The extent of spermatogenic impairment on intracytoplasmic sperm injection (ICSI) outcomes and the risk of major birth defects have been little assessed. In this study, we evaluated the relationship between various spermatogenic conditions, sperm origin on ICSI outcomes, and major birth defects. A total of 934 infertile men attending the Center for Reproductive Medicine of Ren Ji Hospital (Shanghai, China) were classified into six groups: nonobstructive azoospermia (NOA; n = 84), extremely severe oligozoospermia (esOZ; n = 163), severe oligozoospermia (sOZ, n = 174), mild oligozoospermia (mOZ; n = 148), obstructive azoospermia (OAZ; n = 155), and normozoospermia (NZ; n = 210). Rates of fertilization, embryo cleavage, high-quality embryos, implantation, biochemical and clinical pregnancies, abortion, delivery, newborns, as well as major birth malformations, and other newborn outcomes were analyzed and compared among groups. The NOA group showed a statistically lower fertilization rate (68.2% vs esOZ 77.3%, sOZ 78.0%, mOZ 73.8%, OAZ 76.6%, and NZ 79.3%, all P < 0.05), but a significantly higher implantation rate (37.8%) than the groups esOZ (30.1%), sOZ (30.4%), mOZ (32.6%), and OAZ (31.0%) (all P < 0.05), which was similar to that of Group NZ (38.4%). However, there were no statistically significant differences in rates of embryo cleavage, high-quality embryos, biochemical and clinical pregnancies, abortions, deliveries, major birth malformations, and other newborn outcomes in the six groups. The results showed that NOA only negatively affects some embryological outcomes such as fertilization rate. There was no evidence of differences in other embryological and clinical outcomes with respect to sperm source or spermatogenic status. Spermatogenic failure and sperm origins do not impinge on the clinical outcomes in ICSI treatment.

Reciprocal communication between Sertoli and Leydig cells occurs in the testes; however, the detailed mechanisms involved are not completely understood. Exosomes can communicate within neighboring or distant cells to regulate cell function. Our aim was to determine whether exosomes released from Sertoli cells can regulate the survival of Leydig cells. We found that exosomes released from rat primary Sertoli cells could be internalized by Leydig cells in vitro, and promote the survival of Leydig cells, as assessed by optical density at 450 nm, compared to untreated control (mean ± SD: 0.95 ± 0.04 vs 0.79 ± 0.03, P < 0.05). When the exosomes were injected into the interstitial area of rat testis, they could also be internalized by Leydig cells in vivo. To investigate if exosomes released from Sertoli cells can reach Leydig cells in vivo, exosomes were injected into the efferent duct, from where they entered the interstitial space from seminiferous tubules, which indicated that they may cross the blood-testis barrier (BTB). Further in vitro studies found that exosomes released from Sertoli cells significantly increased CC-chemokine ligand 20 (Ccl20) mRNA (mean ± SD: 2.79 ± 0.08 vs 0.98 ± 0.04, P < 0.01) and protein (mean ± SD: 1.08 ± 0.06 vs 0.53 ± 0.05 ng/ml, P < 0.01) levels in Leydig cells, compared to the untreated Leydig cells. CCL20 promoted the phosphorylation of AKT (protein kinase B) in Leydig cells, compared to untreated control (mean ± SD: 0.074 ± 0.002 vs 0.051 ± 0.002, P < 0.01). In conclusion, our results demonstrated that exosomes released by Sertoli cells may cross the BTB and promote the survival of Leydig cells. The findings may add new evidence for Sertoli-Leydig cell communication.

BACKGROUND:Varicocoele-associated stressors, such as hypoxia and heat, can damage cell function and viability, and some exosomal biomarkers released from impaired cells may reflect the cell status in testis.OBJECTIVES:To find if seminal exosomal microRNAs can reflect the Sertoli cell function in varicocoele.MATERIALS AND METHODS:Experimental left varicocoele rat model was established (n = 24), and patients with different grades of varicocoele (n = 104) were enrolled. Primary rat Sertoli cells were isolated with enzymatic hydrolysis. Exosomes were isolated from primary rat Sertoli cells, rat epididymis tissue, and human seminal plasma with polymer-based precipitation method. Exosomal microRNAs were quantified with qPCR. Inhibin-B was detected with enzyme immunoassay. The correlation analysis between microRNA and inhibin-B was evaluated with Spearman's correlation.RESULTS:We screened 12 previously reported hypoxia-responsive microRNAs in the primary rat Sertoli cells and found that 4 exosomal microRNAs increased significantly in response to in vitro hypoxia treatment (P < .05). Of the 4 microRNAs, only miR-210-3p was upregulated in the rats with experimental varicocoele (P < .01). In the patients with varicocoele, we found that seminal exosomal miR-210-3p significantly increased in patients with grade II and III varicocoele (P < .01), and miR-210-3p negatively correlated with sperm count (P < .01) and seminal inhibin-B expression (r = -0.39, P < .01). For the 30 patients with microsurgical varicocelectomy, the operation notably decreased miR-210-3p (P < .01).DISCUSSION AND CONCLUSION:Seminal exosomal miR-210-3p may be a novel, sensitive, and non-invasive biomarker of Sertoli cell damage in varicocoele.

Background The purpose of the study was to compare clinical, laboratory, histological features, microdissection testicular sperm extraction (MicroTESE) and intracytoplasmic sperm injection (ICSI) treatment outcomes of nonobstructive azoospermia (NOA) patients of various etiologies, and to investigate ICSI outcomes using fresh and frozen thawed sperms from MicroTESE, so to explore an optimal MicroTESE-ICSI procedure for NOA couples. Methods A retrospective analysis was made in 595 NOA patients undergoing MicroTESE from January 2013 to December 2017. The men were classified into six groups based on etiology. Patients' age, history, hormone profile, testis volume, testicular histology, sperm retrieval, fertile and pregnancy outcomes of ICSI were included for analysis. Results A total of 595 NOA patients were included in this study, with 446 (75.0%) were idiopathic NOA, 66 (11.1%) were Klinefelter syndrome (KS), 34 (5.7%) with microdeletion of the AZFc, 33 (5.5%) cases had the history of cryptorchidism, 13 (2.2%) had a history of mumps orchitis, and 3 (0.5%) cases underwent chemotherapy. The overall sperm retrieval rate (SRR) was 40.3% (240/595), SRR of the cryptorchidism (84.8%, 28/33) and mumps orchitis (84.6%, 11/13) groups were much higher than that of other groups, the SRR of idiopathic group was the lowest (31.8%, 142/446). One hundred and ninety-eight ICSI cycles utilizing MicroTESE sperm were retrospectively analyzed, including 155 fresh MicroTESE ICSI cycles and 43 frozen-thawed MicroTESE ICSI cycles. Fertilization rate, cleavage rate, and clinical pregnancy rate of fresh sperm group were slightly higher than those in frozen thawed MicroTESE sperm group, but high qualified embryo rate of fresh sperm group was lower than frozen thawed group. The differences were of no statistical meaning. Conclusions Etiology may be an effective prognostic factor for SRR in NOA patients. NOA of definite etiology, such as cryptorchidism, has high SRR, while idiopathic NOA, the most common type of NOA, has the lowest SRR. Using of frozen thawed sperm from MicroTESE had similar pregnant outcome to that of fresh sperm, so cryopreservation of testicular sperm seems to be more suitable and of great benefit in these cases and good results can also be expected when oocyte retrieval and ICSI are not performed at the same time.

目的·探索伐地那非预处理对精子冷冻复苏后活力的影响.方法·在上海市人类精子库随机收集30例捐献者的精液标本,并将收集的每例精液平均分成6份,1份用于冷冻前指标测定,其余5份分别用0、0.4、4.0、40.0、400.0 μg/mL的伐地那非孵育5 min.按精液与保护剂2∶1的比例添加保护剂,采用一步熏蒸法进行冷冻,冷冻复苏后分别检测每份标本的前向运动精子、精子活力及正常精子形态等参数.结果·复苏后,0、0.4、4.0、40.0、400.0 μg/mL伐地那非处理后的前向运动精子百分率分别为(41.47±9 80)％、(42.57±9 60)％、(47.77±8.55)％、(37 27±8 47)％、(26.37±6 99)％.与对照组(0μg/mL伐地那非组)比较,4.0 μg/mL伐地那非处理后的精子复苏后活力有显著改善,差异有统计学意义(P=0.034).结论·适当浓度的伐地那非预处理精液能提高精子冷冻复苏后的活力;然而伐地那非浓度过大,可能对精子有毒害作用.

Cryopreservation of few spermatozoa is still a major challenge for male fertility preservation. This study reports use a new micro-straw (LSL straw) for freezing few spermatozoa for intracytoplasmic sperm injection (ICSI). Semen samples from 22 fertile donors were collected, and each semen sample was diluted and mixed with cryoprotectant in a ratio of 1:1, and then frozen using three different straws such as LSL straw (50-100 μl), traditional 0.25 ml and 0.5 ml straws. For freezing, all straws were fumigated with liquid nitrogen, with temperature directly reducing to −130-−140°C. Sperm concentration, progressive motility, morphology, acrosome integrity, and DNA fragmentation index were evaluated before and after freezing. After freezing-thawing, LSL straw group had significantly higher percentage of sperm motility than traditional 0.25 ml and 0.5 ml straw groups (38.5% vs 27.4% and 25.6%, P < 0.003). Sperm motility and acrosomal integrity after freezing-thawing were significantly lower than that of before freezing. However, there was no significant difference in morphology, acrosome, and DNA integrity between the three types of straws (P > 0.05). As LSL straws were thinner and hold very small volume, the freezing rate of LSL straw was obviously faster than 0.25 ml straw and 0.5 ml straws. In conclusion, LSL micro-straws may be useful to store few motile spermatozoa with good recovery of motility for patients undergoing ICSI treatment.

目的 探讨中国汉族人群HLA-DRA基因rs7194位点多态性与非梗阻性无精症(NOA)遗传易感性的关系.方法 采用PCR产物直接测序的方法对275例NOA患者(病例组)和221例健康对照(对照组)进行基因分型,比较两组基因型和等位基因分布频率的差异.采用logistic回归分析方法探讨NOA的易感基因型,并采用meta分析方法进一步验证rs7194位点多态性与NOA疾病是否相关.结果 rs7194位点的基因型频率和等位基因频率在病例组与对照组之间分布的差异均有统计学意义(P =0.003,P=0.001).Logistic回归结果显示,相比于A/A基因型,A/G基因型和G/G基因型与NOA的患病风险增加相关(OR=1.837,P=0.002;OR=1.942,P=0.048).Meta分析结果也表明HLA-DRA基因rs7194位点多态性与NOA相关.结论 HLA-DRA基因rs7194位点的多态性与中国汉族人群NOA易感性有关;携带G等位基因且为A/G或G/G基因型患NOA的倾向性增大,可能是患NOA的风险因素之一.

Piwi-interacting RNAs (piRNAs) are a broad group of noncoding small RNAs that have important biological functions in germline cells and can maintain genome integrity via silencing of retrotransposons. In this study, we aimed to explore the associations between genetic variants of important genes involved in piRNA biogenesis and male infertility with spermatogenic impairment.

Vascular endothelial growth factor (VEGF) plays fundamental roles in testicular development; however, its function on testicular regeneration remains unknown. The objective of this study was to explore the roles VEGF/VEGFR2 signaling plays in mouse germ cells and in mouse testicular regeneration. VEGF and the VEGFR2 antagonist SU5416 were added to culture medium to evaluate their effects on spermatogonial stem cell line (C18-4 cells) proliferation. Testicular cells obtained from newborn male ICR mice were grafted into the dorsal region of male BALB/c nude mice. VEGF and SU5416 were injected into the graft sites to assess the effects of the VEGF and VEGFR2 signaling pathways on testicular reconstitution. The grafts were analyzed after 8 weeks. We found that VEGF promoted C18-4 proliferation in vitro, indicating its role in germ cell survival. HE staining revealed that seminiferous tubules were reconstituted and male germ cells from spermatogonia to spermatids could be observed in testis-like tissues 8 weeks after grafting. A few advantaged male germ cells, including spermatocytes and spermatids, were found in SU5416-treated grafts. Moreover, VEGF enhanced the expression of genes specific for male germ cells and vascularization in 8-week grafts, whereas SU5416 decreased the expression of these genes. SU5416-treated grafts had a lower expression of MVH and CD31, indicating that blockade of VEGF/VEGFR2 signaling reduces the efficiency of seminiferous tubule reconstitution. Collectively, these data suggest that VEGF/VEGFR2 signaling regulates germ cell proliferation and promotes testicular regeneration via direct action on germ cells and the enhancement of vascularization.

目的 初步研究β-catenin蛋白在不同类型睾丸肿瘤中的表达特性,并探讨β-catenin分子的表达与睾丸肿瘤的关系,为临床诊断或治疗睾丸肿瘤提供一定依据.方法 随机收集我院于2012年1月至2012年12月期间确诊的21例睾丸肿瘤组织和1例梗阻性无精子症(OA)患者的睾丸活检组织(作为正常对照,其睾丸生精功能正常);同时,选取5只9周龄的ICR小鼠作为实验补充.采用了免疫组化、Western blots和RT-qPCR等方法来检测β-catenin的表达情况.结果 在梗阻性患者中,β-catenin在精原细胞的胞膜和胞浆中呈现低表达;在小鼠中,β-catenin也仅表达在精原细胞的胞浆和胞膜上.在睾丸肿瘤组织中,睾丸孤立性纤维肿瘤中可检测到β-catenin的mRNA和蛋白的高表达,分别为OA患者的9.4倍和17倍,且其β-catenin主要异位表达在细胞核中,可检测到磷酸化的β-catenin的表达.然而,β-catenin在睾丸精原细胞肿瘤和间质细胞肿瘤中不表达或低表达.结论 本研究初步表明β-catenin与正常的睾丸生精过程及病理的睾丸孤立性纤维肿瘤发生发展相关,而与睾丸精原细胞肿瘤和间质细胞肿瘤之间无明显相关性.但仍需扩大样本数量进行研究,或采用动物实验深入研究.

精子鞭毛是成熟精子的动力器官,其基于微管的轴丝结构在进化过程中高度保守.精子鞭毛结构和功能相关蛋白质的缺陷或缺失,均会导致精子运动功能障碍,进而导致男性不育.该文对精子尾部形成及动力相关分子的最新研究进展进行综述,这类分子结构与功能的障碍与临床许多疾病息息相关.

Recent studies on the eNOS gene and male infertility show that expression of eNOS regulates normal spermatogenesis in the testis, and the eNOS gene variants (T-786C, 4a4b, and G894T) are potentially involved in impairment of spermatogenesis and sperm function. Thus, we conducted this association and meta-analysis study to further validate whether variants of those three loci affected the risk of idiopathic asthenozoospermia (AZS) and male infertility. Approximately 340 Chinese idiopathic AZS patients and 342 healthy men were included for this case-control study, genotyped by gel electrophoresis analysis or direct sequencing of PCR products. The eNOS mRNA isolated from the semen of patients was further examined by quantitative real-time PCR. Also, a meta-analysis of association between eNOS gene polymorphisms and male infertility was performed. A significant association was identified on allelic level between 4a4b variant and AZS in our study (chi-squared = 7.53, corrected P = 0.018, odds ratio (OR) = 1.808), while there were no significant difference of T-786C and G894T for asthenozoospermia in both genotype and allele distributions. In addition, expression of eNOS was up-regulated in patients compared with controls (about 2.4-fold, P < 0.001). Furthermore, the results of the meta-analysis support the conclusion that the T-786C and 4a4b loci were associated with male infertility in both Asian and Caucasian populations. Our study provides genetic evidence for the eNOS gene being a risk factor for idiopathic AZS and male infertility. Considering genetic differences among populations and complex pathogenesis of male infertility, more validating studies using independent samples are suggested in the future.

BACKGROUND:Indian hedgehog (Ihh) signaling pathway is known to play key roles in various aspects of normal endochondral bone development. This study tested the potential roles of high Ihh signaling in the context of injury-induced bone regeneration.METHODS:A rabbit tibia defect model was established to test the effects of the implant of Ihh/mesenchymal stem cells (MSCs)/scaffold complex. Computed tomography (CT), gross observation, and standard histological and immunohistological techniques were used to evaluate the effectiveness of the treatment. In vitro studies with MSCs and C3H10T1/2 cells were also employed to further understand the cellular and molecular mechanisms.RESULTS:We found that the implanted Ihh/MSCs/scaffold complex promoted bone repair. Consistently, in vitro study found that Ihh induced the upregulation of chondrocytic, osteogenic, and vascular cell markers, both in C3H10T1/2 cells and MSCs.CONCLUSIONS:Our study has demonstrated that high Ihh signaling in a complex with MSCs enhanced bone regeneration effectively in a clinically relevant acute injury model. Even though the exact underlying mechanisms are still far from clear, our primary data suggested that enhanced chondrogenesis, osteogenesis, and angiogenesis of MSCs at least partially contribute to the process. This study not only has implications for basic research of MSCs and Ihh signaling pathway but also points to the possibility of direct application of this specific paradigm to clinical bone repair.

Objective The previous genome-wide association study (GWAS) of non-obstructive azoospermia (NOA) in the Han Chinese populations identified two NOA-risk loci (rs498422 and rs3129878) within the HLA region, and provided strong evidence for the genetic influence of male infertility. A further case-control study found that only rs3129878 remained to be significantly associated with NOA in the Japanese population. Therefore, we conducted the association study to further validate whether the risk of NOA caused by these two SNPs was still existed in an independent Han Chinese male population, consisting of 550 NOA cases and 555 normal controls.

近年来，研究发现人类免疫缺陷病毒（Human immunodeficiency virus ，HIV）感染人数增多并呈现低龄化的趋势。HIV感染人体后，主要攻击、破坏CD4+T淋巴细胞，经过一段时间（一般是6~8年）的潜伏期，人体细胞免疫功能下降而导致机体发生一系列机会性感染或肿瘤，最终人体丧失对各种疾病的抵抗能力而导致死亡[1]，同时，大量研究发现HIV感染可造成精子活力、精子活率、精子密度、精子总数降低和畸形率增加[2]；并且HIV可通过父婴垂直传播。HIV病毒也可感染人体各期生殖细胞，其中，病毒颗粒是通过结合在精子细胞膜上和（或）存在于精子胞浆中，使病毒RNA整合到宿主精子基因组内。因而，受精时携带HIV 基因的人类精子会进入正常卵母细胞。在胚胎发育过程中， HIV 基因随宿主细胞分裂复制和表达，从而使子代患病[3]。因此，临床上对精液捐献者进行HIV检测意义重大，它可大大减少供精人工授精及体外受精后代患AIDS的风险，从而在一定程度上可降低HIV的传播。

A previous genomewide association study of nonobstructive azoospermia (NOA) in the Han Chinese population identified three risk loci (rs12097821, rs2477686, and rs10842262) and provided strong evidence for a genetic influence in male infertility. However, recently, a follow-up study of these single nucleotide polymorphism (SNP) loci in the Japanese population showed that none of them were significantly associated with NOA. Therefore, we conducted an association study, consisting of 550 NOA cases and 555 normal controls, to further validate whether the risk of those three SNPs still existed in an independent Han Chinese male population. The association studies did not support the association of rs12097821 and rs2477686 with NOA for both genotype and allele distributions, but rs10842262 in the SOX5 gene was significantly associated with NOA (chi square = 9.31; P = 0.0095 and chi square 9.27; P = 0.0023, respectively). Our study provides genetic evidence for SOX5 polymorphism in NOA, contributing to predicting males at high risk of NOA in Han Chinese population. Considering genetic differences among populations, future validating studies in independent samples are suggested.

A 30-year-old man presented with a left undescended testis, right testicular deficiency and azoospermia. Testicular biopsy revealed an absence of spermatocytes and increased numbers of Leydig cells in the undescended testis. Additional comparative analyses were undertaken to explore Sonic Hedgehog (Shh) immunostaining in the testis of juvenile and adult mice, in the testis of the patient with cryptorchidism, and in archival testicular tissue from a patient with obstructive azoospermia and a patient with prostate cancer. Shh immunostaining was demonstrated in spermatocytes in juvenile and adult mouse testis and in the patients with obstructive azoospermia and prostate cancer, suggesting that Shh signalling is involved in normal spermatogenesis. In the patient with cryptorchidism, Shh immunostaining was localized to the Leydig cells, which suggests that Shh might be involved in the abnormal expansion of the Leydig cell population in the testis. These preliminary data on the appearance of Shh protein during normal spermatogenesis might provide the basis for further investigations to clarify the role of Shh signalling in spermatogenesis during normal and pathogenic testis development.

Cilia serve as an important regulator of various signaling pathways,as well as the Hedgehog(Hh) signaling pathway.Recent studies have revealed that there is a close relationship between Hh signaling pathway and intraflagellar transport of cilia,and further evidences from genetics and cytobiology have also proved the relationship between primary cilia and Hh signaling pathway.All of the core components of Hh signaling pathway are found on the primary cilia,and intraflagellar transport proteins are required for the transmission of information between the signaling molecules that mediate the activity of Hh signaling pathway.Flagella of sperm share the same structure with cilia,and ciliopathies can also lead to the defects of spermatic flagella.Ciliopathies and other diseases caused by the defects of structure and function of cilia could affect Hh signaling.Regulatory molecules such as Foxj1 contribute to the construction of cilia,which brings new ideas in the field of infertility and contraception.The research progress of regulatory mechanism of Hedgehog signal in cilia is introduced in this paper.

OBJECTIVE:To investigate the effect of pre-freezing equilibration on the cryo-survival of human sperm and to optimize the protocol of direct fumigation for the freeze-thawing of human sperm.METHODS:We collected 50 semen samples from healthy donors, each subjected to cryopreservation with 3 different methods: non-equilibration freezing (Group A), 10-min equilibration at room temperature before freezing (Group B), and 10-min equilibration at 4 degrees C before freezing (Group C). We examined all the post-thaw semen samples by computer-assisted semen analysis for the sperm motility parameters, and detected the sperm vitality and deformity index (SDI).RESULTS:The recovery rate of progressive sperm motility was (61.88 +/- 16.94)% in Group C, remarkably higher than in A ([48.61 +/- 16.44]%) and B ([49.41 +/- 13.77]%) (P < 0.05), but with no significant difference between the latter two. And there were no significant differences in sperm vitality and SDI among the three groups.CONCLUSION:Ten-minute equilibration at 4 degrees C before freezing can evidently improve the progressive motility of sperm in addition to its advantages of easy operation and controllable experimental condition.

BACKGROUND: Recently, autologous iliac crest bone graft is considered to be the “gold standard” therapeutic method for bone defects. However, there are some complications in iliae crest bone donor site, and the clinical application is limited. OBJECTIVE: To compiled analyze the published articles and evaluate the types of complications and morbidity fol owing autologous iliac crest bone graft by employing evidence-based medicine method. METHODS: A computer-based online search in PubMed Medline database, Ovid Medline database, Cochrane database and Embase database was performed using key words of “Iliac crest bone graft”,“autologous bone graft”, “bone graft donor site”, “complication” or “morbidity” in English. The published dates were limited between January 2002 and December 2011. A total of 174 articles were searched. Then 30 articles, involving 2476 patients, were selected for the clinical Meta analysis according to retrieval conditions and inclusion criteria. Al relevant data were extracted and analyzed using SPSS 13.0 and R software and its Meta package. RESULTS AND CONCLUSION: The result indicated that the complications of autologous iliac crest bone graft in donor site included chronic pain (≥6 months) [incidence rates=7.88%,95% confidence interval (CI) 4.76-12.79], infection(incidence rate=4.26%, 95% CI 2.95-6.12]), hematoma or seroma (incidence rate=6.55%, 95% CI 4.90-8.70), nerve injury (incidence rates=5.85%, 95% CI 3.46-9.71), sensory disturbance (incidence rate=10.1%, 95% CI 6.07-16.23) respectively. The postoperative complications of autologous iliac crest bone graft are common; therefore, it should be emphasized by clinicians.