精子DNA完整性与体外受精-胚胎移植临床结局的相关性研究

目的 探讨精子DNA完整性与精液参数及体外受精-胚胎移植（IVF-ET）/卵胞浆内单精子注射（ICSI）临床结局的关系.方法 选择2008年6月～2009年6月在解放军105医院生殖医学中心接受IVF/ICSI治疗的179对不育夫妇作为研究对象,采用吖啶橙试验（AOT）对116例实施IVF和63例实施ICSI治疗的男性患者进行精子DNA完整性分析,根据精子DNA碎片指数（DFI）将患者分为DFI≤30%组和DFI＞30%组,比较两组间精液参数、受精率、卵裂率、优胚率、胚胎冷冻率、着床率和临床妊娠率.结果 DFI ＞30%组精子畸形率显著高于≤30%组（P＜0.01）,但两组间精子密度、活动率、前向运动精子（a＋b）均无显著性差异（P＞0.05）;DFI＞30%组IVF和ICSI的优胚率、ICSI的胚胎着床率和临床妊娠率均显著低于DFI≤30%组（P＜0.01,P＜0.05）.结论 精子DNA完整性与精子形态密切相关,精子DNA损伤在IVF/ICSI过程中对胚胎质量有负面影响,并显著影响ICSI的胚胎着床率和妊娠率,建议行ICSI前应对精子DNA完整性进行评估.     

Preventative ibandronate treatment has the most beneficial effect on the microstructure of bone in experimental tumor osteolysis

We investigated the effect of ibandronate on three-dimensional (3-D) microstructure and bone mass in experimentally induced tumor osteolysis. Walker carcinosarcoma cells were implanted into the left femur of female rats that received 26-day ibandronate pretreatment followed by continued therapy or ibandronate posttreatment only. A tumor-only group received isotonic saline. At endpoint, excised femurs were scanned using microcomputed tomography (μCT) to assess bone volume density, bone mineral content, trabecular number/thickness, and separation for cortical plus trabecular bone or trabecular bone alone. Compared with the nonimplanted right femur, bone volume and surface density and trabecular number and thickness were reduced in the distal left femur following tumor cell implantation. μCT analysis revealed greater cortical and trabecular bone mineral content in the preventative and interventional (pre-post tumor) ibandronate group, and the interventional (post-tumor) ibandronate group, versus the tumor-only group. Bone volume density was significantly higher in pre-post and post-tumor groups compared to the tumor-only group. After preventative and interventional ibandronate, bone volume density and trabecular thickness were 13% and 60% greater, respectively, than in the post-tumor treatment group. 3-D μCT images confirmed microstructural changes. We conclude that combined interventional and preventative ibandronate preserves bone strength and integrity more than intervention alone.     

微流控芯片精子分选法对精子常规参数及DNA完整性的影响

目的:研究微流控芯片精子优选技术对精子常规参数及DNA完整性的影响。方法:自行设计制作微流控芯片,利用芯片处理技术和上游法对40例精液标本进行精子优选,通过计算机辅助精液分析系统及染色质扩散试验从精子常规参数及DNA完整性两个方面评价精液体外处理对精子的影响。结果:精液经微流控芯片法和上游法处理后,精子活力、精子正常形态率以及精子尾部肿胀率均有显著提高(P<0.01),精子的DNA损伤率明显降低(P<0.01)。微流控芯片法与上游法相比,优选后前者精子DNA损伤率明显低于后者[(8.4±5.8)%vs(16.4±9.2)%,P<0.01],而其他参数差异无显著性。结论:微流控芯片技术在精子优选中能获得精子DNA损伤程度小的高质量精子。     

A comprehensive work up for an asthenozoospermic man with repeated intracytoplasmic sperm injection (ICSI) failure

Infertility affects about 15-20% couples attempting pregnancy and in about half cases the problem lies in the male. Among the sperm parameters, linear progressive motility is one of the most important predictors of fertility potential. Though genetic and chromosomal abnormalities are important aetiological factors in the pathogenesis of male infertility, the mechanism involved in impaired sperm motility is poorly understood. Here we report mitochondrial DNA (mtDNA) mutations with increased seminal reactive oxygen species (ROS) levels and higher DNA fragmentation level in the sperm resulting in decreased ATP production which plays an important role in sperm motility defect. Thus it is important to understand the aetiology of asthenozoospermia and to distinguish if infertile men harbour nuclear or mtDNA mutation as they are very important prognostic markers. This case study also highlights that routine semen parameters are very modest predictors of fertility outcome but ROS estimation and DNA integrity analysis by Comet assay have better diagnostic and prognostic capabilities. Thus this study is a detailed and comprehensive workup of an infertile asthenozoospermic male.     

Sperm quality improvement after natural anti-oxidant treatment of asthenoteratospermic men with leukocytospermia

Aim： To study the immune-modulating and anti-oxidant effects of beta-glucan, papaya, lactoferrin, and vitamins C and E on sperm characteristics of patients with asthenoteratozoospermia associated with leucocytosis. Methods： Fifty-one patients referred to our Sterility Center for semen analysis were selected. Sperm parameters were assessed before and after patient＇s treatment with beta-glucan, lactoferrin, papaya, and vitamins C and E. DNA damage was assessed by the acridine orange test and sperm structural characteristics were evaluated by transmission electron microscopy. Results： After 90 days of treatment, an increase in the percentage of morphologically normal sperm （17.0 ± 5.2 vs. 29.8 ± 6.5） and total progressive motility （19.0± 7.8 vs. 34.8 ± 6.8） were detected. Structural sperm characteristics as well as chromatin integrity were also improved after treatment. In terms of leukocyte concentration in seminal fluid, a significant reduction was recorded （2.2 ± 0.9 vs. 0.9± 0.2）. Conclusion： The treatment of an inflammatory process by the synergic action of immune modulators and anti-oxidants could protect sperm during maturation and migration, leading to improved sperm function. （Asian JAndrol 2008 Mar;. 10： 201-206）     

精浆转铁蛋白含量与不育的关系

为探讨精浆转铁蛋白 (Tf)含量与男性生育力的关系 ,采用速率散射比浊法 ,对 2 0例正常生育男性和 96例不育男性精浆Tf含量进行了测定 ,同时做精子密度、精子活动率及精子顶体完整率 (PIA)分析。结果表明少精子组 (精子密度 <2 0× 10 6/ml)精浆Tf含量较生育组与正常密度组 (精子密度≥ 2 0× 10 6/ml)显著低 (P <0 .0 1) ;精浆Tf含量与精子活动率无关 ;正常精子密度的不育患者精子PIA <80 %组精浆Tf的含量显著低于PIA≥ 80 %组(P <0 .0 1)。提示精浆Tf含量的下降与男性生育力有关     

Comparison between computerized slow-stage and static liquid nitrogen vapour freezing methods with respect to the deleterious effect on chromatin and morphology of spermatozoa from fertile and subfertile men

The purpose of this study was to determine the negative effects (cryodamage) on human spermatozoa after freeze-thawing and to determine whether freeze-thawing of spermatozoa with a programmed slow freezer is better than freezing with liquid nitrogen vapour (rapid freezing) with regard to alterations in sperm chromatin and morphology in semen from fertile (donor) and subfertile, IVF/ICSI, patients. Ninety-five semen samples were obtained either from patients attending our IVF unit for treatment (n=34) or from donors (n=25) with proven fertility and normal sperm quality according to WHO guidelines. Each semen sample was divided into two parts after liquefaction and addition of the cryoprotectant. The first part was frozen using a programmed biological freezer and the second part was frozen by means of liquid nitrogen vapour. Smears were made before the freezing and after the thawing procedure to assess morphology (strict criteria) and chromatin condensation (Acridine Orange test). The mean percentage of chromatin condensed spermatozoa in the samples from donors (control group) was 92.4 +/- 8.4% before freezing and this decreased significantly (p < 0.0001) to 88.7 +/- 11.2% after freeze-thawing with the computerized slow-stage freezer and to 87.2 +/- 12.3% after using static liquid nitrogen vapour (p < 0.001). The corresponding values for semen obtained from patients was 78.9 +/- 10.3% before freezing which decreased to 70.7 +/- 10.8 and 68.5 +/- 14.8%, respectively (p < 0.001). On the other hand, the mean percentage of normal sperm morphology in the control group decreased from 26.3 +/- 7.5% before freezing to 22.1 +/- 6.4% (p < 0.0001) after thawing with the computerized slow-stage freezer and to 22.2 +/- 6.6% (p < 0.0001) after the use of static liquid nitrogen vapour. In the patient group, the mean percentage of normal morphology decreased from 11.7 +/- 6.1% after freezing with the biological freezer to 9.3 +/- 5.6% and to 8.0 +/- 4.9% after freezing with static liquid nitrogen vapour. This study demonstrates that chromatin packaging and morphology of human spermatozoa decrease significantly after the freeze-thawing procedure, not only after the use of static liquid nitrogen vapour but also after the use of a computerized slow-stage freezer. However, the chromatin of semen samples with normal semen parameters (donor sperm) withstand the freeze-thaw injury better than those with low quality semen samples. Therefore, the computerized slow stage freezer could be recommended for freezing of human spermatozoa, especially for subnormal semen samples, for example, ICSI and ICSI/TESE candidates and from patients with testicular tumours or Hodgkin's disease, in order to avoid further damage to the sperm chromatin structure.     

Meta分析精子DNA完整性对辅助生殖结局的预测价值

目的系统评价精子DNA完整性对于宫腔内人工授精(IUI)、体外受精-胚胎移植(IVF-ET)以及卵胞浆内单精子注射(ICSI)等不同助孕方式妊娠结局的预测价值。方法采用电子检索和手工检索相结合的方式进行文献初检,检索Medline(via.OvidSP)、PubMed、Embase、CNKI、VIP等数据库,检索时限为建库至2016年3月,并手工检索相关会议资料。由2名评价者依据纳入与排除标准独立筛选研究文献、提取数据资料和评价方法学质量,然后采用RevMan 5.3软件进行Meta分析。结果最终纳入27篇队列研究,合计7 302个周期。Meta分析结果显示:DNA碎片指数(DFI)的增加会降低IUI临床妊娠率(RR=0.22,95%CI:0.12-0.41)、IVF受精率(RR=0.90,95%CI:0.86-0.95)、IVF优质胚胎率(RR=0.86,95%CI:0.82-0.91)、IVF临床妊娠率(RR=0.86,95%CI:0.76-0.98),并增加IVF流产率(RR=1.65,95%CI:1.05-2.57)和ICSI流产率(RR=1.57,95%CI:1.03-2.39)(P均0.05);且DFI的检测方法中只有TUNEL法能预测IVF的临床妊娠率(RR=0.74,95%CI:0.55-0.99,P0.05),DFI对ICSI受精率、优质胚胎率、临床妊娠率的影响无统计学意义(P0.05)。结论 DFI水平高低对辅助生殖治疗的结局有一定的预测价值,DFI较高时选择ICSI的治疗方式可能更优于常规的IVF。但因纳入研究数量和质量的限制,上述结论还需开展更大规模流行病学研究和临床研究进一步验证。     

Effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males with asthenozoospermia

The aim of this work was to study the possible beneficial effect of repeated sequential ejaculation on sperm DNA integrity in subfertile males and its possible implementation in assisted reproduction. The study included 20 infertile males with idiopathic asthenozoospermia or oligoasthenozoospermia. They underwent detailed history taking, complete clinical assessment and hormonal assessment. Patients were asked to bring two semen samples (taken within 1-3 h). Two consecutive samples were assessed with regard to semen volume, sperm count, motility grading, and morphology and sperm DNA integrity using the comet assay. There was a significant improvement in the sperm motility pattern and DNA integrity in the second sample in comparison with the first sample. Therefore, it is concluded that due to its positive impact on sperm motility and DNA integrity, repeated sequential ejaculation is recommended in subfertile males with idiopathic asthenozoospermia who pursue assisted reproduction.