David Battaglia

Human Embryonic Stem Cells Derived by Somatic Cell Nuclear Transfer

Reprogramming somatic cells into pluripotent embryonic stem cells (ESCs) by somatic cell nuclear transfer (SCNT) has been envisioned as an approach for generating patient-matched nuclear transfer (NT)-ESCs for studies of disease mechanisms and for developing specific therapies. Past attempts to produce human NT-ESCs have failed secondary to early embryonic arrest of SCNT embryos. Here, we identified premature exit from meiosis in human oocytes and suboptimal activation as key factors that are responsible for these outcomes. Optimized SCNT approaches designed to circumvent these limitations allowed derivation of human NT-ESCs. When applied to premium quality human oocytes, NT-ESC lines were derived from as few as two oocytes. NT-ESCs displayed normal diploid karyotypes and inherited their nuclear genome exclusively from parental somatic cells. Gene expression and differentiation profiles in human NT-ESCs were similar to embryo-derived ESCs, suggesting efficient reprogramming of somatic cells to a pluripotent state.

Towards germline gene therapy of inherited mitochondrial diseases

Mutations in mitochondrial DNA (mtDNA) are associated with severe human diseases and are maternally inherited through the egg’s cytoplasm. Here we investigated the feasibility of mtDNA replacement in human oocytes by spindle transfer (ST; also called spindle–chromosomal complex transfer). Of 106 human oocytes donated for research, 65 were subjected to reciprocal ST and 33 served as controls. Fertilization rate in ST oocytes (73%) was similar to controls (75%); however, a significant portion of ST zygotes (52%) showed abnormal fertilization as determined by an irregular number of pronuclei. Among normally fertilized ST zygotes, blastocyst development (62%) and embryonic stem cell isolation (38%) rates were comparable to controls. All embryonic stem cell lines derived from ST zygotes had normal euploid karyotypes and contained exclusively donor mtDNA. The mtDNA can be efficiently replaced in human oocytes. Although some ST oocytes displayed abnormal fertilization, remaining embryos were capable of developing to blastocysts and producing embryonic stem cells similar to controls.

Correction of a pathogenic gene mutation in human embryos

Genome editing has potential for the targeted correction of germline mutations. Here we describe the correction of the heterozygous MYBPC3 mutation in human preimplantation embryos with precise CRISPR–Cas9-based targeting accuracy and high homology-directed repair efficiency by activating an endogenous, germline-specific DNA repair response. Induced double-strand breaks (DSBs) at the mutant paternal allele were predominantly repaired using the homologous wild-type maternal gene instead of a synthetic DNA template. By modulating the cell cycle stage at which the DSB was induced, we were able to avoid mosaicism in cleaving embryos and achieve a high yield of homozygous embryos carrying the wild-type MYBPC3 gene without evidence of off-target mutations. The efficiency, accuracy and safety of the approach presented suggest that it has potential to be used for the correction of heritable mutations in human embryos by complementing preimplantation genetic diagnosis. However, much remains to be considered before clinical applications, including the reproducibility of the technique with other heterozygous mutations.

Mitochondrial replacement in human oocytes carrying pathogenic mitochondrial DNA mutations

Maternally inherited mitochondrial (mt)DNA mutations can cause fatal or severely debilitating syndromes in children, with disease severity dependent on the specific gene mutation and the ratio of mutant to wild-type mtDNA (heteroplasmy) in each cell and tissue. Pathogenic mtDNA mutations are relatively common, with an estimated 778 affected children born each year in the United States. Mitochondrial replacement therapies or techniques (MRT) circumventing mother–to–child mtDNA disease transmission involve replacement of oocyte maternal mtDNA. Here we report MRT outcomes in several families with common mtDNA syndromes. The mother’s oocytes were of normal quality and mutation levels correlated with those in existing children. Efficient replacement of oocyte mutant mtDNA was performed by spindle transfer, resulting in embryos containing >99% donor mtDNA. Donor mtDNA was stably maintained in embryonic stem cells (ES cells) derived from most embryos. However, some ES cell lines demonstrated gradual loss of donor mtDNA and reversal to the maternal haplotype. In evaluating donor–to–maternal mtDNA interactions, it seems that compatibility relates to mtDNA replication efficiency rather than to mismatch or oxidative phosphorylation dysfunction. We identify a polymorphism within the conserved sequence box II region of the D-loop as a plausible cause of preferential replication of specific mtDNA haplotypes. In addition, some haplotypes confer proliferative and growth advantages to cells. Hence, we propose a matching paradigm for selecting compatible donor mtDNA for MRT.

Statistical analysis of factors affecting fertilization rates and clinical outcome associated with intracytoplasmic sperm injection

OBJECTIVE To identify and evaluate the statistically significant predictors of intracytoplasmic sperm injection (ICSI) fertilization rates and clinical pregnancy in a single population using appropriate statistical techniques. DESIGN Retrospective study. SETTING Fertility and Endocrinology Center, University of Washington Medical Center, Seattle, Washington. PATIENT(S) Four hundred forty-one patients undergoing their first attempt at IVF-ICSI from January 1, 1999, to May 21, 2001. INTERVENTION(S) Each ICSI procedure for an individual patient was performed by a single operator. Sperm parameters, oocyte age, culture condition, ICSI technique, and ICSI operator were assessed as variables influencing the fertilization rate. We also assessed the impact of patient age, serum E(2) concentration on the day of hCG administration, embryo quality, and number of embryos transferred on the probability of achieving a clinical pregnancy. MAIN OUTCOME MEASURE(S) Fertilization rate and clinical pregnancy. RESULT(S) The 2 pronuclei (2PN) rate was significantly correlated with sperm motility, and there were significant differences in the 2PN rates among the ICSI operators. There was no difference in the 2PN rate among different sperm types or among the eight laboratory incubators or whether the eggs were cultured individually or in groups. Patient age, serum E(2) concentration on the day of hCG administration, embryo quality, and number of embryos transferred were all statistically significant predictors of clinical pregnancy. CONCLUSION(S) In our program, sperm motility and ICSI operator are the two most important predictors for the ICSI fertilization rate in vitro. Patient age, serum E(2) concentration on the day of hCG administration, embryo quality, and number of embryos transferred were all statistically significant predictors of clinical pregnancy.

Storage of cryopreserved reproductive tissues: evidence that cross-contamination of infectious agents is a negligible risk.

A misconception in the field of reproductive medicine is that there is a significant risk of cross-contamination during gamete or embryo cryostorage. This article is a review of the available literature on animal models and human IVF and it suggests otherwise. There is a negligible risk of cross-contamination in IVF working conditions.

Functional Human Oocytes Generated by Transfer of Polar Body Genomes

Oocyte defects lie at the heart of some forms of infertility and could potentially be addressed therapeutically by alternative routes for oocyte formation. Here, we describe the generation of functional human oocytes following nuclear transfer of first polar body (PB1) genomes from metaphase II (MII) oocytes into enucleated donor MII cytoplasm (PBNT). The reconstructed oocytes supported the formation of de novo meiotic spindles and, after fertilization with sperm, meiosis completion and formation of normal diploid zygotes. While PBNT zygotes developed to blastocysts less frequently (42%) than controls (75%), genome-wide genetic, epigenetic, and transcriptional analyses of PBNT and control ESCs indicated comparable numbers of structural variations and markedly similar DNA methylation and transcriptome profiles. We conclude that rescue of PB1 genetic material via introduction into donor cytoplasm may offer a source of oocytes for infertility treatment or mitochondrial replacement therapy for mtDNA disease.

Erratum: Human embryonic stem cells derived by somatic cell nuclear transfer (Cell (2013) 153 (1228-1238))

Masahito Tachibana, Paula Amato, Michelle Sparman, Nuria Marti Gutierrez, Rebecca Tippner-Hedges, Hong Ma, Eunju Kang, Alimujiang Fulati, Hyo-Sang Lee, Hathaitip Sritanaudomchai, Keith Masterson, Janine Larson, Deborah Eaton, Karen Sadler-Fredd, David Battaglia, David Lee, Diana Wu, Jeffrey Jensen, Phillip Patton, Sumita Gokhale, Richard L. Stouffer, Don Wolf, and Shoukhrat Mitalipov* *Correspondence: mitalipo@ohsu.edu http://dx.doi.org/10.1016/j.cell.2013.06.042

Intracytoplasmic Sperm Injection

As illustrated in this book, many medical and surgical options improve male factor infertility and permit couples to conceive naturally, or with minimal medical intervention. But there are still many conditions where sperm function will remain compromised and thus prevent the fertilization of an egg. Even standard in vitro fertilization (IVF) is ineffective with significant male factor situations. Through the introduction of intracytoplasmic sperm injection (ICSI), Palermo et al. (1) made it possible for many male complications to be overcome in the laboratory. Since that time, ICSI has arguably become one of the most important tools in the IVF laboratory. ICSI has provided the successful treatment of many levels of male infertility that were originally untreatable by any available technology.