WO2014143989A1

WO2014143989A1 - Fetal well being surveillance using fetal specific cell free dna

Info

Publication number: WO2014143989A1
Application number: PCT/US2014/028205
Authority: WO
Inventors: Aoy Tomita Mitchell; Michael Mitchell
Original assignee: Medical College Of Wisconsin, Inc.
Priority date: 2013-03-15
Filing date: 2014-03-14
Publication date: 2014-09-18

Abstract

This invention relates to compositions and methods for assessing fetal well being (FWB). In one aspect, 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. In another aspect, 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.

Description

FETAL WELL BEING SURVEILLANCE USING FETAL SPECIFIC CELL FREE

DNA

RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S. Provisional Application No. 61/800,329, filed March 15, 2013, the entire contents of which are incorporated by reference herein.

FIELD OF INVENTION

This invention relates to compositions and methods for assessing fetal well being

(FWB).

SUMMARY OF INVENTION

In one aspect, 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. In another aspect, 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. In another aspect, 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. In embodiments of any one of the methods provided herein, a change from a baseline level in the subject is indicative of the condition of the fetus. In any one of these embodiments, 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. In one embodiment of any one of the methods provided herein, the method further comprises determining, suggesting or providing a treatment for the fetus and/or pregnant subject, or information in regard thereto.

In another aspect, a method of determining a treatment for a fetus or a pregnant subject, the method 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. In any one of these embodiments, 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.

In one embodiment of any one of the methods provided herein, the condition is not the presence or absence of fetal aneuploidy, the gender of the fetus or the Rh blood type of the fetus. In another embodiment of any one of the methods provided herein, 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. In another embodiment of any one of the methods provided herein, the pregnant subject has or is at risk of having chorioamnionitis or preterm labor.

In another embodiment of any one of the methods provided herein, the method further comprises obtaining the biological sample from the pregnant subject. In another embodiment of any one of the methods provided herein, the pregnant subject is considered to have a high- risk pregnancy. In another embodiment of any one of the methods provided herein, the pregnant subject is not considered to have a high-risk pregnancy.

In another embodiment of any one of the methods provided herein, the method further comprises performing one or more additional tests on the pregnant subject or fetus. In another embodiment of any one of the methods provided herein, the decision to perform one or more additional tests on the pregnant subject or fetus is based on the comparison. In another embodiment of any one of the methods provided herein, the one or more tests comprise performing an ultrasound. In another embodiment of any one of the methods provided herein, 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. In another embodiment of any one of the methods provided herein, 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. In another embodiment of any one of the methods provided herein, the method is for the purpose of monitoring the condition of or the progression of the condition in the fetus. In any one of these embodiments, 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. In one embodiment of any one of the methods provided herein, the method further comprises determining, suggesting or providing a treatment for the fetus and/or pregnant subject, or information in regard thereto.

In another embodiment of any one of the methods provided herein, 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. In another embodiment of any one of the methods provided herein, 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. In any one of these embodiments, 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.

In another embodiment of any one of the methods provided herein, the method further comprises providing a treatment to the pregnant subject or providing information about one or more treatments to the pregnant subject. In another embodiment of any one of the methods provided herein, the treatment is an in utero intervention. In another embodiment of any one of the methods provided herein, the treatment comprises early delivery of the fetus. In another embodiment of any one of the methods provided herein, the method further comprises providing a postnatal treatment or providing information about one or more postnatal treatments.

In another embodiment of any one of the methods provided herein, 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.

In another embodiment of any one of the methods provided herein, the amount of fetal specific cell free DNA, such as the percent or ratio of fetal specific cell free DNA relative to the total cell free DNA or non-fetal specific cell free DNA, is determined with a method comprising quantitative genotyping. In another embodiment of any one of the methods provided herein, the method comprising quantitative genotyping comprises cycling temperature capillary electrophoresis (CTCE). In another embodiment of any one of the methods provided herein, the method comprising quantitative genotyping comprises real time quantitative PCR. In another embodiment of any one of the methods provided herein, the method comprising quantitative genotyping comprises next generation sequencing.

In another embodiment of any one of the methods provided herein, determining the amount of fetal specific cell free DNA, such as the percent or ratio as described herein, 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.

In another embodiment of any one of the methods provided herein, 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.

In another embodiment of any one of the methods provided herein, 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. In another embodiment of any one of the methods provided herein, 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. In another embodiment of any one of the methods provided herein, the statistical distribution comprises a binomial distribution. In another embodiment of any one of the methods provided herein, 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.

In another embodiment of any one of the methods provided herein, the biological sample comprises blood, plasma, serum or urine from the pregnant subject.

In another embodiment of any one of the methods provided herein, 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. In any one of these embodiments, 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.

In another embodiment of any one of the methods provided herein, 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. In any one of these embodiments, 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.

In another embodiment of any one of the methods provided herein, 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;

calculating an estimated allele frequency of a first allele at the at least one informative locus using a statistical distribution; determining an amount of cell free DNA of the fetus based on the estimated allele frequency; and determining the presence or absence of the condition or progression of the condition of the fetus based on the determined amount of the cell free DNA of the fetus in the cell free DNA. In another embodiment of any one of the methods provided herein, the method further comprises extracting the cell free DNA from the biological sample. In another embodiment of any one of the methods provided herein, the first allele comprises a minor allele. In another embodiment of any one of the methods provided herein, 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. In another embodiment of any one of the methods provided herein, the first allele comprises a minor allele and the second allele comprises a major allele. 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 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.

In another aspect, at least one computer-readable storage medium storing computer- executable instructions that, when executed by at least one processor, cause a computing device to perform any one of the methods provided herein is provided. In one embodiment of any one of the methods or media provided herein, 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. In any one of these embodiments, the determined amount is compared to a threshold, such as a baseline level. In any one of these embodiments, 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.

In another embodiment of any one of the methods or media provided herein, the plurality of loci is identified by analyzing the nucleic acids using high-throughput DNA sequencing or quantitative genotyping.

In another embodiment of any one of the methods or media provided herein, the first allele comprises a minor allele. In another embodiment of any one of the methods or media provided herein, 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. In another embodiment of any one of the methods or media provided herein, the first allele comprises a minor allele and the second allele comprises a major allele. In another embodiment of any one of the methods or media provided herein, 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 or media 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.

In another embodiment of any one of the methods or media provided herein, 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.

In another embodiment of any one of the methods or media provided herein, 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.

In another aspect, 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. In such aspects, the sample is obtained from a subject having or suspected of having the foregoing instead of a pregnant woman. In one embodiment of any one of these methods, the risk is the presence or absence of the tumor or cancer or metastasis rather than a risk associated with a fetal condition. In another embodiment of any one of these methods, the fetal specific cell free DNA is instead tumor or cancer specific cell free DNA. In another embodiment of any one of these methods, the comparison is indicative of the risk of having the cancer or tumor or recurrence or metastasis thereof. In another embodiment of any one of these methods, 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. In another embodiment of any one of these methods, the first nucleic acids are of the subject. In another embodiment of any one of these methods, the second nucleic acids are specific to the cancer or tumor. In one embodiment of any one of these methods, a treatment is determined, suggested or provided (or information in regard to the treatment is provided) to the subject. In another embodiment of any one of these methods, the treatment or therapy is an anti-cancer or anti-tumor therapy. In another embodiment of any one of these methods, the subject has or is at risk of having any of the cancers or tumors provided herein or a recurrence or metastasis thereof. In another embodiment of any one of these methods, one or more additional tests are performed. In another embodiment of any one of these method, the tests are for assessing the presence or absence of a cancer or tumor or a recurrence or metastasis thereof.

In another aspect, a computer-readable storage media is provided, wherein the medium stores computer-executable instructions for performing any one of the methods provided herein.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. The figures are illustrative only and are not required for enablement of the disclosure.

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.

DETAILED DESCRIPTION OF THE INVENTION Despite decades of research, non-invasive antenatal assessment of fetal well being remains a challenge. There is a particularly critical need for methods of assessment in pregnancies with antenatal anomalies that have an increased risk of fetal loss. Availability of methods to determine fetal compromise before the development of advanced signs of distress would provide a window of time for the appropriate intervention or early delivery. Currently, the only methods available to monitor fetal well being are indirect and late measurements by methods such as ultrasound or biophysical profile, which alerts physicians to problems, such as hydrops fetalis, after they have developed. At this point too much damage may have been done, and fetal recovery, if possible, is difficult and requires tremendous societal resources. There is no currently available method to measure early stages of fetal illness and predict when a fetus is at risk for the development of hydrops or demise before it occurs. A compelling need exists for the methods provided herein which allow for the non-invasive determination of fetal well being. Such methods and related compositions can improve pregnancy outcomes and decrease newborn mortality. While the need is particularly acute in high risk pregnancies, the methods provided can be used as a screening test, such as for preterm labor, in all pregnancies.

Individuals carry non-native DNA sources in health, pregnancy, and in situations of the presence of tumor, metastatic cancer, and following organ transplantation and conditions related thereto as well as in other clinical scenarios. 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. These data demonstrate that changes in levels of circulating cff-DNA can be a viable early indicator of the development of preterm labor, hydrops fetalis, and fetal loss, as well as other conditions as provided herein (examples of fetal outcome), which ultimately can lead to earlier clinical intervention and improved outcomes.

The methods provided herein, therefore, can be used to identify and/or monitor a variety of fetal conditions. As used herein, generally a "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. In some embodiments, 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, accordingly, can be used in a variety of situations to assess the condition of the fetus. For example, fetal well being can be assessed in a fetus that has been diagnosed with a congenital anomaly, such as congenital heart disease. As another example, fetal well being can be assessed in pregnant women with fetal gastroschisis and fetal bradycardia syndrome. In high or low risk pregnancies, 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. Such methods and compositions will allow for the further understanding of pre-term labor and fetal demise and decrease the rate of prematurity and neonatal mortality, which unfortunately, remain unacceptably high. Accordingly, 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. In 10-15% of pregnancies with gastroschisis, there is a sudden, unpredictable intrauterine death. Despite close ultrasound surveillance with measurements of fetal growth, heart rate, and bowel dilation, these factors do not predict when there may be an in utero demise. There are currently no predictive factors to help guide treatment to recommend early delivery in these patients.

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.

One indication for intervention is large congenital pulmonary adenomatoid

malformations (CPAMs) that result in 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. This intervention allows the large cyst in the fetal lung to be decompressed into the amniotic cavity and allows resolution of hydrops. Twin-twin transfusion syndrome (TTTS) 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. There are five stages of TTTS, with progressive changes in amniotic fluid levels, cardiac dysfunction and doppler changes, presence of hydrops, and eventual death of one or both twins. When the amount of amniotic fluid becomes threateningly low in the donor twin such that the bladder is no longer visible on ultrasound, it is considered Stage 2 and is an indication for fetal intervention. Without intervention, the mortality of both twins is 90-100%. The current approach to TTTS is careful monitoring for the progression of disease by watching the amniotic fluid levels and cardiac function of both twins. However, there is currently no clinical tool to predict when the disease will progress.

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.

Further examples where a 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 (e.g., heart valve problems, hypertension, sickle cell disease, asthma, lupus, or rheumatoid arthritis) can also be considered high risk. Other clinical indicators of high-risk pregnancies are known to those of ordinary skill in the art.

As 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. For example, levels or amounts, such as percentages, fractions or ratios, of fetal specific cf-DNA (relative to total cf-DNA or non-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.

Generally, the amount and/or changes in the amount (e.g., percent or ratio of fetal specific cf- DNA as provided herein) relative to a threshold, such as a baseline, of fetal specific cf-DNA 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. The work described suggests that changes from baseline levels of circulating cff-DNA are an early indicator of decreased fetal metabolism, development of fetal compromise and potentially subsequent fetal loss. It has been demonstrated that altered levels of circulating fetal specific cf-DNA are associated with the development of hydrops fetalis, in utero fetal demise, and future spontaneous abortion. Quantification of the fractions (or percent or ratio) of fetal specific cf-DNA in maternal serum, therefore, can be performed in order to monitor normal development, decrease preterm labor and allow for timely fetal intervention or planned pre-term delivery before the development of hydrops fetalis, intrauterine death or other adverse conditions. 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, therefore, 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. Provided herein, accordingly, are methods using amounts of the fetal specific cf-DNA from biological samples, preferably relative to total cell free DNA or non-fetal specific cf-DNA, in the samples, in order to assess the condition of a subject, such as the condition of a fetus in a pregnant subject. 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. As used herein, 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. In any one of the methods provided herein, an increase or decrease relative to the baseline is the indicator of fetal well being. In any one of the methods provided herein changes in the difference relative to the baseline at two or more time points is the indicator of fetal well being. Accordingly, in any one of the methods provided herein 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. In any one of the methods provided herein 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. In any of the methods provided herein, 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. In some embodiments 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. In some embodiments, the pregnant subject is at risk. In some embodiments, the fetus has had or is at risk of having distress. In some embodiments, the pregnant subject is at risk for a condition including, for example, chorioamnionitis or preterm labor. From the examples provided herein, it has been demonstrated that levels of fetal specific cf-DNA (cff-DNA), such as the percent or ratio of cff-DNA, can be decreased compared to a baseline whereby the decrease is indicative of an adverse condition of the fetus. Thus, 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. As used herein, "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. The level of fetal specific cf-DNA can be measured at any point during pregnancy or at multiple time points throughout a pregnancy.

As used herein, 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.

Generally, as provided herein, 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. As used herein, "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. As used herein, "minor allele" refers to the allele that is less frequent in the population of nucleic acids for a locus. A "major allele", on the other hand, refers to the more frequent allele in a population. In some embodiments, 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. In some embodiments, 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. For example, the estimated allele frequency can be calculated by modeling allele read counts using a binomial distribution. In some embodiments, 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. In some embodiments, 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. These amounts can be compared relative to a threshold (such as a baseline level) and/or changes in such values can be monitored over time. For example, 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", as used herein, 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. As another example, a threshold value can be determined from baseline values before the presence of a condition or risk or after a course of treatment. As another example, 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.

Accordingly, 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. In some embodiments, 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. In some embodiments, 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.

It is expected that the amount (e.g., percentage or ratio) of fetal specific cf-DNA can change with clinical intervention. Accordingly, as provided herein 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. In some embodiments, the information includes written materials containing the information. Written materials can include the written information in electronic form. In any one of the methods provided herein, 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. In some embodiments of any one of the methods provided herein, 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. In some embodiments, the recommending comprises providing information regarding a suggested treatment, such as fetal intervention and/or delivery options to the pregnant woman.

In some embodiments of any one of the methods provided herein, 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 suitable therapy may be selected based on the correlation or comparison and/or the amount of the therapy

administered to the pregnant subject may be increased or decreased. Alternatively, no change or no therapy may be determined based on the correlation or comparison. Choice of therapies and dosing involved with such therapies are within the skill of those in the art. In embodiments, 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. In still other embodiments, 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. In some embodiments, 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. In some embodiments, the information is provided in written form. In some embodiments, the information may be provided as computer-readable instructions.

In some embodiments, at least some acts of the methods provided may be

implemented as computer-readable instructions stored on one or more non-transitory computer-readable storage media. The computer-readable instructions, when executed by one or more processors, may cause a computing device to execute the acts of the method. Fig. 4 is an exemplary computer system on which some embodiments of the invention may be employed.

An illustrative implementation of a computer system 500 that may be used in connection with any of the embodiments of the invention described herein is shown in Fig. 4. 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. To perform any of the functionality described herein, 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.

In this respect, it should be appreciated that 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. In addition, it should be appreciated that 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.

While much of the description provided herein focuses on fetal well being and risks associated with various possible fetal conditions, the methods and computer-implemented methods or computer-readable storage media could be used for subjects with or at risk of having cancer or a tumor, recurrence of cancer or a tumor, or metastasis of a cancer or tumor rather than on a pregnant subject. Cancers include, but are not limited to, prostate cancer, bladder cancer, pancreatic cancer, lung cancer, kidney cancer, breast cancer, or colon cancer. Similarly, 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.

Various aspects of the present invention may be used alone, in combination, or in a variety of arrangements not specifically discussed in the embodiments described in the foregoing and are therefore not limited in their application to the details and arrangement of components set forth in the foregoing description or illustrated in the drawings. For example, aspects described in one embodiment may be combined in any manner with aspects described in other embodiments.

Also, 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.

Use of ordinal terms such as "first," "second," "third," etc., in the claims to modify a claim element does not by itself connote any priority, precedence, or order of one claim element over another or the temporal order in which acts of a method are performed. Such terms are used merely as labels to distinguish one claim element having a certain name from another element having a same name (but for use of the ordinal term).

The phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," "having,"

"containing", "involving", and variations thereof, is meant to encompass the items listed thereafter and additional items.

Having described several embodiments of the invention in detail, various

modifications and improvements will readily occur to those skilled in the art. Such modifications and improvements are intended to be within the spirit and scope of the invention. Accordingly, the foregoing description is by way of example only, and is not intended as limiting. The following description provides two examples of the implementation of the described technique.

EXAMPLES

Studies on cf-DNA have focused on the measurement of fetal specific cf-DNA in maternal blood for fetal diagnosis of aneuploidy and other anomalies. However, it has been surprisingly found that quantification of fetal specific cf-DNA in maternal circulation allows for the monitoring of fetal compromise (or fetal distress or fetal well being). It is believed that there have not been any other studies that have evaluated the quantitative relationship between fetal compromise as described herein and fetal specific cf-DNA.

Example 1 - Human Study

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 assay³'⁴. 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 greater¹⁵. White triangles represent patients with fetal hydrops, gray square represents in utero fetal demise (absent heart beat), and 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

homozygous (where mother and fetus have the same genotype at a given probe) and the maximum likelihood calculation of baseline and the blue curve and triangles depict genotypes that were informative (or heterozygous, where mother and fetus have different genotypes) and could be used to calculate percent fetal DNA. Note that the colored points representing hydrops, fetal death, or death within three weeks are all significantly lower than the average percent fetal DNA seen in standard high risk pregnancies.

Example 2 - Human Study (Prophetic) Pregnant women 18 years and older at gestational age 10 weeks or greater with a singleton pregnancy are selected. A time course of samples from mothers carrying fetuses with gastroschisis (n=25 patients) and with fetal bradycardia syndrome (n=15 patients) are followed to determine the relevance of these changes in fetal specific cf-DNA as the diseases progress. Maternal blood samples are taken at diagnosis and every 4 weeks until 28 weeks gestation in gastroschisis at the same time point during ultrasound surveillance. Maternal samples are obtained weekly beginning at 28 weeks and continue to delivery or 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 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.

All samples are collected in BCT tubes optimized for extraction of cf-DNA in plasma¹⁶. Extracted DNA are coded and deidentified. Next generation targeted sequencing using the DANSR assay are performed to determine human fetal fraction and counts³'⁴'¹⁵. Raw count data undergo QC and analysis in a blinded manner. Specifically, fetal specific 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 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.

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. Generally, there are 15 patients each year with congenital pulmonary adenomatoid malformations (CPAMs), out of which 4 are expected to receive in utero thoraco amniotic shunt placement. 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. Ultrasound findings including amniotic fluid levels of both twins are then recorded as are the presence or absence of the donor twin's bladder, and the reversal of any doppler changes that have occurred. The samples are collected and processed as described elsewhere herein or otherwise known to those of ordinary skill in the art.

It is expected that changes similar to what is seen with fetal compromise under other conditions provided herein are seen for babies with CPAM who develop hydrops and for babies with TTTS who develop low amniotic fluid volume and poor cardiac function. It is expected that fetal specific cf-DNA levels revert to baseline as the babies improve after intervention. A primary outcome is the 7 day fetal DNA . Changes from diagnosis and over time are assessed.

Example 3- Ovine Study (Prophetic)

Six pregnant sheep containing a singleton male fetus at 120 days gestation, term gestation being 145 days, are obtained. Singleton male fetuses allow for the avoidance of confounding effects of fetal specific cf-DNA from the twin fetus, and the male gender allows for the use of more rapid PCR based methods to quantify the fetal specific cf-DNA. One day after arrival in the facility, a blood sample from the ewe is obtained for measurement of baseline fetal specific cf-DNA in maternal blood prior to any intervention. 3-5 days after arrival (125 days gestation), sterile instrumentation is performed on the ewe for the insertion of 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, PaO₂, PaCO₂ 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, PaO₂, PaCO₂ 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 described¹⁶. 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 increase in fetal specific cf-DNA in maternal blood can indicate fetal compromise initiated by umbilical cord occlusion. This increase is expected to occur early with fetal compromise as fetal hypoxia leads to decreases in HR and cardiac output and fetal acidosis. Although the mechanism of fetal cf-DNA leak to maternal compartment requires further investigation, these studies can establish a direct relationship between fetal stress and changes in fetal specific cf-DNA.

Example 4 - Ovine Study (Prophetic)

An assay targeting 18s (Ovis Aries) 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. Although 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. Although, a number of fetal anomalies in pregnancies complicated by anomalies largely have decreased fetal cardiac output as the basis for distress. Finally, 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

For the experiments, the 24 hour and 48 hour fetal % to baseline can be compared using a paired t-test. With 6 sheep, using an alpha of 0.025 (adjusting for two outcomes) there is an at least 80% power to detect a difference of 1.7 standard deviations (SD). The change over time can be examined using a mixed model.

References for Examples 1-4

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3. Selective analysis of cell-free DNA I maternal blood for evaluation of fetal trisomy. Sparks AB, Wang ET, Struble CA, Tomita-Mitchell A, Mitchell M et al. PrenatDiagn. 2012 Jan; 32.

4. Sparks AB, Struble CA Wang ET et al. Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. . Am Journal of Obstet Gynecol. 2012, 206(4):319.el-9.

5. Harrison M. The fetus with CCAM. Atlas of fetal surgery. Kentucky: International Publishing. 1996. p 76.

6. Crombleholme TM, Coleman B, Hedrick MH, et al. Cystic adenomatoid

malformation volume ratio predicts outcome in prenatally diagnosed cystic adenomatoid malformation of the lung. J PediatrSurg 2002, 37:331-8. 7. Adzick NS, Harrison MR, Crombleholme TM, et al. Fetal lung lesions: management and outcome. Am J ObstetGynecol 1998; 179:884-9.

8. Gonsoulin W, Moise KJ, Kirshon B, et al. Outcome of twin-twin transfusion diagnosed before 28 weeks of gestation. Obstet Gynecol. 1990;73:214-6.

9. Snyder CL. Outcome analysis for gastroschisis. J Pediatr Surg 1999; 34: 1253-6.

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11. Daniel SS, Yeh MN, Bowe ET, Fukunaga A, James LS. Renal response of the lamb fetus to partial occlusion of the umbilical cord. J Pediatr. 1975 Nov;87(5):788-94

12. Konduri GG, Mattei J. Role of oxidative phosphorylation and ATP release in mediating birth-related pulmonary vasodilation in fetal lambs.Am J Physiol Heart Circ Physiol. 2002 Oct;283(4):H1600-8

13. Konduri GG, Ou J, Shi Y, Pritchard KA Jr. Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension. Am J Physiol Heart Circ Physiol. 2003 Jul;285(l):H204-l l

14. Louey S, Cock ML, Stevenson KM, Harding R. Placental insufficiency and fetal growth restriction lead to postnatal hypotension and altered postnatal growth in sheep. Pediatr Res. 2000;48(6):808-14.

15. Norton ME, Brar H, Weiss J, Karimi A, Laurent LC, Caughey AB, Rodriguez MH, Williams J 3rd, Mitchell ME, Adair CD, Lee H, Jacobsson B, Tomlinson MW, Oepkes D, Hollemon D, Sparks AB, Oliphant A, Song K.Non-Invasive Chromosomal Evaluation (NICE) Study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol. 2012 Jun 1. [Epub ahead of print]PMJD: 22742782

16. Hidestrand M, Stokowski R, Song K, Oliphant A, Deavers J, Goetsch M, Simpson P, Kuhlman R, Ames M, Mitchell M, Tomita-Mitchell A. Influence of temperature during transportation on cell-free DNA analysis. Fetal Diagn Ther. 2012;31(2): 122-8. Epub 2012 Jan 19. PMJO: 22261730 Example 5-Ovine Study (Prophetic)

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 0₂ content difference as a measure of fetal metabolism. For example, 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 0₂ content difference to estimate 0₂ 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. A single episode of umbilical cord occlusion for 30 minutes at 60% gestation has been shown to result in fetal hydrops over 3 days, despite recovery of fetal heart rate and BP ⁹. These studies suggest that fetal hypoxia/acidosis can precede hydrops and if recognized, provide a window of time for intervention.

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. Despite close ultrasound surveillance with measurements of fetal growth, heart rate, and bowel dilation, these factors do not predict in utero demise. 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.

Previous efforts to monitor fetal health relied on physiological indices and are limited by the need for expensive technology, which is not readily available or require obtaining fetal blood samples. Development of new approaches for monitoring is limited by the relative inaccessibility of the fetus. Provided herein are techniques based on the detection and quantification of fragmented cell free DNA of fetal origin in maternal blood. While this technology has been proposed for the detection of fetal genetic defects and copy number variation, it has surprisingly been found that the fraction of fetal specific cf-DNA in maternal blood varies as a function of fetal health. The techniques provided can be used for monitoring all pregnancies, including high-risk pregnancies. 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.

Example 6 - Ovine Study

To demonstrate feasibility of detecting total cf-DNA and fetal specific cf-DNA in an animal model of fetal compromise, 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. 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). Without wishing to be bound by any particular theory, it is believed that a decrease in fetal metabolism in response to fetal hypoxia or ischemia from a variety of causes leads to a decrease in fetal DNA synthesis and circulating fetal specific cf-DNA levels. These changes are expected to precede the appearance of an abnormal heart rate and biophysical profile changes. Example 7 - Ovine Study (Prophetic)

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. However, it has surprisingly been found that 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 0₂ difference, a measure of 0₂ extraction and lactate/pyruvate ratio as metabolic parameters can be measured. Increases in 0₂ extraction and lactate relative to pyruvate levels suggest an effect of induced stress on fetal metabolism.

10 pregnant sheep are obtained containing a singleton male fetus at 120 days gestation, term gestation being 145 days. The study can be focused on a singleton male fetus, that can allow for the avoidance of the confounding effects of fetal specific cf-DNA from the twin, and the male gender allows for the use of a more rapid PCR based method to quantify the fetal specific cf-DNA. One day after arrival in the facility, a blood sample from the ewe is obtained for measurement of baseline fetal specific cf-DNA in maternal blood prior to any intervention. This initial fetal specific cf-DNA test can verify the fetal gender, estimated by ultrasound. 3-5 days after arrival (125 days gestation), 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, PaO₂, PaCO₂, blood lactate/pyruvate ratio and arterial and venous O₂ 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, Pa0₂, PaC0₂, blood lactate/pyruvate ratio and AV 0₂ 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 0₂ 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 0₂ 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. One study has indicated severe bradycardia leading to asystole after 15 min occlusion, followed by recovery with the release of occlusion. Fetal HR and arterial pressure are continuously measured. The fetal arterial blood gases, lactate/pyruvate ratio, AV 0₂ 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 0₂ 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. It is expected that there will be a cumulative increase in fetal acidosis with periodic episodes of fetal bradycardia during sustained cord occlusion. It is also expected that an initial spike in fetal specific cf-DNA immediately after cord occlusion will occur, coinciding with release of fetal DNA from injury, as previously observed with injury to donor transplanted heart 13. However, a steady decrease in fetal specific cf-DNA is then expected with the decrease in fetal metabolism, which can be measured by using lactate/pyruvate ratio and AV 0₂ content difference. This decrease is expected to be amplified with sustained fetal distress for 12 hours.

In some studies 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. Finally, 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. Also, 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. With 6, 8 or 12 sheep, using an alpha of 0.025 (adjusting for two outcomes) there will be an at least 80% power to detect a difference of 1.7, 1.3 or 1 standard deviations. Changes over time can be examined using a mixed model.

Example 8 - Human Study (Prophetic)

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. A time course of samples from mothers carrying fetuses with gastroschisis (n=25 patients) and with fetal arrhythmia syndrome (n=25 patients) are followed and changes in fetal specific cf-DNA as the diseases progress are determined. 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

gastroschisis. 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.

Changes in cjf-DNA with fetal intervention: Baseline and serial levels of fetal specific cf-DNA are obtained in patients undergoing fetal intervention for CPAM or TTTS. Baseline fetal specific cf-DNA levels are also obtained at the time of diagnosis for patients with CPAMs. Samples are then obtained every two weeks until the development of hydrops. Hydropic fetuses with CPAMs are candidates for fetal intervention with a thoracoamniotic 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 the degree of mediastinal shift and hydrops fetalis is recorded. Additionally, levels of fetal specific cf- DNA in pregnancies complicated by TTTS are obtained. Samples are then similarly obtained every two weeks until Stage II TTTS has been reached, at which time fetal intervention can be offered. Samples are then obtained immediately pre-intervention and post-intervention day 1, 3, and 7. Ultrasound findings are recorded, including amniotic fluid levels of both twins, presence or absence of the donor twin's bladder, and reversal of any Doppler changes that occurred.

Analysis of plasma 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. With 25 mothers, 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.

References for Examples 5-8

1. Birch, L., et al. Accurate and robust quantification of circulating fetal and total DNA in maternal plasma from 5 to 41 weeks of gestation. Clin Chem 51, 312-320 (2005).

2. Ghanta, S., et al. Non-invasive prenatal detection of trisomy 21 using tandem single nucleotide polymorphisms. PLoS One 5, el3184 (2010).

3. Sparks, A.B., et al. Selective analysis of cell-free DNA in maternal blood for evaluation of fetal trisomy. Prenat Diagn, 1-7 (2012).

4. Sparks, A.B., Struble, C.A., Wang, E.T., Song, K. & Oliphant, A. Noninvasive prenatal detection and selective analysis of cell-free DNA obtained from maternal blood: evaluation for trisomy 21 and trisomy 18. Am J Obstet Gynecol 206, 319 e311-319 (2012).

5. Adzick, N.S., Harrison, M.R., Crombleholme, T.M., Flake, A.W. & Howell, L.J. Fetal lung lesions: management and outcome. Am J Obstet Gynecol 179, 884-889 (1998). 6. Crombleholme, T.M., et al. Cystic adenomatoid malformation volume ratio predicts outcome in prenatally diagnosed cystic adenomatoid malformation of the lung. J Pediatr Surg 37, 331-338 (2002).

7. Harrison, M. Atlas of fetal surgery, ( Chapman & Hall New York, 1996).

8. Reid, K.P., Dickinson, J.E. & Doherty, D.A. The epidemiologic incidence of congenital gastroschisis in Western Australia. Am J Obstet Gynecol 189, 764-768 (2003).

9. O'Connell, A.E., Boyce, A.C., Lumbers, E.R. & Gibson, K.J. The effects of asphyxia on renal function in fetal sheep at midgestation. J Physiol 552, 933-943 (2003).

10. Castillo-Melendez, M., Chow, J. A. & Walker, D.W. Lipid peroxidation, caspase-3 immunoreactivity, and pyknosis in late-gestation fetal sheep brain after umbilical cord occlusion. Pediatr Res 55, 864-871 (2004).

11. de Haan, H.H., Gunn, A.J. & Gluckman, P.D. Fetal heart rate changes do not reflect cardiovascular deterioration during brief repeated umbilical cord occlusions in near-term fetal lambs. Am J Obstet Gynecol 176, 8-17 (1997).

12. Hidestrand, M., et al. Influence of temperature during transportation on cell-free DNA analysis. Fetal Diagn Ther 31, 122-128 (2012).

13. Norton, M.E., et al. Non-Invasive Chromosomal Evaluation (NICE) Study: results of a multicenter prospective cohort study for detection of fetal trisomy 21 and trisomy 18. Am J Obstet Gynecol 207, 137 el31-138 (2012).

14. Hidestrand, M., et al. Using donor specific cell free DNA to monitor rejection in cardiac transplant patients. 36th Annual Midwest Pediatric Cardiology Society Scientific Session (2012).

15. Gonsoulin, W., et al. Outcome of twin-twin transfusion diagnosed before 28 weeks of gestation. Obstet Gynecol 75, 214-216 (1990).

16. Daniel, S.S., Yeh, M.N., Bowe, E.T., Fukunaga, A. & James, L.S. Renal response of the lamb fetus to partial occlusion of the umbilical cord. J Pediatr 87, 788-794 (1975).

17. Konduri, G.G. & Mattei, J. Role of oxidative phosphorylation and ATP release in mediating birth-related pulmonary vasodilation in fetal lambs. Am J Physiol Heart Circ Physiol 283, H1600-1608 (2002).

18. Konduri, G.G., Ou, J., Shi, Y. & Pritchard, K.A., Jr. Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary

hypertension. Am J Physiol Heart Circ Physiol 285, H204-211 (2003). 19. Louey, S., Cock, M.L., Stevenson, K.M. & Harding, R. Placental insufficiency and fetal growth restriction lead to postnatal hypotension and altered postnatal growth in sheep. Pediatr Res 48, 808-814 (2000).

Claims

1. A method of assessing the condition of a fetus, comprising:

determining an amount of fetal specific cell free DNA in a biological sample obtained from a pregnant subject, and

comparing the amount with one or more baseline levels, wherein a change from a baseline level in the subject is indicative of the condition of the fetus.

2. The method of claim 1, wherein the condition is not the presence or absence of fetal aneuploidy, the gender of the fetus or the Rh blood type of the fetus.

3. The method of claim 1 or 2, further comprising obtaining the biological sample from the pregnant subject.

4. The method of any one of claims 1-3, wherein the pregnant subject is considered to have a high-risk pregnancy.

5. The method of any one of claims 1-3, wherein the pregnant subject is not considered to have a high-risk pregnancy.

6. The method of any one of claims 1-5, wherein 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.

7. The method of any one of claims 1-6, wherein the pregnant subject has or is at risk of having chorioamnionitis or preterm labor.

8. The method of any one of claims 1-7, wherein the method further comprises performing one or more additional tests on the pregnant subject or fetus.

9. The method of claim 8, wherein the one or more tests comprise performing an ultrasound.

10. The method of claim 8 or 9, wherein the 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.

11. The method of any one of claims 1-10, wherein the method comprises 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.

12. The method of claim 11, wherein the amount of fetal specific cell free DNA is compared to one or more baseline levels to assess the condition or the progression of the condition of the fetus at the one or more additional time points.

13. The method of any one of claims 1-12, wherein 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.

14. The method of any one of claims 1-13, wherein the method further comprises providing a treatment to the pregnant subject or providing information about one or more treatments to the pregnant subject.

15. The method of claim 14, wherein the treatment is an in utero intervention.

16. The method of claim 14, wherein the treatment comprises early delivery of the fetus.

17. The method of any one of claims 1-16, wherein the method further comprises providing a postnatal treatment or providing information about one or more postnatal treatments.

18. The method of any one of claims 1-17, wherein the amount of fetal specific cell free DNA is determined by extracting cell free DNA from the biological sample.

19. The method of any one of claims 1-18, wherein the fetal specific cf-DNA is determined with a method comprising quantitative genotyping.

20. The method of claim 19, wherein the method comprising quantitative genotyping comprises cycling temperature capillary electrophoresis (CTCE).

21. The method of claim 19, wherein the method comprising quantitative genotyping comprises real time quantitative PCR.

22. The method of claim 19, wherein the method comprising quantitative genotyping comprises next generation sequencing.

23. The method of any one of claims 1-22, wherein determining the amount of fetal specific cf-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.

24. The method of claim 23, wherein 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.

25. The method of claim 23, wherein 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.

26. The method of any one of claims 23-25, wherein 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.

27. The method of any one of claims 23-26, wherein the statistical distribution comprises a binomial distribution.

28. The method of any one of claims 23-27, wherein 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.

29. The method of any one of claims 1-28, wherein the biological sample comprises blood, plasma, serum or urine from the pregnant subject.

30. The method of any one of claims 1-29, wherein a decrease in the determined amount of the fetal specific cf-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.

31. The method of any one of claims 1-29, further comprising, based on the determined amount of the fetal specific cf-DNA, evaluating an effect of a treatment on the fetus by correlating an increased amount of the determined amount of the fetal specific cf-DNA as compared to one or more baseline levels as indicative of effectiveness of the treatment.

32. The method of any one of claim 1-22, wherein determining the amount of fetal specific cf-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;

calculating an estimated allele frequency of a first allele at the at least one informative locus using a statistical distribution;

determining an amount of cell free DNA of the fetus based on the estimated allele frequency; and

determining the presence or absence of the condition or progression of the condition of the fetus based on the determined amount of the cell free DNA of the fetus in the cell free DNA.

33. The method of claim 32, further comprising:

extracting the cell free DNA from the biological sample.

34. The method of claim 32 or 33, wherein the first allele comprises a minor allele.

35. The method of any one of claims 32-34, wherein 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.

36. The method of claim 35, wherein the first allele comprises a minor allele and the second allele comprises a major allele.

37. The method of any one of claims 32-36, wherein :

the first allele comprises a minor allele; and

the estimated allele frequency of the minor allele is calculated using a binomial distribution.

38. The method of any one of claims 32-36, wherein:

the first allele comprises a minor allele; and

the estimated allele frequency of the minor allele is calculated using an expectation- maximization algorithm.

39. A method of determining a treatment for a fetus or a pregnant subject, the method 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 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.

40. The method of claim 39, wherein the treatment is an in utero intervention.

41. The method of claim 39, wherein the treatment is early delivery.

42. At least one computer-readable storage medium storing computer-executable instructions that, when executed by at least one processor, cause a computing device to perform a method comprising:

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;

determining an amount of DNA of the fetus in the cell free DNA based on the estimated allele frequency;

comparing the amount with one or more baseline levels; and

determining a condition of the fetus based on the comparison.

43. The at least one computer-readable storage medium of claim 42, wherein the method 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.

44. The at least one computer-readable storage medium of claim 42, wherein the method 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 with a positive effect of the treatment.

45. The at least one computer-readable storage medium of any one of claims 42-44, wherein:

the plurality of loci is identified by analyzing the nucleic acids using high-throughput DNA sequencing or quantitative genotyping.

46. The at least one computer-readable storage medium of any one of claims 42-45, wherein:

the first allele comprises a minor allele.

47. The at least one computer-readable storage medium of any one of claims 42-46, wherein the at least one informative locus is selected by:

48. The at least one computer-readable storage medium of any one of claims 42-47, wherein the first allele comprises a minor allele and the second allele comprises a major allele.

49. The at least one computer-readable storage medium of claim any one of claims 42-48, wherein: the first allele comprises a minor allele; and

50. The at least one computer-readable storage medium of any one of claims 42-49, wherein:

the first allele comprises a minor allele; and