OBJECTIVE: There is no consensus on optimum time for freezing pronucleate stage embryos (2PN's). Additionally, little is known regarding any possible adverse effects of long-term storage on implantation potential. Given the dynamic events that occur during syngamy, time-related sensitivities to freezing may exist and may differ for IVF and ICSI 2PN's. The present study was designed to test the hypotheses that: 1) survival and quality of frozen 2PN's is affected by time interval between insemination (IVF) or ICSI and freezing; and 2) that shorter storage time improves 2PN survival and implantation potential. DESIGN: Retrospective cohort study at our academic center. MATERIALS AND METHODS: Autologous cycles were included that resulted in a 2PN freeze-all from 1/1998 through 5/2007 with at least one subsequent cycle in which some of these frozen 2PN's were thawed and transferred on day 3 (CET; n=282; mean age at retrieval: 34.2±4.1y). 2PN's were frozen between 15-25 h post-IVF or ICSI, using Leibo's standard slow freezing and thaw protocol. Since ICSI results in earlier PN formation, cycles were stratified and analysed by type (IVF, n=174; ICSI, n=108). Linear regression analyses were performed to assess the association between time of freezing (i.e. time interval between IVF/ICSI and freeze) and 2PN survival, implantation rate and live-birth rate. Interaction between time to freeze and storage was also investigated. Wald p-values are two-sided. RESULTS: No significant associations were identified for either IVF or ICSI cycles between time of freezing or any of the outcome variables (overall %survival: 86.0±17.4, mean±SD, implantation rate: 20.2%, live birth rate: 39.7%). Similarly, there were no associations between duration of storage and any of these outcome variables. There was also no interaction between time of freezing and duration of storage (range from 0.05 to 6.27 y) for either IVF or ICSI. The only significant observation was an inverse relation between storage time and the number of transferred embryos having at least 6-cells (p=0.03). CONCLUSIONS: Our data show that time of freezing for IVF or ICSI zygotes does not affect survival, implantation or live birth rates. Furthermore, storage of zygotes (at least up to 6.27y) does not affect clinical outcome even when time interval between IVF/ICSI and freeze was considered. Our observation that quality of IVF embryos decreased with increasing years of storage warrants further investigation.

We investigated the frequencies of abnormalities involving either chromosome 1, 16, 18, or 21 in failed-fertilized human oocytes. Although abnormalities involving chromosome 16 showed an age-dependent increase, results for the other chromosomes did not show statistically significant differences among the three age groups, <35 years, 35–39 years, and >39 years. The scoring of four chromosomes is likely to underestimate the true rate of aneuploid cells. Therefore, for a pilot study investigating a more-comprehensive analysis of oocytes and their corresponding first polar bodies, we developed a novel eight-probe chromosome enumeration scheme using fluorescence in situ hybridization and spectral imaging analysis.

Numerous studies have shown that embryo morphology plays a key role in IVF outcome. Development to at least the 8-cell stage has been correlated with improved implantation potential, thereby providing a potentially useful marker for evaluating laboratory performance. However, it is essential to identify what variables impact embryo cleavage when using this type of marker for quality assessment so that relevant target values are identified for each technique being assessed. This study was undertaken to test the hypothesis that culture media type and patient age both influence development of the embryo to at least the 8-cell stage and therefore must be taken into consideration when assessing laboratory performance using ≥ 8-cell embryos as a marker. Retrospective data analysis. 7,030 ART cycles performed in our institution from January 1, 1998 to December 31, 2003 were retrospectively reviewed. The study dataset included 3,678 IVF cycles with or without assisted hatching. Oocytes were inseminated in Ham's F10+5% HSA or 10% SSS and evaluated 16–18 hr later for PN formation. Diploid zygotes were then transferred to one of the following culture media: a) Vitro Life Series: IVF500, n=4,248; G1.2, n=7,323; G1.3, n=5,329; or b) Irvine Scientific: P1, n=10,805. Embryos were stratified according to growth medium used and patient age (<35y and ≥35y), and evaluated for development to the ≥ 8-cell stage. Data were analyzed using Chi square with p<0.05 considered statistically significant. Media type significantly impacted development of embryos to ≥ 8-cell stage (IVF500: 27.7%; G1.2: 34.7%; G1.3: 38.7%; P1: 43.3%; p<0.0001). Of note, there was a significant improvement in the % of embryos at ≥8-cells as each new generation of Vitro Life medium was introduced (p<0.0001). As shown in the table, within age group, medium type continued to impact development to at least the 8-cell stage (p<0.0001 for both age groups). However, this impact was not consistently observed within across age groups and within medium. Our data indicate that cleavage rate is influenced by patient age and culture medium type. This relationship should be taken into consideration when establishing quality assurance target values. For example, our current day 5 inclusion criteria were developed using the IVF500 system. Given our current medium system (G1.3) and the results of this analysis, we must now revise our Day 5 inclusion criteria because the relevant target values have changed. Constant surveillance of relevant target values must be maintained in the IVF laboratory since improvements are continually being made in ART systems as demonstrated by the improvement across the generations of Vitro Life media.

Objective: To determine whether or not patients with unexpectedly good or sub-optimal sperm wash parameters have a better clinical outcome using conventional insemination or ICSI. Design: Retrospective matched pair analysis of 116 cycles were identified in which couples underwent split IVF/ICSI treatment. Materials and Methods: All cycles performed from January 2000 through March 2003 (n = 1622) were screened to identify those patients who underwent an ART cycle in which the oocytes were randomly split to conventional insemination (IVF group) or ICSI (ICSI group). These 116 patients comprised those who were ordered ICSI based on pre-cycle semen analysis, yet produced semen consistent with conventional IVF therapy on day of retrieval or, conversely, were ordered IVF, yet had a semen sample that was either sub-optimal or washed poorly. Additional inclusion criteria were >11 eggs, no freeze alls and cycles with > 5 mature oocytes. Immediately after retrieval, oocyte-cumulus complexes for each patient were randomly split between IVF and ICSI. Fertilization and embryo quality (% 8 cells, % 8 cells with <10 % fragmentation, % with early compaction, and % vacuolated) were determined. Pregnancy rates were derived exclusively from those cycles involving transfer of only those embryos arising from IVF or ICSI. Data were analyzed using Chi-square or Wilcoxon Signed Rank tests, as appropriate, with p < 0.05 considered significant. Results: Fertilization failure occurred following standard insemination in 5 cases (4.3%). No fertilization failures occurred following ICSI. Significantly higher diploid fertilization rates (p = 0.0018) and more embryos were achieved with ICSI (p = 0.0014). Standard insemination resulted in an increase in the triploidy rate (p = 0.0018). There was no difference in the % 8-cell embryos between the 2 insemination groups. However, significantly more embryos had early compaction following ICSI (p = 0.016) although the incidence of vacuolated embryos was higher in this group (p = 0.048). More embryos were selected for transfer from the ICSI group. More cycles had embryos transferred exclusively from the ICSI group, although ongoing pregnancy rates were not different according to the source of the embryos. Tabled 1 Conclusions: Compared with standard insemination, ICSI resulted in a higher fertilization rate, more total embryos, and more embryos selected for transfer. While there was no difference between the 2 groups regarding embryo quality with respect to cell number and fragmentation, the incidence of vacuolated blastomeres and early compaction was increased following ICSI. However, pregnancy rates were the same when 100% of the embryos were from either group. Collectively, the present results suggest that standard insemination can be performed when ICSI is being considered based on semen parameters on the day of egg retrieval.

Objectives: Numerical chromosome aberrations are detrimental to embryo survival and thus cause spontaneous abortions. We used fluorescence in situ hybridization (FISH) to determine the incidence of aneuploidy for chromosomes 1, 16, 18, and 21 in human eggs that failed to fertilize following either IVF or ICSI.Design: A total of 201 failed fertilized eggs from 91 patients were available for this study. Chromosome-specific aneuploidy, including both hypoploidy (nullisomy/monosomy) and hyperploidy (trisomy/tetrasomy), was determined for each of the four chromosomes with respect to patient age (<35 yrs, 35–39 yrs, >39 yrs).Materials and Methods: Institutional Review Board Approval was obtained for this study. Following fixation with ethanol:glacial acetic acid (3:1, v/v) and Wright's staining, suitable spreads were destained in methanol, pretreated with 2×SSC and pepsin, then post-fixed in 1% formaldehyde. The DNA denaturation was performed in 70% formamide/2×SSC at 75°C. DNA probes were purchased (CEP18, Vysis) or prepared in-house (pUC1.77, RMC16L001, LSP21). Slides were hybridized for 40–48 hr with probe mixture. Following removal of unbound probes, signals were scored by fluorescence microscopy. Statistical analyzes were performed using Chi square and Fisher's Exact Tests.Results: A total of 163 spreads were considered suitable for FISH analysis. Informative results were obtained from 114 spreads. There was a statistically significant age-related difference in aneuploidy frequency for chromosome 16 (13.2, 30 and 40% in the three age groups, respectively; P<0.05). Overall aneuploidy rates for chromosomes 1, 18 and 21 were 19.3, 18.8 and 24.3%, respectively, and did not show significant age-dependent increases. Frequencies for hypo- vs. hyperploidy showed chromosome-specific differences (% hypoploidy/total aneuploidy): 1: 59.1%; 16: 57.7%; 18: 66.7%; 21: 69.2%, although these differences did not reach statistical significance.Conclusion: There was an overall high frequency of aneuploidy (∼20%) among failed fertilized human eggs (including chromosome 1) and an age-related increase for chromosome 16. The tendency towards a higher ratio of hypoploidy for chromosomes 18 and 21 could represent a loss either of chromatids during fixation or of signals during FISH for these smaller chromosomes, or a loss of chromatids during meiosis I in the oocyte itself. These findings contribute to our understanding of the origin and fate of aneuploidy in humans. Supported by UC BioSTAR Grant and UCSF/Stanford Enterprise Fund (RAP) and BWH Ob/Gyn IVF Discretionary Funds (CR). Objectives: Numerical chromosome aberrations are detrimental to embryo survival and thus cause spontaneous abortions. We used fluorescence in situ hybridization (FISH) to determine the incidence of aneuploidy for chromosomes 1, 16, 18, and 21 in human eggs that failed to fertilize following either IVF or ICSI. Design: A total of 201 failed fertilized eggs from 91 patients were available for this study. Chromosome-specific aneuploidy, including both hypoploidy (nullisomy/monosomy) and hyperploidy (trisomy/tetrasomy), was determined for each of the four chromosomes with respect to patient age (<35 yrs, 35–39 yrs, >39 yrs). Materials and Methods: Institutional Review Board Approval was obtained for this study. Following fixation with ethanol:glacial acetic acid (3:1, v/v) and Wright's staining, suitable spreads were destained in methanol, pretreated with 2×SSC and pepsin, then post-fixed in 1% formaldehyde. The DNA denaturation was performed in 70% formamide/2×SSC at 75°C. DNA probes were purchased (CEP18, Vysis) or prepared in-house (pUC1.77, RMC16L001, LSP21). Slides were hybridized for 40–48 hr with probe mixture. Following removal of unbound probes, signals were scored by fluorescence microscopy. Statistical analyzes were performed using Chi square and Fisher's Exact Tests. Results: A total of 163 spreads were considered suitable for FISH analysis. Informative results were obtained from 114 spreads. There was a statistically significant age-related difference in aneuploidy frequency for chromosome 16 (13.2, 30 and 40% in the three age groups, respectively; P<0.05). Overall aneuploidy rates for chromosomes 1, 18 and 21 were 19.3, 18.8 and 24.3%, respectively, and did not show significant age-dependent increases. Frequencies for hypo- vs. hyperploidy showed chromosome-specific differences (% hypoploidy/total aneuploidy): 1: 59.1%; 16: 57.7%; 18: 66.7%; 21: 69.2%, although these differences did not reach statistical significance. Conclusion: There was an overall high frequency of aneuploidy (∼20%) among failed fertilized human eggs (including chromosome 1) and an age-related increase for chromosome 16. The tendency towards a higher ratio of hypoploidy for chromosomes 18 and 21 could represent a loss either of chromatids during fixation or of signals during FISH for these smaller chromosomes, or a loss of chromatids during meiosis I in the oocyte itself. These findings contribute to our understanding of the origin and fate of aneuploidy in humans. Supported by UC BioSTAR Grant and UCSF/Stanford Enterprise Fund (RAP) and BWH Ob/Gyn IVF Discretionary Funds (CR).

OBJECTIVE:To study the relationship between the presence of one-pronuclear oocytes in in vitro fertilization (IVF) patients and ovulation-induction response, oocyte and embryo development, and clinical outcome. DESIGN:Retrospective analysis of 535 consecutive IVF retrievals. Retrievals in which one or more oocytes exhibited one pronucleus were compared with retrievals in which no one-pronuclear oocytes (control) were observed. The following one-pronuclear versus control subgroups were also examined: leuprolide acetate/human menopausal gonadotropin (LA/hMG) ovulation inductions, high estradiol (E2) response cases, and retrievals in which a large number of oocytes (greater than or equal to 15) were recovered. SETTING:Brigham and Women's Hospital, a tertiary care, university-affiliated hospital. PATIENTS:Three hundred forty-six IVF patients were treated between January 1989 and May 1991. MAIN OUTCOME MEASURES:Parameters examined included E2 concentration and number of follicles with maximum diameter greater than or equal to 12 mm on day of human chorionic gonadotropin administration; number of total and mature oocytes retrieved; total fertilization rates; number of embryos; and percent per retrieval of embryo transfers (ETs), clinical pregnancies, and ongoing-livebirths. RESULTS:The one-pronuclear patients had higher E2 levels and larger number of follicles, yielded significantly more total and mature oocytes, had a higher overall fertilization rate, produced more embryos, and had higher ET, clinical pregnancy and ongoing-livebirth rates per retrieval than did the control patients. Analysis of the subgroup populations revealed no significant differences in the majority of the main outcome measures studied; however, the one-pronuclear patients yielded significantly more total and mature oocytes per retrieval. CONCLUSIONS:Although there was an increase in the clinical and ongoing-livebirth pregnancy rates (PRs) in one-pronuclear patients, this was probably associated with an improved ovulation-induction response in the one-pronuclear patients. They achieved significantly higher E2 levels, recruited a larger number of follicles, and yielded more oocytes and embryos per retrieval than the control patients. When only the LA/hMG, E2 greater than or equal to 1,500 pg/mL, or the greater than or equal to 15 oocytes/case retrievals were analyzed, the PRs were no longer different; however, the one-pronuclear patients still yielded significantly more total and mature oocytes per retrieval than the controls. Therefore, the appearance of one-pronuclear oocytes is probably associated with the maturation stage of the oocytes obtained and is indicative of an ovulation induction in which a large number of preovulatory, metaphase II oocytes have been recruited.

Annals of the New York Academy of SciencesVolume 248, Issue 1 p. 36-45 GUINEA PIG NEUROPHYSIN: ISOLATION, DEVELOPMENTAL ASPECTS, BIOSYNTHESIS IN ORGAN CULTURE H. Sachs, H. Sachs Roche Institute of Molecular Biology Nutley, New Jersey 07110 Department of Anatomy, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.Search for more papers by this authorD. Pearson, D. Pearson Roche Institute of Molecular Biology Nutley, New Jersey 07110 Department of Animal Science, School of Veterinary Medicine, University of California, Davis, California 95616.Search for more papers by this authorA. Nureddin, A. Nureddin Roche Institute of Molecular Biology Nutley, New Jersey 07110 The Mayo Clinic, Endocrine Research, Rochester, Minnesota 55901.Search for more papers by this author H. Sachs, H. Sachs Roche Institute of Molecular Biology Nutley, New Jersey 07110 Department of Anatomy, Case Western Reserve University School of Medicine, Cleveland, Ohio 44106.Search for more papers by this authorD. Pearson, D. Pearson Roche Institute of Molecular Biology Nutley, New Jersey 07110 Department of Animal Science, School of Veterinary Medicine, University of California, Davis, California 95616.Search for more papers by this authorA. Nureddin, A. Nureddin Roche Institute of Molecular Biology Nutley, New Jersey 07110 The Mayo Clinic, Endocrine Research, Rochester, Minnesota 55901.Search for more papers by this author First published: February 1975 https://doi.org/10.1111/j.1749-6632.1975.tb34175.xCitations: 23 AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Citing Literature Volume248, Issue1Neurophysins: Carriers of Peptide HormonesFebruary 1975Pages 36-45 RelatedInformation