Analysis of a preimplantation genetic test for aneuploidies in 893 screened blastocysts using KaryoLite BoBs: a single-centre experience.

INTRODUCTION: Does euploidy of trophectoderm (TE) biopsies correlate with conventional blastocyst morphological, maternal age and implantation potential? METHODS: This is a one-centre, retrospective, observational study. RESULTS: Eight hundred and ninety-three blastocysts were biopsied; 57.73% were euploid. The euploidy rate was found to be significantly higher for the embryos with good morphology of inner cell mass (ICM) and TE. Between ICM and TE morphology variables, TE was more predictive of the euploidy rate. When broken down into different age groups, the percentage of good morphology embryos remained similar across all age groups, while the percentage of euploid embryos dropped with increasing age. These results suggest that the correlation between blastocyst morphology and ploidy status was present but poor. Faster growing day 5 blastocysts showed a significantly higher euploidy rate than slower growing day 6 or 7 blastocysts. The number of good-quality blastocysts per cycle, euploid blastocysts per cycle and the euploidy rate were strongly associated with maternal age. A trend towards an increased implantation rate was found with euploid embryo transfers compared to the control group without preimplantation genetic test for aneuploidies (PGT-A). CONCLUSIONS: Blastocyst morphology, rate of development and maternal age were found to be significantly associated with euploidy rate. There is a trend that suggests PGT-A may help to improve the pregnancy rate, but it is not statistically different, and therefore, PGT-A remains an unproven hypothesis. Due to the limitation of a small size of the control group, further studies with more data are needed.

Integration-free induced pluripotent stem cells from three endangered Southeast Asian non-human primate species.

Advanced molecular and cellular technologies provide promising tools for wildlife and biodiversity conservation. Induced pluripotent stem cell (iPSC) technology offers an easily accessible and infinite source of pluripotent stem cells, and have been derived from many threatened wildlife species. This paper describes the first successful integration-free reprogramming of adult somatic cells to iPSCs, and their differentiation, from three endangered Southeast Asian primates: the Celebes Crested Macaque (Macaca nigra), the Lar Gibbon (Hylobates lar), and the Siamang (Symphalangus syndactylus). iPSCs were also generated from the Proboscis Monkey (Nasalis larvatus). Differences in mechanisms could elicit new discoveries regarding primate evolution and development. iPSCs from endangered species provides a safety net in conservation efforts and allows for sustainable sampling for research and conservation, all while providing a platform for the development of further in vitro models of disease.

Live birth from vitrified-warmed human oocytes fertilized with frozen-thawed testicular spermatozoa.

A couple with male infertility due to non-obstructive azoospermia were referred to the fertility centre for treatment. Testicular biopsy was performed on the male partner and testicular samples were frozen. The female partner underwent ovarian stimulation and 31 mature oocytes were recovered by ultrasound-guided vaginal aspiration. Twelve oocytes were cryopreserved by the Cryotop vitrification method and 19 oocytes were inseminated by intracytoplasmic sperm injection (ICSI) using frozen-thawed testicular spermatozoa. Nine out of 19 oocytes were fertilized and the resulting embryos were cryopreserved by slow freezing. Four months later, two out of six thawed embryos were transferred, but no pregnancy resulted. One year later, the couple decided to attempt pregnancy using vitrified oocytes and frozen testicular spermatozoa. Six vitrified-warmed oocytes were injected with frozen-thawed testicular spermatozoa and four were fertilized. On the day of transfer, two cleavage stage embryos (4-cell, 2-cell) were obtained. Serum beta-HCG test 14 days after embryo transfer was positive. Hormonal support for the established pregnancy was maintained with oestradiol and progesterone orally until 12 weeks of gestation. A healthy baby boy weighing 3.09 kg was delivered by elective Caesarean section at 38 weeks of gestation. This case report demonstrates that oocyte cryopreservation by the Cryotop vitrification method does not compromise oocyte developmental competence.

Cumulus-specific genes are transcriptionally silent following somatic cell nuclear transfer in a mouse model.

This study investigated whether four cumulus-specific genes: follicular stimulating hormone receptor (FSHr), hyaluronan synthase 2 (Has2), prostaglandin synthase 2 (Ptgs2) and steroidogenic acute regulator protein (Star), were correctly reprogrammed to be transcriptionally silent following somatic cell nuclear transfer (SCNT) in a murine model. Cumulus cells of C57xCBA F1 female mouse were injected into enucleated oocytes, followed by activation in 10 micromol/L strontium chloride for 5 h and subsequent in vitro culture up to the blastocyst stage. Expression of cumulus-specific genes in SCNT-derived embryos at 2-cell, 4-cell and day 4.5 blastocyst stages was compared with corresponding in vivo fertilized embryos by real-time PCR. It was demonstrated that immediately after the first cell cycle, SCNT-derived 2-cell stage embryos did not express all four cumulus-specific genes, which continually remained silent at the 4-cell and blastocyst stages. It is therefore concluded that all four cumulus-specific genes were correctly reprogrammed to be silent following nuclear transfer with cumulus donor cells in the mouse model. This would imply that the poor preimplantation developmental competence of SCNT embryos derived from cumulus cells is due to incomplete reprogramming of other embryonic genes, rather than cumulus-specific genes.

Dynamic changes in microtubules and early development of reconstructed embryos after somatic cell nuclear transfer in a non-human primate.

In order to improve somatic cell nuclear transfer (SCNT) efficiency and to understand cellular changes in SCNT, the dynamic changes in microtubules/DNA and early development of SCNT embryos with single or multiple pronuclei were investigated, along with activation timing on efficiency of SCNT, were studied in the Cynomolgus monkey. The confocal images showed that microtubules assembled around condensed DNA at 1h after cell injection; normal or abnormal reconstructed spindle formed at 2 h after cell injection; and reconstructed spindle separated at 2 h after activation. The results of nuclear formation showed that 61.3% of the reconstructed embryos did not form pronuclei; 19.3% formed a single nucleus, and 11.9% and 7.5% formed two and more than two reconstructed pronuclei, respectively. The cleavage and 8-cell development rates of SCNT embryos with pronuclei were significantly higher than those without pronuclei, but there was no difference in development rates among NT embryos with single, two and more then two pronuclei. Activation at 2 h after cell injection yielded more embryos with pronuclei and yielded 8-cell NT embryos more reliably than did activation at 3-4 h. In conclusion, microtubules assembled around condensed DNA at 1-2 h after cell injection, and formed a spindle at 2 h after SCNT, which separated at 2 h after activation; early development was affected by activation time, but no different between single and multiple pronuclei.

Early development of reconstructed embryos after somatic cell nuclear transfer in a non-human primate.

To improve efficiency and assess variation in nuclear transfer techniques in non-human primates, we investigated the following factors: type of donor cell, interval between enucleation and cell injection, activation after electrical pulsing and cytokinesis inhibitors. An average of 16.4 oocytes were recovered from 91 retrievals; however, 15 (14%) additional retrieval attempts yielded no oocytes due to a failure of follicular stimulation. Oocyte maturation rates at 36, 38 and 40 h post-hCG were 46.2, 52.6 and 61.2%, respectively. The MII spindle could be seen clearly using polarized microscopy in 89.1% (614/689) of oocytes. Nuclei were seen in 42% of the NT couplets, 53% of those cleaved to the 2-cell stage and 63% of the 2-cell embryos developed to the 8-cell stage by Day 3. There was no difference in the occurrence of nuclear formation between couplets created using fibroblasts or cumulus cells, although embryos were more reliably produced with fibroblasts. The interval (2, 3 and 4 h) between enucleation and cell injection did not affect NT efficiency. Ethanol treatment after electrical pulses yielded more 2-cell NT embryos than did treatment with ionomycin, but the frequency of nuclear formation and development to the 8-cell stage was not different. Treatment of couplets with cycloheximide and cytochalasin B for 5 h after activation had no impact on NT efficiency.

"Waste" follicular aspirate from fertility treatment--a potential source of human germline stem cells?

Fertility clinics worldwide routinely produce a large volume of 'waste' follicular aspirate, which is potentially an abundant source of immature ovarian follicles. Current attempts to cultivate these further in vitro to yield viable mature oocytes for fertility treatment have not yet achieved much success. Instead, recent lines of evidence have emerged that are suggestive of a potential stem cell niche within such immature ovarian follicles. The recent discovery of follicular renewal and putative germ-line stem cells within the postnatal mammalian ovary shook the foundations of reproductive biology by challenging the established dogma that mammalian females lose the capacity for germ cell renewal during fetal life, such that a fixed reserve of germ cells (oocytes) enclosed within follicles is endowed at birth. More intriguingly, another recent study in the Drosophila model provided compelling evidence that somatic progenies (nurse cells) of germ-line stem cells had the ability to revert back to the stem-cell-like state. This introduces the exciting possibility that within the mammalian ovarian follicle, similar somatic progenies of germ-line stem cells may also possess a greater intrinsic ability to revert back into functional stem cells. If this is the case, then a favored candidate would be the cumulus/granulosa of immature ovarian follicles, since such cells are true homologues of nurse cells found within the Drosophila ovary. The successful elucidation of a human germ-line stem cell niche within immature ovarian follicles is likely to have huge ramifications in stem cell biology and regenerative medicine.

Reprogramming autologous skeletal myoblasts to express cardiomyogenic function. Challenges and possible approaches.

Cell transplantation therapy is emerging as a promising mode of treatment following myocardial infarction. Of the various cell types that can potentially be used for transplantation, autologous skeletal myoblasts appear particularly attractive, because this would avoid issues of immunogenicity, tumorigenesis, ethics and donor availability. Additionally, skeletal myoblasts display much higher levels of ischemic tolerance and graft survival compared to other cell types. There is some evidence for improvement in heart function with skeletal myoblast transplantation. However, histological analysis revealed that transplanted myoblasts do not transdifferentiate into functional cardiomyocytes in situ. This is evident by the lack of expression of cardiac-specific antigens, and the absence of intercalated disc formation. Instead, there is differentiation into myotubes that are not electromechanically coupled to neighboring cardiomyocytes. This could in turn limit the clinical efficacy of treatment. This review would therefore examine the various challenges faced in attempting to reprogram autologous skeletal myoblast to express cardiomyogenic function, together with the various possible strategies that could be employed to achieve this objective.

Influence of cysteamine supplementation and culture in portable dry-incubator on the in vitro maturation, fertilization and subsequent development of mouse oocytes.

The need to transport oocytes and embryos between two laboratories have prompted us to evaluate the effects of in vitro maturation of immature mouse oocytes in a CO2-deficient dry heat portable incubator and subsequent in vitro development of these fertilized mouse oocytes in a standard CO2 incubator. In addition, the effects of cysteamine supplementation on maturation rate and embryonic development during in vitro maturation (IVM) and culture of embryos in the portable incubator were also investigated. Germinal vesicle stage mouse oocytes, recovered at 40-h post-FSH from 6- to 8-week-old C57BL/6xCBA F1 healthy female mice, were matured in vitro in a modified TCM-199 supplemented with or without 100 microM cysteamine in a standard incubator (5% CO2; 37 degrees C) or cultured in a CO2-deficient dry heat portable incubator for 5 h at 37 degrees C and thereafter transferred to a standard incubator for further culture. The addition of cysteamine in the IVM medium significantly improved maturation rates of the GV mouse oocytes to metaphase II stage. However, cysteamine supplementation in the culture medium did not significantly improve fertilization and blastocyst formation rates of IVM and ovulated oocytes, and in vivo-derived zygotes. Culture conditions in a CO2-deficient dry heat portable incubator did not adversely affect the developmental competence of in vivo-derived zygotes and in vitro matured mouse oocytes after IVF or parthenogenetic activation. Cysteamine supplement in the IVM medium could enhance nuclear maturation of these immature oocytes during shipment.

Comments about possible use of human embryonic stem cell-derived cardiomyocytes to direct autologous adult stem cells into the cardiomyogenic lineage.

Several studies have shown that cell-transplantation therapy following myocardial infarction has some efficacy in aiding myocardial repair and subsequent recovery of heart function. Large-scale production of human embryonic stem cell-derived cardiomyocytes can potentially provide an abundant supply of donor cells for myocardial transplantation. There are, however, immunological barriers to their use in human clinical therapy.A novel approach would be to look at utilizing human embryonic stem cell-derived cardiomyocytes to reprogram autologous adult stem cells to express cardiomyogenic function, instead of using these directly for transplantation. This could be achieved through a number of novel techniques. Enucleated cytoplasts generated from human embryonic stem cell-derived cardiomyocytes could be fused with autologous adult stem cells to generate cytoplasmic hybrids or cybrids. Adult stem cells could also be temporarily permeabilized and exposed to cytoplasmic extracts derived from these cardiomyocytes. Alternatively, intact cells or enucleated cytoplasts from human embryonic stem cell-derived cardiomyocytes could be co-cultured with adult stem cells in vitro, to provide the cellular contacts and electrical coupling that might enable some degree of trans-differentiation to take place. This review would therefore examine the potential advantages and disadvantages of such a novel approach, in comparison to other more conventional techniques such as the use of exogenous cytokines/growth factors or the use of genetic modulation.

In vitro fertilization outcome in severe ovarian hyperstimulation syndrome: An age-matched contemporaneous control study.

Aim: Ovarian hyperstimulation syndrome (OHSS) is a potentially life-threatening, iatrogenic complication of assisted reproduction and has been associated with poor in vitro fertilization outcome. The aim of the present study was to evaluate the pregnancy rate and outcome following severe OHSS, at a single center over a three-year period. Methods: The incidence of severe OHSS at the IVF Center, National University of Singapore, in Singapore, was 4% (48 cases over 1200 cycles) during the period of 1997-2000. The present retrospective study compared 48 cases of severe OHSS to 144 age-matched, contemporaneous controls without OHSS. Results: The total gonadotropin required for severe OHSS group was found to be lower than for that of controls (2664.06 ± 768.29 IU vs 3349.58 ± 2003.73 IU), although duration of stimulation was similar. The OHSS group was associated with a fivefold increase (OR 5.293, 95% CI: 2.116-13.238) in pregnancy rate compared to controls (87.5% vs 56.9%; P < 0.05). Late OHSS was more common (38/48 cases) and had a pregnancy rate of 97% per embryo transfer. There was no significant difference in the multiple pregnancy (54% vs 48%; P > 0.05) and miscarriage rates (14% vs 7.3%; P > 0.05) between the groups. Conclusion: Severe OHSS at our center were mostly late onset. The pregnancy rate was significantly higher, but multiple pregnancy and miscarriage rates were not significantly increased when compared to the age-matched contemporaneous controls. (Reprod Med Biol 2005; 4: 207-211).

Strategies for directing the differentiation of stem cells into the cardiomyogenic lineage in vitro.

Most studies on stem cell transplantation therapy on myocardially infarcted animal models and phase-I human clinical trials have focused on the use of undifferentiated stem cells. There is a strong possibility that some degree of cardiomyogenic differentiation of stem cells in vitro prior to transplantation would result in higher engraftment efficiency, as well as enhanced myocardial regeneration and recovery of heart function. Additionally, this may also alleviate the probability of spontaneous differentiation of stem cells into undesired lineages and reduces the risk of teratoma formation, in the case of embryonic stem cells. The development of efficient protocols for directing the cardiomyogenic differentiation of stem cells in vitro will also provide a useful model for molecular studies and genetic manipulation. This review therefore critically examines the various techniques that could possibly be used to direct and control the cardiomyogenic differentiation of stem cells in vitro.

Potential utility of cell-permeable transcription factors to direct stem cell differentiation.

Application of embryonic and adult stem cells in regenerative medicine will require efficient protocols for directing stem cell differentiation into well-defined lineages. Differentiation induced by exogenous cytokines, growth factors, or extracellular matrix components will require extended in vitro culture that would delay autologous transplantation and may well alter the immunogenicity of cultured cells. Genetic modulation to direct stem cell differentiation may obviate prolonged culture, but safety concerns preclude clinical application of genetically altered cells in the foreseeable future A novel alternative would be to incorporate protein transduction domains (PTDs) into recombinant transcription factors that play important roles in somatic differentiation. Such protein-engineered transcription factors would then have the ability to translocate across the cell membrane and be internalized within the cytosol, where they would act as paracrine signaling molecules. Upon internalization, the recombinant transcription factors would only have a limited active half-life, so that their effects may only be transient. However, this could provide sufficient stimulus for initiating stem cell differentiation into a required lineage.

Ovarian hyperstimulation syndrome is associated with reversible impairment of vascular reactivity.

OBJECTIVE: To determine if there is a loss of normal peripheral arteriolar vasoconstrictor reactivity in women with severe ovarian hyperstimulation syndrome (OHSS). SETTING: Prospective controlled study. DESIGN: National University Hospital, Singapore. PATIENT(S): Forty-three women undergoing treatment in an IVF program: 22 women with severe OHSS and 21 normal women without OHSS. INTERVENTION(S): Measurement and quantification of the cutaneous arteriolar vasoconstrictor response using laser Doppler fluximetry. MAIN OUTCOME MEASURE(S): Changes in blood flow as an index of vasoconstrictor response before and after treatment for OHSS with albumin. RESULT(S): The mean (+/-SE) percentage (%) change in normal control women was -68.3% +/- 4.7. This was significantly different from women with OHSS (-32.3% +/- 11.1). The response in women with OHSS changed significantly after treatment with IV albumin (-60.4% +/- 5.7) and was no different than the control response. The reproducibility of the technique was -13.6% (+/-2.8). CONCLUSION(S): Women with OHSS have impaired vascular reactivity when compared with normal women.

Effects of granulosa coculture on in-vitro oocyte meiotic maturation within a putatively less competent murine model.

A less competent murine in vitro maturation (IVM) model was achieved by shortening the standard duration of in vivo PMSG stimulation from 48 to 24 h and selecting only naked/partially naked GV oocytes from a mixture of large and small follicles. Porcine granulosa coculture enhanced meiotic maturation within such a less competent model (37.3% versus 23.1%, P<0.05), while no significant enhancement was observed with macaque and murine granulosa coculture. Culture of porcine granulosa on extracellular matrix (ECM) gel resulted in a more differentiated morphology, but did not significantly further enhance the beneficial effects it already had on meiotic maturation. Increased concentrations of serum as well as the supplementation of gonadotrophins and follicular fluid within the culture milieu did not enhance IVM under both cell-free and coculture conditions. Porcine granulosa-conditioned medium also enhanced meiotic maturation (36.5% versus 26.7%, P<0.05), which was not diminished upon freeze-thawing (35.8% versus 22.6%, P<0.05). Enhancement of meiotic maturation by porcine granulosa coculture did not however translate to significant improvements in developmental competence, as assessed by in vitro fertilization (IVF) and embryo culture to the blastocyst stage, followed by total cell counts. ECM gel had a detrimental effect on fertilization and developmental competence, even though it had no detrimental effect on meiotic maturation itself.

Intracytoplasmic injection of frozen-thawed epididymal spermatozoa in a nonhuman primate model, the cynomolgus monkey (Macaca fascicularis).

Intracytoplasmic sperm injection (ICSI) with frozen-thawed epididymal spermatozoa was performed in the cynomolgus monkey (Macacafascicularis) to produce embryos in vitro. Eleven sexually mature females were hyperstimulated with an GnRH agonist (1.8 mg active triptorelin per 2 kg body weight), followed (2 weeks later) by rFSH (37.5 IU per 2 kg daily) for 12 days, and finally 1000 IU of hCG. Epididymal spermatozoa were collected from a single adult male monkey. The first stimulation cycle resulted in 90 oocytes; 70% of which were metaphase II (MII). Sixty-four percent of these MII oocytes were fertilized. Comparing ovarian response of five monkeys that underwent a second stimulation cycle there was an increase in oocyte quantity (13.2 versus 9.2 oocytes per monkey) but the percentage of MII oocytes remained the same at 58%. Fertilization and cleavage rates were also reduced but there was an increase in the number of embryos available for transfer. Overall, four monkeys became pregnant resulting in the birth of two healthy infants and two abortions. These findings show that ovarian stimulation by GnRH-rFSH did not compromise the developmental competence of the oocytes; effective combination of cryopreservation of epididymal spermatozoa and ICSI is possible in nonhuman primate reproduction, and thus has potential application in the conservation of highly endangered nonhuman primate species, and the cynomolgus monkey is a reliable biomedical research model to study the potential risks and benefits associated with assisted reproductive techniques prior to approval for clinical trials on humans.

FISH analysis of Y chromosome long arm deletions in subfertile men considering ICSI. A report of two cases.

BACKGROUND: Men with Y chromosome long arm deletions, resulting in infertility, form a very significant group of patients, with a view to treatment. With recent advances in assisted reproductive techniques, it is expected that if these patients undergo intracytoplasmic sperm injection, their male offspring will inherit the same deleted regions of Y chromosomes. Hence, characterization of these deleted Y regions provides information, allowing patients to make informed decisions about reproduction. Fluorescence in situ hybridization (FISH), with probes along the Y chromosome, is very helpful in identification of the exact breakpoints. CASES: Two men with complaints of infertility on examination showed low or no sperm in their semen samples. Routine cytogenetic analysis of the peripheral blood showed a small marker chromosome. This marker was identified as the Y chromosome by FISH. Subsequently, other probes along the Y long arm were used to characterize the extent of the deletion. CONCLUSION: With the use of fluorescence-labelled probes, it was possible to identify the extent of the Y chromosome deletion. The distal Yq11 region had been lost in both patients, resulting in oligospermia and azoospermia. Characterizing markers by FISH gave good guidelines to the patients about the possible effects on offspring, allowing them to make appropriate decisions.

Exposure of mouse cumulus cell nuclei to porcine ooplasmic extract eliminates TATA box protein binding to chromatin, but has no effect on DNA methylation.

PURPOSE: The low cloning efficiency with SCNT is due to incomplete or partial reprogramming of the donor somatic cell nuclei after microinjection into the enucleated oocyte. A possible solution may be to initiate nuclear reprogramming prior to SCNT. METHODS: Pre-exposure of donor somatic cell nuclei to a novel porcine ooplasmic extract prior to microinjection could possibly extend the duration of exposure to ooplamic nuclear reprogramming factors. The effects of the porcine ooplamic extract on two major markers of nuclear preprogramming: (1) TATA box protein binding to chromation and (2) DNA methylation was investigated. RESULTS: The results showed that pre-exposure of mouse cumulus cell nuclei to porcine ooplamic extract drastically reduced TATA box protein binding to chromatin, but had no effect on DNA methylation. CONCLUSIONS: Pre-exposure to the porcine ooplasmic extract had some limited effects on nuclear reprogramming. Whether this can lead to enhanced cloning efficiency needs to be further investigated.