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Purpose: To determine whether maternal age and number of transferred embryos influence early pregnancy losses in twin pregnancies compared to singletons following IVF/ICSI. Methods: We compared the pregnancy loss rates in singleton (n = 549) and twin (n = 252) gestations, stratified by maternal age (≤35 and > 35 years) and the number of transferred embryos (1–3 and 4–9). Results: Loss rates of singleton pregnancies were significantly higher than that in twins (OR 3.0, 95% CI 1.9, 4.9), especially among singletons conceived after transfer of 4–9 embryos (OR 5.0, 95% CI 2.2, 11.9). Younger mothers of twins had lower loss rates (OR 0.3, 95% CI 0.1, 0.9). Conclusion: Twins have a significantly reduced spontaneous miscarriage rate compared with singletons following IVF/ICSI. Higher implantation rates per cycle (i.e., development of twins rather than one live embryo) may represent a better capacity of the uterus for early embryonic development.

To determine whether pregnancies after IVF, with and without intracytoplasmic sperm injection (ICSI), have different early spontaneous loss rates.Retrospective analysis of IVF/ICSI dataset.The Center of Reproductive Medicine, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy.Women undergoing IVF with or without ICSI.First-trimester sonography at 6-7 weeks to count the number of embryos with positive heartbeat. The number of embryos lost was calculated from a second-trimester sonogram.Embryonic loss rates related to the initial number of embryos, maternal age35 oror =35 years, and IVF procedure.In vitro fertilization and ICSI had similar embryonic loss rates (odds ratio [OR] 1.2, 95% confidence interval [CI] 0.9-1.7, and OR 1.3, 95% CI 0.9-1.8 for women aged35 years andor =35 years, respectively). Younger women had fewer losses after IVF (OR 0.7, 95% CI 0.5-0.9). Multiples had lower loss rates compared with singleton pregnancies.In vitro fertilization and ICSI have similar spontaneous embryonic loss rates. Factors other than the initial number of embryos, maternal age, and IVF technique, such as embryo quality or uterine environment, might be involved in the outcome of multiple pregnancies in assisted reproductive technology procedures.

Objective: To evaluate the ability of women affected by functional hypothalamic secondary amenorrhea (FHSA) or polycystic ovary syndrome (PCOS) to adapt to stress. Design: Controlled clinical study. Setting: University hospital. Patient(s): Thirty-one patients affected by FHSA, 29 patients with PCOS, and 30 eumenorrheic women. Intervention(s): The subjects took the Stroop Color Word (Stroop CW) test and underwent blood sampling. Main Outcome Measure(s): Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP), and serum cortisol levels. Result(s): The healthy controls had better Stroop CW scores than patients with FHSA. Serum cortisol levels significantly increased during Stroop CW with respect to the baseline in patients with FHSA or PCOS but not in the healthy controls. The SBP, DBP, and HR of the controls as well as SBP and DBP of patients with PCOS were significantly higher than those measured in patients with FHSA both at the baseline and during Stroop CW. Conclusion(s): Patients with FHSA do not cope as well as healthy patients, and their autonomic response to stress is worse than both controls and patients with PCOS.

Survivors of the "vanishing" twin syndrome, which occurred in dichorionic twins only, and singletons that began as singletons, had similar mean gestational duration and birth weights, as well as similar frequencies of maternal and neonatal complications. This similarity persisted when conventional IVF and IVF plus ICSI cases were separately evaluated.

Serum plasma activin-A is measurable in the maternal circulation of healthy pregnant women, increases in specimens collected during the third trimester of gestation, and is highest at parturition. Hormone abnormalities are known to be associated with preterm labor or diabetes in pregnancy. Therefore, in the present study serum activin-A levels in normal controls were compared to those in pregnant women with preterm labor or gestational diabetes. In some cases, values were obtained before and after insulin therapy. In other controls and patients with preterm labor, the activin-A concentration in cord serum was also studied. A newly developed two-site immunotest was used to determine activin-A levels. Subjects included normal controls (n = 7), who were sampled throughout gestation every 5 weeks; pregnant women at term (38-40 weeks) not in labor (n = 22); pregnant women at term in spontaneous labor (< 3.0 cm dilated; n = 42); women in preterm labor (25-35 weeks; n = 38); and women with gestational diabetes (20-39 weeks; n = 9). In control women, serum activin-A levels increased from 4.8 +/- 5.5 micrograms/L (mean +/- SD) at 20 weeks to 25.4 +/- 27.8 micrograms/L at 40 weeks (P < 0.01), and values correlated with gestational age. Pregnant women in preterm labor had serum activin-A concentrations (89.04 +/- 173.31 micrograms/L) higher than those in normal controls (P < 0.01), and no significant correlation to gestational age was found in this group of pregnant women. Healthy women in labor showed serum activin-A concentrations higher than those in women at term but not in labor (P < 0.01). Diabetic patients had serum activin-A concentrations (52.39 +/- 23.32 micrograms/L) significantly higher than those in normal controls. In these patients, maternal serum activin-A concentrations significantly decreased to the range in healthy controls at the same gestational age after insulin therapy (9.48 +/- 3.82 micrograms/L). The present study shows that preterm labor is associated with increased concentrations of activin-A in the maternal circulation and cord serum. Hypersecretion of activin-A is also shown in same patients with gestation diabetes; this reverts to normal after insulin treatment.

Objective To investigate the hypothesis that Z-score criteria represent a reliable predictor of implantation rate and pregnancy outcome in in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) cycles, excluding the possibility of embryo selection before the embryo transfer. Design Retrospective clinical study. Setting Centre of Reproductive Medicine, Department of Obstetrics and Gynecology, Arcispedale S. Maria Nuova (ASMN), Reggio Emilia, Italy. Patient(s) We analyzed 393 pregnancies obtained by IVF or ICSI cycles. Intervention(s) Morphologic evaluations of Z-score in pre-embryos obtained from IVF or ICSI cycles. Main Outcome Measure(s) Evaluations of Z-scores, implantation rate, and clinical pregnancy outcome. Result(s) We did not find any statistically significant correlation between the Z-score of 1032 embryos transferred in 393 embryo transfers and the implantation rate or the pregnancy outcome. In particular, the best Z-score identified (Z1, 7.2%) did not seem to correlate with embryo implantation rate or pregnancy outcomes any better than those with worse scores (Z2, 6.9% and Z3, 85.9%). Conclusion(s) Our results seem to confirm that Z-score alone cannot be considered a better tool than standard morphologic criteria for identifying, controlling, or selecting embryos with a better chance of successful ongoing pregnancy.

The new Italian law on assisted reproduction technology rules that no more than three oocytes can be fertilized at one time, and that all embryos obtained must be transferred to the maternal uterus simultaneously. The aim of the present study was to investigate the influence of the new law on spontaneous embryonic/fetal losses through comparison of data collected over an identical period of time: the first year of application of the new law compared with the same time period 1 year before (March 10, 2004 to March 9, 2005 versus March 10, 2003 to March 9, 2004). A total of 271 clinical pregnancies were analysed. In the post-law period, a significantly lower percentage of total spontaneous embryonic losses compared with the pre-law period, and a higher percentage of surviving embryos in singletons and twins was observed. In conclusion, the impact of the limitations imposed by the new legislation regulating assisted reproduction in Italy seems to exert positive effects on spontaneous embryonic loss both in singletons and multiple pregnancies. These findings are in contrast to international predictions on this issue and, in general, are counter-intuitive. This suggests that further investigations on a larger cohort of women are required to confirm these preliminary results.

Objective To document outcomes of oocyte cryopreservation performed as a routine procedure in an IVF program. Design Describing the rate of oocyte survival, embryo transfer (ET), implantation, and live births of IVF–intracytoplasmic sperm injection performed on thawed oocytes. Setting Reproductive medicine center in Italy. Patient(s) Women (n = 696) who failed to conceive after IVF–intracytoplasmic sperm injection with fresh oocytes. Intervention(s) Surplus oocytes obtained during a failed cycle with fresh oocytes were frozen and then were thawed, micromanipulated, and transferred in a later cycle. Main Outcome Measure(s) Rates of oocyte survival, ETs, implantation, and live births were calculated in the entire cohort and in patients aged ≤38 and >38 years. Result(s) There were 29 pregnancies, for a total implantation rate of 6.3% (95% CI: 4.3, 9.0) per 456 ET cycles. The clinical-pregnancy rate was 19 (4.2%; 95% CI: 2.6, 6.4) of 456 ET cycles, with a take-home-baby rate of 7 (1.5%; 95% CI: 0.7, 3.0) of 456 ET cycles. Conclusion(s) Cryopreservation performed as a routine procedure for so-called salvaging of surplus oocytes is associated with poor implantation rates and with a probability of 1 live birth in 65 ET cycles.

Objective To investigate a possible correlation between interleukin-12 (IL-12) and IL-13 levels and lymphocyte subsets in the preovulatory follicles of patients with and without polycystic ovarian syndrome (PCOS). Design Controlled clinical study. Setting University hospital. Patient(s) Seventy-eight infertile women undergoing IVF-embryo transfer. Intervention(s) The subjects underwent blood sampling, ovum retrieval, and embryo transfer. Main outcome measure(s) Follicular fluid levels of T, androstenedione (A); IL-12, IL-13, activated T cells, T helper, and T-suppressor lymphocytes. Result(s) The level of IL-12 detected in follicular fluid (FF) was significantly lower in patients with PCOS than in normally ovulating women (mean: 1.47 ± 0.3 pg/mL vs. 2.25 ± 0.7 pg/mL, respectively); in contrast, FF IL-13 concentrations were significantly higher in the patients with PCOS than in the normally ovulating women (mean: 32.5 ± 3.7 pg/mL vs. 19.6 ± 2.5 pg/mL, respectively), as was the total number of activated T lymphocytes (11.5% ± 1.5% vs. 4.8% ± 0.4%). A significant correlation was observed between FF activated T-cell concentrations and FF IL-12, IL-13, T, and A levels. No significant differences were observed when these data were compared with embryological parameters. Conclusion(s) The present study shows significant differences in the correlation between FF IL-12 and IL-13 levels and T lymphocyte numbers in the subset of patients with PCOS as compared to normally ovulating women.

Objective: To evaluate the possible correlation between immunological changes and implantation rates in patients who undergo in vitro fertilization–embryo transfer (IVF-ET). Design: Controlled clinical study. Setting: University hospital. Patient(s): Forty infertile women undergoing IVF-ET. Intervention(s): Stroop Color Word (CW) test, State-Trait Anxiety Inventory (STAI) test, blood sampling. Main Outcome Measure(s): Heart rate and systolic and diastolic blood pressure responses to Stroop CW; circulating T, B, T-helper (CD4), and T-suppressor (CD8) lymphocytes. Result(s): The total number of T lymphocytes increased significantly during superovulation, resulting in significantly higher levels in subjects achieving embryo implantation than in those showing a failure of implantation. An opposite trend was observed for the activated T cells. The number of T-helper lymphocytes and the T-helper/T-suppressor ratio showed a significant increase from baseline to the time of pick-up only in patients with implantation. Conclusion(s): A prolonged condition of stress, which causes a decreased ability to adapt and a transitory anxious state, is associated with high amounts of activated T cells in the peripheral blood. Such a condition, in turn, is associated with a reduced implantation rate in women undergoing IVF-ET.

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte stimulating hormone (alpha-MSH) and beta-endorphin were used to localize, by immunohistochemistry, proopiomelanocortin (POMC)-derived peptides in the skin excised from different regions of the African lungfish Protopterus annectens. Immunoreactivity was observed in the epidermis mainly in the germinal layer. Using human POMC cDNA as hybridization probe, POMC-like mRNA was identified in situ in epidermal cells. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of opiate hormones.

The distribution of neurones expressing POMC mRNA in the cerebral ganglion of the protochordate ascidian, Styela plicata, was investigated using a non-radioactive in situ hybridization technique. Nerve cell bodies of mono and bipolar types expressing POMC mRNA, were observed mainly in the outer layer of the ganglion. Discrete groups of neurones containing POMC mRNA were also localized in the inner portion of the ganglion, and few small monopolar perykaria expressing POMC mRNA were visible at the emergence of the main nerve trunks. POMC mRNA labeling was also found at level of the cytoplasm of previtellogenic and vitellogenic oocytes, and of follicular cells. Our results demonstrate the expression of one or more genes in the cerebral ganglion and ovary, that may be similar to one or more regions of the mammalian POMC gene. Therefore POMC-related molecules seem to be involved in neuromodulatory pathways and regulatory mechanisms of the oogenesis of ascidians.

Antisera against adrenocorticotropic hormone (ACTH), alpha-melanocyte-stimulating hormone (alpha MSH) and beta-endorphin were used to localize pro-opiomelanocortin (POMC)-derived peptides in the ovary of the African lungfish Protopterus annectens by immunohistochemistry. Immunoreactivity was observed in the granulosa and the internal theca of the vitellogenic follicles. No immunoreactivity was observed in immature follicles. Using human POMC cDNA as the hybridization probe POMC-like mRNA was identified in situ in cells of the granulosa and internal theca of the vitellogenic follicles. No labeling was observed in primordial follicles. The demonstration in the same cells of POMC mRNA and POMC-related peptides immunoreactivity indicates a local production of the opiate hormones.

Chronic stress affects the reproductive function by modifying the neuroendocrine homeostasis. The aim of the present study was to clarify the neuroendocrine and the gonadal changes following chronic intermittent stress in male rats and the action of a neuroactive drug, acetyl-l-carnitine (ALC). The effect of two different stressors, cold water swimming or ether, on central beta-endorphin (beta-EP) and GnRH contents, and on plasma testosterone levels was investigated. In addition, the response to an acute stress in chronically stressed rats, treated or untreated with ALC (10 mg/day/rat p.o.), was evaluated. The stressors were applied twice a day for 10 days, and rats were killed before, during and after the last stress session. Mediobasal hypothalamus (MBH) beta-EP and GnRH contents, and plasma testosterone levels were evaluated by radioimmunoassay. The following results were obtained: (1) both chronic swimming and ether stress caused a decrease in hypothalamic beta-EP contents; (2) MBH GnRH contents increased after chronic swimming stress but not after ether stress; (3) chronic swimming stress induced a twofold decrease in plasma testosterone levels, while no changes were observed after ether stress; (4) the treatment with ALC prevented the decrease in plasma testosterone levels after chronic swimming stress, and (5) acute stress in chronically stressed animals caused an increase in MBH-beta-EP. The present data showed that chronic swimming stress reduces the reproductive capacity and impairs the capacity to respond to the acute stress and that ALC modulates the hormonal changes to physical stress and prevents the antireproductive effect of chronic cold swimming.

The aim of the present study was to investigate the possible production, localization, and action of follistatin in human placenta, fetal membranes (amnion, chorion), and maternal decidua. Four different experimental approaches were used: 1) Southern blot analysis following reverse polymerase chain reaction to identify follistatin messenger RNA (mRNA) in tissue homogenates; 2) immunohistochemistry to localize immunoreactive (ir-) follistatin in the various intrauterine tissues; 3) measurement by RIA of ir-follistatin levels in culture medium of placental cells; and 4) possible action of follistatin on human CG (hCG) and progesterone release from cultured placental cells. Placental and decidual cells collected during first trimester or at term gestation express follistatin mRNA; fetal membranes (amnion, chorion) at term also express follistatin mRNA. Immunoreactive follistatin is localized in syncytial cells of placental villi at term as well as in large decidual cells, in amnion epithelium, and in chorionic cells. The placental secretion of follistatin has been confirmed by the evidence of measurable levels of ir-follistatin in the medium of cultured placental cells at term; the release is time dependent and is not modified by the addition of forskolin or progesterone. The addition of increasing doses of recombinant human follistatin does not significantly influence the release of hCG or progesterone from cultured placental cells, whereas the activin A-induced hCG and progesterone release are completely reversed. The present data showed that 1) human placenta, fetal membranes, and decidua express follistatin mRNA; 2) ir-follistatin is localized and released from placental cells at term; and 3) follistatin has a functional role in the local control system regulating placental hormone production.

Immune and neuroendocrine systems interact at various levels. In particular, either cytokines activate the hypothalamus-pituitary-adrenal axis (HPA) or corticotropin-releasing hormone (CRH) induces the release of beta-endorphin from peripheral human mononuclear cells. The aim of the present study was to investigate whether CRH may affect cytokine production and activity in human peripheral blood mononuclear cells (PBMC). Primary cultures of human PBMC and monocytes were used. They were incubated in presence of different doses of synthetic human CRH. Media were collected and interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) levels were measured by ELISA, while interferon-gamma (IFN-gamma) levels were measured by bioassay. In addition, phytohemoagglutinin-induced lymphocyte proliferation was evaluated by testing [3H]thymidine incorporation in the presence of various doses of CRH. CRH significantly increased IL-6 release from PBMC (p < 0.01). The addition of CRH to PBMC significantly decreased IFN-gamma levels, in a dose dependent manner (p < 0.01). No significant effect of CRH was observed on lymphocyte proliferation or IL-1 beta production. The present results suggest a role for CRH as a paracrine mediator for human immune cells, increasing the evidence of a clear correlation between immune and neuroendocrine system.