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STUDY QUESTION Which substances and signal transduction pathways are potentially active downstream to the effect of FSH and LH in the regulation of human oocyte maturation in vivo ? SUMMARY ANSWER The regulation of human oocyte maturation appears to be a multifactorial process in which several different signal transduction pathways are active. WHAT IS KNOWN ALREADY Many studies in animal species have provided insight into the mechanisms that govern the final maturation of oocytes. Currently, these studies have identified several different mechanisms downstream to the effects of FSH and LH. Some of the identified mechanisms include the regulation of cAMP/cGMP levels in oocytes involving C-type natriuretic peptide (CNP), effects of epidermal growth factor (EGF)-related peptides such as amphiregulin (AREG) and/or epiregulin (EREG), effect of TGF-β family members including growth differentiation factor 9 (GDF9) and morphogenetic protein 15 (BMP15), activins/inhibins, follicular fluid meiosis activating sterol (FF-MAS), the growth factor midkine (MDK), and several others. However, to what extent these pathways and mechanisms are active in humans in vivo is unknown. STUDY DESIGN, SIZE, DURATION This prospective cohort study included 50 women undergoing fertility treatment in a standard antagonist protocol at a university hospital affiliated fertility clinic in 2016–2018. PARTICIPANTS/MATERIALS, SETTING, METHODS We evaluated the substances and signalling pathways potentially affecting human oocyte maturation in follicular fluid (FF) and granulosa cells (GCs) collected at five time points during the final maturation of follicles. Using ELISA measurement and proteomic profiling of FF and whole genome gene expression in GC, the following substances and their signal transduction pathways were collectively evaluated: CNP, the EGF family, inhibin-A, inhibin-B, activins, FF-MAS, MDK, GDF9, and BMP15. MAIN RESULTS AND THE ROLE OF CHANCE All the evaluated substances and signal transduction pathways are potentially active in the regulation of human oocyte maturation in vivo except for GDF9/BMP15 signalling. In particular, AREG, inhibins, and MDK were significantly upregulated during the first 12–17 h after initiating the final maturation of follicles and were measured at significantly higher concentrations than previously reported. Additionally, the genes regulating FF-MAS synthesis and metabolism were significantly controlled in favour of accumulation during the first 12–17 h. In contrast, concentrations of CNP were low and did not change during the process of final maturation of follicles, and concentrations of GDF9 and BMP15 were much lower than reported in small antral follicles, suggesting a less pronounced influence from these substances. LARGE SCALE DATA None. LIMITATIONS, REASONS FOR CAUTION Although GC and cumulus cells have many similar features, it is a limitation of the current study that information for the corresponding cumulus cells is not available. However, we seldom recovered a cumulus–oocyte complex during the follicle aspiration from 0 to 32 h. WIDER IMPLICATIONS OF THE FINDINGS Delineating the mechanisms governing the regulation of human oocyte maturation in vivo advances the possibility of developing a platform for IVM that, as for most other mammalian species, results in healthy offspring with good efficacy. Mimicking the intrafollicular conditions during oocyte maturation in vivo in small culture droplets during IVM may enhance oocyte nuclear and cytoplasmic maturation. The primary outlook for such a method is, in the context of fertility preservation, to augment the chances of achieving biological children after a cancer treatment by subjecting oocytes from small antral follicles to IVM. Provided that aspiration of oocytes from small antral follicles in vivo can be developed with good efficacy, IVM may be applied to infertile patients on a larger scale and can provide a cheap alternative to conventional IVF treatment with ovarian stimulation. Successful IVM has the potential to change current established techniques for infertility treatment. STUDY FUNDING/COMPETING INTEREST(S) This research was supported by the University Hospital of Copenhagen, Rigshospitalet, the Independent Research Fund Denmark (grant number 0134-00448), and the Interregional EU-sponsored ReproUnion network. There are no conflicts of interest to be declared. [ABSTRACT FROM AUTHOR]

Follicular fluid meiosis-activating sterol (FF-MAS) exerts beneficial effects on the meiotic resumption of mammalian oocytes and their subsequent early embryonic development, but the signaling pathway underlying these effects has not been elucidated. Therefore, the objective of the present study was to investigate whether the mitogen-activated protein kinase (MAPK) pathway is involved in the FF-MAS-induced in vitro resumption of meiosis in porcine oocytes. Porcine cumulus oocyte complexes (COCs) were allocated in several groups cultured in TCM-199 medium with different concentration of AY 9944-A-7 (20, 30, 40 μmol/L) or ketoconazole (20 μmol/L) to increase or decrease endogenous accumulation of FF-MAS. Each experimental condition was repeated at least six times. After maturation for 44 h, the resumption of meiosis was assessed by the frequency of germinal vesicle breakdown (GVBD) and the first polar body (PBI) extrusion, The relative expressions of related genes in MAPK pathway [c-mos, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase 1/2 (ERK1/2)] at both transcriptional and translational levels were detected to investigate the kinetic trend of expression throughout oocyte maturation in vitro in response to the addition of AY 9944-A-7 or ketoconazole to the maturation medium. Results indicated that AY 9944-A-7 promoted, while ketoconazole inhibited, the in vitro maturation (IVM) of porcine oocytes. Relative expression of meiosis related genes was upregulated by AY 9944-A-7 and downregulated by ketoconazole. With extended culturing time, c-mos mRNA expression levels reached their peak at 12 h of maturation and decreased gradually thereafter, while MEK , ERK1 and ERK2 expression increased after an initial decrease peaking at 44 h of culture in the AY 9944-A-7-group. And the trend of the protein expression of c-mos, MEK, ERK1/2 was basically consistent with the mRNA expression of these genes. These results imply that the endogenous accumulation of FF-MAS is beneficial to resumption of meiosis in porcine oocytes and that MAPK signaling is involved in FF-MAS-induced meiotic resumption. • The endogenous accumulation of FF-MAS is beneficial to resumption of meiosis in porcine oocytes. • The c-mos , MEK , ERK1 and ERK2 and protein mRNA relative expression in porcine oocytes was affected by the endogenous FF-MAS. • MAPK signaling pathway is involved in FF-MAS-induced IVM of porcine oocytes. [ABSTRACT FROM AUTHOR]

Follicular fluid meiosis-activating sterol (FF-MAS) is a small molecule compound found in FF, named for its ability to induce oocyte resumption of meiosis. Granulosa cells (GCs) within the follicle are typically located in a hypoxic environment under physiologic conditions due to limited vascular distribution. Previous research suggests that hypoxia-induced cell cycle arrest and apoptosis in GCs may be crucial triggering factors in porcine follicular atresia. However, the impact of FF-MAS on GCs within follicles has not been explored so far. In this study, we uncovered a novel role of FF-MAS in facilitating GC survival under hypoxic conditions by inhibiting STAT4 expression. We found that STAT4 expression was upregulated in porcine GCs exposed to 1% O2. Both gain and loss of function assays confirmed that STAT4 was required for cell apoptosis under hypoxia conditions, and that the GC apoptosis caused by hypoxia was markedly attenuated following FF-MAS treatment through inhibition of STAT4 expression. Correlation analysis in vivo revealed that GC apoptosis was associated with increased STAT4 expression, while the FF-MAS content in follicular fluid was negatively correlated with STAT4 mRNA levels and cell apoptosis. These findings elucidate a novel role of FF-MAS-mediated protection of GCs by inhibiting STAT4 expression under hypoxia, which might contribute to the mechanistic understanding of follicular development., (© The Author(s) 2024. Published by Oxford University Press on behalf of the American Society of Animal Science. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

SUMOylation, a process of posttranslational modification of proteins by the small ubiquitin-related modifier (SUMO) family of proteins, is known to be involved in yeast and mammalian somatic cell-cycle regulation. However, the identities of the SUMO-modified oocyte targets are largely unknown and the functional role(s) for SUMOylation during mammalian oocyte maturation remains unclear. On the basis of studies in non-germline cells, protein kinase B/Akt is a potential SUMOylation target in the mouse oocyte, where it plays an essential role in cell-cycle resumption and progression during maturation. This study investigated the temporal patterns and prospective role(s) for interactions between SUMOylation and Akt serine-phosphorylation during oocyte meiotic resumption. Pharmacological inhibition of SUMOylation significantly decreased follicular fluid meiosis-activating sterol-induced cell-cycle resumption in oocytes matured in vitro and negatively affected the phosphorylation and nuclear translocation of Akt. Similarly, nuclear localization of cyclin D1, a downstream target of Akt activation, was significantly decreased following SUMOylation inhibition. Together these data show that SUMO and the posttranslational process of SUMOylation are involved in cell-cycle resumption during murine oocyte maturation and exert a regulatory influence on the Akt pathway during germinal vesicle breakdown. [ABSTRACT FROM AUTHOR]

Sterol intermediates of the cholesterol biosynthetic pathway have drawn attention for novel biological activities. Follicular fluid meiosis activating sterol (FF-MAS) is a LXRα ligand and a potential modulator of physiologic processes regulated by nuclear receptors, such as lipid homeostasis and cell proliferation. In this work, we established a model to selectively accumulate FF-MAS in HepG2 cells, by using a combination of the inhibitors AY9944 and 17-hydroxyprogesterone to block C14-sterol reductases and the downstream C4-demethylase complex. We investigated the effects produced by altered levels of cholesterol biosynthesis intermediates, in order to dissect their influence on LXRα signaling. In particular, endogenously accumulated FF-MAS was able to modulate the expression of key genes in cholesterol metabolism, to activate LXRα nuclear signaling resulting in increased lipogenesis, and to inhibit HepG2 cells proliferation. Moreover, a fluorescent ester derivative of FF-MAS localized in nuclear lipid droplets, suggesting a role for these organelles in the storage of signaling lipids interacting with nuclear partners. [ABSTRACT FROM AUTHOR]

Follicular fluid meiosis-activating sterol (FF-MAS), an intermediate in the cholesterol biosynthetic pathway, has been identified as a compound that induces the resumption of meiosis in mammalian oocyte. Follicular fluid meiosis-activating sterol is converted to testis meiosis–activating sterol by a sterol Δ14-reductase. An inhibitor of Δ14-reductase and Δ7-reductase, AY9944 A-7, causes accumulation of FF-MAS by inhibiting its metabolism. The objective of this research was to investigate the specific contribution of AY9944 A-7 on gonadotropin-induced meiotic resumption and its interactive effects with FSH or LH on meiotic maturation of oocytes and preimplantation development of parthenogenetic embryo in sheep by addition of AY9944 A-7 during IVM to cause accumulation of FF-MAS. First, ovine cumulus–oocyte complexes (COCs) were cultured in the presence of FSH (10 μg/mL), LH (10 μg/mL), AY9944 A-7 (20 μmol/L), FSH (10 μg/mL)+AY9944 A-7 (20 μmol/L), or LH (10 μg/ml) + AY9944 A-7 (20 μmol/L) with an inhibitor hypoxanthine (Hx) to prevent spontaneous meiosis of oocytes. The resumption of meiosis was assessed by the frequency of germinal vesicle breakdown and the first polar body (PBI) extrusion. The kinetics of gonadotropin and AY9944 A-7–induced meiotic resumption in vitro was also evaluated in the study. The numbers of oocytes resuming meiosis and undergoing germinal vesicle breakdown were counted after the COCs were cultured for 2, 4, 8, 12, 16, 20, and 24 hours. Matured oocytes extruding PBI were selected for parthenogenetic activation, and the percentages developing to the two-cell stage and blastocyst stage were recorded as indicators of parthenogenetic embryo developmental competence. It was observed that FSH could induce the resumption of meiosis of ovine COCs cultured in the presence of Hx, but LH could not. AY9944 A-7 had a synergistic effect with FSH on nuclear maturation and developmental competence of embryos produced by parthenogenetic activation, whereas it had no added advantage on LH action. However, the kinetics of meiotic resumption after AY9944 A-7 stimulation was remarkably delayed when compared with FSH-induced maturation. In conclusion, the current study suggested that AY9944 A-7 supplementation in IVM medium optimized the beneficial effects of FSH on meiotic maturation of ovine oocytes and subsequent developmental competence of embryos produced by parthenogenetic activation. This work had important potential for developing a novel technique in IVM of ovine oocytes. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the safety of applying follicular-fluid meiosis-activating sterol (FF-MAS) in vitro to immature human oocytes. Design: Phase I bicenter, randomized, parallel-group, controlled, partially blinded trial. Setting: Third-level referral academic centers, including reproductive biology and genetics laboratories. Patients: Endocrinologically normal women with a medical indication for IVF or intracytoplasmic sperm injection, or healthy volunteers. Intervention(s): Subjects were randomized at a ratio 1 to 6 into either conventional GnRH-agonist and recombinant FSH stimulation (IVO) for oocyte retrieval, or minimally stimulated in vitro maturation (IVM) with the use of recombinant FSH. Retrieved immature oocyte cumulus complexes were cultured for 30 or 36 hours in one of six IVM culture conditions containing FF-MAS (range, 0.1–20 μM). Polar body-extruded oocytes from the IVO and IVM groups were processed for chromosomal analysis. Main Outcome Measure(s): The primary endpoint was the incidence of metaphase II stage oocytes with numeric chromosomal abnormalities, using full (spectral karyotyping) or partial (fluorescent in situ hybridization with seven probes) karyotyping or Giemsa count. A secondary objective was to document the frequence of metaphase II oocytes after IVM with FF-MAS supplements. Result(s): Oocyte cumulus complexes obtained from the IVO (mean, 8.9) and IVM (mean, 6.2) groups had equal maturation rates. Compared to IVO, exposure of germinal-vesicle oocytes for a maturation period of 30 hours did not increase aneuploidy. An exposure period of 36 hours doubled the aneuploidy rate, but this was significant only for the 20-μM dose of FF-MAS. Conclusion: Inclusion of 1–10 μM FF-MAS in a 30-hour IVM protocol is safe. [Copyright &y& Elsevier]

Purpose: Studies in mice and humans have shown that Follicular Fluid — Meiosis Activating Sterol (FF-MAS) induces meiotic maturation of immature oocytes in vitro. A multicenter, prospectively randomised study evaluated chromosomal status of embryos from FSH/hCG primed human immature oocytes, cultured with or without FF-MAS. Methods: Denuded immature oocytes (n=365) were randomly allocated into inert control, FF-MAS 5 μMor 20 μM. Seventy ±2 hours after ICSI on matured oocytes, all cleaved embryos were fixed for fluorescence in situ hybridisation analysis. Results:Only 15% of oocytes resulted in cleaved embryos. GV oocytes matured at significantly lower rates (14% and 7%) in the two FF-MAS groups compared to the inert control group (47%). High rates of chromosomal abnormalities were found in all groups. Conclusion: Immature oocytes showed poor development with high rates of embryo chromosomal abnormalities. Exposure to FF-MAS in the concentrations, duration and/or formulation used in this study did not improve the results. [ABSTRACT FROM AUTHOR]

Abstract: Inhibition of fungal lanosterol-14α-demethylase (CYP51) is the working principle of the antifungal activity of azoles used in agriculture and medicine. Inhibition of human CYP51 may result in endocrine disruption since follicular fluid-meiosis activating steroid (FF-MAS), the direct product of lanosterol demethylation, is involved in the control of meiosis. To investigate the specificity of antifungal agents for the fungal enzyme, assays to determine inhibitory potencies of 13 agricultural fungicides and 6 antimycotic drugs were established. FF-MAS product formation was measured by LC–MS/MS analysis in the incubations using lanosterol as substrate. Recombinant human enzyme (hCYP51) was available from BD Gentest. CYP51 of Candida albicans (cCYP51) was co-expressed with Candida tropicalis oxidoreductase in the baculovirus system. IC50 values of 13 fungicides for cCYP51 ranged about six-fold (0.059–0.35μM); for hCYP51 the range was about 30-fold (1.3–37.2μM). The most favourable IC50 ratio human to Candida was observed for imazalil (440-fold), while the specificity of epoxiconazole and tebuconazole for cCYP51 was only by a factor of 10. For the antimycotic drugs, the range of IC50 values for cCYP51 was similar to those of fungicides (0.039–0.30μM). For the inhibition of hCYP51, IC50 values split into two classes: the newer drugs fluconazole and itraconazole showed little inhibition (≥30μM) while the older drugs were even more potent than the agricultural fungicides, with miconazole being the most potent (0.057μM). No correlation was seen between the IC50 values determined for the two enzymes, indicating that a housekeeping gene can show significant diversity if inhibition is concerned. Our data indicate that fungicide residues in food are unlikely to exert a relevant inhibition of CYP51 in humans whereas systemic use of some antimycotic drugs, e.g. ketoconazole or miconazole, should be carefully considered regarding disturbance of human steroid biosynthesis. [Copyright &y& Elsevier]

Objective: To evaluate the effect of adding follicular-fluid meiosis-activating sterol (FF-MAS) in a novel 0.2% recombinant human albumin–based formulation to cumulus-enclosed oocytes on chromosomal status and development of pre-embryos. Design: Multicenter, prospective, randomized, open (double-blind for vehicle and FF-MAS groups), four parallel groups, controlled trial. Setting: Four public IVF clinics in Denmark. Patient(s): Two hundred eighteen women undergoing IVF donated 483 oocytes. Intervention(s): Follicle-stimulating hormone/hCG-primed cumulus-enclosed oocytes randomized to 4 hours of exposure to medium with 1 or 10 μmol/L of FF-MAS dissolved in 0.2% recombinant human albumin, medium with 0.2% recombinant human albumin (vehicle control), or medium alone (control) before insemination. Main Outcome Measure(s): Primary endpoint: incidence of human pre-embryos with chromosomal abnormalities. Secondary endpoint: fertilization rate, cleavage rate, and pre-embryo quality assessed after 68 hours of culture. Result(s): At pre-embryo level, the overall abnormality rates in the control, vehicle control, and 1- and 10-μmol/L FF-MAS groups were 53%, 39%, 42%, 53%, respectively, and at blastomere level 49%, 44%, 44%, and 48%, respectively. After 20 and 26 hours, the fertilization rates were between 67% and 71% in all groups. No differences in the cleavage rates were observed. Conclusion(s): The concentrations of FF-MAS in a novel 0.2% recombinant human albumin–based formulation of FF-MAS did not increase the risk of chromosomal abnormalities in pre-embryos or blastomeres. No statistically significant differences in fertilization rate, cleavage rate, or number of good quality pre-embryos were found among the four groups. [Copyright &y& Elsevier]

A simple and sensitive method has been developed and validated for purity determination of FF-MAS (also known as (3β,20R)-4,4-dimethylcholesta-8,14,24-trien-3-ol an endogenous substance usually present in the pre-ovulatory follicular fluid) at very low concentrations (200ng per unit) in pharmaceutical formulations containing RECOMBUMIN® (recombinant human albumin) as the matrix. The paper focuses on development of the sample preparation for the product containing recombinant human albumin.After removal of recombinant human albumin by precipitation using a mixture of water and ethanol, the FF-MAS was concentrated by evaporation using a vacuum centrifuge and the prepared sample was analyzed. The purity method was based on a reversed-phase high performance liquid chromatography (RP-HPLC) with ultraviolet absorption detection at 250nm.The method was validated according to ICH guidelines. The method indicated a significant degree of specificity with good selectivity and no significant effect from the matrix. The limit of detection was found to be 0.3–0.8% (depending on the impurity) corresponding to 1.9–5.1ng. The limit of quantification was found to be 0.8–2.5% (depending on the impurity) corresponding to 5.2–16ng. The recovery was found to be between 90 and 101% for the FF-MAS, and 100–129% for the six known impurities. The tested range for FF-MAS was from 320 to 960ng corresponding to 50–150% of the nominal concentration (640ng, injection volume is 100μl). The linearity of each compound (FF-MAS and the six impurities) was investigated. The squared correlation coefficient (r2) was 0.999 for FF-MAS (50–150% level) and 0.977–0.998 for the six known impurities (at four levels: 0.20, 0.50, 1.00, 2.00%). The R.S.D. in the repeatability study was found to be 9.2% for the total amount of impurities, and 10.4% for single impurities. The R.S.D. in the intermediate precision study was found to be 10.9% for total impurities, and 12.0% for single impurities.The validation results showed that the method was suitable for the purity analysis. The validated method was then ready for use for samples analysis of phase II clinical studies and the stability investigations of the pharmaceutical product. [Copyright &y& Elsevier]

Follicular fluid meiosis-activating sterol (FF-MAS) is regarded as an important compound relevant to meiotic resumption in mammalian oocytes. The objective of this study was to investigate the influence of FF-MAS on germinal vesicle breakdown (GVBD) and first polar body (PBI) extrusion with regard to culture conditions, state of the oocyte and mouse strain. Denuded oocytes (DO) and cumulus-enclosed oocytes (CEO) were retrieved from PMSG-primed Quackenbush or C57BL/6J x DBA/2 (C57) mice and cultured for 20 h in α-MEM medium under the following conditions: (i) 250 μmol/1 dibutyryl cAMP (dbcAMP) ± EGF, 1 ng/ml or FF-MAS, 20 μmol/l; (ii) 4 mmol/1 hypoxanthine (HX) ± EGF or FF-MAS; (iii) HX + EGF + FF-MAS; and (iv) HX + FF-MAS 5 h priming and subsequent culture with HX + EGF. Oocyte GVBD and PBI emission were recorded and stained with Hoechst 33342. Very limited meiotic inhibition was observed in Quackenbush mice in comparison with C57 mice. FF-MAS promoted maturation in C57 DO and CEO and Quackenbush DO. In Quackenbush DO and CEO and C57 DO a significant increase in atypical PBI extrusion occurred, but not in C57 CEO as well as in EGF-treated Quackenbush CEO primed or co-cultured with FF-MAS. These results support a meiosis resumption function for FF-MAS and suggest that in its presence, the quality of the MII oocytes retrieved appears to be influenced by the strain of the mice, the state of the oocyte and the presence or absence of growth factors in the culture medium. INSET: Heart graphs are becoming more common. [ABSTRACT FROM AUTHOR]

: ObjectiveTo evaluate the effect of follicular fluid meiosis activating sterol (FF-MAS) in a 0.2% ethanol formulation on chromosomal status and development of preembryos.: DesignMulticenter, prospective, randomized, double-blind, five parallel group, controlled trial.: SettingPublic and private IVF-clinics in Denmark and Sweden.: Patient(s)Two hundred ten women undergoing IVF treatment donated 310 oocytes.: Intervention(s)FSH/hCG primed cumulus-enclosed oocytes randomized to 4 hours exposure of medium with 0.1, 1, or 10 μM FF-MAS dissolved in 0.2% ethanol, medium with ethanol 0.2%, or medium with water for injection (control) before insemination.: Main outcome measure(s)Primary: incidence of human preembryos with chromosomal abnormalities. Secondary: fertilization rate, cleavage rate, and preembryo quality after 68 hours of culture.: Result(s)At the preembryo level, no significant differences in chromosomal abnormality rate were found among any of the groups. At the blastomere level, a significant increased abnormality rate was observed in the ethanol group and the combined FF-MAS groups compared with the control group. No significant differences in fertilization rate, cleavage rate, or preembryo quality were observed among any of the groups and the control group except for a significant reduction in the number of embryos with ≥2 cells at 26 hours in the ethanol group.: Conclusion(s)No negative effect of FF-MAS was observed. However, addition of ethanol 0.2% to standard IVF-medium with or without FF-MAS for culturing cumulus-enclosed oocytes for 4 hours before insemination increased the chromosomal abnormality rate at the blastomere level. Further studies of FF-MAS in a nonethanol formulation are under way. [Copyright &y& Elsevier]

In-vitro studies in mouse oocytes have shown that the C-29 endogenously occurring sterol FFMAS (follicular fluid meiosis-activating sterol) is a potent inducer of meiotic maturation leading to increased fertilization rates. We have used synthetic FF-MAS to induce meiotic maturation in immature human oocytes aspirated from polycystic ovarian syndrome patients. The patients were asked to give written consent to donate half of their aspirated oocytes to investigate the influence of culture conditions on maturation kinetics. The oocytes were aspirated from follicles 8–12 mm in diameter under ultrasound guidance after initial treatment with a gonadotrophin-releasing hormone agonist and s.c. injections of recombinant FSH for 3 days. The other half of the oocytes remained outside this present study. They were reserved for the patients’ benefit and were fertilized with appropriate embryo stages being transferred. Fertilization and transfer were not attempted for the study oocytes. Synthetic sterol FF-MAS was added to the culture media at a concentration of 20 μmol/l and nuclear maturation was compared to a control group of oocytes cultured in media only supplemented with vehicle (TCM- 199 supplemented with 0.2% ethanol v/v); thus no additional hormones, growth factors, serum or follicle fluid were added. In 31 cycles, oocytes were randomly allocated to one of seven treatment groups: fixed immediately upon aspiration (0 h group) or after in-vitro maturation culture in the presence or absence of FF-MAS for 22, 30 or 40 h respectively. A total of 81 oocytes were processed for light microscopy. The optimal timing of maturation was observed following 30 h of in-vitro culture, when 67% of FF-MAS-treated oocytes had completed nuclear maturation to the metaphase-II stage compared to 29% in the control group. The maturation time of 30 h appeared significantly superior to both 22 and 40 h, but only in the presence of FFMAS. Cumulus expansion was most profound in the FF-MAS group after 30 h whereas all oocytes had shed the cumulus investment after 40 h. Our observations indicate that FF-MAS positively influences the absolute frequency and the kinetics of human oocytes undergoing nuclear maturation. [ABSTRACT FROM PUBLISHER]