Pregnancy and Fertility Testing

Pregnancy and Fertility Testing

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Pregnancy and Fertility Testing Marie Berry, BScPharm, BA, LLB Date of Revision: February 2013

Pregnancy Testing Introduction

For an overview of the female reproductive cycle, see Contraception. Once an ovum is fertilized, it may take about 9 days before it is implanted in the endometrial wall. Human chorionic gonadotropin (HCG) is detectable in the blood and urine once the ovum is fertilized and implanted. In healthy women, HCG is a specific marker for pregnancy, because it is only produced by the placenta. HCG, a glycoprotein produced by the trophoblastic cells of the placenta, maintains the corpus luteum. It replaces luteinizing hormone (LH). HCG can be detected as soon as 6–8 days after conception, and is highest in concentration between 9 a.m. and 12 p.m. Its concentration in the blood doubles about every 2 days, reaching a peak in 60–70 days. It then decreases to a lower level for the rest of the pregnancy. The half-life of HCG is about 5.6 hours. Following parturition it returns to baseline within 10 days. The detection of HCG is the basis of pregnancy testing kits.1 Initial pregnancy tests can be performed on urine samples and later confirmed with a blood test. Detectable HCG levels start at 5 mIU/mL during the first week of gestation and rise to a peak of about 100 000 mIU/mL. Blood tests can detect levels as low as 1 mIU/mL and most urine tests are able to detect 20–100 mIU/mL. Prior to the advent of a biological assay for HCG, pregnancy tests were performed by injecting a woman's urine into a female rabbit. After 5 days, the rabbit was sacrificed and its uterus examined. Since HCG causes swelling of the corpus luteum and of the uterus, a heavy/enlarged rabbit's uterus meant that the woman was most likely pregnant. In the 1960s, immunoassay technology allowed synthesis of an antibody that combined with HCG to produce a precipitant or change a coloured substrate. The pregnancy testing kits in use today represent the third generation in terms of pregnancy testing technology. For product selection, consult Compendium of Products for Minor Ailments. Home Testing Products: Pregnancy Tests.

First-generation Tests

These tests used polyclonal antibodies, which recognized multiple binding sites on HCG. Unfortunately, these antibodies also reacted with other substances such as LH and follicle-stimulating hormone (FSH), resulting in falsepositive results. This generation of tests has been replaced by second- and third-generation tests, which do not require sample collection and preparation, an incubation period or technical skill.

Second-generation Tests

Modern pregnancy tests employ monoclonal technology and are more specific. In second-generation testing kits, the anti-HCG antibody is bound to a solid surface such as a stick, bead or filter paper. If HCG is present, it forms a complex with the antibody to produce a change in colour of a chromogen-reactive enzyme. The HCG becomes sandwiched between the 2 antibodies, one attached to the test surface and the other attached to the colour-producing enzyme. Second-generation tests can detect HCG as early as the first day of a missed menstrual period and take a shorter time (1–30 minutes) to perform.

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Third-generation Tests

The tests available today for home use are third-generation tests (e.g., Clearblue, First Response) in which the technology is even more refined. One anti-beta-subunit HCG monoclonal antibody is linked to a coloured substrate. If present, HCG binds to this antibody and the resulting complex binds to a second monoclonal antibody bound to a solid surface. The second monoclonal antibody is the alpha-subunit and elicits the colour change. Ease of use and accuracy are significant advantages of third-generation tests, so much so that one American study estimates that one third of women in the US have used third-generation pregnancy tests.2 Pregnancy testing kits afford early detection of pregnancy in privacy, and earlier detection can permit earlier prenatal care.3 They also enable earlier avoidance of harmful chemicals, x-rays, drugs and elective surgery, all of which could potentially harm a developing fetus. The tests are easy to use and readily available with numerous generic versions. They offer speed, convenience and confidentiality, along with economical cost.

Proper Use

False-positive and false-negative results are possible regardless of which generation of test is used. Human error (e.g., holding wrong end of wand or stick in urine stream, exposing the wand or stick to the urine for less than the required length of time) is usually the cause of erroneous results. The easier a test is to use, the less likely errors will occur. Most third-generation tests have control windows to indicate whether the test was conducted correctly. Timing of the pregnancy test is essential in that accuracy is more closely related to ovulation than to intercourse. Sperm can remain viable in the fallopian tubes up to 5 days and implantation can occur up to 12 days later. Falsenegative results are possible up to 17 days after insemination. Other errors leading to false results include: Using an expired testing kit

Not following the timing recommendations (waving the wand/stick through the urine stream too quickly) Testing too early after conception when HCG levels are too low to be detected Testing too late (after 60–70 days when HCG levels decline) Soap residue, blood or protein in the urine sample Cloudy, pink or red urine Strong urine odour

Warm or hot water rinses of the test surface before or after sampling

Use of fertility drugs containing HCG (note: clomiphene will not affect tests), hormones, corticosteroids Ectopic production of HCG by nontrophoblastic tumours Conducting the test after missed or incomplete abortion

Often pregnancy testing kits are claimed to be 99% accurate and able to detect pregnancy as early as the first day of a missed period; however, a study showed that of 7 testing kits, only 2 would have detected 95% and 80% of pregnancies on the first day of the missed period.4 Caution needs to be exercised when interpreting pregnancy test results.

Fertility Testing

Methods Used to Predict Ovulation

Table 1 compares various methods of predicting the time of ovulation, including prediction methods other than those that are hormone based. Table 1: Methods Used to Predict Ovulation

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Test

Indicators of Ovulation

Basal body temperature

Elevation of basal temperature (0.5°F or 0.28°C) over up to 3 days, lasting at least 11 days, usually continuing to day 1 of menses5,6

Chloride ion

Chloride ions levels increase about 6 days prior to ovulation (device is a wrist band with a microcomputer chip which detects chloride ions in perspiration on the skin).7 This test is not sold in Canada.

Cervical mucus

Abundant watery discharge; clear, elastic and stretchable mucus (tested with fingers); changes occur about 3–4 days prior to ovulation5

Saliva

Fern-like pattern on glass; changes occur about 3–4 days prior to ovulation (saliva is licked on a slide which then is examined under a microscope)5

Endometrial biopsy (late luteal phase)

Histology within 2 days of chronological cycle day based on LH surge1

Menstrual cycle history

Most cycles 26–30 days (range 23–35 days) with ovulation mid-cycle, (e.g., around day 14 for a 28-day cycle). The postovulatory phase is usually more likely to be consistently around 14 days (e.g., in a 35-day cycle, ovulation might occur around day 21)6

Midluteal serum progesterone

Values greater than 10 ng/mL1

Urinary LH kits (ovulation prediction kits)

Colour change 20–48 h before ovulation ;8 specific time range is product-dependent

Basal Thermometers

For product selection, consult Compendium of Products for Minor Ailments. Home Testing Products: Thermometers. Basal body temperature is the temperature that occurs prior to rising in the morning. It can be taken orally, rectally or vaginally but must be measured the same way and at the same time each day, consistently. A basal thermometer measures a more narrow range of temperatures than a fever thermometer (36–38°C or 96–99°F) and is thus used to detect small fluctuations in the basal temperature. Basal thermometers are easier to read than fever thermometers, and digital versions measuring to 2 decimal points may be even more user friendly. Electronic basal thermometers are available and combine data about menstrual cycle history with the measured temperatures to provide information about the timing of fertility. These devices are more expensive and are available via the internet. Progesterone is thermogenic; elevated levels cause a rise in body temperature. About 12–24 hours prior to ovulation, a drop in temperature occurs; however, it is not always possible to detect the drop. When ovulation occurs, the corpus luteum releases progesterone, causing a significant rise in temperature that is detectable and lasts for several days. The temperature increase is about 0.5°F or 0.28°C and is measurable by a basal thermometer.9 This temperature rise occurs over a period of up to 3 days and is usually maintained until the first day of menses—day 1 of the next cycle. It is not the temperature itself that is significant, but the maintained high temperature. A woman records her basal temperatures daily on a graph. With several cycles of data, a pattern may emerge, e.g., the usual day of ovulation in a woman's cycle.5,6

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To be relevant, the basal body temperature needs to be measured in a consistent manner. Sleep disturbances can affect the temperature, and at least 4 hours of uninterrupted sleep is required before taking the temperature. Air travel, especially with a change in time zones, or jet lag can adversely affect the measurement, as can any factor that may on its own impact temperature, e.g., alcohol consumption, stress, anxiety, illness including infections. Being able to identify a rise in temperature enables a woman to determine when she is most fertile and likely to conceive. Sexual intercourse (ideally every 2 days) during this time will increase the chance of conception. Conversely, a woman will also know when to avoid sexual intercourse if she does not want to conceive. (For more information on contraception, see Contraception.) Some women learn to use sympto-thermal charting, which combines symptoms (e.g., mucus, pain, breast tenderness, spotting) and basal body temperatures, allowing the fertile period to be more accurately pinpointed.

Ovulation Prediction Kits

For product selection, consult Compendium of Products for Minor Ailments. Home Testing Products: Ovulation Predictor Test Kits. Ovulation prediction kits are not intended to be used as contraception.

Urine-based Kits

While basal thermometers identify the rise in temperature that accompanies progesterone release, ovulation prediction tests identify the luteinizing hormone (LH) surge that precedes ovulation by measuring its concentration in the urine. A woman will know when she is most likely to ovulate and can plan her sexual activity to increase the chances of conception.10 These tests employ polyclonal and monoclonal antibody technology. One antibody is bound to a test surface and another to an enzyme. If LH is present, it becomes sandwiched between the two antibodies and produces a colour change on the test surface. With no LH in the urine, the second antibody bound to the enzyme is washed away and no colour change occurs. The colour intensity depends upon the amount of LH present. Most ovulation tests are mixtures of polyclonal and monoclonal antibodies. The polyclonal antibodies may bind with either the alpha- or beta-subunit of LH or even the entire molecule. The monoclonal antibodies are usually specific for the beta-subunit, which is a more accurate identifier of LH.11 More recently, tests are beta-subunit specific antibodies bound to coloured latex particles. The second monoclonal antibody is bound to the test surface. Without LH, the antibody bound to the coloured latex is washed away. Usually kits contain 5 daily tests that require 3–60 minutes to perform. Using the average length of her cycle, a woman uses a chart to determine the day of her cycle on which she should begin testing. Some test results are compared to baseline colour charts, some to the previous test and others to a control window. These tests are up to 98.3% accurate if performed properly.12 Because of its effects on estrogen levels, women taking clomiphene should not start testing for the LH surge until 3 days after taking their last tablet. Otherwise, the test may not accurately indicate the peak fertility time.

Fertility Monitors

Fertility testing technology has advanced to include monitors containing software programs that more specifically pinpoint fertility. These test kits combine the use of monitors with urine testing. The kits contain from 5–20 urine tests and the software program tracks the cycle day, indicating when a urine test should be performed. It usually reports the degree of fertility as low, high or peak. These advanced tests use monoclonal technology, and not only test for LH, but also for estrone-3-glucuronide (E3G), which is a urinary metabolite of estradiol. Estradiol stimulates the secretion of cervical mucus that is favourable for the survival and transport of sperm. The rise in estradiol levels corresponds to the appearance of

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sperm-supportive cervical mucus. Estradiol levels gradually rise in the early stage of the cycle, reaching a threshold that triggers the LH surge. With these tests, the stick is not read visually, but rather inserted into the monitor, which optically reads the test and displays the result.13 For all of these kits, urine should be collected at the same time each day, and while some tests are to be done on the first urine in the morning, others can be performed anytime during the day as long as there is a sufficient volume to ensure an effective concentration of LH. Human error accounts for the majority of false readings. The inclusion of test controls and comparison of the results to the previous days' results reduce the chance of errors. Test results can be affected by tetracycline and other drugs that affect the menstrual cycle such as hormonal contraceptives, fertility treatments that contain HCG or LH and hormone therapy. Pregnancy, breastfeeding, polycystic ovary syndrome and menopause may also affect results.8 Once a woman discontinues hormonal contraceptives or other hormonal therapy, or after pregnancy or breastfeeding, she should experience 2 consecutive menstrual cycles before she can begin to accurately test for LH surge.

Saliva-based Kits

Saliva-based testing detects a fern-like pattern in dried saliva seen under a microscope at a 40× to 60× magnification. Prior to ovulation, estrogen levels rise causing an increase in the salinity of saliva which, when dried, produces fern-like patterns. Testing is recommended to be performed first thing in the morning before eating, drinking, smoking or brushing the teeth. The saliva sample needs to be dried for at least 5 minutes and bubbles in the sample should be avoided. While the fern-like patterns are usually seen 3–4 days prior to ovulation, daily testing is sometimes recommended to detect changes. Note that not all women's saliva produces a fern-like pattern, and not all women are able to recognize this pattern.

Proper Use

The choice of a fertility testing method or methods depends upon personal preference, motivation of the woman or couple, physical factors (e.g., regularity of the menstrual cycle) and/or the complexity of the testing method. While some studies of accuracy have shown urinary LH tests to be superior to other fertility testing methods,8 factors such as human error, variability in menstrual cycles and concurrent medical conditions can have a significant impact on the accuracy of any method. Home diagnostic kits give an approximate time of ovulation and an indication of when a woman may be more fertile. Each kit has a chart or graph that can be used to determine, based on the woman's cycle, when testing should begin. These instructions should be followed for best results. Couples using home ovulation predictor kits and/or basal body temperature charts to detect ovulation should be instructed in the interpretation of the results. Intercourse should begin before the expected day of ovulation and should occur at approximately 2-day intervals. This timing takes advantage of the fact that sperm survive for up to 5 days in the female reproductive tract, especially in good cervical mucus. Because sperm reserves of the male require at least 2 days for replenishment, more frequent intercourse may result in small volumes and a slightly lower sperm count. The use of artificial lubricants (e.g., K-Y jelly) during intercourse may interfere with fertility because they decrease sperm motility.

References

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1. Stanford JB, White GL, Hatasaka H. Timing intercourse to achieve pregnancy: current evidence. Obstet Gynecol 2002;100:1333-41. 2. Pray WS, Pray GE. Detecting pregnancy and ovulation with home test kits. US Pharm2012;37(9):12–15. 3. Gannon K. Who is most apt to turn to a home pregnancy test? Drug Topics 1992;136:46. 4. Cole LA, Sutton-Riley JM, Khanlian SA et al. Sensitivity of over-the-counter pregnancy tests: comparison of

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utility and marketing messages. J Am Pharm Assoc (2003) 2005;45:608-15. 5. Pyper CM. Fertility awareness and natural family planning. Eur J Contracept Reprod Health Care 1997;2:131-46. 6. Moghissi KS. Accuracy of basal body temperature for ovulation detection. Fertil Steril 1976;27:1415-21. 7. Lennard J. Advanced technology for fertility prediction. US Pharm 2006;12:49-54. 8. Eichner SF, Timpe EM. Urinary-based ovulation and pregnancy: point-of-care testing. Ann Pharmacother 2004;38:325-31. 9. Downs KA, Gibson M. Basal body temperature graph and the luteal phase defect. Fertil Steril 1983;40:466-8. 10. Corsan GH, Ghazi D, Kemmann E. Home urinary luteinizing hormone immunoassays: clinical applications. Fertil Steril 1990;53:591-601. 11. Engle JP. Ovulation predictors. Am Drug 1993;207:55. 12. When the test really counts: part two: the fertility window. Consum Rep 2003;68:48-50. 13. Miller PB, Soules MR. The usefulness of a urinary LH kit for ovulation prediction during menstrual cycles of normal women. Obstet Gynecol 1996;87:13-7. CPhA assumes no responsibility for or liability in connection with the use of this information. For clinical use only and not intended for for use by patients. Once printed there is no quarantee the information is up-to-date. [Printed on: 03-03-2016 02:20 PM] RxTx, Compendium of Therapeutics for Minor Ailments © Canadian Pharmacists Association, 2016. All rights reserved

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