Problems of sperm fertility: A reproductive biologist's view

For natural fertilization to occur successfully, millions of spermatozoa must be deposited in the lower end of the female genital tract during mating. Considerably fewer spermatozoa are required for fertilization when spermatozoa are deposited in the upper region of the female tract. The extreme case of assisted fertilization is the direct injection of a single spermatozoon into an oocyte in vitro, which is referred to as intracytoplasmic sperm injection or ICSI. All that is required to fertilize an oocyte is a single spermatozoon with a genetically and epigenetically normal nucleus. Even spermatozoa with grossly misshapen heads and no motility at all are able to produce normal offspring by ICSI as long as their nuclei are normal. There is a distinct difference between natural and ICSI fertilization. In ICSI the sperm plasma membrane and the acrosome, which never enter the oocyte's cytoplasm during natural fertilization, are injected into an oocyte. There are reports that the oocytes injected with acrosome-less spermatozoa develop better than those injected with acrosome-intact spermatozoa. At least in the mouse, pre-spermatozoal cells (e.g., round spermatids) are able to produce fertile offspring by injection into oocytes. Whether ‘spermatozoa’ produced from embryonic stem (ES) or induced pluri-potent stem (iPS) cells are functional remains to be determined. Will assisted reproduction increase overall male infertility by spreading genes involved in infertility? This is most unlikely in view of the widespread propagation of spontaneous mutation in the general population. When assisted reproduction technologies are perfected, pregnancy through assisted reproduction will become as safe as pregnancy through natural conception.     

ULTRASTRUCTURAL ANALYSIS OF TESE AND SEMEN SPERM TAILS FROM PATIENTS EXHIBITING ABSOLUTE IMMOTILITY ON THE DAY OF ICSI

Sperm flagellar pathology was found to be the underlying cause of motility disorders that lead to male infertility. Conventional in vitro fertilization (IVF) procedures will fail when sperm show a total absence of motility. In such difficult cases intracytoplasmic sperm injection (ICSI) is the only available technique to fertilize an oocyte. Fertilization rates are low and may also be reduced when immotile sperm are used for ICSI from ejaculate of other than epididiymal or testicular origin.Presence of totally immotile sperm in the ejaculate on the day of ICSI if spermatogenesis is normal testicular sperm recovery can improve ICSI outcomes. But for patients having severe morphological or functional sperm defects embryos of lower quality tend to be produced when totally immotile sperm are used. In this study the 2 patients exhibiting totally immotile sperm in their ejaculates and TESE samples on the day of ICSI showed the same ultrastructural abnormalities. Peri-axonemal and axonemal abnormalities that were seen in association with sperm nucleus structural defects suggested that the source of sperm has no effect on morphologic characteristics and also reflects abnormality in both spermatogenesis and spermiogenesis. In this study the two patients who presented with oligoteratozoospermia with total immotility, using either ejaculate or TESE sperm fertilization and embryo development, can be obtained with ICSI, but no pregnancies were established after embryo transfers.     

附睾精子抽吸术结合卵细胞浆内单精子注射治疗阻塞性无精子症所致男性不育的研究

目的:探讨附睾精子抽吸术(ESA)结合卵细胞内单精子注射(ICSI)技术治疗阻塞性无精子症所致男性不育的治疗效果。方法:选择2002年1月~2003年12月到我院治疗不育症确诊为阻塞性无精子症男性不育患者,采用ESA方法吸取男性附睾液,分离精子用于ICSI;同时按常规体外受精-胚胎移植方法(IVF-ET),采用GnRH-α+FSH/hMG+hCG促排卵方案对女性进行促排成熟卵细胞(M II)用于显微注射授精,受精卵体外培养3 d后移植回子宫内。结果:采用MESA结合ICSI技术治疗32周期阻塞性无精子症所致不育的夫妇,所获成熟卵(M II)162个,受精率66.28%,卵裂率62.21%,临床妊娠率31.20%。结论:采用ESA结合ICSI技术治疗阻塞性无精子症所致男性不育获得良好的效果,该方法为阻塞性无精子症男性不育患者提供了一种快速、方便、无痛、有效的治疗方法。     

Are sperm chromatin and DNA defects relevant in the clinic?

There has been an increase in the use of sperm DNA and chromatin integrity tests in the evaluation of the infertile man with the hypothesis that these tests may better diagnose infertility and predict reproductive outcomes. This review discusses the etiology of sperm DNA damage, briefly describing the tests of sperm DNA damage, and evaluates the relationship between sperm DNA damage and reproductive outcomes. A systematic review of the literature allows us to conclude that sperm DNA damage is associated with lower natural, intra-uterine insemination (IUI), and in vitro fertilization (IVF) pregnancy rates. Studies to date have not shown a clear association between sperm DNA and chromatin defects and pregnancy outcomes after intra-cytoplasmic sperm injection (ICSI). However, we cannot exclude the possibility that very high levels of DNA damage will impact on ICSI outcomes. In couples undergoing IVF or ICSI, there is evidence to show that sperm DNA damage is associated with an increased risk of pregnancy loss. A limitation of this systematic review and meta-analysis is that it does not address the heterogeneity of the individual study characteristics. Although the clinical utility of tests of sperm DNA damage remains to be firmly established, the data suggest that there is clinical value in testing couples prior to assisted reproductive technologies (ARTs IUI, IVF, and ICSI) and in those couples with recurrent miscarriages. Additional, well-designed prospective studies are needed before testing becomes a routine part of patient care.     

Meiotic recombination errors,the origin of sperm aneuploidy and clinical recommendations

Since the early 1990s male infertility has successfully been treated by intracytoplasmic sperm injection (ICSI), nevertheless concerns have been raised regarding the genetic risk of ICSI. Chromosome aneuploidy (the presence of extra or missing chromosomes) is the leading cause of pregnancy loss and mental retardation in humans. While the majority of chromosome aneuploidies are maternal in origin, the paternal contribution to aneuploidy is clinically relevant particularly for the sex chromosomes. Given that it is difficult to study female gametes investigations are predominantly conducted in male meiotic recombination and sperm aneuploidy. Research suggests that infertile men have increased levels of sperm aneuploidy and that this is likely due to increased errors in meiotic recombination and chromosome synapsis within these individuals. It is perhaps counterintuitive but there appears to be no selection against chromosomally aneuploid sperm at fertilization. In fact the frequency of aneuploidy in sperm appears to be mirrored in conceptions. Given this information this review will cover our current understanding of errors in meiotic recombination and chromosome synapsis and how these may contribute to increased sperm aneuploidy. Frequencies of sperm aneuploidy in infertile men and individuals with constitutional karyotypic abnormalities are reviewed, and based on these findings, indications for clinical testing of sperm aneuploidy are discussed. In addition, the application of single nucleotide arrays for the analysis of meiotic recombination and identification of parental origin of aneuploidy are considered.     

卵胞浆内单精子显微注射技术的临床应用

目的:探讨男性因素不育症和常规体外受精(IVF)失败者采用卵胞浆内单精子显微注射(ICSI)治疗的临床效果.方法:对51例男性因素不育症和常规体外受精(IVF)失败者共计59个ICSI治疗周期进行了回顾性分析.结果:59个ICSI治疗周期共取卵596个,MⅡ期卵母细胞471个,MⅡ期卵母细胞正常受精率82.21%,卵裂率89.04%,临床妊娠19例,周期临床妊娠率为32.20%.结论:对男性因素不育症和常规体外受精(IVF)失败者ICSI是一种有效的治疗方法.     

A rapidly progressive defective spermatogenesis in a Mexican family affected by spino-bulbar muscular atrophy

Spino-bulbar muscular atrophy (SBMA) is an X-linked recessive adult progressive disorder affecting motor neurons. It is caused by a poly-glutamine tract expansion in the androgen receptor (AR) which generates protein aggregates that cannot be processed by proteasomes. A secondary mild androgen resistance is developed by AR dysfunction and patients present endocrine abnormalities including gynecomastia and poor function of testosterone in tissues; however, normally they are fertile. In this report we describe a Mexican family with three affected brothers with primary infertility caused by a progressive impairment of spermatogenesis leading to azoospermia before 40 years of age. They presented common features associated to patients affected by SMBA, such as gynecomastia, high level of CPK, muscle cramps, fasciculations, muscle wastage, and impaired swallowing. Two intracytoplasmic sperm injection (ICSI) cycles were performed in one of the patients resulting in fertilization failure. Molecular analysis of AR gene exon 1 revealed 54 CAG repeats in DNA extracted from leukocytes in affected patients and 22 repeats in the fertile non-affected brother. Severe impaired spermatogenesis of rapid progression has not been associated before to SBMA. This is the first report of assisted reproduction techniques indicated by male infertility in patients with this rare disorder. Further studies are required to confirm the unusual result of intracytoplasmic sperm injection cycles. We discuss the implications and possible pathogenesis of these unique features of SBMA in this family.     

原因不明性不孕患者同胞卵母细胞行IVF和ICSI结果比较

目的:探讨原因不明性不孕患者行常规IVF、ICSI对受精、受精后胚胎的影响。方法:回顾性分析2005年6月~2007年12月间非男性因素不孕的夫妇采用同胞卵和同一份精液行常规IVF和ICSI的受精情况、受精后胚胎的发育潜能。结果:50例原因不明性不孕患者中11例常规IVF受精完全失败,IVF完全受精失败率为22%,ICSI组无完全受精失败。IVF、ICSI均受精组ICSI受精率(82%)高于常规IVF(79%),差异无显著性意义(P>0.05)。均受精组和仅ICSI受精组患者的年龄、不孕年限、基础FSH、BMI以及精液标本分析均无显著性差异。比较移植不同受精方式的胚胎着床率、妊娠率差异均无显著性意义。结论:对于原因不明性不孕的夫妇选择施行IVF-ICSI split既可避免受精失败所致的周期取消,保证一定的成功率,又可为下一次施行IVF时决定受精方式提供依据,值得推广。尚需要大样本研究原因不明性不孕患者年龄、不孕年限和受精完全失败的关系,为选择ICSI提供依据。     

Problems of sperm fertility: a reproductive biologist's view

For natural fertilization to occur successfully, millions of spermatozoa must be deposited in the lower end of the female genital tract during mating. Considerably fewer spermatozoa are required for fertilization when spermatozoa are deposited in the upper region of the female tract. The extreme case of assisted fertilization is the direct injection of a single spermatozoon into an oocyte in vitro, which is referred to as intracytoplasmic sperm injection or ICSI. All that is required to fertilize an oocyte is a single spermatozoon with a genetically and epigenetically normal nucleus. Even spermatozoa with grossly misshapen heads and no motility at all are able to produce normal offspring by ICSI as long as their nuclei are normal. There is a distinct difference between natural and ICSI fertilization. In ICSI the sperm plasma membrane and the acrosome, which never enter the oocyte's cytoplasm during natural fertilization, are injected into an oocyte. There are reports that the oocytes injected with acrosome-less spermatozoa develop better than those injected with acrosome-intact spermatozoa. At least in the mouse, pre-spermatozoal cells (e.g., round spermatids) are able to produce fertile offspring by injection into oocytes. Whether 'spermatozoa' produced from embryonic stem (ES) or induced pluri-potent stem (iPS) cells are functional remains to be determined. Will assisted reproduction increase overall male infertility by spreading genes involved in infertility? This is most unlikely in view of the widespread propagation of spontaneous mutation in the general population. When assisted reproduction technologies are perfected, pregnancy through assisted reproduction will become as safe as pregnancy through natural conception.     

Infertility, infertility treatment and psychomotor development: the Danish National Birth Cohort

Babies born of infertile couples, regardless of treatment, have a higher risk of preterm birth and low birthweight, conditions associated with delayed development. We examined developmental milestones in singletons as a function of parental infertility [time to pregnancy (TTP) > 12 months] and infertility treatment. From the Danish National Birth Cohort (1997–2003), we identified 37 897 singletons born of fertile couples (TTP ≤ 12 months), 4351 born of infertile couples conceiving naturally (TTP > 12 months), and 3309 born after infertility treatment. When the children were about 18 months old, mothers reported 12 developmental milestones by responding to structured questions. We defined a failure to achieve the assessed milestone or the minimal numbers of milestones in a summary (motor, or cognitive/language skills) as delay.
Naturally conceived children born of infertile couples had a pattern of psychomotor development similar to that of children born of fertile couples, but increasing TTP correlated with a modest delay. When the analysis was restricted to infertile couples (treated and untreated), children born after treatment showed a slight delay in cognitive/language development (odds ratio 1.24, [95% confidence interval 1.01, 1.53]) for not meeting at least three out of six cognitive/language milestones); children born after intracytoplasmic sperm injection (ICSI) had the highest estimated relative risk of delay for most milestones, especially motor milestones. These results suggest that a long TTP may be associated with a modest developmental delay. Infertility treatment, especially ICSI, may be associated with a slight delay for some of these early milestones.     

Are sperm chromatin and DNA defects relevant in the clinic?

There has been an increase in the use of sperm DNA and chromatin integrity tests in the evaluation of the infertile man with the hypothesis that these tests may better diagnose infertility and predict reproductive outcomes. This review discusses the etiology of sperm DNA damage, briefly describing the tests of sperm DNA damage, and evaluates the relationship between sperm DNA damage and reproductive outcomes. A systematic review of the literature allows us to conclude that sperm DNA damage is associated with lower natural, intra-uterine insemination (IUI), and in vitro fertilization (IVF) pregnancy rates. Studies to date have not shown a clear association between sperm DNA and chromatin defects and pregnancy outcomes after intra-cytoplasmic sperm injection (ICSI). However, we cannot exclude the possibility that very high levels of DNA damage will impact on ICSI outcomes. In couples undergoing IVF or ICSI, there is evidence to show that sperm DNA damage is associated with an increased risk of pregnancy loss. A limitation of this systematic review and meta-analysis is that it does not address the heterogeneity of the individual study characteristics. Although the clinical utility of tests of sperm DNA damage remains to be firmly established, the data suggest that there is clinical value in testing couples prior to assisted reproductive technologies (ARTs IUI, IVF, and ICSI) and in those couples with recurrent miscarriages. Additional, well-designed prospective studies are needed before testing becomes a routine part of patient care.     

Teratozoospermia with amorphous sperm head associate with abnormal chromatin condensation in a Chinese family

Male infertility affects approximately 7% of the male population. In about 40% of affected patients, the etiology remains unknown. Here, we report the cases of two infertile brothers who have a uniquely prevalent sperm phenotype with completely amorphous sperm heads. To investigate the mechanisms of familial teratozoospermia with amorphous sperm heads, chromatin condensation was assessed by aniline blue staining, western blot, sperm chromatin structure assay and atomic force microscopy in both the two brothers and 40 control fertile donors. Our results showed an abnormal condensation of chromatin with amorphous headed sperm. We suggest that abnormal chromatin condensation which was induced by disturbances in the process of histone–protamine replacement may be a possible cause of familial teratozoospermia with amorphous head, and the elasticity of sperm nuclei could be a new index to assess sperm quality. Additionally, for the first time, the current study provided a new biomechanics strategy for evaluating pathological sperm contributes to our understanding of teratozoospermia.Abbreviations: SCSA: sperm chromatin structure assay; AFM: atomic force microscopy; ICSI: intracytoplasmic sperm injection; HDS: high DNA stainability; DFI: DNA fragmentation index; PBS: phosphate-buffered saline; DTT: dithiothreitol; FITC: fluorescein isothiocyanate; DAPI: 4?,6-diamidino-2-pheneylindole; SSC: standard saline citrate     

Relationship between phospholipase C-zeta,semen parameters,and chromatin status

The need for additional tests to complement basic sperm analysis in clinics is well appreciated. In this regard, a number of tests such as sperm DNA integrity test as a tool in diagnosis and treatment of infertility are suggested. But recent studies have focused on main sperm factors involved in oocyte activation such as phospholipase C-zeta (PLCζ) that initiate intracellular Ca²⁺ signaling and embryogenesis. Therefore, this study aimed to investigate the relationship between PLCζ, basic semen parameters, sperm DNA fragmentation (SDF), and protamine deficiency in men with normal (n=32) and abnormal (n=23) semen parameters. Unlike SDF and protamine deficiency, as negative factors related to fertility, the mean value of PLCζ as positive factor related to infertility was significantly lower in men with abnormal semen parameters compared to men with normal semen parameters. Significant correlations were also observed between sperm concentration, motility, and abnormal morphology with the percentage of PLCζ positive spermatozoa. In addition, logistic regression analysis revealed that sperm morphology is more predictive than sperm motility and concentration for PLCζ presence. In addition, a statistically significant negative relationship was observed between the percentage of PLCζ positive spermatozoa and SDF. These findings suggested during ICSI, selection of sperm based on morphology has a profound effect on its ability to induce oocyte activation based on the likelihood of PLCζ expression. Therefore, assessment of PLCζ as an index for fertilization potential of a semen sample in men with severe teratozoospermia may define individuals who are candidates for artificial oocyte activation (AOA) and may avoid failed fertilization post ICSI.     

Meiotic recombination errors, the origin of sperm aneuploidy and clinical recommendations

Since the early 1990s male infertility has successfully been treated by intracytoplasmic sperm injection (ICSI), nevertheless concerns have been raised regarding the genetic risk of ICSI. Chromosome aneuploidy (the presence of extra or missing chromosomes) is the leading cause of pregnancy loss and mental retardation in humans. While the majority of chromosome aneuploidies are maternal in origin, the paternal contribution to aneuploidy is clinically relevant particularly for the sex chromosomes. Given that it is difficult to study female gametes investigations are predominantly conducted in male meiotic recombination and sperm aneuploidy. Research suggests that infertile men have increased levels of sperm aneuploidy and that this is likely due to increased errors in meiotic recombination and chromosome synapsis within these individuals. It is perhaps counterintuitive but there appears to be no selection against chromosomally aneuploid sperm at fertilization. In fact the frequency of aneuploidy in sperm appears to be mirrored in conceptions. Given this information this review will cover our current understanding of errors in meiotic recombination and chromosome synapsis and how these may contribute to increased sperm aneuploidy. Frequencies of sperm aneuploidy in infertile men and individuals with constitutional karyotypic abnormalities are reviewed, and based on these findings, indications for clinical testing of sperm aneuploidy are discussed. In addition, the application of single nucleotide arrays for the analysis of meiotic recombination and identification of parental origin of aneuploidy are considered.     

THE GENETICS OF MALE INFERTILITY: A FIELD OF STUDY WHOSE TIME IS NOW

Idiopathic male infertility is often associated with genetic and epigenetic abnormalities. Such abnormalities include chromosome translocations and aneuploidies, Y chromosome microdeletions, and mutations of the CFTR gene. The unraveling of the human genome and ongoing animal transgenic studies have identified numerous other genes likely to be associated with male infertility. Initial reports from human studies have identified several candidate genes, including the protamine genes, SPO11, EIF5A2, USP26, ACT, and others. In addition to gene mutations and polymorphisms, damage to the chromatin resulting in single and double strand DNA breaks affects fertility. Recent studies are highlighting the role of such abnormalities in male infertility, and point to protamine defects as one cause of DNA damage. Epigenetic abnormalities also are being investigated, including the role of residual sperm mRNA in embryogenesis, and the effects of abnormal spermatogenesis on gene imprinting. These studies are pointing to complex etiologies and clinical ramifications in many infertile men.     

SPERM CHROMOSOME ANEUPLOIDY AS RELATED TO MALE FACTOR INFERTILITY AND SOME ULTRASTRUCTURE DEFECTS

Some men have elevated levels of sperm chromosome aneuploidy. In this study, we have evaluated and summarized sperm aneuploidy rates in male infertility patients and control groups. The mean aneuploidy rate for five chromosomes (X, Y, 13, 18, 21) was 1.2 ± 0.1 for fertile controls, 1.4 ± 0.1 for a general population control group, and 5.8 ± 1.14 for the patients. When the patients were classified by the type of male factor infertility, the total aneuploidy rate was 2.6 ± 0.3 in men with moderately diminished semen quality (n = 7), 4.0 ± 0.3 patients with severe teratoasthenooligozoospermia, and 15.9 ± 3.8 for men with rare ultrastructure defects such as round head only syndrome or severe tail agenesis. Some infertility patients have a severely elevated level of sperm chromosome aneuploidy, which may contribute to infertility or diminish the likelihood of a successful outcome from IVF/ICSI. The severity of sperm chromosome aneuploidy appears to be proportional to the severity of abnormal semen quality: in particular, abnormal morphology. The high rates of aneuploidy in patients with severe ultrastructure defects suggest that caution should be employed in counseling those patients prior to IVF/ICSI.     

胞浆内单精子注射临床应用指征及安全性分析

胞浆内单精子注射(ICSI)技术自1992年起为男性不育症的临床治疗带来了巨大的改变,其在人类辅助生育技术领域的应用逐渐广泛,不同生殖中心对于该技术的应用指征观点不一。多数学者认为在非男性因素、获卵数少、单纯性畸精症方面不推荐使用ICSI受精方式;对于临界精液质量、常规体外受精失败或低受精及不明原因不孕的患者,应综合评估总活动精子数及受精失败原因后酌情应用该技术。多数地区的随访研究表明,ICSI后代的总体缺陷率与正常人群无显著差异,但其潜在遗传学风险可能导致某些特殊疾病的发生。     

MSOME及IMSI技术在辅助生殖领域中的应用进展

精子形态与功能密切相关,精子形态异常是导致男性不育的重要原因之一。自1992年卵母细胞胞浆内单精子注射（ICSI）技术首次获得妊娠以来,许多男性因素不育夫妇通过该技术获得较为满意的妊娠结局。但在×400放大倍数下常规ICSI挑选的看似正常的精子仍可能存在超微结构的异常,这可能是导致ICSI周期失败的原因之一。活精子细胞器形态学检测（MSOME）技术是Bartoov在2001年创立的一项新技术,能在高放大倍数下更深入地观察精子细微结构,有助于挑选出形态正常的精子。精子形态选择性胞浆内单精子注射（IMSI）技术是在MSOME基础上结合传统ICSI技术发展而来,为卵母细胞ICSI提供了一个新方法。通过对MSOME和IMSI的文献进行综述,探讨其临床应用价值。     

Sperm epigenetics in the study of male fertility,offspring health,and potential clinical applications

The mammalian sperm contains a highly unique and specialized epigenetic landscape that offers a great degree of interesting research opportunities. One key discriminating feature of the mature sperm epigenome is that it, in theory, represents both remnant marks used throughout spermatogenesis to generate sperm cells competent to perform their function, but also marks that appear to be useful beyond fertilization. Key questions must be asked about the utility of these marks and the multiple purposes that may be served. It is this unique epigenetic landscape that has driven some labs to begin to study the links between aberrant sperm epigenetic patterns and various forms of infertility, from idiopathic to alterations in sperm motility, morphology, and viability and fertilization capacity. Because of the unique nature of the sperm epigenome and the patterns found in mature sperm that appear to reflect perturbations in spermatogenesis that may ultimately have effect on pregnancy outcomes, some researchers believe that these marks may provide predictive insight that can be exploited. Indeed, there is emerging data suggesting that the predictive power of DNA methylation and RNA signatures in sperm likely exceeds that which can be found with traditional assessments of male infertility. This review will focus on the utilization of the sperm epigenome as a potential diagnostic tool in the context of male infertility, as well as the potential difficulties associated with such an approach.     

湖州地区2010年2810例不育男性精液质量分析

目的:分析2010年湖州地区2810例不育男性精液质量状况,探讨精液检查在男性不育诊断中的重要性。方法:严格按照WHO《人类精液及精子-宫颈粘液相互作用实验室检验手册》第四版技术规范,采用纯手工的方法,对2810例男性患者精液进行常规分析。结果:(1)检测指标全部正常的精液样本数286例,所占比例10.2%。精液质量有一项或多项异常者2524例,占精液检测总数的89.8%。(2)精子膜表面抗体IgG总的检测数量为1459例,异常例数为105例,阳性率为7.2%。结论:男性的精液常规检查既方便又快捷,在不孕症夫妇诊疗中非常重要,如能及早查出或排除男方不育因素,必要时配合女方治疗,有利于提高不孕症夫妇在不孕症治疗过程中的成功率。