The Acridine Orange test: determining the relationship between sperm morphology and fertilization in vitro.

The prediction of human fertilization is an important aspect of research protocols dealing with male fertility. Sperm DNA has been reported to be an indicator of human sperm fertility potential. The Acridine Orange test for evaluation of sperm DNA has been employed during the present study to determine its relationship with human sperm morphology and fertilization in vitro. Seventy-six patients from the in-vitro fertilization (IVF) and/or gamete intra-Fallopian transfer (GIFT) programme were randomly selected for the study. All patients had a routine semen analysis, sperm DNA evaluation and underwent standard IVF procedures at the time of the study. The results indicated a moderate positive correlation (r = 0.38, P = 0.0006) between results of the Acridine Orange test and normal sperm morphology. Patients with an Acridine Orange test value exceeding 24% had significantly higher oocyte fertilization rates than patients with lower values, for metaphase I (74 versus 51%, P = 0.0008) and for metaphase II oocytes (88 versus 60%, P = 0.0001). Sperm morphology, however, proved to be a more significant predictor of fertilization in vitro compared to the Acridine Orange test.     

A simple method for the morphological analysis of human ova: zona penetration in oocytes failing to fertilize or fertilizing abnormally in vitro

A simple and rapid technique is described whereby several unfertilized oocytes and embryos can be processed simultaneously for embedding and sectioning for histological analysis. Oocytes are encased in a serum matrix and thereafter can be handled with ease. A total of 112 unfertilized oocytes and abnormal embryos following insemination in vitro have been examined. Only 31% of oocytes showed pyknotic nuclei after a mean of 4.0 +/- 1.24 days in culture and 37% had recognizable metaphase II chromosomes. Twenty per cent showed evidence of fertilization, but spermatozoa had failed entirely to penetrate the zonae pellucidae of 34% of oocytes. The mean percentages of penetrating sperm were not significantly different between oocytes which did and did not fertilize. Under these in-vitro conditions, approximately 1-2% of spermatozoa reaching the oocyte penetrated into the zona pellucida and a lesser number to the perivitelline space. Polar bodies were also observed and the incidence of divisions, retention and parthenogenesis estimated. This technique may advance our understanding of the reasons for the success or failure of fertilization in vitro.     

Andrology: Disordered acrosome reaction of spermatozoa bound to the zona pellucida: a newly discovered sperm defect causing infertility with reduced sperm-zona pellucida penetration and reduced fertilization in vitro

It is believed that during the process of human fertilization,acrosome-intact spermatozoa bind to the surface of the zonapellucida which triggers the acrosome reaction and the enzymesreleased facilitate sperm penetration through the zona pellucida.We describe here reduced frequency of the acrosome reactionon the zona pellucida as a cause of infertility in 10 coupleswith long durations of infertility (average 6 years) and low(<15%, n= 3) or zero (n= 7) fertilization rates in vitro.Sperm concentration, motility, velocity (Hamilton-Thorn), morphologyand DNA normality were within the normal range in all the patients.Electron microscopy of spermatozoa did not reveal any specificultrastructural defects. All couples were negative for antispermantibodies by immunobead tests. Oocytes from other patientswhich failed to fertilize in in-vitro fertilization and normaldonor spermatozoa were used as controls for sperm-zona pellucidabinding and penetration experiments. Acrosome status of spermatozoabound to the zona pellucida was assessed with a fluorescentlectin and electron microscopy. The mean number of spermatozoabound to the zona pellucida was not significantly differentbetween patients and controls. However, the acrosome reactionof spermatozoa bound to the zona pellucida after 2 h incubationwas significantly lower (P< 0.001) in the patients (mean5%, range 0–16) than in the controls (mean 68%, range44–96). No zona pellucida (out of 40) was penetrated bypatient spermatozoa whereas most (39/40) zonae were penetratedby control spermatozoa (average 27 spermatozoa/four zonae pellucidae).The spontaneous acrosome reaction of spermatozoa in inseminationmedium was not different between patients (4%) and controls(3%), the acrosome reaction induced by calcium ionophore waslow (21 and 43% respectively) in six of the eight patients examined.In conclusion, these patients have spermatozoa with a disorderof the zona pellucida-induced acrosome reaction that resultsin failure of sperm-zona pellucida penetration and explainstheir infertility.     

Regulators of sperm function: Composition of the human zona pellucida and modifications following fertilization

The composition of individual human zonae pellucidae and modificationsto this extracellular coat both before and after fertilizationwere analysed using a rapid, sensitive, non-radioactive biotinylation-or lectin-based detection system; these assays use commerciallyavailable reagents and can be performed on fragments of individualzonae pellucidae. The zona pellucida from unfertilized eggsis composed of three glycoprotein species designated as huZP1,huZP2 and huZP3. Under non-reducing conditions, the molecularweights of these proteins are 150 kDa, 100 kDa, and 55–65kDa respectively. Following fertilization, huZP1 was not detectedunder either non-reducing or reducing conditions. In contrast,after fertilization huZP2 was detected under non-reducing conditions,but not under reducing conditions. The ability to detect pre-and postfertilization changes in a single human zona pellucidais discussed in relation to its value in assessing deficienciesin clinical and laboratory protocols used for in-vitro fertilization.     

Sperm morphological assessment based on strict criteria and in-vitro fertilization outcome.

One-hundred-and-twenty-three in-vitro fertilization (IVF) cycles were analysed in order to clarify the influence of strictly normal morphology (SNM) of spermatozoa on IVF outcome. SNM was defined using strict criteria according to Kruger with our modifications. The IVF cycles studied were divided into three groups: %SNM less than 12% (13 cycles), 12 less than 40% (68 cycles), greater than or equal to 40% (42 cycles). The cleavage rates per oocyte were higher in the groups with 12-40% and greater than or equal to 40% of %SNM than in the group with %SNM less than 12%. The embryo transfer rate per cycle increased with increasing %SNM. The overall pregnancy rate per cycle increased with increasing %SNM (7.7% in %SNM less than 12%, 22.1% in 12-40% of %SNM, and 40.5% in %SNM greater than or equal to 40%). The ongoing pregnancy rate per cycle also increased with increasing %SNM (7.7% in %SNM less than 12%, 14.7% in 12-40% of %SNM, and 31.0% in %SNM greater than or equal to 40%). The miscarriage rate was lower in %SNM greater than or equal to 40% (23.5%) than in 12-40% of %SNM (33.3%). It was suggested that %SNM is a good predictor of IVF outcome.     

Blocking effect of antisera to recombinant zona pellucida proteins (r-ZPA) on in vitro fertilization

PROBLEM: The zona pellucida is a good target antigen for contraceptive vaccines due to its strong immunogenicity and high tissue specificity. However, this contraceptive effect is inevitably associated with ovarian failure. Therefore, it is necessary to define an epitope of the zona antigen to which the antibody produced inhibits fertilization without any undesirable side effects. METHOD OF STUDY: The DNA fragment coding for the NH2-terminal region of porcine zona pellucida proteins (ZPA) (1-198 amino acids) and human ZPA (1-206 amino acids) was prepared to produce recombinant porcine ZPA (r-ZPA), r-pZPA1-198 and r-hZPA1-206. Using Freund's complete adjuvant. antisera against these proteins were raised in rabbits. RESULTS: The resultant antisera to r-pZPA1-198 and r-hZPA1-206 were cross-reacted with each other on enzyme-linked immunosorbent assay and immunofluorescent staining. The antiserum to r-pZPA1-198 inhibited in vitro fertilization in pigs, but not human sperm binding to the zona pellucida, while the antiserum to r-hZPA1-206 inhibited the human sperm-binding assay. CONCLUSIONS: Antiserum to r-ZPA inhibited fertilization in the animal species possessing a homologous amino-acid sequence as an immunogen. The recombinant protein, r-hZPA1-206 seems to be a feasible candidate for the development of contraceptive vaccines for humans.     

The hypo-osmotic swelling test and the sperm mucus penetration test in determining fertilization of the human oocyte

The usefulness of the hypo-osmotic swelling (HOS) test and thesperm mucus penetration (SMP) test as sperm function tests forin-vitro fertilization was analysed in 56 couples. Using logisticregression analysis only the SMP test was independently relatedto fertilization (P = 0.004), no false negative results wereobtained, i.e. no fertilization if sperm from the ejaculatefailed to penetrate mucus. The HOS test was of no predictivevalue. The results justify a further examination of the SMPtest in other IVF centres.     

Immunology: Assessment of the relevance of zona pellucida antibodies in follicular fluid of in-vitro fertilization (IVF) patients

The presence of anti-zona pellucida antibodies in the follicularfluid of 11 women who underwent in-vitro fertilization (IVF)and embryo transfer was analysed. Only infertile couples withtubal or unexplained pathologies were included in our study,which was aimed at investigating the relationship between anti-zonapellucida antibodies in follicular fluid and failed fertilization.Whether or not these antibodies were present in some or allfollicles in the same patient was also investigated. Out of55 follicular fluids analysed, 36.3% were positive to the testand no fertilization was observed in oocytes from these follicles,while 63.6% were negative, and the oocyte fertilization rateassociated with these was 51.4%. The presence of anti-zona pellucidaantibodies was positively correlated with the degree of fertilizationfailure (P <0.001 x² test).     

精子DNA碎片率与男性年龄、精子活动力和体外受精结局关系的研究

目的 探讨精子DNA碎片率(DNA fragmentation index,DFI)与男性年龄、精液检查指标、体外受精(in vitro fertilization,IVF)的受精率、优质胚胎率、周期妊娠率和胚胎着床率等关系.方法 随机选取本院生殖中心111例IVF患者,使用流式细胞仪行精子DFI测定,DFI值按不同界...     

Usefulness of the hypo-osmotic swelling test for evaluation of human sperm fertilization.

We examined spermatozoa from 135 male patients consulting for infertility to evaluate the usefulness of the hypo-osmotic swelling (HOS) test of sperm fertility for the swim-up washing method. HOS test values significantly improved following the treatment of spermatozoa using the swim-up washing method. There were no differences in the rate of sperm motility between the normal group and the oligozoospermic group following treatment, but the HOS test identified significant differences in swelling rates in both groups before and after treatment. The results of the HOS test following swim-up washing were higher during fertilization attempts for 11 cases of successful intra-uterine insemination (IUI) than during unsuccessful IUI attempts; there were no significant differences between the control group and the successful IUI cases. Moreover, using the lower-normal limits for overall sperm swelling (52%) and g-type sperm swelling (30%) obtained from a normal control group, we found that swelling rates were higher when IUI was successful. These findings indicate that the HOS test is an effective measure of sperm fertilizing function when the swim-up washing method is used for sperm treatment. The score of g-type sperm swelling can be used as a substitute for overall sperm swelling.     

Efficacy of the sperm survival test for the prediction of oocyte fertilization in culture

The present study was carried out to investigate the predictivevalue of the sperm survival test (SST) with respect to the fertilizationof oocytes in culture. In general, our laboratory uses a totalof 50 000–150 000 motile spermatozoa to inseminate eachoocyte. The remaining material is evaluated for motility beforeand after 24 h of incubation at 37°C in a 5% CO₂ atmosphere.A total of 250 oocytes from 50 cases (mean ± SD, 5.0± 2.4 oocytes per retrieval) were inseminated and thefinal rate of cleaved embryos obtained was 52.5%. The SST (%)was considered normal when the ratio (final density of progressingspermatozoa after 24 h x 100/initial density of progressingspermatozoa) was 50% or more. Any other result was consideredabnormal. Cases presenting one or more cleaved embryos (n =40) were separated from those in which no embryo formation occurred(n = 10) and the results were compared in terms of the respectivesperm survival rates over a period of 24 h: normal SST (oneor more cleaved embryos, 37; none, five), abnormal SST (oneor more cleaved embryos, three; none, five). The specificityof the SST was 0.92 and sensitivity 0.50, the predictive valueof the abnormal test was 0.62 and the predictive value of thenormal test 0.88. The efficacy of the test was estimated at0.71, which was better than the conventional parameters of spermanalysis. A receiver — operating characteristics curvefor SST confirmed that the test can be useful for the predictionof fertilizability of oocytes in the laboratory.     

Multiple proteins present in purified porcine sperm apical plasma membranes interact with the zona pellucida of the oocyte

An important step in fertilization is the recognition and primary binding of the sperm cell to the zona pellucida (ZP). Primary ZP binding proteins are located at the apical plasma membrane of the sperm head. In order to exclusively study primary zona binding proteins, plasma membranes of sperm heads were isolated, highly purified and subsequently solubilized with a mild or a strong solubilization procedure. Native, highly purified ZP ghosts were used as the binding substrate for solubilized sperm plasma membrane proteins, and a proteomic approach was employed to identify ZP binding proteins. Two-dimensional gel electrophoresis of ZP fragments with bound sperm proteins showed very reproducibly 24 sperm protein spots to be associated to the zona ghosts after mild plasma membrane solubilization whereas only three protein spots were detected after strong plasma membrane solubilization. This indicates the involvement of multiple sperm proteins in ZP binding. The three persistently bound proteins were identified by a tandem mass spectrometry as isoforms of AQN-3 and probably represent the main sperm protein involved in ZP binding. P47, fertilin beta and peroxiredoxin 5 were also conclusively identified. None of the identified proteins has a known acrosomal origin, which further indicated that there was no sample contamination with secondary ZP binding proteins from the acrosomal matrix. In this study, we showed and identified multiple zona binding proteins involved in primary sperm-zona binding. Although we were not able to identify all of the proteins involved, this is a first step in understanding the event of primary sperm-zona interactions and the relevance of this for fertilization is discussed.     

A new sperm collection method for in-vitro fertilization: collection at tail of epididymis.

We have developed a method for the collection of spermatozoa from the tail of the epididymis in patients with severe asthenozoospermia. The treatment was applied to 25 cycles, of which 16 cycles provided spermatozoa in good condition with concentration and motility greater than 50 x 10(4)/ml and greater than 50% respectively. Spermatozoa thus obtained were subjected to IVF in 11 cycles and to ZIFT in five cycles; five IVF cases reached cleavage and two of those cases resulted in pregnancy. Two of the five ZIFT cases also achieved a pregnancy. Despite limits on frequency, the sperm collection process is thought to be very useful for the treatment of severe asthenozoospermia.     

Infertility: The prognosis for assisted conception treatment after unexpected failure of fertilization in vitro: a comparative study

The relative prognosis for further assisted conception treatment(without micro-injection) after initial unexpected failure offertilization in apparently favourable couples undergoing in-vitrofertilization (IVF) treatment was assessed. After their firstcycle of treatment, 481 consecutive couples were grouped accordingto their fertilization (including cleavage) rate per oocyteinto five bands. Proportions of couples proceeding to furthercycles of treatment by IVF or gamete intra-Fallopian transfer(GIFT) and resulting fertilization and pregnancy rates werecompared. Pregnancy rates in the first cycle of treatment weresignificantly related to fertilization rate. The fertilizationrate was zero in 13 couples (3%) and only 1–24% in 18(4%). There were no significant differences between these groupsin the proportions proceeding to further treatment (31, 50%)compared with others (overall 37%, including some treated byGIFT), or in their median fertilization rates (75, 60% comparedwith 67% – IVF cycles only), pregnancy rates (20, 38%of cycles compared with 37% – IVF or GIFT) or birth rates(20, 38% of cycles compared with 31% – IVF or GIFT). Amongstcouples whose initial fertilization rate was 50% there wasno fertilization in 4% of subsequent IVF cycles. We concludethat in couples with well defined favourable conditions, includingtests of sperm function for assisted conception treatment, whohave unexpected failure of fertilization, the prognosis forfurther treatment remains favourable without resort to morecomplex investigations or micro-injection methods. Such failureoccurs infrequently and generally as a random event, and shouldhave no appreciable effect on life-table calculation of cumulativepregnancy and birth rates in this group of patients.     

Andrology: A new predictive test for in-vitro fertilization based on the induction of sperm acrosome reaction by N-acetylglucosamine-neoglycoprotein

Neoglycoproteins with N-acetylglucosamine residues (BSA-GIcNAc)induced specifically the acrosome reaction (AR) in human spermatozoa.Our objective was to investigate the relationship between thisphenomenon and the in-vitro fertilization (IVF) rate. Spermsuspensions from IVF protocols were incubated with BSA-GlcNAc(t), using calcium ionophore (i) or medium alone (c) as positiveor negative controls. When the normalized AR percentage ratio(STIM) (%ARt-%ARc): (%ARi-%ARc) was compared with fertilizationrate in 31 couples from our IVF programme, a positive correlationwas found (r = 0.46, P < 0.01). The fertilization rate inpatients with STIM 0.2 was higher than in non-responders (STIM< 0.2); 72 ± 7% compared with 5 ± 3%. The overallpredictive value of this test for adequate fertilization rate(>30%) was 87%, sensitivity 91% and specificity 78%. Falsepositives were 9% and false negatives 22%. For successful fertilizationrates (>60%), the results were: overall predictive value,84%; sensitivity 100%; specificity 64%. False positives were23% and no false negatives were found. The results indicatedthat the induction of AR in human spermatozoa by GlcNAc-neoglycoproteinscould be used to predict their fertilizing ability in vitro.     

人抗卵透明带抗体阳笥血清对小鼠体外受精和早期胚发育的影响

本研究在小鼠体外受精和早期胚胎发育中添加抗卵透明带抗体阳性不孕妇女血清(n＝11）,结果显示：与对照组相比较,6例完全阻断受精和发育（P＜0.01）;2例仅阻断受精,不明显抑制发育;3例则与对照组无显著性差异（P＞0.05）。提示人抗卵透明带抗体的主要效应是阻断受精,并可以进一步抑制胚胎早期发育,但效应的程度表现出个体差异。     

Effects of sera from infertile women with sperm immobilizing antibodies on fertilization and embryo development in vitro in mice

PROBLEM: This study was performed to investigate if patients' sera with anti-human sperm antibodies show inhibitory effects on in vitro fertilization (IVF) and embryo development in mice. METHOD OF STUDY: Patients' sera were collected from eight infertile women having sperm immobilizing antibodies and 17 infertile women without the antibodies. Male ICR mice and female F1 mice (BALB/c X C57BL/6J) were used. In mouse IVF, pre-incubated sperm were cultured in the medium containing patient's serum with or without sperm immobilizing antibodies, or bovine serum albumin (BSA) as a control. The fertilization rates and the incidences of blastocyst formation were compared. RESULTS: A mouse sperm immobilization test was established. Five (62.5%) of eight serum samples with sperm immobilizing antibodies and nine (52.9%) of 17 serum samples without the antibodies showed sperm immobilizing activities in mice. There was no significant difference between the two groups. Five sera with sperm immobilizing activities in human and mice, and five sera without sperm immobilizing activities in human or mice were used for the further experiments. The fertilization rates in BSA, patient's serum with sperm immobilizing antibodies, and that without the antibodies were 82.5% (746/904), 43.6% (508/1165), and 64.5% (669/1037), respectively. There were significant differences between the groups. The incidences of blastocyst formation were 59.9% (447/746), 31.7% (161/508), and 47.7% (319/669), respectively. There were also significant differences between the groups. CONCLUSIONS: Some of the patient's serum with and without sperm immobilizing antibodies could immobilize sperm with complement. However, as compared with control, sera with sperm immobilizing activities against human and mouse sperm significantly blocked IVF and inhibited embryo development in mice. Further studies are required to investigate the mechanisms of the blocking effects of antisperm antibodies on fertilization and embryo development using the mouse model.     

Acrosome status and morphology of human spermatozoa bound to the zona pellucida and oolemma determined using oocytes that failed to fertilize in vitro

It is known that only acrosome-reacted spermatozoa can fusewith the oolemma during normal fertilization with zona pellucida-intactoocytes. The aim of this study was to determine if the oolemmaof human zona pellucida-free oocytes selectively binds spermatozoawith normal morphology and a reacted acrosome. Oocytes thatfailed to fertilize in vitro because of severe sperm defectswere used. The zona pellucida was removed with acidic (pH 2–3)saline. Sperm samples were obtained from normal fertile donorsand normozoospermic men. Motile spermatozoa were selected witha swim-up technique and 2x10⁶/ml incubated with oocytes. Theresults from 23 experiments showed that at 2 h there was a significantlyhigher mean percentage of acrosomereacted spermatozoa boundto the zona pellucida (mean ±SD, 42±22) than inthe insemination medium (27 ± 12). In contrast, all spermatozoabound to the oolemma at 2h were acrosome reacted. Furthermore,each fresh zona pellucida had>100 spermatozoa bound comparedwith an average of 28 (range 4–81) spermatozoa bound perzona pellucida-free oocyte. There was no significant differencein the zona pellucida-induced acrosome reaction between fresh(45 ± 21)and salt-stored (35 ± 22) zonae pellucidae.The percentage with normal morphology was significantly higherfor spermatozoa bound to the zona pellucida (84 ± 13)and oolemma of zona pellucida-free oocytes (71 ± 25)than for spermatozoa in insemination medium (39 ± 11)(P<0.01). Extending the time of incubation of spermatozoawith zona pellucida-intact oocytes increased the proportionof spermatozoa undergoing the acrosome reaction (n=6, 2 h, 41± 23; 3 h, 53 ± 31; 4 h, 61 ± 34). However,there was a large variation in the percentage of acrosome reactionsamong spermatozoa bound to the zona pellucida between individualsperm samples. In conclusion, only acrosome-reacted spermatozoacan bind to the oolemma of zona pellucida-free oocytes. Bothfresh and salt-stored human zonae pellucidae are equally effectivein inducing the acrosome reaction. Both the zona pellucida andthe oolemma of zona pellucida-free oocytes selectively bindspermatozoa with normal morphology     

部分卵胞浆内单精子显微注射112例分析

目的分析本中心行部分卵胞浆内单精子显微注射的病例,探讨half-ICSI的指征的可行性。方法选择2004年1月至2008年12月在本中心行half-ICSI的112个周期,按适应症分三组,A组为男方中度少弱畸精子症,B组为前次IVF受精失败或低下(30%),C组为不明原因不孕,以同期常规IVF治疗的1377个周期为对照,分别比较三组中发生受精失败或低下发生率(受精率30%)情况的比例,以及half-ICSI的效果。结果 half-ICSI的112个周期共获卵1633个,行IVF766个卵,受精率为54.7%,其中完全不受精27个周期,受精低下(30%)11个周期,行ICSI部分867个卵,MII765个,受精645个,受精率84.3%,移植周期临床妊娠率为39.4%(其中IVF完全不受精者临床妊娠率为32.0%)。112个周期有38个周期发生受精失败或低下,发生率为33.9%(38/112),其中A组发生率31.8%,B组50.0%,C组30.8%。对照组发生受精失败或低下率为7.0%(96/1377),结果有显著性差异(P0.05)。结论对存在高危因素的病人在同一治疗周期中行half-ICSI可明显减少因不受精无胚胎移植的情况出现,从而提高妊娠率,又因有IVF胚胎移植,可减少ICSI子代遗传和先天缺陷的风险。