ABSTRACT
Uroplakin (UP) tetraspanins and their associated proteins are major mammalian urothelial differentiation products that form unique two-dimensional crystals of 16-nm particles ("urothelial plaques") covering the apical urothelial surface. Although uroplakins are highly expressed only in mammalian urothelium and are often referred to as being urothelium specific, they are also expressed in several mouse nonurothelial cell types in stomach, kidney, prostate, epididymis, testis/sperms, and ovary/oocytes. In oocytes, uroplakins colocalize with CD9 on cell-surface and multivesicular body-derived exosomes, and the cytoplasmic tail of UPIIIa undergoes a conserved fertilization-dependent, Fyn-mediated tyrosine phosphorylation that also occurs in Xenopus laevis eggs. Uroplakin knockout and antibody blocking reduce mouse eggs' fertilization rate in in vitro fertilization assays, and UPII/IIIa double-knockout mice have a smaller litter size. Phylogenetic analyses showed that uroplakin sequences underwent significant mammal-specific changes. These results suggest that, by mediating signal transduction and modulating membrane stability that do not require two-dimensional-crystal formation, uroplakins can perform conserved and more ancestral fertilization functions in mouse and frog eggs. Uroplakins acquired the ability to form two-dimensional-crystalline plaques during mammalian divergence, enabling them to perform additional functions, including umbrella cell enlargement and the formation of permeability and mechanical barriers, to protect/modify the apical surface of the modern-day mammalian urothelium.
Subject(s)
Genetic Speciation , Oocytes/metabolism , Ovary/metabolism , Uroplakins/genetics , Urothelium/metabolism , Zygote/metabolism , Animals , Cell Differentiation , Female , Fertilization/genetics , Gene Expression Regulation , Litter Size , Male , Mice , Mice, Knockout , Oocytes/cytology , Ovary/cytology , Parthenogenesis/genetics , Phosphorylation , Phylogeny , Proto-Oncogene Proteins c-fyn/genetics , Proto-Oncogene Proteins c-fyn/metabolism , Signal Transduction , Testis/cytology , Testis/metabolism , Tetraspanin 29/genetics , Tetraspanin 29/metabolism , Uroplakins/classification , Uroplakins/metabolism , Urothelium/cytology , Xenopus laevis , Zygote/cytologyABSTRACT
OBJECTIVE: To identify the incidence, risk factors, and obstetric/perinatal outcomes associated with monozygotic twins (MZTs) after IVF. DESIGN: Nested case-control. SETTING: University-based center. PATIENT(S): The IVF cycles eventuating in pregnancy from 2000-2009. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The MZT incidence, chorionicity/zygosity, pregnancy/neonatal outcome. RESULT(S): Of 6,223 gestations, 131 MZTs were diagnosed (2.1% incidence; 2.0% in autologous and 2.7% in donor IVF cycles), 10 were dichorionic, and 121 were monochorionic. Controlling for all risk factors, young oocyte age, extended culture (noncleavage embryos transferred on/after day 4), and year of IVF treatment cycle were significantly associated with MZT. When assessing factors associated with specific MZT placentation, day 3 assisted hatching correlated more with dichorionic MZT, whereas extended culture and advanced day 5 embryonic stage correlated with monochorionic MZT. Comparing monozygotic to dizygotic multigestation outcomes, MZT fared worse; however, once controlling for triplet gestation, only gestational age at delivery remained significantly compromised in the monozygotic group. CONCLUSION(S): After IVF the incidence of MZT is high, with young oocyte age, year of treatment, and extended culture (or embryo stage at transfer) conferring greatest risk. Regarding MZT type, assisted reproductive technology (ART) procedures may influence the timing of embryonic splitting (i.e., division in early embryonic development may be influenced by zona pellucida [ZP] manipulation whereas later splitting may occur during delayed implantation). Poor obstetric/perinatal outcome is significantly impacted by the presence of an "extra" fetus, as high-order multiple gestation concurrent with an MZT conveyed the worst prognosis.
Subject(s)
Fertilization in Vitro/adverse effects , Infertility/therapy , Pregnancy, Twin , Twinning, Monozygotic , Twins, Monozygotic , Adult , Embryo Culture Techniques , Embryo Transfer , Female , Fertility , Gestational Age , Hospitals, University , Humans , Incidence , Infertility/physiopathology , Male , New York City/epidemiology , Pregnancy , Pregnancy Complications/epidemiology , Pregnancy Outcome , Retrospective Studies , Risk Assessment , Risk Factors , Time Factors , Treatment OutcomeABSTRACT
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ABSTRACT
OBJECTIVE: To characterize incidence, chorionicity, amnionicity, and pregnancy outcome for monozygotic twin pregnancy (MZT) after IVF. DESIGN: Retrospective review. SETTING: University-based fertility center. PATIENT(S): Autologous and oocyte donation IVF cycles eventuating in 4,976 clinical gestations from 2000 to 2007. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): MZT incidence, chorionicity, zygosity, pregnancy outcome. RESULT(S): Ninety-eight MZTs were diagnosed after first-trimester ultrasound evaluation (2% incidence). The incidence in cycles transfering autologous oocytes was 1.7% but was 3.3% with donor oocytes; however, women <35 years old using their own oocytes displayed a similar rate (3.1%) to women using donor oocytes. Eighty MZTs occurred after fresh day-5 transfer; only 14 followed fresh day-3 transfer (2.6% vs. 1.2%). The MZT incidence in day-3 transfers without hatching was not different from those with hatching (1.3% vs. 1.1%). In addition, MZT incidence did not differ significantly whether or not ICSI was performed (2.4% vs. 2.0%). Four MZTs occurred after frozen-thawed embryo transfer (0.8% incidence). Ninety-five percent of all placental arrangements were confirmed as monochorionic-diamniotic on obstetric ultrasounds. CONCLUSION(S): These findings confirm a higher incidence of MZT after IVF. Monochorionic-diamniotic implantations were increased, whereas monochorionic-monoamniotic were not. The MZT risk factors included young age and extended culture, but not zona penetration or cryopreservation.
Subject(s)
Embryo Transfer/statistics & numerical data , Fertilization in Vitro/statistics & numerical data , Pregnancy Outcome/epidemiology , Twinning, Monozygotic , Amnion/cytology , Blastocyst/cytology , Chorion/cytology , Female , Humans , Incidence , Male , Oocyte Donation/statistics & numerical data , Pregnancy , Retrospective Studies , Risk Factors , Ultrasonography, PrenatalABSTRACT
OBJECTIVE: To determine whether there is a difference in pregnancy outcomes, stratified by age, between women undergoing elective single blastocyst transfer (eSBT) versus those undergoing double blastocyst transfer (2BT). DESIGN: Retrospective analysis. SETTING: University IVF center. PATIENT(S): A total of 1,141 nondonor IVF cycles in women aged <40 years from January 2004-March 2007. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Eggs retrieved, embryos cryopreserved, implantation rates, clinical pregnancy rates (PR), live birth rates (LBR), spontaneous abortion rates (SAB). RESULT(S): Pregnancy outcomes in 52 cycles of women <40 years of age who underwent eSBT were compared with 1,086 cycles of women who underwent 2BT in fresh IVF cycles from January 2004-March 2007. Overall, the eSBT was associated with a statistically significant 92% reduction in the twinning rate (from 25%-2%) while maintaining a high clinical PR (63% in the eSBT group vs. 61% in the 2BT group). CONCLUSION(S): Women who are <40 years of age undergoing nondonor fresh IVF cycles can electively choose to transfer a single blastocyst for the purpose of significantly reducing their risk of multiples without compromising their PR.
Subject(s)
Embryo Transfer/methods , Pregnancy Outcome , Adult , Age Factors , Elective Surgical Procedures , Embryo Disposition , Female , Fertilization in Vitro/methods , Humans , Infertility, Female/therapy , Pregnancy , Pregnancy Rate , Retrospective Studies , Single Embryo Transfer/methodsABSTRACT
Cancer patients produce similar numbers of oocytes after ovarian hyperstimulation compared with age-matched infertile controls, suggesting that malignancy does not adversely affect ovarian response.
Subject(s)
Cryopreservation/methods , Gonadotropins/pharmacology , Infertility, Female/therapy , Neoplasms/physiopathology , Oocytes/drug effects , Ovulation Induction/methods , Reproductive Techniques, Assisted , Adult , Breast Neoplasms/physiopathology , Case-Control Studies , Chorionic Gonadotropin/pharmacology , Cohort Studies , Drug-Related Side Effects and Adverse Reactions , Estrogens/blood , Female , Follicle Stimulating Hormone , Hodgkin Disease/physiopathology , Humans , Infertility, Female/etiology , Oocyte Retrieval/methods , Oocytes/cytology , Radiotherapy/adverse effects , Retrospective Studies , Uterine Neoplasms/physiopathologyABSTRACT
OBJECTIVE: To determine whether early measurement of the serum cytokines interleukin-2 receptor (IL-2R), IL-6, and IL-8 along with human chorionic gonadotropin (hCG) and progesterone (P(4)) can differentiate an ectopic from an intrauterine gestation. DESIGN: Retrospective analysis. SETTING: University-based fertility center. PATIENT(S): 75 women who underwent treatment with in vitro fertilization (IVF) and subsequently had an ectopic gestation (n = 15), spontaneous abortion (SAB) (n = 30), or term delivery (TD) (n = 30). INTERVENTION(S): Serum samples were obtained 14 (day 28) and 21 (day 35) days after oocyte retrieval. MAIN OUTCOME MEASURE(S): Serum concentrations of IL-2R, IL-6, IL-8, P(4), and hCG. RESULT(S): Median hCG readings on day 28 and day 35 were statistically significantly lower in the ectopic gestation group than in those with spontaneous abortion or term delivery. On day 28, median IL-8 levels were lower in the ectopic gestation group when compared with all intrauterine gestations combined. No statistically significant differences in IL-2R or IL-6 levels were noted between groups. Despite P(4) supplementation, median day-35 P(4) levels were lower in ectopic gestation than in the spontaneous abortion and term delivery cycles. CONCLUSION(S): In the setting of a rise or plateau in hCG levels, low day-28 IL-8 and day-35 P(4) levels suggested an extrauterine implantation. This assay combination may facilitate earlier diagnosis of an ectopic gestation when pregnancy location is unclear.
Subject(s)
Embryo Implantation , Fertilization in Vitro , Interleukin-8/blood , Pregnancy, Ectopic/blood , Abortion, Spontaneous/blood , Adult , Biomarkers/blood , Chorionic Gonadotropin/blood , Female , Gestational Age , Humans , Interleukin-6/blood , Live Birth , Predictive Value of Tests , Pregnancy , Pregnancy Outcome , Pregnancy, Ectopic/diagnosis , Progesterone/blood , Receptors, Interleukin-2/blood , Retrospective Studies , Time Factors , Up-RegulationABSTRACT
OBJECTIVE: To assess the rates of select obstetric outcomes in oocyte donation (OD) recipients aged <35 years and > or =40 years and compare them to similarly aged IVF patients. DESIGN: Retrospective anonymous questionnaire study. SETTING: University-based IVF center. PATIENT(S): Live-birth outcome was experienced by 199 OD recipients and 488 IVF patients <35 or > or =40 years. INTERVENTION(S): The OD or IVF cycles. MAIN OUTCOME MEASURE(S): Response rate, pregnancy outcome, and complications. RESULT(S): Response rate was 60%. The OD recipients had significantly higher rates of pregnancy-induced hypertension (PIH) than their IVF counterparts. The OD <35 years had the highest rate (42%), followed by OD > or =40 years (26%), IVF > or =40 years (14%), and IVF patients <35 years (12%). In twin pregnancies, the rates of PIH remained higher in the OD groups: OD <35 years (56%), OD > or =40 years (36%), IVF > or =40 years (25%), and IVF <35 years (22%). Twin pregnancy rate was lowest in IVF patients > or =40 years (19%) and a lower preterm delivery rate (16%) reflected this difference. The cesarean section rates were 50% for singleton and 78% for twin deliveries in the OD patients <35 year; in OD patients > or =40 years, the rates were 75% and 84%, respectively. CONCLUSION(S): The OD recipients are at higher risk for untoward obstetric outcomes than their IVF counterparts. Young OD recipients reported the highest rate of PIH, warranting further investigation into an association between early loss of ovarian function and PIH.
Subject(s)
Fertilization in Vitro , Hypertension, Pregnancy-Induced/etiology , Oocyte Donation , Adult , Cesarean Section/statistics & numerical data , Female , Humans , Hypertension, Pregnancy-Induced/epidemiology , Maternal Age , Obstetric Labor, Premature/epidemiology , Pregnancy , Retrospective Studies , Risk Factors , TwinsABSTRACT
OBJECTIVE: To reevaluate clinical management of isolated teratozoospermia, in couples initiating IVF. DESIGN: Retrospective analysis of fertility indices in 535 cycles. SETTING: A large, university-based fertility center. PATIENT(S): Consecutive couples (n = 495) who had a semen analysis using Kruger/Tyberberg strict criteria at our center within 12 months before undergoing their first and/or second IVF cycle in 2002-2004 with >2 million postwash, motile sperm on the day of egg retrieval. INTERVENTION(S): Eggs were fertilized either by conventional IVF or ICSI. Semen analysis and gamete/embryo manipulation was standardized in all cases. MAIN OUTCOME MEASURE(S): Fertilization, fertilization failure, pregnancy, and live birth rates. RESULT(S): There was no statistical difference in fertilization, fertilization failure, pregnancy, and live birth rates in the first or second IVF cycle when comparing couples with isolated teratozoospermia (<5% normal morphology) to those with a normal semen analysis. Furthermore, no improvement in these outcomes was noted when ICSI was used to treat these teratozoospermic couples. CONCLUSION(S): Because isolated teratozoospermia generally does not impact on the major indices of IVF, these patients need not be subjected to the unnecessary cost and potential risks of ICSI. Future studies, however, should focus on different sperm morphologic and biochemical parameters to determine if they are important for clinical management in IVF.
Subject(s)
Fertilization in Vitro , Infertility, Male/therapy , Sperm Injections, Intracytoplasmic , Spermatozoa/abnormalities , Adult , Birth Rate , Cell Shape , Female , Humans , Infertility, Male/etiology , Male , Pregnancy , Retrospective Studies , Spermatozoa/cytology , Treatment OutcomeABSTRACT
To assess whether the use of extended embryo culture can reduce the incidence of high-order multiple gestations, a retrospective analysis of 7,418 fresh ETs performed in a university-based IVF clinic from 1997-2003 was conducted, comparing program results before and after institution of a protocol to select patients for extended culture of in vitro fertilized embryos. The incidence of triplet pregnancies was significantly reduced in patients at highest risk for high-order multiple gestations, i.e., those at <35 years of age (16.8% versus 6.8%), those at 35-37 years of age (13.0% versus 5.6%), and recipients of donated oocytes (11.2% versus 4.5%).
Subject(s)
Embryo Culture Techniques/methods , Embryo Transfer , Fertilization in Vitro/methods , Pregnancy Rate , Pregnancy, Multiple , Adult , Embryo Implantation , Female , Humans , Maternal Age , Pregnancy , Retrospective Studies , Triplets , TwinsABSTRACT
OBJECTIVE: To assess the clinical value of transabdominal ultrasound (TAS)--assisted embryo transfer on outcomes of in vitro fertilization--embryo transfer (IVF-ET) in comparison to the "clinical touch" method of transcervical embryo transfer by one physician and to determine if transabdominal ultrasound should be applied to all cases of embryo transfer in this practice. DESIGN: A retrospective comparison study. SETTING: A university-based IVF practice. PATIENT(S): Two hundred forty-nine patients who underwent transcervical transfer of fresh embryos created using autologous oocytes performed by the same physician from July 1, 2003, to June 30, 2004. INTERVENTION(S): On selected days, at time of embryo transfer, transabdominal ultrasound was performed to guide catheter placement depth approximately 1 cm from the uterine fundus. MAIN OUTCOME MEASURE(S): The presence of at least one gestational sac on ultrasound was compared in the two study groups; additionally, the clinical pregnancy rate (presence of fetal cardiac activity observed on ultrasound), the ectopic pregnancy rate, the biochemical pregnancy rate, and the implantation rate (number of gestational sacs identified on ultrasound per number of embryos transferred) between groups was evaluated. Characteristics of the two cohorts were analyzed to verify similarities between the treatment and control groups, including age of recipient, prior IVF history, day of transfer (day 3 or day 5 after retrieval), difficulty of transfer, the use of a tenaculum, and the quality and number of embryos transferred. RESULT(S): No statistical difference was seen in the presence or number of gestational sacs following embryo transfer with or without transabdominal ultrasound guidance. CONCLUSION(S): No additional advantage is conferred when using transabdominal ultrasound to perform embryo transfer. In experienced hands, the "clinical touch" method of embryo transfer yields equivalent results to transabdominal ultrasound-guided embryo placement. However, in patients with a prior history of difficult uterine sounding or embryo transfer, transabdominal ultrasound guidance may still play a role.
Subject(s)
Abdomen/diagnostic imaging , Catheterization/methods , Embryo Transfer , Pregnancy Outcome , Adult , Female , Fertilization in Vitro , Humans , Middle Aged , Pregnancy , Retrospective Studies , UltrasonographyABSTRACT
In mammals, the dynamic reprogramming of DNA methylation begins during gametogenesis and continues through embryogenesis. Recently, immunofluorescence staining with an antibody against 5-methylcytosine (anti-5-MeC) has revealed active demethylation of the male pronucleus in zygotes beginning at 4-6 h after fertilization. In this study, we characterized the DNA methylation patterns in mouse zygotes and in human tripronucleate (3 PN) zygotes discarded after conventional fertilization or following ICSI. Pronuclei were subjected to fluorescence in-situ hybridization to identify the X and/or Y chromosomes and then stained with anti-5-MeC. In diandric 3 PN zygotes from conventional IVF, we consistently observed one strongly and two weakly stained pronuclei. In contrast, the majority of 3 PN ICSI zygotes, mainly digynic zygotes, displayed two strongly and one weakly stained pronuclei. Two zygotes from ICSI failed to show any staining difference among the three pronuclei. Our results indicate that the active demethylation of male pronuclei occurs in both mouse and human zygotes. It is possible that the abnormal methylation patterns resulting from a dysfunctional cytoplasm may occur in a small number of oocytes and may affect embryonic viability.
Subject(s)
Cell Nucleus/genetics , Chromosomes, Human, X/metabolism , Chromosomes, Human, Y/metabolism , DNA Methylation , Zygote/cytology , Animals , Cell Nucleus/metabolism , Cell Nucleus/ultrastructure , Chromosomes, Human, X/chemistry , Chromosomes, Human, X/genetics , Chromosomes, Human, Y/chemistry , Chromosomes, Human, Y/genetics , DNA/analysis , DNA/genetics , DNA/metabolism , Embryo, Mammalian/cytology , Embryo, Mammalian/metabolism , Female , Fertilization/genetics , Humans , In Situ Hybridization, Fluorescence , Male , Mice , Mice, Inbred Strains , Sperm Injections, Intracytoplasmic , Zygote/chemistry , Zygote/metabolismABSTRACT
BACKGROUND: Germinal vesicle (GV) oocytes constitute a potential resource but their developmental competence is questionable especially when surrounding cumulus cells are removed. The intercellular factors/mechanisms underlying such poor embryonic competence may originate at a nuclear and/or ooplasmic level. METHODS: Immature or mature oocytes were obtained from three mouse strains following pregnant mare serum gonadotropin (PMSG) or PMSG+ human chorionic gonadotropin (hCG) treatment. Immature oocytes were denuded of cumulus cells prior to in vitro maturation. Pronuclear (PN) transfer was used to examine nuclear-ooplasmic interplay on resultant embryonic development and Oct-4 immuno-staining patterns. RESULTS: Embryos arising from ooplasts of in vivo matured oocytes displayed significant increases in blastocyst formation rates and total blastomere numbers when compared to those created from ooplasts of denuded oocytes. Oct-4 staining was more pronounced and restricted to the inner cell mass (ICM) in blastocysts arising from the ooplasm of in vivo matured zygotes than in those created from denuded oocytes. CONCLUSIONS: Developmental defect(s) appear to develop primarily in the ooplasm of oocytes that are denuded of their cumulus cells prior to in vitro maturation. Such oocytes result in embryos with poor developmental competence. These defects result in anomalies in cell number and Oct-4 expression during the morula-blastocyst developmental transition.
Subject(s)
DNA-Binding Proteins/metabolism , Oocytes/growth & development , Oocytes/metabolism , Transcription Factors/metabolism , Animals , Blastocyst/cytology , Blastocyst/metabolism , Chorionic Gonadotropin/administration & dosage , Embryonic Development/drug effects , Female , Fertilization in Vitro , Gonadotropins, Equine/administration & dosage , In Vitro Techniques , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred DBA , Morula/cytology , Morula/metabolism , Nuclear Transfer Techniques , Octamer Transcription Factor-3 , Oocytes/drug effects , OogenesisABSTRACT
Cell allocation and subsequent lineage commitment in the human embryo may be established as early as in the unfertilized oocyte. This phenomenon might be the result of subtle differences of gene expression and protein distribution. To assess whether gene expression profiling by reverse transcription-polymerase chain reaction could be a suitable tool for the detection of cell allocation and lineage commitment, the expression pattern of the putative inner cell mass marker gene Oct-4 and the trophectodermal marker genes beta-HCG and beta-LH were correlated in individual blastomeres of preimplantation human embryos. In 2- to 5-cell stage embryos, expression of beta-HCG and Oct-4 mRNA was negatively correlated in all blastomeres with statistical significance, suggesting that cell allocation can be assessed by those markers at early stages. In 7- to 10-cell stage embryos, expression of beta-LH and Oct-4 mRNA was negatively correlated in some blastomeres without statistical significance, suggesting that more experiments are necessary to decide if lineage commitment can be assessed in some cells by those markers at later stages.
Subject(s)
Blastocyst/metabolism , Genetic Markers , Chorionic Gonadotropin/genetics , Chorionic Gonadotropin/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Luteinizing Hormone/genetics , Luteinizing Hormone/metabolism , Octamer Transcription Factor-3 , RNA, Messenger/metabolism , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
In human embryos, blastomeres differentiate into trophectoderm (TE) cells and inner cell mass (ICM) cells of blastocysts. Although morphologically indistinguishable, blastomeres at early cleavage stages are likely to undergo changes on a molecular level that make them destined to become ICM or TE cells. While the transcription factor Oct-4 might serve as a marker for totipotent ICM cells, human chorionic gonadotrophin might be used as the equivalent for TE cells. This study reports a reverse transcription-polymerase chain reaction procedure to assess human beta-HCG mRNA concentrations as well as ploidy in individual blastomeres from normally and abnormally fertilized human embryos. beta-HCG mRNA was detected in both euploid and aneuploid cells and in oocytes. Surprisingly, beta-LH mRNA was also detected in some euploid blastomeres. In regard to preimplantation genetic diagnosis, assessment of expression levels of beta-HCG and Oct-4 mRNA in individual biopsied cells might serve as a tool to identify embryogenic blastomeres in combination with testing for chromosome and single gene abnormalities.
Subject(s)
Blastomeres/physiology , Chorionic Gonadotropin, beta Subunit, Human/genetics , Embryonic and Fetal Development/genetics , Luteinizing Hormone, beta Subunit/genetics , Ploidies , RNA, Messenger/genetics , Base Sequence , Cell Nucleus/genetics , Cytoplasm/genetics , DNA Primers , DNA, Complementary/genetics , Embryo, Mammalian/physiology , Fertilization in Vitro , Humans , Karyotyping , Oocytes/cytology , Polymerase Chain Reaction/methodsABSTRACT
BACKGROUND: Cytoplasmic defects are thought to cause aneuploidies in oocytes and embryos and oocyte 'reconstruction' by germinal vesicle (GV) transfer may circumvent such defects. In mice 'reconstructed' oocytes undergo meiosis and fertilize normally, but early embryonic development is compromised if their ooplasm matured in vitro. This study employs sequential MII spindle and/or pronucleus (PN) transfer to assess the embryonic potential of MII nuclei that form following GV transfer. METHODS AND RESULTS: Mouse embryos generated by these procedures were transferred to the oviducts of pseudopregnant mice to monitor pregnancy outcome. Following GV transfer, the resultant metaphase II (MII) nuclei were activated either in situ or transferred and activated in ooplasts from in-vivo matured oocytes. When exchanged with the female PN of a fertilized zygote, only the PNs that developed in in-vivo matured ooplasts generated live offspring. Viable offspring also resulted when MII nuclei were transferred to in-vivo matured ooplasts and fertilized by insemination with sperm or by artificial activation and male PN transfer. Significantly, the offspring displayed normal fertility as adults. CONCLUSION: This report of live births following GV transfer in mice illustrates the importance of the maturational history of the ooplasm at PN formation for normal embryonic and fetal development.
Subject(s)
Cell Nucleus/physiology , Embryonic and Fetal Development , Metaphase , Micromanipulation , Nuclear Transfer Techniques , Oocytes/physiology , Animals , Birth Rate , Female , Fertility , Male , Mice , Mice, Inbred Strains , Pregnancy , Spindle Apparatus/transplantationABSTRACT
BACKGROUND: We assessed the maturational competence and the chromosomal pattern of mouse oocytes reconstructed by germinal vesicle (GV) transfer technique using nuclear and/or cytoplasmic components from cryopreserved GV stage oocytes. METHODS: From 657 GV oocytes (326 fresh and 331 frozen/thawed), four groups of reconstructed oocytes were obtained by micromanipulation and electrofusion: fresh GV-fresh cytoplast (FF), thawed GV-thawed cytoplast (TT), fresh GV-thawed cytoplast (FT), thawed GV-fresh cytoplast (TF). All reconstructed oocytes were cultured in vitro to metaphase II. RESULTS: Survival rate after manipulation and electrofusion, as well as progression to metaphase II, did not differ significantly among the four groups. Comparing reconstructed oocytes with fresh and thawed control pools, the only difference was a slightly but significantly higher maturation rate in the TT pool versus matched controls (P < 0.01). Cytogenetic analysis of 25 reconstructed oocytes showed the expected number of 20 chromosomes in 88% of them. CONCLUSIONS: We conclude that both nuclear and cytoplasmic components derived from cryopreserved immature oocytes are suitable for GV transfer procedure, and generate chromosomally normal oocytes able to progress to metaphase II in vitro. The possibility of using cryostored immature oocytes as a source of nuclei and cytoplasm could help in applying GV transfer procedure, both in research and clinical settings.