Evaluating an external quality assurance program for semen analysis in China during 2009-2020.

INTRODUCTION: Semen analysis (SA) plays a key role in guiding treatments of male reproductive diseases and infertility due to male factors; however, it remains challenging to conduct an accurate SA due to lack of standardization, highly subjective assessments, and problems with automated procedures. Therefore, quality assurance (QA) and teaching courses are essential for making the laboratory results more consistent. MATERIALS AND METHODS: The external quality assurance (EQA) scheme was organized by national human sperm bank technology training bases in Guangdong province in China between 2009 and 2020. Until 2020, 124 laboratories from China participated in the EQA program. The EQA scheme per year has been organized involving two semen aliquots for sperm concentration, two video recordings for motility, and two smears for sperm morphology. All samples used in the EQA scheme were obtained from different healthy donors or patients. RESULTS: We estimated that the median coefficient of variation (CV) of sperm concentration, ignoring the method used, was 26.6%. Using a 100 µm deep counting chamber led to a decreasing CV of 13.6%. For sperm motility, the median CV of nonprogressive motility was high (50.8%), but the CV of progressive motility (13.2%), immotile sperm (14.3%), and total motility (11.8%) were acceptable. The morphology assessment revealed large variability (44.4%) irrespective of the classification criteria. DISCUSSION: The reduction of interlaboratory variability is still a challenge during SA in China. Therefore, it is critical to increase awareness of joining EQA schemes and establish standardized training centers to follow WHO-recommended procedures toward Chinese standards.

Influence of sperm morphology on pregnancy outcome and offspring in in vitro fertilization and intracytoplasmic sperm injection: a matched case-control study.

Sperm morphology was once believed as one of the most predictive indicators of pregnancy outcome in assisted reproductive technology (ART). However, the impact of teratozoospermia on in vitro fertilization (IVF)/intracytoplasmic sperm injection (ICSI) outcomes and its offspring remains inconclusive. In order to evaluate the influence of teratozoospermia on pregnancy outcome and newborn status after IVF and ICSI, a retrospective study was conducted. This was a matched case-control study that included 2202 IVF cycles and 2574 ICSI cycles and was conducted at the Reproductive and Genetic Hospital of CITIC-Xiangya in Changsha, China, from June 2013 to June 2018. Patients were divided into two groups based on sperm morphology: teratozoospermia and normal sperm group. The pregnancy outcome and newborn outcome were analyzed. The results indicated that couples with teratozoospermia had a significantly lower optimal embryo rate compared to those with normal sperm morphology in IVF (P = 0.007), while there were no statistically significant differences between the two groups in terms of the fertilization rate, cleavage rate, implantation rate, and pregnancy rate (all P > 0.05). Additionally, teratozoospermia was associated with lower infant birth weight in multiple births after IVF. With regard to ICSI, there was no significant difference in both pregnancy outcome and newborn outcome between the teratozoospermia and normal groups (both P > 0.05). Furthermore, no increase in the risk of birth defects occurred in the teratozoospermia group after IVF/ICSI. Consequently, we believe that teratozoospermia has limited predictive value for pregnancy outcomes in IVF/ICSI, and has little impact on the resulting offspring if multiple pregnancy is avoided.

Novel micro-straw for freezing small quantities of human spermatozoa.

OBJECTIVE: To evaluate a novel micro-straw as an efficient, simple method for freezing a small number of human spermatozoa for intracytoplasmic sperm injection (ICSI). DESIGN: Prospective cohort study. SETTING: Sperm bank. PATIENT(S): Men with severe oligozoospermia or azoospermia undergoing a total of 143 ICSI cycles at the CITIC-Xiangya Hospital of Reproduction and Genetics from June 1, 2015, to June 31, 2019, and 20 donors at the Hunan Province Human Sperm Bank from 2001 to 2016. INTERVENTION(S): Analysis of sperm samples and clinical outcomes after sperm use. MAIN OUTCOME MEASURE(S): Clinical information, including number of motile sperm before and after freezing, freeze-thaw survival rates, two-pronuclear fertilization rates, clinical pregnancy, and early pregnancy loss rates after sperm use. RESULT(S): In the feasibility experiment using the micro-straw, we found a freeze-thaw survival rate of 73% ± 8.3% and no difference in normal sperm morphology, normal acrosome integrity, or DNA fragmentation index between the micro-straw and 1.8-mL cryotubes. The prospective cohort included 1,325 cases, and we collected sperm from testicular, epididymis, and ejaculation sources. We observed motile sperm in 1,294 (97.6%) of 1,325 frozen-thawed samples. Postthaw sperm were available for ICSI in 140 (97.9%) of 143 of cycles. The fertilization, cleavage, and high-quality embryo rates were 1,007 (81.7%) of 1,233; 995 (98.8%) of 1,007; and 537 (53.9%) of 995, respectively. Sixty-nine (49%) clinical pregnancies were achieved, and the miscarriage rate was 6 (8.6%) of 69. CONCLUSION(S): The micro-straw is suitable and clinically useful for the cryopreservation of small numbers of spermatozoa.

Emerging spotted fever group rickettsiae in ticks, northwestern China.

We report Rickettsia conorii subsp. indica, Candidatus R. barbariae and R. massiliae in Rhipicephalus turanicus from sheep around the Taklamakan desert, northwestern China. The topology of the phylogenetic trees produced from the maximum likelihood (ML) analyses of the ompA-gltA-rrs-geneD-ompB concatenated sequence data was very similar to that of the neighbor joining (NJ) tree, and with total support of 69%-100% bootstrap values for the inclusion of the rickettsiae in Rh. turanicus within the clade that contained R. conorii subsp. indica; Candidatus R. barbariae and Rickettsia sp. Tselentii; R. massiliae str. AZT80; and R. massiliae MTU5, respectively. Studies suggest that the co-existence of these spotted fever group rickettsiae is a threat to public health in China. Work is important in exploring novel and emerging pathogens.

Rickettsia raoultii in Haemaphysalis erinacei from marbled polecats, China-Kazakhstan border.

We found Rickettsia raoultii DNA in 2 out of 32 (6.25 %) Haemaphysalis erinacei ticks. Result showed that the sequences of five genes (17-kDa, gltA, ompA, rrs, and ompB) were 100 % identity with that of R. Raoultii in GenBank. This study is the first report on the presence of R. raoultii in H. erinacei from wild marbled polecat, Vormela peregusna. Our findings suggest that H. erinacei parasitizing wild marbled polecat may serve as reservoir and carriers for R. raoultii in areas around the China-Kazakhstan border. The transmission of tick-borne diseases originated from wildlife should not be underestimated in border region.

Differential protein expression in Phalaenopsis under low temperature.

A comparative proteomic analysis was carried out to explore the molecular mechanisms of responses to cold stress in Phalaenopsis after treated by low temperature (13/8 °C day/night) for 15 days. Differentially expressed proteins were examined using two-dimensional electrophoresis (2-DE) and matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-TOF/MS). Among 85 differentially expressed proteins, 73 distinct proteins were identified. Comparative analysis revealed that the identified proteins mainly participate in photosynthesis, protein synthesis, folding and degradation, respiration, defense response, amino acid metabolism, energy pathway, cytoskeleton, transcription regulation, signal transduction, and seed storage protein, while the functional classification of the remaining four proteins was not determined. These data suggested that the proteins might work cooperatively to establish a new homeostasis under cold stress; 37 % of the identified cold-responsive proteins were associated with various aspects of chloroplast physiology, and 56 % of them were predicted to be located in the chloroplasts, implying that the cold stress tolerance of Phalaenopsis was achieved, at least partly, by regulation of chloroplast function. Moreover, the protein destination control, which was mediated by chaperones and proteases, plays an important role in tolerance to cold stress.

Rapamycin reduces renal hypoxia, interstitial inflammation and fibrosis in a rat model of unilateral ureteral obstruction.

PURPOSE: The purpose of this study was to explore effects of rapamycin on renal hypoxia, interstitial inflammation and fibrosis, and the expression of transforming growth factor ß1 (TGF-ß1), vascular endothelial growth factor (VEGF), Flk-1 and Flt-1 in a rat model of unilateral ureteral obstruction (UUO). METHODS: Male Sprague-Dawley rats (n=36) were randomly divided into three groups (n=12 per group): sham surgery, UUO and UUO plus rapamycin (0.2 mg/kg/d). Serum creatinine (Scr), blood urea nitrogen, uric acid, triglycerides, cholesterol and 24-h urine protein levels were measured. The extent of interstitial fibrosis was determined by Masson's trichrome staining. ED-1 positive macrophages, type III collagen, hypoxia, TGF-1, VEGF, Flk-1, and Flt-1 mRNA and protein expressions were detected using immunohistochemical staining, real-time PCR and Western blot. RESULTS: UUO induced an elevation in Scr, renal hypoxia, inflammation, interstitial fibrosis, TGF-ß1, VEGF, Flk-1, and Flt-1 mRNA and protein expression levels (P < 0.05). Rapamycin alleviated the UUO-induced renal hypoxia, infiltration of inflammatory cells and tubulointerstitial fibrosis (at days 3 and 7). Rapamycin also down-regulated the UUO-induced elevated expression levels of TGF-ß1 and Flt-1 mRNA and protein (P < 0.05). Rapamycin decreased VEGF mRNA and protein expression at day 3, and increased Flk-1 mRNA and protein expression at day 7, compared with the UUO group (P < 0.05). CONCLUSION: Rapamycin shows beneficial effects by reducing UUO-induced renal hypoxia, inflammation and tubulointerstitial fibrosis.

Evaluation of internal control for gene expression in Phalaenopsis by quantitative real-time PCR.

The selection of appropriate reference genes is one of the most important steps to obtain reliable results for normalizing quantitative real-time reverse transcriptase polymerase chain reaction (qRT-PCR) of MADS-box gene in Phalaenopsis. In this study, we cloned 12 candidate reference genes including 18S ribosomal RNA (18S), elongation factor 1 alpha (EF1α), cytoskeletal structural protein actin (ACT1, ACT2, ACT3, ACT4, ACT5), ubiquitin protein (UBQ1 and UBQ2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the cytoskeletal structural proteins α-tubulin (TUA) and ß-tubulin (TUB) in Phalaenopsis and evaluated their expression reliability. The expression of these candidate reference genes was analyzed using geNorm and normFinder software packages; the results showed that ACT2 and ACT4 were the highest stability reference genes for all experiment sets based on normFinder, followed by ACT1 or ACT3, while ACT3 and ACT4 were the highest stability reference genes for most experiment sets based on geNorm, then TUB or others. Taken together, Actin genes were the higher stability reference genes for all tissues at total developmental stages, and similar results came from analysis by normFinder. According to geNorm analysis, ACT3 and ACT4 were the most stable reference genes for all tissues tested and tissues at reproductive stages; TUB and ACT5 or ACT4 were the most stable reference genes for vegetative tissues or roots. The most stable reference genes for all vegetative tissues and only leaves were ACT4 and ACT5, ACT2 and ACT3, respectively; ACT1 and ACT3 were the most stable genes and sufficient for reliable normalization of flower tissues. While EF1α, UBQ1, UBQ2, and GAPDH were found to be unsuitable as a reference gene in our analysis for flower tissues, total tissues, and reproductive stages; UBQ2 and 18S were identified as the least stable reference genes for vegetative tissues at different stages, different tissues at vegetative stages; TUA and 18S were the least reliable reference genes for the samples from roots at all developmental stages. This is the first systematic report on the selection of reference genes in Phalaenopsis, and these data will facilitate future work on gene expression in orchid.

[The prevalence and risk factors of kidney disease in type 2 diabetic patients in rural Shanghai].

OBJECTIVE: To identify the prevalence and etiology of kidney disease and the related risk factors in type 2 diabetic patients in rural Shanghai. METHODS: A cross-sectional study in type 2 diabetic patients was conducted in a community of Shanghai. Questionnaire, clinical examination and laboratory tests were completed to collect the information about sociodemographic and healthcare characteristics. RESULTS: A total of 1421 eligible patients with complete information were screened from 1487 type 2 diabetic patients between November 2008 and March 2009. Of them, 40.75% were men, 59.25% were women, aged 37 - 86 (61.33 ± 9.65) years old, with diabetic duration of 0.25 - 43.92 (7.85 ± 6.34) years. Among them, 43.42% had diabetic retinopathy, 21.18% had neuropathy; 69.95% met the screening definition for hypertension, 76.07% for hyperlipidemia, 15.55% for hyperuricemia and 23.65% for cardiovascular disease. The control rates of fasting blood glucose, glycosylated hemoglobin, blood pressure and serum cholesterol were 57.71%, 33.99%, 14.22% and 2.46%, respectively. The prevalence of kidney disease, diabetic nephropathy and non-diabetic renal disease was 41.31%, 18.51% and 13.44%, respectively; and 9.36% were diagnosed as renal insufficiency of unknown reasons. Age, diabetic duration, hyperuricemia, diabetic retinopathy and poor control of blood pressure were independently associated with kidney disease; age and poor control of blood pressure were independently associated with diabetic nephropathy; age and hyperuricemia were independent risk factors of renal insufficiency in patients with diabetic nephropathy. CONCLUSIONS: Although the diabetic duration of these subjects is relatively short, the prevalence of complications including diabetic nephropathy is high. The high prevalence of non-diabetic renal disease shows the importance of further screening and diagnoses for prevention. Strict control of blood glucose, blood pressure, serum cholesterol and serum uric acid are key points of cutting down the prevalence of diabetic nephropathy and chronic kidney disease.

Renal protection by delayed ischaemic preconditioning is associated with inhibition of the inflammatory response and NF-kappaB activation.

Brief and sublethal ischaemia renders an organ tolerant to subsequent prolonged ischaemia, which is called ischaemic preconditioning (IPC). In regard to the beneficial effects and endogenous mechanisms of renal delayed IPC, few data are available. In this study, we aim at determining reno-protective effects of delayed IPC against ischaemia-reperfusion (I/R) injury, and illustrating whether these effects are associated with suppressing inflammation and nuclear factor-kappaB (NF-kappaB) activation. I/R injury was induced by clamping both renal pedicles for 40 min, followed by 24 h of reperfusion. The rats were subjected to ischaemia for 20 min (preconditioning) or sham surgery (non- preconditioning) at day 4 before I/R. Functional and morphological parameters were evaluated at 24 h after reperfusion. At the same time, macrophage (ED-1(+)) infiltration, and the expression of intercellular adhesion molecule-1 (ICAM-1) and tumor necrosis factor-alpha (TNF-alpha) were assessed by immunohistochemistry. Moreover, I kappa B-alpha degradation and NF-kappaB/DNA binding activity were analyzed. Compared with rats exposed to I/R injury, preconditioned rats had a significant decrease in levels of serum creatinine (Scr, 384.3 +/- 21.8 micromol/L vs. 52.5 +/- 21.7 micromol/L; p<0.001), blood urea nitrogen (BUN, 40.4 +/- 2.7 mmol/L vs. 15.9 +/- 4.2 mmol/L; p<0.001) and serum aspartate aminotransferase (AST, 486.7 +/- 58.6 IU/L vs. 267.3 +/- 43.9 IU/L; p<0.001). Parallel to the above changes, preconditioned rats preserved structural integrity and decreased tubulointerstitial damage scores (3.4 +/- 0.3 vs. 0.2 +/- 0.05; p<0.001) and ED-1(+) cell infiltration (25.3 +/- 3.5 vs. 6.2 +/- 1.2 cells/HPF, p<0.01). Furthermore, our results showed that the expression of ICAM-1 and TNF-alpha, the degree of I kappa B-alpha degradation, and NF-kappaB/DNA binding activity were reduced by IPC. Taken together, our results demonstrated that delayed IPC offered both functional and histological protection, which may be related to suppression of inflammation in preconditioned kidneys.

[Association of cardiovascular risk factors and small arterial elasticity].

OBJECTIVE: To investigate the major cardiovascular risk factors affecting small arterial elasticity and the effect of combined multiple risk factors on it. METHODS: Arterial elasticity indexes (C(1)-large artery and C(2)-small artery) were measured with CVProfilor DO-2020. The status of insulin resistance was evaluated with HOMA (homeostasis model assessment). Subjects were categorized into abnormal C(2) group and control group according to the level of C(2). The former group was further divided into four subgroups (0 to 3) based on the number of risk factors. RESULTS: (1) The levels of age, total cholesterol (TC), low density lipoprotein- cholesterol (LDL-C), fasting blood glucose (FBG), systolic blood pressure (SBP) and diastolic blood pressure (DBP) in abnormal C(2) group were higher than those in control group, whereas C(2) itself was lower than that in control group (P all < 0.05). Age, TC, LDL-C, FBG, SBP and DBP were significantly inversely correlated with C(2). (2) With the clusters of risk factors increasing, C(2) was decreasing (6.5 +/- 2.6 vs 5.4 +/- 2.3 vs 4.7 +/- 2.7 vs 3.1 +/- 1.6, P < 0.001). C(2) decreased significantly in subjects with multiple risk factors (subgroup 3). (3) Fasting plasma insulin and HOMA-IR (insulin resistance index) were significantly higher in subgroup 3 than in the other subgroups (P < 0.05, P < 0.001 respectively). CONCLUSIONS: The elevations of age, TC, LDL-C, FBG, SBP and DBP were the major cardiovascular risk factors on the reduction of C(2), and the effects on it were continuously. With their concurrent effects, multiple risk factors could decrease small arterial elasticity much more significantly. Insulin resistance seems to be closely related to the clusters of multiple risk factors.