Routine screening for Coxiella burnetii infection during pregnancy: a clustered randomised controlled trial during an outbreak, the Netherlands, 2010.

Between 2007 and 2010, the Netherlands experienced one of the largest outbreaks of Q fever. Since asymptomatic Coxiella burnetii infection has been associated with maternal and obstetric complications, evidence about the effectiveness of routine screening during pregnancy in outbreak areas is needed. We performed a clustered randomised controlled trial during the Dutch outbreak, in which 55 midwife centres were randomised to recruit pregnant women for an intervention or control strategy. In both groups a serum sample was taken between 20 and 32 weeks of gestation. In the intervention group (n=536), the samples were analysed immediately by indirect immunofluorescence assay for the presence of IgM and IgG (phase I/II) and treatment was given during pregnancy in case of an acute or chronic infection. In the control group (n=693), sera were frozen for analysis after delivery. In both groups 15% were seropositive. In the intervention group 2.2% of the women were seropositive and had an obstetric complication, compared with 1.4% in the control group (Odds ratio: 1.54 (95% confidence interval 0.60-3.96)). During a large Q fever outbreak, routine C. burnetii screening starting at 20 weeks of gestation was not associated with a relevant reduction in obstetric complications and should therefore not be recommended.

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.

Is the fertility treatment itself a risk factor for early pregnancy loss?

This longitudinal multicentre cohort study aimed to identify the role of the conception mode in infertile couples with an early pregnancy loss (EPL). All couples referred to the fertility clinic for the first time in the period 2002-2006 because of infertility were followed up to their first clinical pregnancy (n=1809). EPL was the outcome of 286 (15.8%) pregnancies. EPL rates for the different conception modes were as follows: spontaneous 14.5% (125/864), ovulation induction 15.8% (42/266), intrauterine insemination 25.0% (5/20), intrauterine insemination combined with ovarian stimulation 18.2% (37/203), IVF 16.3% (31/190), intracytoplasmic sperm injection (ICSI) 14.9% (30/202) and frozen embryo transfer (FET) 26.2% (16/61). After adjusting for female age, male age, hospital, obstetric history, female smoking habit, male alcohol use, menstrual cycle type and infertility diagnosis, the EPL rate after FET was significantly increased (odds ratio 2.2, 95% CI 1.14-4.19) compared with spontaneous conception. Embryo quality was comparable in fresh and frozen embryos. Other fertility treatments showed no increased miscarriage rate. Therefore, it is concluded that even after adjustment for confounding factors conception through FET remained an independent risk factor for EPL. Other modes of conception were not related with EPL.

Identification of candidate genes in ulcerative colitis and Crohn's disease using cDNA array technology.

Inflammatory bowel disease (IBD) follows a multigenic mode of inheritance, encompassing the clinically discrete phenotypes of ulcerative colitis (UC) and Crohn's disease (CD). The risk of malignant transformation of the colon increases with the duration and extent of IBD and is particularly high for patients with a longstanding history of UC. We wished to identify candidate genes that might be involved in disease pathogenesis based on functional plausibility and their putative role in IBD carcinogenesis. Polyadenylated mRNA (PolyA+ mRNA) preparation from inflamed intestinal mucosa of patients with a longstanding history of UC and CD was performed with subsequent hybridization of alpha phosphorus [alpha-32P]-deoxyadenotriphosphate-labeled complementary deoxyribonucleic acid (DNA) populations to nucleic acid arrays. Of 588 different human gene transcripts arrayed, secreted apoptosis-related protein 1 (Sarp1), frizzled (fz) homologues, and disheveled (dvl) were differentially expressed, being elevated in UC as compared to CD. These genes encode proteins involved in the Wingless-type (Wnt)/beta-catenin signaling pathway. The autonomous expression of Sarp1 and Sarp1-compatible fz receptor genes suggests that the Wnt pathway may be involved in UC carcinogenesis.

Wingless-type frizzled protein receptor signaling and its putative role in human colon cancer.

We wish to identify new candidate genes involved in the pathogenesis of human colon cancer to better understand the diversity of phenotype presentation that varies from individual to individual. Our working hypothesis is that genetic polymorphism of genes in the Wingless-type (Wnt) frizzled protein receptor pathway is associated with the susceptibility to develop colon cancer. The putative role of the Wnt pathway in sporadic human malignancy of the colon suggests involvement in inherited cancer as well. beta-catenin is the crucial messenger in frizzled receptor signaling, transmitting Wnt-ligand signals such as signals from secreted apoptosis-related proteins to the nucleus. It functions as a genome denunciator by initiating amplification of oncogenes. The net effect of beta-catenin depends on the magnitude of its accumulation in the cytoplasm and, therefore, upon expression profiles of genes in the Wnt pathway. We propose that variations in allelic frequencies of genes involved in the beta-catenin cascade may either promote or impede malignant transformation of the colon. If certain polymorphisms in Wnt signaling through beta-catenin predispose to colon cancer, this might manifest as decreased binding affinity of proteins such as axin or the adenomatous polyposis coli protein to beta-catenin. Association studies are proposed to test the hypothesis, which could serve as an initial step toward understanding the complexity of tumor biology. The clinical rationale in unraveling the genetic susceptibility to cancer lies in identification of a subgroup of individuals who may benefit from beta-catenin targeting agents, which could potentially overcome this genetic instability.

The potential role of estrogen in aromatase regulation in the breast.

Aromatase is expressed in both normal and malignant breast tissues. Aromatase activity in the breast varies over a wide range. Our previous studies have demonstrated that in situ aromatization contributes to the estrogen content of breast tumors to a major extent. Consequently, alterations of aromatase activity could serve as a major determinant of tissue estradiol content. However, the mechanisms and extent of aromatase regulation in breast tissues have not been fully established. We have observed an inverse correlation between tumor aromatase activity and estrogen content in nude mice bearing xenografts of MCF-7 cells transfected with the aromatase gene. To investigate the potential role of estrogen in aromatase regulation in the breast, studies were carried out in an in vitro model. In this model, MCF-7 cells were cultured long term in estrogen-deprived medium and called by the acronym, LTED cells. We found that long-term estrogen deprivation enhanced aromatase activity by 3-4-fold when compared to the wild-type MCF-7 cells. Re-exposure of LTED cells to estrogen reduced aromatase activity to the levels of the wild-type MCF-7 cells. We also measured aromatase activity in 101 frozen breast carcinoma specimens and compared tumor aromatase activities in pre-menopausal patients versus post-menopausal patients and in post-menopausal patients with or without hormone replacement therapy (HRT). Although statistically not significant, there was a trend paralleling that observed in the in vitro studies. Aromatase activity was higher in breast cancer tissues from the patients with lower circulating estrogen levels. Our data suggest that estrogen may be involved in the regulation of aromatase activity in breast tissues.

Aromatase within the breast.

In situ aromatization and enhanced uptake of estradiol from plasma are two potential mechanisms for maintenance of high concentrations of estradiol found in breast tumors of postmenopausal patients. To test the relative importance of these two mechanisms, a nude mouse model was established by inoculating aromatase (A+) and/or sham (A-) transfected MCF-7 cells into ovariectomized mice. Postmenopausal hormonal status was simulated by providing estradiol Silastic implants which clamped plasma estradiol levels at 5-20 pg/ml. We demonstrated that in situ aromatization rather than the uptake mechanism is the key determinant of tumor estradiol levels and tumor growth rate under conditions reflecting the postmenopausal state. The importance of intratumoral aromatase was also suggested by the findings that long-term estrogen deprivation increases sensitivity to estradiol and enhances aromatase activity in MCF-7 cells. The results of our in vivo and in vitro studies suggest that complete blockade of in situ aromatization in the breast would provide added benefit to postmenopausal breast cancer patients, especially those who relapse from antiestrogen therapy.

In situ aromatization enhances breast tumor estradiol levels and cellular proliferation.

The high concentrations of estradiol (E2) found in breast tumors of postmenopausal women could be the result of enhanced uptake from plasma or in situ aromatization of androgens to estrogens. To test the relative importance of these two mechanisms, a model system allowing precise distinction between each is required. Such a model was established using aromatase (A+)- and sham (A-)-transfected MCF-7 cells inoculated into ovariectomized (OVX) nude mice. To validate the model, the confounding effect of peripheral aromatization was first excluded experimentally. A- cells were inoculated into OVX mice as homoimplants (A- cells on both flanks) or heteroimplants (A- cells on one flank and A+ cells on the other), and growth of A- cells in response to exogenous aromatase substrate, androstenedione (delta4A), was evaluated. A- cells did not grow in either group during the 8 weeks of observation, indicating the lack of peripheral aromatization in OVX mice. The biological effects of in situ aromatization were then directly examined. We found that A+ cells in the heteroimplant group grew rapidly, and that the average weight of A+ tumor was 7.6-fold larger and tissue E2 concentration was 3-4-fold higher than A- tumors grown in the same animals. These results demonstrate that in situ aromatization rather than uptake can be a determinant of tumor E2 content and growth stimulation. An additional experiment was then designed to evaluate the relative importance of in situ synthesis versus uptake under conditions reflecting postmenopausal physiology. Groups of OVX mice bearing A+ cells received E2 Silastic implants to clamp plasma levels at 5, 7, 10, and 20 pg/ml or delta4A by injection. The highest tumor E2 concentration and growth rate were found in the group receiving delta4A. E2 delivered by Silastic implants always produced lower tissue E2 levels and tumor growth rates than resulted from in situ synthesis. These data provide direct evidence that under physiological conditions reflecting those in postmenopausal women, in situ aromatization in breast tumor makes a major contribution to tissue E2 content. As further validation that our experimental paradigm models the postmenopausal state, we studied OVX animals not given delta4A as substrate. A+ cells also grew under these conditions, and the aromatase inhibitor 4-hydroxyandrostenedione reduced both tumor E2 level and growth rate, providing additional evidence of the importance of in situ synthesis. These studies provide the first direct evidence that in situ synthesis of E2 in breast tumors, as opposed to peripheral aromatization and uptake from plasma, can enhance tissue E2 levels and stimulate tumor growth.

Controlled comparison of interceed and amniotic membrane graft in the prevention of postoperative adhesions in the rabbit uterine horn model.

OBJECTIVE: To evaluate the effects of Interceed TC7 (Johnson & Johnson Medical, New Brunswick, NJ) and gamma-irradiated human amniotic membrane graft in prevention of postoperative adhesions in the rabbit uterine horn model. SETTING: A conventional animal laboratory. SUBJECTS: Virgin, female New Zealand white rabbits. INTERVENTIONS: Both uterine horns were subjected to clean transverse cuts 1 cm apart at the antimesenteric border. The left uterine horn was left uncovered in each case to act as a control. The right uterine horn was randomly covered with either Interceed TC7 or human amniotic membrane. OUTCOME MEASURES: Adhesion score was evaluated 4 weeks after the initial surgery. Samples of each uterine horn were collected for histopathological studies, and subserosal inflammatory score. RESULTS: Interceed resulted in higher adhesion scores and subserosal inflammation score than the control side (P = 0.03 and 0.05, respectively). A similar difference was found for amniotic membrane when compared with the uncovered control side (P = < 0.01 and 0.01, respectively). CONCLUSION: Interceed TC7 failed to have the preventive effect on adhesion formation it is claimed to possess. Amniotic membrane performed poorly too. Therefore, gentle tissue handling, irrigation and good surgical skills remain the most important prerequisite for adhesion prevention.

The value of luteal support with progesterone in gonadotropin-induced cycles.

OBJECTIVE: To evaluate the effect of luteal support with vaginal P suppositories in hMG- and hCG-induced cycles. DESIGN: Between March 1988 and February 1989 patients did not receive luteal support, while between March 1989 and January 1990 P was given routinely in the luteal phase. Induction protocol and patient selection remained otherwise unchanged. SETTING: Infertility clinic of a tertiary care hospital. PATIENTS: Twenty-seven patients with hypogonadotropic amenorrhea (World Health Organization [WHO] group I) (11 women with luteal support, 16 women without) and 102 patients with euprolactinemic clomiphene citrate (CC)-resistant anovulation (WHO group II) (52 women with luteal support, 50 women without). INTERVENTION: Vaginal P suppositories 200 mg/d in the luteal support group. MAIN OUTCOME MEASURES: Pregnancy rate (PR), pregnancy outcome. RESULTS: The overall PR in 118 cycles with luteal support was 26.3% whereas 10.4% pregnancies were achieved in 115 cycles of the control group. The influence of luteal support was more pronounced in patients with CC-resistant anovulation (25.2% versus 6.9%) than in patients with hypogonadotropic amenorrhea (33.3% versus 21.4%, not significant). The abortion rate was not significantly changed. CONCLUSION: Luteal support with P increases the PR after hMG and hCG induction. The need for supplementary P seems to be related to the underlying cause of ovarian disturbance.

Different follicle stimulating hormone/luteinizing hormone ratios for ovarian stimulation.

The aim of the present study was to investigate whether reducing the amount of luteinizing hormone (LH) in gonadotrophic preparations impairs follicular growth in in-vitro fertilization (IVF) cycles during suppression of endogenous LH levels. A selected group of 20 IVF patients was randomly divided into two groups. One group was treated with Org 31338 [follicle stimulating hormone (FSH)/LH 3:1], the other group with Metrodin (purified FSH), both during pituitary down-regulation with buserelin. A fixed daily dose of 150 IU FSH i.m. was given. Serum concentrations of FSH, LH, oestradiol and progesterone were determined frequently and serial ultrasound examinations were performed. Multiple follicular growth with concomitant rise of oestradiol levels was observed in all cycles. The duration of the stimulation phase was shorter in the group treated with Org 31338 than in the group treated with Metrodin. The number of follicles and oocytes and the fertilization rate was larger and the mean embryo quality was higher in the Org 31338 group, but the differences did not reach statistical significance. No significant differences were found in hormonal values. In women with normal endocrine profiles, lowering of the LH activity in gonadotrophic preparations during gonadotrophin-releasing hormone agonist treatment results in adequate ovarian stimulation. However, a preparation with some LH needed a shorter stimulation than a purified FSH preparation. Whether the other beneficial effects of Org 31338 also occur in a larger population needs further investigation.

Percoll semen preparation enhances human oocyte fertilization in male-factor infertility as shown by a randomized cross-over study.

The aim of this study was to compare two methods of semen preparation: multiple tube swim-up and Percoll separation, using a randomized cross-over clinical study, in which sperm parameters, oocyte fertilization rates, embryo quality and cell stage were analysed. Overall, there was no difference between the two preparation methods in the normozoospermic cycles. In the male-factor cycles, Percoll extracted a higher total number of spermatozoa (P = 0.02), increased the concentration of motile spermatozoa (P = 0.02), increased the total number of motile spermatozoa per sample (P = 0.02), and enhanced the recovery rate of motile spermatozoa (P = 0.04) compared to swim-up. There was a significant improvement in fertilization rates (P = 0.0006), in the percentage of embryos over 2-cell stage on day of transfer (P = 0.004), and in the number of replaced embryos per transfer (P = 0.01) in the Percoll as compared to swim-up cycles. There was no significant difference in embryo quality. We conclude, therefore, that in advanced reproductive procedures where sperm dysfunction exists, semen preparation with Percoll should replace the swim-up technique.

Artificial endometrial stimulation for frozen embryo replacement.

In this study, 18 cycles initiated for the transfer of cryopreserved embryos. Endometrium was prepared using a uniform protocol of exogenous E and P. This approach eliminated cycle cancellations caused by ovulatory dysfunction and gave complete cycle control. Fourteen cycles had embryo division and replacement resulting in three pregnancies. In these conception cycles, E was discontinued at 12 days and P at 26 days after ET. One aborted at 6 weeks gestation, whereas the other two pregnancies progressed uneventfully into the third trimester.

Longitudinal ultrasonographic study of the ovarian suppressive activity of a low-dose triphasic oral contraceptive during correct and incorrect pill intake.

A longitudinal study was conducted to evaluate the ability of a low-dose triphasic oral contraceptive to suppress ovulation as documented by frequent ultrasonographic scanning and progesterone determinations, even in the event of a missed pill. The extent of follicular growth and maturation, the incidence of escape ovulation, and the effect of correct and incorrect pill intake were assessed in 30 evaluable women during two consecutive spontaneous menstrual cycles. After the first cycle, 11 of 30 women (36.6%) had follicle-like structures of at least 10 mm in diameter. Ten of 11 structures gradually disappeared during the second cycle, with one persistent structure remaining through the second cycle. Seven of 30 women (23%) developed follicle-like structures during the second cycle. Of these, one woman had a probable ovulation, and another had an elevated progesterone level without follicle rupture, suggesting the luteinized unruptured follicle syndrome. Both of these women missed a pill on day 1 of the second cycle. In all cases cervical scores indicating hostility were noted. Thus, although suppression of ovarian activity may have been incomplete when oral contraceptives were incorrectly taken, secondary mechanisms of contraception remained operant. When they were correctly taken, low-dose triphasic oral contraception consistently prevented ovulation.

Ovulatory disturbances in patients with luteal insufficiency.

In order to study ovulatory disturbances in patients with luteal insufficiency we evaluated 210 cycles of 170 women from infertile couples by ultrasonographic follicle measurements and hormonal determinations. Only cycles with evidence of luteinization were included into the study. Mid-luteal progesterone (P) levels were determined in relation to the ultrasonographic ovulation time or, where the follicle failed to rupture, in relation to the LH peak. In spontaneous cycles with a mid-luteal P level below 32 nmol/l (10 ng/ml) a luteinized unruptured follicle (LUF) was found in 71.1% of cycles, whereas in spontaneous cycles with a mid-luteal P level above 32 nmol/l only 7.9% cycles exhibited a failure of the follicle to rupture. The same phenomenon was encountered in cycles in which agents had been given to induce ovulation. The incidence of LUF cycles in an average infertility population could be calculated to be as high as 50% if the mid-luteal P level is below 32 nmol/l and as low as 4% if the mid-luteal P level is above 32 nmol/l. The geometric mean mid-luteal P level in spontaneous LUF cycles was 32.5 nmol/l, compared to 55.2 nmol/l in spontaneous ovulatory cycles (P less than 0.001). We conclude that in patients with luteal insufficiency a high incidence is found of a failure of the follicle to rupture. The entrapment of the oocyte in the unruptured follicle constitutes an important cause of infertility in these patients.

Ovulatory disorders and inflammatory adnexal damage: a neglected cause of the failure of fertility microsurgery.

Failure of reconstructive tubal surgery is usually attributed to pre-existing endosalpingeal damage, recurrent adhesion formation or progressive inflammatory disease. We studied ovarian follicle development and ovulation by ultrasound in 25 infertile patients with laparoscopically proven sequelae of pelvic inflammatory disease. Failure of the follicle to rupture was seen in 13, although luteinization occurred. The mean mid-luteal progesterone concentration in cycles with an unruptured follicle was significantly lower than the concentrations both in ovulatory cycles in the same patients and in a comparison group of 45 normally ovulating women (P less than 0.01). Microsurgical adhesiolysis did not seem to influence the occurrence of the ovulation disorder.

Follicle growth curves and hormonal patterns in patients with the luteinized unruptured follicle syndrome.

A prospective longitudinal and standardized study is presented, dealing with ultrasonographic and hormonal characteristics of the luteinized unruptured follicle (LUF) syndrome. Among 600 cycles monitored in 270 infertility patients, 40 cycles in 27 patients showed no evidence of follicle rupture, in spite of signs of luteinization, as reflected by basal body temperature recordings and progesterone determinations. In this study, 20 LUF cycles in 20 infertile patients were compared with 45 ovulatory cycles in 45 control women. During the follicular phase, no substantial difference in follicle growth was found, but after the luteinizing hormone peak, LUF follicles, instead of rupturing, showed a typical accelerated growth pattern. Both mean luteinizing hormone peak levels and midluteal progesterone levels were significantly lower in LUF cycles than in the control cycles. However, the duration of the luteal phase was not affected. Both central and local factors can be held responsible for the lack of follicle rupture. Ultrasound offers new possibilities as a noninvasive method in diagnosing the LUF syndrome.