www.nih.gov/women

#SexinScience | @JanineClaytonMD | @NIH_ORWH

Janine A. Clayton, M.D.NIH Associate Director for Research on Women’s HealthDirector, NIH Office of Research on Women’s Health

May 18, 2015 | NIH Center for Scientific Review Advisory Council Meeting

Studying Both Sexes in NIH-Funded Preclinical Research

• 25th anniversary in 2015: Founded in 1990• 1991: Women’s Health Initiative• 1993: NIH mandate to include women and minority

groups in clinical research• 2015: NIH focal point for research on sex/gender

influences on health

… Benefits girls, women, boys, men

NIH Office of Research on Women’s Health

NIH in 2015: Beyond InclusionSex/Gender Influences on Health and Disease

What is “default” biology?

• In the clinic: 70-kg male

• In the lab: male cells and animals

Fundamental is not only that which is shared

• Fundamental biology = same and different in males, females

Reproducibility and Transparency

• Every experiment is part of larger system, the quest to understand fundamental basic living systems

• Approaches and results should be consistent and free of bias at the outset

Every System is Exquisitely Tuned to Get the Results it Gets

Zucker, Beery, Nature : 465, 690 : 2010

An Imbalance: Males Still Dominate Animal Studies

• Size• Age • Sex • Genetic strain• Source of origin (animal, cell)• Passage number• Serum• Group housing• Lighting conditions• Temperature• Time• Order of experimentation• And so on …

Sex is One of Many Biological Variables

Biological/Disease Impact of Experimental Design

McCullough LD, et al. Journal of Cerebral Blood Flow & Metabolism (2005) 25, 502–512.

The effects of the selective poly-ADP ribose polymerase (PARP-1) inhibitor PJ-34 in wild-type (WT) mice of both genders. Treatment with PJ-34 at ischemic onset reduced total infarction in male mice compared with saline-treated controls (* P<0.001). A significant increase in ischemic damage was seen in PJ-34-treated females compared with control (* P<0.001).

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Study Both SexesSex is a Biological Variable

• Basic Research on Human Health and Disease

• Translational Research and Clinical Studies

• Funding for Training and Biomedical Workforce Development

Turning Discovery into Health

• Scientific progress in academic labs• Leveraging current investments with

supplements• Congressional interest and support• Journal policies to improve reporting standards

(including information about sex)• NIH policy for considering sex as a biological

variable in preclinical research • Resources for the scientific community, NIH

staff, reviewers

Enhancing Study of Male and Female Biology in Preclinical Research: It Takes a Village

Better With Both: Encouraging Scientists to Study Both Sexes

Better with Both: Improving Understanding of Female and Male Biology Across Diseases

Of special interest are studies to understand the influence of biological sex on cells, including primary cell cultures, in vitro cell cultures, explants and transformed cells

New!• Robust response: Applications received from 21 NIH

Institutes and Centers• Earliest funding: July 2015 • Different from last year: asked IC reviewers to

identify not only alignment with strategic plan goal/objective 1-3 but also to identify the “sex-based” approach used:• Add the opposite sex• Increase sample size• Conduct new comparative analyses• Single-sex study (with justification)

FY 2015 Administrative Supplements

Huang GZ, Woolley CS (2012) Neuron 74(5):801-8.

“… this is the first demonstration of a sex-specific mechanism of synaptic modulation in a non-reproductive region of the brain.” –C.W.

“The ORWH administrative supplement had a huge impact on the direction of research in my lab … my competitive renewal will focus on continuing studies started begun with the supplement.”

-- Dr. Catherine Woolley

Enabling a Re-Search of a Scientific Question

“The supplement allowed us to expand our mouse colony to include female Mecp2 mutants to perform experiments. Female mutants represent the best pre-clinical model of Rett syndrome because they display a mosaic pattern of neural cells expressing WT and mutant Mecp2 alleles.”

--administrative supplement grantee

Rett syndrome:

• Autism spectrum disorder caused by loss-of-function mutations in MeCP2 gene on X chromosome

• Almost exclusively affects girls and leading cause of intellectual disability in females

Expanding Generalizability of Animal Models

NIH is “directed to report on preclinical research [in their biennial report] in terms of the proportion of studies that incorporate sex as a biological variable and of those studies which analyze data by sex as part of grant review, award, and oversight processes” by Institute and Center across NIH

H.R.83 - Consolidated and Further Continuing Appropriations Act, 2015

• The agreement recommends to NIH: • institute requirements that investigators utilize valid

experimental design including consideration of sex as a biological variable in preclinical research on animals, cells, and human subjects, as scientifically appropriate

• expand policy to require indication of sex in preclinical grant applications, progress reports and subsequent publications

• for investigators studying both sexes, require analysis of preclinical data by sex, when scientifically appropriate

• give priority to proposals that include adequate numbers of women and men, and a robust plan for analysis and distribution of findings

Cromnibus: Sex as a Biological Variable

New Journal Policies to Enhance Reproducibility

“Eight- to 16-week old female Foxn1/Foxn1 (nude) mice (Harlan) were injected subcutaneously with 1 X 106 cells in two to four spots on flanks.”

“Cohorts of male and female EGFRT790M;L858R;CCSP-rtTA were put on a doxycy-cline diet at 6 weeks of age to induce the expression of mutant EGFR, while male and female KrasG12D/+;p53Δ/Δ mice received intranasal adeno-Cre between 6 and 8 weeks of age.”

• RFI released (September 2014)• SABV policy to be released this fall

(FY 2016 submissions for FY 2017 funding)

• Placement, criteria, and language of sex as a biological variable in grant applications and instructions to reviewers

Other current efforts• Developing resources for the

scientific community• Developing plans for evaluation of

the policy• Informing/developing study section

training materials• Already hearing anecdotes of

study sections talking about SABV in reviews

Sex as a Biological Variable (SABV): NIH Policy Activities

• Does considering SABV affect the reproducibility, rigor, and/or generalizability of research findings?

• What are the areas of science or phases of research that might benefit from consideration of SABV?

• What are the main impediments of considering SABV?

• How can NIH facilitate considering SABV?• Other comments?

Request for Information: Considering Sex as a Biological Variable (SABV)

Uncertain, 1% No, 5%

Yes, with some qualifications,

31%

Yes, and will lead to better outcomes, 19%

Yes, 44%

Most RFI Respondents Agreed that Considering Sex as a Biological Variable Affects Rigor, Transparency, Generalizability

• Individual interpretation of the proposed NIH policy frames public feedback

• Pre-formed assumption by some that a blanket policy is forthcoming that will require all NIH-funded researchers to double the number of animals

• Major impediments include cost and constraints on methodological and experimental design

• Favor a flexible approach to policy implementation

• Encourage NIH to promote best practices and awareness of sex as a biological variable in research design and analysis

Public Perspectives on Policy Change

Potential RFA language:

Comparison of male and female populations or subjects on the desired outcome is highly desired. Of particular interest are research activities that increase the understanding of sex and gender differences and factors in health and disease, to support implementation of the NIH Strategic Plan for Women’s Health Research, available at http://orwh.od.nih.gov/research/strategicplan/index.asp.

Encouraging SABV in Research Grant Applications

• RFI released (September 2014)• SABV policy to be released this fall

(FY 2016 submissions for FY 2017 funding)

• Placement, criteria, and language of sex as a biological variable in grant applications and instructions to reviewers

Other current efforts• Developing resources for the

scientific community• Developing plans for evaluation of

the policy• Informing/developing study section

training materials• Already hearing anecdotes of

study sections talking about SABV in reviews

Sex as a Biological Variable (SABV): NIH Policy Activities

Miller et al. An Aging Interventions Testing Program: study design and interim report. Aging Cell. 2007;6:565-75.

Rapamycin:

• Extended lifespan in both sexes

NDGA, Aspirin:

• Extended lifespan extension in males only

Green Tea Extract (secondary analysis):

• Diminished risk of midlife deaths in females only

• Standardized preclinical evaluation of health-span prolonging interventions

• Test subjects = male and female genetically heterogeneous mice, bred as 4-way cross

• Compares multiple experimental agents to two control groups

• Sufficient numbers of male and female mice tested: • 80% chance of detecting a 10%

increase/decrease in lifespan

NIH Programs that Promote the Study of Both Sexes:NIA Interventions Testing Program

Tang T, et al. A mouse knockout library for secreted and transmembrane proteins. Nat Biotechnol. 2010;28:749-55.

• Rigor, generalizability, and utility Broad phenotypic screen of 472 knock-out lines

o Validated assays, relevant to therapeutic areaso All animals screened at the same age, same order of assays

“Eight homozygous mice (equally divided between males and females) per assay… as we were most interested in seeing effects shared by the sexes.”o Results reported for M/F aggregated, M alone, and F alone

• One of many approaches to account for sex as a biological variable

NIH Programs that Promote the Study of Both Sexes:KOMP

https://www.mousephenotype.org/phenoview/

http://commonfund.nih.gov/KOMP2/

• Eps8l1 mutation results in female deafness, while males are unaffected

• Pcks1 null females travel less distance in an open field than their male counterparts

• Baiap2l2 null males and females both exhibit enhanced auditory brain stem response, but the phenotype is more striking in males

• 15 percent of KOMP datasets show sex-specific differences

Knockout Mouse Project (KOMP) Results

NIH Resources to Promote the Study of Both Sexes:Cell Resources

The 4 Cs of Studying Sex to Strengthen Science

#SexinScience | @JanineClaytonMD | @NIH_ORWHwww.nih.gov/women

Thank you.