A high oocyte yield for intracytoplasmic sperm injection treatment is associated with an increased chromosome error rate

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1 A high oocyte yield for intracytoplasmic sperm injection treatment is associated with an increased chromosome error rate Thomas Haaf, M.D., a Antje Hahn, a Anne Lambrecht, a B arbel Grossmann, Ph.D., a Eva Schwaab, M.D., b Omar Khanaga, Ph.D., c Thomas Hahn, M.D., c Achim Tresch, Ph.D., d and Martin Schorsch, M.D. c a Institute for Human Genetics and d Institute of Medical Biostatistics, Epidemiology and Informatics, Johannes Gutenberg University Mainz, Mainz; and b Human Genetic Practice and c Fertility Center, Wiesbaden, Germany Objective: To compare the chromosome error rate among from stimulated ovaries after retrieval of 1 5, 6 10, and >10. Design: Retrospective cohort study. Setting: A university-based human genetic institute in collaboration with a private fertility center. Patient(s): Nine hundred thirty-three women undergoing intracytoplasmic sperm injection (ICSI) with a poor prognosis. Intervention(s): Oocyte collection with ovarian stimulation. Polar body testing of ICSI for common chromosome errors. Main Outcome Measure(s): Chromosome error rate in, as determined by five-color fluorescence in situ hybridization. Result(s): In women less than 35 years and women between 35 and 40 years undergoing the first ICSI cycle, from the high-yield group had an increased likelihood for detectable chromosome errors (50.9% and 54.6%, respectively), compared to the intermediate-yield group (34.9% and 43.8%) and the low-yield group (23.3% and 41.2%). The overall high rate (R50%) of chromosomally abnormal in women more than 40 years appeared to be mainly due to the maternal age effect and increased only slightly with oocyte yield. Conclusion(s): Oocyte yield may be considered as an indicator of ovarian response to hormone stimulation. In women up to 40 years a high yield of after superovulation is associated with an increased chromosome error rate. (Fertil Steril Ò 2009;91: Ó2009 by American Society for Reproductive Medicine.) Key Words: Chromosome error, IVF/ICSI, oocyte yield, ovarian stimulation, polar body diagnosis Infertility affects approximately 15% of couples trying to conceive. In Europe, IVF and intracytoplasmic sperm injection (ICSI) are widely applied for infertility treatment and already account for 2% 3% of all births (1). The main cause for the relatively low efficiency of human reproduction appears to be the high incidence of chromosomal abnormalities in and embryos (2, 3). Most human aneuploidies originate from maternal meiosis I errors and increase with maternal age (4, 5). A large number of cytogenetic studies have been performed on human ; however, because of considerable differences as to oocyte donor population, oocyte collection, spreading and fixation, and chromosome analysis the reported incidences of chromosomal abnormalities are highly variable (<10% to >50%). Essentially, the best estimate is that 15% 20% of human are endowed with an abnormal karyotype. In addition to classic first and second meiotic nondisjunction, premature chromatid separation contributes Received August 13, 2007; revised January 2, 2008; accepted January 3, Supported by the Merck Serono Company. Conducted in collaboration with the Fertility Center Wiesbaden, a commercial provider of IVF/ICSI services. Reprint requests: Thomas Haaf, M.D., Institute for Human Genetics, Johannes Gutenberg University Mainz, Langenbeckstrasse 1, Bldg. 601, Mainz, Germany (FAX: ; haaf@humgen. klinik.uni-mainz.de). to the occurrence of chromosome errors (for review, see Rosenbusch and Pellestor et al.) (6, 7). Approximately 30% of human embryos are lost after implantation (8); chromosomal abnormalities accounting for approximately 50% of the first trimester abortions (2, 9). Fluorescence in situ hybridization (FISH) on interphase nuclei allows one to investigate and preimplantation embryos for aneuploidies of specific chromosomes. Preimplantation genetic screening (PGS) by multicolor FISH can avoid the transfer of aneuploid embryos in women undergoing IVF/ICSI and therefore should improve the chance for an ongoing pregnancy. Chromosomes 13, 16, 18, 21, and 22 are predominantly screened, because these chromosomes are most frequently involved in aneuploidies in preimplantation embryos (10, 11) and spontaneous abortions (2, 12). Indications for PGS include advanced maternal age, recurrent implantation failure, and recurrent miscarriages; however, the clinical benefits remain controversial. In a recent multicenter randomized controlled study PGS significantly reduced the pregnancy rates (PR) after IVF in women of advanced maternal age (13). Overall, the PRs of women undergoing infertility treatment are still disappointingly low. Because the German Embryo Protection Law (14) does not allow PGS on blastomeres, polar body diagnosis of is the only possibility for aneuploidy testing in /09/$36.00 Fertility and Sterility â Vol. 91, No. 3, March doi: /j.fertnstert Copyright ª2009 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 Germany. According to this law an embryo is created at the moment the two pronuclei fuse. This embryo can only be used for establishing a pregnancy but not for quality assessment. The first polar body is extruded at the end of the first meiotic division in the process of oocyte maturation and contains a set of haploid chromosomes (with two chromatids per chromosome). The second polar body is extruded at the end of the second meiotic division shortly after fertilization and contains one set of chromatids. The FISH analysis of the first and second polar bodies provides indirect information on the chromosomal content of the oocyte and, therefore, can be applied for preselection of aneuploidy-free (15 17). Similar to PGS, the clinical benefits of polar body diagnosis have not yet been convincingly demonstrated. MATERIALS AND METHODS Patients All patients were recruited from the Fertility Center at Wiesbaden from Polar body diagnosis was mainly advocated for women undergoing ICSI with a poor prognosis because of advanced maternal age (R35 years) or previous ICSI cycle(s) not resulting in an embryo transfer. The majority (59%) of women in our study population were between 35 and 40 years old, 13% were younger than 35 years, and 29% were older than 40 years. Women with known constitutional chromosome abnormalities (i.e., translocations or inversions) were excluded. A conventional high dose exogenous FSH regimen and GnRH agonist (GnRH-a) co-treatment was used to induce multiple follicle growth, resulting in multiple for retrieval. Briefly, pituitary down-regulation was achieved by using a GnRH-a (Enantone, Takeda Pharma, Aachen, Germany) between cycle days 18 and 21. Stimulation of follicular development in the next cycle was induced by daily injections with IU of recombinant FSH (Gonal- F, Merck Serono, Unterschleissheim, Germany). When the follicles were suitably developed (at least one follicle had reached a diameter of 17 mm), a single dose of 250 mg of hcg alfa (Ovitrelle, Merck Serono) was administered to induce final oocyte maturation. Oocytes were retrieved approximately 36 hours later by transvaginal ultrasound-guided puncture of follicles. Sperm preparation and ICSI were performed according to standard protocols. Polar Body Diagnosis With Five-Color FISH Biopsy of the first and second polar bodies was performed 8 10 hours after sperm injection. The zona pellucida (ZP) was opened with an OCTAX infrared laser module (OCTAX Microscience, Altdorf, Germany) at a wavelength of 1.48 mm and the polar bodies were aspirated in a special capillary. After two short washing steps in Quinn s advanced IVF medium and high-performance liquid chromatography (HPLC)-cleaned water the polar bodies were dropped onto a precleaned glass slide. After air-drying they were fixed with a freshly prepared ice cold 3:1 mixture of methanol and glacial acetic acid. The MultiVysion Probe set (Abbott, Wiesbaden, Germany) was used for aneuploidy testing of chromosomes 13, 16, 18, 21, and 22. It contains differentially fluorescent-labeled probes for chromosome 13q14 (LSI 13, Spectrum red), chromosome 16q12.1-q13 (CEP 16, Spectrum aqua), chromosome 18p11.1 (CEP 18, Spectrum blue), chromosome 21q22.13-q22.2 (LSI 21, Spectrum green), and chromosome 22q11.2 (LSI 22, Spectrum gold). The probes in the hybridization mixture and the polar bodies on the glass slide were denatured together for 8 minutes at 70 C and then hybridized for 4 hours in a humid chamber at 37 C. The slides were washed for 7 minutes in 0.7 saline sodium citrate (SSC), 0.3% Tween 20 at 73 C and for 1 minute in 2 SSC, 0.1% Tween 20 at room temperature, air-dried, and counterstained with 6-diamino-2-phenylindole (DAPI). The hybridization signals were evaluated by eye through an epifluorescence microscope (Leica DM RA 2; Leica Microsystems, Wetzlar, Germany), which was equipped with the appropriate filter sets (Abbott). On the basis of the FISH results in both polar bodies, the were categorized as chromosomally normal if two discrete hybridization spots (chromatids) of each tested chromosome were present in the first polar body and one spot (chromatid) in the second polar body and chromosomally abnormal if a different signal distribution was found. In terms of outcome, two unbalanced chromatid separation events (i.e., indicated by the presence of three chromatid spots in the first polar body and none in the second polar body) can correct TABLE 1 Maternal age-related increase of chromosome errors in from women with one or more ICSI cycles. <35 years Women of years >40 years No. of first ICSI cycles No. of euploid No. of abnormal Chromosome error rate No. of 2nd 4th ICSI cycles No. of euploid No. of abnormal Chromosome error rate % 44.5% 50.0% % 53.5% 54.8% 734 Haaf et al. Oocyte yield and chromosome error rate Vol. 91, No. 3, March 2009

3 themselves. Ten to 20% of the that were not used for transfer had experienced multiple meiotic errors that would result in an euploid oocyte (at least for the tested chromosomes). For 35% 45% of the retrieved the chromosomal constitution could not be determined because of absence of the first or the second polar body after hybridization or failure of the FISH procedure. Statistical Analysis We calculated a logistic regression, followed by a Wald test to assess the influence of the variables donor age and oocyte yield on the binary end point oocyte chromosome error. Several c 2 tests were then used to compare the rate of chromosomally abnormal between selected groups in an exploratory manner. RESULTS A population of infertile couples undergoing ICSI treatment in at the Fertility Center Wiesbaden because of advanced maternal age or previous implantation failure was offered polar body diagnosis of for five common aneuploidies. Altogether, 3,688 from 933 ICSI cycles, for which both polar bodies could be evaluated, were included in this study. Only without detectable nondisjunction and premature chromatid separation events were considered to be chromosomally normal. The only factor that has been irrefutably associated with human aneuploidy so far is increasing maternal age (4, 5). To determine the maternal age effect we compared the chromosome error rates among women who are younger than 35 years, women between 35 and 40 years, and women who are older than 40 years (Table 1). Oocytes from the first ICSI cycle displayed a dramatic increase (by 10%) in aneuploidy (P<.001) between women less than 35 years and women years old. The aneuploidy rate increased further (by 5%; P<.03) in women more than 40 years. In women with more than one ICSI cycle the maternal age-related increase in aneuploidy was less dramatic and occurred mainly between the groups <35 years and years (P<.05). Couples with previous implantation failure(s) may be especially at risk of generating aneuploid embryos (18). In women between 35 and 40 years, from the second to fourth ICSI cycle had a significantly higher (P<.001) aneuploidy rate (53.5%) than from the first cycle (44.5%). The observed increases in aneuploidy in women with more than one ICSI cycle less than 35 years and more than 40 years were not statistically significant. TABLE 2 Relationship between chromosome error rate and oocyte yield in women with one ICSI cycle. <35 years Women of years >40 years No. of women with Total number of retrieved No. of without PBD result Percentage of without PBD result 35.3% 3.4% a 36.8% 2.7% 42.0% 2.1% No. of euploid No. of abnormal Chromosome error rate b 23.3% 5.1% a 41.2% 2.9% 49.6% 4.0% No. of women with Total number of retrieved No. of without PBD result Percentage of without PBD result 38.3% 3.1% 35.5% 2.4% 39.7% 2.7% No. of euploid No. of abnormal Chromosome error rate 34.9% 3.7% 43.8% 2.1% 50.0% 4.0% No. of women with > Total number of retrieved No. of without PBD result Percentage of without PBD result 39.8% 3.7% 38.7% 2.5% 43.1% 3.6% No. of euploid No. of abnormal Chromosome error rate 50.9% 5.6% 54.6% 4.2% 54.1% 12.2% Note: PBD ¼ polar body diagnosis. a Standard deviations reflect variation of the PBD failure and chromosome error rates among women within the same group. b Error rates refer only to with PBD result. Fertility and Sterility â 735

4 Several studies suggested an effect of ovarian stimulation, which has become an integral component of IVF, on mitotic segregation errors in early human embryos, leading to mosaicism (19 21). The occurrence of postzygotic segregation errors, both in vivo and in vitro, may explain why the aneuploidy rates in preimplantation embryos (10, 11), as determined by FISH analysis of 1 2 blastomeres, are considerably higher than those in (6, 7). To find out whether the number of retrieved after superovulation affects their chromosomal competence, women within the same age group were divided into three subgroups with low oocyte yield (1 5), intermediate oocyte yield (6 10), and high oocyte yield (>10). To compare patient populations who are as homogeneous as possible, we looked at women with one ICSI cycle (Table 2) and more than one ICSI cycle (Table 3) separately. For these two populations, we could show a clear positive impact of both donor age (P<.001 for women with one ICSI cycle and P¼.045 for women with more than one ICSI cycle) and oocyte yield (P<.001 and P¼.030, respectively) on the chromosome error rate (Table 4). These effects seemed to be independent of each other and cannot be explained by one variable alone, as the donor age and oocyte yield were even weakly anticorrelated (Spearman correlation 0.16). Encouraged by this result, we examined the situation in more detail. In women less than 35 years undergoing the first ICSI cycle (Table 2) the chromosome error rate in the high oocyte yield group (50.9% 5.6%) was approximately two times higher than that in the low-yield group (23.3% 5.1%) (P<.001). Standard deviations reflect variation of the oocyte error rate among women within the same group. In women between 35 and 40 years, there was no significant increase between the low-yield and intermediate-yield groups. However, the aneuploidy rate increased by >10% between intermediate-yield and high oocyte yield group (P<.01). In women more than 40 years, who already have an age-related increase in chromosome errors, the number of chromosomally abnormal eggs increased only slightly from the lowyield (49.6% 4.0%) to the high-yield group (54.1% 12.2%). In this context, it is important to emphasize that the in a given category were not independent of each other. However, we can largely exclude the possibility that single women with especially high or low oocyte error rates were responsible for the observed effects. First, only women with similar oocyte yields were combined in the same category. Second, standard deviations for the measured chromosome error rates were comparable among the different TABLE 3 Relationship between chromosome error rate and oocyte yield in women with 2 4 ICSI cycles. <35 years Women of years >40 years No. of women with Total number of retrieved No. of without PBD result Percentage of without PBD result 34.8% 4.1% a 36.8% 3.2% 42.0% 3.0% No. of euploid No. of abnormal Chromosome error rate b 26.7% 7.0% 46.5% 3.2% 55.0% 3.8% No. of women with Total number of retrieved No. of without PBD result Percentage of without PBD result 37.8% 3.5% 35.3% 2.2% 39.7% 2.8% No. of euploid No. of abnormal Chromosome error rate 48.2% 6.0% 53.3% 2.5% 53.5% 3.0% No. of women with > Total number of retrieved No. of without PBD result Percentage of without PBD result 41.2% 3.7% 38.7% 3.3% 43.1% 3.9% No. of euploid No. of abnormal Chromosome error rate 43.9% 6.5% 56.6% 4.1% 59.1% 8.2% Note: PBD ¼ polar body diagnosis. a Standard deviations reflect variation of the PBD failure and chromosome error rates among women within the same group. b Error rates refer only to the with PBD result. 736 Haaf et al. Oocyte yield and chromosome error rate Vol. 91, No. 3, March 2009

5 TABLE 4 Logistic regression of oocyte chromosome error rate versus donor age and oocyte yield. Women with one ICSI cycle Women with more than one ICSI cycle Variable Regression coefficient Standard error Odds P value Regression coefficient Standard error Odds P value Donor age < Oocyte yield < categories. Third, the percentages (35% 45%) of retrieved that were complete drop outs due to technical failure(s) did not vary with donor age or oocyte yield. In women with more than one ICSI cycle (Table 3) the chromosome error rates may also increase with the number of retrieved. However, because relatively few from women less than 35 and more than 40 years with repeated ICSI cycles were available for analysis, the observed differences were not statistically significant. In women between 35 and 40 years and with repeated ICSI cycles, there was a strong (10%) increase in aneuploidy between the low-yield and high oocyte yield group (P¼.05). DISCUSSION In this study we provide evidence that oocyte yield after superovulation affects maternal meiotic segregation errors. Ovarian stimulation in IVF/ICSI treatments induces the development and growth of multiple follicles, resulting in high numbers of for retrieval. Although all women in this study were undergoing a similar superovulation regimen, the oocyte yields were widely different, ranging from 1 to >15. This reflects interindividual differences in ovarian response. The observation that a higher oocyte yield, in particular in women less than 35 years with one ICSI cycle and in women between 35 and 40 years with one or more ICSI cycles was associated with an increased chromosome error rate may be explained by unphysiological ovarian stimulation, interfering with chromosome segregation behavior during maternal meiosis. Lower oocyte yields may represent a more appropriate response to ovarian stimulation, allowing only the most competent follicles and to develop and, therefore, reducing oocyte error rates. In contrast, women more than 40 years showed high (R50%) chromosome error rates in all analyzed groups, irrespective of oocyte yield and number of ICSI cycles. A low number of retrieved in women more than 40 years may not necessarily indicate a physiological response to ovarian stimulation, but rather be suggestive of ovarian aging (22, 23). Our results are in agreement with a recent study showing that milder ovarian stimulation for IVF reduces the likelihood of postzygotic segregation errors during early embryo cleavages (20). Collectively, these observations support the conclusion that treatment strategies for infertility should avoid maximum oocyte yields. REFERENCES 1. Andersen AN, Gianaroli L, Felberbaum R, de Mouzon J, Nygren KG. 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6 17. Montag M, Limbach N, Sabarstinski M, van der Ven K, Dorn C, van der Ven H. Polar body biopsy and aneuploidy testing by simultaneous detection of six chromosomes. Prenat Diagn 2005;25: Pehlivan T, Rubio C, Rodrigo L, Romero J, Remohi J, Simon C, et al. Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients. Reprod Biomed Online 2003;6: Munne S, Magli C, Adler A, Wright G, de Boer K, Mortimer D, et al. Treatment-related chromosome abnormalities in human embryos. Hum Reprod 1997;12: Katz-Jaffe MG, Trounson AO, Cram DS. Chromosome 21 mosaic human preimplantation embryos predominantly arise from diploid conceptions. Fertil Steril 2005;84: Baart EB, Martini E, Eijkemans MJ, Van Opstal D, Beckers NG, Verhoeff A, et al. Milder ovarian stimulation reduces aneuploidy in the human preimplantation embryo: a randomized controlled trial. Hum Reprod 2007;22: Beckers NG, Macklon NS, Eijkemans MJ, Fauser BC. Women with regular menstrual cycles and a poor response to ovarian hyperstimulation for in vitro fertilization exhibit follicular phase characteristics suggestive of ovarian ageing. Fertil Steril 2002;78: De Boer EJ, den Tonkelaar I, te Velde ER, Burger CW, Klip H, van Leeuwen EE. A low number of retrieved at in vitro fertilization treatment is predictive of early menopause. Fertil Steril 2002;77: Haaf et al. Oocyte yield and chromosome error rate Vol. 91, No. 3, March 2009

Outcome of intracytoplasmic sperm injection with and without polar body diagnosis of oocytes

Outcome of intracytoplasmic sperm injection with and without polar body diagnosis of oocytes Thomas Haaf, M.D., a Achim Tresch, Ph.D., b Anne Lambrecht, M.D., a B arbel Grossmann, Ph.D., a Eva Schwaab,