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.     

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.     

Differential FSH exposure in preantral follicle culture has marked effects on folliculogenesis and oocyte developmental competence

BACKGROUND: We investigated at what stage early cultured preantral mouse follicles become dependent on a minimal effective FSH dose (10 mIU/ml) and analysed the influence of implementing FSH at several time-points during in vitro culture. METHODS: Two-layered mouse follicles were cultured for 12 days under seven different FSH exposure regimens and ovulated on day 13 by hCG/EGF. Ovulated cumulus-oocyte complexes were fertilized and embryos were cultured up to the blastocyst stage. RESULTS: When FSH was absent or added only once at the start of culture, follicle survival was significantly reduced (22 and 52% respectively versus 95% when FSH was continuously present, P < 0.01) and oocyte quality was compromised, providing few oocytes for embryo culture (19 and 7% versus 71% in continuous presence of FSH, P < 0.01). Optimal follicle survival rates can be ensured by implementing FSH at the latest from day 4 of culture. By introducing FSH later than day 4, follicle survival rates and number of ovulated oocytes decreased. Estradiol production and luteinization were strongly related to the moment of introducing FSH in culture. Fertilization and preimplantation embryo development rate were found to be highest in cultures where FSH support was implemented during the preantral stage. CONCLUSION: Exposure to FSH before formation of the antral-like cavity had a positive effect on follicle survival and oocyte robustness.     

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.     

Purine levels and metabolism in human follicular fluid

Adenosine (ADO) in low micromolar levels and hypoxanthine (HYP) in millimolar levels have been shown to inhibit maturation of cumulus-enclosed oocytes. To determine the effect of ovarian stimulation with gonadotrophins on follicular purine metabolism, we measured ADO, HYP, inosine (INO), adenine (ADE) and cyclic AMP (cAMP) levels in follicular fluid (FF) from natural (n = 7) or human menopausal gonadotrophin/human chorionic gonadotrophin (HMG/HCG)-stimulated (n = 35) cycles. Purines were extracted immediately (natural cycles) or within 30 min of recovery (HMG/HCG cycles) and analysed by high pressure liquid chromatography (HPLC). The concentration of all ADE purines in FF was in the low micromolar range (1-20 microM); cAMP levels were markedly increased (greater than 100 microM) in FF of HMG/HCG-treated patients. While ADO levels were within the range effective for inhibition of oocyte maturation, those of HYP were not. No correlation was found between purine levels in FF and ovum maturation. Purine levels in FF of natural cycles were uniformly lower than those of stimulated cycles. Significant conversion of 5'-AMP into ADO, ADO into INO and INO into HYP occurred within 1 h when FF was incubated at 25 but not at 4 degrees C. These purine levels in human FF confirm our previous findings with bovine FF and suggest a possible role of ADO, but not of HYP, in the inhibition of oocyte maturation in the human.     

Follicular fluid and serum concentrations of myo-inositol in patients undergoing IVF: relationship with oocyte quality

BACKGROUND: The follicular microenvironment is an important determinant of oocyte development. The aim of this study was to examine whether the myo-inositol (MI) content in human follicular fluid (FF) was associated with better oocyte quality. METHODS: A total of 53 patients treated with IVF was recruited to a prospective observational study. FF and serum samples collected were divided into two groups: group A consisted of FF associated with matured and fertilized oocytes, whilst group B was from follicles with immature and unfertilized oocytes. RESULTS: Patient's age, total ampoules of HMG used, days of stimulation, basal levels of FSH, estradiol (E(2)) levels on the day of HCG, and serum MI content were not significantly different between the two groups. FF volume and its MI content were significantly higher in group A compared with group B (P < 0.05). The levels of MI in FF were positively correlated with the amount of E(2) in their corresponding FF samples and also correlated with embryo quality. CONCLUSIONS: We propose that higher concentrations of MI and E(2) in human FF appear to play a role in follicular maturity and provide a marker of good quality oocytes.     

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.     

Effect of a partially purified 30.1 kDa ovine follicular fluid protein on ovine follicle and ovarian somatic cell growth, and oocyte maturation in vitro

Aim: Regulation of folliculogenesis and oocyte–somatic cell interactions in the ovarian follicles is under the control of gonadotrophins and various local factors. In the present study, an attempt was made to isolate and examine the biological activities of ovarian follicular fluid protein(s) in sheep in vitro. Methods: Follicular fluids aspirated from ovarian follicles of slaughterhouse‐derived ovaries were made cell free by centrifugation (5000 g for 30 min) and steroid free by charcoal treatment. The follicular fluid was then subjected to ammonium sulphate precipitation and gel filtration chromatography using G‐75 Sephadex. Protein detection was performed using a UV spectrophotometer at 280 nm. The 35–50% fraction yielded a detectable peak and a protein of 30.1 kDa as examined by SDS‐PAGE. The effect of increasing doses of the 30.1 kDa ovine follicular fluid protein (oFFP) was tested at different doses on pre‐antral and antral follicle growth; cumulus cell expansion; oocyte maturation; changes in protein, calcium and phosphorus levels of oocytes after culture in media containing different levels of isolated protein; mural granulosa cell, polar granulosa cell (cumulus cell), oviductal epithelial cell monolayer formation and granulosa cell proliferation in vitro. Results: The oFFP significantly inhibited antral follicle growth, cumulus expansion, oocyte maturation and somatic cell growth in vitro in a dose‐dependent manner. The oFFP did not have a significant effect on the pre‐antral follicle growth in vitro. The protein, calcium and phosphorus contents of oocytes were found to decrease in oocytes cultured in maturation medium containing the oFFP. Conclusion: The present study demonstrates a follicular fluid factor regulating folliculogenesis and oocyte maturation in sheep.     

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.     

Influence of blood clots in the cumulus complex on oocyte fertilization and cleavage

Since multiple ovarian punctures are performed during oocyteretrieval, the likelihood of blood contaminating the follicularfluid is high. The purpose of this study was to determine whetherthe presence of blood clots, which are frequently seen in thecumulus of oocytes retrieved under ultrasound guidance, hasany effect on fertilization and subsequent embryo cleavage andto identify oocyte characteristics which may predict these events.Oocytes were morphologically graded and the presence of bloodclots in the cumulus was recorded. Cases in which the male partnerhad a total motile sperm count < 20 ? 10⁶ on the day of inseminationwere excluded from the logistic regression analysis. The presenceof blood clots in the cumulus was a negative predictor of cleavage[odds ratio (OR) = 0.54]. The results indicate that an oocytewith optimal quality is one which is spherical (OR = 5.45),has an expanded corona (OR = 3.80) and has no blood clots inthe cumulus complex.     

Physiology: Follicular volume and number during in-vitro fertilization: association with oocyte developmental capacity and pregnancy rate

This study examined the effect of the number of follicles aspiratedduring egg retrieval on pregnancy likelihood during an ovulationinduction, in-vitro fertilization (IVF) programme. In addition,the volume of each individual follicle was related to the probabilityof obtaining an oocyte from that follicle. Its capacity to befertilized, the incidence of polyspermic fertilization, thequality of early embryos derived from that oocyte and the influenceof patient age were the other outcomes studied. Large follicularnumber and high mean follicular volume related positively topregnancy outcome. Successful egg retrieval, normal fertilizationand good embryo quality were more likely with increasing individualfollicular volume. A model was constructed to quantify the predictivevalue of follicular fluid volume on the developmental potentialof individual oocytes and embryos derived from that follicle.Successful outcome of IVF is more likely if ovulation inductionresults in many follicles, particularly if they have a highmean volume. Individual oocyte and embryo quality can be tracedback to the follicular level.     

人类白细胞抗原-G与肝炎病毒感染的相关性研究进展

人类白细胞抗原-G(human leukocyte antigen G,HLA-G)属于非经典主要组织相容性复合体Ⅰ类分子,选择性表达在母胎交界面的细胞滋养层细胞使得妊娠期间形成母体对胎儿的免疫耐受。但是,HLA-G的有限多态性和异常表达与多种病理改变有关,例如肿瘤、自身免疫病和感染性疾病等。近来有研究表明,乙型肝炎病毒和丙型肝炎病毒感染过程中HLA-G的表达均会发生改变,而且HLA-G的表型与病毒的易感性相关。但是,至于HLA-G分子在乙型肝炎和丙型肝炎疾病发展中的作用目前仍不完全清楚。HLA-G与病毒性肝炎的相关性仍需深入研究。     

In-vitro oocyte maturation: some questions concerning the initiation and prevention of this process in humans

Human oocytes isolated from healthy non-preovulatory antralfollicles which had not been stimulated spontaneously resumedmeiosis at a low rate (31%) when compared with those from atreticfollicles (73%). Human follicular fluid did not inhibit spontaneousmaturation of rat oocytes in vitro, regardless of the qualityor maturity of the follicle or the treatment of the patient.In the human, oocyte maturation may involve a stimulatory processas well as a removal of inhibition.     

Follicular growth and oocyte competence in the in vitro cultured mouse follicle: effects of gonadotrophins and steroids

Although there have been extensive studies on the effects of gonadotrophins and steroids on follicular development, less is known as to the effects these hormones have on the acquisition of oocyte developmental competence. This study investigates the effect of altering the gonadotrophin or steroidal environment on follicular development and on oocyte viability and DNA methylation. Oocytes were obtained from pre-ovulatory follicles after individual follicle culture from the pre-antral stage; gonadotrophin or steroid levels were manipulated during the culture period. Oocytes obtained from follicles grown in gonadotrophin free conditions were able to fertilize and develop to the blastocyst stage despite their impaired follicle development. There was no effect of luteinizing hormone or steroids on follicular growth. Altering the steroidal environment did, however, affect oocyte development. The oocytes of follicles exposed to high estrogen levels had lower fertilization rates, regardless of the presence or absence of high androgen levels. The combined presence of high levels of both steroids altered the level of global methylation. This study demonstrates that gonadotrophins and steroids influence the acquisition of developmental competence of the oocyte and suggests that optimal steroid exposure during follicle development is required for the oocyte to mature correctly.     

Therapeutic effects of soluble human leukocyte antigen G2 isoform in lupus-prone MRL/lpr mice

Human leukocyte antigen (HLA)-G, a non-classical HLA class I molecule, has one of the splicing isoforms, HLA-G2, which lacks one domain (α2) and forms a non-covalent homodimer. HLA-G2 is expressed on placental cells, regulatory T cells, tumor cells, and virus-infected cells, and is involved in immunosuppression. The major isoform of HLA-G, HLA-G1, binds to leukocyte immunoglobulin (Ig)-like receptor (LILR) B1 and LILRB2, on the contrary, HLA-G2 binds to only LILRB2. We previously reported that HLA-G2 bound LILRB2 more strongly than HLA-G1 and also to paired Ig-like receptor (PIR)-B, a mouse homolog of LILRBs. Furthermore, HLA-G2 showed immunosuppressive effects in both collagen-induced arthritis (CIA) and atopic dermatitis-like model mice. In this study, we examine in vivo effects of HLA-G2 in systemic lupus erythematosus (SLE) model mice. HLA-G2 showed the suppression of the typical SLE symptoms such as serum anti-dsDNA antibody level and urinary albumin index. Furthermore, HLA-G2 tended to downregulate B-lymphocyte stimulator (BLyS) production. This is the first observation of the immunosuppressive effects of HLA-G2 isoform in SLE model mice, suggesting that HLA-G2 could be a useful therapeutic agent for SLE.     

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.     

Preiinplantation diagnosis: Ultrasound derived indices of follicular blood flow before HCG administration and the prediction of oocyte recovery and preimplantation embryo quality

The principal aim of the study was to relate ultrasound-derivedindices of blood flow in individual follicles on the day of,but before, the administration of human chorionic gonadotrophin(HCG) to the subsequent recovery of oocytes and the productionof preimplantation embryos. Data were obtained from 21 women(aged 29–43 years) with bilateral tubal occlusion, whowere undergoing treatment by in-vitro fertilization (FVF) andembryo transfer. Transvaginal ultrasonography with colour Dopplerimaging and pulsed Doppler spectral analysis were used to measurefollicular volume and derive indices of blood flow. The end-pointsfor each follicle were the volume, peak systolic velocity (PSV),pulsatility index (PI), and the recovery or non-recovery ofan oocyte, the subsequent production or non-production of apreimplantation embryo and the morphological grade of each embryo.A total of 94 follicles were studied; 74 oocytes were recovered(79%) and 40 embryos (33 grade I or II) were produced. Therewere four clinical pregnancies (pregnancy rate 25.0% per transfer,19.0% per patient). There was a significant correlation betweenwhether or not follicular blood flow was detected and whetheror not an oocyte was recovered (P <0.05, x² test). The valuesfor volume and PI were not clinically useful. The PSV (cm/s,mean ± SD) was higher in follicles that were associatedwith the production of an embryo (12.7 ± 5.9) comparedwith those that were not (8.5 ± 5.0; P <0.05, Student'st-est). The probability of producing a grade I or grade II embryowas 75% if the PSV was 10 cm/s. The corresponding value was40% if the PSV was <10 cm/s and 24% if blood flow was notdetected (i.e. PSV <3 cm/s). There was a significant increase(P <0.05, Student's t-test) in the PSV before aspirationin those follicles associated with the subsequent productionof an embryo. We conclude that the value for PSV, before theadministration of HCG, can be used to identify follicles witha high probability of producing an oocyte and a high grade preimplantationembryo. The information may also be used to time the administrationof HCG to achieve the optimum number and quality of embryosfor patient management.     

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.     

Human cumulus-free oocyte maturational profile and in vitro developmental potential after stimulation with recombinant versus urinary FSH

BACKGROUND: This study compares the influence of recombinant (r)FSH and urinary (u)FSH stimulation on oocyte and embryo quality in patients undergoing ICSI. METHODS AND RESULTS: Denuded oocyte maturity in ICSI patients was graded on a scale from metaphase II (MII) to prophase for nuclear maturation of oocytes. The relationships of cumulus-free oocyte maturational profiles with in vitro outcome parameters were evaluated. In the study population with an unknown distribution of FSH receptor polymorphisms, the ovarian response to rFSH stimulation was significantly different from that of uFSH stimulation, including lower number of oocytes retrieved/oocytes in MII, higher fertilization rates and higher good quality embryo rates. In the study population with a similar distribution of FSH receptor polymorphisms, the ovarian responses to rFSH were lower numbers of oocytes in MII, higher fertilization rates and higher good quality embryo rates, but the total number of oocytes retrieved was not influenced, in comparison with ovarian stimulation with uFSH. CONCLUSIONS: rFSH stimulation appears to influence oocyte quality and subsequent embryo quality in comparison with uFSH stimulation. FSH receptor polymorphisms seem to be an intrinsic factor influencing the ovarian response to FSH stimulation.     

Human leukocyte antigen G is associated with esophageal squamous cell carcinoma progression and poor prognosis

Human leukocyte antigen G (HLA-G) is a non-classical HLA class I molecule thought to play a key role in maternal-fetal tolerance and cancer immune evasion. This study aimed to investigate the HLA-G expression in lesion sections and plasma sHLA-G levels of primary esophageal squamous cell carcinoma (ESCC) patients and its clinical significance in diagnosis and prognosis of ESCC. 60 ESCC patients and 28 healthy controls were recruited, and the positive expression of HLA-G in ESCC lesions and adjacent normal tissues were 70% (42/60) and 8.6% (5/60) (P < 0.05), respectively, while no expression was found in normal controls. HLA-G1 and HLA-G5 were determined to be dominating isoforms measured by RT-PCR. There was a significant difference in plasma sHLA-G levels between patients with ESCC (15.04 U/ml, range 4.33–250.00 U/ml) and healthy controls (6.81 U/ml, range 0–29.27 U/ml) (P < 0.01). The plasma IL-10 level was higher in ESCC patients than the controls (23.86 pg/ml vs. 12.81 pg/ml, P < 0.01). HLA-G expression in lesion tissues was correlated with cancer cell differentiation (P = 0.033), lymph node metastasis (P = 0.035) of ESCC. However, no obvious correlations were demonstrated between the plasma sHLA-G levels and the clinicopathological parameters. There was a significant correlation between sHLA-G and IL-10 expression (r = 0.353, P = 0.006) in patients with Esophageal squamous cell carcinoma. HLA-G positive expression showed poorer prognosis of ESCC. HLA-G positive expression might serve as a potential marker in the diagnosis or prediction of ESCC.