Follicular Oocytes Research Articles

Immunolocalization of activin and inhibin at different stages of follicular development in the lizard Sceloporus torquatus

The activins and inhibins are glycoproteins with a role in the follicular development of vertebrates, that are found in follicular fluid and somatic follicular cells, with a different pattern among taxa. The principal function of activin (Act) is to modulate the follicle-stimulating hormone (FSH) synthesis and secretion, whereas inhibin (Inh) downregulates it. Both factors are modulators of intraovarian follicular recruitment, oocyte maturation, cell proliferation, and steroidogenic activity. Our aim was to characterize the immunolocalization of Act and Inh in the ovarian follicles during the reproductive cycle of the lizard Sceloporus torquatus. Act was detected in the granulosa cells and oocyte cortex in the different stages of follicular development. On the other hand, we identified Inh in the oocyte cortex and the cytoplasm of pyriform and small cells of previtellogenic follicles. Also, we found immunoreactivity in the oocyte cortex, theca, and small cells of vitellogenic and preovulatory follicles. Our data provide evidence that Act and Inh have changes related to the stage of follicular development. This dynamic appears to be conserved among vertebrates and is fundamental to ensure an adequate follicular development in this specie.

Assessment of the health status of Lake Nokoué (Benin, West Africa) exposed to multiple pollutants during flood season

ABSTRACT This study used the levels of pollutants and the histopathological analysis of the gonads and liver of Sarotherodon melanotheron sampled in Lake Nokoué and Lake Toho, both in Benin (West Africa) to assess the health status of the ecosystems. Sixty adults (30 males and 30 females) of this species were caught during the flooding seasons from both Lake Nokoué, and Lake Toho (as a reference). The physicochemical parameters of the water and the heavy metal contents (Pb, Cd, Hg) in water/sediment/fish of three sites (S1, S2, S3) in Lake Nokoué and two sites (S4, S5) in Lake Toho were analysed. Results confirm very strong organic status with organic pollution index ranging from 1.25 to 1.5. Pb, Cd, and Hg levels in the water/sediment/fish muscle tissue of Lake Nokoué were far exceeding international standards. The histopathology of gonads revealed testis-ova up to 40% in males and follicular atretic oocytes up to 33.33% in females, indicated oestrogenic effects from the pollutants with a delay of spermatogenesis and stimulation of oogenesis. The histopathology of the liver revealed the presence of regressive changes. The findings call for actions to protect the ecosystem from pollution and attention in terms of health of humans consuming fish products from Lake Nokoué.

Follicle Stimulating Hormone (FSH) as a Predictor of Decreased Oocyte Yield in Patients with Normal Anti-Müllerian Hormone (AMH) and Antral Follicle Count (AFC).

The purpose of the current study was to determine the utility of early follicular phase follicle-stimulating hormone (FSH) testing in patients undergoing in vitro fertilization (IVF). This was a retrospective review of patients from 2012 to 2015 at Mayo Clinic in Rochester, Minnesota, USA. Included subjects had a normal anti-Müllerian hormone (AMH) of 1 to 9 ng/ml and antral follicle count (AFC) of 10 to 29. Patients were stratified by FSH level when associated estradiol was less than 50 ng/ml. In total, 225 patients were categorized into three groups: high FSH (FSH ≥10 IU/L; n= 36), normal FSH (>5 IU/L and <10 IU/L; n=170), and low FSH (FSH ≤5 IU/L; n= 19). ANOVA and multiple logistic regression were used for statistical comparisons and for evaluation of the relationships between variables; significance level was set at <0.05. There were no significant differences in demographics, IVF cycle type, or peak estradiol level between the groups. Patients with a high basal FSH level had a similar clinical pregnancy rate and live birth rate compared to controls and patients with low FSH. High FSH level was associated with decreased follicular development (17 versus 22; p<0.01), oocyte yield (15 versus 18; p=0.02), and embryo yield (8 versus 10; p=0.04) despite higher total doses of gonadotropins. Patients with normal AMH and AFC levels could be further stratified into lower responders and starting doses of medications can be adjusted based on high basal FSH levels. Therefore, it is suggested to counsel patients on pregnancy outcomes which seem to be quite similar regardless of the FSH level.

Effects of follicle-stimulating hormone, insulin-like growth factor 1, fibroblast growth factor 2, and fibroblast growth factor 9 on sirtuins expression and histone deacetylase activity in bovine granulosa cells

Granulosa cells (GC) are critical regulators of fertility. During the process of ovarian folliculogenesis, these cells undergo profound changes while producing steroid hormones that are important to control follicular growth, oocyte maturation, and ovulation. Sirtuins are enzymes that regulate several biological processes and have been associated with control of GC function. However, how sirtuins are regulated in GC during ovarian folliculogenesis remains to be unveiled. The present study was designed to investigate effects of hormones that control GC proliferation, differentiation, and steroidogenesis on expression of the seven members of the mammalian sirtuins family (SIRT1-7) and on histone deacetylase activity of nuclear sirtuins (SIRT1, 6, and 7) in GC. Bovine granulosa cells were isolated from small antral follicles (1–5 mm) and were treated with or without follicle-stimulating hormone (FSH), insulin-like growth factor 1 (IGF-1), and fibroblast growth factors 2 (FGF2) and 9 (FGF9). Following treatments, cell proliferation was determined via a cell analyzer, estradiol synthesis and histone deacetylase activity were determined via ELISA, and sirtuins mRNA expression was determined via qPCR. Treatments with FSH and IGF-1 stimulated cell proliferation while addition of FGF2 or FGF9 suppressed estradiol production stimulated by FSH plus IGF-1. In terms of treatments that regulated sirtuins expression in GC, fibroblast growth factors were the most impactful: FGF2 alone increased SIRT1 mRNA expression in comparison to several treatments and increased mRNA abundance of SIRT2 and SIRT7 when added to the combination of FSH and IGF-1; the addition of FGF9 to the combination of FSH and IGF-1 increased mRNA expression of SIRT2, SIRT3, SIRT4, SIRT6, and SIRT7 and increased mRNA expression of SIRT5 in comparison to the negative control group that received no treatment. Also, FGF2 alone increased histone deacetylase activity of sirtuins in comparison to all treatments that contained FSH and/or IGF-1. Furthermore, several correlations were observed between treatments and sirtuins expression and activity, between estradiol or GC numbers and sirtuins expression, and between expression of sirtuins. As FGF2 and FGF9 are considered anti-differentiation factors of GC that stimulate GC proliferation while suppressing estradiol production in combination with FSH and IGF-1, data of this study suggest that sirtuins are associated with control of differentiation of bovine GC.

Serotonin Transporter Activity in Mouse Oocytes Is a Positive Indicator of Follicular Growth and Oocyte Maturity.

Serotonin (5-hydroxytryptamine, 5-HT) is known to be a regulator of oocyte maturation in a large number of animal species. In maturing mammalian oocytes, the accumulation of exogenous, maternal serotonin occurs due to the activity of the membrane transporter SERT. In this work, we investigated how SERT activity in oocytes correlates with indicators of follicular selection and oocyte maturity. An immunohistochemical study showed that the difference in the 5-HT intake activity in oocytes does not correlate with the marker of apoptosis in follicular cells, but positively correlates with markers of follicular growth, such as granulosa proliferation and follicle size. Functional analysis of oocytes at different stages of maturation showed that the expression and activity of SERT increases with oocyte maturation. An in vivo experiment on administration of the selective serotonin reuptake inhibitor fluoxetine (20 mg/kg) for 7 days showed a significant decrease in the content of serotonin in both growing GV-oocytes and ovulated mature MII-oocytes. The data obtained clearly indicate that the mechanism of specific membrane transport of serotonin normally ensures the accumulation of serotonin in maturing oocytes, and can be considered as a promising positive marker of their mature status.

Detection of La Crosse Virus In Situ and in Individual Progeny to Assess the Vertical Transmission Potential in Aedes albopictus and Aedes aegypti.

La Crosse virus (LACV) is circulating in the midwestern and southeastern states of the United States and can cause human encephalitis. The main vector of the virus is the eastern tree-hole mosquito, Aedes triseriatus. Ae. albopictus has been also described as a natural LACV vector, while Ae. aegypti has been infected with the virus under laboratory conditions. Here, we compare the vertical transmission potential of LACV in Ae. albopictus and Ae. aegypti, with emphasis given to the ovarian infection patterns that the virus generates in both species. Both mosquito species received artificial bloodmeals containing LACV. At defined time points post-infection/bloodmeal, midguts, head tissue, and ovaries were analyzed for the presence of virus. Viral infection patterns in the ovaries were visualized via immunofluorescence confocal microscopy and immunohistopathology assays using an LACV-specific monoclonal antibody. In Ae. aegypti, LACV was confronted with midgut infection and escape barriers, which were much less pronounced in Ae. albopictus, resulting in a significantly higher prevalence of infection in the latter. Following the ingestion of a single virus-containing bloodmeal, no progeny larvae were found to be virus-infected. Regardless, females of both species showed the presence of LACV antigen in their ovariole sheaths. Furthermore, in a single Ae. albopictus female, viral antigen was associated with the nurse cells inside the primary follicles. Following the ingestion of a second non-infectious bloodmeal at 7- or 10-days post-ingestion of an LACV-containing bloodmeal, more progeny larvae of Ae. albopictus than of Ae. aegypti were virus-infected. LACV antigen was detected in the egg chambers and ovariole sheaths of both mosquito species. Traces of viral antigen were also detected in a few oocytes from Ae. albopictus. The low level of vertical transmission and the majority of the ovarian infection patterns suggested the transovum rather than transovarial transmission (TOT) of the virus in both vector species. However, based on the detection of LACV antigen in follicular tissue and oocytes, there was the potential for TOT among several Ae. albopictus females. Thus, TOT is not a general feature of LACV infection in mosquitoes. Instead, the TOT of LACV seems to be dependent on its particular interaction with the reproductive tissues of a female.

Pubertal sexual development and endpoints for disrupted spermatogenesis in the model Xenopus tropicalis

Peripubertal models to determine effects of anti-androgenic endocrine disrupting chemicals are needed. Using the toxicological model species Xenopus tropicalis, the aims of the study were to 1) provide data on sexual maturation and 2) characterise effects of short-term exposure to an anti-androgenic model substance. Juvenile (2.5 weeks post metamorphosis old) X. tropicalis were exposed to 0, 250, 500 or 1000 µg flutamide/L (nominal) for 2.5 weeks. Upon exposure termination, histology of gonads and Müllerian ducts was characterised in detail. New sperm stages were identified: pale and dark spermatogonial stem cells (SSCs). The testes of control males contained spermatozoa, indicating pubertal onset. The ovaries were immature, and composed of non-follicular and pre-vitellogenic follicular oocytes. The Müllerian ducts were more mature in females than males indicating development/regression in the females and males, respectively. In the 500 µg/L group, the number of dark SSCs per testis area was decreased and the number of secondary spermatogonia was increased. No treatment effects on ovaries or Müllerian ducts were detected. To conclude, our present data provide new knowledge on spermatogenesis, and pubertal onset in X. tropicalis. New endpoints for evaluating spermatogenesis are suggested to be added to existing assays used in endocrine and reproductive toxicology.

P-641 Clinical significance of differential expression of miRNA642-a/miRNA5739/miRNA3648 in the follicular microenvironment in patients with polycystic ovary syndrome

Abstract Study question What is the relationship between differential expression of miRNA in the follicular microenvironment,follicular fluid hormone and oocyte quality in polycystic ovary syndrome (PCOS) patients. Summary answer In PCOS patients, differential expression of miRNA in the follicular microenvironment is associated with abnormal hormone levels in the follicular fluid and oocyte quality. What is known already PCOS has abnormalities in local ovarian morphology and function, and studies have shown that the expression of local miRNA in the ovaries will be accompanied by dynamic changes in follicular recruitment, oocyte growth, follicular microenvironment changes, etc., which may affect the quality of oocytes. Study design, size, duration 19 patients with PCOS (PCOS group) and 17 patients with infertility due to male factors alone (control group) aged 22-35 years was included in the study. The main results included differential expression of miRNA642-a/miRNA5739/miRNA3648, MII egg rate, high-quality embryo rate, testosterone and insulin levels in follicular fluid. Participants/materials, setting, methods From April 2017 to January 2019, 19 PCOS patients (PCOS group) and 17 patients (control group) who underwent Intracytoplasmic sperm injection-embryo transfer at the Reproductive Center of the Affiliated Hospital of Shandong University of Traditional Chinese Medicine were collected. RT-PCR was used to detect the differential expression levels of miRNA642-a/miRNA5739/miRNA3648 in follicular fluid and granule cells in PCOS group and control group, and Chemiluminescence was used to detect follicular fluid T and insulin levels. Main results and the role of chance The levels of testosterone and insulin in follicular fluid in PCOS group were significantly higher than those in the control group, and the difference was statistically significant (P &amp;lt; 0.05). The MII egg rate and optimal embryo rate in the PCOS group were both lower than those in the control group, and the difference was significant (P &amp;lt; 0.05). The expression level of miR-642a-3p in follicular fluid and granule cells was consistent, and the PCOS group was lower than that in the control group. The difference was statistically significant (P &amp;lt; 0.05). The expression levels of miRNA-5739 and miRNA-3648 in follicular fluid were higher than those in the control group, but the expression levels in granule cells were lower than those in the control group, and the difference was significant (P &amp;lt; 0.05). Correlation analysis showed that the expression levels of miRNA-3648 and miRNA-5739 in follicular fluid were closely related to the normal fertilization rate of PCOS patients, and the expression levels of miRNA-5739 were closely related to the optimal embryo rate of PCOS patients. The expression levels of miRNA-642a-3p, miRNA-5739 and miRNA-3648 in granule cells were closely related to the MII egg rate and 2PN cleavage rate of PCOS patients. Limitations, reasons for caution MiRNAs participate in follicle development and maturation and hormone secretion through multiple signaling pathways. We only examined miRNA-642a-3p, miRNA-5739, miRNA-3648 in follicular fluid and granule cells, and did not detect the exosomal miRNAs. Dynamic changes in miRNA in the follicular microenvironment are not fully revealed. Wider implications of the findings Our results further demonstrate that the abnormal expression of miRNA in follicular fluid and granule cells in women with polycystic ovary syndrome (PCOS) is closely related to the local abnormal hormone secretion and follicle development of PCOS ovaries. Trial registration number not applicable

P-638 Impact of amphiregulin on oocyte maturation and embryo quality in patients with the polycystic ovarian syndrome.

Abstract Study question Does the concentration of amphiregulin in the follicular fluid affect oocyte maturation and embryo quality in patients with polycystic ovarian syndrome? Summary answer The concentration of amphiregulin affects oocyte maturity and embryo quality in patients with polycystic ovarian syndrome and can be used as a predictive biomarker. What is known already Polycystic ovary syndrome (PCOS) is a common metabolic dysfunction and heterogeneous endocrine disorder in women of reproductive age. Epidermal growth factor receptors and their ligands, such as amphiregulin, which are expressed in female reproductive tissues, have been shown to regulate various important reproductive functions. Amphiregulin is reportedly involved in oocyte maturation through autocrine and paracrine mechanisms, however, the impact of amphiregulin on oocyte maturity end embryo quality in patients diagnosed with POCS which goes under controlled ovarian stimulation remains unknown. Determination of potential biomarkers might be the key to successful IVF outcomes. Study design, size, duration The study consisted of a total of 87 oocytes obtained from 30 patients who underwent ICSI at UKS Homburg (Germany) between October 2021 and March 2022. Fifteen patients were diagnosed with polycystic ovary syndrome while the other fifteen were fertile without any diagnosis. For the study purpose, follicles were aspirated separately, and the follicular fluid was centrifuged for 5 min at 1,500×g. After the preparation, the supernatant was removed and stored at -20 °C until analysis. Participants/materials, setting, methods Concentrations of amphiregulin in follicular fluid were determined by using a commercially available sandwich ELISAkit (Duo Set; R&amp;D Systems Inc., Minneapolis) in accordance with the manufacturer’s instructions. Good embryo quality (GQE) on day 3 was defined as a 7-9 cell stage embryo with less than 25% of fragmentation and with equally sized blastomeres. Between-group comparisons were conducted with a Mann-Whitney test. Logistic regression analysis was used to determine the influence of amphiregulin on studied parameters. Main results and the role of chance The mean age of all patients was 33.5 (±5.2) years. Patients included in the study were stimulated according to a gonadotropin-releasing hormone (GnRH) antagonist protocol. Out of the 87 oocytes, 59 (67.8%) were at MII stadium, 13 (14.9%) were at MI and 8 (9.1%) were at GV stadium. ICSI was done only with MII and MI oocytes and the fertilization rate was 70.1% (61 oocytes were fertilized out of 72). Obtained results showed that concentrations of amphiregulin were significantly lower in follicular fluid of PCOS patients [(107.00(51.95-131.00) ng/mL vs 115.27(43.19-209.51) ng/mL) respectively; p = 0.024]. Data comparisons between mature and immature oocytes indicated that amphiregulin concentration was significantly higher in follicles where mature oocytes were developed [(117.50 (65.99-209.51) ng/mL vs 81.75(43.19-128.20) ng/mL) respectively; p &amp;lt; 0.0001]. Additionally, logistic regression analysis confirmed the positive effect of amphiregulin concentration on oocyte maturity (p &amp;lt; 0.001). Out of the 61 fertilized oocytes, 36 (59.0%) formed good-quality embryos. Additional analysis indicated that oocytes that developed into good-quality embryos came from follicles where amphiregulin concentrations were significantly higher compared to those that developed into poor-quality embryos [(124.05 (91.88-209.51) ng/mL. vs 86.72(43.19-109.40) ng/mL) respectively; p &amp;lt; 0.001]. Logistic regression analysis confirmed the positive effect of amphiregulin concentration on embryo quality (p &amp;lt; 0.01). Limitations, reasons for caution The limitation of the presented study was the relatively small patient cohort. Although the pregnancy rate in the presented study was 27.8 % , single embryo transfer was not performed, therefore further research should link amphiregulin concentrations with the pregnancy rate as well. Wider implications of the findings Results obtained in the study might be useful for further handling of PCOS patients since the literature already confirmed that PCOS patients produce an increased number of mature oocytes, so culture medium-containing amphiregulin might be a future solution for those patients. Trial registration number 146/19

P-637 pseudo-targeted metabolomics of follicular fluid reveals ovarian GPD1L-mediated glycerophospholipid metabolism dysfunction in patients with biochemical premature ovarian insufficiency

Abstract Study question Does the metabolic profile of follicular fluid from patients with biochemical premature ovarian insufficiency (bPOI) differ from that of healthy women? Summary answer The metabolic signature of follicular fluid reveals a significant decrease of phosphatidic acid (PA), phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC) and lysophosphatidylethanolamine (lysoPE) in bPOI patients. What is known already Metabolites in follicular fluid have attracted extensive attention as they are important components of the microenvironment during follicular development and oocyte maturation. Studies on follicular fluid metabolomics may enhance the understanding of the pathogenesis of ovarian aging, particularly premature ovarian insufficiency. Although a variety of metabolites have been suggested to be related to cell senescence, their specific roles in regulating follicular atresia remain unclear. Study design, size, duration An observational study of follicular fluid samples collected from 120 women undergoing in vitro fertilization and embryo transfer was conducted. All enrolled women were classified into two groups: one with 60 bPOI patients and the other with 60 healthy controls. Each group was further subdivided into two subgroups (young and aged group, 30 women for each section). Participants/materials, setting, methods The follicular fluids of mature follicles (diameter over 17 millimeters) were isolated and collected for pseudo-targeted metabolic analysis. The intensities of captured metabolites were utilized for further bioinformatic analysis. The correlation between the significant differential metabolites screened and their clinical outcomes were analyzed. In vitro experiments were used to demonstrate the cellular function of mainly regulated signaling pathway. Main results and the role of chance A total of 608 metabolites were identified in 120 follicular fluid samples. There were significant differences in 143 metabolites between the bPOI and healthy controls. As revealed by metabolic pathway analysis, glycerophospholipid metabolism was the most enriched pathway in bPOI follicular fluids. The intensities of several glycerophospholipid-associated metabolites PA, PC, lysoPC and lysoPE were positively correlated with the ovarian reserve indicator anti-Müllerian hormone (AMH), female age, as well as the expression level of glycerol-3-phosphatedehydrogenase 1-like (GPD1L), a key enzyme of this pathway, in granulosa cells. Furthermore, knockdown of GPD1L induced mitochondria dysfunction and granulosa cell apoptosis via GPD1L/PKCε/ATF2/NEAT1 pathway, as well as led to follicular atresia and impaired the quality of oocytes. Limitations, reasons for caution The validity of differential intensified metabolites we detected as biomarkers for clinical diagnosis needs to be confirmed in a large-scale study. And the multiple effects of glycerophospholipid metabolic pathway posed on follicular development need to be study in-depth. Wider implications of the findings Our results suggested that a highly coordinated follicular metabolism exhibits a strong relationship with the quality of follicles. An understanding of the mechanisms underlying ovarian aging will provide a theoretical basis, as well as new ideas for POI diagnosis, treatment and intervention. Trial registration number Not applicable.

Influence of secretome obtained from preovulatory follicular fluid on energy metabolism and meiotic competence of equine cumulus–oocyte complexes

Secretome is composed by proteins and microvesicles secreted by the granulosa cells that line the follicular wall. These bioactive compounds promote follicular growth and oocyte maturation, influencing oocyte metabolism. Hence, we hypothesized that addition of secretome from preovulatory follicular fluid (FF) to maturation medium may influence the meiotic competence and metabolism of cumulus-oocyte-complexes (COCs) obtained post mortem. FF was retrieved from preovulatory follicles (2 mares) after 32h of hCG administration. Three ml of FF from both mares were pooled, diluted 1:1 in PBS and centrifuged (4000g, 1h at 4°C) using a 10K Amicon® Ultra-15 Centrifugal Filter Unit and protein concentration was measured. A total of 186 COCs were retrieved from post mortem ovaries and were subjected to in vitro maturation (IVM) in TCM-199 medium, 10% of FBS and 5 mU/ml of FSH (Control; 59 oocytes) or with 20 µg/ml of secretome (Secretome20; 66 oocytes) or with 40 µg/ml of secretome (Secretome40; 61 oocytes) for 28 h in 5% CO2/95% air at 38.2°C. Oocytes were denuded, fixed in 4% formaldehyde and stained with Hoechst 33342 to assess chromatin conformation by fluorescence microscopy. Also, selected metabolites of the maturation medium prior to andafter IVM were assessed by magnetic proton nuclear resonance spectroscopy. After IVM, a trend towards glucose consumption and pyruvic acid production was observed compared to their controls prior IVM; a significant increase in lactic acid production in all treatments was observed (Table 1; p < 0.05). Our results show a tendency for quicker meiosis resumption in COCs incubated with Secretome40 (Table 2). In conclusion, secretome obtained from preovulatory follicular fluid added during IVM does not change COCs’ energy metabolism nor maturation rate of equine cumulus-oocyte complexes in our in vitro culture system. Funding: MCCIN/AEI (PID2020-112723RB-I00; RYC-2017-21545; RYC2020-028915-I), Junta de Extremadura-FEDER (IB20005).

Perspective: Can early embryonic losses be reduced in lactating dairy cows?

Perspective: Can early embryonic losses be reduced in lactating dairy cows?

New Technique for Human Sperm Cryopreservation Using Emptied Sheep's Ovarian Follicles.

This study aimed to investigate the efficacy of emptied ovarian follicles of sheep as a container for cryopreservation of human spermatozoa to preserve the presence of low concentrations of spermatozoa at the post-thawing stage. This research was performed on 30 semen samples from oligozoospermic patients and 10 samples from normozoospermic males. They were diagnosed according to the standard criteria of the World Health Organization 2010. Semen samples were classified into four groups of G1-G4 according to sperm concentration: 3-5 million/mL, 6-10 million/mL, 11-15 million/mL, and 16-20 million/mL, respectively. Each sample was divided into two equal parts. One part was cryopreserved without cryoprotectant, while the other was diluted 1:1 with 10% glycerol-based cryosolution. The ovarian follicles of sheep were obtained from a local slaughterhouse and prepared by slicing the ovaries and evacuating the follicular fluid and oocyte. The emptied follicles were injected with the prepared semen samples. After cryopreservation and thawing, the semen mixture aspired outside the follicles, and sperm parameters were measured, namely concentration, progressive motility, total motility, and normal morphology. Sperm concentration and progressive and total sperm motility were significantly (P<0.01) decreased in all groups at the post-thawing stage, compared to the pre-freezing stage. The sperm concentration was significantly higher (P<0.01) in samples cryopreserved without cryoprotectant, compared to that in those cryopreserved with glycerol. However, progressive and total motility were significantly (P<0.01) higher in samples cryopreserved with glycerol, compared to that in the samples cryopreserved without cryoprotectant in all groups. Moreover, no significant difference was found between the pre-freezing and post-thawing stages in terms of normal morphology. Emptied ovarian follicles are an appropriate carrier for cryopreservation of human sperms, especially for patients with oligozoospermia. The best sperm survival rate in this technique was observed when using glycerol-based cryosolution.

Investigation of Ovarian and Non-ovarian Associated Factors Related to Follicular Population and Oocyte Maturation of Chadian Cattle Breeds

A cow can give birth to an average of 6-7 calves in her entire reproductive period. The remaining oocytes could be used for the in vitro production of embryos. The present study was conducted to evaluate the effects of ovarian and non-ovarian factors on the follicular population and oocyte maturation of three Chadian cattle breeds (Arab, Kouri, and Toupouri). For this purpose, the ovaries of 166 cycled cows were collected at the Farcha slaughterhouse of Chad and placed individually in labeled conical tubes containing 0.9% NaCl and 0.5 mg/ml penicillin-streptomycin. After clearing the ovaries of tissue debris, they were weighed, and the follicles were counted. The diameter of each follicle was measured and classified into three categories. A total of 2734 oocytes were collected in 28 days with a minimum of 97 per day by the slicing method using a 10X stereoscope. They were then classified into four groups according to the structure of their cumulus oophorus. Immature oocytes (class 1 and 2 [1455]) were placed in different culture media consisting of Minimum Essential Medium (MEM) alone, MEM with 10% follicular fluid, and MEM with 50% follicular fluid for oocyte maturation. The results indicated that the mean follicular population and mean oocyte yield were 24.71 ± 0.88 and 11.65 ± 0.94, respectively. The mean oocyte index and the number of cultivable oocytes for in vitro embryo production (class 1 and 2) were 1.03 ± 0.23 and 1.65 ± 0.94, respectively. The number of follicles observed in the age group of 6-9 years was higher than in other age groups. Oocyte yield was significantly higher in cows with a body condition score of 4-5 compared to average and lean cows. Among the different culture media used for oocyte maturation, the medium consisting of MEM plus 10% follicular fluid recorded a higher maturation rate than the other culture media. Cows aged 6-9 years had a higher maturation rate than other age groups. In conclusion, the good follicle (follicle that produced oocyte) and appropriate oocyte performance were observed in cows with body condition score 3-5 and an age range of 6-9 years.

Effects of IL-11/IL-11 Receptor Alpha on Proliferation and Steroidogenesis in Ovarian Granulosa Cells of Dairy Cows.

Granulosa cells (GCs) are essential for follicular growth, oocyte maturation, and steroidogenesis in the ovaries. Interleukin (IL)-11 is known to play a crucial role in the decidualization of the uterus, however, the expression of the IL-11 system (IL-11, IL-11Rα, and gp130) in the bovine ovary and its exact role in GCs have not been extensively studied. In this study, we identified the IL-11 signaling receptor complex in the bovine ovary and investigated the regulatory effects and underlying mechanism of IL-11Rα on the proliferation and steroidogenesis of GCs. We observed that the IL-11 complex was highly expressed in the GCs of large follicles. IL-11Rα knockdown significantly inhibited GC proliferation by inducing cell cycle arrest at the G1 phase, along with a significant downregulation of proliferating cell nuclear antigen (PCNA) and Cyclin D1 (CCND1) protein, and induced GC apoptosis by significantly upregulating the ratio of BCL-2-associated X protein (BAX) and B-cell lymphoma-2 (BCL-2). In addition, IL-11Rα knockdown attenuated the Janus kinase (JAK) 1-signal transducer and activator of transcription 3 (STAT3) signaling, which is related to cell proliferation and apoptosis. Furthermore, the enzyme-linked immunosorbent assay (ELISA) indicated that IL-11Rα silencing decreased the basal and forskolin (FSK)-stimulated secretions of estradiol and progesterone in GC culture medium concomitantly with a remarkable decrease in cytochrome P450 family 19 subfamily A member 1 (CYP19A1) and steroidogenic acute regulatory protein (StAR). We subsequently determined that this reduction in steroidogenesis was in parallel with the decrease in phosphorylations of protein kinase A (PKA) substrates, cAMP-response element binding protein (CREB), extracellular regulated protein kinase (ERK) 1/2, and p38 mitogen-activated protein kinase (MAPK). Taken together, these data indicate that the effects of IL-11/IL-11Rα on the proliferation and steroidogenesis in bovine GCs is mediated by the JAK1-STAT3, PKA-CREB, p38MAPK, and ERK1/2 signaling pathways. Our findings provide important insights into the local action of the IL-11 system in regulating ovarian function.

Circulating maternal prorenin and oocyte and preimplantation embryo development: the Rotterdam Periconception Cohort.

Could circulating maternal prorenin serve as a proxy for oocyte and preimplantation embryo development, assessed by time-lapse parameters and clinical treatment outcomes? High circulating maternal prorenin concentrations after ovarian stimulation associate with a larger oocyte area, faster cleavage divisions from the five-cell stage onwards and increased chance of successful implantation. After ovarian stimulation, circulating prorenin (renin's precursor), is largely ovary-derived. Prorenin may contribute to ovarian angiotensin synthesis, which is relevant in reproduction given its role in follicular development and oocyte maturation. Prospective observational cohort study including couples requiring fertility treatment from May 2017 as a subcohort of the ongoing Rotterdam Periconception Cohort conducted in a tertiary referral hospital. Between May 2017 and July 2020, 309 couples with an indication for IVF treatment or ICSI were included. Resulting embryos (n = 1024) were submitted to time-lapse embryo culture. Time of fertilization (t0), pronuclear appearance (tPNa), and fading (tPNf) as well as the exact timing of reaching the two- to eight-cell stage (t2-t8), the start of blastulation (tSB), reaching the full (tB), and expanded blastocyst (tEB) were retrospectively recorded. Oocyte area was measured at t0, tPNa, and tPNf. Prorenin was determined at the day of embryo transfer. After adjustment for patient- and treatment-related factors, linear mixed modeling showed that higher prorenin concentrations associate with a larger oocyte area at tPNa (β 64.45 µm2, 95% CI 3.26; 125.64, P = 0.04), and faster progression from five-cell stage onwards (e.g. β8-cell -1.37 h, 95% CI -2.48; -0.26, P = 0.02). Prorenin associated positively with pre-transfer outcomes (e.g. βfertilized oocytes 2.09, 95% CI 1.43; 2.75, P < 0.001) and implantation (odds ratio+β-hCG-test: 1.79, 95% CI 1.06; 3.08, P = 0.03), but not with live birth. This prospective observational study provides associations and therefore residual confounding cannot be excluded and causality has to be shown in intervention studies. Theca cell-derived factors, such as prorenin, may help to clarify the underlying endocrine mechanism of oocyte maturation and embryo development, with a special focus on the (patho)physiological reproductive role of prorenin and the identification of factors influencing its secretion and activity, which is of great added value for improving embryo selection and predicting implantation and pregnancy outcomes. This will bring us to investigate which determinants of oocyte quality and embryo development should take center stage in developing preconception care strategies. This research was funded by the Department of Obstetrics and Gynecology of the Erasmus MC, University Medical Center, Rotterdam, the Netherlands, and the Erasmus MC Medical Research Advisor Committee's 'Health Care Efficiency Research' program (OZBS72.16080). The authors have no competing interests to disclose. N/A.

Follicular Fluid Components in Reduced Ovarian Reserve, Endometriosis, and Idiopathic Infertility.

Follicular fluid (FF) molecules, and their increase or decrease, can contribute to appropriate follicular growth and oocyte maturation, thus being related to female infertility conditions. In this paper, we studied the changes and the relationships of some biochemical components, hormones, antioxidant enzymes, F2-Isoprostanes (F2-IsoPs), and resolvin (Rv) D1 in the FF of infertile women with different reproductive conditions such as endometriosis, reduced ovarian reserve, and idiopathic infertility during assisted reproductive techniques (ART). In the whole population, positive correlations between albumin (ALB)/iron (Fe), ALB/beta-2-microglobulin (B2MG), and F2-IsoPs/RvD1 were detected in the FF. In FF from aged women, increased levels of follicle stimulating hormone (FSH) and reduced anti-Müllerian hormone (AMH) levels were associated with a worse oocyte quality. The negative ART outcome was influenced by patient age and AMH, B2MG, and FSH levels. Moreover, the reduced ovarian reserve condition was characterised by a significant decrease in oocyte number and quality, AMH amount, and lactate dehydrogenase (LDH) activity, as well as by an increase in age and FSH levels. In the presence of endometriosis, high levels of MDA and RvD1 were detected in FF, with a decrease in luteinising hormone (LH). Finally, among the molecules examined, none characterised the condition of idiopathic infertility. These data could support the identification of new FF markers in different reproductive disorders, suggesting the need for personalised therapeutic approaches and optimised ART outcomes. In particular, the evaluation of resolvins and lipid mediators in FF could be a promising field of investigation with which to understand the entity of oxidative stress and inflammation in some female infertility conditions.

AMPK-like proteins and their function in female reproduction and gynecologic cancer.

Serine-threonine kinase (STK11), also known as liver kinase B1 (LKB1), is a regulator of cellular homeostasis through regulating the cellular ATP-to-ADP ratio. LKB1 is classified as a tumor suppressor and functions as the key activator of AMP-activated protein kinase (AMPK) and a family of serine-threonine kinases called AMPK-like proteins. These proteins include novel (nua) kinase family 1 (NUAK1 and 2), salt inducible kinase (SIK1), QIK (known as SIK2), QSK (known as SIK3 kinase), and maternal embryonic leuzine zipper kinase (MELK) on tightly controlled and specific residual sites. LKB1 also regulates brain selective kinases 1 and 2 (BRSK1 and 2), additional members of AMPK-like protein family, which functions are probably less studied. AMPK-like proteins play a role in variety of reproductive physiology functions such as follicular maturation, menopause, embryogenesis, oocyte maturation, and preimplantation development. In addition, dysfunctional activity of AMPK-like proteins contributes to apoptosis blockade in cancer cells and induction of the epithelial-mesenchymal transition required for metastasis. Dysregulation of these proteins occurs in ovarian, endometrial, and cervical cancers. AMPK-like proteins are still undergoing further classification and may represent novel targets for targeted gynecologic cancer therapies. In this chapter, we describe the AMPK-like family of proteins and their roles in reproductive physiology and gynecologic cancers.

EMBRYOLOGICAL INDICATORS AND INCIDENCE OF PREGNANCY IN WOMEN WITH INFERTILITY UNDERGOING ASSISTED REPRODUCTIVE TECNOLOGY PROGRAMS WITH THE USE OF MELATONIN

The technology of intracytoplasmic sperm injection into the egg cell (ICSI) has taken the mail place among assisted reproductive technologies (ART) in the treatment of infertility. Improving the effectivenessof ART remains a complex medical problem. To a large extent, the success of ICSI depends on the quality of theoocytes, which, in its turn, depends on the number of follicles, which is determined by the ovulatory = ovarianreserve (OV) and effective preparation of patients for ART. The markers of OV are well known, and attention has been drawn to melatonin as a possible prognostic factor for the effectiveness of ART and OV in the last ten years. The indicators of melatonin content in the follicular fluid of stimulated ovaries have not been studied. At the same time, the assessment of embryological indicators of fertilization when using the medication melatonin for the preparation of patients and the effectiveness of ART programs in general can be useful for the furtherdevelopment of treatment algorithms for patients with infertility. The objective of the study. Evaluation of embryological indicators, the effectiveness of ICSI programs andthe incidence of pregnancy in women with infertility during their preparation with melatonin. Materials and methods. 67 women with various infertility factors participated in the prospective study.All patients were prepared for controlled ovarian stimulation (COS), and subsequently for follicular puncture, oocyte retrieval and fertilization by ICSI. All obtained blastocysts were cryopreserved, stored in liquid Nitrogen and thawed when embryo transfer was necessary. Embryological indicators, the number of transfers and the effectiveness of pregnancy incidence were evaluated. According to the purpose of the task, the patients were divided into two groups by the method of even-odd numbers: group 1 – 29 patients with infertility, who received the preparation "Vita-melatonin" 3 mg orally before bedtime, during one month before the follicles puncture,group 2 – 38 patients with infertility, who did not receive this medicine. Statistical processing of the obtainedresults was carried out using the licensed programs "Microsoft Excel" and "Statistica".The research design and all the methods we used in this prospective study were reviewed and approved by the bioethics commission of the higher education institution "Bukovinian State Medical University" (protocol No. 7 dated 04/21/2022). SRW "Preservation and restoration of reproductive health of women and girls with obstetric andgynecological pathology" (state registration number 0121U110020. Implementation period – 01.2021-12.2025). Results of the research. The incidence of pregnancy after thawing of blastocysts and embryo transfers ingeneral in women who received melatonin was significantly (p ˂ 0.05) higher compared to women who didnot receive melatonin, respectively, 86.2 ± 6.41% and 76.3 ± 6.95%. For all performed embryo transfers, theincidence of pregnancy was 56.8 ± 8.24% and 52.8 ± 7.62% (p &gt; 0.05), respectively, for the first embryo transfer 58.1 ± 8 .85% and 55.3 ± 8.15% (р &gt; 0.05), for the second - 43.5 ± 16.53% and 33.3 ± 21.14% (р &gt; 0.05), for the third – 100.0 ± 7.00% and 80.0 ± 17.95% (р &gt; 0.05).When assessing embryological indicators, it was found that in women who received melatonin, the number of mature oocytes was 83.4 ± 6.94%, and in women who did not receive melatonin, 81.5 ± 6.36%, but there was no significant (p &gt; 0.05) difference. Out of mature oocytes in the process of incubation after the ICSI technology,49.5 ± 9.54% and 47.2 ± 8.72 (р &gt; 0.05) blastocysts were formed, respectively, and from them blastocysts of class I – 48.6 ± 9 .36% and 46.4 ± 8.17% (р &gt; 0.05), class II – 36.8 ± 8.94% and 44.4 ± 8.13% (р &gt; 0.05), class III – 14.6 ± 6.67% and 9.3 ± 4.78% (р &gt; 0.05).Conclusion. The use of melatonin in the preparation of women with infertility for the use of assisted reproductive technologies has shown its effectiveness in terms of pregnancy incidence.

SARS-CoV-2 RNA in Follicular Fluid, Granulosa Cells, and Oocytes of COVID-19 Infected Women Applying for Assisted Reproductive Technology

Background: The Coronavirus disease 2019 (COVID-19) pandemic has raised concerns regarding the application of assisted reproductive technology (ART) in the world. Many ART programs have been delayed or continued with new precautions due to the ambiguity about vertical transmission and pregnancy safety. Regarding the possible risks of SARS-CoV-2 infection on ART and the resultant embryos, this study aimed to investigate the presence of SARS-CoV-2 in follicular fluid, granulosa cells, and oocytes of COVID-19-infected women undergoing ART. Materials and Methods: COVID-19-positive polymerase chain reaction tests were reported for five women undergoing ART cycles on the day of oocyte retrieval. SARS-CoV-2 tests were performed on oocytes, granulosa cells, and follicular fluid obtained from these COVID-19-infected women. Results: SARS-CoV-2 RNA was detected only in one follicular fluid sample; however, other follicular fluid samples, granulosa cells, and oocytes were negative regarding viral RNA. Conclusion: Given the unknown effects of COVID-19 on human reproduction and ART, strict precautions should be taken during the COVID-19 pandemic.