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.     

Different protein patterns derived from follicular fluid of mature and immature human follicles

The purpose of our study was to compare the protein patternsoriginating from fluids of mature and immature human folliclesin order to gain further insight into their biochemical composition.A total of 10 patients were stimulated for in-vitro fertilization(IVF) using different stimulation protocols. Follicular fluidswere aspirated trans-vaginally and analysed microscopicallyfor the presence of oocytes. Follicular fluids were stored at-18°C. Samples of 500 µl were processed for two-dimensionalgel electrophor-esis. Up to 60 proteins in various groups couldbe detected. Seven protein spots were selected for chemicalanalysis by cutting them out of the gels and subjecting themto internal amino acid sequencing procedures. Our results canbe summarized as follows: (i) major differences were not detectedbetween the protein patterns from the various mature folliclesof a particular patient, nor were significant differences observedin the proteins derived from follicular fluids collected fromthe seven patients with mature follicles; (ii) considerabledifferences were observed in the protein patterns derived fromfluids of immature compared with mature follicles. Fluid fromthe three patients with immature follicles contained many fewerproteins, some of which were expressed at low levels. We concludethat the observed variations in protein composition of folliclesof different developmental age reflect their physiological conditionand serve as biomedical markers for follicular maturity.     

Gonadotrophic control of human granulosa cell glycolysis

Follicular fluid lactate levels were measured in women undergoinginfertility surgery during the follicular phase or oocyte recoveryfor in-vitro fertilization (IVF). In the largest ovulatory folliclelactate levels were low in the mid-follicular phase (group 1),1.6-fold higher just prior to the onset of the luteinizing hormone(LH) surge (group 2) and a further 2.5-fold higher after theonset of the LH surge (group 3). In IVF patients mean lactatelevels in all aspirated follicles were similar to those in group3 subjects, but the levels within ertch patient were variableand were positively correlated with follicular volume. Basalgranulosa cell lactate accumulation in vitro was 3-fold higherin group 3 compared with group 2 subjects, but stimulation byFSH or HCG was higher in group 2 (2- to 3-fold) compared withgroup 3 (1.4- to 2-fold). These results demonstrate that humanfollicular fluid lactate levels increase as a function of thematurity and size of the developfng follicle. Granulosa celllactate accumulation in vitro is under gonadotrophic control,which suggests that the effects observed in vivo reflect changesin granulosa cell glycolysis in response to gonadotrophic stimulation.Our findings support the concept that low molecular weight energymetabolites transduce gonadotrophin signals that regulate oocytematuration.     

Coasting acts through downregulation of VEGF gene expression and protein secretion

BACKGROUND: This study was conducted to investigate the mechanisms by which coasting may be effective in decreasing the incidence of ovarian hyperstimulation syndrome (OHSS). METHODS: A total of 160 women (patients and oocyte donors) undergoing coasting and 116 controls were included in the study. Serum, follicular fluid and granulosa cells were collected on the day of oocyte retrieval. Vascular endothelial growth factor (VEGF) concentrations were determined using an enzyme-linked immunosorbent assay (ELISA). Real-time PCR was performed to evaluate VEGF gene expression in granulosa cells. Cell death was studied by flow cytometry using annexin V-fluorescein isothiocyanate (FITC) and counterstaining by propidium iodide, and double staining with CD45 monoclonal antibody was performed to distinguish the contamination of apoptotic leukocytes. RESULTS: Follicular cells aspirated from coasted patients showed a ratio in favour of apoptosis, especially in smaller follicles (48 versus 26%, P < 0.05). Follicular fluid determinations confirmed that coasting reduces VEGF protein secretion (1413 versus 3538 pg/ml, P < 0.001) and gene expression (2-fold decrease) in granulosa cells. Follicular fluid VEGF protein levels positively correlated with follicular size (r = 0.594, P = 0.001) and estradiol production (r = 0.558, P = 0.038). Women who underwent coasting showed a comparable IVF cycle outcome; however, a higher cancellation rate was found in cycles that were coasted. CONCLUSIONS: Coasting affects all follicles through apoptosis, especially immature follicles, without affecting oocyte/endometrial quality. The significant decrease found in VEGF expression and secretion explains why coasting is clinically effective in reducing the incidence and severity of OHSS.     

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.     

Measurement of the speed of sound in follicular fluid

BACKGROUND: Measurement of ovarian follicles by ultrasound is common practice in fertility treatment. However, the effect of the speed of sound is not taken into account. We present results from a study aimed at measuring this. METHODS: The speed of sound was measured in samples of follicular fluid aspirated from patients undergoing fertility treatment. The transmitted and received pulses from a single element ultrasound transducer were recorded using a digital oscilloscope for a pulse passed through a sample of the fluid. The distance over which the pulse travelled was known from calibration with pure water. Variation with temperature was investigated in the range 25-45 degrees C. Dependence on ultrasound frequency, patient and time from aspiration were also investigated. RESULTS: The speed of sound in follicular fluid was found to be 1550+/-3 m/s at 37.3 degrees C using 5.0 MHz ultrasound. The speed varied from 1528+/-3 m/s at 24.8 degrees C to 1561+/-3 m/s at 44.8 degrees C. Variation with patient, time and frequency were not detected. CONCLUSION: The speed of sound in follicular fluid at body temperature is 1550 m/s. This small difference from the speed assumed by the ultrasound machine corresponds to the systematic bias in volume measurement evident in previously published results.     

Adjuvant L-arginine treatment in controlled ovarian hyperstimulation: a double-blind, randomized study

BACKGROUND: Enhanced vascularization appears to be important for follicular selection and maturation in both spontaneous and stimulated IVF cycles. Nitric oxide, formed in vivo from L-arginine, may play a key role in follicular maturation and ovulation. METHODS: To evaluate the role of L-arginine supplementation in controlled ovarian hyperstimulation, 37 IVF patients were divided into two groups according to ovarian stimulation protocols: group I, GnRH agonist plus pure (p)FSH plus oral L-arginine (n = 18); and group II, GnRH agonist plus pFSH plus placebo (n = 19). Hormonal, ultrasonographic and Doppler evaluations were performed, and plasma and follicular fluid nitrite/nitrate concentrations were monitored. RESULTS: Thirty-two patients completed the study. In group I (n = 16), plasma L-arginine concentrations increased from (basal) 87 +/- 12 micromol to 279 +/- 31 micromol (P = 0.002) on the day of beta-HCG administration. In this group, pFSH treatment was shorter (P = 0.039) than in group II (n = 16). The number of the follicles > or =17mm was lower (P = 0.038) in group I than group II. The "good quality" embryos were fewer in number (P = 0.034) and pregnancy rate, both per patient (P = 0.024) and per embryo transfer (P = 0.019), was lower in group I. In the L-arginine group, an increased follicular fluid concentration of nitrite/nitrate was observed. On day 8 of the cycle, elevated plasma estradiol levels were associated with decreased blood flow resistances of perifollicular arteries. Follicular fluid concentrations of nitrite/nitrate were inversely correlated with embryo quality (r = -0.613; P = 0.005) and perifollicular artery pulsatility index (r = -0.609; P = 0.021). CONCLUSIONS: L-Arginine supplementation may be detrimental to embryo quality and pregnancy rate during controlled ovarian hyperstimulation cycles.     

The effect of ovarian follicular fluid and peritoneal fluid on Fallopian tube ciliary beat frequency

BACKGROUND: The Fallopian tube undergoes well-recognized changes during the ovarian cycle. Ciliary beat frequency (CBF) increases during the secretory phase of the cycle. The stimulus is unknown, although CBF is known to be hormone responsive. At ovulation, follicular fluid is released into the peritoneal cavity and enters the Fallopian tube. We hypothesized that this fluid may provide the stimulus for the increase in CBF detected after ovulation. METHODS: Using a technique which records changes in light intensity, we have studied the effect of pre-ovulatory follicular fluid on CBF of Fallopian tube epithelial cells, and compared this with the effect of either peritoneal fluid or culture medium alone. Follicular fluid samples from 13 women undergoing IVF were collected by selective aspiration of individual follicles. Peritoneal fluid was collected from six women undergoing laparoscopic sterilization. Fallopian tubes were collected from 10 women who underwent hysterectomy for benign conditions. RESULTS: After 24 h incubation, there was a highly significant difference in CBF between the Fallopian tube samples bathed in follicular fluid (mean CBF +/- SEM: 6.34 +/- 0.02 Hz) compared with explants bathed in either medium (4.20 +/- 0.06 Hz) or peritoneal fluid (5.24 +/- 0.03 Hz) (P < 0.005). There was also a significant difference in CBF between tissues bathed in secretory (5.47 +/- 0.03 Hz) compared with proliferative phase peritoneal fluid (4.75 +/- 0.02 Hz) (P < 0.005). CONCLUSIONS: The increase in CBF detected after ovulation may aid ovum pick-up and transport along the Fallopian tube. Factor(s) within human follicular fluid and secretory phase peritoneal fluid may be responsible for this increase in CBF.     

Physiology: Healthy and atretic follicles: vaginosonographic detection and follicular fluid hormone profiles

The health status of human preovulatory follicles was prospectivelystudied by non-invasive transvaginal ultrasound. Daily ultrasoundscans were performed from the time when the follicle measured15 mm in diameter until formation of corpus luteum or oocyteretrieval. Based on the ultrasound scans two types of follicleswere defined: type A follicles showed a cloud visualized asa cone projecting into the follicular fluid with the base ofthe cone positioned on the follicle wall. A light spot seenat the tip of the cloud in the majority of scans was presumedto be the oocyte-cumulus complex. Type B follicles showed anecho free space without a cloud. Two groups of infertility patientswere studied: Group I received intrauterine insemination; 106patients undergoing a total of 263 cycles (188 spontaneous cyclesand 75 clomiphene citrate cycles). Group II received in-vitrofertilization (IVF); 22 patients undergoing a total of 52 cycles(31 spontaneous cycles and 21 clomiphene cycles). In the firstgroup, the ovulatory potential of the follicle is associatedwith the ultrasound characteristics. From the IVF patients thefollicular fluid was harvested and assessed for the free concentrationof the following steroids: oestradiol, progesterone, testosteroneand androstendione. The endocrine health status of the folliclewas associated with the ultrasound characteristics of the follicle.In patients of group 1, 288 type A follicles ovulated out ofa total of 298 follicles (97%). None of the type B folliclesovulated (0/14). In patients of group II, oocytes were obtainedin 79% of the type A follicles (50/63), whereas 7% of the typeB follicles yielded an oocyte (1/14). The follicular fluid hormoneprofile showed significantly lower free oestradiol, free oestradiol/testosteroneratio and oestradiol/androstenedione ratio and higher free testosteroneand free androstenedione in type B follicles compared to typeA follicles. The hormone status and the high oocyte recoveryrate suggest that type A follicles are healthier than type Bfollicles, which seem to have undergone atretic changes. Itis concluded that non-invasive transvaginal ultrasound witha good degree of accuracy predicts the health status of preovulatoryfollicles. Healthy follicles have a cloud with a light spotat the tip, whereas almost all atretic follicles lack this characteristic.     

Ovary and ovulation: Nitric oxide concentrations in the follicular fluid and apoptosis of granulosa cells in human follicles

To study the relationship between follicular atresia, apoptosis,and nitric oxide (NO) generation in follicular development,steroidogenesis, NO levels in follicular fluid and apoptosiswere analysed in the various sized follicles of women receivingovarian stimulation with human meno-pausal gonadotrophin (HMG)—humanchorionic gonado-trophin (HCG) treatments for in-vitro fertilization(IVF)-embryo transfer. The follicles were divided into threegroups by diameter: large follicle, 18 mm; medium follicle,12 and 15 mm; small follicle, 10 mm. Follicular fluid was obtainedfrom 20 women 34 h after HCG administration, and the concentrationsof oestradiol, progesterone and testosterone, and nitrite, nitrate,arginine and citrulline were measured. Granulosa cells obtainedfrom each group of follicular fluid were stained with Hoechstdye, and nuclear morphology was examined by a fluorescence microscopy.Oestradiol and progesterone concentrations in large follicleswere significantly (P < 0.01) higher than those in mediumor small follicles, and testosterone concentrations in smallfollicles were significantly (P < 0.01) higher than thosein large follicles. There were no significant differences inthe concentrations of nitrite, nitrate, arginine and citrullineamong three groups. The percentage of apoptotic cells with nuclearfragmentation was significantly (P < 0.01) higher in smallfollicles than in large follicles. The present results suggestedthat small follicles with poor response to HMG may undergo atresiathrough apoptosis. No significant difference in the follicularNO level between large and small follicles led us to speculateon a different responsiveness to NO in these two types of follicles.     

Presence of Leukemia Inhibitory Factor and Interleukin-12 in Human Follicular Fluid During Follicular Growth

PROBLEM: Cytokines have been shown to be present in human follicular fluid and have regulatory functions on follicular maturation. The presence of leukemia inhibitory factor (LIF) and interleukin (IL)-12 in human follicular fluid obtained at different stages of maturation was investigated. METHOD OF STUDY: Follicular fluids and granulosa cells were obtained from preovulatory and immature follicles. Follicular fluids from both groups were assayed for IL-12 and LIF by enzyme-linked immunosorbent assay. Granulosa cells from preovulatory and immature follicles were treated with human chorionic gonadotropin (hCG) in vitro and subsequent LIF and IL-12 production were measured. RESULTS: The average concentration of LIF was significantly higher in preovulatory follicles (7.6 ± 1.3 pg/ml, n = 24) than in immature follicles (2.0 ± 1.3 pg/ml, n = 6). The concentration of IL-12 was significantly higher in follicular fluid obtained from immature follicles (10.9 ± 5.0 pg/ml) than in preovulatory follicles (1.3 ± 0.4 pg/ml). hCG only stimulated LIF production from mature granulosa cells; it had no effect on IL-12 production. CONCLUSIONS: IL-12 and LIF are present in follicular fluid and their levels are regulated differently during follicular maturation. hCG stimulates LIF production from granulosa cells in vitro.     

Prolonged HCG action affects angiogenic substances and improves follicular maturation, oocyte quality and fertilization competence in patients with polycystic ovarian syndrome

BACKGROUND: The aim of this study was to determine whether, in polycystic ovarian syndrome (PCOS) patients, HCG action prolonged for 4 h improves the action of angiogenic substances [ovarian renin angiotensin system and vascular endothelial growth factor (VEGF)], and consequently follicular maturation, oocyte quality and oocyte fertilization competence. METHODS: In this prospective study 20 patients with PCOS undergoing IVF were included. Oocyte retrieval was carried out either 34 or 38 h after HCG administration. Each follicle was analysed for prorenin, active renin, VEGF and estradiol. Oocytes were evaluated for quality (mature, immature, degenerated oocytes), as were the embryos (low or high). RESULTS: In the HCG +38 h group there were 245 follicles, and in the HCG +34 h group 240 follicles. In the HCG +38 h group, log active renin was lower (2.78 +/- 0.20 versus 2.91 +/- 0.25; P < 0.001) and VEGF higher (2276.0 +/- 790.1 versus 1946.6 +/- 954.5 pg/ml; P < 0.001). The odds ratio for obtaining oocytes from follicles was 1.6 [95% confidence interval (CI) 1.1-2.6; P = 0.02], and for developing high quality embryos 7.6 (95% CI 2.8-20.9; P < 0.001) in favour of the HCG +38 h group. CONCLUSIONS: Follicular maturation and oocyte quality are related to the intrafollicular influences of active renin and VEGF in a time-dependent manner after HCG administration, whereas fertilization competence is related to VEGF only.     

Cortisol concentrations in follicular fluid of 'low responder' patients

The study was undertaken to examine any differences existing in total cortisol concentrations in the follicular fluid (FF) of pre-ruptured follicles between 'low responder' patients (group 1, n = 20) and 'good responder' patients (group 2, n = 15). The groups were defined according to how many oocytes had been retrieved during the previous in- vitro fertilization procedure (group 1: three or fewer; group 2: more than three) and total oestradiol concentration at previous in-vitro fertilization (IVF) (group 1: < or = 500 pg/ml; group 2: > 500 pg/ml). All patients were aged 36-43 years (group 1 mean +/- SD: 38.2 +/- 4.7; group 2: 32.1 +/- 3.8 years) and were diagnosed with tubal or unexplained infertility. The total FF cortisol concentrations obtained in conjunction with an IVF procedure were assayed and related to oocyte fertilization. Follicular fluid was analysed for total cortisol content. Only follicles between 19 and 20 mm diameter were analysed in both groups. After aspiration of blood-free FF, total cortisol concentrations were measured by radioimmunoassay, designed for the quantitative measurement of cortisol, and related to oocyte fertilization. Total cortisol concentration in FF from fertilized oocytes was 9.7 +/- 0.6 microg/ml (mean +/- SD) in group 1 compared to 9.2 +/- 4.4 microg/ml in group 2 (not statistically significant). Total cortisol concentrations were not associated with oocyte fertilization and no difference between the groups was found in total cortisol concentrations in the FF of unfertilized oocytes or empty follicles.      

Insulin-like growth factor (IGF)-1 and IGF binding protein-1 and -3 in the follicular fluid of infertile patients with endometriosis

BACKGROUND: Endometriosis is associated with pituitary-ovarian axis dysfunction. The study of the follicular fluid in patients with endometriosis is important to elucidate the pathophysiological mechanism of this disease. The objective of this present paper was to analyse the dosages of insulin-like growth factor-1 (IGF-1) and IGF binding protein-1 and 3 (IGFBP-1 and IGFBP-3) in the follicular fluid environment of infertile patients with endometriosis. METHODS: A total of 41 infertile patients undergoing IVF between January 1999 and January 2000 participated in the cross-sectional prospective study. Patients were divided into three groups: group I, minimal/mild endometriosis (n = 12); group II, moderate/severe endometriosis (n = 10); and group III, tubal obstruction (n = 19). The ultra-short protocol was used in association with recombinant FSH for ovulation induction. Follicular fluid analysis was performed using radioimmunoassay with specific kits. RESULTS: Follicular fluid IGF-1 and IGFBP-3 levels were not significantly different among the groups; however, follicular fluid IGFBP-1 levels were lower in those patients with moderate/severe endometriosis (P < 0.05). Comparison of ovulation induction time, number of recombinant FSH units, number of follicles, oocytes and embryos, and fertilization and gestation/cycle rates showed non-significant differences. CONCLUSION: Infertile patients with moderate/severe endometriosis, which is associated with ovulatory dysfunction, presented lower levels of IGFBP-1 in the follicular fluid when undergoing IVF.     

Follicular fluid responds endothermically to aqueous dilution.

BACKGROUND: Studies suggest that ovarian follicles are cooler than their surrounding tissues. The mechanism of this remarkable phenomenon is unclear. We postulate that endothermic reactions accompany the growth-associated hydration of follicular fluid. METHODS: We performed two types of experiment, using human and animal follicular fluids. In the first, saline (50 microl) was injected into follicular fluid (500 microl) held in an equilibrated incubator, with monitoring of sample temperature. In the second, an adiabatic microcalorimeter recorded thermal shifts after injection of buffer (10 microl) into previously dialysed samples (1.4 ml). The relevance of changes observed was assessed by mathematical modelling. RESULTS: In the incubator study, 9/17 bovine and 6/12 human fluids showed a temperature fall (0.05-0.2 degrees C). Cooling was delayed by up to 2 min but sustained for 7-25 min. Remaining fluids showed no change. In the microcalorimeter, 4/9 human, 4/6 bovine, 5/5 porcine and 1/4 equine samples showed an endothermic response. Remaining samples showed either no response (bovine) or exothermy (human, equine). Pre-concentration of human follicular fluid amplified the endothermy or reversed the exothermy. Modelling indicated that the incubator-type response was of appropriate magnitude to explain follicular hypothermy. CONCLUSION: Follicular fluid responds endothermically to aqueous dilution and may contribute to follicular cooling during growth.     

Dimeric inhibins and activin A in human follicular fluid and oocyte-cumulus culture medium.

The concentrations of inhibin A, inhibin B and activin A in follicular fluid and oocyte culture medium were analysed to investigate the production of these peptide hormones by ovarian granulosa cells and oocyte-cumulus complexes, as well as their potential as possible biochemical markers for oocyte quality and fertilizing capacity. Follicular fluids were collected from individual follicles during oocyte retrieval for in-vitro fertilization (IVF). Oocyte-cumulus culture media were collected after in-vitro insemination. The concentrations of dimeric inhibin A, inhibin B and activin A were measured using two-site enzyme-linked immunosorbent assays in the follicular fluid and matched oocyte culture medium. Hormone concentrations were compared with oocyte quality and fertilizing capacity. The concentration of inhibin A in follicular fluid increased while that of inhibin B decreased with increasing follicle size. Follicular fluid concentrations of inhibin A inhibin B and activin A were not significantly different in follicles with differing oocyte quality. Oocyte culture medium concentrations of activin A were significantly higher in morphologically good quality oocytes. There was no relationship between the concentrations of the three hormones and oocyte fertilizing capacity. This study confirms that follicular fluid concentrations of inhibin A may prove to be a marker of follicular growth and maturation. Higher concentrations of activin A produced by good quality oocyte-cumulus complexes suggest that activin A may play a role in oocyte maturation.     

Relationship between follicle size and gonadotrophin surge attenuating factor (GnSAF) bioactivity during spontaneous cycles in women.

BACKGROUND: We have previously demonstrated that follicles < or =11 mm diameter from women undergoing IVF contain higher concentrations of gonadotrophin surge attenuating factor (GnSAF) bioactivity than large follicles from the same ovaries. METHODS: To determine whether this finding is relevant to spontaneous cycles, follicular fluid aspirated from 37 follicles between 3 and 25 mm in diameter from 14 pairs of ovaries from regularly cycling women undergoing total abdominal hysterectomy and bilateral salpingoophorectomy for benign gynaecological disease was pooled into size categories (3 + 4, 5 + 6, 7 + 8, 9 + 10, 11 + 12, 14 + 15, 18 and 25 mm). These pools were bioassayed for GnSAF and inhibin-A, inhibin-B and activin-A concentrations were determined. RESULTS: Follicles of 5 + 6 mm diameter contained the highest concentrations of GnSAF bioactivity (reducing GnRH-induced LH secretion to 38 +/- 8% of control, P < 0.001), while those of 25 mm diameter contained one quarter of this concentration (reducing GnRH-induced LH secretion to 72 +/- 2% of control, P < 0.05). GnSAF bioactivity was closely related to follicle size (r = -0.836, P < 0.01), but not to inhibin-A, inhibin-B or activin-A concentrations. CONCLUSIONS: The finding that small follicles contain high concentrations of GnSAF bioactivity, which fall as folliculogenesis progresses during spontaneous cycles, support the hypothesis that GnSAF has a role in preventing the premature onset of the LH surge in women.     

Dynamics of human follicular growth and in-vitro oocyte maturation

The physiological trigger for meiotic resumption in the human oocyte is the surge of luteinizing hormone, but it can also occur spontaneously if oocytes are released from antral follicles and cultured in vitro. The development of novel techniques for the culture of murine oocytes has raised the possibility of growing human oocytes to maturity in vitro. Such a system could open the door to a number of techniques with revolutionary consequences. It would clearly be of benefit in basic physiological studies of follicular development, as well as being used to test the effect of toxicological substances on oocyte maturation. More significantly, such a system could provide a source of human oocytes for in-vitro fertilization (IVF) where immature or germinal vesicle oocytes are cultured to maturity before being fertilized. If this can be achieved, it might facilitate oocyte cryopreservation, where surplus oocytes are stored, thus avoiding the need for repeated superovulation. A combination of immature oocyte cryopreservation for later maturation and IVF will provide the opportunity to establish oocyte banks and help overcome some of the practical and ethical dilemmas that are currently shadowing the field of reproductive medicine.     

Dynamics of FSH-induced follicular growth in subfertile women: relationship with age, insulin resistance, oocyte yield and anti-Mullerian hormone

BACKGROUND: During excess FSH treatment, different categories of follicles can be discerned: those responding and appearing to grow immediately (FolsS8) and those appearing subsequently during the follicular phase (Fols/d). These follicular categories were explored in cycles of assisted reproduction in the context of follicular biology, including primordial follicle pool (PFP) depletion, age, insulin resistance and potential markers. METHODS: Follicular cohorts were examined in 365 conventional ART cycles and related to patient insulin sensitivity, plasma FSH and anti-Mullerian hormone (AMH). RESULTS: Age had no influence upon the FolsS8 category but was associated with a significant (P < 0.005) decline in the Fols/d. In contrast, insulin-resistant polycystic ovary syndrome (IR-PCOS) showed a significant (P = 0.005) increase in FolsS8. Circulating AMH correlated strongly with oocyte yield and Fols/d. CONCLUSION: Age showed little impact on the initial follicular cohort, but a significant impact upon the secondary cohort, while insulin resistance appeared to promote the former category alone. The disturbance to follicular dynamics and AMH in IR-PCOS reflected a larger stockpile of FSH-sensitive follicles. Circulating AMH appears to represent all categories of antral follicles observed.     

Amino acid, ammonia and urea concentrations in human pre-ovulatory ovarian follicular fluid

BACKGROUND: This study aimed to determine amino acid (AA), ammonia and urea concentrations in human ovarian follicular fluid and to compare these concentrations with those in the circulation. METHODS: Samples of pre-ovulatory follicular fluid and peripheral venous blood were obtained from 14 IVF patients. High-performance liquid chromatography (HPLC) measurements of 25 AAs were the main outcome measures. RESULTS: There was a significant gradient of most AAs from plasma to follicular fluid, with the exception of glutamate, which demonstrated a three-fold increase in follicular fluid concentration (70.0 +/- 3.80 microM) compared with plasma (23.18 +/- 2.20 microM; P < 0.001). The plasma-to-follicular fluid concentration difference for glutamine (81.83 +/- 9.2 microM) was greatest among all AAs. Among essential AAs, this difference was greatest for the branched-chain AAs, isoleucine, leucine and valine. Ammonia concentrations in follicular fluid and blood were 38.87 +/- 2.23 and 22.11 +/- 1.96 microM, respectively (P < 0.001). Urea concentration in follicular fluid was 3.37 +/- 0.18 mM, a value not significantly different from plasma concentration (3.36 +/- 0.22 mM; P = 0.911). CONCLUSIONS: These plasma-follicular fluid differences may reflect both the utilization of AAs and the transport characteristics of the follicular cells. There is accumulation of glutamate and ammonia in pre-ovulatory follicular fluid. The data for urea are consistent with transport by passive diffusion, with no evidence of an active urea cycle in the cells of the follicle.