Levent Keskintepe

Bovine Blastocyst Development from Oocytes Injected with Freeze-Dried Spermatozoa

Abstract Pronuclear formation, and the chromosomal constitution and developmental capacity of bovine zygotes formed by intracytoplasmic sperm injection with freeze-dried (lyophilized) spermatozoa were evaluated. Frozen-thawed spermatozoa were selected, freeze-dried, and stored at 4°C until use. After 22–24 h of in vitro maturation oocytes were denuded and injected singly with a lyophilized spermatozoon. Injected oocytes were activated by treatment with 10 μM ionomycin (5 min) alone and in combination with 1.9 mM 6-dimethylaminopurine (DMAP) for 4 h. Ionomycin plus DMAP activation treatment resulted in a significantly higher proportion of sperm-injected oocytes with two pronuclei than was found after activation with ionomycin alone (74% vs. 56%; P < 0.03). The rates of cleavage, morula, and blastocyst development of sperm-injected oocytes treated with ionomycin plus DMAP were higher than after activation with ionomycin alone (63.3%, 34.2%, and 29.6% vs. 44.7%, 18.7%, and 10.6%, respectively; P < 0.05). Seventy-three percent of blastocysts produced with lyophilized sperm were diploid. These results demonstrate that in vitro-matured bovine oocytes can be fertilized with freeze-dried sperm cells, and that resultant zygotes can develop into karyotypically normal blastocysts.

Derivation and comparison of C57BL/6 embryonic stem cells to a widely used 129 embryonic stem cell line

Typically, embryonic stem (ES) cells derived from 129 mouse substrains are used to generate genetically altered mouse models. Resulting chimeric mice were then usually converted to a C57BL/6 background, which takes at least a year, even in the case of speed congenics. In recent years, embryonic stem cells have been derived from various mouse strains. However, 129 ES cells are still widely used partially due to poor germline transmission of ES cells derived from other strains. Availability of highly germline-competent C57BL/6 ES cells would enormously facilitate generation of genetically altered mice in a pure C57BL/6 genetic background by eliminating backcrossing time, and thus significantly reducing associated costs and efforts. Here, we describe establishment of a C57BL/6 ES cell line (LK1) and compare its efficacy to a widely used 129SvJ ES cell line (GSI-1) in generating germline chimeras. In contrast to earlier studies, our data shows that highly germline-competent C57BL/6 ES cell lines can be derived using a simple approach, and thus support broader use of C57BL/6 ES cell lines for genetically engineered mouse models.

The effect of the biochemical marker soluble human leukocyte antigen G on pregnancy outcome in assisted reproductive technology—a multicenter study

OBJECTIVE To determine whether the presence of soluble human leukocyte antigen G (sHLA-G) affects implantation and pregnancy outcomes in vitro. DESIGN A multicenter retrospective study. SETTING Six certified in vitro fertilization (IVF) units. PATIENT(S) Embryos obtained from 2,040 patients from six different IVF clinics. INTERVENTION(S) Soluble HLA-G determination on day-2 embryos after intracytoplasmic sperm injection, with embryos transferred on day 3 using the sHLA-G data. MAIN OUTCOME MEASURE(S) Ongoing pregnancy rate (10- to 12-week ultrasound finding). RESULT(S) All embryos were individually cultured, and a chemiluminescence enzyme-linked immunosorbent assay was used to detect the presence of sHLA-G in the culture medium surrounding the embryos. Embryos were selected based on a positive sHLA-G result and a graduated embryo scoring (GES) score >70, or on embryo morphology if the test was negative. In all centers, a positive sHLA-G result was associated with an increase in the odds of an ongoing pregnancy. The incidence of an ongoing pregnancy was 2.52 times greater in embryos transferred on day 3 with a positive sHLA-G test result than the incidence of an ongoing pregnancy in embryos with a negative sHLA-G test result. CONCLUSION(S) Data from this multicenter study confirm that sHLA-G expression is a valuable noninvasive embryo marker to assist in improving pregnancy outcomes, with the theoretical potential to reduce multiple pregnancies.

Embryo selection criteria based on morphology VERSUS the expression of a biochemical marker (sHLA-G) and a graduated embryo score: prediction of pregnancy outcome

PurposeTo compare pregnancy and implantation rates when embryos are selected based on a single Day 3 (D 3) morphology score vs. a GES score plus sHLA-G expression.MethodsA prospective randomized study (n = 214) undergoing fresh ICSI cycles. Embryos were selected for transfer based on either Day 3 morphology score (Group A) or GES-scoring plus sHLA-G expression (Group B).ResultsClinical [35/107 (33%) vs. 52/107 (49%)] and ongoing pregnancy [20/107 (19%) vs. 52/107 (49%)] rates were significantly different between Group A and Group B (p < 0.05). Implantation rates were not significantly different between Group A [52/353 (15%)] and Group B [73/417 (18%)] (p < 0.05). The number of pregnancies lost during the first trimester was nearly 12 times higher in Group A [25/52 (48%)].ConclusionThe miscarriage rate was significantly lower in Group B than Group A and the pregnancy results were superior when embryos were selected based on GES plus sHLA-G expression.

Reanalysis of human blastocysts with different molecular genetic screening platforms reveals significant discordance in ploidy status

ObjectiveThe objective of this study is to determine mosaicism and its effect on blastocysts; abnormal blastocysts determined by molecular testing were sequentially biopsied and retested.Material and methodWe re-biopsied 37 blastocyst-stage abnormal embryos from eight patients, which were reanalyzed to determine the level of concordance between biopsies and inter-laboratory congruence between reputable commercial PGS laboratories.ResultsThe main outcome measures were intra-embryo variation between sequential embryo biopsies and inter-laboratory variation between two PGS laboratories. The compatibility between both aCGH and NGS was found to be 11 % (3/27). Importantly, 9/27 (33 %) of embryos originally reported to be aneuploid, upon repeat assessment, were found to be euploid. The concurrence for SNP array and NGS was 50 % (3/6), and 17 % (1/6) of these abnormal embryos tested normal upon re-evaluation with NGS. NGS resulted 41 % (11/27) normal results when 27 of CGH abnormal embryos were retested. Concordance between aCGH and NGS was 4 % (1/27) whereas in three instances, gender discrepancy was observed with NGS when aCGH abnormal embryos were reanalyzed.ConclusionsThe results of these studies reinforce the prevalence of inconsistencies during PGS evaluation of trophectoderm biopsies possibly due to variations in platform sensitivity and heightening concerns over the clinical tractability of such technology in human ARTs..

Use of Cryopreserved Pronuclear Embryos for the Production of Transgenic Mice

Abstract A series of experiments was conducted to test the hypothesis that an improved cryopreservation protocol for pronuclear stage mouse embryos will produce transgenic (Tg) mice by pronuclear gene injection at a rate not significantly different from noncryopreserved embryos. In the first experiment, three cryoprotective agents (CPAs) (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG]) and two cryopreservation protocols, currently used for pronuclear embryos, were compared in regard to their ability to maintain post-thaw morphological integrity and in vitro developmental competence. In the second and third experiments, the optimal cryopreservation protocol determined from the first experiment was used to evaluate in vitro developmental competence of pronuclear embryos following green fluorescence protein gene injection and in vivo developmental competence as well as the gene integration rates. Survival (morphological integrity and development to two cells) of embryos cryopreserved in the presence of DMSO was higher (P < 0.05) than those cryopreserved with either PG or EG. Postinjection developmental competence (development to two cells) of cryopreserved CBA, C57B6/JxCBA-F1 and noncryopreserved (control) embryos was not different (P > 0.05). Postinjection blastocyst formation rate of cryopreserved and noncryopreserved C57B6/JxCBA-F1 embryos was similar (P > 0.05); however, noncryopreserved CBA embryos resulted in a higher blastocyst formation than controls (P < 0.05). While there was no difference in the percentage of transgenic fetuses between cryopreserved and control CBA embryos (P > 0.05), cryopreserved C57B6/JxCBA-F1 embryos resulted in lower transgenic fetuses than control (P < 0.05). These results indicate that the use of cryopreserved mouse pronuclear embryos can be a useful and efficient approach to the production of Tg mice.

A Linear Karyotypic Association between PB-I, PB-II and Blastomere Using Sequentially Performed Comparative Genome Hybridization with No Association Established between Karyotype, Morphologic, Biochemical (sHLA-G Expression) Characteristics, Blastocyst Formation and Subsequent Pregnancy Outcome

Background: The importance of oocyte/embryo ploidy to achieve implantation and a subsequent pregnancy. Aim: To correlate first and second polar bodies and day-3 blastomere ploidy, embryo morphology and biochemical (sHLA-G) characteristics with blastocyst development and in vitro pregnancy outcome. Materials and Methods: All oocytes/zygotes and embryos were individually cultured to the blastocyst stage. PB-I, PB-II and blastomeres underwent complete karyotyping and sHLA-G expression was measured on day 2. Results: 57 mature (MII) donor oocytes were obtained, 33/57 (57.9%) were aneuploid, 21/57 (36.8%) were euploid, and 3/57 (5%) were ‘inconclusive’. No correlation was found between comparative genomic hybridization (CGH) status of PB-I, PB-II and the graduated embryo score. Furthermore, no correlation was established between PB-I CGH results and blastocyst morphology grade. There was a significant correlation between PB-I CGH and blastomere CGH results. Euploid and aneuploid PB-I developed into 58 and 67% blastocysts, respectively. ĸ statistics (>0.7) revealed a positive correlation between the ploidy of PB-I, PB-II and the blastomeres. Conclusion: Following ICSI and sequential genetic karyotyping of the oocyte/zygote and subsequent blastomeres, the majority of oocytes fertilized and subsequent zygotes developed into blastocysts, despite their ploidy status. We therefore conclude that blastocyst development is not associated with ploidy.

Timing of blastocyst transfer relative to the thaw: a critical factor in outcome following FET

Objective: To evaluate effects of different blastocyst freezing and thawing protocols on pregnancy and implantation rate. Design: Retrospective cohort study. Setting: Multi-site, private practice. Patients: Infertile women ages between 27-51 years (n 95) undergoing IVF. Interventions: Women undergoing in vitro fertilization (IVF) between January and December 2002 had surviving expanded supernumerary blastocysts cryopreserved 5 and 6 days following fertilization. All embryos were cultured in P1 (Irvine Scientific) medium until day 3 and thereupon in Blastocyst medium (Irvine Scientific) until day 5 or 6. Patients were allocated to Group 1 or Group 2 based on the method of cryopreservation and the time interval between the thawing process and the frozen embryo transfer (FET). In Group 1, the cryopreservation process involved treatment with 5% and 9% Glycerol 0.2 M sucrose for 10 minutes followed by freezing through exposure of the blastocysts to 7°C for 17 min, and thereupon lowering the temperature by 0.3°C/min to 38°C. Prior to the FET the embryos were thawed in a water bath at 33°C, treated with 0.5 M and 0.2 M sucrose for 10 min and then immediately transferred to the uterine cavity. In Group 2, the same method of exposure of the blastocysts to glycerol and sucrose as described above was employed at 20°C. Thereupon the temperature was progressively reduced by 2°C/min to 6.5°C and maintained at this point for 17 min. Thereafter the temperature was lowered at a rate of 0.3°C/min to 38°C. However, in contrast with Group 1 patients whose blastocysts were transferred immediately upon thawing, the timing of FET’s in Group 2 was predicated upon whether day 5 or day 6 blastocysts were being thawed; whereas day 5 blastocysts were thawed, then treated with 0.5 and 0.2 M sucrose for 10 min, cultured overnight and transferred the following afternoon. Blastocysts frozen on day 6 were thawed similarly on the morning of the scheduled FET, cultured for at least 4 hours and then transferred. Primary measures of outcome: Ongoing pregnancy rate (beyond 12 weeks) and implantation rate (number of viable gestations per transferred blastocyst). Results: The ongoing pregnancy and implantation rates were 16% and 11% for Group 1 as compared to 32% and 20% for Group 2 (P 0.05). Conclusions: Blastocyst transfer has been shown to be associated with improved pregnancy and implantation rates. Recent advances in embryo culture media and techniques have brought about improved embryo quality and survival to the blastocyst stage in the IVF setting. The resulting increase in availability of blastocysts has allowed for an ever increasing degree of discretion in the selection of only the best quality supernumerary blastocysts for cryopreservation. Our data strongly suggests that while the protocol of blastocyst cryopreservation does not affect FET outcome, the timing of the thaw, relative to the transfer could be a critical consideration.