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Background: Follicular aspirates represent a snapshot in time of conditions within the follicle at oocyte retrieval in women undergoing in vitro fertilization and embryo transfer. This clinical material has been much investigated and yet its cellular composition remains unclear. In this study we investigated the origin and profile of leukocytes found within follicular aspirates., Methods: We performed morphological and immunohistochemical analyses of follicular aspirates and peripheral blood obtained concurrently at oocyte retrieval., Results: There was no correlation between erythrocyte and leukocyte numbers in follicular aspirates. The profile of leukocyte subtypes within follicular aspirates was variable and differed significantly from the peripheral circulation in a significant proportion of the analysed samples. A subset of follicular aspirates displayed a marked increase in monocytes/macrophages and an apparent concomitant reduction in polymorphonuclear leukocytes compared with peripheral blood., Conclusions: Leukocytes within follicular aspirates cannot be accounted for solely as a result of blood vessel damage during oocyte retrieval. The variation in leukocyte subtypes observed in some follicular aspirates may reflect a coordinated infiltration of these cells, characteristic of progressive inflammatory responses in other systems. The possibility that leukocyte variation is indicative of follicular maturation deserves further investigation due to its potential relevance in optimizing oocyte selection.

Background: Poor ovarian response limits IVF success but assessing interventions is difficult because of the wide variation in definition. This study attempts to derive objective definitions of poor response., Methods: A retrospective study of a consecutive series of 1190 patients aged <40 years undergoing their first IVF/ICSI cycle was undertaken. Factors adversely affecting implantation, including advanced female age, were excluded. Clinical outcome in cycles reaching oocyte retrieval (n = 1036) were evaluated with respect to gonadotrophin dose used and oocyte number. Cancelled cycles (n = 154) were analysed in relation to the stimulation dose at cancellation and outcome of their subsequent cycle., Results: Cycle cancellation for patients on >/=300 IU FSH/day compared to those on a lower dose was associated with a significantly worse outcome in the subsequent cycle. If <3000 IU FSH/cycle were administered, clinical pregnancy rates remained favourable if <4 eggs were recovered (29 versus 33% for >/=5 eggs). By contrast, if >/=3000 IU FSH was required, the pregnancy rate was 25% if >/=5 eggs were recovered but declined to 7% if <4 were obtained., Conclusions: Definitions of poor response should include the degree of ovarian stimulation used. A low oocyte number is only detrimental if the cumulative dose is >3000 IU FSH. Cancellation at >/=300 IU FSH/day is associated with a significantly worse prognosis and could define poor response.

Background: Insulin-like growth factor-1 (IGF-1) is known to play a role in ovarian follicular development augmenting the action of FSH. Low intrafollicular concentrations have been detected in women who respond poorly to gonadotrophins. This study addresses the relationship between serum IGF-1 levels following pituitary desensitization and ovarian response to gonadotrophin stimulation., Methods: This is a case-control study of 78 patients undergoing IVF-embryo transfer treatment. Thirty-nine strictly-defined poor responder patients requiring 50 or more ampoules (75 IU FSH) to reach oocyte retrieval were compared with 39 age-matched normal responders, requiring fewer than 50 ampoules. IGF-1 concentrations were determined by extraction radioimmunoassay on serum samples obtained after pituitary desensitization but prior to gonadotrophin stimulation., Results: Despite highly significant differences in measures of ovarian response between groups, the mean serum IGF-1 concentration was not statistically significantly different between poor and normal responders [(31.5 nmol/l [95% confidence interval (CI) 28.5-34.5] versus 34.5 nmol/l (95% CI 31.8-37.2)] respectively. No correlation between oocyte number or total gonadotrophin used and serum IGF-1 concentration was observed., Conclusion: Whilst IGF-1 influences ovarian follicular development this study suggests that serum IGF-1 does not predict ovarian response and does not differentiate between critically-defined poor and normal responders.

Background: Conception following gonadotrophin-stimulated IVF and embryo transfer has been associated with a higher intrafollicular cortisol:cortisone ratio and decreased metabolism of cortisol to cortisone. The role of glucocorticoids in human oocyte maturation is not fully understood, but active glucocorticoid (cortisol) may be important. This study relates intrafollicular cortisol and cortisone concentrations to oocyte fertilization and embryo implantation in unstimulated cycles., Methods: Patients aged <40 years with favourable sperm underwent unstimulated IVF-embryo transfer. Study 1 related intrafollicular cortisol levels to oocyte and IVF outcome: (i) fertilized, pregnant (n = 9); (ii) fertilized, not pregnant (n = 21); and (iii) unfertilized (n = 12). Study 2 was a case-control study of 27 patients (same outcome groups of equal size) which measured intrafollicular cortisol, cortisone and the cortisol:cortisone ratio., Results: Conception cycles demonstrated higher cortisol concentrations compared with the fertilized group (study 1) [median (95% confidence interval): 299 (249-330) versus 227 nmol/l (185-261); P < 0.05] and higher cortisol:cortisone ratios when compared with the unfertilized group (study 2) [7.38 (5.23-9.19) versus 3.56 (1.75-7.46) respectively; P = 0.02]. Of the women with cortisol:cortisone ratios greater than the outcome independent mean of 5.90, 58% conceived compared with only 13% with ratios <5.90 (P < 0.02)., Conclusion: Higher cortisol:cortisone ratios in conception cycles suggest that active glucocorticoid may be important for final oocyte maturation and embryo implantation in unstimulated cycles.

Background: The present study addresses the issue of biological ageing of the oocyte (as indicated by basal serum FSH levels) versus chronological ageing., Methods: 1019 infertile but ovulating women were studied in their first cycle of IVF treatment. A series of logistic regression models were developed to assess statistical significance of effects of age and FSH on implantation rates and live babies born., Results: The number of oocytes retrieved and embryos available for transfer declined with increasing age and basal serum FSH concentrations. Fertilizing ability of oocytes increased with advancing age but was not affected by FSH concentrations. Although the number of oocytes or embryos available for transfer had no independent effect on implantation rates, the implanting ability of fertilized oocytes (embryos) was inversely related to increasing age and independently to FSH. The chance of a baby being born, however, was determined more by age than by serum FSH., Conclusions: Ovarian ageing affecting oocyte quality and fecundity can occur independently of chronological age. This has important practical implications whereby serum basal FSH measurement may be a valuable prognostic index, though chronological age remains important.

Background: Cancellation of assisted conception cycles because of poor ovarian response to gonadotrophins is a significant problem in assisted reproduction. Various adjuvant treatments have been suggested to improve responsiveness. This study reports on the potential benefits of low dose dexamethasone., Methods: Patients <40 years of age were invited to participate in a twin centre prospective double blind randomized placebo controlled study. A total of 290 patients were recruited and computer randomized using sealed envelopes to receive either 1 mg dexamethasone (n = 145) or placebo tablets (n = 145) in addition to a standard long protocol gonadotrophin-releasing hormone analogue with gonadotrophin stimulation regime., Results: A significantly lower cancellation rate for poor ovarian response was observed in the dexamethasone group compared with controls (2.8 versus 12.4% respectively, P < 0.002). Further comparisons between the dexamethasone group and controls were made of median fertilization rates (60 versus 61% respectively, NS), implantation rates (16.3 versus 11.6% respectively, NS) and pregnancy rate per cycle started (26.9 versus 17.2%, NS). The benefit was apparent in patients both with polycystic and normal ovaries., Conclusion: Low dose dexamethasone co-treatment reduces the incidence of poor ovarian response. It may increase clinical pregnancy rates and should be considered for inclusion in stimulation regimes to optimize ovarian response.

There is growing evidence that the pathogenic effects of bacterial vaginosis may not be confined to the lower genital tract. Possible associations with infertility and effects on fertilization and implantation were studied in patients undergoing in-vitro fertilization (IVF) treatment. High vaginal swabs taken at the time of oocyte collection were assessed by Gram staining. The prevalence of bacterial vaginosis and of intermediate and normal flora in 301 patients was 25.6, 14.0 and 60.4% respectively. Bacterial vaginosis was more prevalent in patients with tubal (31.5%, n = 149) compared with non-tubal (19.7%, n = 152) infertility (odds ratio (OR) 1.87, CI 1.11-3.18, P = 0.02). Bacterial vaginosis did not have an adverse effect on fertilization rate. Further, no significant difference in implantation rates was seen when comparing bacterial vaginosis (15. 8%, OR 1.03, CI 0.66-1.61) and intermediate flora (13.1%, OR 0.82, CI 0.45-1.52) with normal flora (15.5%). Though confidence intervals around the observations were relatively wide, the findings suggest that routine screening for bacterial vaginosis in the hope of improving the success of IVF treatment is not justified. The prevention of complications in pregnancy associated with bacterial vaginosis might be a more relevant indication for screening at the time of IVF treatment, in particular patients with tubal disease, if treatment were shown to be effective for that particular purpose. However, antibiotic treatment before IVF has been shown to be positively disadvantageous for IVF by encouraging other organisms.

This review examines recent evidence suggesting a role for the immune system, in particular cytokines, in the pathogenesis of ovarian hyperstimulation syndrome (OHSS). Ovarian tissue is known to contain cells capable of producing a range of immunological mediators and the concentrations of these have been shown to be elevated in serum and ascitic fluid from women with established OHSS. Available evidence points to a role for vascular endothelial growth factor and interleukin-2, possibly acting through other intermediary cytokines, in the pathogenesis of OHSS. However, each individual has a unique cytokine profile and several cytokines may share biological actions, making it difficult to interpret data on isolated cytokine concentrations from relatively small numbers of patients. Improved understanding of the role of the immune system in the development of OHSS may have implications for the prediction, prevention and management of this iatrogenic condition.

We questioned the policy of routine microbiological culture of semen prior to in-vitro fertilization (IVF) with a view to prescribing antibiotics to reduce the risk of introducing seminal infection into the embryo culture system. An initial retrospective study examined serum microbiology reports of 449 couples undergoing IVF or gamete intra-Fallopian transfer (GIFT). In semen samples taking >/=1 days to reach the microbiology laboratory compared with same-day delivery there was increased frequency of significant culture of enterococci (27 versus 15%, P < 0.01). In samples taking >/=2 days there was increased frequency of significant culture of Gram-negative bacilli (31 versus 12%, P < 0.01) and of overall culture of other potentially pathogenic organisms (26 versus 14%, P < 0.01). We questioned diagnostic accuracy and relevance. Therefore, in a prospective study, semen and high vaginal swabs obtained on the day of oocyte collection were cultured from 100 couples having IVF or GIFT, of whom 52 male partners had been treated with antibiotics following positive pre-IVF semen culture. The presence of bacteria in semen samples used only for IVF (n = 90) did not reduce fertilization rates nor lead to infection of the embryo culture system. However, there was an increased incidence of significant culture of vaginal Gram-negative bacilli in patients with treated partners compared with untreated partners [15/52 (29%) versus 5/48 (10%), P < 0.05]. Thus antibiotic therapy in the male partner may increase the likelihood of inoculation of antibiotic-resistant pathogenic bacteria from the vagina into the embryo culture system during vaginal oocyte collection. In asymptomatic patients, microbiological screening of semen samples prior to IVF treatment and subsequent treatment with antibiotic therapy in those with positive cultures appears to be unnecessary and may be detrimental to IVF outcome.

This study investigated the development of functional ovarian cysts during pituitary down-regulation prior to in-vitro fertilization (IVF), and identified 16 cases of cysts in 288 IVF cycles studied. Comparing the patients with functional ovarian cysts to the other 272 IVF cycles, there was no significant difference in age or incidence of endometriosis but significantly (P < 0.01) more patients with cysts had ovulatory dysfunction. The serum progesterone was < 5.7 nmol/l in all 16 patients with cysts on day 4 of the IVF cycle, and in eight of these patients the serum progesterone was < 5.7 nmol/l on the day buserelin was commenced. In 10 of the 16 patients with cysts, serum oestradiol concentrations remained elevated despite the prolonged use of buserelin, and the cysts were aspirated. The aspirate in all cases was clear without any suggestion of endometriosis. The cyst aspirates had significantly lower progesterone (P < 0.001), higher androstenedione (P < 0.01) and similar oestradiol concentrations to 10 follicular fluid samples collected at the time of oocyte retrieval. This study suggests that functional ovarian cysts may develop during pituitary down-regulation, and these cysts are follicular cysts rather than persistent corpora lutea or endometriomata.

Reviewing 780 in-vitro fertilization (IVF) cycles, where buserelin was commenced in the preceding luteal phase and human menopausal gonadotrophin on day 4 of the ensuing menses, 53 cycles were identified with sonolucent cysts (30-50 mm diameter). Of the latter 53 cycles, the serum oestradiol was significantly greater on day 4 in 22 cycles abandoned for poor follicular development than in 31 cycles which proceeded to oocyte retrieval (P less than 0.05). Of the 31 cycles proceeding to oocyte retrieval, nine had a day 4 serum oestradiol greater than 200 pmol/l (95th centile for day 4 oestradiol in patients without apparent cysts), and these cycles produced significantly fewer grade 1 embryos than the cycles with day 4 oestradiol levels less than or equal to 200 pmol/l (P less than 0.05). Six of the 53 cycles with cysts resulted in conception, and all of these cycles had a day 4 serum oestradiol less than 200 pmol/l. Among the 53 cycles with ovarian cysts, the serum progesterone on the day of abandonment in four cycles and on the day of human chorionic gonadotrophin administration in one non-abandoned cycle, was above the range established for 104 cycles without cysts. No significant difference was seen in day 4 serum androstenedione levels, and the day 4 serum progesterone was less than 5 nmol/l in all but one patient. Functional activity of ovarian cysts is associated with an adverse influence on IVF cycles.

Biochemical monitoring was undertaken in 22 treatment cycles for women with normal ovarian function who underwent pituitary suppression with buserelin and administration of exogenous oestradiol (E2) and progesterone (P) for cryopreserved embryo transfer (ET). Eighteen transfers of 1-4 thawed embryos, on the third day of exposure to progesterone, resulted in five clinical pregnancies (27.8%) and one biochemical pregnancy. There was no difference between pregnant and non-pregnant patients in the number and quality of embryos transferred, age, weight or infertility diagnosis. Serum E2 level from days 10-17 (the late proliferative phase) of the therapy cycle were significantly higher in the pregnant group compared with the non-pregnant group (P less than 0.05--P less than 0.005). There were no significant differences in P levels between the two groups from the onset of progesterone administration to the end of the cycle. However, as might be expected, the mean E2/P molar ratio in the pregnant group was significantly higher at the time of ET (P less than 0.02). It is concluded that biochemical monitoring during the embryo replacement cycle is necessary to tailor drug dosages for individual requirements to achieve adequate E2 levels before ET. Alternative routes of oestradiol administration need to be considered in patients with poor E2 profiles.

This study identifies a group of 87 patients, who demonstrated a 'poor' response to a standard buserelin/human menopausal gonadotrophin (HMG) regime. The subsequent outcome in 61 of these 'poor' responders when treated with a higher dose of HMG to achieve a satisfactory response was compared with 250 patients, who showed a 'good' response to the standard regime. 'Poor' responders were significantly older than 'good' responders (P less than 0.001), but no significant difference was demonstrated in the indication for in-vitro fertilization (IVF). Even on higher doses of HMG, 'poor' responders took longer for their follicles to achieve maturity than the 'good' responders (P less than 0.01). 'Poor' responders produced 8.9 oocytes per oocyte collection compared to 11.8 in the 'good' responders (P less than 0.01). The fertilization rate was significantly lower in the 'poor' responders compared to the 'good' responders (P less than 0.01). Although there was no significant difference in morphometric grading between 'poor' responder embryos and 'good' responder embryos, the rate of cell division was significantly slower in embryos of the 'poor' responders than the 'good' responders (P less than 0.01). The pregnancy rate per oocyte retrieval was 9% in the 'poor' responders compared to 29% in the 'good' responders (P less than 0.01). The implantation rate in the 'poor' responders was 4.4% compared to 16.1% in the 'good' responders (P less than 0.001).

The circulating levels of placental protein 14 (PP14) and progesterone were measured in three pregnancies resulting from the transfer of cryopreserved embryos. Two of these women had suppressed ovarian activity as a result of pituitary down-regulation with the luteinizing hormone-releasing hormone agonist (buserelin) prior to treatment with exogenous oestradiol and progesterone. After 14 days of oral oestradiol treatment and if the endometrial thickness was greater than 7 mm, progesterone was given intramuscularly for a further 14 days with embryo transfer on the third day of this treatment. On confirmation of pregnancy by human chorionic gonadotrophin analysis, progesterone administration was altered to transvaginal pessaries for maintenance of adequate progesterone levels and endometrial support. In the two women with ovarian suppression, PP14 levels remained below the 2.5th centile of the normal range for pregnancy. In the third pregnancy, embryo transfer was performed 3 days after a spontaneous luteinizing hormone surge in a normal menstrual cycle. In this pregnancy, PP14 levels were within the normal range. Ultrasonic examination confirmed three normal ongoing singleton pregnancies. These results suggest that the majority of PP14 production in normal pregnancy is under ovarian or anterior pituitary control and that the influence of progesterone is of a secondary nature.