Inventors: Cuckle; Howard S. (Harrogate, GB); Iles; Raymond K. (West Drayton, GB); Chard; Timothy (London, GB) Assignee: Yale University (New Haven, CT) Appl. No.: 675152 Filed: July 3, 1996

Primary Examiner: Achutamurthy; Ponnathapura Assistant Examiner: Nelson; Brett Attorney, Agent or Firm: Lauder; Leona L. United States 6,025,149 February 15, 2000

Parent Case Text
This application claims the benefit of U.S. Provisional Application Ser. No. 60/000,945 filed Jul. 7, 1995.

Claims
What we claim is:

1. A method for determining a pregnant woman's risk of carrying a fetus with fetal aneuploidy during the second or third trimester of her pregnancy comprising:

(a) assaying a urine sample from said pregnant woman for .beta.-core-hCG level, wherein said urine sample is obtained during the second or third trimester of her pregnancy;

(b) comparing the level of .beta.-core-hCG in said urine sample to reference levels of .beta.-core-hCG in urine samples from pregnant women carrying normal fetuses at about the same gestational age as the pregnancy under analysis, said comparison being indicative of said pregnant woman's risk of carrying a fetus with fetal aneuploidy, wherein a higher or lower level of .beta.-core-hCG in the pregnant woman's urine sample than the reference levels is indicative of a risk of the pregnant woman carrying a fetus with fetal aneuploidy.

2. The method according to claim 1 wherein the pregnant woman is in the second trimester of her pregnancy.

3. The method according to claim 1 wherein the urine sample is assayed for .beta.-core-hCG level by immunoassay.

4. The method according to claim 1 wherein the fetal aneuploidy is selected from the group consisting of Down syndrome, Edwards syndrome, triploidy, Turner syndrome, Klinefelter syndrome, and triple-X.

5. The method according to claim 4 wherein the level of .beta.-core-hCG in said urine sample is higher than the reference levels, and the fetal aneuploidy is selected from the group consisting of Down syndrome, Turner syndrome, Klinefelter syndrome and triple-X.

6. The method according to claim 4 wherein the level of .beta.-core-hCG in said urine sample is lower than the reference levels, and the fetal aneuploidy is selected from the group consisting of triploidy and Edwards syndrome.

7. The method according to claim 3 wherein said immunoassay is in a sandwich format or in a competitive assay format.

8. The method according to claim 7 wherein said immunoassay is automated.

9. The method according to claim 3 wherein the level of .beta.-core-hCG in said sample is higher than the reference levels, and said fetal aneuploidy is selected from the group consisting of Down syndrome, Turner syndrome, Klinefelter syndrome and triple-X.

10. The method according to claim 9 wherein said fetal aneuploidy is Down syndrome.

11. The method according to claim 3 wherein the pregnant woman is in the second trimester of her pregnancy.

12. The method according to claim 10 wherein the pregnant woman is in the second trimester of her pregnancy.

13. The method according to claim 4 wherein the pregnant woman is in the second trimester of her pregnancy.

14. The method according to claim 1 further comprising assaying said urine sample for a second marker;

wherein the level of said second marker in said sample is compared to reference levels of said second marker in urine samples from pregnant women carrying normal fetuses whose pregnancies are at about the same gestational age as the pregnancy under analysis; and

wherein said comparison is also indicative of said pregnant woman's risk of carrying a fetus with fetal aneuploidy, wherein a higher or lower level of said second marker in said sample than the reference levels for said marker is indicative of a risk of fetal aneuploidy.

15. The method according to claim 14 wherein said second marker is selected from the group consisting of total estrogen (tE), alpha-hydroxydehydroepiandrosterone sulphate (16.alpha.-OH-DHAS), pregnancy-associated plasma protein A (PAPP-A), dimeric inhibin, unconiugated estriol (uE.sub.3), alpha-fetoprotein (AFP), total estriol (tE.sub.3) and proform of eosinophilic major basic protein proMBP).

16. The method according to claim 15 wherein said second marker is selected from the group consisting of PAPP-A, 16 .alpha.-OH-DHAS, dimeric inhibin and tE.

17. The method according to claim 1 further comprising determining the pregnant woman's age and average age of women carrying normal fetuses, and comparing the age of the pregnant woman to said average age, wherein if the pregnant woman's age is older than said average age, there is a risk of the pregnant woman carrying a fetus with fetal aneuploidy.

18. The method according to claim 1 further comprising assaying a serum sample from said Dregnant woman for a marker level, comparing the level of said marker in said serum sample to reference levels of said marker in serum samples from pregnant women carrying normal fetuses whose pregnancies are at about the same gestational age as the pregnancy under analysis, said comparison being further indicative of said pregnant woman's risk of carrying a fetus with fetal aneuploidy, wherein a higher or lower level of said marker in said serum sample than said reference levels for said marker is indicative of a risk of the pregnant woman carrying a fetus with fetal aneuploidy.

19. The method according to claim 18 wherein said serum marker is selected from the group consisting of: pregnancy-associated plasma protein A (PAPP-A), major basic protein, proform of eosinophilic major basic protein (proMBP), intact hCG, free .alpha.-hCG, free .beta.-hCG, alpha-fetoprotein (AFP), unconlugated estriol (uE.sub.3, 16.alpha.-hydroxydehydroepiandrosterone sulphate (16.alpha.-OH-DHAS.L, dimeric inhibin and nondimeric inhibin.

20. The method according to claim 1 further comprising performing ultrasonography to visualize the fetus carried by said pregnant woman, wherein an abnormal ultrasonograph is indicative of a risk of fetal aneuploidy.

21. The method according to claim 1 wherein the .beta.-core-hCG levels are corrected for variability in urine concentrations by assaying the urine sample for creatinine levels and dividing the .beta.-core-hCG level of each urine sample by the creatinine level for that sample.

22. The method according to claim 1 wherein said .beta.-core-hCG level is determined by methods selected from the group consisting of: chromatography, nuclear magnetic resonance (NMR), fluorometric detection, assaying using non-antibody receptors specific for .beta.-core-hCG, and assaying using binding proteins specific for .beta.-core-hCG.

23. The method according to claim 3 wherein said immunoassay comprises using antibodies which bind specifically to .beta.-core-hCG, and which are directly or indirectly linked to a detectable marker.

24. The method according to claim 23 wherein said immunoassay further comprises using antibodies which bind specifically to .beta.-core-hCG, and which are linked to a solid phase.

25. The method according to claim 23 wherein said detectable marker is selected from the group consisting of radionuclides, fluorescers, bioluminescers, chemiluminescers, dyes, enzymes, coenzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, enzyme subunits, metal ions, and free radicals.

26. The method according to claim 25 wherein the detectable marker is either selected from the group consisting of acridinium esters, acridinium sulfonyl carboxamides, fluorescein, luminol, umbelliferone, isoluminol derivatives, photoproteins, and luciferases, or is produced by an enzymatic reaction upon a substrate.

27. The method according to claim 23 wherein the detectable marker is either an acridinium ester or is produced by an enzymatic reaction with a chemiluminescent substrate and an enzyme selected from the group consisting of alkaline phosphatase, glucose oxidase, glucose 6-phosphate dehydrogenase, .alpha.,.beta.-galactosidase, horseradish peroxidase, and xanthine oxidase.

28. The method according to claim 24 wherein said solid phase comprises magnetic or paramagnetic particles.

29. The method according to claim 28 wherein said immunoassay is automated, and wherein said detectable marker is an acridinium ester.

Description
FIELD OF THE INVENTION

The present invention is in the field of prenatal diagnosis. It concerns non-invasive methods to screen prenatally for fetal Down syndrome and other fetal aneuploidies by determining the levels of urinary .beta.-core-hCG [also known as urinary gonadotropin peptide (UGP), .beta.-core, .beta.-core fragment, or urinary gonadotropin fragment (UGF)] in maternal urine samples, alone or in conjunction with other markers.

BACKGROUND OF THE INVENTION

Prenatal tests to detect fetal aneuploidies, such as Down syndrome (trisomy 21), Edwards syndrome (trisomy 18), triploidy (69 chromosomes), Klinefelter syndrome (47, XXY), triple X (47,XXX) and Turner syndrome (45,X) among other aneuploidies, by amniocentesis or chorionic villus sampling (CVS) have been available since the late 1960s. Amniocentesis is the most common invasive prenatal diagnostic procedure. In amniocentesis, amniotic fluid is sampled by inserting a hollow needle through the mother's anterior abdominal and uterine walls into the amniotic cavity by piercing the chorion and amnion. It is usually performed in the second trimester of pregnancy. CVS is performed primarily during the first trimester, and involves collecting cells from the chorion which develops into the placenta.

Another invasive prenatal diagnostic technique is cordocentesis or percutaneous umbilical cord blood sampling, commonly known as fetal blood sampling. Fetal blood sampling involves obtaining fetal blood cells from vessels of the umbilical cord, and is often performed about the 20th gestational week.

Amniocentesis is used selectively because it presents a risk of about 1% of inducing spontaneous abortion. CVS and fetal blood sampling carry a similar or higher risk of inducing abortion, and there is also concern that those procedures may lead to fetal limb malformations in some cases. Thus, amniocentesis, CVS and fetal blood sampling are procedures that are only employed if a pregnancy is considered at high risk for a serious congenital anomaly. Thus, some means is required to select those pregnancies that are at a significant risk of an aneuploidy, such as, Down syndrome, to justify the risks of such invasive prenatal diagnostic procedures, as amniocentesis, CVS and fetal blood sampling.

Prior to 1983, the principal method for selecting pregnancies that had an increased risk for genetic defects was based on maternal age, that is, the older the age of the mother, the higher the risk that the pregnancy would be affected by aneuploidy. In 1974, biochemical screening for neural tube defects by measuring alpha-fetoprotein (AFP) began. In 1984, the use of the AFP screen was additionally adopted for the detection of Down syndrome. Since the early 1990s, a multiple marker blood test has been used to screen for that disorder. A common version of that test is the three marker triple test. The triple screen measures AFP, human chorionic gonadotropin (hCG) and unconjugated estriol (uE.sub.3) in the serum of pregnant women.

The triple screen provides a means to screen the population of pregnant women to determine which pregnancies are at risk for Down syndrome and other serious genetic defects. The risk is calculated based on the results of the screen, along with other cofactors, such as, maternal age, to determine if the risk is high enough to warrant an invasive diagnostic procedure, such as, amniocentesis, CVS or fetal blood sampling. Such prenatal screens, as the triple screen, can be used either to reduce the need for amniocentesis or to increase genetic defect detection for the same amount of amniocentesis. "The efficiency of the Triple test is projected to be one case of fetal Down syndrome detected for every 50 amniocenteses performed." [Canick and Knight, "Multiple-marker Screening for Fetal Down Syndrome," Contemporary OB/GYN, pp. 3-12 (April 1992).]

Although pregnant women who are 35 years or older are the standard high risk group for fetal Down syndrome affected pregnancies, screening also needs to be applied to young women because although they are at lower risk, most affected pregnancies are in young women. Approximately 80% of babies born with Down syndrome are born to mothers under 35. ["Down Syndrome Screening Suggested for Pregnant Women under 35," ACOG Newsletter, 38(8): 141 (Aug. 1994).]

The triple screen combines the analysis of three markers from serum to reduce false positive results (which result in the performance of unnecessary invasive procedures) and false negatives (in which serious genetic defects, such as, trisomy 21, go undetected). In women under 35, the double screen (AFP and hCG) can pick up about half of Down syndrome cases and a large proportion of other chromosome defects during the second trimester. The triple screen (AFP, hCG and uE.sub.3) increases the detection rate by 5-10% of Down syndrome and a further increase in the detection of all other serious chromosome defects, thus decreasing the number of false-positives. However, such rates mean that the double and triple screens still fail to detect a significant number of Down syndrome and other aneuploidy affected pregnancies.

Although the triple screen has a suggested screening period of 15 to 20 weeks gestation, such screening has been recommended between weeks 16-18 to maximize the window for spinal bifida detection. [Canick and Knight, supra (April 1992).] A 1992 survey of prenatal maternal serum screening for AFP alone or for multiple analyses reported that very few such screenings occurred in the thirteenth or earlier week of gestation. [Palomaki et al., "Maternal Serum Screening for Fetal Down Syndrome in the United States: A 1992 Survey," Am. J. Obstet. Gynecol., 169(6): 1558-1562 (1992).] The triple screen thus suffers from the additional problem that once a risk of a genetic defect is predicted, and amniocentesis or another invasive prenatal definitive diagnostic procedure is performed to diagnose the genetic defect, such as Down syndrome, it is at an advanced date of gestation, when termination of a pregnancy can be more physically and emotionally trying for the mother, and when certain less traumatic abortion procedures, such as, vacuum curettage, may not be available.

The limitations of the triple screen and the adverse consequences of unnecessary, potentially harmful and expensive invasive prenatal diagnostic procedures, such as, amniocentesis, have led to a search for more discriminatory markers for prenatal screening of Down syndrome and other aneuploidies. Of the maternal serum markers in routine use, human chorionic gonadotropin (hCG) is by far the most discriminatory. HCG is a glycopeptide hormone produced by the syncytiotrophoblasts of the fetal placenta, and has a molecular weight of about 38 kilodaltons (kd). It can be detected by immunoassay in the maternal urine within days after fertilization and thus provides the basis of the most commonly used pregnancy tests.

The intact hCG molecule is a dimer comprising a specific .beta. subunit (145 amino acids) non-covalently bound to an .alpha. subunit (92 amino acids), which is common to other glycoproteins. Maternal serum levels of both intact hCG and the free .beta.-subunit are elevated on average in Down syndrome but the extent of elevation is greater for free .beta.-hCG [Spencer, K., Clin. Chem., 37: 809-814 (1991); Spencer et al., Ann. Clin. Biochem., 29: 506-518 (1992); Wald et al., Br. J. Obstet. Gynaecol., 100: 550-557 (1993)]. HCG is detected in the serum and urine of pregnant women, as are the free .alpha.- and .beta.-subunits of hCG, and degradation products of hCG and of free .beta.-subunit hCG.

The terminal degradation product of the .beta.-subunit of hCG is called .beta.-core-hCG, or alternatively .beta.-core fragment, .beta.-core, urinary gonadotropin peptide (UGP), or urinary gonadotropin fragment (UGF). .beta.-core-hCG is excreted into urine [Nislua et al., J. Steroid. Biochem., 33: 733-737 (1989); Cole et al., J. Clin. Endocrinol. & Metab., 76: 704-710 (1993)].

.beta.-core-hCG has been found in the urine of pregnant women carrying normal fetuses, and also in the urine of patients with gestational trophoblastic and non-trophoblastic malignancies [Cole et al., "Urinary Human Chorionic Gonadotropin Free B-subunit and B-core Fragment: A New Marker of Gynecological Cancers," Cancer Res., 48: 1356-1360 (1988); Cole et al., "Urinary Gonadotropin Fragments (UGF) in Cancers of the Female Reproductive System," Gynecol. Oncol., 31: 82-90 (1988); O'Connor et al., "Development of Highly Sensitive Immunoassays to Measure Human Chorionic Gonadotropin, Its .beta.-subunit, and .beta.-core Fragment in the Urine: Application to Malignancies," Cancer Res., 48: 1361-1366 (1988); and Akar et al, "A Radioimmunoassay for the Core Fragment of the Human Chorionic Gonadotropin .beta.-subunit," J. Clin. Endocrinol. and Metab., 66: 538-545 (1988).] .beta.-core-hCG has also been found to be associated with certain ovarian cancers. [Cole and Nam, "Urinary Gonadotropin Fragment (UGF) Measurements in the Diagnosis and Management of Ovarian Cancer," Yale J. Bio. and Med., 62: 367-378 (1989).]

The invention disclosed herein concerns the finding that .beta.-core-hCG levels in urine samples taken from pregnant women can be used for prenatal screening to detect fetal aneuploidies. .beta.-core-hCG levels in such maternal urine samples were found on average to be elevated above normal in pregnancies affected by fetal Down syndrome, Turner syndrome, Klinefelter syndrome and triple-X, most notably in Down syndrome cases, and to be reduced in the presence of other serious aneuploidies such as, Edwards syndrome and triploidy. The observed median level in Down syndrome affected pregnancies in the second trimester (6.11 MOM: 95% confidence interval 3.7 to 10.0) has been found to be over three times greater than the corresponding median level for intact hCG in maternal serum (2.0 MOM; 1.9-2.1) and free .beta.-hCG (2.3 MOM; 2.1-2.5).

Further, the urinary screening methods of this invention provide higher detection rates of aneuploidies than the maternal serum screening methods. For example, as shown herein (Example 2), testing for .beta.-core-hCG levels in the second trimester alone generates a detection rate of fetal Down syndrome of about 80% at a 5% false-positive rate. Further development of the methods of this invention is expected to increase that detection rate. In comparison, teting for maternal serum hCG or free .beta.-hCG alone generates no more than a 45% detection rate at a 5% false-positive rate [Wald et al., Br. Med. J., 297: 883 (1988); Spencer, K., Clin. Chem., 37: 809 (1991)]. The present standard methods of maternal serum screening with multiple biochemical markers, which include various combinations of AFP, hCG, free .beta.-hCG, free .alpha.-hCG and uE.sub.3 used in conjunction with maternal age can optimally detect 72% of Down syndrome cases at a 5% false-positive rate [Wald et al., Prenat. Diagn. 14: 707 (1994)].

This invention in providing a means of prenatal screening using urinalysis instead of serum testing has important advantages. Urine tests are less expensive than serum testing, avoid the safety issues and handling risks associated with the collection and storage of blood samples, as well as the invasiveness and discomfort of phlebotomy. Urine samples can be easily collected and shipped, if necessary, where women have limited access to medical testing facilities because of geography or socio-economic status. .beta.-core-hCG is stable to changes in temperature, pH, and storage time at -20 and 40.degree. C. Thus, the methods of this invention provide for more discriminatory, cheaper, less invasive and more geographically accessible means for prenatal screening for fetal aneuploidies, than had been provided by former methods of screening based on maternal serum markers.

Further, the maternal urine screening methods of the instant invention can be used not only in the second trimester as maternal serum screening methods are predominantly used, but also in the first trimester. As indicated above, there are disadvantages to second trimester testing, in that delays in confirming a fetal aneuploidy diagnosis result in more traumatic abortion procedures being necessitated. Also, the emotional attachment and expectations of the pregnant woman and her family for a healthy baby, grow during the pregnancy, making the abortion decision more difficult later in the gestational term. The instant invention provides the benefits of urinalysis and may also avoid the problems of second trimester prenatal screening. Thus, the instant invention represents a significant advance in the field of prenatal diagnosis.

SUMMARY OF INVENTION

The instant invention provides improved methods of prenatal screening for fetal aneuploidies by detecting and quantitating .beta.-core-hCG in maternal urine samples and comparing the levels to those found in maternal urine samples from normal pregnancies at the same gestational age. The methods for determining .beta.-core-hCG levels in maternal urine samples can comprise antibody and non-antibody methods, but preferably comprise the use of immunoassays.

The examples herein show that urinary .beta.-core-hCG levels are elevated on average in. pregnancies affected by fetal Down syndrome and may be reduced or elevated in the presence of other less common but serious aneuploidies. In Example 1, the observed median level in Down syndrome (6.11 MOM; 95% confidence interval 3.7-10.0) is considerably greater than the corresponding median level for maternal serum intact hCG (2.0 MOM; 1.9-2.1) and free .beta.-hCG (2.3 MOM; 2.1-2.5) derived from all published studies combined [Cuckle and Lilford, Br. Med. J., 305: 1017 (1992)]. The findings thus indicate that urinalysis is a candidate as a routine screening modality for prenatal screening for aneuploidy.

Representative fetal aneuploidies for which the methods of the instant invention screen include Down syndrome, Edwards syndrome, triploidy, Klinefelter syndrome, Turner syndrome, and triple-X. On average, .beta.-core-hCG levels are elevated above normal in maternal urine samples from pregnancies affected by Down syndrome, Turner syndrome, Klinefelter syndrome and triple-X, and .beta.-core-hCG levels are lower than normal in maternal urine samples from pregnancies affected by Edwards syndrome and triploidy.

Maternal urine samples are preferably tested according to the methods of this invention in the first or second trimesters. The risk assessment of fetal aneuploidy may be based upon the .beta.-core-hCG level in a maternal urine sample alone or in conjunction with the level of one or more other urinary markers in said urine sample and/or in conjunction with the level of one or more serum markers in a serum sample from the woman carrying the pregnancy under analysis, wherein an abnormal level of any of the urinary or of any of the serum markers indicates an increased risk of fetal aneuploidy. Further, the risk assessment may be made based upon the level of .beta.-core-hCG in said urine sample in conjunction with other factors, as for example, maternal age wherein the older the maternal age, the greater the risk of fetal aneuploidy. A further example of other factors that can be used in conjunction with UGP levels in maternal urine samples to assess the risk of pregnancies being affected by fetal aneuploidies are ultrasound markers, wherein an abnormal ultrasound marker indicates an increased risk of fetal aneuploidy.