Einat Aizenman

Derivation, propagation and controlled differentiation of human embryonic stem cells in suspension

Undifferentiated human embryonic stem cells (hESCs) are currently propagated on a relatively small scale as monolayer colonies. Culture of hESCs as floating aggregates is widely used for induction of differentiation into embryoid bodies. Here we show that hESC lines can be derived from floating inner cell masses in suspension culture conditions that do not involve feeder cells or microcarriers. This culture system supports prolonged propagation of the pluripotent stem cells as floating clusters without their differentiation into embryoid bodies. HESCs cultivated as aggregates in suspension maintain the expression of pluripotency markers and can differentiate into progeny of the three germ layers both in vitro and in vivo. We further show the controlled differentiation of hESC clusters in suspension into neural spheres. These results pave the way for large-scale expansion and controlled differentiation of hESCs in suspension, which would be valuable in basic and applied research.

At what age can human oocytes be obtained

OBJECTIVE To determine whether oocyte retrieval and in vitro maturation (IVM) is effective in girls undergoing fertility preservation before cancer treatment. DESIGN Cohort study. SETTING Tertiary university medical center. PATIENT(S) Patients <or=20 years old before gonadotoxic chemotherapy undergoing ovarian cortex cryopreservation. INTERVENTION(S) Before ovarian cortex cryopreservation, oocytes in all observed follicles were aspirated, matured in vitro, and cryopreserved. MAIN OUTCOME MEASURE(S) Maturation of oocytes. RESULT(S) One hundred seventy-nine oocytes were detected in 17/19 patients (89%) aged 5-20 years. We found 7, 8, and 17 oocytes in patients 5, 8, and 10 years old, respectively. The median number of oocytes per patient was 9 (0-37). Maturation rate was 45/133 oocytes (34%). In total, 81 oocytes were cryopreserved. We cryopreserved 4 of 12 detected, 4 of 9 detected, 1 of 8 detected, and 4 of 9 detected IVM oocytes for patients aged 5-10, 11-14, 15-17, and 18-20 years old, respectively. CONCLUSION(S) Patients undergoing ovarian cryopreservation could benefit from supplementary oocyte aspiration from the cortex. Surprisingly, oocytes were detected even in young premenarcheal girls. The number of oocytes detected, matured, and cryopreserved was not age dependent. Retrieved oocytes can be matured in vitro and cryopreserved. Because no pregnancy has yet resulted from this procedure it should be considered to be experimental. We describe the youngest patients to undergo ovum collection, IVM, and oocyte cryopreservation.

Derivation of xeno-free and GMP-grade human embryonic stem cells--platforms for future clinical applications.

Clinically compliant human embryonic stem cells (hESCs) should be developed in adherence to ethical standards, without risk of contamination by adventitious agents. Here we developed for the first time animal-component free and good manufacturing practice (GMP)-compliant hESCs. After vendor and raw material qualification, we derived xeno-free, GMP-grade feeders from umbilical cord tissue, and utilized them within a novel, xeno-free hESC culture system. We derived and characterized three hESC lines in adherence to regulations for embryo procurement, and good tissue, manufacturing and laboratory practices. To minimize freezing and thawing, we continuously expanded the lines from initial outgrowths and samples were cryopreserved as early stocks and banks. Batch release criteria included DNA-fingerprinting and HLA-typing for identity, characterization of pluripotency-associated marker expression, proliferation, karyotyping and differentiation in-vitro and in-vivo. These hESCs may be valuable for regenerative therapy. The ethical, scientific and regulatory methodology presented here may serve for development of additional clinical-grade hESCs.

Preimplantation genetic diagnosis for BRCA1/2—a novel clinical experience

To describe our 2‐year experience with preimplantation genetic diagnosis (PGD) for carriers of mutations in the genes BRCA1 and BRCA2, the dilemmas incurred and the lessons learned.

Hyaluronic acid can successfully replace albumin as the sole macromolecule in a human embryo transfer medium.

OBJECTIVE To examine the effect on pregnancy and implantation rates when highly purified, fermentation-based hyaluronic acid was the only macromolecule supplement to the transfer medium in a human IVF program. DESIGN Prospective randomized study. SETTING In vitro fertilization center in an academic medical institution. PATIENT(S) Eighty patients were included in this prospective randomized double blind study. Inclusion criteria were age </=35 years, the availability of at least three embryos eligible for transfer on day 3 after fertilization, and no more than three previous embryo transfer attempts. INTERVENTION(S) All embryos were cultured in P1 medium containing 10% synthetic serum substitute (SSS) until day 3. Patients were randomly allocated to two groups; in treatment group A (40 patients), embryos were transferred to P1 medium supplemented with 0.5 mg/mL hyaluronic acid for 5-10 min before their intrauterine transfer. In the control group B (40 patients), embryos were transferred, as routinely performed, in P1 medium containing 10% SSS. MAIN OUTCOME MEASURES Clinical pregnancy and implantation rates. RESULT(S) The mean age of the female partner was 28.7 +/- 3.3 years and 29.7 +/- 3.8 years for groups A and B, respectively. In group A, 103 embryos were transferred and in group B, 97 embryos were transferred for a similar mean number of 2.6 +/- 0.6 and 2.4 +/- 0.5 embryos/transfer, respectively. Twenty-five pregnancies were achieved in group A, and 21 pregnancies in group B. This led to a comparable clinical pregnancy and implantation rates of 62.5% and 34% as compared to 52% and 26.8% for groups A and B, respectively. CONCLUSION(S) Hyaluronic acid can successfully replace albumin as a sole macromolecule in a human embryo transfer medium and result in high pregnancy and implantation rates. The use of this supplement is an important step in the development of human embryo culture media free of blood-derived additives.

Human granulosa luteal cell oxidative phosphorylation function is not affected by age or ovarian response

OBJECTIVE To safely prepare a functional autologous mitochondrial concentrate (MC) from follicular fluid (FF) cells, and to determine the effect of age and ovarian response on the oxidative phosphorylation (OXPHOS). DESIGN The nontoxicity of the MC was confirmed in human and mouse oocytes. The OXPHOS function was assessed by measuring the activity of succinate dehydrogenase (SDH) and cytochrome c oxidase (COX), and adenosine triphosphate (ATP) production in comparison with citrate synthase. The integrity of the mitochondrial DNA (mtDNA) was demonstrated by polymerase chain reaction (PCR). SETTING Tertiary hospital. PATIENT(S) A total of 40 patients undergoing IVF of heterogeneous ages and ovarian response. ANIMAL(S) Superovulated 8- to 12-week-old female B(6)C(3)F(1) mice. INTERVENTION(S) None. MAIN OUTCOME MEASURE(S) A system for the preparation of functional nontoxic MC was established. The correlation between the mitochondrial mass and function to age and ovarian response was calculated. The integrity of mtDNA was demonstrated. RESULT(S) After injection into mouse oocytes, the MC did not interfere with parthenogenetic development. The MC OXPHOS function was intact. Total activity of SDH and COX was in correlation with the retrieved oocytes number, and in reverse correlation with age. However, after correction to the mitochondrial mass, COX and SDH activities were constant, unaffected by age or ovarian response. The mtDNA was intact in all samples, regardless of age and ovarian response. CONCLUSION(S) The function of the respiratory chain in mitochondria of FF cells is constant, unaffected by age or ovarian response.

Monozygotic multiple gestation after intracytoplasmic sperm injection and preimplantation genetic diagnosis.

OBJECTIVE To report a possible association between intracytoplasmic sperm injection (ICSI)-preimplantation genetic diagnosis (PGD) and monozygotic multiple gestation. DESIGN Small case series. SETTING In vitro fertilization unit in an academic medical center. PATIENT(S) Three patients were treated with ICSI-PGD for sexing as well as selection against a known translocation. INTERVENTION(S) Transfer of day 4 embryos to the uterus. MAIN OUTCOME MEASURE(S) Clinical pregnancy. RESULT(S) Two pairs of monozygotic twins and a triplet pregnancy. CONCLUSION(S) Repeated manipulation of the zona pellucida as well as extended embryo culture during ICSI-PGD treatments may result in monozygotic twin and triplet pregnancies.

Familial Dysautonomia (FD) Human Embryonic Stem Cell Derived PNS Neurons Reveal that Synaptic Vesicular and Neuronal Transport Genes Are Directly or Indirectly Affected by IKBKAP Downregulation

A splicing mutation in the IKBKAP gene causes Familial Dysautonomia (FD), affecting the IKAP protein expression levels and proper development and function of the peripheral nervous system (PNS). Here we found new molecular insights for the IKAP role and the impact of the FD mutation in the human PNS lineage by using a novel and unique human embryonic stem cell (hESC) line homozygous to the FD mutation originated by pre implantation genetic diagnosis (PGD) analysis. We found that IKBKAP downregulation during PNS differentiation affects normal migration in FD-hESC derived neural crest cells (NCC) while at later stages the PNS neurons show reduced intracellular colocalization between vesicular proteins and IKAP. Comparative wide transcriptome analysis of FD and WT hESC-derived neurons together with the analysis of human brains from FD and WT 12 weeks old embryos and experimental validation of the results confirmed that synaptic vesicular and neuronal transport genes are directly or indirectly affected by IKBKAP downregulation in FD neurons. Moreover we show that kinetin (a drug that corrects IKBKAP alternative splicing) promotes the recovery of IKAP expression and these IKAP functional associated genes identified in the study. Altogether, these results support the view that IKAP might be a vesicular like protein that might be involved in neuronal transport in hESC derived PNS neurons. This function seems to be mostly affected in FD-hESC derived PNS neurons probably reflecting some PNS neuronal dysfunction observed in FD.

Frozen-Thawed Embryo Transfer Success Rate is Affected by Age and Ovarian Response at Oocyte Aspiration Regardless of Blastomere Survival Rate

OBJECTIVE To identify the factors influencing the success of frozen-thawed embryo transfers, whether originating directly from current cycles or from their matching fresh cycles. METHODS Analysis of 273 frozen-thawed embryo transfer cycles and their matching fresh embryo transfer cycles, with respect to maternal, embryological and clinical factors, comparing successful to unsuccessful cycles. RESULTS The cumulative clinical pregnancy and live birth rates following fresh ET and corresponding FETs were 50.5% and 38.8%, respectively. No outcome measure differed between fresh and frozen ETs. Only maternal age, number of oocytes retrieved and fertilized, and number of cleaved embryos in the fresh cycle were correlated with a higher pregnancy or live birth rate in the FET cycle. None of the other parameters had any effect on the outcome. Pre-freezing embryo quality and blastomere survival rate had no effect on pregnancy/live birth rates. CONCLUSION Clinical pregnancy and live birth rates of fresh and frozen ETs were not significantly different. The only parameters that affected FET success were those resulting from the patients age and ovarian reserve at the time of oocyte aspiration. Post-thawing blastomere survival rate and type of endometrial preparation for FET did not affect the success rate.