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[The effect of the micromanipulations used for transgenosis on mouse development].

Non-specific effects of micromanipulation techniques used for producing transgenic mice on processes of embryonic development were studied. Zygotes obtained from C57BL and BALBxDD mice were treated as follows: (1) incubated in culture medium; (2) the male pronucleus punctured with a glass microneedle; (3) microinjected with a buffer solution; and (4) DNA (mouse P-35 oncogene with human insulin gene promoter) injected into the male pronucleus. Then zygotes were transferred into oviducts of syngeneic or allogeneic pseudopregnant females. Such treatment resulted in the intrauterine death of embryos, as well as in birth of the dead or non-viable offspring with numerous defects of development. Zygote pronucleus puncturing is the most damaging manipulation, since its effect exceeds that of the zygote incubation and is comparable with the effect of buffer of DNA injections.

Animals↗
PubMed1996

The effects of prior gravidity on the outcomes of ovum donor and own oocyte cycles.

OBJECTIVE: To determine the effects of prior gravidity on hormonal parameters, medication regimen, oocyte parameters, fertilization, and clinical pregnancy rates (PRs) in donor and own oocyte cycles. DESIGN: A retrospective study of 64 first-attempt ovum donor cycles and 102 first-attempt IVF and ZIFT cycles using own oocytes conducted during a 2.5-year time period. Analyses of covariance and t-tests using gravidity of oocyte source (gravida versus nulligravida) and controlling for sperm parameters were used to assess differences in hormonal, endometrial, medication, and demographic parameters and were performed separately for donor cycles and for own oocyte cycles. SETTING: Private fertility center. PATIENTS: In ovum donation cycles, oocyte parameters, medication administered, and hormonal parameters of 64 oocyte donors between the ages of 21 and 35, 34 of whom were never pregnant, i.e., nulligravida and 30 who had ever been pregnant, regardless of the outcome of that pregnancy, i.e., gravida, were studied. In own oocyte cycles, oocyte parameters, medication administered, and hormonal parameters of 102 women, 54 nulligravida and 48 gravida, between the ages of 23 and 44 were studied. MAIN OUTCOME MEASURE: Medication requirements, hormonal response, seminal parameters, oocyte quality, fertilization, and clinical PRs. RESULTS: For patients using their own oocytes, there were no significant differences in any of the parameters studied. In contrast, compared with their nulligravida counterparts, gravida oocyte donors had fewer poor quality oocytes, had more high quality oocytes that fertilized, had a higher proportion of their oocytes fertilize, and had a higher PR per transfer. CONCLUSION: A prior history of gravidity is an important predictor of clinical pregnancy in donor oocyte cycles but not in cycles in which patients use their own oocytes. Oocyte recipients may wish to consider donor gravidity in selecting their donor.

Adult↗
PubMed2004

[Efficiency of cryoembryotransfer using embryos frozen in the pronuclear stage].

OBJECTIVE: To evaluate results of cryoembryotransfers in cycles with estrogen-progestin preparation of the endometrium. DESIGN: Prospective study. SETTING: Centre of Assisted Reproduction, Dept. of Obstetrics and Gynecology, Palacký University Medical School, Olomouc. METHODS: The study included 51 patients in whom in the period of January to August 2003 altogether 42 cycles the embryotransfer with frozen and thawed embryos were performed. The embryos for cryopreservation were obtained in the classical IVF (n = 21) or ICSI (n = 30) cycles and were cryopreserved in the pronuclear stage (PN). The storage interval varied from 3 months to 5 years. The endometrial preparation was performed in all cycles with oral estrogen--progestin substitution. Embryos in the PN stage were examined for survival (rate of intact embryos after thawing) and progress of their further development. Endometrial thickness, number of transferred embryos, pregnancy rate (PR), implantation rate (IR) and abortions (AB) were evaluated as outcome parameters. RESULTS: A total of 210 thawed embryos were evaluated. Survival was 79% without regard to the method of fertilization. For 42 cryoembryotransfers 131 embryos (62%) in 6 to 10 blastomeres after 48 hours cultivation in G 1.2 and G 2.2 fy Vitrolife were selected. Further development was more regular in embryos obtained in classical IVF cycles, where 41% of the embryos were in 4 cells stage after 24 hours of cultivation and 36% of the embryos were in 8 cells stage after 48 hours while after ICSI 32% 4 cells embryos and 26% 8 cells embryos respectively. Endometrial thickness in the day of transfer in all patients was 10.9 +/- 1.3 mm, the average number of transferred embryos was 2.4 embryos/transfer, PR was 28.5 (IVF 42.9% and ICSI 20.7%), IR 12.0% (IVF 13.0% and ICSI 11.3%) and AB 41.6% (IVF 33.3% and ICSI 50.0%). CONCLUSION: Improvement of cryopreservation procedures, freezing of embryos in the pronuclear stage and last but not least the optimal endometrial preparation can positively influence the cumulative efficiency of assisted reproduction.

Cryopreservation↗
PubMed1992

Pregnancy after zygote intra-fallopian transfer using spermatozoa from a patient with Kallmann's syndrome.

A successful zygote intra-Fallopian transfer has been performed using spermatozoa from a patient with Kallmann's syndrome. Spermatogenesis was induced by hormonal treatment but even so, semen quality remained slightly impaired and no pregnancy occurred over 4 years, despite ovulation induction in the patient's spouse. Finally she conceived after assisted procreation by in-vitro fertilization and delivered a healthy baby.

Adult↗
PubMed1997

Intracytoplasmic sperm injection for treatment of the infertile male.

Intracytoplasmic sperm injection (ICSI) with in vitro fertilization represents one of the most significant advances in fertility technology. In this relatively new procedure, a single viable sperm is microinjected into an oocyte that has been extracted transvaginally. After fertilization occurs, the embryo is transferred into the uterus. This procedure now affords men who were previously thought to be irreversibly infertile the chance to initiate their own biologic pregnancy. However, because of the procedure's significant costs and its potential risk to the mother, careful selection of couples following a thorough male factor evaluation is mandatory.

Congenital Abnormalities↗
PubMed1997

Clinical outcome of cryopreserved human pronuclear stage embryos resulting from intracytoplasmic sperm injection.

OBJECTIVE: To compare the survival rate and pregnancy rate (PR) of embryos from intracytoplasmic sperm injection (ICSI) or conventional IVF, which were cryopreserved at the pronuclear stage in cycles where fresh transfer was deferred. DESIGN: Comparative observational study. SETTING: University-associated IVF center. PATIENT(S): Ninety-nine patients who deferred ET and had all their embryos cryopreserved at the pronuclear stage after 153 oocyte retrievals. Thirty-nine patients had their oocytes inseminated by ICSI and 60 patients had conventional IVF insemination. INTERVENTION(S): All embryos were frozen-thawed at the two pronuclear stage and allowed to cleave for 2 days before transfer. MAIN OUTCOME MEASURE(S): Survival rate (morphologically intact after thaw), cleavage rate (cleaved by time of transfer), and the clinical PR after frozen ET. RESULT(S): In the ICSI group, 205 embryos were thawed for use in 57 frozen ETs; in the IVF group, there were 527 embryos thawed for use in 149 frozen ETs. There was no significant difference in any of the outcome measures by insemination method: survival rates (ICSI, 93.2%; IVF, 94.8%); cleavage rates (ICSI, 95.2%; IVF, 94.7%), and clinical PR (ICSI, 14.0%; IVF, 17.4%). CONCLUSION(S): Pronuclear embryos resulting from ICSI can be cryopreserved successfully, thawed, and the survival rate and PR are comparable to conventional IVF.

Adult↗
PubMed2001

Evaluation of gestational deficiencies in cloned sheep fetuses and placentae.

Sheep fetal development at 35 days of gestation was examined following natural mating, in vitro production (IVP) of fertilized embryos, or somatic cell nuclear transfer (NT). Five crossbred (Blackface x Black Welsh) and four purebred (Black Welsh) fetuses and their associated placentae produced by natural mating were morphologically normal and consistent with each other. From 10 ewes receiving 21 IVP embryos, 17 fetuses (81%) were recovered, and 15 of these (88%) were normal. The NT fetuses were derived from two Black Welsh fetal fibroblast cell lines (BLW1 and 6). Transfer of 21 BLW1 and 22 BLW6 NT embryos into 12 and 11 ewes, respectively, yielded 7 (33%) and 8 (36%) fetuses, respectively. Only three (43%) BLW1 and two (25%) BLW6 NT fetuses were normal, with the rest being developmentally retarded. The NT fetal and placental deficiencies included liver enlargement, dermal hemorrhaging, and lack of placental vascular development reflected by reduced or absent cotyledonary structures. Fibroblasts isolated from normal and abnormal cloned fetuses did not differ in their karyotype from sexually conceived fetuses or nuclear donor cell lines. Our results demonstrate that within the first quarter of gestation, cloned fetuses are characterized by a high incidence of developmental retardation and placental insufficiency. These deficiencies are not linked to gross defects in chromosome number.

Animals↗
PubMed1993

Characterization of the first cell cycle in human zygotes: implications for cryopreservation.

OBJECTIVE: To study the temporal pattern of deoxyribonucleic acid (DNA) synthesis leading up to the first mitotic division in human one-cell stage zygotes. SETTING: In vitro fertilization program of a university hospital. PATIENTS: Couples donating spare embryos before the existence of an embryo freezing program. DESIGN: Incorporation of 3H-thymidine was examined between 9 to 27 hours after insemination in 72 untransferred human zygotes containing two pronuclei. Microscopic observations on an additional 978 transferred zygotes extended the 3H-thymidine incorporation data. RESULTS: The first thymidine incorporation was seen 9 to 10 hours after insemination, and the heaviest incorporation occurred between 11 and 13 hours. Subsequently, thymidine labeling declined, and chromosomal condensation and cell division first occurred approximately 19 to 20 hours. At 20 hours after insemination, 89% of the zygotes had two visible pronuclei (PN). In contrast, by 24 hours, 41% had no visible PN, whereas 5% had cleaved to the two-cell stage. By 27 hours, 38% had cleaved to the two-cell stage, and only 25% still had two visible PN. CONCLUSIONS: These results suggest that the DNA-synthetic, S-phase of the human zygote is initiated by approximately 9 to 10 hours after insemination and is completed approximately 3 to 5 hours later. The duration of G2 phase and mitosis is in the range of 4 to 6 and 3 to 3.5 hours, respectively. Because zygotes may be particularly susceptible to damage during the S phase of the cell cycle, these findings suggest that the optimal time for freezing of human zygotes may be approximately 20 to 22 hours after insemination when the majority of zygotes should have entered the G2 phase, before pronuclear dissolution and chromosome condensation.

Adult↗
PubMed2000

Evidence for placental abnormality as the major cause of mortality in first-trimester somatic cell cloned bovine fetuses.

The production of cloned animals is, at present, an inefficient process. This study focused on the fetal losses that occur between Days 30-90 of gestation. Fetal and placental characteristics were studied from Days 30-90 of gestation using transrectal ultrasonography, maternal pregnancy specific protein b (PSPb) levels, and postslaughter collection of fetal tissue. Pregnancy rates at Day 30 were similar for recipient cows carrying nuclear transfer (NT) and control embryos (45% [54/120] vs. 58% [11/19]), although multiple NT embryos were often transferred into recipients. From Days 30-90, 82% of NT fetuses died, whereas all control pregnancies remained viable. Crown-rump (CR) length was less in those fetuses that were destined to die before Day 90, but no significant difference was found between the CR lengths of NT and control fetuses that survived to Day 90. Maternal PSPb levels at Days 30 and 50 of gestation were not predictive of fetal survival to Day 90. The placentas of six cloned and four control (in vivo or in vitro fertilized) bovine pregnancies were compared between Days 35 and 60 of gestation. Two cloned placentas showed rudimentary development, as indicated by flat, cuboidal trophoblastic epithelium and reduced vascularization, whereas two others possessed a reduced number of barely discernable cotyledonary areas. The remaining two cloned placentas were similar to the controls, although one contained hemorrhagic cotyledons. Poor viability of cloned fetuses during Days 35-60 was associated with either rudimentary or marginal chorioallantoic development. Our findings suggest that future research should focus on factors that promote placental and vascular growth and on fetomaternal interactions that promote placental attachment and villous formation.

Animals↗
PubMed2001

A prospective randomized clinical trial of differing starter doses of recombinant follicle-stimulating hormone (follitropin-beta) for first time in vitro fertilization and intracytoplasmic sperm injection treatment cycles.

OBJECTIVE: Comparison of the efficacy of differing starter doses of recombinant follicle stimulating hormone (rFSH) for IVF and intracytoplasmic sperm injection cycles when the treatment is administered both subcutaneously and intramuscularly. DESIGN: Single center 1-year prospective randomized study. SETTING: Academic teaching hospital. PATIENT(S): 345 couples in first cycle. INTERVENTION(S): Treatment with subcutaneous or intramuscular rFSH, followed by E(2) and ultrasound follicle tracking, with later oocyte collection and zygote transfer. MAIN OUTCOME MEASURE(S): Ovarian response and other clinically dependent variables. RESULT(S): Group 1 patients, with day-3 FSH levels of less than 8.5 U/L, were randomized to begin treatment with rFSH at 150 IU (n = 146) or 200 IU (n = 151). The total dose of the drugs used was significantly lower in 150 IU group, as was the number of ICSI metaphase II oocytes. No other significant differences found. The dosage was increased in 9% on day 5. Group 2 patients, with day-3 FSH levels of greater than 8.5 U/L, were randomized to treatment with rFSH at 300 IU (n = 24) or 400 IU (n = 24). No significant outcome differences found between the two subgroups. Pregnancy rates for this group were half that of Group 1.Intramuscular administration was significantly more likely to result in a need for increased dosage than was subcutaneous administration. The level of E(2) at the time of hCG treatment was significantly lower in the intramuscular 150 IU group. CONCLUSION(S): In the main study total dosage used, the ICSI metaphase II oocyte numbers were significantly lower and there was a trend toward a need for a dosage increase on day 5 when 150 IU rFSH was the starter dosage, as compared to a starting dosage of 200 IU. Otherwise, there is little advantage to using the higher dosage.

Adult↗
PubMed2001

Nuclear transfer protocol affects messenger RNA expression patterns in cloned bovine blastocysts.

The successful production of embryos by nuclear transfer (NT) employing cultured somatic donor cells depends upon a variety of factors. The objective of the present study was to investigate the effects 1) of two different activation protocols, 2) the use of quiescent or nonquiescent donor cells (G(0) or G(1) of the cell cycle), and 3) passage number of donor cells on the relative abundance (RA) of eight specific mRNAs (DNA methyltransferase, DNMT; mammalian achaete-scute homologue, Mash2; glucose transporter-1, Glut-1; heat shock protein 70.1, Hsp; desmocollin II, Dc II; E-cadherin, E-cad; interferon tau, IF; insulin-like growth factor 2 receptor, Igf2r) in single blastocysts employing a semiquantitative reverse transcription-polymerase chain reaction assay. The results were compared with those for their in vitro (IVP)- and in vivo-generated noncloned counterparts. In experiment 1, employing either FBA (fusion before activation) or AFS (fusion and activation simultaneously) to generate NT blastocysts, Hsp mRNAs were not found in NT embryos from either protocol, whereas Hsp transcripts were detectable in IVP embryos. The relative abundance (RA) of IF transcripts was significantly increased in the AFS and IVP groups compared to the FBA treatment. In experiment 2, the use of either G(0) or G(1) donor cells to produce cloned embryos both significantly reduced the relative amount of DNMT transcripts and significantly increased the RA of Mash2 compared to the IVP embryos. In addition, IF transcript levels were significantly elevated in NT blastocysts employing G(1) donor cells for NT compared to IVP embryos and those generated using G(0) cells. In experiment 3, donor cells, either from passsage 5/6 or 8, were employed for NT. DNMT transcripts were significantly decreased, whereas Mash2 transcripts were significantly increased in both NT groups compared to their IVP counterparts. The amount of IF mRNA was significantly higher in P8-derived than in P5/6 and IVP embryos. In experiment 4, the RA of DNMT transcripts was decreased in in vivo-derived blastocysts compared to those produced in vitro. Mash2 expression was increased in in vivo embryos and those IVP embryos produced in medium containing Sigma BSA. The RA of Hsp was higher in IVP embryos produced in serum containing medium than in those produced in Sigma BSA or in vivo. In vivo embryos and those produced in Life Technologies BSA had the lowest expression of IF transcripts. Expression of all other genes was not affected by variation in NT methodology or IVP culture systems throughout experiments 1-4. In conclusion, depending on steps of the cloning procedure NT-derived embryos display marked differences from their IVP- and in vivo-derived counterparts. An aberrant expression pattern in NT embryos was found with respect to genes thought to be involved in stress adaptation, trophoblastic function, and DNA methylation during preimplantation development.

Animals↗
PubMed2001

Effects of cryopreservation of pronuclear-stage rabbit zygotes on the morphological survival, blastocyst formation, and full-term development after DNA microinjection.

The objectives of this study were to examine the freezing sensitivity of pronuclear-stage rabbit zygotes and to produce transgenic rabbits using the cryopreserved zygotes. Zygotes were cryopreserved either by one of two vitrification protocols or by one of the two conventional freezing protocols. The morphological survival rates of zygotes subjected to two-step freezing in 1.5 M ethylene glycol and 0.1 M sucrose (74%) or to vitrification in 7.2 M ethylene glycol and 1.0 M sucrose (81%) were higher than those subjected to freezing in 1.5 M DMSO (46%) or to vitrification in a mixture of 2.0 M DMSO, 1.0 M acetamide, and 3.0 M propylene glycol (41%). But the in vitro development into blastocysts of zygotes cryopreserved by vitrification (17%) or to a lesser extent by freezing (52%) was impaired, when compared to that of fresh control zygotes (89%). Next, a fusion gene composed from bovine aS1-casein promoter and a human GH structural gene (2.8 kb) was microinjected into the pronucleus of rabbit zygotes frozen-thawed in ethylene glycol and sucrose. Then, the presence of exogenous DNA in the genome of newborn offspring was determined by PCR. The post-injection survival of frozen zygotes (97%) was the same as that of fresh control zygotes (96%). However, of 18 offspring derived from 414 frozen-thawed and DNA-injected zygotes, no transgenic rabbits were produced. Of 52 offspring derived from 403 DNA-injected fresh zygotes, 3 transgenic rabbits were found. Here we report the first rabbit offspring resulting from zygotes cryopreserved at the pronuclear-stage, although the cryopreservation procedure employed must be improved if zygotes are to be used for systematic production of transgenic rabbits.

Animals↗
PubMed2005

Mouse zygotes with one diploid pronucleus formed as a result of ICSI can develop normally beyond birth.

A mouse spermatozoon was injected into mouse secondary oocytes (ICSI) in the vicinity of the metaphase spindle. In 22% of oocytes injected successfully, the maternal chromatin (the haploid chromatids formed after the second meiotic division) and paternal chromatin (from the sperm nucleus) were surrounded by a common nuclear envelope to form one diploid bi-parental pronucleus. However, the use of spermatozoa in which BrdU had been incorporated into DNA during spermatogenesis revealed, that maternal and paternal chromatin occupied two separate compartments within the one pronucleus. In the living state, the diploid pronucleus could be distinguished from a haploid one by its distinctly larger size and by a greater number of "nucleolus-like bodies"-criteria confirmed karylogically at the 1st cleavage division. Such zygotes with one diploid pronucleus were able to develop in vitro into blastocysts as often as those with two haploid pronuclei [11/29 (38%) vs. 14/35 (40%)]. Seventy nine 2-cell embryos developing in vitro from zygotes with one diploid pronucleus were transplanted to the oviducts of pseudopregnant recipients: two females had six foetuses when killed on the 17th day, and two females gave birth to nine young, eight of which survived and developed into normal fertile animals.

Animals↗
PubMed1998

The economic cost of infertility-related services: an examination of the Massachusetts infertility insurance mandate.

OBJECTIVE: To examine the costs and outcomes of infertility-related services in Massachusetts during a time of expanded use of assisted reproductive technology (ART). DESIGN: Cost data were obtained from the Massachusetts Department of Insurance Rate-Setting Commission and 9 large group insurance plans for the period 1986-1993. Utilization and success rates of ART were examined, and the cost per live delivery with the use of ART in 1993 was estimated. SETTING: The state of Massachusetts, in which access to infertility-related services has been mandated by law since 1989. PATIENT(S): The study population consisted of 8 large health maintenance organization plans and the Blue Cross/Blue Shield indemnity plan. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Per capita infertility-related expenditures, infertility-related expenditures as a percentage of total expenditures, live deliveries per initiated ART cycle, and cost per live delivery. RESULT(S): Expenditures for infertility services increased at a rate similar to or slower than inflation during the years 1988-1992. Increases were slowest in health maintenance organizations, probably as a result of provider arrangements. Infertility services accounted for 0.41% of total expenditures within the indemnity plan in 1993 (approximately $1.71 per contract-month). Examination of ART utilization showed no evidence of overutilization by patients with a low chance of success. The cost per live delivery with the use of ART in 1993 was $59,484. CONCLUSION(S): Mandated infertility coverage was associated with increased use of ART but not with excessive increases in consumer cost for infertility insurance coverage.

Adult↗
PubMed1997

Effect of encapsulation on development of mouse pronuclear stage embryos in vitro.

Pronuclear stage embryos with intact (ZI), slit (ZS) or completely removed (ZF) zona pellucida were encapsulated with an artificial zona pellucida (AZP) made of 1.5% sodium alginate. Embryos were cultured in KSOM medium with or without protein and their development in vitro to the blastocyst stage was recorded. AZP significantly (P < 0.05) improved the development of embryos to the blastocyst stage regardless of the presence of the natural zona pellucida. The encapsulated embryos developed at a higher rate (P < 0.05) in the absence of protein as compared with non-encapsulated embryos. Furthermore, the cell contacts at the 4-cell stage were significantly improved (P < 0.05) with encapsulation. AZP improved (P < 0.05) the development of pronuclear stage embryos with a slit zona pellucida to morula and blastocyst stages as compared with ZS embryos. It is concluded that AZP improves the in vitro development of pronuclear stage embryos with intact or completely removed zona pellucida as well as micromanipulated embryos to the blastocyst stage.

Alginates↗