ING2 (inhibitor of growth protein-2) plays a crucial role in preimplantation development.

ING2 (inhibitor of growth protein-2) is a member of the ING-gene family and participates in diverse cellular processes involving tumor suppression, DNA repair, cell cycle regulation, and cellular senescence. As a subunit of the Sin3 histone deacetylase complex co-repressor complex, ING2 binds to H3K4me3 to regulate chromatin modification and gene expression. Additionally, ING2 recruits histone methyltransferase (HMT) activity for gene repression, which is independent of the HDAC class I or II pathway. However, the physiological function of ING2 in mouse preimplantation embryo development has not yet been characterized previously. The expression, localization and function of ING2 during preimplantation development were investigated in this study. We showed increasing expression of ING2 within the nucleus from the 4-cell embryo stage onwards; and that down-regulation of ING2 expression by endoribonuclease-prepared small interfering RNA (esiRNA) microinjection results in developmental arrest during the morula to blastocyst transition. Embryonic cells microinjected with ING2-specific esiRNA exhibited decreased blastulation rate compared to the negative control. Further investigation of the underlying mechanism indicated that down-regulation of ING2 significantly increased expression of p21, whilst decreasing expression of HDAC1. These results suggest that ING2 may play a crucial role in the process of preimplantation embryo development through chromatin regulation.

Sall4 modulates embryonic stem cell pluripotency and early embryonic development by the transcriptional regulation of Pou5f1.

Embryonic stem (ES) cells are pluripotent cells that can self-renew or differentiate into many cell types. A unique network of transcription factors and signalling molecules are essential for maintaining this capability. Here, we report that a spalt family member, Sall4, is required for the pluripotency of ES cells. Similarly to Oct4, a reduction in Sall4 levels in mouse ES cells results in respecification, under the appropriate culture conditions, of ES cells to the trophoblast lineage. Sall4 regulates transcription of Pou5f1 which encodes Oct4. Sall4 binds to the highly conserved regulatory region of the Pou5f1 distal enhancer and activates Pou5f1 expression in vivo and in vitro. Microinjection of Sall4 small interfering (si) RNA into mouse zygotes resulted in reduction of Sall4 and Oct4 mRNAs in preimplantation embryos and significant expansion of Cdx2 expression into the inner cell mass. These results demonstrate that Sall4 is a transcriptional activator of Pou5f1 and has a critical role in the maintenance of ES cell pluripotency by modulating Oct4 expression. The data also indicates that Sall4 is important for early embryonic cell-fate decisions.

[Effects of testosterone on insulin sensitivity in liver cells and related molecular mechanisms].

OBJECTIVE: To explore the regulation of insulin sensitivity in liver cells by androgen signaling. METHODS: Eleven adult female C57BL/6 mice were injected daily with testosterone (group T) for 24 weeks. And 10 control mice received sesame oil only (group Con). HepG2 liver cells were initially pretreated with different doses of testosterone (10(-9)-10(-5) mol/L ) for 0-36 h or with 10(-7) mol/L testosterone for 0-96 h followed by a stimulation of 100 nmol/L insulin for 15 min. Later HepG2 cells were pretreated with 10(-7) mol/L testosterone for 36 h followed by a stimulation of 100 nmol/L insulin for 15 min and then a restimulation of 100 nmol/L insulin for 15 min at 4 h and 6 h interval respectively. Phosphorylation and protein expression of Akt and GSK3ß in C57BL/6 mice liver tissues and HepG2 cells were analyzed by Western blot. RESULTS: The 24-week treatment of testosterone decreased the phosphorylation of Akt and GSK3ß in C57BL/6 adipose and liver tissues (43.1% ± 3.2% vs 77.1% ± 6.7%, 14.7% ± 6.7% vs 82.3% ± 2.0% respectively, P < 0.05). Pretreatment with 10(-8)-10(-6) mol/L testosterone within 36 h obviously increased the phosphorylation of Akt and GSK3ß (P < 0.05). However pretreatment with 10(-5) mol/L within 36 h or with 10(-7) mol/L for 96 h had no effect on the phosphorylation of Akt and GSK3ß compared with control group (P > 0.05). Pretreatment with 10(-7) mol/L testosterone for 36 h followed by insulin stimulation and restimulation after 6 h interval obviously decreased the phosphorylation of Akt and GSK3ß (P < 0.05). CONCLUSION: Androgen signaling may contribute to insulin resistance in liver cells.

[Effects of electroacupuncture on pregnancy outcome in poor ovarian response patients of kidney essence deficiency and undergoing in vitro fertilization-embryo transfer].

OBJECTIVE: To observe the effects of electroacupuncture (EA) on ovarian reaction, egg and embryo quality, as well as pregnancy rate in poor ovarian response (POR) patients of kidney essence deficiency and undergoing in vitro fertilization-embryo transfer (IVF-ET). METHODS: Ninety-six patients who met the inclusion criteria were randomly divided into an EA group and a control group, with 48 cases in each group. Before IVF-ET, the patients in the EA group received EA, once daily, 2 or 3 treatments a week for 12 weeks. Before and after the treatment, traditional Chinese medicine (TCM) syndrome score and clinical pregnancy rate were assessed in two groups. The concentrations of serum follicle-stimulating hormone (FSH), luteinsing hormone, estradiol, progesterone and anti-mullerian hormone were detected by chemiluminescence; the contents of serum insulin-like growth factor-1, serum inhibin B (INHB) and Kisspeptin in follicular fluid were determined by enzyme linked immunosorbent assay (ELISA); the antral follicle counting (AFC) was detected by color Doppler ultrasonography; and the egg and embryo conditions were observed under microscope. Fourteen days after embryo transfer, the positive rate of serum hemchoriconic gonadotropin (HCG) and clinical pregnancy rate were calculated. RESULTS: After the treatment, the TCM syndrome score and level of serum FSH were reduced (P<0.05); the INHB in serum and AFC were increased (P<0.05) when compared with those before the treatment in the EA group. After the treatment, in comparison with the control group, the TCM syndrome score and level of serum FSH were lower (P<0.05); and the contents of serum INHB, AFC, the numbers of MⅡ eggs and high-quality embryos, as well as serum HCG positive rate were all increased (P<0.05) in the EA group. CONCLUSION: EA can relieve the clinical symptoms of TCM in POR patients of kidney essence deficiency and undergoing IVF-ET, increase the ovarian reserve, reduce the serum FSH level, and improve the content of serum INHB, and the quality of eggs and embryos. This therapy tends to improve the clinical pregnancy rate and clinical pregnancy outcome.

Apoptotic mechanism of premature ovarian failure and rescue effect of Traditional Chinese Medicine: a review.

Social and environmental factors render premature ovarian failure (POF) as a major cause of decline or loss of female fertility. The natural pregnancy rate of patients with POF is only 5%-10%. Follicular atresia is the main factor in the pathogenesis of POF. Due to the unique ovarian physiological environment and follicular developmental processes, the apoptosis of ovarian granulosa cells and oocytes together cause follicular atresia, which involves the apoptosis-related internal and external pathways. Furthermore, during POF, apoptosis and oxidative stress forms a ""vicious circle"", which involves a variety of changes between the molecules. The existing pharmaceutical preparations such as gonadal hormones are the basic methods for the treatment of POF, and the curative effect was affirmative; however, it was ineffective after withdrawn, while the long-term application led to adverse reactions. Traditional Chinese Medicine (TCM) has a history of treating gynecological diseases and infertility and has gained increasing attention. Studies have shown that compounds isolated from herbal medicine exerted a positive effect on follicular atresia caused by cell apoptosis that also improved the POF. The present study reviewed the mechanisms underlying the apoptosis in POF and elaborated the internal mechanism of TCM in the treatment of the condition.

Reduction in exposure of human embryos outside the incubator enhances embryo quality and blastulation rate.

Embryo quality is strongly dependent on the in-vitro culture environment. Conventionally, IVF/intracytoplasmic sperm injection (ICSI) embryos are examined microscopically every morning (from day 1 to day 6) to assess fertilization, cleavage and embryo quality. Consequently, the frequent exposure to non-optimal conditions outside the incubator may adversely affect embryonic viability and quality. Hence, this study investigated whether reduction of observation frequency outside the incubator can enhance blastocyst formation rate. A total of 285 IVF/ICSI cycles were divided into two groups. Embryos in the control group (103 cycles) were assessed out-of-incubator every day after insemination (day 1 to day 6; six times). In the experimental group (182 cycles), embryos were assessed four times, on days 1, 3, 5 and 6. The total blastocyst formation rate, day-5 blastocyst formation rate, proportion of good blastocysts and number of cryopreserved blastocysts per patient were significantly lower for the control group compared with the experimental group (42.5%, 31.4%, 50.7%, 1.72+/-1.55 versus 52.6%, 40.7%, 60.1%, 2.64+/-2.59, respectively, P<0.05); although there were no significant differences in the proportions of good embryos on day 3, blastocyst formation rate on day 6, clinical pregnancy rate and implantation rate. Hence, reduction of the observation frequency of embryos outside the incubator can enhance embryo quality and blastocyst formation rate.

Effect of cryotop vitrification on preimplantation developmental competence of murine morula and blastocyst stage embryos.

Vitrification is an effective method for the cryopreservation of mammalian embryos. Nevertheless, it is unclear which embryonic developmental stage is the most suited for vitrification and would ensure maximal developmental competence upon subsequent warming. This study, therefore, compared the effects of cryotop vitrification on the developmental competence of murine morula and blastocyst stage embryos. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in two hatched blastocyst groups derived from vitrified morulae and blastocysts, respectively. The post-vitrification survival rates for mouse embryos at the morula and blastocyst stage were 95.4% (186/195) and 96.5% (195/202), respectively. The blastocyst formation rate was significantly lower for vitrified morulae (90.3%) compared with the non-vitrified control group (98.4%) (P < 0.05). The hatching rates were similar between the vitrified morula (79.6%) and the vitrified blastocyst (81.0%) groups. When further development to the fully hatched blastocyst stage was compared, fully hatched blastocysts derived from vitrified morulae had significantly higher cell counts for both the ICM and TE lineage, as compared with hatched blastocysts derived from vitrified blastocysts (P < 0.001). Cryotop vitrification of mouse embryos at the morula stage rather than blastocyst stage would thus ensure a higher degree of post-warming developmental competence.

Vitrification of mouse embryos at 2-cell, 4-cell and 8-cell stages by cryotop method.

PURPOSE: The objective of this study was to investigate the effects of vitrification on the preimplantation developmental competence of mouse 2-cell, 4-cell and 8-cell stage embryos. METHODS: Mouse 2-cell, 4-cell and 8-cell stage embryos were cryopreserved using the cryotop vitrification method and subsequently warmed on a later date. The embryos were then assessed by their morphology, blastocyst formation and hatching rates. Additionally, trophectoderm (TE) and inner cell mass (ICM) cell numbers were compared in hatched blastocysts from the control and experimental groups. RESULTS: Vitrified embryos at the 2-cell, 4-cell and 8-cell stages appeared morphologically normal after warming. The overall survival rate of vitrified embryos at various stages after warming was 96.7% and there were no significant differences among 2-cell stage (96.0%), 4-cell stage (96.8%) and 8-cell stage (97.1%) embryos (P > 0.05). The blastocyst formation rate (69.4%) and hatching rate (52.6%) of vitrified 2-cell embryos were significantly lower than that from the control group and vitrified 8-cell embryos (P < 0.05). In the vitrified 4-cell embryo group, the blastocyst formation rate (90.3%) was similar to the 8-cell group (91.2%), but the hatching rate (60.0%) was significantly lower than that of the non-vitrified control ( 84.1%) and vitrified 8-cell embryo (78.4%) groups (P < 0.05). When further development to the fully hatched blastocyst stage was compared, hatched blastocysts derived from vitrified 2-cell, 4-cell and 8-cell embryos had significantly lower cell counts both in the ICM and TE, as compared to fresh blastocysts (P < 0.05). Among the vitrified 2-cell, 4-cell and 8-cell embryo groups, there were no significant differences in the cell counts of ICM and TE (P > 0.05). CONCLUSIONS: Although cryotop vitrification was suitable for the cryopreservation of mouse embryos from the 2-cell stage, 4-cell stage and 8-cell stage without significant loss of survival, vitrification had an adverse effect on the development of 2-cell embryos. Mouse embryos at the 8-cell stage had the best tolerance for vitrification and would yield the highest level of post-vitrification developmental competence among early cleavage stage embryos. Nevertheless, it is unclear how these findings can be extrapolated to human embryos.

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.

The egg-sharing model for human therapeutic cloning research: managing donor selection criteria, the proportion of shared oocytes allocated to research, and amount of financial subsidy given to the donor.

Recent advances in human therapeutic cloning made by Hwang and colleagues have opened up new avenues of therapy for various human diseases. However, the major bottleneck of this new technology is the severe shortage of human donor oocytes. Egg-sharing in return for subsidized fertility treatment has been suggested as an ethically justifiable and practical solution to overcome the shortage of donor oocytes for therapeutic cloning. Because the utilization of shared oocytes in therapeutic cloning research does not result in any therapeutic benefit to a second party, this would necessitate a different management strategy compared to their use for the assisted conception of infertile women who are unable to produce any oocytes of their own. It is proposed that the pool of prospective egg-sharers in therapeutic cloning research be limited only to younger women (below 30 years of age) with indications for either male partner sub-fertility or tubal blockage. With regards to the proportion of the shared gametes being allocated to research, a threshold number of retrieved oocytes should be set that if not exceeded, would result in the patient being automatically removed from the egg-sharing scheme. Any excess supernumerary oocyte above this threshold number can be contributed to science, and allocation should be done in a randomized manner. Perhaps, a total of 10 retrieved oocytes from the patient may be considered a suitable threshold, since the chances of conception are unlikely to be impaired. With regards to the amount of subsidy being given to the patient, it is suggested that the proportion of financial subsidy should be equal to the proportion of the patient's oocytes being allocated to research. No doubt, the promise of future therapeutic benefit may be offered to the patient instead of financial subsidy. However, this is ethically controversial because therapeutic cloning has not yet been demonstrated to be a viable model of clinical therapy and any promises made to the patient might turn out to be illusionary. Hence, it is proposed that a tangible financial subsidy on the medical fees might be the better option for the patient's welfare.

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.

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.

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.

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.

Clinical outcome of fresh and vitrified-warmed blastocyst and cleavage-stage embryo transfers in ethnic Chinese ART patients.

OBJECTIVES: This study sought to evaluate the outcome of fresh and vitrified-warmed cleavage-stage and blastocyst-stage embryo transfers in patients undergoing ART treatment within an ethnic Chinese population. STUDY DESIGN: We compared the clinical results of embryo transfer on the 3rd (cleavage stage) or 5th (blastocyst stage) day after oocyte retrieval, including clinical pregnancy rates, implantation rates and multiple pregnancy rates. RESULTS: Our data showed that blastocyst transfer on day 5 did not significantly increase clinical pregnancy rate (41.07% vs 47.08%, p>0.05) and implantation rate (31.8% vs 31.2%, p>0.05) in patients under 35 years of age, in comparison with day 3 cleavage stage embryo transfer. In patients older than 35 years of age, the clinical pregnancy rate after blastocyst transfer was slightly decreased compared with cleavage stage embryo transfer (33.33% vs 42.31%, p>0.05). Unexpectedly, It was found that vitrified-warmed blastocyst transfer resulted in significantly higher clinical pregnancy rate (56.8%) and implantation rate (47%) compared with fresh blastocyst transfer in controlled stimulation cycles (41.07% and 31.8%, respectively). For patients under 35 years of age, the cumulative clinical pregnancy rate combining fresh and vitrified-warmed blastocyst transfer cycles were significantly higher compared to just cleavage-stage embryo transfer (70.1% versus 51.8%, p<0.05). However, the cumulative multiple pregnancy rates showed no significant difference between the two groups. CONCLUSIONS: In an ethnic Chinese patient population, fresh blastocyst transfer does not significantly increase clinical pregnancy rate. However, subsequent vitrified-warmed blastocyst transfer in a non-controlled ovarian hyperstimulation cycle dramatically improves clinical outcomes. Therefore, blastocyst culture in tandem with vitrified-warmed blastocyst transfer is recommended as a favourable and promising protocol in human ART treatment, particularly for ethnic Chinese patients.

Vitrified-warmed blastocyst transfer cycles yield higher pregnancy and implantation rates compared with fresh blastocyst transfer cycles--time for a new embryo transfer strategy?

OBJECTIVE: To compare the clinical outcome of fresh versus vitrified-warmed blastocyst transfer (BT) cycles. DESIGN: Retrospective study. SETTING: Medical university affiliated hospital. PATIENT(S): Women aged less than 40 years undergoing BT cycles. INTERVENTION(S): Vitrification and warming of blastocyst with the Cryotop system. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (CPR), implantation rate (IR), and multiple pregnancy rate (MPR). RESULT(S): In 110 fresh BT cycles versus 136 vitrified-warmed BT cycles performed from January 2007 to March 2010, the IR and CPR of vitrified-warmed BT cycles were 37.0% and 55.1%, respectively, which were statistically significantly higher than the corresponding values of 25.2% and 36.4% obtained for fresh BT cycles. Additionally, the MPR was not statistically significantly different between vitrified-warmed and fresh BT cycles when a similar number of blastocysts was transferred to patients. CONCLUSION(S): Vitrified-warmed BT cycles resulted in statistically significantly higher CPR and IR compared with fresh BT cycles. A new embryo transfer strategy is therefore proposed whereby fresh BT would be avoided in the initial ovarian stimulation cycle. Instead, all the patient's available blastocysts would be vitrified-warmed and transferred in subsequent cycles.