Biomedical subjects

D Moore

Publications and source records attributed to D Moore.

At least 73 records · Page 4Linked to original sources

PubMed1999

MHC class II alpha/beta heterodimeric cell surface molecules expressed from a single proviral genome.

Transplantation tolerance to renal allografts can be induced in large animal preclinical models if the donor and recipient have identical major histocompatibility complex (MHC) class II loci. Such class II matching is, however, not clinically achievable owing to the extreme diversity of class II sequences. With the ultimate goal of creating a somatic class II match in the bone marrow of an allograft recipient, the aim of the study is to develop a double-copy retrovirus construct to express both chains of the MHC class II DQ glycoprotein on a single transduced cell. Analysis of the expression patterns of the retroviral DQ transgenes in both virus producer and transduced fibroblasts revealed correct transcription and stable surface expression of the DQ heterodimers. In addition, we demonstrate that both the DQA and DQB sequences are functional within the same proviral copy, a prerequisite for efficient induction of transplantation tolerance following transduction of bone marrow precursor cells. The DQ double-copy retrovirus vector showed efficient expression of the transferred class II cDNA in murine colony-forming units for the granulocyte-monocyte lineage (CFU-GM), indicating that it is suitable for gene therapy of multimeric proteins in hematopoietic cells.

3T3 Cells↗

PubMed1999

Trench-shaped binding sites promote multiple classes of interactions between collagen and the adherence receptors, alpha(1)beta(1) integrin and Staphylococcus aureus cna MSCRAMM.

Most mammalian cells and some pathogenic bacteria are capable of adhering to collagenous substrates in processes mediated by specific cell surface adherence molecules. Crystal structures of collagen-binding regions of the human integrin alpha(2)beta(1) and a Staphylococcus aureus adhesin reveal a "trench" on the surface of both of these proteins. This trench can accommodate a collagen triple-helical structure and presumably represents the ligand-binding site (Emsley, J., King, S. L., Bergelson, J. M., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517; Symersky, J., Patti, J. M., Carson, M., House-Pompeo, K., Teale, M., Moore, D., Jin, L., Schneider, A., DeLucas, L. J., Höök, M., and Narayana, S. V. L. (1997) Nat. Struct. Biol. 4, 833-838). We report here the crystal structure of the alpha subunit I domain from the alpha(1)beta(1) integrin. This collagen-binding protein also contains a trench on one face in which the collagen triple helix may be docked. Furthermore, we compare the collagen-binding mechanisms of the human alpha(1) integrin I domain and the A domain from the S. aureus collagen adhesin, Cna. Although the S. aureus and human proteins have unrelated amino acid sequences, secondary structure composition, and cation requirements for effective ligand binding, both proteins bind at multiple sites within one collagen molecule, with the sites in collagen varying in their affinity for the adherence molecule. We propose that (i) these evolutionarily dissimilar adherence proteins recognize collagen via similar mechanisms, (ii) the multisite, multiclass protein/ligand interactions observed in these two systems result from a binding-site trench, and (iii) this unusual binding mechanism may be thematic for proteins binding extended, rigid ligands that contain repeating structural motifs.

Adhesins, Bacterial↗

PubMed1999

Structural basis of profactor D activation: from a highly flexible zymogen to a novel self-inhibited serine protease, complement factor D.

The crystal structure of profactor D, determined at 2.1 A resolution with an Rfree and an R-factor of 25.1 and 20.4%, respectively, displays highly flexible or disordered conformation for five regions: N-22, 71-76, 143-152, 187-193 and 215-223. A comparison with the structure of its mature serine protease, complement factor D, revealed major conformational changes in the similar regions. Comparisons with the zymogen-active enzyme pairs of chymotrypsinogen, trypsinogen and prethrombin-2 showed a similar distribution of the flexible regions. However, profactor D is the most flexible of the four, and its mature enzyme displays inactive, self-inhibited active site conformation. Examination of the surface properties of the N-terminus-binding pocket indicates that Ile16 may play the initial positioning role for the N-terminus, and Leu17 probably also helps in inducing the required conformational changes. This process, perhaps shared by most chymotrypsinogen-like zymogens, is followed by a factor D-unique step, the re-orientation of an external Arg218 to an internal position for salt-bridging with Asp189, leading to the generation of the self-inhibited factor D.

Amino Acid Sequence↗

PubMed1999

Intraperitoneal interferon-alpha in residual ovarian carcinoma: a phase II gynecologic oncology group study.

OBJECTIVE: The purpose of this study was to confirm the activity of interferon-alpha intraperitoneally in minimal residual epithelial ovarian cancer in a Phase II multi-institutional trial and to investigate the activity of the agent based on prior response to first-line platinum compounds. METHODS: Ninety-two patients with minimal residual (<0.5 cm) epithelial ovarian cancer at reassessment laparotomy were entered into a multicenter trial of intraperitoneal interferon-alpha given for 12 cycles unless disease progression or unacceptable toxicity occurred first. Patients were considered favorable if they were platinum sensitive and/or relapsed 6 months or longer after completing treatment and unfavorable if they were platinum insensitive and/or relapsed shorter than 6 months after completing treatment and/or had stable or progressive disease during initial therapy. A third-look laparotomy was performed within 12 weeks of completion of treatment in those patients who were in clinical remission. RESULTS: Eighty patients were clinically evaluable for toxicity only (48 favorable, 32 unfavorable) and 46 of them were evaluable for response, of whom 25 were favorable (platinum sensitive) and 21 unfavorable (platinum resistant). In the favorable group, there was a 28% surgically documented response rate (7/25 patients): 16% (4/25) had complete responses (negative reassessment operation), 12% (3/25) had partial responses, 32% (8/25) were nonresponders, and 40% (10 patients) developed progressive disease before planned reassessment operation. In the unfavorable group, there were no responding patients: 6 were nonresponders at reassessment operation and 15 developed progressive disease before planned reassessment operation. Of the 80 patients evaluable for toxicity, the most common adverse effects that were more than grade 2 were gastrointestinal (12; 15%), fever (8; 10%), neutropenia (7;9%), and leukopenia (6; 8%). Grade 4 toxicity was seen in 5 patients; each had fever and gastrointestinal toxicity, and 1 each had neutropenia and thrombocytopenia. CONCLUSIONS: Interferon-alpha is an active and generally well-tolerated agent in favorable patients with minimal residual epithelial ovarian cancer at second-look surgery. These results are comparable to those achieved with cytotoxic chemotherapy. If Phase III trials are considered in the patients with minimal residual ovarian cancer, they should be limited to the platinum-sensitive patient population.

Adult↗

PubMed1999

FIGO stage IIIC endometrial carcinoma with metastases confined to pelvic lymph nodes: analysis of treatment outcomes, prognostic variables, and failure patterns following adjuvant radiation therapy.

OBJECTIVES: This study was undertaken to evaluate the prognostic significance of isolated positive pelvic lymph nodes on survival and to analyze other prognostic variables, overall survival, and failure patterns in surgically staged endometrial carcinoma patients with positive pelvic lymph nodes and negative para-aortic lymph nodes following radiation therapy (RT). METHODS: Between January 1, 1987, and December 31, 1997, 782 women underwent primary treatment for uterine cancer at Indiana University Medical Center. Through a review of the medical records, we identified 58 patients with pathologic stage IIIA, 27 patients with pathologic stage IIIB, and 77 patients with pathologic stage IIIC endometrial carcinoma. Patients with pathologically positive or unsampled para-aortic lymph nodes and patients who received preoperative radiation therapy were excluded, leaving a study group of 17 patients with nodal metastases confined to pelvic lymph nodes. Thirteen patients received adjuvant pelvic RT using AP-PA or four-field technique. A median dose of 5040 cGy was delivered. Four patients received whole abdominal irradiation (WAI) delivering a median dose of 3000 cGy. Two patients received vaginal cuff boosts of 1000 and 3560 cGy to 0.5 cm from the vaginal surface mucosa via Cs-137 brachytherapy. Two patients also received adjuvant chemotherapy (cis-platinum and doxorubicin) and/or hormonal therapy (megestrol acetate). Disease-free and overall survivals were estimated using the Kaplan-Meier method of statistical analysis and prognostic variables were analyzed using the log-rank test. RESULTS: With a median follow-up of 51 months the actuarial 5-year disease-free survival was 81% and the actuarial 2-year and 5-year overall survival rates were 81 and 72%, respectively. Univariate analysis revealed that positive peritoneal cytology in conjunction with positive pelvic lymph nodes imparts a greater risk of recurrence and decreased overall survival. There were no pelvic and/or upper abdominal failures, but there were recurrences in the para-aortic lymph nodes (two patients) and distantly (two patients). CONCLUSION: Surgery followed by postoperative pelvic RT is a viable treatment option for pathologically staged stage IIIC endometrial carcinoma with disease confined to the pelvic lymph nodes. Failures in the para-aortic region suggest a possible role for extended-field RT. Patients with positive peritoneal cytology in conjunction with nodal metastasis fared poorly with pelvic RT. Studies evaluating the efficacy of WAI are ongoing. Finally, substages within FIGO stage IIIC are recommended in an effort to better understand and define treatment strategies which might be appropriate for these patients.

Actuarial Analysis↗

PubMed1999

Mechanism of protracted metabolic effects of fatty acid acylated insulin, NN304, in dogs: retention of NN304 by albumin.

AIMS/HYPOTHESIS: The provision of stable, reproducible basal insulin is crucial to diabetes management. This study in dogs examined the metabolic effects and interstitial fluid (ISF) profiles of fatty acid acylated insulin, Lys(B29)-tetradecanoyl, des-(B30) human insulin (NN304). METHODS: Euglycaemic clamps were carried out under inhalant anaesthesia during equimolar intravenous infusions (3.6 pmol. min(-1) x kg(-1) for 480 min) of human insulin or NN304 (n = 8 per group). RESULTS: Steady-state total NN304 (albumin-bound and unbound) was considerably higher in plasma compared with human insulin (1895 +/- 127 vs 181 +/- 10 pmol/l, p < 0.001) and increased in interstitial fluid (163 +/- 14 vs 106 +/- 9 pmol/l, p < 0.01). The halftime for appearance of NN304 in interstitial fluid was slower than human insulin (92 vs 29 min, p < 0.001). Yet, equivalency of action was shown for glucose turnover; steady-state glucose uptake (Rd) of 7.28 +/- 0.55 and 6.76 +/- 0.24 mg. min(-1). kg(-1) and endogenous glucose production of 0.11 +/- 0.12 and 0.22 +/- 0.03 mg x min(-1) x kg(-1) (p > 0.40; NN304 and human insulin, respectively). Similar to interstitial fluid, half times for Rd and endogenous glucose production were delayed during NN304 infusion (162 vs 46 min and 80 vs 31 min, respectively; p < 0.01 vs human insulin). CONCLUSION/INTERPRETATION: Firstly equivalency of steady-state action is found at equimolar physiologic infusions of human insulin and NN304. Secondly NN304 binding to plasma albumin results in slower NN304 appearance in the interstitial compartment compared with human insulin. Thirdly the delay in appearance of NN304 in interstitial fluid may not in itself be a source of the protracted action of this insulin analogue. The protracted effect is due primarily to albumin binding of the insulin analogue NN304. [Diabetologia (1999) 42: 1254-1263]

Animals↗

PubMed1999

Three-dimensional spiral CT scanning in children with acute torticollis.

Three-dimensional spiral CT scanning is now becoming a common investigation in children who have a history of acute torticollis. In the last year, 21 consecutive children who came to our unit with a history of acute torticollis were assessed using standard plain radiographs and a 3-dimensional spiral CT scan. Ten patients had a history of recent trauma. Spiral CT scanning revealed that 13 children had atlanto-axial rotatory subluxation (AARS). Plain radiographs had only a sensitivity of 33% and specificity of 71% in detecting AARS. Sixteen children were treated using a Halter traction. Four failed to resolve clinically and were put on a halo traction after 3-dimensional CT scanning again confirmed residual AARS. Two children remained symptomatic after halo traction, with persisting rotatory and anterior subluxation on repeat spiral CT. They both underwent a posterior in-situ fusion, with no attempt at open reduction. Plain radiography is limited in investigating acute torticollis in children. Spiral 3-dimensional CT reconstruction has an important role to play in both the investigation and management of children who present with acute torticollis.

Acute Disease↗

PubMed1999

Safe mobilization of normal progenitors in advanced chronic myeloid leukemia with intensive chemotherapy and granulocyte-colony stimulating factor.

Twenty-one patients with advanced chronic myeloid leukemia (late chronic phase (n = 8), accelerated phase (n = 11) and blast crisis (n = 2)) were treated with idarubicin, cytarabine, and etoposide followed by G-CSF and subsequent collection of peripheral blood progenitor cells in the early recovery phase. Treatment was reasonably well tolerated with no deaths or intensive care admissions. Despite the advanced phase of disease and heavy pretreatment with cytotoxics and interferon-alfa, 11 of 21 patients (52%) achieved a cytogenetic response. Of the nine major cytogenetic responses (complete (n = 3) and partial (n = 6)), seven achieved adequate progenitor collections for consideration for autologous transplantation. The only predictor of response was disease duration (P = 0.02). With a median follow-up of 1171 days from treatment it appears unlikely that G-CSF contributed to disease progression. Survival post-IcE was predicted by disease stage (P = 0.0001). Intensive chemotherapy followed by G-CSF allowed adequate yields of predominantly Philadelphia chromosome negative progenitor cells to be obtained from one-third of patients with advanced CML.

Adult↗

PubMed1999

Serotonergic responses of the siphons and adjacent mantle tissue of the zebra mussel, Dreissena polymorpha.

Bivalve siphons have important functional roles in nutritional physiology, defense, and reproductive mechanisms, yet little is known about their neuromuscular control. In the present study, tension measurements and video observations of siphons and adjacent mantle tissue were used to investigate responses to serotonin (5-HT). 5-HT caused relaxation at 10(-6) M and 10(-5) M, contraction or biphasic responses at 10(-4) M and 10(-3) M, and siphon opening at 10(-4) M and 10(-3) M. Responses were slow and lasted 5-10 min after much shorter (20 s) applications of 5-HT. The relaxation phase was enhanced in high potassium medium. Contractile responses could be mimicked by alpha-1-methyl-5-HT and 2-methyl-5-HT but not by 8-OH-DPAT. The responses were not affected by methiothepin, TFMPP, 1-(1-naphthyl)piperazine, metergoline, NAN-190, mianserin, cyproheptadine, and ketanserin. The pharmacology of the 5-HT receptor(s) mediating these siphon/mantle responses is, therefore, different from previously described 5-HT receptors involved in spawning. The siphon/mantle contains previously undescribed longitudinal muscle fibers in the mantle and circular muscle fibers around the siphons. Serotonergic innervation of the siphon margins and mantle tissue was observed by immunohistology. The presence of 5-HT in the siphon/mantle tissue and the responsiveness of these preparations to 5-HT suggest that 5-HT may be a physiological regulator of mantle and siphon movements in the zebra mussel.

Animals↗

PubMed1999

Examination of tumor and tumor-like conditions of bone.

Surgical pathology specimens composed of bone, ranging from core biopsy to limb amputation specimens, require special attention, processing, and often unique equipment. This readily translates into additional handling steps and time, especially when one factors in clinical correlation with the surgeon and radiologic review of all images with a knowledgeable musculoskeletal radiologist. When these factors are superimposed on the rarity of these lesions in routine practice, it is not surprising that most trainees, as well as seasoned pathologists, are wary of these lesions. In this report, we use a case of osteofibrous dysplasia (Campanacci's disease) to demonstrate the dissection of such a surgical specimen and complete the report with a brief discussion of the entity.

Adolescent↗

PubMed1999

Liver transplantation for citrullinaemia improves intellectual function.

BACKGROUND: Arginosuccinic acid synthetase (ASA) (EC 6.3.4.5) deficiency (citrullinaemia) (McKusick 215700) is a well-recognized cause of neonatal hyperammonaemic coma with poor long-term intellectual function, despite good medical management. METHODS: Cadaveric hepatic transplantation was performed in a 12-year-old boy with citrullinaemia under poor biochemical control. Subsequent development of fulminant hepatic failure necessitated a second cadaveric transplant. Psychometric assessments before and after transplantation were performed using a variety of age-appropriate tests. RESULTS: Normalization of plasma ammonium in our patient post transplantation has resulted in dramatic improvement in mental functioning and well-being and he now enjoys a normal diet. Psychometric assessment confirmed decline in his abilities prior to transplantation with particular post-transplantation improvement in perceptual organization and visuospatial abilities; these did not, however, return to normal. His family report considerable reduction in stress associated with the unpredictable nature of previous hyperammonaemic crises and recurrent hospitalization. CONCLUSIONS: Liver transplantation should be considered as an early therapeutic option in children with citrullinaemia to prevent ongoing cerebral insult associated with hyperammonaemia.

Amino Acid Metabolism, Inborn Errors↗

PubMed1999

Mathematical modelling of morphogenesis in fungi: a key role for curvature compensation ('autotropism') in the local curvature distribution model.

The assumption that the mushroom stem has the ability to undergo autonomic straightening enables a mathematical model to be written that accurately mimics the gravitropic reaction of the stems of Coprinus cinereus. The straightening mechanism is called curvature compensation here, but is equivalent to the 'autotropism' that often accompanies the gravitropic reactions of axial organs in plants. In the consequently revised local curvature distribution model, local bending rate is determined by the difference between the 'bending signal' (generated by gravitropic signal perception systems) and the 'straightening signal' (proportional to the local curvature at the given point). The model describes gravitropic stem bending in the standard assay with great accuracy but has the virtue of operating well outside the experimental data set used in its derivation. It is shown, for example, that the mathematical model can be fitted to the gravitropic reactions of stems treated with metabolic inhibitors by a change of parameters that parallel the independently derived physiological interpretation of inhibitor action. The revised local curvature distribution model promises to be a predictive tool in the further analysis of gravitropism in mushrooms.

Calcimycin↗

PubMed1999

Spatial organization of the gravitropic response in plants: applicability of the revised local curvature distribution model to Triticum aestivum coleoptiles.

The revised local curvature distribution model, which provides accurate computer simulations of the gravitropic response of mushroom stems, was found to produce accurate simulations of the gravitropic reaction of wheat (Triticum aestivum) coleoptiles. The key feature of the mathematical model that enables it to approach universality of application is the assumption that the stem has an autonomic straightening reaction (curvature compensation or 'autotropism'). In the model, the local bending rate for any segment of the organ is determined by the difference between the 'bending signal' (generated by the gravitropic signal perception system) and a 'straightening signal' (which is proportional to the local curvature of the segment). The model reveals three major differences between the gravitropic reactions of wheat coleoptiles and Coprinus mushroom stems. First, in Coprinus, the capacity for autonomic straightening is much more concentrated in the apical region of the stem. Second, local perception of the gravitropic signal, which is necessary for exact simulation in Coprinus, is not needed in wheat coleoptiles (the corresponding constant in the model can be set to zero). Third, the transmission rate of the gravitropic signal is about seven times faster in wheat coleoptiles than in the mushroom stem. Thus, we demonstrate that a single model, depending on the values given to its parameters, is able to simulate the spatial organization of the gravitropic reaction of wheat coleoptiles and Coprinus mushroom stems. The model promises to be a valuable predictive tool in guiding future research into the gravitropic reaction of axial organs of all types.

Computer Simulation↗

PubMed1999

Topical gene delivery to murine skin.

We topically applied naked plasmid DNA containing the luciferase or chloramphenicol acetyltransferase cDNA directly to mouse skin. Gene expression was detected in skin samples as early as 4 h after DNA application, plateaued from 16 to 72 h post-application, and had decreased significantly by 7 d post-application. Reporter gene activity following topical DNA delivery was comparable with that produced by intradermal injection of DNA. Plasmid DNA at concentrations > or =0.25 microg per microl were required to achieve maximal expression levels. Reporter gene expression following topical administration was largely confined to the superficial layers of the epidermis and to hair follicles. Surprisingly, certain cationic liposomes inhibited the efficiency of cutaneous gene transfer. This technique provides a simple, clinically relevant approach to deliver genes to the skin, with potential application in treating a variety of cutaneous disorders.

Administration, Topical↗

PubMed1999

Fifteen-year experience of transperitoneal management of inflammatory abdominal aortic aneurysms.

OBJECTIVES: to assess the long-term outcome of patients with inflammatory abdominal aortic aneurysms. MATERIALS AND METHODS: over a fifteen-year period 598 cases of abdominal aortic aneurysm were treated and, of these, 32 cases (5.3%) were inflammatory in nature. The diagnosis was made on preoperative (CT) computed tomography in fifteen cases. Twenty-six patients were symptomatic on presentation and ten cases were repaired on an emergency basis. Only six were repaired electively. The transabdominal transaortic approach without dissection on the nearby adherent structures was used routinely. RESULTS: there was one postoperative death from a respiratory arrest leading to a thirty-day mortality of 3.1%. Early graft thrombosis occurred in three cases (9.3%) and all underwent successful thrombectomy. Colonic ischaemia was encountered in one patient who later developed an aortoenteric fistula. Two patients suffered a non-fatal myocardial infarction postoperatively leading to an overall morbidity of 18.7%. CONCLUSIONS: patients with inflammatory aortic aneurysms fare worse than patients with aortic aneurysms in general. Preoperative suspicion assists in planning surgery. We believe that the transperitoneal approach with an anterolateral aortotomy and minimal dissection of adherent structures offers excellent results in dealing with this difficult group of patients.

Aged↗

PubMed1999

Gender traits and identities in a "masculine" organization: the Israeli police force.

Gender and occupational identities were examined within the Israeli police force, a stereotypically masculine organization. The principal hypothesis was that women in this organizational setting did not reject their gender identity. Rather, they self-attributed more traditionally masculine traits in addition to their feminine traits. This was especially so among women going through particularly intense occupational and organizational socialization needed for field jobs. The findings only partially confirmed the hypothesis. Most women in the sample considered themselves to be highly feminine, even though they self-attributed masculine traits. Although the women, like their male colleagues, ranked occupational identity higher than gender identity, their feminine identity (usually in contrast with the masculine organizational context) was not repressed and their gender identity was as strong as that of the men.

Adult↗

PubMed1999

Background ionizing radiation plays a minor role in the production of chromosome translocations in a control population.

PURPOSE: To obtain a relationship between background chromosome translocation frequency and age with translocation frequency measured to a high statistical precision, and to identify the role of background ionizing radiation in the production of chromosome translocations in a control population. MATERIALS AND METHODS: Lymphocytes from 35 healthy control individuals (15 females and 20 males) were scored, using fluorescence in situ hybridization, for the presence of chromosomal translocations. Translocation frequencies were measured to a high statistical precision (s.d. 25% or less for each individual). These control subjects were of varying ages, ranging from 0 (cord blood) to 98 years. RESULTS: In a total of 521,492 metaphases (203,754 genome equivalent cells) scored, an average of 5,822 genome equivalent cells per individual, 764 translocations were observed in the 35 individuals. The translocation frequencies ranged from 0 (for cord blood) to 0.0167 (for a 98 year old) translocations per cell. The average age and translocation frequency was 50 years and 0.004 translocations per cell, respectively. The best fit of the relationship between translocations and age was: Y=7x10(-4)+6.9x10(-6)A+1.35x10(-6)A2, which does not obey the linear relationship expected from chronic background radiation alone. The curvilinear relationship observed clearly shows that other endogenous and exogenous clastogens or clastogenic events, in addition to radiation, serve to generate chromosome translocations in control populations. CONCLUSION: The background translocation frequency in control individuals follows a curvilinear relationship with age. No significant variation was observed between individuals of the same age. Clastogenic processes of normal aging and physiological factors in additional to ionizing radiation play a major role in the production of chromosome translocations in a control population. Background radiation, however, appears to play a minor role in chromosome translocation production in control individuals living near sea level.

Adult↗

PubMed1999

Preliminary clinical experience with human blastocyst development in vitro without co-culture.

This preliminary analysis was designed to quantify blastocyst development of supernumerary embryos without the use of feeder cells, conditioned medium or whole serum. Embryos derived from in-vitro fertilization (IVF) that were not transferred or cryopreserved were included in this study. Ova were harvested for IVF after a standard ovarian stimulation with gonadotrophin-releasing hormone agonist/ human menopausal gonadotrophin (GnRHa/HMG) or follicle-stimulating hormone (FSH). Ova were collected and culture in 150 microliters droplets of P1 medium under mineral oil, in groups at 37 degrees C under 5% CO2, 5% O2, 90% N2 (group A) or under 5% CO2 in air (group B) environment. Embryo transfer was performed 72 h post-harvest. Viable embryos not transferred or cryopreserved were placed in blastocyst medium and cultured for an additional 48 h in 5% CO2 in air. Embryos that exhibited an expanded blastocoelic cavity and well-defined inner cell mass at 120 h were counted. Of 838 supernumerary embryos cultured, 448 (53.5%) reached the expanded blastocyst stage by 120 h of culture. Patients were given the option of cryopreservation at that time. The embryos were cryopreserved using a standard protocol with serial addition of glycerol. Embryos reaching the blastocyst stage after more than 120 h of culture were not included. There was no difference in the proportions of blastocyst development between group A, 217/410 (53.5%) and group B, 231/428 (54%). To date, 16 patients have each had up to three thawed blastocysts transferred, out of whom seven became pregnant. This report demonstrates that a simple system of sequential culture generated acceptable, viable blastocyst development (54%) with supernumerary embryos, without the use of feeder cells, conditioned medium or whole serum. Recognizing the differential metabolic requirements of early and late cleavage stage embryos has enabled the application of a glucose/phosphate-free simple culture medium (P1) for up to 72 h of culture and a complex, glucose-containing medium (blastocyst medium) for subsequent blastocyst development.

Blastocyst↗