Comparison of embryo implantation potential between time-lapse incubators and standard incubators: a randomized controlled study.

RESEARCH QUESTION: What are the potential clinical benefits of embryo culture and assessment in a time-lapse incubator compared with a standard incubator using static assessment? DESIGN: This large multicentre, single-blinded, randomized controlled study included 1224 participants randomly assigned (1:1) to the time-lapse or standard incubator group. In all patients one or two embryos were transferred on day 3. The primary outcome was the implantation rate in the first embryo transfer cycle. Secondary outcomes included the cumulative implantation rate, live birth rate in the first embryo transfer cycle and cumulative live birth rate. RESULTS: Among 1224 participants recruited, 1182 underwent embryo transfer. The number of successfully implanted embryos in the first transfer cycle was significantly higher in the time-lapse incubator group (time-lapse group: 52.35%, standard incubator group: 47.11%, P = 0.014). The implantation rate in the first embryo transfer cycle was still significantly higher in the time-lapse group than the standard incubator group after adjusting for age, body mass index, medical centre and embryo status (relative risk 1.11, 95% confidence interval 1.02-1.20, P = 0.020). However, the cumulative implantation rate, live birth rate in the first embryo transfer cycle and cumulative live birth rate were not statistically different between the groups. CONCLUSIONS: The implantation rate in the first embryo transfer cycle was significantly improved in the time-lapse group, but the effect of the time-lapse system on the cumulative implantation rate or cumulative live birth rate was not significant. The embryo assessment method offered by time-lapse systems rather than an undisturbed environment may play an important role in improving the implantation rate in the first embryo transfer cycle. These results are only applicable to young patients.

Effect of ambient air pollutants on in vitro fertilization-embryo transfer pregnancy outcome in Zhengzhou, China.

With the acceleration of China's urbanization and industrialization, air pollution has become a major environmental problem. Retrospective data analysis of 6564 patients who underwent IVF-ET in the center for reproductive medicine of the First Affiliated Hospital of Zhengzhou University from 2015 to 2020. Different stages were selected from 90 days before oocyte retrieval to 35 days after transfer and divided into five exposure periods. Multivariate logistic regression was used to analyze the relationship between six ambient air pollutants (PM2.5, PM10, NO2, SO2, CO and O3) and the IVF-ET pregnancy outcome. The results showed that air pollutants can significantly affect the IVF pregnancy outcome. The harmful effects of ambient air pollutants are more obvious in the patients aged < 35 years, single embryo transfer and cleavage stage embryo transfer.

Propranolol inhibits infantile hemangioma by regulating the miR-424/vascular endothelial growth factor-A (VEGFA) axis.

BACKGROUND: Infantile hemangioma (IHA) is the most common tumor in infancy. We aimed to explore the effect of propranolol on the expression of microRNA (miR)-424 in IHA tissues and XPTS-1 cells, as well as its molecular mechanism of inhibiting XPTS-1 cell activity. METHODS: Tumor tissues and peritumoral tissue were collected from 13 IHA patients in Lishui Municipal Central Hospital. The level of miR-424 were detected using real-time quantitative reverse transcription polymerase chain reaction (RT-PCR). Cell counting kit-8 (CCK-8) was used to measure XPTS-1 cell viability. Flow cytometry and transwell were used to detect the apoptosis level and invasion ability of XPTS-1 cells. Western blot was used to measure the protein level of vascular endothelial growth factor-A (VEGFA). The luciferase reporter gene assay detected the targeting relationship between miR-424 and VEGFA. RESULTS: Compared with normal tissues and human umbilical vein endothelial cells, the expression level of miR-424 in IHA tissues and XPTS-1 cells was significantly reduced (P<0.05). As the concentration of propranolol increased, XPTS-1 cell viability gradually decreased (P<0.05), and the expression level of VEGFA decreased (P<0.05). The expression of miR-424 increased with the time of propranolol treatment (P<0.05). Compared with the control group, treatment with an miR-424 inhibitor resulted in a significant increase in XPTS-1 cell viability and invasion ability (P<0.05), and a decrease in apoptosis (P<0.05). However, both propranolol and miR-424 inhibitor treatment resulted in a partial decrease in XPTS-1 cell viability (P<0.05), and a partial increase in the level of apoptosis (P<0.05). MiR-424 directly targeted VEGFA; the overexpression of miR-424 resulted in a decrease in the VEGFA protein level (P<0.05), while inhibition of miR-424 resulted in an increase in the VEGFA protein level (P<0.05). Compared with the propranolol group, the XPTS-1 cell viability and invasion ability in the propranolol + VEGFA-si group were significantly decreased (P<0.05), while the level of apoptosis increased (P<0.05). Meanwhile, simultaneous miR-424 inhibitor treatment resulted in no difference in cell viability and apoptosis levels compared with the propranolol group, and the invasion ability was partially restored (P<0.05). CONCLUSIONS: Propranolol affects the malignant biological behavior of IHA cells by regulating the miR-424/VEGFA axis.

Analysis of Related Causes for No Embryos Transferred and Corresponding Coping Measures in Assisted Reproductive Technology.

OBJECTIVE: To analyze the related causes for no embryos transferred in assisted reproductive technology (ART) in order to provide corresponding coping measures for infertile couples. STUDY DESIGN: The data of 607 couples who underwent ART and had no embryos transferred in our reproductive center between January 2010 and January 2014 were retrospectively analyzed. RESULTS: The cycles of no embryos transferred accounted for 3.99% (607/15,224) of total cycles. Of those, complete fertilization failure, oocyte retrieval failure, and complete abnormal fertilization accounted for 28.3% (172/607), 25.7% (156/607) and 22.24% (135/607), respectively. The incidence of complete abnormal fertilization was higher in IVF than in ICSI (p<0.05). In both IVF and ICSI cycles, the incidences of no embryos transferred were higher in the patients retrieving ≤3 oocytes than in the patients retrieving >3 oocytes (p<0.05). In IVF cycles the incidences of no embryos transferred were higher in the patients with primary infertility than in those with secondary infertility (p<0.05). CONCLUSION: The main causes of no embryos transferred are complete fertilization failure, oocyte retrieval failure, and complete abnormal fertilization. Retrieving adequate number of mature oocytes is the key to success of ART. Patients who experienced complete abnormal fertilization in IVF or the patients with primary infertility who experienced complete fertilization failure or normal fertilization without cleavage should receive ICSI in the next treatment.

Reduced microRNA-188-3p expression contributes to apoptosis of spermatogenic cells in patients with azoospermia.

BACKGROUND AND AIMS: Human mutL homologl (MLH1) works coordinately in sequential steps to initiate repair of DNA mismatches, and aberrant MLH1 expression is related to spermatogenetic malfunction. In the present study, MLH1 expression in patients with azoospermia was investigated, and moderating effects of miR-188-3p on MLH1 expression and spermatogenesis were identified. METHODS: Testicular tissues from 16 patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA), and tissues of eight healthy patients were collected. Real-time PCR, Western blotting and immunohistochemical staining were used to detect MLH1 expression. Chromatin immunoprecipitation assay and luciferase reporter assay were performed to evaluate histone acetylation level of miR-188-3p and relationships between miR-188-3p and MLH1. RESULTS: Testicular MLH1 expression at mRNA and protein levels was significantly increased, while miR-188-3p expression was lower in patients with OA and NOA than that in controls. Reduced histone acetylation level of miR-188-3p promoter was observed in patients with azoospermia. Overexpression/inhibition of HDAC1, but not HDAC2, contributed to the significant reduction/increase of miR-188-3p expression. miR-188-3p targeted 3' UTR of MLH1 and regulated MLH1 expression. miR-188-3p inhibitor led to elevation of apoptotic level of spermatogenic cells in mice, while this effect was reversed by si-MLH1. CONCLUSION: Down-regulation of miR-188-3p by reducing histone acetylation up-regulated MLH1 expression and contributed to promotion of apoptosis in spermatogenic cells, in patients with azoospermia.

Effects of Vitrification on Outcomes of In Vivo-Mature, In Vitro-Mature and Immature Human Oocytes.

BACKGROUND/AIMS: To observe the effects of vitrification on spindle, zona pellucida, embryonic aneuploidy and DNA injury in in vivo-maruted, in vitro-mature and immature human oocytes. METHODS: Between January 2009 and February 2015, 223 immature oocytes from 450 infertile patients, and 31 in vivo-mature oocytes from 3 infertile couples were collected. Of the 223 immature oocytes, 113 were used for in vitro culture before vitrification. Some oocytes were randomly divided into in vivo-mature group (group A, n = 15), in vitro-mature group (group B, n = 88) and immature group (group C, n = 85), and then the oocytes with spindle in these three groups after freezing-thawing were selected to use for Polscope imaging, embryonic aneuploidy screening and embryo development evaluation. Other oocytes were randomly divided into group A (n = 16), group B (n = 25) and group C (n = 25) for detecting DNA injury. RESULTS: After thawing, spindle occurrence rate, spindle Retardance value, and cleavage rate were significantly higher in groups A and B than in group C (all P < 0.05), but there were no statistical differences in fertility rate, high-quality embryo rate, blastulation rate and aneuploidy rate amongst the three groups (all P > 0.05). Zona pellucida density (ZPD) was significantly lower in group A than in groups B and C both before and after vitrification (all P < 0.05). ZPD was significantly higher after thawing than before vitrification (all P < 0.05), but zona pellucida thickness (ZPT) was not significantly changed in all the three groups (all P > 0.05). Rate of comet cells was significantly lower in group A than in groups B and C (all P < 0.01). Comet tail was significantly longer in group C than in groups B and A (all P < 0.05). CONCLUSION: In vivo- and in vitro-mature human oocytes are more suitable to vitrification than immature human oocytes. Spindle Retardance value has more predictive value for embryonic development potential than ZPD and ZPT.

The effects of anticancer drugs TSA and GSK on spermatogenesis in male mice.

OBJECTIVE: The effect of anticancer drugs Trichostation A (TSA) and GSK2126458 (GSK) on genetic recombination of sperm meiosis in mice was investigated, and their clinical feasibility of fertility preservation in cancer patients was also assessed. METHODS: Eighteen Kunming mice were randomly given TSA or GSK at the concentrations of 0, 0.1 and 0.2 umol/L for three months. Immunofluorescence was used to evaluate the genetic recombination of homologous chromosomes and fidelity of chromosome synapsis. Sperm density, motility and viability were also examined to investigate the spermatogenic function. RESULTS: The average number of MLH1 foci in each spermatocyte was greatly higher in TSA (0.1) group than that in control (P<0.05), but no difference with the TSA (0.2) group (P>0.05). The frequency of SC with no MLH1 foci was lower while the frequency of SC with one MLH1 foci was higher in spermatocyte of mice with different doses of TSA compared with controls (P<0.05). The weight of left testis in TSA (0.1) group was significant decreased compared with that in control (P<0.05). However, no significant differences were observed in average number of MLH1, frequency of SC with 0-3 MLH1 foci, spermatocyte percentage of XY chromosomes containing MLH1 foci and percentages of cells containing gaps and splits among groups with or without the treatment of GSK. Furthermore, there were no statistical differences in body weight, testicular weight, sperm density, sperm motility and sperm viability among the three groups. CONCLUSION: TSA increased genetic recombination frequency of spermatocyte meiosis. GSK had no significant effect on genetic recombination frequency of spermatocyte meiosis and spermatogenic function.

Comparison of vitrified outcomes between human early blastocysts and expanded blastocysts.

We compared the vitrified outcomes between early and expanded blastocysts with or without laser drilling. The grade III embryos from the patients undergoing in vitro fertilization-embryo transfer (IVF-ET) in our reproductive center from September 2009 to February 2015 were incubated into early blastocysts and expanded blastocysts. The early blastocysts and expanded blastocysts were, respectively, divided into laser group (vitrification after laser drilling), non-laser group (direct vitrification), and control group (fresh non-vitrified blastocysts). After thawing, the blastular anabiosis rate, expansion rate, hatching rate, and apoptosis were observed in each group and then were compared amongst groups. This study indicated that the blastular expansion rate (all P < 0.01) and hatching rate (all P < 0.01) were significantly lower, but the blastular apoptosis (all P < 0.05) was significantly higher in both laser and non-laser groups than in the control group in the early blastocysts. In the expanded blastocysts, the blastular anabiosis rate was significantly higher in the laser group than in the non-laser group (P < 0.01), and the blastular expansion rate was significantly higher, but the blastular apoptosis was significantly lower in both laser group and control group than in the non-laser group (all P < 0.05). The blastular expansion rate (all P < 0.01) and hatching rate (all P < 0.01) were significantly higher, but the blastular apoptosis (all P < 0.05) was significantly lower in the expanded laser group than in both early laser and early non-laser groups. We conclude that vitrification for laser-drilling expanded blastocysts can achieve the best outcomes.

Blastocoele expansion degree predicts live birth after single blastocyst transfer for fresh and vitrified/warmed single blastocyst transfer cycles.

OBJECTIVE: To evaluate the independent effects of the degree of blastocoele expansion and re-expansion and the inner cell mass (ICM) and trophectoderm (TE) grades on predicting live birth after fresh and vitrified/warmed single blastocyst transfer. DESIGN: Retrospective study. SETTING: Reproductive medical center. PATIENT(S): Women undergoing 844 fresh and 370 vitrified/warmed single blastocyst transfer cycles. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Live-birth rate correlated with blastocyst morphology parameters by logistic regression analysis and Spearman correlations analysis. RESULT(S): The degree of blastocoele expansion and re-expansion was the only blastocyst morphology parameter that exhibited a significant ability to predict live birth in both fresh and vitrified/warmed single blastocyst transfer cycles respectively by multivariate logistic regression and Spearman correlations analysis. Although the ICM grade was significantly related to live birth in fresh cycles according to the univariate model, its effect was not maintained in the multivariate logistic analysis. In vitrified/warmed cycles, neither ICM nor TE grade was correlated with live birth by logistic regression analysis. CONCLUSION(S): This study is the first to confirm that the degree of blastocoele expansion and re-expansion is a better predictor of live birth after both fresh and vitrified/warmed single blastocyst transfer cycles than ICM or TE grade.

[Percutaneous permeability of rhein at shenque acupoint in vitro].

To study rhein's permeative properties of acupoint and non-acupoint and different species' transdermal administration in vitro. Cumulative permeation amount and steady-state infiltration rate were taken as evaluative indexes to assess the permeability difference. The Valia-Chien diffusion cell method was used to conduct the permeability test, with fresh acupoint and non-acupoint skin of rat, rabbit and swine in vitro as permeation barriers, and blank 20% EtOH saline as absorption liquid. HPLC was used to determine the rhein. The absorption difference was observed by confocal laser scanning microscopy. The 24-hour cumulative permeation amount through acupoint skin in rats was (102.63±9.60) µg•cm⁻², the steady-state infiltration rate was 4.307 µg•cm⁻²â€¢h⁻¹, both were higher than that through non-acupoint skin. The thickness of acupoint skin in rat was thinner than that in rabbit and swine. The cumulative permeation amount and steady-state infiltration rate of rhein in acupoint of rat were signally higher than those in rabbit and swine. The absorption difference can be clearly observed through an accumulation of fluorescence. In conclusion, species and acupoint all affect the permeability of rhein in vitro. The permeation amount and rate of rhein on Shenque acupoint were better than that on non-acupoint skin, which could verify that treatment through Shenque acupoint is superior to that through non-acupoint. The preliminary mechanism may be the drug delivery through Shenque acupoint as a channel and carrier, which is a visual verification the specificity and superiority of clinical application through Shenque acupoint in treating diseases.

Role of PAFAH1B1 in human spermatogenesis, fertilization and early embryonic development.

Spermatogenesis, fertilization and subsequent embryonic development are complex processes that require tight regulation. The PAFAH1B1 gene plays important roles in these reproductive events in mice, but its expression and roles in human reproduction have not been investigated. Expression analysis of testicular tissue by reverse transcription quantitative PCR and immunohistochemistry revealed varied expression levels among samples of different spermatogenic abilities (as assessed by the Johnsen score), with protein expression restricted to spermatogonia, spermatocytes and spermatids. Immunofluorescence on spermatozoa showed expression over the acrosome and midpiece regions of ejaculated samples, whereas a high proportion of percutaneous epididymal sperm aspiration-derived spermatozoa showed expression restricted to the midpiece. Analysis for PAFAH1B1 mRNA also revealed different expression levels among unfertilized oocytes, zygotes, cleavage stage embryos and blastocysts, with protein localized at the membrane level in oocytes and zygotes, and gradually distributing within the cytoplasm of cleavage stage embryos and blastocysts. Interestingly, microinjection of PAFAH1B1 siRNA into zygotes significantly (P = 0.024) increased fragmentation formation rates in subsequent embryonic development stages. Altogether, these are the first results to support a role for PAFAH1B1 in human spermatogenesis and early embryonic development.

Impact of oxygen concentrations on fertilization, cleavage, implantation, and pregnancy rates of in vitro generated human embryos.

The aim of the present study was to determine the impact of oxygen concentration during in vitro culture of human oocytes and embryos on fertilization, cleavage, implantation, pregnancy, multiple gestation and abortion rates. Women 20-48 years old presenting for infertility treatment and accounting for 3484 in vitro fertilization/intracytoplasmic sperm injection cycles were included in the study. Oocytes/embryos were randomly allocated to be incubated under three different oxygen tension environments: (1) 20% O2 in air; (2) initially 20% O2 in air, followed on day 2 (2-4 cells stage) by 5% CO2, 5% O2 and 90% N2; and (3) 5% CO2, 5% O2 and 90% N2 throughout. Interestingly, IVF-derived embryos cultured in 5% O2 yielded higher rates of fertilization and implantation as compared to those incubated in 20% O2 (P < 0.05). Conversely, embryos in 20% O2 yielded higher rates of fertilization, high quality embryo and implantation than those in the 20%-5% O2 group (P < 0.05). Moreover, ICSI-derived embryos cultured in 20% O2 resulted in lower rates of cleavage as compared to those from the 20%-5% O2 group (P < 0.05). These results are consistent with in vitro and subsequent in vivo embryo development being more susceptible to O2 tension fluctuations rather than the degree of O2 tension itself during culture.

Embryo developmental potential of microsurgically corrected human three-pronuclear zygotes.

We explored the embryo development potential of human three-pronuclear (3PN) zygotes reduced to two-pronuclear (2PN) zygotes (3 → 2PN zygotes) by micropuncture. In this study, there were three groups, the 3 → 2PN group (338 zygotes), the non-corrected 3PN group (381 zygotes), and the normal 2PN group (359 zygotes). The first cleavage mode (2-cell cleavage or 3-cell cleavage), 6-8 cell embryogenesis rate, high-quality embryogenesis rate and Day 5/Day 6 blastulation rate were compared between the three groups. The success rate of enucleation was 92.9%. The 2-cell cleavage rate was significantly higher in the 3 → 2PN group (74.3%) than in the 3PN group (36.4%) (P < 0.05), but had no statistical difference compared with the 2PN group (86.0%) (P > 0.05). The 6-8 cell embryogenesis rate was significantly higher in the 3 → 2PN group (91.1%) as compared to the 2PN group (85.6%) (P < 0.05), but had no statistical difference compared with the 3PN group (95.0%) (P > 0.05). Total blastulation rate was significantly higher in the 2PN group (58.8%) as compared to the 3PN group (21.5%) (P < 0.01), and in the 3 → 2PN group as compared to the 3PN group (5.6%) (P < 0.01). Also D5 blastulation rate was significantly higher in the 2PN group (53.7%) as compared to the 3 → 2PN group (8.9%) (P < 0.01), and in the 3 → 2PN group as compared to the 3PN group (1.9%) (P < 0.01). In 3 → 2PN zygotes, the first cleavage mode is mainly 2 cells which is significantly higher than that in 3PN zygotes. Compared with 3PN zygotes, the embryo developmental potential of 3 → 2PN zygotes is improved, but still is lower than that in 2PN zygotes.

Expression of CD11c+HLA-DR+dendritic cells and related cytokines in the follicular fluid might be related to pathogenesis of ovarian hyperstimulation syndrome.

OBJECTIVE: To explore the expressions of CD11c+HLA-DR+dentritic cells in the follicular fluid of patients with OHSS and their significances. SUBJECTS: 100 individuals. TREATMENT: embryos were observed. The distribution of dentritic cells in follicular fluid and the levels of IL-10, IL-12, IL-18 and IL-23 in follicular fluid were detected. METHODS: There were ovarian hyperstimulation syndrome (OHSS) group and control group in this study. The OHSS group consisted of 50 patients with OHSS and the control group consisted of 50 patients who underwent in vitro fertilization-embryo transfer (IVF-ET) only due to male factors. The statuses of embryos were compared between the two groups. The distribution of dentritic cells in follicular fluid was determined with flow cytometry, and the levels of IL-10, IL-12, IL-18 and IL-23 in follicular fluid were detected with enzyme-linked immunosorbent assay (ELISA) in all patients. RESULTS: The two-pronuclear (2PN) fertility rate, high-quality embryo rate and available embryo rate were all significantly lower in OHSS group than in control group (all P<0.05). The number of CD11c+HLA-DR+dentritic cells (P<0.05) and the levels of IL-10, IL-12, IL-18 and IL-23 were all significantly higher in OHSS group than in control group (all P<0.01). CONCLUSION: The follicular fluid of the patients with OHSS is in an inflammatory status, the inflammatory status may be involved in OHSS and the microenvironment of follicular fluid may affects oocyte quality and embryo development.

An research on the isolation methods of frozen-thawed human ovarian preantral follicles.

OBJECTIVE: To explore the effective isolation method for preantral follicles from human frozen-thawed ovarian tissue. METHODS: The ovarian cortical tissue was frozen by direct cover vitrification (DCV). The frozen-thawed ovarian tissue was used for isolation of preantral follicles with collagenase combined with mechanical method and mechanical method alone, respectively. RESULTS: 1. There was no statistical difference in the survival rates of follicles in various stages between before and after freezing (P > 0.05). 2. The survival rate of secondary follicles was higher, but the survival rate of primordial follicles was lower in mechanical method alone than in collagenase combined with mechanical method (all P < 0.05). 3. The diameters of follicles were larger and E2 levels were higher in mecha-nical method alone than that in collagenase combined with mechanical method (all P < 0.05). CONCLUSION: After the frozen-thawed ovarian tissue was cultured for 6 days, compared with collagenase combined with mechanical method, mechanical method alone can obtain higher survival rate of secondary follicles, greater follicular diameter and higher E2 level, which are conducive to follicular subsequent development.

[Synergetic taste masking of lipid coating and beta-cyclodextrin inclusion].

Paracetamol was used as a model drug in this study to investigate the synergetic effects of lipid coating and beta-cyclodextrin (beta-CD) inclusion for masking the bitter taste of poorly soluble drugs. To control the concentration as low as possible of the free drug which produced a bitter taste, a kinetic model was established to calculate the drug distribution theoretically among the free drug in medium, lipid coated particles and molecular inclusion on the basis of the preparation and characterization of the lipid microspheres, so as to select the proper amount of beta-CD. Finally, the synergetic drug delivery systems were prepared and characterized by 1H nuclear magnetic resonance (1H NMR), molecular simulation and the electronic tongue. As a result, the drug release rate constant (k) of the lipid microspheres coated with octadecanol was determined as 0.001 270 s(-1). Then, the synergetic drug delivery systems were prepared with the ratio of 6.74 : 1 (w/w) for beta-CD and paracetamol. The chemical shift values for the fingerprint peaks of paracetamol all increased and hydrogen bonds were formed between the oxygen on the phenolic hydroxyl group, the nitrogen on the imino in paracetamol and the hydrogens on the hydroxyl groups in beta-CD. The results tested by the electronic tongue indicated that the paracetamol, lipid microspheres, beta-CD inclusion and their mixture showed different taste characteristics, with the bitterness order of the synergetic drug delivery systems approximately lipid microspheres < beta-CD inclusion < paracetamol, which confirmed the synergetic taste masking effects of lipid coating and beta-CD molecular inclusion. In summary, the synergetic taste masking was jointly achieved through the retard of the drug release by the lipid coating and the inclusion of the free paracetamol by beta-CD through hydrogen bonds.

Expression and potential roles of HLA-G in human spermatogenesis and early embryonic development.

As one of the non-classical major histocompatibility complex(MHC)-1 antigens, Human Leukocyte Antigen G (HLA-G), has been suggested as a prognostic marker to identify the embryo developmental potential. In the present study, we investigated the potential roles of HLA-G in human spermatogenesis and early embryonic development. Quantitative real-time PCR analysis revealed that HLA-G's expression was increased with increased Johnsen score in testicular tissues. There was no significant difference in HLA-G mRNA expression between testicular tissues with Johnsen score of 8-9 and normal sperm from ejaculated semen. HLA-G mRNA expression was detected in human zygotes, embryos and blastocysts but not in unfertilized oocytes. In testicular tissues where sperm was obtained by testicular sperm extraction (Johnsen score was 8 to 9), there were no correlations between HLA-G mRNA expression and fertilization, cleavage and high-quality embryo rates. At 48-72 h post-fertilization, HLA-G expression was higher in fast growing embryos. HLA-G specific siRNA injection into zygotes not only slowed down embryonic cleavage rate at 48 h post-fertilization, but also down-regulated the expression of embryo metabolism related gene (SLC2A1) and cell cycle-regulated gene (CCND2). Taken together, our findings suggested that HLA-G plays significant roles in human spermatogenesis and early embryonic development.

Effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection in mouse mature oocytes.

Better pregnancy outcomes can be obtained by human mature oocyte vitrification, but many problems remain to be resolved in human mature oocyte vitrification. Since mature oocyte development possesses its own maturity cycle, there should be the optimal timing for mature oocyte vitrification. The purpose of this study was to observe the effects of frozen timing on the spindle density, the angle between the polar body and spindle, and embryo development of intracytoplasmic sperm injection (ICSI) in vitrified mouse mature oocytes and explore its possible mechanism. Mouse oocytes were randomly divided into three groups according to different frozen timing including Groups A, B, and C in which oocytes were vitrified within 2 h after ovum pick-up, and 3-4 and 5-6 h after ovum pick-up, respectively. Spindle-related parameters were measured, ICSI was performed. The spindle occurrence rate of vitrified-thawed oocytes was 98.4% in Group A, 82.3% in Group B, and 75.8% in Group C, without statistical differences between pre-vitrification and post-thawing and among the three groups (P > 0.05). The angles between the polar body and spindle were larger after thawing than before vitrification (P < 0.01). The spindle retardance values were lower after thawing than before vitrification in Groups B and C (P < 0.05), but higher in Group A (P < 0.05). The spindle retardance values before vitrification were higher in Group B than in Groups A and C (P < 0.05), but the spindle retardance value, oocyte survival and two-cell rate after thawing were higher in Group A than in Groups B and C (P < 0.05). There were no statistical differences in ICSI fertility rate between the three groups (P > 0.05). The damage on the spindle is the slightest and embryo quality is the highest in the mouse oocytes vitrified within 2 h after ovum pick-up. The spindle retardance value is more valuable than the spindle occurrence rate in the evaluation of vitrified-thawed oocyte quality, and is positively correlated with embryo quality.

[Preparation and characterization of dihydroartemisinin/ hydroxypropyl-beta-cyclodextrin inclusion complex].

To optimize the preparation method of the complex of dihydroartemisinin (DHA) included by hydroxypropyl-beta-cyclodextrin (HP-beta-CD), the molar ratio of DHA and HP-beta-CD, inclusion temperature and inclusion time were optimized by the orthogonal design method with the inclusion drug yield and drug loading as the evaluation indexes. The IR spectrum, DSC and PXRD analyses were employed to characterize the complex and the molecular simulation was processed to investigate the tendency of complex formation. The optimized molar ratio of DHA and HP-beta-CD was 1 : 5, and the optimized preparation was performed under 50 degrees C for 1 h. The IR spectrum, DSC and PXRD analyses indicated the formation of the complex. The low binding free energy and the high solvent accessible surface obtained by molecular simulation showed that DHA could be included by HP-beta-CD and its solubility could be improved significantly. In conclusion, the optimized conditions for the preparation of DHA-HP-beta-CD complex provide a theoretical and experimental basis for further scale-up research.

[Multidimensional spatial triangular area as an index for the evaluation of the release-absorption correlation of multiple component traditional Chinese medicines].

The paper is aimed to provide a novel index, named as multidimensional spatial triangular area, for the evaluation of the release-absorption correlation of multiple component traditional Chinese medicines (TCMs). The applicability of the method was demonstrated by the example data. The method and standard practice for evaluation of the release-absorption correlation for western medicines with single compound could not be applied to TCMs with multiple components. The release percentage or absorption percentage of the multiple components for TCMs at the sampling time was a point in the multidimensional space. The area of the triangle formed byt the sequential three points represented the changing characteristics of the components' release and absorption kinetics. The side lengths of the triangle could be calculated from the spatial distances between each two of the sequential three points. Then the triangle area could be obtained by the side lengths. The in vitro release-in vivo absorption correlation of the multiple components could be represented by the correlation between the integrating values of the release triangle areas and that of the absorption triangle areas. The results of the examples indicated that the multidimensional spatial triangular area method could treat the multiple components in a holistic way, in line with the holism the hi he TCMs. Therefore, the multidimensional spatial triangular area method provided new methodology for the release-absorption correlation of the TCMs with multiple components.