Biomedical subjects

S E Lanzendorf

Publications and source records attributed to S E Lanzendorf.

At least 19 recordsLinked to original sources

PubMed1999

Pregnancy following transfer of ooplasm from cryopreserved-thawed donor oocytes into recipient oocytes.

OBJECTIVE: To determine if frozen-thawed donor oocytes could be used to provide cytoplasm for transfer into patients' oocytes to improve subsequent embryonic development. DESIGN: Prospective evaluation of the procedure in consenting IVF patients. SETTING: Assisted reproductive technology program. PATIENT(S): The study was open to consenting IVF patients (of any age) with a history of poor embryo quality or those couples in which the wife's age was > or = 40 years. INTERVENTION(S): Transfer of donor egg cytoplasm from frozen-thawed oocytes into the oocytes of infertile recipients. MAIN OUTCOME MEASURE(S): Donor oocyte survival following cryopreservation, fertilization following cytoplasmic transfer into recipient oocytes, embryo quality, and pregnancy outcome. RESULT(S): Oocytes collected from four donors were cryopreserved and 61% (28/46) survived the thaw procedure. Cytoplasmic transfer was performed on the eggs of four patients, with fertilization occurring in 70.3% (26/37). Twin pregnancy was established in one patient (35 years of age) with a history of poor embryo quality. CONCLUSION(S): Cryopreserved donor oocytes may provide a source of cytoplasm for transfer into recipient oocytes, eliminating the need for cycle synchronization between donor and infertile patient.

Adult↗

PubMed1998

Impact of fertilization history and semen parameters on ICSI outcome.

PURPOSE: The objectives of this study were (1) to investigate intracytoplasmic sperm injection (ICSI) outcome according to its indications, i.e., a history of failed or poor fertilization and unsuitable sperm parameters for conventional IVF, and (2) to examine the impact of a female's age, sperm concentration, motility, morphology, presence of antisperm antibodies, and hemizona assay (HZA) results on overall outcome. METHODS: Two hundred seventy-nine ICSI cycles performed in 207 couples were retrospectively evaluated. RESULTS: Clinical pregnancy and delivery rates were 36.8 and 29.8% for patients with prior failed fertilization, 23.2 and 17.8% for patients who had prior poor fertilization, and 28.6 and 21.3% for patients with unsuitable sperm parameters. The differences among all groups were found to be insignificant. There was a significant, negative correlation between a female's age and pregnancy results. No difference was found in the three basic sperm parameters between those patients who produced and those who did not produce a pregnancy, but the fertilization rate was significantly higher in patients with more adequate sperm parameters. Although there was a trend toward a better fertilization rate in patients with a hemizona index (HZI) greater than 30 (indicative of a superior sperm-zona pellucida binding capacity) than in those with a HZI less than 30, the difference was not significant. There were no differences in fertilization rate according to the presence or absence of antisperm antibodies. CONCLUSIONS: Fertilization history in a conventional IVF cycle has no effect on success rates following ICSI, and there is no correlation among the basic sperm parameters, the presence of antisperm antibodies, and pregnancy rates.

Age Factors↗

PubMed1998

A prospective, randomized, double-blind study for the evaluation of assisted hatching in patients with advanced maternal age.

The objective of this study was to determine if assisted hatching improved the rates of implantation, clinical pregnancy and ongoing pregnancy for in-vitro fertilization (IVF) patients aged > or =36 years. On the day of oocyte aspiration, consenting patients were randomized according to whether all embryos underwent the hatching procedure (hatched; n = 41) or all embryos remained unhatched (controls; n = 48). Patients in both groups were treated with methylprednisolone and doxycycline starting on the day of oocyte retrieval and continuing for 4 days. The hatching procedure was performed approximately 55 h after insemination on all potential embryos for transfer and employed the release of acidified acid Tyrode's medium against the zona pellucida to create an opening approximately 20 microm in diameter. No significant differences were noted in the mean age, number of oocytes aspirated and number of embryos transferred between the hatched and control groups. In addition, no significant differences were observed in the rates of implantation (11.1 versus 11.3%), clinical pregnancy (39.0 versus 41.7%) and ongoing pregnancy (29.3 versus 35.4%) between the hatched and control groups respectively. These results suggest that assisted hatching may have no significant impact on IVF success rates in the patient population studied.

Adult↗

PubMed1997

Effects of sperm viability on fertilization and embryo cleavage following intracytoplasmic sperm injection.

PURPOSE: In the human, intracytoplasmic sperm injection is typically performed using "viable" sperm which has been mechanically rendered nonmotile. The purpose of the present study was to determine the ability of nonviable sperm to fertilize human oocytes and the early developmental normalcy of the resulting embryos. METHODS: In this study, immature, prophase I oocytes from a total of 27 consenting patients were matured in vitro and then randomized into two groups: injection with a viable human sperm or injection with a sperm rendered nonviable by freeze-thawing in liquid nitrogen. The rates of fertilization and cleavage were compared between the two groups. RESULTS: The results demonstrated a significantly higher two-pronuclear fertilization rate when oocytes were injected with viable sperm (62.2%) compared to when oocytes were injected with nonviable sperm (16.2%). Oocytes injected with viable sperm also demonstrated a higher cleavage rate (91 vs 33%). CONCLUSIONS: These findings suggest that while the intracytoplasmic injection of nonviable human sperm can result in normal fertilization, it does so at a much reduced rate compared to viable sperm and may not result in normally cleaving embryos.

Cell Survival↗

PubMed1997

Follicle stimulating hormone effects on immature human oocytes: in vitro maturation and hormone production.

PURPOSE: Our purpose was (1) to determine if in vitro maturation of unstimulated oocytes could be improved with the addition of urofollitropin; (2) to evaluate the output of estradiol, testosterone, progesterone, and androstenedione by the cultured oocyte-cumulus complex; and (3) to ascertain if steroid hormone production of the oocyte-cumulus complex correlates with final oocyte maturation stage. METHODS: Fifty-eight immature oocytes were obtained from 11 regularly cycling women undergoing oophorectomy. The oocyte-cumulus complexes were randomly assigned to control medium (Ham's F-10 supplemented with 7.5% fetal bovine serum) or test medium (control medium supplemented with 75 mIU/ml of urofollitropin). RESULTS: (1) The addition of urofollitropin to oocyte culture medium does not significantly increase the ability of the oocyte to achieve the metaphase II stage; (2) the addition of urofollitropin significantly increases the production of progesterone, testosterone, and androstenedione by the oocyte-cumulus complex; and (3) there is no difference in the production of estradiol, progesterone, testosterone, and androstenedione by the oocyte-cumulus complex at the germinal vesicle, metaphase I or metaphase II stage of oocyte maturation. CONCLUSIONS: This information is of importance in the use of oophorectomy specimens for patients who must undergo an oophorectomy but desire to attempt pregnancy using their oocytes, in the use of oophorectomy specimens for donor oocytes, or for patients undergoing in vitro fertilization using immature oocyte collection.

Androstenedione↗

PubMed1996

Polymerase chain reaction amplification specificity: incidence of allele dropout using different DNA preparation methods for heterozygous single cells.

PURPOSE: The purpose was to evaluate methods of DNA preparation in a single cell to determine the ability to amplify and correctly diagnose a targeted gene. METHODS: One- or two-cell lymphoblasts (n = 100/group), heterozygous for the normal and 4-base pair insertion on exon 11 of the beta-hexosaminidase A gene, were collected and prepared under the following conditions: (1) freeze-thaw liquid nitrogen, then boiling (LN2); (2) potassium hydroxide/dithiothreitol, heated to 65 degrees C, followed by acid neutralization (KOH); (3) boiling only (Bl); and (4) water only (H2O). Cells were analyzed by polymerase chain reaction using nested primers. RESULTS: The total number of cells amplifying [in brackets] and the cells with amplification for both alleles (heterozygous), the normal allele, or the mutant allele were as follows, respectively: LN2 [38], 11, 16, 11; KOH [97], 91, 5, 1; Bl [41], 17, 13, 11; and H2O [85], 41, 16, 28. With two cells per reaction tube the results were as follows: LN2 [85], 53, 14, 18; and KOH [97], 96, 1, 0. CONCLUSIONS: KOH lysis was significantly greater than with all other methods (P < 0.006) and should be used for single cells. This study also demonstrates the importance of using heterozygous cells to determine the ability to amplify both alleles as a method of quality control for single-cell analysis.

Alleles↗

PubMed1996

The effect of coculture on the postfertilization development of in vitro-matured monkey oocytes.

OBJECTIVE: To determine if the developmental potential of embryos resulting from in vivo- and in vitro-matured monkey oocytes could be increased through the use of a coculture system. DESIGN: Randomized prospective comparison of embryos resulting from either in vitro- or in vivo-matured oocytes cocultured with Vero cells or cultured in medium alone (control). SETTING: Basic research laboratory. MAIN OUTCOME MEASURES: In vitro embryo development to the blastocyst stage and blastocyst hatching. RESULTS: No significant difference in development was noted between coculture and control groups with embryos resulting from in vivo-matured oocytes. However, coculture was found to improve significantly the development of monkey embryos resulting from in vitro-matured oocytes. CONCLUSIONS: These results demonstrate that primate embryos resulting from in vitro-matured and in vitro-fertilized oocytes differ in their culture requirement when compared with embryos resulting from in vivo-matured oocytes.

Animals↗

PubMed1996

Messenger ribonucleic acid kinetics in human oocytes--effects of in vitro culture and nuclear maturational status.

OBJECTIVE: To study the effects of different nuclear maturational status (prophase I [PI] versus metaphase II [MII]) and in vitro culture on the kinetics of maternal messenger ribonucleic acid (mRNA) in human oocytes. DESIGN: Molecular biology on excess oocytes obtained from our clinical IVF program. INTERVENTIONS: The oocytes, classified as either PI or MII at collection, were used as such or cultured in vitro for an additional 24 hours. The relative levels of c-mos and cyclin-B1 were measured using semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: The mean levels of c-mos and cyclin-B1 transcripts were indistinguishable between the PI, MII, PI oocytes matured in vitro, PI oocytes failing to mature, and MII oocytes cultured for additional 24 hours. The variability in the levels of these transcripts increased during the vitro culture. CONCLUSIONS: The level of c-mos and cyclin-B1 transcripts were not different in PI versus MII oocytes, therefore, differences seen in the clinical outcome of PI and MII oocytes may be unrelated to levels of these gene products. C-mos and cyclin B1 mRNA were maintained in vitro, thus degradation of maternal RNA is not activated in excess during the 24-hour culture.

Base Sequence↗

PubMed1995

Primate reproductive organs reveal a novel pattern of proto-oncogene c-mos and transcription factor Oct-3 mRNA expression.

In mice, expression of the transcription factor Oct-3 and the proto-oncogene c-mos is limited to germ cells, suggesting a specific role for these factors in gamete physiology and early embryonic development. We have studied the expression pattern of Oct-3 and c-mos in various reproductive as well as control tissues in the cynomolgus monkey, using reverse transcriptase polymerase chain reaction (RT-PCR) and Northern analysis. Analogously with the data from the mouse model, strong expression of Oct-3 and c-mos could be detected in monkey ovary and oocytes. Unexpectedly, strong expression of c-mos was demonstrable in the pituitary gland and the amount of mRNA expression in the pituitary was roughly equal to that found in the ovary. Of the tissues examined, the testicular expression of c-mos was the most intense. Weak signal for c-mos mRNA was also seen in hypothalamus and brain; however, all other tissue types examined were negative for c-mos expression. In addition to the oocytes, expression of Oct-3 mRNA was detected in the ovarian granulosa cells, fallopian tube, myometrium, cervix, breast, liver, adrenal gland, pituitary, hypothalamus, brain cortex, prostate, and in testis. Thus, in the cynomolgus monkey, Oct-3 is predominantly, but not specifically, expressed in reproductive tissues. In the female monkey reproductive organs, the expression of c-mos seems to be germ cell specific. Therefore, further characterization of c-mos and Oct-3 functions in primate reproductive physiology, especially in gametogenesis and early embryonic development, is highly warranted.

Animals↗

PubMed1995

Strategies to respond to polymerase chain reaction deoxyribonucleic acid amplification failure in a preimplantation genetic diagnosis program.

OBJECTIVES: Our purpose was to identify and evaluate practical methods within a preimplantation genetic diagnosis program that will increase the percentage of embryos for which a genetic diagnosis can be obtained, including clinical responses after failure of deoxyribonucleic acid amplification has occurred. STUDY DESIGN: Known human lymphoblast cell lines and human embryo blastomeres were evaluated in a single-cell, nested primer polymerase chain reaction system with primer sequences for the specific locus surrounding the four base pair insertion mutation on exon 11 of beta-hexosaminidase A-Tay-Sachs disease, the delta F508 mutation of cystic fibrosis, and the sex-determining region on the Y chromosome. Reamplification polymerase chain reaction with standard polymerase chain reaction and primer extension preamplification was performed in deoxyribonucleic acid preparations after previous polymerase chain reaction amplification attempts had resulted in failure of amplification. RESULTS: The amplification efficiency of Tay-Sachs disease, 51% (97/187), was significantly lower than that for cystic fibrosis, 85% (87/107), and for the sex-determining region on the Y chromosome, 85% (77/90). Tay-Sachs disease polymerase chain reaction amplification occurred in 51% of one-cell lymphoblasts, 89% of two-cell lymphoblasts, and 94% of samples when more than two cells were processed together. When previous amplification failure had occurred, standard Tay-Sachs disease polymerase chain reaction resulted in an amplification efficiency of 16% (three of 19), whereas primer extension preamplification polymerase chain reaction for Tay-Sachs disease resulted in amplification of 52% (31/59) lymphoblasts and 54% (13/24) of polyspermic human blastomeres. Four of six human blastomeres in which amplification failure occurred in a Tay-Sachs disease preimplantation genetic diagnosis cycle amplified by primer extension preamplification polymerase chain reaction, which increased the diagnostic information obtained from four to six of the seven embryos on which biopsy was performed. CONCLUSIONS: We suggest that practical approaches for consideration within a clinical preimplantation genetic diagnosis program to limit the net effect of amplification failure (i.e., reduced embryo transfer number) include increasing the deoxyribonucleic acid content in the polymerase chain reaction tube by using more than one blastomere and by using primer extension preamplification when the initial attempt at amplification fails.

Base Sequence↗

PubMed1995

The relationship between size and maturation in vitro in the unstimulated human oocyte.

OBJECTIVE: To determine if the size of human oocytes at collection from unstimulated ovaries is related to their ability to resume meiosis and undergo maturation in vitro. DESIGN: A comparative study of oocyte diameter at collection. SETTING: Department of Obstetrics and Gynecology, Northwestern University Medical School. PATIENTS: Women age 25 to 39 years of age undergoing gynecological procedures yielding oophorectomy specimens. INTERVENTION: Oocytes obtained from ovarian tissue were cultured in Ham's F-10 and fetal bovine serum for 72 hours and observed two times per day. MAIN OUTCOME MEASURE: The oocytes ability to resume meiosis and complete maturation based on their diameter at collection. RESULTS: Chi-squared analysis revealed a significant difference in oocytes measuring 86 to 105 microns versus those measuring 106 to 125 microns. CONCLUSION: The unstimulated human oocyte appears to have a size-dependent ability to resume meiosis and complete maturation.

Adult↗

PubMed1995

Preimplantation genetic diagnosis for Tay-Sachs disease: successful pregnancy after pre-embryo biopsy and gene amplification by polymerase chain reaction.

OBJECTIVE: To determine the ability to apply preimplantation genetic diagnostic techniques to screen for and prevent Tay-Sachs disease (TSD). DESIGN: A couple, both carriers for the 4 base pair (bp) insertion in exon 11 of the beta-hexosaminidase A gene, which results in TSD, underwent IVF, pre-embryo biopsy, polymerase chain reaction (PCR) DNA amplification of the biopsied blastomeres, and pre-embryo transfer. One to two blastomeres were aspirated using a biopsy pipette that was inserted through an opening in the zona formed with acidified phosphate buffer. Polymerase chain reaction was performed on the individual blastomeres for 20 cycles followed by an additional 30 cycles using nested primers. This yielded amplified DNA products of 272 and 276 bp for the normal and mutant gene, respectively. Heteroduplex formation was used for identification of normal, homozygous affected, and heterozygous pre-embryos. RESULTS: Seven of 13 oocytes fertilized normally and were biopsied at the four- to eight-cell stages. Deoxyribonucleic acid amplification occurred in four of seven pre-embryos (one homozygous affected and three homozygous normal pre-embryos). The three normal pre-embryos that continued to cleave after biopsy were transferred on the evening of day 3 after retrieval. Subsequently, a single gestational sac was observed and the genetic diagnosis was confirmed at amniocentesis. CONCLUSION: A successful pregnancy and birth were accomplished after preimplantation genetic diagnostic screening for the prevention of TSD.

Base Sequence↗

PubMed1995

Evaluation of the meiotic spindle apparatus in oocytes undergoing intracytoplasmic sperm injection.

OBJECTIVE: To determine whether intracytoplasmic sperm injection has an adverse effect on the meiotic spindle of oocytes after injection of a sperm into the ooplasm. DESIGN: Hamster oocytes were injected with human sperm (Test group) and evaluated for meiotic spindle and chromosome morphology using immunofluorescent staining. Results were compared with control uninjected oocytes exposed to the microscope environment (CS group) and untreated oocytes remaining in the incubator (CI group). SETTING: Basic research center at a medical school. RESULTS: No significant differences were noted in spindle appearance and chromosome alignment between Test (13 abnormal/68 normal) and both control groups (CS 9/73 and CI 12/71; P = 0.602). CONCLUSION: Our results demonstrate that injection of human sperm into the cytoplasm of hamster oocytes may not result in a significant increase in damage to the meiotic spindle provided care is taken to orient the polar body away from the site of injection.

Animals↗

PubMed1995

Cell cycle genes c-mos and cyclin-B1 are expressed in a specific pattern in human oocytes and preimplantation embryos.

Little is known about the molecular mechanisms governing the development of human oocyte and pre-embryo. We characterized the expression pattern of c-mos, cyclin B1 and beta-actin mRNA in oocytes and granulosa cells from human and monkey, and in human early embryos, using both qualitative and semi-quantitative reverse transcriptase polymerase chain reaction. The proto-oncogene c-mos was expressed in an oocyte-specific manner and no mRNA for c-mos could be detected in the granulosa cells. Similarly, strong expression of cyclin-B1 was seen in the oocytes. In human pre-embryos, the expression of cyclin-B1 and beta-actin increased from the 6-cell stage onwards, indicating active transcription and thus activation of embryonic genome either at or before the 6-cell stage. The expression of c-mos was transient and very little c-mos mRNA could be detected in the human embryos beyond the 6-cell stage. Thus, both its time-specific and site-specific expression suggest meiosis-specific functions for the proto-oncogene c-mos in human oocytes. As judged by the disappearance of c-mos, the maternal pool of mRNA seems to be degraded towards the 6- to 8-cell stage. The transient expression of c-mos and high levels of cyclin-B1 mRNA suggest that mechanisms similar to those found in lower organisms govern the growth and development of the human oocyte and preimplantation embryo.

Actins↗

PubMed1995

Evaluation of the spindle apparatus of in-vitro matured human oocytes following cryopreservation.

The present study was conducted to determine if the cryopreservation of immature human oocytes has a deleterious effect on the meiotic spindle following maturation in vitro. Oocytes were obtained in excess from in-vitro fertilization patients and divided into four groups. Groups 1 (n = 98) and 2 (n = 80) consisted of immature oocytes cryopreserved before or after maturation in vitro respectively. Groups 3 (n = 37) and 4 (n = 9) served as non-frozen controls and included oocytes matured in vitro and in vivo respectively. The meiotic spindle was identified after incubation in anti-tubulin monoclonal antibody (1 h, 37 degrees C) and fluorescein-conjugated goat anti-mouse immunoglobulin G (IgG) (1 h, RT). Chromosomes were counterstained with 4',6'-diamidino-2-phenylindole. Following cryopreservation, group 1 oocytes demonstrated a 63% survival rate and 68% maturation rate in vitro. In all, 58% of the oocytes in group 2 survived the thaw. The number of oocytes with normal spindles in group 1 (81.0%) was not significantly different from control groups 3 (83.8%) and 4 (88.9%), while the number of group 2 oocytes with normal structures (43.5%) was significantly lower than groups 1 (P = 0.0004), 3 (P = 0.0002), and 4 (P = 0.025). These results suggest that cryopreservation of the prophase I human oocyte does not significantly increase abnormalities in the resulting meiotic spindle.

Cell Survival↗

PubMed1994

Fertilization and in vitro development of cryopreserved human prophase I oocytes.

OBJECTIVE: To determine the potential for in vitro maturation, fertilization, and cleavage after cryopreservation of immature, prophase I human oocytes. DESIGN: Immature oocytes obtained in excess of the number required by the patient were randomized and cryopreserved at the prophase I stage or cultured as control. After thawing and maturation in vitro, test and control oocytes were inseminated with husband's sperm and evaluated for fertilization and cleavage in vitro. SETTING: In vitro fertilization program. PATIENTS: Consenting patients undergoing controlled ovarian hyperstimulation for the purposes of IVF. MAIN OUTCOME MEASURES: Rates of maturation to metaphase II, fertilization, and cleavage were compared between control and cryopreserved oocytes. RESULTS: Upon thaw, 58.5% (72/123) of prophase I oocytes were viable. Control oocytes demonstrated a 74.8% (98/131) maturation rate to metaphase II, a 56.5% (52/92) fertilization rate, and an 11.5% (6/52) blastocyst rate. Cryopreserved oocytes showed a 83.3% (60/72) rate of maturation, a 57.7% (30/52) fertilization rate, and a 3.3% (1/30) blastocyst rate. No significant differences were noted between any of these parameters. CONCLUSIONS: These results demonstrate that prophase I oocytes from stimulated IVF cycles are able to survive cryopreservation and resume meiosis to achieve full nuclear maturation post-thaw. In addition, cryopreserved oocytes retain the same capacity for fertilization and development as control oocytes.

Adult↗

PubMed1994

Cryopreservation of human prophase I oocytes collected from unstimulated follicles.

OBJECTIVE: To evaluate the cryopreservation of immature human oocytes obtained from unstimulated ovarian tissue. DESIGN: Immature prophase I oocytes were obtained from unstimulated follicles and were either cryopreserved or cultured as controls. Cryopreservation was performed in a programmable freezing machine using one of two protocols. Method I (n = 133) used a one-step addition of cryoprotectant followed by a slow freeze and thaw protocol. With method II (n = 95), the cryoprotectant was added in a stepwise manner with cryopreservation performed in the presence of 0.2 M sucrose followed by rapid freezing and thawing. SETTING: Basic research center at a medical school. PATIENTS: Patients undergoing oophorectomy for nonovarian pathology. MAIN OUTCOME MEASURES: Rates of survival and maturation to metaphase II were compared between control oocytes and oocytes cryopreserved with methods I and II. RESULTS: With method I, a survival rate of 15.6% was obtained with 58.3% of surviving oocytes reaching metaphase II after culture compared with 50.0% of nonfrozen control oocytes. Method II produced a survival rate of 43.3% with 27.3% maturing to metaphase II. Maturation of control oocytes for method II was 46.4%. Although the survival rate with method II was significantly higher than with method I, the rate of in vitro maturation to metaphase II showed no difference. CONCLUSIONS: These results demonstrate that human prophase I oocytes obtained from unstimulated antral follicles are capable of meiotic maturation after cryopreservation.

Cell Survival↗

PubMed1992

Cortical granule complements in human oocytes undergoing partial zona dissection.

This study was performed to evaluate the effects of mechanical stimulation and sucrose treatment on the oocyte activation process. Fresh and aged human oocytes were exposed to sucrose and zonae were dissected with microneedles before fixation and quantitative analysis of cortical granules by transmission electron microscopy. Examination of the mean number of cortical granules/analyzed segment revealed no significant differences between control oocytes or oocytes treated with sucrose or sucrose treatment followed by zona dissection. A significant decline in the number of cortical granules/segment was observed for oocytes undergoing prolonged culture after dissection (P less than 0.05). Thus, zona dissection and sucrose exposure of freshly aspirated mature human oocytes do not result in classical oocyte activation.

Cells, Cultured↗