High level of chromosome aneuploidy in the spermatozoa of man with severe oligoasthenoteratozoospermia (OAT): case report

DESIGN: Retrospective analysis. MATERIALS AND METHODS: Three EZP with several blastomeres remaining inside after hatching were selected. Each of EZP (#1, #2 and #3) after visual confirmation of the absence of cumulus cells on the outside, was gently rinsed in a hypotonic solution (1% sodium citrate in 0.2 mg/ mL HSA) and transferred into 3–5 ul drop of solution of 1% Tween 20 in 0.01 N HCl onto a glass microscope slide, which was then allowed to evaporate completely. Thereafter, several drops of a 1:3 mixture of acetic acid/methanol were placed on the slide over the specimen. Fixed nuclei were immediately marked and recorded. Slides were processed for hybridization without any additional treatment. Hybridization was accomplished using a standard protocol with probes for chromosomes X, Y, 13, 18 and 21. RESULTS: Three, two and three nuclei were obtained from blastomeres left inside after the EZP #1, EZP #2 and EZP #3 fixations respectively. FISH results were available for all recorded nuclei. The numerical distribution of fluorescent signals for chromosomes X, Y, 13, 18 and 21 of each nucleus was - (XX, 0 signals for13, 2 for18, 1 for 21), (XX, 0 for13, 2 for18, 2 for 21), (XX, 2 for 13, 3 for 18, 2 for 21) for EZP #1; (XY, 2 for13, 1 for 18, 2 for 21), (XY, 2 for 13, 0 for 18, 2 for 21) for EZP #2 and lastly (XX, 2 signals for13, 2 for18, 2 for 21), (XX, 2 for13, 2 for18, 2 for 21), (XX, inconclusive for 13, 2 for 18, 2 for 21) for EZP #3. CONCLUSIONS: Results for tested chromosomes were discordant for all nuclei of EZP #1 and EZP #2 showing aneuploid mosaic status and normal status of EZP #3 with the exception of the one nucleus with inconclusive result for chromosome 13. Available literature about chromosomal mosaicism among blastomeres integrated into human blastocysts leads us to believe that aneuploidy may not be the reason for the occasional exclusion of several blastomeres form blastocyst formation. If excluded blastomeres represent genetic status of related embryos then it can be used as additional or sometimes the only source of nuclei for testing. We demonstrated a new source and method for obtaining nuclei for the purpose of PGD from blastomeres remaining inside zonae pellucidae after hatching however, further investigation with larger number of observations is needed to estimate true diagnostic value of this otherwise lost material. Supported by: None. P-383 CORRELATION BETWEEN PREIMPLANTATION GENETIC DIAGNOSIS RESULTS AND SOLUBLE HLA G CONCENTRATIONS IN EMBRYO CULTURE MEDIA. C. B. Coulam, R. G. Roussev, S. Lerner, Y. Verlinsky, I. Tur-Kapsp. CARI Reproductive Institute, Chicago, IL; Pregnancy Success Center, Rinehart Center for Reproductive Medicine, Evanston, IL; Reproductive Genetics Institute, Chicago, IL. OBJECTIVE: The transfer of a single embryo is the goal of assisted reproductive technology programs to minimize the risk of multiple gestations. The difficulty has been to decide which embryo to transfer. Measurement of soluble HLA-G (sHLA-G) concentrations in culture media has been reported to predict implantability of embryos. The question arose whether HLA-G culture media concentrations were associated with chromosomal complement of the embryo. To answer this question, culture media from embryos undergoing preimplantation genetic diagnosis (PGD) were analyzed for sHLA-G concentration. The results of the sHLA-G concentrations were compared with the results of the PGD analysis. DESIGN: Prospective comparative study. MATERIALS AND METHODS: Culture media from 100 embryos obtained 2 days after fertilization by intracytoplasmic sperm injection were analyzed for sHLA G concentrations using an ELISA kit (Ex Bio/BioVendor, CARI, Chicago, IL). A sHLA G concentration of R2 U/ml was considered a positive result. On the morning of day 3 of development, embryos were biopsied in calcium and magnesium free Global media. Fluorescent in situ hybridization with directly labeled probes (Abbott/Vysis; Des Plaines, IL) for chromosomes 13, 14,15,16,17, 18, 21, 22, X and Y was performed in two cycles. RESULTS: Soluble HLA G concentration R2 U/ml was found in 45 (45%) and <2 UI/ml in 55 (55%) of embryo culture media. PDG results were interpreted as normal chromosomal complement for those chromosomes studied in 59 (59%) and abnormal in 41 (41%) of embryos. Of 41 embryos reported to have a normal karyotype, 16 (40%) had sHLA G concentrations R2 U/ml and 25 (60%) had sHLA G concentrations <2 U/ ml. Among the 45 embryos that had culture media concentrations of sHLA G R2 U/ml, 24 (54%) displayed normal PGD results and 21 (46%) had abnormal PGD results. sHLA G concentrations were <2 U/ml in culture media of 55 embryos among which 33 (60%) had normal and 22 (40%) had abnormal PGD results.

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Abstracts

CONCLUSIONS: No correlation between concentrations of sHLA-G in embryo culture media and PGD results were observed. Supported by: Supported in part by a research grant from Ferring Pharmaceutical Company. P-384 DEVELOPING A SINGLE CELL OLIGONUCLEOTIDE ARRAYCGH TEST FOR ANEUPLOID SCREENING. J. B. Spencer, W. Shang, W. Sun, D. R. Session, D. H. Ledbetter, C. Lese Martin. Division of Reproductive Endocrinology & Infertility, Dept of Gynecology & Obstetrics, Emory University School of Medicine, Atlanta, GA; Department of Human Genetics, Emory University School of Medicine, Atlanta, GA. OBJECTIVE: The most commonly used technique for preimplantation aneuploid screening of embryos in IVF is fluorescence in situ hybridization (FISH). FISH analysis typically only includes 5–10 chromosome specific probes, which excludes the remaining 13–18 chromosomes from analysis. Comparative genomic hybridization (CGH) can be performed using an oligonucleotide microarray which allows the detection of copy number imbalances across the genome from genomic DNA. The purpose of this study is to develop an oligonucleotide array-CGH method that could potentially be used for a more comprehensive aneuploid screen in preimplantation genetic diagnosis (PGD). DESIGN: Experimental study. MATERIALS AND METHODS: With IRB approval, single lymphoblasts from internally obtained lymphoblastoid cell lines (47, XY, þ21 with 46, XX as an opposite sex control) were isolated with a micromanipulator and placed in individual PCR tubes in 1ul of TE. Whole genome amplification (WGA) was then performed on the single cells with a commercially available kit (GenomePlexÒ, Sigma-Aldrich). After amplification, DNA was digested, labeled and hybridized according to a modified manufacturer’s protocol, testing several modifications (4  44B Human Genome CGH Microarray Kit, Agilent Technologies, Santa Clara, CA). After 24 hour hybridization, the slides were washed and scanned using the GenePix Autoloader 4200AL (Molecular Devices, Sunnyvale, CA). BlueFuse software was used (BlueGnome Limited, Cambridge, UK) to detect copy number alterations using set thresholds for the channel ratios based on 4 SDs from the median of the autosomes. RESULTS: WGA from a single cell was successful 90% of the time (N ¼ 40, with DNA product sizes between 200–1000 bp), giving a final yield of 1.4 mg to 9.3 mg (the majority of the samples were 5 mg). Of the arrays that had successful hybridization (N ¼ 9), all detected Trisomy 21. Two of these erroneously called other copy number abnormalities. Three arrays detected the loss of X and 3 others detected the gain of Y, but none of the 9 arrays detected both. All arrays to date show high background. CONCLUSIONS: We have shown that oligo array-CGH can be performed on a single cell. 100% of the arrays that successfully hybridized detected the expected Trisomy 21. We are currently optimizing this protocol to reduce background and improve detection of other aneuploidies. Supported by: Laboratory development fund. P-385 HIGH LEVEL OF CHROMOSOME ANEUPLOIDY IN THE SPERMATOZOA OF MAN WITH SEVERE OLIGOASTHENOTERATOZOOSPERMIA (OAT): CASE REPORT. S. A. S. Fonseca, C. C. Rocha, V. S. Pereira, J. Cardoso, R. Abdelmassih, S. Abdelmassih. ART Lab, Clinic and Research Center on Human Reproduction Roger Abdelmassih, Sao Paulo, Brazil; Stem Cell Lab, Clinic and Research Center on Human Reproduction Roger Abdelmassih, Sao Paulo, Brazil. OBJECTIVE: ICSI is used to overcome problems of severe male factor, improving the chances of achieving pregnancy. Even though, attempts have been made to select good quality spermatozoa based on morphology, it is not possible to avoid the transmission of chromosomal abnormalities to the conceptus. It has been demonstrated that some patients with severe teratozoospermia present high levels of aneuploidy in spermatozoa, which can result in aneuploid embryos. This study aims to evaluate the rate of aneuploid spermatozoa in a patient with severe OAT during ICSI treatment. DESIGN: Prospective study with a couple with seven years of infertility submitted to ICSI treatment. MATERIALS AND METHODS: Semen samples were analyzed to evaluate concentration, motility, and morphology according to the Worth Health Organization (WHO) and strict criteria (Kruger) parameters. Sperm DNA fragmentation was measured by flow cytometry using acridine-orange

Vol. 88, Suppl 1, September 2007

staining. FISH analyses, using specific probes for sex chromosomes and chromosomes 13, 16, 18, 21 and 22, were performed to identify any chromosomal aneuploidy. At least 500 spermatozoa were analysed for each probe. RESULTS: Semen samples (sperm concentration <5106/ml, spermatozoa with total progressive motility 5–10%) were regarded as oligoasthenozoospermia. The morphological analysis showed acrosomal deficiency (hypoplasia or aplasia) in 100% of the spermatozoa and the fragmentation test presented 53% of DNA fragmentation. After four cycles of ICSI, the fertilization rate observed was 50% and almost all embryos had poor quality. No pregnancy was achieved in any cycle. FISH analyses in spermatozoa showed a significantly higher aneuploidy of sexual chromosomes (8%) and autosomes (12%), when compared to normal ones (<1%). The aneuploidy rates observed in this work are raiser than similar studies (6, 7%-Strassburger et al., 2007). The most common types of aneuploidy observed in this sample were diploidy (46) and disomy XY (24, XY). CONCLUSIONS: Although a low fertilization rate and poor embryo quality were expected as a result of acrosomal deficiency, we suggest that this could be related to the high abnormal chromosome set observed within the spermatozoa. Our data, as well as other publications, support an existing correlation between poor semen parameters, including severe OAT, with an increased aneuploidy rate of chromosomes analyzed. In conclusion, we suggest PGD analyses in embryos for severe teratozoospermia patients to avoid abnormal embryo implantation and enhance the implantation rate. Supported by: None. P-386 DOES IVF WITH PGD AND BLASTOCYST TRANSFER AFFECT OFFSPRING’S SEX-RATIO? I. Tur-Kaspa, N. Katz, I. Kuznyetsova, Y. Verlinsky. Institute for Human Reproduction (IHR), Chicago, IL; Clinical IVF-PGD Program, Reproductive Genetics Institute (RGI), Chicago, IL. OBJECTIVE: Almost 30 years since the first IVF newborn, conception through ART should still be followed for unknown consequences. A shifting of male to female ratio following IVF and blastocyst transfer has been recently suggested. We used PGD results of gender to investigate whether the sex ratio was altered and if gender affects embryo development in vitro, implantation, and eventually delivery rate. DESIGN: Retrospective data analysis. MATERIALS AND METHODS: 143 consecutive cycles of IVF with PGD for aneuploidy were analyzed. 48.2% (466/966) of the embryos were normal for 5–9 chromosomes and the gender was known. In vitro embryo development until day 5–6, implantation rate, miscarriage rate, and delivery rate by gender were evaluated. RESULTS: 46.8% of the embryos tested by PGD were diagnosed as males (n ¼ 218) and 53.2% (n ¼ 248) as females (P¼NS). There were no significant differences in their potential development to early or advanced blastocyst stages. 111 male blastocysts and 129 female blastocysts were transferred. Implantation rates were comparable, 32.4% and 31%, respectively, and 78% of the implanted embryos in each group were delivered. CONCLUSIONS: Male and female embryos generated by IVF-PGD treatment have similar developmental, implantation and delivery potential. This is the first study to use PGD results to follow in vitro development and implantation of embryos by gender. Our data suggest that ART with extended blastocyst culture is not associated with sex-ratio imbalance. Supported by: None. P-387 EMBRYO CULTURE TO THE BLASTOCYST STAGE DOES NOT SELECT FOR GENDER. J. L. Eaton, M. R. Hacker, D. Harris, K. Thornton, A. Penzias. Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA; Boston IVF, Waltham, MA. OBJECTIVE: Reports of increased developmental rates in male embryos have resulted in concerns that day 5 blastocyst transfer may lead to gender selection. We compared blastocyst development and prevalence of euploidy in XX vs. XY embryos. DESIGN: Retrospective analysis. MATERIALS AND METHODS: We identified all women who underwent in vitro fertilization (IVF) with their first preimplantation genetic diagnosis (PGD) cycle for recurrent miscarriage, recurrent IVF failure, advanced maternal age, or other indications between January 1 and December 31, 2006. Fluorescence in situ hybridization was utilized to analyze 9 or 12 chromosomes. Embryos with abnormal sex chromosomes were excluded. P values were adjusted to account for lack of independence among embryos from the same woman.

FERTILITY & STERILITYÒ

RESULTS: A total of 481 embryos from 99 cycles in 99 patients with complete results for the 9-chromosome panel were analyzed. 235 (49%) were XX and 246 (51%) were XY. The prevalence of euploidy was similar between the XX and XY embryos (30% vs. 32%, P¼0.73). XX and XY embryos were equally likely to develop to the blastocyst stage by day 5 (59% vs. 60%, P¼0.84), and those that became blastocysts had similar proportions of euploidy (41% vs. 37%, P¼0.43). Results were similar in a subset of embryos in which all 12 chromosomes were analyzed. Abnormal numbers of individual autosomes were no more likely to occur in XX than XY embryos. CONCLUSIONS: XX and XYembryos are equally likely to develop to the blastocyst stage by day 5. Moreover, XX and XY blastocysts are equally likely to be euploid. There is no predilection for aneuploidy of any single autosome to occur with greater frequency in XX vs. XY embryos. Our data suggest that day 5 blastocyst culture does not result in gender selection and that euploidy and aneuploidy are not gender dependent. Supported by: None. P-388 BLASTOCYST TROPHECTODERM BIOPSY: A CASE STUDY. A. R. Anderson, K. J. Graff, J. L. Crain. Embryology, REACH, Charlotte, NC. OBJECTIVE: The purpose of this case study is to discuss how a failed analysis for an inversion was rescued with a trophectoderm biopsy on day 6 hatching blastocysts. DESIGN: A case study where the day 3 analysis for an inversion on chromosome 8 (inv(8)(p21q24.1)) failed to yield appropriate results. Thus salvaging the cycle by biopsying the resulting blastocysts. MATERIALS AND METHODS: The patient in this study underwent an antagonist controlled ovarian hyperstimulation where 14 oocytes were retrieved, 13 underwent intracytoplasmic sperm injection, and 7 normally fertilized zygotes were cultured to day 3. One cell from all 7 embryos were biopsied and fixed to a superfrost glass slide and sent to an off site reference laboratory for analysis of an inversion on chromosome 8 between p21 and q24.1. Only 2 embryos yielded result where both were abnormal and could not be transferred. Three of the 5 embryos that could not be evaluated from the day 3 biopsy developed to the blastocyst stage. All 3 embryos underwent a biopsy of the trophectoderm. The embryos were biopsied in Sage HEPES buffered medium with a Hamilton-Thorne laser removal of 3–5 cells into a Cook biopsy pipette. The cells retrieved from the trophectoderm were fixed onto a superfrost slide while the remaining blastocysts were immediately frozen in 10% glycerol/0.2 M sucrose. The patient in this study underwent a hormone replacement therapy cycle. On day 12 of estrogen replacement therapy progesterone in oil was administered. On the 6th day of progesterone one normal embryo was thawed and transferred. RESULTS: From the embryos biopsied on day 3 only 2 embryos yielded a result and no normal embryos were available for ET. From the 3 blastocysts biopsied on day 6, 2 embryos were normal and 1 embryo was abnormal. Upon thaw for the FET an elective single embryo was thawed as well as the abnormal embryo. Both embryos survived. A single embryo was transferred leaving one normal embryo in storage for a later cycle. Pregnancy is pending. CONCLUSIONS: The ability to biopsy and freeze a blastocyst with efficiency opens up several opportunities for alternative methods for evaluation of preimplantation embryos. The possibilities for microarray analysis, Comparative Genome Hybridization (CGH), and Fluorescent In Situ Hybridization (FISH) of several cells from the trophectoderm are endless. Multiple cells would reduce errors in FISH. Additionally, embryos from one biopsy could for aneuploidy as well as single gene abnormalities in the case of CGH. Tested. Supported by: None. P-389 MULTIPLE DISPLACEMENT AMPLIFICATION (MDA) A NEW APPROACH FOR PREIMPLANTATION GENETIC DIAGNOSIS (PGD): OUR EXPERIENCE. J. Llacer, B. Lledo, R. Morales, A. Rodriguez, J. Ten, R. Bernabeu. Reproductive Medicine, Instituto Bernabeu, Alicante, Spain; Molecular Biology, Instituto Bernabeu, Alicante, Spain. OBJECTIVE: For single-gene disorders, the development and validation of highly sensitive amplification strategies are required, often using nestedPCR, whole-genome amplification (WGA), or fluorescent PCR methods. There is a need for a technique that would be able to amplify the single-cell DNA with a high fidelity that suits the diagnosis of any known single-gene

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