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Background: Infertility is a health problem that affects approximately 10-15% of couples trying to achieve pregnancy worldwide. Various studies have shown that Nigella sativa (N. Sativa) has many health benefits, including reproductive health. However, studies examining the effectiveness of N. Sativa in preventing folliculogenesis disorders have never been reported. Purpose: This study aims to determine the benefits of administering N. Sativa in preventing folliculogenesis disorders as measured by the diameter of the antral follicles and the thickness of the granulosa cell layer. Methods: This research design uses an experiment with a randomized posttest-only controlled group design approach. There were 27 experimental animals (mice) divided into three groups, namely the negative control group (9 animals) and the positive control group (9 animals), as well as the treatment group (9 animals). The positive control group was exposed to two cigarettes of cigarette smoke per day. The treatment group was exposed to cigarette smoke and N. Sativa 300 mg/KgBW/day for 28 days. After that, the mice were sacrificed to take their ovaries. Then, the ovarian organ samples were stained with Hematoxylin-eosin and examined for the diameter of the antral follicles and the thickness of the granulosa cell layer in the antral follicles with photo slides at 400x magnification in 5 fields of view, statistical analysis using the Independent T-test. Results: There were significant differences in antral follicle diameter and granulosa cell thickness in mice exposed to cigarette smoke and N. Sativa. Administration of N. Sativa extract can increase granulosa cell thickness through estrogenic function. Administration of N. Sativa extract showed estrogenic activity by increasing serum estradiol levels. N. Sativa heterotrophic activity cannot be separated from the flavonoid and phenolic compounds contained in N. Sativa, which have been proven to have high estrogenic activity. Conclusion: N. Sativa extract is efficacious in improving the diameter of antral follicles and the thickness of granulosa cells caused by exposure to cigarettes.

Abstract Background An unexpected impaired ovarian response pertains to an insufficient reaction to controlled ovarian hyperstimulation. This deficient reaction is identified by a reduced count of mature follicles and retrieved oocytes during an IVF cycle, potentially diminishing the likelihood of a successful pregnancy. This research seeks to examine whether the characteristics of antral follicles can serve as predictive indicators for the unexpected impaired ovarian response to controlled ovarian stimulation (COS). Methods This retrospective cohort study was conducted at a tertiary university hospital. The electronic database of the ART (assisted reproductive technologies) center was screened between the years 2012–2022. Infertile women under 35 years, with normal ovarian reserve [anti-Müllerian hormone (AMH) > 1.2 ng/ml, antral follicle count (AFC) > 5] who underwent their first controlled ovarian stimulation (COS) cycle were selected. Women with

The development of the ovarian antral follicle is a complex, highly regulated process. Oocytes orchestrate and coordinate the development of mammalian ovarian follicles, and the rate of follicular development is governed by a developmental program intrinsic to the oocyte. Characterizing oocyte signatures during this dynamic process is critical for understanding oocyte maturation and follicular development. Although the transcriptional signature of sheep oocytes matured in vitro and preovulatory oocytes have been previously described, the transcriptional changes of oocytes in antral follicles have not. Here, we used single-cell transcriptomics (SmartSeq2) to characterize sheep oocytes from small, medium, and large antral follicles. We characterized the transcriptomic landscape of sheep oocytes during antral follicle development, identifying unique features in the transcriptional atlas, stage-specific molecular signatures, oocyte-secreted factors, and transcription factor networks. Notably, we identified the specific expression of 222 genes in the LO, 8 and 6 genes that were stage-specific in the MO and SO, respectively. We also elucidated signaling pathways in each antral follicle size that may reflect oocyte quality and in vitro maturation competency. Additionally, we discovered key biological processes that drive the transition from small to large antral follicles, revealing hub genes involved in follicle recruitment and selection. Thus, our work provides a comprehensive characterization of the single-oocyte transcriptome, filling a gap in the mapping of the molecular landscape of sheep oogenesis. We also provide key insights into the transcriptional regulation of the critical sizes of antral follicular development, which is essential for understanding how the oocyte orchestrates follicular development. [ABSTRACT FROM AUTHOR]

Background: An unexpected impaired ovarian response pertains to an insufficient reaction to controlled ovarian hyperstimulation. This deficient reaction is identified by a reduced count of mature follicles and retrieved oocytes during an IVF cycle, potentially diminishing the likelihood of a successful pregnancy. This research seeks to examine whether the characteristics of antral follicles can serve as predictive indicators for the unexpected impaired ovarian response to controlled ovarian stimulation (COS). Methods: This retrospective cohort study was conducted at a tertiary university hospital. The electronic database of the ART (assisted reproductive technologies) center was screened between the years 2012–2022. Infertile women under 35 years, with normal ovarian reserve [anti-Müllerian hormone (AMH) > 1.2 ng/ml, antral follicle count (AFC) > 5] who underwent their first controlled ovarian stimulation (COS) cycle were selected. Women with < 9 oocytes retrieved (group 1 of the Poseidon classification) constituted the group A, whereas those with ≥ 9 oocytes severed as control (normo-responders) one (group B). Demographic, anthropometric and hormonal variables together with COS parameters of the two groups were compared. Results: The number of patients with < 9 oocytes (group A) was 404, and those with ≥ 9 oocytes were 602 (group B). The mean age of the group A was significantly higher (30.1 + 2.9 vs. 29.4 + 2.9, p = 0.01). Group A displayed lower AMH and AFC [with interquartile ranges (IQR); AMH 1.6 ng/ml (1-2.6) vs. 3.5 ng/ml (2.2–5.4) p < 0.01, AFC 8 (6–12) vs. 12 (9–17), p < 0.01]. The number of small antral follicles (2–5 mm) of the group A was significantly lower [6 (4–8) vs. 8 (6–12) p < 0.01), while the larger follicles (5–10 mm) remained similar [3 (1–5) vs. 3(1–6) p = 0.3] between the groups. Conclusion: The propensity of low ovarian reserve and higher age are the main risk factors for the impaired ovarian response. The proportion of the small antral follicles may be a predictive factor for ovarian response to prevent unexpected poor results. [ABSTRACT FROM AUTHOR]

Background: An endometrioma is a cyst created when endometrial tissue proliferates in the ovaries. It affects women during the reproductive years and may result in persistent pelvic pain related to menstruation. The phrase "ovarian reserve" refers to the ovary's ability to produce an ovum that can be fertilized to produce a successful and healthy pregnancy. Currently, the two techniques most frequently employed to evaluate ovarian functional reserve are Serum anti-Mullerian hormone (AMH) and antral follicle count (AFC). The aim of this work is to determine the possible effects of ovarian endometrioma on the antral follicle count and serum anti-Müllerian hormone through the present case-control study. Methods: Out of 100 women, 50 had ovarian endometrioma, and the remaining 50 were chosen as the control group. On the second day of the menstrual cycle, we completed a thorough clinical examination and an entire history taking, including an ultrasound and measurements of the anti-Müllerian hormone in serum. Results: Endometriomas were more common on the right side (46%), and those with them had lower anti-müllerian hormone levels and total antral follicles. Conclusions: When the endometrioma volume is high, the number of antral follicles is further decreased. [ABSTRACT FROM AUTHOR]

Simple Summary: Oocytes are the foundation of life in mammals. They develop in small sacs called follicles in the ovaries. Each follicle holds one oocyte and numerous accompanying cells, and the final stage of oocyte development happens in a follicle called the antral follicle. The successful growth and maturation of oocytes depend on complex interactions with neighboring cells, mainly granulosa cells. Understanding the differences among these granulosa cells within the antral follicle and how they communicate with each other is essential for comprehending how mammals reproduce and grow. In this study, we collected cells from these follicles in pigs and utilized single-cell RNA sequencing to profile the transcriptomic features of these cells. The study discovered diverse subpopulations of granulosa cells within antral follicles, and they were found to communicate extensively. These findings provide us with valuable insights into how follicles develop and oocytes mature in pigs. This could potentially help improve pig reproduction efficacy and advance our knowledge in human reproductive medicine. The antral follicle stage is a critical period in mammalian oocyte maturation, marked by complex interactions between oocyte development and neighboring granulosa cells. Understanding the heterogeneity and communication patterns of granulosa cells within antral follicles is crucial for deciphering their roles in follicle development and oocyte maturation. Here, we employed single-cell RNA-sequencing to explore the molecular and cellular characteristics of porcine antral follicles. Our analysis revealed distinct subpopulations within mural and cumulus granulosa cells, indicating diverse cellular states and functions within the follicles. Functional enrichment analysis unveiled the involvement of specific subpopulations in steroid biosynthesis, cumulus expansion, and cellular communication. Moreover, comparing mature and less mature follicles highlighted differences in cell distribution and functions, indicating developmental-specific variations. Our findings shed light on the intricate cellular heterogeneity and communication network within porcine antral follicles, providing valuable insights into the regulation of follicle development and oocyte maturation in pigs. These results hold promise for improving pig reproductive efficiency and advancing human reproductive medicine. [ABSTRACT FROM AUTHOR]

Summary: Preantral to early antral follicles transition is a complex process regulated by endocrine and paracrine factors, as well as by a precise interaction among oocyte, granulosa cells and theca cells. Understanding the mechanisms that regulate this step of folliculogenesis is important to improve in vitro culture systems, and opens new perspectives to use oocytes from preantral follicles for assisted reproductive technologies. Therefore, this review aims to discuss the endocrine and paracrine mechanisms that control granulosa cell proliferation and differentiation, formation of the antral cavity, estradiol production, atresia, and follicular fluid production during the transition from preantral to early antral follicles. The strategies that promote in vitro growth of preantral follicles are also discussed. [ABSTRACT FROM AUTHOR]

Porcine ovaries are a biological material frequently used in studies that analyze follicular development processes, oocyte maturation in close connection with the stages of the estrus cycle. Ovarian follicles and their degree of development represent the point of departure in obtaining good quality oocytes, capable of being used in in vitro fertilization processes. The present study aims to identify the possible correlations between some biometric parameters and the degree of development of the follicles in the ovaries of pigs harvested after slaughtering the animals. The study was carried out on a number of 36 ovaries from 2 slaughters that were carried out in different stages of the estrus cycle, grouped into two experimental groups. In the laboratory, 3 biometric parameters weight (g), length (mm) and width (mm) were determined, respectively they were identified, photographed, numerically quantified and analyzed to establish the type of ovarian follicle. Based on the determinations made, we can conclude that the number of follicles and their type is changed in the ovaries of the two experimental groups, these two parameters are closely related to the stage of the estrous cycle and do not cause significant changes in the biometric parameters studied - weight, length, width. [ABSTRACT FROM AUTHOR]

The study objectives were to determine whether ovarian morphology can distinguish between women with regular menstrual cycles, normo-androgenic anovulation (NA-Anov), and PCOS and whether body mass index (BMI)-specific thresholds improved diagnostic potential. Women with PCOS (biochemical and/or clinical hyperandrogenism and irregular cycles; N = 66), NA-Anov (irregular cycles without clinical and/or biochemical hyperandrogenism; N = 64), or regular cycles (controls; cycles every 21–35 days in the absence of clinical or biochemical hyperandrogenism; N = 51) were evaluated. Participants underwent a reproductive history, physical exam, transvaginal ultrasound, and a fasting blood sample. Linear regression analyses were used to assess the impact of BMI on ovarian morphology across groups. The diagnostic performance of ovarian morphology for anovulatory conditions, and by BMI (lean: <25 kg/m2; overweight: ≥25 kg/m2), was tested using Receiver Operating Characteristic (ROC) curves. Follicle number per ovary (FNPO) and ovarian volume (OV), but not follicle number per cross-section (FNPS), increased across controls, NA-Anov, and PCOS. Overall, FNPO had the best diagnostic performance for PCOS versus controls (AUCROC = 0.815) and NA-Anov and controls (AUCROC = 0.704), and OV to differentiate between PCOS and NA-Anov (AUCROC = 0.698). In lean women, FNPO best differentiated between PCOS and controls (AUCROC = 0.843) and PCOS versus NA-Anov (AUCROC = 0.710). FNPS better distinguished between NA-Anov and controls (AUCROC = 0.687), although diagnostic performance was lower than when thresholds were generated using all participants. In women with overweight and obesity, OV persisted as the best diagnostic feature across all analyses (PCOS versus control, AUCROC = 0.885; PCOS versus NA-Anov, AUCROC = 0.673; NA-Anov versus controls, AUCROC = 0.754). Ovarian morphology holds diagnostic potential to distinguish between NA-Anov and PCOS, with marginal differences in diagnostic potential when participants were stratified by BMI suggesting that follicle number may provide better diagnostic performance in lean women and ovarian size in those with overweight. [ABSTRACT FROM AUTHOR]

Porcine ovaries are a biological material frequently used in studies that analyze follicular development processes, oocyte maturation in close connection with the stages of the estrus cycle. Ovarian follicles and their degree of development represent the point of departure in obtaining good quality oocytes, capable of being used in in vitro fertilization processes. The present study aims to identify the possible correlations between some biometric parameters and the degree of development of the follicles in the ovaries of pigs harvested after slaughtering the animals. The study was carried out on a number of 36 ovaries from 2 slaughters that were carried out in different stages of the estrus cycle, grouped into two experimental groups. In the laboratory, 3 biometric parameters weight (g), length (mm) and width (mm) were determined, respectively they were identified, photographed, numerically quantified and analyzed to establish the type of ovarian follicle. Based on the determinations made, we can conclude that the number of follicles and their type is changed in the ovaries of the two experimental groups, these two parameters are closely related to the stage of the estrous cycle and do not cause significant changes in the biometric parameters studied - weight, length, width.

The antral follicle stage is a critical period in mammalian oocyte maturation, marked by complex interactions between oocyte development and neighboring granulosa cells. Understanding the heterogeneity and communication patterns of granulosa cells within antral follicles is crucial for deciphering their roles in follicle development and oocyte maturation. Here, we employed single-cell RNA-sequencing to explore the molecular and cellular characteristics of porcine antral follicles. Our analysis revealed distinct subpopulations within mural and cumulus granulosa cells, indicating diverse cellular states and functions within the follicles. Functional enrichment analysis unveiled the involvement of specific subpopulations in steroid biosynthesis, cumulus expansion, and cellular communication. Moreover, comparing mature and less mature follicles highlighted differences in cell distribution and functions, indicating developmental-specific variations. Our findings shed light on the intricate cellular heterogeneity and communication network within porcine antral follicles, providing valuable insights into the regulation of follicle development and oocyte maturation in pigs. These results hold promise for improving pig reproductive efficiency and advancing human reproductive medicine.

Context: The FMR1 gene consists of 17 exons and codes for the FMRP protein. FMR1 is involved in four genetic disorders depending on the CGG repeats length in its 5′UTR: the full mutation is responsible for the Fragile X syndrome while the premutation is associated with the Fragile X-associated Tremor/Ataxia Syndrome, Fragile X-associated Primary Ovarian Insufficiency (FXPOI) and Fragile X-associated neuropsychiatric disorders. FMR1 presents multiple isoforms resulting from skipping of exons 12 and 14 and the use of alternative splice sites in exons 15 and 17. Aims: To investigate the expression of Fmr1 splicing variants during folliculogenesis in the rat. Methods: We used preantral, early antral and preovulatory follicles to isolate RNA and characterise, by fluorescent PCR followed by sequencing, all the isoforms present in the different follicular stages. Key results: We identified two isoforms resulting from splicing of exon 12, six isoforms resulting from splicing of exon 14 and 15 and one isoform for exon 17. Conclusions: The expression levels of the isoforms vary within each follicular stage but not between different stages of folliculogenesis. Importantly, we identify for the first time in rat, an isoform that contains exon 12 and two isoforms, one that includes and one that excludes exon 14 and use the third acceptor site in exon 15. Implications: Characterisation of the different FMR1 variants expressed during folliculogenesis will help to understand the potential distinct cellular roles of each of them and the possible implication in the development of FXPOI. The main genetic cause of isolated Primary Ovarian Insufficiency (POI) is due to an expansion of a CGG repeat located on the 5′UTR of the FMR1 gene responsible for Fragile X-associated POI (FXPOI). The gene can potentially produce multiple protein isoforms through alternative splicing of the transcript although no information is available regarding splice variants in the ovary or their implication in the pathology. Here, we analyse Fmr1 isoforms in a rat model of follicular development. We found differential expression of the variants and identified three new isoforms in the rat ovary. [ABSTRACT FROM AUTHOR]

Guidelines regarding the optimal use and timing of anti-Mullerian hormone (AMH) screening in childhood cancer survivors to evaluate for the risk of premature ovarian insufficiency or reduced fertility potential are lacking. We conducted a systematic review of the current evidence supporting AMH screening of female childhood cancer survivors with the overall objective to identify gaps in the literature needing further study, to allow for future data-driven recommendations. Search terms included "cancer, fertility, and anti-Mullerian hormone." We included original research articles that had ≥20 female childhood cancer survivors and excluded studies not including pediatric oncology survivors (≤18 years of age), did not include raw AMH values, were a mixed pediatric/young adult population which were minority pediatric, or did not separate pediatric from adult AMH data. In total, 17 studies (8 case-control, 5 cross-sectional, and 4 longitudinal prospective cohorts), encompassing 1106 total survivors met inclusion criteria and were further evaluated. Three studies evaluated the relationship of AMH to antral follicle count with generally good concordance. Four studies analyzed longitudinal changes in AMH with chemotherapy demonstrating that most patients will have an acute drop in AMH during therapy, and recovery of AMH over time is dependent on treatment intensity. No studies evaluated the optimal timing or interval of AMH testing. AMH correlates well with other markers of ovarian reserve, but there is insufficient data regarding the utility of AMH to predict the ability to conceive or timing of menopause. Optimal AMH screening initiation, duration, and intervals also require further study.

Antral follicle size is a useful predictive marker of the competency of enclosed oocytes for yielding an embryo following in vitro maturation and fertilization. However, the molecular mechanisms underpinning oocyte developmental potential during bovine antral follicle growth are still unclear. Here, we used a modified single-cell multi-omics approach to analyze the transcriptome, DNA methylome, and chromatin accessibility in parallel for oocytes and cumulus cells collected from bovine antral follicles of different sizes. Transcriptome profiling identified three types of oocytes (small, medium, and large) that underwent different developmental trajectories, with large oocytes exhibiting the largest average follicle size and characteristics resembling metaphase-II oocytes. Differential expression analysis and real-time polymerase chain reaction assay showed that most replication-dependent histone genes were highly expressed in large oocytes. The joint analysis of multi-omics data revealed that the transcription of 20 differentially expressed genes in large oocytes was associated with both DNA methylation and chromatin accessibility. In addition, oocyte-cumulus interaction analysis showed that inflammation, DNA damage, and p53 signaling pathways were active in small oocytes, which had the smallest average follicle sizes. We further confirmed that p53 pathway inhibition in the in vitro maturation experiments using oocytes obtained from small antral follicles could improve the quality of oocytes and increased the blastocyte rate after in vitro fertilization and culture. Our work provides new insights into the intricate orchestration of bovine oocyte fate determination during antral folliculogenesis, which is instrumental for optimizing in vitro maturation techniques to optimize oocyte quality., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Context: The oocyte-secreted factors growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) play essential roles in follicle development and oocyte maturation, and aberrant regulation might contribute to the pathogenesis of polycystic ovary syndrome. Objective: Are there measurable differences in concentrations of GDF9, BMP15, and the GDF9/BMP15 heterodimer in small antral follicle fluids from women with and without polycystic ovaries (PCO)? Design and Setting: Follicle fluids (n = 356) were collected from 4- to 11-mm follicles in unstimulated ovaries of 87 women undergoing ovarian tissue cryopreservation for fertility preservation. Patients: Twenty-seven women with PCO were identified and 60 women without PCO-like characteristics (non-PCO women) were matched according to age and follicle size. Main outcome measures: Intrafollicular concentrations of GDF9, BMP15, GDF9/BMP15 heterodimer, anti-Mullerian hormone (AMH), inhibin-A and -B, total inhibin, activin-B and -AB, and follistatin were measured using enzyme-linked immunosorbent assays. Results: The detectability of GDF9, BMP15, and the GDF9/BMP15 heterodimer were 100%, 94.4%, and 91.5%, respectively, and concentrations were significantly negatively correlated with increasing follicle size (P < 0.0001). GDF9 was significantly higher in women with PCO (PCO: 4230 ± 189 pg/mL [mean ± SEM], n = 188; non-PCO: 3498 ± 199 pg/mL, n = 168; P < 0.03), whereas BMP15 was lower in women with PCO (PCO: 431 ± 40 pg/mL, n = 125; non-PCO: 573 ± 55 pg/mL, n = 109; P = 0.10), leading to a significantly higher GDF9:BMP15 ratio in women with PCO (P < 0.01). Significant positive associations between BMP15 and AMH, activins, and inhibins in non-PCO women switched to negative associations in women with PCO. Conclusions: Intrafollicular concentrations of GDF9 and BMP15 varied inversely in women with PCO reflecting an aberrant endocrine environment. An increased GDF9:BMP15 ratio may be a new biomarker for PCO. [ABSTRACT FROM AUTHOR]

Although follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol (E2), antimullerian hormone (AMH), and ovarian volume are commonly used to assess ovarian reserve and ovarian response to stimulation, no test directly indicates this. We aimed to observe how ovarian reserve tests help us in the diagnosis and treatment of women of late reproductive age. We included 190 patients in this prospective cohort study. We took FSH, LH, E2 and AMH blood samples on the third day of menstruation to biochemically evaluate the ovarian reserve of the patients who were planned for hysterectomy and bilateral salpingo-oopherectomy at late reproductive age. We recorded antral follicle counts (AFC) and ovarian volumes by transvaginal ultrasonography for ultrasonographic evaluation and weighed ovarian weights postoperatively. The data of 190 women aged 39-44.9 years revealed that AMH and ovarian volume, ovarian volume and AFS show significant positive correlations, whereas FSH, LH, and E2 levels do not indicate a significant relationship with AFS. AMH, ovarian volume and ovarian weight, and AFC may show us the ovarian reserve with the highest reliability in women of late reproductive age. [ABSTRACT FROM AUTHOR]

The aim of this study was to verify the association between ovarian size and blood AMH levels in HF cows. Sixty multiparous HF cows from three herds were included in this study. The data required for calculating the ovarian volume included the “major axis (length),” “minor axis (width),” and “thickness” of the ovary. All ultrasonography (US) images were acquired at the outermost ends/poles of both the ovaries and of the follicles (>8 mm) and corpus luteum (CL); concomitantly, the blood was sampled from the jugular or coccygeal vein. Based on the ovarian images of each cow, the following ovarian size patterns were calculated using an image analysis software: (1) total area of both the left and right ovaries, (2) individual size of the large ovary, and (3) individual size of the small ovary. For each ovary area pattern, two properties were assessed: (A) presence of follicles (>8 mm) and CL, which may not secret AMH, in the ovaries and (B) absence of follicles (>8 mm) and CL in the ovaries. Serum AMH levels were measured using enzyme-linked immunosorbent assay. The correlation between ovary size and serum AMH levels was measured in terms of the aforementioned patterns and was evaluated statistically. The results of our preliminary study with ovaries from slaughter-house cows (n = 22) revealed that the “thickness” of the ovary was not necessary for estimating ovarian volume and that length and width were sufficient. A strong correlation was observed among ovarian length, width, and thickness (r > 0.96). No significant difference was observed (p > 0.05) in the mean ages or parities among the three herds. Among the ovary sizes measured in this study, the highest correlation was found between the total size of an individual large ovary (including follicular and luteal size) and AMH levels (r = 0.387, p = 0.002). This is the first study to demonstrate the correlation between total size of individual large ovaries and serum AMH levels in HF cows. US observations of the ovaries will allow for estimation of differences in AMH levels and help predict ovarian activity and superovulation performance of cows.

Knowledge of the growth and maturation of human antral follicles is based mainly on concepts and deductions from clinical observations and animal models. To date, new experimental approaches and in vitro data contributed to a deep comprehension of gonadotropin receptors' functioning and may provide new insights into the mechanisms regulating still unclear physiological events. Among these, the production of androgen in the absence of proper LH levels, the programming of follicular atresia and dominance are some of the most intriguing. Starting from evolutionary issues at the basis of the gonadotropin receptor signal specificity, we draw a new hypothesis explaining the molecular mechanisms of the antral follicular growth, based on the modulation of endocrine signals by receptor-receptor interactions. The "heteromer hypothesis" explains how opposite death and life signals are delivered by gonadotropin receptors and other membrane partners, mediating steroidogenesis, apoptotic events, and the maturation of the dominant follicle. [ABSTRACT FROM AUTHOR]

Final antral follicle development and future ovulation are mediated by gonadotropin-induced changes with spatio-temporally regulated expression of genes. Here, we aimed to quantify the relative mRNA abundance of bta-miR-222 and its predicted target, LHCGR, in granulosa cells (GCs) from follicles, after follicle deviation, as well as from GCs cultured in vitro with follicle stimulating hormone (FSH) and/or insulin. Thus, to study the impact of follicle deviation, Nelore heifers (n = 10; Bos taurus indicus) were hormonally synchronized and slaughtered 3 days after ovulation. Then, GCs from the dominant follicle (DF) and its respective subordinate follicle (SF) were recovered for RT-qPCR. For in vitro analysis, small follicles (2–5 mm) were dissected from bovine ovaries collected from a local abattoir. The GCs were isolated and cultured in serum-free medium, or treated with insulin (1 ng/mL or 10 ng/mL) alone or in combination with human recombinant FSH (1 ng/mL), for 6 days. Our findings showed that the relative mRNA abundance of LHCGR in GCs was higher in the DF compared to the SF (p = 0.01). Inversely, bta-miR-222 expression was lower in the DF compared to the SF (p = 0.01). Furthermore, GCs cultured with FSH and insulin together resulted in a higher abundance of LHCGR and a lower abundance of bta-miR-222 (p ≤ 0.05) when compared to GCs cultured with insulin alone. In conclusion, we found that the LHCGR upregulation in GCs from the DF is inversely related to bta-miR-222 expression. We also suggest the involvement of FSH in bta-miR-222 suppression in healthy bovine GCs. • We showed the inverse expression of mir-222 and its possible target LHCGR on bovine granulosa cells on follicle deviation. • The influence of insulin and FSH during granulosa cells culture from subordinate follicles on mir-222 and LHCGR abundance. [ABSTRACT FROM AUTHOR]

We evaluated the relationship between plasma levels of anti-Müllerian hormone (AMH) and the number of antral follicles at the restart of the follicular wave in crossbred Holstein cows reared under extensive grazing systems over 2500 m above sea level. The study included 140 cows from 15 farms that were in average at the 75.3 ± 2.10 d post partum. Animals were synchronized according to the following regime: day 0 = intravaginal progesterone releasing device (IPD) + estradiol benzoate (EB); day 7: withdrawal of IPD + prostaglandin; and day 8: EB, for restart of the follicular wave on day 11. On this day 11, antral follicle counts (AFCs) were made by transrectal ultrasound, and a plasma sample was taken for the determination of AMH. The mean AMH plasma level was 0.06 ± 0.03 ng/ml and the mean AFC was 17.26 ± 0.38 follicles. A strong positive linear correlation was found between these two variables (r = 0.783, r = 0.613, P < 0.0001). Cows were categorized according to AMH concentration as high (>0.09 ng/ml), intermediate (0.09–0.05 ng/ml) or low (<0.05 ng/ml). Cows with high AMH presented a higher AFC (25.0 ± 2.21 follicles) than those with low AMH (14.08 ± 2.68 follicles; P < 0.001. Our results suggest that the cut-off value of AMH = 0.09 ng/ml may be useful for selecting donors in multiple ovulation embryo transfer programs involving cows with these characteristics. Our data further suggest that AMH plasma concentration correlates with AFC and can be used as an endocrine biomarker of the number of antral follicles present at a given moment of the estrous cycle in crossbred Holstein cows raised at altitudes above 2500 m. [ABSTRACT FROM AUTHOR]

Objective: We investigated the impact of vitamin D3 (VD3) supplementation during mouse preantral follicle culture in vitro and the mRNA expression of 25-hydroxylase (CYP2R1), 1-alpha-hydroxylase (CYP27B1), and vitamin D receptor {VDR) in mouse ovarian follicles at different Methods: Preantral follicles were retrieved from 39 BDF1 mice (7-8 weeks old) and then cultured in vitro for 12 days under VD3 supplementation (0,25, and 50 pg/mL). Follicular development and the final oocyte acquisition were assessed. Preantral follicles were retrieved from 15 other BDF1 mice (7-8 weeks old) and cultured without VD3 supplementation. Three stages of mouse ovarian follicles were obtained (preantral, antral, and ruptured follicles). Total RNA was extracted from the pooled cells (from 20 follicles at each stage), and then reverse transcrip-tase-polymerase chain reaction was performed to identify mRNA for CYP2R1, CYP27B1, and VDR. Results: The survival of preantral follicles, rates of antrum formation and ruptured follicles (per initiated follicle) and the number of total or mature oocytes were all comparable among the three groups. Both CYP2R1 and CYP27B1 were expressed in antral and ruptured follicles, but not in preantral follicles. VDR was expressed in all three follicular stages. Conclusion: VD3 supplementation in vitro (25 or 50 pg/mL) did not enhance mouse follicular development or final oocyte acquisition. Follicular stage-specific expression of CYP2R1, CYP27B1, and VDR was observed. [ABSTRACT FROM AUTHOR]

Objectives: To evaluate the ovarian reserve by measuring AMH level after the both treatment of drug and surgery for Endometrioma. Methods: We studied 129 patients who have diagnosed with ovarian endometrioma, aged 20-46 years, using case-control study design. There are 4 groups with medication and surgeries. Results: There were significant reduction of pain in patients who received synthetic progestin (p = 0.001) groups. Serum AMH were 3.48 ± 0.9 before treatment and 3.41 ± 1.0 after treatment respectively (p = 0.456). Prior treatment of laparoscopic surgery with progestin 3 months, it decreases abdominal lower pain (p = 0.001) and dysmenorrhea (p = 0.001). Serum AMH level were 3.11 ± 1.8 before surgery and 2.21 ± 0.2 after surgery, respectively (p = 0.005). Conclusions: Laparoscopic surgery might be decrease ovarian reserve which leads to infertility later life in women who has ovarian endometrioma with ovarian cystectomy.

• PicoAMH for predicts ovarian hyperstymulation syndrome better than antral follicle counts alone. • PicoAMHis useful to optimize stimulation protocols of in-vitro fertilization. • PicoAMH studies have to be done in larger populations for utility in PCOS. Our study aims to evaluate the diagnostic performance of a high-sensitivity picoAnti-Müllerian Hormone (picoAMH) for predicting ovarian response in women undergoing controlled ovarian hyperstimulation and occurrence of ovarian hyperstimulation syndrome. Retrospective cohort study at a single academic fertility center including all patients with picoAMH ELISA who underwent controlled ovarian hyperstimulation. The primary outcome was the number of oocytes retrieved, and secondary outcomes included cycle cancellation and ovarian hyperstimulation syndrome. Patients were grouped into poor, normal, and hyper-responders based on number of oocytes retrieved. The mean AMH and antral follicle count (AFC) were significantly different between normal response vs. hyper response group (p < 0.0001). Only serum AMH and not AFC was significantly increased in patients diagnosed with ovarian hyperstimulation syndrome (OHSS). For prediction of OHSS, receiver operating characteristic (ROC) analysis revealed that AMH (area under the ROC curve [AUC] = 0.85) was significantly better than the AFC (AUC = 0.64). The serum AMH cut-off at sensitivity of 80% for predicting OHSS among hyper responders from ROC curve was 3.67 ng/ml. Serum AMH measured by picoAMH ELISA showed superior correlation to number of oocytes retrieved when compared to AFC in the age group over 40 years old (r2 = 0.74 and r2 = 0.4, respectively) This study shows great utility of picoAMH ELISA for predicting ovarian response to controlled ovarian hyperstimulation (COH). Diagnostic performance of picoAMH for prediction of OHSS is superior to the AFC in our cohort. [ABSTRACT FROM AUTHOR]

This study demonstrates the effect of a single high-intensity interval training (HIIT) session on the redox status of rat ovaries with excess adiposity. Forty Wistar female rats (mean (±s.e.m.) weight 94.40 ± 13.40 g) were divided into two groups and fed either a standard diet (SD) or a high-fat diet (HFD) for 62 days. At the end of this period, the rats were subjected to a single HIIT session and were killed 24 h after exercise. Both groups subjected to exercise (SDex and HFDex) generated a significantly higher antioxidant environment by presenting a higher thiol content, which represents a lower oxidation rate of GSH than their respective controls (SD and HFD). The percentage of morphologically normal primary follicles decreased, whereas that of antral follicles increased, in the SDex group. In addition, the HFD group had a higher percentage of degenerated antral follicles than the SD and SDex groups. Cells immunoreactive for α-smooth muscle actin were seen in the cortical stroma and thecal layer enclosing late secondary and tertiary follicles in all groups. Moreover, heme oxygenase and cytochrome P450 family 19 subfamily A member 1 (Cyp19A1) labelling was seen in all antral follicles. Progesterone concentrations were significantly higher in the HFDex than SDex group. In conclusion, this study indicates that a single session of HIIT may result in an improvement in ovary redox status because of metabolic muscle activity by inducing physiological adaptation after exercise in a paracrine manner. Although the mechanisms by which obesity causes infertility are not yet well known, weight loss in a short period of time has been the main treatment for infertility in cases of overweight or obesity. This study shows that physical exercise using a high-intensity interval training (HIIT) protocol may be more beneficial for maintaining redox balance and promoting follicle development in ovaries from high-fat diet-fed rats. Such findings set a precedent for possible treatments of infertility caused by overweight or obesity. [ABSTRACT FROM AUTHOR]

Objectives: To determine the level of agreement across assessments of follicle number per ovary (FNPO) and classifying of polycystic ovarian morphology (PCOM; FNPO ≥25) with the use of various real-time (RT) and off-line two-dimensional (2D) and three-dimensional (3D) ultrasonographic methods.Design: Method comparison study.Setting: University-based clinical research unit.Patient(s): Sixteen women with and without PCOM.Intervention: Thirty-two ovaries were analyzed with the use of eight ultrasonographic methods: 2D-Grid (reference method), 2D-RT, 2D-RT with Grid, multiplanar view (MPV), MPV-RT, tomographic ultrasound imaging (TUI), TUI-RT, and semiautomated volume calculation (SonoAVC).Main Outcome Measure(s): FNPO, PCOM status, and time to obtain FNPO. Clinical feasibility, defined as the time taken to obtain FNPO, also was evaluated.Result(s): 2D-RT overestimated FNPO versus 2D-Grid (3 ± 9 follicles) owing to overcounting in non-PCOM ovaries (6 ± 6 follicles). However, systematic bias was not detected when a grid overlay was incorporated (2D-RT with Grid). SonoAVC underestimated FNPO (-3 ± 5 follicles), particularly in PCOM ovaries (-4.1 ± 5.0 follicles). No bias in FNPO was detected between MPV, TUI, or TUI-RT versus 2D-Grid. 2D-RT significantly misclassified ovaries as PCOM. All methods except MPV took less time to complete FNPO assessments compared with 2D-Grid.Conclusion(s): Variability in FNPO across ultrasonographic methods limits their interchangeable use, particularly when a precise metric is needed. 2D-RT may be problematic owing to its propensity to misclassify PCOM. 2D-RT with Grid and MPV-RT could represent clinically feasible alternatives to obtain FNPO and classify PCOM. Efforts to reduce variation in FNPO will clarify the relevance of PCOM in women's health. [ABSTRACT FROM AUTHOR]

Objective: Premature ovarian insufficiency (POI) is a refractory disease that seriously affects female fertility. Growing body of evidence has indicated mesenchymal stem cells (MSCs) as promising resources in regenerative medicine. In this study, we treated POI patients with umbilical cord‐derived MSCs (UCMSCs) and then investigated the restoration of ovarian function and clinical outcomes through follow‐ups. Materials and methods: Sixty‐one patients diagnosed with POI participated in this study. UCMSCs were isolated and cultured according to GMP standards, and then transplanted to the patients' ovary by orthotopic injection under the guidance of vaginal ultrasound. We monitored side effects, vital signs and changes in clinical and collected haematological and imaging parameters during the follow‐ups. Results: All patients showed normal clinical behaviour without serious side effects or complications relevant to the treatment. Transplantation of UCMSCs rescued the ovarian function of POI patients, as indicated by increased follicular development and improved egg collection. POI patients who experienced shorter amenorrhoea durations (<1 year) seemed to obtain mature follicles more easily after stem cell therapy, and patients with better ovarian conditions (pre‐operative antral follicles) were more likely to derive the better outcomes by UCMSC injection. Four successful clinical deliveries were obtained from POI patients after UCMSC transplantation, and all of these babies are developed normally. Conclusions: The clinical trial result sugggests a possible therapy for POI by UCMSC transplantation. [ABSTRACT FROM AUTHOR]

Background: Antral follicle count (AFC) has been labeled as the most accurate biomarker to assess female fecundity. Unfortunately, no baseline Indian data exists, and we continue using surrogate values from the Western literature (from studies on women, grossly different than Indian women in morphology and genetic makeup). Aim: To establish the role of AFC as a function of ovarian reserve in fertility-proven and in subfertile Indian women. Materials and methods: Thirty patients undergoing workup for infertility were included and compared to an equal number of controls (women with proven fertility). The basal ovarian volume and AFC were measured by endovaginal USG the relevant clinical data and AFC, ovarian volume was charted for every patient. Results: Regression analysis revealed the highest correlation of AFC and age in fertile and infertile patients with the difference in mean AFC of both the groups. A comparison of the data recorded for cases and controls showed no significant difference in the mean ovarian volume. Conclusion: AFC has the closest association with chronological age in normal and infertile Indian women. The same is lower in infertile women than in matched controls. Baseline and cut-off values in south Indian women are lower than those mentioned in Western literature. [ABSTRACT FROM AUTHOR]

Summary: The activation, growth, and maturation of oocytes to an ovulatory phase, termed folliculogenesis, is governed by the orchestrated activity of multiple specialized cell types within the ovary; yet, the mechanisms governing diversification and behavior of discrete cellular sub-populations within follicles are poorly understood. We use bulk and single-cell RNA sequencing to distinguish the transcriptional signature of prospectively isolated granulosa and theca/stroma cell subsets within human antral follicles derived from xenografts or ovaries. The analysis deconstructs phenotypic diversification within small (

Aim: To determine whether performing incision on the surface of the ovarian cortex in rats advances follicular development. Methods: Five to seven separate superficial incisions were performed on the surface of right ovaries of 6‐7–month‐old albino Wistar rats. Daily 40 IU of gonadotropins were administered for 14 days. On the 15th day, both ovaries of the rats were removed. The right (incised) ovaries were compared with the contralateral ovaries in terms of ovary's weight, numbers of primordial, primary, secondary and antral follicles, their mean percentages and mean Ki‐67 proliferation indices. Results: A total of 22 ovaries were evaluated, with 11 right ovaries (incised) and 11 left ovaries (non‐incised). The mean weight of ovaries was greater in the right ovaries than in the left ovaries; however, no statistical difference was found between them (0.77 ± 1.22 vs. 0.22 ± 0.08 gr, P = 0.159). The numbers of secondary and antral follicle were statistically higher in the right ovaries than in the left ovaries (4.4 ± 1.5 vs. 2.1 ± 1.6, P = 0.003 and 18.6 ± 8.7 vs. 11.3 ± 7.5, P = 0.046, respectively). The right ovaries also significantly differed from the left ovaries in terms of mean percentages of primordial and antral follicles (P < 0.05 for both). The mean Ki‐67 proliferation index had a marginal difference between the groups (P = 0.064). Conclusion: Performing incisions on the surface of the ovarian cortex in rats may advance the ovarian follicular development. Future animal studies may provide more evidence regarding potential benefits of mechanical stimulation to the ovaries. [ABSTRACT FROM AUTHOR]

Glutathione (GSH), the most abundant intracellular nonprotein thiol, is critical for many cellular functions. The rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL), a heterodimer composed of a catalytic (GCLC) and a modifier (GCLM) subunit. The tissue-specific regulation of GSH synthesis is poorly understood. We showed previously that gonadotropin hormones regulate ovarian GSH synthesis. In the present study, we sought to clarify the ovarian cell type-specific effects of follicle-stimulating hormone (FSH) and estradiol on GSH synthesis. Immature female rats were treated with estradiol to stimulate development of small antral follicles. Granulosa cells (GCs) from these follicles or whole follicles were cultured in serum-free media, with or without FSH and 17beta-estradiol. The GSH and GCLC protein and mRNA levels increased in GCs treated with FSH alone. The effects of FSH on GCLC and GCLM protein and mRNA levels, GCL enzymatic activity, and GSH concentrations in GCs were significantly enhanced by the addition of estradiol. Estradiol alone had no effects on GSH. Dibromo-cAMP mimicked and protein kinase A (PKA) inhibitors prevented FSH stimulation of GCL subunit protein levels. In cultured small antral follicles, FSH stimulated estradiol synthesis and robustly increased GCL subunit mRNA and protein levels and GSH concentrations. The GCL subunit mRNA expression increased in both the granulosa cells and theca cells of follicles with FSH stimulation. These data demonstrate that maximal stimulation of GSH synthesis by FSH in granulosa cells and follicles requires estradiol. Without estradiol, FSH causes lesser increases in GCL subunit expression via a PKA-dependent pathway.

Objective: In this study, we aimed to investigate if the gonadotropin-releasing hormone (GnRH) agonist and antagonist have any effect on the follicle-stimulating hormone (FSH)-induced-growth of early antral follicles of mice, cultured in vitro, and expressing cognate receptors for FSH and GnRH. For this purpose, small antral follicles were isolated from mouse ovaries and randomly assigned to the groups as control, FSH only, FSH + GnRH agonist, and FSH + GnRH antagonist, and they were cultured for five days. Methods: The ovaries of C57BL/6 mice (n=24), which were 21 days old, were removed after euthanasia. Small antral follicles measuring ~200ì in diameter were mechanically isolated after the enzymatic digestion of the ovaries with collagenase and DNase-I. The expression of FSH and GnRH receptors in these follicles was validated by quantitative real time-polymerase chain reaction. Isolated follicles were randomly assigned into four different groups, each consisting of 20-30 follicles: control, FSH only, FSH + GnRH agonist, and FSH + GnRH antagonist. Results: The FSH treatment significantly enhanced the in vitro growth of the follicles compared to those cultured without FSH after five days of the culture period. The antrum formation was markedly enhanced, and cumulus-oophorus complexes were more easily visible in the FSH-treated follicles compared to control follicles. The mean diameters of follicles treated with the FSH + GnRH agonist or the FSH + GnRH antagonist were not significantly different from those treated with FSH only, but they were significantly greater than control follicles. Conclusion: These results may suggest that the GnRH agonist and antagonist do not appear to adversely affect the FSH-induced proliferation of mitotic non-luteinizing granulosa cells and the growth of early antral follicles of mice in vitro. [ABSTRACT FROM AUTHOR]

Lead impairs female reproductive health because it can induce oxidative stress. Zinc as an antioxidant produces an enzyme system that helps neutralize free radicals. α-Tocopherol has an antagonistic effect that reduces oxidative stress. This study aimed to demonstrate the effects of zinc (Zn) and α-tocopherol on the ovarian endogenous antioxidants and antral follicles of albino rats (Rattus norvegicus) exposed to lead acetate (Pb(C 2 H 3 O 2) 2). Twenty-five female Wistar rats were divided into five groups, namely groups K (control), P0, P1, P2, and P3. Following exposure and treatment for 21 days with different combinations, the albino rats were necropsied, and their ovaries were removed for subsequent histopathological preparations and endogenous antioxidant analysis. Observations were made on the ovary, including an antral follicle count and diameter calculations. Analysis of the superoxide dismutase (SOD) levels (560 nm wavelength) and malondialdehyde MDA-TBA (532 nm wavelength) were performed by a spectrophotometer. The data were analyzed using a one-way ANOVA and least significant difference (LSD) test with the SPSS V24 software. The highest SOD enzyme expression in the albino rat ovaries was in P0 (17.23 ± 5.34), and the lowest was in P3 (4.21 ± 0.76). The lowest MDA level was observed in the control group (K) and P3 compared to the other groups. The highest average antral follicle count and diameter were found in the albino rats exposed to 1.5 mg/kg BW lead acetate, and treated with 0.54 mg/kg BW zinc sulfate and 100 mg/kg BW α-tocopherol (group P3) compared to the other groups. The mechanisms of action of zinc and α-tocopherol work synergistically to decrease free radicals and ovarian damage. The results showed that a combination of 0.54 mg/kg BW zinc (Zn) and 100 mg/kg BW α-tocopherol can maintain the number and diameter of the antral follicles and reduce ovarian SOD expression and MDA levels in albino rats exposed to lead acetate. • Lead impairs ovarian damage due to oxidative stress triggers. • Antioxidant produces an enzyme system that reduce oxidative stress. • Zinc and α-Tocopherol protect the antral follicles from lead exposure. • Mechanisms action of zinc and α-tocopherol work synergistically to decrease ovarian damage. [ABSTRACT FROM AUTHOR]