Empirical antibiotic therapy for pneumonia in intensive care units: a multicentre, retrospective analysis of potentially pathogenic microorganisms identified by endotracheal aspirates cultures.

The purpose of this investigation was to explore the presumed relationship between the days of hospitalisation and microorganisms identified by endotracheal aspirate cultures in relation to adequate empirical treatment strategies of pneumonia in the intensive care unit (ICU). All potentially pathogenic microorganisms identified by (surveillance) cultures of endotracheal aspirates obtained in the ICUs of two Dutch teaching hospitals in 2007 and 2012 were retrospectively collected and analysed. Antibiotic susceptibilities to 11 antibiotics were calculated for several time points (days or weeks) after hospital admission and expressed per patient-day. In total, 4184 potentially pathogenic microorganisms identified in 782 patients were analysed. Prevalence of the classic early-onset pneumonia-causing microorganisms decreased from 55 % on the first four days to 34 % on days 4-6 after hospital admission (p < 0.0001). Susceptibility to amoxicillin/clavulanic acid was below 70 % on all days. Except for days 0 and 12, susceptibility to ceftriaxone was below 80 %. The overall susceptibility to piperacillin/tazobactam was 1518/1973 (77 %) in 2007 vs. 727/1008 (67 %) in 2012 (p < 0.0001). After day 8 of hospital admission, susceptibility to piperacillin/tazobactam therapy was below 80 % in 2012. After one week of hospital admission, susceptibilities to antibiotics were lower in the hospital that included that antibiotic in the local empirical treatment protocols as compared to the hospitals in which that antibiotic was not or infrequently included: 90/434 (21 %) vs. 117/398 (29 %); p = 0.004 for amoxicillin/clavulanic acid and 203/433 (47 %) vs. 253/398 (64 %); p < 0.001 for ceftriaxone. No cut-off in the number of days after hospital admission could be identified to distinguish early-onset from late-onset pneumonia. Consequently, the choice of empirical antibiotics should probably not be based on the time of onset.

Risk factors for mortality in patients with pulmonary infections with non-tuberculous mycobacteria: a retrospective cohort study.

BACKGROUND: Infections with non-tuberculous mycobacteria (NTM) represent an increasing problem. Their clinical relevance is still largely unknown as well as predictors for mortality in affected patients. The objective was to describe prevalence and clinical relevance of different NTM and to identify risk factors for mortality. METHODS: Retrospective cohort study of 124 patients with NTM detection between January 2001 and December 2011. Clinical characteristics like symptoms and radiological appearance were assessed at presentation. The primary outcome was all cause mortality during the follow-up period. Univariate and multivariate survival analyses using Cox proportional hazard models were employed for statistical analysis. RESULTS: Over the study period, the frequency of NTM isolation varied from 4 to 12 patients per year. Twenty-nine out of 124 patients (23%) had a clinically relevant infection, according to the criteria of the American Thoracic Society (ATS). Mycobacterium avium was isolated most frequently, but Mycobacterium kansasii, Mycobacterium malmoense and Mycobacterium xenopi had the highest clinical relevance. Symptoms were mostly diverse and non-specific. On radiology, cavities were observed more frequently than a nodular-bronchiectatic variant or consolidation. In 75% of all patients, follow up time was more than two years. Median survival was 6.5 years (95%CI = 2.7-10.3). Factors significantly influencing survival time were haemoptysis (HR = 0.2, 95%CI = 0.1-0.6) and a consolidation on imaging (HR = 5.1, 95%CI 1.4-18.2). CONCLUSIONS: The presentation of an infection with NTM can be diverse and depends mainly on the causative NTM pathogen. The most important predictor for increased mortality is the radiological appearance of a consolidation.

A clinical prediction model to assess the risk of operative delivery.

OBJECTIVE: To predict instrumental vaginal delivery or caesarean section for suspected fetal distress or failure to progress. DESIGN: Secondary analysis of a randomised trial. SETTING: Three academic and six non-academic teaching hospitals in the Netherlands. POPULATION: 5667 labouring women with a singleton term pregnancy in cephalic presentation. METHODS: We developed multinomial prediction models to assess the risk of operative delivery using both antepartum (model 1) and antepartum plus intrapartum characteristics (model 2). The models were validated by bootstrapping techniques and adjusted for overfitting. Predictive performance was assessed by calibration and discrimination (area under the receiver operating characteristic), and easy-to-use nomograms were developed. MAIN OUTCOME MEASURES: Incidence of instrumental vaginal delivery or caesarean section for fetal distress or failure to progress with respect to a spontaneous vaginal delivery (reference). RESULTS: 375 (6.6%) and 212 (3.6%) women had an instrumental vaginal delivery or caesarean section due to fetal distress, and 433 (7.6%) and 571 (10.1%) due to failure to progress, respectively. Predictors were age, parity, previous caesarean section, diabetes, gestational age, gender, estimated birthweight (model 1) and induction of labour, oxytocin augmentation, intrapartum fever, prolonged rupture of membranes, meconium stained amniotic fluid, epidural anaesthesia, and use of ST-analysis (model 2). Both models showed excellent calibration and the receiver operating characteristics areas were 0.70-0.78 and 0.73-0.81, respectively. CONCLUSION: In Dutch women with a singleton term pregnancy in cephalic presentation, antepartum and intrapartum characteristics can assist in the prediction of the need for an instrumental vaginal delivery or caesarean section for fetal distress or failure to progress.

Long-term outcomes in women with polycystic ovary syndrome initially randomized to receive laparoscopic electrocautery of the ovaries or ovulation induction with gonadotrophins.

BACKGROUND: Long-term effects of laparoscopic electrocautery of the ovaries are unknown. To study the long-term effects of laparoscopic electrocautery of the ovaries and gonadotrophins, we followed women with clomiphene-resistant polycystic ovary syndrome (PCOS) randomly allocated to one of these treatments until 8-12 years after their initial treatment. METHODS: Between February 1998 and October 2001 168 women with clomiphene citrate-resistant PCOS were included in a randomized controlled trial comparing an electrocautery strategy to a strategy starting with rFSH. In 2009 these women were contacted about their reproductive outcome and menstrual cycle regularity. Analysis was by intention-to-treat. We compared time to conception resulting in live birth, subsequent pregnancies, ectopic and multiple pregnancies, menopause, as well as minimal and maximal menstrual cycle length. RESULTS: After 8-12 years, the cumulative proportion of women with a first child was 86% in women who had been allocated to electrocautery versus 81% in women who had been allocated to immediate rFSH [relative ratio (RR): 1.1; 95% confidence interval (CI): 0.92-1.2]. Treatment with electrocautery resulted in a significantly lower need for stimulated cycles to reach a live birth; 53% after electrocautery versus 76% after rFSH (RR: 0.69; 95% CI: 0.55-0.88).The cumulative proportion of women with a second child was 61% after electrocautery versus 46% after immediate rFSH (RR: 1.4; 95% CI: 1.00-1.9). Overall, there were 7 twins out of 134 deliveries (5%) after electrocautery versus 10 twins out of 124 deliveries (8%) in the rFSH group (RR: 0.65; 95% CI: 0.25-1.6). Fifty-four per cent of the women allocated to electrocautery had a regular menstrual cycle 8-12 years after randomization versus 36% in those allocated to rFSH (RR: 1.5; 95% CI: 0.87-2.6). CONCLUSION: In women with clomiphene-resistant PCOS, laparoscopic electrocautery of the ovaries is as effective as ovulation induction with FSH treatment in terms of live births, but reduces the need for ovulation induction or ART in a significantly higher proportion of women and increases the chance for a second child. Clinicians may use these data when informing clomiphene-resistant anovulatory women about treatment options.

The value of medical history taking as risk indicator for tuboperitoneal pathology: a systematic review.

BACKGROUND: Guidelines recommend diagnostic laparoscopy in subfertile women with known co-morbidities in their medical history. Aggregated evidence underpinning these recommendations is, however, currently lacking. OBJECTIVE: The objective of this study was to perform a systematic review and meta-analysis of the available evidence on the association between items reported during medical history taking and tuboperitoneal pathology. SEARCH STRATEGY: MEDLINE (from 1966 to May 2007), EMBASE (from 1960 to January 2007) and bibliographies of retrieved primary articles. SELECTION CRITERIA: All relevant studies that compared medical history with the presence or absence of tubal pathology. DATA COLLECTION AND ANALYSIS: Studies comparing medical history with the presence or absence of tubal pathology were included. A diagnosis of tubal pathology had to be made by hysterosalpingography, laparoscopy or a combination of both. In the absence of invasive tubal testing, tuboperitoneal pathology was considered to be absent in case of intrauterine pregnancy. Homogeneity between studies was assessed, and the association between medical history and tubal pathology was expressed as a common odds ratio with a 95% CI. No language restriction was applied. MAIN RESULTS: We included 32 studies. In cohort studies, strong associations were found for a history of complicated appendicitis (OR 7.2, 95% CI 2.2-22.8), pelvic surgery (OR 3.6, 95% CI 1.4-9.0) and pelvic inflammatory disease (PID) (OR 3.2, 95% CI 1.6-6.6), and in case-control studies, for a history of complicated appendicitis (OR 3.3, 95% CI 1.8-6.3), PID (OR 5.5, 95% CI 2.7-11.0), ectopic pregnancy (OR 16.0, 95% CI 12.5-20.4), endometriosis (OR 5.9, 95% CI 3.2-10.8) and sexually transmitted disease (OR 11.9, 95% CI 4.3-33.3). AUTHOR'S CONCLUSIONS: Subfertile women reporting a history of PID, complicated appendicitis, pelvic surgery, ectopic pregnancy and endometriosis are at increased risk of having tuboperitoneal pathology. In these women, diagnostic laparoscopy should be offered early in the fertility work-up.

Genetic diversity of methicillin-resistant Staphylococcus aureus in a tertiary hospital in The Netherlands between 2002 and 2006.

The aim of this study was to investigate the methicillin-resistant Staphylococcus aureus (MRSA) clones isolated in a Dutch university hospital, situated near the borders of Belgium and Germany, between 2002 and 2006. MRSA strains (n = 175) were characterized using spa and SCCmec typing. The presence of Panton Valentine leukocidin (PVL) was determined. Between 2002 and 2005, ST5-MRSA-IV was predominant, and the spa type of ST5-MRSA-IV changed from t002 to t447. ST5-MRSA-I, ST5-MRSA-II, ST228-MRSA-I, and ST247-MRSA-I were also observed in this period. From 2004, the MRSA genetic background became more diverse, and in 2006, ST5-MRSA-IV was only sporadically observed. From 2005, ST5-MRSA-II, ST8-MRSA-IV, ST22-MRSA-IV, and ST45-MRSA-IV were increasingly observed. Several other MRSA clones, such as ST239-MRSA-III, were found sporadically. Four PVL-positive MRSA isolates were observed, associated with ST80-MRSA-IV and ST8-MRSA-IV. ST5-MRSA-I, ST5-MRSA-II, ST5-MRSA-IV, and ST228-MRSA-I have not been described previously in The Netherlands.

Removal of cervical mucus: effect on pregnancy rates in IVF/ICSI.

Cervical mucus may cover the embryo transfer catheter during passage of the cervical canal, interfering with the correct placement of the embryo(s) into the uterine cavity. The effect of removal of cervical mucus prior to embryo transfer in IVF/ intracytoplasmic sperm injection (ICSI) on live birth rate was studied. The study was set up as a single blind randomized controlled trial. Couples undergoing IVF/ICSI were randomly allocated to either removal of cervical mucus prior to embryo transfer, or a mock procedure. Randomization was done with stratification for age, cycle number and method of treatment. Primary outcome was live birth rate. A total of 317 couples were included and underwent 428 cycles, of which the outcome of 3 cycles was unknown. Baseline characteristics of both groups were comparable. Live birth occurred in 52 of 220 (24%) cycles in the treatment group and 42 of 205 (21%) cycles in the control group (risk difference 3%, 95% confidence interval-5- 11%). It is unlikely that removal of cervical mucus prior to embryo transfer has a significant effect on live birth rate. A small effect, however, cannot be excluded.

Routine Chlamydia antibody testing is of limited use in subfertile women with anovulation.

The Chlamydia antibody titre (CAT) is a test used to identify subfertile couples at increased risk for tubal pathology. The usefulness of the routine performance of CAT was evaluated in a multicentre prospective cohort study, in women without regular ovulation. Consecutive couples presenting with subfertility due to an irregular menstrual cycle or amenorrhoea were included. A total of 711 women were studied, all of whom underwent CAT. Tubal status was verified in 190 of these women. Two-sided tubal pathology was found in 5% of these women, and one-sided occlusion in 10%. Of all the women in the study group, 33 (4.6%) had an abnormal CAT, of which 21 underwent further tubal testing. Tubal pathology was found in two (10%) of these 21 patients. The sensitivity and specificity of CAT were respectively 20% and 89%. Correction for verification bias increased the specificity to 96% with a drop of the sensitivity to 9%. In subfertile couples with anovulation, the performance of CAT is not useful. It is proposed that testing for tubal disease in these women is delayed until treatment with clomiphene citrate has failed.

Pregnancy is predictable: a large-scale prospective external validation of the prediction of spontaneous pregnancy in subfertile couples.

BACKGROUND: Prediction models for spontaneous pregnancy may be useful tools to select subfertile couples that have good fertility prospects and should therefore be counselled for expectant management. We assessed the accuracy of a recently published prediction model for spontaneous pregnancy in a large prospective validation study. METHODS: In 38 centres, we studied a consecutive cohort of subfertile couples, referred for an infertility work-up. Patients had a regular menstrual cycle, patent tubes and a total motile sperm count (TMC) >3 x 10(6). After the infertility work-up had been completed, we used a prediction model to calculate the chance of a spontaneous ongoing pregnancy (www.freya.nl/probability.php). The primary end-point was time until the occurrence of a spontaneous ongoing pregnancy within 1 year. The performance of the pregnancy prediction model was assessed with calibration, which is the comparison of predicted and observed ongoing pregnancy rates for groups of patients and discrimination. RESULTS: We included 3021 couples of whom 543 (18%) had a spontaneous ongoing pregnancy, 57 (2%) a non-successful pregnancy, 1316 (44%) started treatment, 825 (27%) neither started treatment nor became pregnant and 280 (9%) were lost to follow-up. Calibration of the prediction model was almost perfect. In the 977 couples (32%) with a calculated probability between 30 and 40%, the observed cumulative pregnancy rate at 12 months was 30%, and in 611 couples (20%) with a probability of >or=40%, this was 46%. The discriminative capacity was similar to the one in which the model was developed (c-statistic 0.59). CONCLUSIONS: As the chance of a spontaneous ongoing pregnancy among subfertile couples can be accurately calculated, this prediction model can be used as an essential tool for clinical decision-making and in counselling patients. The use of the prediction model may help to prevent unnecessary treatment.

Outbreak of a susceptible strain of Acinetobacter species 13 (sensu Tjernberg and Ursing) in an adult neurosurgical intensive care unit.

Between December 1999 and June 2000, an outbreak caused by Acinetobacter emerged on the neurosurgical intensive care unit of our hospital. It was shown using automated ribotyping using Eco RI and pulsed-field gel electrophoresis that the outbreak was caused by spread of a single strain, which was identified by ribotyping and amplified ribosomal DNA restriction analysis as Acinetobacter DNA group 13TU (sensu Tjernberg and Ursing). The outbreak strain, which showed no antibiotic resistance, was identified in 23 patients, five of whom developed an infection. The organism was also isolated from various environmental sites. Cross-transmission among patients continued despite contact isolation of colonized patients and reinforcement of basic disinfection procedures. Eventually, after implementation of additional stringent measures such as cohorting of positive patients and daily disinfection of the floor, the outbreak was brought under control. This study demonstrates that apart from Acinetobacter baumanii, Acinetobacter 13TU strains, even when they are fully susceptible, may cause outbreaks that are difficult to control. Correct identification to the species level of Acinetobacter by genotypic methods is necessary to get insight in the importance of the different Acinetobacter genomic species in hospital epidemiology.

Elevated serum levels of free insulin-like growth factor I in polycystic ovary syndrome.

Polycystic ovary syndrome (PCOS) is the most common cause of anovulation in women. Previous studies suggest that the pathogenesis of PCOS may involve interrelated abnormalities of the insulin-like growth factor (IGF) and ovarian steroidogenesis systems. We investigated this hypothesis in fasting serum samples from 140 women with PCOS (age, 27.4 +/- 0.4 yr; body mass index, 26.3 +/- 0.5 kg/m2; mean +/- SEM). IGF-related parameters were also studied in a group of normoovulatory women (n = 26; age, 26 +/- 4 yr; body mass index, 23.6 +/- 4.3 kg/m2). For the PCOS group, the mean testosterone (T) level was 2.5 +/- 0.1 nmol/L, and it was significantly correlated with LH (r = 0.41; P < 10(-6)), estrone (r = 0.33; P = 0.016), estradiol (r = 0.18; P = 0.04), and androstenedione (AD; P < 10(-6)), but not with dehydroepiandrosterone sulfate (P = 0.71), a marker of adrenal steroidogenesis. T and AD were also related to total ovarian follicle number and ovarian size, as previously found with normoovulatory women (1). There were no differences between the PCOS subjects and the normoovulatory group for total IGF-I, IGF-II, or IGF-binding protein-3 (IGFBP-3). However, IGFBP-1 levels were significantly decreased in the PCOS group (1.0 +/- 0.2 vs. 7.3 +/- 1.1 ng/mL; P < 0.001) and were inversely correlated with serum insulin levels (r = -0.50; P < 10(-8)). Serum levels of free IGF-I (fIGF-I) were elevated (5.9 +/- 0.3 vs. 2.7 +/- 0.3 ng/mL; P < 0.001) in inverse relation with IGFBP-1 (r = -0.31; P = 0.046). Serum fIGF-I levels were related to total follicle number (r = - 0.35; P < 10(-4)) and to the ratio of sex hormone-binding globulin to T (r = -0.23; P = 0.009). However, these relationships were not independent of other variables. Despite the more than 2-fold elevation in fIGF-I levels, significant relationships between fIGF-I and markers of ovarian steroidogenesis (T, AD, estradiol, and estrone) could not be demonstrated. In conclusion, although we confirmed correlations between LH and hyperandrogenemia and have found abnormalities in the IGF system in a large cohort of PCOS subjects, a direct relationship between hyperandrogenism and the IGF system could not be shown. Previous studies suggest that elevated LH and hyperinsulinemia lead to excess ovarian androgen synthesis in PCOS and that the intraovarian IGF system is important for normal follicle development and may be important in the arrested state of follicle development in PCOS. However, the data presented in this cross-sectional study suggest that insulin-related changes in circulating IGFBP-1 and subsequent elevation of fIGF-I reflect insulin resistance and have little enhancing effects on ovarian steroidogenesis in this disorder.

[Extrauterine pregnancy in Netherlands: patient characteristics, treatment, and pregnancy prognosis]. / Extra-uteriene graviditeit in Nederland: patiëntkenmerken, behandeling en zwangerschapsprognose.

OBJECTIVE: To determine the probability of pregnancy after a finished extrauterine pregnancy (EUP) and the length of time in between. DESIGN: Prospective multicentric cohort study. METHOD: Of all patients with an EUP between May 1990 and October 1993, data were collected using a questionnaire from surgeons in five university hospitals and 30 general training and non-training hospitals. During the subsequent 3 years, the patients semi-annually reported on their pregnancy or wish to become pregnant using reply cards. RESULTS: A total of 665 patients with an EUP were reported their mean age was 30.7 years (SD: 4.9). There were 341 patients who during the follow-up desired pregnancy, did not start an IVF procedure and supplied complete follow-up data 207 of them (61%) became pregnant after a median interval of 12 months. Age above 35, previous fertility problems, a Chlamydia antibody titre > or = 1:64 and adnexitis in the anamnesis were correlated with a longer interval until a subsequent pregnancy. The nature of the treatment (laparotomy versus laparoscopy, conservative versus radical and surgical versus pharmaceutical) did not affect the duration of the interval. If the contralateral tube was judged to be abnormal by the operator, pregnancy was still possible, but the occurrence of the pregnancy was delayed. CONCLUSION: The probability of pregnancy after an earlier EUP averages 61%; the interval until the next pregnancy, if any, depends mostly on factors that cannot be influenced at the time of the diagnosis of EUP.

Insulin-like growth factors and insulin-like growth factor binding proteins in androgen-dominant ovarian follicles from testosterone-treated female-to-male trans-sexuals.

OBJECTIVE: To determine insulin-like growth factor (IGF)-I and IGF-II levels, IGF binding protein (IGFBP) profile, and IGFBP-4 protease activity in androgen-dominant follicular fluid (FF) from female-to-male trans-sexuals and to compare with those in follicles from normocycling women. DESIGN: Follicular fluid samples were obtained from four female-to-male trans-sexuals and 15 women with normo-ovulatory cycles at the Dijkzigt Academic Hospital. Western ligand blot analysis and protease assays were used to determine IGFBP profile, and immunoradiometric assays were used to detect IGF levels. SETTING: The study was performed in two academic medical centers. PATIENT(S): Female-to-male trans-sexuals and women with normo-ovulatory cycles. INTERVENTIONS: None. MAIN OUTCOME MEASURE(S): Determination of IGF levels and IGFBP profile. RESULT(S): Insulin-like growth factor I levels in FF from female-to-male trans-sexuals were not significantly different compared with levels in androgen-dominant FF and estrogen-dominant FF. Significantly lower levels of IGF-II were observed in FF from female-to-male trans-sexuals than in estrogen-dominant FF, whereas IGF-II levels in FF from female-to-male trans-sexuals were not significantly different than those in androgen-dominant FF. Similar IGFBP profiles from FF from female-to-male trans-sexuals and androgen-dominant FF were noted, with markedly elevated levels of the 31- and 24-kd IGFBPs and a 28-kd IGFBP, compared with estrogen-dominant FF. An IGFBP-4-specific metalloserine protease that is active in estrogen-dominant FF likewise was undetected in FF from female-to-male trans-sexuals. CONCLUSION(S): Follicles developing under the influence of exogenous androgens in ovaries in female-to-male trans-sexuals appear to be similar to androgen-dominant follicles in normo-ovulatory women with regard to IGF-I and IGF-II levels, IGFBP profile, and the absence of IGFBP-4 protease activity.

Successful induction of ovulation in normogonadotrophic clomiphene resistant anovulatory women by combined naltrexone and clomiphene citrate treatment.

Patients suffering from normogonadotrophic anovulation and infertility are initially treated with clomiphene citrate. Those who do not respond to clomiphene citrate usually receive gonadotrophin treatment which is labour-intensive, expensive, and associated with an increased risk of multiple pregnancies and ovarian hyperstimulation syndrome. We treated 22 patients with clomiphene resistant normogonadotrophic anovulation with naltrexone (an opioid receptor blocker) alone or naltrexone in combination with an antioestrogen. In 19 patients ovulation and resumption of a regular menstrual cycle was achieved and in 12 out of 19 a singleton pregnancy was observed. In conclusion, ovulation can be induced successfully using naltrexone alone or naltrexone in combination with an anti-oestrogen in clomiphene citrate resistant anovulatory patients. Compared to gonadotrophin induction of ovulation, this method is safe, simple and inexpensive.

Serum and follicular fluid levels of insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding protein-1 and -3 during the normal menstrual cycle.

Insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) have important regulatory functions in ovarian follicular development. Although most studies have investigated the IGF system in ovarian cells in vitro, investigation of the IGF system in the peripheral circulation and in follicles of varying sizes throughout the menstrual cycle in large numbers of subjects has been lacking. In the current study we performed daily IGF-I, IGF-II, IGFBP-1, and IGFBP-3 measurements in 9 healthy regularly cycling volunteers throughout the menstrual cycle. In addition, we investigated IGF-I, IGF-II, IGFBP-1, and IGFBP-3 levels in 13 samples of androgen-dominant follicular fluid [FFa androstenedione to estradiol (AD:E2) ratio, > 4] and 19 samples of estrogen-dominant follicular fluid (FFe; AD:E2 ratio, 4) obtained from 21 regularly cycling subjects and in 18 samples of fluid from luteinizing follicles obtained from patients undergoing in vitro fertilization (IVF) treatment (FFivf). IGF-I, IGF-II, IGFBP-1, and IGFBP-3 were measured using two-site immunoradiometric assays. No significant day to day differences were observed in IGF-I, IGF-II, IGFBP-1, and IGFBP-3 levels across the menstrual cycle. Median IGF-II levels in FFe (630 ng/mL; range, 212-1000) were significantly higher compared to those in FFa (474 ng/mL; range, 272-603; P = 0.002). Median IGFBP-3 levels in FFe (2955 ng/mL; range, 388-3448) were also significantly higher than those in FFa (2352 ng/mL; range, 756-2604; P = 0.003). Median IGF-I (192 ng/mL; range, 29-256) and IGFBP-1 (12 ng/mL; range, 2-281) levels in FFe were not significantly different from those in FFa [149 (range, 22-232) and 21 (range, 5-32) ng/mL, respectively). In contrast, significantly lower IGFBP-1 levels were found in FFe compared to FFivf (79 ng/mL; range, 57-234; P = 0.002), whereas there was no significant difference between FFe and FFivfe IGF-I, IGF-II, or IGFBP-3 levels, respectively. IGF-II levels were correlated with follicle diameter (r = 0.52; P = 0.002), cycle day (r = 0.47; P = 0.0065), E2 levels (r = 0.53; P = 0.003), AD:E2 ratio (r = -0.58; P = 0.001), and P concentrations (r = 0.60; P = 0.001) in all follicles, whereas no such correlations were found with IGF-I. In conclusion, as circulating levels of IGF-I, IGF-II, IGFBP-1, and IGFBP-3 are not menstrual cycle dependent, it is unlikely that these growth factors and these binding proteins play an endocrine role in cyclic ovarian follicle development, although both cycle-dependent delivery to the ovary and modification of their actions locally within the ovary cannot be excluded. With regard to FF1 the findings that IGF-II levels in FF1 are elevated compared to those in FFa and correlate with follicular functional status support a role for IGF-II during development of the dominant follicle. In addition, as IGFBP-3 in estrogen-dominant follicles mirrors the rise of IGF-II, this IGFBP may be a primary regulator of IGF-II action within the estrogen-dominant follicle. Finally, the finding of elevated levels of IGFBP-1 in luteinizing (IVF) follicles suggests an important role for this peptide in corpus luteum regulation.

Circulating immunoreactive and bioactive follicle stimulating hormone concentrations in anovulatory infertile women and during gonadotrophin induction of ovulation using a decremental dose regimen.

Our purpose was to determine whether decreased follicle stimulating hormone (FSH) activity, either systemic or at the follicular level, is involved in impaired follicle growth associated with normogonadotrophic anovulation. To differentiate between the possible levels of disturbance, bioactive (BIO-FSH; using the in-vitro rat granulosa cell aromatase bioassay) and immunoreactive (IRMA-FSH) FSH serum concentrations of three groups of subjects were compared: (i) 172 normogonadotrophic anovulatory infertile women during baseline conditions, (ii) 22 clomiphene-resistant polycystic ovary syndrome patients undergoing ovulation induction by exogenous gonadotrophins using a decremental dose regimen, and (iii) nine regularly cycling controls. BIO-FSH [13.2 (range 0.8-29.5) IU/l] and IRMA-FSH [4.4 (range 1.2-9.3) IU/l] concentrations in anovulatory women during baseline conditions were significantly lower than maximum concentrations reached during the follicular phase in controls [18.7 (13.2-23.4) and 6.4 (5.7-10.0) IU/l respectively], but were not significantly different from initial concentrations in controls [10.4 (7.2-19.6) and 4.8 (2.8-8.2) IU/l respectively]. Moreover, concentrations of IRMA-FSH and BIO-FSH were negatively correlated (r = -0.25, P = 0.01, and r = -0.24, P = 0.02 respectively) with the interval between last vaginal bleeding and blood sampling. Maximum concentrations of IRMA-FSH [7.6 (3.9-10.9) IU/l] during ovulation induction by gonadotrophins were not significantly different from maximum [6.4 (5.7-10.0) IU/l] concentrations in controls, whereas maximum BIO-FSH concentrations [13.5 (8.7-17.4) versus 18.7 (13.2-23.4) IU/l] were significantly lower. Our findings suggest that (i) circulating FSH does not reach concentrations that are sufficient to induce normal follicle development in anovulatory women during baseline conditions, and (ii) the FSH threshold for ovarian stimulation of this patient group is not different from normal.

Normal human follicle development: an evaluation of correlations with oestradiol, androstenedione and progesterone levels in individual follicles.

OBJECTIVE: The mechanism of dominant follicle selection remains obscure. We have investigated the association between follicle diameter and follicular steroid levels in individual human ovarian follicles throughout the menstrual cycle. DESIGN: Fluid from ovarian follicles (n = 326) was obtained in vivo during surgery from 55 regularly cycling women with proven fertility. Follicles were divided into dominant (diameter >9 mm, n = 45) and non-dominant (diameter < or = 9 mm, n = 281) based on ultrasound measurements. MEASUREMENTS: Fluid was assayed for oestradiol (E2), androstenedione (AD), and progesterone (P). RESULTS: Median P and E2 levels were significantly lower (P < 0.0001) and AD levels significantly higher (P = 0.03) in non-dominant as compared to dominant follicles. In non-dominant follicles AD (r = 0.14, P = 0.02), but not P and E2, levels were correlated to follicular diameter, and significant changes in steroid concentrations across the menstrual cycle were absent. In dominant follicles, diameter was positively correlated with P and E2 (P < 0.001) levels, and inversely correlated with AD concentrations (P = 0.01). CONCLUSIONS: Results indicate that (1) intrafollicular oestradiol concentrations rise only in follicles exceeding 9 mm in diameter and correlate with the diameter of these dominant follicles, suggesting that significant increase in aromatase enzyme activity occurs only in the dominant follicle (2) a cycle-independent accumulation of androstenedione with size occurs in non-dominant follicles, and (3) progesterone production occurs in the largest dominant follicles only, suggesting a limited, if any, role for progesterone during follicle development.

Estrogen- but not androgen-dominant human ovarian follicular fluid contains an insulin-like growth factor binding protein-4 protease.

Estrogen-dominant follicular fluid (FFe) and granulosa-luteal cell conditioned media, in contrast to androgen-dominant FF (FFa), contain barely detectable levels of insulin-like growth factor binding protein-4 (IGFBP-4) by ligand binding techniques. The current study was designed to evaluate the possibility of an IGFBP-4 protease in FFe, which may alter the affinity of IGFBP-4 for insulin-like growth factors (IGFs), rendering IGFBP-4 undetectable by ligand binding techniques. FFe and FFa were obtained from regularly menstruating women, and FFe was also obtained during in vitro fertilization procedures. Mixing experiments were performed by using human recombinant IGFBP-4 or IGFBP-4 in nonpregnancy serum (NPS) as substrate and FF as the source of the putative protease. Incubation of NPS at 37C for 5 h in the presence of FFe resulted in the reduction of IGFBP-4 to barely detectable levels when analyzed by Western ligand blotting, with no change occurring in the levels of the other binding proteins present in NPS. In contrast, incubation of FFa with NPS under similar conditions had no effect on the levels of IGFBP-4. The disappearance of IGFBP-4 when NPS was mixed with FFe exhibited optimal pH-dependence at pH 7-9. Inhibition of the putative protease by aprotinin, ethylenediaminetetraacetic acid, and 1,10-phenanthroline supports its identification as a metalloserine protease. Western immunoblot analysis detected the presence of a proteolytic fragment of approximately 17-18 kDa after incubation of recombinant IGFBP-4 in the presence of FFe but not in the presence of FFa. Similar incubation of other recombinant human IGFBPs did not reveal their degradation, further suggesting that the protease in FFe is specific for IGFBP-4. These data demonstrate the presence of an IGFBP-4-specific metalloserine protease in FFe but not in FFa, and they suggest that proteolytic cleavage may be responsible for effectively decreasing levels of inhibitory IGFBP-4 and thus increasing bioavailability of IGF peptides within estrogen-dominant follicles. The importance of this mechanism may lie in providing the dominant follicle with more available IGFs to synergize with gonadotropins in stimulating estradiol production and in inhibiting this synergy in androgen-dominant and atretic follicles.

Circulating and ovarian IGF binding proteins: potential roles in normo-ovulatory cycles and in polycystic ovarian syndrome.

IGFs function as co-gonadotropins in the ovary, facilitating steroidogenesis and follicle growth. IGFBP-1 to -5 are expressed in human ovary and mostly inhibit IGF action in in vitro ovarian cell culture systems. In the clinical disorder of polycystic ovarian syndrome (PCOS), which is characterized by hyperandrogenemia, polycystic ovaries and anovulation, follicles have a higher androgen: estradiol (A : E2) content and growth is arrested at the small antral stage. In the PCOS follicle, follicle stimulating hormone (FSH) and IGF levels are in the physiologic range, and even in the face of abundant androstenedione (AD) substrate, aromatase activity and E2 production are low. When PCOS granulosa are removed from their ovarian environment, they respond normally or hyperrespond to FSH. It has been postulated that an inhibitor of IGF's synergistic actions with FSH on aromatase activity may be one (or more) of the IGFBPs, which contributes to the arrested state of follicular development commonly observed in this disorder. High levels of IGFBP-2 and IGFBP-4 are present in follicular fluid (FF) from androgen-dominant follicles (FFa) from normally cycling women and in women with PCOS. This is in marked contrast to the near absence of these IGFBPs in estrogen-dominant FF (FFe), determined by Western ligand blotting. Regulation of granulosa-derived IGFBPs is effected by gonadotropins and insulin-like peptides. In addition, an IGFBP-4 metallo-serine protease is present in FFe, but not in FFa in ovaries from normally cycling women and those with PCOS, although the IGFBP-4 protease is present in PCOS follicles hyperstimulated for in vitro fertilization. Recent studies demonstrate that IGF-II in FFe is higher than in FFa' whereas IGF-I, IGFBP-3 and IGFBP-1 levels do not differ, underscoring the importance of local IGF-II production by the granulosa and the importance of IGFBP-4 and IGFBP-2 in regulation of IGF-II action within the follicle during its developmental pathway as an E2- or A-dominant follicle. In the androgen-treated female-to-male transsexual (TSX) model for PCOS, IGF-I, IGF-II, IGFBP-3 and IGFBP-1 levels do not differ.