Determining Ovarian Reserve A Review of the Tools, Tests, and Techniques

Ovarian Reserve: When the News is Bad
The results of ovarian reserve testing are often viewed by patients as either good news or bad. The bad news is that, for patients with low ovarian reserve, implantation rates are generally poor and the possibility of successful pregnancy is very limited. When faced with such information, couples are often devastated. They may have a variety of reactions - including, anger, denial or depression, all of which are normal and natural expressions of grief.

When couples receive a diagnosis of poor ovarian reserve, it is often helpful for them to spend time talking to each other, examining their parenting needs. They may want to ask themselves, "What is the most important thing to us about having a family? Is it the opportunity to provide love and guidance to a child? Is the genetic link essential? Is it important to experience pregnancy and childbirth? Is childfree living a realistic alternative?" By answering such questions, the couple will be in a better position to assess other options. Many patients have enlisted the aid of support groups, therapists, or compassionate physicians to help them through this crucial time. Whatever the outcome, patients receiving such shocking news will need time to mourn their loss and find a way toward acceptance.

Methods of Assessing Ovarian Reserve
Since a woman's chronological age is the single most important factor in predicting a couple's reproductive potential, age has often guided infertility treatment choices. However, age alone doesn't tell the whole story. Consequently, researchers have developed (and are continuing to develop) more refined methods of predicting a couple's response to infertility treatment. Some of the more sophisticated tools for assessing fertility potential include the measurement of FSH, LH, estradiol, and inhibin-B [Table 1]. Additionally, because patients should not be subjected to all tests, decisions regarding which method(s) to use are guided by practitioner experience.

Even though several sophisticated tools exist for measuring ovarian reserve, most fall short of what we consider ideal sensitivity and specificity. Also, how best to interpret ovarian reserve tests is controversial, since clinical experience with these tests is still evolving. Even so, most infertility patients should be periodically evaluated for the possibility of impaired ovarian reserve before pursuing any advanced fertility treatment.

Finite Supply of Eggs
Even before birth, a woman's eggs begin to diminish in number. During the 20th gestation week, a female embryo contains about seven million eggs. At birth, the number of eggs has already dropped to about 200,000. The number of eggs continues to decline as the woman ages, until no eggs remain (menopause).

Fortunately, women are naturally equipped with an ample supply of extra eggs. The number of eggs a woman has at birth far exceeds the average number of menstrual cycles she will have during her lifetime. Therefore, when women undergo fertility treatment to boost egg production, the risk for premature menopause is no different than it would be for other women [1].

The Effects of Maternal Age on Ovarian Reserve
 Menstrual cycles that occur near the end of the ovaries' lifetime are associated with older eggs of poorer quality [2]. In general, ovarian age parallels chronological age. But since that is not always the case, it is vitally important for clinicians to assess an infertility patient's ovarian reserve. This is particularly true for women over the age of 35.

Conception and childbirth in women of advanced age has always been uncommon. This fact was recognized from the beginning of recorded human history [3]. Later, studies looking at communal societies found solid evidence of reduced fertility associated with older women [4]. More recent work has validated these findings [5,6]. The father's age, however, appears to have only a marginal influence on fertility [7].

The effect of maternal age on fertility has been the subject of considerable research. In one such study, pregnancy success rates as a result of timed therapeutic insemination (intrauterine insemination; IUI) were evaluated in terms of maternal age. The poorest outcomes were consistently seen among women 36 years of age or older [8]. This observation was confirmed by research in an in vitro fertilization (IVF) setting, where women 37 years of age or older had a 9% ongoing pregnancy rate compared to a 26% ongoing pregnancy rate in patients younger than 30 [9].

In our practice, IVF treatments involving women aged 40 or older have been associated with fewer oocytes obtained per cycle, low estradiol (E2) on the day of hCG administration, and considerably lower embryo implantation rates when compared to women 32 years of age or younger. Even if pregnancy does occur in women who are 40 or older, there is a high risk for unfavorable outcomes. As maternal age increases, miscarriage occurs more frequently, as does the chance of fetal chromosomal abnormalities [12].

Our experience with donor-egg cycles offers further evidence that infertility and live birth rates are strongly influenced by the age of oocytes. Younger oocytes from donors under the age of 35 are typically used in donor-egg cycles. This greatly improves the reproductive outcome[13,14]. While the recipient's age does have some negative impact on implantation rates, the effects appear limited. In fact, even if the recipient is over 40, clinical pregnancy rates approaching 59% have been achieved when donor (younger) oocytes are used [15].

Passive Ovarian Reserve Testing: Measuring FSH and LH
The methods for assessing ovarian reserve are classified into two groups: passive testing and dynamic testing. The goal of both approaches is to provide information regarding oocyte (egg) quality and quantity. We'll begin this section by examining passive testing methods.

As clinicians gained experience with IVF in the 1980's, it became apparent that early follicular-phase FSH levels played an important role in pregnancy outcomes. We soon discovered that day 3 FSH (measured by a blood test) could be very useful in predicting response to ovulation induction and IVF.
As a woman ages, FSH becomes elevated in an attempt to force the aging ovary to respond. However, the exact mechanism responsible for this adaptive response remains unknown. A rise in early follicular-phase FSH is also accompanied by a decline in oocyte quality, and some investigators have linked such FSH elevations to fetal abnormalities [18]. In fact, it has been theorized that subtle but measurable increases in FSH precede menopause by approximately five years in some women [19].

Since FSH has such high predictive value, should FSH always be measured? And if so, what values are important? It is difficult to establish absolute values that define how high an FSH level can be and still achieve pregnancy due to variations in laboratory assessments and treatment methods. Adding to this uncertainty is the fact that no data exists which describes FSH patterns in a "control" population of fertile women [20].

Arbitrarily applying day 3 FSH tests to all women (even those with no infertility history) is controversial, and can offer confusing results. Some clinicians have therefore questioned the usefulness of widespread FSH screening [21]. It is our opinion that ovarian reserve screening should be an integral part of every infertility patient's workup.

While it is unwise to rely on a single test to fully assess ovarian reserve, considerable data exists that provides some general guidelines about which FSH values are most significant. In one center, women undergoing IVF with a day 3 FSH of less than 15 mIU/ml were twice as likely to conceive than women with FSH values between 15 and 24.9 mIU/ml [22]. Other investigators confirmed these results, and FSH values emerged as superior to maternal age as a method for determining reproductive outcome in IVF [23]. Indeed, one series reported that when day 3 FSH levels exceed 20 IU/L, conception rates fell sharply [24].

Traditionally, clinicians have relied on cycle day 3 FSH test results to help assess ovarian function. However, since FSH fluctuates only slightly during cycle days 2 through 5, testing does not have to be done exactly on cycle day 3. More flexible FSH testing may be done over a range of dates [25].

While FSH values may not change significantly from days 2 through 5 within a given cycle, fluctuations of day 3 FSH from cycle to cycle are more important to detect. When FSH does fluctuate, subsequent menstrual cycles will likely produce oocytes of varying quality. This principle has emerged as a fundamental belief in human reproductive physiology [26]. Patients with low FSH values (suggesting satisfactory ovarian reserve) generally show the least fluctuation, while those with elevated FSH levels have broader ranges. Wide FSH fluctuations from month to month present a difficult "moving target" for laboratory assessment. In such cases, it is difficult to precisely estimate ovarian reserve

A single measurement of day 3 FSH may not represent actual ovarian reserve. When testing reveals elevated FSH, this result should be confirmed in a later cycle. However, interpretation of fluctuations across multiple cycles is controversial. Among patients with a series of day 3 FSH values that include at least one unfavorable (elevated) FSH test, a low response to ovulation induction has been observed [27]. Another analysis revealed that patients with both high and low FSH values across multiple cycles performed as low responders during IVF [28]. However, other investigators regard variable FSH results differently. Some consider relatively low day 3 FSH values permissive for IVF or other fertility treatments during that cycle. Preliminary data suggest that if FSH returns to a "normal" level after an abnormal (high) test in a previous month, conception rates for IVF may be approximately 35% for patients under the age of 40 [29].

Passive Ovarian Reserve Testing: Measuring Estradiol
Investigators initially thought estradiol (E2) would be a more specific marker for ovarian reserve than either FSH or LH. Unfortunately, subsequent research has shown a weak relationship between E2 and ovarian response to fertility treatments. Evaluation of cycle day 3 E2 in IVF patients revealed no clear association between E2 and treatment outcome [22].

In a population of IVF patients without pre-treatment GnRH-a (i.e., Lupron) suppression, cycle day 3 E2 and FSH were compared to reproductive outcome. The researchers observed that even when FSH values were less than 20mIU/mL, no pregnancy occurred when day 3 E2 was greater than 75pg/mL [33]. This result was supported by others who observed better outcomes for women aged 38-42 when day 3 E2 was less than 80pg/mL and FSH was normal [34]. From these studies it seems that evaluating both E2 and FSH was a better predictor of ovarian reserve than using either measurement alone.

Low day 3 E2 levels, combined with normal FSH, have been associated with improved stimulation response, higher pregnancy rates [35], and lower cycle cancellation rates [36]. Interestingly, researchers reported that measurement of E2 one day earlier (on cycle day 2) did not enhance the predictive value of ovarian response [37].

High levels of E2 early in the menstrual cycle suggest an inappropriately advanced stage of follicular development. This may occur as the ovary ages, or when ovarian follicular cysts remain from a prior menstrual cycle. The follicular cysts can interfere with egg "recruitment" in the treatment cycle, naturally leading lead to a poor response to fertility treatment.

Passive Ovarian Reserve Testing: Measuring Progesterone
A decline in ovarian reserve has also been associated with a short follicular phase, early LH surge, and premature elevation of progesterone (P4) [38]. Initially, it was though P4 might be a useful tool for ovarian screening. However, daily E2 and P4 testing performed in volunteers with ovulatory cycles revealed no differences in E2 or P4 as a function of age [31]. Researchers then turned their attention from "static" P4 assessment to the study of P4 patterns in the context of dynamic testing (see next section). In that setting, some investigators found high P4 levels (*1.1 ng/mL) on day 10 of clomiphene citrate challenge tests (CCCT) to be associated with short follicular phases, diminished ovarian reserve, and reduced potential to achieve pregnancy. [39].

Passive Ovarian Reserve Testing: Measuring Inhibin-B
Inhibin-B is an ovarian hormone that inhibits FSH release [40]. Although present in ovulating women, it is not normally found in postmenopausal women. As early as 1932, researchers suspected that a non-steroidal regulator of FSH secretion might exist [41], but it was not until 1976 that this hypothesis was actually confirmed [42].

Unfortunately, the initial enthusiasm for inhibin-B as an ovarian reserve screening tool was tempered by the lack of a satisfactory way to study it. If measured by standard FSH-release methods, more than seven active forms of inhibin, as well as inactive inhibin constituents, are found in human serum [46]. Studies are currently underway to uncover more information about inhibin-B [47,48].

Although measuring inhibin-B is still considered investigational as a way to screen ovarian reserve, a number of advances have helped make measurement of inhibin-B a clinical reality. Nevertheless, it is critical for clinicians using inhibin-B in ovarian reserve testing to understand exactly which assay is being used, and acknowledge limitations in measurement methods.

Inhibin-B may prove to be a beneficial marker for ovarian reserve assessment because it fluctuates during the menstrual cycle, and is significantly reduced in women over the age of 35 [51]. One center observed that when day 3 inhibin-B was less than 45pg/mL, the response to fertility treatment was lower, the cancellation rate was higher, the number of retrieved oocytes was less, and the pregnancy rate was significantly reduced when compared to subjects with day 3 inhibin-B values greater than or equal to 45pg/mL [52]. In an effort to broaden the diagnostic capability of inhibin-B, some researchers have proposed including this test as an experimental component of the clomiphene citrate challenge test (see next section) [53].

Although these early reports confirm that inhibin-B can enhance current tools that measure ovarian reserve, more data are needed before meaningful normal ranges for inhibin-B can be routinely applied in clinical practice [54,55].

Passive Ovarian Reserve Testing: Transvaginal Ultrasound
Diminished ovarian reserve means that fewer follicles are available for stimulation and recruitment by fertility drugs. By allowing physicians to view the ovaries and assess the number of follicles, transvaginal ultrasound can aid in the assessment of ovarian volume.

Previous investigators have documented that the ovary reduces in size with increasing age, regardless of whether the woman has given birth [56]. Other researchers have found that the lower the ovarian volume, the greater the dose of fertility drugs required to stimulate the ovaries [57]. Ultrasound ovarian volume has also been used to predict the risk for ovarian hyperstimulation syndrome [58]. However, it has been theorized that ultrasound ovarian volume done at the beginning of the treatment cycle is more closely related to the number of follicles found during the pre-treatment period rather than the number of oocytes developed during treatment [59]. It is uncertain that ovarian size or follicular number is a better indicator of ovarian reserve. The more follicles you have, the larger the number of eggs retrieved during the treatment cycle. Is it better to measure prior to any medical therapy or after GnRH-a is administered but prior to gonadotrophin injections? Studies to clarify the relative predictive value of follicular number and ovarian size have yet to be done.

Among patients taking GnRH-a (Lupron) before a treatment cycle, abnormally high E2 levels are often associated with large ovarian cysts (identified by transvaginal ultrasound). Such E2 elevations may be improved by short-term continuation of GnRH-a. Using this approach, the cyst frequently resolves and the treatment cycle can continue. It is important to note that the additional GnRH-a exposure during this brief interval has not been associated with significantly reduced response to fertility drugs, nor has it adversely affected pregnancy rates [62].

Dynamic Ovarian Reserve Testing: Clomiphene Citrate Challenge Test
In contrast to the static measurements of ovarian reserve mentioned previously, the clomiphene citrate challenge test (CCCT) is a dynamic approach. Its purpose is to stimulate the ovary to initiate egg production in response to a fertility drug called clomiphene (Clomid or Serophene). In theory, the CCCT was designed to detect low ovarian reserve that would not be discovered by a single FSH and/or E2 measurements.

The CCCT is based on the assumption that adequate ovarian reserve is associated with a healthy group of developing follicles. This healthy group of follicles should be capable of producing enough inhibin and E2 to suppress FSH production and resist the effects of clomiphene.

Clomiphene works by shutting down the estrogen receptors on the hypothalamus and tricking the hypothalamus into thinking the patient doesn't have enough estrogen. In response, the hypothalamus works harder to induce the pituitary gland to produce more FSH and LH. This, in turn, initiates follicular growth.

When undergoing CCCT, the first step is to measure day 3 FSH and E2. Then 100mg of clomiphene is administered on cycle days 5 through 9, and FSH and E2 measurements are repeated on cycle day 10 [63]. In general, a high day 10 FSH suggests poor ovarian reserve.

In the original report describing the CCCT, 18 patients out of 51 had abnormal responses. Of those with abnormal responses, only one pregnancy resulted (1 of 18, or 6%). The pregnancy rate among those with normal CCCT response was substantially higher (14 of 33, or 42%) [63]. Several other investigators have confirmed the good predictive value of CCCT before treatment [64-66].

Evaluation of a large number of infertility patients found a 10% prevalence of abnormal CCCT responders [67]. Another report found CCCT to be a better predictor of ovarian reserve than day 3 FSH measurement alone [68].

Dynamic Ovarian Reserve Testing: Gonadotropin-releasing Hormone Agonist Stimulation Test
A gonadotropin-releasing hormone agonist (such as Lupron) initially elevates E2, then profoundly suppresses both FSH and LH [69]. This is sometimes called a "flare-effect". More than a decade ago, it was theorized that low ovarian reserve might be detected by evaluating differences in LH, FSH, and E2 levels following the administration of GnRH-a during IVF [24]. This approach was later formalized as a diagnostic tool known as the GnRH-a stimulation test, or GAST [70].

The purpose of GAST is to evaluate changes in E2 on cycle day 2 and 3 following administration of leuprolide acetate (Lupron). Patients with greater elevations of E2 have correspondingly higher pregnancy rates. Four GAST E2 patterns have emerged:

1) prompt E2 elevation, then decrease by cycle day 4
2) delayed E2 rise with fall by cycle day 6
3) persistent E2 elevation
4) no E2 response after GnRH-a

Clinical pregnancy rates for these groups were strikingly different: 46%, 38%, 16%, and 6% were observed in patterns 1 through 4, respectively [9].

In summary, GAST has been a better predictor of the functional abilities of the ovary than either FSH or age [70]. Because the GnRH-a is costly and involves an injection and repeated blood tests, the GAST is not widely used in clinical practice.

The Value of Medical Records
Clinicians can often uncover valuable information about possible ovarian reserve from records describing a patient's response to earlier ovulation induction attempts. Unlike any of the other assessment tools described, knowing how a patient responded previously shows reproductive performance over the broadest possible range. When available, such records represent the ultimate dynamic test of ovarian status.

Obviously, records would not be available for patients who have no prior ovulation induction attempts. However, the availability of old stimulation records does not mean that repeat assessment of ovarian reserve can be left out. Prior stimulation records can guide both the estimation of ovarian reserve and the selection of the appropriate treatment plan in an upcoming cycle.

Summary
Several methods have been developed to estimate the functional or biological age of the ovary. Since ovarian reserve can vary over time, any results suggesting limited ovarian reserve should be confirmed by further testing in subsequent months. It may be that inflammatory, infectious or autoimmune conditions are contributing to the abnormal results. Such potentially reversible causes of low ovarian reserve should be corrected, particularly in younger woman.

When further testing confirms compromised ovarian reserve, it is important to point out that the likelihood of success with infertility treatment is low. Such patients may want to consider other options, such as donor-egg treatment. However, we realize that donor eggs will not be acceptable to everyone with poor ovarian reserve.

We have found that when ovarian reserve is low, the best course of action for the physician is compassionate honesty. After all, time is precious by the time a couple reaches infertility specialists, so little can be gained by creating false hope or continuing down a fruitless path. Instead, physicians can provide invaluable assistance by helping the couple re-evaluate their parenting needs and determine what, if any, other options they might pursue. In the world of infertility, some doors shut while others open. The door may not reveal what the couple thought they wanted, but for many, it might reveal something that is even better.

References:

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