Precision of progesterone measurements with the use of automated immunoassay analyzers and the impact on clinical decisions for in vitro fertilization.

OBJECTIVE: To compare the precision of progesterone measurements obtained with the use of immunoassays and of liquid chromatography-tandem mass spectrometry (LC-MS/MS). DESIGN: Comparative study. SETTING: Academic, private practice, and in vitro fertilization (IVF) research centers. PATIENT(S): A total of 189 human serum samples were collected during controlled ovarian hyperstimulation and early pregnancy in women undergoing IVF. INTERVENTION(S): Serum progesterone pools (n = 10; 0.2-4 ng/mL) were sent to four laboratory centers that used four different automated immunoassay analyzers. Progesterone was measured by immunoassay in triplicate at three separate time points (n = 9 per pool) and by LC-MS/MS in triplicate once (n = 3 per pool). MAIN OUTCOME MEASURE(S): Inter- and intraassay coefficients of variation (CVs) of progesterone measurements were compared for each analyzer and LC-MS/MS. RESULT(S): Progesterone measurements by immunoassay were highly correlated with those by LC-MS/MS. Only two analyzers had intraassay CVs <10% at all three experimental time points, and only two analyzers had an interassay CV <10%. Mean progesterone levels by the analyzers were different across multiple progesterone pools. CONCLUSION(S): Our results indicate that progesterone threshold measurements used for IVF clinical decisions should be interpreted cautiously and based on laboratory- and method-specific data. A validated progesterone standard incorporated into daily immunoassays could improve medical decision accuracy.

In vivo delivery of FTY720 prevents radiation-induced ovarian failure and infertility in adult female nonhuman primates.

OBJECTIVE: To determine whether sphingosine-1-phosphate (S1P), or the S1P mimetic FTY720 shields ovaries of adult female rhesus monkeys from damage caused by 15 Gy of targeted radiotherapy, allowing for the retention of long-term fertility, and to evaluate whether S1P protects human ovarian tissue (xenografted into mice) from radiation-induced damage. DESIGN: Research animal study. SETTING: Research laboratory and teaching hospital. PATIENT(S): Adult female rhesus macaques (8-14 years of age; n = 21) and two women (24 and 27 years of age) undergoing gynecologic surgery for benign reasons, after informed consent and approval. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Ovarian histologic analysis, ovarian reserve measurements, and fertility in mating trials. RESULT(S): Rapid ovarian failure was induced in female macaques by ovarian application of 15 Gy of radiation. Females given S1P or FTY720 by direct intraovarian cannulation for 1 week before ovarian irradiation rapidly resumed menstrual cycles because of maintenance of follicles, with greater beneficial effects achieved using FTY720. Monkeys given the S1P mimetic before ovarian irradiation also became pregnant in mating trials. Offspring conceived and delivered by radioprotected females developed normally and showed no evidence of genomic instability, as measured by micronucleus frequency in reticulocytes. Adult human ovarian cortical tissue xenografted into mice also exhibited a reduction in radiation-induced primordial oocyte depletion when preexposed to S1P. CONCLUSION(S): S1P and its analogs hold clinical promise as therapeutic agents to preserve ovarian function and fertility in female cancer patients exposed to cytotoxic treatments.

Hypothalamic expression of KISS1 and gonadotropin inhibitory hormone genes during the menstrual cycle of a non-human primate.

Kisspeptin, the product of the KISS1 gene, stimulates gonadotropin-releasing hormone (GnRH) secretion; gonadotropin inhibitory hormone (GnIH), encoded by the RF-amide-related peptide (RFRP) or NPVF gene, inhibits the reproductive axis. In sheep, kisspeptin neurons are found in the lateral preoptic area (POA) and the arcuate nucleus (ARC) and may be important for initiating the preovulatory GnRH/luteinizing hormone (LH) surge. GnIH cells are located in the ovine dorsomedial hypothalamic nucleus (DMN) and paraventricular nucleus (PVN), with similar distribution in the primate. KISS1 cells are found in the primate POA and ARC, but the function that kisspeptin and GnIH play in primates has not been elucidated. We examined KISS1 and NPVF mRNA throughout the menstrual cycle of a female primate, rhesus macaque (Macaca mulatta), using in situ hybridization. KISS1-expressing cells were found in the POA and ARC, and NPVF-expressing cells were located in the PVN/DMN. KISS1 expression in the caudal ARC and POA was higher in the late follicular phase of the cycle (just before the GnRH/LH surge) than in the luteal phase. NPVF expression was also higher in the late follicular phase. We ascertained whether kisspeptin and/or GnIH cells project to GnRH neurons in the primate. Close appositions of kisspeptin and GnIH fibers were found on GnRH neurons, with no change across the menstrual cycle. These data suggest a role for kisspeptin in the stimulation of GnRH cells before the preovulatory GnRH/LH surge in non-human primates. The role of GnIH is less clear, with paradoxical up-regulation of gene expression in the late follicular phase of the menstrual cycle.

Estradiol increases alpha7 nicotinic receptor in serotonergic dorsal raphe and noradrenergic locus coeruleus neurons of macaques.

Acetylcholine, acting on presynaptic nicotinic receptors (nAChRs), modulates the release of neurotransmitters in the brain. The rat dorsal raphe nucleus (DR) and the locus coeruleus (LC) receive cholinergic input and express the alpha7nAChR. In previous reports, we demonstrated that estradiol (E) administration stimulates DR serotonergic and LC noradrenergic function in the macaque. In addition, it has been reported that E induces the expression of the alpha7nAChR in rats. We questioned whether E increased the expression of the alpha7nAChR in the macaque DR and LC. We utilized double immunostaining to study the effect of a simulated preovulatory surge of E on the expression of the alpha7nAChR in the DR and the LC and to determine whether alpha7nAChR colocalizes with serotonin and tyrosine hydroxylase (TH) in macaques. There was no difference in the number of alpha7nAChR-positive neurons between ovariectomized (OVX) controls and OVX animals treated with a silastic capsule containing E (Ecap). However, supplemental infusion of E for 5-30 hours to Ecap animals (Ecap + inf) significantly increased the number of alpha7nAChR-positive neurons in DR and LC. In addition, supplemental E infusion significantly increased the number of neurons in which alpha7nAChR colocalized with serotonin and TH. These results constitute an important antecedent for study of the effects of nicotine and ovarian steroid hormones in the physiological functions regulated by the DR and the LC in women.

Progress with nonhuman primate embryonic stem cells.

Embryonic stem cells hold potential in the fields of regenerative medicine, developmental biology, tissue regeneration, disease pathogenicity, and drug discovery. Embryonic stem (ES) cell lines are now available in primates, including man, rhesus, and cynomologous monkeys. Monkey ES cells serve as invaluable clinically relevant models for studies that can't be conducted in humans because of practical or ethical limitations, or in rodents because of differences in physiology and anatomy. Here, we review the current status of nonhuman primate research with ES cells, beginning with a description of their isolation, characterization, and availability. Substantial limitations still plague the use of primate ES cells, such as their required growth on feeder layers, poor cloning efficiency, and restricted availability. The ability to produce homogenous populations of both undifferentiated as well as differentiated phenotypes is an important challenge, and genetic approaches to achieving these objectives are discussed. Finally, safety, efficiency, and feasibility issues relating to the transplantation of ES-derived cells are considered.

Differentiation of monkey embryonic stem cells into neural lineages.

Embryonic stem (ES) cells are self-renewing, pluripotent, and capable of differentiating into all of the cell types found in the adult body. Therefore, they have the potential to replace degenerated or damaged cells, including those in the central nervous system. For ES cell-based therapy to become a clinical reality, translational research involving nonhuman primates is essential. Here, we report monkey ES cell differentiation into embryoid bodies (EBs), neural progenitor cells (NPCs), and committed neural phenotypes. The ES cells were aggregated in hanging drops to form EBs. The EBs were then plated onto adhesive surfaces in a serum-free medium to form NPCs and expanded in serum-free medium containing fibroblast growth factor (FGF)-2 before neural differentiation was induced. Cells were characterized at each step by immunocytochemistry for the presence of specific markers. The majority of cells in complex/cystic EBs expressed antigens (alpha-fetal protein, cardiac troponin I, and vimentin) representative of all three embryonic germ layers. Greater than 70% of the expanded cell populations expressed antigenic markers (nestin and musashi1) for NPCs. After removal of FGF-2, approximately 70% of the NPCs differentiated into neuronal phenotypes expressing either microtubule-associated protein-2C (MAP2C) or neuronal nuclear antigen (NeuN), and approximately 28% differentiated into glial cell types expressing glial fibrillary acidic protein. Small populations of MAP2C/NeuN-positive cells also expressed tyrosine hydroxylase (approximately 4%) or choline acetyltransferase (approximately 13%). These results suggest that monkey ES cells spontaneously differentiate into cells of all three germ layers, can be induced and maintained as NPCs, and can be further differentiated into committed neural lineages, including putative neurons and glial cells.