Transdermal testosterone pretreatment in poor responders undergoing ICSI: a randomized clinical trial.

STUDY QUESTION: Does pretreatment with transdermal testosterone increase the number of cumulus-oocyte complexes (COCs) retrieved by more than 1.5 in poor responders undergoing intracytoplasmic sperm injection (ICSI), using recombinant follicle stimulating hormone (FSH) and gonadotrophin releasing hormone agonists (GnRHa)? SUMMARY ANSWER: Testosterone pretreatment failed to increase the number of COCs by more than 1.5 as compared with no pretreatment in poor responders undergoing ICSI (difference between medians: 0.0, 95% CI: -1.0 to +1.0). WHAT IS KNOWN ALREADY: Androgens are thought to play an important role in early follicular development by enhancing ovarian sensitivity to FSH. In a recent meta-analysis, testosterone pretreatment resulted in an increase of 1.5 COCs as compared with no pretreatment. However, this effect was based on the analysis of only two randomized controlled trials (RCTs) including 163 patients. Evidently, there is a need for additional RCTs that will allow firmer conclusions to be drawn. STUDY DESIGN, SIZE, DURATION: The present RCT was designed to detect a difference of 1.5 COCs (sample size required = 48 patients). From 02/2014 until 04/2015, 50 poor responders fulfilling the Bologna criteria have been randomized (using a randomization list) to either testosterone pretreatment for 21 days ( ITALIC! n = 26) or no pretreatment ( ITALIC! n = 24). PARTICIPANTS/MATERIALS, SETTING, METHODS: All patients underwent a long follicular GnRHa protocol. Recombinant FSH stimulation was started on Day 22 following GnRHa initiation. In the testosterone pretreatment group, a daily dose of 10 mg of testosterone gel was applied transdermally for 21 days starting from GnRHa initiation. Results are expressed as median (interquartile range). MAIN RESULTS AND THE ROLE OF CHANCE: No differences in baseline characteristics were observed between the two groups compared. Testosterone levels [median (interquartile range)] were significantly higher in the testosterone pretreatment on the day of initiation of FSH stimulation [114 (99.5) ng/dl versus 20 (20) ng/dl, respectively, ITALIC! P < 0.001]. Duration of FSH stimulation [median (interquartile range)] was similar between the groups compared [12.5 (3.0) days versus 12 (3.0) days, respectively, ITALIC! P = 0.52]. The number of COCs retrieved [median (interquartile range)] was not different between the testosterone pretreatment and the no pretreatment groups [3.5 (4.0) versus 3.0 (3.0), 95% CI for the median: 2.0-5.0 versus 2.7-4.3, respectively; difference between medians: 0.0, 95% CI: +1.0 to -1.0). Similarly no differences were observed regarding fertilization rates [median (interquartile range)] [66.7% (32.5) versus 66.7% (42.9), respectively, ITALIC! P = 0.97] and live birth rates per randomized patient (7.7% versus 8.3%, respectively, rate difference: -0.6%, 95% CI: -19.0 to +16.9). LIMITATIONS, REASONS FOR CAUTION: The study was not powered to detect differences less than 1.5 COCs, although it is doubtful whether these differences would be clinically relevant. Moreover, due to sample size restrictions, no conclusions can be drawn regarding the probability of live birth. WIDER IMPLICATIONS OF THE FINDINGS: The results of this randomized clinical trial, suggesting that pretreatment with 10 mg of transdermal testosterone for 21 days does not improve ovarian response by more than 1.5 oocytes, could be used to more accurately consult patients with poor ovarian response. However, an improvement in IVF outcome using a higher dose of testosterone or a longer pretreatment period cannot be excluded. STUDY FUNDING/COMPETING INTEREST: The study was partially funded by a Scholarship from the Academy of Athens. C.A.V. reports personal fees and non-financial support from Merck, Sharp and Dome, personal fees and non-financial support from Merck Serono, personal fees and non-financial support from IPSEN Hellas S.A., outside the submitted work. B.C.T. reports grants from Merck Serono, grants from Merck Sharp & Dohme, personal fees from Merck Serono, personal fees from Merck Sharp & Dohme, personal fees from IBSA & Ferring, outside the submitted work. TRIAL REGISTRATION NUMBER: NCT01961336. TRIAL REGISTRATION DATE: 10 October 2013. DATE OF FIRST PATIENT'S ENROLLMENT: 02/2014.

Chorionic villi derived mesenchymal like stem cells and expression of embryonic stem cells markers during long-term culturing.

Mesenchymal stem cells (MSCs) can be obtained from a variety of human tissues. MSCs derived from placental chorionic villi of the first trimester are likely to resemble, biologically, embryonic stem cells (ESC), due to the earlier development stage of placenta. In the present study long-term cultures of MSC-like cells were assessed in order to evaluate MSCs multipotent characteristics and molecular features during the period of culture. CV-cells obtained from 10 samples of chorionic villus displayed typical fibroblastoid morphology, undergone 20 passages during a period of 120 days, maintaining a stable karyotype throughout long term expansion. The cells were positive, for CD90, CD73, CD105, CD29, CD44, HLA ABC antigens and negative for CD14, CD34, AC133, and HLA DR antigens as resulted from the flow cytometry analysis. CV-cells were differentiated in adipocytes, osteoblasts, chondrocytes and neuronal cells under specific culture conditions. The expression of the ESC-gene markers POU5F1 (Oct-4) and NANOG was observed at earliest stages (4-12 passages) and not at the late stages (14-20 passages) by RT-PCR analysis. ZFP42 and SOX2 expression were not detected. Moreover, CV-cells were found to express GATA4 but not NES (Nestin). Chorionic villi-derived cells possess multipotent properties, display high proliferation rate and self-renew capacity, share common surface antigens with adult MSCs and express certain embryonics stem cells gene markers. These characteristics highlight chorionic villi as an attractive source of MSCs for the needs of regenerative medicine.

Genes and Conditions Controlling Mammalian Pre- and Post-implantation Embryo Development.

Embryo quality during the in vitro developmental period is of great clinical importance. Experimental genetic studies during this period have demonstrated the association between specific gene expression profiles and the production of healthy blastocysts. Although the quality of the oocyte may play a major role in embryo development, it has been well established that the post - fertilization period also has an important and crucial role in the determination of blastocyst quality. A variety of genes (such as OCT, SOX2, NANOG) and their related signaling pathways as well as transcription molecules (such as TGF-ß, BMP) have been implicated in the pre- and post-implantation period. Furthermore, DNA methylation has been lately characterized as an epigenetic mark since it is one of the most important processes involved in the maintenance of genome stability. Physiological embryo development appears to depend upon the correct DNA methylation pattern. Due to the fact that soon after fertilization the zygote undergoes several morphogenetic and developmental events including activation of embryonic genome through the transition of the maternal genome, a diverse gene expression pattern may lead to clinically important conditions, such as apoptosis or the production of a chromosomically abnormal embryo. The present review focused on genes and their role during pre-implantation embryo development, giving emphasis on the various parameters that may alter gene expression or DNA methylation patterns. The pre-implantation embryos derived from in vitro culture systems (in vitro fertilization) and the possible effects on gene expression after the prolonged culture conditions are also discussed.

Sperm DNA fragmentation measured by Halosperm does not impact on embryo quality and ongoing pregnancy rates in IVF/ICSI treatments.

Sperm DNA fragmentation (SDF) has been proposed to be one of the main markers regarding male infertility. A prospective study was performed to assess primarily whether sperm DNA damage has any impact on embryological data and secondarily on pregnancy rates. This prospective study evaluated the sperm DNA damage in fresh ejaculated sperm samples from couples undergoing IVF/ICSI treatments, using the improved SCD method, known as Halosperm(®) . The results were evaluated by performing statistical analysis with the statistical package of SPSS v17. A total of 156 fresh semen samples derived from 156 couples undergoing 156 IVF/ICSI cycles. From the 156 couples, 139 finally reached the embryo transfer (ET) procedure. Overall, SDF did not correlate with embryological data, while ongoing pregnancy rate/ET was 21.6%. SDF only correlated with sperm characteristics. After the categorisation of SDF (≤35% and >35%), according to the specific references of the method used, embryological data were comparable as also ongoing pregnancy rates. Using the SCD method, sperm DNA damage is associated neither with embryological data nor to pregnancy rates. However, we should not rule out the fact that extremely high DNA damages are associated with total pregnancy failure.

Inhibitory effect of submaximal doses of ghrelin on gonadotropin secretion in women.

The effect of ghrelin on gonadotropin secretion has been equivocal. Recent data have shown an inhibitory effect of repeated injections of ghrelin on nocturnal LH and FSH secretion in women. The aim of this study was to investigate the effect of submaximal doses of ghrelin on the diurnal secretion of gonadotropins. Ten normally cycling women received 2 consecutive dosages of ghrelin (0.15 µg/kg and 0.30 µg/kg) intravenously in the early and late follicular phases of the cycle. Saline was injected in the preceding cycle. Blood samples in relation to ghrelin or saline administration (time 0 and 90 min) were taken at -15, 0, 30, 90, 120, 150, and 180 min. Serum estradiol concentrations were significantly higher in the late than in the early follicular phase. Following ghrelin administration, serum LH and FSH levels decreased significantly, in relation to the saline injection, in the late (p<0.01), although FSH values showed a within the group decrease also in the early follicular phase (p<0.05). The study suggests a differential action of ghrelin on diurnal gonadotropin secretion throughout the follicular phase of the cycle.

The impact of cigarette smoking and alcohol consumption on sperm parameters and sperm DNA fragmentation (SDF) measured by Halosperm(®).

PURPOSE: Both cigarette smoking and alcohol consumption are somehow implicated in sperm function, but the impact of these two lifestyle factors on sperm parameters remains controversial. The present study is focused on the impact of cigarette smoking and alcohol consumption separately and combined on sperm parameters and sperm DNA fragmentation (SDF). METHODS: The study included 207 consecutive semen samples derived from men who were seeking semen analysis for fertility purposes in our IVF Unit. RESULTS: Semen volume, percent of degenerated spermatozoa and SDF were significantly correlated with the various smoking status. The percent of spermatozoa with small halos significantly correlated with the alcohol status. The smoking status of the men was correlated with the alcohol status. CONCLUSIONS: Cigarette smoking and alcohol consumption separately and combined were found to have deleterious effect on sperm parameters and SDF. It is suggested that both habits may contribute to infertility problems.

Ghrelin accelerates in vitro maturation of bovine oocytes.

Ghrelin, apart from its metabolic role, is nowadays considered as a basic regulator of reproductive functions of mammals, acting at central and gonadal levels. Here, we investigated for possible direct actions of ghrelin on in vitro maturation of bovine oocytes and for its effects on blastocyst yield and quality. In experiment 1, cumulus oocyte complexes (COCs) were matured in the presence of four different concentrations of ghrelin (0, 200, 800 and 2000 pg/ml). In vitro fertilization and embryo culture were carried out in the absence of ghrelin, and blastocyst formation rates were examined on days 7, 8 and 9. In experiment 2, only the 800 pg/ml dose of ghrelin was used. Four groups of COCs were matured for 18 or 24 h (C18, Ghr18, C24 and Ghr24), and subsequently, they were examined for oocyte nuclear maturation and cumulus layer expansion; blastocysts were produced as in experiment 1. The relative mRNA abundance of various genes related to metabolism, oxidation, developmental competence and apoptosis was examined in snap-frozen cumulus cells, oocytes and day-7 blastocysts. In experiment 1, ghrelin significantly suppressed blastocyst formation rates. In experiment 2, more ghrelin-treated oocytes matured for 18 h reached MII compared with controls, while no difference was observed when maturation lasted for 24 h. At 18 and 24 h, the cumulus layer was more expanded in ghrelin-treated COCs than in the controls. The blastocyst formation rate was higher in Ghr18 (27.7 ± 2.4%) compared with Ghr24 (17.5 ± 2.4%). Differences were detected in various genes' expression, indicating that in the presence of ghrelin, incubation of COCs for 24 h caused over-maturation (induced ageing) of oocytes, but formed blastocysts had a higher hatching rate compared with the controls. We infer that ghrelin exerts a specific and direct role on the oocyte, accelerating its maturational process.

FSH modulatory effect on human granulosa cells: a gene-protein candidate for gonadotrophin surge-attenuating factor.

Previous evidence indicates a homology of gonadotrophin surge-attenuating factor (GnSAF) to the carboxyl terminal of human serum albumin (HSA) and the ability of human granulosa cells to produce mRNA transcripts corresponding to this fragment, but the underlying mechanism is still unknown. This study investigated the role of FSH in vitro in the expression of the carboxyl terminal of HSA by human luteinized granulosa cells. Cells were cultured on poly-l-lysine-coated microscope slides in the absence or presence of 10 ng/ml FSH, followed by in-situ hybridization and immunocytochemistry. In the presence of FSH, mRNA transcripts corresponding to the carboxyl terminal of the HSA gene and corresponding protein could be detected in comparable intensity to that seen by hepatic HepG2 cells (positive control). Significantly lower expression was detected in granulosa cells cultured without FSH addition (P<0.01), but no expression was detected in HeLa cells. These results demonstrate for the first time that FSH stimulates the expression of the carboxyl terminal fragment of the HSA gene and corresponding protein in human luteinized granulosa cells. Therefore, the carboxyl terminal of HSA has a functional role in the ovary and this further supports the notion that this HSA fragment is a GnSAF-bioactive entity.

Effect of ghrelin and metoclopramide on prolactin secretion in normal women.

BACKGROUND: Administration of ghrelin to women stimulates the secretion of PRL but the mechanism is not known. AIM: The aim of the study was to investigate the effect of the dopamine receptor blocker, metoclopramide, on ghrelin-induced PRL release. SUBJECTS AND METHODS: Ten healthy normally cycling women were studied in the midluteal phase of 4 menstrual cycles. A single dose of normal saline (cycle 1), ghrelin (1 µg/kg) (cycle 2), metoclopramide (20 mg) (cycle 3), and ghrelin plus metoclopramide (cycle 4) was given to the women iv. Blood samples in relation to the iv injection (time 0) were taken at -15, 0, 15, 30, 45, 60, 75, 90, and 120 min. The response of PRL and GH was assessed. RESULTS: Following ghrelin administration (cycles 2 and 4), plasma ghrelin and serum PRL and GH levels increased rapidly, peaking at 30 min (p<0.001). PRL was also increased after the injection of metoclopramide (p<0.001, cycle 3), but the increase was much greater than after the administration of ghrelin. The combination of ghrelin and metoclopramide stimulated PRL secretion to the same extent with metoclopramide alone. No changes in GH and PRL levels were seen after saline injection. CONCLUSIONS: These results demonstrate that the stimulating effect of ghrelin on PRL secretion is not additive with that of metoclopramide, although a dose range study might provide further information.

Effect of ghrelin and thyrotropin-releasing hormone on prolactin secretion in normal women.

It is known that ghrelin stimulates the secretion of prolactin in women. The aim of this study was to examine the effect of exogenous thyrotropin-releasing hormone (TRH) on ghrelin-induced prolactin release. Ten healthy normally cycling women were studied in four menstrual cycles. The women were injected intravenously in late follicular phase (follicle size 16-17 mm) with a single dose of normal saline (cycle 1), ghrelin (1 microg/kg) (cycle 2), thyrotropin-releasing hormone (200 microg) (cycle 3), and ghrelin plus thyrotropin-releasing hormone (cycle 4). Blood samples in relation to saline or drugs injection (time 0) were taken at -15, 0, 15, 30, 45, 60, 75, 90, and 120 min. The prolactin and growth hormone responses were assessed. After ghrelin administration (cycles 2 and 4), plasma ghrelin, serum prolactin, and growth hormone levels increased rapidly, peaking at 15-30 min (p<0.001). The injection of thyrotropin-releasing hormone (cycle 3) stimulated prolactin secretion markedly (p<0.001), but reduced growth hormone levels significantly (p<0.05). Ghrelin induced a smaller prolactin increase than thyrotropin-releasing hormone (p<0.05). The combination of ghrelin and thyrotropin-releasing hormone induced a similar increase in prolactin levels as with thyrotropin-releasing hormone alone. No changes in growth hormone and prolactin levels were seen after saline injection. These results demonstrate that the stimulating effect of ghrelin on prolactin secretion is not additive with that of thyrotropin-releasing hormone.

The effect of estrogens on plasma ghrelin concentrations in women.

BACKGROUND: Data regarding the possible effects of estrogen on ghrelin secretion in humans are limited and contradictory. AIM: To investigate the effect of estradiol (E2) on ghrelin levels in normal pre- and post-menopausal women. SUBJECTS AND METHODS: A total of 21 women divided into 3 groups, i.e.13 normally cycling women (no.=7, group 1 and no.=6, group 2) and 8 post-menopausal women (group 3). Women of group 1 received increasing doses of E2 through skin patches from cycle days 3 to 5. Women of group 2, underwent total abdominal hysterectomy plus bilateral salpingo-oophorectomy (TAH+BSO) on cycle day 3. Women of group 3 received po increasing doses of E2 valerate for 15 days. Acylated ghrelin and E2 were measured in all blood samples. RESULTS: In group 1, plasma ghrelin levels did not show any significant changes for the week following cycle day 3. In group 2, ghrelin levels were similar before and after TAH+BSO and remained stable during the first 7 post-operative days. In group 3, no significant changes in plasma ghrelin levels were seen during the 15 days of E2 administration. CONCLUSIONS: The present study demonstrates for the first time that ghrelin values were not affected either by exogenous short-term estrogen administration to pre- and post-menopausal women or following ovariectomy in pre-menopausal women. It is suggested that ovarian hormones are not involved in the regulation of ghrelin secretion in women.

Effect of an OT antagonist on the control of the midcycle LH surge in women.

OBJECTIVE: Data in women regarding the role of OT in LH secretion during the LH surge are conflicting. As in previous studies blood samples for LH measurements were taken infrequently, we re-examined this matter in women with a fully characterized midcycle LH surge. DESIGN: Normal women were studied over two cycles. When the dominant follicle reached a diameter of either 16-17 mm (Group 1) or 18-19 mm (Group 2), the women were infused intravenously for 3 h with normal saline (cycle-1) or atosiban (cycle-2). PATIENTS: Fifteen women (10 in group 1 and 5 in group 2) aged 23-35 years. MEASUREMENTS: Blood samples were obtained every 6 h to characterize the midcycle LH surge. RESULTS: The time interval (mean +/- SEM) from the start of the infusion to the onset of the LH surge in the two cycles was 46.8 +/- 4.8 and 45.6 +/- 9.6 h in group 1 and 6.0 +/- 2.4 and 7.5 +/- 2.8 h, respectively, in group 2. LH values during the LH surge were similar in the two cycles except in group 1 at the point of 30 h at which LH value in cycle-2 (41.2 +/- 4.6 mIU/ml) was significantly lower than in cycle-1 (52.8 +/- 3.4 mIU/ml, P < 0.05). Nevertheless, in each group, the area under the curve for LH was similar in the two cycles. CONCLUSIONS: Antagonism of endogenous OT action by atosiban does not alter the LH profile during a fully characterized midcycle LH surge, suggesting that OT is not a major regulator of LH secretion in women.

Luteal-phase endocrinology.

The corpus luteum is formed from the pre-ovulatory follicle under the action of the mid-cycle LH surge. LH is the main luteotrophic hormone in women controlling luteal structure and function during the normal menstrual cycle. Local factors, however, including progesterone are also involved. If conception does not take place, luteolysis occurs as a physiological apoptotic process. Human chorionic gonadotrophin, secreted after implantation, is able to rescue the corpus luteum and extend its lifespan. In ovulation-induction cycles, the negative feedback effect of the ovarian steroids on the pituitary is markedly potentiated, leading to the suppression of endogenous LH secretion during the whole menstrual cycle. The marked suppression of LH secretion disrupts corpus luteum function regardless of the treatment regimen.

Endothelin-3 and PRL levels in the maternal and fetal circulation at delivery.

OBJECTIVE: The aim of the present cross-sectional study was to test the hypothesis that endothelin-3 (ET-3) is involved in PRL secretion via systemic hormonal interaction during labor. MATERIALS AND METHODS: Fifty healthy pregnant women with singleton pregnancies were included in the present study. At delivery, blood samples were drawn from umbilical vein and artery. At the same time, a blood sample was obtained from a peripheral vein of the mother. In all blood samples, plasma ET-3 and serum PRL concentrations were determined. The main outcome measures were the differences between maternal peripheral blood, umbilical artery and vein in terms of ET-3 and PRL levels, and the associations between ET-3 and PRL levels. RESULTS: ET-3 values (mean+/-SEM) in umbilical artery did not differ significantly from those in umbilical vein (4.94+/-0.27 vs 5.05+/-0.32 pg/ml) but were in both vessels significantly higher than in maternal vein (1.14+/-0.56 pg/ml, p<0.001). Serum PRL values showed similar patterns. There was a significant positive correlation of the ET-3 levels between umbilical artery and vein (r=0.906, p<0.001), but not between maternal peripheral venous blood and the umbilical vessels. Similar correlations were found for PRL values. However, no significant correlations were found between ET-3 and PRL levels in all vessels studied. CONCLUSIONS: The present study demonstrates for the first time that ET-3 levels are higher in fetal than in maternal circulation at term. The lack of correlation between ET-3 and PRL levels suggests that ET-3 does not play an important endocrine role in the control of maternal and fetal PRL secretion during labor.

Evidence that termination of the estradiol-induced luteinizing hormone surge in women is regulated by ovarian factors.

BACKGROUND: The endogenous LH surge is the result of the estrogen-positive feedback effect. However, the factors that are responsible for the termination of LH surge are not known. OBJECTIVE: The objective of the study was to investigate the mechanism that terminates the LH surge in women. SUBJECTS AND METHODS: Eight normally cycling women (aged 42-48 yr) were investigated in two cycles, i.e. cycle 1 (control) and cycle 2. In cycle 2 total abdominal hysterectomy plus bilateral salpingooophorectomy was performed on d 3. In both cycles, estradiol was administered transdermally at the dose of 100 microg on d 3 and 150 microg on d 4 and 5. Blood samples were obtained every 12 h from d 3 to 5 and every 6 h thereafter until d 9. RESULTS: In both cycles, after suppression of gonadotropins, the women displayed an endogenous LH surge. The time intervals between the commencement of estradiol treatment and the LH surge onset (73.5 +/- 1.5 vs. 76.5 +/- 2.5 h) and peak LH values (11.4 +/- 1.9 vs. 12.4 +/- 3.1 IU/liter) were comparable in the two cycles (mean +/- sem). After peaking, LH values decreased gradually in cycle 1, whereas in cycle 2 they remained stable and were higher than the corresponding values in cycle 1 (P < 0.05). Before the LH surge onset, estradiol values showed in both cycles a preovulatory pattern of changes, but starting 24 h after the onset of the LH surge, they were lower in cycle 2 (P < 0.05). Progesterone levels were similar in both cycles until the day of the LH surge onset, but in cycle 2 they declined thereafter and were lower than in cycle 1 (P < 0.05). CONCLUSIONS: It is suggested that ovarian factors rather than exhaustion of pituitary reserves are important for termination of the endogenous LH surge during the normal menstrual cycle.

Blood ghrelin, adiponectin and resistin levels during controlled ovarian stimulation in IVF cycles.

The aim of the present study was to investigate changes of blood ghrelin, adiponectin and resistin levels in IVF/ICSI-ET cycles. Twenty women were stimulated with recombinant FSH in a GnRH agonist short protocol for IVF/ICSI. Blood samples were taken on cycle day 2 before the commencement of injections, on cycle day 6 and on the days of HCG injection, oocyte pick up (OPU), embryo transfer (ET) as well as 7 and 12 days post-ET. Serum E2 levels increased during the stimulation, peaking on the HCG day and declined thereafter (p<0.001). Serum progesterone levels started to increase on the OPU day, peaking on the ET day (p<0.001) and decreased on days 7 and 12 post-ET. Plasma ghrelin remained unchanged during the whole cycle. Serum adiponectin levels remained stable during the stimulation period until the ET day and decreased on days 7 and 12 post-ET (p<0.001). Serum resistin levels increased until the ET day (p<0.05), remained unchanged on day 7 post-ET and decreased on day 12 post-ET (p<0.05). The present study shows for the first time that ghrelin levels did not change significantly during IVF/ICSI-ET cycles. Resistin levels increased during the stimulation period while adiponectin levels remained stable decreasing during the luteal phase.

Ghrelin suppresses the GnRH-induced preovulatory gonadotropin surge in dairy heifers.

Ghrelin, a known growth hormone (GH) secretagogue, alters gonadotropin secretion in many species. Our objectives were to study the effects of ghrelin, on GH, LH, FSH secretion, and on luteal function of the ensuing estrous cycle in cattle. The estrous cycles of eight heifers were synchronized with progesteron releasing intravaginal device, and ovulation was induced with GnRH. Eight animals were treated with 1.5 µg kg(-1) bovine ghrelin (group Ghr, n = 4) or saline (group C, n = 4). Starting with the first ghrelin injection, 13 blood samples were collected over a 4-hour period for the determination of ghrelin, GH, LH, and FSH concentration. Progesterone levels were measured in samples collected every other day after estrus expression. Data were analyzed by repeated measures of ANOVA followed by Bonferroni post hoc testing and t test. In group Ghr, ghrelin concentration increased significantly 15 minutes after the first injection and remained in elevated levels until the 90th minute after the last injection. At the time of third ghrelin injection, GH was significantly higher in the Ghr group compared with C (17.1 ± 1.3 vs. 2.6 ± 0.3 ng mL(-1), P < 0.0001). Similar differences were found for the next three samples collected 15, 30, and 60 minutes later; no difference was evident after 90 minutes. In group Ghr, the area under the curve for LH and FSH were significantly reduced compared with the ones of group C (266 ± 10.3 vs. 331.9 ± 7.3, P = 0.007 and 102.3 ± 2.0 vs. 134.9 ± 5.5, P < 0.005 for LH and FSH respectively). At particular time points the concentration of the two gonadotrophins in group Ghr was significantly lower than those of group C (15, 30, 45, 75, and 90 and 60, 75, 90, 120, and 150 minutes after GnRH administration for LH and FSH respectively). The duration of the following estrous cycle was shorter (P = 0.004) in group Ghr (19.0 ± 0.4 days) compared with C (21.8 ± 0.5 days). In days 4, 6, 8, 10, and 14, progesterone concentration was lower (P < 0.05) in group Ghr compared with C; similarly the progesterone area under the curve for group Ghr (113.1 ± 4.8) was suppressed (P = 0.007) compared with that of C (141 ± 4.8). These results imply that ghrelin acts on pituitary causing impaired response to the GnRH stimulus, and it is likely to affect luteinization of the cellular compartment of the preovulatory follicle, and/or to suppress steroidogenetic activity of the luteal cells.

Epidemiology of congenital anomalies in different ethnicities.

PURPOSE OF INVESTIGATION: To study the prevalence and the epidemiologic characteristics of major congenital anomalies (MCAs) in two different populations in Thrace-Greece. METHODS: The ethnic origin of all mothers who delivered in our department and the types of MCAs were determined. We compared the frequencies of MCAs between Christians and Muslims. The chi-square test, t-test, binary and multinomial logistic regression analysis were performed. RESULTS: The prevalence of MCAs was significantly higher in Muslims as compared to Christians (51/4,028 (12.78%) vs 49/5,994 (8.17%), p = 0.035). However, the frequencies of each type of MCA in the total number of deliveries between the two groups did not differ significantly. The central nervous system malformations were most frequently associated with perinatal mortality. CONCLUSION: This is the first study in Greece showing that there is probably a higher prevalence of MCAs in Muslims as compared to Christians, although it can not be elucidated whether this increased risk is related to specific differences between them.

Cystosarcoma phyllodes in a 13-year-old Muslim girl treated with conservative surgery: a case report.

Phyllodes tumor of the breast is an unusual tumor with an incidence of 1 in 100,000. In particular, it is a very rare neoplasm in adolescent girls and young women. The authors present a case of a 13-year-old adolescent girl with a large unilateral palpable mass in her right breast. The diagnosis of cystosarcoma phyllodes was made in a frozen section after wide local excision. The management and the cytological and histological characteristics are described with particular reference to the very unusual clinical presentation in this patient.

Removal of endometrial polyps by use of grasping forceps and curettage after diagnostic hysteroscopy.

PURPOSE OF INVESTIGATION: To determine the therapeutic efficacy of the use of gall stone forceps and curettage in endometrial polyps removal, after their detection with diagnostic hysteroscopy. METHODS: From 1997 to 2001, we conducted a prospective study in 53 patients who presented at our department for menstrual disorders, infertility problems or postmenopausal bleeding and in whom endometrial polyps were detected by hysteroscopy. All patients received general anesthesia and after hysteroscopic detection of the polyps' location, their removal was attempted by use of Desjardins gall stone forceps and curettage. Immediately after the procedure, a second hysteroscopy was performed in order to detect remnants of the polyps. RESULTS: Fifty patients presented with only one polyp, two with two polyps and one with three polyps. The mean diameter of the polyps ranged from 0.5 to 3 cm. The hysteroscopic appearance of all polyps was not suggestive of malignancy. During the second hysteroscopy we found parts or whole polyps in five and two cases, respectively, accounting for a therapeutic success of 86.8%. The hospitalization time for all patients was 24 hours and occurred no intraoperative or postoperative complications. CONCLUSION: Our method seems to be safe, with low cost and sufficient therapeutic outcome and could be used in hospitals with availability of diagnostic hysteroscopy only.