best result was observed when frozen ovarian tissue was cultured in the presence of FSH. The best result was observed in group 3 and 4 with FSH. The percentages of normal primordial, primary, and preantral follicles were: 1) TCM 199 + 10% FBS + FSH = 53.5 ± 3.1, 39.7 ± 3.8a, 28.5 ± 3.2; 2) TCM 199 + 10% NOS + FSH = 49.4 ± 2.3a, 36.7 ± 3.3a, 25.3 ± 4.1; 3) TCM-Hepes + 10% FBS + FSH = 66.3 ± 2.5, 45.7 ± 3.9, 35.1 ± 3.8; 4) TCM-Hepes + 10% NOS + FSH = 86.5 ± 3.8b, 75.4 ± 4.2b, 45.7 ± 3.5; 5) DMEM + 10% FBS + FSH = 42.1 ± 3.5a, 33.7 ± 2.9a, 21.3 ± 4.9, 20.7 ± 3.9; 6) DMEM + 10% NOS + FSH = 41.3 ± 3.9a, 32.9 ± 2.5a; control group = 98.2 ± 1.1, 93.7 ± 1.7, 90.3 ± 1.9 (ab, P &lt; 0.01). The majority of follicles in groups without FSH were degenerated. In group 4) TCM-Hepes + 10% NOS without FSH, a damaged structure of primordial follicles was observed.

Histological and immunohistochemical study of the ovaries and oviducts of the juvenile female of Caiman latirostris (Crocodilia: Alligatoridae)

The purpose of this study was to examine the ovaries and the oviduct of juvenile females of the broad-snouted caiman, Caiman latirostris (Daudin, 1802), using light microscopy, histochemical and immunocytochemical techniques. The ovarian cortex was observed to contain groups of germinative cells and abundant previtellogenic follicles at different stages of development. In previtellogenic follicles, the oocyte was surrounded by a single cuboidal layer of granulosa cells, supported by the theca layer. The theca was formed by a concentric layer containing collage nous fibers, predominantly type I, and several smooth muscle fibers. The inner perivitelline layer appeared as a narrow basophilic region between the oocyte and the granulosa. The ovarian medullary region of loose connective tissue contained blood and lymphatic vessels, as well as numerous lacunae, being covered by a simple squamous epithelium, supported by a thin layer of connective tissue. The oviduct of C. latirostris showed five histologically distinct regions: infundibulum, magnum, isthmus, uterus and vagina. The infundibulum was composed of an irregularly folded mucosa covered by a simple columnar epithelium with the presence of ciliated cells, weakly reactive to PAS staining. In the magnum, the mucosa was highly folded, and pseudostratified columnar epithelium contains mucous cells, which reacted positively to PAS staining. The isthmus was lining by an epithelium of ciliated and non-ciliated secretory cells, but no gland was visualized in mucosa. In the uterus, the folded mucosa was composed of a simple epithelium of high cylindrical cells and the lamina propria, which was predominantly formed by strongly birefringent fibers, but yellow-green weakly birefringent fibers were also present. The epithelium of the vagina contained intensely ciliated and non-ciliated cells, both of which were positive to PAS staining and no gland was observed in this region. Although the ovary and the oviduct showed some morphological particularities, they are similar to other crocodilians.

Re-evaluation of Tellervotrema katadara (Kuramochi, 2001) Kuramochi, 2009 (Opecoelidae: Plagioporinae) and supplementary morphological data for T. beringi (Mamaev, 1965) Gibson & Bray, 1982 with new host and locality.

The trematode genus Tellervotrema Gibson & Bray, 1982 was erected for Podocotyle-like species that parasitize archybenthal macrourid fishes (also known as grenadiers or rattails) and that possess no vitelline follicles dorsal to the ceca but do have a symmetrical pair of isolated groups of vitelline follicles in the posterior forebody. Tellervotrema katadara (Kuramochi, 2001) Kuramochi, 2009 is resurrected as a valid species based on an examination and re-description of holotype and paratype specimens collected from the intestine of the bathygadine macrourid Gadomus colletti Jordan & Gilbert from 518-582 m depth in Tosa Bay, off the Pacific coast of southern Japan. Tellervotrema beringi (Mamaev, 1965) Gibson & Bray, 1982 is re-described from specimens originally identified as T. katadara, collected from the intestine of the longfin grenadier, Coryphaenoides longifilis Günther, and found at 1,196 m depth off the Pacific coast of the Tōhoku region, northern Honshu, Japan. New host and locality records for T. beringi are presented along with a brief listing of museums housing type and voucher specimens of the three species now recognized in Tellervotrema. A comprehensive listing is given of all parasites reported from the two macrourid species relevant to this study and a key is presented for members of Tellervotrema. Finally, we hypothesize that the life cycles for T. beringi and T. katadara in the deep waters of the North Pacific Ocean off Japan most likely include a gastropod as a first intermediate host, one or more of a variety of invertebrates (amphipods, decapods, mysids) and/or finfish as second intermediate hosts, and the grenadiers, C. longifilis and G. colletti, as definitive hosts, respectively.

The effects of human recombinant granulocyte-colony stimulating factor treatment during in vitro maturation of porcine oocyte on subsequent embryonic development

Granulocyte colony-stimulating factor (G-CSF) is required for proliferation, differentiation, and survival of cells. It is also a biomarker of human oocyte developmental competence for embryo implantation. In humans, the G-CSF concentration peaks during the ovulatory phase of the ovarian cycle. In this study, the expressions of G-CSF and its receptor were analyzed by polymerase chain reaction in granulosa cells (GCs), CL, cumulus cells (CCs), and oocytes. Cumulus–oocyte complexes were aspirated from antral follicles of 1 to 3 mm (small follicles) and 4 to 6 mm (medium follicles). Cumulus–oocyte complexes from two kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of G-CSF (0, 10, and 100 ng/mL). By real-time polymerase chain reaction, the expressions of G-CSF and its receptor were detected in GCs, CL, CCs, and oocytes. Interestingly, the G-CSF transcript levels were significantly lower in oocytes than in the other cell types, whereas the G-CSF receptor transcript levels in oocytes were similar to those in GCs. After 44 hours of IVM, no differences in the rate of nuclear maturation were detected; however, the intracellular reactive oxygen species levels in oocytes from both groups of follicles matured with 10 ng/mL of human recombinant G-CSF (hrG-CSF) groups were significantly lower (P < 0.05). After parthenogenetic activation, the cleavage rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF–treated small (63.3%) follicles than in 0, 10 ng/mL hrG-CSF–treated small (38.6% and 49.0%, respectively) follicles and 0 ng/mL hrG-CSF–treated medium (52.1%) follicles, and the cleavage rates were significantly (P < 0.05) higher in 10 ng/mL hrG-CSF–treated medium (76.3%) follicles than in all other groups. The blastocyst formation rates were significantly (P < 0.05) higher in 100 ng/mL hrG-CSF–treated small (31.2%) follicles than in 0 and 10 ng/mL hrG-CSF small (10.4% and 15.6%, respectively) follicles, and the 10 ng/mL hrG-CSF medium (45.7%) follicle was significantly (P < 0.05) higher than in all other groups. The total cell number in blastocysts from the 10 ng/mL hrG-CSF medium (106.5) follicles was significantly (P < 0.05) increased compared to 0, 10, 100 ng/mL hrG-CSF small (55.0, 73.7 and 59.5, respectively) follicles and 0, 100 ng/mL hrG-CSF–treated medium (82.5 and 93.5, respectively) follicles. After IVF, the blastocysts stage was significantly (P < 0.05) increased in 10 ng/mL hrG-CSF–treated medium (36.4%) follicles. Fertilization efficiency was significantly high in 100 ng/mL of small (29.1%) and 10 ng/mL of medium (44.0%) follicles. We also examined the Bcl2 and ERK2 transcript levels and found that they were significantly higher in the small and medium follicle treatment groups. In conclusion, these results indicate that hrG-CSF improve the viability of porcine embryos.

In Vitro Mouse Ovarian Follicle Growth and Maturation in Alginate Hydrogel: Current State of the Art

This review describes the main factors affecting the in vitro development of mouse ovarian follicles under conditions of three-dimensional alginate hydrogel system. The factors discussed include concentration of alginate hydrogel, presence of additives (collagen, fibrin) influencing substrate rigidity; culture conditions; composition of culture media; substances that act like antioxidants (salts of ascorbic acid, glutathione) and contribute to the improvement of lipid metabolism (L-carnitine), hormones and growth factors. The methods for follicle group cultivation in alginate hydrogel and cocultivation of different cell populations with follicles encapsulated in alginate hydrogel are covered in the present article.

The effect of delaying intrauterine insemination till 48 h after hCG injection on pregnancy rate

Abstract Objective: Intrauterine insemination (IUI) is a simple procedure which has been used to treat unexplained infertility. There is no agreement about the optimum time for IUI. An interval of only a few hours exists in which it can be performed. The aim of this study was to find out whether there is a difference in pregnancy rate if IUI is performed 48 h rather than later than 36 h after Human chorionic gonadotropin (hCG) Injection. Setting: infertility clinic, Kasr Alainy, Cairo University Hospital. Materials and methods: A randomized case-control prospective study in which subjects in Group A will perform IUI 48 h after hCG injection after controlled ovarian stimulation, in comparison with Group B (control) in which the IUI will be performed 36 h after hCG injection. Main outcome measures: Pregnancy rate in group A was 22.6% while group B was 10.6% ( p = 0.049). The mean Number of mature follicles in group A was 2.6 ± 1 follicles, while that of group B was 2.1 ± 0.9 follicles ( p = 0.002). Conclusions: A lot of controversy surrounds the correct timing of IUI. Our study showed a statistically significant increase in pregnancy rate up to 22.6% in patients performing IUI 48 h after hCG trigger, compared to the 10.6% in the patients having the procedure done 12 h earlier.

Potential synergistic effects of human placental extract and minoxidil on hair growth-promoting activity in C57BL/6J mice.

Human placenta extract (HPE) has been used to alleviate tiredness and promote wound healing, and for its antiageing functions; however, it has not yet been studied for its effects on hair growth. In the present study, we evaluated the in vitro effect of HPE on hair growth by observing its actions on human dermal papilla cells (DPCs). To define how HPE promotes induction of anagen hair growth during the telogen phase, and to understand the synergistic molecular mechanisms of HPE and minoxidil (MXD) actions on hair growth. We examined the effects of HPE and MXD on C57BL6/J mice using haematoxylin and eosin staining, quantitative histomorphometry, hair growth scoring, immunohistochemistry and immunofluorescence on the dorsal skins of C57BL/6J mice. We found that HPE synergistically augmented the effects of MXD, a promoter of hair growth. In particular, histomorphometric analysis data indicated that subcutaneous injection of HPE induced an earlier anagen phase and prolonged the anagen phase. It also stimulated increases in both the number and size of hair follicles in groups treated with HPE alone and HPE+MXD. From our data, we conclude that HPE increases β-catenin and Wnt3a expression levels. Overall, our findings suggest that HPE in combination with MXD has hair growth-promoting activity and is a potential novel therapeutic treatment for alopecia or baldness in humans.

303 HUMAN RECOMBINATION GRANULOCYTE-COLONY STIMULATING FACTOR (HRG-CSF) HAVE BENEFICIAL EFFECTS ON PORCINE OOCYTES QUALITY DURING IN VITRO MATURATION AND SUBSEQUENT VIABILITY OF EMBRYONIC DEVELOPMENT

Granulocyte colony-stimulating factor (G-CSF), also known as colony-stimulating factor 3 (CSF3), is required for the proliferation, differentiation, and survival of cells. In humans, G-CSF is a biomarker of human oocyte developmental competence for embryo implantation. Furthermore, G-CSF concentration increases during the menstrual cycle and levels were significantly higher during ovulatory phase than the other phases. In this study, we examined G-CSF and its receptor gene expression in the porcine granulosa cells, corpus luteum, cumulus cells, and oocytes. The cumulus-oocyte complexes (COC) were aspirated from antral follicles 1 to 3 mm (small follicle) and 3 to 6 mm (medium follicle). The COC from 2 kinds of follicles were matured in protein-free maturation medium supplemented with various concentrations of hrG-CSF (0, 10, and 100 ng mL–1, respectively). Statistical analyses were done by one-way analysis of variance (ANOVA) followed by Duncan's multiple range tests. After real time-PCR was performed, the CSF3 and its receptor (CSF3R) were observed all of granulosa cells, corpus luteum, cumulus cells, and oocytes. Interestingly, the CSF3 transcript levels were significantly lower in oocytes compared with other cell types, but the CSF3R transcript levels in oocytes were almost similar with granulosa cells. After 44 h of IVM, the rates of nuclear maturation had no difference, and the intracellular ROS levels of oocytes from both kind of follicle groups matured with 10 ng mL–1 were significantly decreased compared to other groups (P &lt; 0.05). After PA, the cleavage and blastocyst formation rates were significantly (P &lt; 0.05) increased for the 100 ng mL–1 of small follicle (SF; 63.29 and 31.18%) group compared to control and 10 ng mL–1 of SF (38.64, 10.4, and 49.0, 15.6%, respectively) group, and significantly (P &lt; 0.05) increased in the 10 ng mL–1 of medium follicle (MF; 76.32 and 45.61%) group compared with control and 100 ng mL–1 of MF (52.1, 32.8 and 61.3, 33.9%, respectively). The total cell numbers of blastocyst from SF and MF groups were significantly increased in the 10 ng mL–1 (73.67 and 106.52) groups. At IVF, the blastocysts formation rates were significantly increased in the 10 ng mL–1 of MF group compared to control, 100 ng mL–1 of SF, and control of MF (21.1, 22.8, and 27.8%, respectively). We also examined the Bcl2 and ERK2 transcript levels, which were significantly increased at 100 ng mL–1 of SF and 10 ng mL–1 of MF. These results suggest that hrG-CSF improved the quality of porcine oocyte and embryonic viability.This was supported, in part, by a grant from the National Research Foundation of Korea Grant Government (NRF-2012R1A1A4A01004885, NRF-2013R1A2A2A04008751), Republic of Korea.

11 FERTILITY RESPONSE IN SUCKLED BEEF COWS SUPPLEMENTED WITH LONG-ACTING PROGESTERONE AFTER TIMED ARTIFICIAL INSEMINATION

Corpus luteum (CL) and progesterone (P4) secretion are affected by preovulatory follicle (POF) size. Increased circulating P4 during early diestrus has a positive effect on embryo development in beef cattle. However, the combined effects of the POF size and P4 supplementation during early diestrus on fertility of beef cows are not known. The objective was to evaluate the effects of POF size and supplementation of long-acting P4 after timed-AI on pregnancy rates (P/AI). Suckled Nelore cows (n = 596) were evaluated twice by transrectal Doppler ultrasonography (US) 10 days apart to detect the cyclic status. In Study 1, anestrous cows (absence of CL on both exams) received an intravaginal P4 device and an oestradiol benzoate (EB) injection on Day –10 (day of the second US). Devices were removed and sodium cloprostenol [prostaglandin F2α (PGF2α)], oestradiol cypionate, and eCG were given on Day –2. Cows were timed-AI on Day 0 and assigned to receive placebo (control group, n = 187) or 150 mg of long-acting P4 on Day 4 (P4 group, n = 189). In Study 2, cyclic cows (presence of CL) received a PGF2α injection on Day –20 (first US). Cows with a new CL on Day –10 received an intravaginal P4 device and an injection of EB and were split to receive an injection of PGF2α [large follicle (LF); n = 109] or not [small follicle (SF); n = 111]. Devices were removed and PGF2α was injected on Day –2. Ovulation was induced with buserelin acetate, and cows were timed-AI on Day 0 and split to receive placebo (LF/control group, n = 55, and SF/control group, n = 55) or 150 mg of long-acting P4 on Day 4 (LF/P4 group, n = 56, and SF/P4 group, n = 54). Ultrasonographic scanning was done on Days 0, 4, and between 35 and 40 to detect the POF and CL sizes and P/AI, respectively. Data were analysed using PROC GLIMMIX (SAS Institute Inc., Cary, NC). In anestrous cows, P/AI was reduced in POF with &lt;11 mm. The P/AI was greater in the P4-treated group than in the control group for all cows (55.6% v. 46.0%; P = 0.05) and for ovulated cows (59%, 105/178 v. 49%, 86/173; P = 0.08). For cyclic cows, POF size (mm) on Day 0 (13.5 ± 0.3 v. 11.2 ± 0.2), ovulation rate (90% v. 77%), and CL area (cm2) on Day 4 (1.46 ± 0.05 v. 1.25 ± 0.05) were greater (P &lt; 0.007) in the LF group than in the SF group. There was a main effect of follicle group on P/AI (54%, LF group v. 38%, SF group; P &lt; 0.01). Moreover, P/AI were greater (P &lt; 0.05) in the LF/control (56%) and LF/P4 (52%) groups than in the SF/control group (31%), whereas no difference was detected between the SF/P4 group (45%) and the other groups. Among cows that ovulated, P/AI was lower (P = 0.05) in the SF/control group (41%, 17/41) compared with the LF/control group (62%, 31/50) and were similar for the SF/P4 group (56%, 25/45) and LF/P4 group (57%, 28/49) compared with others. We suggest that P4-stimulated embryotrophic effects improved fertility in anestrous beef cows supplemented with long-acting P4 on Day 4 after timed-AI. Also, the presence of a functional CL during follicle growth results in smaller POF and CL and reduces the ovulatory and P/AI rates in cyclic cows. Post-AI P4 supplementation may attenuate the negative effects of small POF/CL. Research was supported by CNPq, FAPESP, Ouro Fino Agronegócio, and Innovare.

Intraoperative blood loss in female patients with adolescent idiopathic scoliosis during different phases of the menstrual cycle.

BackgroundThe vast majority of AIS patients who require surgical intervention are women. Blood loss is a major concern during the operation.MethodsThe medical records of all female AIS patients who underwent posterior correction and fusion operations using the all-pedicle screw system from January 2012 to January 2014 were reviewed. Patients with irregular menstruation; underwent osteotomy; use coagulants were excluded from the study. The remaining patients were divided into 4 groups according to the operation date in the menstrual cycle (A: premenstrual group, 24–30 d; B: follicle group, 6–11 d; C: ovulatory group, 12–17 d; D: luteal group, 18–23 d). The information of patients from the 4 groups was reviewed. The data was analyzed using analysis of variance, the Student-Newman-Keels test and Kruskal-Wallis Test.ResultsA total of 161 patients were included in this study. There were 40 patients included in group A, 38 patients in group B, 41 patients in group C and 42 patients in group D. The 4 groups were matched in age (P = 0.238), body height (P = 0.291), body weight (P = 0.756), Risser sign (P = 0.576), mean curve Cobb angle (P = 0.520), and bending flexibility index (P = 0.547), the number of levels fused (P = 0.397). The activated partial thromboplastin time (P = 0.235) and prothrombin time (P = 0.074) tended to be higher in group A, but the difference was not statistically significant. The fibrinogen level was lower in group B than the other 3 groups (P = 0.039). Blood loss and normalized intraoperative blood loss (NBL) was significantly higher in group A than the other 3 groups (P<0.01).ConclusionsThe hemostatic function tended to be lower in the premenstrual phase. The fibrinogen level was lowest in the mid-follicle phase. Female AIS patients tended to endure more intraoperative blood loss when the operation was performed in the premenstrual phase during the menstrual cycle.

The expression pattern of microRNAs in granulosa cells of subordinate and dominant follicles during the early luteal phase of the bovine estrous cycle.

This study aimed to investigate the miRNA expression patterns in granulosa cells of subordinate (SF) and dominant follicle (DF) during the early luteal phase of the bovine estrous cycle. For this, miRNA enriched total RNA isolated from granulosa cells of SF and DF obtained from heifers slaughtered at day 3 and day 7 of the estrous cycle was used for miRNAs deep sequencing. The results revealed that including 17 candidate novel miRNAs, several known miRNAs (n = 291–318) were detected in SF and DF at days 3 and 7 of the estrous cycle of which 244 miRNAs were common to all follicle groups. The let-7 families, bta-miR-10b, bta-miR-26a, bta-miR-99b and bta-miR-27b were among abundantly expressed miRNAs in both SF and DF at both days of the estrous cycle. Further analysis revealed that the expression patterns of 16 miRNAs including bta-miR-449a, bta-miR-449c and bta-miR-222 were differentially expressed between the granulosa cells of SF and DF at day 3 of the estrous cycle. However, at day 7 of the estrous cycle, 108 miRNAs including bta-miR-409a, bta-miR-383 and bta-miR-184 were differentially expressed between the two groups of granulosa cell revealing the presence of distinct miRNA expression profile changes between the two follicular stages at day 7 than day 3 of the estrous cycle. In addition, unlike the SF, marked temporal miRNA expression dynamics was observed in DF groups between day 3 and 7 of the estrous cycle. Target gene prediction and pathway analysis revealed that major signaling associated with follicular development including Wnt signaling, TGF-beta signaling, oocyte meiosis and GnRH signaling were affected by differentially expressed miRNAs. Thus, this study highlights the miRNA expression patterns of granulosa cells in subordinate and dominant follicles that could be associated with follicular recruitment, selection and dominance during the early luteal phase of the bovine estrous cycle.

The influence of nutrition on the insulin-like growth factor system and the concentrations of growth hormone, glucose, insulin, gonadotropins and progesterone in ovarian follicular fluid and plasma from adult female horses (Equus caballus).

BackgroundFeed intake affects the GH-IGF system and may be a key factor in determining the ovarian follicular growth rate. In fat mares, the plasma IGF-1 concentration is high with low GH and a quick follicular growth rate, in contrast to values observed in thin mares. Nothing is known regarding the long-term effects of differential feed intake on the IGF system. The objective of this experiment was to quantify IGFs, IGFBPs, GH, glucose, insulin, gonadotropin and progesterone (P4) in blood and in preovulatory follicular fluid (FF) in relation to feeding levels in mares.MethodsThree years prior to the experiment, Welsh Pony mares were assigned to a restricted diet group (R, n = 10) or a well-fed group (WF, n = 9). All mares were in good health and exhibited differences in body weight and subcutaneous fat thickness. Follicular development was scanned daily and plasma was also collected daily. Preovulatory FF was collected by ultrasound-guided follicular aspiration. Hormone levels were assayed in FF and plasma with a validated RIA.ResultsAccording to scans, the total number of follicles in group R was 53% lower than group WF. Insulin and IGF-1 concentrations were higher in WF than in R mares. GH and IGF-2 concentrations were lower in plasma from WF mares than from R mares, but the difference was not significant in FF. The IGFBP-2/IGFBP-3 ratio in FF was not affected by feeding but was dramatically increased in R mare plasma. No difference in gonadotropin concentration was found with the exception of FSH, which was higher in the plasma of R mares. On the day of puncture, P4 concentrations were not affected by feeding but were higher in preovulatory FF than in plasma.ConclusionsThe bioavailability of IGF-1 or IGF-2, represented by the IGFBP2/IGFBP3 ratio, is modified by feed intake in plasma but not in FF. These differences partially explain the variability in follicular growth observed between well-fed mares and mares on restricted diets.

EFECTO INHIBITORIO DE LA PROGESTERONA SOBRE EL DESARROLLO DE LA ONDA FOLICULAR EN LLAMAS.

The effect of the exogenous progesterone (P4) on the development of follicular waves in llamas was studied in twenty-five females. According to dose and regimen for P 4 administration, animals were allocated to 5 groups (n=5): 00 (control group ), G 1 (30 mg P /day for 2 days); G2 (30 mg P/day for 4 days); G3 (50 mg P /day for 2 days) and G4 (50 mg P /da y for 4 days ). Development of dominant ():. 7 mm) and subordinated follicles (&gt;3 mm and &lt; 7 mm) were monitored daily by transrectal ultrasonography. The minimun size reachedbythedorninantfollicleingroupsGl (4.6±0.9mm), G3(5.1±1.5 mm) andG4(5.3 ± 1.6 mm) were smaller (P&lt; 0.05) than group 00 (8.5 ± 1.0 mm). The whole follicular regression of the dominant follicle in groups G 1 (5.5 ± 2.0 mm), G2 (5.9 ± 1.4 mm) and G3 (5.3 ± 3. 3 mm) were larger (P&lt; 0.05) than group 00 (0.9 ± 1.4 mm). The number of subordinated follicles hada significative increase (P&lt; 0.05) in groups G 1 and G4, on days 2 and 3 in relation to days 7 and 8 post-treatment. Results demonstrate that exogenous progesterone inhibits the development of the dominant and subordinate follicles in lla-mas during treatment days; however, immediately after the end of the treatment, the · number of subordinate follicles increases, due to the regression of the dominant follicle in order to allow the emergence of a new follicular wave.

Control of ovulation after prostaglandin treatment by means of ultrasonography and effect of the time of ovulation on conception rate in dairy cows

Primiparous and multiparous lactating crossbred dairy cows (after Day 40 postpartum) with a mature corpus luteum (CL) (diameter of ≥ 17 mm determined by ultrasonography) and having a follicle with a diameter of ≥ 10 mm were treated with natural prostaglandin F2α (n = 80). Those from the cows that showed oestrus within 5 days after treatment were inseminated (Group 1: n = 39). Other group of cows showing oestrus without treatment (Group 2: n = 41) were inseminated and served as controls. The ovaries of each cow were scanned by transrectal ultrasonography from the day of detected oestrus (Day -1p.m.) until ovulation, to measure the changes in the areas of the CL and the largest follicle and to determine the occurrence of ovulation. Although no significant differences were found between the treated and untreated cows in terms of a reduction in the area of the corpora lutea and of an increase in the area of the dominant follicles, the mean area of these follicles in Group 2 was somewhat greater than in Group 1. The highest conception rate was achieved if AI was performed at the same day as ovulation occurred in both groups (conception rate in treated group was: 62.5%, in untreated group: 66.6%, respectively) between Day 0a.m. to Day 0p.m.. In Group 1, 54.5% conception rate has been achieved if ovulation occurred between Day 0p.m. to Day 1p.m., or 50% between Day 1p.m. to Day 2p.m. after AI, and 53.3% and 44.4% in Group 2, respectively. The conception rate for cows that ovulated before AI in Group 2 was 25%. No ovulation occurred in 7 cows until Day 2p.m. after AI and none of them became pregnant. The overall conception rate was approximately 50% in both groups, but when the cows had ovulated too early or too late relative to the time of AI, the conception rate was significantly lower, thus determination of the optimal time for AI is of great practical importance in dairy herds.