Embryonic Development and Pregnancy Test Sensitivity: The Importance of Earlier Pregnancy Detection

Abstract

Embryogenesis research provides information on a time of heightened vulnerability in embryo development: the time from conception to the day a woman misses her menstrual period. During this period, it is vital for the woman to be aware of exposures, behaviors and nutritional factors that could negatively impact the developing embryo. This article discusses this critical, often-overlooked window of development and will review the various types of teratogens that affect pregnancy. Over-the-counter pregnancy tests are widely used to determine pregnancy status. Earlier test models detected only the pregnancy hormone human chorionic gonadotropin in the urine, but it is now known that there are other key forms of human chorionic gonadotropin that are relevant when determining pregnancy status. This article will explain why early knowledge of pregnancy status is important for both woman and embryo, and the role that patient education and pregnancy test choice can have on normal embryonic development.

Keywords

embryonic development home pregnancy test hyperglycosylated hCG teratogen

Early knowledge of pregnancy status is important for the health of the woman and the developing embryo. The sooner a woman knows her pregnancy status, the sooner she can begin making informed choices for herself and her embryo, and the sooner she can seek prenatal care. This is especially important in women who were not planning to become pregnant, for these patients have most likely taken no steps toward healthy conception and are most in need of prenatal care. 60 million unintended pregnancies occur each year [1,2]. The prevalence of unintended pregnancies is high, for instance it is 50% of all pregnancies in the USA [3], 33% of those in France [4], and 28% of those in Edinburgh, Scotland [5,6].

Healthcare providers (HCPs) should stress to their patients of childbearing age the importance of being aware of their pregnancy status. For those planning on becoming pregnant, organizations such as the March of Dimes and the American College of Obstetricians and Gynecologists (ACOG) emphasize the importance of preconception care – interventions designed to lower risks prior to conception [7,101]. In a planned pregnancy, women can initiate an appropriate diet and avoid foods, medications and chemicals that may be harmful to a developing fetus. In addition, they can adopt healthy behaviors and actions, such as taking folic acid supplements at least 1 month prior to conception [8]. Daily intake of folic acid has been shown to reduce the occurrence of neural tube defects by 50–70% [102], and recent research points to reduced rates of other congenital abnormalities among babies born to women who took folic acid supplements [9].

Planning for pregnancy allows women to adopt healthy behaviors early in the embryo's development; however, not all pregnancies are planned [1,2], which is why HCPs should give all patients of childbearing potential some basic education regarding pregnancy, particularly if they are in a high-risk group for pregnancy failure or are receiving treatment that may have an adverse effect upon embryonic development [7,10]. Included in this patient education should be information pertaining to the use of over-the-counter (OTC) home pregnancy tests, particularly with regard to their levels of sensitivity and reliability.

Embryogenesis

The moment of conception and the 2 weeks following have been referred to as the ‘all or nothing’ period. It is assumed that most cells of the conceptus, if damaged, will be replaced by normal cells, eventually leading to normal development, or that if normal development is not possible embryonic death will occur [10 –12]. However, several studies indicate that the first few days of gestation can result in malformations, not just a simple live-or-die end point [11,12]. This 2-week period can be considered the foundation of the embryo, from which a fetus will eventually evolve. Thus, it is vital that the initial building blocks be as healthy and viable as possible.

During the second week post-conception, some important developments occur. On days 7–8, the embryo begins to develop and separate into the ectodermal (the outermost of the three layers of the embryo) and endodermal (the innermost of the three layers from which will come the digestive and lower respiratory tracts) layers. On days 9–10, the cytotrophoblast (i.e., extravillous cytotrophoblast) cells work their way into the maternal tissue in the uterus to form the outermost fetal component of the placenta. It is the cytotrophoblast cells that make human chorionic gonadotropin (hCG), which promotes metalloproteinases and controls the invasion process [13,14]. On days 11–12, the trophoblast begins to be molded into villi, which will constitute the developing placenta. It is at this time that the mesodermal layer (which will be the source of bone, muscle, connective tissue and dermal development) arises between the ectodermal and endodermal layers [12,15,16].

Teratogens

A teratogen is an agent that can cause structural or functional abnormalities, including birth defects, in a developing embryo or fetus [10]. HCPs should counsel women of childbearing age on the dangers of teratogens and of the different categories of agents that exist (such as medications, behaviors/exposures, chronic/infectious diseases and genetic conditions) [10,17]. Some patients should be made aware that genetic counseling may be necessary if they are planning to become pregnant after 35 years of age, or if they are in a particular ethnic group that has a proclivity for certain genetic conditions [103]. Unfortunately, because many pregnancies are unplanned [1,2] women often do not have the information necessary to protect the health of the developing embryo, and by the time a pregnant woman presents for her first early prenatal visit, most fetal organs have already been formed and, thus, it may be too late to intervene and minimize birth defects [7].

Medications

Lewis B Holmes, Professor of Pediatrics at Harvard Medical School and Chief of the Genetics and Teratology Unit, Pediatric Services, at Massachusetts General Hospital, Boston, MA, USA (where the first North American registry for pregnant women taking antiepileptic drugs is being maintained) has written extensively on medication-induced teratogens and notes that while many are well-documented, ‘any list of probable human teratogens is arbitrary, reflects value judgments, and must be updated frequently’ [18]. A very partial list of teratogenic medications and their associated birth defects can be found in Table 1. These medications are proven to result in severe birth defects, for example, isotretinoin is considered so teratogenic that even a single dose can cause major birth defects [104].

Table 1.

Teratogenic drugs.

Medication	Teratogenic risk	Ref.
Coumarin derivatives	Effects on phalange and limb morphogenesis, marked nose hypoplasia	[18]
Isotretinoin	Hydrocephaly, microencephaly, mental retardation, ear and eye abnormalities, cleft palate and other facial abnormalities, heart defects, learning disabilities	[19]
Misoprostol	Cranial nerve hypoplasia, terminal transverse defects in arms and leg, club foot, other risks	[18]
Thalidomide	Effects on limb and phalange morphogenesis, ear malformations, heart defects, bowel atresia, cranial nerve dysfunction, abnormal teeth	[18]
Valproic acid derivatives (antiepileptics)	Effects on phalange and limb morphogenesis, stiffness of the distal interphalangeal joint, spina bifida, midface hypoplasia, heart defects, abdominal wall defects, limb defects	[18]

Behaviors

Teratogenic behaviors include: eating disorders, illicit drug use, cigarette smoking and alcohol consumption [7]. Despite a major public health campaign in the USA alerting women to the dangers of smoking while pregnant, more than 13% of women continue to do so [20]. In Germany, one study found that 25% of women continue to smoke during pregnancy [21] and a survey in the UK found that 17% of pregnant women reported smoking during their pregnancy [105]. Cigarette smoking during pregnancy has been associated with a wide range of fetal and birth abnormalities, among them cognitive dysfunction, oral clefts, abnormal placentation, growth retardation, club foot, and in later years an increased risk of asthma [18,22]. As with cigarette smoking, heavy alcohol consumption during pregnancy is associated with increased frequency of several malformations, a well-delineated pattern of facial features, growth retardation and microencephaly [18]. Unfortunately, despite attempts to educate the public regarding the dangers of drinking while pregnant, in the USA approximately one in 12 pregnant women continue to drink alcohol during pregnancy, with almost one in 30 consuming five or more drinks at a time [23]. This type of heavy drinking greatly increases alcohol-related damage to the fetus [23]. Drinking while pregnant is a global problem. In a study of almost 19,000 women in Japan, 44.6% drank before receiving confirmation of pregnancy and 4.6% continued to drink even after confirmation [24]. In the UK, one study reported that 70% of mothers continued to drink during pregnancy, although most of these (71%) consumed on average less than 1 unit of alcohol per week [106].

Chronic diseases

A number of chronic diseases, including heart disease, high blood pressure, diabetes, obesity and asthma (see Figure 1 for their prevalence), should be carefully considered during preconception as they can have an adverse impact on fetal development [7]. Infectious diseases (Table 2) and certain genetic disorders (Box 1) can also have adverse effects upon fetal development. During the preconception and prenatal period, HCPs should weigh the benefits and consequences for the woman and the fetus when treating preexisting conditions [10]. In some cases, substituting the patient's current pharmacologic regimen with agents that are safe for the growing fetus may be a viable solution.

Table 2.

Teratogens: infectious diseases*.

Vaccine-preventable diseases	Non-vaccine-preventable diseases
Hepatitis B	Cytomegalic inclusion virus
Influenza‡	HIV/AIDS
Rubella	Periodontal disease
Tetanus	Sexually transmitted diseases (including chlamydia and syphilis)
Varicella	Toxoplasmosis

This list is not exhaustive.

‡

Not 100% effective.

Data taken from [7].

Figure 1.

Prevalence of chronic conditions in women of reproductive age (18 to 44 years).

Diabetes

Both Type 1 and Type 2 diabetes are a growing problem in the USA, and diabetes is also a significant concern in pregnant women. Pre-existing diabetes is often exacerbated by pregnancy and almost 4% of nondiabetic women become diabetic during pregnancy [26,27,108,109]. In one study, rates of perinatal mortality (25/1000) and congenital malformations (99/1000) were found to be significantly higher among women with treated Type 2 diabetes compared with healthy pregnant women [28]. Although measures for controlling maternal glucose levels exist and it is known that they can help decrease birth defects, overall rates of congenital malformations have not dropped substantially [29]. Women with a family history of diabetes or those with high risks for diabetes should be particularly cautious about preparing for and detecting early pregnancies.

Asthma

Maternal asthma during pregnancy has been shown to increase rates of perinatal mortality, pre-eclampsia, preterm delivery and low birth weight [30], and has been reported to affect 3.7-8.4% of pregnant women in the USA [31]. National Heart, Lung, and Blood Institute (NHLBI) guidelines emphasize the importance of adequate asthma control during pregnancy, and recommend that pregnant women be treated with asthma medication rather than experience asthma symptoms and exacerbations [30]. There are also published guidelines that recommend specific medications and individualized approaches to caring for pregnant women with asthma [30,32].

Hypertensive disorders

Coronary and vascular disorders in pregnancy are another major cause of maternal, fetal and neonatal morbidity and mortality [33].

Box 1.

Teratogens: genetic and inherited disorders*.

Canavan's disease [25]

Cystic fibrosis [7]

Down syndrome [7]

Fragile X syndrome [7]

Hearing and vision loss associated with genetic predisposition [7]

Muscular dystrophy [7]

Sickle cell anemia [7]

Tay-Sachs disease [7]

Thalassemia [7]

This list is not exhaustive.

Hypertension in pregnancy is classified into one of five categories, depending on the severity of the hypertension and whether hypertension onset preceded pregnancy or was pregnancy induced [33]. Severe chronic hypertension has been reported to lead to significant maternal mortality and fetal loss rates of 50% at less than 37 weeks of gestation. Pre-eclampsia occurs in approximately 25% of women with chronic hypertension and is associated with intrauterine growth restriction and fetal distress, as well as maternal morbidities such as hemolysis, elevated liver enzymes, low platelet count and deteriorating renal function [33]. Hypertensive disorders require aggressive treatment as early as possible during and throughout pregnancy [33]. Several medications used to treat high blood pressure, such as angiotensin-converting enzyme inhibitors, angiotensin-receptor blockers and renin inhibitors are not recommended during pregnancy and should be discontinued prior to conception or as soon as pregnancy is confirmed [33,110]. The National Committee on the Prevention, Detection, Evaluation, and Treatment of High Blood Pressure recommends methyldopa or β-blockers – antihypertensives that have been proven to be safe during pregnancy [33].

Obesity

Weight can be an issue in women even before they start gaining due to pregnancy. In 1999–2002, 54.3% of US women of reproductive age (18–44 years) were overweight or obese [107]. During pregnancy, being overweight (body mass index [BMI] = 25–30 kg/m²) and obesity (BMI −30–34.9 kg/m²) are significant risk factors for the woman and her fetus [34,111]. Compared with normal-weight patients (BMI = 19.8-25 kg/m²), obese patients have a higher rate of cesarean delivery, gestational hypertension, gestational diabetes, neural tube defects and possible birth defects [35,36]. The fetuses of obese women are at higher risk of late fetal death and macrosomia (which increases their susceptibility to obesity in adulthood) [37,111]. HCPs can help women prior to becoming pregnant in achieving a healthy body weight with a BMI between 18.5 and 25 kg/m² [112]. During preconception or during the very early stages of her pregnancy, a woman should take key steps to improve pregnancy outcomes [35,113]. Women should be counseled on caloric needs, and on the importance of eating a balanced, nutritious diet [101]. Dietary supplements are recommended for pregnant women who may not be getting the necessary amounts of certain nutrients in their diet [101], such as folic acid, which has been shown to reduce the occurrence of neural tube defects by 50–70%, as well as to reduce the incidence of other congenital abnormalities [9,102]. In addition, pregnant women should avoid unpasteurized dairy (which carries the risk of listeriosis, that can result in miscarriage, fetal death or neonatal morbidity) [38], limit caffeine intake (as consumption >200 mg/day makes women twice as likely to miscarry) [39], limit consumption of fish that could contain mercury (high levels can affect a developing nervous system) [40], and avoid raw or lightly cooked eggs (salmonella bacteria can cause illness in pregnant women) [114,115].

Over-the-counter home pregnancy tests

After a missed period, OTC pregnancy tests are the first source of pregnancy detection for most women [41]. While they are intended for use at home, HCPs should still play an active role in educating their patients about the use of these tests [42]. All OTC pregnancy tests detect hCG in the urine of women [14]. The association of hCG and pregnancy was first reported in 1927 based upon the detection of hCG activity partially purified from human urine [43]. Subsequent techniques built upon this biological association until the advent of immunoassays in the 1960s, which allowed the direct detection of hCG in pregnancy urine [44,45]. In recent years, monoclonal antibodies have further advanced the detection of hCG and allowed the development of more complex and sensitive assays. However, the form of hCG that has been traditionally associated with pregnancy is not the only form of the molecule found in urine during early pregnancy [14,41,46]. Other key molecules in the early detection of pregnancy include: hyperglycosylated hCG (hCG-H), the principal variant of hCG early in pregnancy; free β-subunit hCG; free β-subunit core fragments; and other hCG degradation products. Only a few OTC pregnancy tests detect a majority of these molecules, in addition to the traditional form of hCG [14,42]. A growing body of evidence points to pregnancy tests sensitive to hCG alone as not being as effective as tests that detect hCG plus these other hCG molecules [14,46]. In one study, urine samples were assayed for hCG, free-β subunit, and β-core fragment, plus the combined measurement of these hCG forms during the first 6 weeks postconception [46]. Researchers assayed first morning urine samples from 37 women who had conceived naturally and whose pregnancies all ended in singleton births. According to study results, hCG and free β-subunit showed the most day-to-day variability, and even 10 days after pregnancy was confirmed, these analytes were only transiently detectable. After studying doubling times for each analyte, the authors concluded that the most stable estimate of doubling time, and therefore the best predictor of early pregnancy, is the combined measurement of hCG, free-β subunit and β-core fragment [46].

Healthcare providers should also understand the limitations of OTC tests, as many make claims of accuracy up to 99%, but these are based on testing reproduced under laboratory conditions, not real-life conditions [47]. Studies investigating the accuracy of home pregnancy tests revealed that when used in real-life settings, accuracy and effectiveness may decrease [48,49]. Therefore, when choosing a pregnancy test, the two most important things to consider are: what molecules will it detect and at what levels; and, will the tests deliver false readings.

Molecule detection & sensitivity

Studies have been performed to gauge the effectiveness of OTC pregnancy tests. In a study by Butler and colleagues, almost 600 early pregnancy urine samples of hCG and hCG-H were tested with 15 OTC pregnancy tests and it was determined that these tests vary significantly in detection limits for regular hCG (6.3 IU/l to 50 IU/l), and that nine of the 15 devices had poorer detection limits for hCG-H than for hCG [41]. This is important as earlier work concluded that hCG-H is the primary molecule produced during the 5–6 weeks after gestation [50,51]. These critical findings were later corroborated by Cole and colleagues who stated that ‘if an hCG test is to be considered a pregnancy test, it needs to detect regular hCG and hyperglycosylated hCG equally’ [14].

In a 2005 study, Cole and colleagues compared the sensitivity of seven OTC pregnancy tests using the first day of a woman's missed menstrual period as a model to create surrogate urine samples containing a mixture of hCG-related molecules [42]. Specifically, urine was prepared to closely resemble the urine of women on the first day of missed menses: hCG, hCG-H, free β-subunit and β-core hCG were added to non-pregnant urines [42]. This study showed that the pregnancy tests had a range of sensitivity (mIU/ml) of less than 6.3 to more than 100. Confidence in the test results ranged from 22 to 100% for positive results, and from 26 to 100% for negative results. Faulty devices were found in up to 8% of one of the pregnancy test types.

A more recent study assessed commercially available OTC pregnancy tests to determine their sensitivity o hCG, hCG-β, hCG-β core fragment, hCG-H, and a mixture of these hCG-related molecules. Approximately 900 kits were tested and the range of molecule levels that these tests detected were (in mIU/ml): hCG (2.8 to 25.7), hCG-β (7.8 to >22.3), hCG-H (7.4 to >23.5), and ‘blend’ hCG (3.9 to >23.9) (Table 3).

Table 3.

Over-the-counter pregnancy test sensitivity to key forms of hCG (mlU/ml).

Over-the-counter pregnancy test	hCG	hCG-β	hCG-β core fragment*	hCG-H	‘Blend’ hCG
Clearblue® Easy Early Results	13.7	>22.3	>25.5	>23.5	>23.9
Clearblue® Easy Digital	13.7	>22.3	>25.5	>23.5	>23.9
Private label ‘A’	13.7	>22.3	>25.5	>23.5	>23.9
Private label ‘B’	13.7	>22.3	>25.5	>23.5	>23.9
e.p.t®	25.7	>22.3	>25.5	>23.5	>23.9
e.p.t® Certainty®	25.7	>22.3	>25.5	>23.5	>23.9
First Response® Early Result	2.8	7.8	>25.5	7.4	3.9
First Response® Gold™ Digital	6.5	7.8	>25.5	7.4	3.9

hCG-β core fragment was undetectable up to 25.5 mIU/ml.

Determination of the sensitivity of pregnancy testing devices to hCG and hCG-related molecules when present in isolation or in combination: final report. Data taken from Church & Dwight Co., Inc. Princeton, New Jersey, NJ, USA. January 2009. Data on file. hCG: Human chorionic gonadotropin.

The wide limits of detection seen in these two studies of commercially available, OTC pregnancy tests underscore the need to carefully choose a test that is effective at detecting low levels of these molecules.

False results

Confidence in results is another important aspect of choosing an OTC pregnancy test. False-positives, where the woman is considered to be pregnant but is not, can carry an emotional toll, especially on those who have been trying to conceive but have been unsuccessful. In the real world, apparent false-positive results cannot be avoided because a large number of conceptions end spontaneously as normal events where a viable fetus would not be produced. Some studies have estimated that only 30% of all fertilized eggs continue to term to result in a live birth [52,53]. Couples should be counseled that such events are not necessarily bad and that women who experience such events are highly likely to conceive successfully in the near future without therapy. A false-negative result, however, may be dangerous because if a woman believes that she is not pregnant, she may continue to consume alcohol or expose herself to teratogenic agents, which could harm the developing embryo [14].

Pregnancy counseling

All HCPs should counsel patients to receive prenatal care once pregnancy has been confirmed [25]. Babies born to mothers who do not get prenatal care are three-times more likely to have a low birth weight and five-times more likely to die than babies whose mothers get prenatal care [116].

Preconception and the first few weeks of pregnancy are when women should initiate steps for improving pregnancy outcomes. The importance of diet should be emphasized, both in what they should and should not be eating, as pregnancy complications or birth defects can result from certain foods [38 –40,114,115]. Pregnant women should also consider lifestyle modifications such as avoiding hot tubs and saunas [54], and exposure to chemicals, such as insecticides, cleaners and paint thinners [116]. Prenatal care should include a thorough medical history, a physical examination, a pelvic exam, and an estimation of delivery date [116].

Conclusions

The preconception, peri-implantation and prenatal periods are critical. During preconception, and later during the prenatal period, a woman has the ability to make behavioral choices that can affect her health and the health of her developing embryo and fetus. HCPs should explain to their patients the many reasons that knowing their pregnancy status as soon as possible can help ensure a healthy outcome for mother and baby. The first 3 weeks are considered an important period for the embryo where a wide range of implantational defects can occur that may negatively impact the pregnancy in later stages. HCPs should also emphasize that preconception preparation is the safest approach because it empowers women to address potential teratogens and to adopt healthy behaviors prior to conception. In this way, risk is minimized during the very early stages of embryogenesis, a time of heightened fetal vulnerability.

In order to determine pregnancy status as early as possible, almost all women rely on OTC home pregnancy tests. New research shows that pregnancy tests vary in their ability to detect pregnancy and the relevant forms of the pregnancy hormone early. HCPs should educate women about these substantial differences in available pregnancy tests. While all pregnancy tests detect the molecule hCG, the sensitivity level at which they detect hCG varies. Moreover, pregnancy tests that detect several molecules found in the urine of pregnant women – hCG, hCG-H, free β-subunit hCG and free β-subunit core fragments – further increase test sensitivity and improve their ability to detect pregnancy status after missed menses. A more sensitive pregnancy test is important for women who want to determine their pregnancy status and begin to take steps to ensure a healthy pregnancy. In addition, HCPs should advise patients to select the most accurate pregnancy test in order to avoid false-negative readings, which can lead to a lapse of critical surveillance.

Future perspective

Any conversation between an HCP and a patient regarding pregnancy should begin with the basic question of whether the patient is having unprotected sex. Surprisingly, some patients who are not intending to get pregnant do not make the connection that unprotected sex can lead to pregnancy. To them, they are two separate issues, one of intimacy versus procreation. Once this basic element has been addressed, HCPs can begin to provide more in-depth education to patients who are intending to become pregnant and to those with childbearing potential. These patients need to understand the risks that certain diseases/conditions and the medications that treat them can have on pregnancies. Those intending to get pregnant should also be made aware of the various teratogenic agents and behaviors (particularly smoking and alcohol consumption) that can negatively impact embryonic development. The first step in prenatal care is the knowledge that the patient is pregnant, and OTC pregnancy tests are the way that most patients discover their pregnancy status. HCPs can play a pivotal role in discussing the relative benefits of available pregnancy tests and emphasize the importance of early knowledge of pregnancies.

Considering the importance of knowing as early as possible if pregnancy has been achieved, and the increased knowledge that has been gained regarding the molecules released in the urine during early pregnancy, pregnancy test regulations are limited and out-of-date. Currently, all OTC pregnancy tests are only required to detect hCG and not its variants [117]. There is a need for better regulation of this marketplace, and all pregnancy tests should be highly sensitive to the various hCG molecules (such as hCG-H, free β-subunit hCG and free β-subunit core fragments). These additional molecules are present in the urine in early pregnancy, and their detection can help hone the accuracy of pregnancy results.

The benefits of detecting hCG variants are clear: more sensitive pregnancy tests and potentially fewer false-negative results. Given the proven benefits of better pregnancy tests, and the tangible improvements in pregnancy outcomes when women are involved early in their pregnancies, there is a clear imperative to revise applicable standards for pregnancy tests.

Executive summary

Embryogenesis

The first 2 weeks of embryonic development are vital to fetal development.

Background

Healthcare providers should provide some prenatal education to women of childbearing potential, especially in those with chronic disorders or on pharmacologic regimens that could adversely affect a developing embryo.

There are 60 million unplanned pregnancies each year.

Teratogens

There are several types of teratogenic agents: medications, behaviors/exposures, chronic/infectious diseases and genetic disorders. Over-the-counter home pregnancy tests

Over-the-counter pregnancy tests are used by almost all women to determine pregnancy status.

There are several molecules that can be used to determine pregnancy.

Not all tests detect these different molecules.

Not all tests have equal sensitivity.

The greater number of molecules that a test can detect, the more accurate it is considered to be.

Conclusions

Knowledge of pregnancy, as early as possible, can have a significant impact on the health of the woman and the developing embryo.

Footnotes

Mary Jane Minkin is on the speaker's bureaus of Bayer, Novogyne and Schering Plough; and is a consultant for Church and Dwight, Co., Wyeth Ayerst, and Ortho-McNeil. The author has no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. Editorial services provided by BioScience Communications via funding from Church & Dwight Co. Inc., of Princeton, NJ, USA (manufacturers of the First Response line of pregnancy tests).

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