Embryoid body formation of human amniotic fluid stem cells depends on mTOR.

Human amniotic fluid stem cells (hAFSCs) harbor high proliferative capacity and high differentiation potential and do not raise the ethical concerns associated with human embryonic stem cells. The formation of three-dimensional aggregates known as embryoid bodies (EBs) is the principal step in the differentiation of pluripotent embryonic stem cells. Using c-Kit-positive hAFSC lines, we show here that these stem cells harbor the potential to form EBs. As part of the two kinase complexes, mTORC1 and mTORC2, mammalian target of rapamycin (mTOR) is the key component of an important signaling pathway, which is involved in the regulation of cell proliferation, growth, tumor development and differentiation. Blocking intracellular mTOR activity through the inhibitor rapamycin or through specific small interfering RNA approaches revealed hAFSC EB formation to depend on mTORC1 and mTORC2. These findings demonstrate hAFSCs to be a new and powerful biological system to recapitulate the three-dimensional and tissue level contexts of in vivo development and identify the mTOR pathway to be essential for this process.

Contraceptive efficacy and safety of a low-dose oral contraceptive, (0.03 mg ethinyl oestradiol and 2 mg chlormadinone acetate) Belara, over three medication cycles.

OBJECTIVE: To describe the modulation of ovarian function during three medication cycles with 0.03 mg ethinyl oestradiol (EE) and 2 mg chlormadinone acetate (CMA), leading to inhibition of conception in healthy women. METHODS: Phase II, single-centre, open, non-controlled trial. The main outcome measure was inhibition of ovarian activity, assessed by frequent monitoring of the presence, size and persistence of follicle-like structures using ultrasonography. Secondary parameters included: cervical reaction score (CRS-probability of fertilization), endometrial thickness (probability of nidation), and serum levels of the sex hormones oestradiol, progesterone, luteinizing hormone and follicle stimulating hormone. Safety was primarily assessed by monitoring the occurrence of adverse events. RESULTS: Thirty-three subjects were eligible for the trial and were included in the efficacy assessment (per protocol analysis, PPA). All subjects ovulated during the pretreatment cycle, but none during the three medication cycles. Follicular growth was profoundly suppressed during the medication phase, with residual ovarian activity occurring in only 12/83 (14.5%) treatment cycles. The CRS was negative during each medication cycle and endometrial thickness was suppressed on each medication day, with median values of 4.0-6.0 mm. EE/ CMA was well tolerated, with few adverse events reported; most were typically cycle-related and included headache, breast discomfort, nausea and vomiting. CONCLUSION: During the administration of EE/CMA follicular development, cervical reaction and endometrial thickness are profoundly suppressed, resulting in unfavourable conditions for fertilization, implantation and, thus, pregnancy.

[Recurrent miscarriage or failed in-vitro fertilization: antibodies against annexin V, cardiolipin, beta-2-glycoprotein-1 and APC-resistance]. / Rezidivierende Aborte und frustrane In-vitro-Fertilisation. Antikörper gegen Annexin-V, Cardiolipin und beta-2-Glykoprotein-1, oder APC-Resistenz.

UNLABELLED: Antibodies against annexin-V, a potent anticoagulant abundant in placental tissues, were recently controversially reported to be associated with recurrent miscarriages or failures of in-vitro-fertilization (IVF) attempts. PATIENTS, MATERIAL, METHODS: We screened 56 women (34.7 +/- 4.3 years of age: mean +/- 1 SD) with recurrent IVF failures and/or early pregnancy losses for resistance against activated protein C, lupus anticoagulant and antibodies against annexin V, cardiolipin or beta(2)-glycoprotein-1. Among them the prevalence of APC-R (8/56, 14%) and elevated levels of IgG- or IgM-anti-cardiolipin antibodies (7/56, 12%) were more common than elevated levels of (IgG or IgM) antibodies against beta(2)-glycoprotein-1 (3/56, 5%) or annexin-V (1/56, 2%). 42 (75%) of the women had another IVF-attempt after this haemostaseological evaluation and received low molecular weight heparin and/ or acetylsalicylic acid in the case of positivity for APC-resistance, lupus anticoagulant or antibodies against annexin V, cardiolipin or beta(2)-glycoprotein-1. RESULTS: The outcome of these IVF-attempts were 19 pregnancies (34%): 4 early miscarriages (7%) and 15 so far uncomplicated pregnancies (27%). The only woman with an elevated anti-annexin V (IgG) level had had 7 IVF before and received 40 mg Enoxaparin (Lovenox) subcutaneously once daily during the 8(th) IVF, which resulted in a healthy pregnancy. DISCUSSION, CONCLUSION: Our findings suggest that among women with recurrent IVF failures anti-annexin V antibody positivity is less prevalent than APC-resistance, lupus anticoagulant (LA) or elevated levels of antibodies against cardiolipin, beta(2)-glycoprotein-1 and that the IVF-result of women with APC-R, LA or with elevated levels of antibodies against annexin V, cardiolipin or beta(2)-glycoprotein might be positively influenced by low molecular weight heparin.

Chromosome banding in Amphibia. XXVIII. Homomorphic XY sex chromosomes and a derived Y-autosome translocation in Eleutherodactylus riveroi (Anura, Leptodactylidae).

Extensive cytogenetic analyses on a population of the leptodactylid frog Eleutherodactylus riveroi in northern Venezuela revealed the existence of multiple XXAA male/XYAA female/XAA(Y) female sex chromosomes. The XAA(Y) karyotype originated by a centric (Robertsonian) fusion between the original, free Y chromosome and an autosome. 46.2% of the male individuals in this population are carriers of this Y-autosome fusion. In male meiosis the XAA(Y) sex chromosomes pair in the expected trivalent configuration. In the same population 53.8% of the male animals still possess the original, free XY sex chromosomes. E. riveroi is only the second vertebrate species discovered in which a derived Y-autosome fusion coexists with the ancestral free XY sex chromosomes. The free XY sex chromosomes, as well as the multiple XA(Y) sex chromosomes are still in a very primitive (homomorphic) stage of differentiation. With no banding technique applied it is possible to distinguish the Y from the X. Various banding techniques and in situ hybridizations have been carried out to characterize the karyotypes. DNA flow cytometric measurements show that the genome size of E. riveroi resembles that of other Eleutherodactylus species. The cytogenetic data obtained in E. riveroi are compared with those of the sole other vertebrate known to possess the extremely rare, multiple XXAA male/XYAA female/XAA(Y) female sex chromosomes. Surprisingly enough, this vertebrate again is a frog belonging to the genus Eleutherodactylus [E. ((maussi) biporcatus] which lives exactly in the same habitat in northern Venezuela as does E. riveroi.

The rare human fragile site 16B.

The rare human fragile site 16B (FRA16B) has been found to occur spontaneously. Its expression in lymphocyte cultures can also be induced or greatly enhanced by addition of chemicals which are known to bind to AT-rich DNA regions. Following optimal treatment with 150 microg/ml berenil 24 h prior to fixation, the heterozygote frequency of FRA16B is found to be about 5% in populations of European descent. Thus, FRA16B represents the most common of the rare fragile sites. Consistent with cytogenetic observations, the molecular characterization of FRA16B revealed that it is an amplified 33-base pair AT-rich minisatellite repeat. These interindividually variable, extremely large repeat expansions of 15-70 kb in size do not seem to interfere with the expression of genes essential for human development since heterozygotes and homozygotes for FRA16B are normal.

Comparative chromosome painting of chicken autosomal paints 1-9 in nine different bird species.

In a Zoo-FISH study chicken autosomal chromosome paints 1 to 9 (GGA1-GGA9) were hybridized to metaphase spreads of nine diverse birds belonging to primitive and modern orders. This comparative approach allows tracing of chromosomal rearrangements that occurred during bird evolution. Striking homologies in the chromosomes of the different species were noted, indicating a high degree of evolutionary conservation in avian karyotypes. In two species, the quail and the goose, all chicken paints specifically labeled their corresponding chromosomes. In three pheasant species as well as in the American rhea and blackbird, GGA4 hybridized to chromosome 4 and additionally to a single pair of microchromosomes. Furthermore, in the pheasants fission of the ancestral galliform chromosome 2 could be documented. Hybridization of various chicken probes to two different chromosomes or to only the short or long chromosome arm of one chromosome pair in the species representing the orders Passeriformes, Strigiformes, and Columbiformes revealed translocations and chromosome fissions during species radiation. Thus comparative analysis with chicken chromosome-specific painting probes proves to be a rapid and comprehensive approach to elucidate the chromosomal relationships of the extant birds.

Chromosome banding in amphibia. XXIII. Giant W sex chromosomes and extremely small genomes in Eleutherodactylus euphronides and Eleutherodactylus shrevei (Anura, Leptodactylidae).

Highly differentiated, heteromorphic ZZ female symbol /ZW male symbol sex chromosomes were found in the karyotypes of the neotropical leptodactylid frogs Eleutherodactylus euphronides and E. shrevei. The W chromosomes are the largest heterochromatic, female-specific chromosomes so far discovered in the class Amphibia. The analyses of the banding patterns with AT- and GC base-pair specific fluorochromes show that the constitutive heterochromatin in the giant W chromosomes consists of various categories of repetitive DNA sequences. The W chromosomes of both species are similar in size, morphology and banding patterns, whereas their Z chromosomes exhibit conspicuous differences. In the cell nuclei of female animals, the W chromosomes form very prominent chromatin bodies (W chromatin). DNA flow cytometric measurements demonstrate clear differences in the DNA content of male and female erythrocytes caused by the giant W chromosome, and also shows that these Eleutherodactylus genomes are among the smallest of all amphibian genomes. The importance of the heteromorphic ZW sex chromosomes for the study of Z-linked genes, the similarities and differences of the two karyotypes, and the significance of the exceptionally small genomes are discussed.

Chromosome banding in Amphibia. XXII. Atypical y chromosomes in Gastrotheca walkeri and Gastrotheca ovifera (Anura, Hylidae).

The chromosomes of the rare South American marsupial frogs Gastrotheca walkeri and G. ovifera were extensively reexamined with various banding techniques. The karyotypes of both species are distinguished by a new category of XY female symbol /XX male symbol female sex chromosomes. The unusual Y chromosomes are characterized by containing the least amount of constitutive heterochromatin in the karyotypes. This is in contrast to all previously known amphibian Y chromosomes and does not fit the evolutionary model of early XY differentiation in vertebrates. In male meiosis, the heteromorphic XY chromosomes of both species still exhibit the same pairing configurations as the autosomes. DNA flow cytometric measurements show the nuclear DNA amount of G. walkeri to be 10.90 pg. The significance of the XY/XX sex chromosomes of these marsupial frogs, the various classes of constitutive heterochromatin detected, and the data obtained from meiotic analyses are discussed in detail.

X chromosomes of American marsupials contain minimal amounts of euchromatin.

The karyotypes of four South American didelphid marsupials, representing diploid numbers of 2n = 14 and 18, have been analyzed by a variety of banding techniques. The 2n = 14 karyotypes display a high degree of homoeology, but there also exist distinct similarities between the 2n = 14 and 2n = 18 karyotypes. The interspecific differences found are due to centric fissions, pericentric inversions, and variations in the amount and composition of the constitutive heterochromatin. Contrary to the evolutionary conservation of the banding patterns in all autosomal arms, there are multiple differences in the number and chromosomal location of the nucleolus organizer regions. In species with X-linked nucleolus organizers, the 18S + 28S ribosomal RNA genes escape inactivation in female cells. Measurements on the X chromosomes of Marmosa fuscata and Micoureus demerarae unexpectedly reveal the lowest quantities of euchromatin so far determined in the X chromosomes of mammals: 1.5% and 1.8%, respectively, of their haploid female genomes. This is significantly less than the amount of euchromatin in the basic X chromosomes of other marsupials (3%) or eutherians (5%).

Chromosome banding in Amphibia. XXVI. Coexistence of homomorphic XY sex chromosomes and a derived Y-autosome translocation in Eleutherodactylus maussi (Anura, Leptodactylidae).

A 15-year cytogenetic survey on one population of the leaf litter frog Eleutherodactylus maussi in northern Venezuela confirmed the existence of multiple XXAA male symbol /XAA(Y) female symbol sex chromosomes which originated by a centric (Robertsonian) fusion between the original Y chromosome and an autosome. 95% of the male individuals in this population are carriers of this Y-autosome fusion. In male meiosis the XAA(Y) sex chromosomes pair in the expected trivalent configuration. In the same population, 5% of the male animals still possess the original, free XY sex chromosomes. In a second population of E. maussi analyzed, all male specimens are characterized by these ancestral XY chromosomes which form normal bivalents in meiosis. E. maussi apparently represents the first vertebrate species discovered in which a derived Y-autosome fusion still coexists with the ancestral free XY sex chromosomes. The free XY sex chromosomes, as well as the multiple XA(Y) sex chromosomes are still in a very primitive (homomorphic) stage of differentiation. With no banding technique applied it is possible to distinguish the Y from the X. DNA flow cytometric measurements show that the genome of E. maussi is among the largest in the anuran family Leptodactylidae. The present study also supplies further data on differential chromosome banding and fluorescence in situ hybridization experiments in this amphibian species.

IVF-patients with nonmale factor "to ICSI" or "not to ICSI" that is the question?

PURPOSE: Intracytoplasmic sperm injection (ICSI) guarantees high fertilization rates and could theoretically lead to higher implantation rates as well. Furthermore injection into oocyte creates a hole in the zona pellucida similar to the procedure of assisted hatching. We were therefore interested to assess such a potential benefit for infertile IVF patients without male factor. MATERIALS AND METHODS: Open randomized prospective study according to the rules "Good Clinical Practice" with informed consent of the patients and institutional review board approval. Ninety-one consecutively seen patients with tubal infertility or hostile cervical mucus were randomized to undergo either ICSI (44 patients) or IVF (45 patients). In two patients fertilization of oocytes failed and so a repeated ICSI had to be performed. All these patients were stimulated with the same protocol, using the gonadotropin releasing hormone-agonist (GnRH-a) buserelin acetate in an ultrashort flair-up protocol together with pure follicle stimulating hormone (rFSH). The two study groups did not differ in terms of age, BMI, and all baseline hormone levels. RESULTS: The total pregnancy rate was 42% in the normal IVF group with 33% ongoing pregnancies. The ICSI group had a total pregnancy rate of 39% with 23% ongoing pregnancies. The implantation rate per transferred embryo was higher for normal IVF but not significant (18% versus 11%). The variables, fertilization rate, age, body mass index, baseline hormone levels, endometrial thickness, embryo score, and the highest grade embryo per transfer were very similar in both groups. CONCLUSION: ICSI should be applied only when conventional IVF fails, that is, for male factor patients and for patients with unexplained infertility.

Seasonal influences on in vitro fertilization and embryo transfer.

PURPOSE: The study was aimed to investigate the influence of time factors (day, week, month) on pregnancy rates and in vitro fertilization (IVF) and embryo transfer (ET) parameters. METHODS: 8,184 IVF-ET cycles, taking place in two IVF centers from 1992 to 1999, were analyzed. Multiple logistic and linear regression methods were performed as statistical analyses. RESULTS: Oocyte pickup on Tuesdays achieved a significantly higher mean number of oocytes (median: 7) and the highest pregnancy rate (PR) (33.4%) per ET There was a significant variation over the year, with the lowest PR/ET in July (25.71%) and the highest in December (35.5%). Concerning outcomes, the age factor and the number of embryos transferred had the highest significant (p < 0.0001) influence. CONCLUSIONS: The observed weekly rhythm for oocyte pick-ups is certainly due to preprogrammed ovarian stimulation used in our IVF programs. Age as well as the number of embryos transferred are the main influencing factors on a positive outcome and more predictive than seasonal aspects.

The effect of single-dose oxytocin application on time to ejaculation and seminal parameters in men.

PURPOSE: To report the effect of a single dose of oxytocin on ejaculation time and seminal parameters. METHODS: The prospective randomized clinical study was performed at a private assisted reproduction technology center and included 103 consecutive healthy men (semen donors or husbands of IVF patients). Oxytocin (16 IU) were administered intranasally to 49 subjects, randomly assigned to the study group just before masturbation to ejaculation. The time needed for ejaculation as well as the semen parameters was recorded according to the WHO standards, and compared to the control group of 54 subjects with no prior oxytocin treatment. RESULTS: No significant difference was noted between the two groups concerning ejaculation time and semen parameters. CONCLUSIONS: Although the administration of oxytocin stimulates sexual behavior and performance in mammalian species, our data indicate that oxytocin has no detectable effect on ejaculation time and seminal parameters after intranasal application in normal, healthy men.

Number and size of antral follicles as predictive factors in vitro fertilization and embryo transfer.

PURPOSE: The purpose of the study was to evaluate whether number and size of antral follicles can predict the outcome of in vitro fertilization-embryo transfer. METHODS: A total of 113 patients were prospectively included into this study. After 19 days of down-regulation, number and size of follicles were determined by using recent three-dimensional transvaginal ultrasound technology. Before application of gonadotropin, all follicles had been defined as antral follicles. According to size, antral follicles were categorized into four different groups: group I included antral follicles < 5 mm, group II follicles 5-10 mm; group III 11-20 mm; and group IV > 20 mm. Pregnant and non-pregnant patients were compared in terms of their number of antral follicles of group I-IV. These four groups were then compared regarding implantation rate, number of retrieved oocytes, endometrium thickness, and age. RESULTS: Pregnant patients showed an significant higher number of follicles with the size between 5 and 10 mm (P = 0.04). A significant correlation was found between number of retrieved oocytes and antral follicle size of 5-10 mm (P = 0.0001). Antral follicles with a diameter between 5 and 10 mm decreased significantly with age (P = 0.008). In group III and IV, a significant correlation was found between antral follicle size (P = 0.016) and serum estradiol level after gonadotropin-releasing hormone-agonist down-regulation (P = 0.011). CONCLUSIONS: We demonstrated that patients with a higher number of follicles between 5 and 10 mm showed a significantly higher pregnancy rate, whereas patients with a dominant number of antral follicles > 11 mm have a higher cancellation rate due to ovarian low response.

Spectral karyotyping of the human colon cancer cell lines SW480 and SW620.

The cell lines SW480 and SW620, derived from different stages of colon carcinoma in the same patient, have been used for a number of biochemical, immunological, and genetic studies on colon cancer. A comparative analysis of their karyotypes may identify chromosomal aberrations that might represent markers for metastatic spread. In the present study spectral karyotyping (SKY) was applied to these two colon cancer cell lines. Compared to previously reported G-banded karyotypes, 9 (SW480) and 7 (SW620) markers were identical, 3 (SW480) and 3 (SW620) markers could be redefined, 5 (SW480) and 8 (SW620) markers were newly identified, and 4 (SW480) and 5 (SW620) of the previous described markers could not be confirmed. The redefined aberrations include very complex rearrangements, such as a der(16) t(3;16;1;16;8;16; 1;16;10) and a der(18)t(18;15;17)(q12; p11p13;??) in SW620 and a der(19)t(19;8;19;5) in SW480, that have not been identified by conventional banding techniques. The resulting chromosome gains (5q11-->5q15, 7pter-->q22, 11, 13q14-->qter, 20pter-->p12, X) and losses (8pter-->p2, 18q12-->qter, Y) found in both SW480 and SW620 were in good agreement with those frequently described in colorectal tumors as primary changes in the stem cell. Abnormalities found exclusively in SW620 cells only (gains of 5pter-->5q11, 12q12-->q23, 15p13-->p11, and 16q21-->q24 and losses of 2pter-->2p24, 4q28-->qter, and 6q25-->qter) can be viewed as changes that occurred in a putative metastatic founder cell.

[Painfulness of transvaginal follicle aspiration for oocyte retrieval in in-vitro-fertilization]. / Schmerzhaftigkeit vaginaler Punktionen im Rahmen der In-vitro-Fertilisation.

OBJECTIVE: The aim of this study was to analyze the painfulness of a transvaginal ultrasound-guided follicle punction. MATERIAL AND METHODS: Patients could choose to have either a neuroleptanalgesia or to be supported by the partner or a psychotherapist during the oocyte pick up. Based on a questionnaire, 277 patients tried to describe their level of pain in a horizontal alphanumeric scale divided in 15 categories, while other painful physical interacts could be quantified, too. Furthermore, patients were asked for the cause of sterility, former IVF-ET treatments and whether the partner or the psychotherapist looked after them during the oocyte retrieval. Statistical analysis was performed using a standardized computer program (Stat View, Abacus Concepts, USA). RESULTS: The neuroleptanalgesia reduced the intensity of pain (average value of pain: 1.21 vs 9.26 without analgesia) in a significant way (p = 0.0001). Some patients compared the pain at oocyte retrieval with a bone fracture (8.08) or surgeries (10.12). More painful was a colic (13.67), infections (13.5), some diagnostic surgeries (12.09) or a delivery (11.91). Younger woman and patients with dysmenorrhea felt significantly more pain than others. Neither the presence of the partner or psychotherapist during punction nor the indication, number of previous IVF-ET treatments or a following pregnancy had any influence on the statistics. CONCLUSION: Age as well as the rate of problems with menstruation should help the patient to decide on whether to have the follicle punction with or without anesthesia.

Cumulus assessment by three-dimensional ultrasound for in vitro fertilization.

OBJECTIVE: To correlate the appearances of intrafollicular cumulus-like structures using three-dimensional (3D) ultrasound technology with the recovery of mature oocytes. MATERIALS AND METHODS: A prospective, observational study using 3D ultrasound examination was performed in 50 women undergoing in vitro fertilization (IVF) after ovarian stimulation. Examination was performed on the day of human chorionic gonadotropin (hCG)-ovulation induction. Estradiol-17-beta (E2) levels were measured on the same day. The ovaries were examined for the presence of cumuli in all follicles greater than 15 mm in diameter. Only cumuli demonstrable in all three planes, or in a computer-animated 3D reconstruction, were recorded as 'visible'. The number, maturity, and fertilization rate of retrieved oocytes were compared with the pre-ovulatory assessment of cumuli by 3D ultrasound. STATISTICS: Correlation coefficients (Pearson) between the variables were calculated and stepwise regression analyses performed. RESULTS: A total of 262 cumuli were visualized within 318 follicles from which 296 mature oocytes were retrieved and 218 fertilized. There was a significant correlation between the number of detected cumuli and the number of retrieved oocytes (r2 = 0.83; P < 0.0001), mature oocytes (r2 = 0.78; P < 0.0001), and the number of the fertilized oocytes (r2 = 0.65; P < 0.0001). There was a marked correlation between E2 and the number of follicles and the number of fertilized oocytes. CONCLUSIONS: Cumulus visualization by 3D ultrasound appears to be an indicator for mature oocytes and successful fertilization. Follicles in which the cumulus cannot be visualized in all three planes are unlikely to contain mature oocytes or oocytes in which fertilization is achieved.