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Introduction The transplantation of mesenchymal stem cells (MSCs) in patients with premature ovarian failure (POF) could lead to clinical improvement. The transplantation to the ovaries among other transplantation methods have been reported in various animal models, however, there is little evidence regarding the optimal method, including the clinical safety and the efficiency for the treatment of age associated ovarian hypofunction. Objectives To establish the most effective transplantation route of MSCs, explore the resistance to therapy, its safety and role in the natural aging process of the ovaries. Methods Highly purified MSCs were injected intraperitoneally, directly into the ovaries or tail-intravenously in mice animal model. The ovarian function, quantity and quality of oocytes, cell viability/apoptosis, were evaluated, applying chemiluminescence analysis (CLIA), western blotting, immunofluorescence staining, transmission electron microscope (TEM), TdT mediated dUTP Nick End Labeling (TUNEL) assay and other techniques. The organ tumorigenicity was also evaluated by long-term observation and histopathological examination. The efficiency of MSCs was further verified in non-human primates by the most effective transplantation route. Results The 32nd week was ultimately determined as the time point of MSCs transplantation. Our results showed that the intra-ovarian injection was the best transplantation method with a more conspicuous effect. With deeper investigations, we found that the transplanted MSCs showed an effective influence on the follicular number, promoted follicle maturation and inhibited cell apoptosis, which was further verified in non-human primates. In addition, the long-term observation and the histopathological examinations ruled out neoplasms or obvious prosoplasia after MSCs transplantation. Conclusion MSCs transplantation by intra-ovarian injection could within a month exert the most conspicuous anti-age-associated ovarian hypofunction effects, which may improve the quantity and quality of oocytes by changing the mitochondrial structure, regulating mitochondrial function and attenuating cell apoptosis to increase the storage of the follicle pool without a remarkable potential of tumorigenicity.

Recently, we reported the differential expression of kisspeptinergic system in the bubaline hypothalamus and corpus luteum. Here, we document the expression of kisspeptin (Kp) and its receptor (Kiss1r) in the ovarian follicles of the buffalo with respect to the functional status. Follicles of ≥10 to ≤13 mm diameter (n = 45) were retrospectively categorized into active (n = 18), intermediate (n = 16) and atretic (n = 11) follicles based on the concentrations of intrafollicular progesterone and estradiol. The P4:E2 ratio was significantly lower in the active follicle (0.43 ± 0.08) than that of the intermediate (3.46 ± 0.53) and atretic (28.4 ± 10.6) follicles (P

Intra-follicular oocyte transfer (IFOT) is a promising and innovative technique for in vivo embryo production previously described for equines and bovines. The aim of this study was to assess the feasibility of IFOT in the ovine species. Two preliminary in vivo and in vitro trials were performed to test the optimal procedures and timing for IFOT. In the in vivo trial, follicular growth was monitored with transrectal ultrasonography in ten adult ewes to preliminarily determine the ovulation and ideal timing for IFOT. The in vitro trial assessed i) the optimal inner diameter of the injection needle and ii) the recovery rate and integrity of injected cumulus-oocyte complexes (COCs) after follicle aspiration. For IFOT and embryo collection, five ewes were synchronized by CIDR insertion. Forty hours after CIDR removal, in ewes under sedation and general anesthesia, the ovaries were exposed by laparotomy, and the preovulatory follicle was injected with COCs previously collected from ovaries obtained from an abattoir. At 4 h after surgery, fully recovered ewes were housed in a paddock with a ram of proven fertility. Crayon marking on ram's chest was used to detect mating. Ovulation was assessed 40 h after the transfer of oocytes by transrectal ultrasonography. On day 6 after IFOT, embryo collection was performed by uterine flushing. In the in vitro testing, injection of >5 mm follicles with a 28 G needle loaded with 30 COCs in a 5 μL volume resulted in higher recovery rates and better preservation of COCs integrity. In the in vivo trial, ultrasound scanning revealed that ovulation occurred between 60 and 72 h after CIDR removal in all animals. In one ewe subjected to IFOT, 22/24 oocytes were effectively injected into the preovulatory follicle, but no embryos were collected after flushing. In the remaining four animals, 85/102 oocytes were injected, and six cleaved embryos, 12 morulae and 1 blastocyst were collected, including native embryos. This preliminary investigation indicated that IFOT in ovine species resulted in ovulation, fimbrial capture, tubal transport of heterologous oocytes and in vivo embryo production. Further studies are needed to optimize the embryo recovery rate and develop less invasive techniques for oocyte injection and uterine flushing, such as through a laparoscopic or transcervical approach.

5 fluorouracil (5FU), an antineoplastic drug, is often utilized in the therapeutic regimen for several types of cancer, including the hepatoblastoma in children. The effects of 5FU on the population of ovarian preantral follicles, which is the largest oocyte reservoir, is still poorly understood. The integrity of the ovarian preantral follicle pool is important for lifelong fertility. The better understanding of such effects may favor intervention strategies to protect fertility in 5FU-treated children and women coping with cancer. To analyze the effects of 5FU on isolated murine secondary follicles in vitro, ovaries were collected from young mice (28-30 days old), and secondary follicles were isolated and cultured for 12 days in basic culture medium, with or without 5FU at concentrations of 0.3 mM, 1 mM, 3 mM, 10 mM, and 30 mM. In the in vitro study, we analyzed the percentage of morphologically normal follicles, antrum formation, follicular diameter, and hormone production. On day 12, oocytes were recovered for in vitro maturation. 5FU treatment did not alter the percentage of morphologically normal follicles. On day 12, only 1, 10, and 30 mM 5FU significantly reduced the percentage of antrum. From day 4 onwards, 5FU treatments significantly reduced follicle diameter. The meiosis resumption rate was significantly lower in all 5FU treatments. 5FU concentrations ≥3 mM reduced estradiol levels. In conclusion, 5FU does not affect follicular morphology. However, 5FU deleteriously affects follicular growth, estradiol production, and oocyte maturation in isolated ovarian follicles.

Phthalates are a family of chemicals that can be found in plastic and personal care products used by consumers every day and they are known endocrine disrupting chemicals that can disrupt female reproduction. In previous studies, an environmentally relevant phthalate mixture was shown to affect female reproduction in a transgenerational manner. However, limited information was available on the effect of phthalate mixtures on ovarian steroidogenesis and folliculogenesis. Ovarian steroidogenesis is important for producing hormones needed for reproduction and ovarian regulation, and folliculogenesis is essential for the development of ovarian follicles and successful fertility. Thus, this study tested the hypothesis that prenatal exposure to an environmentally relevant phthalate mixture adversely affects ovarian steroidogenesis and folliculogenesis in the F1 generation of adult female mice. Pregnant dams (F0 generation) were orally dosed with vehicle control or a phthalate mixture (20 μg/kg/day-500 mg/kg/day) daily from gestational day 10 to birth, and the adult F1 females were the offspring of the dosed dams. The ovaries of the F1 generation were collected at postnatal day 60. One ovary was used for histological examination of the numbers and percent of different follicle types. The other ovary was used to measure expression of theca and granulosa cell enzymes. Additionally, sera were collected for measuring hormone levels. The results indicate that prenatal exposure to the phthalate mixture decreases hormone levels and gene expression, alters the transitioning of follicle types, and leads to a higher incidence of atresia in the F1 generation offspring.

In vitro systems capable of reconstituting the process of mouse oogenesis are now being established to help develop further understanding of the mechanisms underlying oocyte/follicle development and differentiation. These systems could also help increase the production of useful livestock or genetically modified animals, and aid in identifying the causes of infertility in humans. Recently, we revealed, using an in vitro system for recapitulating oogenesis, that the activation of the estrogen signaling pathway induces abnormal follicle formation, that blocking estrogen-induced expression of anti-Müllerian hormone is crucial for normal follicle formation, and that the production of α-fetoprotein in fetal liver tissue is involved in normal in vivo follicle formation. In mouse fetuses, follicle formation is not carried out by factors within the ovaries but is instead orchestrated by distal endocrine factors. This review outlines findings from genetics, endocrinology, and in vitro studies regarding the factors that can affect the formation of primordial follicles in mammals.

Although it is well known that the ovulation occurs during a period of time after LH surge in dogs, there are few reports of observing the entire process of development, ovulation and luteinization of each follicle. This study aimed to detect the ovulation kinetics by ultrasonography in combination with progesterone monitoring and therefore identify the time-range of the ovulation process in a dog. Daily transabdominal ultrasonography and progesterone monitoring were conducted for 24 natural oestrus cycles of Labrador Retrievers. Ovarian follicles were observed as anechoic structure with contours before ovulation. Ovulation (follicular collapse) was defined as when follicles became cloudy and contours obscure by transabdominal ultrasonography. Ultrasound imaging was capable of identifying the day of ovulation for 94.7% (178/188) of the follicles through the appearance of collapsed follicle or corpus luteum. Ovulation was observed between LH 0 (the day of LH surge) and LH 5, with 48.0%, 33.5% and 15.0% for LH 2, LH 3 and LH 1, respectively. The total number of ovulations on LH 2 and LH 3 accounted for 81.5% (141/173) of the total ovulation in 24 cycles examined. Ovulation occurred in 12 cycles for 2 d and for 3 d in 12 cycles. Seventeen cycles (70.8%) with multiple days of ovulation showed the largest number of ovulations on LH 2. The average follicle diameter 3 d before the LH surge was less than 5 mm, then exceeded 5 mm 2 d before the LH surge. The average follicle diameter at the time of ovulation (follicular collapse) was 6.1 ± 1.0 mm (n = 118). On the day before ovulation, the average diameters of the follicles ovulated on LH 1, LH 2 and LH 3 were 5.0 ± 0.7 mm, 5.8 ± 1.2 mm and 6.2 ± 1.3 mm, respectively. There was a significant difference in the follicle diameter between LH1 and LH2 (p

BACKGROUND: In vitro follicular maturation (IVFM) of ovarian follicles is an emerging option for fertility preservation. Many paracrine factors and two-dimensional or three-dimensional (3D) environments have been used for optimization. However, since most studies were conducted using the murine model, the physiological differences between mice and humans limit the interpretation and adaptation of the results. Marmoset monkey is a non-human primate (NHPs) with more similar reproductive physiology to humans. In this study, we attempted to establish a 3D matrix (Matrtigel)-based IVFM condition for marmoset ovarian follicles in combination with anti-apoptotic factor, X-linked inhibitor of apoptosis protein (XIAP). METHODS: Marmoset follicles were isolated as individual follicles and cultured in a single drop with the addition of 0, 10, and 100 μg/mL of XIAP molecules. Matured oocytes and granulosa cells from mature follicles were collected and analyzed. The average number of isolated follicles was less than 100, and primordial and antral follicles were abundant in the ovaries. RESULTS: IVFM of marmoset follicles in 3D matrix conditions with XIAP increased the rates of survival and in vitro follicle development. Furthermore, oocytes from the 3D cultures were successfully fertilized and developed in vitro. The addition of XIAP increased the secretion of estradiol and aromatase. Furthermore, expression of granulosa-specific genes, such as bone morphogenetic protein 15, Oct4, and follicle-stimulating hormone receptor were upregulated in the in vitro-matured follicles than in normal, well-grown, and atretic follicles. Apoptosis-related B-cell lymphoma-2 was highly expressed in the atretic follicles than in the XIAP-treated follicles, and higher caspase-3 was localized in the XIAP-treated follicles. CONCLUSION: In this study, we attempted to establish a 3D-matrix-based marmoset IVFM condition and demonstrated the synergistic effects of XIAP. The use of a 3D matrix may be applied as an optimal culture condition for marmoset ovarian follicles.

Polycystic ovary syndrome is a disorder involving prolonged menstrual cycle, and often excess androgen level normally occurs in several women at the time of their reproductive age. This causes impotence along with gynaecomastia and hirsutism. Studying these kinds of condition in women is a major problem which can be resolved by analysing ultrasound images which have the necessary details like number of follicles, size, and position. However, there is a lack of solid objective test that can provide absolute affirmative to diagnose and understand PCOS. This motivates us to think about finding a method to diagnose PCOS at early stages preventing further complications. An automatic PCOS diagnosing tool would help to save the actual time spent on manual tracing of follicles and measuring the geometric features of every follicle. The proposed method was able to achieve classification with accuracy greater than 97% using a KNN classifier. The classifier will improve the time spent on diagonising PCOS and improve its accuracy, reducing the risk of the fatal complications that can be caused by delayed diagnosis.

Background Germinal vesicle (GV) chromatin configurations of oocytes are proposed to be related to oocyte competence and may reflect the quality of oocyte. Currently, a limited number of published studies investigated the GV chromatin configurations of guinea pig oocytes. Objective In this study on the in vitro maturation (IVM) of guinea pig oocytes, we examined the changes in their GV chromatin configurations during meiotic progression. Methods Based on the degree of chromatin compaction, the GV chromatin configurations of guinea pig oocytes could be divided into three categories depending on whether the nucleolus‐like body (NLB) was surrounded or partly surrounded by compacted chromatin, namely the uncondensed (NSN), the intermediate type (SN‐1) and the compacted type (SN‐2). Results The percentage of cells displaying the SN‐2 configuration increased with the growth of guinea pig oocytes, suggesting that this configuration presents the potential for maturation in oocytes. Oocytes derived from larger follicle exhibited increased meiotic potential. Serum starvation affected the GV chromatin configurations of guinea pig oocytes. Conclusions Collectively, these results suggest that the SN‐2 type might be a more mature form of configuration in guinea pig oocyte, whose proportion was associated with the follicle size and susceptible to the environment (e.g. serum concentration)., According to the degree of chromatin compaction and the visibility of nucleoli and nuclear membranes, the GV chromatin configurations of guinea pig oocytes were divided into three types, the uncondensed type (NSN‐type), the intermediate type (SN‐1‐type), and the compacted type (SN‐2‐type). The NSN‐types exhibited chromatin that did not surround the nucleolus and was instead dispersed throughout the nucleoplasmic region. The SN‐1‐type oocytes showed that a part of chromatin was condensed around the nucleolus, and some chromatin dispersed in the nucleoplasm. The SN‐2‐type oocytes showed that almost all chromatin was condensed around the nucleolus.

SUMMARY OBJECTIVE: This study evaluates the effects of a ketogenic diet on morphology and follicle reserve. METHOD: Sixteen Sprague-Dawley rats were randomized into two groups: standard diet group (n=8) and ketogenic diet group (n=8). Rats were time mated. Dams were permitted to deliver spontaneously. The animals were monitored for the onset of puberty. All the rats were weighed and anesthetized, serum anti-Müllerian hormone level was measured, and the oviducts were removed. The morphological characteristics of follicles were determined and total ovarian volumes were calculated. RESULTS: The mean ovarian volume was statistically significantly lower in the ketogenic diet group compared to the standard diet group (14.41±0.99 mm3 versus 18.89±1.28 mm3) (p=0.000). The mean number of antral follicles was 13.63±1.80 in the standard diet group and 4.462±0.760 in the ketogenic diet group. The mean ovarian weight of the ketogenic diet group was significantly lower than that of the standard diet group (0.42±0.06 g versus 0.815±107 g). The mean anti-Müllerian hormone levels were significantly higher in the standard diet group compared to the ketogenic diet group (1.023±4.75 ng/mL versus 0.69±0.07 ng/mL) (p=0.000). The mean percentage of staining of Ki-67 was 35.28±4.75 in the standard diet group and 16.98±3.33 in the ketogenic diet group (p=0.000). CONCLUSION: Maternal ketogenic diet reduces ovarian follicular reserve in female offspring and has important implications for maintaining reproductive potential at a population level.

An experiment was carried out at the Experimental Center La Raya-Universidad Nacional del Altiplano Puno, to determine the effect of copulation duration on ovulation and pregnancy in alpacas. 47 adult females were used (postpartum time ≥ 20 days and presence of a follicle ≥ 7 mm) distributed in three groups, according to copulation time: 20, 35 and 50 min, for G1 (n = 15), G2 (n = 16) and G3 (n = 16), respectively. For the mating, 6 reproducers of proven fertility were used, interrupting the copulation at the established time. Ovulation and pregnancy rates were evaluated on days 7 and 30 post service respectively (by ultrasound and receptivity of the female), the data were analyzed using X2 and simple correspondence. Both the ovulation rate (G1: 73.33%, G2: 81.25% and G3: 81.25%); and pregnancy (G1: 53.33%, G2: 81.25% and G3: 75%) were not different (P> 0.05). Pregnancy rates between G1 and G2 tended to be different (P

Ghrelin has previously been proven to have anti-inflammatory and antioxidant properties in preventing cisplatin-induced ovarian damage. The aim of this study was to evaluate the potential effects of this hormone in preventing this damage in rats using histopathological and biochemical methods. Twenty-eight Wistar-albino rats were randomly divided into four groups. While no drug was given to Group 1 (sham group), acylated ghrelin was intraperitoneally administered to Group 2 at 0.5 nmol/kg and Group 3 at 2 nmol/kg for 21 days. Group 4 received only saline solution. On the 15th day, a single dose of 5 mg/kg cisplatin was intraperitoneally administered to each rat in Groups 2, 3 and 4. Serum anti-Mullerian hormone (AMH) values were measured on days 0, 15 and 21. Then, laparotomy and bilateral oophorectomy were performed, and the ovaries were histopathologically examined. The number of primordial and primary follicles was significantly higher in Group 3 than in the saline solution + cisplatin group. In Group 4, cisplatin caused significantly higher follicle damage in the primordial, primary and secondary phases compared to the sham group. The AMH level of the SF + cisplatin group was significantly lower than that of the sham group and the high-dose ghrelin + cisplatin group, and the AMH level of the sham group was significantly higher than that of the low-dose ghrelin + cisplatin group. High-dose ghrelin was effective in preventing cisplatin-induced ovarian damage by preserving the number of primordial and primary follicles. Larger randomized studies are needed to determine the optimal dosage and duration of ghrelin.

Genetic differences have been suggested as a possible cause for variation in responses to exogenous hormones. Here we evaluated the effect of follicle ablation, exogenous FSH and coasting time prior to ovum pick-up (OPU) on the number of follicles suitable for aspiration, oocyte quality, and cleavage rate in Ethiopian Boran cows. The experiment was carried out in three parts, I) Cows were synchronized using 500µg PGF2α given 11 days apart. Cows were then subjected to a biweekly ovum pickup session before ovulation (n=5) or starting Day 7 after ovulation (n=4) for three weeks. II) Cows were similarly synchronized and all visible follicles were ablated on the first days of overt estrus which were then further grouped into cows that received a divided dose of 350IU FSH (n=5) or 175IU FSH (n=5) over three days. In both groups OPU was carried out weekly starting 48h after the last FSH for six weeks. III) A similar protocol as in part II was carried out but coasting period was increased to 72hrs for cows that received 350IU FSH as divided dose (n=5) and 48hrs coasting period for single 350IU FSH dose (n=5). The covariates of follicles and oocyte were not affected (P>0.05) by corpus luteum presence at OPU. The mean number of medium (7.36±0.57) and large (8.28±0.96) follicles were significantly higher (P

AIM One of the most important ways to understand the ovarian biology is studding the initiation of primordial follicle development and subsequent folliculogenesis control. In this study, proliferating cell nuclear antigen (PCNA) presentation was used as a marker of follicular development in the thawed ovarian tissue (OT) following transplantation onto chick embryo chorioallantoic membrane (CAM) using two methods of freezing of slow freezing and vitrification. METHODS Samples of OT from 10 patients were subjected to slow freezing and vitrification. After warming, CAM transplantation was done and PCNA proliferation index (PI; percent of PCNA-positive granulosa cells) was calculated for each follicle stage. Image J software was used to determine the mean staining intensity. RESULTS PCNA was positive for granulosa cells and oocytes nuclei, but negative for ooplasm. There were no remarkable PCNA staining in the granulosa cells of primordial follicles, but increased significantly as follicle progression (p

Objective: The present work aimed to study the effect of progesterone (CIDR)-based GnRH treatments plus eCG (1000 or 500 IU) on inducing and synchronizing the onset of puberty in buffalo-heifers. Methods: Buffalo-heifers (n=28) were randomly allocated into four treatment protocols (7 each): (G1, CIDR- eCG1000- GnRH), (G2, CIDR- eCG 500- GnRH), (G3, CIDR-GnRH) and (G4, CIDR- alone). The largest follicle diameter (LFD) and antral follicles counts (AFC) of small (≤ 4mm), medium (> 4mm- ≤8mm) and large follicles (>8mm) were estimated by transrectal ultrasonography on Days 0, 8, and 11. Also, the LFD was estimated on Day 13 as well. Results: On Day 11, the AFC of both small and medium-sized follicles was higher in G1 compared with G2 (P < 0.05) and with both G3 and G4 (P < 0.01). The AFC of large follicles was the greatest (P < 0.05) in G1compared with all groups but did not differ among G2, G3, and G4 on Day 11. On Day 13, the LFD showed non-significant variation between G1 and G2 as well as between G3 and G4. It showed a significant increase in G1 (P < 0.01) and G2 (P < 0.05) compared with either G3 or G4. The conception rate was the highest (P

Objective: To study the effect of Yu Linzhu on ovarian function and mitochondria in natural aging mice. Methods: Female BALB/c mice were selected as normal group at 7–8 weeks and natural aging group at 9 months. The natural aging group was divided into Yu Linzhu intervention group and non-intervention group by intragastric administration once a day for 6 weeks. The morphology and blood flow of ovary were observed by ultrasound. Ovarian morphology and follicle were observed by HE staining. Hormone levels were analyzed by ELISA. Serum oxidative stress were detected by radioimmunoassay. The distribution of mitochondria in oocytes was observed by fluorescence staining. The ultrastructure of oocytes and the morphology of mitochondria were observed under electron microscope. The mitochondrial membrane potential was detected by JC-1. Results: Two groups of aging mice had serious disturbance of estrus cycle. The ovarian area of the mice in the aging non-intervention group was smaller than that in the normal group, and the ovarian area of the mice in the aging intervention group recovered. The ovarian blood flow was weak or even disappear in the aging non-intervention group, and the blood flow in the intervention group was improved. The ovarian volume of mice in the non-intervention group was smaller than that in the normal group. Some ovarian tissues were adhered to the surrounding tissues. While in the intervention group, the ovarian volume increased, the degree of adhesion decreased, the infiltration of ovarian interstitial lymphocytes decreased, and the zona pellucida recovered. Granular cell arrangement returned neatly, egg cell shape recover regular and the number also increased. In the non-intervention group, E2 (Estrogen), AMH (Anti-Mullerian hormone) decreased (P = 0.0092 and P = 0.0334, respectively), FSH (Follicle stimulating hormone) increased (P < 0.0001). In the intervention group, FSH decreased (P = 0.0002), LH (luteinizing hormone) decreased and E2, AMH increased. In the non-intervention group, GSH-Px (Glutathione peroxidase) decreased (P = 0.0129), SOD (Superoxide dismutase) decreased, ROS (reactive oxidative species), MDA (Malondialdehyde) increased. In the aging intervention group, ROS, MDA decreased and GSH-Px increased. In the non-intervention group, mitochondrial expression was scattered at the concentrated distribution point, the length of mitochondria was mostly long and the average volume increased, the density decreased, the number decreased and some mitochondria fused, and lesions such as swelling, vacuolar degeneration and inclusion body formation, membrane potential decreased (P = 0.0002). In the aging intervention group, mitochondria were evenly distributed, the mitochondria were basically round, the distribution density was moderate, the inner ridge was clear, and the membrane potential of the aging intervention group increased. Conclusion: Yu Linzhu can improve the ovarian function of natural aging mice by improving the mitochondrial function of oocytes.

Ovarian tissue vitrification is a promising method to preserve follicles and gametes, but can be improved with mineral supplementation to the vitrification medium. The objective of this study was to determine the effects of supplementing 0.5 mg/mL zinc chloride to the media during cryopreservation of pig ovarian tissue. After thawing, the following criteria were evaluated: (1) follicular development and damage, (2) in vitro fertilization (IVF) characteristics, and (3) embryonic development. The number of damaged antral follicles (72.0 ± 3.8%) was less (p < 0.05) in ovarian tissue vitrified in media supplemented with zinc chloride compared to those not supplemented with zinc chloride (86.7 ± 4.1%). Oocytes obtained from the antral follicles on ovarian tissue vitrified in media supplemented with zinc chloride had less (p < 0.05) polyspermic penetration and higher (p < 0.05) male pronuclear formation during IVF than oocytes obtained from ovarian tissue not supplemented with zinc chloride. There were no statistical differences in embryonic development rates. Based on these results, supplementing zinc chloride during the vitrification protocol improves follicular development and subsequent IVF in pigs.

Proper follicle development is very important for the production of mature oocytes, which is essential for the maintenance of female fertility. This complex biological process requires precise gene regulation. The most abundant modification of mRNA, N6-methyladenosine (m6A), is involved in many RNA metabolism processes, including RNA splicing, translation, stability, and degradation. Here, we report that m6A plays essential roles during oocyte and follicle development. Oocyte-specific inactivation of the key m6A methyltransferase Mettl3 with Gdf9-Cre caused DNA damage accumulation in oocytes, defective follicle development, and abnormal ovulation. Mechanistically, combined RNA-seq and m6A methylated RNA immunoprecipitation sequencing (MeRIP-seq) data from oocytes revealed, that we found METTL3 targets Itsn2 for m6A modification and then enhances its stability to influence the oocytes meiosis. Taken together, our findings highlight the crucial roles of mRNA m6A modification in follicle development and coordination of RNA stabilization during oocyte growth.

Di(2-ethylhexyl) phthalate (DEHP) is a chemical commonly used as a plasticizer to render polyvinyl chloride products more durable and flexible. Although exposure to DEHP has raised many health concerns due to the identification of DEHP as an endocrine disruptor, it is still used in consumer products, including polyvinyl chloride plastics, medical tubing, car interiors, and children’s toys. To investigate the impact of early life exposure to DEHP on the ovary and testes, newborn piglets were orally dosed with DEHP (20 or 200 mg/kg/day) or vehicle control (tocopherol-stripped corn oil) for 21 days. Following treatment, ovaries, testes, and sera were harvested for histological assessment and measurement of steroid hormone levels. In male piglets, progesterone and pregnenolone levels were significantly lower in both treatment groups compared to control, whereas in female piglets, progesterone was significantly higher in the 20 mg group compared to control, indicating sex-specific effects in a non-monotonic manner. Follicle numbers and gene expression of steroidogenic enzymes and apoptotic factors were not altered in treated ovaries compared to controls. In DEHP-treated testes, germ cell migration was impaired and germ cell death was significantly increased compared to controls. Overall, the results of this study suggest that neonatal exposure to DEHP in pigs leads to sex-specific disruption of the reproductive system.

Follicle-stimulating hormone (FSH), an important hypothalamic-pituitary-gonadal axis (HPG) hormone, is secreted by the pituitary gland. This study confirms that FSH is expressed in chicken follicles at different stages, and positive FSHβ mRNA signals were stronger (P0.05) was observed between the number of granulosa cells treated with FSHβ siRNA and in exogenous FSH. However, the number of granulosa cells without FSHβ siRNA transfection was significantly higher (P

Objective: To determine correlation between serum levels of Follicle Stimulating Hormone and Graafian Follicle vascularity on Doppler ultrasound in infertile females of Lahore, Pakistan. Study Design: Cross sectional study. Place and Duration of Study: Prime Ultrasound center, 2-Shalamrar Link Road, Mughalpura, Lahore, Pakistan From Mar 2018 to Sep 2019. Methodology: A total of 100 infertile females using simple random sampling were taken. All the married women of reproductive age and married for >12 months who have not conceived despite regular unprotected intercourse, having no other uterine and endometrial or ovarian pathology were taken. The patients who fail to provide serum hormone laboratory reports were excluded. Transvaginal ultrasonography was done for all females and graafian follicle vascularity on Doppler was measured on day 12 of the cycle. Results: The Median age of females was 28 (30-25) years range from 19 and 46 years. The median Follicle Stimulating Hormone levels at day 12 was 6 (8.25-5.22) mlU/ml with minimum and maximum FSH values as 3.20 and 40 mlU/ml. The median follicle size was 13.65 (16.04-10.84) (mm) with minimum and maximum size as 10 mm and 26 mm. There were 61% females who had Grade–1 perifollicular vascularity, 24% females had grade-2 and 15% females had grade-3 of perifollicular vascularity. There was significant positive correlation between perifollicular vascularity grades and Follicle-stimulating hormone levels day 12 (mm) i.e., r=0.309, p-value

The objective of this study was to test the effect of low circulating concentrations of progesterone (P4) on pre-ovulatory follicle development in heifers as part of an overarching objective to develop a model to understand this phenomenon in dairy cattle without the confounding factors of lactation. Holstein heifers between 12 and 13 mo of age were pre-synchronized to ensure all heifers were on d 6 of the estrous cycle at the start of the Ovsynch program. Only heifers with CL regression and ovulation to the following pre-treatment strategy were used in the study: 0.5 mg cloprostenol (PGF2α), 2 d later, 0.1 mg GnRH, 6 d later, GnRH (G1; 1st GnRH of Ovsynch). Heifers (n = 159) responding to pre-treatment were randomly assigned to 4 groups and completed the Ovsynch program: high P4 control (HPC), low P4 control (LPC; PGF2α 24 h after G1), PG2 (PGF2α 24 and 48 h after G1) and PG3 (PGF2α 24, 48, and 96 h after G1). Only heifers that had ovulation to G1 remained in the study. Blood samples were collected in all heifers on d 7 (n = 157) and in a subset of heifers on d 1, 2, 3, 4 (n = 82) after G1 to measure serum P4. Pre-ovulatory follicle size at G1 (13.0 ± 0.1 mm; P = 0.53) and mean serum P4 24 h after G1 (d 1; 3.62 ± 0.11 ng/mL; P = 0.46) did not differ among treatments. HPC heifers had greater (P 0.10). On d 7, LPC heifers had greater (P

Aim: The patient was followed up in consultation with the orthodontist, and after a long-term follow-up, it was found that the dental germ developed, but the position of the tooth was horizontal. When the follow-ups were continued, it was observed that the position of the tooth also improved, and it progressed smoothly along the tooth eruption path. Methodology: In the panoramic radiography of an 8-year-old male patient who applied to the clinic complaining about the left upper first primary molar tooth, the right lower permanent second premolar tooth follicle developed, but there was no tooth development in the follicle. Results: When follicle formation is observed, only following the patients without directing them to orthodontic treatment, which is expensive and difficult, positively affects their dental structure. Following patients without affecting their lives can be significant. Conclusion: We emphasize the importance of long-term follow-up when follicle formation was observed. We predicted that this situation was caused by the disconnection in the interaction of transcription factors and signals in the tooth development stage. How to cite this article: Sukurica Y, Çakır A. Long-term follow-up in lower right second premolar tooth with a dental follicle but no tooth bud: A case report. Int Dent Res 2021;11(Suppl.1):312-5. https://doi.org/10.5577/10.5577/intdentres.2021.vol11.suppl1.47 Linguistic Revision: The English in this manuscript has been checked by at least two professional editors, both native speakers of English.

Certain chemotherapeutic drugs are toxic to ovarian follicles. The corpus luteum (CL) is normally developed from an ovulated follicle for producing progesterone (P4) to support early pregnancy. To fill in the knowledge gap about effects of chemotherapy on the CL, we tested the hypothesis that chemotherapy may target endothelial cells and/or luteal cells in the CL to impair CL function in P4 steroidogenesis using doxorubicin (DOX) as a representative chemotherapeutic drug in mice. In both mixed background mice and C57BL/6 mice, a single intraperitoneal injection of DOX (10 mg/kg) on 0.5-day postcoitum (D0.5, postovulation) led to ~58% D3.5 mice with serum P4 levels lower than the serum P4 range in the phosphate buffer saline-treated control mice. Further studies in the C57BL/6 ovaries revealed that CLs from DOX-treated mice with low P4 levels had less defined luteal cords and disrupted collagen IV expression pattern, indicating disrupted capillary, accompanied with less differentiated luteal cells that had smaller cytoplasm and reduced StAR expression. DOX-treated ovaries had increased granulosa cell death in the growing follicles, reduced proliferating cell nuclear antigen-positive endothelial cells in the CLs, enlarged lipid droplets, and disrupted F-actin in the luteal cells. These novel data suggest that the proliferating endothelial cells in the developing CL may be the primary target of DOX to impair the vascular support for luteal cell differentiation and subsequently P4 steroidogenesis. This study fills in the knowledge gap about the toxic effects of chemotherapy on the CL and provides critical information for risk assessment of chemotherapy in premenopausal patients.

The aim of the study was to explore the effect and mechanism of a low-level laser on hair follicle stem cells in full-thickness skin wound healing in mice. Full-thickness skin defects were generated by a 5-mm punch biopsy tool on the backs of depilated C57/BL6N mice, which were randomly divided thereafter into a low-dose laser treatment group (LLLT-Low), a high-dose laser treatment group (LLLT-High), and a control group (control). From the day of modeling to the day before the skin samples were taken, the wound area and wound edge of the mice in the LLLT-Low and LLLT-High groups were irradiated with a laser comb every 24 h, and the energy density was 1 J/cm2 and 10 J/cm2, respectively. The control group was irradiated with an ordinary fluorescent lamp. At 0, 3, 5, 10, and 14 days after modeling, pictures of each wound were taken, and the percent wound closure was analyzed. At 3, 5, 10, and 14 days after modeling, the samples were observed by hematoxylin and eosin (HE) and immunofluorescence (IF) staining. Whole transcriptome sequencing (RNA-Seq) was performed on the samples on day 10. Gene Ontology (GO) analysis was performed, and the results were validated by Western blot analysis and enzyme-linked immunosorbent assay (ELISA). The analysis of the percent of wound closure showed that healing was accelerated (significantly from 5 to 10 days) in the LLLT-Low group, but there was no clear change in the LLLT-High group. HE staining showed that the LLLT-Low group had an increasing number of hair follicles and a tendency to migrate to the center of the wound. There was no significant increase in the number of hair follicles and no obvious migration in the LLLT-High group. Immunofluorescence staining showed that the total number of CK15 + hair follicle stem cells in the LLLT-Low group was higher than that in the control group and LLLT-High group at all time points. The number and farthest migration distance of CK15 + hair follicle stem cells increased significantly with time, and after 5 days, they were significantly higher than those in the control group and LLLT-High group. RNA-Seq and Western blot analysis showed that the expression of related genes in hair follicle stem cells, including CK15, in the LLLT-Low group was upregulated. GO analysis and ELISA showed that the expression of many cytokines, represented by IL34, in the LLLT-Low group was upregulated. Low-level laser treatment can promote the proliferation, differentiation, and migration of CK15 + hair follicle stem cells by upregulating the cytokine IL34, thereby promoting skin wound healing in mice.

Background: Most available therapeutics to address alopecia are based on singular targets and mechanism of action based on the assumption of sub-classification to reflect morphological or etiological sub-classes such as hereditary vs acquired factors, anti-inflammatory vs. non-inflammatory factors or scarring vs. nonscarring factors. Aims and Objective: Impact of oral consumption of Diabliss Hair Water, a multi-component herbal water supplement (HWS) on hair fall reduction, hair growth, hair density, hair thickness was investigated in a single arm, exploratory, pilot open label clinical study among healthy male and female subjects of age group between 35-50 years, with hair fall and hair thinning concerns. Materials and Methods: The study was conducted under dermatological control and included hair fall by comb test, hair pull test, hair thinning measurement using photo numerical scale (modified Norwood and Ludwig scale) and assessment for hair quality along with the phototrichogram assessment using Trichoscan® with Cutiscope. Results: The Diabliss HWS was efficacious in improving hair growth rate and density as per TrichoScan® assessment, improvement in hair thickness as per Caslite-Nova software. The Diabliss HWS was also efficacious in improving the hair tensile strength (break force by Chatillon Force gauge), reducing hair fall and improving the hair quality. By the end of the study the Diabliss HWS was found to show an improvement in the entire study population in all of the assessments parameters. Dermatologist assessment parameters of hair texture and hair shine also showed improvements in the entire study population. Conclusion: The Diabliss HWS was found to be efficacious in providing hair fall control and hair growth benefits as per the dermatological and instrumental evaluations using TrichoScan®. The product was well tolerated with no product related AE or SAE with regular consumption for 06 months. Keywords: Herbal Supplement, Hair C

AIMS The aim of this study was to evaluate the relationship between follicular anti-Mullerian hormone (fAMH) regarding follicle size, the presence of an oocyte, sociodemographic parameters, and other hormones and vitamins in follicular fluid. MATERIALS AND METHODS This prospective exploratory cohort study included 376 follicles from 61 women undergoing IVF/ICSI cycles. The size of each follicle was measured individually, and they were divided into a large and a small group according to their size. The presence of oocytes was detected on the day of oocyte retrieval. Sociodemographic factors were documented during the course of preliminary testing. Then, other parameters of patients' serum and follicular fluid were measured. RESULTS Small follicles contained a significantly higher concentration of fAMH than large ones did. We showed that the presence of an oocyte in a follicle is associated with a significantly higher fAMH level than in those without one (p

In the current study, the hypothesis of the effects of luteinizing hormone (LH) and follicular fluid (FF) derived from follicles of varying size on in vitro embryo production of the Shami goat breed were tested. The caprine follicular fluid (cFF) was obtained from healthy female’s ovaries by aspiration method and classified into two main classes (follicles with a diameter of ≤ 2mm and ≥3mm). The resulting cFF was added to the culture medium TCM-199 through six Treatments (A, B and C with a source of follicle size of ≤ 2mm; D, E and F with a source of size of ≥3mm). LH was added only to four of the previous Treatments with the levels of 50 µg ml-1 (B and E) and100 µg ml-1 (C and F). Results of the study showed that the oocytes incubated in Treatment F achieved a clear superiority (p=0.001) in the rates of maturation (87.0%), fertilization (80.0%) and cleavage (82.3%). The oocytes incubated in the same Treatment (F) continued to outperform (p= 0.006) by achieving the best rates across cleavage stages at 2-16 cell (16%; the lower value of arrest) and blastocyst (42%). Significant differences (P=0.03) were observed among the rates of Type 1embryos (the highest rate: 45.3%; Treatment F) and Type 3 embryos (the highest rate: 45.1%; Treatment A). No significant differences were observed in the rates of morula and Type 2 embryos. It is advised to add 15% of the cFF derived from follicles with a diameter of ≥3mm and 100 µg of LH ml-1 in the maturation media to obtain higher rates of maturation and cleavage of goat oocytes.

During an ovulatory follicular wave in the monovulatory species of heifers, mares, and women, the two largest follicles deviate in diameter at the end of a common follicle growth phase. The largest follicle before deviation becomes the future ovulatory follicle in most ovulatory waves. In 10-30% of the ovulatory waves, the destiny of the two follicles switches just before or at deviation so that the second-largest follicle becomes the future ovulatory follicle, and the largest follicle becomes a subordinate. In FSH-driven switching in heifers, mares, and women, the wave-stimulating FSH surge decreases to a low concentration before the largest follicle has developed the ability to utilize the low concentrations. The concentrations of FSH then increase (mares, women) or cease to decrease (heifers), and the next largest follicle acquires the capability of becoming the future ovulatory follicle. Luteolysis-driven switching has been reported in heifers but not in mares and women. The switching in heifers occurs during ovulatory wave 3 of three wave interovulatory intervals (IOI) when the wave of follicles is in the common growth phase in synchrony with the time of luteolysis. Regression of the CL during the common growth phase of ovulatory wave 3 is accompanied by decreased activity of follicles that are adjacent to the regressing CL but not when follicles and CL are separated or in opposite ovaries. The role of luteolysis in switching in heifers has been tested by treating with PGF2α when the largest follicle of wave 2 was near the end of the common growth phase. Switching in destiny of the largest follicle from the expected future dominant to a future subordinate occurred in most waves (10 of 17) when the largest follicle and regressing CL were in the same ovary and adjacent but not when separated in the same ovary or when in opposite ovaries (0 of 11). The newly selected future ovulatory follicle may develop in the opposite ovary. Thereby, frequency of the contralateral vs ipsilateral relationship between the preovulatory follicle and CL in heifers is greater in three-wave IOI than in two-wave IOI. In summary, the second largest predeviation follicle becomes the postdeviation dominant follicle when the decreasing FSH is out of phase with the largest predeviation follicle in heifers, mares, and women or when luteolysis and predeviation are in synchrony in heifers.

Frozen-thawed human ovarian tissue endures large-scale follicle loss in the early post-grafting period, characterized by hypoxia lasting around 7 days. Tissue revascularization occurs progressively through new vessel invasion from the host and neoangiogenesis from the graft. Such reoxygenation kinetics lead to further potential damage caused by oxidative stress. The aim of the present manuscript is to provide a systematic review of proangiogenic growth factors, hormones and various antioxidants administered in the event of ovarian tissue transplantation to protect the follicle pool from depletion by boosting revascularization or decreasing oxidative stress. Although almost all investigated studies revealed an advantage in terms of revascularization and reduction in oxidative stress, far fewer demonstrated a positive impact on follicle survival. As the cascade of events driven by ischaemia after transplantation is a complex process involving numerous players, it appears that acting on specific molecular mechanisms, such as concentrations of proangiogenic growth factors, is not enough to significantly mitigate tissue damage. Strategies exploiting the activated tissue response to ischaemia for tissue healing and remodelling purposes, such as the use of antiapoptotic drugs and adult stem cells, are also discussed in the present review, since they yielded promising results in terms of follicle pool protection.

BackgroundEstradiol is an important marker of ovarian response to ovarian stimulation in ART cycles. The study tries to find the correlation of serum estradiol on the day of HCG trigger to the number of follicles, the number of oocytes retrieved, and the number of mature oocytes, and also, to correlate estradiol per follicle and estradiol per oocyte on the day of HCG, to the number of oocytes retrieved, and to the number of mature oocytes. It is a cross sectional study using retrospective data.ResultsThe data of 232 patients were analyzed. Our study showed a positive correlation between estradiol levels and the number of follicles (NF) (r= 0.592,p< 0.001), number of retrieved oocytes (NRO) (r= 0.576,p< 0.001), and number of mature oocytes (NMO) (r= 0.554,p< 0.001). E/follicle ratio did not have a significant correlation with NRO and NMO. E/Oocyte ratio had a strong negative correlation with NMO (r= −0.280,p< 0.001)ConclusionsSerum estradiol had a positive correlation with NF, NRO, and NMO. But E/O had a strong negative correlation with NMO. These results indicate that estradiol levels can be used as an important clinical tool in the prediction of oocyte and mature oocyte yield in ART cycles. Reproductive outcome in ART cycles is largely dependent on the number of oocytes and mature oocyte yield. Estradiol levels on the day of HCG appear to strongly correlate with the outcome of ART cycles.

BACKGROUND: This study aimed to explore the effects of triptorelin and leuprolide on serum hormone levels and the clinical efficacy of girls with idiopathic central precocious puberty (ICPP). METHODS: Retrospective analysis was performed on 128 girls with ICPP who were diagnosed and treated in our hospital from January 2017 to January 2020, including 71 girls in the leuprolide group and 57 girls in the triptorelin group. The differences of serum sex hormone level, ovarian volume, uterine volume, follicle diameter, bone age, growth rate (height change within half a year), maturity (bone age/living age), and other aspects between the two groups of girls were compared. RESULTS: Before treatment, there was no significant difference in the baseline levels of sex hormones [estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH)] between the triptorelin group and the leuprorelin group (P>0.05). After 1 year of treatment, serum levels of E2 and FSH in the triptorelin group were lower than those in the leuprolide group (P0.05). At baseline, there was no significant difference in the ovarian volume, follicle diameter, and uterine volume between the triptorelin group and the leuprolide group (P>0.05). After 1 year of treatment, the ovarian volume, follicle diameter, and uterine volume of the girls in the triptorelin group were all lower than those in the leuprolide group (P0.05). After 1 year of treatment, the growth rate and maturity of participants in the triptorelin group were lower than those in the leuprolide group (P0.05). CONCLUSIONS: For girls with ICPP, triptorelin is superior to leuprolide in reducing sex hormone level, reducing uterine volume, follicle diameter, ovarian volume, slowing down the growth rate, and decreasing maturity. Triptorelin should be selected as a priority for the treatment of girls with ICPP.

This study was conducted to (i) evaluate the requirement for the administration of GnRH coincident with insertion of a progesterone-releasing intravaginal device (PRID) and (ii) the effect of supplementing with 400 IU eCG at PRID removal on pregnancy per AI (P/AI) in spring and autumn calving suckled beef cows, subjected to a 7-d CO-Synch + PRID timed artificial insemination (TAI) program. Suckled beef cows (n = 1408) on 62 commercial farms were enrolled and randomly assigned to either of three treatments: 1) cows received a PRID and 100 μg GnRH on Day −10, followed by 25 mg PGF2α at PRID removal (Day −3) and 100 μg GnRH 72 h later (Day 0) at TAI (Treatment 1; n: spring = 236, autumn = 248); 2) as Treatment 1, but without GnRH at PRID insertion on Day −10 (Treatment 2; n: spring = 232, autumn = 227); 3) as Treatment 1, but cows also received 400 IU eCG at PRID removal on Day −3 (Treatment 3; n: spring = 233, autumn = 232). At Day −10, ovaries were examined by ultrasonography to evaluate the presence or absence of a corpus luteum (CL) and follicle(s) ≥ 10 mm in diameter. Body condition score (BCS) was assessed on a scale of 1–5. Pregnancy diagnosis was carried out 30–35 d after TAI by transrectal ultrasonography. Data were analyzed using the GENMOD and LOGISTIC procedures of SAS. There was a treatment by season interaction for P/AI (P 0.05) by treatment (50.1 v 53.7 v 48.7% for Treatments 1, 2 and 3, respectively). Overall, eCG had a positive effect on P/AI for cows lacking a CL at treatment initiation (P

The Drosophila ovary is regenerated from germline and somatic stem cell populations that have provided fundamental conceptual understanding on how adult stem cells are regulated within their niches. Recent ovarian transcriptomic studies have failed to identify mRNAs that are specific to follicle stem cells (FSCs), suggesting that their fate may be regulated post-transcriptionally. We have identified that the RNA-binding protein, Musashi (Msi) is required for maintaining the stem cell state of FSCs. Loss of msi function results in stem cell loss, due to a change in differentiation state, indicated by upregulation of Lamin C in the stem cell population. In msi mutant ovaries, Lamin C upregulation was also observed in posterior escort cells that interact with newly formed germ cell cysts. Mutant somatic cells within this region were dysfunctional, as evidenced by the presence of germline cyst collisions, fused egg chambers and an increase in germ cell cyst apoptosis. The msi locus produces two classes of mRNAs (long and short). We show that FSC maintenance and escort cell function specifically requires the long transcripts, thus providing the first evidence of isoform-specific regulation in a population of Drosophila epithelial cells. We further demonstrate that although male germline stem cells have previously been shown to require Msi function to prevent differentiation this is not the case for female germline stem cells, indicating that these similar stem cell types have different requirements for Msi, in addition to the differential use of Msi isoforms between soma and germline. In summary, we show that different isoforms of the Msi RNA-binding protein are expressed in specific cell populations of the ovarian stem cell niche where Msi regulates stem cell differentiation, niche cell function and subsequent germ cell survival and differentiation.