High bone morphogenetic protein-15 level in follicular fluid is associated with high quality oocyte and subsequent embryonic development

BACKGROUND: Bone morphogenetic protein-15 (BMP-15) has been shown to influence oocyte maturation and quality. However, no relationship has been established between BMP-15 and oocyte quality/embryonic development in humans. The aim of this study is to investigate BMP-15 level in human follicular fluid (FF) and its possible role in determining oocyte quality and developmental potential. METHODS: A total of 79 oocytes and their corresponding FF from 79 women undergoing ICSI were examined. Individual oocytes were inseminated and subsequently assessed on the basis of their fertilization, cleavage and preimplantation development. BMP-15, FSH, estradiol (E(2)) and progesterone levels of FF were also analysed via the techniques of western blot or radioimmunoassay. RESULTS: Higher FF BMP-15 levels were observed in the fertilized and cleaved groups versus the unfertilized and uncleaved groups, respectively (P < 0.05). The best (Grade I) embryo morphology was associated with higher FF BMP-15 levels than Grade II or III embryos (P < 0.01). A significant positive correlation was found between BMP-15 and E(2) levels in the same follicle. CONCLUSION: The present study demonstrates that the BMP-15 level in FF appears to be a potential factor in predicting oocyte quality and subsequent embryo development, and is correlated with E(2) level, which may additionally be a valuable predictor of oocyte fertilization.     

Cortisol and cortisone in human follicular fluid and serum and the outcome of IVF treatment

BACKGROUND: The glucocorticoid status of ovarian follicular fluid has been linked to oocyte quality. The aim of this study was to examine whether the concentrations of cortisone and cortisol and their calculated ratios in the follicular fluid and serum samples are predictive of IVF outcome. METHODS: In the prospective study of 387 patients (420 treatment cycles) undergoing IVF treatment the concentrations of cortisone and cortisol were measured with specific assays, and their calculated ratios in the follicular fluid and serum samples obtained after ovarian stimulation and induced ovulation were determined. RESULTS: In 75 patients, treatment resulted in clinical pregnancy and was associated with significantly lower follicular cortisone (24+/-12 versus 29+/-16 nmol/l, P<0.002) and higher cortisol/cortisone ratio (7.24+/-2.22 versus 6.45+/-2.17 nmol/l, P<0.007). In addition, the ratios of serum cortisone and cortisol to follicular cortisone and cortisol were significantly higher in those women who became pregnant. CONCLUSIONS: We propose that the follicular fluid glucocorticoid concentration resulting from the conditions in the circulation and the course of the intrafollicular cortisol-cortisone interconversion appear to play a role in the outcome of IVF.     

Granulosa cell metabolism and the assessment of oocyte quality in IVF

The progesterone production of the granulosa cells of the cumulus– oocyte complex correlates very well with the cleavagepotential of embryos in an IVF system. The method is simpleand can be easily performed by any laboratory associated withIVF. Furthermore, high intratubal progesterone levels in theimmediate post-ovulatory period are probably important in prolongingthe intra-ampullary residence of the oocyte or embryo untilthe uterine endometnum is optimal for implantation.     

Endocrine profile in serum and follicular fluid differs after ovarian stimulation with HP-hMG or recombinant FSH in IVF patients

BACKGROUND: Highly purified menotrophin (HP-hMG) has been associated with fewer oocytes retrieved and a higher proportion of top-quality embryos compared with recombinant FSH (rFSH). METHODS: A randomized, assessor-blind, multinational trial in 731 women undergoing IVF after stimulation with HP-hMG (MENOPUR) (n = 363) or rFSH (GONAL-F) (n = 368) following a long GnRH agonist protocol was conducted. Blood was collected before, during and after stimulation. Fluid was collected from follicles > or =17 mm. RESULTS: Serum androstenedione, total testosterone and free androgen index (FAI) were higher (P < 0.001) with HP-hMG than with rFSH after starting stimulation. At the end of stimulation, serum estradiol was higher (P = 0.031) with HP-hMG, whereas progesterone was higher (P < 0.001) with rFSH, even after adjusting for ovarian response. Serum LH was not different between treatments. Mean mid- and end-follicular hCG levels in the HP-hMG group were 2.5 and 2.9 IU/l, respectively. Follicular fluid levels of FSH, LH, hCG, androstenedione, testosterone, FAI and estradiol and ratios of estradiol:androstenedione, estradiol:total testosterone and estradiol:progesterone were higher (P < 0.001) with HP-hMG, whereas progesterone was higher (P < 0.001) with rFSH. CONCLUSION: Major differences in serum and follicular fluid endocrine profile exist after stimulation with HP-hMG or rFSH. Exogenous LH activity induces a differential endocrine environment influencing oocyte quantity and quality, which may be of relevance for clinical outcome.     

Propofol concentrations in follicular fluid during general anaesthesia for transvaginal oocyte retrieval

Propofol (Diprivan) is an i.v. anaesthetic used for general anaesthesia. The purpose of this study was to measure the propofol concentration in arterial blood and follicular fluid in patients during transvaginal oocyte retrieval. After approval by the University Ethics Committee, 30 women participated in this prospective study. Following induction of anaesthesia with 0.5 mg alfentanil and 2 mg.kg-1 propofol i.v., a continuous infusion of propofol at 10 mg.kg-1.h-1 was used for maintenance of anaesthesia. Follicular fluid and arterial blood samples were aspirated simultaneously at fixed intervals during the surgical procedure and propofol assayed by high pressure liquid chromatography (HPLC). The mean follicular fluid concentration of propofol increased linearly with time from 0.10 +/- 0.02 microgram.ml-1 to 0.57 +/- 0.06 microgram.ml-1 and was strongly related to the cumulative dose of propofol administered. The absorption of propofol was time-dependent. There was no correlation between the concentration of propofol in the follicular fluid and the arterial blood concentration of the drug. In conclusion, a propofol-based anaesthetic technique resulted in significant concentrations of this agent in follicular fluid, related to the dose administered and to the duration of propofol administration.     

Follicular fluid prostaglandin E2:prostaglandin F2{alpha} ratio in relation to outcome of matched oocyte

The prostaglandins PGE₂ and PGF₂ and the steroid hormones oestradiol(E₂) and progesterone (P) were measured in 345 follicular fluidsof patients undergoing ovarian hyperstimulation for in-vitrofertilization (IVF). The measured concentrations were analysedin relation to the outcome of the matched oocyte. Progesteronelevels were significantly tower in the unfertilized group (P<0.005)compared to the fertilized group but there was no differencebetween ‘pregnancy’ and ‘no pregnancy’.No differences were shown in either E₂ levels or the E₂:P ratio.No significant differences were shown among the groups in theconcentration of either PGE₂ or PGF₂ but there were highly significantdifferences shown when the PGE₂:PGF₂ ratios were compared. ThePGE₂:PGF₂ ratio fell within a much narrower range for the ‘pregnancy’group compared with any of the other groups. The ratio was significantlylower (P<0.001) when ‘pregnancy’ was comparedwith ‘no pregnancy’. The range of the prostaglandinratio found for the ‘pregnancy’ group may reflectthe moment when conditions are optimal within the follicle forthe associated oocyte to go on to establish pregnancy.     

Relationship between human oocyte maturity, fertilization and follicular fluid growth factors

Follicular fluid concentrations of growth hormone (GH), insulin-likegrowth factor-I (IGF-I), epidermal growth factor (EGF) and oestradiolwere related to diversities in oocyte maturation and fertilizationamong oocytes obtained for invitro fertilization (IVF). Follicularfluid GH, IGF-I and oestradiol concentrations were significantlycorrelated with increasing follicular size. Follicles with immatureoocytes had concentrations of oestradiol that were significantlylower when compared to follicles with intermediate and matureoocytes. Follicular fluid EGF concentration was similar forall oocyte maturational stages. In follicular fluids with matureoocytes we found IGF-I and GH concentrations were significantlyhigher compared to those of follicular fluid with atretic oocytes.Follicular fluids with Immature and intermediate oocytes hadsimilar concentrations of GH and IGF-I to follicular fluid containingmature oocytes and higher concentrations than follicular fluidwith atretic oocytes. No statistically significant differencewas found between fertilized and unfertilized oocytes. We concludethat maturation of oocytes Is associated with higher concentrationsof GH, IGF-I and oestradiol, but follicular fluid IGF-I andGH concentrations cannot serve as a predictor for IVF.     

Antioxidants and reactive oxygen species in follicular fluid of women undergoing IVF: relationship to outcome

BACKGROUND: The role of free radicals and reactive oxygen species (ROS) in female reproductive function is still unclear. The present study was designed to investigate their relationship with ovulation, fertilization and conception. METHODS: Follicular aspirates obtained from women undergoing IVF following controlled ovarian stimulation were evaluated using the ferric reducing antioxidant power (FRAP) assay for baseline total antioxidant capacity (TAC). Both the baseline TAC and the decline in TAC over 72 h (two-point assay) were used as markers of oxygen radical activity. RESULTS: A total of 303 follicular aspirates from 63 women were analysed. Two hundred and eighteen (71.9%) yielded oocytes, 169 (77.5%) of these fertilized and 134 (79.3%) of these embryos survived until the time of embryo transfer. Baseline TAC was no different in follicular fluid whether the follicle contained an oocyte or not, but was significantly higher in fluid from follicles whose oocyte successfully fertilized and significantly lower in fluid from follicles where the resultant embryo survived to transfer. The decline in TAC was lower when the oocytes fertilized and higher in association with embryo viability, but the differences were not statistically significant. CONCLUSIONS: These results provide further evidence that ROS play a role in female reproductive function.     

Glucose and lactate in human follicular fluid: concentrations and interrelationships

The concentrations of glucose, lactate, progesterone and oestradiol have been measured in 122 follicular fluids obtained from women attending a natural cycle IVF programme. Some women received low-dose clomiphene. Fluids were recovered 28-35 h after the onset of the spontaneous LH surge. Mean follicular fluid volumes were greater for stimulated (4.46 ml) than spontaneous (3.11 ml) cycles. Mean glucose and lactate concentrations were similar in the two groups, with overall means of 3.29 mM (glucose) and 6.12 mM (lactate). Total glucose and lactate were greater in the fluids from stimulated (14.1 and 29.5 mol, respectively) than from the spontaneous cycles (11.5 and 20.2 mmol, respectively). There were negative correlations between follicular fluid volume and glucose concentration (r = -0.348) and between glucose concentration and lactate concentration (r = -0.367) but no relationship between follicular fluid volume and lactate concentration (r = 0.086). A model is presented to account for these findings in terms of the movement of pyruvate, glucose and lactate from the vascular theca into follicular fluid, and of glycolysis occurring in the granulosa cells.     

Follicular fluid markers of oocyte developmental potential

BACKGROUND: Concentrations of certain substances in follicular fluid (FF) are related to fertilization outcome and early post-fertilization development. The study aim was to identify FF markers with which to predict embryo implantation potential. METHODS: Concentrations of selected hormones, cytokines and growth factors in individual FF samples obtained during assisted reproduction treatment were related with treatment outcomes. RESULTS: Mean concentrations of LH, growth hormone (GH), prolactin (PRL), 17beta-estradiol (E2) and insulin-like growth factor (IGF)-I were higher, and that of interleukin-1 (IL-1) was lower, in treatment attempts leading to a clinical pregnancy as compared with those in which no pregnancy was established. Concentrations of FSH, progesterone, tumour necrosis factor-alpha and IGF-II were similar in successful and unsuccessful attempts. In successful attempts, LH and GH levels were higher in those follicles from which oocytes giving rise to transferred embryos (i.e. embryos with best morphology and fastest cleavage rate) originated, as compared with other follicles from which a mature oocyte was recovered but was cryopreserved for later use. CONCLUSIONS: FF levels of LH, GH, PRL, E2, IGF-I and IL-1 may serve to analyse cases of repeated assisted reproduction failures and to assess effects of modifications of the ovarian stimulation protocol.     

Few instead of many: human follicle collection from follicular aspirates at oocyte retrieval

BACKGROUND: The aim of this study was to determine if follicular aspirates obtained during oocyte retrieval for IVF were a good source of ovarian follicles for research purposes. METHODS: Follicular aspirates from 86 patients were collected and examined for the presence of follicles, and histological examination of tissue sample found was undertaken. RESULTS: Follicles were only obtained from aspirates of seven out of a total of 86 patients. From these samples a total of 14 follicles was found. The follicles were primordial, primary or secondary, 40-80 microm in diameter. Three of these recovered follicles were cultured and all degenerated within 2 days. In all aspirates some groups of granulosa cells that did not contain follicles or oocytes were found, as was vaginal epithelium that was also identified and verified by histology. CONCLUSIONS: Follicular aspirates are not a useful source of human follicles. Some structures found in the aspirates may be erroneously identified as follicles.     

Human chorionic gonadotrophin and steroid concentrations in follicular fluid: the relationship to oocyte maturity and fertilization rates in stimulated and natural in-vitro fertilization cycles

The study investigates the relationship of follicular fluidsteroids and human chorionic gonadotrophin to oocyte maturityand fertilization rates in stimulated and natural cycles. Oestradiol,progesterone, testosterone and human chorionic gonadotrophinwere quantified in 129 samples of follicular fluid and the progesterone:oestradiol ratio calculated. Both stimulated cycles (short andlong luteinizing hormone-releasing hormone/human menopausalgonadotrophin regimens) and natural cycles were compared. Atotal of 60 women were studied, 20 in each group. In the naturalcycles, testosterone was significantly lower in follicles withintermediate oocytes (P = 0.015). Both oestradiol and testosteronewere significantly lower in stimulated cycles compared to naturalcycles (P = 0.032 and P = 0.034 respectively). In the ovarianstimulation cycles, the progesterone: oestradiol ratio was significantlyhigher when oocytes fertilized (P = 0.052). Moreover, in thestimulated cycles, oestradiol and human chorionic gonadotrophinwere significantly lower in the short protocol compared to thelong protocol. The data demonstrate that the hormonal milieuof the follicle is altered in downregulated stimulated cyclesto varying degrees, depending partially on the type of protocolused. Furthermore, the progesterone: oestradiol ratio, ratherthan individual hormone concentrations, may be a useful predictorof the fertilizing capacity of the oocytes.     

Human follicular fluid from pre-ovulatory follicles in patients undergoing in-vitro fertilization inhibits the in-vitro growth of Gram-positive micro-organisms

In this study, we assessed the antibacterial activity of humanfollicular fluid obtained from patients undergoing oocyte aspirationfor in-vitro fertilization and embryo transfer. Gram-positiveand Gram-negative microorganisms and Candida albicans were addedto the follicular fluid and broth cultures which served as controls.Follicular fluid was shown to be inhibitory against Gram-positivemicroorganisms; this inhibition was probably due to lysosymeand progesterone. Growth of Gram-negative micro-organisms andC.albicans were, however, supported by follicular fluid incubation.     

Propofol anaesthesia for ultrasound guided oocyte retrieval: accumulation of the anaesthetic agent in follicular fluid.

Propofol (2,6-diisopropylphenol, Diprivan, ICI-Pharmaceuticals, Manchester, UK) is widely used either as an adjunct in general anaesthesia or as sole anaesthetic agent by the continuous intravenous route and intermittent bolus injections for minor surgical interventions. For several years, we have been using this kind of anaesthesia in transvaginal oocyte retrieval for in-vitro fertilization (IVF), allowing a completely painless puncture on an out-patient basis. From in-vitro studies on mouse oocytes, it appeared that propofol could be deleterious for fertilization in a dose- and time-dependent manner. We therefore investigated the concentrations of propofol in follicular fluid during oocyte retrieval in women. We measured propofol levels in serum and follicular fluid of nine patients at fixed intervals during ultrasound guided oocyte retrieval. Serum levels fluctuated randomly, due to interference from top-off doses of propofol. In follicular fluid, however, we found a steady increase of propofol levels, which was proportional to the total dose of propofol administered. These data indicate that propofol accumulates in follicular fluid. Although it seems unlikely that propofol as used in the present protocol exerts a clinically significant unfavourable effect on IVF, we suggest that the oocyte retrieval procedure should be kept as short as possible, in order to limit the accumulation of the anaesthetic in follicular fluid.     

Follicular fluid concentrations of adrenomedullin, vascular endothelial growth factor and nitric oxide in IVF cycles: relationship to ovarian response

Marked granulosa cell proliferation along with important changes in the vascular bed of the ovary characterize IVF cycles associated with multiple follicular growth and maturation. The present report investigated follicular fluid (FF) and circulating concentrations of adrenomedullin, vascular endothelial growth factor (VEGF) and nitric oxide (NO) in 70 IVF patients (14 of whom became pregnant); these three vasoactive substances may be implicated in extensive ovarian tissue remodelling. Serum and FF concentrations of oestradiol and progesterone were also measured in the 70 IVF cycles studied. Follicular fluid concentrations of VEGF and adrenomedullin but not nitrite/nitrate (the two stable oxidation products of NO metabolism) were significantly higher (P < 0.0001) than the corresponding circulating concentrations. Follicular fluid concentrations of oestradiol and progesterone were not correlated with those of adrenomedullin, VEGF or nitrite/nitrate. No relationship existed between circulating concentrations of adrenomedullin, VEGF or nitrite/nitrate on the day of oocyte aspiration and parameters of ovarian response to gonadotrophin stimulation. In contrast, FF adrenomedullin concentration showed a direct relationship with day 3 FSH serum concentration (r = 0.53, P < 0.01) and the number of ampoules of gonadotrophin administered (r = 0.36, P < 0.005), but an inverse correlation with the total number of oocytes retrieved (r = -0.29, P < 0.01) and the number of mature oocytes (r = -0.25, P < 0. 05). A positive correlation was found for FF VEGF concentration and chronological age (r = 0.29, P < 0.05) and ampoules of gonadotrophins administered (r = 0.30, P < 0.05). There was no relationship between nitrite/nitrate FF concentrations and parameters of ovarian response. Neither serum concentrations nor FF concentrations of adrenomedullin, VEGF or nitrite/nitrate were correlated with IVF outcome. This study suggested for the first time that increased FF concentrations of adrenomedullin can be a marker of decreased ovarian response in IVF. Our results also provide further evidence favouring an association between FF VEGF and patient's age, while on the basis of our findings NO measurements are not a useful marker of ovarian response.     

Reduction of progesterone receptor expression in human cumulus cells at the time of oocyte collection during IVF is associated with good embryo quality

BACKGROUND: It has been reported that the progesterone receptor (PR) level is transiently increased within the follicle by LH stimulation and controls cumulus cells in follicles and oocyte maturation. The purpose of this study was to predict developmental competence of human oocytes during IVF via analysis of PR in cumulus cells surrounding mature oocytes. METHODS: Prior to oocyte retrieval, the follicular diameter was measured and follicular fluid was collected from each mature follicle. Cumulus cells were manually separated from the oocyte-cumulus complex under a microscope. PR and PR mRNA were assessed by immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR) measurement in human cumulus cells. RESULTS: Immunoreactive PR-A was mainly localized in the cytoplasm and PR-B was localized in the nuclei. There was no significant relationship between PR expression and follicular diameter, follicular fluid concentration of steroids, or LH. There was no significant relationship between expression of PRs and fertilization or cleavage rate. However, PR expression was lower in the good morphology group (blastomeres > or =7 cells with fragmentation > or =5% on day 3) when compared to the other groups (P<0.05). CONCLUSIONS: These results suggest that follicular LH or steroids do not affect PR expression, and full reduction of total PR expression on cumulus cells at the time of oocyte collection is associated with good morphology in human oocytes.     

Supraphysiological estradiol levels do not affect oocyte and embryo quality in oocyte donation cycles.

BACKGROUND: The study aim was to determine whether supraphysiological estradiol (E(2)) levels reduce oocyte/embryo quality in oocyte donation cycles. METHODS: A retrospective analysis of 330 consecutive fresh oocyte donation cycles was performed in an assisted reproductive treatment programme between January 1996 and December 2000. Throughout the study period, oocyte donors and recipients followed a standard synchronization regimen that did not vary. A serum E(2) level (peak E(2)) was obtained from all oocyte donors on the morning of HCG administration. Peak E(2) values were grouped by 33rd percentile (group I, <1500 pg/ml; group II, 1500-3000 pg/ml; and group III, >3000 pg/ml). All embryo transfers were performed on day 3 after oocyte recovery. RESULTS: Comparisons between groups revealed no significant differences in the quality of oocytes retrieved, and in fertilization rates. Higher peak E(2) levels were directly correlated with a greater number of oocytes retrieved, embryos available for transfer and cryopreservation, and higher average embryo quality scores (P < 0.005). Compared with group I, group III had significantly higher embryo implantation rates (P < 0.05). CONCLUSIONS: Sustained supraphysiological E(2) levels do not adversely affect the quality of developing oocytes and embryos. On the contrary, elevated E(2) levels are associated with a larger number of oocytes and embryos and high-grade embryos for transfer/cryopreservation and, consequently, improved implantation rates.     

Follicular fluid pH changes following intraperitoneal exposure of Graafian follicles to carbon dioxide: a comparative study with follicles exposed to ultrasound

Follicular aspiration to obtain oocytes is generally performedvia laparoscopy after creating a pneumoperitoneum with carbondioxide. Such a procedure has been shown to reduce the rateof in-vitro fertilization of human oocytes and affect the rateof cleavage of rabbit embryos. These adverse effects may becaused by a reduction in follicular fluid pH due to diffusionof carbon dioxide into the folilde. In laparoscopic oocyte retrievals,a negative correlation was observed between duration of CO₂exposure and follicular fluid pH, whereas In ultrasound-guidedretrievals, the pH remaIned unchanged. The mean pH In 78 folliclesaspirated at laparoscopy was 7.22 ± 0.03 compared with7.62 ± 0.01 in 35 follicles aspirated under ultrasoundguidance (P = 0.0003). The results also indicate that oocytesin preovulatory follicles are surrounded by fluid that is morealkaline than plasma. Hence, the acidic environment treatedby CO₂ may be deleterious to sub sequent reproductive functionof the oocyte.     

Endocrinology: Follicular fluid insulin-like growth factor-I and insulin-like growth factor-II concentrations vary as a function of day 3 serum follicle stimulating hormone

We determined follicular fluid concentrations of insulin-likegrowth factor (IGF)-I, IGF-II and inhibin as a function of day3 serum follicle stimulating hormone (FSH) in 16 women undergoingfollicular fluid aspiration in preparation for in-vitro fertilizationand embryo transfer. Follicular fluid concentrations of IGF-Iand IGF-II were significantly less in the ‘low’FSH group as compared to the ‘high’ FSH group. Themean IGF-I concentration was 67.6 ng/ml [confidence intervals(CI) 51.6–92.5] in the ‘low’ FSH group comparedto 87.1 ng/ml (CI 72.8–104.2; P < 0.025) in the ‘high’FSH group. Mean IGF-II concentrations were 354.8 ng/ml (CI 297.8–422.9)in the ‘low’ FSH group compared to 489.8 ng/ml (CI384.6–624.5; P < 0.05) in the ‘high’ FSHgroup. Follicular fluid inhibin concentrations did not differbetween groups. These differences in follicular fluid IGF asa function of day 3 FSH may raise questions regarding the rolegrowth factors play in the physiological processes of the ageingfollicle.     

Effect of prednisolone on serum and follicular fluid androgen concentrations in women with polycystic ovary syndrome undergoing in-vitro fertilization.

Increased androgen concentrations are thought to be detrimental to oocyte quality and reproductive potential. Adjuvant treatment with glucocorticoids has been tried to suppress androgens in women undergoing infertility treatment. In the present study 20 infertile women with polycystic ovary syndrome were prospectively randomized in a placebo-controlled study to receive either placebo or prednisolone 10 mg at night, during standard in-vitro fertilization (IVF) treatment. Serum samples for assays of gonadotrophins, steroids and sex hormone-binding globulin (SHBG) were collected before treatment, at down-regulation, and at oocyte retrieval. Up to five follicles in each ovary were analysed separately regarding follicular fluid and oocytes, the rest according to the clinic's routines. In the placebo group, serum dehydroepiandrosterone (DHEA) and dehydroepiandrosterone-sulphate (DHEA-S) did not change between down-regulation and oocyte retrieval, whereas adjuvant prednisolone resulted in a significant decrease. In follicular fluid, adjuvant prednisolone resulted in significantly lower concentrations of DHEA-S as compared to placebo, no other significant differences were found. No significant differences were found in embryo characteristics or pregnancy rates between the groups.