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) 435-0714, 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.

Weekly TOC for this Announcement
NIH Funding Opportunities and Notices

	Office of Extramural Research (OER)			National Institutes of Health (NIH) 9000 Rockville Pike Bethesda, Maryland 20892			Department of Health and Human Services (HHS)
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