Long-term comparisons of the efficacy, safety, and pregnancy outcomes of adjuvant tamoxifen plus ovarian function suppression in premenopausal Han and Zhuang Chinese patients with hormone receptor-positive early breast cancer.

OBJECTIVE: To compare the efficacy, safety, and pregnancy outcomes of tamoxifen plus ovarian function suppression (OFS) between Han and Zhuang women with hormone receptor-positive breast cancer. METHODS: A total of 236 Han and 101 Zhuang women with hormone receptor-positive breast cancer who received tamoxifen plus OFS were analyzed retrospectively. Long-term disease-free survival (DFS) and overall survival (OS) were evaluated by Kaplan-Meier analysis, and adverse events and pregnancy outcomes were assessed by χ2 and Fisher's exact-probability tests. RESULTS: There was no significant difference in DFS or OS between Han and Zhuang women (5-year DFS 74.57% and 77.23%, OS 85.59% and 90.01%, respectively). The incidences of endometrial hyperplasia, ovarian cysts, nausea and vomiting, fatty liver, retinitis, and thrombocytopenic purpura were similar in both groups, but Zhuang women had significantly more allergic reactions (6.93% vs. 2.12%). Pregnancy rates among women who attempted pregnancy were similar (Han, 7/138, 5.07%; Zhuang, 2/46, 4.35%). CONCLUSIONS: OFS plus tamoxifen resulted in similar DFS and OS among premenopausal Han and Zhuang women with hormone receptor-positive breast cancer. However, Zhuang women were more likely to experience an allergic reaction. For women with fertility concerns, OFS plus tamoxifen was associated with similar pregnancy rates in Zhuang and Han women.

The available time window for embryo transfer in the rhesus monkey (Macaca mulatta).

Much effort has been focused on improving assisted reproductive technology procedures in humans and nonhuman primates (NHPs). However, the pregnancy rate after embryo transfer (ET) has not been satisfactory, indicating that some barriers still need to be overcome in this important procedure. One of the key factors is embryo­uterine synchronicity, which is little known in NHPs. The objective of this study was to investigate the available ET time window in rhesus monkey (Macaca mulatta). Eighty-two adult female rhesus monkeys were superovulated with recombinant human FSH. Ovarian phases were identified according to estrogen (E2) and progesterone (P4) levels as well as ovarian examination by ultrasonography and laparoscopy. A total of 259 embryos were transferred by the laparoscopic approach into the oviducts of 63 adult female monkeys. Ovarian phases were divided into late follicular and early luteal phases. Similar pregnancy rates (30­36.4%) were obtained from recipients receiving ET either in their late follicular or early luteal phases, regardless of embryo developmental stages. This study indicates that the available time window for ET in rhesus monkeys is from the late follicular to early luteal phases.

Folic acid deficiency inhibits neural rosette formation and neuronal differentiation from rhesus monkey embryonic stem cells.

Evidence from epidemiological studies has proved that periconceptional use of folic acid (FA) can significantly reduce the risk of neural tube defects (NTDs). However, it is hard to explore when and how FA plays roles in neurogenesis and brain development in vivo, especially in human or other nonhuman primate systems. Primate embryonic stem cell (ESC) lines are ideal models for studying cell differentiation and organogenesis in vitro. In the present study, the roles of FA in neural differentiation were assessed in a rhesus monkey ESC system in vitro. The results showed no significant difference in the expression of neural precursor markers, such as nestin, Sox-1, or Pax-6, among neural progenitors obtained from different FA concentrations or with the FA antagonist methotrexate (MTX). However, FA depletion decreased cell proliferation and affected embryoid body (EB) and neural rosette formation, as well as neuronal but not neuroglia differentiation. Our data imply that the ESC system is a suitable model for further exploring the mechanism of how FA works in prevention of NTDs in primates.

Reproductive traits of polycystic ovary syndrome in female rhesus monkeys.

The objective of this study was to set up a rhesus monkey model of polycystic ovary syndrome (PCOS), which is globally prevalent among reproductive-aged human women, and to understand the reproductive traits of PCOS female monkeys. Six adult female rhesus monkeys aged 6-10 a, were divided into a PCOS group and a control group. The PCOS group were given two cycles of subcutaneous injections of propionic acid testosterone (PAT), 3.5 mg/kg body weight, on day 1, day 3, and day 5 of the menstrual cycle, respectively, and then given muscle injections of human chorionic gonadotropin (HCG), 350 IU/kg body weight, on day 7, day 9, and day 11, respectively. Results showed that high levels of serum LH and T [(5.35±0.17) IU/L and (7.58±0.14) ng/mL, respectively], and a high ratio value of LH/FSH (5.35/1.30=4.12) were observed in the PCOS group. No significant differences were found in serum FSH, E2, and P in the PCOS group compared with those of the control. Polycystic ovaries in the PCOS monkeys were recorded by live ultrasound. The blastocysts rates of the PCOS vs. the control were 23.53% vs. 66.67%, and there was a significant difference between the two groups. This study shows that PAT coupled with HCG can induce PCOS in rhesus monkeys in the short term. The reproductive features of PCOS monkeys were similar to those of PCOS patients.

Generation of trophoblast stem cells from rabbit embryonic stem cells with BMP4.

Trophoblast stem (TS) cells are ideal models to investigate trophectoderm differentiation and placental development. Herein, we describe the derivation of rabbit trophoblast stem cells from embryonic stem (ES) cells. Rabbit ES cells generated in our laboratory were induced to differentiate in the presence of BMP4 and TS-like cell colonies were isolated and expanded. These cells expressed the molecular markers of mouse TS cells, were able to invade, give rise to derivatives of TS cells, and chimerize placental tissues when injected into blastocysts. The rabbit TS-like cells maintained self-renewal in culture medium with serum but without growth factors or feeder cells, whilst their proliferation and identity were compromised by inhibitors of FGFs and TGF-ß receptors. Taken together, our study demonstrated the derivation of rabbit TS cells and suggested the essential roles of FGF and TGF-ß signalings in maintenance of rabbit TS cell self-renewal.

Impairments in embryonic genome activation in rhesus monkey somatic cell nuclear transfer embryos.

Somatic cell nuclear transfer (SCNT) is a remarkable process in which a somatic cell nucleus is acted upon by the ooplasm via mechanisms that today remain unknown. Here we show the developmental competence (% blastocyst) of embryos derived from SCNT (21%) was markedly (p < 0.05) impaired compared with those derived from in vitro fertilization (IVF) (42.1%) in rhesus monkey. Also, SCNT embryos were abnormal in their time course of embryonic development. SCNT produced embryos reached the eight-cell stage faster than did IVF produced embryos. We compare the transcription patterns of five nucleolar-related proteins-nucleolin, nucleophosmin, fibrillarin, PAF53, and UBF-in single IVF and SCNT blastocysts by RT-PCR. The SCNT embryos showed abnormal gene transcription. Immunolocalization of fibrillarin was undetectable in 8-cell and 16-cell SCNT embryos, indicating embryonic genomic activation was delayed in monkey embryos produced by SCNT compared to their IVF-derived counterparts. Some of SCNT embryos appeared to relative higher developmental potential and fibrillarin expression by prolonged exposure of incoming nuclei to a cytoplasm. Thus, our data show that SCNT embryos are characterized by abnormal cleavage and the timely onset of embryonic genome transcription, deficits that may explain their reduced pre- and postimplantation developmental capacity.

A comparison of the protective action of added egg yolks from five avian species to the cryopreservation of bull sperm.

Cryopreservation of domestic animal sperm has been widely used for artificial insemination (AI), and egg yolk is one of the most commonly used cryoprotectants during the freezing-thawing process. The objectives of this study were to compare the effectiveness of egg yolk from five avian species (domestic chicken, domestic duck, domestic goose, Japanese quail or domestic pigeon) and to optimize the concentration of egg yolk on the cryopreservation of bull sperm in terms of frozen-thawed sperm progressive motility and viability. The results were two-fold. First, they showed that pigeon egg yolk provided the best cryoprotective effects on the cryopreservation of bull sperm, compared with egg yolk of chicken, quail, goose or duck. Second, the best concentration of pigeon egg yolk in extender was 20% during cryopreservation among five concentrations of 5, 10, 20, 30 or 40%. The results suggest that pigeon egg yolk could be used as an alternative to chicken egg yolk in extender but requires further testing in fertility trials.

Generation and characterization of rabbit embryonic stem cells.

We described the derivation of four stable pluripotent rabbit embryonic stem cell (ESC) lines, one (RF) from blastocysts fertilized in vivo and cultured in vitro and three (RP01, RP02, and RP03) from parthenogenetic blastocysts. These ESC lines have been cultivated for extended periods (RF >1 year, RP01 >8 months, RP02 >8 months, and RP03 >6 months) in vitro while maintaining expression of pluripotent ESC markers and a normal XY or XX karyotype. The ESCs from all lines expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), and the tumor-related antigens (TRA-1-60 and TRA-1-81). Similar to human and mouse ESCs, rabbit ESCs expressed pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and signaling pathway genes (fibroblast growth factor, WNT, and transforming growth factor pathway). Morphologically, rabbit ESCs resembled primate ESCs, whereas their proliferation characteristics were more like those seen in mouse ESCs. Rabbit ESCs were induced to differentiate into many cell types in vitro and formed teratomas with derivatives of the three major germ layers in vivo when injected into severe combined immunodeficient mice. Our results showed that pluripotent, stable ESC lines could be derived from fertilized and parthenote-derived rabbit embryos.

Epigenetic marks in cloned rhesus monkey embryos: comparison with counterparts produced in vitro.

Until now, no primate animals have been successfully cloned to birth with somatic cell nuclear transfer (SCNT) procedures, and little is known about the molecular events that occurred in the reconstructed embryos during preimplantation development. In many SCNT cases, epigenetic reprogramming of the donor nuclei after transfer into enucleated oocytes was hypothesized to be crucial to the reestablishment of embryonic totipotency. In the present study, we focused on two major epigenetic marks, DNA methylation and histone H3 lysine 9 (H3K9) acetylation, which we examined by indirect immunofluorescence and confocal laser scanning microscopy. During preimplantation development, 67% of two-cell- and 50% of eight-cell-cloned embryos showed higher DNA methylation levels than their in vitro fertilization (IVF) counterparts, which undergo gradual demethylation until the early morula stage. Moreover, whereas an asymmetric distribution of DNA methylation was established in an IVF blastocysts with a lower methylation level in the inner cell mass (ICM) than in the trophectoderm, in most cloned blastocysts, ICM cells maintained a high degree of methylation. Finally, two donor cell lines (S11 and S1-04) that showed a higher level of H3K9 acetylation supported more blastocyst formation after nuclear transfer than the other cell line (S1-03), with a relatively low level of acetylation staining. In conclusion, we propose that abnormal DNA methylation patterns contribute to the poor quality of cloned preimplantation embryos and may be one of the obstacles to successful cloning in primates.