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     

精子功能检测与男性不育诊治的新进展

传统的精液常规分析是用于判断男性生育力的最基本临床指标,但是,只依靠精液分析的结果来预测男性生育状况仍是很不准确的。精子功能正常与否,对临床选择IVF还是ICSI治疗不育症极为重要。因为IVF需要功能完全正常的精子才能受精,而ICSI的受精只需要精子的正常核DNA,不需要其它的精子功能。在发明ICSI以前,患者IVF受精失败或低下(<30%)发生率很高(20%~35%)。研究证明,这些IVF受精失败的患者主要与精子功能障碍有关。常见的是少精子症,弱精子症和畸形精子症。但是有很多患者,精液分析结果仍正常。为了提高临床对精子功能测定的准确性,文献里有很多新的精子功能试验的研究报导,比如丫啶橙(AO)测定精子DNA、精子与透明带结合和穿透、顶体诱发精子顶体反应和精子与透明质酸结合试验。精子形态测定是常规精液分析中最重要的临床指标之一。但精子形态又是最难测定准确和稳定。IVF/ICSI受精失败的人卵可以用来测定精子功能。人卵透明带选择性地与正常形态和顶体完整的精子结合,透明带诱发的顶体反应与精子穿透明带的能力有很强的相关性。在不明原因的男性不育患者中,由于透明带诱发顶体反应障碍所导致的不育症占25%左右。少精子症(精子计数<2×106/ml)和严重精子形态畸形症(严格正常形态<5%)的男性不育患者,精子-透明带结合反应缺陷的发生率很高(>70%)。这类患者用IVF治疗受精率会很低,因此只能用ICSI治疗。精子与透明质酸结合试验与精子活力和形态有很强的相关性,但它不是很有用的精子功能试验。AO测定精子DNA对预测ART的受精和妊娠率的临床意义目前还没有肯定的结论,需要进一步研究。总之,在常规精液分析时,增加一些新的精子功能试验,在临床ART中对男性不育患者的诊治会有很大的帮助。     

The human acrosome reaction

We developed tests of sperm-oocyte interaction: sperm-zona binding, zona-induced acrosome reaction, spermzona penetration and sperm-oolemma binding, using oocytes which failed to fertilise in clinical in vitro fertilization (IVF). Although oocyte defects contribute to failure of sperm oocyte interaction, rarely are all oocytes from one woman affected. Low or zero fertilization in standard IVFwas usually caused by sperm abnormalities. Poor sperm-zona pellucida binding was frequently associated with failure of standard IVF and obvious defects of sperm motility or morphology. The size and shape of the acrosome is particularly important for sperm binding to the oocyte. The proportion of acrosome intact sperm in the insemination medium was related to the IVF rate. Inducing the acrosome reaction with a calcium ionophore reduced sperm-zona binding. Blocking acrosome dispersal with an acrosin inhibitor prevented spermzona penetration. Sperm-zona penetration was even more highly related to IVF rates than was sperm-zona binding. Some patients had low or zero fertilization rates with standard IVF but normal sperm by conventional tests and normal sperm-zona binding. Few of their sperm underwent the acrosome reaction on the surface of the zona and none penetrated the zona. In contrast, fertilization and pregnancy rates were high with intracytoplasmic sperm injection. We call thiscondition defective zona pellucida induced acrosome reaction. Discovery of the nature of the abnormalities in the signal transduction and effector pathways of the human zona pellucida induced acrosome reaction should result in simpler tests and treatments for the patients and also provide new leads for contraceptive development.     

体外受精中的受精失败

受精是雌雄配子结合形成合子的过程,它标志着一个新生命的开始,是自然界最神秘和复杂的事件之一.受精失败（Total fertilization failures, TFF）特指在体外受精（in vitro fertilization, IVF）培养过程的一定时段内[包括常规受精和卵胞浆内单精子注射（ICSI）],精卵没能结合为合子,形态学上没有看到精原核和卵原核形成的状态.     

Differential effects of polysulphates between mouse and hamster during in vitro fertilization

The interaction between zona pellucida polysulphates and sperm receptors appears to be a widespread mechanism used by mammals during gamete interaction. In this work, the effect of heparin on binding, penetration and fertilization of mouse and hamster oocytes was assessed. We found that heparin inhibited oocyte penetration and fertilization in both species. Heparin as well as fucoidan (a fucose-sulphate polymer) inhibited the proteolytic activity of acrosomal enzymes in both species. Our results suggest that zona pellucida penetration in both species may be modulated by polysulphates acting on either the proteolytic rate of degradation of the zona and/or its interaction with acrosome-reacted sperm (secondary binding).     

Seminal reactive oxygen species as predictors of fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization and intracytoplasmic sperm injection

High seminal reactive oxygen species (ROS) are related to poor semen quality and impaired fertilization. We aimed at finding whether there is an association between ROS and fertilization, embryo quality and pregnancy rates after conventional in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI). In prepared semen of 147 male partners of infertile couples, ROS were assessed with luminol chemiluminescence. Spermiogram was assessed in native semen. ROS were negatively correlated with standard sperm characteristics and testicular volume, and positively with abnormal sperm head morphology. Fertilization rate and embryo morphology on day 2 and on day 4 were assessed in 41 IVF and 106 ICSI cycles. The influence of maternal (female age and number of oocytes) and paternal (sperm motility, morphology and ROS) factors on fertilization and embryo quality were assessed by means of regression analyses. After IVF, fertilization and pregnancy rates were negatively associated with ROS level (p = 0.031 and 0.041, respectively). In case of higher ROS, significantly fewer ICSI-derived embryos (p = 0.036) reached the morula-blastocyst stage on day 4. High seminal ROS levels are associated with impaired sperm fertilizing ability and lower pregnancy rates after IVF. In ICSI, a negative association of ROS with embryo development to the blastocyst stage has been observed.     

Correlation between sperm penetration into the human zona pellucida and in vitro fertilization rates

Summary.  Sperm penetration into the zona pellucida of unfertilized oocytes, and its correlation with in vitro fertilization rates of the sibling oocytes, were assessed. This was performed in order to evaluate the prediction rate of the sperm penetration test into the zona pellucida. Unfertilized oocytes ( n =1872) from 371 cycles were pipetted through a microcapillary, and the remaining sperm cells penetrating the zona pellucida were counted. The mean (±SD) number of spermatozoa that penetrated the zona pellucida of unfertilized oocytes was 12.9±16.37. A significant correlation was found between the fertilization rate and the mean number of spermatozoa that penetrated into the zona pellucida of the unfertilized sibling oocytes (r = 0.48; P < 0.001), or the percent of unpenetrated zonae pellucidae in a cohort (r= —0.43; P < 0.001). However, a distinct variation in the number of spermatozoa that penetrated into the zona pellucida was detected. A step-wise regression analysis proved the number of spermatozoa penetrating the zona pellucida to be more predictive for fertilization rates than the variable of percent of unpenetrated zonae pellucidae. The results imply that although there is interdependence between penetration into the zona pellucida and fertilization rate, the predictive value of sperm penetration test for prognosis and future management after the first in vitro fertilization attempt, is limited.     

Predictive value of selected sperm parameters for classical in vitro fertilization procedure of oocyte fertilization

A proportion of fertilized oocytes during classical in vitro fertilization (IVF) procedure was analysed depending on the following factors: number of mature oocytes, seminological criteria such as sperm morphology in raw semen and after its selection in a density gradient (six structural defects of a male gamete were taken into consideration), sperm concentration, motility parameters according to World Health Organization criteria and the functional tests: hypo-osmotic swelling assay and acrosomal reaction induced by calcium ionophore. Evaluation of DNA content in sperm by image cytometry and determination of malonyldialdehydes in seminal plasma were also performed. Seventy-nine semen samples from patients undergoing IVF were assessed. Apart from significant correlations obtained for selected semen parameters and proportion of fertilized eggs, logistic regression analysis showed that the best predictive factors for oocyte fertilization were normal morphology of sperm before and after gradient selection, grade B and C of sperm movement in raw semen, and DNA content after density gradient centrifugation, which all accounted for 76.7% of fertilization predictive value.     

优选前后精子顶体酶活性与IVF受精率相关性的研究

目的探讨优选处理前和处理后精子顶体酶活性的变化,及与体外受精(IVF)受精率的相关性。方法采用分光光度比色法,对接受IVF治疗的53例不育夫妇男方精液,分别测定优选处理前和处理后精子顶体酶活性,分析其与IVF受精率的相关性。结果优选处理后精子顶体酶活性与优选前比较有显著性差异(P<0.05);达到常规IVF标准并选择常规IVF治疗者,优选后的精子顶体酶活性与IVF受精率有相关性,精子顶体酶活性降低与IVF受精率降低有关。结论精子顶体酶活性与IVF受精率有相关性,并且通过精子顶体酶活性可以预测IVF受精率。     

短时受精与受精结局

受精是一个复杂的过程,特征表现为多个连续步骤的串联反应.主要包括精子获能,精子和卵黄膜融合,卵母细胞恢复并完成减数分裂Ⅱ及排出第二极体,精子和卵母细胞的核开始去凝集,雄性和雌性原核形成,此后不久配子融合完成.     

Value of sperm characteristics and the result of in-vitro fertilization for predicting the outcome of assisted reproduction

The outcome of procedures for assisted reproduction, namely in-vitro fertilization (IVF) and gamete intrafallopian transfer (GIFT) were used as end-points to assess the capacity of particular sperm characteristics to predict the fertilizing potential of semen. In addition, the result of IVF performed with spare oocytes from the GIFT procedure, was used to predict the pregnancy outcome of GIFT. The power of sperm characteristics to predict the outcome of assisted reproduction procedures was poor. With regard to the outcome of IVF, sperm morphology was the best discriminant. The threshold value of 16% normal sperm morphology could be used as a good predictor of successful IVF, but could not be applied as a criterion to reject an IVF trial. For the latter purpose, the limit of 5% sperm with normal morphology should be used. The proportion of sperm with grade A motility in native semen provided the best discrimination between GIFT-fertile and GIFT-infertile cases. A negative result from IVF performed with spare oocytes was 93% accurate in predicting unsuccessful outcome of GIFT. Hence, diagnostic IVF is the most sensitive assay for male fertility. The occurrence of oocyte fertilization during IVF predicts a successful outcome of GIFT in only 50% of cases. Hence, a negative result in IVF indicates that semen is almost certainly infertile, but a positive result does not guarantee the successful outcome of GIFT.     

Lignosulfonic acid blocks in vitro fertilization of macaque oocytes when sperm are treated either before or after capacitation

Lignin-derived macromolecules (LDMs) are biologically active compounds that affect a variety of cell-to-cell interactions including the inhibition of fertilization and embryo development in a number of nonmammalian species. The effect of ligno-sulfonic acid (LSA), a highly sulfonated LDM, on cynomolgus macaque sperm-oocyte interaction was evaluated with a zona pellucida binding assay and by in vitro fertilization (IVF). Sperm were treated with LSA (1.5 mg/mL) either before washing or after capacitation. Capacitation included centrifugation through 80% Percoll followed by 2 consecutive washes with medium, overnight incubation, and activation with dibutyryl cyclic adenosine monophosphate and caffeine. The zona binding assay was performed using immature oocytes that had adhered to the center of glass "binding chambers." The number of capacitated sperm that attached to the zona over a 3-minute period was recorded. Sperm attachment was significantly inhibited by LSA as compared to controls whether treatment occurred after capacitation (92.5%; P <.001) or before washing (82.5%; P <.001). When sperm were treated similarly with fucoidin, a sulfated polysaccharide known to inhibit sperm-oocyte interaction, sperm-zona binding was significantly inhibited by postcapacitation treatment but not by prewash treatment. Treatment of sperm with LSA consistently blocked fertilization over 4 IVF cycles both before washing and after capacitation. Fertilization rate for controls was 65% +/- 17%. No LSA-treated sperm were observed on the surface of lightly rinsed oocytes after 4 hours of coincubation. Localization of biotinylated LSA showed labeling over the entire sperm surface with the greatest intensity observed over the head and midpiece. LSA treatment had no effect on the percentage of motile sperm or quality of sperm motility. Due to the antifertility properties of this nontoxic molecule, LSA appears to have potential as a vaginal contraceptive.     

In vitro tests for essential sperm functions using the phyto-oestrogen genistein as a test substance

Sperm motility, binding of spermatozoa to the zona pellucida and induction of the acrosome reaction are prerequisites for successful oocyte fertilization. Examination of the physiological and nonphysiological effects of particular compounds on sperm functions requires high-quality in vitro test systems. In this short methodological overview, a reliable combined in vitro test system with bovine gametes is described. The purpose of the study was to evaluate whether aliquots of pooled post-thaw spermatozoa are suitable for examination of environmental substances that affect essential sperm functions. The combined test system includes a number of known methods for the assessment of sperm vitality and motion parameters, acrosomal status, inducibility of acrosome reaction and sperm zona pellucida binding. First observations indicate that genistein inhibits the induction of acrosomal exocytosis and binding of spermatozoa to the zona pellucida. Motility parameters and the viability of bovine spermatozoa were not affected by this substance. It is concluded that genistein, a phyto-oestrogen which is abundant in several plants, can be used as a test substance for the evaluation of effects upon essential bovine sperm functions in vitro.     

In vitro sperm-binding assay to distinguish differences in populations of human sperm or damage to sperm resulting from cryopreservation.

Annually, >1.3 million men are members of couples seeking help because of infertility. Semen from many of these men contains reasonable numbers of motile and normal sperm, but for a subset of individuals, many sperm are deficient in ability to bind to the zona pellucida during in vitro fertilization. Diagnosis of this defect has been hampered by lack of a low-cost test. Molecular similarity exists between the perivitelline membrane of a hen's egg and the mammalian zona pellucida. These facts and some preliminary data led to evaluation of binding of human sperm during incubation for 60 minutes at 37 degrees C to an extract of chicken perivitelline membrane coated in microwell assay plates. The sperm-binding assay had inter- and intraassay plate variations of 21 and 12%, respectively, using washed fresh sperm. All seminal samples were normal, except a few that had 36 to 50% motile sperm with a low rate of sperm movement (if there is a low rate of movement, World Health Organization [WHO] criterion for normalcy is >50% motile). Nevertheless, this sperm-binding assay detected differences among individuals in percentage of sperm bound. Based on data for two to four ejaculates from each of eight occasional sperm donors, the coefficient of variation for ejaculates within donor averaged 31%, and means for the donors differed (P < 0.02). Percentage of sperm bound ranged from <1 to 38% for fresh semen from 57 men and from <1 to 13% for frozen-thawed semen from 34 men. Percentage of motile sperm accounted for <30% of the variation in percentage of sperm bound. In a direct comparison based on 17 ejaculates, aliquots evaluated fresh averaged 13% sperm bound, versus 2% for frozen-thawed aliquots. We concluded that the egg membrane substrate used in these microwell assay plates might serve as the basis for a diagnostic assay. However, it remains to be established whether samples of human semen with a low percentage of sperm binding indeed have relatively low fertilizing potential.     

Seminal elastase-inhibitor complex, a marker of genital tract inflammation, and negative IVF outcome measures: role for a silent inflammation?

There is no consensus on whether and how male genital tract inflammation affects sperm fertilizing potential. The aims of this prospective study were to evaluate the elastase-inhibitor complex in seminal plasma (s-EI) level, a marker of male genital tract inflammation, in men undergoing in vitro fertilization (IVF) having no clinical signs of inflammation, and its association with semen characteristics, and the predictive role of s-EI for the outcome of IVF in terms of fertilization, embryo development to the blastocyst stage and pregnancy. The study involved 104 male partners of infertile couples with normal spermiogram undergoing IVF. On the day of oocyte retrieval, spermiogram and s-EI assessment using homogeneous immunoassay were performed. The outcome of IVF according to the s-EI level was assessed in 85 cycles with two or more oocytes. In 67 cycles embryos were cultured to the blastocyst stage. Spearman's and Pearson's correlation tests, chi-square test, and multiple linear and logistic regression analyses were used for statistical evaluation. Increased s-EI level (> or =250 microg/L) was detected in semen of 32% of men, and in 24% of men without leucocytospermia. We found an association between the s-EI level and semen leucocytes (r = 0.49, p = 0.004) but not with classical sperm characteristics. No correlation between the s-EI level and fertilization was observed. Increased s-EI levels were associated with a poorer blastocyst development rate (p = 0.03) and a higher number of arrested embryos (p = 0.04). Extended embryo culture to the blastocyst stage shows a negative effect of clinically silent male genital tract inflammation on embryo developmental potential.     

Effect of bariatric surgery on in vitro fertilization in infertile men with obesity

BackgroundObesity has previously been related to reduced female fertility, with prolonged waiting time to pregnancy among women with a body mass index (BMI) >35 kg/m² but there are few studies investigating the relationship between high BMI, bariatric surgery, and male fertility.ObjectivesThe primary objective of this article was to investigate the effect of bariatric surgery on in vitro fertilization (IVF) outcomes in a cohort of men with morbid obesity who underwent sleeve gastrectomy (SG).SettingUniversity hospital, bariatric surgery unit.MethodsPre- and postsurgery data on patient age, body mass index (BMI), and variables related to male fertility (semen volume, concentration, progressively motile sperm count, and sperm morphology) were collected; assisted reproductive technology outcomes before and after bariatric surgery were measured by the number of metaphase II oocytes; the number of top-quality oocytes and embryos; the number of fertilized oocytes; the number of transferred embryo; the implantation rate; the pregnancy rate; the live birth rate and the miscarriage rate.ResultsThirty-five men with obesity and idiopathic infertility were included in this study. We found a significant increase, after bariatric surgery, in semen volume, total sperm concentration, progressively motile sperm count, and sperm morphology. Considering IVF outcomes, mean number of top-quality oocytes, mean number of fertilized oocytes, mean number of embryos obtained, and top-quality embryos were significantly increased after bariatric procedure.ConclusionBariatric surgery is confirmed to be safe and effective in increasing the outcomes of assisted reproductive technology treatment also in case of infertile men with obesity, both in terms of pregnancy and live birth rate.     

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.     

Teratozoospermia and its effect on male fertility potential

Teratozoospermia and its significance for male fertility problems is discussed. Different points of view and available information concerning the phenomenon of teratozoospermia have been presented. Ongoing debate about the influence of sperm morphology on male infertility and IVF results emphasizes the fact that when sperm morphology is evaluated using strict criteria, this parameter is thought to have an excellent predictive value and shows a significant positive correlation with successful fertilization. Selective abilities of zona pellucida to differentiate between morphologically normal and abnormal spermatozoa, the superior role of acrosomal morphology assessment in the prediction of IVF outcome, and the involvement of cell surface molecules in the adhesion of spermatozoa to oocytes are described. The usefulness of the hamster oocyte penetration assay (HOPA), which is important not only because of its predictive value of sperm function, but also because of its role as a tool for visualizing sperm chromosomes, was found to be questionable in cases of teratozoospermia. Cytogenetic findings related to teratozoospermia are controversial as classical methodology of visualizing sperm chromosomes (through HOPA) may cause selection of sperm metaphases which can be obtained only from good-penetrating spermatozoa. There are few reports presenting chromosome complements of abnormal sperm visualized with the help of micromanipulation. There is clear evidence that a variety of genetic defects can affect spermatogenesis. The possible genetic background of teratozoospermia is discussed.     

The hemizona assay (HZA) as an experimental model to evaluate the inhibition of sperm binding to the murine zona pellucida by isolated zona pellucida protein

Compelling evidence has demonstrated that zona binding represents gamete recognition by sperm binding with high affinity and specificity to complex glycoproteins of the zona pellucida. In the present study we evaluated the hemizona assay (HZA) in the investigation of the interaction of mouse spermatozoa with unfertilized murine oocytes and hemizonae after exposure to solubilized murine zonae pellucidae proteins. The zonae pellucidae were isolated from ovarian tissue following described mincing techniques. The sperm binding characteristics of murine spermatozoa were studied by using SDS-PAGE isolated ZP2 (+/- 120 Kd) and ZP3 (+/- 83 Kd) components of the zona pellucida. Sperm receptor activity was examined in a competitive gamete binding fashion using the HZA as an indicator of sperm/zona interaction. The results illustrated that isolated, solubilized ZP2 and ZP3 glycoprotein moieties of the zona pellucida inhibited sperm binding to hemizonae and oocytes when compared to controls, and that the HZA can be utilized as an internally controlled homologous bioassay to evaluate the effects of zona pellucida proteins on tight binding of spermatozoa to the zona pellucida.     

Decline of semen quality during IVF is not associated with subjective male stress

The aim of the present study was to assess if semen quality declines during in vitro fertilization （IVF） and whether or not this phenomenon is triggered by chronic male stress. In order to test this hypothesis, we first investigated a retrospective cohort of 155 male IVF patients （testing cohort）. Subsequently, we started a prospective cohort study in men undergoing their first IVF and assessed semen quality and subjective male chronic stress using a validated tool, i.e. the Fertility Problem Inventory （FPI） questionnaire. The association between stress and sperm quality decline measured 4-6weeks before the start of IVF （T1） and at the day of oocyte retrieval （T2） was the primary outcome. Live birth rate, first trimester abortion and rate of poor responders were secondary outcomes. In the testing cohort, mean progressive motility, but not mean sperm density significantly declined. There were 78/154 （51%） men who showed a decline in semen density and 50/154 （32%） men who showed a decline in progressive motility. In the validation cohort, progressive motility declined, whereas, sperm density increased from T1 to T2. Of 78 men, 27 men had increased stress （FPI-score 〉 146）. Sperm density and progressive motility were not significantly different in men with and without stress. However, in the presence of male stress, couples had a higher rate of poor responders, miscarriages and a lower rate of live births. Subjective stress is not associated with a decline in semen quality observed during IVF but may be associated with adverse ore~nancv outcome.