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Detection of alternatively spliced transcripts of human leukocyte antigen-g in single human preimplantation blastocysts using nested RT-PCR
going in vitro fertilization. Paulo M. Perin, Mariangela Maluf, Carlos E. Czeresnia, Patricia D. Sousa. DIASON - Sonographic Diagnosis / Div of Reproductive Medicine, Sa˜ o Paulo, Brazil. Objective: To compare oocyte/embryo quality and treatment outcome in patients undergoing IVF/ICSI using recombinant human luteinizing hormone (rhLH) or human menopausal gonadotropin (hMG) as a supplement to recombinant human follicle-stimulating hormone (rhFSH) during controlled ovarian hyperstimulation. Design: Forty-nine women undergoing their first IVF cycle were prospectively randomized into two age-matched patient groups. Materials and Methods: All patients underwent standard down-regulation with GnRH agonist. After pituitary desensitization, patients were randomized to receive a fixed dose of either rhFSH (225 IU/d) plus hMG (75 IU/d) (group A, n ⫽ 28) or rhFSH (225 IU/d) plus rhLH (75 IU/d) (group B, n ⫽ 21). ICSI was performed in all cycles due to male factor infertility. Oocyte (Van Blerkom’s classification), zygote (Tesarik & Greco’s classification) and embryo quality (Veeck’s classification) along with the embryo fragmentation score (1 ⫽ ⱕ10%; 2 ⫽ 11-30%; 3 ⫽ ⬎30%) were determined by the same embryologist. Results: Patient demographics (age, infertility duration, body mass index, gonadotropin ampoules, duration of stimulation) were similar in both groups. Mean E2 and P levels and endometrial thickness on the day of hCG administration were similar in both groups. The mean number of M2 oocytes recovered showed no difference between groups. However, the mean number of normal/abnormal M2 oocytes was 4.5⫾3.10/ 2.8⫾3.13 for group A and 2.4⫾1.47/4.0⫾3.70 for group B, a significant difference between groups (p ⫽ 0.006;p ⫽ 0.01; respectively). The incidence of major cytoplasmic abnormalities (dark/granular, clustered, SER) was similar in both groups. Nonetheless, the incidence of minor cytoplasmic abnormalities (vesiculated, necrotic, polarized, vacuolated) was significantly higher (p ⫽ 0.000) in group B (28.1%) than in group A (9.4%). The fertilization rate was similar in both groups (79.0%,86.0%; groups A and B respectively), yet the number of zygotes judged normal (pattern 0) was significantly higher (p ⫽ 0.02) in group A (2.4⫾2.40) when compared to group B (1.2⫾1.76). The embryo development rate (number of blastomeres/embryo), morphology and fragmentation scores were similar in the groups for both the day two and day three evaluations. The mean number of embryos, embryo quality and fragmentation scores before day three transfer were similar in both groups. Overall clinical pregnancy/implantation rates were 46.4% (13/ 28)/25.3% (22/87) in group A and 38.1% (8/21)/17.1% (13/76) in group B. The trend toward better pregnancy outcomes among patients in group A did not reach statistical significance. The miscarriage rate was similar in both groups (7.1%, 4.8%; groups A and B respectively). Conclusion: Our study suggests that the addition of recombinant luteinizing hormone instead of human menopausal gonadotropin to recombinant FSH throughout ovulation induction in down-regulated women undergoing IVF not only does not improve ovarian response, it has a negative impact on oocyte/zygote quality. The result is a trend toward poorer treatment outcome.
Wednesday, October 15, 2003 11:15 A.M. O-201 Detection of alternatively spliced transcripts of human leukocyte antigen-g in single human preimplantation blastocysts using nested RTPCR. Y. Q. Yao, D. H. Barlow, I. L. Sargent. Univ of Oxford, Oxford, United Kingdom. Objective: Human leukocyte antigen G (HLA-G) is a virtually nonpolymorphic HLA class I gene expressed by extravillous cytotrophoblast, which is believed to play a key role in maternal immune tolerance of the fetus. HLA-G mRNA can be alternatively spliced into 6 transcripts, which encode 4 membrane isoforms (G1, G2, G3, G4) and 2 soluble isoforms (G5 and G6). HLA-G is expressed by some human pre-implantation embryos and the levels of soluble HLA-G secreted may relate to implantation success (1).
FERTILITY & STERILITY威
However, which of the mRNA isoforms are expressed and in what proportion of embryos they are found is unknown. Design: Laboratory study to detect alternatively spliced mRNA isoforms of HLA-G in human blastocysts using nested RT-PCR. Materials and Methods: Human embryos were donated with informed consent by couples attending the Oxford Fertility Unit and the study was approved by the Central Oxford Research Ethics Committee and the Human Fertilisation & Embryology Authority. On Day 6 of culture, single blastocysts were transferred to lysis buffer and stored at -70°C prior to mRNA isolation. 8 pairs of specific primers were designed; one for first round RT-PCR to amplify all HLA-G mRNA isoforms, 6 for second round PCR to distinguish G1, G2, G3, G4, G5, G6 mRNA isoforms and 1 for all HLA-G mRNAs. The JEG-3 cell line was used as a positive control and amplicons of the nested PCR were sequenced for confirmation. Results: Twenty five day 6 expanded blastocysts (grade 4) were examined. All were found to express HLA-G, but the isoforms were differentially expressed. G1 was detected in 20/25 (80%) blastocysts, G2 in 3/25 (12.0%), G3 in 25/25 (100%) and G4 in 24/25 (96.0%). The expression of soluble HLA-G mRNAs was relatively lower than membrane forms with G5 mRNA detected in 6/25 (24.0%) and G6 9/25 (36.0%) blastocysts. Conclusion: Our results demonstrate that human blastocysts express the alternatively spliced transcripts of HLA-G, but the patterns of expression varies between embryos. This differential expression may be important to the success of early human embryo development and implantation. Supported by: the Oxford Fertility Unit. (1) Fuzzi et al. (2002) Eur J Immunol. 32:311-315.
Wednesday, October 15, 2003 11:30 A.M. O-202 Blastomere transplantation in early human embryos. Norbert Gleicher, YaXu Tang. The Centers for Human Reproduction (CHR) New York & Illinois & The Fdn for Reproductive Medicine, Chicago, IL. Objective: We previously reported the successful transplantation and integration of donor blastomeres into recipient embryos (ESHRE, Madrid, Spain, July 1, 2003). The objective of this study was to determine whether the degree of integration of the donor transplant into the recipient embryo changed, based on the number of blastomeres transplanted. Design: This study was approved by the CHR’s IRB, after external legal review, and, involved only embryos which, by written consent, were explicitly donated “to research”. In a chimera transplantation model, male (xy) blastomeres were transplanted into female (xx) recipient embryos and the distribution of y-chromosome markers was then followed in recipient embryos into the blastocyst stage of development. Materials and Methods: 16 xy-embryos were donors and 21 xx-embryos were recipients, which received one to three blastomere transplants. All embryos in this study had been cryopreserved and were thawed prior to gender determination. Embryo gender was determined utilizing routine preimplantation genetic diagnostic (PGD) techniques and standard fluorescent in situ hybridization (FISH) methodology. The transplantation of blastomeres into recipient embryos utilized standard PGD instrumentation and the initial biopsy site for the insertion of the cells. Distribution of y-chromosome at the blastocyst stage in recipient embyos was also assessed using standard FISH technology. Results: 4/19 (19%) of recipient embryos failed to reach blastocyst stage and only 12/19 (57%) reached what was considered a morphologically normal blastocyst stage. As the table demonstrates, normal blastocyst stage recipient embryos uniformly demonstrated an even and well distributed y-staining pattern throughout inner cell mass and trophoectoderm. Moreover, staining intensity appeared also to correlate with the number of blastomeres transplanted.
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Wednesday, October 15, 2003 11:15 A.M. O-201 Detection of alternatively spliced transcripts of human leukocyte antigen-g in single human preimplantation blastocysts using nested RTPCR. Y. Q. Yao, D. H. Barlow, I. L. Sargent. Univ of Oxford, Oxford, United Kingdom. Objective: Human leukocyte antigen G (HLA-G) is a virtually nonpolymorphic HLA class I gene expressed by extravillous cytotrophoblast, which is believed to play a key role in maternal immune tolerance of the fetus. HLA-G mRNA can be alternatively spliced into 6 transcripts, which encode 4 membrane isoforms (G1, G2, G3, G4) and 2 soluble isoforms (G5 and G6). HLA-G is expressed by some human pre-implantation embryos and the levels of soluble HLA-G secreted may relate to implantation success (1).
FERTILITY & STERILITY威
However, which of the mRNA isoforms are expressed and in what proportion of embryos they are found is unknown. Design: Laboratory study to detect alternatively spliced mRNA isoforms of HLA-G in human blastocysts using nested RT-PCR. Materials and Methods: Human embryos were donated with informed consent by couples attending the Oxford Fertility Unit and the study was approved by the Central Oxford Research Ethics Committee and the Human Fertilisation & Embryology Authority. On Day 6 of culture, single blastocysts were transferred to lysis buffer and stored at -70°C prior to mRNA isolation. 8 pairs of specific primers were designed; one for first round RT-PCR to amplify all HLA-G mRNA isoforms, 6 for second round PCR to distinguish G1, G2, G3, G4, G5, G6 mRNA isoforms and 1 for all HLA-G mRNAs. The JEG-3 cell line was used as a positive control and amplicons of the nested PCR were sequenced for confirmation. Results: Twenty five day 6 expanded blastocysts (grade 4) were examined. All were found to express HLA-G, but the isoforms were differentially expressed. G1 was detected in 20/25 (80%) blastocysts, G2 in 3/25 (12.0%), G3 in 25/25 (100%) and G4 in 24/25 (96.0%). The expression of soluble HLA-G mRNAs was relatively lower than membrane forms with G5 mRNA detected in 6/25 (24.0%) and G6 9/25 (36.0%) blastocysts. Conclusion: Our results demonstrate that human blastocysts express the alternatively spliced transcripts of HLA-G, but the patterns of expression varies between embryos. This differential expression may be important to the success of early human embryo development and implantation. Supported by: the Oxford Fertility Unit. (1) Fuzzi et al. (2002) Eur J Immunol. 32:311-315.
Wednesday, October 15, 2003 11:30 A.M. O-202 Blastomere transplantation in early human embryos. Norbert Gleicher, YaXu Tang. The Centers for Human Reproduction (CHR) New York & Illinois & The Fdn for Reproductive Medicine, Chicago, IL. Objective: We previously reported the successful transplantation and integration of donor blastomeres into recipient embryos (ESHRE, Madrid, Spain, July 1, 2003). The objective of this study was to determine whether the degree of integration of the donor transplant into the recipient embryo changed, based on the number of blastomeres transplanted. Design: This study was approved by the CHR’s IRB, after external legal review, and, involved only embryos which, by written consent, were explicitly donated “to research”. In a chimera transplantation model, male (xy) blastomeres were transplanted into female (xx) recipient embryos and the distribution of y-chromosome markers was then followed in recipient embryos into the blastocyst stage of development. Materials and Methods: 16 xy-embryos were donors and 21 xx-embryos were recipients, which received one to three blastomere transplants. All embryos in this study had been cryopreserved and were thawed prior to gender determination. Embryo gender was determined utilizing routine preimplantation genetic diagnostic (PGD) techniques and standard fluorescent in situ hybridization (FISH) methodology. The transplantation of blastomeres into recipient embryos utilized standard PGD instrumentation and the initial biopsy site for the insertion of the cells. Distribution of y-chromosome at the blastocyst stage in recipient embyos was also assessed using standard FISH technology. Results: 4/19 (19%) of recipient embryos failed to reach blastocyst stage and only 12/19 (57%) reached what was considered a morphologically normal blastocyst stage. As the table demonstrates, normal blastocyst stage recipient embryos uniformly demonstrated an even and well distributed y-staining pattern throughout inner cell mass and trophoectoderm. Moreover, staining intensity appeared also to correlate with the number of blastomeres transplanted.
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