Follicles In Group Research Articles

Identification of functional circRNAs regulating ovarian follicle development in goats

BarkgroundCircular RNAs (circRNAs) play important regulatory roles in a variety of biological processes in mammals. Multiple birth-traits in goats are affected by several factors, but the expression and function of circRNAs in follicular development of goats are not clear. In this study, we aimed to investigate the possible regulatory mechanisms of circRNA and collected five groups of large follicles (Follicle diameter > 6 mm) and small follicles (1 mm < Follicle diameter < 3 mm) from Leizhou goats in estrus for RNA sequencing.ResultsRNA sequencing showed that 152 circRNAs were differentially expressed in small and large follicles. Among them, 101 circRNAs were up-regulated in large follicles and 51 circRNAs were up-regulated in small follicles. GO and KEGG enrichment analyses showed that parental genes of the differential circRNAs were significantly enriched in important pathways, such as ovarian steroidogenesis, GnRH signaling pathway, animal autophagy and oxytocin signalling pathway. BioSignal analysis revealed that 152 differentially expressed circRNAs could target 91 differential miRNAs including miR-101 family (chi-miR-101-3p, chi-miR-101-5p), miR-202 family (chi-miR-202-5p, chi-miR-202-3p),60 circRNAs with translation potential. Based on the predicted sequencing results, the ceRNA networks chicirc_008762/chi-miR-338-3p/ARHGAP18 and chicirc_040444/chi-miR-338-3p/STAR were constructed in this study. Importantly, the new gene circCFAP20DC was first discovered in goats. The EDU assay and flow cytometry results indicated that circCFAP20DC enhanced the proliferation of follicular granulosa cells(GCs). Real-time quantitative PCR and western blotting assays showed that circCFAP20DC activated the Retinoblastoma(RB) pathway and promoted the progression of granulosa cells from G1 to S phase.ConclusionDifferential circRNAs in goat size follicles may have important biological functions for follicular development. The novel gene circCFAP20DC activates the RB pathway, promoting the progression of GCs from G1 to S phase. This, in turn, enhances the proliferation of follicular GCs in goats.

Application of small animal ultrasound imaging technology for identification of polycystic ovary syndrome in a mouse model

Background and AimsPolycystic ovary syndrome (PCOS) is a hormonal disorder common among women of reproductive age, characterized by irregular menstrual periods, elevated levels of androgens, and polycystic ovaries, leading to various symptoms and complications such as infertility, metabolic issues, and increased risk of diabetes and heart disease. This study aimed to compare traditional histological methods and ultrasound imaging for consistency in identifying PCOS in a mouse model. The shortest time to construct the PCOS model using letrozole was determined. MethodsFemale C57/BL mice were randomly divided into three groups: Group A received normal saline and a regular diet; Group B received 1 mg/kg/day of letrozole with a regular diet; and Group C received 1 mg/kg/day of letrozole with a high-fat diet. All mice were administered letrozole by intragastric gavage daily for five weeks. The traditional identification method included measuring body weight, examining vaginal smears, monitoring the estrous cycle, measuring serum androgen levels, and performing H&E staining of ovarian tissues. The PCOS model was evaluated using ultrasound imaging to identify and monitor follicles. The significance of the difference between the traditional identification method and the ultrasonic method was calculated using the nonparametric McNemar test, and consistency between the two methods was assessed with the kappa-coefficient test. On this basis, the ultrasound imaging technology was used to monitor the model-making process for 2, 3 and 4 weeks, and to monitor the parameters of the ovary and follicles to judge the shortest time that gavage letrozole caused the appearance of vesicular follicles in the mouse ovary. ResultsThe traditional identification method showed no PCOS phenotype in group A mice, while groups B and C showed multiple ovarian cystic follicles, indicating successful model induction. The ultrasound imaging results were consistent with the traditional method, showing no PCOS in group A and multiple cystic follicles in groups B and C. The McNemar test revealed no significant difference between the traditional and ultrasonic identification methods. The kappa-coefficient test assessed consistency, yielding a value of 0.903, indicating strong agreement between the methods. The ovarian area, diameter, and the number and diameter of cystic follicles were not significantly changed at two weeks in the letrozole group compared with the control group. At three weeks, there were significant increases in the number and in the diameter of vesicular follicles compared with control cells. At four weeks, the number and diameter, the maximum cross-sectional area and diameter of the ovary were significantly increased compared with the control group. ConclusionsThe ultrasound and traditional methods provide consistent results for identifying PCOS in a mouse model. Construction of the PCOS model by letrozole gavage takes at least three weeks.

Local Regulatory Changes of HSD11B1 and NR3C1 in the Follicular and Luteal Tissue During Experimentally Induced Ovulation in the Cow.

The objective of the study was to characterise the expression patterns of the two key components of cortisol action namely HSD11B1 (11-beta-hydroxysteroid dehydrogenase type 1) and NR3C1 (nuclear receptor subfamily 3, group C, member 1, also known as the glucocorticoid receptor) in superovulation induced bovine follicles during the periovulation and subsequent corpus luteum (CL) formation. Bovine ovaries containing preovulatory follicles or CL were timely defined during induced ovulation as follows: 0 h before GnRH (Gonadotropin-releasing hormone) application, and 4, 10, 20, 25 (follicles) and 60 h (early CL) after GnRH. The low mRNA expression of HSD11B1 and NR3C1 in the follicle group before the GnRH application increased significantly in the follicle group 20 h after GnRH and remained high afterward also in the early CL group. In contrast, the high NR3C1 mRNA decreased in follicles 25 h after GnRH (close to ovulation) and significantly increased again after ovulation (early CL). Our results indicated the involvement of HSD11B1 and NR3C1 as the two key components of cortisol action in the local mechanisms coordinating final follicle maturation, ovulation, follicular-luteal transition and CL development in the cow.

MicroRNA expression profiles reveal wool development and fineness regulation in Gansu alpine fine-wool sheep

The development of wool has a complex regulatory mechanism both influenced by genetic and environmental factors. MicroRNAs (miRNA) were involved in various biological processes of animals, and may play an important role in the regulation of wool development. In this study, we comprehensively analyzed and identified the histological parameters of hair follicles, as well as the miRNAs, target genes, pathways, and Gene Ontology terms related to wool fineness regulation and wool growth and development using HE staining and RNA-Seqs methods. Both coarse (group C, mean fiber diameter (MFD) = 22.26 ± 0.69 μm, n = 6) and fine (group F, MFD = 16.91 ± 0.29 μm, n = 6) of Gansu alpine fine-wool sheep with different wool fineness were used in this study. The results showed that the primary follicle diameter and secondary wool fiber diameter in group C were significantly higher than those in group F (P < 0.05). And the number of primary and secondary hair follicles in group C was significantly lower than that in group F (P < 0.05). Furthermore, a total of 67 DE miRNAs and 290 potential DE miRNAs target genes were screened in the skin tissues of sheep from groups F and C, and some potential target genes related to wool fineness regulation were screened, such as CDH2, KRT82, FOXN1, LOC101106296, KRT20, MCOLN3, KRT71, and TERT. These genes were closely related to Glutathione metabolism, epidermal cell differentiation, keratinization, and regulation of hair cycle. Moreover, the regulatory network of miRNAs-mRNAs suggested that miRNAs (miR-129-x, novel m0079-3p, miR-2484-z, novel m0025-5P, etc.) may play a key role in the wool development and wool fineness regulation of Gansu alpine fine-wool sheep. In summary, this study expands the existing miRNAs database and provides new information for studying the regulation of wool development in Gansu alpine fine wool sheep.

The Role of Mannitol and Vitamin D in Ovarian Ischemia/Reperfusion Injury in Rats with Acute Abdominal.

This study was designed to investigate the effects of vitamin D and mannitol in an experimental rat ovarian torsion model. Thirty-two female Wistar albino rats were randomly classified as group 1: (sham), group 2: (detorsion), group 3: (detorsion + mannitol), group 4: (detorsion + vitamin D) and group 5: (detorsion + mannitol + vitamin D) (for each group n = 8). All groups were subjected to bilateral adnexal torsion for 2 h except for group 1. Bilateral adnexal detorsion was performed in all groups except for group 1. Groups 3 and 5 intraperitoneally received the injection of mannitol at a dose of 0.3 mg/kg 30 min before detorsion. Also, the group's 4 and 5 orally received vitamin D in a dose of 500 IU/kg/day for two weeks before torsion. Total oxidant status (TOS), total antioxidant status (TAS), oxidative stress index (OSI) and proliferating cell nuclear antigen (PCNA) levels were analyzed. According to the histopathological analyses, ovarian tissue damage and follicle counting were evaluated. TOS, OSI and histopathologic score values of ovarian tissue were significantly lower in group 5 than groups 2, 3 and 4 (p < 0.05). The PCNA level was significantly higher in group 5 than in groups 2, 3 and 4 (p < 0.05). A strong negative correlation was found between OSI and PCNA in groups 2, 3, 4 and 5 (r = -0.92, p = 0.01; r = -0.98, p < 0.0001; r = -0.98, p < 0.0001 and r = -0.96, p = 0.0002, respectively). The numbers of primordial follicles in group 5 (p < 0.001) and primary follicles in group 4 (p < 0.001) were significantly higher when compared to group 2. Based on the results of this study, it could be suggested that combination treatment of mannitol with vitamin D is more effective in reversing tissue damage induced by ischemia-reperfusion (I/R) injury in the ovarian torsion model than administration of only an agent.

Effect of the gonadotropin surge on steroid receptor regulation in preovulatory follicles and newly formed corpora lutea in the cow

The objective of the study was to characterize the mRNA expression patterns of specific steroid hormone receptors namely, estrogen receptors (ESRRA—estrogen related receptor alpha and ESRRB—estrogen related receptor beta) and progesterone receptors (PGR) in superovulation-induced bovine follicles during the periovulation and subsequent corpus luteum (CL) formation. The bovine ovaries (n = 5 cow / group), containing preovulatory follicles or early CL, were collected relative to injection of the gonadotropin-releasing hormone (GnRH) at (I) 0 h, (II) 4 h, (III) 10 h, (IV) 20 h, (V) 25 h (preovulatory follicles) and (VI) 60 h (CL, 2–3 days after induced ovulation). In this experiment, we analyzed the steroid receptor mRNA expression and their localization in the follicle and CL tissue. The high mRNA expression of ESRRA, ESRRB, and PGR analyzed in the follicles before ovulation is significantly reduced in the group of follicles during ovulation (25 h after GnRH), rising again significantly after ovulation in newly formed CL, only for ESRRA and PGR (P < 0.05). Immunohistochemically, the nuclei of antral follicles' granulosa cells showed a positive staining for ESRRA, followed by higher activity in the large luteal cells just after ovulation (early CL). In contrast, the lower PGR immunopresence in preovulatory follicles increased in both small and large luteal cell nuclei after follicle ovulation. Our results of steroid receptor mRNA expression in this experimentally induced gonadotropin surge provide insight into the molecular mechanisms of the effects of steroid hormones on follicular–luteal tissue in the period close to the ovulation and subsequent CL formation in the cow.

Effect of acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) on PI3K/AKT/FOXO3a pathway in mice with poor ovarian response

To observe the protective effect of acupuncture at "Zhibian" (BL 54) through "Shuidao (ST 28)" based on the PI3K/AKT/FOXO3a pathway in mice with poor ovarian response (POR), and to explore the possible mechanism of acupuncture in inhibiting ovarian granulosa cells apoptosis in POR. A total of 45 mice with regular estrous cycles were randomly divided into a blank group, a model group and an acupuncture group, with 15 mice in each group. Mice in the model group and the acupuncture group were given triptolide suspension (50 mg•kg-1•d-1) by gavage for 2 weeks to establish POR model. After successful modeling, mice in the acupuncture group were given acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) for 2 weeks, once a day, 20 min each time. Ovulation induction was started the day after the intervention ended, and samples were taken from each group after ovulation induction. Vaginal smears were used to observe changes in the estrous cycle of mice. The number of oocytes retrieved, ovarian wet weight, final body weight, and ovarian index were measured. The levels of anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), estradiol (E2), and luteinizing hormone (LH) in serum were detected by ELISA. The morphology of ovarian tissue was observed by HE staining. The apoptosis of ovarian granulosa cells was detected by TUNEL staining. The mRNA expression of PI3K, AKT, and FOXO3a in ovarian tissue was detected by real-time fluorescence quantitative PCR. The protein expression of Bcl-2 associated X protein (BAX), caspase-3, phosphorylated phosphatidylinositol 3-kinase (p-PI3K), and phosphorylated protein kinase B (p-AKT) in ovarian tissue was detected by Western blot. Compared with the blank group, the rate of estrous cycle disorder in the model group was increased (P<0.01); compared with the model group, the rate of estrous cycle disorder in the acupuncture group was decreased (P<0.01). Compared with the blank group, the number of oocytes retrieved, ovarian wet weight, ovarian index, and final body weight in the model group were decreased (P<0.01); compared with the model group, the number of oocytes retrieved, ovarian index, and ovarian wet weight were increased (P<0.01, P<0.05), and there was no significant difference in final body weight (P>0.05) in the acupuncture group. Compared with the blank group, the serum levels of FSH and LH were increased (P<0.01), and the serum levels of AMH and E2 were decreased (P<0.01) in the model group; compared with the model group, the serum levels of FSH and LH were decreased (P<0.01, P<0.05), and the serum levels of AMH and E2 were increased (P<0.01, P<0.05) in the acupuncture group. Compared with the blank group, the number of normal developing follicles in ovarian tissue in the model group was decreased and the morphology was poor, while the number of atretic follicles increased; compared with the model group, the number, morphology, and granulosa cell structure of follicles in the acupuncture group improved to varying degrees, and the number of atretic follicles decreased. Compared with the blank group, the apoptosis rate of ovarian granulosa cells in the model group was increased (P<0.01); compared with the model group, the apoptosis rate of ovarian granulosa cells in the acupuncture group was decreased (P<0.01). Compared with the blank group, the FOXO3a mRNA expression and caspase-3 and BAX protein expression in ovarian tissue in the model group were increased (P<0.01), and the mRNA expression of PI3K and AKT and the protein expression of p-PI3K, p-AKT, and p-FOXO3a in ovarian tissue were decreased (P<0.01); compared with the model group, the mRNA expression of FOXO3a and protein expression of caspase-3 and BAX in ovarian tissue in the acupuncture group were decreased (P<0.05, P<0.01), and the mRNA expression of PI3K and AKT and the protein expression of p-PI3K, p-AKT, and p-FOXO3a in ovarian tissue were increased (P<0.01, P<0.05). Acupuncture at "Zhibian" (BL 54) through "Shuidao" (ST 28) could inhibit ovarian cell apoptosis, and improve ovarian function in POR mice, and its mechanism may be related to the regulation of key factors in the PI3K/AKT/FOXO3a pathway.

The impact of heterotopic ovarian tissue transplantation with varied volumes on reproductive endocrine function in oophorectomized rats

Background This study aimed to investigate the effects of different volumes of ovarian tissue transplantation on the reproductive endocrine function of rats after oophorectomy. Methods Female rats were selected to establish a castration model and then underwent different volumes of ovarian tissue transplantation. Group I served as the sham operation group. The transplantation group was divided into five subgroups based on the calculated ratio of ovarian weight to body weight in normal female rats, δ = (2.52 ± 0.17) ×10−4: Group II: transplanted ovarian volume was δ; Group III: 0.75δ; Group IV: 0.5δ; Group V: 0.25δ; Group VI: without ovarian transplantation. The post-transplant oestrous cycle recovery was observed, and blood samples were collected every 2 weeks to measure serum hormone levels. Histological evaluation was performed at the end of the observation period. Results Rats in Group V exhibited disrupted oestrous cycles after transplantation, which were significantly longer than those in Group I. Rats in Groups II, III, and IV showed no cyclic changes. At 6 weeks post-transplantation, rats in Group V had lower E2 and AMH levels and higher FSH levels compared to Group I. The uterine wet weight and the number of normal follicles in Group V were significantly lower than those in Group I, but the number of atretic follicles was higher than in Group I. Conclusion The larger ovarian tissue transplantation resulted in a faster recovery with a higher survival rate of the uterus and normal follicles, compared to smaller ovarian tissue transplantation.

Long-Term Protective Effects of Lamotrigine in a Rat Ovarian Ischemia-Reperfusion Model

Ovarium torsion is a gynecological emergency that is common in women of reproductive age and requires early diagnosis and intervention. In this study, we aimed to investigate the long-term anatomical, histological and biochemical protective effects of lamotrigine in ovariums in the ischemia – reperfusion (I–R) model created experimentally in rats. A total of 40 female Sprague-Dawley rats, 14 weeks old, weighing 220-270 grams were used in the study. The subjects were randomly distributed to form 4 groups named SHAM group, I - R group, I - R + Lamotrigine (LTG) group and R + LTG group. Under general anesthesia, the ovaries of the rats were reached and ischemia was created for 3hours with vascular clamps. 20mg / kg LTG was administered intraperitoneally (ip.) to group 3 30minutes before ischemia and to group 4 30minutes before reperfusion. At the third hour of ischemia, the vascular clamps were opened and the abdomen of the rats was closed according to the surgical procedure. The rats were followed up for 28 days postoperatively and the ovarium tissues taken on the 28th day were examined anatomically and histologically. Biochemically, estradiol (E2), follicle stimulating hormone (FSH) and antimullerian hormone (AMH) levels were measured from blood samples taken from their hearts. Granulosa cells with diffuse vaculations were observed in degenerative follicles in group I-R. Again in this group, severe hemorrhage, fibrosis and edematous areas were observed in the ovarium stroma (Ovarian Histopathological Scoring (OHS): 7). In the I - R + LTG group, OHS was statistically significantly lower than the I - R group (OHS: 2; p <0.000). In the R + LTG group, although the OHS score was calculated to be lower than the I - R group, there was no statistically significant difference (OHS: 6; p> 0.05). The protective effect of LTG against experimentally created ischemia and reperfusion damage was determined anatomically and histologically. No protective effect of LTG was observed in terms of FSH, E2 and AMH values measured from the blood sera of rats. These findings may provide a basis for future studies using LTG to treat ovarian ischemia-reperfusion injury.

Effect of Coenzyme Q10a and Clomiphene Citrate for Ovulation Induction in Patients with PCOS as Compared to Clomiphene Citrate alone

To evaluate the effects of coenzyme Q10A and clomiphene citrate taken in addition to other drugs to stimulate ovulation in people with polycystic ovarian syndrome. The Department conducted the study of Obstetrics and Gynecology, Pakistan Ordinance Factory (POF), Wah Cantonment, Rawalpindi from Mar 2019 to Feb 2021 By using a lottery, all of the patients were split into two random groups. Group A was given Coenzyme Q10A 60mg daily from day 1 of the cycle thrice a day and clomiphene citrate 150mg/day from days 2-6 of the cycle, and Group B was given only clomiphene citrates 150mg/day from 2-6 day of cycle for 5 days. The follicular growth and maturation were observed on days 10, 12, and 14 of the cycle with transvaginal ultrasound. Ovulation was confirmed by measuring serum progesterone level on day 21 of the cycle. Patients were followed for 3 cycles.The mean number of mature follicles in Group A was 1.63+0.49 and 1.3+0.47 in Group B (p-value=0.01). The analysis of either of the groups' ovulation statuses revealed that in 70% of patients (n=21) in Group A and 36.67% (n=11) in Group B, ovulation was initiated (p value= 0.01). We concluded that the outcome of combined coenzymeQ10A and clomiphene citrate is significantly better for patients with polycystic ovarian syndrome; inducing ovulation is more effective with clomiphene citrate alone.

THU199 Using Primary Human Ovarian Stromal Cells To Elucidate Stromal Cell Influences On Egg Quality Across Pubertal Development

Abstract Disclosure: E. Tsui: None. M.M. Laronda: None. Follicles, the functional unit of the ovary with the potential to develop into mature eggs, are composed of a single germ cell and somatic support cells (i.e., granulosa and theca cells). While animal studies have demonstrated a positive effect of ovarian stromal cells to follicle development, the ovarian stromal compartment remains largely undescribed. Moreover, egg quality is maximized after the pubertal transition, but the impact of puberty on ovarian stromal cells and their influence on egg quality is not well understood. To elucidate the relationship between puberty, ovarian stromal cells, and follicle development, we utilized a follicle growth assay in which murine ovarian secondary follicles were cultured in three groups: alone (control), with pre-pubertal derived primary human ovarian stromal cells, or with post-pubertal derived primary human ovarian stromal cells. Follicles (n = 40 per group, N = 4 independent experiments) were monitored over 8 days by brightfield microscopy and evaluated for diameter and survival. No difference in follicle diameter or survival was detected between follicles grown with either pre-pubertal or post-pubertal derived stromal cells (diameter: p = 0.93 for days 2-8; survival: p &amp;gt; 0.9999 at day 8), however, each group of follicles co-cultured with stromal cells grew to larger diameters than follicles grown alone at days 2 (pre-pubertal and post-pubertal, p = 0.005), 4 (pre-pubertal, p = 0.0006; post-pubertal, p = 0.0002), 6 (pre-pubertal, p = 0.03; post-pubertal, p = 0.02), and 8 (pre-pubertal, p = 0.001; post-pubertal, p = 0.004). Survival between follicles grown alone or co-cultured with stromal cells trended towards significance (p = 0.0526 for both pre-pubertal and post-pubertal stroma groups), and all groups demonstrated average survival at day 8 above 70%. Follicle rupture was more commonly observed in follicles cultured with stromal cells (control, 10%; pre-pubertal, 40%; post-pubertal, 80%), with additional maturation studies in progress. Concurrently, the cellular landscape of ovarian stromal cells across puberty was evaluated with single cell RNA sequencing (n = 11, age range = 0.34 to 16.09 years, Tanner stages 1 and 3-5). Analyses revealed 12 sub-clusters representing five major cell types: endothelial cells, theca cells, vascular smooth muscle cells, granulosa cells, and immune cells. This data is screened through existing human secretome databases to identify candidate secreted factors that will be investigated in future follicle growth assays. These results demonstrate a benefit of human ovarian stromal cells in our follicle growth assay and, in conjunction with ongoing maturation and sequencing experiments, validate the utility of this assay to further understand how puberty affects the stromal microenvironment to impact egg quality, insight that can be used to develop and improve future reproductive technologies. Presentation: Thursday, June 15, 2023

The effect of unexpected follicular development in artificial cycles on the clinical outcomes in frozen thawed embryo transfer based on propensity score matching

Objective: To study the effect of unexpected follicular development in artificial cycles on the clinical outcomes in frozen thawed embryo transfer based on propensity score matching(PSM). Methods: The retrospective cohort study analyzed the clinical data of 7 064 cycles (5 716 patients) of artificial cycle frozen-thawed embryo transfer (AC-FET) in the Reproduction Center of the Third Affiliated Hospital of Zhengzhou University from January 1, 2016 to December 31, 2020. The clinical data were divided into three groups according to the degree of follicular development in AC-FET: no follicular growth group (group A, 6 349 cycles), small follicular growth group (group B, 248 cycles), and large follicular growth group (group C, 467 cycles). Differences in clinical outcomes between the small follicle growth group (Group B) and the large follicle growth group (Group C) were compared with the no follicle growth group (Group A) after PSM and logistic regression to adjust for confounding factors at baseline. A binary logistic regression model was used to analyze the factors related to the unanticipated follicular development in AC-FET. Results: Age [M(Q1,Q3)] was [31.0 (28.0, 36.0)] years in Group A, [34.5 (30.0, 40.0)] years in Group B, and [36.0 (31.0, 41.0)] years in Group C. After adjusting for confounders, the differences between Groups A and B in clinical pregnancy rate (P=0.169), live birth rate (P=0.318), early abortion rate (P=0.470), and miscarriage rate (P=0.783) were not statistically significant. The differences in clinical pregnancy rate (P=0.743), live birth rate (P=0.486) and miscarriage rate (P=0.080) between Groups A and C were not statistically significant, while early miscarriage rate (P=0.034) differences were statistically significant. The age, BMI, basal AFC, AMH and starting dose of estrogen were correlates of the emergence of non-expected small follicles in Groups B and A. The adjusted OR (AOR) values (95%CI) were 1.03 (1.01-1.06), 0.93 (0.90-0.98), 0.97 (0.95-0.99), 0.96 (0.95-0.97), and 0.59 (0.45-0.77), all P<0.05. Age, basal AFC, AMH and starting dose of estrogen were the associated factors of the appearance of non-expected large follicles in Groups C and A. The AOR values (95%CI) were 1.03 (1.01-1.05), 0.93 (0.91-0.95), 0.96 (0.95-0.97), and 0.52 (0.42-0.64), all P<0.05. Conclusions: In AC-FET, the clinical outcome of small follicular growth is similar to that of unfollicular growth; Compared with the growth without follicles, the growth and development of large follicles can reduce the early abortion rate; Patients with older age, less AFC, lower AMH, and lower initial dose of estrogen could be more likely to have unanticipated follicular development during endometrial preparation.

Does the addition of metformin to carboplatin treatment decreases ovarian reserve damage associated with carboplatin usage?

BackgroundWe aimed to determine whether adding metformin to carboplatin treatment would reduce the damage to ovarian reserve associated with carboplatin use.MethodsWe included 35 adult female non-pregnant albino Wistar rats approximately three months old, weighing 220–310 g. The rats were divided into five groups of seven rats according to the treatment they received. Carboplatin and salin was given to Group 2, and carboplatin plus metformin was given to Group 3. Group 4 was administered only metformin. Group 5 was administered only salin. Carboplatin was given to Groups 2 and 3 as a single dose on the 15th day, while metformin was given to Groups 3 and 4 during the 28-day experiment. After oophorectomy, histopathologic analyses of primordial, primary, secondary, and tertiary Graff follicles according to the epithelial cells surrounding the oocyte and total follicular number were conducted per section. Serum Anti-Mullerian Hormone (AMH), tissue catalase, and malonyl dialdehyde levels were measured and compared within each group.ResultsThe baseline and 15th-day serum AMH values of the menstrual cycle were compared among the groups, and no statistically significant differences were observed (p > 0.05). Group 3, which was given both carboplatin and metformin, had statistically significantly higher 28th-day AMH levels than Group 2, which was given only carboplatin and saline (p < 0.001). The number of primordial follicles in Group 3 was found to be statistically significantly higher than in Group 2 (p < 0.001). Tissue catalase enzyme levels in Group 3 were statistically significantly higher than in Group 2 (p < 0.001). Tissue malondialdehyde levels in Group 2 were statistically significantly higher than tissue malondialdehyde levels in Groups 3 and 4 (p < 0.001).ConclusionsMetformin may attenuate carboplatin-induced ovarian damage, possibly through its antioxidative effects.

Evaluation of cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles in jennies.

Reference values of follicular fluid components at different stages of follicular development in jennies are unknown. It is important to estimate such values because the biochemical composition of follicular fluid provides a suitable microenvironment for oocyte development, growth and maturation. The experimental protocol was carried out in the Equine Reproduction Clinic of the FMVZ-UNAM and was approved by an Institutional Sub-committee for the Care and Use of Experimental Animals (SICUAE.MMVZ-2020/4-2). Seven healthy female donkeys “Mexican burro” with average body condition score of 3 on a scale of 1 to 5 (Svendsen et al. The professional Handbook of the Donkey. 2008), were monitored by transrectal ultrasonography through the Veterinary Digital Diagnostic Ultrasound System 5 MHz (Model, DUS 60 VET) with a multi-frequency linear transducer. The ovum pick up (OPU) by transvaginal aspiration (TVA) was performed in a total of 70 preovulatory follicles, 10 for each jenny using a real- time B- mode ultrasound vaginal 6.5 MHz (Sonovet 600) with curvilinear transducer and 12 gauge (12 G) needle. Follicular fluid components were measured and analyzed by Luminex Multiplex Assays at the Universidad Nacional Autónoma de México. The relationship between cortisol, thyroxine (T3) and triiodothyronine (T4) concentrations in preovulatory follicles and follicular size was assessed. The preovulatory follicles are classified in three groups accordingto the diameter: small (27-30 mm), medium (31-35 mm) and large (36-38 mm). Concentrations of cortisol were lower in the large follicles group (18.8 ng/ml) compared with small (721.44 ng/ml) and medium follicles (361 ng/ml). The concentrations of thyroxine were higher in the medium follicle group (49 ng/ml) than in the small (21ng/ml) and large follicle groups (15.9 ng/ml). Finally, the concentration of triiodothyronine was higher in the small follicles group (1043 ng/ml) followed by the medium (545 ng/ml) and the lowest concentration was in the large follicles group (22 ng/ml). It is concluded that thyroxine concentrations are lower than T4 concentrations in preovulatory follicles irrespective of size as reported for human follicular fluid (Wakim. et. al. Fertil Steril 1993; 59(6) 1187–1190). Furthermore, no relationship was found between preovulatory follicular size and thyroxine hormone concentrations. It has been suggested that cortisol and triiodothyronine concentrations are related to preovulatory follicle size. Cortisol concentrations are higher in preovulatory follicles that in subordinate follicles and cortisol is a mediator of apoptosis in granulosa cells, differentiation, development and maturation of tissues, specifically in the oocyte (Acosta & Miyamoto. Anim Reprod Sci 2004; 82:127-140, Scarlett. et al. Dom Anim Endocrinol 2016; 59:11-22). The role of cortisol within the different sizes of preovulatory follicles is unknown.

Hair Follicle Transcriptome Analysis Reveals Differentially Expressed Genes That Regulate Wool Fiber Diameter in Angora Rabbits

Wool fiber diameter (WFD) is an important index of wool traits and the main determinant of wool quality and value. However, the genetic determinants of fiber diameter have not yet been fully elucidated. Here, coarse and fine wool of Wan strain Angora rabbits and their hair follicle traits were characterized. The results indicated significant differences in the diameters of wool fibers and their hair follicles. The RNA sequencing (RNA-Seq) technique was used to identify differences in gene expression in hair follicles between coarse and fine wool. In total, 2574 differentially expressed genes (DEGs) were found between the two hair follicle groups. Transcription factors, keratin-associated protein (KAP) and keratin (KRT) families, and ECM-related genes may control the structure of fine fibers in rabbits. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that skin development, epidermal cell and keratinocyte differentiation, epithelium development, and Notch and ribosome signaling pathways were significantly enriched, respectively. GSEA further filtered six important pathways and related core genes. PPI analysis also mined functional DEGs associated with hair structure, including LEF1, FZD3, SMAD3, ITGB6, and BMP4. Our findings provide valuable information for researching the molecular mechanisms regulating wool fiber and could facilitate enhanced selection of super-fine wool rabbits through gene-assisted selection in the future.

Phosphoproteomic analysis on ovarian follicles reveals the involvement of LSD1 phosphorylation in Chicken follicle selection

BackgroundFollicle selection in chickens refers to the process of selecting one follicle from a group of small yellow follicles (SY, 6–8 mm in diameter) for development into 12–15 mm hierarchal follicles (usually F6 follicles), which is controlled by sex hormones including follicle-stimulating factor (FSH), estrogen and progesterone. Follicle selection is a critical process impacting egg production in chicken, therefore, is the focus of many studies. Phosphorylation is important for the proper function of proteins, thus, needs to be analyzed by proteomic level.ResultIn this study, we compared the phosphoproteomes of SY and F6 follicles in laying hens and identified 2,386 phosphoproteins and 5,940 phosphosites, of which 4,235 sites of 1,963 phosphoproteins were quantified. From SY to F6 follicles, 190 phosphorylation sites of 149 proteins changed significantly, among which the phosphorylation level of lysine demethylase 1 A (LSD1) at the conserved 54th serine (LSD1Ser54p) was significantly upregulated in F6 follicles compared to SY follicles (p < 0.05); however, the expression of chicken LSD1 were not significantly different on both mRNA and protein levels. LSD1Ser54p is mainly located in the nucleus of both SY and F6 follicles, and was higher in F6 follicles than that of SY follicles revealed by both immunofluorescence and Western blotting. LSD1Ser54p level increased after treatment with 5 ng/mL and 10 ng/mL of FSH in the theca cells and the granulosa cells of pre-hierarchal follicles, and with 50 ng/mL in granulosa cells of hierarchal follicles. In the theca cells of hierarchal follicles, estrogen stimulated the level of LSD1Ser54p in a dosage-dependent manner, and in granulosa cells of pre-hierarchal follicles, 10 ng/mL of estrogen increased LSD1Ser54p expression. Treatment with 50 ng/mL of progesterone increased LSD1Ser54p expression in theca cells of pre-hierarchal follicles, and with 10 to 100 ng/ml enhanced LSD1Ser54p expression in the granulosa cells of hierarchal follicles.ConclusionThe expression dynamics of LSD1Ser54p in follicles from SY to F6 and its regulation by sex hormones suggest that it is involved in chicken follicle selection.

Effect of follicular size on clinical outcomes of frozen-thawed embryo transfer induced by hCG on ovulation in natural cycles

Objective: To investigate the effect of follicular size on the clinical outcomes of frozen-thawed embryo transfer induced by human chorionic gonadotropin (hCG) of natural cycles on ovulation. Methods: Clinical data of 427 cycles of frozen-thawed single blastocyst transfer in Nanjing Drum Tower Hospital from January 2016 to December 2019 were retrospectively analyzed. The patients were divided into 15-16 mm group (15≤diameter≤16 mm, n=66), 16-17 mm group (16<diameter≤17 mm, n=101), 17-18 mm group (17<diameter≤18 mm, n=125), 18-20 mm group (18<diameter≤20 mm, n=109),>20 mm group (diameter>20 mm, n=26), according to the maximum follicle diameter on the induction day of hCG ovulation induction. The estradiol and luteinizing hormone (LH) levels, and clinical pregnancy rate, abortion rate and live birth rate were compared in five groups. Results: There were statistically significant differences in estradiol and LH levels among the five groups on the day of hCG induction (all P<0.05). Estradiol levels in 15-16 mm group to >20 mm group gradually increased on the day of hCG induction, and estradiol level in 15-16 mm group was significantly lower than those in 17-18 mm group, 18-20 mm group and >20 mm group (median: 1 002.3 vs 1 103.3 vs 1 171.2 vs 1 539.0 pmol/L), with statistical significances (P=0.034, P<0.001, P=0.002). On the day of hCG induction, LH levels in 15-16 mm group to >20 mm group showed a decreasing trend, and LH level in 15-16 mm group was significantly higher than those in 17-18 mm group and >20 mm group (median: 37.73 vs 28.24 vs 24.11 U/L), with statistically significant differences (P=0.007, P=0.006). There were no significant differences in clinical pregnancy rate, abortion rate and live birth rate in 15-16 mm group to >20 mm group (all P>0.05). Conclusion: In the natural cycle protocol of hCG induced ovulation, the small follicle group could achieve similar clinical outcomes compared with normal sized follicles in the single blastocyst transfer of frozen-thawed embryos.

Live birth associated with peak serum estradiol levels in letrozole intrauterine insemination cycles

To identify whether the serum estradiol (E2) level on the day of human chorionic gonadotropin (hCG) trigger or luteinizing hormone (LH) surge (hCG-LH) was associated with the live birth rate (LBR) during ovulation induction (OI) or controlled ovarian hyperstimulation with letrozole followed by intrauterine insemination (IUI). Retrospective cohort study. Large, multicenter private practice. A total of 2,368 OI-IUI cycles in patients treated with letrozole followed by IUI were evaluated from January 1, 2014, to July 31,2019. Ovulation induction with letrozole, followed by autologous IUI. The primary outcome measure was the LBR as a function of the serum E2 level at the time of hCG administration or LH surge, adjusting for age, body mass index, the largest follicle diameter, and the number of follicles ≥14 mm in diameter. The clinical pregnancy rate as a function of the E2 level, pregnancy rate as a function of the lead follicle diameter, and pregnancy loss rates were the secondary outcome variables. A total of 2,368 cycles met the inclusion criteria. Outcomes were evaluated at the 25th (E2 level, 110 pg/mL), 50th (157 pg/mL), 75th (225 pg/mL), and 90th (319 pg/mL) percentiles. The LBRs ranged from 9.4% to 11.1% in the lower E2 cohorts and from 12.5% to 13.5% in the higher E2 cohorts. The LBR was significantly greater in the cohort of women with higher E2 levels in all percentile comparisons except for the 90th percentile. The mean periovulatory follicle diameter of ≥20 or <20 mm was not independently associated with the LBR or clinical pregnancy rate, despite a significantly higher mean E2 level in the larger follicle group. In letrozole OI cycles followed by IUI, lower LBRs and clinical pregnancy rates were found in women with lower E2 levels than in those with higher E2 levels at the 25th, 50th, and 75th percentile E2 level quartiles. Where possible, delaying hCG trigger until the E2 level increases after aromatase inhibition and approaches the physiologic periovulatory level may improve the pregnancy rates with letrozole followed by IUI.

The Effects of Sub-chronic Administration of the Ethanolic Extract of Chromolaena odorata Leaves on the Immune System and Spleen of Male Wistar Rats

Chromolaena odorata, a neglected weed with wide spectrum is a multipurpose plant that contains high concentration of anti-inflammatory, anti-diarrheal, anti-hypertensive agents that protects against tissue damage. This study was designed to evaluate the effect of ethanolic leaf extract of Chromolaena odorata on the immune system and spleen of Albino Wistar Rats. 41 adult male rats weighing between 110g -140g were used for this study. LD50 was carried out using 13 rats while 28 rats were used for the experiment proper. The rats used for the experiment proper were divided into four groups of 7 animals each. Group A served as control group and received animal feed and distilled water only, Group B received 1000mg/kg of the leaf extract and water, Group C received 3000mg/kg of the leaf extract and water, and Group D received 6000mg/kg of the leaf extract and water. The ethanolic leaf extract of Chromolaena odorata was administered 4-times per week for 13 weeks. The animals were killed using diethyl ether suffocation, blood samples were collected by ocular puncture for haematological analysis of RBC, WBC and differentials, Platelets, CD4 and CD8 cells count, the spleen and peyer’s patches harvested, weighed and the spleen immediately fixed in 10% formol saline for histological analysis. Data was considered significant at p&lt;0.05. Result showed a significant increase (p&lt;0.05) in body weight in the groups when compared to the control group A. Peyer’s patches had a significant decrease (p&lt;0.05) in group B and C, while group D had a significant increase (p&gt;0.05). Hemoglobin concentration, and PCV revealed a significant (p&lt;0.05) decrease when compared to the control group. RBC result showed a significant (p&lt;0.05) decrease when compared to group A. Platelet count revealed a significant (p&lt;0.05) increase in the groups. CD4 and CD8 count also revealed a significant increase. Histopathological study showed reactive lymphoid follicles in group B, circumferential zone of necrosis around a disc shaped eosinophilic, foreign material was seen in group C, and group D had necrosis when compared to control group. In conclusion, the ethanolic leaf extract of Chromolaena odorata have toxic effect on hematological indices of RBC, PCV with no effect on immune cell functions of Cd4 and CD8 cell counts and white blood cells.

RESULTS OF IN VITRO FERTILIZATION IN STERILITY PATIENTS USING MELATONIN

The objective – to evaluate the results of in vitro fertilization in patients with infertilitywhen using melatonin.Material and methods. The study of 67 women with infertility was conducted on the basis of«Medical Center of Infertility Treatment» (Chernivtsi). Group A included 29 patients who tookinternally daily 1 tab. (3 mg) of the drug «Vita-melatonin» produced by Kyiv Vitamin Plant for one month before sleep two weeks before and during the controlled ovarian stimulation(COS), group B included 38 patients who did not take this drug. There were no womenamong the patients who worked night shifts. ELISA kits by IBL company (Germany) wereused to determine melatonin levels. Melatonin levels were determined in blood plasmaand follicular fluid obtained during the puncture at 9:00 am.The criteria for inclusion of patients in the study were as follows: age 22-41 years (meanage – 31.4 years), no hereditary disease or severe forms of pathozoospermia in partner,controlled ovarian stimulation (COS) using gonadotropin- releasing hormone antagonists,work with oocytes at M2 stage.The following indicators were assessed in this prospective study: the incidence ofpreembryonic development to the 4-8th cell stage, compaction and formation of blastocystsin the period from the 2nd to the 6th day of embryonic development.Statistical processing of the obtained results was performed on a personal computerusing the licensed programs «Microsoft Exelle» and «Statistica».Results. When puncturing follicles in group A, we obtained 451 oocytes during puncture,and in group B – 616. When evaluating the obtained mature oocytes, it should be notedthat we did not observe a significant difference in their quantity: 83.4 ± 6.94 % in groupA. and 81.5 ± 6.36 % in group B. From mature oocytes in the incubation process afterfertilization in group A 185 cells developed into blastocysts, which made up to 49.2± 9.54 %, and in group B – 237, which amounted to 47.2 ± 8.72 %. In the process ofblastocyst development we evaluated them by classes: class I in group A was 48.6 ±9.36 %, and in group B – 46.4 ± 8.17 %, respectively, class II – 36.8 ± 8.94 % and 44.4 ±8.13 %, class III – 14.6 ± 6.67 % and 9.3 ± 4.78 %. At first sight, it seems that in groupA there were more mature oocytes, blastocysts in general and blastocysts in class I, inclass II – less blastocysts. But, we emphasize, that a significant difference was not found.The incidence of pregnancy for all transfers in patients of group A, where women usedmelatonin in ART programs, was 56.8 ± 8.25 %, and in group B – 52.8 ± 7.62 % (p &gt;0.05). Considering the onset of pregnancy in general, it should be noted that in group A itwas 86.2 ± 6.41 %, which was statistically different from the indicators of group B, wherethe incidence was 76.3 ± 6.95 % (p ˂ 0.001).Conclusions. The analysis of melatonin use in patients with infertility for two weeksbefore the expected menstruation and during controlled ovarian stimulation showedthat the incidence of pregnancy was generally significantly higher. Considering theembryological parameters, it should be noted that in general in this group with melatoninuse we received more mature oocytes and more blastocysts of class I, although there wasno significant difference.