Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage

BACKGROUND: A correlation between morphology, developmental competence and chromosome abnormalities is established. However, since absolute correlations are rare, embryo selection remains one of the most arduous tasks in assisted reproduction. This study was undertaken in order to determine which chromosomal abnormalities are compatible with development to the blastocyst stage. METHODS: Embryos diagnosed by preimplantation genetic diagnosis (PGD) as chromosomally abnormal or unsuitable for transfer were cultured to day 5 or 6. Morphology and development were observed daily. After extended culture, embryos were fixed and analysed by two rounds of FISH with the same probes used for PGD. RESULTS: Some types of numerical chromosome abnormalities do not preclude full differentiation in vitro. For instance, extensive mosaicism was detected in blastocysts and trisomic embryos reached the blastocyst stage with a frequency of 37%. Interestingly, only those monosomies compatible with first trimester development (monosomy X and 21) were detected at blastocyst stage. CONCLUSION: Even though there is a strong selection against chromosomally abnormal embryos, extended culture to day 5 or 6 cannot be used as a reliable tool to select against clinically relevant chromosome abnormalities such as trisomies.     

Polarity of the mouse embryo is established at blastocyst and is not prepatterned

Polarity formation in mammalian preimplantation embryos has long been a subject of controversy. Mammalian embryos are highly regulative, which has led to the conclusion that polarity specification does not exist until the blastocyst stage; however, some recent reports have now suggested polarity predetermination in the egg. Our recent time-lapse recordings have demonstrated that the first cleavage plane is not predetermined in the mouse egg. Here we show that, in contrast to previous claims, two-cell blastomeres do not differ and their precise future contribution to the inner cell mass and/or the trophectoderm cannot be anticipated. Thus, all evidence so far strongly suggests the absence of predetermined axes in the mouse egg. We observe that the ellipsoidal zona pellucida exerts mechanical pressure and space constraints as the coalescing multiple cavities are restricted to one end of the long axis of the blastocyst. We propose that these mechanical cues, in conjunction with the epithelial seal in the outer cell layer, lead to specification of the embryonic-abembryonic axis, thus establishing first polarity in the mouse embryo.     

Fertiliza1tion and early embryology: Four indications for embryo transfer at the blastocyst stage

The transfer of blastocysts obtained by co–culture with‘Vero’ (African green monkey kidney) cells was offeredto infertile couples with the following indications: (i) repeatedfailure of implantation, (ii) patients in whom multiple pregnancieshad to be avoided (malformed uterus or risk of descending uterus),(iii) patients where embryo development potential had to beassessed, and (iv) replacement of supernumerary embryos frozenat the blastocyst stage. In the 142 cycles analysed, the pregnancyrates per transfer were 37.2, 36.3, 13.0 and 13.6% respectivelyfor the couples with indications i-iv. The respective implantationrates per blastocyst were 20.0, 16.7, 7.1 and 9.3%. In patientsin whom multiple pregnancies had to be avoided, the transferof a maximum of two blastocysts gave a pregnancy rate per cycleof 23.5% without any multiple pregnancies. The freezing of supernumeraryembryos at the blastocyst stage allowed us to replace them usingsimple protocols and to avoid cancellation of the transfer cycles.Embryo co-culture has been found to be an interesting techniquefor selected indications, making available a good number ofblastocysts for transfer. The transfer of blastocysts allowedus to reduce the number of embryos transferred per patient andtherefore also reduce the rate of multiple pregnancies (therewere no triplet pregnancies in this study). These results needto be confirmed by larger, randomized studies with comparisonsto control groups to evaluate the effectiveness of blastocysttransfers.     

Cryopreservation of biopsied embryos at the blastocyst stage

BACKGROUND: The availability of an efficient cryopreservation program is especially important in the case of embryos that have undergone blastomere biopsy for PGD. Unfortunately, the freezing/thawing of biopsied embryos has given disappointing results when performed at the cleavage stage. In this study, embryos diagnosed as normal after PGD were grown to the blastocyst stage, frozen and thawed for successive frozen embryo transfer. METHODS: A total of 34 patients performed a thawing cycle in which 47 blastocysts were thawed. The cryopreservation solutions were based on HEPES-buffered medium supplemented with human serum albumin (HSA), sucrose and 1,2-propanediol. The same protocol was applied to embryos from 88 IVF/ICSI patients, which underwent 92 thawing cycles with 150 thawed blastocysts. RESULTS: The survival rate was similar in the two groups (53% after PGD and 58% in IVF/ICSI cycles), as well as the cumulative pregnancy rate per patient (59% after PGD versus 47% in IVF/ICSI cycles), despite a higher maternal age and a lower proportion of embryos available for transfer or cryopreservation in the PGD group. CONCLUSIONS: Neither the survival rate nor the subsequent development and chances of implantation, differed between embryos frozen at the blastocyst stage following biopsy and those frozen intact.     

Proliferation of blastomeres from biopsied cleavage stage human embryos in vitro: an alternative to blastocyst biopsy for preimplantation diagnosis

Normally fertilized human embryos were biopsied at cleavagestages on the third day after in-vitro fertilization (IVF).One or two blastomeres at the 8-cell stage were removed andco-cultured with the biopsied embryos. Embryos and blastomereswere assessed daily for morphological development until day6, when the number of cells were counted by labelling the nuclei.In all, 53% of the biopsied embryos (25 out of 47) reached theblastocyst stage between day 5 and 6 and the proportion wasthe same irrespective of the number of cells removed. Therewas no significant difference between biopsied embryos fromwhich one or two blastomeres respectively had been removed withregard to total cell numbers at the blastocyst stage (56.2 ±3.0 and 64.7 ± 5.5), number of trophectoderm (45.4 ±3.5 and 44.0 ± 5.7) and inner cell mass cells (14.0 ±1.2 and 16.6 ± 1.8). Overall, 72% of the isolated blastomeresdivided at least once over 3 days in culture and 50% dividedmore than once. The mean overall cell number after 3 days inculture was 3.7 ± 0.48 per blastomere (range 1–8cells) if one cell was removed and 6.9 ± 1.0 if two cellswere removed. If the undivided blastomeres are excluded, themean cell number was 4.8 ± 0.51 and 8.3 ± 1.0respectively. Over this period, 55% of the blastomeres cavitated.Of the blastomeres taken from embryos that developed to theblastocyst stage, 92% divided and 76% cavitated. In those fromarrested embryos, 50% divided (P < 0.002) and 32% cavitated(P < 0.003). From the first group 8% of blastomeres and fromthe second group 41% of blastomeres neither divided nor cavitated(P < 0.008). We conclude that as a more consistent alternativeto blastocyst biopsy, cryopreserving biopsied cleavage stageembryos and culturing blastomeres would increase the numberof cells available for genetic analysis. This could facilitatepreimplantation diagnosis of inherited disease by improvingreliability and possibly allowing combined detection of chromosomaland single cell defects. Further studies will investigate thegenetics of proliferated blastomeres.     

Human preimplantation development in vitro is not adversely affected by biopsy at the 8-cell stage

Normally fertilized human embryos biopsied 3 days after in-vitro fertilization (IVF) have been examined for effects on viability and development in vitro after removal of one or two cells at the 8-cell stage (1/8 and 2/8) from each embryo. A high proportion of 7/8 and 6/8 biopsied and unmanipulated embryos developed to the blastocyst stage between days 5 and 6 (79, 71 and 59%, respectively), and many biopsied embryos (56%) hatched from the zona pellucida in vitro. The viability of biopsied embryos which developed to the blastocyst stage was assessed by daily non-invasive measurement of the uptake of two energy substrates, glucose and pyruvate. Uptake of both substrates was generally lower in 7/8 and 6/8 biopsied embryos but only in proportion to the reduced cellular mass. The total cell number and the numbers of both trophectoderm (TE) and inner cell mass (ICM) cells in biopsied embryos at the blastocyst stage, counted by differential labelling of their nuclei, were also reduced in proportion but the ratio of ICM to TE cells was maintained in both 7/8 and 6/8 biopsied embryos. We conclude that removal of one or two cells at the 8-cell stage, while reducing the cellular mass, does not adversely affect the preimplantation/development of biopsied embryos in vitro and suggest that this approach could be used for preimplantation diagnosis of genetic defects.     

Expression of intercellular junctions during preimplantation development of the human embryo

A total of 74 human embryos were stained with gap junction proteinspecific anti-peptide antibodies and antibodies to the desmosomalprotein desmoplakin to reveal the expression pattern of intercellularjunctions during preimplantation development. Prior to implantation,the human embryo expresses predominantly connexin (Cx43)-containinggap junctions. Gap junctions were first detected in apposingcell membranes at the 4-cell stage and became increasingly organizedas development proceeded. In normal blastocysts, trophectoderm(TE) cells were linked by dense arrays of gap junctions whileinner cell mass (ICM) cells were linked by small, punctate gapjunctions. Gap junctions containing Cx32 or Cx26 were observedoccasionally in the TE of late blastocysts. Desmosomes appearedbetween outer cells prior to cavitation and were retained inthe TE, but not in the ICM. Levels of gap junction protein expressionwere variable in morphologically normal embryos at the samestage, suggesting that a normal appearance may not be a reliableindicator of future viability. Morphologically normal embryosoften possessed multinucleate, apoptotic and decompacting cells.They could show either extensive, disorganized over-expressionor reduced expression of gap junction protein. The results fitthe view that only embryos destined to survive display an organizedpattern of intercellular junctions. blastocyst/connexin/desmosome/gap junction/immunocytochemistry     

Characterization of telomerase activity in the human oocyte and preimplantation embryo

Telomerase, a ribonucleoprotein, has been described as an essential component of highly proliferative cells as it stabilizes the telomeres and avoids cellular senescence. The objective of this study was to modify the polymerase chain reaction-based telomeric repeat amplification protocol to detect telomerase activity in the single cell and to characterize the activity expressed in the human oocyte through to the blastocyst stage embryo. A comparative evaluation of telomerase activity and developmental stage was conducted using discarded or donated human oocytes and embryos. Telomerase activity was detected in all developmental stages evaluated from immature oocytes through to blastocyst stage embryos. Immature oocytes and blastocysts had similar levels of telomerase activity; however, both groups had significantly (P < 0.05) higher activity than zygote through to pre-morula stage embryos. Seventy-five thawed zygotes were cultured to day 3, biopsied by removing 1-2 cells, and the biopsied embryos were cultured to blastocyst stage. There was no difference (P < 0.05) in telomerase activity between cells biopsied from embryos that reached the blastocyst stage and cells from those that arrested in growth. This study has shown that human oocytes through to blastocyst stage embryos express telomerase activity, but that the level of telomerase activity in biopsied blastomeres, of the day 3 cleavage stage embryo, is not predictive of embryonic growth potential.     

Blastocyst biopsy versus cleavage stage biopsy and blastocyst transfer for preimplantation genetic diagnosis of beta-thalassaemia: a pilot study

BACKGROUND: Trophectoderm biopsy at the blastocyst stage is an emerging approach in preimplantation genetic diagnosis (PGD). This study aimed to compare genotyping success and implantation rates in PGD cycles for beta-thalassaemia following biopsy at the cleavage versus the blastocyst stage, with transfer of blastocysts. METHODS: This pilot study included 20 cycles: Group A: 10 cycles, day 3 blastomere biopsy, day 5 transfer; Group B: 10 cycles, day 5 trophectoderm biopsy, day 6 transfer. Standard-assisted reproduction and laser biopsy procedures were used. Biopsied cells were genotyped using real-time PCR multiplexed with fluorescent microsatellite analysis. RESULTS: In Group A, 131 fertilized eggs developed to 101 embryos suitable for single blastomere biopsy; 76/101 blastomeres were diagnosed (75.2%), 30 unaffected blastocysts were transferred resulting in six pregnancies (eight fetal hearts, 26.7% implantation rate). In Group B, 128 fertilized eggs developed to 53 blastocysts for trophectoderm biopsy (four to five cells), with 50/53 blastocysts diagnosed (94.3%), 21 unaffected blastocysts transferred and 6 pregnancies initiated (10 fetal hearts, 47.6% implantation rate). Overall, nine pregnancies reached >10 weeks gestation and were confirmed unaffected by prenatal diagnosis, with 12 healthy babies born. CONCLUSIONS: This pilot study suggests that trophectoderm biopsy and blastocyst transfer may be more advantageous than cleavage stage biopsy with respect to outcome of PGD for monogenic diseases.     

The first appearance of the future cerebral hemispheres in the human embryo at stage 14

Summary Thirty-five embryos of stage 14 (32 days) were studied in detail and graphic reconstructions of four of them were prepared. Characteristic features of this stage include the beginning formation of the future cerebral hemispheres and the cerebellar plates.The ventral boundary between telencephalon medium and diencephalon is the preoptic recess. Although a velum transversum is not yet distinguishable as a dorsal boundary, its site is indicated by a change in the thickness of the roof of the forebrain. As the cerebral vesicles (future hemispheres) begin to evaginate, a di-telencephalic sulcus and a corresponding lateral ventricle and ventricular ridge (torus hemisphericus) develop. The telencephalic wall is mainly ventricular layer but three areas show advanced differentiation: olfactory area, future amygdaloid body (which lies at first mainly in the diencephalon), and primordium of the hippocampus. The telencephalon is growing in length, and the forebrain now occupies almost one quarter of the total length of the brain.Abbreviations Aff. Common afferent tract - A-H Adenohypophysial pouch - Amn Amnion - Amyg. Area of the future amygdaloid body - B.c. Boundary cap - B.v. Blood vessel - Cer. Cerebellum - Cer.v. Cerebral vesicle - Ch. Chiasmatic plate - Comm. Commissural plate - D-t S. Di-telencephalic sulcus - D Diencephalon - End. Endolymphatic duct - Hip. Hippocampus - Hyp. C. Hypothalamic cell cord - H-Th. Hypothalamothalamic tract - I Infundibulum - Int.st. Interstitial nucleus - L.L. Lateral longitudinal fasciculus - L.T. Adult lamina terminalis - M Mesencephalon - Ma. Mamillary area - Med.E. Medial eminence - MLF Medial longitudinal fasciculus - Nas. Nasal plate - N.Cr. Neural crest - Not. Notochord - Olf. Olfactory area - Olf.E. Olfactory eminence - Opt. Optic vesiole - Opt.G. Optic Groove - Opt.St. Optic stalk - Ot Otic vesicle - Pr.-H-T Preopticohypothalamotegmental tract - Rh. Rhombomere - S.L. Sulcus limitans - S. med. Sulcus medius - St. Stomach - Syn. Synencephalon - Tel. Telencephalon - Tel.m. Telencephalon medium - d.Th. Dorsal Thalamus - v.Th. ventral Thalamus - T.hem. Torus hemisphericus - Tr. Trachea - VLF Ventral longitudinal fasciculus - Vel. Velum transversum Supported by research grant No. HD-16702, Institute of Child Health and Human Development, National Institutes of Health (USA)Numbers 5 to 12 cranial nerves. Where cranial nerves and rhombomeres are labelled in the same figure, the latter are in boldface numbers. The asterisk in Figs. 3 and 8 indicates the borderline between rhombencephalon and spinal cord     

Pronuclear, cleavage and blastocyst histories in the attempted preimplantation diagnosis of the human hydatidiform mole.

We report a study of fertilization, syngamy and embryonic development in 14 oocytes from a woman with four previous pregnancies involving complete hydatidiform moles. Serial observations of pronuclear movements and syngamy were compared to those in a group of 10 multipronucleate embryos from other patients. One embryo and possibly two others developed normally or near-normally. The others displayed immediate cleavage or had one or three pronuclei. The tripronucleate eggs displayed various anomalous forms of growth. The unipronucleate eggs passed through a double form of syngamy, which might have involved chromosome doubling, and could have developed as androgenetic diploids. We suggest a hypothesis to explain these unusual observations.     

Development of human embryos to the hatched blastocyst stage in the presence or absence of a monolayer of Vero cells

In a prospective randomized study, excess embryos from 100 womenundergoing in-vitro fertilization were cultured from the 2-cellto the hatched-blastocyst stage in the presence or absence ofa confluent monolayer of Vero cells. The frequencies of fragmentation,developmental arrest, multi-nucleation and blastocyst formationwere observed for 254 embryos over 7 days in culture. The numberof nucleated cells, and fine structure of trophectoderm andinner cell mass were analysed at the expanded blastocyst stageon day 5.5 post-insemination. The frequency of hatching fromthe zona pellucida was determined between days 6 and 7 post-insemination.With respect to these developmental parameters, the findingsindicate that no overt or statistically significant improvementin early human embryogenesis occurs in the co-culture system.     

Transfer at the blastocyst stage of embryos derived from testicular round spermatid injection

BACKGROUND: Intracytoplasmic injection of testicular round spermatids has been suggested as a salvage treatment in couples when testicular sperm extraction does not yield any mature sperm. However, the success of the procedure is debatable, and controversy surrounds issues such as the presence and (if present) identification of spermatids in testicular tissue. Progression rate to the blastocyst stage of spermatid-derived embryos appears to be low. METHODS: In this study, we investigated the feasibility and outcome of blastocyst stage embryo transfer after round spermatid injection (ROSI). ROSI was undertaken in 58 couples who did not yield mature or elongated sperm to testicular sperm extraction. RESULTS: The incidence of blastocyst formation from two pronuclear oocytes was 7.6%. A total of 16 blastocysts were transferred in 12 patients (20.7%). None of the patients conceived. CONCLUSIONS: The results of this study indicate that the blastocyst stage is reached by only very few ROSI-derived embryos and these embryos do not implant.     

The first appearance of the neural tube and optic primordium in the human embryo at stage 10

Summary Thirteen embryos of stage 10 (22 days) were studied in detail and graphic reconstructions of most of them were prepared. The characteristic feature of this stage is 4–12 pairs of somites. Constantly present are the prechordal and notochordal plates (the notochord sensu stricto is not yet apparent), the neurenteric canal or at least its site, the thyroid primordium, probably the mesencephalic and rhombencephalic neural crest and the adenohypophysial primordium. During this stage, the following features appear: terminal notch, optic sulcus, initial formation of neural tube, oropharyngeal membrane, pulmonary primordium, cardiac loop, aortic arches 1–3, intersegmental arteries, and laryngotracheal groove. The primitive streak is still an important feature.Graphic reconstructions have permitted the detection of the telencephalic portion of the forebrain, for the first time at such an early stage. It is proposed that the remainder of the forebrain comprises two subdivisions: D1, which becomes largely the optic primordium during stage 10, and D2, which is the future thalamic region. The optic sulcus is found in D1 but does not extent into D2, as has been claimed in the literature. An indication of invagionation of the otic disc appears towards the end of the stage. As compared with the previous stage, the prosencephalon has increased in length, the mesencephalon has remained the same, the rhombencephalon has decreased, and the spinal part of the neural plate has increased fivefold in length. The site of the initial closure of the neural groove is rhombencephalic, upper cervical, or both. The neural plate extends caudally beyond the site of the neurenteric canal. Cytoplasmic inclusions believed to indicate locations of great activity were always detected in the forebrain (especially in the optic primordium), and also in the rhombencephalon, spmal part, and mesencephalon.Supported by research grant No. HD-16702, Institute of Child Health and Human Development, National Institutes of Health (USA)