Sperm functional changes and fertilization in vitro in co-culture with human skin fibroblasts

This study was undertaken to evaluate the effects of human skinfibroblast monolayers on human sperm function and fertilizationin vitro. Sperm function was evaluated using the hamster oocytepenetration assay (HOPA) and zona binding assay (ZBA) in mediumalone and in co-culture with human skin fibroblast monolayersand suspensions. The ZBA was also studied in fibroblast conditionedmedium and in bovine oviduct cell monolayers and suspensions.Fertilization was measured both in in-vitro fertilization (IVF)couples with a normal semen analysis (first study; randomized)and in IVF couples with subnormal semen analysis (second study;each patient served as its own control). The HOPA results werenot significantly different with or without fibroblasts. Inall co-culture situations and in conditioned medium the ZBAscored significantly lower than medium alone. No significantdifferences with respect to IVF were observed between the co-cultureand the control group in either study. The mean fertilizationrate per patient was 60% in the group with normal semen analysisand 25% in the group with abnormal semen analysis. From thisstudy we conclude that although co-culture with human skin fibroblastsand epithelial cells influences the results of some sperm functiontests, it does not influence fertilization in vitro.     

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.     

MCP-1在胚胎种植中的作用

趋化因子是一类对中性粒细胞、单核细胞、嗜碱性粒细胞等起趋化和激活作用的多功能糖蛋白,介导炎症和免疫反应,单核细胞趋化蛋白-1（MCP-1）属于CC类趋化因子家族的一员。近年来研究表明,在激素的调节作用下,MCP-1在胚胎种植中发挥重要作用,其机制主要为趋化并激活单核巨噬细胞,参与Th2型免疫偏移及促进血管生成。     

Differential effects of human granulocytes and lymphocytes on human fibroblasts in vitro

Polymorphonuclear leucocytes (PMN) were found to damage human fibroblast monolayers. Allogeneic and autochthonous PMN were equally efficient and monolayer destruction (plaque-formation) occurred within 24 hr, as a rule, in the absence of agglutinating substances such as phytohaemagglutinin. Living PMN were not necessary for plaque-formation, since cells heated to 56°C for 30 min or disintegrated by ultrasound were still competent to produce plaques. It is suggested that enzymes enclosed in cytoplasmic granules in the PMN are responsible for plaque-formation. Although the monolayer was destroyed, the target cells were not killed after treatment with PMN, but detached from the surface of the culture vessel into the medium and could be recultivated from the supernatant. Heparin, chondroitin sulphate and trypan blue suppressed plaque-formation by intact and disintegrated PMN, while a variety of metabolic inhibitors or X-rays had no effect.

Lymphocytes from non-immunized donors were also competent to cause destruction of fibroblast monolayers, but this reaction required the presence of phytohaemagglutinin. Heating of the lymphocytes to 48·5°C or ultrasound disintegration abolished the plaque-forming ability, suggesting that living lymphocytes were required. In contrast to PMN, lymphocytes were shown to kill the target cells. PHA-treated lymphocytes were equally cytotoxic to allogeneic and autochthonous fibroblasts and it is suggested that actual target destruction is immunologically non-specific and caused by transformed lymphocytes, which acquire the ability to kill all fibroblast targets regardless of their genotype and the event triggering lymphocyte transformation.

     

Effects of taurine on human embryo development in vitro.

Glutamine and taurine are reported to be beneficial for mouse embryo development in vitro, and we have recently shown that glutamine improves human blastocyst formation in vitro. This randomized study compared the development of supernumerary human embryos in the presence of 1 mmol/l glutamine and/or 5 mmol/l taurine from the 2-4-cell stage to the blastocyst stage. Blastocyst development and cell numbers were similar in the presence of glutamine or taurine: 52.6% and 58.3% of the embryos reached the blastocyst stage, respectively. Pyruvate uptake was similar in the presence of glutamine or taurine throughout development, as was lactate production after the 8-cell stage. Before this stage, lactate production was 4-fold higher in the presence of taurine (P < 0.001). The proportion of embryos reaching the blastocyst stage was similar with glutamine alone or with glutamine and taurine (62.5% and 47.2% respectively), as were the blastocyst cell numbers (63.0 +/- 4.6 and 61.0 +/- 5.1 respectively). In conclusion, taurine supports development of 2-4-cell human embryos to the blastocyst stage, although it does not further augment the beneficial effects of glutamine.     

Toxic effects of oxygen on human embryo development

The toxic effects of oxygen on the embryos of various animal species are reviewed. Methodologies for assessing embryonic damage are discussed and possible ways of preventing the damage are explored. Three methods of potentially minimizing oxidative damage to human embryos were tested using gametes, zygotes, and embryos from a clinical IVF programme: (i) decreasing the oxygen tension in the gas phase used for culture during insemination, fertilization, and embryo growth; (ii) changing the formulation of culture media to include some components designed to protect against oxidative damage; and (iii) reducing the duration of insemination to minimize the effect of oxidative damage caused by spermatozoal metabolism. Fertilization, cleavage, embryo utilization, pregnancy, and embryo implantation rates were used to monitor these changes. Although all three methods gave an increase in success rates, there was still a dramatic decrease in success with patient age. It is suggested that, although the system of handling and culturing embryos can be optimized with respect to embryonic mitochondrial function, there are inherent age-related defects in oocytes and embryos that are still more fundamental than the environmental conditions of the embryo.     

Fertilization and early embryology: Effect of different co-culture systems in early human embryo development

The objective of this study was to examine the effects of differentculture systems on the development of early human embryos invitro. A total of 460 fertilized oocytes from 82 cycles of patientswere transferred into one of four systems: (1) into dropletsof Ham's F10 medium+12% normal human serum (NHS); (2) co-culturedon a human granulosa monolayer; (3) co-cultured with bovineoviductal epithelial cells (BOEC); or (4) co-cultured with bovineuterine epithelial cells (BUEC). The percentage of deavage andthe morphological appearance of embryos were recorded dailyfor 72 h in each system using an inverted phase-contrast microscope.The results showed that the proportions of the fertilized oocyteswhich developed to the four-cell stage 48 h after retrievalwere, by culture system: (1) 70% (84/120); (2) 74% (85/115);(3) 78% (91/117); and (4) 76% (82/108). At 72 h after retrieval,the proportions of the eight-cell stage were, by culture system:(1) 45% (38/84); (2) 62% (53/85); (3) 75% (68/91); and (4) 70%(57/82). We conduded that a higher proportion of fertilizedoocytes developed to embryos at the eight-cell stage in systems2, 3 and 4 than in system 1. This indicates the beneficial effectof co-culture of human embryos with granulosa cell, BOEC andBUEC monolayers, which may be due to various factors.     

细胞因子抑制剂吡非尼酮对体外培养人瘢痕成纤维细胞增殖的影响

背景：有研究表明细胞因子抑制剂吡非尼酮通过调控多种细胞因子,抑制成纤维细胞的生物学活性,其在内脏器官的抗纤维化作用的研究和应用取得了良好的进展,但对于皮肤增生性瘢痕及成纤维细胞是否有影响及其机制尚不清楚。 目的：观察细胞因子抑制剂吡非尼酮对人增生性瘢痕成纤维细胞增殖的影响。 方法：采用组织块法培养人增生性瘢痕成纤维细胞,实验取第3-6代生长状态良好的对数生长期细胞。根据吡非尼酮不同质量浓度分为对照组(吡非尼酮0 g/L),吡非尼酮0.15,0.3,1 g/L组,共干预12,36,48 h。 结果与结论：MTT,反转录-聚合酶链式反应和酶链免疫吸附实验结果显示,与对照组相比,吡非尼酮0.15,0.3,1 g/L组细胞增殖情况、转化生长因子β1 mRNA的表达、Ⅰ、Ⅲ型胶原蛋白的分泌量均降低(P < 0.05),其中吡非尼酮1 g/L组降低最明显(P < 0.05)。干预24,48,72 h后,吡非尼酮0.15,0.3,1 g/L组间细胞增殖抑制率、Ⅰ、Ⅲ型胶原蛋白的分泌量均差异有显著性意义(P < 0.05)。结果证实,细胞因子抑制剂吡非尼酮对体外培养的人增生性瘢痕成纤维细胞胶原蛋白的分泌、转化生长因子β1的表达及细胞增殖活性有明显的抑制作用。 中国组织工程研究杂志出版内容重点：干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程     

人子宫内膜蜕膜化间质细胞与囊胚体外共培养建立人胚胎着床模型

目的建立有效的体外人胚胎着床模型,为体外研究人胚胎着床过程提供条件。方法将人子宫内膜蜕膜化的间质细胞与囊胚共同培养,光镜下观察囊胚在细胞上的定位、粘附及侵入过程;免疫荧光法测定共同培养系统中的角蛋白,以确定着床模型的建立。结果与蜕膜细胞共培养5—10h后,囊胚开始黏附在细胞层上,48h后侵入蜕膜细胞间;共培养48h后,胚胎及周围的内膜细胞表达角蛋白阳性。结论囊胚与蜕膜化细胞共同培养,可以成功建立体外胚胎着床模型,更好地模拟体内着床时状态,是较理想的体外研究模型。     

The role of the endometrium and embryo in human implantation

Despite many advances in assisted reproductive technologies (ART), implantation rates are still low. The process of implantation requires a reciprocal interaction between blastocyst and endometrium, culminating in a small window of opportunity during which implantation can occur. This interaction involves the embryo, with its inherent molecular programme of cell growth and differentiation, and the temporal differentiation of endometrial cells to attain uterine receptivity. Implantation itself is governed by an array of endocrine, paracrine and autocrine modulators, of embryonic and maternal origin. Implantation failure is thought to occur as a consequence of impairment of embryo developmental potential and/or impairment of uterine receptivity and the embryo-uterine dialogue. Therefore a better comprehension of implantation, and the relative importance of the factors involved, is warranted. New techniques for monitoring changes in the endometrium and/or the embryo at the level of gene regulation and protein expression may lead to the identification of better markers for implantation. Moreover, the use of predictive sets of markers may prove to be more reliable than a single marker. Continuing refinements to ART protocols, such as optimizing ovarian stimulation regimens, the timing of human chorionic gonadotrophin injection, or the timing of embryo transfer, should help to increase implantation rates further.     

Improved development of human embryos in vitro by a human oviductal cell co-culture system.

This study was undertaken to determine the effect of co-culture with human oviductal cells on human embryos. Spare embryos from gamete intra-Fallopian transfer (GIFT), pronuclear stage transfer (PROST) and in-vitro fertilization/embryo transfer (IVF/ET) programmes were either cultured in serum-supplemented Earle's balanced salt solution alone, or co-cultured in the same solution with oviductal cells from the pronuclear stage (day 1 post-insemination) or two- to four-cell stage (day 2 post-insemination). The co-cultured embryos appeared to have a higher developmental potential (higher rate of blastocyst formation and lower fragmentation rate), although there was no statistical difference in their rate of development, degree of fragmentation and stages attained, when compared with conventionally cultured embryos. The percentage of hatching blastocysts was significantly higher (P less than 0.05, Fisher's exact test) for embryos co-cultured from day 1 post-insemination (38%) than for embryos which had not been co-cultured (7%). The blastocyst hatching rate for embryos co-cultured from day 2 post-insemination was 15%. It was therefore concluded that co-culture of human embryos with oviductal cells could improve the development of the embryos in vitro. The degree of improvement was more pronounced when the co-culture started at an earlier stage.     

Implication of HLA-G in human embryo implantation

The mechanisms underlying the processes of human embryo implantation and maternal immunotolerance to the fetus remain incompletely understood. Growing evidence indicates that one of the nonclassic human leukocyte antigen (HLA) genes, HLA-G, is expressed selectively on the surface of the extravillous throphoblast and plays an important role in the adaptations of the maternal immune system to pregnancy; however the implication of this molecule in the process of human embryo implantation is also plausible. Therefore the aim of the present article is to review the available literature specifically focused on the possible relationship between HLA-G and human embryo implantation. In particular, studies investigating HLA-G expression on human preimplantation embryos and in the endometrium, as well as its levels in embryo culture supernatants and circulating maternal blood, are discussed.     

3Development of an in vitro model for human embryo implantation and progesterone regulation

Aim:  Develop an in vitro 3D-model for human embryo implantation on receptive endometrial construct.
Material and Methods:  Three-dimensional in vitro endometrial construct (n=12) was developed by culturing isolated human endometrial stromal cells in collagen matrix overlaid with epithelial cells. After priming hormonally, human blastocysts were placed and cultured in alpha-medium. Attachment of embryo was confirmed using cytokeratin-7 and expression of endometrial receptivity markers using immunohistochemistry. Antiprogestin mifepristone was used to test implantation and receptivity.
Results:  Embryos attached to receptive endometrial construct . Epithelial and stromal cells of endometrial construct expressed ER-α, ER-β, PR-(A+B), PR-B, VEGF, LIF, IL-1beta and COX-2. Epithelial cells expressed AR, integrinα_Vβ₃ and MUC1. Mifepristone inhibited blastocyst attachment, up-regulated epithelial ER-β, PR-B; down regulated stromal VEGF, MUC1 and integrin α_Vβ₃ as observed in vivo .
Conclusion:  An in vitro human embryo implantation model, regulated by progesterone was developed and tested. This could be further used to understand molecular basis of human embryo implantation .     

Fertilization and early embryology: Granulosa cell co-culture enhances human embryo development and pregnancy rate following in-vitro fertilization

A preliminary study and related clinical trial were performedto evaluate the effects of granulosa-lutein cell co-cultureon human embryo development and pregnancy rates for in-vitrofertilization (IVF). In the study, sibling two-pronuclear zygoteswere randomly allocated to culture with (co-culture) or without(control) autologous granulosalutein cells. After 24 h, embryoswere examined for blastomere number and degree of fragmentation.Co-culture had no effect on the average number of blastomeresper embryo at 24 h; however, fragmentation was significantlydecreased in co-cultured embryos (0.7 ± 0.1) comparedwith controls (1.3 ± 0.2; P < 0.05). In the subsequentclinical trial, all two-pronuclear zygotes were co-culturedfor 48 h prior to embryo transfer. The live birth rate per embryotransfer was 43.4% with an implantation rate per embryo of 17.6%.Of the untransferred embryos, 68% developed to the blastocyststage and were cryopreserved. We conclude that the simple systemof autologous granulosa-lutein cell co-culture improves embryodevelopment, implantation and subsequent pregnancy rates forIVF.     

Vero cell co-culture counteracts the detrimental effects of hydrosalpinx fluid on the development of mouse embryos in vitro

Recent studies have suggested that the hydrosalpinx has a negative effect on pregnancy outcome, with markedly diminished implantation and increased early pregnancy loss. Fluid from the hydrosalpinx may leak into and accumulate in the uterine cavity. It is not clear, however if this creates a hostile local environment in the uterus for embryo implantation or exerts a direct embryotoxic effect. This study was conducted to investigate the detrimental effects of hydrosalpinx fluid (HSF) on the development of mouse embryos in vitro and to demonstrate whether Vero cells overcome these adverse effects. HSF was collected from three women with bilateral hydrosalpinx at the time of laparoscopic surgery. Collected fluid was centrifuged and the supernatant was frozen at -20 degrees C. For co-culture, Vero cells were commercially obtained in a frozen state and cultured using Ham's F10 medium. Single-cell mouse embryos (B6CBAF1) were cultured for 5 days in 0, 0.4, 0.8, and 1.2% of HSF in media with and without Vero cells and examined daily to record the number of embryos reaching expanded blastocyst and hatching stage. Co-culture of mouse embryos with Vero cells at 0.8% HSF concentration significantly enhanced embryo development, but not at 1.2% hydrosalpinx fluid concentration. These results suggest that HSF is highly embryotoxic and Vero cells are likely to overcome these detrimental effects to some degree.     

Fertilization and early embryology: Granulosa cells improve human embryo development in vitro

A total of 17 couples with repetitive implantation failure aftertransfer of fresh or frozen—thawed embryos had half oftheir zygotes cultured in standard conditions and frozen atday 2 after insemination, and the other half cocultured withautologous granulosa cells and transferred at the morula orblastocyst stage at day 5 or 6 after oocyte retrieval. At theend of the culture period, supernatants of cocultures were recoveredfor steroid assays. Monolayers were stained for granulosa cellgrowth and morphological assessment. We observed that granulosacells improve embryo development in vitro since 32 out of 60(53%) reached the morula stage and 18 (30%) the blastocyst stage,leading to a total of 83% embryos available for transfer (comparedwith 3% without coculture). The ongoing pregnancy rate of thesepatients who were selected because they had at least three previousimplantation failures, is only 5.9%, however, which is similarto the control group without coculture (6.3%). To conclude,granulosa cells improve embryo development but not the pregnancyrate after transfer of cocultured embryos in patients with multipleprevious implantation failures.     

Propagation of hepatitis A virus in human embryo fibroblasts

human diploid fibroblasts and human primary liver cell carcinoma cells (PLC/PRF/5) were infected with hepatitis A virus (HAV) adapted to growth in cell culture or derived directly from human stool. Viral antigen was expressed in PLC/PRF/5 cells 28 days after infection with cell culture-adapted HAV, and 50 days after infection with virus from human stool. In human fibroblasts the periods until first expression of viral antigen were 90 and 210 days, respectively. During further passages of HAV in fibroblasts the time of first appearance of antigen decreased to about 28 days. Biophysical properties of HAV extracted from infected fibroblasts were comparable to those of HAV derived directly from human stool. Immunofluorescence studies showed that the antigen was located exclusively within the cytoplasm of the infected fibroblasts. Kinetics of antigen production indicated that an equilibrium between virus multiplication and cell metabolism was reached in persistently infected fibroblasts.