Objective: To study the value of E-2 production during controlled ovarian hyperstimulation (COH) in predicting IVF-ET outcome.Design: Historical cohort.Setting: Academic infertility center.Patient(s): A cohort of 270 patients who completed 324 consecutive IVF-ET treatment cycles.Intervention(s): None.Main Outcome Measure(s): Area under the curve for E-2 levels (AUC-E-2) from the first day of COH until the day of hCG administration was calculated and cycles grouped into low, average, and high AUC-E-2 groups. Clinical pregnancy rates per cycle were compared among the three groups, and correlation with AUC-E-2 values were calculated for all patients and after sub-grouping according to age, COH protocol and infertility diagnosis.Result(s): Cycles with low and high AUC-E-2 values had significantly lower pregnancy rates particularly in patients 35 years or older. There was a positive correlation between AUC-E-2 and pregnancy rates up to a certain AUC-E-2 level above with a negative correlation was found. The turning point between positive and negative correlation occurred at a significantly lower AUC-E-2 level in patients 35 years or older.Conclusions: Estradiol production during OCH correlates with IVF-ET outcome. Women > 35 years of age seem more vulnerable to high E-2 levels.

PURPOSE:Most studies reported estradiol (E2) levels attained on day of hCG administration when investigating effect of E2 on IVF outcome. We studied whether a relationship exists between the area under the curve for E2 levels (AUC-E2) and E2 levels on hCG day during IVF-ET.METHODS:Retrospectively, we analyzed data for 313 patients who completed one IVF-ET cycle each. Patients were sorted according to AUC-E2 levels. Then we compared between each patient's own AUC-E2 and the corresponding E2 level on hCG day for the same patient.RESULTS:Although overall AUC-E2 correlated positively with E2 levels on hCG day, there was no consistent correlation between individual patients.CONCLUSIONS:AUC-E2 reflects more accurately the amount of E2 produced by the follicles during controlled ovarian hyperstimulation. The absence of a uniform correlation between AUC-E2 and E2 on hCG day may result in different conclusions when studying outcomes of IVF treatment.

Objective: To report a live birth after transfer of cryopreserved blastocysts derived from testicular sperm that were cryopreserved 15 hours after biopsy due to inclement weather.Design: Case report.Setting: University-affiliated private clinic.Patient(s): Couple undergoing lVF with intracytoplasmic sperm injection (ICSI) using testicular extracted sperm for male factor infertility due to unexplained obstructive azoospermia.Intervention(s): An IVF,case involving testicular biopsy, cryopreservation, and thawing of testicular tissue, ICSI, embryo freezing, and transfer of cyropreserved blastocysts.Main Outcome Measure(s): The ability to use cryopreserved testicular tissue in which optimal time from biopsy to freezing had been delayed because the weather was inclement.Result(s): In spite of a 15-hour delay in processing and cryopreservation of the testicular biopsy, subsequent thawing yielded viable sperm that resulted in viable embryos after ICSI. Transfer of fresh embryos was unsuccessful. However, subsequent use of supernumerary viable blastocysts that were frozen resulted in the live birth of a baby.Conclusion(S): A 15-hour delay in processing and cryopreservation of a testicular biopsy did not affect the viability of the extracted sperm after thawing. Although it is desirable to freeze the testicular specimen as soon as possible, the delay in processing in this case due to inclement weather did not affect the potential for fertilization and implantation of a viable cryopreserved blastocyst. (C)2005 by American Society for Reproductive Medicine.

There is mounting evidence that endometriosis may have an environmental origin, possibly exposure to hormonally active agents such as dioxin and other compounds that bioaccumulate. Among candidate materials are polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs). Both animal and human studies support a role for PCBs and the development of endometriosis, an estrogen-dependent disorder. The present study examined the relationship, if any, between endometriosis and 62 individual PCBs. Eighty-four women 18 to 40 yeas of age who were scheduled for laparoscopy constituted the study group. Most were white, married, college-educated women; their mean age was approximately 32 years. Endometriosis was visually confirmed in 32 women. Women with endometriosis had fewer pregnancies and live births than did their unaffected peers. Both women with and those without endometriosis had relatively low serum concentrations of PCBs. Total serum PCBs ranged from 0.5 to 5.3 ng/g in women with endometriosis and from 0.2 to 5.6 ng/g serum for those without endometriosis. The sum of estrogenic PCB congeners ranged from 0.07 to 0.9 and from 0.4 to 1.7 ng/g serum, respectively. The respective figures for antiestrogenic congeners were 0.01 to 0.2 and from zero to 0.3 ng/g serum. On logistic regression analysis, a significantly elevated odds ratio for endometriosis of 3.8 was noted in women in the third tertile of antiestrogenic PCBs (95% confidence interval [CI], 1.1–12.7). The association remained significant at 3.3 (95% CI, 0.9–12.5) after controlling for gravidity, current cigarette smoking, and serum lipids. Gravidity and cigarette smoking were both associated with a significantly reduced risk of endometriosis. These findings suggest an association between antiestrogenic PCBs and the development of endometriosis.

BACKGROUND:Hormonally active environmental agents have recently been associated with the development of endometriosis.METHODS:We undertook a study to assess the relationship between endometriosis, an estrogen-dependent gynaecological disease, and 62 individual polychlorinated biphenyl (PCBs) congeners. We enrolled 84 eligible women aged 18-40 years undergoing laparoscopy for study, which included an interview and blood specimen (n=79; 94%). Thirty-two women had visually confirmed endometriosis at laparoscopy while 52 did not. Blood specimens were run in batches of 14 including four quality control samples for toxicological analysis. Each PCB congener was adjusted for recovery; batch-specific reagent blanks were subtracted. All PCB concentrations were log transformed and expressed in ng/g serum first as a sum and then as tertiles by purported estrogenic or anti-estrogenic activity of PCB congeners.RESULTS:Using unconditional logistic regression analysis, a significantly elevated odds ratio (OR) was observed for women in the third tertile of anti-estrogenic PCBs [OR 3.77; 95% confidence interval (CI) 1.12-12.68]. Risk remained elevated after controlling for gravidity, current cigarette smoking and serum lipids (OR 3.30; 95% CI 0.87-12.46).CONCLUSIONS:These data suggest that anti-estrogenic PCBs may be associated with the development of endometriosis.

Several factors have been suggested to explain for the poor outcome of pregnancies achieved after in vitro fertilization (IVF-ET) compared to spontaneous pregnancies. These include maternal age, exposure of the gametes and embryos to in-vitro conditions as well as the infertility factor itself. Controlled ovarian hyperstimulation (COH) with the inevitably associated supraphysiologic estradiol (E2) levels, is believed to explain at least in part for the poor obstetric outcome after IVF-ET. The objective of this study was to study the value of E2levels attained during COH in correlating the risk for pregnancy loss after IVF-ET. Retrospective analysis of 236 consecutive pregnancies achieved after IVF-ET. E2production during COH has been calculated as area under the curve for E2levels (AUC-E2) from the first day of COH until the day of hCG administration. Correlation between E2 production and rate of pregnancy loss has been calculated for all pregnancies and after controlling for age, COH protocol, and infertility diagnosis. Correlations have been calculated for total AUC-E2and for AUC-E2per administered FSH (AUC-E2/FSH) and AUC-E2 per follicle (AUC-E2/Follicle), separately and after combining AUC-E2/FSH and AUC-E2/Follicle (combined formula calculated as AUC-E2/FSH over AUC-E2/Follicle). Pregnancy cycles have been divided into low, average and high groups according to E2 production (AUC-E2, AUC-E2/FSH, AUC-E2/Follicle and combined formula values). Rates of pregnancy loss have been compared among the three groups for all patients and after controlling for age, stimulation protocol and infertility factor. Both low and high AUC-E2values were associated with higher pregnancy loss. The group of average AUC-E2 has been associated with lowest risk for pregnancy loss. This was true for all pregnancy cycles combined together and after controlling for age, COH protocol and infertility diagnosis. AUC-E2/Follicle positively correlated with pregnancy loss while AUC-E2/FSH had a negative correlation. The combined formula had the strongest negative correlation with pregnancy loss. Estradiol production during COH correlates with the risk for pregnancy loss. Low E2 production may reflect reduced ovarian reserve with anticipated poor quality oocytes and higher pregnancy loss, while high E2 levels seem to be associated with higher pregnancy loss potentially due to possible deleterious effects on the endometrium, developing oocyte/embryo or other possible targets. Optimizing COH protocols to achieve optimum E2 levels may reduce pregnancy loss after IVF-ET.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

To study correlation between E2 levels attained during COH and birth weight in pregnancies achieved after IVF-ET Retrospective analysis The study included 99 consecutive live births achieved in 61 singleton and 19 twin pregnancies after IV-ET. 9 singleton and 15 twin pregnancies ended in preterm labor (<37 wks). Gestational age was calculated from day of embryo transfer till day of delivery considering embryo transfer day as gestation day 17 (when day 3 embryos were transferred and day 19 when day 5 embryos were transferred). Birth weight was calculated as percentage from the birth weight expected according to gestational age (expected birth weight was calculated from a control population group similar to Western New York region that matched with the patients who underwent IVF-ET). E2 production during COH was calculated as area under the curve for E2levels (AUC-E2) from first day of COH until the day of hCG administration. The mean for birth weights (expressed as % from expected weight) was compared among singletons and twins after subdividing according to gender and gestational age (full term and preterm). Correlations have been calculated between AUC-E2and birth weight (expressed as % from expected weight) for all singletons, twins both preterm (<37 wks and full term deliveries). Overall birth weight was lower than expected for gestational age in almost all subgroups, particularly in singletons compared to twins, preterm compared to full term deliveries and in males compared to females. Although the differences were not statistically significat, the pattern was maintained in all groups. AUC-E2values negatively correlated with birth weight (-0.25 to -0.38). The negative correlation was true for all studied groups. A significant negative correlation was found between AUC-E2attained after COH and birth weight. This may explain for what was previously reported on COH as an independant risk for lower birth weight after infertility treatment. The inevitably supraphysiologic E2 levels associated with COH may lead to low birth weight by an effect on the early development of the placenta and/or an effect on the developing oocyte/embryo. Studies including larger number of live births are needed to confirm or contradict our findings.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

There is evidence that infertility treatment, including in vitro fertilization and embryo transfer (IVF-ET), is associated with less favorable obstetric outcome, specifically, higher pregnancy loss, preterm labor and low birth weight (BW). Vanishing-twin is a phenomenon in which one or more of implanted embryos fail to continue. We studied outcome of singleton pregnancies after vanishing-twin compared to pregnancies that started as singletons. Retrospective analysis of 61 consecutive live-births from singleton pregnancies in women undergoing IVF-ET. Five pregnancies started as twins and ended as singletons due to vanishing-twin; 56 pregnancies started as singletons. Vanishing-twin was diagnosed by ultrasound at 6-wks (2 gestational sacs with 2 heartbeats) followed by another ultrasound showing loss of one of the twins at 8 to 12 wks. Gestational age (GA) was calculated from ET day until delivery. BW was calculated as percentage from BW expected according to GA (expected BW was calculated from control population similar to Western New York region matching patients who underwent IVF-ET). Vanishing-twin pregnancies were associated with significantly lower BW and shorter GA. There was no significant difference regarding age, gravity, infertility factor and duration, ovarian stimulation protocol, or in vitro conditions (oocyte insemination, day and number of embryos transferred, fertilization or implantation rate). Pregnancies associated with vanishing-twin after IVF-ET are associated with higher risk for low BW and short GA.Tabled 1P value < .05 considered significantVanishing twin pregnanciesSingleton pregnanciesP valueAge(y)34 ± 233 ± 3.4Gravity.8 ± .8.7 ± .8.4Infertility duration (mo)31 ± 1731 ± 17.5GA(wk)32 ± 536 ± 2.001%BW88 ± 1299 ± 11.03Data presented as mean ± SD Open table in a new tab

Objective: To study the value of E2 concentrations attained during COH (controlled ovarian hyperstimulation) in predicting the outcome of treatment in women undergoing IVF-ET. Design: Retrospective study. Materials and Methods: We analyzed data of 270 infertile women who completed 324 cycles of IVF-ET during the period of January 2001 and July 2002 at a tertiary referral academic IVF center. The AUC for E2 concentrations was calculated from the available daily E2 concentrations along the follicular phase starting on the first day of COH until the day of hCG administration. We studied the value of AUC in predicting treatment outcome by comparing clinical pregnancy rates among three patient groups defined according to mean AUC. Group 1 (low responders) included patients with AUC Mean + 1SD and group 3 (average responders) included patients with AUC between Mean minus 1SD to Mean plus 1SD. We also compared pregnancy rates among groups of patients divided according to AUC at increments of (3000 pg/ml/stimulation). Correlation between AUC and clinical pregnancy rates was calculated using linear regression analysis. The analysis was done for all patients and for patients <35 and >35 years separately. Results: Both low and high AUC (low and high responders groups were associated with significantly lower pregnancy rates than average responders (P <.05). The difference was more significant in patients > 35 years (P <.01). There was a positive correlation between AUC and pregnancy rates until a certain AUC value above which, a negative correlation was noticed. The turning AUC level between positive and negative correlation occurred at a significantly lower level in patients >35 years. Conclusions: AUC for E2 levels attained during COH are better predictors of treatment outcome after IVF-ET. There might be an optimum range of AUC for E2 that is associated with the best outcome. Levels below (low responders) and above (high responders) the optimum range are associated with poor treatment outcome. In women > 35 years, the negative correlation occurred at a lower AUC level when compared to women < 35 years indicating a higher vulnerability for the possible deleterious effects of high estrogen levels in this age group. Optimizing AUC for E2 levels during COH may help in improving the treatment outcome after IVF-ET by using less aggressive stimulation protocols as well as other possible agents such as aromatase inhibitors that might lower E2 levels without affecting the number of mature follicles. Clinical Pregnancy Rates Among Three Study Groups Tabled 1∗ = P value was < .05 when compared with the corresponding low and high responders separately View Large Image Figure ViewerDownload (PPT) ∗ = P value was < .05 when compared with the corresponding low and high responders separately

Objective: To study the effect of age on the pattern of estrogen production per mature ovarian follicle and in response to FSH stimulation during different ovarian stimulation protocols Design: Retrospective. Materials and Methods: Patients undergoing ovarian stimulation in conjunction with intrauterine insemination or for IVF-ET (in vitro fertilization and embryo transfer) have been included from two tertiary referral academic infertility centers in the period from January 2000 and July 2002. Patients were divided into two groups, first group included patients who underwent COH (controlled ovarian hyperstimulation) for IVF-ET (226 patients had 254 cycles). Both standard and microdose flare protocol were applied for COH. The second group included 357 patients who received different ovarian stimulation protocols in conjunction with IUI (intrauterine insemination) in 493 cycles. Clomiphene citrate was used alone in 176 cycles and in conjunction with FSH in 28 cycles, FSH injection alone in 87 cycles, letrozole (an aromatase inhibitor) was used alone in 84 cycles and in conjunction with FSH in 118 cycles). The AUC for E2 production was calculated from available daily E2 levels along the follicular phase starting on the first day of ovarian stimulation until the day of hCG administration (or endogenous LH surge in IUI cycles). AUC for E2 production per FSH unit administered (AUC/FSH) as well as the AUC for E2 produced per mature follicle (AUC/follicle) were calculated. Patients were sub-grouped according to the age in ascending matter. Each patient group was analyzed separately and after subdividing them according to the protocol of ovarian stimulation. Results: Group 1 (IVF) patients had a positive correlation between AUC/follicle and advancing age (P<0.05) and a negative correlation between AUC/FSH and advancing age (P<0.05). This was true when all patients were analyzed together and after subgrouping according to the stimulation protocol (long standard and microdsoe flare up protocol). The same findings were found in the second patient group when all patients were analyzed together and when subdivided into different ovarian stimulation protocol. Pregnancy rates negatively correlated well with advancing age as expected and was also negatively correlating with increasing AUC/follicle and decreasing AUC/FSH. The effect of age on AUC/follicle, AUC/FSH and Pregnancy Rate in IVF-ET cycles Tabled 1 View Large Image Figure ViewerDownload (PPT) Conclusions: High AUC/follicle and low AUC/FSH seem to be features of advancing age irrespective of type of ovarian stimulation. Both parameters also might serve as predictors for poor treatment outcome.

Objective: There is a controversy regarding the effect of supraphysiologic estradiol (E2) levels attained during controlled ovarian hyperstimulation (COH) in patients undergoing assisted reproductive technology (ART) cycles on the outcome of treatment. Some investigators suggested unfavorable outcome associated with high responders who attained significantly high E2 levels while others failed to find such an effect. Most of the studies observed E2 concentrations attained on the day of hCG administration rather than the area under the curve for E2 concentrations which is expected to reflect more accurately the amount of E2 produced during COH. In this report we studied the relationship between AUC for E2 and E2 concentration on the day of hCG administration for E2 concentrations attained during COH in patients undergoing IVF-ET. Design: Retrospective study. Materials and Methods: Retrospectively we analyzed data from 313 patients who completed one IVF-ET cycle each. We sorted the patients according to the AUC for E2 levels comparing each patient's own AUC with the corresponding E2 concentration on the day of hCG administration. Results: Although the overall AUC for E2 concentrations positively correlated with the E2 concentrations attained on the day of hCG administration, there was no uniform correlation between successive individual patients. Figures 1 and 2 show representative E2 concentrations in two samples (pregnant and non-pregnant cycles), each consisted of 21 patients selected around the mean levels of AUC. View Large Image Figure ViewerDownload (PPT) Conclusions: In calculating AUC, the duration of ovarian stimulation as well as several E2 measurements are taken into consideration, which is expected to reflect, more accurately, the amount of E2 attained during ART cycles. The absence of a uniform correlation between AUC and E2 on hCG day among individual patients suggests that AUC rather than E2 concentration on the day of hCG administration should be used when studying the effect of E2 on the outcome of ART treatment.

Endometriosis is an estrogen dependent gynecologic disorder affecting approximately 10% of females of reproductive age. Its etiology remains elusive though hormonal and immunological factors have been postulated as underlying mechanisms. Of late, there is increasing speculation that hormonally active environmental agents may be associated with endometriosis. We investigated 61 polychlorinated biphenyl (PCBs) congeners and 7 pesticides (i.e., mirex, hexachlorobenzene (HCB), b-BHC, Oxychlordane, trans-nonachlor (t-Nonachlor), Aldrin, and 1,1-dichloro-2,2-bis(p-chlorophenyl) ethylene (p,p-DDE)) in relation to risk of incident endometriosis among women undergoing laparoscopy for either diagnostic or sterilization purposes, 1998–1999. Among the 100 eligible women identified, 84 agreed to be interviewed prior to laparoscopy. Blood specimens were collected from 80 (95%) consenting women. Thirty-two women had visually confirmed endometriosis while 52 did not. Among women with endometriosis, severity ranged from AFS-R Stage 1 (minimal, n = 15), Stage II (mild, n = 5). Stage III (moderate, n = 6), and Stage IV (severe, n = 6). All bloods were analyzed using gas chromatography with electron-capture. PCB congeners and pesticides were run in batches of 14 including four quality control samples (i.e., reagent control, serum control, control with 15 calibration standards at known values, and a duplicate participant sample). Congeners were calculated from standard curves for the 15 calibration standards, and the remaining congeners from response factors that were laboratory generated. Each congener and pesticide was adjusted for percent recovery and lipids; batch specific reagent blanks were subtracted as well. All PCB concentrations were expressed as ng/g serum lipid and summed into a total PCB concentration. Women with moderate/severe (Stages III and IV) endometriosis had a significantly higher mean concentration of total PCBs than women without disease (1.45 and 1.20 ng/g, respectively). No significant difference in mean PCB concentration was observed between women with mild/moderate (Stages I and II) and women without disease (1.44 and 1.20 ng/g, respectively). An elevated risk of endometriosis was observed for log of lipid adjusted total PCBs (OR = 1.98; 95% CI = 0.84,4.65) but not for DDE (OR = 0.93; 95% CI = 0.50,1.77) after controlling for household income, current cigarette smoking and parity. Risk of endometriosis remained for total PCBs after adjusting for DDE (OR = 2.13; 95% CI = 0.88,5.17). These data are suggestive of a possible association between PCBs and, possibly, more severe endometriosis but await confirmation in larger populations.

N-Acylethanolamines (NAEs) are an important family of lipid-signaling molecules. Arachidonylethanolamide (anandamide) (AEA), palmitoylethanolamide (PEA), and oleoylethanolamide (OEA) are co-produced from similar phospholipid precursors when neurons are stimulated. AEA is an endogenous agonist (endocannabinoid) for cannabinoid receptors. It binds with higher affinity to type CB1 than to type CB2 cannabinoid receptors. PEA does not bind to CB1, while the hypothesis that it reacts with putative CB2-like receptors has been questioned. OEA does not activate currently known cannabinoid receptors, but it mimics the effects of AEA and cannabinoids in reducing the fertilizing capacity of sea urchin sperm. OEA and PEA also act as entourage compounds by inhibiting the hydrolysis of AEA by fatty acid amide hydrolase. Cannabinoid receptors and/or AEA are present in mammalian reproductive organs including the testis, epididymis, prostate, ovary, uterus, sperm, preimplantation embryo and placenta, as well as prostatic and mammary carcinomas. We now report that analysis by high-performance liquid chromatography/mass spectrometry (HPLC/MS) shows the presence of AEA, PEA, and OEA in human seminal plasma, mid-cycle oviductal fluid, follicular fluid, amniotic fluid, milk, and fluids from malignant ovarian cysts. Previous studies showed that AEA-signaling via cannabinoid receptors regulates capacitation and fertilizing potential of human sperm, early embryonic development and blastocyst implantation into the uterine mucosa of rodents, as well as proliferation of human mammary and prostatic carcinomas. Current results imply that NAEs also may modulate follicular maturation and ovulation, normal and pathological ovarian function, placental and fetal physiology, lactation, infant physiology, and behavior. Collectively, these findings suggest that NAEs in human reproductive fluids may help regulate multiple physiological and pathological processes in the reproductive system, and imply that exogenous cannabinoids delivered by marijuana smoke might impact these processes. This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adolescents and adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids.

Ejaculated mammalian sperm require several hours exposure to secretions in female reproductive tracts, or incubation in appropriate culture medium in vitro, before acquiring the capacity to fertilize eggs. Arachidonylethanolamide (AEA), also known as anandamide, is a novel lipid-signal molecule that is an endogenous agonist (endocannabinoid) for cannabinoid receptors. We now report that AEA is present in human seminal plasma, mid-cycle oviductal fluid, and follicular fluid analyzed by high-performance liquid chromatography/mass spectrometry. Sperm are sequentially exposed to these reproductive fluids as they move from the vagina to the site of fertilization in the oviduct. Specific binding of the potent cannabinoid agonist [H-3]CP-55,940 to human sperm was saturable (K-D 9.71 +/- 1.04 nM), suggesting that they express cannabinoid receptors. R-methanandamide [AM-356], a potent and metabolically stable AEA analog, and (-)Delta(9) tetrahydrocannabinol (THC), the major psychoactive constituent of Cannabis, modulated capacitation and fertilizing potential of human sperm in vitro. AM-356 elicited biphasic effects on the incidence of hyperactivated sperm motility (HA) between 1 and 6 hr of incubation: at (2.5 nM) it inhibited HA, while at (0.25 nM) it stimulated HA. Both AM-356 and THC inhibited morphological alterations over acrosomal caps between 2 and 6 hr (IC50 5.9 +/- 0.6 pM and 3.5 +/- 1.5 nM, respectively). Sperm fertilizing capacity, measured in the Hemizona Assay, was reduced 50% by (1 nM) AM-356. These findings suggest that AEA-signaling may regulate sperm functions required for fertilization in human reproductive tracts, and imply that smoking of marijuana could impact these processes. This study has potential medical and public policy ramifications because of the incidence of marijuana abuse by adults in our society, previously documented reproductive effects of marijuana, and the ongoing debate about medicinal use of marijuana and cannabinoids.

Tumor angiogenesis, a major requirement for tumor outgrowth and metastasis formation, is regulated by pro- and anti-angiogenic factors. We have studied the expression of a panel of angiogenic factors, and of the angiogenesis inhibitor angiostatin, in a panel of human melanoma cell lines giving rise to xenografts with different vascular densities. Angiogenic-factor expression was analyzed in vitro (cell lines) and in vivo (xenografts), both at mRNA (RT-PCR and Northern blot) and at protein level (ELISA and Western blot). In vitro angiostatin generation was assessed by Western-blot analysis. Expression of bFGF and VEGF was clearly correlated with a high degree of vascularization, confirming the importance of these factors for tumor angiogenesis. In addition, there was exclusive or elevated in vitro expression of angiogenic factors IL-8, PDGF-AB, and, to a lesser extent, midkine in cell lines that formed highly vascularized tumors. A similar angiogenic-factor-expression pattern was found in the corresponding xenografts, with the exception of VEGF. in most cell lines, this factor had low expression in vitro which was strongly enhanced in vivo. Although all 8 melanoma cell lines were able to excise the angiostatin fragment from the plasminogen parent molecule in vitro, cell lines BLM and M14 showed the most potent angiostatin generation. In vitro angiostatin generation by cell lysates prepared from melanoma xenografts was comparable in all xenograft types. Thus, in our model system we found no correlation between angiostatin generation and vascular density. Our study has limited the number of pro-angiogenic factors that may be involved in melanoma angiogenesis, and provides evidence for the notion that regulation of tumor angiogenesis is dependent on multiple factors. Inhibition of angiogenesis for therapeutic purposes, therefore, should preferably not concentrate on a single factor. (C) 2000 Wiley-Liss, Inc.

Objective: To test the hypothesis that in women with polycystic ovary syndrome (PCOS), adrenal cytochrome P450c 17 alpha activity is different after physic,logic vs. pharmacologic ACTH stimulation and that ovarian activity promotes adrenal hyperactivity that is different after physiologic vs. pharmacologic ACTH stimulation.Design: Prospective controlled pilot study.Setting: Reproductive endocrinology unit of an academic medical center.Patient(s): Six women with PCOS who had adrenal hyperandrogenism were compared with four women with normal ovulation.Intervention(s): Adrenal dynamic blood sampling was performed before and after 6 months of GnRH agonist administration.Main Outcome Measure(s): Comparison of physic,logic and pharmacologic ACTH-stimulated levels of progesterone, 17-hydroxyprogesterone, and androgens before and after ovarian steroid modulation.Result(s): In women with PCOS, exaggerated responses of androstenedione and 11 beta-hydroxyandrostenedione as well as elevated ratios of 17-hydroxyprogesterone to progesterone and of androstenedione to 17-hydroxyprogesterone after physiologic ACTH stimulation did not persist after GnRH-agonist administration. Three of the six women with PCOS had an increased response of androstenedione and a ratio of androstenedione to 17-hydroxyprogesterone that were >2 SD above the mean of those in the women with normal ovulation after pharmacologic ACTH stimulation; this finding persisted after GnRH-agonist administration.Conclusion(s): In women with PCOS, increases in adrenal androgen sensitivity after physiologic ACTH stimulation reflected in both arms of cytochrome P450c 17 alpha activity may be influenced by ovarian activity. However, 17,20-lyase hyperactivity in a subset after pharmacologic ACTH stimulation may be an intrinsic adrenal disorder. (Fertil Steril(R) 1999;71:439-44. (C)1999 by American Society for Reproductive Medicine.).