A variety of tests have been used to predict the reproductive potential of women seeking fertility care. These include levels of circulating early follicular phase follicle stimulating hormone (FSH) and estradiol, antimüllerian hormone, and ultrasound measurement of antral follicles, among others.1 However, the predictive value of ovarian reserve testing has been somewhat controversial. Although it would appear that abnormalities of antimüllerian hormone, antral follicles, and FSH are predictive of poor response, oocyte yield, and cycle cancellation, these tests have limited predictive value for pregnancy.2–8 Attempts to interpret these tests independent of patient age are typically not helpful.9,10 The fundamental question, therefore, is what is the true meaning of diminished ovarian reserve? Do ovarian reserve tests predict a more fundamental oocyte defect?
It has been well-established that the frequency of aneuploid oocytes (abnormal chromosome copy numbers) increases with advancing maternal age.11,12 This increase parallels diminishing ovarian reserve and decrease of fertility in older women.13 There are conflicting data regarding whether compromised ovarian reserve may be associated with an increase in aneuploid pregnancies.14–18
Comprehensive chromosomal screening is a validated means by which the entire complement of chromosomes within an embryo can be analyzed.19–21 Clinical application of this approach after biopsy of blastocyst stage embryos results in extremely favorable live birth rates after transfer of euploid embryos.22
The objective of this investigation is to estimate the relationship between abnormal hormonal ovarian reserve parameters in patients undergoing in vitro fertilization and the incidence of aneuploid blastocyst-stage embryos diagnosed using comprehensive screening techniques.
MATERIALS AND METHODS
This investigation evaluated outcomes of 372 patients who were offered comprehensive chromosome screening from 2007 to 2011 at the Colorado Center for Reproductive Medicine. Indications for comprehensive chromosome screening included patients with advanced maternal age (39 years or older), history of recurrent unsuccessful in vitro fertilization cycles (two or more previous cycles with adequate embryo quality), and unexplained recurrent pregnancy loss (two or more). The study was conducted with the approval of the Health One Institutional Review Board.
As part of a standard evaluation, all patients underwent ovarian reserve screening including assessment of serum antimüllerian hormone levels, cycle day 2 or 3 serum FSH and estradiol levels, and baseline follicular phase ultrasound evaluation, including calculation of the number of antral follicles in both ovaries. All patients also underwent endometrial cavity assessment with office-based diagnostic hysteroscopy. Lesions were surgically corrected before embryo transfer.
Serum FSH was measured by using an electrochemiluminescent immunoassay on cycle day 2 or 3. A normal level was considered to be less than 10 milli-international units/mL.1,17 The coefficient of variation at a value of 11.1 milli-international units/mL was 2.6%. Serum antimüllerian hormone was measured by using an enzyme-linked immunosorbent assay (Gen II ELISA). A level more than 1 ng/mL regardless of cycle day was considered to be normal.7 The coefficient of variations for patient replicates was 15% or less for values of 0.1 ng/mL and less than 20% for standard curve replicates. Antral follicle count was determined by transvaginal ultrasound examination performed during the early follicular phase by calculating the total number of follicles in both ovaries with mean diameters between 2 and 7 mm measured in three planes.23 Diminished ovarian reserve was associated with a total count of less than six.1
Patients were initially divided into two groups based on the results of serum ovarian reserve testing. Group 1 consisted of 279 patients with normal ovarian reserve. Group 2 consisted of 93 patients with abnormal ovarian reserve defined as cycle day 2 or 3 serum FSH more than 10 milli-international units/mL, serum antimüllerian hormone 1 ng/mL or less, or both. Group 2 patients with abnormal ovarian reserve were further subdivided into three subgroups for additional analysis. Group A included day 3 FSH more than 10 milli-international units/mL and antimüllerian hormone 1 ng/mL or less (n=25 patients). Group B included day 3 FSH more than 10 milli-international units/mL and antimüllerian hormone more than 1 ng/mL (n=34 patients). Group C included day 3 FSH less than 10 milli-international units/mL and antimüllerian hormone 1 ng/mL or less (n=34 patients). Antral follicle count was not used as an independent variable for subgroup analysis because of the prerequisite for a number of oocytes retrieved (more than six) to allow for development of an adequate number of blastocysts for analysis. However, a close correlation between antral follicle count and serum antimüllerian hormone levels has been previously described.5
Standard protocols for controlled ovarian hyperstimulation were used. Assignments of the specific protocol and dose required for individual patients were based on age, previous response, and the results of ovarian reserve testing. In general, patients who were anticipated to be normal or high responders were administered either mid-luteal phase GnRH agonist for 7 days before initiation of gonadotropin stimulation or a GnRH antagonist initiated after the start of gonadotropin stimulation and when the leading follicle had reached a mean diameter of 14 mm. Those with anticipated or previous poor responses were typically administered microdose flare, luteal phase antagonist and estradiol priming, or clomiphene and GnRH antagonist protocols as previously described.24–27 Human chorionic gonadotropin (hCG) was administered with attainment of appropriate estradiol levels and a leading follicle of at least 18 mm in mean diameter. When indicated, high responders were alternatively administered leuprolide acetate in place of hCG.28 Transvaginal ultrasound--guided oocyte aspiration was performed 35 hours after hCG or initial leuprolide acetate administration.
In vitro fertilization, embryo culture, and blastocyst biopsy techniques were performed as previously described.22 Intracytoplasmic sperm injection was performed routinely to remove potential sperm or cumulus cell DNA contamination. Trophectoderm biopsy was performed on all expanding or fully expanded blastocysts on postretrieval day 5 or 6, depending on the rate at which individual embryos reached this developmental stage.29
Each trophectoderm biopsy sample underwent comprehensive chromosome screening analysis performed at commercial PGD reference and research laboratories. Metaphase comparative genomic hybridization was performed by Reprogenetics for cycles from 2007 to 2008,19,22 and single nucleotide polymorphism microarray was performed by Reproductive Medicine Associates of New Jersey for cycles from 2009 to 2011.20 Metaphase comparative genomic hybridization is a very lengthy and laboratory-intensive platform requiring up to 4 weeks at times for patient results. With the development in 2009 of time-efficient, laboratory-friendly, microarray-based platforms, comprehensive chromosome screening results are now available to patients within 48 hours. No differences in accuracy, reliability, “no call” rates, frequency and incidence of chromosome errors, or reproductive outcomes were noted between the techniques. While awaiting comprehensive chromosome screening results, all biopsied blastocysts were vitrified individually using Cryotops.30
Only euploid embryos were warmed and subsequently transferred after adequate endometrial preparation and luteal support had been achieved as reported previously.22 Embryo transfer was performed 3 to 4 hours after blastocyst warming under ultrasound guidance using standard Colorado Center for Reproductive Medicine techniques.31 Embryo survival from vitrification was determined immediately after warming and surviving blastocysts were reassessed just before embryo transfer. The number of embryos transferred was based on American Society for Reproductive Medicine guidelines.32
An initial serum pregnancy test was obtained 9 days after embryo transfer. If an appropriate hCG increase was documented, an initial ultrasound examination was performed 17–21 days later and repeated as appropriate. Implantation rate was defined as the number of intrauterine gestational sacs with cardiac activity noted on ultrasound examination per number of embryos transferred. Data were analyzed by Student t tests, Fisher exact test, unpaired t test, analysis of variance, and χ2 analyses as appropriate. P<.05 was considered to be statistically significant.
Baseline clinical characteristics and indications for comprehensive chromosome screening for groups 1 and 2 are displayed in Table 1. As would be expected, group 2 consisted of a slightly older group of patients (38.8 compared with 37.5 years for group 1; P=.003), with a greater proportion undergoing comprehensive chromosome screening for the primary indication of advanced maternal age (45.2% compared with 28.3% for group 1; P=.003). Not surprisingly, a significantly higher mean antral follicle count was noted in group 1 patients with normal ovarian reserve than in group 2 patients with diminished ovarian reserve (P<.001).
The outcomes of controlled ovarian hyperstimulation, chromosome analysis, and embryo transfer of euploid embryos in the two groups are shown in Table 2. As anticipated, patients with compromised ovarian reserve required a significantly higher mean total gonadotropin dose (P<.001), resulting in a lower number of oocytes retrieved and number of blastocysts available for biopsy compared with patients with normal ovarian reserve (P<.001). There were significant differences in the stimulation protocols used, with a higher percentage of group B patients receiving more aggressive approaches (data not shown; P<.005). All patients had expanded blastocysts available for trophectoderm biopsy and analysis.
A significantly higher percentage of aneuploid embryos was identified in women with abnormal ovarian reserve (66% compared with 51.7%; P=.048), which was associated with a significantly higher percentage of cycles with all aneuploid embryos (35.1% compared with 14.3%; P<.001). The most common errors involved chromosomes 15, 16, 21, and 22, which accounted for approximately 40% of the aneuploidies observed in both groups. There were no differences in outcome based on presenting diagnosis or comprehensive chromosome screening technique used. It is interesting to note that there were no differences in implantation or miscarriage rates between the two groups once euploid embryos were transferred (Table 2).
Clinical characteristics of the three subgroups of patients with diminished ovarian reserve are displayed in Table 3. In this circumstance, there were no differences in age among the groups. Antral follicle counts were lower in group A patients with both abnormal FSH and antimüllerian hormone levels (P<.001).
Cycle outcomes for the diminished ovarian reserve subgroups are shown in Table 4. Significantly greater mean gonadotropin doses were needed for group A patients with both increased FSH and decreased antimüllerian hormone levels (P<.001). Although a higher number of oocytes were retrieved in group B (P<.001), which correlates with the increased antral follicle count (P<.001), similar numbers of blastocysts were available for biopsy among the groups (P=.075). Interestingly, the percentage of aneuploid blastocysts was increased only in group A with both abnormal FSH and antimüllerian hormone levels (P=.048). A trend toward an increased percentage of all aneuploid blastocyst cycles, which did not achieve statistical significance, was observed in groups A and C. As noted in the previous analysis, once euploid embryos were transferred, there were no differences in implantation rates among the groups (Table 4).
In this prospective study, we have demonstrated that patients undergoing in vitro fertilization with compromised serum parameters of ovarian reserve as assessed by both abnormal serum FSH and antimüllerian hormone levels have a significantly higher proportion of aneuploid blastocysts after comprehensive chromosome screening than patients with normal values. However, once a blastocyst was identified as euploid, the implantation potential after transfer was equivalent for all groups, independent of ovarian reserve parameters.
Investigators have demonstrated that independent of age, currently used tests of ovarian reserve are poorly predictive of pregnancy. In a meta-analysis of 21 studies comparing basal FSH levels with in vitro fertilization outcomes, Bancsi et al2 calculated an extremely low predictive value for nonpregnancy and only a moderately predictive value for poor response. Broer et al,5 in a meta-analysis reporting on the predictive values of serum antimüllerian hormone levels and antral follicle count, noted no significant differences between the two in predicting poor responses to gonadotropin stimulation, but both performed poorly in predicting nonpregnancy. In another meta-analysis comparing antral follicle count with day 3 FSH levels, the former was noted to be more accurate in predicting poor response, but both performed poorly in predicting the likelihood of pregnancy as well.3 This general consensus has been confirmed by others.6–8
If ovarian reserve testing does not truly predict the likelihood of pregnancy, then could abnormal tests reflect a more fundamental defect at the level of the oocyte? The increased incidence of aneuploidy in blastocysts derived from patients with diminished serum markers of ovarian reserve in this trial may reflect this phenomenon. The striking similarity of implantation rates among all groups when euploid embryos were transferred would suggest that the increased incidence of aneuploid oocytes may represent the primary predictive nature of ovarian reserve testing.
The unique aspects of the current investigation include the fact that all 23 chromosome pairs were evaluated after trophectoderm biopsy of embryos that had demonstrated the developmental competence to reach the expanded blastocyst stage. Schoolcraft et al22,33 reported a euploid blastocyst implantation rate 50% higher than that for contemporary patients undergoing transfer of untested blastocysts resulting in high live birth rates and low miscarriage rates for infertility patients.
There are several confounding variables associated with the current investigation. The mean age of patients with normal ovarian reserve (group 1) was lower than that of patients with diminished ovarian reserve (group 2). The incidence of aneuploidy clearly increases with age.13 Therefore, some component of the increased incidence of aneuploid embryos may be attributable to this phenomenon as well. However, it is unlikely that the clinical significance of differences in mean ages of 37.5 and 38.8 years in the two groups would be great. In addition, there was no significant difference in mean age among the three subgroups with diminished ovarian reserve, although the incidence of aneuploidy was significantly different among the groups.
Patients in groups 1 and 2 were exposed to different controlled ovarian hyperstimulation protocols and required different mean total gonadotropin doses. Patients were assigned a stimulation protocol based on the results of ovarian reserve testing and previous response. The fact that a lower number of oocytes and blastocysts resulted and a higher overall gonadotropin dose was required in group 2 patients with diminished ovarian reserve, which was further accentuated in group A patients with two abnormal parameters, is not surprising. The question of whether stimulation protocol or gonadotropin dose in otherwise matched patients would have an affect on aneuploidy has not been definitively answered.34–36 The design of the present investigation does not allow for resolution of this issue in the patient populations studied.
At this point, we cannot extrapolate the results of this evaluation to the general population as a whole. This study solely evaluated patients who were already candidates for comprehensive chromosome screening because of advanced maternal age, a history of unexplained recurrent pregnancy loss, or recurrent implantation failure. It is possible that the results may differ if this approach were applied to the broader infertility population or to those attempting to conceive spontaneously.
In summary, in this prospective cohort trial, we have demonstrated that patients with abnormal serum ovarian reserve parameters are more likely to produce aneuploid blastocysts than women in a control group with normal ovarian reserve when all 23 chromosome pairs are evaluated. Abnormally low antimüllerian hormone levels, particularly when combined with abnormally evaluated FSH levels, increase the likelihood that the majority of blastocysts will be aneuploid. There is a clear need for appropriately designed randomized clinical trials before comprehensive chromosome screening should be considered to be clinically applicable outside of a research setting. However, these data, if confirmed, would allow clinicians to counsel patients regarding the true meaning of diminished ovarian reserve testing as reflecting, at least in part, an enhanced incidence of chromosomal abnormalities within the occyte. This will provide that patient with a more accurate explanation of one of the root causes of her infertility and provide enhanced predictive value regarding the outcomes of not only comprehensive chromosome screening but also in vitro fertilization in general.
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