A Critique on the Application of the Principle of Subsidiarity Concerning Human Embryonic Stem Cell Research in South Africa

Researchers from all around the world emphasize on the enormous possible benefits that stem cells may have for the treatment of diseases. However, this technology is considered morally problematic when the source of the stem cell is from a human embryo. Nonetheless, there is a consensus that of all the types of stem cells, hESC （human embryonic stem ceils） are the most promising for particular and important research and therapies. Yet, there are controversial issues regarding the ＂killing＂ of the human embryo for stem cell derivation. There are two general ethical conditions that should govern the instrumental use of embryo. One of them, the principle of subsidiarity, which is defined as ＂a state we have that we have to choose the less contentious means of achieving the intended goal＂. Based on this principle, we ought only to use hESC when there are no other alternatives, which are less morally controversially. Subsidiarity is based on the assumption that there is something ethically unsound about the use ofhESC. However, this principle only makes sense if it is based on consistently upheld views of the moral status of embryo, moreover, the law should also not limit or prohibit hESC research based on this principle. In this paper, I argue---using the South African law for hESC technology--that criterion for deciding which type of stem cells to use should be based on their potential and suitability for advancing scientific knowledge and development of new therapies which will be greatly beneficial in alleviating human suffering.

Effects of Fluoride on Lipid Peroxidation, DNA Damage and Apoptosis in Human Embryo Hepatocytes

Objective To investigate the effects of fluoride on lipid peroxidation, DNA damage and apoptosis in human embryo hepatocyte L-02 cells. Methods Lipid peroxide (LPO) level, reduced glutathione (GSH) content, DNA damage, apoptosis, and cell cycle analysis were measured after in vitro cultured L-02 cells were exposed to sodium fluoride at different doses (40 μg/mL, 80 μg/mL, and 160 μg/mL) for 24 hours. Results Fluoride caused an increase of LPO levels and a decrease of GSH content in L-02 cells. There appeared to be an obvious dose-effect relationship between the fluoride concentration and the observed changes. Fluoride also caused DNA damage and apoptosis and increased the cell number in S phase of cell cycle in the cells tested. There was a statistically significant difference in DNA damage and apoptosis when comparing the high dose of fluoride treated cells with the low dose of fluoride treated cells. Conclusion Fluoride can cause lipid peroxidation, DNA damage, and apoptosis in the L-02 cell experimental model and there is a significant positive correlation between fluoride concentration and these pathological changes.

Total saponins of Panax ginseng effects on proliferation and differentiation of human embryonic neural stem cells and in a Parkinson's disease mouse model

BACKGROUND： Total saponins of Panax ginseng （TSPG） exhibits neuroprotection against Parkinson＇s disease in the substantia nigra. OBJECTIVE： To investigate the effects of TSPG on human embryonic neural stem cells （NSCs） proliferation and differentiation into dopaminergic neurons using in vitro studies, and to observe NSC differentiation in a mouse model of Parkinson＇s disease, as well as behavioral changes before and after transplantation. DESIGN, TIME AND SETTING： In vitro neural cell biology trial and in vivo randomized, controlled animal trial were performed at the Institute of Basic Medical Sciences, Chongqing Medical University between September 2004 and December 2007. MATERIALS： TSPG （purity 〉 95%） was isolated, extracted, and identified by Chongqing Academy of Chinese Materia Medica. Recombinant human basic fibroblast growth factor （bFGF） and recombinant human epidermal growth factor （EGF） were purchased from PeproTech, USA. A total of 25 C57/BL6J mice, aged 18-20 weeks were included. Twenty were used to establish a Parkinson＇s disease model with i.p. injection of MPTP （1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine） and TSPG alone or combined with interleukin-1 （IL-1）-treated NSCs prior to transplantation into the corpus striatum. The remaining five mice were pretreated for 3 days with TSPG prior to MPTP injection, serving as the TSPG prevention group. METHODS： Primary NSCs were isolated, cultured and purified from embryonic cerebral cortex. Immunocytochemistry was employed to detect specific antigen expression in the NSCs. In vitro experiment： （1） to induce proliferation, NSCs were treated with TSPG, EGF＋bFGF, or TSPG＋EGF＋bFGF, respectively; （2） to induce dopaminergic neuronal differentiation, NSCs were treated with TSPG, IL-1, or TSPG＋IL-1, respectively. MAIN OUTCOME MEASURES： In vitro experiment： the effects of TSPG on NSCs proliferation were evaluated with flow cytometry and MTT assay. Tyrosine hydroxylase expression was determined by immunocytochemistry assay to observe effects of TSPG on dopaminergic neuronal differentiation. In vivo experiment： differentiation of grafted NSCs in the mouse brain was determined by immunohistochemical staining. Behavioral changes were evaluated by spontaneous activity frequency, memory function, and score of paralysis agitans. RESULTS： （1） NSCs were cultured and passaged for more than three passages. Immunocytochemistry revealed positive nestin staining, as well as neurofilament protein and glial fibrillary acidic protein. （2） TSPG significantly increased NSC proliferation, in particular when combined with EGF and bFGF, which was twice as effective as FGF or bFGF alone. TSPG also induced dopaminergic differentiation in NSCs, in particular when TSPG was added together with IL-1, resulting in an effect five times greater than that of IL-1 alone. （3） At day 30 following transplantation, most NSCs in the TSPG prevention group differentiated into dopaminergic neurons, and the scores of paralysis agitans, spontaneous activity, and memory function were significantly increased compared with TSPG alone or TSPG＋IL-1 groups （P 〈 0.05）. CONCLUSION： TSPG stimulated NSC proliferation, in particular when combined with FGF and bFGF. TSPG significantly induced dopaminergic neuronal differentiation of NSCs, and the effect was greater when combined with IL-1. In addition, TSPG greatly improved behavior in the Parkinson＇s disease mouse model following NSC transplantation. Following NSC transplantation, TSPG pretreatment exhibited superior efficacy over either TSPG alone or TSPG in combination with IL-1, in terms of behavioral improvements in the Parkinson＇s disease mouse model.

Neuroprotective effects of human telomerase reverse transcriptase on beta-amyloid fragment 25-35-treated human embryonic cortical neurons

BACKGROUND： Numerous current studies have suggested that human telomerase reverse transcriptase （hTERT） gene has neuroprotective effects and can inhibit apoptosis induced by various cytotoxic stresses; however, the mechanism of action remains unknown. OBJECTIVE： To evaluate the neuroprotective effects and possible mechanism of action of hTERT gene transfection in human embryonic cortical neurons treated with beta-amyloid fragment 25-35 （AI325-35）. DESIGN, TIME AND SETTING： The randomized, controlled and molecular biological studies were performed at the Department of Anatomy and Brain Research, Zhongshan School of Medicine, Sun Yat-sen University, China, from September 2005 to June 2008. MATERIALS： AdEasy-1 Expression System was gifted by Professor Guoquan Gao from Sun Yat-Sen University, China. Human cortical neurons were derived from 12-20 week old aborted fetuses, obtained from the Guangzhou Maternal and Child Health Hospital, China. Mouse anti-Odk5 and mouse anti-p16 monoclonal antibodies （Lab Vision, USA）, and mouse anti-hTERT monoclonal antibody （Epitomics, USA）, were used in this study. METHODS： （1） Recombinant adenovirus vectors, encoding hTERT （Ad-hTERT） and green fluorescent protein （Ad-GFP）, were constructed using the AdEasy-1 Expression System. Human embryonic cortical neurons in the Ad-hTERT group were transfected with Ad-hTERT for 1-21 days. Likewise, human embryonic cortical neurons in the Ad-GFP group were transfected with Ad-GFP for 1-21 days. Human embryonic cortical neurons in the control group were cultured as normal. （2） Human embryonic cortical neurons in the Ad-hTERT group were treated with 10 pmol/L Aβ25-35 for 24 hours. Normal human embryonic cortical neurons treated with 10 pmol/Lβ25.35 for 24 hours served as a model group. Human embryonic cortical neurons in the Ad-GFP and control groups were not treated with Aβ25-35. MAIN OUTCOME MEASURES： Expression of hTERT in human embryonic cortical neurons was evaluated by immunocytochemical staining and Western blot assay. Telomerase activity was measured using a PCR-based telomeric repeat amplification protocol （TRAP） ELISA kit. Neural activity in human embryonic cortical neurons was examined by MTT assay; apoptosis was measured using TUNEL assay; and Cdk5 and p16 protein expressions were measured by Western blot. RESULTS： Expression of hTERT protein was significantly increased and peaked at day 3 post-transfection in the Ad-hTERT group. No hTERT expression was detected in the Ad-GFP and control groups. Telomerase activity was significantly greater in the Ad-hTERT group compared with the Ad-GFP and control groups （P 〈 0.01）. Compared with the control group, cell activity was significantly decreased （P 〈 0.05）, and cell apoptotic rate, Cdk5 and p16 expression were significantly increased （P 〈 0.01） in the model group. Compared with the model group, cell activity was increased in the Ad-hTERT group, and peaked at day 3 post-transfection （P 〈 0.05）. Neuroprotective effects also peaked at day 3 post-transfection; and the apoptotic rate, Cdk5 and p16 expression significantly decreased （P 〈 0.01）. CONCLUSION： Expression of hTERT in human embryonic cortical neurons can relieve Aβ25-35-induced neuronal apoptosis. The possible mechanism by which hTERT produces these neuroprotective effects may be associated with inhibition of Cdk5 and p16 expression.

Human cytomegalovirus induces alteration of β-actin mRNA and microfilaments in human embryo fibroblast cells

Objective: To investigate the infection of human embryo fibroblast cell line HF cells by CMV as well as the effects of CMV on β-actin mRNA and microfilaments. Methods: HF cells shape was observed after the infection of CMV.RT-PCR assay was used to detect the mRNA expression of CMV immediate early (IE) gene, β-actin and GAPDH genes of HF cells infected by CMV. CMV particles and cell microfilaments were detected with electron microscope. Results: Shape of HF cell changed after the infection by CMV. HF cells infected by CMV could express IE mRNA and the expression of β-actin mRNA decreased in a time-and titer-dependent manner compared with the uninfected HF cells whose expression of GAPDH mRNA did not change much. CMV particles were found with electron microscope in the cells. Microfilaments were ruptured and shortened after the infection of CMV. Conclusion: CMV can not only infect human embryo fibroblast cells line HF cells and replicate in the cells, but can also affect the expression of β-actin mRNA and the microfilaments.

Location and expression of neurotrophin-3 and its receptor in the brain of human embryos during early development

BACKGROUND： Cell culture in vitro trials have demonstrated that neurotrophin-3 （NT-3） can enhance the survival of sensory neurons and sympathetic neurons, and can also support embryo-derived motor neurons. This effect is dependent on nerve growth factor on the surface of cells. Understanding the role of NT-3 and its receptor in the early development of human embryonic brains will help to investigate the correlation between early survival of nerve cells and the microenvironment of neural regeneration. OBJECTIVE： To observe the proliferation of cerebral neurons in the development of human embryonic brain, and to investigate the location, expression and distribution of NT-3 and its receptor TrkC during human brain development. DESIGN, TIME AND SETTING： An observation study on cells was performed in the Department of ttuman Anatomy, Histology and Embryology, Chengdu Medical College in September 2007. MATERIALS： Fifteen specimens of flesh human embryo, aged 6 weeks, were used in this study. METHODS： The proliferation of cerebral neurons was detected using proliferating cell nuclear antigen, and the immunocytochemistry ABC technique was applied to observe the location, expression and distribution of NT-3 and its receptor TrkC in the brain of the human embryo. MAIN OUTCOME MEASURES： Location, expression and distribution of NT-3 and its receptor in the brain of the human embryo. RESULTS： In the early period （aged 6 weeks） of human embryonic development, proliferating cell nuclear antigen-positive reactive substances were mainly observed in the nucleus of the forebrain ventricular zone and subventricular zone, and the intensity was stronger in the subventricular zone than the forebrain ventricle. NT-3 positive reactive substance was mainly distributed in the cytoblastema of the forebrain neuroepithelial layer and nerve cell process, while TrkC was mainly distributed in the cell membrane of the forebrain ventricular zone and subventricular zone. During embryonic development, NT-3 and TrkC showed a positive immune reaction to a greater or lesser extent in ependymal epithelium. CONCLUSION： During early human embryonic development, cerebral nerve cells proliferate in the ventricular zone and subventricular zone, and NT-3 is expressed in the neural axon. The results show that the highly expressed NT-3 could promote the proliferation of neural axons and maintain the neuron body＇s survival.

Apoptotic gene expression in the neural tube during early human embryonic development

Neural tube development comprises neural induction, neural epithelial cell proliferation, and apoptosis, as well as migration of nerve cells. Too much or too little apoptosis leads to abnormal nervous system development. The present study analyzed expression and distribution of apoptotic-related factors, including Fas, FasL, and caspase-3, during human embryonic neural tube development. Experimental results showed that increased caspase-3 expression promoted neural apoptosis via a mitochondrial-mediated intrinsic pathway at 4 weeks during early human embryonic neural tube development. Subsequently, Fas and FasL expression increased during embryonic development. The results suggest that neural cells influence neural apoptosis through synergistic effects of extrinsic pathways. Therefore, neural apoptosis during the early period of neural tube development in the human embryo might be regulated by the death receptor induced apoptotic extrinsic pathways.

Human Embryo Neuronal Culture <i>in Vitro</i>: A Model to Study Cellular Physiology, Receptors, Power and Toxicity of Cytostatic Drugs for Human Use

Neural cells cultures from human embryo brain of 9° - 11°W gestational age have been used to study ERα (Estrogens Receptor α) and to perform toxicity test for Mitomycin C and Methotrexate. Histochemical confirmation of cellular neuronal phenotype was based on histochemical evidence of NSE (Neuron Specific Enolase).The detection of ERα in neuronal cells was performed with a rabbit Monoclonal Antibody. ERα was absent both on neurons grown in vitro and on tissue brain specimens. This finding is apparently in contrast with the positive immunoreactivity of ERα and ERβ reported by other Authors on foetal and adult CNS (Central Nervous System). The absence of nuclear ERα on neurons in culture and in brain tissue specimens in our experiment is not in contrast with the relevant physiologic role of estrogens on nervous central system, but it could be correlated to the embryonic period of life and could represent a protection of male brain from an undue estrogens imprinting. The mitomycin C, alkylation agent, has shown in our experiment a major neurotoxic and cytostatic power in comparison with methotrexate. Our conclusion is that human embryo neuronal culture in vitro is a powerful instrument for physiology and human therapy for cancer and neurodegenerative diseases.

Correction of β-thalassemia mutant by base editor in human embryos

β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 （A〉G） mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor （BE） system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 （A〉G） mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 （A〉G） homozygous mutation. Data showed that base editor could precisely correct HBB -28 （A〉G） mutation in the patient＇s primary cells. To model homozygous mutation disease embryos, we consb＇ucted nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured （IVM） oocytes.Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.

Exploring the role of autophagy during early human embryonic development through single-cell transcriptome and methylome analyses

Early human embryogenesis is a very sophisticated process due to its unique gene regulatory network.Autophagy has been suggested to play an important role in mediating the development of early embryonic cells in mammals.However,evidence showing how autophagy regulates early human embryogenesis remains to be further explored.In this study,we systematically investigated the human transcriptome and methylome patterns of autophagy-related(ATG)genes in early embryonic cells at single-cell resolution.We analyzed the transcriptomic data of 365 cells and methylome data of 265 cells.The results showed that most ATG genes remained epigenetically active and were expressed stably throughout early embryogenesis,whereas the dynamics varied among different developmental stages.This evidence indicated that the autophagy pathway was constitutively activated and exerted a fundamental role in early human embryo development.Our work,for the first time,comprehensively reveals the features of autophagy during early human embryo development.

Co-Culture of Early Embryo with Human Decidual Stromal Cells in vitro by Improvement of Early Embryo Development

Summary: An early embryo co-culture system with human decidual stromal cells was established to study its effect on early embryonic cleavage and growth in vitro. Three hundred and eight 2-cell mouse embryos were co-cultured with human decidual stromal cell monolayer in MEM+0. 4 % bovine serum albumin (BSA) and 163 embryos cultured in MEM+15 % FCS alone as control. Among the mouse 2-cell embryos co-cultured with human decidual stromal cells, 72.73 % developed to the morula stage and 67.21 % cavitated to blastocysts with 59. 74 % hatching, as compared with 61. 34 % to morula stage, 48. 47 % to blastocysts and none hatching in the controls, respectively. Co-cultured embryos cleaved slightly faster than controls and showed no or less fragmentation than those in the control. These results suggested that human decidual stromal cells can support early embryonic development and yield a reasonable number of embryos with good quality up to blastocyst stage.

Estimating zona pellucida hardness under microinjection to assess oocyte/embryo quality: Analytical and experimental studies

The precise determination of zona pellucida (ZP) hardness is largely unknown due to the lack of appropriate measuring and modelling methods. In this study, we have used experimental and theoretical models to describe the mechanical behavior of a single oocyte cell to improve the assisted reproductive technology (ART) outcomes by assessing oocyte/embryo quality. This paper presents the development of: i) a microinjection model to estimate the force of ZP penetration, ii) a micropipette aspiration model to determine the corresponding hardness, and iii) an experimental procedure to generate the required data for these two models. Our results show that the estimated penetration force provides a performance target for the penetration process during intracytoplasmic sperm injection (ICSI), while the estimated corresponding hardness serves as an indicator of the extent of deformation sustained by the oocyte prior to penetration. Evaluation of these results shows that a routine assessment of ZP hardness under microinjection would allow for the identification of certain oocyte pools for which further manipulation is recommended in order to improve injection, hatching and finally ART outcomes.

Evaluation on Sensitivity of the Human Sperm Motility Assay for Detecting Endotoxin in Culture Medium

Objective To investigate the sensitivity of the human sperm motility assay for detecting endotoxin in culture mediumMaterials &. Methods Motile sperm were separated and exposed to different concentrations of endotoxin (0.5 ng/mL, 1ng/mL, 10ng/mL, 1000ng/mL, 10 000ng/ mL, and 50 000ng/mL), and sperm motility was determined after incubation. Effects of endotoxin on sperm motility in media without albumin were also examined. In addition, at the same concentrations of endotoxin (0. 5ng/mL, 1 ng/mL, and 10 ng/ mL ) , the sensitivity of the human sperm motility assay was compared to those of 1-cell and 2-cell mouse embryo bioassays.Results At levels of 0. 5ng/mL-1000ng/mL endotoxin in media with 2mg/mL albumin, sperm did not show significant change in motility during 24 h of incubation when compared with the control (P>0. 05). However, the sperm motility was significantly inhibited at endotoxin dosages of 10 000 and 50 000 ng/mL. In the absence of albumin supplementation, at endotoxin levels of 50 000ng/mL, and 1 000ng/mL, there was a marked decrease in sperm motility compared with the control after 2 h or 8 h of incubation, respectively (P<0. 01). In media containing 0. 5 ng/mL and 1 ng/ mL endotoxin, 1-cell and 2-cell mouse embryos had significantly reduced developmental rates in all developmental stages, and at the level of 10ng/mL, the development of the embryos was arrested. Conclusion The human sperm motility assay could detect high levels of endotoxin in culture medium but its sensitivity to endotoxin would be inferior to that of the 1-cell or 2-cell mouse embryo bioassay. In the absence of albumin supplementation, the sensitivity of the sperm motility assay could be improved.

Evaluation of Endotoxin in Culture Medium for Human in vitro Fertilization

To determine whether the presence of bacterial endotoxin in the commercial culture media utilized for human in vitro fertilization （IVF）, and evaluate the difference in detecting endotoxin in culture medium between the human sperm motility assay and the 2-cell mouse embryo assay. Methods Thirty-six batches of culture media commonly used in IVF laboratories from 3 manufacturers were determined for thepresence ofendotoxin before using the medium for the assisted reproductive programs （group A）. After being used, 25 specimens among above media were also tested （group B）. The chromogenic limulus amoebocyte lysate （LAL） test was used for quantification the content of endotoxin. In addition, the human sperm motility assay was compared with the 2-cell mouse embryo assay to evaluate the difference in detecting endotoxin in culture medium. Results Endotoxin was not detected in group A. However, 2 samples were positive in group B, Sperm did not show significant change in motility in group A during 24 h of incubation when compared with the control （P〉0. 05）. However, in group A the 2-cell embryo development to blastocyst was suppressed in 3 batches of media. Conclusions Regular screening of each batch of culture medium should be performed if possible although there was no evidence of endotoxin contamination in commercially prepared pre-tested media. Culture environment should be stringently controlled in case the medium is polluted. The sensitivity of the sperm motility assay was lower than that of the mouse embryo assay for detecting low levels of endotoxin or toxic compounds in the medium.

A Plasmid vector encoding functional human keratinocyte growth factor gene in vitro—Functional human KGF gene expression in vitro

In this study, we cloned human KGF (hKGF) genes using RT-PCR techniques and developed a eukaryotic expression plasmid vector capable of directing the expression of functional hKGF. Monolayer culture of human embryo lung fibro-blast (HLF) was used for isolation of total RNA. Then the total RNA was purified and reverse- transcribed into cDNA using an oligo (dT) primer. A full PCR fragment for hKGF was generated and cloned. Restriction digestion and nucleo-tide sequence analysis validated the complete hKGF transcription. The hKGF cDNA fragment was inserted into pEGFP-C2 vector by means of recombinant DNA technology and verified by restriction analysis and sequencing. We have constructed pEGFP-C2-hKGF encoding the green fluorescent protein (GFP). Furthermore, hKGF had the effect on AEC II proliferation. These results suggest that the potential appli-cation of a hKGF plasmid of gene expression should be useful for sustained AEC proliferation, and its in vivo efficacy needs to be validated. Keywords:

Predictive Value of Initial Serum Human Chorionic Gonadotropin Levels for Pregnancies after Single Fresh and Frozen Blastocyst Transfer

As one of the earliest markers for predicting pregnancy outcomes, human chorionic gonadotropin(h CG) values have been inconclusive on reliability of the prediction after frozen and fresh embryo transfer(ET). In this retrospective study, patients with positive h CG(day 12 after transfer) were included to examine the h CG levels and their predictive value for pregnancy outcomes following 214 fresh and 1513 vitrified-warmed single-blastocyst transfer cycles. For patients who got clinical pregnancy, the mean initial h CG value was significantly higher after frozen cycles than fresh cycles, and the similar result was demonstrated for patients with live births(LB). The difference in h CG value existed even after adjusting for the potential covariates. The area under curves(AUC) and threshold values calculated by receiver operator characteristic curves were 0.944 and 213.05 m IU/m L for clinical pregnancy after fresh ET, 0.894 and 399.50 m IU/m L for clinical pregnancy after frozen ET, 0.812 and 222.86 m IU/m L for LB after fresh ET, and 0.808 and 410.80 m IU/mL for LB after frozen ET with acceptable sensitivity and specificity, respectively. In conclusion, single frozen blastocyst transfer leads to higher initial h CG values than single fresh blastocyst transfer, and the initial h CG level is a reliable predictive factor for predicting IVF outcomes.

Serum Beta-hCG of 11 Days after Embryo Transfer to Predict Pregnancy Outcome

Objective To assess the clinic value of a single maternal serum beta-human chorionic gonadotropin （β-hCG） assay 11 d after embryo transfer in ART pregnancies and to predict pregnancy outcome. Methods A total of 384 pregnancies after embryo transfer were included. Inviable pregnancies were defined as biochemical pregnancies, ectopic pregnancies and first trimester abortions. Ongoing pregnancies were defined as singleton pregnancies and multiple pregnancies whose gestation were achieved more than 12 weeks. Serum β- hCG concentrations were compared among different groups. Results On the post embryo transfer d 11, the mean β-hCG concentration of the ongoing pregnancy group （323. 7±285.2 mIU/ml） was significantly higher than that of the inviable pregnancy group （81.4±68.1 mmIU /ml） （P〈 0. 001）. In multiple gestations, the levels of β-hCG were significantly higher compared with singleton pregnancies. If the β-hCG level was between 10 mIU/ml and 50 mIU/ml, the positive predictive value of biochemical pregnancies and ectopic pregnancies was 81.8%, the negative predictive value was 94.4%. If the level was less than 100 mIU/ml, the positive predictive value of first trimester abortions was 80.8%, the negative predictive value was 77.8%. If the level was greater than 250 mIU/ml, the positive predictive value of multiple pregnancies was 83.3%, the negative predictive value was 74.4%. Conclusions A single serum β-hCG level on d 11 after embryo transfer has good predictive value for clinical pregnancy outcome in controlled ovarian stimulation cycles and helps to plan the subsequent follow-up.

Physiological Roles of Platelet-activating Factor in Mammalian and Human Reproduction

This review described origination, biosynthesis and functions of platelet-activating factor （PAF） in the reproductive system of mammals and human beings. The article mainly focused on biological roles of the phospholipid mediator in sperm fertilization and embryonic implantation. As an autocrine product of sperm and embryos, PAF markedly stimulates sperm motility and fertilization and serves as a capacitation factor in a ligand-receptor manner, After fertilization, embryo-derived PAF improves its own development, especially from fertilized ova to blastocyst stage and is thought to act as an embryo growth factor in the same manner as on sperm. Its mechanism of action was also clarified. At the end, it was presented some advances in its clinical application, followed by discussion of some issues possibly concerning in its current application.

Sensitivity of sperm motility assay for detecting endotoxin effect on human sperm in vitro

Objective: To evaluate the sensitivity of sperm motility assay for detecting the endotoxin effect on human sperm in vitro. Methods: Motile human sperm were separately incubated for up to 24 hours with different concentrations of endotoxin (0.5, 1, 10, 1000, 10 000 and 50 000 ng/mL). Then the sperm motility was determined. The effect of endotoxin on the sperm motility in media without albumin was also determined. In addition, at the endotoxin concentrations of 0.5, 1 and 10 ng/mL, the sensitivity of the assay was compared to those of 1-cell and 2-cell mouse embryo bioassays. Results: At levels of 0.5-1 000 ng/mL endotoxin in media with 2 mg/mL albumin, sperm did not show significant change in motility after 24 h of incubation (P>0.05), while it was significantly inhibited at endotoxin levels of 10 000 and 50 000 ng/mL. In media without albumin, endotoxin levels of 50 000 and 1 000 ng/mL, markedly inhibited the sperm motility after 2 or 8 h of incubation (P<0.01). With media containing 0.5 and 1 ng/mL endotoxin, there was a significant reduction in the development rate at all developmental stages with 1-cell and 2-cell mouse embryo assays and at the level of 10 ng/mL, the embryo development was completely arrested. Conclusion: The sperm motility assay could detect high levels of endotoxin effect in vitro, but its sensitivity is low as compared with the 1-cell or 2-cell mouse embryo bioassay.

拮抗剂方案双扳机次日血清β-hCG水平对新鲜胚胎移植结局的影响

目的:探讨拮抗剂方案双扳机次日血清β-人绒毛膜促性腺激素(β-human chorionic gonadotropin,β-hCG)水平对体外受精/卵胞质内单精子注射(in vitro fertilization/intracytoplasmic sperm injection,IVF/ICSI)新鲜胚胎移植周期妊娠结局的预测价值。方法:回顾性分析2017年1月-2024年1月于南京医科大学附属妇产医院生殖中心行拮抗剂方案促排卵患者共717个周期的临床资料,所有周期均采用双扳机(hCG+促性腺激素释放激素激动剂)诱导排卵,按照扳机次日血清β-hCG水平分为5组,A组(42个周期)β-hCG≤50 U/L,B组(282个周期)50 U/L<β-hCG≤100 U/L,C组(250个周期)100 U/L<β-hCG≤150 U/L,D组(92个周期)150 U/L<β-hCG≤200 U/L,E组(51个周期)β-hCG>200 U/L。比较各组的促排卵实验室结局及妊娠结局。结果:5组间体质量指数、基础卵泡刺激素、促性腺激素天数及总量、hCG日雌孕激素水平及高评分囊胚数的差异均有统计学意义(均P<0.05),其中体质量指数越高,扳机次日β-hCG水平越低。5组可利用胚胎数、高评分囊胚率、临床妊娠率、流产率及活产率比较,差异均无统计学意义(均P>0.05)。结论:在拮抗剂方案新鲜胚胎移植周期中,双扳机次日血清β-hCG水平并不能预测IVF/ICSI治疗周期的妊娠结局。