Abstract Objectives: In this study, we aimed at determining whether human immature dental pulp stem cells (hIDPSC) would be able to contribute to different cell types in mouse blastocysts without damaging them. Also, we analysed whether these blastocysts would progress further into embryogenesis when implanted to the uterus of foster mice, and develop human/mouse chimaera with retention of hIDPSC derivates and their differentiation. Materials and Methods: hIDPSC and mouse blastocysts were used in this study. Fluorescence staining of hIDPSC and injection into mouse blastocysts, was performed. Histology, immunohistochemistry, fluorescence in situ hybridization and confocal microscopy were carried out. Results and Conclusion: hIDPSC showed biological compatibility with the mouse host environment and could survive, proliferate and contribute to the inner cell mass as well as to the trophoblast cell layer after introduction into early mouse embryos (n = 28), which achieved the hatching stage following 24 and 48 h in culture. When transferred to foster mice (n = 5), these blastocysts with hIDPSC (n = 57) yielded embryos (n = 3) and foetuses (n = 6); demonstrating presence of human cells in various organs, such as brain, liver, intestine and hearts, of the human/mouse chimaeras. We verified whether hIDPSC would also be able to differentiate into specific cell types in the mouse environment. Contribution of hIDPSC in at least two types of tissues (muscles and epithelial), was confirmed. We showed that hIDPSC survived, proliferated and differentiated in mouse developing blastocysts and were capable of producing human/mouse chimaeras.

Prophylactic use of cabergoline has been associated with a decrease in the severity of ovarian hyperstimulation syndrome (OHSS). A prospective randomized study was designed to evaluate the potential of cabergoline to decrease the incidence of OHSS in high-risk patients undergoing assisted reproductive technology treatment; 166 patients with oestradiol concentrations over 4000 pg/ml on the day of human chorionic gonadotrophin (HCG) administration were evaluated. They all received 20 g routine preventive intravenous human albumin on the day of oocyte retrieval. They were then randomized into two groups: group A (n = 83) received 0.5 mg oral cabergoline per day for 3 weeks beginning on the day after oocyte retrieval, and group B (n = 83) received no medication.'Early' OHSS was defined as being when the onset of the syndrome was initiated during the first 9 days after HCG administration, and 'late' OHSS was defined as being when the onset of the syndrome was initiated from 10 days after HCG administration. In group A, no patients progressed to 'early' OHSS and nine patients (10.8%) developed 'late' OHSS; in group B, 12 patients (15.0%) progressed to 'early' OHSS and three (3.8%) to 'late' OHSS. Although the risk of 'early' OHSS decreased significantly (P < 0.001), the risk of 'late' onset OHSS did not. The two groups presented no changes in pregnancy, implantation or miscarriages rates.

We evaluated the efficiency of microdissection testicular sperm extraction (MicroTESE) in patients with nonobstructive azoospermia (NOA) and their pregnancy outcomes in a programme based on in vitro fertilisation (IVF)/intracytoplasmic sperm injection (ICSI). Fifty-six MicroTESE procedures were performed in 53 patients with NOA. Pre-operative levels of luteinising hormone, follicle-stimulating hormone (FSH), testosterone and prolactin were obtained and a Doppler sonography examination was conducted. Sperm retrieval rate, mean age of female partner, mean ICSI and fertilisation rate, number and quality of embryos transferred, implantation, pregnancy and miscarriage rates were calculated. Samples for testicular histological analysis were taken trans-operatively in every case. Sperm retrieval rate, mean ICSI per case and fertilisation rate were 57.1%, 7.4% and 58.4% respectively. A significant difference in pre-operative testicular volume (P = 0.001), serum FSH (P = 0.008) and total testosterone levels (P = 0.021) was found in patients from whom sperm could be retrieved. Mean 1.9 type A embryos were transferred per cycle. Implantation, clinical pregnancy and miscarriage rates were 20%, 40% and 18.7% respectively. It is concluded that MicroTESE is a viable option for men with NOA, offering excellent results in couples undergoing IVF-ICSI. Pre-operative serum FSH, testicular volume and total testosterone levels may have a prognostic value, although more data are needed to determine their significance and whether or not patients should be excluded from an initial sperm retrieval attempt.

Pioneer work in male mouse embryonic stem (ES) cells differentiation into germ cells (GC) showed generations of male or female gametes in separate experiments, using genetically manipulated or preselected ES cells. In an attempt to produce both types of gametes from male mouse ES cells without any genetic manipulation or preselection, we induce the differentiation by retinoic acid (RA) within nonadherent embryoid bodies (EB). It seems that gamete-like cell formation occurs in the correct manner based on the expression of early and late GC-specific genes such as Oct-4, Mvh, Stella, Dazl, Piwil 2, Pdrd 1, Rex 14, Rnf 17, Bmp8b, Acrosin, Stra-8, Haprin, LH-R, Gdf9, Zp3, Zp2, Sycp1, and Sycp3. Immunofluorescence analysis of morphologically well-formed GC and presumptive gametes showed positive labeling for SSEA1, Oct-4, EMA-1, FE-J1, Dazl, Fragilis, Mvh, Acrosin, and acetylated alpha-tubulin. Conventional cytogenetic and FISH analysis indicated a chromosome reduction in ES-derived GC. Our data suggest that ES cells with XY chromosomes can produce under the same experimental conditions both types of presumptive gametes, and this production depends on their positional and temporal information within the EB context.

BACKGROUND Sperm aster organization during bovine and human fertilization requires a paternally-derived centriole that must first disengage from the sperm tail connecting-piece. We investigated the participation of the 26S proteasome in this process. METHODS Proteasome localization and enzymatic activity were studied in normal and pathological human spermatozoa by immunocytochemistry and enzyme-substrate assays. The role of proteasomes during bovine zygote development was investigated using a pharmacological proteasome-inhibitor, MG132, and with anti-proteasome antibodies delivered by Streptolysin O-permeabilization or with the Chariot reagent. Human zygotes discarded after ICSI failures (n = 28) were also examined. RESULTS Proteasomes were localized in the sperm acrosome and connecting-piece, as well as in the pronuclei of bovine and human zygotes. Proteasomal enzymatic activities were decreased in defective human spermatozoa. Disrupted sperm aster formation and pronuclear development were found after pharmacological and immunological block of proteasomes in human/bovine spermatozoa and oocytes, as well as in 28 discarded human post-ICSI fertilization failures. CONCLUSIONS Specific proteasome inhibition disrupts sperm aster formation and pronuclear development/apposition in bovine and human zygotes. Human spermatozoa with defective centriolar/pericentriolar structures have decreased proteasomal enzymatic activity. Release of a functional sperm centriole that acts as a zygote microtubule-organizing center probably relies on selective proteasomal proteolysis. These findings suggest an important role of sperm proteasomes in zygotic development.

The objective of this study was to assess whether positioning the embryo transfer (ET) catheter guide at the time of embryo expulsion, before or beyond the internal os, has an impact on IVF cycle outcome. We performed a retrospective study comparing IVF outcomes in relation to the ET guide position relative to the internal uterine os. We analyzed ultrasound-guided ETs in IVF-intracytoplasmic sperm injection (ICSI) cycles, performed with the tip of the ET catheter guide just before the internal os (group 1) and beyond the internal os (group 2). Implantation and pregnancy rates were significantly better in group 1 than in group 2, at 14.8% versus 11.8% and 57.3% versus 43.1, respectively. We conclude that passing the ET catheter guide beyond the internal os reduces implantation and pregnancy rates.

OBJECTIVE: To determine whether the injection of testicular spermatozoa results in more viable embryos (higher implantation rate) than injection of epididymal spermatozoa in cases of obstructive azoospermia. DESIGN: Retrospective analysis of 265 cases of testicular sperm aspiration (TESA) and percutaneous sperm aspiration (PESA), including 185 cases of obstructive azoospermia. SETTING: Private Infertility clinic. PATIENT(S): None, charts review. INTERVENTION(S): None, charts review. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (PR), implantation rate. RESULT(S): Although fertilization rates were higher in the PESA group, implantation rates were significantly better in the TESA group. There was also a trend to higher ongoing PR and lower miscarriage rates in TESA cases. CONCLUSION(S): In cases of obstructive azoospermia, embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa.

During mitosis, a spindle checkpoint detects chromosome misalignment and halts the cell cycle progression. In meiosis of female germ cells, however, it is debatable whether such a checkpoint is present. This research employed a unique model in the mouse, mitotic chromosomes transferred to meiotic cytoplasts to investigate whether a meiotic oocyte's microtubule apparatus can effectively separate mitotic metaphase chromosomes, and whether a spindle checkpoint exists during its division. The intact germinal vesicle (GV) oocytes, enucleated GV cytoplasts, and enucleated GV cytoplasts at 15 h in-vitro maturation were transferred with a metaphase fibroblast cell. When mitotic chromosomes were transferred into enucleated or intact mouse GV oocytes, the first bipolar meiotic spindles were established and the reconstructed oocytes were able to extrude polar bodies. However, none of the reconstructed oocytes showed complete and accurate alignment of chromosomes, except the enucleated GV cytoplasts reconstructed after maturation. The spindle formation and polar body extrusion suggest that the first meiotic spindle was functional, and the chromosome misalignment did not prevent the onset of anaphase. The data indicate that a spindle checkpoint, providing surveillance of misaligned chromosomes, was overridden or compromised by the incompatibility between somatic chromosomes and meiotic spindles during the first meiotic division.

PURPOSE: To compare the efficacy and safety of u-hCG with r-hCG in IVF cycles. METHODS: A prospective, investigator-blind, randomized, comparative study. Patients (n = 100)< or =35 years with IVF indication were randomly assigned on the day of hCG administration for oocyte maturation to receive either u-hCG (10,000 IU) or r-hCG (250 microg). RESULTS: No statistical differences were found between groups in relation to total number of oocytes retrieved, percentage of mature oocytes, number of injected oocytes, fertilization rates and number of embryos transferred. The data indicate a possible trend toward a higher incidence of pregnancy in the r-hCG group. Adverse events, predominantly injection-site reactions, were significantly more common in the u-hCG group. CONCLUSIONS: r-hCG is at least as effective for inducing final stages of oocyte maturation as 10,000 IU u-hCG and is also associated with significantly better patient tolerance and thus higher patient acceptability.

Fertilization rates increased continuously with the time elapsed after administration of hCG, reaching a peak of 84% when intracytoplasmic sperm injection (ICSI) was performed >41 hours after hCG administration. However, the highest implantation rate, 24%, was achieved when ICSI was performed 37-41 hours after hCG administration.