Evaluation of an alternative heterotopic transplantation model for ovarian tissue to test pharmaceuticals improvements for fertility restoration.

BACKGROUND: Ovarian tissue cryopreservation and transplantation (OTCTP) is currently the main option available to preserve fertility in prepubertal patients undergoing aggressive cancer therapy treatments. However, a major limitation of OTCTP is follicle loss after transplantation. The mouse is a model of choice for studying ovarian function and follicle development after ovarian tissue grafting in vivo. In these mouse models, ovarian tissue or ovaries can be transplanted to different sites. Our aim was to evaluate a new alternative to heterotopic transplantation models that could be useful to test pharmaceutical improvement for ovarian grafts after OTCTP. METHODS: Slow frozen murine whole ovaries were transplanted into the mouse ears (between the external ear skin layer and the cartilage). Ovarian transplants were recovered after 3, 14 or 21 days. Grafts were analyzed by immunohistochemistry and follicle density analyses were performed. RESULTS: An increase of ovarian vascularization (CD31 and Dextran-FITC positive staining), as well as cellular proliferation (Ki67 staining) were observed 3 weeks after transplantation in comparison to 3 days. Fibrosis density, evaluated after Van Gieson staining, decreased 3 weeks after transplantation. Furthermore, transplantation of cryopreserved ovaries into ovariectomized mice favored follicle activation compared to transplantation into non-ovariectomized mice. CONCLUSION: The present study indicates that surgical tissue insertion in the highly vascularized murine ear is an effective model for ovarian grafting. This model could be helpful in research to test pharmaceutical strategies to improve the function and survival of cryopreserved and transplanted ovarian tissue.

Transcriptome analysis of eutopic endometrium in adenomyosis after GnRH agonist treatment.

BACKGROUND: Adenomyosis is a chronic gynecological disease characterized by invasion of the uterine endometrium into the muscle layer. In assisted reproductive technology (ART), gonadotropin-releasing hormone agonist (GnRHa) is often used to improve pregnancy rates in patients with adenomyosis, but the underlying mechanisms are poorly understood. METHODS: Eutopic endometrial specimens were collected from patients with adenomyosis before and after GnRHa treatment in the midsecretory phase. RNA sequencing (RNA-Seq) of these specimens was performed for transcriptome analysis. The differentially expressed genes (DEGs) of interest were confirmed by real-time PCR and immunohistochemistry. RESULTS: A total of 132 DEGs were identified in the endometrium of patients with adenomyosis after GnRHa treatment compared with the control group. Bioinformatics analysis predicted that immune system-associated signal transduction changed significantly after GnRHa treatment. Chemokine (C-C motif) ligand 21 (CCL21) was found to be highly expressed in the eutopic endometrium after GnRHa treatment, which may be involved in the improvement of endometrial receptivity in adenomyosis. CONCLUSION: This study suggests that molecular regulation related to immune system-associated signal transduction is an important mechanism of GnRHa treatment in adenomyosis. Immunoreactive CCL21 is thought to regulate inflammatory events and participate in endometrial receptivity in adenomyosis.

The influence of ethnicity on outcomes of ovulation induction with clomifene citrate in women with PCOS.

RESEARCH QUESTION: What is the influence of ethnicity on the outcome of ovulation induction with clomifene citrate in women with polycystic ovary syndrome (PCOS)? DESIGN: This was a retrospective cohort study. In total, 420 women diagnosed with PCOS who were of Northern European, Mediterranean, African, South-East Asian or South American descent, and who started ovulation induction treatment with clomifene citrate, were included. All women were treated with clomifene citrate according to a standardized treatment regimen. The minimal effective dose of clomifene citrate and prevalence of clomifene resistance (CRA) were assessed, and the chance of becoming ovulatory was predicted. RESULTS: Differences were observed in body mass index (P = 0.008), waist circumference (P = 0.036) and serum LH, insulin and androgen concentrations (all P < 0.001) in women of different ethnicities with PCOS. Compared with women of Northern European descent, the minimal effective dose of clomifene citrate in women of other ethnic groups was not significantly different. The prevalence of CRA (P = 0.574) was similar in all ethnic groups A similar chance of ovulation (P = 0.504) was predicted for the different ethnic groups. CONCLUSIONS: This is the first study aiming to link ethnicity to ovulation induction outcome in PCOS. Although women of different ethnicities who have PCOS exhibit a variation in phenotypic expression, there do not appear to be differences in the prevalence of clomifene-resistant anovulation or the minimal effective dose of clomifene citrate. Furthermore, a prediction model revealed no significant differences in the predicted chance of ovulation. A larger cohort is needed to validate these findings.

Metformin, clomiphene citrate and flutamide effects on oocyte ultrastructure status and quality in PCOS mouse model.

RESEARCH QUESTION: Polycystic ovary syndrome (PCOS) alters oocyte developmental competence and so the treatment of PCOS patients with assisted reproductive techniques is a major challenge for an infertility specialist. Does ovulation induction therapy with clomiphene citrate, metformin and flutamide affect abnormal PCOS follicle gene expression, its quality, and nuclear and cytoplasmic maturation in the oocyte matured in vitro? DESIGN: Fifty NMRI mice (7-8 weeks old) were randomly divided into five groups, including non-PCOS, PCOS and PCOS groups treated with clomiphene citrate (18 mg/kg body weight for 2 days), metformin (50 mg/100 g body weight for 30 days) and flutamide (10 mg/kg body weight injection for 15 days). The oocytes were collected for quantitative real-time polymerase chain reaction analysis (the evaluation of genes associated with oocyte maturation), transmission electron microscopy (the evaluation of cytoplasmic maturation) and in-vitro maturation (IVM) and IVF outcomes. RESULTS: The dysregulated expression of some genes of insulin-like growth factor (IGF) families involved in oocyte competence and steroid metabolism (e.g. Cyp19a1 and Cyp11a1), transforming growth factor beta (TGF-ß) family (e.g. Tgfb1, Gdf9, Bmp15, Inhbb and Bmpr1a), and oestrogen receptor signalling (e.g. Ncoa3, Pgr) was, to some extent, restored in the PCOS follicles following their with clomiphene citrate, metformin and flutamide. The improved cytoplasmic maturation was observed particularly in the PCOS mice treated with clomiphene citrate. The better IVM-IVF outcomes were also observed in the clomiphene citrate and metformin groups compared with the PCOS group. CONCLUSIONS: This study opens up a new perspective on knowing the effect of ovulation induction therapy on not only nuclear maturation but also ultrastructural characteristics, IVM-IVF outcomes and PCOS oocyte developmental competence.

The regulatory effects of clomiphene and tamoxifen on mTOR and LC3-II expressions in relation to autophagy in experimental polycystic ovary syndrome (PCOS).

BACKGROUND: Polycystic ovary syndrome (PCOS) is a metabolic disease that causes infertility due to anovulation in women in reproductive age. It is known that clomiphene citrate (CC) and tamoxifen citrate (TMX) induce ovulation in women with PCOS. In this study, we aimed to investigate the effects of CC and TMX on the autophagy pathway in PCOS. METHODS AND RESULTS: Experimental PCOS model was induced by letrozole (1 mg/kg) in rats by gavage for 21 days. After the last letrozole administration, rats were treated TMX (1 mg/kg) or CC (1 mg/kg) for 5 days. At the end of the experimental procedures, rats in all groups were sacrificed and ovarian tissues were removed. It was observed that mRNA and protein expressions of LC3-II were significantly higher in TMX and CC groups than control and PCOS groups (p < 0.05), while mRNA and protein expressions of mTOR in TMX and CC groups were found significantly lower than control and PCOS groups (p < 0.05). CONCLUSIONS: In conclusion, present study suggests that TMX and CC induce autophagy in ovaries with PCOS. Autophagy is a promising target for understanding pathophysiology of this disease and for developing more effective and safe new protocols for the treatment of PCOS-related anovulation.

Ovarian stimulation and exogenous progesterone affect the endometrial miR-16-5p, VEGF protein expression, and angiogenesis.

Angiogenesis, where vascular endothelial growth factor (VEGF) is critically involved, is an important factor in endometrial receptivity. Angio-miRNAs form a special class of microRNAs (miRNAs) that target angiogenic genes and regulate angiogenesis. Various studies have shown that ovarian stimulation and exogenous progesterone affect endometrial vascular density. The present research aimed to assess the impact of HMG/HCG and progesterone on miR-16-5p, VEGF protein expression, and angiogenesis in the mouse endometrium during the preimplantation period. Forty adult female mice were divided into four groups: 1) control, 2) ovarian stimulation (HMG and 48 h after HCG IP), 3) progesterone (progesterone IP for 3 days), 4) ovarian stimulation + progesterone (HMG and 48 h after HCG IP) + (progesterone IP for 3 days) groups.The mice were sacrificed 96 h following HCG administration. miR-16-5p, VEGF protein expression, and CD31-positive cell (Endothelial cell) density were specified.The results showed that endothelial cell density,VEGF protein, and miR-16-5p expression increased in all treatment groups, with the maximum increase belonging to the ovarian stimulation + progesterone group. This study provides evidence that ovarian stimulation and progesterone administration enhance endometrial angiogenesis through VEGF protein upregulation. Furthermore, except for miR-16-5p, other miRNAs and molecules appear to be involved in angiogenic pathways, thereby requiring further studies.

Endometrial preparation for vitrified-warmed embryo transfer with or without GnRH-agonist pre-treatment in patients with polycystic ovary syndrome: a randomized controlled trial.

RESEARCH QUESTION: What are the effects on pregnancy outcome in patients with polycystic ovary syndrome (PCOS) in endometrial preparation cycles for vitrified-warmed embryo transfer with or without gonadotrophin releasing hormone (GnRH) agonist pre-treatment? DESIGN: A total of 212 patients with PCOS referred to Royan Institute, Tehran, Iran, between 20 August 2017 to 20 June 2018 were included. The patients were randomly assigned to receive oestradiol after downregulation with GnRH agonist (group A) or without GnRH agonist down-regulation (group B). RESULTS: A total of 188 patients with PCOS completed the trial, 93 patients in group A and 95 patients in group B. Basal oestradiol and LH levels were significantly higher in group B (26.66 versus 41.61, P = 0.01 and 0.93 versus 5.33, P < 0.0001, respectively). Clinical pregnancy rates were not significantly different in both groups (31.2% versus 33.7%). Similarly, no significant differences were found between groups A and B in miscarriage (9.7% versus 11.6%), implantation (0.58 versus 0.51) and live birth (21.7% versus 22.1%) rates and for medical complications during pregnancy and neonatal anomalies. CONCLUSIONS: Our findings indicate that endometrial preparation for frozen-thawed embryo transfer with and without ovarian suppression by GnRH agonist provides similar results.

A single ovarian stimulation, as performed in assisted reproductive technologies, can modulate the anxiety-like behavior and neuronal activation in stress-related brain areas in rats.

Infertility affects about 8 to 12% of couples of childbearing age around the world, and is recognized as a global public health issue by the WHO. From a psychosocial perspective, infertile individuals experience intense psychological distress, related to emotional disorders, which have repercussions on marital and social relationships. The symptoms persist even after seeking specialized treatment, such as assisted reproductive technologies (ART). While the stress impact of ART outcome has been comprehensively studied, the role of supraphysiological concentrations of gonadal hormones on stress response, remains to be elucidated. This study aimed to evaluate the effect of a single ovarian stimulation on the stress response in rats. To mimic the context of ART in rodents, female rats were submitted to the superovulation (150 UI/kg of PMSG and 75 UI/kg of hCG) and then to psychogenic stress (restraint stress for 30 min/day, repeated for three days). Anxiety-like behavior was evaluated in the elevated plus-maze, and neuronal activation in the stress-related brain areas assessed by Fos protein immunoreactivity. Corticosterone, estradiol, progesterone and corpora lutea were quantified. Data were analyzed using Generalized Linear Model (GzLM). Our findings indicate anxiolytic-like and protective effects of supraphysiological concentrations of gonadal hormones induced by a single ovarian stimulation on stress response. An activation of hypothalamus-pituitary-adrenal response inhibitory pathways, with participation of the prefrontal cortex, basomedial amygdala, lateral septum, medial preoptic area, dorsomedial and paraventricular hypothalamus, was detected.

Morphometric, subcellular, in vitro fertilisation and embryonic developmental assessment of mouse oocytes produced by anti-inhibin serum or pregnant mare serum gonadotrophin superovulation.

This study compared the morphometric, subcellular characteristics, in vitro fertilisation (IVF) and embryonic developmental potential of metaphase II (MII) mouse oocytes obtained from females superovulated with either anti-inhibin serum-human chorionic gonadotrophin (AIS-hCG) or pregnant mare serum gonadotrophin (PMSG)-hCG. The oocyte's quantity, quality, zona pellucida (ZP) thickness, perivitelline space (PVS), diameter, microtubules, F-actin, cortical granules (CGs) and mitochondrial distribution were determined. Superovulation using AIS-hCG resulted in a higher numbers of oocyte/donor compared with PMSG-hCG (P=0.002). There was no difference in morphologically normal and abnormal oocytes between AIS-hCG and PMSG-hCG (P=0.425 and P=0.194, respectively). The morphometric measurements showed no difference in oocyte diameter between AIS-hCG and PMSG-hCG (P=0.289). However, the thickness of the ZP of oocytes from AIS-hCG females was decreased compared with PMSG-hCG (P<0.001). The PVS of oocytes from the AIS-hCG was larger than with PMSG-hCG (P<0.001). The microtubules of oocytes from both AIS-hCG and PMSG-hCG were normal, although there was an increased fluorescence intensity in the AIS-hCG oocytes (P<0.001). The F-actin and CGs distribution in oocytes from both AIS-hCG and PMSG-hCG were similar (P=0.330 and P=0.13, respectively). Although the oocytes from PMSG-hCG females had homogenously distributed mitochondria, AIS-hCG oocytes showed more peripheral distribution with no differences in fluorescence intensity (P=0.137). The blastocyst development rates after IVF with fresh sperm showed no difference between AIS-hCG and PMSG-hCG (P=0.235). These data suggested that AIS-hCG superovulation produces high numbers of morphologically normal oocytes that also possess normal subcellular structures, good morphological characteristics and had high invitro embryonic developmental potential.

Gonadotrophin-releasing hormone agonist triggering may improve central oocyte granularity and embryo quality.

This study aimed to describe outcomes in four women aged 28-34 years with central cytoplasmic granulation (CCG) of the oocytes who underwent in vitro fertilization/intracytoplasmic sperm injection (ICSI) using gonadotrophin-releasing hormone (GnRH) agonist to replace human chorionic gonadotrophin (hCG) as a trigger of final oocyte maturation. The initial ICSI procedure showed that all four women had CCG of the ooplasm and poor quality embryos. Subsequent ICSI used an antagonist protocol with a GnRH agonist trigger replacing the agonist protocol, plus hCG triggered ovulation. Ooplasm and embryo quality were improved in all four patients. All four became pregnant and gave birth to live infants. This study provides GnRH agonist triggering that may improve ooplasm granularity and embryo quality.

Single injection of triptorelin or buserelin acetate in saline solution induces ovulation in mares the same as a single injection of hCG.

The aim of this study was to assess the efficacy of different doses of buserelin acetate and another GnRH agonist, triptorelin acetate, in saline solution in a single subcutaneous injection, to induce ovulation of growing pre-ovulatory follicle in mare and compare it with the classical treatment of a single injection of hCG. The study is split into 3 experiments over different breeding seasons in the same stud with a random distribution of treatment. The first one was to compare the injection of 6 mg of buserelin with 1,500 IU of hCG; the second one consisted of comparing different doses of buserelin (6 mg and 3 mg); and the third one compared three different doses of buserelin (3, 2 and 1 mg), 0.1 mg of triptorelin with 1,500 IU of hCG as a control group. The results of all experiments showed the same efficacy between all treatments with mares ovulating between 24 and 48 hr after injection: experiment 1: hCG (78% n = 41) and buserelin 6 mg (90% n = 50); experiment 2: buserelin 6 mg (78,1% n = 192) and buserelin 3 mg (78% n = 341); and experiment 3: hCG (87% n = 106), buserelin 3 mg (84,7% n = 137), buserelin 2 mg (82,7% n = 104), buserelin 1 mg (87% n = 54) and triptorelin 0.1 mg (84,7% n = 72). In conclusion, this study contributes to erasing the dogma that has been established since 1975 that a single injection in solution without any long-acting excipient of a GnRH agonist cannot induce ovulation in the mare. This study also shows that a injection of 0.1 mg of triptorelin in solution is a good alternative for ovulation induction and is comparable to small doses of buserelin acetate in solution (1 mg) and 1,500 IU of the gold standard trigger hCG, mainly in countries where human formulation of buserelin is not available.

Effects of letrozole and clomiphene citrate on Wnt signaling pathway in endometrium of polycystic ovarian syndrome and healthy women†.

Polycystic ovary syndrome (PCOS) is an endocrine disorder in women of reproductive age. In addition to anovulation, endometrial dysfunction can reduce fertility in PCOS. The cyclical changes of endometrium are controlled by estrogen and progesterone via modulating the Wnt/B-catenin pathway. Clomiphene citrate (CC) and letrozole are used to induce ovulation; unlike letrozole, there is a discrepancy between ovulation and pregnancy rates in CC-treated cycles. Because of the anti-estrogenic effects of CC on endometrium, we compared the expression of the key molecules of the Wnt/B-catenin pathway in the endometrium of women taking CC and letrozole. This study included PCOS and healthy women divided into the groups stimulated with letrozole (5 mg) or CC (100 mg) as well as NO-treatment groups. The endometrial thickness and hormonal profile were measured on day 12 of the menses. Using real-time polymerase chain reaction and western blot, we evaluated mRNA and protein expression of B-catenin, glycogen synthase kinase 3 beta (GSK3B), dickkopf Wnt signaling pathway inhibitor 1 (DKK1), and estrogen receptor 1 (ESR1) in the endometrial samples. Significantly, the mean serum estrogen and progesterone were lower and higher, respectively, in letrozole than CC groups. The endometrial thickness was significantly reduced in CC. The proteins expression of active B-catenin, inactive GSK3B, and ESR1 were significantly decreased in CC-treated groups. The mRNA and protein assessment of DKK1 showed significantly higher expression in CC. Our results indicate that letrozole can provide an acceptable activation of the Wnt/B-catenin pathway, resulting in adequate proliferation of endometrium in the women receiving letrozole compared to CC.

An estrogen antagonist, cyclofenil, has anti-dengue-virus activity.

Dengue virus (DENV) infections are a major cause of morbidity and mortality in tropical and subtropical areas. Several compounds that act against DENV have been studied in clinical trials to date; however, there have been no compounds identified that are effective in reducing the severity of the clinical manifestations. To explore anti-DENV drugs, we examined small molecules that interact with DENV NS1 and inhibit DENV replication. Cyclofenil, which is a selective estrogen receptor modulator (SERM) and has been used clinically as an ovulation-inducing drug, showed an inhibitory effect on DENV replication in mammalian cells but not in mosquito cells. Other SERMs also inhibited DENV replication in mammalian cells, but cyclofenil showed the weakest cytotoxicity among these SERMs. Cyclofenil also inhibited the replication of Zika virus. A time-of-addition assay suggested that cyclofenil may interfere with two stages of the DENV life cycle: the translation-RNA synthesis and assembly-maturation stages. However, the level of intracellular infectious particles decreased more drastically after treatment with cyclofenil than the viral RNA level did, indicating that the assembly-maturation stage might be the main target of cyclofenil. In electron microscopy analysis, many aggregated particles were detected in DENV-infected cells in the presence of cyclofenil, supporting the possibility that cyclofenil impedes the process of assembly and maturation of DENV.

Ulmus minor bark hydro-alcoholic extract ameliorates histological parameters and testosterone level in an experimental model of PCOS rats.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is a common and multifactorial disease associated with female factor infertility. Ulmus minor bark (UMB) is one of the medicinal plants used in Persian folklore as a fertility enhancer. In the current study, we aimed to elucidate the effect of UMB hydro-alcoholic extract on histological parameters and testosterone condition in an experimental model of PCOS rats. METHODS: Thirty female rats were randomly divided into five groups: (1) control, (2) vehicle, (3) PCOS/50 mg [6 mg/kg dehydroepiandrosterone (DHEA) + 50 mg/kg UMB hydro-alcoholic extract], (4) PCOS/150 mg (6 mg/kg DHEA + 150 mg/kg UMB hydro-alcoholic extract), and (5) PCOS (6 mg/kg DHEA). All interventions were performed for 21 days. Afterwards, stereological analysis was done for determination of ovarian volume and follicle number. The serum level of testosterone was measured by ELISA kit. RESULTS: UMB hydro-alcoholic extract improved the total number of the corpus luteum in the treatment groups when compared to the PCOS group (p<0.05). PCOS/150 mg and PCOS/50 mg groups showed significantly lower total number of the primordial, primary, and secondary follicles as well as testosterone level compared to the PCOS group (p<0.05). The total number of antral follicles and volume of ovary did not differ significantly between groups. CONCLUSION: UMB extract may be an effective and good alternative in improving PCOS histo-logical and testosterone disturbances although further studies are warranted to confirm the safety of UMB plant in human.

Effects of metabolic abnormalities, hyperandrogenemia and clomiphene on liver function parameters among Chinese women with polycystic ovary syndrome: results from a randomized controlled trial.

PURPOSE: To investigate the effects of metabolic abnormalities, hyperandrogenemia and ovulation induction by clomiphene/acupuncture on liver function parameters among women with polycystic ovary syndrome (PCOS). METHODS: This is a secondary analysis of a randomized controlled trial. All 1000 subjects were diagnosed as PCOS by modified Rotterdam criteria. Liver function parameters, metabolic panel and hormone profile were measured at baseline and after treatment. The relationship between liver parameters with metabolic, hormonal parameters and ovulation induction was examined. RESULTS: PCOS women with metabolic syndrome had higher liver enzyme levels but lower bilirubin and bile acid levels than without. PCOS women with hyperandrogenemia had higher liver enzyme, bilirubin levels than without. Correlation analyses showed that worsening of metabolic parameters was associated with higher liver enzyme levels but lower bilirubin and bile acid levels, while increased androgen levels were associated with higher liver enzyme, bilirubin and bile acid levels. Ovulation induction with clomiphene citrate could decrease bilirubin and bile acid levels, while acupuncture had no obvious effect on liver function. CONCLUSIONS: Among PCOS women, metabolic abnormalities and hyperandrogenemia impaired different liver function parameters. Clomiphene could decrease the bilirubin and bile acid levels while acupuncture had no obvious effect on liver function.

Comparing cervical mucus changes in response to an oral progestin or oestrogen withdrawal in ovarian-suppressed women: a clinical pilot.

Purpose: Prior studies evaluating the effect of administered progestogens on peak cervical mucus have not controlled for the influence of endogenous hormones. To address this, we treated women with a gonadotropin-releasing hormone (GnRH) agonist to suppress the hypothalamus-pituitary-ovarian (HPO) axis and used transdermal oestradiol replacement to stimulate peak cervical mucus and then evaluated the effects of an oral progestin or oestradiol withdrawal. Materials and methods: We used a crossover design to examine cervical mucus changes in women receiving transdermal oestradiol replacement following intramuscular administration of leuprolide acetate. After increasing oestradiol patches to mid-cycle levels, subjects were assigned to either 0.35 mg oral norethindrone with continuation of the patches (NET) or oestradiol withdrawal by patch removal (E2WD). We collected serum and cervical mucus samples at 0, 2, 4, 6, 22 and 24 h following the intervention. Results: We analysed 12 cycles (6 NET, 6 E2WD) from three subjects. Baseline cervical mucus scores were favourable to sperm penetration [NET median 11, interquartile range (9-12), E2WD 13 (12-13)]. Two hours after removal of oestradiol patch or administration of norethindrone, cervical mucus scores declined [NET 8.5 (4-9), E2WD 10.5 (10-12)]. Low cervical mucus scores persisted at 24 h with NET [8.0 (7-8)] but not E2WD [10.5 (8-11)]. Conclusions: We observed a rapid decline in cervical mucus Insler scores following administration of a single dose of oral norethindrone, and scores remained lower and unfavourable through 24 h. Oestradiol withdrawal did not result in similar unfavourable changes.

Sonic hedgehog signaling mediates resveratrol to improve maturation of pig oocytes in vitro and subsequent preimplantation embryo development.

The beneficial effects of resveratrol on in vitro maturation (IVM) have been explained mainly by indirect antioxidant effects and limited information is available on the underlying mechanism by which resveratrol acts directly on porcine cumulus oocyte complexes (COCs). Recently, several studies reported that sonic hedgehog (SHH) signaling mediates resveratrol to exert its biological activities. Furthermore, SHH is an important signaling molecule for follicle development, oocyte maturation, and embryo development. Therefore, to elucidate the relationship between resveratrol and SHH signaling, we designed three groups: (i) control; (ii) resveratrol; and (iii) resveratrol with cyclopamine (SHH signaling inhibitor). We evaluated the effects of these agents on cumulus expansion, oocyte maturation, embryo development after parthenogenetic activation, expression levels of mRNAs in cumulus cells, oocytes and blastocysts, and protein expression in COCs. Resveratrol significantly increased the proportion of COCs exhibiting complete cumulus expansion (degree 4), oocyte nuclear maturation, cleavage and blastocyst formation rates and total cell numbers, which were blocked in the presence of cyclopamine. At the same time, a significant increase in the expression levels of mRNAs related to cumulus expansion, oocyte maturation and SHH signaling-related mRNAs and proteins from the resveratrol treatment group was also inhibited by simultaneous addition of cyclopamine. In conclusion, our results indicate that SHH signaling mediates resveratrol to improve porcine cumulus expansion, oocyte maturation, and subsequent embryo development.

Production of inbred offspring by intracytoplasmic sperm injection of oocytes from juvenile female mice.

The aim of the present study was to determine whether the use of oocytes from juvenile female mice would improve the efficiency of intracytoplasmic sperm injection (ICSI). In the present study, 15 adult and 14 juvenile C57BL6/J female mice were superovulated, with 17.8 oocytes per mouse harvested from adults, significantly lower than the 40.2 harvested from juveniles (P<0.01). Sixty and 233 oocytes were harvested from C57BL/6J adult and juvenile mice respectively, activated in 10mM SrCl2+5µgmL-1 cytochalasin B for 5-6h and cultured in potassium simplex optimisation medium (KSOM) for 3.5 days, with no differences in morula and blastocyst rates between groups (91.7% vs 96.6%; P>0.05). Twelve hours after injection of human chorionic gonadotrophin, oocytes were harvested from C57BL/6J juvenile mice into KSOM, randomly divided into groups and activated with the same method mentioned above at 0, 2, 4 or 6h and then cultured in KSOM for 3.5 days. There was no significant difference in morula and blastocyst rates among the different groups (P>0.05). Oocytes from juvenile mice activated in 10mM SrCl2 for 2h were subjected to ICSI and the rates of pronuclear formation and Day 1 cleavage were significantly improved compared with the control group (P<0.01). ICSI combined with activation of oocytes from inbred mouse strains (C57BL/6J, C57BL/6N and 129Svev) successfully produced pups. The fertility of some these mice resulting from ICSI was tested, and the animals proved fertile. In conclusion, superovulated juvenile mice can yield more useable oocytes than adult mice, but additional activation is essential for full development of ICSI oocytes harvested from juvenile inbred mice.

In vitro growth and development of isolated secondary follicles from vitrified caprine ovarian cortex.

The aim of this study was to evaluate the viability, antrum formation and in vitro development of isolated secondary follicles from vitrified caprine ovarian cortex in a medium previously established for fresh isolated secondary follicles, in the absence (α-minimum essential medium (α-MEM+) alone) or presence of FSH and vascular endothelial growth factor (VEGF; α-MEM++FSH+VEGF). Ovarian fragments were distributed among five treatments (T1 to T5): fresh follicles were fixed immediately (T1), follicles from fresh tissue were cultured in vitro in α-MEM+ (T2) or α-MEM++FSH+VEGF (T3) and follicles from vitrified tissue were cultured in vitro in α-MEM+ (T4) or α-MEM++FSH+VEGF (T5). After 6 days of culture, treated follicles (T2, T3, T4 and T5) were evaluated for morphology, viability and follicular development (growth, antrum formation and proliferation of granulosa cells by Ki67 and argyrophilic nucleolar organiser region (AgNOR) staining). The levels of reactive oxygen species (ROS) in the culture media were also assessed. Overall, morphology of vitrified follicles was altered (P<0.05) compared with the fresh follicles. Follicular viability, antrum formation and ROS were similar between treatments (P>0.05). The average overall and daily follicular growth was highest (P<0.05) in T3. Granulosa cells in all treatments (T1, T2, T3, T4 and T5) stained positive for Ki67. However, fresh follicles from T3 had significantly higher AgNOR staining (P<0.05) compared with follicles of T1, T2, T4 and T5. In conclusion, secondary follicles can be isolated from vitrified and warmed ovarian cortex and survive and form an antrum when growing in an in vitro culture for 6 days.

Beneficial effects of melatonin on in vitro embryo production from juvenile goat oocytes.

Melatonin is a universal antioxidant that improves in vitro embryo production in several species. The aims of this study were to determine the melatonin concentration in the ovarian follicular fluid (FF) of juvenile goats and the effect of melatonin during in vitro maturation (IVM) on embryo development. The FF melatonin concentration was 0.57--1.07×10-9 M, increasing with follicular diameter. Oocytes were matured, fertilised and cultured under conventional conditions. Blastocyst development, embryo quality and levels of reactive oxygen species (ROS) and reduced glutathione were assessed. In Experiment 1 different melatonin concentrations (10-3, 10-7, 10-9, 10-11 M) were added to the IVM medium, which contained cysteamine as antioxidant, and no differences were observed. In Experiment 2, melatonin (10-7 M) was tested in the presence or absence of cysteamine (experimental groups: melatonin, cysteamine, melatonin+cysteamine, non-antioxidant). The melatonin group presented a higher blastocyst rate than the non-antioxidant group (28.9 vs 11.7%; P<0.01) and a higher total cell number than the cysteamine group (225.1 vs 129.0; P<0.05). Oocytes from the melatonin and cysteamine groups had lower ROS levels than those from the non-antioxidant group. This study shows that melatonin is an interesting tool for improving oocyte competence in juvenile goats as it increases embryo production and quality.