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

OBJECTIVE: The 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 METHOD: We 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. RESULTS: The 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. CONCLUSIONS: The 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..

Comparison of aneuploidy frequencies between in vitro matured and unstimulated cycles oocytes by metaphase comparative genomic hybridization (mCGH).

A common observation after in vitro matured oocyte is that they yield poorer embryo quality compared to their in vivo counterparts. This study was designed to assess chromosomal status with metaphase comparative genomic hybridization after in vitro maturation (IVM) in unstimulated cycles and compare the results with those obtained after in vivo maturation. Patients without any obstetrical or gynecological pathology were admitted into the study. IVM oocytes were collected 36 h post hCG and matured in vitro at 37°C in 5% O(2), 6% CO(2), and 89% air for 36 h. All matured (metaphase II) oocytes were subject to polar body 1 (PB-1) biopsy and vitrified individually. PB-1 samples were transferred into 0.25 cc PCR tubes containing 2.5 µl of PBS. PB-1 samples from 12 IVM patients were studied. Twenty-six out of 63 PB-1 samples (41%) were determined as euploid and 37 samples (59%) were aneuploid, whereas these values were 42% euploid and 58% aneuploid in the control group (in vivo matured oocytes). No statistical differences were found between the IVM and the control groups for euploid-aneuploid samples (P = 0.900). More aneuploidy was observed on chromosomes 11, 13, 15, 21, and 22 after IVM. Results show a non-significant rate of abnormal PB-1 formation after IVM compared to in vivo maturation. More aneuploidy was observed in chromosomes 11, 13, 15, 21, and 22 in the IVM group.

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.

Preservation of Mammalian Sperm by Freeze-Drying.

Long-term preservation of mammalian sperm at suprazero temperatures is desired to save storage and space costs, as well as to facilitate transport of preserved samples. This can be accomplished by the freeze-drying of sperm samples. Although freeze-drying results in immotile and membrane-compromised sperm, intracytoplasmic sperm injection (ICSI) can be used to introduce such an immotile sperm into an oocyte and thus start the fertilization process. So far, it has been shown that improved freeze-drying protocols preserve chromosomal integrity and oocyte-activating factor(s) in rodent and mammalian species at 4 °C for several years and at ambient temperature for up to 1 year depending on species, which permits shipping freeze-dried samples at ambient temperature. This chapter concisely reviews freeze-drying of mammalian sperm first and then presents a simple freeze-drying protocol.

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

PURPOSE: To 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. METHODS: A 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). RESULTS: Clinical [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%)]. CONCLUSION: The 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.

Vitrification of human embryos subjected to blastomere biopsy for pre-implantation genetic screening produces higher survival and pregnancy rates than slow freezing.

PURPOSE: Cryopreservation of blastocysts, especially those subjected to the trauma due to blastomere biopsy for the purposes of pre-implantation genetic screening (PGS), requires significant optimization. Laboratory and clinical outcomes were compared to determine the effect of two different cryopreservation techniques on the development of human pre-implantation embryos that underwent blastomere biopsy and blastocoel drainage prior to cryopreservation. DESIGN: Retrospective clinical study. PATIENT(S): Women who requested cryotransfer of supernumerary blastocysts were analyzed by FISH. RESULTS: The main outcome measures were post-thaw survival (SR), pregnancy (PR), and implantation (IR). The SR of slowly frozen blastocysts was 83% compared to 97% for vitrified blastocysts. In 160 cases where biopsied embryos were cryotransferred, the results for slowly frozen versus vitrified blastocysts were: SR (71% vs. 95%), PR (23% vs. 37%), and IR (26% vs. 36%, P < 0.05), respectively. CONCLUSION: The results revealed that vitrified blastocysts provided higher SR, PR and IR as compared to slowly frozen counterparts.

Freeze-drying of mammalian sperm.

Long-term preservation of mammalian sperm at suprazero temperatures is desired to save storage and space costs as well as to facilitate transport of preserved samples. This can be accomplished by the freeze-drying of sperm samples. Although freeze-drying results in immotile and membrane-compromised sperm, intracytoplasmic sperm injection (ICSI) can be used to introduce such an immotile sperm into an oocyte and thus start the fertilization process. So far, it has been shown that improved freeze-drying protocols preserve chromosomal integrity and oocyte-activating factor(s) at 4 °C for several years and at ambient temperature for approximately 1 month, which permits shipping freeze-dried samples at ambient temperature. This chapter concisely reviews freeze-drying of mammalian sperm first and then presents a simple freeze-drying protocol.

The graduated embryo score predicts the outcome of assisted reproductive technologies better than a single day 3 evaluation and achieves results associated with blastocyst transfer from day 3 embryo transfer.

OBJECTIVE: To evaluate the graduated embryo score (GES) for predicting assisted reproductive technology (ART) outcome compared to a single morphologic evaluation on day 3 of culture (grade A: > or =7 cells; <20% fragmentation). DESIGN: Prospective cohort analysis. SETTING: Private practice. PATIENT(S): Women aged <40 years with a normal uterine cavity treated with ART (n = 106). INTERVENTION(S): Embryos were graded by GES and by day 3 morphologic characteristics alone before ET. Cycle outcomes were compared with embryo grade. MAIN OUTCOME MEASURE(S): Ongoing gestation and implantation rates. RESULT(S): Overall ongoing gestation and implantation rates were 48% and 26%, respectively. With 1+ embryo GES > or =70 (n = 77), the rates were 62% and 36%, respectively, which were significantly higher than for those with 0 embryos GES > or =70 (n = 29). With 1+ grade A embryo (n = 102), the rates were 50% and 27%, respectively. Transfer of more than one embryo GES > or =70 did not improve the pregnancy rate, but did increase the risk of multiple gestations. A single day 3 evaluation had an extremely low specificity (7%) compared to GES (47%). Graduated embryo scoring (GES) was an excellent predictor of pregnancy and implantation rates from blastocyst transfer. Day of transfer did not affect pregnancy rates, although implantation was higher from day 5 embryo transfer (ET) than from day 3 ET, as fewer embryos were transferred. CONCLUSION(S): Transfer of one or more embryo GES > or =70 predicts pregnancy and implantation rates better than a single morphologic evaluation on day 3 and achieves ART outcomes associated with blastocyst transfer from day 3 ET, making extended culture unnecessary for most patients.

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.

Genetic analysis of human embryos by metaphase comparative genomic hybridization (mCGH) improves efficiency of IVF by increasing embryo implantation rate and reducing multiple pregnancies and spontaneous miscarriages.

OBJECTIVE: To assess the benefit of selecting blastocysts for cryotransfer based upon prior comparative genomic hybridization (CGH) karyotyping of blastomeres derived from their cleaved embryos of origin. Implantation and birth rates per transfer of previtrified CGH-tested blastocysts were compared with those following the transfer of nonCGH-tested fresh and warmed embryos. DESIGN: In vitro studies. SETTING: Private infertility clinic. PATIENT(S): Women undergoing infertility treatment. INTERVENTION(S): Three groups of women with similar clinical and demographic characteristics were compared. Group A underwent transfer of warmed blastocysts derived from CGH-normal day 3 embryos. Group B underwent embryo transfer of warmed blastocysts derived from nonkaryotyped vitrified embryos. Group C underwent fresh transfers with non-CGH-tested blastocysts. MAIN OUTCOME MEASURE(S): Implantation and birth rates per embryo after the cryotransfer of CGH-tested blastocysts. RESULT(S): The birth rate per transferred blastocyst in group A was 48%, versus 15% for group B and 19% for group C. The birth rate per embryo transfer was 60% for group A, and 33% for group B and 36% for group C. The miscarriage rate was 4% in group A, 8% in group B, and 12% in group C. CONCLUSION(S): The transfer of previously vitrified blastocysts derived from CGH-normal embryos significantly improves implantation and birth rates per embryo transferred and reduces the miscarriage rate. Vitrification does not compromise this enhancement.

High survival rate of metaphase II human oocytes after first polar body biopsy and vitrification: determining the effect of previtrification conditions.

OBJECTIVE: To use metaphase II (MII) bovine oocytes as a model for MII human oocyte cryopreservation and to determine the effect of different previtrification equilibration temperatures, vitrification solutions, zona slitting, and first polar body biopsy on in vitro and in vivo developmental competence of MII human oocytes after the CryoLoop vitrification method. DESIGN: In vitro and in vivo studies. SETTING: A private infertility clinic. PATIENT(S): Women undergoing infertility treatment. INTERVENTION(S): Metaphase II stage bovine and MII human oocytes underwent first polar body biopsy before cryopreservation in different vitrification conditions, and human oocytes were fertilized by intracytoplasmic sperm injection after warming. The resulting embryos were transferred into women undergoing infertility treatment. MAIN OUTCOME MEASURE(S): Postvitrification morphologic survival, in vitro blastocyst development, and clinical outcome after ET. RESULT(S): The equilibration temperature had a significant effect on cryosurvival of both bovine and human oocytes. High (97%-99%) postvitrification survival was achieved for both MII bovine and human oocytes, and high fertilization (90%-97%) at 35 degrees C to 37 degrees C, blastocyst development (18%-45%), and pregnancy (50%) rates were achieved at 35 degrees C with 5.0 mol/L ethylene glycol + 1.3 mol/L dimethyl sulfoxide for MII human oocytes that underwent first polar body biopsy. CONCLUSION(S): Previtrification equilibration temperature had a profound effect on the postthaw developmental competence of MII human oocytes in vitro and in vivo. The CryoLoop vitrification of first polar body-biopsied MII human oocytes in the presence of 5 mol/L ethylene glycol plus 1.3 mol/L dimethyl sulfoxide gave the best results in terms of fertilization, embryo development, and implantation rates.

Gonadotropin-releasing hormone agonist/antagonist conversion with estrogen priming in low responders with prior in vitro fertilization failure.

OBJECTIVE: To evaluate gonadotropin-releasing hormone (GnRH) agonist/antagonist conversion with estrogen priming (AACEP) in low responders with prior IVF failure. DESIGN: Descriptive. SETTING: Private practice. PATIENT(S): Women aged 20% fragmentation on day 3 (n = 137; <38: n = 63; 38-42: n = 74). In addition to unexplained poor response to stimulation (n = 52), diagnoses included elevated follicle-stimulating hormone (FSH >9.0 mIU/mL; n = 40), advanced age (>41 years; n = 26), endometriosis (III-IV; n = 12), and decreased ovarian reserve (AFC <5; n = 7). INTERVENTION(S): Patients received sequential GnRH agonist, low-dose GnRH antagonist, and estradiol valerate followed by recombinant FSH, 600 IU/day (n = 72) or 750 IU/day (n = 65). MAIN OUTCOME MEASURE(S): Pregnancy, ongoing gestation, implantation rates. RESULT(S): Although women aged <38 years and those on 600 IU/day produced more mature eggs and fertilized embryos than women aged 38 to 42 years, there were no differences in peak estradiol, endometrial lining, or embryos transferred. Outcomes were similar for all patients regardless of age or FSH dosage. Ongoing gestation rates were 27% (37 out of 137) for all patients, 25% (16 out of 63) for age <38 years, and 28% (21 out of 74) for ages 38 to 42 years. CONCLUSION(S): Women aged

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.

Graduated Embryo Score and soluble human leukocyte antigen-G expression improve assisted reproductive technology outcomes and suggest a basis for elective single-embryo transfer.

OBJECTIVE: To evaluate assisted reproductive technology (ART) outcomes by using Graduated Embryo Score (GES) and soluble human leukocyte antigen-G (sHLA-G) expression to select embryos for transfer on day 3. DESIGN: Prospective cohort. SETTING: Private practice. PATIENT(S): Women undergoing fresh ART cycles (n = 209). INTERVENTION(S): In vitro fertilization using standard protocols. Embryos scoring GES of > or =70 using were selected for transfer on the basis of sHLA-G expression in the culture media on day 2. MAIN OUTCOME MEASURE(S): Pregnancy, implantation, and multiple-gestation rates. RESULT(S): Ongoing gestations increased with the number of embryos expressing sHLA-G (37%, 42%, 58%, and 56% with 0, 1, 2, or 3 sHLA-G(+), respectively). With at least two sHLA-G(+) embryos, ongoing gestation and implantation rates were higher than those with fewer than two sHLA-G(+). Differences were even higher for women aged < or =37 years. With at least two sHLA-G(+) embryos, the odds ratio (95% confidence interval) was 1.59 (1.51-1.68) for ongoing gestation compared with the case of fewer than two sHLA-G(+). Age was the most important predictor of outcome; the odds ratio (95% confidence interval) was 2.07 (1.98-2.16) for ongoing gestation in women aged < or =37 years with at least two sHLA-G(+) embryos, compared with the case of women aged 38-40 years. CONCLUSION(S): Day 3 embryo transfer using GES and sHLA-G improves ART outcomes by increasing predictive accuracy. High twin rates suggest that couples with at least two sHLA-G(+) embryos consider elective single-embryo transfer.

Oocyte karyotyping by comparative genomic hybridization [correction of hybrydization] provides a highly reliable method for selecting "competent" embryos, markedly improving in vitro fertilization outcome: a multiphase study.

OBJECTIVE: To assess the karyotypic relationship between prefertilized/postfertilized oocytes and embryos using comparative genomic hybridization (CGH) on polar body-1 (PB-1), PB2, and blastomere biopsies and to evaluate IVF outcomes after transfer of blastocysts derived from euploid oocytes. DESIGN: Prospective cohort. SETTING: Medical center. PATIENT(S): Phase1: Fourteen oocyte donors (23-29 years). Phase 2: Forty-one healthy embryo recipients aged 29-43 years free of endometrial implantation dysfunction. In 30 cases own eggs were used. Eleven women used donated oocytes. INTERVENTION(S): Phase 1: PB-1 biopsies followed intracytoplasmic sperm injection (ICSI), PB-2, and day 3 blastomere biopsies. Phase 2: PB-1 biopsy followed by ICSI using normal sperm and the subsequent embryo transfer of < or =2 blastocysts derived from euploid oocytes. Comparative genomic hybridization on all DNA derived from phase 1 and 2 biopsies. MAIN OUTCOME MEASURE(S): Pregnancy and implantation rate. RESULT(S): Phase 1: 39% of oocytes and 88% of zygotes were euploid; >95% progressed to blastocysts. Mosaicism as evidenced by euploid oocytes developing into aneuploid zygotes or embryos occurred in 13% of concepti. Phase 2: Six of 30 women using own eggs, who failed to produce euploid oocytes, were cancelled. Thirty-five women underwent embryo transfers with < or =2 (mean, 1.3 +/- 0.7) blastocysts derived from euploid oocytes. The ongoing pregnancy/implantation rates per embryo transfer were 74% and 82%, respectively. CONCLUSION(S): Transferring euploid embryos markedly improved IVF outcome. These findings, if corroborated, could initiate a paradigm shift in assisted reproductive technology (ART).

Soluble human leukocyte antigen G expression in phase I culture media at 46 hours after fertilization predicts pregnancy and implantation from day 3 embryo transfer.

OBJECTIVE: To compare pregnancy and implantation rates after ART when embryos for day 3 embryo transfer were selected based on soluble HLA-G (sHLA-G) expression in the culture media at 46 hours after fertilization by intracytoplasmic sperm injection (ICSI). DESIGN: Prospective cohort study. SETTING: Private practice. PATIENT(S): One hundred seven patients undergoing ART aged <39 years with normal ovarian reserve, a normal uterine cavity, and two or more embryos scoring > or =70 by the graduated embryo scoring (GES) method, transferred on day 3. INTERVENTION(S): Patients were divided into two groups. In group A (n = 51) all embryos transferred expressed sHLA-G above the geometric mean (sHLA-G+), whereas in group B (n = 56) all embryos transferred were sHLA-G-ve. MAIN OUTCOME MEASURE(S): Viable pregnancy rate (patients with fetal heart activity at 8 weeks of gestation per embryo transfer procedure), and implantation rate (viable gestational sacs per total embryos transferred). RESULT(S): When all embryos transferred were sHLA-G+ve the pregnancy and implantation rates were 75% (38/51) and 44% (51/116), respectively, compared to 23% (13/56) and 14% (20/143) when all embryos transferred were sHLA-G-ve. CONCLUSION(S): Pregnancy and implantation rates after day 3 embryo transfer are improved when sHLA-G expression in phase I culture media at 46 hours after fertilization by ICSI is used prospectively as a criterion for selecting optimal embryos for transfer.

Expression of sHLA-G in supernatants of individually cultured 46-h embryos: a potentially valuable indicator of 'embryo competency' and IVF outcome.

A retrospective cohort study was conducted on 201 women aged 28-44 years, each of whom underwent one cycle of IVF-embryo transfer with fresh, intracytoplasmic sperm injection (ICSI)-derived 7- to 10-cell embryos, transferred 72 h after oocyte retrieval. Samples of media surrounding separately cultured embryos were collected 46 h post-ICSI and stored for subsequent specific enzyme-linked immunosorbent assay. A total of 594 embryos (from own or donor oocytes) were transferred to 201 women. Group A comprised 159 recipients under 39 years and group B compromised 42 recipients aged 39-44 years. Groups A-1 and B-1 recipients had at least one embryo that tested above the geometric mean for soluble human leukocyte antigen-G (sHLA-G) ('positive expression') transferred. In groups A-2 and B-2, all embryos transferred expressed sHLA-G below the geometric mean ('negative expression'). In group A-1, 72/101 women (71%) achieved ultrasound confirmed (clinical), viable (cardiac activity observed) pregnancies. The implantation rate per embryo (IR) was 38%. In group A-2, 13/58 (22%) achieved viable clinical pregnancies. The IR was 9%. In group B-1, the viable clinical pregnancy rate was 52% (15/29) and the IR was 25% compared with a viable clinical pregnancy rate of 15% (2/13) and an IR of 5% in group B-2. The results of this study suggest that by selecting specific embryos for transfer based on their individual sHLA-G expression, pregnancy and implantation rates can be maximized while the number of embryos transferred can be reduced, thereby minimizing the incidence of high-order multiple pregnancies.

Bovine blastocyst development from oocytes injected with freeze-dried spermatozoa.

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 degrees 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 microM 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.