John D. Brannian

Relationship between the outcomes of assisted reproductive techniques and sperm DNA fragmentation as measured by the sperm chromatin structure assay

OBJECTIVEnTo investigate how moderate and/or high levels of DNA fragmentation (DFI), as measured by the sperm chromatin structure assay (SCSA), affect either IVF or IVF with intracytoplasmic sperm injection (ICSI) fertilization, cleavage, blastulation, implantation, and pregnancy.nnnDESIGNnRetrospective clinical study.nnnSETTINGnAcademic human reproduction laboratory.nnnPATIENT(S)nEighty-nine couples undergoing IVF with conventional fertilization or ICSI.nnnINTERVENTION(S)nSperm chromatin structure assay testing (SCSA) of semen aliquot taken from ejaculate used for assisted reproductive technology (ART).nnnMAIN OUTCOME MEASURE(S)nRelated DFI to conventional semen parameters and cycle-specific outcomes after ART.nnnRESULT(S)nNo patients achieved clinical pregnancy if SCSA values exceeded the DFI (27%, P<.01), moderate DFI (15%, P<.01), or high DFI (15%, P<.05) thresholds. Dividing the DFI sperm population into moderate-fragmentation and high-fragmentation categories did not improve the prognostic value of the SCSA. No coefficient of determination (r(2)) between SCSA parameters and conventional parameters exceeded 0.29.nnnCONCLUSION(S)nSperm chromatin structure assay identified thresholds for negative pregnancy outcome after ART not identified using conventional semen parameters. This is the first study analyzing the clinical value of sperm DFI to [1] include a large number of ART patients (n = 89), [2] perform SCSA analysis on a semen aliquot from the ejaculate used for ART, and [3] examine how the extent (moderate and high DFI) of DFI influenced ART outcomes.

DNA microarray analysis of gene expression markers of endometriosis

OBJECTIVEnTo use DNA microarray technology to examine differential gene expression in uterine endometrium versus endometriosis implants.nnnDESIGNnPilot study.nnnSETTINGnVolunteers in an academic research environment.nnnPATIENT(S)nPremenopausal women scheduled for surgery for suspected endometriosis.nnnINTERVENTION(S)nSurgical excision of endometriosis tissue and uterine endometrial biopsy.nnnMAIN OUTCOME MEASURE(S)nGene expression.nnnRESULT(S)nThe expression of eight genes from a total of 4,133 genes on the DNA microarray was increased in endometriosis implants compared with uterine endometrium. The eight genes were beta-actin, alpha-2 actin, vimentin, 40S ribosomal protein S23, Ig-lambda light chain, Ig germline H chain G-E-A region gamma-2 constant region gene, major histocompatibility complex class 1,C, and complement component 1 S subcomponent.nnnCONCLUSION(S)nThe data demonstrate that the DNA microarray is an effective tool for the identification of differentially expressed genes between uterine and ectopic endometrium; further study of the genes identified herein will expand our understanding of the nature of endometriosis and assist in the eventual development of new treatments for endometriosis.

Differential gene expression in human granulosa cells from recombinant FSH versus human menopausal gonadotropin ovarian stimulation protocols

BackgroundThe study was designed to test the hypothesis that granulosa cell (GC) gene expression response differs between recombinant FSH and human menopausal gonadotropin (hMG) stimulation regimens.MethodsFemales < 35 years-old undergoing IVF for tubal or male factor infertility were prospectively randomized to one of two stimulation protocols, GnRH agonist long protocol plus individualized dosages of (1) recombinant (r)FSH (Gonal-F) or (2) purified human menopausal gonadotropin (hMG; Menopur). Oocytes were retrieved 35 h post-hCG, and GC were collected. Total RNA was extracted from each GC sample, biotinylated cRNA was synthesized, and each sample was run on Human Genome Bioarrays (Applied Microarrays). Unnamed genes and genes with <2-fold difference in expression were excluded.ResultsAfter exclusions, 1736 genes exhibited differential expression between groups. Over 400 were categorized as signal transduction genes, ~180 as transcriptional regulators, and ~175 as enzymes/metabolic genes. Expression of selected genes was confirmed by RT-PCR. Differentially expressed genes included A kinase anchor protein 11 (AKAP11), bone morphogenetic protein receptor II (BMPR2), epidermal growth factor (EGF), insulin-like growth factor binding protein (IGFBP)-4, IGFBP-5, and hypoxia-inducible factor (HIF)-1 alpha.ConclusionsResults suggest that major differences exist in the mechanism by which pure FSH alone versus FSH/LH regulate gene expression in preovulatory GC that could impact oocyte maturity and developmental competence.

Progressive obesity leads to altered ovarian gene expression in the Lethal Yellow mouse: a microarray study

BackgroundLethal yellow (LY; C57BL/6J Ay/a) mice exhibit adult-onset obesity, altered metabolic regulation, and early reproductive senescence. The present study was designed to test the hypothesis that obese LY mice possess differences in expression of ovarian genes relative to age-matched lean mice.Methods90- and 180-day-old LY and lean black (C57BL/6J a/a) mice were suppressed with GnRH antagonist (Antide®), then stimulated with 5 IU eCG. cRNA derived from RNA extracts of whole ovarian homogenates collected 36 h post-eCG were run individually on Codelink Mouse Whole Genome Bioarrays (GE Healthcare Life Sciences).ResultsFifty-two genes showed ≥ 2-fold differential (p < 0.05) expression between 180-day-old obese LY and lean black mice. LY mice exhibited elevated ovarian expression of agouti (350×), leptin (6.5×), and numerous genes involved in cholesterol/lipid transport and metabolism, e.g. lanosterol synthase, Cyp51, and steroidogenic acute regulatory protein (Star). Fewer genes showed lower expression in LY mice, e.g. angiotensinogen. In contrast, none of these genes showed differential expression in 90-day-old LY and black mice, which are of similar body weight. Interestingly, 180-day-old LY mice had a 2-fold greater expression of 11beta-hydroxysteroid dehydrogenase type 1 (Hsd11b1) and a 2-fold lesser expression of 11beta-hydroxysteroid dehydrogenase type 2 (Hsd11b2), differences not seen in 90-day-old mice. Consistent with altered Hsd11b gene expression, ovarian concentrations of corticosterone (C) were elevated in aging LY mice relative to black mice, but C levels were similar in young LY and black mice.ConclusionThe data suggest that reproductive dysfunction in aging obese mice is related to modified intraovarian gene expression that is directly related to acquired obesity.

Pioglitazone administration alters ovarian gene expression in aging obese lethal yellow mice

BackgroundWomen with polycystic ovary syndrome (PCOS) are often treated with insulin-sensitizing agents, e.g. thiazolidinediones (TZD), which have been shown to reduce androgen levels and improved ovulatory function. Acting via peroxisome proliferator-activated receptor (PPAR) gamma, TZD alter the expression of a large variety of genes. Lethal yellow (LY; C57BL/6J Ay/a) mice, possessing a mutation (Ay) in the agouti gene locus, exhibit progressive obesity, reproductive dysfunction, and altered metabolic regulation similar to women with PCOS. The current study was designed to test the hypothesis that prolonged treatment of aging LY mice with the TZD, pioglitazone, alters the ovarian expression of genes that may impact reproduction.MethodsFemale LY mice received daily oral doses of either 0.01 mg pioglitazone (n = 4) or an equal volume of vehicle (DMSO; n = 4) for 8 weeks. At the end of treatment, ovaries were removed and DNA microarrays were used to analyze differential gene expression.ResultsTwenty-seven genes showed at least a two-fold difference in ovarian expression with pioglitazone treatment. These included leptin, angiopoietin, angiopoietin-like 4, Foxa3, PGE1 receptor, resistin-like molecule-alpha (RELM), and actin-related protein 6 homolog (ARP6). For most altered genes, pioglitazone changed levels of expression to those seen in untreated C57BL/6J(a/a) non-mutant lean mice.ConclusionTZD administration may influence ovarian function via numerous diverse mechanisms that may or may not be directly related to insulin/IGF signaling.

Hydrogen peroxide suppresses low-density lipoprotein (LDL) uptake and LDL-supported steroidogenesis by porcine luteal cells.

The present study tested the hypothesis that hydrogen peroxide (H2O2) inhibits low-density lipoprotein (LDL) uptake and LDL-supported steroidogenesis by luteal cells. LDL uptake: dispersed porcine luteal cells from mid-cycle (days 6-11, estrus = day 0) were incubated for 0-120 min at 37 degrees C in F-10 medium + 0.1% BSA containing various concentrations of H2O2 (0-1000 microM). Cells were washed with catalase (2800 U/ml), and then with fresh medium. Cell viability based on trypan blue exclusion was unaltered by H2O2 exposure through 60 min. H2O2-exposed cells were incubated with fluorescent-tagged-LDL (Dil-LDL; 1 microgram/ml) for 10 min at 37 degrees C. Fluorescence of small (SLC) and large (LLC) luteal cells was analyzed by flow cytometry (n = 6 experiments). H2O2 (> or = 10 microM) caused a progressive reduction (P < 0.01) in mean fluorescence intensity (MFI) of SLC and LLC indicative of up to a 30-35% decline in LDL uptake. Progesterone (P) production: dispersed luteal cells (4 x 10(4)/0.2 ml) were pre-cultured in DMEM/F-12 medium overnight (approximately 18 h) in 96-well culture plates. Wells were rinsed and fresh media (0.2 ml) containing H2O2 (0-500 microM) was added. After 30 min, the following treatments were added: human(h)LDL (0 or 50 micrograms/ml), human chorionic gonadotropin (hCG; 0 or 100 ng/ml), hCG + LDL, or 22R-hydroxycholesterol (22[OH]-C; 0 or 25 micrograms/ml). Cells were incubated for an additional 4 h, and P concentrations in final media samples were measured by RIA.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene Expression Profiling in the Aging Ovary

DNA microarray is an important discovery technology that allows the analysis of the expression of thousands of genes at a time. Data from DNA microarrays elucidate fundamental biological processes through discovery of differential expression of genes not previously known or predicted to be involved in a particular process. In the ovary and other hormone-responsive tissues, the technology can be used to examine the effects of gene mutations, pharmaceutical agents, disease, hormones, developmental changes, or changes in gene expression related to aging.

Impact of Leptin on Ovarian Folliculogenesis and Assisted Reproduction

With the discovery in the late 1990s that leptin receptors are expressed in high levels within maturing follicles, it became clear that leptin may regulate ovarian function at multiple levels. Not only does leptin participate in the control of gonadotropin secretion via its hypothalamic actions, but circulating or locally produced leptin may also provide direct modulation of thecal, granulosa, and oocyte function. Addition of leptin to the growing list of cytokines and growth factors involved in follicular development and oocyte maturation extends our appreciation of the complex integration of basic cellular and metabolic processes with reproductive function and fertility.