Lack of cell cycle checkpoints in human cleavage stage embryos revealed by a clonal pattern of chromosomal mosaicism analysed by sequential multicolour FISH

Multicolour fluorescence in situ hybridisation (FISH) analysis of interphase nuclei in cleavage stage human embryos has highlighted a high incidence of postzygotic chromosomal mosaicism, including both aneuploid and ploidy mosaicism. Indeed, some embryos appear to have a chaotic chromosomal complement in a majority of nuclei, suggesting that cell cycle checkpoints may not operate in early cleavage. Most of these studies, however, have only analysed a limited number of chromosomes (3-5), making it difficult to distinguish FISH artefacts from true aneuploidy. We now report analysis of 11 chromosomes in five sequential hybridisations with standard combinations of two or three probes and minimal loss of hybridisation efficiency. Analysis of a series of arrested human embryos revealed a generally consistent pattern of hybridisation on which was superimposed frequent deletion of one or both chromosomes of a specific pair in two or more nuclei indicating a clonal origin and continued cleavage following chromosome loss. With a binucleate cell in a predominantly triploid XXX embryo, the two nuclei remained attached during preparation and the chaotic diploid/triphoid status of every chromosome analysed was the same for each nucleus. Furthermore, in each hybridisation the signals were distributed as a mirror-image about the plane of attachment, indicating premature decondensation during anaphase consistent with a lack of checkpoint control.     

Incidence of chromosomal mosaicism in human embryos at different developmental stages analyzed by fluorescence in situ hybridization

Chromosomal mosaicism has been reported in in vitro-cultured embryos at early cleavage stages, as well as in morulae and blastocysts. We have assessed the incidence and pattern of mosaicism during in vitro development of human embryos from early-cleavage stages to morula and blastocyst. Fifty spare embryos were fixed for fluorescence in situ hybridization (FISH) analysis for chromosomes X, Y, 13, 18, and 21 on days 2 or 3 (4- to 10-cell stage) (n = 16), on day 4 (morula stage) (n = 14), on day 5 (pre-expanded blastocyst) (n = 5), and the expanded blastocyst stages (n = 15). Blocked embryos (no cleavage observed within the last 24 hr) were not included. A total of 2367 cells were analyzed. Four early-cleavage stage embryos were found uniformly diploid; all of the others were mosaic for the chromosomes analyzed (mean diploid nuclei 48.3% +/- 28.7). All of the embryos at more advanced developmental stages, except one fully normal morula, had mosaic chromosome constitutions, with an increase in the percentage of diploid cells in morulae, pre-expanded, and expanded blastocysts, respectively (mean diploid nuclei 78.6% +/- 11.7, 66.0% +/- 20.8, 79.6% +/- 12.8), in comparison with earlier stages. Hypotheses about the origin of mosaicism and embryo regulation mechanisms will be discussed.     

From oocyte to embryo: a model, deduced from in vitro fertilization, for natural selection against chromosome abnormalities

A cytogenetical analysis was performed on 151 unfertilized oocytes, 22 fertilized eggs at the pronuclear stage, and 108 cleaved embryos obtained in the course of in vitro fertilization (IVF). Thirty-two per cent of unfertilized oocytes were abnormal, carrying nullisomies or disomies, mainly of D and G chromosomes, and a structural anomaly (Gq-) in one case. Fertilized eggs showed frequent asynchronism in the development of pronuclei and only 2 out of 8 karyotyped pronuclei were normal. Cleaved embryos were classified according to the number of pronuclei observed 17 hours after insemination. One per cent displayed a single pronucleus, and haploid chromosome complements were found in the corresponding cleaved embryos which were considered to be parthenotes. The rate of chromosome abnormalities of diploid eggs depended on their morphological aspect. Healthy cleaved embryos carried 12.5% of anomalies while this rate reached 37% in fragmented embryos (p less than 0.05). Lastly, 6% of fertilized eggs displayed three pronuclei or more. Only 41% of the corresponding embryos were triploid. Diploidy or diploidtriploid mosaicism were often encountered. This leads to a 21% rate of abnormalities in the preimplantation embryos. Parental karyotyping and HLA typing were carried out in a series of eight couples with in vitro idiopathic infertility or recurrent embryo degeneration in vitro. No abnormality was noted. According to these results, a model of natural selection of normal conceptuses is proposed.     

Increase of ICSI efficiency with hyaluronic acid binding sperm for low aneuploidy frequency in pig

The present study was designed to evaluate the ability of hyaluronic acid binding sperm (HABS) in increasing the efficiency of intracytoplasmic sperm injection (ICSI) in terms of the production of chromosomally normal porcine embryos. Porcine embryos were produced by in vitro fertilization (IVF), ICSI and ICSI using hyaluronic acid binding sperm (ICSI-HABS). Chromosome aneuploidy in sperm and embryos was evaluated using chromosome 1 submetacentric probe for fluorescence in situ hybridization (FISH) analysis. No significant differences were observed in the blastocysts rates (18.6, 23.6 and 23.8%) and cell numbers (61.8+/-12.5, 55.5+/-7.3 and 59.3+/-9.6) among embryos derived from IVF, ICSI, and ICSI-HABS. However, the frequency of normal diploidy in ICSI-HABS (75.5%) was significantly higher (P<0.05) than that in IVF (57.0%) and ICSI (68.2%). Embryos from ICSI-HABS showed significantly lower chromosome abnormality rate (P<0.05). Both ICSI and IVF embryos showed higher rates of polyploidy, and hence chromosomally abnormal embryos, in comparison to ICSI-HABS embryos. In addition, we investigated the chromosomal complement of porcine spermatozoa by FISH. The rate of chromosome number abnormality in porcine sperm was found to be 6.25% (70/1120). Thus, we conclude that the use of hyaluronic acid binding sperm is superior to morphological sperm selection for ICSI in producing chromosomally normal embryos and increasing the ICSI efficiency by lowering the aneuploidy frequency. Our results indicate that the selection of normal sperm with hyaluronic acid binding assay might help to reduce the early embryonic mortality due to chromosomal aneuploidy thereby increasing the success rate of embryo transfer technology in pigs.     

Frequency of sex chromosomal mosaicism in bovine embryos and its effects on sexing using a single blastomere by PCR

Assessment of nuclear status is important when a biopsied single blastomere is used for embryo sexing. In this study we investigated the nuclear status of blastomeres derived from 8- to 16-cell stage in vitro fertilised bovine embryos to determine the representativeness of a single blastomere for embryo sexing. In 24 embryos analysed, the agreement in sex determination between a biopsied single blastomere and a matched blastocyst by polymerase chain reaction (PCR) was 83.3%. To clarify the discrepancies, karyotypes of blastomeres in 8- to 16-cell stage bovine embryos were analysed. We applied vinblastine sulfate at various concentrations and for different exposure times for metaphase plate induction in 8- to 16-cell stage bovine embryos. The 1.0 mg/ml vinblastine sulfate treatment for 15 h was selected as the most effective condition for induction of a metaphase plate (> 45%). Among 22 embryos under these conditions, only 8 of 10 that had a normal diploid chromosome complement showed a sex chromosomal composition of XX or XY (36.4%) and 2 diploid embryos showed mosaicism of the opposite sex of XX and XY in blastomeres of the embryo (9.1%). One haploid embryo contained only one X-chromosome (4.5%). Four of another 11 embryos with a mixoploid chromosomal complement contained a haploid blastomere with a wrong sex chromosome (18.2%). In conclusion, assessment of nuclear status of 8- to 16-cell stage bovine embryos revealed that morphologically normal embryos had a considerable proportion of mixoploid blastomeres and sex chromosomal mosaicism; these could be the cause of discrepancies in the sex between biopsied single blastomeres and matched blastocysts by PCR.     

Regional and developmental characteristics of human embryo mosaicism revealed by single cell sequencing

Chromosomal mosaicism is common throughout human pre- and post-implantation development. However, the incidence and characteristics of mosaicism in human blastocyst remain unclear. Concerns and confusions still exist regarding the interpretation of chromosomal mosaicism on preimplantation genetic testing for aneuploidy (PGT-A) results and embryo development. Here, we aimed to estimate the genetic concordance between trophectoderm (TE), inner cell mass (ICM) and the corresponding human embryonic stem cells (hESCs), and to explore the characteristics of mosaicism in human blastocyst and hESCs on a single cell level. The single cell sequencing results of TE cells indicated that 65.71% of the blastocysts were mosaic (23 in 35 embryos), while the ICM sequencing results suggested that 60.00% of the blastocysts were mosaic (9 in 15 embryos). The incidence of mosaicism for the corresponding hESCs was 33.33% (2 in 6 embryos). No significant difference was observed between the mosaic rate of TE and that of ICM. However, the mosaic rate of the corresponding hESCs was significantly lower than that of TE and ICM cells, suggesting that the incidence of mosaicism may decline during embryonic development. Upon single cell sequencing, we found several “complementary” copy number variations (CNVs) that were usually not revealed in clinical PGT-A which used multi-cell DNA sequencing (or array analysis). This indicates the potential diagnostic risk of PGT-A based multi-cell analysis routinely in clinical practice. This study provided new insights into the characteristics, and considerable influences, of mosaicism on human embryo development, as well as the clinical risks of PGT-A based on multi-cell biopsies and bulk DNA assays.     

Mosaicism diminishes the value of pre-implantation embryo biopsies for detecting CRISPR/Cas9 induced mutations in sheep

The production of knock-out (KO) livestock models is both expensive and time consuming due to their long gestational interval and low number of offspring. One alternative to increase efficiency is performing a genetic screening to select pre-implantation embryos that have incorporated the desired mutation. Here we report the use of sheep embryo biopsies for detecting CRISPR/Cas9-induced mutations targeting the gene PDX1 prior to embryo transfer. PDX1 is a critical gene for pancreas development and the target gene required for the creation of pancreatogenesis-disabled sheep. We evaluated the viability of biopsied embryos in vitro and in vivo, and we determined the mutation efficiency using PCR combined with gel electrophoresis and digital droplet PCR (ddPCR). Next, we determined the presence of mosaicism in?~?50% of the recovered fetuses employing a clonal sequencing methodology. While the use of biopsies did not compromise embryo viability, the presence of mosaicism diminished the diagnostic value of the technique. If mosaicism could be overcome, pre-implantation embryo biopsies for mutation screening represents a powerful approach that will streamline the creation of KO animals.     

The genetic screening of preimplantation embryos by comparative genomic hybridisation

Comparative genomic hybridization (CGH) is an indirect DNA-based test which allows for the accurate analysis of aneuploidy involving any of the 24 types of chromosomes present (22 autosomes and the X and Y sex chromosomes). Traditionally, embryos have been screened using fluorescence in situ hybridization (FISH)--a technique that was limited in the number of chromosomes able to be identified in any one sample. Early CGH reports on aneuploidy in preimplantation embryos showed that any of the 24 chromosomes could be involved and so FISH methods were going to be ineffective in screening out abnormal embryos. Our results from routine clinical application of array CGH in preimplantation genetic diagnosis (PGD) patients confirm previous reports on patterns of chromosomal contribution to aneuploidy. The pregnancy outcomes following embryo transfer also indicate that despite the requirement to freeze embryos, rates are encouraging, and successful ongoing pregnancies can be achieved.     

Preimplantation genetic diagnosis: State of the ART 2011

For the last 20 years, preimplantation genetic diagnosis (PGD) has been mostly performed on cleavage stage embryos after the biopsy of 1–2 cells and PCR and FISH have been used for the diagnosis. The main indications have been single gene disorders and inherited chromosome abnormalities. Preimplantation genetic screening (PGS) for aneuploidy is a technique that has used PGD technology to examine chromosomes in embryos from couples undergoing IVF with the aim of helping select the chromosomally ‘best’ embryo for transfer. It has been applied to patients of advanced maternal age, repeated implantation failure, repeated miscarriages and severe male factor infertility. Recent randomised controlled trials (RCTs) have shown that PGS performed on cleavage stage embryos for a variety of indications does not improve delivery rates. At the cleavage stage, the cells biopsied from the embryo are often not representative of the rest of the embryo due to chromosomal mosaicism. There has therefore been a move towards blastocyst and polar body biopsy, depending on the indication and regulations in specific countries (in some countries, biopsy of embryos is not allowed). Blastocyst biopsy has an added advantage as vitrification of blastocysts, even post biopsy, has been shown to be a very successful method of cryopreserving embryos. However, mosaicism is also observed in blastocysts. There have been dramatic changes in the method of diagnosing small numbers of cells for PGD. Both array-comparative genomic hybridisation and single nucleotide polymorphism arrays have been introduced clinically for PGD and PGS. For PGD, the use of SNP arrays brings with it ethical concerns as a large amount of genetic information will be available from each embryo. For PGS, RCTs need to be conducted using both array-CGH and SNP arrays to determine if either will result in an increase in delivery rates.     

Mechanisms of aneuploidy induction in human oogenesis and early embryogenesis

The mechanisms of aneuploidy induction in human oogenesis mainly involve nondisjunction arising during the first and second meiotic divisions. Nondisjunction equally affects both whole chromosomes and chromatids, in the latter case it is facilitated by "predivision" or precocious centromere division. Karyotyping and CGH studies show an excess of hypohaploidy, which is confirmed in studies of preimplantation embryos, providing evidence in favour of anaphase lag as a mechanism. Preferential involvement of the smaller autosomes has been clearly shown but the largest chromosomes are also abnormal in many cases. Overall, the rate of chromosomal imbalance in oocytes from women aged between 30 and 35 has been estimated at 11% from recent karyotyping data but accruing CGH results suggest that the true figure should be considerably higher. Clear evidence has been obtained in favour of germinal or gonadal mosaicism as a predisposing factor. Constitutional aneuploidy in embryos is most frequent for chromosomes 22, 16, 21 and 15; least frequently involved are chromosomes 14, X and Y, and 6. However, embryos of women under 37 are far more likely to be affected by mosaic aneuploidy, which is present in over 50% of 3-day-old embryos. There are two main types, diploid/aneuploid and chaotic mosaics. Chaotic mosaics arise independently of maternal age and may be related to centrosome anomalies and hence of male origin. Aneuploid mosaics most commonly arise by chromosome loss, followed by chromosome gain and least frequently by mitotic nondisjunction. All may be related to maternal age as well as to lack of specific gene products in the embryo. Partial aneuploidy as a result of chromosome breakage affects a minimum of 10% of embryos.     

Statistical models predicting embryo survival to term in cattle after embryo transfer

Embryo survival to term in recipient cattle is highly variable. We examined calving data in the published literature to determine whether a model of binomial independence or a model which includes an embryo (e) and recipient term (r), adequately explain observed embryo survival rates following attempts to induce twin calving using transfer of two embryos. To achieve this we examined 32 published papers which provided us with 47 sets of data concerning 4560 recipients with either 0, 1 or 2 calves born. In each set of data, the observed embryo survival rate to term (p) (number of calves born/number of embryos) was calculated and the expected number of recipients with either 0, 1 or 2 calves born was determined, assuming a binomial distribution. Parameters for the second model were estimated using maximum-likelihood procedures. The model of embryo independence was rejected in 85% of the sets of data, suggesting that factors other than the embryo are important sources of variation in embryo survival or loss. The proposed e and r model of embryo survival adequately describes the published data in recipients receiving either single or twin embryos. In general, only 50-70% of embryos and recipients are sufficiently competent to result in a calving. Variation among laboratories producing either in vitro or in vivo derived embryos was due to variation in recipient and not embryo competence. It is argued that e rather than observed embryo survival rate, and r rather than observed pregnancy rate, should be used to compare differences among embryo treatments and groups of recipients, respectively. Acceptance of this proposition should permit faster progress in identifying the biology of superior embryos and recipients, which is a prerequisite to improving embryo survival rate in cattle. Collectively, the published data are not consistent with a model of embryo independence, and that a model of embryo survival to term which recognises recipient as well as embryo contributions to embryo survival may be more appropriate in cattle.     

A retrospective study of preimplantation embryos diagnosed with monosomy by fluorescence in situ hybridization (FISH)

This report is a retrospective study of preimplantation embryos diagnosed with monosomy for chromosomes 13, 15, 16, 18, 21, 22, X and Y on day 3 to determine the rate of true positives, false positives and/or mosaicism and to assess if these embryos are suitable for in vitro fertilization (IVF) transfer. In a one year period, 80 patients went through preimplantation genetic diagnosis for aneuploidy screening (PGD-AS). Monosomy was diagnosed in 51 embryos. Fluorescence in situ hybridization (FISH) was then performed on the blastomeres at day 5-7 with commercially available probes using the same probe set that initially identified monosomy for chromosomes 13, 16, 21 and 22 or chromosomes 15, 18, X and Y. Based on FISH analysis, the monosomy diagnosed during routine PGD-AS analysis was confirmed in 17 of the 51 embryos. A euploid result for the specific chromosomes tested was observed in 16 of the 51 embryos while mosaicism was found in the remaining 18 embryos. This results in an estimated false positive rate of 3.8% for a diagnosis of monosomy. Reanalysis of these embryos demonstrates that the majority of monosomy diagnoses represents true monosomy or mosaicism and should be excluded for transfer in IVF. Furthermore, improved understanding from recent emerging data regarding the fate of oocytes in women with advanced maternal age undergoing IVF to the development of early embryos may provide a valuable insight into the mechanism of chromosome mosaicism.     

The role of structural integrity of the fertilising spermatozoon in early human embryogenesis.

While the fertilising spermatozoon supplies the active centre directing the human zygote's first mitotic division, the relative contributions of the sperm head and tail (as well as the importance of the sperm's general structural integrity) to subsequent developmental processes remain incompletely studied. The sperm nucleus contains paternal chromatin necessary for restoration of a diploid genome, but the functional role of the sperm tail (either attached or dissected) in early human embryonic growth is not known. In this investigation using oocytes donated by in vitro fertilisation patients, human oocytes were injected with isolated sperm heads (n = 73), isolated sperm flagella (n = 11) or both (dissected sperm heads + free sperm tails, n = 26). The formation of bipronucleate zygotes was recorded for each method. Among oocytes surviving injection with isolated sperm heads, 44 of 66 (67%) formed two pronuclei. Of oocytes receiving only sperm tails, 2 of 11 (18%) displayed two pronuclei, but a single polar body was evident in both cases. When dissected spermatozoa parts (head + tail) were jointly injected, 12 of 26 (46%) developed two pronuclei. From embryos resulting from each of these three fertilisation regimes, blastomere biopsies were obtained and subjected to multiprobe fluorescent in situ hybridisation (FISH) analysis to detect mosaicism or aneuploidy arising from these experimental treatments. Only embryos with growth sufficient to permit sampling of at least two blastomeres were evaluated, and FISH analysis was successful in 25 of 29 (86%) embryos tested. Of 12 embryos derived from injection of an isolated sperm head, only one was normal diploid; the remaining 11 were mosaic. Both embryos resulting from injection of an unattached sperm tail were mosaic. Of 11 embryos generated from oocyte injection with sperm head + tail segments, 10 (91%) were mosaic and only one was normal diploid. Results from this study show that injection of isolated sperm segments can permit oocyte activation and bipronuclear formation. However, a high rate of mosaicism in human embryos originating from disrupted sperm or sperm components suggests that more than a 'sum of parts' is needed for later development. The structural integrity of the intact fertilising spermatozoon appears to contribute to normal human early embryogenesis.     

FISH analysis reveals aneuploidy and continual generation of chromosomal mosaicism in Leishmania major

The protozoan parasite Leishmania is generally considered to be diploid, although a few chromosomes have been described as aneuploid. Using fluorescence in situ hybridization (FISH), we determined the number of homologous chromosomes per individual cell in L. major (i) during interphase and (ii) during mitosis. We show that, in Leishmania, aneuploidy appears to be the rule, as it affects all the chromosomes that we studied. Moreover, every chromosome was observed in at least two ploidy states, among monosomic, disomic or trisomic, in the cell population. This variable chromosomal ploidy among individual cells generates intra-strain heterogeneity, here precisely chromosomal mosaicism. We also show that this mosaicism, hence chromosome ploidy distribution, is variable among clones and strains. Finally, when we examined dividing nuclei, we found a surprisingly high rate of asymmetric chromosome allotments, showing that the transmission of genetic material during mitosis is highly unstable in this 'divergent' eukaryote: this leads to continual generation of chromosomal mosaicism. Using these results, we propose a model for the occurrence and persistence of this mosaicism. We discuss the implications of this additional unique feature of Leishmania for its biology and genetics, in particular as a novel genetic mechanism to generate phenotypic variability from genomic plasticity.     

Chromosome analysis of blastomeres from human embryos by using comparative genomic hybridization

Karyotypic studies of aborted fetuses have been used to draw the inference that the proportion of conceptuses with chromosome abnormalities is very high. Fluorescent in situ hybridization (FISH) studies of blastomeres from early cleavage embryos have provided some support for this inference but they are limited to the study of a few chromosomes. We describe the novel application of comparative genomic hybridization (CGH) to the study of numerical and structural abnormalities of single blastomeres from disaggregated 3-day-old human embryos. CGH results were obtained for 63 blastomeres from 12 embryos. Identification of all chromosomes with the exception of chromosomes 17, 19, 20 and 22 was possible. The embryos divided into four groups: (1) embryos with a normal CGH karyotype seen in all blastomeres; (2) embryos with consistent aneuploidy suggesting meiotic non-disjunction had occurred; (3) embryos that were mosaic generally with one or more cells showing aneuploidy for one or two chromosomes but some with cells showing extensive aneuploidy; and (4) one embryo with extensive aneuploidy in all blastomeres. The extensive aneuploidy in group 4 is interpreted as corresponding to the random aneuploidy seen in "chaotic" embryos reported by using interphase FISH. Partial chromosome loss and gain following chromosome breakage was observed in one embryo. Our analysis provides basic biological information on the occurrence of constitutional and post-zygotic chromosome abnormalities in early human embryos. Used in conjunction with embryo biopsy, diagnostic CGH should allow the exclusion of a proportion of embryos that appear normal but that have a poor probability of survival and, therefore, may improve the implantation rate after in vitro fertilization.     

Relationship between morphology and chromosomal constitution in human preimplantation embryo

In in vitro fertilization (IVF) procedures, morphologic embryo grading is the sole criteria for selection of embryos transferable in utero. Cytogenetic analysis of preimplantation embryos was performed to investigate the relationship between chromosomal status and morphologic quality of preimplantation eggs. Aneuploidy was the most frequently observed abnormality. In addition, various types of aberrations such as polyploidy, haploidy, mosaicism, and fragmentation were also found. Our results, pooled with data drawn from previous reports, demonstrated the prognostic value of the embryo grading system as a means for eliminating chromosomally abnormal embryos. In contrast, data suggested that some aspects of the IVF process might be responsible for the occurrence of these abnormalities. © 1994 Wiley-Liss, Inc.     

人-兔异种核移植构建克隆胚的实验研究

“治疗性克隆”是人类最关注的课题之一,而人体细胞核移植是治疗性克隆的基础和前提。异种核移植的方法虽已被引入人体细胞克隆胚的构建,但供体细胞的类型、培养代数及准备方法与其效率之间的关系尚有待探讨。本实验以不同培养代数和不同准备方法的人卵丘细胞、皮肤成纤维细胞和软骨细胞为供体构建了克隆胚,对其发育情况的比较表明,以卵丘细胞为供体时重构胚的体外发育率高于其余二者,差异显著（P〈0．05）;不同培养代数的成纤维细胞克隆胚和不同冷藏天数供体细胞克隆胚体外发育率无明显差异。此外,本实验还尝试用荧光原位杂交法检测所构建的异种克隆胚核遗传物质的来源,结果显示来自人体细胞。本研究表明,人一兔异种核移植构建克隆胚切实可行;体细胞的类型与核移植效率相关;供体细胞的体外培养传代对克隆胚的发育并无影响;而冷藏是一种简便有效的供体细胞准备方法;此外,用FISH方法对重构胚进行核遗传物质的鉴定切实可行。     

Experimental cloning of embryos through human-rabbit inter-species nuclear transfer

Therapeutic cloning,which is based on human somatic cell nuclear transfer,is one of our major research objectives.Though inter-species nuclear transfer has been introduced to construct human somatic cell cloned embryos,the effects of type,passage,and preparation method of donor cells on embryo development remain unclear.In our experiment,cloned embryos were reconstructed with different passage and preparation methods of ossocartilaginous cell,skin fibroblast,and cumulus cells.The cumulus cell embryos showed significantly higher development rates than the other two (P＜0.05).The development rate of embryos reconstructed with skin fibroblasts of different passage number and somatic cells of different chilling durations showed no significant difference.Also,fluorescence in situ hybridization (FISH)was conducted to detect nuclear derivation of the embryos.The result showed that the nuclei of the inter-species cloned embryo cells came from human.We conclude that (1)cloned embryos can be constructed through human-rabbit interspecies nuclear transfer;(2)different kinds of somatic cells result in different efficiency of nuclear transfer,while in vitro passage of the donor does not influence embryo development;(3)refrigeration is a convenient and efficient donor cell preparation method.Finally,it is feasible to detect DNA gcnotype through FISH.     

Single embryo transfer in IVF to prevent multiple pregnancies.

As the success rates of IVF clinics improve, one of the adverse consequences is the increased incidence of twins, due largely to the number of embryos transferred. Even if the number of embryos transferred is restricted to two, the twinning rate can exceed 40% of the pregnancies. An obvious way to reduce this high twin rate would be to transfer only one embryo. This would require that cryopreservation of the supernumerary embryos be efficacious enough so that the chance of achieving an ongoing pregnancy is not diminished by transferring a single embryo in the stimulated cycle. Previous studies utilising embryos on day 2 and 3 of development have shown that the pregnancy rates can be acceptable (about 40%) and that the cumulative rate can be up to 60%. Most of these studies, however, do not include a comparison with the cumulative pregnancy rate with two embryos transferred in the stimulated cycle. Therefore, the efficacy has not been proven. We present clinical data from the past few years to illustrate the increase in success rates and the concomitant increase in twinning rates. The increased success in the cryopreservation program has enabled us to trial a single embryo transfer program and compare the results to the transfer of two embryos. The results strongly suggest that the transfer of a single embryo is the better clinical option.