Overnight ovarian tissue transportation for centralized cryobanking: a feasible option.

RESEARCH QUESTION: Is overnight transportation of ovarian tissue before cryopreservation in a centralized cryobank from the FertiPROTEKT network feasible? DESIGN: Data from 1810 women with cryopreserved ovarian tissue after overnight transportation from December 2000 to December 2017 were analysed with a focus on transportation, tissue activity parameters and pregnancy, and delivery rates after transplantation. RESULTS: A total of 92.4% of tissue samples arrived at ideal temperatures of 2-8°C, 0.4% were transported at temperatures lower than ideal and 6.4% were transported at temperatures that were too high, generally due to mishandling of the inlayed cool packs of the transportation boxes. In 62 women, 78 tissue transplantations were carried out. A subgroup of 30 women who underwent a single orthotopic transplantation with fulfilled criteria of a complete follow-up after transplantation until the end of study, a premature ovarian insufficiency after gonadotoxic therapy as well as the absence of pelvic radiation, was further analysed. In this group, transplantations into a peritoneal pocket accounted for 90%. Transplants were still active at 1 year and above after transplantation in 93.3%. Pregnancy and delivery rates were 46.7% and 43.3%, respectively, with one ongoing pregnancy at the end of the study. CONCLUSIONS: Overnight transportation for central cryobanking is a feasible concept that results in high reproducible success rates through standardized professional tissue freezing and storage.

Melatonin status in pediatric intensive care patients with sepsis.

OBJECTIVE: Considering the potential immunomodulatory role of melatonin and its direct antioxidant activity, disturbances of the melatonin secretion pattern in the septic conditions could be particularly unfavorable. The aim of this study was to evaluate the nocturnal melatonin concentration and total 24-hr excretion of 6-sulfatoxymelatoninsulfate, melatonin's major urinary metabolite, in children with sepsis in the pediatric intensive care unit. DESIGN: Prospective observational pilot study. SETTING: A pediatric intensive care unit. PATIENTS: Twenty septic and 20 nonseptic children admitted between February 2008 and January 2010. INTERVENTIONS: None. MEASUREMENT AND MAIN RESULTS: Blood and urine samples were obtained from each patient on days 1, 2, 3, 5, and 10. There were no significant differences between the groups concerning age and gender. The median nocturnal melatonin concentrations were not significantly different between septic and nonseptic patients during the study period (p > .05). A subgroup analysis in septic patients showed that the nocturnal melatonin concentrations in nonsurvivors were significantly higher than in survivors, whereas total 6-sulfatoxymelatoninsulfate excretions in nonsurvivors were significantly lower than in survivors (p = .001 and p = .015, respectively). Furthermore, nocturnal melatonin concentrations of septic patients in septic shock state were statistically significantly higher than those of septic patients without septic shock state (p = .002). The 24-hr 6-sulfatoxymelatoninsulfate excretions in septic patients with liver dysfunction were found significantly lower than those in septic patients without liver dysfunction (p = .015). The presence of sedation and mechanical ventilation had no effect on the nocturnal melatonin concentrations in septic patients (p = .953 and p = .922, respectively). CONCLUSION: The present study shows that, in contradiction to results in adult patients, the nocturnal melatonin concentrations are not decreased in septic pediatric intensive care unit patients despite severe disease. Further investigations are needed to identify whether treatment with melatonin may have beneficial effects in pediatric intensive care unit patients with sepsis/septic shock.

Use of nocturnal melatonin concentration and urinary 6-sulfatoxymelatonin excretion to evaluate melatonin status in children with severe sepsis.

The aim of this study was to evaluate whether nocturnal melatonin concentration (NMC) and urinary 6-sulphatoxymelatonin (aMT6s) excretion can predict melatonin status in patients with severe sepsis in the pediatric intensive care unit (PICU). Blood samples for the determination of NMC were obtained from each patient at 3 a.m. Urine samples for the determination of aMT6s excretion were obtained from each patient at 12 h intervals. We obtained 89 blood and 178 urine samples from 23 septic patients, and 52 blood and 104 urine samples from 13 non-septic patients. The NMC of septic patients in a state of septic shock was significantly higher than that of septic patients not in septic shock (p = 0.017) and those of non-septic patients (p = 0.019). In contrast, there was no significant difference for nocturnal (NaMT6s) and total aMT6s (TaMT6s) excretion between septic patients with and without septic shock and non-septic patients (p > 0.05). The NMC was significantly higher in septic patients in shock with and without hepatic dysfunction (HD) than in non-septic patients (p = 0.004 and p = 0.024, respectively). NaMT6s and TaMT6s excretion was significantly lower in septic patients with HD than in septic patient without HD (p = 0.040 and p = 0.029, for NaMT6s and TaMT6s, respectively). Our results showed that an elevated NMC may not reflect an increased MT production in septic patients in septic shock. It seems that, to evaluate the melatonin status of septic PICU patients, it is necessary to collect both serum and urine samples.

Effect of mono-(2-ethylhexyl) phthalate on steroid production of human granulosa cells.

The phthalate ester mono-(2-ethylhexyl) phthalate (MEHP) is the active metabolite of di-(2-ethylhexyl) phthalate, a high-production-volume chemical used as a plasticizer and solvent in numerous consumer products. MEHP has been demonstrated to be a reproductive toxicant in rodents decreasing estradiol and progesterone production in preovulatory granulosa cells. In the present study, we examined the effect of MEHP on steroid production of human granulosa-lutein (GL) cells. Human GL cells collected from women undergoing in vitro fertilization were cultured in medium containing FSH, hCG and 8-Br-cAMP, respectively, together with various concentrations of MEHP (0-500 micromol L(-1)). After incubation for 48 h estradiol and progesterone were assayed in the spent culture medium. Furthermore, aromatase activity and mRNA levels of GL cells were determined. Basal as well as FSH-, hCG- and 8-Br-cAMP-stimulated estradiol production of GL cells was suppressed by MEHP in a dose-dependent manner (IC(50)=105 micromol L(-1), 138 micromol L(-1), 49 micromol L(-1) and 78 micromol L(-1)). Furthermore aromatase activity and mRNA levels were reduced in GL cells cultured with MEHP. In contrast, MEHP did not alter the production of progesterone up to a concentration of 167 micromol L(-1). The present data indicate that MEHP is a specific inhibitor of estradiol production in human GL cells with a post-cAMP site of action. The inhibition of estradiol production obviously results from a reduction of aromatase activity on the transcript level. As the in vitro effective doses of MEHP are within the range of real environmental exposure levels an inhibitory effect on estrogen production in vivo seems to be possible.

Concept Paper on the Technique of Cryopreservation, Removal and Transplantation of Ovarian Tissue for Fertility Preservation.

The cryopreservation of ovarian tissue with subsequent transplantation of the tissue represents an established method of fertility protection for female patients who have to undergo gonadotoxic therapy. The procedure can be performed at any point in the cycle and thus generally does not lead to any delay in oncological therapy. With the aid of this procedure, more than 130 births to date worldwide have been able to be recorded. The birth rate is currently approximately 30% and it can be assumed that this will increase through the further optimisation of the cryopreservation and surgical technique. The concept paper presented here is intended to provide guidance for managing cryopreservation and transplantation of ovarian tissue to German-speaking reproductive medicine centres.

Human ovarian tissue preservation: is vitrification acceptable method for assisted reproduction?

The aim of this study was to evaluate the most successful vitrification protocol. The ovarian tissue pieces were randomly distributed into seven groups including fresh control. Each experimental group was divided into three subgroups according to the following cooling modes: a) in 1.8 ml cryo-vials with 1ml vitrification medium, b) in 1.8 ml cryo-vials with 0.1 ml vitrification medium, or c) by direct dropping with 0.05 ml vitrification medium into liquid nitrogen. The best results were observed in the protocol using 2.62 M dimethylsulphoxide + 2.6 M acetamide + 1.31 M propylene glycol + 0.0075M polyethylene glycol in combination with direct dropping of ovarian tissue pieces into liquid nitrogen. The vitrified and rewarmed samples after in vitro culture with this protocol showed 86 percent normally developed follicles, compared with 92 percent in fresh non-treated control. The concentrations of hormones in spent medium from culture of the same samples were 319 pg/ml for 17beta-estradiol and 2.6 ng/ml for progesterone compared with fresh non-treated control (253 pg/ml and 6 ng/ml, respectively). The results obtained by vitrification of ovarian tissue with this protocol were compatible with those of the fresh ovarian tissue.

Detection of human antibodies generated against therapeutic antibodies used in tumor therapy.

Application of monoclonal antibodies (MAb) for therapeutic purpose may induce the formation of human antibodies directed against the immunogenic epitopes, which are presented on the therapeutic MAb. Formation of such human antibodies mostly is an undesired side effect, but in the case of newly developed immunotherapeutic tumor treatment strategies it represents the underlying therapeutic effect. Especially the formation of so-called "internal image" antibodies, which are directed against the antigen-combining site (paratope) of the therapeutic antibody, is supposed to evoke specific immune responses against tumor antigens mediated via idiotype-anti-idiotype interactions within the immunoregulatory network. For the monitoring of the immune response after antibody application, the newly formed human antibodies can be measured with immunoassay procedures involving the applied therapeutic antibody as test antibody. Because the original antigen is directed against the therapeutic antibody and inhibits the binding of "internal image" antibodies, a special assay design is needed to avoid interferences with samples containing the antigen. We describe an immunoassay procedure that allows the correct quantification of antiidiotypic antibodies including "internal image" antibodies that are not affected by the original antigen or other serum components that may interact with the therapeutic antibody.

Cryopreservation of human ovarian tissue: comparison of rapid and conventional freezing.

Cryopreservation, which is the most important procedure in ovarian tissue banking, can be divided into two methods: conventional freezing and rapid freezing. In previous study, the higher effectiveness of rapid freezing in comparison with the conventional freezing for human oocytes and embryos was shown. Data on comparison of these two methods for human ovarian tissue are limited. The aim of this study was to compare conventional freezing and rapid freezing for human ovarian tissue. Ovarian tissue fragments from 14 patients were transported to the laboratory within 22-25 h in a special, isolated transport box, which can maintain a stable temperature of between 5 and 8 degrees C for 36 h. Small pieces of ovarian tissue (1 x 1-1.5 x 0.7-1mm) were randomly distributed into four groups: Group 1: control, fresh pieces immediately after receiving transport box, Groups 2 and 3: experimental pieces after rapid freezing/warming, and Group 4: experimental pieces after conventional freezing/thawing. All pieces were cultured in vitro for 14 days. The viability of the tissue by in vitro production of hormones and development of follicles after culture was evaluated. The level of estradiol 17-beta and progesterone was measured using heterogeneous competitive magnetic separation immunoassay. For histological analysis, the number of viable and damaged follicles was counted. After culture of fresh tissue pieces (Group 1), rapidly frozen/warmed pieces (Groups 2 and 3), and conventionally frozen/thawed pieces (Group 4), the supernatants showed estradiol 17-beta concentrations of 358, 275, 331, and 345 pg/ml, respectively, and progesterone concentrations of 3.02, 1.77, 1.99, and 2.01 ng/ml, respectively. It was detected that 96%, 36%, 39%, and 84% follicles for Groups 1, 2, 3, and 4, respectively, were normal. For cryopreservation of human ovarian tissue, conventional freezing is more promising than rapid freezing.

Melatonin Concentrations and Antioxidative Capacity of Human Breast Milk According to Gestational Age and the Time of Day.

BACKGROUND: While changes in the composition of breast milk throughout the lactation period are well known, little is known about the antioxidative capacity of breast milk and its regulation as a function of time of day. OBJECTIVE: The aim of this study was to evaluate the antioxidative capacity in breast milk and its regulation by time of day. METHODS: Melatonin, superoxide dismutase (SOD), glutathione peroxidase 3 (Gpx3) concentrations, and the total antioxidative capacity (TAOC) were analyzed in 105 breast milk samples and 12 maternal serum samples from 21 healthy nursing mothers. RESULTS: Comparison between daytime breast milk (collected from 1000-2200 h) and nighttime breast milk (collected from 2200-1000 h) revealed significantly higher concentrations of melatonin and Gpx3 in nighttime milk (melatonin: 1.5 pg/mL [1.0-2.1] day vs 7.3 pg/mL [3.8-13.6] night, median [quartiles], with an estimated mean night-to-day ratio of 5.2 [3.9, 7.1], P < .001; Gpx3: 1436 ng/mL [765-2060] day vs 1800 ng/mL [1242-2297] night, night-to-day difference 192.1 [0.6, 383.7], P = .049). Subgroup analysis showed that melatonin had a circadian rhythm in both preterm and term milk, with a significantly higher nighttime concentration ( P < .001), while antioxidant enzymes had a circadian rhythm only in preterm milk, with a significantly higher nighttime concentration for Gpx3 and a significant higher daytime concentration for SOD and TAOC ( P = .041 and P = .049, respectively). We found no significant correlation between the concentration of melatonin and the concentration of SOD, Gpx3, or TAOC. Moreover, there were no significant correlations observed between gestational age and the concentration of melatonin and antioxidant enzymes. CONCLUSION: Because of its higher melatonin and Gpx3 content, future research is needed to determine if preterm nighttime milk ought to be the first choice in the feeding of high-risk preterm infants.

HLA-G is a potential tumor marker in malignant ascites.

PURPOSE: Molecular approaches as supplements to cytological examination of malignant ascites may play an important role in the clinical management of cancer patients. HLA-G is a potential tumor-associated marker and that one of its isoforms, HLA-G5, produces a secretory protein. This study is to assess the clinical utility of secreted HLA-G levels in differential diagnosis of malignant ascites. EXPERIMENTAL DESIGN: We used ELISA to assess whether secretory HLA-G (sHLA-G) could serve as a marker of malignant ascites in ovarian and breast carcinomas, which represent the most common malignant tumors causing ascites in women. RESULTS: On the basis of immunohistochemistry, 45 (61%) of 74 ovarian serous carcinomas and 22 (25%) invasive ductal carcinomas of the breast demonstrated HLA-G immunoreactivity ranging from 2 to 100% of the tumor cells. HLA-G staining was not detected in a wide variety of normal tissues, including ovarian surface epithelium and normal breast tissue. Revese transcription-PCR demonstrated the presence of HLA-G5 isoform in all of the tumor samples expressing HLA-G. ELISA was performed to measure the sHLA-G in 42 malignant and 18 benign ascites supernatants. sHLA-G levels were significantly higher in malignant ascites than in benign controls (P < 0.001). We found that the area under the receiver-operating characteristic curve for sHLA-G was 0.95 for malignant versus benign ascites specimens. At 100% specificity, the highest sensitivity to detect malignant ascites was 78% (95% confidence interval, 68-88%) at a cutoff of 13 ng/ml. CONCLUSIONS: Our findings suggest that measurement of sHLA-G is a useful molecular adjunct to cytology in the differential diagnosis of malignant versus benign ascites.

Antioxidative status and oxidative stress in the fetal circulation at birth: the effects of time of delivery and presence of labor.

BACKGROUND: There are important physiological changes in the maternal, placental, and fetal compartments during pregnancy and labor. Increased oxidative stress has been demonstrated during labor. Melatonin has been reported to serve as an indirect antioxidant via the stimulation and induction of antioxidant enzymes as superoxide dismutase (SOD) and glutathione peroxidase (Gpx) in several tissues. AIM: : To assess whether the melatonin status, presence of labor at the time of birth and the time of delivery influence the extracellular antioxidative enzymes and DNA oxidative stress in newborns. METHODS: The extracellular antioxidative status and oxidative stress were analyzed by measuring the concentrations of the SOD3, Gpx3 and 8-hydroxydeoxyguanosine (8-OHdG) in the cord blood of 135 newborns. Newborns delivered during the day and at night and newborns delivered by spontaneous vaginal delivery (labor group) or elective caesarean section delivery (no labor group) were studied. OUTCOME MEASURES: The concentration of melatonin, SOD3, Gpx3 and 8-OHdG. RESULTS: Independent of the time of delivery, we found significantly higher melatonin, SOD3 and Gpx3 but lower 8-OHdG concentrations in the labor group than in the no labor group. We did not observe a correlation between the concentration of melatonin and SOD3, Gpx3 or 8-OHdG, or a day-night difference in SOD3, Gpx3 or 8-OHdG. CONCLUSION: Our findings suggest that oxidative stress during labor leads to an elevation of melatonin, SOD3 and Gpx3 in the fetal circulation, protecting the newborn from serious impairment, which is reflected by lower 8-OHdG levels. The melatonin status at the time of birth does not influence the extracellular SOD3 or Gpx3 concentrations.

The impact of culture conditions on early follicle recruitment and growth from human ovarian cortex biopsies in vitro.

OBJECTIVE: To investigate the effects of a dynamic fluidic culture system on early in vitro folliculogenesis in standardized ovarian cortex biopsies. DESIGN: Cortical small strips were cultured for 6 days in a conventional static or in a dynamic fluidic culture system. SETTING: University-affiliated laboratory with an associated cryobank facility. PATIENT(S): Ovarian cortex from postpuberal female cancer patients (26.1 ± 1.3 y) who opted for cryopreservation of their tissue for fertility protection before gonadotoxic cancer therapy. With informed consent of the Institutional Ethics Committee, part of the tissue was available for patient-related research studies. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The viability and proliferative capacity of the cortex biopsies were evaluated by chemiluminescent microparticle immunoassay for detection of in vitro produced E2 and P in the supernate, by viable follicle counting via calcein staining, by histologic analyses, and by total RNA preparation and reverse transcription for real-time polymerase chain reaction of selected early folliculogenesis genes. RESULT(S): The data support the notion that early follicle development can be better achieved in vitro in a dynamic fluidic culture system. The findings are based on the presence of more viable follicles, higher expression levels of early folliculogenesis genes KIT-L, INHB, and GDF9, and the absence of premature luteinization of follicles. CONCLUSION(S): This study provides evidence that dynamic fluidic culture is a promising approach for investigating early follicular recruitment and growth in cortical biopsies. It may serve as a first step in a multistep culture system to design a complex in vitro system for complete folliculogenesis.

Melatonin concentration in umbilical cord blood depends on mode of delivery.

BACKGROUND AND AIMS: Melatonin (MT) is rapidly transferred from the maternal to fetal circulation in humans. There is little knowledge about factors which influence the MT concentration (MTc) in the umbilical cord (UC) blood during delivery. The aim of our study was to evaluate the MT status in the UC blood according to the time and mode of delivery. SUBJECTS AND METHODS: Blood samples from umbilical artery (UA) and vein (UV) were collected from spontaneous vaginal deliveries (SVD, n=122) and cesarean section deliveries (CSD, n=188). MTc was measured using a commercially available radioimmunoassay. RESULTS: The MTc was not significantly different between UA and UV blood both at daytime and at nighttime (p=0.216 and p=0.440, respectively). Both in UA and in UV, the MTc was significantly higher at nighttime than at daytime (p<0.0001). Compared with the CSD group, MTc in the SVD group was significantly higher both at night- and daytime (p<0.05). MTc both in UA and in UV was found to be not significantly different between patients with and without risk factors for stress including pregnancy complications (e.g., preeclampsia) and intrapartum complications (e.g., emergency section, pathological doppler, and pathological cardiotocography) (p>0.05). CONCLUSION: Our study revealed for the first time that MTc both in UA and in UV depends on modus of labor. In agreement with other studies, we found a clear circadian MT rhythm in the UC blood of neonates. The results of our study may suggest to a physiological role of MT at the onset of labor.

Methylation at global LINE-1 repeats in human blood are affected by gender but not by age or natural hormone cycles.

Previously, we reported on inter-individual and gender specific variations of LINE-1 methylation in healthy individuals. In this study, we investigated whether this variability could be influenced by age or sex hormones in humans. To this end, we studied LINE-1 methylation in vivo in blood-derived DNA from individuals aged 18 to 64 years and from young healthy females at various hormone levels during the menstrual cycle. Our results show that no significant association with age was observed. However, the previously reported increase of LINE-1 methylation in males was reconfirmed. In females, although no correlation between LINE-1 or Alu methylation and hormone levels was observed, a significant stable individual specific level of methylation was noted. In vitro results largely confirmed these findings, as neither estrogen nor dihydrotestosterone affected LINE-1 or Alu methylation in Hek293T, HUVEC, or MDA-kb2 cell lines. In contrast, a decrease in methylation was observed in estrogen-treated T47-Kbluc cell lines strongly expressing estrogen receptor. The very low expression of estrogen receptor in blood cells could explain the observed insensitivity of methylation at LINE-1 to natural hormonal variations in females. In conclusion, neither natural cycle of hormones nor age has a detectable effect on the LINE-1 methylation in peripheral blood cells, while gender remains an important factor.

Utility of salivary melatonin measurements in the assessment of the pineal physiology in newborn infants.

SUBJECTS: The aim of this study was to assess whether salivary melatonin could be used as a reliable alternative to serum melatonin to study the pineal physiology in newborn infants. DESIGN AND METHODS: The 95 newborn infants were allocated to four groups according to the time of sampling (09-11am, 03-05pm, 09-11pm, and 03-05am). RESULTS: The median melatonin levels in serum and saliva were not significantly different between groups: median (interquartile range), 18.4pg/mL (13.9-26.0pg/mL) and 10.6pg/mL (7.5-14.9pg/mL); 13.3pg/mL (11.5-19.0pg/mL) and 9.1pg/mL (7.8-14.2pg/mL); 16.0pg/mL (12.4-18.7pg/mL) and 12.3pg/mL (8.2-16.8pg/mL); 13.0pg/mL (8.8-27.4pg/mL) and 11.2pg/mL (7.7-16.6pg/mL) for groups 1, 2, 3, and 4, respectively (p>0.05). The results revealed a highly significant correlation between the serum and salivary melatonin levels (Pearson's correlation coefficient, r=0.763; P<0.001). CONCLUSION: Melatonin levels in saliva reflect those in serum at any time of the day and like serum melatonin levels do not increase at night.

Saliva as a valid alternative in monitoring melatonin concentrations in newborn infants.

BACKGROUND AND AIMS: Pineal physiology is not completely elucidated in newborn infants. As melatonin in pharmacological doses has been reported to reduce oxidative stress in neonates with asphyxia, respiratory distress syndrome, or sepsis, there is the need to better understand the physiological role of melatonin in the neonatal period. The aim of this study was to evaluate a new saliva sampling method suitable for newborn infants and to assess whether salivary melatonin could be used as a reliable, non-invasive, pain-free alternative to serum melatonin to study the pineal physiology in newborn infants. SUBJECTS AND METHODS: In 86 healthy term newborn infants, a serum sample was collected by venipuncture after 36h of life during blood sampling for newborn screening, immediately after a saliva sample had been collected. RESULTS: Of the 86 saliva samples, 62 were 'ideal' samples for the analysis. The median serum and salivary melatonin levels of the 62 newborns were 14.4pg/mL (11.5-20.5pg/mL) and 10.8pg/mL (7.5-16.1pg/mL), respectively. There was no significant difference between serum and salivary melatonin levels. The results revealed a highly significant correlation between the serum and salivary melatonin levels (Pearson's correlation coefficient, r=0.793; p<0.001). Linear regression analysis showed that the equation for the relationship between serum (x) and saliva (y) was y=1.12x-5.58pg/mL. CONCLUSION: To study pineal function in newborn infants, saliva collection using cotton buds and measurement of melatonin in saliva offers a valid, non-invasive, pain-free and practical alternative to blood sampling and determination of serum melatonin.

Cryopreservation of human ovarian tissue: effect of spontaneous and initiated ice formation.

This investigation compared conventional freezing of human ovarian tissue using either spontaneous or initiated ('seeded') ice formation. Biopsies of ovarian tissue were obtained from women with indications for chemotherapy or radiotherapy. Small pieces of experimental tissue were randomly distributed into three groups that were then subjected to different treatments prior to culture in vitro for 16 days: the control group, no treatment, cultured immediately after biopsy (group 1); cryopreservation/thawing with spontaneous ice formation (group 2); and cryopreservation/thawing with initiated ice formation (group 3). Follicle viability and hormonal activity were then evaluated. There was no significant difference between groups regarding the concentration of oestradiol 17-beta in the culture supernatant, whereas progesterone concentration was significantly (P < 0.05) higher in group 1 compared with group 2 or 3. There was a significant (P < 0.05) difference in primordial and primary follicle density between all of the groups (group 1 having the highest and group 2 having the lowest) and group 2 had significantly (P < 0.05) fewer normal grade follicles than the other two groups. For optimal cryopreservation of human ovarian tissue, the protocol of conventional freezing should therefore include a step of initiated ice formation.

Pitfalls in assessment of progesterone production by granulosa cells cultured in contact with silicone rubber or paraffin oil.

BACKGROUND: The influence of silicone templates, used for compartmentalization of culture dishes, on progesterone accumulation in granulosa cell cultures is studied and compared with the effect of paraffin oil, which is frequently used to cover oocyte/embryo cultures. METHODS: Human granulosa-lutein cells were cultured in culture dishes compartmentalized by silicone templates, or in polystyrene plates under paraffin oil. Progesterone concentrations in the culture supernatant were compared with controls cultured in polystyrene plates. RESULTS: The progesterone concentration in culture supernatant was grossly reduced in silicone template cultures (2+/-0.7% of control). No inhibitory activity was identified in medium conditioned by preincubation with silicone rubber, but progesterone was absorbed from spiked medium incubated in silicone templates (recovery <2%). Progesterone concentration in culture supernatant was also reduced by a paraffin oil overlay (38+/-3% of control). From steroid spiked microdrops under oil, <2% of progesterone and 85+/-4% of estradiol was recovered. CONCLUSION: The steroidogenesis of cells cultured in silicone templates or under oil cannot be assessed correctly. It has to be considered that the concentration of lipophilic compounds may be grossly changed due to absorption by silicone rubber or paraffin oil.

Leptin, VEGF, IGF-1, and IGFBP-3 concentrations in serum and follicular fluid of women undergoing in vitro fertilization.

We studied the influence of recombinant follicle-stimulating hormone (rFSH) stimulation on the concentration of leptin, vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) in serum and follicular fluid (FF) in women undergoing assisted reproduction. To test the hypothesis that these hormones could predict successful implantation and that the levels correlate with pregnancy rate. Sequential serum samples were drawn at the beginning of stimulation and on the day of embryo transfer (ET) from 84 women undergoing IVF. The follicular fluid (FF) obtained during oocyte retrieval was collected and the concentration of leptin, VEGF, IGF-1 and IGFBP-3 were measured in all samples. The hormones were measured by commercially available IRMA, RIA or EIA. Patients' characteristics and results of the assisted reproductive cycles were registered. Serum concentrations of VEGF, IGF-1 and IGFBP-3 significantly decreased during rFSH treatment. In contrast, serum leptin significantly increased after rFSH treatment. A strong correlation was found between the FF - levels of IGF-1, IGFBP-3, leptin and respective serum levels. With regard to IVF outcome, higher serum concentrations of IGF-1, IGFBP-3 and VEGF on the day of oocyte retrieval were observed in conception cycles vs. non-conception cycles. No such difference, however, was apparent at the beginning of the stimulation cycle. There was no association between FF levels of any of these hormones and IVF outcome. Our results demonstrate that VEGF, IGF-1, IGFBP-3 and leptin levels are affected by rFSH during controlled ovarian hyperstimulation and that there is a direct association between serum and FF levels, albeit without clinical implications