Sperm deoxyribonucleic acid fragmentation assessment in normozoospermic male partners of couples with unexplained recurrent pregnancy loss: a prospective study.

OBJECTIVE: To determine whether sperm DNA integrity in normozoospermic male partners plays a role in idiopathic recurrent pregnancy loss (RPL). DESIGN: Prospective, cohort study. SETTING: Academic tertiary care center. PATIENT(S): Group I: 26 male partners of women with unexplained RPL. Group II: 31 normozoospermic males with proven fertility. INTERVENTION(S): Semen samples were collected by masturbation after 48-72 hours of abstinence. After liquefaction at room temperature, semen analysis was performed according to World Health Organization standards. Only samples with >20 × 10(6) spermatozoa/mL with at least 50% progressive sperm motility and 30 % normal morphology were selected for the study. DNA fragmentation of the sperm was assessed with TUNEL assay followed by flow cytometric analysis. MAIN OUTCOME MEASURE(S): Sperm DNA fragmentation in both groups. RESULT(S): Mean DNA fragmentation (mean ± SD) was significantly more in men with RPL (36.8 ± 5) compared with controls (9.4 ± 2.7). CONCLUSION(S): Sperm DNA fragmentation may play a role in unexplained RPL despite normal semen analysis parameters.

Single-cell analysis of circulating tumor cells identifies cumulative expression patterns of EMT-related genes in metastatic prostate cancer.

BACKGROUND: Prostate tumors shed circulating tumor cells (CTCs) into the blood stream. Increased evidence shows that CTCs are often present in metastatic prostate cancer and can be alternative sources for disease profiling and prognostication. Here we postulate that CTCs expressing genes related to epithelial-mesenchymal transition (EMT) are strong predictors of metastatic prostate cancer. METHODS: A microfiltration system was used to trap CTCs from peripheral blood based on size selection of large epithelial-like cells without CD45 leukocyte marker. These cells individually retrieved with a micromanipulator device were assessed for cell membrane physical properties using atomic force microscopy. Additionally, 38 CTCs from eight prostate cancer patients were used to determine expression profiles of 84 EMT-related and reference genes using a microfluidics-based PCR system. RESULTS: Increased cell elasticity and membrane smoothness were found in CTCs compared to noncancerous cells, highlighting their potential invasiveness and mobility in the peripheral circulation. Despite heterogeneous expression patterns of individual CTCs, genes that promote mesenchymal transitioning into a more malignant state, including IGF1, IGF2, EGFR, FOXP3, and TGFB3, were commonly observed in these cells. An additional subset of EMT-related genes (e.g., PTPRN2, ALDH1, ESR2, and WNT5A) were expressed in CTCs of castration-resistant cancer, but less frequently in castration-sensitive cancer. CONCLUSIONS: The study suggests that an incremental expression of EMT-related genes in CTCs is associated with metastatic castration-resistant cancer. Although CTCs represent a group of highly heterogeneous cells, their unique EMT-related gene signatures provide a new opportunity for personalized treatments with targeted inhibitors in advanced prostate cancer patients.

Assisted reproductive technology in nonhuman primates.

Nonhuman primates (NHP) are the closest animal species to humans and have been widely used for studying human reproductive physiology. Assisted reproductive technology (ART) in Old World NHPs provides great opportunity for studying fertilization, embryo development, embryonic stem cell (ESC) derivation for regenerative medicine, somatic cell nuclear transfer (cloning), and transgenic NHP models of inherited genetic disorders. Here we present two ART protocols developed for rhesus monkey (Macaca mulatta) and baboon (Papio cynocephalus).

Derivation and characterization of novel nonhuman primate embryonic stem cell lines from in vitro-fertilized baboon preimplantation embryos.

The development of nonhuman primate (NHP) embryonic stem cell (ESC) models holds great promise for cell-mediated treatment of debilitating diseases and to address numerous unanswered questions regarding the therapeutic efficacy of ESCs while supplanting ethical considerations involved with human studies. Here we report successful establishment and characterization of 3 novel baboon (Papio cynocephalus) ESC lines from the inner cell mass of intracytoplasmic sperm injection-derived blastocysts. Embryos were cultured in an improved baboon embryo in vitro culture protocol. The inner cell mass of blastocyst was laser-dissected and plated on mouse embryonic fibroblast feeder cell monolayer in the NHP ESC culture medium. Three cell lines with characteristic ESC morphology have been cultured through an extended period (>14 months), with 2 male cell lines (UT-1 and -2) and 1 female cell line (UT-3) displaying normal baboon karyotypes. Reverse transcription-polymerase chain reaction analysis confirmed that all 3 lines express primate ESC pluripotency markers, including OCT-4, NANOG, SOX-2, TERT, TDGF, LEFTYA, and REX-1. All 3 lines demonstrated positive immunocytochemical staining for OCT-4, stage-specific embryonic antigen-3, stage-specific embryonic antigen-4, TRA-1-60, and TRA-1-81. Baboon ESCs injected into NOD/SCID mice formed teratomas with all 3 germ layers. In addition, embryoid body-like spherical structures were derived and initial outgrowth was observed when embedded into extracellular matrix Matrigel. The ESC lines established in this NHP model have the potential to extend our knowledge in the fields of developmental biology, regenerative medicine, and future applications, including preclinical safety assessment of in vivo stem cell therapy.

Ovarian stimulation, in vitro fertilization, and effects of culture conditions on baboon preimplantation embryo development.

OBJECTIVE: To evaluate the effects of ovarian stimulation and intracytoplasmic sperm injection (ICSI)-induced fertilization and efficacy of various culture systems on in vitro development of baboon embryos. DESIGN: In vitro study, animal model. SETTING: Research laboratory. ANIMAL(S): Baboons in laboratory animal research facility. INTERVENTION(S): Baboons received FSH (75 IU daily) for 7 to 8 days and FSH/LH (75/75 IU daily) for 3 days, followed by hCG (2,000 IU). Oocytes were retrieved laparoscopically 36 hours after hCG. Intracytoplasmic sperm injection was performed on metaphase II (MII) oocytes. Fertilized embryos were placed into different culture conditions and feeder cell coculture. Embryo development was observed through the most advanced stages, including blastocyst formation. MAIN OUTCOME MEASURE(S): Oocytes retrieved, fertilization rates, multicell embryo rates, and blastocyst rates. RESULT(S): Baboon oocytes (n = 1,924, from 49 cycles) were retrieved. Significant heterogeneity was seen in ovarian response to exogenous gonadotropins and subsequent oocyte maturation. The percentage of MII oocytes showed no significant difference among individual female baboons and stimulation cycles. Nearly two thirds of MII oocytes were successfully fertilized with ICSI. Blastocyst rates varied significantly among embryos in different treatments. Coculture with feeder cells in P1/Blast, Quinn's Advantage, and Sydney IVF media generated better blastocyst rates. CONCLUSION(S): We tested multiple media and feeder cell combinations to optimize culture conditions in baboon embryo culture and obtained a high blastocyst rate similar to those reported for rhesus monkey embryos cultured in vitro, but still lower than with assisted reproductive technologies in women.

Trophoblast stem cells: models for investigating trophectoderm differentiation and placental development.

The placenta is an ephemeral organ containing diverse populations of trophoblasts that are all derived from the embryonic trophectoderm but have morphological, functional, and molecular diversity within and across species. In hemochorial placentation, these cells play especially important roles, interfacing with and modifying the cells of the maternal decidua. Within the rapidly growing placenta, it has been shown that there are trophoblast stem cells well characterized in the mouse and postulated but not well understood in primates. This review will discuss the characteristics of candidates for human and nonhuman primate trophoblast stem cells, present the diverse methods of their generation, and propose future prospects for experimental systems in which they can shed light on developmental and pathophysiological processes in human pregnancy.