Relationships between timing of syngamy, female age and implantation potential in human invitro-fertilised oocytes

2007 ◽  
Vol 19 (3) ◽  
pp. 482 ◽  
Author(s):  
Celine Lawler ◽  
H. W. Gordon Baker ◽  
David H. Edgar
Keyword(s):  
Multiple Logistic Regression Analysis ◽  
Implantation Rate ◽  
Subsequent Development ◽  
Multiple Logistic Regression ◽  
Predictors Of Outcome ◽  
Cell Stage ◽  
Female Age ◽  
Early Entry ◽  
Early Developmental

Although early developmental markers are frequently used to select embryos for transfer in human assisted reproduction, their value as independent predictors of outcome is often unclear. In this study, the value of using early syngamy and first cleavage as predictors of implantation potential of Day 2 embryos was investigated by examining their interrelationships with subsequent development, female age and implantation. Implantation rates were higher when syngamy occurred before 23–24 h post insemination even when all embryos analysed were transferred 42 h post insemination at the 4-cell stage (25.8 v. 11.9% for the later syngamy group; P < 0.01). Although there was a significant (r = 0.682; P < 0.001) relationship between earlier entry into syngamy and female age, earlier syngamy was still associated with a significantly higher implantation rate in Day 2 embryos with four blastomeres in women under 36 years of age (31.4 v. 15.4% for the later syngamy group; P < 0.05). The ability of timing of syngamy to predict implantation independent of other variables was confirmed by multiple logistic regression analysis. Although related to both subsequent embryo development and female age, early entry into syngamy is a predictor of implantation potential independent of both correlates in human Day 2 in vitro-fertilised embryos.

In vitro fertilisation of Chinese hamster oocytes by spermatozoa that have undergone ionophore A23187-induced acrosome reaction, and their subsequent development into blastocysts

Zygote ◽  
1996 ◽  
Vol 4 (2) ◽  
pp. 93-99 ◽  
Author(s):  
Hiroyuki Tateno ◽  
Yujiroh Kamiguchi
Keyword(s):  
Gas Phase ◽  
Acrosome Reaction ◽  
Chinese Hamster ◽  
Subsequent Development ◽  
In Vitro Fertilisation ◽  
Ionophore A23187 ◽  
Cell Stage ◽  
Developmental Arrest ◽  
Potential Use

SummaryTo enhance potential use of the Chinese hamster, Cricetulus griseus, in developmental and cytogenetic studies of mammalian gametes and embryos, techniques for in vitro fertilisation and embryo culture were developed in the species. Spermatozoa were recovered from the vasa deferentia of mature males, and incubated in modified TYH medium for 1 h at 37°C under 5% CO2 in air. They were then treated with ionophore A23187 (20¼M) for 10min to induce the acrosome reaction. Following ionophore treatment, superovulated oocytes were collected from hormonally stimulated females and incubated with the acrosome-reacted spermatozoa for 2 h at 37°C under 5% CO2 in air. In this study, 245 oocytes ova (98.0%) were determined to be monospermic. The monospermic ova were then cultured in TYH supplemented with 1mM hypotaurine under the same gas phase. Within 30h of fertilisation, 182 ova (93.8%) cleaved to the 2-cell stage, and subsequently 163 ova (84.0%) developed beyond the 2-cell stage. Thus, obstinate developmental arrest at the 2-cell stage(‘2-cell block’) was not observed in this species. Ultimately, 65.5% of monospermic ova reached morula to blastocyst stages.

82 EARLY PORCINE EMBRYO ENERGY PREFERENCE AND SUBSEQUENT DEVELOPMENT

2016 ◽  
Vol 28 (2) ◽  
pp. 170
Author(s):  
L. D. Spate ◽  
B. K. Redel ◽  
R. S. Prather
Keyword(s):  
Energy Source ◽  
Subsequent Development ◽  
Cell Number ◽  
Total Cell ◽  
Control Medium ◽  
Blastocyst Development ◽  
Total Cell Number ◽  
Cell Stage ◽  
Porcine Embryo

Early porcine embryo metabolism in vitro is not completely understood. It has been suggested that before embryo genome activation (4-cell stage), the preferred energy source of the embryo is pyruvate. In our porcine zygote culture medium (MU1), the energy sources are 0.2 mM pyruvate and 2.0 mM calcium lactate. Three experiments were performed with in vitro-matured and IVF embryos to examine the effect on blastocyst development after withholding pyruvate and/or lactate during the first 48 h of culture. In Experiment 1, embryos were cultured without lactate for 48 and then cultured to Day 6 in control medium containing lactate. Control embryos were cultured in medium with lactate starting after fertilization to Day 6. All data were analysed by using SAS 9.3 with a GENMOD procedure used for the blastocyst data and a GLM procedure used for the cell number data. On Day 6, the percentage of embryos that formed blastocysts was 30.2% for control and 26.5% for embryos cultured for 48 h without lactate (n = 490, 4 replications). The difference was not significant P > 0.05. In Experiment 2, embryos were cultured without pyruvate for 48 and then cultured to Day 6 in control medium containing pyruvate. Control embryos were cultured in medium with pyruvate starting after fertilization to Day 6. On Day 6, the percentage of embryos that formed blastocysts was 31.1% for control and 30.5% for embryos cultured for 48 h without pyruvate (n = 385, 3 replications). In Experiment 3, embryos were cultured in control medium for the first 48 h and then cultured to Day 6 in medium without pyruvate, thus forcing the embryos to use lactate instead of pyruvate. On Day 6, the percentage of embryos that formed blastocysts in the pyruvate free medium increased from 28.6%a ± 1.0 to 33.9%b ± 1.0; P ≤ 0.05 (n = 490, 4 replications) compared with the control and total cell number increased from 30.7a ± 1.5 to 41.3b ± 1.8 cells, respectively; P ≤ 0.05 (n = 65, 4 replications). The results from Experiments 2 and 3 were unanticipated as it was believed that the embryo would be more dependent on pyruvate for energy up to the blastocyst stage. We believed in Experiment 2 that from zygote to 4 cells the embryos were not as capable of using lactate and that removing the pyruvate would hinder further development. In Experiment 3, forcing the embryo to use lactate from Day 2 to Day 6 significantly improved blastocyst development and total cell number, suggesting that the embryo is not dependent on a specific energy source or that there are adequate pyruvate stores in the oocyte to 4-cell stage, to promote development to blastocyst. Funding was provided by Food for the 21st Century, the University of Missouri, and the National Institutes of Health (OD011140).

Preimplantation development of microsurgically obtained haploid and homozygous diploid mouse embryos and effects of pretreatment with Cytochalasin B on enucleated eggs

Development ◽  
1980 ◽  
Vol 60 (1) ◽  
pp. 153-161
Author(s):  
Jacek A. Modliński
Keyword(s):  
Survival Rate ◽  
Cytochalasin B ◽  
Single Case ◽  
Cell Stage ◽  
Morula Stage ◽  
Homozygous Diploid ◽  
Haploid Embryos ◽  
Early Developmental

Haploid embryos were obtained by microsurgical removal of one pronucleus, followed by doubling of the haploid chromosome set with Cytochalasin B (CB), either at the first or second mitosis. This procedure provides a source of fully homozygous diploid embryos, which were grown in vitro or in vivo. The effect of CB treatment before and during operation on the course of enucleation and further development of embryos was studied. Out of 81 eggs made diploid at 2-cell stage and transplanted into the oviducts of immature or pseudopregnant recipients 27 morulae and blastocysts were recovered, but not a single case of implantation occurred by the eighth or ninth day of development. After 72–80 h of in vitro culture, most of the homozygous embryos were morulae but after an additional 24 h the majority of them transformed into blastocysts. The rate of development of homozygotes was markedly better than that of haploids, which progressed beyond morula stage. The immediate survival rate of operated eggs was dependent on whether or not the eggs were pre-incubated and the enucleation was performed in the presence of CB. In the former case the immediate survival rate was nearly twice as high as in the absence of CB, but more of the treated eggs underwent fragmentation and early developmental arrest.

Vitrification of biopsied mouse embryos

2005 ◽  
Vol 53 (1) ◽  
pp. 103-112 ◽  
Author(s):  
B. Baranyai ◽  
Sz. Bodó ◽  
◽  
Keyword(s):  
Solid Surface ◽  
Birth Rate ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Mouse Embryos ◽  
The Novel ◽  
Cell Stage ◽  
Cryopreservation Protocol

Solid surface vitrification (SSV) was compared with in-straw vitrification for cryopreservation of biopsied mouse embryos. Eight-cell stage embryos were zona drilled and one blastomere was removed. Developed morulae or blastocysts were vitrified in microdrop (35% EG + 5% PVP + 0.4 M trehalose) or in straw (7.0 M EG + 0.5 M sucrose). Following recovery, embryos were cultivated in vi tro or transferred into recipients. Cryopreservation had an effect not only on the survival of biopsied embryos but also on their subsequent development in vitro. Cryosurvival of biopsied morulae vitrified in straw was significantly inferior to SSV. The post-warm development of biopsied and non-biopsied morulae was delayed on Day 3.5 and 4.5 in both vitrification groups. A delay in development was observed on Day 5.5 among vitrified non-biopsied blastocysts. The percentage of pups born from biopsied morulae or blastocysts following cryopreservation did not differ from that of the control. No significant differences could be detected between methods within and between embryonic stages in terms of birth rate. The birth rate of biopsied embryos vitrified in straw was significantly lower compared to the non-biopsied embryos. The novel cryopreservation protocol of SSV proved to be effective for cryopreservation of morula- and blastocyst-stage biopsied embryos.

34 DEVELOPMENTAL ABILITY OF ZONA-FREE PORCINE CLONED EMBRYOS RECONSTRUCTED BY SOMATIC CELLS AND CENTRIFUGED CYTOPLASTS

2006 ◽  
Vol 18 (2) ◽  
pp. 125
Author(s):  
M. Fahrudin ◽  
K. Kikuchi ◽  
N. W. K. Karja ◽  
M. Ozawa ◽  
T. Somfai ◽  
...  
Keyword(s):  
Somatic Cell ◽  
Gradient Centrifugation ◽  
Somatic Cells ◽  
Subsequent Development ◽  
Blastocyst Stage ◽  
Blastocyst Development ◽  
Cell Stage ◽  
In Vitro Development ◽  
Vitro Development

The combination of bulk enucleation and zona-free cloning will offer simplification of the conventional nuclear transfer technique. A bulk enucleation method such as enucleation by centrifugation could reduce the time of manipulation that is necessary for removing genetic materials from the oocytes. The present study was conducted to examine the ability of cytoplasts obtained by centrifugation of zona-free in vitro maturation (IVM) porcine oocytes to support remodeling of the somatic cell nucleus and the subsequent development in vitro of somatic cell nuclear transferred (SCNT) embryos. A primary culture of cumulus cells was used as the source of donor cells, and recipient cytoplasts were derived from IVM oocytes that were cultured for 48 h, denuded of zonae pellucidae, and subjected to gradient centrifugation in Percoll solution to separate the ooplasm into fragments. Fragments were stained with Hoechst-33342 and cytoplasts were selected under an epifluorescence microscope. Then two or three cytoplasts were aggregated with a single somatic cell in phytohemagglutinin solution (500 �g/mL). Fusion between somatic cell and cytoplasts was induced by two DC pulses of 1.5 kV/cm for 20 �s, and activation was accomplished by two DC pulses of 0.8 kV/cm for 30 �s at 1 h after fusion in 0.28 M mannitol solution supplemented with 0.05 mM CaCl2 and 0.1 mM MgSO4. The resultant embryos were transferred to a WOW culture system (Vajta et al. 2000 Mol. Reprod. Dev. 55, 256-264) and cultured in glucose-free NCSU-37 containing 4 mg/mL BSA supplemented with 0.17 mM sodium pyruvate and 2.73 mM sodium lactate from Days 0 to 2; from Days 2 to 7 they were cultured in NCSU-37 supplemented with 5.55 mM {D}-glucose and 5% FCS. Some of the reconstructed embryos were fixed at 1, 10, and 24 h after activation and stained with 1% (w/v) orcein to display the morphology of the transferred somatic nuclei. The results showed that 53.6% (30/56) of the SCNT embryos underwent premature chromosome condensation at 1 h, 90.9% (50/55) formed pseudo-pronuclei at 10 h, and 21% (19/90) of them cleaved to the two-cell stage at 24 h after the activation. The development to the blastocyst stage of the embryos that were reconstructed by quartet cells (three cytoplasts and one somatic cell; 8.9%, 10/112) was significantly higher (P < 0.05) than that of the triplet ones (2.2%, 3/139). However, these blastocyst rates were significantly lower (P < 0.05) than the blastocyst development rate of parthenogenetic embryos with the intact zonae pellucidae (28.3%, 17/60). These results suggest that (1) cytoplasts obtained by gradient centrifugation could support reprogramming of somatic cells and in vitro development of SCNT embryos to the blastocyst stage, and (2) the volume of cytoplasts apparently affects their in vitro development in pigs.

scholarly journals Control of nuclear remodelling and subsequent in vitro development and methylation status of porcine nuclear transfer embryos

Reproduction ◽  
2008 ◽  
Vol 135 (5) ◽  
pp. 649-656 ◽  
Author(s):  
D J Kwon ◽  
C K Park ◽  
B K Yang ◽  
H T Cheong
Keyword(s):  
Nuclear Transfer ◽  
Apoptotic Cell ◽  
Formation Rate ◽  
Methylation Status ◽  
Subsequent Development ◽  
Treated Group ◽  
High Rate ◽  
Cell Stage ◽  
Methylation Patterns

We attempted to control the nuclear remodelling of somatic cell nuclear transfer embryos (NTs) and examined their subsequent development and DNA methylation patterns in pigs. Porcine foetal fibroblasts were fused to enucleated oocytes treated with either 5 mM caffeine for 2.5 h or 0.5 mM vanadate for 0.5 h. After activation, NTs were cultured in vitro for 6 days to examine their development. The nuclear remodelling type of the reconstituted embryos was evaluated 1 h after fusion. Methylated DNA of in vitro-fertilised (IVF) embryos and NTs at various developmental stages and of donor cells was detected using a 5-methylcytosine (5-MeC) antibody. Caffeine-treated NTs induced premature chromosome condensation at a high rate (P<0.05), whereas most vanadate-treated NTs formed a pronucleus-like structure. Although cleavage rates to the two-cell stage did not differ among groups, delayed cleavage was observed in the vanadate-treated group. The blastocyst formation rate was significantly reduced by vanadate treatment compared with caffeine-treated and non-treated (control) NT groups (P<0.05). The apoptotic cell index of NT blastocysts was lower in the caffeine-treated group than in other groups (P<0.05). The methylation patterns were similar among NTs, but more hypermethylated DNA was observed at the four-cell stage of control and vanadate-treated NTs when compared with that in IVF embryos (P<0.05). Thus, the nuclear remodelling type controlled by caffeine or vanadate treatment can affect in vitro development and the methylation status of NTs in relation to nuclear reprogramming.

scholarly journals 315THE REDUCTION OF MATURATIONAL COMPETENCE BY STREPTOMYCIN DURING IN VITRO MATURATION OF GOAT FOLLICULAR OOCYTES

2004 ◽  
Vol 16 (2) ◽  
pp. 277 ◽  
Author(s):  
J.K. Kang ◽  
J.H. Yang ◽  
K. Naruse ◽  
C.S. Park ◽  
K.S. Min ◽  
...  
Keyword(s):  
Oocyte Maturation ◽  
In Vitro Maturation ◽  
Subsequent Development ◽  
Oocyte Activation ◽  
Cell Stage ◽  
Parthenogenetic Activation ◽  
Maturation Medium ◽  
Significant Difference ◽  
Activation Potential

Antibiotics are commonly added to mammalian oocyte maturation media, but their effects on oocytes maturation have not been examined thoroughly. Goat follicular oocytes were used to investigate whether penicillin, streptomycin or gentamycin affect maturational competence of oocytes and subsequent parthenogenetic activation potential in vitro. Cumulus-oocyte complexes collected from a local abattoir were matured for 24h in five treatments, and matured oocytes were cultured for 48h in five treatments after parthenogenetic activation by treatment with ionomycin, followed by immediate exposure to 6-diethlaminopurine; (1) Control: TCM-199 medium with no antibiotics, (2) TCM-199 with 100IU/mL−1 penicillin (P-4687, Sigma, St. Louis, MO, USA), (3) TCM-199 with 50μgmL−1 streptomycin (S-1277, Sigma), (4) TCM-199 with 50μgmL−1 gentamycin (G-1264, Sigma) and (5) TCM-199 with both 100IUmL−1 penicillin and 50μgmL−1 streptomycin. Maturation rates at 24h post-in vitro maturation and parthenogenetic cleavage development at 48h post-activation were evaluated. Data were analyzed by ANOVA and Student’s t-test. Penicillin and gentamicin treatment groups did not affect maturation rates and percentages of cleavage to 2–4 cell stage at 48h post-chemical oocyte activation. However, when streptomycin was present in the maturation medium, the percentages of matured oocytes at 24h post-in vitro maturation of immature goat oocytes were significantly lower than those from the other groups. However, among the five treatments, there was no significant difference in cleavage rates of matured oocytes at 48h post-activation (Table 1). Therefore, streptomycin did interfere with the maturation of immature goat oocytes, but did not affect the subsequent development of matured goat oocytes. The mechanism by which streptomycin affects the maturation of goat follicular oocytes needs to be investigated further. We conclude that streptomycin in oocyte maturation medium can be detrimental during in vitro maturation of goat follicular oocytes. Table 1 Effect of antibiotics on maturational competence of goat follicular oocytes and subsequent parthenogenetic activation potential in vitro

45 NUCLEAR LAMIN A/C EXPRESSION IN BOVINE PARTHENOTES AND NUCLEAR TRANSFER EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 131
Author(s):  
R. D. W. Kelly ◽  
R. Alberio ◽  
K. H. S. Campbell
Keyword(s):  
Nuclear Transfer ◽  
Expression Patterns ◽  
Subsequent Development ◽  
Lamin A ◽  
Santa Cruz ◽  
Cell Stage ◽  
Aberrant Expression ◽  
Fetal Fibroblasts ◽  
Bovine Oocytes

Despite the apparent successes of nuclear transfer (NT) technology, numerous recent reports have indicated de-regulation of key gene expression patterns in NT embryos as compared to their in vivo and IVF counterparts. Aberrant expression of lamin A/C has been reported in mouse (Moreira et al. 2003 J. Cell Sci. 116, 3713-3720) and bovine (Sullivan et al. 2004 Biol. Rep. 70, 146-153) NT embryos, leading to the hypothesis that the presence of lamin A/C might affect subsequent development. Lamin A/C expression is a potential marker for reprogramming due to the induced expression and remodeling during differentiation. Previously using immunofluorescence in bovine IVF embryos, we have demonstrated the persistence of lamin A/C until the 2-cell stage (Kelly et al. 2005 Reprod. Fertil. Dev. 17, 205-206). The present study was initiated to further characterize lamin A/C expression in bovine parthenogenetic and NT embryos using a monoclonal antibody specific to lamin A/C. Bovine oocytes were matured in vitro as previously described (Fouladi-Nashta et al. 1998 Biol. Rep. 59, 255-262). NT embryos were constructed using lamin A/C-positive primary bovine fetal fibroblasts and in vitro-matured, enucleated MII bovine oocytes. Oocyte cell couplets were fused at 24 h post onset of maturation 1 h prior to activation. Oocyte activation was achieved with 7% ethanol for 7 min followed by a 6 h incubation in mSOF containing 10 �g/mL cycloheximide and 7.5 �g/mL cytochalasin B for the production of both NT and parthenogenetic embryos. Embryos were cultured in mSOF supplemented with 10% FCS and collected at various stages for immunofluorescence staining. Prior to fixation, embryos were incubated in 2 mg/mL protease to remove the zona pellucida. Samples were fixed in 100% methanol at -20�C for 20 min and then blocked for 1 h (4% goat serum in PBS) at RT. Embryos were then incubated overnight at 4�C with mouse anti-lamin A/C antibody (IgM; Santa Cruz Biotechnology, Santa Cruz, CA, USA) or with blocking solution as a control. Following the primary incubation, embryos were washed extensively in 1% BSA in PBS and then incubated with Cy3 goat anti-mouse IgM (1:400) (Chemicon International, Inc., Temecula, CA, USA) for 1 h at RT. Unbound secondary antibody was removed by washing with 1% BSA in PBS, and embryos were mounted in VectaShield mounting medium containing 42,6-diamidino-1-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Images were viewed using epifluorescence (Leica DMR; Leica Microsystems, Wetzlar, Germany) and confocal microscopy (Leica TCS). Inhibiting protein synthesis during the activation period with cycloheximide had no effect on lamin A/C assembly in 6 h post activation (hpa) parthenogenetic (35/35) and NT (7/7) embryos. The pronuclei of parthenogenetic (30/30) and NT (15/15) zygotes at 22 hpa were also positively labeled for lamin A/C. Nuclear labeling was observed in both parthenogenetic (25/25) and NT (12/12) 2-cell embryos. All parthenogenetic and NT embryos examined from the 4-cell stage through to blastocysts were stained negatively for lamin A/C. These results suggest that lamin A/C present in bovine NT zygotes is not due to aberrant reprogramming and that remodeling of the nuclear lamina occurs correctly in bovine NT embryos.

Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

1974 ◽  
Vol 32 ◽  
pp. 320-321
Author(s):  
J. P. Revel
Keyword(s):  
Developmental Stages ◽  
Three Dimensional ◽  
Cell Movement ◽  
Cellular Interactions ◽  
Serial Sections ◽  
Cell Sheets ◽  
Freeze Etching ◽  
Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

The experience of holding the cycles of assisted reproductive technology with defrost, a biopsy, genetic study and refreezing of embryos in patients with multiple unsuccessful implantations

2016 ◽  
pp. 166-170
Author(s):  
Y.V. Masliy ◽  
◽  
I.O. Sudoma ◽  
P.S. Mazur ◽  
D.A. Mykytenko ◽  
...  
Keyword(s):  
Chromosomal Rearrangements ◽  
Genetic Diagnosis ◽  
Morphological Characteristics ◽  
Implantation Rate ◽  
Ovarian Response ◽  
Reproductive Technologies ◽  
Comparative Genome Hybridization ◽  
Comparative Genome ◽  
Vitro Fertilization

The objective: to study the possibility of using frozen blastocysts for biopsy and genetic testing and performance measurement transfer euploeded 5–7-day-old embryos after thawing, biopsies, refreezing and thawing in patients with unsuccessful implantation. Patients and methods. The object of the study was the group of patients with repeated failure of implantation (4) in programs of auxiliary reproductive technologies (ART), subject to transfer to the uterus in total (i.e. in all the programs) for at least 6 good quality embryos based on morphological characteristics). All women had sufficient ovarian reserve. The patient was treated for infertility within the ART programs of the clinic of reproductive medicine "Nadiya" in the period from 2006 to 2016. The sample included couples who were not carriers of chromosomal rearrangements, without anomalies of the uterus (congenital and acquired: a doubling of the uterus, one-horned uterus, intrauterine membrane, synechia, submucous myoma of the uterus). All women had a positive ovarian response to controlled stimulation with gonadotropins (at least 7 oocytes) and a sufficient number of cryopreserved embryos. The first group (G1) included 64 women who trophectodermal a biopsy was performed on fresh blastocysts (in a loop controlled ovarian hyperstimulation). The second group (G2) were included 31 women who underwent thawing previously cryopreserved blastocysts trophectodermal re-biopsy and vitrification of blastocysts. Results. It was found that the performance of transfers euploid embryos that were vitrified, bioptrone and revitriphted, a little lower than those that were bioptrone fresh and vitrified only once. At the same time computationa genetic diagnosis previously vitrified blastocysts using comparative genome hybridization in patients with recurrent failed implantation allows to obtain a reasonable pregnancy rate (58%), implantation rate (33.3 %) and the birth of living children (45.1 %). Conclusion. Reprising biopropane embryos does not cause significant destructive impact and allows you to achieve pregnancy and birth of the alive child. Key words: in vitro fertilization, reusable unsuccessful implantation, a method of comparative genome hybridization, refreezing.

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