R.S Williams

Modulation of mouse sperm-egg interaction, early embryonic development and trophoblastic outgrowth by activated and unactivated macrophages.

Exposure of mouse spermatozoa and oocytes duringin vitro fertilization (IVF) to lipopolysaccharide (LPS) and phorbol myristate acetate (PMA) activated macrophages (U937 cell line), but not unactivated macrophages cultureconditioned medium or control medium (RMPI+DMEM with 0.5% FBS) resulted in inhibition of IVF (87.2%), first cleavage (90.8%) and total blastocyst formation 97.5%). The direct coculture of the activated macrophages with 2-cell stage embryos resulted in arrested development (91.2%), an effect that was significantly diminished in the presence of monolayer of human endometrial stromal cells in the coculture (58.3%). In contrast, the majority of 2-cell embryos developed to blastocysts when exposed to unactivated macrophages, or macrophage-stromal cell cocultures (94.1%). The majority of 2-cell embryos cultured in control medium (DMEM/Ham’s F12 with 2% FBS) developed to morulae (96.2%), then underwent growth arrest and degeneration. Furthermore, culturing blastocyst stage embryos in the above groups resulted in a significant enhancement of trophoblast outgrowth, particularly in coculture with activated macrophages as compared to any other group (P<0.005). There was a significant increase in the levels of TGF-β, GM-CSF, IL-1α, IL-1β, TNF-α, PGE2, TXB2 and LTB4 released into the culture conditioned medium of activated macrophages compared to unactivated macrophages (P<0.001). These results suggest that the secretory products of activated macrophages, among them those determined in this study, in a stage-specific manner can directly effect sperm-egg interaction, early embryonic development and trophoblastic outgrowth. This data provides further support for the hypothesis that in endometriosis-associated infertility, continuous exposure of spermatozoa, oocytes and early embryos to activated macrophage-derived factors may play a vital role in their survival during transportation and fertilization as well as development during early embryonic stage.

Neem oil inhibits two-cell embryo development and trophectoderm attachment and proliferation in vitro

PurposeThe in vitro effect of neem oil was studied on the development of mouse two-cell embryos and trophectodermal cell attachment and proliferation.MethodFemale mice were primed with gonadotropins for superovulation and caged with male mice. Early embryos, at the two-cell and the blastocyst stages, were recovered at 40 and 88 hr post-hCG from the oviducts and the uteri, respectively. In the first experiment, two-cell embryos were exposed to culture medium containing different concentrations of neem oil for 1, 12, and 24 hr and then grown in neem oil-free culture medium and assessed for the formation of total and hatching blastocysts at 96 hr. In the second experiment, partially hatching blastocysts were cocultured with human endometrial stromal cell monolayers in culture medium containing different concentrations of neem oil and assessed for the attachment and proliferation of trophectodermal cells at 96 hr.ResultsExposure of two-cell embryos to neem oil concentrations of 0.050–0.500% for 1 hr, 0.010–0.250% for 12 hr, and 0.005–0.100% for 24 hr caused significant inhibition of the formation of total and hatching blastocysts, in a dose-dependent manner. Neem oil at 0.050–0.100% concentrations inhibited, in a dose-dependent manner, the in vitro attachment and proliferation of trophectodermal cells of partially hatching blastocysts cocultured with human endometrial stromal cells monolayers.ConclusionNeem oil inhibits the development of two-cell embryos and attachment and proliferation of the trophectodermal cells of partially hatching blastocysts in vitro. The study encourages the use of this herbal product as a postcoital contraceptive that warrants further research.

Results on single cell PCR for Huntington's gene and WAVE™ product analysis for preimplantation genetic diagnosis

Triple repeat base pair amplification is the basis for a number of prevalent genetic diseases such as Huntingtons, Fragile X, Myotonic Dystrophy and others. We have chosen to investigate the use of PCR to amplify a portion of the Huntingtons gene in single cells in order to develop a clinical test system for preimplantation genetic diagnosis (PGD). Amplification of CAG triple repeat sequences poses difficulties due to resistance of GC melting for amplification. Special PCR modifications are necessary to carry out the amplification of GC rich areas found in most triple base pair expansions. We have used a modified polymerase chain reaction (PCR) protocol to amplify the expanded repeat sequence of the Huntingtons gene with satisfactory efficiency. Detection of the amplified expanded CAG repeats is shown to be possible using both agarose gel electrophoresis and high definition denaturing high pressure liquid (DHPLC) chromatography. The incidence of allele dropout (ADO) is documented.

Primer System for Single Cell Detection of Double Mutation for Tay-Sachs Disease

AbstractPurpose: Nearly 100% of infantile Tay-Sachs disease isproduced by two mutations occurring in the alpha chain ofthe lysosomal enzyme beta-N-acetylhexosaminidase (HEXA)in the Ashkenazi Jewish population. Although others havedescribed primer systems used to amplify both sitessimultaneously, few discuss the allele dropout problems inherent inthis test. Our goal was to construct a more robust testenabling stronger signal generation for single cellpreimplantation genetic diagnosis and to investigate theoccurrence of allele dropout. Methods: New nested primers were designed to optimizedetection of both major Tay-Sachs mutations. Four hundredfifty-seven single cells, including normal cells and thosecarrying mutations of either the 4bp insertion exon 11 orsplice-site intron 12 defects, were used to screen a newprimer system. Results: Based on PCR amplified product analysis, totalefficiency of amplification was 85.3%, (390/457). The alleledropout rate for the 4bp insertion mutation in exon 11 andsplice-site mutation in intron 12 was 4.8% and 5.8%,respectively. Conclusons: Multiple mutation detection and analysiswithin the Tay-Sachs disease gene (HEXA) is possible usingsingle cells for clinical preimplantation genetic diagnosis.Alternative PCR primers and conditions offer variousmethods for developing systems compatible to specificprogram requirements.

Simultaneous Single-Cell Detection of Two Mutations for Cystic Fibrosis

AbstractPurpose: A single-cell diagnosis procedure using polymerase chain reaction (PCR) technology was developed to simultaneously detect two cystic fibrosis (CF) mutations (DF-508, W1282X). Methods: Human viable, arrested, and nonviable embryos and immature, and nonfertilized oocytes donated by our patients were used to detect apoptosis by Tunel labeling, annexin staining, and single-cell reverse transcriptase–polymerase chain reaction (RT-PCR). Results: Using cells carrying the DF-508 mutation, the PCR signal efficiency for the affected homozygous, normal homozygous, and carrier heterozygote cell populations were 91%, 81%, and 92%, respectively. The total combined PCR efficiency was 87.7% and the ADO rate was 5.7%. For W1282X carrier heterozygote cells, the PCR signal efficiency was 82.0% and the ADO rate was 8.7%. Conclusions: Methods have been developed to detect two common mutations simultaneously for CF in single-cell assays. The high signal efficiencies and low ADO rates obtained in these tests allow those embryos from couples wishing to avert the transmission of this serious genetic disease to their offspring to be screened by preimplantation genetic diagnosis.