RBMOnline - Vol 18 No 1. 2009 29-36 Reproductive BioMedicine Online; www.rbmonline.com/article/3465 on web 14 November 2008 Article Natural cycle IVF and oocyte in-vitro maturation in polycystic ovary syndrome: a collaborative prospective study Dr Moncef Benkhalifa obtained his PhD in 1992 from the Clermont Ferrand University and qualified in Reproductive Biology and Medical Cytogenetics from the school of Medicine there. He has been involved in assisted reproduction and genetics for more than 15 years, in collaboration with Professor Yves Ménézo, and is involved with different research groups in Europe and the Middle East. In 2007 he was appointed as Scientific Consultant on Reproductive Medicine and Genetics in the Eylau Laboratory, Paris and the UNILABS Group, Geneva. Dr Benkhalifa is author or co-author of more than 50 national and international publications and his main research interest is in embryology and genetics. Dr Moncef Benkhalifa M Benkhalifa 1,2, A Demirol 3, Y Ménézo 1,2,7, E Balashova 4, AK Abduljalil 5, S Abbas 5, I Giakoumakis 6, T Gurgan 3 1 ATL R&D, Reproductive Biology and Genetics Laboratory, 4 Rue Louis Lormand, 78320 La Verriere, 78320 France; 2 Unilabs Laboratories, Geneva, Switzerland; 3 Women s Health Clinic, IVF and Genetics, Ankara, Turkey; 4 IVF Dept, National Institute of Surgery, Moscow, Russia; 5 Dr Samir Abbas Medical Center, Jeddah, Saudi Arabia; 6 Mediterranean Fertility Centre and Genetics Services, Chania, Crete, Greece 7 Correspondence: Tel: +33 130 480 178; Fax: +33 130 571 934; e-mail: Yves.menezo@gmail.com Abstract In-vitro maturation (IVM) was performed in 350 cycles for 262 unstimulated patients diagnosed with polycystic ovary syndrome who were primed with human chorionic gonadotrophin (HCG) before oocyte retrieval. In order to improve nuclear and cytoplasmic maturation, growth hormone was added to the maturation medium. Oocytes were recovered in 94.8% of the cycles, with a mean number of nine cumulus oocyte complexes retrieved. Within 28 h, 62% of the oocytes reached the metaphase II (MII) stage, and 17.6% were MII after a further 20 h in culture. An ongoing pregnancy rate of 15.2% was obtained, but with a high miscarriage rate, 28% of the total with a positive HCG test assessed after embryo transfer. Cytogenetic and DNA fragmentation analysis of the embryos was not fundamentally different from what is classically observed in routine IVF. This observation implies that the results are not necessarily due to compromised oocyte quality after IVM, and that endometrial receptivity should also be considered, especially in IVM cycles where the follicular phase is dramatically shortened. Keywords: aneuploidy, DNA fragmentation, IVM, miscarriages, PCOS, pregnancy Introduction In-vitro maturation (IVM) protocols are now a valuable alternative to conventional IVF techniques, as a strategy to prevent ovarian hyperstimulation syndrome. In addition to clinical indications, invitro maturation in natural cycles can be considered as a social and economic alternative to classical assisted reproduction techniques on the basis of cost effectiveness. The first attempt at IVM was reported by Pincus and Enzmann (1934) for rabbit oocytes, and Edwards et al. (1965) published the first reports on human oocyte maturation in vitro. The first birth of a baby after oocyte IVM was reported in 1983 (Veeck et al., 1983). The first real large-scale experiments in assisted reproduction programmes started in the early 1990s (Trounson et al., 1994), with an increase in interest after the turn of the century, especially for polycystic ovary syndrome (PCOS) patients (Cha et al., 2000; Chian, 2004). In fact, for this special population, ovarian stimulation often means hyperstimulation. The maturation and success rates after IVM are affected by the number of collected cumulus oocyte complexes (COC) and the oocyte maturation rate during the first 24 h. However, the presence of the first polar body does not guarantee that the oocyte is capable of undergoing activation and further sustaining the capacity of the zygote to undergo genomic activation at the appropriate time. An inappropriate concentration and storage of key cytoplasmic factors such as housekeeping mrnas may lead to early developmental arrest. Based on previous animal experiments showing that growth hormone (GH) increases the rate of oocyte maturation (Izadyar et al., 1996, 1998; Pantaleon et al., 29 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK
30 1997) and improves maturation of naked human oocytes (Hassan et al., 2001; Ménézo, 2006), GH was added to the culture media in order to improve nuclear and cytoplasmic maturation. The GH receptor is constantly present in human oocytes, cumulus cells and early developing embryos (Ménézo et al., 2003). IVM protocols generally lead to significant miscarriage rates, but they are not associated with an additional risk to the outcome of an established pregnancy (Mikkelsen, 2005). Buckett et al. (2007), comparing the obstetric outcome in pregnancies conceived after IVM, IVF and intracytoplasmic sperm injection (ICSI), did not observe differences with those in spontaneously conceived controls. After oocyte fertilization, maternal and paternal factors (Ménézo, 2006; Dubey et al., 2008) can impair early embryonic development; thus apoptosis and chromosomal anomalies are classical features observed in embryos obtained through assisted reproduction technology. Therefore an investigation was undertaken into the degree of DNA fragmentation and chromosomal aneuploidy in arrested zygotes and embryos after IVM of oocytes retrieved from unstimulated PCOS patients. Materials and methods Patients This study included 350 cycles carried out in 262 patients who had been diagnosed with PCOS. Patients were recruited between September 2005 and September 2007, and were given complete information regarding the goals and the methodology of the procedure. The criteria of the Rotterdam 2003 consensus were used to define polycystic ovaries, i.e. when at least one ovary demonstrates a volume greater than 10 cm 3, or 12 or more follicles measuring 2 9 mm in diameter (European Society for Human Reproduction and Embryology [ESHRE]/ American Society for Reproductive Medicine [ASRM], 2004). The Farriman Galloway score was not performed. Hyperandrogenaemia was defined by (total serum testosterone >75 ng/dl [2.6 nmol/l] and calculated free testosterone >1.4 ng/ dl [50 pmol/l]). Testosterone concentration was measured by radioimmunoassay using a commercially available kit (ICN Biomedicals; Inc., Costa Mesa, CA, USA). The intra- and interassay coefficients of variation for testosterone were 9.6% and 11.6% respectively. Other androgen excess or related disorders (21-hydroxylase deficient non-classic adrenal hyperplasia, androgen-secreting neoplasms, androgenic/anabolic drug use or abuse, Cushing syndrome, the syndromes of severe insulin resistance, thyroid dysfunction, and hyperprolactinaemia) were excluded. The patients were included in the IVM programme after having signed a consent form if they satisfied the following criteria: baseline ultrasound scan at the beginning of the follicular phase showed an antral follicle count of more than 17, using transvaginal ultrasound (General Electric, Voluson 730 Pro). The age of the patient was older than 36 years of age, and sperm parameters were suitable for classical IVF or ICSI. Clinical preparation of patients undergoing IVM If the patients had irregular or no menstrual cycle, menstrual bleeding was induced with progesterone (Prometrium 300 mg/ day for 10 days or Provera 5 mg twice daily for 5 days). Once the medication was stopped, menstrual bleeding occurred within 3 days. If the patient had regular cycles, she was monitored after the onset of menses. During the first 2 3 days of the menstrual cycle, a baseline transvaginal ultrasound examination was performed to ensure that no ovarian cysts were present. The number and size of follicles on both ovaries were recorded. Between days 6 to 9, a second ultrasound scan was performed to confirm follicular development. Follicle size measurement, ensuring that the leading follicle did not exceed 10 12 mm and the lining of the endometrium was not less than 6 mm in thickness. When the patient was scheduled for IVM procedure, 10,000 IU human chorionic gonadotrophin (HCG; Profasi, Serono, Italy) was administered subcutaneously 36 h before oocyte retrieval. Immature oocyte retrieval Oocyte retrieval was usually performed between days 9 11, with oocyte collection scheduled 36 h after HCG administration. Immature oocytes were retrieved from the ovary under ultrasound guidance. The Cook IVM ovum aspiration needle (Cook, Australia) was used with an aspiration pressure of approximately 7.5 8.0 kpa (85 100mmHg). Each follicle was carefully punctured and the follicular fluid aspirated; three or four follicles were aspirated sequentially. The follicular fluid was collected in 14 ml Falcon tubes (Falcon 2001) containing approximately 2 3 ml of warm heparin saline solution (2 IU/ ml heparin). Follicular aspirates were examined for COC under a stereo-microscope. After examination of the aspirates, the fluid was poured through a 70 m cell strainer (Falcon 2350). COC were transferred to the wash dish containing warm IVM washing medium at 37 C. In order to ascertain maturity of the oocyte, the presence of a germinal vesicle or the extruded first polar body in the perivitelline space was assessed by tilting the Petri dish slightly to stretch the cumulus. Mature oocytes, if any, were subjected to ICSI within 3 h of collection after denudation. Immature oocytes were washed several times before being transferred to the IVM media (Medicult, Denmark) containing 15% patient serum collected at the time of oocyte retrieval, and 100 miu of recombinant GH (Saizen, Serono) oocyte maturation medium. The oocytes were incubated for 26 48 h (37 C, 5% CO 2 ) for maturation to take place. After 26 28 h, all the COC were denuded and maturation was assessed. Cumulus denudation was performed using a drawnout pipette after 1 min exposure to hyaluronidase (Syn vitro Hyadase, Medicult). ICSI was performed on mature oocytes and immature oocytes remained in culture for another 20 h. Sperm preparation and ICSI Semen was collected and prepared for insemination on the day of oocyte retrieval if at least one mature oocyte had been retrieved from the dominant follicle. If no mature oocyte was retrieved, semen collection and preparation was performed the following day. ICSI dishes were prepared at least 1 h before the procedure, and kept at 37 C in an incubator. After ICSI, the oocytes were transferred into 40 l drops of IVM maintenance medium and
cultured in an incubator (37 C, 5% CO 2 ). A maximum of four COC were cultured in each drop. Approximately 16 18 h after ICSI, oocytes were examined for fertilization; oocytes with two distinct pronuclei (2PN) and two polar bodies were considered as fertilized. Fertilized oocytes were cultured in 40 l drops (maximum of four/drop). The remaining oocytes were reassessed 48 h after collection, and mature oocytes were ICSI injected. Fertilized oocytes were cultured for 1 or 2 days, depending on the number and quality of embryos available. Since the oocytes might have been inseminated at different times post collection, embryos with more advanced development, originating from the oocytes with a shorter maturation time, were preferred for transfer. A maximum of three embryos were transferred. The first pregnancy test was performed 20 days post transfer. Endometrial preparation for embryo transfer Patients were administered oestradiol valerate tablets (Estrace) from the day of the oocyte retrieval, in order to prepare the uterine endometrium for embryo transfer. The dose was adjusted according to the status of the endometrium, i.e. a daily oral dose of 6 mg if the endometrial lining was >7 mm and 8 10 mg daily if the endometrium was <7 mm. Progesterone was given intravaginally (200 mg of Prometrium, administered three times daily) or subcutaneously, (100 mg daily injections), starting from the day after oocyte retrieval (or day of ICSI) and continued until the first pregnancy test performed after 20 days. If the test was positive, progesterone was continued until 12 weeks of gestation. If the endometrium was <7 mm on the day of embryo transfer, the embryos were cryopreserved for transfer after thawing in a subsequent cycle. Arrested zygotes and embryo analysis Embryo cleavage was assessed between 18 and 72 h post ICSI. Arrested zygotes and embryos during this period were analysed. Preparation for cytological and cytogenetic evaluation was performed according to a modification of the air-drying techniques. Briefly, cells were transferred (whether oocytes or arrested zygotes) one by one into a drop of 1% sodium citrate solution over a 2 3 min period, and then to a second drop of HCl Tween 20 for total cell lysis, until the appearance of the nucleus and/or chromosomes. After allowing the preparation to dry, four or five drops of fixative (methanol/acetic acid: 3/1, v/v) were added. The slides were dried for a minimum of 1 h at room temperature, then rinsed three times for 5 min with a phosphatebuffered saline (PBS) solution and treated with a pepsin solution at 37 C for 5 min (0.5 mg in 0.01 mol/l HCl). After rinsing in PBS, they were post-fixed in paraformaldehyde and dehydrated with alcohol solutions (70%, 80%, 90%, 100%). Assessment of DNA fragmentation DNA fragmentation was assessed using the In Situ Death Detection Kit (1684795 Roche Diagnostics, USA). After treatment, slides were covered with aluminium foil and placed into a humidity chamber for incubation (37 C, 45 min). After incubation, they were washed in PBS three times (1 min each) and air dried. Before evaluation, 8 l of DAPI was applied to each slide, and these were examined under a fluorescence microscope before being processed for fluorescence in-situ hybridization (FISH). Fluorescent in-situ hybridization After initial denaturation for 5 min at 73 C, the slides were hybridized with 3 l of probe mix at 37 C. FISH analyses were performed using DNA probes for chromosomes 13, 18, 21, X and Y. Following the hybridization step in a hybridization chamber (Hybrite, Vysis Ltd, UK) at 37 C for approximately 8 h, posthybridization washes were performed at room temperature. After drying the slides, 10 l of DAPI was added to each slide, and evaluation was performed under a fluorescence microscope with the recommended filters. Results Oocytes were retrieved from 332 of the 350 aspiration cycles (94.9%), with a minimum of four COC and an average of nine COC per patient: a total of 2988 COC were collected (Table 1). All patients obtained a minimum of one metaphase II (MII) oocyte after 28 h and one MII after 34 h of culture in vitro. Maturation rate after 28 h was 62.1%, and a further 17.6% matured during the subsequent 20 h. From an initial 1856 MII oocytes obtained after 28 h of in-vitro maturation, 1475 (79.4%) exhibited fertilization (2PN) 20 h post ICSI. Of the 1475 zygotes, 1258 (85.3%) reached the first cleavage stage. From 526 late-matured oocytes (maturing after 46 48 h), only 321 (61.0%) were fertilized and 176 (54.8%) of these reached cleavage. Ultimately, only 13.1% of these latematured oocytes resulted in embryos suitable for freezing or transfer (Figure 1). Outcomes of cycles in which oocytes were retrieved are shown in Table 2. From the 332 cycles with oocytes retrieved, 289 cycles (87.0%) led to embryo transfer on day 3 or 4. A total of 751 embryos were transferred (average of 2.6 embryos per transfer). Positive HCG tests were obtained in 81 cycles with transfer (28.0%). At 12 weeks, only 57 cycles (19.7%) remained positive. After 32 33 weeks, there were 44 ongoing pregnancies or deliveries (15.2%), with four twin gestations (9.1%) and an ongoing implantation rate per embryo of 6.4%. Analysis of 156 arrested zygotes that originated from a short maturation time revealed mainly a lack of syngamy (17%), premature chromosome condensation (PCC; 23%) and arrest at first somatic division, with partial or total DNA fragmentation (12%) (Figure 2a c). After 28 h maturation, 102 FISH analyses were informative: 13 zygotes (12.7%) showed an aneuploid status (five simple trisomy, three simple monosomy, two double trisomy, one double monosomy and two with complex aneuploidy). Testing of 110 arrested embryos revealed that 32.7% were aneuploid and 16% had total or partial nuclear DNA fragmentation (Figure 2d). Out of 110 FISH analyses of arrested embryos between days 2 to 4, 86 were informative for the five chromosomes and 32.7% were abnormal (16 aneuploid with nullisomy and/or monosomy and/or trisomy, six complex aneuploidy, four polyploidy and two haploid). After late maturation, 96 intact zygotes exhibited asynchrony between paternal and maternal nucleus decondensation 31
Table 1. Maturation and early embryonic development rates. Number % Patients 262 Cycles 350 100.0 Cycles with positive aspiration 332 94.9 Cumulus oocytes retrieved 2988 Mean cumuli/positive aspiration 9 Matured after 26 28 h 1856 62.1 Matured after 28 34 h 526 17.6 26 28 h in-vitro maturation Oocytes reaching MII (% of total) 1856 Oocytes fertilized (2PN) 1475 79.5 Embryos cleavage from 2PN 1258 85.3 46 48 h in-vitro maturation Oocytes reaching MII (% of total) 526 Oocytes fertilized (2PN) 321 61.0 Embryos cleavage from 2PN 176 54.8 Total embryos obtained on day 2 1434 Embryos suitable for transfer 886 61.8 MII = metaphase II; PN = pronuclei. 32 Figure 1. (A) Global appearance of collected cumulus oocyte complexes. (B) Mature oocytes obtained after 24 28 h. (C) 2-pronuclear zygotes after intracytoplasmic sperm injection. (D) Day-3 embryo from 28 h oocyte culture. (E) Abnormal zygote generated from oocyte matured for 48 h. (F) Slow-growing day-3 embryo.
Table 2. Transfers and pregnancies after in-vitro maturation. Parameter Value (%) No. of cycles with positive aspiration 332 No. of cycles with transfers 289 (87) No. of embryos transferred 751 No. of embryos/transfer 2.6 No. of positive HCG a 81 (28) Positive HCG (3 months) 57 (19.7) Ongoing pregnancies (>33 weeks) 44 (15.2) (4 twins) Miscarriage rate 27/81 (33.3) HCG = human chorionic gonadotrophin. Figure 2. (A) Arrested zygote at the first somatic stage, with complex aneuploidy (B) and DNA fragmentation in both nuclei (C). (D) Day-3 arrested embryos with three apoptotic and one partial apoptotic nucleus. (E) Normal euploid nucleus for five chromosomes, with 4 6 apoptotic nuclei (F). (12%), PCC (29%) and pulverized DNA (31%). After 48 h maturation, 55 of 96 zygotes were informative after FISH analysis: 10 zygotes (18.2%) were aneuploid (three carrying simple trisomy, two simple monosomy, two double monosomy and three with complex aneuploidy). The 78 blocked embryos between days 2 4 showed a high number of abnormal embryos (76.3%) with degenerated micronuclei combined with a chaotic nuclear status linked to complex aneuploidies (49.3%) and partial or total nuclear DNA fragmentation (27%) (Figure 2e,f). Sixty-one of 78 analysed embryos exhibited informative data after FISH: 49.3% of the embryos were abnormal. Twelve were aneuploid with nullisomy and/or monosomy and/or trisomy, nine exhibited complex aneuploidy, six were polyploid and three haploid (Table 3). 33
Table 3. DNA integrity and chromosomal aneuploidy in arrested zygotes and embryos. 28 h maturation time No. of arrested zygotes 156 Lack of syngamy 17.0 Premature chromosome condensation 23.0 Arrested at first cleavage stage 12.0 No. of arrested embryos 110 Aneuploid embryos 32.7 Embryos with partial or total fragmented nucleus 16.0 48 h maturation time No. of arrested zygotes 96 Asynchronous DNA decondensation 12.0 Premature chromosome condensation 29.0 Pulverized DNA 31.0 No. of arrested embryos 78 Complex aneuploidy 49.3 Embryos with partial or total fragmented nucleus 27.0 Values are percentages, unless otherwise stated. 34 Discussion Human IVM is now becoming an effective tool as an adjunct to assisted reproduction procedures, although mainly for selected patients in relation to clinical management (Suikkari and Söderström-Anttila, 2007), or to special regulations (Dal Canto et al., 2006). Nevertheless, the procedure avoids stimulating the ovaries and side effects of medical treatment as well as other risks such as ovarian hyperstimulation syndrome (Chian, 2004; Rao and Tan, 2005). An oocyte maturation rate of over 75% can readily be observed, but this study s data confirm that, although the fertilization rate is not affected by the duration of IVM (Kadoch et al., 2007), oocytes with a shorter duration of IVM have a better developmental potential. However, the ongoing implantation rate, i.e. the delivery rate per embryo transferred, is low, approximately half of that obtained after routine IVF/ ICSI (Kadoch et al., 2007). The complex intracellular processes involved in cytoplasmic maturation of the human oocyte are not precisely known. For example, the impact of low-dose FSH priming prior to the emergence of the dominant follicle is not clear, nor is the impact of HCG priming before retrieval. Activation of the transforming growth factor (TGF- ) superfamily (Gilchrist and Thompson, 2007) and the epidermal growth factor (EGF) network (Conti et al., 2006) are FSH and/ or LH (HCG) dependent. FSH priming might thus be more efficient in this respect. LH is necessary for protein synthesis in the oocyte (Panigone et al., 2008), and this explains the significance of HCG priming, as well as the effect of growth hormone, which is a potent stimulator of oocyte maturation. All of these effectors interact with transmembrane receptors that are associated with kinase activity, which is involved in the paracrine/autocrine loops that allow the oocyte to regulate its own environment through secretion of oocyte-secreted factors (Gilchrist and Thompson, 2007). The regulation of developmental competence is more probably dependent on the action of epiregulin and amphiregulin (EGF network ligands), and the TGF- superfamily ligands, growth differentiation factor 9 (GDF-9) or bone morphogenic protein 15 (BMP-15). LH (HCG) also up-regulates glutathione synthesis, which affects sperm decondensation and subsequently protects against reactive oxygen species during and after the fertilization process. The cumulative impact of in-vivo priming and in-vitro conditions are of major consequence, and supplementation with (hormonal) additives rather than composition of the culture media is also important. The supplements affect the rate and, therefore, the quality of IVM (Filali et al., 2008): addition of GH in the culture medium resulted in a shorter duration of maturation time, and an increased total maturation rate when compared with routine conditions. GH is a partner in the IVM process, acting either directly on the oocyte or through the cumulus cells. Supplementation of exogenous GH in patients with low circulating GH improves IVF outcomes (Rajesh et al., 2007) The positive effect on the speed of maturation is interesting as late matured oocytes are rather of poor quality. The question of epigenetic regulation is open, but in all animal species studied, GH has a positive effect on oocyte competence; as far as imprinting is concerned, epigenetic (i.e. methylation) marks are acquired after fertilization (El-Maari et al., 2001). GH signalling uses at least two different pathways: the camp response element-binding, mitogen-activated protein (CREB- MAP) kinase pathway and the signal transducer and activator of transcription (STAT) pathway. For a successful outcome, the oocyte must achieve two levels of maturation: nuclear and cytoplasmic. Low ongoing implantation rates are not necessarily due to inadequate cytoplasmic oocyte maturation. When blastocyst culture is attempted, embryos produced after IVM of human oocytes are often arrested at pronuclear or 4 8-cell stages (Barnes et al., 1996). In IVM programmes, embryos obtained after IVM have usually been transferred at the 2 8-cell stage, and their developmental potential to the blastocyst stage is relatively unknown. However, in this study, one of the major observations in arrested
zygotes is the lack of syngamy between maternal and paternal genome and premature DNA condensation and fragmentation. Nuclear asynchrony and pulverized or fragmented DNA were observed in both arrested zygotes and preimplantation embryos. Nuclear asynchrony and pulverized or fragmented DNA were observed in blocked zygotes and preimplantation embryos and could be related to degeneration or fragmentation of cells (Bongso et al., 1991; Fujini et al., 1996). This aspect of DNA degradation can be explained by apoptosis or by the necrosis phenomenon. Inadequate culture conditions could increase the occurrence of incomplete oocyte maturation and competency. This is of concern and long-term studies are needed to determine the safety of performing IVM. The high rate of embryo arrest between days 2 and 5 can be directly related to these phenomena, and they may also be directly responsible for the high rate of chromosome aneuploidies in early embryos if they manage to overcome the potential arrest. This may lead to implantation failure and repeated abortion. The rate of chromosome abnormalities in arrested embryos is not significantly higher than that reported in the literature for preimplantation embryos obtained with classical IVF/ICSI (Benkhalifa et al., 2003). For an average of 40% of abnormal arrested embryos, 22% had partial or total nuclear fragmentation, in parallel with simple or complex aneuploidy. Analysis of routine IVF embryos not selected for transfer (Bielanska et al., 2002) exhibited 48% of mosaic embryos. In other reports (Xu et al., 2000), 50% of the IVF embryos are chromosomally mosaic and the frequency of mosaicism increases from 18% to 60.5% between early cleavage and the 8-blastomere stage. An even higher incidence of chromosomal abnormalities can be observed in slow-growing embryos; this was the case in this study (49.3% with complex aneuploidy and 27% with fragmented nucleus). The ongoing implantation rates per embryo are low when compared with classical IVF, especially in relation to the high rate of miscarriages. However, it should be noted that the patient population consists of difficult PCOS patients, where the miscarriage risks are significant, and therefore the miscarriage rate is not necessarily due to IVM oocyte quality. Implantation, endometrial maturation and receptivity of the uterus are always a black box, even in IVF. Assessment of the endometrium has its limitations, and improving the conditions for implantation is still a challenge. Shortening the follicular phase does not help in this respect, and clinical practice must be based upon an understanding of the basic principles involved. 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