Extensive monitoring of the natural menstrual cycle using the serum biomarkers estradiol, luteinizing hormone and progesterone.

Expected values for estradiol (E2), luteinizing hormone (LH), and progesterone determined in serum allow accurate assessment of menstrual cycle phase. Automated immunoassays demonstrate variable degrees of bias, emphasizing the need to establish method-specific reference values. We therefore established method-specific reference intervals for the Elecsys® LH assay and new generation Elecsys Estradiol III and Progesterone III assays (cobas e 801 analyzer) in 85 apparently healthy women aged 22-37 (US)/18-37 (EU) years over one natural menstrual cycle. Cycle length and day of ovulation were standardized; phases were defined by LH surge and/or progesterone/E2 levels. Median (5th-95th percentile) concentrations (follicular/ovulation/luteal) were E2: 198 â€‹pmol/L (114-332), 757 â€‹pmol/L (222-1959) and 412 â€‹pmol/L (222-854); LH: 7.14 IU/L (4.78-13.2), 22.6 IU/L (8.11-72.7) and 6.24 IU/L (2.73-13.1); progesterone: 0.212 â€‹nmol/L (0.159-0.616), 1.81 â€‹nmol/L (0.175-13.2) and 28.8 â€‹nmol/L (13.1-46.3). Sub-phase (early/intermediate/late) reference values were also determined for follicular and luteal phases. This multicenter study established reliable, method-specific E2, LH and progesterone reference values that could assist clinical decision-making in women with fertility disorders and monitoring of natural cycles in assisted reproductive treatment.

A novel neurotropic expression vector based on the avirulent A7(74) strain of Semliki Forest virus.

Semliki Forest virus (SFV), an enveloped alphavirus of the family Togaviridae, infects a wide range of mammalian host cells. Most strains are neurotropic but differ in virulence. The authors took advantage of the nonpathogenic properties of SFV strain A7(74), cloned recently in their laboratory, and constructed a replication-proficient expression vector to target the central nervous system (CNS) for heterologous gene expression. The vector, termed VA7, was engineered to drive expression of foreign inserts through a second subgenomic promoter inserted in the viral 3' nontranslated region (NTR). Infectious virus was obtained by in vitro transcription and transfection into BHK cells, and was shown to direct synthesis of heterologous proteins in several mammalian cell lines. Although novel expression vehicle is not applicable for targeting specific cell populations within the CNS in its present form, in cultured rat hippocampal slices, VA7 encoding enhanced green fluorescent protein (EGFP) efficiently transduced pyramidal cells, interneurons, and glial cells. With prolonged time post infection, the number of EGFP-expressing neurons in hippocampal slices increased. Mice infected intraperitoneally with the recombinant virus remained completely asymptomatic but showed CNS expression of EGFP as evidenced by immunohistochemistry. SFV A7(74) is a nonintegrating virus, which gives rise to a randomly distributed, patchy infection of the adult CNS that is cleared within 10 days. With the advantage of noninvasive administration, the expression vector described in this work is thus applicable for short-term gene expression in the CNS.

Semliki Forest virus A7(74) transduces hippocampal neurons and glial cells in a temperature-dependent dual manner.

In central nervous system (CNS) tissue preparations, wild-type Semliki Forest virus (SFV) mainly infects neurons, and in vivo it causes lethal encephalitis in neonatal and adult rodents. The SFV strain A7(74), by contrast, is avirulent in adult rodents, triggering only limited CNS infection. To examine A7(74) infection in hippocampal tissue, the authors constructed a replicon, termed SFV(A774nsP)-GFP, expressing green fluorescent protein. The results were compared to replication-proficient recombinant A7(74) encoding GFP, named VA7-EGFP. As nonstructural gene mutations can confer temperature sensitivity, the authors also tested whether infection was temperature-dependent. Indeed, at 31 degrees C both viral recombinants transduced significantly more baby hamster kidney cells than at 37 degrees C. When rat hippocampal slices and dissociated cells were incubated at 37 degrees C, SFV(A774nsP)-GFP transduced glial cells but virtually no neurons-the opposite of conventional SFV. For VA7-EGFP at 37 degrees C, the preferred GFP-positive cells in hippocampal slices were also non-neuronal cells. At 31 degrees C, however, a more wild-type phenotype was found, with 33% and 94% of the GFP-positive cells being neurons for SFV(A774nsP)-GFP in slices and dissociated cells, respectively, and 94% neurons for VA7-EGFP in slices. Immunochemical and electrophysiological analyses confirmed that at 37 degrees C virtually all cells transduced by SFV(A774nsP)-GFP in slices were astrocytes, while at 31 degrees C they also contained neurons. These results show that in addition to the developmental age, the temperature determines which cell type becomes infected by A7(74). Our data suggest that A7(74) is avirulent in adult animals because it does not readily replicate in mature neurons at body temperature, whereas it still does so at lower temperatures.