Effects of cyclophosphamide administration on the in vitro fertilization of mice.

PURPOSE: To evaluate the oocyte fertilization ability and embryo growth after cyclophosphamide (CPA) treatment in mice. METHODS: Mice were treated with CPA at different doses (0-800 mg/kg body weight). The oocytes then were retrieved and evaluated for their in vitro fertilization efficiency. RESULTS: The average number of metaphase II (MII) oocytes significantly decreased by ≥400 mg/kg CPA administration. The fertilization rate also decreased in the group that was treated with ≥400 mg/kg CPA. However, after fertilization, the embryos demonstrated normal growth ability. Two weeks after CPA administration, the number of mice from which the oocytes could be retrieved markedly decreased, but the fertilization rate and development of morphological features in the embryos were similar to those of the controls. One month after CPA administration, the number of mice from which the oocytes could be retrieved, fertilization rate, and development of the morphological features in the embryos were similar to those of the controls. CONCLUSION: The number of oocytes decreased as the CPA administration level increased; however, the oocytes' potential for fertilization and development to the blastocyst stage was not significantly affected. One month after CPA administration, the number of oocytes and the potential for development into blastocysts were recovered.

Prediction of the in vitro developmental competence of early-cleavage-stage human embryos with time-lapse imaging and oxygen consumption rate measurement.

PURPOSE: To assess an embryo's ability to develop into a good-quality blastocyst during the early-cleavage stage using time-lapse imaging and the oxygen consumption rate. METHODS: In total, 942 zygotes had their oxygen consumption rates measured. In total, 282 zygotes were assessed by using time-lapse imaging. In total, 121 zygotes were examined by using both their oxygen consumption rate and time-lapse imaging. RESULTS: The embryos with moderate respiration rates of between 0.41 and 0.61 (×1014/mol s-1) on day 3 had a 22.1% chance of becoming good-quality blastocysts; those outside that range had a 14.3% chance. With the time-lapse system, when the first division was within 24 hours, 22.3% of the embryos grew to good blastocysts. After 24 hours, the rate dropped to 8.6%. The intervals between two consecutive cleavages were calculated and the duration of the second cell cycle was defined. When the time was between nine hours and 13 hours, there was a higher rate of good blastocysts. Regarding both criteria, when the embryos had progressed in the optimal range, a high percentage of them had become good blastocysts; it was 8.0% outside of that range. CONCLUSION: Individual embryos with the potential to develop into good-quality blastocysts could be selected at day 3 of culture using these systems.

DNA methylation errors at imprinted loci after assisted conception originate in the parental sperm.

There is an increased prevalence of imprinting disorders, such as Beckwith-Wiedemann syndrome, associated with human assisted reproductive technologies (ART). Work on animal models suggests that in vitro culture may be the source of these imprinting errors. However, in this study we report that, in some cases, the errors are inherited from the father. We analyzed DNA methylation at seven autosomal imprinted loci and the XIST locus in 78 paired DNA samples. In seven out of seventeen cases where there was abnormal DNA methylation in the ART sample (41%), the identical alterations were present in the parental sperm. Furthermore, we also identified DNA sequence variations in the gene encoding DNMT3L, which were associated with the abnormal paternal DNA methylation. Both the imprinting errors and the DNA sequence variants were more prevalent in patients with oligospermia. Our data suggest that the increase in the incidence of imprinting disorders in individuals born by ART may be due, in some cases, to the use of sperm with intrinsic imprinting mutations.

The efficacy of the transfer of twice frozen-thawed embryos with the vitrification method.

OBJECTIVE: To confirm the clinical benefits of recryopreserved, twice-thawed embryo transfer (ET). DESIGN: Retrospective study. SETTING: Private fertility clinic. PATIENT(S): Forty-nine women whose embryos had been refrozen after a previous frozen-thawed ET. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Comparison of implantation and pregnancy rates of twice-cryopreserved, twice-thawed embryos versus once-cryopreserved, once-thawed embryos. RESULT(S): The pregnancy rate per ET cycle was 27.8% in the refrozen group and 25.9% in the control group (no statistically significant difference). The implantation rate was 25.0% in the refrozen group and 19.3% in the control group (no statistically significant difference). CONCLUSION(S): The refreezing of supernumerary embryos can prevent ovarian hyperstimulation syndrome in stimulated patients and in those who have experienced repeated failed pregnancies. If unexpected supernumerary embryos are available for recryopreservation after frozen-thawed ET, these embryos may be revitrified for a future transfer.

Chromosome analysis of human refrozen embryos following fluorescence in situ hybridization.

PURPOSE: Several recent reports have discussed refrozen and thawed embryo transfer; however, the process may cause a degree of chromosomal damage and subtle genomic mutation. In view of this possibility, the purpose of this study was to investigate the incidence of aneuploidy in refrozen embryos. METHODS: In order to investigate the incidence of aneuploidy and mosaicism observed in chromosome 1, fluorescent in situ hybridization (FISH) was used on surviving embryos that first underwent one freeze-thaw cycle, then were allowed to develop to the blastocyst stage, and subsequently survived a second freeze-thaw cycle. RESULTS: Of 1,132 blastomeric nuclei analyzed from 15 refrozen embryos, disomy was found in 82.9%. In contrast, for the 11 blastocysts subjected to only one freeze-thaw cycle, disomy was noted in 78.4%. Of the 197 blastomeric nuclei analyzed in all arrested embryos, disomy was found in 51.8%. CONCLUSIONS: The refreezing process did not increase aneuploidy. The good and fair morphology groups demonstrated a higher percentage of disomy than the poor morphology group regardless of whether they were frozen once or twice.

Distribution of N-Acylhomoserine Lactone-Producing Fluorescent Pseudomonads in the Phyllosphere and Rhizosphere of Potato (Solanum tuberosum L.).

Four hundred and fifty nine isolates of fluorescent pseudomonads were obtained from the leaves and roots of potato plants. Of these, 20 leaf isolates and 28 root isolates induced violacein production in two N-acylhomoserine lactone (AHL)-reporter strains-Chromobacterium violaceum CV026 and VIR24. VIR24 is a new reporter strain for long N-acyl-chain-homoserine lactones, which can not be detected by CV026. Thin-layer chromatography revealed that the isolates produced multiple AHL molecules. We compared the 16S rRNA gene sequences of these isolates with sequences from a known database, and examined phylogenetic relationships. The AHL-producing isolates generally separated into three groups. Group I was mostly composed of leaf isolates, and group III, root isolates. Group II comprised both leaf and root isolates. There was a correlation between the phylogenetic cluster and the AHL molecules produced and some phenotypic characteristics. Our study confirmed that AHL-producing fluorescent pseudomonads could be distinguished in the phyllosphere and rhizosphere of potato plants.

Aberrant DNA methylation of imprinted loci in sperm from oligospermic patients.

Recent studies suggest that assisted reproductive technologies (ART), which involve the isolation, handling and culture of gametes and early embryos, are associated with an increased incidence of rare imprinting disorders. Major epigenetic events take place during this time and the process of ART may expose the epigenome to external influences, preventing the proper establishment and maintenance of genomic imprints. However, the risks of ART cannot be simply evaluated because the patients who receive ART may differ both demographically and genetically from the general population at reproductive age. In this study, we examined the DNA methylation status of seven imprinted genes using a combined bisulphite-PCR restriction analysis and sequencing technique on sperm DNA obtained from 97 infertile men. We found an abnormal paternal methylation imprint in 14 patients (14.4%) and abnormal maternal imprint in 20 patients (20.6%). The majority of these doubly defective samples were in men with moderate or severe oligospermia. These abnormalities were specific to imprinted loci as we found that global DNA methylation was normal in these samples. The outcome of ART with sperm shown to have an abnormal DNA methylation pattern was generally poor. However, one sample of sperm with both paternal and maternal methylation errors used in ICSI produced a child of normal appearance without any abnormalities in their imprinted methylation pattern. Our data suggest that sperm from infertile patients, especially those with oligospermia, may carry a higher risk of transmitting incorrect primary imprints to their offspring, highlighting the need for more research into ART.

Assessment of human oocyte developmental competence by cumulus cell morphology and circulating hormone profile.

The predictive value of the morphology of the cumulus--oocyte complex (COC) has not yet been explored as a possible factor contributing to the success of human in-vitro maturation (IVM). In the present study, development-supporting competency of oocytes encircled in a large ( > or = 5) (grade A), moderate (3 approximately 4) (grade B) or small ( < or = 2) (grade C) number of cumulus cell layers was assessed, together with changes in hormonal profile following a truncated course of 150 IU pure FSH administration for 3 days prior to aspiration on laparoscopy indicated for endometriosis. FSH priming increased the number of COC aspirated without changing the proportion of the three morphological types of COC, which were then subjected to IVM in the presence of 200 mIU/ml FSH plus 1000 mIU/ml human chorionic gonadotrophin, followed by intracytoplasmic sperm injection. The highest development-supporting competence was observed not with oocytes in grade A COC harvested from natural cycles, but with oocytes in grade B COC from FSH-primed cycles. Hormonal profiles in patients bearing grade B COC were characterized by moderate response in oestradiol and progesterone production following FSH, with LH/FSH ratio being below 1.0. It is concluded that an optimal window of hormonal profile(s) may exist for follicle aspiration to obtain grade B COC in FSH-stimulated human IVM cycles.

Developmentally retarded frozen blastocysts can be rescued by synchronizing culture prior to transfer.

Asynchrony between embryo development and endometrial differentiation is the limiting step of successful pregnancy in assisted reproduction. The aim of this study was to investigate whether or not post-thaw synchronization culture of day 5-6 frozen embryos, prior to transfer, with endometrial differentiation resulted in pregnancy. A total of 142 cycles of 134 patients were transferred in three protocols. Blastocysts with cavities larger than half of the entire blastocyst volume were transferred without synchronizing culture on day 5 or 6 of progesterone commencement (P5/6) in hormone replacement treatment cycles (protocol 1). Blastocysts with cavitation below half of the entire blastocyst were cultured for 1 or 2 days after thawing prior to transfer on P5 or P6 (protocol 2). Morulae and very early stage blastocysts were thawed on the days corresponding to P5 and P6, and only the embryos that reached expanded or hatching blastocysts were transferred on P7 without synchronizing culture (protocol 3). Pregnancy rate in protocol 2 (32.0%) was comparable with that of protocol 1 (35.0%). It is concluded that developmentally retarded frozen embryos can be rescued with synchronizing culture prior to transfer by evading asynchrony.

Developmental potential and chromosomal constitution of embryos derived from larger single pronuclei of human zygotes used in in vitro fertilization.

Blastocysts that develop from larger 1 pronuclear zygotes are suspected to be diploid and are comparable in development potential to 2 pronuclear zygotes.