Iowa Newborn Screening Program for Cystic Fibrosis
Pilot Project Proposal
Miles Weinberger, M.D.
Professor of Pediatrics
Director, Pediatric Allergy & Pulmonary Division
Director, University of Iowa Cystic Fibrosis Center
Introduction: Cystic fibrosis (CF) is the most common fatal genetic disease of Caucasians. It
also occurs in other ethnic groups with variable though lesser frequency. A recessive trait, the
gene frequency in those of Northern European descent, is estimated to be as high as five percent
of population. There are no adverse effects for heterozygous carriers of a CF mutation; the
potential for disease only occurs when a CF mutation is inherited from both parents. Although a
specific cure is still wanting, aggressive treatment at specialized Cystic Fibrosis Centers has
increased the average survival from less than ten years, when the disease was first identified over
40 years ago, to 16 years about 25 years ago, to its current mean approaching the mid-thirties.
Quality of life has similarly progressed during this period.
The disease: Cystic fibrosis is a disease of exocrine glands. Due to a defect in a single gene
locus on chromosome 7, misinformation is provided regarding the production of a protein called
the cystic fibrosis transmembrane regulator (CFTR). This CFTR serves as a chloride channel,
and the abnormal CFTR results in failure of sodium chloride, salt, to be reabsorbed in various
exocrine secretions thereby disrupting normal functioning in some critical organs. The resulting
high salt content in sweat serves as the basis for the diagnosis. Sweat chloride concentrations are
generally under 40 meq/L in normals and over 60 meq/l in people with cystic fibrosis. When
done appropriately, this is a highly accurate means of determining the presence or absence of
cystic fibrosis. The major clinical manifestations of the disease are pancreatic exocrine
insufficiency, resulting in malabsorption, and chronic pulmonary infection due to a host defense
defect in the mucous membrane of the airway associated with the high salt concentrations in
airway secretions. Normally bacteriologically sterile, despite frequent contamination from the
upper airway, airways in people with cystic fibrosis permit bacteria to survive and thrive,
resulting in chronic infection and inflammation of the airways. This eventually results in
progressive damage with erosion of the airways to form sacs that retain mucopurulent material
and make treatment progressively difficult and ineffective. This type of lung damage is called
bronchiectasis, which once present is irreversible. The end result is progressive loss of
functioning lung tissue, crippling chronic lung disease, respiratory failure, and death at an early
Treatment: Over 90 percent of patients have pancreatic insufficiency resulting in malabsorption.
Pancreatic enzyme replacements from animal sources taken orally before each meal provide
partial correction of malabsorption sufficient to permit normal or near normal growth and weight
gain for most patients. Treatment of the pulmonary disease involves an early and sustained
program of bronchial toilet using physical means to promote evacuation of mucous and
mucopurulent material accumulating in the lungs combined with antibiotics to control the
chronic infection. While improving outcome, this treatment has had obvious limitations since
progressive lung damage is still the major cause of early death in those with cystic fibrosis.
Identification of immunologically mediated inflammation in response to the chronic infection led
to controlled trials of anti-inflammatory therapy to slow the progression of lung disease. When
instituted in early stages of the lung disease prior to development of advanced bronchiectasis,
separate studies over four-year periods using alternate-morning prednisone and high dose
ibuprofen demonstrated slowing of the progression of lung disease with systemically
administered anti-inflammatory therapy. More recently, azithromycin has demonstrated benefit
in three different six month-long studies through what may also be an anti-inflammatory
A major predictor of increased rate of progression of lung disease has been chronic infection
with Pseudomonas species of bacteria. Once well established in the lung, this infection cannot
be eradicated. However, recent experience has found that eradication is possible early in the
course of Pseudomonas infection. This has led to aggressive efforts to identify early infection
with Pseudomonas and treat vigorously in an attempt at eradication. While reinfection will
eventually occur, further eradication appears possible so long as vigorous treatment is instituted
early. It is expected that these efforts to delay chronic infection with Pseudomonas will delay the
more rapid deterioration in lung function that follows.
A major impediment to beginning aggressive treatment prior to the development of poor
growth and permanent changes in the lung has been delayed diagnosis. The average age of
diagnosis is greater than two years of age, and the data from Wisconsin indicates that 20 percent
of those not diagnosed by neonatal screening were not diagnosed till age four. This delay can
result in irreversible bronchiectatic changes in the lungs, which further encourages chronic
infection in progressive lung damage.
Objective of the Screening: Identification of most patients with cystic fibrosis will be
accomplished prior to the development of pulmonary disease and malnutrition. This will permit
early referral to a CF Foundation certified center where aggressive early intervention will permit
normal growth and delay the onset of lung damage from infection and inflammation.
Rationale for newborn screening: Studies done by us1and others2 have identified that chronic
inflammation and infection of the airways precedes symptoms in most cases. Therefore,
treatment for many patients is delayed because the diagnostic test is not performed until after
lung disease has already resulted in chronic infection and irreversible damage to the airways.
Even when lung disease is not prominent, delayed diagnosis results in decreased growth and
weight gain compared with those in whom the diagnosis is made through neonatal screening.3
Increasing emphasis has been placed on early intervention and prophylactic therapy. Since
.Balough K, McCubbin M, Weinberger M, Smits W, Ahrens R, Fick R. The relationship between infection
and inflammation in the early stages of lung disease from cystic fibrosis. Pediatr Pulm 1995; 20:63-70.
.Khan TZ, Wagener JS, Bost T, Martinez J, Accurso FJ, Riches DW. Early pulmonary inflammation in infants
with cystic fibrosis. Am J Respir Crit Care Med 1995; 151:1075-82.
. Farrell PM et al. Nutritional benefits of neonatal screening for cystic fibrosis. N Eng J Med 1997; 337:963-9.
diagnosis is often delayed, interest in the potential value of newborn screening led to pilot
programs in Wisconsin and Colorado. The program in Wisconsin, a neighboring state with
similar demographics to Iowa, has been in place since 1985. A report of that program in the
October 2, 1997 issue of the New England Journal of Medicine indicated improved outcome
from identification of the disease through neonatal screening in Wisconsin. An accompanying
editorial concluded “...the evidence has accumulated that early diagnosis and treatment (of cystic
fibrosis) are beneficial and that screening probably saves money and carries no harm of its own.
The results of this new study provide further evidence that the time has come for routine neonatal
screening for cystic fibrosis.”4 This position was reiterated in a subsequent editorial.5 Benefits
from programs elsewhere have been substantiated in multiple reports,6, 7,8,9,10,11 and an argument
has been made that a CF newborn screening program provides the potential for a cost-saving
alternative to the traditional method of diagnosing CF only after symptoms are readily
Method for newborn screening: Newborn screening first became feasible in 1979 through the
identification that neonates with the disease have an elevated blood immunoreactive trypsinogen
(IRT) level. This test suffers from a high false positive rate if set sufficiently low to provide
optimal sensitivity. The subsequent identification of the gene defect led to a two-tiered test.
Both tiers of the test can be run from whole blood spots collected as part of current newborn
screening procedures. In the Wisconsin experience from 1991 to 1994, 132 (0.13%) of 104,308
had a positive 2 tiered screen of whom 22 (15.9%) were confirmed to have cystic fibrosis.
Currently, it is now possible to screen for multiple CFTR mutations so that patients and carriers
with less common mutations than the ∆F508 can be identified in the screening procedure with no
greater effort that was previously required for the ∆F508 alone. Based on the Wisconsin
experience and adjusting for the differences in population between the two states, we anticipate
about 50 newborns will require referral for evaluation yearly with about eight being confirmed
with a diagnosis of CF; the remainder will be carriers.
.Dankert-Roelse JE, Meerman GJ. Screening for cystic fibrosis – time to change our position. N Eng J Med
.Wilcken B. Neonatal screening for cystic fibrosis: It is time. Pediatric Pulmonology 1998;26:219-21.
.Dankert-Roelse JE, Meerman GJ. Long term prognosis of patients with cystic fibrosis in relation to early
detection by neonatal screening and treatment in a cystic fibrosis centre. Thorax 1995;50:712-8.
.Waters DL, et al. Clinical outcomes of newborn screening for cystic fibrosis. Arch Dis child fetal neonatal
.Scotet V, et al. Neonatal screening for cystic fibrosis in Brittany, France: assessment of 10 years’ experience
and impact on prenatal diagnosis. Lancet 2000;356:789-94.
.Farrell PM, et al. Early diagnosis of cystic fibrosis through neonatal screening prevents severe malnutrition
and improves long-term growth. Pediatrics 2001;107:1-13.
.Wang SS, et al. The impact of early cystic fibrosis diagnosis on pulmonary function in children. J Pediatr
.Siret D, et al. Comparing the clinical evolution of cystic fibrosis screened neonatally to that of cystic fibrosis
diagnosed from clinical symptoms: a 10-year retrospective study in a French region (Brittany). Pediatr Pulmonol
.Lee DS et al. Analysis of the costs of diagnosing cystic fibrosis with a newborn screening program. J
Diagnostic confirmation: Infants having a positive newborn screen (an IRT > 170ng/ml and no
detectable mutations, an IRT in the top five percent and one detectable mutation or an IRT in the
top five percent and two detectable mutations) will require confirmation by measurement of
sweat chlorides. This must be done by the classical quantitative pilocarpine iontophoresis
(QPIT) method at centers that perform sufficient numbers of tests with adequate quality
assurance. For Iowa and contiguous areas, Cystic Fibrosis Centers certified by the Cystic
Fibrosis Foundation, which includes certification of appropriate sweat chloride methodology and
experience, include: 1) UIHC in Iowa City and its Affiliate Program at the McFarland Clinic in
Ames, 2) Iowa Methodist (Blank Children’s hospital), 3) the University of Nebraska (Omaha), 4)
the University of South Dakota (Sioux Falls), and 5) the University of Wisconsin (Madison).
While commercial tests for sweat chlorides are marketed and used at some hospitals and
laboratories, those other than the QPIT are insufficiently reliable to either make or exclude the
diagnosis. Patients with positive screens will be advised to have the diagnostic tests performed
at one of the CF Foundation certified Cystic Fibrosis Centers. Since a small percentage of CF
patients with some of the less common mutations have sweat chlorides in a borderline or even
normal range, more extensive genetic testing and a procedure known as nasal potential
measurement may be needed in selected patients. Abnormalities of ion transport in respiratory
epithelia of patients with CF are associated with a different pattern of nasal potential difference
compared to normal epithelia. A raised basal nasal potential difference is strong evidence for the
diagnosis of CF. The presence of nasal polyps or inflamed mucosa, however, alters the
bioelectric properties and may yield a false-negative result. Nasal potential difference can be
measured in patients as young as a few hours of life. Extensive genetic testing for over 900
mutations is now available through a commercial laboratory available to all centers. Nasal
potential measurement is a procedure available only at a few centers in the U.S., with the
University of Iowa Center being one of them. The modest increase in volume of sweat tests that
will result from the testing can be readily managed at the existing centers, which are also capable
of providing genetic counseling for families of both patients and carriers.
For those with a confirmed diagnosis of CF, ongoing assessment and treatment at a CF
Foundation certified CF center will be strongly encouraged. Families will be advised that the
standard of care is a visit every three months to a CF Foundation certified CF center. The
treatment will be provided by the CF centers that serve Iowans including: 1) the University of
Iowa Hospital and Clinic in Iowa city with its Affiliate Program at the McFarland Clinic in
Ames, 2) Blank Hospital and clinic in Des Moines, 3) University of Nebraska in Omaha, 4)
University of S. Dakota in Sioux Falls, and 5) University of Wisconsin in Madison.
Benefits of Screening: Improved nutritional outcome has been now well documented in a
controlled clinical trial.3 While improved survival from the lung disease will take many years to
fully document; the value of aggressive intervention of the lung disease is a well-established
practice. Currently applied measures at University of Iowa Hospital and Clinic CF Center and
elsewhere identify early evidence of lung inflammation and infection prior to the onset of
symptoms and consequent lung damage. However, early identification of lung disease and early
intervention requires identification of the disease, which is delayed to beyond one year of life in
well over half of all patients. Some are misdiagnosed even though symptomatic for many years.
Experience in the Netherlands indicates that neonatal screening is permitting therapeutic
measures instituted sufficiently early to not only improve growth but also delay the onset of
progressive lung inflammation and damage.6
Risks of Screening and Management: The primary risk is the anxiety induced from a positive
screen. The transient anxiety can be effectively managed by prompt referral to a CF Center
where a sweat chloride is performed in duplicate by quantitative pilocarpine iontophoresis.
Other medical evaluation is then offered if indicated by a positive sweat chloride result, and
adequate counseling regarding the implications of the carrier state is provided if CF is not
confirmed by the sweat test. For those subsequently identified as having the disease, the
anticipation of improved outcome by having earlier effective intervention at a CF Center
provides benefit that far outweighs concerns about the natural anxiety of having a serious
disease.13 For those subsequently identified as being only a carrier, genetic counseling will be
essential, and this will generally be done by the CF Center personnel at the center performing the
sweat test. Prompt communication of results and interpretation with the patient’s primary care
physician will also be important. It is anticipated that current facilities at the CF Centers can
manage the increase in volume anticipated by this additional screening.
.Farrell MH, Farrell PM. Newborn screening for cystic fibrosis: ensuring more good than harm. J Pediatr
PILOT SCREENING PROTOCOL
EXPECTATION: Estimates of cystic fibrosis (CF) identified by newborn screening in
Caucasian populations have ranged from about 1 in 2500 to almost 1 in 4000. About 1 in 29 will
be a carrier of a CF mutation.
OBJECTIVE: Capture all babies with CF in a pool of newborns identified through newborn
screening and keep the number of non-CF babies within the pool as low as possible.
STRATEGY: Use a two-tiered screening approach to meet the objective. Immunoreactive
trypsinogen (IRT) will be used as an initial screen test to capture most/all CF babies
(unfortunately, a large number of false positives for CF are also identified). If the IRT level is
in the highest five percent of the daily IRT results, DNA will be extracted from the same
specimen card and analysis of the Cystic Fibrosis Transmembrane Conductance Regulator
(CFTR) gene to detect CF related mutations would be performed. An algorithm using the results
from both tests will be used to reduce the number of babies referred for confirmatory sweat
chloride testing by pilocarpine iontophoresis.
SPECIMEN: Dried blood spot specimens already submitted to the laboratory for mandated
IRT Vender: The laboratory will use the time-resolved fluoroimmunoassay kits produced by
Perkin Elmer for neonatal IRT.
Principle of IRT assay: The DELFIA neonatal immunoreactive trypsinogen (IRT) assay
is a solid phase, two-site fluoroimmunometric assay based on the direct sandwich
technique in which two monoclonal antibodies (derived from mice) are directed against
two separate antigenic determinants on the IRT molecule. Standards, controls and test
specimens containing IRT are reacted simultaneously with immobilized monoclonal
antibodies directed against a specific antigenic site on the IRT molecule and europium-
labeled monoclonal antibodies (directed against a different antigen site) in the Neo IRT
Assay Buffer. The buffer elutes IRT from the dried blood spots on the filter paper discs.
Enhancement Solution dissociates europium ions from the labeled antibody into solution
where they form highly fluorescent chelates with components of the Enhancement
Solution. The fluorescence in each well is then measured. The fluorescence of each
sample is proportional to the concentration of IRT in the sample.
Staff and Equipment: This test utilizes the same methodology as others tests currently
performed in the laboratory. Although no additional staff training will be required, a
second instrument will be needed for testing.
DNA Analysis Vender: The laboratory will use the Linear Array CF Gold 1.0, an analyte
specific reagent produced by Roche Molecular Biochemicals, for the detection of the most
common CF mutations, including the 25 pan-ethnic mutation panel recommended by the
American College of Medical Genetics.
Background information and DNA assay: The CFTR gene is located on the long arm of
chromosome 7. The CF GOLD 1.0 Primer Mix contains 30 different 5' biotin labeled primers
that simultaneously amplify 15 different regions of this gene. The following table describes the
targeted region, amplicon size, and the mutations amplified by each primer.
Size Amplicon Mutation (Polymorphism)
598 Exon 13 2184delA
548 Exon 9 A455E
intron 8 (5/7/9 T)
482 Exon 10 _ I507, _F508, (F508C, I507V, I506V)
433 exon 11 G542X, G551D, R553X, R560T
420 Exon 19 R1162X, 3659delC
397 exon 21 N1303K
359 Exon 20 W1282X
328 exon 7 R334W, R347P, 1078delT
288 Exon 4 R117H, I148T
intron 4 621+1G>T
248 intron 14 b 2789 + 5G>A
237 intron 19 3849 + 10kbC>T
210 exon 3 G85E
198 Exon 12 1898+1G>A
166 intron 5 711 + 1G>T
139 intron 16 3120 + 1G>A
Post amplification, the PCR products are denatured and then hybridized with the CF GOLD 1.0
Linear Array Panel (LAp). After hybridization, the CF GOLD 1.0 LAps are washed and then
incubated with streptavidin-POD. After an additional wash step, substrate is added and the color
developed. Once this is complete, the LAps are washed again and aligned with the reference
guide to identify the alleles in the sample.
Staff and Equipment: Although this test introduces new methodology into the newborn
screening laboratory the expertise already exists in other sections within the Hygienic
Laboratory. The MSAFP section is using this same test method for the CF carrier-screening
program. Initial training will be provided to the NBS staff utilizing MSAFP personnel.
Equipment necessary to support this additional testing is listed in the proposed budget.
REPORTING OF SCREENING RESULTS: During the pilot study, normal screening results
will not be reported out. Only those infants requiring follow-up will be reported to the CF
consulting physician. The laboratory will call and fax both test results (IRT and CFTR mutation
analysis) and the patient information collected with the routine NBS specimen to the medical
consultant or designee. The laboratory will not report pilot screening results to the birthing
facility or to the attending physician.
IRT > 170 ng/ml
One mutant allele (and top 5% of daily IRT results)
Two mutant alleles (and top 5% of daily IRT results)
Screening for cystic fibrosis using the two-tiered IRT / DNA approach cannot always distinguish
babies who are CF carriers from babies who are affected. Sweat chloride testing by the CF-
approved quantitative pilocarpine iontophoresis method is recommended for all babies with
either one or two identified mutations. Sweat testing will also be recommended for babies with
no detectable mutations but with an extremely elevated IRT > 170 ng/mL.
Based on this information the laboratory anticipates reporting three categories of infants to the
consultant for CF follow-up. These include:
1. Possible Cystic Fibrosis: IRT: > 170 ng/mL
No CFTR mutations detected
Laboratory interpretation: An elevated trypsinogen result may be indicative of Cystic Fibrosis
due to mutant alleles other than the 25 mutations included in the screening panel.
2. Possible Cystic Fibrosis: One CFTR mutation detected
Laboratory interpretation: The presence of one CFTR mutant allele is indicative of a Cystic
Fibrosis carrier, although this does not rule out CF disease due to the possibility of a second
mutant allele other than the 25 mutations included in the screening panel.
3. Definite Cystic Fibrosis: Two CFTR mutations detected
Laboratory interpretation: The presence of two CFTR mutant alleles is consistent with Cystic
DETECTION RATE: It is estimated that screening with the two-tiered approach for 25 CF
mutations will detect 99 percent of babies with CF.
SCREENING PRACTICE CONSIDERATIONS: Primary care providers should continue to
consider Cystic Fibrosis as a potential diagnosis in patients with negative newborn screening
results and persistent diarrhea, poor weight gain, and chronic cough or respiratory problems.
Newborns with meconium ileus may demonstrate normal IRT levels. If a newborn presents with
meconium ileus, contact should be made with a CF specialist.
Questions related to the Laboratory proposal please contact:
Dr. Stanton L. Berberich Laurie Hazelwood
(319) 335-4500 (515) 243-0141
Top 5% of Lower 95%
daily results of daily
< 170 ng/mL Report
> 170 ng/mL Possible
analysis of One CFTR 2.
the CFTR mutant allele Possible
gene detected Cystic
Two CFTR 3.
EXPERIENCES OF OTHER STATES
Currently, ten states in the U.S. perform CF newborn screening. These states are Colorado,
Connecticut, Massachusetts, Mississippi, Montana, New Jersey, New York, Pennsylvania,
Wisconsin, and Wyoming. Oklahoma and South Carolina are mandated to screen for Cystic
Fibrosis and will soon implement the testing in their states. CF newborn screening is mandated
and performed on all newborns in Colorado, Mississippi, New Jersey, New York, Wisconsin,
and Wyoming. Massachusetts also performs universal screening but the CF screening is a not a
mandated disorder currently. The other states screen a select population or offer it as a
supplement to their routine screening panel. Seven of the states use the two-tiered IRT/DNA
approach for their screening method. Wisconsin and Massachusetts are the only states in which
genetic counseling is routinely available. The Massachusetts program reports 83-87 percent
compliance with genetic counseling when it is the same day as the sweat test but only 32 percent
when it is on a different day.
FOLLOW-UP AND EDUCATION
The Iowa Neonatal Metabolic Screening Program will be establishing a new consultation branch
with cystic fibrosis newborn screening. The consultants will be personnel from the Cystic
Fibrosis Foundation certified CF Centers in Iowa. The CF follow-up coordination will take
place at the University of Iowa under the direction of Dr. Weinberger, the CF Clinic Director and
will be performed by CF clinic Coordinator, Beth Dowd. Confirmatory sweat chloride testing,
family education and genetic counseling will be performed at the certified center closest to the
family. There are certified centers in Iowa City and Des Moines as well as an affiliate center in
Ames. For some families, the closest certified CF center will be in a bordering state. The CF
follow-up consultants will provide public and professional education about cystic fibrosis
newborn screening prior to implementation of the pilot.
FOLLOWUP OF ABNORMAL SCREENING RESULTS:
There are three types of abnormal cystic fibrosis newborn screening results requiring follow-up
by the CF consultants:
1. Extremely elevated trypsinogen level equal to or greater than 170 ng/mL but no CFTR
gene mutations detected. Possible CF
2. Elevated trypsinogen level and one CFTR gene mutation detected. Possible CF
3. Elevated trypsinogen level and two CFTR gene mutations detected. Definitive CF
Primary Care Provider Notification
All abnormal results will be called to the primary care provider by the CF follow-up coordinator
and recommendations provided. Patients with one of these screening results should have sweat
chloride testing at a certified CF center to confirm or rule out the diagnosis of CF. A follow-up
letter will be faxed to the primary care provider by the CF follow-up coordinator with the results,
recommendations, the contact information for the nearest certified CF center and a letter for the
parents describing the results. The purpose of the parent letter is to help allay anxiety. The CF
follow-up coordinator will then contact the CF clinic coordinator of the CF center recommended
to the provider to perform the testing.
Successful collection of sweat chloride can be performed in infants at and less than two weeks of
age. Sweat testing will be scheduled within seven days of notification if the family is seen at a
CF center site in Iowa. All families will receive education about the screening results and
confirmatory testing procedures by a CF clinic coordinator the day of the testing. Test results
will be shared with the family the same day and tailored education will be provided. Families
will also be given written materials and sent a follow-up letter.
Family Education/Genetic Counseling
Family education/genetic counseling about sweat testing, cystic fibrosis, and genetic risk
assessment is a critical and essential component to CF newborn screening. It can assist families
in understanding the nature of their CF results as well as identify decisions that need to be made
and guide families through the process. Education about the genetics of CF and carrier risk
assessment will be provided to families the same day as the sweat testing. CF clinic coordinators
will provide the family education/genetic counseling. The CF follow-up coordinator will
provide training to the CF clinic coordinators in the Des Moines and Ames sites to ensure that
the information is provided uniformly and correctly. Families will be provided with written
materials and follow-up letter. Parents of a child identified to be a CF carrier or to have CF will
be provided with the opportunity to have genetic testing. The 25- mutation panel will be
available through the CF Carrier Screening Program if the family selects to have the testing. CF
clinic coordinators will not provide genetic counseling and testing for other family members
other than the immediate family. These individuals will be provided with contact information
for the Regional Genetics Consultation Service and other genetic counseling services available in
PUBLIC AND PROFESSIONAL EDUCATION
A large component of the CF newborn screening pilot activities will be devoted to the
development and review of educational materials and statewide education outreach for families,
the general public, and health care professionals. A CF review workgroup of health care
professionals and parents will be established to review the drafted materials to assure that the
information is easy to understand and clear. The education development and review activities
will be in the first phase of the pilot study (outlined in the attached timeline) and is anticipated to
take four to five months. The second phase of the pilot study will focus on professional
education. Articles will be submitted to the Perinatal Letter and the Iowa Chapter of the
American Academy of Pediatrics and IDPH Focus newsletters. Grand round presentations will
be scheduled and in-hospital trainings will be performed. CF educational materials will be added
to Center for Congenital and Inherited Disorders website. All primary care providers, birthing
hospitals and insurance companies will be informed by letter prior to the commencement of CF
newborn screening by the Newborn Screening Laboratory.