The Examination of Peroxidase-Positive Leukocytes in Semen.

Leukocytospermia can lead to decreased spermatozoa motility, increased spermatozoa morphological abnormalities, elevated spermatozoa DNA fragmentation index, impairment of the spermatozoa acrosome function, and even affected embryonic development. It is a common andrological disease in clinical practice and one of the important causes of male infertility. When determining whether male reproductive tract inflammation exists, andrologists often choose to examine round cells or seminal plasma elastase in the semen as a clinical diagnostic basis. However, the examination of round cells is easily influenced by sloughed spermatogenic cells and reproductive tract epithelial cells, which do not contribute to reducing the indiscriminate and unnecessary use of antibiotics. At the same time, the detection process of elastase is relatively complicated, time-consuming, and slow in reporting results, which is not beneficial for early diagnosis and treatment of diseases such as male genital tract infections (MGTIs). We have innovatively applied the examination of peroxidase-positive leukocytes in semen assisted by a computer-assisted semen analysis (CASA) system as a diagnostic criterion for leukocytospermia, successfully solving these problems. This examination only requires the addition of the operating fluid consisting of four reagents into the specimen, and the total reaction time at room temperature can be controlled within 20-30 min. With the subsequent smear and microscopic examination, the concentration of peroxidase-positive leukocytes in semen can be obtained within a total of 60 min, which can be used to diagnose whether the inflammation of the male reproductive tract existed.

The effect of luteinizing hormone changes in GnRH antagonist protocol on the outcome of controlled ovarian hyperstimulation and embryo transfer.

BACKGROUD: To investigate the effect of Luteinizing hormone (LH) level changes on the outcomes of controlled ovarian hyperstimulation (COH) and embryo transfer (ET) in gonadotropin-releasing hormone antagonist (GnRH-ant) protocol. METHODS: A total of 721 patients undergoing GnRH-ant protocol COH for the first IVF/ICSI cycles were retrospectively analyzed. COH process were divided into 2 stages, before (stage 1) and after (stage 2) the GnRH-ant initiation, and each with 5 groups basing on LH levels: LH decreased more than 50% (groups A1, A2), decreased 25-50% (groups B1, B2), change less than 25% (groups C1, C2), increased 25-50% (groups D1, D2), and increased more than 50% (groups E1, E2). RESULTS: There were no significant differences among groups of stage1 regarding COH and ET outcomes. For stage 2, the more obvious the decrease of LH level, the more the number of oocytes retrieved, mature oocytes, fertilized oocytes, embryos cleavaged and the numbers of embryo available (P < 0.05), but without significant differences regarding ET outcomes. We also found the freeze-all rate in Group A2 was higher (P < 0.001). CONCLUSION: LH level changes before GnRH-ant addition were not related to COH and ET outcomes. LH level changes after the addition of GnRH-ant were related to the outcome of COH, and no significant differences were found relating to ET outcomes.

Hematologic and spirometric characteristics of Tajik and Kyrgyz highlanders in the Pamir Mountains.

OBJECTIVES: In this study, we measured the hematologic and spirometric parameters of native Tajik and Kyrgyz highlanders in the Pamir Mountains to investigate adaptations to high altitude stressors. METHODS: Hematological parameters including arterial oxygen saturation (SaO2 ), red blood cell (RBC) counts, and hemoglobin (Hb) concentration were measured on Sarikoli Tajik (n = 80; 3100 m), Wakhi Tajik (n = 48; 3500 m), and Kyrgyz (n = 64; 3250 m) in comparison to lowland Uyghurs (n = 50; 1300 m). Spirometric parameters including forced vital capacity (FVC), the first second of forced expiration (FEV1), and forced expiratory flow between 25% and 75% (FEF25-75) were measured. We also reported mountain sickness symptoms in these highlanders and conducted a multivariate regression analysis to analyze the association between these symptoms and the measured parameters. RESULTS: SaO2 of Sarikoli Tajik, Wakhi Tajik, and Kyrgyz (91%-93.5%) are significantly lower than lowland Uyghurs, yet are comparable to other native highlanders at a similar altitude. RBC counts and Hb concentrations of all three highland populations are significantly increased compared to Uyghurs. FVC is lower in Sarikoli Tajik, Wakhi Tajik, and Kyrgyz (male: 3.48-3.86 L, female: 2.47-2.78 L) compared to Uyghurs. Combined with normal FEV1, elevated FEV1/FVC ratio, and FEF25-75, the spirometric patterns of these highlanders indicate restrictive lung disease. A high prevalence of mountain sickness symptoms such as headache and nausea was found in all three highland populations, and are attributed to low FVC and aging by regression analysis. CONCLUSION: Tajik and Kyrgyz highlanders showed adaptation in SaO2 , RBC, and Hb level, but poor performance in spirometry, which causes mountain sickness.

Thawed embryo transfer and ectopic pregnancy: a meta-analysis.

PURPOSE: To examine whether thawed embryo transfers can reduce the rate of EP. METHODS: The PubMed, EMBASE, Cochrane Library databases and two randomized controlled trials registration centers were thoroughly searched until March 2017. The clinical outcomes of IVF/ICSI cycles were compared between thawed and fresh embryo transfer. RESULTS: Twenty-one articles were included in this meta-analysis. There were 801,464 pregnancies totally (thawed-ET: n = 158,967, fresh-ET: n = 642,497). The ectopic pregnancy rate was significantly lower in the group of thawed-ET than that in the group of fresh-ET (OR 0.69, 95% CI 0.57-0.82; I2 = 83%). We subdivided the data into subgroups for D3 embryo transfer and D5 embryo transfer. We also found that the ectopic pregnancy rate was significantly lower with thawed-ET on D3 than that with fresh-ET (OR 0.67, 95% CI 0.53-0.85; I2 = 0%). The risk of ectopic pregnancy was significantly decreased with thawed-ET on D5 than that with fresh-ET (OR 0.57, 95% CI 0.50-0.64; I2 = 45%). CONCLUSION: Our results indicate that in contrast to fresh embryo transfers, thawed D3 or D5 embryo transfers can reduce the rate of EP.

A Universal Approach to Correct Various HBB Gene Mutations in Human Stem Cells for Gene Therapy of Beta-Thalassemia and Sickle Cell Disease.

Beta-thalassemia is one of the most common recessive genetic diseases, caused by mutations in the HBB gene. Over 200 different types of mutations in the HBB gene containing three exons have been identified in patients with ß-thalassemia (ß-thal) whereas a homozygous mutation in exon 1 causes sickle cell disease (SCD). Novel therapeutic strategies to permanently correct the HBB mutation in stem cells that are able to expand and differentiate into erythrocytes producing corrected HBB proteins are highly desirable. Genome editing aided by CRISPR/Cas9 and other site-specific engineered nucleases offers promise to precisely correct a genetic mutation in the native genome without alterations in other parts of the human genome. Although making a sequence-specific nuclease to enhance correction of a specific HBB mutation by homology-directed repair (HDR) is becoming straightforward, targeting various HBB mutations of ß-thal is still challenging because individual guide RNA as well as a donor DNA template for HDR of each type of HBB gene mutation have to be selected and validated. Using human induced pluripotent stem cells (iPSCs) from two ß-thal patients with different HBB gene mutations, we devised and tested a universal strategy to achieve targeted insertion of the HBB cDNA in exon 1 of HBB gene using Cas9 and two validated guide RNAs. We observed that HBB protein production was restored in erythrocytes derived from iPSCs of two patients. This strategy of restoring functional HBB gene expression will be able to correct most types of HBB gene mutations in ß-thal and SCD. Stem Cells Translational Medicine 2018;7:87-97.

The impact of luteinizing hormone supplementation in gonadotropin-releasing hormone antagonist cycles: a retrospective cohort study.

The impact of exogenous luteinizing hormone (LH) supplementation to patients undergoing controlled ovarian stimulation with gonadotropin-releasing hormone (GnRH) antagonists on cycle outcomes is controversial. Here, we present a retrospective cohort study including cycles from December 2015 to December 2016. Totally 320 cycles were divided into two groups according to with or without exogenous LH supplementation. No significant differences regarding the number of retrieved oocytes, the number of good-quality embryos, and clinical pregnancy rate between the two groups were found. The logistic regression analysis revealed that LH supplementation was not independently associated with clinical pregnancy rate (OR = 0.577, 95% CI: 0.272-1.222, p = .58) or a biochemical pregnancy rate (OR = 0.922, 95% CI: 0.444-1.916, p = .83). When patients were divided into subgroups based on age, more retrieved oocytes (5.60 vs. 3.97, p = .04) and good-quality embryos (3.07 vs. 1.93, p = .01) were achieved in cycles with exogenous LH supplementation for 40 years and over group. We conclude that for aged women (40 years old and over), LH supplementation has a positive impact on the number of retrieved oocytes and good-quality embryos in GnRH antagonist cycles.

Mitochondrial genomes uncover the maternal history of the Pamir populations.

The Pamirs, among the world's highest mountains in Central Asia, are one of homelands with the most extreme high altitude for several ethnic groups. The settlement history of modern humans on the Pamirs remains still opaque. Herein, we have sequenced the mitochondrial DNA (mtDNA) genomes of 382 individuals belonging to eight populations from the Pamirs and the surrounding lowlands in Central Asia. We construct the Central Asian (including both highlanders and lowlanders) mtDNA haplogroup tree at the highest resolution. All the matrilineal components are assigned into the defined mtDNA haplogroups in East and West Eurasians. No basal lineages that directly emanate from the Eurasian founder macrohaplogroups M, N, and R are found. Our data support the origin of Central Asian being the result of East-West Eurasian admixture. The coalescence ages for more than 93% mtDNA lineages in Central Asians are dated after the last glacial maximum (LGM). The post-LGM and/or later dispersals/admixtures play dominant roles in shaping the maternal gene pool of Central Asians. More importantly, our analyses reveal the mtDNA heterogeneity in the Pamir highlanders, not only between the Turkic Kyrgyz and the Indo-European Tajik groups, but also among three highland Tajiks. No evidence supports positive selection or relaxation of selective constraints in the mtDNAs of highlanders as compared to that of lowlanders. Our results suggest a complex history for the peopling of Pamirs by multiple waves of migrations from various genetic resources during different time scales.

Deriving cells expressing markers of female germ cells from premature ovarian failure patient-specific induced pluripotent stem cells.

AIM: We proposed a two-step protocol for deriving cells expressing markers of female germ cells (FGCs) from premature ovarian failure patient-specific induced pluripotent stem cells (POF-iPSCs). MATERIAL & METHODS: We cultured POF-iPSCs in suspension and pretreated them with TGFß-1 (1 ng/ml) for 2 days and continued with both TGFß-1 and BMP4 (50 ng/ml) for 5 more days. Then changed to media containing retinoic acid (1 µM) and 5% follicular fluid for another 7 days. Expression of markers of different stages of FGCs were detected. RESULTS: c-KIT, STELLA/DPPA3, VASA/DDX4, SCP3, GDF9 and ZP3 were positively detected and statistically significant different when compared with control groups. CONCLUSION: Our in vitro system was beneficial for POF-iPSCs differentiated cells to express STELLA, VASA and SCP3, which were the markers of meiosis initiation of FGCs.

Genome editing systems in novel therapies.

Genome editing is the process in which DNA sequences at precise genomic locations are modified. In the past three decades, genome editing by homologous recombination has been successfully performed in mouse for generating genetic models. The low efficiency of this process in human cells, however, had prevented its clinical application until the recent advancements in designer endonuclease technologies. The significantly improved genome editing efficiencies aided by ZFN, TALEN, and CRISPR systems provide unprecedented opportunities not only for biomedical research, but also for developing novel therapies. Applications based on these genome editing tools to disrupt deleterious genes, correct genetic mutations, deliver functional transgenes more effectively or even modify the epigenetic landscape are being actively investigated for gene and cell therapy purposes. Encouraging results have been obtained in limited clinical trials in the past two years. While most of the applications are still in proof-of-principle or preclinical development stages, it is anticipated that the coming years will see increasing clinical success in novel therapies based on the modern genome editing technologies. It should be noted that critical issues still remain before the technologies can be translated into more reliable therapies. These key issues include off-target evaluation, establishing appropriate preclinical models and improving the currently low efficiency of homology-based precise gene replacement. In this review we discuss the preclinical and clinical studies aiming at translating the genome editing technologies as well as the issues that are important for more successful translation.

Doctors' Preferences for Controlled Ovarian Stimulation Protocols in Intrauterine Insemination.

INTRODUCTION: Intrauterine insemination (IUI) is an important treatment for infertility. IUI combined with controlled ovarian stimulation (COS) is widely used because of the higher pregnancy rates compared to IUI cycles without COS. MATERIAL AND METHODS: We retrospectively analyzed a single center data from 458 patients underwent the first IUI cycle and had only 1 mature follicle from May 2009 to January 20144. 48 cycles were performed with Clomiphene citrate/Letrozole (CC/LE), 244 cycles with gonadotropins (Gn), 71 cycles with CC/LE+Gn, and 95 cycles in NC group. RESULTS: Results showed that doctors preferred Gn protocol (53.3%) (p<0.05). Older patients were more likely to be allocated to CC/LE or NC group. 98.95% patients in NC group had regular menstruation cycle, with only 49.3% in CC/LE+Gn group (p<0.05). Estradiol (E2) level was much higher in COS groups than in NC group (p<0.05, for one mature follicle patients), and no significant differences were found within the COS groups. Duration of reaching follicles maturation was the shortest in Gn group and the longest in NC group, and NC group has the smallest follicular diameter (p<0.05). CONCLUSION: No significances were found regarding the IUI outcomes. To sum up, doctors prefer COS for IUI. Patients' age, menstruation cycle, infertile etiology and ovary function were the main factors affecting doctors' selection of COS protocols.

Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis.

In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Forensic Identification for Erectile Dysfunction: Experience of a Single Center.

OBJECTIVE: To analyze the characteristics of forensic identification cases and evaluate the importance of integrating penile erection length, angle, and rigidity in diagnosing erectile dysfunction (ED). METHODS: Retrospective analysis of the forensic identification cases between Jan 2009 and May 2013. Correlation between ED diagnosis and nocturnal penile tumescence (NPT) test result or the site of injury was analyzed. RESULTS: In total, 148 patients came for forensic identification of sexual function because of rape charges, divorce, medical accidents, or injury: 126 of 148 (85.1%) because of injury, of which 95 (75.4%) resulted from traffic accidents. There was a significant correlation between the site of injury and ED diagnosis; pelvic fracture with urethral or perineum injury was the most common. Our data showed that ED diagnosis was in general significantly associated with NPT results. However, we also identified three cases of diagnosed organic ED with normal NPT reactions. Our analyses showed that abnormal length and/or angle of the erectile penis were contributing factors to the diagnoses in these cases. CONCLUSION: In addition to NPT test, which measures the rigidity of the erectile penis, the length and angle of the erectile penis should also be considered in diagnosing ED, particularly in the case of forensic identification of sexual function.

Effect of sperm count on success of intrauterine insemination in couples diagnosed with male factor infertility.

OBJECTIVE: To exam semen parameters in predicting intrauterine insemination (IUI) outcomes in couples with male factor. STUDY DESIGN: This retrospective study was performed at department of infertility and sexual medicine from September 2007 to February 2014. 307 couples with male factor infertility were included and 672 IUI cycles were analyzed. RESULTS: From 672 inseminations performed on 307 couples, there are 27.36% couples get pregnancy (84 out of 307) and the overall pregnancy rate was 12.95% (87 out of 672) of IUI. With the increase of post total progressive sperm count, the clinical pregnancy rate increased. When the initial progressive sperm count was lower than 5*10(6), there was no pregnant in the IUI cycle. At the end of the third cycle, 85 clinical pregnancies had been achieved (97.70%). CONCLUSIONS: The initial total progressive sperm count lower than 5*10(6) means the poor outcome of IUI in the infertile couples with male factor. If the infertile couples with male factor don't get pregnancy after three IUI cycles, the couples should receive re-assessment or other artificial reproductive technology.

Treatment of penile deep dorsal venous leakage of erectile dysfunction by embedding the deep dorsal vein of the penis: a single center experience with 17 patients.

INTRODUCTION: The common surgery for venous leakage was not very successful; unsatisfactory long-term results have reduced the indications for venous surgery for erectile dysfunction (ED). AIMS: To assess the outcomes of embedding the deep dorsal vein of the penis (EDDVP), a new surgical technique used in patients with penile deep dorsal venous leakage of ED. METHODS: Between December 2001 and November 2007, 17 patients diagnosed with penile deep dorsal venous leakage of ED underwent embedding the deep dorsal vein of the penis. MAIN OUTCOME MEASURES: All cases were available for follow up by using the abridged 5-item version of the International Index of Erectile Function (IIEF-5) scoring system and penile color Doppler ultrasound. Dynamic cavernosography were also assessed in three patients at 3 months postoperatively. RESULTS: After surgery, 14 patients were able to achieve satisfactory intercourse and three had sufficient erection after oral sildenafil (50-100 mg). The IIEF-5 scoring changed from a preoperative mean IIEF-5 score of 8.8 +/- 3.9 to 20.8 +/- 4.1 (P < 0.05). Peak systolic velocity (average of right and left cavernosal arteries) changed from 41.9 +/- 7.7 cm/second to 44.2 +/- 9.2 cm/second (P > 0.05), resistance index changed from 0.79 +/- 0.1 to 1.00 +/- 0.0 (P < 0.05), and venous velocity changed from 8.4 +/- 4.0 cm/second to 0.0 +/- 0.0 cm/second (P < 0.05). Dynamic cavernosography demonstrated a smooth flow of the deep dorsal vein during the flaccid phase. During the tumescent phase, the deep dorsal vein of the penis was compressed between the dilated sinusoidal spaces and the tunica albuginea and resulted in venous drainage blockade. And then the hardness of erection was improved and maintained. CONCLUSIONS: The new surgical technique of EDDVP is a simple operative procedure, which seems to be efficient in the treatment of penile deep dorsal venous leakage of ED.

Promoting human embryonic stem cell renewal or differentiation by modulating Wnt signal and culture conditions.

We previously showed that Wnt3a could stimulate human embryonic stem (hES) cell proliferation and affect cell fate determination. In the absence of feeder cell-derived factors, hES cells cultured under a feeder-free condition survived and proliferated poorly. Adding recombinant Wnt3a in the absence of feeder cell derived-factors stimulated hES cell proliferation but also differentiation. In the present study, we further extended our analysis to other Wnt ligands such as Wnt1 and Wnt5a. While Wnt1 displayed a similar effect on hES cells as Wnt3a, Wnt5a had little effect in this system. Wnt3a and Wnt1 enhanced proliferation of undifferentiated hES cells when feeder-derived self-renewal factors and bFGF are also present. To explore the possibility to promote the proliferation of undifferentiated hES cells by activating the Wnt signaling, we overexpressed Wnt3a or Wnt1 gene in immortalized human adult fibroblast (HAFi) cells that are superior in supporting long-term growth of undifferentiated hES cells than primary mouse embryonic fibroblasts. HAFi cells with or without a Wnt transgene can be propagated indefinitely. Over-expression of the Wnt3a gene significantly enhanced the ability of HAFi feeder cells to support the undifferentiated growth of 3 different hES cell lines we tested. Co-expression of three commonly-used drug selection genes in Wnt3a-overpressing HAFi cells further enabled us to select rare hES clones after stable transfection or transduction. These immortalized engineered feeder cells (W3R) that co-express growth-promoting genes such as Wnt3a and three drug selection genes should empower us to efficiently make genetic modified hES cell lines for basic and translational research.