Human ovarian granulosa cells and follicular fluid indices: the relationship to oocyte maturity and fertilization in vitro

The study investigates the correlation between oocyte maturity and fertilization and a variety of hormonal parameters in follicular fluid and ovarian granulosa cells. A methodology for purification of granulosa cells from contaminating blood cells is also established. A total of 63 follicular aspirates were collected at oocyte retrieval from 30 women superovulated using the long luteinizing hormone- releasing hormone (LHRH analogue)/human menopausal gonadotrophin regimen. Oestradiol, progesterone, testosterone and human chorionic gonadotrophin (HCG) were quantified in follicular fluid and granulosa cells were immunostained for human chorionic gonadotrophin. Immunopurification of granulosa cells from contaminating blood cells was performed. HCG in follicular fluid was significantly high in follicles yielding immature (grade 3) oocytes (P=0.002); there was no correlation with fertilization. Aspirates from follicles containing mature (grade 1) oocytes and oocytes that subsequently fertilized had significantly more granulosa cells immunobound to HCG (P < 0.001, P=0.02). Moreover, the immunomagnetic purification technique provided >98% pure population of granulosa cells. The data demonstrate that HCG in follicular fluid and on granulosa cells may help to predict oocyte maturity and fertilization. Furthermore, immunomagnetic beads provide a reliable procedure for the purification of ovarian granulosa cells.      

The clinical role of interleukin-6 and interleukin-6 soluble receptor in human follicular fluids

In order to investigate the role of interleukin-6 (IL-6) and interleukin-6 soluble receptor (sR) in human ovulation, we evaluated the concentrations in human follicular fluid and analyzed the correlation of IL-6 and IL-6 sR with oocyte maturation. The oocytes were obtained from the follicular fluid of 45 women undergoing in vitro fertilization and embryo transfer. The concentrations of IL-6 and IL-6 sR in follicular fluid were measured by ELISA. In addition, granulosa cells obtained from the follicular fluid were cultured and treated with forskolin and 12-o-tetradecanoylphorbol 13-acetate for 24–48 h. The concentration of IL-6 was significantly higher in the follicular fluid than in the serum (P<0.01). In contrast, the concentration of IL-6 sR was significantly lower in the follicular fluid than in the serum (P<0.001). The concentrations of IL-6 and IL-6 sR were significantly higher in the follicular fluid containing mature oocytes than in fluid containing immature oocytes (P<0.05). The production of IL-6 was markedly increased over the basal level after 24 h of treatment with forskolin(P<0.001) and 48 h of treatment (P<0.01) with cultured granulosa cells. Our data suggest that IL-6 and IL-6 sR may play an important role in follicular growth and development in human preovulatory processes. It is possible that IL-6 in particular may be regulated by cAMP. IL-6 and IL-6 sR might also be valuable biochemical markers in the evaluation of oocyte maturation. Received: 6 July 2002 / Accepted: 18 December 2002 Correspondence to Y. Kawano     

Endocrinology: Growth hormone, oestradiol, progesterone and testosterone concentrations in follicular fluid after ovarian stimulation with various regimes for assisted reproduction

Follicular fluid samples and oocytes were obtained from 75 women(87 cycles), who participated in an assisted conception programme.Determinations of the concentration of oestradiol, progesterone,testosterone and growth hormone were performed in all follicularfluid samples. Patients were stimulated with the following regimes:group A (24 cycles, 94 samples), human menopausal gonadotrophin(HMG) (three ampoules/day) and human chorionic gonadotrophin(HCG); group B (23 cycles, 53 samples), HMG/HCG with prednisolone(7.5 mg/day) after cycle programming with oral contraceptives;group C (40 cycles, 60 samples), buserelin with HMG/HCG. Oestradiolconcentrations (mean ± SEM) were significantly higher(P < 0.05) in group A (320.1 ± 27.3 ng/ ml) and thoseof growth hormone in both groups A and C (3.8 ± 0.2 and3.2 ± 0.15 ng/ml, respectively), as compared to the othergroups, whereas progesterone and testosterone concentrationswere similar in all groups. The mean concentrations of oestradiol,progesterone, testosterone and growth hormone were significantlyhigher (P < 0.01) in follicular fluid with oocytes of intermediatematurity than with mature oocytes (382.5 ng/ml, 7847.5 ng/ml,1704.5 ng/dl and 3.7 ng/ml versus 217.8 ng/ml, 5488.4 ng/ml,1313.6 ng/dl and 2.7 ng/ml, respectively). On the other hand,only oestradiol concentrations were significantly higher infollicular fluid of fertilized compared to non-fertilized oocytes.Concentrations of the other hormones analysed, except growthhormone, were similar in follicular fluid from pregnant andnon-pregnant women after assisted reproduction. Growth hormone,on the other hand, was significantly lower (P < 0.05) infollicular fluid from pregnant compared to non-pregnant women(2.8 versus 3.5 ng/ml). It is concluded that intermediate maturityoocytes and oocytes which will be subsequently fertilized arefound in follicles with higher follicular fluid concentrationsof growth hormone and steroids. Moreover, oocytes leading topregnancy after in-vitro fertilization and embryo transfer arederived from follicles with lower growth hormone concentrationsin follicular fluid.     

异丙酚麻醉不同阶段对卵细胞受精和对早期胚胎质量的影响

目的探讨异丙酚静脉麻醉下取卵手术中卵泡抽吸液中异丙酚的浓度变化及各阶段对卵细胞受精和早期胚胎质量的影响.方法 17例实施超促排卵下体外受精-胚胎移植术的妇女预测卵泡至少有15个,按取卵的先后分3组(早、中、晚)分别培养.记录从静脉给异丙酚到每个卵泡取出时间.检测卵泡抽取液中异丙酚的浓度及观察卵细胞的受精率及卵裂率等.结果取出第G一个卵泡的平均时间是200秒,取出卵泡的平均速率是17.6s/ 个.所有患者的最后一个卵泡抽吸液中异丙酚的浓度明显高于第一个卵泡抽吸液中异丙酚的浓度(P<0.01).除未成熟的卵细胞占总卵泡百分比在3组间有明显的区别外,受精率和卵裂率无明显区别.结论随着麻醉的进展,卵泡抽吸液中异丙酚的浓度逐渐增加,但与所给剂量或持续的时间之间无必然的关联.我们不能显示卵泡抽吸液中升高的异丙酚浓度对卵细胞质量的不利影响.     

Cotinine levels in follicular fluid and serum of IVF patients: effect on granulosa-luteal cell function in vitro

Cigarette smoking is accepted as a risk factor for pregnancybut its effect on fertility is uncertain. In this study we determinedthe concentration of cotinine, a nicotine metabolite, in follicularfluid and serum from women participating in an In-vitro fertilizationand embryo transfer (IVF-ET) programme. Cotinine was undetectableIn serum and follicular fluid of non-smokers but ranged from<5 to 371 ng/ml in follicular fluid and from 24 to 245 ng/mlin serum of smokers. Granulosa-luteal cells, obtained from IVFpatients and cultured for 4 days, secreted progesterone and,when an aromatizable androgen was added, oestradiol-17. Theaddition of cotinine or nicotine did not alter progesteroneor oestradlol-17 secretion. However, the presence of cotininein follicular fluid of women smokers provides evidence for accessof at least one component of cigarette smoke to the developinggamete and the cells of the follicle. Further work is requiredto determine whether fertllity is compromised by the presence,In follicular fluid, of contaminants derived from cigarettesmoke.     

Relationship between fertilization results after intracytoplasmic sperm injection, and intrafollicular steroid, pituitary hormone and cytokine concentrations

Previous studies relating hormone and cytokine concentrations in follicular fluid to oocyte fertilizability were flawed by the uncertainty about the actual oocyte maturity status at the time of recovery and by the possible contribution of the male factor to failures of conventional in-vitro fertilization. This is the first study in which oocyte maturity was assessed immediately after recovery and only mature oocytes were selected for treatment by intracytoplasmic sperm injection. Fertilization outcomes were related to follicular fluid concentrations of 17beta-oestradiol, progesterone, follicle stimulating hormone, luteinizing hormone (LH), growth hormone (GH), prolactin (PRL), interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF alpha). Those oocytes that subsequently showed normal fertilization were harvested from follicles with higher concentrations of progesterone, GH, PRL, IL-1 and TNF alpha as compared with those of oocytes that failed to fertilize. Among the normally fertilized oocytes, low GH concentrations were associated with the failure of cleavage and with poor morphology of cleaving embryos, whereas rapidly cleaving embryos developed from oocytes recovered from follicles with high concentrations of LH and IL-1. These data suggest important roles for GH, IL-1 and TNF alpha, and of residual LH after pituitary suppression, as positive regulators of the final phase of oocyte intrafollicular development.     

Interdependent influence of follicular fluid oestradiol concentration and motility characteristics of spermatozoa on in-vitro fertilization results.

Methods are presented for an objective assessment of the quality of both gametes in an in-vitro fertilization programme. The concentrations of oestradiol and progesterone in follicles whose oocytes did or did not fertilize, were measured and assessed as potential markers of oocyte maturity. There was no difference in the mean concentrations of either steroid in follicular fluid of fertilized and unfertilized oocytes. However, a highly significant inverse correlation was observed between the oestradiol concentration in follicles of oocytes becoming fertilized and the number of spermatozoa added for fertilization (P less than 0.001). Follicular fluid concentrations of progesterone did not correlate with the number of inseminated spermatozoa. The correlation between follicular oestradiol concentrations and the number of spermatozoa needed for fertilization was then used to identify movement characteristics of spermatozoa in the culture medium which were relevant for fertilization. Sufficient numbers of spermatozoa having specific values of head cross frequency, lateral head displacement, linearity and curvilinear velocity were critical for the occurrence of fertilization in vitro.     

Blastocyst development and pregnancies after IVF of mature oocytes retrieved from unstimulated patients with PCOS after in-vivo HCG priming.

A major side-effect of controlled ovarian stimulation (COS) in patients with polycystic ovarian syndrome (PCOS) is the risk of ovarian hyperstimulation syndrome (OHSS). In-vitro maturation (IVM) of immature oocytes represents a potential alternative for the fertility treatment of these patients. Two patients at high risk of OHSS were primed with 10,000 IU HCG 36 h before oocyte retrieval. After retrieval, oocyte maturity was evaluated. Oocytes considered to be mature at the time of collection were inseminated by IVF or ICSI, and the resulting embryos were cultured to blastocysts. Transfer of these blastocysts resulted in pregnancy in both patients. Immature oocytes were cultured in YS medium supplemented with 30% human follicular fluid, 1 IU/ml rFSH, 10 IU/ml HCG and 10 ng/ml epidermal growth factor (rhEGF). After in-vitro maturation of the oocytes, ICSI was performed. Two and five expanded blastocysts were obtained after 5 day culture and were cryopreserved. This report indicates that mature oocytes can be collected at the time of retrieval using only in-vivo HCG priming in women with PCOS, and clinical pregnancy can be established by transfer of blastocysts derived from the mature oocytes. This approach opens a potential for a new dimension in the management of patients with PCOS.     

Ovarian follicular concentration of IL-12, IL-15, IL-18 and p40 subunit of IL-12 and IL-23

BACKGROUND: The aim of the study was to determine the presence of interleukin (IL)-12, IL-15, IL-18 and p40 subunit of IL-12/IL-23 in follicular fluid from spontaneous cycles and the relation between the concentration of selected cytokines and IVF-embryo transfer outcome. METHODS: IVF-embryo transfer and enzyme immunoassay (EIA) (R&D Systems, Minneapolis, MN, USA and MBL, Nagoya, Japan) were used. RESULTS: Follicular fluid of women included in the IVF-embryo transfer procedure contained common p40 subunit of IL-12/IL-23 (median 70.1 pg/ml), IL-15 (median 1.3 pg/ml) and IL-18 (median 38.2 pg/ml). There was a significant negative correlation between follicular fluid concentrations of IL-15 and IL-18 (R=-0.392, P=0.003). Significantly higher concentrations of common p40 subunit of IL-12/IL-23 (median 79.8 pg/ml) were found in the follicular fluid taken from follicles containing oocytes, when compared with those without an oocyte (median 44.5 pg/ml, P=0.006). Patients who achieved clinical pregnancy had significantly decreased concentration of IL-15 (median 0.8 pg/ml) compared with patients without successful IVF-embryo transfer outcome (median 1.4 pg/ml, P=0.047). CONCLUSION: Follicular fluid collected from spontaneous cycles contains detectable levels of p40 subunit of IL-12/IL-23, IL-15 and IL-18. Increased concentrations of p40 subunit of IL-12/IL-23 in follicles containing oocytes suggest an important role of this cytokine in reproduction. Possible negative value of IL-15 as a predictor of IVF-embryo transfer success remains to be determined.     

Fibronectin and glycosaminoglycans in human preovulatory follicular fluid and their correlation to follicular maturation

The purpose of this study was to investigate the involvementof two extracellular matrix constituents-fibronectin (FN) andglycosaminoglycans (GAG)–in the development of human ovarianfollicles. One-hundred-and-one samples of human follicular fluid(HFF) aspirated from patients participating in an IVF–ETprogramme were assayed for FN, GAG, protein, progesterone (P)and oestradiol (E₂. FN/protein and FN/GAG ratios increased significantlywith volume of HFF and with follicular P levels. In contrast,GAG/protein ratios decreased significantly with HFF volume,follicular P and E₂. Ratios of FN/protein correlated positivelywith maturity of oocytes; 3.1 ? 0.5 with dysmature, 4.2 ? 0.5with immature, 8.5 ? 1.0 with intermediate and 7.8 ? 0.5 withmature oocytes. Ratios of GAG/protein were inversely correlatedwith maturity of oocytes; 11.0 / 1.0 with dysmature, 12.5 /1.4 with immature, 8.9 ? 0.9 with intermediate and 9.2 ? 0.5with mature oocytes. Ratios of FN/GAG correlated with maturityof oocytes; 0.3 ? 0.02 with dysmature, 0.4 ? 0.1 with immature,1.2 ? 0.2 with intermediate and 1.1 ? 0.1 with mature oocytes.Furthermore, follicles leading to oocytes which fertilized showedsignificantly higher FN/protein and FN/GAG ratios than thoseyielding oocytes which remained unfertilized. In contrast, GAG/proteinratios were significantly lower in follicles with which oocytesfertilized than in those with oocytes which did not fertilize.These results suggest that FN and GAG can be useful markersfor follicular development and the potential of the oocyte tobe fertilized.     

Inhibin A, inhibin B and activin A in follicular fluid of infertile women with tubal damage, unexplained infertility and emdometriosis

PROBLEM: To measure and compare concentrations of inhibin A, inhibin B, activin A and oestradiol in the follicular fluid of women with endometriosis, tubal damage and unexplained infertility with oocyte quality and fertilising capacity. Also, to assess whether impaired follicular function in women with endometriosis might be related to altered inhibin or activin concentrations and whether this correlated. METHOD OF STUDY: Follicular fluids were collected from individual follicles during oocyte retrieval for in vitro fertilisation (IVF) in natural cycles. Inhibin A, inhibin B and activin A were measured using two-site enzyme immunoassay, and oestradiol was assayed by fluoro-immunometric method. RESULTS: Follicular fluid inhibin A levels were found to be significantly higher in women with endometriosis. Inhibin A was directly correlated with follicle size. There was no correlation between the levels of inhibin A, inhibin B, activin A and oocyte quality or fertilising capacity in the three groups of women. CONCLUSIONS: Follicular fluid concentration of inhibin A is elevated in follicles of women with endometriosis and is positively correlated with follicle maturation. However, we were unable to demonstrate any association between the follicular fluid concentrations of inhibin A, inhibin B, activin A or oestradiol and the quality and fertilisation capacity of oocytes in women with tubal damage, unexplained infertility or endometriosis.     

The effect of propofol anaesthesia on oocyte fertilization and early embryo quality

Propofol, frequently used for i.v. induction of anaesthesia in assisted reproduction procedures, has been suspected of damaging oocytes. Concentrations of propofol have recently been shown to increase in follicular fluid during oocyte retrieval. Our study was designed to assess whether exposure to increasing concentrations of propofol has a measurable effect on in-vitro fertilization, cleavage and embryo development. A cohort of 130 women underwent i.v. anaesthesia using propofol and fentanyl. Time of anaesthesia from i. v. injection of propofol was measured, as were the doses of the two drugs. In 32 women expected to have more than 15 oocytes retrieved, first, middle and last oocytes were cultured separately. The mean time from i.v. injection to first follicle aspiration was 200 s. The mean time for the aspiration of each additional oocyte was 17.6 s. In 10 out of 11 cases where follicular fluid concentrations of propofol were measured, there was an increase from the first to the last follicle, but no difference was found in the ratio of mature to immature oocytes. Nor were any differences found in fertilization, cleavage and embryo cell number. In so far as in-vitro development reflects embryo quality, we conclude that the time elapsed between retrieval of the first and last oocyte does not affect oocyte quality.     

The production and clinical evaluation of macrophage colony-stimulating factor and macrophage chemoattractant protein-1 in human follicular fluids

PROBLEM: In order to investigate the role of macrophage colony-stimulating factor (M-CSF) and macrophage chemoattractant protein-1 (MCP-1) in human ovulation, we measured the concentrations of M-CSF and MCP-1 in human follicular fluids (FFs) and correlated them with oocyte maturation. METHOD OF STUDY: The oocytes were obtained from the FFs of 46 women undergoing in vitro fertilization and embryo transfer (IVF ET). The concentrations of M-CSF and MCP-1 in the FFs were measured by enzyme-linked immunosorbent assay. In addition, granulosa cells obtained from the FFs of IVF patients were cultured and treated with forskolin and 12-O-tetradecanoylphorbol 13-acetate (TPA) for 24 48 hr. RESULTS: Concentrations of M-CSF and MCP-1 were significantly higher in the FFs than in the serum (P < 0.01). M-CSF concentrations tended to be higher, while MCP-1 concentrations were significantly higher in the FFs containing mature oocytes than in FFs containing immature oocytes (P < 0.05). The production of M-CSF was markedly increased over the basal level after treatment with forskolin (10 microM) for 24 (P < 0.02) and 48 hr (P < 0.01); however, the production of MCP-1 was unchanged. CONCLUSIONS: Our data suggest that M-CSF and MCP-1 may play an important role in human preovulatory processes and that M-CSF, in particular, may be regulated by cyclic adenosine monophosphate. M-CSF and MCP-1 may also be valuable biochemical markers in the evaluation of oocyte maturation.     

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.     

Leukaemia inhibitory factor expression in human follicular fluid and ovarian cells

Leukaemia inhibitory factor (LIF) is a 43 kDa glycoprotein with a remarkable range of biological actions in different tissue systems. LIF improves the rate of fertilization of mouse oocytes in vitro and up- regulates aromatase enzyme. We postulated that LIF may be an important modulator of ovarian function and may also improve embryo quality in humans. Follicular fluid samples from patients undergoing in-vitro fertilization (IVF) and embryo transfer (n = 123), from women undergoing ovarian stimulation (n = 4) and from women undergoing laparoscopy for tubal ligation during their follicular phase (n = 3) were used. Follicular fluid LIF, oestradiol, and progesterone were measured and embryo quality was assessed. Granulosa-lutein cells were cultured for 3 days in Ham's F-12:Dulbecco's modified Eagle's medium (DMEM). Ovarian stromal cells, isolated by enzymatic dispersion of ovarian tissue, were also cultured in the same medium. Following experimental treatments, LIF mRNA and protein concentrations were quantified. The concentration of LIF was 0.8 +/- 0.3 (mean +/- SEM) pg/ml in pre-human chorionic gonadotrophin (HCG) follicular fluid samples and 13.0 +/- 1.1 pg/ml in post-HCG follicular fluid samples (P < 0.05). LIF levels were undetectable in three follicular fluid samples obtained during unstimulated follicular phase. There was a correlation between follicular fluid LIF and follicular fluid oestradiol concentrations (r = 0.36; P = 0.0001) and the number of grade I embryos (r = 0.62; P = 0.01). LIF mRNA and the protein were expressed constitutively but in low amounts in the ovarian stromal cell cultures. The concentrations of LIF mRNA as well as protein were increased by interleukin (IL)-1alpha and tumour necrosis factor alpha (TNF alpha) in a time- and concentration-dependent manner. Purified granulosa-lutein cells expressed low amounts of LIF mRNA and protein which were not significantly increased by IL-1alpha or TNF alpha. Our findings suggest that HCG stimulates the expression of LIF in follicular fluid. Both granulosa-lutein and ovarian stromal cells express the LIF mRNA and produce the protein. Modulation of LIF in these cells may play an important role in the physiology of ovulation and early embryo development.      

Increase in transforming growth factor beta1 in ovarian follicular fluid following ovarian stimulation and in-vitro fertilization correlates to pregnancy

We have analysed the content of the growth and differentiation regulating peptide, transforming growth factor beta1 (TGFbeta1), in follicular fluid from patients undergoing in-vitro fertilization (IVF), and correlated concentrations of TGFbeta1 with the outcome of the IVF treatment and the concentrations of 17beta oestradiol in serum at ovum retrieval. A total of 88 women with infertility of >3 years duration and age <38 years participated in the study. During IVF treatment, follicular fluid and matched serum samples were collected at ovum retrieval and analysed for TGFbeta1, oestradiol, progesterone, follicle- stimulating hormone (FSH) and luteinizing hormone (LH) using radioimmunoassay and enzyme-linked immunosorbent assay. We found that the TGFbeta1 content in the follicular fluid at the time of oocyte retrieval correlated positively with subsequent pregnancy. In 29 women who became pregnant following IVF, follicular fluid TGFbeta1 values were significantly higher (P=0.005) than in 59 women where IVF was unsuccessful. In the pregnant group, TGFbeta1 values correlated positively with oestradiol at ovum retrieval. TGFbeta1 also correlated positively with the number of fertilized oocytes. TGFbeta1 may thus be important for successful human pre-embryo development, contribute to successful embryo implantation and development and may be necessary for the establishment of pregnancy.      

The paradox of declining fertility but increasing twinning rates with advancing maternal age

BACKGROUND: Advancing female age is associated with declining fertility potential due to decreasing numbers and quality of oocytes but also with a distinct increase in dizygotic twinning rates, a phenomenon that has never been explained. METHOD: An analysis of follicle development was made in 959 spontaneous ovulatory cycles of 507 women. RESULTS: Multiple ovarian follicular development (>1 follicle >14 mm) and, by implication, multiple rather than single ovulations occurred in 105 women whose mean age (36.1 versus 34.6 years) and mean basal FSH concentrations (10.3 versus 7.7 IU/l) were significantly greater than those with monofollicular development (P < 0.01). The prevalence of multifollicular development increased with age. CONCLUSIONS: Dizygotic twinning must be associated with the development of >1 large follicle, which we found to be a significantly more frequent occurrence in older women. It is hypothesized that the response of pituitary release of FSH to the decreased negative feedback induced by impending ovarian failure often 'overshoots', causing multiple follicular development. In the presence of two good-quality oocytes, a twin pregnancy may result.     

人卵泡液氨基酸谱影响卵母细胞发育潜能的初步研究

目的分析体外受精（IVF）患者单卵泡液氨基酸谱含量与卵母细胞发育潜能的相关性。方法利用高效液相色谱（HPLC）分析技术检测49份IVF患者优势卵泡的单卵泡液氨基酸谱,并追踪所获卵母细胞发育结局。依据取卵后第三天（D3）胚胎评分分为可用胚胎组（A组）与不可用胚胎组（B组）,比较两组氨基酸谱含量差异。结果①两组患者的年龄、不孕年限、体重指数、基础性激素及用药支数均无统计学差异（P〉0.05）;②A组患者的取卵数、受精率及卵裂率略高于B组,但无统计学意义（P〉0.05）;A组患者的可用胚胎数及可用胚胎率（7.41±4.64、65.15%）显著高于B组（5.00±3.38、49.75%）（P〈0.05）;③A组卵泡液中丙氨酸（Ala）含量（335.86±70.79μmol/ml）明显高于B组（293.56±67.30μmol/ml）（P〈0.05）,其余氨基酸含量无统计学差异（P〉0.05）。④利用ROC曲线确定卵泡液中Ala含量预测可用胚胎形成的临界值为325.4μmol/ml,灵敏度63%,特异度81.8%。结论优势卵泡的发育可在一定程度上反映IVF患者的总体情况,卵泡液中Ala含量可作为预测卵母细胞受精后早期胚胎发育的参考指标之一。     

Possible contribution of follicular interleukin-1beta to nitric oxide generation in human pre-ovulatory follicles

The aim of this study was to investigate the relationships between follicular nitric oxide (NO) metabolite concentrations and several related variables, with special reference to follicular interleukin- 1beta (IL-1beta). The follicular fluid from the leading and secondary follicles was collected individually from 20 women undergoing in-vitro fertilization (IVF) treatment, and the concentrations of nitrite (NO2-) and nitrate (NO3-) were determined fluorometrically using 2,3- diaminonaphthalene. Both follicular nitrite (r = 0.42, P < 0.01) and nitrate (r = 0.49, P < 0.001) were found to be significantly correlated with follicular IL-1beta concentrations. There were also significant positive correlations between follicular nitrate and the number of oocytes retrieved (P < 0.01) and serum oestradiol concentration on the day of human chorionic gonadotrophin (HCG) administration (P < 0.05). When follicular cells were incubated in vitro with 10 ng/ml of IL-1beta for 24 h, nitrate generation was significantly (P < 0.01) elevated compared with the control. In conclusion, our study demonstrates that follicular IL-1beta and the number of developing follicles are significant variables that affect follicular NO concentrations, and points to the possible contribution of IL-1beta to NO generation in human preovulatory follicles.      

Effects of gonadotrophin-releasing hormone agonists on human ovarian steroid secretion in vivo and in vitro-results of a prospective, randomized in-vitro fertilization study

The aim of this prospective randomized study was to compare the effects of two gonadotrophin-releasing hormone (GnRH) agonists, buserelin and triptorelin, on human ovarian follicular steroidogenesis, oocyte fertilization and IVF treatment outcome. Ovulatory, healthy women undergoing IVF were treated either with human menopausal gonadotrophin (HMG) alone or with HMG and one of the two GnRH agonists. Serum and follicular fluid hormonal concentrations and cultures of luteinizing granulosa cells obtained during follicular aspiration were analysed. GnRH agonist treatment significantly affected steroidogenesis both in serum and follicular fluid. In follicular fluid, progesterone and oestradiol concentrations were significantly elevated while testosterone concentrations were significantly lower in the triptorelin group. The ratios of testosterone/progesterone, oestradiol/progesterone but not oestradiol/testosterone concentrations were significantly affected by GnRH agonist administration. Similarly, the steroidogenic activity of luteinizing granulosa cells in vitro was significantly decreased in women treated with GnRH agonists. Women treated with GnRH agonists had significantly more fertilized oocytes and cleaving embryos. The results indicate a marked effect of GnRH agonists on the pattern of ovarian follicular steroidogenesis that cannot be explained solely by changes in gonadotrophin concentrations.