Permanence of de novo segmental aneuploidy in sequential embryo biopsies.

STUDY QUESTION: Is de novo segmental aneuploidy (SA) a biological event or an artifact that is erroneously interpreted as partial chromosome imbalance? SUMMARY ANSWER: The detection of de novo SA in sequential biopsies of preimplantation embryos supports the biological nature of SA. WHAT IS KNOWN ALREADY: Although some SAs are detected in oocytes and in blastocysts, the highest incidence is observed in cleavage-stage embryos. Based on these findings, we can postulate that the majority of cells affected by SAs are eliminated by apoptosis or that affected embryos mainly undergo developmental arrest. STUDY DESIGN, SIZE, DURATION: This retrospective study includes 342 preimplantation genetic testing for aneuploidy (PGT-A) cycles performed between January 2014 and December 2018. Chromosome analysis was performed on 331 oocytes, 886 cleavage-stage embryos and 570 blastocysts (n = 1787). From 268 expanded blastocysts, the blastocoelic fluid (BF) was also analyzed (resulting in 2025 samples in total). In cases of SAs involving loss or gain in excess of 15 Mb, embryos were not considered for transfer and sequential biopsies were performed at following stages. This resulted in 66 sets where the initial diagnosis of SAs (4 made in polar bodies, 25 in blastomeres and 37 in trophectoderm (TE) cells) was followed up. PARTICIPANTS/MATERIALS, SETTING, METHODS: A total of 2082 samples (2025 + 27 whole embryos) were processed by whole genome amplification followed by array comparative genomic hybridization. MAIN RESULTS AND THE ROLE OF CHANCE: The incidence of SAs was 6.3% in oocytes, increased to 16.6% in cleavage-stage embryos (P < 0.001) and decreased to 11.2% in blastocysts (P < 0.025 versus oocytes; P < 0.01 versus cleavage-stage embryos). The highest incidence of SAs was found in BFs (26.1%, P < 0.001). The analysis of 66 sets of sequential biopsies revealed that the initial finding was confirmed in all following samples from 39 sets (59.1% full concordance). In 12 additional sets, SAs were detected in some samples while in others the interested chromosome had full aneuploidy (18.2%). In three more sets, there was a partial concordance with the initial diagnosis in some samples, but in all TE samples the interested chromosome was clearly euploid (4.5%). In the remaining 12 sets, the initial SA was not confirmed at any stage and the corresponding chromosomes were euploid (18.2% no concordance). The pattern of concordance was not affected by the number of SAs in the original biopsy (single, double or complex) or by the absence or presence of concomitant aneuploidies for full chromosomes. LIMITATIONS, REASONS FOR CAUTION: Chromosome analyses were performed on biopsies that might not be representative of the true constitution of the embryo itself due to the occurrence of mosaicism. WIDER IMPLICATIONS OF THE FINDINGS: The permanence of SAs throughout the following stages of embryo development in more than half of the analyzed sets suggests for this dataset a very early origin of this type of chromosome imbalance, either at meiosis or at the first mitotic divisions. Since SAs remained in full concordance with the initial diagnosis until the blastocyst stage, a corrective mechanism seems not to be in place. In the remaining cases, it is likely that, as for full chromosome aneuploidy, mosaicism derived from mitotic errors could have occurred. In following cell divisions, euploid cell lines could prevail preserving the embryo chances of implantation. Due to the scarcity of data available, the transfer of embryos with SAs should be strictly followed up to establish possible clinical consequences related to this condition. STUDY FUNDING/COMPETING INTEREST(S): No specific funding was obtained. There are no conflicts of interest.

Correction to: Contribution of cryopreservation to the cumulative live birth rate: a large multicentric cycle-based data analysis from the Italian National Registry.

The original article unfortunately contained a mistake. The names of the collaborators were captured as authors of the article.

Contribution of cryopreservation to the cumulative live birth rate: a large multicentric cycle-based data analysis from the Italian National Registry.

PURPOSE: To estimate the contribution of cryopreservation to the cumulative live birth rate (CLBR) after law modification in Italy in the era of vitrification and freeze-all. METHODS: The Italian National Registry performed a cycle-based data collection. Nine Italian IVF clinics were involved incorporating a total of 10,260 fresh cycles performed between January 2015 and April 2016 resulting in 9273 oocyte retrievals and 3266 subsequent warming cycles from the same oocyte retrievals performed up to December 2016. Mean female age was 37 ± 4.3 years. Primary outcome measure was CLBR per oocyte retrieval. Confounding factors were tested in multivariate regression analysis, and the relative impact of cryopreservation to the CLBR in different patient categories was calculated. RESULTS: CLBR per oocyte retrieval was 32.6%, 26.5%, 18.7%, 13.0%, and 5.5% for women younger than 36, aged 36-39, 40-41, and older than 41 years, respectively. The total relative contribution of oocyte/embryo cryopreservation was 40.6% (95% CI 38.41-42.75). An association between maternal age, number of oocytes retrieved, fertilization rate, cryopreservation, and cumulative live birth was shown. When adjusted for confounders, a 2.3-fold increase was observed in the chance of live birth when cryopreservation was performed (OR 2.3; 95% CI 1.99-2.56). In high responder patients (> 15 oocytes retrieved) where freeze-all was applied in 67.6% of cycles to avoid the risk of hyper stimulation syndrome, the relative contribution of vitrification to the CLBR was 80.6%. CONCLUSIONS: Cryopreservation is essential in IVF and should always be available to patients to optimize success rates. Multicentric, cycle-based data analyses are crucial to provide infertile couples, clinicians, and regulatory bodies with accurate information on IVF effectiveness including fresh and cryopreserved cycles.

Using Clinical Signs and Symptoms for Medical Management of Radiation Casualties - 2015 NATO Exercise.

The utility of early-phase (≤5 days) radiation-induced clinical signs and symptoms (e.g., vomiting, diarrhea, erythema and changes in blood cell counts) was examined for the prediction of later occurring acute radiation syndrome (ARS) severity and the development of medical management strategies. Medical treatment protocols for radiation accident victims (METREPOL) was used to grade ARS severities, which were assigned response categories (RCs). Data on individuals (n = 191) with mild (RC1, n = 45), moderate (RC2, n = 19), severe (RC3, n = 20) and fatal (RC4, n = 18) ARS, as well as nonexposed individuals (RC0, n = 89) were generated using either METREPOL (n = 167) or the system for evaluation and archiving of radiation accidents based on case histories (SEARCH) database (n = 24), the latter comprised of real-case descriptions. These data were converted into tables reflecting clinical signs and symptoms, and submitted to eight teams representing five participating countries. The teams were comprised of medical doctors, biologists and pharmacists with subject matter expertise. The tables comprised cumulated clinical data from day 1-3 and day 1-5 postirradiation. While it would have reflected a more realistic scenario to provide the data to the teams over the course of a 3- or 5-day period, the logistics of doing so proved too challenging. In addition, the team members participating in this exercise chose to receive the cumulated reports of day 1-3 and 1-5. The teams were tasked with predicting ARS incidence, ARS severity and the requirement for hospitalization for multiple cases, as well as providing the certainty of their diagnosis. Five of the teams also performed dose estimates. The teams did not employ harmonized methodologies, and the expertise among the members varied, as did the tools used and the means of analyzing the clinical data. The earliest report time was 3 h after the tables were sent to the team members. The majority of cases developing ARS (89.6% ± 3.3 SD) and requiring hospitalization (88.8% ± 4.6 SD) were correctly identified by all teams. Determination of ARS severity was particularly challenging for RC2-3, which was systematically overestimated. However, RC4 was correctly predicted at 94-100% by all teams. RC0 and RC1 ARS severities were more difficult to discriminate. When reported RCs (0-1 and 3-4) were merged, on average 89.6% (±3.3 SD) of all cases could be correctly classified. Comparisons on frequency distributions revealed no statistically significant differences among the following: 1. reported ARS from different teams (P > 0.2); 2. cases generated based on METREPOL or SEARCH (P > 0.5); or 3. results reported at day 3 and 5 postirradiation (P > 0.1). Dose estimates of all teams increased significantly along with ARS severity (P < 0.0001) as well as with dose estimates generated from dicentric chromosomal-aberration measurements available for SEARCH cases (P < 0.0001). In summary, early-phase radiation-induced clinical signs and symptoms proved to be useful for rapid and accurate assessment, with minor limitations, toward predicting life-threatening ARS severity and developing treatment management strategies.

Estrogen receptor signaling in the ferutinin-induced osteoblastic differentiation of human amniotic fluid stem cells.

AIMS: Ferutinin is a diaucane sesquiterpene with a high estrogenic activity. Since ferutinin is able to enhance osteoblastic differentiation of human amniotic fluid stem cells (hAFSCs), the aim of this study was to evaluate the role of the estrogen receptors α (ERα) and G-protein coupled receptor 30 (GPR30) in ferutinin-mediated osteoblastic differentiation. Moreover, it was investigated if MEK/ERK and PI3K/Akt signaling pathways are involved in ferutinin-induced effects. MAIN METHODS: hAFSCs were cultured in a standard medium or in an osteoblastic medium for 14 or 21days and ferutinin was added at 10-8M. Immunofluorescence techniques and Western-blot 21analysis were used to study estrogen receptors and signaling pathways. KEY FINDINGS: In both undifferentiated and differentiated hAFSCs we identified ERα and GPR30 with a nuclear or cytoplasmatic localization, respectively. The presence of ferutinin in the osteoblastic medium leads to an increase in ERα expression. To dissect the role of estrogen receptors, MPP and G15 were used to selectively block ERα and GPR30, respectively. Notably, ferutinin enhanced osteoblastic differentiation in cells challenged with G15. Ferutinin was able to increase ERK and Akt phosphorylations with a different timing activation. These phosphorylations were antagonized by PD0325901, a MEK inhibitor, and wortmannin, a PI3K inhibitor. Both MPP and G15 inhibited the ferutinin-induced MEK/ERK and PI3K/Akt pathway activations. In the osteoblastic condition, PD0325901, but not wortmannin, reduced the expression of OPN and RUNX-2, whereas ferutinin abrogated the down-modulation triggered by PD0325901. SIGNIFICANCE: PI3K/Akt pathways seems to mediate the enhancement of hAFSCs osteoblastic differentiation triggered by ferutinin through ERα.

Enrichment in c-Kit improved differentiation potential of amniotic membrane progenitor/stem cells.

INTRODUCTION: Human term placenta has attracted increasing attention as an alternative source of stem cells for regenerative medicine since it is accessible without ethical objections. The amniotic membrane (AM) contains at least two stem cell types from different embryological origins: ectodermal amniotic epithelial stem cells, and mesodermal mesenchymal stromal cells. Among the second group we studied the characteristics of amniotic mesenchymal cells (AMC) versus the ones enriched for the commonly used surface marker c-Kit (amniotic progenitor/stem cells-ASC), a stem cell factor receptor with crucial functions in a variety of biological systems and presents in early progenitors of different origin, as been already demonstrated in the enriched chorionic stem cells. METHODS: After isolation, cells from the amniotic membranes (amniotic cells-AC) were selected for c-Kit (ASC) and compared these cells with c-Kit unselected (AMC), evaluating the expression of other stem cell markers (Oct-4, Tra-1-81, SSEA-4), CD271 and Slug. RESULTS: Immunofluorescence analysis showed that ASC cells exhibited greater stem cell marker expression and included more CD271 and Slug positive cells. This was consistent with the interpretation that c-Kit enriched AC show greater stemness capacity compared to c-Kit unselected AMC. DISCUSSION: AMC and ASC can both differentiate into various cell types including adipogenic, osteogenic, chondrogenic, neurogenic and hepatic lineages, but the enrichment in c-Kit improved stemness and differentiation potential of ASC.

Effects of physical exercise on the female reproductive system.

The excess in physical activity could be closely linked to considerable negative consequences on the whole body. These dysfunctions called as "female athlete triad"' by the American College of Sports Medicine (ACSM) include amenorrhea, osteoporosis and disorder eating. The female athlete triad poses serious health risks, both on the short and on the long term, to the overall well-being of affected individuals. Sustained low energy availability can impair health, causing many medical complications within skeletal, endocrine, cardiovascular, reproductive and central nervous system. On the contrary, several studies have shown, that physical activity improves cardiovascular risk factors, hormonal profile and reproductive function. These improvements include a decrease in abdominal fat, blood glucose, blood lipids and insulin resistance, as well as improvements in menstrual cyclicity, ovulation and fertility, decreases in testosterone levels and Free Androgen Index (FAI) and increases in sex hormone binding globulin (SHBG). Other studies reported that physical activity improved self-esteem, depression and anxiety. Thus, the aim of this review is to elucidate the effect of physical exercise on female reproductive system and viceversa the impact of hormonal status on physical activity and metabolism. In addition this review supports the idea that physical exercise is a helpful tool for the management of obesity, prevention of cardiovascular, metabolic diseases and female reproductive organs related diseases (e.g. breast cancer). When the excess in physical activity leads up to the female athlete triad, it is imperative to treat each component of the triad by employing both pharmacological and non pharmacological treatments.

Macroscopic and microscopic findings of the placenta in women with polycystic ovary syndrome.

STUDY QUESTION: Do patients with polycystic ovary syndrome (PCOS) have macroscopic and/or microscopic placental alterations? SUMMARY ANSWER: The placental structure in patients with PCOS, even in those with uncomplicated pregnancy, is altered. WHAT IS KNOWN ALREADY: The spectrum of pregnancy complications seems to have a common denominator: a defective trophoblast invasion and placentation. In women with PCOS, alterations in endovascular trophoblast invasion related to insulin resistance and hyperandrogenism have been observed. STUDY DESIGN, SIZE, DURATION: For this prospective case-control study, 30 pregnant patients with PCOS (cases) and 60 healthy pregnant women without PCOS features (controls) were enrolled and studied until delivery. Clinical, biochemical, ultrasonographic and obstetric data were recorded. The baseline clinical and biochemical data for screening for PCOS and for inclusion/exclusion were obtained before the seventh week of gestation. At delivery, placentas were collected and detailed macroscopic and microscopic analyses were performed. PARTICIPANTS, SETTING, METHODS: Cases and controls were matched for age and BMI (all <30 kg/m(2)). The matching procedure was one-to-two. Only subjects with spontaneous conception and uncomplicated pregnancies were included in the final analysis. MAIN RESULTS AND THE ROLE OF CHANCE: Placental weight (P = 0.04), thickness (P = 0.02), density (P = 0.02) and volume (P = 0.01) were significantly inferior in women with, compared with those without PCOS. The placentas from patients with PCOS more frequently had an irregular shape (P = 0.03) and a higher cord coiling index (P = 0.02). Differences between cases and controls also concerned the extent of villous (P = 0.04) and intervillous (P = 0.01) spaces, the extent of fibrosis (P = 0.03), endovascular trophoblast (depth, extension and morphometry) (P < 0.05) and mitotic activity (P = 0.01). The percentage of patients with lesions [22/30 (73.3%) versus 25/60 (41.7%), respectively; P = 0.01] and the mean number of placental lesions (3.5 ± 2.1 versus 1.4 ± 1.1, respectively; P = 0.02) were higher in the PCOS than the control group. The odds ratio for placental alterations, adjusted for weight gain, was 2.8 (95% confidence interval 1.3-9.9). LIMITATIONS, REASONS FOR CAUTION: The main limitation of the study was the selection of a specific PCOS sample, which is probably not representative of the PCOS phenotype as a whole. In fact, we excluded patients with PCOS who were obese and who achieved a pregnancy following the use of ovulation inductors or assisted reproduction techniques. WIDER IMPLICATIONS OF THE FINDINGS: The present study is the first to demonstrate that the morphology and microscopic structure of placenta in patients with PCOS with an uncomplicated pregnancy are altered. Further studies are needed to assess a correlation of these changes with the increased risk of obstetric complications observed in some pregnancies of women with PCOS. STUDY FUNDING/COMPETING INTEREST(S): The authors declare no conflict of interest and no financial support for the research. TRIAL REGISTRATION NUMBER: N/A.

Enrichment in c-Kit+ enhances mesodermal and neural differentiation of human chorionic placental cells.

OBJECTIVE: Human term placenta (HTP) has attracted increasing attention as an alternative source of stem cells for regenerative medicine since the amniochorionic membrane harbors stem cells populations that are easily accessible, abundantly available without ethical objections. In the chorionic side of HTP we found a progenitor perivascular "niche" in which rare cells co-express Oct-4 and c-Kit. We investigated the stem cell characteristics and differentiation potential of a chorionic derived population enriched in c-Kit(+) cells and compared this to the unenriched population. STUDY DESIGN: Cells, isolated from the chorion of HTP, were expanded and enriched in c-Kit(+) cells (Chorionic Stem Cells-CSC). Histological staining, immunofluorescence, Western blot and flow cytometry were used to verify the stem cells characteristics of the populations and to compare the differentiation capability towards mesodermal and neural lineages in vitro. RESULTS: The expression of the pluripotent marker Oct-4 was greater in the CSCs compared to the unselected cells (Chorionic Cell-CC) but both Oct-4 and c-Kit expression decreased during passages. After differentiation, CSC displayed stronger chondrogenic and osteogenic potential and a greater adipogenic forming capacity compared to unselected ones. CSC differentiated better into immature oligodendrocytes while CC showed a neuronal progenitor differentiation potential. Moreover, both populations were able to differentiate in hepatogenic lineage. CONCLUSION: CSC display improved Oct-4 expression and a high differentiation potential into mesodermal lineages and oligodendrocytes.

Effects of metformin in women with polycystic ovary syndrome treated with gonadotrophins for in vitro fertilisation and intracytoplasmic sperm injection cycles: a systematic review and meta-analysis of randomised controlled trials.

BACKGROUND: Metformin is widely used for treating women with polycystic ovary syndrome (PCOS), and many patients with PCOS who are infertile receive gonadotrophins while being treated with metformin. OBJECTIVES: To assess the effects of metformin administration in infertile patients with PCOS who receive gonadotrophins for in vitro fertilisation (IVF) and intracytoplasmic sperm injection (ICSI) cycles. SEARCH STRATEGY: We searched international scientific databases, websites for the registration of trials, and bibliographies of retrieved articles, books, and review articles up to August 2012. SELECTION CRITERIA: Randomised controlled trials (RCTs). DATA COLLECTION AND ANALYSIS: Authors independently reviewed and extracted the data. MAIN RESULTS: Ten RCTs (with a total of 845 women with PCOS) were included in the final analysis. Metformin administration in IVF/ICSI cycles had no effect on the rates of pregnancy (OR 1.20, 95% CI 0.90-1.61) and live birth (OR 1.69, 95% CI 0.85-3.34). No effect of metformin dose, metformin pretreatment duration, and stopping time of metformin administration was observed on these reproductive end points. Metformin administration reduced the risk of ovarian hyperstimulation syndrome (OHSS; OR 0.27, 95% CI 0.16-0.46) and of miscarriage (OR 0.50, 95% CI 0.30-0.83), while increased that of implantation (OR 1.42, 95% CI 1.24-2.75). AUTHOR'S CONCLUSIONS: In infertile patients with PCOS treated with gonadotrophins for IVF/ICSI cycles, metformin exerts no clinical effect on rates of pregnancy or live birth, but it reduces the risk of OHSS, and improves the rates of miscarriage and implantation. Further RCTs are needed to assess the reproductive effect of metformin in young well-selected patients with PCOS and specific phenotypes and features.

Protein microarrays on midtrimester amniotic fluids: a novel approach for the diagnosis of early intrauterine inflammation related to preterm delivery.

Novel technologies that allow simultaneous assessment of multiple biomarkers provide new and promising diagnostic/prognostic approaches. By protein microarrays, here we analyzed amniotic fluids (AF) from 50 women with preterm delivery (PTD) and 50 control women, who delivered at term. In detail, cytokines, chemokines, matrix metalloproteinases and antigen-specific antibodies were assessed. The AF analysis showed significant differences between women with preterm and term delivery in the levels of IL-1alpha, IL-1beta, IL-4, IL-6, IL-8, MCP-1, IFN-gamma and anti-HSV2 IgG. No significant differences were observed in the levels of TNF-alpha, MMP-2, MMP-9 and specific IgG for seven vertically transmitted pathogens. In conclusion, we demonstrated the feasibility of protein microarrays in the diagnosis of early intrauterine inflammation. The significant association between the increased levels of certain cytokines and preterm delivery argues on their relevance as early pathogenetic markers for identification of risk patients.

Diagnostic accuracy of sonohysterography and transvaginal sonography as compared with hysteroscopy and endometrial biopsy: a prospective study.

AIM: The aim of the study was to compare the diagnostic accuracy between transvaginal sonography (TVS) and sonohysterography (SHG) versus hysteroscopy (Hys) plus endometrial biopsy (EB) to evaluate uterine cavity. METHODS: One hundred and sixteen patients were enrolled. These presented with infertility and/or abnormal uterine bleeding and/or suspicious uterine cavity pathology. Women consecutively underwent during the same day, to TVS, SHG and Hys plus EB by three different operators. RESULTS: TVS shows excellent specificity (95.7%) in uterine polyps detection, good sensitivity (85,7%) and specificity (89.2%) in investigating endometrial hyperplasia, and excellent NPV (92.2%) in the diagnosis of submucous myomas. Diagnostic accuracy of TVS for synechiae is not evaluable. SHG demonstrates high specificity (92.8%) in the detection of uterine polyps, and high sensitivity (92.9%) and specificity (96.8%) in the diagnosis of endometrial hyperplasia. In addition it shows high sensitivity (90%), specificity (99%), PPV (92.2%), and NPV (99%) for detection of submucous myomas. Finally, SHG shows high PPV (100%) and NPV (100%) for synechiae assessment. CONCLUSION: TVS could be used as first step investigation to exclude uterine pathologies. TVS could reduce the number of diagnostic Hys normally performed in women with normal uterine cavity. Furthermore SHG should be useful to diagnose the pathologies and to decide between operative Hys in-office or resectoscopic treatment.

Intrapartum translabial three-dimensional ultrasound visualization of levator trauma.

OBJECTIVES: The aim of this study was to visualize levator trauma by three-dimensional (3D) ultrasound performed during labor and soon after the crowning of the fetal head and to determine how often levator trauma occurs. METHODS: This was a prospective, observational study of 66 women enrolled during the first stage of labor. The women underwent intrapartum 3D transperineal ultrasound examination during the first and second stages of labor and within 12 h after delivery. Volume datasets were acquired and analyzed to determine the presence of levator trauma. RESULTS: Data from 10 of the 66 women were excluded from analysis-nine because they underwent Cesarean section in the first or second stage of labor and one because she underwent hysterectomy and no postpartum volumes were collected. Thus our study group comprised 56 women-35 nulliparous and 21 parous. A total of 504 volumes were collected in the 56 women (three volumes for each stage of labor). One hundred and twenty levator volumes were excluded from analysis, but volumes of acceptable quality were available for all three stages of labor in all women. Eleven (31.4%) of the 35 nulliparae had levator lesions detected postpartum and none of them had levator lesions before delivery. Five (23.8%) of the 21 parous women had a levator tear detected in their postpartum volumes. In two of these five women the levator tear was also present in both volumes taken during labor. CONCLUSIONS: Visualization of the levator ani during labor by 3D ultrasound examination is feasible. Comparison of volumes obtained during labor and within the first 2 h after delivery supports the theory that crowning of the head is the immediate cause of avulsion of the levator ani muscle.

Ovarian function and gastrointestinal motor activity.

Gastrointestinal disorders are strictly related to the ovary function. In fact, it is noted that the prevalence of visceral pain disorders such as irritable bowel syndrome, gastroesophageal reflux disease, gallbladder and biliary tract diseases are significantly higher in women. Furthermore, symptom such as nausea, vomiting, abdominal pain, distension, satiety, bloating, diarrhoa or constipation, frequently appears in relation with pregnancy, luteal phase of the menstrual cycle or perimenopausal and menopausal states. Further support for the contribution of ovarian steroids to functional gastrointestinal disorders comes from studies demonstrating that pharmacological ovariectomy reduces abdominal pain symptoms. Therefore, addressing the influence of sex and sex hormones in the modulation of visceral pain appears critical to develop new strategies of diagnosis and therapy sex-directed for gastro-intestinal disorders.

Choroidal melanoma metastasized to the ovary: case report and review of the literature.

BACKGROUND: Malignant melanoma metastases to the female genital tract in only 2.5% of cases. Melanoma is characterized by clinical variability and unpredictable biological behavior with long remissions and relapses that develop rapidly. CASE AND REVIEW: A 57-year-old woman was admitted for hypogastric pain and weight loss. She had presented enucleation of the right eye six years before for malignant choroid melanoma. Gynaecological examination revealed enlarged ovaries. Bilateral salpingo-oophorectomy, hysterectomy, and omentectomy were performed. Final pathology diagnosed a choroidal metastatic melanoma (CMM). The patient died seven months later. Only seven cases of CMM have been reported in the literature. Patients affected by CMM ranged in age from 38 to 83 years (median 51.2 years), the time to relapse ranged from 3-25 years (median 51.2 years), the size of the cysts ranged from 4-17 cm (median 9.7 cm) and the survival period ranged from 2-14 months (median 8.1 months). CONCLUSION: Malignant melanoma is misdiagnosed because of lack of discriminatory symptoms, increased tumor markers, characteristic imaging findings and the capacity to mimic other tumors. Today CMM still represents a challenge for gynecologic oncologists.

The effect of the 2004 Italian law on outcomes of assisted reproduction technology in severe male factor infertility.

The Italian law regulating assisted reproductive technologies that came into force in 2004 restricts the number of fertilized oocytes per cycle to three, obliges the subsequent transfer of all resulting embryos and prohibits the freezing of surplus embryos. This study evaluates the impact of the law on severe oligozoospermic, cryptozoospermic, obstructive azoospermic and non-obstructive azoospermic patients. Intracytoplasmic sperm injection outcomes of 1066 cycles performed in the 4years before the passing of the law were compared with 804 cycles performed in the 4years after the law came to pass. Globally, analysis of clinical and obstetric outcomes showed a significant decrease in terms of pregnancy and delivery rates per cycle (17.8% versus 10.9% and 14.2% versus 8.5%, respectively) and per embryo transfer (18.8% versus 13.8% and 15.0% versus 10.7%, respectively), and a significant drop in multiple deliveries (35.1% versus 17.6%) in the post-law period. Cryptozoospermic and azoospermic couples were affected by the Italian law more than severe oligozoospermic couples. The results showed that the Italian law limits the efficiency of assisted reproduction treatment in couples with severe male factor. It is hoped that the Italian assisted reproductive technologies law is altered as soon as possible, allowing the insemination of more than three oocytes.

MATER protein as substrate of PKCepsilon in human cumulus cells.

High activity of the phosphoinositide 3-kinase/Akt pathway in cumulus cells plays an important role in FSH regulation of cell function and Protein Kinase C epsilon (PKCepsilon) collaborates with these signalling pathways to regulate cell proliferation. Relevant roles in follicular development are played by Maternal Antigen That Embryos Require (MATER) that is a cumulus cell- and oocyte-specific protein dependent on the maternal genome. We recently demonstrated that human MATER localizes at specific domains of oocytes and, for the first time, also in cumulus cells. MATER contains a carboxy-terminal leucine-rich repeat domain involved in protein-protein interactions regulating different cellular functions. Here we investigated the functional role of MATER. Thus, we performed coimmunoprecipitation experiments using HEK293T cells expressing human MATER; a similar approach was then followed in human cumulus/follicular cells. In MATER(+)HEK293T cells, we observed that this protein acts as a phosphorylation substrate of PKCepsilon. Western blot experiments indicate that, unlike oocytes, human cumulus cells express PKCepsilon. Immunoprecipitation and confocal analysis suggest for the first time that MATER protein interacts with this protein kinase in cumulus cells under physiological conditions. Since PKCepsilon is known to collaborate with antiapoptotic signalling pathways, this suggests a novel mechanism for the function of MATER in follicular maturation.

Estrogen receptor-mediated transcriptional activity of genistein in the mouse testis.

Here we show that genistein, through an estrogen receptor-mediated action, modulates gene expression in the mouse testis throughout development. Genistein passed from the lactating mother to the suckling offspring at levels sufficient to activate gene expression in the testis of the pups. Testis are already responsive to genistein as well as to estradiol at day 14.5 of fetal development. Activation of luciferase correlates with an activation of cell proliferation. In conclusion, our results show that genistein affects reproductive organs of male mice at all developmental ages.

Different glycoforms of the human GPI-anchored antigen CD52 associate differently with lipid microdomains in leukocytes and sperm membranes.

CD52 is a human GPI-anchored antigen, expressed exclusively in the immune system and part of the reproductive system (epididymal cells). Sperm cells acquire the antigen from the epididymal secretions when transiting in the epididymal corpus and cauda. The peptide backbone of CD52, consisting of only 12 aminoacids, is generally considered no more than a scaffold for post-translational modifications, such as GPI-anchor and especially N-glycosylation which occur at the third asparagine. The latter modification is highly heterogeneous, especially in the reproductive system, giving rise to many different glycoforms, some of which are tissue specific. A peculiar O-glycan-containing glycoform is also found in reproductive and immune systems. We determined to locate CD52 in microdomains of leukocytes and sperm membranes using two antibodies: (1) CAMPATH-1G, the epitope of which includes the last three aminoacids and part of the GPI-anchor of glycoforms present in leukocytes and sperm cells; (2) anti-gp20, the epitope of which belongs to the unique O-glycan-bearing glycoform also present in both cell types. Using a Brij 98 solubilization protocol and sucrose gradient partition we demonstrated that the CD52 glycoforms recognized by both antibodies are markers of typical raft microdomains in leukocytes, whereas in capacitated sperm the O-glycoform is included in GM3-rich microdomains different from the cholesterol and GM1-rich lipid rafts with which CAMPATH antigen is stably associated. The importance of the association between GM3 and O-glycans for formation of specialized microdomains was confirmed by heterologous CD52 insertion experiments. When prostasomes from human seminal fluid were incubated with rat sperm from different epididymal regions, the CD52 glycoform recognized by anti-gp20 decorated rat epididymal corpus and cauda sperm, associated with the same low-cholesterol GM3-rich sperm membrane fractions as in human sperm. The glycoforms recognized by CAMPATH-1G were not found in rat sperm. The relationship between this differential insertion and differences in glycosylation of rat and human CD52 is discussed.

Epitope analysis of immunoglobulins against gp20, a GPI-anchored protein of the human sperm surface homologous to leukocyte antigen CD52.

Gp20 is a sialylglycoprotein of the human sperm surface related to maturation and capacitation and is homologous to CD52, a glycosyl- phosphatidyl-inositol (GPI)-anchored protein highly expressed in lymphocytes, monocytes, eosinophils, and epididymal cells, described by the monoclonal antibody family CAMPATH. The CAMPATH antigen is characterized by a very short peptide (12 amino acids) and an N-linked oligosaccharide chain bound to the asparagine located in the third position and a GPI anchor bound to the C-terminal serine. The CAMPATH epitope includes three amino acids at the C-terminus and part of the GPI anchor. It has been suggested that anti-gp20 interacts with the same peptide recognized by CAMPATH antibodies but with a different epitope, since it describes the corresponding antigen in a different way. For example, it localizes the corresponding antigen in the equatorial region of the sperm head when sperm are capacitated, whereas CAMPATH antibodies bind all over the sperm surface. Our results indicate that the anti-gp20 epitope does not include the peptide backbone, the GPI anchor, or the N-glycans but consists of O-linked oligosaccharide chains bound to a unique CD52 glycoform present both in sperm and leukocytes. This is suggested by results obtained using many different approaches, such as immunoblot analysis of gp20 after removal of N- and O-glycans and after jacalin (Artocarpus integrifolia agglutinin)-affinity chromatography.