Five years experience of preimplantation genetic diagnosis in the Parisian Center: outcome of the first 441 started cycles

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1 Five years experience of preimplantation genetic diagnosis in the Parisian Center: outcome of the first 441 started cycles Estelle Feyereisen, M.D., a Julie Steffann, M.D., b Serge Romana, M.D., b Marc Lelorc h, Ph.D., b Pierre Ray, Ph.D., b Violaine Kerbrat, B.Sc., a Gérard Tachdjian, M.D., c René Frydman, M.D., a and Nelly Frydman, Pharm.D. c a Service de Gynécologie-Obstétrique et de Médecine de la Reproduction, Hôpital Antoine Béclère, Clamart; b Département de Génètique, Hôpital Necker Enfants Malades, Paris; c Service de Biologie et Génétique de la Reproduction, Hôpital Antoine Béclère, Clamart, France Objective: To investigate the evolution of techniques and strategies and to evaluate the results of preimplantation genetic diagnosis (PGD) from January 2000 to December 2004 in chromosomal, monogenic and mitochondrial DNA disorders treated at our institution. Design: Retrospective study. Setting: Single French Parisian PGD center. Patient(s): Patients at risk of transmitting a serious genetic disorder to their offspring. Intervention(s): 171 couples enrolled in the program undergoing stimulated and frozen embryo replacement cycles with PGD. Main Outcome Measure(s): Results of the 441 first PGD cycles performed for various genetic conditions. Result(s): During 5 years, 416 stimulation and 25 frozen embryo replacement cycles were started, among which 52 clinical and 47 ongoing pregnancies occurred. In stimulation cycles, the overall ongoing pregnancy rate was 24% per embryo transfer, 11% per started cycle, and 27% per couple. The implantation rate was 16%. Conclusion(s): These encouraging results demonstrate that PGD might be considered as a valid alternative to prenatal diagnosis. Nevertheless, couples referred for PGD must be selected and counseled appropriately, considering the complexity of the treatment and the relatively low take-home baby rate. (Fertil Steril 2007;87: by American Society for Reproductive Medicine.) Key Words: Preimplantation genetic diagnosis (PGD), translocations, chromosomal rearrangements, single gene defects, X-linked disorders, mitochondrial DNA disorders Preimplantation genetic diagnosis (PGD) resulted from the evolution of assisted reproductive technology, the development of molecular biology, and advances in genetic knowledge. The first pregnancies after PGD in couples at risk of X-linked disorders were reported in London (1). Since then, PGD techniques have been improved, and pregnancies and births reported worldwide in cases of translocations (2, 3) and monogenic disorders (4 6). Diagnosing genetic diseases at the embryonic level is possible with PGD, and it may be considered as a very early form of prenatal diagnosis. One or two individual cells are removed from day-3 preimplantation embryos obtained in vitro, followed by genetic analysis; only unaffected embryos are replaced in the uterus on day 4. Embryos without structural or numerical chromosomal disorders can be identified by fluorescent in situ hybridization (FISH) analysis and without single gene defects by polymerase chain reaction (PCR) assay. Received November 18, 2005; revised and accepted May 27, Reprint requests: Nelly Frydman, Pharm.D., Service de Biologie et Génétique de la Reproduction, Hôpital Antoine Béclère, 157 rue de la Portede-Trivaux, Clamart Cedex, France (FAX: ; nelly.frydman@abc.aphp.fr). Couples who are referred for PGD often have a complex reproductive history, including pregnancies terminations, birth of an affected child, or neonatal death. Structural chromosomal aberrations such as balanced translocations are also often associated with recurrent miscarriages and even infertility (7). Until recently, these carrier couples could opt for gamete or embryo donation, adoption, or prenatal diagnosis with the possible recurrence of pregnancy termination. These options carried genuine emotional morbidity. New applications and methodology are being introduced regularly, and PGD is now a concrete alternative that avoids the psychological burden of classic prenatal diagnosis (8). Nevertheless, the relative complexity of this assisted reproduction treatment and the relatively low take-home baby rate must be stressed through a rigorous information program, especially with fertile couples (9). The first successful PGD cycle was reported more than a decade ago, but French legislation has allowed the practice of PGD only since 2000 in couples who are at risk of transmitting a serious genetic disorder to their offspring. The Beclère Hospital (Clamart), associated with the Necker Hospital (Paris), is one of the three French centers licensed for PGD. Although aneuploidy screening (where the number of several key chro- 60 Fertility and Sterility Vol. 87, No. 1, January /07/$32.00 Copyright 2007 American Society for Reproductive Medicine, Published by Elsevier Inc. doi: /j.fertnstert

2 mosomes are checked to increase the pregnancy rates in poor prognosis IVF patients) has seen exponential growth elsewhere, it is still prohibited in France (10, 11). The objective of this retrospective study was to evaluate the results of PGD for the various indications in our center and to investigate the evolution of techniques and strategies over 5 years. We describe our 2000 to 2004 experience of PGD in the Parisian center with couples who are carriers of a preexisting genetic abnormality and are at risk of transmitting a particularly serious incurable disease to their offspring. MATERIALS AND METHODS Patients We retrospectively analyzed the patients selected for PGD in our center from 1999 to During this period, 780 couples with a high probability of giving birth to a genetically affected child were referred to our team, including 343 (44%) chromosomal rearrangement carriers and 437 (56%) single gene disorder carriers. Referrals were received from geneticists or gynecologists; a few patients were selfreferrals. The patients were screened at the weekly PGD meeting that involved specialists in each area of PGD. After confirmation that PGD was ethically acceptable and technically feasible for their genetic disorder, 586 couples were invited for a consultation in the assisted conception unit. An initial multidisciplinary consultation with a geneticist, a gynecologist, an embryologist, and a psychologist was performed for 284 couples with structural chromosomal abnormalities (48.5%) and 302 (51.5%) with monogenic diseases, including autosomal or X-linked disorders (12, 13). Extensive information about the PGD procedure, the expected results, and the risk of misdiagnosis was provided, and informed consent was obtained from all patients. Blood samples were taken as required for DNA analysis or karyotyping. The medical investigation focused especially on the day-3 ovarian reserve (basal hormonal level and ultrasound examination with antral follicular count), status of the uterus (vaginal ultrasound scan, hysteroscopy) and sperm parameters (spermogram, spermocytogram, spermoculture). As a result of the initial consultation and the subsequent investigation, 71 of the couples (47 chromosomal rearrangements carriers and 24 single gene disorder carriers) were advised to consider alternate approaches such as prenatal diagnosis, gamete donation, or adoption. In these couples, PGD was not indicated because of maternal age 40 years, evidence of poor ovarian reserve, or severe medical disorders that contraindicated IVF or pregnancy. Incidentally, 164 couples, including 81 initially referred for chromosomal abnormalities and 83 for single gene defects, chose to start spontaneous pregnancies with prenatal diagnosis; 75 others, including 18 translocations carriers and 57 single gene defects carriers, decided against further treatment because of the procedure s complexity. From 2000 to 2004, of the 138 couples in which one partner carried a chromosomal rearrangement were eligible for PGD, 77 went through the program, for a total of 194 initiated cycles; 61 couples had yet to start treatment. From 2000 and 2004, among the 138 eligible couples with single gene defects, 94 were enrolled in our PGD program, for a total of 222 initiated cycles; 44 couples had yet to start treatment. In Vitro Fertilization Protocol Appropriate consent forms were signed before each IVF. The ovarian stimulation protocol was carried out by using 3 mg IM of Decapeptyl (Ipsen Biotech, Paris, France), a depot formulation of gonadotropin-releasing hormone (GnRH) agonist, on day 3 of the menstrual cycle. After 18 days, the patients underwent a transvaginal ultrasound screening and an assay of serum estradiol concentrations to document their pituitary desensitization. After confirmation of ovarian quiescence, gonadotropin ( IU) was administered daily, usually urinary Menopur (Ferring, Gentilly, France) or the recombinant follicle-stimulating hormone (FSH) Gonal F (Serono, Boulogne, France) or Puregon (Organon, Puteaux, France). The gonadotropin dose after day 6 was adjusted to the ovarian response. Since 2003, after recombinant FSH was used, 75 IU daily of recombinant LH (Luveris, Serono, Boulogne, France) was systematically added from day 6 of stimulation until the day of human chorionic gonadotropin (hcg) administration. Monitoring of the cycle was done by assessment of plasma level of LH, E 2, and progesterone in conjunction with ultrasonography starting on day 8 of stimulation. The regimen continued until clinical parameters for hcg administration were achieved. Human Chorionique Gonadotrophine Endo (Organon, Puteaux, France), 10,000 IU, was administered when at least five follicles 16 mm were observed by ultrasound and the E 2 plasma level was greater than 1000 pg/ml. In cases at risk for ovarian hyperstimulation syndrome, only 5000 IU of hcg was injected. A patient s cycle was canceled if the E 2 level did not reach 1000 pg/ml, the progesterone level was over 2 ng/ml, or fewer than eight growing follicles were observed after 5 days of ovarian stimulation. Transvaginal ultrasound-guided oocyte retrieval under local or general anesthesia was scheduled for 36 hours after hcg administration. All oocytes obtained were inseminated by intracytoplasmic injection of spermatozoon (ICSI) to prevent residual contamination with sperm (14). In cases of cystic fibrosis with congenital bilateral absence of the vas deferens, epididymal sperm was used. After fertilization, the zygotes were cultured in specific culture media ISMI and ISM2 (Medi-Cult, Lyon, France) until the biopsy samples were obtained. Biopsy Procedure The biopsy procedure was carried out on cleavage stage at day 3 in ISM2 medium (Medi-Cult, Lyon, France) using a no-contact laser (Fertilase; MTG Medical Technology Vertriebs- GmbH, Altdorf, Germany) for zona drilling (15). To reduce Fertility and Sterility 61

3 the possibility of misdiagnosis, two blastomeres were systematically removed and used for PGD analysis if the embryo had at least six cells or whenever possible (16). Preimplantation Genetic Diagnosis Techniques Polymerase chain reaction analysis The amplification protocols were described by Ray et al. (16 19) and Burlet et al. (20). Results were obtained 6 to 12 hours after embryo biopsy samples has been obtained. Nested polymerase chain reaction, usually performed for the analysis of single gene defects, was used for all autosomal monogenic disorders and whenever feasible (identified mutation) for X-linked disorders (21). For the NARP mitochondrial DNA (mtdna) disorder, PGD required the semiquantitative fluorescent PCR described by Gigarel et al. (22). Fluorescent in situ hybridization analysis When a specific PCR diagnosis for recessive X-linked disorders was not technically possible at the single-cell level, an embryo sexing by FISH analysis was performed (23, 24). In these situations, PGD aimed at selecting potentially affected XY male embryos to avoid replacing them in the uterine cavity. The probes were specific centromeric probes for X and Y chromosomes, labeled with different fluorochromes. Centromeric probes for X were labeled with green fluorescein (FITC-12-dUTP), whereas centromeric Y probes were labeled with red rhodamine (tetramethyl-rhodamine-5-dutp). The biopsied blastomeres were spread on slide (Superfrost Plus; Fisher Scientific, Illkirch, France) by the HCL-Tween method according to Coonen et al. (25). Following the hybridization, the slides were washed and analyzed using a Leica fluorescence microscope (Leica Microsystems AG, Wetzlar, Germany), fitted with a Photometrics camera (Roper Scientific, Photometrics, Tucson, AZ). Only the blastomeres with two evident green signals were considered normal. We also applied FISH in cases with structural chromosomal rearrangements (26, 27). The efficiency of the FISH technique and the specificity of the probes had been previously tested on interphase nuclei and metaphase chromosome spreads of karyotypically normal individuals prepared to an efficiency 95%. After analysis of the karyotype of both members of the couple, the probe set was tested on their metaphase chromosome spreads to verify the translocation and detect possible hybridization polymorphisms. The probe sets selected depended on the chromosome translocation. To detect all types of segregation, robertsonian translocations need only two probes, one probe by chromosome. For the reciprocal translocations, our strategy used three subtelomeric probes with different fluorochromes: two on both sides of the first chromosome implicated in the translocation, and the third probe distal or proximal to the other breakpoint. All probes used in this study were homemade, and each was screened on genome bioinformatics sites such as the University of California at Santa Cruz (UCSC) or National Center of Biotechnology Information (NCBI; Bethesda, MD). The clones were cultured, and DNA was extracted. The probes were labeled by nick translation with fluorescein (FITC-12-dUTP), tetramethyl-rhodamine-5-dutp, or biotin-16-dutp (Roche Diagnostics, Basel, Switzerland) detected with Cy5-labeled streptavidin (Amersham Biosciences, Uppsala, Sweden). The two blastomeres from an embryo had to demonstrate the same pattern; if not, the embryo was considered to be mosaic and was not transferred. Embryo Transfer Procedure The embryo transfers (ET) were carried out on day 4 using a classic Frydman catheter 4.5 (CCD, Paris, France). Since 2003, the procedure has been guided by vaginal ultrasonography. As in regular IVF cycles, the age of the patient, the number of previous attempts, and the embryo quality (28) determined the number of embryos transferred; a maximum of three embryos were replaced. Supernumerary unaffected embryos were cryopreserved. Luteal phase support was systematically given by daily vaginal administration of micronized progesterone (Estima G; EFFIK, Bievres, France) until a negative serum pregnancy test or until 8 weeks of gestation. A clinical pregnancy was noted when an intrauterine gestational sac with a fetal heartbeat was seen on vaginal ultrasound 5 weeks after embryo replacement. An ongoing pregnancy was defined as a clinical pregnancy with a fetal heartbeat at 12 weeks. Prenatal diagnosis was discussed at the beginning of the pregnancy follow-up, according to each indication. The follow up of children born after PGD had been prospectively organized since the beginning of our study. Data Analysis We recorded the outcome of stimulated and frozen embryo replacement cycles performed over a period of 5 years. We detailed the disorders treated in our center, and the results that had been obtained in PGD for robertsonian and reciprocal translocations, autosomal monogenic diseases, X-linked defects, and mitochondrial DNA pathologies. We kept track of information on oocyte retrieval, fertilization, embryo biopsies, embryo transfers, and pregnancy rates for each genetic condition. An analysis of the results for the different female age groups was also conducted. The data were analyzed using the chi-square test; P.05 was considered to be statistically significant. RESULTS Between 2000 and 2004, 171 couples were enrolled in our PGD program, including 77 for translocations, 56 for autosomal single gene defects, 37 for X-linked disorders, and 1 for a mitochondrial DNA disorder. The mean age of the female partner was 32.7 ( 3.5; range 23 to 41 years) and was comparable in each indication group. The rank of at- 62 Feyereisen et al. Five years of PGD activity in a French center Vol. 87, No. 1, January 2007

4 tempts per couple varied between 1 and 5. The 171 couples started 416 stimulated cycles. Among them, 16 had frozen embryos from a previous classic IVF attempt and had started 16 frozen embryos replacement cycles before the stimulation for PGD. In 11 cases, unaffected embryos were frozen after PGD in our center, followed by nine replacement cycles. Stimulated Cycles The data of the 416 stimulated started cycles were summarized in Tables 1 and 2. Among them, 194 cycles were started for structural chromosomal abnormalities, 182 for single gene defects (including X-linked disorders when a specific diagnosis was possible), one for a mitochondrial DNA disorder, and 32 for X-linked disorders (sexing); seven were double PGD, defined as the combination of a single gene defect diagnosis and a sexing. Because of poor ovarian response, 136 cycles (33%) were canceled. Among the cancellations, 118 were due to insufficient ovarian responses (87%). Only four cycles were canceled because of a high response associated with major risk of ovarian hyperstimulation syndrome (3%); 14 (10%) were canceled for other reasons, including lack of desensitization (four cases), the couple s decision (four cases), an increase of progesterone plasmatic level (two cases), mistakes in the treatment administration (two cases), or scarce endometrial thickness (two cases). Two hundred and eighty oocyte retrievals were performed (67% of started cycles). The mean number of oocytes retrieved was 12.5, and 67% of the oocytes inseminated yielded cleaved embryos. Embryo biopsies were feasible in 239 cases (85% of oocyte retrievals and 57% of started cycles), and 189 ET were realized (45% of started cycles). The mean number of embryos transferred was 2.02, and 51 clinical pregnancies resulted (27%/ET), including five miscarriages and 46 ongoing pregnancies (24%/ET) that led to the birth of 57 unaffected babies. Among the 46 ongoing pregnancies, 10 were twin pregnancies, and one was a triplet pregnancy that reduced spontaneously. The rate of multiple pregnancies was 23%. The implantation rate (IR) was 16%. The average take-home baby rate was 27% per couple. Chromosomal Abnormalities Of the 77 couples who started stimulated cycles for chromosomal abnormalities, 42 were carriers of a robertsonian translocation, and 35 were carriers of a reciprocal translocation. The details of translocations treated in our center were reported in Table 3. We started 113 cycles for robertsonian translocations and 81 for reciprocal translocations. The outcome of these cycles is found in Table 4. For robertsonian translocations, 81 oocyte retrievals were performed. The mean number of oocytes retrieved was We obtained 548 embryos, of which 346 (63%) were biopsied. The successful diagnosis rate was 79%; among the diagnosed embryos, 50% were unbalanced. We performed 57 ET (mean 1.96 embryos transferred), leading to 16 ongoing pregnancies. The IR in robertsonian translocations was 22%. For reciprocal translocations, 53 oocyte retrievals were performed. The mean number of oocytes retrieved was We obtained 383 embryos, of which 240 (62%) were biopsied. The successful diagnosis rate was 80%, and 78% of embryos diagnosed were unbalanced. We performed 23 ET (mean 1.56 embryos transferred), yielding five ongoing pregnancies. The IR in reciprocal translocations was 19%. Autosomal Single Gene Defects Fifty six couples were enrolled in our program for autosomal single gene defects, including 34 for recessive diseases and 22 for dominant pathologies. The seven double PGDs performed were counted here as single gene defects. The detailed list of the autosomal monogenic defects treated at our institution is found in Table 5. The most common indications treated were cystic fibrosis (59% of couples enrolled for recessive disorders) and myotonic dystrophy (more than 95% of couples enrolled for dominant diseases). The outcome of stimulated PGD cycles for single gene defects is described in Table 6. Eighty five cycles were initiated for recessive disorders, yielding 50 oocyte retrievals. The cancellation rate was 41% in these indications; the mean number of oocytes retrieved was We obtained 313 embryos, of which 233 (74%) were biopsied. The successful diagnosis rate was 79%, and 33% of embryos diagnosed were affected. We performed 40 ET (mean 2.35 embryos transferred), yielding 10 ongoing pregnancies. The IR in recessive autosomal monogenic diseases was 15%. During the same period, 48 cycles were started for dominant disorders, leading to 37 oocyte retrievals. The cancellation rate was 23% in these indications. The mean number of oocytes retrieved was We obtained 225 embryos, of which 156 (69%) were biopsied. The successful diagnosis rate was 81%, and 66% of embryos diagnosed were affected. We performed 22 ET (mean 1.59 embryos transferred), leading to five ongoing pregnancies. The IR in dominant autosomal monogenic diseases was 14%. X-linked Disorders We provide the details of X-linked pathologies treated in our unit in Table 5. Thirty seven couples were enrolled in these indications: 13 had sexing by FISH because a specific disease diagnosis was not available at the single-cell level, and 24 had a specific diagnosis by PCR. Thirty two cycles were started for sexing, yielding 21 oocyte retrievals. The cancellation rate was 34%. The mean number of oocytes retrieved was We obtained 216 Fertility and Sterility 63

5 TABLE 1 Overview of the first 416 stimulated cycles, Year Stimulated started cycles (SC) Chromosomal abnormalities Single gene defects Mitochondrial DNA disorder Sex diagnosis (sexing) Double PGD a a Cycles with a double-pgd performed, including both a single gene defect diagnosis and a sexing. embryos, of which 137 (63%) were biopsied. The successful diagnosis rate was 85%, and 62% of embryos were potentially affected (i.e., male embryos). We performed 15 ET (mean 2.13 embryos transferred), yielding five ongoing pregnancies. The IR in this group was 16%. Fifty six cycles were initiated for specific diagnosis of X- linked diseases, leading to 38 oocyte retrievals. The cancellation rate was 32%. The mean number of oocytes retrieved was We obtained 293 embryos, of which 222 (75%) were biopsied. The successful diagnosis rate was 83%, and 41% of embryos were affected. We performed 31 ET (mean 2.25 embryos transferred), and 10 ongoing pregnancies occurred. The IR in this population was 9% (Table 7). Mitochondrial DNA Disorder The parents of a boy affected with Leigh syndrome requested PGD because they wanted to avoid a termination of pregnancy of an affected fetus. The mother was an asymptomatic carrier of the m.8993t G NARP mitochondrial DNA mutation, involving a mitochondrial gene encoding the subunit 6 of the ATPase. Her affected son carried the NARP mutation in muscle at a homoplasmic state. One cycle was performed, leading to one oocyte retrieval. Five oocytes were retrieved, and four were inseminated, resulting in three embryos. All embryos were biopsied: one embryo was shown to be affected (100% of mutant mitochondrial DNA), and two were unaffected (no mutant mitochondrial DNA detected). These latter were transferred, and one ongoing pregnancy occurred. A healthy boy was born. Frozen Embryo Replacement Cycles Twenty five couples started 25 frozen embryo replacement cycles. Sixteen of them had supernumerary embryos from a previous classic IVF attempt. Embryo biopsies were successfully carried out on thawed embryos in only seven cases, according to the embryo quality. Six ET were performed; two biochemical and one ongoing pregnancy ensued. The ongoing pregnancy occurred after the transfer of three unaffected embryos in woman who is a Duchenne muscular dystrophy carrier. In the remaining cycles, the embryos did not survive the thawing process in a state suitable for biopsy. TABLE 2 Overall stimulation cycle data collection, Couples 171 Started cycles (SC) 416 SC per couple 2.43 Canceled cycles 136 (33%) Cycles to oocyte 280 (67%) retrieval (OR) Oocytes retrieved 3506 Oocytes retrieved/or 12.5 Oocytes inseminated 2966 Embryos obtained 1982 Embryo biopsies 239 (85%/OR) Embryos biopsied (%) 1337 (67%) Embryos diagnosed (%) 1083 (81%) Embryos affected (%) 584 (54%) Embryo transfers (ET) 189(45%/SC) (67%/OR) Embryos transferred 381 Embryos transferred/et hcg 67 Clinical pregnancies 51 Twins 10 Triplets 1 Implantation rate 16% Miscarriages 5 Ongoing pregnancies 46 (16%/OR) (24%/ET) (11%/SC) (27%/couple) 64 Feyereisen et al. Five years of PGD activity in a French center Vol. 87, No. 1, January 2007

6 TABLE 1 Continued. Cancellations Oocyte retrievals Embryo biopsies Embryo transfers (ET) Clinical pregnancies Ongoing pregnancies Children born (33%/SC) 280 (67%/SC) 239 (57%/SC) 189 (45%/SC) 51 (27%/ET) 46 (24%/ET) 57 Exceptionally, 11 couples had supernumerary unaffected embryos after PGD. They were grown to the blastocyst stage for freezing. Nine frozen embryo replacement cycles have been initiated, but none of the embryos survived the thawing process. Influence of Maternal Age on the Outcome of PGD For all indications, we performed an analysis of the results of the 416 PGD stimulated cycles in three female age groups: 32, 33 37, and 38 years of age. Ninety three women were 32 years of age and started 222 stimulated cycles; 61 were between 33 and 37 years of age and started 155 cycles; 17 were 38 years of age and started 39 cycles. Cancellation rates were similar in the three groups (35%, 32%, and 26%, respectively). The outcome of the cycles was comparable in the two groups of women younger than 38 years with respect to IR (18% in the 32-year-old group, and 16% in the year-old group) and ongoing pregnancy rate (26%/ET in both groups). After 38 years of age, only one ongoing pregnancy occurred; very low IR (4.5%) and ongoing pregnancy rate (6%/ET) were noted. A statistically significant difference was found with regard to the ongoing pregnancy rate per couple between the 32-year-old women (30%) and the 38-year-old women (6%) (P.03). DISCUSSION The clinical outcome of the cycles demonstrates that PGD is a valid technique that can be offered to prevent the transmission of inheritable disorders. The range of chromosomal disorders and single gene defects for which PGD was available in our center increased over the 5 study years. For autosomal dominant diseases and reciprocal translocations, PGD has been performed since 2001, and referrals for these conditions have increased over the years. We reported 52 clinical pregnancies and 47 ongoing pregnancies. In stimulation cycles, 51 clinical pregnancies and 46 ongoing pregnancies (24% per ET) occurred, with a 16% implantation rate. Twenty seven percent of the couples enrolled in our PGD program achieved an ongoing pregnancy. The outcome of PGD cycles in our series is similar to those reported by the PGD ESHRE Consortium (11). Although differences were observed for the cycle characteristics among the groups of disorders, the ongoing pregnancy rates were similar. Only reciprocal translocations yielded a poor outcome (five ongoing pregnancies of 81 started cycles) with a low take-home baby rate (14% per couples). Despite the presence of normal fertility in many of the women treated, the prevalence of cycles canceled was unexpectedly high in our series (33%). This is the most striking difference between our results and those reported by other teams; the cancellation rate reported by Pickering et al. (29) was 18%, and by the European Consortium only 6.6% (11). The average number of oocytes retrieved was high (12.5) because the women were strictly selected, and was comparable for each indication group. The fertilizing capacity of the oocytes and spermatozoa did not seem to be affected by the genetic condition. Sixty-seven percent of oocytes inseminated yielded cleaved embryos. Embryo biopsies were not performed on poor quality embryos; the phase of biopsy was reached in 85% of cases after oocyte retrieval. The rate of multiple pregnancies (23%) was higher than in classic IVF but was comparable with reports by other teams. This might related to our decision to transfer two embryos whenever possible (for a maximum of three embryos replaced) because of the poor outcome of unaffected frozen-thawed embryos. The first birth following PGD performed by a French team occurred at our institution in It was the first specific genetic diagnosis reported for ornithine transcarbamylase deficiency (17). During the 5 years, specific diagnoses for X-linked disorders were further developed. In these disorders, only half of the male embryos will carry the mutation, so specific diagnosis is preferred over sexing. When specific diagnosis was not available, only sexing was possible, and the unaffected Fertility and Sterility 65

7 TABLE 3 Details of robertsonian and reciprocal translocations treated, Translocations Number of patients (couples) Number of oocyte retrievals Robertsonian translocations 45,XX,der(13;14)(q10;q10) ,XX,der(14;21)(q10;q10) ,XX,der(13;22)(q10;q10) ,XX,der(13;21)(q10;q10) 1 1 (Total female robertsonian translocations) (19) (32) 45,XY,der(13;14)(q10;q10) ,XY,der(14;21)(q10;q10) ,XY,der(13;15)(q10;q10) ,XY,der(13;22)(q10;q10) ,XY,der(13;21)(q10;q10) 1 2 (Total male robertsonian translocations) (23) (49) Total robertsonian translocations Reciprocal translocations 46,XY,t(8;11)(p12;q21) ,XY,t(5;13;14)(q23;q21;q31) ,XY,t(11;22)(q23;q11) ,XY,t(6;9)(p22;p24) ,XY,t(8;13)(p21;q34) ,XY,t(8;22)(q24;q11) ,XY,t(14;18)(q22;q21) ,XY,t(2;14)(q36;q23) ,XY,t(5;15)(q35;q24) ,XY,t(2;22)(q24;p11) ,XY,t(9;14)(p21;p11) ,XY,t(1;3)(p22;p24) ,XY,t(8;14)(q22;q23) ,XY,t(18;21)(q21;q22) ,XY,t(4;17)(p16;q11.2) ,XY,t(1;16)(q10;q10) ,XY,t(3;7)(q13;p21) ,XY,t(7;14)(p21;q12) ,XY,t(9;17)(p13;q11.2) ,XY,t(11;21)(q14.1;q22.3) ,XY,t(15;20)(p11.1;p11.1) ,XY,t(4;13)(q23;q31) 1 1 (Total male reciprocal translocations) (22) (34) 46,XX,t(3;14)(p22;q32) ,XX,t(2;8)(q37;q21) ,XX,t(2;8)(q33;p23) ,XX,t(13;14)(q33;q13) ,XX,t(8;16)(p22;p12) ,XX,t(6;9)(p22;p22) ,XX,t(11;22)(q23;q11) ,XX,t(14;16)(q2;p11) ,XX,t(10;13)(p13;q34) ,XX,t(5;10)(q33;q23) ,XX,t(9;12)(q22;q13) Feyereisen et al. Five years of PGD activity in a French center Vol. 87, No. 1, January 2007

8 TABLE 3 Continued. Translocations Number of patients (couples) Number of oocyte retrievals 46,XX,t(10;13)(q24;q32) ,XX,t(9,21)(q11,q22) 1 1 (Total female reciprocal translocations) (13) (19) Total reciprocal translocations male embryos were lost for transfer. Surprisingly, we obtained a better outcome in sexing than in specific diagnosis, perhaps due to the less rigorous selection of the women with regard to their ovarian potential. Another hypothesis is that we had recently started cycles for fragile-x syndrome, in which women are known to be at risk of premature ovarian failure. Affected women or carriers of some particular pathology exhibited a relatively hindered response to controlled ovarian hyperstimulation. This could make it difficult to carry out the PGD process with reasonable chances of success. Carriers of X-fragile syndrome are well known as having ovarian defects and premature ovarian failure (30, 31). Nevertheless, PGD for fragile X is probably feasible for a number of couples after estimation of a sufficient ovarian reserve (32), although some centers have stopped offering it after a few initial cycles (33, 34). At the present time, our experience for this condition is limited; we recently started the first cycles in fragile-x syndrome carriers of mutations or premutations. After 2004, only three couples were enrolled in the program, TABLE 4 Outcome of PGD cycles for translocations, Robertsonian Reciprocal Total Couples Started cycles (SC) No. of oocyte retrievals (OR) Cancellation rate (%) 28% 35% 31% Oocytes retrieved Oocytes retrieved/or Oocytes inseminated Embryos obtained Embryos biopsied 346 (63%) 240 (62%) 586 (63%) Embryos diagnosed 274 (79%) 193 (80%) 467 (79%) Embryos affected 137 (50%) 152 (78%) 289 (62%) No. of embryo transfers (ET) Embryos transferred Embryos transferred/et Clinical pregnancies Ongoing pregnancies (OP) Twin pregnancies Triplet pregnancies Gestational sacs Implantation rate 22% (24/112) 19% (7/ 36) 21% (31/ 148) OP/ET 16/57 (28%) 5/23 (22%) 21/80 (26%) OP/OR 16/81 (20%) 5/53 (9%) 21/134 (16%) OP/SC 16/113 (14%) 5/81 (6%) 21/194 (11%) Fertility and Sterility 67

9 TABLE 5 Details of autosomal single gene defects and X-linked disorders treated, Number of patients (couples) Number of oocyte retrievals Autosomal single gene defects Autosomal recessive Cystic fibrosis Spinal muscular atrophy 8 11 Diastrophic dysplasia 1 3 Sickle-cell anemia 2 2 Phosphoribosyl-pyrophosphate synthetase superactivity 1 1 Severe combined immunodeficiency 1 1 Osteogenesis imperfecta 1 1 (Total autosomal recessive) (34) (50) Autosomal dominant Myotonic dystrophy Perth hemolytic anemia (unstable hemoglobin Perth) 1 3 (Total autosomal dominant) (22) (37) Total autosomal single gene defects X-linked disorders Sexing Mental retardation 5 12 Duchenne muscular dystrophy 2 2 Adrenoleukodystrophy 2 2 Hemophilia 1 2 Hydrocephaly 1 1 Androgen receptor mutation 1 1 Leber hereditary optic neuropathy 1 1 (Total sexing) (13) (21) Specific diagnosis 8 11 Duchenne muscular dystrophy Becker muscular dystrophy 3 6 Fragile-X syndrome 3 5 Hemophilia 3 3 Hunter syndrome 2 3 Adrenoleukodystrophy 1 3 Ornithine transcarbamoylase deficiency 2 4 Pelizaeus Merzbacher disease 1 2 Lissencephaly 1 1 (Total specific diagnosis) (24) (38) Total X-linked disorders and seven cycles were started, yielding five oocyte retrievals and four embryo transfers; no pregnancies occurred. Incidentally, a significantly decreased ovarian response was also found in women affected by myotonic dystrophy (35). In another study, an increased risk for poor ovarian response to ovarian stimulation was described in female carriers of balanced translocations (36). We cannot confirm this point from our experience, but we did note that 31% (14 of 45) of couples achieved a pregnancy when the male partner was the translocation carrier versus 22% (7 of 32) when the female partner was the carrier (P.05). A female translocation carrier might also involve a greater risk of conceiving a chromosomally abnormal embryo and a subsequent lower pregnancy rate. Another questionable point is the validity of PGD in reciprocal translocations. As we previously noted, PGD cy- 68 Feyereisen et al. Five years of PGD activity in a French center Vol. 87, No. 1, January 2007

10 TABLE 6 Outcome of PGD cycles for monogenic autosomal disorders, Recessive Dominant Total Couples Started cycles (SC) No. of oocyte retrievals (OR) Cancellation rate (%) 41% 23% 38% Oocytes retrieved Oocytes retrieved/or Oocytes inseminated Embryos obtained Embryos biopsied 233 (74%) 156 (69%) 389 (72%) Embryos diagnosed 186 (79%) 126 (81%) 312 (80%) Embryos affected 62 (33%) 83 (66%) 145 (46%) No. of embryo transfers (ET) Embryos transferred Embryos transferred/et Clinical pregnancies (CP) Ongoing pregnancies (OP) Twin pregnancies Triplet pregnancies Gestational sacs Implantation rate 15% (14/94) 14% (5/35) 14% (19/129) OP/ET 10/40 (25%) 5/22 (23%) 15/62 (24%) OP/OR 10/50 (20%) 5/37 (13%) 15/87 (17%) OP/SC 10/85 (12%) 5/48 (10%) 15/133 (11%) OP/couple 10/34 (29%) 5/22 (23%) 15/56 (27%) cles performed at our institution for these indications led to a low pregnancy rate per cycle (but not per transfer). Other teams reported a similar experience in PGD for reciprocal translocations, even if some groups obtained satisfying clinical results (37, 38). This poor outcome is easily explained by the well-known gradual loss of the absolute number of oocytes and embryos after each step of the PGD procedure, resulting in a particularly low number of available embryos for ET, as 78% of embryos are diagnosed as unbalanced in reciprocal translocations (39). Our high abnormality rate is comparable with two previous studies (11, 37) that reported an abnormality rate of 79.1% (749 of 946) and 76.9% (546 of 710). Most PGD patients who are carriers of reciprocal translocation have experienced a catastrophic reproductive history, with repetitive miscarriages or serial terminations of pregnancy, justifying their inclusion in our PGD program. Consequently, we can speculate that this high rate observed could be expected in view of the nature of the population treated. Unfortunately, because of the occurrence of multiple miscarriages or infertility in these patients, PGD is often one of the only ways to achieve an ongoing, healthy pregnancy. Consequently, these couples must be informed of the poor outcome and may be directed to other options. Of the seven cases of double-pgd for a double medical indication, one ongoing twin pregnancy was obtained which led to the birth of two healthy baby girls after PGD for cystic fibrosis combined with gender determination (19). Recently, we also reported the world s first ongoing pregnancy after PGD in a case of mitochondrial DNA disorder, using the single cell quantification of the m.8993t G NARP mitochondrial DNA mutation by fluorescent PCR (22, 40). Poor results were obtained in older women despite the mean number of started cycles per couple, cancellation rate, number of embryo biopsies, and number of ETs being similar to the other two age groups. For comparative purposes, between 2000 and 2004, 3040 stimulated IVF cycles were started at our institution for various indications, leading to 2265 oocyte retrievals and 766 clinical pregnancies (pregnancy rate 34%). In the group of patients having exclusive tubal factor infertility, 295 oocyte retrievals were performed (123 oocyte retrievals in women 32 years of age, 119 in women years of age, and 53 in women 38 years of age), leading to 98 clinical pregnancies (pregnancy rate 33%). Among these pregnancies, 56 occurred in women 32 years of age, 35 in women years of age, and Fertility and Sterility 69

11 TABLE 7 Outcome of PGD cycles for X-linked disorders, Sex diagnosis/fish Specific diagnosis/pcr Total Couples Started cycles (SC) No. of oocyte retrievals (OR) Cancellation rate (%) 34% 32% 33% Oocytes retrieved Oocytes retrieved/or Oocytes inseminated Embryos obtained Embryos biopsied 137 (63%) 222 (75%) 359 (70%) Embryos diagnosed 117 (85%) 184 (83%) 301 (84%) Embryos affected 73 (62%) 76 (41%) 149 (49.5%) No. of embryo transfers (ET) Embryos transferred Embryos transferred/et Clinical pregnancies (CP) Ongoing pregnancies (OP) Twin pregnancies Triplet pregnancies Gestational sacs Implantation rate 16% (5/32) 9% (6/70) 11% (11/102) OP/ET 5/15 (33%) 5/31 (16%) 10/46 (22%) OP/OR 5/21 (24%) 5/38 (13%) 10/59 (17%) OP/SC 5/32 (16%) 5/56 (9%) 10/88 (11%) OP/couple 5/13 (38%) 5/24 (21%) 10/37 (27%) seven in women 38 years of age. The pregnancy rates were 46%, 29%, and 13%, respectively, for each age group. The difference in the rates among the three groups is statistically significant. These results remain acceptable for woman older than 37 years in classic IVF, but they are insufficient in PGD. Incidentally, women 38 years represented only 10% of our PGD population, and they were strictly selected for their adequate ovarian reserve by day-3 ultrasonographic and hormonal parameters. Despite the selection procedure to pick the women with a good prognosis from this population, the poor clinical outcome observed in this group has led to us only accepting patients younger than 36 years at the first attempt. This stringent new policy began January As has been noted by others, the frozen embryo replacement cycles yielded poor clinical results (41, 42). Of the 16 cycles started, only seven embryo biopsies and six embryo transfers were possible. Only one birth was obtained, previously reported as the first one after PGD on thawed embryos (43). Moreover, we have had exceptionally unaffected supernumerary embryos after PGD. We tried nine times to freeze and thaw blastocysts after PGD, but none of them resisted the thawing process. Australian groups have reported successful pregnancies with frozen thawed PGD embryos, and this seemed related to the composition of the freezing medium (44). When we have started our PGD activity in 2000, we took into consideration the published experience from Brussels (41) of more than 5 five years of PGD activity with ET performed at day 4. Subsequent studies confirmed those results (38, 27, 29, 37). Furthermore, the guidelines for clinical PGD (9) recommend that extended embryo culture be carried out if the culture procedures are familiar to the IVF laboratory; unfortunately, that was not the case in our IVF center. On the one hand, we recognized that extended culture of whole, unaffected embryos until the blastocyst stage would be a useful tool for selecting the best embryos for transfer and decreasing the number of embryos transferred. On the other hand, the number of available embryos for ET obtained in the majority of cases was so low that designing an extended culture process was precarious. The survival rate of nonbiopsied blastocysts in our clinic during the same period was 67.6% (67 of 99). Fifty four cycles of blastocyst thawing yielded 48 blastocyst transfers (1.4 per transfer), from which six ongoing pregnancies were obtained (five single and one double). This reported pregnancy rate was lower than our usual (45) and may be 70 Feyereisen et al. Five years of PGD activity in a French center Vol. 87, No. 1, January 2007

12 attributed to the small size of the treatment sample. The lack of survival of thawed blastocysts previously biopsied at cleavage stage could be attributed to the biopsy procedure; the disruption of the zona pellucida during the biopsy may affect the capacity of the embryo to resist to the cryopreservation process. One misdiagnosis in our series must be discussed. A PGD cycle was carried out for a couple in which the male was carrying a robertsonian translocation involving the chromosomes 13 and 14 45,XY,der(13;14)(q10;q10). When the product of the spontaneous first trimester miscarriage was analyzed, the fetus was found to have a trisomy 13. The misdiagnosis had occurred in spite of our rules to reduce the levels of uncertainty in PGD. First, as previously described, ICSI was performed in all cases. In addition, we rigorous programmed the PGD activity to avoid performing several PGD analyses on the same day, to decrease the risk of a potential misdiagnosis. When the embryo had at least six available cells, we routinely removed two cells during embryo biopsy; these embryos were transferred only when concordant results had been obtained from both cells. The removal of only one blastomere was accepted in cases of slowly developing embryos that had not reached 6 cells by day 3, to ensure that a sufficient number of cells remained to allow normal development (46). It has been well demonstrated that the reliability of the diagnosis increases when two blastomeres are analyzed (41, 47, 48), but two-cell biopsy remains controversial because it adds a further level of stringency to the PGD procedure yet cannot eliminate totally the risk of misdiagnosis (28). The implantation rate also may be detrimentally affected by the significant reduction of the embryo s cellular mass (29, 49). Each center had its own policy regarding one-cell or two-cell biopsy, according to the accuracy of the diagnostic test used (9) and the ensuing prenatal diagnosis. To reduce the risk of allele dropout and amplification failure, PCR conditions had to be optimized. In the future, the incorporation of linked polymorphic microsatellite markers, the use of multiplex PCR (50, 51), and the improvement of the accuracy of FISH probes are probably better ways to control misdiagnoses and avoid the removal of two cells. Other new methods for diagnosis are being developed at a fast rate; at the single cell level, real-time PCR, microarrays, and comparative genomic hybridization (CGH) are promising (52). Patients were informed of the low but existent risk of misdiagnosis before they enrolled in our PGD program; they were told that PGD did not exclude prenatal diagnosis. When a pregnancy occurred, the prenatal diagnosis was reevaluated for the accuracy of PGD in each situation (the test performed, the number of blastomeres analyzed, and the results) and for the genetic pathology (ultrasonographic follow-up reliable or not). In all cases, we attempted to offer amniocentesis in the third trimester to reduce pregnancy loss because we believe that third trimester amniocentesis may be an option to avoid fetal loss in selected cases (53), with a low rate of obstetrical and neonatal complications when the expected risk is low. This alternative strategy may provide a form of late reassurance for women in countries such as France where legislation permits late termination of pregnancies for severe fetal anomalies. Only 10 couples have had a prenatal diagnosis, given that the pregnancy was particularly precious after the complex PGD treatment and that the course of pregnancy was reassuring. Among these 10 amniocenteses, five were performed because of ultrasonographic abnormalities, and two for advanced maternal age; only three couples opted for a systematic prenatal diagnosis to confirm the PGD procedure. In all cases, the fetuses were unaffected. All the amniocenteses were performed at 32 weeks of gestation, except one that was performed earlier (17 weeks) at the couple s request. The policy of our team to avoid prenatal diagnosis may be controversial, as several groups are convinced it is always necessary to control PGD results (29, 41). We preferred an obstetric follow-up performed in our center that was adjusted to each situation. In cases of X-linked disorders, the determination of fetal sex in maternal blood was offered to couples during the first trimester of gestation (54), but only three couples opted for it. A confirmation was always carried out on cord blood at childbirth. From the beginning of our study, we did follow-up evaluations on the children born after PGD. This prospective, comparative study used children born after ICSI as controls. As of this point in time, the course and the outcome of the pregnancies have been similar in both groups. Follow-up evaluations of the children are scheduled until they reach their 7th birthday, and they include a clinical examination at 1 year, a questionnaire every year until the 6th year, and a second clinical examination at the start of school (7 years old). All these examinations are performed by an experimented pediatrician. At this time, two major malformations have been noted among the PGD group: one case of spina bifida and one case of Fallot tetralogy. In view of reported data (11) and our preliminary results, the malformation rate at birth seems to be in the expected range, similar to controls. However, follow-up evaluations of the PGD children must be organized by each center to detect other pathologic, psychological, or developmental abnormalities. Between 2000 and 2004, 47 of 171 couples achieved 47 ongoing pregnancies after PGD at our institution, with deliveries of 58 unaffected babies. Although PGD is an alternative for couples who want to avoid prenatal diagnosis (52), it may not be an appropriate option for all patients. Couples who are referred for a PGD cycle should be counseled about the success rate, which depends largely on the genetic condition and on the number of cumulus oocyte complexes retrieved (55). That is why the initial multidisciplinary consultation screening is very important for assessing the chances of a successful response to controlled ovarian hyperstimulation and counseling couples appropriately. Accu- Fertility and Sterility 71

13 rate information and communication must be provided to patients, not false optimism (56). CONCLUSION The present report is the first complete study from a single French center. Encouraging results with regard to pregnancy rates demonstrate that PGD is a valid, realistic alternative to prenatal diagnosis. The catalog of disorders that can be diagnosed in the preimplantation embryo is increasing in parallel with the growing demand. Nevertheless, PGD is not an easy solution for patients who are at risk of transmitting serious genetic diseases; this is even more true if they are fertile. Thus, couples referred for PGD must be selected and counseled appropriately about the complexity of the IVF treatment, their genetic condition, and the take-home baby rate. When they have been selected appropriately, patients undergoing PGD have an acceptable pregnancy rate. Advances in techniques will allow us to increase the number of diagnoses available for couples and to improve the accuracy of the genetic tests. Acknowledgments: The authors thank all personnel for their help and assistance. The authors are indebted to many colleagues of the clinical, scientific, laboratory, nursing, secretarial and technical staff of the Antoine Beclère Hospital Centre for Reproductive Medicine and of the Necker Hospital Department of Medical Genetics, without whom the PGD program could not function. The authors thank Nadia Prisant, M.D., for his helpful suggestions made while editing and translating the English text. REFERENCES 1. Handyside AH, Kontogianni EH, Hardy K, Winston RM. Pregnancies from biopsied human preimplantation embryos sexed by Y-specific DNA amplification. Nature 1990;344: Conn CM, Harper JC, Winston RM, Delhanty JD. Infertile couples with Robertsonian translocations: preimplantation genetic analysis of embryos reveals chaotic cleavage divisions. Hum Genet 1998;102: Pierce KE, Fitzgerald LM, Seibel MM, Zilberstein M. Preimplantation genetic diagnosis of chromosome balance in embryos from a patient with a balanced reciprocal translocation. Mol Hum Reprod 1998;4: Liu J, Lissens W, Silber SJ, Devroey P, Liebaers I, Van Steirteghem A. Birth after preimplantation diagnosis of the cystic fibrosis delta F508 mutation by polymerase chain reaction in human embryos resulting from intracytoplasmic sperm injection with epididymal sperm. JAMA 1994;272: Ao A, Ray P, Harper J, Lesko J, Paraschos T, Atkinson G, et al. Clinical experience with preimplantation genetic diagnosis of cystic fibrosis (delta F508). Prenat Diagn 1996;16: Blaszczyk A, Tang YX, Dietz HC, Adler A, Berkeley AS, Krey LC, Grifo JA. Preimplantation genetic diagnosis of human embryos for Marfan s syndrome. J Assist Reprod Genet 1998;15: Campana M, Serra A, Neri G. Role of chromosome aberrations in recurrent abortion: a study of 269 balanced translocations. Am J Med Genet 1986;24: Katz MG, Fitzgerald L, Bankier A, Savulescu J, Cram DS. Issues and concerns of couples presenting for preimplantation genetic diagnosis (PGD). Prenat Diagn 2002;22: Thornhill AR, dedie-smulders CE, Geraedts JP, Harper JC, Harton GL, Lavery SA, et al. ESHRE PGD Consortium Best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Hum Reprod 2002;20: Wilton L. Preimplantation genetic diagnosis for aneuploidy screening in early human embryos: a review. Prenat Diagn 2002;22: ESHRE PGD Consortium Steering Committee. ESHRE Preimplantation Genetic Diagnosis (PGD) Consortium: data collection IV: May December Hum Reprod 2005;20: Geraedts JP, Harper J, Braude P, Sermon K, Veiga A, Gianaroli L, et al. Preimplantation genetic diagnosis (PGD), a collaborative activity of clinical genetic departments and IVF centres. Prenat Diagn 2001;21: Flis-Treves M, Achour-Frydman N, Kerbrat V, Munnich A, Vekemans M, Frydman R. Preimplantation genetic diagnosis and its psychological effects [in French]. J Gynecol Obstet Biol Reprod (Paris) 2003;32: Liebaers I, Sermon K, Staessen C, Joris H, Lissens W, Van Assche E, et al. Clinical experience with preimplantation genetic diagnosis and intracytoplasmic sperm injection. Hum Reprod 1998;13(Suppl 1): De Vos A, Van Steirteghem A. Aspects of biopsy procedures prior to preimplantation genetic diagnosis. Prenat Diagn 2001;21: Ray PF, Ao A, Taylor DM, Winston RM, Handyside AH. Assessment of the reliability of single blastomere analysis for preimplantation diagnosis of the delta F508 deletion causing cystic fibrosis in clinical practice. Prenat Diagn 1998;18: Ray PF, Gigarel N, Bonnefont JP, Attie T, Hamamah S, Frydman N, et al. First specific preimplantation genetic diagnosis for ornithine transcarbamylase deficiency. Prenat Diagn 2000;20: Ray PF, Vekemans M, Munnich A. Single cell multiplex PCR amplification of five dystrophin gene exons combined with gender determination. Mol Hum Reprod 2001;7: Ray PF, Frydman N, Attie T, Hamamah S, Kerbrat V, Tachdjian G, et al. Birth of healthy female twins after preimplantation genetic diagnosis of cystic fibrosis combined with gender determination. Mol Hum Reprod 2002;8: Burlet P, Frydman N, Gigarel N, Bonnefont JP, Kerbrat V, Tachdjian G, et al. Improved single-cell protocol for preimplantation genetic diagnosis of spinal muscular atrophy. Fertil Steril 2005;84: Gigarel N, Frydman N, Burlet P, Kerbrat V, Steffann J, Frydman R, et al. Single cell co-amplification of polymorphic markers for the indirect preimplantation genetic diagnosis of hemophilia A, X-linked adrenoleukodystrophy, X-linked hydrocephalus and incontinentia pigmenti loci on Xq28. Hum Genet 2004;114: Gigarel N, Ray PF, Burlet P, Frydman N, Royer G, Lebon S, et al. Single cell quantification of the 8993T G NARP mitochondrial DNA mutation by fluorescent PCR. Mol Genet Metab 2005;84: Harper JC, Coonen E, Ramaekers FC, Delhanty JD, Handyside AH, Winston RM, Hopman AH. Identification of the sex of human preimplantation embryos in two hours using an improved spreading method and fluorescent in-situ hybridization (FISH) using directly labelled probes. Hum Reprod 1994;9: Staessen C, Van Assche E, Joris H, Bonduelle M, Vandervorst M, Liebaers I, Van Steirteghem A. Clinical experience of sex determination by fluorescent in-situ hybridization for preimplantation genetic diagnosis. Mol Hum Reprod 1999;5: Coonen E, Dumoulin JC, Dreesen JC, Bras M, Evers JL, Geraedts JP. Clinical application of FISH for sex determination of embryos in preimplantation diagnosis of X-linked diseases. J Assist Reprod Genet 1996;13: Scriven PN, Handyside AH, Ogilvie CM. Chromosome translocations: segregation modes and strategies for preimplantation genetic diagnosis. Prenat Diagn 1998;18: Scriven PN, O Mahony F, Bickerstaff H, Yeong CT, Braude P, Mackie Ogilvie C. Clinical pregnancy following blastomere biopsy and PGD for a reciprocal translocation carrier: analysis of meiotic outcomes and embryo quality in two IVF cycles. Prenat Diagn 2000;20: Frydman N, Fanchin R, Le Du A, Bourrier MC, Tachdjian G, Frydman R. Improvement of IVF results and optimisation of quality control by using intermittent activity. Reprod Biomed Online 2004;9: Pickering S, Polidoropoulos N, Caller J, Scriven P, Ogilvie CM, Braude P. Preimplantation Genetic Diagnosis Study Group. Strategies and 72 Feyereisen et al. Five years of PGD activity in a French center Vol. 87, No. 1, January 2007