Linda C. Giudice

Science and policy on endocrine disrupters must not be mixed: a reply to a “common sense” intervention by toxicology journal editors

The “common sense” intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU.

CLINICAL PRACTICE: Endometriosis

A healthy 25-year-old woman presents with worsening dysmenorrhea, pain of recent onset in the left lower quadrant, and dyspareunia. She has regular menstrual cycles, and her last menstrual period was 3 weeks before presentation. How should this patient be evaluated and treated?

Expression Profiling of Endometrium from Women with Endometriosis Reveals Candidate Genes for Disease-Based Implantation Failure and Infertility

Endometriosis is clinically associated with pelvic pain and infertility, with implantation failure strongly suggested as an underlying cause for the observed infertility. Eutopic endometrium of women with endometriosis provides a unique experimental paradigm for investigation into molecular mechanisms of reproductive dysfunction and an opportunity to identify specific markers for this disease. We applied paralleled gene expression profiling using high-density oligonucleotide microarrays to investigate differentially regulated genes in endometrium from women with vs. without endometriosis. Fifteen endometrial biopsy samples (obtained during the window of implantation from eight subjects with and seven subjects without endometriosis) were processed for expression profiling on Affymetrix Hu95A microarrays. Data analysis was conducted with GeneChip Analysis Suite, version 4.01, and GeneSpring version 4.0.4. Nonparametric testing was applied, using a P value of 0.05, to assess statistical significance. Of the 12,686 genes analyzed, 91 genes were significantly increased more than 2-fold in their expression, and 115 genes were decreased more than 2-fold. Unsupervised clustering demonstrated down-regulation of several known cell adhesion molecules, endometrial epithelial secreted proteins, and proteins not previously known to be involved in the pathogenesis of endometriosis, as well as up-regulated genes. Selected dysregulated genes were randomly chosen and validated with RT-PCR and/or Northern/dot-blot analyses, and confirmed up-regulation of collagen alpha2 type I, 2.6-fold; bile salt export pump, 2.0-fold; and down-regulation of N-acetylglucosamine-6-O-sulfotransferase (important in synthesis of L-selectin ligands), 1.7-fold; glycodelin, 51.5-fold; integrin alpha2, 1.8-fold; and B61 (Ephrin A1), 4.5-fold. Two-way overlapping layer analysis used to compare endometrial genes in the window of implantation from women with and without endometriosis further identified three unique groups of target genes, which differ with respect to the implantation window and the presence of disease. Group 1 target genes are up-regulated during the normal window of implantation but significantly decreased in women with endometriosis: IL-15, proline-rich protein, B61, Dickkopf-1, glycodelin, N-acetylglucosamine-6-O-sulfotransferase, G0S2 protein, and purine nucleoside phosphorylase. Group 2 genes are normally down-regulated during the window of implantation but are significantly increased with endometriosis: semaphorin E, neuronal olfactomedin-related endoplasmic reticulum localized protein mRNA and Sam68-like phosphotyrosine protein alpha. Group 3 consists of a single gene, neuronal pentraxin II, normally down-regulated during the window of implantation and further decreased in endometrium from women with endometriosis. The data support dysregulation of select genes leading to an inhospitable environment for implantation, including genes involved in embryonic attachment, embryo toxicity, immune dysfunction, and apoptotic responses, as well as genes likely contributing to the pathogenesis of endometriosis, including aromatase, progesterone receptor, angiogenic factors, and others. Identification and validation of selected genes and their functions will contribute to uncovering previously unknown mechanism(s) underlying implantation failure in women with endometriosis and infertility, mechanisms underlying the pathogenesis of endometriosis and providing potential new targets for diagnostic screening and intervention.

Insulin-like growth factor binding protein-1: recent findings and new directions.

Abstract In 1988, insulin-like growth factor-binding protein-1 (IGFBP-1) became the first characterized member of a group of structurally related soluble proteins which specifically bind and modulate the actions of the IGFs. Since then, a wealth of information has accumulated regarding the physiology of this dynamic serum protein. In this review, we update our 1993 summary (Lee PDK et al. Proc Soc Exp Biol Med 204:4-29) of the status of IGFBP-1 research. The IGFBP-1 protein sequence contains 12 N-terminal and 6 C-terminal cysteine residues which are conserved in other mammalian IGFBP-1 sequences and amongst other IGFBPs; both of the cysteine-rich regions are required for optimal IGF binding. The nonconserved IGFBP-1 midregion may act as both a hinge which defines ligand binding characteristics and as a specific target for protease activity. Integrin-binding and phosphorylation sites within the IGFBP-1 sequence have functional significance in vitro, but their physiologic relevance in vivo have not been defined. The human IGFBP-1 and IGFBP-3 genes are contiguous and located in close proximity to the homeobox A (HOXA) gene cluster on chromosome 7. The other IGFBP genes, located on chromosomes 2, 12, and 17, are also associated with HOX clusters, suggesting evolutionary linkage of the IGFBP and HOX gene families. Similarities between the hlGFBP-1 and phosphoenolpyruvate kinase (PEPCK) promoters, including regions conferring insulin, glucocorticoid, and cyclic adenosine-monophosphate responses, are consistent with our previous hypothesis that IGFBP-1 is involved in regulation of glucose metabolism. The tissue-specific patterns of IGFBP-1 gene expression in liver, kidney, decidua, and ovary may be due to stimulation of IGFBP-1 transcription by hepatic nuclear factor 1 (HNF1) proteins. Clinical and basic studies of IGFBP-1 physiology have been aided by several recently developed assay methods. Numerous investigations have confirmed that insulin, via inhibition of IGFBP-1 transcription, is the primary determinant of IGFBP-1 expression both in vitro and in viva IGF-I and IGF-II also have specific inhibitory effects on IGFBP-1 expression. Glucocorticoids and cAMP stimulate IGFBP-1 transcription, but these effects are observed only in conditions of low or absent insulin effect. Other stimulants of IGFBP-1 expression include thyroid hormones and epidermal growth factor. Phorbol ester stimulation of IGFBP-1 expression can supersede the effects of insulin in vitro; however, the mechanism and in vivo correlates of this effect have not been determined. Cytokines and, perhaps, growth hormones may affect IGFBP-1 expression, perhaps by altering the regulatory actions of insulin; this effect may have important clinical relevance. IGFBP-1 expression is upregulated in liver and (nonhuman) kidney during postinjury regeneration. The IGF-inhibitory actions of IGFBP-1 has been confirmed by numerous in vitro studies and several in vivo animal investigations, including administration of recombinant IGFBP-1 and IGFBP-1 transgenic models. IGFBP-1 has been shown to inhibit somatic linear growth, weight gain, tissue growth, and glucose metabolism. Moreover, IGFBP-1 appears to be a primary determinant of free IGF-I levels in serum. Excess levels of IGFBP-1 may contribute to growth failure in intrauterine growth restriction and in pediatric chronic renal failure, while low IGFBP-1 levels are associated with obesity and with cardiovascular risk factors in insulin resistance syndromes. Serum IGFBP-1 measurements may be useful biochemical marker in these pathologic conditions. IGFBP-1 is expressed in decidualized stromal cells of the uterine endometrium and in ovarian granulosa cells. IGFBP-1, together with IGFs, insulin, ovarian steroids, cytokines, and other factors, is involved in a complex system which regulates menstrual cycles, ovulation, decidualization, blastocyst implantation, and fetal growth. Models for the role of IGFBP-1 in female reproductive physiology are presented, and evidence for pathophysiologic roles in pre-eclampsia, polycystic ovarian syndrome, and uterine malignancy are reviewed. Very recent data indicates that IGFBP-1 undergoes regulated expression in human osteoblasts. Limited information also suggests that IGFBP-1 may be present in peripheral neurons, and that serum IGFBP-1 may increase during exercise and in critical ilIness. In summary, two major roles for IGFBP-1 in normal physiology can be constructed from current data: (i) As an “endocrine” factor, IGFBP-1 regulates the bio-availability of serum IGF-I, thereby modulating IGF-mediated tissue metabolism. The dominant regulation of IGFBP-1 expression by meal-related changes in hepatic insulin concentrations provides a dynamic link to substrate availability. (ii) As an autocrine/paracrine factor, IGFBP-1 appears to play a crucial role in the female reproductive system and, in particular, the sequence of events leading from ovulation to implantation to successful fetal outcome. Future investigations will further delineate the manner in which IGFBP-1 participates in these and other physiologic processes, and the mechanisms by which IGFBP-1 may be involved in clinical pathophysiology.

Pathogenesis and pathophysiology of endometriosis

Originally described over three hundred years ago, endometriosis is classically defined by the presence of endometrial glands and stroma in extrauterine locations. Endometriosis is an inflammatory, estrogen-dependent condition associated with pelvic pain and infertility. This work reviews the disease process from theories regarding origin to the molecular basis for disease sequelae. A thorough understanding of the histopathogenesis and pathophysiology of endometriosis is essential to the development of novel diagnostic and treatment approaches for this debilitating condition.

Patterns of matrix metalloproteinase expression in cycling endometrium imply differential functions and regulation by steroid hormones.

Matrix metalloproteinases are a highly regulated family of enzymes, that together can degrade most components of the extracellular matrix. These proteins are active in normal and pathological processes involving tissue remodeling; however, their sites of synthesis and specific roles are poorly understood. Using in situ hybridization, we determined cellular distributions of matrix metalloproteinases and tissue inhibitor of metalloproteinase-1, an inhibitor of matrix metalloproteinases, in endometrium during the reproductive cycle. The mRNAs for all the metalloproteinases were detected in menstrual endometrium, but with different tissue distributions. The mRNA for matrilysin was localized to epithelium, while the others were detected in stromal cells. Only the transcripts for the 72-kD gelatinase and tissue inhibitor of metalloproteinases-1 were detected throughout the cycle. Transcripts for stromelysin-2 and the 92-kD gelatinase were only detected in late secretory and menstrual endometrium, while those for matrilysin, the 72-kD gelatinase, and stromelysin-3 were also consistently detected in proliferative endometrium. These data indicate that matrix metalloproteinases are expressed in cell-type, tissue, and reproductive cycle-specific patterns, consistent with regulation by steroid hormones, and with specific roles in the complex tissue growth and remodeling processes occurring in the endometrium during the reproductive cycle.

Growth factors and growth modulators in human uterine endometrium: their potential relevance to reproductive medicine * †

OBJECTIVE To provide an up-to-date, comprehensive review on the presence and regulation of growth factors (GFs), GF receptors, and GF regulatory proteins in human endometrium in an effort to understand the potential roles of these proteins in endometrial cell mitosis and differentiation and in endometrial-trophoblast interactions. DESIGN Relevant studies were identified through a computerized bibliographic search (MEDLINE; BRS Information Technologies, a division of Maxwell Online, Inc., McLean, VA) and through manual scanning of recent relevant journals. RESULTS Several GFs, their receptors, and regulatory proteins have been identified in endometrium, and cellular localization and steroid-dependence of these proteins as well as action of several growth modulators on endometrial cell function have been studied. Epidermal growth factor, transforming growth factor (TGF)-alpha, platelet-derived growth factor, insulin-like growth factors (IGFs) and their binding proteins, fibroblast growth factor (FGF), TGF-beta, colony-stimulating factor (CSF)-1, and interferon-gamma regulate mitosis of endometrial cellular components in vitro. Endothelin-1 may participate in vasoconstriction and FGF may participate in angiogenesis in this tissue in vivo. Interleukins-1 and -6 are believed to be involved in endometrial T-cell activation, and TGF-beta, CSF-1, the interleukins, and the IGFs likely mediate endometrial-trophoblast interactions. The role of tumor necrosis factor in endometrium remains uncertain. CONCLUSIONS Current evidence supports the thesis that GFs play a central role in cyclic mitosis and differentiation of endometrial cellular components, recruitment of macrophages in decidualizing endometrium, endometrial-trophoblast interactions, early pregnancy maintenance, tissue shedding in the absence of implantation, and endometrial functionalis regeneration.

Female reproductive disorders: the roles of endocrine-disrupting compounds and developmental timing

OBJECTIVE To evaluate the possible role of endocrine-disrupting compounds (EDCs) on female reproductive disorders emphasizing developmental plasticity and the complexity of endocrine-dependent ontogeny of reproductive organs. Declining conception rates and the high incidence of female reproductive disruptions warrant evaluation of the impact of EDCs on female reproductive health. DESIGN Publications related to the contribution of EDCs to disorders of the ovary (aneuploidy, polycystic ovary syndrome, and altered cyclicity), uterus (endometriosis, uterine fibroids, fetal growth restriction, and pregnancy loss), breast (breast cancer, reduced duration of lactation), and pubertal timing were identified, reviewed, and summarized at a workshop. CONCLUSION(S) The data reviewed illustrate that EDCs contribute to numerous human female reproductive disorders and emphasize the sensitivity of early life-stage exposures. Many research gaps are identified that limit full understanding of the contribution of EDCs to female reproductive problems. Moreover, there is an urgent need to reduce the incidence of these reproductive disorders, which can be addressed by correlative studies on early life exposure and adult reproductive dysfunction together with tools to assess the specific exposures and methods to block their effects. This review of the EDC literature as it relates to female health provides an important platform on which womens health can be improved.

Discovery of New Inducible Genes in in vitroDecidualized Human Endometrial Stromal Cells Using Microarray Technology

A prerequisite for implantation in humans is differentiation (decidualization) of stromal cells in the endometrium, believed to be stimulated by progesterone (P) and/or cAMP. In the current study, advances in microarray technology have allowed us to investigate genes differentially expressed in human endometrial stromal cells decidualized in vitro in response to P or cAMP, compared to nondecidualized cells. Endometrial stromal cells were isolated from endometrial biopsy tissue and cultured without steroid hormones, with 1 microM P (after E2 priming), or 1 mM 8-bromo-cAMP. Total RNA was isolated and reverse transcribed to synthesize 32P-labeled cDNA probes using primers corresponding to genes represented on the Clontech Human Atlas cDNA Expression Array. After hybridization, signals were quantified by phosphor imaging densitometry and were normalized to GAPDH and ubiquitin. Of the 588 genes screened, marked upregulation was observed of cytokines, growth factors, nuclear transcription factors, members of the cyclin family, and mediators of the cAMP signal transduction pathway. Additional mRNAs expressed unexpectedly and regulated by P and cAMP, include the insulin receptor, some neurotransmitter receptors, neuromodulators, the FSH receptor, inhibin/activin betaA subunit, inhibin alpha, and TNF-related apoptosis-inducing ligand (TRAIL). Expression of previously unrecognized genes regulated in decidualized human endometrial stromal cells suggests mechanisms not yet appreciated in the endometrium during decidualization. In addition, marked upregulation of cytokines, chemokines, growth factors, apoptosis modulators, and their receptors in decidualized stromal cells supports a major role for paracrine interactions between the stroma and other endogenous and transient cell populations within the endometrium and during early pregnancy.

Microbes on the human vaginal epithelium

Using solely a gene-based procedure, PCR amplification of the 16S ribosomal RNA gene coupled with very deep sequencing of the amplified products, the microbes on 20 human vaginal epithelia of healthy women have been identified and quantitated. The Lactobacillus content on these 20 healthy vaginal epithelia was highly variable, ranging from 0% to 100%. For four subjects, Lactobacillus was (virtually) the only bacterium detected. However, that Lactobacillus was far from clonal and was a mixture of species and strains. Eight subjects presented complex mixtures of Lactobacillus and other microbes. The remaining eight subjects had no Lactobacillus. Instead, Bifidobacterium, Gardnerella, Prevotella, Pseudomonas, or Streptococcus predominated.