Relationship of acrosin activity to sperm function tests

Summary.  Acrosin activity of spermatozoa from infertile patients and fertile volunteers was measured. Acrosin activity of spermatozoa from asthenozoospermic patients and patients with unexplained infertility was lower than that from fertile volunteers. We utilize the zona-free hamster egg sperm penetration test to select candidates for conventional in vitro fertilization among unexplained infertile patients. The zona-free hamster egg sperm penetration test, however, requires several hours and special equipment which are not used in the clinical setting. It is preferable that other sperm function tests or a combination of them can replace this test. Thus three distinct tests of sperm function, namely, acrosin activity, Penetrak® test, hypo-osmotic swelling test, were compared with the hamster test, using spermatozoa from patients with unexplained infertility.
A combination of the Penetrak® test and measurement of acrosin activity could predict the results of the zona-free hamster egg sperm penetration test with 88.2% accuracy. Thus, the hamster test should be done when either Penetrak® test or measurement of acrosin activity showed abnormal values.     

An evaluation of the hypo-osmotic sperm swelling test as a predictor of fertilizing capacity in vitro

The ability of sperm to swell in hypo-osmotic conditions was examined in 211 semen samples from the partners of patients about to undergo oocyte retrieval for in-vitro fertilization (IVF). The test was performed using aliquots of semen, the remainder of which was then prepared for IVF. No significant difference was found, in either the percentage of swollen sperm or the type of swelling response, between samples that achieved fertilization in vitro and those that did not, or between any of the diagnostic categories of infertility (tubal damage, unexplained infertility, oligospermia). In samples which achieved fertilization in vitro there were correlations between sperm swelling and sperm motility (r = -0.51) and abnormal morphology (r = 0.33), but no such correlations were demonstrated in samples that failed to achieve fertilization. Moreover, there was no significant difference between the percentage of swollen sperm in semen (mean motility 64%), in samples immediately after preparation for IVF (mean motility 96%) or in capacitated sperm 24 h after preparation (mean motility 91%). These results demonstrate that the hypo-osmotic sperm swelling test does not assist in the prediction of the fertilizing capacity of human sperm in vitro.     

补救的卵胞浆内单精子注射方法的评估

目的探讨对常规体外受精(IVF)后完全受精失败(TFF)的卵母细胞进行补救的卵胞浆内单精子注射(L-ICSI)方法的效果。方法自2002年1月至2007年2月对86对夫妇常规IVF后TFF的卵母细胞行L-ICSI,对取卵数目达到12个的原发不育,尤其是对原因不明和子宫内膜异位症不育的14对夫妇采用半数卵母细胞ICSI(Half-ICSI),并进行评估。结果L-ICSI与取卵日ICSI的受精率和分裂胚胎率差异非常显著(P<0.01),而且可移植胚胎数目减少,临床妊娠率极低,仅获得1.2%(1/86)的临床妊娠与分娩。31对L-ICSI的夫妇在本中心行第二次助孕治疗选择取卵日ICSI,获得41.9%(13/31)的妊娠率。Half-ICSI与L-ICSI比较,受精率、分裂胚胎率相似,但临床妊娠率(50%比1.2%)差异非常显著(P<0.01)。Half-ICSI受精率67.6%,临床妊娠率50%(5/10),与取卵日ICSI比较,差异无显著性(P>0.05)。结论L-ICSI作为补救措施作用甚小,建议对合适的人群采用Half-ICSI以避免TFF,提高临床妊娠率。     

Semen analysis in 21st century medicine： the need for sperm function testing

Sperm function testing, once commonly performed for the infertile couple before employing assisted reproductive technology （ART）, has fallen out of favour in many reproductive medicine centers throughout the world. Indeed, the most recent addition of the ＇World Health Organisation （WHO） Laboratory Manual for the Examination and Processing of Hu- man Semen＇ now groups many of these procedures into a section termed Research Procedures. In large part, this reflects the current clinical practice of bypassing the in-depth evaluation of the male partner, while assuming that if a spermatozoon can be found for intracytoplasmic sperm injection （ICSI）, it must be a healthy cell capable of achieving fertilization. Never- theless, sperm function testing can provide valuable clinical insights into defects causing male infertility. Admittedly, in some cases, functional sperm deficiencies can be overcome using an ART. In other cases, couples will be empowered by the knowledge of the cause of their infertility, and for some couples, perhaps even the likelihood of ICSI success （relative to the spermatozoa）. The knowledge allows them to make truly informed reproductive decisions, including （perhaps） the de- cision to seek donor insemination, to adopt or to remain childless. Knowledge of the cause of their infertility may provide closure for couples and a sense of confidence regarding their choice of reproductive treatment.     

Relationship between human sperm lipid peroxidation, comprehensive quality parameters and IVF outcome

The membranes of human spermatozoa contain an extremely high concentration of polyunsaturated fatty acids and are therefore susceptible to lipid peroxidation damage. The aim of this study was to retrospectively determine the association between the lipid peroxidation levels of washed spermatozoa, as indicated by thiobarbituric-acid-reactive substance concentration, and: (a) semen quality evaluated by basic routine, biochemical, cytological and quantitative ultramorphological analyses; (b) IVF fertilization rate. Semen samples from 45 male partners of couples who had been referred for IVF treatment due to a female infertility factor were evaluated for quality as well as for thiobarbituric-acid-reactive substance concentrations. The latter were found to have a negative correlation with total sperm count, semen volume, zinc/fructose ratio, and the integrity of sperm acrosome and axonema. It was suggested that lipid peroxidation has a deleterious effect on the ultramorphological status of the sperm cells and, thereby, on the male fertilization potential. The content of the seminal fluid, about 30% of which is produced by the prostate, may protect spermatozoa from this destructive process. A negative correlation was also found between thiobarbituric-acid-reactive substance concentrations and IVF fertilization rate. When the patients were subdivided into fertilizing (fertilization rate > 0%) and nonfertilizing (fertilization rate = 0%) subgroups (n = 33 and n = 12, respectively), the former exhibited significantly lower thiobarbituric-acid-reactive substance concentrations than the latter. A new IVF fertilization index based on the lipid peroxidation level was established. This index had a predictive power of 93% (94% sensitivity and 92% specificity). The clinical value of this index should be further verified.     

Tales of the tail and sperm head aches: changing concepts on the prognostic significance of sperm pathologies affecting the head, neck and tail

This article presents an update on the variable prognostic significance of different sperm pathologies in patients with severe male factor infertility due to morphology and motility disorders. Severe asthenozoospermia is one of the leading causes of male infertility as spermatozoa cannot reach the oocyte and/or penetrate normally. Identifying structural causes of sperm immotility was of great concern before the advent of intracytoplasmic sperm injection (ICSI), because immotility was the limiting factor in the treatment of these patients. In these cases, in vitro methods are used to identify live spermatozoa or stimulate sperm motility to avoid selection of non-viable cells. With these advances, fertilization and pregnancy results have improved dramatically. The identification of genetic phenotypes in asthenozoospermia is important to adequately inform patients of treatment outcomes and risks. The one sperm characteristic that seriously affects fertility prognosis is teratozoospermia, primarily sperm head and neck anomalies. Defects of chromatin condensation and acrosomal hypoplasia are the two most common abnormalities in severe teratozoospermia. The introduction of microscopic methods to select spermatozoa and the development of new ones to evaluate sperm quality before ICSI will assure that ultrastructural identification of sperm pathologies will not only be of academic interest, but will also be an essential tool to inform treatment choice. Herein, we review the differential roles played by sperm components in normal fertilization and early embryo development and explore how assisted reproductive technologies have modified our concepts on the prognostic significance of sperm pathologies affecting the head, neck, mid-piece and tail.     

Alterations in sperm protein phosphorylation in male infertility

Protein phosphorylation is involved in sperm capacitation, so the effect of protein phosphatase inhibitors on the capacitation of spermatozoa of males with unexplained infertility was investigated. d-mannose ligand specific receptor expression in fresh, living spermatozoa, capacitated or treated with calyculin A (an inhibitor of protein phosphatases 1 and 2A), was studied in three groups of men: pre-vasectomy (fertile) males, males in couples with male infertility, and males in couples with infertility of unknown aetiology. Flow cytometry showed significant differences between infertile couples with a male factor and fertile couples (P < 0.05), both after capacitation and after treatment with calyculin A. In the group of couples with infertility of unknown aetiology (n = 15), d-mannose receptor expression was diminished in six cases after classical capacitation. However, when the spermatozoa of these six men were treated with calyculin A, five showed an increased specific d-mannose receptor expression. From these results it is suggested that in vitro treatment of spermatozoa with inhibitors of protein phosphatases may be of great value in some cases of unexplained infertility.     

Body mass index is not associated with sperm-zona pellucida binding ability in subfertile males

Lifestyle factors, such as weight and nutritional status may affect male fertility, including sperm fertilization ability. The objective of this retrospective study was to evaluate the association between body mass index （BMI） and sperm-zona pellucida binding ability assessed according to the zona binding （ZB） test, which has been described to be a relevant diagnostic tool for the prediction of in vitro fertilization （IVF） ability. Three hundred and six male patients from couples diagnosed with primary idiopathic or mild male factor infertility were included. Correlations between BMI and semen parameters according to ZB test indices were assessed, together with frequencies of positive and negative tests across the BMI categories. In this selected population, BMI was not related to conventional semen parameters or sperm quality assessed according to the ability of spermatozoa to bind to the zona pellucida. The previously described poor outcomes of IVF procedures in cases of male obesity could be due to other sperm defects, such as alterations of sperm capacitation or acrosome reaction. The link between male BMI and biological outcomes during IVF procedures, such as fertilization rates, should be further evaluated.     

Influence of motility and vitality in intracytoplasmic sperm injection with ejaculated and testicular sperm

The vitality of spermatozoa used for intracytoplasmic sperm injection (ICSI) is a crucial factor for fertilization, establishment and outcome of a pregnancy in assisted reproductive technique cycles. The sperm origin may also be a limiting factor, although little is known about this issue. It is known that the motility of injected spermatozoa and their origin from ejaculate or testicular biopsies are important predictors in terms of fertilization, pregnancy and birth rates. Oocytes of patients in 2593 cycles were retrieved in our in vitro fertilization programme and inseminated via ICSI. We used motile (group 1, n = 2317) or immotile ejaculated spermatozoa (group 2, n = 79), motile sperm retrieved from testicular biopsies (group 3, n = 62) and immotile spermatozoa from testicular biopsies (group 4, n = 135). Female age and number of oocytes retrieved did not differ significantly among the groups. The fertilization rates were as follows: 67.1% in group 1, 49.8% in group 2, 68.3% in group 3 and 47.8% in group 4. The pregnancy rates in cases where three embryos had been transferred amounted to 35.7% in group 1, 17.3% in group 2, 38.3% in group 3 and 20.5% in group 4. The embryo quality showed no differences between groups 1 and 3 (14.5), and between groups 2 (11.8) and 4 (10.8). The abortion rate was similar in groups 1-3, but increased in group 4 (26.6%, 27.3%, 31.6% and 55.5%). Irrespective of their origin, the fertilization potential of injected spermatozoa was found to be influenced by motility. The resulting pregnancy and birth rates, i.e. the potential of the resulting embryos to implant and to achieve viable pregnancies, seem to be additionally dependent on the sperm origin. This was well shown by declining rates when spermatozoa in a relatively early stage of maturity had been used. We see increasing evidence that the degree of sperm maturity has an important impact on the outcome of ICSI. In obstructive azoospermia, spermatozoa retrieved from the epididymis should be used rather than testicular biopsy spermatozoa, or testicular sperm should be preincubated in culture medium before ICSI.     

精子缺陷和受精失败

不育症是当今一个重要的健康问题,有大约15％的夫妇都在为生育寻求临床治疗。在所有不能自然受孕的夫妇中,多达三分之二的夫妇是由男性生育力的下降导致的。尽管我们在生物学和生理学上对于受精的认识已有很大进步,但仍然不能解释受精失败的潜在原因。即使是在不育的男性中发现有精索静脉曲张、生殖器感染或抗精子抗体,这些因素对于不育症病因的解释价值尚不明确。     

Comparison of the outcome of intracytoplasmic sperm injection in obstructive and non-obstructive azoospermia in the first cycle: a report of case series and meta-analysis

To investigate the outcome of intracytoplasmic sperm injection with fresh and cryopreserved-thawed testicular spermatozoa in the first cycle in patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA), a total of 90 cases, 48 OA and 42 NOA were studied. All patients underwent sperm retrieval by testicular sperm extraction (TESE) while their wives received conventional ovarian hyperstimulation. The hormone levels, testicular histology, the rates of sperm retrieval, fertilization, implantation and pregnancy were analysed and evaluated. This study and other four similar studies were subjected to meta-analysis. Sperm retrieval was successful in 100% OA and 61% NOA. Fresh spermatozoa were used in 87.5% and 92.4% of OA and NOA cases respectively; while cryopreserved-thawed spermatozoa were used in 12.5% and 7.6% of OA and NOA, respectively. The fertilization, implantation and clinical pregnancy rates were 65.5%, 15% and 25% respectively in OA group, and 54.2%, 5% and 23.1% respectively in NOA group. Sperm status (fresh or thawed), male partner's age, female age and male serum follicle-stimulating hormone had no significant effect upon fertilization rate, implantation rate, or pregnancy rate per embryo transfer. The results of meta-analysis indicate that there is no statistically significant difference in clinical pregnancy rates between the two groups. There was a significantly higher fertilization rate among OA patients in all analysed studies (95% CI = 14.29-15.71, d.f. 832, T = 1.96). In conclusion, although the fertilization rate was significantly higher in the OA group in our study and from the given meta-analysis, there were some differences as regards pregnancy rates. Although the overall effect was more or less similar pregnancy rates in both subtypes of azoospermia, this may not be true if non-male infertility variables were controlled for in all studies.     

IVF过程受精失败的预测

在发明和常规应用卵胞浆内单精子注射（ICSI）之前,十多年的临床体外受精（IVF）治疗实践中,受精率低下很常见,大约有20％～35％的IVF患者受精率很低（〈35％的卵子受精）和受精完全失败（所有卵子都不受精）。虽然受精失败与精子或卵子的质量有关,但相当一部分患者受精失败与精液质量或精子功能低下有很密切相关性。最常见的是严重的少精,弱精和畸形精子症患者。     

Influence of the Concentration of Motile Sperm Inseminated on the Ovum Penetration Assay Results: Toward a Standardized Method

The influence of the concentration of motile sperm inseminated on the zona-free hamster egg penetration rate has been studied. In 3 fertile men, the higher the concentration of motile sperm inseminated, the higher the ovum penetration rate. This correlation emphasizes the usefulness of standardizing the technical conditions under which the ovum penetration assay is performed. It is also suggested to refer to a standard curve for results analysis. In the 22 patients studied, the outcome of the heterologous ovum assay was in most cases related to the initial sperm parameters. However, some disagreement was also observed. In the absence of a correlation between the initial sperm parameters and the ovum penetration rates, further investigation is needed in order to either more precisely determine the aetiology of unexplained couple infertility, or, in case of intrauterine insemination or in vitro fertilization, to better understand the conditions under which spermatozoa are capacitated in vitro.     

Surgical recovery of sperm in non-obstructive azoospermia

The development of intracytoplasmic sperm injection (ICSI) opened a new era in the field of assisted reproduction and revolutionized the assisted reproductive technology protocols for couples with male factor infertility. Fertilisation and pregnancies can be achieved with spermatozoa recovered not only from the ejaculate but also from the seminiferous tubules. The most common methods for retrieving testicular sperm in non-obstructive azoospermia (NOA) are testicular sperm aspiration (TESA: needle/fine needle aspiration) and open testicular biopsy (testicular sperm extraction: TESE). The optimal technique for sperm extraction should be minimally invasive and avoid destruction of testicular function, without compromising the chance to retrieve adequate numbers of spermatozoa to perform ICSI. Microdissection TESE (micro-TESE), performed with an operative microscope, is widely considered to be the best method for sperm retrieval in NOA, as larger and opaque tubules, presumably with active spermatogenesis, can be directly identified, resulting in higher spermatozoa retrieval rates with minimal tissue loss and low postoperative complications. Micro-TESE, in combination with ICSI, is applicable in all cases of NOA, including Klinefelter syndrome (KS). The outcomes of surgical sperm retrieval, primarily in NOA patients with elevated serum follicle-stimulating hormone (FSH) (NOA including KS patients), are reviewed along with the phenotypic features. The predictive factors for surgical sperm retrieval and outcomes of treatment were analysed. Finally, the short- and long-term complications in micro-TESE in both 46XY males with NOA and KS patients are considered.     

The current perspective on genetic and epigenetic factors in sperm maturation in the epididymis

Male infertility affects approximately 30% of infertile couples. As spermatozoa mature in the epididymal lumen, their potential for mobility increases, and their protein, lipid and small RNA (sRNA) content changes, whereas capacitation and fertilisation take place in the female reproductive tract. Both of the latter processes are affected by maturation, because impaired maturation causes premature capacitation and fertilization. The epididymis produces a suitable environment for sperm maturation via ion transport, vesicle secretion and protein matrix formation. The microenvironment for sperm maturation varies in three broad segments: the caput, the corpus and the cauda epididymis. Epididymosomes transfer proteins, lipids and sRNAs from the epididymal epithelium to spermatozoa and genetic alterations of epididymal genes can lead to decreased sperm motility, morphological abnormalities of spermatozoa and subfertility. Genetic factors are involved in all aetiological categories in male infertility. However, studies conducted on the genes involved in epididymal functions are limited. The sRNA content of spermatozoa changes during epididymal migration, and these sRNAs play a role in embryo development and epigenetic inheritance. This review aims to clarify the role of the epididymal epithelium in the maturation of spermatozoa in light of the current molecular genomic knowledge.     

精子染色质结构分析的临床意义

目的探讨女方不明原因流产和不明原因不孕与其配偶精子染色质性状之间的关系;方法通过精子染色质结构分析(SCSA)对22例不明原因流产和36例不明原因不孕妇女配偶精子,以及20例正常对照者精子进行DNA完整性检测,并比较它们之间的差异;结果对照组与不明原因流产组以及不孕组之间精子DNA完整性存在差异,且差异具有统计学意义(P<0．05),而不明原因流产组与不孕组之间精子DNA完整性比较无明显差异(P>0．05);结论精子染色质结构分析有可能作为不明原因流产和不孕的辅助诊断方法在临床应用。     

Sperm chromatin integrity in young men with no experiences of infertility and men from idiopathic infertility couples

Damage to the genetic component of spermatozoa seems to play the main role in a majority of cases where current approaches fail to reveal the specific cause of male infertility. In this study, we compared semen quality in men assigned to two defined groups: men from couples with unexplained infertility – idiopathic infertility (A) and young men with no experiences of infertility (B). All samples were examined by standard ejaculate analysis and sperm chromatin structure assay (SCSA). Sperm chromatin damage was significantly higher in men from group A than in those from group B. Similar results were obtained by comparison of men from group A (all men were normozoospermic) with normozoospermic men from group B. According to these results, we can suppose that chromatin disorders may be the causal factor of subfertility or infertility in some of these men. No evidence for a strong association between chromatin disorders and standard parameters of ejaculates was found. We failed to confirm a relationship between smoking and sperm quality in men from any of the investigated groups. SCSA is a method that facilitates the identification of infertile men who otherwise show normal semen variables.     

Correlation between the motility of frozen–thawed epididymal spermatozoa and the outcome of intracytoplasmic sperm injection

The purpose of this study was to investigate if the outcome of ICSI was influenced by epididymal sperm motility in frozen-thawed specimens. A total of 18 ICSI treatment cycles using spermatozoa retrieved by microsurgical epididymal sperm aspiration (MESA) were analysed retrospectively. Cryopreservation of epididymal spermatozoa was performed when enough epididymal aspirates were collected. Sixty-nine out of 126 oocytes injected with spermatozoa retrieved by MESA were fertilized, giving a fertilization rate of 54.8%. Out of 18 embryo transfer cycles, 6 (33.3%) achieved pregnancies. Fresh epididymal spermatozoa were used in 5 cycles while frozen-thawed epididymal spermatozoa were used in 13 cycles for ICSI. The fertilization rates were 68.6% (35/51) in the former group and 45.3% (34/75) in the latter group, respectively. There was a significant difference between the two groups (p < 0.05). In ICSI treatments using fresh epididymal spermatozoa, the cells used for injection were all motile. However, motile epididymal spermatozoa could be used in only five ICSI treatment cycles after freeze-thawing. In 6 cycles, only immotile sperm were used for injection of frozen-thawed spermatozoa. The fertilization rate in each group was 68.4% (13/19) and 31.6% (12/38), respectively. There was a significant difference between these groups (p < 0.01). These results indicate that the outcome of ICSI was influenced by sperm motility in frozen-thawed epididymal specimens. When no sperm motility could be recovered after freeze-thawing even with chemical treatments, consideration should be given to retrieving fresh epididymal spermatozoa again to achieve a better fertilization rate in such patients.     

Human sperm movement assessed with the Hamilton-Thorn motility analyzer and in vitro fertilization

Summary. The relationship between sperm movement characteristics obtained by computerized analysis and the in vitro fertilization rates of human oocytes was studied. In 144 consecutive in vitro fertilization treatments a sample of prepared semen was analysed by a Hamilton-Thorn Motility Analyzer. In addition a visual estimation of sperm count and motility was made. Significant correlations with the fertilization rate were found for all visual parameters. Of the computerized measurements, the mean velocities of motile spermatozoa and the concentration of motile cells were significantly correlated. The average path velocity correlated best ( r = 0.42, P < 0.001). There was no relationship between the percentage of motile sperm showing hyperactivated movement and the fertilization rate. A forward stepwise logistic regression analysis selected the following variables of predictive value for fertilization: average path velocity, male factor infertility as indication for in vitro fertilization, motility and concentration, as measured by the Hamilton-Thorn analyzer. A logistic regression model to predict the cases with low (< 0.2) or high fertilization rates, included the average path velocity as a significant variable and classified the samples with 90% overall accuracy. In conclusion: movement characteristics of spermatozoa in culture medium, especially the average path velocity are of prognostic value in prediction of human oocyte fertilization rates.     

Evaluation and assessment of semen for IVF/ICSI

Evaluation and assessment of semen is very important for both diagnosis of male infertility and selection of patients for treatment with IVF or ICSI. In standard IVF, sperm function is essential for normal fertilization: sperm must be able to bind to zona pellucida (ZP), undergo the acrosome reaction and penetrate the ZP and fuse with the oolemma before fertilization takes place. In contrast, most sperm functions are not required for fertilization in ICSI since sperm bypass the ZP and oolemma by injection of a single sperm directly into cytoplasm of oocyte. Therefore, the clinical decision on treatment of patients with either IVF or ICSI is mostly dependent on results of sperm tests. However, conventional semen analyses do not provide accurate information about sperm fertilizing ability since many patients with subtle sperm defects can not be detected. More advanced sperm function tests are required to detect sperm defects that may lead to failure of fertilization in standard IVF. In the last 15 years w