2DefinitionsInfertility – Inability to conceive after one year of unprotected coitusAssisted reproductive technologies – Infertility procedures involving the fertilization of gametes outside the human bodyIn 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
3Infertility Diagnoses in Patients Utilizing ART Centers for Disease Control and Prevention Assisted Reproductive Technology Success Rates, 2001.
4Infertility 1995 National Survey of Family Growth Statistics 10.2% of reproductive age women (6 million) had impaired fecundity15.4% of reproductive age women (9 million) had ever utilized some type of infertility service1.2 million visits in 1995 for infertility2001 CDC/Society of Assisted Reproductive Technology Statistics107,587 ART cycles performed in 200140,687 live babies born as a resultCenters 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.
5Utilization of Assisted Reproductive Technologies Centers for Disease Control and Prevention Assisted Reproductive Technology Success Rates, 2001.
6Aging and Female Infertility Fertility both in natural and assisted reproductive cycles declines with ageThe pool of female gametes is fixed and progressively declines during growth and developmentMaximum number of oocytes achieved in fetal life is 6-7 million300,000 oocytes present by beginning of pubertyAfter age 35 the progressive loss of oocytes becomes more rapidRichardson, S. et al. Follicular depletion during the menopausal transition: evidence for acceleratedloss and ultimate exhaustion. JCEM 1987;65:
7Aging and Female Infertility Quality of oocytes also appears to decline with increasing ageIncrease in frequency of euploid and aneuploid spontaneous miscarriage with ageMeiotic spindle assembly more frequently abnormal in oocytes from older womenGreater proportion of degenerative oocytes in older women isolated from IVFNot a problem of age-related uterine changesLim A et al. Age-related decline in fertility: a link to degenerative oocytes? Fertility and Sterility 1997;68:Battaglia DE et al. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women.Human Reproduction 1996;11:
8Aging and Female Infertility Growing proportion of women delay childbearing into their thirtiesSome 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 fertilityCBSNews.com, August 17, 2003.
9Aging and Female Infertility Centers for Disease Control and Prevention Assisted Reproductive Technology Success Rates, 2001.
11Aging and Female Infertility The major locus of reproductive aging in women is the ovarian follicleThe decline in age-related reproductive potential correlates with follicular depletion and diminished quality of the oocyteThe native oocyte endowment combined with the reproductive potential of those oocytes represents a woman’s ovarian reserve
12Premature 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 poolThese women are characterized as having a clinical condition called diminished ovarian reserve
13Premature Ovarian Aging Diminished ovarian reserve has been associated with:Suboptimal response to ovulation inductionDiminished pregnancy rates after ART independent of ageIncreased risk of miscarriage and fetal aneuploidyBukulmez O et al. Assessment of ovarian reserve. Curr Opinion in Obstetrics and Gynecology 2004;16:Nasseri A et al. Elevated day 3 serum follicle stimulating hormone and/or estradiol may predict fetal aneuploidy.Fertility and Sterility 1999;71:
14Premature Ovarian Aging Centers for Disease Control and Prevention Assisted Reproductive Technology Success Rates, 2001.
15Premature Ovarian Aging The etiology of diminished ovarian reserve is unknownExposures that cause rapid atresia of ovarian folliclesChemotherapy, RadiotherapyOvarian surgery for benign conditions that removes or damages significant portions of ovarian cortexGenetic or acquired mechanisms may predispose some women to more rapid than normal follicular atresia
17Testing 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, ScotlandChronological age and ultrasonagraphic ovarian characteristics are incorporated into the model to determine the “reproductive age” of the patientModel is designed to tell a patient when she may transition into menopauseTime to menopause may not provide an accurate assessment of years of fertility remaining
18Ovarian ReserveReproductive 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 successFalsely discouraging patients from such treatments that might result in pregnancy
19Testing Ovarian Reserve Screening for ovarian reserve was an outgrowth of assisted reproductive technologiesTests were initially designed to determine prognosis of in vitro fertilization in terms of follicle recruitment and odds of pregnancyFew tests have been validated in the general infertility population
20Testing 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 fecundityIn 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 attemptedBarnhart K et al. Follicle stimulating hormone as a predictor of fertility. Curr Opinion in Obstetrics and Gynecology 1998;10:
21Testing Ovarian Reserve DiseasePPV=A/A+BThe probability of not achieving pregnancy given a positive testNPV=D/C+DThe probability of achieving pregnancy given a negative test-+TestABCD+-
22Testing Ovarian Reserve Day 3 Follicle Stimulating Hormone (FSH)First proposed as a screening test in 1988FSH is at its maximum in the early part of the menstrual cycle when estrogen levels are at their nadirElevated values suggest that ovarian follicles are not able to adequately suppress FSHValues greater than 10mIU/ml correlate with poor prognosis for those going through IVFBarnhart K. Curr Opinion in Obstetrics and Gynecology 1998;10:
23Day 3 Follicle Stimulating Hormone Advantages:NoninvasiveRelatively inexpensiveBetter predictor of IVF outcomes than age aloneHigh positive predictive value (as high as 97%)High specificity (as high as 98.7%)Disadvantages:High intercycle variabilityPoor negative predictive value (as low as 17%)Poor sensitivity (as low as 8%)Positive predictive value highly sensitive to population prevalence of infertilityBarnhart K. Curr Opinion in Obstetrics and Gynecology 1998;10:
24Clomiphene Challenge Test Provocative test of ovarian reserve first proposed as a screening test in 1989Clomiphene Citrate is an estrogen antagonist at the pituitary gland and causes a rise in FSH levelsThe test aims to determine if ovarian follicles are sufficient in quantity and quality to produce enough hormone to bring FSH back down to normal
26Clomiphene Challenge Test FSH is checked on day 3 of cycleClomiphene Citrate (100 mg) is administered from days 5-9 of the cycleFSH is rechecked on day 10 of cycleScott RT et al. Prognostic assessment of ovarian reserve. Fertility and Sterility 1995;63:1-11.
27Clomiphene Challenge Test Advantages:Non-invasiveInexpensiveSensitivity and negative predictive value improved over basal day 3 FSH testing alone (26%, 42%)Validated in IVF patients and general infertility populationDisadvantages: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:
28Inhibin B Hormone produced by granulosa cells of the ovary Negative feedback on FSH secretionDecreasing inhibin production by aging or rapidly declining ovarian follicles may explain relationship between elevated FSH levels and declining fertilityEarly investigations suggest that women with normal values are 6.8 times more likely to conceive even when age and day 3 FSH are accounted forBarnhart K. Curr Opinion in Obstetrics and Gynecology 1998;10:
29Testing Ovarian Reserve Optimal test may be an index that incorporates several serum markers and non-serologic dataInterpretation of screening test must take into consideration the population its being utilized in
30SEARCHING FOR CAUSES OF DIMINISHED OVARIAN RESERVE
31Diminished Ovarian Reserve Main target cell in the ovarian follicleOocyte versus the granulosa cellCandidate mediatorsReactive oxygen species (ROS)Ubiquitous and naturally occuringLinked to DNA damage, irreversible cell injury and aging in multiple organ systemsHigh follicular fluid ROS levels have been associated with diminished fertilization and embryo quality in IVFCandidate molecular targetsSeino 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:
33Telomeres and Telomerase A potential mechanism for aging is the shortening of telomeres with successive cell divisionsCells with critically short telomeres are unable to divide and may become lost as a result of apoptosisTelomerase maintains telomeric length preventing replicative senescence but is only active in germ cells and early embryonic cellsBuys C. Telomeres, Telomerase and Cancer. NEJM 2000;342:Hahn W. Role of Telomeres and Telomerase in the Pathogenesis of Human Cancer. J of Clinical Oncology 2003;21:
34Telomeres, Telomerase and Aging Shortened telomere lengths have been found in a number of premature aging syndromes including:Hutchinson-Gilford progeriaDown syndromeWerner syndromeJennings, B et al. Nutrition, oxidative damage, telomere shortening and cellular senescence: individual or connected agents of aging? Molecular Genetics and Metabolism, :32-42.
35Telomeres, 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 developmentIt 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 oocytediminished oocyte viability and/or qualityan accelerated loss of the oocyte pool
36Study HypothesesTelomere length will be shorter in the granulosa cells of women with decreased ovarian reserve compared to women other infertility diagnoses undergoing IVFTelomerase activity will be lower in granulosa cells of women with decreased ovarian reserve compared to women with other diagnoses undergoing IVF
37MethodsAll patients going through IVF at Penn Fertility Care are approached for study participationFollicular fluid obtained during oocyte retrieval is used to isolate granulosa cellsGranulosa cell telomere length and telomerase activity are determinedTelomerase activity is assessed using a commercially available PCR-based assayTo test for telomere length, Southern blot electrophoresis method is used (average telomere length calculated)
38Telomeres, 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 timePreliminary results have been promising for an association between telomerase inactivity and diminished ovarian reserve
39Diminished Ovarian Reserve Preliminary ResultsInfertility DxTelomerasePositiveTelomerase NegativeProportion(Negative/Total)Diminished Ovarian Reserve(7)1686%Male Factor(16)41275%Tubal Factor(9)333%Χ² test for trend=5.03 p=0.025
40Summary 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 infertilityThe use of assisted reproductive technologies is increasing in this country particularly among these groups of womenExpensive of proceduresRisks associated with ART are not trivialMedications, minor surgery, high order multiples
41SummaryReasonable screening tools exist that provide insight to the odds of fertility with reproductive technologies but they are not without limitationsPublic 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
42Future DirectionsTo optimize screening tools that can provide women with an accurate assessment of their fertility potentialTo improve our understanding of the etiology of premature ovarian agingInherited genetic underpinnings could be determined early in a woman’s reproductive lifePreventable or modifiable exposures may be discovered that impact the process of premature ovarian agingEnvironmental exposuresBehaviorsDietary exposures