Clinical course of human immunodeficiency virus type 1 associated pulmonary tuberculosis during short-course antituberculosis therapy

PURPOSE: It was reported that Matrigel improved hatching of mouse blastocysts produced in vitro from F1 hybrid-derived zygotes. We investigated whether Matrigel would be similarly beneficial with outbred strain-derived embryos, which exhibit a "two-cell" block similar to the developmental blocks of other species. METHODS: Mouse embryo development was assessed with or without Matrigel in KSOM medium, which supports the development of blocking strain zygotes in vitro, and in human tubal fluid (HTF) medium, which normally does not but which is used for human IVF. RESULTS: Matrigel severely inhibited the development of zygotes to blastocysts in KSOM and did not improve culture in HTF. There was no effect on development from the two-cell stage. We were not able to replicate the previous finding of Matrigel's beneficial effect on hatching of F1-derived zygotes. CONCLUSIONS: Matrigel may be a deleterious addition to embryo culture or coculture systems.     

A prospective randomized trial of blastocyst culture and transfer in in-vitro fertilization

The effectiveness of blastocyst culture and transfer in human in-vitro fertilization (IVF) was evaluated in a prospective randomized trial in patients having a moderate to good response to gonadotrophin stimulation. Embryos were transferred either on day 3 after culture to around the 8-cell stage in Ham's F-10 medium supplemented with fetal cord serum, or on day 5 after culture to the blastocyst stage in the sequential serum-free media G 1.2 and G 2.2. The pregnancy rates after transfer on day 3 or day 5 were equivalent, 66 and 71% respectively; however, significantly more embryos were transferred on day 3 (3.7) than on day 5 (2.2). The number of blastocysts transferred did not affect the implantation rate, and pregnancy rates when either two or three blastocysts were transferred were 68 and 87% respectively. The implantation rate of the blastocysts (50.5% fetal heart beat) was significantly higher compared to the cleavage stage embryos transferred on day 3 (30.1%). The percentage of blastocyst development was not affected by the number of 2-pronuclear embryos, or by maternal age. Irrespective of the number of blastocysts formed, pregnancy rates were similar. Furthermore, the pregnancy rate following blastocyst transfer in patients with 10 or more follicles at the time of human chorionic gonadotrophin administration was not affected by patient age. More than 60% of patients having blastocyst culture and transfer had supernumerary embryos for cryopreservation. The establishment of a pregnancy following thaw and transfer confirmed the viability of cryopreserved blastocysts cultured in the absence of serum or co-culture. The ability to transfer just two blastocysts while maintaining high pregnancy rates will therefore help to eliminate high order multiple gestations and improve the overall efficiency of human IVF.     

Increased detection and yield of adenovirus in caffeine-stabilised KB cell monolayers

In human leukocyte cultures set up with Eagle's MEM and stimulated with Difco's PHA M, DNA synthesis and mitotic indices were analyzed by means of 3(H)-thymidine autoradiography and cell counting from 23 up to 52 h after culture initiation. Considerable amounts of DNA synthesis and mitoses were found in this time span. This resembles the results found with Ham's F-10 medium. However, the DNA synthesis pattern and the distribution of mitotic indices a higher yield of asynchrony in Eagle's MEM as compared with Ham's F-10 cultures. Proportions of first, second, and third mitoses at 72 h culture time were determined with different methods.     

Effect of free amino acids and vitamins on cleavage and developmental rate of bovine zygotes in vitro

Due to the complicated media used for culturing bovine embryos, most of the nutrient requirements are unknown. Recently, we developed a simple, serum-free medium (CR1) that allows bovine embryos to develop in vitro. Therefore, our objective was to determine whether development of bovine embryos would be improved by the addition of free amino acids and vitamins to CR1. Oocytes were recovered from slaughterhouse ovaries and matured 22 +/- 2 h, following which the oocytes were randomly allotted to treatment. The experiment was a randomized block design with a 2 x 5 factorial treatment structure. The oocytes were fertilized with or without cumulus cells intact. The five fertilization media were 1) Control (CR1 +/- 10 micrograms/mL of phenol red); 2) control + basal medium Eagle (BME) essential amino acids (EAA) + minimum essential medium (MEM) nonessential amino acids (NEA) + MEM vitamins (VIT); 3) control + EAA + NEA; 4) control + EAA + VIT; and 5) control + NEA + VIT. Cleavage rate was greater (P < .001) when cumulus cells remained on the oocytes during fertilization (51.7 vs 73.2% without and with cumulus cells, respectively). The frequency of blastocysts was increased (P < .001) when EAA or NEA were added to CR1; however, adding VIT had no effect or tended (P = .12) to decrease the frequency of embryos attaining the blastocyst stage. This experiment demonstrates that development of bovine embryos in vitro can be improved by the addition of free amino acids to a simple medium. Contrary to work in rodents, the mixture of vitamins in MEM was not beneficial for bovine embryos.     

Effect of certain diuretics on thyroid function in rats

The effects of isolated protein fractions from rabbit uteri (prealbumin, albumin, uteroglobin, and beta-glycoprotein), unfractionated uterine proteins, progesterone, oestradiol-17beta, and prostaglandin F-2a on the development of rabbit embryos in vitro were investigated. When exposed to individual protein fractions obtained from Day-6 uteri, 8-cell embryos did not develop into early blastocysts; morulae readily developed into early blastocysts, but further development was retarded. Progesterone (10(-5)-10(-11)M) and prostaglandin F-2a (0-1-10 ng/ml) added to the medium slowed development of blastocysts to advanced stages. Growth of 8- to 16-cell embryos, morulae, and Day-4 blastocysts was stimulated by unfractionated uterine proteins obtained from Day-5 uterine flushings. Although embryos cultured in medium containing BSA had similar rates of blastocyst formation and, ultimately similar blastocyst expansion as did the embryos cultured in medium with unfractionated proteins, the radial and immediate expansion of the early blastocysts cultured in the latter approximated that found in utero.     

Presence of dioxins in human follicular fluid: their possible stage-specific action on the development of preimplantation mouse embryos

Examination of human follicular fluid revealed the presence of polychlorinated dibenzodioxins (PCDDs) and dibenzofurans (PCDFs) at concentrations of approximately 1 pg/ml (0.01 pg TEQ/ml). To study their possible action, two-cell mouse embryos were cultured in the presence of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) at concentrations between 0.5 and 100 pM and evaluated at 24-h intervals for their development to the eight-cell and blastocyst stages. The percentage of eight-cell embryos exposed to TCDD at 1, 2, and 5 pM concentrations was significantly lower than that of controls. However, blastocyst formation of the surviving eight-cell embryos was accelerated, with the number of cells in the blastocysts increased in a dose-dependent manner. Findings suggest that PCDDs and PCDFs may be present in human reproductive fluid and may exert some stage-specific effects on early embryonic development.     

Role of endometrial factors in regulating secretion of components of the immunoreactive human embryonic interleukin-1 system during embryonic development

In the study reported here, we localized at the protein level the major components of the interleukin (IL)-1 system in the human embryo, and we investigated the endometrial factors influencing their secretion during embryonic development. To localize these components, we performed immunohistochemical experiments in 44 oocytes and 78 embryos. The following primary antibodies were used: monoclonal mouse anti-human IL-1 receptor type I (IL-1R tl), monoclonal mouse anti-human IL-1 beta, and polyclonal rabbit anti-human IL-1 receptor antagonist (IL-1ra). For embryo culture, human embryos at different developmental stages were cultured in 100-microliters drops of Ham's F-10 medium + 4 mg/ml BSA (n = 33), in 100-microliters drops of Menezo B2 culture medium (n = 18), or in wells with 1 ml of Menezo B2 culture medium (n = 8). For embryo coculture, endometrial stromal cells (ESC) and endometrial epithelial cells (EEC) were isolated from human secretory endometrium and cultured until confluence in 75% Dulbecco's Modified Eagle's Medium and 25% MCDB-105 containing antibiotics and supplemented with 10% charcoal-Dextran-treated fetal bovine serum. Individual human embryos were cocultured with experimental EEC and ESC (n = 23 and n = 4, respectively) for 5 days in 600-microliters drops of Menezo B2 medium, and conditioned medium was removed every 24 h. Human embryos were also cultured with EEC-conditioned medium (n = 9). IL-1 alpha, IL-1 beta, and IL-1ra levels were determined by ELISA in the 24-h culture- or coculture-conditioned media. Immunostaining confirmed the presence of IL-1 beta, IL-1ra, and IL-1R tl in oocytes and embryos in all stages analyzed, with no statistical differences. IL-1 alpha, IL-1 beta, and IL-1ra were absent in conditioned media of cultured embryos and embryos cocultured with ESC. However, when human embryos were cocultured with EEC or with EEC-conditioned medium alone, two different populations of embryos were observed: IL-1 producers (57% and 56%) and IL-1 nonproducers (43% and 44%, respectively). Finally, the IL-1 profile of a single human embryo cocultured with maternal EEC which successfully implanted and developed is presented, this pattern being similar to that described in the IL-1 producer population. These results demonstrate the presence of the IL-1 system in the human embryo. However, the selective release of IL-1 only when embryos were cocultured with EEC or EEC-conditioned medium indicates an obligatory role of the endometrium in the regulation of the embryonic IL-1 system. Furthermore, the differential embryonic production of IL-1 may be related to the implantation capability of the embryos.     

Current and future methodology for monitoring sleep

The present study examined the effect of follicular shell pieces (FSP) during in vitro maturation (IVM) of porcine oocytes on 1) in vitro fertilization (IVF) parameters, 2) subsequent embryo development, 3) oocyte glutathione (GSH) concentration, and 4) viability after embryo transfer. Cumulus-oocyte complexes were cultured in North Carolina State University (NCSU) 23 medium containing porcine follicular fluid, cysteine, and hormonal supplements and with or without FSP for 20-22 h. They were then cultured in the same medium but without hormonal supplements for an additional 20-22 h. After culture, cumulus-free oocytes were coincubated with frozen-thawed spermatozoa for 5-6 h. Putative zygotes were transferred to NCSU 23 containing 0.4% BSA and cultured for 144 h. In comparisons between the presence and absence of FSP, no differences were observed in fertilization parameters. At 48 h, no mean differences were found in cleavage rates. However, at 144 h, the proportion of embryos that developed to the blastocyst stage was significantly (p < 0.01) higher (18% vs. 36%) for oocytes cocultured with FSP. A significantly (p < 0.05) higher GSH concentration was found in oocytes matured with FSP as determined by dithionitrobenzoic acid-glutathione disulfide (DTNB-GSSG) reductase recycling assay. Transfer of embryos to 9 recipients resulted in 5 pregnancies with the birth of 18 live piglets. The results provide clear evidence of the beneficial effect of FSP during IVM of pig oocytes cultured in the presence of cysteine on subsequent embryo development to the blastocyst stage. The birth of piglets confirms the viability of IVM-IVF-derived embryos.     

Arsenic levels in drinking water and the prevalence of skin lesions in West Bengal, India

OBJECTIVE: To evaluate the suitability and efficiency of human follicular fluid (HFF) as a protein supplement in human IVF programs. DESIGN: Comparative study of the effects of HFF and other protein supplements on the in vitro development of mouse oocytes and on the pregnancy rate in human IVF programs. SETTING: In Vitro Fertilization Center, Hanna Women's Clinic, Seoul, Korea. PATIENT(S): Three hundred twenty-seven patients (388 cycles) who were down-regulated with GnRH agonist and stimulated with hMG. INTERVENTION(S): The suitability was evaluated with the results of animal studies and the efficiency of HFF was investigated with the results of human IVF programs. MAIN OUTCOME MEASURE(S): Meiotic maturation of mouse oocytes, development of mouse embryos, morphological grades of human embryos, pregnancy rate in human IVF programs, and electrophoresis. RESULT(S): Human follicular fluid significantly stimulated meiotic resumption in mouse oocytes, even in the presence of meiotic inhibitors, and enhanced the developmental potential of mouse embryos in vitro. Compared with human fetal cord serum, human follicular fluid also improved the morphological grade of human embryos by reducing cytoplasmic fragmentation. In conventional IVF cycles of human IVF programs, use of HFF significantly increased the clinical PR (109/234 cycles, 46.5%; P < .05), compared with use of human fetal cord serum (14/52 cycles, 26.9%). However, in intracytoplasmic sperm injection cycles, there was no difference in PRs between use of HFF (38/85 cycles, 44.7%) and use of human fetal cord serum (7/17 cycles, 41.1%). When the protein compositions of human fetal cord serum and HFF were investigated by electrophoresis, a protein of 21 kD was detected specifically in HFF. CONCLUSION(S): Human follicular fluid is suitable for use as a protein supplement and is effective in increasing the pregnancy rate in human IVF programs.     

Co-culture, oviduct secretion and the function of oviduct-specific glycoproteins

Co-cultures of embryos with somatic cells, usually in the form of monolayers, or conditioned medium from these somatic cells, results in development past the early stage blocks and the formation of hatched blastocysts. Optimum rates of development are not achieved, however, and the task is to investigate components of the oviduct that are obligatory or facilitative for embryo development. Glycine and alanine are amino acids present in much higher concentrations in oviduct fluid than in serum or culture media. Glycoproteins specifically produced by the oviduct around oestrus bind to embryos and aid development but are absent from most culture media. These glycoproteins are induced by oestrogen in vivo but not in vitro. It is our contention that co-cultures of mammalian embryos should include appropriate concentrations of amino acids and a source of embryotrophic glycoproteins as an additive or by including stromal cells in addition to epithelial cells.     

Embryotoxicity of leukocyte extracts and its relationship to intrauterine contraception in humans

Twelve human uteri containing intrauterine contraceptive devices (IUDs) and ten uteri without IUDs were obtained at hysterectomy. Samples of fluid were collected from the uterine lumina by absorbing the fluid in small pieces of lens paper. In the samples of luminal fluid we measured the concentration of beta-galactosidase, an enzyme which is present in human neutrophilic leukocytes and whose concentration in luminal fluid should correlate with the local inflammatory response to the intrauterine foreign body. In the samples of fluid from IUD-bearing uteri, the concentration of beta-galactosidase was significantly (P less than 0.0005) greater than that in luminal fluid from control uteri, the averages of the two groups differing by 3.8 units. To determine whether a foreign-body response of this magnitude could have any effect on preimplantation embryos, we cultured mouse embryos from day 4 to day 7 of development in culture media to which extracts of human leukocytes were added. All mouse embryos were killed when the culture media contained enough leukocyte extract to give beta-galactosidase concentrations of 0.5 unit or higher. Thus mouse embryos were killed by leukocyte extracts whose beta-galactosidase concentrations were actually less than the concentration of this marker enzyme measured in IUD uterine fluid. This comparison indicates that the luminal fluid in IUD-bearing uteri contains leukocyte break-down products in sufficient concentration to be lethal for preimplantation embryos.     

Amino acids in maturation medium and presence of cumulus cells at fertilization promote male pronuclear formation in porcine oocytes matured and penetrated in vitro

The present study was conducted to examine the ability of porcine oocytes to achieve male pronuclear (MPN) formation when they are matured and penetrated in vitro under various culture conditions. When cumulus-enclosed oocytes were cultured for 24-48 h in modified Whitten's medium (pH 7.4) supplemented with 10% porcine follicular fluid, 10 IU eCG/ml, and 10 IU hCG/ml (designated mWM-FG), nuclear maturation of oocytes reaching metaphase II was completed by 36 h after the start of culture. However, there were no differences in the proportions (94-95%) of oocytes penetrated in vitro by cryopreserved ejaculated spermatozoa or in the rates (35-45%) of MPN formation between oocytes cultured for 36 and 48 h. When cumulus-enclosed oocytes were cultured for 36 h in mWM-FG supplemented with 2% (v:v) minimal essential medium (MEM) essential amino acids (EAA) with the addition of 0.1 mM glutamine and/or 1% (v:v) MEM nonessential amino acids (NEAA) and inseminated in vitro, 93-97% of oocytes were penetrated regardless of the presence of amino acids during maturation, but the rates of MPN formation were higher in the presence (79-84%) than in the absence (51%) of any amino acids. The addition of EAA+NEAA and/or 0.57 mM cysteine to mWM-FG also did not affect sperm penetration in vitro, while it promoted MPN formation (76-83%) in penetrated oocytes as compared with those matured in the absence of amino acids and cysteine (53%). When oocytes were freed from cumulus cells after culture in mWM-FG, sperm penetration rates were not different between cumulus-enclosed (100%) and cumulus-free (92%) oocytes, but the rate of MPN formation was higher in cumulus-enclosed (53%) than in cumulus-free (28%) oocytes. When EAA+NEAA+cysteine was added to mWM-FG, MPN formation was not improved in cumulus-free oocytes but was much improved (78%) in cumulus-enclosed oocytes. These results indicate that MPN formation in porcine oocytes is promoted by the addition of amino acids and/or cysteine in simple maturation medium and by the presence of cumulus cells at fertilization in vitro.     

A new source of human oocytes: preliminary report on the identification and maturation of human preantral follicles from follicular aspirates

This study aimed to investigate the development of human preantral follicles and oocyte maturation in vitro. Preantral follicles were obtained from follicular aspirates during egg retrieval carried out during an in-vitro fertilization (IVF) programme. They were first incubated in Ham's F10 medium with 15% fetal cord serum (FCS). After 28 days, the medium was supplemented with different doses of human menopausal gonadotrophin (HMG), human follicular fluid (hFF) and epidermal growth factor (EGF) by orthogonal design. Promotion of final maturation was completed in the presence of HMG and hFF. Development from preantral to antral follicles was found within 6-12 days of culture. With time, the proportion of follicles with diameters of >300 microm increased at 21-28 days of culture (P < 0.005). The maximum number of oocytes extruded, and first polar body formation, occurred in the presence of 0.15 IU/ml HMG 40% (v/v) hFF and 6 ng/ml EGF. We conclude that follicular aspirates obtained during egg retrieval in an IVF programme contain many preantral follicles which could develop into antral follicles with extrusion of oocytes in culture, and that the oocytes can mature in vitro. Hence, a new source of human oocytes is available.     

Monoclonal antibodies to mammalian heat shock proteins impair mouse embryo development in vitro

Two-cell mouse embryos (B6D2F1) were cultured in the presence or absence of 100 microg/ml monoclonal antibodies specific for the mammalian 60 kDa (HSP60), 70 kDa (HSP70) and 90 kDa (HSP90) heat shock proteins. Embryo development was evaluated after 3, 5 and 7 days in culture by determining the number of blastocysts, hatched blastocysts and outgrown trophoblasts at the successive time points. At day 3, only 29% (22/75) of the embryos cultured with anti-HSP60 antibody developed to the blastocyst stage (P < 0.0001) as compared to 67% (31/46) of the embryos cultured with anti-HSP70, 72% (43/60) cultured with anti-HSP90, and 79% (49/62) in medium plus mouse IgG1. By day 5, hatched embryos were present in 28% (13/ 46) of the cultures containing anti-HSP70 (P < 0.0001), as opposed to 57% (34/60) containing anti-HSP90 and 73% (45/62) containing IgG1. At day 7, outgrown trophoblasts were observed in 9% (4/46) of cultures containing anti-HSP70 (P < 0.0001), 45% (27/60) containing anti-HSP90 (P < 0.01) and 66% (41/62) cultured in medium plus IgG1. Antibodies to different heat shock proteins exerted a detrimental effect on mouse embryo development at unique development stages. Immune sensitization to heat shock proteins may be a cause of reproductive failure.     

Aggressive behavior of psychiatric patients among geriatric population

The effects of protein supplements and culture dish type on in vitro fertilization (IVF) and embryo development in culture were examined in the domestic cat. In Experiment I, follicular oocytes were fertilized and cultured in either 1) modified Earle's balanced salt solution, designated MK-1, supplemented with one of the following: 10% human serum (HS), 10% FCS or 0.4% BSA, or 2) Medium 199 (M-199) supplemented with 10% FCS. Fertilization rates were lower (P < 0.01) in MK-1 + BSA (74.4%), MK-1 + FCS (56.1%), and M-199 + FCS (51.4%) than in MK-1 + HS (94.7%). A greater (P < 0.01) percentage of blastocysts was obtained in MK-1 + HS (50.0%) than in other treatment groups (range, 4.3-17.2%). In Experiment II, the effect of dish type (tissue culture dish, TCD, versus suspension culture dish, SCD) on embryo development was evaluated in MK-1 supplemented with either HS or BSA. Significantly higher proportions of IVF-derived embryos developed to blastocysts at 120 and 144 hr post-insemination, respectively, when cultured in HS/SCD (47.2 and 71.7%) than in BSA/SCD (11.4 and 27.3%) or BSA/TCD (10.4 and 25.0%). At 120 hr post-insemination, there was a lower (P < 0.01) percentage of blastocysts in HS/TCD (22.2%) than in HS/SCD. In Experiment III, six embryos per cat were transferred to the uterine horns of 17 recipients at 144 hr after hCG treatment. Five of 7 recipients which received late morulae cultured in MK-1 + BSA (SCD) for 120 hr became pregnant (71.4%). Eight of 10 recipients which received early blastocysts cultured in MK-1 + HS (SCD) for 120 hr became pregnant (80.0%). We conclude that MK-1 containing HS is highly beneficial for overcoming the in vitro developmental block of IVF-derived feline embryos and increasing the success rate of IVF/ET.     

Effects of glycosaminoglycans on the development of in vitro-matured and -fertilized porcine oocytes to the blastocyst stage in vitro

We examined the effects of four glycosaminoglycans (GAGs) on the development of in vitro-matured (IVM) and -fertilized (IVF) porcine oocytes to the blastocyst stage. IVM and IVF oocytes were cultured in Whitten's medium supplemented with hyaluronic acid, chondroitin sulfate A, dermatan sulfate, or heparin at 38.5 degrees C in an atmosphere of 5% CO2 in humidified air for up to 6 days. After 2 days in culture, 28-34% of the inseminated oocytes cleaved to the 2- to 8-cell stage, and the GAGs showed no significant effect on development. After 6 days in culture, blastocysts were observed in all groups. The percentage of blastocysts was significantly higher in hyaluronic acid-supplemented medium (14%) than in dermatan sulfate-supplemented (5%), heparin-supplemented (2%), or nonsupplemented (2%) media. In addition, the percentage of blastocysts was significantly higher in chondroitin sulfate A-supplemented medium (11%) than in heparin-supplemented and nonsupplemented media, although the number of blastocysts in chondroitin sulfate A was not significantly different from that in hyaluronic acid- and dermatan sulfate-supplemented media. There were no significant differences in the mean number of nuclei per blastocyst cultured in any group. The effects of hyaluronic acid and chondroitin sulfate A on development to the blastocyst stage was examined at various concentrations. After 6 days in culture, development of IVM and IVF oocytes to the blastocyst stage was best supported in 0.5 mg/ml hyaluronic acid-supplemented (17%) and in 0.1 or 0.5 mg/ml chondroitin sulfate A-supplemented (10% or 9%, respectively) media. It is concluded from these results that hyaluronic acid and chondroitin sulfate A supported the development of porcine oocytes matured and fertilized in vitro to the blastocyst stage.     

A mutation in the neurofibromatosis type 2 tumor-suppressor gene, giving rise to widely different clinical phenotypes in two unrelated individuals

Polychlorinated biphenyls (PCBs) are industrial chemicals that are long-lasting global environmental contaminants. PCBs have been reported to adversely affect reproduction in laboratory and wild animals by reducing the incidence of breeding and the survival rate of young. The present study was undertaken to determine the toxic potential of PCBs on in vitro fertilization (IVF) in the mouse. Aroclor 1221, 1254, and 1268, and 3, 3', 4, 4'-tetrachlorobiphenyl (TCB), a PCB congener, were added to IVF medium at various concentrations (0.01, 0.1, 1, and 10 micrograms/mL). Cumulus masses containing oocytes were obtained from superovulated B6D2F1 mice and cultured in medium containing PCB to which capacitated sperm were added. Oocytes were assessed for fertilization 20 to 24 h after insemination. A-1221, A-1268, and TCB reduced the fertilization rate at the 1 microgram/mL and 10 micrograms/mL doses, while inhibition of fertilization by A-1254 reached significance at 0.1 microgram/ml. Furthermore, all of these chemicals caused an increased incidence of degenerative ova and abnormal 2-cell embryos at the higher dose levels (1 microgram/mL and 10 micrograms/mL). The results suggest that higher dosages of PCB and TCB adversely affect fertilization and cause an increased incidence of degeneration of oocytes and abnormality in the early mouse embryos.     

Evidence for the requirement of autocrine growth factors for development of mouse preimplantation embryos in vitro

The mitotic stimuli in the early mammalian embryo have not been unequivocally identified. One hypothesis is that the embryo releases autocrine growth factors (GFs) that have a role in such growth. To determine whether such putative GFs were limited by dilution, and hence secreted, development was observed at various embryo concentrations in culture. Embryos were collected at the zygote or 2-cell stage. Zygotes were produced by fertilization in situ (ISF) or in vitro (IVF). Two-cell-stage embryos had a high rate of development to the blastocyst stage across an embryo concentration range of 1/microl-0.001/microl. By contrast, zygotes produced by either ISF or IVF were adversely affected by reducing the embryo concentration over this range (p < 0.001), with approximately 80% of ISF zygotes developing to blastocysts at the highest concentration but only 26% at the lowest. For IVF zygotes the corresponding results were 64% and 6%. For all three embryo types, the number of cells in each blastocyst was significantly lower with reduced embryo concentration. The major determinant of zygote development was the concentration of embryos in culture rather than the absolute volume of culture medium or the actual number of embryos present. A concentration of 1 embryo/microl (in the form of 10 embryos/10microl) gave the best development rates and highest cell numbers per blastocyst. Varying the albumin concentration influenced development rates; a 10-fold reduction in BSA concentration (to 0.3 mg/ml) resulted in significantly more IVF zygotes developing to the blastocyst stage. Platelet-activating factor (PAF) is released by embryos, and albumin can act as a competitive inhibitor of PAF's action on cells. ISF embryos released more PAF (p < 0.05) into media than did similarly treated IVF embryos. There was no difference in the amount of PAF remaining associated with the resulting 2-cell embryos. The amount of PAF released by both these groups was markedly less (p < 0.001) than the amount released by 2-cell embryos collected fresh from the reproductive tract and cultured for 24 h. PAF supplementation of media caused a significant increase in the rate of blastocyst development of IVF zygotes at embryo concentrations of 0.1/microl (1 ng/ml) and 0.01/microl (100 ng/ml). Insulin-like growth factor (IGF)-I (30 ng/ml) and IGF-II (1 ng/ml) also stimulated development of IVF zygotes when cultured at an embryo concentration of 1/10 microl. Epidermal growth factor was without effect over the range 0.2-2000 ng/ml. Supplementation of media with both PAF and IGF-II gave no additional benefit over that caused by IGF-II alone, but this treatment was marginally better (p < 0.05) than PAF treatment alone. The results show that factors necessary for normal embryo development are diluted to suboptimal levels during culture at low embryo concentration. The ability of PAF, IGF-I, and IGF-II to partially compensate for the adverse effects of low embryo concentration during culture is consistent with their having roles as autocrine embryotrophic factors. The use of IVF and low embryo concentrations in culture may provide a functional multiple ablation model that will help to define the range of GFs required for normal embryo development.     

Is hydrosalpinx fluid cytotoxic?

Accumulation of oviductal fluid in the ampullar lumen as a result of occlusion of the infundibulum is referred to as hydrosalpinx. A low pregnancy rate (10%) after in-vitro fertilization (IVF) in hydrosalpinx patients and a relatively high incidence (50%) of abortions during the first trimester suggested that leakage of this fluid into the uterine cavity may exert a cytotoxic effect on the developing embryo. To examine this possibility, we analysed the composition of the hydrosalpinx fluid and tested its effect on human granulosa cells and embryos. Hydrosalpinx fluids and granulosa cells were collected from IVF patients at ovum pick-up. IVF eggs containing three pronuclei (3PN) were employed for this study. Analysis of hydrosalpinx fluids revealed electrolyte concentrations similar to those in serum with lower amounts of total protein and albumin. No blood cells were detected and bacterial cultures were negative. Granulosa cells incubated in hydrosalpinx fluid-containing medium (diluted 1:1) were not morphologically different and showed a steroidogenic capacity that was higher than that of cells incubated in its absence. Fertilized 3PN eggs incubated in IVF culture medium successfully developed into 6- to 8- and 8- to 16-cell embryos within 48 and 72 h, respectively. This rate of embryonal development was not impaired by hydrosalpinx fluid (at either 50 or 100% concentration). In the absence of a demonstrable detrimental effect we suggest that the low implantation rate in hydrosalpinx IVF patients may not be due to an embryotoxic effect. We further suggest that constant passage of fluid into the uterine cavity in these patients could possibly introduce some mechanical interference that may result in implantation failure.     

Culturing human embryos with and without glucose

OBJECTIVE: To review published data and to compare pregnancy rates (PRs) after culturing human embryos with and without glucose and phosphate. DESIGN: Comparison of results from various programs. SETTING: Assisted Reproductive Technology Program. PATIENT(S): Patients were enrolled in various studies. INTERVENTION(S): Human embryos were cultured with and without glucose and phosphate. MAIN OUTCOME MEASURE(S): Pregnancy rates after different techniques of embryo culture. RESULT(S): Some studies reported higher PRs in patients undergoing IVF after embryos were cultured in media without glucose and phosphate versus media with glucose and phosphate. One study showed that PRs were lower when embryos were cultured in media lacking glucose and phosphate compared with media containing glucose and phosphate. Some studies have also shown similar PRs with the two types of culture media. CONCLUSION(S): The PRs in IVF patients will not necessarily be enhanced if the embryos are cultured in media without glucose and phosphate.