Premature Ovarian Aging and Infertility

Definitions Infertility – Inability to conceive after one year of unprotected coitus Assisted reproductive technologies – Infertility procedures involving the fertilization of gametes outside the human body In vitro fertilization (IVF) Gamete intrafallopian transfer (GIFT) Zygote intrafallopian transfer (ZIFT) Intracytoplasmic sperm injection (ICSI) Fecundity – The per cycle probability of conception resulting in a live birth

Infertility Diagnoses in Patients Utilizing ART Centers for Disease Control and Prevention Assisted Reproductive Technology Success Rates, 2001.

Infertility 1995 National Survey of Family Growth Statistics 10.2% of reproductive age women (6 million) had impaired fecundity 15.4% of reproductive age women (9 million) had ever utilized some type of infertility service 1.2 million visits in 1995 for infertility 2001 CDC/Society of Assisted Reproductive Technology Statistics 107,587 ART cycles performed in 2001 40,687 live babies born as a result Centers for Disease Control and Prevention Assisted Reproductive Technology Success Rates, 2001. National Center for Health Statistics, Fertility, family planning, and women’s health: new data from the 1995 National Survey of Family Growth, Report No. 19; Series 23, 1997.

Utilization of Assisted Reproductive Technologies Centers for Disease Control and Prevention Assisted Reproductive Technology Success Rates, 2001.

Aging and Female Infertility Fertility both in natural and assisted reproductive cycles declines with age The pool of female gametes is fixed and progressively declines during growth and development Maximum number of oocytes achieved in fetal life is 6-7 million 300,000 oocytes present by beginning of puberty After age 35 the progressive loss of oocytes becomes more rapid Richardson, S. et al. Follicular depletion during the menopausal transition: evidence for accelerated loss and ultimate exhaustion. JCEM 1987;65:1231-1237.

Aging and Female Infertility Quality of oocytes also appears to decline with increasing age Increase in frequency of euploid and aneuploid spontaneous miscarriage with age Meiotic spindle assembly more frequently abnormal in oocytes from older women Greater proportion of degenerative oocytes in older women isolated from IVF Not a problem of age-related uterine changes Lim A et al. Age-related decline in fertility: a link to degenerative oocytes? Fertility and Sterility 1997;68:265-71. Battaglia DE et al. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Human Reproduction 1996;11:2217-2222.

Aging and Female Infertility Growing proportion of women delay childbearing into their thirties Some lack of awareness of aging as a risk factor for infertility “I know medically you could get pregnant up to the age of 50” “I plan to be super fit, super in shape when I’m 40, 50. And if I’m physically able to do it, then I will have a child at 55” Expectations about the potential of reproductive technologies to circumvent barriers to fertility CBSNews.com, August 17, 2003.

Aging and Female Infertility Centers for Disease Control and Prevention Assisted Reproductive Technology Success Rates, 2001.

Aging and Female Infertility The major locus of reproductive aging in women is the ovarian follicle The decline in age-related reproductive potential correlates with follicular depletion and diminished quality of the oocyte The native oocyte endowment combined with the reproductive potential of those oocytes represents a woman’s ovarian reserve

Premature Ovarian Aging Although age predicts loss of reproductive function, some women behave as if they are reproductively older than their chronological age due to a more rapid than normal depletion of the ovarian follicular pool These women are characterized as having a clinical condition called diminished ovarian reserve

Premature Ovarian Aging Diminished ovarian reserve has been associated with: Suboptimal response to ovulation induction Diminished pregnancy rates after ART independent of age Increased risk of miscarriage and fetal aneuploidy Bukulmez O et al. Assessment of ovarian reserve. Curr Opinion in Obstetrics and Gynecology 2004;16:231-237. Nasseri A et al. Elevated day 3 serum follicle stimulating hormone and/or estradiol may predict fetal aneuploidy. Fertility and Sterility 1999;71:715-18.

Premature Ovarian Aging Centers for Disease Control and Prevention Assisted Reproductive Technology Success Rates, 2001.

Premature Ovarian Aging The etiology of diminished ovarian reserve is unknown Exposures that cause rapid atresia of ovarian follicles Chemotherapy, Radiotherapy Ovarian surgery for benign conditions that removes or damages significant portions of ovarian cortex Genetic or acquired mechanisms may predispose some women to more rapid than normal follicular atresia

TESTING OVARIAN RESERVE

Testing Ovarian Reserve A mathematical model for assessment of a woman’s reproductive lifespan has been proposed by a group of investigators from the University of Andrews, Scotland Chronological age and ultrasonagraphic ovarian characteristics are incorporated into the model to determine the “reproductive age” of the patient Model is designed to tell a patient when she may transition into menopause Time to menopause may not provide an accurate assessment of years of fertility remaining

Ovarian Reserve Reproductive endocrinologists face the challenge of providing patients with an adequate prognosis of their fertility potential at a given age to avoid the following: Exposing patients to expensive and labor-intensive treatments that have little chance of success Falsely discouraging patients from such treatments that might result in pregnancy

Testing Ovarian Reserve Screening for ovarian reserve was an outgrowth of assisted reproductive technologies Tests were initially designed to determine prognosis of in vitro fertilization in terms of follicle recruitment and odds of pregnancy Few tests have been validated in the general infertility population

Testing Ovarian Reserve Ideal screening test for ovarian reserve should have the following characteristics: Capture all patients who are infertile (high sensitivity) Correctly identify those patients who are fertile (high specificity) Should test that component of the reproductive axis felt to be primarily responsible for decrease in fecundity In the case of testing ovarian reserve, high positive predictive value (PPV) and negative predictive value (NPV) are most important since the accuracy of the test can only be confirmed after pregnancy has been attempted Barnhart K et al. Follicle stimulating hormone as a predictor of fertility. Curr Opinion in Obstetrics and Gynecology 1998; 10:227-32.

Testing Ovarian Reserve Disease PPV=A/A+B The probability of not achieving pregnancy given a positive test NPV=D/C+D The probability of achieving pregnancy given a negative test - + Test A B C D + -

Testing Ovarian Reserve Day 3 Follicle Stimulating Hormone (FSH) First proposed as a screening test in 1988 FSH is at its maximum in the early part of the menstrual cycle when estrogen levels are at their nadir Elevated values suggest that ovarian follicles are not able to adequately suppress FSH Values greater than 10mIU/ml correlate with poor prognosis for those going through IVF Barnhart K. Curr Opinion in Obstetrics and Gynecology 1998;10:227-32.

Day 3 Follicle Stimulating Hormone Advantages: Noninvasive Relatively inexpensive Better predictor of IVF outcomes than age alone High positive predictive value (as high as 97%) High specificity (as high as 98.7%) Disadvantages: High intercycle variability Poor negative predictive value (as low as 17%) Poor sensitivity (as low as 8%) Positive predictive value highly sensitive to population prevalence of infertility Barnhart K. Curr Opinion in Obstetrics and Gynecology 1998;10:227-32.

Clomiphene Challenge Test Provocative test of ovarian reserve first proposed as a screening test in 1989 Clomiphene Citrate is an estrogen antagonist at the pituitary gland and causes a rise in FSH levels The test aims to determine if ovarian follicles are sufficient in quantity and quality to produce enough hormone to bring FSH back down to normal

Clomiphene Challenge Test

Clomiphene Challenge Test FSH is checked on day 3 of cycle Clomiphene Citrate (100 mg) is administered from days 5-9 of the cycle FSH is rechecked on day 10 of cycle Scott RT et al. Prognostic assessment of ovarian reserve. Fertility and Sterility 1995;63:1-11.

Clomiphene Challenge Test Advantages: Non-invasive Inexpensive Sensitivity and negative predictive value improved over basal day 3 FSH testing alone (26%, 42%) Validated in IVF patients and general infertility population Disadvantages: Slight decrease in positive predictive value compared to day 3 FSH especially when used in low risk population (96%) Slight decrease in specificity compared to day 3 FSH (98%) Barnhart K. Curr Opinion in Obstetrics and Gynecology 1998;10:227-32.

Inhibin B Hormone produced by granulosa cells of the ovary Negative feedback on FSH secretion Decreasing inhibin production by aging or rapidly declining ovarian follicles may explain relationship between elevated FSH levels and declining fertility Early investigations suggest that women with normal values are 6.8 times more likely to conceive even when age and day 3 FSH are accounted for Barnhart K. Curr Opinion in Obstetrics and Gynecology 1998;10:227-32.

Testing Ovarian Reserve Optimal test may be an index that incorporates several serum markers and non-serologic data Interpretation of screening test must take into consideration the population its being utilized in

SEARCHING FOR CAUSES OF DIMINISHED OVARIAN RESERVE

Diminished Ovarian Reserve Main target cell in the ovarian follicle Oocyte versus the granulosa cell Candidate mediators Reactive oxygen species (ROS) Ubiquitous and naturally occuring Linked to DNA damage, irreversible cell injury and aging in multiple organ systems High follicular fluid ROS levels have been associated with diminished fertilization and embryo quality in IVF Candidate molecular targets Seino T et al. Eight-hydroxy-2”-deoxyguanosine in granulosa cells is correlated with the quality of oocytes and embryos in an in vitro fertilzation-embryo transfer program. Fertility and Sterility 2002;77:1184-90.

Telomeres and Telomerase

Telomeres and Telomerase A potential mechanism for aging is the shortening of telomeres with successive cell divisions Cells with critically short telomeres are unable to divide and may become lost as a result of apoptosis Telomerase maintains telomeric length preventing replicative senescence but is only active in germ cells and early embryonic cells Buys C. Telomeres, Telomerase and Cancer. NEJM 2000;342:1282-63. Hahn W. Role of Telomeres and Telomerase in the Pathogenesis of Human Cancer. J of Clinical Oncology 2003;21:2034-43.

Telomeres, Telomerase and Aging Shortened telomere lengths have been found in a number of premature aging syndromes including: Hutchinson-Gilford progeria Down syndrome Werner syndrome Jennings, B et al. Nutrition, oxidative damage, telomere shortening and cellular senescence: individual or connected agents of aging? Molecular Genetics and Metabolism, 2000. 71:32-42.

Telomeres, Telomerase and Premature Ovarian Aging Telomerase has been demonstrated in bovine granulosa cells and its presence is thought to permit their proliferation supporting normal follicle development It is possible that diminished granulosa cell telomerase activity in concert with unusually short telomeres could lead to: The sub-optimal proliferation of granulosa cells surrounding the oocyte diminished oocyte viability and/or quality an accelerated loss of the oocyte pool

Study Hypotheses Telomere length will be shorter in the granulosa cells of women with decreased ovarian reserve compared to women other infertility diagnoses undergoing IVF Telomerase activity will be lower in granulosa cells of women with decreased ovarian reserve compared to women with other diagnoses undergoing IVF

Methods All patients going through IVF at Penn Fertility Care are approached for study participation Follicular fluid obtained during oocyte retrieval is used to isolate granulosa cells Granulosa cell telomere length and telomerase activity are determined Telomerase activity is assessed using a commercially available PCR-based assay To test for telomere length, Southern blot electrophoresis method is used (average telomere length calculated)

Telomeres, Telomerase, Premature Ovarian Aging This investigation examines the possible association of diminished granulosa cell telomerase activity/telomeric shortening and premature ovarian aging for the first time Preliminary results have been promising for an association between telomerase inactivity and diminished ovarian reserve

Diminished Ovarian Reserve Preliminary Results Infertility Dx Telomerase Positive Telomerase Negative Proportion (Negative/Total) Diminished Ovarian Reserve (7) 1 6 86% Male Factor (16) 4 12 75% Tubal Factor (9) 3 33% Χ² test for trend=5.03 p=0.025

Summary Aging represents a significant risk to fertility in women In a subset of women who have diminished ovarian reserve, a more rapid than normal decline in oocyte quantity and quality occurs and these women are at particular risk for infertility The use of assisted reproductive technologies is increasing in this country particularly among these groups of women Expensive of procedures Risks associated with ART are not trivial Medications, minor surgery, high order multiples

Summary Reasonable screening tools exist that provide insight to the odds of fertility with reproductive technologies but they are not without limitations Public awareness of the effect of age on fertility and the realistic capabilities of assisted reproductive technologies may be suboptimal but the timing of family initiation should ultimately rest with the individual’s personal readiness

Future Directions To optimize screening tools that can provide women with an accurate assessment of their fertility potential To improve our understanding of the etiology of premature ovarian aging Inherited genetic underpinnings could be determined early in a woman’s reproductive life Preventable or modifiable exposures may be discovered that impact the process of premature ovarian aging Environmental exposures Behaviors Dietary exposures