A nomogram to assist blastocyst selection in vitrified-warmed embryo transfer cycles.

AIM: The purpose of the study was to establish a mathematical model to help rank the order of blastocysts and assist selection of which blastocysts to warm in vitrified-thawed embryo transfer cycles. DESIGN: A total of 2862 women who underwent first vitrified-thawed single blastocyst transfer (SBT) between July 2015 and July 2019 were retrospectively recruited and randomized into a training set (n = 2289) and testing set (n = 573). Least absolute shrinkage and selection operator (LASSO) regression was used to screen the factors critical to live birth (LB). Subsequently, a nomogram model was established to convert the effect of each factor on LB into a measurable score. The efficacy of the model was then evaluated by the receiver operating characteristic and calibration curve. The performance of the model was also internally tested in the testing set. RESULTS: Maternal age, endometrial thickness, oocyte number, day-3 embryo quality, blastocyst morphology, and blastulation day were selected as the critical predictors of LB in the vitrified-thawed SBT cycle and fitted into a nomogram model. The area under the curve (AUC) of the model was 0.67, and the AUC in the testing set was 0.64, which indicates moderate discrimination. The calibration curve showed good concordance between prediction and observation. Importantly, the score of each variable in the nomogram helped to rank the order of the blastocysts resulting in LB. CONCLUSIONS: The nomogram model can provide guidance for embryo selection in vitrified-thawed blastocyst transfer cycles, which may help to optimize the LB rate.

Endogenous hydrogen sulfide promotes human preimplantation embryonic development by regulating metabolism-related gene expression.

Hydrogen sulfide (H2S) as an endogenous gaseous signaling molecule had been proved to play a vital role in gametes physiology, covering meiosis, maturation and aging. However, little is known about H2S involvement in embryonic development. The present study explored the positive effect of H2S on human early embryonic development. Results validated that the two H2S producing enzymes, CBS and CSE mRNA and proteins were identified in donated human cleavage and blastocyst-stage embryos. The l-cysteine incubation produced endogenous H2S in human blastocysts. NaHS positively affected in vitro blastulation. Single-cell RNA-seq analysis identified 228 differentially expressed genes (DEGs) after NaHS treatment versus the control. The Gene Ontology (GO) enrichment analysis of DEGs showed that genes for protein modification and metabolism were significantly enriched in the NaHS treatment group. For the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, 2-oxocarboxylic acid metabolism, glycosaminoglycan biosynthesis-keratan sulfate, steroid biosynthesis, carbon metabolism, and biosynthesis of amino acids were significantly enriched. Six DEGs, including Neural EGFL like 1 (NELL1), aconitase 1 (ACO1), phosphoglycerate mutase 1 (PGAM1), TP53 induced glycolysis regulatory phosphatase (TIGAR), UDP-GlcNAc:betaGal beta-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2), and carbohydrate Sulfotransferase 4 (CHST4) were validate by real-time RT-PCR. These findings suggest that H2S is a positive regulator of early embryonic development and may alter the transcription of embryonic genes for protein modification and metabolism in human embryos.

Granulocyte-Macrophage Colony Stimulating Factor in Single Blastocyst Conditioned Medium as a Biomarker for Predicting Implantation Outcome of Embryo.

Background: It is highly desirable to develop new strategies based on secretomics to more accurately selection of embryos with the highest developmental potential for transfer. Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been reported to promote embryo development and pregnancy establishment. However, the predictive value of GM-CSF in single blastocyst selection remains unclear. This study is to determine the concentration of GM-CSF in human single-blastocyst conditioned medium (SBCM) and to evaluate its association with embryo quality and pregnancy outcome. Methods: The patients with ≤38 years of age receiving the first cycle of assisted reproductive therapy were included in this study. The patients who had <4 top-quality embryos formed by the fertilized two pronuclear zygotes on day 3 were excluded. A total of 126 SBCM samples (SBCMs) were included, of which blastocysts from 77 SBCMs were later transferred in subsequent frozen-thawed embryo transfer. The concentrations of GM-CSF were detected by single-molecule array (SIMOA) and analyzed for their possible association with embryo quality and pregnancy outcomes. The top-quality embryo (TQ), positive HCG (HP), clinical pregnancy (CP), and ongoing pregnancy (OP) rates were determined and compared between groups divided based on GM-CSF concentrations. Results: The detection rate of GM-CSF was found to be 50% in all SBCMs. There were significant differences in TQ rate, HP rate, CP rate and OP rate among high concentration group, medium concentration group and low concentration group. Both GM-CSF alone or GM-CSF combined with the morphological score (MS) had a greater AUC of ROC curve than that of MS alone to predict the pregnancy outcome, and GM-CSF combined with MS had the highest AUC. Conclusions: The concentration of GM-CSF in SBCM was detected at fg/ml levels, which was associated with embryo quality and pregnancy outcome. Collectively, GM-CSF may be used as a biomarker for prediction of pregnancy outcome and selection of embryos with high developmental potential for transfer in assisted reproductive technology (ART).

A visualized clinical model predicting good quality blastocyst development in the first IVF/ICSI cycle.

RESEARCH QUESTION: Is it possible to establish a visualized clinical model predicting good quality blastocyst (GQB) formation for patients in their first IVF/intracytoplasmic sperm injection (ICSI) cycle? DESIGN: A total of 4783 patients in their first IVF/ICSI cycle between January 2015 and December 2019 were retrospectively included and randomly divided into the training set (n = 3826) and the testing set (n = 957) in an 8:2 ratio. The least absolute shrinkage and selection operator (LASSO) regression was adopted to select the most critical predictors for GQB formation to construct a visualized nomogram model based on the data of patients in the training set. Receiver operating characteristic and calibration curves were used to evaluate the predictive accuracy and discriminative ability. The performance of the model was also validated on independent data from patients treated in the testing set. RESULTS: Maternal age, maternal serum anti-Müllerian hormone (MsAMH) concentration and the number of oocytes retrieved were highlighted as critical predictors of GQB development and were incorporated into the nomogram model. Based on the area under the curve (AUC) values, the predictive ability for ≥1, ≥3 and ≥5 GQB were 0.831, 0.734 and 0.748, respectively. The calibration curve also showed high concordance between the observed and predicted results. The AUC for predicting ≥1, ≥3 and ≥5 GQB in the testing set were 0.805, 0.695 and 0.707, respectively, which were similar to those for the training set. CONCLUSIONS: The visualized nomogram model provides great predictive value for GQB development in patients in their first IVF/ICSI cycle and can be used to improve clinical counselling.

Perinatal and neonatal outcomes of pregnancies after early rescue intracytoplasmic sperm injection in women with primary infertility compared with conventional intracytoplasmic sperm injection: a retrospective 6-year study.

BACKGROUND: Early rescue intracytoplasmic sperm injection (ICSI) has been used in clinic as appropriate currently. While the outcomes of children born after this method were not well assessed. The purpose of this study was to evaluate the effect of early rescue ICSI on women with primary infertility. METHODS: Fresh embryo transfer cycles after rescue (n = 214) and conventional (n = 546) ICSI were retrospectively evaluated from women with primary infertility who underwent their first assisted reproductive technology cycles at our center in 2012-2017. The conventional ICSI group was subdivided into ICSI-1 (semen suitable for in vitro fertilization, IVF) and ICSI-2 (poor semen quality) to minimize bias from differences in semen quality. Pregnancy, delivery and neonatal outcomes were compared between groups. RESULTS: There was a higher rate of polyspermy and a lower rate of top-quality embryos (TQE) on day 3 for oocytes subject to rescue ICSI compared with conventional ICSI. This reduced the total number of TQE and the number of TQE transferred in the rescue ICSI group. There was no significant difference between groups in clinical pregnancy, ongoing pregnancy, early miscarriage and live birth. For pregnant women, gestational age, route of delivery, risk of preterm birth and gestational diabetes mellitus were also comparable. Neonatal outcomes including sex ratio, birth weight, neonatal intensive care unit admission and birth defects were also similar after rescue and conventional ICSI. Moreover, no differences were observed with the different ICSI subgroups. CONCLUSIONS: For women with primary infertility who have a high risk of IVF fertilization failure (FF), rescue ICSI provides a safe and efficient alternative to minimize FF after initial IVF, but results in fewer TQE on day 3.

Comparative secretome profile analysis of cultured immortalized human endometrial stromal cells supplemented with implanted versus nonimplanted blastocyst-conditioned medium: A preliminary analysis.

AIM: Human endometrial stromal cells (HESCs) were previously shown to be capable of discriminating embryos with different qualities. Here we aimed to compare the specific response of the HESC secretome to implanted blastocyst-conditioned medium (BCM) versus nonimplanted medium and identify cytokine candidates useful for the assessment of blastocyst implantation. METHODS: Cleavage embryos were individually cultured in one microdrop of medium for blastocyst formation. The BCM was collected after fresh blastocyst transfer on day 5 and used to supplement HESC culture medium. A high-throughput antibody array covering 440 cytokines was used to detect the secretory proteins of HESCs supplemented with implanted or nonimplanted BCM. RESULTS: A total of 22 differentially expressed proteins were found out of 440 cytokines in the supernatant of HESCs supplemented with BCM from the implanted group compared to the nonimplanted group, including seven upregulated and 15 downregulated proteins. Gene Ontology enrichment analysis showed that the differentially expressed proteins were mainly involved in cell chemotaxis and motility, and ERK1/2 cascade regulation. Kyoto Encyclopedia of Genes and Genomes analysis suggested that the mitogen-activated protein kinase and phosphatidylinositol 3 kinase/Akt pathways were mainly involved. CONCLUSION: HESCs specifically responded to BCM from different quality blastocysts, a finding that can be used to develop a novel approach for blastocyst quality assessment.

Detection of IL-6, IL-10, and TNF-α level in human single-blastocyst conditioned medium using ultrasensitive Single Molecule Array platform and its relationship with embryo quality and implantation: a pilot study.

PURPOSE: The purpose was to investigate the association between embryonic development or implantation and the content of interleukin-6 and 10 (IL-6, IL-10) and tumor necrosis factor-α (TNF-α) in single-blastocyst conditioned medium (SBCM). METHODS: Thirty-eight SBCM samples (SBCMs) were collected from blastocysts with different morphological scores. IL-6, IL-10, and TNF-α concentration in 38 SBCMs was detected by Single Molecule Array and compared according to the blastocyst quality: top-quality (TQ) and non-top quality (NTQ), or blastulation time: day 5 (D5) and day 6 (D6). In another experiment, 61 SBCMs were collected from TQ blastocyst transplanted on D5, and IL-6 concentration in SBCM was compared based on whether embryos are implanted or not (implanted and non-implanted). RESULTS: In the first experiment, IL-6, IL-10, and TNF-α concentration was not significantly different between the TQ-SBCM and NTQ-SBCM. The D6-SBCM had a higher IL-6 concentration compared with the D5-SBCM, while IL-10 and TNF-α concentration was not significantly different between the D5-SBCM and D6-SBCM. The IL-6 concentration in D5-NTQ or D6-TQ SBCM was higher than that in D5-TQ or D6-NTQ SBCM (P < 0.05), respectively. Furthermore, the spearman analysis demonstrated that IL-6 concentration in SBCM was negatively correlated with the blastocyst quality on D5 and positively correlated with the blastocyst quality on D6. In the second experiment, no significant difference in IL-6 concentration was found between SBCM from implanted and non-implanted blastocyst. CONCLUSION: IL-6 concentration in SBCM was associated with embryo quality depending on the blastulation time, although it might not be associated with the blastocyst implantation.

Association between the number of top-quality blastocysts and live births after single blastocyst transfer in the first fresh or vitrified-warmed IVF/ICSI cycle.

RESEARCH QUESTION: Is there an association between the total number of top-quality blastocysts (TQB) developed in the first IVF/intracytoplasmic sperm injection cycle (ICSI) and live births after a single blastocyst transfer (SBT)? DESIGN: Pregnancy outcomes from 1336 infertile women who had undergone their first IVF/ICSI treatment and accepted a first-time embryo transfer with a single fresh or vitrified-warmed blastocyst between January 2016 and August 2018 were assessed retrospectively. The restricted cubic splines method was used to evaluate the association between the number of TQB, and ongoing pregnancies and live births. RESULTS: A significant non-linear functional form was found between the number of TQB and the ongoing pregnancies and live births (P < 0.05). The odds of an ongoing pregnancy or live birth were similar, at about 11% or higher for each additional TQB up to five TQB (odds ratio [OR] 1.11; 95% confidence interval [CI] 1.01-1.21). After this, pregnancy outcomes nearly plateaued, indicating that the number of TQB was not related to pregnancy when it was greater than five. CONCLUSIONS: The quantity of TQB available for transfer or cryopreservation can provide important predictors for pregnancy and live birth after the first embryo transfer cycle with a single blastocyst. This valuable information may assist with the future application of SBT.

Initial serum HCG levels are higher in pregnant women with a male fetus after fresh or frozen single blastocyst transfer: A retrospective cohort study.

OBJECTIVE: Substantial previous studies have almost reached an agreement on the gender effect on maternal serum human chorionic gonadotropin (MsHCG) in and after the late first trimester of pregnancy. However, there is little knowledge of the sex-related difference in MsHCG level at the preliminary stage of pregnancy. The purpose of this study is to reveal this difference in women after fresh or frozen single blastocyst transfer (SBT). MATERIALS AND METHODS: A total of 252 fresh SBT cycles and 1486 frozen-thawed SBT cycles collected between June 1, 2014 and May 30, 2017 were retrospectively analyzed in our center. Patients with MsHCG level ≥5 IU/L on day 11 after transfer, achieving a singleton intrauterine pregnancy and subsequent live birth were included. We compared MsHCG levels between women gave birth to a male neonate and those gave birth to a female one in fresh or frozen SBT cycles, respectively. RESULTS: A total of 136 neonates including 57 females and 79 males were born following fresh SBT. The male-female ratio was 1.39:1. The average MsHCG level of male fetuses was higher than that of female fetuses on day 11 after transfer (549.82 ± 253.24 IU/L versus 439.03 ± 198.41 IU/L, P < 0.05). Correspondingly, a total of 431 infants was born after frozen SBT, containing 188 females and 243 males. The male-female ratio was 1.29:1. Initial MsHCG level remained higher in women with a male neonate than the counterparts with a female neonate (894.43 ± 622.17 IU/L versus 758.05 ± 624.33 IU/L, P < 0.05). It was also found the pregnant women following frozen-thawed SBT exhibited higher initial MsHCG level than those following fresh SBT in whether male-bearing or female-bearing gestations. CONCLUSIONS: MsHCG levels are higher in pregnant women with a male fetus than those with a female one on day 11 after fresh or frozen SBT. A sex-specific response to the stress in the process of in vitro embryo culture was suggested.

Clinical outcomes after transfer of blastocysts derived from frozen-thawed cleavage embryos: a retrospective propensity-matched cohort study.

PURPOSE: To evaluate the clinical outcomes after fresh transfer of blastocysts cultured from vitrified-thawed cleavage embryos (VTCE) compared with conventional frozen-thawed blastocysts transfer (FBT), or with the usual fresh blastocysts transfer (FRBT). METHODS: A total of 155 cycles undergoing fresh transfer of VTCE blastocysts, 4904 cycles undergoing FBT, and 1014 cycles undergoing FRBT were retrospectively analyzed from August 2014 to July 2017. Pregnancy, delivery, and neonatal outcomes were compared after propensity score matching. RESULTS: VTCE blastocysts' transfer resulted in a lower risk of early miscarriage (8.82% versus 19.70%, P < 0.05) and a decreased fetal birth weight (2611.90 ± 618.65 g versus 2931.86 ± 546.52 g, P < 0.01) compared to FBT. No significant difference was found regarding live birth rate, gestational age, and cesarean section. Correspondingly, VTCE blastocysts' transfer led to significantly compromised pregnancy outcomes regarding clinical pregnancy rate and implantation, and even a slightly compromised live birth rate when compared with FRBT. Moreover, a higher occurrence of cesarean Section (88.89% versus 71.29%, P < 0.05) and a shorter gestational age (262.04 ± 14.99 days versus 268.06 ± 14.07, P < 0.05) were also found. Nevertheless, the risk of small for gestational age and large for gestational age, and the neonatal birth weight were comparable. CONCLUSIONS: VTCE blastocysts' transfer results in a comprehensively moderate outcome, which is an acceptable option for patients. Our results can provide efficient value for patients' counseling. Furthermore, these findings indicate directions for exploring the mechanisms of low birth weight and short gestational age.

Obstetric and perinatal outcomes of pregnancies according to initial maternal serum HCG concentrations after vitrified-warmed single blastocyst transfer.

RESEARCH QUESTION: Do pregnancy, obstetric and perinatal outcomes differ according to initial maternal serum human chorionic gonadotrophin (HCG) level measured on day 11 after single blastocyst transfer? DESIGN: Vitrified-warmed single blastocyst transfer cycles (n = 640) were collected between 1 January 2013 and 30 April 2017 with positive HCG values and retrospectively analysed by receiver operating characteristic curves to predict clinical pregnancy, ongoing pregnancy and delivery. Cycles were divided into a low HCG group (n = 155) and high HCG group (n = 485) based on cut-off value of live birth prediction. Cycles in the HCG group were subdivided into a low-high subgroup (n = 162), medium-high subgroup (n = 162) and high-high subgroup (n = 161) based on tertile points. Pregnancy rates and obstetric and perinatal outcomes were compared. RESULTS: The area under curves for clinical pregnancy, ongoing pregnancy and live birth prediction were 0.95, 0.81 and 0.79, respectively; corresponding cut-off values were 152.2 IU/l, 211.9 IU/l and 211.9 IU/l; HCG less than 211.9 IU/l indicated an extremely low clinical pregnancy rate (34.84%), a high early miscarriage rate (61.11%) and a low live birth rate (12.26%). Rates of gestational diabetes mellitus (GDM) (P = 0.007) and female neonates (P = 0.001) were significantly higher in the LHG group compared with the HHG group; no significant differences were observed in the low versus high HCG group overall. CONCLUSIONS: Lower initial maternal serum HCG levels indicated poorer clinical outcomes. Within the high HCG group, a lower initial maternal HCG level was found to be associated with GDM occurrence and proportion of female neonates.

[IVOS â ¡ versus Sperm Class Analyzer in the results of semen analysis].

OBJECTIVE: To compare the results obtained from the computer-aided sperm analysis (CASA) systems of the two fully-automated commercial sperm quality analyzers, Hamilton-Thorn IVOS Ⅱ (IVOS Ⅱ) and Spanish Sperm Class Analyzer (SCA). METHODS: A total of 99 semen samples were collected in the Center of Reproduction of Shenzhen Zhongshan Urology Hospital from September 2018 to October 2018 and, according to the sperm concentration, divided into groups A (<15 ×106/ml), B (15－50 ×106/ml) and C (>50 ×106/ml). IVOS Ⅱ, SCA and manual microscopy were used for the examination of each sample, followed by comparison of the sperm concentration, sperm motility and percentage of progressively motile sperm (PMS) obtained from IVOS Ⅱ and SCA. RESULTS: The sperm concentrations derived from IVOS Ⅱ and SCA were significantly higher than that from manual microscopy in group A (ï¼»10.24 ± 4.60ï¼½ and ï¼»10.20 ± 5.11ï¼½ vs ï¼»8.45 ± 4.15ï¼½ ×106/ml, P < 0.05), but showed no statistically significant difference in group B (ï¼»30.95 ± 11.84ï¼½ and ï¼»31.81 ± 12.90ï¼½ vs ï¼»29.14 ± 10.65ï¼½ ×106/ml, P > 0.05) or C (ï¼»102.14 ± 45.97ï¼½ and ï¼»109.48 ± 46.32ï¼½ vs ï¼»104.74 ± 41.87ï¼½ ×106/ml, P > 0.05). Significant differences were not observed between IVOS Ⅱ and SCA in the percentage of PMS (ï¼»24.21 ± 14.62ï¼½% vs ï¼»23.92 ± 15.42ï¼½%, P > 0.05) or sperm motility (ï¼»37.48 ± 19.34ï¼½% vs ï¼»37.69 ± 16.61ï¼½%, P > 0.05) in group B, nor in group C (PMS: ï¼»30.80 ± 12.06ï¼½% vs ï¼»32.98 ± 16.10ï¼½%, P > 0.05; sperm motility: ï¼»44.50 ± 15.62ï¼½% vs ï¼»47.26 ± 17.46ï¼½%, P > 0.05). Both the percentage of PMS and sperm motility obtained from IVOS Ⅱ were remarkably lower than those derived from SCA in group A (PMS: ï¼»18.54 ± 12.96ï¼½% vs ï¼»22.90 ± 12.88ï¼½%, P < 0.05; sperm motility: ï¼»26.97 ± 14.05ï¼½% vs ï¼»34.90 ± 15.18ï¼½%, P < 0.05). IVOS Ⅱ and SCA both showed a high repeatability (CV <15%), and the former exhibited an even higher one than the latter, in detection of sperm concentration, sperm motility and the percentage of PMS. CONCLUSIONS: IVOS Ⅱ and SCA both had a good consistency in the results of sperm concentration, motility and progressive motility, but showed a poor comparability with low-concentration semen samples.

The sperm quality and clinical outcomes were not affected by sY152 deletion in Y chromosome for oligozoospermia or azoospermia men after ICSI treatment.

Azoospermia factor (AZF) microdeletion plays a key role in the genetic etiology of male infertility. The relationship between sY152 deletion in the AZFc region and clinical outcomes is still unclear. This study was to determine the effects of sY152 deletion on the sperm parameters and clinical outcomes of non-obstructive azoospermia or oligozoospermia men after intracytoplasmic sperm injection (ICSI) treatment. A total of 61 infertile men with AZFc microdeletion of the Y chromosome from January 2008 to December 2012 were recruited in the present study. They were divided into two groups, the sY152 group (n=12) and the AZFc group (n=49), based upon whether they have deleted single sY152 marker or all AZFc markers. Fifty azoospermia or oligozoospermia patients without Y chromosome microdeletion were included as the control group. The sperm quality and clinical data were compared among the three groups. Retrospective cohort-control study was performed. The sperm concentration and motility in sY152 group were better than AZFc group (P<0.05), and were comparable to the control group (P>0.05); the morphology, seminal zinc, seminal fructose and seminal carnitine were similar among the three groups (P>0.05). Patients in both sY152 and AZFc groups had lower fertilization rates (68.40% and 70.63%, respectively) than those in the control group (74.91%), and the differences were statistically significant (P<0.05). No significant differences were found in terms of MII oocyte, high-grade embryo rate, 2PN zygote, number of available embryos and transferred embryos, clinical pregnancy rate, implantation rate, miscarriage rate, multiple pregnancy rate, delivery rate, preterm rate and the male/female ratio among the three groups (P>0.05). Single sY152 deletion might cause a lower fertilization rate, but no adverse effects on sperm quality and clinical outcomes were found. Our study may provide more information for consultation in these patients.

Laser-assisted hatching improves clinical outcomes of vitrified-warmed blastocysts developed from low-grade cleavage-stage embryos: a prospective randomized study.

The aim of this study was to evaluate the effects of quarter zona-pellucida (ZP) opening by laser-assisted hatching (QLAH) on the clinical outcomes following transfer of vitrified-warmed blastocysts developed from low-grade cleavage-stage embryos in patients with all high-grade and fair-grade cleavage-stage embryos transferred without achieving pregnancy. Patients were randomized into two groups: QLAH (n=101) and control (n=102). The implantation and clinical pregnancy rates were significantly higher in the QLAH group compared with the control group (P=0.021 and P=0.034, respectively). The live birth rate of the QLAH group was also higher, although not significantly. When the clinical outcomes according to the day of blastocyst vitrification were compared between the groups, the implantation, clinical pregnancy and live birth rates of the QLAH group were significantly higher (P<0.05) than those of the control group for day 6 blastocysts, but not for day 5 or day 5/day 6 blastocysts. These results suggest that QLAH improves the clinical outcomes of vitrified-warmed blastocysts, especially of day 6 vitrified blastocysts, developed from low-grade cleavage-stage embryos.

Biodiversity, abundance, and activity of nitrogen-fixing bacteria during primary succession on a copper mine tailings.

Microorganisms are important in soil development, inputs and biogeochemical cycling of nutrients and organic matter during early stages of ecosystem development, but little is known about their diversity, distribution, and function in relation to the chemical and physical changes associated with the progress of succession. In this study, we characterized the community structure and activity of nitrogen-fixing microbes during primary succession on a copper tailings. Terminal fragment length polymorphism (T-RFLP) and clone sequencing of nifH genes indicated that different N(2) -fixing communities developed under primary succession. Phylogenetic analysis revealed a diversity of nifH sequences that were mostly novel, and many of these could be assigned to the taxonomic divisions Proteobacteria, Cyanobacteria, and Firmicutes. Members of the Cyanobacteria, mostly affiliated with Nostocales or not closely related to any known organisms, were detected exclusively in the biological soil crusts and represented a substantial fraction of the respective diazotrophic communities. Quantitative PCR (and statistical analyses) revealed that, overall, copy number of nifH sequences increased with progressing succession and correlated with changes in physiochemical properties (including elementary elements such as carbon and nitrogen) and the recorded nitrogenase activities of the tailings. Our study provides an initial insight into the biodiversity and community structure evolution of N(2) -fixing microorganisms in ecological succession of mine tailings.

Spatial and temporal analysis of the microbial community in the tailings of a Pb-Zn mine generating acidic drainage.

Analysis of spatial and temporal variations in the microbial community in the abandoned tailings impoundment of a Pb-Zn mine revealed distinct microbial populations associated with the different oxidation stages of the tailings. Although Acidithiobacillus ferrooxidans and Leptospirillum spp. were consistently present in the acidic tailings, acidophilic archaea, mostly Ferroplasma acidiphilum, were predominant in the oxidized zones and the oxidation front, indicating their importance to generation of acid mine drainage.

Intraspecific variation in cadmium tolerance and accumulation of a high-biomass tropical tree Averrhoa carambola L.: implication for phytoextraction.

Averrhoa carambola L., a high-biomass tropical tree, has recently been shown to be a strong accumulator of cadmium (Cd) and has great potential for Cd phytoextraction. In the present study, field studies and a controlled-environment experiment were combined to establish the extent of variation in Cd tolerance and accumulation at the cultivar level using 14 to 19 cultivars of A. carambola. The results indicated that all cultivars tested could accumulate Cd at high but different levels, and that Cd tolerance also varied greatly between these cultivars. It is confirmed that the high Cd tolerance and accumulation capacity are species-level and constitutional traits in A. carambola. However, no correlation was detected between tolerance index and accumulation of Cd in different cultivars, suggesting that the two traits are independent in this woody Cd accumulator. More importantly, cultivar Wuchuan Sweet (WCT) was shown to have the highest Cd-extraction potential; it yielded a high shoot biomass of 30 t ha(-1) in 230 d, and extracted 330 g ha(-1) Cd in the aerial tissues grown in Cd-contaminated field soil, which accounted for 12.8% of the total soil Cd in the top 20 cm of the soil profile.

Biodiversity and population dynamics of microorganisms in a full-scale membrane bioreactor for municipal wastewater treatment.

The total, ammonia-oxidizing, and denitrifying Bacteria in a full-scale membrane bioreactor (MBR) were evaluated monthly for over one year. Microbial communities were analyzed by denaturing gradient gel electrophoresis (DGGE) and clone library analysis of the 16S rRNA and ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ) genes. The community fingerprints obtained were compared to those from a conventional activated sludge (CAS) process running in parallel treating the same domestic wastewater. Distinct DGGE profiles for all three molecular markers were observed between the two treatment systems, indicating the selection of specific bacterial populations by the contrasting environmental and operational conditions. Comparative 16S rRNA sequencing indicated a diverse bacterial community in the MBR, with phylotypes from the α- and ß-Proteobacteria and Bacteroidetes dominating the gene library. The vast majority of sequences retrieved were not closely related to classified organisms or displayed relatively low levels of similarity with any known 16S rRNA gene sequences and thus represent organisms that constitute new taxa. Similarly, the majority of the recovered nosZ sequences were novel and only moderately related to known denitrifiers from the α- and ß-Proteobacteria. In contrast, analysis of the amoA gene showed a remarkably simple ammonia-oxidizing community with the detected members almost exclusively affiliated with the Nitrosomonas oligotropha lineage. Major shifts in total bacteria and denitrifying community were detected and these were associated with change in the external carbon added for denitrification enhancement. In spite of this, the MBR was able to maintain a stable process performance during that period. These results significantly expand our knowledge of the biodiversity and population dynamics of microorganisms in MBRs for wastewater treatment.