Blastomere Biopsy Research Articles

P-936: Are chromosomal abnormalities compatible with development to the blastocyst stage?

Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy on D3, followed by genetic analysis and transfer of unaffected embryos later on D3 or D4. We evaluate a strategy involving embryo biopsy on D3, genetic analysis on D4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on D5. Moreover, this study was undertaken in order to determine whether chromosomal abnormalities are compatible with development to the blastocyst stage.

P-947: Does indication for PGD influence outcome?

Preimplantation Genetic Diagnosis (PGD) has become an invaluable tool in the diagnostic and therapeutic management of many couples facing reproductive challenges. In general, PGD may benefit four distinct patient groups: 1) those undergoing aneuploidy screening due to recurrent miscarriages, repeated IVF failures, or advanced maternal age; 2) those undergoing PGD for gender selection due to X linked recessive disorders or elective family balancing 3) couples with translocations due to the increased risk they have of producing unbalanced gametes and 4) couples that are carriers of monogenic mutations. PGD allows the transfer of only those embryos identified as being free of genetic abnormalities, and in some patients, may theoretically improve implantation rates and decrease miscarriage rates. The objectiveof this analysis is to determine the clinical outcome for each PGD indication. Retrospective analysis Eighty patients undergoing PGD cycles between January 2004 and March 2006 were included. Standardized ovarian stimulation was performed using recombinant FSH, HMG or a combination of the two. Final oocyte maturation was achieved by the administration of hCG and oocyte retrieval was perfomed 36 hours after. Insemination was performed with ICSI. Embryo morphology was assessed daily, and the blastomere biopsy performed on day 3 from a cleaving embryo. During the biopsy, the zona pellucida was chemically digested with acidified Tyrode’s solution, and one or two blastomeres removed from each cleaving embryo. The blastomeres were sent to one of three genetic laboratories. After cell fixation, multiprobe, multicolor fluorescence in situ hybridization (FISH) was performed for chromosome aberrations or polymerase chain reaction (PCR) for DNA analysis for single gene defects. FISH results were received, and selected embryos were transferred on day 5. Statistical analysis was performed using ANOVA for continuous data and Chi Square for categorical data. The characteristics of PGD cases are described in Table 1.Tabled 1 The results of our study suggest that acceptable implantation and pregnancy rates per transfer can be achieved after PGD, regardless of the indication. Patients undergoing PGD for gender selection had a significantly higher number of euploidic embryos. Given that the euploid embryo rate is significantly lower in patients with translocations, they generally have fewer embryos available for transfer. These results have been consistent with previous reports, as we found that only 4 out of 9 translocation cases had an embryo transfer, with an euploidy rate of only 13.8%. Given these findings, translocation couples should be informed and counseled about the increased incidence of abnormal embryos and about the fewer availability of embryos for transfer.

P-935: A human blastomere chromosome assay system using electro-fusion with in vitro matured human oocytes

In our study “Reconsidering IVF Preimplantation Genetic Screening” using multi-colored fluorescent In situ hybridization (FISH), it became clear that almost all 3-day-old human embryos show chaotic mosaicism. This suggested that a more precise cytogenetic assessment system for blastomeres was needed during biopsy diagnosis at the metaphase stage. Therefore, we developed a new assay system to diagnose human biopsied blastomeres that have been electrically fused with human in vitro matured (IVM) oocytes.

Cryopreservation of biopsied embryos at the blastocyst stage

The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact.

Pre-embryonic diagnosis for Sandhoff disease

Embryos found to be abnormal during preimplantation genetic diagnosis (PGD) are discarded or analysed to confirm the diagnosis. To overcome this limitation, which is unacceptable in some communities and ethnic groups, pre-embryonic genetic diagnosis has been introduced, involving sequential first and second polar body analysis followed by transfer of embryos deriving from the mutation-free oocytes, while removing from culture and freezing the mutant oocytes at the pronuclear stage. The technique is applied here to PGD of Sandhoff disease caused by 16-kb deletion of the hexosaminidase B gene for a couple with a religious objection to discarding embryos irrespective of embryo genotype. Of 16 oocytes tested in a standard IVF protocol for 16-kb deletion, simultaneously with five linked polymorphic markers, eight were predicted mutant and frozen prior to syngamy, with the remaining eight, found to be free of mutation, further cultured and confirmed unaffected using blastomere biopsy. The transfer of two of these embryos resulted in birth of an unaffected child, demonstrating feasibility of pre-embryonic diagnosis to avoid embryo discard.

201 FACTORS AFFECTING THE SURVIVAL OF BIOPSIED RHESUS MACAQUE EMBRYOS

The rhesus macaque (Macaca mulatta) is widely used for the study of human disease. Increasingly assisted reproductive techniques are applied to the propagation of this species to identify specific genotypes, to generate animals with genetic modifications, or to preserve valuable genetic resources. A significant advantage in this endeavor would be the ability to identify embryos with desired genetic characteristics by pre-implantation genetic diagnosis (PGD) prior to their transfer into a surrogate. The objectives of this study, therefore, were to assess the feasibility of performing blastomere biopsies on rhesus embryos and to determine the effects of (a) oxygen tension during culture, (b) transient cryopreservation, and (c) the number of blastomeres removed (one or two) on subsequent development. A total of 17 oocyte aspirations were performed, which resulted in 206 8- to 10-cell embryos that were selected for biopsy. Embryos were cultured in HECM-9 medium and cryopreserved with propylene glycol. The chi-square test was used for data analysis. For the first experiment, 160 embryos were used to assess the effect of oxygen tension and transient cryopreservation. Oxygen tension during culture had no effect on the number of embryos proceeding to the blastocyst stage (26.0 vs. 25.0%) following blastomere removal. Similarly, transient freezing and storage in liquid nitrogen did not affect subsequent development to the blastocyst stage (26.6 vs. 24.7%). For the second experiment, 46 embryos were biopsied and assigned to removal of either one or two blastomeres. Subsequent development was not affected by the number of blastomeres removed, and a similar percentage of embryos reached the blastocyst stage (38.1 vs. 20%). These results demonstrate that rhesus embryos are tolerant of biopsies and proceed to advanced stages of development at the same rate as control embryos. Funding was provided by the Tulane National Primate Research Center base grant NIH 5P51 RR00164-44.

Outcomes of Preimplantation Genetic Diagnosis Therapy in Treatment of β‐Thalassemia: A Retrospective Analysis

Thalassemia is one of the most common single-gene disorders that can be cured by hematopoietic stem cell transplantation (HCT) from a human leukocyte antigen (HLA)-identical sibling donor. In families that have an affected child, preimplantation genetic diagnosis (PGD) can be used to select an unaffected, HLA-identical embryo. In brief, this procedure requires in vitro fertilization, oocyte retrieval, fertilization, and blastomere biopsy for identification of unaffected HLA-identical embryos. After delivery, umbilical cord blood from the sibling donor is collected for HCT. The objective of this study was to determine the outcomes of families using PGD therapy for cure of beta-thalassemia and to review the limitations of PGD therapy. Families affected with beta-thalassemia who attempted PGD therapy were retrospectively identified and reviewed for indication, attempted cycles, successful pregnancy, and transplantation outcomes. Eight identified families affected by thalassemia underwent PGD. The diagnosis of their affected children included six cases of beta-thalassemia major and two cases of transfusion-dependent hemoglobin E-beta-thalassemia patients. A total of 14 cycles of PGD were attempted, ranging from one to four attempts per family. Following successful identification of HLA-identical cells, two pregnancies occurred, of which one resulted in engraftment of a beta-thalassemia child. PGD therapy offers the possibility of recruiting a suitable donor for HCT, yet is limited by financial cost due to labor-intensive techniques, low probability of obtaining an HLA-matched unaffected embryo, variable implantation capacity, and significant emotional impact. Improvements in PGD therapy's efficacy and cost will make this a more viable option for affected families.

The Blastocyst Embryo Cryopreservation After Blastomere Biopsy on Day 3 for Preimplantation Genetic Diagnosis (PGD)

The Blastocyst Embryo Cryopreservation After Blastomere Biopsy on Day 3 for Preimplantation Genetic Diagnosis (PGD)

Impact of Embryo Quality on Developmental and Aneuploidy Rates After Blastomere Biopsy for PGD

To examine the impact of cleavage stage embryo quality on subsequent embryonic development and aneuploidy rates following blastomere biopsy. Retrospective. Embryology data and PGD records for 189 embryos, which underwent day 3 blastomere biopsy for PGD of aneuploidy at our center were reviewed. Embryos were divided into euploid or aneuploid based on FISH analysis of 9 chromosomes (X, Y, 13, 15, 16, 17, 18, 21 and 22). The Mean patient’s age for all cases in the study (n=22) was 37.0±5.6. Assessment of embryo morphology, blastomere biopsies and nuclei fixation were performed by the same embryologist. All biopsies were performed on day 3 and all FISH analysis were performed at Reprogenetics, N.J. Our laboratory uses a strict embryo morphology assessment system whereby embryos are evaluated systematically once every 24 hr period and assigned a quality grade ranging from A to E. Top quality embryos fall in categories A/B, Mid quality embryos fall in category C, Low quality embryos are assigned scores D/E. All data were compared using the Fisher’s exact test. Out of 189 embryos that were biopsied 88% had one blastomere removed, and 12% required an additional blastomere to be obtained. The distribution of 5 to 8 cell embryos at 60-62 hrs in culture was similar among those that were subsequently reported as aneuploid or euploid (94% vs. 93%, respectively). There was no difference in blastulation rate in embryos with one vs. those with two blastomeres removed. Overall, blastulation rates for Day-3 Top, Mid and Poor quality embryos were 65% (43/66), 56% (62/111), 17% (2/12), respectively. Accurate FISH results were available in 77% of all embryos biopsied. In this cohort, 72% (105/146) were determined aneuploid and 28% (41/146) euploid. Day 3 Top quality euploid embryos had higher blastulation rate, 83% (19/23), as compared to their aneuploid counter parts, 56% (18/32), (P=0.04). There was no significant difference in the blastulation rate of Mid and Low quality Day 3 euploid embryos when compared to their aneuploid cohorts. The table below summarizes aneuploidy / euploidy rates for Top, Mid and Low quality blastocysts. Tabled 1 In our initial experience, biopsy of one or two blastomeres does not appear to affect blastulation rate. However, day 3 embryo quality was associated with blastulation rate following biopsy. Based on FISH results, Top quality day 3 euploid embryos demonstrated greater blastulation potential. Approximately 50% of Top quality blastocysts were aneuploid. Furthermore, the aneuploidy rate was higher in Mid and Low quality blastocysts. Therefore, FISH results are necessary for adequate selection of euploid embryos for transfer.

Preimplantation Genetic Diagnosis for Aneuploidy Screening in Patients With Poor Reproductive Outcome

Aneuploidies of embryos are related to implantation failure or miscarriage. The risk of aneuploid embryo increases in advanced maternal age or parental karyotype abnormality and it results in poor reproductive outcomes such as recurrent spontaneous abortion (RSA) or recurrent implantation failure (RIF). Preimplantation genetic diagnosis for aneuploidy screening (PGD-AS) can be applied to these patients with poor reproductive outcome for better ART outcome by selecting chromosomally normal embryos. The aim of this study is to evaluate the clinical outcome of PGD-AS and which group can get much benefit from PGD-AS among the patients expected to have poor reproductive outcome. Retrospective anaylsis of PGD data. PGD-AS was performed in 48 cycles of 31 patients with poor reproductive outcome from May 1999 to December 2003. Patients were divided into 3 groups: group I - RIF more than 3 times (n=11, mean age =42.2 yrs.), group II - RSA (≥ 2 times) associated with aneuploidy (n=19, mean age =38.9 yrs.), group III - parental sex chromosome abnormality (n=18, mean age =29.6 yrs.) including Turner syndrome (45,X/46,XX or 47,XXX/45,X/46,XX), Klinefelter syndrome (47,XXY or 47,XXY/46,XY or 48,XXXY/47,XXY/46,XY) and 47,XYY. Ovarian stimulation was done using GnRH agonist/FSH/HMG midluteal long protocol. Oocytes were inseminated by ICSI and the blastomeres were biopsied at 6-10 cell stage by drilling with Acid Tyrode’s. PGD was performed by using FISH for chromosome 13, 16, 18, 21, X and Y in group I and II, and chromosome X, Y and 18/17 in group III. The embryos diagnosed to be chromosomally normal were replaced 4 days after oocyte retrieval. Blastomere biopsy was successful in 481 embryos and FISH efficiency was 93.1%. The proportions of transferable embryos for the tested probes were 27.9%, 20.5% and 51.2%, respectively in each group showing higher normal rate in group III (group II vs. III, p<0.05). The number of transferred embryos was 3.9±1.5, 2.0±1.2 and 3.1±1.5 (mean±SD), respectively. The clinical pregnancy rate per transfer was 0%, 35.7% and 16.7%, respectively and significantly higher in group II (p<0.05). The overall pregnancy rate was 18.6 % (8/43). Only 2 biochemical pregnancies occurred in group I. Eight healthy babies (one twin pregnancy) were born with normal karyotype and one case was aborted with normal karyotype. Our data suggest that PGD-AS provides advantages in patients with poor reproductive outcome, especially with previous RSA associated with aneuploidy or sex chromosome abnormality. PGD-AS does not seem to be beneficial in RIF group in our study due to advanced maternal age and other detrimental factors involved in implantation. Further comprehensive analysis for other autosomes and in more cases of proper indication might be needed.

Preimplantation Genetic Diagnosis (PGD) for Heritable Neoplasia

Especially applicable for heritable neoplasia, preimplantation genetic diagnosis (PGD) is possible for any Mendelian disorder whose gene has been localized, whether the molecular basis is known or not. PGD requires DNA from gametes (oocytes) or embryos before 6 days postconception, when implantation occurs. Approaches include 1) polar body biopsy, 2) blastomere biopsy (aspiration of one or two cells from the six- to eight-cell embryos at 2 or 3 days), and 3) trophectoderm biopsy, which allows recovery of 20 or more cells (20-50) from 125- to 150-cell, 5- to 6-day blastocysts. Of some 6000 PGD cycles worldwide, approximately 1500 have been performed for Mendelian indications. The approximately 25% live birth rates following PGD parallel the general U.S. experience for assisted reproductive technology. PGD has been accomplished for both cancer-specific disorders like adenomatous polyposis coli (APC), BRCA1, retinoblastoma, Li-Fraumeni syndrome, and von Hippel-Lindau syndrome (VHL), as well as disorders predisposing to neoplasia (Fanconi anemia, Wiskott-Aldrich syndrome). PGD also makes possible the identification and, hence, transfer of embryos of specific HLA genotypes. This allows cord blood harvesting for stem cell implantation into a moribund child, often an older sibling of the fetus. PGD is a complex, but achievable, approach especially applicable to Mendelian forms of neoplasia. PGD is an attractive addition to the prenatal diagnostic armamentarium, especially relevant to heritable neoplasia. PGD also makes possible novel indications having special relevance to heritable neoplasia.

Using BAC clones to characterize unbalanced chromosome abnormalities in interphase cells

Carriers of balanced translocations usually carry alterations in gene sequences, which lead to dysfunction during early and late embryogenesis. Related spatial rearrangement causes either cumulative delay in cell cycles and/or anomalies in transcription and translation. This has also an important impact at the time of genomic activation, the longest cell cycle of preimplantation development. Carrier patients may be affected either with primary infertility or by repeated miscarriages [Hum. Reprod. 12 (1997) 2019.]. Assuming that a fraction of the germ cells is karyotypically normal, these patients would greatly benefit from efficient procedures for generation and use of breakpoint-specific DNA hybridisation probes in preconception and preimplantation genetic diagnosis (PGD) after polar body or blastomere biopsy of the embryos. The objective of this research was to design an approach of patient-specific probes for preimplantation diagnosis of chromosome translocations and which could be used eventually to select chromosomally normal embryos from balanced or unbalanced interphase cells prior to their transfer to the mother's womb. This approach was used for a couple, where the female partner was a carrier of a balanced translocation 46,XX,t(4;12)(p16.1;q24.31). First, BAC/PAC clones were chosen from specific chromosome bands from the genome sequence that hybridise adjacent to the chromosomal breakpoints or span them. Then, the probes and hybridisation conditions were optimised using unrelated normal amniocytes, lymphoblastoid cells and lymphocytes from the carrier to increase hybridisation signal intensity and decrease background. Finally, the probes were tested on target cells before they were used for mimicking preconception or preimplantation genetic diagnosis. Three slides were analysed blindly from a normal karyotype, an unbalanced and a balanced translocation. A total of 78 cells were analysed of which in the slide A 19/22 (86%) were found to be unbalanced, in slide B 25/31 (81%) were normal and in slide C 21/25 (84%) were balanced. Thus, it was demonstrated that cells with known structural abnormalities could be detected, based on hybridisation of breakpoint spanning bacterial artificial chromosome (BAC) DNA probes in interphase cells.

Birth of two healthy females after preimplantation genetic diagnosis for familial amyloid polyneuropathy

Familial amyloid polyneuropathy (FAP), Portuguese type, is a late onset, high penetrance, autosomal dominant Mendelian disorder caused by a V30M substitution in the transthyretin (TTR) protein. A genetic diagnosis was developed using fluorescent single cell polymerase chain reaction (PCR) on lymphocytes from patients and controls. Ovarian stimulation and oocyte retrieval were carried out using conventional protocols in a couple in whom the female was heterozygous for the mutation TTR V30M. Blastomere biopsy was performed on day 3 after intracytoplasmic sperm injection. PCR was then performed for a segment of the TTR gene encompassing the V30M mutation. The transfer of three embryos at day 4 resulted in a twin pregnancy, confirmed as healthy females by amniocentesis at 16 weeks of gestation; the birth took place at 37 weeks of gestation. With this report, FAP, TTR related, joins the lengthening list of genetic conditions for which preimplantation genetic diagnosis has been successfully carried out.

Cryopreservation of biopsied cleavage stage human embryos

The aim was to develop a method to optimize cryopreservation of biopsied multi-celled human embryos. Human day 3 embryos that were donated to research, along with those found to be chromosomally abnormal after blastomere biopsy and fluorescence in-situ hyridization (FISH), were cryopreserved using a slow-freezing protocol in either standard embryo cryopreservation solution [embryo transfer freezing medium (ETFM), a conventional sodium-based medium] or CJ3 (a choline-based, sodium-free medium). After thawing, the number of intact cells was recorded and the previously biopsied embryos were re-analysed using FISH. Biopsied embryos had a lower proportion of intact blastomeres after cryopreservation as compared with intact embryos. However, a significantly (P < 0.05) higher proportion of blastomeres from intact and biopsied embryos cryopreserved in CJ3 (84.1 and 80.1% respectively) survived after thaw than those in ETFM (73.6 and 50.5% respectively). The proportion of aneuploid and mosaic embryos was not statistically different between the two groups. In addition, the frequency of lost cells by aneuploid and mosaic embryos was similar. This study describes a new method that improves the survival of cryopreserved biopsied embryos, and shows that it may also be beneficial for the storage of intact human multi-celled embryos.

Comparison of effects of zona drilling by non-contact infrared laser or acid Tyrode's on the development of human biopsied embryos as revealed by blastomere viability, cytoskeletal analysis and molecular cytogenetics

Use of a non-contact infrared laser (IRL) or acid Tyrode's for zona drilling before embryo biopsy was compared by assessing blastomere viability using various fluorescent markers or culture of the single biopsied blastomere, and, by cytoskeletal and molecular cytogenetic analysis of the biopsied embryos following culture to the blastocyst stage. There was no significant difference in the proportion of biopsied embryos that showed no damage in both the biopsied blastomere and in the remaining embryo (acid Tyrode's: 75% versus IRL: 68%), or in the proportion of single biopsied blastomeres that divided in culture (P > 0.05). However, single biopsied blastomeres from laser drilled embryos showed a greater tendency to form miniblastocysts. The proportion of laser or acid Tyrode's biopsied embryos that reached the blastocyst stage by day 6 was similar, although evident earlier (day 5) in the laser biopsied embryos. Spindle abnormalities at the blastocyst stage included tripolar and tetrapolar spindles, but their incidence was not significantly different from controls. In addition, no significant difference was observed in the incidence of chromosomal abnormalities and mosaicism between the two groups. It is concluded that using an IRL at a safe working distance does not cause adverse immediate or longer term effects on the development of human biopsied embryos, although damage can occur if drilling within this distance is unavoidable. Acid Tyrode's drilling can also cause damage, and tended to retard blastocyst development.

Efficient blastomere biopsy for mouse embryo splitting for future applications in human assisted reproduction

The objective of the current study was to establish a safe, efficient biopsy procedure for embryo splitting using the mouse model for future applications in human assisted reproduction. From mouse embryos at the 2-, 4-, 6- and 8-cell stage, half the number of blastomeres were microsurgically biopsied and transferred into empty mouse zonae pellucidae. Twin embryonic development was monitored during in-vitro culture. Blastocyst developmental rate using 2-, 4-, 6-, and 8-cell splitting was 74.4, 75.0, 66.7 and 38.4 respectively, with corresponding hatching rates of 94.9, 97.5, 92.7 and 83.8%. Blastocysts from 2-, 4-, and 6-cell splitting resulted in elevated hatching rates compared with non-operated blastocysts (87.5%), due to the Tyrode-assisted hatching effect. Blastocyst morphology was superior from 2- and 4-cell splitting when compared with 6- and 8-cell splitting. Furthermore, outgrowth of twin blastocysts from 2- and 4-cell splitting showed well-developed colonies with trophoblast cells and clusters of ICM cells, whereas those obtained from 6- and 8-cell splitting frequently formed small-sized colonies. Due to the high twinning success rate obtained under the experimental conditions employed in this study, it appears that with further modifications and proper safeguards, such embryo splitting efforts could have potential applications in humans.

Impact of the presence of one or more multinucleated blastomeres on the developmental potential of the embryo to the blastocyst stage

A retrospective study of 5,982 embryos in 619 blastocyst-stage embryo transfer cycles revealed that detection of multinucleated blastomeres either on day 2 or 3 signifies a poor prognosis for blastocyst formation and that no good-quality blastocyst can be expected from an embryo with more than one multinucleated blastomere. Patients should be counseled regarding a poor prognosis when multiple embryos with multinucleated blastomeres are present.

154 EFFECTS OF BLASTOMERE BIOPSY AND OXYGEN CONCENTRATION DURING CULTURE ON THE DEVELOPMENT AND INTERFERON-TAU SECRETION OF BOVINE EMBRYOS

Pre-implantation genetic diagnosis has become a powerful tool in human in vitro fertilization. Yet, for ethical reasons, controlled experiments assessing the effects of embryo biopsies on human embryos are difficult to perform. Therefore, a series of experiments was performed to examine the effects of blastomere biopsies on subsequent development of IVF-derived bovine embryos cultured under either atmospheric or low (5%) oxygen. Embryos were generated by IVF of in vitro-matured oocytes and cultured in CR1aa medium containing 5% bovine fetal serum. The first experiment was designed to assess the optimal time for blastomere removal. One blastomere was removed from each of a large number of embryos at either 48 or 72 h after IVF. Biopsy at 48 h resulted in 17.2% (10/58) of embryos proceeding to the blastocyst stage, which was significantly lower than when biopsies were performed at 72 h (37.5% (33/88), P &lt; 0.05). In the second experiment, embryos were cultured under either atmospheric or 5% O2 following blastomere removal. Biopsies at 72 h had no effect on rate of blastocyst formation with 38.5% (110/286) of controls and 33.7% (60/178) of biopsied embryos proceeding to the blastocyst stage. However, culture under 5% O2 significantly increased the number of blastocysts from 29.9% (69/231) to 43.3% (101/233). This effect was significant in both biopsied and control embryos. In contrast, staining of blastocysts for cell numbers did not reveal any effects of biopsies or culture conditions. In the final experiment, biopsies were again performed at 72 h and embryos were cultured as previously. Blastocysts were collected and cultured individually for 48 h in 50-μL-medium droplets in their respective O2 concentrations after which time the medium was assayed for concentration of interferon-tau (IFN-τ). Previous work has shown that IFN-τ secretion can be affected by genetic and environmental factors. Reduced O2 concentration again significantly increased blastocyst formation from 24.9 to 36.9% (49/197 vs. 75/203, P &lt; 0.05), while blastomere removal had no effect on development. IFN-τ secretion did not differ between biopsied and control blastocysts, although culture under atmospheric O2 resulted in significantly increased IFN-τ concentration in medium droplets (12,690.1 ± 2715.6 vs. 4726.8 ± 2488.5 pM, P &lt; 0.05). This work was supported by a grant (HMK) from the National Institutes of Health (HD 36421).

Using BAC clones to characterize unbalanced chromosome abnormalities in interphase cells

Abstract Carriers of balanced translocations usually carry alterations in gene sequences, which lead to dysfunction during early and late embryogenesis. Related spatial rearrangement causes either cumulative delay in cell cycles and/or anomalies in transcription and translation. This has also an important impact at the time of genomic activation, the longest cell cycle of preimplantation development. Carrier patients may be affected either with primary infertility or by repeated miscarriages [Hum. Reprod. 12 (1997) 2019.]. Assuming that a fraction of the germ cells is karyotypically normal, these patients would greatly benefit from efficient procedures for generation and use of breakpoint-specific DNA hybridisation probes in preconception and preimplantation genetic diagnosis (PGD) after polar body or blastomere biopsy of the embryos. The objective of this research was to design an approach of patient-specific probes for preimplantation diagnosis of chromosome translocations and which could be used eventually to select chromosomally normal embryos from balanced or unbalanced interphase cells prior to their transfer to the mother’s womb. This approach was used for a couple, where the female partner was a carrier of a balanced translocation 46,XX,t(4;12)(p16.1;q24.31). First, BAC/PAC clones were chosen from specific chromosome bands from the genome sequence that hybridise adjacent to the chromosomal breakpoints or span them. Then, the probes and hybridisation conditions were optimised using unrelated normal amniocytes, lymphoblastoid cells and lymphocytes from the carrier to increase hybridisation signal intensity and decrease background. Finally, the probes were tested on target cells before they were used for mimicking preconception or preimplantation genetic diagnosis. Three slides were analysed blindly from a normal karyotype, an unbalanced and a balanced translocation. A total of 78 cells were analysed of which in the slide A 19/22 (86%) were found to be unbalanced, in slide B 25/31 (81%) were normal and in slide C 21/25 (84%) were balanced. Thus, it was demonstrated that cells with known structural abnormalities could be detected, based on hybridisation of breakpoint spanning bacterial artificial chromosome (BAC) DNA probes in interphase cells.

The Outcomes of Preimplantation Genetic Diagnosis Therapy in Treatment of β Thalassemia - a Retrospective Analysis.

Abstract BACKGROUND: β thalassemia is one of the most common single gene disorders. Hematopoietic stem cell transplantation (HCT) from a human leukocyte antigen (HLA)-identical sibling donor is a curative option that minimizes the risk of graft-versus-host disease, compared to alternative donor HCT. In families that have an affected child, preimplantation genetic diagnosis (PGD) can be utilized to select an unaffected embryo that is HLA-identical. Briefly, this procedure requires in vitro fertilization, oocyte retrieval, fertilization and blastomere biopsy for preimplantation analysis and identification of unaffected HLA-identical embryos. After delivery, umbilical cord blood from the sibling donor is collected for HCT. In our institution, PGD has been pursued as a therapeutic option by families with thalassemia. The estimated cost of this uninsured procedure is $20,000 per cycle. METHODS: Families affected with β thalassemia who attempted PGD were identified and reviewed for indication, attempted cycles, successful pregnancy and transplantation outcome. RESULTS: Eight identified families affected by thalassemia underwent PGD. The diagnosis of their affected children included: 6 cases of β thalassemia major and 2 cases of transfusion dependent E β thalassemia patients. A total of 14 cycles of PGD were attempted, ranging from 1–4 attempts per family. Following successful identification of HLA-identical cells, 2 pregnancies occurred (1 early miscarriage, 1 successful delivery). This pregnancy resulted in the engraftment of a β thalassemia child. CONCLUSION: PGD including selection of HLA-identical sibling embryos is a novel, therapeutic approach for patients with β thalassemia. While this offers the possibility of recruiting a suitable donor for HCT, it is limited by significant financial and emotional burdens that it places on families affected with β thalassemia. Improvements in its efficiency and cost will make this a more viable option for affected families.