Clinical analysis of the patients with single fair cleavage-stage embryo on day 3.

OBJECTIVE: This study aimed to explore an appropriate selection for the patients with single fair cleavage-stage embryo on day 3. METHODS: This study included 469 fresh transfers and 220 frozen-thawed transfers from January 2014 to June 2016. Furthermore, in 72 patients who have only 4-6 fair embryos (4-5 blastomeres) on day 3, the blastocysts were cultured to day 5 for transfer. RESULTS: In the fresh transfers, the clinical pregnancy rate of 4-5 blastomeres group was significantly lower than 6-7 and 8-10 blastomeres group (5.88 vs. 30.13%, p<.001and 5.88 vs. 26.09%, p < .001). In the frozen-thawed transfers, the clinical pregnancy rate of 4-5 blastomeres group was also significantly lower than 6-7 and 8-10 blastomeres group (10.00 vs. 28.57%, p = .040 and 10.00 vs. 33.33%, p = .005). For the blastocyst transfers derived from fair embryos with 4-5 blastomeres, the clinical pregnancy rate was significantly higher than single and double fair embryo transfers of similar quality (44.44 vs. 7.04%, p < .001 and 44.44 vs. 28.09%, p = .013). CONCLUSIONS: For the patients with single fair embryo (6-7 blastomeres or 8-10 blastomeres), transfer at the cleavage stage is feasible. For the patients with single fair embryo (4-5 blastomeres), transfer of single fair embryo at the blastocyst stage or accumulating two fair embryos might be worthy of consideration.

An insulin signaling feedback loop regulates pancreas progenitor cell differentiation during islet development and regeneration.

As one of the key nutrient sensors, insulin signaling plays an important role in integrating environmental energy cues with organism growth. In adult organisms, relative insufficiency of insulin signaling induces compensatory expansion of insulin-secreting pancreatic beta (ß) cells. However, little is known about how insulin signaling feedback might influence neogenesis of ß cells during embryonic development. Using genetic approaches and a unique cell transplantation system in developing zebrafish, we have uncovered a novel role for insulin signaling in the negative regulation of pancreatic progenitor cell differentiation. Blocking insulin signaling in the pancreatic progenitors hastened the expression of the essential ß cell genes insulin and pdx1, and promoted ß cell fate at the expense of alpha cell fate. In addition, loss of insulin signaling promoted ß cell regeneration and destabilization of alpha cell character. These data indicate that insulin signaling constitutes a tunable mechanism for ß cell compensatory plasticity during early development. Moreover, using a novel blastomere-to-larva transplantation strategy, we found that loss of insulin signaling in endoderm-committed blastomeres drove their differentiation into ß cells. Furthermore, the extent of this differentiation was dependent on the function of the ß cell mass in the host. Altogether, our results indicate that modulation of insulin signaling will be crucial for the development of ß cell restoration therapies for diabetics; further clarification of the mechanisms of insulin signaling in ß cell progenitors will reveal therapeutic targets for both in vivo and in vitro ß cell generation.

Origin and determination of inhibitory cell lineages in the vertebrate retina.

Multipotent progenitors in the vertebrate retina often generate clonally related mixtures of excitatory and inhibitory neurons. The postmitotically expressed transcription factor, Ptf1a, is essential for all inhibitory fates in the zebrafish retina, including three types of horizontal and 28 types of amacrine cell. Here, we show that specific types of inhibitory neurons arise from the cell-autonomous influence of Ptf1a in the daughters of fate-restricted progenitors, such as Ath5 or Vsx1/2-expressing progenitors, and that in the absence of Ptf1a, cells that would have become these specific inhibitory subtypes revert to the histogenetically appropriate excitatory subtypes of the same lineage. Altered proportions of amacrine subtypes respecified by the misexpression of Ptf1a in the Ath5 lineage suggest that Ath5-expressing progenitors are biased, favoring the generation of some subtypes more than others. Yet the full array of inhibitory cell subtypes in Ath5 mutants implies the existence of Ath5-independent factors involved in inhibitory cell specification. We also show that an extrinsic negative feedback on the expression of Ptf1a provides a control mechanism by which the number of any and all types of inhibitory cells in the retina can be regulated in this lineage-dependent way.

Secondary embryonic axis formation by transplantation of D quadrant micromeres in an oligochaete annelid.

Among spiral cleaving embryos (e.g. mollusks and annelids), it has long been known that one blastomere at the four-cell stage, the D cell, and its direct descendants play an important role in axial pattern formation. Various studies have suggested that the D quadrant acts as the organizer of the embryonic axes in annelids, although this has never been demonstrated directly. Here we show that D quadrant micromeres (2d and 4d) of the oligochaete annelid Tubifex tubifex are essential for embryonic axis formation. When 2d and 4d were ablated the embryo developed into a rounded cell mass covered with an epithelial cell sheet. To examine whether 2d and 4d are sufficient for axis formation they were transplanted to an ectopic position in an otherwise intact embryo. The reconstituted embryo formed a secondary embryonic axis with a duplicated head and/or tail. Cell lineage analyses showed that neuroectoderm and mesoderm along the secondary axis were derived from the transplanted D quadrant micromeres and not from the host embryo. However, endodermal tissue along the secondary axis originated from the host embryo. Interestingly, when either 2d or 4d was transplanted separately to host embryos, the reconstituted embryos failed to form a secondary axis, suggesting that both 2d and 4d are required for secondary axis formation. Thus, the Tubifex D quadrant micromeres have the ability to organize axis formation, but they lack the ability to induce neuroectodermal tissues, a characteristic common to chordate primary embryonic organizers.

Nuclear transfer to prevent mitochondrial DNA disorders: revisiting the debate on reproductive cloning.

Preclinical experiments are currently performed to examine the feasibility of several types of nuclear transfer to prevent mitochondrial DNA (mtDNA) disorders. Whereas the two most promising types of nuclear transfer to prevent mtDNA disorders, spindle transfer and pronuclear transfer, do not amount to reproductive cloning, one theoretical variant, blastomere transfer does. This seems the most challenging both technically and ethically. It is prohibited by many jurisdictions and also the scientific community seems to avoid it. Nevertheless, this paper examines the moral acceptability of blastomere transfer as a method to prevent mtDNA disorders. The reason for doing so is that most objections against reproductive cloning refer to reproductive adult cloning, while blastomere transfer would amount to reproductive embryo cloning. After clarifying this conceptual difference, this paper examines whether the main non-safety objections brought forward against reproductive cloning also apply in the context of blastomere transfer. The conclusion is that if this variant were to become safe and effective, dismissing it because it would involve reproductive cloning is unjustified. Nevertheless, as it may lead to more complex ethical appraisals than the other variants, researchers should initially focus on the development of the other types of nuclear transfer to prevent mtDNA disorders.

Inter-species transplantation and migration of primordial germ cells in cyprinid fish.

Primordial germ cells (PGCs) are the only cells in developing embryos that can transmit genetic information to the next generation. PGCs therefore have considerable potential value for gene banking and cryopreservation, particularly via production of donor gametes using germ-line chimeras. In some animal species, including teleost fish, the feasibility of using PGC transplantation to obtain donor-derived offspring, within and between species, has been demonstrated. Successful use of PGC transplantation to produce germ-line chimeras is absolutely dependent on the migration of the transplanted cells from the site of transplantation to the host gonadal region. Here, we induced germ-line chimeras between teleost species using two different protocols: blastomere transplantation and single PGC transplantation. We evaluated the methods using the rate of successful migration of transplanted PGCs to the gonadal region of the host embryo. First, we transplanted blastomeres from zebrafish, pearl danio, goldfish, or loach into blastula-stage zebrafish embryos. Some somatic cells, derived from donor blastomeres, were co-transplanted with the PGCs and formed aggregates in the host embryos; a low efficiency of PGC transfer was achieved. Second, a single PGC from the donor species was transplanted into a zebrafish embryo. In all inter-species combinations, the donor PGC migrated toward the gonadal region of the host embryo at a comparatively high rate, regardless of the phylogenetic relationship of the donor and host species. These transplantation experiments showed that the mechanism of PGC migration is highly conserved beyond the family barrier in fish and that transplantation of a single PGC is an efficient method for producing inter-species germ-line chimeras.

Transplantation of GFP-expressing blastomeres for live imaging of retinal and brain development in chimeric zebrafish embryos.

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed.

Generation of germ-line chimera zebrafish using primordial germ cells isolated from cultured blastomeres and cryopreserved embryoids.

Primordial germ cells (PGCs) are the only cells in developing embryos with the potential to transmit genetic information to the next generation. In our previous study, a single PGC transplanted into a host differentiated into fertile gametes and produced germ-line chimeras of cyprinid fish, including zebrafish. In this study, we aimed to induce germ-line chimeras by transplanting donor PGCs from various sources (normal embryos at different stages, dissociated blastomeres, embryoids, or embryoids cryopreserved by vitrification) into host blastulae, and compare the migration rates of the PGCs towards the gonadal ridge. Isolated, cultured blastomeres not subject to mesodermal induction were able to differentiate into PGCs that retained their motility. Moreover, these PGCs successfully migrated towards the gonadal ridge of the host and formed viable gametes. Motility depended on developmental stage and culture duration: PGCs obtained at earlier developmental stages and with shorter cultivation periods showed an increased rate of migration to the gonadal ridge. Offspring were obtained from natural spawning between normal females and chimeric males. These results provide the basis for new methods of gene preservation in zebrafish.

Vitrification of isolated human blastomeres.

This article describes a new methodology for preserving and banking isolated human blastomeres, whose originality is based on packing the blastomere into an emptied zona pellucida before vitrification. After warming, 75.7% of blastomeres survived and developed at a rate comparable to that in noncryopreserved blastomeres (62.5% cleavage, 26.6% compaction, and 20.3% cavitation).

Frozen-thawed embryo transfer cycles.

OBJECTIVE: To review the outcomes of frozen-thawed embryo transfer cycles. DESIGN. Retrospective review. SETTING: Tertiary assisted reproduction centre, Hong Kong. PATIENTS: Subfertile patients undergoing frozen-thawed embryo transfer between July 2005 and December 2007. MAIN OUTCOME MEASURES: Clinical and ongoing pregnancy rates. RESULTS: A total of 983 frozen-thawed embryo transfer cycles performed during the study period were reviewed. The clinical pregnancy and ongoing pregnancy rates were 35% and 30%, respectively. Factors associated with successful outcome included younger maternal age (<=35 years) and 4 or more blastomeres at replacement, but not the method of insemination, the cause of subfertility, or the type of frozen-thawed embryo transfer cycle. The overall multiple pregnancy rate was 18%. For cycles with a single embryo replaced, embryos having 4-cell or higher stages at replacement gave an ongoing pregnancy rate of 25%, whereas those with less than 4 cells had a significantly lower ongoing pregnancy rate of 5% only. Blastomere lysis after thawing significantly reduced the clinical pregnancy and ongoing pregnancy rates of cycles with one embryo replaced. CONCLUSIONS: Clinical pregnancy and ongoing pregnancy rates of frozen-thawed embryo transfer cycles were 35% and 30%, respectively. Higher pregnancy rates were associated with younger maternal age (<=35 years), blastomere numbers of 4 or more, and no blastomere lysis after thawing.

Donor-recipient pairs to evaluate the effect of day 3 embryos having at least six blastomeres on pregnancy outcome.

PURPOSE: To determine if having all embryos transferred with at least six blastomeres improves pregnancy rates compared to women having an embryo transfer with at least one embryo with less than six cells. METHODS: Donor-recipient pairs were used to help remove the confounding effect of egg quality. Four donor-recipient pair types were evaluated: (1) both donor and recipient had all embryos with at least six cells, (2) neither donor nor recipient had all embryos with > or =6 cells, (3) donor but not recipient had all > or = six cell embryos, (4) recipient but not donor had all embryos with > or = six cells. Combining donor and recipients there was a significantly higher pregnancy rate per transfer when all embryos had at least six blastomeres (50/92 or 54.3%) vs. the group with at least one embryo with <6 cells (46/121 or 38.8%). Similarly the implantation rate was significantly higher (37.8% vs. 20.3%). CONCLUSIONS: These data corroborate conclusions made by evaluating single embryo transfer in women with diminished egg reserve that the presence of at least six blastomeres is associated with a better chance of a given embryo to implant

Culture medium preferences of pre-implantation cloned mouse embryos.

Somatic cloning technology involves the transfer of a somatic cell nucleus into an enucleated oocyte, followed by activation and in vitro culture. Efficiency in terms of live offspring generally remains very low. Little attention has been devoted so far to the impact of culture environment on cloned embryo development. Failure of genomic reprogramming of the donor nucleus in nuclear transfer (NT) experiments could lead to an altered phenotype in these cloned embryos that could be manifested by different medium preferences of the NT embryos. We describe here the application of sequential culture media to support preimplantation development of mouse embryos reconstructed using conventional NT techniques. Embryo-quality analysis was performed on NT blastocysts obtained. Additionally, NT embryos that arrested during development also were analyzed.

Are there any normal clones?

An exhaustive study of the fidelity of a clone to its parent is prohibitive because of cost and the necessary scope of experimental design. Therefore, these data must be gathered from existing observational evidence. This in itself cannot provide a definitive accounting of the abnormalities and variation found among clones or between clones and parents because there is no standardization in the data points collected between one study and another. This literature survey shows that clone developmental abnormalities, variation among clones, and variation between clone and parent are prevalent at most stages of development (cleavage, placental, fetal, neonatal, maturity), and that occasionally the observed variation greatly exceeds that which might be expected. Some variation can be explained by differences in protocols and procedures between studies. The choice of nuclear donor cell is particularly influential of variation observed between a clone and its parent. In general, however, it appears that there is an inherent stochastic response to nuclear transfer that results in clone infidelity and variation. The survey of characteristics of clone infidelity to parent and documentation of abnormalities provided here should not be viewed as exhaustive or limiting in the recording of such data from future studies. Because controlled hypothesis testing of clone fidelity or clone health may not be possible, meticulous documentation of such observational evidence is a valuable contribution to the field.

Generation of transgenic rabbits by the novel technique of chimeric somatic cell cloning.

A novel technique of chimeric somatic cell cloning was applied to produce a transgenic rabbit (NT20). Karyoplasts of transgenic adult skin fibroblasts with Tg(Wap-GH1) gene construct as a marker were microsurgically transferred into one, previously enucleated, blastomere of 2-cell non-transgenic embryos, while the second one remained intact. The reconstructed embryos either were cultured in vitro up to the blastocyst stage (Experiment I) or were transferred into recipient-females immediately after the cloning procedure (Experiment II). In Experiment I, 25/102 (24.5%) embryos formed blastocysts from whole embryos and 46/102 (44.12%) embryos developed to the blastocyst stage from single non-operated blastomeres, while the reconstructed blastomeres were damaged and degenerated. Thirteen (12.7%) embryos did not exceed 3- to 4-cell stages and 18 (17.7%) embryos were inhibited at the initial 2-cell stage. Out of 14 blastocysts which were subjected to molecular analysis, the transgene was detected in the cells of 4 blastocysts. In Experiment II, 163/217 (75.0%) embryos were transferred into 9 pseudopregnant recipient-rabbits (an average of 18 embryos per recipient). Four recipient-females (44.4%) became pregnant and delivered a total of 24 (14.7%) pups. Molecular analysis confirmed that two pups (1.2%), one live and one stillborn, showed a positive transgene signal. Live transgenic rabbit NT20 appeared healthy and anatomically as well as physiologically normal. The results of our experiments showed that transgenic adult skin fibroblast cell nuclei, which have been introduced into the cytoplasmic microenvironment of single enucleated blastomeres from 2-cell stage rabbit embryos, are able to direct the development of chimeric embryos not only to the blastocyst stage but also up to term.

Towards defining parameters for a successful single embryo transfer in frozen cycles.

BACKGROUND: Twin pregnancies in IVF should be avoided by transferring embryos one at a time, even for frozen cycles. In this study, we investigated the effect of blastomere lysis and cleavage in singleton frozen embryo transfer (sFET) cycles. Outcomes were compared with the transfer of two embryos in frozen transfer cycles (dFET). METHODS: A retrospective analysis was performed on 891 FET cycles, involving 404 sFET and 487 dFET cycles. RESULTS: Overall, in sFET cycles, the pregnancy and implantation rates were 8.9 and 8.7%. When blastomere lysis was more than 25% but no greater than 50%, the pregnancy and implantation rates were 3.2%. If blastomere lysis was greater than 50% there were no pregnancies. If blastomere lysis was less than 25%, but with no cleavage, the pregnancy and implantation rates were 4.1%. The results significantly improved (P = 0.007) in the group with less than 25% lysis, when cleavage occurred. The pregnancy and implantation rates for this group were 17.3 and 16.6%. This was not significantly different from unselected two embryo transfers (22 and 12.7%,P = 0.2 and 0.19, respectively). There were 21 twins with dFET (19.6% of pregnancies) and none in sFET. CONCLUSION: Both blastomere lysis and cleavage affect the outcome in sFET. To avoid the risk of twins, sFET should be considered when the embryo shows less than 25% blastomere lysis and at least one blastomere cleaves.

Blastomere transplantation in human embryos may be a treatment for single gene diseases.

OBJECTIVE: To determine whether human embryos accept blastomere transplants and integrate them normally into the architecture of the developing embryo. DESIGN: A human blastomere transplantation model, involving 44 cryopreserved embryos that were specifically donated to research. SETTING: Academically affiliated private infertility center. PATIENT(S): Forty-four human embryos. INTERVENTION(S): In 21 experiments, one, two, or three blastomeres were transplanted, using standard microsurgical techniques that are widely used in preimplantation genetic diagnosis (PGD). Embryos were thawed and gender was determined, using established PGD techniques. Male (xy) blastomeres were then transplanted into female (xx) day 3 embryos, and the xy cells were tracked through blastocyst stage (days 5-6) and into the hatching period (day 6), using fluorescent in situ hybridization (FISH). MAIN OUTCOME MEASURE(S): Degree and location of xy cell integration into xx embryos. RESULT(S): High-quality recipient embryos (with 4 to 10 cells) developed uniformly into normal blastocyst stage embryos in 12 of 12 experiments (100%) and integrated donor blastomeres into their architecture, with apparently even distribution of daughter cells; this integration was documented in inner cell mass as well as in trophoectoderm. The intensity of this distribution appeared to correlate with the number of blastomeres transferred. Among nine abnormally developing embryos, only three (33%) demonstrated a normal distribution of offspring donor cells. CONCLUSION(S): High-quality embryos appear to have the ability to integrate donor blastomeres. Because the treatment of single gene diseases does not require successful treatment of all cells, blastomere transplantation could be explored as a treatment option, which also would greatly enhance efficiency and utilization of preimplantation genetic diagnosis.

Pluripotency of cryopreserved blastomeres of the goldfish.

To examine the pluripotency of cryopreserved blastomeres, we transplanted them into blastula. Donor blastomeres were prepared from blastula of goldfish (Carassius auratus) and cryopreserved in liquid nitrogen for two months. Fifty-five percent and 44% of blastomeres survived after thawing. Cryopreserved blastomeres were transplanted to the blastula of triploid crucian carp (C. a. longsdorfii), which reproduces gynogenetically in nature. At four days after the operation, resultant chimeric embryos transplanted with cryopreserved blastomeres showed a survival rate (41.6%) lower than that of embryos transplanted with unfrozen blastomeres (57.1%). Transplanted blastomeres were histologically identified in various organs derived from all three germ layers. A primordial germ cell differentiated from a cryopreserved blastomere was detected in one of the 32 chimeric fish examined. These results suggest blastomeres that survive after cryopreservation retain their pluripotency and are able to differentiate into both somatic and germ cell lines.

Transfer of nonselected transferable day 3 embryos in low embryo producers.

OBJECTIVE: To examine the implantation potential of embryos from assisted reproductive technology cycles with low embryo production and to assess the effects of clinical variables and embryo scores (ES) on pregnancy outcome. DESIGN: Prospective clinical study. SETTING: Assisted reproductive technology unit in a tertiary medical center. PATIENT(S): From July 1998 to December 2001, 280 cycles in 229 infertile couples produced a limited number of one, two, or three embryos 3 days after oocyte retrieval and underwent fresh embryo transfer (ET). INTERVENTION(S): Embryos with two or more blastomeres were scored and transferred. MAIN OUTCOME MEASURE(S): ES and implantation rate per ET. RESULT(S): Of 863 fresh ET cycles during the study period, 32.4% (280) were low embryo producers. Among them, there were no significant differences in average ES of individual embryos in single, dual, or triple ET or in embryos obtained from patients with low or high E2 responses, or young or old age. Embryos derived from conventional IVF had a better ES than those derived from intracytoplasmic sperm injection. The clinical pregnancy rate was strongly correlated with the cumulative ES. Implantation rates were similar among and between groups, with an average rate of 15.9%. CONCLUSION(S): Embryos of low embryo producers had an inherently low implantation potential that appeared to be unrelated to the number of embryos transferred, female age, ovarian E2 genesis, or fertilization method. The cumulative ES can serve as a predictor of pregnancy.

Production of germ-line chimeras in rainbow trout by blastomere transplantation.

We describe a technique for producing germ-line chimeric rainbow trout, Oncorhynchus mykiss, by microinjection of the isolated blastomeres. FITC-labeled donor cells and non-labeled recipient embryos at various developmental stages between the early blastula and early gastrula stages were used for cell transplantation. The chimera formation rate and the degree of donor cell distribution in recipient embryos were evaluated at both the late gastrula stage (5 days post fertilization (dpf)) and the 40-somite stage (10 dpf). Among the six combinations of developmental stages of donor and recipient embryos, the combination of midblastula (2.5 dpf) donor cells and early blastula (1.5 dpf) recipient embryos gave the highest chimera formation rate and the best distribution pattern of donor cells. Using this combination, chimeric rainbow trout were produced with donor blastomeres from dominant orange-colored mutant embryos and wild-type recipient embryos. Of the 238 chimeric embryos produced, 28 (12%) hatched normally and 14 of the 28 fry (50%) had donor-derived orange body color. To test for germ-line transmission of donor cells, gametes obtained from the matured chimeras were fertilized with gametes from wild-type fish. Of the 19 matured chimeras, 6 (32%) yielded donor-derived orange-colored progeny, in addition to wild-type siblings. The contribution rates of donor cells in the germ-line ranged from 0.3 to 14%. This technique for producing germ-line chimeras should be a powerful tool for cell-mediated gene transfer in rainbow trout. Especially, if body color mutants are used for either donor cells or the host embryos, it will be possible to easily concentrate F1 transgenic embryos derived from transplanted donor cells by body color screening. Mol. Reprod. Dev. 59: 380-389, 2001.