Article The impact of in-vitro maturation of oocytes on aneuploidy rate

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1 RBMOnline - Vol 18 No Reproductive BioMedicine Online; on web 1 April 2009 Article The impact of in-vitro maturation of oocytes on aneuploidy rate Antonio Requena achieved his MD at Complutense University of Madrid, and his obstetric and gynaecology specialism at La Paz Hospital (Autonoma University of Madrid). He undertook post-doctoral training in in-vitro oocyte maturation at John Eppig s laboratory (Bar Harbour, USA). He joined IVI in 1996 and is now co-director of IVI-MADRID as well as the medical coordinator of EQUIPO IVI. He is an active member of ESHRE and the Spanish Fertility Society. His main areas of interest are gynaecologic endocrinology and IVM. Dr Antonio Requena Antonio Requena 1, Fernando Bronet, Alfredo Guillén, David Agudo, Carmen Bou, Juan Antonio García-Velasco IVI-Madrid, Santiago de Compostela 88, E Madrid, Spain 1 Correspondence: arequena@ivi.es Abstract Chromosome abnormalities in embryos obtained through in-vitro maturation (IVM) of oocytes from 11 oocyte donors were compared with embryos from women undergoing fluorescence in-situ hybridization (FISH) analysis for sex selection. Thirtythree oocytes had reached metaphase II stage at h (65%) and 27 were successfully fertilized by intracytoplasmic sperm injection. Blastomere biopsy was performed in 20 embryos (74%). For five embryos, two blastomeres were analysed, three of which were mosaic. FISH study revealed aneuploidies of chromosomes 13, 15, 16, 18, 21, 22, X and Y in 12 embryos (60%) and euploidy in the remaining eight (40%). The percentage of aneuploidies in the control group was 33%. Differences between IVM and control embryos were not statistically significant. The high incidence of chromosome abnormalities in embryos resulting from the IVM protocol may account for the low implantation rates reported by others. Although a greater incidence of miscarriage or congenital abnormalities in babies born alive following IVM versus conventional IVF has not been observed in previous studies, preimplantation genetic aneuploidy screening or prenatal chromosome studies may be recommended to these patients on the basis of the present results. Keywords: aneuploidy, in-vitro maturation, oocytes, prenatal chromosome studies Introduction A successful pregnancy outcome after IVF treatment during a natural cycle (Steptoe and Edwards, 1978) was promptly followed by the introduction of ovarian stimulation programmes with gonadotrophins, with the aim of optimizing the IVF technique (Lopata et al., 1978; Jones et al., 1982). The use of these regimens is associated with side effects, ovarian hyperstimulation syndrome being the most common (Chen et al., 2003). On the other hand, the potential long-term effect of these treatments remains unclear (Brinton et al., 2005). Although the original study of in-vitro maturation (IVM) of human oocytes was published by Edwards in 1965 (Edwards, 1965), it was not until 1983 when the first studies of IVM and fertilization of immature oocytes in an IVF programme were reported (Veeck et al., 1983). These initial experiences were followed by IVM and fertilization of oocytes collected from unstimulated cycles (Cha et al., 1991; Trounson et al., 1994). The technique has been progressively developed over the past decade with a substantial number of newborn infants (Chian et al., 2003), and with miscarriage rates, length of gestation and birth weight of the infants in pregnancies conceived by IVM comparable to those treated by conventional IVF (Cha et al., 2005). Accordingly, IVM is an important option with clear advantages to be considered in a selected group of patients, including those at risk for ovarian hyperstimulation syndrome, such as patients with polycystic ovary syndrome, patients with any contraindication for the use of gonadotrophins or patients who wish to avoid using drugs for ovarian stimulation. Moreover, in comparison with routine IVF, IVM treatments have a reduced cost and are less complicated. Pregnancy rates have increasingly improved up to current rates that exceed 35% per cycle, but implantation rates per embryo continue to be low (Al-Sunaidi et al., 2007), so that a Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 778 higher number of embryos per treatment should be transferred to achieve acceptable pregnancy rates. Chromosome abnormalities, including aneuploidy, polyploidy and haploidy, in preimplantation embryos after IVF could explain the relatively low implantation rate (Gianaroli et al., 1999; Kahraman et al., 2000; Pehlivan et al., 2003; Voullaire et al., 2007). Although many groups have reported their experience with IVM, to date there has been no reference to possible chromosome abnormalities of in-vitro-matured oocytes (Edwards, 2007). Therefore, the purpose of this study was to assess the incidence of chromosome abnormalities in embryos obtained through IVM. Materials and methods Study population Between January 2007 and August 2007, 11 women from an oocyte donation programme were included in the study. The study was carried out in a private university-affiliated assisted reproduction institute in Madrid, Spain. This project was approved by the Institutional Review Board, and all of the patients gave written informed consent. Prior to inclusion in the oocyte donation programme, all women underwent all work-up studies required by the Spanish law of assisted reproduction techniques and were eligible to be included in the study. Moreover, high-resolution karyotyping is routinely performed in the institute in all oocyte and semen donors, excluding those in which any type of numerical or structural chromosome abnormality has been detected. Criteria for inclusion in the semen donation programme required a semen sample with a sperm concentration > /ml, progressive motility >50%, with >12% normal forms (strict criteria for normal morphology of Kruger), and post-thaw sperm survival >50%. In addition, none of the women included in the study had polycystic ovary syndrome or echographic appearance of multicystic ovaries. All women had normal menstrual patterns and day-3 serum basal FSH concentration <10 IU/l. A control group of couples undergoing preimplantation genetic diagnosis (PGD) during the same time period because of the risk of sex-linked diseases and without other infertility problems was included to compare the incidence of abnormalities for each chromosome with the IVM group. The control group included 25 patients with mean (SD) age of 33.1 (2.5) years and mean number of previous miscarriages of 0.3 ± 0.6. In all cases, oocytes were fertilized by intracytoplasmic sperm injection (ICSI) because this is the routine method used in the institute for PGD programmes. The fluorescence in-situ hybridization (FISH) protocol was similar in the control and study group. IVM treatment All donors underwent an unstimulated IVM treatment cycle, as previously described (Cobo et al., 1999). A baseline ultrasound scan of the ovaries was obtained between days 2 and 3 of the menstrual cycle to measure the number and sizes of antral follicles, the endometrial thickness, and to exclude the presence of ovarian cysts. Exclusion criteria included 5 antral follicles, cyst >20 mm, oestradiol concentrations >60 pg/ ml, or an endometrial thickness >4 mm. A second ultrasound scan was performed between days 6 and 8 of the cycle to assess the lining of the endometrium and to confirm follicular development. The oocyte retrieval was scheduled within 24 h when a leading follicle of mm in mean diameter and endometrial thickness of 5 mm were confirmed. No human chorionic gonadotrophin (HCG) priming was given. Oocyte retrieval was performed under ultrasound guidance with a single-lumen aspiration needle (Ovum aspiration needle, K-OPS-7035-RWH-ET; needle 19 g, 35 cm length, total aspiration lumen volume 0.71 ml; Cook, Australia) and 80 mmhg aspiration pressure. Cumulus oocyte complexes (COC) were collected in 10 m culture tubes (Falcon) containing 1 3 ml heparinized warm flushing medium (Medi-Cult, Jillinge, Denmark). After the oocyte retrieval, each tube of follicular aspirate was poured through a 70 m cell strainer immediately after collection. The COC contained in the cell strainer were collected, and then transferred into Petri dishes to assess the maturity immediately. Thereafter, COC were transferred to oocyte washing medium (Medi-Cult) to wash several times. The oocytes were identified, classified, and collected in a four-well culture dish (NUNC, DK-6000, Roskilde, Denmark) containing 0.5 ml pre-equilibrated Lag Medium (vial 1 of IVM system medium; Medi-Cult) incubated at 37 C and 5% CO 2 atmosphere for 2 3 h. Then, oocytes were transferred to a culture dish with micro-drops with 0.5 ml IVM medium (vial 2 of IVM system medium; Medi-Cult) supplemented with recombinant FSH IU/ml, HCG 0.1 IU/ml (Serono, UK), and 10% maternal serum (obtained from the patient before being anaesthetized for the oocyte retrieval). Oocytes were cultured in this medium for h and all COC were checked for identification of oocyte maturity. Subsequently, the oocytes were denuded using a fine pipette following exposure to a commercially available hyaluronidase solution (SAGE IVF, Inc., Cooper Surgical, Trumbull, USA). Metaphase II (MII) oocytes were assessed by the presence of the first polar body. Oocytes were fertilized by ICSI as previously described (Mínguez et al., 1996). Embryo biopsy and genetic assay Embryo biopsy was performed on day 3. Briefly, embryos were placed on a droplet containing Ca 2+ - and Mg 2+ -free medium (G-PGD; VitroLife, Göteborg, Sweden) and the zona pellucida was perforated using Tyrode s solution (ZD-10; VitroLife) or laser technology (Octax, Herbron, Germany) (Rubio et al., 2003). Only embryos with five or more nucleated blastomeres and 25% fragmentation were biopsied, and one or two blastomeres were withdrawn depending on the cell number. In embryos with more than seven cells, two blastomeres were biopsied, and in embryos with less than seven cells only one blastomere was retrieved. Two cells were analysed in five embryos, and in the remaining 15 embryos one cell was analysed. All blastomeres were fixed individually (to minimize signal overlap and loss of micronuclei) under an inverted microscope, using a slightly modified Tarkowski s protocol without hypotonic pretreatment (Tarkowski, 1966). Mosaic embryos were classified as those having different FISH results when two cells were analysed (five embryos). Fixed cells were analysed by the genetics unit

3 laboratory. The biopsied embryos were cultured until they reached the blastocyst stage (day 5). The FISH procedure included two consecutive hybridization rounds. In the first round, a cocktail of probes (Multivysion PB; Vysis, Inc., Downers Grove, IL, USA) that includes chromosomes 13 (LSI 13 region 13q14; locus RB, spectrum red), 16 (CEP16, satellite II, region 16p11.1 q11.1; locus D16Z2, spectrum aqua), 18 (CEP 18; alpha satellite, region 18p11 q11; locus D18Z1, spectrum blue), 21 (LSI 21; region 21q22.13 q22.2; loci D21S259, D21S341, D21S342, spectrum green) and 22 (LSI 22; region 22q11.2; locus bcr, spectrum gold) were analysed. In a second round and after signal elimination (Vidal et al., 1998), nuclei were hybridized with centromeric probes for chromosomes X (CEP X, alpha satellite, region Xp11 q11; locus DXZ1, spectrum green) and Y (CEP Y, alpha satellite, region Yq12; locus DYZ1, spectrum aqua) and locus specific probe for chromosome 15 (LSI 15, region 15p11.1 q11.1; locus D15Z4, spectrum orange). Finally, a third round with subtelomeric DNA probes was performed for embryos with ambiguous signals in the previous rounds, to decrease the risk of false monosomies and not informative embryos (L. Rodrigo, personal communication). All probes were commercially available from Vysis, Inc. The risk of FISH errors was assumed, but the risk was considered to be homogeneously distributed in all groups and did not interfere in the statistical analysis of the results. Hybridization efficiency was 96% for the blastomeres analysed in the IVM group and up to 95% in the control group. Hybridization efficiencies for each probe were similar in both groups, independent of the order in which the probes were used. Therefore, technical artefacts could appear equally frequently in the two groups, and would not be responsible for any increased aneuploidy rate in the IVM group compared with controls. The percentage of abnormal embryos in each group was estimated as the number of affected embryos divided by the number of informative embryos for the probe employed. Embryos without chromosome abnormalities reaching the blastocyte stage were donated to women on the waiting list of our oocyte donation program. Endometrial preparation for embryo implantation has been described in detail elsewhere (Remohí et al., 1997). Statistical analysis Differences in the aneuploidy rate between IVM and control group embryos were assessed with the chi-squared ( 2 ) test. Statistical significance was set at P < The Statistics Package for Social Sciences (SPSS, Inc., USA) was used for data analysis. Results The 11 woman included in the study group had a mean (SD) age of 22.1 (2.3) years, body mass index (BMI) of 22.2 (2.1) kg/m 2, mean serum FSH concentration on day 3 of 6.2 (2.1) IU/l, and LH concentration of 4.7 (2.1) IU/l. Although the mean (SD) age of women in the control group was greater (33 (25) years), differences were not statistically significant. The mean number of antral follicles documented in the basal echographic study on day 3 was 7.1 (1.7). In one patient, no oocytes were recovered. In the remaining cases, a total of 51 oocytes were retrieved (mean 5.1 oocytes per ovarian puncture). Number of COC and rates of maturation, fertilization, embryo biopsy, and chromosomally normal embryos are shown in Table 1. A total of 33 oocytes had reached the MII stage at h (65%) and 27 (82%) of them were successfully fertilized after insemination by means of ICSI. Blastomere biopsy could be performed in 20 embryos (74%) fulfilling criteria for biopsy. In five embryos, two blastomeres were analysed, three of which were found to be mosaic. FISH study revealed aneuploidies of chromosomes 13, 15, 16, 18, 21, 22, X and Y in 12 embryos (60%). In the remaining eight embryos (40%), no chromosome abnormalities were observed. The percentage of aneuploidies in the control group was 33%. Differences between IVM and ICSI embryos were not statistically significant. The percentage of abnormalities for each chromosome in both the study and control groups is shown in Table 2. Chromosomes 21, 18, 16 and 22 were those more frequently affected. Chromosome abnormalities found in each embryo in the study group are detailed in Table 3. In the control group, a total of 200 embryos for chromosomes 13, 15, 16, 18, 21, 22, X and Y were analysed. Table 1. Number of cumulus oocyte complexes (COC) and rates of maturation, fertilization, embryo biopsy and chromosomally normal embryos in the study group. Parameter Number (%) COC 51 (100) Oocytes matured 33 (65) Oocytes fertilized 27 (82) Embryos biopsied 20 (74) Chromosomally normal embryos 8 (40) Table 2. Anomalies distributed by chromosome in embryos resulting from in-vitro-matured (IVM, n = 20) and control oocytes (n = 200). Percentage of embryos with anomalies IVM Control Chromosome Chromosome Chromosome Chromosome Chromosome Chromosome Sex chromosomes

4 Table 3. Description of the chromosomes analysed in the study group. Embryo Chromosome X Y a b 2 b Table 4. Embryo development after blastomere biopsy in the study group. Embryos with Embryos with normal chromosome chromosomes abnormalities (n = 8) (n = 12) Blastocyst stage 5 (63) 3 (25) Arrest before 3 (38) 9 (75) blastocyst stage Values are n (%). There were no statistically significant differences in the development of embryos with normal or abnormal chromosomes. a Mosaicism: the cell with abnormalities is indicated, the other cell was diploid XY. b Mosaicism: 15 monosomy (cell a), 15 trisomy (cell b). 780 Three of the eight normal embryos showed developmental arrest before reaching the blastocyst stage. The remaining five embryos reached the blastocyst stage with optimal embryo quality to be frozen. Of the 12 embryos with chromosome abnormalities, nine showed arrest before the blastocyst stage. The blastocyst stage was reached in only three cases: two mosaicisms and one embryo with chromosome 18 and 21 monosomy and nullisomy for chromosome 22. Development of embryos after blastomere biopsy in the study group is shown in Table 4. Embryos without chromosome abnormalities were transferred to the assigned woman but no pregnancies were achieved. Discussion Although IVM of oocytes is a promising technique for the patients because gonadotrophins are not used for ovarian stimulation, implantation rates are not comparable to those obtained with ovarian stimulation protocols. After the first reports of babies who were born alive, pregnancy rates in the 1990s were relatively low (Barnes et al., 1996; Trounson et al., 1998). With the administration of HCG before oocyte retrieval, pregnancy rates over 35% per cycle were obtained (Chian et al., 2000), but the rate of embryo implantation was around 15% (Son et al., 2007). This low implantation rate may be related to altered endometrial development and for this reason endometrial priming with oestrogen and progesterone was established when embryo transfer was performed in the same cycle in which immature oocytes had been obtained (Russell et al., 1997). Other reasons related to the low implantation rates may be related to abnormalities experienced by the oocyte during IVM, which would be translated into less competent embryos for implantation and for continuing the development process. In particular, the incidence of chromosome abnormalities in embryos obtained with this technique may be another factor related to the low implantation rate. In a previous study, Nogueira et al. (2000) analysed embryos after IVM maturation by investigating their nuclear status and cytogenetic constitution. A high incidence of multinuclear blastomeres and aneuploidy, suggesting abnormal cytokinesis or genetic abnormalities, was observed. However, in contrast to the present study, analysed oocytes were obtained after ovarian stimulation and IVM of denuded immature oocytes. In the present study, the incidence of chromosomal abnormalities was higher in the IVM group but differences as compared with ICSI embryos were not statistically significant. This may be explained by the limited number of analysed embryos, but the lack of statistical significance should be taken into account in the interpretation of the present data. Both groups were similar with regard to age of the participants (differences were not statistically significant, although the control group showed a higher mean age) and semen characteristics. Chromosomes 21, 18, 16, and 22 were the most frequently affected. These chromosomes are also the most commonly affected in common indications for PGD, such as implantation failure (Pehlivan et al., 2003). These results would be consistent with the study of Emery et al. (2005) in which a high level of aneuploidies in IVM cycles compared with standard IVF was observed. In the present study, the control group included embryos fertilized by ICSI after ovarian stimulation. Although it has also been shown that a large number of IVF embryos present chromosome abnormalities, some of which are as a result of non-disjunction during meiosis, premature sister chromatic separation or failures during fertilization and/or the first mitotic division (Gianaroli et al., 2002; Munné et al., 2002), given the characteristics of the sample, it was considered that this was the better control group. The spindle has a crucial role during meiosis. It has been observed that meiotic spindle retardance may cause misalignment of chromosomes (Liu et al. 2000). Some authors have suggested that spindle alterations may predispose oocytes to aneuploidy or maturation arrest. Thus, perturbation of the cytoskeletal integrity of oocytes may critically influence the

5 fate of the embryo (Pickering et al., 1988; Eichenlaub-Ritter et al., 2002). The high incidence of chromosome abnormalities among IVM embryos may be explained by an altered meiotic spindle causing anomalous chromosomal segregation, although due to the design of the study this potential link was not directly examined. In mouse oocytes, intra- and extrafollicular changes (ph, perifollicular microvasculature, oxidative stress, toxins) can disturb the orderly sequence of oocyte maturation and predispose oocytes to faulty chromosome segregation. These factors can arrest formation of spindle microtubules, resulting in depolymerization of the microtubules and disruption of the spindle structure (Pickering et al., 1990; Mailhes et al., 2000; Zenzes et al., 2002; Sun et al., 2004; du Plessis et al., 2008). Injury to the second meiotic spindle can lead to non-disjunction of chromatids, resulting in polyploidy and aneuploidy of chromosomes, delay of segregation, and aneuploidy embryos (Van der Elst et al., 1993; Eroglu et al., 1998). It has been shown that the incidence of aneuploidy is higher in mouse preantral follicles cultured using an in-vitro follicle culture protocol than in in-vivo-matured control oocytes (Carrell et al., 2005). In addition, the influence of different culture media on nuclear and cytoplasmic maturation of the oocyte should be considered (Filali et al., 2008). Nutrients such as glucose (el Mouatassim S et al., 1999) or hormones such as growth hormone may have a positive effect on oocyte maturation (Ménézo et al., 2006). Roberts et al. (2004), using a mouse model, found that FSH accelerates oocyte maturation and significantly increases glycolysis in COC undergoing IVM. In another study the same authors showed that the exposure to high concentrations of FSH during IVM can accelerate nuclear maturation and induce chromosomal abnormalities, suggesting that high FSH concentrations facilitate the maturation of oocytes that have pre-existing chromosomal errors (Roberts et al., 2005). In a mouse model, FSH, both in vivo and in vitro, affected the number of oocyte centrosomes, suggesting that the hormonal environment can modulate the dynamics of centrosome-based microtubule assembly, including spindle formation (Albertini, 1992). Although the doses of FSH used in the IVM culture media are not high, the effect on the oocyte of these factors should be considered. The effect of the follicular environment on the oocyte (Russell and Robker, 2007) and the role of the interaction between granulosa cells and oocyte on the development and maturation process are important. Growth-differentiation factor 9 and bone morphogenetic protein 15 are two oocyte-secreted factors involved in this interaction. Studies conducted in murine and bovine embryos have shown that supplementing oocyte IVM media with exogenous oocyte-secreted factors improves oocyte developmental potential, as shown by enhanced pre- and postimplantation embryo development (Gilchrist et al., 2008). Therefore, extended in-vitro culture conditions may have profound effects on the genetic competence of in-vitro-matured oocytes. During IVM, any disturbances in the orderly sequence of oocyte maturation may predispose the oocytes to faulty chromosome segregation; they can disrupt spindle assembly and chromosomal alignment during meiosis I and, if meiosis proceeds, predispose the oocyte to inaccurate segregation of the chromosomes and aneuploidy. It is largely documented in literature that nature exerts a strong selection against chromosomally abnormal embryos. In fact, the aneuploidy rate decreases as gestation advances, with an incidence of 0.6% in newborns and between 50% and 70% in spontaneous abortions (Hassold et al., 1980; Eiben et al., 1990). De-novo numerical chromosomal anomalies are the most common cause of spontaneous abortion (Strom et al., 1992; Stephenson et al., 2002). Other studies have shown high aneuploidy rates in patients with recurrent miscarriage (Vidal et al., 1998; Munné et al., 2003). Moreover, an increase in aneuploidy rate as a possible cause of implantation failure has also been suggested (Pehlivan et al., 2003; Wilding et al., 2004). The higher rate of chromosome abnormalities found in the present study is in contrast with data reported of a similar miscarriage rate and incidence of congenital disorders in newborn infants after IVM and conventional IVF protocols (Chian et al., 2003; Cha et al., 2005). Therefore, it may be postulated that the higher incidence of chromosome abnormalities, together with other possible cytoplasm alterations due to altered maturation of organelles, may account for early cleavage arrest and the low implantation rate. However, once the embryo has been implanted, subsequent development would not differ from embryo development following ovarian stimulation regimens. In a previous study including 6936 embryos from 1245 women undergoing PGD, normal euploid embryos showed significantly higher blastocyst rates (68.2%) compared with chromosomally abnormal embryos (42.8%) (Rubio et al., 2007). In summary, this study shows a higher incidence of chromosome abnormalities in embryos resulting from the IVM protocol compared with the control group, although differences were not statistically significant. The high aneuploidy rate may account for the low implantation rates found in these women. 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