Pregnancies after fertilization and cleavage in human coculture.
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Cleavage Stage, Ovum↗
Biomedical subjects
S Ratnam
Publications and source records attributed to S Ratnam.
Preimplantation genetics: chromosomes of fragmented human embryos.
STUDY OBJECTIVE: To examine the chromosome makeup of fragmented human embryos. DESIGN: Prospective. SETTING: Assisted reproductive technology (ART) program. PATIENTS: One hundred twenty-one poor-quality embryos from 58 patients 31 to 40 years of age admitted for an ART program were examined for chromosome makeup. RESULTS: Chromosome anomalies were observed in 31.9% (29/91) of poor-quality embryos, 19.8% (18/91) displayed mosaicism (diploid/haploid, diploid/triploid, diploid/aneuploid), 5.5% (5/91) showed polyploidy, 2.2% (2/91) had pulverized chromosomes, 2.2% (2/91) revealed aneuploidy, 1.1% (1/91) had prematurely condensed chromosomes, and 1.1% (1/91) had structural rearrangements involving chromosome number 2. The mean age of patients showing anomalies (36.5 years) was not significantly higher than the mean for the entire group (35.5 years). CONCLUSIONS: The incidence of chromosome anomalies in fragmented human embryos is high. These anomalies originate either in the gametes or through mitotic nondisjunction within the embryos. It is not advisable to replace such embryos into patients going through in vitro fertilization.
Adult↗
Cocultures: a new lead in embryo quality improvement for assisted reproduction.
One of the contributory causes to poor PRs in assisted reproduction has been the decreased viability of transferred embryos and the transfer of four-cell embryos into an environment that naturally would be receptive only to 5-day-old blastocysts. In this paper, we have reviewed our own work and that of others on the role of tubal ampullary cells (cocultures) to mimic the in vivo environment to bring about improved embryo quality and an increased number of blastocysts for replacement in IVF patients. The establishment, maintenance, and behavior of human tubal cell lines is first presented, followed by their use as cocultures for fertilization and cleavage of embryos. The mode of action, specificity, and cryopreservation of ampullary cells are also discussed. The currently available results of pregnancies after cocultures are presented together with future aspects of research that are necessary to refine the coculture system. The ultimate aim is to mimic in vivo conditions in vitro, so that at least the PRs of assisted conception can be parallel to normal fecundity in the human. Therefore, a very attractive future includes the freezing of blastocysts generated from coculture, thawing, and replacing them in natural cycles.
Animals↗
Etiologic role of Hafnia alvei in human diarrheal illness.
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Diarrhea↗
Ultrastructure of preimplantation human embryos co-cultured with human ampullary cells.
Ova with two pronuclei were co-cultured with established human ampullary cell lines and various stages of preimplantation embryonic development were monitored by Nomarski optics and then assessed by transmission electron microscopy (TEM). Fifteen embryos ranging from the 2-cell stage to blastocyst hatching were examined for normal and abnormal features. Their ultrastructure was similar to that of embryos cultured in Whittingham's T6 medium, reported previously. Seven embryos were evidently morphologically normal and showed good organization of fine structure. Most cellular organelles underwent progressive changes during early development. There was evidence of enhanced embryonic genome activation at the 8-cell stage. Invariably, all embryos had few too many fragments, some internalized, which were later segregated into the blastocoele or found outside the trophoblast of the late morula and blastocysts. Six grossly 'normal' embryos assessed by Nomarski had multiple nuclei of various dimensions, which highlights the subjectivity of embryo assessment in the IVF laboratory. Incomplete incorporation of chromatin into nuclei and formation of micronuclei were evident in some blastomeres. The results are discussed in relation to early embryonic loss, prevalent in IVF. Significant events reported include the detection of centrioles at the 8-cell stage, cavitation of the early blastocyst and the initiation of blastocyst hatching visualized by TEM.
Blastocyst↗
Co-cultures: their relevance to assisted reproduction.
Assisted reproductive techniques have contributed significantly to alleviating subfertility in the childless couple. However, the take-home baby rates have been very low throughout the world. One of the contributory causes has been the reduced viability of replaced embryos brought about by suboptimal in vitro conditions. The culture of human embryos in the presence of passaged human tubal ampullary monolayers (co-cultures) is an attractive approach to improving the viability of embryos for assisted reproduction. Seventy per cent of blastocysts can be produced in human ampullary co-culture as compared to 33% in standard culture media. This paper discusses the various roles of human co-cultures in assisted reproduction and provides an opinion as to how the transfer of blastocysts produced via the co-culture system could enhance pregnancy rates. Particular emphasis is placed on human oviductal characteristics, the various co-culture systems, screening of co-cultures for microbes, freezing of ampullary cells, growth factors and embryonic blocks, specificity of co-cultures, sperm hyperactivation in co-culture and pregnancy rates. The first patient on a clinical trial who had four of her oocytes fertilized and grown in human ampullary co-culture and then replaced into her uterus became pregnant. The co-culture system may have tremendous potential in supporting human embryonic growth via embryotrophic factors.
Animals↗
Regulation of sialyltransferase activity in intestinal segments of rats.
A differential distribution of sialyltransferase (ST) in different regions of intestine has been shown. Jejunum and ileum homogenates from rats showed almost exclusive presence of alpha-2-3 ST (to Gal in Gal beta-1-4GlcNAc and/or to Gal in Gal beta-1-3GalNAc). In contrast, colon homogenates showed the presence of both alpha-2-3 ST (as above) and alpha-2-6 ST. Incubation of intestinal slices in presence of heat-inactivated horse serum (HHS) showed a time- and temperature-dependent secretion of soluble ST into the medium. Both jejunum and ileum slices showed high rates of secretion of alpha-2-3 ST. Colon slices, though rich in alpha-2-6 ST, secreted only alpha-2-3 ST. Colchicine, an anti-mitotic drug, injected into rats caused about 10-fold increase of the serum ST level. Jejunum slices from colchicine-treated rats showed an increased secretion of alpha-2-6 ST, suggesting that intestine undergoes a change in the expression of normal secretion of alpha-2-3 ST to a secretion of alpha-2-6 ST. The secretion of ST from incubated intestinal slices was inhibited by heparin. Certain protein factors (anti-proteases) in HHS bind to heparin-sepharose column and these protein factors are responsible for causing the secretion of ST into the medium. It has also been found that a supernatant fraction of the colon homogenate activated ST. Gel chromatography on HPLC produced 3-4 protein fractions from the colon cytosol and one of this fraction bearing high molecular weight proteins produced the maximum activation of ST.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Lesions and immune responses produced in hamsters and guinea pigs inoculated with some strains of leptospira.
Pathogenic lesions and immune responses in hamsters and guinea pigs produced by three leptospiral serovars, viz. autumnalis, grippotyphosa, and pomona, and their pool were experimentally studied. Hepatic lesions precede renal localisation. The infections were documented by the demonstration of leptospires and histopathological study. The 2-Me sensitive IgM was responsible for MAT titres in the early immune response.
Animals↗
Epidemiologic features of hepatitis B virus infection in northern Labrador.
We studied the epidemiologic features of hepatitis B virus (HBV) infection in northern Labrador to determine the prevalence of the infection and to obtain a database to develop a vaccination strategy. The study population included seven communities in which five ethnic groups were represented: Inuit, Innu, mixed Inuit and European ancestry ("settler"), nonnative/nonsettler transient population ("white") and people of Innu-white or Innu-Inuit origin ("mixed"). Blood samples from 2156 people (62% of the area residents) were tested for antibody to HBV core antigen (anti-HBc), HBV surface antigen (HBsAg), HBV e antigen (HBeAg), anti-HBc IgM and antibody to the surface antigen (anti-HBs). The overall crude prevalence rate of HBV seromarkers was 14.7% and the HBsAg carrier rate at least 3.2%; the rates were highest for Inuit (26.4% and 6.9% respectively), followed by settler (10.0% and 1.9% respectively) and Innu (7.6% and 0.4% respectively); the white and mixed groups had the lowest overall rates (2.5% and 3.3% respectively). Although the overall prevalence rates were about the same for the two sexes, the HBsAg carrier rate was higher in males (male:female ratio 1.6:1.0). No HBV carriers were positive for HBeAg or anti-HBc IgM antibody. The rate of exposure to HBV was 4% for those below the age of 20 years and reached a peak for those aged 45 to 54 years (85% for Inuit, 40% for settlers and 37% for Innu). There was also a wide variation in the age-standardized prevalence rates (0% to 27.9%) among the ethnic groups in the seven communities surveyed.
Adolescent↗
Anti-idiotypic antibodies elicited by pterin recognize active site epitopes in dihydrofolate reductases and dihydropteridine reductase.
Monoclonal antibodies (mAbs) against antipterin immunoglobulin and dihydropteridine reductase (DHPR) and also polyclonal antibodies against human dihydrofolate reductase (DHFR) were obtained. The anti-idiotypic mAbs and anti-DHPR mAbs bind specifically to human DHFR, Escherichia coli DHFR, soybean seedling DHFR, and human DHPR in solid-phase immunoassays. Further, the mAbs bind to the native but not to the denatured forms of DHFRs. The monoclonal antibodies also inhibit the enzymatic activity of human DHFR but not that of human DHPR. Competitive solid-phase immunoassays show stoichiometric inhibition by methotrexate and partial inhibition by NADPH of mAb binding to human DHFR. Cyanogen bromide fragments derived from human DHFR (residues 15-52 and 53-111), containing several active site residues, bind partially to some of the monoclonal antibodies. Accordingly, polyclonal antibodies to peptide 53-111 of human DHFR cross-react to some extent with human DHPR. Data from competitive immunoassays in which the binding of the various mAbs was tested singly and in combination with other mAbs suggest that these antibodies bind to a common region on human DHFR. The results also indicate that the mAbs display some heterogeneity with respect to specific epitopes. These data suggest that despite the absence of significant amino acid sequence homologies among the various DHFRs and DHPR, they have a fundamentally similar topography at the site of binding of the pterin moiety that is recognized by the anti-idiotypic mAbs generated by pterin. In the relatively simple structure of the pterin ring system there are different substituent groups at positions C4 and C6 in methotrexate, 7,8-dihydrofolate, and 7,8-dihydrobiopterin, suggesting that these antibodies are specific for regions on various proteins that interact with the remainder of the pterin moiety. These mAbs and similar mAbs specified by substituent groups on pterin may thus be used as specific probes or inhibitors of various folate-dependent enzymes and transport proteins. They should also provide insights into some of the general features of antibody recognition of protein antigens.
Antibodies, Anti-Idiotypic↗
Improved sperm concentration, motility, and fertilization rates following Ficoll treatment of sperm in a human in vitro fertilization program.
Duplicate sperm samples from the spouses of 54 patients admitted to an in vitro fertilization program were prepared by the swim-up and a simplified procedure using Ficoll. Cellsoft (CRYO Resources, New York, NY) sperm curvilinear velocities (microns/sec) and mean amplitude of lateral head displacement values (microns) equivalent to grade 1 and 2 visual sperm motility were significantly higher for Ficoll as compared with swim-up samples (P less than 0.01). Fertilization rates were significantly higher in the Ficoll as compared with the swim-up group for poor semen samples (grade less than 2: 58% versus 24%; P less than 0.01) and normal semen samples (grade greater than or equal to 2: 85% versus 78%; P less than 0.05). Ficoll sperm separation appears to be an excellent method of yielding increased fertilization rates in in vitro fertilization programs.
Female↗
Transfer of human sperm into the perivitelline space of human oocytes after zona-drilling or zona-puncture.
To evaluate the transfer of sperm from severely oligozoospermic men into the perivitelline space of mature oocytes, zona-drilling with acid phosphate-buffered saline (PBS) and direct zona-puncture in the presence of cytochalasin D were studied. Zona-drilling also was done for eggs from patients with previous failed in vitro fertilization (IVF). Forty-seven eggs from seven patients with oligozoospermia and three patients with failed IVF had a mean of between 2.6 and 3.6 sperm transferred into the perivitelline space. In the group whose eggs had zona-drilling with acid PBS, 1 of 13 eggs fertilized from the oligozoospermic category, while there was no fertilization from the failed IVF category. Karyotyping of the unfertilized eggs after zona-drilling revealed a high incidence (2/5 and 3/7, respectively) of possible arrest at anaphase II after reinitiation of meiosis. In the group whose eggs were directly punctured through the zona in the presence of cytochalasin D, there was no fertilization in 23 undamaged eggs. Two of the 15 interpretable karyotypes were aneuploid, but this incidence is within that observed for our unfertilized eggs after IVF. Hence, the use of acid PBS for zona-drilling is not advised. Moreover, transfer of sperm from men with previous failed fertilization resulted in poor fertilization rates.
Cytochalasin D↗
Improved quality of human embryos when co-cultured with human ampullary cells.
Cultured human, ampullary, epithelial cells obtained from fertile women undergoing hysterectomy were evaluated for the support of human embryonic cleavage and growth in vitro. Twelve patients provided 23 embryos for co-culture with subcultured ampullary cells grown in T6 + 15% patient's serum and 18 embryos for growth in T6 + 15% patient's serum alone (controls). Of embryos co-cultured with ampullary cells, 78% cleaved to the compacted embryo stage and 69% cavitated as compared with 50 and 33% respectively for controls (P less than 0.01). Only 30% of co-cultured embryos reached the expanded blastocyst and 26% underwent hatching as compared with 28% for both stages in controls. At the 2 - 4- and 6 - 8-cell stages, 91 and 87% of co-cultured embryos showed an absence or slight fragmentation as compared with 72 and 61% respectively for embryos grown in medium alone (P less than 0.01). None of the co-cultured embryos showed unequal-sized blastomeres while 22% of controls showed unequal cleavage. Embryos grown with ampullary cells cleaved slightly faster than controls. Scanning electron micrographs showed that ampullary cells collected from co-cultures were all of the secretory type with several microvilli and apical protrusions. It is clear that subcultured human ampullary cells support human embryonic cleavage and yield a reasonable number of good quality embryos up to the cavitation stage. Development past the expanded blastocyst and hatching stages seems to involve another critical phase with its own specific requirements.
Cells, Cultured↗
Simple office methods to predict ovulation: the clinical usefulness of a new urine luteinizing hormone kit compared to basal body temperature, cervical mucus and ultrasound.
A new urine luteinizing hormone (LH) kit, First Response (Tambrands Inc., Palmer, MA) was compared with basal body temperature (BBT), cervical mucus scoring and abdominal ultrasound follicular scanning in their ability to predict ovulation to within 2 days of the serum LH peak. BBT was kept daily. From day 10 daily ultrasound scanning and cervical mucus examination were performed and serum oestradiol, luteinizing hormone, follicular stimulating hormone and progesterone were assayed. First Response was significantly more accurate than BBT and cervical mucus when compared in their ability to predict ovulation to within 2 days of the LH peak (p less than 0.05). First Response pinpointed 93% (27/29) of the ovulatory cycles compared to 72% (18/25) and 61% (19/31) for BBT and cervical mucus respectively. It was better but not significantly so against abdominal ultrasound which predicted 77% (24/31). The implications of this finding and the value of the other simple office tests in clinical practice are discussed.
Ambulatory Care↗
Latex agglutination test for detection of Escherichia coli serotype O157.
The value of a latex agglutination test (Escherichia coli O157 latex test; Oxoid Ltd.) for rapid presumptive detection of E. coli serotype O157:H7 was determined by laboratory trials and during an outbreak of hemorrhagic colitis. The latex test was found to be a simple, highly efficient and reliable test in detecting E. coli O157:H7 with 100% sensitivity and specificity. It was also found that sorbitol-MacConkey agar cultures were not as useful for food samples as they were for fecal specimens in screening for E. coli O157:H7, but the use of the latex screen was particularly efficient in this setting.
Colitis↗
False-positive results with third-generation monoclonal hepatitis B surface antigen enzyme immunoassay.
Nonspecific hepatitis B surface antigen reactions with a third-generation enzyme immunoassay (Auszyme Monoclonal; Abbott Laboratories, North Chicago, Ill.) were investigated with 9,577 serum specimens in a clinical laboratory setting. Of the 196 serum specimens found reactive in Auszyme screen by the overnight procedure, 103 turned out to be true-positives, 71 were nonrepeatably reactive, and 22 were repeatably reactive but actually falsely positive (false-positive rate, 22 of 196, or 11.2%). Verification of the 196 screen reactives by the Auszyme 3-h incubation assay detected all but 4 true-positives, with a false-negative rate of 3.9% (4 of 103), and was negative for the rest. These observations reinforce the need for retesting all reactive specimens and confirming repeatedly reactive samples when the Auszyme Monoclonal test is used to detect hepatitis B surface antigen.
Carrier State↗
Chromosome analysis of two-cell mouse embryos frozen by slow and ultrarapid methods using two different cryoprotectants.
Three freezing methods (slow-1,2 propanediol; ultrarapid-dimethyl sulphoxide; ultrarapid-1,2 propanediol) were used to assess the effects of low temperature storage on morphologic features and chromosome make-up of 2-cell mouse embryos. Weekly batches (15 weeks) of 2-cell mouse embryos were frozen for a period of 7 days using these three freezing methods, then thawed and subjected to chromosome analysis. After thawing, all three freezing regimens yielded 70.2% to 75.8% of healthy intact 2-cell embryos, and 5.5% to 8.1% of embryos with one healthy blastomere intact, respectively, out of 817 embryos examined. Chromosome analysis was possible in all 1- and 2-cell embryos. The incidence of aneuploidy and polyploidy in frozen-thawed embryos of all three methods (2.8% to 3.4%; 4.0% to 6.5%) was not significantly different from that of control unfrozen embryos (3.0%; 6.0%) (P greater than 0.01). Mitotic crossing over was observed in 3.5% of embryos frozen-thawed by the ultrarapid-dimethyl sulphoxide method only. It is not clear how the mitotic crossing over was induced by ultrarapid freezing in the presence of dimethyl sulphoxide.
Animals↗
Chromosome anomalies in human oocytes failing to fertilize after insemination in vitro.
Three-hundred-and-two unfertilized oocytes left over from successful in-vitro fertilization (IVF) attempts in 143 women (27-42 years) on a follicular stimulating hormone-human menopausal gonadotrophin (FSH-HMG) stimulation regime were subjected to chromosome analysis. Ten oocytes were degenerated with no visible chromosomes and 41 metaphases had chromosomes that were clumped together which could not be interpreted either numerically or structurally. Of the remaining oocytes, 76.6% (192/251) had a normal haploid complement (n = 23), 13% (33/251) were hypohaploid (n = 19-22), 8% (20/251) were hyperhaploid (n = 24-26), 2% (5/251) were diploid (2n = 46) and 0.4% (1/251) had structural rearrangements. The 21% aneuploidy was from 24 different patients and hypohaploid sets had chromosomes missing mainly from the A, B, C, D and G groups while the hyperhaploid sets had extra chromosomes from A, B, D, G and E groups of the human karyotype. The mean age of patients showing aneuploid oocytes was 36.7 years which was above the mean for the entire group. The aneuploidy may have been brought about by errors in oogenesis (anaphase lagging or non-disjunction) and may offer one explanation for fertilization failure and overall low pregnancy rates after IVF.
Adult↗