Investigation on the mechanisms of human sperm DNA damage based on the proteomics analysis by SWATH-MS.

BACKGROUND: Spermatozoa have the task of delivering an intact paternal genome to the oocyte and supporting successful embryo development. The detection of sperm DNA fragmentation (SDF) has been emerging as a complementary test to conventional semen analysis for male infertility evaluation, but the mechanism leading to SDF and its impact on assisted reproduction remain unclear. Therefore, the study identified and analyzed the differentially expressed proteins of sperm with high and low SDF. METHODS: Semen samples from men attended the infertility clinic during June 2020 and August 2020 were analyzed, and sperm DNA fragmentation index (DFI) was detected by the sperm chromatin structure assay. Semen samples with low DFI (< 30%, control group) and high DFI (≥ 30%, experimental group) were optimized by density gradient centrifugation (DGC), and the differentially expressed proteins of obtained sperm were identified by the Sequential Window Acquisition of All Theoretical Mass Spectra Mass Spectrometry (SWATH-MS) and performed GO and KEGG analysis. RESULTS: A total of 2186 proteins were identified and 1591 proteins were quantified, of which 252 proteins were identified as differentially expressed proteins, including 124 upregulated and 128 downregulated. These differentially expressed proteins were involved in metabolic pathways, replication/recombination/repair, acrosomal vesicles, kinase regulators, fertilization, tyrosine metabolism, etc. Western blotting results showed that the expression levels of RAD23B and DFFA proteins and the levels of posttranslational ubiquitination and acetylation modifications in the experimental group were significantly higher than those in the control group, which was consistent with the results of proteomics analysis. CONCLUSIONS: Proteomic markers of sperm with high DNA fragmentation can be identified by the SWATH-MS and bioinformatic analysis, and new protein markers and posttranslational modifications related to sperm DNA damage are expected to be intensively explored. Our findings may improve our understanding of the basic molecular mechanism of sperm DNA damage.

Investigation of the mechanisms leading to human sperm DNA damage based on transcriptome analysis by RNA-seq techniques.

RESEARCH QUESTION: What are the molecular mechanisms leading to human sperm DNA damage? DESIGN: Semen samples were collected and the sperm DNA fragmentation index (DFI) was assessed. Differentially expressed RNA in spermatozoa with a high (DFI ≥30%, experimental group) or normal (DFI <30%, control group) DFI were identified by RNA-sequencing (RNA-seq) technology, and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was performed. Three differentially expressed RNA related to sperm DNA damage and repair, namely PMS1, TP53BP1 and TLK2, were validated using real-time quantitative (RT-qPCR). RESULTS: A total of 19,970 expressed RNA were detected in the two groups. Compared with the control group, the expression levels of 189 RNA in the experimental group were significantly increased and those of 163 genes decreased. Gene Ontology enrichment analysis showed that these RNA were mainly concentrated in the ATPase-dependent transmembrane transport complex, extracellular exosome, somatic cell DNA recombination, protein binding, cytoplasm and regulation of localization. KEGG pathway analysis showed that these RNA were mainly related to the PI3K-Akt signalling pathway, endocytosis, p53 signalling pathway and cGMP-PKG signalling pathway. The RT-qPCR results showed that the expression levels of PMS1, TP53BP1 and TLK2 in the experimental group were significantly lower than in the control group (P = 0.01, 0.015 and 0.004, respectively), which was identical to the results of RNA sequencing. CONCLUSIONS: Differentially expressed RNA related to sperm DNA damage and repair may be identified by RNA-seq technology, which provides new insights into the understanding of sperm DNA damage and repair, and will help to discover new biomarkers related to sperm DNA damage.

Performance evaluation of sperm concentration, motility, and morphological analysis for GSA-810 series of sperm quality analysis system.

BACKGROUND: The performance evaluation of each computer-assisted sperm analysis (CASA) system may provide a basis for the interpretation of clinical results and further improvement of the CASA system. METHODS: The accuracy of the GSA-810 CASA system was evaluated by detecting latex bead quality control products. The precision of sperm concentration, morphology, and percentages of progressively motile sperm (PR) were evaluated by coefficient of variation (CV). Three samples with sperm concentration of about 100 × 106 /mL were diluted to evaluate the linear range. RESULTS: The detection values of latex beads were within the range of target values. The CVs of sperm concentration and PR were significantly and negatively correlated with sperm concentration (r = -0.561, p = 0.001) and PR value (r = -0.621, p < 0.001), respectively. The R2 values of the linear range of sperm concentration were ≥0.99. There was no significant difference in sperm motility and PR within 1-10 min at 36.5°C ± 0.5°C. The coincidence rates of sperm morphology and sperm head morphology for 36 semen samples analyzed by the GSA-810 system and manual method were 99.40% and 99.67%, respectively. The CVs of the percentage of sperm with abnormal morphology and percentage of sperm with abnormal head morphology were less than 5%. CONCLUSION: The GSA-810 system can accurately analyze normal semen samples, but the repeatability of the results is poor for oligozoospermia and asthenozoospermia samples. The future CASA system for analyzing sperm morphology should focus on recognizing the middle and tail segments of a spermatozoon.

Effects of six kinds of sperm staining methods on human sperm size and evaluation of their staining effects.

BACKGROUND: Large- and small-headed sperm are common morphological abnormalities. If different sperm staining methods affect sperm size, they will make a difference in the accuracy of sperm morphological analysis results. In this case, the normal reference values of sperm head parameters for different staining methods should be established. METHODS: Six sperm staining methods, including Papanicolaou, Diff-Quik, Shorr, Hematoxylin-eosin (HE), Wright, and Wright-Giemsa staining, were used to stain the sperm smears of 25 semen samples, respectively. Sperm head parameter's length (L), width (W), area (A), perimeter, acrosomal area (Ac), and the derived values L/W and Ac/A of 2500 sperm (100 for each specimen) per staining method were measured by a computer-aided sperm morphological analysis system. RESULTS: The highest sperm head length and width were observed with the Wright-Giemsa and Wright staining, followed by the Diff-Quik. The lowest sperm head length and width were observed with the Papanicolaou staining, and the sperm head length and width of HE and Shorr staining were between those of Papanicolaou and Diff-Quik staining. There was the same trend in changes in sperm head area and perimeter. Diff-Quik and Shorr staining could clearly distinguish acrosome and nucleus, followed by HE staining, whereas the boundary between acrosome and nucleus was not evident in Papanicolaou, Wright, and Wright-Giemsa staining. CONCLUSION: Different staining methods influence sperm size, and the normal reference values of sperm head parameters of each staining method should be established. Diff-Quik and Shorr staining may be suitable methods for routine sperm morphological analysis.

High DNA stainability (HDS) should not be recommended as a marker for the detection of sperm DNA damage.

There was a marker of high DNA stainability (HDS) in the detection of sperm DNA damage, which was defined as the sperm with high green stainability (HIGRN). The sperm with normal double-stranded DNA was stained green by acridine orange (AO). However, the sperm with high green fluorescence or HDS were thought of as immature sperm or the sperm with poor chromatin condensation. Some previous literature reported that the proportion of sperm with HDS increased with age, and had a certain correlation with the poor outcome of assisted reproductive technology. Recently, several articles reported that the marker of HDS decreased linearly with age, which was obviously inconsistent with that reported by the previous literature. In this case, what kind of marker is HDS? Is it worth studying? After extensively reading the literature related to HDS and flow cytometry related books and performing a series of studies related to the detection of sperm DNA damage, we believe that the establishment of HDS in the detection of sperm DNA damage has no theoretical basis and also no support of evidence-based medicine and that using HDS as a marker in the detection of sperm DNA integrity is inappropriate.

[Standardization and quality control for detection of sperm DNA damage by flow cytometry: A preliminary investigation].

OBJECTIVE: To investigate preliminarily the standardization and quality control for the detection of sperm DNA damage by flow cytometry. METHODS: Semen samples were randomly selected and observed for the effects of acid denaturation time, acridine orange (AO) staining time, semen sample refrigeration, freezing and repeated freezing-thawing on the results of sperm DNA fragmentation index (DFI). RESULTS: Sperm DFI increased gradually with the prolongation of acid denaturation time, significantly at 2 minutes in comparison with that at 30 seconds (P<0.05). There was no significant difference in sperm DFI after AO staining for 5, 20, 40, 60, 100 and 140 minutes. Sperm DFI also exhibited an evident increase with the prolongation of the refrigeration of the semen samples at 2℃－8℃, significantly at 2 days. The semen samples could be frozen directly at －20℃, and three times of repeated freezing and thawing produced little effect on the results of sperm DFI, except for some inadequate stability. Based on the data obtained from freezing the semen samples after sub-packed and tested 2 days for 1 month and simulation of inter-laboratory quality control, the calculated CV value was 7.13%. CONCLUSIONS: In detection of sperm DFI, it is very important to ensure the accuracy of acid denaturation time, which cannot exceed 1 minute at most. The time of or after AO staining does not significantly affect the results of sperm DFI. The samples for detection of sperm DFI should be fresh and not exceed 1 day in case of refrigeration. Directly frozen semen samples can be used as the materials for inter-laboratory quality control for detection of sperm DFI. Whether cryoprotectants can make frozen semen samples more stable and how to prepare and transport the materials for inter-laboratory quality control are the key problems to be solved in the future.

[Establishment and evaluation of a flow cytometry technique reflecting the severity of human sperm DNA damage].

OBJECTIVE: To establish a flow cytometry (FC) technique reflecting the severity of human sperm DNA and evaluate its performance. METHODS: We analyzed the red and green fluorescence peaks of normal sperm in 1 165 human semen samples, defined the average lower limit of 5% of green fluorescence and the average upper limit of 95% of red fluorescence as the red and green fluorescence limits of the four-quadrant gate, and established an FC technique for detection of the sperm DNA fragmentation index (DFI), analysis of its repeatability and linear range and determination of the reference value of normal fertile men. We also analyzed the correlations of the sperm DFI, mild DNA damage marker (DFIm) and severe DNA damage marker (DFIs) with sperm concentration and motility in 122 men from the Infertility Clinic of Zhongda Hospital. RESULTS: With the established FC technique based on the four-quadrant gate, the sperm DFI, DFIm and DFIs were clearly distinguished among different groups of males, and the coefficients of variation obtained in 10 repeated examinations of the semen samples with a high, medium or low DFI using the FC technique were all lower than 5%. The sperm DFI showed a very good correlation within the range of 8.93%－3.90% (r > 0.99). With the upper limit of 95% as the range of normal reference value, the sperm DFI of 274 of the normal fertile males was ≤ 25.50%. The sperm DFI was remarkably negatively correlated with sperm motility and the percentage of progressively motile sperm (PMS) but exhibited no significant correlation with sperm concentration. The DFIs showed significantly higher related coefficients with sperm motility and PMS (r = －0.592 and －0.543) than DFIm (r = －0.323 and －0.236). Both DFIs and DFIm were markedly higher in the patients with decreased sperm motility and PMS than in the normal fertile men, the former even more significantly (P < 0.01) than the latter (P < 0.05). CONCLUSIONS: Compared with the existing FC technique, ours can reflect the severity of sperm DNA damage and is more suitable for clinical application. DFIs may be more closely related to male fertility.

Identification and preliminary study of immunogens involved in autoimmune prostatitis in human males.

BACKGROUND: Experimental models have confirmed that autoimmunity is an important factor in the onset of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS); however, there is no conclusive evidence on whether autoimmune prostatitis exists in human males. METHODS: Rabbits were immunized with either human prostate tissue homogenates or normal saline and the antiserum was collected. Two-dimensional electrophoresis (2-DE) was performed on the homogenates and Western blotting was conducted on the sera. The identified human prostate tissue immunodominant antigens (HPTIAs) were detected by mass spectrometry. The serum immunoglobulin (Ig)G from the immunized rabbits was purified with protein A-agarose, and the purified IgG was linked with Sepharose to purify HPTIAs by affinity chromatography. Non-obese diabetic (NOD) mice were immunized with the purified HPTIAs, and the levels of serum antibodies, INF-γ, and histopathological changes in their prostate tissues were detected. The purified HPTIAs were coated into polystyrene pores and serum autoantibodies in CP/CPPS patients were detected by enzyme-linked immunosorbent assay (ELISA). Meanwhile, serum interleukin 2 (IL-2), interferon gamma (IFNγ), and tumor necrosis factor alpha (TNFα) levels in CP/CPPS patients were also determined by ELISA. RESULTS: Sixteen HPTIAs were identified. Among them, three types were reported to be associated with prostatic diseases. Prostatitis was induced in mice immunized with the 16-HPTIA complex, with positive serum autoantibody and increased prostatic IFN-γ levels. The positive rate of serum autoantibodies against HPTIAs was significantly higher in CP/CPPS patients (23.1%, 18/78) than in the control (2.7%, 2/75). But there was no significant difference in serum TNFα, IFNγ, and IL-2 levels between the CPPS patients with positive and negative autoantibodies against HPTIAs. CONCLUSIONS: Autoantibodies against HPTIAs exist in part in CP/CPPS patients, which implies that autoimmunity and the 16 HPTIAs are important factors in the onset of CP/CPPS. The detection of serum autoantibodies could be applied in clinical diagnoses of autoimmune prostatitis; treatment protocols might change. Additional studies are needed to determine which of the 16 HPTIAs is the most important.

Analysis of human sperm DNA fragmentation index (DFI) related factors: a report of 1010 subfertile men in China.

BACKGROUND: Many factors may lead to sperm DNA damage. However, it is little known that the correlations of sperm DNA damage with obesity-associated markers, and reproductive hormones and lipids levels in serum and seminal plasma. METHODS: In our prospective study, a total of 1 010 subfertile men, aged from 18 to 50 years old, were enrolled from August 2012 through June 2015. Their obesity-associated markers, semen parameters, sperm acrosomal enzyme activity, seminal plasma biochemical markers, and reproductive hormones and lipids levels in serum and seminal plasma were detected. Sperm DNA fragmentation index (DFI) was determined by sperm chromatin structure assay. The correlations between DFI and each of the above-mentioned variables were analyzed. RESULTS: Spearman correlation analysis showed that sperm DFI was positively related to age and abstinence time (P<0.001). Sperm DFI was also positively related to semen volume and percent of abnormal sperm head (P<0.001), while negatively related to sperm concentration, progressive motility (PR), sperm motility, total normal-progressively motile sperm count (TNPMS), percent of normal sperm morphology (NSM), percent of intact acrosome and acrosomal enzyme activity (P<0.001). Sperm DFI was positively related to seminal plasma zinc level (P<0.001) but unrelated to seminal plasma total α-glucotase, γ-glutamyl transpeptidase (GGT) and fructose levels. There was no any correlation between sperm DFI and obesity-associated markers such as body mass index (BMI), waist-to-hip ratio (WHR), waist circumference (WC) and waist-to-height ratio (WHtR), and serum lipids levels, but there was positive correlation between sperm DFI and seminal plasma triglyceride (TG) and total cholesterol (TC) levels (P<0.001). Sperm DFI was positively related to serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels and seminal plasma FSH and estradiol (E2) levels (P<0.001), but unrelated to serum and seminal plasma testosterone (T) levels. The multivariate regression analysis for the variables which were significantly correlated with sperm DFI in Spearman correlation analysis showed that age, semen volume, sperm concentration, progressive motility, TNPMS and intact acrosome were independently correlated with sperm DFI. CONCLUSIONS: There are many potential factors associated with sperm DFI, including age, abstinence time, spermatogenesis and maturation, seminal plasma lipids and reproductive hormones levels. However, the potential effects of seminal plasma lipids and reproductive hormones on sperm DNA damage need still to be demonstrated by the studies with scientific design and a large size of samples.

[Automatic detection and clinical application of semen biochemical markers].

Human seminal plasma is rich in potential biological markers for male infertility and male reproductive system diseases, which have an application value in the diagnosis and treatment of male infertility. The methods for the detection of semen biochemical markers have been developed from the manual, semi-automatic to the present automatic means. The automatic detection of semen biochemical markers is known for its advantages of simple reagent composition and small amount of reagents for each test, simple setting of parameters, whole automatic procedure with few errors, short detection time contributive to batch detection and reduction of manpower cost, simple calibration and quality control procedure to ensure accurate and reliable results, output of results in the order of the samples in favor of clinical diagnosis and treatment, and open reagents applicable to various automatic biochemistry analyzers. At present, the automatic method is applied in the detection of such semen biochemical markers as seminal plasma total and neutral alpha-glucosidase, acid phosphatase, fructose, γ-glutamyl transpeptidase, zinc, citric acid, uric acid, superoxide dismutase and carnitine, sperm acrosin and lactate dehydrogenase C4, and semen free elastase, which can be used to evaluate the secretory functions of the epididymis, seminal vesicle and prostate, sperm acrosome and energy metabolism function, seminal plasma antioxidative function, and infection or silent infection in the male genital tract.

Relationship Between Amyloid Precursor Protein in Seminal Plasma and Abnormal Penile Sympathetic Skin Response in Lifelong Premature Ejaculation.

INTRODUCTION: Hyperactivity of the sympathetic nervous system can play an important role in lifelong premature ejaculation (PE). Our previous study found that amyloid precursor protein (APP) levels in seminal plasma of patients with PE were clearly increased. Amyloid-ß (Aß) is derived from APP. Excessive Aß, especially Aß42, can cause neuronal dysfunction. AIM: To determine whether APP and Aß42 are associated with an abnormal penile sympathetic skin response (PSSR). METHODS: From November 2015 to April 2016, 24 patients with lifelong PE (mean age = 29.2 ± 5.3) with self-estimated intravaginal ejaculatory latency time no longer than 2 minutes and 10 control subjects (mean age = 28.0 ± 5.5) were enrolled consecutively from andrology clinics. PSSR was measured in patients with lifelong PE. APP and Aß42 levels in seminal plasma were determined. MAIN OUTCOME MEASURES: PSSR in patients with lifelong PE and APP and Aß42 levels in all subjects. RESULTS: Patients with PE presented 1.5-fold higher levels of APP (P = .004) than control subjects. Seminal plasma protein concentration (C) in the PE group was lower than that in the control group (P = .007). APP divided by C (APP/C) was 2.0-fold higher (P < .001) in the PE group. Aß42 level was not different between the PE and control groups, but Aß42 divided by C (Aß42/C) was significantly higher in the PE group (P < .001). No differences in APP and APP/C were found between patients with PE in the abnormal and normal PSSR groups. The abnormal PSSR group presented significantly higher Aß42 (P = .007) and Aß42/C (P < .001) levels. The latency of PSSR was negatively correlated with Aß42/C (r = -0.436; P = .033). CONCLUSION: These results showed that patients with lifelong PE had higher APP and Aß42 levels in seminal plasma. Abnormal PSSR was related to a higher Aß42 level. Drugs that decrease Aß could be treatment of PE.

[Association of abnormal length of Y chromosome with semen quality and outcome of assisted reproductive technology in humans].

OBJECTIVE: To investigate the association of the abnormal length of human Y chromosome with semen quality and the outcome of assisted reproductive technology (ART). METHODS: Based on the karyotype, we assigned the patients undergoing ART to a normal control, a long Y chromosome (Y>18), and a short Y chromosome group (Y<22). We compared the semen parameters and numbers of embryos and high-quality embryos among the three groups of patients and performed statistical analysis of the obtained data using Chi-square distribution and t-test. RESULTS: Compared with the control, the Y>18 group showed a significantly lower incidence rate of asthenozoospermia (31.03% vs 8.33%, P <0.05) and a larger number of high-quality embryos (5.46 ± 4.54 vs 7.40 ± 5.49, P<0.05). Both the incidence rate of azoospermia and number of total embryos were remarkably lower in the control than in the Y<22 group (1.87% vs 16.47%, P <0.05; 8.60 ± 7.03 vs 10.00 ± 6.58, P<0.05). No statistically significant differences were found in the pregnancy rate between the Y>18 and Y<22 groups (P>0.05). CONCLUSIONS: Short Y chromosome may affect spermatogenesis, but the length of Y chromosome does not negatively influence the outcome of ART.

[Animal models of autoimmune prostatitis and their evaluation criteria].

Chronic prostatitis is a highly prevalent disease of unclear etiology. Researches show that autoimmune reaction is one cause of the problem. An effective animal model may help a lot to understand the pathogenesis and find proper diagnostic and therapeutic strategies of the disease. Currently used autoimmune prostatitis-related animal models include those of age-dependent spontaneous prostatitis, autoimmune regulator-dependent spontaneous prostatitis, self antigen-induced prostatitis, and steroid-induced prostatitis. Whether an animal model of autoimmune prostatitis is successfully established can be evaluated mainly from the five aspects: histology, morphology, specific antigens, inflammatory factors, and pain intensity.

[Related factors of sperm DNA damage: Advances in studies].

The detection of sperm DNA damage, as an important supplement to semen routine examination strategies, has been applied in some clinical andrology laboratories. What factors may lead to sperm DNA damage remains one of the concerns among many andrologists. Present studies show a variety of factors of sperm DNA damage, including age, environmental pollutants such as organophosphorus and organochloride pesticides, plasticizer, heavy metals such as lead, carcinogens such as polycyclic aromatic hydrocarbons (c-PAHs) and zearalenone (ZEA), male reproductive system diseases or systemic diseases such as varicocele, infection, tumor, spermatogenesis and maturation dysfunction, spinal cord injury and endocrine disorders, seasons and temperature, lifestyle, abstinence time, semen refrigeration, semen handling in vitro, and certain medications. Among them, spermatogenesis and sperm maturation dysfunction may be the most secretive factors, which are involved in the molecular mechanisms of sperm chromatin packaging and restructuring, such as the transformation of histone to protamine, single nucleotide polymorphism of genes, and the role of telomere, which may be one of the hotspots in the future studies of sperm DNA damage. Relevant researches in the future are expected to focus on the prevention of sperm DNA damage and clarification of its specific pathogenic mechanisms so as to provide some evidence for its treatment.

[Correlations of 24 biochemical markers in seminal plasma with routine semen parameters].

OBJECTIVE: To investigate the correlations of 24 biochemical markers in the seminal plasma with routine semen parameters. METHODS: According to the WHO5 standards, we analyzed the routine semen parameters of 66 subfertile men, including the semen volume, sperm concentration, total sperm count, sperm motility, and the percentage of progressively motile sperm (PR). Based on the calibration and quality control measures and using an automatic biochemistry analyzer or electrolyte analyzer, we detected 24 biochemical markers in the seminal plasma of the patients, including total protein (TP), albumin (Alb), globulin (Glb), uric acid (UA), alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (AKP), γ-glutamyltransferase (GGT), lactate dehydrogenase (LDH), creatine kinase (CK), alpha hydroxybutyrate dehydrogenase (αHBDH), adenosine deaminase (ADA), glucose (Glu), triglyeride (TG), total cholesterol (TC), urea nitrogen (UN), creatinine (Cr), high-sensitive C-reactive protein (hsCRP), K+, Na+, Cl- , Ca, Mg, and phosphorus (P). Then we analyzed the correlations of the 24 biochemical markers with routine semen parameters. RESULTS: The levels of the TP, Alb, and Glb proteins in the seminal plasma were positively correlated with sperm concentration, so was that of Alb with the total sperm count, and the AST and LDH activities with sperm concentration and total sperm count. The AKP activity in the seminal plasma was correlated negatively with the semen volume, but positively with sperm motility. The αHBDH activity exhibited a positive correlation with both sperm concentration and total sperm count, with a coefficient of correlation (r) above 0.7. The UN level was correlated negatively with the semen volume, so was the Cr level with the semen volume, sperm concentration, and total sperm count, and the Glu level with sperm concentration and total sperm count. The TG level was correlated positively with the semen volume, but negatively with sperm motility. The levels of seminal plasma ALT, GGT, ADA, UA, TC, CK, and hsCRP showed no correlation with the above-mentioned semen parameters. None of the seminal plasma K+, Na+, Ca, Mg, and P levels was found correlated with semen parameters except the Cl- level, which was negatively correlated with the semen volume. CONCLUSION: Many biochemical markers in the seminal plasma are closely related to routine semen parameters, indicating that these biochemical components may play roles in spermatogenesis, sperm maturation, and physiological metabolism.

Hyperlipidemia is negatively associated with pregnancy outcomes in patients following frozen-thawed embryo transfer: A retrospective study.

Background and Aims: It is demonstrated that lipid metabolism disorders are associated with the reproductive performances of assisted reproductive technology. However, it is little known whether hyperlipidemia is associated with the endometrial receptivity and pregnancy outcomes of patients undergoing frozen-thawed embryo transfer (FET). Methods: This was a retrospective analysis involving 554 infertile women undergoing FET. The patients were divided into the hyperlipidemia group (n = 224) and control group (n = 320) based on the levels of serum lipids. The clinical and laboratory indexes between the two groups were compared. Meanwhile, the stratified analysis based on body mass index (BMI) and endometrial preparation protocols was performed. The independent samples t-test, Mann-Whitney U test, χ2 test and multiple logistic regression analysis were used to compare and analyze the data. Results: The patients with hyperlipidemia had significantly higher serum lipids levels and BMI and lower clinical pregnancy and implantation rates than those with normal blood lipids (p < 0.05). The impact of hyperlipidemia on pregnancy outcomes was independent of BMI. The multiple logistic regression analysis showed that higher cholesterol was associated with lower pregnancy rate and implantation rate (p < 0.05). Regardless of blood lipid levels, the patients undergoing the hormone replacement therapy (HRT) protocol had higher estradiol levels and lower progesterone levels compared with the stimulated cycles (STC) (p < 0.05). Moreover, the clinical pregnancy rate and implantation rate of the HRT protocol were higher than those of the STC, although there was no significant difference between the two. Conclusion: Hyperlipidemia especially higher cholesterol has a negative effect on the pregnancy outcomes of the patients undergoing FET. Actively implementing lipid-lowering treatment and the HRT protocol seem more friendly for these patients.

[Analysis of sperm morphology: yes or no?].

The analysis of sperm morphology can be used to evaluate sperm fertilizing ability and spontaneous conception status, and especially the overall analysis of the sperm head, neck and tail, along with the patient's living habits, occupation and clinical manifestations, may contribute to the primary diagnosis of the patients potentia generandi. It can also be employed to assess the effects of the treatment of semen samples. Although oocyte fertilization can be achieved by the technologies of intracytoplasmic sperm injection (ICSI), motile sperm organelle morphology examination (MSOME) and intracytoplasmic morphologically selected sperm injection (IMSI) regardless of sperm morphology and / or motility, which may somewhat weaken the clinical application of sperm morphology analysis, the standardized procedure and the practice of quality control for the analysis of sperm morphology can significantly improve the accuracy of its results and largely promote its clinical application. Therefore, it is of positive necessity as well as clinical application value to perform sperm morphology analysis in andrology laboratories, reproductive centers, sperm banks and the department of laboratory medicine.

[Establishment and evaluation of an automatic method for seminal plasma gamma-L-glutamyl transpeptidase detection].

OBJECTIVE: To establish an automatic method for seminal plasma gamma-L-glutamyl transpeptidase (GGT) detection and evaluate its accuracy, repeatability and linear range. METHODS: We detected the GGT activity in the seminal plasma by rate assay, and established the detection parameters on an automatic biochemical analyzer. Then, we evaluated the reagent blank absorbance, accuracy, repeatability and linear range of the automatic method, and compared the results obtained from the method and the seminal plasma GGT detection kit (Xindi Biological Pharmaceutical Engineering Co., Ltd, Nanjing, China) commonly used in clinical laboratories. RESULTS: The average absorbance of reagent blank was 0.0476, and the average change rate of blank absorbance (deltaA/min) was 0.000168. The coefficients of variation (CV) for 3 seminal plasma samples with high, middle and low GGT activity detected for 10 times, respectively, were 0.26%, 4.83% and 1.60%. The accuracy of the automatic method was evaluated by a comparison test, and the relative deviation for each concentration point of 40 seminal plasma samples ranged from 13.38% to 11.05%, which met the requirement of < 15%. There was a good linear relationship (r > 0.99) when the seminal plasma GGT activity was between 299 and 1 833 U/L. A significant positive correlation was found between the seminal plasma GGT detection kit (a colorimetric method) as the control and the automatic method as the test reagent in the results of 115 seminal plasma samples (r = 0.981, P < 0.01), with a Kappa value of 0.776 (P < 0.05) and a coincidence rate of 90.43%. CONCLUSION: The established automatic method to detect seminal plasma GGT activity has a low reagent blank, good repeatability and accuracy, and fine concordance with the colorimetric method commonly used in clinical laboratories. It is simple, rapid and suitable for screening large numbers of samples, avoids the necessity of diluting the seminal plasma sample, and saves a lot of manpower and reagents.

[Influence of the depth of the sperm counting chamber on sperm motility].

OBJECTIVE: To investigate the influence of the depth of the sperm counting chamber on sperm motility. METHODS: We measured the depths of sperm counting chambers using the Filmetrics F20 Spectral Reflectance Thin-Film Measurement System. Then, according to the WHO5 manual, we analyzed 36 semen samples for the percentages of progressively motile sperm (PR) and non-progressively motile sperm (NP) and sperm motility (PR + NP) with the Ruiqi CFT-9201 computer-aided sperm analysis system, and compared the results of analysis. RESULTS: The depths of the 4 sperm counting chambers were 9.8, 12.7, 15.7 and 19.9 microm, respectively, and the obtained PR were (44.00 +/- 11.63), (41.96 +/- 12.62), (40.86 +/- 11.71) and (37.78 +/- 11.38)%, NP (13.54 +/- 3.01), (14.13 +/- 2.94), (14.91 +/- 3.02) and (16.53 +/- 2.77)%, and sperm motility (57.53 +/- 11.06), (56.08 +/- 11.97), (55.78 +/- 11.55) and (54.31 +/- 12.11)% from the 4 chambers, respectively. The depth of the sperm counting chamber was correlated negatively with PR (r = -0.993, P < 0.05) and sperm motility (r = -0.978, P < 0.05), but positively with NP (r = 0.989, P < 0.05). There were statistically significant differences between the 9.8 microm and 19.9 microm deep chambers in PR and NP (P < 0.05) though not in sperm motility among the 4 chambers of different depths. CONCLUSION: The impact of the depth of the sperm counting chamber on sperm motility should not be ignored, for the deviation of the results from the chambers of different depths may lead clinicians to incorrect diagnosis and consequently inappropriate therapeutic approaches. Different reference ranges of sperm motility need to be normalized in correspondence to the depths of sperm counting chambers.

Effect of hyperlipidemia on the outcome of in vitro fertilization in non-obese patients with polycystic ovary syndrome.

Introduction: It is little known whether hyperlipidemia alone has adverse effects on the outcome of in vitro fertilization (IVF) in patients with polycystic ovarian syndrome (PCOS). Methods: The PCOS patients with body mass index (BMI) < 30 kg/m2 were performed IVF or intracytoplasmic sperm injection treatment, including 208 fresh cycles and 127 frozen embryo transfer (FET) cycles. All the patients were divided into hyperlipidemia and control groups, and embryo quality and pregnancy outcomes between the two groups were compared. Results: In the fresh cycles, total gonadotropin dosage in the control group was significantly lower than that in the hyperlipidemia group, and serum estradiol levels on trigger day were reversed (P < 0.05). The embryo fragment score was positively correlated with serum low-density lipoprotein level (r = 0.06, P < 0.05) and negatively with serum high-density lipoprotein (HDL) and lipoprotein A levels (r = -0.489 and -0.085, P < 0.01). Logistic regression analysis found that HDL was beneficial for clinical pregnancy (OR = 0.355, 95% CI: 0.135-0.938, P < 0.05). In the FET cycles, there were no differences in pulse index, systolic/diastolic ratio and serum estradiol and progesterone levels between the two groups, but resistance index in the hyperlipidemia group was significantly higher than that in the control group (P < 0.05). Conclusion: Hyperlipidemia may increase the dosage of gonadotropin and have adverse effect on the embryo quality, endometrial receptivity, and clinical outcomes of lean PCOS patients. It is recommended that the non-obese patients with hyperlipidemia and PCOS perform lipid-lowering treatment before undergoing embryo transfer.