The diagnosis of pregnancy has taken on greater importance in recent years as advanced reproductive technologies have become more commonplace and the ability to medically treat early extrauterine pregnancies has become a safe reality. Sensitive biochemical assays and high-resolution ultrasonography now make the diagnosis of pregnancy highly sensitive and specific. This chapter reviews the clinical signs and symptoms of pregnancy, salient features of human implantation, human chorionic gonadotropin (hCG) hormone synthesis and secretion in normal and aberrant pregnancies, and other biochemical and technical markers for the diagnosis of pregnancy.

The patient history and physical examination play important roles in the diagnosis of pregnancy. Clinical signs and symptoms are often the earliest indication of pregnancy and should be considered first in the evaluation of patients. Abnormalities in menstruation, specifically amenorrhea, serve as the most common clinical marker for pregnancy in women who typically have regular menstrual cycles. Menstrual irregularities other than amenorrhea can also occur in pregnancy, and pregnancy should be a consideration in any woman who displays menstrual aberrations. More importantly, if irregular menses occur in the face of pregnancy, one must consider an extrauterine or nonviable intrauterine pregnancy. Biochemical testing and ultrasonography can help in the differentiation of these conditions. Abdominal enlargement, caused by the growing uterus, reaches the umbilicus by 20 weeks' gestation. Fetal movement can usually be perceived by 18 weeks' gestation. Breast tenderness, nausea, vomiting, and urinary complaints can also occur (Table 1). However, physical symptoms are not sufficiently reliable to diagnose pregnancy.¹

TABLE 1. Clinical Symptoms of Pregnancy

  Amenorrhea
  Abdominal enlargement
  Fetal movement
  Breast tenderness
  Nausea
  Vomiting
  Urinary complaints

Positive signs of pregnancy include identification of fetal heartbeat, maternal perception of fetal movement, and ultrasonographic demonstration of pregnancy (Table 2). The demonstration of a fetal heartbeat by auscultation, Doppler technology, or sonography suffice as a positive sign of pregnancy. Auscultation of a fetal heartbeat can usually be achieved by 19 weeks’ gestation in most pregnancies, and fetal heartbeat can be discerned by 10 weeks’ gestation using Doppler ultrasound devices. Because of the relative rapidity of the fetal heart rate, fetal and maternal heartbeats are easily differentiated.

TABLE 2. Clinical Signs of Pregnancy

  Identification of fetal cardiac action
  Perception of fetal movements
  Ultrasonic recognition of pregnancy

Another sign of pregnancy is the perception of fetal movement by the examiner through the placement of his or her hands on the maternal abdomen. Fetal movement can be detected after the 20th week of pregnancy. Fetal movements are variable in intensity and can occasionally be visualized.

A third positive sign is the ultrasonographic demonstration of pregnancy. Ultrasonographic evidence of pregnancy can be seen as early as 4 to 5 weeks’ gestation using menstrual dates. Fetal cardiac activity can be seen by 6 menstrual weeks’ gestation, and the fetal brain can be seen by 8 weeks. The crown-rump length can be used accurately to assess gestational age within 4 days up to approximately 12 weeks.

Implantation, or the process by which the embryo comes in contact, adheres, and penetrates the endometrium, is necessary prior to the diagnosis of pregnancy.² First contact between the blastocyst and the endometrium occurs 6 days after fertilization. This is known as apposition. Soon after apposition, the blastocyst becomes adherent to the endometrium, and the process of implantation has begun. Various molecules play an active role in this process. Laminin, a basement membrane glycoprotein involved in tumor invasion and possibly embryo adhesion and implantation, is expressed in human embryos by day 3 after fertilization.³ In humans, the first signs of blastocyst attachment occur on day 8.⁴ β₁-Integrin, a receptor subunit that interacts with extracellular matrix proteins, is expressed in human embryos from day 2 and is also involved with invasion of the cytotrophoblast.⁵

Human Chorionic Gonadotropin Hormone Synthesis and Secretion in Normal and Aberrant Pregnancies

hCG is a glycoprotein secreted by the syncytiotrophoblast with a molecular weight of about 36,700 d. The molecule contains about 30% carbohydrate, which is the highest concentration of carbohydrate moiety of any human hormone.⁶ hCG is the hormone that classically has been measured to diagnose pregnancy. It is composed of an α and β subunit, which are noncovalently linked. The β subunit confers specific activity to the hormone and is the subunit most commonly measured in most pregnancy assays. There exists much homology between hCG and luteinizing hormone (LH), especially with respect to the first 121 amino acids of the β subunits of both hormones, which have about 80% homology. hCG has a 24-amino acid extension on the carboxyl-terminal end that is lacking in the LH β subunit.⁷ hCG can be detectedby molecular techniques in human embryo culture at the 8-cell stage; however, detection of hCG in the plasma is not possible until implantation has occurred, approximately 10 days after the LH surge. Typically, β-hCG doubles approximately every 36 hours and peaks at about 100,000 mIU/ml at 10 weeks' gestation,⁶ after which the level decreases to about 20,000 mIU/ml by midpregnancy, where it remains until term. β-hCG is not diagnostic of only normal pregnancy (Table 3). Abnormal elevations, plateaus, or decreasing titers of β-hCG raises the possibility of ectopic pregnancy or miscarriage. The use of the assay in this context typically requires other modalities such as ultrasound, serum progesterone levels, or both.

TABLE 3. Characteristics of Human Chorionic Gonadotropin

  Glycoprotein
  Composed of 30% carbohydrate
  α and β Subunits covalently bonded
  80% Homology with luteinizing hormone
  Produced by embryo at 8-cell stage
  Produced by syncytiotrophoblast 10 days after luteinizing hormone surge
  Peaks at about 10 weeks’ gestation (~100,000 mIU/ml)
  Level falls after 10 weeks until term (~20,000 mIU/ml)

Human Chorionic Gonadotropin Hormone Assays

The first international standard for hCG was established in 1938; however, when stock standards ran low, the second international standard was established.⁸ However, because of its relative impurity and heterogeneity, the more pure International Reference Preparation (IRP) was developed in 1980.⁹ The numerical value of the IRP in international units (IU) is about twice that of the second international standard.⁹

The early hCG assays were bioassays that consisted of injecting an animal, usually a rabbit, with the urine of the possibly pregnant women. Various end points such as increased prostate weight, seminal vesicle weight, and gain in uterine weight (depending on the gender of the rabbit) were measured. These assays were expensive and time consuming and lacked reliability.

The immunoassay provided greater ease compared with the bioassay, but it was plagued with high cross-reactivity. The standard assay was performed by mixing a known amount of anti-hCG with the patient's urine. If hCG was present in the urine, it would bind to the anti-hCG and leave no free anti-hCG to bind to hCG-coated red blood cells. In this situation no agglutination would occur, indicating a positive test result. A negative test result would be marked by agglutination of the red cells, indicating binding between the added anti-hCG and the hCG-coated red cells. The sensitivity of this assay was 150 mIU/ml.

The radioimmunoassay (RIA) was perhaps the most popular assay for hCG. This assay involved competition between radioactively labeled and unlabeled antigens for binding sites on an antibody that was highly specific for the hCG antigen.¹¹ The disadvantage of the RIA is a relatively long turnaround time of 4 to 6 hours.

The enzyme-linked immunosorbent assay (ELISA) is a quick, easy method of hCG determination. This assay uses highly specific monoclonal antibodies for hCG. The sensitivity of this assay is as low as 5 mIU/ml, and hCG can be detected several days before a missed menses. The ELISA is based on a reaction between anti-α-hCG monoclonal antibody that is attached to a solid phase and the α subunit of hCG in the patient's urine. This reaction creates a complex that leaves the β-hCG subunit exposed. An enzyme-linked monoclonal antibody to the β subunit is then added to the reaction, forming an antibody-hCG-antibody-enzyme complex. Excess reagent is washed away, allowing the remaining enzyme to react with its substrate to form a colored product. The colorimetric reaction is then quantitated. Most qualitative tests use this technology.¹² Most home pregnancy tests are based on urinary ELISA technology. Concern has been expressed regarding the accuracy of such kits, but this probably reflects the technique of the user rather than inadequacy of the kit itself.¹³ It is estimated that home pregnancy tests have a 10% false-positive and false-negative rate.

Urinary and Serum Follicle-Stimulating Hormone

Total urinary and serum follicle-stimulating hormone (FSH) β subunit levels in the postovulatory period are lower in conception cycles that nonconception cycles. It has been shown by Qui and colleagues that mean serum and urinary FSH levels rose significantly above the postovulatory baseline by day 10 to 12 following the midcycle LH peak in nonconception cycles but did not rise at any time following ovulation in conception cycles.¹⁴ It was reported that sensitivity and specificity of urinary FSH to detect pregnancy were 88.9% and 89.3%, respectively.

Early Pregnancy Factor

Early pregnancy factor (EPF) has been studied as an alternative to β-hCG determination, because it can be detected in the blood prior to hCG.¹⁵ EPF is a placental protein and is one of the first pregnancy markers known to appear in the blood.

Modern ultrasound technology, especially transvaginal techniques, have markedly enhanced the diagnosis and prognosis of early pregnancy. Although ultrasound alone has not replaced biochemical testing for the diagnosis of early pregnancy, in has greatly improved the differentiation of normal versus abnormal intrauterine pregnancies and the determination of extrauterine pregnancies.¹⁶

Sonographic Methodology

Transvaginal ultrasound allows early detection of pregnancy. A chorionic gestational sac can be detected with this method at a discriminatory zone of about 1400 mIU/ml and 6500 mIU/ml by transabdominal scanning^17,18 by the IRP. Practitioners must be aware of the assay standard being used for β-hCG determination in their laboratory. The second international standard will have a discriminatory zone of about 50% less than the IRP. With this in mind and a keen sense of clinical awareness, aberrant pregnancies can be reliably detected.

Normal and Aberrant Pregnancy

Using transvaginal ultrasonography, Bree and colleagues were able to discern a gestational sac, yolk sac, and fetal cardiac activity at β-hCG levels of 1025, 7200, and 10,800 mIU/ml IRP, respectively²⁰ (Table 4). More recently, ultrasonography of the chorionic rim, which is the echogenic border of the gestational sac, has been used to diagnose early intrauterine pregnancies. This sonographic technique demonstrates a sensitivity of 80% and a specificity of 97% in diagnosing early intrauterine pregnancy.²¹

TABLE 4. Sensitivity of Transvaginal Ultrasound in the Detection of Pregnancy

Ectopic pregnancies can now be diagnosed much earlier than was previously possible with the advent of transvaginal ultrasonography. This technology has a sensitivity of 100%, a specificity of 98%, a positive predictive value of 98%, and a negative predictive value of 98%.²² Lack of a gestational sac by day 35 of the menstrual cycle or when the β-hCG level is 1025 mi/ml is associated with an increased risk of ectopic pregnancy. Pseudogestational sacs, which represent a fluid collection within the endometrial cavity in conjunction with an ectopic pregnancy, must be considered and should not be confused with a normal gestational sac which should be located immediately adjacent to the endometrial cavity.²³

New Diagnostic Methods of Pregnancy for Assisted Reproduction

Ultrasonographic assessment of endometrial thickness in patients undergoing in vitro fertilization (IVF) predict higher pregnancy rates when the endometrial thickness is 10 mm.²⁴ Sonography has also been used to predict uterine receptivity in women undergoing assisted reproduction. Based on the uterine receptivity index, Serafini and colleagues demonstrated a mathematical equation involving the sonographic endometrial pattern, thickness, diastolic blood flow, and resistance index to be predictive of pregnancy outcome in women undergoing ovulation induction with luprolide acetate and human menopausal gonadotropins.²⁵

The diagnosis of pregnancy begins with astute clinical awareness. The signs and symptoms of the condition stimulate suspicion, which should then be followed by a qualitative and/or quantitative biochemical assay for confirmation. Ultrasound will verify the location of the pregnancy and rule out aberrant implantation. Newer biochemical tests are on the horizon for the early, effective diagnosis of pregnancy.

1. Bastian LA, Piscitelli JT. Is this patient pregnant? Can you reliably rule in or rule out early pregnancy by clinical examination? JAMA 1997;278:586–91.

2. Enders AC, Schlafke S. Cytological aspects of trophoblast-uterine interaction in early implantation. Am J Anat 1969;125:1–30.

3. Turpeeniemi-Hujanen T, Ronnberg L, Kauppila A, Puistola U. Laminin in the human embryo implantation: analogy to the invasion by malignant cells. Fertil Steril 1992;58: 105–13.

4. Enders AC, Schlafke S. Implantation in nonhuman primates and in the human. Comp Primate Biol 1986;3: 291–310.

5. Damsky CH, Fitzgerald ML, Fisher SJ. Distribution patterns of extracellular matrix components and adhesion receptors are intricately modulated during first trimester cytotrophoblast differentiation along the invasive pathway in vivo. J Clin Invest 1992;89:210–22.

6. Cunningham FG, MacDonald PC, Gant NF, eds. The placental hormones. In: Williams’ Obstetrics, 19th ed. Norwalk, CT, Appleton and Lange, 1993;139–64.

7. Jameson JL, Hollenberg AN. Regulation of chorionic gonadotropin gene expression. Endocr Rev 193;14:203–21.

8. Bangham DR, Grab B. The second international standard for chorionic gonadotropin. Bull WHO 1964;31:111.

9. Storring PL, Gaines-Das RE, Bangham DR. International reference preparation of human chorionic gonadotropin for immunoassay: potency estimates in various bioassay and protein binding assay systems; and international reference preparations of the alpha and beta subunit of human chorionic gonadotropin for immunoassay. J Endocrinol 1980;84: 295.

10. Rasor JR, Farber S, Braunstein GD. An evaluation of 10 kits for the determination of human chorioniogonadotropin in serum. Clin Chem 1983;29:1828.

11. Vaitukaitis JL, Braunstein GD, Ross GT. A radioimmunoassay which specifically measures human chorionic gonadotropin in the presence of human luteinizing hormone. Am J Obstet Gynecol 1972;113:751.

12. Bandi ZL, Schoen I, DeLara M. Enzyme-linked immunosorbent urine pregnancy test. Am J Clin Pathol 1987;87:236.

13. Hicks JM, Iosefsohn M. Reliability of home pregnancy-test kits in the hands of laypersons [letter]. N Engl J Med 1989;320:320.

14. Qui Q, Overstreet JW, Todd H, Nakajima ST, Stewart DR, Lasley BL. Total urinary follicle stimulating hormone as a biomarker for detection of early pregnancy and periimplantation spontaneous abortion. Environ Health Perspect 1997;105:862–6.

15. Sinosich MJ, Grudzinskas JG, Saunders DM. Placental proteins in the diagnosis and evaluation of the “elusive” early pregnancy. Obstet Gynecol Surv 1985;40:273.

16. Fossum GT, Davajan V, Kletzky OA. Early detection of pregnancy with transvaginal ultrasound. Fertil Steril 1988; 49:788.

17. Kadar N, DeVore G, Romero R. Discriminatory hCG zone: its use in the sonographic evaluation for ectopic pregnancy. Obstet Gynecol 1981;58:156.

18. Nyberg DA, Filly RA, Mahony BS. Early gestation: correlation of hCG levels and sonographic identification. AJR 1985;144:951.

19. Bratzer FR, Weiner S, Corson SL. Landmarks during the first forty days of gestation demonstrated by the beta-subunit of human chorionic gonadotropin and ultrasound. Am J Obstet Gynecol 1983;146:973.

20. Bree RL, Edward M, Bohm-Velez M. Transvaginal sonography in the evaluation of normal early pregnancy: correlation with HCG level. AJR Am J Roentgenol 1989; 153:75.

21. Parvey HR, Dubinsky TJ, Johnston DA, Maklad NF. The chorionic rim and low impedance intrauterine arterial flow in the diagnosis of early intrauterine pregnancy: evaluation of efficacy. AJR Am J Roentgenol 1996;167:1479- 85.

22. Timor-Tritsch IE, Yeh MN, Peisner DB. The use of transvaginal ultrasonography in the diagnosis of ectopic pregnancy. Obstet Gynecol 1989;161:157.

23. Sabbagha RE, Cohen LS, Karins MR, Longley JV. Early pregnancy evaluations. In: Sabbagha RE, ed. Diagnostic Ultrasound Applied to Obstetrics and Gynecology. Philadelphia, JB Lippincott, 1993.

24. Rinaldi L, Lisi F, Floccari A, Lisi R, Pepe R, Fishel S. Endometrial thickness as a predictor of pregnancy after in vitro fertilization but not intracytoplasmic sperm injection. Hum Reprod 1996;11:1538–41.

25. Serafini P, Nelson J, Batzofin J, Olive D. Preovulatory sonographic uterine receptivity index (SURI): usefulness as a predictor of pregnancy in women undergoing assisted reproductive treatments. J Ultrasound Med 1995;14:751–5.