The Diagnosis of Pregnancy

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's 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 should be easily differentiated.

Table 2. Clinical signs of pregnancy

  Identification of fetal cardiac action
  Perception of fetal movements
  Ultrasonographic 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 4–5 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 with, adheres to, 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 A host of molecular mediators are involved and are integral to the implantation process which is central to the diagnosis of pregnancy.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 Da. 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 detected by 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, the level of β-hCG doubles approximately every 36 hours and peaks at about 100,000 mIU/ml at 10 weeks' gestation,⁶ after which it 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 Nevertheless, 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.¹⁰ The disadvantage of the RIA is a relatively long turnaround time of 4–6 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.¹¹ 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.¹² 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 than in 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 10–12 following the midcycle LH peak in nonconception cycles but did not rise at any time following ovulation in conception cycles.¹³ 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.¹⁴ 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.¹⁵

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 at about 6500 mIU/ml by transabdominal scanning^{16, 17} 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, respectively¹⁸ (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.¹⁹

Table 4. Sensitivity of transvaginal ultrasound in the detection of pregnancy

β-hCG, β human chorionic gonadotropin; IRP, International Reference Preparation.

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%.²⁰ Lack of a gestational sac by day 35 of the menstrual cycle or when the β-hCG level is 1025 mIU/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.²¹

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.²² 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 leuprolide acetate and human menopausal gonadotropins.²³

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.