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

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1 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, M.D., c Omar Khanaga, Ph.D., d Thomas Hahn, M.D., d and Martin Schorsch, M.D. d a Institute for Human Genetics, Johannes Gutenberg University, Mainz; b Gene Center Munich, Ludwig Maximilians University, Munich; c Human Genetic Practice, Wiesbaden; and d Fertility Center, Wiesbaden, Germany Objective: To compare the reproductive outcome of women undergoing intracytoplasmic sperm injection (ICSI) with or without polar body diagnosis of oocytes. Design: Nonrandomized retrospective study. Setting: University-based human genetic institute in collaboration with a private fertility center. Patient(s): Six hundred seven women undergoing ICSI with polar body diagnosis and 591 women undergoing ICSI without polar body diagnosis at the same time in the same fertility center. Intervention(s): Polar body testing of ICSI oocytes by five-color fluorescence in situ hybridization. Main Outcome Measure(s): Pregnancy rate (positive fetal heartbeats) and live-birth rate (of at least one child). Result(s): The pregnancy and live-birth rates were significantly lower in women undergoing ICSI with polar body diagnosis than in women without polar body diagnosis. The negative effects of polar body diagnosis were evident in all analyzed subgroups, that is, women of different age groups, with one ICSI cycle, with transfer of a high-quality embryo, and with male factor infertility as indication for ICSI. Conclusion(s): Within the legal restrictions of the German embryo protection law aneuploidy testing of oocytes may not improve reproductive outcome. (Fertil Steril Ò 2010;93: Ó2010 by American Society for Reproductive Medicine.) Key Words: Assisted reproduction, FISH, ICSI, live-birth rate, polar body diagnosis, pregnancy rate The percentage of aneuploid oocytes that originate from both meiotic nondisjunction and premature chromatid division increases significantly with maternal age (1 3). Because of postzygotic segregation errors the aneuploidy rates in preimplantation embryos are even higher than in oocytes (4). The high rate of aneuploidy in human oocytes and embryos is associated with low implantation and pregnancy rates and a high risk for spontaneous abortions and trisomic offspring (5, 6). Because most chromosomally abnormal embryos are eliminated before clinical recognition of pregnancy (7 9), it is plausible to assume that aneuploidy is the main cause of fertility problems in women with poor pregnancy prognosis. In vitro fertilization and intracytoplasmic sperm injection (ICSI) are widely applied for infertility treatment, already accounting for 2% to 3% of all births in Europe (10). Preimplantation genetic screening has been developed to select against chromosomally abnormal embryos. The transfer of euploid embryos (at least for the chromosomes tested) generally is Received June 9, 2008; revised January 19, 2009; accepted February 20, 2009; published online April 1, T.H. has nothing to disclose. A.T. has nothing to disclose. A.L. has nothing to disclose. B.G. has nothing to disclose. E.S. has nothing to disclose. O.K. has nothing to disclose. T.H. has nothing to disclose. M.S. has nothing to disclose. Supported by the Merck Serono Company. 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). thought to improve implantation and pregnancy rates, to reduce spontaneous abortions, and to prevent children having numerical chromosome abnormalities. Indications for preimplantation genetic screening include advanced maternal age, recurrent implantation failure, and recurrent miscarriages, but the clinical benefits remain controversial. Several nonrandomized studies comparing the same patients with and without preimplantation genetic screening demonstrated improved implantation rates and/or fewer pregnancy losses after aneuploidy testing (11 16). However, the case numbers in those studies usually were not sufficient to demonstrate a significant increase in ongoing pregnancy and/or live-birth rates in the preimplantation genetic screening group. Two randomized clinical trials found no difference in reproductive outcome between preimplantation genetic screening and control patients (17, 18), whereas another randomized study provided evidence for preimplantation genetic screening significantly reducing the pregnancy rates in women of advanced maternal age (19). Overall, the pregnancy and live-birth rates of women undergoing infertility treatment still are disappointingly low. Although more than 20,000 preimplantation genetic screenings have been performed worldwide (20), so far there are no generally accepted criteria for patient selection and methodology, which makes it difficult to compare results from different centers. Preimplantation genetic screening usually is performed by fluorescence in situ hybridization (FISH) on one or two blastomeres from biopsied day 3 embryos. Chromosomes 13, 16, 18, 21, and 22 are predominantly tested, because these chromosomes are involved most frequently in aneuploidies in /10/$36.00 Fertility and Sterility â Vol. 93, No. 2, January 15, doi: /j.fertnstert Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 preimplantation embryos (4, 17) and spontaneous abortions (21, 22). In Germany and other countries, where preimplantation genetic screening of embryonic blastomeres is forbidden, biopsy and FISH analysis of polar bodies can be used for aneuploidy testing of oocytes (3, 23 26). Polar body diagnosis can be considered as a preconception analysis, because according to the German embryo protection law the embryo is created at the moment the two pronuclei fuse. The first polar body, which contains a haploid set of chromosomes, is extruded at the end of the first meiotic division during oocyte maturation; the second polar body is extruded shortly after fertilization and contains one set of chromatids from the second meiotic division. In this context, it is important to emphasize that both first and second polar body have to be analyzed to obtain safe information on the chromosomal content of the oocyte. Similar to preimplantation genetic screening, the clinical benefits of polar body diagnosis have not yet been demonstrated convincingly. Here we performed a nonrandomized retrospective study to assess the outcome after ICSI with and without polar body diagnosis. MATERIALS AND METHODS Assisted Reproductive Technologies A conventional high-dose exogenous FSH regimen and GnRH agonist cotreatment were used to induce multiple follicle growth, resulting in multiple oocytes for retrieval. In brief, pituitary down-regulation was achieved by using a GnRH agonist (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 to 225 IU recombinant FSH (Gonal-F; Merck Serono, Darmstadt, Germany). When the follicles were suitably developed (at least one follicle had reached a diameter of 17 mm), a single dose of 250 mg 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. Biopsy of the first and second polar body was performed 8 to 10 hours after sperm injection. The zona pellucida was opened with an OCTAX infrared laser module (OCTAX Microscience, Altdorf, Germany) at a wave length 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 cleaned water, the polar bodies were fixed with an ice-cold 3:1 mixture of methanol and glacial acetic acid on a precleaned glass slide. Fluorescence in situ hybridization with the MultiVysion probe set (Abbott, Wiesbaden, Germany) was used for aneuploidy testing of chromosomes 13, 16, 18, 21, and 22. The hybridization signals of first and second polar bodies were evaluated by eye through a Leica DM RA 2 epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) that was equipped with the appropriate filter sets (Abbott). The oocytes 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. It is important to emphasize that, 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 themselves. Oocytes that had experienced multiple meiotic errors resulting in an euploid oocyte (at least for the tested chromosomes) were considered as chromosomally unstable and, therefore, not used for transfer. Study Design Women undergoing infertility treatment at the Fertility Center Wiesbaden from 2002 to 2005 were recruited retrospectively. The study group included 607 women undergoing ICSI with polar body diagnosis of oocytes. The control group included 591 women undergoing ICSI without polar body diagnosis. In the control group, oocytes were selected on the basis of morphologic criteria, that is, pronuclear disposition and nucleolar organization (27, 28). Before transfer, each embryo/blastocyst was graded by using a systematic scoring system (29, 30). Because the costs for polar body diagnosis are not covered by the German health insurance system, the patients had to choose between ICSI with and ICSI without polar body diagnosis. Polar body diagnosis was advocated mainly for women undergoing ICSI with a poor prognosis because of advanced maternal age (R35 years), implantation failure in previous IVF-ICSI cycles, and/or recurrent miscarriages. However, during the study period the tenet was that ICSI with polar body diagnosis would improve the pregnancy rates. In fact, many women already had learned about the supposed benefits of polar body diagnosis before they were counseled by us and specifically requested to perform ICSI with polar body diagnosis. To determine whether aneuploidy testing of oocytes has any effects on reproductive outcome, women undergoing ICSI with and without polar body diagnosis at the same time in the same fertility center were stratified by maternal age and number of ICSI cycles. Because oocyte aneuploidy is known to increase with maternal age (3, 5, 6), we decided to group women into three age strata: <35 years, between 35 and 40 years, and >40 years. Because previous implantation failure(s) also may be associated with an increased rate of aneuploid oocytes (3, 31), we looked at women undergoing the first ICSI cycle and women with more than one ICSI cycle separately. Women in the second, third, or fourth ICSI cycle already had undergone one to three ART cycles that did not result in a pregnancy. Biochemical pregnancy was defined as a serum b-hcg level of at least 20 IU/L 2 weeks after ET. Clinical pregnancy was defined by the presence of a fetal sac with positive heartbeat at 6 weeks after follicular puncture. The pregnancy rate was calculated by the number of women with at least one fetal heartbeat divided by the number of ICSI cycles. Women who had achieved a clinical pregnancy were followed up. 406 Haaf et al. ICSI with and without polar body diagnosis Vol. 93, No. 2, January 15, 2010

3 Only 38 of 411 clinical pregnancies (1,198 ICSI cycles) had an unknown outcome of pregnancy and therefore were excluded from further analyses. The live-birth rate represents the ratio between the number of live births of at least one child and the number of ICSI cycles. The pregnancy loss rate was measured as lost fetal heartbeats per ICSI cycle. Statistical Analysis Generally, a P<.05 was considered significant. Descriptions of continuous variables are given as mean SD. The influence of binary variables (polar body diagnosis, embryo quality, embryo stage) on the binary outcomes (pregnancy, live birth) was tested with a Fisher exact test. The distributions of continuous variables (age, numbers of retrieved and fertilized oocytes) within the study and control group, respectively, were compared with use of a Kolmogoroff-Smirnov test. A two-sided Mann-Whitney test was applied to test for location differences of continuous variables between two groups (e.g., number of retrieved oocytes in the polar body diagnosis vs. the non-polar body diagnosis group). Logistic regressions were calculated to assess the influence of polar body diagnosis and other variables (maternal age, number of ICSI cycles, numbers of retrieved and fertilized oocytes, number of transferred embryos, and embryo quality) on the dependent variables pregnancy rate and live-birth rate. RESULTS For this study we recruited 607 women undergoing ICSI with polar body diagnosis and 591 women without polar body diagnosis. Thirteen women had performed two ICSI cycles. Their second attempts were removed from the analysis; thus each individual contributed only one observation to the analyzed dataset. In the study group 70% of the couples had primary infertility and 30% had secondary infertility. This is significantly different (two-sided Fisher test, P<.001) from the control group with 58% primary and 42% secondary infertility. However, primary versus secondary infertility had no or only slight effects on reproductive outcome: 36% and 29% of women in the primary infertility group had a pregnancy versus a live birth, respectively, compared with 32% and 23% in the secondary infertility group (Fisher test, P¼.11 vs. P¼.02). Male factor infertility was the predominant indication for ICSI in both study (61%) and control group (63%). Female infertility, combined male and female infertility, and idiopathic infertility accounted in approximately equal shares for the remaining cases. Figure 1A demonstrates that the age distributions differ substantially between study and control group. The average age was 38 years in the polar body diagnosis group versus 34 years in the control group (P<.001). The most likely explanation for this age bias may be that older women with poorer pregnancy prognosis more often opted for the more expensive ICSI with polar body diagnosis than younger women. To account for the effects of this bias, we performed an age standardization with age groups spanning 2 years each (38 39, 39 40, and so on). However, the standardization procedure essentially did not change the expected pregnancy and live-birth rates (Fig. 1C). The standardized pregnancy and live-birth rates were lower in women with polar body diagnosis (18.4% and 12.5%, respectively) than in women without polar body diagnosis (51.6% and 43.8%). As expected, the age globally had a highly significant negative impact on both endpoints (P<.001 in a logistic regression; odds ratio per year for pregnancy rate and for live-birth rate). Consequently, the variable age was included into a subsequent multivariate regression analysis. Both study and control groups exhibited very similar distributions for the numbers of retrieved oocytes (mean SD; vs ), the numbers of fertilized oocytes ( vs ), and the fertilization rates ( vs ) (Fig. 2). A Kolmogoroff-Smirnov test, which at these sample sizes is a rather sensitive method, did not detect any differences between the two groups (P>.4 in each case). Nevertheless, the oocyte number had a significant positive effect on pregnancy rate and livebirth rate. The mean number of retrieved oocytes was compared with in women who achieved a pregnancy versus a live birth, respectively, against 9.13 in the women without pregnancy (two-sided Mann-Whitney test, P¼.001) and 9.24 in women without live birth (P¼.019). The mean number of fertilized oocytes was 6.42 in the pregnant group against 5.69 in the nonpregnant group (two-sided Mann- Whitney test, P¼.003). Similarly, the number of fertilized oocytes was 6.35 in the group with live birth and 5.79 in the group without live birth (P¼.021). According to the German embryo protection law, at most three fertilized oocytes can be selected before pronuclear fusion for further culturing and transfer. Of the 607 women in the polar body diagnosis group, 14 women (2.3%) had no embryo, 118 (19.4%) had one embryo, 318 (52.4%) had two embryos, and 157 (25.9%) had three embryos transferred. Of the 591 women in the control group without polar body diagnosis, 1 (0.2%) had no embryo, 46 (7.8%) had one embryo, 379 (64.1%) had two embryos, and 165 (27.9%) had three embryos transferred (Fig. 3A). As expected, the number of replaced embryos was significantly lower (P<.001) in the polar body diagnosis group ( ) than in the control group ( ). The number of transferred embryos has a positive influence on reproductive outcome. On average 2.22 embryos were transferred in the group of pregnant women, compared with 2.05 in the group without pregnancy (P<.001). The same kind of dependence (P¼.016) was observed for the group with live birth (mean number of transferred embryos 2.20) versus the group without live birth (mean 2.07). We defined a binary variable that indicates whether at least one top-scoring embryo (29, 30) was transferred or not. There was a clear dependence between polar body diagnosis and embryo quality, with the polar body diagnosis group having a lower embryo quality. Only 51.5% of women undergoing ICSI with polar body diagnosis received a high-quality embryo, compared with 72.7% of women without polar body Fertility and Sterility â 407

4 FIGURE 1 (A) Age distribution of 1,198 women undergoing ICSI without polar body diagnosis (PBD) (control group) or with PBD (study group). (B) Standardization factor for women of a given age in the study and control group, respectively. (C) Pregnancy and live-birth rates in women with and without PBD, compared with standardized pregnancy and live-birth rates. (D) Age distribution of a subpopulation of 712 women in which the two groups age profiles match perfectly. diagnosis (two-sided Fisher test, P<.001). At the same time the embryo quality turned out to be a crucial factor for the reproductive success. The percentages of women who became pregnant and had a live birth were significantly higher for the group with high-quality embryo (42.3% and 33.1%, respectively) than for the group without high-quality embryo (21.8% and 17.2%) (two-sided Fisher test, P<.001 for both endpoints). In addition, we defined a binary variable indicating whether a blastocyst(s) or an earlier preimplantation stage embryo(s) was transferred. There was a slight but significant dependence between polar body diagnosis and embryo stage: 49.9% of women with polar body diagnosis but 57.6% of women without polar body diagnosis had a blastocyst transfer (two-sided Fisher test, P¼.009). The stage of the transferred embryo had a highly significant impact on the pregnancy rate and live-birth rate (two-sided Fisher test, P<.001 for both endpoints): 40.4% and 31.2% of women with blastocyst transfer became pregnant or had an infant, respectively, compared with only 28.0% and 22.2% of women without blastocyst transfer. The mean number of ICSI cycles significantly (P<.001) varied between the study (2.30) and control (1.86) groups (Fig. 3B). The impact on pregnancy and live-birth rate was highly significant (P<.001 in both cases). The mean number 408 Haaf et al. ICSI with and without polar body diagnosis Vol. 93, No. 2, January 15, 2010

5 FIGURE 2 Distributions of retrieved oocyte numbers (A), fertilized oocyte numbers (B), and fertilization rates (C) in women undergoing ICSI without polar body diagnosis (PBD) (control group) and women undergoing ICSI with PBD (study group). of ICSI cycles was 1.73 versus 2.26 in the pregnant versus the nonpregnant group and 1.62 versus 2.25 in the live birth versus the non-live birth group. A multivariate regression analysis of all analyzed cases revealed that polar body diagnosis, number of ICSI cycles, and lack of a high-quality embryo all had a highly significant (P<.001) negative impact on the pregnancy rate, whereas the number of transferred embryos had a positive impact (P<.001). The same effects were evident on the live-birth rate, although the P values (< ) were slightly higher. Detailed results containing estimates of the respective effect sizes are presented in Table 1. There the exponential of the logistic regression coefficients of each independent variable is reported as an estimate of the odds ratio. Because the number of ICSI cycles had been shown to exert a major negative impact (<.001) on both endpoints, the logistic regression was redone for the subgroup of 663 women who had their first ICSI cycle (Table 1). This rigorously removes bias from this variable instead of trying to account for the influence of the number of ICSI cycles in the regression model. The results were confirmed in this subgroup, although the degree of evidence (as indicated by the P values) was slightly lower. Similarly, we calculated regression analyses for the subgroup of women who received a high-quality embryo and for another subgroup in which male factor infertility was the indication for ICSI (Table 1). In all analyzed subgroups polar body diagnosis turned out to have a negative effect (P<.001) on reproductive outcome. To finally exclude the possibility that the observed effects were due to the age difference between study and control groups we selected randomized matched pairs (according to their age) of members of both groups. This resulted in a subpopulation of 712 women in which the two groups age profiles matched perfectly (Fig. 1D). When we repeated all regression analyses in this subpopulation with matching age profiles, our previous results were confirmed (Table 2). In Tables 3 through 6 we present detailed analyses of subgroups of women with respect to age (<35 years, years, and >40 years) and number of ICSI cycles (first versus multiple attempts). In women of all three age groups Fertility and Sterility â 409

6 FIGURE 3 (A) Distribution of number of transferred embryos in polar body diagnosis (PBD) and control group. (B) Number of ICSI cycles in women with and without PBD. undergoing the first ICSI cycle, the clinical pregnancy rate in the polar body diagnosis group was significantly lower than in the control group (Table 3). In women undergoing the second through fourth ICSI cycle, a decreased pregnancy rate after polar body diagnosis was observed in women younger than 35 years of age and older than 40 years of age. The negative impact of polar body diagnosis in women between 35 and 40 years of age was not significant. The main goal of fertility treatment is live birth of a child. The polar body diagnosis group yielded 66 positive outcomes (58 singletons and 8 twins), whereas the control group produced 254 positive outcomes (200 singletons, 50 twins, and 4 triplets). For calculation of the live-birth rate we distinguished only between birth of at least one child and no child. Consistent with the decreased pregnancy rates, women of all age groups undergoing the first ICSI cycle with polar body diagnosis had significantly lower live-birth rates than women in the control group without polar body diagnosis (Table 4). A significantly lower live-birth rate also was seen in women younger than 35 years of age and between 35 and 40 years of age undergoing the second through fourth ICSI cycle with polar body diagnosis. For women older than 40 years with more than one ICSI cycle, the live-birth rate did not differ significantly between study and control group. To exclude the formal possibility that the low pregnancy and live-birth rates in the polar body diagnosis group were due to insufficient practice in the starting phase, we compared the reproductive outcome per calendar year (Table 5). Regression analysis did not reveal a significant effect of the calendar year on pregnancy (P¼.16) or live-birth rate (P¼.60) in women undergoing ICSI with polar body diagnosis. Another aim of polar body diagnosis is to reduce the number of pregnancy losses. In this study, we calculated the pregnancy loss rate by the number of lost fetal heartbeats divided by the number of ICSI cycles. We observed similar rates of lost fetal heartbeats in study and control groups (Table 6). Only for women older than 40 years with more than one ICSI cycle, who have the poorest pregnancy prognosis of all studied subgroups, polar body diagnosis significantly reduced the risk for spontaneous abortions. DISCUSSION To have a positive impact on reproductive outcome, the benefit of selecting against chromosomally abnormal embryos must outweigh possible negative effects of polar body diagnosis. The Fertility Center Wiesbaden was founded in 1996 and is one of the largest providers of IVF-ICSI services in Germany. Before introducing polar body diagnosis as a diagnostic tool in August 2002, we acquired experience with state-of-the art biopsy techniques in a well-respected center (20). Each of our two embryologists performed biopsies on several hundred oocytes before the polar bodies were used for aneuploidy testing, and the results were included in this study. A noncontact diode laser system was used to introduce an opening into the zona pellucida through which polar bodies were withdrawn by blunt-ended capillaries. This technique avoids the use of toxic Tyrode s solution and large zona openings. To the extent of present knowledge, laser-assisted biopsy does not seem to interfere with further development of embryos (24, 32). Comparison of the pregnancy and live-birth rates in 2002, 2003, 2004, and 2005 largely excluded unwanted effects of a polar body diagnosis learning curve. Because of legal regulations in Germany, polar body diagnosis has to be performed within a very short time window between polar body biopsy (8 10 hours after ICSI) and pronuclear envelope breakdown (20 hours after ICSI). At most three oocytes can be selected for further culturing and transfer. In a routine laboratory setting, it is very difficult to perform two rounds of FISH analysis to screen more than five chromosomes. Safe conclusions on the chromosomal content of the oocyte can be made only if both the first and second polar body give optimum hybridization results. Our laboratory has a long-standing expertise (>10 years) in different applications of interphase FISH, including single-blastomere analysis (33 35). Even for experienced molecular cytogeneticists, evaluation of the FISH signals in polar bodies is difficult and time consuming. To minimize FISH misdiagnoses, usually two observers have analyzed the hybridization patterns. In a pilot study, when we analyzed first polar bodies and the corresponding unfertilized oocytes, we always obtained matching results. Using this accurate and safe procedure, a relatively large percentage (30% 40%) of the biopsied oocytes remained without complete diagnosis for all five tested chromosomes in both polar bodies (3, 26). However, it is worth emphasizing that in most of these oocytes we could score either a subset of the tested chromosomes in both polar bodies or all five chromosomes in one polar body (the other polar body was missing). The percentage of oocytes without any diagnosis was <10%. These undetermined oocytes were used for transfer only when there were no normal embryos available. 410 Haaf et al. ICSI with and without polar body diagnosis Vol. 93, No. 2, January 15, 2010

7 TABLE 1 Quantification of the effects of independent variables on pregnancy rate and live-birth rate. Clinical pregnancy rate Live-birth rate P value Effect size P value Effect size Logistic regression analysis of all cases (N ¼ 1,198 women) Polar body diagnosis < < Maternal age No. of ICSI cycles < < No. of retrieved oocytes No. of fertilized oocytes No. of transferred embryos < No high-quality embryo < No blastocyst embryo Constant of women in the first ICSI cycle Polar body diagnosis < < Maternal age No. of retrieved oocytes No. of fertilized oocytes No. of transferred embryos No high-quality embryo No blastocyst stage Constant with transfer of a high-quality embryo Polar body diagnosis < < Maternal age Primary vs. secondary infertility No. of retrieved oocytes No. of fertilized oocytes No blastocyst stage < Constant with male factor infertility only Polar body diagnosis < < Maternal age Primary vs. secondary infertility No. of retrieved oocytes No. of fertilized oocytes No high-quality embryo No blastocyst stage Constant Depending on maternal age group and number of ICSI cycles, 35% to 55% of analyzed oocytes in the polar body diagnosis group were chromosomally abnormal (Table 3) and thus excluded from transfer. The observed aneuploidy rates may appear high because only five chromosomes (13, 16, 18, 21, and 22) were tested; however, they are within the expected range. A large number of FISH studies have been performed on human oocytes (for review, see Pellestor et al. [2]). Because of considerable differences between oocyte donor populations and technical limitations, the reported incidences of chromosomal abnormalities are highly variable. The best estimate from polar body studies comparable to our setup is that 32% to 52% of IVF-ICSI oocytes are aneuploid. In our study >50% of the chromosomally abnormal oocytes (by five-color FISH) exhibit two or more aneuploidies (26). It is reasonable to assume that the vast Fertility and Sterility â 411

8 TABLE 2 Quantification of the effects of independent variables on pregnancy rate and live-birth rate in a subpopulation of women with matching age profiles between study and control group. Clinical pregnancy rate Live-birth rate P value Effect size P value Effect size Logistic regression analysis of all matched pairs (N ¼ 712 women) Polar body diagnosis < < Maternal age No. of ICSI cycles < < No. of retrieved oocytes No. of fertilized oocytes No. of transferred embryos No high-quality embryo No blastocyst embryo Constant of matched pairs in the first ICSI cycle Polar body diagnosis < < Maternal age No. of retrieved oocytes No. of fertilized oocytes No. of transferred embryos No high-quality embryo No blastocyst stage Constant with transfer of a high-quality embryo Polar body diagnosis < < Maternal age No. of ICSI cycles No. of retrieved oocytes No. of fertilized oocytes No. of transferred embryos No blastocyst stage Constant with male factor infertility only Polar body diagnosis < < Maternal age No. of ICSI cycles No. of retrieved oocytes No. of fertilized oocytes No. of transferred embryos No high-quality embryo No blastocyst stage Constant majority of abnormal oocytes that are responsible for the low pregnancy rates in women of advanced maternal age and/or repeated implantation failure are in fact aneuploid for multiple (both tested and nontested) chromosomes. Although in the control group without polar body diagnosis at least 35% to 55% chromosomally abnormal embryos were replaced, there were significantly higher pregnancy and live-birth rates but in most subgroups no higher incidence of miscarriages. This is consistent with the view that in both groups (with and without polar body diagnosis) a strong natural selection 412 Haaf et al. ICSI with and without polar body diagnosis Vol. 93, No. 2, January 15, 2010

9 TABLE 3 Clinical pregnancy rate after ICSI without and with polar body diagnosis. ICSI with PBD Maternal age (y) ICSI without PBD, pregnancy rate (%)(No.) Aneuploidy rate a (%) Pregnancy rate (%) (No.) P value, Fisher test First ICSI cycle <35 68 b (156/230) b (4/16) b (82/142) b (28/155).001 >40 39 b (9/23) b (10/102).002 Second through fourth ICSI cycle <35 30 b (16/53) b (9/64) (32/104) (40/163).322 >40 28 b (11/39) b (14/107).046 Note: PBD ¼ polar body diagnosis. a Data from Haaf et al. 3 b Significantly different between study and control group. against abnormal embryos occurs before recognition of a pregnancy (7 9). Evidently, the benefit of an added selection by aneuploidy testing of oocytes before transfer did not compensate for the negative effects of polar body diagnosis, that is, misdiagnosis and damage of embryos. Because the number of transferred embryos was not dramatically different between study and control group, it is unlikely that the significantly reduced pregnancy and live-birth rates after polar body diagnosis are due mainly to FISH errors and exclusion of a large number of euploid embryos from transfer. It appears to be more likely that micromanipulation and biopsy of the human oocyte decreased the developmental potential of the resulting embryo. Indeed, in our analysis significantly fewer women in the polar body diagnosis group than in the control group had a high-quality embryo transferred. Embryo quality has a substantial effect on reproductive outcome. However, when we looked at the subgroup of women with a high-quality embryo, the highly significant negative impact of polar body diagnosis on pregnancy rate and live-birth rate remained. As outlined above, polar body biopsy and FISH were performed by embryologists and molecular cytogeneticists with long-standing expertise in their fields. It is our opinion that the discouraging results of this study are not due to substandard application of polar body diagnosis such as a high biopsy damage rate, poor FISH technique, or inappropriate patient selection in our center. There rather may be a problem(s) inherent to polar body diagnosis, at least within the legal regulations of the German embryo protection law. TABLE 4 Live-birth rate after ICSI without and with polar body diagnosis. Maternal age (y) ICSI without PBD, live-birth rate (%)(No.) ICSI with PBD, live-birth rate (%)(No.) P value, Fisher test First ICSI cycle <35 60 a (132/221) 25 a (4/16) a (72/138) 13 a (19/152) <.001 >40 30 a (7/23) 7 a (7/102).004 Second through fourth ICSI cycle <35 29 a (15/52) 11 a (7/64) a (23/99) 13 a (20/154).040 >40 13 (5/38) 9 (9/104).525 Note: PBD ¼ polar body diagnosis. a Significantly different between study and control group. Fertility and Sterility â 413

10 TABLE 5 Pregnancy and live-birth rates after ICSI with polar body diagnosis per calendar year. Year Pregnancy rate (%) Live-birth rate (%) In such a nonrandomized retrospective study it is difficult to exclude confounding factors entirely. All women underwent the same superovulation regimen and were treated in the same fertility center. Although all women received similar information and counseling before infertility treatment, their decision whether or not to perform polar body diagnosis was not made by chance only, but also, for example, on their assessment of the benefits of polar body diagnosis and their financial resources. The observed difference in the age distributions between study and control groups may be explained by the fact that in general older women have a poorer pregnancy prognosis but better financial resources than younger women and therefore more often opted for polar body diagnosis. We accounted for possible age bias by performing an age standardization procedure and by analyzing a subpopulation of women with matching age profiles. This did not change our conclusions. Because advanced maternal age and previous implantation failure are known to impair reproductive success, we stratified the analysis of women in the polar body diagnosis and control group by maternal age and number of ICSI cycles. Clearly, the highly significant negative impact of polar body diagnosis on pregnancy and live-birth rates in our study is not due to an age bias. In any subgroup of women analyzed, the effect of polar body diagnosis on reproductive outcome was negative. It has been reported that the cumulative probability of achieving a pregnancy is higher in women with secondary infertility than in women with primary infertility (36). In our study, we did not find a difference in pregnancy rates in favor of secondary infertility versus primary infertility. This may be due to the fact that most women were in the first ICSI cycle and no women had more than four ICSI cycles. In addition, we looked at the reproductive outcome of a single ICSI attempt and not on cumulative pregnancy rates. However, to demonstrate that our findings are not limited to the policy and/or technical issues at our center, data from other institutions will be needed. Our results are consistent with those of a recent study (19) demonstrating that preimplantation genetic screening may decrease the ongoing pregnancy rate in women of advanced maternal age and may add arguments to the following controversy on the benefits of aneuploidy testing (20, 37, 38). In our experience, the largest benefit of polar body diagnosis is the prevention of trisomic offspring. In our center many women still opt for polar body diagnosis to avoid prenatal diagnosis and having to terminate a pregnancy with trisomy 13, 18, or 21, even when knowing that the pregnancy rate may be decreased. REFERENCES 1. Rosenbusch B. The incidence of aneuploidy in human oocytes assessed by conventional cytogenetic analysis. Hereditas 2004;141: Pellestor F, Andreo B, Anahory T, Hamamah S. The occurrence of aneuploidy in human: lessons from cytogenetic studies of human oocytes. Eur J Med Genet 2006;49: Haaf T, Hahn A, Lambrecht A, Grossmann B, Schwaab E, Khanaga O, et al. A high oocyte yield for intracytoplasmic sperm injection treatment is associated with an increased aneuploidy rate. Fertil Steril 2009; 91: Munne S. Chromosome abnormalities and their relationship to morphology and development of human embryos. Reprod Biomed Online 2006;12: Griffin DK. The incidence, origin, and etiology of aneuploidy. Int Rev Cytol 1996;167: TABLE 6 Pregnancy loss rate after ICSI without and with polar body diagnosis. Maternal age (y) ICSI without PBD, pregnancy loss rate (%) (No.) ICSI with PBD, pregnancy loss rate (%) (No.) P value, Fisher test First ICSI cycle <35 7 (15/221) 0 (0/16) (6/138) 4 (6/152) >40 9 (2/23) 3 (3/102).228 Second through fourth ICSI cycle <35 0 (0/52) 3 (2/64) (4/99) 7 (11/154).418 >40 13 a (5/38) 2 a (2/104).015 Note: PBD ¼ polar body diagnosis. a Significantly different between study and control group. 414 Haaf et al. ICSI with and without polar body diagnosis Vol. 93, No. 2, January 15, 2010

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