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.

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.

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.

Safety and effectiveness of alveolar recruitment maneuvers and positive end-expiratory pressure during general anesthesia for cesarean section: a prospective, randomized trial.

INTRODUCTION: During cesarean section, the supine position reduces functional residual capacity and worsens lung compliance. We tested the hypothesis that alveolar recruitment maneuvers and positive end-expiratory pressure improve lung compliance in women undergoing general anesthesia for cesarean section. METHODS: Ninety women undergoing cesarean section were randomly assigned to one of two groups in a prospective, double-blind trial. In the alveolar recruitment maneuver group, pressure-control ventilation was used and inspiratory time was increased to 50% after delivery; positive end-expiratory pressure was increased to 20cmH2O and peak airway inspiratory pressure gradually increased to 45-50cmH2O. Volume-control ventilation was then used with low tidal volumes (6mL/kg) and positive end-expiratory pressure was reduced stepwise to 8cmH2O. In the control group, alveolar recruitment maneuvers were not used. Data were collected before and 3, 10 and 20min after the alveolar recruitment maneuver, before extubation and postoperatively at 10 and 20min. RESULTS: Dynamic compliance, peak airway inspiratory pressure, PaO2 and PaO2/FiO2 were significantly different in the alveolar recruitment maneuver group compared to controls at all time points during surgery except at baseline. Oxygen saturation was significantly greater in the alveolar recruitment maneuver group at 10 and 20min and before extubation. Dynamic compliance was 29.7-42.5% higher and peak airway inspiratory pressure 3.6-10.2% lower in the alveolar recruitment maneuver group compared to controls. The PaO2, PaO2/FiO2 and oxygen saturation were higher (9.4-12%, 10.3-11.9% and 0.4-1.3%, respectively) in the alveolar recruitment maneuver group. Postoperatively, PaO2 and oxygen saturation were significantly higher in the alveolar recruitment maneuver group compared to controls (PaO2 9.2% at 10min and 8.4% at 20min, oxygen saturation 0.8% at 10min and 1.1% at 20min). There were no significant differences in hemodynamic stability or adverse events between groups. CONCLUSION: Compared to standard care, the alveolar recruitment maneuver with positive end-expiratory pressure and low tidal volumes appears safe and effective in improving lung compliance and both intraoperative and postoperative oxygenation in women undergoing general anesthesia for elective cesarean section.

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.

Follicular fluid oocyte/cumulus-free DNA concentrations as a potential biomolecular marker of embryo quality and IVF outcome.

The present prospective study examined the follicular fluid oocyte/cumulus-free DNA concentrations (ff o/c-free DNA) during ovarian stimulation and the possible association between ff o/c-free DNA and embryological results such as embryo quality and pregnancy rate. Eighty-three women undergoing IV/ICSI-ET treatments were prospectively included in this study. ff o/c-free DNA was determined by conventional quantitative real time PCR-Sybr green detection approach. The 83 ff samples were categorized in two groups: group 1 (n = 62) with cumulus oocytes complexes (CoCs) ≥2 and group 2 (n = 21) with CoCs = 1. Group 1 revealed significant higher embryo quality in terms of mean score of embryo transfer (MSET), but lower ff o/c-free DNA concentrations compared to group 2. The two groups showed comparable pregnancy rates (positive hCG and clinical pregnancy). The higher the ff o/c-free DNA concentration, the lower the number of produced oocytes. ff o/c-free DNA did not seem to have any direct role in the IVF outcome. Further research is required to clarify whether ff o/c-free DNA is a biomolecular marker of embryo quality and IVF outcome.

The BMI of men and not sperm parameters impact on embryo quality and the IVF outcome.

It has been reported that increased body mass index (BMI) of men influences fecundity but it is not clear if it impacts on sperm parameters. Whether or not BMI of men influence sperm parameters and subsequently in vitro fertilization (IVF) result remains to be clarified. The aim of the present study was primarily to investigate the relationship between the BMI of men and sperm parameters (volume, concentration and motility) and whether or not it impacts on embryo quality and IVF outcome. Secondly, to investigate the impact of BMI of both men and women, in combination with their age, on IVF result. Three hundred and one couples were categorized according to their BMI. Group 1 (n = 64, both men and women had BMI l ≤ 25 kg/m(2) ), group 2 (n = 79, both men and women had BMI > 25 kg/m(2) ), group 3 (n = 142, men had BMI > 25 kg/m(2) and their wives had BMI ≤ 25 kg/m(2) ) and group 4 (n = 16, men had BMI ≤ 25 kg/m(2) and their wives had BMI > 25 kg/m(2) ). Overall (n = 301) BMI and age of men did not correlate with sperm parameters. Group 1 and group 4, regardless of the BMI of their women, demonstrated the highest quality of embryos and consequently the highest percentage of pregnancy. Furthermore, the score of the combination of both BMI and age of both men and women resulted in a threshold level of less than 800 with a relative high per cent of pregnancy. BMI of men does not correlate with sperm parameters, but influences the quality of produced embryos in such a way that impacts on pregnancy rate.

Elimination half-life of anti-Müllerian hormone.

CONTEXT: Anti-Müllerian hormone (AMH) is a glycoprotein that is secreted by the granulosa cells in the human ovary. In the postpubertal female, circulating AMH reflects the number of follicles within the ovary. It is mandatory to know the serum elimination half-life (t(1/2)) of AMH to study in vivo short-term changes of the hormone. OBJECTIVE: Our objective was to determine the kinetics of decay of AMH in the human female. PATIENTS, DESIGN, AND SETTING: Premenopausal women undergoing total abdominal hysterectomy plus bilateral salpingo-oophorectomy participated in this cohort study (n = 21) at an academic tertiary referral center. INTERVENTIONS: Serum samples were obtained immediately before surgery and in 12-h intervals thereafter for 8 d. MAIN OUTCOME MEASURE: AMH elimination was calculated, applying a one-compartment model with first-order kinetics. RESULTS: Mean preoperative AMH levels were 0.67 ng/ml (range, 0.1-1.78 ng/ml) and dropped to 0.08 ng/ml within 84 h after surgery. The AMH decay followed first-order kinetics. The mean terminal t(1/2) of AMH was calculated as 27.6 ± 0.8 h. CONCLUSION: AMH elimination reaches approximately 84% after 3 d, approximately 91% after 4 d, approximately 95% after 5 d, and can be considered complete after 8 d.

Effect of the position of the polar body during ICSI on fertilization rate and embryo development.

This prospective study was designed to evaluate and clarify further whether the position of the polar body (PB) in relation to injection site during intracytoplasmatic sperm injection (ICSI) has an impact on fertilization and developmental rates and consequently clinical pregnancy outcome. The study included 264 patients undergoing 306 ICSI cycles from September 2007 to January 2009 performed by the same practitioner. Of all oocytes retrieved, 1736 were in metaphase II (MII). From every woman reaching ovum pick up, all MII-collected oocytes were allocated to 1 of the 4 groups according to PB orientation. In group A, MII oocytes were injected with the PB at 6 o'clock, group B with the PB at 7 o'clock, group C with the PB at 11 o'clock, and a group D with the PB at 12 o'clock. A significantly higher proportion of fertilized oocytes were produced from oocytes that had been injected with the PB at 11 o'clock (79.2%) as compared to those at 6 o'clock (70.5%), 7 o'clock (64.4%), and 12 o'clock (68.8%). Furthermore, embryos derived from oocytes that were injected with the PB at 11 o'clock appeared to be of higher quality score than those of the other groups of oocytes. A higher clinical pregnancy rate (28.7%) was obtained after the transfer of embryos from oocytes that had been injected with the PB at 11 o'clock. Given the higher fertilization, developmental, and pregnancy rate in the 11 o'clock group, it is suggested that this may be the preferred position of the PB at ICSI.