Age And Infertility:
The Biological Clock: Fact Or Fiction?

Michael Fox, M.D.
Michael Fox, M.D. is a Reproductive Endocrinologist with North Florida Gynecologic
Specialists in Jacksonville. He is also an Assistant Professor, Department of Obstetrics
and Gynecology, University of Florida Health Science Center / Jacksonville.

With recent dramatic advances in infertility treatment, age related infertility remains as one of our most difficult challenges. Reproductive specialists have known for years that the pregnancy rate is inversely related to the female partner's age. Early explanations for this trend included decreased coital frequency, diminished desire for childbearing, decreased overall time to try for a pregnancy, and diminished ovarian reserve with advancing age.^1,2 Today, we would add increased spontaneous abortion, oocyte depletion, and oocyte aging to the list.³ Coupling this issue of age with our current societal trend of increased numbers of women who delay childbearing for educational and career goals, we now have a dramatic increase in age related infertility. For those providing primary care to women, it is now much more important to carefully counsel patients regarding family planning issues, especially with regards to advancing age and diminished pregnancy rates. Patients who are in their early to mid thirties or beyond who are considering pregnancy or have been trying for any length of time without success warrant an early referral for evaluation.

General Infertility Rates

Strictly defined, infertility is failure to conceive a pregnancy in a 12 month period for patients under 35 years of age and failure to conceive in a 6 month period for the over 35 age group. Using this definition, approximately 14% of couples have not conceived in the first 12 months. At 24 months, 4% and at 36 months only 1.9 % of the original group have not conceived. From this, we know that a significant proportion of patients who are not pregnant after one year will achieve a pregnancy with continued attempts. Therefore, patients are most commonly considered subfertile and not sterile. Therapies used to treat infertility are thus designed to shorten the interval to pregnancy in this group. This is especially important in the older age group where a two or three year trial of pregnancy would be unreasonable, especially in couples who desire more than one pregnancy. Additionally, it is generally agreed that there has not been a significant increase in infertility in the general population over time.

Population Studies Of Infertility

The widespread use of contraception and many other confounding variables for reproduction in industrialized societies, and poor nutrition in developing countries, has significantly reduced our ability to establish natural fertility rates. For these reasons, historical population studies using unencumbered societies that did not limit reproduction have served to provide us with estimates of fertility and sterility rates. While overall fertility rates may vary some from historical population to population, the demonstrated trends have been strikingly consistent when age or age of marriage and fertility rates are compared² (Figure 1). From these studies a noticeable decline in fertility begins between age 30 and 33 and a much steeper decline begins at 35-38 years of age for the female partner. The decline in male fertility are much less dramatic and only become significant in the late forties and early fifties. In these studies, when age at first marriage for nulliparous women was specifically evaluated, the risk of never conceiving for the 35-40 year age group was 30 % and for the 40-44 year age group, 64%. Infertility, on the other hand, in longitudinal studies is less common. For the Hutterite population, 11% demonstrated infertility from age 35-39, 33% at age 40 and 87% by age 45. This population had an overall sterility rate of 2.4% which is exceptionally low. This could be explained by the low tubal disease in such a closely knit religious community. Other studies looking at age and IVF pregnancy rate show an extremely low per cycle pregnancy rate (1% or less) in women 44 and older. In summary, on average women go from a near normal pregnancy rate at age 35 to nearly zero at age 43, a short 8 years.

As a direct result of the above evidence, in population terms, the age range 15-44 years has been chosen to represent the "reproductive age group." Unfortunately, in our media laden society, patients seem to hear with great frequency about women in their late forties with pregnancies. Because of the rarity, they are newsworthy, but in reality these events often represent undisclosed donor oocyte pregnancies. This has created a false sense of security to those women who are delaying childbearing.

The historical studies are probably biased by the fact that some women failed to report abortions or the use of "non-culturally" appropriate birth control. Therefore, these studies may overestimate the risk of sterility with advancing age.^2,3 Studies looking at populations today, where there is a large proportion of women utilizing surgical or hormonal contraception, also have significant biases. If the group studied is only the non-contraceptive group, then the assumption is that there is no infertility in the contraceptive group which potentially results in either an increase or decrease in the overall estimate of infertility. Therefore, the application of these statistics from such subgroups of the population to women who have delayed childbearing is not wholly accurate. The trends with age, however, are real and must be heeded by patients and physicians alike.

Donor Insemination Vs. Maternal Age

A second way to "control" or isolate maternal age is to look at donor sperm cycles in couples where the woman has no identifiable infertility factor or risks. A multicenter French study involving 2,193 patients with azoospermic husbands demonstrated a significant decline in pregnancy rates for women aged 30 and older. The cumulative success rates after 12 cycles of insemination were 73% for women under age 25, 74% in women aged 26-30, 61% for ages 31-35, and 54% in the over 35 age group. This was the first time a significant reduction in fertility between the ages of 31 and 35 was clearly demonstrated. The infertility rate is lower in the azoospermic group than in a group including oligospermic males.⁴ A second study found similar results for pregnancy rates and demonstrated a significant increase in months to conception with increasing age from 6 to 9-10 months.⁵

Menopause And Ovarian Embryology

The average age of menopause in the U.S. is 51.4 years and is not related to race, socioeconomic status, education, height, weight, age at menarche, or age at the last pregnancy.⁶ The only significant risk factor is cigarette smoking, which significantly reduces the age of menopause. Recently, cigarette smoking has been shown to hasten the development of diminished ovarian reserve. On average there are very few pregnancies beyond age 43, yet these women for the most part have regular ovulatory cycles for the roughly eight years preceding menopause. In fact, in cursory hormonal analysis of these cycles, spot estrogen measurements and follicle stimulating hormone (FSH) values will be quite normal. What then is the cause of infertility in this group? The answer is not specifically known but assumed by indirect evidence to be the result of oocyte dysfunction.

In contrast to males, females at birth have their entire oocyte compliment of one million which declines to approximately 400,00 by puberty. Recent work suggests that a cohort of 30-100 follicles "come due" every day or so regardless of pregnancy, the use of birth control pills, etc. Only when the hormonal milieu is appropriate will one or more follicles proceed to maturity and ultimately to ovulation while unselected follicles undergo atresia. Thus, hundreds to thousands of follicles become atretic each month of reproductive life. Serial sections of ovaries in perimenopausal and menopausal women show that there is a dramatic progressive decline in egg numbers from normally cycling perimenopausal women, to irregularly cycling perimenopausal women, to menopausal women who only have rare oocytes.⁷ In addition, the oocyte is frozen in prophase I of meiosis at puberty and remains so until ovulation at which time it resumes meiosis. Therefore, the later in life an oocyte is recruited, the longer it has been frozen in a relatively unstable state which may allow for chromosomal or spindle damage, each known to result in failure to achieve pregnancy, increased incidence of spontaneous abortion or, more rarely, chromosomally abnormal children. It is also felt that the more sensitive or more "functional" oocytes are recruited at an earlier age leaving the most dysfunctional for the waning years of reproduction life. Further than these superficial ideas, not much is known about the reproductive failure of this period between ages 44 and 51. For lack of a better term, this period has been dubbed the period of "diminished ovarian reserve."

Diminished Ovarian Reserve

As with any distribution in medicine, there is a bell curve and the same is true for the period of diminished ovarian reserve (Figure 2). Using the CCCT we are able to test for this period. Today we are identifying patients in their teens, twenties, thirties and early forties whose ovarian function is the equivalent of the average 44 year old. Therefore, an extrapolation of this concept leads us now to think in terms of "ovarian age" as opposed to chronologic age; the two are often not synonymous.

Diminished ovarian reserve refers to a group of patients at any age that are cycling regularly but whose ovaries, and the eggs contained within, have a markedly decreased ability to produce pregnancies. Risk factors for this entity include age > 35, previous ovarian surgery, single ovary, unexplained infertility, and history of poor stimulation with injectable ovulation drugs (3 or fewer follicles developed and or peak estradiol of less than 1000 pg/ml). The patients exhibiting diminished ovarian reserve have identifiable clinical characteristics (Table 1). The patient's cycle intervals become subtly but progressively shorter with 21 to 27 day cycle intervals common. This is due primarily to a shortened follicular phase (first 14 days of the cycle) and correlates with patient histories of positive ovulation predictor kits on cycle day 9 or 10 (cycle day one is the first day of menses).⁸ Premenstrual syndrome symptoms seem to be more common in this group especially those of emotional swings and anger. The rate of spontaneous abortion is higher. In fact, over the last 6 months, we have tested all our patients for this condition who present with recurrent pregnancy loss for this condition and have found an unusually high incidence of diminished reserve in this group (authors unpublished observation). Another personal observation in these patients has been unusually high numbers of patients with any gray hair. Luteal phase defect has become a questionable entity in the diagnosis and treatment of infertility, yet remains a diagnostic possibility in the recurrent pregnancy loss patient. Of our last five patients with documented recurrent pregnancy loss and luteal phase defect as the only discernable cause, all have had diminished ovarian reserve (author's unpublished findings). Lastly, when these patients are stimulated with gonadotropins (injectable ovulation drugs) their response is universally poor and in some cases there is no response despite maximum dosages. Based on the above, several diagnostic categories in reproductive medicine, such as luteal phase defect, which have been formerly of questionable significance, and hard to study or characterize, may have at least part of their basis in diminished ovarian reserve.

Early Testing Methods And Physiological Basis for Ovarian Reserve Screening

The earliest tests of ovarian response were the cycle day 3 FSH and later the addition of cycle day 3 estradiol tests.^9,10 If the FSH or the estradiol level is high on day 3, it was found that patients had poor stimulation with gonadotropins and had greatly reduced pregnancy rates both with in vitro fertilization (IVF) or insemination cycles. This is due to the aging follicle's failure to produce adequate amounts of inhibin, a hormone that feeds back to the pituitary to suppress FSH production. The estradiol is elevated due to the elevation of FSH earlier in the cycle. An earlier and more marked rise in FSH, results in earlier follicular recruitment and a shortened follicular phase of the cycle. It was also known that if patients did not respond well to gonadotropins with high numbers of follicles and a robust estradiol response than the overall pregnancy outcome was poor. It was not until the late 1980's when the clomiphene challenge test (CCCT) was introduced, that the diagnosis of diminished ovarian reserve could be detected with a high degree of sensitivity.

The Clomiphene Citrate Challenge Test (CCCT)

In short, clomiphene citrate is given at a dose of 100 mg on days 5 through 9 of the cycle and FSH levels are determined on days 3 and 10. An estradiol level can be done on cycle day 3 as well to add supportive diagnosis. Various "cut off" values have been reported in the literature which is confusing due to various lab techniques (radioimmunoassay vs. newer fluorometric assays) and the evolution of international standards for FSH since the time the test was introduced.³ In the author's experience, with the newer fluorometric assays used by most commercial labs in our area, a value of greater than 10 for either cycle day 3 or 10 indicates in an abnormal test. The rationale for the test is simple. The cycle day 3 value is unstimulated and represents the same basal value used in the cycle day 3 FSH screening as described above. Clomiphene stimulates an increased release of FSH early in the follicular phase which improves follicular function and when normal the follicle would produce enough inhibin and estradiol to negatively feed back to the pituitary and suppress FSH production by cycle day 10.¹¹ Clomiphene adds a provocative element which uncovers patients who might otherwise not be detected with basal FSH screening. In our experience the positive predictive value of the test has been exceptional.

The CCCT And The General Fertility Population

The test has been validated both in selected infertility patients as well as the general infertility population. In one study of 236 unselected infertile women, abnormal CCCT was noted in 2 of 61 (3%) for women under 30 years of age, in 5 of 72 (7%) aged 30-34, 7 of 68 (10%) aged 35-39 and 9 of 35 (26%) aged 40 or older.¹² Pregnancy rates were dramatically reduced in the abnormal test group (2 of 23 (8.7%) vs. 92 of 213 (43%) in the normal group (Figure 3). Fifty-two percent of patients with abnormal CCCT had unexplained infertility, while 38% of study patients with the diagnosis of unexplained infertility had abnormal CCCT regardless of age.¹³ In a separate study, life table analysis of pregnancy rates in 589 couples from the general infertility population who were followed for 45 months demonstrated that for patients with an abnormal CCCT, the pregnancy rates were uniformly poor irrespective of age¹⁴ (Figure 4).

Figure 3. Pregnancy Rates Based On Outcome Of Clomiphene Challenge Testing	Figure 4. Pregnancy Rates Based On Outcome Of Clomiphene Challenge Testing
Pregnancy rates in long term prospective evaluation of CCCT in 236 women from the general infertility population (adapted from Scott, et al12)	Comparison of cumulative probability of pregnancy in 539 couples from the general infertility population (adapted from Scott RT, Hoffman GE3).

CCCT And Selected Infertility Patients

Studies of selected infertility groups have shown similar results. In one study, 91 patients who were over age 35, who had previous ovarian surgery or who were diagnosed with previous ovarian endometriosis, 37 (40.7%) had abnormal CCCT.¹⁵ Of the 37 abnormal tests, only 20 had abnormal day 3 FSH levels which would leave 17 ( 46%) of patients with diminished ovarian reserve undetected by traditional day 3 FSH measurement. No pregnancies occurred in the abnormal CCCT group compared to 11% in the normal group. In a second study, 219 women with age greater than 35 years, unexplained infertility, one ovary, or a poor response to gonadotropins, 16% had an abnormal test. For women attempting pregnancy, 49 of 148 (33.1%) of the normal group and 2 of 34 (5.9%) in the abnormal test group achieved pregnancy.¹⁶ Other studies have confirmed the significantly decreased pregnancy rates in patients with abnormal CCCT (in general less than 10%) regardless of age³ (Figures 3 and 4).

How To Manage Test Results

There are several caveats about the test results. First, if the test is normal, say in a 35 year old, it does not mean that the ovaries are "normal for age." The only conclusion that can be drawn is that the ovaries are functioning similarly to a female less than 43 years of age. She could be destined to enter diminished reserve in one or ten years. Secondly, studies that have evaluated the test carefully, have not found a stratification of normals and abnormals; the test is either positive or negative. In women over 40 years of age, there are no reported cases of an abnormal test associated with a successful pregnancy. These patients then can be definitively counseled that alternative treatments to "own-egg IVF" such as donor egg or adoption must be considered. As evidenced above, a positive test at any age portends a poor prognosis to treatment including the use of IVF, the most efficacious infertility treatment available today. Alternative stimulation protocols and other enhancements have been proposed but significant improvements in the pregnancy rate are lacking in patients with diminished ovarian reserve.

Female family history is unusually consistent both with early or late onset of reproductive failure due to diminished ovarian reserve, suggesting a strong genetic basis for these findings. It has been proposed that even prior to a clinically detectable abnormal CCCT, ovarian reserve is declining (the equivalent of age 38 - 43 before the average test would become positive). Thus a significant portion of our unexplained infertility population, roughly 40% who would have a frankly positive test and then an undisclosed number who are nearing the period of frank diminished ovarian reserve, have abnormal ovarian function. It has been suggested that an abnormal CCCT be a diagnostic category of infertility, further reducing the number of patients considered "unexplained." Putting all the evidence together, the 15% of patients with unexplained infertility are at high risk for this most distressing and difficult problem and should be seen early by an infertility specialist so that directed therapies can be offered in a timely fashion.

Age And Pregnancy Outcome

With the widespread application of donor egg to the practice of reproductive medicine and the general trend of delayed childbearing, pregnancies in the >40 age group have become imminently more common. An increase in maternal and neonatal complications has been demonstrated with advanced age. A large Swedish population-based study involving 173,715 women aged 20 and above were studied for perinatal morbidity while controlling for maternal complications.¹⁷ The rate of late stillbirth rose from 3.6/1000 births in women aged 20-24 to 6.5/1000 in the 40 and older group. Simultaneously, the rate of early neonatal death rates for the same age groups were 3.0/1000 births and 4.7/1000 respectively. In a smaller U.S. hospital-based cohort study of 3,917 women who were 20 years of age or older and had singleton pregnancies, there were no significant fetal or neonatal adverse outcomes.¹⁸ Antepartum complications were increased twofold from the 30-34 year age group as compared to the 35 and over group. In a third study, maternal mortality in the 35 years or older group compared to that of 20-34 year age group demonstrated a relative risk of 4.0 with obstetrical hemorrhage and embolism listed as the leading causes of death.¹⁹ Certainly, over time the maternal morbidity and mortality rate has declined significantly for all age groups. The risk of injury to the fetus or neonate is markedly increased but the overall risk is still relatively low and with modern obstetrical care and screening, these risks can be further minimized. There is limited data regarding the outcome of pregnancies in women 45 and older, a group which today is made up largely of donor egg recipients. Until further evidence is produced, we continue to use a conservative approach and recommend a full cardiac evaluation including a stress test, in addition to the normal pre-conceptual screening recommended for this age group.

Summary

In summary, age and infertility is an increasing problem due to general societal trends for women to delay childbearing until later ages. Patients with diminished ovarian reserve demonstrate a clinical picture which can be detected with thorough evaluation and may represent a large portion of our now so-called unexplained infertility group. The CCCT is the best screening test for diminished ovarian reserve and can be administered quickly, safely and easily to any infertility patient. Abnormal testing portends a very poor future pregnancy outcome for the individual patient. The results however, should in no way restrict or exclude patients from care.²⁰ Unfortunately, due to national reporting requirements of Assisted Reproduction programs and the competitive emphasis this places on programs to demonstrate success, some programs have excluded patients based on the results of this and other tests of ovarian function. The author hopes that readers will be more aggressive in the counseling of women who are in the early to mid thirty and beyond age group who consider delaying childbearing. In general, our approach to treatment of patients over age 35 is vastly more aggressive than in younger patients. For unknown reasons, many couples today are under the false impression that normal pregnancy rates extend well into the late thirties and early forties.

REFERENCES

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3. Scott RT, Hofmann GE. Prognostic assessment of ovarian reserve. Fertil Steril. 1995; 63: 1-11.
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5. Virro MR, Shewchuk AB. Pregnancy outcome in 242 conceptions after artificial insemination with donor sperm and effects of maternal age on the prognosis of successful pregnancy. Am J Obstet Gynecol. 1984; 148:518-524.
6. Brenner PF, Mishell DR. Menopause. In: Infertility, Contraception & Reproductive Endocrinology, 3^rd ed. Mishell DR, Davajan V, Lobo RA (eds). Boston: Blackwell, 1991.
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8. Lenton EA, Landgren, BM, Sexton L, Harper R. Normal variation in the length of the follicular phase of the menstrual cycle: effect of chronological age. Br J Obstet Gynecol. 1984;91:681-684.
9. Scott RT, Toner JP, Muasher SJ, et al. Follicle-stimulating hormone levels on cycle day 3 are predictive of invitro fertilization outcome. Fertil Steril. 1989; 51:651-654.
10. Pearlsotone AC, Fournet N, Gambone JC, et al. Ovulation induction in women age 40 and older: the importance of basal follicle-stimulating hormone level and chronological age. Fertil Steril. 1992; 58:674-679.
11. Hofmann GE, Danforth DR, Seifer DB. Inhibin-B: the physiologic basis of the clomiphene citrate challenge test for ovarian reserve screening. Fertil Steril. 1998; 11:474-477.
12. Scott RT, Leonardi MR, Hofmann GE, et al. A prospective evaluation of clomiphene citrate challenge test screening in the general infertility population. Obset Gynecol. 1993; 82:539-545.
13. Scott RT, Illions EH, Kost ER, et al. Evaluation of the significance of the estradiol response during the clomiphene citrate challenge test. Fertil Steril. 1993; 60:242-246.
14. Scott RT, Opshal MS, Leonardi MR, et al. Life table analysis of cumulative pregnancy rates based on the results of ovarian reserve screening and patient age in a general infertility population. Human Reprod. 1995; 10:1706-1710.
15. Tanbo T, Dale PO, et al. Prediction of response to controlled ovarian hyperstimulation: a comparison of basal and clomiphene citrate-stimulated follicle-stimulating hormone levels. Fertil Steril. 1992; 57:819-24.
16. Hofmann GE, Sosnowski J, Scott RT, Thie J. Efficacy of selection criteria for ovarian reserve screening using the clomiphene citrate challenge test in a tertiary fertility center population. Fertil Steril. 1996; 66:49-53.
17. Cnattingius S, Forman MR, Berendes HW, et al. Delayed childbearing and risk of adverse perinatal outcome. JAMA. 1992; 268:886-890.
18. Berkowitz GS, Skovron ML, Lapinski RH, Berkowitz RL. Delayed childbearing and the outcome of pregnancy. N Engl J Med. 1990; 322:659-664.
19. Buehler JW, Kaunitz AM, Hogue JR, et al. Maternal mortality in women aged 35 years or older: United States. JAMA. 1986; 255:53-57.
20. Seifer DB, Gardiner AC, Lambert-Messerlian G, Schneyer AL. Follicle stimulating hormone as a predictor of fertility. Curr Opin Obstet Gynecol. 1998; 81:227-232.

Jacksonville Medicine / May, 2000

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