REPRODUCTIVE GENETICS AND EPIGENETICS RELEASE DATE: January 8, 2004 PA NUMBER: PA-04-049 March 2, 2006 (NOT-OD-06-046) Effective with the June 1, 2006 submission date, all R03, R21, R33 and R34 applications must be submitted through Grants.gov using the electronic SF424 (R&R) application. This announcement will stay active for only the May 1, 2006 AIDS and AIDS-related application submission date for these mechanisms. The non-AIDS portion of this funding opportunity for these mechanisms expires on the date indicated below. Other mechanisms relating to this announcement will continue to be accepted using paper PHS 398 applications until the stated expiration date below, or transition to electronic application submission. Replacement R03 (PA-06-347) and R21 (PA-06-346) funding opportunity announcements have been issued for the submission date of June 1, 2006 and submission dates for AIDS and non-AIDS applications thereafter. EXPIRATION DATE for R03 and R21 Non-AIDS Applications: March 2, 2006 EXPIRATION DATE for R03 and R21 AIDS and AIDS-Related Applications: May 2, 2006 EXPIRATION DATE for All R01 Applications: December 1, 2006 Department of Health and Human Services (DHHS) PARTICIPATING ORGANIZATION: National Institutes of Health (NIH) (http://www.nih.gov) COMPONENT OF PARTICIPATING ORGANIZATION: National Institute of Child Health and Human Development (NICHD) (http://www.nichd.nih.gov/) CATALOG OF FEDERAL DOMESTIC ASSISTANCE NUMBER(S): 93.865 THIS PA CONTAINS THE FOLLOWING INFORMATION o Purpose of this PA o Research Objectives o Mechanisms of Support o Eligible Institutions o Individuals Eligible to Become Principal Investigators o Where to Send Inquiries o Submitting an Application o Peer Review Process o Review Criteria o Award Criteria o Required Federal Citations PURPOSE OF THIS PA This Announcement replaces the NICHD PA-01-005 on Reproductive Genetics (http://grants.nih.gov/grants/guide/pa-files/PA-01-005.html) initially published in October 2000. The purpose of reissuing the Reproductive Genetics PA is to indicate our continued desire to support new studies on the genes, and genetic and epigenetic mechanisms influencing sex determination, fertility, reproductive health and reproductive aging, and other topics in Reproductive Genetics and Epigenetics. Studies submitted under this program announcement are expected to identify and characterize the relevant genes, determine their function in normal human reproduction and reproductive development, identify functional partners or pathways and the nature of the interactions, and further our understanding of the consequences of mutations or dysregulation for human reproductive health. Studies of animal models are integral to this effort and are encouraged along with studies involving human subjects. RESEARCH OBJECTIVES Background With the completion of the human genome project, the focus of genetic research must shift to functional genomics. NICHD encourages scientists interested in reproduction to lead the way in determining the genes and their mechanisms of action involved in the development of the gonads, reproductive ducts and genitalia, the processes of gametogenesis, normal and premature reproductive aging, and reproductive disorders such as infertility, cryptorchidism, endometriosis, and polycystic ovarian syndrome (PCOS). Studies on the genetic epidemiology of reproductive disorders might begin with the collection of large numbers of affected patients and their relatives for linkage analysis, association studies or quantitative trait loci (QTL) analysis. Studies using innovative statistical or technical methods are highly encouraged. We also encourage research into epigenetic mechanisms critical to reproduction, especially areas such as the establishment and maintenance of methylation patterns or imprinted loci in the early embryo, the timing, mechanisms and role of genomic methylation in gametogenesis, the effects of assisted reproductive therapy (ART) on imprinting and genomic methylation, and the reproductive determinants and consequences of X-chromosome inactivation. Reproductive genetics is a broad research area, and the topics discussed and listed below are not meant to be exclusive areas of interest, but rather a sampling of the types of problems that this program announcement intends to address. Research Scope (1) The Genetics of Sex Determination Sex determination is the translation of the chromosomal sex (XX or XY) into the gender-appropriate internal and external reproductive structures. The initial events of sex determination are, therefore, genetically determined. Errors in the process can range in severity from complete sex reversal to gonadal dysgenesis or minor genital abnormalities. Sex determination, as an early embryological event, can help us address basic questions of the regulation of gene expression, cell-fate determination, and hormone signaling. Approximately one in 1,000 newborns has some abnormality of genital and/or gonadal development. In many cases, gonadal dysgenesis is part of a larger pathologic syndrome, such as Frasier syndrome, Deny-Drash syndrome, or campomelic dysplasia, to name a few. The known genes involved in sex determination often act as growth and/or differentiation factors and there is mounting evidence that they may be important in tumorigenesis in the gonads as well as other tissues. Despite the identification of the Y-chromosome gene SRY as the "testis determining factor" almost 15 years ago, the mechanisms and pathways of normal sex differentiation are still not well understood. In particular, although some downstream effects of SRY are known, such as cellular proliferation, Sertoli cell differentiation, and testis-specific vascularization, the direct transcriptional targets of SRY remain unknown. The factors regulating SRY expression remain unknown as well. While genes such as SOX-9, WT-1, DAX-1, DMRT-1, GATA4, FOG2, and SF-1, among others, contribute to sex determination, the nature and timing of their interactions remain unclear, and there are clearly other unknown genes to be identified. A further level of complexity arises with gene dosage effects, such as XY sex reversal caused by duplication of Dax-1. Sex determination can be divided into steps consisting of establishment of the bipotential gonad, formation of the primordial gonad, and differentiation of the gonad. Many of the sex determining genes act in multiple steps, but SRY mainly functions in shaping the primordial gonad into a testis. However, the classic view of SRY as a "switch" that confers maleness is an over- simplification as illustrated by the enormous potential for ambiguity in sex determination, and by evidence suggesting that steps in testis development that were once thought to be tightly coordinated, such as mesonephric cell migration and Leydig cell differentiation, or the formation of testis cords and the inhibition of male germ cell meiosis, can occur independently of each other. Additionally, ovarian development may not be the passive "default" process it was once thought to be. Estrogen may be necessary to maintain the ovarian phenotype, as mice unable to make estrogen (ArKO mice) or bind estrogen develop patches of Sertoli and Leydig cells within their ovaries postnatally. Germ cells play a critical role in the formation of ovaries, although testes can form in their absence. The germ cells migrate into the gonad through the gut, through a process which has yet to be fully characterized. The presence of meiotic germ cells is critical for the formation and maintenance of ovarian follicles while, in contrast, in males the testis cords surround the germ cells and meiosis is inhibited. Germ cell migration and the progression into meiosis are not well understood. There is clear evidence that the genes involved in sex determination have important roles beyond gonadal fate. Some, such as WT-1, are expressed in common embryonic precursors to different organ systems. Mutations in FOXL2, a gene deleted in polled intersex goats, cause the human syndrome BPES that often includes premature ovarian failure. The anti-mullerian hormone, known as Amh or MIS, causes regression of the female duct system in normal males, and in adult males, MIS has inhibitory effects on both Leydig cells and testosterone production. Such examples clearly demonstrate that the continued study of sex determination will not only benefit those born with gonadal dysgenesis or ambiguous genitalia, but will also advance our knowledge of the physiology of the adult reproductive system, and the development and regulation of other organ systems. Specific topics of interest include, but are not limited to: o Identification of the target genes and processes regulated by SRY; o Clarification of the functional interactions between sex determining genes; o Cloning of genes at loci associated with sex reversal, in humans and other species, and elucidation of their function; these studies may entail the collection of affected families or animal models and careful phenotypic description; o Determination of how germ cell migration and meiosis affect sex determination and gonadal development; o Study of the genes and processes regulating the retention or loss of the Wolffian and Mullerian ducts; o Comparing and contrasting mammalian and non-mammalian sex determination systems to better understand the common pathways and genes; o Creation of new cell or tissue culture systems, or animal models (especially transgenic or knock-out mice), to precisely characterize the functions of sex- determining genes. (2) Genes Regulating Fertility, Reproductive Health, and Reproductive Aging Infertility is a major public health problem in our country, affecting 10-15 percent of couples, or about 2.5 million couples in the United States. The annual cost of services to diagnose and combat infertility is now estimated at over one billion dollars. In recent years, great advances have been made in medical and surgical treatments for infertility caused by hormonal or structural defects. However, 30 percent of couples are infertile due to idiopathic or genetic causes, and they may suffer through failed conventional treatments before resorting to assisted reproductive technologies (ART) to conceive their biological children. Given the known and potential problems associated with the use of ART, it is essential that we focus our efforts on identifying and treating the underlying causes of infertility. Studies of human infertility and studies using animal models have revealed many single gene mutations that cause infertility and new phenotypes continually appear in the literature. Each new gene teaches us more about the intricate pathways that contribute to normal fertility and may suggest leads for contraceptives. Epidemiological and family studies of human infertility are now feasible with the advent of genetic databases and new statistical techniques. The most common identifiable cause of human male infertility is Klinefelter's syndrome, occurring in one in 400 live births. The Klinefelter's XXY genotype disrupts testis development and, in combination with high levels of meiotic non-disjunction, low sperm counts and infertility ensue. The Klinefelter's phenotype, along with data showing exclusive expression of several X- chromosome genes in the testes, suggests that the X-chromosome figures prominently in testis physiology. Clearly, loci on the Y-chromosome are also critical to male fertility. Deletions within the male specific region of the Y-chromosome, previously referred to as the non-recombining region, are also a common genetic cause of spermatogenic failure in men. Mutation of specific genes within the AZF (azoospermia factor) regions of the Y-chromosome, most notably DAZ, severely disrupts spermatogenesis. The recent mapping of the male specific region of the Y-chromosome suggests that gene conversion (non- reciprocal recombination), while conserving important testis gene function on the Y-chromosome through evolution, may also predispose to deletions that abolish spermatogenesis. Less dramatic mutations can also render males infertile. Disruption of the action of hypothalamic hormones can delay or prevent puberty, leading to oligospermia or azoospermia. Mutations causing both the X-linked and autosomal dominant forms of Kallmann's syndrome (hypogonadotropic hypogonadism and anosmia), which is more common in males, were recently identified (KAL-1 and FGFR1, respectively). Similarly, mutation of the beta-subunit of the gonadotropin FSH also causes infertility by compromising spermatogenesis. Even when spermatogenesis proceeds smoothly, infertility can result if the chromatin is incorrectly packaged into the sperm head. Mutations that abolish the function of the transition proteins or the protamines that compact sperm chromatin cause infertility. The sperm mitochondrial genome also contributes to fertility. For example, absence of the common form of the POLG allele, encoding a mitochondrial DNA polymerase, is associated with infertility in men. Genetic conditions in which the testes themselves are normal, but the male tract is affected, can render men infertile. Mutations in CFTR (the gene causing cystic fibrosis) can cause congenital bilateral absence of the vas deferens, seen in one percent of infertile men. Cryptorchidism is the most common defect of newborn boys, affecting two three percent. Strong evidence demonstrates a genetic component to cryptorchidism. Mutation of the genes encoding either INSL3 (insulin-like hormone) or its receptor GREAT/LGR8, compromises the transabdominal phase of testicular descent, causing cryptorchidism which, if uncorrected, will result in infertility. However, the known mutations explain only a minority of cases of cryptorchidism, suggesting the involvement of other genes and pathways. The identification of genetic causes of female infertility lags behind, possibly because the female reproductive system is more complex than the male system. Finely tuned cyclic fluctuations in hormones coordinate the follicular development, ovulation, and uterine receptivity for implantation, the components that comprise a normal menstrual cycle. This complexity suggests that there are hundreds of genes, each contributing a small effect on female fertility. Genes involved in regulating the hypothalamic-pituitary-ovarian axis are obvious candidates for female infertility and, while mutations have been reported in the genes encoding FSH-beta and the LH receptor, and the genes associated with Kallmann's syndrome have been identified, these mutations explain only a tiny proportion of cases of female infertility. However, work in highly prolific sheep has identified genes controlling ovulation rate and fertility, as well as ovarian development, which may lead to better understanding of infertility in women. In some breeds of ewes, naturally occurring mutations of genes encoding key players in the transforming growth factor beta signaling pathway increase ovulation rate and twinning. Conversely, homozygous mutation of the gene encoding the TGF signaling molecule BMP15 (GDF9B) causes sterility in the same breed of sheep. Such studies suggest new candidate molecules and pathways to study in human fertility. The disruption of early embryonic development may be an under-estimated cause of infertility. Mammalian oocytes store products necessary for the very early stages of development, until the embryonic genome is activated. Deletion of maternal oocyte products such as MATER, DNMT1o, and Npm2 arrests embryo development and leads to female infertility or sub-fertility in knockout mice. It is not known if mutations in these genes, or insufficient levels of their products, are a cause of human infertility. Reproductive diseases such as endometriosis and polycystic ovarian syndrome are common and can be quite debilitating. Recent research indicates genetic components to these disorders; identification of causative or modifying genes would be of enormous benefit. Both diseases are likely to involve complex interactions between gene products and environment rather than single major genes. Polymorphisms in the insulin gene, the gene CYP11a, and the androgen receptor gene have been associated with hyperinsulinemia and hyperandrogenism in PCOS. Similarly, alterations in the estrogen receptor gene, genes encoding products involved in detoxification, homeobox genes, and the LH-beta gene, have been associated with a small number of cases of endometriosis. Comparative genomic hybridization and gene chip studies of endometriosis have revealed candidate regions and patterns of altered gene expression, but no major genes as yet. Because of the sharp decline in female fertility with age and the increasing number of women who opt to have children later in life, the incidence of infertility is growing. Data from animal models and some human syndromes indicate that the timing of reproductive aging, in a continuum from premature ovarian failure to early menopause and normal menopause, may have genetic components. The genes and mechanisms contributing to reproductive aging have not been well characterized. Given the social trend to delay starting a family and the concerns about the prolonged use of hormone replacement therapy for menopause, understanding the mechanisms of reproductive aging is a high priority. Premature ovarian failure (POF), defined as the cessation of menstruation before the age of 40, affects approximately one percent of women. Most cases of POF are assumed to be genetic and insight into this condition may help us better understand the variation in normal ovarian aging as well. Mutations in the gene encoding the FSH receptor are a rare cause of POF. Women carrying the fragile X premutation have a greater risk for premature ovarian failure, although the mechanism is not known. Mutation in a forkhead transcription factor, FOXL2 (3q23), causes autosomal dominant POF due to follicle depletion in some women affected with the syndrome BPES. FOXL2 mutation results in ovarian phenotypes ranging from streak ovaries to otherwise normal ovaries that lack adequate follicles. Mice lacking Foxo3a, a distant relative of FoxL2, show early depletion of ovarian follicles and sterility shortly after sexual maturity. Other causative genes for POF in women, and perhaps protective genes or alleles, remain to be identified. The accumulation of meiotic errors in aging oocytes contributes strongly to the age-related decrease in women's fertility and the increased risk for chromosomal abnormalities in children born to older mothers. This may be due to the unusual robustness of oocytes to proceed through meiosis despite flaws in the process; there are multiple examples of greater tolerance of meiotic defects in oogenesis as compared to spermatogenesis. For example, male germ cells are unable to progress through meiosis when the synaptonemal complex, which helps to hold homologous chromosomes together during meiosis, is compromised. While male mice bred to lack synaptonemal complex protein 3 are infertile, female SCP-3 knockout mice, though subfertile, are able to reproduce. Because the phenotype of subfertility due to embryo wastage becomes more severe with age, these mice may be a good model system not only for delineating the differences in meiosis in male and female gametes, but also for delineating the interactions between infertility and aging. The phenomenon of reproductive aging in men, or decreased fertility with male age, is under debate and definitive studies are needed. Studies in old male rats demonstrate decreased fertility and an increased risk of siring abnormal offspring. Mutation rates appear to increase with age in male gametes and some genetic diseases, including both recessive X-linked and autosomal dominant conditions, demonstrate a paternal age effect, suggesting that the process of spermatogenesis does change with age in men. This is a phenomenon that needs further characterization and mechanistic study. Specific topics of interest include, but are not limited to: o Identifying specific Y-chromosome genes responsible for oligospermia or azoospermia, and establishing their functions in spermatogenesis; o Identification of major genes, gene interactions or QTLS involved in regulating female fertility or ovarian or uterine function; o Investigations of the heritability of infertility in offspring conceived through ART; o Studies of the genetic mechanisms that establish the pool of primordial follicles and subsequent follicle development or loss; o Identification of the gene mutations underlying inherited disorders of the reproductive organs or tract, such as PCOS, endometriosis, premature ovarian failure, and cryptorchidism, using candidate gene approaches as well as genetic epidemiology and linkage and/or association studies; o Studies to elucidate the processes and mechanisms of the condensation and decondensation of the paternal and maternal genomes during gametogenesis and embryogenesis; o Studies of the mechanisms responsible for the accumulation of meiotic errors in aging oocytes and identification of factors that impede or advance the process; o Studies of similarities and differences in male and female meiosis, and how those contribute to the differential tolerance for meiotic errors; implications for fertility and contraception. (3) Genomic Imprinting and X-Chromosome Inactivation The wealth of gene sequence data generated by the Human Genome Project will significantly improve our ability to detect and treat genetic diseases. However, diseases caused by epigenetic defects, such as improper gene methylation or improper X-chromosome inactivation, clearly demonstrate that in addition to a normal gene sequence, the timing, specificity, degree of gene expression, and even the parental origin of an allele, are critical to normal human development and continued health. The epigenetic processes of imprinting and X-inactivation are intimately tied to reproduction, as the patterns are established during gametogenesis and embryogenesis, and they may in turn affect embryogenesis, gonadal/genital development, and fertility. Imprinting is the phenomenon whereby one of the two autosomal alleles is preferentially expressed, dependent on its parental origin. Current estimates suggest that greater than one percent of all human genes are imprinted. Imprints are thought to be encoded by gene methylation patterns that differ between the maternally- and paternally-derived alleles. Parental imprints from the previous generation are erased in the germ cells at an early stage of development and new sex-specific imprints are established. This appears to occur before the onset of meiosis in male germ cells, but maternal imprints are established later, in growing oocytes arrested at the diplotene stage. Interestingly, the imprints are not all imposed together, as different genes are marked at various stages of oocyte growth. Although a genome-wide wave of demethylation occurs before implantation and de novo methylation re- establishes the pattern shortly after implantation, the core regions of the imprinted genes are somehow protected from these changes. Imprinting centers may play a role in the establishment and maintenance of the appropriate parental imprint, although the mechanism of such events remains unclear. Many imprinted loci encode anti-sense transcripts that have been implicated in the initiation of genomic imprinting, as well as X-chromosome inactivation. Many key molecules regulating genomic methylation and transcriptional silencing have been identified. Methylation generally silences allele expression, as methyl-CpG-binding proteins such as MeCP2, bind to methylated DNA and recruit histone deacetylases. Hypoacetylated DNA is presumably inactive because it is conformationally inaccessible to the transcription machinery. The establishment and maintenance of DNA methylation are regulated by the DNA methyltransferases (Dnmt). Dnmt3A and Dnm3B function in de novo methylation, while Dnmt1 maintains methylation after each round of replication. Deficiency of Dnmt1 is lethal to embryos due to genome-wide demethylation. In contrast, the oocyte-specific form, Dnmt1o, seems to act only on certain genes and only at the eight-cell stage. Dnmt3L is required for the establishment of imprints during oogenesis, but is not necessary for the maintenance of paternal imprints during embryogenesis. BORIS, a paralog of CTCF, may participate in the erasure of parental methylation marks in the male germ line. More studies are needed to determine how the methylation and demethylation machinery correctly recognizes imprinted regions, discriminates between the maternal and paternal marks, and establishes or maintains the appropriate methylation patterns during gametogenesis and early embryogenesis. Methylation of histones, in addition to DNA methylation, may regulate gene expression and the "read-out" of these types of methylation signals remains unclear. In mice lacking the polycomb group gene Eed, a subset of paternally repressed genes is improperly activated and expressed. Such data suggest that other trans-acting factors form an additional layer of regulation of the expression of imprinted genes. Several human syndromes, such as Rett syndrome, ICF, Beckwith-Weidemann syndrome, Prader-Willi syndrome, and Angelman syndrome, are caused by defects in imprinting or in DNA methylation. Dysregulation of imprinted genes often manifests as abnormal growth of the fetus or placenta. One recently discovered example is the unknown locus on chromosome 19q13.4 that causes recurrent biparental complete hydatidiform molar pregnancies, as maternal alleles acquire paternal methylation patterns. Studies suggest that a failure of epigenetic reprogramming, as evaluated by methylation patterns, may underlie the extraordinarily high failure rate of cloning by nuclear transfer. The findings that cloned mouse embryos aberrantly express Dnmt1, while Dnmt1o fails to translocate to the nucleus, provide further support for this hypothesis. Culture conditions can also significantly and selectively alter the expression of imprinted genes, a finding that may be critical to human in vitro fertilization protocols. There is a trend among ART clinics to culture embryos for longer periods to enable selection of "higher quality" embryos; it is not clear if loss of imprinting occurs in such conditions and, if so, what effect it might have on the offspring. It seems likely that other more subtle phenotypes will be linked to defects in imprinting or DNA methylation/demethylation as well; exploration of these processes specifically in reproductive tissues is encouraged. The inactivation of one X-chromosome in females is another type of gene silencing that acts as dosage compensation for the XX vs. XY genotype. Some critical X-linked genes "escape" inactivation and are expressed from both copies of the X-chromosome. Turner syndrome, resulting from a 45, X karyotype, clearly demonstrates the importance of genes on the second X-chromosome for fetal survival, as well as ovarian development. There are two basic processes in X-inactivation: choice of which X-chromosome to inactivate, and implementation of the silencing. While recent studies show that X-inactivation has some mechanistic similarities to autosomal imprinting, X-chromosome inactivation in the embryo is usually random so that in each cell, the maternally- and paternally-derived X-chromosome have an equal probability of inactivation. The molecule Xist, an X-encoded untranslated RNA, is the master regulator of X-chromosome inactivation. Xist is expressed only from the X-chromosome destined to become inactive (X-I). The Xist transcripts coat X-I in cis and soon after, histone 3 is methylated on lysine 9 on the inactive X. The X-chromosome that is destined to remain active (X-A) is protected from Xist by Tsix, the Xist antisense transcript. On X-A, histone 3 is methylated on lysine 4; this differential methylation suggests that a histone code may regulate the transcriptional status of the X-chromosome. The DNA of the inactive X-chromosome is hypermethylated and this is functionally significant as Dnmt1 mutant embryos fail to maintain random X-chromosome inactivation. Other events that mediate the silencing of the Xist-coated X- chromosome remain unknown. Recent data also suggest that there is active selection of both X-I and X-A, rather than one chromosome's state being conferred by default. Although the choice of which X-chromosome to inactivate is random in the embryo, it is imprinted in the extra-embryonic cells of mammals: the paternal X (Xp) chromosome is preferentially inactivated. The mechanisms for imprinted silencing of Xp in the extra-embryonic tissue and random X-chromosome inactivation in the embryo seem to be quite different. For example, Dnmt1 mutant embryos fail to maintain random X-chromosome inactivation in the embryo, but Xp is correctly inactivated in the extra-embryonic cells. Also, homozygous mutant eed mice initiate but fail to maintain imprinted Xp inactivation in the trophectoderm, but maintain normal random X-chromosome inactivation in the embryo itself, suggesting that eed functions only in maintenance of imprinted, but not random, X-chromosome inactivation. Normal X-chromosome inactivation is essential to reproduction. Appropriate imprinted X-inactivation is critical to formation of the trophoblast and, ultimately, the placenta. Both heterozygous and homozygous Tsix knockout females are subfertile, with homozygous females showing a more drastic loss of fertility. Similar to imprinting defects in cloned embryos, cloned or in vitro embryos show disruption of dosage compensation of X-linked genes that may affect embryonic development. The presence of skewed X-chromosome inactivation (XCI), usually defined as greater than 90 percent inactivation of a particular one of the pair of X- chromosomes, is increased in women with recurrent spontaneous abortion. In addition, women with skewed XCI and recurrent spontaneous abortion are more likely to have trisomic losses than women without XCI, but experiencing recurrent spontaneous abortion. Finally, deviations from random choice in X- chromosome inactivation can affect the relative expression of X-linked genes, many of which act in reproduction. Transcriptional silencing of the X-chromosome (as well the Y-chromosome) occurs in males as well, just before meiotic prophase in spermatogenesis. The mechanism of male X-chromosome inactivation is likely completely different from that in the female because Xist mutation does not prevent the silencing in males. This remains a very poorly understood area. Specific topics of interest include, but are not limited to: o Identifying genes and mechanisms important in erasing and re-establishing genomic imprinting and genome-wide methylation during gametogenesis and early embryonic development; o Characterizing the effects of manipulations of gametes or fertilized eggs, especially procedures commonly used in assisted reproductive technology, on gene methylation patterns, imprinting or X-inactivation; o Investigation of defects in imprinting or methylation patterns in abnormal reproductive phenotypes including effects on gametogenesis, fertility or gonadal differentiation and development; o Description of the effects of mutations of the imprinting machinery in gametes and reproductive tissues, and on early embryonic development; o Elucidation of the mechanism of the reversal of X-inactivation in XX primordial germ cells; o Identification of the nature of the imprinting mark of the paternal X- chromosome and the mechanisms of imprinted X-inactivation in extra-embryonic cells; o Studies of the biological significance and the mechanisms leading to X- chromosome inactivation in male meiotic germ cells; o Studies of possible associations between skewed X-inactivation and various reproductive tract development and function, whether having protective or deleterious effects. MECHANISM OF SUPPORT This PA will use the NIH Research Project Grant (R01), Small Grant (R03) and Exploratory/Developmental Grant (R21) award mechanisms. The NIH Small Grant (R03) Program guidelines are available at http://grants.nih.gov/grants/guide/pa-files/PA-03-108.html. The guidelines for the NIH Exploratory/Developmental Research Grant (R21) may be found at http://grants.nih.gov/grants/guide/pa-files/PA-03-107.html. As an applicant you will be solely responsible for planning, directing, and executing the proposed project. This PA uses just-in-time concepts. It also uses the modular as well as the non-modular budgeting formats (see http://grants.nih.gov/grants/funding/modular/modular.htm). Specifically, if you are submitting an application with direct costs in each year of $250,000 or less, use the modular format. Otherwise follow the instructions for non- modular research grant applications. This program does not require cost sharing as defined in the current NIH Grants Policy Statement at http://grants.nih.gov/grants/policy/nihgps_2001/part_I_1.htm. ELIGIBLE INSTITUTIONS You may submit an application if your institution has any of the following characteristics: o For-profit or non-profit organizations o Public or private institutions, such as universities, colleges, hospitals, and laboratories o Units of State and local governments o Eligible agencies of the Federal government o Domestic or foreign institutions/organizations o Faith-based or community-based organizations INDIVIDUALS ELIGIBLE TO BECOME PRINCIPAL INVESTIGATORS Any individual with the skills, knowledge, and resources necessary to carry out the proposed research is invited to work with his/her institution to develop an application for support. Individuals from underrepresented racial and ethnic groups as well as individuals with disabilities are always encouraged to apply for NIH programs. WHERE TO SEND INQUIRIES We encourage your inquiries concerning this PA and welcome the opportunity answer questions from potential applicants. Inquiries may fall into two areas: scientific/research and financial or grants management issues: o Direct your questions about scientific/research issues to: Susan Taymans, Ph.D. Reproductive Sciences Branch National Institute of Child Health and Human Development 6100 Executive Boulevard, Room 8B01, MSC 7510 Bethesda, MD 20892-7510 Telephone: (301) 496-6517 FAX: (301) 496-0962 Email: taymanss@mail.nih.gov o Direct your questions about financial or grants management matters to: Kathy Hancock Grants Management Branch National Institute of Child Health and Human Development 6100 Executive Boulevard, Room 8A17, MSC 7510 Bethesda, MD 20892-7510 Telephone: (301) 435-5482 FAX: (301) 402-0915 Email: kh246t@nih.gov SUBMITTING AN APPLICATION Applications must be prepared using the PHS 398 research grant application instructions and forms (rev. 5/2001). Applications must have a Dun and Bradstreet (D&B) Data Universal Numbering System (DUNS) number as the Universal Identifier when applying for Federal grants or cooperative agreements. The DUNS number can be obtained by calling (866) 705-5711 or through the web site at http://www.dunandbradstreet.com/. The DUNS number should be entered on line 11 of the face page of the PHS 398 form. The PHS 398 is available at http://grants.nih.gov/grants/funding/phs398/phs398.html in an interactive format. For further assistance contact GrantsInfo, Telephone (301) 710-0267, Email: GrantsInfo@nih.gov. The title and number of this program announcement must be typed on line 2 of the face page of the application form and the YES box must be checked. SUPPLEMENTARY INSTRUCTIONS: Applications for the R03 must be prepared following the guidelines presented in NIH PA-03-108 (http://grants.nih.gov/grants/guide/pa-files/PA-03-108.html). Applications for the R21 must be prepared following the guidelines presented in NIH PA-03-107 (http://grants.nih.gov/grants/guide/pa-files/PA-03-107.html). APPLICATION RECEIPT DATES: Applications submitted in response to this program announcement will be accepted at the standard application deadlines, which are available at http://grants.nih.gov/grants/dates.htm. Application deadlines are also indicated in the PHS 398 application kit. SPECIFIC INSTRUCTIONS FOR MODULAR GRANT APPLICATIONS: Applications requesting up to $250,000 per year in direct costs must be submitted in a modular grant format. The modular grant format simplifies the preparation of the budget in these applications by limiting the level of budgetary detail. Applicants request direct costs in $25,000 modules. Section C of the research grant application instructions for the PHS 398 (rev. 5/2001) at http://grants.nih.gov/grants/funding/phs398/phs398.html includes step-by-step guidance for preparing modular grants. Additional information on modular grants is available at http://grants.nih.gov/grants/funding/modular/modular.htm. SPECIFIC INSTRUCTIONS FOR APPLICATIONS REQUESTING $500,000 OR MORE PER YEAR: Applications requesting $500,000 or more in direct costs for any year must include a cover letter identifying the NIH staff member within one of NIH institutes or centers who has agreed to accept assignment of the application. Applicants requesting more than $500,000 must carry out the following steps: 1) Contact the IC program staff at least six weeks before submitting the application, i.e., as you are developing plans for the study; 2) Obtain agreement from the IC staff that the IC will accept your application for consideration for award; and, 3) Identify, in a cover letter sent with the application, the staff member and IC who agreed to accept assignment of the application. This policy applies to all investigator-initiated new (type 1), competing continuation (type 2), competing supplement, or any amended or revised version of these grant application types. Additional information on this policy is available in the NIH Guide for Grants and Contracts, October 19, 2001 at http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-004.html. SENDING AN APPLICATION TO THE NIH: Submit a signed, typewritten original of the application, including the checklist, and five signed photocopies in one package to: Center for Scientific Review National Institutes of Health 6701 Rockledge Drive, Room 1040, MSC 7710 Bethesda, MD 20892-7710 Bethesda, MD 20817 (for express/courier service) APPLICATION PROCESSING: Applications must be mailed on or before the receipt dates described at http://grants.nih.gov/grants/funding/submissionschedule.htm. The CSR will not accept any application in response to this PA that is essentially the same as one currently pending initial review unless the applicant withdraws the pending application. The CSR will not accept any application that is essentially the same as one already reviewed. This does not preclude the submission of a substantial revision of an application already reviewed, but such application must include an Introduction addressing the previous critique. Although there is no immediate acknowledgement of the receipt of an application, applicants are generally notified of the review and funding assignment within eight weeks. PEER REVIEW PROCESS Applications submitted for this PA will be assigned on the basis of established PHS referral guidelines. An appropriate scientific review group convened in accordance with the standard NIH peer review procedures (http://www.csr.nih.gov/refrev.htm) will evaluate applications for scientific and technical merit. As part of the initial merit review, all applications will: o Receive a written critique o Undergo a selection process in which only those applications deemed to have the highest scientific merit, generally the top half of applications under review, will be discussed and assigned a priority score o Receive a second level review by the appropriate national advisory council or board. REVIEW CRITERIA The goals of NIH-supported research are to advance our understanding of biological systems, improve the control of disease, and enhance health. In the written comments, reviewers will be asked to discuss the following aspects of the application in order to judge the likelihood that the proposed research will have a substantial impact on the pursuit of these goals: o Significance o Approach o Innovation o Investigator o Environment The scientific review group will address and consider each of these criteria in assigning the application's overall score, weighting them as appropriate for each application. The application does not need to be strong in all categories to be judged likely to have major scientific impact and thus deserve a high priority score. For example, an investigator may propose to carry out important work that by its nature is not innovative but is essential to move a field forward. SIGNIFICANCE: Does this study address an important problem? If the aims of the application are achieved, how will scientific knowledge be advanced? What will be the effect of these studies on the concepts or methods that drive this field? APPROACH: Are the conceptual framework, design, methods, and analyses adequately developed, well-integrated, and appropriate to the aims of the project? Does the applicant acknowledge potential problem areas and consider alternative tactics? INNOVATION: Does the project employ novel concepts, approaches or methods? Are the aims original and innovative? Does the project challenge existing paradigms or develop new methodologies or technologies? INVESTIGATOR: Is the investigator appropriately trained and well suited to carry out this work? Is the work proposed appropriate to the experience level of the Principal Investigator and other researchers (if any)? ENVIRONMENT: Does the scientific environment in which the work will be done contribute to the probability of success? Do the proposed experiments take advantage of unique features of the scientific environment or employ useful collaborative arrangements? Is there evidence of institutional support? ADDITIONAL REVIEW CRITERIA: In addition to the above criteria, the following items will be considered in the determination of scientific merit and the priority score: PROTECTION OF HUMAN SUBJECTS FROM RESEARCH RISK: The involvement of human subjects and protections from research risk relating to their participation in the proposed research will be assessed. (See criteria included in the section on Federal Citations, below.) INCLUSION OF WOMEN, MINORITIES AND CHILDREN IN RESEARCH: The adequacy of plans to include subjects from both genders, all racial and ethnic groups (and subgroups), and children as appropriate for the scientific goals of the research will be assessed. Plans for the recruitment and retention of subjects will also be evaluated. (See Inclusion Criteria in the sections on Federal Citations, below.) CARE AND USE OF VERTEBRATE ANIMALS IN RESEARCH: If vertebrate animals are to be used in the project, the five items described under Section f of the PHS 398 research grant application instructions (rev. 5/2001) will be assessed. ADDITIONAL REVIEW CONSIDERATIONS SHARING RESEARCH DATA: Applicants requesting more than $500,000 in direct costs in any year of the proposed research are expected to include a data sharing plan in their application. The reasonableness of the data sharing plan or the rationale for not sharing research data will be assessed by the reviewers. However, reviewers will not factor the proposed data sharing plan into the determination of scientific merit or priority score. BUDGET: The reasonableness of the proposed budget and the requested period of support in relation to the proposed research. AWARD CRITERIA Applications submitted in response to a PA will compete for available funds with all other recommended applications. The following will be considered in making funding decisions: o Scientific merit of the proposed project as determined by peer review o Availability of funds o Relevance to program priorities REQUIRED FEDERAL CITATIONS HUMAN SUBJECTS PROTECTION: Federal regulations (45CFR46) require that applications and proposals involving human subjects must be evaluated with reference to the risks to the subjects, the adequacy of protection against these risks, the potential benefits of the research to the subjects and others, and the importance of the knowledge gained or to be gained. http://www.hhs.gov/ohrp/humansubjects/guidance/45cfr46.htm DATA AND SAFETY MONITORING PLAN: Data and safety monitoring is required for all types of clinical trials, including physiologic, toxicity, and dose- finding studies (phase I); efficacy studies (phase II), efficacy, effectiveness and comparative trials (phase III). The establishment of data and safety monitoring boards (DSMBs) is required for multi-site clinical trials involving interventions that entail potential risk to the participants (NIH Policy for Data and Safety Monitoring, NIH Guide for Grants and Contracts, June 12, 1998: http://grants.nih.gov/grants/guide/notice-files/not98-084.html). SHARING RESEARCH DATA: Starting with the October 1, 2003 receipt date, investigators submitting an NIH application seeking $500,000 or more in direct costs in any single year are expected to include a plan for data sharing or state why this is not possible (http://grants.nih.gov/grants/policy/data_sharing). Investigators should seek guidance from their institutions on issues related to institutional policies, local IRB rules, as well as local, state and Federal laws and regulations, including the Privacy Rule. Reviewers will consider the data sharing plan but will not factor the plan into the determination of the scientific merit or the priority score. INCLUSION OF WOMEN AND MINORITIES IN CLINICAL RESEARCH: It is the policy of the NIH that women and members of minority groups and their sub-populations must be included in all NIH-supported clinical research projects unless a clear and compelling justification is provided indicating that inclusion is inappropriate with respect to the health of the subjects or the purpose of the research. This policy results from the NIH Revitalization Act of 1993 (Section 492B of Public Law 103-43). All investigators proposing clinical research should read the "NIH Guidelines for Inclusion of Women and Minorities as Subjects in Clinical Research - Amended, October, 2001," published in the NIH Guide for Grants and Contracts on October 9, 2001 (http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-001.html); a complete copy of the updated Guidelines is available at http://grants.nih.gov/grants/funding/women_min/guidelines_amended_10_2001.htm. The amended policy incorporates: the use of an NIH definition of clinical research; updated racial and ethnic categories in compliance with the new OMB standards; clarification of language governing NIH-defined Phase III clinical trials consistent with the new PHS Form 398; and updated roles and responsibilities of NIH staff and the extramural community. The policy continues to require for all NIH-defined Phase III clinical trials that: a) all applications or proposals and/or protocols must provide a description of plans to conduct analyses, as appropriate, to address differences by sex/gender and/or racial/ethnic groups, including subgroups if applicable; and b) investigators must report annual accrual and progress in conducting analyses, as appropriate, by sex/gender and/or racial/ethnic group differences. INCLUSION OF CHILDREN AS PARTICIPANTS IN RESEARCH INVOLVING HUMAN SUBJECTS: The NIH maintains a policy that children (i.e., individuals under the age of 21) must be included in all human subjects research, conducted or supported by the NIH, unless there are scientific and ethical reasons not to include them. This policy applies to all initial (Type 1) applications submitted for receipt dates after October 1, 1998. All investigators proposing research involving human subjects should read the "NIH Policy and Guidelines" on the inclusion of children as participants in research involving human subjects that is available at http://grants.nih.gov/grants/funding/children/children.htm. REQUIRED EDUCATION ON THE PROTECTION OF HUMAN SUBJECT PARTICIPANTS: NIH policy requires education on the protection of human subject participants for all investigators submitting NIH proposals for research involving human subjects. You will find this policy announcement in the NIH Guide for Grants and Contracts Announcement, dated June 5, 2000, at http://grants.nih.gov/grants/guide/notice-files/NOT-OD-00-039.html. HUMAN EMBRYONIC STEM CELLS (hESC): Criteria for federal funding of research on hESCs can be found at http://stemcells.nih.gov/index.asp and at http://grants.nih.gov/grants/guide/notice-files/NOT-OD-02-005.html. Only research using hESC lines that are registered in the NIH Human Embryonic Stem Cell Registry will be eligible for Federal funding (see http://escr.nih.gov). It is the responsibility of the applicant to provide the official NIH identifier(s) for the hESC line(s) to be used in the proposed research. Applications that do not provide this information will be returned without review. PUBLIC ACCESS TO RESEARCH DATA THROUGH THE FREEDOM OF INFORMATION ACT: The Office of Management and Budget (OMB) Circular A-110 has been revised to provide public access to research data through the Freedom of Information Act (FOIA) under some circumstances. Data that are (1) first produced in a project that is supported in whole or in part with Federal funds and (2) cited publicly and officially by a Federal agency in support of an action that has the force and effect of law (i.e., a regulation) may be accessed through FOIA. It is important for applicants to understand the basic scope of this amendment. NIH has provided guidance at http://grants.nih.gov/grants/policy/a110/a110_guidance_dec1999.htm. Applicants may wish to place data collected under this PA in a public archive, which can provide protections for the data and manage the distribution for an indefinite period of time. If so, the application should include a description of the archiving plan in the study design and include information about this in the budget justification section of the application. In addition, applicants should think about how to structure informed consent statements and other human subjects procedures given the potential for wider use of data collected under this award. STANDARDS FOR PRIVACY OF INDIVIDUALLY IDENTIFIABLE HEALTH INFORMATION: The Department of Health and Human Services (DHHS) issued final modification to the Standards for Privacy of Individually Identifiable Health Information , the Privacy Rule, on August 14, 2002. The Privacy Rule is a federal regulation under the Health Insurance Portability and Accountability Act (HIPAA) of 1996 that governs the protection of individually identifiable health information, and is administered and enforced by the DHHS Office for Civil Rights (OCR). Those who must comply with the Privacy Rule (classified under the Rule as covered entities ) must do so by April 14, 2003 (with the exception of small health plans which have an extra year to comply). Decisions about applicability and implementation of the Privacy Rule reside with the researcher and his/her institution. The OCR website (http://www.hhs.gov/ocr/) provides information on the Privacy Rule, including a complete Regulation Text and a set of decision tools on Am I a covered entity? Information on the impact of the HIPAA Privacy Rule on NIH processes involving the review, funding, and progress monitoring of grants, cooperative agreements, and research contracts can be found at http://grants.nih.gov/grants/guide/notice-files/NOT-OD-03-025.html. URLs IN NIH GRANT APPLICATIONS OR APPENDICES: All applications and proposals for NIH funding must be self-contained within specified page limitations. Unless otherwise specified in an NIH solicitation, Internet addresses (URLs) should not be used to provide information necessary to the review because reviewers are under no obligation to view the Internet sites. Furthermore, we caution reviewers that their anonymity may be compromised when they directly access an Internet site. HEALTHY PEOPLE 2010: The Public Health Service (PHS) is committed to achieving the health promotion and disease prevention objectives of "Healthy People 2010," a PHS-led national activity for setting priority areas. This PA is related to one or more of the priority areas. Potential applicants may obtain a copy of "Healthy People 2010" at http://www.health.gov/healthypeople. AUTHORITY AND REGULATIONS: This program is described in the Catalog of Federal Domestic Assistance at http://www.cfda.gov/ and is not subject to the intergovernmental review requirements of Executive Order 12372 or Health Systems Agency review. Awards are made under the authorization of Sections 301 and 405 of the Public Health Service Act as amended (42 USC 241 and 284) and under Federal Regulations 42 CFR 52 and 45 CFR Parts 74 and 92. All awards are subject to the terms and conditions, cost principles, and other considerations described in the NIH Grants Policy Statement. The NIH Grants Policy Statement can be found at http://grants.nih.gov/grants/policy/policy.htm. The PHS strongly encourages all grant recipients to provide a smoke-free workplace and discourage the use of all tobacco products. In addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking in certain facilities (or in some cases, any portion of a facility) in which regular or routine education, library, day care, health care, or early childhood development services are provided to children. This is consistent with the PHS mission to protect and advance the physical and mental health of the American people.


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