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WO2014143989A1 - Surveillance du bien-être fœtal au moyen de l'adn fœtal libre spécifique - Google Patents

Surveillance du bien-être fœtal au moyen de l'adn fœtal libre spécifique Download PDF

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WO2014143989A1
WO2014143989A1 PCT/US2014/028205 US2014028205W WO2014143989A1 WO 2014143989 A1 WO2014143989 A1 WO 2014143989A1 US 2014028205 W US2014028205 W US 2014028205W WO 2014143989 A1 WO2014143989 A1 WO 2014143989A1
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allele
fetal
fetus
dna
cell free
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Aoy Tomita Mitchell
Michael Mitchell
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Medical College of Wisconsin
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Medical College of Wisconsin
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to compositions and methods for assessing fetal well being
  • a method of assessing the condition of a fetus in a pregnant subject comprising any of the steps provided herein of any one of the methods described or illustrated herein, including in the Examples and figures, is provided.
  • a method of monitoring over a period of time a condition of a fetus in a pregnant subject comprising any of the steps provided herein of any one of the methods described or illustrated herein, including in the Examples and figures, is provided.
  • a method of assessing the condition of a fetus comprising determining an amount of fetal specific cell free DNA (cff-DNA) in a biological sample obtained from a pregnant subject, and comparing the amount with one or more threshold values, such as baseline levels is provided.
  • a change from a baseline level in the subject is indicative of the condition of the fetus.
  • amounts compared are the percent or ratio of fetal specific cell free DNA compared to the total cell free DNA or non-fetal specific cell free DNA.
  • the method further comprises determining, suggesting or providing a treatment for the fetus and/or pregnant subject, or information in regard thereto.
  • a method of determining a treatment for a fetus or a pregnant subject comprising determining an amount of fetal specific cell free DNA in a biological sample obtained from a pregnant subject, comparing the amount with one or more threshold values, such as one or more baseline levels, wherein the result of the comparison is indicative of the condition of the fetus, and providing a treatment to the fetus or pregnant subject or providing information in regard to the treatment to the pregnant subject is provided.
  • amounts compared are the percent or ratio of fetal specific cell free DNA compared to the total cell free DNA or non-fetal specific cell free DNA.
  • the condition is not the presence or absence of fetal aneuploidy, the gender of the fetus or the Rh blood type of the fetus.
  • the fetus has or is at risk of having fetal bradycardia, twin-twin transfusion syndrome (TTTS), gastroschisis, congenital pulmonary airway malformations (CPAMS), hydrops fetalis or fetal arrhythmia.
  • the pregnant subject has or is at risk of having chorioamnionitis or preterm labor.
  • the method further comprises obtaining the biological sample from the pregnant subject.
  • the pregnant subject is considered to have a high- risk pregnancy.
  • the pregnant subject is not considered to have a high-risk pregnancy.
  • the method further comprises performing one or more additional tests on the pregnant subject or fetus.
  • the decision to perform one or more additional tests on the pregnant subject or fetus is based on the comparison.
  • the one or more tests comprise performing an ultrasound.
  • one or more additional tests comprise a test that determines fetal heart rate, amniotic fluid level, fetal or maternal biophysical profile, bowel dilation or development of hydrops.
  • the one or more tests comprise determining the amount of fetal specific cell free DNA in the pregnant subject at one or more additional time points to determine a change from an individual's baseline levels.
  • the method is for the purpose of monitoring the condition of or the progression of the condition in the fetus.
  • amounts compared are the percent or ratio of fetal specific cell free DNA compared to the total cell free DNA or non-fetal specific cell free DNA at one or more time points.
  • the method further comprises determining, suggesting or providing a treatment for the fetus and/or pregnant subject, or information in regard thereto.
  • the amount of fetal specific cell free DNA is compared to one or more baseline levels to assess the condition of or the progression of the condition in the fetus at the one or more additional time points.
  • the amount of fetal specific cell free DNA is determined and compared to one or more baseline levels at one or more time points during gestation, during progression of a condition in the fetus, at fetal intervention and/or when resolution of the condition has occurred or is suspected to have occurred.
  • amounts compared are the percent or ratio of fetal specific cell free DNA compared to the total cell free DNA or non-fetal specific cell free DNA.
  • the method further comprises providing a treatment to the pregnant subject or providing information about one or more treatments to the pregnant subject.
  • the treatment is an in utero intervention.
  • the treatment comprises early delivery of the fetus.
  • the method further comprises providing a postnatal treatment or providing information about one or more postnatal treatments.
  • the amount of fetal specific cell free DNA is determined by extracting cell free DNA from the biological sample. In another embodiment of any one of the methods provided herein, the method further comprises determining the amount of total cell free DNA or non-fetal specific cell free DNA in the sample. In another embodiment of any one of the methods provided herein, the fetal specific cell free DNA, total cell free DNA and/or non-fetal specific cell free DNA is determined with any one of the methods provided herein, including in the Examples and figures, or as would be otherwise known to those of ordinary skill in the art. In another embodiment of any one of the methods provided herein, the amounts of the various types of DNA can be determined with any one of the methods provided in PCT publication no. WO 2013/159035, the contents of which including the methods for determining the level of cell free DNA are incorporated herein by reference.
  • the amount of fetal specific cell free DNA is determined with a method comprising quantitative genotyping.
  • the method comprising quantitative genotyping comprises cycling temperature capillary electrophoresis (CTCE).
  • CTCE cycling temperature capillary electrophoresis
  • the method comprising quantitative genotyping comprises real time quantitative PCR.
  • the method comprising quantitative genotyping comprises next generation sequencing.
  • determining the amount of fetal specific cell free DNA comprises extracting and quantifying cell free DNA from the biological sample, wherein the cell free DNA comprises nucleic acids comprising first nucleic acids of the pregnant subject and second nucleic acids of the fetus; analyzing the nucleic acids to identify a plurality of loci; determining an allele of each of the plurality of loci; selecting at least one locus from the plurality of loci based on the determining of the allele; calculating an estimated allele frequency of a minor allele based on modeling a number of counts of the minor allele at the at least one locus using a statistical distribution; and determining an amount of DNA of the fetus in the cell free DNA based on the estimated allele frequency.
  • the at least one locus of the first plurality of loci is selected by detecting a major allele and the minor allele at the at least one locus of the first plurality of loci; and determining that the first nucleic acids of the pregnant subject are homozygous for the major allele at the at least one locus and the second nucleic acids of the fetus are heterozygous for the minor allele at the at least one locus.
  • the at least one locus of the first plurality of loci is selected by detecting a major allele and the minor allele at the at least one locus of the first plurality of loci; and determining that the first nucleic acids of the pregnant subject are homozygous for the major allele at the at least one locus and the second nucleic acids of the fetus are homozygous for the minor allele at the at least one locus.
  • calculating the estimated allele frequency of the minor allele at the at least one locus comprises calculating the estimated allele frequency of the minor allele at the at least one locus using a maximum likelihood method.
  • the statistical distribution comprises a binomial distribution.
  • calculating an estimated allele frequency of the minor allele based on modeling the number of counts of the minor allele at the at least one locus using the statistical distribution comprises modeling a number of counts of a plurality of different allele combinations at the at least one locus using a binomial distribution.
  • the biological sample comprises blood, plasma, serum or urine from the pregnant subject.
  • a decrease in the determined amount of the fetal specific cell free DNA as compared to one or more baseline levels is indicative of the presence or absence of the condition in the fetus or progression of the condition in the fetus.
  • amounts compared are the percent or ratio of fetal specific cell free DNA compared to the total cell free DNA or non- fetal specific cell free DNA.
  • the method further comprises, based on the determined amount of the fetal specific cell free DNA, evaluating an effect of a treatment on the fetus by correlating an increased amount of the determined amount of the fetal specific cell free DNA as compared to one or more baseline levels as indicative of effectiveness of the treatment.
  • amounts compared are the percent or ratio of fetal specific cell free DNA compared to the total cell free DNA or non-fetal specific cell free DNA.
  • determining the amount of fetal specific cell free DNA comprises analyzing nucleic acids from cell free DNA extracted from a biological sample obtained from the pregnant subject to identify a plurality of loci, the nucleic acids comprising first nucleic acids of the pregnant subject and second nucleic acids of a fetus; determining an allele of each of the plurality of loci; selecting at least one informative locus from the plurality of loci based on the determining of the allele;
  • the method further comprises extracting the cell free DNA from the biological sample.
  • the first allele comprises a minor allele.
  • the at least one informative locus is selected by detecting the first allele and a second allele at the at least one informative locus; and determining that the first nucleic acids of the pregnant subject are homozygous for the second allele at the at least one informative locus and the second nucleic acids of the fetus are heterozygous for the first allele or homozygous for the first allele at the at least one locus.
  • the first allele comprises a minor allele and the second allele comprises a major allele.
  • the first allele comprises a minor allele; and the estimated allele frequency of the minor allele is calculated using a binomial distribution. In another embodiment of any one of the methods provided herein, the first allele comprises a minor allele; and the estimated allele frequency of the minor allele is calculated using an expectation-maximization algorithm.
  • the method or medium comprises determining an allele of each of a plurality of loci identified in nucleic acids from cell free DNA extracted from a biological sample, the nucleic acids comprising first nucleic acids of a pregnant subject and second nucleic acids of a fetus; selecting at least one informative locus from the plurality of loci based on the determining of the allele; calculating an estimated allele frequency of a first allele at the at least one informative locus using a statistical distribution; determining an amount of DNA of the fetus in the cell free DNA based on the estimated allele frequency; and determining a condition of the fetus based on the determined amount of the DNA of the fetus in the cell free DNA.
  • the determined amount is compared to a threshold, such as a baseline level.
  • the amounts compared are the percent or ratio of fetal specific cell free DNA compared to the total cell free DNA or non-fetal specific cell free DNA.
  • the plurality of loci is identified by analyzing the nucleic acids using high-throughput DNA sequencing or quantitative genotyping.
  • the first allele comprises a minor allele.
  • the at least one informative locus is selected by detecting the first allele and a second allele at the at least one informative locus; and determining that the first nucleic acids of the pregnant subject are homozygous for the second allele at the at least one informative locus and the second nucleic acids of the fetus are heterozygous for the first allele or homozygous for the first allele at the at least one locus.
  • the first allele comprises a minor allele and the second allele comprises a major allele.
  • the first allele comprises a minor allele; and the estimated allele frequency of the minor allele is calculated using a binomial distribution.
  • the first allele comprises a minor allele; and the estimated allele frequency of the minor allele is calculated using an expectation-maximization algorithm.
  • the method or medium further comprises, in a case where the amount of the cell free DNA of the fetus in the cell free DNA has negatively changed from baseline, determining that the probability that the fetus has a condition or has a condition that is progressing is increased.
  • the method or medium further comprises, based on the determined amount of the cell free DNA of the fetus, evaluating an effect of a treatment on the fetus or pregnant subject by correlating an increase in the amount of the determined amount of the cell free DNA of the fetus relative to baseline with a positive effect of the treatment.
  • the methods provided herein can be used to assess the risk of having cancer or a tumor, the recurrence of cancer or a tumor, or metastasis of a cancer or tumor.
  • the sample is obtained from a subject having or suspected of having the foregoing instead of a pregnant woman.
  • the risk is the presence or absence of the tumor or cancer or metastasis rather than a risk associated with a fetal condition.
  • the fetal specific cell free DNA is instead tumor or cancer specific cell free DNA.
  • the comparison is indicative of the risk of having the cancer or tumor or recurrence or metastasis thereof.
  • an increase in an amount of tumor or cancer specific cell free DNA or an increase above a threshold is indicative of the presence of the cancer, tumor, recurrence or metastasis or a progression of the cancer in the subject.
  • the first nucleic acids are of the subject.
  • the second nucleic acids are specific to the cancer or tumor.
  • a treatment is determined, suggested or provided (or information in regard to the treatment is provided) to the subject.
  • the treatment or therapy is an anti-cancer or anti-tumor therapy.
  • the subject has or is at risk of having any of the cancers or tumors provided herein or a recurrence or metastasis thereof.
  • one or more additional tests are performed.
  • the tests are for assessing the presence or absence of a cancer or tumor or a recurrence or metastasis thereof.
  • a computer-readable storage media wherein the medium stores computer-executable instructions for performing any one of the methods provided herein.
  • Figure 1 shows a still born fetus with hydrops fetalis.
  • Figure 2 provides data from the analysis of high risk pregnancies.
  • Figure 2A shows the percentage of circulating cell free fetal DNA (cff-DNA) in high risk pregnancies.
  • White triangles represent patients with fetal hydrops; gray square represents in utero fetal demise; white circles represent live fetus at time of sample with spontaneous abortion within three weeks of sample.
  • Figure 2B shows data from an individual with severe fetal hydrops and low calculated of cff-DNA.
  • Figure 3 provides data from an ovine model.
  • Figure 3A presents the total cf-DNA levels pre- and post-ductal occlusion in an ovine model.
  • Figure 3B presents the percent fetal cf-DNA levels pre- and post-ductal occlusion. All pre-ductal occlusion samples were drawn prior to surgery on the pregnant female. Post occlusion samples for Ewes 3 and 9 were collected 6 days after the ductal occlusion with the fetus still viable, but hypoxic. Post occlusion samples for Ewe 12 were collected one day after occlusion with the fetus having perished between the two time points.
  • Figure 4 provides an example computer system.
  • Fragmented cell free fetal DNA (cff- DNA) originating from the fetus and placental trophoblastic tissue circulates in maternal plasma at detectable levels from week 5 onward. 1 It has been surprisingly found that acute changes in fetal well being can result in detectably altered levels of circulating cff-DNA in maternal plasma, particularly as such levels relate to baseline levels. Specifically, it has been found that altered levels of circulating cell free fetal DNA, as compared to baseline levels, are associated with in utero fetal demise, spontaneous abortion, and hydrops fetalis.
  • fetal condition or “condition of a fetus” does not refer to the presence or absence of fetal aneuploidy, the gender of the fetus or the Rh blood type of the fetus, rather it refers to a health condition or assessment of well being that can change over time.
  • the condition of the fetus is the presence or absence or level of fetal distress (or fetal compromise).
  • the approach provided herein includes the detection and quantification of fragmented cell free DNA of fetal origin in maternal blood. It has been surprisingly found that the fraction (or percent or ratio) of cff-DNA in maternal blood varies as a function of fetal health and can be compared to baseline values to assess fetal health. Fetuses undergo cellular injury during distress from, for example, anatomic, metabolic, or issues of maternal environment that can result in fetal hydrops, a compromised newborn, prematurity or spontaneously terminated pregnancy. Fetal conditions include gastroschisis, fetal cardiac arrhythmias, congenital pulmonary adenomatoid malformations and twin-twin transfusion syndrome. The clinical conditions described herein are some examples where detection of fetal compromise remains a challenge with current methods; however, the techniques provided can have broader applicability to detect fetal compromise in other conditions, such as chorioamnionitis and preterm labor.
  • the methods and compositions provided herein can be used in a variety of situations to assess the condition of the fetus.
  • fetal well being can be assessed in a fetus that has been diagnosed with a congenital anomaly, such as congenital heart disease.
  • fetal well being can be assessed in pregnant women with fetal gastroschisis and fetal bradycardia syndrome.
  • the methods and compositions can allow for the recognition of fetal compromise for early intervention or delivery to prevent fetal loss.
  • the methods and compositions therefore, can assist in planning a woman's pregnancy and allow for medical intervention, appropriate timing of delivery, etc.
  • any one of the methods or compositions provided can be for use in any one of the conditions provided herein, such as the foregoing situations or for the foregoing subjects.
  • Gastroschisis a defect in the abdominal wall, in an otherwise normal fetus, resulting in the bowel protruding through the defect and floating in the amniotic fluid throughout gestation, is one prototypical condition in which the fetus is at risk for unpredictable intrauterine fetal death.
  • the survival of live-born fetuses with gastroschisis is 90-95%. After the babies with gastroschisis are born, they undergo an operation (or a series of operations) to reduce the intestines into the abdominal cavity and close the abdominal wall. These children do well and typically have normal lives with high quality of life. Unfortunately, there are subsets of patients with gastroschisis who do not survive to delivery and have the opportunity for operative repair.
  • Fetal cardiac arrhythmias are also associated with an increased risk of fetal hydrops and in utero demise.
  • the weight-adjusted cardiac output in the fetus is higher than during postnatal life to maintain tissue oxygenation despite lower oxygen tension in the fetus.
  • the fetal cardiac output is critically dependent on maintaining normal heart rate (120-160) and is adversely affected when the heart rate is lower ( ⁇ 100) or higher (>200).
  • Fetal bradycardia can occur from a variety of causes, most commonly from maternal collagen vascular diseases, such as Systemic Lupus (SLE). Maternal antibodies in SLE cross placenta and damage the developing conduction system in the fetal heart and lead to sustained heart block in the affected patients.
  • Fetal bradycardia is also associated with some congenital heart defects, such as 1-transposition of great arteries that affect the conduction system in the heart.
  • Sustained fetal bradycardia from heart block results in decreased cardiac output, tissue perfusion, fetal hydrops and increased risk of fetal demise.
  • Current management involves frequent monitoring of the pregnancies with ultrasound to assess cardiac function and evolution of hydrops.
  • Availability of a non-invasive test that can identify fetal compromise early from decreased cardiac function and tissue perfusion can provide a window of time to consider the risk-benefit equation of early delivery and postnatal intervention for these pregnancies.
  • CPAMs fetal mediastinal shift and non-immune hydrops fetalis.
  • CPAMs are a developmental abnormality of the lung that consist of large, cystic structures within the normal lung tissue. They have a variable natural history; they can be stable in size or cause rapid growth, mediastinal shift, and hydrops fetalis. Approximately 33% of fetuses with CPAMs develop hydrops and require intervention. CPAMs associated with hydrops have a near 100% mortality with no intervention.
  • the current approach to a fetus with a large, macrocystic CPAM resulting in hydrops is placement of an in utero thoracoamniotic shunt.
  • Twin-twin transfusion syndrome is also one of the most common indications for fetal intervention. This occurs in monochorionic, diamniotic pregnancies with imbalanced blood flow through placental anastomoses. The result is weight discordance and amniotic fluid discordance with the "donor" twin being small in size with dangerously low amounts of amniotic fluid and the "recipient" twin large in size with cardiac changes, hydrops, and polyhydramnios.
  • the methods provided can be used to monitor any pregnancy, where a fetus has or is suspected of having any one of the conditions provided herein. Accordingly, the methods provided can be used to monitor low- as well as high-risk pregnancies, including intra-uterine growth restriction and maternal vascular diseases. "High-risk pregnancy” is meant to refer to any pregnancy a clinician would deem at risk for one or more complications or conditions associated with such complication(s). For example, a pregnancy can be considered high-risk when there are potential complications that could affect the mother, the baby, or both.
  • pregnancy may be indicated as high-risk include pregnancies where health problems exist (e.g., Diabetes, Cancer, High blood pressure, Kidney disease (e.g., chronic pyelonephritis, chronic pyelonephritis and renal insufficiency), Epilepsy).
  • a pregnancy may also be deemed high risk if the mother uses alcohol or illegal drugs, or smokes; is younger than 17 or older than 35; has a history of multiple pregnancies, has a history of prior miscarriages; or where the fetus is found to have genetic conditions such as Down syndrome, or a heart, lung, or kidney problem.
  • High risk pregnancies also include those who had prior or have current problems in pregnancy (e.g., Preterm labor, Preeclampsia or seizures (eclampsia)); those who have an infection (e.g., HIV, hepatitis C, cytomegalovirus (CMV), chickenpox, rubella, toxoplasmosis, or syphilis); or those taking certain medications (e.g., lithium, phenytoin (such as Dilantin), valproic acid (Depakene), or carbamazepine (such as Tegretol)).
  • Pregnancies where the pregnant woman has certain health problems can also be considered high risk.
  • Other clinical indicators of high-risk pregnancies are known to those of ordinary skill in the art.
  • fetal distress can result in cellular injury
  • changes in circulating fetal specific cf- DNA can be used to identify fetal distress and/or monitor the condition of a fetus.
  • levels or amounts, such as percentages, fractions or ratios, of fetal specific cf-DNA can be determined using any of the methods provided herein, such as with targeted next generation sequencing technology (e.g., DANSR or Tandem SNP), or as otherwise known to those of ordinary skill in the art.
  • the amount and/or changes in the amount can be correlated with fetal distress and related conditions. Based on the work described herein it is believed that acute changes in fetal well being can result in dramatically decreases in the foregoing amounts of fetal specific cf-DNA. It is expected, for example, that a decrease in fetal metabolism secondary to hypoxia/ischemia leads to a decrease in maternal cff-DNA levels and precedes the appearance of traditional measures of fetal compromise.
  • fetal distress or compromise Provided herein are tools that can detect an adverse condition in the fetus, such as fetal distress or compromise, before the development of advanced signs of distress and, thus, can provide a window of time for appropriate intervention or early delivery providing a major advance in improving the health and outcome of children.
  • the fetal specific cf-DNA can be determined using any of the methods provided herein or that would be otherwise apparent to one of ordinary skill in the art.
  • the DNA may be analyzed using any suitable next generation sequencing technique, such as those provided herein. Any one of the methods may be employed over any period of time.
  • the described methods of assessing a risk may be implemented in any suitable manner. For example, the method may be implemented as described below in connection with the Examples and accompanying figures. It should also be appreciated that any one of the methods provided can include a step of correcting the results based on maternal weight and/or gestational age.
  • the methods provided herein can be used to identify and/or monitor fetal well being over several time points during gestation and/or progression of disease. Such monitoring can also occur after fetal intervention. Changes in cff-DNA can be used to correlate with clinical variables and current non-invasive measures of fetal status, including development of hydrops, resolution of hydrops after intervention, and fetal loss.
  • the amount of specific cf-DNA can be given as a ratio or percent or fraction of the total cf-DNA. Whatever the form of the amount, in preferred embodiments, the amount is compared to baseline levels.
  • a “baseline level” includes an amount of specific cf-DNA (such as the percent or ratio of fetal specific cf-DNA relative to the total cf-DNA or non-specific cf-DNA) from a sample from the subject taken at a time prior to a subsequent sample or at a time where the subject (e.g., the pregnant woman or fetus) was or was believed to be in good health, did not have or was not believed to have a condition provided herein, or did have or was believed to have a condition provided herein but the condition was or was believed to be at a stage that did not require treatment or intervention.
  • an increase or decrease relative to the baseline is the indicator of fetal well being.
  • changes in the difference relative to the baseline at two or more time points is the indicator of fetal well being.
  • the amount of fetal specific cf-DNA is determined at one or more time points, and the increase or decrease relative to one or more baselines values is determined.
  • the amount of fetal specific cf-DNA is determined at two or more time points, and the changes in the difference relative to one or more baseline values is determined.
  • the methods can further comprise a step of spiking in an internal standard at known quantities to aid in the quantitation of the specific cf-DNA.
  • a "risk” as provided herein refers to the presence or absence or progression of any undesirable condition (including a disease) in a subject, such as a fetus or pregnant subject where the condition is adverse to the fetus, or an increased likelihood of the presence or absence or progression of such a condition.
  • the fetus has or is at risk of having badycardia, twin-twin transfusion syndrome (TTTS), gastroschisis, congential pulmonary airway malformations (CPAMS), hydrops fetalis or fetal arrhythmia.
  • the pregnant subject is at risk.
  • the fetus has had or is at risk of having distress.
  • the pregnant subject is at risk for a condition including, for example, chorioamnionitis or preterm labor.
  • a condition including, for example, chorioamnionitis or preterm labor.
  • levels of fetal specific cf-DNA cff-DNA
  • the amounts of fetal specific cf-DNA from a cf-DNA sample obtained from a pregnant subject can provide a sensitive and noninvasive way of monitoring the well being of a fetus and allowing for medical intervention or early delivery, if needed.
  • any one of the methods provided can comprise extracting cf-DNA from a biological sample obtained from a pregnant subject.
  • biological sample is any sample that can be obtained from the subject from which cell free DNA can be extracted. Examples of such biological samples include whole blood, plasma, serum or urine.
  • the cf-DNA generally comprises DNA of the pregnant subject and DNA of the fetus, where a decreasing amount of the fetal DNA relative to the pregnant subject or total DNA can be indicative of a risk in the fetus and/or indicative of the presence or progression of an adverse condition in the fetus.
  • the condition of the fetus can be determined by assessing the level or amount of fetal specific cell free-DNA, for example, through the use of high-throughput sequencing, such as next generation sequencing (NGS), real time quantitative PCR, cycling temperature capillary electrophoresis (CTCE), or other type of quantitative genotyping.
  • high-throughput sequencing such as next generation sequencing (NGS), real time quantitative PCR, cycling temperature capillary electrophoresis (CTCE), or other type of quantitative genotyping.
  • the level of fetal specific cf-DNA can be measured at any point during pregnancy or at multiple time points throughout a pregnancy.
  • the "amount of cf-DNA" refers to any quantitative value for the measurement of the cf-DNA and can be given in an absolute or relative amount. Further, the amount can be a total amount, ratio, percentage, etc. As an example, correlating changes in absolute amounts or percentages of circulating fetal specific DNA can provide for sensitive and specific monitoring of a fetal condition, such as fetal well being.
  • the amount, such as the percent or ratio, of fetal specific cf-DNA can be indicative of the presence or absence of a risk associated the fetus or can be indicative of the need for further testing or surveillance.
  • the DNA may be analyzed to identify multiple loci, an allele of each of the loci may be determined and informative loci may be selected based on the determined alleles.
  • informative loci refers to a loci where the native genotype (e.g., pregnant subject genotype) is homozygous for the major allele, while the non-native genotype (e.g, fetus genotype) is homozygous or heterozygous for the minor allele.
  • the informative loci can be determined based on prior genotyping and any one of the methods provided herein can include such a step, such as a step of genotyping the pregnant subject and/or fetus or obtaining or being provided with such genotypes.
  • An estimated allele frequency, such as the estimated minor allele frequency, at the informative loci may then be calculated in a suitable manner.
  • the estimated allele frequency of may be calculated based on modeling the number of counts of the allele, such as the minor allele, at the informative loci using a statistical distribution.
  • the estimated allele frequency can be calculated by modeling allele read counts using a binomial distribution.
  • the peak of such a distribution is determined and is indicative of the percent fetal specific cf-DNA.
  • a frequency of the minor allele (MAF) at the informative loci may also be calculated using a maximum likelihood method.
  • the MAF may be calculated with genotypes from pregnant subject plasma DNA, and fetal genotypes for informative loci may be inferred using expectation maximization.
  • the determined amount of the fetal specific cf-DNA, such as the percent or ratio of fetal specific cf-DNA, in the sample from the pregnant subject may then be used to determine a risk associated with the fetus.
  • a threshold such as a baseline level
  • changes in such values can be monitored over time.
  • a change in the difference from a threshold value (such as a baseline) can be used as a non-invasive clinical indicator. This ratio can allow for the measurement of variations in a clinical state and/or permit calculation of normal values or baseline levels.
  • An increase or decrease above a threshold (e.g., baseline) in the determined amount, or changes in the increase or decrease over time, can indicate an increased or decreased risk in the fetus.
  • “Threshold” refers to any predetermined level that is indicative of the presence or absence of a condition or the presence or absence of a risk.
  • the threshold value can take a variety of forms. It can be single cut-off value, such as a median or mean. In some embodiments of any one of the methods provided herein, the threshold is any of the medians or means provided herein, such as in the Examples, or that are otherwise known in the art. It can be established based upon comparative groups, such as where the risk in one defined group is double the risk in another defined group.
  • It can be a range, for example, where the tested population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quadrants, the lowest quadrant being subjects with the lowest risk and the highest quadrant being subjects with the highest risk.
  • the threshold value can depend upon the particular population selected. For example, an apparently healthy population will have a different 'normal' range.
  • a threshold value can be determined from baseline values before the presence of a condition or risk or after a course of treatment.
  • a threshold values can be a value taken at a prior time point. Such a value can be indicative of a normal or other state in the subject, such as a state not correlated with the risk or condition that is being tested for.
  • the predetermined values selected may take into account the category in which the subject falls. Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.
  • the threshold value can be a baseline value of the subject being tested. In embodiments of any one of the methods provided herein, it is preferred that the comparison is relative to one or more baseline values or changes thereto.
  • Such baseline values can represent an amount of fetal specific cf-DNA, such as the percent or ratio thereof relative to total cf-DNA or non- fetal specific cf-DNA, when the fetus was in good health, prior to the onset of a condition in the fetus or prior to a condition progressing to a deleterious point.
  • Any one of the methods provided can further comprise performing another test on the subject, optionally based on the result of a method provided herein or comparison of a result thereof.
  • Such other tests can be any other test known by one of ordinary skill in the art to be useful in determining the presence or absence of a risk, such as to a fetus.
  • Such tests include those that determine other clinical measures of fetal well being, including ultrasound biometrical parameters, fetal heart rate, arterial pressure, amniotic fluid levels, bowel dilation, development of hydrops, resolution of hydrops after intervention and fetal loss.
  • the pH, Pa02, PaCo2, and/or blood lactate levels are assessed in the fetus and/or the pregnant subject.
  • Another test in some embodiments, can be performing another test with a method provided herein at one or more additional time points.
  • the methods provided and/or the additional test(s) can be performed at any of a number of time points during pregnancy, for example, time points can include during gestation, during progression of a condition, such as a disease, at fetal intervention, and upon resolution of hydrops after intervention.
  • the amount of fetal specific cf-DNA such as the percent or ratio of fetal specific cd-DNA, may also be determined at any other time during the pregnancy, and may be utilized for short- or long-term surveillance. The determination may be performed instead of or in addition to other tests currently used to assess the condition of a fetus.
  • the ability to detect early risk to a fetus such as with a non-invasive method, can offer early intervention and better patient outcomes.
  • any one of the methods provided can include the step of providing a therapy, or providing information regarding therapies, to the pregnant subject based on the determination of an amount relative to a threshold, such as a baseline, or change in the amount relative to a threshold, such as a baseline.
  • the information includes written materials containing the information. Written materials can include the written information in electronic form.
  • the method can further comprise recording the administration of a therapy, the providing of information for a therapy or the suggesting of a therapy to the subject.
  • the approaches provided herein can aid in timing intervention and planning delivery of fetuses at risk for loss or development of hydrops and can also aid in monitoring for supportive care to minimize the risk of preterm labor.
  • the ultimate benefit can be a reduction of fetal loss and reduction in infant mortality.
  • the method can further include a step of treating or providing information regarding a treatment to a pregnant woman. Any one of the methods provided herein, therefore, can further include a step of performing or recommending fetal intervention or delivery of the baby before an intrauterine death occurs for the pregnant woman.
  • the recommending comprises providing information regarding a suggested treatment, such as fetal intervention and/or delivery options to the pregnant woman.
  • the amount of fetal specific cf-DNA in the sample from the pregnant subject may be used to evaluate an effect of a therapy (e.g., positive or negative) on the fetus by correlating (or comparing) a difference or change in the difference in the amount of fetal specific cf-DNA, such as percent or ratio of fetal specific cf-DNA, relative to one or more baseline values.
  • a therapy e.g., positive or negative
  • a suitable therapy may be selected based on the correlation or comparison and/or the amount of the therapy
  • any one of the methods provided herein can include the step of providing a therapy (or treatment) or providing information regarding a therapy (or treatment), to the pregnant subject based on any one or more of the comparisons described herein.
  • any one of the methods can be used to assess the efficacy of a therapy (or treatment) where improved values can indicate less of a need for the therapy, while worsening values can indicate the need for a therapy, a different therapy, or an increased amount of a therapy.
  • any one of the methods provided herein can include the step of evaluating the need or dose of a therapy based on the result of one or more comparisons at one or more time points.
  • the therapy or intervention involves an in utero intervention ⁇ e.g. surgical procedure, administration of a drug) or early delivery.
  • aspects of the invention relate to comparing the amount of fetal specific cf-DNA in a sample of a pregnant subject relative to one or more baseline values and, optionally, treating or providing information in regard to a treatment.
  • the information is provided in written form.
  • the information may be provided as computer-readable instructions.
  • Fig. 4 is an exemplary computer system on which some embodiments of the invention may be employed.
  • the computer system 500 may include one or more processors 510 and one or more computer-readable non-transitory storage media (e.g., memory 520 and one or more nonvolatile storage media 530).
  • the processor 510 may control writing data to and reading data from the memory 520 and the non-volatile storage device 530 in any suitable manner, as the aspects of the present invention described herein are not limited in this respect.
  • the processor 510 may execute one or more computer-executable instructions stored in one or more computer-readable storage media (e.g., the memory 520), which may serve as non-transitory computer-readable storage media storing instructions for execution by the processor 510.
  • one or more computer-readable storage media e.g., the memory 520
  • the processor 510 may execute one or more computer-executable instructions stored in one or more computer-readable storage media (e.g., the memory 520), which may serve as non-transitory computer-readable storage media storing instructions for execution by the processor 510.
  • the above-described embodiments of the present invention can be implemented in any of numerous ways. For example, some aspects of the embodiments may be implemented using hardware, software or a combination thereof. When implemented in software, the software code can be executed on any suitable processor or collection of processors, whether provided in a single computer or distributed among multiple computers. It should be appreciated that any component or collection of components that perform the functions described above can be generically considered as one or more controllers that control the above-discussed functions. The one or more controllers can be implemented in numerous ways, such as with dedicated hardware, or with general-purpose hardware (e.g., one or more processors) that is programmed using microcode or software to perform the functions recited above.
  • one implementation of the embodiments of the present invention comprises at least one non-transitory computer-readable storage medium (e.g., a computer memory, a floppy disk, a compact disk, a tape, etc.) encoded with a computer program (i.e., a plurality of instructions), which, when executed on a processor, performs the above-discussed functions of the embodiments of the present invention.
  • the computer-readable storage medium can be transportable such that the program stored thereon can be loaded onto any computer resource to implement the aspects of the present invention discussed herein.
  • the reference to a computer program which, when executed, performs the above-discussed functions is not limited to an application program running on a host computer. Rather, the term computer program is used herein in a generic sense to reference any type of computer code (e.g., software or microcode) that can be employed to program a processor to implement the above-discussed aspects of the present invention.
  • cancers include, but are not limited to, prostate cancer, bladder cancer, pancreatic cancer, lung cancer, kidney cancer, breast cancer, or colon cancer.
  • the therapies that are provided or for which information is provided can be therapies for treating cancer, a tumor or metastasis.
  • Such therapies include, but are not limited to, antitumor agents, such as docetaxel; corticosteroids, such as prednisone or hydrocortisone; immuno stimulatory agents; immunomodulators; or some combination thereof.
  • Antitumor agents include cytotoxic agents, chemotherapeutic agents and agents that act on tumor neo vasculature. Cytotoxic agents include cytotoxic radionuclides, chemical toxins and protein toxins. The cytotoxic radionuclide or radiotherapeutic isotope can be an alpha-emitting or beta-emitting. Cytotoxic radionuclides can also emit Auger and low energy electrons.
  • Suitable chemical toxins or chemo therapeutic agents include members of the enediyne family of molecules, such as calicheamicin and esperamicin. Chemical toxins can also be taken from the group consisting of methotrexate, doxorubicin, melphalan,
  • chlorambucil ARA-C, vindesine, mitomycin C, cis-platinum, etoposide, bleomycin and 5- fluorouracil.
  • Other antineoplastic agents include dolastatins (U.S. Patent Nos. 6,034,065 and 6,239,104) and derivatives thereof.
  • Toxins also include poisonous lectins, plant toxins such as ricin, abrin, modeccin, botulina and diphtheria toxins.
  • Other chemo therapeutic agents are known to those skilled in the art.
  • embodiments of the invention may be implemented as one or more methods, of which an example has been provided.
  • the acts performed as part of the method(s) may be ordered in any suitable way. Accordingly, embodiments may be constructed in which acts are performed in an order different from illustrated, which may include performing some acts simultaneously, even though shown as sequential acts in illustrative embodiments.
  • DNA extracted from maternal plasma from pregnant women were analyzed for percent fetal DNA in a blinded fashion for 93 high risk pregnancies in Figs. 2A and 2B.
  • Percent fetal DNA was calculated from the quantitative genotyping of 192 probes using the DANSR assay 3 ' 4 .
  • Data are depicted as a standard boxplot with median denoted by the horizontal black line and the inter-quartile range representing the 25th to the 75th percentile denoted by the grey box.
  • Median Percent Fetal DNA of this high risk cohort is 10%; the median and interquartile range of this cohort was consistent with previously published data from a separate prospective cohort study of over 4000 pregnant women at gestational age 10 weeks or greater 15 .
  • White triangles represent patients with fetal hydrops
  • gray square represents in utero fetal demise (absent heart beat)
  • white circles represent live fetus at time of sample with spontaneous abortion within three weeks of sample.
  • the inset is the data analysis of an individual with severe fetal hydrops and a low calculation of fetal DNA (4.3%).
  • the black curve and points in the inset figure depict genotypes that were
  • fetal distress is recorded at each time point including fetal heart rate, amniotic fluid levels, and ultrasound parameters including the development of fetal hydrops in bradycardic fetuses and the degree of bowel dilation in fetuses with gastroschisis.
  • the levels of fetal specific cf-DNA for fetuses that developed an intrauterine fetal death are examined and for those who survived to delivery in patients with gastroschisis. Changes in fetal specific cf-DNA levels for bradycardic fetuses that become hydropic compared for those bradycardic fetuses who do not develop hydrops are also examined.
  • Median fetal specific cf-DNA are expected to be 10%, which are expected to increase after 22 weeks in fetuses who do not develop gastroschisis or hydrops. It is expected that in fetuses who do develop fetal compromise with gastroschisis and bradycardic fetuses that become hydropic, the percent fetal DNA will be significantly lower. Babies with fetal anomalies, such as gastroschisis and fetal bradycardia can have distinct pattern of changes in fetal specific cf-DNA with gestation compared to normal fetuses. These changes can be assessed from baseline measurements obtained at, for example, weekly intervals. It is expected that fetal compromise results in changes that are beyond those caused by baseline variation.
  • a primary endpoint is fetal delivery.
  • the change from diagnosis measurement using a one sample t-test can be compared. With 25(15) mothers, at an alpha of 0.05 there is 80% power to detect a change of 0.6 SD.
  • a secondary endpoint is 28 weeks which can be similarly analyzed. Random regression models with spline fits can be used if necessary to characterize the change in values and include covariates such as baseline measurement and clinical status (time dependent where appropriate).
  • Baseline fetal specific cf-DNA levels for all fetuses with the diagnosis of CPAM or TTTS who are at risk for developing hydrops fetalis or in utero demise are also obtained. These levels can be followed serially to assess development of hydrops or demise preceded by measurable changes in fetal specific cf-DNA levels. Levels are also determined to assess the return to baseline as the fetal hydrops resolves with intervention in babies with CPAM and with improved cardiovascular function in babies with TTTS that undergo intervention.
  • Baseline and serial levels of fetal specific cf-DNA in patients undergoing fetal intervention for CPAM or TTTS are obtained.
  • CPAMs congenital pulmonary adenomatoid malformations
  • 12 patients are also expected to undergo in utero therapy for twin-twin transfusion syndrome (TTTS) each year.
  • Baseline fetal specific cf-DNA levels are obtained at the time of diagnosis for patients with CPAMs.
  • Samples are obtained every two weeks until hydrops develops. Hydropic fetuses with CPAMs are candidates for fetal intervention with a thoraco amniotic shunt. Samples are obtained immediately pre-intervention and post intervention day 1, 3, and 7. Objective ultrasound findings are recorded during these time points and degree of mediastinal shift and hydrops fetalis are recorded. Additionally, levels of fetal specific cf-DNA in pregnancies complicated by TTTS are obtained. Similarly, samples are obtained every two weeks until Stage II TTTS has been reached, at which time fetal intervention is offered. Samples are obtained immediately pre-intervention and post intervention day 1, 3, and 7.
  • fetal arterial catheter in the left carotid artery and an inflatable silicone vascular occluder around the fetal umbilical cord (Size 14-16, In Vivo Metric, Healdsburg, CA) is tethered to the fetal abdominal wall and exteriorized to the mother's back. 11 The fetus is then returned to the uterine cavity, incisions closed and the fetus and ewe allowed to recover for 5 days.
  • Mild to moderate fetal distress At around 130 days gestation (90% of term), baseline samples are obtained to measure the fetal blood pH, PaO 2 , PaCO 2 and blood lactate levels. Maternal blood samples from a venous catheter are also obtained to measure the fetal specific cf-DNA in maternal circulation. Fetal arterial pressure and heart rate are measured from the fetal arterial catheter continuously. Then the inflatable occluder around the umbilical cord is inflated with saline to produce partial compression of the umbilical cord. A 20-point drop in the fetal HR and a 0.15 decrease in pH is expected, indicating mild-moderate distress. 11 The partial cord occlusion is maintained for a period of 30 minutes.
  • Fetal arterial blood samples are obtained every 10 minutes for measurement of fetal pH, PaO 2 , PaCO 2 and blood lactate levels. Maternal blood samples are drawn every 10 minutes for 30 minutes. Following 30 minutes, the occluder is deflated and the fetus allowed to recover from distress. The hemodynamic variables, blood gases and lactate levels are measured every 10 minutes during the recovery. Maternal samples and fetal samples are obtained at 24 and 48 h after cord occlusion to determine the time course of changes in fetal specific cf-DNA. Blood is drawn in 10 ml BCT tubes (Streck, Omaha, NE..USA), and total and fetal specific cf-DNA concentrations are measured by multiplexed quantitative real-time PCR as previously described 16 .
  • Severe fetal distress After a 2-day recovery, the ewe will be brought back to the study area and baseline fetal blood gases and maternal sample for fetal specific cf-DNA obtained. A more complete occlusion of the umbilical cord is then performed by inflating the balloon occluder with a larger volume of saline and target fetal bradycardia (HR ⁇ 100) and more significant acidosis (pH ⁇ 7.00) for another 30 min epoch. Fetal HR and arterial pressure are continuously measured. The fetal arterial blood gases and lactate levels and fetal specific cf- DNA levels in maternal blood are measured every 10 min during the occlusion and during 30 minute recovery after the release of occlusion. Then maternal blood sample and fetal samples are obtained 24 and 48 h after relieving the cord occlusion to determine the time course of recovery and changes in fetal specific cf-DNA during recovery.
  • Recurrent fetal distress After another 2 day recovery period from severe distress, a new baseline is established and a study conducted to induce 3 consecutive epochs of mild- moderate distress lasting 30 minutes, followed by 30 minute recovery. Fetal samples and fetal specific cf-DNA are obtained at the end of each 30 min epoch of cord occlusion and recovery from cord occlusion. A cumulative increase in fetal acidosis and more significant decreases in fetal HR are expected with the recurrent occlusions that will simulate ongoing fetal compromise from recurrent cord occlusions, which can lead to severe fetal compromise.
  • An assay targeting 18s can also be used to determine total cf-DNA and a new assay targeted to sheep SRY (accession number Z30265) designed to determine fetal specific cf-DNA.
  • sheep SRY accession number Z30265
  • studies are initially done on male gender fetus for rapid determination of fetal specific cf-DNA, facilitated by the ability to distinguish the Y chromosome, both male and female fetal lambs can be examined to assess biological differences with respect to tolerance of distress. While in the model fetal distress is caused by vascular compromise, with decreased fetal cardiac output. In clinical situations, the occurrence of fetal distress can have a variety of etiologies, such as, fetal infection and anemia.
  • fetal anomalies in pregnancies complicated by anomalies largely have decreased fetal cardiac output as the basis for distress.
  • fetal compromise can occur over time or by superimposition of a mild stressful event (e.g., anemia) on a fetus already compromised at baseline.
  • a model of acute distress can be modified to cause chronic stress by placental and umbilical cord embolization in utero between 120 and 140 days gestation and the fetal specific cf-DNA levels monitored. 14
  • the 24 hour and 48 hour fetal % to baseline can be compared using a paired t-test.
  • SD standard deviations
  • In utero model of fetal distress The relationship between time of onset, severity of fetal compromise, and change in fetal specific cf-DNA in maternal plasma are confirmed using a fetal lamb model of in utero fetal distress, which can assess the effect of discrete or sustained episodes of fetal distress on fetal specific cf-DNA and on fetal blood gases, lactate/pyruvate ratio and arterio-venous 0 2 content difference as a measure of fetal metabolism.
  • a vascular occluder around the umbilical cord can be later inflated to create a graded degree of fetal compromise 9 ' 10 .
  • Fetal distress can be maintained for a specific length of time and can be done as discrete or repetitive episodes.
  • Maternal samples at different time points are obtained to allow for the study of temporal changes in fetal specific cf-DNA with a timed episode of fetal compromise. Changes in fetal metabolism are also measured using information obtained from blood gases, arterio-venous 0 2 content difference to estimate 0 2 extraction and lactate/pyruvate ratio. These studies allow for the correlation of the change in fetal specific cf-DNA from baseline with the metabolic changes induced by fetal hypoxia and acidosis. The model has been widely used in the past for the investigation of fetal adaptation to hypoxia and acidosis 9 ' 10 .
  • Fetal compromise in fetal anomalies Advances in ultrasound technology have increased the recognition of fetal anomalies. Fetal compromise can occur in these anomalies for a variety of reasons and is potentially preventable when recognized early.
  • the anomalies that can be followed through pregnancy include gastroschisis, fetal bradycardia, TTTS, fetal arrhythmia, and CPAMS.
  • gastroschisis fetal bradycardia
  • TTTS fetal arrhythmia
  • CPAMS CPAMS.
  • Fetal cardiac arrhythmias are also associated with an increased risk of fetal hydrops and in utero demise.
  • Availability of a non-invasive test to identify fetal compromise early, before the onset of hydrops from low cardiac output can provide a window of time to consider the risk- benefit equation of early delivery and postnatal intervention and allow for following the course of these babies in utero.
  • the techniques can also aid in timing the intervention and planning the delivery of fetuses at risk for loss or development of hydrops.
  • the clinical conditions described herein are some examples where detection of fetal compromise remains a challenge and the techniques provided can be used. Additionally, the techniques provided are also broadly applicable to detecting fetal compromise in other conditions, such as chorioamnionitis and preterm labor.
  • total cf-DNA content in each sample was evaluated in triplicate by TaqMan real-time PCR using an assay targeting 18S (Hs99999901_sl, Applied Biosystems, Foster City, CA). These data were obtained from a fetal lamb model of prenatal ductus arteriosus occlusion, which leads to fetal hypoxia and sometimes, fetal death. R esults are presented in ng/ml plasma and show that total cell free-DNA increases post ductus arterious occlusion of the fetus.
  • Total cf-DNA and fetal specific cf-DNA were determined using Cycling Temperature Capillary Electrophoresis (CTCE), a sensitive, quantitative, and cost-effective DNA separation technology, as previously described (Fig. 3A and 3B) 2 .
  • CTCE Cycling Temperature Capillary Electrophoresis
  • a sensitive, quantitative, and cost-effective DNA separation technology as previously described (Fig. 3A and 3B) 2 .
  • the data indicate that fetal specific cf-DNA decreases post ductal occlusion despite an increase in total cf-DNA, suggesting that a fetal percentage change from baseline is associated with fetal stress, and that a dramatic decrease is observed with fetal demise (Fig. 3B, Ewe 12).
  • a decrease in fetal metabolism induced by fetal hypoxia/ischemia can lead to a decrease in fetal specific cf-DNA in maternal plasma.
  • Recent studies on fetal specific cf- DNA have focused on the measurement of fetal specific cf-DNA in maternal blood for the diagnosis of fetal aneuploidy, gender or Rh blood type.
  • quantification of fetal specific cf-DNA in maternal circulation allows for the monitoring of the rate of fetal metabolism and by inference, fetal health.
  • a fetal lamb model allows one to precisely time and vary the severity of fetal distress as the time course of fetal specific cf-DNA changes in maternal blood is measured.
  • the fetal arterio-venous 0 2 difference, a measure of 0 2 extraction and lactate/pyruvate ratio as metabolic parameters can be measured. Increases in 0 2 extraction and lactate relative to pyruvate levels suggest an effect of induced stress on fetal metabolism.
  • a sterile instrumentation on the ewe is performed for the insertion of fetal arterial and venous catheters in the aorta and superior vena cava via the left carotid artery jugular vein and an inflatable silicone vascular occluder around the fetal umbilical cord (Size 14-16, In Vivo Metric, Healdsburg, CA).
  • the catheters are then tethered as well as the occluder to fetal chest and abdominal wall and exteriorized to the mother's back 16 .
  • the fetus is then returned to the uterine cavity, incisions closed and the fetus and ewe allowed to recover for 5 days. This length of time allows the fetal specific cf- DNA released during instrumentation to clear from maternal circulation.
  • Moderate fetal distress At 130 days gestation (90% of term), baseline samples are obtained to measure fetal blood pH, PaO 2 , PaCO 2 , blood lactate/pyruvate ratio and arterial and venous O 2 content from aorta and SVC samples. Maternal blood samples are also obtained from a venous catheter to measure the total cf-DNA and fetal specific cf-DNA in maternal plasma. Fetal arterial pressure and heart rate are measured from the arterial catheter continuously. Then the inflatable occluder around the umbilical cord is inflated with saline to produce partial compression of the umbilical cord. A 20- point drop in the fetal HR and a 0.15 decrease in pH is expected, indicating moderate distress 16 .
  • the partial cord occlusion is maintained for a period of 30 minutes.
  • Fetal arterial blood samples are obtained every 10 minutes for measurement of fetal pH, Pa0 2 , PaC0 2 , blood lactate/pyruvate ratio and AV 0 2 content difference.
  • Maternal blood samples are drawn every 10 minutes for 30 minutes. Following 30 minutes, the occluder is deflated and the fetus allowed to recover from distress.
  • the hemodynamic variables, blood gases, AV 0 2 difference and lactate/pyruvate ratio are measured every 10 min during a 30 min recovery period.
  • Maternal samples and fetal samples are obtained at 8, 24 and 48 h after cord occlusion to determine the time course of changes in fetal specific cf-DNA.
  • Maternal blood is drawn in 10 ml BCT tubes (Streck, Omaha, NE,USA) and total and fetal specific cf-DNA concentrations are measured by multiplexed quantitative real-time PCR 12 and/or using CTCE 2 as elsewhere described.
  • Severe fetal distress After a 2-day recovery, the ewe is brought back to the study area to obtain baseline fetal blood gases, lactate/pyruvate ratio, AV 0 2 contents and maternal sample for fetal specific cf-DNA. A more complete occlusion is then performed of the umbilical cord by inflating the balloon occluder with a larger volume of saline and target fetal bradycardia (HR ⁇ 60) and more significant acidosis (pH ⁇ 7.00) for 15 min.
  • HR ⁇ 60 target fetal bradycardia
  • pH ⁇ 7.00 more significant acidosis
  • the fetal arterial blood gases, lactate/pyruvate ratio, AV 0 2 content difference and fetal specific cf-DNA in maternal blood are measured every 10 min during the occlusion and during 30- minute recovery after the release of occlusion. Then maternal blood sample and fetal samples are obtained 8, 24 and 48 h after relieving the cord occlusion to determine the time course of recovery and changes in fetal specific cf-DNA during recovery.
  • Sustained fetal distress After another 2-day recovery period from severe distress, a new baseline is established and a study conducted to induce partial compression of the umbilical cord to achieve moderate distress, which is sustained for 12 hours. Fetal blood gases, lactate/pyruvate ratio and AV 0 2 difference are then measured every 30 min during the first hour and then every 2 hours for the 12 hours of occlusion. The maternal samples are obtained at the same time points for fetal specific cf-DNA. After release of occlusion, maternal samples and fetal samples are obtained at 2, 8, 12 and 24 hours to determine the time course for changes in fetal specific cf-DNA in relation to fetal metabolic state.
  • the fetal distress is related to vascular compromise with decreased fetal cardiac output.
  • the fetal anomalies of a number of conditions largely have decreased fetal cardiac output as the basis for distress. Nevertheless, clinically, the occurrence of fetal distress may have a variety of etiologies, such as, fetal infection or anemia.
  • fetal compromise can occur over time or by superimposition of a different stressful event on a fetus already compromised at baseline (e.g., anemia). It is possible to use the techniques provided to evaluate such situations.
  • the studies provided can be modified to one of chronic stress by placental and umbilical cord embolization in utero between 120 and 140 days gestation while monitoring the fetal specific cf-DNA levels 19 .
  • Time-dependent changes in fetal specific cf-DNA with occlusions to baseline are compared by ANOVA.
  • alpha of 0.025 adjusting for two outcomes
  • Changes over time can be examined using a mixed model.
  • the development of fetal hydrops or fetal demise can be correlated with changes from baseline levels of circulating fetal specific cf-DNA. Additional changes in response to fetal intervention for hydrops in CP AM and for TTTS in pregnancies with these anomalies are assessed.
  • the relationship between circulating fetal specific cf-DNA and gestational age, development of fetal compromise or in utero demise are evaluated with the methods provided by following its time course in samples from mothers with fetal gastroschisis or with fetal hydrops secondary to fetal cardiac arrhythmia, large CPAMs or TTTS. Samples are taken at the time of diagnosis and at scheduled intervals until delivery. Standard clinical measures of fetal distress, such as, fetal heart rate, biophysical profile, bowel dilation and development of hydrops, need for fetal intervention and response to intervention by ultrasound are recorded at each time point.
  • Gastroschisis and fetal arrhythmia are examples of anomalies that can be identified with the techniques provided.
  • the techniques can also be used to determine fetal specific cf- DNA to assess fetal health after an intervention for CPAM or TTTS.
  • Maternal blood samples are taken at diagnosis and every 4 weeks until 28 weeks gestation in gastroschisis, at the same time that mothers are seen for ultrasound surveillance. A similar schedule are followed in babies with arrhythmias from the time of recognition to 28 weeks gestation.
  • Maternal samples are then obtained weekly beginning at 28 weeks and continue to delivery or fetal demise.
  • Objective clinical measurements of fetal distress are recorded at each time point, including fetal heart rate, amniotic fluid levels, and ultrasound parameters including the development of fetal hydrops in subjects with fetal arrhythmia and the degree of bowel dilation in fetuses with
  • the levels of fetal specific cf-DNA are examined in fetuses that developed an intrauterine fetal death and those who survived to delivery in patients with gastroschisis. Changes in fetal specific cf-DNA levels are examined in arrhythmia fetuses that become hydropic compared to those who do not develop hydrops.
  • cjf-DNA All samples are collected in BCT tubes optimized for quantification of cf-DNA in plasma 12. Plasma and cell free DNA are coded, deidentified and prepared. Real time quantitative PCR (RTQPCR) are performed to calculate levels of total cell free DNA (Hidestrand et al). Next generation targeted sequencing are performed to determine human fetal fraction and counts 3 ' 4,12 . Raw count data undergo QC and analysis in a blinded manner. Specifically, cf-DNA are quantified by performing quantitative genotyping at many (192) distinct genetic loci and counting the major and minor alleles seen. The alleles counted fall into frequency categories corresponding to the two individuals' joint genotype at each probed location. The probes which are determined to be a combination of multiple distinct genotypes are modeled with maximum likelihood methods to a binomial distribution, which determines the relative ratios of each individual's DNA within the sample plasma. Results undergo systematic unblinding and clinical comparison.
  • the mean Percent Fetal DNA is expected to be 10%, which will increase after 22 weeks in fetuses who do not develop gastroschisis or hydrops. In fetuses that develop fetal compromise with gastroschisis and arrhythmia fetuses that become hydropic, it is expected that the percent fetal DNA will be significantly lower. In pregnancies with CP AM and TTTS, it is expected that fetal specific cf-DNA levels will revert to baseline as the babies improve after intervention. Studies can also provide normative data.
  • Fetal cf-DNA% is expected to be different in fetuses with or without distress.
  • the measurements at diagnosis and 28-week measurements between 2 groups are compared using ANOVA.
  • a 7% absolute difference in the mean fetal specific cf-DNA levels (mean of 10% with a range 7-14% in no fetal distress group and mean of 3% with a range 1-5% in fetal distress group from pilot data, Fig. 2A) at an alpha of 0.05 and 90% power can be detected.
  • Random regression models with spline fits can be performed if necessary to characterize the changes.

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Abstract

Cette invention concerne des compositions et des méthodes d'évaluation du bien-être fœtal (FWB). Selon un aspect, l'invention porte sur une méthode d'évaluation de l'état d'un fœtus chez un sujet gravide, comprenant des étapes quelconques parmi celles décrites, et une méthode quelconque parmi celles décrites ou illustrées dans la présente demande, y compris dans les exemples et les figures. Selon un autre aspect, l'invention porte sur une méthode de surveillance pendant une période donnée de l'état d'un fœtus chez un sujet gravide, comprenant des étapes quelconques parmi celles décrites et une méthode quelconque parmi celles décrites ou illustrées dans la présente demande, y compris dans les exemples et figures.
PCT/US2014/028205 2013-03-15 2014-03-14 Surveillance du bien-être fœtal au moyen de l'adn fœtal libre spécifique Ceased WO2014143989A1 (fr)

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US11939634B2 (en) 2010-05-18 2024-03-26 Natera, Inc. Methods for simultaneous amplification of target loci
US12100478B2 (en) 2012-08-17 2024-09-24 Natera, Inc. Method for non-invasive prenatal testing using parental mosaicism data
US12305229B2 (en) 2014-04-21 2025-05-20 Natera, Inc. Methods for simultaneous amplification of target loci
US12203142B2 (en) 2014-04-21 2025-01-21 Natera, Inc. Detecting mutations and ploidy in chromosomal segments
US12260934B2 (en) 2014-06-05 2025-03-25 Natera, Inc. Systems and methods for detection of aneuploidy
US11946101B2 (en) 2015-05-11 2024-04-02 Natera, Inc. Methods and compositions for determining ploidy
US12146195B2 (en) 2016-04-15 2024-11-19 Natera, Inc. Methods for lung cancer detection
EP3449018A4 (fr) * 2016-04-29 2019-11-06 The Medical College of Wisconsin, Inc. Pcr en temps réel d'amplification de mésappariements optimisée multiplexée (moma) pour l'évaluation du bien-être foetal
EP3449019B1 (fr) * 2016-04-29 2023-08-23 The Medical College of Wisconsin, Inc. Nombre de cibles par amplification de mésappariement (moma) optimisée multiplexée (moa)
JP2019514387A (ja) * 2016-04-29 2019-06-06 ザ メディカル カレッジ オブ ウィスコンシン インクＴｈｅ Ｍｅｄｉｃａｌ Ｃｏｌｌｅｇｅ Ｏｆ Ｗｉｓｃｏｎｓｉｎ， Ｉｎｃ． 胎児健康を評価するための多重／最適化ミスマッチ増幅（ｍｏｍａ）−リアルタイムｐｃｒ
EP4306653A3 (fr) * 2016-04-29 2024-04-10 The Medical College of Wisconsin, Inc. Nombre cible d'amplification de désadaptation optimisée multiplexée (moma)
CN109661476A (zh) * 2016-04-29 2019-04-19 威斯康星州立大学医学院 用于评估胎儿健康的多重优化错配扩增(moma)实时pcr
US12460264B2 (en) 2016-11-02 2025-11-04 Natera, Inc. Method of detecting tumour recurrence
US11773434B2 (en) 2017-06-20 2023-10-03 The Medical College Of Wisconsin, Inc. Assessing transplant complication risk with total cell-free DNA
US12084720B2 (en) 2017-12-14 2024-09-10 Natera, Inc. Assessing graft suitability for transplantation
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US12024738B2 (en) 2018-04-14 2024-07-02 Natera, Inc. Methods for cancer detection and monitoring
US12234509B2 (en) 2018-07-03 2025-02-25 Natera, Inc. Methods for detection of donor-derived cell-free DNA
US11931674B2 (en) 2019-04-04 2024-03-19 Natera, Inc. Materials and methods for processing blood samples
CN114391821B (zh) * 2021-12-28 2023-09-22 张雨萌 一种用于宫内缺氧检测胎儿心率与血压自动换算的监测仪
CN114391821A (zh) * 2021-12-28 2022-04-26 张雨萌 一种用于宫内缺氧检测胎儿心率与血压自动换算方法
WO2025160028A1 (fr) * 2024-01-25 2025-07-31 Stavro Medical, Inc. Dialyseur de sang pour nourrissons et procédés d'utilisation
US12486542B2 (en) 2024-06-04 2025-12-02 Natera, Inc. Detecting mutations and ploidy in chromosomal segments

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