- Email: [email protected]
Basal follicle-stimulating hormone as a predictor of fetal aneuploidy
Basal follicle-stimulating hormone as a predictor of fetal aneuploidy Jamie A. M. Massie, M.D., Richard O. Burney, M.D., Amin A. Milki, M.D., Lynn M. Westphal, M.D., and Ruth B. Lathi, M.D. Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Stanford University Medical Center, Stanford, California
Objective: To determine whether an elevated basal FSH concentration is an independent predictor of fetal aneuploidy, as measured in spontaneous abortions (SAB). Design: Retrospective study. Setting: Academic reproductive endocrinology and infertility center. Patient(s): All women with karyotypes of chorionic villi isolated from first trimester spontaneous miscarriages at the time of dilation and curettage from 1999 to 2006. The highest basal serum FSH level in the year preceding dilation and curettage was recorded. Interventions(s): Monitoring of early pregnancy. Main Outcome Measure(s): Fetal karyotype. Results(s): A total of 177 spontaneous miscarriages with karyotypes (70 euploid and 107 aneuploid) were identified, of which 53% were conceived by IVF. The aneuploid cohort consisted of trisomic (87%), teraploid (9.3%), and monosomic (3.7%) gestations. Using logistic regression analysis, basal FSH was not found to be independently predictive of an aneuploid gestation in our data set. Conclusion(s): Our data do not support the hypothesis that an elevated basal FSH concentration is associated with an increase in fetal aneuploidy. Our findings suggest that the association between diminished ovarian reserve and SAB may result from nonkaryotypic factors. (Fertil Steril 2008;90:2351–5. 2008 by American Society for Reproductive Medicine.) Key Words: Aneuploidy, IVF, miscarriage, FSH, E2, ovarian reserve, oocyte quality
The risk of fetal aneuploidy increases with advancing maternal age, and the majority of this risk is believed to be secondary to errors arising at meisois I in the oocyte (1). Two prominent theories have developed to explain this observation—one highlights the influence of chronologic ovarian aging, whereas the ‘‘limited pool hypothesis’’ emphasizes the role of biologic signals on ovarian aging. Several lines of evidence support the impact of chronologic ovarian aging on fetal aneuploidy risk. Meiotic error rates in oocytes are higher in women with advancing maternal age (2), a finding that may result from the prolonged time that oocytes spend arrested in meiosis I before ovulation (3). Meiotic spindle morphology has also been found to change deleteriously with advancing maternal age (4). Finally, the risk of maternal sex chromosome nondisjunction increases with maternal age (5). On the other hand, the limited pool hypothesis holds that ovarian aging rather than chronologic aging contributes to fetal aneuploidy risk. This concept was first proposed by investigators who noted that unilateral oophorectomy in mice Received August 16, 2007; revised October 10, 2007; accepted October 22, 2007. Drs. Massie and Burney contributed equally to this work. Presented at the 2007 Annual Meeting of the Pacific Coast Reproductive Society, Indian Wells, California, April 18–22, 2007. Reprint requests: Ruth Lathi, M.D., Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford University, 300 Pasteur Drive, Stanford, CA 94305 (FAX: 650-723-7737; E-mail: [email protected]).
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.10.041
not only advanced cessation of reproduction, but also increased the incidence of trisomic offspring (6). The human correlate to this observation in mice would imply that women conceiving trisomic pregnancies experience menopause at an earlier age than the population at large. Women with a known trisomic pregnancy enter menopause about 1 year earlier than do those with a chromosomally normal index pregnancy (7). Likewise, mothers of Down syndrome individuals were significantly more likely than controls to have a reduced ovarian complement, either as a result of ovarian surgery or because of congenital absence of one ovary (8). The treatment of infertility and reduction of aneuploidy risk require an ability to identify the highest quality oocytes (9). Several modalities currently exist for evaluating oocyte quality after retrieval, including early embryonic cleavage, multinucleation, morphology of the polar body, and preimplantation genetic diagnosis (10, 11). Unfortunately, a reliable, noninvasive predictor of oocyte quality before oocyte retrieval has yet to be identified (9). An elevated basal FSH concentration is used clinically as a marker for diminished ovarian reserve (12–14). Yet, the correlation between basal FSH and oocyte quality is less clear. It has been hypothesized that increased FSH levels toward the end of reproductive life may correlate with the increased aneuploidy rates seen in oocytes retrieved from older women
Fertility and Sterility Vol. 90, No. 6, December 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
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(15). Elevations in basal FSH levels are also seen in younger women whose reduction in oocyte pool is a result of premature ovarian failure (POF). A role for basal FSH as a marker of oocyte quality in these populations has been suggested (3). An age-independent relationship between elevated basal FSH level and reduced oocyte quality/aneuploidy risk would lend support to the limited pool hypothesis. Clinically, such an association would impact the preconceptual counseling of the younger patient with an elevated basal FSH as to her miscarriage and fetal aneuploidy risks. In this study, we sought to determine whether an elevated basal FSH concentration is an independent predictor of fetal aneuploidy, as assessed by karyotyping of spontaneous abortions (SAB). MATERIALS AND METHODS By retrospective chart review from 1999 to 2006 we identified 177 women experiencing a first trimester SAB who underwent dilation and curettage with karyotyping of chorionic villi and who had a basal FSH level and normal E2 level within 1 year before dilation and curettage. At our center, tandem basal FSH and E2 measurements are assayed by ELISA (Immulite; Diagnostic Products Corporation, Los Angeles, CA). Only basal FSH in the setting of normal (<60 pg/mL) E2 were included, as previous studies have shown that elevated E2 levels can falsely suppress cycle day 3 FSH concentrations (16, 17). Institutional review board (IRB) approval for this study was granted under an exemption protocol. Patient characteristics, such as maternal age, reproductive history, method of conception, and use of intracytoplasmic sperm injection (ICSI), were reviewed. Pregnancies were the result of IVF–embryo transfer (53%), IUI (24%), or spontaneous conception (23%). Donor oocyte and preimplantation genetic diagnosis conceptions were excluded. Cytogenetic testing was performed by the cytogenetics laboratory at Stanford University Medical Center. A technique of tissue selection that has been shown to decrease false-negative results was used to isolate the specimens for testing (18). This involved identifying chorionic villi after rinsing the products of conception. A sample of villi was then carefully dissected from the remaining tissue and sent for chromosomal analysis. Specimens were submitted to the cytogenetics laboratory in complete RPMI (Rosewell Park Memorial Institute) media. Tissue was cultured and chromosomes were analyzed using the GTW banding method (G-banding Trypsin-Wright method). Karyotype abnormalities, such as trisomy, triploidy, and monosomy, were included in the analysis. Tetraploidy (92XXXX, n ¼ 5) was excluded as this finding may represent culture artifact. Statistical analysis consisted of Student’s t-test or c2 tests, with FSH and maternal age evaluated both as continuous and as categorical variables. P values < .05 were considered statistically significant. For categorical analysis, advanced maternal age was defined as age R35 years, as increased rates 2352
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of fetal aneuploidy have been documented in this age group (19). A retrospective review of clinical outcomes at our center established a threshold for diminished ovarian reserve at a basal serum FSH level of R14 mIU/mL (Immulite), and this was the cutoff used when treating basal FSH as a categorical variable. Interestingly, this cutoff is consistent with that published by other investigators using a similar approach (20). Logistic regression was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) relating aneuploidy to each predictor variable. RESULTS For purposes of analysis, patients were divided into those with euploid (n ¼ 70) versus those with aneuploid (n ¼ 107) fetal karyotypes. Due to a higher than expected (61%) ratio of XX karyotypes in the euploid cohort, and the potential for maternal contamination to confound interpretation (21), we considered only 46XY karyotypes (n ¼ 27) as the definitively euploid group. Specimens were not available for differential single nucleotide polymorphism studies to discriminate maternal from fetal DNA. The aneuploid cohort consisted of trisomic (87%), triploid (9.3%), and monosomic (3.7%) gestations. Patient characteristics, including gravity, parity, and E2 levels, were similar among the two groups (Table 1). The distribution of patients who conceived spontaneously, by IVF–embryo transfer, or IUI was not statistically different between the two groups. The number of patients with a history of one or more spontaneous miscarriages was also similar between the groups. Logistic regression analysis revealed basal FSH not to be independently predictive of an aneuploid gestation when analyzed as a continuous variable (Table 2). However, when analyzed as a categorical variable, basal FSH R14 mIU/mL was found to be significantly associated with karyotypically normal pregnancies (P¼.029). When maternal age was evaluated as a continuous variable, no statistically significant association with fetal aneuploidy was identified. However, when advanced maternal age was analyzed as a categorical variable (age R35 years), the association with fetal aneuploidy nearly reached statistical significance (P¼.064). A separate analysis including 46XX karyotypes in the euploid group was also performed and no significant association between basal FSH and aneuploid gestation was demonstrated (data not shown). DISCUSSION This study was designed to determine whether an elevated basal serum FSH level is associated with fetal aneuploidy risk. Because maternal age has a known association with increased meiotic error rates, as well as gradually increasing FSH levels (1), we sought to determine whether FSH acts as an independent marker for increased aneuploidy risk. We found no significant age-independent relationship between elevated basal FSH concentration and fetal aneuploidy, regardless of whether basal FSH level was treated as a continuous or as a categorical (R14 mIU/mL) variable. Using
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TABLE 1 Characteristics of patients with euploid versus aneuploid fetal karyotypes.
Characteristics
Euploid XY (n [ 27)
Aneuploid (n [ 107)
Maternal age (y) 36.67 3.65 38.02 3.96 Gravidity 0.96 1.28 1.40 1.64 Parity 0.12 0.33 0.29 0.52 Prior SAB 0.56 0.93 0.91 1.36 29.28 9.26 33.34 11.33 CD3 E2 (pg/mL) Method of conception Spontaneous (n) 6 24 IUI (n) 7 25 IVF-embryo 14 56 transfer (n) ICSI (n) 4 25 Note: Values are expressed as mean SD. All P values not significant. CD3 ¼ cycle day 3; SAB ¼ spontaneous abortion. Massie. Basal FSH a predictor of fetal aneuploidy. Fertil Steril 2008.
a cutoff of 35 years, a nearly significant association between advanced maternal age (R35 years) and increased fetal aneuploidy rate was found (P¼.064), a trend consistent with other studies (2, 22, 23). Other studies have assessed the value of basal FSH as a marker for oocyte quality as measured by fetal aneuploidy (3, 24). The results of these studies suggested an inverse relationship between basal FSH and oocyte quality, with a significantly greater proportion of age-matched women with abnormally elevated FSH values experiencing miscarriages
TABLE 2 Basal serum FSH and E2 levels, maternal age, and the fetal karyotype. Variables
Euploid XY (n [ 27)
Aneuploid (n [ 107)
P value
Maternal age 36.67 3.65 38.02 3.96 NS (y) Maternal age 55.6% 73.8% .064 >35 y CD3 FSH 9.21 3.38 8.60 6.49 NS (mIU/mL) CD3 FSH 18.5% 5.6% .029 R14 mIU/ mL Note: Values are expressed as mean SD. CD3 ¼ cycle day 3; NS ¼ not significant. Massie. Basal FSH a predictor of fetal aneuploidy. Fertil Steril 2008.
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with abnormal fetal karyotypes. Other investigators have reported an increased risk of Down syndrome in women with elevated basal FSH concentrations measured preconceptually (8, 24). These studies would seem to support the limited pool hypothesis of ovarian aging, in that diminished ovarian reserve and advanced biologic ovarian aging are better predictors of aneuploidy risk than is chronological age. However, more recent studies contradict this conclusion. Havryliuk et al. (25) showed no correlation between basal FSH level and fetal trisomy risk in poor prognostic IVF patients. A separate study found that premature decline in ovarian function, as evidenced by history of ovarian resistance or an elevated basal FSH level, was not associated with an increased risk of aneuploidy (26). Other investigators have reported basal FSH to be particularly poor at predicting aneuploidy risk in younger women (27, 28). In sum, it appears that elevated basal FSH concentration is a consistent marker for decreased ovarian reserve, but a poor indicator of oocyte quality, especially in younger women with premature decline in ovarian reserve (29, 30). Interestingly, our study revealed that spontaneous abortuses in women with a basal FSH level of R14 mIU/mL were more likely to be euploid (5/27) than aneuploid (6/ 107), and this finding reached statistical significance (P¼.029). Logistic regression treating both basal FSH and maternal age as categorical variables (cutoffs as discussed previously) confirmed an association between elevated basal FSH and euploid karyotype of miscarriage products (OR for aneuploidy 0.25, 95% CI 0.07–0.91, P<.04). Thus, women with a basal serum FSH R14 mIU/mL who conceive and experience miscarriage are less likely to have conceived an aneuploid gestation. A subset analysis was attempted to further investigate this finding; however, due to small sample size, no meaningful conclusions could be reached. It is possible that factors other than fetal aneuploidy were responsible for miscarriages within in this group. Such factors could include immunologic, endocrine, and hematologic processes not accounted for within the scope of this study. Smoking has been associated with both an elevated basal FSH level (31) and with miscarriage (32), yet none of the five women with FSH R14 mIU/mL and euploid miscarriages in our study were smokers. This group of women was heterogeneous in term of method of conception (1, spontaneous conception; 2, IUI; 2, IVF), and only one of the five had experienced a previous miscarriage. An elevated basal FSH level has been associated with recurrent pregnancy loss (33, 34), and this association was assumed to be consequent to an increase in fetal aneuploidy. Yet our findings suggest that an elevated basal FSH level may be associated with an abnormality not detectable by conventional karyotype or an altogether nongenetic (i.e., hormonal) abnormality that deleteriously impacts fecundity. Although we did not specifically test patients for skewed X chromosome inactivation (XCI) in our study, this phenomenon may provide an explanation for our finding of an age2353
independent increase in normal karyotypes in women with elevated basal FSH levels. Theoretically, recessively inherited mutations on the X chromosome lethal to the male carrier would result in skewed X chromosome inactivation in female carriers and predispose these women to abortion of 46XY conceptuses receiving the defective X (35). A skewed XCI pattern has been independently reported in women with recurrent SAB (36, 37). It is hypothesized to exist in women with diminished ovarian reserve (38), and would seem a unifying explanation for our observation. Our study design offers several strengths compared to previous reports. First, the design allowed us to capture karyotypic abnormalities that would not progress past the first trimester of pregnancy, and would therefore be missed by study designs that use karyotype information collected through chorionic villus sampling or amniocentesis (39–41). Second, ‘‘46XX’’ karyotypes were excluded from analysis, eliminating the potential for confounding of results by maternal contamination. We believe that this is necessary to improve the fidelity of the euploid cohort data until a reliable and readily available method is developed to discriminate maternal from fetal cytogenetics in the case of 46XX karyotypes. Finally, the large sample size in this study relative to others increases the reliability of our findings. As with any retrospective review there are possible confounding factors that could not be fully addressed by this design. For example, the type of stimulation protocol used in patients undergoing IVF was not addressed by our study. The type of stimulation protocol could potentially affect aneuploidy rates and is an area of current investigation (26). Although most fetal aneuploidy is believed to result from maternal meiotic errors, other factors affecting this outcome may exist that were not addressed by our analysis. In summary, our data do not appear to support the hypothesis that a reduced oocyte pool is associated with an increased meiotic error rate. Continued investigations to identify reliable, noninvasive methods for predicting oocyte and fetal aneuploidy are necessary.
REFERENCES 1. Hassold T, Hunt P. To err (meiotically) is human: the genesis of human aneuploidy. Nat Rev Genet 2001;2:280–91. 2. Munne S, Alikani M, Tomkin G, Grifo J, Cohen J. Embryo morphology, developmental rates, and maternal age are correlated with chromosome abnormalities. Fertil Steril 1995;64:382–91. 3. Nasseri A, Mukherjee T, Grifo JA, Noyes N, Krey L, Copperman AB. Elevated day 3 serum follicle stimulating hormone and/or estradiol may predict fetal aneuploidy. Fertil Steril 1999;71:715–8. 4. Battaglia DE, Goodwin P, Klein NA, Soules MR. Influence of maternal age on meiotic spindle assembly in oocytes from naturally cycling women. Hum Reprod 1996;11:2217–22. 5. Thomas NS, Ennis S, Sharp AJ, Durkie M, Hassold TJ, Collins AR, et al. Maternal sex chromosome non-disjunction: evidence for X chromosome-specific risk factors. Hum Mol Genet 2001;10:243–50. 6. Brook JD, Gosden RG, Chandley AC. Maternal ageing and aneuploid embryos—evidence from the mouse that biological and not chronological age is the important influence. Hum Genet 1984;66:41–5.
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7. Kline J, Kinney A, Levin B, Warburton D. Trisomic pregnancy and earlier age at menopause. Am J Hum Genet 2000;67:395–404. 8. Freeman SB, Yang Q, Allran K, Taft LF, Sherman SL. Women with a reduced ovarian complement may have an increased risk for a child with Down syndrome. Am J Hum Genet 2000;66:1680–3. 9. Combelles CM, Racowsky C. Assessment and optimization of oocyte quality during assisted reproductive technology treatment. Semin Reprod Med 2005;23:277–84. 10. Xia P. Intracytoplasmic sperm injection: correlation of oocyte grade based on polar body, perivitelline space and cytoplasmic inclusions with fertilization rate and embryo quality. Hum Reprod 1997;12: 1750–5. 11. Ebner T, Yaman C, Moser M, Sommergruber M, Feichtinger O, Tews G. Prognostic value of first polar body morphology on fertilization rate and embryo quality in intracytoplasmic sperm injection. Hum Reprod 2000;15:427–30. 12. Muasher SJ, Oehninger S, Simonetti S, Matta J, Ellis LM, Liu HC, et al. The value of basal and/or stimulated serum gonadotropin levels in prediction of stimulation response and in vitro fertilization outcome. Fertil Steril 1988;50:298–307. 13. Scott RT, Toner JP, Muasher SJ, Oehninger S, Robinson S, Rosenwaks Z. Follicle-stimulating hormone levels on cycle day 3 are predictive of in vitro fertilization outcome. Fertil Steril 1989;51:651–4. 14. Toner JP, Philput CB, Jones GS, Muasher SJ. Basal follicle-stimulating hormone level is a better predictor of in vitro fertilization performance than age. Fertil Steril 1991;55:784–91. 15. Roberts R, Iatropoulou A, Ciantar D, Stark J, Becker DL, Franks S, et al. Follicle-stimulating hormone affects metaphase I chromosome alignment and increases aneuploidy in mouse oocytes matured in vitro. Biol Reprod 2005;72:107–18. 16. Evers JL, Slaats P, Land JA, Dumoulin JC, Dunselman GA. Elevated levels of basal estradiol-17beta predict poor response in patients with normal basal levels of follicle-stimulating hormone undergoing in vitro fertilization. Fertil Steril 1998;69:1010–4. 17. Licciardi FL, Liu HC, Rosenwaks Z. Day 3 estradiol serum concentrations as prognosticators of ovarian stimulation response and pregnancy outcome in patients undergoing in vitro fertilization. Fertil Steril 1995;64:991–4. 18. Lathi RB, Milki AA. Tissue sampling technique affects accuracy of karyotype from missed abortions. J Assist Reprod Genet 2002;19:536–8. 19. Spencer K. Aneuploidy screening in the first trimester. Am J Med Genet C Semin Med Genet 2007;145:18–32. 20. Levi AJ, Raynault MF, Bergh PA, Drews MR, Miller BT, Scott RT Jr. Reproductive outcome in patients with diminished ovarian reserve. Fertil Steril 2001;76:666–9. 21. Menasha J, Levy B, Hirschhorn K, Kardon NB. Incidence and spectrum of chromosome abnormalities in spontaneous abortions: new insights from a 12-year study. Genet Med 2005;7:251–63. 22. Taranissi M, El-Toukhy T, Gorgy A, Verlinsky Y. Influence of maternal age on the outcome of PGD for aneuploidy screening in patients with recurrent implantation failure. Reprod Biomed Online 2005;10:628–32. 23. Baart EB, Martini E, van den Berg I, Macklon NS, Galjaard RJ, Fauser BC, et al. Preimplantation genetic screening reveals a high incidence of aneuploidy and mosaicism in embryos from young women undergoing IVF. Hum Reprod 2006;21:223–33. 24. van Montfrans JM, Dorland M, Oosterhuis GJ, van Vugt JM, RekersMombarg LT, Lambalk CB. Increased concentrations of folliclestimulating hormone in mothers of children with Down’s syndrome. Lancet 1999;353:1853–4. 25. Havryliuk Y, Dragisic KG, Rosenwaks Z, Spandorfer S. Baseline serum FSH levels in poor prognostic IVF patients: do abnormal levels predict fetal aneuploidy? Fertil Steril 2006;86:S21. 26. Weghofer A, Barad D, Li J, Gleicher N. Aneuploidy rates in embryos from women with prematurely declining ovarian function: a pilot study. Fertil Steril 2007;88:90–4. 27. Akande VA, Fleming CF, Hunt LP, Keay SD, Jenkins JM. Biological versus chronological ageing of oocytes, distinguishable by raised FSH levels
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in relation to the success of IVF treatment. Hum Reprod 2002;17: 2003–8. Abdalla H, Thum MY. An elevated basal FSH reflects a quantitative rather than qualitative decline of the ovarian reserve. Hum Reprod 2004;19:893–8. van Rooij IA, Bancsi LF, Broekmans FJ, Looman CW, Habbema JD, te Velde ER. Women older than 40 years of age and those with elevated follicle-stimulating hormone levels differ in poor response rate and embryo quality in in vitro fertilization. Fertil Steril 2003;79:482–8. Gleicher N, Barad D. ‘‘Ovarian age-based’’ stimulation of young women with diminished ovarian reserve results in excellent pregnancy rates with in vitro fertilization. Fertil Steril 2006;86:1621–5. Lambalk CB, de Koning CH. Interpretation of elevated FSH in the regular menstrual cycle. Maturitas 1998;30:215–20. Ness RB, Grisso JA, Hirschinger N, Markovic N, Shaw LM, Day NL, et al. Cocaine and tobacco use and the risk of spontaneous abortion. N Engl J Med 1999;340:333–9. Trout SW, Seifer DB. Do women with unexplained recurrent pregnancy loss have higher day 3 serum FSH and estradiol values? Fertil Steril 2000;74:335–7. Gurbuz B, Yalti S, Ficicioglu C, Ozden S, Yildirim G, Sayar C. Basal hormone levels in women with recurrent pregnancy loss. Gynecol Endocrinol 2003;17:317–21.
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35. Lanasa MC, Hogge WA. X chromosome defects as an etiology of recurrent spontaneous abortion. Semin Reprod Med 2000;18:97–103. 36. Lanasa MC, Hogge WA, Kubik CJ, Ness RB, Harger J, Nagel T, et al. A novel X chromosome-linked genetic cause of recurrent spontaneous abortion. Am J Obstet Gynecol 2001;185:563–8. 37. Uehara S, Hashiyada M, Sato K, Sato Y, Fujimori K, Okamura K. Preferential X-chromosome inactivation in women with idiopathic recurrent pregnancy loss. Fertil Steril 2001;76:908–14. 38. Beever CL, Stephenson MD, Penaherrera MS, Jiang RH, Kalousek DK, Hayden M, et al. Skewed X-chromosome inactivation is associated with trisomy in women ascertained on the basis of recurrent spontaneous abortion or chromosomally abnormal pregnancies. Am J Hum Genet 2003;72:399–407. 39. Nicolaidis P, Petersen MB. Origin and mechanisms of non-disjunction in human autosomal trisomies. Hum Reprod 1998;13:313–9. 40. Strom CM, Ginsberg N, Applebaum M, Bozorgi N, White M, Caffarelli M, et al. Analyses of 95 first-trimester spontaneous abortions by chorionic villus sampling and karyotype. J Assist Reprod Genet 1992;9:458–61. 41. van Montfrans JM, van Hooff MH, Huirne JA, Tanahatoe SJ, Sadrezadeh S, Martens F, et al. Basal FSH concentrations as a marker of ovarian ageing are not related to pregnancy outcome in a general population of women over 30 years. Hum Reprod 2004;19:430–4.
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Objective: To determine whether an elevated basal FSH concentration is an independent predictor of fetal aneuploidy, as measured in spontaneous abortions (SAB). Design: Retrospective study. Setting: Academic reproductive endocrinology and infertility center. Patient(s): All women with karyotypes of chorionic villi isolated from first trimester spontaneous miscarriages at the time of dilation and curettage from 1999 to 2006. The highest basal serum FSH level in the year preceding dilation and curettage was recorded. Interventions(s): Monitoring of early pregnancy. Main Outcome Measure(s): Fetal karyotype. Results(s): A total of 177 spontaneous miscarriages with karyotypes (70 euploid and 107 aneuploid) were identified, of which 53% were conceived by IVF. The aneuploid cohort consisted of trisomic (87%), teraploid (9.3%), and monosomic (3.7%) gestations. Using logistic regression analysis, basal FSH was not found to be independently predictive of an aneuploid gestation in our data set. Conclusion(s): Our data do not support the hypothesis that an elevated basal FSH concentration is associated with an increase in fetal aneuploidy. Our findings suggest that the association between diminished ovarian reserve and SAB may result from nonkaryotypic factors. (Fertil Steril 2008;90:2351–5. 2008 by American Society for Reproductive Medicine.) Key Words: Aneuploidy, IVF, miscarriage, FSH, E2, ovarian reserve, oocyte quality
The risk of fetal aneuploidy increases with advancing maternal age, and the majority of this risk is believed to be secondary to errors arising at meisois I in the oocyte (1). Two prominent theories have developed to explain this observation—one highlights the influence of chronologic ovarian aging, whereas the ‘‘limited pool hypothesis’’ emphasizes the role of biologic signals on ovarian aging. Several lines of evidence support the impact of chronologic ovarian aging on fetal aneuploidy risk. Meiotic error rates in oocytes are higher in women with advancing maternal age (2), a finding that may result from the prolonged time that oocytes spend arrested in meiosis I before ovulation (3). Meiotic spindle morphology has also been found to change deleteriously with advancing maternal age (4). Finally, the risk of maternal sex chromosome nondisjunction increases with maternal age (5). On the other hand, the limited pool hypothesis holds that ovarian aging rather than chronologic aging contributes to fetal aneuploidy risk. This concept was first proposed by investigators who noted that unilateral oophorectomy in mice Received August 16, 2007; revised October 10, 2007; accepted October 22, 2007. Drs. Massie and Burney contributed equally to this work. Presented at the 2007 Annual Meeting of the Pacific Coast Reproductive Society, Indian Wells, California, April 18–22, 2007. Reprint requests: Ruth Lathi, M.D., Department of Obstetrics and Gynecology, Stanford University School of Medicine, Stanford University, 300 Pasteur Drive, Stanford, CA 94305 (FAX: 650-723-7737; E-mail: [email protected]).
0015-0282/08/$34.00 doi:10.1016/j.fertnstert.2007.10.041
not only advanced cessation of reproduction, but also increased the incidence of trisomic offspring (6). The human correlate to this observation in mice would imply that women conceiving trisomic pregnancies experience menopause at an earlier age than the population at large. Women with a known trisomic pregnancy enter menopause about 1 year earlier than do those with a chromosomally normal index pregnancy (7). Likewise, mothers of Down syndrome individuals were significantly more likely than controls to have a reduced ovarian complement, either as a result of ovarian surgery or because of congenital absence of one ovary (8). The treatment of infertility and reduction of aneuploidy risk require an ability to identify the highest quality oocytes (9). Several modalities currently exist for evaluating oocyte quality after retrieval, including early embryonic cleavage, multinucleation, morphology of the polar body, and preimplantation genetic diagnosis (10, 11). Unfortunately, a reliable, noninvasive predictor of oocyte quality before oocyte retrieval has yet to be identified (9). An elevated basal FSH concentration is used clinically as a marker for diminished ovarian reserve (12–14). Yet, the correlation between basal FSH and oocyte quality is less clear. It has been hypothesized that increased FSH levels toward the end of reproductive life may correlate with the increased aneuploidy rates seen in oocytes retrieved from older women
Fertility and Sterility Vol. 90, No. 6, December 2008 Copyright ª2008 American Society for Reproductive Medicine, Published by Elsevier Inc.
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(15). Elevations in basal FSH levels are also seen in younger women whose reduction in oocyte pool is a result of premature ovarian failure (POF). A role for basal FSH as a marker of oocyte quality in these populations has been suggested (3). An age-independent relationship between elevated basal FSH level and reduced oocyte quality/aneuploidy risk would lend support to the limited pool hypothesis. Clinically, such an association would impact the preconceptual counseling of the younger patient with an elevated basal FSH as to her miscarriage and fetal aneuploidy risks. In this study, we sought to determine whether an elevated basal FSH concentration is an independent predictor of fetal aneuploidy, as assessed by karyotyping of spontaneous abortions (SAB). MATERIALS AND METHODS By retrospective chart review from 1999 to 2006 we identified 177 women experiencing a first trimester SAB who underwent dilation and curettage with karyotyping of chorionic villi and who had a basal FSH level and normal E2 level within 1 year before dilation and curettage. At our center, tandem basal FSH and E2 measurements are assayed by ELISA (Immulite; Diagnostic Products Corporation, Los Angeles, CA). Only basal FSH in the setting of normal (<60 pg/mL) E2 were included, as previous studies have shown that elevated E2 levels can falsely suppress cycle day 3 FSH concentrations (16, 17). Institutional review board (IRB) approval for this study was granted under an exemption protocol. Patient characteristics, such as maternal age, reproductive history, method of conception, and use of intracytoplasmic sperm injection (ICSI), were reviewed. Pregnancies were the result of IVF–embryo transfer (53%), IUI (24%), or spontaneous conception (23%). Donor oocyte and preimplantation genetic diagnosis conceptions were excluded. Cytogenetic testing was performed by the cytogenetics laboratory at Stanford University Medical Center. A technique of tissue selection that has been shown to decrease false-negative results was used to isolate the specimens for testing (18). This involved identifying chorionic villi after rinsing the products of conception. A sample of villi was then carefully dissected from the remaining tissue and sent for chromosomal analysis. Specimens were submitted to the cytogenetics laboratory in complete RPMI (Rosewell Park Memorial Institute) media. Tissue was cultured and chromosomes were analyzed using the GTW banding method (G-banding Trypsin-Wright method). Karyotype abnormalities, such as trisomy, triploidy, and monosomy, were included in the analysis. Tetraploidy (92XXXX, n ¼ 5) was excluded as this finding may represent culture artifact. Statistical analysis consisted of Student’s t-test or c2 tests, with FSH and maternal age evaluated both as continuous and as categorical variables. P values < .05 were considered statistically significant. For categorical analysis, advanced maternal age was defined as age R35 years, as increased rates 2352
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of fetal aneuploidy have been documented in this age group (19). A retrospective review of clinical outcomes at our center established a threshold for diminished ovarian reserve at a basal serum FSH level of R14 mIU/mL (Immulite), and this was the cutoff used when treating basal FSH as a categorical variable. Interestingly, this cutoff is consistent with that published by other investigators using a similar approach (20). Logistic regression was used to estimate the odds ratios (OR) and 95% confidence intervals (CI) relating aneuploidy to each predictor variable. RESULTS For purposes of analysis, patients were divided into those with euploid (n ¼ 70) versus those with aneuploid (n ¼ 107) fetal karyotypes. Due to a higher than expected (61%) ratio of XX karyotypes in the euploid cohort, and the potential for maternal contamination to confound interpretation (21), we considered only 46XY karyotypes (n ¼ 27) as the definitively euploid group. Specimens were not available for differential single nucleotide polymorphism studies to discriminate maternal from fetal DNA. The aneuploid cohort consisted of trisomic (87%), triploid (9.3%), and monosomic (3.7%) gestations. Patient characteristics, including gravity, parity, and E2 levels, were similar among the two groups (Table 1). The distribution of patients who conceived spontaneously, by IVF–embryo transfer, or IUI was not statistically different between the two groups. The number of patients with a history of one or more spontaneous miscarriages was also similar between the groups. Logistic regression analysis revealed basal FSH not to be independently predictive of an aneuploid gestation when analyzed as a continuous variable (Table 2). However, when analyzed as a categorical variable, basal FSH R14 mIU/mL was found to be significantly associated with karyotypically normal pregnancies (P¼.029). When maternal age was evaluated as a continuous variable, no statistically significant association with fetal aneuploidy was identified. However, when advanced maternal age was analyzed as a categorical variable (age R35 years), the association with fetal aneuploidy nearly reached statistical significance (P¼.064). A separate analysis including 46XX karyotypes in the euploid group was also performed and no significant association between basal FSH and aneuploid gestation was demonstrated (data not shown). DISCUSSION This study was designed to determine whether an elevated basal serum FSH level is associated with fetal aneuploidy risk. Because maternal age has a known association with increased meiotic error rates, as well as gradually increasing FSH levels (1), we sought to determine whether FSH acts as an independent marker for increased aneuploidy risk. We found no significant age-independent relationship between elevated basal FSH concentration and fetal aneuploidy, regardless of whether basal FSH level was treated as a continuous or as a categorical (R14 mIU/mL) variable. Using
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TABLE 1 Characteristics of patients with euploid versus aneuploid fetal karyotypes.
Characteristics
Euploid XY (n [ 27)
Aneuploid (n [ 107)
Maternal age (y) 36.67 3.65 38.02 3.96 Gravidity 0.96 1.28 1.40 1.64 Parity 0.12 0.33 0.29 0.52 Prior SAB 0.56 0.93 0.91 1.36 29.28 9.26 33.34 11.33 CD3 E2 (pg/mL) Method of conception Spontaneous (n) 6 24 IUI (n) 7 25 IVF-embryo 14 56 transfer (n) ICSI (n) 4 25 Note: Values are expressed as mean SD. All P values not significant. CD3 ¼ cycle day 3; SAB ¼ spontaneous abortion. Massie. Basal FSH a predictor of fetal aneuploidy. Fertil Steril 2008.
a cutoff of 35 years, a nearly significant association between advanced maternal age (R35 years) and increased fetal aneuploidy rate was found (P¼.064), a trend consistent with other studies (2, 22, 23). Other studies have assessed the value of basal FSH as a marker for oocyte quality as measured by fetal aneuploidy (3, 24). The results of these studies suggested an inverse relationship between basal FSH and oocyte quality, with a significantly greater proportion of age-matched women with abnormally elevated FSH values experiencing miscarriages
TABLE 2 Basal serum FSH and E2 levels, maternal age, and the fetal karyotype. Variables
Euploid XY (n [ 27)
Aneuploid (n [ 107)
P value
Maternal age 36.67 3.65 38.02 3.96 NS (y) Maternal age 55.6% 73.8% .064 >35 y CD3 FSH 9.21 3.38 8.60 6.49 NS (mIU/mL) CD3 FSH 18.5% 5.6% .029 R14 mIU/ mL Note: Values are expressed as mean SD. CD3 ¼ cycle day 3; NS ¼ not significant. Massie. Basal FSH a predictor of fetal aneuploidy. Fertil Steril 2008.
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with abnormal fetal karyotypes. Other investigators have reported an increased risk of Down syndrome in women with elevated basal FSH concentrations measured preconceptually (8, 24). These studies would seem to support the limited pool hypothesis of ovarian aging, in that diminished ovarian reserve and advanced biologic ovarian aging are better predictors of aneuploidy risk than is chronological age. However, more recent studies contradict this conclusion. Havryliuk et al. (25) showed no correlation between basal FSH level and fetal trisomy risk in poor prognostic IVF patients. A separate study found that premature decline in ovarian function, as evidenced by history of ovarian resistance or an elevated basal FSH level, was not associated with an increased risk of aneuploidy (26). Other investigators have reported basal FSH to be particularly poor at predicting aneuploidy risk in younger women (27, 28). In sum, it appears that elevated basal FSH concentration is a consistent marker for decreased ovarian reserve, but a poor indicator of oocyte quality, especially in younger women with premature decline in ovarian reserve (29, 30). Interestingly, our study revealed that spontaneous abortuses in women with a basal FSH level of R14 mIU/mL were more likely to be euploid (5/27) than aneuploid (6/ 107), and this finding reached statistical significance (P¼.029). Logistic regression treating both basal FSH and maternal age as categorical variables (cutoffs as discussed previously) confirmed an association between elevated basal FSH and euploid karyotype of miscarriage products (OR for aneuploidy 0.25, 95% CI 0.07–0.91, P<.04). Thus, women with a basal serum FSH R14 mIU/mL who conceive and experience miscarriage are less likely to have conceived an aneuploid gestation. A subset analysis was attempted to further investigate this finding; however, due to small sample size, no meaningful conclusions could be reached. It is possible that factors other than fetal aneuploidy were responsible for miscarriages within in this group. Such factors could include immunologic, endocrine, and hematologic processes not accounted for within the scope of this study. Smoking has been associated with both an elevated basal FSH level (31) and with miscarriage (32), yet none of the five women with FSH R14 mIU/mL and euploid miscarriages in our study were smokers. This group of women was heterogeneous in term of method of conception (1, spontaneous conception; 2, IUI; 2, IVF), and only one of the five had experienced a previous miscarriage. An elevated basal FSH level has been associated with recurrent pregnancy loss (33, 34), and this association was assumed to be consequent to an increase in fetal aneuploidy. Yet our findings suggest that an elevated basal FSH level may be associated with an abnormality not detectable by conventional karyotype or an altogether nongenetic (i.e., hormonal) abnormality that deleteriously impacts fecundity. Although we did not specifically test patients for skewed X chromosome inactivation (XCI) in our study, this phenomenon may provide an explanation for our finding of an age2353
independent increase in normal karyotypes in women with elevated basal FSH levels. Theoretically, recessively inherited mutations on the X chromosome lethal to the male carrier would result in skewed X chromosome inactivation in female carriers and predispose these women to abortion of 46XY conceptuses receiving the defective X (35). A skewed XCI pattern has been independently reported in women with recurrent SAB (36, 37). It is hypothesized to exist in women with diminished ovarian reserve (38), and would seem a unifying explanation for our observation. Our study design offers several strengths compared to previous reports. First, the design allowed us to capture karyotypic abnormalities that would not progress past the first trimester of pregnancy, and would therefore be missed by study designs that use karyotype information collected through chorionic villus sampling or amniocentesis (39–41). Second, ‘‘46XX’’ karyotypes were excluded from analysis, eliminating the potential for confounding of results by maternal contamination. We believe that this is necessary to improve the fidelity of the euploid cohort data until a reliable and readily available method is developed to discriminate maternal from fetal cytogenetics in the case of 46XX karyotypes. Finally, the large sample size in this study relative to others increases the reliability of our findings. As with any retrospective review there are possible confounding factors that could not be fully addressed by this design. For example, the type of stimulation protocol used in patients undergoing IVF was not addressed by our study. The type of stimulation protocol could potentially affect aneuploidy rates and is an area of current investigation (26). Although most fetal aneuploidy is believed to result from maternal meiotic errors, other factors affecting this outcome may exist that were not addressed by our analysis. In summary, our data do not appear to support the hypothesis that a reduced oocyte pool is associated with an increased meiotic error rate. Continued investigations to identify reliable, noninvasive methods for predicting oocyte and fetal aneuploidy are necessary.
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